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Ecotoxicological information

Toxicity to microorganisms

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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
Reference:
Composition 0
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
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). The pH of the mixtures was then determined and adjusted to 6.5 - 8.5 with 5N NaOH/HCl as required. 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:
19.3-19.8 °C
pH:
test start: 7.5-7.6
test end: 8.1-8.3
Dissolved oxygen:
6.0-7.2 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): 11.2 mg/L (95% confidence limits 8.8 to 14.4 mg/L)
Reported statistics and error estimates:
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.
The EC50 of the reference substance and 95% confidence limits were calculated using the SAFEstat curvefit programme (SAS Institute, 2002).

Respiration rates were not inhibeted at any test substance concentration (0%).

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: not toxic to microorganisms: inhibition of respiration rates <10% (OECD 209, EU C.11)

Key value for chemical safety assessment

EC10, LC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The EC10 for the test material is much higher than its water solubility.