Paul A. Dinnel

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.

Inter-laboratory comparison of a sediment toxicity test using the marine amphipod Rhepoxynius abronius

Abstract An inter-laboratory comparison of the Swartz et al. (1985) amphipod sediment toxicity test was performed for seven marine sediments of and one a posteriori hypotheses or criteria were tested for three end points (survival, emergence and reburial). The bioassay met the a priori criterion of success, acceptable survival and behavior (emergence and reburial) of controls. It also met two of three a priori hypotheses: acceptable agreement on the rank order of toxicity for all three end points and acceptable agreement on mean values for the end points. The third hypothesis, classification of sediments as toxic or non-toxic, was only met for the emergence end point; however, this was probably due to the narrow range of toxic sediments tested (four of the seven sediments tested were only marginally toxic). Review of these and other amphipod sediment toxicity test data indicates that sediments that are clearly nontoxic (survival is greater than 87%) and those that are clearly toxic (survival is less than 76%) will be accurately classified whereas those of marginal toxicity (survival is between 76% and 87%) can only be classified based on emergence data. An a posteriori comparison indicated that the amphipod sediment toxicity test was more precise in LC50 and EC50 determinations with a reference toxicant (cadmium-amended sediments) than has previously been shown in inter-laboratory comparisons. Based on the results of this study, we recommend the wider use of this toxicity test to determine the toxicity of field-collected marine sediments and for laboratory studies with contaminant-amended sediments.

Effects on sea urchin fertilization and embryogenesis of water and sediment from two rivers in Campania, Italy

Sea urchin embryos and sperm were utilized for evaluating the toxicity of water and sediment from two rivers, the Sarno (S) River and the Volturno (V) River, in the Campania region, Italy. The effects on developing embryos were evaluated by scoring developmental defects, whereas sperm exposure was tested for the effects on fertilization and offspring quality. Ten sampling sites from the rivers (S.1–S.4 and V.1–V.6) were monitored for water and sediment quality. Water sampling was carried out biweekly for a year (1988–1989); the samples were tested at dilutions 10−4 to 10−2 in natural seawater. Sediment tests were carried out on solid phase samples (collected in 1989 to 1992), at concentrations ranging from 2 to 10 mg/ml (dry wt) in seawater. The tests conducted on water samples mostly led to nonsignificant results in either embryo-or spermiotoxicity, possibly due to sharp changes in pollutant levels in the water column. Unlike water, sediment samples displayed clear-cut results both on embryogenesis and on fertilization success. The grain size of sediment failed to reveal any relationship with toxicity, which could only be referred to the presence of toxic contaminants (Melluso et al., 1993). The most polluted sediment samples displayed a dramatic embryo-toxicity, up to approximately 87% developmental arrest in embryos reared in 2 mg/ml of sediment from site S.1 (affected by leather tanning effluent). These results were independent of storage conditions of sediment samples (i.e., at +4°C or −20°C). A decrease in fertilization success was also induced by sediment samples from polluted sites (not by a relatively unpolluted reference sample). Interestingly, sediment spermiotoxicity appeared to be related to storage temperature in samples from two agricultural sites (S.3 and V.6), in that freezing caused a drop in sample spermiotoxicity compared to refrigeration; e.g., a sample from site V.6 stored at +4°C decreased fertilization rate (FR) to 9% versus a control value of 86%, whereas the same sample induced but a minor change following storage at −20°C (FR=68%).In general, a better reliability was observed for sediment bioassays than for water bioassays, thus suggesting that future biomonitoring studies should mainly focus on sediment quality. The use of solid phase sediment in toxicity testing by sea urchin embryos and sperm is warranted by the present results.

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.

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.

An analysis of the relationship between a sand-dollar embryo elutriate assay and sediment contaminants from stations in an urban embayment of puget sound, Washington

Abstract A sand-dollar embryo test was used to assess the toxicity of contaminants in sediment elutriate samples from Puget Sound, Washington. A synoptic chemical data set of priority pollutants was reduced and subjected to combinatorial clustering which grouped stations by the amount of chemicals present. Clustering was done for metals and organic compounds together and separately. Analysis of variance revealed that the embryo test was able to predict the group of stations considered least contaminated by organic chemicals but not for metals, although copper and lead could not be excluded due to confounding effects. The results generally support the additivity hypothesis of toxicity in that as total contamination increased toxicity increased. Due to a possible change in redox conditions or the release of bioorganically bound metals, it was concluded that the elutriate test may not be appropriate for assessment of metal contaminants associated with sediment. A dose-response relationship between embryo abnormality and the degree of elutriate dilution was observed which demonstrates that serial dilutions of an elutriate should be tested to properly characterize a sediments toxicity.