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Ecotoxicological information

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: 16 June 2020 to 06 October 2020.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Remarks:
The test item was dispersed directly in water.
Details on test solutions:
Activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L (three replicates for the test concentration of 1000 mg/L). The test item was dispersed directly in water.

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 sealed with parafilm prior to being subjected to ultrasonication for approximately 15 minutes followed bymagnetic 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 volumeof 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (three replicates at 1000 mg/L).
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 05 October 2019 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK which treats predominantly domestic sewage.


Preparation of inoculum for exposure:

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, determination of the suspended solids level of the activated sewage sludge was carried out. The suspended solids concentration was equal to 2.5 g/L and was adjusted to 3.0 g/L prior to the activated sewage sludge) being fed an aliquot of synthetic sewage.


The pH of the sample on the day of the test was 7.6 measured using a pH meter
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Test temperature:
Between 20 °C and 21 °C
pH:
The pH of the test item dispersions was measured after stirring using Hach HQ40d Flexihandheld meter and adjusted to between pH 7.0 and 8.0.
Dissolved oxygen:
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
Nominal and measured concentrations:
Nominal
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml flasks
- Aeration: continuous aeration
- No. of vessels per concentration (replicates): Triplicate for 1000 mg/L concentration, one each for 100 and 10 mg/L concentrations.
- No. of vessels per control (replicates): four
- Sludge concentration (weight of dry solids per volume): 3.0 g/L prior to use

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

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item stock solutions were measured using a handheld meter and if necessary, adjusted to between pH 7.0 to pH 8.0.
- Photoperiod: The test was conducted under normal laboratory lighting
Reference substance (positive control):
yes
Remarks:
The reference item, 3,5-dichlorophenol, gave a 3-Hour EC50 value of 6.2 mg/L, 95% confidence limits 3.3 to 11 mg/L confirming the acceptability of the inoculum used in the test system.
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Results with reference substance (positive control):
The EC50 value (3-Hour contact time) for the reference item, 3,5-dichlorophenol, was 6.2 mg/L which was within the acceptance criteria range of 2 to 25 mg/L for total respiration. The test is therefore considered valid and operating as expected.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.


 


No statistically significant toxic effects were shown at 1000 mg/L. It was therefore considered justifiable not to perform a definitive test.


 


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

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The NOEC after 3 hours exposure was 1000 mg/L.
Executive summary:

Introduction


A 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 following Guideline:


 



  • OECD Guidelines for the Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".


 


Method


Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (three replicates of the 1000 mg/L test concentration) for a period of 3 hours at a measured temperature of between 20 °C and 21 °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 effect of the test item on the respiration of activated sewage sludge micro-organisms 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.

Description of key information

Toxicity to Microorganisms data summary.

Key value for chemical safety assessment

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

Additional information

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The NOEC after 3 hours exposure was
1000 mg/L.