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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:
other: According to international guideline(s), GLP-compliant, performed in recognized contract research organization, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
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 all vessels was adjusted from pH 6.0-6.1 to pH 7.6. Synthetic sewage feed (16 mL) and of the 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 6.6 to pH 7.5 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: 7.3-7.4
test end: 7.5-7.9
Dissolved oxygen:
7.7-8.2 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 6.0-6.1 to pH 7.6; inoculum: from pH 6.6 to pH 7.5

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
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
Details on results:
The three replicates of the highest loading rate were clearly above the water solubility limit of the test material under the test conditions.

At all tested concentrations up to and including 1000 mg/L, the inhibition of the respiration rate of activated sludge after the incubation period of three hours was <10% compared to the control. For the three replicates of the highest test concentration of 1000 mg/L respiration rates were 92.2, 95.6 and 97.4%, compared to the control.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 4 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. NOEC/ECx calculations were done using the software ToxRat Professional.
The NOEC was derived by per-vessel statistics using the Student t-test. Compared were the three replicates of the highest test item concentration with the four blank control replicates using one-tailed (smaller) hypothesis and α = 0.05.
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.
The 3 hour EC50 of the reference item 3,5 dichlorophenol was calculated by Probit Analysis.
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/g/h and the coefficient of variation was ≤30%. The EC50 of 3,5-dichlorophenol was in the accepted range of 2-25 mg/L.

Description of key information

Toxicity to microorganisms: not toxic to microorganisms: inhibition of respiration rates <10% (OECD 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The results indicate that the test material is not toxic to sewage bacteria up to the highest recommended test concentration and a NOEC and thus a PNEC cannot be determined.