Herbicides are used to control weeds, but they may also be highly toxic to nontarget aquatic plants, which play an important role in natural aquatic ecosystems. However, the difference in species sensitivity among aquatic vascular plants was not well known due to the lack of test method for wide range of aquatic plant species. In the present study, we developed the seed germination and seedling growth test method for determining the difference in species sensitivity of 5 species vascular plants simultaneously. Candidate test species were selected by considering the following 5 factors: 1) belonging to the 17 orders of Japanese major aquatic plants, 2) inclusion of a wide range of taxonomic groups, 3) constantly available for purchase, 4) suitability for culture experiment using a microplate, and 5) suitable seedling shape for quantifying plant biomass by an image analysis. The following 5 vascular plant species were selected: Welsh onion Allium fistulosum (Asparagales), watercress Nasturtium officinale (Brassicales), carnation Dianthus caryophyllus (Caryophyllales), lettuce Lactuca sativa (Asterales), and basil Ocimum basilicum (Lamiales). An efficient test method using the 5 species was developed based on a microplate assay using a 6-well microplate and a test duration of 7 days. Plant biomass was quantified as a green area, which can be automatically measured by image analysis. Test performance was evaluated by conducting bioassays of 3,5-dichlorophenol and 2,4-dichlorophenoxyacetic acid as standard test substances. The 5 species vascular plants were more sensitive than algal species and duckweed to herbicide 2,4-dichlorophenoxyacetic acid.

In Japan, several aquatic organisms such as fish, daphnia, and monocellular algae have been used for assessing the environmental impact of agrochemicals. Recently, Daphnia magna, which is a representative species of arthropods (crustaceans and insects), was confirmed to be less susceptible to some insecticides than many other crustaceans and insects. Therefore, it is of concern that the environmental risk of several pesticides would be underestimated using the present evaluation system, and it is considered that additional species are needed for more accurate risk assessment. In this context, midges (Chironomus sp.) are attracting attention as a suitable species for such testing. Use of Chironomus sp. for testing has been recommended in the OECD Test Guideline No.235 (TG235). However, there are some technical issues involved in the use of Chironomus sp. according to TG235 for toxicity testing; the most important is the issue of how healthy larvae can be consistently collected through the year for the test. We have established a breeding and cultivating methods for Chironomus yoshimatsui on a small-scale and a cheap apparatus with self-made readily available materials in our laboratory. Our methods can provide a constant subculture for the acute toxicity test