Assessment of the Aquatic Release and ... - ACS Publications

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Chapter 22

Assessment of the Aquatic Release and Relevance of Selected Endogenous Chemicals: Androgens, Thyroids and Their in Vivo Metabolites Usman Khan and Jim Nicell* Department of Civil Engineering and Applied Mechanics, McGill University, 817 Sherbrooke Street West, Montreal, Quebec H3A 2K6 *E-mail: [email protected].

Endogenous chemicals released through anthropogenic excretions enter the environment on a continuous basis. Therefore, it is important to establish their rate of introduction into the environment. An approach was developed to establish such environmental loads for any given endogenous chemical that is likely to be released in anthropogenic excretions. The approach developed was used to quantify influent, effluent and surface water loads of 25 endogenous chemicals including androgens, thyroids and their in vivo metabolites, using the United States as an illustrative case. The predicted surface water concentrations matched up well with the limited monitoring data presently available. The environmental relevance of the surface water presence could only be assessed for 5 of the 25 endogenous chemicals due to limited availability of eco-toxicological data. Data available thus far suggest that testosterone, dihydrotestosterone, thyroxine, triiodothyronine are unlikely to pose a risk to the receiving aquatic ecosystem when considered individually. However, androstenedione is predicted to pose a potential risk to the receiving environment in situations where the surface water dilution of the effluents is expected to be low.

© 2010 American Chemical Society Halden; Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Introduction The detectable presence of pharmaceutically active compounds (PhACs) in the aquatic environment has garnered increasing attention from the scientific community as is evident from the relevant literature compilation maintained by the United States Environmental Protection Agency (1), which on its last update cited 8,882 publications related to the subject of pharmaceuticals and personal care products. (PhACs is defined here to include pharmaceuticals, endogenous hormones and their metabolites generated in vivo). Such scientific interest and activity is also gradually leading to the establishment of regulatory guidelines for a number of such contaminants in the form of environmental quality standards (2, 3), water quality standards (4), and acceptable daily intakes for human exposure (5). Among PhACs that are likely to be present in the environment, special attention is paid to those that are likely to interact with the endocrine system. This largely stems from the eco-toxicological evidence available to date which suggests that endocrine-modulating or disrupting PhACs are likely to manifest significant effects at environmentally relevant concentrations (6). Such contaminants are typically released in human urinary and fecal excretions (7). In the present day wastewater treatment context, anthropogenic excretions are diluted by large amounts of grey water; i.e., water that is partially contaminated. This relatively dilute and complex mixture is then transferred to sewage treatment plants where such contaminants, after undergoing varying levels of treatment, are ultimately discharged into the receiving environment. Endocrine-disrupting PhACs that are eventually released into the environment can be broadly categorized into two classes: (1) Endogenously-produced hormones that are naturally produced by and are present in living organisms and therefore logically have high potential for interactions with the endocrine system; and (2) Exogenously-consumed hormones/pharmaceuticals, that are typically not naturally produced and therefore are not naturally present but can nevertheless interact with the endocrine system in either expected or unexpected manners. Perhaps the greatest distinction between the two categories from an environmental standpoint would be that if an exogenous compound is deemed to be detrimental to the receiving ecosystems, its release could be mitigated by switching to a “greener” alternative or an alternative may be developed if none is available. However, no such alternatives are available with respect to the release of endogenous chemicals, which are a fixed characteristic of our human biology. Given this, the presence of endogenous chemicals in anthropogenic excretions, and therefore wastewater treatment effluents and hence aquatic ecosystems, can be considered to be omnipresent. Therefore, it is imperative that one makes an attempt to quantify the environmental release and the environmental relevance of such contaminants. 438 Halden; Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Attempts have been made by others to precisely address such concerns by developing models for the environmental release of endogenous estrogens (8–10). Considerably less attention has been paid to the release and eventual fate of endogenous androgens, thyroid hormones and their in vivo metabolites. In vertebrates, androgens modulate sexual characteristics by interacting with the androgen receptor (11, 12). Similarly, thyroid hormones regulate a wide range of physiological endpoints including, reproductive ones, by interacting with the thyroid receptor (13–16). The presence of such receptors with a high degree of homology to the respective human receptors has been confirmed in eco-toxicologically relevant aquatic taxons such as fish (12, 17, 18). Therefore, from an environmental perspective, the question is not whether excreted endogenous androgens and thyroid hormones making their way into the environment interact with associated receptors in relevant aquatic taxa but, rather, whether the interactions are expected to be significant from an eco-toxicological standpoint. Furthermore, anthropogenic excretions are not only likely to contain active hormones but also their in vivo metabolites. As these in vivo metabolites will also make their way into the receiving environment their release loads and environmental relevance is also worth investigating. In order to address such concerns the first logical step is to develop a model for the environmental release of endogenous chemicals (androgens and thyroids and their in vivo metabolites) originating from anthropogenic excretions. Therefore, the overall objectives of the research presented herein were to: (1) Develop a model for estimating the quantities (i.e., load and concentrations) of the endogenous chemicals of interest here in the influents and effluents of wastewater treatment plants; (2) Where possible to assess the environmental relevance of such contaminants making their way to the environment; and (3) Highlight knowledge gaps that need to be addressed before the next logical iterative refinement of the developed model is undertaken. The application of the proposed methodology will be demonstrated using data corresponding to the Unites States, as an illustrative case.

Model Development The mass balance model suggested by Johnson and Williams (8) for quantifying the load of a particular contaminant expected to reach the influent of a treatment plant can be logically extended to establish such loads for any given endogenous chemical that is likely to be present in anthropogenic excretions. Therefore, the general form of their model is adapted here and modified, where necessary. Appropriate simplifications and enumerations of the various parameters of the general form that is relevant to the chemicals being considered here are included in the model development.

439 Halden; Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Estimating Influent Loads of Endogenous Chemicals


Following modification of the general form of the model of Johnson and William (8), the total amount of an endogenous chemical expected to arrive in the influent of a wastewater treatment plant (HT-inf, in kg/d) can be expressed as:

where kd is the fraction of the endogenous chemical that is degraded during sewer transit; P is the total population of the community; C is the fraction of the total population that is connected to the wastewater treatment and collection train; Ut is the amount of the endogenous hormone that is excreted in the urine of population P on a per capita basis (µg/cap·d); Ft is the amount of the endogenous chemical that is excreted in the fecal matter of population P on a per capita basis (µg/d); G is the potential rate of generation of the endogenous chemical arising from the degradation of another contaminant (µg/d); and X is the contribution to the influent load due to exogenous consumption of the endogenous chemical itself or the consumption of an another chemical/pharmaceutical preparation that may be metabolized by humans to the endogenous chemical being considered (µg/d). Furthermore, Ut can be represented as a summation of the endogenous chemical excreted by relevant human population cohorts:

where: I is the number of population cohorts excreting different amounts of the compound of interest; fi is the fraction of population P that belongs to cohort i; and Ui is the amount of the endogenous chemical that is excreted in urine by cohort i on a per capita basis (µg/cap·d). The amount of a given endogenous chemical that is excreted by a given cohort i in urine on a per capita basis is likely to be composed of three distinct components (11, 19–23): namely, a fraction that is excreted as the free endogenous chemical itself (Fu), a further fraction that is excreted conjugated to glucuronic acid (Gu), and an additional fraction that is excreted as sulphate conjugates (Su). Considering this, equation (2) can be expanded as follows:

where, by definition, the sum of the fractions, Fu + Gu + Su, must be equal to one. Similar development can lead to the expansion of the Ft term in equation (1):



where: Fi is the amount of the endogenous chemical that is excreted in fecal matter by cohort i on a per capita basis (µg/cap·d); Ff is the fraction of Fi that is excreted as the free endogenous chemical itself; Gf is a further fraction of Fi that is excreted conjugated to glucuronic acid; and Sf is an additional fraction of Fi that is excreted as sulfate conjugates. However, data available to-date regarding fecal excretions of androgens (24) and thyroids (25, 26) suggest that such endogenous chemicals are primarily excreted in the form of free chemical in fecal matter. This is largely a consequence of the intestinal flora acting on conjugated fractions to liberate free chemicals (24, 25). Therefore, equation (4) can be simplified to the following:

Further, recognizing that Ff ≈ 1, equation (5) can be simplified to:

The load, X, added due to exogenous use of the chemical itself or another chemical/pharmaceutical-preparation that may be metabolized to the endogenous chemical being considered can be calculated as follows:

where: N is the total per capita daily consumption of the pharmaceutical preparation (µg/cap·d); Xu is the fraction of the administrated daily dose of the preparation that is excreted in urine as the endogenous chemical of interest; Xf is the fraction of the administrated daily dose that is excreted in fecal matter as the endogenous chemical of interest; Mweh is the molecular weight of the endogenous chemical being considered; and Mwp is the molecular weight of the pharmaceutical preparation that is metabolized to the endogenous chemical of interest. Note that the pharmaceutical preparation could be the exogenous form of the endogenous chemical of interest and, in such cases; the molecular weight adjustment (Mweh/Mwp) would be equal to 1. Substitution of equations (7), (6) and (3) into equation (1) leads to the following equation that can be used to estimate the amount of a given endogenous androgen or a thyroid chemical that is expected to reach treatment plants in their influents:


Quantifying Parameters To Estimate Influent Loads of Endogenous Chemicals


Influent Loads In order to quantify influent loads of a given endogenous chemical (HT-inf), the various daily excretions parameters (i.e., values of Ui and Fi) of equation (8) were quantified. This was accomplished by performing a comprehensive literature search for all studies reporting urinary and fecal excretion profiles of endogenous chemicals of interest here (androgens, thyroids and their in vivo metabolites) by mining the following literature databases: MEDLINE, EMBASE, SCOPUS and Scifinder Scholar. No language restrictions were applied in the retrieval of relevant literature and search keywords where kept very generic so as to capture as diverse a body of relevant literature as possible. In the initial phase, all studies that demonstrated one or more of the following criteria were excluded: (1) excreta to be analyzed had been collected for durations of less than 24 hours; and (2) results reported were normalized to creatinine content of the sample without stating the adjustment creatinine level that was used. The former data was excluded because collection durations of less than 24 hours or analysis of spot urine are unlikely to yield accurate estimates of the daily excretion of the endogenous chemical being considered. Additionally, the use of creatinine-adjusted results, where such adjustment levels were not stated in the original reference, would entail that one multiply reported results (i.e., µg/mg of creatinine) by the typical daily creatinine excretion (i.e., mg/24 hr) thereby, adding an additional degree of variability to the daily excretion values of the endogenous chemical being considered. The validity of applying such a correction to data can further be questioned considering the wide range that constitutes “typical” daily excretion of creatinine (27). After initial screening of the studies, the estimated per capita daily excretions of the various chemicals (µg/cap·d) from the remaining studies were compiled. The compiled dataset was used to identify the number of population cohorts excreting different amounts of the compound of interest, I, for each endogenous chemical. Identification of the cohorts allowed one to further refine the collection of reported data. At this level of screening, studies were excluded that reported data encompassing more than one relevant cohort. The screening at this stage is best illustrated with the aid of an example: that is, it is known that testosterone levels in women decreases with age (28), therefore a study like Doberne and New (29) which presented mean testosterone excretion for 14 women whose ages were between 22 and 67 is too broad as it combines many different excreting cohorts into one. From all remaining studies, means and standard deviations (σ’s) were extracted from the reported data. In cases where data was only presented in graphical form, the images were digitized and the dimensions of the extracted images was subsequently used using DigitizeIt™ (30) to estimate means and standard deviations of the reported data. Often, studies reported either the standard error or the range of data rather than the desired σ. In cases where standard error was reported, it was converted to standard deviation by multiplying 442 Halden; Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

it with the square root of the number of observations. For those cases where the range was reported, σ was approximated by dividing the range by four as described by Massart et al. (31). Means from different studies within a relevant population cohort, i, were combined by weighting each mean by the number of subjects monitored in the study. Standard deviations were also combined by the number of subjects, specifically according to the pooled standard deviation method suggested by Massart et al. (31).


Population Parameters Since the case study reported here had the objective of estimate the release of such contaminants to United States waterways, the national population of the United States was used for P. The base simulation year used here is 2005, because it is the latest year for which certain population cohorts of interest have has been enumerated (i.e., number of pregnancies in a given year). The fraction of the national population that is connected to the wastewater treatment and collection trains, C, was reported to be 98.1% (32). Once the appropriate cohorts were identified for the various endogenous chemicals of interest, the fraction of population corresponding to each cohort, fi, were derived from United States Census Bureau (33) data for the simulation base year of 2005.

Degradation and Sewer Transit Parameters Typical sewer transit times reported in literature are on the order of a few hours (34, 35). Furthermore, considering that anaerobic conditions set in fairly early on during the transit process (36), it is reasonable to assume that the contaminants considered here won’t be significantly transformed en-route to the treatment plant. Hence, it is conservatively assumed here that the total daily loads of endogenous androgens, thyroids and their in vivo metabolites released in human urinary and fecal excretions are transmitted to the influents of wastewater treatment plants unchanged (i.e., kd and G ≈ 0). However, the conjugated fraction of the total loads is expected to change during the sewer transit process. Research primarily with conjugated fractions of endogenous estrogens suggests that the glucuronide fraction will be essentially deconjugated to the free form (i.e., Gu → Fu), while the sulphate fraction will survive the transmission (35, 37). These assertions are assumed to hold true here for the excreted conjugated fractions of the endogenous chemicals being considered here.

Exogenous Chemical Parameters Besides being an endogenous hormone that is present in anthropogenic excretions, testosterone is also used exogenously by the human population, primarily by those undergoing treatment for hypogonadism (38). Current 443 Halden; Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


consumption rates of exogenous testosterone were not available for the United States. However, consumption data from Canada (39) and Sweden (40) suggests that the population-normalized consumption of exogenous testosterone is between 73 and 120 µg/cap·d. The mean of this range (≈ 96 µg/cap·d) is used here as N. Canadian data further suggests that (39) on a mass basis testosterone is predominately applied topically or transdermally, with intravenous administration amounting to a minor fraction of the total administered mass. Further, considering that only 10% of topical administered testosterone is absorbed (41) and that up to 98% of a transdermal dose may remain as residue in a used transdermal patch (42), it is conservatively assumed here that the exogenous mass of testosterone utilized enters the sewer unmetabolized in the free form (i.e., Xu+Xf = 1). Levothyroxine, the synthetic form of the endogenous hormone thyroxine, is among the top 10 prescribed drugs in the United States (43) and is primarily used for the treatment of hypothyroidism (38). Again, actual consumption data with respect to the use of Levothyroxine was not available for the United States. However, population-normalized Canadian (39) and French data (44) suggests that the consumption of levothyroxine in those countries ranged between 2.6 and 4.6, µg/cap·d. The mean of this range (3.6 µg/cap·d) is assumed here for N. Reports on disposition of levothyroxine suggest that on average 30% of the administered dose is excreted in fecal matter as thyroxine (Xf = 0.3 for thyroxine) while on average 9% of the administered dose is excreted in urine as thyronine (Xu = 0 for thyroxine and Xu = 0.09 for thyronine) (45, 46). Effluent Loads of Endogenous Chemicals The effluent load of a particular endogenous chemical that is likely to be released to the receiving environment, HT-eff (kg/d), can be calculated from HT-inf by multiplying it by one minus the removal efficiency expected during the treatment process (Eff):

Based on considerations discussed in the preceding section, the influent load (HT-inf) arriving at the influent of the wastewater treatment facility will be composed of endogenous chemicals in free form (which includes the excreted glucuronide fraction that has been assumed to be fully cleaved to the free form) and the sulphate conjugates of the endogenous chemical. Evidence reported to date in literature regarding sulphate conjugates of endogenous estrogens (8, 37) suggests that this fraction is likely to survive the treatment process. Hence, it is assumed here that this will be the case with the sulphate conjugates of endogenous chemicals being considered here. Incorporation of such an assumption leads to the following modification of equation (9):

where HT-infs is the total load of the endogenous chemical that is conjugated to sulphate ions and can be stated as: 444 Halden; Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Since measurements of removal efficiencies (Eff) from full-scale treatment plants entails that specific monitoring studies measuring both influent and effluent concentrations of the contaminant of interest, such data is rare and rarer still for the compounds being considered here, as will become evident below. Therefore, in cases where such data is not available or where the data available is limited to a small number of samples, Eff was estimated using the STPWIN™ software of the USEPA (47). Predicting Surface Water Load and Concentrations of Endogenous Chemicals The expected load of a given endogenous chemical in surface water immediately downstream of wastewater treatment plants, HT-sw, can be estimated by dividing HT-eff by the dilution expected of the effluent in the receiving surface water:

where: DF is the dilution factor and is indicative of the number of times a typical wastewater effluent is diluted in the receiving surface water. Based on the work of Rieses et al. (48), Lyndall et al (32) suggested DF values of between 4 and 130 for environmental exposure assessment purposes for United States waterways. A DF of 4 was suggested to represent a “high-end” exposure scenario while the value of 130 was considered to represent a “typical” exposure scenario. These suggested values of DF were used here. The concentration of a particular endogenous chemical in surface waters immediately downstream of wastewater treatment plants, CT-sw (ng/L), can then be derived from HT-sw using the following relationship:

where W is the average daily water use per capita in the United States, which was estimated to be 677 L/cap·d based on the work by Hutson et al. (49). Environmental Relevance The environmental relevance of any contaminant making its way into the aquatic environment can be assessed by deriving the contaminant’s Predicted No-Effect Concentration (PNEC). A contaminant-specific PNEC is typically derived from the lowest available eco-toxicological endpoint by dividing it by an assessment factor (AF). The magnitude of AF is dependent on the nature of 445 Halden; Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


the lowest available eco-toxicological endpoint (i.e. whether it was obtained after an acute or chronic exposure) and the overall diversity of the eco-toxicological data when chronic eco-toxicological data is available. When the lowest available eco-toxicological endpoint was derived after an acute exposure, a value of 1000 is suggested for AF (50, 51). However, in cases where the lowest eco-toxicological endpoint was established after chronic exposure, the magnitude of AF depends on the number of chronic endpoints available for different taxa. The typical value used for AF when chronic endpoints are available from only one taxon is a 100, and in cases where chronic endpoints are available from two or three taxa the suggested values are 50 and 10, respectively (50, 51). The derived PNEC of a given contaminant could subsequently be compared to its predicted surface water concentration (CT-sw) to derive an environmental risk quotient (RQ):

A risk quotient greater than or equal to one would indicate unacceptable risk of the contaminant being considered, while a value of less than one is indicative of no risk in light of the eco-toxicological data used to derive respective the contaminant’s PNEC.

Results and Discussion Influent Loads Per Capita Release of Androgens and Their in Vivo Metabolites Through the data mining strategy employed here, 19 endogenous androgens and their in vivo metabolites were identified for which varying degrees of information were available with respect to their anthropogenic excretions. The methodology used to quantify influent loads for these is clarified below by illustrating the methodology as applied to three of these compounds. These three represent typical cases of the diversity that were encountered in terms of availability of relevant excretion data. The process for the remaining compound was similar to the three cases illustrated here and has been summarized in tables presented in Appendix A (located in the back of this book).

Case 1 – Example for Which Extensive Data Was Available for All Relevant Cohorts - Testosterone The body of literature reporting the urinary excretion of testosterone indicated that the urinary excretion of testosterone is dependent on the age the subject (52); gender of the subject (53) and the trimester of pregnancy for pregnant women (54), while the variation due to the day of the menstrual cycle and the onset of menopausal state is likely to be insignificant when compared to observed 446 Halden; Contaminants of Emerging Concern in the Environment: Ecological and Human Health Considerations ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


inter-individual variation (56, 57). The urinary excretion of testosterone for both sexes was reported to rise during puberty, peaking in the late-teens to mid-twenties and then decline steadily thereafter (52, 53). It was shown that females excrete considerably lesser amounts throughout their life cycle than males (53). The effect of pregnancy was suggested to be significant only during the third trimester, with significantly higher amounts being excreted during the third trimester while excretions profile during the first two trimesters was suggested to be similar to those of normal (i.e. non-pregnant) women (54). Considering these excretion trends and the resolution of data available, the relevant cohort’s (i’s) for the excretion of testosterone were defined to be: gender, age cohorts within each of the gender cohorts, and pregnant women in the third trimester cohort within the cohort of women that are likely to be pregnant (i.e., assumed to be 15-40 year old females (58)). Urinary excretion (Ui’s) data for each of these cohorts is summarized in Table I. Ut for the whole population was obtained by multiplying Ui values with their respective fi’s (from (33)) and subsequently summing these for all cohorts (i’s), as indicated in equation (2). The analysis (Table I) suggests that, on a demographic normalized basis, the daily urinary excretion of testosterone on a per capita basis is expected to be 28 µg/cap·d (Ut). Of this total per capita load, 20 µg/cap·d is expected to be conjugated to the glucuronide moiety while the majority of the remaining is expected to be conjugated to sulphate ions (i.e., 7.9 µg/cap·d). In contrast to the vast body of literature reporting urinary excretions of testosterone, the fecal excretions of endogenous androgens is rarely studied. To date, only one study has analyzed human fecal excretions for their androgen content (24). In that study, upon analysis of 18 fecal samples of young males, aged 21-36, it was suggested that the average testosterone content of the analyzed human fecal samples was 10 µg/cap·d (24). The average urinary excretion of testosterone for such individuals based on our analysis is expected to be between 66 and 85 µg/cap·d (Table I). If one were to use the mean of this range ( ≈ 75 µg/d) as the likely urinary excretion for such individuals, their reported fecal excretion of 10 µg/cap·d amounts to 13% of the urinary excretions. Since no other data is available regarding fecal excretions, it was assumed here that the fecal excretions for all cohorts of interest amounted to 13% of the respective urinary excretions. Such an approximation allows one to estimate Ft as 3.6 µg/cap·d; this fecal load is expected to be in free form in light of previously mentioned considerations. Therefore, the total per capita daily load of testosterone released by humans in fecal and urinary excretions (i.e., Ut + Ft) amounts to 31.6 µg/cap·d. Recalling that the fraction conjugated to the glucuronide moiety is expected to revert to the free form during sewer transit, 23.8 µg/cap·d of this load is expected to the arrive in the influents of wastewater treatment plants in the free form and therefore would be available for treatment. The remaining 7.9 µg/cap·d, would arrive at the treatment plant “as-is” in the form of sulphate conjugates and, as assumed earlier, would not be available for treatment.



Table I. Total per capita daily urinary excretion of testosterone (Ut) and its conjugated and unconjugated fractions (Fu·Ut, Gu·Ut, Su·Ut) (see Appendix B for larger version of table)

Case 2 – Example for Which a Reasonable Data Set Was Available, but with Information Lacking on Certain Cohorts - Androstenedione As with the excretion of testosterone, the literature suggested that daily urinary excretion of androstenedione was dependent on the age of the subject (69); gender of the subject (69, 76) and the trimester of pregnancy for pregnant women (54). Therefore the relevant cohorts (i’s) for androstenedione were defined to be: gender, age cohorts within each of the gender cohorts, and women in the third trimester within the cohort of women likely to be pregnant. Sufficient data was available to quantify each of the age cohorts for the males; however, this was not possible for all the female cohorts (Table II). For females, limited data was available; the three identified studies reported daily urinary excretion of androstenedione by adult women (20-40 age cohort) (75, 76) and pregnant women in their third trimester (54). The approach taken here to estimate excretion by women of other age cohorts than the 20 to

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