A Model To Estimate Influent and Effluent Concentrations of Estradiol

Environ. Sci. Technol. 2004, 38, 3649-3658

A Model To Estimate Influent and Effluent Concentrations of Estradiol, Estrone, and Ethinylestradiol at Sewage Treatment Works ANDREW C. JOHNSON* AND RICHARD J. WILLIAMS Centre for Ecology and Hydrology, Wallingford, Oxon OX10 8BB, United Kingdom

To predict sewage influent and effluent concentrations of the steroid estrogens 17β-estradiol, estrone, and 17Rethinylestradiol, a review of human excretion was carried out. This included conjugation and metabolism of the natural and synthetic steroid estrogens within the body, together with quantities excreted in the urine and feces by different members of the population. This has been combined with fate and behavior information for conjugated and unconjugated estrogens in the sewage treatment system to enable sewage works influent and effluent concentration predictions to be made. The model has proved to be reasonably accurate when tested against recent measurements of these steroid estrogens in the influent and effluent of sewage treatment works. The model may be used with river dilution ratios to predict which sewage treatment works are most likely to cause the greatest endocrine disruption due to steroid estrogens.

Introduction The possible harm caused to individual fish, and possibly fish populations, by endocrine disrupting chemicals in the aquatic environment continues to concern environmental scientists and regulators (1, 2). An estrogenic signal was first detected in sewage effluent by Purdom et al. (3), who found that caged male rainbow trout (Oncorhynchus mykiss) exposed to effluent had very high plasma vitellogenin (female egg yolk) concentrations. Subsequent fish studies reported the presence of intersex roach (Rutilis rutilis), with oocytes in the testes, downstream of many domestic sewage effluents in the U.K. (1). The occurrence of oocytes in the testes can be induced in the laboratory by exposure to estrogenic chemicals, therefore suggesting that wild roach had been exposed to estrogenic chemicals in the effluent. A toxicity identification and evaluation procedure which examined 8 different sewage treatment works (STW) in the U.K. using an in vitro estrogen yeast assay identified the steroid estrogen component of the effluent as contributing the greatest proportion of the overall estrogenic activity (4). Similar studies in Japan and Germany using the yeast estrogen screen and human breast cancer MCF-7 cells respectively, confirmed steroid estrogens as the predominant endocrine disrupter in sewage effluent (5, 6). While in vitro assays may not perfectly reflect the true in vivo response of fish to the same compound, steroid estrogens appear to be the most potent endocrine disrupters found in sewage effluent. The estrogenic activity of the steroid estrogens has been shown in a variety of in vivo studies conducted in fish and their potency demonstrated to be over a thousand times greater 10.1021/es035342u CCC: $27.50 Published on Web 05/19/2004

 2004 American Chemical Society

than any xenobiotic mimic (7-9). Thus, on the available evidence, steroid estrogens are the most probable common cause of endocrine disruption observed in wild fish. This conclusion underlines an urgent requirement to know what steroid estrogen concentrations are discharged from STW and the impact of these chemicals on wild fish in rivers receiving sewage effluent. Unfortunately, chemical analysis of the steroid estrogens, even at the concentrations predicted to cause endocrine disruption, is both difficult and expensive. There are an increasing number of reports (1, 10) which identify this effluent, or that river reach, as being strongly, weakly, or not estrogenic, but leaving the reader unable to ascertain the significance of the result. In light of this problem there is a clear need to develop a model for assessing steroid estrogen loads reaching and leaving typical STWs. Such a model would be useful for predicting river reaches and catchments most at risk from endocrine disruption due to steroid estrogens. The principal steroidal estrogens of environmental concern are estrone (E1), 17β-estradiol (E2), 17R-ethinylestradiol (EE2), and estriol (E3). Although E3 can be found in effluent at high concentrations (11) it is a much less potent endocrine disrupter to fish (12) and will not be examined further in this study. The high estrogenic potency of EE2 (12) is believed to be related to its persistence in plasma, for example an oral ingestion of 4 mg/d of E2 would be required to give the same contraceptive effect of only 30-50 µg/d EE2 (13). Since the natural and synthetic steroid estrogens are mostly excreted as a variety of largely inactive glucuronide, sulfate or sulfoglucuronide conjugates (14, 15), it might not have been expected that the discharge of these compounds would give rise to endocrine disruption concerns (Figure 1). However, deconjugated steroid estrogens are detected both in the influent and effluent of many STW (4, 11, 16). Since the source of the steroid estrogens entering the STW is the human population, it should be possible to calculate the quantity of steroid estrogens that a population would generate on a daily basis. To achieve a good prediction a wide range of factors need to be assessed: (1) the quantity of each steroid estrogen excreted by different sections of the population; (2) the quantities of different steroid conjugates excreted in urine and feces; (3) the extent of deconjugation during transit in the sewers; (4) the extent of transformation of free steroid estrogens during transit in the sewers; and (5) for 17R-ethinylestradiol (EE2), the amount of compound ingested, together with the proportion transformed in the body must also be known. The information gained from this review improves a model proposed by Johnson et al. (16) to estimate steroid estrogen concentrations arriving at a STW by incorporating additional data sets and including more information regarding conjugate fate.

Model Description The Johnson et al. (16) model was an accounting model where the total of a steroid estrogen arriving at a sewage treatment works was assumed to be equal to the sum of the steroid estrogen excreted in urine from different groups of the human population (eq 1) n

UT )

∑ fU i

(1)

i

i)1

where UT (µg/d) is the total amount of steroid estrogen in urine arriving at the sewage treatment works, n is the number VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. The steroid estrogens 17β-estradiol and estrone and their most commonly reported conjugates. of differently excreting groups in the population, fi is the fraction of the population group i, and Ui is the amount excreted by the ith population group (µg/d). In this paper three main extensions to this model are presented: (1) estrogens excreted in feces are included, (2) the fate of the two main conjugates (glucuronide and sulfate) are accounted for separately, (3) a term for loss of estrogens on transit through the sewers has been added, and (4) the values of Ui have been refined and an estimate of uncertainty added. Details of the improvements and refinements of the model are discussed in the following sections. Estrogen Excretion in Feces. The importance of intestinal metabolism of estrogens was initially revealed by experiments in which steroid conjugates were measured in feces before and after the administration of antibiotics to patients. A range of studies (17-20) has reported that the majority of steroid estrogens in feces from healthy adults are unconjugated. Lombardi et al. (21) spiked freshly prepared fecal samples with E2-3-glucuronide and E1-3-sulfate complete deconjugation appeared to occur following a 24 h aerobic incubation. These data indicate that the conjugated estrogens, both sulfate and glucuronide, excreted from the bile are largely deconjugated by the natural intestinal flora prior to excretion from the bowel (Figure 1). Thus, for the purposes of the model it was assumed that all estrogens excreted in the feces will be in the deconjugated form. Steroid Estrogen Conjugates and Their Fate in Sewer Transit. For the U.K., a typical sewer residence time would be 2 h, with temperature at ∼15 °C, and ∼0.2 g/L solids present and some oxygen available (22). While this does not represent the same favorable conditions for biodegradation as the aeration tank of an activated sludge plant, it does suggest some transformation is likely to take place during transit. Many of the intestinal bacteria, which are able to both deconjugate and transform estrogens, will be present. 3650

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Also it is known that sewers contain their own native biofilms which have a wide range of metabolic capacities (23). Thus, some sorption to, and biodegradation within, biofilms associated with the sewer walls is probable. D’Ascenzo et al. (20) indirectly studied the sewer biodegradation of estrogen conjugates by comparing concentrations leaving a condominium and arriving at the local sewage works in Rome. A change in conjugate composition was noted in which sulfate conjugates came to dominate on reaching the sewage works with very little glucuronide conjugates left. Similarly, in a study of the fate of linear alkylbenzene sulfonate (LAS) as much as 60% appeared to be lost through degradation during sewer transit (22). In laboratory experiments using diluted activated sludge, Ternes et al. (24) found that the glucuronide conjugates of E2 were removed in minutes and transformed to E2 and E1 (Figure 1). Panter et al. (25) found that the E2-3-glucuronide conjugate of E2 was very readily converted to the active hormone E2 both in a semicontinuous activated sludge system and in dechlorinated tap water. Thus, in the model all glucuronide conjugates, whether excreted in the urine or the feces, were assumed to be deconjugated prior to arrival at a STW. As discussed above, significant deconjugation of the sulfate forms occurs in the gut, indicating the presence of steroid-desulfating bacteria. Strictly anaerobic desulfating strains have been isolated from human feces (26) which were capable of cleaving E1-3-sulfate and E2-3-sulfate. The release of sulfate was believed to be associated with its use as a terminal electron acceptor by these bacteria (26). In contrast to the glucuronide conjugates, little research seems to have been performed into the fate and behavior of sulfate conjugates within the sewer system. However, the study by D’Ascenzo et al. (20) suggests that, in contrast to the glucuronides, the sulfate conjugates are much more persistent

in the sewer system and can be detected in large quantities at the STW influent. Indeed this study indicated that E1-3sulfate can be found in the effluent, suggesting at least some of the sulfate conjugates can survive sewage treatment. Perhaps this poor desulfating activity is due to the fact that the principal organisms responsible are obligate anaerobes (26), and much of the sewer and treatment works are aerobic environments. In light of the literature data it was assumed for the model that the sulfate conjugates will survive their sewer transit period and will not contribute to the influent or effluent estrogen load. In the subsequent modeling exercise the potential impact of including the sulfate conjugates in the total estrogen load was also examined. Steroid Estrogen Transformation during Sewer Transit. E2 was found to readily oxidize to E1 within 1-3 h in aerobic batch experiments with 10% diluted activated sludge (24). Similarly Lombardi et al. (21) noted that when E2 was spiked into dilute fecal matter (greater than 10% dilution) up to 64% was converted to E1 in 24 h at 37 °C under aerobic conditions. These results, indicating the ready degradability of E2, suggest that some transformation of E2 to E1 in the sewer is likely. A mean sewer transit time of 2 h for the U.K. (22) and 3-5 h has been reported for Rome (20). However, the amount of transformation that would take place is still a matter of speculation. In this modeling exercise we examine the impact of assuming no E2 to E1 transformation in the sewer and 50% transformation on the predictions. Based on the greater persistence of E1 (24) and EE2 (24, 27) observed in activated sludge microcosms, it was assumed that these molecules will not be transformed during sewer transit. Given the low levels of suspended solids (∼0.2 g/L) a small proportion of the steroid estrogens will associate with this solid phase. Although this possible loss is ignored in the influent prediction part of the model, it is an accepted part of the effluent prediction where removal in sewage treatment incorporates all loss processes (with higher suspended solids ∼3.0 g/L more sorption is expected).

Revised Model The model revised from its earlier form (16) now becomes

ST ) (1 - kT)(UT + FT) + Ss

(2)

where ST is the total of a estrogen in all forms arriving at the STW, FT is the total of the estrogen excreted in feces, kT represents the overall fraction of the steroid lost in transit through the sewerage network, and Ss is the internal generation of the estrogen from other estrogens (e.g. E1 formed by transformation of E2) n

UT )

∑ f (U ′ + U i

i

g i

+ Uis)

(3)

i)1

where U′i is the amount of the estrogen in a particular form excreted by the ith fraction (fi) of the population (µg/d). The superscripts ′, g, and s refer to the free, glucuronide, and sulfate forms of the steroid. The contribution of feces to the estrogen load is given by n

FT )

∑ fF

(4)

i i

i)1

where Fi is the amount of the estrogen excreted by the ith fraction of the population (µg/d). Substituting into eq 2 above gives n

ST ) (1 - kT)

∑ f (U ′ + U i

i)1

i

g i

+ Uis + Fi) + Ss

(5)

For the steroids considered in this paper it has been assumed that kT will be zero for E1 and EE2 and 0.5 for E2, assuming 50% of E2 is degraded in the sewerage system. E1 is a product of the degradation of E2 and all the loss of E2 has been assumed to have resulted in the production of E1; therefore, Ss for E1 is equal to the amount of E2 lost on transit. So explicitly for the three estrogens three equations can be written. For EE2 g s + UEE2 + FEE2) SEE2 ) fEE2(U′EE2 + UEE2

(6)

where fEE2 is the fraction of the population taking the contraceptive pill (the only source of excreted EE2). For E2 n

SE2 ) 0.5

∑ f (U′ i

E2,i

g s + UE2,i + UE2,i + FE2,i)

(7)

i)1

For E1 n

SE1 )

∑ f (U′ i

E1,i

s g + UE1,i + UE1,i + FE1,i) + 0.5SE2 (8)

i)1

In these equations 5 sections of the population have been included as excreting E1 and E2 at different rates and identified by the subscripts 1-5. They are 1. pregnant females, 2. menstrual females 3. menopausal females, 4. females taking hormone replacement therapy (HRT), and 5. males. Estimating Parameter Values for the Model. The discussion above indicates the requirement for information on how much of each estrogen in the urine is present as a glucuronide or sulfate conjugate and how much estrogen is excreted in the feces. However, a wide range of excretion values have been reported. Together with other estrogens and their metabolites, E2 and E1 excretion in women varies with each stage of the menstrual cycle (28) and also with the term of pregnancy (29). Subtle differences also exist due to diet, such as between vegetarians and omnivores (30) and between races (31). Thus, for the purposes of this survey the focus will be on Caucasian omnivorous women. Another difficulty associated with the literature is deciding whether the most reliable values come from studies using the most sophisticated analysis or those with the largest sample size. The approach selected was to use a mean generated from all the literature values weighted by the number of reported observations. In some literature, the studies were broken down into different cohorts; in these cases, each cohort has been taken as a separate experiment. The range about this mean value was calculated as twice the weighted (by number of observations) standard error, which for a normally distributed variable should correspond to 95% confidence limits. In a number of cases, such as with fecal excretion and the proportion of glucuronide to sulfate conjugates excreted, insufficient data existed to do any statistical analysis, and these values have been used in the model without qualification. Excretion of 17r-Ethinylestradiol. Statistics provided for the U.S. Centre for Disease Control indicated 17% of the total female population was taking the contraceptive pill in 1995. This is probably a good starting point for an industrialized Western country, although cultural factors will reduce uptake in some of these countries such as Ireland. In the U.K. the current British National formulation handbook (32) recommends products to general practitioners containing EE2 in the range of 20-40 µg/d, with standard strength preparations being between 30 and 35 µg/d EE2. For the purposes of this study a dose of 35 µg/d was chosen. It was assumed that British medical practice would not differ greatly VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Reported EE2 Urine Excretion Values Used To Compile a Mean and 95% Confidence Limits for the Modela reference

sample size

overall urine excretion

n ) 9 first study then n ) 4 in second study n ) 16

first study 40% second study 53-68% 53-68% of dose in urine over 5 d 23-47% dose in urine over 5 d 19-30% dose in feces 26-35% dose in urine over 5 d 22-27% dose in urine over 3 d

mainly 20-30 yr

(34)

between 28 and 52 yr

(35) (36)

>45 yr hysterectomized mixture of young and hysterectomized mean 65 yr

n)9 n ) 27

46 yr plus 22-30 yr

n)5 n)4

(37) (38) (40) mean dose in urine UEE2 dose in urine as g EE2-glucuronide UEE 2 a

subjects

(33)

n)8

16-28% dose in urine over 3 d, 28-42% in bile over 24 h 18% dose in urine over 3 d 30-44% dose in urine over 5 d 27% 17%

Proportion of dose in urine present as EE2-glucuronide is a mean value based on literature review (33, 34, 36, 37, 38, 40).

from other industrialized Western countries. As oral contraceptives are taken 3 weeks out of four, this can be interpreted as an average intake of 26 µg/d. As hormone replacement therapies containing EE2 appear to have been largely replaced by products containing the natural estrogens (32), the only input considered was contraceptive pill use and that 17% of the total female population are ingesting 26 µg/d EE2. There is a certain amount of variability in the EE2 excretion data due to differences in the age and status of the volunteers, natural variations in metabolism, the mode of administration of the compound (oral or intravenous), and the number of days over which the urine was collected. This can influence the results since the relative concentration of the different metabolites changes with time due to enterohepatic circulation (33). In assessing the literature, much of it reliant on ingestion of a radiolabeled EE2 analogue, it is necessary to examine how much of the label was excreted, and second how much of that label was in the form of free EE2 or as its glucuronide conjugate form. Reviewing the data (33-38) where urine was collected over 3-5 days a mean value of 27% of the dose excreted emerges (Table 1). Given the limitations of the day, researchers were less specific on how much of this radioactive dose found in the urine was due to EE2 rather than its different metabolites. The principal metabolic route as described by Guengerich (39) is through 2-hydroxylation. This product is subject to further metabolism in the body, particularly to 2-methoxy-EE2, a proportion of which is completely mineralized (Figure 2). From examining the data on the percentage of EE2glucuronide forms excreted (33, 34, 36-38, 40) a mean value of 63% in this form is arrived at. The data of Maggs et al. (37) and Reed et al. (34) indicated that 30 and 42% of the labeled dose could be recovered from the bile and feces, respectively. Of this dose recovered in the feces 77% was found to be EE2 (34). Overall the data presented in Table 1 suggests that 27.3 ( 4.8% of the dose is excreted in urine (UEE2 ) 7.1 µg/d, range 5.9-8.3 µg/d). Of this amount 63% is present as the glucuronide conjugate, giving an excretion value (U′EE2 + g UEE2 ) of 4.5 µg/d (range 3.6-5.3 µg/d) for EE2 in urine (17%). For feces, 30% of the EE2 dose is excreted of which 77% is in the form of the parent molecule giving 6 µg/d (Figure 2). The total EE2 excreted by those ingesting EE2 is therefore estimated to be 10.5 µg/d (range 9.6-11.3 µg/d). Since 8.5% of the population are ingesting EE2 (17% of women), the average excretion per head of population, SEE2 is taken as 0.89 µg/d (range 0.82-0.96 µg/d) for the model. Estrone Excretion by Pregnant Females. The most recent data for E1 in the urine (20) suggests 47% will be in the 3652

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glucuronide form and 53% in the sulfate form. It is assumed that all the E1 present in the feces is present in the deconjugated form (17, 18). A mean value of 550 µg/d for the overall literature data (17, 18, 20, 41-45) was then calculated by combining the urine excreted E1 glucuronide conjugates and the total E1 fecal excretion (Table 2). On the basis of the 2001 census data for pregnancies/births provided by the U.K. National Office of Statistics the pregnancy/birth rate is taken to be 1 in 57 females. Naturally where accurate local information exists on pregnancy rates then this value can be amended in the model. Estrone Excretion by Menstrual Females. Key et al. (28) recorded that E1 excretion value rose from 4.6 µg/d in the early follicular stage to 7.3-7.8 µg/d in the following late follicular, early luteal, and late luteal stages in their sample group. Thus, excretion values appear to be fairly consistent past the early follicular stage. D’Ascenzo et al. (20) from a study of 50 females found 71% of the excreted E1 was in the E1-glucuronide form. A mean total excretion value of 11.7 µg/d for the overall literature data (20, 31, 45-47) was then calculated by combining the urine excreted E1 glucuronide conjugates and the total E1 fecal excretion (Table 3). Menstruating females were assumed to be between the ages of 15-59, which from the U.K. population census data for 2001 represent 60% of the female population. Estrone Excretion by Menopausal Females. Reviewing the literature for urine excretion reveals values ranging from 1.4 to 8.5 µg/d E1 excretion for this group of females (20, 28, 46). D’Ascenzo et al. (20) found 71% of the excreted E1 was in the E1-glucuronide form from this group of females. Thus a urine excretion rate, UE1,3 of 2.3 µg/d (range 0.0-7.8 µg/d), was selected with 71% in the glucuronide form to give an s effective urine excretion rate, UE1,3 - UE1,3 , for menopausal females of 1.7 µg/d (range 0.0-5.6 µg/d). In a study of 10 postmenopausal females a mean value of 0.07 µg/d E1 was reported as being present in the feces using radioimmunoassay (FE1,3) (48). Thus, combining the total E1 fecal and urine glucuronide values we obtain a mean total value of 1.8 µg/d (range 0.0-5.7 µg/d). The mean age of the menopause is 51 (49), which, on the basis of the U.K. population census data for 2001 represents about 27% of the female population. A range of hormone replacement therapies (HRT), which contains estrogen and progesterone compounds and estrogen replacement therapies (ERT) are used by some postmenopausal women. The mean age of women starting HRT treatments is 54-55 (50). The total female population in this age group (55+) will represent around 25.6% of the total female population (U.K. 2001 National Census). In the United States, in the most recent survey 22% of this group were current HRT users which would be equivalent to 6% of the total female population (51). It has been reported in the U.K.

FIGURE 2. Fate and excretion of 17r-ethinylestradiol in the body.

TABLE 2. Reported E1 Urine and Fecal Excretion Values for Pregnant Women Used To Compile a Mean and 95% Confidence Limits for the Model reference

subjects

sample size

(41) (18) (42) (17) (43) (44) (45) (20) mean availablea UE1,1 - UEs 1,1 total SE1,1

normal in 8th month normal in 8th month normal in 8th month normal in 8th month late pregnancy 6th-9th month middle term 6th month

n ) 13 n ) 2, 24 h collection n ) 6, 24 h collection mean of 5 d n ) 2, 24 h collection mean of 5 d n ) 22 n)4 n)1 n)1

a

overall fecal excretion (FE1,1) (µg/d)

overall urine excretion (UE1,1) (mean µg/d)

NTb 76-116 NT 96 NT NT NT NT 100 100

670 NT 845 NT 1200 674 1480 650 958 (706-1210) 450 (332-568) 550 (432-668)

Refers to the steroid estrogen being considered free (deconjugated) in the feces or as a glucuronide form in the urine. b NT not tested.

that 1.5 million women (5.2% of the total female population) take HRT (52). If we assume these data are representative for an industrialized Western country, this would indicate around 5-6% of all females are receiving some form of HRT treatment. A wide range of different products are available on the market, from tablets, to sprays, patches, and intrauterine devices (32). A study in the United States indicated a preference for tablets, with 74% of women (4% of all females) on HRT using this method (51). Most of the ERT and HRT preparations contain 2 mg E2 to be taken daily (32). A proportion of the market is taken by products containing conjugated estrogens from the urine of pregnant mares, in which the principal ingredient (50%) is a sulfate conjugate of E1 (Wyeth corporation). It is difficult to give a single value for E1 due to the many different types of HRT products that

are commercially available, but we have selected HRT products containing 2 mg E2/d as being the most representative (32). This may result in a slight overestimate for this group. In a study of 18 postmenopausal women taking 2 mg E2 as an oral preparation, the urine content of E1(UE1,4) was found to be 13.5 µg/8 h (SD 11) which we may assume would translate to 40 µg/d in the urine (53). If we assume, as with untreated postmenopausal women, that 71% is in g the glucuronide form (UE1,4 ), then this would generate 28.4 µg/d (range 24-33 µg/d) potentially releasable E1 into the urine. Estrone Excretion by Males. For males excretion values of 2.8-3.9µg/d E1 have been reported (45-47) in the urine. A mean fecal excretion rate of unconjugated E1 of 0.40 ( 0.2 µg/d can be taken from the available literature (19, 54, 55). VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Reported E1 Urine and Fecal Excretion Values for Menstrual Women Used To Compile a Mean and 95% Confidence Limits for the Model reference

subjects

sample size

(31) (46) (45) (20) (47) mean availablea UE1,2 - UEs 1,2 total SE1,2

mean age 32 not defined single healthy female age 18-52 mean age 42

n ) 12 n ) 12 n)1 n ) 50 n ) 150

a



0.3 NT NT NT NT 0.3 0.3

7.8 8 15 14 7 16.1 (+/-5.6) 11.4 (7.5-15.4) 11.7 (7.5-15.4)

Refers to the steroid estrogen being considered free (deconjugated) in the feces or as a glucuronide form in the urine.

TABLE 4. Reported E2 Urine and Fecal Excretion Values for Pregnant Women Used To Compile a Mean and 95% Confidence Limits for the Model reference

subjects

sample size

(41) (18) (42) (17) (43) (44) (45) (20) mean availablea UE2,1 - UEs 2,1 total SE2,1

normal in 8th month normal in 8th month normal in 8th month normal in 8th month late pregnancy 6th-9th month middle term 6th month

n ) 13 n ) 2, 24 h collection n ) 6, 24 h collection mean of 5 d n ) 2, 24 h collection mean of 5 d n ) 22 n)4 n)1 n)1

a



NT 202 NT 203 NT NT NT NT 202 202

170 NT 312 NT 330 192 360 171 269 +/-74 191 (138-243) 393 (340-445)


TABLE 5. Reported E2 Urine and Fecal Excretion Values for Menstrual Women Used To Compile a Mean and 95% Confidence Limits for the Model reference

subjects

(31) (46) (45) (20) (47) (57) mean availablea UE2,2 - UEs 2,2 total SE2,2

mean age 32 not defined single healthy female age 18-52 mean age 42 age 20-40

a

sample size

n ) 12 n ) 12 n)1 n ) 50 n ) 150 n)4 0.2



0.2 NT NT NT NT NT 0.2 3.0 (1.5-4.4)

2.9 3 5.4 7.7 3.6 6.0 4.6 +/-2.3 3.2 (1.7-4.6)


Thus, a urine excretion rate, UE1,5 of 3.2 ( 0.6 µg/d calculated as an unbiased mean is selected with 71% of this assumed g to be in the glucuronide form (UE1,5 ), which with the fecal contribution, gives an effective excretion rate for males, SE1,5 of 2.6 µg/d (range 1.4-2.9 µg/d). Estrogen Excretion by Prepubescent Males and Females. Any calculation for males on E1 and E2 should differentiate between males pre- and postpuberty. Sex steroid production (and urine production) in children of both sexes is low until puberty (56). Thus, the mean values for males will be a slight overestimate, although conversely the female values, since prepubescent girls were not included, will be a slight underestimate. Either way their inclusion would have little effect on the final prediction. Estradiol Excretion by Pregnant Females. Of the excreted E2 from this group, Andreolini et al. (14) gave 79% present as glucuronide conjugates, while the most recent data (20) suggested 71% is present as glucuronide conjugates with 29% in the sulfate form. So the glucuronide content selected was 3654

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the mean of 75%. Combining the total fecal and urine E2glucronide values yields a mean E2 excretion value of 393 µg/d (range 340-445 µg/d) from this group (17, 18, 20, 4145) (Table 4). Estradiol Excretion by Menstrual Females. D’Ascenzo et al. (20) reported that 65% of the urine excreted E2 was in the E2-glucuronide form for these females (Table 6). Adachi et al. (57) from 4 menstrual females found 64% of the urine E2 forms present as glucuronides, so a mean glucuronide value was taken as 65%. Adlercreutz et al. (31) reported a value of 0.2 µg/d in the feces, which, as discussed previously, is assumed to be completely deconjugated (Table 5). Combining the total fecal and urine E2-glucuronide data (20, 31, 45-47, 57) yields a mean E2 excretion from this group of the population as 3.2 µg/d (range 1.7-4.6 µg/d). Estradiol Excretion by Menopausal Females. Urine excretion values, UE2,3 of 1-4.5 µg/d E2, have been reported for this group of women (20, 28, and 46). D’Ascenzo et al. (20) reported that 72% of this E2 excreted was in the

TABLE 6. Summary of Selected Model Values for E1a

males menstrual females menopausal females menopausal on HRT pregnant “average per head” addition of E1 from E2 0.5SE2 transformation revised average per head SE1 a

% of total population (fE1,i)

mean (range) available E1 excretion (µg/d)

mean (range) production by population group (SE1,i) (µg/d)

50 30 13.5 2 0.88

2.6 (1.4-2.9) 11.7 (7.5-15.4) 1.8 (0.0-5.7) 28.4 (24.0-33.0) 550 (432-668)

1.3 (0.7-1.5) 3.5 (2.3-4.6) 0.2 (0.0-0.8) 0.6 (0.5-0.7) 4.8 (3.8-5.9) 10.5 (7.2-13.4) 3.3 (2.6-4.2) 13.8 (9.9-17.6)

Means given with upper and lower values derived from two standard errors to give 95% confidence interval.

TABLE 7. Summary of Selected Model Values for E2 with Upper and Lower Values Derived from Two Standard Errors To Give 95% Confidence Limits

males menstrual females menopausal females menopausal on HRT pregnant “average per head” SE2 corrected following 50% 0.5SE2 loss in sewers

% of total population (fE2,i)

mean (range) available E2 excretion (µg/d)

mean (range) production by population group (SE2,i) (µg/d)

50 30 13.5 2 0.88

1.8 (1.3-2.4) 3.2 (1.7-4.6) 1.0 (0.0-3.5) 56 (51.5-61.5) 393 (340-445)

0.9 (0.7-1.2) 1.0 (0.5-1.4), 0.1 (0.0-0.5) 1.1 (1.0-1.2) 3.5 (3.1-4.1) 6.6 (5.3-8.4) 3.3 (2.6-4.2)

g glucuronide forms (UE2,3 ). The weighted mean of all the values chosen was 1.2 µg/d (range 0.0-3.5 µg/d) with 72% in the glucuronide form to give an effective E2 excretion rate s for menopausal females (UE2,3 - UE2,3 ) of 0.9 µg/d (range 0.0-2.6 µg/d). In a study of 10 postmenopausal females a mean value of 0.09 µg/d E2 was reported as being present in the feces (FE2,3) using radioimmunoassay (48). Thus, combining the E2 fecal and urine E2-glucuronide values we obtain a mean total excretion value of 1.0 µg/d E2 (range 0.0-3.5 µg/d). In a study of postmenopausal women using an HRT treatment, the use of an oral preparation of 2 mg of E2 resulted in a urine content of 26 µg/8 h (SD 16) for E2 which we may assume would translate to 78 µg/d in the urine (UE2,4) (53). If we assume that, as with untreated postmenopausal women, g 72% is in the glucuronide form (UE2,4 ) this would generate 56.1 µg/d (range 51.5-61.5 µg/d) potentially releasable E2 into the urine from women on HRT. Estradiol Excretion by Males. Urine excretion values of 1.5-2.1 µg/d have been reported for males (46, 57, 58). Adachi et al. (57) from 4 males between 20 and 40 years reported that 78% of that excreted is present as glucuronide forms. Thus, a urine excretion rate, UE2,5 ) 1.5 µg/d (range 1.4-1.6 µg/d), is suggested with 78% in the glucuronide form g (UE2,5 ) to give an effective urine excretion rate for males of 1.2 µg/d (range 1.1-1.3). By combining the studies in this area (19, 55, 56) a mean fecal excretion rate of unconjugated E2 (FE2,5) of 0.63 µg/d (range 0.21-1.05 µg/d) can be taken. Thus a combined E2 value for males (SE2,5) would be 1.8 µg/d (range 1.3-2.4 µg/d) excreted. Summary of Natural Steroid Estrogen Excretion. When forming the summary of E1 excretion for a generalized human population we can at this stage choose to include additional E1, formed from the possible transformation of E2 in the sewers, as discussed earlier (Table 6). Thus, of the E1 detected in the influent of a STW the prediction indicates 34% will have come from pregnant women, with 26% from menstrual females, 10% from males, 2% from menopausal females, 4% from females on HRT, and 24% formed by conversion from E2.

To complete the summary of E2 excretion for a generalized human population we can choose to reduce the actual E2 delivered to the STW by assuming 50% transformation to E1 in the sewers, as discussed earlier (Table 7). The prediction indicates that pregnant women will account for 52% of E2 arriving in the sewers, menstrual females 14%, males 15%, menopausal females 2%, and females taking HRT 17%, excreted prior to loss by conversion to E1 in the sewers (Table 7). This suggests that pregnant women are the most important source for E2 in the raw sewage, and the E2 calculation will be sensitive to errors in predicting the number of pregnant females. Estimating Steroid Estrogen Losses from Sewage Treatment. Although studies that assess both influent and effluent steroid estrogen values are rare, there are some reports where an attempt to quantify average removal performance was made for activated sludge treatment plants (Table 8). There are a number of factors which could influence the biological performance of activated sludge sewage treatment such as hydraulic residence time, sludge residence time, sludge particle content, and temperature (16, 62); however, frequently these details are missing, and it is not possible to relate removal performance with the management of the sewage treatment. Removal from the water stream will include both biodegradation in the water and solid phases and loss through sorption to the solid phase (59, 62, 63). Generally a pattern of greater E2 than E1 removal can be seen (Table 8). So far there are few data on EE2 removal because of the great difficulty in measuring the low concentrations present. Thus, if we were to use these removal rates we could predict (albeit with an additional level of error) the effluent concentrations from head of population and flow data for a STW (Table 9). In the case of EE2 effluent concentration, this can be calculated by starting with the influent prediction of 0.89 µg/head/d and use the predicted 85.2% removal rate to give an effluent prediction number of 0.13 µg/head/d for EE2. To get the 95% confidence limit we can take the upper standard error value for EE2 influent concentration per head, 0.96 µg/head/d, and take the lowest expected removal VOL. 38, NO. 13, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 8. Compilation of Steroid Estrogen Removal Values for Activated Sludge Plantsa

a

country

reference

European av (n ) 8) Italy (n ) 30) Canada (n ) 6) Japan (n ) 27) Italy (n ) 18) Germany (n ) 1) worldwide mean removal

(16) (11) (60)b (61)b (21)b (59)

Mean removal given plus 95% confidence limits.

b

E2% removal

E1% removal

EE2% removal

88 (SD 13) 87 (SD 6) 99c 67 85 98 81.7 (( 10.6)

74 (SD 27) 62 (SD 33) 71 (SD 19) NAd 61 98 64.7 (( 5.8)

NAd 85 (SD 14) NAd NAd NAd 90 85.2 (( 5.1)

Flow values not given. c Removed to below detection level.

TABLE 9. Compilation of Values Used Per Head in the Model To Estimate Influent and Effluent Loads (µg/person/d)a

steroid

mean (range) of multiplication no. for influent (ST) (µg/person/d)

mean (range) multiplication no. for effluent (µg/person/d)

EE2 E2 E1

0.89 (0.82-0.96) 3.3 (2.6-4.2) 13.8 (9.9-17.6)

0.13 (0.08-0.19) 0.6 (0.2-1.2) 4.9 (2.9-7.2)

a

Concentration values are obtained by dividing by the flow (m3/d) to give ng/L.

performance, 80.1% to give an upper limit effluent prediction number of 0.19 µg/head/d. To give the lowest predicted effluent EE2 concentration we take the lower standard error value for EE2 influent concentration per head, 0.82 µg/head/ d, and take the highest expected removal performance, 90.3% to give a lower limit effluent prediction number of 0.08 µg/ head/d. As described previously, these numbers are multiplied by the head of population and then divided by the flow (m3/d) to give ng/L leaving the STW (24 h composite). The same procedure is followed for the other steroid estrogens (Table 9).

Model Corroboration Prediction of Influent Steroid Estrogen Concentrations. One of the most useful data sets available for testing the model is provided by Baronti et al. (11), which reports both influent and effluent concentrations of steroid estrogens at Roman STW (Table 10). The difficulty with making predictions with these data is that while sampling was undertaken in February, March, October, November, and December, only

d

NA not analyzed.

a single flow value was given. It is probable, however, that the flow value on each sampling occasion would have been different. It is noteworthy that for all the determinands and at almost all the STW, the values for February and November are from half to an order of magnitude below numbers collected in the other months. This suggests higher flows were occurring on these dates. The recent paper by Andersen et al. (59) also has influent data and sufficient STW information to permit a prediction comparison (Table 10). When the mean predicted value is divided by observed value for E1 this gave a mean ratio of 0.78 (range 0.56-1.0), 0.85 (range 0.67-1.1) for E2, and 0.80 (range 0.74-0.87) for EE2 (Table 10). These model predicted mean values are all underestimates, but only in the case of EE2 does the range of estimated values not straddle the observed mean value. A regression of the predicted mean values against the observed means gave R2 values of 0.70 (p < 0.001) for E1, 0.66 (p < 0.001) for E2, and 0.53 (p ) 0.06) for EE2. For E1 and E2 there was no significant intercept, but for the EE2 regression an intercept was necessary to produce a reasonable regression, although even in that case the R2 value was not significant at the 95% level. Overall the model provided good predictions of the sewage treatment works influent values. This would not have been the case if significant sorption of the steroid estrogens occurred during sewer transit. By way of comparison, the model was also run assuming that all sulfate conjugates were deconjugated in the sewer and that no E2 was transformed in the sewer. In this case the mean ratio of the estimated mean concentrations to the observed mean concentrations was 1.0 (range 0.71-1.3) for E1, 2.2 (range 1.7-2.7) for E2, and 1.0 (range 0.91-1.1) for EE2. Thus, this approach gave slightly improved estimates for E1 and EE2 but a considerably worse estimate for E2. The E2 prediction was over double the observed values. This

TABLE 10. Mean of Influent Concentrations (ng/L) for Six Roman STW (11) and One German STW (59) Compared to Predicted Values E1 influent

E2 influent

EE2 influent

STW with population and flow

observed mean and range

model mean and range





Cobis (40 000 pop and 10 000 m3/d) Fregene (120 000 pop and 42 000 m3/d) Ostia (350 000 pop and 112 000 m3/d) Roma Sud (1 200 000 pop and 734 000 m3/d) Roma Est (800 000 pop and 265 000 m3/d) Roma Nord (800 000 pop and 354 000 m3/d) Wiesbaden (300 000 pop and 66 000 m3/d)

71 (42-132) 67 (41-87) 51 (44-67) 35 (28-48) 50 (34-68) 37 (30-49) 66 (55-77)

55 (40-70) 40 (25-50) 43 (31-55) 23 (16-29) 42 (30-53) 31 (22-40) 63 (45-80)

16 (8-25) 9 (4-16) 14 (6-22) 9 (5-10) 9 (6-11) 11 (6-14) 16 (12-19)

13 (10-17) 9 (7-12) 10 (8-13) 5 (4-7) 10 (8-13) 8 (6-10) 15 (12-19)

4 (0.5-13) 3.4 (0.5-7) 2.5 (0.5-5) 2.9 (0.5-6) 2.3 (0.5-3) 2.9 (0.5-7) 8 (6-10)

3.6 (3.3-3.8) 2.5 (2.3-2.7) 2.8 (2.6-3.0) 1.5 (1.3-1.6) 2.7 (2.5-2.9) 2.0 (1.9-2.2) 4.0 (3.7-4.4)

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TABLE 11. Mean of Effluent Concentrations (ng/L) for Activated Sludge Plants for Six Roman STW (11) and One British STW (64) Compared to Predicted E1 effluent

E2 effluent

EE2 effluent

STW with population and flow


modeled mean and range





Cobis (40 000 pop and 10 000 m3/d) Fregene (120 000 pop and 42 000 m3/d) Ostia (350 000 pop and 112 000 m3/d) Roma Sud (1 200 000 pop and 734 000 m3/d) Roma Est (800 000 pop and 265 000 m3/d) Roma Nord (800 000 pop and 354 000 m3/d) Gt Billing (215 780 pop and 60 000 m3/d

9.7 (5-17) 4.1 (2-6) 44.6 (13-82) 30.3 (8-51) 7.7 (4-10) 13.8 (6-40) 4.6 (0.8-11.2)

20 (12-29) 14 (8.0-21) 15 (9.0-23) 8 (4.7-12) 15 (9.0-22) 11 (6.6-16) 18 (10-26)

1.5 (0.5-2.9) 0.9 (0.3-2.1) 2.4 (0.7-3.5) 1.9 (0.5-3.1) 0.7 (0.6-0.8) 1.0 (0.4-1.9) 0.9 (

A Model To Estimate Influent and Effluent Concentrations of Estradiol

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