Usually only the pooled aliquot from each spawning was evaluated. Dead eggs and embryos 

 were removed daily and preserved in 10 percent neutral formalin. After 80 to 100 hours, the 

 remaining sample was preserved and H:F and L:H ratios were later determined. 



8. In nearly all cases, the pooled aliquot was used to provide a single determination 

 per spawning of each reproductive success measure. The measures of percent floating eggs 

 and percent fertilization success in the serial aliquots were used in the above-mentioned 

 analysis of variance. 



9. Females had between two and five spawnings, with most females sacrificed after 

 three spawnings. As mentioned above, the mean values of each measure for all spawnings 

 was used to derive one series of reproductive success values for each female. 



Fish Plasma Hormone Analyses 



Plasma samples collected in the field from live fish were frozen at -76°C and shipped 

 to Dr. Peter Thomas at the University of Texas for hormone analyses. Estradiol-17B and 

 testosterone were analyzed by radioimmunoassay (RIA) techniques in P. stellatus. The 

 estradiol-17B antiserum generated against estradiol-17B -3-carboxymethyl-ether BSA 

 (Radioassay Systems Laboratories) cross-reacted 22.3 percent with 16-ketoestradiol, 2.46 

 percent with estriol, and 1.32 percent with estrone. The assay could detect 2.5 picograms 

 (pg) estradiol-17B per assay tube. The testosterone antiserum prepared against testosterone- 

 3-BSA (Cambridge Medical Diagnostics) was relatively specific and cross-reacted 28.2 

 percent with dihydrotestosterone, 17.2 percent with 11-ketotestosterone, and 1.46 percent 

 with androstenedione. The assay could detect 1.25 pg testosterone per assay tube. 



Radioimmunoassay for testosterone and estradiol-17B were performed on the same 

 plasma extract. One hundred microliters of plasma were extracted with 2 mL hexane/ethyl 

 acetate (70:30) in 12 X 75 mm borosilicate tubes. Prior to extraction, tritiated testosterone 

 (1,800 counts per min (cpm)) was added to each sample for determination of extraction 

 efficiency. The extract was dried under a stream of nitrogen and reconstituted in 250 mL of 

 gelatin assay buffer (21.2 micromole (mM) phosphate buffer, pH 7.6). Two aliquots (50 mL 

 and 25 mL) of the reconstituted sample were measured in each RIA to test for parallelism. 

 In the estradiol-17B assay, 50 mL of tritiated steroid tracer (approximately 4,000 cpm, 

 Radioassay Systems Laboratories) and 75 mL of antiserum (1 in 23,200 dilution) were added 

 to the samples and the standards. In the testosterone assay, 25 mL of tritiated steroid tracer 

 (approximately 4,000 cpm, Research Products International) and 25 mL of antiserum (1, in 

 9,000 dilution) were added to the samples and standards. Assay mixtures were incubated 

 overnight at 4°C and bound steroid was separated from free steroid with dextran-coated 

 charcoal. 



The intra-assay coefficients of variation (CV) of replicate determinations of estradiol- 

 17B and testosterone in a plasma control pool were 16.8 and 8.3 percent, respectively 

 (estradiol-17B mean 1.14 ng/mL, s.e.m 0.15, N=3; testosterone 2.12 ng/mL, s.e.m 0.04, N=3). 

 Recovery of steroid standards (40-250 pg/tube) added to the control plasma ranged from 94.5 

 to 119 percent. 



Fish Erythrocyte Micronuclei Analyses 



The number of micronuclei in peripheral erythrocytes of the fish was determined by the 

 SCCWRP and Occidental College. A total of 158 fish were examined from the two sampling 

 periods. 



The micronucleus technique was applied to circulating peripheral erythrocytes in 

 approximately 1 cc of blood removed in a heparinized syringe from the caudal vein. For 

 each fish, three blood smears were prepared immediately with one drop of blood each, 



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