Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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:

from 2007-11-13 to 2007-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Before the start of the test defined amounts of the test item were directly weighed into the test flasks and mechanically dispersed using ultrasonic bath for an hour. The test concentrations (10, 31, 100, 313 and 1000 mg/L) were chosen to permit the determination of the EC50. Concentrations in excess of nominal 1000 mg test item/L were not tested.

Test organisms (species):

activated sludge

Details on inoculum:

Species: Activated sludge, microorganisms from a domestic wastewater treatment plant.
Origin: The activated sludge was supplied from the sewage plant for domestic sewage in Veszprém, Hungary.
Conditioning: The activated sludge used for this study was washed and centrifuged and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution and again centrifuged. This procedure was repeated twice. An aliquot of the final sludge suspension was weighed, dried and a ratio of wet sludge to its dry weight determined. Based on this ratio, calculated amounts of wet sludge were suspended in isotonic saline solution to yield a concentration equivalent to 4 g per litre (on dry weight basis). The pH of the activated sludge inoculum was determined to be pH 7.41. The activated sludge was used directly after conditioning.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.4 – 20.6 °C (during the incubation)
19.9 – 21.0 °C (during oxygen measurement)

pH:

7.37 to 8.70

Dissolved oxygen:

6.8 - 8.1

Nominal and measured concentrations:

nominal: 10; 31; 100; 313 and 1000 mg/L
no measured concentrations

Details on test conditions:

Test Units
Type and Size: Erlenmeyer bottles of approximately 350 ml volume and BOD bottles with special neck of 300 ml volume.
Identification: Each test flask was uniquely identified with study code, code of each group (control and treatment) and replicate number (in case of controls).

Test Conditions
Surrounding Type: Climate chamber (during the incubation) and controlled environment room (during the formulation and oxygen measuring)
Temperature:19.4 – 20.6 °C (during the incubation) and 19.9 – 21.0 °C (during oxygen measurement)
Aeration: With compressed air (approximately 1 litre per minute)
Recording: Test conditions were measured with suitable instruments and documented in the raw data.
Equipment:
Normal laboratory equipments were used in the study,
Self stirring O2 electrode,
Oxygen meter,
Thermometer,
pH meter,
Balance,
Centrifuge,
Moisture analyzer,
Aeration system,
Orbital shaker

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

72.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

27.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

190.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited dose-dependently in the whole examined concentration range, displaying inhibition rates from 4.4 up to 96.5 %.
At the lowest nominal concentration of 10 mg/L, the respiration rate of the activated sludge was inhibited by 4.4 %, at 31 and 100 mg/L, by 9.7 % and 80.5 %, respectively. Furthermore, at the nominal concentrations of 313 and 1000 mg test item/L, the respiration rate was inhibited by 96.5 % and 92.9 %, respectively.
Defined amount of test item were directly weighed into the test flasks, and the low water solubility of the test item could cause the slight deviations from dose-dependent inhibition (Stronger inhibition was observed at the test item concentration of 313 mg/L than at the highest concentration level.).
Based on measured inhibition rates the 3-hour EC20, EC50 and EC80 and their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.
The NOEC was determined to be 31 mg/L. The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were inhibited and influenced dose dependently in the whole concentration range, however the observed slight inhibition (4.4 %) at the concentration level of 10 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.540 was equal the respiration rate calculated at the second control. At the concentration of 31 mg/L the calculated respiration rate (0.510) was in the historical control data range (0.490 ± 0.089), and the deviation from the control within ± 15 % (the inhibition at 31 mg/L is < 10%) can be considered as a biological variability of the test system.

Results with reference substance (positive control):

The following nominal concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 5, 16 and 32 mg/L. In comparison to the controls the respiration rate of the activated sludge was inhibited by 27.4 % at the lowest nominal concentration of 5 mg/L. At the nominal concentrations of 16 and 32 mg/L, the respiration rate was inhibited by 73.5 % and 85.8 %, respectively. The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 9.30 mg/L with 95 % confidence limits of 7.73 to 11.19 mg/L.

Reported statistics and error estimates:

Probit analysis using TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value was determined as 72.6 mg/L. The NOEC was determined to be 31 mg/L.

Executive summary:

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item on the activity of activated sludge by measuring the respiration rate under defined conditions. 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. The inhibitory effect of the test item at the particular concentrations was expressed as percentage of the mean respiration rate of two controls.

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 4.4 % and 96.5 % in the examined nominal test concentration range. The inhibition showed a nearly dose-related tendency. The respiration rate was inhibited with 96.5 % at the concentration level of 313 mg/L and 92.9 % at the highest concentration level, at 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested.

Based on measured inhibition rates the 3-hour EC20, EC50 and EC80 and their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

The NOEC was determined to be 31 mg/L.

The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were inhibited and influenced dose dependently in the whole concentration range, however the observed slight inhibition (4.4 %) at the concentration level of 10 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.540 was equal the respiration rate calculated at the second control. At the concentration of 31 mg/L the calculated respiration rate (0.510) was in the historical control data range (0.490 ± 0.089), and the deviation from the control was within ± 15 % (the inhibition at 31 mg/L is < 10%) can be considered as a biological variability of the test system.

Description of key information

The test item was assessed in a study according to EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test). The EC50 was determined to be 72.6 mg/L and the NOEC was 31 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

72.6 mg/L

EC10 or NOEC for microorganisms:

31 mg/L

Additional information

The purpose of the 3-hour toxicity test was to evaluate the influence of the test item on the activity of activated sludge by measuring the respiration rate under defined conditions. 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. The inhibitory effect of the test item at the particular concentrations was expressed as percentage of the mean respiration rate of two controls.

In comparison to the inoculum controls the respiration rate of the activated sludge was inhibited between 4.4 % and 96.5 % in the examined nominal test concentration range. The inhibition showed a nearly dose-related tendency. The respiration rate was inhibited with 96.5 % at the concentration level of 313 mg/L and 92.9 % at the highest concentration level, at 1000 mg/L. Concentrations exceeding 1000 mg/L nominal were not tested.

Based on measured inhibition rates the 3-hour EC20, EC50 and EC80 and their 95 %-confidence limits were calculated by Probit analysis using TOXSTAT software.

The NOEC was determined to be 31 mg/L.

The NOEC for respiration rates was not based on the results of a statistical analysis, but it is biologically justified. The respiration rates were inhibited and influenced dose dependently in the whole concentration range, however the observed slight inhibition (4.4 %) at the concentration level of 10 mg/L was evaluated as reflecting the biological variability in the test. The calculated respiration rate, 0.540 was equal the respiration rate calculated at the second control. At the concentration of 31 mg/L the calculated respiration rate (0.510) was in the historical control data range (0.490 ± 0.089), and the deviation from the control was within ± 15 % (the inhibition at 31 mg/L is < 10%) can be considered as a biological variability of the test system.