Population relevance of neurotoxic effects in refined and alternative behavior tests with zebrafish (Danio rerio)

Water supplies are widely, but unobtrusively contaminated with numerous substances of largely unknown biological properties. A particularly worrisome group are neurotoxic substances, which may, in the long term, not only affect human health, but also wildlife. Neurotoxic effects have become an issue of emerging concern in ecotoxicology, since they may have multiple underlying mechanisms, are often hard to detect, but have the potential to give rise to a severe adverse outcome. As neurotoxicity is even more difficult to detect without extensive animal testing, it presents a major challenge to modern ecotoxicology which is striving to reduce and replace animal studies. My model species, the zebrafish (Danio rerio), is widely used in aquatic ecotoxicology but room for refinement remains especially where tests are carried out with adult individuals instead of potentially less perceptive early-life stages. Since zebrafish, like many other small fish, naturally form shoals and likely behave differently in isolation, I developed a shoal-based approach. In brief, early-life stage tests according to OECD TG 210 were augmented by two behavior tests that are typically carried out with single adult fish, but could be adapted to groups of juveniles with acceptable limitations: a novel tank test and a predator response assay. The selective serotonin reuptake inhibitor fluoxetine ((RS)-N-Methyl-3-phenyl-3-(4-trifluoromethylphenoxy) propylamine) served as model substance during a proof-of-concept study. In a follow-up study, I verified the suitability of this approach using a selection of other substances with different modes-of-action: carbamazepine (sodium channel inhibition), paraoxon-methyl (acetylcholine esterase inhibition), and tris(1,3-dichlorisopropyl) phosphate (TDCPP; endocrine disruption). Finally, in order to assess whether existing alternative methods correlate to immediately population relevant endpoints, I carried out several other experiments across the life-stages of zebrafish with the same model substances. Fluoxetine produced adverse effects down to concentrations three orders of magnitude below the EC10 from acute fish embryo toxicity tests (OECD TG 236). The known neurotoxicants carbamazepine and paraoxon-methyl caused significant effects on zebrafish behavior both upon release into a novel tank and after presentation of a predator dummy. TDCPP, which is thought to disrupt neural development at much earlier stages than those exposed in my experiments, only caused minor behavioral changes. Histopathology of the test fish confirmed the absence of acute organ damage at the concentrations used (always ≤ EC10 from fish embryo tests). The suitability of shoal-based behavioral changes in juvenile zebrafish as sensitive endpoints of neurotoxicity could thus be confirmed. The deviations in behavior compared to the control groups permit conclusions about the “anxiety state”, which arguably influences the fish’s survival chances in the wild. An early and more abstract behavior endpoint, larval motility (6 dpf), also proved to be very efficient and held up well in a comparison with adult and juvenile behavior tests. Finally, a reproduction assay with adult fish exposed to fluoxetine revealed decreased fecundity as another directly population relevant effect of this chemical. 2 Correlation with embryonic and further adult data from literature revealed the good predictive power of 24-h spontaneous coiling tests for later behavior defects, leading me to propose a set of embryonic tests (FET + coiling) for neurotoxicity range-finding and screening in the future. If the results from these “alternative methods” are negative or inconclusive, in vivo testing is indispensable to assess neurotoxicity; as such, larval motility and juvenile behavior assays might follow