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EC number: 219-941-5 | CAS number: 2579-20-6
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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:
- 02 - 03 December 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study (with certificate) OECD and EC guidelines
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
- 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)
- Specific details on test material used for the study:
- Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: less than -25°C
- Boiling point: 240°C
- Vapour pressure: 34 Pa at 25°C
- Water solubility (under test conditions): greater than 1000 g/l of water
- log Pow: 0.73
- pKa: 10.2 - Analytical monitoring:
- no
- Vehicle:
- yes
- Details on test solutions:
- TEST SYSTEM
- Test vessel: no data
- Aeration: no data
- Renewal rate of test solution (frequency/flow rate): no
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates):1 x 10, 30, 100, 300mg/L. 3x 1000 mg/L
- No. of vessels per control (replicates):2
- No. of vessels per positive control (replicates):1 x 3, 10, 32mg/L
- Biomass loading rate: no data
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis
no other data
OTHER TEST CONDITIONS
- Adjustment of pH: yes, using 11.7M HCl to adjust pH from ~11 to ~7.
- Photoperiod: no data
- Light intensity: no data
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3-3.2 - Test organisms (species):
- activated sludge, domestic
- Details on inoculum:
- - Method of cultivation:
A sample of activated sludge was obtained the day before the start of the test from a sewagr treatment works, which treats predominantly domestic waste. In the laboratory, the sample was maintained under aerobic conditions until required. The concentration of suspended solids in a homogenised sample was determined on the day of collection and immediately before the start of the test.
- Preparation of inoculum for exposure:
On the day of collection, an aliquot (10 mL) of the activated sludge was filtered through a dried and preweighed Whatman GF/C filter paper, which was then dried again at approximately 105°C for at least one hour, then allowed to cool in a desiccator before being reweighed. The mixed liquor suspended solids (MLSS) content of the activated sludge was then calculated. Synthetic sewage (50 mL/L) was added to the stock of activated sludge and this was aerated overnight.
- Pretreatment:
On the day of the test, the MLSS content of the sludge was determined (in triplicate) and adjusted to 4 g/L by the addition of tap water. The pH and temperature of the sludge were also measured. Aliquots (200 mL) were then added to each mixture to give a final MLSS concentration of 1.6g/L.
- Initial biomass concentration:1.6 g/L - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 3 h
- Post exposure observation period:
- Following the exposure period, a well-mixed sample of each mixture was transferred to a biochemical oxygen demand (BOD) bottle (nominal capacity, 270 mL).
The rate of oxygen consumption was measured, over a period of approximately 10 minutes or until the dissolved oxygen concentration fell below ca. 2 mgO2/L using a Yellow Springs Instrument (YSI) dissolved oxygen meter, with temperature probe and self stirring bottle probe connected to a chart recorder.
The pH and temperature of the samples were measured at the start and end of the test. - Hardness:
- no data
- Test temperature:
- 19.6 to 19.8 °C
- pH:
- 7.3 to 8.1.
- Dissolved oxygen:
- 5.5 to 9.1 mgO2/L
- Salinity:
- freshwater
- Nominal and measured concentrations:
- Nominal: 0, 10, 30, 100, 300, 1000 mg/L
- Details on test conditions:
- TEST SYSTEM
- Aeration: The prepared mixtures were aerated for three hours in a thermostatically-controlled water bath, using a glass aerator connected to a laboratory supply of oil-free compressed air (one litre/minute).
- Renewal rate of test solution (frequency/flow rate): no
- No. of vessels per concentration (replicates):1 x 10, 30, 100, 300mg/L. 3x 1000 mg/L
- No. of vessels per control (replicates):2
- No. of vessels per positive control (replicates):1 x 3, 10, 32mg/L
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis
no other data
OTHER TEST CONDITIONS
- Adjustment of pH: yes, using 11.7M HCl to adjust pH from ~11 to ~7 - Reference substance (positive control):
- yes
- Remarks:
- 3,5-Dichlorophenol (DCP) 99%
- Duration:
- 3 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 1 000 mg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth inhibition
- Details on results:
- - Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
No biologically significant inhibition was observed in mixtures containing the test substance at 10, 30 and 100 mg/L.
Respiration rates were decreased by 27% at 300 mg/L and 27% to 52% (mean 37%) at 1000 mg/L.
Although the level of inhibition at 300 and 1000 mg/L was greater than that considered biologically significant (15%), it was not concentration related.
Therefore increasing the dose levels above 1000 mg/L would not give proportionally higher levels of inhibition.
Consequently no further testing was carried out and the results of this study are reported. The three-hour EC50 for 1,3-Bis(aminomethyl)cyclohexane could not be accurately calculated but was considered >1000 mg/L, the highest concentration tested. From these data, it was not possible to obtain an accurate three-hour EC20 or EC80. - Results with reference substance (positive control):
- - Results with reference substance valid? yes
Sludge respiration rates were progressively reduced in the presence of increasing concentrations of 3,5-DCP. The three-hour 50% effect concentration (EC50) for 3,5-DCP was calculated to be 6.0 mg/L (95% confidence limits, 2.4 to 11.4 mg/L).
The specific respiration rates of the control culture established at the end of the test (Control 2, 17.6 mgO2/g/h) was 108% of the rate established at the start (Control 1, 16.3 mgO2/g/h).
These results show that the test was valid and that the sample of activated sludge employed was sensitive to inhibition. - Validity criteria fulfilled:
- yes
- Conclusions:
- The three-hour EC50 for 1,3-Bis(aminomethyl)cyclohexane was >1000 mg/L, the highest concentration tested.
- Executive summary:
Concentration-related inhibition of respiration rates were not observed at any of the 1,3-Bis(aminomethyl)cyclohexane concentrations. Respiration rates were decreased by 27% at 300 mg/L and 27% to 52% (mean 37%) at 1000 mg/L. The three-hour EC50 for 1,3-Bis(aminomethyl)cyclohexane could not be accurately calculated but was considered >1000 mg/L, the highest concentration tested. The three-hour EC50 for 3,5-DCP (6.0 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 5 to 30 mg/L). The validity criterion relating to the respiration rates in the control (variation not greater than 15%) was also satisfied.
Reference
Table 1: Dissolved oxygen concentrations, measurement times, respiration rates and percentage inhibition
Test mixture | Initial DO concentration in the culture (mgO2/L) | Initial measured DO concentration (mgO2/L) (DO(1)) | Final measured DO concentration (mgO2/L) (DO(2)) | Measurement time (minutes) (t) | Specific respiration rate (SRR mgO2/g/h) | % inhibition |
Control (1) | 7.2 | 6.5 | 2.5 | 9.2 | 16.3 | - |
1,3-Bis(aminomethyl) cyclohexane (mg/L) | ||||||
10 | 6.6 | 5.5 | 2.0 | 7.9 | 16.6 | 2 |
30 | 6.7 | 6.0 | 2.5 | 8.2 | 16.0 | 6 |
100 | 7.0 | 6.0 | 3.0 | 7.2 | 15.6 | 8 |
300 | 6.2 | 5.5 | 3.0 | 7.6 | 12.3 | 27 |
1000 | 8.2 | 7.3 | 5.5 | 8.3 | 8.1 | 52 |
1000 | 8.4 | 7.5 | 5.0 | 7.6 | 12.3 | 27 |
1000 | 7.9 | 7.0 | 4.5 | 8.0 | 11.7 | 31 |
3,5-DCP (mg/L) | ||||||
3 | 8.3 | 7.4 | 5.5 | 6.4 | 11.1 | 34 |
10 | 9.1 | 7.9 | 6.5 | 7.8 | 6.7 | 60 |
32 | 9.3 | 9.1 | 8.5 | 7.8 | 2.9 | 83 |
Control (2) | 8.6 | 6.5 | 2.5 | 8.5 | 17.6 | - |
Table 2: Temperature and pH measurements
Test mixture | Temperature (°C) | pH | ||
Initial | Final | Initial | Final | |
Control (1) | 19.6 | 19.7 | 7.4 | 8.0 |
1,3-Bis(aminomethyl) cyclohexane (mg/L) | ||||
10 | 19.7 | 19.8 | 7.4 | 7.9 |
30 | 19.7 | 19.8 | 7.4 | 7.9 |
100 | 19.8 | 19.8 | 7.5 | 7.9 |
300 | 19.8 | 19.7 | 7.4 | 8.0 |
1000 | 19.7 | 19.7 | 7.6 | 8.0 |
1000 | 19.8 | 19.7 | 7.6 | 8.1 |
1000 | 19.7 | 19.7 | 7.6 | 8.0 |
3,5-DCP (mg/L) | ||||
3 | 19.8 | 19.7 | 7.4 | 8.1 |
10 | 19.8 | 19.7 | 7.3 | 8.0 |
32 | 19.8 | 19.7 | 7.3 | 8.0 |
Control (2) | 19.8 | 19.7 | 7.3 | 8.0 |
Description of key information
The three-hour EC50 for 1,3-Bis(aminomethyl)cyclohexane was >1000 mg/L, the highest concentration tested.
Key value for chemical safety assessment
- EC50 for microorganisms:
- 1 000 mg/L
Additional information
Concentration-related inhibition of respiration rates were not observed at any of the 1,3-Bis(aminomethyl)cyclohexane concentrations. Respiration rates were decreased by 27% at 300 mg/L and 27% to 52% (mean 37%) at 1000 mg/L. The three-hour EC50 for 1,3-Bis(aminomethyl)cyclohexane could not be accurately calculated but was considered >1000 mg/L, the highest concentration tested. The three-hour EC50 for 3,5-DCP (6.0 mg/L) fulfilled the validity criterion relating to sensitivity to inhibition (acceptable EC50 range 5 to 30 mg/L). The validity criterion relating to the respiration rates in the control (variation not greater than 15%) was also satisfied.
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