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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Glass slides were loaded with the respective weight of the test material and added to the designated test vessel followed by the addition of deionised water (234 mL). The test material was mixed into the water by intense stirring for 30 minutes at room temperature. No emulsifiers or solvents were used. After stirring, the appearance of the test media was evaluated and the pH measured. The pH of the vessels with the highest loading rate was adjusted from pH 8.4-8.5 to pH 7.5-7.7. Synthetic sewage feed (16 mL) and activated sludge inoculum (250 mL) were added to give a final volume of 500 mL.
- Controls: Four controls without test material were tested in each test series, two at the start and two 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 washed twice with tap water (chlorine free) by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated. A calculated amount of wet sludge was resuspended in tap water to obtain a mixed liquor suspended solids (MLSS) content of 3 g dry material per liter. During the holding period of one day prior to use, the sludge was fed once with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again and readjusted to a MLSS of 3 g dry weight per liter. The pH of the activated sludge inoculum was adjusted from pH 6.8 to pH 7.7 with a diluted sodium hydroxide solution.
- Initial biomass concentration: 3.0 g MLSS/L
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 °C
pH:
test start: 7.1 - 7.5
test end: 7.5 - 8.0
Dissolved oxygen:
7.0 - 7.6 mg O2/L (initial DO concentration in the cultures)
Nominal and measured concentrations:
10, 100, 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 2000-mL beakers (incubation), biological oxygen demand (BOD) bottles (measurement of oxygen consumption rates)
- Aeration: intense stirring
- No. of vessels per concentration (replicates): 10 and 100 mg/L: 1; 1000 mg/L: 3
- No. of vessels per control (replicates): 4
- Biomass loading rate: 1.5 g/L (MLSS, dry weight)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water (<1 mg/L DOC)

OTHER TEST CONDITIONS
- Adjustment of pH: test solutions: from pH 8.4 - 8.5 to pH 7.5 - 7.7 (highest loading rate only); inoculum: from pH 6.8 to pH 7.7

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:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
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
Details on results:
All tested concentrations were clearly above the water solubility limit of the test material under the test conditions.

A clear stimulatory effect was observed for each concentration tested:
At 10 and 100 mg/L, respiration rates of activated sludge were 4 and 5% higher than in the controls.
At 1000 mg/L respiration rates were 13-25% higher than in the controls.
A statistical evaluation of the results was not performed, since no inhibition of the respiration was observed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 9 mg/L (95% confidence limits could not be calculated)
Reported statistics and error estimates:
The percentage inhibition of the respiration was plotted against logarithm of the test substance concentration.
- Test material: The NOEC was derived directly from the raw data. A statistical evaluation of the results was not necessary, since no inhibition of the respiration was observed. The 3 hour EC10, EC20, EC50 and EC80 values of the test material and their 95% confidence limits could not be calculated because of the absence of a toxic effect.
- Reference item: The 3 hour EC50 was calculated by Weibull Analysis using the software ToxRat Professional.

All test results were based on nominal concentrations of the test and reference item.
Validity criteria fulfilled:
yes
Remarks:
The specific respiration rates in the controls were ≥20 mgO2/gh and the coefficient of variation was ≤30%. The EC50 of 3,5-dichlorophenol was in the accepted range of 2-25 mg/L.
Conclusions:
The results indicate that the test material is not toxic to sewage bacteria up to the limits of its water solubility and a NOEC (EC10) and thus a PNEC cannot be determined.

Description of key information

Toxicity to microorganisms: not toxic to microorganisms: EC10 >1000 mg/L (OECD 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

EC10 for microroganisms > 1000 mg/L


The results indicate that the test material is not toxic to sewage bacteria up to the limits of its water solubility and a NOEC and thus a PNEC cannot be determined.