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EC number: 270-171-6 | CAS number: 68412-15-7
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Toxicity to microorganisms
Administrative data
Link to relevant study record(s)
- Endpoint:
- activated sludge respiration inhibition testing
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-05-22 to 2017-05-23
- 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
- Version / remarks:
- 2010-07-22
- Deviations:
- no
- Principles of method if other than guideline:
- In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification by measuring with addition of Allylthiourea.
- GLP compliance:
- yes (incl. QA statement)
- Analytical monitoring:
- no
- Details on sampling:
- not applicable (no analytics)
- Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: weighed out directly in the Erlenmeyer flasks
- Eluate: n.a.
- Differential loading: no
- Controls: reference item concentrations were diluted out of a stock solution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n.a.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported - Test organisms (species):
- activated sludge of a predominantly domestic sewage
- Details on inoculum:
- - Laboratory culture: no
- Name and location of sewage treatment plant where inoculum was collected:
municipal treatment plant of 31137 Hildesheim, Germany
- Method of cultivation: n.a.
- Preparation of inoculum for exposure:
- Pretreatment:
The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Initial biomass concentration:
Dry sludge concentration 3.30 g/L (corresponding to 1.65 g/L suspended solids in the test vessels) - Test type:
- static
- Water media type:
- freshwater
- Remarks:
- Synthetic waste water according to OECD Guideline 209
- Limit test:
- yes
- Total exposure duration:
- 3 h
- Hardness:
- not reported
- Test temperature:
- actually measured 21.6 °C
- pH:
- activated sludge 7.49
sytnthetic waste water 7.34
test solution 7.66 (1000mg/l) - Dissolved oxygen:
- 3 replicates with 6.46, 7.12, 7.16 [mg O2/L] determined at the start of the measurement
- Salinity:
- not reported
- Conductivity:
- not reported
- Nominal and measured concentrations:
- preliminary test: 10, 100, 1000 mg/L
main test: 1000 mg/L - Details on test conditions:
- TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Material, size, headspace, fill volume: glas, 500 mL, 250 mL
- Aeration: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3
- No. of vessels per abiotic control (replicates): n.a.
- Sludge concentration (weight of dry solids per volume): Dry sludge concentration 3.30 g/L (corresponding to 1.65 g/L suspended solids in the test vessels)
- Weight of dry solids per volume of reaction mixture per unit of time: ?
- Nutrients provided for bacteria: Synthetic waste water according to OECD-Guideline 209
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea
- Biomass loading rate: see above
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralised water
- Particulate matter: n.a.
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported
- Light intensity: n.a.
- Details on termination of incubation: not reported
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: preliminary test: 10; main test: limit test
- Justification for using fewer concentrations than requested by guideline:
Based on the results of the preliminary test it could concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in 3 replicates (limit test to determine the NOEC).
- Range finding study: yes
- Test concentrations: 10, 100, 1000 mg/l
- Results used to determine the conditions for the definitive study: no significant inhibition of respiration or nitrification in the definitive study was observed, even the inhibitions of the heterotrophic respiration or nitrification show no appreciable value. - Reference substance (positive control):
- yes
- Remarks:
- Copper (II) sulphate pentahydrate
- 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
- Details on results:
- - Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported
- Effect concentrations exceeding solubility of substance in test medium:
- Adsorption (e.g. of test material to the walls of the test container): not measured / reported
- Blank controls oxygen uptake rate: 25.3 mg mg O2/g x h
- Coefficient of variation of oxygen uptake rate in control replicates: 5.3% - Results with reference substance (positive control):
- - Results with reference substance valid? yes
- Relevant effect levels or copper (II) sulphate pentahydrate: EC50=99.5 mg/L (95 % Confidence Interval = 95.7 - 104 mg/L)
- Other: recommended range of validity of 53 - 155 mg/L - Reported statistics and error estimates:
- Statistical Analysis (NOEC)
One Way Analysis of Variance
Normality Test (Shapiro-Wilk) Failed (P < 0.050)
Equal Variance Test: Passed (P = 0.566)
Group Name N Missing Mean Std Dev SEM
Control 6 0 41.683 2.427 0.991
1000 mg/L 3 0 43.667 0.737 0.426
Source of Variation DF SS MS F P
Between Groups 1 7.867 7.867 1.804 0.221
Residual 7 30.535 4.362
Total 8 38.402
The differences in the mean values among the treatment groups are not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (P = 0.221).
Power of performed test with alpha = 0.050: 0.117
The power of the performed test (0.117) is below the desired power of 0.800.
Less than desired power indicates you are less likely to detect a difference when one actually exists. Negative results should be interpreted cautiously. - Validity criteria fulfilled:
- yes
- Conclusions:
- The NOEC of the test item Octadecanoic acid, reaction products with triethylenetetramine is ≥ 1000 mg/L.
- Executive summary:
For the test item Octadecanoic acid, reaction products with triethylenetetramine a respiration inhibition test with activated sludge according to OECD Guideline No. 209 (22 July 2010) was carried out.
A preliminary test performed with test item concentrations of 10, 100, 1000 mg/L showed no relevant inhibition of the heterotrophic respiration or nitrification (tested with Allylthiourea ATU).
The main test was performed as a limit test with a duration of 3 h. The NOEC resulted to be ≥ 1000 mg/L.
Reference
Preliminary Test (non GLP)
Inhibitions determined in the preliminary test are listed in Table 4 and Table 5.
Table4: Inhibitions in the Preliminary Test
Nominal Test Item Concentration |
pH |
Inhibition |
1stpreliminary test - without pH adjustment, direct aeration |
||
10 |
- |
-5 |
- |
-5 |
|
100 |
- |
-4 |
- |
-4 |
|
1000 |
7.66 |
-5 |
- |
1 |
Table5: Inhibitions in the Preliminary Test (with / without ATU)
Nominal Test Item Concentration |
Inhibition of total respiration |
Inhibition of heterotrophic respiration |
Inhibition of nitrification |
1000 |
-5 |
1 |
-11 |
1 |
-4 |
4 |
Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
From the oxygen depletion the oxygen uptake rates were calculated in mg O2/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O2/g×h. The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison to the oxygen uptake rates of the control. The specific respiration rates are listed inTable 6. The results are listed in Table 7.
Table6: Specific Respiration Rates of the Control Replicates
|
|
Repl. |
Specific Respiration Rate Rs |
|
Control |
1 |
25.2 |
2 |
25.9 |
||
3 |
22.4 |
||
4 |
26.4 |
||
5 |
26.3 |
||
6 |
25.3 |
||
Mean value |
25.3 |
Repl. = Replicate
Suspended solids in the test vessel: 1.65 g/L
Table7: Oxygen Uptake Rates, Inhibition of the Respiration of Control, Test and Reference Item Concentrations
Test and Reference Item Concentration |
Repl. |
Oxygen Uptake Rate |
Inhibition |
Mean Inhibition |
Control |
1 |
41.6 |
- |
- |
2 |
42.8 |
|||
3 |
37.0 |
|||
4 |
43.5 |
|||
5 |
43.4 |
|||
6 |
41.8 |
|||
Test item |
1 |
43.1 |
-3 |
-4 |
2 |
44.5 |
-6 |
||
3 |
43.4 |
-3 |
||
Reference item |
1 |
31.7 |
24 |
26 |
2 |
31.5 |
24 |
||
3 |
29.6 |
29 |
||
100 |
1 |
22.3 |
47 |
48 |
2 |
21.8 |
48 |
||
3 |
20.7 |
50 |
||
180 |
1 |
8.7 |
79 |
79 |
2 |
9.3 |
78 |
||
3 |
8.7 |
79 |
Repl.
= Replicate
Coefficient of variation of oxygen uptake rates of the control: 5.3%
Suspended solids in the test vessel: 1.65 g/L
Description of key information
For Octadecanoic acid, reaction products with triethylenetetramine a respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out.
A preliminary test with 10, 100, 1000 mg/l showed no relevant inhibiting of the heterotrophic respiration or nitrification (tested with Allylurea ATU).
The main test was made as limit test resulting in a 3h-NOEC=1000 mg/l.
Key value for chemical safety assessment
- EC50 for microorganisms:
- 1 000 mg/L
Additional information
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