gonadal maturation, and were thermally conditioned for 4 weeks in unfiltered seawater at 14 

 ±- 1°C. The mussels were fed a daily diet of a marine diatom culture (Phaeodactylum 

 tricornutwn). Prior to spawning, 30 mussels were stored moist at 5°C for 24 hours. 



Spawning was induced by placing the chilled mussels in individual Pyrex™ dishes 

 containing 250 mL of 1 |im filtered seawater at 20°C. Fertilization was accomplished within 

 1 h of spawning initiation by combining eggs and sperm in a 1-L Nalgene beaker. The 

 fertilized eggs were then washed through a 250 urn Nitex screen to remove excess gonadal 

 material and suspended in 2 L of filtered seawater at incubating temperature 20°C. The 

 embryos were kept suspended prior to testing by frequent agitation with a perforated plunger. 

 When microscopic examination of fertilized eggs revealed the formation of polar bodies, 

 triplicate counts were made of the number of eggs in 1.0 mL samples of a 1:99 dilution of the 

 homogeneous egg suspension. 



Elutriate Preparation. Sediment toxicity tests were conducted in clean, distilled water-rinsed 

 1-L polyethylene bottles. Twenty grams (g) (wet weight (ww)) of sediment was added to 

 each bottle and the volume brought up to 1 L with filtered seawater (30 ppt salinity) to 

 make a final concentration in all containers of 20 g (ww) of sediment per liter of seawater. 

 The sediments were suspended by vigorous shaking for 10 seconds and were allowed to settle 

 at incubation temperature for 1 hour prior to adding the embryos. No additional agitation 

 was provided after inoculation. 



Test Procedures. Toxicity testing was conducted following the standardized procedures of 

 Chapman and Morgan (1983), updated by Chapman and Becker (1986). Within 2 hours after 

 fertilization, approximately 15,000 developing mussel embryos were inoculated by automatic 

 pipette into each container, resulting in a concentration of about 15 per mL. The containers 

 were covered and incubated in a temperature-controlled room for 48 hours at 17+ 0.5°C under a 

 14-h light:10-h dark photoperiod. Test vessels were not aerated during the test. After 48 

 hours, surviving larvae were removed from the water column of each container by automatic 

 pipette. Repeated, gentle mixing with a perforated plunger was used to ensure that the 

 larvae were homogeneously suspended prior to removal of a 7-mL aliquot. The bottom 

 sediments were not disturbed during the subsampling as bivalve larvae are pelagic and do not 

 associate with the benthos until metamorphosis occurs. Previous experience has shown that 

 larvae found in the sediments invariably are dead. Live larvae were transferred to 8-mL 

 screw-cap glass vials and preserved in 5 percent buffered formalin. The preserved samples 

 (equal in volume to that containing at least 100 larvae in controls) were examined in 

 Sedgewick-Rafter cells under 100 times magnification. As bivalve larvae sink after 

 preservation (ASTM, 1985), half of the water was discarded from the vials before examining 

 the residual volume containing the larvae. Control sediments collected off West Beach in 

 Puget Sound, Washington (the collection site for the amphipod Rhepoxynius abronius test 

 animals) were tested in the same manner. 



Normal and abnormal prodissoconch I larvae were enumerated to determine percent 

 survival and percent abnormality. Percent survival in the 15 samples was determined as the 

 number of normal and abnormal prodissoconch I larvae surviving in each test container 

 relative to the number surviving in the seawater control, which was assigned a survival 

 value of 100 percent. Larvae which failed to transform to the fully shelled, straight-hinged, 

 "D" shaped prodissoconch I stage were considered abnormal. The weighted rather than 

 arithmetic method was used to calculate mean larval abnormality for a given station 

 (including controls) because numbers of larvae vary within treatments and abnormality tends 

 to increase as mortality increases (ASTM, 1985). This method involves multiplying the 

 percent abnormal value for each replicate by the ratio between the percent abnormals in the 

 replicate and the total percent abnormals for all five replicates. 



Parallel reference toxicant bioassays (in clean seawater without sediment) were 

 conducted using sodium pentachlorophenate (NaPCP). A 100 ppm stock solution of NaPCP 

 was prepared in a 0.04 mol solution of sodium hydroxide using anhydrous grade 

 pentachlorophenol (Sigma Chemicals), following procedures described in Niimi and 

 McFadden (1982). Bioassay concentrations were prepared in duplicate by volumetric dilution 



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