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To carry out a field study to measure the discharge of steroid oestrogens from two STWs and the .... despite both operating activated sludge, treatment systems. .... steroid oestrogens entering river systems via sewage treatment works. ..... the River Aire at Riddlesden or at the River Thames sampling point (Table 2.1). As part ...
Fate and Behaviour of Steroid Oestrogens in Aquatic Systems

Research and Development Technical Report P2-162/TR

Fate and Behaviour of Steroid Oestrogens in Aquatic Systems R&D Technical Report P2-162/TR R J Williams, A C Johnson, J J L Smith, M D Jürgens and K Holthaus

Research Contractor: Centre for Ecology and Hydrology - Wallingford

CONTENTS

Page

LIST OF TABLES

iii

LIST OF FIGURES

V

EXECUTIVE SUMMARY

1

1.

INTRODUCTION

5

2.

LABORATORY STUDIES

6

2.1 2.2 2.3 2.4

Summary of results from previous scoping study Degradation studies Sorption studies Conclusions

6 6 24 35

3.

FIELD SURVEYS

36

3.1 3.2 3.3 3.4 3.5 3.6

Background, reason for work and selection of sites Site descriptions Sampling Methods Analytical methods Results and Discussion Conclusions

36 37 41 42 45 56

4.

MODELLING OF STEROID OESTROGENS IN RIVERS

58

4.1 4.2 4.3 4.4 4.5

Background EXAMS Modelling the River Nene and the River Lea with SIMCAT and TOMCAT Catchment-wide modelling using GREAT-ER Conclusions

58 59 70 77 81

5.

DISCUSSION

83

5.1 5.2 5.3 5.4 5.5 5.6

Background Sewage Treatment Works effluents In-river processes Wider implications of this work Towards risk assessment Conclusions

83 83 84 85 86 87

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Page 6.

RECOMMENDATIONS

88

6.1 6.2 6.3 6.4 6.5 6.6

Sewage Treatment Works effluents Fate and behaviour studies In-river concentrations Bed sediments Biological effects Catchment risk assessment

88 88 88 89 89 89

REFERENCES

90

APPENDICES APPENDIX A FIELD SURVEY RAW DATA

95

APPENDIX B ANALYTICAL QUALITY CONTROL DATA

109

APPENDIX C TOMCAT FILES FOR THE RIVER LEA AND RIVER NENE

113

APPENDIX D

120

SIMCAT FILES FOR THE RIVER LEA AND RIVER NENE

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Page LIST OF TABLES Table 2.1

Degradation rates for 100 µg/l 17β-oestradiol (E2) in river water at 20ºC and water properties

13

Low concentration 17β-oestradiol (E2) degradation in samples from the River Thames, Wallingford

17

Degradation of 17β-oestradiol (E2) in bed-sediments (room temperature, 20±2°C)

21

Properties and sorption/desorption partition coefficients (K d) for bed-sediments with 17β-oestradiol (E2) (l/kg)

30

Sorption and desorption partition coefficients (K d) for bed-sediments with 17α-ethinyloestradiol (EE2) (l/kg)

31

Table 2.6

Characteristics of water and suspended sediment samples

33

Table 2.7

Distribution coefficients (Kd) and standard errors (SE) for 17βoestradiol (E2) and 17α-ethinyloestradiol (EE2) and suspended sediments

34

Table 3.1

Summary descriptions of the sampling sites on the River Nene

38

Table 3.2

Summary descriptions of the sampling sites on the River Lea

40

Table 3.3

Oestrone (E1) and Total organic carbon (TOC) concentrations in bed-sediment and water from the River Nene, with associated Kd values.

52

Oestrone and total organic carbon (TOC) concentrations in bed sediments and water from the River Lea, and associated Kd values.

56

Dimensions of the segments used in the EXAMS model of the River Nene (L refers to the water column and B to the bed sediments).

62

Calculated steroid oestrogen loads (kg/hour) used in the simulation of the River Nene with the EXAMS model.

62

Physico-chemical properties of the steroid oestrogens used in the initial EXAMS modelling

63

Velocities and volumes used for the water column segments in the EXAMS model of the River Lea.

63

Calculated steroid oestrogen loads (kg/hour) used in the simulation of the River Lea with the EXAMS model.

64

Non-parametric distributions for flow, boron and oestrone (E1) for the input flows to the TOMCAT model for the River Nene.

71

Non-parametric distributions for flow, boron and oestrone (E1) for the input flows to the SIMCAT model for the River Nene.

72

Table 2.2 Table 2.3 Table 2.4 Table 2.5

Table 3.4 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Table 4.6 Table 4.7

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Page Table 4.8

Non-parametric distributions used for the upstream flow and discharges entering the TOMCAT model for the River Lea.

73

Non-parametric distributions used for the upstream flow and discharges entering the SIMCAT model for the River Lea.

74

Table A1

Results of preliminary survey of Great Billing STW (20th July 2000)

96

Table A2

Concentrations of oestrone (ng/l) measured in the River Nene during the main survey (August 2000)

97

Concentrations of 17β-oestradiol (ng/l) measured in the River Nene during the main survey (August 2000)

98

Concentrations of 17α-ethinyloestradiol (ng/l) measured in the River Nene during the main survey (August 2000)

99

Concentrations of suspended sediment (mg/l) measured in the River Nene during the main survey (August 2000)

100

Concentrations of dissolved organic carbon (mg/l) measured in the River Nene during the main survey (August 2000)

101

Concentrations of boron (mg/l) measured in the River Nene during the main survey (August 2000)

102

Results of analysis of bed-sediments taken from the River Nene during the main survey (August 2000)

103

Results of preliminary survey of East Hyde and Harpenden STWs (4 September 2000)

104

Concentrations of boron measured in the preliminary survey of the River Lea (4 September 2000)

104

Concentrations of oestrone (ng/l) measured in the River Lea during the main survey (October 2000)

105

Concentrations of 17β-oestradiol (ng/l) measured in the River Lea during the main survey (October 2000)

105

Concentrations of 17α-ethinyloestradiol (ng/l) measured in the River Lea during the main survey (October 2000)

106

Concentrations of suspended sediment (mg/l) measured in the River Lea during the main survey (October 2000)

106

Concentrations of dissolved organic carbon (mg/l) measured in the River Lea during the main survey (October 2000)

107

Concentrations of boron (mg/l) measured in the River Lea during the main survey (October 2000)

107

Results of analysis of bed-sediments taken from the River Lea during the main survey (October 2000)

108

Table 4.9

Table A3 Table A4 Table A5 Table A6 Table A7 Table A8 Table A9 Table A10 Table A11 Table A12 Table A13 Table A14 Table A15 Table A16 Table A17

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Page

LIST OF FIGURES Figure 2.1 Figure 2.2

Figure 2.3 Figure 2.4

Locations of sampling sites on the River Aire, River Calder and River Thames

10

Aerobic degradation of 17β-oestradiol (E2) (100 µg/l) and formation and subsequent degradation of oestrone (E1) in river water from the River Aire at Beal (10 Dec ‘99). Error bars are +/- one standard deviation of three replicate samples. E2 was poorly dissolved in the water at the beginning of the incubation, therefore its concentration lower than the target value (100 µg/l).

11

Half-lives of 17β-oestradiol (E2) in water samples collected from five sites on four occasions in 1999 and 2000.

11

Degradation of 17α-ethinyloestradiol (EE2) compared to 17βoestradiol (E2) in River Thames water. Error bars are +/- one standard deviation of three replicate samples

12

Figure 2.5A Aerobic degradation of 17β-oestradiol (E2) (100 µg/l and 100ng/l) in River Thames water (18 Jan 2000). Error bars are +/- one standard deviation of three replicate samples.

16

Figure 2.5B Evolution of oestrone (E1) from 17β-oestradiol (E2) (100 µg/l and 100 ng/l) in River Thames water (18 Jan 2000). Error bars are +/one standard deviation of three replicate samples.

17

Figure 2.6

Photolytic degradation of 17β-oestradiol (E2) and 17α-ethinyl oestradiol (EE2) in water from the River Thames

19

Anaerobic degradation of 17β-oestradiol (E2) (500 µg/L) in River Thames (Wallingford) bed-sediments. Error bars are +/- one standard deviation of three replicate samples.

20

Percentage mineralisation of 17β-oestradiol (E2) in river water measured by evolution of 14 C. Error bars are +/- one standard deviation of three replicate samples.

23

Loss of 17β-oestradiol (E2) and formation and loss of oestrone (E1) correlated with overall oestrogenicity measured by the YES assay for river water collected from the River Thames. E2 was poorly dissolved in the solution at time zero, hence there is an initial rise in its concentration as it dissolves completely.

24

Figure 2.10 Sorption kinetics of 17β-oestradiol (E2) with bed-sediment samples from the River Aire and River Thames

28

Figure 3.1

Locations of sampling sites on the River Nene

38

Figure 3.2

Locations of sampling sites on the River Lea

40

Figure 2.7

Figure 2.8

Figure 2.9

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Page Figure 3.3

Figure 3.4

Figure 3.5 Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Concentrations of oestrone (E1), 17β-oestradiol (E2), suspended sediment and Dissolved Organic Carbon in the final effluent of Great Billing STW on 20 July 2000. Values below the detection limit (1ng/l or 1mg/l) were plotted at half of that value.

46

Concentrations of 17β-oestradiol (E2), suspended sediment and Dissolved Organic Carbon in the final effluent of East Hyde STW on 4 September 2000. Values below the detection limit (1 ng/l or 1 mg/l) were plotted at half of that value.

47

Flow rates in the River Nene and the Great Billing final effluent over the period of the oestrogen survey.

48

Concentrations of oestrone (E1) and 17β-oestradiol (E2) in the effluent from Great Billing during the 28-day survey. Values of E2 below the detection limit are plotted at half the value (i.e. 0.5 ng/l)

49

Mean concentrations of oestrone (E1) and 17β-oestradiol (E2) in the River Nene around Great Billing STW. The error bars are set at ± 2 standard errors. Concentrations below the detection limit were included in the calculation of the means as a value of half the detection limit (i.e. 0.5 ng/l)

50

Comparison of observed data from the River Nene with estimates of expected oestrone (E1) concentrations arising from dilution effects alone.

51

Flow rates in the River Lea upstream and downstream of East Hyde Sewage Treatment Works effluent discharge during the study period.

53

Figure 3.10 Concentrations of oestrone (E1) and 17β-oestradiol (E2) in the effluent from Harpenden STW during the first 10 days of the followon survey. Values of E2 below the detection limit are plotted at half the value (i.e. 0.5 ng/l).

54

Figure 3.11 Mean concentrations of oestrone (E1) and 17β-oestradiol (E2) in the River Lea around East Hyde and Harpenden STWs. The error bars are set at ± 2 standard errors. Concentrations below the detection limit were included in the calculation of the means as a value of half the detection limit (i.e. 0.5 ng/l)

55

Figure 4.1

Figure 4.2 Figure 4.3 Figure 4.4

Illustration of the arrangement of water column and bed-sediment compartments used within the EXAMS model. The arrows indicate the direction of movement of the compound modelled.

59

Simulation of mean oestrone (E1) concentrations down the River Nene using EXAMS.

64

Simulation of mean 17β-oestradiol (E2) concentrations down the River Nene using EXAMS.

65

Simulation of mean oestrone (E1) concentrations down the River Lea using EXAMS using a range of model parameters.

66

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Page Figure 4.5 Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Simulation of mean 17β-oestradiol (E2) concentrations down the River Lea using EXAMS

67

Simulation of mean oestrone (E1) concentrations down the River Nene using EXAMS. The two lines show the simulation assuming degradation in the water column only (half-life 1.5 days) and the simulation including biodegradation (half-life 1.7 days), photodegradation (half- life 15 days) and sorption (K d = 92 l/kg).

69

Simulation of oestrone (E1) in the River Lea using degradation in the water column only. The half-life of 1.5 days was the value used for the River Nene.

69

Observed (-O) and simulated oestrone (E1) and boron concentrations in the River Nene around GT Billing STW. TC and SC stand for TOMCAT and SIMCAT simulations respectively.

75

Observed and simulated boron concentrations in the River Lea. (Where only one line is visible simulations are identical.)

75

Figure 4.10 Comparison of modelled oestrogen concentrations generated by the SIMCAT (SC) and TOMCAT (TC) models (using two degradation half-lives) with mean observed oestrone (E1) concentrations along the River Lea.

76

Figure 4.11 Predicted mean concentrations of 17β-oestradiol (E2) in the effluents from the STWs in the Aire and Calder Catchments using the GREAT-ER model. The size of the dot increases with the predicted mean discharge.

78

Figure 4.12 Predicted mean concentrations of oestrone (E1) in the effluents from the STWs in the Aire and Calder Catchments using the GREAT-ER model. The size of the dot increases with the predicted mean discharge.

79

Figure 4.13 Predicted mean 17β-oestradiol (E2) concentrations in the Rivers Aire and Calder using the GEAT-ER model. The colour of the river section indicates the predicted concentration (see scale).

80

Figure 4.14 Predicted mean oestrone (E1) concentrations in the Rivers Aire and Calder using the GEAT-ER model. The colour of the river section indicates the predicted concentration (see scale).

81

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EXECUTIVE SUMMARY From previous work in the UK, there is persuasive evidence showing oestrogenic (feminising) endocrine disruption of fish living downstream of sewage treatment works (STW), in particular the observation of intersex male roach. The most oestrogenic fraction (in-vitro) of these effluents has been isolated and three chemicals have been identified in this fraction: 17β-oestradiol (E2), oestrone (E1), both naturally excreted hormones, and ethinyloestradiol (EE2), a synthetic hormone present in the contraceptive pill and other therapies. A scoping study funded by the Environment Agency found that physico-chemical data on these substances in river systems was very limited. Some laboratory and preliminary modelling studies were presented in the scoping study report and recommendations for further work were made. This study takes forward these recommendations and has the following objectives: 1. To provide further data on the degradation (see Box 2.1) and sorption (see Box 2.2) of E1, E2 and EE2 within river systems; 2. To carry out a field study to measure the discharge of steroid oestrogens from two STWs and the subsequent concentrations at a number of downstream sites in the receiving waters; 3. To use the laboratory data and field data within process models to understand the fate and behaviour of these steroid oestrogens in river systems. In addition, to suggest methods that could be used by the Environment Agency in risk assessments of STW effluent concentrations containing steroid oestrogens. To address the first objective, these studies were carried out using material (water, bedsediments or suspended sediments) from three rivers: the Thames, Aire and Calder. The location on the River Thames essentially represented a rural/agricultural area whereas both the River Aire and River Calder drain urban/industrial areas. The experiments determined properties of E2, E1 and EE2 that describe the potential of these steroid oestrogens to bind to sediments or undergo degradation within the river system. All water samples (which were not filtered to remove any suspended sediment) were shown to biodegrade E2 rapidly to form E1 in river water. The degradation half-lives for E2 varied from 0.2 to 8.7 days and from 0.1 to 10.9 days for E1 (see Box 2.1, Section 2). The most rapid biodegradation of E2 was associated with the downstream urban/industrial river stretches of the River Aire and River Calder. E2 was more persistent upstream of the major STW effluent discharges. This suggests that the E2 degradation rate could be related to the overall microbial activity of a river reach. Degradation of E1 also occurred following its formation from E2, although at a slightly slower rate: over all 25 separately collected water samples, the mean half-life for E1 was 3.0 days compared to 2.8 days for E2. E2 could be completely mineralized in river water, although this was slow with only 20-40% of the carbon converted to CO2 after 35 days (dependant on which river water was used). EE2 was much harder to degrade in river water and showed a half-life approximately 10 times that of E2. The degradation rate, in conjunction with the river residence time, will determine the exposure levels downstream of a STW discharge. Although degradation of the steroid oestrogen is of interest in terms of describing its fate in the environment, the environmental effect depends on when the oestrogenic effect is lost.

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Oestrogenic activity was tested using the yeast oestrogen-screen and was shown to be lost following the degradation of E2 and its metabolite E1. This suggests that the degradation products of E1 are not particularly oestrogenic. Anaerobic degradation was tested using bed sediments from the River Calder and River Thames. E2 degraded rapidly to E1 (half life ~8 – 16 hours) but E1 showed no reduction in concentration over a period of 48 hours, suggesting E1 might be persistent under these conditions although more work is required to confirm this hypothesis. Both E2 and EE2 degraded slowly under simulated continuous bright daylight conditions with degradation halflives of ~ 120 hours. Potentially, sorption to bed- and suspended sediments could be an important removal route for steroid oestrogens from the water column (see Box 2.2, Section 2). Studies using natural sediments from the River Thames, River Aire and River Calder showed that E2 and EE2 would sorb to both bed- and suspended sediments. EE2 showed higher partition coefficient values (Kds ranging from 8 – 121 l/kg) than E2 (K ds from 4 – 72 l/kg) by around 50%. Suspended sediments seemed to be slightly more attractive sorbents than bed sediments with Kd values ranging from 21 – 159 l/kg for E2 and 19 – 260 l/kg for EE2. For bed sediments, higher values of Kd were associated with high silt contents and low sand contents, indicating a surface area effect for sorption. No relationships were found between distribution coefficients for suspended sediments and sediment properties. Two field surveys were carried out to assess the concentrations of steroid oestrogens in the River Nene and the River Lea, which both received effluent from STWs known to contain steroid oestrogens. These data represent the first systematic assessment of the concentrations of steroid oestrogens in rivers downstream of a STW in Europe. The 12 km stretch of the River Nene downstream of Northampton runs through farmland and is used extensively for leisure pursuits (fishing, canoeing, narrow boat holidays). There are a number of small towns close to river along the study stretch, which finishes at Wellingborough. The 11 km stretch of the River Lea also runs through an essentially rural landscape, but there are significant reaches flowing through the towns of Harpenden and Wheathampstead. At both field sites, E1 was the dominant steroid oestrogen in STW effluent with concentrations ranging from 0.8 – 10.2 ng/l for Great Billing STW (River Nene), 0.7 for both E2 and EE2 and the bed-sediments). Weaker positive correlations were found for clay content (R2 = 0.5 for E2 and 0.7 for EE2) and organic carbon content (R2 =0.5 for E2 and 0.6 for EE2). Therefore E2 and EE2 were more significantly attracted to particularly fine bed-sediments than to those with a high organic carbon content. This contrasts with Lai et al. (2000) who found good correlations with TOC but less so with particle size, when looking at the sorption of 5 steroid estrogens to 5 different bed-sediments. They also showed however that organic carbon is not a prerequisite for estrogen sorption by demonstrating sorption of estrogens to pure iron oxide. It is of course difficult to clearly distinguish between TOC and particle size, because the two are intimately related in natural systems. From the Kd and the TOC data, the organic carbon normalized partition coefficient (K oc) was calculated for E2 (Table 2.4), but given the relatively poor R2 correlation with TOC, must be treated with caution. Using the relationship between Kow and Koc, described by Di Toro et al. (1991), a Koc value of around 1200 l/kg might be expected for E2. Table 2.4 shows values between 612-2645 l/kg, which is within the suggested uncertainty range of a factor of 2 or 3 (Di Toro et al., 1991). The more hydrophobic compound EE2 was as expected more attracted to organic carbon than E2 with Koc values between 1453-5124 l/kg (Table 2.5).

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Properties and sorption/desorption partition coefficients (Kd) for bed-sediments with 17β -oestradiol (E2) (l/kg)

% Smectite

% TOC

50 20

30 65

20 15

2.4 7.0

Calder 06/09/96 M. Bridge 16/01/97 18/07/97

3.0 19.0 77.0 6.60 55.0 38.4 6.82 49.8 43.4

50 25 60

40 65 30

10 10 10

0.1 5.7 3.3

n.d. 57 (1.6) 36 (1.4)

6.8 (0.2) 41 (0.8) 35 (0.6)

n.d. 980 1091

6800 718 1061

Thames W.ford

5.50 3.0 4.80 6.00 6.20 7.70 9.40

10 10 10 40 55 60 55

10 10 10 15 40 35 40

80 80 80 45 5 5 5

1.8 0.1 2.9 1.1 2.0 0.9 3.7

16 (1.2) n.d. 51 (1.2) 20 (0.1) 34 (0.7) 20 (0.5) 50(2.8)

16 (0.4) 4.3 (0.1) 16 (0.4) 14 (0.3) n.d. n.d. n.d.

889 n.d. 1757 1818 1700 2222 1351

889 4300 552 1273 n.d. n.d. n.d.

Tees SS Tees BS Tyne

05/12/96 23/09/96 15/04/97 27/06/97 31/07/97 31/07/97 01/08/97

38.0 12.0 43.0 41.4 42.6 52.2 76.5

% Sand

9.20 72.9 17.9 7.40 62.8 29.8

Aire F.Weir

% Silt

06/09/96 18/12/96

96/97 72 (3.2) 45 (1.6)

Location Date

% Clay

% Illite

Sorption Kocb

% Kaolinite

Table 2.4

56.0 89.0 51.8 52.6 51.1 40.1 14.1

a

Sorption Kd (SE ) 96/97

a

standard error of the slope (ie of Kd ) organic carbon normalized partition coefficient c not determined b

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99/00 54 (1.3) 57 (1.1)

2645 612

99/00

Desorption (SE)

Kd2

96/97

99/20

2231 812

143 (3.3) n.d.

119 (2.6) 90 (2.3)

n.d. n.d. 80 (1.9)

n.d. 96 (2.1) n.d.

n.d. n.d. n.d. 53 (0.8) 86 (0.8) 38 (1.1) 132 (1.9)

38 (1.3) 11 (0.05) 70 (1.7) 48 (0.7) n.d. n.d. n.d.

Table 2.5

Sorption and desorption partition coefficients (Kd) for bed-sediments with 17β -ethinyloestradiol (EE2) (l/kg)

Location

Date

Aire

FW

Calder

MB

Thames

W.ford

06/09/96 18/12/96 06/09/96 16/01/97 18/07/97 05/12/96 23/09/96 15/04/97 27/06/97

Determined 1999/2000 after 3-4 years storage Desorption Sorption Kd (SEa ) Sorption Kocb Kd2 (SE) 121 102 12 110 108 41 8 67 22

(4.7) (3.8) (0.4) (5.0) (3.3) (1.7) (0.2) (1.7) (0.8)

5000 1453 10000 1926 3273 2330 10000 2326 2037

227 165 n.d.c 166 n.d. 69 29 104 32

(3.6) (6.0) (3.9) (1.3) (1.6) (2.0) (0.4)

a

Standard error of the slope (ie of Kd ) organic carbon normalized partition coefficient c not determined b

2.3.2

Potential for sorption of 17β -oestradiol (E2) and 17α -ethinyloestradiol (EE2) to suspended sediments

Along with bed-sediments, suspended sediments may also have a role to play in removing oestrogens from the aqueous phase by sorption. To understand the extent to which this occurs in different rivers/seasons, a range of experiments were carried out with different sediments. Materials and Methods Suspended matter from the River Aire, River Calder and River Thames was collected on site in spring, summer and winter 1999 and spring 2000 using a continuous flow centrifuge (SediSamp System II, model WSB/103-ENV) to concentrate the suspended sediments. The concentrated suspended sediments were stored for up to 4 days at 4°C. Instantaneous bulk samples of river water were collected in 1l flasks at the same points where suspended sediments were taken. Suspended sediment loads of the water or the concentrated suspended sediment slurry were determined as dry weight after 0.2 µm filtration (cellulose nitrate, Whatman, Maidstone, UK). DOC, TOC and pH measurements were carried out as described earlier. Chlorophyll-a concentration was determined for concentrated and non-concentrated suspended sediments with a UV/vis spectrophotometer at 410, 430, 480, 665 and 750 nm after glass fibre filtration (GF/C Whatman, Maidstone, UK) and extraction of the filters with ethanol overnight at 4°C. The surface area (SA) of suspended sediments was analysed using a Beckman-Coulter SA 3100 instrument as described by Gregg and Sing (1982) after oven drying (16 h, 40°C), gently breaking up with a pestle and mortar and outgassing at 60°C with a nitrogen gas flow to remove any water that evolved.

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For the Spring 2000 suspended sediment samples the organic carbon to nitrogen ratio was determined by further concentrating the suspended sediment slurry with a centrifuge (30 min, 4750 g) and discarding the overlying water. The samples were dried at 60°C overnight and homogenized in an agate mortar mill. To determine the particulate nitrogen (PN) and particulate organic carbon (POC) 10-15 mg sub-samples were weighed accurately (± 0.01 mg) into silver cups (9 x 5 mm). The sub-samples were then acidified with 20 µL 5M HCl and kept at 50-60°C for 30 min to remove inorganic carbon. Acid treatment was repeated until effervescence was no longer observed (generally three times). Water blanks were obtained using silver cups processed the same way but without sample addition. PN was determined on duplicate samples by high temperature oxidation using a Carlo Erba NA 1500 series 2 C/H/N/O/S analyser. Sulphanilamide was used to construct the calibration curve. Carbon and nitrogen content were expressed as percentage of total solid. The average blank level was < 0.5 µg N; detection limits (calculated according to the sensitivity of the instrument for nitrogen) was 0.5 µg for carbon and nitrogen respectively, corresponding to 0.005% N for a 10 mg sample. Analytical precision was ±1.6% of the measured value. To determine sorption coefficients to suspended sediments, a volume of 5 ml concentrated suspended sediments (re-diluted with river water to a suitable concentration if necessary) was added to PTFE centrifuge tubes and spiked with 14 C-E2 or EE2 at five concentrations (1.5-10 µg/l, two replicates each). The samples were then processed and analysed in the same way as described for the bed-sediments except that equilibration was only for 1 h. Assuming that the partitioning of E2 or EE2 between water and suspended sediments with the ambient suspended sediment loads would yield the same Kd as determined from the concentrated suspended sediments, the fraction E2 or EE2 that would sorb to suspended particles at their natural concentration was calculated. Results and discussion The characteristics of the water and suspended sediment samples are shown in Table 2.6. The River Thames showed higher surface area and chlorophyll than the other rivers in spring, which may be related to a higher algal population. The relatively poor algal production in the urban/industrial reaches of the Yorkshire rivers has been noted previously (Pinder et al., 1997). The calculated distribution coefficients for E2 and EE2 and suspended sediments are shown in Table 2.7. There are many factors which could influence the suspended sediment quality, such as rainfall intensity, land use change, inputs from sewage treatment works and industrial source (Walling et al., 1987). The hydrophobicity of the organic matter (Koelmans et al., 1995), as measured by the C/N ratio has been suggested to be particularly important in respect of sorption of organic molecules to suspended sediment, but it is not possible to confirm that based on our spring 2000 samples only. No clear correlations could be found between Kd and the river and sample parameters measured. As can be seen from the flow values for winter 1999, the sediments on that occasion were collected following a period of high rainfall. In such circumstances soil runoff would have led to a high inorganic mineral content which would be a el ss attractive sorbent to hydrophobic molecules and so low Kd values might be expected. As noted previously with the bed-sediments, generally higher Kd values were obtained with EE2 than E2, however this was less clear than with the bed-sediments, with some samples showing even lower Kds for EE2 than E2 (Table 2.7). Overall, suspended

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sediments would be expected to remove less than 1% of steroid oestrogens from the water column (Table 5). Table 2.6

Characteristics of water and suspended sediment samples Aire Aire (Riddle - (Beal) sden)

Calder (Brig-

Calder (Methley Bridge)

Thames (Wallingford)

house)

Winter 1999 Water Average daily flow (m3 /Section) PH DOC [mglL] Suspended sediments Surface area [m2 /g] Chlorophyll-α [µg/L] TOC

67.0 7.5 5.3

137.0 7.3 5.8

12.6 0.7 7.6%

Spring 2000 Water Average daily flow (m3 /Section) PH DOC [mg/l] Suspended sediments Surface area [m2 /g] Chlorophyll-α [µg/L] TOC C/N ratio

28.0 7.1 4.5

47.0 8.1 4.3

8.6 2 7.6%

8.2 0.6 6.9%

9.7 1.3 10.2%

14.9 1.9 6.7%

9.5 8.2 3.1

15.0 7.6 6.5

4.0 7.8 3.2

12.0 7.6 6.0

30.0 8.1 2.9

8.6 3.2 9.9% 5.4

6.5 7.9 10.7% 5.7

6.4 2.5 10.5% 5.8

a

Average daily flow registered by gauging station nearest to river reach Dissolved organic carbon c Total organic carbon b

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65.0 7.4 5.8

33

5.8 6.5 8.8% 7.5

10.1 27.6 7.0% 8.0

Table 2.7

Distribution coefficients (Kd) and standard errors (SE) for 17β -oestradiol (E2) and 17α -ethinyloestradiol (EE2) and suspended sediments E2 Kocc

SS load (mg/l)a

Kd (l/kg) (SEb)

(l/kg)

EE2

Aire

Winter 1999

15.0

50 (1.4)

658

Estimated removal by SSd 0.07%

(Riddlesden)

Spring 2000

8.0

21 (1.4)

212

0.02%

19 (1.1)

192

0.02%

Aire (Beal)

Winter 1999

52.0

122 (2.7)

1605

0.60%

165 (5.8)

2171

0.90%

Spring 2000

12.0

75 (2.7)

701

0.09%

177 (2.5)

1654

0.21%

Calder

Winter 1999

25.0

64 (3.9)

928

0.16%

80 (3.4)

1159

0.20%

(Brighouse)

Spring 2000

7.6

159 (7.2)

1514

0.12%

164 (8.5)

1562

0.12%

Calder

Winter 1999

36.0

44 (1.6)

431

0.16%

107 (5.4)

1049

0.40%

(Methley Bridge)

Spring 2000

10.0

113 (4.0)

1284

0.11%

260 (3.7)

2955

0.30%

Thames

Winter 1999

22.0

38 (1.9)

567

0.08%

65 (1.1)

970

0.14%

(Wallingford)

Spring 2000

8.0

113 (2.3)

1614

0.09%

87 (3.8)

1243

0.07%

a

Ambient suspended sediment loads. The Kd values were determined with the concentrated suspended sediments. Standard error of the slope (ie of Kd ) c Organic carbon normalized partition coefficient d Calculated amount for ambient concentrations of suspended sediments b

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Kd (l/kg) (SE)

Koc (l/kg)

80 (1.3)

1053

Estimated removal by SS 0.12%

e

Not determined

2.4

Conclusions

2.4.1

Degradation studies



E2 at a test concentration of 100 – 500 µg/l can be degraded in river water via E1 under aerobic conditions at 20o C to give half-lives between several hours and nine days. The most rapid biodegradation occurred from the water samples collected in summer.



The most rapid biodegradation of E2 was associated with the downstream urban/industrial river stretches of the River Aire and River Calder. E2 was more persistent further upstream and in the rural stretch of the River Thames.



The degradation rate was similar when the E2 concentrations were reduced from 100 µg/l to 20 – 100 ng/l, indicating that all the degradation results are relevant to ambient river concentrations of steroid oestrogens.



The river water samples were capable of cleaving the steroid ring system of E2.



E2 could be degraded under anaerobic conditions in bed-sediments, but only to E1 over a two-day period.



Oestrogenic activity, as measured by the yeast screen, was lost with the transformation of E2 and its first degradation product, E1.



EE2 was much more resistant to degradation in river water than E2.



E2 and EE2 are susceptible, but only slowly, to photodegradation with an estimated halflife of ten days given clear water and 12 hours of bright sunshine per day.

2.4.2

Sorption studies



A potential exists for E2 and EE2 to sorb to suspended sediments in British rivers. The attractiveness of these particles as sorbents was extremely variable. It is predicted that less than 1% of steroids present would be removed from the water phase by suspended sediments, given ambient concentrations of suspended sediments.



Whilst in general bed-sediments were less attractive sorbents than suspended sediments, given the quantity available binding to bed-sediments could be important.



The bed-sediments with the lowest sand content and highest silt content were the most effective sorbents.



The potential for E1, EE2 and to a lesser extent E2, to persist in sediments can not be ruled out, posing a potential long term sink for these steroids.

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3.

FIELD SURVEYS

3.1

Background, reason for work and selection of sites

The purpose of the field monitoring was two-fold: •

to determine the actual concentrations and distributions of steroid oestrogens (E1, E2 and EE2) within a UK river system;



to provide data to set up and test a computer model able to simulate adequately steroid oestrogen concentrations in UK rivers.

An approach based on short-term intensive monitoring sampling for a period of 2-4 weeks from sites concentrated around a sewage treatment works was adopted following discussions with the project board. The reasons for this approach were: •

The calculation of the steroid oestrogen mass balance down the river system is likely to be reliable;



Given that a good simulation of observed data is obtained, the reliability of the mass balance gives more confidence that the processes represented in the model are appropriate;



The survey can be carried out within a short period of time;



The steroid oestrogen concentrations monitored are likely to be attributable to a specific point source.

The original proposal for the field survey task within this project specified certain criteria for the selection of field sampling sites. These criteria were designed to ensure that a good mass balance of steroid oestrogens and flow could be made over the stretch of river selected and were that: •

ideally there should only be one major sewage treatment works acting as a source of steroid oestrogens within the river reach studied. There must be good data on the flows from this sewage treatment works;



the single discharge should significantly increase the level of steroid oestrogens above the upstream concentrations;



all major discharges need to be known and accessible to sampling personnel. Major refers to a flow that contributes more than 5% to the river low flow, or 5% to the load of any of the water quality variables to be modelled;



a flow gauging station should be available on the river in close proximity to the reach being studied, ideally at the top and bottom of the river section in question;

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the river reach should not be fed by land that is predominantly used for animal husbandry because diffuse sources of steroid oestrogens may act as confounding variables on the mass balance.

Secondary criteria identified a number of other requirements: •

laboratory data on degradation and sorption of steroid oestrogens within the selected reaches;



monitoring data for other variables (particularly organic molecules) for a number of sites or discharges on or into the river reaches;



time of travel data at a range of flow rates;



previous modelling studies over the same or similar reaches;



access to local laboratories for on-site processing and short-term storage of water samples.

In addition to these criteria a previous study of sewage treatment works effluents had indicated a number of works from which measurable amounts of steroid oestrogens were known to be discharged (Anon, 1997). Clearly there could have been benefit in conducting the field study on the River Aire or Calder. The main reason for seeking another site was the difficulty in maintaining the integrity of the water samples. In order to ensure that there was no degradation of steroid oestrogens the samples had to be collected and returned to the laboratory within the same day. This would not have been possible for the River Aire or for the River Calder. Great Billing STW on the River Nene and East Hyde STW and Harpenden STW on the River Lea were amongst those identified in the study and both met the majority of the criteria set out above. These sites therefore became the first choice for inclusion in this study.

3.2

Site descriptions

3.2.1

River Nene at Northampton

Sampling took place at six sites along a 12km stretch of the River Nene around Great Billing STW, between Northampton and Wellingborough, Northamptonshire. The river runs mainly through farmland and is under pressure from fishing, canoeing and narrowboats. There are several locks along this stretch, and in some places the river splits into two or three channels. Sampling took place where the river remains whole where possible and where this was not possible the main channel with the highest flow was sampled. The locations of the sampling sites selected ranged from relatively urban to rural.

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WELLINGBOROUGH Wellingborough Mills

Doddington Lock

NORTHAMPTON

Great Billing STW

Earls Barton Whiston Lock

Billing Mill

Figure 3.1

Locations of sampling sites on the River Nene

Great Billing is a relatively large works and serves a catchment of approximately 300,000 population equivalents. The first sampling site was just upstream of the works, the second in the final effluent channel and the third, fourth, fifth and sixth at intervals downstream. Table 3.1 gives a brief description of each site and Figure 3.1 shows their locations. Table 3.1

Summary descriptions of the sampling sites on the River Nene

Site 1. Billing Mill

2. Great Billing STW

Distance Grid d/s STW Reference

Description

-2.0 km SP 815611

0 km

Upstream of STW at Great Billing. Fairly heavy boat traffic due to close proximity of lake and moorings. Occasional fishing. Sample taken from bridge. Site semi-urban and sometimes flow appeared sluggish. Concrete final effluent channel sampled before river discharge point.

SP 826618

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Site

Distance Grid d/s STW Reference

Description

3. Whiston Lock

1.5 km SP 847618

Rural stretch of river surrounded by arable fields and the Nene Way. River splits for lock – main channel sampled from bridge. Weir just upstream. Usually appeared fairly fast flowing.

4. Earls Barton

3.0 km SP 859621

Rural stretch of river near popular fishing site and canoe launch point. River sampled from bank downstream of a three way split and re-merge.

5. Doddington Lock

7.4 km SP 889644

Rural location near wetland nature reserve. Dairy cows in field next to river. River splits for lock – main channel sampled from bridge.

6.

Wellingborough Mills

3.2.2

10 km

SP 902666

Urban area adjacent to industrial site with open area on opposite bank. Narrowboats often moor here. River fairly shallow and wide. Sampled from bank.

River Lea at Harpenden

The Section of the River Lea studied covers a distance of 11 km and is predominantly rural in character apart from where it flows through the north east corner of Harpenden. The upstream site was 0.5 km above the East Hyde final effluent discharge point, but still within the confines of the works. There were four river sampling sites downstream of East Hyde STW locations and short descriptions of which are given in Figure 3.2 and Table 3.2. Harpenden STW also discharges to this stretch of the River Lea just downstream of Harpenden; this site was shown by the preliminary survey to deliver steroid oestrogens to the river and so it was included in the sampling programme.

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New Mill East Hyde STW

East Hyde Bridge

Harpenden Harpenden Works (STW)

HARPENDEN Leasey Bridge

WHEATHAMPSTEAD Water End Ford

Figure 3.2

Locations of sampling sites on the River Lea

Table 3.2

Summary descriptions of the sampling sites on the River Lea

Site 1. New Mill

Distance Grid d/s STW Reference

Description

-0.5 km

TL 121182

Upstream East Hyde STW but within the compound. Small very shallow stream. Samples taken from bridge.

2. East Hyde STW

0 km

TL 124178

Final effluent discharge from immediately before confluence with the river. Agency compliance monitoring site.

3. East Hyde Bridge

1 km

TL 128172

Site located in uncultivated field. Large stones on streambed. Small side channel with stagnant water.

4. Harpenden Bridge

3.5 km

TL 144153

Location in built up area between shops and commercial estate. Stony river bed some fine material. Samples taken from bridge

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Distance Grid d/s STW Reference

Site

Description

4. Harpenden STW

4.5 km

TL 151147

Final effluent discharge from immediately before confluence with the river. Agency compliance monitoring site. Much smaller than East Hyde.

5. Leasey Bridge

6 km

TL 162144

Tree liner river banks popular with anglers. Sandy/silty bed-sediments. Samples taken from bridge.

6. Waterend Ford

10.5 km

TL 203137

River in rural area, wider and shallower. More fine bed-sediments. Samples taken from bridge upstream of ford.

3.3

Sampling Methods

3.3.1

Preliminary survey

Since the aim of the study was to generate data to further our understanding of the fate and behaviour of steroid oestrogens in rivers, a preliminary assessment of the final effluents was made to determine if there were detectable levels of steroid estrogens present. During the preliminary survey a number of samples at different times of the day were taken to determine the within-day variations in the quality of the final effluents. The preliminary survey took place at Great Billing STW on 20th July 2000. Four sets of two 5 litre samples were taken from the final effluent channel at intervals of approximately three hours, starting at about 08:30. The samples were collected in silanized, clear glass bottles using a stainless steel bucket and funnel; both were rinsed with effluent before each sample was taken. One sample was used to determine the concentration of the three steroid oestrogens, E1, E2 and EE2. The other was used to perform an oestrogen spike recovery test as part of the quality assurance procedure. A 0.5 litre sample was also taken at the same time for analysis of suspended solids and dissolved organic carbon. A similar procedure was carried out at East Hyde on 4th September 2000, but only three samples were taken during the day at 11:30, 14:20 and 15:40. The fourth sample was taken from the final effluent of Harpenden STW, which also discharges to the River Lea within the study section. Two 5 l samples and one 0.5 l sample were taken at 11:15 as for East Hyde STW. 3.3.2

Main survey

The survey of the River Nene started on 28 July 2000 and lasted for four weeks. Samples were taken daily at about the same time at each site (between 10:30 and 13:00) Samples were taken using either a 1 litre stainless steel bucket (for sampling from bridges) or a 0.5 l PTFE R&D TECHNICAL REPORT P2-162/TR

41

container attached to an aluminium pole (for sampling from the bank). The bottles were filled with the aid of a stainless steel funnel; both the funnel and the bucket were rinsed through with river water/sewage effluent from each site prior to the first sample being collected. The following bottles were filled: •

a 5 litre silanized glass bottle for steroid extraction and analysis;



a 0.5 litre plastic bottle for suspended sediment and dissolved organic carbon analysis;



a 30 ml filtered sample (surfactant free cellulose acetate syringe filter) for boron analysis;



an extra 1 litre silanized glass bottle at Great Billing STW for spike recovery tests.

Samples from the effluent stream were analysed for dissolved and particulate steroids, those from the river for total and dissolved steroids. The boron sample was taken because it is a good indicator of sewage effluent and behaves conservatively in river water. It can therefore be used for mass balance calculations. Bed-sediment samples were also taken on three occasions. On 7th August 2000, two 0.5 l glass jars were filled with sediment at each site using a stainless steel 0.5 l grab-sampler. However, it proved difficult to collect bed-sediments at some sites due to rocky substrates so subsequent bed-sediment collections (on 14th and 21st August) were made only at Billing Mill (one sample), Doddington Lock (two samples) and Wellingborough Mills (two samples). These samples were analysed for total steroids and total organic carbon. The main survey on the River Lea started on 29 September with daily samples taken between 10:30 and 13:00 for 2 weeks. The same samples were taken as for the River Nene with the exception of the Harpenden final effluent, where no spike recovery tests were performed and the analysis did not discriminate between dissolved and particulate steroids. Bed-sediment samples were taken on two occasions (2 and 9 October 2000) from four of the river sites on the River Lea. There was no access to the site at East Hyde Bridge. The samples were taken by carefully removing the top 5 cm of bed-sediment with a spade and retaining the amount that would pass through a 2-mm sieve in 0.5-litre glass jars.

3.4

Analytical methods

All the determinands, except boron, were analysed by WRc-NSF under contract T62H05/00C. Boron was analysed at the CEH Wallingford laboratories. Steroid oestrogen analysis methods at the very low environmental concentrations reported here are still in the stages of early development. There is not yet a water industry standard for analysis in water or any other matrix, although there is currently research to develop such methods. 3.4.1

Sample storage

All the samples were kept in cool boxes during the sampling campaigns. The cool boxes were kept cold (4-6 °C) and the samples delivered to the analytical laboratory within 8 hours after collection. Samples were stored in a fridge below 6 °C prior to extraction. Sample extracts were stored in a freezer below –18 °C prior to GC/MS/MS analysis.

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3.4.2

Steroids in river water

Samples were filtered to remove particulate matter and a mixture of deuterated internal standards (d4 -E2, d4 -17β-E2 and d4 -17α-ethynyl E2) was added. The samples were extracted using C18 solid phase extraction cartridges. The cartridges were washed with 50% aqueous methanol prior to elution with aqueous methanol (85:15 methanol:water). The particulate fraction was extracted using solvent extraction using dichloromethane. The particulate and aqueous extracts were combined and the combined extract concentrated to a small volume using a Turbo-Vap concentrator and cleaned-up by HPLC fraction collection. The collected fractions were reduced to dryness, and derivatized with a solution in acetonitrile of a mixture of N-methyl-N-t-butyldimethylsilyl trifluoroacetamide (MTBSTFA) (99%) and tbutyldimethyl-chlorosilane (TBDMCS) (1%). Excess derivatising agent was removed by taking the derivatized extract to dryness and re-dissolving the residue in diethyl ether prior to analysis by GC/MS/MS. Using the MS/MS, selected reaction monitoring was used for quantification on the M.+ and [M-57]+ ion pairs from the target analytes and the internal standards. The [M-57]+ ion corresponds to the loss of the t- butyl group from the molecular ion of each of the steroids. The established method performance of the procedure to determine steroids in river waters gives a reporting limit of 0.4 ng/l for E1 and E2 and 0.5 ng/l for EE2 for a sample in the range 0 – 50 ng/l. The standard deviation of a 1 ng/l sample was in the range 0.12 - 0.17 ng/l. Recovery and analytical variability obtained for the analytical quality control procedural spike samples (for this survey) is detailed in Appendix B. The data were not corrected for recovery efficiency. 3.4.3

Steroids in sewage treatment effluent

Analysis of treated sewage effluent samples for total steroids was undertaken using the same procedure as river water samples. Samples that required analysis of steroids in the aqueous and particulate phases were filtered using GF/F filters. The aqueous sample and the particulate fraction (which was transferred to a 50 ml silanized glass flask) were spiked with a mixture of deuterated internal standards (d4 -E2, d4 -17β-E2 and d4 -17α-ethinyl E2). The aqueous samples were extracted using C18 solid phase extraction cartridges. The cartridges were washed with 50% aqueous methanol prior to elution with aqueous methanol (85:15 methanol:water). The extract was reduced to 1 ml using a Turbo-Vap concentrator, transferred to a 2 ml autosampler vial and further reduced to dryness using a nitrogen blowdown apparatus and finally methanol (100 µl) was added to the vial. The particulate sample was extracted using dichloromethane, which was added to the flask containing the particulate fraction and filter paper. The flask was sonicated for 3 minutes and further extracted by vigorously shaking by hand for a further 3 minutes. The extract was decanted into a silanized 200 ml Turbo-Vap tube. The extraction procedure was repeated and the second extract was combined with the first. The combined extract was reduced to 1 ml using a Turbo-Vap concentrator, transferred to a 2 ml autosampler vial and further reduced to dryness using a nitrogen blow-down apparatus and finally methanol (100 µl) was added to the vial. The particulate and aqueous extracts were cleaned-up by HPLC fraction collection and analysed by GC-MS/MS as before.

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The established method performance of the procedure to determine steroids in treated sewage effluent gave a reporting limit (3 times the signal to noise of standard solutions) of 1 ng/l on an undiluted sample range of 0 – 50 ng/l. 3.4.4

Steroids in sediments

Very little work has been done on the concentrations of steroid oestrogens in river bed sediments. A new analytical method had to be developed by WRc-NSF to analyse the bed sediment samples collected in this study. An aliquot of the wet sediment was sieved through a 2 mm sieve to remove stones and other large matter. A portion (30 g) of the sieved sample was transferred to a 100 ml silanized screw top flask. The sediment was spiked with a mixture of deuterated internal standards (d4 -E2, d4 -17β-E2 and d4 -17α-ethynyl E2). Groundwater (50 ml) and dichloromethane (50 ml) were added to the sample. The flask was sonicated for three minutes and further extracted by vigorously shaking by hand for a further 3 minutes. The upper aqueous layer was removed and the remaining sample and dichloromethane separated using GF/F filter paper in a silanized buchner flask and funnel. The sediment was rinsed with two aliquots (25 ml) of dichloromethane and the filtrate transferred to a 100 ml separating flask. The solvent was drained into a TurboVap tube and concentrated to 1 ml using a TurboVap concentrator. The extract was transferred to a 2 ml autosampler vial and further reduced to dryness using a nitrogen blow-down apparatus and finally methanol (100 µl) was added to the vial. The particulate and aqueous extracts were cleaned-up by HPLC fraction collection and analysed by GC-MS/MS as before. The method performance of the procedure to determine steroids in sediments gave a reporting limit (3 times the signal to noise of standard solutions) of 100 ng/kg on an undiluted sample range of 0 – 5000 ng/kg. 3.4.5

Dissolved organic carbon in river water and treated sewage

Dissolved Organic Carbon (DOC) was determined using a UKAS accredited procedure, in which a portion of the sample was filtered through a GF/C filter, acidified to pH2 with phosphoric acid and purged with argon. Inorganic carbonates and bicarbonates were converted to carbon dioxide and removed. Volatile or purgeable organic carbon was also removed at this stage. A known volume of the purged sample was injected into the instrument, where the organic carbon reacts with acidified potassium persulphate in the presence of UV radiation to generate carbon dioxide. The carbon dioxide is measured using non-dispersive infra red detection. The established method performance of the procedure to determine DOC in river waters and treated sewage effluents gave a reporting limit of 0.2 mg/l C on a sample of 20 mg/l C. The standard deviation on a 5 mg/l C sample was 0.15 mg/l C. 3.4.6

Total organic carbon in sediment

Total organic carbon (TOC) was determined using a UKAS accredited procedure, in which a sub-sample (10 g) of the sediment sampled for steroid analysis was placed into a 100 ml glass beaker and the beaker placed in an oven to dry the sediment. The dried sample was ground using a pestle and mortar and a 0.2 g sub-sample was weighed into another 100 ml glass R&D TECHNICAL REPORT P2-162/TR

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beaker. The sample was treated with sulphurous acid to remove inorganic carbon (carbonates and bicarbonates). The treated sample was placed in an oven to dry overnight. A portion (5 mg) of the dry sample was subjected to high temperature combustion in a pure oxygen environment. Products produced include CO2 , H2 O and N2 gases, which are homogenized and brought to exact conditions of temperature, pressure, and volume. The stabilised gases are allowed to de-pressurise through a column where they are separated and detected by thermal conductivity. The established method performance of the procedure to determine TOC in sediments gave a reporting limit of 0.13 % on a sample in the range 0 – 100%. The standard deviation on a sample of 5 % TOC was 0.5% TOC. 3.4.7

Suspended solids in river water and treated sewage

Suspended solids were determined using a UKAS accredited procedure, in which a measured volume of homogenous sample is filtered under vacuum through a GF/C glass fibre filter which has previously been washed, dried at 105 °C and weighed. The filter with the collected solids is then dried at 105 °C for 2 hours, allowed to cool in a desiccator and reweighed. Suspended solids in the sample were calculated from the weight difference and the volume of sample filtered and expressed in mg/l. The established method performance of the procedure to determine suspended solids gave a reporting limit of 1mg/l for 1-litre of sample filtered. A 100 mg/l standard gave a standard deviation of 1.3 mg/l. 3.4.8

Boron in river and treated sewage effluent samples

The filtered boron samples were stored at 4o C for up to one month prior to analysis. Samples were acidified with 1% nitric acid and analysis was by ICP-AES (Optima 3300 DV) with a wavelength of 249.755 nm. The calibration range was 0 – 1000 µ/l and the detection limit 5 µ/l. Results were blank and drift corrected.

3.5

Results and Discussion

3.5.1

Preliminary survey

River Nene All four samples from the Great Billing STW final effluent showed measurable concentrations of E1 and two samples showed E2 (detection limit 1 ng/l) (Figure 3.3). E2 is known to degrade to E1 in sewage treatment and it is not that surprising that the levels of E1 were found to be higher than those for E2. None of the samples showed concentrations of EE2 above the detection limit (1 ng/l). The E1 concentrations are similar to those reported in a previous Environment Agency report, which gave values in the range 1.4-9.9 ng/l (Anon, 1997). However, the values reported for E2 in the Agency report were similar to those reported for

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E1 and higher than those found in this study. Great Billing works has seen some changes to its operation (including the commissioning of biological phosphorus removal) over the last few years and these may have a bearing on E2 concentrations. River Lea The results from East Hyde sewage treatment works were rather different than those from Great Billing (Figure 3.4). At East Hyde only E2 was found in the final effluent and no E1 (no values greater than 1 ng/l). EE2 was not detected in any of the samples. The one sample that was collected from Harpenden STW effluent interestingly showed measurable concentrations of E2 (1.77 ng/l), E1 (5.27 ng/l) and EE2 (0.56 ng/l). The Agency report discussed above also looked at the effluent from Harpenden and found E2 concentrations slightly higher than those reported here (5.2-8.9 ng/l) and E1 at similar concentrations (3.7-7.1 ng/l). 9 8 7

Concentration

6 5 4 3 2 1

Time of sample

0 Oestrone (ng/l)

Oestradiol (ng/l)

Suspended sediment (mg/l)

Dissolved organic carbon (mg/l)

08:35

7.97

2.24

1.91

8.12

11:30

5.67

0.5

1.49

7.98

14:20

2.78

0.5

1.25

8.14

17:15

5.31

2.45

1.28

8.45

Figure 3.3

Concentrations of oestrone (E1), 17β -oestradiol (E2), suspended sediment and Dissolved Organic Carbon in the final effluent of Great Billing STW on 20 July 2000. Values below the detection limit (1ng/l or 1mg/l) were plotted at half of that value.

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8

7

Concentration

6

5

4

3

2

Time of sample

1

0

Oestradiol (ng/l)

Suspended sediment (mg/l)

Dissolved Organic Carbon (mg/l)

11:30

0.8

3.1

7.3

14:00

1.34

2.7

6.2

15:50

1.32

0.5

6.1

Figure 3.4

3.5.2

Concentrations of 17β -oestradiol (E2), suspended sediment and Dissolved Organic Carbon in the final effluent of East Hyde STW on 4 September 2000. Values below the detection limit (1 ng/l or 1 mg/l) were plotted at half of that value.

Main survey of the River Nene

Flow Conditions The main survey on the River Nene was carried out in August 2000 when the river levels were low. The aim was to select a period when the sewage effluent from Great Billing would form a significant part of the flow in the river. The mean flow over the period of the study measured at South Bridge just upstream of the study section was 1240 l/s. The mean effluent flow rate over the same period was 730 l/s (Figure 3.5)

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2500 River Nene

Great Billing FE

Flow rate (l/s)

2000

1500

1000

500

26/08/00

24/08/00

22/08/00

20/08/00

18/08/00

16/08/00

14/08/00

12/08/00

10/08/00

08/08/00

06/08/00

04/08/00

02/08/00

31/07/00

29/07/00

27/07/00

0

Date

Figure 3.5

Flow rates in the River Nene and the Great Billing final effluent over the period of the oestrogen survey.

Boron occurs in river water as the chemically unreactive species borate, which is a product used in household and industrial detergents. Sewage treatment works are the dominant source of boron in rivers and therefore boron can be used as a conservative tracer of sewage effluent. Because boron is conservative, the change in its concentration downstream of a sewage treatment works indicates the amount of dilution of the effluent (assuming no other boron inputs). The boron data collected in this study showed that Great Billing effluent made up 34% of the flow in the River Nene just below its discharge point. This fell only slightly to 31.6% at Wellingborough, the most downstream end of the study section. These data indicate that there were no significant inputs to the study section other than the sewage treatment works effluent. Steroid oestrogens E1 was the dominant steroid in the effluent from Great Billing STW. It was detectable in all the samples analysed. Concentrations ranged from 0.78 – 11.22 ng/l with a mean value of 4.6 ng/l (Figure 3.6). E2 was detected in about half of the samples analysed (>1 ng/l) with a maximum concentration of 2.01 ng/l (Figure 3.6). EE2 was found in 9 of the 28 effluent samples analysed at concentrations of up to 1.9 ng/l. Figure 3.6 clearly illustrates the variability of the effluent with respect to the concentration of steroid oestrogens. The concentrations of steroid oestrogens associated with the particulate phase were measured for the effluent, but no values above the detection value of 1 ng/l were found.

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12 Oestrone

Oestradiol

Concentration (ng/l)

10

8

6

4

2

23/08/00

21/08/00

19/08/00

17/08/00

15/08/00

13/08/00

11/08/00

09/08/00

07/08/00

05/08/00

03/08/00

01/08/00

30/07/00

28/07/00

0

Date

Figure 3.6

Concentrations of oestrone (E1) and 17β -oestradiol (E2) in the effluent from Great Billing during the 28-day survey. Values of E2 below the detection limit are plotted at half the value (i.e. 0.5 ng/l)

The STW effluent input clearly led to a noticeable rise in the concentration of E1 in the River Nene. The impact on the concentrations of E2 is less clear-cut (Figure 3.7). In fact the E2 concentrations are generally around the limit of detection (the means were calculated including values less than 1 ng/l at half the detection limit value). It is interesting to note that there are already quite high concentrations of E1 and E2 in the River Nene upstream of Great Billing STW. There are 26 small to medium sized STWs upstream of Billing Mill (the largest serves a population equivalent of ~22 000), which discharge either directly in to the River Nene or its tributaries. Johnson et al. (2000) have developed an empirical relationship to determine the input concentrations of E1 and E2 to STWs:

E 2 = P / 263 E1 = P / 114 where E2 and E1 are the loads (mg/day) of E2 and E1 respectively and P is the population served by the works. The same authors have also suggested removal rates for E1 and E2 with STWs of 73% and 88% respectively. Using this it has been possible to estimate the total contribution of steroid oestrogens from the 26 upstream works. Diluting this load into the mean flow at Great Billing and assuming there is no loss of E1 or E2 by degradation in the river (see below and Section 4), The maximum likely concentration of E1 at Great Billing would be 0.9 ng/l and the concentration of E2 would be 0.4 ng/l. These values are close to the observed mean values for E2 and E1 of 1.4 ± 0.4 ng/l and 0.5 ± 0.2 ng/l respectively. A possible additional point source could originate from the caravan park situated 1 km upstream of the Billing Mill site. Steroid oestrogens may also arise from diffuse sources associated with the excreta of farm and wild animals. Such sources are more likely to be active in winter when runoff amounts are larger, rather than in summer when this survey was conducted.

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There is also a small increase in mean value of E2 between the last two monitoring sites, Doddington Lock and Wellingborough Mill; 1.12 ± 0.26 ng/l to 1.20 ± 0.43 ng/l. Given the other data a decrease along this stretch might have been expected. There are two possible sources of E2 along this reach: (1) a very small STW that discharges into the River Nene below Doddington Lock and (2) a small tributary in to which five other very small STWs discharge. Using the same approach as above the effect of these STWs would have been to raise E2 concentrations by a maximum of 0.09 ng/l, which is consistent with the observed increase. E1 concentrations showed a small drop over this stretch, but these values are too close to the detection limit to be significant. The raw data for the survey are shown in appendix A (tables A2 – A8). From the average data, it is possible to compare the observed values of E1 with those that would have occurred because of dilution alone. It is clear from Figure 3.8 that dilution alone would not account for the observed concentrations and that there is a loss of E1 along the river. Section 4 of this report discusses the modelling of these data in detail using a range of approaches including the EXAMS model and the Environment Agency’s TOMCAT and SIMCAT models.

3 E1

E2

Concentration (ng/l)

2

1

0 -2

0

2

4

6

8

10

-1 Distance d/s STW (km)

Figure 3.7

Mean concentrations of oestrone (E1) and 17β -oestradiol (E2) in the River Nene around Great Billing STW. The error bars are set at ± 2 standard errors. Concentrations below the detection limit were included in the calculation of the means as a value of half the detection limit (i.e. 0.5 ng/l)

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3 Measured

Dilution only

Concentration

2

1

0 -4

-2

0

2

4

6

8

10

-1

Distance d/s of STW (km)

Figure 3.8

Comparison of observed data from the River Nene with estimates of expected oestrone (E1) concentrations arising from dilution effects alone.

Bed-sediment The concentrations of E1 found in the bed sediments are shown in Table 3.3. There is a large variation between sites, ranging from