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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
Principles of method if other than guideline:
Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes as it was considered likely that any longer test duration would result in significant losses of test material from the test system. In order to minimise any losses of test material from the test system, rather than vigorous aeration of the test vessels, the test preparations were kept in suspension by stirring via magnetic stirrers.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 19 August 2008 Date of signature:4 March 2009
Analytical monitoring:
no
Details on sampling:
Concentrations: 10, 32, 100, 320 and 1000 mg/L

- Sampling method: Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contact time.

- Sample storage conditions before analysis: not specified in report
Vehicle:
no
Details on test solutions:
Method: Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes as it was considered likely that any longer test duration would result in significant losses of test material from the test system. In order to minimise any losses of test material from the test system, rather than vigorous aeration of the test vessels, the test preparations were kept in suspension by stirring via magnetic stirrers. For the purpose of the test, the test material was dispersed directly in water. Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately added to synthetic sewage (16 mL), activated sewage sludge (200 mL) and water to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L. The control group was maintained under identical conditions but not exposed to the test material.

Eluate: At time "0" 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 1 litre beaker (first control). The mixture was stirred via magnetic stirring. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared. Finally a second control was prepared. As each vessel reached 30 minutes contact time an aliquot was removed from the beaker and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each vessel was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/L and 2.5 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve except for the 32 mg/L test material vessel. Due to the linear portion of the respiration curve not being attained for the 32 mg/L test vessel, the oxygen consumption for this vessel was not calculated. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C. Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contact time.

Differential loading: Not applicable.

Controls: The control group was maintained under identical conditions but not exposed to the test material.

Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that all test concentrations contained a clear colourless water column with an oily layer of test material visible on the surface. At the test concentrations of 320 and 1000 mg/L oil globules of test material were also visible dispersed throughout. Observations of the dosed activated sludge mixtures made after 30 minutes contact time showed the test concentration of 10 mg/L to contain a dark brown dispersion, with the brown/undissolved matter to be attributed to the activated sludge solids and the absence of floating film or suspended micelles of undissolved test material visible. The test concentrations of 32 and 100 mg/L contained the same dark brown dispersions, but with visible floating globules of test material present. The floating globules of undissolved test material were increasingly prominent at the test concentrations of 320 and 1000 mg/L. These observations confirm that the test material was not substantially lost (via volatilization) following 30 minutes of stirring/suspension in the activated sludge test mixtures.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.8 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Remarks on exposure duration:
Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes
Post exposure observation period:
Not applicable
Hardness:
The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 2 (attached in IUCLID).
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.
pH:
The pH values of the test preparations at the start and end of the exposure period are given in Table 2. And were 7.6 to 8.2.
Dissolved oxygen:
In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases, except for the 32 mg/L test material vessel, the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. Due to the linear portion of the respiration curve not being attained for the 32 mg/L test material vessel, the concentration response curve for the test material was plotted using the percentage inhibition values for the remaining four test material concentrations. A line could still be fitted using the Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line. The relatively low initial oxygen readings observed at the two lowest concentrations (10 and 32 mg/L) were likely to be due to increased rates of oxygen consumption resulting from biodegradation of the test material components at concentrations below their toxicity thresholds (i.e., <100 mg/L).
Salinity:
Not applicable
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
Preparation of test system: At time "0" 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 1 litre beaker (first control). The mixture was stirred via magnetic stirring. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared as described in the details of test solutions section. Finally a second control was prepared. As each vessel reached 30 minutes contact time an aliquot was removed from the beaker and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each vessel was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/L and 2.5 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve except for the 32 mg/L test material vessel. Due to the linear portion of the respiration curve not being attained for the 32 mg/L test vessel, the oxygen consumption for this vessel was not calculated. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.

Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contacontact time.

OTHER TEST CONDITIONS

The pH values of the test preparations at the start and end of the exposure period are given in Table 2 (any other information on results section)

- Photoperiod: 30 minutes

- Light intensity: Normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage of the two control respiration rates.
% inhibition = [ 1 – 2RS ÷ RC1 + RC2 ] x 100
where
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line.

The equation of the fitted line for the reference material was as follows:
y = (A + (B*log(x)))
where
A = the vertical shift factor
B = the slope factor. A positive result is returned when y is increasing as x increases. A negative result is returned when y is increasing as x decreases
x = the original concentration values
y = the original inhibition values

The equation of the fitted line for the test material was as follows:
y = ((A/(1(10^((C-log(x))*B))))+ ((100-A)/(1+(10^((D-log(x))*B))))).
where
A = 100 minus the top of curve 1 and the bottom of curve 2
B = the slope factor
C = the log (X2). Value at the middle of the second curve
D = the log (X1). Value at the middle of the first curve
x = the original concentration values
y = the original inhibition values

95% confidence limits were calculated for the EC50 values using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).

Whilst the test guidelines do not specify procedures for determination of a No Observed Effect Concentration (NOEC), a NOEC was determined which is equivalent to the EC15 value determined from a dose response curve and percentage inhibition values for the test material.
The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (30-Minute contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/L.

Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Sigma-Aldrich Batch No. 04621CJ)
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
380 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence limits 290 - 510 mg/l
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1 (any other information on results section). The pH values of the test preparations at the start and end of the exposure period are given in Table 2 (any other information on results section), and observations made on the test preparations throughout the study are given in Table 3 (any other information on results section).
Percentage inhibition is plotted against concentration for the test and reference materials (Figures 1 to 2 - attached in IULCID).

The following results were derived from the described experimental procedure. Whereas some loss of the volatile test material components may have occurred during preparation and incubation of the test mixtures, a similar degree of loss of these materials could be expected upon their discharge to and treatment within a typical activated sludge wastewater treatment plant. Therefore, the results are expected to give a reasonable approximation of the potential effects of the test material on operation of such wastewater treatment processes:

Divinylbeneze 55
ECx (30 Minutes) (mg/L) 95% Confidence Limits (mg/L)
EC20 160 -
EC50 380 290-510
EC80 710 -
NOEC 100 -

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

3,5-dichlorophenol:
ECx (30 Minutes) (mg/L) 95% Confidence Limits (mg/L)
EC20 2.7 -
EC50 8.9 6.1 - 13
EC80 30 -
NOEC 2.2

Variation in respiration rates of controls 1 and 2 after 30 minutes contact time was ± 5%.

The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.

Observations made at 0 hours (see Table 3) prior to the addition of activated sewage sludge and synthetic sewage showed that all test concentrations contained a clear colourless water column with an oily layer of test material visible on the surface. At the test concentrations of 320 and 1000 mg/l oiL globules of test material were also visible dispersed throughout. Observations of the dosed activated sludge mixtures made after 30 minutes contact time showed the test concentration of 10 mg/L to contain a dark brown dispersion, with the brown/undissolved matter to be attributed to the activated sludge solids and the absence of floating film or suspended micelles of undissolved test material visible. The test concentrations of 32 and 100 mg/L contained the same dark brown dispersions, but with visible floating globules of test material present. The floating globules of undissolved test material were increasingly prominent at the test concentrations of 320 and 1000 mg/L. These observations confirm that the test material was not substantially lost (via volatilization) following 30 minutes of stirring/suspension in the activated sludge test mixtures.
Results with reference substance (positive control):
- Results with reference substance valid? Yes

- Relevant effect levels: The reference material gave a 30-Minute EC50 value of 8.9 mg/L, 95% confidence limits 6.1 - 13 mg/L.
Reported statistics and error estimates:
None

Table1              Oxygen Consumption Rates and Percentage Inhibition Values after 30 Minutes Contact Time

Nominal

Concentration

(mg/l)

Initial O2

Reading

(mg O2/L)

Measurent Period

(minutes)

Final O2Reading

(mg O2/L)

O2Consumption Rates

(mg O2/L/min)

% Inhibition

Control

R1

6.3

8

2.4

0.49

-

 

R2

5.8

7

2.0

0.54

-

Test Material

10

2.6

2

1.6

0.50

3

 

32

1.5

1

1.1

[1]

*

 

100

5.9

7

2.8

0.44

15

 

320

7.8

10

4.8

0.30

42

 

1000

7.8

10

7.3

0.05

90

3,5-dichlorophenol

3.2

7.3

10

3.5

0.38

26

 

10

7.2

10

4.6

0.26

50

 

32

7.6

10

6.7

0.09

83


R1– R2= Replicates 1 to 2

[1]Values not calculated due to very low start and finish oxygen concentrations

Table2              pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal

Concentration

(mg/l)

pH

0 Hours

30 Minutes

Control

R1

7.7

7.6

 

R2

7.8

8.0

Test Material

10

7.8

7.8

 

32

7.8

7.7

 

100

7.8

7.9

 

320

7.8

8.2

 

1000

7.8

7.9

3,5-dichlorophenol

3.2

7.7

7.8

 

10

7.7

8.0

 

32

7.7

7.9


R1– R2= Replicates 1 to 2

Table3              Observations on the Test Preparations Throughout the Test Period

Nominal

Concentration

(mg/l)

Observations on Test Preparations

0 Hours Contact Time

30 Minutes Contact Ti

Control

R1

Dark brown dispersion

Dark brown dispersion

 

R2

Dark brown dispersion

Dark brown dispersion

Test Material

10

Clear colourless water column with globules of test material on surface*

Dark brown dispersion, no undissolved test material visible

 

32

Clear colourless water column with globules of test material on surface*

Dark brown dispersion, with a few oily specks of test material visible on surface

 

100

Clear colourless water column with an oily layer of test material on surface*

Dark brown dispersion, with a few oily specks of test material visible on surface

 

320

Clear colourless water column with an oily layer of test material on surface and oily globules visible dispersed throughout*

Dark brown dispersion, with a few small globules of test material visible on surface

 

1000

Clear colourless water column with an oily layer of test material on surface and oily globules visible dispersed throughout*

Dark brown dispersion, with oil globules of test material visible on surface

3,5-dichlorophenol

3.2

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

10

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

32

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible


R1– R2= Replicates 1 to 2

*Observations made prior to the addition of synthetic sewage and activated sewage sludg

Concentration-Response Curve: 3,5-dichlorophenol – 30 Minutes Contact Time see attached figure 1

Concentration-Response Curve: Divinylbenzene 55– 30 Minutes Contact Time see attached figure 2

See Attached Appendix 1 for Certificate of Analysis

See atached Appendix 2 For Typical Water Quality Characteristics

   REFERENCES

Litchfield, J T and Wilcoxon, F (1949) A Simplified Method of Evaluating Dose-Effect Experints. J Pharmacol Exp Ther, 96, 99-113.

Xlfit® 4.2, (2005), ID Business Solutions Ltd.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 30-Minute EC50 of 380 mg/L, 95% confidence limits 290 - 510 mg/L. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was 100 mg/L.
Executive summary:

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes as it was considered likely that any longer test duration would result in significant losses of test material from the test system. In order to minimise any losses of test material from the test system, rather than vigorous aeration of the test vessels, the test preparations were kept in suspension by stirring via magnetic stirrers.  Activated sewage sludge was exposed to an aqueous dispersion of the test material at nominal concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 30 minutes at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 minutes contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 30-minute EC50 of 380 mg/L, 95% confidence limits 290 - 510 mg/L. Whilst the test guidelines do not specify procedures for determination of a No Observed Effect Concentration (NOEC), a NOEC was determined which is equivalent to the EC15 value determined from a two site dose response curve and percentage inhibition values for the test material. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was 100 mg/L. The reference material gave a 30-Minute EC50 value of 8.9 mg/L, 95% confidence limits 6.1 - 13 mg/L.

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 30-Minute EC50 of 380 mg/L, 95% confidence limits 290 - 510 mg/L. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was 100 mg/L.

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
Principles of method if other than guideline:
Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes as it was considered likely that any longer test duration would result in significant losses of test material from the test system. In order to minimise any losses of test material from the test system, rather than vigorous aeration of the test vessels, the test preparations were kept in suspension by stirring via magnetic stirrers.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection:19 August 2008 Date of Signature on GLP certificate:4 March 2009
Analytical monitoring:
no
Details on sampling:
Concentrations: 10, 32, 100, 320 and 1000 mg/L

Sampling method: Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contact time.

Sample storage conditions before analysis: not specified in report
Vehicle:
no
Details on test solutions:
Method: Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes as it was considered likely that any longer test duration would result in significant losses of test material from the test system. In order to minimise any losses of test material from the test system, rather than vigorous aeration of the test vessels, the test preparations were kept in suspension by stirring via magnetic stirrers. For the purpose of the test, the test material was dispersed directly in water. Amounts of test material (5, 16, 50, 160 and 500 mg) were each separately added to synthetic sewage (16 mL), activated sewage sludge (200 mL) and water to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L. The control group was maintained under identical conditions but not exposed to the test material.

Eluate: At time "0" 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 1 litre beaker (first control). The mixture was stirred via magnetic stirring. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared as described above. Finally a second control was prepared. As each vessel reached 30 minutes contact time an aliquot was removed from the beaker and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each vessel was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/L and 2.5 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve except for the 32 mg/L test material vessel. Due to the linear portion of the respiration curve not being attained for the 32 mg/Ltest vessel, the oxygen consumption for this vessel was not calculated. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period. The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.

Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contact time.

Differential loading: Not applicable

Controls: The control group was maintained under identical conditions but not exposed to the test material.

Evidence of undissolved material (e.g. precipitate, surface film, etc): Observations made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10 and 32 mg/L contained a clear colourless water column with oily globules of test material visible on the surface. For the test concentrations of 100, 320 and 1000 mg/L an oily layer of test material was visible on the surface. Observations of the dosed activated sludge mixtures made after 30 minutes contact time showed the test concentration of 10 mg/L to contain a dark brown dispersion with the brown/undissolved matter considered to be attributed to the activated sludge solids and the absence of floating film or suspended micelles of undissolved test material visible. The test concentrations of 32 and 100 mg/L contained similar dark brown dispersions, but with visible floating globules of test material present. The floating globules of undissolved test material were increasingly prominent at the test concentrations of 320 and 1000 mg/L. These observations confirm that the test material was not substantially lost (via volatilization) following 30 minutes of stirring/suspension in the activated sludge test mixtures.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was 7.8 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Remarks on exposure duration:
Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes
Post exposure observation period:
Not applicable
Hardness:
The test water used for the definitive test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 2 (attached).
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.
pH:
The pH values of the test preparations at the start and end of the exposure period were 7.7 to 8.2.
Dissolved oxygen:
In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/L and 2.5 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases, except for the 32 mg/L test material vessel, the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace. Due to the linear portion of the respiration curve not being attained for the 32 mg/L test material vessel, the concentration response curve for the test material was plotted using the percentage inhibition values for the remaining four test material concentrations. A line could still be fitted using the Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line. The relatively low initial oxygen readings observed at the two lowest concentrations (10 and 32 mg/L) were likely to be due to increased rates of oxygen consumption resulting from biodegradation of the test material components at concentrations below their toxicity thresholds (i.e., <100 mg/L).
Salinity:
Not applicable
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
Preparation of test system: At time "0" 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 1 litre beaker (first control). The mixture was stirred via magnetic stirring. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. The test material vessels were prepared as described in the details of test solutions section. Finally a second control was prepared. As each vessel reached 30 minutes contact time an aliquot was removed from the beaker and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each vessel was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/L and 2.5 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve except for the 32 mg/L test material vessel. Due to the linear portion of the respiration curve not being attained for the 32 mg/L test vessel, the oxygen consumption for this vessel was not calculated. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.

Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 30 minutes contacontact time.


OTHER TEST CONDITIONS

Photoperiod: 30 minutes

Light intensity: Normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Evaluation of Data - In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage of the two control respiration rates.

% inhibition = [ 1 – 2RS ÷ RC1 + RC2 ] x 100
where
RS = oxygen consumption rate for test or reference sample
RC1+ RC2 = oxygen consumption rates for controls 1 and 2

The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC15, EC20, EC50 and EC80 values determined from the equation for the fitted line.

The equation of the fitted line for the reference material was as follows:
y = (A + (B*log(x)))
where
A = the vertical shift factor
B = the slope factor. A positive result is returned when y is increasing as x increases. A negative result is returned when y is increasing as x decreases
x = the original concentration values
y = the original inhibition values
The equation of the fitted line for the test material was as follows:
y = ((A+(B*x))+(((C-B)*(1-exp(((-1)*D)*x)))/D))
where
A = the y value when the value of x is 0
B = the initial rate of the curve
C = the steady state of the curve
D = the observed rate of the curve
x = the original concentration values
y = the original inhibition values
95% confidence limits were calculated for the EC50 values using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).

Whilst the test guidelines do not specify procedures for determination of a No Observed Effect Concentration (NOEC), a NOEC was determined which is equivalent to the EC15 value determined from an inhibition response curve and percentage inhibition values for the test material.
The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (30-Minute contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/L.


Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (Sigma-Aldrich Batch No. 04621CJ)
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
190 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence limits 150 - 240 mg/l.
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1. The pH values of the test preparations at the start and end of the exposure period are given in Table 2, and observations made on the test preparations throughout the study are given in Table 3.
Percentage inhibition is plotted against concentration for the test and reference materials (Figures 1 to 2).
The following results were derived from the described experimental procedure. Whereas some loss of the volatile test material components may have occurred during preparation and incubation of the test mixtures, a similar degree of loss of these materials could be expected upon their discharge to and treatment within a typical activated sludge wastewater treatment plant. Therefore, the results are expected to give a reasonable approximation of the potential effects of the test material on operation of such wastewater treatment processes:
Divinylbenzene
ECx (30 Minutes)
(mg/l) 95% Confidence Limits (mg/L)
EC20 70 -
EC50 190 150 - 240
EC80 560 -
NOEC 50 -

3,5-dichlorophenol
ECx (30 Minutes)
(mg/l) 95% Confidence Limits (mg/L)
3.0 -
9.6 6.6 - 14
31 -
2.4 -

Variation in respiration rates of controls 1 and 2 after 30 minutes contact time was ± 1%.
The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.



Results with reference substance (positive control):
- Results with reference substance valid? Yes

- Relevant effect levels: The reference material gave a 30-minute EC50 value of 9.6 mg/L, 95% confidence limits 6.6 - 14 mg/L.
Reported statistics and error estimates:
None.

Table 1: Oxygen Consumption Rates and Percentage Inhibition Values after 30 Minutes Contact Time

Nominal

Concentration

(mg/l)

Initial O2

Reading

(mg O2/L)

Measurement Period

(minutes)

Final O2Reading

(mg O2/L)

O2Consumption Rates

(mg O2/L/min)

% Inhibition

Control

R1

4.9

5

2.5

0.48

-

 

R2

6.5

8

2.6

0.49

-

Test Material

10

3.8

4

1.8

0.50

[3]

 

32

1.1

2

0.4

[1]

*

 

100

6.1

10

2.7

0.34

30

 

320

6.0

10

4.4

0.16

67

 

1000

5.6

10

5.0

0.06

88

3,5-dichlorophenol

3.2

6.2

10

2.4

0.38

22

 

10

7.4

10

5.0

0.24

51

 

32

7.5

10

6.6

0.09

81


R1– R2= Replicates 1 to 2

[1]Values not calculated due to very low start and finish oxygen concentrations

Table 2: pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal

Concentration

(mg/l)

pH

0 Hours

30 Minutes

Control

R1

7.7

7.7

 

R2

7.8

7.8

Test Material

10

7.7

7.8

 

32

7.8

7.8

 

100

7.8

7.9

 

320

7.8

8.0

 

1000

8.2

8.2

3,5-dichlorophenol

3.2

7.7

7.7

 

10

7.7

8.0

 

32

7.7

8.0

 


R1– R2= Replicates 1 to 2

Table 3: Observations on the Test Preparations Throughout the Test Period

Nominal

Concentration

(mg/l)

Observations on Test Preparations

0 Hours Contact Time

30 Minutes Contact Time

Control

R1

Dark brown dispersion

Dark brown dispersion

 

R2

Dark brown dispersion

Dark brown dispersion

Test Material

10

Clear colourless water column with oily globules of test material on surface*

Dark brown dispersion, no undissolved test material visible

 

32

Clear colourless water column with oily globules of test material on surface*

Dark brown dispersion, with a few globules of test material visible on surface

 

100

Clear colourless water column with an oily layer of test material on surface*

Dark brown dispersion, with a few globules of test material visible on surface

 

320

Clear colourless water column with an oily layer of test material on surface *

Dark brown dispersion, with globules of test material visible on surface

 

1000

Clear colourless water column with an oily layer of test material on surface *

Dark brown dispersion, with globules of test material visible on surface

3,5-dichlorophenol

3.2

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

10

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible

 

32

Dark brown dispersion, no undissolved reference material visible

Dark brown dispersion, no undissolved reference material visible


R1– R2= Replicates 1 to 2

*Observations made prior to the addition of synthetic sewage and activated sewage sludge

Concentration-Response Curve: 3,5-dichlorophenol – 30 Minutes Contact Time see attached figure 1

Concentration-Response Curve: Divinylbenzene HP– 30 Minutes Contact Time see attached figure 2

See Attached Appendix 1 for Certificate of Analysis

See atached Appendix 2 For Typical Water Quality Characteristics

REFERENCES

Litchfield, J T and Wilcoxon, F (1949) A Simplified Method of Evaluating Dose-Effect Experints. J Pharmacol Exp Ther 96, 99-113.

Xlfit® 4.2, (2005), ID Business Solutions Ltd.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 30-minute EC50 of 190 mg/L, 95% confidence limits 150 - 240 mg/L. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was 50 mg/L.
Executive summary:

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C.11 of Commission Regulation (EC) No. 440/2008 and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes as it was considered likely that any longer test duration would result in significant losses of test material from the test system. In order to minimise any losses of test material from the test system, rather than vigorous aeration of the test vessels, the test preparations were kept in suspension by stirring via magnetic stirrers. Activated sewage sludge was exposed to an aqueous dispersion of the test material at nominal concentrations of 10, 32, 100, 320 and 1000 mg/L for a period of 30 minutes at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 30 minutes contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 30-minute EC50 of 190 mg/L, 95% confidence limits 150 - 240 mg/L. Whilst the test guidelines do not specify procedures for determination of a No Observed Effect Concentration (NOEC), a NOEC was determined which is equivalent to the EC15 value determined from an inhibition response curve and percentage inhibition values for the test material. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was 50 mg/L. The reference material gave a 30-minute EC50 value of 9.6 mg/L, 95% confidence limits 6.6 - 14 mg/L.

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 30-minute EC50 of 190 mg/L, 95% confidence limits 150 - 240 mg/L. The No Observed Effect Concentration (NOEC) after 30 minutes exposure was 50 mg/L.

Description of key information

GLP-studies according to OECD guideline 209 are available for different grades of the reaction mass of divinylbenzene and ethylstyrene.

Key value for chemical safety assessment

EC50 for microorganisms:
190 mg/L
EC10 or NOEC for microorganisms:
50 mg/L

Additional information

Two studies on toxicity to microorganisms are available for the reaction mass of divinylbenzene and ethylstyrene one conducted with DVB-55 (reaction mass of approximately 55% divinylbenzene and 45% ethylstyrene) and one conducted with DVB-HP (reaction mass of approximately 80% divinylbenzene and 20% ethylstyrene). Both key studies were conducted under GLP and according to OECD guidelines 209. Due to the volatile nature of the test material, the test duration was reduced from 3 hours as specified in the Test Guidelines to 30 minutes as it was considered likely that any longer test duration would result in significant losses of test material from the test system. The DVB-HP grade proved to be more toxic to microorganisms in comparison to DVB-55. The 30 min EC50 for DVB-55 was 380 mg/L, while the 30 min EC50 for DVB-HP was 190 mg/L, the NOECs in these studies were 100 mg/L and 50 mg/L, respectively. No data is available for DVB-63 (reaction mass of approximately 63% divinylbenzene and 37% ethylstyrene). However, as higher concentrations of each of the components are present in the grades tested (DVB-55 and DVB-HP), the toxicity of both components of this reaction mass has been sufficiently investigated by the studies conducted on those grades.