| Front page | | Contents | | Previous | | Next |

Survey of Estrogenic Activity in the Danish Aquatic Environment

1 Background

1.1 Estrogens as an Environmental problem
1.2 Previous Danish studies on estrogens
     1.2.1 Field studies of intersex
     1.2.2 Preliminary survey of WWTP effluents
     1.2.3 Characterisation of WWTP influents and effluents
     1.2.4 Estrogen elimination processes at WWTPs
1.3 Project objectives

1.1 Estrogens as an Environmental problem

The recognition of steroid hormones and other chemical substances with estrogenic activity as environmental contaminants and as an issue with potentially wide ecological implications is rather new.

However, a few isolated examples date more than 20 years back. Already in 1980 visible changes in sexual development and fertility in a population of alligators in Lake Apopka, Florida, was observed following a massive release of the insecticide DDT and the chemically very resembling acaricide dicofol into the lake. Also the use of TBT (tributyltin oxide) as an antifouling agent in ship paints has been shown to severely affect the aquatic environment by impairing the reproduction of oysters and marine snails in coastal waters of e.g. western and northern Europe.

Within the last 10 years a number of studies from Europe, Japan and North America have reported anomalous sexual development and reduced reproductive capacity among species of fish, amphibians, reptiles and molluscs as a result of exposure to chemicals with estrogenic activity.

In Great Britain in particular, a number of field studies as well as controlled experiments have demonstrated the sensitivity of roach and other freshwater fish species to discharges of municipal and industrial wastewater leading to feminisation of the males, a phenomenon known as intersex. E.g., in two rivers heavily polluted by discharges of municipal wastewater almost 100% of the male population of roach was found to exhibit intersex and only half of the males were able to reproduce normally (cfr. Christiansen et al. 2002).

Many of the investigations in the aquatic (freshwater) environment as well as controlled exposure of fish in the field and in the laboratory indicate that the natural steroid hormones of humans (and other mammals, e.g. domestic animals) to a large extent may be responsible for the sexual and reproductive disturbances that have been observed. It is suspected that effluents from municipal sewage treatment plants are major contributors to this type of pollution though other sources such as drainage water or surface runoff from fields fertilised with liquid animal manure may also contribute.

A comprehensive study was recently conducted in the Netherlands (Vethaak et al. 2002), in which field observations were combined with controlled experiments and laboratory tests to give an overview of the problem of estrogens in the Dutch aquatic environment. In was concluded, among others, that municipal wastewater effluents generally exhibited estrogenic activity and that natural and synthetic steroid hormones in domestic sewage were accountable for a major part of the observed activity.

In the USA, a "national reconnaissance" of pharmaceuticals, hormones and other wastewater contaminants was conducted in 1999-2000 by the U.S. Geological Survey (Kolpin et al. 2002a) in streams "susceptible to contamination". The investigation included more than 10 natural and synthetic reproductive hormones, including estrone (E1), 17-β-estradiol (E2) and 17α-ethynylestradiol (EE2). In total, the frequency of detection of reproductive hormones in the streams was 37% (Kolpin et al. 2002b), and the specific detection frequencies of E1, E2 and EE2 were 7%, 10% and 5.7%, with median detectable concentrations of 27 ng/L, 9 ng/L and 94 ng/L, respectively (the latter value being remarkably high).

Also German scientists, e.g. Ternes et al. (1999), quite early started to investigate the emissions of estrogenic substances with discharges from wastewater treatment plants (WWTPs). Körner et al. (2001) found that E2 and EE2 (and to some extent E1) could account for about 90% of the estrogenic activity determined by the E-screen assay in effluents from 16 German WWTPs.

In Italy, D'Ascenzo et al. (2003) studied the fate of the conjugated forms of steroid estrogens (the form on which the estrogens are excreted from the human body with urine) in sewage systems and WWTPs. They found that glucuronide conjugates were easily transformed back to the original, active form in these systems, while a sulphate glucuronide was more resilient to degradation.

Reports of findings as the above did, at a quite early stage, also in Denmark lead to considerable concern among scientists, politicians and the general public. In recent years a number of studies with limited scope have been carried out to provide preliminary information on the possible extent and severity of this issue in the Danish environment. A brief review of the conducted field and laboratory studies is given below.

1.2 Previous Danish studies on estrogens

1.2.1 Field studies of intersex

In 2000, a field study to reveal possible sexual disturbances in populations of roach and trout was conducted in a number of streams and lakes in the County of Århus (Århus Amt 2001). A high frequency (44%) of males with elevated vitellogenin plasma concentrations was observed among trout in the Voel Bæk stream as well as a high blood concentration of vitellogenin in the fish. In another watercourse, Kristrup Landkanal, in which a major part of the flow originates from the effluent of the 75.000 PE WWTP (current actual load) of Randers town, the intersex frequency among roach males was 26%.

A follow-up study (Århus Amt 2003) in the Voel Bæk stream revealed that the observed anomalous sexual development in the trout population could not exclusively be attributed to the outlet from the small WWTP of Voel village as high concentrations of steroid hormones were also observed in two field drains receiving septic tank effluents from single farmhouses. Drains from fields amended with liquid manure were not investigated specifically, but the catchment of Voel Bæk is known to hold a quite high density of cattle and pigs. It was not possible to link the observed intersex effects to exposure from one single source of pollution.

1.2.2 Preliminary survey of WWTP effluents

In the late autumn of 2002, Ingerslev et al. (2003a) conducted a preliminary survey of the contents of steroid hormones in effluents from 19 Danish WWTPs (one sample per WWTP) comprising a range of sizes, technologies and geographical locations. It is concluded by the authors that steroid estrogens could be identified in effluents from 15 out of the 19 investigated plants and that in 8 of the 19 samples the content exceeded the limit of quantification of 2 ng/Liter for E1 and 1 ng/Liter for E2/EE2, respectively.

Generally, estrone was detected in the highest concentrations and, maybe, the WWTPs from the greater Copenhagen area had higher levels of estrogens in their effluents than the WWTP effluents from other parts of the country. Also, as a weak trend, the Copenhagen WWTP effluents contained more EE2 than the other effluents. This pattern in the results could be due to the higher population density in the Copenhagen area but might as well simply be a result of degradation of the labile estrogens in the samples from outside the Copenhagen area, which in general did not arrive at the laboratory until 1-3 days after sampling (in one case as much as five days).

1.2.3 Characterisation of WWTP influents and effluents

A study of 3 steroid estrogens and 10 xeno-estrogens in influents and effluents from Danish WWTPs was reported by Kjølholt et al. (2003), who in the summer/autumn of 2002 carried out three rounds of wastewater influent and effluent sampling at the WWTPs in Avedøre and Usserød (actual load 345.000 PE and 30.000 PE, respectively), which both serve suburban municipalities of Copenhagen. The results are shown in the table below.

Table 1.1 Concentrations of Three estrogens in influent and effluent of two WWTPs in the greater Copenhagen area.

Overall, the influent concentrations correspond quite well whereas it appears that the elimination of the estrogens is more efficient at Usserød WWTP. This could be due to a sand filter installed after the clarifier tank at Usserød, a feature that Avedøre WWTP does not possess. Otherwise, the two plants have been designed according to the same overall principles and mainly differ with respect to size. At both plants, the steroid estrogens could account for far more estrogenic activity than the analysed xenoestrogens; 80-94% and 90-95% of the total (calculated as E2-equivalents), respectively.

1.2.4 Estrogen elimination processes at WWTPs

Andersen et al. (2004) studied the degradation of E1, E2, EE2 and two conjugates of E1 (E1-3Glu and E1-3Sul), in laboratory experiments under aerobic and anaerobic conditions using activated sludge from Egå WWTP (near Århus).

Aerobic degradation was found to take place very fast for E1 and E2 i.e. with half-lives of only a few minutes, while the half-life of EE2 was more than 100 times slower. The glucoronide conjugate of E1 (E1-3GLU) was transformed slightly slower than E1/E2 whereas the transformation rate of the sulphate conjugate (E1-3SUL) resembled that of EE2. In conclusion, the observed order of aerobic degradability was: E2 > E1 > E1-3Glu >> EE2 > E1-3Sul.

Under anaerobic conditions, the degradation rates for E1 and EE2 were considerably (10-20 times) lower than under aerobic conditions while the degradation of E2 was not significantly changed i.e. still only a few minutes or lower depending on sludge concentration.

Predictions of estrogen elimination at Egå WWTP based on the laboratory results indicated that more than 99.9% of E1, E2 and E1-3Glu in the sewage would be removed while about 3.3 % of EE2 would still remain in the effluent. The removal rate of E1-3SUL could not be estimated. The predicted removal efficiencies were higher than typically observed in monitoring studies at WWTPs, an observation that may be explained by the presence of one or more rate determining processes in real WWTPs (e.g. desorption rate of the sorbed fraction) that could not easily be simulated in the laboratory.

1.3 Project objectives

In addition to the international findings and the Danish field and lab studies mentioned above, three review studies on feminisation of fish (Christiansen et al. 2002), analytical chemical methods (Ingerslev & Halling-Sørensen 2003) and biological assays (Kinnberg 2003) were carried out by initiative of the Danish Environmental Protection Agency. On this basis, the Danish minister of environment decided to launch new initiatives to further elucidate the estrogen issue. The initiatives should include generation of Danish data on estrogens in the environment to enable a better national assessment of the potential problem.

Therefore, the present study was initiated with the following two main objectives, which should be reflected in the selected scenarios for investigation and in the choice of locations and sample types for testing and analysis:

• Survey of environmental status - the study must provide sufficient data to assess the general estrogenic activity in the aquatic (freshwater) environment in Denmark including whether the findings are of a general nature or they relate only to special conditions or geographic regions.
   
• Identification of significant sources of pollution - a range of potential pollution sources must be investigated and characterised to the extent necessary for politicians, authorities and others to initiate corrective measures, if considered necessary.

Some minimum requirements to scenarios/types of samples were stated by the DEPA as part of the project description:

• Sewage treatment plants covering a range of technologies and sizes,
• possible contamination sources and freshwater bodies in the "open land" i.e. agricultural areas, and
• water bodies suitable for establishing the background level in Danish freshwater environments.

The strategy for meeting the objectives is described in the following chapter.

| Front page | | Contents | | Previous | | Next | | Top |

Version 1.0 Januar 2005, © Danish Environmental Protection Agency