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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-03-2002 to 20-03-2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
(4 April 1984)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Commission Directive 87/302/EEC
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: February 2000; signature: April 2000
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test item was: (i) dispersed directly in water and also (ii) a single test concentration prepared using a preliminary solution in auxiliary solvent.
Within (i): 500 mg test item 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.
Within (ii): 529 mg test item was dissolved in acetone and the volume adjusted to 10 mL to give a 529 mg/10 mL solvent stock solution. An aliquot (50 µL) of this solvent stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (200 mL) and water, to a final volume of 500 mL, to give the required concentration of 5.29 mg/L. The volumetric flask containing the solvent stock solution was inverted several times to ensure homogeneity of the stock solution.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: Two stock solutions of 50 and 160 mg/L were prepared by dissolving the reference material directly in water with the aid of ultrasonic disruption. Aliquots (10 and 100 mL) of the 160 mg/L stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/L. A 100 mL aliquot of the 50 mg/L stock solution was used to prepare the 10 mg/L concentration.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): for (ii) using preliminary solution in auxiliary solvent: acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): (i) direct dispersion: 1000 mg/L and (ii) preliminary solution in auxiliary solvent: 5.29 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Within (i) at 1000 mg/L observations indicated the test item was visible as an oily slick on the surface. With globules of test item also visible on the surface. This was considered to be due to the insoluble nature of the test item in the test system at a concentration of 1000 mg/L. Within (ii) at 5.29 mg/L, no undissolved test item was visible during the study period.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: A mixed population of activated sewage sludge microorganisms was obtained from the aeration stage of the sewage treatment plant at Loughborough, UK (full details available in the full study report), which treats predominantly domestic sewage wastewater.
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at temperature of 21°C and was used on the day of collection. The pH of the sample was 7.6.
- Pretreatment: Not applicable.
- Initial biomass concentration: The suspended solids concentration in the inoculum was determined to be 4.1 g (dry weight) / L prior to use. In the preparation of the test system 200 mL inoculum was dissolved in 16 mL synthetic sewage and 300 mL water. Applicant assessment indicates: this should yield a final suspended solids concentration of ca. 1.64 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Respiration was measured at 30 minutes and 3 hours using a dissolved oxygen meter BOD probe
Post exposure observation period:
As each vessel reached 30 minutes contact time, an aliquot was removed from the conical flask and poured into the measuring vessel and the rate of respiration measured. 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 8.8 mg O2/1 and 1.0 mg O2/1). After measurement the contents of the BOD bottle were returned to the test vessel. This was repeated after 3 hours contact time.
Test temperature:
21 °C
pH:
3 hours: control: 7.7 and test groups: 7.6 - 7.7 and reference item: 7.8 - 7.9
Dissolved oxygen:
30 minutes: control: 6.9 – 7.1 mgO2/L and test groups: 1000 mg/L direct dispersion: 4.1 – 4.5 mgO2/L ; 5.29 mg/L auxiliary solvent: 5.3 mgO2/L ; solvent control: 6.2 mgO2/L and reference item: 7.3 - 8.7 mgO2/L
3 hours: control: 6.9 – 7.1 mgO2/L and test groups: 1000 mg/L direct dispersion: 4.8 – 5.3 mgO2/L ; 5.29 mg/L auxiliary solvent: 7.0 mgO2/L ; solvent control: 7.3 mgO2/L and reference item: 7.9 - 8.8 mgO2/L
Nominal and measured concentrations:
Range finder: nominal: 0 (control), 10, 100, 1000 mg/L and Reference item was completed: nominal: 3.2 and 32 mg/L
Definitive test (direct dispersion): nominal: 0 (control) and 1000 mg/L
Definitive test (auxiliary solvent): nominal: 0 (control) and 5.29 mg/L
Reference item was completed: nominal: 3.2, 5, 32 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL glass flasks
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Aeration: Yes, with stirring on magnetic stirrers but without foaming.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Definitive test (direct dispersion): Triplicate ; Definitive test (auxiliary solvent): single replicate
- No. of vessels per control (replicates): control: duplicate ; single (negative solvent control) and triplicate (reference item)
- Nitrification inhibitor used (delete if not applicable): Not applicable.
- Biomass loading rate: See table.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 through a Duplex Water Softener giving total hardness 100 mg/L CaCO3. Water quality characteristics is reported in the full study report.
- Culture medium different from test medium: Not applicable.
- Intervals of water quality measurement: Temperature, pH and dissolved oxygen values were determined in all test media and controls; prior to and at the end of the 3 hour incubation period.

OTHER TEST CONDITIONS
- Adjustment of pH: No. pH at end of test was in range 7.6 to 7.7 in controls and treatment groups.
- Light intensity: The test was conducted under normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biological Oxygen Demand. Monitor the oxygen consumed by the test and control mixtures following a 30-minute and 3-hour exposure phase.

TEST CONCENTRATIONS
- Test concentrations: Definitive test (direct dispersion): nominal: 0 (control) and 1000 mg/L
Definitive test (auxiliary solvent): nominal: 0 (control) and 5.29 mg/L
Reference item was completed: nominal: 3.2, 5, 32 mg/L
- Range finding study: Yes.
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Remarks on result:
other: direct dispersion
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
Remarks on result:
other: direct dispersion
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 5.29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: a preliminary solution in auxiliary solvent.
Remarks:
Greater than water solubility limit
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
- Effect concentrations exceeding solubility of substance in test medium: Yes.
- Adsorption (e.g. of test material to the walls of the test container): Not reported.
- Blank controls oxygen uptake rate: 0.69 to 0.79 mgO2/L/min
- Coefficient of variation of oxygen uptake rate in control replicates: The oxygen consumption rates of the two controls differed by 7% (guideline, recommended maximum variation: 15%).
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- Relevant effect levels: the EC50 for 3,5-dichlorophenol was: total respiration: actual 11 (C.I. – ) mg/L. This was within the expected range: 5 to 30 mg/L. Full information is provided in the full study report.

Table 1.0 – Definitive test results: 3 hours contact time

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 min)

% inhibition

Control R1

7.1

7

1.6

0.79

-

Control R2

6.9

8

1.4

0.69

-

Solvent Control

7.3

8

1.4

0.74

-

Test Item

 

 

 

 

 

5.29

7.0

8

1.2

0.73

1

Test Item

 

 

 

 

 

1000 R1

4.8

5

1.3

0.70

5

1000 R2

5.3

6

1.0

0.72

3

1000 R3

5.3

5

1.8

0.70

5

 

 

 

 

 

 

Positive Control

 

 

 

 

 

3.2

7.9

10

1.2

0.67

9

10

8.0

10

4.0

0.40

46

32

8.8

10

7.6

0.12

84

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 3 hour EC50 for total inhibition was > 1000 mg/L sed on nominal test item concentrations prepared by direct dispersion .
Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Following a preliminary range finding test (conducted over a range of test concentrations of 10, 100 and 1000 mg/L), activated sewage sludge was exposed in a definitive limit test to the test item at concentrations of 1000 mg/L (three replicate flasks) prepared by direct dispersion and 5.29 mg/L (single flask) prepared by preliminary solution in an auxiliary solvent (acetone) for a period of 3 hours at 21°C with the addition of synthetic sewage as a respiratory substrate examining total respiration under static conditions.Using this approach the effect of an excess amount of undissolved test item can be assessed in order to determine whether the test item has any effect on the activated sewage sludge microorganisms exoenzymes, or whether the uptake of undissolved test item by processes such as phagocytosis has any adverse effect on the activated sewage sludge. In addition the use of a test vessel at the limit of water solubility can show whether the soluble test item has an adverse effect on the activated sewage sludge. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The variation of oxygen uptake in the inoculum control vessels was 7%. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 11 mg/L. This confirmed the suitability of the activated sludge and method used. All validity criteria were considered to have been satisfied.In the single test vessel prepared using a preliminary solution in auxiliary solvent, to give a test concentration of 5.29 mg/L (the 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 > 5.29 mg/L and the NOEC was 5.29 mg/L.The effect of the test item on the respiration rate of activated sewage sludge at concentrations in excess of the water solubility level of the test item was also assessed using a test concentration of 1000 mg/L prepared by direct dispersion of the test item in water with the aid of ultrasonication. Under the conditions of this study, the test item total respiration EC50 was > 1000 mg/L. The NOEC was 1000 mg/L based on nominal test item concentrations.

Description of key information

ASRIT: EC50 = > 1000 mg/L (nominal), 21°C, OECD TG 209, 2002

ASRIT: NOEC = 1000 mg/L (nominal), 21°C, OECD TG 209, 2002

No effects below water solubility limit.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

Key study : OECD TG 209, 2002: The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. Following a preliminary range finding test (conducted over a range of test concentrations of 10, 100 and 1000 mg/L), activated sewage sludge was exposed in a definitive limit test to the test item at concentrations of 1000 mg/L (three replicate flasks) prepared by direct dispersion and 5.29 mg/L (single flask) prepared by preliminary solution in an auxiliary solvent (acetone) for a period of 3 hours at 21°C with the addition of synthetic sewage as a respiratory substrate examining total respiration under static conditions.Using this approach the effect of an excess amount of undissolved test item can be assessed in order to determine whether the test item has any effect on the activated sewage sludge microorganisms exoenzymes, or whether the uptake of undissolved test item by processes such as phagocytosis has any adverse effect on the activated sewage sludge. In addition the use of a test vessel at the limit of water solubility can show whether the soluble test item has an adverse effect on the activated sewage sludge. The respiration rates of the control, reference item (3,5-dichlorophenol) and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The variation of oxygen uptake in the inoculum control vessels was 7%. The three-hour 50% effect concentration for 3,5-DCP was estimated to be EC50 11 mg/L. This confirmed the suitability of the activated sludge and method used. All validity criteria were considered to have been satisfied.In the single test vessel prepared using a preliminary solution in auxiliary solvent, to give a test concentration of 5.29 mg/L (the 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 > 5.29 mg/L and the NOEC was 5.29 mg/L.The effect of the test item on the respiration rate of activated sewage sludge at concentrations in excess of the water solubility level of the test item was also assessed using a test concentration of 1000 mg/L prepared by direct dispersion of the test item in water with the aid of ultrasonication. Under the conditions of this study, the test item total respiration EC50 was > 1000 mg/L. The NOEC was 1000 mg/L based on nominal test item concentrations.