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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:
03 August 2017 to 17 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Purity: > 90%
- Description: Brown viscous liquid
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Nominal amounts of test item (5, 50 and 500 mg) were each separately weighed onto GF/A filter paper (70 mm) prior to being dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures between 19 ºC and 21 ºC, to maximize the dissolved test item concentration.

Single test vessel replicates were prepared for the two lower concentrations and three replicates were prepared for the highest concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration).

The pH of the test item dispersions was measured after stirring using a handheld pH meter and adjusted to between pH 7.0 and 8.0, as necessary.

A filter paper was added to each control vessel to ensure consistency between the test and control vessels. The control group was maintained under identical conditions, but was not exposed to the test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 19 to 21 ºC and was used on the day of collection. The pH of the sample was 7.5 measured using a handheld pH meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel, which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until two consecutive dry weights within 4% were attained. The suspended solids concentration was equal to 3.0 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 19 °C and 21 °C.
pH:
Adjusted to between pH 7.0 and 8.0.
Dissolved oxygen:
Ranged from 6.1 to 8.7 mg O2/L.
Nominal and measured concentrations:
Nominal test concentrations: 10, 100 and 1000 mg/L.
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
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
Details on results:
No statistically significant toxic effects were shown at any of the test concentrations employed (P <0.05). It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary to test at concentrations in excess of 1000 mg/L.
Results with reference substance (positive control):
The EC50 value for the reference item satisfied the validation criterion.
Validity criteria fulfilled:
yes
Conclusions:
No effects of the test item were observed on the respiration of activated sewage sludge following a 3-hour exposure, resulting in an EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L.
Executive summary:

This study was conducted according to OECD Guideline 209, under GLP conditions, to assess the effect of the test item on the respiration of activated sewage sludge. Activated sewage sludge was exposed to an aqueous dispersion of the test item at nominal concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 19 °C and 21 °C with the addition of a synthetic sewage as a respiratory substrate. A reference item, 3,5 dichlorophenol, and control group were incubated concurrently and the rates of respiration were determined following the exposure period.

The 3-hour EC50 value for the reference item, 3 -5-dichlorophenol, was determined to be 6.5 mg/L, with 95% confidence limits of 4.8 to 8.8 mg/L.

No effects of the test item were observed on the respiration of activated sewage sludge following a 3 hour exposure, resulting in an EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L.  It was considered unnecessary to test at concentrations in excess of 1000 mg/L.

Description of key information

In an OECD 209 study, conducted according to GLP, no effects of the test item were observed on the respiration of activated sewage sludge following a 3 hour exposure, resulting in an EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L (Envigo, 2018).

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information