2,2-bis[(allyloxy)methyl]butyl (3-{[({2,2-bis[(allyloxy)methyl]butoxy}carbonyl)amino]methyl}-3,5,5-trimethylcyclohexyl)carbamate

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:

19 August 2009 to 23 September 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals. 209 Activated Sludge, Respiration Inhibition Test. China Environmental Science Press.

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Remarks:

deionised water

Details on test solutions:

PREPARATION OF TEST MATERIAL SOLUTION
A certain amount test material was weighted and dissolved in 160 mL deionised water, this solution was treated ultrasonically for 10 minutes. Stock solutions of test material with different concentrations were obtained.

PREPARATION OF REFERENCE SUBSTANCE SOLUTION
A solution of 3,5-dichlorophenol was prepared by dissolving 0.5 g 3,5-dichlorophenol in 10 mL of 1 mol/L NaOH, diluting to approximately 30 mL with deionised water and adding, whilst stirring, 0.5 mol/L H2SO4 to the point of incipient precipitation (approximately 8 mL of 0.5 mol/L H2SO4 was required) and finally diluting the mixture to one litre with deionised water. The pH was in the range 7 to 8.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The activated sludge was obtained from Lijiao Sewage Treatment Plant of Guangzhou.
- Preparation of inoculum for exposure: sludge was washed with tap water and after centrifuging the supernatant was decanted. This procedure was repeated three times. A small amount of the washed sludge was weighed and dried. From this result the amount (17.9 g/L) of wet sludge can be calculated that must be suspended in water in order to obtain an activated sludge with a mixed liquor suspended solids level of 4 g/L (± 10 per cent). This level gives a concentration of 1.6 g/L in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

Nominal and measured concentrations:

Nominal: 12.5, 25, 50, 100 and 200 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: cleaned and marked 1 L beakers were used as test vessels.
- Material, size, headspace, fill volume: 400 mL
- Aeration: Clean, oil-free air.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Sludge concentration (weight of dry solids per volume): 1.6 g/L
- Synthetic sewage feed: The synthetic sewage feed was made by dissolving the following amounts of substances in 1 litre of deionised water as follows: Peptone: 16 g, Beef extract: 11 g, Urea: 3 g, NaCl: 0.7 g, CaCI2.2H2O: 0.4 g, MgSO4.7H2O: 0.2 g and K2HPO4: 2.8 g.

METHOD
- At time "0", 12.8 mL of the synthetic sewage was made up to 240 mL with water. 160 mL of microbial inoculum was added and the total mixture of 400 mL was poured into the first vessel (first control C1). Oxygen was continuously supplied by magnetic stirring and puff.
- At "15 minutes", 160 mL of the test material stock solution (corresponding to 5.0 mg test material) and 12.8 mL synthetic sewage were added to vessel, then made up to 240 mL with water. At the end 160 mL microbial inoculum was added to make a final volume of 400 mL (T1). Oxygen was continuously supplied by magnetic stirring. This process was repeated at 15-minute intervals with 200 mL test material stock solution to give a series of vessels containing different amounts of the test material with different concentrations (containing 10.3, 19.6, 40.7 and 80 mg test material respectively).
- Finally, a second control (C2) was prepared.
- After 3 hours, the 10 minutes respiration rate of C1 was measured.
- The 10 minutes respiration rate of T1, T2, T3, T4, T5 and C2 was measured at 15-minute intervals in the same way.
- The respiration inhibition rate of each treatment group was calculated.

DATA PROCESSING
According to the concentration of dissolved oxygen in each group, the respiration rate was calculated, and the EC50 of test material in respiration inhibition rate was derived.

Respiration = (R0 - Rt) x 60/ ¿ t
Inhibition Rate (%) = [1 2Rs / (Rc1 + Rc2)] x 100%

Where:
¿ t - Test time, 10 min
R0 - Dissolved oxygen at 0 min, mg/L
Rt - Dissolved oxygen at 10 min, mg/L
Rs - The speed of depleting oxygen in the concentration of the test material, mg/(L.h);
Rc1 - The speed of depleting oxygen in C1, mg/(L.h)
Rc2 - The speed of depleting oxygen in C2, mg/(L.h)

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

SENSITIVITY TEST
-The results showed that the beginning and the end of controlled trials of respiratory rate were 28 and 27.1 mg/(L.h), the difference between the two sets of control was 1.6% and so falling within the 15% target of the quality assurance.
-The reference material exposed at the concentrations of 5, 10 and 20 mg/L and the test solution of the respiratory rate were 13.4, 11.8 and 4.44 mg/(L.h), respiratory inhibition rates were 51.4, 57.2 and 83.9%, respectively. Through regression analysis, the 3 h EC50 of reference material was 5.5 mg/L.

RANGE-FINDING TEST
- The results showed that at the beginning and the end of the controlled trials, respiratory rates were 25.80 and 24.18 mg/(L.h), the difference between the two sets of controls was 3.2%, falling within the 15% target of the quality assurance.
- At the test material concentrations of 12.5, 25, 50, 100 and 200 mg/L, the respiratory rates were 25.86, 26.22, 23.22, 21.78 and 25.32 mg/(L.h), respiratory inhibition rates were -3.5, -4.9, +7.1, +12.8 and -1.3 respectively. Through regression analysis, the 3 h EC50 of test material was higher than 200 mg/L.

QUALITY ASSURANCE
- The two control respiration rates were within 15% of each other.
- The 3 h EC50 of 3,5-dichlorophenol was 5.5 mg/L (within the accepted range of 5 to 30 mg/L).

Results with reference substance (positive control):

-The 3h- EC50 of 3, 5-dichlorophenol was 5.5 mg/L (within the accepted range of 5 to 30 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the 3 h EC50 of the test material was > 200 mg/L.

Executive summary:

The potential toxicity of the test material to micro-organisms was investigated in accordance with the standardised guidelines "State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 209 Activated Sludge, Respiration Inhibition Test" and OECD 209 and under GLP conditions using the activated sludge respiration inhibition test. The method described in this test was used to assess the effect of the test material on micro-organisms by measuring the respiration rate in the presence of five concentrations of the test material under defined conditions. The tested concentrations were 12.5, 25, 50, 100 and 200 mg/L. The exposure duration was 3 hours and after this period, the respiration rate was measured. Two blank control groups were included in the study as well as three difference concentrations of the reference substance 3,5-dichlorophenol (5, 10 and 20 mg/L). The quality assurance criteria were met as the reference substance produced an EC50 value of 5.5 mg/L that fell in the acceptance range, and the two measured control respiration rates were within 15% of each other. The test material did not affect the respiration rate of the activated sludge, therefore under the conditions of this study the 3 h EC50 of the test material was > 200 mg/L.

Description of key information

Under the conditions of this study the 3 h EC50 of the test material was > 200 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

200 mg/L

Additional information

The potential toxicity of the test material to micro-organisms was investigated in accordance with the standardised guidelines "State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 209 Activated Sludge, Respiration Inhibition Test" and OECD 209 and under GLP conditions using the activated sludge respiration inhibition test. The method described in this test was used to assess the effect of the test material on micro-organisms by measuring the respiration rate in the presence of five concentrations of the test material under defined conditions. The tested concentrations were 12.5, 25, 50, 100 and 200 mg/L. The exposure duration was 3 hours and after this period, the respiration rate was measured. Two blank control groups were included in the study as well as three difference concentrations of the reference substance 3,5-dichlorophenol (5, 10 and 20 mg/L). The quality assurance criteria were met as the reference substance produced an EC50 value of 5.5 mg/L that fell in the acceptance range, and the two measured control respiration rates were within 15% of each other. The test material did not affect the respiration rate of the activated sludge, therefore under the conditions of this study the 3 h EC50 of the test material was > 200 mg/L.