The applicability of the Fish Embryo Toxicity Test (FET) for the testing of chemical substances with particular reference to a possible barrier function of the chorion

Abstract

The Fish Embryo Toxicity Test (FET) has been proposed and adopted by the OECD as a suit-able alternative method for the acute toxicity testing of chemicals in adult fish. The present study was conducted to evaluate boundary parameters of the standard test design and how different modules can be changed to fill in the need for more sophisticated endpoints. The key aspects of this thesis were (1) the definition of impacts of the chemical and physical proper-ties of a substance to cross the chorion, the acellular envelope by which the developing em-bryo is surrounded during the first days of its development; (2) the possibility to assess the toxicity and behavior of highly reactive compounds in fish embryos; and (3) the transferabil-ity of the test design to study the behavior of substances in the environment under controlled conditions. The chorion permeability was studied with differently sized polyethylene glycols (PEGs) which were applied to generate an osmotic gradient strong enough to result in deformed cho-rions, if no uptake across the chorion is feasible. The extent of chorion shrinkage depended on PEG molecular weight, thus size, and the duration of exposure. The barrier function of the zebrafish chorion for molecules larger than 3,000 at 24 hpf and 4,000 Da at 48 hpf was de-fined. For fathead minnow, the critical size to cross the chorion was narrowed down to 4,000 Da. Imaging with nano-sized fluorescent microspheres revealed that besides the size of a molecule the surface properties, e.g. residue groups, are a key factor for uptake. Carbox-ylate-modified microspheres (0.05 to 0.2 μm) of a size small enough to be able to cross the chorion via the pores were completely blocked by the chorion of zebrafish and fathead min-nows. In further testing series with similarly sized microspheres bearing no additional func-tional groups on the surface were shown to be taken up into zebrafish embryos and thus across the chorion. In order to expand the FET beyond the standard test design, as a further variation of the protocol, tests were initiated with differently aged test solutions of highly re-active substances with the ability of self-coupling. To clarify if the mode of action is triggered by the formation of reactive oxygen species or other free radicals, ascorbic acid as a scaven-ger was shown to be effective. The transferability of the standard laboratory protocol to a more environmentally relevant approach was documented by tests both in modified test medi-um and natural waters. Whereas the complexity of aggregation, disaggregation and the for-mation of complexes with naturally occurring organic matters could not be imitated by the amendment of humic acids, the use of natural water samples could be shown to yield results comparable to those expected in the environment.