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Survey of Estrogenic Activity in the Danish Aquatic Environment Part B

3 Results

• 3.1 WWTPs and receiving streams
• 3.2 Septic tank effluents
• 3.3 Field drains
• 3.4 Part A follow-up
  • 3.4.1 Reference lake
  • 3.4.2 Drain from sludge amended field

3.1 WWTPs and receiving streams

The activities of free steroid estrogens in effluents, receiving stream waters and sediments were studied at six Category C (mechanical-biological treatment) WWTPs for which some basic data are presented in Table 3-1.

All of the plants were constructed as closed tank systems which are believed to be based on (aerated) activated sludge processes though this has not in all cases been possible to verify.

The results (total activities) are shown in Table 3-2 while the chemical speciation of the total activity measured in selected stream water samples and sediment samples can be found in Table 3-3 and, respectively.

The sampling dates are also stated in Table 3-2. The weather conditions were dry at all locations immediately before and during sampling.

Table 3-1 General data about the selected WWTPs

* M = Mechanical; B = Biological; C = Chemical precipitation.
( ) very uncertain estimate

Table 3-2 Activity of free estrogens in WWTP effluents, receiving streams and sediments. Levels in E2 equivalents (ng/L)

- = not analysed

Results from chemical analysis are presented as estrogen equivalents calculated as the sum of the concentrations of each of the analytes multiplied with conversion factors for the relative estrogenic activity of each analyte. The conversion factors were determined experimentally with the YES-assay by testing aqueous solutions of E1 (0.29) , EE2 (0.88) and a-E2 (0.04). Only values >LOD were included in the calculation.

As appears from Table 3-2 the estrogenic activity in the effluent samples was generally found to be low, below the detection limit (= neg) in four out of six samples tested biologically, and only the sample from Tisted exhibited an activity level of a magnitude corresponding reasonably to those found in Part A of the survey for the same WWTP category (C) (see section 6.1.2.1 in the Part A report).

In accordance with the effluent results also the levels in the water of the receiving streams are found to be low.

The results of the biological testing and the chemical determinations are found to correspond well to each other.

E1 was detected at low concentrations (i.e. < 1 ng/L) in all the effluent and stream samples that were analysed chemically. However, as the estrogenic activity of E1 is only 0.29 times that of E2, the calculated E2-equivalents were even lower and in two cases below the E2 quantification limit, LOQ, of 0.1 ng/L.

The highest total estrogenic activities among the "chemistry" samples were observed in the samples from Kjellerup-Demstrup WWTP and it receiving stream, Grundel Bæk. These were the only two samples in which the contraceptive agent EE2 appeared in concentrations above the LOQ.

Table 3-3 Chemical speciation of free estrogens in WWTP effluents and receiving streams (downstream 1 position).

In the sediments (Table 3-4), the total estrogenic activity calculated as E2-equivalents was found by the chemical analysis to be in the range from 30 to 1740 ng/kg dw. E1 was found in all samples in concentrations ranging from 100 to 3180 ng/kg dw. E2 was found in 7 out of 10 samples in the range from 40 – 580 ng/kg dw. α-E2, that is related to farming animals, was only found in one sample at a level of 1540 ng/kg sediment.

In the sediment sample EE2 did, however, not appear in the sample from Kjellerup-Demstrup but was found instead in samples from two other locations.

Table 3-4 Chemical speciation of free estrogens in sediments from streams receiving WWTP effluents.

It is noted that all sediment samples showed estrogenic activity according to the results of the chemical analysis and that the rather high levels observed (compared to the water samples) reflect well the lipofilicity of the un-conjugated steroid estrogens and the derived tendency to partition into (the organic fraction of) the sediment phase.

However, it was only possible to detect estrogenic activity in half of the sediment samples when applying the biological assay (Table 3-4). These samples were the same as those with the highest contents of estrogens in the chemical analysis.

3.2 Septic tank effluents

The results of the biological testing and chemical analysis of septic tank effluents stated as E2-equivalents appear from Table 3-5 while the composition of the individual steroid estrogen species in selected samples (those with highest activity) are shown in Table 3-6.

The samples from Egtved were taken at the outlet from the septic tank while the others were sampled in inspection wells of the pipelines transporting the effluents to the discharge or infiltration points. The distance from tank to inspection well ranged from a few metres and up to about 100 metres. All locations were sampled in the second half of June 2005.

Table 3-5 Free estrogenic activity in septic tank effluents.
Values in E2 equivalents (ng/L).

- = not analysed

The levels found in the effluents from Egtved are in line with the results from Part A, while the levels consistently observed in the samples from Karlebo are considered very high. In contrast to the Karlebo samples, the Gunderød samples demonstrate low, even negative, responses in the biological assay.

Four of the five locations have only one household connected to the septic tank system while at the last, Karlebo, seven households are connected. At Karlebo, the higher number of persons connected combined with the fact a woman living in one of the houses was pregnant at the time when sampling took place, may account for the high estrogenic activity observed at this location.

No explanation of the low or even absent estrogenic activity in the Gunderød samples can be given based on the information available about the location including the household.

Table 3-5 Chemical speciation of free estrogens in septic tank effluents

The deviation between biological and chemical results appears to be somewhat bigger for the septic tank effluents than for the WWTP effluent and stream water samples. This could partly be due to presence of other chemical substances with estrogenic activities than the steroid estrogens (e.g. alkylphenols and phthalates) and partly be a matrix effect.

A relatively high level of the synthetic steroid estrogen EE2 (α-ethynylestradiol) was observed in the sample from Egtved while this substance was not detectable in any of the other samples. In Part A of the survey EE2 was only observed in very few samples.

3.3 Field drains

Sampling of drains dewatering fields amended with liquid manure was carried out at three locations, all located in Jutland. At one location (Ejer Bavnehøj), the sampling was by mistake discontinued after one week without replacement of the location by another.

The Ejer Bavnehøj and Odderbæk locations were fields amended with cattle manure while the Løsning field was amended with pig manure. A summary of data about the fields and the application of manure or sludge is presented in Table 3-7.

Table 3-7 Data on the sewage sludge and manure applied fields where drainage water was sampled. All locations area in Jutland.

* 25 tons liquid manure + 5 tons solid manure per hectare.
** Dry matter content: 16 %

In April when the application of manure and sludge took place (week 14 was 4-10 April and week 16 was 18-24 April) the weather was largely dry and it was not until around the turn of the month (April-May) that significant rain episodes occurred.

Heavy rains (12-34 mm) occurred throughout Jutland at the time when the first round of sampling was initiated. During the three weeks when sampling took place several smaller but no further major rain episodes were registered.

The estrogenic activities in the field drains (manure as well as sewage sludge) appear from Table 3-8. Only in one sub-sample detectable, very low estrogenic activity in the biological assay was found while in the others the response was negative.

Table 3-8 Free estrogenic activity in drainage water from fields amended with liquid manure or sewage sludge. Values in E2 equivalents (ng/L).

* due to a perforation of the sampler tube caused by rodent "attack".

The results of the chemical analyses are in good accordance with the biological results although a low activity was observed by chemical analysis in two out of four samples that gave negative response in the biological assay. However, the maximum level of activity measured chemically was only 0.32 ng E2 equiv./L.

The composition of the chemically determined estrogenic activities is presented in Table 3-9. While the presence of α-E2 in the drainage water from Ejer Bavnehøj was expected as the manure applied on this field was from cattle, the presence of this steroid estrogen in the samples from Løsning is strange as the manure at this location should originate from pigs (which do not excrete much of this specific substance)

Table 3-9 Chemical speciation of free estrogens in drainage water from fields amended with liquid manure or sewage sludge

Further, it is noted that in the two samples with the highest activity, the major part of this is caused by the presence of EE2. This could indicate that the field drains may receive contributions from other sources than just domestic animals i.e. humans. Unfortunately, it has not been possible to obtain further any information that could lead to clarification of this question.

3.4 Part A follow-up

3.4.1 Reference lake

The reference lake, Sorte Sø (Funen), in which some estrogenic activity was observed in Part A in spite of the absence of agricultural or domestic contamination sources, was sampled again in May 2005 at exactly the same spot and using the same procedures as in Part A (2004). Further, two stored "bank" samples from Part A (sub-samples of the exactly the same samples from March and June 2004 that were analysed in Part A, see section 3.2) were tested with the YES assay. The results of the biological tests are shown in Table 3-10 while the chemical speciation of steroid estrogens in the June 2004 bank sample is shown in Table 3-11.

Table 3-10 Estrogenic activity in a new sample and re-tested bank samples from the reference lake Sorte Sø (location A1, fig. 2-1). Results of bank samples are compared to original 2004 data.

* Confirmatory test of stored 2004 sub-sample of the same sample tested in 2004.

Table 3-11 Chemical speciation of free estrogens in the June 2004 bank sample from Sorte Sø

Thus, the high estrogenic activity observed in 2004 could not be confirmed by the new sample taken in May 2005. However, the two stored sub-samples of the 2004 sample still showed moderate and high activity, respectively, when re-tested with the YES assay and analysed chemically.

3.4.2 Drain from sludge amended field

A drain from a sludge amended field at Blommenslyst (County of Funen) demonstrated high estrogenic activity when tested with the YES assay in the 2004 survey (Part A) and was sampled again in May 2005. Also in this case two "bank" sub-samples from the same sample that was tested in 2004 were tested again. The results are shown in Table 3-12 and Table 3-13.

Table 3-11 Estrogenic activity determined by the YES assay in a new sample and re-tested bank samples from a sludge field drain at Blommenslyst, County of Funen (location A2, fig. 2-1). Results of bank samples are compared to original 2004 data.

* Confirmatory test of stored 2004 sub-sample of the same sample tested in 2004.

Table 3-12 Chemical speciation of free estrogens in drainage water from the field drain at Blommenslyst sampled in May 2005 (the field was amended with sewage sludge in the spring of 2004).

The observed high estrogenic activity levels are difficult to explain unless the drain also transports e.g. septic effluent. However, a recent inspection at the site by the local Water & Sanitation company has not provided any definitive information on the issue. A nearby farm has a septic tank but it is not obvious that the effluent could affect the field drain inspection well where the drainage water samples were taken.

Some difference between the biological and the chemical result is noted, which may be due to presence of other substances with estrogenic activity in the sample than the few steroid estrogens that were analysed chemically.

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Version 1.0 March 2006, © Danish Environmental Protection Agency