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Survey of Estrogenic Activity in the Danish Aquatic Environment Part B

4 Discussion

• 4.1 Study design and methodology
  • 4.1.1 Study design
  • 4.1.2 Methodology
• 4.2 WWTPs and receiving streams
  • 4.2.1 Estrogenic activity in effluents and streams
  • 4.2.2 Estrogenic activity in sediment
  • 4.2.3 Possibilities of upgrading existing small WWTPs
• 4.3 Septic tanks
• 4.4 Field drains
• 4.5 Part A follow-up

4.1 Study design and methodology

4.1.1 Study design

Part B of the survey of estrogenic activity in the aquatic environment in Denmark was designed based on the results and experience obtained in Part A of the study as reported in Environmental Project 977 (DEPA, 2005).

However, while Part A was intended and designed to provide a broad impression of the "typical" situation with regard to both the state of the environment and the possible sources of contamination, Part B was initiated with the aim to focus specifically on two main aspects:

• investigate the state of vulnerable water bodies (i.e. small streams) receiving effluents from WWTPs with limited ability to remove steroid estrogens (category C and D plants cf. Part A, section 2.2.1), and
• further elucidate the level of estrogenic activity in two categories of contamination sources for which only insufficient data were obtained in Part A i.e. septic tank effluents and drainage water from field drains where manure or sludge had been applied.

4.1.1.1 WWTPs and small streams

Regarding the WWTP and associated streams, the main difference between Part A and Part B in the process of selecting the specific sampling locations was on the "stream side". While in Part A the WWTPs were selected with rather little emphasis on the size of the water body receiving the treated effluent it has been a central requirement in Part B that the effluent volume should be significant relative to the flow in the receiving stream.

In practice this implied that the search for suitable WWTPs was delimited to plants larger than 100 PE. At the same time it was attempted to find locations where the dilution of the effluent upon discharge into the stream would be less than 10 times of the effluent. Further, the sampling was carried out in July-August, which is the time of year when the stream flows are typically lowest.

The selection of sampling sites was carried out as a desk exercise using available GIS and internet information combined with interviews with local authorities and operational staff at the WWTPs or their municipalities. However, very little specific data about the stream flows was available.

Despite this, at five of the six selected locations it was estimated that the dilution was 5-8 times at the time of sampling and only at one (Mygdal) the dilution probably exceeded 10 times. Assuming estrogenic activity levels comparable to those observed in similar WWTP effluents in Part A, the activity level in the downstream water samples should be clearly above the quantification limit.

The WWTPs themselves were, similar to Part A, identified based on the information available in the counties' registrations and they are believed to be fairly representative of plants within the selected categories (C and D, though in reality only C was represented in the final selection of plants). However, there do exist, in particular in Category D, quite many WWTPs that are significantly smaller (fewer PE) than the selected ones and that also differ from these by not discharging continuously.

4.1.1.2 Septic tanks

It was assessed that the geographic variation in the composition of domestic sewage would be limited and therefore no attempt to distribute the required five locations for sampling of septic tank effluents over different regions. One suitable location in Jutland was already identified in Part A while the four others were identified within a small area in northern Zealand.

Four of the five locations have only one household connected to the septic tank system while at the last, Karlebo, seven households are connected. The latter represents a spectrum of households including one family in which there was a pregnancy at the time of sampling. The selected locations are, based on the information available, assessed to be representative of septic tank effluents nationwide.

4.1.1.3 Field drains

All the field drains locations are situated in the eastern half of Jutland, and all on soils with a moderate content of clay (10-15%; soil classes JB5-JB6), which is fairly typical of Danish agricultural soils. All the selected drains are largely rain-fed and normally run dry during summer.

To the extent possible the fields were selected within a number of small catchment areas, which under the national water and environment monitoring system, NOVANA, are designated for monitoring and evaluation of nutrient and contaminant releases to the freshwater environment. The three manure amended fields are situated in such an area while a suitable sludge amended field could only be found at another location.

4.1.2 Methodology

The sampling procedures and methodologies of biological testing and chemical analysis were identical to the ones applied in Part A. However, as sediments were not analysed in Part A the methodology applied for this purpose was new (within the framework of the study). Only free estrogens were tested/analysed in Part B because the conjugated forms were shown in Part A to account only for a minor part of the overall estrogenic activity. Further, in sediments conjugated estrogens are not expected to sorb to sediments due to their higher water solubility than the free estrogens.

The laboratory procedures and methodologies were tested and documented thoroughly in Part A of the survey (DEPA 2005), but also in Part B all work-up, tests and analyses of real samples were accompanied by blank samples and samples with standard addition of steroid estrogens to have a continuous control of possible background contamination and/or malfunction of the equipment and instruments.

As no problems with background contamination or lack of precision or sensitivity was observed when running the blank and standard samples the results of the real samples are considered to be valid.

4.2 WWTPs and receiving streams

4.2.1 Estrogenic activity in effluents and streams

The results of the biological testing and chemical analysis of WWTPs and associated stream samples including sediment are presented in chapter 3, Tables 3-2 and 3-3.

It appears from Table 3-1 that a significant biological estrogenic activity was observed in the effluent from Tisted WWTP (about 20 ng E2-equiv./L) while a somewhat lower activity was found in the effluent from Moseby (about 4 ng E2-equiv./L).

In the effluents at the four remaining WWTPs the activity was below the detection limit. However, by chemical analysis a very low activity could be observed in two of the effluents where the biological assay did not give any response.

The apparent lack of estrogenic activity was unexpected as in Part A the effluents from the same categories of WWTPs all showed significant activity (see Table 4-1, which summarises the WWTP effluent results in Part A). Only the result from Tisted is at a level that is comparable with those found for C/D plants in Part A.

It has only been possible to find a likely explanation of the lacking estrogenic activity at two of the WWTPs, Mygdal and Tjele-Hammershøj, at which we have been informed that the retention time is high (> 2 days).

There are no indications of problems with field or laboratory procedures and follow-up enquiries to the other WWTPs have not revealed any atypical conditions. All samplings took place in dry weather and, hence, dilution by rainwater cannot be an explanation either.

Table 4-1
Summary of tables 6-3 and 6.4 in Part A of the survey: Median and max. content of free estrogenic activity in the effluents from wastewater treatment plants in categories A-F. Measured biologically and chemically and presented as ng Estradiol equivalents/L. N, the number of samples, is separated into values <LOD, values >LOD, and all values.

In accordance with the lacking estrogenic activity in the effluents none or only little and sporadic activity could be detected in the stream samples - not even in Skibsted Å, the stream receiving the discharge from Tisted WWTP, although the effluent volume in this case was rather significant relative to the stream flow at the time of sampling (ratio roughly estimated to about 1 : 5).

In summary, the tests and analyses of stream water downstream the discharge points of small WWTPs have not, neither in Part A or B (results from a total of 18 category C/D plants), given any indication that such discharges significantly increase the estrogenic activity in the streams though a number of small increases (and one significant) were observed in Part A of the survey.

4.2.2 Estrogenic activity in sediment

All sediments demonstrated significant contents of steroid estrogens when analysed chemically and about half of the samples also gave a response in the biological assay. In the majority of samples wihout a biological response the chemically determined contents were below the detection limit of the YES assay (approximately 100 ng E2 equiv./kg dw).

The levels range from 30-1740 ng E2 equiv./kg dw in the chemical analysis and from <100-1160 ng E2 equiv./kg dw (7 out of 10 samples showed estrogenic activity) in the bioassay i.e. a significant enrichment compared to the levels in the stream water. The highest levels were found downstream of the Tisted WWTP discharge point and significant contents were also found in Lyremoseløbet, the stream receiving the effluent from Moseby WWTP.

These results demonstrate that despite the absence of activity in the water samples the WWTPs obviously release estrogens that, due to their lipofilicity (log P = approx. 3.5) when appearing on the free form, tend to sorb to particulates (organic matter) and precipitate in sedimentation sections of the streams. Further, the results indicate that enrichment of sediment with steroid estrogens occur downstream of category C treatment plants.

A comparison of the chemical and biological results shows that assessment of estrogenic activity in sediments only by use of a biological test in some cases may be insufficient due to poorer sensitivity of the latter. 30% of the samples showed no activity in the bioassay while activity was observed in all samples when analysed chemically.

The levels of estrogens found in Danish sediments (see Table 3-4) are comparable to or lower than levels found in other European countries. A Spanish study using chemical analysis showed that in river sediment from the Catalonia region much higher levels were found than in Denmark. E1 was found at the level of 11,900 ng/kg dw and EE2 at 22,400 ng/kg dw. In the present study (i.e. Part B) E1 was found at between 100 and 3,180 ng/kg dw and EE2 between 170 and 440 ng/l (3 out of 10 samples). Thus, the values found in Denmark are roughly a factor 10 lower than those from Spain

In Germany, levels similar to the Danish were found of E1 (up to 2,000 ng/kg dw) and EE2 (900 ng/kg dw) in samples of sediment from a polluted river, the River Rhine (Ternes et al. 2002).

The biological/ecological significance of such sediment levels is at present not possible to assess.

4.2.3 Possibilities of upgrading existing small WWTPs

For assessment of the possibilities of upgrading the existing small WWTPs for better removal rates of estrogenic substances, it is essential to 1) assess the mechanisms in wastewater treatment plants that influence the treatment performance and 2) specify the type and process configurations of the plants that shall be upgraded.

Based on previous investigations, it is assessed that the lower removal rates for estrogenic substances for small treatment plants (MB plants) compared to bigger (MBN and MBND plants) are associated with one or more of the following circumstances:

1. Low sludge age.
   Low sludge age in a wastewater is a disadvantage for slowly growing bacteria, which will be washed-out and hence it will not be possible to sustain some specific bacteria in the biological treatment units. This is a phenomenon that is very essential to bear in mind when designing a WWTP. For nitrifying treatment plants, for instance, it is essential to have a high sludge age, i.e. minimum 15-20 days for Danish conditions, in order maintain nitrifying bacteria in the plant. A similar mechanism might be relevant for removal of estrogenic substances, i.e. bacteria with high potential for decomposing estrogenic substances might be washed out of the biological treatment plant, if the sludge age is too low.
   
2. Low hydraulic retention time.
   Estrogenic substances can be regarded as moderately biodegradable organic matter, meaning that a certain minimum retention time in the process tank is necessary for a high degree of decomposition of the estrogenic substances. For small mechanical-biological treatment plants the hydraulic retention time in the process tank is typically around 4-8 hours, whereas average retention time in bigger MBND plants is close to one day (24 hours). Furthermore, the variation in incoming wastewater flow is bigger for small plants and therefore the retention time in peak hours is less.
3. Poor removal rate for suspended solids.
   Steroid estrogens in their free form tend, when discharged from wastewater treatment plants, to be attached to the suspended solids.
   A poor removal of suspended solids will hence have a direct influence on the removal rates for estrogens. Small treatment plants have, in general, poorer treatment results for suspended solids due to less monitoring and control systems and generally lower attention.
   

Low sludge age and low hydraulic retention time are, of course, to some degree linked together as both can be increased by construction of a bigger process tank at the treatment plant.

Most existing small WWTPs in Denmark of the category mechanical-biological plants (MB) are based on activated sludge technology, i.e. the treatment plants are constructed with the following process configuration:

• screens
• grit and grease chamber
• aerated activated sludge tank
• secondary clarifier
• sludge thickener

A minor part of the small treatment plants are based on fixed film technology, i.e. typically with a trickling filter still followed by a secondary clarifier.

Typically sludge is thickened at the treatment plant and transported to a central WWTP for mechanical dewatering. Some plants have, however, sludge drying beds for final dewatering.

Mechanical plants (M), typically very small treatment plant, are constructed as large septic tanks followed by direct discharge to the recipient or through an infiltration system. Sludge from the plants are emptied regularly (1-2 times per year) and transported to a central WWTP for further treatment.

Based on the above assessments of mechanisms that influence the treatment performance and the type and process configurations of the small treatment plants, the following improvement possibilities of the treatment performance in relation to enhanced removal of estrogenic substances can be proposed:

1. Increase sludge age by:
   • increase process tank volume
   • increase sludge concentration in the existing process tank, if this is possible in relation to the capacity of the secondary clarifier
     
2. Increase hydraulic retention time by:
   • increase process tank volume
     
3. Improve removal of suspended solids by:
   • improve monitoring and control of secondary clarifiers (turbidity meter, sludge blanket level meter)
   • improve performance of secondary clarifiers (installation of lamellas, Flockbee, etc.)
   • increase secondary clarifier volume
   • installation of polishing unit (conventional sand filter, biological sand
     filter, lagoons, etc.)

In general, most of the above suggestions demand a relatively high investment, i.e. typically 20-50% of construction costs for a new treatment plant. Upgrading of the treatment plant by increasing of the process tank volume can typically also improve the treatment performance for ammonia removal (i.e. upgrading to MBN-plant).

Beside the above mentioned improvements, it is expected that a general optimization of the treatment plant operation also will have an effect on the removal rate for estrogenic substances. These optimizations could include improved control of sludge concentration and levels in the different treatment units, improved oxygen control, etc. Measures for improved control and monitoring can, however, be implemented for a substantially lower investment.

4.3 Septic tanks

Significant estrogenic activity was found in septic tank effluents at four out of five investigated locations (see Table 3-4), while at the fifth location the effluent demonstrated none or only relatively low activity. The significant activities are in line with the findings at similar locations in Part A while the low activity at the Gunderød location remains inexplicable.

At one location very high activity levels were observed, about 350-500 ng E2 equiv./L, maybe caused by a pregnancy at the time of sampling in one of the seven families connected to the system. Only in one effluent was the synthetic steroid estrogen EE2 present at a level higher than the limit of detection, namely 9.8 ng/L. This result is in accordance with Part A where EE2 was also only rarely detected in the samples.

The results show, as expected, that the content of steroid estrogens typically remains high in the undiluted sewage after having passed through a septic tank. Hence, if discharged directly into a stream, this type of effluent will require significant dilution to reduce the estrogenic activity to a level below those that in experimental studies have been shown to cause biological effects.

Normally, a sufficient dilution is expected to take place as the effluent volume will be small compared to the flow in most streams. During summer effluents discharged into field drains may even dry out before reaching the stream and not be discharged until late in the autumn where the flow is higher and where the majority of the estrogens have probably undergone degradation by micro-organisms.

4.4 Field drains

The drainage water from two fields where liquid manure (pig and cattle, respectively) had been applied and one field where municipal sewage sludge had been applied was monitored during a period of three weeks in the spring of 2005, while water from a fourth drain at a cattle manure amended field was monitored for one week. The manure and sludge application took place in dry weather around mid April while the monitoring was not initiated until around 1st of May as no significant rain episodes, which could provoke estrogen leaching, occurred until then.

The biological testing revealed no estrogenic activity in the drainage water from any of the locations, except a very low activity (0.1 ng E2 equiv./L) in a single sample from the pig manure amended field.

Chemical analysis was carried out on the samples from the first sampling round at each location plus of the sample where activity was observed in the bioassay. In two of the four "round 1" samples a low content of steroid estrogens was found (0.32 and 0.10 ng E2 equiv./L) and the biological observation of activity in one sample was confirmed by the chemical analysis (0.29 ng E2 equiv./L). In the two samples with the highest activity (as E2 equiv.), the main contribution came from EE2, which was not expected to appear in the samples.

Thus, the quite short monitoring of field drains at a few fields having received either liquid manure or sewage sludge has not given any indication that high levels of estrogenic activity might appear in this possible source of aquatic contamination. In Part A low levels of estrogenic activity was observed in a minor part of such field drains while high levels (up to 36 ng E2 equiv./L) were observed in only one drain - from a sludge amended field at Blommenslyst, which therefore was re-investigated in Part B.

The re-investigation confirmed the previous results but has provided no conclusion as to the cause of this. It is, however, considered unlikely that such high levels can occur as a result of leaching of sludge contaminants through soil. In the specific case it is even more unlikely as the sewage sludge was applied more than one year before the re-sampling of the location took place.

4.5 Part A follow-up

The follow-up on specific Part A samples comprised renewed sampling and testing of a reference lake, Sorte Sø, and of a drain at a field where sewage sludge was applied in the spring of 2005; Blommenslyst. Comments on the latter were given above.

Sorte Sø is a slightly acidic forest lake with an apparently high content of dissolved humic substances. There are no visible anthropogenic sources of contamination of the lake but nevertheless the lake water exhibits a higher than background estrogenic activity.

A confirmatory chemical analysis of one of the bank samples of Sorte Sø water from 2004 (Part A) was included in Part B of the survey. The result showed that half of the total estrogenic activity observed in 2004 could be explained by the (unexpected) presence of steroid estrogens in the lake water and thereby that a hitherto unknown (and not immediately visible) anthropogenic contamination source may exist in the vicinity of the lake.

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Version 1.0 March 2006, © Danish Environmental Protection Agency