A method for embryo-larval development toxicity testing on an endemic and commercially important mussel, <i>Perna canaliculus</i>

Bivalve embryo-larval development tests are widely used as standardized bioassays for assessing the toxicity of chemicals and environmental samples on early life stages. The green-lipped mussel (Perna canaliculus; GLM), however, was unsuitable for toxicity testing due to reliance on suitable quality seawater requiring the use of chelating agents during embryo-larval development. Given the cultural, environmental, and economic importance of this species in Aotearoa New Zealand, we developed a new bioassay to accurately assess toxicity, including trace metals. Using an iterative experimental approach, we examined the role of ethylenediaminetetraacetic acid (EDTA) at different early developmental stages to determine requirements for successful development; tested reconstituted seawater (RSW), which contains no chelating agent, as an alternative to natural seawater (SW); and finally, validated a method with zinc. Aging oocytes and fertilizing gametes in seawater containing minimum 1.2 µM EDTA was essential to successful subsequent larval development. Following exposure to EDTA during fertilization, survival of D-larvae was the same for embryos incubated in SW containing EDTA and in RSW with and without EDTA. This prior treatment of gametes allows standardized embryo-larval development testing to be run in RSW, given that EDTA carry-over remains below 100 nM. A method assessment of this assay was undertaken using RSW and zinc as a model toxicant, and the resulting LC50 values were comparable with embryo-larval development tests of other marine bivalve species. These results confirmed that the protocol using RSW can be applied, providing a critical tool for improved environmental management and protection of the GLM aquaculture industry.