Approximately two-thirds of the world’s freshwater is used to dilute wastewater discharges. The demand for freshwater is expected to rise by 70% by 2050  driving an urgent need to understand the impacts of treated waste effluent discharges on aquatic ecosystems. Wastewater treatment works (WWTW) effluents contain tens of thousands of chemicals, including pharmaceuticals and natural steroid estrogens that are biologically active at low (ng/L) exposure concentrations . However, the long-term consequences of exposure to most of these chemicals on fish health and population sustainability are not known.
There is substantial evidence showing that experimental exposure of fish to WWTW effluents and the estrogens they contain can result in adverse health effects, including effects on reproductive development and breeding output. This has led to concerns that freshwater fish populations might also be affected with cascading consequences for freshwater ecosystems. Feminization of male fish is widespread in stretches of rivers downstream of WWTW outfalls as demonstrated in studies using wild [3, 4] and caged [5, 6] fish. Feminized phenotypes include the presence of vitellogenin, a female-specific protein in the blood of male fish  and the intersex condition: the presence of oocytes and/or female reproductive ducts in otherwise male gonads . Feminization has been attributed to the presence of estrogens in effluents: estradiol (E2) and estrone (E1) from human excretion; 17 alpha-ethinylestradiol (EE2), a component of the female contraceptive pill ; and a large number of other estrogenic chemicals from industrial and domestic effluents. WWTW effluents can also induce genotoxic effects , alterations in immune function , decreased reproductive output , altered stress response  and changes in reproductive behavior .
Concern about estrogens in rivers in the United Kingdom drove a £40 M programme to evaluate the efficacy of various tertiary treatment processes in the removal of estrogens [14, 15]. Implementation of such processes will, however, incur considerable costs and a greater carbon footprint for WWTW [14, 16], emphasising the need to understand better the population-level consequences for exposure to estrogenic and other so-called endocrine disrupting chemicals (EDCs).
A critical question is whether chronic exposure to estrogenic effluents negatively impacts the viability of wild fish populations, but this has been difficult to address experimentally as it requires controlled experiments extending over periods of several years. Limited studies suggest that high concentrations of EE2 (between 3 to 6 ng/L) in the aquatic environment could be a threat to the sustainability of fish populations. For example, a controlled exposure of an entire lake to EE2 in Canada resulted in the collapse of the fathead minnow (Pimephales promelas) population within three years . Likewise, long-term (>204 days) laboratory exposures of a range of fish species have resulted in the absence of breeding males [18–20] and a three-year exposure of roach (Rutilus rutilus L.) to an undiluted WWTW effluent in large tanks resulted in an all-female population . It is not known, however, if this occurs in rivers contaminated by effluents. Female fecundity can also be reduced through estrogen exposure, which can potentially reduce population growth rates . Although the exposure concentrations in these studies were high compared to those typically experienced by wild fish populations , exposures to EE2 at concentrations below 1 ng/L during the period of sexual development, have been shown to result in feminized gonads in roach  and decreased egg fertilization and female-skewed sex ratios in fathead minnows . Evidence from wild roach living in UK rivers has similarly shown that feminized fish (generally less than 10% of males) with large numbers of eggs in their gonads have impaired semen quality  and severely (up to 76%) reduced reproductive success .
While these studies suggest that exposure to high concentrations of effluent could threaten the viability of fish populations, aggregates of cyprinid fish, including roach, are often found in effluent contaminated rivers. However, numbers alone may provide a misleading assessment of population sustainability as these could be sink populations maintained by substantial immigration from less contaminated locations where successful reproduction still occurs. Likewise, effective population sizes (N
e) – related to the breeding population of fish – may be decreased without necessarily impacting on population sizes , as effluent exposure can affect the number of reproducing individuals and can skew reproductive success [21, 26]. Density-dependent growth and survival can also play an important role , so a few reproducing individuals can potentially maintain large adult population sizes. Indeed, studies in several species of marine fish with high fecundity have shown that N
e can be several orders of magnitude smaller than census population sizes. One study found two populations of the exploited New Zealand snapper (Pagrus auratus) to have values of N
e less than 1,000 despite adult census population sizes in the millions . Similarly, a study of striped bass (Morone saxatilis), a freshwater fish species, found cohorts to consist of a few, full sib families, despite an adult census size of over 300,000 . Critically, N
e influences long-term sustainability as it determines the rate at which genetic diversity is lost from a population through genetic drift . High genetic diversity increases the long-term potential for populations to adapt to changes in the environment and also acts to reduce the risk of inbreeding . Small N
e, however, may act to increase the chances of losing some lethal or sub-lethal mutations through genetic purging.
Understanding the impact of estrogenic effluents on the sustainability of fish populations is, therefore, paramount, but has been limited to date by the logistical challenges involved in undertaking long-term exposures to realistic effluent concentrations, and understanding the demographic history of wild fish populations at highly contaminated sites. In this study, we examine evidence for population impacts on wild roach (R. rutilus), a fish species in which feminization is widespread, in southern England. Southern England has some of the highest proportions of WWTW effluent in rivers known globally, and numerous weirs and locks which potentially confine fishes to heavily polluted stretches of river. We have used this system to evaluate whether stretches of river highly contaminated with estrogenic effluents have impaired breeding populations of roach. To do this we undertook analysis of population genetic structures of roach in the region using DNA microsatellite analysis. Microsatellite data were also used to calculate N
e and estimate levels of gene flow to determine the extent to which these populations are maintained through immigration of fish from less contaminated stretches of river.