cytologic/cytogenetic examinations. The embryos in 200 mL of the remaining solution were 

 removed with a plastic screen (44 urn) and extracted for echinochrome pigment measurement. 



Microscopic examination was used to determine abnormalities in development of the 48-h 

 embryos. The numbers of embryos in the prism stage that had a normal pyramid shape, a 

 differentiated gut, and well-developed skeletal rods were counted as normal. Embryos 

 exhibiting an unusual pattern of development, such as exogastrulation, blastula filled with 

 cells, or unhatched embryo with abnormal cleavage were classified as abnormal. Embryos 

 that appeared normal, but at a less advanced stage of development (gastrula or earlier) after 

 48 hours, were counted and classified as retarded. The samples were examined randomly 

 without knowledge of the station identification. 



To determine echinochrome pigment content, 48-h embryos that had been removed from 

 200 mL of the test sample were transferred to a centrifuge tube with seawater and 

 concentrated by centrifugation (1,500 x G for 5 min). The overlying water then was removed 

 by aspiration, the pellet was washed with 95 percent ethanol and re-centrifuged. The 

 ethanol then was removed, and 1 mL of acidified ethanol (5 percent HC1) was added to each 

 tube and mixed to extract the echinochrome. The absorbance of the echinochrome-containing 

 supernatant was measured with a spectrophotometer at 475 nanometer (nm), using the 

 methods of Bay et al. (1983). 



To determine cytologic/cytogenetic abnormalities, preserved 48-h embryos were placed on 

 a glass microscope slide and stained with aceto-orcein solution for 15 min. A glass coverslip 

 was then placed onto the slide and the embryos were squashed into monolayers. Twenty 

 embryos were examined from each replicate sample. The number of mitoses in each embryo 

 was recorded, and all of the cells in each embryo were examined for micronucleated cells, 

 mitotic (anaphase) aberrations, and cytologic abnormalities, following the criteria of Hose 

 (1985). 



Two batches of samples were tested in order to minimize storage of the sediments before 

 testing: the first with samples from the VA, YB, and OA sites; and the second with samples 

 from the TB and SP sites. Each experiment included two controls; a laboratory seawater 

 control from Redondo Beach, California and an elutriate control (laboratory seawater carried 

 through the elutriate preparation steps). 



Sediment Pore Water Toxicity Test with the Polychaete Dinophilus gurociliatus 



Laboratory bioassay data with spiked sediments indicate that sediment toxicity is more 

 highly correlated with interstitial (pore) water contamination than with total sediment 

 concentrations of toxicants (DiToro, in press). This toxicity test had been developed and 

 applied in bioassays of effluents and single chemicals in water (Carr et al, 1986), but had not 

 been used previously in sediment quality assessments. 



Pore water extraction. A 2- to 3-L aliquot of the homogenized sample from each station was 

 transferred to a Zip-Lock bag, labeled, and stored on ice in a cooler or in a refrigerator at 

 approximately 4°C. Sediments were transferred from the container to a pressurized Teflon- 

 lined steel cylinder for extraction of the pore water on the same day that the samples were 

 collected, using the methods of Carr et al, in press. A porous Teflon filter plate and a 0.7 urn 

 porosity borosilicate glass filter were mounted in the base of the cylinder. The system was 

 pressurized with compressed air supplied from a standard SCUBA cylinder. Pressure was 

 applied to the top of the cylinder chamber via a first-stage regulator and manifold, forcing 

 the top plate downward. The pore water samples emitted from the bottom of the cylinder 

 were collected in amber glass bottles with Teflon-lined lids and stored frozen until they were 

 used in the toxicity tests. 



Animal collections. A population of Dinophilus gyrociliatus cultured in the laboratory was 

 used in the toxicity tests. 



11 



