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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:
Experimental Phase: 19 December 2017 to 20 December 2017. Report issue: 14 August 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 (Carbon and Ammonium Oxidation))
Deviations:
yes
Remarks:
One deviation occurred which was considered to have had no effect on the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test item concentrations for the main test were 10, 100 and 1000 mg/L

Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximise the dissolved test item 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.

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 if necessary.
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 19 December 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (9.5 litres) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was determined to be 7.5 using a handheld pH meter.

The suspended solids concentration of the activated sewage sludge was determined to equal to 3.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20 °C to 22 °C
pH:
7.3 to 8.3 (definitive test)
Dissolved oxygen:
5.98 to 8.31 mg O2/L (definitive test after 30 minutes contact time)

Nominal and measured concentrations:
Nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL conical flask
- Aeration: continuous aeration
- No. of vessels per concentration (replicates): 1 vessel for 10 mg/L, 1 vessel for 100 mg/L, 3 vessels for 1000 mg/L test concentration.
- No. of vessels per control (replicates): 4
- No. of vessels per positive control (replicates): 1 per test concentration (3.2, 10 and 32 mg/L)

- Sludge concentration (weight of dry solids per volume): 3.0 g/L
- Nutrients provided for bacteria: Synthetic sewage

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted to between pH 7.0 and 8.0 if necessary.
- Photoperiod: normal laboratory lighting

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
Results with reference substance (positive control):
 The reference item, 3,5-Dichlorophenol, gave a 3-Hour EC50 value of 8.6 mg/L, with 95% confidence limits 6.7 to 11 mg/L.
Reported statistics and error estimates:
Statistical Methods
The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC80 values determined from the equation for the fitted line.

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 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 occurred at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

Validity criteria fulfilled:
yes
Conclusions:
The test item gave a 3-Hour EC50 value of greater than 1000 mg/L for the respiration rate of activated sewage sludge. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
 
Executive summary:

Introduction

The study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

 

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a measured temperature of between 20 °C and 22 °C with the addition of a synthetic sewage as a respiratory substrate.

 

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-Dichlorophenol.

 

Results and Conclusion

The test item gave a 3-Hour EC50 value of greater than 1000 mg/L for the respiration rate of activated sewage sludge. 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 8.6 mg/L, with 95% confidence limits 6.7 to 11 mg/L.

Description of key information

Toxicity of the test substance to activated sludge.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information