Distribution of estrogens, 17β-estradiol and estrone, in Canadian municipal wastewater treatment plants

Science of the Total Environment 336 (2005) 155 – 170 www.elsevier.com/locate/scitotenv

Distribution of estrogens, 17h-estradiol and estrone, in Canadian municipal wastewater treatment plants M.R. Servos a,b,*, D.T. Bennie b, B.K. Burnison b, A. Jurkovic b, R. McInnis b, T. Neheli b, A. Schnell c, P. Seto b, S.A. Smyth b, T.A. Ternes d b

a Department of Biology, University of Waterloo, Waterloo, Ontario, Canada National Water Research Institute, Environment Canada, Burlington, Ontario, Canada c Hydromantis Inc., Cambridge, Ontario, Canada d ESWE-Institute for Water Research and Water Technology, Wiesbaden, Germany

Received 16 February 2003; accepted 25 May 2004

Abstract The distribution of female hormones, 17h-estradiol and estrone, was determined in effluents of 18 selected municipal treatment plants across Canada. Replicate 24-h composite samples were collected from the influent and final effluent of each treatment plant, and the removal efficiency compared to the operational characteristics of the plants. In conventional activated sludge and lagoon treatment systems, the mean concentrations of 17h-estradiol and estrone in influent were 15.6 ng/l (range 2.4 – 26 ng/l) and 49 ng/l (19 – 78 ng/l). In final effluents, the mean concentrations of both 17h-estradiol and estrone were reduced to 1.8 ng/l (0.2 – 14.7 ng/l) and 17 ng/l (1 – 96 ng/l), respectively. 17h-Estradiol was removed effectively, >75% and as high as 98%, in most of the conventional mechanical treatment systems with secondary treatment. The removal of estrone was much more complex with removal varying from 98% to situations where the concentrations in the effluent were elevated above that detected in the influent. The estrogenicity, measured using a transfected estrogen receptor in yeast (YES) assay, was also variable, ranging from high removal to elevations of estrogenicity in final effluent. Although the apparent removals were not statistically correlated with either hydraulic (HRT) or solid (SRT) retention times, plants or lagoons with high SRT were very effective at reducing the levels of hormones. Well-operated plants that achieved nitrification also tended to have higher removal of hormones than those that did not nitrify. Laboratory aerobic reactor experiments confirmed the rapid removal of 17hestradiol, estrone, and estrogenicity when exposed to sewage slurries. D 2004 Elsevier B.V. All rights reserved. Keywords: Endocrine disruptors; Estrogen; Hormones; Removal efficiency; Municipal wastewater

1. Introduction

* Corresponding author. Department of Biology, University of Waterloo, 200 University Ave. W. Waterloo, Ontario, Canada N2L 3G1. Tel.: +1-519-888-4567x6034; fax: +1-519-883-7574. E-mail address: [email protected] (M.R. Servos). 0048-9697/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2004.05.025

The presence of substances in the environment that have the potential to disrupt the normal function of endocrine systems of biota has raised considerable concern in Canada and worldwide (Servos et al., 2001b; WHO, 2002). Subtle alterations in endocrine

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function may lead to altered growth, development, or reproduction in exposed animals, and these changes may be expressed later in the life cycle or even in future generations (Lister and Van Der Kraak, 2001; Metcalfe et al., 2001). Estrogenic substances have been identified and quantified in a wide variety of environments associated with industrial and municipal effluents, as well as urban and agricultural runoff in Canada (Hewitt and Servos, 2001). Discharges from municipal treatment plants represent one of the largest sources of effluent to the Canadian environment, and the potential for these effluents to contain a variety of estrogenic substances has recently raised concerns (Chambers et al., 1997; Servos et al., 2001c). The presence of estrogenic substances in municipal effluents has been linked to a number of biological responses, such as induction of plasma vitellogenin and intersex in fish exposed in the environment immediately adjacent to the outfalls (Purdom et al., 1994; Jobling et al., 1998; Harries et al., 1999). Exposure during critical life stages may result in a variety of biological impacts mediated through endocrine systems. Numerous potential endocrine disrupting substances have been reported in municipal effluents in Canada including natural and synthetic estrogens (Ternes et al., 1999b; Lee and Peart, 1998a), alkylphenol polyethoxylates (Bennie et al., 1998; Lee and Peart, 1998b; Servos et al., 2001a), and bisphenol-A (Lee and Peart, 2000). It has also been established that many of these compounds can be detected in surface waters and sediments that receive wastewater discharges in Canada and elsewhere (Bennie et al., 1997; Kolpin et al., 2002; WHO, 2002). The major contribution to the estrogenicity in effluents has been shown to be related to the presence of natural and synthetic estrogens in several municipal effluents (Desbrow et al., 1998; Routledge et al., 1998). The presence of industrial chemicals, such as alkylphenols, has also been shown to contribute to the estrogenicity of effluents, especially if there is a significant industrial contribution to the effluent (Burnison et al., 2002; Thomas et al., 2001; Sheahan et al., 2002). Even though natural and synthetic estrogens have been reported previously in Canadian sewage effluents (Ternes et al., 1999b), very little information is available about their distribution or fate in Canadi-

an wastewaters. Many factors may influence the relative distribution, fate, and treatability of these chemicals in municipal effluents. The current study examined the distribution of the natural estrogens, 17h-estradiol and estrone, in 18 municipal treatment plants across Canada. The removal of these substances from municipal treatment systems was compared with the characteristics of the plants including basic design and performance parameters collected at the time of sampling. Laboratory batch experiments were conducted to investigate the removal under controlled conditions.

2. Materials and methods 2.1. Site descriptions Eighteen municipal wastewater treatment plants (WWTPs) were selected to represent a wide range of process types, including primary, secondary, tertiary, and lagoon treatment (Table 1). Plant supervisors were interviewed regarding site-specific treatment processes and operations during sampling at each plant (Table 2). During the site sampling periods, daily field recordings were taken for daily flow rates into the plant and through various parallel process stages, routine process information, and any unusual plant conditions. A questionnaire was completed by supervisory staff at each plant to verify plant operating conditions corresponding to the sampling periods. The solid retention times (SRT) reported are for the aeration stage of the biological treatment process. The hydraulic retention times (HRT) are based on aerated basin or the entire treatment system. 2.2. Sample collection Raw sewage influent and final treated effluent samples were collected September through November of 1998 using Isco 6700FR refrigerated automatic samplers (Isco, Lincoln, NE, USA). Samples were 24-h bulk composites of defined volumes every 20 min for plants A, B, D, E, F, H, I, P, and Q, and 24-h flow-proportioned composites for plants C, G, N, O, and R. Influent and effluent samples were staggered by 24 h. Sample temperature in the automatic samplers was maintained at 4 jC during sample

Table 1 Process characteristics for selected Canadian wastewater treatment plants Sampling date

Plant classification

Grit removal

Primary treatment

Secondary treatment

Nitrification

Phosphorus removal

Tertiary filtration

Disinfection (intensity)

A

October 30, 1998

Aerated

Yes

Conventional AS

Yes

Alum

Sand

UV (low)

B

November 6, 1998

Ferric chloride

No

Chlorin.

Yes

Conventional AS, polymer Conventional AS

Partial

November 19, 1998

Aerated ferric chloride polymer Aerated

Yes

C

No

Ferrous chloride

No

Chlorin.

D

September 3, 1998

Vortex

Yes

High AS

Partial

Alum

No

E

September 16, 1998

Alum

Yes

Conventional AS

Partial

Primary

No

F

September 24, 1998

Aerated

Yes

Conventional AS

Partial

Alum

No

G

September 16, 1998

Yes

No

Yes

Alum

Sand

October 13, 1998

Yes

Yes

Yes

I

October 8, 1998

Yes

Yes

Yes

Ferrous chloride, dual point Ferric chloride

Granular anthracite No

Chlorin.

J

October 16, 1998

No

No

No

Alum

No

No

K

October 16, 1998

Lagoon

No

No

No

Alum

Slow sand

No

L

October 16, 1998

Lagoon

No

No

No

Alum when needed

No

No

M

October 16, 1998

Lagoon

No

No

No

Alum, polymer

No

No

N

December 3, 1998

Aerated

Yes

Yes

Alum

No

UV

O

December 9, 1998

Aerated

Yes

No

No

No

No

P

December 9, 1998

Aerated

Yes

No

No

No

No

Q

January 28, 1999

Mechanical Secondary Mechanical Secondary Mechanical Secondary TF/SC Secondary

No

Yes

No

No

No

Chlorin. seasonal

R

February 11, 1999

Primary

Yes

Yes

Extended aeration AS Extended aeration AS Conventional AS, methanol, antifoam Aerated/facultative lagoon, seasonal discharge Aerated/facultative lagoon, seasonal discharge Aerated/facultative lagoon, seasonal discharge Anaerobic lagoon seasonal discharge, Biological nutrient removal (BNR) High rate oxygen AS High rate oxygen AS Trickling filter solids contact process (TF/SC) No

UV (med) seasonal UV (low) seasonal UV (med) seasonal UV (high)

H

Mechanical Tertiary Mechanical Secondary Mechanical Secondary Mechanical Secondary Mechanical Secondary Mechanical Secondary Mechanical Tertiary Mechanical Tertiary Mechanical Secondary Lagoon

No

Alum

No

No 157

AS, activated sludge.

UV (high)

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Plant no.

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Table 2 Operational parameters for selected Canadian wastewater treatment plants corresponding to sampling periods Plant Actual Average daily Wastewater sources no. population flow (m3/day)

Significant industrial discharges

MLSS Mixed liquor SRT Aeration System (mg/l) percentage (days) basin HRT HRT (h) volatility (h)

A B

68,800 124,000

32,872 68,498

60% res, 40% IC 70% res, 30% IC

1780 2000

75 69

5.5 9.6

8.5 6.6

22 15

C

1,226,000

585,667

60% res, 40% IC

2672

73

2.7

6.7

14

D E F G H

46,100 24,800 179,300 3,600 29,000

17,364 6,243 125,248 2,400 14,600

80% 90% 60% 60% 54%

textile 11% meat 2%, chem 1%, steel 1% food 1.4%, brew 1.2%, paper 0.8% metal, food

1210 1860 2174 5000 2490

83 76 83 58 57

0.9 4.1 4.7 13.6 53.0

2.8 8.0 6.5 43.0 16.8

11 – 12 14 13 – 14 61 28

I J K L M N O

10,000 2,000 2,150 6,475 1,600 600,000 382,000

5,074 1,171 850 2,382 432 366,898 185,000

2667 na na na na 3200 2423

64 na na na na 75 75

35.5 >150 >150 >150 >150 12.6 2.7

12.3 >150 >150 >150 >150 11.1 4.1

27 >150 >150 >150 >150 23 12

P Q R

157,000 900,000 1,799,000

47,860 626,000 2,366,208

50% res, 50% IC 85% res, 15% cheese res res res Not available food 1.7%, animal 0.6% Not available slaughter, tannery, malting Not available slaughter, poultry Not available paper 2%, wood, food 85% res, 15% IC food 2%, metal 2%, textile 1%

3275 na na

82 78 na

2.2 1.9 na

2.7 1.0 na

13 6–8 3

res, res, res, res, res,

20% 10% 40% 40% 46%

IC com IC dairy IC

food dairy 40% auto, metal, plastics, feed auto, metal, paper

Notes: Res = residential; IC = industrial/commercial; na = not applicable.

collection. Containers used for sample collection and compositing were 20 l stainless steel canisters, precleaned with Contrad 70 (Decon Laboratories), and rinsed sequentially with reverse-osmosis-filtered water, acetone, and hexane (Caledon Laboratories). At plants in which it was necessary to collect samples from parallel process locations, 24-h composites were collected at each location then blended in proportion to the flow rates through the parallel processes, based on WWTP process information corresponding to the sampling times. Sampling of treated wastewater from the four lagoon processes (plants J, K, L, and M) involved collection of a grab sample from that lagoon cell within 1 month prior to scheduled seasonal discharge (autumn). At plants L and M, a second grab sample was collected at the actual time of discharge. For Plant L, a different cell was sampled, while at Plant M, a sample was taken after the addition of coagulant (polymer/alum mixture). Sample aliquots for the analysis of 17h-estradiol and estrone and the yeast estrogen screening (YES)

bioassay were prepared directly in the field by completely filling new, precleaned amber glass bottles with Teflon-lined caps with a portion of the composite bulk sample. All samples were shipped on ice either the same day or by overnight transport. Sample extractions were performed within 1 day of sampling for the YES assay and normally within 1 day but always less than 3 days for the hormones. Field duplicate samples of wastewater were collected periodically, representing about 10% of the total number of samples analyzed. Water blanks consisting of overnight collections of reverse-osmosis-filtered water into precleaned stainless steel canisters using an autosampler under the same conditions as the effluent samples. 2.3. Analysis Conventional wastewater parameters (5-day carbonaceous biochemical oxygen demand (cBOD5), total suspended solids (TSS), total Kjeldahl nitrogen

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(TKN), total phosphorus, ammonia-nitrogen, and nitrate-nitrogen) were analyzed according to standard methods (A.P.H.A., 1995). 17h-Estradiol and estrone in raw and treated wastewater were measured using a method adapted from Ternes et al. (1999b). To summarize, samples were adjusted to pH 3, spiked with 17h-estradiol acetate, and filtered through a 1.2-Am Whatman GF/C filter. They were then extracted on a vacuum manifold using reverse-phase 1 g C18 (SPE) cartridges (Supelco 5-7054), eluted with acetone, cleaned up on deactivated silica gel, and derivatized with MSTFA/TMSI/DTE (Sigma). Final quantification was done by Wellington Laboratories, Guelph, Ontario, using high resolution GC-MS (Vg70 coupled to a HP5890 Series II GC) with a 60 m JW DB-5 column 0.25 mm id, 0.25 Am film; m/z estrone, 342.2015, 343.2042, 17h-estradiol, 416.2567, 417.2591, estradiol acetate surrogate 386.2277, 387.2305. GC conditions were as follows: 2 Al splitless injection, injector temperature 250 jC, initial oven temperature of 100 jC hold for 1 min, 30 jC/min to 180 jC, 4 jC/min to 290 jC hold for 6 min. Mirex was also used as an internal instrument standard. A con-

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tamination problem with the surrogate prevented the trace level quantification of 17a-ethinylestradiol in environmental samples. Variability of repeated injections of the same sample (based on three plants) was 0.5 F 0.1% and 2.3 F 2.1% for estradiol and estrone, respectively. The variability of duplicate samples (based on eight plants) was 5.8 F 8.9% and 8.9 F 6.5% for estradiol and estrone, respectively. Method detection limits were 0.7 ng/l for estrone and 0.8 ng/ l for 17h-estradiol. The yeast estrogen screening (YES) bioassay used for the detection of possible estrogenic substances was based on Gaido et al. (1997), with minor alterations. The transformed Saccharomyces cerevisiae was kindly donated by Dr. Kevin W. Gaido. The yeast contains two plasmids. One plasmid contains the CUP1 metallothionien promotor and human estrogen receptor cDNA for copper inducible estrogen receptor production. The other plasmid contains two estrogen-responding elements linked to the lacZ gene (the structural gene for the h galactosidase enzyme). Estrogenic substances bind to the estrogen receptor and form dimers, which then bind to the estrogen-responding elements,

Fig. 1. Mean influent and effluent 17h-estradiol concentrations in Canadian wastewater treatment plants. Bars represent the range where applicable.

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Fig. 2. Mean influent and effluent estrone concentrations in Canadian wastewater treatment plants. Bars represent the range where applicable.

which causes the production of the h- galactosidase enzyme. The amount of enzyme produced is then detected by permeabilizing the yeast cells to allow

the h- galactosidase contact with its substrate, orthonitrophenolgalactopyranoside (ONPG), and the color change is quantified.

Fig. 3. Mean influent and effluent YES % response in Canadian wastewater treatment plants. Bars represent the range where applicable.

M.R. Servos et al. / Science of the Total Environment 336 (2005) 155–170 Table 3 Hormone levels and YES response for secondary, tertiary, and lagoon plants (trickling filter and primary plant left out of means) 17h-Estradiol (ng/l)

Estrone (ng/l)

YES response (%)

Influent Effluent Influent Effluent Influent Effluent Mean 15.6 Maximum 26.0 Minimum 2.4

1.8 14.7 0.2

49 78 19

17 96 1

79 145 n.d.

50.1 106.0 n.d.

2.4. Batch aerobic treatability An extended batch aerobic biotreatability experiment was conducted to determine the ultimate extent of biodegradation possible under ideal conditions. Intermediate samples were taken during the experiment to determine the removal and possible biotransformation products. Two cleaned 20 l glass bioreactors were used for the experiment. The baffled reactors included finebubble spargers for aeration, supplemental mixing by an impeller stirrer, and provision for automatic temperature control. To initiate the experiment, each reactor was filled with 12 l of raw sewage and 6 l of return activated sludge (RAS) from Plant A. Since Plant A was nitrifying, alkalinity in the form of 18 g sodium bicarbonate (NaHCO3) was added to each reactor to ensure adequate buffering capacity. After mixing for 10 min, 3 l of the suspension was withdrawn from each reactor, blended, then subdivided for various chemical and biological analyses. This represented the time zero untreated sample. The reactors were reseeded with 2 l of RAS on day 6. A control sample was also prepared and analyzed to determine possible contributions of compounds of interest from the sludge inoculums. The control sample was prepared by mixing 33% RAS with 67% distilled water for 10 min, settling, and then sampling the supernatant. The reactors were then stirred and aerated continuously for 26 days. Temperature was controlled at 25 jC and each reactor was monitored regularly for dissolved oxygen (DO), pH, and temperature. Distilled water was added periodically over the course of the experiment to compensate for evaporative losses. Intermediate samples of 1 L were withdrawn from each reactor and blended for analysis of 17h-estradiol, estrone, and YES response on days 0, 1, 2, 5, 12, and 26.

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3. Results Four main categories of WWTPs were included in this survey: 1 primary treatment plant, 10 secondary plants, 3 tertiary plants, and 4 lagoons (Table 1). One of the secondary plants (Plant Q) utilized a combination trickling filter/solids contact process that combines a high rate attached-growth stage with a high rate suspended growth-activated sludge stage for enhanced treatment; the other nine secondary plants use suspended growth processes (activated sludge). The three tertiary plants used sand or granular media filtration (anthracite) as a tertiary treatment process. Disinfection and phosphorus removal were not considered ‘‘tertiary’’ processes in the classification of plants in this data set. The influent and effluent concentrations of 17hestradiol and estrone, as well as the YES response, in the individual plants varied considerably (Figs. 1 –3). The mean influent and effluent hormone concentrations and the YES percent response values for the nine conventional suspended growth secondary

Table 4 Number of samples collected and analyzed, and mean percent removal of hormones and YES response at each plant Plant

A B C D E F G H I J K L M N O P Q R

17h-Estradiol

Estrone

YES response

na

n

n

Percent removal

1 0 2/3 3 1 3 1 1 1 1 0 1/2 1/2 2 2 2 2 2

3

3 1 3 3 1 3 1 1 1 1 1 1 1 3 3 3/2 2/3 3 a

Percent removal 82.9 96.8 39.5 92.7 98.3 75.9 93.3 98.8 98.2 98.4 80.5 95.9 98.1 94.7 96.1 97.1 18.5 1.0

3 1 3 3 1 3 1 1 1 1 1 1 1 3 3 3 2/3 3

Percent removal 66.7 72.7 54.8 76.7 85.4 45.8 96.5 97.8 95.1 93.3 46.4 95.3 96.1 82.1 80.6 95.1 62.4 28.6

41 84 nd 51 16 100 100 100 ‘‘created’’ ‘‘created’’ 78 50 54 62 10

Influent/effluent sample size; ‘‘created’’= nondetectable levels of YES response in the influent but measurable levels in the effluent.

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Table 5 Correlation (R2) between hormone removal and solid retention time (SRT) or hydraulic retention time (HRT) for conventional secondary and tertiary treatment systems (n = 12) Percent removal (%)

SRT (days)

Aerated basin HRT (h)

System HRT (h)

17h-Estradiol Estrone YES response

0.313 0.390 0.534

0.355 0.399 0.028

0.443 0.491 0.102

plants, the three tertiary plants, and the four lagoons are summarized in Table 3. The mean influent concentrations were 15.6 ng/l for 17h-estradiol and 49 ng/l for estrone. The mean effluent concentrations were considerably lower at 1.8 ng/l for 17h-estradiol and 17 ng/l for estrone. The mean YES response declined from 79% to 50% from the influent to the final effluent. The apparent removal of 17h-estradiol in conventional mechanical treatment systems was generally greater than 75% and as high as 98%, although there were exceptions (Table 4). Plant C (secondary treatment) had a removal only 39.5% and Plant Q that used a trickling filter had essentially no remov-

al of 17h-estradiol. Lagoons were generally very effective at removing 17h-estradiol, with apparent removal ranging from 80% to 98%. Estrone removal is more complex and ranged from as high as 98% to situations were the concentrations in final effluent were elevated above that in the influent. The YES response was also more variable and ranged from almost complete removal in aerated lagoons to higher responses in final effluent compared to influent. There was a considerable range in the HRT in the various secondary treated systems ranging from 6 to 28 h. The HRT for the aeration basins ranged from 2.7 to 43 h, while the HRT for Plant Q (TF/SC) was only 1 day. The one primary treated plant had a HRT of only 3 h. The four lagoons had a HRT of >150 h. Similarly, the SRT varied considerably ranging from 1.9 to 53 days in conventional activated sludge plants and >150 days in lagoons. There was no statistical relationship (R2 < 0.53) between the HRT or SRT and hormone or estrogenicity removal for the nine conventional mechanical secondary plants and the three tertiary plants (Table 5, Figs. 4 and 5). Using the HRT for the aeration basin

Fig. 4. Reduction of estrogens in Canadian wastewater treatment plants relative to solid retention time (SRT). Lagoons with >150 days retention are not included.

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Fig. 5. Reduction of estrogens in Canadian wastewater treatment plants relative to hydraulic retention time (HRT). Lagoons with >150 h retention are not included.

rather than the whole system did not increase the strength of the relationships. However, the plants with high SRT or HRT have relative high apparent removal of both hormones and YES response, while the low SRT plants tend to have more variability and lower removal. The diverse characteristics of the plants and limited data set may affect the strength of any underlying relationships. WWTPs are designed to reduce cBOD5 and TSS, and in some cases total phosphorus, in order to minimize adverse impacts on the receiving waters. For the plants in this survey, the effluent criteria for cBOD5 and TSS were >25 mg/l each. Tables 6a and b summarize the influent and effluent levels of conventional parameters, cBOD5, TSS, total phosphorus, total Kjeldahl nitrogen (TKN), ammonia-nitrogen, and nitrate-nitrogen, providing an illustration of plant performance during the sampling period. Four of the plants in this study (A, G, H, and N) were being operated to nitrify (sufficiently long sludge age and dissolved oxygen levels to promote the growth of autotrophic nitrifying bacteria, which convert ammonia to nitrate). Of these four, plant A was not nitrifying as well as plants G, H, and N. Examination of the

influent and effluent nitrogen species (Table 6b) indicates that in fact plants B, D, E, F, and I were also achieving nitrification. The percent removal values for 17h-estradiol, estrone, and YES response associated with nitrifying and non-nitrifying plants are presented in Fig. 6, along with the mean removal of each hormone from plants grouped according to nitrification. There is a weak pattern suggesting that plants that were nitrifying had elevated apparent removal of the hormones and YES response. The removal of 17h-estradiol and estrone, as well as YES response, was very rapid in batch reactors. More than 95% of the hormones and estrogenicity was removed within 1 day in the reactors (Fig. 7). However, even after 26 days, traces of the hormones were still detectable. The reseeding with RAS on day 6 may be responsible for the slight increase in concentrations seen at the midpoint of the experiments.

4. Discussion Biological treatment appears to be effective in reducing the release of 17h-estradiol by 75 –98% in

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Table 6a Summary of conventional parameters: cBOD5, TSS, and total P Plant 5-day carbonaceous no. biochemical oxygen demand (mg/l)

A B C D E F G H I J K L M N O P Q R

Total suspended solids (mg/l)

Total phosphorus (mg/l)

Influent

Effluent

Influent Effluent Influent Effluent

179 272 229 194 186 288 623 114 90 525 200 197 197 262 279 279 148 103

3 4 6 5 4 8 2 1 3 2 25 2 29 11 16 13 9 51

218 268 310 137 138 195 394 188 146 350 160 146 134 203 269 225 148 98

9 5 4 16 3 22 6 4 12 5 78 3 83 11 19 11 14 30

5.5 7.7 7.8 4.4 6.4 6.4 17.0 6.2 4.3 18.0 6.4 5.8 8.2 6.4 9.3 9.3 5.6 1.9

0.9 0.5 0.3 1.1 0.6 1.0 0.6 0.2 0.2 0.9 1.4 1.3 1.0 1.4 4.1 4.3 2.5 0.6

Notes: plants K and M (lagoons) BOD>25 mg/l, TSS>>25 mg/l.

Canadian municipal treatment plants. The removal of estrone, however, is much more variable. Removal of estrone was as high as 98%, but in several plants, the concentrations in the final effluent were elevated above that observed in the influent. The removal of estrogens from Canadian effluents is consistent with that reported in other countries. In a study of six treatment plants in Italy, repeated sampling of the effluents demonstrated that 87% of the 17h-estradiol was effectively removed during treatment (Baronti et al., 2000; Johnson et al., 2000). In contrast, the removal of estrone was only 61%, and during several sampling periods, the concentrations of estrone were elevated in the final effluent above that in the influent. Over a 5-month period, the inlet concentrations averaged 12 and 52 ng/l for 17h-estradiol and estrone compared to only 1.0 and 9.3 ng/l in the final effluents. Johnson et al. (2000) reported an average of 88% of 17h-estradiol and 74% of estrone being removed in a series of treatment plants in Europe. Nasu et al. (2001) reported that the concentration of 17h-estradiol could increase from influent to primary before declining during biological treatment. Huang and Sedlak (2001) reported levels of 17h-estradiol in

wastewater effluents of 0.2 –4.1 ng/l (measured by ELISA and validated with GC-MS). A study of 27 treatment plants in Japan reported the removal of 17h-estradiol at 70% using an ELISA for qualification (Nasu et al., 2001). Great caution needs to be used when interpreting the relative removal of estrogens during treatment because of analytical limitations and the transformation of estrogen among various forms during collection and treatment. The extraction method used in the current study allowed for the measurement of only the free form of the estrogens and did not include conjugates or the fraction sorbed to particles. A general reduction of estrogenicity (47%) in effluents was observed using the YES assay although there was considerable variability. In most cases, the estrogenicity was decreased, but in several cases, the total estrogenicity increased slightly. Other studies have reported on the estrogenicity in effluents using a variety of in vitro bioassays to estimate the 17hestradiol equivalency. Ko¨rner et al. (1999) used a human estrogen receptor-positive MCF-7 breast cancer cell assay (E-screen assay) to determine the relative estrogenicity of effluent from municipal sewage plants in south Germany. All five effluents

Table 6b Summary of conventional parameters: nitrogen species Plant Total Kjeldahl N no. (mg/l)

A B C D E F G H I J K L M N O P Q R

Ammonia-N (mg/l)

Nitrate-N (mg/l)

Influent

Effluent

Influent Effluent Influent Effluent

28 45 48 28 37 31 28 37 22 45 39 35 51 33 62 44 39 16

5.3 10 22 4.4 2.9 4.7 1.5 1.0 2.0 2.0 6.6 1.2 9.3 2.9 39 31 30 13

14 25 26 20 25 14 11 23 18 21 28 25 42 25 40 29 25 6.0

3.4 8.5 20 1.9 0.82 1.6 0.08 0.22 0.52 0.24 0.39 0.22 1.5 0.16 36 29 21 5.3

1.10 0.04 0.40 0.10 0.01 0.15 0.11 nd 0.01 10.50 0.07 0.05 0.05 0.10 0.12 0.02 0.02 nd

11 9 4.7 14 18 5.0 8 21 24 0.06 0.54 0.51 0.15 11 0.18 0.19 0.03 0.3

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Fig. 6. Estrogen removal in nitrifying and non-nitrifying secondary and tertiary wastewater treatment plants in Canada.

Fig. 7. Removal of estrogens in aerated batch degradation test with a slurry of activated sludge. Reactors were partially reseeded with fresh activated sludge after 144 h.

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strongly induced cell proliferation that could be inhibited by an estrogen receptor antagonist. They estimated the 17h-estradiol equivalency to be between 2.5 and 25 ng/l in these effluents. In a later study at a modern municipal treatment plant in Germany, Ko¨rner et al. (2000) reported the 17h-estradiol equivalency (E-screen) to be reduced from 58 to 70 ng/l in the influent to 6 ng/l in the effluent. The estrogenic potency measured using three in vitro bioassays (ER binding, YES, ER-CALUX) in four municipal wastewater plants in The Netherlands was reduced by 90 – 95% from the influent to final effluent (Murk et al., 2002). Seventy to ninety-five percent of the estrogenicity was removed from the effluent with the exception of one plant that did not have secondary treatment and removed only 7 – 10% of the estrogenicity. Matsui et al. (2000) also observed a decrease in the estrogenicity of sewage effluents during biological treatment using a YES system. Using these in vitro assays is useful because they give an indication of the total estrogenicity of a very complex mixture. They may, however, be difficult to interpret, as in many cases the measured chemicals cannot fully explain the results. Additional unknown chemicals in the effluent may contribute to the response or may act to reduce the response by competing for the receptor. Estrogens are excreted by mammals as glucuronide or sulfate conjugates in urine or in the unmetabolized form in feces (Orme et al., 1983). D’Ascenzo et al. (2003) have shown that although most of the estrogens are excreted in urine as conjugates they are rapidly transformed and degraded in treatment systems. Adler et al. (2001) reported that 50% of 17hestradiol and 58% of estrone were conjugated in raw sewage. The transformation of estrogens appears to start in the collection system. The degradation of estrogens in aerobic batch reactors with a sewage sludge in the current study was very rapid, with 17hestradiol and estrone being reduced by >95% in less than 24 h. However, even after 120 h, traces of estrone and the YES response could be detected. Ternes et al. (1999b) studied the behavior of estrogens in aerobic batch reactors with slurries of activated sludge from a German sewage treatment plant. Under these conditions, 17h-estradiol was rapidly (hours) oxidized to estrone that was then eliminated from the systems, without the appearance of further degrada-

tion products. Layton et al. (2000) also reported that biosolids from municipal plants mineralized 70– 80% of added 14C-17h-estradiol to 14CO2 in 24 h. Batch reactor studies with glucuronides of 17h-estradiol (17h-estradiol-(17 or 3)-h- D -glucuronide) have shown that the glucuronides are rapidly cleaved in contact with diluted activated sludge resulting in the release of 17h-estradiol (Ternes et al., 1999a). After < 15 min, the 17h-estradiol-glucuronide was cleaved and both 17h-estradiol and estrone could be detected. Within 20– 30 h, 70% of the conjugated 17h-estradiol could be detected, primarily as the oxidized form (estrone). Panter et al. (1999) also reported that 17hestradiol-3-glucuronide was rapidly converted to17hestradiol in aqueous systems. Ju¨rgens et al. (2002) also showed that 17h-estradiol was rapidly oxidized to estrone and then was mineralized by natural bacteria in river water without the appearance of other major degradation products. Lee and Liu (2002) examined the fate of 17h-estradiol in aerobic and anaerobic reactors with activated sludge and observed the rapid degradation of 17h-estradiol to estrone but did not observe any other major metabolites. Belfroid et al. (1999) treated effluent samples with h-glucuronidase prior to extraction to release the conjugated hormones but were not able to detect the hormones in this form in final effluents (with one exception). Huang and Sedlak (2001) also detected less than 2% of 17h-estradiol as conjugates in wastewater in California. After 28 h in experimental batch reactors, Ternes et al. (1999a) found traces (3%) of 17hestradiol remaining, suggesting that the cleavage of glucuronides was not complete. Estrone enters the treatment system either directly from excretion of humans (in the free form or as glucuronide or sulfate conjugates) or from the oxidation of 17h-estradiol in the collection or treatment systems. The much higher variability in the concentrations and removal of estrone may be a result of several factors. The creation of estrone early in the treatment process is likely to occur as 17h-estradiol is rapidly oxidized. This would result in an apparent increase in the concentration of estrone in the early stages of the treatment plant. As biological treatment continues, free estrone will be removed from the treatment system. There could also be additional estrone leaving the treatment system as estrone-3sulfate that is not included in the analytical method-

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ology used in this study. D’Ascenzo et al. (2003) observed a lower removal rate of the sulfate conjugate of estrone in treatment systems relative to the free form. The conditions and degree of treatment may therefore greatly influence the relative concentrations of estrone in different parts of the treatment process leading to the apparent high variability and even the apparent creation of estrone in the final effluents in this and other studies. The form of the estrogens greatly influences their estrogenic potency. Matsui et al. (2000) compared the estrogenic activity of various substances using the EC50 of the YES response. Estone showed 0.21 the activity of 17h-estradiol while estriol was only 1.3  10 3 the activity of 17h-estradiol. The conjugated form 17h-estradiol 3-sulfate was 5.3  10 5 and 17h-estradiol 17-h-D-glucuronide and 17h-estradiol 3-h-D-glucuronide were only 5.9  10 7 and 3.1  10 5, respectively, relative to the activity of 17h-estradiol. The estrogenic potentials of the conjugated forms of estrogens are clearly much lower. The cleavage of glucuronide during treatment or in the collection system may therefore greatly increase the estrogenicity of the effluent. The estrogenicity or potential estrogenicity of the effluent may therefore change depending on the relative degradation and form of the estrogens present. The distribution and fate of estrogens in municipal treatment plants are very complex, and no clear patterns associated with the process or treatment characteristics could be established. In general, most of the conventional mechanical activated sludge treatment plants were effective at removing 17hestradiol, while the removal of estrone and estrogenicity was less evident. Two plants in the current study stood out as having particularly low apparent removal of estrogens. Plant R had minimal treatment (primary) and corresponding minimal effectiveness at removing estrogens. Plant Q in this study utilized a trickling filter/solid contact that despite being effective at removing conventional parameters was ineffective at removing estrogens. The trickling filters (attached growth process) oxygenate the settled wastewater and remove soluble carbonaceous BOD by means of the biofilm attached to the support media. These systems therefore have relatively low SRT and HRT. Ternes et al. (1999b) reported poor removal of a trickling filter system in Brazil. Spen-

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gler et al. (2001) noted that the one plant in their study fitted with a trickling filter had elevated levels of estrogens. There were no statistical relationships (R2 < 0.53) between SRT or HRT (total or aerated basin) and the apparent removal of estrogens in the activated sludge plants. There are many factors including the influent and plant characteristics that may mask the importance of these parameters. The two treatment plants with the highest apparent removal both had very high HRT (>27 h) and SRT (>35 days). The two plants with relatively low SRT (2.7 and 4.7 days) are the two plants that had elevated levels of estrone in the final effluent relative to the influent. The lagoons that had extremely long HRT and SRT had consistently high removal of estrogens and estrogenicity (YES response). The current study used a limited number of samples at many different plants with a wide variety of treatment and process characteristics. Additional controlled studies are needed to more fully explore these relationships, especially the potential relationship between extended SRT on removal of estrogens. The addition of advanced treatment (filtration or phosphorous removal) also did not have an apparent effect on increasing the removal of estrogens in the Canadian plants studied. However, additional controlled experiments are needed to examine the potential effects of additional treatment and disinfection (i.e., UV light) in more detail. Kirt et al. (2002) examined the fate of estrogenic activity (YES) in five treatment plants in the United Kingdom and found that most of the removal occurred in the secondary treatment with some additional removal associated with tertiary treatment. In two of the plants they studied, considerable reductions in estrogenicity also occurred during primary treatment. Spengler et al. (2001) observed that the plants equipped with activated sludge followed by an activated carbon filtration had the lowest concentrations. Huang and Sedlak (2001) reported that treatment plants with advanced treatment (filtration, microfiltration, and reverse osmosis) had reduced levels of 17h-estradiol compared to a conventional secondary treatment plant. Although 17h-estradiol and estrone are relatively water soluble, a significant fraction can be associated with organic particles or colloids in the treatment systems, potentially influ-

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encing their degradation and ultimate fate (Huang and Sedlak, 2001; Layton et al., 2000). 17h-Estradiol and estrone have been detected in digested sewage sludge at up to 49 and 37 ng/g, respectively (Ternes et al., 2002). Sorption may be an important removal route and potential alternate pathway for exposure to the environment. In this study, there appeared to be a potential association between the degree of nitrification in the treatment system and apparent removal of the estrogens. This may simply be a reflection of improved biological diversity and growth conditions in these systems resulting in increased biological transformations. Many factors influence the growth of nitrifying bacteria including pH, oxygen, and temperature (Metcalf and Eddy, 1991). Various studies have previously demonstrated the ability of ammonium-oxidizing bacteria to co-metabolize low molecular weight organic compounds (Keener and Arp, 1994; Rashe et al., 1990; Rashe et al., 1991). The activity of nitrifying activated sludge results in hydroxylation of organic compounds resulting in more hydrophilic compounds. Vader et al. (2000) examined the degradability of 17a-ethinylestradiol by activated sludge under nitrifying and non-nitrifying conditions. They found that under non-nitrifying conditions, there was no degradation of 17a-ethinylestradiol, while nitrifying sludge oxidized 17a-ethinylestradiol to more hydrophobic compounds. Layton et al. (2000) also found in laboratory experiments that sludges that failed to nitrify also significantly failed to degrade 17a-ethinylestradiol. Vader et al. (2000) suggest that the seasonal and temperature effects on nitrification may therefore result in changes in the ability of treatment systems to remove 17a-ethinylestradiol and related compounds. The removal of estrogens and estrogenicity in municipal treatment plants is very complex and currently not well understood. Municipal effluents may contain a wide variety of additional potentially estrogenic industrial and domestic contaminants such as alkylphenols, bisphenol-A, and pharmaceuticals. How these complex mixtures of substances interact in effluents and impact on the environment remains poorly understood. Additional studies to determine cost-effective alternatives for the removal of these potentially harmful substances from effluents are urgently needed.

Acknowledgements This work was supported by the former Burlington Environmental Technologies Office (BETO) and the National Water Research Institute of Environment Canada. Mr. David Hay, BETO, was an important contributor to initiating this work and his support was greatly appreciated. The work was done in cooperation with the Wastewater Technology Centre (WTC), Environment Canada, who coordinated sampling and analysis of samples. We thank the numerous staff of WTC who assisted in sample collection, preparation, and analysis. Further, we thank the international office of the BMBF within the bilateral Canadian/German cooperation program. We also acknowledge the cooperation of the numerous municipalities involved in the project.

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