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Toxicity to microorganisms

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Reference
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:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
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:
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.

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 
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.

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.