Abstract Toxic chemical contaminants have a variety of detrimental effects on various species, and the impact of pollutants on ecosystems has become an urgent issue. However, the majority of studies regarding the effects of chemical contaminants have focused on vertebrates. Among aquatic organisms, Daphnia magna has been used extensively to evaluate organism‐ and populationlevel responses of invertebrates to pollutants in acute toxicity or reproductive toxicity tests. Although these types of tests can provide information concerning hazardous concentrations of chemicals, they provide no information about their mode of action. Recent advances in molecular genetic techniques have provided tools to better understand the responses of aquatic organisms to pollutants. In the present study, we adapted some of the techniques of molecular genetics to develop new tools, which form the basis for an ecotoxicogenomic assessment of D. magna . Based on a Daphnia expressed sequence tag database, we developed an oligonucleotide‐based DNA microarray with high reproducibility. The DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to several different chemicals: Copper sulfate, hydrogen peroxide, pentachlorophenol, or β‐naphthoflavone. Exposure to these chemicals resulted in characteristic patterns of gene expression that were chemical‐specific, indicating that the Daphnia DNA microarray can be used for classification of toxic chemicals and for development of a mechanistic understanding of chemical toxicity on a common freshwater organism.
In view of developing a bioassay for use into effluent management system in Japan, short-term toxicity tests using fish, daphnids, and algae were selected as a battery of bioassays, which evaluate the integrated effect of effluent, and "a draft protocol of bioassays for effluent testing" has been prepared. To examine the robustness, sensitivity and reproducibility of the draft protocol, we conducted an inter-laboratory trial where the same effluent was tested by 9 laboratories in Japan. No observed effect concentrations (NOEC) of the test effluent in all the laboratories were within the median +/-1 dilutions, suggesting that the variability between laboratories was at acceptable level. Furthermore, testing of reference chemicals by 10 testing laboratories showed that the reproducibility of the bioassays was relatively higher than that exhibited in the inter-laboratory validation of similar bioassays conducted by US EPA. However, the percentage of minimum significant difference (PMSD) and control coefficient of variation (CV) in the almost all the Japanese laboratories were at acceptable levels (as appears in the US EPA report), indicating that the intra-laboratory precision and the sensitivity of each test was sufficient. We investigated the value of using IC20 for fish, IC25 for daphnids, and IC5 for algae as an alternative to NOEC. With this approach, toxicity survey of 91 effluent samples conducted from 2008 to 2013 found that 36% of effluents were toxic to at least one of the three test species even after 10-fold dilution. The test species that demonstrated toxic effects were different in each sample; thus, we suggest that effluent toxicity evaluation should be based on more than one species due to different sensitivities.
