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Survey of Estrogenic Activity in the Danish Aquatic Environment Part B 2 Study design and methodology
2.1 Overview of investigationsTo meet the overall study objectives, the following categories of estrogen pollution sources and aquatic environments were sampled and tested biologically with the YES assay and/or analysed chemically for contents of the steroid estrogens estrone (E1), 17ß-estradiol (E2), 17α-estradiol (α-E2), and 17α-ethynylestradiol (EE2):
More details on the considerations behind the sampling strategy and selection of locations are given in the following sections. 2.2 Sampling strategy and locationsPart A of "Survey of Estrogenic Activity in the Danish Aquatic Environment" was a broad study for which a large number of locations were selected. The aim was to describe the range of the "typical" or "average" situation both with regard to the aquatic environment and with regard to pollution sources, and not to focus on the "worst case" scenarios. As the conclusion turned out to be that in general the estrogenic activity levels found in the investigated streams, rivers and lakes were relatively low compared to known effect levels, it was decided to focus more on the critical situations in Part B of the study. Further, Part A of the study provided within certain categories, e.g. septic tank effluents and field drains, too few results to allow firm conclusions to be drawn. Therefore, Part B should also include such sample types to enable a better assessment of their potential importance in relation to contamination of the aquatic environment with steroid estrogens. Specific comments on each sample category are given below. 2.2.1 WWTPs and receiving streamsThe total of six WWTPs to be selected for Part B should all belong to either category C (mechanical-biological treatment) or category D (mechanical treatment) defined in Part A of the survey as these plants generally demonstrated low ability to remove steroid estrogens from the wastewater prior to discharge. However, in Part A the effluents from C/D-plants only had a low or moderate impact on receiving stream water quality, probably due to the normally relatively high stream flow in comparison to WWTP effluent volume. Therefore, it was decided to select a new set of C/D-WWTPs for Part B having more critical ratios between effluent volume and stream flow i.e. ideally rather big C/D-plants with very small receiving streams (preferably max. ratio effluent/stream flow of 1:10). Ideally, the discharge should take place continuously. In reality, these requirements limited the possible WWTPs to category C as most category D plants would be too small and/or operating (discharging) too infrequently. Further, the sampling should take place in the periods when the stream flow was as close to base flow as possible within the timeframe available. On the other hand, sampling of stream water should be also possible throughout the year, i.e. the streams must not dry out during summer, and sampling should be possible upstream the discharge point as well as downstream (up to a distance of at least up to 100 times the stream width). By this requirement a significant number of locations had to be given up as it is very common that small streams/ditches are covered and pipelined in some sections of their course. Specific locations were selected based on a review of the county-wise listings of existing C/D WWTPs and subsequent personal contact to the responsible departments in the county administrations to obtain
more detailed information about the status of the WWTPs and their receiving streams. In many cases further follow-up by contact to technical/WWTP administrations at municipality level turned out to be
necessary to obtain the required updated baseline information. 2.2.2 Septic tank effluentsSuitable locations for sampling of septic tank effluents from isolated houses or farms in the countryside turned out in Part A of the survey to be difficult to find. Hence, this type of potential source of estrogen contamination was not fully examined in Part A and it was decided instead to further explore the issue in Part B as a significant number of such potential point sources exist in the open land in Denmark. The composition of domestic sewage will differ from one household to another depending on the structure of the families (including sex and age). On the other hand, no significant regional differences in the domestic sewage composition were anticipated. Therefore, for practical reasons the identification of specific locations was largely restricted to an area in North Zealand where the consultant had long working experience within the sector including collaboration with the local authorities together with whom the specific locations were then selected. One location in Jutland from Part A of the survey was, however, re-selected for the sampling at the very outlet from the tank while the other samples were taken in inspection wells at a relatively short distance from the tanks (sampling directly in the outlet would have required re-configuration of the system). Only tanks discharging into tight pipelines and not into porous field drains were selected for sampling to avoid possible drying out during the summer period. 2.2.3 Field drainsPlanning of sampling in field drains within a limited time period is extremely difficult since the flow in the drains depend not only on the local soil and hydro-geological conditions but also strongly on the actual weather conditions, in particular the frequency, intensity and duration of rain episodes. Field drains of relevance for this study should largely be rain-fed to enable detection of steroid estrogens percolating through the topsoil and into the drains. However, such drains are likely to dry out already during the spring and usually flow does then not re-appear until late in the autumn. On the other hand manure or sludge cannot be applied before the fields are dry enough to allow traffic with heavy vehicles. Therefore, in a study like this with just a few months available for the field activities, only a narrow "window" is open for obtaining the required samples. Originally, it was planned to sample for a relatively short period at four locations for each type of waste material (manure and sludge). However, following consultations with the Geological Survey of Denmark and Greenland (GEUS), and learning from their experience with operating field drain sampling stations established with the aim to develop an early warning system for pesticide leaching (VAP), it was decided rather to select only two locations of each type and monitor each of them for a somewhat longer period (3 weeks). The monitoring should start shortly after application of the manure/sludge in order to catch possible "first flush" events caused by leaching through different types of macropores. The drains for sampling from manure amended field were selected in collaboration with the counties responsible for the environmental monitoring in the so-called "LOOP" catchment areas (a number of particularly well described small catchments under the national water environment surveillance programme), as it was considered important to work in areas with well described physical environments in this particular part of the study. Unfortunately, it was not possible to locate sewage sludge amended fields in any of the LOOP areas, and therefore contact was made to a company distributing sewage sludge to farmers who suggested possible locations. Based on farmer interviews a presumably suitable location for sampling was selected. 2.2.4 Follow-up on Part A samplesIn Part A of the survey relatively high levels of estrogenic activity (up to 6.2 ng E2 equiv./L) were observed in a forest lake selected as reference lake (i.e. no known contamination sources). A confirmatory biological test of the stored "bank" samples as well as a renewed spot sampling at the location was performed upon request from DEPA. One bank sample was also analysed chemically. Likewise, DEPA requested a confirmatory analysis of the bank sample plus renewed (spot) sampling and testing at a field drain location (sludge amended field) which was found in Part A to exhibit inexplicably high estrogen activity (up to 36 ng E2-equiv./L). Finally, DEPA requested three sediment samples (one upstream and two downstream) to be taken in receiving streams from category C WWTPs at two locations. It was agreed with DEPA that these two locations could be selected among the locations to be identified for the sampling programme for WWTP effluents and receiving waters described in Section 2.2.1. 2.3 Sampling programme and methodologyThe strategy for and process of selecting the different sampling locations was described in the preceding section and in Figure 2-1 the locations of the individual stations within each category are shown. The sampling programme can be briefly described as follows:
Figure 2-1 Location of sampling sites in Part B of the survey of estrogenic activity in the Danish aquatic environment. The spot samples were taken by the procedure known as "qualified spot sampling" (combined sample of five sub-samples) as described in part A section 3.1. For description of on-site conservation, storage and pre-treatment of aqueous samples, please see Part A sections 3.1 and 3.2.1. The sediment samples were not preserved on-site but were frozen immediately upon arrival to the laboratory and stored at -20 °C until work-up and subsequent testing/analysis. An overview of the number of samples taken within each category as well as the associated numbers of biological tests and chemical analyses is given in Table 2-1 below. Table 2-1 Overview of programme for sampling of the different sample categories and the associated biological testing and chemical analysis.
* = number of locations x number of samples per location. The sampling was carried out in the following sequential order:
2.4 Biological testing and chemical analysisIn Part A both the estrogenic activity from steroid estrogens in their free form (the active form) and the potential total activity of the free plus the conjugated forms (glucoronides and sulphates) were tested (by applying an enzymatic de-conjugation step in the sample pre-treatment to convert the conjugated estrogens into their free form). It was, however, a general observation in Part A that most of the estrogenic activity in the different samples was due to the estrogens in their free form whereas the conjugated forms only accounted for a minor fraction of the potential total activity. This was observed both in the biological test and in the chemical analysis. Therefore, it was decided to test and analyse only the free forms of the estrogens in Part B of the survey and, hence, the results to be presented in Chapter 3 of this report only regard the free estrogens. 2.4.1 Selection of samples for biological testing and chemical analysisThe number of samples within the different sample categories that were tested biologically and/or analysed chemically in Part B are stated in Table 2-1. Sampling, testing and analysis of WWTP effluents were not included in the original programme. However, such testing/analysis was found to be highly relevant in order to enable interpretation of the results from the receiving stream samples, and was made possible as the number of field drain samples taken turned out to be lower than originally planned. All samples were tested biologically while only selected samples were analysed chemically. Only in the case of sediments all samples were also analysed chemically. For all other samples the selection of samples for chemical analysis was not made until the results of the biological testing was available. Selection of samples for chemical analysis: WWTP effluents: Two locations showing no estrogenic activity in the biological assay were selected for chemical analysis to confirm this result. WWTP receiving streams: The downstream 1 samples. WWTP receiving stream sediments: All samples. Septic tank effluents: The sample at each location exhibiting the highest estrogenic activity. Field drains (manure + sludge): The first sample in each series + the single sample in which estrogenic activity above the detection limit was found (Løsning, week 2, 2. sample). Part A samples: The renewed sample from the field drain at Blommenslyst. 2.4.2 Methodology - Aqueous samplesThe methodologies and procedures applied in Part B for pre-treatment, biological testing and chemical analysis of aqueous samples were identical to the methodologies and procedures applied in Part A of the survey. This means that the samples were cleaned up by solid phase extraction (SPE) using C18-cartridges (+ silica gel for the sub-samples for chemical analysis), and subsequently tested biologically by the in vitro yeast cell assay known as the YES-assay and/or analysed chemically by GC-MS/MS after derivatisation with a mixture of MSDTFA, TMSI and DTE. The methodologies and procedures are described more detailed in the Part A report, section 3.2 and appendices referred to in that section. 2.4.3 Methodology - Sediment samplesTesting and analysis of sediments were not included in Part A and therefore the applied method for sample preparation is described in detail below: A sediment sample of approximately 30 g was dried at 25 °C. For each analysis a sub-sample of approximately 5.0 g dried sediment was weighed out and mixed with 20 g Ottawa sand. Internal standards, deuteriated E1 and E2 (20 ng per sample), were added through the methanolic stock solution. The ASE-extraction cells for the pressurised liquid extraction (PLE) applied were lined with two cellulose filters in the bottom. Extraction was performed using methanol/acetone (1:1; v/v) and the following parameters: preheat (0 min); heat (5 min); static (5 min); flush (60%); purge (60 sec); cycles (2); pressure (500 psi); temperature (100°C). The ASE extract was evaporated to dryness under a stream of nitrogen and reconstituted in 5 ml acetonitrile/MilliQ-water (2:3, v/v). The sample was then centrifuged at 3000 rpm for 3 minutes. The supernatant was transferred to a 250 ml blue-cap bottle, while another 5 ml acetonitrile/MilliQ-water (2:3, v/v) was added to the precipitate. The sample was mixed and centrifuged, and the supernatant again transferred to the same blue-cap bottle. The procedure was repeated once more. The combined extract in the blue-cap bottle was diluted with 150 ml MilliQ-water resulting in a MeCN content below 5%, which is required for the subsequent SPE (C18) procedure. pH was adjusted to pH =3. The methodology for the subsequent biological testing and chemical analysis of sediment samples was identical to the one described above for the aqueous samples. 2.4.4 Detection and quantification limits2.4.4.1 Biological assayThe detection limit in the YES assay was approximately 0.05 ng per litre in water samples and 100 to 200 ng per kg dry weight sediment (assuming that the absolute number of picogrammes (pg) estrogenic activity detectable are identical in the water and the sediment samples). 2.4.4.2 Chemical analysisIn part A an experimentally generated general LOD for the chemical analyses was determined to 0.1 ng E2 equiv./L for each component. In this determination, tap water was used instead of real samples (i.e. sewage effluent or surface waters) though, obviously, more analytical problems due to effects from the matrix could be expected if real samples were analysed. However, by comparing chromatograms from sewage effluent and surface water with those obtained with tap water, matrix effects generally appeared to have only minor importance and therefore the LOD in Part B is also assessed to be 0.1 ng E2 equiv./L. There were, however, situations where such problems occurred and resulted in elevated detection limits. LOD for the sediment chemical analysis was determined to 25 ng/kg dry sediment. This was based on the same determination on tap water used as explained above. Also here chromatograms from sediment were compared with tap water and again matrix effects generally appeared to be negligible.
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