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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:
8 July, 2016
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))
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
FORMULATION
Just before the start of the experiment a concentrated stock solution (3200 mg/L) was prepared by dissolving test item in deionised water using ultrasonic bath.

The test solutions used in the test were freshly prepared by dilution of the stock solution by mechanical dispersion at the beginning of the experiment, in the testing laboratory.

The pH of the test solutions was checked but not adjusted, because they were in the range of 7–8 at the start of the experiment.
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: Veszprém, Hungary
- Method of cultivation: The activated sludge was supplied by the sewage plant for domestic sewage two days before the start of the experiment. During holding prior to use the sludge was fed daily with 50 mL synthetic sewage per litre and kept aerated at 20 ± 2°C until use.
- Preparation of inoculum for exposure: The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in chlorine-free tap water and again centrifuged. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in chlorine-free tap water to yield a concentration equivalent to 3 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.8. The activated sludge was used
directly after conditioning.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
20.3 – 21.7 °C
pH:
Range 7.47 - 8.22
Nominal and measured concentrations:
10, 100, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Appropriate glass beakers for 500 mL volume and BOD bottles with 300 mL volume.
- Aeration: With compressed air (about 1 L/min)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5
- No. of vessels per abiotic control (replicates): 5
- Sludge concentration (weight of dry solids per volume): 3 g/L
- Nutrients provided for bacteria: 12.8 mL of a solution of feed comprising Peptone 16 g/L; meat extract 11 g/L; urea 3 g/L; NaCl 0.7 g/L; CaCl2.2H2O 0.4 g/L; MgSO4.7H2O 0.2 g/L; K2HPO4 2.8 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water
- Particulate matter: n/a
OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration rate
For the measurement of the respiration rate a well-mixed sample of each treatment was poured into a BOD flask after about 3 hours incubation time, and was not further aerated. The oxygen concentration was measured for about fifteen minutes, with an O2 electrode and was recorded in every half minute. The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
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
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
Validity criteria fulfilled:
yes
Conclusions:
The 3-hour EC50: > 1000 mg/L. The test item had no effect on the total respiration rate of the activated sludge microorganisims at 1000 mg/L.
Executive summary:

A test was carried out according to OECD 209 to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

The test concentrations (10, 100 and 1000 mg/L) were chosen to permit the determination of the approximate EC50.

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of the test item was the following:

The 3-hour EC50: > 1000 mg/L

Based on the results of this study, the test item had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.

Description of key information

The 3-hour EC50: > 1000 mg/L (OECD 209)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

A test was carried out according to OECD 209 to evaluate the effect of the test item on microorganisms by measuring the respiration rate.

The test concentrations (10, 100 and 1000 mg/L) were chosen to permit the determination of the approximate EC50.

The reference control results showed that in this study, the inoculum and methodology used provided a good measure of inhibition of activated sludge respiration rate.

Under the conditions of this Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) the observed endpoint for the effect of the test item was the following:

The 3-hour EC50: > 1000 mg/L

Based on the results of this study, the test item had no inhibition effect on the total respiration rate of the microorganisms of the activated sludge at 1000 mg/L. No further testing is necessary.