Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine-active compounds. At age 2 weeks post-fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week-old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst-like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false diagnoses of 'intersex gonads' in zebrafish.

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Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP.

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.

This study presents an overview of the distribution of proliferative kidney disease (PKD) in wild and farmed fish in Switzerland based on two sources: (1) the results of routine diagnostic work at the National Fish Disease Laboratory, and (2) the results of a country‐wide survey for PKD. The first case of PKD in Switzerland was diagnosed in 1979 and since 1981, a few cases have been found every year. Affected species were rainbow trout, brown trout and grayling from rivers and fish farms. Most of the diseased fish were found in the lower altitude regions of the country (Swiss midlands). A seasonal distribution of the cases was evident: PKD‐positive fish occurred only from June to November withprominent peaks in August and September. Among infected fish all length classes were present, but the highest numbers of affected fish were found in the length classes <10 cm and 16–20 cm. The evaluation of the samples from a survey of the occurrence of PKD in 2000/2001 revealed a much broader geographical distribution of the infection than expected from routine diagnostic observations. Fish from 56 of 139 rivers analysed by histological examination were positive for parasites. Fish from 131 of these sites were examined macroscopically. Of these, trout from 45 sites showed gross alterations indicative for PKD. Most of the positive fish were found in the Swiss midlands north of the Alps. Fish from six of 65 fish farms investigated were found to be PKD‐positive by histopathological analysis, and in four of these cases fish could be identified as infected on the basis of gross examination.

The functional status of organ systems involved into the processing of exogenous food is critical for the survival and growth of fish early life stages. The present study on laboratory-reared larval turbot, ScophthaImus maximus, provides an overview on the ontogeny of structure and functions involved in digestion, absorption and metabolism of nutrients. At start of exogenous feeding the intestine of larval turbot is anatomically differentiated, with enterocytes displaying an adult-type ultrastructure and being able to process lipids. At the microvillous border of the enterocytes, enzymes of contact digestion such as aminopeptidase are found. The ultrastructure of the exocrine pancreatic cells is fully differentiated from hatching onwards. Likewise, substantial activities of trypsintype proteases are present. A stomach anlage exists in firstfeeding larvae; however, the stomach becomes functional (appearance of gastric glands and pepsin secretion) only during metamorphosis. Liver parenchymal cells already display a functional ultrastructure during the endotrophic phase; with onset of exogenous feeding they develop pronounced diet-related changes of their energy stores. Larval respiration is not executed by the gills since respiratory surface of these structures develops only towards metamorphosis. The energy generation of larval muscle tissue depends on aerobic metabolism, whereas glycolytic activCommunicated by O. Kinne, Oldendorf/Luhe H. Segner I (m) 9 W. Kloas

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1. The uptake of heavy metals via the alimentary tract can be an important factor for the metal budget of fish. 2. Concepts such as biomagnification, bioaccumulation, biotransference, or concentration factors, convey little information about the real threat originating from heavy metals in an aquatic food chain. 3. In polluted aquatic ecosystems the transfer of metals through food chains can be high enough to bring about harmful concentrations in the tissues of fish. This relationship is called the food chain effect. 4. Two kinds of ecological factors influence the food chain effect: firstly, high levels of contamination of the food, and, secondly, the reduction of species diversity. When susceptible species are eliminated, metal-tolerant food organisms may become dominant. Their tolerance may be based either on their ability to accumulate excessive amounts of metals or to exclude heavy metals from the tissues. These two strategies represent feedback mechanisms which may enhance or weaken the food chain effect. 5. It is concluded that future investigations on transference of heavy metals to fish must take into more careful consideration the specific ecological situation of a given environment.