Fish Hepatic Aryl Hydrocarbon Hydroxylase Activity 



Slices <5 mm thick were removed from the posterior liver for the in-vitro assay of 

 microsomal enzyme activity. These were immediately frozen on dry ice and transferred 

 within an hour to a freezer maintained at -76°C. One or more liver slices were later used to 

 prepare a microsomal pellet. 



These microsomes were assayed for AHH activity using the method of Nebert and 

 Gelboin (1968) as described previously (Spies et al., 1982). A portion of the hepatic 

 microsomes from each fish was re-assayed with 10"^ M 7,8-BF. This concentration was 

 determined to cause maximal suppression of AHH activity. A more optimal assay 

 temperature was determined to be 25°C rather than 18 or 37°C. At 25°C, reaction kinetics 

 were linear for 10 min. An Aminco Bowman spectrofluorometer was used to quantify the 3- 

 OH benzo(a)pyrene metabolite. The spectrofluorometer was calibrated with quinine sulfate 

 standards. In addition, a standard of microsomes from 3-methylchoIanthrene-induced mice 

 was assayed with each batch of microsomes as a control for assay conditions. Fluoresence 

 values of assays were corrected according to the quinine sulfate standard. Based on 

 duplicate and triplicate assays from seven fish, the mean coefficient of variation was 14.6 

 percent for AHH activity, and 2 percent for the mean percent change in AHH activity with 

 the addition of the AHH inhibitor, 7,8-BF. Protein concentrations were determined by the 

 method of Lowry et al. (1951) using bovine serum albumin (BSA) as the standard. Hepatic 

 AHH specific activities, are reported as picomoles 3-OH-benzo(a)pyrene (B(a)p) milligrams 

 (mg) protein"! min"* . 



Fish Reproductive Success 



For the November-December collections, a small section of the gonad of each female 

 was removed during necropsy and preserved in Davidson's fixative. Appropriate subsections 

 were taken from female gonads in these samples and made up into paraffin sections at 6 \im, 

 mounted on microscope slides and stained with hematoxylin and eosin. These were later 

 examined to evaluate the predominant egg stages present (Yamamoto, 1956) and for the 

 occurrence of atretic oocytes. 



Fish captured in the January-February sampling period were taken live to the LLNL to 

 be spawned. Spawning success of several stages in reproduction was determined. 



Fish handling. The spawning methods of Policansky and Sieswierda (1979), the only known 

 procedure for spawning Platichthys stellatus, were adopted. After capture by trawl from San 

 Francisco Bay in the winter months, 1 to 3 days were allowed for acclimation to the 

 laboratory seawater system that is maintained at 11 to 13°C and at a salinity of 29 to 30 

 ppt. The holding aquaria measured 58 x 58 x 47 cm (158.1 L) and two to three females were 

 placed in each aquarium. Males were generally maintained separately. Gonadally mature 

 females were then started on a course of intramuscular injections of freeze-dried pituitaries 

 from spawning carp (Crescent Biochemicals) reconstituted in physiological saline, 1 mg 

 pituitary kg'^d"^. Over 90 percent of females, captured mainly in the months of January and 

 February, responded by eventually spawning. The mean time to spawning in all fish (n=49) 

 captured in previous research and so treated in 1983-1985, was 27 ± 13 days. Females 

 generally spawned between two and five times. Females that had not spawned within 43 

 days were eliminated from analyses because of the positive relationship observed between 

 numbers of days of injections and hepatic AHH activity after 47 d (Rice et al, in press). It 

 was not always necessary to administer pituitary injections as males, unlike females, were 

 often captured in San Francisco Bay in a condition in which sperm could be stripped from 

 them and with a photoperiod of less than 8 h d'l males came into spawning condition in the 

 laboratory. 



22 



