An assessment of estrogenic organic contaminants in Canadian wastewaters

A suite of 30 primarily estrogenic organic wastewater contaminants was measured in several influent/effluent wastewater samples from four municipal wastewater treatment plants and effluents from one bleached kraft pulp mill (BKME) using an ultra-trace analytical method based on gas chromatography-high resolution mass spectroscopy (GC-HRMS). In vitro recombinant yeast assay detection of the estrogenic equivalent (EEq) on whole and solid phase extracted (SPE) and fractionated wastewater was also performed. 19-norethindrone was the most frequently detected and abundant (26-224 ng/L) of all the synthetic estrogens/progesterones in the influent samples. 17alpha-ethinylestradiol was the more frequently detected synthetic estrogen/progesterone in the effluents occurring at or below 5 ng/L with some sporadic occurrences of up to 178 ng/L. The greatest levels of steroidal estrogens in municipal effluents were E1>E2>E3 which were all <20 ng/L. Nonylphenol and di(2-ethylhexyl) phthalate were found to be the highest non-steroidal synthetic compounds surveyed in both municipal influent and effluent samples, both occurring at 6-7 microg/L in municipal effluents. BKME contained relatively large amounts of the plant sterol stigmasterol (4 microg/L) but low amounts of fecal sterols, and steroidal estrogens (E2 only at 6 ng/L) when compared to the municipal effluents. In vitro EEq in the wastewater surveyed ranged from 9-106 ng E2/L and ranked from municipal influent>municipal effluent approximately BKME, with most of the estrogenicity fractionating in the 100% methanol SPE fraction followed by a secondary amount in the diethyl ether (for municipal) or methyl-tert butyl ether (for BKME) SPE fractions. Most correlations between chemical and in vitro estrogenic equivalency were weak (p>0.05 in most cases). Unexpected inverse correlations between in vitro estrogenic activity and concentrations of the estrogenic contaminant bisphenol A were found which likely contributed to the weakness of these correlations. A modified toxicity identification and evaluation procedure was continued with the SPE extracts from the more potent 100% methanol SPE fractions of municipal effluent. High performance liquid chromatography band elution retention times, based on in vitro estrogen detection, indicated that steroidal estrogens such as E2 were responsible for most of the estrogenicity of the samples. Subsequent collection and GC-MS analysis of active bands did not confirm the presence of steroidal estrogens, but expanded the possibility of phthalate esters (i.e. dibutyl phthalate) and natural sterols (i.e. beta-sitosterol) contributing to the overall estrogenic load.     

Do sewage treatment plant discharges substantially impair fish reproduction in polluted rivers?

Sewage treatment plants are frequently associated with the release of xenobiotics and, consequently, with alterations of the reproductive function induced by many of these substances in aquatic organisms. In order to assess the impacts of sewage treatment plant (STP) discharges in polluted rivers, two sentinel species (gudgeon Gobio gobio and stoneloach Barbatula barbatula) were caught during their reproductive cycle upstream and downstream two STPs (STP1--Goffontaine, STP2--Wegnez). Gonadosomatic index, histological (testicular and ovarian stages, atretic follicles, intersexuality) and endocrine (sex steroids, aromatase activity, alkali-labile phosphorus) parameters were assayed. In brief, the results revealed no systematic significant differences (p<0.05) between upstream and downstream sites, whatever the STP, species or sampling period. However, stoneloach females displayed some signs of reproductive impairment and endocrine disruption downstream STP1 (reduced GSI, oocyte diameter and ALP concentrations, increased proportion of atretic follicles) and STP2 (changes in gonadal aromatase activity and plasma levels of 11-KT and T). Few significant changes were observed for gudgeon males and females while there were no significant differences between upstream and downstream sites for stoneloach males. Moreover, plasma E(2) concentrations recorded in gudgeon males sampled in all sites were as high as in females and this was confirmed by high ALP levels. Besides, spermatogenesis of gudgeon males was delayed in STP1 upstream and downstream sites compared to the corresponding sites in STP2. These observations for gudgeon males do not seem related to STP discharge but to a probable estrogenicity of the river. Therefore, as shown by the results, stoneloach seemed more sensitive than gudgeon to STP discharges. In the present study, sewage treatment plant discharges do not substantially impair fish reproduction. In this respect, caution is required when generalising negative impacts of STP discharges.     

Avoidance of bleached kraft mill effluent by pre-exposed Coregonus albula L.

The ability of vendace (Coregonus albula L.) to avoid different concentrations of bleached kraft mill effluent (BKME) was studied in the laboratory with fish pre-exposed to BKME for 1 week and non-pre-exposed fish. Pre-exposure to BKME in sublethal concentrations reduced the avoidance of BKME by vendace. Throughout the range of exposure concentrations (0.13–4.50 v/v%), pre-exposed fish tended to be observed less in the clean water than non-pre-exposed fish or even to prefer contaminated water. In field conditions, changes in behaviour might lead to a chain reaction: the fish come into contact with concentrations of BKME lower than the threshold level for avoidance and their threshold level increases gradually due to this exposure, which makes exposure to higher concentrations possible and weakens the ability to return to clean water. Desensitization caused by pre-exposure and long distances between pronounced BKME concentration differences in the field together makes it improbable that the vendace can show avoidance responses in the field. Analyses of the various waste-water samples support the conclusion that the most toxic components in BKME, resin acids and chlorophenols, are of minor importance to the avoidance reaction.     

Comparative study of the estrogenic responses of mirror carp (Cyprinus carpio) exposed to treated municipal sewage effluent (Lisbon) during two periods in different seasons

To assess the estrogenic potency of the treated domestic sewage effluent from a sewage treatment plant (STP) located in Lisbon (Chelas), 60 mirror carp (Cyprinus carpio) were exposed to different concentrations of the sewage effluent (0%, 25%, 50% and 100%) for two periods of 28 days in two different seasons (winter/spring). Vitellogenin induction in males was used as a biomarker of exposure to xenoestrogens. At the end of the experiment, blood samples were taken for vitellogenin analysis and the fish were sacrificed and dissected. Gonad samples were taken for histological evaluation of the sewage effects. The results showed an increase in vitellogenin induction in exposed fish, both males and females, depending on the different dilutions of the sewage effluent. In comparison with controls, the gonadosomatic index decreased significantly (P < 0.05) in fish exposed to 100% treated effluent. Although statistically not significant, the hepatosomatic index (HSI) was high in all exposed fish. Histological abnormalities in fish gonads were evaluated and related to the different percentages of sewage effluent. Seasonal variations found in estrogenic responses were attributed to weather influences on sewage dilution.     

The interaction of treated bleached kraft mill effluent and dissolved oxygen concentration on the survival of the developmental stages of the sheepshead minnow (Cyprinodon variegatus)

The interactions of treated bleached kraft mill effluent (BKME) and dissolved oxygen concentration (DO) on the survival of the embryo, fry, juvenile and adult stages of the sheepshead minnow, Cyprinodon variegatus, were studied under continuous-flow 96 h bioassay conditions. Embryo survival was dependent on effluent concentration only and showed no interaction at nominal DO concentrations of 1.0, 3.0 and 5.0 mg l⁻¹. Survival of the fry was related solely to DO concentration with no interaction with BKME concentrations up to 100%. Juvenile sheepshead minnows showed increased sensitivity to BKME at a nominal DO concentration of 1.0 mg l⁻¹. Adult fish were not affected by BKME at any of the DO concentrations tested. This study shows that the acute toxicity of BKME effluent to sheepshead minnows is a function of the developmental stage of the organism and DO concentration in the receiving stream.     

Plasma vitellogenin levels and gonadal morphology of wild carp (Cyprinus carpio L.) in a receiving rivers downstream of sewage treatment plants

A field study was carried out in order to assess, in a wild cyprinid fish, the biological effects related to the exposure to estrogenic compounds. The gonadosomic index (GSI), plasma vitellogenin (VTG) level and histological observations of gonads were performed to evaluate the reproductive health in common carp (Cyprinus carpio) from three rivers receiving sewage treatment plant (STP) effluents (Guadarrama, Henares and Jarama rivers, Spain) and from a reference site (Lozoya river). No significative differences in male GSI between sites were described. Only in the 18% of fish from Guadarrama river were elevated plasma VTG levels detected. The presence of oocytes in the testis was not found in any of males examined. The results suggest that the released estrogenic compounds by STPs effluents are not enough capable to adversely affect the gonadal development of the fish studied. Additional studies will be required to assess the risk of estrogenic compounds on fish from other sites of this area.     

Analysis of environmental endocrine disrupting chemicals using the E-screen method and stir bar sorptive extraction in wastewater treatment plant effluents

Endocrine disrupting chemicals (EDCs) have become a major issue in the field of environmental science due to their ability to interfere with the endocrine system. Recent studies show that surface water is contaminated with EDCs, many released from wastewater treatment plants (WWTP). This pilot study used biological (E-screen assay) and chemical (stir bar sorptive extraction-GC-MS) analyses to quantify estrogenic activity in effluent water samples from a municipal WWTP and in water samples of the recipient river, upstream and downstream of the plant.The E-screen assay was performed on samples after solid phase extraction (SPE) to determine total estrogenic activity; the presence of estrogenic substances can be evaluated by measuring the 17-β-estradiol equivalency quantity (EEQ). Untreated samples were also assayed with an acute toxicity test (Vibrio fischeri) to study the correlation between toxicity and estrogenic disruption activity.Mean EEQs were 4.7 ng/L (± 2.7 ng/L) upstream and 4.4 ng/L (± 3.7 ng/L) downstream of the plant, and 11.1 ng/L (± 11.7 ng/L) in the effluent. In general the WWTP effluent had little impact on estrogenicity nor on the concentration of EDCs in the river water. The samples upstream and downstream of the plant were non-toxic or weakly toxic (0 < TU < 0.9) while the effluent was weakly toxic or toxic (0.4 < TU < 7.6). Toxicity and estrogenic activity were not correlated.At most sites, industrial mimics, such as the alkylphenols and phthalates, were present in higher concentrations than natural hormones. Although the concentrations of the detected xenoestrogens were generally higher than those of the steroids, they accounted for only a small fraction of the EEQ because of their low estrogenic potency. The EEQs resulting from the E-screen assay and those calculated from the results of chemical analyses using estradiol equivalency factors were comparable for all samples and closely correlated.     

Comparison of six different sewage treatment processes—Reduction of estrogenic substances and effects on gene expression in exposed male fish

Treated sewage effluents often contain a mixture of estrogenic compounds in low concentrations. The total combined activity of these, however, may be sufficiently high to affect the reproduction of aquatic vertebrates. The introduction of advanced treatment technologies has been suggested as a way to remove micro-contaminants, including estrogenic substances. In this study, one municipal influent was treated with six different processes in parallel on a semi-large scale in order to assess their potential to reduce substances that could contribute to estrogenic effects in male fish. The effluent from a conventional, activated sludge treatment line was compared to a similarly treated effluent with a final sand-filtering step. The addition of ozonation (15 g O₃/m³), a moving bed biofilm reactor (MBBR) or both in combination was also evaluated. There was also a separate treatment line that was based on a membrane bioreactor. A small battery of hepatic estrogen-responsive genes was measured in the exposed fish using quantitative PCR. Concentrations of steroid estrogens and estrogenic phenols in the effluents were measured by GC-ECNI-MS. The ozonated effluents were the only tested effluents for which all measured biological effects in exposed fish were removed. Chemical data suggested that the MBBR technology was equally effective in removing the analyzed estrogens; however, elevated expression of estrogen-responsive genes suggested that some estrogenic substances were still present in the effluent. The membrane bioreactor removed most of the measured estrogens and it reduced the induction of the estrogen-responsive genes. However, fish exposed to this effluent had significantly enlarged livers. Given that the same influent was treated in parallel with a broad set of technologies and that the chemical analyses were combined with an in vivo assessment of estrogenic responses, this study provides valuable input into the assessment of advanced treatment processes for removing estrogenic substances.     

The use of modelling to predict levels of estrogens in a river catchment: How does modelled data compare with chemical analysis and in vitro yeast assay results?

Effluent discharges at Rodbourne sewage treatment works (STWs) were assessed using chemical and in vitro biological analysis as well as modelling predictions. Results showed that Rodbourne STW discharged less estrone (E1) than expected, but similar 17β-estradiol (E2) and 17α-ethinyl estradiol (EE2) to those predicted by a widely cited effluent prediction model. The Exposure Analysis Modelling System (EXAMS) model was set up using measured effluent concentrations as its starting point to predict estrogen concentrations along a 10 km length of the receiving water of the River Ray. The model adequately simulated estrogen concentrations along the river when compared to July 2007 measured data. The model predicted combined estrogen equivalents in reasonable agreement with estrogenicity as measured by passive sampler (POCIS) extracts using the yeast estrogen screen. Using gauged mean flow values for 2007 the model indicated that the most important determinand for estrogen exposure in the Ray was not season, but proximity to the Rodbourne effluent. Thus, fish in the first 3 km downstream of Rodbourne were typically exposed to two or even three times more estrogens than those living 7-10 km further downstream. The modelling indicated that, assuming the effluent estrogen concentrations measured in February 2008 were typical, throughout the year the whole length of the Ray downstream of Rodbourne would be estrogenic, i.e. exceeding the 1 ng/L E2 equivalent threshold for endocrine disruption.     

Disruption of precopulatory behavior in the amphipod Anisogammarus pugettensis upon exposure to bleached kraft pulpmill effluent

Marine amphipods in precopula, Anisogammarus pugettensis (Dana), were used in static 117 h bioassays with neutralized, filtered, bleached kraft pulp mill effluent (BKME) and daily solution replacement. The effluent interfered with precopula behavior; high concentrations (40% of full strength BKME) resulted in rapid release of paired amphipods. A concentration-related time to 50% release of paired individuals was observed. For one batch of BKME, the behavioral threshold concentration (EC₅₀) was estimated at 10–15% of full strength BKME. The estimated 96 h LC₅₀ for underyearling coho salmon (Oncorhynchus kisutch) for the same effluent tested in freshwater was 36% BKME—illustrating a greater sensitivity of the amphipod behavioral bioassay in comparison to the salmonid acute lethal toxicity test. The amphipod precopula bioassay for toxicity testing has some merit in that it is moderately quantitative under given test conditions and does not require elaborate equipment.     

An evaluation of the potential toxicity of treated bleached kraft mill effluent to striped bass (Morone saxatilis walbaum) larvae exposed through metamorphosis to the juvenile stage

Twenty-day-old striped bass (Morone saxatilis) larvae were exposed to a range of treated bleached kraft mill effluent (BKME) concentrations from 0 to 20% effluent by volume (v/v) under continuous flow test conditins. The experimental test concentrations in the 2–20% BKME test aquaria had a BOD₅ which ranged from < 1 to 5 mg l⁻¹, TSS 12–17 mg l⁻¹, and true color 35–416 mg l⁻¹. Bleached kraft mill effluent did not kill larvae exposed to it for 20 days through metamorphosis to the juvenile stage. The BKME did not alter growth in length, weight or condition factor in larvae over the 20-day exposure period as determined by multiple regression analysis. A linear regression analysis on the dry weight data at Day 20 only, however, indicated a trend of decreasing weight with increasing BKME concentration. Effluent-exposed larvae also developed normally during the 20-day study. All individuals examined completed the transformation from postlarvae to juveniles by the age of 40 days.     

Multiscale approach of fish responses to different types of environmental contaminations: a case study

In order to study the responses of wild fish to environmental contaminations, different sites (two references and three contaminated) were sampled across the Walloon hydrographical network (southern Belgium). The status of fish communities was characterized according to an index of biotic integrity (IBI). Furthermore, population structure, reproductive parameters and biochemical assays were performed on chub (Leuciscus cephalus) as sentinel species. The results showed impaired responses in fish sampled downstream paper mill effluents (BKME), in a polychlorinated biphenyls (PCB) contaminated site, and in a river receiving pesticides (i.e. atrazine and hexachlorocyclohexane). High level of hepatic 7-ethoxyresorufin-Odeethylase (EROD) activity were evidence of direct toxicant exposure, while population structures showed unbalanced size-frequency distributions. Moreover, in the pesticide contaminated site, a higher proportion of atretic oocytes was found in females and abnormally high plasma alkali-labile phosphorus (ALP) values were noticed in males, compared to reference sites, suggesting that chubs were exposed to estrogenic compounds. IBI scores from both sites were also lower than in reference sites. On the other hand, no particular response was found in hepatic glutathione-S-transferase (GST) activities, hepatic heat-shock protein HSP70 concentrations and plasma sex steroids (estradiol-17beta, 11-ketotestosterone and testosterone) in chubs from any sites. By using a multiparametric approach, a better understanding of response mechanisms to pollution in fish may be achieved in order to improve the ecological status of river ecosystems.     

Simultaneous colour and DON removal from sewage treatment plant effluent: Alum coagulation of melanoidin

The aim of this study was to detect and characterise melanoidin in sewage treatment plant (STP) effluent, and to study the ability of alum coagulation to remove the colour and dissolved organic nitrogen (DON) associated with melanoidin. The melanoidin is non-biodegradable due to the complex cyclic based structure and thus it directly contributes to effluent nitrogen concentrations from the sewage treatment plant (STP). Lowering of effluent total nitrogen limits and the link between colour and chlorinated disinfection by-products have therefore driven a need to understand the structure, properties and treatability of DON species found in STP effluent.The focus of this paper is the refractory coloured, organic nitrogen compound melanoidin. Wetalla STP effluent has relatively high colour (170 mg-PtCo L⁻¹) and DON (2.5 mg L⁻¹) for a biological nutrient removal STP, owing to an industrial supply of melanoidin containing molasses fermentation wastewater. Alum coagulation jar tests were performed on synthetic melanoidin solution, STP effluent containing melanoidin (Wetalla, Toowoomba, Australia) and STP effluent free of melanoidin (Merrimac, Gold Coast, Australia) to examine the treatability of melanoidin and its associated colour and DON content when present in STP effluent.The removal of melanoidin from STP effluent resulted in significant colour and DON reduction. An alum dose of 30 mg L⁻¹ as aluminium was sufficient to reach maximum removal of colour (75%), DON (42%) and dissolved organic carbon (DOC) (30%) present in melanoidin containing STP effluent. Alum was shown to preferentially remove DON with a molecular weight >10 kDa over small molecular weight DON. Fluorescence excitation-emission matrix examination of the humic compounds present in the STP effluent indicated that melanoidin type humic compounds were more readily removed by alum coagulation than other humic compounds.     

Identification of estrogen receptor agonists in sediments from Wenyu River, Beijing, China

Assignment of ecological impacts of contamination to specific classes of contaminants is a prerequisite for risk assessment and remediation. In this study, the combination of polarity-based fractionation, two-hybrid yeast bioassay, and chemical analysis were used to evaluate and identify estrogen receptor agonists (ER-agonists) in sediments from Wenyu River, Beijing, China. By bioassay, organic raw sediment extracts could induce significant estrogenicity and the bioassay-derived 17β-estradiol equivalents (EEQs) of raw extracts (EEQ_raws) ranged from 0.8 to 19.8 ng/g dry weight. By polarity-based fractionation, the raw extracts were separated into three fractions, i.e. non-polar, moderately polar, and polar fractions, which were subjected to bioassay and chemical analysis. The highest estrogenicity was observed in the polar fraction, which accounted for more than 78% of the total. The medium polar fraction contains PAHs and OCPs, and the estrogenic activities in this fraction contributed 3%-12% of the total in raw extract. An estrogenic activity of non-polarity fraction was negligible in compare to other two fractions. By chemical analysis and toxic equivalent calculation, major part of the estrogenicity in polar fraction could be attributed to six natural/synthetic estrogens (16%-63%), i.e. 17β-estradiol, estrone, estriol, 17α-ethynylestradiol, diethylstilbestrol, and β-estradiol-17-valerate, and to nonylphenols (26%-55%). The proposed approach has been successfully used for characterization of ER-agonists in this case study.     

Effects of biologically-active chemical mixtures on fish in a wastewater-impacted urban stream

Stream flow in urban aquatic ecosystems often is maintained by water-reclamation plant (WRP) effluents that contain mixtures of natural and anthropogenic chemicals that persist through the treatment processes. In effluent-impacted streams, aquatic organisms such as fish are continuously exposed to biologically-active chemicals throughout their life cycles. The North Shore Channel of the Chicago River (Chicago, Illinois) is part of an urban ecosystem in which > 80% of the annual flow consists of effluent from the North Side WRP. In this study, multiple samplings of the effluent and stream water were conducted and fish (largemouth bass and carp) were collected on 2 occasions from the North Shore Channel. Fish also were collected once from the Outer Chicago Harbor in Lake Michigan, a reference site not impacted by WRP discharges. Over 100 organic chemicals with differing behaviors and biological effects were measured, and 23 compounds were detected in all of the water samples analyzed. The most frequently detected and highest concentration (> 100 μg/L) compounds were ethylenediaminetetraacetic acid and 4-nonylphenolmono-to-tetraethoxycarboxylic acids. Other biologically-active chemicals including bisphenol A, 4-nonylphenol, 4-nonylphenolmono-to-tetraethoxylates, 4-tert-octylphenol, and 4-tert-octylphenolmono-to-tetraethoxylates were detected at lower concentrations (< 5 μg/L). The biogenic steroidal hormones 17β-estradiol, estrone, testosterone, 4-androstene-3,17-dione, and cis-androsterone were detected at even lower concentrations (< 0.005 μg/L). There were slight differences in concentrations between the North Side WRP effluent and the North Shore Channel, indicating minimal in-stream attenuation. Fish populations are continuously exposed to mixtures of biologically-active chemicals because of the relative persistency of the chemicals with respect to stream hydraulic residence time, and the lack of a fresh water source for dilution. The majority of male fish exhibited vitellogenin induction, a physiological response consistent with exposure to estrogenic compounds. Tissue-level signs of reproductive disruption, such as ovatestis, were not observed.     

Removal of estrogenicity in Swedish municipal sewage treatment plants

The human estrogen receptor alpha-test, hosted in a yeast strain, was used to quantify estrogenicity in three-week composite samples of untreated and treated effluents from 20 Swedish municipal sewage treatment plants. The treatment plants were selected to represent different treatment processes regarding chemical precipitation and microbial procedures. The discharge from Swedish domestic sewage treatment plants contained estrogenic compounds corresponding to <0.1-15 ng estradiol equivalents/L. Low levels of estrogenic activity were also found in a river receiving municipal effluents, 3.5-35 km downstream the outlet from a sewage treatment works. The range of estrogenicity in untreated, raw sewage effluents was found to be 1-30 ng estradiol equivalents/L. Generally, wastewater treatment reduced the estrogenicity and extended biological treatment was most effective in its removal. Activated sludge treatment tended to be more effective than trickling filters, whereas chemical precipitation using iron or aluminium salts without biological treatment showed little effectivity. The study showed that treatment methods in current use are able to eliminate or largely reduce estrogenicity in domestic wastewater.