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EC number: 205-181-1 | CAS number: 135-16-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Nanomaterial pour density
- Nanomaterial photocatalytic activity
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- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2017-11-24 to 2018-02-21
- 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))
- Version / remarks:
- 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Analytical monitoring:
- no
- Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weight out directly in the Erlenmeyer flasks
- Controls: Reference item for total respiration: Copper (ll) sulphate pentahydrate p.a.; Reference item for Nitrification: N-Methylaniline also directly weight out in the Erlenmeyer flasks. - Test organisms (species):
- activated sludge
- Details on inoculum:
- - Pretreatment:
The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L s 10 %. The sludge was used within 24 h after sampling.
- Initial biomass concentration: 2.98 g/L, corresponding to 1.49 g/L in the test vessel for replicates without ATU and 2.83 g/L, corresponding to 1.43 g/L in the test vessel for replicates with ATU (N-allylthiourea). - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 3 h
- Test temperature:
- 20.1 °C
- pH:
- The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
- Dissolved oxygen:
- above 60 - 70 % saturation
- Nominal and measured concentrations:
- 10, 32, 100, 320, 1000 mg a.i../L (corresponding to 11.1, 35.6, 111, 356, 1111 mg test item/L)
- Details on test conditions:
- TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): Triplicates at the beginning of the test + triplicates at the end of the replicates without ATU (N-allylthiourea). Triplicates at the beginning of the replicates with ATU (N-allylthiourea) + triplicates at the end of the test
- Sludge concentration (weight of dry solids per volume): 3.0 g/L ±10 %
- Nutrients provided for bacteria: Synthetic waste water according to OECD Guideline 209
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU)
OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Respiration Inhibition Test with activated sludge to determine the inhibition of total, heterotrophic and ammonium respiration in %.
TEST CONCENTRATIONS
- Range finding study
- Test concentrations: In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. One replicate of 10, 100, 1000 mg/L were measured with addition of N-allylthiourea (ATU). A second preliminary test (non GLP) was carried out with the concentrations of the test item Tetrahydrofolic acid of 500 - 1000 mg/L with one replicate each and one replicate each with pH adjustment to determine the pH effect and the inhibition of nitrification.
- Results used to determine the conditions for the definitive study: Based on the results of the preliminary test the definitive study was carried out with 5 concentrations within the range of 10 to 1000 mg a.i./L (with and without ATU) in a geometrical series and with a dilution factor of 3.2. - Reference substance (positive control):
- yes
- Remarks:
- Reference item for total respiration: Copper (ll) sulphate pentahydrate p.a.; Reference item for Nitrification: N-Methylaniline
- Key result
- Duration:
- 3 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 32 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- inhibition of total respiration
- Remarks on result:
- other: Due to an inhibition of 7% at 100 mg/L and lack of biological relevance, 100 mg/L should be regarded as NOEC
- Key result
- Duration:
- 3 h
- Dose descriptor:
- EC10
- Effect conc.:
- 146 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- inhibition of total respiration
- Key result
- Duration:
- 3 h
- Dose descriptor:
- EC50
- Effect conc.:
- 671 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- 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:
- act. ingr.
- Basis for effect:
- inhibition of heterotrophic respiration
- Key result
- Duration:
- 3 h
- Dose descriptor:
- EC10
- Effect conc.:
- > 1 000 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- inhibition of heterotrophic respiration
- Duration:
- 3 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 1 000 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- inhibition of heterotrophic respiration
- Key result
- Duration:
- 3 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 32 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- inhibition of nitrification rate
- Remarks on result:
- other: Due to an inhibition of 7% at 100 mg/L and lack of biological relevance, 100 mg/L should be regarded as NOEC
- Key result
- Duration:
- 3 h
- Dose descriptor:
- EC10
- Effect conc.:
- 39 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- inhibition of nitrification rate
- Key result
- Duration:
- 3 h
- Dose descriptor:
- EC50
- Effect conc.:
- 246 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- act. ingr.
- Basis for effect:
- inhibition of nitrification rate
- Details on results:
- The NOEC of the total respiration was found to be 32 mg/L. At 100 mg a.i./L, 7% inhibition was observed. Although this inhibition was found to be statistically significant, this inhibition might not be considered to be biologically relevant. Accordingly, when factoring in the biological relevance of an inhibition of only 7%, the statistically significant result should be considered an artefact. Therefore, 100 mg/L should be considered as NOEC.
- Results with reference substance (positive control):
- - Results with reference substance valid? yes
- Relevant effect levels: The EC50 of the reference item copper (ll) sulphate pentahydrate was 116 mg/L and is in the range of 53 - 155 mg/L. - Reported statistics and error estimates:
- The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05. The EC-values of the test item and the reference item were calculated by sigmoidal dose-response regression (biphasic regression where necessary) and linear regression, respectively, using software GraphPadPrism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.
- Validity criteria fulfilled:
- yes
- Conclusions:
- A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Tetrahydrofolic acid. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 -320 - 1000 mg a.i./L with and without ATU.
The NOEC of Tetrahydrofolic acid for total respiration and nitrification is 32 mg a.i./L.
The EC10 for total respiration is 146 mg a.i./L and the EC50 is 671 mg a.i./L. ,
The NOEC for heterotrophic respiration is > 1000 mg a.i./L.
The EC10 and EC50 for heterotrophic respiration are >1000 mg a.i./L.
The EC10 for nitrification is 39.0 mg a.i./L and the EC50 is 246 mg a.i./L.
Reference
Table 6: Oxygen Uptake Rates and Specific Respiration Rates of the total Respiration of the Control
|
Repl. |
Oxygen Concentration* [mg O2/L] |
Oxygen uptake Rate R [mg O2/(L x h)] |
Specific Respiration rate Rs [mg O2/g x h] |
Control |
1 |
7.27 |
34.2 |
23.0 |
2 |
6.90 |
34.0 |
22.8 |
|
3 |
6.50 |
34.5 |
23.2 |
|
4 |
7.58 |
33.0 |
22.1 |
|
5 |
7.67 |
31.6 |
21.2 |
|
6 |
7.49 |
32.8 |
22.0 |
|
|
Mean value |
33.4 |
22.4 |
|
Standard deviation |
1.0 |
|
||
CV [%] |
3.0 |
Table 7: Oxygen Uptake Rates and Inhibition of the total Respiration of the Test item Concentrations
Test item concentration [mg a.i./L] |
Repl. |
Oxygen Concentration* [mg O2/L] |
Oxygen uptake Rate R [mg O2/(L x h)] |
Inhibition [%] |
Mean Inhibition [%] |
10 |
1 |
6.43 |
33.8 |
-1 |
2 |
2 |
6.51 |
33.5 |
0 |
||
3 |
6.88 |
34.2 |
-2 |
||
4 |
7.12 |
30.1 |
10 |
||
5 |
6.65 |
33.0 |
1 |
||
32 |
1 |
7.17 |
33.4 |
0 |
-2 |
2 |
6.31 |
33.5 |
0 |
||
3 |
6.34 |
34.8 |
-4 |
||
4 |
6.47 |
33.4 |
0 |
||
5 |
6.47 |
35.3 |
-6 |
||
100 |
1 |
7.18 |
31.2 |
7 |
7 |
2 |
6.68 |
29.4 |
12 |
||
3 |
6.96 |
31.3 |
6 |
||
4 |
6.63 |
30.1 |
10 |
||
5 |
6.79 |
33.1 |
1 |
||
320 |
1 |
7.21 |
26.3 |
21 |
25 |
2 |
7.44 |
23.8 |
29 |
||
3 |
7.06 |
25.4 |
24 |
||
4 |
7.18 |
25.5 |
24 |
||
5 |
7.01 |
24.2 |
28 |
||
1000 |
1 |
7.67 |
10.9 |
67 |
65 |
2 |
7.89 |
11.1 |
67 |
||
3 |
7.76 |
12.4 |
63 |
||
4 |
7.51 |
11.7 |
65 |
||
5 |
7.99 |
11.9 |
64 |
Table 8: Oxygen Uptake Rates of the heterotrophic Respiration of the Control Replicates with ATU
|
Repl. |
Oxygen Concentration* [mg O2/L] |
Oxygen uptake Rate R [mg O2/(L x h)] |
Control |
1 |
8.09 |
12.9 |
2 |
8.13 |
11.7 |
|
3 |
8.15 |
12.9 |
|
4 |
7.91 |
13.9 |
|
5 |
7.85 |
13.1 |
|
6 |
8.15 |
12.3 |
|
|
Mean value |
12.8 |
|
Standard deviation |
0.7 |
||
CV [%] |
5.3 |
Table 9: Oxygen Uptake Rates and lnhibition of the heterotrophic Respiration of the Test Item Concentrations
Test Concentration [mg a.i./L] |
Repl. |
Oxygen Concentration* [mg O2/L] |
Oxygen uptake Rate R [mg O2/(L x h)] |
Inhibition [%] |
Mean Inhibition [%] |
|
Test item |
10 + ATU |
1 |
8.08 |
12.9 |
-1 |
-12 |
2 |
7.74 |
15.0 |
-17 |
|||
3 |
7.77 |
14.9 |
-16 |
|||
4 |
7.59 |
14.1 |
-10 |
|||
5 |
7.58 |
15.0 |
-17 |
|||
32 + ATU |
1 |
8.22 |
13.8 |
-8 |
-10 |
|
2 |
8.18 |
14.1 |
-10 |
|||
3 |
8.33 |
14.2 |
-11 |
|||
4 |
7.96 |
14.4 |
-13 |
|||
5 |
8.38 |
13.8 |
-8 |
|||
100 + ATU |
1 |
8.00 |
16.6 |
-30 |
-20 |
|
2 |
7.60 |
15.0 |
-17 |
|||
3 |
7.47 |
15.8 |
-23 |
|||
4 |
7.15 |
14.3 |
-12 |
|||
5 |
7.12 |
15.3 |
-20 |
|||
320 + ATU |
1 |
7.97 |
16.0 |
-25 |
-32 |
|
2 |
7.42 |
17.3 |
-35 |
|||
3 |
7.44 |
17.3 |
-35 |
|||
4 |
7.33 |
16.8 |
-31 |
|||
5 |
7.88 |
17.4 |
-36 |
|||
1000 + ATU |
1 |
7.66 |
11.8 |
8 |
0 |
|
2 |
7.69 |
12.7 |
1 |
|||
3 |
7.57 |
14.6 |
-14 |
|||
4 |
7.43 |
11.4 |
11 |
|||
5 |
7.49 |
13.3 |
-4 |
*) determined at start of the measurement
Table 10: Calculated Oxygen Uptake Rates of the Nitrification of the Control
|
Repl. |
Oxygen Concentration* [mg O2/L] |
Control |
1 |
21.3 |
2 |
22.3 |
|
3 |
21.6 |
|
4 |
19.1 |
|
5 |
18.5 |
|
6 |
20.5 |
|
Mean value |
20.6 |
|
Standard deviation |
1.4 |
|
CV [%] |
6.6 |
Table 11: Calculated Oxygen Uptake Rates and Inhibition of the Nitrification of the Test Item Concentrations
Test Concentration [mg a.i./L] |
Repl. |
Oxygen uptake Rate R [mg O2/(L x h)] |
Inhibition [%] |
Mean Inhibition [%] |
|
Test item |
10 |
1 |
20.9 |
-1 |
10 |
2 |
18.5 |
10 |
|||
3 |
19.3 |
6 |
|||
4 |
16.0 |
22 |
|||
5 |
18.0 |
13 |
|||
32 |
1 |
19.6 |
5 |
3 |
|
2 |
19.4 |
6 |
|||
3 |
20.6 |
0 |
|||
4 |
19.0 |
8 |
|||
5 |
21.5 |
-4 |
|||
100 |
1 |
14.6 |
29 |
24 |
|
2 |
14.4 |
30 |
|||
3 |
15.5 |
25 |
|||
4 |
15.8 |
23 |
|||
5 |
17.8 |
14 |
|||
320 |
1 |
10.3 |
50 |
61 |
|
2 |
6.5 |
68 |
|||
3 |
8.1 |
61 |
|||
4 |
8.7 |
58 |
|||
5 |
6.8 |
67 |
|||
1000 |
1 |
-0.9 |
104 |
106 |
|
2 |
-1.6 |
108 |
|||
3 |
-2.2 |
111 |
|||
4 |
0.3 |
99 |
|||
5 |
-1.4 |
107 |
Description of key information
- OECD 209 guideline study, GLP, respiration inhibition test with activated non-adapted sludge, 3h exposure to 10, 32, 100, 320 and 1000 mg a.i./L of the test item; NOEC for total respiration and nitrification is 32 mg a.i../L (100 mg/L should be considered as NOEC for total respiration). EC10 for total respiration = 146 mg a.i./L; EC50 = 671 mg a.i./L. ,NOEC for heterotrophic respiration = > 1000 mg a.i./L. The EC10 and EC50 for heterotrophic respiration = >1000 mg a.i./L. The EC10 for nitrification = 39.0 mg a.i./L and the EC50 = 246 mg a.i./L.
Key value for chemical safety assessment
- EC50 for microorganisms:
- 671 mg/L
- EC10 or NOEC for microorganisms:
- 146 mg/L
Additional information
A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Tetrahydrofolic acid. The test was carried out under static conditions with the test item concentrations 10 - 32 - 100 -320 - 1000 mg a.i./L with and without ATU. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of total respiration for the test item replicates ranged from - 2 % to 65 %. The heterotrophic respiration was not inhibited. The mean inhibition of nitrification for the test item replicates ranged from 3 % to 106 %.
The NOEC of the total respiration was found to be 32 mg/L. At 100 mg/L test item, 7% inhibition was observed. Although this inhibition was found to be statistically significant, this inhibition might not be considered to be biologically relevant. Accordingly, when factoring in the biological relevance of an inhibition of only 7%, the statistically significant result should be considered an artefact. Therefore, 100 mg/L should be considered as NOEC.
The NOEC of Tetrahydrofolic acid for total respiration and nitrification is 32 mg a.i./L
(100 mg/L should be considered as NOEC for total respiration, for details see text).
The EC10 for total respiration is 146 mg a.i./L and the EC50 is 671 mg a.i./L. ,
The NOEC for heterotrophic respiration is > 1000 mg a.i./L.
The EC10 and EC50 for heterotrophic respiration are >1000 mg a.i./L.
The EC10 for nitrification is 39.0 mg a.i./L and the EC50 is 246 mg a.i./L.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.