The Handbook of Environmental Chemistry Vol. 5, Part O (2005): 1– 24 DOI 10.1007/b98605 © Springer-Verlag Berlin Heidelberg 2005
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil Marina Kuster · Maria J. López de Alda (✉) · Damià Barceló Department of Environmental Chemistry, IIQAB-CSIC, Jordi Girona 18–26, 08034 Barcelona, Spain
[email protected] Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
2 Usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Human Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Animal Farming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 4 4
3 Sources and Fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Environmental Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 7
4
Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
5
Toxicity Identification Evaluation (TIE) Approaches . . . . . . . . . . . . . . .
14
6 6.1 6.2 6.3
Analytical Methods . . . . Sampling . . . . . . . . . . Sample Pretreatment . . . Analyses . . . . . . . . . .
. . . .
16 19 19 20
7
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
1
References
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
Abstract Estrogens and progestogens are two classes of female steroidal hormones whose presence in the environment has been associated with the appearance of certain alarming reproductive and development effects, such as feminization, decreased fertility, and hermaphroditism, in living organisms exposed to these compounds. Synthetic chemicals resembling these natural hormones are now well established in human medicine (mainly as contraceptives and for treatment of hormonal disorders) and in animal farming practices (usually as growth promoters). They are therefore produced on a large scale every year. Mainly due to unsuccessful removal in wastewater treatment plants, they are continuously released into the aquatic environment.Adverse effects on aquatic wildlife at concentrations as low as ~1 ng L–1 have been reported. Studies have also shown that estrogens and progestogens are easily distributed in the environment and may accumulate in river sediments. However, little is known about their long-term environmental impact. In this chapter, the main sources of estrogens and progestogens, their principal pathways into the aquatic environment, and the primary routes of exposure to these compounds are discussed. This chapter also reviews the methods described so far for the analysis of estrogens and progestogens in wastewater, sludge, sediments, and soils as well as the environmental levels found in these compartments. Keywords Estrogens · Progestogens · Environmental analysis · Occurrence · Fate
2
M. Kuster et al.
Abbreviations APCI Atmospheric-pressure chemical ionization EC European Community EDC Endocrine disrupting compound EEF Molar-based 17b-estradiol equivalency factor ELISA Enzyme-linked immunosorbent assay ER-CALUX Estrogen receptor-mediated chemically activated luciferase gene expression assay ESI Electrospray ionization EXAMS Exposure assessment modeling system FDA United States Food and Drug Administration GC Gas chromatography GPC Gel-permeation chromatography HPLC High-performance liquid chromatography LC Liquid chromatography LLE Liquid–liquid extraction LOD Limit of detection LOQ Limit of quantitation MCF-7 Cell proliferation (E-screen) MS Mass spectrometry MSTFA N-methyl-N-(trimethylsilyl)trifluoroacetamide NI Negative ion PFPA Pentafluoropropionic anhydride PI Positive ion RAM Restricted access material SIM Selected ion monitoring SPE Solid-phase extraction SRM Selected reaction monitoring STP Sewage treatment plant TIE Toxicity identification and evaluation USEPA United States Environmental Protection Agency WW Wastewater WWTP Wastewater treatment plant YES Yeast-based recombinant estrogen receptor–reporter assay
1 Introduction Chemicals used in a wide range of applications in our modern society are produced on a large scale worldwide. Because of their physical and chemical properties, many of these substances or their metabolites end up in the environment, where they can induce adverse effects on wildlife organisms. The environmental presence of endocrine disrupting compounds has become a hot topic to the point that it competes with other priority health concerns such as the environmental pollution by carcinogenic compounds [1]. Among the various categories of substances with reported endocrine disrupting properties – polychlorinated organic compounds, pesticides, organotins, alkyl phenols and
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil
3
alkyl phenol ethoxylates, phthalates, bi-phenolic compounds, fitoestrogens and microestrogens, etc. – the group of female sexual hormones and related synthetic steroids stands out because of their estrogenic potency. Many studies have confirmed the presence of estrogens and progestogens at concentrations of toxicological concern in the aquatic environment. Already at very low concentrations of ~1 ng L–1 endocrine disrupting effects, such as decreased fertility, feminization, and hermaphroditism of aquatic organisms, are assigned to this class of steroidal hormones [2–5]. Due to their strong endocrine disrupting potency, special attention has been given to the natural estrogens estradiol and estrone, as well as to the synthetic estrogen ethynylestradiol [6]. Synthetic chemicals, resembling the action of natural hormones, find wide application in both human and veterinary medicine and in animal farming practices. Both natural and synthetic estrogens and progestogens are eliminated, either as free compounds or in their conjugated form, primarily through the urine but also in the feces. These substances enter the aquatic environment mainly via wastewater treatment plant (WWTP) effluents (after incomplete removal in the plant) and untreated discharges, and through runoff of sewage sludge used in agriculture [7, 8]. Once in the waterways they may undergo a series of processes, such as photolysis, biodegradation, and sorption to bedsediments, where estrogens and progestogens may persist for long periods [9]. At present, the environmental occurrence of these substances is not subjected to regulation. However, there are concrete indications that the presence of the most active estrogens in the aquatic environment will be regulated in the near future. This calls for efficient and reliable analytical methods for routine monitoring and control. Since the consequences linked to the presence of these compounds in the environment were first made public, numerous analytical methods for their quantification in different environmental matrices have been developed. Most of these methods have focused on surface waters, while wastewater (WW), sludge, and principally sediments and soils, have received comparatively less attention, probably due to the complexity of these matrices. This chapter reviews the most advanced methods applied to the analysis of estrogens and progestogens in these complex matrices, together with the environmental levels found in these natural systems. The main sources of the most environmentally relevant estrogens and progestogens, their physicochemical properties, their principal pathways into the aquatic environment, the primary routes of exposure to these compounds, and data regarding their activity as endocrine disruptors are discussed in this chapter.
2 Usage Large quantities of pharmacologically active substances are used annually in human medicine for diagnosis, treatment, and prevention of illness or to avoid unwanted pregnancy. In animal and fish farming, drugs are mostly adminis-
4
M. Kuster et al.
tered as food additives for preventing illness, as growth promoters or parasiticides [10]. During the last five decades, the consumption of estrogens and progestogens for some of these purposes has experienced a steady growth, and this fact, together with the discovery of their negative ecological effects, has contributed to the current concern about their occurrence in the environment. 2.1 Human Medicine In terms of binding to the human estrogen receptor, estradiol is the principal endogenous phenolic steroid estrogen, which is oxidized in the metabolic processes to estrone and further transformed to estriol. The natural hormones are rapidly metabolized and are therefore orally inactive or active only at very high concentrations. Blocking the oxidation to estrone by, for instance, introducing an ethynyl group in position 17a or 17b of estradiol leads to much more stable products, which remain longer in the body. The consequence of this increased stability is that the so-formed synthetic steroid ethynylestradiol is excreted up to 80% unchanged in its conjugated form [11]. Estradiol also forms the backbone structure used in the engineering of other synthetic estrogens, such as mestranol and estradiol valerate, also utilized in human hormone treatments [11]. One of the main applications of estrogens and progestogens is in contraceptives. The estrogen content in birth control pills is usually in the range of 20 to 50 mg daily [12]. As for the progestogenic content, it varies depending on the type of contraceptive. Thus, in combined oral formulations the progestogenic content is in the range of 0.25 to 2 mg daily, whereas in progestogen-only contraceptives, it is lower (30–500 mg daily). Besides contraception, the uses of estrogens can largely be put into three main groups: the management of the menopausal and postmenopausal syndrome (its widest use); physiological replacement therapy in deficiency states; and the treatment of prostatic cancer and of breast cancer in postmenopausal women. In the same way as estrogens, progestogens are used in the treatment of several other conditions such as infertility, endometriosis, in the management of certain breast and endometrial cancers, and either alone or in combination with estrogens in the treatment of menstrual disorders, among others. The therapeutic doses required in the treatment of many of these diseases are often significantly larger than those employed in contraception. 2.2 Animal Farming Estrogens and progestogens are mainly used as growth promoters in animal farming, and for the development of single-sex populations of fish in aquaculture. Some naturally occurring sexual steroids such as estradiol, progesterone,
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil
5
and testosterone, and synthetic chemicals such as zeranol (estrogenic), melengestrol acetate (gestagenic), and trenbolone acetate (androgenic) have growthpromoting effects. Due to the improvement of weight gain and feed efficiency in meat-production animals, administration of sex steroids to cattle has been a common practice for many years in several meat-exporting countries, including the USA. The most widely used substances are estrogens, either in the form of 17b-estradiol, estradiol benzoate, or the synthetic zeranol. Progesterone, testosterone, and the two synthetic hormones trenbolone acetate and melengestrol acetate are generally used in combination with estrogens [13]. In contrast, no hormone applications for use in commercial-level poultry have been United States Food and Drug Administration (FDA)-approved since the agency’s withdrawal of the cancer-causing hormone diethylstilbestrol in the 1950s. In the European Community (EC), the use of hormonal substances for the promotion of animal growth is prohibited (Directive 96/22/EC). The ban was applied without discrimination internally and to imports from third countries as from January 1, 1989. As a result, countries wishing to export bovine meat and meat products to the EC were required either to have an equivalent legislation or to follow a hormone-free cattle program [14]. In aquaculture, steroidal compounds are used to develop single-sex populations of fish to optimize growth. Sex determination in fish is primarily under genetic control but may be influenced by various environmental conditions, such as temperature, social environment, pH, stocking density, and exposure to exogenous hormones or hormone-like chemicals [15]. Thus, all-male [16] and all-female [17] fish stocks may be obtained through exposure to androgens and estrogens, respectively. The potencies of sex steroids to induce sex reversal are different for each steroid. Functional sex reversal from female to male is carried out by using 17a-methyltestosterone, 19-norethynyltestosterone, or methylandrosterone (concentration range: 0.1–100 mg/kg diet). 11-Ketotestosterone and androsterone have also been used but the dosage required is higher than those of synthetic androgens. Phenotypical feminization is induced successfully by using estradiol, although estrone and ethynylestradiol are used as well [18].
3 Sources and Fate Figure 1 shows the principal routes of environmental exposure to estrogens and progestogens. The most relevant ways by which these compounds enter the environment and reach aquatic systems or the food chain are through WWTP effluents, untreated discharges, and runoff of manure and sewage sludge used in agriculture [7, 8, 10, 19–21, 45]. Human excretion is thought to be the principal source of estrogens and progestogens. These compounds are readily adsorbed from the gastrointestinal tract and through the skin or mucous membranes, and are metabolized in the
6
M. Kuster et al.
Fig. 1 Routes of environmental exposure to estrogens and progestogens
liver with some undergoing enterohepatic recycling. Excreted hormones and their metabolites are found in urine, usually as water-soluble conjugates, and a small amount of “free” estrogens occur in feces [12, 22]. The normal daily estrogen secretion of women is 24–100 mg, depending on the menstrual cycle, and can rise to 30 mg toward the end of pregnancy. The excreted hormones and metabolites collected in the sewer systems end up in WWTPs, where different processes of varying efficiency are applied. Field data suggest that the activated sludge treatment process can consistently remove over 85% of estradiol, estriol, and ethynylestradiol, and a lower, variable percentage of estrone [23]. On the contrary, the concentration of unconjugated steroids in the effluent of WWTPs has occasionally been found to be higher than that in the corresponding influent. Thus, many studies suggest that the conjugated forms (mainly glucuronides and sulfates) are readily converted to the more active free compounds in both the sewer system and WWTPs, as a result of the activity of the b-glucuronidase and arylsulfatase enzymes present in these systems [7, 24–27]. In activated sludge treatment works the principal mechanisms for removal of these compounds are likely to be sorption and biodegradation. Based on the log Kow, sorption to sludge is predicted to play an important role in the removal of hydrophobic compounds (e.g., mestranol) from the aqueous phase. However, this does not seem to be the case for more hydrophilic compounds, such as estriol, estrone, and their glucuronide and sulfate conjugates. With regards to biodegradation, the extent of which depends on factors such as nitrifying bacteria, sludge retention times, aeration, and temperature, some laboratory test studies indicate that estradiol is more readily mineralized than ethynylestradiol
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil
7
or estrone and that synthetic estrogens in general exhibit greater recalcitrance in the activated sludge process [23, 27, 28]. More advanced water purification techniques, utilizing UV-irradiation, ozonization, or activated charcoal, may significantly improve the removal of these compounds, but these techniques are not broadly applied due to their high cost. Thus, current European activated sludge treatment plants, with a hydraulic residence time not greater than 14 h, can in most cases not completely eliminate all the estrogens and progestogens from the effluent [23]. As previously mentioned, contamination of water resources by estrogens and progestogens may also occur through runoff from manure and sewage sludge used in agriculture. Most of the drugs used for animals end up in their urine and feces.When this manure, or the sludge from sewage treatment plants, is dispersed onto the field, the unmetabolized drugs present or their metabolites, depending on their mobility in the soil system, may reach the groundwater (as a result of leaching from fields) or the surface water in the vicinity (through runoff) and affect terrestrial and aquatic organisms [29]. Other disposal options for the sewage sludge are landfill, dumping at sea (forbidden in the EU since 1998) [30], and incineration. The most popular for solid waste disposal is landfill. However, many of the disposal sites are open dumps without protective barriers or leachate-collection systems, which represent a potential risk to the quality of the nearby groundwater. Another increasingly important source of estrogens and progestogens in the environment is, as mentioned before, fish farming. Treatment with steroids is usually carried out by feeding, although in species where male sex differentiation is initiated before feeding commences (e.g., salmon), other procedures are used, such as immersion of alevins [18]. Drugs used in aquaculture as feed additives are discharged directly into the water. It has been estimated that around 70% of the drugs administered end up in the environment surrounding the farm, due to overfeeding, loss of appetite by diseased fish, and poor adsorption of the drugs [31]. 3.1 Environmental Distribution The introduction of estrogens and progestogens into the environment is a function of the way several factors are combined. The manufactured quantity and the dosage applied (amount, frequency, and duration) combined with the excretion efficiency of the compound and its metabolites, the capability of adsorption and desorption on soil, and the metabolic decomposition in sewage treatment are examples of necessary factors to assess environmental exposure. In general the fate and effect of a substance in the environment is dependent on the distribution into the different natural systems, such as air, water, and solids (soil, particles, sediment, and biota). Information on the physical and chemical properties (KH, Kd, and Kow vapor pressure) of a compound may help determine whether it is likely to concentrate in the aquatic, terrestrial, or atmospheric
8
M. Kuster et al.
environment. Table 1 lists the main physicochemical characteristics of the most relevant estrogens and progestogens from the environmental point of view.With regards to the water solubility, it might be worth pointing out that the steroid solubility in WW may be markedly lower than that in distilled water [32]. Once estrogens and progestogens have reached the waterways, a series of processes, such as, photolysis, biodegradation, and sorption to bed-sediments, can contribute to their elimination from the environmental water. Given the relatively low polarity of these compounds, with octanol–water partition coefficients mostly between 103 and 105, sorption to bed-sediments appears to be a likely process. Kd values calculated for estriol, norethindrone, and progesterone in a Spanish river (479, 128, and 204, respectively) as the ratio between the sediment concentration (ng kg–1) and the water concentration (ng L–1) indicate that, in fact, these compounds exhibit a general tendency to accumulate in sediments. Jurgens et al. [33] carried out a series of laboratory experiments to study the behavior of estrogens in the aquatic environment and set up a model to estimate their likely environmental concentrations in the water column and bed-sediments. According to this study, between 13 and 92% of the estrogens entering a river system would end up in the bed-sediment compartment with the majority of sorption occurring within the first 24 h of contact. A similar approach conducted by Lai et al. [9] to investigate the partitioning of estrogens from water to sediments, kinetics of sorption, and the influence of various environmental variables (salinity, total organic carbon, etc.) indicated that sorption takes place rapidly within the first half hour, slows down within the next half hour, and steadily decreases afterward. Furthermore, the synthetic estrogens (mestranol and ethynylestradiol), with their higher Kow values, were shown to partition to the sediment to a greater extent than the natural estrogens. At higher estrogen concentrations, there was a decrease in estrogen removal from the aqueous phase, while higher levels of sediment induced greater removal. The sorption of estrogens to sediments correlated to the total organic carbon content. However, the presence of organic carbon was not a prerequisite for sorption. Tests performed with laboratory saline water resulted in an increase of estrogen removal from the water phase compared to unsalted waters, which is consistent with partitioning experiments using actual field water samples. The addition of estradiol valerate, with a particularly high Kow, suppressed sorption of other estrogens, suggesting that it competed with other compounds for the binding sites. A series of experiments was also conducted by Bowman et al. [34] to ascertain the effects of differing environmental factors on the sediment–water interactions of natural estrogens (estradiol and estrone) under estuarine conditions. Sorption onto sediment particles was in this case relatively slow, with sorption equilibrium being reached in about 10 and 170 h for estrone and estradiol, respectively. On the other hand, true partition coefficients calculated on colloids were found to be around two orders of magnitude greater that those on sediment particles. Hence, it was concluded that under estuarine conditions, and in comparison to other more hydrophobic compounds, both estrone and estradiol
c
b
Values are at 25 °C if not specified. Estimated data. Experimental data.
000050-28-2 000050-27-1 000053-16-7 000057-63-6 000056-53-1 000068-22-4 000057-83-0 000797-63-7
Estradiol Estriol Estrone Ethinyl estradiol Diethylstilbestrol Norethindrone Progesterone D-Norgestrel
a
CAS number
Compound
272.39 288.39 270.37 296.41 268.36 298.43 314.47 312.46
Molecular weight
3.6 (27 °C) 441b 30 11.3 (27 °C) 12 7.04 8.81 2.05b
Water solubility a, c (mg L–1) 4.01 2.45 3.13 3.67 5.07 2.97 3.87 3.48
Log Powc
1.26E-008 1.97E-010 1.42E-007 2.67E-009 1.41E-008 7.31E-009 1.3E-006 3.93E-010
Vapor pressure a, b (mm Hg)
3.64E-011 1.33E-012 3.8E-010 7.94E-012 5.8E-012 5.8E-010 6.49E-008 7.7E-010
Henry’s law constant a, b (atm-m3 mole–1)
Table 1 Physico-chemical Properties of selected Estrogens and Progestogens [(http://esc.syrres.com/interkow/physdemo.htm)]
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil 9
10
M. Kuster et al.
would be expected to remain mainly in the dissolved phase and to have a strong tendency for bioaccumulation. Nevertheless, it is not clear yet whether sorption or biodegradation processes play a major role. Studies conducted with activated sludge [25, 35] pointed at biodegradation as the mechanism contributing the most to the elimination of estrogens from the aqueous phase, while losses by sorption effects were considered rather unlikely. However, Jurgens et al. [33], based on a designed exposure assessment modeling system (EXAMS) model, postulate that degradation processes in rivers are unimportant under average flow conditions, as they account for only 2–8% of the input loading. This is in agreement with the theory presented by Huang et al. [36], according to which the main removal mechanism for hormones in WWTPs would be sorption onto particles and not biotransformation. Degradation studies carried out in waters from five English rivers indicate that estradiol has a half-life of 3–27 days [33]. Estrone was found to be the first degradation product of estradiol but no investigations of the subsequent byproducts were conducted. The poorest degradation rates were observed in the estuary river water samples, where the high salt content might have inhibited microbial degradation. Furthermore, ethynylestradiol (half-life 46 days) was found to be more stable than 17b-estradiol (half-life 4 days, e.g., in the River Thames). These half-life values might correspond to ideal summer temperatures. However, under winter conditions these compounds could be twice as persistent. In activated sludge, the synthetic estrogens ethynylestradiol and mestranol have been shown to remain stable and intact over 5 days, while progestogens are already up to more than 50% disintegrated after 48 h [32]. Under the anaerobic, dark conditions normally present in the subsurface layers of river sediments, these compounds are expected to undergo a slow photodecomposition and biodegradation. On the other hand, desorption from sediments has been shown to be significantly less important than sorption, with desorption distribution coefficients two to three times lower than those obtained for the sorption process [33]. In an environment like this, river sediments can therefore act as sinks where estrogens and progestogens may persist for long periods, be transported to other areas, and be eventually released by diffusion across the sediment water-column interface or by scouring in storm events [9]. The concentration of estrogens and progestogens in bed-sediments is predicted to increase over time; thus, bed-sediments can be anticipated as environmental reservoirs from where these substances may eventually become bioavailable [37].
4 Occurrence Most research of estrogens and progestogens has been conducted on water samples and less frequently on solid samples. Soils and sediments, in particular, have received very little attention and thus literature data on these matrices are very
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil
11
scarce. The same applies to the analytes investigated. Whereas free estrogens, both natural (e.g., estradiol, estrone, estriol) and synthetic (e.g., ethynylestradiol, mestranol, diethylstilbestrol), have often been investigated, estrogen conjugates [38, 39] and progestogens [40] have seldom been studied, probably due to their lower estrogenic potency. Figure 2 shows the chemical structure of the estrogens and progestogens most frequently investigated in environmental samples. Table 2 summarizes the literature data available on the occurrence of these two classes of steroidal compounds in WW, sludge, and sediments. Natural hormones (and their metabolites) have always been present in the environment. The growing use of both natural and synthetic estrogens and progestogens in human medicine and in livestock farming (see Sect. 2, Usage) has led to an increase of their occurrence in natural systems. Due to steady population growth and regional population density, an irregular distribution of these pollutants is found. Particular concern is given to certain areas where high levels were detected, e.g., areas adjacent to agricultural and animal farms.
Table 2 Environmental occurrence of estrogens and progestogens
Matrix (location)
Compounds
Concentration (ng L–1 or ng g–1)
Ref.
Estrogens and progestogens Natural and synthetic estrogens Natural and synthetic estrogens Natural and synthetic estrogens
0.4–188 Æ 0.3–82.1
