the site of capture of flounder in the mersey estuary ( and in nearby liverpool bay ) , the method of capture , and the method of sample collection are described in previous articles ( allen et al .
data on vtg concentrations used in the present study were taken from these previous studies . however , the analysis of data by stage of maturation in the present article is new .
the histological sections of gonads were also prepared during the course of the previous studies ( allen et al .
this determination was based upon the presence of the most advanced stage of gamete development that could be seen in histological sections of the gonads described below .
ovaries : f1 , primary oocytes only ; f2 , cortical alveoli - stage oocytes as well as primary oocytes ; f3a , a mixture of cortical alveoli - stage and secondary ( i.e. , vitellogenic ) oocytes ; f3b , predominantly secondary oocytes .
testes : m1 , spermatogonia only ; m2a , secondary spermatogonia ; m2b , primary spermatocytes through to spermatids ; m3a , some spermatozoa in addition to spermatocytes and spermatids ; m3a , spermatozoa only . to increase the numbers of fish in each grouping ( for statistical purposes ) , we combined the subcategories ( i.e. , a and b ) for the analyses of vtg , e2 , and 11-kt concentrations . for steroid assay , plasma aliquots ( 100 l )
were shaken vigorously with 50 l distilled water and 4 ml diethyl ether for 2 min .
the solvent phase was poured into a glass tube ( 12 mm  75 mm ) , then evaporated in a water bath at 45c .
the residue was redissolved in 400 l radioimmunoassay ( ria ) buffer and measured as described previously for e2 and 11-kt ( scott et al .
two 100-l aliquots were used for assay of e2 , and an additional two 50-l aliquots were used for assay of 11-kt . a plasma sample  spiked  with 2 ng / ml
sixty plasma samples from flounder collected in 1997 were also assayed twice ( and by two different people ) 4 years apart .
the correlation coefficient between values generated by the two assays was 0.93 , and the slope of the relationship was 1.03 .
data were statistically analyzed by analysis of variance followed by comparison of means using duncan s multiple range test at a significance level of 0.05 .
the numbers of fish caught at each maturation stage and at each sampling time in the mersey estuary between 1996 and 2003 are shown in table 1 . for the purpose of analyzing steroid and vtg concentrations
, we divided fish of both sexes into three groups that roughly equated to  immature ,   on the borderline between immaturity and maturity ,  and  mature . 
this last category encompassed the whole period of time from the first appearance of secondary oocytes and secondary spermatocytes up until the fish were ready to spawn .
the histological and sex steroid data indicate that the secondary maturation phase in flounder lasts from september until march .
mean concentrations of e2 , 11-kt , and vtg in males and females , categorized by sampling date and stage of maturation , are summarized in tables 2 and 3 . when only one or two fish were at a particular stage of maturation , the data were not included in the tables .
the data in tables 2 and 3 were used to explore the relationship between sex , stage of maturation , and time of year versus vtg and sex steroid concentrations in flounder .
mean e2 concentrations in males were low ( between 0.08 and 0.22 ng / ml , table 2 ) and displayed no obvious relationship to the time of year or stage of sexual maturity .
most important , there was no evidence that high vtg concentrations were caused by high e2 concentrations ; in fact , there was no relationship ( r = 0.03 ; n = 158 ) between e2 and vtg concentrations in males ( figure 1 ) .
we found no obvious trend in vtg concentrations of either mature or immature males throughout the year ( figure 2 ) and no statistical difference between fish at different stages of maturation ( table 2 ) .
in contrast , mean 11-kt concentrations in males showed a regular month - on - month increase between september and march ( figure 3 ) and were also different between mature and immature fish ( see statistics in table 2 ) .
mean e2 concentrations in females were strongly influenced by stage of maturation ( table 3 ) .
e2 concentrations in immature females were never higher than those concentrations in either immature or mature males .
in mature females , however , concomitant with the beginning of secondary oocyte development in september , e2 concentrations started to rise and reached a peak of 20 ng / ml at spawning time in march ( figure 4 ) .
as with males , we saw no obvious trend or pattern in vtg concentrations between september and march ( figure 5 ) .
values fluctuated between 2,800 and 18,000 g / ml . these concentrations were , nevertheless , in all cases higher than those found in immature females sampled at the same time ( table 3 ) . also , vtg concentrations in immature females were never at any sampling time significantly different from vtg concentrations in mature or immature males ( analysis not shown ) .
mature males caught in december 1996 had significantly lower mean 11-kt concentrations than mature males caught in december 2002 ( table 4 ) . however , there was no significant difference between mean 11-kt concentrations in males caught in february 2001 and those caught in february 2003 ( table 4 ) .
mature females caught in december 1996 had significantly lower e2 concentrations than mature females caught in december 2002 ( table 4 ) .
we also observed a significant difference between e2 concentrations in mature females caught in february 2001 and those caught in february 2003 ( table 4 ) . in all cases ,
the lower e2 concentrations were found in the years when vtg concentrations in males were higher , which suggests that the presence of edcs is associated with lower endogenous steroid synthesis .
we firmly concluded from this study that the vtg found in the plasma of male flounder in the mersey estuary is not induced by endogenous e2 .
although this conclusion seems nave , substantial concentrations of e2 ( > 1 ng / ml ) have been found in blood plasma of reproductively mature males of a closely - related species , the north sea plaice ( pleuronectes platessa ) ( scott et al . 2000 ; wingfield and grimm 1977 ) .
the concentrations of e2 in male flounder in the present study , however , were < 0.22 ng / ml , never higher than those found in immature females , and thus were unlikely to be a trigger for vtg synthesis .
exogenous compound(s ) , acting directly on the estrogen receptor , are the more likely cause .
the number of fish caught at each maturity stage , plus steroid concentration data , in each month clearly indicates that the maturity cycle in both males and females begins about september and culminates in spawning ( in the open sea ) in march and april .
2004a ) of females caught in both the river mersey and the river dee estuaries between 1998 and 2000 .
unlike in those studies , vtg concentrations in mature females in the present study showed no relationship to month of capture .
the reason for this finding is probably that our samples have been collected over a period of 7 years , during which the level of estrogenic contamination has varied dramatically from a high in 1996 to a relative low in 2003 ( kirby et al .
this high variability in contamination has probably obscured the cycle . a previous observation that estrogen - induced vtg concentrations in male flounder also have a seasonal cycle ( kirby et al .
2004c ) was also obscured by the high year - to - year variations in estrogenic edcs in the mersey .
if , as seems likely , elevated vtg concentrations are mainly caused by estrogenic edcs , then one might hypothesize that these same edcs have a negative feedback effect on the secretion of gonadotropin from the pituitary and , hence , on 11-kt secretion by the testis in the males .
the high dependence of 11-kt concentrations on stage of maturation and month of capture meant that , despite the large overall number of observations , it was only possible to examine this hypothesis via two pairs of data with the same month of capture and with a sufficient number of mature males for statistical analysis ( table 4 ) . in december 1996 ,
when vtg concentrations were high , 11-kt concentrations were significantly lower than in december 2002 , when vtg concentrations were 20 times lower . in february 2001 and 2003
though , when vtg concentrations were much closer together ( although still significantly different ) , 11-kt concentrations were not significantly different from each other . in females in september ,
december , and february , mean e2 concentrations were significantly lower in the years when mean vtg concentrations were higher .
although supported by statistical analysis , the finding that mean steroid concentrations are in most cases lower in the years when the effects of exogenous estrogens are higher must be interpreted with caution . at the moment
, it is an association only and needs to be tested in a laboratory experiment .
there are many other factors that could be the cause of 2- to 3-fold differences in steroid concentrations between years .
the sampling in the present study covered 7 years ; therefore , another possible reason is that the differences were caused by deterioration of steroids during long - term storage .
however , as indicated in  materials and methods ,  there was no evidence of any deterioration of immunoreactivity of e2 in plasma samples that were stored for 4 years .
bearing in mind that in no case is there necessarily a causal link , high plasma concentration of vtg in flounder have so far been associated with higher incidences of testicular malformation ( gill et al .
1997 , 1998 ) , higher amounts of oocyte malformation ( lye et al . 1998 ) , more sperm abnormalities ( gill et al . 2002 ) , more pathological lesions in the liver and kidney ( simpson et al .
however , there has been no clear link with degree of intersex ( allen et al . 1999a ; kirby et al .
we stress the lack of a causal link because there are many other xenobiotics in estuaries ( not just estrogens ) that work through different mechanisms .
these other compounds could cause the observed changes ( without themselves affecting vtg concentrations ) .
an important practical outcome of the present study was that vtg concentrations in immature females ( i.e. , those shown by histological examination to possess only primary oocytes ) were not statistically different from those found in males .
the importance of this finding is that , until now , estuary surveys ( kirby et al .
2004a ) have largely ignored data on vtg concentrations in females because of the uncertainty about the possible role of endogenous e2 .
however , our data suggest that , provided females can be proved immature by histological examination , their vtg concentrations can be pooled with those of males to increase the overall number of observations at each site .
finally , an important lesson we have learned from the present study is that future field surveys should , if possible , be rigidly planned to catch flounder at the same places and at the same times each year . only by ensuring such consistency is there any point in measuring sex steroid concentrations in flounder .
we have also already concluded that only with rigid scheduling of field surveys is it possible to obtain clear - cut data on long - term trends in estrogenic edc contamination in estuaries ( kirby et al .
few studies have been conducted on the population consequences of estrogenic edcs on marine species . in the present article
, we show that high vtg concentrations in male and female flounder caught in the mersey estuary are not caused by endogenous e2 .
rather , evidence indicates that elevated vtg concentrations are associated with a reduction in the synthesis of e2 by females and of 11-kt by males .
several mechanisms are possible , but the most likely is that the exogenous estrogenic edcs not only enhance the production of vtg by the liver but also lower sex steroid production by negative feedback on the hypothalamic  pituitary axis .
work on other species suggests that reductions of sex steroid levels may have an adverse effect on fecundity and fertility .
 high concentrations of vitellogenin ( vtg ; egg yolk protein ) have previously been found in male flounder ( platichthys flesus ) from several uk estuaries ; these levels have been ascribed to the presence of estrogenic endocrine - disrupting compounds ( edcs ) . 
 gonadal abnormalities , including intersex , have also been recorded in these estuaries . 
 however , there is no firm evidence to date that these two findings are causally linked or that the presence of estrogenic edcs has any adverse population effects . in the present study 
 , we examined the relationship between concentrations of vtg and sex steroids ( 11-oxotestosterone in males and 17-estradiol in females ) in specimens of flounder captured from the estuary of the river mersey . 
 we first questioned whether the high concentrations of vtg in male and immature female flounder were indeed caused by a direct effect of exogenous edcs and not indirectly via the endogenous secretion of 17-estradiol . 
 the data favored the direct involvement of estrogenic edcs . 
 we then questioned whether the presence of estrogenic edcs not only stimulated inappropriate vtg synthesis but whether it might also have had a negative effect on endogenous steroid secretion . 
 it should be noted that the predicted consequences of a drop in steroid secretion include smaller gonads , smaller oocytes , fewer numbers of sperm , and depressed spawning behavior . 
 this question was more difficult to answer because of the strong effect of the seasonal reproductive cycle and stage of maturation on steroid concentrations . 
 however , matched by month of capture and stage of maturation , both 17-estradiol in females and 11-keto - testosterone in males were in most cases significantly lower in those years when vtg concentrations were higher . 
Materials and Methods
Results
Discussion
Conclusions