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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Information as provided by the Supplier (Sigma-Aldrich).
Identification: 3,5-Dichlorophenol
Physical state/Appearance: pale brown crystalline solid
Batch: MKBV7274V
Purity: 99.6%
Expiry Date: 01 September 2017
Storage Conditions: room temperature
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms was obtained on 27 January 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
Test type:
not specified
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
Activated sewage sludge was exposed for a period of 3 hours.
Post exposure observation period:
No
Hardness:
N/A
Test temperature:
between 19 and 20 °C
pH:
The pH of the synthetic sewage stock was 7.2. The pH value was measured using a Hach HQ40d Flexi handheld meter.
Dissolved oxygen:
The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).
Salinity:
N/A
Nominal and measured concentrations:
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dissolved directly in water.
Details on test conditions:
A nominal amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 5 minutes and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock
solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity.

The control group was maintained under identical conditions but not exposed to the test item.
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
Details on results:
The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after3 hours exposure was 1000 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
Results with reference substance (positive control):
The reference item gave a 3-Hour EC50 value of 9.3 mg/L, 95% confidence limits 7.3 to 12 mg/L.
Reported statistics and error estimates:
95% confidence limits were calculated for the reference item EC50 value using the method of
Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949). In order to determine the NOEC, one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the range-finding test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).
No statistically significant toxic effects were shown at all of the test concentrations employed.
Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.
The reference item gave a 3-Hour EC50 value of 9.3 mg/L, 95% confidence limits 7.3 to 12 mg/L.
Executive summary:

A study was performed to assess the effect of the test item on the respiration of activatedsewage sludge. The method followed was designed to be compatible with the OECDGuidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, RespirationInhibition Test (Carbon and Ammonium Oxidation)".


Activated sewage sludge was exposed to an aqueous solution of the test item atconcentrations 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 and 20 °C with the additionof a synthetic sewage as a respiratory substrate.The rate of respiration was determined after 3 hours contact time and compared to data forthe control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after3 hours exposure was 1000 mg/L.It was considered unnecessary and unrealistic to test at concentrations in excess of1000 mg/L.The reference item gave a 3-Hour EC50value of 9.3 mg/L, 95% confidence limits 7.3 to12 mg/L

Description of key information

The effect of the test item on the respiration of activated sewage sludge gave a

The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information