1 Department of Biology, Faculty of Science, SDU2 CEFAS3 Department of Biology, Faculty of Science, SDU
The OECD has successfully validated reproductive toxicity test guidelines with fish and frogs for diverse chemicals (including endocrine disrupters). Since molluscs are especially sensitive to chronic exposure to a number of chemicals, the OECD supports the complementary development and validation of a mollusc reproduction test guideline. An ad hoc mollusc expert group has been formed in Europe to validate methods that can meet this need. Currently, a key species for use in this context is the freshwater gastropod Lymnaea stagnalis. An important aspect of this work is to first develop a specific pathogen free defined strain of L. stagnalis and second to establish a historical database of growth and reproductive rates under defined culturing conditions. A mass culture of the RENILYS® strain of L. stagnalis have been established at INRA (France) since 2002 and has been distributed to research laboratories in Denmark, Germany and the UK for the OECD pre-validation work to date. Laboratory cultures of L. stagnalis are traditionally fed fresh (preferably organic) lettuce; however, interrupted supplies of fresh lettuce in some countries in 2011 highlighted a potential problem for the draft OECD test guideline. Therefore, we have evaluated other diets based on a review of the published literature and report here the results for different feeds: namely, cabbage leaves, fish flakes, lettuce leaves or sliced sweet potato. For the feeding trial, 5 snails (shell size ca. 2.6 cm) were held in 1 L freshwater. Aeration was provided in all vessels, and for all the vegetable diets treatments concentrations of ammonia, nitrate and nitrite, the dissolved oxygen concentrations (>6 mg/L), pH (6.5-8.5) and temperature (20±1ºC) ranges remained acceptable. However, the fish flake diet gave low dissolved oxygen levels (< 10% saturation). In this 28-d study, mean specific growth rates were highest for snails fed lettuce ad libitum (0.09 mm/snail/day) followed by snails fed fish flake>sweet potato>cabbage leave diets (0.03 mm/snail/day). Similarly, in a 56-d ring-test study with 7 laboratories, the lettuce diet gave mean specific growth rates of 0.06-0.09 mm/snail/day. Fecundity in the same test ranged from 519-1424 total embryos per snail. These data will be used towards developing a reference database of growth and reproduction for L. stagnalis and to aid the statistical optimization of the draft OECD test guideline for partial and full lifecycle testing of chemicals.