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EC number: 224-632-3 | CAS number: 4432-31-9
- 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
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- Nanomaterial pour density
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- Nanomaterial catalytic activity
- Endpoint summary
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- Environmental data
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- 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
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- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
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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:
- 2017-09-26 to 2018-05-08
- 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:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Analytical monitoring:
- no
- Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In the main test the test item concentration (as a limit concentration) of 1080 mg/L was examined with five replicates. The defined amount (323 mg) of the test item was (administered) measured directly into empty containers that were filled up with water and synthetic sewage, just before the inoculation. Concentrations in excess of nominal 1080 mg test item/L were not tested. Based on the preliminary experience, the test item does not affect adversely pH within the test system; therefore any pH adjustment was not performed prior to inoculum addition. - Test organisms (species):
- activated sludge, domestic
- Details on inoculum:
- - Source: domestic sewage in Balatonfüred, Hungary
- Preparation of inoculum for exposure: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation (at 4000 rpm that corresponds to 3488 g at the given centrifuge) after adding isotonic saline solution and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice. 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 amount of wet sludge was suspended in isotonic saline solution to yield a concentration equivalent to about 3 g per litre (on dry weight basis). The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature for about 24 hours and fed with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.32), pH adjustment of the inoculum was considered not necessary. - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- yes
- Total exposure duration:
- 3 h
- Hardness:
- not specified
- Test temperature:
- 20.1 - 21.0 °C
- pH:
- before addition of inoculum: 7.65 - 7.68
at the start of the 3h aeration: 7.41 - 7.94
at the end of the 3h aeration: 7.85 - 8.29
(across all controls groups)
Test substance:
before addition of inoculum: 5.39 - 5.48
at the start of the 3h aeration: 6.09 - 6.24
at the end of the 3h aeration: 7.36 - 7.48 - Dissolved oxygen:
- at the beginning of the selected section of the linear phase: 7.01 - 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:
- not applicable
- Conductivity:
- not specified
- Nominal and measured concentrations:
- Nominal conc.: 1080 mg/L
Measured conc.: Not specified - Details on test conditions:
- TEST SYSTEM
- Test vessel (type and size): Erlenmeyer glass bottles of approximately 300 mL volume
- Aeration: yes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8
- No. of vessels per abiotic control (replicates): 3
- Sludge concentration (weight of dry solids per volume): 3 g/L (based on dry weight)
- Nutrients provided for bacteria: synthetic sewage
- Nitrification inhibitor used: N-allylthiourea
OTHER TEST CONDITIONS
- Adjustment of pH: no
EFFECT PARAMETERS MEASURED: activated sludge respiration rate
TEST CONCENTRATIONS:
- Range finding study: yes
- Test concentrations: 1000 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes - Reference substance (positive control):
- yes
- Remarks:
- 3,5-dichlorophenol
- Key result
- Duration:
- 3 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 1 080 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- inhibition of total respiration
- Duration:
- 3 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 1 080 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- inhibition of total respiration
- Key result
- Duration:
- 3 h
- Dose descriptor:
- EC10
- Effect conc.:
- > 1 080 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- inhibition of total respiration
- Details on results:
- - Any observations that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no
- Blank controls oxygen uptake rate: approx. 56 mg O2/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 (95 % confidence limits: 10.21 - 31.42 mg/L) - Reported statistics and error estimates:
- - 2-sample test to compare the test substance with control
- variability measure: coefficient of variation (CV) - Validity criteria fulfilled:
- yes
- Conclusions:
- Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as higher than 1080 mg/L. Based on the statistical evaluation in this test the NOEC was determined to be 1080 mg/L.
- Executive summary:
The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions in a study according to OECD TG 209 under GLP. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. A preliminary test was performed to estimate the range of concentrations of the test item needed in the definite (main) test for determining the inhibition of oxygen consumption. In the preliminary test of this study the test item was investigated at the nominal concentrations of 10.8; 108 and 1080 mg/L. These test item concentrations were chosen taking into account a correction for the water content of the test item. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. Based on the absence of any inhibitory effect in the preliminary experiment, the test item was investigated at the limit concentration of nominal 1080 mg/L in the main test. Similarly to the preliminary test, defined amounts of the test item were added directly into the test vessels. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control at concentration levels of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The definite test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1080 mg/L and no remarkable abiotic oxygen consumption was noticed. In accordance to the preliminary experiment, the test item does not affect adversely the pH within the test system; therefore, the main test was performed without additional pH adjustment. All validity criteria of the study were met. The observed oxygen consumption rates and consequently the specific respiration rates in the examined test item (limit) concentration remained in the range of the blank controls (the average specific respiration rate at 1080 mg/L: 37.13 mg O2/gh), no remarkable inhibitory effect of the test item was observed (the obtained 1.17 % inhibition was considered as being within the range of biological variability of the applied test system). The specific respiration rates at the examined test item concentration of 1080 mg/L were compared with the blank control values using 2-Sample T-test (α = 0.05). No statistical significant differences were noticed in the comparison of blank control and test item treated groups. Under the test conditions, the EC10 and EC50 values of test item were determined as higher than 1080 mg/L. Based on the statistical evaluation in this test the NOEC was 1080 mg/L. In conclusion this study demonstrated the absence of inhibition of oxygen consumption by the test item up to and including the limit concentration of 1080 mg/L.
Reference
Validity of the Study
The specific respiration rate of the blank controls (without the test substance or reference substance) was 37.57 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 2.71 %. The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 16.82 mg/L and, thus, within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).
Toxicity of 3,5-Dichlorophenol
The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 9.39 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 19.50 % and 76.86 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 16.82 mg/L, (95 % confidence limits: 10.21-31.42 mg/L).
Nitrification Controls
An additional nitrification control was examined in the test with three parallels to check the possible nitrification potential of the applied activated sludge batch. With applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 56.36 mg/Lh, the heterotrophic respiration (RH) was 56.36 mg/Lh, the nitrification respiration (RN): 0.00 mg/Lh was calculated according to the equation: RN= RT-RH. There was no difference between the total and heterotrophic respiration, the obtained zero value was consequently lower than the 5 % of RT (2.82 mg/Lh) in blank controls. In this test the heterotrophic oxygen uptake equals the total uptake.
Table 1: The Specific Respiration Rate (RS) in the Test Item and Control Groups
Identification | Concentration (mg/L) | Specific Respiration Rate (mg O2/gh) | Average RS (mg O2/gh) |
cba | 0.00 | 39.20 | 37.57 CV(%)= 2.71 |
cbb | 37.45 | ||
cbc | 37.31 | ||
cbd | 38.32 | ||
cbe | 37.60 | ||
cbf | 35.60 | ||
cbg | 37.75 | ||
cbh | 37.36 | ||
cna | 11.6 mg ATU/L | 37.53 | 37.58
CV(%)= 0.78 |
cnb | 37.31 | ||
cnc | 37.89 | ||
R1a | 2 mg 3,5-DCP/L | 35.27 | 34.04
CV(%)= 4.35 |
R1b | 34.47 | ||
R1c | 32.40 | ||
R2a | 7 mg 3,5-DCP/L | 30.71 | 30.25
CV(%)= 4.96 |
R2b | 28.57 | ||
R2c | 31.47 | ||
R3a | 24.5 mg 3,5-DCP/L | 8.68 | 8.69
CV(%)= 2.53 |
R3b | 8.48 | ||
R3c | 8.92 | ||
T1a | 1080 mg Test Item/L | 37.38 | 37.13 NS
CV(%)= 1.12 |
T1b | 36.58 | ||
T1c | 37.38 | ||
T1d | 36.80 | ||
T1e | 37.53 |
3,5-DCP: 3,5-dichlorophenol
ATU: N-allylthiourea
CV: Coefficient of variation
NS: There is no statistically significant difference when compared to the control (2-Sample t-Test; α = 0.05)
Description of key information
Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item were determined as higher than 1080 mg/L. Based on the statistical evaluation in this test the NOEC was 1080 mg/L (reference 6.1.7-1).
Key value for chemical safety assessment
- EC50 for microorganisms:
- 1 080 mg/L
- EC10 or NOEC for microorganisms:
- 1 080 mg/L
Additional information
The purpose of the 3-hour test was to evaluate the influence of the test item on the activity of the activated sludge by measuring the respiration rate under defined conditions in a study according to OECD TG 209 under GLP. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. A preliminary test was performed to estimate the range of concentrations of the test item needed in the definite (main) test for determining the inhibition of oxygen consumption. In the preliminary test of this study the test item was investigated at the nominal concentrations of 10.8; 108 and 1080 mg/L. These test item concentrations were chosen taking into account a correction for the water content of the test item. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. Based on the absence of any inhibitory effect in the preliminary experiment, the test item was investigated at the limit concentration of nominal 1080 mg/L in the main test. Similarly to the preliminary test, defined amounts of the test item were added directly into the test vessels. In parallel with the test item treatments 3,5-Dichlorophenol was used as positive reference control at concentration levels of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control and nitrification controls were investigated. The definite test was performed without abiotic controls, based on the results of the preliminary test where abiotic controls were tested at the test item concentration of 1080 mg/L and no remarkable abiotic oxygen consumption was noticed. In accordance to the preliminary experiment, the test item does not affect adversely the pH within the test system; therefore, the main test was performed without additional pH adjustment. All validity criteria of the study were met. The observed oxygen consumption rates and consequently the specific respiration rates in the examined test item (limit) concentration remained in the range of the blank controls (the average specific respiration rate at 1080 mg/L: 37.13 mg O2/gh), no remarkable inhibitory effect of the test item was observed (the obtained 1.17 % inhibition was considered as being within the range of biological variability of the applied test system). The specific respiration rates at the examined test item concentration of 1080 mg/L were compared with the blank control values using 2-Sample T-test (α = 0.05). No statistical significant differences were noticed in the comparison of blank control and test item treated groups. Under the test conditions, the EC10 and EC50 values of test item were determined as higher than 1080 mg/L. Based on the statistical evaluation in this test the NOEC was 1080 mg/L. In conclusion this study demonstrated the absence of inhibition of oxygen consumption by the test item up to and including the limit concentration of 1080 mg/L.
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