Thomas H. Hutchinson

Screening and Testing for Endocrine Disruption in Fish—Biomarkers As “Signposts,” Not “Traffic Lights,” in Risk Assessment

Biomarkers are currently best used as mechanistic “signposts” rather than as “traffic lights” in the environmental risk assessment of endocrine-disrupting chemicals (EDCs). In field studies, biomarkers of exposure [e.g., vitellogenin (VTG) induction in male fish] are powerful tools for tracking single substances and mixtures of concern. Biomarkers also provide linkage between field and laboratory data, thereby playing an important role in directing the need for and design of fish chronic tests for EDCs. It is the adverse effect end points (e.g., altered development, growth, and/or reproduction) from such tests that are most valuable for calculating adverseNOEC (no observed effect oncentration) or adverseEC10 (effective concentration for a 10% response) and subsequently deriving predicted no effect concentrations (PNECs). With current uncertainties, biomarkerNOEC or biomarkerEC10 data should not be used in isolation to derive PNECs. In the future, however, there may be scope to increasingly use biomarker data in environmental decision making, if plausible linkages can be made across levels of organization such that adverse outcomes might be envisaged relative to biomarker responses. For biomarkers to fulfil their potential, they should be mechanistically relevant and reproducible (as measured by interlaboratory comparisons of the same protocol). VTG is a good example of such a biomarker in that it provides an insight to the mode of action (estrogenicity) that is vital to fish reproductive health. Interlaboratory reproducibility data for VTG are also encouraging; recent comparisons (using the same immunoassay protocol) have provided coefficients of variation (CVs) of 38–55% (comparable to published CVs of 19–58% for fish survival and growth end points used in regulatory test guidelines). While concern over environmental xenoestrogens has led to the evaluation of reproductive biomarkers in fish, it must be remembered that many substances act via diverse mechanisms of action such that the environmental risk assessment for EDCs is a broad and complex issue. Also, biomarkers such as secondary sexual characteristics, gonadosomatic indices, plasma steroids, and gonadal histology have significant potential for guiding interspecies assessments of EDCs and designing fish chronic tests. To strengthen the utility of EDC biomarkers in fish, we need to establish a historical control database (also considering natural variability) to help differentiate between statistically detectable versus biologically significant responses. In conclusion, as research continues to develop a range of useful EDC biomarkers, environmental decision-making needs to move forward, and it is proposed that the “biomarkers as signposts” approach is a pragmatic way forward in the current risk assessment of EDCs.

Impacts of Climate Change on Indirect Human Exposure to Pathogens and Chemicals from Agriculture

Objective Climate change is likely to affect the nature of pathogens and chemicals in the environment and their fate and transport. Future risks of pathogens and chemicals could therefore be very different from those of today. In this review, we assess the implications of climate change for changes in human exposures to pathogens and chemicals in agricultural systems in the United Kingdom and discuss the subsequent effects on health impacts. Data sources In this review, we used expert input and considered literature on climate change; health effects resulting from exposure to pathogens and chemicals arising from agriculture; inputs of chemicals and pathogens to agricultural systems; and human exposure pathways for pathogens and chemicals in agricultural systems. Data synthesis We established the current evidence base for health effects of chemicals and pathogens in the agricultural environment; determined the potential implications of climate change on chemical and pathogen inputs in agricultural systems; and explored the effects of climate change on environmental transport and fate of different contaminant types. We combined these data to assess the implications of climate change in terms of indirect human exposure to pathogens and chemicals in agricultural systems. We then developed recommendations on future research and policy changes to manage any adverse increases in risks. Conclusions Overall, climate change is likely to increase human exposures to agricultural contaminants. The magnitude of the increases will be highly dependent on the contaminant type. Risks from many pathogens and particulate and particle-associated contaminants could increase significantly. These increases in exposure can, however, be managed for the most part through targeted research and policy changes.

Analysis of the ECETOC aquatic toxicity (EAT) database. II - Comparison of acute to chronic ratios for various aquatic organisms and chemical substances

Environmental risk assessment often requires prediction of potential chronic effects in aquatic species from acute toxicity data. The scientific rationale for using extrapolation factors for this continues to be debated. As a contribution to the debate this paper analyses the acute-chronic-ratios (ACRs) based on acute EC50s (or LC50s) and (sub)chronic NOEC values using information in the ECETOC Aquatic Toxicity (EAT) data base. The ACRs were calculated separately for all aquatic species available and the results presented according to specific substance classes. It was unusual to find more than three species giving ACRs for a single chemical but eight of the 28 species in the analysis gave ACRs for five or more chemicals. Based on the commonly used 90%-ile ACR (whose usefulness is underlined by the data presented here), 90%-ile ACRs were observed from 192 (for metals/organometals to 20 for ‘other inorganics’. For organic chemicals resembling those which may be submitted for registration under the European Community 7th Amendment Directive (92/32/EEC), the results were further analysed based on time-specific ACRs and gave the 90%-ile as 24.5: the maximum ACR observed for such organic substances was 28.3. ACRs for individual taxonomic groups (e.g. fish, daphnids) were also calculated. Except for three cases an ACR derived from studies on Pimephales promelas was within a factor of 2 of the ACRs of five other fish species. In contrast, the ACRs for Daphnia magna varied to a greater extent from the ACRs for other invertebrates. For a quarter of cases the ratios differed by 5.0 or more. The results are discussed with respect to current environmental risk assessment procedures.

Impact of natural and synthetic steroids on the survival, development and reproduction of marine copepods (Tisbe battagliai)

Abstract Given recent reports suggesting that natural and synthetic steroids (namely, oestradiol, oestrone and ethynylestradiol) may be present in sewage effluent at levels which may impact on fish, it is pertinent to extend the ecological hazard evaluation for such substances to aquatic invertebrates. Studies have therefore been undertaken to address whether 17β-oestradiol, oestrone and 17α-ethynylestradiol can inhibit survival, development or reproductive output in Tisbe battagliai (Crustacea, Copepoda, Harpacticoida). This sexually reproducing species was selected since it is representative of a widespread group of aquatic Crustacea, is sensitive to environmental contaminants and is highly amenable to laboratory life-cycle studies. Newly released ( T. battagliai nauplii were exposed to individual steroids dissolved in sea water (using the ecdysteroid, 20-hydroxyecdysone, as a positive control) and effects monitored in terms of survival, development and sex ratio after 10 days at 20±1°C. Adult males and females were then paired and exposures continued to investigate effects on reproductive output (21 days total exposure). In summary, the lowest 21 day No Observed Effect Concentrations based on these life-cycle parameters were: 20-hydroxyecdysone: 8.7 μg·l −1 ; oestrone: ≥100 μg·l −1 ; 17β-oestradiol: ≥100 μg·l −1 ; and 17α-ethynylestradiol: ≥100 μg·l −1 (all based on nominal concentrations). These data are relevant for the development of an ecological risk assessment for oestrogenic steroids in the aquatic environment and should be extended to other invertebrate groups. As novel analytical techniques allow, future bioassay studies should be ideally supported by steroid analyses wherever possible.

Toxicity of cadmium, hexavalent chromium and copper to marine fish larvae (Cyprinodon variegatus) and copepods (Tisbe battagliai)

Abstract For comparative purposes, the toxicity of cadmium, hexavalent chromium and copper to marine fish larvae (Cyprinodon variegatus) and copepods (Tisbe battagliai) has been evaluated. Toxicity to fish larvae was measured in terms of survival and growth over 7 days, whilst toxicity to copepods was assessed in terms of survival and reproduction after 8 days exposure. For fish larvae, 96 h LC50 values (based on mean measured concentrations of total metal ion) were 1.23 mg Cd/litre, 31.6 mg Cr6+/litre and >0.22 mg Cu/litre. Subchronic values (SChVs) for larval fish survival and growth after 7 days were 0.75 mg Cd/litre, 24.0 mg Cr6+/litre and 0.16 mg Cu/litre. For copepod nauplii and adults, 96 h LC50 values were as follows: 0.46 mg Cd/litre and 0.34 mg Cd/litre, respectively; 1.60 Cr6+/litre, and 5.9 mg Cr6+/litre respectively; and 0.064 mg Cu/litre and 0.088 mg Cu/litre, respectively. SChVs for naupliar survival and adult survival or reproduction after 8 days were 0.024 mg Cd/litre, 0.42 mg Cr6+/litre and 0.008 mg Cu/litre.

Effects of Ammonium Perchlorate on Thyroid Function in Developing Fathead Minnows, Pimephales promelas

Perchlorate is a known environmental contaminant, largely due to widespread military use as a propellant. Perchlorate acts pharmacologically as a competitive inhibitor of thyroidal iodide uptake in mammals, but the impacts of perchlorate contamination in aquatic ecosystems and, in particular, the effects on fish are unclear. Our studies aimed to investigate the effects of concentrations of ammonium perchlorate that can occur in the environment (1, 10, and 100 mg/L) on the development of fathead minnows, Pimephales promelas. For these studies, exposures started with embryos of < 24-hr postfertilization and were terminated after 28 days. Serial sectioning of thyroid follicles showed thyroid hyperplasia with increased follicular epithelial cell height and reduced colloid in all groups of fish that had been exposed to perchlorate for 28 days, compared with control fish. Whole-body thyroxine (T4) content (a measure of total circulating T4) in fish exposed to 100 mg/L perchlorate was elevated compared with the T4 content of control fish, but 3,5,3′-triiodothyronine (T3) content was not significantly affected in any exposure group. Despite the apparent regulation of T3, after 28 days of exposure to ammonium perchlorate, fish exposed to the two higher levels (10 and 100 mg/L) were developmentally retarded, with a lack of scales and poor pigmentation, and significantly lower wet weight and standard length than were control fish. Our study indicates that environmental levels of ammonium perchlorate affect thyroid function in fish and that in the early life stages these effects may be associated with developmental retardation.

Analysis of the ecetoc aquatic toxicity (EAT) database IV — Comparative toxicity of chemical substances to freshwater versus saltwater organisms

Abstract In contrast to the substantial quantity of information available on the ecotoxicity of chemical substances to freshwater organisms, there are relatively few data on the effects of many such substances to marine and estuarine organisms. While it is desirable to improve the ecotoxicity database for saltwater (viz. estuarine and marine) species, there is an immediate need to conduct risk assessments for those substances which may enter the marine environment, and in some cases, this requires the extrapolation of freshwater ecotoxicity data to saltwater species. In order to examine the rationale for this extrapolation, a comparison has been made of the sensitivities of related freshwater and saltwater fish and invertebrates to various substances, based on the ECETOC Aquatic Toxicity (EAT) database. In summary: (1) for fish, freshwater and saltwater species were within a factor of 10 in their sensitivity to 91% and 93% of all substances (based on EC50 and NOEC values, respectively); (2) for invertebrates, freshwater and saltwater species were within a factor of 10 in their sensitivity to 33% and 83% of all substances (based on EC50 and NOEC values, respectively). Given the limited number of data available, the preliminary nature of these conclusions is emphasised. Insufficient data were available to allow a similar comparison between freshwater and saltwater plants and algae. There is a need for more high quality data on the effects of a wide range of chemical substances to both freshwater and saltwater organisms, and especially algae and plant species.

Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna

Highlights ► Daphnia magna have a detectable activity towards ethoxyresorufin. ► This activity is unaffected by 24 h exposure to dimethylsulfoxide or methanol. ► EROD activity in zebrafish is inhibited by 24 h exposure to both DMSO and methanol. ► DMSO and methanol exposures (24 h) decreased expression of CYP and UGT genes in zebrafish larvae. ► A maximum solvent concentration of 0.01% v/v is recommended for use where possible in zebrafish.

Metabolism of ibuprofen in zebrafish larvae.

The application of zebrafish (Danio rerio) larvae to drug discovery assays and toxicity testing, and the occurrence of pharmaceuticals in the environment, has resulted in a need to understand the extent of the metabolic capabilities in the early life stages of this species. The aims of this study were to determine if zebrafish larvae absorbed, metabolized and excreted the model pharmaceutical, ibuprofen. Zebrafish larvae (72 h post fertilization) were exposed to ibuprofen (100 µg/L), 14C-ibuprofen (100 µg/L) or a solvent control (ethanol) for ≤24 h. Water samples and larval extracts were assessed for metabolites of ibuprofen using liquid chromatography mass spectrometry (LC–MS–MS). Fractions from the separation of the samples treated with 14C-ibuprofen were collected after chromatography and analysed for 14C content by scintillation counting. Assessment of larval extracts and water samples by LC–MS–MS at 24 h resulted in the identification of hydroxy-ibuprofen in both water samples and larval extracts (8.2 and 0.08% of the total detected 14C, respectively). A second putative hydroxy-ibuprofen moiety was also observed in water samples at trace levels, and a third minor unknown metabolite was detected in larval extracts only by scintillation counting (0.02% of the total 14C detected). This study provides evidence that zebrafish larvae can metabolize and excrete ibuprofen in a manner known to be cytochrome P450-dependent in mammals, and the similarity to the mammalian pathway supports the use of this system as a surrogate in toxicity and efficacy screening.

Advancing the 3Rs in regulatory ecotoxicology: A pragmatic cross‐sector approach

The ecotoxicity testing of chemicals for prospective environmental safety assessment is an area in which a high number of vertebrates are used across a variety of industry sectors. Refining, reducing, and replacing the use of animals such as fish, birds, and amphibians for this purpose addresses the ethical concerns and the increasing legislative requirements to consider alternative test methods. Members of the UK-based National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) Ecotoxicology Working Group, consisting of representatives from academia, government organizations, and industry, have worked together over the past 6 y to provide evidence bases to support and advance the application of the 3Rs in regulatory ecotoxicity testing. The group recently held a workshop to identify the areas of testing, demands, and drivers that will have an impact on the future of animal use in regulatory ecotoxicology. As a result of these discussions, we have developed a pragmatic approach to prioritize and realistically address key opportunity areas, to enable progress toward the vision of a reduced reliance on the use of animals in this area of testing. This paper summarizes the findings of this exercise and proposes a pragmatic strategy toward our key long-term goals-the incorporation of reliable alternatives to whole-organism testing into regulations and guidance, and a culture shift toward reduced reliance on vertebrate toxicity testing in routine environmental safety assessment. Integr Environ Assess Manag 2016;12:417-421.