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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January to 09 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 209 with GLP statement:
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 17 June 2015 / signed on 24 September 2015)
Specific details on test material used for the study:
No additional information
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- The test item was dispersed directly in water.
Nominal amounts of test item (5.0, 16, 50, 160 and 500 mg (five replicates of each)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20 °C and 21 °C, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 32, 100, 320 and 1000 mg/L (five replicates of each). The pH of the test item dispersions were measured after stirring using Hach HQ40d Flexi handheld meter and adjusted to between pH 7.0 to pH 8.0 if necessary.
- Controls: Control group was maintained under identical conditions but not exposed to the test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected:
A mixed population of activated sewage sludge micro-organisms was obtained on 14 January 2016 for the range-finding test and on 8 February 2016 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Preparation of inoculum for exposure:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C overnight prior to use in the test. On the day of collection the activated sewage sludge (10 liters) was fed synthetic sewage (500 mL). The pH of the sample on the day of the test was 7.7 measured using a Hach HQ40d Flexi handheld 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 deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0g/L prior to use.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
Not applicable
Test temperature:
ca. 21 °C
pH:
0 hours: 7.0-7.6
3 hours: 7.2-7.5
Dissolved oxygen:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.
Salinity:
Not applicable
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flask
- At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control).
- Aeration: The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- Sludge concentration (weight of dry solids per volume): Suspended solids concentration was equal to 3.0g/L prior to use

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the range-finding and definitive tests was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
- Synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate: 16 g Peptone; 11 g Meat extract; 3 g Urea; 0.7 g NaCl; 0.4 g CaCl2.2H2O; 0.2 g MgSO4.7H2O; 2.8 g K2HPO4 dissolved in a final volume of 1 liter of water with the aid of ultrasonication.

OTHER TEST CONDITIONS
- pH: In the range-finding test, the pH of the synthetic sewage stock was pH 7.4. In the definitive test, the pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.4 and the pH of the synthetic sewage stock used for the definitive test was pH 7.4. The pH values were measured using a Hach HQ40d Flexi handheld meter.
- Incubation: Test was conducted under normal laboratory lighting in a temperature controlled room at measured temperature of approximately 21 ºC.

EFFECT PARAMETERS MEASURED
- Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.
- pH Measurements: The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3 hour incubation period using a Hach HQ40d Flexi handheld meter.
- Oxygen Concentration: The oxygen concentrations in all vessels were measured after 30 minutes contact time.
- Measurement of the Respiration Rates: As each vessel reached 3 hours contact time an aliquot was removed from the conical flask 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 flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 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. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

TEST CONCENTRATIONS
- Range finding study: In the range-finding test activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L.
- Results used to determine the conditions for the definitive study: The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L. No statistically significant toxic effects were shown at the test concentration of 10 mg/L, however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L. Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain both a NOEC and an EC10 and EC50 value for the test item. Based on this information test concentrations of 10, 32, 100, 320 and 1000 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (3.2, 10 and 32 mg/L)
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: inhibition of respiration rate
Details on results:
- No statistically significantly toxic effects were shown at the test concentrations of 10 and 32 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentrations of 100, 320 and 1000 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 32 mg/L. It was not possible to determine an EC50 or EC80 value for the test item as no concentration tested resulted in greater than 50% inhibition.
- In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.
- The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.
Results with reference substance (positive control):
The reference item gave a 3 hour EC50 value of 8.1 mg/L, 95% confidence limits 6.4 to 10 mg/L.
Reported statistics and error estimates:
The percentage inhibition values were plotted against concentration, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.
The EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups in order to determine where the NOEC lies. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 6.1.7/1: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Definitive Test

Nominal Concentration (mg/L)

 

Replicates

Initial O2 Reading (mg O2/L)

 

Measurement Period (minutes)

 

Final O2 Reading (mg O2/L)

 

O2 Consumption Rates (mg O2/L/hour)

 

% Inhibition

 

Control

R1

4.6

4

1.5

46.50

-

R2

4.8

4

1.8

45.00

-

R3

5.0

4

2.1

43.50

-

R4

4.0

3

1.7

46.00

-

R5

6.4

7

2.0

37.71

-

R6

4.7

4

2.0

40.50

-

Test substance

10

R1

4.0

3

1.7

46.00

[6]

R2

5.6

5

2.2

40.80

6

R3

3.9

3

1.7

44.00

[2]

R4

4.1

3

1.9

44.00

[2]

R5

4.6

4

1.8

42.00

3

32

R1

4.1

3

1.9

44.00

[2]

R2

3.0

1

2.2

48.00

[11]

R3

3.6

2

2.1

45.00

[4]

R4

5.0

4

2.0

45.00

[4]

R5

3.1

2

1.7

42.00

3

100

R1

3.5

2

2.2

39.00

10

R2

4.1

4

1.9

33.00

24

R3

3.5

2

2.2

39.00

10

R4

5.7

7

2.0

31.71

27

R5

3.2

10

2.0

36.00

17

320

R1

4.1

4

2.0

31.50

27

R2

4.5

5

2.0

30.00

31

R3

4.6

5

2.1

30.00

31

R4

4.5

5

2.1

28.80

33

R5

5.1

6

2.0

31.00

28

1000

R1

6.7

10

2.8

23.40

46

R2

4.0

4

2.3

25.50

41

R3

5.6

8

2.2

25.50

41

R4

5.9

9

2.3

24.00

44

R5

5.4

8

2.1

24.75

43

3,5-dichlorophenol

3.2

R1

6.3

4

1.9

33.00

24

10

R1

6.8

8

3.6

19.20

56

32

R1

7.8

10

6.9

5.40

88

R1 – R5 = Replicates 1 to 5

R1 – R6 = Replicates 1 to 6

[Increase in respiration rate as compared to controls] 

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 8.0% and the specific respiration rate of the controls was 28.80 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

The validation criterion for the reference item EC50 value was also satisfied.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge gave a 3 hour EC10 value of 75 mg/L and a 3 hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 32 mg/L.
Executive summary:

An activated sludge respiration inhibition test was performed according to OECD Guideline 209 with GLP compliance.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 32, 100, 320 and 1000 mg/L (5 replicates) for a period of 3 hours at a measured temperature of approximately 21 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L. No statistically significantly toxic effects were shown at the test concentrations of 10 and 32 mg/L, however statistically significantly toxic effects (P < 0.05) were shown at the test concentrations of 100, 320 and 1000 mg/L. It was not possible to determine an EC50 or EC80 value for the test item as no concentration tested resulted in greater than 50% inhibition.

The reference item gave a 3 hour EC50 value of 8.1 mg/L, 95% confidence limits 6.4 to 10 mg/L. The validation criterion for the reference item EC50 value was satisfied.

Under the test conditions, the effect of the test item on the respiration of activated sewage sludge gave a 3 hour EC10 value of 75 mg/L and a 3 hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 32 mg/L.

Description of key information

OECD Guideline 209, GLP, key study, validity 1:

3h-EC10 (activated sludge) = 75 mg/L;

3h-EC50 (activated sludge) > 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
75 mg/L

Additional information

One valid key study is available to assess the toxicity of the registered substance to microorganisms.

In this study, the effect of registered substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours, according to OECD Guideline 209 with GLP statement. Following a preliminary range-finding test, the final test was performed with five loading rates: 10, 32, 100, 320 and 1000 mg/L. Five replicates per loading rate and six replicates for an untreated control group were tested. No statistically significant inhibition of the respiration rate of the activated sewage sludge was recorded at 10 and 32 mg/L, however statistically significantly toxic effects were shown at the test concentrations of 100, 320 and 1000 mg/L.

In conclusion, the effect of the test substance on the respiration of activated sewage sludge gave a 3 hour EC10 value of 75 mg/L and a 3 hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 32 mg/L. NOECs are increasingly contested in ecotoxicology because the NOEC is determined as the concentration directly below the LOEC (Lowest Observed Effect Concentration) and depend upon the choice of the test concentrations and is not a statistically interpolated value. Therefore, to use a less selective method, the EC10 value was taken into account for the chemical safety assessment.