Publications by year
In Press
Brask JB, Croft D, Edenbrow M, James R, Bleakley BH, Ramnarine IW, Heathcote R, Tyler C, Hamilton P, Dabelsteen T, et al (In Press). Evolution of non-kin cooperation: social assortment by cooperative phenotype in guppies.
Royal Society Open Science Full text.
Witherall L, Wagley S, Butler C, Tyler C, Temperton B (In Press). Genome sequences of four Vibrio parahaemolyticus strains isolated from the English Channel and the River Thames.
Microbiology Resource Announcements Full text.
Stevens CH, Croft DP, Paull, Tyler (In Press). Stress and welfare in ornamental fishes: what can be learned from aquaculture?.
Jounral of Fish Biology Full text.
Edenbrow M, Bleakley BH, Darden SK, Tyler CR, Ramnarine IW, Croft DP (In Press). The evolution of cooperation: Interacting phenotypes among social partners.
The American Naturalist Full text.
2021
Pinheiro JPS, Windsor FM, Wilson RW, Tyler CR (2021). Global variation in freshwater physico-chemistry and its influence on chemical toxicity in aquatic wildlife.
Biol Rev Camb Philos Soc,
96(4), 1528-1546.
Abstract:
Global variation in freshwater physico-chemistry and its influence on chemical toxicity in aquatic wildlife.
Chemical pollution is one of the major threats to global freshwater biodiversity and will be exacerbated through changes in temperature and rainfall patterns, acid-base chemistry, and reduced freshwater availability due to climate change. In this review we show how physico-chemical features of natural fresh waters, including pH, temperature, oxygen, carbon dioxide, divalent cations, anions, carbonate alkalinity, salinity and dissolved organic matter, can affect the environmental risk to aquatic wildlife of pollutant chemicals. We evidence how these features of freshwater physico-chemistry directly and/or indirectly affect the solubility, speciation, bioavailability and uptake of chemicals [including via alterations in the trans-epithelial electric potential (TEP) across the gills or skin] as well as the internal physiology/biochemistry of the organisms, and hence ultimately toxicity. We also show how toxicity can vary with species and ontogeny. We use a new database of global freshwater chemistry (GLORICH) to demonstrate the huge variability (often >1000-fold) for these physico-chemical variables in natural fresh waters, and hence their importance to ecotoxicology. We emphasise that a better understanding of chemical toxicity and more accurate environmental risk assessment requires greater consideration of the natural water physico-chemistry in which the organisms we seek to protect live.
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2020
Lange A, Paris JR, Gharbi K, Cézard T, Miyagawa S, Iguchi T, Studholme DJ, Tyler CR (2020). A newly developed genetic sex marker and its application to understanding chemically induced feminisation in roach (. Rutilus rutilus. ).
Molecular Ecology Resources,
20(4), 1007-1022.
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Mintram KS, Maynard SK, Brown AR, Boyd R, Johnston ASA, Sibly RM, Thorbek P, Tyler CR (2020). Applying a mechanistic model to predict interacting effects of chemical exposure and food availability on fish populations.
Aquat Toxicol,
224Abstract:
Applying a mechanistic model to predict interacting effects of chemical exposure and food availability on fish populations.
The potential environmental impacts of chemical exposures on wildlife are of growing concern. Freshwater ecosystems are vulnerable to chemical effects and wildlife populations, including fish, can be exposed to concentrations known to cause adverse effects at the individual level. Wild fish populations are also often subjected to numerous other stressors simultaneously which in temperate climates often include sustained periods of food limitation. The potential interactive effects of chemical exposures and food limitation on fish populations are however difficult to establish in the field. Mechanistic modelling approaches can be employed to help predict how the physiological effects of chemicals and food limitation on individuals may translate to population-level effects. Here an energy budget-individual-based model was developed and the control (no chemical) model was validated for the three-spined stickleback. Findings from two endocrine active chemical (EAC) case studies, (ethinyloestradiol and trenbolone) were then used to investigate how effects on individual fecundity translated into predicted population-level effects for environmentally relevant exposures. The cumulative effects of chemical exposure and food limitation were included in these analyses. Results show that effects of each EAC on the population were dependent on energy availability, and effects on population abundance were exacerbated by food limitation. Findings suggest that chemical effects and density dependent food competition interact to determine population responses to chemical exposures. Our study illustrates how mechanistic modelling approaches might usefully be applied to account for specific chemical effects, energy budgets and density-dependent competition, to provide a more integrated evaluation of population outcomes in chemical risk assessments.
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Thornber K, Verner-Jeffreys D, Hinchliffe S, Rahman MM, Bass D, Tyler CR (2020). Evaluating antimicrobial resistance in the global shrimp industry.
Reviews in Aquaculture,
12(2), 966-986.
Abstract:
Evaluating antimicrobial resistance in the global shrimp industry
Antimicrobial resistance (AMR) is a growing threat to global public health, and the overuse of antibiotics in animals has been identified as a major risk factor. With high levels of international trade and direct connectivity to the aquatic environment, shrimp aquaculture may play a role in global AMR dissemination. The vast majority of shrimp production occurs in low- and middle-income countries, where antibiotic quality and usage is widely unregulated, and where the integration of aquaculture with family livelihoods offers many opportunities for human, animal and environmental bacteria to come into close contact. Furthermore, in shrimp growing areas, untreated waste is often directly eliminated into local water sources. These risks are very different to many other major internationally-traded aquaculture commodities, such as salmon, which is produced in higher income countries where there are greater levels of regulation and well-established management practices. Assessing the true scale of the risk of AMR dissemination in the shrimp industry is a considerable challenge, not least because obtaining reliable data on antibiotic usage is very difficult. Combating the risks associated with AMR dissemination is also challenging due to the increasing trend towards intensification and its associated disease burden, and because many farmers currently have no alternatives to antibiotics for preventing crop failure. In this review, we critically assess the potential risks the shrimp industry poses to AMR dissemination. We also discuss some of the possible risk mitigation strategies that could be considered by the shrimp industry as it strives for a more sustainable future in production.
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Hinchliffe S, Butcher A, Rahman MM, Guilder J, Tyler C, Verner‐Jeffreys D (2020). Production without medicalisation: Risk practices and disease in Bangladesh aquaculture. The Geographical Journal, 187(1), 39-50.
2019
Windsor FM, Durance I, Horton AA, Thompson RC, Tyler CR, Ormerod SJ (2019). A catchment-scale perspective of plastic pollution.
Glob Chang BiolAbstract:
A catchment-scale perspective of plastic pollution.
Plastic pollution is distributed across the globe, but compared with marine environments, there is only rudimentary understanding of the distribution and effects of plastics in other ecosystems. Here, we review the transport and effects of plastics across terrestrial, freshwater and marine environments. We focus on hydrological catchments as well-defined landscape units that provide an integrating scale at which plastic pollution can be investigated and managed. Diverse processes are responsible for the observed ubiquity of plastic pollution, but sources, fluxes and sinks in river catchments are poorly quantified. Early indications are that rivers are hotspots of plastic pollution, supporting some of the highest recorded concentrations. River systems are also likely pivotal conduits for plastic transport among the terrestrial, floodplain, riparian, benthic and transitional ecosystems with which they connect. Although ecological effects of micro- and nanoplastics might arise through a variety of physical and chemical mechanisms, consensus and understanding of their nature, severity and scale are restricted. Furthermore, while individual-level effects are often graphically represented in public media, knowledge of the extent and severity of the impacts of plastic at population, community and ecosystem levels is limited. Given the potential social, ecological and economic consequences, we call for more comprehensive investigations of plastic pollution in ecosystems to guide effective management action and risk assessment. This is reliant on (a) expanding research to quantify sources, sinks, fluxes and fates of plastics in catchments and transitional waters both independently as a major transport routes to marine ecosystems, (b) improving environmentally relevant dose-response relationships for different organisms and effect pathways, (c) scaling up from studies on individual organisms to populations and ecosystems, where individual effects are shown to cause harm and; (d) improving biomonitoring through developing ecologically relevant metrics based on contemporary plastic research.
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Nomiri S, Hoshyar R, Ambrosino C, Tyler CR, Mansouri B (2019). A mini review of bisphenol a (BPA) effects on cancer-related cellular signaling pathways.
Environmental Science and Pollution Research,
26(9), 8459-8467.
Abstract:
A mini review of bisphenol a (BPA) effects on cancer-related cellular signaling pathways
Bisphenol a (BPA) is a plasticizer used widely in many industrial products and is now well established as an endocrine-disrupting chemical (EDC). BPA readily leaches out from these products into the environment and into foodstuffs (from packaging materials) and human exposure can be considerable. Many studies have shown that BPA exposure is associated with a range of chronic human health conditions, including diabetes, cardiovascular disorders, polycystic ovarian disease, hepatotoxicity, and various types of cancer. BPA exerts its effects through deregulating cell signaling pathways associated with cell growth, proliferation, migration, invasion, and apoptosis. Previous studies on the molecular mechanisms of BPA have illustrated a variety of pathways impaired at very low exposure concentrations and that stimulate cellular responses relating to tumorigenesis both in cancer onset and progression. In this mini review, the recent advancements made through in vitro analyses are reported on for the effect of BPA on various cellular signaling pathways focusing on the signaling pathways that play a major role in carcinogenesis.
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Godfray HCJ, Stephens AEA, Jepson PD, Jobling S, Johnson AC, Matthiessen P, Sumpter JP, Tyler CR, McLean AR (2019). A restatement of the natural science evidence base on the effects of endocrine disrupting chemicals on wildlife.
Proceedings of the Royal Society B: Biological Sciences,
286(1897).
Abstract:
A restatement of the natural science evidence base on the effects of endocrine disrupting chemicals on wildlife
Endocrine disrupting chemicals (EDCs) are substances that alter the function of the endocrine system and consequently cause adverse effects to humans or wildlife. The release of particular EDCs into the environment has been shown to negatively affect certain wildlife populations and has led to restrictions on the use of some EDCs. Current chemical regulations aim to balance the industrial, agricultural and/or pharmaceutical benefits of using these substances with their demonstrated or potential harm to human health or the environment. A summary is provided of the natural science evidence base informing the regulation of chemicals released into the environment that may have endocrine disrupting effects on wildlife. This summary is in a format (a ‘restatement’) intended to be policy-neutral and accessible to informed, but not expert, policy-makers and stakeholders.
Abstract.
Foreman A (2019). Adopting a gene regulatory network approach to investigate toxicity through the adaptive stress response in teleost fish species.
Abstract:
Adopting a gene regulatory network approach to investigate toxicity through the adaptive stress response in teleost fish species.
Given current risks of pollutant exposures in aquatic environments, there is a growing need to generate reliable computational risk assessment methods to establish how adverse outcomes can be produced across exposure organisms. The adaptive stress response is widely targeted by pollutants of concern and includes transcription factors including nuclear factor (erythroid- derived 2)-like 2 (Nrf2), hypoxia inducible factor (HIF-1α), heat shock factor (HSF1), nuclear factor kappa-light-chain-enhancer of activated -B cells (NFkB), metal transcription factor 1 (MTF1), the aryl hydrocarbon receptor (AhR) and tumor protein P53 (P53). While these TFs are known to be activated by distinct inducers, less is understood about the regulatory links between factors, particularly at the transcription factor (TF) DNA-binding level.
In this thesis, a gene regulatory network (GRN) of adaptive-stress response factors that are key targets of chemical toxicity was constructed based on experimental evidence from mammalian cell-lines. The GRN was modeled using boolean logic and this identified a number of response outcomes that could be attributed to the activation of pathways including antioxidant defence processes and glucose metabolism. The GRN model illustrated that the activation of Nrf2, HIF-1α, AhR, MTF1 and HSF1 led to the same adverse outcomes, suggesting canalisation in stress response pathways.
The ability to use GRNs across different species is widely supported by the identification of TF binding sites (TFBS) within target genes. To assess the efficiency of using the mammalian GRN across teleost fish species, a comprehensive analysis of validated binding sites for the AhR, MTF1, HIF-1α and Nrf2 was conducted across fish-species in comparison to the mammalian consensus binding sequence. This showed variations in binding site composition across validated TFBS for HIF-1α and Nrf2 in fish compared to the mammalian consensus, preventing the identification of the functional sequences for these factors using traditional methods. To establish if such changes affected the efficiency to predict positive downstream target genes for Nrf2 and HIF-1α in mammals and across teleost fish species, random
1
forest classification models were used to compare the efficiency of multiple positional weight matrices (PWM) motifs of TFBS for Nrf2 and HIF-1α. Whilst the result from this analysis identified discrepancies in the ability to predict target genes based on the mammalian motif file used, mammalian motifs were able to predict target genes across fish species. Validated binding sites in fish species were then aligned to generate PWM motifs and sites were predicted across shared target genes hsp70 and hmox1 using both fish based and mammalian based models. This showed that whilst there was some overlap in identified sites across species, fish-specific motifs identified unique sites from mammalian models.
To validate the GRN, gene-expression responses across exposures traditionally associated with activating distinct adaptive stress response factors were collated across the literature. This showed support for some of the key responses identified in the model. Chemical exposure studies were then undertaken in vivo in embryo-larval zebrafish (2 and 4 dpf) to identify potential connectivity between the TFs NFkB, MTF1 and HIF-1α with Nrf2, a key factor in the adaptive stress-response and a regulator of antioxidant response processes. The inducer of Nrf2, tert-butylhydroquinone (tBHQ), was used to determine if there was a change in transcriptional output of mtf1, hif1a and nfkb1 over time and with exposure concentration. This showed a significant difference in expression for nfkb1 and alterations in expression of mtf1 over prolonged exposure scenarios. In addition, the developmental expression of nrf2a, mtf1, hif1α and nfkb1 from 2 hpf to 96 hpf showed differences between transcript levels with hif1α and nfkb1 having the highest levels of expression compared to nrf2a and mtf1.
Overall, the research presented in this thesis provides a novel approach to assess the initiation of adaptive stress-response factors from molecular interactions. The research goes some way in establishing the feedback loops and connections between NFkB, MTF1, Nrf2, AhR, HIF-1α, HSF1 and P53. In doing so, the model generated in this thesis provides a novel approach of establishing outcomes under toxicant exposures.
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Pinion J (2019). Concordant spatio-temporal patterns of brain activation in zebrafish exposed to compounds with similar pharmacodynamics or with similar seizurogenic potential.
Abstract:
Concordant spatio-temporal patterns of brain activation in zebrafish exposed to compounds with similar pharmacodynamics or with similar seizurogenic potential.
Abstract
Drug development is a highly resource intensive process that uses large numbers of animals for assessing the safety and efficacy of drugs prior to clinical testing. Improving the efficiency of drug development in terms of financial expenditure and number of animals used is therefore of utmost concern, not only to industry, but also to animal welfare organisations such as the NC3Rs. Poor efficiency in drug development largely stems from drug attrition, particularly attrition in the latter stages of the testing due to the large amount of resources expended at the point of failure. It is therefore imperative that deleterious off-target effects are identified as early as possible. However, typically, identification of seizure as a side-effect of drugs is performed in the later stages of development due to the highly intensive and low-throughput nature of seizure assays. At which point, if a compound fails, a large amount of resources have been squandered. There therefore exists a need for high-throughput and relatively inexpensive seizure liability assays that can be used early in drug development to prevent compounds destined for failure undergoing unnecessary resource intensive testing.
In this thesis we propose a refined approach using non-invasive imaging techniques in non-protected life stage zebrafish as a method for the detection of seizurogenic compounds early in drug development. In addition, we highlight its utility for elucidating the pharmacodynamics of compounds. In this study, a transgenic zebrafish line containing a GCaMP6s calcium sensor under the control of the pan-neuronal promoter elavl3 was used for functional profiling of compounds with varied pharmacologies. Light sheet microscopy was used to record fluorescent activity in three spatial dimensions over time (4-dimensions) from the zebrafish brain after exposure to forty-three different compounds with varied pharmacodynamics and seizure liability profiles. Hierarchical clustering was employed in order to assess if compounds with seizurogenic activity or similar pharmacodynamics elicited specific functional brain activity. It was found that compounds with dopaminergic and serotonergic mechanisms of action elicited highly specific and similar brain activity patterns and that non-seizurogenic drugs also clustered separately from seizurogenic ones. Subsequent analyses, focussed on the utilisation of machine learning techniques, developing a model that could be used to discriminate between compounds with and without potentially seizurogenic effects. It is clear, from the analyses presented here, that drugs do in fact elicit specific brain patterns in zebrafish and that these brain patterns are effectively detected using light sheet microscopy. This system is highly applicable for use within the drug industry and even in its relatively preliminary stages provided an accurate method of discriminating between compounds based on their physiological effects in zebrafish.
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Mintram K (2019). Development and Application of Modelling Approaches for Realistic Assessments on Population Impacts of Endocrine Disruption in Fish.
Abstract:
Development and Application of Modelling Approaches for Realistic Assessments on Population Impacts of Endocrine Disruption in Fish.
Chemical exposures threaten the health of freshwater ecosystems worldwide. In particular, endocrine disrupting chemicals (EDCs) are of concern because of their ability to cause sub-lethal effects on organisms at low, including environmentally relevant, concentrations. The susceptibility of fish populations to the effects of these chemicals depends on exposure risk, physiological susceptibility, and population resilience. Population models can explicitly incorporate these factors into environmental risk assessments (ERAs) to improve realism and identify potentially vulnerable species.
In this thesis, modelling tools were developed and evaluated for the three-spined stickleback (Gasterosteus aculeatus) to advance understanding of the ecological relevance of EDC effects.The thesis begins with a critical review of the current status of EDCs in freshwater ecosystems worldwide and their effects on individual fish and their populations. The potential for different modelling techniques to provide realistic ecological assessments for EDCs is then explored. Individual-based models (IBMs) are used throughout this thesis to provide case study specific chemical assessments. Reproductive endpoints, including disruption of breeding behaviours, are used to extrapolate EDC effects from individuals to the population level. Findings included the importance of considering behavioural endpoints within chemical assessments, since disruption of breeding behaviours caused significant reductions in population abundance. Moreover, it was identified that the breeding strategy of the stickleback makes it particularly vulnerable to chemicals which directly affect reproductive output. The chemical exposure regime and density dependent processes determined whether the population recovered post-exposure.
Empirical experimental exposures were used to investigate the interactive
effects of EDC exposure and food limitation on somatic growth in early life stages. An energy budget model was then developed and used to further explore the mechanisms underlying this observed effect. The combined empirical and modelling results suggested that fish can adapt their physiology (by reducing physical activity) to cope with the effects of multiple stressors. Finally, in order to explicitly incorporate environmental conditions into population level assessments, a model was developed combining the energy budget model with the stickleback IBM. This model allows analyses on direct effects of the EDC as well as the additional metabolic effects associated with the chemical exposure. The effects of two case study EDCs (an oestrogen and an androgen) on individual fecundity were simulated in low and high food availability environments in order to explore how environmental conditions affect population susceptibility. The findings illustrate that the underlying mechanism of the EDC effect and environmental conditions can affect the susceptibility of populations to EDC exposures.
This thesis has developed novel models for use by both researchers and risk assessors for application in realistic population level assessments of chemical risks. The findings presented have important implications for understanding the ecological relevance of EDC exposures for fish populations.
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Windell D (2019). Dylan Windell.
Abstract:
Dylan Windell
Gold nanoparticles (AuNPs) are increasingly being used in biomedicine for enhancing drug delivery and facilitating imaging, as well as directly in applications such as cancer photothermal therapy. Despite this, very little is known about their potential impacts on the environment. Importantly, the effectiveness of AuNPs in such applications depends on a range of particle specifications that have been optimised during development to serve their specific purpose. The manipulations include their size and the addition of coatings, usually to enhance biocompatibility. How these different particle characteristics affect their bioavailability and biological effects on wildlife once released into the environment, has received very little research attention. Consequently, the main aims of this thesis were to investigate, principally via imaging methods, how the characteristics of size and surface coatings of gold nanoparticle (AuNP) affected bioavailability, tissue distribution and potential for toxicity using zebrafish (Danio rerio) as an experimental model.
To undertake this work, a second major objective of the thesis work was to construct an imaging platform to trace the uptake and biodistribution of the gold nanoparticles. For this, I constructed an open source selective-plane illumination microscopy system (OpenSPIM) which allowed me to quantitatively trace fluorescently tagged AuNPs in exposed zebrafish embryos with relatively high-throughput. Applying SPIM through multiple angles, the fluorescence tag on the AuNPs was detectable throughout the zebrafish embryo body enabling concentration-response uptake analyses. Having built the OpenSPIM system from the ground up, careful method developments were undertaken to optimise the SPIM which included the imaging of multiple transgenic models of zebrafish such as the MPO:GFP model with fluorescent neutrophils, the elavl3:GCaMP6s utilised in chapter 6 to measure neural activity, and the casper model which was utilised in all uptake studies. Studies into the toxicity of the AuNPs were carried out with models for detecting assess oxidative stress responses in the 3EpRE:hsp70:mCherry model (using the Zeiss inverted microscope) and the pod::NTR-mCherry/l-fabp::VDBP-GFP model to assess for damage to the pronephric glomerular filtration barrier (using the EVOS fluorescent microscope). The OpenSPIM system was applied to image uptake and distribution of quantum dots (CdTe) in the casper model which indicated minimal uptake in the entire zebrafish embryo. Extensive oxidative stress was detected for exposures to aqueous levels of CdTe QDs down to 10 µg/L in the lateral line neuromasts, fionocytes and pronephros (undeveloped kidney) but there were no effects to the glomerular filtration barrier. Aqueous exposure to AuNPs illustrated a size selective uptake with the highest uptake for 40-80 nm AuNPs across a size range between 10 to 100 nm. This was principally found accumulating in the pronephros and gut with some fluorescence visualised in the heart. The effect of AuNP functionalisation on uptake in the casper model demonstrated varied levels of uptake depending on surface modification. Modifications of the AuNPs resulted in enhanced uptake levels for coatings with PEG and TNFα but not for NHS/PAMAM where there was decreased uptake. Depuration studies with all AuNP studies indicated that the AuNPs were cleared (but not completely) from the zebrafish embryos over a period of 4 days. No toxic effects were seen in any study with AuNP exposure in concentrations that exceed environmentally relevant doses (2 mg/L).
This body of work illustrates the utility of SPIM for detecting the uptake and fate of selected NPs in exposed fish embryos. When combined with transgenic models that allow for functional analyses across specific tissue targets and effect mechanisms, thus providing a powerful tool for nanoparticle screening. Finer level details relating to the biological effects of NPs, however, require imaging methods that allow for sub-cellular resolution to analyse cell mechanics and how NPs influence cellular functions.
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Lee CJ, Paull GC, Tyler CR (2019). Effects of environmental enrichment on survivorship, growth, sex ratio and behaviour in laboratory maintained zebrafish Danio rerio.
J Fish Biol,
94(1), 86-95.
Abstract:
Effects of environmental enrichment on survivorship, growth, sex ratio and behaviour in laboratory maintained zebrafish Danio rerio.
Environmental enrichment involves increasing the complexity of a fish's environment in order to improve welfare. Researchers are legally obliged to consider the welfare of laboratory animals and poor welfare may result in less robust data in experimental science. Laboratory zebrafish Danio rerio are usually kept in bare aquaria for ease of husbandry and, despite being a well-studied species, little is known about how laboratory housing affects their welfare. This study shows that environmental enrichment, in the form of the addition of gravel substratum and plants into the tank, affects survivorship, growth and behaviour in laboratory-maintained D. rerio. Larvae reared in enriched tanks had significantly higher survivorship compared with larvae reared in bare tanks. Effects of the tank conditions on growth were more variable. Females from enriched tanks had a higher body condition than females maintained in bare tanks, but intriguingly this was not the case for males, where the only difference was a more variable body condition in males maintained in bare tanks. Sex ratio in the rearing tanks did not differ between treatments. Resource monopolisation was higher for fish in enriched tanks than for those in bare tanks. Fish from enriched tanks displayed lower levels of behaviours associated with anxiety compared with fish from bare tanks when placed into a novel environment. Thus, this study demonstrates differences in welfare for D. rerio maintained under different environmental conditions with enhancements in welfare more commonly associated with tank enrichment.
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Le Page G (2019). Environmental Risk Assessment of Antibiotics: Investigations into Cyanobacteria Interspecies Sensitivities and Establishing Appropriate Protection Limits.
Abstract:
Environmental Risk Assessment of Antibiotics: Investigations into Cyanobacteria Interspecies Sensitivities and Establishing Appropriate Protection Limits
Antibiotics have been described as a ‘wonder drug’ that have transformed medicine since their discovery at the beginning of the 20th century and are used globally in safeguarding human and animal health. Environmental risk assessment (ERA) aims to ensure their environmental safety by setting protection limits that seek to prevent adverse effects upon populations and ecosystem function. In the case of antibiotics however, there is concern that ERA may not be fully protective of bacterial populations.
This thesis examines the ERA of antibiotics and highlights that protection limits may in some cases be under-protective or over-protective for bacteria populations (including cyanobacteria), depending on the antibiotic mode of action and the species on which the protection limit is based. The first section of the thesis contains a systematic review including a meta-analysis of all publicly available aquatic ecotoxicity data. The results illustrate that generally bacteria are the most sensitive taxa to antibiotics compared with eukaryotes but that interspecies variability in sensitivity among bacteria can range by up to five orders of magnitude. This far exceeds the assessment factor of 10 used to account for such uncertainty in protection limits. It also shows that the costly testing on fish may not be required and in accordance with the principle of the 3Rs could be excluded from the ERA of antibiotics, as they are not likely to drive the protection limit. Further, it demonstrates that protection limits established for antimicrobial resistance (AMR) may not always be protective of environmental health and that both protection limits should be determined in ERA.
Next, the thesis reports on the development and validation of a microplate assay for the rapid screening of chemical effects (here antibiotics) on cyanobacteria. The microplate assay is optimised to allow for the direct comparison of species sensitivity, ensuring consistent test conditions and thus limiting differences in antibiotic behaviour between assays. Reference toxicity testing with potassium dichromate demonstrates reproducibility over time and comparability with the standard shake flask test used in ERA.
The microplate assay is then used to experimentally confirm the findings of the meta-analysis. In this work, interspecies sensitivity across eight cyanobacteria species was demonstrated to vary by up to 70 fold following exposure to β-lactam antibiotics but only by an order of magnitude for macrolides. Cyanobacteria were not sensitive to sulfonamides and thus are not likely to be suitable for the setting of protection limits for this antibiotic class.
Finally in this thesis, species sensitivity distributions were created to examine how effective the protection limit currently derived in ERA is for antibiotics. For cephalosporins, there was a higher probability of under-protection whilst the protection limits were over-protective for classes of antibiotics with less interspecies differences in sensitivity, such as macrolides. Further, a probabilistic ecological risk assessment suggested that 60 to 100% of cyanobacteria species might be adversely affected at the higher measured environmental concentrations in the literature, while no significant risk was found at average concentrations.
The findings from this thesis illustrate that protection limits, as currently determined in ERA for antibiotics, may not be suitable for the adequate protection of cyanobacteria populations and most likely other bacterial taxa. The thesis proposes several approaches for improving ERA, including incorporating greater bacterial diversity in ecotoxicity testing, the inclusion of functional and/or community testing and the use of probabilistic methods to derive protection limits.
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Cooper R (2019). Investigating the health effects of estrogenic chemicals individually and within wastewater treatment works effluents using an ERE-GFP transgenic zebrafish (Danio rerio) model.
Abstract:
Investigating the health effects of estrogenic chemicals individually and within wastewater treatment works effluents using an ERE-GFP transgenic zebrafish (Danio rerio) model.
It is now well established globally that oestrogens are a major environmental contaminant in WwTW effluent discharging into receiving waterways. Even in developed countries despite advances in wastewater treatment and a general reduction in the incidence of gross point source pollution, effluent from most WwTWs contain detectable estrogenic activity; around a third of English rivers currently are thought to contain levels of oestrogenic chemicals sufficient to cause some endocrine disruption in fish. These disruptions in fish present themselves in a
number of different ways including a sex ratio biased in favour of females, the presence intersex individuals; delayed sexual maturation; and alterations in reproductive behaviour. This illustrate the need for a deeper understanding on how oestrogenic chemicals act within the body to affect wildlife health.
Using a transgenic oestrogen response element (ERE), green fluorescent protein (GFP) zebrafish model, this thesis work set out to determine tissue targets and the potential for health impacts of environmental oestrogens and their mixtures within effluents from WwTWs and how these responses may vary both seasonally and for exposures for different developmental life stages. This was measured through a
combination of biological endpoints including mortality/hatching rates, length, weight and condition factor (in adults), vitellogenin (vtg) induction (mRNA) in whole body embryos and in the livers of adults, and also through GFP quantification (measured by fluorescence microscopy on target tissues, Western blotting for whole body GFP induction and GFP mRNA induction).
The body of thesis work also sought to further establish how fluorescence responses in the transgenic zebrafish model compared in terms of sensitivity for the detection of oestrogens with vitellogenin (VTG) induction, a well-established biomarker for oestrogen exposure in fish. The ontogeny of endogenous GFP expression in individual fish was also followed from early life stages through the period of sexual differentiation using fluorescent microscopy, to determine if endogenous GFP expression could be used as a sex marker in our zebrafish
model.
In the WwTW effluent studies, differences in GFP tissue patterning (and of other biological endpoints such as vtg induction) were found to be reflective of the varying concentrations of steroidal oestrogens found in the different wastewaters, (both temporally and between treatment works), and they also illustrated the considerable dynamic nature of the oestrogenic potencies of the WwTW effluents. For chronic effluent exposures, biological responses to both the oestrogenic effluent and the synthetic oestrogen EE2 were differed between the sexes particularly in terms of overall growth and sex differentiation parameters.
For my investigation into endogenous GFP expression, I found varying levels of fluorescence, throughout the various life stages of our zebrafish to 30dpf. There were four different patterns of GFP expression across the different body tissues recorded, based on the tissues expressing GFP and the timing and duration of that expression. Gonadal sex (measured via histology at 60dph) however was associated only with sex for one of the recorded GFP expression patterns (GFP expression in the liver at 30dph – corresponding to the female phenotype).
Overall, the results from the studies presented in this thesis illustrate the potential for differing health effects of both individual exogenous oestrogens and for a single WwTW effluent over time/season and for effluents from different WwTW. The work also highlights the importance in the timing (developmental stage) of exposure on the biological effects seen. The ability of ERE-GFP transgenic zebrafish model to allow for the analysis of individual tissues, such as the heart or brain, through non-destructive fluorescence microscopy, further demonstrates its significant
advantages for screening and testing for environmental oestrogens.
Whilst these results suggest that GFP induction is less sensitive than VTG
induction per se, related (in part) to the greater magnitude of response for VTG induction, there was a good positive correlation between the two variables illustrating further the utility of the GFP induction as a bio-monitor to detect for the presence of exogenous oestrogens contained in real world effluents. These findings further highlight the importance of integrating biomonitoring approaches to complement analytical chemistry techniques in the monitoring of oestrogenic chemicals in wastewater effluents.
Differences, in the levels of endogenous GFP expression across the different body tissues during early life in our zebrafish model, and prior to gonadal differentiation, are likely a reflection of the varying functional roles oestrogen receptors perform during development (i.e. they are under spatial-temporal control) and are not thought to provide an accurate marker for the determination of sex in the zebrafish
model but could be utilised as a phenotypic marker for identifying females at 30dpf.
With a view towards the future, additional steps now need to be taken in order to further corroborate these findings and to maximise the potential of the transgenic ERE-GFP zebrafish model system. These include the need for identifying the molecular mechanisms of oestrogenic chemicals and of oestrogen responsive genes and pathways, particularly in the responsive target tissues identified in this thesis, to better inform on adverse health outcomes.
Ultimately, the results of this thesis further highlight how the development and application of transgenic fish models can offer huge potential for more integrative health effects assessments, allowing for an improved understanding of the risks posed by oestrogenic chemicals, and for tailoring future approaches to ERA strategies, specifically in respect of AOP frameworks.
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Windsor FM, Tilley RM, Tyler CR, Ormerod SJ (2019). Microplastic ingestion by riverine macroinvertebrates.
Science of the Total Environment,
646, 68-74.
Abstract:
Microplastic ingestion by riverine macroinvertebrates
Although microplastics are a recognised pollutant in marine environments, less attention has been directed towards freshwater ecosystems despite their greater proximity to possible plastic sources. Here, we quantify the presence of microplastic particles (MPs) in river organisms upstream and downstream of five UK Wastewater Treatment Works (WwTWs). MPs were identified in approximately 50% of macroinvertebrate samples collected (Baetidae, Heptageniidae and Hydropsychidae) at concentrations up to 0.14 MP mg tissue −1 and they occurred at all sites. MP abundance was associated with macroinvertebrate biomass and taxonomic family, but MPs occurred independently of feeding guild and biological traits such as habitat affinity and ecological niche. There was no increase in plastic ingestion downstream of WwTW discharges averaged across sites, but MP abundance in macroinvertebrates marginally increased where effluent discharges contributed more to total runoff and declined with increasing river discharge. The ubiquity of microplastics within macroinvertebrates in this case study reveals a potential risk from MPs entering riverine food webs through at least two pathways, involving detritivory and filter-feeding, and we recommend closer attention to freshwater ecosystems in future research.
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Parsons A, Lange A, Hutchinson TH, Miyagawa S, Iguchi T, Kudoh T, Tyler CR (2019). Molecular mechanisms and tissue targets of brominated flame retardants, BDE-47 and TBBPA, in embryo-larval life stages of zebrafish (Danio rerio).
Aquatic Toxicology,
209, 99-112.
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Mourabit S, Fitzgerald JA, Ellis RP, Takesono A, Porteus CS, Trznadel M, Metz J, Winter MJ, Kudoh T, Tyler CR, et al (2019). New insights into organ-specific oxidative stress mechanisms using a novel biosensor zebrafish.
Environment International,
133Abstract:
New insights into organ-specific oxidative stress mechanisms using a novel biosensor zebrafish
Background: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity. Objectives: Our aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS. Methods: to achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity. Results: We produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity. Discussion: Our model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer.
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Windsor FM, Pereira MG, Tyler CR, Ormerod SJ (2019). Persistent contaminants as potential constraints on the recovery of urban river food webs from gross pollution.
Water Research,
163Abstract:
Persistent contaminants as potential constraints on the recovery of urban river food webs from gross pollution
Urban areas contribute substantially to xenobiotic contaminant loads in rivers, but their effects have been investigated more for individual organisms and sensitive taxa, rather than through the emergent properties of communities. Here, we use replicated, catchment-scale sampling of benthic invertebrates and novel multivariate techniques to assess whether urban wastewater contaminants affected the structure and function of river food webs. We postulated that the continued occurrence of selected contaminants in river systems might explain the incomplete recovery of urban rivers from legacy gross pollution. Benthic invertebrate communities were sampled monthly over a year (2016–2017) at 18 sites across 3 river systems in South Wales (United Kingdom). Contaminant sources were characterised using remote sensing, water quality data from routine monitoring and measured concentrations of selected persistent xenobiotic pollutants (polychlorinated biphenyls and polybrominated diphenyl ethers). Urban wastewater discharges had relatively limited effects on river water quality, with small increases in nitrate, phosphate, temperature, conductivity and total dissolved solids in urban systems. Concentrations of polychlorinated biphenyls and polybrominated diphenyl ethers in invertebrates, however, were significantly higher under greater urban land cover and wastewater discharge. Food webs at the most highly contaminated urban sites were characterised by: (i) reduced taxonomic and functional diversity; (ii) simplified food web structure with reduced network connectance; and (iii) reductions in the abundance of prey important for apex predators such as the Eurasian dipper (Cinclus cinclus). Although correlative and partially confounded by other effects, these data provide support for the hypothesis that impairment to food webs resulting from urban pollutants might explain population, community and ecosystem-level effects in urban river systems, and hence incomplete recovery from past pollution.
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Gunnarsson L, Snape JR, Verbruggen B, Owen SF, Kristiansson E, Margiotta-Casaluci L, Österlund T, Hutchinson K, Leverett D, Marks B, et al (2019). Pharmacology beyond the patient - the environmental risks of human drugs.
Environ Int,
129, 320-332.
Abstract:
Pharmacology beyond the patient - the environmental risks of human drugs.
BACKGROUND: the presence of pharmaceuticals in the environment is a growing global concern and although environmental risk assessment is required for approval of new drugs in Europe and the USA, the adequacy of the current triggers and the effects-based assessments has been questioned. OBJECTIVE: to provide a comprehensive analysis of all regulatory compliant aquatic ecotoxicity data and evaluate the current triggers and effects-based environmental assessments to facilitate the development of more efficient approaches for pharmaceuticals toxicity testing. METHODS: Publicly-available regulatory compliant ecotoxicity data for drugs targeting human proteins was compiled together with pharmacological information including drug targets, Cmax and lipophilicity. Possible links between these factors and the ecotoxicity data for effects on, growth, mortality and/or reproduction, were evaluated. The environmental risks were then assessed based on a combined analysis of drug toxicity and predicted environmental concentrations based on European patient consumption data. RESULTS: for most (88%) of the of 975 approved small molecule drugs targeting human proteins a complete set of regulatory compliant ecotoxicity data in the public domain was lacking, highlighting the need for both intelligent approaches to prioritize legacy human drugs for a tailored environmental risk assessment and a transparent database that captures environmental data. We show that presence/absence of drug-target orthologues are predictive of susceptible species for the more potent drugs. Drugs that target the endocrine system represent the highest potency and greatest risk. However, for most drugs (>80%) with a full set of ecotoxicity data, risk quotients assuming worst-case exposure assessments were below one in all European countries indicating low environmental risks for the endpoints assessed. CONCLUSION: We believe that the presented analysis can guide improvements to current testing procedures, and provide valuable approaches for prioritising legacy drugs (i.e. those registered before 2006) for further ecotoxicity testing. For drugs where effects of possible concern (e.g. behaviour) are not captured in regulatory tests, additional mechanistic testing may be required to provide the highest confidence for avoiding environmental impacts.
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Tyler CR, Parsons A, Rogers NJ, Lange A, Brown AR (2019).
Plasticisers and Their Impact on Wildlife.Abstract:
Plasticisers and Their Impact on Wildlife
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Cross RK, Tyler CR, Galloway TS (2019). The fate of cerium oxide nanoparticles in sediments and their routes of uptake in a freshwater worm.
Nanotoxicology,
13(7), 894-908.
Abstract:
The fate of cerium oxide nanoparticles in sediments and their routes of uptake in a freshwater worm
The relative importance of ingestion and transdermal uptake of nanomaterials is poorly understood, particularly in sediment dwelling organisms, where diet has the potential to contribute significantly to particle accumulation. In aquatic sediments, nanoparticles may partition to bind with the solid fraction of sediment, be freely mobile in the pore water or, for certain metal/metal oxides, undergo dissolution, each of which could influence the route of nanoparticle uptake. Here, we used the freshwater worm Lumbriculus variegatus as a model species. We took advantage of its unique feeding and non-feeding life-stages to assess the contribution of dietary and transdermal uptake in the bioaccumulation of cerium oxide nanoparticles (CeO2 NP) and soluble Ce(III)NO3. Distribution of cerium between the solid, colloidal and soluble fractions in the sediments was determined through sediment separations using micro and ultrafiltration techniques. We assessed particles of differing sizes (10, 28 and 615 nm CeO2) and stabilizing surfactants (10 nm electrostatic Citrate-CeO2 and steric stabilized PEG-CeO2). Soluble Ce(III)NO3, was found to accumulate readily across the skin of the worms whilst nanoparticles were not. Sediments reduced the uptake of CeIII by limiting the presence of dissolved species of cerium in the pore waters. Neither particle size nor the coatings studied altered the distribution of nanoparticles between solid and colloidal fractions of the sediment, with ∼99% associated to the solid phase. Any uptake of CeO2 nanoparticles into worms was only through ingestion. Stabilized 10 nm particles were retained even after gut clearance, indicating that these particles may translocate across the gut wall.
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Le Page G, Gunnarsson L, Trznadel M, Wedgwood KCA, Baudrot V, Snape J, Tyler CR (2019). Variability in cyanobacteria sensitivity to antibiotics and implications for environmental risk assessment.
Science of the Total Environment,
695Abstract:
Variability in cyanobacteria sensitivity to antibiotics and implications for environmental risk assessment
Once released into the environment antibiotics can kill or inhibit the growth of bacteria, and in turn potentially have effects on bacterial community structure and ecosystem function. Environmental risk assessment (ERA) seeks to establish protection limits to minimise chemical impacts on the environment, but recent evidence suggests that the current regulatory approaches for ERA for antibiotics may not be adequate for protecting bacteria that have fundamental roles in ecosystem function. In this study we assess the differences in interspecies sensitivity of eight species of cyanobacteria to seven antibiotics (cefazolin, cefotaxime, ampicillin, sufamethazine, sulfadiazine, azithromycin and erythromycin) with three different modes of action. We found that variability in the sensitivity to these antibiotics between species was dependent on the mode of action and varied by up to 70 times for β-lactams. Probabilistic analysis using species sensitivity distributions suggest that the current predicted no effect concentration PNEC for the antibiotics may be either over or under protective of cyanobacteria dependent on the species on which it is based and the mode of action of the antibiotic; the PNECs derived for the macrolide antibiotics were over protective but PNECs for β-lactams were generally under protective. For some geographical locations we identify a significant risk to cyanobacteria populations based upon measured environmental concentrations of selected antibiotics. We conclude that protection limits, as determined according to current regulatory guidance, may not always be protective and might be better derived using SSDs and that including toxicity data for a wider range of (cyano-) bacteria would improve confidence for the ERA of antibiotics.
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2018
Foreman AL, Foreman A (2018). Adopting a gene regulatory network approach to investigate toxicity through the adaptive stress response in teleost fish species.
Abstract:
Adopting a gene regulatory network approach to investigate toxicity through the adaptive stress response in teleost fish species.
Given current risks of pollutant exposures in aquatic environments, there is a growing need to generate reliable computational risk assessment methods to establish how adverse outcomes can be produced across exposure organisms. The adaptive stress response is widely targeted by pollutants of concern and includes transcription factors including nuclear factor (erythroid- derived 2)-like 2 (Nrf2), hypoxia inducible factor (HIF-1α), heat shock factor (HSF1), nuclear factor kappa-light-chain-enhancer of activated -B cells (NFkB), metal transcription factor 1 (MTF1), the aryl hydrocarbon receptor (AhR) and tumor protein P53 (P53). While these TFs are known to be activated by distinct inducers, less is understood about the regulatory links between factors, particularly at the transcription factor (TF) DNA-binding level.
In this thesis, a gene regulatory network (GRN) of adaptive-stress response factors that are key targets of chemical toxicity was constructed based on experimental evidence from mammalian cell-lines. The GRN was modeled using boolean logic and this identified a number of response outcomes that could be attributed to the activation of pathways including antioxidant defence processes and glucose metabolism. The GRN model illustrated that the activation of Nrf2, HIF-1α, AhR, MTF1 and HSF1 led to the same adverse outcomes, suggesting canalisation in stress response pathways.
The ability to use GRNs across different species is widely supported by the identification of TF binding sites (TFBS) within target genes. To assess the efficiency of using the mammalian GRN across teleost fish species, a comprehensive analysis of validated binding sites for the AhR, MTF1, HIF-1α and Nrf2 was conducted across fish-species in comparison to the mammalian consensus binding sequence. This showed variations in binding site composition across validated TFBS for HIF-1α and Nrf2 in fish compared to the mammalian consensus, preventing the identification of the functional sequences for these factors using traditional methods. To establish if such changes affected the efficiency to predict positive downstream target genes for Nrf2 and HIF-1α in mammals and across teleost fish species, random
1
forest classification models were used to compare the efficiency of multiple positional weight matrices (PWM) motifs of TFBS for Nrf2 and HIF-1α. Whilst the result from this analysis identified discrepancies in the ability to predict target genes based on the mammalian motif file used, mammalian motifs were able to predict target genes across fish species. Validated binding sites in fish species were then aligned to generate PWM motifs and sites were predicted across shared target genes hsp70 and hmox1 using both fish based and mammalian based models. This showed that whilst there was some overlap in identified sites across species, fish-specific motifs identified unique sites from mammalian models.
To validate the GRN, gene-expression responses across exposures traditionally associated with activating distinct adaptive stress response factors were collated across the literature. This showed support for some of the key responses identified in the model. Chemical exposure studies were then undertaken in vivo in embryo-larval zebrafish (2 and 4 dpf) to identify potential connectivity between the TFs NFkB, MTF1 and HIF-1α with Nrf2, a key factor in the adaptive stress-response and a regulator of antioxidant response processes. The inducer of Nrf2, tert-butylhydroquinone (tBHQ), was used to determine if there was a change in transcriptional output of mtf1, hif1a and nfkb1 over time and with exposure concentration. This showed a significant difference in expression for nfkb1 and alterations in expression of mtf1 over prolonged exposure scenarios. In addition, the developmental expression of nrf2a, mtf1, hif1α and nfkb1 from 2 hpf to 96 hpf showed differences between transcript levels with hif1α and nfkb1 having the highest levels of expression compared to nrf2a and mtf1.
Overall, the research presented in this thesis provides a novel approach to assess the initiation of adaptive stress-response factors from molecular interactions. The research goes some way in establishing the feedback loops and connections between NFkB, MTF1, Nrf2, AhR, HIF-1α, HSF1 and P53. In doing so, the model generated in this thesis provides a novel approach of establishing outcomes under toxicant exposures.
Abstract.
Johnston HJ, Verdon R, Gillies S, Brown DM, Fernandes TF, Henry TB, Rossi AG, Tran L, Tucker C, Tyler CR, et al (2018). Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials.
Crit Rev Toxicol,
48(3), 252-271.
Abstract:
Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials.
Assessing the safety of engineered nanomaterials (NMs) is paramount to the responsible and sustainable development of nanotechnology, which provides huge societal benefits. Currently, there is no evidence that engineered NMs cause detrimental health effects in humans. However, investigation of NM toxicity using in vivo, in vitro, in chemico, and in silico models has demonstrated that some NMs stimulate oxidative stress and inflammation, which may lead to adverse health effects. Accordingly, investigation of these responses currently dominates NM safety assessments. There is a need to reduce reliance on rodent testing in nanotoxicology for ethical, financial and legislative reasons, and due to evidence that rodent models do not always predict the human response. We advocate that in vitro models and zebrafish embryos should have greater prominence in screening for NM safety, to better align nanotoxicology with the 3Rs principles. Zebrafish are accepted for use by regulatory agencies in chemical safety assessments (e.g. developmental biology) and there is growing acceptance of their use in biomedical research, providing strong foundations for their use in nanotoxicology. We suggest that investigation of the response of phagocytic cells (e.g. neutrophils, macrophages) in vitro should also form a key part of NM safety assessments, due to their prominent role in the first line of defense. The development of a tiered testing strategy for NM hazard assessment that promotes the more widespread adoption of non-rodent, alternative models and focuses on investigation of inflammation and oxidative stress could make nanotoxicology testing more ethical, relevant, and cost and time efficient.
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Le Page G, Gunnarsson L, Snape J, Tyler CR (2018). Antibiotic risk assessment needs to protect both environmental and human health.
Environ Int,
115, 397-399.
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Mintram KS, Brown AR, Maynard SK, Liu C, Parker SJ, Tyler CR, Thorbek P (2018). Assessing population impacts of toxicant-induced disruption of breeding behaviours using an individual-based model for the three-spined stickleback.
Ecological Modelling,
387, 107-117.
Abstract:
Assessing population impacts of toxicant-induced disruption of breeding behaviours using an individual-based model for the three-spined stickleback
The effects of toxicant exposure on individuals captured in standard environmental risk assessments (ERA) do not necessarily translate proportionally into effects at the population-level. Population models can incorporate population resilience, physiological susceptibility, and likelihood of exposure, and can therefore be employed to extrapolate from individual- to population-level effects in ERA. Here, we present the development of an individual-based model (IBM) for the three-spined stickleback (Gasterosteus aculeatus) and its application in assessing population-level effects of disrupted male breeding behaviour after exposure to the anti-androgenic pesticide, fenitrothion. The stickleback is abundant in marine, brackish, and freshwater systems throughout Europe and their complex breeding strategy makes wild populations potentially vulnerable to the effects of endocrine disrupting chemicals (EDCs). Modelled population dynamics matched those of a UK field population and the IBM is therefore considered to be representative of a natural population. Literature derived dose-response relationships of fenitrothion-induced disruption of male breeding behaviours were applied in the IBM to assess population-level impacts. The modelled population was exposed to fenitrothion under both continuous (worst-case) and intermittent (realistic) exposure patterns and population recovery was assessed. The results suggest that disruption of male breeding behaviours at the individual-level cause impacts on population abundance under both fenitrothion exposure regimes; however, density-dependent processes can compensate for some of these effects, particularly for an intermittent exposure scenario. Our findings further demonstrate the importance of understanding life-history traits, including reproductive strategies and behaviours, and their density-dependence, when assessing the potential population-level risks of EDCs.
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Winter MJ, Windell D, Metz J, Matthews P, Pinion J, Brown JT, Hetheridge MJ, Ball JS, Owen SF, Redfern WS, et al (2018). Author Correction: 4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.
Sci Rep,
8(1).
Abstract:
Author Correction: 4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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Lee CJ, Tyler CR, Paull GC (2018). Can simple tank changes benefit the welfare of laboratory zebrafish Danio rerio?.
J Fish Biol,
92(3), 653-659.
Abstract:
Can simple tank changes benefit the welfare of laboratory zebrafish Danio rerio?
This study examined the effects of simple changes in the tank environment on the wellbeing of laboratory-maintained zebrafish Danio rerio. Groups of D. rerio were either housed in stable environments (where they were maintained in the same tanks throughout the study) or in environments subject to change (where they were periodically moved to novel but identical tanks) and the effects of these treatments on morphometry, reproductive success and aggressive behaviour assessed. No effect of simple tank changes was found on body condition, reproductive output or aggression, for the periods of time studied, indicating that more complex scenarios in housing tank conditions are required for significant welfare benefits for captive D. rerio.
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Mintram KS, Brown AR, Maynard SK, Thorbek P, Tyler CR (2018). Capturing ecology in modeling approaches applied to environmental risk assessment of endocrine active chemicals in fish.
Crit Rev Toxicol,
48(2), 109-120.
Abstract:
Capturing ecology in modeling approaches applied to environmental risk assessment of endocrine active chemicals in fish.
Endocrine active chemicals (EACs) are widespread in freshwater environments and both laboratory and field based studies have shown reproductive effects in fish at environmentally relevant exposures. Environmental risk assessment (ERA) seeks to protect wildlife populations and prospective assessments rely on extrapolation from individual-level effects established for laboratory fish species to populations of wild fish using arbitrary safety factors. Population susceptibility to chemical effects, however, depends on exposure risk, physiological susceptibility, and population resilience, each of which can differ widely between fish species. Population models have significant potential to address these shortfalls and to include individual variability relating to life-history traits, demographic and density-dependent vital rates, and behaviors which arise from inter-organism and organism-environment interactions. Confidence in population models has recently resulted in the EU Commission stating that results derived from reliable models may be considered when assessing the relevance of adverse effects of EACs at the population level. This review critically assesses the potential risks posed by EACs for fish populations, considers the ecological factors influencing these risks and explores the benefits and challenges of applying population modeling (including individual-based modeling) in ERA for EACs in fish. We conclude that population modeling offers a way forward for incorporating greater environmental relevance in assessing the risks of EACs for fishes and for identifying key risk factors through sensitivity analysis. Individual-based models (IBMs) allow for the incorporation of physiological and behavioral endpoints relevant to EAC exposure effects, thus capturing both direct and indirect population-level effects.
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Brown AR, Green J, Moreman J, Gunnarsson L, Mourabit S, Ball J, Winter M, Trznadel M, Correia A, Hacker C, et al (2018). Cardiovascular Effects and Molecular Mechanisms of Bisphenol a and its Metabolite MBP in Zebrafish.
Environmental Science and Technology Full text.
David A, Lange A, Tyler CR, Hill EM (2018). Concentrating mixtures of neuroactive pharmaceuticals and altered neurotransmitter levels in the brain of fish exposed to a wastewater effluent.
Science of the Total Environment,
621, 782-790.
Abstract:
Concentrating mixtures of neuroactive pharmaceuticals and altered neurotransmitter levels in the brain of fish exposed to a wastewater effluent
Fish can be exposed to a variety of neuroactive pharmaceuticals via the effluent discharges from wastewater treatment plants and concerns have arisen regarding their potential impacts on fish behaviour and ecology. In this study, we investigated the uptake of 14 neuroactive pharmaceuticals from a treated wastewater effluent into blood plasma and brain regions of roach (Rutilus rutilus) after exposure for 15 days. We show that a complex mixture of pharmaceuticals including, 6 selective serotonin reuptake inhibitors, a serotonin-noradrenaline reuptake inhibitor, 3 atypical antipsychotics, 2 tricyclic antidepressants and a benzodiazepine, concentrate in different regions of the brain including the telencephalon, hypothalamus, optic tectum and hindbrain of effluent-exposed fish. Pharmaceuticals, with the exception of nordiazepam, were between 3–40 fold higher in brain compared with blood plasma, showing these neuroactive drugs are readily uptaken, into brain tissues in fish. To assess for the potential for any adverse ecotoxicological effects, the effect ratio was calculated from human therapeutic plasma concentrations (HtPCs) and the measured or predicted fish plasma concentrations of pharmaceuticals. After accounting for a safety factor of 1000, the effect ratios indicated that fluoxetine, norfluoxetine, sertraline, and amitriptyline warrant prioritisation for risk assessment studies. Furthermore, although plasma concentrations of all the pharmaceuticals were between 33 and 5714-fold below HtPCs, alterations in serotonin, glutamate, acetylcholine and tryptophan concentrations were observed in different brain regions of effluent-exposed fish. This study highlights the importance of determining the potential health effects arising from the concentration of complex environmental mixtures in risk assessment studies.
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Verbruggen B, Gunnarsson L, Kristiansson E, Österlund T, Owen SF, Snape JR, Tyler CR (2018). ECOdrug: a database connecting drugs and conservation of their targets across species.
Nucleic Acids Res,
46(D1), D930-D936.
Abstract:
ECOdrug: a database connecting drugs and conservation of their targets across species.
Pharmaceuticals are designed to interact with specific molecular targets in humans and these targets generally have orthologs in other species. This provides opportunities for the drug discovery community to use alternative model species for drug development. It also means, however, there is potential for mode of action related effects in non-target wildlife species as many pharmaceuticals reach the environment through patient use and manufacturing wastes. Acquiring insight in drug target ortholog predictions across species and taxonomic groups has proven difficult because of the lack of an optimal strategy and because necessary information is spread across multiple and diverse sources and platforms. We introduce a new research platform tool, ECOdrug, that reliably connects drugs to their protein targets across divergent species. It harmonizes ortholog predictions from multiple sources via a simple user interface underpinning critical applications for a wide range of studies in pharmacology, ecotoxicology and comparative evolutionary biology. ECOdrug can be used to identify species with drug targets and identify drugs that interact with those targets. As such, it can be applied to support intelligent targeted drug safety testing by ensuring appropriate and relevant species are selected in ecological risk assessments. ECOdrug is freely accessible and available at: http://www.ecodrug.org.
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Green JM, Lange A, Scott A, Trznadel M, Wai HA, Takesono A, Brown AR, Owen SF, Kudoh T, Tyler CR, et al (2018). Early life exposure to ethinylestradiol enhances subsequent responses to environmental estrogens measured in a novel transgenic zebrafish.
Sci Rep,
8(1).
Abstract:
Early life exposure to ethinylestradiol enhances subsequent responses to environmental estrogens measured in a novel transgenic zebrafish.
Estrogen plays fundamental roles in a range of developmental processes and exposure to estrogen mimicking chemicals has been associated with various adverse health effects in both wildlife and human populations. Estrogenic chemicals are found commonly as mixtures in the environment and can have additive effects, however risk analysis is typically conducted for single-chemicals with little, or no, consideration given for an animal's exposure history. Here we developed a transgenic zebrafish with a photoconvertable fluorophore (Kaede, green to red on UV light exposure) in a skin pigment-free mutant element (ERE)-Kaede-Casper model and applied it to quantify tissue-specific fluorescence biosensor responses for combinations of estrogen exposures during early life using fluorescence microscopy and image analysis. We identify windows of tissue-specific sensitivity to ethinylestradiol (EE2) for exposure during early-life (0-5 dpf) and illustrate that exposure to estrogen (EE2) during 0-48 hpf enhances responsiveness (sensitivity) to different environmental estrogens (EE2, genistein and bisphenol A) for subsequent exposures during development. Our findings illustrate the importance of an organism's stage of development and estrogen exposure history for assessments on, and possible health risks associated with, estrogen exposure.
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Collison EJ, Hird H, Tyler CR, Cresswell JE (2018). Effects of neonicotinoid exposure on molecular and physiological indicators of honey bee immunocompetence.
Apidologie,
49(2), 196-208.
Abstract:
Effects of neonicotinoid exposure on molecular and physiological indicators of honey bee immunocompetence
Bee declines have been associated with various stressors including pesticides and pathogens. We separately exposed immune-challenged adult worker honey bees (Apis mellifera L.) to two neonicotinoid pesticides, thiamethoxam (10 ppb) and imidacloprid (102 ppb), by dietary delivery. We found that whereas neonicotinoid exposure weakly affected transcriptional responses of antimicrobial genes, it did not detectably affect the physiological antimicrobial response as measured by a lytic clearance assay of haemolymph. Our findings add to the evidence that transcriptional responses in immune-related genes are not yet reliable indicators of pesticide impacts on bee health, which suggests caution in their future use as biomarkers in pesticide risk assessment.
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Windsor FM, Ormerod SJ, Tyler CR (2018). Endocrine disruption in aquatic systems: up-scaling research to address ecological consequences.
Biol Rev Camb Philos Soc,
93(1), 626-641.
Abstract:
Endocrine disruption in aquatic systems: up-scaling research to address ecological consequences.
Endocrine-disrupting chemicals (EDCs) can alter biological function in organisms at environmentally relevant concentrations and are a significant threat to aquatic biodiversity, but there is little understanding of exposure consequences for populations, communities and ecosystems. The pervasive nature of EDCs within aquatic environments and their multiple sub-lethal effects make assessments of their impact especially important but also highly challenging. Herein, we review the data on EDC effects in aquatic systems focusing on studies assessing populations and ecosystems, and including how biotic and abiotic processes may affect, and be affected by, responses to EDCs. Recent research indicates a significant influence of behavioural responses (e.g. enhancing feeding rates), transgenerational effects and trophic cascades in the ecological consequences of EDC exposure. In addition, interactions between EDCs and other chemical, physical and biological factors generate uncertainty in our understanding of the ecological effects of EDCs within aquatic ecosystems. We illustrate how effect thresholds for EDCs generated from individual-based experimental bioassays of the types commonly applied using chemical test guidelines [e.g. Organisation for Economic Co-operation and Development (OECD)] may not necessarily reflect the hazards associated with endocrine disruption. We argue that improved risk assessment for EDCs in aquatic ecosystems urgently requires more ecologically oriented research as well as field-based assessments at population-, community- and food-web levels.
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Moreman J, Takesono A, Trznadel M, Winter MJ, Perry A, Wood ME, Rogers NJ, Kudoh T, Tyler CR (2018). Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol a (BPA) and its Metabolite 4-Methyl-2,4-bis( p-hydroxyphenyl)pent-1-ene (MBP) in Zebrafish.
Environ Sci Technol,
52(11), 6656-6665.
Abstract:
Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol a (BPA) and its Metabolite 4-Methyl-2,4-bis( p-hydroxyphenyl)pent-1-ene (MBP) in Zebrafish.
Environmental exposure to Bisphenol a (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the bulbus arteriosus were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 μg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures.
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Orton F, Säfholm M, Jansson E, Carlsson Y, Eriksson A, Fick J, Uren Webster T, McMillan T, Leishman M, Verbruggen B, et al (2018). Exposure to an anti-androgenic herbicide negatively impacts reproductive physiology and fertility in Xenopus tropicalis.
Sci Rep,
8(1).
Abstract:
Exposure to an anti-androgenic herbicide negatively impacts reproductive physiology and fertility in Xenopus tropicalis.
Amphibians are threatened on a global scale and pollutants may be contributing to population declines, but how chemicals impact on their reproduction is poorly understood. We conducted a life cycle analysis to investigate the impacts of early life exposure to two anti-androgens (exposure until completion of metamorphosis;stage 66): flutamide, (50 µg/L)/linuron (9 and 45 µg/L)) on sexual development and breeding competence in Xenopus tropicalis. Our analyses included: mRNA levels of dmrt1, cyp17, amh, cyp19, foxl2 and ar (tadpoles/metamorphs), gonadal histomorphology (metamorphs/adults), mRNA levels of ar/gr (adult male brain/gonad/forelimb), testosterone/corticosterone levels (adult males), secondary sexual characteristics (forelimb width/nuptial pad: adult males) and breeding competence (amplexus/fertility: adult males). Compared to controls, feminised sex ratios and increased number of spermatogonia (adults) were observed after exposure to flutamide and the lower linuron concentration. Exposure to the lower linuron concentration also resulted in demasculinisation of secondary sexual characteristics and reduced male fertility. Flutamide exposure resulted in masculinisation of the nuptial pad and elevated mRNA levels of dmrt1, cyp17, amh and foxl2 in brains (metamorphs). Testosterone levels were higher in all treatment groups, however, overall few effects were observed in response to the higher linuron concentration. Our findings advance understanding of reproductive biology of X. tropicalis and illustrate negative effects of linuron on reproductive processes at a concentration measured in freshwater environments.
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Holder PJ, Jones A, Tyler CR, Cresswell JE (2018). Fipronil pesticide as a suspect in historical mass mortalities of honey bees.
Proc Natl Acad Sci U S A,
115(51), 13033-13038.
Abstract:
Fipronil pesticide as a suspect in historical mass mortalities of honey bees.
Mass mortalities of honey bees occurred in France in the 1990s coincident with the introduction of two agricultural insecticides, imidacloprid and fipronil. Imidacloprid, a neonicotinoid, was widely blamed, but the differential potency of imidacloprid and fipronil has been unclear because of uncertainty over their capacity to bioaccumulate during sustained exposure to trace dietary residues and, thereby, cause time-reinforced toxicity (TRT). We experimentally quantified the toxicity of fipronil and imidacloprid to honey bees and incorporated the observed mortality rates into a demographic simulation of a honey bee colony in an environmentally realistic scenario. Additionally, we evaluated two bioassays from new international guidance for agrochemical regulation, which aim to detect TRT. Finally, we used analytical chemistry (GC-MS) to test for bioaccumulation of fipronil. We found in demographic simulations that only fipronil produced mass mortality in honey bees. In the bioassays, only fipronil caused TRT. GC-MS analysis revealed that virtually all of the fipronil ingested by a honey bee in a single meal was present 6 d later, which suggests that bioaccumulation is the basis of TRT in sustained dietary exposures. We therefore postulate that fipronil, not imidacloprid, caused the mass mortalities of honey bees in France during the 1990s because it is lethal to honey bees in even trace doses due to its capacity to bioaccumulate and generate TRT. Our results provide evidence that recently proposed laboratory bioassays can discriminate harmful bioaccumulative substances and, thereby, address evident shortcomings in a regulatory system that had formerly approved fipronil for agricultural use.
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Ogino Y, Tohyama S, Kohno S, Toyota K, Yamada G, Yatsu R, Kobayashi T, Tatarazako N, Sato T, Matsubara H, et al (2018). Functional distinctions associated with the diversity of sex steroid hormone receptors ESR and AR.
J Steroid Biochem Mol Biol,
184, 38-46.
Abstract:
Functional distinctions associated with the diversity of sex steroid hormone receptors ESR and AR.
Sex steroid hormones including estrogens and androgens play fundamental roles in regulating reproductive activities and they act through estrogen and androgen receptors (ESR and AR). These steroid receptors have evolved from a common ancestor in association with several gene duplications. In most vertebrates, this has resulted in two ESR subtypes (ESR1 and ESR2) and one AR, whereas in teleost fish there are at least three ESRs (ESR1, ESR2a and ESR2b) and two ARs (ARα and ARβ) due to a lineage-specific whole genome duplication. Functional distinctions have been suggested among these receptors, but to date their roles have only been characterized in a limited number of species. Sexual differentiation and the development of reproductive organs are indispensable for all animal species and in vertebrates these events depend on the action of sex steroid hormones. Here we review the recent progress in understanding of the functions of the ESRs and ARs in the development and expression of sexually dimorphic characteristics associated with steroid hormone signaling in vertebrates, with representative fish, amphibians, reptiles, birds and mammals.
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Holder PJ, Jones A, Tyler C, Cresswell J (2018). Holder et al (PNAS): Datasets for dietary exposures of mixed-age honey bees to fipronil, imidacloprid, thiamethoxam and cypermethrin.
Abstract:
Holder et al (PNAS): Datasets for dietary exposures of mixed-age honey bees to fipronil, imidacloprid, thiamethoxam and cypermethrin
Feeding and longevity endpoints for laboratory exposures of caged honey bees to various dietary pesticides. Analysis of these data are presented in Holder et al. 2018 (PNAS).
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Stevens C (2018). Patterns in Stress and Mortality in Small Ornamental Aquarium Fish and Interventions for Improving Health and Well-being.
Abstract:
Patterns in Stress and Mortality in Small Ornamental Aquarium Fish and Interventions for Improving Health and Well-being
The ornamental fish trade is an industry of significant size and scope, trading over 1.5 billion fishes each year, and worth approximately over 370 million USD. Ornamental fishes are kept world-wide, and are one of the most popular pets in UK households. The industry is currently experiencing steady growth, and has done since the FAO began keeping records in the 1970s. Despite this, the welfare of fishes within the industry remains one of the least-studied areas in the field of animal welfare. Mortality rates of fishes within the industry are debated, with estimates ranging from less than 2% to over 70%; however, a lack of clear data means that the accuracy of these figures is difficult to determine. Where mortality is believed to be high, stressors in the supply chain are thought to be a significant contributing factor. In this thesis, I explored some possible interventions designed to reduce the stress experienced by ornamental fishes.
Stress in fishes can be measured in a variety of ways, but the most common way is probably measurement of cortisol release rates. However, cortisol has often previously been measured in fishes by taking a blood sample – a technique which cannot be applied to many ornamental species as they are too small to obtain enough blood. Instead, cortisol released by small fishes can be measured in the fish holding water. I carried out a study to validate the use of this method in my study species and found that cortisol can be detected in the holding water of all three species, although I did not find clear differences between stressed fishes and controls. This highlighted the importance of using a variety of measures of stress, including behavioural measures, which are one of the most cost-effective ways to assess stress, and can easily be implemented in the ornamental fish supply chain.
Based on the literature, personal observations of industry practices, and the results of my analyses, a number of interventions intended to help reduce stress in ornamental species were developed. These involved training handlers to catch fish more effectively, providing neon tetras with environmental choices to allow them to select conditions which might promote welfare, and conditioning guppies to associate handling events with a reward or a predictable signal. I found fish which were not handled but were exposed to trained handlers showed fewer behavioural signs of stress than those exposed to untrained handlers, and that neon tetras showed preferences for particular tank backgrounds over others. However, I did not find any evidence that trained handlers caused less stress in handled fish, or that conditioning led to lower stress in handled fish.
the results of this project suggest that there are a number of sources of stress and poor welfare in the ornamental fish industry which may be contributing to high mortality rates. However, many of these sources can be addressed, either through application of current best-practice guidelines or by introduction of training programmes which encourage understanding and empathy for fishes. Further work aimed at developing interventions including enrichment strategies, conditioning regimes, and other areas of research, will likely help to further reduce stress and mortality, and improve fish welfare.
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2017
Winter MJ, Windell D, Metz J, Matthews P, Pinion J, Brown JT, Hetheridge MJ, Ball JS, Owen SF, Redfern WS, et al (2017). 4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.
Sci Rep,
7(1).
Abstract:
4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.
Functional neuroimaging, using genetically-encoded Ca2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.
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Moreman J, Lee O, Trznadel M, David A, Kudoh T, Tyler CR (2017). Acute Toxicity, Teratogenic, and Estrogenic Effects of Bisphenol a and its Alternative Replacements Bisphenol S, Bisphenol F, and Bisphenol AF in Zebrafish Embryo-Larvae.
Environ Sci Technol,
51(21), 12796-12805.
Abstract:
Acute Toxicity, Teratogenic, and Estrogenic Effects of Bisphenol a and its Alternative Replacements Bisphenol S, Bisphenol F, and Bisphenol AF in Zebrafish Embryo-Larvae.
Bisphenol a (BPA), a chemical incorporated into plastics and resins, has estrogenic activity and is associated with adverse health effects in humans and wildlife. Similarly structured BPA analogues are widely used but far less is known about their potential toxicity or estrogenic activity in vivo. We undertook the first comprehensive analysis on the toxicity and teratogenic effects of the bisphenols BPA, BPS, BPF, and BPAF in zebrafish embryo-larvae and an assessment on their estrogenic mechanisms in an estrogen-responsive transgenic fish Tg(ERE:Gal4ff)(UAS:GFP). The rank order for toxicity was BPAF > BPA > BPF > BPS. Developmental deformities for larval exposures included cardiac edema, spinal malformation, and craniofacial deformities and there were distinct differences in the effects and potencies between the different bisphenol chemicals. These effects, however, occurred only at concentrations between 1.0 and 200 mg/L which exceed those in most environments. All bisphenol compounds induced estrogenic responses in Tg(ERE:Gal4ff)(UAS:GFP) zebrafish that were inhibited by coexposure with ICI 182 780, demonstrating an estrogen receptor dependent mechanism. Target tissues included the heart, liver, somite muscle, fins, and corpuscles of Stannius. The rank order for estrogenicity was BPAF > BPA = BPF > BPS. Bioconcentration factors were 4.5, 17.8, 5.3, and 0.067 for exposure concentrations of 1.0, 1.0, 0.10, and 50 mg/L for BPA, BPF, BPAF, and BPS, respectively. We thus show that these BPA alternatives induce similar toxic and estrogenic effects to BPA and that BPAF is more potent than BPA, further highlighting health concerns regarding the use of BPA alternatives.
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Hamilton PB, Rolshausen G, Uren Webster TM, Tyler CR (2017). Adaptive capabilities and fitness consequences associated with pollution exposure in fish.
Philos Trans R Soc Lond B Biol Sci,
372(1712).
Abstract:
Adaptive capabilities and fitness consequences associated with pollution exposure in fish.
Many fish populations are exposed to harmful levels of chemical pollution and selection pressures associated with these exposures have led to the evolution of tolerance. Our understanding of the physiological basis for these adaptations is limited, but they are likely to include processes involved with the absorption, distribution, metabolism and/or excretion of the target chemical. Other potential adaptive mechanisms include enhancements in antioxidant responses, an increased capacity for DNA and/or tissue repair and alterations to the life cycle of fish that enable earlier reproduction. Analysis of single-nucleotide polymorphism frequencies has shown that tolerance to hydrocarbon pollutants in both marine and estuarine fish species involves alteration in the expression of the xenobiotic metabolism enzyme CYP1A. In this review, we present novel data showing also that variants of the CYP1A gene have been under selection in guppies living in Trinidadian rivers heavily polluted with crude oil. Potential costs associated with these adaptations could reduce fitness in unpolluted water conditions. Integrating knowledge of local adaptation to pollution is an important future consideration in conservation practices such as for successful restocking, and improving connectivity within river systems.This article is part of the themed issue 'Human influences on evolution, and the ecological and societal consequences'.
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Bickley LK, van Aerle R, Brown AR, Hargreaves A, Huby R, Cammack V, Jackson R, Santos EM, Tyler CR (2017). Bioavailability and Kidney Responses to Diclofenac in the Fathead Minnow (Pimephales promelas).
ENVIRONMENTAL SCIENCE & TECHNOLOGY,
51(3), 1764-1774.
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Bickley LK, van Aerle R, Brown AR, Hargreaves A, Huby R, Cammack V, Jackson R, Santos EM, Tyler CR (2017). Bioavailability and Kidney Responses to Diclofenac in the Fathead Minnow (Pimephales promelas).
Environ Sci Technol,
51(3), 1764-1774.
Abstract:
Bioavailability and Kidney Responses to Diclofenac in the Fathead Minnow (Pimephales promelas).
Diclofenac is one of the most widely prescribed nonsteroidal anti-inflammatory drugs worldwide. It is frequently detected in surface waters; however, whether this pharmaceutical poses a risk to aquatic organisms is debated. Here we quantified the uptake of diclofenac by the fathead minnow (Pimephales promelas) following aqueous exposure (0.2-25.0 μg L-1) for 21 days, and evaluated the tissue and biomolecular responses in the kidney. Diclofenac accumulated in a concentration- and time-dependent manner in the plasma of exposed fish. The highest plasma concentration observed (for fish exposed to 25 μg L-1 diclofenac) was within the therapeutic range for humans. There was a strong positive correlation between exposure concentration and the number of developing nephrons observed in the posterior kidney. Diclofenac was not found to modulate the expression of genes in the kidney associated with its primary mode of action in mammals (prostaglandin-endoperoxide synthases) but modulated genes associated with kidney repair and regeneration. There were no significant adverse effects following 21 days exposure to concentrations typical of surface waters. The combination of diclofenac's uptake potential, effects on kidney nephrons and relatively small safety margin for some surface waters may warrant a longer term chronic health effects analysis for diclofenac in fish.
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Lange A, Corcoran J, Miyagawa S, Iguchi T, Winter MJ, Tyler CR (2017). Development of a common carp (Cyprinus carpio) pregnane X receptor (cPXR) transactivation reporter assay and its activation by azole fungicides and pharmaceutical chemicals.
Toxicol in Vitro,
41, 114-122.
Abstract:
Development of a common carp (Cyprinus carpio) pregnane X receptor (cPXR) transactivation reporter assay and its activation by azole fungicides and pharmaceutical chemicals.
In mammals, the pregnane X receptor (PXR) is a transcription factor with a key role in regulating expression of several genes involved in drug biotransformation. PXR is present in fish and some genes known to be under its control can be up-regulated by mammalian PXR ligands. Despite this, direct involvement of PXR in drug biotransformation in fish has yet to be established. Here, the full length PXR sequence was cloned from carp (Cyprinus carpio) and used in a luciferase reporter assay to elucidate its role in xenobiotic metabolism in fish. A reporter assay for human PXR (hPXR) was also established to compare transactivation between human and carp (cPXR) isoforms. Rifampicin activated hPXR as expected, but not cPXR. Conversely, clotrimazole (CTZ) activated both isoforms and was more potent on cPXR, with an EC50 within the range of concentrations of CTZ measured in the aquatic environment. Responses to other azoles tested were similar between both isoforms. A range of pharmaceuticals tested either failed to activate, or were very weakly active, on the cPXR or hPXR. Overall, these results indicate that the cPXR may differ from the hPXR in its responses and/or sensitivity to induction by different environmental chemicals, with implications for risk assessment because of species differences.
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David A, Lange A, Abdul-Sada A, Tyler CR, Hill EM (2017). Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics.
Environ Sci Technol,
51(1), 616-624.
Abstract:
Disruption of the Prostaglandin Metabolome and Characterization of the Pharmaceutical Exposome in Fish Exposed to Wastewater Treatment Works Effluent As Revealed by Nanoflow-Nanospray Mass Spectrometry-Based Metabolomics.
Fish can be exposed to a complex mixture of chemical contaminants, including pharmaceuticals, present in discharges of wastewater treatment works (WwTWs) effluents. There is little information on the effects of effluent exposure on fish metabolism, especially the small molecule signaling compounds which are the biological target of many pharmaceuticals. We applied a newly developed sensitive nanoflow-nanospray mass spectrometry nontargeted profiling technique to identify changes in the exposome and metabolome of roach (Rutilus rutilus) exposed to a final WwTWs effluent for 15 days. Effluent exposure resulted in widespread reduction (between 50% and 90%) in prostaglandin (PG) profiles in fish tissues and plasma with disruptions also in tryptophan/serotonin, bile acid and lipid metabolism. Metabolite disruptions were not explained by altered expression of genes associated with the PG or tryptophan metabolism. of the 31 pharmaceutical metabolites that were detected in the effluent exposome of fish, 6 were nonsteroidal anti-inflammatory drugs but with plasma concentrations too low to disrupt PG biosynthesis. PGs, bile acids, and tryptophan metabolites are important mediators regulating a diverse array of physiological systems in fish and the identity of wastewater contaminants disrupting their metabolism warrants further investigation on their exposure effects on fish health.
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Galloway TS, Dogra Y, Garrett N, Rowe D, Tyler CR, Moger J, Lammer E, Landsiedel R, Sauer UG, Scherer G, et al (2017). Ecotoxicological assessment of nanoparticle-containing acrylic copolymer dispersions in fairy shrimp and zebrafish embryos.
Environmental Science: Nano,
4(10), 1981-1997.
Abstract:
Ecotoxicological assessment of nanoparticle-containing acrylic copolymer dispersions in fairy shrimp and zebrafish embryos
Nanoparticle-containing polymer dispersions are widely used, but little is known of their environmental effects. We studied the bioavailability, uptake, tissue localisation and effects of nanoparticle-containing acrylic copolymer (ACP) dispersions (mean nanoparticle sizes: 80 nm and 110 nm) in aquatic invertebrates (Thamnocephalus platyurus; fairy shrimp) and Danio rerio zebrafish embryos after aquatic exposures. Dietary exposure tests were enabled using Casper zebrafish that lack skin pigmentation allowing for bio-imaging of uptake and internal distribution. Aqueous exposures of 1000 and 2500 mg L-1 80 nm-ACP or 110 nm-ACP showed no acute toxicity in fairy shrimp or zebrafish, constituting a non-Toxic classification according to the United Nations Globally Harmonised System of Classification and Labelling of Chemicals threshold (100 mg L-1). Similarly, dietary exposures resulted in no ecotoxicological effects. In Casper zebrafish fed with 80 nm-ACP-spiked food, hyperspectral signals derived using coherent Raman scattering (CRS) indicated that test material was present in the intestine, and possibly in the liver, but not in other organs. CRS imaging indicated that the chemical composition of the yolk sac of an 80 nm-ACP exposed zebrafish (aquatic exposure) was altered, attributed to a change in lipid metabolism, although we could not confirm with certainty that the test material was physically present in the yolk sac. These results illustrate how CRS microscopy can be used to investigate the bioaccumulation of organic nanomaterials, provided that they induce hyperspectral profiles distinct from the biological samples. In conclusion, both 80 nm-and 110 nm-ACP dispersions are internalised through dietary exposure, but are not associated with significant toxic effects.
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Tohyama S, Ogino Y, Lange A, Myosho T, Kobayashi T, Hirano Y, Yamada G, Sato T, Tatarazako N, Tyler CR, et al (2017). Establishment of estrogen receptor 1 (ESR1)-knockout medaka: ESR1 is dispensable for sexual development and reproduction in medaka, Oryzias latipes.
Dev Growth Differ,
59(6), 552-561.
Abstract:
Establishment of estrogen receptor 1 (ESR1)-knockout medaka: ESR1 is dispensable for sexual development and reproduction in medaka, Oryzias latipes.
Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptor (ESR) in all vertebrates. Most vertebrates have two ESR subtypes (ESR1 and ESR2), whereas teleost fish have at least three (Esr1, Esr2a and Esr2b). Intricate functionalization has been suggested among the Esr subtypes, but to date, distinct roles of Esr have been characterized in only a limited number of species. Study of loss-of-function in animal models is a powerful tool for application to understanding vertebrate reproductive biology. In the current study, we established esr1 knockout (KO) medaka using a TALEN approach and examined the effects of Esr1 ablation. Unexpectedly, esr1 KO medaka did not show any significant defects in their gonadal development or in their sexual characteristics. Neither male or female esr1 KO medaka exhibited any significant changes in sexual differentiation or reproductive activity compared with wild type controls. Interestingly, however, estrogen-induced vitellogenin gene expression, an estrogen-responsive biomarker in fish, was limited in the liver of esr1 KO males. Our findings, in contrast to mammals, indicate that Esr1 is dispensable for normal development and reproduction in medaka. We thus provide an evidence for estrogen receptor functionalization between mammals and fish. Our findings will also benefit interpretation of studies into the toxicological effects of estrogenic chemicals in fish.
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Uren Webster TM, Williams TD, Katsiadaki I, Lange A, Lewis C, Shears JA, Tyler CR, Santos EM (2017). Hepatic transcriptional responses to copper in the three-spined stickleback are affected by their pollution exposure history.
Aquat Toxicol,
184, 26-36.
Abstract:
Hepatic transcriptional responses to copper in the three-spined stickleback are affected by their pollution exposure history.
Some fish populations inhabiting contaminated environments show evidence of increased chemical tolerance, however the mechanisms contributing to this tolerance, and whether this is heritable, are poorly understood. We investigated the responses of two populations of wild three-spined stickleback (Gasterosteus aculeatus) with different histories of contaminant exposure to an oestrogen and copper, two widespread aquatic pollutants. Male stickleback originating from two sites, the River Aire, with a history of complex pollution discharges, and Siblyback Lake, with a history of metal contamination, were depurated and then exposed to copper (46μg/L) and the synthetic oestrogen ethinyloestradiol (22ng/L). The hepatic transcriptomic response was compared between the two populations and to a reference population with no known history of exposure (Houghton Springs, Dorset). Gene responses included those typical for both copper and oestrogen, with no discernable difference in response to oestrogen between populations. There was, however, some difference in the magnitude of response to copper between populations. Siblyback fish showed an elevated baseline transcription of genes encoding metallothioneins and a lower level of metallothionein induction following copper exposure, compared to those from the River Aire. Similarly, a further experiment with an F1 generation of Siblyback fish bred in the laboratory found evidence for elevated transcription of genes encoding metallothioneins in unexposed fish, together with an altered transcriptional response to 125μg/L copper, compared with F1 fish originating from the clean reference population exposed to the same copper concentration. These data suggest that the stickleback from Siblyback Lake have a differential response to copper, which is inherited by the F1 generation in laboratory conditions, and for which the underlying mechanism may include an elevation of baseline transcription of genes encoding metallothioneins. The genetic and/or epigenetic mechanisms contributing to this inherited alteration of metallothionein transcription have yet to be established.
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Sherman KD, King RA, Dahlgren CP, Simpson SD, Stevens JR, Tyler CR (2017). Historical processes and contemporary anthropogenic activities influence genetic population dynamics of Nassau grouper (Epinephelus striatus) within the Bahamas.
Frontiers in Marine Science,
4(DEC).
Abstract:
Historical processes and contemporary anthropogenic activities influence genetic population dynamics of Nassau grouper (Epinephelus striatus) within the Bahamas
Severe declines of endangered Nassau grouper (Epinephelus striatus) across the Bahamas and Caribbean have spurred efforts to improve their fisheries management and population conservation. The Bahamas is reported to hold the majority of fish spawning aggregations for Nassau grouper, however, the status and genetic population structure of fish within the country is largely unknown, presenting a major knowledge gap for their sustainable management. Between August 2014-February 2017, 464 individual Nassau grouper sampled from the Bahamas were genotyped using 15 polymorphic microsatellite loci to establish measures of population structure, genetic diversity and effective population size (Ne). Nassau grouper were characterized by mostly high levels of genetic diversity, but we found no evidence for geographic population structure. Microsatellite analyses revealed weak, but significant genetic differentiation of Nassau grouper throughout the Bahamian archipelago (Global FST 0.00236, p = 0.0001). Temporal analyses of changes in Ne over the last 1,000 generations provide evidence in support of a pronounced historic decline in Bahamian Nassau grouper that appears to pre-date anthropogenic fishing activities. M-ratio results corroborate significant reductions in Ne throughout the Bahamas, with evidence for population bottlenecks in three islands and an active fish spawning aggregation along with apparent signs of inbreeding at two islands. Current estimates of Ne for Nassau grouper are considerably lower compared with historic levels. These findings represent important new contributions to our understanding of the evolutionary history, demographics and genetic connectivity of this endangered species, which are of critical importance for advancing their sustainable management.
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Le Page G, Gunnarsson L, Snape J, Tyler CR (2017). Integrating human and environmental health in antibiotic risk assessment: a critical analysis of protection goals, species sensitivity and antimicrobial resistance.
Environ Int,
109, 155-169.
Abstract:
Integrating human and environmental health in antibiotic risk assessment: a critical analysis of protection goals, species sensitivity and antimicrobial resistance.
Antibiotics are vital in the treatment of bacterial infectious diseases but when released into the environment they may impact non-target organisms that perform vital ecosystem services and enhance antimicrobial resistance development with significant consequences for human health. We evaluate whether the current environmental risk assessment regulatory guidance is protective of antibiotic impacts on the environment, protective of antimicrobial resistance, and propose science-based protection goals for antibiotic manufacturing discharges. A review and meta-analysis was conducted of aquatic ecotoxicity data for antibiotics and for minimum selective concentration data derived from clinically relevant bacteria. Relative species sensitivity was investigated applying general linear models, and predicted no effect concentrations were generated for toxicity to aquatic organisms and compared with predicted no effect concentrations for resistance development. Prokaryotes were most sensitive to antibiotics but the range of sensitivities spanned up to several orders of magnitude. We show reliance on one species of (cyano)bacteria and the 'activated sludge respiration inhibition test' is not sufficient to set protection levels for the environment. Individually, neither traditional aquatic predicted no effect concentrations nor predicted no effect concentrations suggested to safeguard for antimicrobial resistance, protect against environmental or human health effects (via antimicrobial resistance development). Including data from clinically relevant bacteria and also more species of environmentally relevant bacteria in the regulatory framework would help in defining safe discharge concentrations for antibiotics for patient use and manufacturing that would protect environmental and human health. It would also support ending unnecessary testing on metazoan species.
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Bruintjes R, Harding HR, Bunce T, Birch F, Lister J, Spiga I, Benson T, Rossington K, Jones D, Tyler CR, et al (2017). Shipbuilding Docks as Experimental Systems for Realistic Assessments of Anthropogenic Stressors on Marine Organisms.
BIOSCIENCE,
67(9), 853-859.
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2016
Lillicrap A, Springer T, Tyler CR (2016). A tiered assessment strategy for more effective evaluation of bioaccumulation of chemicals in fish.
Regul Toxicol Pharmacol,
75, 20-26.
Abstract:
A tiered assessment strategy for more effective evaluation of bioaccumulation of chemicals in fish.
There is currently limited guidance available for regulators and risk assessors on how to use data from non-guideline methods when assessing the bioaccumulation potential of a chemical. Furthermore, bioaccumulation assessments can be more subjective than they need to be due to the lack of a guidance framework on how to use/include the range of information that may be available for a substance. Under some circumstances, in silico, in vitro and/or in vivo non-test guideline data may be sufficient to classify whether a substance is bioaccumulative without the need for further animal testing. Classifying the bioaccumulative potential of a substance is especially difficult when the bioconcentration factor (BCF) is close to the threshold for defining it as bioaccumulative/very bioaccumulative (B/vB), and a more structured process is required to reduce uncertainty in the BCF estimates. In these situations, in silico and in vitro data can, and should, be used to provide greater confidence in classifying these substances. To aid future evaluations of bioaccumulation data, a proposed tiered assessment strategy is presented incorporating all available data on the bioaccumulative properties of a substance. In addition, a revised scheme is recommended for improving the classification of the bioaccumulative potential of a substance.
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Dogra Y, Arkill KP, Elgy C, Stolpe B, Lead J, Valsami-Jones E, Tyler CR, Galloway TS (2016). Cerium oxide nanoparticles induce oxidative stress in the sediment-dwelling amphipod Corophium volutator.
Nanotoxicology,
10(4), 480-487.
Abstract:
Cerium oxide nanoparticles induce oxidative stress in the sediment-dwelling amphipod Corophium volutator.
Cerium oxide nanoparticles (CeO2 NPs) exhibit fast valence exchange between Ce(IV) and Ce(III) associated with oxygen storage and both pro and antioxidant activities have been reported in laboratory models. The reactivity of CeO2 NPs once they are released into the aquatic environment is virtually unknown, but this is important to determine for assessing their environmental risk. Here, we show that amphipods (Corophium volutator) grown in marine sediments containing CeO2 NPs showed a significant increase in oxidative damage compared to those grown in sediments without NPs and those containing large-sized (bulk) CeO2 particles. There was no exposure effect on survival, but significant increases in single-strand DNA breaks, lipid peroxidation and superoxide dismutase activity were observed after a 10-day exposure to 12.5 mg L(-1) CeO2. Characterisation of the CeO2 NPs dispersed in deionised or saline exposure waters revealed that more radicals were produced by CeO2 NPs compared with bulk CeO2. Electron energy loss spectroscopy (EELS) analysis revealed that both CeO2 NPs were predominantly Ce(III) in saline waters compared to deionised waters where they were predominantly Ce(IV). In both types of medium, the bulk CeO2 consisted mainly of Ce(IV). These results support a model whereby redox cycling of CeO2 NPs between Ce(III) and Ce(IV) is enhanced in saline waters, leading to sublethal oxidative damage to tissues in our test organism.
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Tohyama S, Miyagawa S, Lange A, Ogino Y, Mizutani T, Ihara M, Tanaka H, Tatarazako N, Kobayashi T, Tyler CR, et al (2016). Evolution of estrogen receptors in ray-finned fish and their comparative responses to estrogenic substances.
J Steroid Biochem Mol Biol,
158, 189-197.
Abstract:
Evolution of estrogen receptors in ray-finned fish and their comparative responses to estrogenic substances.
In vertebrates, estrogens play fundamental roles in regulating reproductive activities through estrogen receptors (ESRs), and disruption of estrogen signaling is now of global concern for both wildlife and human health. To date, ESRs of only a limited number of species have been characterized. We investigated the functional diversity and molecular basis or ligand sensitivity of ESRs among ray-finned fish species (Actinopterygii), the most variable group within vertebrates. We cloned and characterized ESRs from several key species in the evolution of ray-finned fish including bichir (Polypteriformes, ESR1 and ESR2) at the basal lineage of ray-finned fish, and arowana (Osteoglossiformes, ESR1 and ESR2b) and eel (Anguilliformes, ESR1, ESR2a and ESR2b) both belonging to ancient early-branching lineages of teleosts, and suggest that ESR2a and ESR2b emerged through teleost-specific whole genome duplication, but an ESR1 paralogue has been lost in the early lineage of euteleost fish species. All cloned ESR isoforms showed similar responses to endogenous and synthetic steroidal estrogens, but they responded differently to non-steroidal estrogenic endocrine disrupting chemicals (EDCs) (e.g. ESR2a exhibits a weaker reporter activity compared with ESR2b). We show that variation in ligand sensitivity of ESRs can be attributed to phylogeny among species of different taxonomic groups in ray-finned fish. The molecular information provided contributes both to understanding of the comparative role of ESRs in the reproductive biology of fish and their comparative responses to EDCs.
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Author URL.
Green JM, Metz J, Lee O, Trznadel M, Takesono A, Brown AR, Owen SF, Kudoh T, Tyler CR (2016). High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.
Environ Sci Technol,
50(12), 6536-6545.
Abstract:
High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.
Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates.
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Author URL.
Sherman KD, Dahlgren CP, Stevens JR, Tyler CR (2016). Integrating population biology into conservation management for endangered Nassau grouper Epinephelus striatus.
Marine Ecology Progress Series,
554, 263-280.
Abstract:
Integrating population biology into conservation management for endangered Nassau grouper Epinephelus striatus
Groupers are a phylogenetically diverse group and include many ecologically and economically valuable predatory marine fishes that have experienced drastic population declines. Reproduction via spawning aggregations increases the vulnerability of grouper species such as Nassau grouper Epinephelus striatus to overfishing, and this is likely to be a major contributing factor to population declines. However, the lack of information pertaining to population structure and dynamics of Nassau grouper spawning aggregations has impeded effective ecosystem-based fisheries management for remaining stocks. Worldwide, the Bahamas has the largest number of known Nassau grouper spawning aggregations, yet very little is known about the overall status of groupers in the region. Landings of Nassau grouper in the Bahamas have declined by 86% in the last 20 years from a peak of 514 t in 1997. Available data suggest that existing management measures are failing in their attempts to prevent further declines. Effective management strategies are urgently needed that balance ecological and socioeconomic considerations to enable a sustainable Nassau grouper fishery. This review provides an analysis of the reproductive and population biology of Nassau grouper and a suggested framework to direct future research efforts for enhancing conservation management of this endangered marine fish species.
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Collison E, Hird H, Cresswell J, Tyler C (2016). Interactive effects of pesticide exposure and pathogen infection on bee health – a critical analysis.
Biological Reviews,
91(4), 1006-1019.
Abstract:
Interactive effects of pesticide exposure and pathogen infection on bee health – a critical analysis
Bees are fundamentally important for pollination services and declines in populations could have significant economic and environmental implications. Pesticide exposure and pathogen infection are recognised as potential stressors impacting upon bee populations and recently there has been a surge in research on pesticide–disease interactions to reflect environmentally realistic scenarios better. We critically analyse the findings on pesticide–disease interactions, including effects on the survival, pathogen loads and immunity of bees, and assess the suitability of various endpoints to inform our mechanistic understanding of these interactions. We show that pesticide exposure and pathogen infection have not yet been found to interact to affect worker survival under field-realistic scenarios. Colony-level implications of pesticide effects on Nosema infections, viral loads and honey bee immunity remain unclear as these effects have been observed in a laboratory setting only using a small range of pesticide exposures, generally exceeding those likely to occur in the natural environment, and assessing a highly selected series of immune-related endpoints. Future research priorities include the need for a better understanding of pesticide effects on the antimicrobial peptide (AMP) component of an individual's immune response and on social defence behaviours. Interactions between pesticide exposure and bacterial and fungal infections have yet to be addressed. The paucity of studies in non-Apis bee species is a further major knowledge gap.
Abstract.
Verbruggen B, Bickley LK, van Aerle R, Bateman KS, Stentiford GD, Santos EM, Tyler CR (2016). Molecular Mechanisms of White Spot Syndrome Virus Infection and Perspectives on Treatments.
Viruses,
8(1).
Abstract:
Molecular Mechanisms of White Spot Syndrome Virus Infection and Perspectives on Treatments.
Since its emergence in the 1990s, White Spot Disease (WSD) has had major economic and societal impact in the crustacean aquaculture sector. Over the years shrimp farming alone has experienced billion dollar losses through WSD. The disease is caused by the White Spot Syndrome Virus (WSSV), a large dsDNA virus and the only member of the Nimaviridae family. Susceptibility to WSSV in a wide range of crustacean hosts makes it a major risk factor in the translocation of live animals and in commodity products. Currently there are no effective treatments for this disease. Understanding the molecular basis of disease processes has contributed significantly to the treatment of many human and animal pathogens, and with a similar aim considerable efforts have been directed towards understanding host-pathogen molecular interactions for WSD. Work on the molecular mechanisms of pathogenesis in aquatic crustaceans has been restricted by a lack of sequenced and annotated genomes for host species. Nevertheless, some of the key host-pathogen interactions have been established: between viral envelope proteins and host cell receptors at initiation of infection, involvement of various immune system pathways in response to WSSV, and the roles of various host and virus miRNAs in mitigation or progression of disease. Despite these advances, many fundamental knowledge gaps remain; for example, the roles of the majority of WSSV proteins are still unknown. In this review we assess current knowledge of how WSSV infects and replicates in its host, and critique strategies for WSD treatment.
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Hamilton PB, Cowx IG, Oleksiak MF, Griffiths AM, Grahn M, Stevens JR, Carvalho GR, Nicol E, Tyler CR (2016). Population-level consequences for wild fish exposed to sublethal concentrations of chemicals – a critical review.
Fish and Fisheries,
17(3), 545-566.
Abstract:
Population-level consequences for wild fish exposed to sublethal concentrations of chemicals – a critical review
Concentrated chemical spills have been shown to impact adversely on fish populations and even cause localized population extinctions. Evaluating population-level impacts of sublethal exposure concentrations is, however, complex and confounded by other environmental pressures. Applying effect measures derived from laboratory-based chemical exposures to impacts in wild fish populations is constrained by uncertainty on how biochemical response measures (biomarkers) translate into health outcomes, lack of available data for chronic exposures and the many uncertainties in available fish population models. Furthermore, wild fish show phenotypic plasticity and local adaptations can occur that adds geographic and temporal variance on responses. Such population-level factors are rarely considered in the chemical risk assessment process and can probably be derived only from studies on wild fish. Molecular technologies, including microsatellite and SNP genotyping, and RNASeq for gene expression studies, are advancing our understanding of mechanisms of eco-toxicological response, tolerance, adaptation and selection in wild populations. We examine critically the application of such approaches with examples including using microsatellites that has identified roach (Rutilus rutilus) populations living in rivers contaminated with sewage effluents that are self-sustaining, and studies of stickleback (Gasterosteus aculeatus) and killifish (Fundulus heteroclitus) that have identified genomic regions under selection putatively related to pollution tolerance. Integrating data on biological effects between laboratory-based studies and wild populations, and building understanding on adaptive responses to sublethal exposure are some of the priority research areas for more effective evaluation of population risks and resilience to contaminant exposure.
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Osborne OJ, Mukaigasa K, Nakajima H, Stolpe B, Romer I, Philips U, Lynch I, Mourabit S, Hirose S, Lead JR, et al (2016). Sensory systems and ionocytes are targets for silver nanoparticle effects in fish.
Nanotoxicology,
10(9), 1276-1286.
Abstract:
Sensory systems and ionocytes are targets for silver nanoparticle effects in fish.
Some nanoparticles (NPs) may induce adverse health effects in exposed organisms, but to date the evidence for this in wildlife is very limited. Silver nanoparticles (AgNPs) can be toxic to aquatic organisms, including fish, at concentrations relevant for some environmental exposures. We applied whole mount in-situ hybridisation (WISH) in zebrafish embryos and larvae for a suite of genes involved with detoxifying processes and oxidative stress, including metallothionein (mt2), glutathionine S-transferase pi (gstp), glutathionine S-transferase mu (gstm1), haem oxygenase (hmox1) and ferritin heavy chain 1 (fth1) to identify potential target tissues and effect mechanisms of AgNPs compared with a bulk counterpart and ionic silver (AgNO3). AgNPs caused upregulation in the expression of mt2, gstp and gstm1 and down regulation of expression of both hmox1 and fth1 and there were both life stage and tissue-specific responses. Responding tissues included olfactory bulbs, lateral line neuromasts and ionocytes in the skin with the potential for effects on olfaction, behaviour and maintenance of ion balance. Silver ions induced similar gene responses and affected the same target tissues as AgNPs. AgNPs invoked levels of target gene responses more similar to silver treatments compared to coated AgNPs indicating the responses seen were due to released silver ions. In the Nrf2 zebrafish mutant, expression of mt2 (24 hpf) and gstp (3 dpf) were either non-detectable or were at lower levels compared with wild type zebrafish for exposures to AgNPs, indicating that these gene responses are controlled through the Nrf2-Keap pathway.
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2015
Song L, Vijver MG, Peijnenburg WJGM, Galloway TS, Tyler CR (2015). A comparative analysis on the in vivo toxicity of copper nanoparticles in three species of freshwater fish.
Chemosphere,
139, 181-189.
Abstract:
A comparative analysis on the in vivo toxicity of copper nanoparticles in three species of freshwater fish.
Copper nanoparticles (CuNPs) are used extensively in a wide range of products and the potential for toxicological impacts in the aquatic environment is of high concern. In this study, the fate and the acute toxicity of spherical 50nm copper nanoparticles was assessed in juvenile rainbow trout (Oncorhynchus mykiss), fathead minnow (Pimephales promelas) and zebrafish (Danio rerio) for in vivo aqueous exposures following standardized OECD 203 guideline tests. The fate of the CuNPs in the aqueous media was temperature dependent. At the higher study temperature (26±1°C), there was both an enhanced particle aggregation and higher rate of dissolution compared with that at the lower study temperature (15±1°C). 96h LC50s of the CuNPs were 0.68±0.15, 0.28±0.04 and 0.22±0.08mg Cu/L for rainbow trout, fathead minnow and zebrafish, respectively. The 96h lowest-observed-effect concentration (LOEC) for the CuNPs were 0.17, 0.023 and
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Miyagawa S, Lange A, Tohyama S, Ogino Y, Mizutani T, Kobayashi T, Tatarazako N, Tyler CR, Iguchi T (2015). Characterization of Oryzias latipes glucocorticoid receptors and their unique response to progestins.
Journal of Applied Toxicology,
35(3), 302-309.
Abstract:
Characterization of Oryzias latipes glucocorticoid receptors and their unique response to progestins
Various receptor bioassays, including estrogens, androgens and thyroid hormones, have been developed and applied successfully for assessing hormone function in a wide range of animal species, including fish. In fish, corticosteroids play a pivotal role in physiology as they do in mammals, but far less is known about the corticosteroid receptor system in fish compared with in mammals. Here we established a transient transactivation assay using the Japanese medaka, Oryzias latipes, glucocorticoid receptors (olGRs) and mineralocorticoid receptor to analyse their functional properties in a fish. We found that olGR2 was highly responsive to glucocorticoids, similar to the human GR, whereas the olGR1 subtype was minimally responsive. Thus, olGR2 most likely mediates glucocorticoid signaling in medaka. We further tested crosstalk between GRs and other steroid hormones, and found that progestins could activate or inactivate olGR2-mediating transcription, depending on the presence or absence of cortisol. The transactivation assays developed for medaka GRs provide tools to gain useful insights into corticosteroid signaling in fish and for in vitro screening of environmental substances activating GRs.
Abstract.
Brown AR, Owen SF, Peters J, Zhang Y, Soffker M, Paull GC, Hosken DJ, Wahab MA, Tyler CR (2015). Climate change and pollution speed declines in zebrafish populations.
Proc Natl Acad Sci U S A,
112(11), E1237-E1246.
Abstract:
Climate change and pollution speed declines in zebrafish populations.
Endocrine disrupting chemicals (EDCs) are potent environmental contaminants, and their effects on wildlife populations could be exacerbated by climate change, especially in species with environmental sex determination. Endangered species may be particularly at risk because inbreeding depression and stochastic fluctuations in male and female numbers are often observed in the small populations that typify these taxa. Here, we assessed the interactive effects of water temperature and EDC exposure on sexual development and population viability of inbred and outbred zebrafish (Danio rerio). Water temperatures adopted were 28 °C (current ambient mean spawning temperature) and 33 °C (projected for the year 2100). The EDC selected was clotrimazole (at 2 μg/L and 10 μg/L), a widely used antifungal chemical that inhibits a key steroidogenic enzyme [cytochrome P450(CYP19) aromatase] required for estrogen synthesis in vertebrates. Elevated water temperature and clotrimazole exposure independently induced male-skewed sex ratios, and the effects of clotrimazole were greater at the higher temperature. Male sex ratio skews also occurred for the lower clotrimazole exposure concentration at the higher water temperature in inbred fish but not in outbred fish. Population viability analysis showed that population growth rates declined sharply in response to male skews and declines for inbred populations occurred at lower male skews than for outbred populations. These results indicate that elevated temperature associated with climate change can amplify the effects of EDCs and these effects are likely to be most acute in small, inbred populations exhibiting environmental sex determination and/or differentiation.
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Verbruggen B, Bickley LK, Santos EM, Tyler CR, Stentiford GD, Bateman KS, van Aerle R (2015). De novo assembly of the Carcinus maenas transcriptome and characterization of innate immune system pathways.
BMC Genomics,
16Abstract:
De novo assembly of the Carcinus maenas transcriptome and characterization of innate immune system pathways.
BACKGROUND: the European shore crab, Carcinus maenas, is used widely in biomonitoring, ecotoxicology and for studies into host-pathogen interactions. It is also an important invasive species in numerous global locations. However, the genomic resources for this organism are still sparse, limiting research progress in these fields. To address this resource shortfall we produced a C. maenas transcriptome, enabled by the progress in next-generation sequencing technologies, and applied this to assemble information on the innate immune system in this species. RESULTS: We isolated and pooled RNA for twelve different tissues and organs from C. maenas individuals and sequenced the RNA using next generation sequencing on an Illumina HiSeq 2500 platform. After de novo assembly a transcriptome was generated encompassing 212,427 transcripts (153,699 loci). The transcripts were filtered, annotated and characterised using a variety of tools (including BLAST, MEGAN and RSEM) and databases (including NCBI, Gene Ontology and KEGG). There were differential patterns of expression for between 1,223 and 2,741 transcripts across tissues and organs with over-represented Gene Ontology terms relating to their specific function. Based on sequence homology to immune system components in other organisms, we show both the presence of transcripts for a series of known pathogen recognition receptors and response proteins that form part of the innate immune system, and transcripts representing the RNAi, Toll-like receptor signalling, IMD and JAK/STAT pathways. CONCLUSIONS: We have produced an assembled transcriptome for C. maenas that provides a significant molecular resource for wide ranging studies in this species. Analysis of the transcriptome has revealed the presence of a series of known targets and functional pathways that form part of their innate immune system and illustrate tissue specific differences in their expression patterns.
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Orton F, Tyler CR (2015). Do hormone-modulating chemicals impact on reproduction and development of wild amphibians?.
Biol Rev Camb Philos Soc,
90(4), 1100-1117.
Abstract:
Do hormone-modulating chemicals impact on reproduction and development of wild amphibians?
Globally, amphibians are undergoing a precipitous decline. At the last estimate in 2004, 32% of the approximately 6000 species were threatened with extinction and 43% were experiencing significant declines. These declines have been linked with a wide range of environmental pressures from habitat loss to climate change, disease and pollution. This review evaluates the evidence that endocrine-disrupting contaminants (EDCs) - pollutants that affect hormone systems - are impacting on wild amphibians and contributing to population declines. The review is limited to anurans (frogs and toads) as data for effects of EDCs on wild urodeles (salamanders, newts) or caecilians (limbless amphibians) are extremely limited. Evidence from laboratory studies has shown that a wide range of chemicals have the ability to alter hormone systems and affect reproductive development and function in anurans, but for the most part only at concentrations exceeding those normally found in natural environments. Exceptions can be found for exposures to the herbicide atrazine and polychlorinated biphenyls in leopard frogs (Rana pipiens) and perchlorate in African clawed frogs (Xenopus laevis). These contaminants induce feminising effects on the male gonads (including 'intersex' - oocytes within testes) at concentrations measured in some aquatic environments. The most extensive data for effects of an EDC in wild amphibian populations are for feminising effects of atrazine on male gonad development in regions across the USA. Even where strong evidence has been provided for feminising effects of EDCs, however, the possible impact of these effects on fertility and breeding outcome has not been established, making inference for effects on populations difficult. Laboratory studies have shown that various chemicals, including perchlorate, polychlorinated biphenyls and bromodiphenylethers, also act as endocrine disrupters through interfering with thyroid-dependent processes that are fundamental for amphibian metamorphosis. Perchlorate has also been shown to induce these effects in wild anuran populations from perchlorate-contaminated environments. Overall, the published data available suggest that some health effects observed in wild anuran populations, most notably intersex, likely have a chemical aetiology; however they derive only from very few anuran species and for a few pesticides at field sites in the USA. To understand better the impacts of EDCs on wild anuran populations, as well as other amphibian groups, assessment of fertility in exposed animals are required. Development of non-destructive biomarkers that are indicative of specific EDC-effect mechanisms are also needed to allow the study of vulnerable populations. This will help to distinguish the effects of EDCs from other environmental and/or genetic influences on development and reproduction.
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Rolshausen G, Phillip DAT, Beckles DM, Akbari A, Ghoshal S, Hamilton PB, Tyler CR, Scarlett AG, Ramnarine I, Bentzen P, et al (2015). Do stressful conditions make adaptation difficult? Guppies in the oil-polluted environments of southern Trinidad.
Evol Appl,
8(9), 854-870.
Abstract:
Do stressful conditions make adaptation difficult? Guppies in the oil-polluted environments of southern Trinidad.
The ability of populations to rapidly adapt to new environments will determine their future in an increasingly human-modified world. Although meta-analyses do frequently uncover signatures of local adaptation, they also reveal many exceptions. We suggest that particular constraints on local adaptation might arise when organisms are exposed to novel stressors, such as anthropogenic pollution. To inform this possibility, we studied the extent to which guppies (Poecilia reticulata) show local adaptation to oil pollution in southern Trinidad. Neutral genetic markers revealed that paired populations in oil-polluted versus not-polluted habitats diverged independently in two different watersheds. Morphometrics revealed some divergence (particularly in head shape) between these environments, some of which was parallel between rivers. Reciprocal transplant experiments in nature, however, found little evidence of local adaptation based on survival and growth. Moreover, subsequent laboratory experiments showed that the two populations from oil-polluted sites showed only weak local adaptation even when compared to guppies from oil-free northern Trinidad. We conclude that guppies show little local adaptation to oil pollution, which might result from the challenges associated with adaptation to particularly stressful environments. It might also reflect genetic drift owing to small population sizes and/or high gene flow between environments.
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Goodhead RM, Johnston BD, Cole PA, Baalousha M, Hodgson D, Iguchi T, Lead JR, Tyler CR (2015). Does natural organic matter increase the bioavailability of cerium dioxide nanoparticles to fish?.
Abstract:
Does natural organic matter increase the bioavailability of cerium dioxide nanoparticles to fish?
Abstract.
Jennings V, Goodhead R, Tyler CR (2015). Ecotoxicology of Nanomaterials in Aquatic Systems.
,
8, 3-45.
Abstract:
Ecotoxicology of Nanomaterials in Aquatic Systems
The unique properties of manufactured nanomaterials (NMs), conveyed by their small size that are exploited for novel uses, may also confer different toxicological effects compared with their larger (bulk) counterparts. In this first chapter we provide a critical review on current toxicity data for NMs in aquatic organisms and illustrate thorough characterisation of NMs, which is essential for making comparisons on effects data. Adverse effects of NMs have been shown for a wide range of NMs and in diverse aquatic organisms but, with a few exceptions, only for concentrations that exceed current levels in the natural environment predicted by modelling studies. For some NMs, there appear to be particle-induced biological effectsthat include oxidative stress. For some metal NMs most evidence indicates that biological effects derive principally from metal ions due to dissolution. For carbon-based NMs some effects have been shown to derive from materials associated with the preparation of those NMs. The physicochemistry of the aquatic environment fundamentally effects the form of NMs altering their toxicity, and any standardised tests for assessing 'nano'-related toxicity in aquatic wildlife will need comprehensive characterisation on the dynamics of NMs in relevant test media. Some nanomedicines are being designed specifically to penetrate cell membranes and induce altered biological function and they may warrant particular attention for risk analysis in the near future.
Abstract.
Hamilton PB, Lange A, Nicol E, Bickley LK, De-Bastos ESR, Jobling S, Tyler CR (2015). Effects of Exposure to WwTW Effluents over Two Generations on Sexual Development and Breeding in Roach Rutilus rutilus.
Environ Sci Technol,
49(21), 12994-13002.
Abstract:
Effects of Exposure to WwTW Effluents over Two Generations on Sexual Development and Breeding in Roach Rutilus rutilus.
Exposure to environmental estrogens in wastewater treatment works (WwTW) effluents induces feminized responses in male fish, including the development of eggs in male testes. However, the impacts on the offspring of exposed fish are not well understood. In this study, we examined whether roach (Rutilus rutilus) from mothers that had been exposed to an undiluted WwTW effluent from early life to sexual maturity had altered susceptibility to gonadal feminization and an impaired capacity to reproduce. For males from both WwTW effluent exposed mothers and dilution water exposed mothers, effluent exposure for up to 3 years and 9 months induced feminized male gonads, although the intersex condition was relatively mild. There was no difference in the severity of gonadal feminization in roach derived from either WwTW effluent exposed or dilution water exposed mothers. Furthermore, a breeding study revealed that roach with effluent-exposed mothers reproduced with an equal success as roach with mothers exposed to clean water. Roach exposed to the effluent for 3 years in this study were able to reproduce successfully. Our findings provide no evidence for impacts of WwTW effluent exposure on reproduction or gonadal disruption in roach down the female germ line and add to existing evidence that male roach with a mild intersex condition are able to breed competitively.
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Corcoran J, Winter MJ, Lange A, Cumming R, Owen SF, Tyler CR (2015). Effects of the lipid regulating drug clofibric acid on PPARα-regulated gene transcript levels in common carp (Cyprinus carpio) at pharmacological and environmental exposure levels.
Aquat Toxicol,
161, 127-137.
Abstract:
Effects of the lipid regulating drug clofibric acid on PPARα-regulated gene transcript levels in common carp (Cyprinus carpio) at pharmacological and environmental exposure levels.
In mammals, the peroxisome proliferator-activated receptor α (PPARα) plays a key role in regulating various genes involved in lipid metabolism, bile acid synthesis and cholesterol homeostasis, and is activated by a diverse group of compounds collectively termed peroxisome proliferators (PPs). Specific PPs have been detected in the aquatic environment; however little is known on their pharmacological activity in fish. We investigated the bioavailability and persistence of the human PPARα ligand clofibric acid (CFA) in carp, together with various relevant endpoints, at a concentration similar to therapeutic levels in humans (20mg/L) and for an environmentally relevant concentration (4μg/L). Exposure to pharmacologically-relevant concentrations of CFA resulted in increased transcript levels of a number of known PPARα target genes together with increased acyl-coA oxidase (Acox1) activity, supporting stimulation of lipid metabolism pathways in carp which are known to be similarly activated in mammals. Although Cu,Zn-superoxide dismutase (Sod1) activity was not affected, mRNA levels of several biotransformation genes were also increased, paralleling previous reports in mammals and indicating a potential role in hepatic detoxification for PPARα in carp. Importantly, transcription of some of these genes (and Acox1 activity) were affected at exposure concentrations comparable with those reported in effluent discharges. Collectively, these data suggest that CFA is pharmacologically active in carp and has the potential to invoke PPARα-related responses in fish exposed in the environment, particularly considering that CFA may represent just one of a number of PPAR-active compounds present to which wild fish may be exposed.
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Lange A, Sebire M, Rostkowski P, Mizutani T, Miyagawa S, Iguchi T, Hill EM, Tyler CR (2015). Environmental chemicals active as human antiandrogens do not activate a stickleback androgen receptor but enhance a feminising effect of oestrogen in roach.
Aquat Toxicol,
168, 48-59.
Abstract:
Environmental chemicals active as human antiandrogens do not activate a stickleback androgen receptor but enhance a feminising effect of oestrogen in roach.
Sexual disruption is reported in wild fish populations living in freshwaters receiving discharges of wastewater treatment works (WwTW) effluents and is associated primarily with the feminisation of males by exposure to oestrogenic chemicals. Antiandrogens could also contribute to the feminisation of male fish, but there are far less data supporting this hypothesis and almost nothing is known for the effects of oestrogens in combination with antiandrogens in fish. We conducted a series of in vivo exposures in two fish species to investigate the potency on reproductive-relevant endpoints of the antiandrogenic antimicrobials triclosan (TCS), chlorophene (CP) and dichlorophene (DCP) and the resin, abietic acid (AbA), all found widely in WwTW effluents. We also undertook exposures with a mixture of antiandrogens and a mixture of antiandrogens in combination with the oestrogen 17α-ethinyloestradiol (EE2). In stickleback (Gasterosteus aculeatus), DCP showed a tendency to reduce spiggin induction in females androgenised by dihydrotestosterone (DHT), but these findings were not conclusive. In roach (Rutilus rutilus), exposures to DCP (178 days), or a mixture of TCS, CP and AbA (185 days), or to the model antiandrogen flutamide (FL, 178 days) had no effect on gonadal sex ratio or on the development of the reproductive ducts. Exposure to EE2 (1.5ng/L, 185 days) induced feminisation of the ducts in 17% of the males and in the mixture of antiandrogens (TCS, CP, AbA) in combination with EE2, almost all (96%) of the males had a feminised reproductive ducts. In stickleback androgen receptor (ARα and ARβ) transactivation assays, the model antiandrogens, FL and procymidone inhibited 11-ketotestosterone (11-KT) induced receptor activation, but none of the human antiandrogens, TCS, CP, DCP and AbA had an effect. These data indicate that antimicrobial antiandrogens in combination can contribute to the feminisation process in exposed males, but they do not appear to act through the androgen receptor in fish.
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Author URL.
Pountney A, Filby AL, Thomas GO, Simpson VR, Chadwick EA, Stevens JR, Tyler CR (2015). High liver content of polybrominated diphenyl ether (PBDE) in otters (Lutra lutra) from England and Wales.
Chemosphere,
118, 81-86.
Abstract:
High liver content of polybrominated diphenyl ether (PBDE) in otters (Lutra lutra) from England and Wales.
Polybrominated diphenyl ethers (PBDEs), used as flame retardants since the 1970s, are being phased out of use, but are persistent and widespread in the environment. Historical declines in Eurasian otter (Lutra lutra) populations have been associated with exposure to dieldrin and polychlorinated biphenyls (PCBs), but links with other persistent organic pollutants have not been explored. In this study, liver samples from 129 otters, collected across England and Wales from 1995-2006, were analysed for PBDEs, together with PCBs, DDT breakdown products, and hexachlorobenzene. Associations with geographical location and life history parameters were explored. Concentrations of PBDEs in otters (∑BDE 12-70000ngg(-1) lipid) paralleled those measured in marine mammals, with PBDE-47 the dominant congener and high levels of PBDE-99 and -100. Otter livers contained high concentrations of PBDE-153 and -209, typical of terrestrial top predators. Inter-individual variation in PBDE concentrations was high and correlated with geographical location. ∑PBDE was 25% of ∑PCB, and comparable with ∑DDT, identifying PBDEs as a major contaminant in otter populations in England and Wales.
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Heindel JJ, Newbold RR, Williams CJ, Iguchi T, Tyler CR (2015). Lou Guillette--in memorandum.
Mol Reprod Dev,
82(10), Fmi-v.
Author URL.
Goodhead RM, Moger J, Galloway TS, Tyler CR (2015). Tracing engineered nanomaterials in biological tissues using coherent anti-Stokes Raman scattering (CARS) microscopy - a critical review.
Nanotoxicology,
9(7), 928-939.
Abstract:
Tracing engineered nanomaterials in biological tissues using coherent anti-Stokes Raman scattering (CARS) microscopy - a critical review.
Nanomaterials (NMs) are used in an extremely diverse range of products and are increasingly entering the environment, driving a need to better understand their potential health effects in both humans and wildlife. A major challenge in nanoparticle (eco)toxicology is the ability to localise NMs post exposure, to enable more targeted biological effects analyses. A range of imaging techniques have been applied to do so, but they are limited, requiring either extensive processing of the material, staining or use of high intensity illumination that can lead to photo damage and/or have limited tissue penetration. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique, providing contrast based on the intrinsic molecular vibrations of a specimen, circumventing the need for chemical perturbation by exogenous labels. CARS uses near infra-red excitation wavelengths which allow microscopy at depths of several hundred microns in intact tissues and minimises photo-damage to live and delicate samples. Here we provide an overview of the CARS process and present a series of illustrative examples demonstrating its application for detecting NMs within biological tissues, ranging from isolated cells to whole organisms and including materials spanning metals to polymers. We highlight the advantages of this technique which include chemically selective live imaging and substantial depth penetration, but we also discuss its limitations when applied to nanotoxicology, which most notably include the lack of resolution for studies on single nanoparticles.
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Cross RK, Tyler C, Galloway TS (2015). Transformations that affect fate, form and bioavailability of inorganic nanoparticles in aquatic sediments.
Abstract:
Transformations that affect fate, form and bioavailability of inorganic nanoparticles in aquatic sediments
Abstract.
Lee O, Green JM, Tyler CR (2015). Transgenic fish systems and their application in ecotoxicology.
Crit Rev Toxicol,
45(2), 124-141.
Abstract:
Transgenic fish systems and their application in ecotoxicology.
The use of transgenics in fish is a relatively recent development for advancing understanding of genetic mechanisms and developmental processes, improving aquaculture, and for pharmaceutical discovery. Transgenic fish have also been applied in ecotoxicology where they have the potential to provide more advanced and integrated systems for assessing health impacts of chemicals. The zebrafish (Daniorerio) is the most popular fish for transgenic models, for reasons including their high fecundity, transparency of their embryos, rapid organogenesis and availability of extensive genetic resources. The most commonly used technique for producing transgenic zebrafish is via microinjection of transgenes into fertilized eggs. Transposon and meganuclease have become the most reliable methods for insertion of the genetic construct in the production of stable transgenic fish lines. The GAL4-UAS system, where GAL4 is placed under the control of a desired promoter and UAS is fused with a fluorescent marker, has greatly enhanced model development for studies in ecotoxicology. Transgenic fish have been developed to study for the effects of heavy metal toxicity (via heat-shock protein genes), oxidative stress (via an electrophile-responsive element), for various organic chemicals acting through the aryl hydrocarbon receptor, thyroid and glucocorticoid response pathways, and estrogenicity. These models vary in their sensitivity with only very few able to detect responses for environmentally relevant exposures. Nevertheless, the potential of these systems for analyses of chemical effects in real time and across multiple targets in intact organisms is considerable. Here we illustrate the techniques used for generating transgenic zebrafish and assess progress in the development and application of transgenic fish (principally zebrafish) for studies in environmental toxicology. We further provide a viewpoint on future development opportunities.
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Author URL.
Tohyama S, Miyagawa S, Lange A, Ogino Y, Mizutani T, Tatarazako N, Katsu Y, Ihara M, Tanaka H, Ishibashi H, et al (2015). Understanding the Molecular Basis for Differences in Responses of Fish Estrogen Receptor Subtypes to Environmental Estrogens.
Environmental Science and Technology,
49(12), 7439-7447.
Abstract:
Understanding the Molecular Basis for Differences in Responses of Fish Estrogen Receptor Subtypes to Environmental Estrogens
Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p′-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.
Abstract.
2014
David A, Abdul-Sada A, Lange A, Tyler CR, Hill EM (2014). A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatography-nanoelectrospray ionisation mass spectrometry.
J Chromatogr A,
1365, 72-85.
Abstract:
A new approach for plasma (xeno)metabolomics based on solid-phase extraction and nanoflow liquid chromatography-nanoelectrospray ionisation mass spectrometry.
Current metabolite profiling methods based on liquid chromatography-mass spectrometry (LC-MS) platforms do not detect many of the components present at trace concentrations in extracts of plasma due to their low ionisation efficiency or to interference from highly abundant compounds. Nanoflow LC-nanospray MS platforms, which are commonly used in proteomics, could overcome these limitations and significantly increase analytical sensitivity and coverage of the plasma (xeno)metabolome (i.e. metabolites and xenobiotics), but require small injection volumes (94% of the predominant phospholipid/lysophospholipid species from plasma, whilst absolute recoveries of 63 selected (xeno)metabolites from spiked plasma were generally between 60 and 104%. After a further SPE step, recoveries of test compounds were between 50 and 81%. Studies revealed that both the sample preparation methodology and nUHPLC-nESI-TOFMS analyses gave acceptable repeatability. A qualitative comparison of SPE methods revealed that sample concentration by either polymer or mixed mode ion-exchange SPE gave comprehensive metabolite coverage of plasma extracts, but the use of cation exchange SPE significantly increased detection of many cationic compounds in the sample extracts. Method detection limits for steroid, eicosanoid and bile metabolites were
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Author URL.
Brown AR, Gunnarsson L, Kristiansson E, Tyler CR (2014). Assessing variation in the potential susceptibility of fish to pharmaceuticals, considering evolutionary differences in their physiology and ecology.
Philosophical Transactions of the Royal Society B: Biological Sciences,
369(1656).
Abstract:
Assessing variation in the potential susceptibility of fish to pharmaceuticals, considering evolutionary differences in their physiology and ecology
© 2014 the Author(s) Published by the Royal Society. All rights reserved.Fish represent the planet’s most diverse group of vertebrates and they can be exposed to a wide range of pharmaceuticals. For practical reasons, extrapolation of pharmaceutical effects from ‘model’ species to other fish species is adopted in risk assessment. Here, we critically assess this approach. First, we show that between 65% and 86% of human drug targets are evolutionarily conserved in 12 diverse fish species. Focusing on nuclear steroid hormone receptors, we further show that the sequence of the ligand binding domain that plays a key role in drug potency is highly conserved, but there is variation between species. This variation for the oestrogen receptor, however, does not obviously account for observed differences in receptor activation. Taking the synthetic oestrogen ethinyloestradiol as a test case, and using life-table-response experiments, we demonstrate significant reductions in population growth in fathead minnow and medaka, but not zebrafish, for environmentally relevant exposures. This finding contrasts with zebrafish being ranked as more ecologically susceptible, according to two independent life-history analyses. We conclude that while most drug targets are conserved in fish, evolutionary divergence in drug-target activation, physiology, behaviour and ecological life history make it difficult to predict population-level effects. This justifies the conventional use of at least a 10× assessment factor in pharmaceutical risk assessment, to account for differences in species susceptibility.
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Corcoran J, Lange A, Cumming RI, Owen SF, Ball JS, Tyler CR, Winter MJ (2014). Bioavailability of the imidazole antifungal agent clotrimazole and its effects on key biotransformation genes in the common carp (Cyprinus carpio).
Aquat Toxicol,
152, 57-65.
Abstract:
Bioavailability of the imidazole antifungal agent clotrimazole and its effects on key biotransformation genes in the common carp (Cyprinus carpio).
Clotrimazole (CTZ) is a persistent imidazole antifungal agent which is frequently detected in the aquatic environment and predicted to bio-concentrate in fish. Common carp (Cyprinus carpio) were exposed to mean measured concentrations of either 1.02 or 14.63μgl(-1) CTZ for 4 and 10 days, followed by a depuration period of 4 days in a further group of animals. Following each exposure regimen, plasma and liver CTZ concentrations were measured. Mean measured plasma concentrations of CTZ in animals exposed to the lower concentration of CTZ were 30 and 44μgl(-1) on days 4 and 10, respectively, and in the higher concentration were 318 and 336μgl(-1). Mean measured liver levels in the same animals were 514, 1725, 2111 and 7017μgl(-1) suggesting progressive hepatic accumulation. Measurement of CTZ in plasma after depuration suggested efficient elimination within 4 days, but appreciable levels of CTZ remained in the liver after depuration suggesting a degree of persistence in this tissue. In addition we measured responses of a number of key hepatic detoxification gene targets in the liver associated with the transcription factor pregnane X receptor (PXR); namely cyp450s 2k and 3a, glutathione-S-transferases a and p (gsta and p), and drug transporters multidrug resistance protein1 (mdr1), and MDR-related protein2 (mrp2). CTZ is a potent ligand of the PXR in humans and there is some evidence of PXR activation following exposure to CTZ in fish. The highest concentration of CTZ was adopted to explore the potential for alterations to detoxification gene expression in fish at a pharmacologically relevant dose level, and the lower concentration is within the range reported in effluents from waste water treatment works (WWTW). The genes for all biotransformation enzymes were up-regulated after exposure to the higher concentration of CTZ for 10 days, and alterations in expression occurred for the drug transporter genes mdr1 and mrp2 following exposure to the lower concentration of 1.02μgl(-1) CTZ (mean measured concentration). These data support the potential for CTZ to induce alterations in biotransformation and drug transporter genes associated with PXR in fish at concentrations measured in some WWTW effluents.
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Author URL.
Miyagawa S, Lange A, Tohyama S, Ogino Y, Mizutani T, Kobayashi T, Tatarazako N, Tyler CR, Iguchi T (2014). Characterization of Oryzias latipes glucocorticoid receptors and their unique response to progestins. Journal of Applied Toxicology
Morrissey CA, Stanton DW, Tyler CR, Pereira MG, Newton J, Durance I, Ormerod SJ (2014). Developmental impairment in eurasian dipper nestlings exposed to urban stream pollutants. Environmental Toxicology and Chemistry
Morrissey CA, Stanton DWG, Tyler CR, Pereira MG, Newton J, Durance I, Ormerod SJ (2014). Developmental impairment in eurasian dipper nestlings exposed to urban stream pollutants.
Environmental Toxicology and Chemistry,
33(6), 1315-1323.
Abstract:
Developmental impairment in eurasian dipper nestlings exposed to urban stream pollutants
Avian studies of endocrine disruption traditionally have focused on reproductive impairment, given that many environmental contaminants affect sex steroid hormones. There is also increasing interest in altered thyroid function, and associated early development, particularly in altricial species with extended developmental windows. Both types of effect are relevant under the complex pollutant conditions created in streams draining urban areas, but case studies are scarce. Therefore, the authors measured breeding performance, as well as nestling growth, condition, and plasma thyroid hormones, in 87 Eurasian dipper (Cinclus cinclus) nests on 36 urban and rural streams in south and mid-Wales (UK); invertebrate prey data were also collected. The objective of the present study was to evaluate whether urban stream pollution or food scarcity might affect reproduction or development in this specialized aquatic songbird. Clutch sizes and egg fertility were similar on rural and urban streams, whereas nest success was actually higher at urban sites and food abundance was not significantly reduced. However, subtle but important differences were apparent. Urban nestlings were significantly lighter than rural nestlings for their body size (condition index), and brood sex ratios were increasingly male biased with increasing urbanization. The nestling thyroid hormone profile closely reflected urban land use, whereas depressed triiodothyronine (T3) hormones and poorer body condition were associated with higher exposure to polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) at urbanized sites. These data suggest that PCBs, PBDEs, and/or accompanying contaminants in urban streams could be affecting dipper nestling development, with potential consequences for the birds' fitness. Environ Toxicol Chem 2014;33:1315-1323. © 2014 SETAC.
Abstract.
Miyagawa S, Lange A, Hirakawa I, Tohyama S, Ogino Y, Mizutani T, Kagami Y, Kusano T, Ihara M, Tanaka H, et al (2014). Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor.
Environmental Science and Technology,
48(9), 5254-5263.
Abstract:
Differing species responsiveness of estrogenic contaminants in fish is conferred by the ligand binding domain of the estrogen receptor
Exposure to estrogenic endocrine disrupting chemicals (EDCs) induces a range of adverse effects, notably on reproduction and reproductive development. These responses are mediated via estrogen receptors (ERs). Different species of fish may show differences in their responsiveness to environmental estrogens but there is very limited understanding on the underlying mechanisms accounting for these differences. We used custom developed in vitro ERα reporter gene assays for nine fish species to analyze the ligand- and species-specificity for 12 environmental estrogens. Transcriptonal activities mediated by estradiol-17β (E2) were similar to only a 3-fold difference in ERα sensitivity between species. Diethylstilbestrol was the most potent estrogen (∼10-fold that of E2) in transactivating the fish ERαs, whereas equilin was about 1 order of magnitude less potent in all species compared to E2. Responses of the different fish ERαs to weaker environmental estrogens varied, and for some considerably. Medaka, stickleback, bluegill and guppy showed higher sensitivities to nonylphenol, octylphenol, bisphenol a and the DDT-metabolites compared with cyprinid ERαs. Triclosan had little or no transactivation of the fish ERαs. By constructing ERα chimeras in which the AF-containing domains were swapped between various fish species with contrasting responsiveness and subsequent exposure to different environmental estrogens. Our in vitro data indicate that the LBD plays a significant role in accounting for ligand sensitivity of ERα in different species. The differences seen in responsiveness to different estrogenic chemicals between species indicate environmental risk assessment for estrogens cannot necessarily be predicted for all fish by simply examining receptor activation for a few model fish species. © 2014 American Chemical Society.
Abstract.
Southam AD, Lange A, Al-Salhi R, Hill EM, Tyler CR, Viant MR (2014). Distinguishing between the metabolome and xenobiotic exposome in environmental field samples analysed by direct-infusion mass spectrometry based metabolomics and lipidomics.
Metabolomics,
10(6), 1050-1058.
Abstract:
Distinguishing between the metabolome and xenobiotic exposome in environmental field samples analysed by direct-infusion mass spectrometry based metabolomics and lipidomics.
Environmental metabolomics is increasingly used to investigate organismal responses to complex chemical mixtures, including waste water effluent (WWE). In parallel, increasingly sensitive analytical methods are being used in metabolomics studies, particularly mass spectrometry. This introduces a considerable, yet overlooked, challenge that high analytical sensitivity will not only improve the detection of endogenous metabolites in biological specimens but also exogenous chemicals. If these often unknown xenobiotic features are not removed from the "biological" dataset, they will bias the interpretation and could lead to incorrect conclusions about the biotic response. Here we illustrate and validate a novel workflow classifying the origin of peaks detected in biological samples as: endogenous, xenobiotics, or metabolised xenobiotics. The workflow is demonstrated using direct infusion mass spectrometry-based metabolomic analysis of testes from roach exposed to different concentrations of a complex WWE. We show that xenobiotics and their metabolic products can be detected in roach testes (including triclosan, chloroxylenol and chlorophene), and that these compounds have a disproportionately high level of statistical significance within the total (bio)chemical changes induced by the WWE. Overall we have demonstrated that this workflow extracts more information from an environmental metabolomics study of complex mixture exposures than was possible previously.
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Author URL.
Kermorgant M, Lancien F, Mimassi N, Tyler CR, Le Mével J-C (2014). Effects of intracerebroventricular administered fluoxetine on cardio-ventilatory functions in rainbow trout (Oncorhynchus mykiss).
Gen Comp Endocrinol,
205, 176-184.
Abstract:
Effects of intracerebroventricular administered fluoxetine on cardio-ventilatory functions in rainbow trout (Oncorhynchus mykiss).
Fluoxetine (FLX) is a selective serotonin (5-HT) reuptake inhibitor present in the aquatic environment which is known to bioconcentrate in the brains of exposed fish. FLX acts as a disruptor of various neuroendocrine functions in the brain, but nothing is known about the possible consequence of FLX exposure on the cardio-ventilatory system in fish. Here we undertook to investigate the central actions of FLX on ventilatory and cardiovascular function in unanesthetized rainbow trout (Oncorhynchus mykiss). Intracerebroventricular (ICV) injection of FLX (dosed between 5 and 25 μg) resulted in a significantly elevated total ventilation (VTOT), with a maximum hyperventilation of +176% (at a dose of 25μg) compared with vehicle injected controls. This increase was due to an increase in ventilatory amplitude (VAMP: +126%) with minor effects on ventilatory frequency. The highest dose of FLX (25 μg) produced a significant increase in mean dorsal aortic blood pressure (PDA: +20%) without effects on heart rate (ƒH). In comparison, intra-arterial injections of FLX (500-2,500 μg) had no effect on ventilation but the highest doses increased both PDA and ƒH. The ICV and IA cardio-ventilatory effects of FLX were very similar to those previously observed following injections of 5-HT, indicating that FLX probably acts via stimulating endogenous 5-HT activity through inhibition of 5-HT transporter(s). Our results demonstrate for the first time in fish that FLX administered within the brain exerts potent stimulatory effects on ventilation and blood pressure increase. The doses of FLX given to fish in our study are higher than the brain concentrations of FLX in fish that result from acute exposure to FLX through the water. Nonetheless, our results indicate possible disrupting action of long term exposure to FLX discharged into the environment on central target sites sensitive to 5-HT involved in cardio-ventilatory control.
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Author URL.
Baker TJ, Tyler CR, Galloway TS (2014). Impacts of metal and metal oxide nanoparticles on marine organisms.
Environmental Pollution,
186, 257-271.
Abstract:
Impacts of metal and metal oxide nanoparticles on marine organisms
Increasing use of metal and metal oxide nanoparticles [Me(O)NPs] in products means many will inevitably find their way into marine systems. Their likely fate here is sedimentation following hetero-aggregation with natural organic matter and/or free anions, putting benthic, sediment-dwelling and filter feeding organisms most at risk. In marine systems, Me(O)NPs can absorb to micro-organisms with potential for trophic transfer following consumption. Filter feeders, especially bivalves, accumulate Me(O)NPs through trapping them in mucus prior to ingestion. Benthic in-fauna may directly ingest sedimented Me(O)NPs. In fish, uptake is principally via the gut following drinking, whilst Me(O)NPs caught in gill mucus may affect respiratory processes and ion transport. Currently, environmentally-realistic Me(O)NP concentrations are unlikely to cause significant adverse acute health problems, however sub-lethal effects e.g. oxidative stresses have been noted in many organisms, often deriving from dissolution of Ag, Cu or Zn ions, and this could result in chronic health impacts. © 2013 Published by Elsevier Ltd. All rights reserved.
Abstract.
Baker TJ, Tyler CR, Galloway TS (2014). Impacts of metal and metal oxide nanoparticles on marine organisms.
Environ Pollut,
186, 257-271.
Abstract:
Impacts of metal and metal oxide nanoparticles on marine organisms.
Increasing use of metal and metal oxide nanoparticles [Me(O)NPs] in products means many will inevitably find their way into marine systems. Their likely fate here is sedimentation following hetero-aggregation with natural organic matter and/or free anions, putting benthic, sediment-dwelling and filter feeding organisms most at risk. In marine systems, Me(O)NPs can absorb to micro-organisms with potential for trophic transfer following consumption. Filter feeders, especially bivalves, accumulate Me(O)NPs through trapping them in mucus prior to ingestion. Benthic in-fauna may directly ingest sedimented Me(O)NPs. In fish, uptake is principally via the gut following drinking, whilst Me(O)NPs caught in gill mucus may affect respiratory processes and ion transport. Currently, environmentally-realistic Me(O)NP concentrations are unlikely to cause significant adverse acute health problems, however sub-lethal effects e.g. oxidative stresses have been noted in many organisms, often deriving from dissolution of Ag, Cu or Zn ions, and this could result in chronic health impacts.
Abstract.
Author URL.
Hazlerigg CRE, Tyler CR, Lorenzen K, Wheeler JR, Thorbek P (2014). Population relevance of toxicant mediated changes in sex ratio in fish: an assessment using an individual-based zebrafish (Danio rerio) model.
Ecological Modelling,
280, 76-88.
Abstract:
Population relevance of toxicant mediated changes in sex ratio in fish: an assessment using an individual-based zebrafish (Danio rerio) model
Ecological risk assessments (ERAs) of toxicants are predominantly based on data from laboratory tests on individuals. However, the protection goal is generally at the population level. Ecological modelling has the potential to link individual-level effects to population-level outcomes. Here we developed an individual-based zebrafish population model to study the possible population-level relevance of toxicant-mediated changes in sex ratio. The model was structured with sub-models based on empirical data (e.g. growth, reproduction, mortality) derived from a combination of our own laboratory and field experiments, the literature and theoretical concepts. The outputs of the default model were validated against size distributions for wild populations of zebrafish sampled in Bangladesh. Sensitivity analysis showed that population abundance was most sensitive to changes in density-dependent survival and the availability of refugia for juveniles.The model was then used to determine the population-level relevance of changes in sex ratio caused by an androgenic (dihydrotestosterone) and oestrogenic (4-tert-octylphenol) substance. Both were investigated under acute (10 day) and chronic (1 year) exposure regimes. Acute exposures to the test chemicals had little effect on population-level endpoints at any of the concentrations tested. Chronic exposures decreased population abundance at higher concentrations for both chemicals and most strongly with DHT. However, these concentrations were far in excess of environmentally realistic levels. Our study demonstrated that ecological models can be applied to link laboratory derived ecotoxicity data at the individual level to impacts at the population level and in our study we found different modes of action and potencies caused different levels of population perturbation. Ecological models can therefore help in assessing the ecological relevance of different organism-level effects of toxicants aiding future environmental protection strategies. © 2014 Elsevier B.V.
Abstract.
Hazlerigg CRE, Tyler CR, Lorenzen K, Wheeler JR, Thorbek P (2014). Population relevance of toxicant mediated changes in sex ratio in fish: an assessment using an individual-based zebrafish (Danio rerio) model. Ecological Modelling
Hamilton PB, Nicol E, De-Bastos ESR, Williams RJ, Sumpter JP, Jobling S, Stevens JR, Tyler CR (2014). Populations of a cyprinid fish are self-sustaining despite widespread feminization of males.
BMC Biol,
12Abstract:
Populations of a cyprinid fish are self-sustaining despite widespread feminization of males.
BACKGROUND: Treated effluents from wastewater treatment works can comprise a large proportion of the flow of rivers in the developed world. Exposure to these effluents, or the steroidal estrogens they contain, feminizes wild male fish and can reduce their reproductive fitness. Long-term experimental exposures have resulted in skewed sex ratios, reproductive failures in breeding colonies, and population collapse. This suggests that environmental estrogens could threaten the sustainability of wild fish populations. RESULTS: Here we tested this hypothesis by examining population genetic structures and effective population sizes (N(e)) of wild roach (Rutilus rutilus L.) living in English rivers contaminated with estrogenic effluents. N(e) was estimated from DNA microsatellite genotypes using approximate Bayesian computation and sibling assignment methods. We found no significant negative correlation between N(e) and the predicted estrogen exposure at 28 sample sites. Furthermore, examination of the population genetic structure of roach in the region showed that some populations have been confined to stretches of river with a high proportion of estrogenic effluent for multiple generations and have survived, apparently without reliance on immigration of fish from less polluted sites. CONCLUSIONS: These results demonstrate that roach populations living in some effluent-contaminated river stretches, where feminization is widespread, are self-sustaining. Although we found no evidence to suggest that exposure to estrogenic effluents is a significant driving factor in determining the size of roach breeding populations, a reduction in N(e) of up to 65% is still possible for the most contaminated sites because of the wide confidence intervals associated with the statistical model.
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Full text.
Filby AL, Ortiz-Zarragoitia M, Tyler CR (2014). The vas:: Egfp transgenic zebrafish: a practical model for studies on the molecular mechanisms by which environmental estrogens affect gonadal sex differentiation.
Environmental Toxicology and Chemistry,
33(3), 602-605.
Abstract:
The vas:: Egfp transgenic zebrafish: a practical model for studies on the molecular mechanisms by which environmental estrogens affect gonadal sex differentiation
The vas::egfp transgenic zebrafish (Danio rerio) could significantly enhance studies on the mechanisms by which environmental estrogens disrupt sexual differentiation, because the developing gonad can be visualized during early life via fluorescence detection. There are methodological challenges regarding dissecting out the gonads in early-life-stage fish, however, and transgene responses to estrogen exposure have not been tested. The authors exposed vas::egfp transgenic zebrafish and their wild-type siblings to the model estrogen 17α-ethinylestradiol (EE2; 0.62ng/L and 3.33ng/L measured concentrations) during sexual development (20-60 d posthatch) and used enhanced green fluorescent protein (egfp) fluorescence to identify and dissect single gonads (at 40 d posthatch) to provide sufficient RNA for individual gene expression analyses, retaining the remaining gonad in the body cavity for histological analyses of sex and stage of development. Genotyping confirmed that all transgenic control fish were phenotypically egfp positive (showed green fluorescence). Interestingly, however, in a few transgenic fish exposed to EE2, no phenotypic egfp signal was seen, most notably for the 3.33ng/L EE2 exposure. It was subsequently found that gonadal vasa expression was reduced by this concentration of EE2. Hepatic vitellogenin expression demonstrated that the vas::egfp and wild-type lines responded to estrogen with an equivalent sensitivity. The authors conclude that the vas::egfp zebrafish provides an enhanced and practical system for mechanistic studies on the effects of environmentally relevant concentrations of estrogens on gonad development. Environ Toxicol Chem 2014;33:602-605. © 2013 SETAC.
Abstract.
Filby AL, Ortiz-Zarragoitia M, Tyler CR (2014). The vas::egfp transgenic zebrafish: a practical model for studies on the molecular mechanisms by which environmental estrogens affect gonadal sex differentiation.
Environ Toxicol Chem,
33(3), 602-605.
Abstract:
The vas::egfp transgenic zebrafish: a practical model for studies on the molecular mechanisms by which environmental estrogens affect gonadal sex differentiation.
The vas::egfp transgenic zebrafish (Danio rerio) could significantly enhance studies on the mechanisms by which environmental estrogens disrupt sexual differentiation, because the developing gonad can be visualized during early life via fluorescence detection. There are methodological challenges regarding dissecting out the gonads in early-life-stage fish, however, and transgene responses to estrogen exposure have not been tested. The authors exposed vas::egfp transgenic zebrafish and their wild-type siblings to the model estrogen 17α-ethinylestradiol (EE2; 0.62 ng/L and 3.33 ng/L measured concentrations) during sexual development (20-60 d posthatch) and used enhanced green fluorescent protein (egfp) fluorescence to identify and dissect single gonads (at 40 d posthatch) to provide sufficient RNA for individual gene expression analyses, retaining the remaining gonad in the body cavity for histological analyses of sex and stage of development. Genotyping confirmed that all transgenic control fish were phenotypically egfp positive (showed green fluorescence). Interestingly, however, in a few transgenic fish exposed to EE2, no phenotypic egfp signal was seen, most notably for the 3.33 ng/L EE2 exposure. It was subsequently found that gonadal vasa expression was reduced by this concentration of EE2. Hepatic vitellogenin expression demonstrated that the vas::egfp and wild-type lines responded to estrogen with an equivalent sensitivity. The authors conclude that the vas::egfp zebrafish provides an enhanced and practical system for mechanistic studies on the effects of environmentally relevant concentrations of estrogens on gonad development.
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Author URL.
Watts AJR, Lewis C, Goodhead RM, Beckett SJ, Moger J, Tyler CR, Galloway TS (2014). Uptake and retention of microplastics by the shore crab Carcinus maenas.
Environ Sci Technol,
48(15), 8823-8830.
Abstract:
Uptake and retention of microplastics by the shore crab Carcinus maenas.
Microplastics, plastics particles
Abstract.
Author URL.
2013
Katsu Y, Lange A, Miyagawa S, Urushitani H, Tatarazako N, Kawashima Y, Tyler CR, Iguchi T (2013). Cloning, expression and functional characterization of carp, cyprinus carpio, estrogen receptors and their differential activations by estrogens.
Journal of Applied Toxicology,
33(1), 41-49.
Abstract:
Cloning, expression and functional characterization of carp, cyprinus carpio, estrogen receptors and their differential activations by estrogens
Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ERα, ERβ1 and ERβ2) and applied an estrogen-responsive (ERE)-luciferase reporter assay system to characterize the interactions of these receptors with steroidal and synthetic estrogens. DNA fragments encoding all three ERs in carp, ERα, ERβ1 and ERβ2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% (α vs β1), 49% (α vs β2) and 53% (β1 vs β2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cERβ2 showed a higher sensitivity to the natural steroid oestrogen, 17β-estradiol, than cERα. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution. Copyright © 2011 John Wiley & Sons, Ltd. Environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. We cloned all three carp Cyprinus carpio, estrogen receptors [ER; ERa, ERb1 and ERb2] and applied a transient transfection ERE-luciferase reporter assay system using mammalian cells. cERb2 showed a higher sensitivity to the natural steroid oestrogen, 17b-estradiol, than cERa. This is a powerful assay for toxicology, and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen disrupting mechanisms in carp. © 2011 John Wiley & Sons, Ltd.
Abstract.
Ankley GT, Tyler CR (2013). Development of methods to detect occurrence and effects of endocrine-disrupting chemicals: Fueling a fundamental shift in regulatory ecotoxicology. Environmental Toxicology and Chemistry, 32(12), 2661-2662.
Osborne OJ, Johnston BD, Moger J, Balousha M, Lead JR, Kudoh T, Tyler CR (2013). Effects of particle size and coating on nanoscale Ag and TiO₂ exposure in zebrafish (Danio rerio) embryos.
Nanotoxicology,
7(8), 1315-1324.
Abstract:
Effects of particle size and coating on nanoscale Ag and TiO₂ exposure in zebrafish (Danio rerio) embryos.
Manufactured metal (oxide) nanoparticles are entering the aquatic environment with little understanding on their potential health impacts for exposed organisms. Adopting an integrative approach, we investigated effects of particle size and coating on biological responses for two of the most commonly used metal (oxide) nanoscale particles, silver (Ag) and titanium dioxide (TiO₂) in zebrafish embryos. Titanium dioxide nanoparticles (nominally, 4 nm, 10 nm, 30 nm and 134 nm) had little or no toxicity on the endpoints measured. Ag both in nano form (10 nm and 35 nm) and its larger counterpart (600-1600 nm) induced dose-dependent lethality and morphological defects, occurring predominantly during gastrula stage. of the silver material tested 10 nm nanoparticles appeared to be the most toxic. Coating Ag nanoparticles with citrate or fulvic acid decreased toxicity significantly. In situ hybridisation analysis identified the yolk syncytial layer (YSL) as a target tissue for Ag-nano toxicity where there was a significant induction of the heavy metal stress response gene, metallothionein 2 (Mt2) at sub-lethal exposures. Coherent Anti-stroke Raman Scattering (CARS) microscopy provided no evidence for silver particles crossing the chorionic membrane in exposed embryos. Collectively, our data suggest that silver ions play a major role in the toxicity of Ag nanoparticles.
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Author URL.
Hawkes LA, McGowan A, Godley BJ, Gore S, Lange A, Tyler CR, Wheatley D, White J, Witt MJ, Broderick AC, et al (2013). Estimating sex ratios in Caribbean hawksbill turtles: Testosterone levels and climate effects.
Aquatic Biology,
18(1), 9-19.
Abstract:
Estimating sex ratios in Caribbean hawksbill turtles: Testosterone levels and climate effects
Evolutionary theory predicts that male and female offspring should be produced at a 1:1 ratio, but this may rarely be the case for species in which sex is determined during incubation by temperature, such as marine turtles. Estimates of primary sex ratio suggest that marine turtle sex ratios are highly skewed, with up to 9 females per male. We captured juvenile hawksbill turtles Eretmochelys imbricata in waters around Anegada, British Virgin Islands, a regionally important foraging aggregation, and analysed concentrations of plasma testosterone and oestradiol- 17β from 62 turtles to estimate sex ratio. There were 2.4 to 7.7 times more females than males. Testosterone concentrations correlated with sampling date and sea surface temperature (SST), with higher con centrations in the late summer when SST was highest, suggesting that assigning sex through threshold values of sex hormones must be carried out cautiously. The sex ratio in the juvenile foraging aggregation around Anegada is more male biased than at other locations, suggesting that turtles at Anegada have resilience against feminising effects of climate change. Future work should (1) integrate the relative contributions of different genetic stocks to foraging aggregations and (2) investigate the annual and seasonal cycles of sex hormones, and differences among individuals and life history stages. © Inter-Research 2013.
Abstract.
Morrissey CA, Stanton DWG, Pereira MG, Newton J, Durance I, Tyler CR, Ormerod SJ (2013). Eurasian dipper eggs indicate elevated organohalogenated contaminants in urban rivers.
Environmental Science and Technology,
47(15), 8931-8939.
Abstract:
Eurasian dipper eggs indicate elevated organohalogenated contaminants in urban rivers
Many urban European streams are recovering from industrial, mining, and sewage pollution during the 20th century. However, associated recolonization by clean water organisms can potentially result in exposure to legacy or novel toxic pollutants that persist in the environment. Between 2008 and 2010, we sampled eggs of a river passerine, the Eurasian dipper (Cinclus cinclus), from 33 rivers in South Wales and the English borders (UK) which varied in catchment land use from rural to highly urbanized. Dipper egg δ15N and δ13C stable isotopes were enriched from urban rivers while δ34S was strongly depleted, effectively discriminating their urban or rural origins at thresholds of 10% urban land cover or 1000 people/km2. Concentrations of total polychlorinated biphenyls (PCBs) and polybrominated biphenyl ethers (PBDEs) were positively related to urban land cover and human population density while legacy organochlorine pesticides such as p,p′-DDE, lindane, and hexachlorobenzene were found in higher concentrations at rural sites. Levels of PBDEs in urban dipper eggs (range of 136-9299 ng/g lw) were among the highest ever reported in passerines, and some egg contaminants were at or approaching levels sufficient for adverse effects on avian development. With the exception of dieldrin, our data shows PCBs and other organochlorine pesticides have remained stable or increased in the past 20 years in dipper eggs, despite discontinued use. © 2013 American Chemical Society.
Abstract.
Segner H, Casanova-Nakayama A, Kase R, Tyler CR (2013). Impact of environmental estrogens on Yfish considering the diversity of estrogen signaling.
General and Comparative Endocrinology,
191, 190-201.
Abstract:
Impact of environmental estrogens on Yfish considering the diversity of estrogen signaling
Research on endocrine disruption in fish has been dominated by studies on estrogen-active compounds which act as mimics of the natural estrogen, 17β-estradiol (E2), and generally exert their biological actions by binding to and activation of estrogen receptors (ERs). Estrogens play central roles in reproductive physiology and regulate (female) sexual differentiation. In line with this, most adverse effects reported for fish exposed to environmental estrogens relate to sexual differentiation and reproduction. E2, however, utilizes a variety of signaling mechanisms, has multifaceted functions and targets, and therefore the toxicological and ecological effects of environmental estrogens in fish will extend beyond those associated with the reproduction. This review first describes the diversity of estrogen receptor signaling in fish, including both genomic and non-genomic mechanisms, and receptor crosstalk. It then considers the range of non-reproductive physiological processes in fish that are known to be responsive to estrogens, including sensory systems, the brain, the immune system, growth, specifically through the growth hormone/insulin-like growth factor system, and osmoregulation. The diversity in estrogen responses between fish species is then addressed, framed within evolutionary and ecological contexts, and we make assessments on their relevance for toxicological sensitivity as well as ecological vulnerability. The diversity of estrogen actions raises questions whether current risk assessment strategies, which focus on reproductive endpoints, and a few model fish species only, are protective of the wider potential health effects of estrogens. Available - although limited - evidence nevertheless suggests that quantitative environmental threshold concentrations for environmental protection derived from reproductive tests with model fish species are protective for non-reproductive effects as well. The diversity of actions of estrogens across divergent physiological systems, however, may lead to and underestimation of impacts on fish populations as their effects are generally considered on one functional process only and this may underrepresent the impact on the different physiological processes collectively. © 2013 Elsevier Inc.
Abstract.
Bickley LK, Brown AR, Hosken DJ, Hamilton PB, Le Page G, Paull GC, Owen SF, Tyler CR (2013). Interactive effects of inbreeding and endocrine disruption on reproduction in a model laboratory fish.
Evolutionary Applications,
6(2), 279-289.
Abstract:
Interactive effects of inbreeding and endocrine disruption on reproduction in a model laboratory fish
Inbreeding depression is expected to be more severe in stressful environments. However, the extent to which inbreeding affects the vulnerability of populations to environmental stressors, such as chemical exposure, remains unresolved. Here we report on the combined impacts of inbreeding and exposure to an endocrine disrupting chemical (the fungicide clotrimazole) on zebrafish (Danio rerio). We show that whilst inbreeding can negatively affect reproductive traits, not all traits are affected equally. Inbreeding depression frequently only became apparent when fish were additionally stressed by chemical exposure. Embryo viability was significantly reduced in inbred exposed fish and there was a tendency for inbred males to sire fewer offspring when in direct competition with outbred individuals. Levels of plasma 11-ketotestosterone, a key male sex hormone, showed substantial inbreeding depression that was unaffected by addition of the fungicide. In contrast, there was no effect of inbreeding or clotrimazole exposure on egg production. Overall, our data provide evidence that stress may amplify the effects of inbreeding on key reproductive traits, particularly those associated with male fitness. This may have important implications when considering the consequences of exposure to chemical pollutants on the fitness of wild populations. Journal compilation © 2013 Blackwell Publishing Ltd62 February 2013 10.1111/j.1752-4571.2012.00288.x Original Article Original Articles © 2012 Blackwell Publishing Ltd. This is an open access article under the terms of the Creative Commons Attribution Non Commercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
Abstract.
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van Aerle R, Lange A, Moorhouse A, Paszkiewicz K, Ball K, Johnston BD, de-Bastos E, Booth T, Tyler CR, Santos EM, et al (2013). Molecular mechanisms of toxicity of silver nanoparticles in zebrafish embryos.
Environ Sci Technol,
47(14), 8005-8014.
Abstract:
Molecular mechanisms of toxicity of silver nanoparticles in zebrafish embryos.
Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity, however, remain unclear. We examined the effects of exposure to silver in nano-, bulk-, and ionic forms on zebrafish embryos (Danio rerio) using a Next Generation Sequencing approach in an Illumina platform (High-Throughput SuperSAGE). Significant alterations in gene expression were found for all treatments and many of the gene pathways affected, most notably those associated with oxidative phosphorylation and protein synthesis, overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down-regulation of this pathway at 24 h of exposure, but with a recovery at 48 h. This finding was consistent with a dose-dependent decrease in oxygen consumption at 24 h, but not at 48 h, following exposure to silver ions. Overall, our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms.
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Santos EM, Hamilton PB, Coe TS, Ball JS, Cook AC, Katsiadaki I, Tyler CR (2013). Population bottlenecks, genetic diversity and breeding ability of the three-spined stickleback (Gasterosteus aculeatus) from three polluted English Rivers.
Aquatic Toxicology,
142-143, 264-271.
Abstract:
Population bottlenecks, genetic diversity and breeding ability of the three-spined stickleback (Gasterosteus aculeatus) from three polluted English Rivers
Pollution is a significant environmental pressure on fish populations in both freshwater and marine environments. Populations subjected to chronic exposure to pollutants can experience impacts ranging from altered reproductive capacity to changes in population genetic structure. Few studies, however, have examined the reproductive vigor of individuals within populations inhabiting environments characterized by chronic pollution. In this study we undertook an analysis of populations of three-spined sticklebacks (Gasterosteus aculeatus) from polluted sites, to determine levels of genetic diversity, assess for evidence of historic population genetic bottlenecks and determine the reproductive competitiveness of males from these locations. The sites chosen included locations in the River Aire, the River Tees and the River Birket, English rivers that have been impacted by pollution from industrial and/or domestic effluents for over 100 years. Male reproductive competitiveness was determined via competitive breeding experiments with males and females derived from a clean water site, employing DNA microsatellites to determine parentage outcome. Populations of stickleback collected from the three historically polluted sites showed evidence of recent population bottlenecks, although only the River Aire population showed low genetic diversity. In contrast, fish collected from two relatively unpolluted sites within the River Gowy and Houghton Springs showed weak, or no evidence of such bottlenecks. Nevertheless, males derived from polluted sites were able to reproduce successfully in competition with males derived from clean water exposures, indicating that these bottlenecks have not resulted in any substantial loss of reproductive fitness in males. © 2013 Elsevier B.V.
Abstract.
Bergman A, Andersson AM, Becher G, Van Den Berg M, Blumberg B, Bjerregaard P, Bornehag CG, Bornman R, Brandt I, Brian JV, et al (2013). Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.
Environmental Health: a Global Access Science Source,
12(1).
Abstract:
Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors
The "common sense" intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU. © 2013 Bergman et al.; licensee BioMed Central Ltd.
Abstract.
2012
Moreman J, Lee O, Kudoh T, Tyler CR (2012). Application of ERE-transgenic biosensor zebrafish to identify target tissues and effect mechanisms of the environmental estrogen, bisphenol A.
Author URL.
Lange A, Sebire M, Rostkowski P, Horwood J, Miyagawa S, Mizutani T, Iguchi T, Hill EM, Tyler CR (2012). Bioavailable environmental antiandrogens and their potential effects on endpoints relevant to reproduction in fish.
Author URL.
Lee OH, Takesono A, Tada M, Tyler CR, Kudoh T (2012). Biosensor zebrafish provide new insights into potential health effects of environmental estrogens.
Environ Health Perspect,
120, 990-996.
Full text.
Baalousha M, Ju-Nam Y, Cole PA, Gaiser B, Fernandes TF, Hriljac JA, Jepson MA, Stone V, Tyler CR, Lead JR, et al (2012). Characterization of cerium oxide nanoparticles-Part 1: Size measurements.
Environmental Toxicology and Chemistry,
31(5), 983-993.
Abstract:
Characterization of cerium oxide nanoparticles-Part 1: Size measurements
The present study gives an overview of some of the major aspects for consideration in the characterization of nanomaterials (NMs). Part 1 focuses on the measurement of particle size and size-related parameters using several analytical techniques such as transmission electron microscopy, atomic force microscopy, dynamic light scattering, X-ray diffraction, and Brunauer, Emmett, and Teller surface area measurements as applied to commercially available cerium oxide nanoparticles (NPs) and microparticles (MPs). Part 2 (see companion paper) considers nonsize-related characterization and analysis. The results are discussed in relation to the nature of the sample and preparation, and the analytical principles, limitations, and advantages of each technique. Accurate information on the particle size of the different fractions of a sample can be obtained by using a combination of different types of microscopy, spectroscopy, separation, and other techniques; this should inform ecotoxicological and environmental studies. The good agreement between the measured primary particle size of the NPs (~15nm) by atomic force microscopy, transmission electron microscopy, X-ray diffraction, and Brunauer, Emmett, and Teller suggests that the primary particles are formed of semispherical single crystals. For MPs, all measurements agree that they are large particles in the range above the NPs (100nm), with some difference between the measured sizes, possibly as a result of polydispersity effects. Additionally, our findings suggest that atomic force microscopy and transmission electron microscopy prepared by centrifugation methods provide consistent data at low concentrations when dynamic light scattering fails. © 2012 SETAC.
Abstract.
Baalousha M, Ju-Nam Y, Cole PA, Hriljac JA, Jones IP, Tyler CR, Stone V, Fernandes TF, Jepson MA, Lead JR, et al (2012). Characterization of cerium oxide nanoparticles-Part 2: Nonsize measurements.
ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY,
31(5), 994-1003.
Author URL.
Baalousha M, Ju-Nam Y, Cole PA, Hriljac JA, Jones IP, Tyler CR, Stone V, Fernandes TF, Jepson MA, Lead JR, et al (2012). Characterization of cerium oxide nanoparticles-Part 2: Nonsize measurements.
Environmental Toxicology and Chemistry,
31(5), 994-1003.
Abstract:
Characterization of cerium oxide nanoparticles-Part 2: Nonsize measurements
Part 1 (see companion paper) of the present study discussed the application of a multimethod approach in characterizing the size of cerium oxide nanoparticles (NPs). However, other properties less routinely investigated, such as shape and morphology, structure, chemical composition, and surface properties, are likely to play an important role in determining the behavior, reactivity, and potential toxicity of these NPs. The present study describes the measurement of the aforementioned physicochemical properties of NPs (applied also to nanomaterials [NMs]) compared with micrometer particles (MPs). The authors use a wide range of techniques, including high resolution-transmission electron microscopy, scanning electron microscopy, atomic force microscopy, X-ray diffraction, X-ray energy dispersive spectroscopy, electron energy loss spectroscopy, X-ray photoelectron spectroscopy, and electrophoresis, and compare these techniques, their advantages, and their limitations, along with recommendations about how best to approach NM characterization, using an application to commercial cerium oxide NPs and MPs. Results show that both cerium oxide NPs and MPs are formed of single polyhedron or truncated polyhedron crystals. Cerium oxide NPs contain a mixture of Ce 3+ and Ce 4+ cations, whereas the MPs contain mainly Ce 4+, which is potentially important in understanding the toxicity of cerium oxide NPs. The isoelectric point of cerium oxide NPs was approximately pH 8, which explains their propensity to aggregate in aqueous media (see companion paper). © 2012 SETAC.
Abstract.
Baalousha M, Ju-Nam Y, Cole PA, Gaiser B, Fernandes TF, Hriljac JA, Jepson MA, Stone V, Tyler CR, Lead JR, et al (2012). Characterization of cerium oxide nanoparticles-part 1: Size measurements. Environmental Toxicology and Chemistry
Baalousha M, Ju-Nam Y, Cole PA, Hriljac JA, Jones IP, Tyler CR, Stone V, Fernandes TF, Jepson MA, Lead JR, et al (2012). Characterization of cerium oxide nanoparticles-part 2: Nonsize measurements. Environmental Toxicology and Chemistry
Katsu Y, Lange A, Miyagawa S, Urushitani H, Tatarazako N, Kawashima CR, Iguchi T (2012). Cloning, expression and functional characterization of carp, Cyprinus carpio, estrogen receptors and their differential activations by estrogens.
Journal of Applied ToxicologyAbstract:
Cloning, expression and functional characterization of carp, Cyprinus carpio, estrogen receptors and their differential activations by estrogens
Environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. We cloned all three carp Cyprinus carpio, estrogen receptors [ER; ERa, ERb1 and ERb2] and applied a transient transfection ERE-luciferase reporter assay system using mammalian cells. cERb2 showed a higher sensitivity to the natural steroid oestrogen, 17b-estradiol, than cERa. This is a powerful assay for toxicology, and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen disrupting mechanisms in carp.
Abstract.
Söffker M, Stevens JR, Tyler CR (2012). Comparative breeding and behavioral responses to ethinylestradiol exposure in wild and laboratory maintained zebrafish (Danio rerio) populations.
Environ Sci Technol,
46(20), 11377-11383.
Abstract:
Comparative breeding and behavioral responses to ethinylestradiol exposure in wild and laboratory maintained zebrafish (Danio rerio) populations.
Genetic variation has a significant effect on behavior, fitness, and response to toxicants; however, this is rarely considered in ecotoxicological studies. We compared fitness-related behavioral traits, breeding activity, and the effects of exposure to the environmental estrogen ethinylestradiol (EE(2)) on reproduction in a laboratory (Wild Indian Karyotype, WIK) strain and a wild-caught population (Bangladesh, BLD01) of Danio rerio (zebrafish). In WIK fish, males with higher observed heterozygocity were more active reproductively and more successful in securing parentage, but these relationships were not apparent in the BLD01 fish. The frequency of reproductive behaviors increased in WIK zebrafish for exposure to 0.4 ng/L EE(2), which was not apparent in the BLD01 zebrafish. The different strains showed the same threshold for hepatic vitellogenin gene (vtg) induction (2.2 ng EE(2)/L), but results suggested an elevated response level in the BLD01. There were no effects on total egg production up to 2.2 ng EE(2)/L in either population; however, there was reduced egg fertilization rate at 2.2 ng EE(2)/L in the BLD01 fish. These results show consistency in the general responses to EE(2) between these two genetically divergent strains of zebrafish, but also illustrate differences in their breeding biology and response sensitivities. These findings highlight the need for due consideration of the source (and genetics) of populations used in ecological risk assessment for accurate comparisons among studies.
Abstract.
Author URL.
Lange A, Katsu Y, Miyagawa S, Ogino Y, Urushitani H, Kobayashi T, Hirai T, Shears JA, Nagae M, Yamamoto J, et al (2012). Comparative responsiveness to natural and synthetic estrogens of fish species commonly used in the laboratory and field monitoring.
Aquat Toxicol,
109, 250-258.
Abstract:
Comparative responsiveness to natural and synthetic estrogens of fish species commonly used in the laboratory and field monitoring.
Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ERα activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17α-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). of the fish species tested, zebrafish ERα was found to be the most responsive and carp and stickleback ERα the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ERα-mediated response sensitivity order of zebrafish > medaka > roach > fathead minnow > carp > stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ERα activation assays) > zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ERα compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ERα-mediated responses indicative of estrogenic responses (VTG induction) in vivo.
Abstract.
Author URL.
Hazlerigg CRE, Lorenzen K, Thorbek P, Wheeler JR, Tyler CR (2012). Density-dependent processes in the life history of fishes: Evidence from laboratory populations of zebrafish Danio rerio.
PLoS ONE,
7(5).
Abstract:
Density-dependent processes in the life history of fishes: Evidence from laboratory populations of zebrafish Danio rerio
Population regulation is fundamental to the long-term persistence of populations and their responses to harvesting, habitat modification, and exposure to toxic chemicals. In fish and other organisms with complex life histories, regulation may involve density dependence in different life-stages and vital rates. We studied density dependence in body growth and mortality through the life-cycle of laboratory populations of zebrafish Danio rerio. When feed input was held constant at population-level (leading to resource limitation), body growth was strongly density-dependent in the late juvenile and adult phases of the life-cycle. Density dependence in mortality was strong during the early juvenile phase but declined thereafter and virtually ceased prior to maturation. Provision of feed in proportion to individual requirements (easing resource limitation) removed density dependence in growth and substantially reduced density dependence in mortality, thus indicating that 'bottom-up' effects act on growth as well as mortality, but most strongly on growth. Both growth and mortality played an important role in population regulation, with density-dependent growth having the greater impact on population biomass while mortality had the greatest impact on numbers. We demonstrate a clear ontogenic pattern of change in density-dependent processes within populations of a very small (maximum length 5 mm) fish, maintained in constant homogeneous laboratory conditions. The patterns are consistent with those distilled from studies on wild fish populations, indicating the presence of broad ontogenic patterns in density-dependent processes that are invariant to maximum body size and hold in homogeneous laboratory, as well as complex natural environments. © 2012 Hazlerigg et al.
Abstract.
Lee O, Tyler CR, Kudoh T (2012). Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos.
BMC Biotechnol,
12Abstract:
Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos.
BACKGROUND: Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein). RESULTS: the response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1-2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally. CONCLUSION: Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.
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Brown AR, Bickley LK, Ryan TA, Paull GC, Hamilton PB, Owen SF, Sharpe AD, Tyler CR (2012). Differences in sexual development in inbred and outbred zebrafish (Danio rerio) and implications for chemical testing.
Aquat Toxicol,
112-113, 27-38.
Abstract:
Differences in sexual development in inbred and outbred zebrafish (Danio rerio) and implications for chemical testing.
Outbred laboratory animal strains used in ecotoxicology are intended to represent wild populations. However, breeding history may vary considerably between strains, driving differences in genetic variation and phenotypes used for assessing effects of chemical exposure. We compared a range of phenotypic endpoints in zebrafish from four different "breeding treatments" comprising a Wild Indian Karyotype (WIK) zebrafish strain and a WIK/Wild strain with three levels of inbreeding (F(IT)=n, n+0.25, n+0.375) in a new Fish Sexual Development Test (FSDT). There were no differences between treatments in terms of egg viability, hatch success or fry survival. However, compared with WIKs, WIK/Wild hybrids were significantly larger in size, with more advanced gonadal (germ cell) development at the end of the test (63 days post fertilisation). Increasing the levels of inbreeding in the related WIK/Wild lines did not affect body size, but there was a significant male-bias (72%) in the most inbred line (F(IT)=n+0.375). Conversely, in the reference WIK strain there was a significant female-bias in the population (80% females). Overall, our results support the use of outbred zebrafish strains in the FSDT, where one of the core endpoints is sex ratio. Despite increased variance (and reduced statistical power) for some endpoints, WIK/Wild outbreds (F(IT)=n) met all acceptance criteria for controls in this test, whereas WIKs failed to comply with tolerance limits for sex ratio (30-70% females). Sexual development was also more advanced in WIK/Wild outbreds (cf. WIKs), providing greater scope for detection of developmental reproductive toxicity following chemical exposure.
Abstract.
Author URL.
Cresswell JE, Page CJ, Uygun MB, Holmbergh M, Li Y, Wheeler JG, Laycock I, Pook CJ, de Ibarra NH, Smirnoff N, et al (2012). Differential sensitivity of honey bees and bumble bees to a dietary insecticide (imidacloprid).
Zoology,
115(6), 365-371.
Abstract:
Differential sensitivity of honey bees and bumble bees to a dietary insecticide (imidacloprid)
Currently, there is concern about declining bee populations and the sustainability of pollination services. One potential threat to bees is the unintended impact of systemic insecticides, which are ingested by bees in the nectar and pollen from flowers of treated crops. To establish whether imidacloprid, a systemic neonicotinoid and insect neurotoxin, harms individual bees when ingested at environmentally realistic levels, we exposed adult worker bumble bees, Bombus terrestris L. (Hymenoptera: Apidae), and honey bees, Apis mellifera L. (Hymenoptera: Apidae), to dietary imidacloprid in feeder syrup at dosages between 0.08 and 125μgl-1. Honey bees showed no response to dietary imidacloprid on any variable that we measured (feeding, locomotion and longevity). In contrast, bumble bees progressively developed over time a dose-dependent reduction in feeding rate with declines of 10-30% in the environmentally relevant range of up to 10μgl-1, but neither their locomotory activity nor longevity varied with diet. To explain their differential sensitivity, we speculate that honey bees are better pre-adapted than bumble bees to feed on nectars containing synthetic alkaloids, such as imidacloprid, by virtue of their ancestral adaptation to tropical nectars in which natural alkaloids are prevalent. We emphasise that our study does not suggest that honey bee colonies are invulnerable to dietary imidacloprid under field conditions, but our findings do raise new concern about the impact of agricultural neonicotinoids on wild bumble bee populations. © 2012 Elsevier GmbH.
Abstract.
Cresswell JE, Page CJ, Uygun MB, Holmbergh M, Li Y, Wheeler JG, Laycock I, Pook CJ, de Ibarra NH, Smirnoff N, et al (2012). Differential sensitivity of honey bees and bumble bees to a dietary insecticide (imidacloprid). Zoology
Corcoran J, Lange A, Winter MJ, Tyler CR (2012). Effects of Pharmaceuticals on the Expression of Genes Involved in Detoxification in a Carp Primary Hepatocyte Model.
Environmental Science and Technology,
46(11), 6306-6314.
Abstract:
Effects of Pharmaceuticals on the Expression of Genes Involved in Detoxification in a Carp Primary Hepatocyte Model
Fish in many surface freshwaters are exposed to a range of pharmaceuticals via wastewater treatment works effluent discharges. In mammals the pregnane X receptor (PXR) plays a key role in the regulation of a suite of genes involved in drug biotransformation, but information on the role of this response pathway in fish is limited. Here we investigated the effects of exposure of carp (Cyprinus carpio) primary hepatocytes to the human PXR agonist rifampicin (RIF) on expression of target genes involved in phase I (cyp2k, cyp3a) and phase II (gstα, gstπ) drug metabolism and drug transporters mdr1 and mrp2. RIF induced expression of all target genes measured and the PXR antagonist ketoconazole (KET) inhibited responses of cyp2k and cyp3a. Exposure of the primary carp hepatocytes to the pharmaceuticals ibuprofen (IBU), clotrimazole (CTZ), clofibric acid (CFA) and propranolol (PRP), found responses to IBU and CFA, but not CTZ or PRP. This is in contrast with mammals, where CTZ is a potent PXR-agonist. Collectively our data indicate potential PXR involvement in regulating selected genes involved in drug metabolism in fish, but suggest some divergence in the regulation pathways with those in mammals. The carp primary hepatocyte model serves as a useful system for screening for responses in these target genes involved in drug metabolism.
Abstract.
Söffker M, Tyler CR (2012). Endocrine disrupting chemicals and sexual behaviors in fish--a critical review on effects and possible consequences.
Crit Rev Toxicol,
42(8), 653-668.
Abstract:
Endocrine disrupting chemicals and sexual behaviors in fish--a critical review on effects and possible consequences.
Endocrine disrupting chemicals (EDCs) enter aquatic ecosystems through discharged effluents, mainly from wastewater treatment works and diffuse run off from land, and affect a wide range of aquatic biota, including fish. Evidence for altered physiology in fish as a consequence of endocrine disruption is global, with some of the most widely reported effects on sexual development and function. In recent years, research has shown that fish behaviors can also be affected by EDCs which potentially has wide implications for individual fitness and population level outcomes. This review presents a critical assessment on reported effects of EDCs on behavior in fish, focusing on behaviors associated with reproduction. We investigate commonalities and differences in sexual behaviors between fish species most commonly applied in ecotoxicology, drawing out common principles for impacts of EDCs and then reviewing the evidence for, and implications of, disruptions of these behaviors after exposures to EDCs. In an analysis of the reported effects of exposure to the estrogen ethinylestradiol, we show that life-stage at exposure is a key factor determining behavioral responses of affected populations. EDC-induced changes in behavior occur at similar concentrations as for established biomarker responses (e.g. vitellogenin induction for estrogens), indicating behavior is equally sensitive (and noninvasive) as an indicator of EDC exposure. Adopting behaviors in fish as indicators of chemical exposure and effects, however, still has many technical and interpretation challenges and there is very little information available on how behaviors under laboratory conditions equate with those occurring in wild populations.
Abstract.
Author URL.
Filby AL, Paull GC, Searle F, Ortiz-Zarragoitia M, Tyler CR (2012). Environmental estrogen-induced alterations of male aggression and dominance hierarchies in fish: a mechanistic analysis.
Environ Sci Technol,
46(6), 3472-3479.
Abstract:
Environmental estrogen-induced alterations of male aggression and dominance hierarchies in fish: a mechanistic analysis.
Environmental estrogens have been shown to affect aspects of fish behavior that could potentially impact on wild populations, but the physiological mechanisms underpinning these effects are unknown. Using small colonies of zebrafish (Danio rerio), we evaluated the impacts of estrogen exposure on the aggression of dominant males, the associated implications for their social status and reproductive success, and their signaling mechanisms. The aggression of dominant males exposed to 17α-ethinylestradiol (EE(2); 10 ng/L nominal) was reduced significantly, and half of these fish subsequently lost their dominance, behavioral changes that were reflected in their reproductive success. Plasma androgen and the expression of genes involved in sex steroid production/signaling (cyp19a1b, cyp17, hsd11b2, hsd17b3, ar) and aggression (avplrv1b, tph1b, htr1a, sst1, sstr1, th, slc6a3, ar) were higher in control dominant versus subordinate males, but suppressed by EE(2) exposure, such that the differences between the social ranks were not retained. The expression levels of avpl (brain), which promotes aggression and dominance, and ar and cyp17 (gonad) were elevated in nonexposed males paired with EE(2)-exposed males. Our findings illustrate that disruptions of behaviors affecting social hierarchy, and in turn breeding outcome, as a consequence of exposure to an environmental estrogen are signaled through complex interconnecting gonadal and neurological control mechanisms that generally conform with those established in mammalian models. The extensive molecular, genetic, physiological, and behavioral toolbox now available for the zebrafish makes this species an attractive model for integrated analyses of chemical effects spanning behavior to molecular effect mechanisms.
Abstract.
Author URL.
Tyler CR, Hamilton PB, Lange A, Filby AL, Soffkar M, Lee O, Takesono A, Kudoh T, Paull GC, Iguchi T, et al (2012). Health impacts of exposure to environmental oestrogens in fish.
Author URL.
Gaiser BK, Fernandes TF, Jepson MA, Lead JR, Tyler CR, Baalousha M, Biswas A, Britton GJ, Cole PA, Johnston BD, et al (2012). Interspecies comparisons on the uptake and toxicity of silver and cerium dioxide nanoparticles.
Environmental Toxicology and Chemistry,
31(1), 144-154.
Abstract:
Interspecies comparisons on the uptake and toxicity of silver and cerium dioxide nanoparticles
An increasing number and quantity of manufactured nanoparticles are entering the environment as the diversity of their applications increases, and this will lead to the exposure of both humans and wildlife. However, little is known regarding their potential health effects. We compared the potential biological effects of silver (Ag; nominally 35 and 600-1,600nm) and cerium dioxide (CeO 2; nominally
Abstract.
Filby AL, Paull GC, Searle F, Ortiz-Zarragoitia M, Tyler CR (2012). Investigating the mechanisms by which environmental oestrogens affect male aggression and dominance hierarchies in fish.
Author URL.
Fabrega J, Tantra R, Amer A, Stolpe B, Tomkins J, Fry T, Lead JR, Tyler CR, Galloway TS (2012). Sequestration of zinc from zinc oxide nanoparticles and life cycle effects in the sediment dweller amphipod Corophium volutator.
Environ Sci Technol,
46(2), 1128-1135.
Abstract:
Sequestration of zinc from zinc oxide nanoparticles and life cycle effects in the sediment dweller amphipod Corophium volutator.
We studied the effects of ZnO nanoparticles [ZnO NPs, primary particle size 35 ± 10 nm (circular diameter, TEM)], bulk [160 ± 81 nm (circular diameter, TEM)], and Zn ions (from ZnCl(2)) on mortality, growth, and reproductive endpoints in the sediment dwelling marine amphipod Corophium volutator over a complete lifecycle (100 days). ZnO NPs were characterized by size, aggregation, morphology, dissolution, and surface properties. ZnO NPs underwent aggregation and partial dissolution in the seawater exposure medium, resulting in a size distribution that ranged in size from discrete nanoparticles to the largest aggregate of several micrometers. Exposure via water to all forms of zinc in the range of 0.2-1.0 mg L(-1) delayed growth and affected the reproductive outcome of the exposed populations. STEM-EDX analysis was used to characterize insoluble zinc precipitates (sphaerites) of high sulfur content, which accumulated in the hepatopancreas following exposures. The elemental composition of the sphaerites did not differ for ZnO NP, Zn(2+), and bulk ZnO exposed organisms. These results provide an illustration of the comparable toxicity of Zn in bulk, soluble, and nanoscale forms on critical lifecycle parameters in a sediment dwelling organism.
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Author URL.
Croft DP, Hamilton PB, Darden SK, Jacoby DMP, James R, Bettaney EM, Tyler CR (2012). The role of relatedness in structuring the social network of a wild guppy population. Oecologia, 1-9.
Al-Sahli R, Abdul-Sada A, Lange A, Tyler CR, Hill EM (2012). The xenometabolome and novel contaminant biomarkers in fish exposed to a wastewater treatment works effluent.
Author URL.
Al-Salhi R, Abdul-Sada A, Lange A, Tyler CR, Hill EM (2012). The xenometabolome and novel contaminant markers in fish exposed to a wastewater treatment works effluent.
Environmental Science and Technology,
46(16), 9080-9088.
Abstract:
The xenometabolome and novel contaminant markers in fish exposed to a wastewater treatment works effluent
Organisms exposed to wastewater treatment works (WwTW) effluents accumulate complex mixtures of xenobiotics but there is a scarcity of information on the nature and impacts of these chemical mixtures. We applied metabolomics techniques as a novel approach to identify xenobiotics and their metabolites (the xenometabolome) that bioconcentrate in fish exposed to a WwTW effluent. Exposed juvenile rainbow trout (Oncorhynchus mykiss) accumulated surfactants, naphthols, chlorinated xylenols, and phenoxyphenols, chlorophenes, resin acids, mefenamic acid, oxybenzone, and steroidal alkaloids in the bile or plasma, and there were perturbations in the plasma concentrations of bile acids and lipids. Exposure of adult roach (Rutilus rutilus) to 50% or 100% concentrations of the same effluent resulted in dose-dependent increases in plasma concentrations of xenometabolites as well as cyprinol sulfate and taurocholic acid, lysophospholipids, and a decrease in sphingosine levels (a key component of cell membrane lipids). Our findings reveal the highly complex nature of xenobiotics accumulating in effluent-exposed fish, and the great potential of metabolomics for both identifying plasma marker (bio)chemicals for monitoring exposure to wastewater effluents, and for targeting studies on potential consequent impacts on fish health.
Abstract.
Larner F, Dogra Y, Dybowska A, Fabrega J, Stolpe B, Bridgestock LJ, Goodhead R, Weiss DJ, Moger J, Lead JR, et al (2012). Tracing bioavailability of ZnO nanoparticles using stable isotope labeling.
Environ Sci Technol,
46(21), 12137-12145.
Abstract:
Tracing bioavailability of ZnO nanoparticles using stable isotope labeling.
Zinc oxide nanoparticles (ZnO NPs) are widely used in commercial products and knowledge of their environmental fate is a priority for ecological protection. Here we synthesized model ZnO NPs that were made from and thus labeled with the stable isotope (68)Zn and this enables highly sensitive and selective detection of labeled components against high natural Zn background levels. We combine high precision stable isotope measurements and novel bioimaging techniques to characterize parallel water-borne exposures of the common mudshrimp Corophium volutator to (68)ZnO NPs, bulk (68)ZnO, and soluble (68)ZnCl(2) in the presence of sediment. C. volutator is an important component of coastal ecosystems where river-borne NPs will accumulate and is used on a routine basis for toxicity assessments. Our results demonstrate that ionic Zn from ZnO NPs is bioavailable to C. volutator and that Zn uptake is active. Bioavailability appears to be governed primarily by the dissolved Zn content of the water, whereby Zn uptake occurs via the aqueous phase and/or the ingestion of sediment particles with adsorbed Zn from dissolution of ZnO particles. The high sorption capacity of sediments for Zn thus enhances the potential for trophic transfer of Zn derived from readily soluble ZnO NPs. The uncertainties of our isotopic data are too large, however, to conclusively rule out any additional direct uptake route of ZnO NPs by C. volutator.
Abstract.
Author URL.
2011
Brown AR, Bickley LK, Le Page G, Hosken DJ, Paull GC, Hamilton PB, Owen SF, Robinson J, Sharpe AD, Tyler CR, et al (2011). Are toxicological responses in laboratory (inbred) zebrafish representative of those in outbred (wild) populations? - a case study with an endocrine disrupting chemical.
Environ Sci Technol,
45(9), 4166-4172.
Abstract:
Are toxicological responses in laboratory (inbred) zebrafish representative of those in outbred (wild) populations? - a case study with an endocrine disrupting chemical.
Laboratory animals tend to be more inbred and less genetically diverse than wild populations, and thus may differ in their susceptibility to chemical stressors. We tested this hypothesis by comparing the responses of related inbred (theoretical inbreeding F(IT) = n + 0.25) and outbred (F(IT) = n) zebrafish (Danio rerio) WIK/Wild family lines to an endocrine disrupting chemical, clotrimazole. Exposure of inbred and outbred zebrafish to 2.9 μg clotrimazole/L had no effect on survival, growth, or gonadal development. Exposure of both lines to 43.7 μg clotrimazole/L led to male-biased sex ratios compared with controls (87% versus 55% and 92% vs 64%, for inbred and outbred males, respectively), advanced germ cell development, and reduced plasma 11-ketotestosterone concentrations in males. However, outbred males (but not inbred males) developed testis that were more than twice the weight of controls, which corresponded with a proliferation of Leydig cells and maintenance of the expression (rather than down-regulation occurring in inbreds) of gonadal aromatase (cyp19a1a) and insulin-like growth factor (igf1). Our results illustrate that the effects of an endocrine disrupting chemical (clotrimazole) on some end points (here testis development) can differ between inbred and outbred zebrafish. This highlights the need for reporting pedigree/genetic information and consistency in the responses of laboratory animals (e.g. by using model compounds as positive controls).
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Author URL.
Rostkowski P, Horwood J, Shears JA, Lange A, Oladapo FO, Besselink HT, Tyler CR, Hill EM (2011). Bioassay-directed identification of novel antiandrogenic compounds in bile of fish exposed to wastewater effluents.
Environmental Science and Technology,
45(24), 10660-10667.
Abstract:
Bioassay-directed identification of novel antiandrogenic compounds in bile of fish exposed to wastewater effluents
The widespread occurrence of feminized male fish downstream of some UK Wastewater Treatment Works (WwTWs) has been associated with exposure to estrogenic and potentially antiandrogenic (AA) contaminants in the effluents. In this study, profiling of AA contaminants in WwTW effluents and fish was conducted using HPLC in combination with in vitro androgen receptor transcription screens. Analysis of extracts of wastewater effluents revealed complex profiles of AA activity comprising 21-53 HPLC fractions. Structures of bioavailable antiandrogens were identified by exposing rainbow trout to a WwTW effluent and profiling the bile for AA activity using yeast (anti-YAS) and mammalian-based (AR-CALUX) androgen receptor transcription screens. The predominant fractions with AA activity in both androgen receptor screens contained the germicides chlorophene and triclosan, and together these contaminants accounted for 51% of the total anti-YAS activity in the fish bile. Other AA compounds identified in bile included chloroxylenol, dichlorophene, resin acids, napthols, oxybenzone, 4-nonylphenol, and bisphenol A. Pure standards of these compounds were active in the androgen receptor screens at potencies relative to flutamide of between 0.1 and 13.0. Thus, we have identified, for the first time, a diverse range of AA chemicals in WwTWs that are bioavailable to fish and which need to be assessed for their risk to the reproductive health of these organisms and other aquatic biota. © 2011 American Chemical Society.
Abstract.
Gaiser BK, Biswas A, Rosenkranz P, Jepson MA, Lead JR, Stone V, Tyler CR, Fernandes TF (2011). Effects of silver and cerium dioxide micro- and nano-sized particles on Daphnia magna.
Journal of Environmental Monitoring,
13(5), 1227-1235.
Abstract:
Effects of silver and cerium dioxide micro- and nano-sized particles on Daphnia magna
Acute (96 h) and chronic (21 d) exposures of Daphnia magna neonates were carried out with nano- and micro-sized Ag and CeO2 particles to assess the influence of both material and size of particles on mortality and moulting. Mortality rates for silver in the acute exposures were: AgNP, 56.7 ± 23.3% at 0.1 mg L-1 and 100 ± 20% at 1 mg L -1, and micro-Ag, 13.3 ± 6.7% at 0.1 mg L-1 and 80 ± 20% at 1 mg L-1. CeO2 was not acutely toxic at concentrations up to 10 mg L-1. Mortality for Ag over 21d at concentrations of up to 0.05 mg L-1 was low, while mortality of 30% was observed for 0.001 mg L-1 of nano-Ag. CeO2, with the exception of the 10 mg L-1 of nano-CeO2 (100% mortality by day 7), was non-toxic. Inhibition of moulting and growth in the acute study occurred at toxic concentrations (Ag particles), and at 10 mg L-1 of nano-CeO2. The chronic study revealed reduced moulting at 0.001 mg L-1 of nano-Ag and 0.01 and 0.05 mg L-1 of both sizes of Ag, but there was no impact on D. magna size, and no effects of CeO2. The toxicity of nano-CeO2 may be attributed to reduced feeding and physical interference with the daphnids' carapace, resulting in reduced swimming ability. Our results suggest that Ag NPs in particular have the potential to be harmful to aquatic invertebrates after release into the environment, whereas CeO2 particles appear to cause little adverse effects, and only at environmentally irrelevant concentrations. © 2011 the Royal Society of Chemistry.
Abstract.
Lange A, Paull GC, Hamilton PB, Iguchi T, Tyler CR (2011). Implications of persistent exposure to treated wastewater effluent for breeding in wild roach (Rutilus rutilus) populations.
Environ Sci Technol,
45(4), 1673-1679.
Abstract:
Implications of persistent exposure to treated wastewater effluent for breeding in wild roach (Rutilus rutilus) populations.
Feminized responses are widespread in wild populations of roach, Rutilus rutilus, living in UK rivers, and some of these responses have been shown to arise as a consequence of exposure to wastewater treatment works (WwTW) effluent discharges and the endocrine disrupting chemicals (EDCs) they contain. The causation of the ovotestis condition in wild roach, however, has yet to be established. Furthermore, the impact of long-term exposure to WwTW effluents on the reproductive fitness of wild fish populations is not known, and this information is crucial for population level effect assessments. We undertook a chronic exposure of roach to a treated estrogenic wastewater effluent for up to 3.5 years to assess principally for effects on subsequent reproductive fitness, as determined through parentage analysis on offspring from a competitive breeding study. In generating the fish for the breeding study we found that exposure to full strength WwTW effluent until sexual maturity resulted in sex reversal in almost all males in the population; 98% of the exposed fish were phenotypic females, containing ovaries. Furthermore, fish exposed to a 50% dilution of WwTW effluent contained ovotestis (21% of the male roach) that was absent from the control population. In competitive breeding studies, and applying DNA microsatellites to assess parentage, we show that presumptive females exposed to sexual maturity to WwTW effluent bred normally, albeit in the absence of nonexposed females, but putative sex-reversed males breeding as females contributed poorly, if at all, in a breeding population, depending on the competition. These novel findings on sex reversal add a new dimension for impact assessments of exposure to WwTW effluents on fish populations.
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García-López Á, Sánchez-Amaya MI, Tyler CR, Prat F (2011). Mechanisms of oocyte development in European sea bass (Dicentrarchus labrax L.): investigations via application of unilateral ovariectomy.
Reproduction,
142(2), 243-253.
Abstract:
Mechanisms of oocyte development in European sea bass (Dicentrarchus labrax L.): investigations via application of unilateral ovariectomy.
Unilateral ovariectomy (ULO) was performed in European sea bass (Dicentrarchus labrax L.) during late pre-vitellogenesis/early vitellogenesis. Plasma steroid levels and the expression of a suite of potential oogenesis-relevant genes in the ovary, brain, and pituitary were evaluated with the aim of understanding their involvement in the compensatory oocyte development occurring within the remaining ovarian lobe. After 69 days of surgery the remaining ovarian lobe in ULO fish was gravimetrically equivalent to an intact-paired ovary of sham operated, control fish. This compensatory ovarian growth was based on an increased number of early perinucleolar oocytes and mid-late stage vitellogenic follicles without an apparent recruitment of primary oocytes into the secondary growth phase. Plasma steroid levels were similar in ULO and control females at all time points analyzed, suggesting an increased steroid production of the remaining ovarian lobe in hemi-castrated females. Results of the gene expression survey conducted indicate that the signaling pathways mediated by Fsh and Gnrh1 constitute the central axes orchestrating the observed ovarian compensatory growth. In addition, steroid receptors, Star protein, Igfs, and members of the transforming growth factor beta superfamily including anti-Mullerian hormone and bone morphogenetic protein 4 were identified as potentially relevant players within this process, although their specific actions and interactions remain to be established. Our results demonstrate that ULO provides an excellent in vivo model for elucidating the interconnected endocrine and molecular mechanisms controlling oocyte development in European sea bass.
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Author URL.
Southam AD, Lange A, Hines A, Hill EM, Katsu Y, Iguchi T, Tyler CR, Viant MR (2011). Metabolomics reveals target and off-target toxicities of a model organophosphate pesticide to roach (Rutilus rutilus): Implications for biomonitoring.
Environmental Science and Technology,
45(8), 3759-3767.
Abstract:
Metabolomics reveals target and off-target toxicities of a model organophosphate pesticide to roach (Rutilus rutilus): Implications for biomonitoring
The ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored. Fenitrothion, an organophosphate pesticide, is categorized as a "red list" pollutant, being particularly hazardous to aquatic life. It acts primarily as a cholinesterase inhibitor, but evidence suggests it can also act as an androgen receptor antagonist. Whole-organism fenitrothion-induced toxicity is well-established, but information regarding target and off-target molecular toxicities is limited. Here we study the molecular responses of male roach (Rutilus rutilus) exposed to fenitrothion, including environmentally realistic concentrations, for 28 days. Acetylcholine was assessed in brain; steroid metabolism was measured in testes and plasma; and NMR and mass spectrometry-based metabolomics were conducted on testes and liver to discover off-target toxicity. O-demethylation was confirmed as a major route of pesticide degradation. Fenitrothion significantly depleted acetylcholine, confirming its primary mode of action, and 11-ketotestosterone in plasma and cortisone in testes, showing disruption of steroid metabolism. Metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes. On the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity, we propose that chronic pesticide exposure induces an adapting phenotype in roach, which may have considerable implications for interpreting molecular biomarker responses in field-sampled fish. © 2011 American Chemical Society.
Abstract.
Sebire M, Katsiadaki I, Taylor NGH, Maack G, Tyler CR (2011). Short-term exposure to a treated sewage effluent alters reproductive behaviour in the three-spined stickleback (Gasterosteus aculeatus).
Aquat Toxicol,
105(1-2), 78-88.
Abstract:
Short-term exposure to a treated sewage effluent alters reproductive behaviour in the three-spined stickleback (Gasterosteus aculeatus).
Some UK sewage treatment work (STW) effluents have been found to contain high levels of anti-androgenic activity, but the biological significance of this activity to fish has not been determined. The aim of this study was to investigate the effects of exposure to a STW effluent with anti-androgenic activity on the reproductive physiology and behaviour of three-spined sticklebacks (Gasterosteus aculeatus). Fish were exposed to a STW effluent (50 and 100%, v/v) with a strong anti-androgenic activity (328.56±36.83 μgl(-1) flutamide equivalent, as quantified in a recombinant yeast assay containing the human androgen receptor) and a low level of oestrogenic activity (3.32±0.66 ngl(-1) oestradiol equivalent, quantified in a recombinant yeast assay containing the human oestrogen receptor) for a period of 21 days in a flow-through system in the laboratory. Levels of spiggin, an androgen-regulated protein, were not affected by the STW effluent exposure, nor were levels of vitellogenin (a biomarker of oestrogen exposure), but the reproductive behaviour of the males was impacted. Males exposed to full strength STW effluent built fewer nests and there was a significant reduction in male courtship behaviour for exposures to both the 50 and 100% STW effluent treatments compared with controls. The effect seen on the reproduction of male sticklebacks may not necessarily have been as a consequence of the endocrine active chemicals present in the STW effluent alone, but could relate to other features of the effluent, such as turbidity that can impair visual signalling important for courtship interactions. Regardless the specific causation, the data presented show that effluents from STW have an impact on reproductive behaviour in male sticklebacks which in turn affects reproductive performance/outcome. The study further highlights the use of fish behaviour as a sensitive endpoint for assessing potential effects of contaminated water bodies on fish reproduction.
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Fabrega J, Luoma SN, Tyler CR, Galloway TS, Lead JR (2011). Silver nanoparticles: behaviour and effects in the aquatic environment.
Environ Int,
37(2), 517-531.
Abstract:
Silver nanoparticles: behaviour and effects in the aquatic environment.
This review summarises and evaluates the present knowledge on the behaviour, the biological effects and the routes of uptake of silver nanoparticles (Ag NPs) to organisms, with considerations on the nanoparticle physicochemistry in the ecotoxicity testing systems used. Different types of Ag NP syntheses, characterisation techniques and predicted current and future concentrations in the environment are also outlined. Rapid progress in this area has been made over the last few years, but there is still a critical lack of understanding of the need for characterisation and synthesis in environmental and ecotoxicological studies. Concentration and form of nanomaterials in the environment are difficult to quantify and methodological progress is needed, although sophisticated exposure models show that predicted environmental concentrations (PECs) for Ag NPs in different environmental compartments are at the range of ng L(-1) to mg kg(-1). The ecotoxicological literature shows that concentrations of Ag NPs below the current and future PECs, as low as just a few ng L(-1), can affect prokaryotes, invertebrates and fish indicating a significant potential, though poorly characterised, risk to the environment. Mechanisms of toxicity are still poorly understood although it seems clear that in some cases nanoscale specific properties may cause biouptake and toxicity over and above that caused by the dissolved Ag ion. This review concludes with a set of recommendations for the advancement of understanding of the role of nanoscale silver in environmental and ecotoxicological research.
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Harris CA, Hamilton PB, Runnalls TJ, Vinciotti V, Henshaw A, Hodgson D, Coe TS, Jobling S, Tyler CR, Sumpter JP, et al (2011). The consequences of feminization in breeding groups of wild fish.
Environ Health Perspect,
119(3), 306-311.
Abstract:
The consequences of feminization in breeding groups of wild fish.
BACKGROUND: the feminization of nature by endocrine-disrupting chemicals (EDCs) is a key environmental issue affecting both terrestrial and aquatic wildlife. A crucial and as yet unanswered question is whether EDCs have adverse impacts on the sustainability of wildlife populations. There is widespread concern that intersex fish are reproductively compromised, with potential population-level consequences. However, to date, only in vitro sperm quality data are available in support of this hypothesis. OBJECTIVE: the aim of this study was to examine whether wild endocrine-disrupted fish can compete successfully in a realistic breeding scenario. METHODS: in two competitive breeding experiments using wild roach (Rutilus rutilus), we used DNA microsatellites to assign parentage and thus determine reproductive success of the adults. RESULTS: in both studies, the majority of intersex fish were able to breed, albeit with varying degrees of success. In the first study, where most intersex fish were only mildly feminized, body length was the only factor correlated with reproductive success. In the second study, which included a higher number of more severely intersex fish, reproductive performance was negatively correlated with severity of intersex. The intersex condition reduced reproductive performance by up to 76% for the most feminized individuals in this study, demonstrating a significant adverse effect of intersex on reproductive performance. CONCLUSION: Feminization of male fish is likely to be an important determinant of reproductive performance in rivers where there is a high prevalence of moderately to severely feminized males.
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2010
Southam AD, Hines A, Viant MR, Lange A, Tyler CR, Hill EM (2010). A metabolomic investigation to identify molecular changes induced in roach after exposure to the anti-androgen organophosphate pesticide fenitrothion.
Author URL.
Scown TM, Goodhead RM, Johnston BD, Moger J, Baalousha M, Lead JR, Van Aerle R, Iguchi T, Tyler CR (2010). Assessment of cultured fish hepatocytes for studying cellular uptake and (eco)toxicity of nanoparticles.
Environmental Chemistry,
7(1), 36-49.
Abstract:
Assessment of cultured fish hepatocytes for studying cellular uptake and (eco)toxicity of nanoparticles
Relatively little is known regarding the fate and possible toxic effects of engineered nanoparticles (ENPs) in the aquatic environment. We assessed the suitability of isolated trout hepatocytes for high throughput toxicity screening of ENPs, exposing them to a variety of metal and metal oxide nanoparticles and their bulk counterparts. We found no effects of the ENPs on cell viability, or on lipid peroxidation, with the exception of exposure to ZnO nanoparticles, or on glutathione-S-transferase (GST) levels, for exposure concentrations up to 500 g mL-1. All ENPs, however, were internalised in the cultured hepatocytes, as shown by coherent anti-Stokes Raman scattering (CARS) as an imaging technique. Our findings suggest that fish hepatocyte cultures are suitable for studies investigating the cellular uptake of ENPs, but they do not appear to be sensitive to ENP exposure and thus not a good in vitro model for nanoparticle toxicity screening. © 2010 CSIRO.
Abstract.
Johnston BD, Scown TM, Moger J, Cumberland SA, Baalousha M, Linge K, van Aerle R, Jarvis K, Lead JR, Tyler CR, et al (2010). Bioavailability of nanoscale metal oxides TiO(2), CeO(2), and ZnO to fish.
Environ Sci Technol,
44(3), 1144-1151.
Abstract:
Bioavailability of nanoscale metal oxides TiO(2), CeO(2), and ZnO to fish.
Nanoparticles (NPs) are reported to be a potential environmental health hazard. For organisms living in the aquatic environment, there is uncertainty on exposure because of a lack of understanding and data regarding the fate, behavior, and bioavailability of the nanomaterials in the water column. This paper reports on a series of integrative biological and physicochemical studies on the uptake of unmodified commercial nanoscale metal oxides, zinc oxide (ZnO), cerium dioxide (CeO(2)), and titanium dioxide (TiO(2)), from the water and diet to determine their potential ecotoxicological impacts on fish as a function of concentration. Particle characterizations were performed and tissue concentrations were measured by a wide range of analytical methods. Definitive uptake from the water column and localization of TiO(2) NPs in gills was demonstrated for the first time by use of coherent anti-Stokes Raman scattering (CARS) microscopy. Significant uptake of nanomaterials was found only for cerium in the liver of zebrafish exposed via the water and ionic titanium in the gut of trout exposed via the diet. For the aqueous exposures undertaken, formation of large NP aggregates (up to 3 mum) occurred and it is likely that this resulted in limited bioavailability of the unmodified metal oxide NPs in fish.
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Paull GC, Filby AL, Giddins HG, Coe TS, Hamilton PB, Tyler CR (2010). Dominance hierarchies in zebrafish (Danio rerio) and their relationship with reproductive success.
Zebrafish,
7(1), 109-117.
Abstract:
Dominance hierarchies in zebrafish (Danio rerio) and their relationship with reproductive success.
The zebrafish has considerable potential for use as a model in the study of behavior in social systems, particularly dominance hierarchies, which are widespread in nature and can affect the lifelong success of individuals. There is, however, a paucity of information relating to the characterization of social groups and significance of dominance hierarchies in the zebrafish model. This study set out to bridge this knowledge gap and better characterize dominance and its implications for reproductive success in both male and female zebrafish in colonies comprising of two males and two females. Analyses of four aggressive behaviors (chase, bite, repel, spar) were conducted twice daily over a 5-day period, and fertilized eggs were collected for parentage analyses using DNA microsatellite markers. Dominant-subordinate relationships occurred both between males and between females, and in both sexes, dominance was associated with a greater body size and higher levels of aggression. During the spawning period, dominant females were, however, less aggressive toward their subordinates than dominant males to their subordinates. Aggressive behaviors employed for maintaining dominance did not differ between the sexes, but in females, in contrast with males, the level of aggression directed toward the subordinate fish increased over the study period. Overall, dominance resulted in a greater total reproductive success in males but not in females; however, dominant females sired more offspring with the dominant male. The findings illustrate that energy invested in dominance behavior appears beneficial for both sexes in zebrafish.
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Author URL.
Panter GH, Hutchinson TH, Hurd KS, Bamforth J, Stanley RD, Wheeler JR, Tyler CR (2010). Effects of a weak oestrogenic active chemical (4-tert-pentylphenol) on pair-breeding and F1 development in the fathead minnow (Pimephales promelas).
Aquatic Toxicology,
97(4), 314-323.
Abstract:
Effects of a weak oestrogenic active chemical (4-tert-pentylphenol) on pair-breeding and F1 development in the fathead minnow (Pimephales promelas)
A fish full life-cycle (FFLC) is the most comprehensive test to determine reproductive toxicity of chemicals to fish and this is likely to apply equally to endocrine active chemicals (EACs). However, FFLC tests use large numbers of animals, are expensive and time consuming. Alternative chronic tests, to the FFLC, potentially include sensitive life-stage windows of effect, such as sexual differentiation, early gonadal development and reproduction. In this paper, a fish pair-breeding study was applied to assess the biological effects of a weak environmental oestrogen, 4-tert-pentylphenol (4TPP), on reproduction and subsequent development of the F1 generation. The results of this study were then compared with the results for a published FFLC study, with this chemical. Fathead minnows (Pimephales promelas) were held in pairs and their reproductive performance assessed over two concurrent 21-day periods, the first without exposure to the test chemical, followed by the second with exposure to the test chemical, in a flow-through system at 25 ± 1 °C. Embryos from two pairs, per treatment, were subsequently grown up in clean water until 90 days post-hatch to assess developmental effects of the parental exposure on the F1 generation. Nominal (measured geometric mean, time weighted) test concentrations of 4TPP were 56 (48), 180 (173) and 560 (570) μg l-1. A significant decrease in fecundity was observed in all 4TPP exposed fish (mean number of eggs spawned per pair and number of spawns per pair) when compared to the solvent control. Vitellogenin (VTG) was significantly elevated in F0 males exposed to 560 μg 4TPP l-1. Somatic endpoints, secondary sexual characteristics (SSC) and gonadosomatic index (GSI) were not affected by the 4TPP exposure. In the F1 generation, there were no treatment-related effects on hatching success, survival, growth, SSC or GSI. Histological examination of the gonads of the F1 fish revealed no treatment-related effects on sex ratio, sexual differentiation or sexual development. However, plasma VTG concentrations were significantly elevated in F1 male fish, derived from parents that had previously been exposed to 4TPP at concentrations of ≥180 μg l-1. These data show that the reproductive performance test is suitable for detecting weak environmental oestrogenic chemicals and that exposure of adult fish to oestrogens can result in altered biomarker expression (VTG) of the F1 generation. Our findings indicate that the reproductive performance test was as sensitive for detecting effects on reproduction when compared with a published FFLC test for 4TPP. © 2010 Elsevier B.V. All rights reserved.
Abstract.
Filby AL, Shears JA, Drage BE, Churchley JH, Tyler CR (2010). Effects of advanced treatments of wastewater effluents on estrogenic and reproductive health impacts in fish.
Environ Sci Technol,
44(11), 4348-4354.
Abstract:
Effects of advanced treatments of wastewater effluents on estrogenic and reproductive health impacts in fish.
Whether the implementation of additional treatments for the removal of estrogens from wastewater treatment works (WwTWs) effluents will eliminate their feminizing effects in exposed wildlife has yet to be established, and this information is crucial for future decisions on investment into WwTWs. Here, granular activated carbon (GAC), ozone (O(3)), and chlorine dioxide (ClO(2)) were investigated for their effectiveness in reducing steroidal estrogen levels in a WwTW effluent and assessments made on the associated estrogenic and reproductive responses in fathead minnows (Pimephales promelas) exposed for 21 days. All treatments reduced the estrogenicity of the standard-treated (STD) effluent, but with different efficacies; ranging between 70-100% for total estrogenicity and 53-100% for individual steroid estrogens. In fish exposed to the GAC- and ClO(2)- (but not O(3)-) treated effluents, there was no induction of plasma vitellogenin (VTG) or reduction in the weight of the fatpad, a secondary sex character in males, as occurred for fish exposed to STD effluent. This finding suggests likely benefits of employing these treatment processes for the reproductive health in wild fish populations living in rivers receiving WwTW discharges. Exposure of pair-breeding minnows to the GAC-treated effluent, however, resulted in a similar inhibition of egg production to that occurring for exposure to the STD effluent (34-40%). These data, together with a lack of effect on egg production of the estrogen, ethinylestradiol (10 ng/L), alone, suggest that chemical/physical properties of the effluents rather than their estrogenicity were responsible for the reproductive effect and that these factor(s) were not remediated for through GAC treatment. Collectively, our findings illustrate the importance of assessing integrative biological responses, rather than biomarkers alone, in the assessment and improvement of WwTW technologies for the protection of wild fish populations.
Abstract.
Author URL.
Scown TM, Santos EM, Johnston BD, Gaiser B, Baalousha M, Mitov S, Lead JR, Stone V, Fernandes TF, Jepson M, et al (2010). Effects of aqueous exposure to silver nanoparticles of different sizes in rainbow trout.
Toxicol Sci,
115(2), 521-534.
Abstract:
Effects of aqueous exposure to silver nanoparticles of different sizes in rainbow trout.
Despite increasing application of silver nanoparticles (NPs) in industry and consumer products, there is still little known about their potential toxicity, particularly to organisms in aquatic environments. To investigate the fate and effects of silver NPs in fish, rainbow trout (Oncorhynchus mykiss) were exposed via the water to commercial silver particles of three nominal sizes: 10 nm (N(10)), 35 nm (N(35)), and 600-1600 nm (N(Bulk)), and to silver nitrate for 10 days. Uptake into the gills, liver, and kidneys was quantified by inductively coupled plasma-optical emission spectrometry, and levels of lipid peroxidation in gills, liver, and blood were determined by measurements of thiobarbituric acid reactive substances. Expression of a suite of genes, namely cyp1a2, cyp3a45, hsp70a, gpx, and g6pd, known to be involved in a range of toxicological response to xenobiotics was analyzed in the gills and liver using real-time PCR. Uptake of silver particles from the water into the tissues of exposed fish was low but nevertheless occurred for current estimated environmental exposures. of the silver particles tested, N(10) were found to be the most highly concentrated within gill tissues and N(10) and N(Bulk) were the most highly concentrated in liver. There were no effects on lipid peroxidation in any of the tissues analyzed for any of the silver particles tested, and this is likely due to the low uptake rates. However, exposure to N(10) particles was found to induce expression of cyp1a2 in the gills, suggesting a possible increase in oxidative metabolism in this tissue.
Abstract.
Author URL.
Fenlon KA, Johnson AC, Tyler CR, Hill EM (2010). Gas-liquid chromatography-tandem mass spectrometry methodology for the quantitation of estrogenic contaminants in bile of fish exposed to wastewater treatment works effluents and from wild populations.
Journal of Chromatography A,
1217(1), 112-118.
Abstract:
Gas-liquid chromatography-tandem mass spectrometry methodology for the quantitation of estrogenic contaminants in bile of fish exposed to wastewater treatment works effluents and from wild populations
Fish can be exposed to a complex mixture of chemical contaminants arising from the exposure to wastewater treatment works (WwTWs) effluents. Some of these contaminants are estrogenic and have been associated with feminisation of male fish and the presence of populations containing intersex individuals. However the detection of trace levels (ng/L) of estrogenic chemicals surface waters can be difficult and does not give information on the exposure of aquatic organisms to these contaminants. In this study we assessed whether the analysis of estrogenic substances that bioconcentrate in fish bile can be used to detect the exposure of fish to feminising contaminants in receiving waters and effluents, and thus facilitate their monitoring of these substances in aquatic environments. Estrogenic metabolites in bile were deconjugated using enzymatic hydrolysis and partially purified by solid phase extraction. Steroidal and xenoestrogens were derivatized to their trimethylsilyl ethers and quantified by gas-liquid chromatography-mass spectrometry (GC-MS/MS) using multiple reaction monitoring. The method was validated using spiked bile samples from immature female rainbow trout (Oncorhynchus mykiss) as well as bile from sexually mature roach (Rutilus rutilus) that had been exposed to either tap water or an undiluted estrogenic effluent for 10 days or captured from a river site downstream of a WwTWs effluent discharge. The mean recovery of target analytes from spiked bile was between 86 and 99% and the limit of detection was between 0.1 and 0.7 ng/mL bile for bisphenol a (BPA), 17β-estradiol (E2), estrone (E1) and 17α-ethinylestradiol (EE2), and 11, 60 and 327 ng/mL bile for branched nonyl chain isomeric mixtures of 4-nonylphenolethoxylate (NP1EO), 4-nonylphenol (NP) and 4-nonylphenoldiethoxylate (NP2EO), respectively. All target analytes were detected in bile from roach exposed directly to a WwTWs effluent, with concentrations between 6-13 μg/mL bile for NP, 18-21 μg/mL for NP1EO, 75-135 μg/mL for NP2EO, 0.7-2.5 μg/mL for BPA, E2 and E1 and 17-29 ng/mL for EE2. With the exception of NP2EO, all analytes were detected in at least 2 out of the 5 fish sampled from the River Thames. BPA and NP1EO were detected in all three reference fish held in tap water indicating possible contamination from laboratory plastics. The work shows that analysis of 20-100 μL quantities of bile could be a useful approach in detecting exposure to mixtures of estrogenic contaminants taken up by fish from WwTW effluents and has the potential for monitoring the efficacy of remediation strategies that may be adopted for reduction of these endocrine disrupting chemicals in the aquatic environment. © 2009 Elsevier B.V. All rights reserved.
Abstract.
Katsiadaki I, Williams TD, Ball JS, Bean TP, Sanders MB, Wu H, Santos EM, Brown MM, Baker P, Ortega F, et al (2010). Hepatic transcriptomic and metabolomic responses in the Stickleback (Gasterosteus aculeatus) exposed to ethinyl-estradiol.
Aquat Toxicol,
97(3), 174-187.
Abstract:
Hepatic transcriptomic and metabolomic responses in the Stickleback (Gasterosteus aculeatus) exposed to ethinyl-estradiol.
An established three-spined stickleback (Gasterosteus aculeatus) cDNA array was expanded to 14,496 probes with the addition of hepatic clones derived from subtractive and normalized libraries from control males and males exposed to model toxicants. Microarrays and one-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy, together with individual protein and gene biomarkers were employed to investigate the hepatic responses of the stickleback to ethinyl-estradiol (EE(2)) exposure. Male fish were exposed via the water to EE(2), including environmentally relevant concentrations (0.1-100ng/l) for 4 days, and hepatic transcript and metabolite profiles, kidney spiggin protein and serum vitellogenin concentrations were determined in comparison to controls. EE(2) exposure did not significantly affect spiggin concentration but significantly induced serum vitellogenin protein at the threshold concentration of 32ng/l. (1)H NMR coupled with robust univariate testing revealed only limited changes, but these did support the predicted modulation of the amino acid profile by transcriptomics. Transcriptional induction was found for hepatic vitellogenins and choriogenins as expected, together with a range of other EE(2)-responsive genes. Choriogenins showed the more sensitive responses with statistically significant induction at 10ng/l. Real-time polymerase chain reaction (PCR) confirmed transcriptional induction of these genes. Phosvitinless vitellogenin C transcripts were highly expressed and represent a major form of the egg yolk precursors, and this is in contrast to other fish species where it is a minor component of vitellogenic transcripts. Differences in inducibility between the vitellogenins and choriogenins appear to be in accordance with the sequential formation of chorion and yolk during oogenesis in fish.
Abstract.
Author URL.
Santos EM, Ball JS, Williams TD, Wu H, Ortega F, van Aerle R, Katsiadaki I, Falciani F, Viant MR, Chipman JK, et al (2010). Identifying health impacts of exposure to copper using transcriptomics and metabolomics in a fish model.
Environ Sci Technol,
44(2), 820-826.
Abstract:
Identifying health impacts of exposure to copper using transcriptomics and metabolomics in a fish model.
Copper (Cu) is a micronutrient essential for the biochemical functioning of numerous processes in vertebrates but is also often present in the aquatic environment at concentrations able to cause adverse health effects in aquatic organisms. This study investigated the signaling pathways mediating the effects of exposure to Cu using a toxicogenomic approach in a fish model, the stickleback ( Gasterosteus aculeatus ). Freshwater-acclimated male fish were exposed via the water to Cu, including at environmentally relevant concentrations (3.2-128 microg of Cu/L for 4 days), and the biological responses explored through analyses of the hepatic transcriptome and metabolome and phenotypic end points, including assessment of DNA damage in blood cells. The Cu exposures resulted in DNA strand breaks in blood cells at all exposure concentrations and alterations in hepatic gene expression and metabolite concentrations in a concentration-dependent manner (from 10 microg of Cu/L). Genes associated with the cholesterol biosynthesis pathway were significantly over-represented and consistently down-regulated (at 128 microg of Cu/L), similar to that occurring in a mouse model for Wilson's disease. Additionally, inductions in metallothionein and catalase were also observed. The concentrations of NAD(+) and lactate increased significantly with the Cu exposure, consistent with a shift toward anaerobic metabolism, and these aligned closely with changes observed in gene expression. The pathways of Cu toxicity identified in our study support the conserved mechanisms of Cu toxicity from lower vertebrates to mammals, provide novel insights into the deleterious effects of Cu in fish, and further demonstrate the utility of fish as environmental sentinels for chemical impacts on both environmental and human health.
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Author URL.
Coe TS, Söffker MK, Filby AL, Hodgson D, Tyler CR (2010). Impacts of early life exposure to estrogen on subsequent breeding behavior and reproductive success in zebrafish.
Environ Sci Technol,
44(16), 6481-6487.
Abstract:
Impacts of early life exposure to estrogen on subsequent breeding behavior and reproductive success in zebrafish.
Impacts of exposure to environmental estrogens on reproductive development are well documented, but recently wider concern has been raised due to evidence that such exposures can disrupt normal patterns of reproductive behavior, dominance, and parentage, with potential population level implications. It is fundamental therefore to understand any such effects for effective risk assessment. This study investigated the impact of a transient exposure to ethinylestradiol (EE(2)) during early life (from 20-60 days post fertilization), including at a dosing level within the environmental range, on the subsequent reproductive behavior and success in both male and female zebrafish (Danio rerio) in competitive breeding scenarios. There were no obvious effects of the early life EE(2) exposures on the subsequent gonadal phenotypes in either mature males or females. In fact, reproductive success in males exposed to 2.76 ng EE(2)/L was increased in competitive spawning scenarios. In contrast, exposure of females to EE(2) (9.86 ng/L) during early life reduced their subsequent reproductive success in competitive spawning scenarios. Mate choice experiments suggested this was a consequence of the females' diminished courting behavior toward males, rather than any male preference for unexposed females. Reproductive capability of females is generally considered a key determinant in population demographics and dynamics, and therefore the effect of exposure to EE(2) on female reproductive success may have significant implications for exposed fish populations.
Abstract.
Author URL.
Tyler CR, Goodhead RM (2010). Impacts of hormone-disrupting chemicals on wildlife. In (Ed)
Silent Summer: the State of Wildlife in Britain and Ireland, 125-140.
Abstract:
Impacts of hormone-disrupting chemicals on wildlife
Abstract.
Van Aggelen G, Ankley GT, Baldwin WS, Bearden DW, Benson WH, Chipman JK, Collette TW, Craft JA, Denslow ND, Embry MR, et al (2010). Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.
Environ Health Perspect,
118(1), 1-5.
Abstract:
Integrating omic technologies into aquatic ecological risk assessment and environmental monitoring: hurdles, achievements, and future outlook.
BACKGROUND: in this commentary we present the findings from an international consortium on fish toxicogenomics sponsored by the U.K. Natural Environment Research Council (Fish Toxicogenomics-Moving into Regulation and Monitoring, held 21-23 April 2008 at the Pacific Environmental Science Centre, Vancouver, BC, Canada). OBJECTIVES: the consortium from government agencies, academia, and industry addressed three topics: progress in ecotoxicogenomics, regulatory perspectives on roadblocks for practical implementation of toxicogenomics into risk assessment, and dealing with variability in data sets. DISCUSSION: Participants noted that examples of successful application of omic technologies have been identified, but critical studies are needed to relate molecular changes to ecological adverse outcome. Participants made recommendations for the management of technical and biological variation. They also stressed the need for enhanced interdisciplinary training and communication as well as considerable investment into the generation and curation of appropriate reference omic data. CONCLUSIONS: the participants concluded that, although there are hurdles to pass on the road to regulatory acceptance, omics technologies are already useful for elucidating modes of action of toxicants and can contribute to the risk assessment process as part of a weight-of-evidence approach.
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Author URL.
Corcoran J, Lange A, Tyler CR, Winter MJ (2010). Investigating drug-metabolising cytochrome P450 (cyp) isoforms in a fish hepatocyte model.
Author URL.
Corcoran J, Winter MJ, Tyler CR (2010). Pharmaceuticals in the aquatic environment: a critical review of the evidence for health effects in fish.
Crit Rev Toxicol,
40(4), 287-304.
Abstract:
Pharmaceuticals in the aquatic environment: a critical review of the evidence for health effects in fish.
The authors review the current data on the presence and reported biological effects in fish of some of the most commonly detected pharmaceuticals in the aquatic environment; namely nonsteroidal anti-inflammatory drugs (NSAIDs), fibrates, beta-blockers, selective serotonin reuptake inhibitors (SSRIs), azoles, and antibiotics. Reported biological effects in fish in the laboratory have often been shown to be in accordance with known effects of pharmaceuticals in mammals. Water concentrations at which such effects have been reported, however, are generally, between microg L(-1) and mg L(-1), typically at least 1 order of magnitude higher than concentrations normally found in surface waters (ng L(-1)). There are exceptions to this, however, as for the case of synthetic oestrogens, which can induce biological effects in the low ng L(-1) range. Although generally effect levels for pharmaceuticals are higher than those found in the environment, the risks to wild fish populations have not been thoroughly characterised, and there has been a lack of consideration given to the likely chronic nature of the exposures, or the potential for mixture effects. As global consumption of pharmaceuticals rises, an inevitable consequence is an increased level of contamination of surface and ground waters with these biologically active drugs, and thus in turn a greater potential for adverse effects in aquatic wildlife.
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Author URL.
Filby AL, Paull GC, Bartlett EJ, Van Look KJW, Tyler CR (2010). Physiological and health consequences of social status in zebrafish (Danio rerio).
Physiol Behav,
101(5), 576-587.
Abstract:
Physiological and health consequences of social status in zebrafish (Danio rerio).
Social status affects access to food, mates and shelter and has consequences for the physiology of individuals and their health status. In the zebrafish (Danio rerio), an emerging model for studies into animal behavior, the possible consequences of social hierarchy to an individual's physiology and health are unknown. To address this, in this species we assessed the effects of social interaction (for periods of 1-5days) on growth, stress, immune function and reproductive condition. Wide-ranging differences in physiology occurred between the social ranks, some of which were sex-related and time-dependent. In both sexes, dominant fish were larger than subordinates and dominant males had a higher growth rate during the trials. Subordinates had higher plasma cortisol and in males higher telencephalic corticotrophin-releasing hormone, neuropeptide y and glucocorticoid receptor gene expression. Splenic cytokine expression suggested differences in immune status between ranks in both sexes and hematocrit was elevated in subordinate males. In both sexes, dominants and subordinates differed in the expression of genes for various gonadal sex steroid receptors and steroidogenic enzymes and in dominant females the ovary was larger relative to body mass compared with in subordinates. Dominant males had higher plasma 11-ketotestosterone than subordinates and there was an increase in the number of spermatids in their testes over the duration of the study that was not seen in subordinate males. The wide-ranging physiological differences seen between dominant and subordinate zebrafish as a consequence of their social status suggest negative health impacts for subordinates after prolonged durations in those hierarchies.
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Author URL.
Hill EM, Evans KL, Horwood J, Rostkowski P, Oladapo FO, Gibson R, Shears JA, Tyler CR (2010). Profiles and some initial identifications of (anti)androgenic compounds in fish exposed to wastewater treatment works effluents.
Environ Sci Technol,
44(3), 1137-1143.
Abstract:
Profiles and some initial identifications of (anti)androgenic compounds in fish exposed to wastewater treatment works effluents.
Exposure of fish to wastewater treatment works (WwTWs) effluents can result in reproductive anomalies consistent with exposure to estrogenic compounds. However, UK WwTWs effluents also contain compounds with androgen receptor activities which may contribute to reproductive dysfunction in fish. A toxicity identification and evaluation (TIE) approach was used to profile (anti)androgenic compounds in bile of fish exposed to two WwTWs effluents. Extracts of bile from exposed fish and effluent were fractionated by liquid chromatography and tested for (anti)androgenic activity using a yeast androgen receptor transcription screen (YAS). A number of bile fractions contained (anti)androgenic activity unique to the effluent-exposed fish. Some of these fractions contained di(chloromethyl)anthracene or dichlorophene, and these contaminants showed antagonistic activity in the YAS when tested as pure compounds. No androgenic activity was detected in the effluents, but TIE analysis of bile revealed a number of androgenic fractions which contained testosterone metabolites that were unique to effluent-exposed fish. This is the first work reported on the nature of some of the (anti)androgenic compounds that bioaccumulate in fish from WwTWs effluents and indicates that other contaminants, besides estrogenic substances, need to be considered for their potential to contribute to the disruption of reproductive system of fish in UK waters.
Abstract.
Author URL.
Scown TM, van Aerle R, Tyler CR (2010). Review: Do engineered nanoparticles pose a significant threat to the aquatic environment?.
Crit Rev Toxicol,
40(7), 653-670.
Abstract:
Review: Do engineered nanoparticles pose a significant threat to the aquatic environment?
Nanotechnology is a rapidly growing industry of global economic importance, exploiting the novel characteristics of materials manufactured at the nanoscale. The properties of engineered nanoparticles (ENPs) that make them useful in a wide range of industrial applications, however, have led to concerns regarding their potential impact on human and environmental health. The aquatic environment is particularly at risk of exposure to ENPs, as it acts as a sink for most environmental contaminants. This paper critically evaluates what is currently known about sources and discharge of ENPs to the aquatic environment and how the physicochemical characteristics of ENPs affect their fate and behaviour and thus availability for uptake into aquatic organisms, and assesses reported toxicological effects. Having reviewed the ecotoxicological information, the conclusion is that whilst there are data indicating some nanoparticles have the potential to induce harm in exposed aquatic organisms, there is insufficient evidence for harm, for known/modelled environmental concentrations for almost all ENPs considered. This conclusion, however, must be balanced by the fact that there are significant gaps in our understanding on the fate and behaviour of ENPs in the aquatic environment. Greater confidence in the assessments on ENP impacts in aquatic systems to enable effective comparisons across studies urgently requires more standardised approaches for ENP hazard identification, and critically, more thorough characterisations on the exposed particles. There is also an urgent need for the advancement of tools and techniques that can accurately quantify and visualise uptake of nanoparticles into biological tissues.
Abstract.
Author URL.
Filby AL, Paull GC, Hickmore TF, Tyler CR (2010). Unravelling the neurophysiological basis of aggression in a fish model.
BMC Genomics,
11Abstract:
Unravelling the neurophysiological basis of aggression in a fish model.
BACKGROUND: Aggression is a near-universal behaviour with substantial influence on and implications for human and animal social systems. The neurophysiological basis of aggression is, however, poorly understood in all species and approaches adopted to study this complex behaviour have often been oversimplified. We applied targeted expression profiling on 40 genes, spanning eight neurological pathways and in four distinct regions of the brain, in combination with behavioural observations and pharmacological manipulations, to screen for regulatory pathways of aggression in the zebrafish (Danio rerio), an animal model in which social rank and aggressiveness tightly correlate. RESULTS: Substantial differences occurred in gene expression profiles between dominant and subordinate males associated with phenotypic differences in aggressiveness and, for the chosen gene set, they occurred mainly in the hypothalamus and telencephalon. The patterns of differentially-expressed genes implied multifactorial control of aggression in zebrafish, including the hypothalamo-neurohypophysial-system, serotonin, somatostatin, dopamine, hypothalamo-pituitary-interrenal, hypothalamo-pituitary-gonadal and histamine pathways, and the latter is a novel finding outside mammals. Pharmacological manipulations of various nodes within the hypothalamo-neurohypophysial-system and serotonin pathways supported their functional involvement. We also observed differences in expression profiles in the brains of dominant versus subordinate females that suggested sex-conserved control of aggression. For example, in the HNS pathway, the gene encoding arginine vasotocin (AVT), previously believed specific to male behaviours, was amongst those genes most associated with aggression, and AVT inhibited dominant female aggression, as in males. However, sex-specific differences in the expression profiles also occurred, including differences in aggression-associated tryptophan hydroxylases and estrogen receptors. CONCLUSIONS: Thus, through an integrated approach, combining gene expression profiling, behavioural analyses, and pharmacological manipulations, we identified candidate genes and pathways that appear to play significant roles in regulating aggression in fish. Many of these are novel for non-mammalian systems. We further present a validated system for advancing our understanding of the mechanistic underpinnings of complex behaviours using a fish model.
Abstract.
Author URL.
Mehinto AC, Hill EM, Tyler CR (2010). Uptake and biological effects of environmentally relevant concentrations of the nonsteroidal anti-inflammatory pharmaceutical diclofenac in rainbow trout (Oncorhynchus mykiss).
Environ Sci Technol,
44(6), 2176-2182.
Abstract:
Uptake and biological effects of environmentally relevant concentrations of the nonsteroidal anti-inflammatory pharmaceutical diclofenac in rainbow trout (Oncorhynchus mykiss).
Diclofenac, a nonsteroidal anti-inflammatory drug, is widely detected in surface waters and can potentially cause deleterious effects in fish. Here, we investigated the biological effects of 21-day exposure to waterborne diclofenac at environmentally relevant concentrations (0, 0.5, 1, 5, and 25 μg/L) in rainbow trout Accumulation of diclofenac in the bile was measured and responses in selected tissues were assessed via changes in the expression of selected genes (cytochrome P450 (cyp) 1a1, cyclooxygenase (cox) 1 and 2, and p53) involved in metabolism of xenobiotics, prostaglandin synthesis, and cell cycle control, respectively, together with histopathological alterations in these tissues. Diclofenac accumulated in the bile by a factor of between 509 ± 27 and 657 ± 25 and various metabolites were putatively identified as hydroxydiclofenac, diclofenac methyl ester, and the potentially reactive metabolite hydroxydiclofenac glucuronide. Expression levels of both cox1 and cox2 in liver, gills, and kidney were significantly reduced by diclofenac exposure from only 1 μg/L. Expression of cyp1a1 was induced in the liver and the gills but inhibited in the kidney of exposed fish. Diclofenac exposure induced tubular necrosis in the kidney and hyperplasia and fusion of the villi in the intestine from 1 μg/L. This study demonstrates that subchronic exposure to environmental concentrations of diclofenac can interfere with the biochemical functions of fish and lead to tissue damage, highlighting further the concern about this pharmaceutical in the aquatic environment.
Abstract.
Author URL.
2009
Oehlmann J, Schulte-Oehlmann U, Kloas W, Jagnytsch O, Lutz I, Kusk KO, Wollenberger L, Santos EM, Paull GC, VanLook KJW, et al (2009). A critical analysis of the biological impacts of plasticizers on wildlife.
Philosophical Transactions of the Royal Society B: Biological Sciences,
364(1526), 2047-2062.
Abstract:
A critical analysis of the biological impacts of plasticizers on wildlife
This review provides a critical analysis of the biological effects of the most widely used plasticizers, including dibutyl phthalate, diethylhexyl phthalate, dimethyl phthalate, butyl benzyl phthalate and bisphenol a (BPA), on wildlife, with a focus on annelids (both aquatic and terrestrial), molluscs, crustaceans, insects, fish and amphibians. Moreover, the paper provides novel data on the biological effects of some of these plasticizers in invertebrates, fish and amphibians. Phthalates and BPA have been shown to affect reproduction in all studied animal groups, to impair development in crustaceans and amphibians and to induce genetic aberrations. Molluscs, crustaceans and amphibians appear to be especially sensitive to these compounds, and biological effects are observed at environmentally relevant exposures in the low ng 1 -1 to μg 1-1 range. In contrast, most effects in fish (except for disturbance in spermatogenesis) occur at higher concentrations. Most plasticizers appear to act by interfering with the functioning of various hormone systems, but some phthalates have wider pathways of disruption. Effect concentrations of plasticizers in laboratory experiments coincide with measured environmental concentrations, and thus there is a very real potential for effects of these chemicals on some wildlife populations. The most striking gaps in our current knowledge on the impacts of plasticizers on wildlife are the lack of data for long-term exposures to environmentally relevant concentrations and their ecotoxicity when part of complex mixtures. Furthermore, the hazard of plasticizers has been investigated in annelids, molluscs and arthropods only, and given the sensitivity of some invertebrates, effects assessments are warranted in other invertebrate phyla.
Abstract.
Gaiser BK, Fernandes TF, Jepson M, Lead JR, Tyler CR, Stone V (2009). Assessing exposure, uptake and toxicity of silver and cerium dioxide nanoparticles from contaminated environments.
Abstract:
Assessing exposure, uptake and toxicity of silver and cerium dioxide nanoparticles from contaminated environments
Abstract.
Tyler CR, Filby AL, Bickley LK, Cumming RI, Gibson R, Labadie P, Katsu Y, Liney KE, Shears JA, Silva-Castro V, et al (2009). Environmental health impacts of equine estrogens derived from hormone replacement therapy.
Environ Sci Technol,
43(10), 3897-3904.
Abstract:
Environmental health impacts of equine estrogens derived from hormone replacement therapy.
Many factors have been considered in evaluations of the risk-benefit balance of hormone replacement therapy (HRT), used for treating menopausal symptoms in women, but not its potential risks for the environment We investigated the possible environmental health implications of conjugated equine estrogens (CEEs), the most common components of HRT, including their discharge into the environment, their uptake, potency, and ability to induce biological effects in wildlife. Influents and effluents from four U.K. sewage treatment works (STWs), and bile of effluent-exposed fish, were screened for six equine estrogens. In vitro estrogen receptor (ER) activation assays were applied in humans and fish to compare their potencies, followed by in vivo exposures of fish to equine estrogens and evaluation of bioaccumulation, estrogenic responses, and ER gene expression. The equine estrogen equilenin (Eqn), and its metabolite 17beta-dihydroequilenin (17beta-Eqn), were detected by tandem GC-MSMS in all STW influent samples and 83% of STW effluent samples analyzed, respectively, at low concentrations (0.07-2.6 ng/L) and were taken-up into effluent-exposed fish. As occurs in humans, these estrogens bound to and activated the fish ERs, with potencies at ERalpha 2.4-3490% of thatfor 17beta-estradiol. Exposure of fish for 21 days to Eqn and 17beta-Eqn induced estrogenic responses including hepatic growth and vitellogenin production at concentrations as low as 0.6-4.2 ng/L. Associated with these effects were inductions of hepatic ERalpha and ERbeta1 gene expression, suggesting ER-mediated mechanism(s) of action. These data provide evidence for the discharge of equine estrogens from HRT into the aquatic environment and highlight a strong likelihood that these compounds contribute to feminization in exposed wildlife.
Abstract.
Author URL.
Thorpe KL, Maack G, Benstead R, Tyler CR (2009). Estrogenic wastewater treatment works effluents reduce egg production in fish.
Environ Sci Technol,
43(8), 2976-2982.
Abstract:
Estrogenic wastewater treatment works effluents reduce egg production in fish.
Estrogenic chemicals found within wastewater treatment work (WwTW) effluents have been shown individually to inhibit reproduction in fish, but the impact of the WwTW effluents themselves and the complex mixtures of environmental estrogens and other endocrine disrupting chemicals they contain has not been established. In this investigation, the effect of exposure to three WwTW effluents, with differing levels of estrogenic activity, was assessed on egg production in pair-breeding fathead minnow. Exposure to two of the three effluents tested resulted in a reduced egg production (by 28% for effluent I at a dilution of 50% and by 44% for effluent III at full strength), which was proportional to the estrogenic content of the effluents. The test effluents, however, had a greater effect on egg production than might be expected, on the basis of both the response they induced for induction of vitellogenin (an estrogen exposure biomarker) and when compared with an equivalent estrogen exposure to EE2. These data show that reliance on relatively simple biomarker responses for estrogenic activity alone, such as vitellogenin, can significantly underestimate the impacts of estrogenic WwTW effluents on fitness parameters such as reproductive health that are regulated by more complex estrogenic (and other endocrine) signaling mechanisms.
Abstract.
Author URL.
Bickley LK, Lange A, Winter MJ, Tyler CR (2009). Evaluation of a carp primary hepatocyte culture system for screening chemicals for oestrogenic activity.
Aquat Toxicol,
94(3), 195-203.
Abstract:
Evaluation of a carp primary hepatocyte culture system for screening chemicals for oestrogenic activity.
The presence of endocrine disrupting chemicals (EDCs) in the environment has driven the development of screening and testing assays to both identify chemicals with hormonal activity and evaluate their potential to cause adverse effects. As the number of animals used for research and regulatory purposes rises, and set against a desire to reduce animal testing, there is increased emphasis on the development and application of in vitro techniques to evaluate chemical risks to the environment. Induction of vitellogenin (VTG) in isolated fish liver cells has been used successfully to identify a wide range of EDCs, including both natural and synthetic oestrogens and a variety of other xenoestrogens. However, the vitellogenic response reported for hepatocytes in culture has been shown to vary widely, making comparisons between studies difficult. The work presented in this paper explored the variability of the vitellogenic response in primary cultures of common carp (Cyprinus carpio) hepatocytes following exposure to the model oestrogenic compound, 17beta-oestradiol (E2). As expected, variability in the vitellogenic response was observed, both in terms of the sensitivity and magnitude of VTG induction, for hepatocytes isolated from different fish. An apparent difference was observed in the response of isolated hepatocytes based on the sex of the donor fish; maximum levels of E2-stimulated VTG synthesis in hepatocytes derived from females appeared higher (1962 ng mL(-1)+/-487 [n=9] compared with 1194 ng mL(-1)+/-223 for hepatocytes from males [n=9]) and EC(50) values lower (1.61+/-0.4 microM E2 for females and 2.12+/-0.2 microM E2 for males). However, these differences were not statistically significant, likely in part due to the variation observed in the vitellogenic response. In particular, hepatocytes derived from female fish showed more variation than their male counterparts (the co-efficient of variation for females was 77% compared to 28% for males). Despite the variation observed in the vitellogenic response between different cultures, data from the different donor fish could be compared by standardising responses relative to the maximum VTG induction in each culture following exposure to E2. Adopting this approach in the future will allow for data from different hepatocyte cultures and from donor fish of different sexes, age and stage of maturity to be compared with greater consistency. Measurement of vtg mRNA expression was relatively more sensitive to the oestrogenic effects of E2 exposure than measurement of VTG protein (the LOEC at the transcriptome level was 10-fold lower [0.01 microM E2] than at the protein level [0.1 microM E2]) and changes in vtg mRNA expression showed less variation between individual hepatocyte isolations. Measurement of vtg mRNA in the hepatocyte culture system therefore may offer the most sensitive and consistent option for the screening of chemicals with oestrogenic activity in fish primary hepatocyte cultures.
Abstract.
Author URL.
Coe TS, Hamilton PB, Griffiths AM, Hodgson DJ, Wahab MA, Tyler CR (2009). Genetic variation in strains of zebrafish (Danio rerio) and the implications for ecotoxicology studies.
Ecotoxicology,
18(1), 144-150.
Abstract:
Genetic variation in strains of zebrafish (Danio rerio) and the implications for ecotoxicology studies.
There is substantial evidence that genetic variation, at both the level of the individual and population, has a significant effect on behaviour, fitness and response to toxicants. Using DNA microsatellites, we examined the genetic variation in samples of several commonly used laboratory strains of zebrafish, Danio rerio, a model species in toxicological studies. We compared the genetic variation to that found in a sample of wild fish from Bangladesh. Our findings show that the wild fish were significantly more variable than the laboratory strains for several measures of genetic variability, including allelic richness and expected heterozygosity. This lack of variation should be given due consideration for any study which attempts to extrapolate the results of ecotoxicological laboratory tests to wild populations.
Abstract.
Author URL.
Brown AR, Hosken DJ, Balloux F, Bickley LK, LePage G, Owen SF, Hetheridge MJ, Tyler CR (2009). Genetic variation, inbreeding and chemical exposure--combined effects in wildlife and critical considerations for ecotoxicology.
Philos Trans R Soc Lond B Biol Sci,
364(1534), 3377-3390.
Abstract:
Genetic variation, inbreeding and chemical exposure--combined effects in wildlife and critical considerations for ecotoxicology.
Exposure to environmental chemicals can have negative consequences for wildlife and even cause localized population extinctions. Resistance to chemical stress, however, can evolve and the mechanisms include desensitized target sites, reduced chemical uptake and increased metabolic detoxification and sequestration. Chemical resistance in wildlife populations can also arise independently of exposure and may be spread by gene flow between populations. Inbreeding-matings between closely related individuals-can have negative fitness consequences for natural populations, and there is evidence of inbreeding depression in many wildlife populations. In some cases, reduced fitness in inbred populations has been shown to be exacerbated under chemical stress. In chemical testing, both inbred and outbred laboratory animals are used and for human safety assessments, iso-genic strains (virtual clones) of mice and rats are often employed that reduce response variation, the number of animals used and associated costs. In contrast, for environmental risk assessment, strains of animals are often used that have been selectively bred to maintain heterozygosity, with the assumption that they are better able to predict adverse effects in wild, genetically variable, animals. This may not necessarily be the case however, as one outbred strain may not be representative of another or of a wild population. In this paper, we critically discuss relationships between genetic variation, inbreeding and chemical effects with the intention of seeking to support more effective chemical testing for the protection of wildlife.
Abstract.
Author URL.
Paull GC, Filby AL, Tyler CR (2009). Growth rate during early life affects sexual differentiation in roach (Rutilus rutilus).
Environmental Biology of Fishes,
85(4), 277-284.
Abstract:
Growth rate during early life affects sexual differentiation in roach (Rutilus rutilus)
Many environmental factors have been shown to influence sex differentiation in fish, resulting in sex-biased populations, but the effects of growth rate have received limited attention. We conducted a controlled laboratory experiment in which growth rate and population density were manipulated in roach (Rutilus rutilus) during early development, and the subsequent effects on sex ratio determined. Significant differences in growth rate between fish populations were induced through provision of three different ration levels. In the slowest growing population there were fewer females compared within the fastest growing population (19% compared to 36% females), suggesting that in roach it may be more advantageous to become a small male than a small female when growth potential is limited. This may result from the fact that fecundity is limited by body size in female roach and that male roach are able to reproduce at a significantly smaller body size than females. In contrast, where roach were kept at different stocking densities, and there were no differences in growth rate, the subsequent proportion of females did not vary. Our data highlight the importance of controlling for growth rate in research on sexual differentiation in this species, notably when assessing for the effects of endocrine disrupting chemicals and other environmental factors, and have implications for fisheries management and aquaculture. The underlying mechanism for the influence of growth rate on sex differentiation has yet to be determined but is likely to have a strong endocrinological basis. © Springer Science+Business Media B.V. 2009.
Abstract.
Williams TD, Wu H, Santos EM, Ball J, Katsiadaki I, Brown MM, Baker P, Ortega F, Falciani F, Craft JA, et al (2009). Hepatic transcriptomic and metabolomic responses in the stickleback (Gasterosteus aculeatus) exposed to environmentally relevant concentrations of dibenzanthracene.
Environ Sci Technol,
43(16), 6341-6348.
Abstract:
Hepatic transcriptomic and metabolomic responses in the stickleback (Gasterosteus aculeatus) exposed to environmentally relevant concentrations of dibenzanthracene.
A three-spined stickleback (Gasterosteus aculeatus) cDNA array and one-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy-based metabolomics approach, together with individual biomarkers,were employed to investigate the responses of male sticklebacks to polycyclic aromatic hydrocarbon exposure. Fish were exposed to 1,2:5,6-dibenzanthracene (DbA) at concentrations between 0.01 and 50 microg per liter dissolved in the ambient water for four days, and hepatic transcript and metabolite profiles were determined in comparison with those of solvent-exposed controls. Induction of gene expression was apparent for cytochrome P450 1A (CYP1A) and CYP2-family monooxygenases and these responses were strongly correlated with DbA exposure concentrations (for CYP1A r > 0.996). Expression of suites of genes related to bile acid biosynthesis, steroid metabolism, and endocrine function were also affected, as demonstrated by gene ontology analyses. Expression changes in selected genes were confirmed by real-time PCR. Metabolomics highlighted notable changes in concentrations of taurine, malonate, glutamate, and alanine. These statistically significant responses to environmentally relevant concentrations of DbA at the transcriptomic and metabolomic levels provided sensitive markers characteristic of environmentally relevant low-level DbA exposure. Metabolic pathways were identified where both gene expression and metabolite concentrations were altered in response to DbA.
Abstract.
Author URL.
Scown TM, van Aerle R, Johnston BD, Cumberland S, Lead JR, Owen R, Tyler CR (2009). High doses of intravenously administered titanium dioxide nanoparticles accumulate in the kidneys of rainbow trout but with no observable impairment of renal function.
Toxicol Sci,
109(2), 372-380.
Abstract:
High doses of intravenously administered titanium dioxide nanoparticles accumulate in the kidneys of rainbow trout but with no observable impairment of renal function.
Our recent work suggests limited uptake of unstabilized metal oxide nanoparticles via water into fish, however, some other studies have indicated such exposures can induce oxidative stress. To investigate tissue distribution and toxicity of titanium dioxide (TiO(2)) nanoparticles that may enter into fish, we conducted a series of injection studies. Rainbow trout (Oncorhynchus mykiss) were intravenously injected with 100 microg TiO(2) nanoparticles and the content of titanium in blood, brain, gills, liver, and kidney quantified at time points between 6 h and 90 days using inductively coupled plasma optical emission spectroscopy. Injected Ti was concentrated in the kidneys and remained there up to 21 days, however, there was evidence of clearance of TiO(2) at 90 days. Ti accumulation in the liver was 15 times lower than in the kidney with no apparent clearance. Using TEM we showed nanoparticles were localized in tissue vesicles surrounding the kidney tubules. In a second injection study, rainbow trout were injected with 100 microg TiO(2) and plasma samples from individual fish analyzed for total protein and creatinine content at time points between 6 h and 21 days to assess for possible effects on kidney function. No effect of TiO(2) on total plasma protein content or creatinine concentrations were found indicating that neither urine production nor glomerular filtration rate were affected. We conclude that in trout upon a single high dose exposure of TiO(2) nanoparticles via the bloodstream, TiO(2) accumulates in the kidneys but has minimal effect on kidney function.
Abstract.
Author URL.
Coe TS, Hamilton PB, Hodgson D, Paull GC, Tyler CR (2009). Parentage outcomes in response to estrogen exposure are modified by social grouping in zebrafish.
Environ Sci Technol,
43(21), 8400-8405.
Abstract:
Parentage outcomes in response to estrogen exposure are modified by social grouping in zebrafish.
Evidence has recently emerged that endocrine-disrupting chemicals (EDCs) can affect various behaviors, including dominance and aggression in social groups, including fish. This study investigated the effect of short-term exposure of male adult zebrafish to 17alpha-ethinylestradiol (EE(2)) on subsequent reproductive output and parentage in colonies with differing numbers of competing males. It was predicted that impacts of EDCs might differ in social groups of fish of differing size because of the greater costs of maintaining dominance hierarchies in large groups. Adult male zebrafish were exposed for 14 days to clean water, 2 ng/L EE(2) or 10 ng/L via the water, prior to placement into colonies in clean water with unexposed females. Exposure to EE(2) at the concentrations adopted prior to the breeding trials did not significantly affect subsequent colony reproductive output. The reproductive success of the most reproductively successful (MRS) male within colonies containing two males (relative to controls) was also unaffected. There was, however, a significant impact of previous EE(2) exposure in tanks containing four males, resulting in a reduction in paternity for the most successful male. Hence, nonlethal behavioral impacts of even short-term exposure to EDCs can have significant impacts on social dominance hierarchies and population genetic diversity.
Abstract.
Author URL.
Lange A, Paull GC, Coe TS, Katsu Y, Urushitani H, Iguchi T, Tyler CR (2009). Sexual reprogramming and estrogenic sensitization in wild fish exposed to ethinylestradiol.
Environ Sci Technol,
43(4), 1219-1225.
Abstract:
Sexual reprogramming and estrogenic sensitization in wild fish exposed to ethinylestradiol.
Globally, feminization responses in wild male freshwater fish are caused by exposure to estrogenic chemicals, including natural and synthetic estrogens, contained in effluentsfromwastewater treatment works. In U.K. rivers, feminization responses, including intersex, are widespread in wild roach (Rutilus rutilus) populations, and severely affected fish have a reduced reproductive success. We exposed roach to environmentally relevant concentrations of the contraceptive estrogen 17alpha-ethinylestradiol (EE2) for up to 2 years, including intermittent and repeated exposures,to determine effects on sexual development and subsequent responsiveness to estrogen. Exposure of roach to EE2 (at 4 ng/L) for 2 years resulted in sex reversal in males, leading to an all-female population with two cohorts in terms of their stages of ovarian development one paralleling the control females and one at a significantly less advanced stage, which we propose were sex-reversed males. Differing developmental and maturing rates of the putative sex-reversed males compared with control females would question their functional capability as females in the wild. Early-life exposure to environmentally relevant concentrations of EE2 sensitized females to estrogen, as determined by the measurement of the responses of estrogen-sensitive genes in a further EE2 challenge 398 days after the original exposure. In the wild, exposure to environmentally relevant concentrations of EE2 during early life has significantly wider implications for the sexual physiology in fish than has thus far been determined.
Abstract.
Author URL.
Full text.
Jobling S, Burn RW, Thorpe K, Williams R, Tyler C (2009). Statistical modeling suggests that antiandrogens in effluents from wastewater treatment works contribute to widespread sexual disruption in fish living in English rivers.
Environ Health Perspect,
117(5), 797-802.
Abstract:
Statistical modeling suggests that antiandrogens in effluents from wastewater treatment works contribute to widespread sexual disruption in fish living in English rivers.
BACKGROUND: the widespread occurrence of feminized male fish downstream of some wastewater treatment works has led to substantial interest from ecologists and public health professionals. This concern stems from the view that the effects observed have a parallel in humans, and that both phenomena are caused by exposure to mixtures of contaminants that interfere with reproductive development. The evidence for a "wildlife-human connection" is, however, weak: Testicular dysgenesis syndrome, seen in human males, is most easily reproduced in rodent models by exposure to mixtures of antiandrogenic chemicals. In contrast, the accepted explanation for feminization of wild male fish is that it results mainly from exposure to steroidal estrogens originating primarily from human excretion. OBJECTIVES: We sought to further explore the hypothesis that endocrine disruption in fish is multicausal, resulting from exposure to mixtures of chemicals with both estrogenic and antiandrogenic properties. METHODS: We used hierarchical generalized linear and generalized additive statistical modeling to explore the associations between modeled concentrations and activities of estrogenic and antiandrogenic chemicals in 30 U.K. rivers and feminized responses seen in wild fish living in these rivers. RESULTS: in addition to the estrogenic substances, antiandrogenic activity was prevalent in almost all treated sewage effluents tested. Further, the results of the modeling demonstrated that feminizing effects in wild fish could be best modeled as a function of their predicted exposure to both antiandrogens and estrogens or to antiandrogens alone. CONCLUSION: the results provide a strong argument for a multicausal etiology of widespread feminization of wild fish in U.K. rivers involving contributions from both steroidal estrogens and xenoestrogens and from other (as yet unknown) contaminants with antiandrogenic properties. These results may add further credence to the hypothesis that endocrine-disrupting effects seen in wild fish and in humans are caused by similar combinations of endocrine-disrupting chemical cocktails.
Abstract.
Author URL.
Sebire M, Scott AP, Tyler CR, Cresswell J, Hodgson DJ, Morris S, Sanders MB, Stebbing PD, Katsiadaki I (2009). The organophosphorous pesticide, fenitrothion, acts as an anti-androgen and alters reproductive behavior of the male three-spined stickleback, Gasterosteus aculeatus.
Ecotoxicology,
18(1), 122-133.
Abstract:
The organophosphorous pesticide, fenitrothion, acts as an anti-androgen and alters reproductive behavior of the male three-spined stickleback, Gasterosteus aculeatus.
Fenitrothion (FN) is a widely used organophosphorous pesticide that has structural similarities with the clinical anti-androgen flutamide. The potential for FN to act as an anti-androgen (at exposures of 1, 50, and 200 microg FN/l over a 26-day period) was assessed in male three-spined sticklebacks, Gasterosteus aculeatus, by measuring kidney spiggin concentration, nest-building, and courtship behavior. Spiggin is the glue protein that male sticklebacks use to build their nests and is directly controlled by androgens. FN exposure significantly reduced spiggin production as well as nest-building activity. It also adversely affected courtship--especially the 'zigzag dance' and biting behavior of the males. FN thus appears to have anti-androgenic effects on both the physiology and behavior of the male stickleback.
Abstract.
Author URL.
2008
Thorpe KL, Benstead R, Eccles P, Maack G, Williams T, Tyler CR (2008). A practicable laboratory flow-through exposure system for assessing the health effects of effluents in fish.
Aquat Toxicol,
88(3), 164-172.
Abstract:
A practicable laboratory flow-through exposure system for assessing the health effects of effluents in fish.
The knowledge that exposure to estrogenic wastewater treatment work (WwTW) effluents induces a range of reproductive abnormalities in fish has highlighted the need to understand the wider health effects of effluents. Access to laboratory-based testing systems for WwTW effluents could greatly facilitate this endeavour. In this investigation, a laboratory-based test system was developed and applied for WwTW effluents using fathead minnows (Pimephales promelas). Sexually maturing fathead minnows were exposed, under flow-through conditions in the laboratory, for up to 21 days to graded concentrations of effluent from three different UK (temperate) WwTWs. The stability of the estrogenic component within the test system was assessed via measurements for estradiol and estrone concentrations in the effluent, and through determining estrogenic responses in an in vitro recombinant yeast estrogen screen (rYES) and in fish (plasma vitellogenin induction). The estrogen component of the effluents was stable within the holding system used (chilled
Abstract.
Author URL.
Lange A, Katsu Y, Ichikawa R, Paull GC, Chidgey LL, Coe TS, Iguchi T, Tyler CR (2008). Altered sexual development in roach (Rutilus rutilus) exposed to environmental concentrations of the pharmaceutical 17alpha-ethinylestradiol and associated expression dynamics of aromatases and estrogen receptors.
Toxicol Sci,
106(1), 113-123.
Abstract:
Altered sexual development in roach (Rutilus rutilus) exposed to environmental concentrations of the pharmaceutical 17alpha-ethinylestradiol and associated expression dynamics of aromatases and estrogen receptors.
Wild roach (Rutilus rutilus) inhabiting UK rivers contaminated with estrogenic effluents from wastewater treatment works show altered sexual development, including intersex, and this can impact negatively on their reproductive capabilities. The molecular events underlying these disruptions in gender assignment, however, are still poorly understood. In this study, two isoforms of aromatase (cyp19a1a and cyp19a1b) were cloned from the roach, and effects of exposure to 17alpha-ethinylestradiol (EE(2)) during early life were determined on the expression of both aromatases and on the estrogen receptors (ERs) (subtypes esr1 and esr2b) and analyzed against effects on the progression of gonadal sex differentiation. Exposure to environmentally relevant concentrations of EE(2) during the critical period of sex differentiation resulted in gonadal feminization and all roach exposed to 4 ng EE(2)/l were females. These effects on gonadal development were associated with alterations in the expression of both esr and cyp19a1 genes in bodies and heads of exposed fish with the most marked effects on the expression of esr1 and cyp19a1b. Our findings show that both aromatase isoforms and both ER subtypes are associated with sexual differentiation in roach, and alterations in their expression can signal for disruptions in sexual development.
Abstract.
Author URL.
Coe TS, Hamilton PB, Hodgson D, Paull GC, Stevens JR, Sumner K, Tyler CR (2008). An environmental estrogen alters reproductive hierarchies, disrupting sexual selection in group-spawning fish.
Environ Sci Technol,
42(13), 5020-5025.
Abstract:
An environmental estrogen alters reproductive hierarchies, disrupting sexual selection in group-spawning fish.
There is global concern regarding the potential impacts of endocrine-disrupting chemicals (EDCs) on the health of wildlife and humans. Exposure to some estrogens, at concentrations found in the environment impairs reproductive function and behavior. However, nearly all work on endocrine disruption has investigated the effects of exposure on individuals and there is an urgent need to understand impacts on populations. Many fish have mating systems with complex social structures and it is not known whether EDCs will exaggerate or buffer the reproductive skews caused by the dominance hierarchies that normally occur for these species. This study investigated the impact of exposure to the pharmaceutical estrogen ethinylestradiol (EE2) on reproductive hierarchies and sexual selection in group-spawning fish. Breeding zebrafish were exposed to environmentally relevant concentrations of EE2, and effects were determined on reproductive output, plasma androgen concentrations (in males), and reproductive success through microsatellite analyses of the offspring. Reproductive hierarchies in breeding colonies of zebrafish were disrupted by exposure to EE2 at a concentration that did not affect the number of eggs produced. The effect was a reduction in the skew in male paternity and increased skew in female maternity. This disruption in the reproductive hierarchy in group spawning fish, if it occurs in the wild, has potentially major implications for population genetic diversity. Reproductive success in male zebrafish was associated with elevated plasma concentrations of the male sex hormone 11-ketotestosterone and this hormone was suppressed in EE2-exposed males.
Abstract.
Author URL.
Williams TD, Brown M, Chipman JK, Falciani F, Ortega F, Geoghegan F, Craft JA, Katsiadaki I, Ball J, Tyler CR, et al (2008). Development of a stickleback (Gasterosteus aculeatus) cDNA microarray and gene expression responses to dibenzanthracene, ethinyl-estradiol and copper.
MARINE ENVIRONMENTAL RESEARCH,
66(1), 140-140.
Author URL.
Benstead, R. Eccles, P. Maack, G. (2008). Effective assessment of the oestrogenic activity of effluents using a laboratory flow-through exposure system. Aquatic Toxicology
Routledge, E. van Aerle, R. (2008). Estrogenic effects of treated sewage effluent on fish: steroids and surfactants in English rivers. In DiGiulio R, Hinton D (Eds.) The Toxicology of Fishes.
van Aerle R, Kille P, Lange A, Tyler CR (2008). Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish.
Peptides,
29(1), 57-64.
Abstract:
Evidence for the existence of a functional Kiss1/Kiss1 receptor pathway in fish.
In mammals, the Kiss1 receptor (Kiss1r) and its kisspeptin ligands are key factors regulating the onset of puberty. In fish, however, the mechanisms underlying the initiation of puberty are poorly understood and the role of the Kiss1r/kisspeptin pathway in this process has not been established. In this study, a bioinformatics approach was used to identify the genes for Kiss1 and Kiss1r in five teleost genomes and the information used to clone the corresponding transcripts from zebrafish. Zebrafish kiss1r was expressed predominantly in the brain, with a minor level of expression in the eye, and zebrafish kiss1 was expressed in brain, intestine, adipose tissue and testis. Analysis of the chromosome region containing the kiss1 locus showed high synteny across vertebrate genomes. In contrast to their mammalian homologues, teleost Kiss1 protein sequences were poorly conserved with the exception of the region representing kisspeptin-10. Signal peptide sequences and likely cleavage and amidation sites in the teleost Kiss1 sequences were determined and found to be similar to those in mammalian Kiss1. This is the first report of the existence and characterization of the Kiss1 gene outside the mammalian taxa, suggesting that a functional Kiss1/Kiss1 receptor pathway is conserved across vertebrate species.
Abstract.
Author URL.
Tyler CR, Filby AL, van Aerle R, Lange A, Ball J, Santos EM (2008). Fish toxicogenomics. In (Ed) Advances in Experimental Biology, Elsevier.
Johnston, B.J. Tyler, C.R. (2008). Imaging metal oxide nanoparticles in biological structures with CARS microscopy. Optical Express, 16(5), 3408-3419.
Handy RD, Henry TB, Scown TM, Johnston BD, Tyler CR (2008). Manufactured nanoparticles: their uptake and effects on fish--a mechanistic analysis.
Ecotoxicology,
17(5), 396-409.
Abstract:
Manufactured nanoparticles: their uptake and effects on fish--a mechanistic analysis.
There is an emerging literature reporting toxic effects of manufactured nanomaterials (NMs) and nanoparticles (NPs) in fish, but the mechanistic basis of both exposure and effect are poorly understood. This paper critically evaluates some of the founding assumptions in fish toxicology, and likely mechanisms of absorption, distribution, metabolism and excretion (ADME) of NPs in fish compared to other chemicals. Then, using a case study approach, the paper compares these assumptions for two different NPs; TiO2 and C60 fullerenes. Adsorption of NPs onto the gill surface will involve similar processes in the gill microenvironment and mucus layer to other substances, but the uptake mechanisms for NPs by epithelial cells are more likely to occur via vesicular processes (e.g. endocytosis) than uptake on membrane transporters or by diffusion through the cell membranes. Target organs may include the gills, gut, liver and sometimes the brain. Information on metabolism and excretion of NPs in fish is limited; but hepatic excretion into the bile seems a more likely mechanism, rather than mainly by renal or branchial excretion. TiO2 and C60 share some common chemical properties that appear to be associated with some similar toxic effects, but there are also differences, that highlight the notion that chemical reactivity can inform toxic effect of NPs in a fundamentally similar way to other chemicals. In this paper we identify many knowledge gaps including the lack of field observations on fish and other wildlife species for exposure and effects of manufactured NMs. Systematic studies of the abiotic factors that influence bioavailability, and investigation of the cell biology that informs on the mechanisms of metabolism and excretion of NMs, will greatly advance our understanding of the potential for adverse effects. There are also opportunities to apply existing tools and techniques to fundamental studies of fish toxicology with NPs, such as perfused organs and fish cell culture systems.
Abstract.
Author URL.
Ibañez, A. Tyler, C.R. Prat, F. (2008). Molecular characterisation of growth differentiation factor 9 (gdf9) and bone morphogenetic protein 15 (bmp15) and their patterns of gene expression during the ovarian reproductive cycle in the European sea. bass. Molecular and Cellular Endocrinology
Paull GC, Lange A, Henshaw AC, Tyler CR (2008). Ontogeny of sexual development in the roach (Rutilus rutilus) and its interrelationships with growth and age.
J Morphol,
269(7), 884-895.
Abstract:
Ontogeny of sexual development in the roach (Rutilus rutilus) and its interrelationships with growth and age.
The roach (Rutilus rutilus) has become a sentinel species for the study of sexual disruption in wild fish populations as a consequence of exposure to endocrine disrupting chemicals (EDCs). Little is known, however, about the normal ontogeny of sexual development in this species. Here, we analyzed the ontogeny of sexual development in captive-bred roach and assessed how growth rate and fish size affected the timing of both sexual differentiation and sexual development over a 2-year period. Ovarian differentiation was first recorded at 68 days post-fertilization (dpf) and this preceded testicular differentiation (first recorded at 98 dpf). In contrast, sexual maturation occurred at an earlier age in males (300 dpf) compared with females (728 dpf). No differences in body size (length or weight) were recorded between male and female roach until the fish were 415 dpf. Studies on three populations of roach which grew at different rates showed that the timing of sexual differentiation was highly variable and more related to fish size than to fish age. Time to sexual maturation was also variable among populations but, subsequent to their first year of life, gonadal status was less well associated with fish size. Interestingly, the sex ratio of the population was biased towards females in populations that grew more rapidly during early life. The findings presented here provide a valuable foundation of work to support both field- and laboratory-based assessments on the effects of EDCs, and other stressors, on sexual differentiation and development in the roach.
Abstract.
Author URL.
Tyler CR, Jobling S (2008). Roach, sex, and gender-bending chemicals: the feminization of wild fish in English rivers.
BioScience,
58(11), 1051-1059.
Abstract:
Roach, sex, and gender-bending chemicals: the feminization of wild fish in English rivers
Feminization of the male roach, Rutilus rutilus, a freshwater, group-spawning fish, is widespread in English rivers; among the causative agents are natural and synthetic steroidal estrogens and chemicals that mimic estrogens. In feminized male roach, concentrations of the egg-yolk protein vitellogenin are elevated, sex steroid hormone dynamics are altered, and gonad development is disrupted (most notably, a female reproductive duct or developing eggs [oocytes] are present in the testis). In some English rivers containing high levels of estrogens, all male roach sampled have been feminized to varying degrees. In the more severely affected males, individuals produce low-quality sperm with a reduced capability for fertilization. Laboratory studies have shown that the environmental estrogens responsible for inducing gonadal feminization in roach can also alter reproductive behavior, disrupting normal breeding dynamics (parentage) in the zebrafish, another group-spawning fish. Together these findings indicate that feminization of wild roach may result in adverse population-level effects, but this hypothesis has yet to be fully addressed. © 2008 American Institute of Biological Sciences.
Abstract.
Kille, P. Workman, V.L. Paull, G.C. (2008). Sexually dimorphic gene expression in the brains of mature zebrafish. Comparative Biochemistry and Physiology Part A
Tyler C, Lange A, Santos E, Coe T, Paull G, Filby A, Hamilton P (2008). The feminization of fish in English rivers: Causation, mechanisms, and significance.
Author URL.
Filby AL, van Aerle R, Duitman J, Tyler CR (2008). The kisspeptin/gonadotropin-releasing hormone pathway and molecular signaling of puberty in fish.
Biol Reprod,
78(2), 278-289.
Abstract:
The kisspeptin/gonadotropin-releasing hormone pathway and molecular signaling of puberty in fish.
The mechanisms underlying the initiation of puberty in fish are poorly understood, and whether the Kiss1 receptor (Kiss1r; previously designated G protein-coupled receptor 54; GPR54) and its ligands, kisspeptins, play a significant role, as has been established in mammals, is not yet known. We determined (via real-time PCR) temporal patterns of expression in the brain of kiss1r, gnrh2, and gnrh3 and a suite of related genes in the hypothalamo-pituitary-gonadal (HPG) axis and analyzed them against the timing of gonadal germ cell development in male and female fathead minnow (Pimephales promelas). Full- or partial-length cDNAs for kiss1r (736 bp), gnrh2 (698 bp), and gnrh3 (804 bp) cloned from fathead minnow were found to be expressed only in the brain, testis, and ovary of adult fish. Localization of kiss1r, gnrh2, and gnrh3 within the brain provided evidence for their physiological roles and a likely hypophysiotropic role for GnRH3 in this species (which, like other cyprinids, does not appear to express gnrh1). In both sexes, kiss1r expression in the brain increased at the onset of puberty and reached maximal expression in males when spermatagonia type B appeared in the testis and in females when cortical alveolus-stage oocytes first appeared in the ovary, the timings of which differed for the two sexes. However, kiss1r expression was considerably lower during more advanced stages of spermatogenesis and oogenesis. The expression of kiss1r closely aligned with that of the gnrh genes (gnrh3 in particular), suggesting the Kiss1r/kisspeptin system in fish has a similar role in puberty to that occurring in mammals, and this hypothesis was supported by the induction of gnrh3 (2.25-fold) and kiss1r (1.5-fold) in early-mid pubertal fish injected with mammalian kisspeptin-10 (2 nmol/g wet weight). An intriguing finding, and contrasting that in mammals, was an elevated expression of esr1, ar, and cyp19a2 (genes involved in sex steroid signaling) in the brain at the onset of puberty, and in females slightly in advance of the elevation in the expression of kiss1r.
Abstract.
Author URL.
Paull, G.C. van Look, K.J.W. Santos, E.M. Filby A.L. Gray D.M. Nash, J.P. & Tyler, C.R (2008). Variability in measures of reproductive success in laboratory-kept colonies of zebrafish (Danio rerio) and implications for studies addressing population-level effects of environmental chemicals. Aquatic Toxicology
2007
Tyler, C.R. (2007). Cloning and Characterisation of cDNAs for hormones and/or receptors of growth hormone, insulin-like growth factor-I, thyroid hormone, and corticosteroid and the gender- and tissue- and developmental-specific expression of their mRNA transcripts in fathea. General & Comparative Endocrinology, 150, 151-163.
Katsu, Y. Urushitani, H. Lange, A. (2007). Developmental effects of exposure to. pharmaceutical steroids in the aquatic environment: Studies in Mosquitofish (Gambusia affinis), Roach. (Rutilus rutilus) and Medaka (Oryzias latipes. Journal of Marine Science and Technology
Hetheridge, M. Hutchinson, T.H. Benstead, R. (2007). An optimized experimental test procedure for measuring chemical effects on reproduction in the fathead minnow (<em>Pimephales promelas</em>). Aquatic Toxicology, 8(1), 90-98.
Filby AL, Tyler CR, Iguchi T, Kramer V, Larsson DGJ, van Leewen K, Viant M, Tillit DE (2007). Application of genomics to tiered testing. In (Ed) Molecular Biology and Risk Assessment: Evaluation of the Potential Roles of Genomics in Regulatory Ecotoxicology, SETAC Press, 33-62.
Filby AL, Tyler CR (2007). Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish.
BMC Mol Biol,
8Abstract:
Appropriate 'housekeeping' genes for use in expression profiling the effects of environmental estrogens in fish.
BACKGROUND: Attempts to develop a mechanistic understanding of the effects of environmental estrogens on fish are increasingly conducted at the level of gene expression. Appropriate application of real-time PCR in such studies requires the use of a stably expressed 'housekeeping' gene as an internal control to normalize for differences in the amount of starting template between samples. RESULTS: We sought to identify appropriate genes for use as internal controls in experimental treatments with estrogen by analyzing the expression of eight functionally distinct 'housekeeping' genes (18S ribosomal RNA [18S rRNA], ribosomal protein l8 [rpl8], elongation factor 1 alpha [ef1a], glucose-6-phosphate dehydrogenase [g6pd], beta actin [bactin], glyceraldehyde-3-phosphate dehydrogenase [gapdh], hypoxanthine phosphoribosyltransferase 1 [hprt1], and tata box binding protein [tbp]) following exposure to the environmental estrogen, 17alpha-ethinylestradiol (EE2), in the fathead minnow (Pimephales promelas). Exposure to 10 ng/L EE2 for 21 days down-regulated the expression of ef1a, g6pd, bactin and gapdh in the liver, and bactin and gapdh in the gonad. Some of these effects were gender-specific, with bactin in the liver and gapdh in the gonad down-regulated by EE2 in males only. Furthermore, when ef1a, g6pd, bactin or gapdh were used for normalization, the hepatic expression of two genes of interest, vitellogenin (vtg) and cytochrome P450 1A (cyp1a) following exposure to EE2 was overestimated. CONCLUSION: Based on the data presented, we recommend 18S rRNA, rpl8, hprt1 and/or tbp, but not ef1a, g6pd, bactin and/or gapdh, as likely appropriate internal controls in real-time PCR studies of estrogens effects in fish. Our studies show that pre-validation of control genes considering the scope and nature of the experiments to be performed, including both gender and tissue type, is critical for accurate assessments of the effects of environmental estrogens on gene expression in fish.
Abstract.
Author URL.
Benstead, R. Hutchinson, T.H. Tyler, C.R. (2007). Associations between altered vitellogenin concentrations and adverse health effects in fathead minnow (Pimephales promelas. Aquatic Toxicology
Trudeau V, Tyler C (2007). Endocrine disruption.
Gen Comp Endocrinol,
153(1-3), 13-14.
Author URL.
Schulz RW, Bogerd J, Male R, Ball J, Fenske M, Olsen LC, Tyler CR (2007). Estrogen-induced alterations in amh and dmrt1 expression signal for disruption in male sexual development in the zebrafish.
Environmental Science and Technology,
41(17), 6305-6310.
Abstract:
Estrogen-induced alterations in amh and dmrt1 expression signal for disruption in male sexual development in the zebrafish
Dmrt1 and amh are genes involved in vertebrate sex differentiation. In this study, we cloned dmrt1 and amh cDNAs in zebrafish (Danio rerio) and investigated the effects of exposure to 17α-ethinylestradiol (EE 2), during early life on their patterns of expression and impact on the subsequent gonadal phenotype. Expression of both amh and dmrt1 in embryos was detected as early as at 1 day post fertilization (dpf) and enhanced expression of amh from 25 dpf was associated with the period of early gonadal differentiation. Sex-dependent differences in enhanced green fluorescent protein transgene expression driven by the promoter of the germ cell-specific vas gene were exploited to show that at 28dpf and 56dpf both amh and dmrt1 mRNA were overexpressed in males compared with females. Exposure during early life to environmentally relevant concentrations of EE2 had a suppressive effect on the expression of both amh and dmrt1 mRNAs and this was associated with a cessation/retardation in male gonadal sex development. Our findings indicate that estrogen-induced suppression in expression of dmrt1 and amh during early life correlate with subsequent disruptive effects on the sexual phenotype in males. © 2007 American Chemical Society.
Abstract.
Bickley L, Lange A, Tyler C, Winter M (2007). Fish hepatocyte cultures as an alternative to in vivo tests for screening oestrogen receptor active chemicals.
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY,
146(4), S72-S72.
Author URL.
Jobling, S. (2007). Fish, Sex and Gender-Bending Chemicals – the Feminization of Fish Living in English Rivers. Biosciences
Lange, A. Ichikawa, R. Urushitani, H.G.C. (2007). Functional associations between two estrogen receptors, environmental oestrogens, and sexual disruption in the roach (<em>Rutilus rutilus</em>). Environmental Science and Technology, 41(9), 3368-3374.
Thorpe, K.L. Maack, G. Tyler, C.R. (2007). Gene expression profiles revealing the mechanisms of anti-androgen- and oestrogen- induced feminisation in fish. Aquatic Toxicology, 81(2), 219-231.
Santos, E.M. Thorpe, K.L. Maack, G. (2007). Gene expression profiling for understanding chemical causation of biological effects of complex mixtures: a Case Study for Estrogens. Environmental Science and Technology
Santos, E.M. Paull, G.C. Van Look, K.J.W. Workman, V.L. Holt, W.V. van Aerle, R. Kille, P. & Tyler, C.R (2007). Gonadal transcriptome responses and physiological consequences of exposure to oestrogen in breeding zebrafish (Danio rerio). Aquatic Toxicology, 83(2), 134-142.
Tyler CR, Filby AL, Galloway TS, Neuparthe T (2007). Health impacts of estrogens in the environment, considering complex mixture effects.
Full text.
Tyler, C.R. (2007). Identification of microsatellite loci for parentage analysis in roach Rutilus rutilus and 8 other cyprinids by cross-species amplification, and a novel test for detecting hybrids between roach and other cyprinids. Molecular Ecology Notes
Workman, V.L. Paull, G.C. Filby, A.L. (2007). Molecular basis of sex and reproductive status in breeding zebrafish. Physiological Genomics, 30(2), 111-122.
Boegard, J. Male, R. Ball, J. (2007). Oestrogen-induced alterations in the expression of AMH and DMRT-1 during sexual differentiation signal for impacts on male sexual development in the zebrafish (<em>Danio rerio</em>). Environmental Science and Technology, 41(17), 6305-6310.
Tyler C, Lange A, Santos E, Filby A (2007). The application of transcriptomics and other molecular approaches in advancing our understanding of endocrine disruption.
TOXICOLOGY LETTERS,
172, S27-S27.
Author URL.
Katsu, Y. Kato, Y. Tooi, O. (2007). The medaka (Oryyzias latipes) for assessing developmental effects of endocrine disrupting chemicals with special reference to gonadal intersex (testis-ova. Environmental Science
Tyler CR, Lange A, Paull GC, Katsu Y, Iguchi T (2007). The roach (Rutilus rutilus) as a sentinel for assessing endocrine disruption.
Environ Sci,
14(5), 235-253.
Abstract:
The roach (Rutilus rutilus) as a sentinel for assessing endocrine disruption.
Alterations in development and reproduction as a consequence of exposure to endocrine-disrupting chemicals (EDCs) have been demonstrated in many wildlife species. Animals living in, or closely associated with, the aquatic environment are particularly vulnerable to endocrine disruption because thousands of chemicals are actively disposed into rivers, estuaries and seas. Fish have thus been a focus in endocrine disruption studies, and some of the most comprehensive studies on the disruption of sexual development and function are on the roach (Rutilus rutilus). This paper provides a critical analysis of the roach as a sentinel for studies into endocrine disruption. The paper starts by describing what is known on the basic reproductive biology of the roach, information essential for interpreting chemical effect measures on sexual development and function. We then analyze where and how the roach has been applied to improve our understanding of the estrogenic nature of discharges from wastewater treatment works (WWTWs) and describe the phenomenon of feminized male roach in UK rivers. In this paper, the causation of these effects and issues of relative susceptibility and sensitivity of the roach to the effects of estrogenic EDCs are addressed. The paper then describes the ongoing work on the development of genetic and genomic resources for roach and analyses how these are being applied in studies to understand the mechanisms of disruption of sexual development. Finally, the paper addresses the biological significance of sexual disruption and intersex for the individual and discusses the possible implications for wild populations.
Abstract.
Author URL.
2006
Hutchinson, T.H. Hurd, K.S. Bamforth, J. (2006). Development of chronic tests for endocrine active chemicals. Part I. An extended fish early- life stage test for oestrogen active chemicals in the fathead minnow <em>Pimephales promelas</em>. Aquatic Toxicology, 77, 279-290.
Kennedy, S. Snape, J. Tillet, D. (2006). An Evaluation of the Potential Role of Genomics in Regulatory Ecotoxicology. Environmental Science and Technology, 40(13), 4055-4065.
Gross-Sorokin, M. Johnson, I. Brighty, G. (2006). An assessment of the model of concentration addition for predicting the estrogenic activity of chemical mixtures in wastewater treatment works effluent. Environmental Health Perspectives, 114, 90-97.
Schulte-Oehlmann, U, Albanis T, Allera A, Bachmann J, Berntsson P, Beresford N, Carnevali DC, Ciceri F, Dagnac T, Falandysz J, et al (2006). COMPRENDO: Focus and approach. Environmental Health Perspectives, 114(1), 98-100.
Eidem JK, Kleivdal H, Kroll K, Denslow N, van Aerle R, Tyler C, Panter G, Hutchinson T, Goksoyr A (2006). Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin. Aquatic Toxicology, 78(2), 202-206.
Santos E, Paull G, Filby A, Tyler C, Van Look K, Holt W, Workman V, Kille P (2006). Gender and stage-specific transcriptomic profiles of gonads and brains in breeding zebrafish.
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY,
143(4), S101-S101.
Author URL.
tsgallo1, Hagger JA, Liney KE, Tyler CR (2006). Health effects in fish of long-term exposure to effluents from wastewater treatment works.
Environmental Health Perspectives,
114(S1), 81-89.
Full text.
Jobling S, Tyler CR (2006). Introduction: the ecological relevance of chemically induced endocrine disruption in wildlife.
ENVIRONMENTAL HEALTH PERSPECTIVES,
114, 7-8.
Author URL.
Thorpe, K.L. Tyler, C.R. (2006). Multiple molecular effect pathways for an environmental. oestrogen in fish. Journal of Endocrinology, 37, 121-134.
Jobling,S. Williams, R. Johnson, A. Taylor, A. Gross-Sorokin, M.. Nolan, M. Tyler, C.R.. van Aerle, R. Santos, E.M.& Brighty, G (2006). Predicted exposures to steroid oestrogens in UK Rivers correlate with widespread sexual disruption in. wild fish populations. Environmental Health Perspectives, 114, 29-32.
Ankley, G.T. Segner, H. Tyler, C.R. (2006). Screening and testing for endocrine. disruption in fish -. biomarkers as signposts not traffic lights in risk assessment. Environmental Health Perspectives, 114, 106-114.
Filby A, Thorpe K, Tyler C (2006). The developmental expression of three oestrogen receptor subtypes and their differential responses to oestrogen in fish.
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY,
143(4), S101-S101.
Author URL.
Tyler, C.R. (2006). The ecological relevance of chemically-induced endocrine disruption in wildlife. Environmental Health Perspectives, 114, 7-8.
Ankley GT, Daston GP, Degitz SJ, Denslow ND, Hoke RA, Kennedy SW, Miracle AL, Perkins EJ, Snape J, Tillitt DE, et al (2006). Toxicogenomics in regulatory ecotoxicology.
Environmental Science and Technology,
40(13), 4055-4065.
Abstract:
Toxicogenomics in regulatory ecotoxicology
Interest in the application of genomics to toxicology has grown rapidly since the term "toxicogenomics" was coined in 1999. The authors explore the roles of toxicogenomics in the field of regulatory ecotoxicology, the current limitations in the science and practice of genomics, and possible avenues to approach and resolve some of the major challenges. © 2006 American Chemical Society.
Abstract.
2005
Tyler CR, Spary C, Gibson R, Santos EM, Shears J, Hill EM (2005). Accounting for differences in estrogenic responses in rainbow trout (Oncorhynchus mykiss: Salmonidae) and roach (Rutilus rutilus: Cyprinidae) exposed to effluents from wastewater treatment works.
Environ Sci Technol,
39(8), 2599-2607.
Abstract:
Accounting for differences in estrogenic responses in rainbow trout (Oncorhynchus mykiss: Salmonidae) and roach (Rutilus rutilus: Cyprinidae) exposed to effluents from wastewater treatment works.
Effluents from wastewater treatment works (WwTWs) contain estrogenic substances that induce feminizing effects in fish, including vitellogenin (VTG) synthesis and gonadal intersex. Fish vary in their responsiveness to estrogenic effluents, but the physiological basis for these differences are not known. In this study, uptake of estrogen from two WwTW effluents (measured in hydrolyzed bile) and estrogenic response (VTG induction) were compared in a salmonid (rainbow trout, Onchorhynchus mykiss) and a cyprinid fish (roach, Rutilus rutilus). Immature rainbow trout were more responsive than maturing roach to the estrogenic effluents. The more potent of the two estrogenic effluents (containing between 24.3 and 104.1 ng estradiol-17beta equivalents/L [E2eq/L]) resulted in a 700-fold and 240-fold induction of plasma VTG in male and female trout, respectively, but only a 4-fold induction in roach (and in males only). The less potent effluent (varying between 4.1 and 6.8 ng E2eq/L) induced VTG in the trout only, with a 4-fold and 18-fold induction in males and females, respectively. In fish exposed to tap water, the estrogenicity of the hydrolyzed bile was 0.03+/-0.01 ng E2eq/microL (for both sexes in trout), 0.18+/-0.04 ng E2eq/microL in male roach, and 0.88+/-0.15 ng E2eq/microL in female roach. The higher bile content of estrogen in control roach reflected their more advanced sexual status (and thus higher endogenous estrogen) compared with the immature female trout. In trout maintained in effluents, the bile content of estrogen was 100-fold and 30-fold higher than controls at WwTW a and B, respectively. Bioconcentration factors (BCFs) for estrogenic activity in bile were between 16 344 and 46 134 in trout and between 3543 and 60 192 in roach (no gender differences were apparent). There were strong correlations between VTG induction and the estrogenic activity of bile extracts for both trout and roach. The results confirm that estrogenic contaminants bioconcentrate to a high degree in fish bile and that the level (and nature) of this accumulation may accountfor responsiveness to the endocrine disruptive effects of estrogenic effluents. Immature fish were the more appropriate life stage for quantifying estrogen exposure and uptake in bile, as they contain little circulating endogenous oestrogen compared with sexual maturing fish. The nature of the estrogenic contaminants is detailed in an accompanying paper.
Abstract.
Author URL.
Tyler, C.R. Hill, E.M. (2005). An analytical methodology for the identification of estrogenic contaminants in fish bile. Journal of Chromatography A, 1066, 33-40.
Jobling, S. Shears, J. Simpson, P. (2005). Assessing the sensitivity of different life stages. for sexual disruption in roach (<em>Rutilus rutilus</em>) exposed to effluents from wastewater treatment works. Environmental Health Perspectives, 113(10), 1299-1307.
Fenske, M. Verslycke, T. Tyler, C.R. (2005). Development of a quantitative enzyme- linked immunosorbent assay for vitellin and its application in a preliminary study on vitellogenesis in the mysid, Neomysis intege. Comparative Biochemistry and Physiology Part B
Ghekiere A, Fenske M, Verslycke T, Tyler C, Janssen C (2005). Development of a quantitative enzyme-linked immunosorbent assay for vitellin in the mysid Neomysis integer (Crustacea: Mysidacea).
Comp Biochem Physiol a Mol Integr Physiol,
142(1), 43-49.
Abstract:
Development of a quantitative enzyme-linked immunosorbent assay for vitellin in the mysid Neomysis integer (Crustacea: Mysidacea).
Mysid crustaceans have been put forward by several regulatory bodies as suitable test organisms to screen and test the potential effects of environmental endocrine disruptors. Despite the well-established use of mysid reproductive endpoints such as fecundity, egg development time, and time to first brood release in standard toxicity testing, little information exists on the hormonal regulation of these processes. Control of vitellogenesis is being studied intensively because yolk is an excellent model for studying mechanisms of hormonal control, and vitellogenesis can be chemically disrupted. Yolk protein or vitellin is a major source of nourishment during embryonic development of ovigorous egg-laying invertebrates. The accumulation of vitellin during oocyte development is vital for the production of viable offspring. In this context, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for vitellin of the estuarine mysid Neomysis integer. Mysid vitellin was isolated using gel filtration, and the purified vitellin was used to raise polyclonal antibodies. The ELISA was sensitive within a working range of 4 to 500 ng vitellin/mL. Serial dilutions of whole body homogenates from female N. integer and the vitellin standard showed parallel binding curves, validating the specificity of the ELISA. The intra- and interassay coefficients of variation were 8.2% and 13.8%, respectively. Mysid vitellin concentrations were determined from ovigorous females and eggs at different developmental stages. The availability of a quantitative mysid vitellin ELISA should stimulate further studies on the basic biology of this process in mysids. Furthermore, it could provide a means to better understand and predict chemically induced reproductive effects in mysids.
Abstract.
Author URL.
Shears, J. van Aerle, R. (2005). Endocrine (sexual) disruption is not a prominent feature in the pike (Esox lucius), a top predator, living in English waters. Environmental Toxicology and Chemistry, 24(6), 1436-1443.
Smith, M.D. Spary, C. Tyler, C.R. (2005). Mixtures of oestrogenic contaminants in bile in fish exposed to wastewater treatment works effluents. Environmental Science and Technology, 39(8), 2461-2471.
Tyler, C.R. (2005). Molecular characterization of estrogen receptors 1, 2a and 2b and their tissue and ontogenic expression profiles in fathead minnow (<em>Pimephales promelas</em>). Biology of Reproduction, 73, 648-662.
Williams, R. Johnson, A. (2005). Predicted exposures to steroid estrogens in UK Rivers correlate with widespread sexual disruption in wild fish populations. Environmental Health Perspectives, 32-39.
2004
Sauer, A. Shears, J. Tyler, C.R. (2004). Androgenic and oestrogenic effects of the synthetic androgen, 17-methyltestosterone on sexual development and reproductive performance in the fathead minnow (<em>Pimephales promelas</em>) determined using a gonadal recrudescence assay. Aquatic Toxicology, 68(3), 277-291.
Jobling S, Casey D, Rodgers-Gray T, Oehlmann J, Schulte-Oehlmann U, Pawlowski S, Baunbeck T, Turner AP, Tyler CR (2004). Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent (vol 65, pg 205, 2003).
AQUATIC TOXICOLOGY,
66(2), 205-+.
Author URL.
Jobling S, Casey D, Rogers-Gray T, Oehlmann J, Schulte-Oehlmann U, Pawlowski S, Baunbeck T, Turner AP, Tyler CR (2004). Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent.
Aquat Toxicol,
66(2), 207-222.
Abstract:
Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent.
It is now well established that there is a diverse array of chemical discharged into the environment that can mimic or antagonise the action of hormones. These endocrine-disrupting chemicals (EDCs) can thus interact with physiological systems and cause alterations in development, growth and reproduction in wildlife that are exposed to them. As yet, however, there is little information on the relative sensitivities of different wild life groups to these chemicals and/or mixtures of them (e.g. estrogenic effluents) and hence, there are fundamental shortfalls in our knowledge of the ecological chemicals (17alpha-ethinylestradiol; EE2, bisphenol-A, and 4-tert octylphenol) and a mixture containing these chemicals (treated sewage effluent) on embryo production in the prosobranch mollusc, Potamopyrgus antipodarum, were studied and compared with the effects of EE2 and the same estrogenic effluent on vitellogenin induction and/or egg production in various species of freshwater fish (fathead minnow; Pimaphales promelas, rainbow trout (Oncorhynchus mykiss); Cyprinus carpio, carp; Cyprinus carpio). The lab-based studies demonstrated that all of the tested chemicals (known to be estrogenic and to cause reproductive effects in fish) also affected embryo production in P. antipodarum. Furthermore, exposure to EE2 induced similar reproductive responses in the snails as in the fathead minnow (Pimephales promelas), stimulating egg/embryo production at low doses (up to 1 ng/l in the minnow and 25 ng/l in the snail) and causing inhibitory effects at higher doses. A similar pattern of embryo production occurred in P. antipodarum when it was exposed to a graded concentration of treated sewage effluent containing mixtures of estrogenic EDCs and hence, the total number of new embryos produced by the snails increased steadily over the 9 week exposure period in treated snails. Plasma vitellogenin concentrations in two species of male fish (the rainbow trout and the carp) also increased over the same time period. These data indicate that both the nature of the response and the relative sensitivities to environmental estrogens in P. antipodarum and three different fish species fish are comparable. P. andtipodarum is thus, potentially a sensitive test organism for assessing estrogenicity of chemicals with a relevance to their activity in vertebrates.
Abstract.
Author URL.
Tyler CR, van Aerle R, Santos EM (2004). ELISAs for detecting vitellogenin in the fathead minnow (Pimephales promelas)-a critical analysis. Response to Mylchreest et al. Comp Biochem Physiol C 134: 251-257, 2003.
Comp Biochem Physiol C Toxicol Pharmacol,
138(4), 531-532.
Author URL.
van Aerle, R. Tyler, C.R. (2004). Effects of 17 alpha-ethinylestradiol in a fathead minnow (Pimephales promelas) gonadal recrudescence assay. Ecotoxicology and Environmental Safety, 57(3), 330-345.
Spano, L. Tyler, C.R. van Aerle R, Devos, P. Mandiki SNM, Silvestre F, Thome JP, Kestemont P (2004). Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish (Carassius auratus). Aquatic Toxicology, 66(4), 369-379.
Jobling S, Casey C, Rodgers-Gray T, Oehlmannd J, Schulte-Oehlmann U, Pawlowski S, Baunbeck T, Turner AP, Tyler CR (2004). Erratum: Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent (Aquatic Toxicology (2003) 65 (205-220)). Aquatic Toxicology, 66(2), 205-222.
Thorpe KL, Tyler CR (2004). Estrogenic effects of the alkylphenol ethoxylates andtheir biodegradation products. In (Ed)
Handbook of Detergents, Part B: Environmental Impact, 447-466.
Abstract:
Estrogenic effects of the alkylphenol ethoxylates andtheir biodegradation products
Abstract.
Tyler, C.R. Palazzi, X. Laillet, B. (2004). Impacts of. 17- estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-juvenile and adult-life stages in zebrafish (<em>Danio rerio</em>). Aquatic Toxicology, 68(3), 193-217.
Nash, J.P. Kime, D.E. Van der Ven, L.T.M. (2004). Long-term exposure to environmental concentrations of the pharmaceutical ethinyloestradiol cause \r
reproductive failure in fish.
Environmental Health Perspectives,
112(17), 1725-1733.
Full text.
van Aerle, R. Runnalls, T. & Tyler, C.R (2004). Ontogeny of gonadal sex development relative to growth in. fathead minnow (<em>Pimephales promelas</em>). Journal of Fish Biology, 64, 355-369.
van Aerle, R, Runnalls, T.J. Tyler, C.R. (2004). Ontogeny of gonadal sex development relative to growth in fathead minnow. Journal of Fish Biology, 64(2), 355-369.
Benstead, R. Hutchinson, T.H. Cummings, R.I. (2004). Reproductive effects of. exposure to oestrone in the fathead minnow. Fish Physiology and Biochemistry, 28, 451-452.
Hutchinson, T.H. Hurd, K.S. Sherren, A. (2004). Successful detection of (anti-) androgenic and aromatase inhibitors in pre-spawning fathead minnows (<em>Pimephales promelas</em>) using easily measured endpoints of sexual development. Aquatic Toxicology, 70, 11-21.
2003
Kwon, J. Rand-Weaver, M. Sumpter, J.P. (2003). Cloning and gene expression of 17,20- lyase cDNA in the gonads and brain of the fathead minnow (<em>Pimephales promelas</em>). General & Comparative Endocrinology, 130, 256-266.
Casey, D. Rodgers-Gray, T. Oehlmann, J. (2003). Comparative responses of molluscs and fish to environmental oestrogens and an oestrogenic effluent. Aquatic Toxicology, 65, 205-220.
Thomas-Jones E, Thorpe K, Harrison N, Thomas G, Morris C, Hutchinson T, Woodhead S, Tyler C (2003). Dynamics of estrogen biomarker responses in rainbow trout exposed to 17beta-estradiol and 17alpha-ethinylestradiol.
Environ Toxicol Chem,
22(12), 3001-3008.
Abstract:
Dynamics of estrogen biomarker responses in rainbow trout exposed to 17beta-estradiol and 17alpha-ethinylestradiol.
We have investigated the response dynamics of the estrogen-dependent genes vitellogenin (VTG) and the vitelline envelope proteins (VEPs) as well as circulating VTG in immature female rainbow trout (Oncorhynchus mykiss) exposed to 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) for periods of 7 and 14 d. Gene responses were quantified by measurement of messenger RNA (mRNA) in liver extracts using a chemiluminescent hybridization protection assay. Circulating VTG was measured by a homologous enzyme-linked immunosorbent assay. Exposure to both E2 and EE2 induced concentration-dependent increases in all biomarkers. The data presented indicate that VEP genes may be more sensitive to estrogens than the VTG gene. The biomarker lowest-observed-effect concentrations (biomarkerLOEC) in the 14-d study with E2 were 14 ng/L (VTG protein, VTG mRNA, VEPbeta, and VEPgamma) or 4.8 ng/L (VEPalpha). The EE2 was 5- to 66-fold more potent depending on the biomarker studied. In the 7-d study, all biomarkers were elevated after 48-h exposure to E2, with biomarkerLOECs of 30 ng/L (VTG protein, VTG mRNA, and VEPgamma) or 9.7 ng/L (VEPalpha and VEPbeta). Vitellogenin mRNA was induced up to 1,000-fold above baseline, and this translated into an increase of approximately 50,000-fold in circulating VTG. In conclusion, all biomarkers responded to estrogen exposure at environmentally relevant concentrations.
Abstract.
Author URL.
Tyler, C.R. (2003). Endocrine Disruption in Wild Freshwater Fish. Pure and Applied Chemistry, 75(11-12), 2219-2234.
Jobling S, Tyler CR (2003). Endocrine disruption, parasites and pollutants in wild freshwater fish.
Abstract:
Endocrine disruption, parasites and pollutants in wild freshwater fish.
Abstract.
Author URL.
Tyler, C.R. (2003). Impacts of Endocrine Disruption, Parasites and Pollution in Freshwater Fish. Journal of Parasitology, 126, 103-108.
Segner, H. Tyler, C.R. (2003). Ontogeny of sexual differentiation in different strains of zebrafish (<em>Danio rerio</em>). Fish Physiology and Biochemistry, 28, 125-128.
Tyler CR, Hetheridge MJ, Scholze M, Thorpe K (2003). Relative potencies and combination effects of steroidal oestrogens in fish. Environmental Science & Technology, 37(6), 1142-1149.
Pickford, K.A. Wheale, B.Thomas,-Jones, E. Tyler, C.R. & Sumpter, J.P (2003). Route of exposure affects the oestrogenic responses of fish to 4-tert nonylphenol. Aquatic Toxicology, 65, 267-279.
2002
Tyler CR, Beresford N, Jobling S, Nolan M (2002). Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents. Biology of Reproduction, 66(2), 272-281.
Kirk LA, Tyler CR, Lye CM, Sumpter JP (2002). Changes in estrogenic and androgenic activities at different stages of treatment in wastewater treatment works.
Abstract:
Changes in estrogenic and androgenic activities at different stages of treatment in wastewater treatment works.
Abstract.
Author URL.
Lye, C. Tyler, C.R. Sumpter, J.P. (2002). Effects of treatment processes on the endocrine activity of various influents as they pass through waster water treatment works\r. Environmental Toxicology and Chemistry, 21(5), 972-979.
Tyler CR, Ball J, Maack G, Paull G, Santos EM (2002). Endocrine disruption in vertebrate wildlife - Today's story.
Author URL.
Rand-Weaver, M. Sumpter, J.P. Tyler, C.R. (2002). Exposure to exogenous oestradiol 17 disrupts P450 aromatase B mRNA expression in the brain and ovary of adult fathead minnow (<em>Pimephales promelas</em>).\r. Aquatic Toxicology, 60, 285-299.
Tyler CR, van Aerle R, Nilsen MV, Blackwell R, Maddix S, Nilsen BM, Berg K, Hutchinson TH, Goksøyr A (2002). Monoclonal antibody enzyme-linked immunosorbent assay to quantify vitellogenin for studies on environmental estrogens in the rainbow trout (Oncorhynchus mykiss).
Environ Toxicol Chem,
21(1), 47-54.
Abstract:
Monoclonal antibody enzyme-linked immunosorbent assay to quantify vitellogenin for studies on environmental estrogens in the rainbow trout (Oncorhynchus mykiss).
Vitellogenin (VTG) induction has proved to be a valuable biomarker for assessing exposure to environmental estrogens in fish. The widespread use of VTG in this regard has lead to the need for standardized assays to quantify VTG, and monoclonal antibodies have the potential to help accomplish this. A VTG enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody prepared against Atlantic salmon (Salmo salar) VTG (MAb BN-5) and its ability to quantify VTG in the rainbow trout (Oncorhynchus mykiss) compared with a rainbow trout vitellogenin (rt-VTG) ELISA that employed homologous polyclonal antibodies (PAb). In routine protocols, the working range of the homologous rt-PAb VTG ELISA was between 9 ng/ml and 70 ng/ml (80- 20% relative maximum binding [B/Bo]) with a 50% B/Bo of 25+/-0.9 ng/ml and inter- and intraassay variations at 50% B/Bo of 7% (n = 7) and 8% (n = 15), respectively. The working range of the MAb BN-5 VTG ELISA was between 60 ng/ml and 850 ng/ml (80-20% B/Bo) with a 50% B/Bo of 227+/-22 ng/ml and inter- and intraassay variations at 50% B/Bo of 5% (n = 10) and 9% (n = 12), respectively. In the routine protocols, detection limits for measurement of plasma VTG in rainbow trout (at 80% B/Bo; and given the requirement to dilute plasma to a minimum of 1:10 for the assays) were 90 ng/ml for the polyclonal rt-VTG assay and approximately 600 ng/ml in the monoclonal antibody assay. In juvenile female rainbow trout exposed to a series of doses of estradiol-17beta (E2) and 4-tert nonylphenol (4-NP), there were no differences in the vitellogenic responses measured in the PAb and MAb BN-5 VTG ELISAs. The monoclonal MAb BN-5 VTG ELISA is likely to be of considerable value for studies on environmental estrogens in juvenile female rainbow trout in standardized tests.
Abstract.
Author URL.
Tyler, C.R. Maddix, S. Karbe, L. (2002). Plasma biomarkers in fish provide evidence for endocrine modulation in the Elbe River, Germany. Environmental Science and Technology, 36(11), 2311-2321.
Campbell, P.M. Hutchinson, T.H. Kime, D.E. (2002). Utility of. a fathead. minnow juvenile fish screening assay for detecting. (anti-) oestrogenic substances. Environmental Toxicology and Chemistry, 21, 319-326.
Jobling S, Coey S, Whitmore JG, Kime DE, Van Look KJW, McAllister BG, Beresford N, Henshaw AC, Brighty G, Tyler CR, et al (2002). Wild intersex roach (Rutilus rutilus) have reduced fertility.
Biol Reprod,
67(2), 515-524.
Abstract:
Wild intersex roach (Rutilus rutilus) have reduced fertility.
Endocrine-disrupting chemicals, known to be present in the environment, have great potential for interfering with reproductive health in wildlife and humans. There is, however, little direct evidence that endocrine disruption has adversely affected fertility in any organism. In freshwater and estuarine fish species, for example, although a widespread incidence of intersex has been reported, it is not yet known if intersexuality influences reproductive success. The purpose of this study was, therefore, to determine gamete quality in wild intersex roach (Rutilus rutilus) by assessing sperm characteristics, fertilization success, and ability to produce viable offspring. The results clearly demonstrate that gamete production is reduced in intersex roach. A significantly lower proportion of moderately or severely feminized fish (17.4% and 33.3%, respectively) were able to release milt compared with normal male fish from contaminated rivers (in which 97.6% of the males were able to release milt), reference male fish (97.7%), or less severely feminized intersex fish (experiment 1: 85.8%, experiment 2: 97%). Intersex fish that did produce milt produced up to 50% less (in terms of volume per gram of testis weight) than did histologically normal male fish. Moreover, sperm motility (percentage of motile sperm and curvilinear velocity) and the ability of sperm to successfully fertilize eggs and produce viable offspring were all reduced in intersex fish compared with normal male fish. Male gamete quality (assessed using sperm motility, sperm density, and fertilization success) was negatively correlated with the degree of feminization in intersex fish (r = -0.603; P < 0.001) and was markedly reduced in severely feminized intersex fish by as much as 50% in terms of motility and 75% in terms of fertilization success when compared with either less severely feminized intersex fish or unaffected male fish. This is the first evidence documenting a relationship between the morphological effects (e.g. intersex) of endocrine disruption and the reproductive capabilities of any wild vertebrate. The results suggest that mixtures of endocrine-disrupting substances discharged into the aquatic environment could pose a threat to male reproductive health.
Abstract.
Author URL.
van Aerle R, Pounds N, Hutchinson TH, Maddix S, Tyler CR (2002). Window of sensitivity for the estrogenic effects of ethinylestradiol in early life-stages of fathead minnow, Pimephales promelas.
Ecotoxicology,
11(6), 423-434.
Abstract:
Window of sensitivity for the estrogenic effects of ethinylestradiol in early life-stages of fathead minnow, Pimephales promelas.
Sexual differentiation in fish occurs after hatching during early life-stages and is believed to be a time when the gonad has a heightened sensitivity to disruption by chemicals that mimic hormones. In this study fathead minnows (Pimephales promelas) were exposed to an environmentally relevant concentration of ethinylestradiol (EE2) for short intervals in fish early life-stages and vitellogenic and gonadal responses were measured at 30 and 100 dph (sexual maturity), respectively. All EE2 exposure regimes induced vitellogenin (VTG) synthesis and disruption in duct development (a feminization) in males, with a window of enhanced sensitivity between 10 and 15 dph (where 60% of the males had feminized ducts). There was an altered pattern in sex cell development in males (inhibition of spermatogenesis) in the solvent controls (ethanol 0.1 ml/l) and all EE2 treatments when compared with the dilution water controls. Furthermore, fewer spermatozoa were observed in the testis of males exposed to EE2 from 15 to 20 dph and fertilized eggs (
Abstract.
Author URL.
van Aerle, R.. Pounds, N. Maddix, S. Hutchinson, T.H. and Tyler, C.R. (2002). Windows of sensitivity for the oestrogenic effects of an environmentally relevant concentration of ethinyloestradiol\r
in the fathead minnow (<em>Pimephales promelas</em>). \r. Ecotoxicology, 11, 423-434.
2001
Jobling, S. Sumpter, J.P. Brighty, G. (2001). A histological. description of intersexuality in the roach. Journal of Fish Biology, 58, 160-176.
Tyler CR, Hetheridge M, Scholze M, Thorpe K (2001). Assessing the Biological Potency of Binary Mixtures of Environmental Estrogens using Vitellogenin Induction in Juvenile Rainbow Trout (Oncorhynchus mykiss) . Environmental Science & Technology, 35(12), 2476-2481.
Rand-Weaver, M. Sumpter, J.P. Tyler, C.R. (2001). Cloning and molecular characterisation of an ovarian-derived (brain-like) P450-aromatase. and development. of a. competitive rtPCR bioassay to quantify its expression in the fathead minnow (Pimephales promelas ). Fish Physiology and Biochemistry, 24, 49-62.
van Aerle, R. Brack, S. (2001). Development and validation of a homologous zebrafish (Danio rerio Hamilton-Buchanan) vitellogenin enzyme-linked immunosorbent assay (ELISA) and its application for studies on estrogenic chemicals. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology, 129(3), 217-232.
Rand-Weaver, M. Tyler, C.R. (2001). Development and validation of radioimmunoassays for follicle stimulating hormone and its alpha and beta sub-units and their seasonal plasma and pituitary concentrations in the rainbow trout (<em>Oncorhynchus mykiss</em>). Biology of Reproduction, 65, 288-294.
van Aerle, R. Jobling, S. Christiansen. L. Sumpter, J.P and Tyler, C.R. (2001). Evidence for sexual disruption in a second species of freshwater fish (<em>gudgeon, Gobio gobio</em>) living in UK rivers receiving sewage treatment works effluent. Environmental Toxicology and Chemistry, 20, 2841-2847.
Jobling, S. Kelly, C. (2001). Exposure of juvenile roach, <em>Rutilus rutilus</em>, to treated sewage effluent induces dose-dependent and persistent disruption in gonadal development. Environmental Science and Technology, 35(3), 462-470.
Santos, E. Pottinger, T.G. Tyler, C.R. (2001). Exposure to 4 tert –nonylphenol alters gonadotrophin dynamics. in the pituitary and. plasma of. female rainbow trout (Oncorhynchus mykiss ). Environmental Science and Technology, 35, 2909-2916.
Santos EM, Rand-Weaver M, Tyler CR (2001). Follicle-stimulating hormone and its alpha and beta subunits in rainbow trout (Oncorhynchus mykiss): purification, characterization, development of specific radioimmunoassays, and their seasonal plasma and pituitary concentrations in females.
Biol Reprod,
65(1), 288-294.
Abstract:
Follicle-stimulating hormone and its alpha and beta subunits in rainbow trout (Oncorhynchus mykiss): purification, characterization, development of specific radioimmunoassays, and their seasonal plasma and pituitary concentrations in females.
Gonad development in fish, as in mammals, is regulated by two gonadotropins (GTHs), FSH and LH. The function of LH in fish has been clearly established; however, the function(s) of FSH is less certain. The lack of specific and sensitive assays to quantify FSH and its alpha and beta subunits has hindered studies to assess physiological function. In this study, gel filtration chromatography, ion exchange chromatography, and HPLC were employed to purify FSH and its subunits from pituitary glands of rainbow trout (Oncorhynchus mykiss), and the identities of the isolates were confirmed by amino acid analysis. Polyclonal antibodies were raised against the free GTHalpha2 and free FSHbeta subunits to develop specific RIAs. The sensitivities of the intact FSH, GTHalpha2, and FSHbeta assays were 1 ng/ml, 0.2 ng/ml, and 0.1 ng/ml, respectively, and the cross-reaction of these molecules with each other and with intact LH in the heterologous assays was 90% throughout. Measurement of plasma and pituitary concentrations of intact FSH in female rainbow trout confirmed the established seasonal profiles. Concentrations of free GTHalpha2 subunit were elevated both in the plasma and in the pituitary in females at ovulation (maximum concentrations: 34.93 +/- 6.3 ng/ml in plasma; 37.63 +/- 5.79 microg/pituitary). In both the plasma and the pituitary, free FSHbeta subunit was present throughout the reproductive cycle but at very low concentrations when compared with both free GTHalpha2 and intact FSH. The presence of free GTHalpha2 subunit in the plasma similarly occurs in mammals, but its functional significance in fish has yet to be established.
Abstract.
Author URL.
Cavaco, J.E.B. Rebers, F.E.M. Vischer, H.F. (2001). Gonadotrophins, their receptors, and regulation of testicular functions in fish. Comparative Biochemistry and Physiology Part B, 129, 407-417.
Schulz RW, Vischer HF, Cavaco JEB, Santos EM, Tyler CR, Goos HJT, Bogerd J (2001). Gonadotropins, their receptors, and the regulation of testicular functions in fish.
Author URL.
Prat, F. Randall, C. Tyler, C.R. (2001). Molecular characterisation of putative yolk processing enzymes and their expression during oogenesis and embryo development in the rainbow trout, <em>Oncorhynchus mykiss</em>. Biology of Reproduction, 65, 1701-1709.
Tyler, C.R. van Aerle, R. Nilsen, M. Maddix, S. Nilsen,B. Hutchinson, T. and Goksoyr, A. (2001). Monoclonal antibody enzyme-linked immunosorbant assay to quantify vitellogeinin in the rainbow trout, <em>Oncorhynchus mykiss</em>, and its application for studies on environmental oestrogens. Environmental Toxicology and Chemistry, 21, 47-54.
Harris CA, Santos EM, Janbakhsh A, Pottinger TG, Tyler CR, Sumpter JP (2001). Nonylphenol affects gonadotropin levels in the pituitary gland and plasma of female rainbow trout.
Environ Sci Technol,
35(14), 2909-2916.
Abstract:
Nonylphenol affects gonadotropin levels in the pituitary gland and plasma of female rainbow trout.
Female rainbow trout (Oncorhynchus mykiss) were exposed to 4-nonylphenol (NP) at (mean measured) concentrations of 0.7, 8.3, and 85.6 micrograms/L for 18 weeks, during early ovarian development. Fish were sampled sublethally every six weeks, and terminal samples were taken at 18 weeks. NP induced an estrogenic effect (the synthesis of vitellogenin) at concentrations of 8.3 and 85.6 micrograms/L. An effect on gonadotropin synthesis and secretion was also observed. Plasma follicle stimulating hormone (FSH) levels and FSH gene expression in the pituitary were the most sensitive endpoints assessed, being reduced at the lowest dose employed (0.7 microgram NP/L). Pituitary gland luteinizing hormone (LH) content was significantly lower in fish exposed to 85.6 micrograms NP/L, and LH gene expression was suppressed in fish exposed to 8.3 and 85.6 micrograms NP/L. In contrast, plasma LH concentration increased in these fish, but by a very minor absolute amount, and returned to control levels by the final sampling time. Gonadal development ceased in the fish exposed to 85.6 micrograms NP/L, and steroidogenesis in these fish was also markedly inhibited. Although the mechanisms underlying these responses are unknown, this study demonstrates that NP has adverse effects on pituitary function that can result in inhibition of ovarian development.
Abstract.
Author URL.
Tyler, C.R. Toy, R. Caunter, J. (2001). Reproductive effects of long term exposure to Bisphenol a in the fathead minnow (Pimephales promelas ). Environmental Science and Technology, 35, 2917-2925.
Van Aerle R, Christiansen LB, Jobling S, Tyler CR (2001). Sexual disruption in a second species of wild cyprinid fish (the Gudgeon, Gobio gobio) in United Kingdom freshwaters. Environmental Toxicology and Chemistry, 20(12), 2841-2847.
2000
Runnalls, T. Harris, C. Hill, E. (2000). Development of a reproduction performance test for endocrine-disrupting chemicals in the fathead minnow (Pimephales promelas. Environmental Science and Technology, 34, 3003-3011.
Hutchinson, T.H. Hetheridge, M. Sumpter, J.P. (2000). Development of an in vivo. screening assay for estrogenic chemicals using juvenile rainbow trout, Oncorhynchus mykiss. Environmental Toxicology and Chemistry, 19, 2812-2820.
Brion, F. Palazzi, X. Babut, M. (2000). Estrogenic effects in chub (Leuciscus cephalus): Induction of vitellogenin and alterations in testicular structure. Ecotoxicology, 9, 127-135.
Flammarion P, Brion F, Babut M, Garric J, Migeon B, Noury P, Thybaud E, Palazzi X, Tyler CR (2000). Induction of fish vitellogenin and alterations in testicular structure: Preliminary results of estrogenic effects in chub (Leuciscus cephalus).
Abstract:
Induction of fish vitellogenin and alterations in testicular structure: Preliminary results of estrogenic effects in chub (Leuciscus cephalus)
Abstract.
Jobling, S. Morris, S. Kelly, C. (2000). Long-term temporal changes in the oestrogenic composition of treated sewage effluent and its biological effects in fish. Environmental Science and Technology, 34, 1521-1528.
Tyler, C.R. Beresford, N. van der Woning, M. Sumpter, J.P. & Thorpe, K (2000). Metabolism and. environmental degradation of pyrethroid. insecticides produce compounds with endocrine activities. Environmental Toxicology and Chemistry, 19, 801-809.
May, D. Rand-Weaver, M. Tyler, C.R. (2000). Molecular characterisation of the first non-mammalian p73 cDNA. Comparative Biochemistry and Physiology Part B, 126, 49-57.
1999
Tyler, C.R. van Aerle, R. Hutchinson, T. Maddix, S, & Trip, H (1999). An in vivo. testing system for endocrine disrupters in fish early life stages using induction of vitellogenin. Environmental Toxicology and Chemistry, 18(2), 337-347.
Perazzolo, L. Coward, K. Davail, B. Normand, E. Tyler, C.R. Pakdel, F. Schneider, W. & Le Menn, F (1999). Expression and localisation of messenger ribonucleic acid for the vitellogenin receptor in ovarian follicles throughout oogenesis in the rainbow trout, <em>Oncorhynchus mykiss</em>. Biology of Reproduction, 60, 1057-1068.
May, D. Rand-Weaver, M. Tyler, C.R. (1999). Fish p53 - a possible biomarker for genotoxins in the aquatic environment?. Environmental and Molecular Mutagenesis, 33, 144-151.
Bhaskaran A, May D, Rand-Weaver M, Tyler CR (1999). Fish p53 as a possible biomarker for genotoxins in the aquatic environment.
Environ Mol Mutagen,
33(3), 177-184.
Abstract:
Fish p53 as a possible biomarker for genotoxins in the aquatic environment.
The p53 gene is a tumour suppressor gene which has a fundamental role in cell cycle control and division, and in mammals certain genotoxic agents induce specific mutations in p53, leading to tumourigenesis. Fish have been investigated as models for studying carcinogens, but as yet very little data exists that links exposure to specific chemicals with the aetiology of tumours found in wild populations. In this study, p53 was sequenced from five species of fish with a view to the possible use of mutations in the highly conserved domains of p53 to identify genotoxins in the aquatic environment. A 0.8 kb fragment of the cDNA encompassing the conserved DNA-binding domain of p53 was sequenced in three Oncorhynchus salmonid fish: coho (O. kisutch), chum (O. keta), and chinook (O. tshawytscha) and full-length p53 cDNAs were sequenced in the puffer fish (Tetraodon miurus) and the barbel (Barbus barbus). The full-length puffer fish and barbel p53 cDNAs were 1834 bp and 1790 bp in length, encoding a 367 aa protein and a 369 aa protein, respectively. The deduced aa sequences of the p53 cDNA in the Oncorhynchus salmon shared a 100% identity in the five conserved regions (I-V). Comparisons of the deduced aa sequences for puffer fish and barbel p53 with other fish p53s revealed a high homology within the conserved DNA binding domain (68-86% for puffer fish and between 66-88% for barbel). "Conserved" domain I was not highly conserved in fish, as it is in mammals, and, therefore, conserved domains II-V are most likely to provide the valuable sequences in fish p53 for use in mutational studies to fingerprint genotoxins in the aquatic environment.
Abstract.
Author URL.
Jambakhsh, A. Jobling, S. Matthiessen, P. (1999). Oestrogenic potency of effluent from two sewage-treatment works in the United Kingdom. Environmental Toxicology and Chemistry, 18, 932-937.
1998
Jobling, S. Sumpter, J.P. (1998). Endocrine disruption in wildlife: a critical review of the. evidence\r. Critical Reviews in Toxicology, 28(4), 319-361.
Coward, K. Sumpter, J.P. Tyler, C.R. (1998). Molecular characterisation and expression of two ovarian lipoprotein receptors in the rainbow trout, <em>Oncorhynchus mykiss</em>. Biology of Reproduction, 58, 1146-1153.
Routledge, E. (1998). Oestrogenic effects in fish in English rivers with evidence for causation. Pure and Applied Chemistry, 70, 1795-1804.
Nolan, M. Tyler, C.R. Brighty, G. (1998). Widespread sexual disruption in wild fish. Environmental Science and Technology, 32, 2498-2506.
1997
Tyler, C.R. Sumpter, J.P. (1997). Egg quality in fish; what makes a good egg? \r. Reviews in Fish Biology and Fisheries, 7, 387-416.
Tyler, C.R. Carnevali, O. Coward, K. (1997). Molecular characterisation of ovarian cathepsin D in the rainbow trout, Oncorhynchus mykiss. Gene, 201, 45-54.
Brooks S, Tyler CR, Sumpter JP (1997). Quality in fish: what makes a good egg?.
REVIEWS IN FISH BIOLOGY AND FISHERIES,
7(4), 387-416.
Author URL.
Pottinger, T.G. Coward, K. Prat, F. (1997). Salmonid follicle stimulating hormone (GtH I) mediates. vitellogenic development of oocytes in the rainbow trout, Oncorhynchus mykiss. Biology of Reproduction, 57, 1238-1244.
1996
Nagler JJ, Scott AP, Tyler CR, Sumpter JP (1996). Gonadotropins I and II do not stimulate the in vitro secretion of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one 20-sulphate by rainbow trout gonads during final sexual maturation.
FISH PHYSIOLOGY AND BIOCHEMISTRY,
15(2), 149-156.
Author URL.
Lubberink, K. (1996). Identification of four ovarian receptor proteins that bind vitellogenin, but not other homologous plasma lipoproteins, in the rainbow trout, <em>Oncorhynchus mykiss</em>. Journal of Comp. Physiol.B, 166, 11-20.
Tyler, C.R. van der Eerden, Sumpter, J.P. Jobling, S. & Panter, G (1996). Measurement of vitellogenin, a biomarker for exposure to oestrogen, in a wide variety of cyprinids. Journal of Comp. Physiol.B, 166, 418-426.
Tyler CR, Pottinger TG, Santos E, Sumpter JP, Price SA, Brooks S, Nagler JJ (1996). Mechanisms controlling egg size and number in the rainbow trout, Oncorhynchus mykiss.
Biol Reprod,
54(1), 8-15.
Abstract:
Mechanisms controlling egg size and number in the rainbow trout, Oncorhynchus mykiss.
Female rainbow trout (Oncorhynchus mykiss) produce a single batch of eggs each year; synchronous growth of oocytes, all of which are ovulated at the same time, occurs in the two ovaries. To examine the regulatory mechanisms controlling egg size and number, virgin female rainbow trout were subjected to unilateral ovariectomy (ULO) during early vitellogenesis, and oocyte recruitment and growth in the remaining ovary were monitored. The study also set out to determine whether the presence of a second population of smaller oocytes in the maturing pool (induced by ULO) affected the timing of ovulation and/or the size of the eggs ovulated. Two months after ULO, there was no difference in the gonadosomatic index between ULO fish and controls. Compensatory ovarian hypertrophy resulted from the recruitment of a second population of primary oocytes into the vitellogenic pool. This population of smaller maturing oocytes in the ULO fish displayed growth rates up to twice those of the population of larger oocytes in the same ovary and of oocytes in controls. The growth rate of the population of larger oocytes in the ULO fish was not altered by the recruitment of a second maturing population. One month after ULO, fish had a lower concentration of plasma estradiol-17 beta than did controls; subsequently the concentrations of plasma estradiol-17 beta in the ULO and control groups were similar. After ULO, plasma levels of vitellogenin in the ULO fish did not differ from those in the control group throughout the study. At or close to ovulation, the fecundity of ULO fish was 75-80% that of controls. In the control group, oocytes appeared to reach a certain critical size before they were ovulated, and fish with higher fecundity ovulated later than their less fecund counterparts. ULO did not affect the timing of ovulation, and ULO fish ovulated eggs with a considerably greater size-range than did controls.
Abstract.
Author URL.
Sumpter, J.P. (1996). Oocyte growth and development in teleosts. Reviews in Fish Biology and Fisheries, 6, 287-318.
Sumpter, J.P. Tyler, C.R. (1996). Validation of radioimmunoassays for two salmon gonadotropins (gonadotropins I and II) and their plasma concentrations throughout the reproductive cycle in male and female rainbow trout, Oncorhynchus mykiss. Biology of Reproduction, 54, 1375-1382.
1994
Tyler, C.R. (1994). Developmental expression and modulation of the vitellogenin receptor in ovarian follicles of the rainbow trout, <em>Oncorhynchus mykiss</em>. Journal of Experimental Zoology, 269, 458-466.
Nagler, J.J. Pottinger, T.P. Turner, M. (1994). Effects of unilateral ovariectomy on recruitment and growth of follicles in the rainbow trout, <em>Oncorhynchus mykiss</em>. Fish Physiology and Biochemistry, 13, 309-316.
Purdom CE, Hardiman PA, Bye VJ, Eno NC, Tyler CR, Sumpter JP (1994). Estrogenic Effects of Effluents from Sewage Treatment Works.
Chemistry and Ecology,
8(4), 275-285.
Abstract:
Estrogenic Effects of Effluents from Sewage Treatment Works
The occurrence of hermaphrodite fish in the lagoons of sewage treatment works led us to hypothesize that sewage effluent might contain a substance, or substances, estrogenic to fish. To test this hypothesis, we placed cages containing rainbow trout in the effluent from sewage-treatment works, and one to three weeks later measured the vitellogenin concentration in the plasma of the fish. Vitellogenin is a protein synthesized by the liver of oviparous fish in response to estradiol stimulation; it is then conveyed by the blood to the ovary, where it is sequestered by oocytes to form the yolk. Thus, the presence of vitellogenin in the plasma is indicative of estrogenic stimulation of the liver. An initial study, at a sewage-treatment works, showed that plasma vitellogenin concentrations rose rapidly and very markedly (over 1000-fold in three weeks) when trout were maintained in the effluent. An extensive nationwide survey was then conducted. Results were obtained from fifteen sewage-treatment works distributed throughout England. In all cases, exposure of trout to effluent resulted in a very pronounced increase (500 to 100,000-fold, depending on site) in the plasma vitellogenin concentration. Induction of vitellogenesis was also observed in carp, but to a much lesser extent than in trout. The identity of the estrogenic substance is unknown. It is suggested that the two most likely possibilities are ethynylestradiol, originating from pharmaceutical use, or alkylphenoLethoxylates (APE), originating from the biodegradation of surfactants and detergents during sewage treatment. Laboratory studies on the potency of ethynylestradiol demonstrated that levels as low as 1 to 10 ng f' could generate the response shown by the caged fish and that positive responses may arise at 0.1 to 0.5 ng Ll. Further work is in progress on the potency of APE. © 1994, Taylor & Francis Group, LLC. All rights reserved.
Abstract.
Hardiman, P.A. Bye, V. Eno, N.C. (1994). Oestrogenic effects of effluent from sewage treatment works. Chemistry and Ecology, 8, 275-285.
Tyler, C.R. Sumpter, J.P. (1994). Ovarian follicles of rainbow trout (<em>Oncorhynchus mykiss</em>) cultured within lamellae survive well, and sequester and process vitellogenin. Journal of Experimental Zoology, 269, 45-52.
1993
Tyler, C.R. (1993). Electrophoretic patterns of yolk proteins throughout ovarian development and their relationship to vitellogenin in the rainbow trout, <em>Oncorhynchus mykiss</em>. Comparative Biochemistry and Physiology Part B, 106, 321-329.
Everett, S. (1993). Gross morphological disorders in barbel (<em>Barbus barbus</em>) in three rivers in southern England. Journal of Fish Biology, 43, 739-748.
Tyler CR, Everett S (1993). Incidences of gross morphological disorders in barbel (Barbus barbus) in three rivers in southern England.
Journal of Fish Biology,
43(5), 739-748.
Abstract:
Incidences of gross morphological disorders in barbel (Barbus barbus) in three rivers in southern England
Incidences of gross morphological disorders in barbel (Barbus barbus) were investigated in three rivers in southern England. The water quality at the sites chosen for study on the Rivers Teme, Kennet and Lee were classified (according to the criteria of the National Water Council, 1978) as 1A, IB and 2, respectively. Generally, low levels of gross morphological abnormalities were seen in fish caught in the Teme and Kennet, although there were incidences of ocular disorders and extra barbules in 10 and 7%, respectively, of the Kennet fish. No tumours were found in barbel from the River Teme whereas there was a 2% incidence in the Kennet fish. In contrast barbel from the River Lee showed high levels of all the gross morphological disorders, especially haemorrhagic patches on the ventral surface (36%) and ocular disorders (72%). Tumours were seen in 25% of the barbel caught in the River Lee. The incidences of most of these gross morphological disorders did not appear to be age related. The possibility that these disorders were related to water quality is discussed. Copyright © 1993, Wiley Blackwell. All rights reserved
Abstract.
Tyler CR, Lancaster P (1993). Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout, Oncorhynchus mykiss.
Journal of Comparative Physiology B,
163(3), 225-233.
Abstract:
Isolation and characterization of the receptor for vitellogenin from follicles of the rainbow trout, Oncorhynchus mykiss
The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (125iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (Kd=8.2·10-9 mol·1-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin. © 1993 Springer-Verlag.
Abstract.
1991
Tyler CR, Sumpter JP, Kawauchi H, Swanson P (1991). Involvement of gonadotropin in the uptake of vitellogenin into vitellogenic oocytes of the rainbow trout, Oncorhynchus mykiss.
Gen Comp Endocrinol,
84(2), 291-299.
Abstract:
Involvement of gonadotropin in the uptake of vitellogenin into vitellogenic oocytes of the rainbow trout, Oncorhynchus mykiss.
The effects of two fully characterized, structurally distinct gonadotropins, GtH I and GtH II, on the uptake of vitellogenin (VTG) into oocytes of the rainbow trout, Oncorhynchus mykiss, were investigated both in vivo and in vitro. GtH I, injected into maturing vitellogenic females at a dose of 10 micrograms.kg body wt-1, increased the rate of [3H]VTG uptake into oocytes by more than two-fold, effectively doubling their rate of growth. Ovaries from females similarly treated with GtH II sequestered VTG at rates similar to controls. In vitro, GtH I stimulated VTG uptake in a dose-dependent manner. At a GtH I concentration of 100 ng.ml-1 and above, the rate of VTG uptake was significantly greater than that of the controls and at 1000 ng.ml-1 the rate of uptake was more than doubled. GtH II did not significantly increase VTG sequestration into isolated oocytes at concentrations up to, and including, 1000 ng.ml-1. These data provide the first evidence that GtH I has a primary function in stimulating VTG uptake and strongly support the contention that at least two functionally distinct GtHs occur in fish.
Abstract.
Author URL.
Sumpter, J.P. Campbell, P.M. (1991). Uptake of vitellogenin into oocytes during early vitellogenic development in the rainbow trout, <em>Oncorhynchus mykiss</em> (Walbaum). Journal of Fish Biology, 38, 681-689.
1990
Tyler CR, Sumpter JP, Bromage NR (1990). An in vitro culture system for studying vitellogenin uptake into ovarian follicles of the rainbow trout, Salmo gairdneri.
Journal of Experimental Zoology,
255(2), 216-231.
Abstract:
An in vitro culture system for studying vitellogenin uptake into ovarian follicles of the rainbow trout, Salmo gairdneri
The development of a short‐term culture system to study vitellogenin (VTG) sequestration in vitro into vitellogenic follicles of the rainbow trout, Salmo gairdneri, is described. The outer epithelial cell layer of the trout follicle restricted VTG uptake considerably and its removal was a prerequisite to study VTG uptake in vitro. Vitellogenic follicles divested of the surface epithelium sequestered VTG at rates of up to 170 ng·mm2 follicle surface−1·h−1 (ng·mm−2·h−1). Uptake of VTG over an 8 h period of incubation was linear. Increasing the titre of VTG in the culture medium induced greater rates of sequestration in a dose‐dependent manner; follicles cultured in medium containing 45 mg VTG·ml−1 sequestered 170 ± 9ng·mm−2·h−1 whereas those follicles cultured in medium containing 2 mg·ml−1 only incorporated 6 ± 0.5 ng·mm−2·h−1. Uptake of VTG was temperature dependent with higher temperatures inducing higher levels of incorporation. Sequestration of VTG persisted at temperatures below 5°C, showing that the uptake mechanisms are adapted to low temperatures in this species. Size of follicles also had a considerable bearing on the rate of VTG sequestration; follicles of less than 1 mm diameter (pre‐vitellogenic follicles) did not sequester VTG in amounts that were detectable. All follicles measuring 1 mm or greater in diameter sequestered VTG. As the follicles increased in size between approximately 1 and 3 mm in diameter there was an increase in both the total amount of VTG sequestered and the rate of uptake of VTG relative to surface area. Limited data suggested that in follicles closely approaching ovulation the rate of VTG sequestration was considerably reduced. Copyright © 1990 Wiley‐Liss, Inc. a Wiley Company
Abstract.
