The fish embryo toxicity (FET) biotest has gained popularity as one of the alternative approaches to acute fish toxicity tests in chemical hazard and risk assessment. Despite the importance and common acceptance of FET, it is still performed in multiwell plates and requires laborious and time-consuming manual manipulation of specimens and solutions. This work describes the design and validation of a microfluidic Lab-on-a-Chip technology for automation of the zebrafish embryo toxicity test common in aquatic ecotoxicology. The innovative device supports rapid loading and immobilization of large numbers of zebrafish embryos suspended in a continuous microfluidic perfusion as a means of toxicant delivery. Furthermore, we also present development of a customized mechatronic automation interface that includes a high-resolution USB microscope, LED cold light illumination, and miniaturized 3D printed pumping manifolds that were integrated to enable time-resolved in situ analysis of developing fish embryos. To investigate the applicability of the microfluidic FET (μFET) in toxicity testing, copper sulfate, phenol, ethanol, caffeine, nicotine, and dimethyl sulfoxide were tested as model chemical stressors. Results obtained on a chip-based system were compared with static protocols performed in microtiter plates. This work provides evidence that FET analysis performed under microperfusion opens a brand new alternative for inexpensive automation in aquatic ecotoxicology.

Extreme environments are often considered a predation refuge for organisms living in them. In southern Mexico several species of poeciliid fishes are undergoing incipient speciation in a variety of extreme (i.e. permanently dark and/or sulphidic) freshwater systems, and previous research has demonstrated reproductive isolation between populations from sulphidic and adjacent benign habitats. In the present study, we investigated bird predation rates (measured as successful captures per minute) in two sulphidic surface and several benign surface habitats, to test the hypothesis that extreme habitats are predation refuges. We found capture rates to be approximately 20 times higher in sulphidic environments: probably facilitated by extremophile poeciliids spending most of their time at the water surface, where they engage in aquatic surface respiration as a direct response to hypoxia. Even birds that are usually not considered major fish predators regularly engage in fish predation in the toxic habitats of southern Mexico. Our results demonstrate that extreme environments do not necessarily represent a refuge from predation, and we discuss the general importance of predation in driving incipient speciation in these systems. Finally, we hypothesize that natural selection via avian predation may play an important role in maintaining reproductive isolation between divergent poeciliid populations. Plath M, Parzefall J, Schlupp I. 2003. The role of sexual harassment in cave-and surface-dwelling populations of the Atlantic molly, Poecilia mexicana (Poeciliidae, Teleostei). Behavioral Ecology and Sociobiology 54: 303-309. Plath M, Hauswaldt S, Moll K, Tobler M, García de León FJ, Schlupp I, Tiedemann R. 2007a. Local adaptation and pronounced genetic differentiation in an extremophile fish, Poecilia mexicana, inhabiting a Mexican cave with toxic hydrogen sulfide. Molecular Ecology 16: 967-976. Plath M, Tobler M, Riesch R, García de León FJ, Giere O, Schlupp I. 2007b. Survival in an extreme habitat: the roles of behaviour and energy limitation. Naturwissenschaften 94: 991-996. Price IM, Nickum JG. 1995. Aquaculture and birds: the context for controversy. Colonial Waterbirds 18: 33-45. Reiffenstein R, Hulbert W, Roth S. 1992. Toxicology of hydrogen sulfide. Annual Reviews of Pharmacology and Toxicology 1992: 109-134. Reznick DN, Bryga H, Endler JA. 1990. Experimentally induced life-history evolution in a natural population. Nature 346: 357-359. Riesch R, Duwe V, Herrmann N, Padur L, Ramm A, Scharnweber K, Schulte M, Schulz-Mirbach T, Ziege M, Plath M. 2009. Variation along the shy-bold continuum in extremophile fishes (Poecilia mexicana, P. sulphuraria). Behavioral Ecology and Sociobiology 63: 1515-1526. Riesch R, Plath M, García de León FJ, Schlupp I. 2010a. Convergent life-history shifts: toxic environments result in big babies in two clades of poeciliids. Naturwissenschaften 97: 133-141. Riesch R, Plath M, Schlupp I. 2010b. Toxic hydrogen sulfide and dark caves: life history adaptations in a livebearing fish (Poecilia mexic...

Adaptation to ecologically heterogeneous environments can drive speciation. But what mechanisms maintain reproductive isolation among locally adapted populations? Using poeciliid fishes in a system with naturally occurring toxic hydrogen sulfide, we show that (a) fish from non-sulfidic sites (Poecilia mexicana) show high mortality (95 %) after 24 h when exposed to the toxicant, while locally adapted fish from sulfidic sites (Poecilia sulphuraria) experience low mortality (13 %) when transferred to non-sulfidic water. (b) Mate choice tests revealed that P. mexicana females exhibit a preference for conspecific males in non-sulfidic water, but not in sulfidic water, whereas P. sulphuraria females never showed a preference. Increased costs of mate choice in sulfidic, hypoxic water, and the lack of selection for reinforcement due to the low survival of P. mexicana may explain the absence of a preference in P. sulphuraria females. Taken together, our study may be the first to demonstrate independent-but complementary-effects of natural and sexual selection against immigrants maintaining differentiation between locally adapted fish populations.