Weathered Alaska North Slope crude oil (ANS 521) was stirred for 2 and 14 days in 20‰ salinity sterile seawater or for 14 days in seawater with nutrients and a group of three (GO3) microorganisms from Prince William Sound, Alaska, that were capable of biodegrading hydrocarbons. A total of 0.65 and 0.69 mg/L water soluble fraction (WSF) of neutral fraction hydrocarbons was recovered from the 2- and 14-day stirred sterile systems, respectively. In comparison, a total of 7.5 mg/L WSF neutral fraction hydrocarbons was recovered from systems containing ANS 521 that were stirred and biodegraded by the GO3 microbes for 14 days. Toxicity/teratogenicity tests were conducted with neutral fraction hydrocarbons recovered from the sterile and biodegraded systems using embryonic inland silversides, Menidia beryllina. Hydrocarbons from the sterile systems did not cause statistically significant teratogenic responses at concentrations of 1%, 10%, and 100% (w/v) of recovered fractions (redissolved in 20‰ salinity sterile seawater). Counts of heart contraction rates were significantly lower (α≤0.05) at the 100% WSF concentration of hydrocarbons on days 5 and 6 of embryogenesis. In contrast, recovered and redissolved neutral fraction hydrocarbons from ANS 521, that were biodegraded for 14 days, caused statistically significant (α≤0.05) teratogenic responses at the 1, 10, and 100% WSF concentrations. Measurement of heart contraction rates showed statistically significant (α≤0.05) reductions at the 100% WSF concentration on days 2 through 6 of embryogenesis, compared to controls.

Sand fiddler crabs, Uca panacea, were exposed in laboratory habitats to measured concentrations of ULV-grade fenthion via simulated aerial spray at 5% and 50% of field rate application of 6–12 mg fenthion/m2 (0.05–0.10 lbs fenthion/acre). Two habitats served as controls, and two habitats served as replicates for each of the test exposures. The aerial application was repeated 12 times between July 7, 1997, and August 15, 1997, during the period of the most active larval production of the crabs. The resulting measured concentrations of fenthion at the surface and in the water were consistently lower than what the application rate would have suggested. Statistical analysis of total, daily larval counts showed a periodicity of approximately 14 days that did not appear to be affected by fenthion, at least at the 5% application rate, where a 15% reduction in larval production was noted by the end of the second hatching cycle and a 25% reduction by the end of the third. Larval production in the habitats exposed to the 50% application rate was reduced by 18% at the end of the first hatching cycle, 77% at the end of the second, and 100% at the end of the third hatching cycle. At the end of the third hatching cycle, adult crab mortality observed at the surface was 0%, 3%, and 20% for the control, 5%-, and 50%-exposure habitats, respectively. Three weeks after the final fenthion application, survival of adult crabs was 100% in the control habitats, 75% in the 5%-exposure habitats, and 3% in the 50%-exposure habitats. Survival of unfed first-stage zoeae released during the night following the sprays averaged 4.6 ± 0.9, 3.7 ± 0.9 and 1.7 ± 0.6 days for control, 5%-, and 50%-exposure habitats, respectively. Forty-eight-hour LC-50s (nominal) for fenthion exposure of adult crabs submerged in water and for first-stage zoeae were 215 μg fenthion/L and 0.8 μg fenthion/L, respectively.

Renewed interest in the use of Metarhizium anisopliae and its toxins for insect control prompted the following safety assessment. A neutral extract (methylene chloride, pH 7.2), derived from M. anisopliae cultures, was evaluated for toxicity and mutagenicity using aquatic animal bioassays and the Ames test. The average LC50 of the neutral extract obtained in static, acute 96-h tests conducted with ≤24-h-old Mysidopsis bahia was 2.41 mg L−1. By partially purifying destruxins from the neutral extract, it was shown that destruxins alone were not responsible for the observed toxicity in mysids. The neutral extract was fetotoxic to developing grass shrimp, Palaemonetes pugio, and frog, Xenopus laevis, embryos; the LC50 values were 52 and 32 mg L−1, respectively. Eye spot abnormalities were observed in shrimp and frog embryos exposed to the neutral extract. In extract-exposed frog embryos, moderate to severe cranial, facial, and gut malformations were also observed. The neutral extract was toxic to juvenile mosquito fish, Gambusia affinis, at an LC50 value of 141 mg L−1. Adult female G. affinis surviving a 24-h exposure to 200 μg ml−1 of the neutral extract produced healthy broods. After 3 months, no mortalities or adverse effects were observed in adult G. affinis fed a diet partially composed of a freeze-dried M. anisopliae culture. The neutral extract did not show mutagenicity in the Ames test using strains TA98 and TA100 with and without metabolic activation by rat liver S9. Significant (p  ≤ 0.05) mortalities were obtained when embryos of grass shrimp and inland silverside fish, Menidia beryllina, were exposed to the same lot of M. anisopliae conidiospores. Exposure of frog embryos to M. anisopliae conidiospores did not cause significant (p > 0.05) mortalities or malformations.

Weathered Alaska North Slope crude oil (ANS 521) was subjected to biodegradation in vigorously stirred incubations for 14 days at 15 ± 1°C in 20‰ salinity sterilized seawater, amended with nutrients and inoculated with a hydrocarbon-degrading microorganism (EI2V) isolated from an oil-contaminated beach in Prince William Sound, Alaska. A total of 13.7 mg/L water-soluble neutral fraction (WSF) was recovered from the incubation of weathered ANS 521. Toxicity/teratogenicity tests were conducted with WSF recovered from the biodegradation system using embryonic and larval Pacific herring, Clupea pallasi. Exposures were begun at 4, 48, and 96 h postfertilization of herring eggs. Exposure concentrations were 1, 10, and 100% of the original concentration of WSF recovered from incubations (redissolved in 20‰ salinity sterile seawater at 15 ± 1°C). Sterile 20‰ salinity seawater without the addition of redissolved neutral fraction was used as a control. Significant (p ≤ 0.05) embryo mortality or teratogenic responses were observed at WSF concentrations of 10 and 100%. On days 5 through 8 of embryogenesis, counts of heart contraction rates were significantly lower (p ≤ 0.05) at the 100% WSF concentration for embryos exposed beginning at 4 and 48 h postfertilization. Grow-out of larvae from selected exposures was conducted. High mortality was noted in larvae exposed to the 10% WSF concentration beginning at 4 and 48 h postfertilization. Most of these larvae died 5 to 8 days after hatching when they elicited vertebral displacements at a time concurrent with the onset of feeding behavior.

To determine whether a 7- to 10-d embryo toxicity/teratogenicity test with the inland silverside fish, Menidia beryllina, is a sensitive indicator for evaluation of bleached kraft mill effluents, we compared this test with the Microtox® 15-min acute toxicity test and the Ceriodaphnia dubia 7-d chronic toxicity test. Water samples used in each test were collected from three areas in a bleached kraft pulp and paper mill using a 100% chlorine dioxide bleaching process: 1) river water prior to use in the mill; 2) the combined acid/base waste stream from the pulping process prior to biological treatment in the aerated stabilization basin (ASB); and 3) the effluent from the ASB with a retention time of approximately 11 d. Relative toxicity determined by the three tests for each water sampling location was compared. All three toxicity tests were predictive indicators of toxicity; however, the C. dubia and M. beryllina tests were the more similar and sensitive indicators of toxicity. Process water (ASB influent) prior to biological treatment in the ASB was toxic at all concentrations using the Microtox® and C. dubia tests. The fish embryo test showed no toxicity at 1% concentrations, slight toxicity at 10%, and acute toxicity at the 100% ASB influent concentration. Tests with biologically-treated ASB effluent indicated a substantial reduction in observed toxicity to Microtox® bacteria, C. dubia, and M. beryllina. No toxic responses were observed in any test at a 1% ASB effluent concentration which was the approximate effluent concentration in the receiving river following mixing. No relationship was found among any toxicological response and effluent levels of adsorbable organic halides, polychlorinated phenolic compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, total suspended solids, color, chemical oxygen demand, or total organic carbon.

This chapter contains sections titled: Introduction Biology of the Atheriniformes Toxicity Tests with Cyprinodon Variegatus, Menidia Beryllina, M. Menidia, Leurestfles Tenuis, and Atherinops Afflnis Culture of the Alga, Isochrysis Galbana, and Mixohaline Rotifer, Brachionus Plicatilis Culture of the Rotifer, Brachionus Plicatilis Hatching of Brine Shrimp, Artemia SP. Suggested Indicator Species from other Geographical Regions Acknowledgements References

Developing embryos of the inland silverside fish, Menidia beryllina, and grass shrimp, Palaemonetes pugio, were exposed to conidiospores of the fungal weed control agent, Colletotrichum gloeosporioides f. sp. aeschynomene and the entomopathogen, Metarhizium anisopliae. Only Metarhizium anisopliae caused significant (p≤0.05) mortalities in the exposed embryos. Colletotrichum gloeosporioides did, however, cause fatal infections in adults when conidia were injected into the peritoneum of fish or the hemocoel of shrimp.

Developing embryos of the inland silverside fish Menidia beryllina were exposed to conidiospores of the entomopathogenic fungus Metarhizium anisopliae. Several adverse effects were observed in both embryos and newly hatched larvae. These included transitory effects on the heart resulting in decreased cardiac output or circulation velocity, rupture of the chorion, fungal growth on the mandibles of larvae, focal vertebral abnormalities in larvae and teratogenic expressions in embryos and larvae. An ordinal ranking system was used to enumerate responses to conidiospores. This ranking system allowed significance to be determined by nonparametric analysis of variance. Responses were highly variable with significant (p less than or equal to 0.05) adverse effects observed in 5 of the 6 experiments conducted. Heat-killed spores failed to cause significant adverse effects indicating that viable spores were required for the adverse effects.

Embryonic inland silversides, Menidia beryllina, in the early blastula stage were exposed to the water-soluble fraction (WSF) of No. 2 Fuel oil and the oil dispersants Corexit 7664® and 9527®, singly and in combination. An ordinal ranking system was used to score observed daily craniofacial, cardiovascular, and skeletal responses in control embryos and those exposed to 1%, 10%, and 100% concentrations of the WSF of No. 2 Fuel oil, the dispersants Corexit 7664® and 9527® applied at the recommended field application concentrations, and the combination of No. 2 Fuel oil and respective dispersants in seawater. The non-parametric Kruskal-Wallis analysis of variance (ANOVA) and post hoc analyses were used to identify statistically significant differences for control embryos and those exposed to No. 2 Fuel oil and dispersants.

Chemical analyses and biological response data were used to assess the efficacy of a field-scale hyperfiltration unit in the removal of polycyclic aromatic hydrocarbons (PAHs) and other organic compounds from creosote- and pentachlorophenol (PCP)-contaminated ground water. The hyperfiltration unit consisted of four modules containing porous stainless steel tubes which were coated with a formed-in-place zirconium hydrous oxide-polyacrylic acid (ZOPA) membrane. A fivefold concentration of the feed water (80% volume reduction) with up to 97% removal of high molecular weight PAHs was achieved during pre-demonstration and field-demonstration runs of the hyperfiltration unit. Approximately 68% of PCP was removed by the unit. Removal of phenolics averaged 27% and 36%, respectively, for the two runs.

Developing embryos of the inland silverside fish, Menidia beryllina, were exposed to conidiospores of the insect pathogenic fungus, Beauveria bassiana, that possessed activity against the migratory grasshopper, Melanoplus sanguinipes. Various adverse effects were observed in Menidia beryllina embryos and larvae. They included rupture of the chorion, embryo death, developmental defects (vertebral abnormalities) in the embryo or hatched larvae, and fungal infections on the mandibles of larvae. Although there was little evidence of a definitive dose-response trend based on densities of viable conidiospores, statistically significant (p≤0.01) responses were observed in tests in which conidiospore densities were as low as 7.1×103/ml and as high as 1.3×106/ml. Viable spores were required for adverse effects to occur; heat-killed spores failed to cause significant adverse effects.

A two-stage, sequential inoculation bioreactor strategy for the bioremediation of groundwater contaminated with creosote and pentachlorophenol (PCP) was evaluated at bench scale (1.2 L) and pilot scale (454 L). Bioreactor performance using specially selected microorganisms was assessed according to chemical analyses of system influent, effluent, and bioreactor residues, a chemical mass balance evaluation, and comparative biological toxicity and teratogenicity measurements. During pilot-scale operations, the concentration of creosote constituents was reduced from ca. 1000 ppm in the groundwater feed (flow rate 114 L/day) to <9 ppm in the system effluent (total removal efficiency of >99%). Notably, the cumulative concentration of carcinogenic polycyclic aromatic hydrocarbons was reduced from 368 ppm in the feed to 5.2 ppm in the system effluent. Moreover, the toxicity and teratogenicity of the bioreactor effluent were significantly reduced. In general, field data correlated well with those obtained from bench-scale studies.

A Gram-negative bacterium,Pseudomonas sp. strain SR3, was isolated from soil at a former wood treatment plant in north central Florida. The ability of this bacterium to degrade pentachlorophenol (PCP) was confirmed by growing cells in a basal salts medium in which PCP was the only source of carbon and energy. Degradation from a measured concentration of 39–40 μg PCP/ml to 0.0006 μg PCP/ml was observed within 120 h of incubation in the presence of PCP-induced cells ofPseudomonas sp. strain SR3. The initial cell density in these cultures was 6 x 106 cfu/ml. Microtox® 5 min EC50 toxicity tests revealed that aqueous solutions of PCP, measured concentrations 39–40 p μ/ml were toxic but that final biodegraded samples, 0.0006 μ/g PCP/ml were nontoxic. However, bioassays with embryonic inland silversides,Menidia beryllina, showed that the biodegraded samples were embryotoxic or teratogenic. Water containing added PCP at concentrations up to 30 times higher than measured in the final biodegraded samples was less toxic/teratogenic. These results indicate that while biodegradation of PCP was nearly complete, intermediate metabolites of the degradation process or undegraded impurities in PCP were toxic or teratogenic. Thus, theM. beryllina bioassay allows extremely sensitive assessment of toxicity associated with biodegraded environmental pollutants and may be a useful criterion for determining whether bioremediated water or soil is safe for discharge back into the environment.

Flow-through acute and early-life-stage (ELS) toxicity tests were conducted with topsmelt (Atherinops affinis), a Pacific coast saltwater fish, and fenvalerate, a synthetic pyrethroid insecticide. The 96-h LC50 for juvenile fish was 0.66-mu-g/L. In the 30-d ELS test with laboratory-spawned embryos, average measured fenvalerate concentrations were nondetectable (< 0.075-mu-g/L) in two control treatments, 0.14, 0.34, 0.82, 1.5, and 3.2-mu-g/L. Survival of embryos to hatching ranged from 94 to 100%, with no statistically significant differences among treatments. No fry survived exposure to fenvalerate concentrations greater-than-or-equal-to 0.82-mu-g/L; overall survival in lower concentrations and control treatments ranged from 86 to 97%. There were no consistent concentration-dependent differences in weight between fish in the carrier-control treatment and fish exposed to fenvalerate. Mean wet weights of surviving fish ranged from 16.9 mg in 0.34-mu-g/L to 20.3 mg in 0.14-mu-g/L. The average bioconcentration factor for fish exposed to 0.14 and 0.34-mu-g fenvalerate per liter was 315.

The reproductive ecology of the inland silverside, Menidia beryllina, was studied during Feb. 1988-March 1989 at Robinson Point, Blackwater Bay, Florida. Environmental variables including pH, rainfall, salinity, water temperature, and dissolved oxygen were measured weekly or biweekly. Fish were sampled weekly with a seine designed to catch adult, juvenile, and young-of-the-year (YOY) individuals. Most reproductive activity occurred during Feb.-April 1988. The maximum mean weekly female gonadosomatic index (GSI) of 12.5 occurred in April. Fecundity ranged from 63 to 419 hydrated eggs/female. The maximum mean weekly male GSI of 6.1 occurred in early March. Catches of YOY individuals 7.6-37.5 mm SL were greatest in May. Some of these YOY individuals matured in July-Sept. and spawned. This reproductive activity resulted in recruitment of a second group of YOY fish into the population during Aug.-Oct. Growth rates of YOY in May-July, calculated by regression methods from weekly frequency distributions of standard length, was 0.34 mm/day for females and 0.31 mm/day for males.The reproductive pattern of M. beryllina from Blackwater Bay, Florida indicates that qualitatively it is an r-strategist with rapid growth of YOY, sexual maturation at an early age, relatively high fecundity, and multiple spawnings within the first reproductive period for YOY fish in July-Sept. and again as 1- to 1-plus-year-old individuals.

Larval topsmelt (Atherinops affinis) and inland silversides (Menidia beryllina) were exposed in 96-h static acute toxicity tests to 11 chemicals to determine the relative sensitivity of the two atherinid species. High to low LC50 ratios for endosulfan, methoxychlor, carbophenothion, chlorpyrifos, terbufos, fenvalerate, permethrin, 4-nitrophenol, and sodium lauryl sulfate were within a factor of < 2 for the two species. Atherinops affinis was more sensitive to both azinphos-methyl and 2,4-dinitrophenol by factors of 6.7 and 4.4, respectively. Comparisons of the relative sensitivity of Atherinops affinis with three freshwater fish species (Lepomis macrochirus, Oncorhynchus mykiss, Pimephales promelas) and one estuarine fish species (Cyprinodon variegatus) are presented. Sensitivities were similar between Atherinops affinis and the two most sensitive freshwater species, Lepomis macrochirus and Oncorhynchus mykiss. Atherinops affinis is easily transported, cultured, and maintained in the laboratory and readily adaptable for use in toxicological studies.

Topsmelt (Atherinops affinis) were spawned repeatedly in the laboratory from May to July 1989. A periodic "temperature spike" from the holding temperature of approximately 18 up to approximately 20.5-degrees-C, introduced at 7- to 9-d intervals, resulted in maximum production of viable embryos on the fourth morning after the spike. Examination of embryonic stages and comparison to known developmental rates for Atherinops affinis embryos revealed that spawning was generally nocturnal, occurring between 1900 and 0500 h. Survival of embryonic and larval Atherinops affinis cultured at 21 +/- 1-degrees-C and 20 +/- 2 parts per thousand salinity was excellent (> 80%). A larval growth curve was developed for the first 24 d post-hatch.

A chemical toxicity and teratogenicity test was adapted to assess potential adverse effects of a microbial pest control agent on a nontarget fish. Developing embryos of the inland silverside, Menidia beryllina, were exposed to conidiospores of the insect-pathogenic fungus Beauveria bassiana. Embryo rupture and death were observed. Embryo rupture did not always result in death, nor was death always associated with embryo rupture. Adherence of spores to the chorion, followed by germination and penetration by the germ tube, probably caused the embryos to rupture. Statistically significant (P less than or equal to 0.05) responses were observed in tests in which conidiospore concentrations were greater than or equal to 8.3 x 10(4) or less than or equal to 1.5 x 10(6)/ml. Conidiospores treated with a dispersant (biological detergent) showed significantly less binding (P less than or equal to 0.01) to embryos than did untreated spores. Both detergent-treated and heat-killed spores failed to cause significant adverse effects.

An ovarian mycosis in a topsmelt Atherinops affinis (Ayres), collected from Elkhorn Slough, Monterey County, California, USA, is reported. The fungus caused a considerable host response resulting in numerous granulomas and follicular atresia. Based on morphologic characteristics, the fungus was probably a member of the class Oomycetes. This is the first report of an invasive ovarian mycosis in a marine fish.