1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

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:

2011-07-07 to 2011-09-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

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

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

- Due to the low solubility of the test item in test water, adequate amounts of the test item were directly dosed into each test flask after adding the test water.
- Stock Solution of 3,5-Dichlorophenol: 1 g of 3,5-dichloro-phenol was dissolved in 1000 mL deionised water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it had cooled to room temperature. The final pH was 7.2 and therefore in the range of 7 to 8. NaOH was used for the adjustment of the pH.
- Stock Solution of N-allylthiourea (ATU): . 2.5 mL of this stock solution were added to an incubation mixture of final volume of 500 mL. This resulted in a final concentration of 11.6 mg ATU/L.

Test organisms (species):

activated sludge

Details on inoculum:

- Source: municipal sewage treatment plant Bensheim, Germany
- Preparation of inoculum for exposure: used as collected, but coarse particles were removed by settling for a short period (10 minutes) and then the upper layer decanted.
- Pretreatment: During holding prior to use the sludge was fed with 50 mL synthetic sewage (see below) per litre and kept aerated at room temperature overnight. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined.
- Initial biomass concentration: Based on the sludge dry matter, calculated amounts of wet sludge were suspended in tap water to yield a concentration equivalent to about 3.0 g/L on dry weight basis. This level gives a concentration of 1.2 g/L suspended solids in the test medium.
- The pH of the activated sludge inoculum was 7.6

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20-21 °C during pre-incubation (3 h)
17-23 °C during evaluation period

pH:

7.4 - 8.3

Dissolved oxygen:

7.2 - 9.4 mg/L

Nominal and measured concentrations:

Nominal: 10, 32, 100, 320, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: 1L glass flasks, 500 mL fill volume
- Aeration: yes, compressed air (0.945 L/min)
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water / tap water

SYNTHETIC SEWAGE
- 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 • 2H2O, 0.2 g MgSO4 • 7H2O, 2.8 g K2HPO4 => filled up to 1 litre with deionised water

OTHER TEST CONDITIONS
- Adjustment of pH: no


EFFECT PARAMETERS MEASURED
- A well-mixed sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time and was aerated shortly before measurement.
- The oxygen concentration was measured with an oxygen electrode and recorded for about ten minutes.
- During measurement, the samples were continuously stirred on a magnetic stirrer.
- The oxygen consumption (in mg O2 L-1 minute-1) was determined from the most linear part of the respiration curve in the range between approx 7.0 - 2.5 mg O2/L.
- both the respiration rates with nitriffication and without nitrification (herotrophic respiration) were evaluated

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenole, purity 99.7%

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

91.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: CL 56-123 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

3 024 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: CL 252.4-361.9 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

123.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: CL 36.5-197 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

405.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: CL 285.6-584.1 mg/L

Results with reference substance (positive control):

- The 3-hour EC50 of the reference item 3,5-dichlorophenol for the total respiration was 5.1 mg/L and therefore in the range of 2 to 25 mg/L for the used activated sludge batch. The inhibition of the activated sludge treated with the ref-erence item was in the range of 41.9% to 86.9% for the test concentrations from 3.2 until 32 mg/L.
- The 3-hour EC50 of the reference item 3,5-dichlorophenol for the heterotrophic respiration without nitrification was 11.4 mg/L and therefore also in the range of 5 to 40 mg/L for the used activated sludge batch. The inhibition of the activated sludge treated with the ref-erence item was in the range of 22% to 80% for the test concentrations from 3.2 until 32 mg/L.
- The 3-hour EC50 of the reference item 3,5-dichlorophenol for the oxygen uptake due to nitrification was 1.4 mg/L and therefore in the range of
0.1 to 10 mg/L for the used activated sludge batch.

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited at the test concentrations of 10 and 32 mg/L. The respiration rate of the activated sludge was inhibited by 9.3% at 100 mg/L and 55.1% at 320 mg/L. At a nominal concentration of 1000 mg/L, the respiration rate was inhibited by 86.9% by a maximum. Concentrations exceeding 1000 mg /L test item nominal were not tested.

 

Respiration without nitrification (Heterotrophic Respiration):

In comparison to the inoculum controls the respiration rate of the activated sludge was not inhibited at the test concentrations of 10, 32 and 100 mg/L. The respiration rate of the activated sludge was inhibited by 44.3% at 320 mg/L. At a nominal concentration of 1000 mg/L, the respiration rate was inhibited by 79.8% by a maximum. Concentrations exceeding 1000 mg/L nominal were not tested.

 

The 3-hour EC50 for total respiration was calculated to be 302.4 mg/L 1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine. For heterotrophic respiration the 3-hour EC50 was calculated to be 406.9 mg/L. For the oxygen uptake due to nitrification the 3-hour EC50 was calculated to be 119.1 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The results of the study indicate that the test substance does not effect aquatic microorganisms up to a concentration of 32 mg/L. From 100 mg/L 1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine onwards the respiration ratge of the activated sludge was inhibited. The EC50 was 302.4 mg/L for the overall respiration and 405.9 mg/L for the heterotrophic respiration (without nitrification).

Executive summary:

The influence of 1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to OECD 209 and EU method C.11. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. Five concetrations ranging from 10.0 to 1000 mg/L were tested. The results of the study indicate that the test substance does not effect aquatic microorganisms up to a concentration of 32 mg/L. From 100 mg /L onwards the respiration ratge of the activated sludge was inhibited. The EC50 was 302.4 mg/L for the overall respiration and 405.9 mg/L for the heterotrophic respiration (without nitrification).

Description of key information

The influence of the test item 1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to OECD 209 and EU method C.11. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. Five concentrations ranging from 10.0 to 1000 mg/L were tested. The results of the study indicate that the test substance does not affect aquatic microorganisms up to a concentration of 32 mg/L. From 100 mg/L onwards the respiration rate of the activated sludge was inhibited. The EC50 was 302.4 mg/L for the overall respiration and 405.9 mg/L for the heterotrophic respiration (without nitrification).

Key value for chemical safety assessment

EC50 for microorganisms:

302.4 mg/L

EC10 or NOEC for microorganisms:

91.2 mg/L