Reaction products of acrylonitrile and 2,2’-Iminodi(ethylamine), hydrogenated

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr 2019 to Sep 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

Principles of method if other than guideline:

NA (study was performed according to guideline).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of acrylonitrile and 2,2’-Iminodi(ethylamine), hydrogenated

EC Number:

810-418-2

Cas Number:

1703784-30-8

IUPAC Name:

Reaction products of acrylonitrile and 2,2’-Iminodi(ethylamine), hydrogenated

Test material form:

liquid

Specific details on test material used for the study:

PU-2018-821 (batch number: 2298695)
Test item PU-2018-821
Trade name Ortegol LA 2
Batch number 2298695
CAS No. 1703784-30-8
CAS name 2-propenenitrile, reaction products with diethylenetriamine, hydrogenated
Composition: 100%
Molecular weight: 217 g/mol
Molecular formula: C10H27N5
Appearance Colorless - light yellowish liquid
Density 0.96 g/cm³
pH 9 - 10
Water solubility Soluble
Expiry date: 2019-12-31
Recommended storage Room temperature, protection from light, under nitrogen in the tightly closed container

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Measurement of the Respiration Rates:
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: All concentrations were diluted from a stock solution.
- Controls: Test medium without test or reference item. Test medium without test or reference item with ATU
- Other relevant information: The reference item N-Methylanilin was tested with three concentrations within the concentration range 0.1 – 1.8 – 32 mg/L and a dilution factor of 18 (with and without ATU).
The reference item Copper (II)-sulfate pentahydrate was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of approximately 1.8 (without ATU).

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro organisms was obtained on 08 April 2019 from the aeration stage of the Municipal sewage treatment plant of 31137 Hildesheim, Germany which treats predominantly domestic sewage.

Preparation of Inoculum:
The sludge was washed twice and adjusted with chlorine free tap water if necessary. The sludge was used within 24 h after sampling.
Dry sludge concentration 3.21 g/L, corresponding to 1.61 g/L in the test vessel for replicates without ATU and 3.19 g/L, corresponding to 1.60 g/L in the test
vessel for replicates with ATU.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

NA

Post exposure observation period:

NA

Test conditions

Hardness:

Not specified

Test temperature:

20°C

pH:

7.26 to 8.57

Dissolved oxygen:

5.91 to 8.19 mg O2/L (>60% dissolved oxygen saturation in all vessels)

Salinity:

NA

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations: 4 - 16 - 64 - 256 - 1000 mg/L

Details on test conditions:

Based on the results of the preliminary test (see Annex I for details) the definitive study was carried out with 5 concentrations within the
range of 4 to 1000 mg/L (with and without ATU) in a geometrical series and with a dilution factor of 4. Additionally, replicates with
1000 mg/L were pH adjusted with 1m HCL. All concentrations were diluted from a stock solution.
Triplicates of the control (without ATU) without test item were included at the beginning and at the end of the replicates without ATU.
Triplicates of the control (with ATU) without test item were included at the beginning of the replicates with ATU and at the end of the test.
The reference item Copper (II)-sulfate pentahydrate was tested with three concentrations within the concentration range 58 - 180 mg/L
and a dilution factor of approximately 1.8 (without ATU).
The reference item N-Methylanilin was tested with three concentrations within the concentration range 0.1 – 1.8 – 32 mg/L
and a dilution factor of 18 (with and without ATU).
A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice
and adjusted with chlorine free tap water to a dry sludge concentration of 3.0 g/L ± 10 %.


pH The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined
prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
Oxygen After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of
oximeter every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.
Temperature The temperature was determined in the first replicate with and without ATU at test start with the pH-meter. The room temperature
during the test was recorded continuously.
Atmospheric pressure The atmospheric pressure was determined with a barometer before the first replicate was measured (needed for measurement of the
oxygen concentration).

Reference substance (positive control):

yes

Remarks:

N-Methylaniline

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

160 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

36.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 3

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 4

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

556 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Effect concentrations 5

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

55.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Effect concentrations 6

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Effect concentrations 7

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

98.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Effect concentrations 8

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Effect concentrations 9

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Results with reference substance (positive control):

The 3-hour EC50 value for the reference item N- Methylaniline, was EC50 = 2.32 mg/L with 95% Confidence Interval = 1.79 – 3.03 mg/L, thus confirming the acceptability of the inoculum used in the test system.

In the reference test the EC50-value for copper (II) sulphate pentahydrate was determined by linear regression with: EC50 = 86.8 mg/L with 95% Confidence Interval = 83.9 – 89.7 mg/L.
This is in the recommended range of validity of 53 - 155 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of PU-2018-821 for total respiration is 16 mg/L.
The EC10 for total respiration is 36.8 mg/L and the EC50 is 160 mg/L.
The NOEC for heterotrophic respiration is 16 mg/L.
The EC10 for heterotrophic respiration is 55.8 mg/L and the EC50 is 556 mg/L.
The NOEC for nitrification is 16 mg/L.
The EC10 for nitrification is 35.0 mg/L and the EC50 is 98.2 mg/L