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Pure Appl. Chem., Vol. 75, Nos. 11–12, pp. 2219–2234, 2003.
© 2003 IUPAC
2219
Topic 4.3
Endocrine disruption in wild freshwater fish*
Susan Jobling1,‡ and Charles R. Tyler2
1Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8
3PH, UK; 2School of Biological Sciences, Hatherly Laboratory, Exeter University,
Exeter, Devon, EX4 4PS, UK
Abstract: Endocrine disruption has been reported in freshwater fish populations around the
world. This phenomenon ranges from subtle changes in the physiology and sexual behavior
of fish to permanently altered sexual differentiation and impairment of fertility. Despite wide-
spread reports of endocrine disruption in fish (and this is well characterized at the individual
level), few studies have demonstrated population-level consequences as a result of exposure
to endocrine-disrupting chemicals (EDCs). An exception to this is in Lake Ontario Lake trout
where precipitous declines in the population have been linked with periods of high exposure
to organochlorine chemicals (known EDCs). Recently, it has been established that roach
(Rutilus rutilus) exposed to treated sewage effluent (that contains complex mixtures of
EDCs) in UK rivers, have a reduced reproductive capacity. This, in turn, may have popula-
tion-level consequences.
Evidence for a link between exposure to effluents from kraft mill (BKME) and sewage
treatment works (STW) and altered reproductive function in freshwater fish is compelling. In
most cases, however, a causal link between a specific chemical and a physiological effect has
not been established. Indeed, identifying specific chemical(s) responsible for adverse effects
observed in the wild is difficult, given that tens of thousands of man-made chemicals enter
the aquatic environment and that mixtures of chemicals can have combination (e.g., additive)
effects. Some EDCs are known to act at a number of different body targets to affect a variety
of physiological processes, further complicating the identification of the causative agent(s).
Endocrine disruption appears to be particularly widespread in freshwater fish popula-
tions. There is little evidence, however, to suggest fish are more susceptible to EDCs relative
to other wildlife. Notwithstanding this, there are some features of the endocrine physiology
of fish that may be particularly susceptible to the effects of EDCs, including the processes of
sex-determination and smoltification (in salmonids). Furthermore, their aquatic existence
means that fish can be bathed constantly in a solution containing pollutants. In addition, up-
take of chemicals readily occurs via the gills and skin, as well as via the diet (the major ex-
posure route for most EDCs in terrestrial animals). The exposure of fish early life stages to
the cocktail of EDCs present in some aquatic environments may be of particular concern,
given that this is an especially vulnerable period in their development.
The challenge, from the point of view of ecological risk assessment, is to determine ef-
fects of EDCs on freshwater fish populations and freshwater ecosystems. In order to meet
this challenge, high-quality data are required on the population biology of freshwater fish, on
the effects of EDCs on their various life history characteristics, and comprehensive and ap-
*Report from a SCOPE/IUPAC project: Implication of Endocrine Active Substances for Human and Wildlife (J. Miyamoto and
J. Burger, editors). Other reports are published in this issue, Pure Appl. Chem. 75, 1617–2615 (2003).
‡Corresponding author
propriate population models. Basic information on the population biology of most species of
wild freshwater fish is, however, extremely limited, and needs significant improvement for
use in deriving a sound understanding of how EDCs affect fish population sustainability.
Notwithstanding this, we need to start to undertake possible/probable predictions of popula-
tion level effects of EDCs using data derived from the effects found in individual fish.
Furthermore, information on the geographical extent of endocrine disruption in freshwater
fish is vital for understanding the impact of EDCs in fish populations. This can be derived
using published statistical associations between endocrine disruption in individual fish and
pollutant concentration in receiving waters. Simplistic population models, based on the ef-
fects of EDCs on the reproductive success of individual fish can also used to model the likely
population responses to EDCs. Wherever there is sufficient evidence for endocrine disruption
in freshwater fish and the need for remediation has been established, then there is a need to
focus on how these problems can be alleviated. Where industrial chemicals are identified as
causative agents, a practical program of tighter regulation for their discharge and/or a switch
to alternative chemicals (which do not act as EDCs) is needed. There are recent examples
where such strategies have been adopted, and these have been successful in reducing the im-
pacts of EDCs from point source discharges on freshwater fish. Where EDCs are of natural
origin (e.g., sex steroid hormones from human and animal waste), however, remediation is a
more difficult task. Regulation of the release of these chemicals can probably be achieved
only by improvements in treatment processes and/or the implementation of systems that
specifically remove and degrade them before their discharge into the aquatic environment.
INTRODUCTION
Endocrine disruption has been reported in freshwater fish populations in various parts of the world [re-
viewed in 1,2]. This phenomenon ranges from subtle changes in the physiology and sexual behavior of
fish, to permanently altered sexual differentiation and impairment of fertility. Most of the data comes
from studies in Europe and America, although evidence for endocrine disruption in freshwater fish has
also been reported in Australia [3] and Japan [4]. Biological effects in wild freshwater fish that have
been attributed to the effects of endocrine disruptors include the inappropriate production of the blood
protein vitellogenin (VTG; the female-specific and estrogen-dependent egg yolk protein precursor) in
male and juvenile fish, inhibited ovarian or testicular development, abnormal blood steroid concentra-
tions, intersexuality and/or masculinization or feminization of the internal or external genitalia, im-
paired reproductive output, precocious male and/or female maturation, increased ovarian atresia (in fe-
male fish), reduced spawning success, reduced hatching success and/or larval survival, altered growth
and development (thyroid hormone-like effects) and alterations in early development (altered rate or
pattern) [2]. These effects may arise due to disruption of a range of endocrine-mediated mechanisms
(including receptor-mediated processes, and/or interference with steroid metabolism and/or excretion),
although nonendocrine toxicity could also explain some of these effects. Overall, current scientific ev-
idence strongly suggests that certain effects observed in freshwater fish can be attributed to cocktails of
chemicals that mimic and/or disrupt hormone function/balance. In most cases, however, the evidence of
a causal link between a specific physiological disruptor and a specific effect is weak, largely due to the
fact that freshwater fish and, indeed, all other wildlife, are exposed to a wide range of chemicals, that
act at a number of different body targets, to affect a variety of physiological processes. Sewage treat-
ment works (STWs), for example, (which often receive domestic, industrial and/or agricultural waste)
release a complex (and ill-defined) mixture of natural and synthetic chemicals into the aquatic envi-
ronment, following their partial or complete biodegradation during the treatment process. It is estimated
that 60 000 man-made chemicals are in routine use worldwide and most of these enter the aquatic en-
vironment [5]. Identifying specific chemical(s) responsible for adverse effects observed in the wild is,
thus, difficult and requires extensive laboratory studies to support the hypotheses drawn from field stud-
S. JOBLING AND C. R.TYLER
© 2003 IUPAC, Pure and Applied Chemistry 75, 2219–2234
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ies. Moreover, very recent studies investigating the interactive effects of mixtures of estrogenic chemi-
cals in fish, using vitellogenin induction as an endpoint, have shown that combinations of steroid es-
trogens, alkylphenolic chemicals and a pesticide (methoxychlor) are additive in their effect [6]. This
highlights the fact that even chemicals that are have slight effects on the endocrine system should be
taken into consideration when assessing the effects of chemical mixtures in freshwater fish. A weak link
in establishing whether observed adverse effects in freshwater fish are caused by exposure to EDCs is
the lack of data documenting what freshwater fish are actually exposed to and what they take up into
their bodies. Moreover, there is often a large discrepancy between the relatively high levels of pollu-
tants generally used in laboratory studies and the low levels of these pollutants that actually occur in the
aquatic environment. Exposures of fish to environmentally relevant concentrations of EDCs (and at the
relevant life stages) are essential to adequately evaluate exposure/response relationships in field studies
and produce credible risk assessments.
CURRENT EVIDENCE FOR ENDOCRINE DISRUPTION IN FRESHWATER FISH
Although there is a considerable amount of evidence for endocrine disruption in wild freshwater fish,
only in a very few cases has a causal link between the presence of EDCs in freshwaters and altered en-
docrine function in exposed fish populations been demonstrated. In order to determine causality be-
tween an EDC and a particular perturbation, clearly, a relationship between exposure to the putative
stressor and the effect of concern needs to be firmly established (e.g., decline in the population or re-
duced fertility). For a chemical to be designated an endocrine disrupter, exposure to the stressor has to
result in an endocrine-mediated event (and at the relevant exposure concentration that occurs in the en-
vironment) that ultimately results in an effect of concern. In the following section of the review, docu-
mented examples of endocrine disruption in wild freshwater fish are described. Those examples for
which there is considerable evidence for a link between exposure and effect are described first, followed
by cases where the evidence is less convincing and/or where further research is much needed in order
to provide a definitive association.
Reproductive abnormalities in freshwater fish living downstream of pulp- and paper-
mill effluents
Over the last 10 years, a number of species of freshwater fish in Canada (white sucker, Catostomus com-
mersoni; longnose sucker, Catostomus catostomus; lake whitefish, Coregonus clupea formis [7–15])
and Europe (perch, Perca fluviatilis; roach, Rutilus rutilus; [16,17]) living downstream of pulp- and
paper-mill effluents have been found to exhibit an array of altered features in their reproductive devel-
opment, including reductions in gonadal growth, inhibition of spermatogenesis, depressed sex steroids,
reduced pituitary hormone concentrations, and delayed sexual maturity. In the studies on perch (but not
for the suckers) viability of the developing larvae was also affected [18]. Lowered egg production and
delayed reproduction have also been induced in fathead minnows in life-long exposures to bleached
kraft mill effluents (BKMEs) [19]. Furthermore, the endocrine changes seen in wild fish are less severe
during periods of reduced effluent discharge [20] and decrease with increasing distance from the efflu-
ent outfalls into the rivers. There is, thus, very strong evidence to suggest that something in the BKME
is causing the adverse effects seen. The causative agents responsible for these reproductive effects in
fish in Canada and Europe have, however, not been identified [21], although in a very recent in vivo
study, using a toxicity identification and evaluation approach, Hewitt et al. [22] were able to provide the
first evidence that at least one of the effects (the depression in steroid hormone concentrations) seen in
wild fish in the vicinity of pulp mills may be due to products of the degradation of lignin. The authors
showed that these chemicals were present in active fractions of the effluent that caused depressions in
serum testosterone concentrations in mummichogs both in vitro and in vivo. Moreover, although not
proven, other studies have suggested that the reproductive effects may (at least in part) be mediated
© 2003 IUPAC, Pure and Applied Chemistry 75, 2219–2234
Endocrine disruption in wild freshwater fish 2221
through disruption of the process of steroidogenesis, by affecting the availability of cholesterol and
pregnenolone and thus impairing steroid production by the gonads [23,24]. Still other in vitro studies
suggest that mixtures of both estrogenic (e.g., β-sitosterol, lignans, stilbenes, and resin acids [25,26])
and androgenic chemicals (e.g., stigmastanol and a β-sitosterol degradation products [27]), together
with Ah-receptor agonists (e.g., polychlorinated dibenzofurans and thianthrenes, dibenzothiphenes, and
diphenyl sulfides), are found in these effluents, and these studies are supported by in vivo studies that
show that white suckers, living in the vicinity of BKME discharges, rapidly accumulate chemicals that
bind to the estrogen receptor, androgen receptor, and sex steroid binding protein [28]. Another study
showed that during the spawning migration of white sucker in Jackfish Bay in Canada, returning fish
were found to have altered pituitary function, as determined by depressed levels of luteinizing hormone
(LH) in males and females compared with control fish from a reference location [29]. When taken to-
gether, the evidence shows clearly that the endocrine disrupters within BKME act at many targets in the
hypothalamic-pituitary-gonad axis. Although it has not (thus far) been possible to link endocrine dis-
ruption (leading to deleterious effects on reproduction and development) in these various species of fish
to a specific chemical or group of chemicals, it is clear that the endocrine effects are clearly linked to
the constituents of pulp-mill effluents.
Interestingly, the multiplicity of androgenic-, estrogenic-, and steroidogenesis-inhibiting chemi-
cals in paper-mill effluents reported for BKMEs in Canada has not been reported for BKMEs in Florida,
USA. Instead, in Florida, only androgenic effects have been identified. In these studies, development of
a male gonopodium was observed in female mosquito fish exposed to BKME (an androgenic effect
[30,31]), but no apparent feminizing effects were seen in males. A recent in vitro study by Parks et al.
[32] determined that the pulp-mill effluent from a Florida mill exhibited androgenic activity (deter-
mined by transcriptional activity of the androgen receptor) at levels sufficient to account for the mas-
culinization of the female mosquitofish. It is not yet known whether the differences in effects of BKME
on fish in Florida and Canada are due to differences in species sensitivities, or to different substances
discharged into the BKME in Canada compared with that in Florida. Further characterization of the ef-
fluents is needed to more fully understand causation. The ecological significance of the physiological
effects of BKME are not known, but could be hypothesized to result in the gradual impairment and
eventual loss of reproductive function after continued BKME exposure. These seemingly intuitive pop-
ulation-level predictions have not, however, been observed directly in any wild population of fish ex-
posed to BKME. Indeed, some recent evidence suggests the contrary, LeBlanc et al. [33], for example,
recently observed a reduction in the intensity and duration of the spawning period in Fundulus hetero-
clitus exposed to BKME in the Mirichami Estuary, New Brunswick, Canada, but they also reported a
simultaneous marked increase in reproductive investment and increased fecundity in these individuals.
Reproductive abnormalities in freshwater fish living downstream of sewage treatment
works discharges
There is considerable (and increasing) evidence for endocrine disruption in freshwater fish populations
living in stretches of river downstream of treated sewage effluent discharges in Europe [34–40], Canada
and America [41–43] as well as more recent evidence of endocrine disruption in riverine carp in Osaka
in Japan [4]. In the original work on freshwater fish, conducted in the United Kingdom, it was estab-
lished that effluents from treated sewage effluents were estrogenic, inducing the production of vitel-
logenin, in male fish [44]. Vitellogenin is normally synthesized by the liver in female oviparous (egg-
laying) vertebrates in response to estrogen and is sequestered by developing oocytes and stored as yolk
to act as a nutrient reserve for the subsequent development of the embryo [45]. The production of VTG,
therefore, is usually restricted to females. Male fish however, do contain the VTG gene(s), and expo-
sure to both natural and synthetic estrogens can trigger its expression, resulting in the secretion of VTG
in the blood plasma [46]. Vitellogenin is now one of the most widely used biomarkers for exposure to
estrogen(s) in fish in freshwaters and it has been detected in the blood of both male and juvenile fish in
S. JOBLING AND C. R.TYLER
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rivers, lakes and streams contaminated by effluents from STWs and/or mixtures of estrogens [reviewed
in 47]. Although almost all effluents tested in the United Kingdom have been shown to be estrogenic,
causing induction of VTG in exposed male fish, there are some STW effluents in the United States that
do not appear to be estrogenic to fish (they do not induce VTG [48,49]), probably due to the large di-
lution that occurs when the effluent reaches the receiving river and/or to the more extensive sewage
treatment processes that are in place at these sites.
In addition to VTG production, exposure to treated sewage effluents has also been associated with
deleterious effects on gonad differentiation and development [3,4,34–43,50] in various species of fish
and with the abnormal development (feminization) of secondary sexual characteristics in male
mosquitofish (Gambusia affinis) in Australia [3]. The most thoroughly studied effects are concerned
with the widespread incidence of intersex reported in some species of freshwater fish in the United
Kingdom, parts of Continental Europe, and the United States. Freshwater fish species in which an oc-
currence of intersex has been reported and deemed to be abnormal include the roach (R. rutilus [37]),
bream (Abramis abramis [34,35]), the chub [38], gudgeon (Gobio gobio [36]), the barbel (Barbus ple-
bejus [39]), the perch (Perca fluviatilis), the stickleback (Gasterosteus aculeatus [40]), and the shovel-
nose sturgeon (Scaphirhynchus platyorynchus [41]). Intersex as a consequence to exposure to effluent
has been most intensively studied in the roach, a cyprinid fish common throughout lowland rivers in the
United Kingdom and Europe. At some river sites downstream from large STW discharges in the United
Kingdom, all of the “male” roach population has been reported to be intersex [37]. Intersex roach often
have both male and female reproductive ducts, and many also have female germ cells (oocytes) within
a predominantly male “testis”. The number, pattern, and developmental stage of oocytes within testic-
ular tissue in intersex roach vary greatly; the condition ranges from the presence of single primary
oocytes scattered randomly throughout testicular tissue in a mosaic fashion, to a condition in the more
severely feminized fish, where large areas of ovarian tissue occur that are clearly separated from testic-
ular tissue [50]. Intersex roach also often have an altered endocrine status (altered plasma sex steroid
hormone concentrations), and an elevated concentration of plasma VTG relative to normal male fish
[51] and gonadal growth is often inhibited in severely intersex roach. More recent studies suggest that
intersex roach (R. rutilus) also have impaired fertility relative to normal male fish from reference sites.
Small numbers of wild roach in UK rivers were found that could not produce any gametes at all due to
the presence of severely disrupted gonadal ducts [52]. Fertilization and hatchability studies have further
shown that intersex roach (even with a low level of gonadal disruption—“mildly intersex”) are com-
promised in their reproductive capacity and produce fewer offspring than fish from uncontaminated
sites. In these studies, an inverse correlation was demonstrated between reproductive performance (de-
fined as the ability to produce viable offspring) in intersex roach and severity of gonadal intersex. This,
in turn suggests that the intersex condition is quite likely to have population level consequences, al-
though further studies on wild populations are necessary to confirm or refute this.
In contrast to the effects observed in male roach, effects in female roach living in rivers contam-
inated by treated sewage effluents in UK rivers were less obvious [52]: There was a higher incidence of
oocyte atresia and a slight, but statistically significant, lower fecundity in effluent-exposed fish com-
pared with females from the reference sites. Interestingly, at some river sites, small proportions (up to
14 %) of the adult female fish (aged between 3 and 7 years) were sexually immature or sexually indif-
ferent, and, although not proven, it is possible that these effects are also due to endocrine disruption.
There is substantive evidence (principally from lab-based studies, see below) to support that hy-
pothesis that gonadal disruption in wild freshwater fish, inhabiting rivers that receive treated sewage ef-
fluents, is caused by estrogenic substances contained within these effluents. Moreover, the statistical as-
sociations between the various gonadal abnormalities that occur in wild freshwater fish and plasma
VTG concentrations [34,37,51], adds further weight to the evidence that suggests these effects are all
caused by estrogenic factors within the effluent.
Analyses of treated sewage effluents using toxicity, identification, and evaluation (TIE) ap-
proaches have shown that estrogens and their mimics are present in most, if not all, treated sewage ef-
© 2003 IUPAC, Pure and Applied Chemistry 75, 2219–2234
Endocrine disruption in wild freshwater fish 2223
fluents [53–55]. Studies in the United Kingdom have indicated that alkylphenolic compounds (e.g.,
nonylphenol, NP) and low levels of natural and synthetic steroidal estrogens (estradiol-17βestrone and
17αethynylestradiol) are the primary estrogenic constituents of sewage effluents [53]. Moreover, lab-
oratory studies have shown that the concentrations of the 17αethynylestradiol, estradiol-17β, and es-
trone [6,44,55–58] or (in some industrial effluents) alkylphenolic chemicals [6,52], present in STW ef-
fluent in England are sufficient to explain the induction of vitellogenin synthesis in caged fish placed
close to effluent discharges. Many rivers (in which the fish live) contain more dilute STW effluent, and
thus the concentrations of estrogens in these rivers may not be high enough to induce plasma VTG that
is seen in wild fish (based on short-term exposures). Longer-term exposures of freshwater fish to efflu-
ents have, however, been shown to reduce the threshold level for effect; a study by Rodgers-Gray et al.
[59] found that exposure of roach to a STW effluent for 1 month induced a vitellogenic response at an
effluent concentration of 37.9 +/– 2.3 %, but at an effluent concentration of only 9.4 +/– 0.9 %, after a
4-month exposure. The abnormal occurrence of VTG in wild freshwater fish is thus likely to occur, in
many cases, as a result of long-term exposure to mixtures of estrogens present in effluents. It is proba-
ble that natural and synthetic estrogens, and in some instances, alkylphenolic chemicals found in STW
effluents, also cause the effects on gonadal development and differentiation, and play a part in the evo-
lution of intersexuality in wild fish; both groups of chemicals have been shown to do this under labo-
ratory conditions. Concentrations of steroid estrogens and/or xenoestrogens required to induce these ef-
fect on the gonad, however, are higher than found in most effluents [e.g., 60–62], with the exception of
some highly polluted rivers, and/or in times of drought (when river flow is low and the contribution
made by effluent is high). Few studies have investigated whether environmentally relevant concentra-
tions of estrogens within effluents, or indeed, the effluents themselves can cause the effects on the
gonad duct seen in wild freshwater fish populations. In a study in which juvenile roach were exposed
to a treated sewage effluent, it was proven that feminization of the development of the gonadal duct
(prevalent in wild roach in UK and European rivers) occurs as a consequence of exposure to treated
sewage effluent during the period of sexual differentiation [63]. Furthermore, a lab-based study has
shown that gonad duct disruption can be induced in fish exposed to ethinyloestradiol at a concentration
found in some treated effluents, when the exposure occurs during early life [64]. Although it is theo-
retically possible to produce an intersex or sex-reversed fish by exposure to sex steroid hormones or
alkylphenols (usually during early life), in relatively short-term exposures, even higher concentrations
are required to do so than for those inducing duct disruption. Furthermore, induction of altered sex cell
development has not been shown in fish exposed to sewage effluents in controlled experiments. The rea-
sons for this might be that the effluents used for the exposures [63] did not contain a sufficient concen-
tration of the causative agent(s) and/or that the appropriate life stages have not been exposed and/or that
the fish were not exposed long enough to cause this effect (the maximum duration of these exposures
was 4 months). In our own unpublished studies on wild roach, we have found a positive correlation be-
tween the age of the fish (length of the exposure) and the severity of the intersex condition. This sug-
gests that in real exposure scenarios (such as roach living in an effluent contaminated river in the United
Kingdom), the longevity of the exposure might be of greater importance for disruptions in sex cell de-
velopment (inducing oocytes in the testis), than the window in development during which the exposure
occurs. In support of this hypothesis, NP has been shown to induce ovo-testes in the medaka, at a con-
centration of only 17 µg/l in the water when the exposure was life-long [65].
In summary, it seems that exposure of freshwater fish in the wild to natural steroidal and synthetic
estrogens and, in some instances, alkylphenols cause inappropriate VTG induction and disruptions in
the development of the reproductive ducts. Although not yet proven, it seems likely that these chemi-
cals are also responsible for (or at least significantly contribute to) the occurrence of oocytes in the
testes of male fish, for retarded testicular and ovarian development and delayed maturation.
S. JOBLING AND C. R.TYLER
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Early life stage mortality syndrome and blue sac disease
There are very few studies that have demonstrated that freshwater fish are being impacted at the popu-
lation level by exposure to a specific chemical (including EDCs). One such case, however, is for lake
trout (Salvelinus namaycush) living in Lake Ontario where exposure to tetrachlorodibenzodifuran
(TCDD) and coplanar polychlorinated biphenyls (PCBs) caused population declines because of nega-
tive impacts on reproductive success and early life survival [reviewed in 66]. The organochlorines in-
duced a condition called blue sac disease, which is characterized by yolk sac edema, hemorrhaging,
cranofacial abnormalities, and mortality in early larval development. Lake trout populations in Ontario
declined precipitously during the 1950s when environmental concentrations of organochlorine chemi-
cals were the at their highest. Subsequent, retrospective studies (based on measured PCB, PCDF, and
PCDD residues in dated sediment cores) have established a strong relationship between the concentra-
tions of TCDD, PCDDs, and PCDFs and the observed historical trends in lake trout reproduction, in-
cluding the more recent signs of successful reproduction [67,68]. Laboratory studies have also shown
that exposure to Ah (aryl-hydrocarbon) receptor agonists, including TCDD and coplanar PCBs, induces
blue sac disease [69], but there is no evidence to show that these effects occur through an endocrine-
mediated mechanism.
Reduced hatching success, low embryo survival, and slower rates of development in fry have also
been reported in lake trout in the Great Lakes [70,71] and in Arctic char (Salvelinus alpinus) in Lake
Geneva [72] and causally linked with exposure to coplanar PCBs, TCDDs, and PCDFs. Other condi-
tions found in the Great Lakes fish during the 1960–1980s including early mortality syndrome and (in
Baltic salmon) M74, resulting from thiamine deficiency, were thought to have a chemical etiology [73].
Like blue sac disease, M74 affects fry and is characterized by a loss of equilibrium, spiral swimming,
lethargy, hemorrhaging, and death. There are data correlating incidences of M74 in Baltic salmon to el-
evated body burdens of PCDFs and coplanar PCBs and DDT [e.g., 74,75]. In none of these examples
on fish in the Great Lakes, however, is there sufficient evidence to link the effects seen to a specific en-
docrine disruptor and/or their mixtures. Furthermore, the mechanisms via which these effects occur are
generally unknown, and thus ascribing these effects to endocrine disruption at this time would be inap-
propriate.
Thyroid dysfunction in Great Lakes fish
Alterations in thyroid function have been reported in several wild populations of fish as a consequence
of disruptions in their endocrine systems. Epizootics of thyroid hyperplasia and hypertrophy (affecting
the whole population) have been reported in various species of salmonids in heavily polluted regions of
the Great Lakes in the United States [76–79]. Although enlargement of the thyroid gland can occur as
a result of iodine deficiency in the diet, this has been ruled out as a causative factor in the case of these
salmonids. It was originally hypothesized that organochlorine contaminants, functioning as EDCs
might be responsible for these effects [79]. Studies in the laboratory have shown that goiters and de-
pressed thyroid hormone concentrations can be induced in rodents fed with contaminated fish from the
Great Lakes, although fish fed with the same contaminated fish did not develop thyroid lesions [80,81].
Laboratory-based studies, however, have failed to identify the causative chemicals of these thyroid dis-
ruptions in the wild fish [reviewed in 82]. In summary, more than 40 years after the discovery of the
thyroid dysfunctions in salmonids in the Great Lakes, although a chemical etiology has been estab-
lished, the mechanism (endocrine, or otherwise) via which these effects occur is still uncertain. Very re-
cently, thyroid abnormalities were also reported in mummichogs (Fundulus heteroclitus) from a pol-
luted site (Piles Creek, New Jersey, USA) in the United States [83]. These effects have been loosely
associated with exposure to a range of contaminants, especially mercury and petroleum hydrocarbons.
When taken together, these studies suggest that thyroid function in fish appears to be sensitive to con-
taminant exposure generally.
© 2003 IUPAC, Pure and Applied Chemistry 75, 2219–2234
Endocrine disruption in wild freshwater fish 2225
Disruptions in adrenal physiology
There is a limited amount of evidence to suggest that environmental contaminants chronically stress
fish, resulting in a compromised responsiveness of the HPI axis [84–89]. For example, Hontela et al.
[84–86] demonstrated that yellow perch (Perca flavescens) and Northern pike (Esox lucius) from sites
in Canada contaminated with heavy metals, PCBs, and PAHs were unable to produce cortisol in re-
sponse to acute handling stress. Moreover, the adrenocorticotrophic hormone (ACTH)-producing cells
(corticotrophs) in these fish were severely atrophied. Other studies by Hontela [87] have shown that
both corticotrophs and the interrenal steroid producing cells undergo atrophy when fish are exposed to
PAHs, PCBs, and heavy metals. It was speculated by the authors that the atrophy of the cells was a re-
sult of prolonged secretory hyperactivity of the cells. This hypothesis was later supported by studies on
brown trout living in metal-contaminated waters that were shown to be hyper secreting ACTH and cor-
ticotrophin-releasing hormone [88,89]. More research is necessary to establish if the effects seen on the
interrenal axis during exposure to specific contaminants have consequences to the health of affected fish
populations.
ARE FRESHWATER FISH MORE SUSCEPTIBLE TO ENDOCRINE DISRUPTORS THAN
OTHER ANIMALS?
Endocrine disruption appears to be particularly widespread in freshwater fish populations. There is lit-
tle evidence, however, to suggest that fish are more susceptible to EDCs relative to other wildlife.
Indeed, the evidence available on receptor binding affinities for chemicals that mimic sex steroid hor-
mone, thyroid, and retinoic acid receptors suggests that vertebrates are likely to be similarly sensitive
to environmental EDCs. Furthermore, there are many more similarities between the endocrine systems
of fish and other higher vertebrates, notably with respect to the nature of the hormones, their receptors,
and in the regulatory control of their endocrine system [7]. Notwithstanding this, there are more than
10 000 species of freshwater fish worldwide, displaying a high degree of heterogeneity in their physi-
ology, anatomy, behavior, and ecology, and there are some features of the endocrine physiology of
freshwater fish that may be particularly susceptible to the effects of EDCs, including those that deter-
mine sex (sex determination in fish has been shown to be especially sensitive to steroid hormones) and
the process of smoltification in salmonids.
Living in the aquatic environment, fish can be bathed constantly in a solution of chemical pollu-
tants. Furthermore, uptake of chemicals into fish can readily occur via the gills and skin, as well as via
the diet (the major route of exposure to EDCs in terrestrial animals) [90]. Features of the gills includ-
ing thin epithelial membranes and a large surface area coupled with the relatively high ventilation rates
that occur in fish, facilitate the uptake of compounds from the water and their transfer into the blood
stream. Some freshwater fish species are also top-predators and thus, are likely to bioconcentrate EDCs
to a greater degree than other organisms at lower trophic levels. Freshwater fish are hypo-osmotic with
their surroundings and thus a considerable movement of water into their bodies occurs down an osmotic
gradient (taking chemicals with it). A major route of exposure to EDCs in fish during early life is from
contaminants that have accumulated in lipid reserves within the egg as a consequence of maternal trans-
fer during ovary development. These contaminants that have accumulated in the egg are mobilized
when the lipid reserves are metabolized to fuel embryo development, exposing early life stages to es-
pecially high concentrations of EDCs at a time of greatest vulnerability to disruptions in their develop-
ing endocrine system. Furthermore, early life stages of fish have a limited capacity to metabolize and
excrete contaminants, including EDCs. In situ exposures of fish have been used to assess both the
bioavailability of EDCs, contained within complex mixtures, such as treated sewage effluents, and to
determine non-point sources of pollution (agricultural run-off) and their biological effects. In such stud-
ies on rainbow trout, Larsson et al. [91] reported significant bioconcentration factors (in bile) for natu-
ral and synthetic sex steroid hormones (17β-estradiol, estrone, 17αethinylestradiol) of up to 10 000-
S. JOBLING AND C. R.TYLER
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2226
fold after a 3-week exposure, whereas xenoestrogens (e.g., nonylphenol and bisphenol) bioconcentrate
by several hundred to 1000-fold. Hewitt et al. [28] similarly obtained evidence for a very rapid uptake
of EDCs in fish exposed to BKME, but here they also demonstrated that a rapid depuration of these
chemicals occurs too. Apart from these two studies, however, there is very little information on the bio-
availabilty of EDCs in wild fish or caged fish exposed to effluent discharges.
Another important issue that complicates determination of cause–effect relationships for EDCs,
that is sometimes overlooked, is the possible time lag between the time of exposure and the biological
response. Fish for example, living in the vicinity of sewage effluent outfalls will accumulate harmful
contaminants in their tissues which may not cause any immediate deleterious effects, but which might
affect the embryo development of their subsequent offspring. The biological responses in freshwater
fish are very often especially influenced by physical environmental features, and concentrations of con-
taminants in the aquatic environment can vary widely temporally, and hence responses and effects may
vary with season. All of these considerations are rarely taken into account in the analysis and interpre-
tation of field-simulated exposures.
SCALE OF THE PROBLEM OF ENDOCRINE DISRUPTION IN FRESHWATER FISH
Endocrine disruption has only been studied in a small proportion of freshwater fish species, and data on
cyprinids and salmonids dominate the literature. The differences in the sensitivities of different fish
species to the effects of EDCs has not been comprehensively examined, although studies on the effects
of pulp mill [92] and STW effluents [93], respectively, suggest that inter-species differences in sensi-
tivity are likely to exist, between some fish species. Moreover, given the fact that endocrine disruption
is commonly associated with exposure to effluents from domestic or industrial processes that enter
rivers and streams, it seems likely that endocrine disruption in freshwater fish is more widespread than
is currently documented. In the United Kingdom, for example, there are more than 70 000 consented
discharges and 6500 of these are STW. Worldwide, each year, more than 5000 km3of water are used
[94], and this figure is increasing every year. Furthermore, in some rivers, at times of low flow, up to
80 % of the river volume is made up of STW effluent discharge and this figure can be even higher in
periods of drought. Using established statistical associations between endocrine disruption in freshwa-
ter fish and effluent dilution in receiving waters, theoretical predictions of the geographical extent of
endocrine disruption can be estimated. In the United Kingdom, our own unpublished predictions, based
on a statistical association between the concentration and dilution of the sewage effluent and the degree
of feminization in wild fish exposed to the effluent, indicate that intersex fish are likely to exist at more
than 50 % of 464 river sites that have effluent discharges with a population equivalent of more than
10 000. These predictions can be made using simple associative data from surveys of endocrine disrup-
tion in fish from a limited number of rivers (eight, in this case). Obviously, the more rivers from which
one collects data, the better the predictions are likely to be. In our studies on roach in eight UK rivers,
the following equation has been derived linking intersex in roach with effluent concentration in the
rivers from where they were sampled: log (y+ 1) = (0.000 002 88 * x) + 0.203 where y= the intersex
index and x= the concentration of effluent in the river (calculated by dividing the population equiva-
lent of the effluent by the dilution factor for that effluent upon its entry into the river). The intersex index
is a numerical index used to describe the degree of feminization of the gonads, based on their histolog-
ical appearance [37]. The results from the UK analyses in roach for predicting intersexuality are illus-
trated in Fig. 1. This predictive map is now being validated, through determining the actual (observed)
incidence of intersex at 46 of these study sites.
Additional studies in the roach have shown that the intersex index is correlated with fertility of
intersex fish and hence, a predictive map of gamete quality can be constructed using the information on
the intersex index and its relationship with fertility. Using this approach, we have estimated that at ap-
proximately 13 % of the 464 river sites selected for study in the United Kingdom (that receive effluent
discharges with a PE of more than 10000), the degree of intersexuality is estimated to be severe enough
© 2003 IUPAC, Pure and Applied Chemistry 75, 2219–2234
Endocrine disruption in wild freshwater fish 2227
S. JOBLING AND C. R.TYLER
© 2003 IUPAC, Pure and Applied Chemistry 75, 2219–2234
2228
Fig. 1 Predicted intersex indices for populations of roach living downstream of sewage treatment works in selected
rivers throughout the UK. The results were obtained using the following equation that links intersex in roach with
effluent concentration in the rivers from where they were sampled: log (y+ 1) = (0.000 002 88 * x) + 0.203 where
y= the intersex index and x= the concentration of effluent in the river (calculated by dividing the population
equivalent of the effluent by the dilution factor for that effluent upon its entry into the river). The correlation
between the intersex index and sperm quantity and quality predicts the following effects on fertilization success.
Intersex index: 0–1 = little effect, 1–3 = slight effects, 3–4 = moderate effects, 4–>6: severe effects.
to have deleterious effects on gamete quality (fertilization success of intersex individuals would be pre-
dicted to be less than 60 %). At a further 16 % of the sites, the gamete quality of intersex and/or male
fish would be predicted to be impaired, relative to male fish from reference sites. This system could be
extended to other fish populations exposed to sewage and other types of effluent in order to provide a
predictive map of the reproductive effects of endocrine disruption in freshwater fish populations world-
wide.
Notwithstanding this, in any predictive study of ED in freshwater fish, it is important to establish
the influence of age or longevity of exposure upon the effect that one is measuring. Our own investiga-
tions into the inter-relationships between age and intersexuality, for example, suggest that the intra-site
variability in the degree of feminization that one observes in wild intersex roach is severely influenced
by the ages of the fish collected, as older fish are more feminized than their younger counterparts
(Jobling, unpublished data). Consequently, a perceived difference in the incidence and severity of in-
tersex in fish collected from several different sites may be due to the differential age distributions of the
fish sampled, rather than to differences in the endocrine-disrupting potencies of the various waters from
whence the fish were collected.
POPULATION-LEVEL EFFECTS OF ENDOCRINE DISRUPTION
Much of the research on the effects of EDCs in freshwater fish has focused on effects at the individual
level. A major challenge, from the point of view of ecological risk assessment, is to determine effects
of endocrine disruption on populations and ecosystems. In order to meet this challenge, high-quality
data on the population biology of freshwater fish, effects of EDCs on their various life history charac-
teristics, and comprehensive and appropriate population models are needed. Basic information on the
population biology of most species of wild freshwater fish is, however, extremely limited and it needs
significant improvement for use in deriving a sound understanding of how EDCs affect fish population
sustainability. Population growth rate in fish is determined by the balance between birth rate and the
mortality rate. Collective fecundity and mortality thus predict the population’s fate. In a large popula-
tion with a stable age structure and sex ratio, future population size can be predicted from life table and
fecundity data. This assumes, however, that each individual has an equal chance of contributing genes
to the next generation, and this rarely happens in practice due to unequal sex ratios, differences in in-
dividual fertility, nonrandom mating, and variation in age structure. All of these factors influence the
number of breeding individuals and hence, variation in the effective population size. In fish, juveniles
are often not recruited to the adult population until they are 2–4 years old, and hence juvenile mortal-
ity and the rate of sexual maturity have a major bearing on the number of breeding individuals. Various
external factors enhance population growth and others limit, and even prevent, population growth, and
many of these are dependent on the density of the population. The most common density-dependent fac-
tors that limit population growth are food supply, space, predators, disease, and parasitism. Population-
limiting factors in fish that are independent of population density include abiotic factors such as drought
or floods.
Many of the parameters affecting fish population growth rate and sustainability are difficult to
measure accurately in the field and are consequently, poorly understood. Whilst endocrine disrupters
are known to affect factors such as individual fertility, and rate of sexual maturation and fecundity, a
thorough assessment of these effects would require very extensive studies on the general life history and
population biology of the exposed species compared with a reference population. Moreover, population
declines are not usually caused by only one factor alone, but occur because of the effects of a multitude
of factors.
Notwithstanding this, there are some examples in freshwater fish where there is substantial in-
formation on the consequences of endocrine disruption—on key reproductive parameters at the indi-
vidual level, either from studies of wild populations, or from laboratory studies in which fish have been
exposed to concentrations of EDCs known to be present in freshwaters. In the roach, for example, UK
© 2003 IUPAC, Pure and Applied Chemistry 75, 2219–2234
Endocrine disruption in wild freshwater fish 2229
studies have provided sufficient evidence to show that widespread intersexuality, as a result of exposure
to estrogenic sewage effluents, results in reduced fertility; there is a negative correlation between the
degree of feminization of the intersex fish and their fertilization success (r= –0.603; p< 0.001). This
information could be used albeit simplistically, to model the likely population responses to endocrine
disruptors in the wild (in the absence of other factors that might also affect the population). Basic pop-
ulation models are already available, for this purpose. In a recent paper, Gleason and Nacci [95] at-
tempted to model the effects of exposure to 17β-estradiol on populations of fathead minnow based
(based on laboratory studies that showed egg production by females was negatively correlated with
plasma VTG concentrations in the exposed males). The model predicted a negative linear correlation
between the population growth rate of populations of fathead minnow and plasma vitellogenin produc-
tion in males. Although these predictions are based on simple density-independent population models
that require verification in realistic settings, they nevertheless provide a starting place for projecting
population responses to EDCs from lab-based studies.
RECOMMENDATIONS
This account illustrates that there is very good evidence for endocrine disruption in freshwater fish. In
order to more comprehensively assess the importance of endocrine disruption in freshwater fish, how-
ever, it will be necessary to put endocrine disruption into context with other environmental pressures
that face freshwater fish populations. In our opinion, this will require research to define the global ex-
tent of the problem by expanding studies of endocrine disruption to other parts of the world and stud-
ies to extrapolate effects on individual fish to predict effects on populations and higher levels of bio-
logical organization.
Assessment of the extent of the problem of endocrine disruption in freshwater fish requires a more
widespread sampling of a variety of wild populations of fish, ideally using nondestructive sampling
methodologies and biomarkers. For biomarkers to be meaningful in this regard, efforts need to be di-
rected at determining how they are related to the health of both individuals and populations. The bio-
markers available for monitoring endocrine disruption are rather limited, and development of novel bio-
markers should be encouraged to extend beyond those for estrogenic effects, with an emphasis on
biomarkers that are indicative of reproductive and/or developmental effects and/or population re-
sponses. The presence of VTG in male fish, for example, is known to be negatively correlated with tes-
ticular growth and maturation [96]. In intersex fish, VTG is positively correlated with the degree of go-
nadal feminization [51], and hence also with their perceived reproductive success (which declines with
increased degree of feminization [52]). A widespread assessment of VTG concentrations or of the de-
gree of gonadal feminization in freshwater fish in a particular catchment could thus provide predictive
information on the likely state of the testes or of their likely reproductive success, respectively.
Vitellogenin concentration, however, could not (on its own) be used to directly predict the perceived fer-
tility of a population of fish because even male fish, when exposed to estrogen for short periods of time,
exhibit elevated plasma VTG concentrations, and little is known about the relationship between the tim-
ing and longevity of exposure to estrogen and the manifestation of gonadal feminization. Predictive
maps and models thus need to be interrogated (by conducting both field and lab studies) to establish
their validity. Information on what extent freshwater fish are exposed to EDCs in the wild is lacking,
and hence more information is needed on what EDCs (and their concentrations) are present in the en-
vironment and to what extent they are absorbed and metabolized by freshwater fish. Moreover, the re-
sponses of fish to environmentally relevant mixtures of chemicals (containing EDCs) require further
study and understanding.
Even with more widespread field data, research on endocrine disruption in wild freshwater fish is
likely to be limited to those species in which large numbers of individuals are easily obtainable. A more
global assessment of endocrine disruption in freshwater fish should ideally include the more rare and
vulnerable species, although this is less practical. Current research on endocrine disruption in freshwa-
S. JOBLING AND C. R.TYLER
© 2003 IUPAC, Pure and Applied Chemistry 75, 2219–2234
2230
ter fish is limited to studies on a very few species, and there has been little or no attention given to the
comparative sensitivities of different species of fish to EDCs, or indeed, of other animal species, on
which freshwater fish may be dependent (e.g., many invertebrate species). Furthermore, current risk as-
sessment strategies for endocrine disruption in freshwater fish are based on the responses of laboratory
fish species and are unlikely to represent the full range of fish species that may be at risk in the wild.
Laboratory studies will, therefore, need to be targeted at species with different life histories and differ-
ent reproductive strategies, in order to compare the sensitivities of different fish species to EDCs and
their mixtures. Moreover, an assessment of the implications of endocrine disruption on wild freshwater
fish will require a comprehensive understanding of their physiology, endocrinology, and population bi-
ology, and thus a further recommendation for future research is to develop this information for sentinel
species.
Wherever there is substantive evidence for endocrine disruption in freshwater fish, and the need
for remediation has been established, there is a requirement to focus on how these problems can be al-
leviated. Where high quantities of industrial chemicals are used that are known to cause/contribute to
endocrine disruption in freshwater fish, a program of tighter regulation for their discharge and/or a
switch to alternative greener chemicals (which do not impact the endocrine system) is needed. The suc-
cess of such programs can be illustrated by schemes implemented in both the United States and United
Kingdom that have reduced the concentrations of EDCs discharged (either as a consequence of changes
in industrial processes [97–98], or due to closure of a treatment plant [30]), which subsequently resulted
in concomitant decreases in endocrine disruption in the exposed fish. Many known EDCs cannot, how-
ever, easily be eliminated at source, because they are of natural origin (e.g., sex steroid hormones from
human and animal waste). For these types of contaminants, regulation of their release is likely to be
achieved by improvements in treatment processes and/or the implementation of systems that specifi-
cally remove and degrade them. EDCs also enter the freshwater environment through non-point
sources, but there has been very little study to assess the risk posed by these sources to freshwater fish.
Studies of this nature are also needed.
ACKNOWLEDGMENTS
We gratefully acknowledge the U.K. Environment Agency for provision of data on effluent dilution and
Richard Williams of CEH, Wallingford for his production of the predictive map in Fig. 1.
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