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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: 08 January 2018 to 14 March 2018. Report issued: 01 August 2018.
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:
yes
Remarks:
The deviations were not considered to have impacted on the conclusions or validity of the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. 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 dispersed directly in water.


For the definitive test nominal amounts of test item (50, 160 and 500 mg (five replicates of each)) 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 100, 320, 1000, mg/L (five replicates of each).

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

The control group was maintained under identical conditions but not exposed to the test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: A mixed population of activated sewage sludge micro-organisms was obtained on 08 January 2018 for the range-finding test and on 13 March 2018 for the definitive test from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- Method of cultivation: On the day of collection the activated sewage sludge (7 litres) was fed synthetic sewage (350 mL).
- 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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Test temperature:
20 -21 °C
pH:
7.3 - 8.0 at the start of the test and 7.1 - 7.9 at 3 hours (end of the test)
Dissolved oxygen:
5.41 - 8.39 mg O2/L
Nominal and measured concentrations:
Definitive test concentrations were 100, 320 and 1000 mg/L of test substance based on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml conical flasks
- Aeration: continuous aeration
- No. of vessels per concentration (replicates): Five for each test concentration (100, 320 and 1000 mg/L)
- No. of vessels per control (replicates): Six
- No of vesseels referecne substance: one for each concentration.
- 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:
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 EC50 value (3-hhour contact time) for the reference item, 3,5-Dichlorophenol was 9.0 mg/L, 95% confidence limits 7.3 to 11 mg/L.

Range-finder Test

No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L. Based on these results and after discussion with the Sponsor, it was considered necessary to perform a definitive test in order to obtain a NOEC value for the test item. Test concentrations of 100, 320 and 1000 mg/L were selected for the definitive test.

Validity criteria fulfilled:
yes
Conclusions:
The test substance gave a 3-Hour EC50 value of greater than 1000 mg/L when exposed to activated sludge. The No Observed Effect Concentration (NOEC) after 3 hours exposure was also > 1000 mg/L.
Executive summary:

Introduction

The study performed to assess the effect of the test substance 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 solution of the test item at concentrations of 100, 320 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 test substance gave a 3-Hour EC50 value of greater than 1000 mg/L when exposed to activated sludge. The No Observed Effect Concentration (NOEC) after 3 hours exposure was also > 1000 mg/L.


The reference item gave a 3-Hour EC50 value of 9.0 mg/L, 95% confidence limits 7.3 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