1,3-Cyclohexanedimethanamine

Toxicity to microorganisms

Link to relevant study record(s)

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

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.

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.