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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 April 2017 to 13 April 2017
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
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
Specific details on test material used for the study:
No additional information
Analytical monitoring:
no
Details on sampling:
Not applicable
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, 50 and 500 mg (in triplicate)) 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 a temperature of 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, 100 and 1000 mg/L (3 replicates).
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexi handheld meter (see Table 1).
- 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 12 April 2017 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 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (4.5 liters) was fed synthetic sewage (225 mL). The pH of the sample on the day of the test was 7.5 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 ºC 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 3.0 g/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:
Between 20 and 21 °C
pH:
0 hours: 7.3-7.4
3 hours: 7.3-7.7
Dissolved oxygen:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
Salinity:
Not applicable
Nominal and measured concentrations:
10, 100 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): 1 for 10 mg/L; 1 for 100 mg/L and 3 replicates for 1000 mg/L
- No. of vessels per control (replicates): 4
- 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: The pH of the synthetic sewage stock used to feed the activated sewage sludge was pH 7.3 and the pH of the synthetic sewage stock used for the range finder test was pH 7.3. 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: No statistically significant toxic effects were shown at all of the test concentrations employed in the range-finding test. It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
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)

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

5.8

6

1.9

39.00

-

R2

5.4

6

1.5

39.00

-

R3

4.8

4

2.3

37.50

-

R4

5.4

5

2.1

39.60

-

Test item

10

5.3

5

2.0

39.60

[2]

100

4.5

4

2.0

37.50

3

1000 R1

4.4

4

1.9

37.50

3

1000 R2

5.4

5

2.3

37.20

4

1000 R3

5.6

5

2.7

34.80

10

3,5-dichlorophenol

3.2

6.0

8

2.3

27.75

28

10

7.0

10

4.3

16.20

58

32

8.0

10

7.2

4.80

88

R1 – R4 = Replicates 1 to 4

[Increase in respiration rate as compared to controls] 

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 2.31% and the specific respiration rate of the controls was 25.85 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 EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
Executive summary:

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

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 20 and 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 effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

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

The reference item gave a 3-Hour EC50 value of 7.4 mg/L, 95% confidence limits 5.7 to 9.6 mg/L.

Description of key information

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

No statistically significant toxic effects were shown at all of the test concentrations employed.

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

3h-EC10 (activated sludge) = 1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 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. Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 20 and 21 °C with the addition of a synthetic sewage as a respiratory substrate. No statistically significant toxic effects were shown at all of the test concentrations employed. The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

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