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Degradation of Estrogens in Sewage Treatment Processes

3 Estrogens in sewage and sewage treatment processes

This chapter serves as an introduction to the following chapters describing the experimental work and results, which constitute the core of this project. The text in this chapter provides a brief summary of selected, newer journal articles and reports on the occurrence and fate of estrogens in municipal STPs and on laboratory simulation studies of their behaviour in treatment processes. The intention is provide an overview of newer, representative data rather than a comprehensive review of existing knowledge.

3.1 Estrogenic substances and their excretion

The main active estrogenic substance in humans is 17β-estradiol (E2) but the primary metabolite of E2, estrone (E1), also exhibits some estrogenic activity. An even less active metabolite, estriol (E3), is also produced to a significant extent. 17α-ethinylestradiol (EE2), which is the most widely used active substance in contraceptive pills, also has a high estrogenic activity. Hence, the substances that appear to be the most obvious to focus on in the context of this project are E1, E2 and EE2.

In order for the human body to inactivate and excrete estrogenic substances, which are hydrophobic and thus hard to eliminate, they must be transformed into more soluble forms that can be excreted via the kidneys. Therefore, the estrogens are transformed by the metabolic system into soluble conjugates - a reaction by which they also loose their estrogenic activity.

The conjugation process is enzymatically mediated and generates sulphate- and glucuronic acid esters of the hydroxyl groups in the 3- and 7-position of the basic steroid structure. For E2 and EE2, which both have two hydroxyl groups, eight different conjugates are theoretically possible, while for E1 there are only two. In human urine, natural estrogens only occur in their conjugated form, which, however, can be transformed back into the parent substances by microorganisms and enzymes in sewage.

3.2 Levels of estrogens in untreated and treated sewage

A number of studies on the occurrence and levels of estrogens in municipal sewage have been published in recent years. Some of the studies report contents of estrogens in sewage only based on their activity as measured by in vitro assays (e.g. the YES assay), while others have determined single substances by chemical analytical methods.

In Denmark, only very limited data exist where in the same study both influent and effluent concentrations of estrogens have been studied. DEPA (2003a) recently published a report on the occurrence of the estrogens E1, E2 and EE2 in influents and effluents sampled at selected public STPs by qualified grab sampling (sample composed of five sub-samples taken with few minutes intervals). The levels observed at the two plants where most samples were taken (Avedøre STP and Usserød STP) were comparable to those reported from other countries (Table 2-2).

Table 3-1
Selected estrogens (E1, E2 and EE2) in influents and effluents at two Danish public STPs (DEPA 2003a).

In the same study also the contents of a number of xenoestrogens (alkylphenols, bisphenol A, phthalates etc.) were determined analytically, and their share of the total estrogenicity was calculated based on E2-equivalence factors (EEQs) in the YES-assay (an in vitro assay based on a genetically modified yeast strain). The xenoestrogens were found to account only for a limited part of the total estrogenic activity (only the influent samples were possible to calculate; Table 3-2). This is consistent with observations from other studies.

It should be mentioned that the estrogenicity of a substance determined by an in vitro assay such as e.g. the YES-assay is not necessarily the same as the estrogenicity in vivo. Thus, the estrogenic activities of E2 and EE2 in in vitro assays are typically about the same, while EE2 in laboratory experiments with fish has been shown to be 14-130 times more active (stated as LOEC-values) than E2 (Metcalfe et al. 2001, Thorpe et al. 2003).

Table 3-2
The estrogenic activity of natural and synthetic estrogens (E1, E2 and EE2), and of selected xenoestrogens, in influents of two Danish STPs (3 sampling rounds) stated as E2-equivalents, and the xenoestrogens' share of the total estrogenic activity (among the measured substances) (DEPA 2003a).

* EEQ-E = sum of E1, E2 and EE2
** EEQ-XE = sum of NPE, NP, OP, Bisphenol A, DEP, DBP, BBP, DEHP and DOP.

Table 3-3 shows examples of levels of estrogenic compounds (stated as E2-equivalents) observed in influents and effluents at public STPs in some European countries outside Denmark and in the USA (adapted from DEPA 2003a).

Table 3-3
Levels of estrogens in influents and effluents at public STPs in various countries, stated as E2-equivalents (DEPA 2003a).

l.o.d. = Limit of detection.
* One, very deviating result (46.7 ng/L) was omitted.

A review and assessment report on possible analytical methods for the determination of estrogens in the environment was recently published by DEPA (2003b). The report also includes a brief review of data (primarily from Germany and Belgium) on the levels of a number of estrogens in raw and treated sewage and in sewage sludge. The data are shown in Table 3-4.

Table 3-4
Representative selection of expected and measured environmental concentrations in STP influents, effluents and sludge (<LOD and >LOQ indicate that estrogens were detected at concentrations below limits of detection or quantification) (from DEPA 2003b).

a) Concentrations in sewage effluent are estimated assuming 50-95% removal.
b) N.D. indicates that no data are available in literature.

Svenson et al. (2003) performed in 1999 sampling of raw and treated sewage at 20 Swedish public STPs ranging from simple to advanced, complex technologies and found at the latter (which resemble typical, modern Danish plants) influent levels from approximately 3-25 ng EEQ/L and effluent levels from <0.1-5 ng EEQ/L (measured as total estrogenic activity in a yeast-based screening assay).

3.3 The fate of estrogens in sewage treatment plants

Only rather recently, i.e. since the late 1990ies, have reports on studies of the fate of estrogens in sewage treatment plants begun to appear in the international literature. Some of the key publications and their main findings are briefly reviewed below.

Ternes et al. studied a number of natural and synthetic estrogens in sewage at a municipal STP near Frankfurt/Main (Ternes et al. 1999a), and found that about 2/3 of the incoming 17β-estradiol (E2) and 16α-hydroxyestrone was eliminated in the STP whereas the elimination efficiencies for estrone (E1) and 17α-ethinylestradiol (EE2) were low (<10%). In subsequent laboratory experiments with activated sludge from the same plants, Ternes et al. (1999b) confirmed the persistence of EE2 under aerobic conditions while both E1 and E2 were degraded fairly rapidly under these conditions (E2 via E1). Glucuronides of E2 were rapidly cleaved thus producing free E2.

In a recent study at 20 STPs in Sweden (Svenson et al. 2003), removal efficiencies of estrogenic activity (yeast-based assay) in the whole range from practically 0% to more than 99% were observed. Simple treatment with only chemical precipitation using Fe- or Al-salts resulted in no removal at all while precipitation with lime appeared to be more effective (73%, only one plant). Trickling filters were less effective than activated sludge systems to eliminate estrogenic activity, and the highest removal rates were obtained at plants with comprehensive treatment technologies i.e. combined biological and chemical removal of organic matter, nitrogen and phosphorus. Samples filtered at 20 µm were also studied, and it was found that practically no estrogenicity was associated with particles larger than this size.

Andersen et al. (2003) investigated the fate of E1, E2 and EE2 at the STP of Wiesbaden, Germany. They observed an overall elimination efficiency of E1 and E2 of >98%, while EE2 elimination was slightly lower; about 90%. E1 and E2 were found to be degraded in the activated sludge system while EE2 primarily was degraded only in the nitrifying tank. In the effluent, however, all concentrations were below 1 ng/L.

The fate of selected estrogens in sewage from six municipal STPs in Rome (dominated by domestic sewage) was recently studied by D'Ascenzo et al. (2003). They found that conjugated estrogens were easily cleaved by the sewage, presumably by the â-glucuronidase enzyme produced by fecal coliform bacteria. However, the degradation of the studied sulphate, E1-3-sulphate (E1-3Sul), was somewhat less. The elimination of free and conjugated estrogens at the STPs was 84-97% except for E1-3Sul, which was only reduced by 64%.

Matsui et al. (2000) found in a study at three STPs in Japan that in the influent E2 accounted for 34% of the total estrogenicity while in the effluent it was almost 100%. Matsui et al. also observed that primary treatment contributed only little to the observed overall removal of estrogenicity, which primarily took place in the activated sludge part of the system. It was found that during the dewatering of sludge, large amounts of estrogens were released to the water phase (which is afterwards recycled back into the plant). Concentrations close to 500 ng EEQ/L were observed in the water phase.

In another investigation of estrogens and other endocrine disruptors at Japanese STPs, Tanaka et al. (2000) reported that at most of the plants studied (total of 20) the total estrogenicity (as determined by the YES assay) of the treated wastewater could be attributed to E2 to a very high degree. However, in the raw sewage there was at many STPs a significant content of estrogenicity that could not be explained by the concentrations measured chemically of 44 known or suspected endocrine disruptors.

In a review article, Johnson & Sumpter (2001) conclude based on field data that the activated sludge treatment process appear to consistently be able to remove more than 85% of E2, E3 and EE2 whereas the removal of E1 is less and more variable. Researchers from the same group (Kirk et al. 2002) describe results of studies at five public STPs in the UK, where removal rates for estrogens (measured only as the sum of estrogenic activity by a yeast-based in vitro assay) were generally found to be 70% or higher. Very limited removal occurred in the primary treatment step while the activated sludge treatment resulted in significant reductions. Only the fate of free (i.e. not conjugated) estrogens could be studied due to the detection method applied.

A recent Danish literature review on substances causing feminisation of fish (DEPA 2002) also includes a review of observations on the fate of estrogens in STPs. It is concluded that the advanced STPs generally cope better with estrogens than more simple plants (e.g. only mechanical treatment) and that a high hydraulic retention time and a high sludge retention time in the activated sludge treatment process has a positive influence on the ability of an STP to remove estrogens.

To summarise the above review of selected recent literature, it has generally been observed that primary treatment alone results in no or only limited removal of estrogens from sewage while secondary treatment involving activated sludge very significantly reduces the levels of all estrogens. A long sludge retention time appears to have a positive influence on the ability of the activated sludge system to eliminate estrogens. It appears that the substances E2 and E3 are very efficiently removed in the latter systems while the removal rate of E1 is somewhat lower and more variable and EE2 even more so as it only undergoes significant degradation in the nitrification step. Conjugated forms of estrogens are easily cleaved in sewage and STP processes to produce the free, active forms of the estrogens. However, there is a lack of studies describing in more detail the relative influence of the presumed two main removal processes involved in the activated sludge system i.e. sorption to sludge particles and microbiological degradation.

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Version 1.0 November 2004, © Danish Environmental Protection Agency