EthoCRED: a framework to guide reporting and evaluation of the relevance and reliability of behavioural ecotoxicity studies.

Behavioural analysis has been attracting significant attention as a broad indicator of sub-lethal toxicity and has secured a place as an important subdiscipline in ecotoxicology. Among the most notable characteristics of behavioural research, compared to other established approaches in sub-lethal ecotoxicology (e.g. reproductive and developmental bioassays), are the wide range of study designs being used and the diversity of endpoints considered. At the same time, environmental hazard and risk assessment, which underpins regulatory decisions to protect the environment from potentially harmful chemicals, often recommends that ecotoxicological data be produced following accepted and validated test guidelines. These guidelines typically do not address behavioural changes, meaning that these, often sensitive, effects are not represented in hazard and risk assessments. Here, we propose a new tool, the EthoCRED evaluation method, for assessing the relevance and reliability of behavioural ecotoxicity data, which considers the unique requirements and challenges encountered in this field. This method and accompanying reporting recommendations are designed to serve as an extension of the "Criteria for Reporting and Evaluating Ecotoxicity Data (CRED)" project. As such, EthoCRED can both accommodate the wide array of experimental design approaches seen in behavioural ecotoxicology, and could be readily implemented into regulatory frameworks as deemed appropriate by policy makers of different jurisdictions to allow better integration of knowledge gained from behavioural testing into environmental protection. Furthermore, through our reporting recommendations, we aim to improve the reporting of behavioural studies in the peer-reviewed literature, and thereby increase their usefulness to inform chemical regulation.

New insights on in vitro biotransformation of anticoagulant rodenticides in fish.

The assessment of the bioaccumulation potential of chemicals is an essential and mandatory part of their regulatory environmental risk and hazard assessment. So far, in vitro data on fish metabolism is rarely available for biocidal active substances such as anticoagulant rodenticides. In this case study we present in vitro biotransformation rates of eight biocidal and one pharmaceutical anticoagulants in rainbow trout (Oncorhynchus mykiss) liver subcellular S9 fraction (RT-S9) determined following the Organisation for Economic Co-operation and Development test guideline 319B method at two different incubation temperatures (i.e., 12 ± 1 °C and 23 ± 2 °C). Furthermore, we address challenges associated with the usability and interpretation of in vitro data to support the decision making within the regulatory bioaccumulation assessment in bridging the gap between in silico methods and in vivo studies. According to our results, four of the tested substances (i.e., chlorophacinone, coumatetralyl, bromadiolone, and difenacoum) exhibited significant intrinsic clearance (p < .001) in the RT-S9 assay. Overall, the observed metabolism was (very) slow and clearance rates were temperature-dependent. Whether the determined in vitro biotransformation rate had a substantial influence on the predicted bioconcentration factor during extrapolation was subject to the lipophilicity of the test substance. Further improvements of existing concepts are needed to overcome uncertainties in the prediction of bioconcentration factors for chemicals such as anticoagulants.

The Role of Behavioral Ecotoxicology in Environmental Protection.

For decades, we have known that chemicals affect human and wildlife behavior. Moreover, due to recent technological and computational advances, scientists are now increasingly aware that a wide variety of contaminants and other environmental stressors adversely affect organismal behavior and subsequent ecological outcomes in terrestrial and aquatic ecosystems. There is also a groundswell of concern that regulatory ecotoxicology does not adequately consider behavior, primarily due to a lack of standardized toxicity methods. This has, in turn, led to the exclusion of many behavioral ecotoxicology studies from chemical risk assessments. To improve understanding of the challenges and opportunities for behavioral ecotoxicology within regulatory toxicology/risk assessment, a unique workshop with international representatives from the fields of behavioral ecology, ecotoxicology, regulatory (eco)toxicology, neurotoxicology, test standardization, and risk assessment resulted in the formation of consensus perspectives and recommendations, which promise to serve as a roadmap to advance interfaces among the basic and translational sciences, and regulatory practices.

Development of a capillary electrophoresis-mass spectrometry method for the analysis of metformin and its transformation product guanylurea in biota.

A method with capillary electrophoresis coupled to mass spectrometry was optimized to determine the uptake of metformin and its metabolite guanylurea by zebrafish (Danio rerio) embryos and brown trout (Salmo trutta f. fario) exposed under laboratory conditions. Metformin was extracted from fish tissues by sonication in methanol, resulting in an absolute recovery of almost 90%. For the extraction of guanylurea from brown trout, solid-phase extraction was implemented with a recovery of 84%. The use of a mixture of methanol and glacial acetic acid as a non-aqueous background electrolyte was vital to achieve robust analysis using a bare fused-silica capillary with an applied voltage of +30 kV. Problems with adsorption associated with an aqueous background electrolyte were eliminated using a non-aqueous background electrolyte made of methanol/acetic acid (97:3) with 25 mM ammonium acetate (for zebrafish embryos) or 100 mM ammonium acetate (for brown trouts), depending on the sample complexity and matrix influences. High resolution and high separation selectivity from matrix components were achieved by optimization of the ammonium acetate concentration in the background electrolyte. An extensive evaluation of matrix effects was conducted with regard to the complex matrices present in the fish samples. They required adapting the background electrolyte to higher concentrations. Applying this method to extracts of zebrafish embryos and brown trout tissue samples, limits of detection for both metformin and guanylurea in zebrafish embryos (12.2 µg/l and 15 µg/l) and brown trout tissues (15 ng/g and 34 ng/g) were in the low µg/l or ng/g range. Finally, metformin and guanylurea could be both quantified for the first time in biota samples from exposure experiments.

Effects of guanylurea, the transformation product of the antidiabetic drug metformin, on the health of brown trout (Salmo trutta f. fario).

BACKGROUND: Guanylurea is the main transformation product of the antidiabetic drug metformin, which is one of the most prescribed pharmaceuticals worldwide. Due to the high rate of microbial degradation of metformin in sewage treatment plants, guanylurea occurs in higher concentrations in surface waters than its parent compound and could therefore affect aquatic wildlife. In this context, data for fish are scarce up to now which made us investigate the health of brown trout (Salmo trutta f. fario) in response to guanylurea. METHODS: In two experiments, eggs plus developing larvae and juvenile brown trout were exposed to three different concentrations of guanylurea (10, 100 and 1,000 µg/L) and, as a negative control, filtered tap water without this compound. Low internal concentrations were determined. The investigated parameters were mortality, length, weight, condition factor, tissue integrity of the liver and kidney, levels of stress proteins and lipid peroxides, as well as behavioural and developmental endpoints. It was found that guanylurea did not significantly change any of these parameters in the tested concentration range. RESULTS: In conclusion, these results do not give rise to concern that guanylurea could negatively affect the health or the development of brown trout under field conditions. Nevertheless, more studies focusing on further parameters and other species are highly needed for a more profound environmental risk assessment of guanylurea.

Does the antidiabetic drug metformin affect embryo development and the health of brown trout (Salmo trutta f. fario)?

BACKGROUND: Due to the rising number of type 2 diabetes patients, the antidiabetic drug, metformin is currently among those pharmaceuticals with the highest consumption rates worldwide. Via sewage-treatment plants, metformin enters surface waters where it is frequently detected in low concentrations (µg/L). Since possible adverse effects of this substance in aquatic organisms have been insufficiently explored to date, the aim of this study was to investigate the impact of metformin on health and development in brown trout (Salmo trutta f. fario) and its microbiome. RESULTS: Brown trout embryos were exposed to 0, 1, 10, 100 and 1000 µg/L metformin over a period from 48 days post fertilisation (dpf) until 8 weeks post-yolk sac consumption at 7 °C (156 dpf) and 11 °C (143 dpf). Chemical analyses in tissues of exposed fish showed the concentration-dependent presence of metformin in the larvae. Mortality, embryonic development, body length, liver tissue integrity, stress protein levels and swimming behaviour were not influenced. However, compared to the controls, the amount of hepatic glycogen was higher in larvae exposed to metformin, especially in fish exposed to the lowest metformin concentration of 1 µg/L, which is environmentally relevant. At higher metformin concentrations, the glycogen content in the liver showed a high variability, especially for larvae exposed to 1000 µg/L metformin. Furthermore, the body weight of fish exposed to 10 and 100 µg/L metformin at 7 °C and to 1 µg/L metformin at 11 °C was decreased compared with the respective controls. The results of the microbiome analyses indicated a shift in the bacteria distribution in fish exposed to 1 and 10 µg/L metformin at 7 °C and to 100 µg/L metformin at 11 °C, leading to an increase of Proteobacteria and a reduction of Firmicutes and Actinobacteria. CONCLUSIONS: Overall, weight reduction and the increased glycogen content belong to the described pharmaceutical effects of the drug in humans, but this study showed that they also occur in brown trout larvae. The impact of a shift in the intestinal microbiome caused by metformin on the immune system and vitality of the host organism should be the subject of further research before assessing the environmental relevance of the pharmaceutical.