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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
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
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test substance was administered to empty containers, which were subsequently filled up with water and sewage
- Eluate: not applicable
- Differential loading: not applicable
- Controls: according to guideline
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 1000mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none
Test organisms (species):
activated sludge, domestic
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: domestic sewage in Balatonfüred, Hungary, on 25.09.2017 (one day before the test)
- Method of cultivation: not applicable
- Preparation of inoculum for exposure: Coarse particles were removed after 10 min of settling and the upper layer of finer solids were decanted. The sludge was washed by centrifugation (at 3488 g) after adding isotonic saline solution and the supernatant was decanted. The solid material was re-suspended in isotonic saline solution with shaking and centrifuged again. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to dry weight was determined. Based on this ratio, wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to about 3g/L (on dw basis).
- Pretreatment: aeration (2/L minute) at test temperature for 24h and fed with 50 mL synthetic sewage/activated sludge.
- Initial biomass concentration: 1000 mg/L
- pH adjustment: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
no information
Test temperature:
20.1 - 21.0 °C
pH:
before addition of inoculum: 7.64 - 7.98
at the start of the 3h aeration: 7.41 - 7.99
at the end of the 3h aeration: 7.85 - 8.29
(across all controls and test substance groups)
Dissolved oxygen:
at the beginning of the selected section of the linear phase: 7.01 - 7.7.58
at the end of the selected section of the linear phase: 2.00 - 5.41
(across all controls and test substance groups)
Salinity:
no information
Conductivity:
no information
Nominal and measured concentrations:
not applicable
Details on test conditions:
TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: no further information
- Aeration: yes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates): not applicable
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3 g/L (on dw basis)
- Nutrients provided for bacteria: synthetic sewage
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (in preliminary experiment only)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: no information
- Light intensity: no information
- Details on termination of incubation: not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : respiration rate

TEST CONCENTRATIONS : 1000 mg/L
- Spacing factor for test concentrations: not applicable
- Justification for using fewer concentrations than requested by guideline: no effects in preliminary test
- Range finding study : yes
- Test concentrations: 1000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no
- Adsorption (e.g. of test material to the walls of the test container): no information
- Blank controls oxygen uptake rate: approx. 56 mg O/Lh
- Coefficient of variation of oxygen uptake rate in control replicates: 2.7%
Results with reference substance (positive control):
- Results with reference substance 3,5-Dichlorophenol valid? Yes
- Relevant effect levels: EC50: 16.82 mg/L
- Other:

The toxicity of the substance on microorganisms was investigated in an activated sludge respiration inhibition test according to OECD Test Guideline 209 under GLP-conditions.

The test concentration of 1000 mg/L was chosen based on the results of a preliminary test, in which the substance did not show and inhibitory effects, did not affect the pH did not consume oxygen abiotically.

The main test was conduct as a limit test with the nominal concentration of 1000 mg/L, without abiotic controls and without pH adjustment, but with a nitrification control. The blank and positive controls (3,5 -dichlorophenol) met all validity criteria. The respiration rates of the examined concentration did not differ (statistically) from those observed for the blank control after 3 hours of incubation.

Therefore, the 3 -hour EC10 was determined as > 1000 mg/L and the NOEC as 1000 mg/L.

Detailed results: Specific respiration rate R in the test substance and control groups

 Identification  concentration (mg/L)  specific respiration rate (mg O2/gh)  average R (mg O2/gh)
 Cba  0  39.20  37.57
 Cbb  0  37.45  CV(%) = 2.71
 Cbc  0  37.31  
 Cbd  0  38.32  
 Cbe  0  37.60  
 Cbf  0  35.60  
 Cbg  0  37.75  
 Cbh  0  37.36  
 Cna   11.6 mg ATU/L  37.53  37.58
 Cnb   11.6 mg ATU/L  37.31  CV(%) = 0.78
 Cnc   11.6 mg ATU/L  37.89  
 R1a   2 mg 3,5 -DCP/L  35.27  34.04
 R1b   2 mg 3,5 -DCP/L  34.47  CV(%) = 4.35
 R1c   2 mg 3,5 -DCP/L  32.40  
 R2a   7 mg 3,5 -DCP/L  30.71  30.25
 R2b   7 mg 3,5 -DCP/L  28.57  CV(%) = 4.96
 R2c   7 mg 3,5 -DCP/L  31.47  
 R3a   24.5 mg 3,5 -DCP/L  8.68  8.69
 R3b   24.5 mg 3,5 -DCP/L  8.48  CV(%) = 2.53
 R3c   24.5 mg 3,5 -DCP/L  8.92  
 T1a   1000 mg/L test item  35.64  36.54 (ns)
 T1b   1000 mg/L test item  37.16  CV(%) = 2.33
 T1c   1000 mg/L test item  37.60  
 T1d   1000 mg/L test item  35.78  
 T1e   1000 mg/L test item  36.51  

Cb...: blank control; Cn...: nitrification control n-allylthiourea; R...: reference control 3,5 -dichlorophenol; T...: test substance; ns: not-significant (t-test vs blank control)

Validity criteria fulfilled:
yes
Executive summary:

The toxicity of the substance on microorganisms was investigated in an activated sludge respiration inhibition test according to OECD Test Guideline 209 under GLP-conditions.

The test concentration of 1000 mg/L was chosen based on the results of a preliminary test, in which the substance did not show and inhibitory effects, did not affect the pH did not consume oxygen abiotically.

The main test was conduct as a limit test with the nominal concentration of 1000 mg/L, without abiotic controls and without pH adjustment, but with a nitrification control. The blank and positive controls (3,5 -dichlorophenol) met all validity criteria. The respiration rates of the examined concentration did not differ (statistically) from those observed for the blank control after 3 hours of incubation.

Therefore, the 3 -hour EC10 was determined as > 1000 mg/L and the NOEC as 1000 mg/L.

Description of key information

The toxicity of the substance on microorganisms was investigated in an activated sludge respiration inhibition test according to OECD Test Guideline 209 under GLP-conditions.

In a limit test with the nominal concentration of 1000 mg/L no (statistically significant) inhibitory effect of the substance was observed as compared to the blank control after 3 hours of incubation.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information