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Overview
Bivalve fertilization and developmental tests are used in toxicology to determine the toxicity of chemicals in marine and freshwaters. Bivalves are important for toxicological studies because they can live in both saltwater and freshwater environments. Developmental tests on bivalves were originally developed by Woelk [1972] and are used to assess water quality as an indicator of bivalve reproductive success and stunted embryo development. Fertilization tests are another test method used in both saltwater and freshwater toxicology testing on Bivalves. Fertilization tests have been used to determine the effects of environmental and chemical stressors on the reproduction of bivalves. Some common bivalves used for saltwater fertilization testing are Pacific oyster (Crassostrea gigas), Eastern oyster (Crassostrea virginica), and the blue mussel (Mytilus edulis), although the organism used may vary based on the type of test being done. . The EPA and the Office of Prevention, Pesticides and Toxic Substances (OPPTS) have developed a standardized method for an acute 48 hour static toxicity test to determine EC50 values. The four major families of freshwater bivalves used for toxicity testing are Corbiculidae, Dreissenida, Sphariidae, and Uniionidae.

Bivalve Reproduction and Development (how does it work)
Fertilization success in sessile non-hermaphroditic bivalves is completely dependent upon gametes making physical contact and is affected by many aspects of water quality. Spawning may be constant or seasonal with environmental cues such as temperature, light and sperm density in water triggering mass spawning events. Fertilized eggs usually hatch within 48-hrs. The larvae, known as trochophores, feed on phytoplankton in the surface waters until hinged shells begin to develop. Trochophores are completely dependent on nutrients from the yolk of the egg until feeding. Most of the yolk material is from the egg which is substantially more biologically costly than sperm resulting in being produced. Sperm motility directly affects probability of sperm-egg contact and is a proxy measurement for reproductive success. In general, bivalves have a high fecundity with small populations able to repopulate given optimum water conditions.

Bivalves as Test Organisms
Many species of bivalves, especially oysters and mollusks, are widely distributed and sessile. As filter feeders, bivalves participate in sediment filtration and are actively used for porewater toxicity testing. Bivalves are also larger than most filter feeders so they are easy to collect after they are deployed or sampled. They are also easy to culture and keep alive in a lab. In a lab setting the spawning of bivalves can be controlled by temperature and other water properties .It is inexpensive to hold bivalves in a lab because if they are held at an appropriate temperature prior experimentation they can live for days or weeks without food. If held in good conditions holding the bivalves without food should not affect the viability of their gametes. Most importantly, bivalves are tolerant to a wide range of abiotic factors and are sensitive to a number of contaminants ranging from organometallic to toxic metals and endocrine disrupting chemicals (EDC's) .Effects of stresses in bivalves monitored in the field do not vary much from effects seen in the lab but most tests are done in a lab for cost effectiveness. .

Developmental Testing
Embryo and larval bivalve testing is used to test acute toxicity and 48-hr EC50 as a regulated FIFRA test by the EPA. Bivalve gametes can be purchased from labs or collected from field samples. Developmental testing must take place within 4 hrs of fertilization and monitors the embryo (2-8 cells) 48-hr development into a shelled veliger. ELS tests are important for surveying species health as damage to organisms in the larval stage can greatly impact growth and development.

Juveniles exposed to a range of chemicals, such as EDCs and PPCP, can adversly effect sexual development by causing early sexual maturation resulting in low sperm motility and egg size, sex reversal from males to females or females development of penis and a variety of other known and unknown effects which disrupt successful reproduction. These effects are highly variable depending on toxicant and species. Effects of EDC on mollusks, specifically tributyltin (TBT) have been observed to cause sex-reversal of larval European flat oyster (Ostrea edulis) males into females and cause penis development in female Dog whelk's (Nucella lappillus) in concentrations lower than 1 ng L-1, yet TBT has shown no significant effect on sexual development of Crassostrea virginica.

Fertilization Testing
While gamete function tests have been run in lab they are less common. Toxicants can have a variety of adverse effects on both sperm and egg function. Estrogen and estrogen-mimicking compounds can cause production of vitellogenin-like proteins in male oysters, scallops and clams. Nutrient rich vitellogenin proteins are deposited in oocytes and responsible for yolk development. Sperm motility is seldom observed in lab, but can be greatly affected by early exposure to toxicants.