laminated seawater. One hour after fertilization, 

 three replicates of 25-35 eggs for each treatment 

 level were placed in glass incubation jars (12.8 X 5.4 

 cm) which were wrapped with black tape and aerated 

 with Pasteur pipettes connected to air pumps. Each 

 jar contained 100 ml of decontaminated seawater to 

 which various levels of BaP had been added as de- 

 scribed above. In all cases, sand was excluded as an in- 

 cubation medium. Dissolved BaP concentrations 

 were measured when the California grunion eggs 

 were introduced into the glass jars and on alternate 

 days until day 15, using fluorescence spectroscopy 

 (365 nm excitation, 405 nm emission) (Felton et al. 

 1982). Seawater samples (2 or 4 ml) were analyzed by 

 Aminco-Bowman spectrophotofluorometer sensi- 

 tive to 2 ng BaP. 



Hatching and morphological abnormalities were 

 observed and photographed at intervals over a 14-d 

 period using a Wild M5 dissecting microscope and a 

 Zeiss 5 photomicroscopy attachment. The signifi- 

 cance of arc sin-transformed percentages of abnor- 

 malities was tested using analysis of variance 

 followed by the Student-Newman-Keuls multiple 

 range test (Sokal and Rohlf 1969). Notochord length 

 of embryo and yolk-sac larvae was measured using a 

 calibrated ocular micrometer. Notochord length was 

 defined as the distance from the tip of the snout to the 

 tip of the notochord before flexion and was always 

 measured on the left side of the embryos and larvae. 

 Deformed, circular-shaped embryos were measured 

 from the posterior tip of the deformed notochord to 

 the opposite side of the embryo, and this diameter 

 was used to calculate the circumference which was 

 considered to be equal to the notochord length. All 

 values were recorded to the nearest 0.1 mm. Dif- 

 ferences were tested using analyses of variance and 

 the Student-Newman-Keuls test. 



To measure accumulation of BaP by California grun- 

 ion embryos, two additional series of incubation jars 

 were prepared containing similar BaP concen- 

 trations and to which was added a small amount (6.7 

 nCi) of {al , 10- U C) benzo(a)pyrene (Amersham/ 

 Searle Corp., Arlington Heights, III; 21.7 mCi/mmol, 

 997( chemical and radiochemical purity). On alter- 

 nate days, three replicate samples of two eggs each 

 were taken from the 14 C-BaP series to measure BaP 

 accumulation using the method of Hose et al. (1981). 

 Radioactivity was measured using a Beckman LS250 

 scintillation counter with an efficiency of 80% at 4°C. 

 Total radioactivity was calculated using a series of 

 solubilized embryos as the quenched standards. 



FISHERY BULLETIN. VOL. 81, NO. 3 



RESULTS 

 BaP Determinations 



Added BaP levels of 5, 10, 100, 500, 1,000, and 

 5,000 ppb to seawater yielded initial BaP concen- 

 trations in the incubation jars of 4, 7, 24, 297, 361, 

 and 869 ppb, respectively, when measured at time 

 "0" when the California grunion eggs were in- 

 troduced into the jars. Dissolved BaP levels declined 

 thereafter with a half- life of 3.0±0.1 d (x±SD) until 

 steady-state levels of 24 ppb (361 ppb initial), 9 ppb 

 (297 ppb initial), 5 ppb (24 ppb initial), 3 ppb (7 ppb 

 initial), and 2 ppb (ppb initial) were reached within 4- 

 10 d (Fig. 1). Stable BaP levels occurred most rapidly 

 at lowest doses, while BaP concentrations in jars 

 receiving the highest initial dose of 869 ppb de- 

 creased throughout the experimental period and did 

 not achieve steady-state. 



Accumulation of 14 C-BaP 



The amount of BaP plus its metabolites in each egg 

 corresponding to measured l4 C increased from day 1 



1000 



5 Reference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



DAY 



FIGURE 1.— Dissolved benzo(a)pyrene (BaP) concentrations follow- 

 ing addition of 5-5,000 ppb BaP during the 1 4-d incubation period 

 for the California grunion. 



474 



