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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 2006-01-31 to 2006-03-06
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
Version / remarks:
1984
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
Commission Directive 88/302/EEC: "Activated Sludge Respiration Inhibition Test", Official Journal of the European Communities No. L 133/118-122, May 30, 1988
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Due to the low solubility of the test item in test water, adequate amounts of the test item were directly weighed into each test flask.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: sewage plant Darmstadt, Germany
- Preparation of inoculum for exposure: The washed sludge suspension was made up with tap water and 50 mL synthetic sewage feed per litre was added and the sludge was kept at room temperature under continuous aeration for two days until use. Immediately before use, the dry weight of the activated sludge was determined, and diluted to 2 g/L with tap water. The pH of the activated sludge was 7.2.
- Pretreatment: The sludge was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and again centrifuged. The latter procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge (g) to its dry weight (g) determined.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.0 - 22.9 °C
pH:
7.1 - 8.1
Dissolved oxygen:
6.9 - 8.5 mg/L
Nominal and measured concentrations:
Nominal concentrations: 10, 32, 100, 320, 1000 mg/L
No measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks of approximately 1 litre volume and Karlsruher flasks of 250 mL volume
- Aeration: with compressed air (approximately 0.720 litre per minute) before use, during the 3-h exposure the test flasks were not aerated

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED: Oxygen concentration was measured with an oxygen electrode.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
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:
inhibition of total respiration
Remarks:
respiration rate
Key result
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 on result:
other: % inhibition ranged between -13.8 and 6.9 (i. e. always < 10%) for all tested concentrations and is thus considered to reflect biological variation rather than a test item effect. Moreover, there was no concentration-response relationship observed.
Results with reference substance (positive control):
The following nominal concentrations of the positive control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 32, 10 and 3.2 mg/L. In comparison to the controls the respiration rate of the activated sludge was moderately inhibited by 13.8 % at the lowest nominal concentration of 3.2 mg/L. At the nominal concentrations of 10 and 32 mg test item/L, the respiration rate was inhibited by 41.4 and 82.8 %, respectively. The 3-hour EC50 of the positive control 3,5-Dichlorophenol, which was tested in the same way as the test substance, was found to be 12 mg/L and is within the range of 5 - 30 mg/L recommended by the test guidelines; thus, confirming suitability of the activated sludge.

Toxicity of Test Item:

In comparison to the inoculum controls the respiration rates of the activated sludge was slightly inhibited between -13.8 % and 6.9 % up to the highest tested concentration of nominal 1000 mg/L.

Table 1. Influence of the test item on Oxygen Consumption of Activated Sludge

Flask
No.

Treatment

Concentration

mg/L

Oxygen
consumption

mg O2/L min

Inhibition
%

pH-values

Oxygen
concentration

mg O2/L

start*

end*

start*

end*

1

control

-

0.313

-

7.1

8.1

8.0

7.9

10

control

-

0.267

-

7.1

81

8.2

7.1

 

 

mean

0.290

 

 

 

 

 

 

replicate deviation

14.7

 

 

 

 

 

5

test item

1000

0.300

-3.4

7.1

7.9

8.0

6.7

6

test item

320

0.290

0

7.1

8.0

8.1

7.2

7

test item

100

0.270

6.9

7.1

7.9

8.0

6.9

8

test item

32

0.330

-13.8

7.1

8.0

8.1

7.2

9

test item

10

0.300

-3.4

7.1

8.0

8.2

7.1

* start and end of 3-hour aeration

3-hour EC20/80 and EC50 of Test Substance:

Based on measured inhibition rates, the 3-hour EC20/80 and EC50 could not be quantified because up to the highest nominal test concentration of 1000 mg/L less than 10 % inhibition was noted after three hours incubation. Nevertheless, the 3-hour EC20/80 and EC50 are clearly higher than 1000 mg/L under the present test conditions. Since the % inhibition ranged between -13.8 and 6.9 and was always < 10 % for all tested concentrations, it is considered to reflect biological variation rather than a test item effect. Moreover, there was no concentration-response relationship observed. Thus, the NOEC is considered to be >= 1000 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The toxic effects of the test substance on the oxygen consumption rate of aerobic microorganisms (activated sludge) after of 3 h was determined according to the OECD 209 (1984). The EC50 was determined to be > 1000 mg/L.
Executive summary:

The objective of this study performed according to OECD TG 209 (1984) under GLP conditions was to determine the toxic effects of the test substance on the oxygen consumption rate of aerobic microorganisms (activated sludge) after short-term exposure of 3 h, compared with the blank inoculum control. The test was performed in nominal concentrations of 0 (control), 10, 32, 100, 320 and 1000 mg/L. No concentrations were measured analytically. The inoculum was activated sludge from the municipal waste treatment plant of Darmstadt in Germany. The sludge concentration was 2 g/L (dry weight) and a mineral medium according to the guideline was used. A reference substance, 3,5-dichlorphenol, was used as positive control. The EC50 value of the reference substance 3,5-dichlorphenol was determined to be 12 mg/L, which was within the acceptable limits of 5.0 to 30 mg/L, as specified in OECD TG 209. All validation criteria were fulfilled. The EC50 was determined to be > 1000 mg/L. The NOEC was determined to be >= 1000 mg/L.

Description of key information

The toxic effects of the test substance on the oxygen consumption rate of aerobic microorganisms (activated sludge) after of 3 h was determined according to the OECD 209 (1984). The EC50 was determined to be > 1000 mg/L. The NOEC was determined to be >= 1000 mg/L (reference 6.1.7 -1).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The objective of this study performed according to OECD TG 209 (1984) under GLP conditions was to determine the toxic effects of the test substance on the oxygen consumption rate of aerobic microorganisms (activated sludge) after short-term exposure of 3 h, compared with the blank inoculum control. The test was performed in nominal concentrations of 0 (control), 10, 32, 100, 320 and 1000 mg/L. No concentrations were measured analytically. The inoculum was activated sludge from the municipal waste treatment plant of Darmstadt in Germany. The sludge concentration was 2 g/L (dry weight) and a mineral medium according to the guideline was used. A reference substance, 3,5-dichlorphenol, was used as positive control. The EC50 value of the reference substance 3,5-dichlorphenol was determined to be 12 mg/L, which was within the acceptable limits of 5.0 to 30 mg/L, as specified in OECD TG 209. All validation criteria were fulfilled. The EC50 was determined to be > 1000 mg/L and the NOEC was determined to be >= 1000 mg/L (reference 6.1.7-1).