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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance is poorly soluble in water. At test initiation, nominal concentrations of the test substance were established by the addition of weights of the test substance to the respective vessel followed by the addition of RO (reverse osmosis) water (284 mL). Additions of synthetic sewage (16 mL) and microbial inoculums (200 mL) were made to give a final volume of 500 mL.
- Controls: Two controls without test material were tested in each test series, one at the start and one at the end.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Preparation of inoculum for exposure: A sample of activated sludge was obtained the day before the start of the test. The mixed liquor suspended solids (MLSS) content was determined, synthetic sewage (50 mL/L) was added and the mixture was aerated overnight.
- Pretreatment: none
- Initial biomass concentration: ca. 4.0 g MLSS/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
19-20 °C
pH:
test start: 7.3-8.0
test end: 7.9-8.6
Dissolved oxygen:
7.0-8.8 mg O2/L (initial DO concentration in the cultures)
Nominal and measured concentrations:
10, 30, 100, 300, 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers (test bottles) and 270 mL biological oxygen demand (BOD) bottles
- Aeration: oil-free compressed air at 1 L/min
- No. of vessels per concentration (replicates): 10-300 mg/L: 1; 1000 mg/L: 3
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1.6 g/L (MLSS)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis (RO) water

OTHER TEST CONDITIONS
- Adjustment of pH: to 6.5-8.5 with 5 N NaOH or HCl as required

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen concentration after 3 hours contact time
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:
other: EC20
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:
other: EC80
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 7.6 mg/L (95% confidence limits, 5.4 to 10.5 mg/L)
Reported statistics and error estimates:
The EC20, EC50 and EC80 of the test substance and EC50 of the reference substance and their 95% confidence limits were calculated using the SAFEstat curvefit programme (SAS Institute, 2002). Since the permitted maximum difference for specific respiration rates in control beakers established at the beginning and end of the test period was 15%, this value was used to define the criteria for biologically significant levels of inhibition in mixtures containing the test substance.

Respiration rates of samples of activated sludge were decreased by 1% to 5% between 10 and 300 mg/L. In triplicate mixtures at 1000 mg/L, the respiration rates were decreased by 10% to 25% (mean 19%). Consequently, the EC20, EC50 and EC80 could not be calculated as these were greater than 1000 mg/L.

Although inhibition at 1000 mg/L was considered to be biologically significant, concentrations above 1000 mg/L was not likely to give an EC50 because inhibition would most likely to have been limited by the water solubility of the test substance. Therefore, no further testing was carried out.

Table 1 Percentage inhibition

Test mixture

% Inhibition

Control 1

-

WS400152 (mg/L)

10

1

30

3

100

5

300

5

1000

22

1000

25

1000

10

Control 2

-

 

Validity criteria fulfilled:
yes
Remarks:
Variations in respiration rates of controls <15%. The EC50 of 3,5-dichlorophenol was in the accepted range of 5-30 mg/L.

Description of key information

Toxicity to microorganisms: mean inhibition of respiration rates 19% at nominal 1000 mg/L; EC50 > 1000 mg/L (OECD 209, EU C.11).

Key value for chemical safety assessment

Additional information

EC50 and EC20 for microorganisms: > 1000 mg/L.

The EC50 for the test material is much higher than its water solubility. A NOEC cannot be determined.