Estrogenic activity and steroid hormones in swine wastewater through a lagoon constructed-wetland system

Anaerobic lagoons and treatment wetlands are used worldwide to treat wastewater from dense livestock production facilities; however, there is very limited data on the hormonal activity of the wastewater effluent produced by these treatment systems. The objectives of this experiment were to measure (1) the hormonal activity of the initial effluent and (2) the effectiveness of a lagoon-constructed wetland treatment system for producing an effluent with a low hormonal activity. Wastewater samples were taken in April, July, and November 2004 and July 2005 from a lagoon-constructed wetland system at a swine farrowing facility. Estrogenic activity (in vitro E-screen assay), 17 beta-estradiol (E2), and testosterone concentrations (LC/MS-MS) were measured. A high correlation was found between estradiol equivalents determined by E-screen and LC/MS-MS (R2 = 0.82). Nutrient removal was measured to ensure that the wetlands were functioning in a manner similar to literature reports. Nutrient removals were typical for treatment wetlands: TKN 59-75% and orthophosphate 0-18%. Wetlands decreased estrogenic activity by 83-93%. Estrone was the most persistent estrogenic compound. Constructed wetlands produced effluents with estrogenic activity below the lowest equivalent E2 concentration known to have an effect on fish (10 ng/L or approximately 37 x 10(-12) M).     

Transport of chemical and microbial compounds from known wastewater discharges: potential for use as indicators of human fecal contamination

The quality of drinking and recreational water is currently (2005) determined using indicator bacteria. However, the culture tests used to analyze forthese bacteria require a long time to complete and do not discriminate between human and animal fecal material sources. One complementary approach is to use chemicals found in human wastewater, which would have the advantages of (1) potentially shorter analysis times than the bacterial culture tests and (2) being selected for human-source specificity. At 10 locations, water samples were collected upstream and at two successive points downstream from a wastewaster treatment plant (WWTP); a treated effluent sample was also collected at each WWTP. This sampling plan was used to determine the persistence of a chemically diverse suite of emerging contaminants in streams. Samples were also collected at two reference locations assumed to have minimal human impacts. Of the 110 chemical analytes investigated in this project, 78 were detected at least once. The number of compounds in a given sample ranged from 3 at a reference location to 50 in a WWTP effluent sample. The total analyte load at each location varied from 0.018 microg/L at the reference location to 97.7 microg/L in a separate WWTP effluent sample. Although most of the compound concentrations were in the range of 0.01-1.0 microg/L, in some samples, individual concentrations were in the range of 5-38 microg/L. The concentrations of the majority of the chemicals present in the samples generally followed the expected trend: they were either nonexistent or at trace levels in the upstream samples, had their maximum concentrations in the WWTP effluent samples, and then declined in the two downstream samples. This research suggests that selected chemicals are useful as tracers of human wastewater discharge.     

Occurrence of estrogenic compounds in and removal by a swine farm waste treatment plant

The total estrogenic activity of the wastewater from a swine farm in Japan was quantitatively characterized, and the compounds responsible for the estrogenic activity were identified and quantified. The wastewater treatment process consisted of a series of an up-flow anaerobic sludge blanket (UASB) and a trickling filter. Samples were collected at each treatment step, and the total estrogenic activity was determined by use of an in vitro gene expression assay (MVLN; MCF-7 human breast cancer cell stably transfected with the pVit-tk-LUC receptor plasmid). Individual estrogenic compounds were identified and quantified using liquid chromatography-mass spectrometry (LC/MS) and liquid chromatography-tandem mass spectrometry (LC/ MS/MS). To further identify the compounds contributing to the estrogenic activity in the wastewater, the sample extracts were fractionated into 12 fractions (fractions 1-12) by HPLC. The rate of removal of estrogenic activity between the effluent and the influent was greater than 97%. The trickling filter removed the majority of the estrogenic activity. The removal rates of specific estrogenic compounds ranged from 44 to 99%. Estrogenic activity was detected mainly in the fractions containing estrone (El), 17beta-estradiol (betaE2), 17alpha-estradiol (alpha E2), estriol (E3), bisphenol A (alphaPA), and equol (EQ0). The ratios of betaE2-EQc (betaE2 equivalents derived from chemical analysis) to betaE2-EQB (betaE2 equivalent derived from bioassay) in the 12 fractions collectively were contributed by El (17-30%), betaE2 (23-30%), acE2 (<1%), E3 (1-2%), BPA (<1%), and EQO (2-3%) in the influent and El (16-37%), PE2 (<1-7%), alphaE2 (<1%), E3 (<1-3%), BPA (<1%), and EQO (<1%) in the effluent. The compounds responsible for most of the estrogenic activity measured in the bioassay were natural estrogens such as El and betaE2.     

Lagrangian mass-flow investigations of inorganic contaminants in wastewater-impacted streams

Understanding the potential effects of increased reliance on wastewater treatment plant (WWTP) effluents to meet municipal, agricultural, and environmental flow requires an understanding of the complex chemical loading characteristics of the WWTPs and the assimilative capacity of receiving waters. Stream ecosystem effects are linked to proportions of WWTP effluent under low-flow conditions as well as the nature of the effluent chemical mixtures. This study quantifies the loading of 58 inorganic constituents (nutrients to rare earth elements) from WWTP discharges relative to upstream landscape-based sources. Stream assimilation capacity was evaluated by Lagrangian sampling, using flow velocities determined from tracer experiments to track the same parcel of water as it moved downstream. Boulder Creek, Colorado and Fourmile Creek, Iowa, representing two different geologic and hydrologic landscapes, were sampled under low-flow conditions in the summer and spring. One-half of the constituents had greater loads from the WWTP effluents than the upstream drainages, and once introduced into the streams, dilution was the predominant assimilation mechanism. Only ammonium and bismuth had significant decreases in mass load downstream from the WWTPs during all samplings. The link between hydrology and water chemistry inherent in Lagrangian sampling allows quantitative assessment of chemical fate across different landscapes.     

Positive effects of wastewater ozonation displayed by in situ bioassays in the receiving stream

Tertiary treatment methods, like ozonation, are currently under discussion to improve removal efficiencies of micropollutants by municipal wastewater treatment plants (WWTPs). In order to assess the effects of a full-scale wastewater ozonation at WWTP Wu?eri, Switzerland, on the receiving stream, a total of seven in situ bioassays with Gammarus fossarum that lasted 7-days were conducted during an overall period of 33 months. Caged gammarids were exposed between 150 m up- and 400 m downstream of a WWTP effluent before, during and following the operation of the full-scale wastewater ozonation. During the release of nonozone treated wastewater, gammarid feeding was significantly reduced by up to 90% 50 and 150 m downstream of the WWTP effluent. In contrast, during the operation period of the ozonation, no significant alterations in feeding were observed downstream. The results of these bioassays were incorporated into a mathematical simulation of the release of nonozone treated wastewater in a Central European region, suggesting a 40% reduction in leaf litter breakdown and hence in energy provision for the remaining aquatic food web downstream of WWTP effluents, while the release of ozone treated wastewater did not affect this important ecosystem function.     

Accounting for differences in estrogenic responses in rainbow trout (Oncorhynchus mykiss: Salmonidae) and roach (Rutilus rutilus: Cyprinidae) exposed to effluents from wastewater treatment works

Effluents from wastewater treatment works (WwTWs) contain estrogenic substances that induce feminizing effects in fish, including vitellogenin (VTG) synthesis and gonadal intersex. Fish vary in their responsiveness to estrogenic effluents, but the physiological basis for these differences are not known. In this study, uptake of estrogen from two WwTW effluents (measured in hydrolyzed bile) and estrogenic response (VTG induction) were compared in a salmonid (rainbow trout, Onchorhynchus mykiss) and a cyprinid fish (roach, Rutilus rutilus). Immature rainbow trout were more responsive than maturing roach to the estrogenic effluents. The more potent of the two estrogenic effluents (containing between 24.3 and 104.1 ng estradiol-17beta equivalents/L [E2eq/L]) resulted in a 700-fold and 240-fold induction of plasma VTG in male and female trout, respectively, but only a 4-fold induction in roach (and in males only). The less potent effluent (varying between 4.1 and 6.8 ng E2eq/L) induced VTG in the trout only, with a 4-fold and 18-fold induction in males and females, respectively. In fish exposed to tap water, the estrogenicity of the hydrolyzed bile was 0.03+/-0.01 ng E2eq/microL (for both sexes in trout), 0.18+/-0.04 ng E2eq/microL in male roach, and 0.88+/-0.15 ng E2eq/microL in female roach. The higher bile content of estrogen in control roach reflected their more advanced sexual status (and thus higher endogenous estrogen) compared with the immature female trout. In trout maintained in effluents, the bile content of estrogen was 100-fold and 30-fold higher than controls at WwTW A and B, respectively. Bioconcentration factors (BCFs) for estrogenic activity in bile were between 16 344 and 46 134 in trout and between 3543 and 60 192 in roach (no gender differences were apparent). There were strong correlations between VTG induction and the estrogenic activity of bile extracts for both trout and roach. The results confirm that estrogenic contaminants bioconcentrate to a high degree in fish bile and that the level (and nature) of this accumulation may accountfor responsiveness to the endocrine disruptive effects of estrogenic effluents. Immature fish were the more appropriate life stage for quantifying estrogen exposure and uptake in bile, as they contain little circulating endogenous oestrogen compared with sexual maturing fish. The nature of the estrogenic contaminants is detailed in an accompanying paper.     

Combined sewer overflows: an environmental source of hormones and wastewater micropollutants

Data were collected at a wastewater treatment plant (WWTP) in Burlington, Vermont, USA, (serving 30,000 people) to assess the relative contribution of CSO (combined sewer overflow) bypass flows and treated wastewater effluent to the load of steroid hormones and other wastewater micropollutants (WMPs) from a WWTP to a lake. Flow-weighted composite samples were collected over a 13 month period at this WWTP from CSO bypass flows or plant influent flows (n = 28) and treated effluent discharges (n = 22). Although CSO discharges represent 10% of the total annual water discharge (CSO plus treated plant effluent discharges) from the WWTP, CSO discharges contribute 40-90% of the annual load for hormones and WMPs with high (>90%) wastewater treatment removal efficiency. By contrast, compounds with low removal efficiencies (<90%) have less than 10% of annual load contributed by CSO discharges. Concentrations of estrogens, androgens, and WMPs generally are 10 times higher in CSO discharges compared to treated wastewater discharges. Compound concentrations in samples of CSO discharges generally decrease with increasing flow because of wastewater dilution by rainfall runoff. By contrast, concentrations of hormones and many WMPs in samples from treated discharges can increase with increasing flow due to decreasing removal efficiency.     

Temporal variation in the estrogenicity of a sewage treatment plant effluent and its biological significance

Daily variation in the estrogenic activity of effluent released by a modern sewage treatment plant (STP) was measured and its effects on the physiology, behavior, and reproductive success of male fish were evaluated. As measured by an estrogen receptor binding assay, the daily estrogenic activity of this effluent was both high and extremely variable (42 +/- 25.4 [mean +/- SD] ng 17beta-estradiol (E2) equivalents/L; n = 18). Liver VTG mRNA expression in male fathead minnows (FHM) covaried with the binding assay estimates, suggesting that these fluctuations are biologically relevant. Tests which exposed male FHMs to either fluctuating levels of E2, a constant concentration of E2 (time-weighted to reflect average concentrations), or control (no E2) demonstrated that while the estrogenic activity of this effluent was detrimental to male spawning success, the fact that its concentration varied in a daily manner was without additional influence. The variability of the effluent's estrogenicity suggests that studies concerned with the effects of STP effluents should collect multiple daily samples and then test them on an appropriate time-weighted basis.     

Reproductive responses of common carp (Cyprinus carpio) exposed in cages to influent of the Las Vegas Wash in Lake Mead, Nevada, from late winter to early spring

The Las Vegas Wash (LW) delivers tertiary-treated municipal wastewater effluent, nonpotable shallow groundwater seepage, and runoff from the urbanized Las Vegas Valley to Las Vegas Bay (LX) of Lake Mead. To investigate the potential for contaminants in LW influent to produce effects indicative of endocrine disruption in vivo, adult male and female common carp (Cyprinus carpio) were exposed in cages for 42-48 d at four sites in Lake Mead: LW, LX, and two reference locations in the lake. End points examined included gonadosomatic index; gonad histology; concentrations of plasma vitellogenin (VTG) and plasma sex steroids (17beta-estradiol (E2), testosterone (T), 11-ketotestosterone (11-KT)); plasma estrogen:androgen ratios (E2:T, E2:11-KT), in vitro production of T by gonad tissue, and hepatopancreas ethoxyresorufin O-deethylase activity. Few differences among fish caged at different sites were potentially attributable to exposure to contaminants     

Fate, transport, and biodegradation of natural estrogens in the environment and engineered systems

Natural steroidal estrogen hormones, e.g., estrone (E1), 17beta-estradiol (E2), estriol (E3), and 17alpha-estradiol (17alpha), are released by humans and livestock in the environment and are the most potent endocrine disrupters even at nanogram per liter levels. Published studies broadly conclude that conventional wastewater treatment is efficient in the removal of 17beta-estradiol (85-99%), but estrone removal is relatively poor (25-80%). The removal occurs mainly through sorption by sludge and subsequent biodegradation. The long solids retention time in wastewater treatment systems enhances estrogen removal due to longer exposure and the presence of a diverse microbial community, particularly nitrifiers. In spite of the treatment, the effluent from conventional biological wastewater treatment systems still contains estrogenic compounds at a level that may cause disruption of endocrine systems in some species. Advanced wastewater treatment systems such as membrane processes remove the estrogen compounds mainly through physical straining of particle-bound estrogens. Another major source, which accounts for 90% of the estrogen load, is animal manure from concentrated animal-feeding operations (CAFOs). Manure is not required to be treated in the United States as long as it is not discharged directly into water bodies. Thus, there is an urgent need to study the fate of animal-borne estrogens from these facilities into the environment. A number of studies have reported the feminization of male aquatic species in water bodies receiving the effluents from wastewater treatment plants (WWTPs) or surface runoff from fields amended with livestock manure and municipal biosolids. Estrogenicity monitoring studies have been conducted in more than 30 countries, and abundant research articles are now available in refereed journals. This review paper focuses on estrogen contributions by wastewater and livestock manure, their removal rate and mechanisms in an engineered system, and their transport and ultimate fate in an engineered system and the environment. The review aims to advance our understanding of fate, transport, and biodegradation of estrogen compounds and outlines some directions for future research.     

Alkylphenol and alkylphenol-ethoxylates in carp,water, and sediment from the Cuyahoga River,Ohio

The occurrence of alkylphenol and alkylphenol ethoxylates (APEs) was determined over a 74-mile length of the Cuyahoga River, Ohio. Measurable levels of both the octyl and nonyl forms of these abundantly used nonionic surfactants were observed with the nonylphenol (NP) plus nonylphenol ethoxylates (NPEs) typically accounting for greater than 90% of the total APEs in each sample. For all media (water, fish, and sediment) the total NPE (NP + NPE) concentrations were higher in the more urbanized downstream section of the river. Maximum water and fish values were observed immediately downstream (2.1 miles) from the discharge of the Akron WWTP located 35.31 miles from the river mouth and the sediment maxima occurred at the most downstream site near Cleveland. The ranges in concentration fortotal NPEs and their ethoxylate (EO) makeup were as follows: 32-920 ug/kg wet wt (NP 0 to 2 EO) for carp; 0.13-1.0 ug/L (NP 0 to 3 EO) for water; and 250-1020 ug/kg dry wt (NP 0 to 5 EO) for sediment. When the higher ethoxymers (NP 6 to 17 EO) were added to these sediment totals, the average total estimated NPE concentrations were 1.3-1.8 times higher.     

Evidence for the migration of steroidal estrogens through river bed sediments

Estrogenic substances discharged from wastewater treatment plants have been detected in surface sediments of receiving waters, but little is known of their vertical migration through buried sediments and their potential to contaminate subsurface waters. The vertical profiles of estrogenic chemicals were investigated in sediment cores at an alluvial freshwater site (Ditchling) and a clay-rich estuarine site (Lewes), both of which are downstream of wastewater discharges into the River Ouse (Sussex, U.K.). Estrone (E1) was the predominant estrogen detected in surface and buried sediments at both sites and was detected in undisturbed clay sediments > 120 years old. Profiles of E1 at Ditchling were characterized by a prominent subsurface peak of E1 at the alluvium/clay interface (-15 cm) at a concentration (28.8 +/- 6.0 ng/g of dry wt) that was 9-fold higher than in the surface sediment. In contrast, a steady downcore decline in E1 concentrations was observed in the clay-rich Lewes core. This work provides the first in situ evidence of estrogen migration through river bed sediments and reveals that movement of estrogens through unconsolidated sediment can result in penetration to the underlying substrata and therefore the potential for groundwater contamination.     

Characterization of environmental estrogens in river water using a three pronged approach: active and passive water sampling and the analysis of accumulated estrogens in the bile of caged fish

Estrogenicity of river water is highly variable and it is difficult to obtain an average measure of the estrogenicity. Consequently it is difficult to tie the estrogenic effects observed in fish to their level of exposure to estrogens. To get a better handle on average estrogenic exposure we tested a recently developed passive sampling system (polar organic chemical integrative sampler, POCIS). In addition, we investigated the bioaccumulation of estrogens in caged brown trout and measured plasma vitellogenin in males as a bioindicator of estrogenic effects. We developed a mini-caging method to suit the hydrological conditions in small rivers and to improve upon the often poor survival of salmonids in caging trials. POCISs were positioned upstream and downstream of 5 sewage treatment works' discharges and left on site for 3 weeks (as were the caged fish), during which period 3 water grab samples were taken at each site. Concentrations of estrogens were determined using a yeast-based reporter gene assay and chemical analysis. Results from grab sampling, passive sampling, and bioaccumulation were correlated; however, plasma vitellogenin concentrations were elevated at only 1 of 5 sites. POCISs provide an integrated and biologically meaningful measure of estrogenicity in thatthey accumulate estrogens in a pattern similar to that of brown trout. Mini-caging appears a significant methodological advance; no fish were lost, moreover, all fish survived in excellent health.     

Monitoring the effects of pulp and paper effluent is restricted in genetically distinct populations of common bully (Gobiomorphus cotidianus)

The common bully (Gobiomorphus cotidianus), a small-bodied New Zealand native fish species, was used to monitor population impacts of multiple effluents in the Tarawera River, New Zealand. In an initial survey, the absence of reproductive development at the expected spawning time for common bullywas observed in a population downstream of effluent discharges. Subsequently, we examined the hypotheses that the observed changes were due to effluent exposure, migratory patterns, or genetic differences between populations. Liver detoxification enzyme activity and stable isotopes provided evidence against upstream migration of sexually mature bully. The observed presence of developed gonads in the downstream population during winter season resulted in the rejection of the hypothesis that reproductive failure was due to effluent exposure, and itwas concluded that there were substantial differences in reproductive timing. Genetic analyses of two upstream, one downstream, and one population from a nearby coastal river indicated the upstream (reference) and downstream (effluent exposed) bully in the river formed genetically distinct populations. The identification of a nearby river population with similar reproductive timing and high genetic similarity to the effluent-exposed population suggests that the observed differences in the genetics of the downstream population were not caused by effluent exposure. The genetic analysis did highlight the lack of downstream dispersion and gene flow in the river which could possibly be related to anthropogenic stress.     

Fate of 4-nonylphenol and 17β-estradiol in the Redwood River of Minnesota

The majority of previous research investigating the fate of endocrine-disrupting compounds has focused on single processes generally in controlled laboratory experiments, and limited studies have directly evaluated their fate and transport in rivers. This study evaluated the fate and transport of 4-nonylphenol, 17β-estradiol, and estrone in a 10-km reach of the Redwood River in southwestern Minnesota. The same parcel of water was sampled as it moved downstream, integrating chemical transformation and hydrologic processes. The conservative tracer bromide was used to track the parcel of water being sampled, and the change in mass of the target compounds relative to bromide was determined at two locations downstream from a wastewater treatment plant effluent outfall. In-stream attenuation coefficients (k(stream)) were calculated by assuming first-order kinetics (negative values correspond to attenuation, whereas positive values indicate production). Attenuation of 17β-estradiol (k(stream) = -3.2 ± 1.0 day(-1)) was attributed primarily due to sorption and biodegradation by the stream biofilm and bed sediments. Estrone (k(stream) = 0.6 ± 0.8 day(-1)) and 4-nonylphenol (k(stream) = 1.4 ± 1.9 day(-1)) were produced in the evaluated 10-km reach, likely due to biochemical transformation from parent compounds (17β-estradiol, 4-nonylphenolpolyethoxylates, and 4-nonyphenolpolyethoxycarboxylates). Despite attenuation, these compounds were transported kilometers downstream, and thus additive concentrations from multiple sources and transformation of parent compounds into degradates having estrogenic activity can explain their environmental persistence and widespread observations of biological disruption in surface waters.     

Sterols and anionic surfactants in urban aerosol: emissions from wastewater treatment plants in relation to background concentrations

Aerosol particles that are emitted from aeration tanks of wastewater treatment plants (WWTPs) can be enriched with environmentally relevant wastewater constituents. In this study, aerosol particles were sampled simultaneously at the pre-aeration tank of a municipal WWTP and at two urban locations approximately 1 km away from the WWTP to evaluate the significance of these aerosol emissions. Moreover, aerosol particles were sampled at a small wastewater irrigation facility and at a rural site. In aerosol particles and wastewater, six sterols (cholesterol, coprostanol, campesterol, beta-sitosterol, stigmasterol, stigmastanol) and anionic surfactants (expressed in terms of methylene blue active substances, MBAS) were quantified. The results showed significantly higher concentrations of sterols and MBAS at the WWTP than atthe urban locations. At the WWTP, average concentrations of cholesterol (848 +/- 321 pg m(-3)), coprostanol (1132 +/- 565 pg m(-3)), and MBAS (132 +/- 43 ng m(-3)) in aerosol were approximately twice as high. This can be attributed to emissions from the treatment tank. Coprostanol, a unique tracer for wastewater, was detected only occasionally at the urban locations. The results of this study show that the aeration of wastewater is a continuously operating local source for organic compounds in aerosol. The wastewater irrigation facility was a minor source of aerosol-bound sterols (coprostanol, 287 +/- 218 pg m(-3)) and anionic surfactants (64 +/- 32 ng m(-3)). Except for coprostanol, all compounds were also present in samples of rural aerosol.     

Quantitative determination of fluorinated alkyl substances by large-volume-injection liquid chromatography tandem mass spectrometry-characterization of municipal wastewaters

A quantitative method was developed for the determination of fluorinated alkyl substances in municipal wastewater influents and effluents. The method consisted of centrifugation followed by large-volume injection (500 microL) of the supernatant onto a liquid chromatograph with a reverse-phase column and detection by electrospray ionization and tandem mass spectrometry. The fluorinated analytes studied include perfluoroalkyl sulfonates, fluorotelomer sulfonates, perfluorocarboxylates, and select fluorinated alkyl sulfonamides. Recoveries of the fluorinated analytes from wastewater treatment plant (WWTP) raw influents and final effluent were in the ranges of 82-100% and 86-100%, respectively. The lower limit of quantitation was 0.5 ng/L depending on the analyte. The method was applied to flow-proportional composites of raw influent and final effluent collected over a 24 h period from 10 WWTPs nationwide. Fluorinated alkyl substances were observed in wastewater at all treatment plants, and each plant exhibited unique distributions of fluorinated alkyl substances despite similarities in treatment processes. In 9 out of the 10 plants sampled, at least one class of fluorinated alkyl substances exhibited increased concentrations in the effluent as compared to the influent concentrations. In some instances, decreases in certain fluorinated analyte concentrations were observed and attributed to sorption to sludge.