Johnson et al.: Fecundity and egg weight in Pleuronectes vetulus 
235 
sampled fish. Concentrations of biliary protein were 
determined by the method of Lowry et al. (1951) with 
bovine serum albumin (BSA) as the standard. 
Liver and ovary tissue were analyzed for PCB’s by 
following the method described by MacLeod et al. ( 1985 ) 
and modifications later described in Stein et al. ( 1987). 
Tissue samples (approximately 2 g) were ground with 
10 g of silica and then added to a column (270 x 23 mm) 
containing 3 g of activated silica gel (Amicon Corp., 
Danvers, MA) held in place by a glass wool plug. PCB’s 
were eluted with pentane: methylene chloride (90:10, 
V/V). The first 50 mL of eluant were collected, concen- 
trated, and exchanged with 1 mL of hexane prior to 
analysis by gas chromatography with electron capture 
detection (GC/ECD) (MacLeod et al., 1985). Selected 
samples of ovary tissue required the removal of lipids 
by size-exclusion HPLC (Krahn et al., 1988) prior to 
analysis with GC/ECD. 
Plasma estradiol- 17/? concentrations were deter- 
mined by radioimmunoassay as described by Sower 
and Schreck (1982). Plasma glucose and triglycer- 
ides were determined as described by Casillas et al. 
(1983) and Casillas and Ames (1986), respectively. 
Fish age was estimated from length by using site- 
specific age-length curves calculated from length and 
age data collected from female English sole sampled 
during previous studies in Puget Sound. Fish ages 
were determined from otolith analysis (Chilton and 
Beamish, 1982). Site-specific growth relationships were 
fitted by using the von Bertalanffy growth curve (Ricker, 
1987), and age was then estimated with the formula 
age = t - ((ln( 1 - length / ))) / K , 
where t = time at which length = 0; 
L m = asymptotic length; and 
K = Brody’s growth coefficient. 
Substituting site-specific values for t , L m , and K into 
the general formula, age-length equations for female 
sole from the specific sites were as follows: 
age Port Susan = - 3 - 41 - (dn(l-length/487)))/ 
0.096; 
a £ e Smciair inlet = -1-82 - ((ln( 1-length/ 445)))/ 
0.200; 
^Duwaimsh Waterway = ~ 2 - 87 ~ ((ln(l-fe/lgffc/586)))/ 
0.085; 
a^Eagie Harbor = -2.4 1 - (( ln( l-length/394 )))/ 
0.209. 
Statistical analyses 
Data were initially analyzed to identify the major 
biological factors affecting fecundity and egg weight 
so that potential confounding factors could be ad- 
justed before evaluating the impacts of sampling site 
and contaminant exposure on these endpoints. Analy- 
sis of variance ( ANOVA), and Fisher’s protected least- 
significant difference multiple-comparison test 
(Fisher’s PLSD) were used to examine the effects of 
site, year, and month of capture on fecundity and egg 
weight. Intersite differences in ovarian atresia sever- 
ity, an ordinal variable, were compared by using the 
nonparametric Kruskall-Wallis test. Linear regression 
analysis was used to examine the relationships of fe- 
cundity and egg weight with biological variables (i.e. 
fish size, condition, and gonadosomatic index (GSI). 
Stepwise multiple-regression analysis was subse- 
quently used to assess the relationships of fecundity 
and egg weight with indicators of contaminant expo- 
sure (e.g. tissue PCB levels, biliary FAC’s, and site of 
capture) after adjusting for relevant biological factors 
identified in initial regression analyses. Data were log- 
transformed as necessary prior to statistical analyses 
to normalize data and reduce heteroscedasticity. These 
standard statistical analyses are described in detail in 
Sokal and Rohlf ( 1981 ) and Dowdy and Wearden ( 1991 ). 
For all statistical tests, a was set at 0.05. 
Results 
Biological factors affecting egg production 
The number of eggs produced by sole from our sam- 
pling sites ranged from approximately 120,000 in a 
28-cm TL fish to approximately 1.2 million in a 43- 
cm TL fish. In multiple regression analysis (Table 
1), fish length was the strongest predictor of fecun- 
dity, but GSI (an indicator of the level of gonadal de- 
velopment) also showed significant associations with 
fecundity. Fish length explained the highest propor- 
tion (48%) of variation in fecundity; GSI accounted 
for 8% of variation in fecundity. Fish age was also 
positively correlated with fecundity (r 2 =0.41, 
P=0.0001, /?=47), but the association was not as 
strong as the association between fecundity and 
length because of the high variability in size, and 
consequently, in egg production, among fish of the 
same age class. After the influences of fish length 
and GSI had been accounted for, fish age and sam- 
pling time had weak but significant negative rela- 
tionships with fecundity, a finding that suggests a 
tendency for fecundity to decline in older animals 
and at the end of the sampling season. Sampling time 
and age accounted for approximately 3%’ and 4% of 
variation in fecundity, respectively. 
In contrast to fecundity, egg weight was not highly 
correlated with either fish size or age but was re- 
