Development of a methodology for successful multigeneration life-cycle testing of the estuarine sheepshead minnow, Cyprinodon variegatus.

Evaluation of effects on fish reproduction and development during chemical exposures lasting for multiple generations is sometimes limited by variable reproductive responses and the time required for the exposure. Established testing methods and the short life cycle of the sheepshead minnow, Cyprinodon variegatus, make this species particularly suitable for use in identifying potential impacts of contaminants in estuarine and marine environments. This study describes the refinement of life-cycle exposure methods that increased the reliability of reproduction in sheepshead minnows and reduced the time to maturation for larvae and juvenile fishes. A test of three spawning chamber designs, three sex ratios, and two photoperiods identified conditions that reduced the coefficient of variation in egg production from >100% to as little as 32%. The most reliable results were produced with groups of three female and two male fishes (all of similar size) when they were placed in a rectangular chamber and acclimated for 12 days. A test water temperature of 26.5 +/- 2 degrees C and a 14L:10D photoperiod resulted in fish producing a mean of 74 embryos per female per day, with a coefficient of variation of 31.8%. Egg fertility exceeded 90%, with a hatch rate of 95% for normal embryos (>or=80% yolk) and a hatch rate of or=2.7 cm standard length) was critical for spawning readiness. Adult fish were prepared for the spawning assessment by adding frozen brine shrimp to their diet. Results of these experiments provide methods that are of particular interest in assessment of endocrine-disrupting chemicals that are known to affect reproduction.

Comparative embryonic and larval developmental responses of estuarine shrimp (Palaemonetes pugio) to the juvenile hormone agonist fenoxycarb.

Grass shrimp (Palaemonetes pugio) were reared separately through both embryonic and total larval development during exposure to fenoxycarb at measured concentrations of <2.2 to 888 microg L(-1). A fenoxycarb concentration of 888 microg L(-1) significantly (p < 0.05) inhibited embryonic development to larval hatching and extended the embryonic developmental period from 11.9 to 12.7 days. Exposure to fenoxycarb concentrations < or = 502 microg L(-1) had no significant (p > 0.05) effect on complete embryonic development. Significantly fewer shrimp successfully metamorphosed to postlarvae when exposed through complete larval development to fenoxycarb concentrations > or = 4 microg L(-1). Larval development of grass shrimp was therefore >2 orders of magnitude more sensitive to this juvenile hormone agonist than was embryonic development. Viability of larvae developing in fenoxycarb was concentration dependent. Development beyond third zoeal stage was significantly inhibited at fenoxycarb concentrations > or = 190 microg L(-1), whereas development beyond fourth zoeal stage was inhibited by a concentration of > or = 45 microg L(-1). Fenoxycarb exposure of developing larvae did not alter either the duration of total larval development or the total number of larval stages before metamorphosis. Rearing of fenoxycarb-exposed embryos through larval development without further exposure had no significant effect on number of larval stages, larval development rate, or metamorphic success of larvae. Similarities in the sensitivity of grass shrimp larvae and mosquito larvae to fenoxycarb suggests that the use of a bioassay protocol measuring the metamorphic success of crustacean larvae would be a valuable adjunct to the hazard assessment of newly developed pesticides that target endocrine control of metamorphosis in insects and possibly other endocrine-disrupting xenobiotics as well.

Effects of fenoxycarb exposure on complete larval development of the xanthid crab, Rhithropanopeus harrisii.

Pest control agents, such as juvenile hormone analogues (JHA), have been developed to limit effects on non-target organisms that co-inhabit insect pest habitats. Rhithropanopeus harrisii, an estuarine xanthid crab, was used to observe the impacts of the JHA, fenoxycarb, on the pattern of complete larval development as well as survival of larvae and successful metamorphosis to first crab stage. Significant mortality occurred in the first of four zoeal stages (after 2-3 days of exposure) at the highest treatment of 240 microg fenoxycarb/l and in megalopae exposed to 48 microg fenoxycarb/l. The time required to metamorphose to the first crab stage was significantly increased for megalopae in all treatments 48 microg/l. This delay in development was sufficient to significantly prolong the entire developmental period from zoea to crabs. Unexposed larvae developed to crabs in an average of 16 days; larvae exposed to >/=48 microg/l required 19-20 days. Reduced survival and extended duration of developing larval stages in the life history of a benthic invertebrate may alter the population dynamics of these organisms in the estuary.

Early life-stage toxicity test methods for gulf toadfish (Opsanus beta) and results using chlorpyrifos.

Gulf toadfish (Opsanus beta) were continuously exposed as embryos, sac fry, and juveniles to technical chlorpyrifos in two 49-day early life-stage toxicity tests. Survival was significantly (alpha = 0.05) reduced only in 150 micrograms/liter. However, toadfish exposed to chlorpyrifos concentrations from 3.7 to 150 micrograms/liter weighted significantly less than control fish: 9% lower in 3.7 micrograms/liter to 62% lower in 150 micrograms/liter. The 96-hr LC50 for juvenile fish was 520 micrograms/liter. Concentrations of chlorpyrifos in toadfish and bioconcentration factors increased with increasing exposure concentration, a condition not generally observed with other marine fishes and other test chemicals. These results demonstrated the procedures for, and the practicality of, early life-stage tests with this marine species. We recommend the use of the gulf toadfish for comparative toxicity testing and for evaluating the toxicity of substances in conjunction with ontogenetical, physiological, and histological investigations of this considerably studied genus. We do not recommend it for routine effects testing.

A new early life-stage toxicity test using the California grunion (Leuresthes tenuis) and results with chlorpyrifos.

California grunion were continuously exposed as embryos and fry to technical chlorpyrifos in two toxicity tests conducted in the same exposure apparatus. The first test, a 35-day early life-stage (ELS) test, began with approximately 2.5-day-old embryos that were exposed in flow-through aquaria in darkness until hatching was stimulated on Day 9 of exposure. The second toxicity test, a fry test, began with newly hatched fry and lasted 26 days. Test water temperature ranged from 23 to 26 degrees C and salinity from 24.5 to 34.0%. Results of the two tests were similar, indicating that exposure of embryos added little to the overall toxicity of chlorpyrifos to grunions. Percentage hatch of embryos was unaffected by the chlorpyrifos concentrations tested. Fry survival was apparently reduced in nominal concentrations greater than or equal to 1.0 micrograms/liter in both tests, but significantly so in concentrations greater than or equal to 1.0 micrograms/liter in the ELS test and greater than or equal to 2.0 micrograms/liter in the fry test. When compared with carrier controls, mean fish weights were significantly reduced in nominal chlorpyrifos concentrations greater than or equal to 0.5 micrograms/liter in the ELS test and greater than or equal to 1.0 micrograms/liter in the fry exposure. Mean bioconcentration factors were 770X for fish that survived the ELS test and 190X for those that survived the fry test. Results demonstrate the practicality of conducting ELS tests for the first time with a marine fish from the Pacific coastal waters of the United States.