Short-term test for the toxicogenomic assessment of ecotoxic modes of action in Myriophyllum spicatum.

In environmental risk assessment of substances, the 14-day growth inhibition test following OECD test guideline 239 is employed to assess toxicity in the macrophyte Myriophyllum spicatum. Currently, this test evaluates physiological parameters and does not allow the identification of the mode of action (MoA) by which adverse effects are induced. However, for an improved ecotoxicity assessment of substances, knowledge about their ecotoxic MoA in non-target organisms is required. It has previously been suggested that the identification of gene expression changes can contribute to MoA identification. Therefore, we developed a shortened three-day assay for M. spicatum including the transcriptomic assessment of global gene expression changes and applied this assay to two model substances, the herbicide and photosynthesis inhibitor bentazone and the pharmaceutical and HMG-CoA reductase inhibitor atorvastatin. Due to the lack of a reference genome for M. spicatum we performed a de novo transcriptome assembly followed by a functional annotation to use the toxicogenomic results for MoA discrimination. The gene expression changes induced by low effect concentrations of these substances were used to identify differentially expressed genes (DEGs) and impaired biological functions for the respective MoA. We observed both concentration-dependent numbers and differentiated patterns of DEGs for both substances. While bentazone impaired genes involved in the response to reactive oxygen species as well as light response, and also genes involved in developmental processes, atorvastatin exposure led to a differential regulation of genes related to brassinosteroid response as well as potential metabolic shifts between the mevalonate and methyl erythritol 4-phosphate pathway. Based on these responses, we identified biomarker candidates for the assessment of MoA in M. spicatum. Utilizing the shortened assay developed in this study, the investigation of the identified biomarker candidates may contribute to the development of future MoA-specific screening approaches in the ecotoxicological hazard prediction using aquatic non-standard model organisms.

A microcosm study to support aquatic risk assessment of nickel: Community-level effects and comparison with bioavailability-normalized species sensitivity distributions.

The aquatic risk assessment for nickel (Ni) in the European Union is based on chronic species sensitivity distributions and the use of bioavailability models. To test whether a bioavailability-based safe threshold of Ni (the hazardous concentration for 5% of species [HC5]) is protective for aquatic communities, microcosms were exposed to 5 stable Ni treatments (6-96 µg/L) and a control for 4 mo to assess bioaccumulation and effects on phytoplankton, periphyton, zooplankton, and snails. Concentrations of Ni in the periphyton, macrophytes, and snails measured at the end of the exposure period increased in a dose-dependent manner but did not indicate biomagnification. Abundance of phytoplankton and snails decreased in 48 µg Ni/L and 96 µg Ni/L treatments, which may have indirectly affected the abundance of zooplankton and periphyton. Exposure up to 24 µg Ni/L had no adverse effects on algae and zooplankton, whereas the rate of population decline of the snails at 24 µg Ni/L was significantly higher than in the controls. Therefore, the study-specific overall no-observed-adverse-effect concentration (NOAEC) is 12 µg Ni/L. This NOAEC is approximately twice the HC5 derived from a chronic species sensitivity distribution considering the specific water chemistry of the microcosm by means of bioavailability models. Thus, the present study provides support to the protectiveness of the bioavailability-normalized HC5 for freshwater communities.

Multi-criteria decision analysis of test endpoints for detecting the effects of endocrine active substances in fish full life cycle tests.

Fish full life cycle (FFLC) tests are increasingly required in the ecotoxicological assessment of endocrine active substances. However, FFLC tests have not been internationally standardized or validated, and it is currently unclear how such tests should best be designed to provide statistically sound and ecologically relevant results. This study describes how the technique of multi-criteria decision analysis (MCDA) was used to elicit the views of fish ecologists, aquatic ecotoxicologists and statisticians on optimal experimental designs for assessing the effects of endocrine active chemicals on fish. In MCDA qualitative criteria (that can be valued, but not quantified) and quantitative criteria can be used in a structured decision-making process. The aim of the present application of MCDA is to present a logical means of collating both data and expert opinions on the best way to focus FFLC tests on endocrine active substances. The analyses are presented to demonstrate how MCDA can be used in this context. Each of 3 workgroups focused on 1 of 3 species: fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio). Test endpoints (e.g., fecundity, growth, gonadal histopathology) were scored for each species for various desirable features such as statistical power and ecological relevance, with the importance of these features determined by assigning weights to them, using a swing weighting procedure. The endpoint F1 fertilization success consistently emerged as a preferred option for all species. In addition, some endpoints scored highly in particular species, such as development of secondary sexual characteristics (fathead minnow) and sex ratio (zebrafish). Other endpoints such as hatching success ranked relatively highly and should be considered as useful endpoints to measure in tests with any of the fish species. MCDA also indicated relatively less preferred endpoints in fish life cycle tests. For example, intensive histopathology consistently ranked low, as did measurement of diagnostic biomarkers, such as vitellogenin, most likely due to the high costs of these methods or their limited ecological relevance. Life cycle tests typically do not focus on identifying toxic modes and/or mechanisms of action, but rather, single chemical concentration-response relationships for endpoints (e.g., survival, growth, reproduction) that can be translated into evaluation of risk. It is, therefore, likely to be an inefficient use of limited resources to measure these mechanism-specific endpoints in life cycle tests, unless the value of such endpoints for answering particular questions justifies their integration in specific case studies.

Environmental effect assessment for sexual endocrine-disrupting chemicals: Fish testing strategy.

Current standard testing and assessment tools are not designed to identify specific and biologically highly sensitive modes of action of chemicals, such as endocrine disruption. This information, however, can be important to define the relevant endpoints for an assessment and to characterize thresholds of their sublethal, population-relevant effects. Starting a decade ago, compound-specific risk assessment procedures were amended by specifically addressing endocrine-disrupting properties of substances. In 2002, the Conceptual Framework, agreed upon by OECD's Task Force on Endocrine Disrupters Testing and Assessment, did not propose specific testing strategies, and appropriate testing methods had not yet been developed and approved. In the meantime, the OECD Test Guidelines Programme has undertaken important steps to revise established and to develop new test methods, which can be used to identify and quantify effects of endocrine-disrupting chemicals on mammals, birds, amphibians, fish, and invertebrates. For fish testing of endocrine-disrupting chemicals, the first Test Guidelines have recently been adopted by the OECD and validation of further test systems is under progress. Based on these test systems and the experience gained during their validation procedures, we propose a 3-step fish testing strategy: 1) Weight-of-evidence approach for identifying potential sexual endocrine-disrupting chemicals; even after advanced specification of systematic criteria, this step of establishing initial suspicion will still require expert judgment; 2) in vivo evaluation of sexual endocrine-disrupting activity in fish by applying in vivo fish screening assays; sufficient data are available to diagnose the aromatase-inhibition and estrogen-receptor agonist mechanisms of action by indicative endpoints (biomarkers), whereas the ability of the respective biomarkers in the screening assay to identify the estrogen-receptor antagonists and androgen-receptor agonists and antagonists requires further validation; 3) characterization of sexual endocrine-mediated adverse effects including threshold concentrations; in cases when the most sensitive population-relevant endpoints and the most sensitive time window for exposure are known for the mechanisms of action, the fish full life-cycle or 2-generation test, which are the normal definitive tests, might be abbreviated to, e.g., the fish sexual development test. In the European Union, the measurement of indicative endpoints in the definitive test might be crucial for the authorization procedure under REACH and plant-protection products. The results of the definitive tests can be used in existing schemes of compound-specific environmental risk assessments.

An overview of hazard and risk assessment of the OECD high production volume chemical category--long chain alcohols [C(6)-C(22)] (LCOH).

This review summarizes the findings of the assessment report for the category, long chain alcohols (LCOH) with a carbon chain length range of C(6)-C(22) covering 30 substances, and >1.5million tonnes/year consumed globally. The category was evaluated under the Organization for Economic Co-operation and Development (OECD) high production volume chemicals program in 2006. The main findings of the assessment include: (1) no unacceptable human or environmental risks were identified; (2) these materials are rapidly and readily biodegradable; (3) a parabolic relationship was demonstrated between carbon chain length and acute and chronic aquatic toxicity; (4) category-specific (quantitative) structure-activity relationships were developed enabling prediction of properties across the entire category; (5) LCOH occur naturally in the environment in an equilibrium between synthesis and degradation; (6) industry coming together and sharing resources results in minimizing the need for additional animal tests, produces cost savings, and increases scientific quality of the assessment.