1 Department of Environmental Engineering, Technical University of Denmark
New and updated test methods to detect and characterise endocrine disrupting chemicals are urgently needed for the purpose of environmental risk assessment. Although endocrine disruption in invertebrates has not been studied as extensive as in vertebrates, in particular in fish, numerous reports on effects of potential endocrine disrupters on crustacean development and reproduction have been published since the beginning of the nineties. However, most crustacean toxicity test protocols routinely used so far have not been designed with endocrine-specific endpoints in mind. The main objectives of the present Ph.D. project were: 1) to develop a fully synthetic saltwater medium suitable for laboratory culturing of marine copepods including their feeding organism as well as for toxicity testing 2) to identify sensitive endpoints related to growth, development and reproduction of the pelagic calanoid copepod Acartia tonsa 3) to elucidate the applicability of those endpoints for testing sublethal effects of model compounds 4) to investigate these model compounds in a second species, the benthic harpacticoid copepod Nitocra spinipes 5) to study the model compounds in vitro for ecdysteroid agonistic/antagonistic activity with the ecdysteroid-responsive Drosophila melanogaster BII cell line 6) to draft an OECD guideline proposal for testing of chemicals based on the experimental work performed within this study In preliminary investigations with A. tonsa were studied various parameters related to processes regulated by hormones such as growth, molting, sexual maturation and reproduction. The primary endpoints were larval development ratio, egg production and sex ratio. Exposure experiments were conducted with naturally occurring and synthetic vertebrate and invertebrate hormones as well as compounds known to act as endocrine disrupters in vertebrates. Larval development ratio was identified to be a remarkably sensitive endpoint. The larval development test with A. tonsa is rapid, cost-effective, easily to perform and results in full concentration-response relationships allowing the determination of effective concentrations (ECx). After having demonstrated that larval development of A. tonsa was a very sensitive endpoint for evaluating effects of chemicals that might interfere with the endocrine system of crustaceans, the larval development test has been applied to two groups of emerging environmental contaminants, brominated flame retardants (BFRs) and synthetic musks, known or suspected to be potential endocrine disrupters. In parallel, these compounds were investigated in a full life-cycle test with N. spinipes. This test allows studying larval development ratio as well as other individual life-table endpoints, which enable the calculation of population-level endpoints, e.g. the intrinsic rate of natural increase. In N. spinipes, larval development ratio has been proven to be the most sensitive endpoint too. The knowledge about the impact of BFRs on aquatic organisms is still limited. A primary contribution of the present work with BFRs was to establish data on their (sub)chronic toxicity towards marine copepods. To discriminate between general toxicological and endocrine-mediated toxic effects, the model compounds were assessed in vitro for ecdysteroid agonistic/antagonistic activity using the Drosophila melanogaster BII cell line assay. The pentabrominated diphenyl ethers BDE-99 and BDE-100 showed weak ecdysteroid antagonistic activity. Thus, they are able to interfere with a crucial physiological mechanism in arthropods, a property that only is known for a very few substances. The combination of in vitro assays and (sub)chronic copepod tests, as applied in this study, is a valuable tool when screening chemicals suspected to be specifically toxic, in particular, to interfere with the endocrine system. The results of the experimental work as well as the literature survey demonstrated clearly that marine copepods such as Acartia tonsa and Nitocra spinipes are suitable and very sensitive test organisms to study sublethal effects of specific toxic chemicals including compounds suspected to be potential EDCs. Another outcome of this study, which is of particular importance for environmental risk assessment of chemicals, is the OECD Draft Guideline for Testing of Chemicals - Proposal for a New Guideline, Calanoid Copepod Development and Reproduction Test with Acartia tonsa.