Importance of sperm density in assessing the toxicity of metals to the fertilization of broadcast spawners.

Many marine invertebrates reproduce through broadcast spawning, where sperm and eggs are released into the water column and are vulnerable to toxicants present in the environment. The potential impacts of toxicants on spawning success are often assessed through laboratory-based fertilization tests. In most cases, these tests assess toxicant impacts at a single, pre-defined sperm density, based on a sperm:egg ratio that ensures high fertilization success (≥70-80%) in a filtered seawater control. Here we show that use of a single sperm density can considerably underestimate toxicity and that assessments over a range of sperm densities can provide more ecologically relevant, conservative and informative toxicity data. Fertilization assays were run for the polychaete Galeolaria caespitosa exposed to four heavy metals (Cu, Zn, Pb, Cd) across multiple sperm densities. There was a three-fold difference in the toxicity of copper and zinc when calculated at sperm densities of 10 and 10 sperm mL, both of which result in over 80% fertilization in FSW controls. By testing across multiple sperm densities, we identified that metals impact the sperm of G. caespitosa during the fertilization process. Assessing toxicity across multiple sperm densities is not always practical. This is due to the increased effort required to complete full fertilization curves, across enough concentrations of a toxicant, to establish a concentration-response relationship. In such cases, we provide recommendations for adopting aspects of fertilization assays that will improve on standard spermiotoxicity tests but which are still manageable for routine toxicity testing.