Quentin J. Stober

Improved methodology for a sea urchin sperm cell bioassay for marine waters

A simple sperm/fertilization bioassay, primarily using sea urchin (and sand dollar) gametes, was improved to yield a quick, sensitive, and cost-effective procedure for measuring toxicity in marine waters. Standard sperm bioassays are conducted by exposing sperm cells to test solutions for 60 min prior to addition of eggs to the test solution for fertilization. Reduced fertilization success (as indicated by the presence or absence of the obvious fertilization membrane) is used as an indicator of toxic effects on sperm viability and/or the fertilization response. This study, in conjunction with earlier work, has shown that the results of sperm bioassays can be affected by a number of factors including temperature, pH, salinity, sperm:egg ratios, sperm exposure times, test materials, and echinoid species. Each of these factors have been considered in designing the “standard” conditions for the improved test. Examples of the effect of these factors on the test results are illustrated, using silver as a reference toxicant.

Comparative sensitivity of sea urchin sperm bioassays to metals and pesticides.

A simple sperm/fertilization bioassay, primarily using sea urchin gametes, has been developed and used by a variety of laboratories. This assay was recently refined into a standard test and is now being used by the U.S. Environmental Protection Agency and others for toxicity testing in marine waters. One factor that has lagged behind the development of this assay is the comparison of its sensitivity to various common toxicants as compared to other bioassay systems and life stages of other marine organisms. The objective of this study was to compare the sensitivity of a standardized sea urchin sperm/fertilization assay to the responses of embryo, larval, and adult marine organisms to metals (Ag, Cd, Cu, Pb, Zn) and pesticides (DDT, Dieldrin, Endrin, Endosulfan) added to natural seawater. The results, although highly variable, generally showed that sperm/fertilization and embryo assays were quite sensitive to the metals tested, but that the larval and adult assays were more sensitive to the pesticides. These comparative data, together with other studies of complex effluents, show that the standardized sperm/ fertilization bioassay is an especially quick and useful tool for biomonitoring of marine waters.

Effects of Urban Development on Fish Population Dynamics in Kelsey Creek, Washington

Abstract A 30-month study of the comparative dynamics of the fish populations inhabiting Kelsey Creek, located in the City of Bellevue, Washington, and a nearby pristine control stream suggest that urban development has resulted in a restructuring of the fish community. Environmental perturbations, including habitat alteration, increased nutrient loading, and degradation of the intragravel environment appeared to have a greater impact on coho salmon Oncorhynchus kisutch and nonsalmonid fish species than on cutthroat trout Salmo clarki. Although the total biomass (g/m2) of fish in each stream was similar, its composition differed markedly. Ages 0 and I cutthroat trout were the majority of the fish community inhabiting Kelsey Creek, whereas the control stream supported a diverse assemblage of salmonids of various ages and numerous nonsalmonids. The rapid growth and greater biomass of salmonids in Kelsey Creek (a 2-year mean of 3.51 g/m2 versus 2.03 g/m2 in the control stream) resulted in a total annual net ...

Development of a Sperm Cell Toxicity Test for Marine Waters

Preliminary methods for conducting a quick and sensitive sperm cell toxicity test for marine waters have been developed. This paper presents a simple static test in which sea urchin or sand dollar sperm cells are exposed to test or control solutions for short periods of time (typically 15 to 60 min). Eggs are added to the test containers after the sperm exposure period has ended, and a standard time is allowed for fertilization to take place before the eggs are fixed with formalin. Samples are then tabulated for fertilization success by noting the presence or absence of the fertilization membrane. The results are analyzed by standard statistical methods to yield a time-dependent median effective concentration (EC 5 0 ) (that is, the concentration of the test material that is effective in reducing fertilization success by 50 percent) after a specified period of exposure. Sperm cell toxicity tests with two reference toxicants (silver nitrate and the pesticide endosulfan) showed that toxic responses were directly related to sperm exposure time. The 60-min EC 5 0 s and 95 percent fiducial limits for green sea urchin (Strongylocentrotus droebachiensis) sperm were 76 (57 to 103) and 168 (99 to 285) μg/litre for silver and endosulfan, respectively. The 60-min EC 5 0 s for sand dollar (Dendraster excentricus) sperm were 45 (34 to 61) and 147 (123 to 175) μg/litre for silver and endosulfan, respectively. Matrix bioassays using temperature, salinity, and toxicant concentration as variables showed that the toxic response to both silver and endosulfan generally increased with increasing temperature and decreasing salinity. One test series of silver and endosulfan in combination showed that toxicity was additive. Comparative sea urchin and sand dollar embryo tests (72 to 96 h) showed embryo sensitivity to be similar to sperm sensitivities with levels of effect (retardation of development) of 50 and 25 μg/litre, respectively, for silver and 253 and 158 μg/litre for endosulfan. Additional development and refinement of this sperm cell toxicity test should yield a rapid and sensitive procedure for screening large numbers of toxicants in marine waters, aid in the prediction of ecological effects, and provide an additional tool for the biomonitoring of receiving water quality.

Application of the sea urchin sperm bioassay to sewage treatment efficiency and toxicity in marine waters

Abstract A newly standardized 60-minute sea urchin (and sand dollar) sperm bioassay was used to measure the toxicity of sewage at each stage in the treatment process. The sensitivity of the sperm bioassay was compared to the results of 48- to 96-hour bioassays with sea urchin and oyster embryos and crab zoeae in side-by-side tests of each sewage type. These data were then used to estimate the dilutions necessary to protect sensitive life stages of marine animals around a proposed sewage outfall in Puget Sound and to select the best bioassay procedure for a post-discharge program for monitoring water column toxicity. Results showed that very little acute toxicity was removed by the primary treatment process but that toxicity was greatly reduced by secondary treatment. A high degree of toxicity was reintroduced by chlorination but this source of toxicity was essentially eliminated by dechlorination. The simple and rapid sperm assay proved more sensitive to all stages of sewage than did the longer embryo and larval assays. Based on the results of the comparative testing and the ease, rapidity and year-round availability of the sea urchin sperm assay (sea urchins in winter, sand dollars in summer), this test was recommended for post-discharge monitoring of sewage effluent-related toxicity in the receiving waters.

Sea urchin sperm bioassay for sewage and chlorinated seawater and its relation to fish bioassays

Abstract Bioassays were conducted with sea urchin and sand dollar sperm to determine the toxicity of chlorinated and unchlorinated sewage effluent and chlorinated and brominated seawater. The sperm cells were exposed to seawater dilutions of each toxicant for 5–15 min. The fertilisation of eggs served as the indicators of sperm viability. The effective concentrations which reduced fertilisation success by 50% (EC50) averaged 2·2 and 4·8% chlorinated and unchlorinated sewage in seawater, respectively. The sperm cells were extremely sensitive to chlorinated seawater at concentrations from 0·002 to 0·020 mg/litre total residual oxidant (TRO). Brominated seawater proved toxic to sperm in one test at 0·015 mg/litre TRO. Results of the sperm bioassays are compared with previous acute and chronic bioassays with fish.

Toxicity of Chlorine and Heat to Pink (Oncorhynchus gorbuscha) and Chinook Salmon (O. tshawytscha)

Abstract The toxicity of chlorine to juvenile chinook salmon (Oncorhynchus tshawytscha) was tested at five residual concentrations ranging from 0.05 to 1.0 mg/liter and at four temperatures (Δt 0, 2.6, 4.9, and 10 C) in a matrix design. Juvenile pink salmon (O. gorbuscha) were tested at three temperatures (Δt 0, 5.0, and 9.9 C). Each matrix was stressed at four exposure periods (7.5, 15, 30 and 60 min). An array of LC50 and LT50 values are presented for each of the conditions tested. A decrease in the tolerance of both species to residual chlorine was demonstrated with increasing temperature and exposure time. The 60-min exposure period and the 9.9 and 10 C Δt were the most acutely toxic to each species. The equilibrium loss response for chinook salmon exposed to heat and chlorine preceded the LT50 by at least 55%. The LC50 was 0.045 mg/liter for both species after exposure to fluctuating chlorine concentrations during 2-hr periods. The LT50 occurred in about 100 min at approximately 0.5 mg/liter residual...

Acute toxicity and behavioral responses of coho salmon (Oncorhynchus kisutch) and shiner perch (Cymatogaster aggregata) to chlorine in heated sea-water

Abstract The acute toxicity and behavioral response to chlorinated and heated sea-water was determined for coho salmon smolts and 1–3 month old shiner perch. LC50s were determined for 7.5, 15, 30 and 60 min exposure times; 13, 16 and 20°C (Δt = 0, 3, 7°C) temperatures and total residual oxidant (TRO) concentrations ranging from 0.077 to 1.035 mg l−1. The mean 60 min LC50 for shiner perch was significantly reduced (P ≤ 0.05) from 308 μg l−1 TRO at 13°C to 230 μg l−1 TRO at 20°C. The 60 min LC50 for coho salmon decreased from 208 μg l−1 TRO at 13°C to 130 μg l−1 at 20°C. The LC50s for coho salmon in chlorinated sea-water averaged 55% of those for shiner perch. The relationship between TRO concentration, exposure time, and percent survival in chlorinated sea-water at 13°C is presented for both species. A significant (P ≤ 0.01) avoidance threshold for coho salmon occurred at 2 μg l−1 TRO and was reinforced with increasing temperature. A significant (P ≤ 0.01) avoidance threshold for shiner perch occurred at 175 μg l−1 TRO, while a significant preference (P ≤ 0.05 or 0.01) response at 16°C and 20°C occurred at 10, 25, 50 and 100 μg l−1 TRO. The ecological implications of the toxicity tests and the behavioral responses are discussed.

Barrier net to reduce entrainment losses of adult kokanee from Banks Lake, Washington

Abstract A barrier net 1,341 m long was developed to reduce the entrainment of mature kokanee (Oncorhynchus nerka) into the main irrigation canal intake of Banks Lake in Washington. The dacron net was constructed of 83-mm mesh (stretch measure) and relied on the visual avoidance response exhibited by salmonids. The net was hauled by machine and cleaning was done with high-pressure water jets. The screening efficiency of the barrier was evaluated by numerous methods including sampling the fishes entrained in the irrigation canal with large nets, mark and recapture of adult kokanee in the reservoir, estimates of the number of beach spawners, sonic tracking near the barrier, census of the sport fishery, and mortality of kokanee gilled in the barrier. The annual canal entrainment of kokanee declined from an average of 64% before installation of the net to 10% afterwards, based on 4 years of catch data. An estimated 35,391 adult kokanee, based on mark-and-recapture estimates, were retained in the lake during t...

Man's Impact on the Columbia River Stocks of Salmon*

ABSTRACT The impact of man on the salmon and steelhead of the Columbia River includes 35 years of effort to prevent declines in the stocks. Between 1866 and 1940, the commercial fishery harvested an annual average of 29 million pounds of salmon and steelhead, but in the next 35 years the annual average harvest dropped to 8 million pounds. During these 35 years the habitat for anadromous fishes dropped from 163,200 to 72,800 square miles. Various approaches to mitigation were tried, but the cumulative effects of the impacts were not compensated for and the runs declined. The productivity in terms of returning adults per spawner has become less than 1 for the upper river stocks. The management flexibility of allowing additional losses at any stage in the life history of these stocks has been lost. Thus, a policy of zero impact for all new developments has been adopted.