Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th January 2002 (range finding test); 24th January 2002 (definitive test)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: Experimental 11334-18

Data relating to the identity, purity and stability of the test material are the responsibility of the Sponsor.

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
Not applicable

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: For the purpose of the definitive test, the test material was dispersed directly in water or prepared using a preliminary solution in dimethylformamide.
An amount of test material (500 mg) was dispersed in approximately 250 mL of water and subjected to ultrasonication (approximately 30 minutes). Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentration of 1000 mg/L. This method of preparation was performed for each of 3 replicate vessels.
Additionally, the definitive test also consisted of a single test concentration prepared using a preliminary solution in dimethylformamide. An amount of test material (47.6 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give an 47.6 mg/10 mL stock solution from which a dilution was made to give a 4.76 mg/10 mL solvent stock solution. An aliquot (50 µL) of the 4.76 mg/10 mL stock solution was dispersed with activated sewage sludge (200 mL), synthetic sewage (16 mL) and water, to a final volume of 500 mL to give the test concentration of 0.0476 mg/L.
The volumetric flasks containing the solvent stock solutions were inverted several times to ensure homogeneity of the stock solutions.
Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.
A standard synthetic sewage was added to each test vessel to act as a respiratory substrate.
- Controls: At time "0" 16 mL of synthetic sewage was diluted to 300 mL with water and 200 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added. A solvent control vessel was prepared containing 16 mL synthetic sewage, 300 mL water, 50 µL of dimethylformamide and 200 mL of inoculum in a 500 mL conical flask. Finally a second control was prepared.
- Chemical name of vehicle (organic solvent): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not specified
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The 0.0476 mg/L test samples were dark brown dispersions with no undissolved test material visible. The 1000 mg/L test samples were dark brown dispersions with many white particles of undissolved test material on the surface and dispersed throughout the test medium.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Not applicable
- Source: A mixed population of activated sewage sludge micro-organisms was obtained on 24 January 2002 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 21°C and was used on the day of collection. The pH of the sample was 7.6 measured using a WTW pH 320 pH 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 piece of pre-weighed GFIA filter paper using a Buchner funnel. The paper was then dried in an oven at approximately 110°C for approximately 1 hour and allowed to cool before re-weighing. The suspended solids was equal to 3.9 g/L prior to use.
- Pretreatment: None specified
- Initial biomass concentration: Not specified

Study design

Test type:
other: aerobic
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
Not performed

Test conditions

Hardness:
Mean: 176 mg/L CaCO3
Test temperature:
21 °C
pH:
pH of the inoculum: 7.6
pH values of the test preparations at the end of the exposure period:7.6-7.9
Dissolved oxygen:
Not specified
Salinity:
Not specified
Nominal and measured concentrations:
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.
Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/L (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.
In addition to this a test concentration of 0.0476 mg/L was prepared using a preliminary solution in auxiliary solvent to confirm that at the limit of water solubility (data supplied by the Sponsor) no effect on respiration of the activated sewage sludge was observed.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (open / closed): Not specified
- Material, size, headspace, fill volume: Standard glass conical flasks of 500 mL capacity.
- Aeration: The mixtures were aerated with compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: Not applicable
- No. of vessels per concentration (replicates): 3 replicates at the 1000 mg/L dose level; 1 vessel at the 0.0476 mg/L dose level in the definitive test.
- No. of vessels per control (replicates): 2 replicates
- No. of vessels per vehicle control (replicates): 1 vessel (the solvent control was exposed to 100 µL/L of dimethylformamide)
- Biomass loading rate: Not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for both the range-finding and definitive tests was laboratory tap water dechlorinated by passage through an activated carbon filter and partly softened.
- Total organic carbon: Mean value: 1 mg/L
- Particulate matter: Not specified
- Metals: Various metals are present in the water at differing concentrations
- Pesticides: <0.01 µg/L
- Chlorine: Total chlorine (mean value): 0.156 mg/L
- Alkalinity: Mean pH: 7.804
- Ca/mg ratio: Ca: 54 mg/L; Mg: 10 mg/L
- Conductivity: Mean value: 413267 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Observations were made on the test preparations throughout the test period, and the pH of the control, reference material and test material preparations were measured at the end of the exposure period prior to measurement of the oxygen consumption rate.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: None specified
- Light intensity: Normal laboratory lighting

EFFECT PARAMETERS MEASURED:
As each vessel reached 30 minutes 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 for an approximate 10 minute period (between approximately 8.9 mg O2/L and 1.3 mg O2/L). After measurement the contents of the BOD bottle were returned to the test vessel.
This procedure was repeated after 3 hours contact time.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
- Justification for using less concentrations than requested by guideline: The definitive study was performed as a limit test.
- Range finding study
- Test concentrations: 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. The test material was dispersed directly in water. Amounts of test material (5.0, 50 and 500 mg) were separately dispersed in approximately 250 mL of water and subjected to ultrasonication (approximately 30 minutes). Synthetic sewage (16 mL), activated sewage sludge (200 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L.
The control group was maintained under identical conditions but not exposed to the test material.
A reference material, 3.5-dichlorophenol, was included in the range-finding test at concentrations of 3.2 and 32 mg/L in order to confirm the suitability of the inoculum.
Oxygen consumption rates were measured after 3 hours contact time only.
- Results used to determine the conditions for the definitive study: Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the range-finding test are given in Table 1.
No significant effect on respiration was observed at any of the test concentrations employed. Based on this information, a single test concentration, in triplicate, of 1000 mg/L was selected for the definitive test.
Data supplied by the Sponsor indicated the water solubility of the test material to be 0.0476 mg/L therefore in order to satisfy the requirements of all Competent Authorities worldwide a single test concentration of 0.0476 mg/L prepared using a preliminary solution in auxiliary solvent was also included in the study.
Using this approach the effect of an excess amount of undissolved test material can be assessed in order to determine whether the test material has any effect on the activated sewage sludge micro-organisms exoenzymes, or whether the uptake of undissolved test material by processes such as phagocytosis has any adverse effect on the activated sewage sludge. In addition the use of a test vessel at the maximum attainable test concentration that is a solution can show whether soluble test material has an adverse effect on the activated sewage sludge.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 0.048 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: Test performed at the limit of water solubility
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations made throughout the study period, showed that at the test concentration of 1000 mg/L, white particles of undissolved test material were visible on the surface and dispersed throughout the test media at 0 hours, 30 minutes and 3 hours contact time. This was considered to be due to the insoluble nature of the test material in the test media. However at the test concentration of 0.0476 mg/L no undissolved test material was visible in a dark brown dispersion at 0 hours, 30 minutes and 3 hours contact time.
- Effect concentrations exceeding solubility of substance in test medium: The 1000 mg/L test samples exceeded the solubility of the test material in the test medium.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes
- Relevant effect levels:
EC50 (30 minutes): 20 mg/L
EC50 (3 hours): 13 mg/L
Reported statistics and error estimates:
No data available

Any other information on results incl. tables

Variation in respiration rates of controls 1 and 2 after 30 minutes: 2%.

Variation in respiration rates of controls 1 and 2 after 3 hours: ± 4%.

The validation criteria for the control respiration rates and reference material EC50 values have been satisfied.

Table 1: Oxygen Consumption Rates and Percentage Inhibition Values in the Range-finding Test

Nominal concentration (mg/L)

Initial O2reading (mg O2/L)

Measurement period (minutes)

Final O2reading (mg O2/L)

O2consumption rates (mg O2/L/min)

% inhibition

Control

R1

6.9

9

2.7

0.47

-

 

R2

7.1

9

2.6

0.50

-

Test material

10

7.1

8

2.6

0.56

[15]

 

100

6.6

8

2.3

0.54

[11]

 

1000

6.1

7

2.5

0.51

[5]

3,5-dichlorophenol

3.2

6.5

9

2.5

0.44

9

 

32

8.1

10

7.2

0.09

81

Table 2:Oxygen Consumption Rates and Percentage Inhibition Values in theDefinitive Test after 3 Hours Contact Time

Nominal concentration (mg/L)

Initial O2reading (mg O2/L)

Measurement period (minutes)

Final O2reading (mg O2/L)

O2consumption rates (mg O2/L/min)

% inhibition

Control

R1

7.1

8

2.3

0.60

-

R2

6.7

8

2.3

0.55

-

Solvent control

7.3

9

2.2

0.57

-

Test material

0.0476

7.2

10

1.9

0.53

7

1000 R1

6.5

9

1.5

0.56

3

1000 R2

6.3

8

1.8

0.56

3

1000 R3

6.3

9

1.3

0.56

3

3,5-dichlorophenol

3.2

6.8

8

2.4

0.55

4

10

7.9

10

4.2

0.37

36

32

8.9

10

8.0

0.09

84

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3 hour EC50 of greater than 1000 mg/L. The NOEC after 3 hours exposure was 1000 mg/L.
In the single test vessel prepared using a preliminary solution in auxiliary solvent to give a test concentration of 0.0476 mg/L (limit of water solubility), no significant inhibition of the respiration rate of activated sewage sludge was observed and hence the 3-Hour EC50 value was determined to be greater than 0.0476 mg/L. Correspondingly the NOEC determined from this vessel was 0.0476 mg/L. These results show that at the limit of water solubility no effect on the respiration rate of activated sewage sludge micro-organisms was observed.
Executive summary:

The inhibitory effect of Millad 3988 on the respiration of activated sewage sludge was assessed in a study performed in accordance with OECD Guideline No. 209.

In the single test vessel prepared using a preliminary solution in auxiliary solvent to give a test concentration of 0.0476 mg/L (the limit of water solubility supplied by the Sponsor) no significant inhibition of the respiration rate of activated sewage sludge was observed and hence the 3-Hour EC50 value was determined to be greater than 0.0476 mg/L. Correspondingly the NOEC determined from this vessel was 0.0476 mg/L. These results show that at the limit of water solubility of the test material, the test material has no effect on the respiration rate of activated sewage sludge.

The effect of the test material on the respiration rate of activated sewage sludge at concentrations in excess of the water solubility level of the test material was also assessed using a single test concentration of 1000 mg/L prepared by direct dispersion of the test material in water with the aid of ultrasonication. At a test concentration in excess of the water solubility level of the test material inhibition of respiration of the activated sewage sludge did not occur and the 3-Hour EC50 value was determined to be greater than 1000 mg/L. Correspondingly the NOEC was determined to be 1000 mg/L. These results show that at a concentration in excess of the water solubility level of the test material, the test material does not have an effect on the respiration rate of activated sewage sludge.