METHODS 



Sediment Sampling 



The NS&T Program protocols include collection of sediments at each of three stations per 

 sampling site. Accordingly, in this evaluation, three stations, generally separated by 50 to 

 100 meters, were sampled at each site. Samples were collected with a O.lm^ Young grab 

 sampler (similar to a modified Van Veen grab sampler). Multiple (usually 6 to 10) grab 

 samples were taken at each station and the upper 1 centimeter (cm) of sediment was removed 

 with a Teflon-lined, stainless-steel, calibrated scoop. These 1-cm thick samples were 

 collected in a stainless steel, Teflon-lined basin until about 7 liters (L) of sediment had been 

 accumulated and composited from each station. The sediments then were homogenized for 

 approximately 5 min. with a Teflon-lined steel spoon until the composited sample appeared 

 homogeneous. Portions of varying sizes of the composited sample from each station then were 

 removed for each of the chemical and sedimentological analyses and toxicity tests. Care was 

 taken to avoid contamination of the samples. Sampling was conducted in February 1987. 



All toxicity tests were performed with five laboratory replicates or aliquots of the 

 composited sediments per station. The sediment samples for chemical analyses were frozen 

 at -40°C and stored for a maximum of 60 days until the analyses were performed. The 

 toxicity tests were performed on three phases of the sediments: solid, elutriate, and pore 

 water. All except the pore water test were conducted on nonfrozen samples held for no more 

 than 5 days. 



Fish Sampling 



Starry founder (Platichthys stelktus) were collected twice: in November/ December 1986 

 when fish were anticipated to be late in the reproductive cycle but not yet ready to spawn, 

 and January/February 1987 when the fish were expected to be sexually mature. Fish were 

 captured with 5- and 7-m otter trawls towed for 20 min. in water depths of 2.5 to 7 meters 

 behind a research vessel. A target of 30 and 10 to 15 fish per site was set for each sampling 

 period, respectively, to facilitate determinations of between-site differences in measures of 

 effects. Fish less than 20 cm were not retained. When more than 15 fish were caught at a 

 station, more of the larger, sexually mature individuals were kept. 



Immediately upon capture of the fish, they were bled from either the caudal vein, gill 

 arch, or heart to obtain samples for the micronuclei and hormone analyses. A 1-milliliter 

 (mL) blood sample was centrifuged in the field and frozen at -76°C for the future hormone 

 analyses. A blood smear was prepared on a slide, fixed in alcohol, and kept in cold storage 

 for the micronuclei analyses. 



Captured fish were maintained on the vessel in flowing bay water until transported to 

 the recirculating marine aquaria at LLNL. Fish were sacrificed the day following capture. 

 Solvent-rinsed tools were used to remove livers and the gonads, which were weighed and 

 aliquots of each put aside for subsequent analyses. For each fish, the gonadosomatic index 

 (GSI) was calculated as [gonad weight/(body weight - gonad weight)] x 1,000. The 

 hepatosomatic index (HSI) was calculated similarly. Standard length was determined for 

 each fish. The fish were kept alive until just before necropsy. Liver and ovary were 

 removed and frozen at -76°C for enzyme and chemical analyses. An additional subsample of 

 ovary was collected and fixed in Davidson's fluid for histological examination. 



Female fish captured in January/February were taken to the laboratory in Livermore to 

 be spawned for an evaluation of measures of reproductive success. 



