N,N-bis(2,3-epoxypropyl)aniline

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 February 2013 - 01 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was performed according with OECD Guideline No. 209 and in accordance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

(2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2007)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

EC Number:

275-662-9

EC Name:

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

Cas Number:

71604-74-5

Molecular formula:

C15H19NO4

IUPAC Name:

3-(oxiran-2-ylmethoxy)-N,N-bis(oxiran-2-ylmethyl)aniline

Test material form:

liquid: viscous

Details on test material:

Description: Clear slightly yellow very viscous liquid
Test substance storage: In refrigerator (2-8°C) in the dark

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

- Method: The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 10 minutes. Subsequently, 16 ml synthetic medium, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring

- Controls: a blank-control, an abiotic control and the highest loading rate with a nitrification inhibitor.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge). The pH was 7.1 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Performance of the test: The synthetic medium (16 ml) and an appropriate amount of the test substance stock were mixed and made up to 250 ml with Milli-RO water in a 1 litre bottle. The pH was determined. Thereafter 250 ml activated sludge was added.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

- After the 3-hour contact time, the oxygen consumption was recorded for a period of 10-11 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.

Test conditions

Test temperature:

18 to 20°C

pH:

- Before addition of sludge was between 7.4 and 7.5.
- After the 3 hour exposure period the pH was between 7.4 and 8.1.

Dissolved oxygen:

- At the start of the test the dissolved oxygen concentration was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C)

- The respiration rate (R) from each vessel, in mg O2/l.h was calculated or interpolated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/l.

- Controls: 42 mg O2/l h (mean value)
T1 (a loading rate of 10 mg/l): 41 mg O2/l h
T2 (a loading rate of 100 mg/l): 46 mg O2/l h
T3 (a loading rate of 1000 mg/l): 30 mg O2/l h (mean value)

Nominal and measured concentrations:

- Nominal: Blank-control and loading rates of 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass bottles
- Type: open
- Material, size, headspace, fill volume: All-glass, 1 L capacity, filled with 500 ml of test solution
- Aeration: Yes. The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 3 replicates for the 1000 mg/L test concentration and 1 replicate for the other test concentrations
- No. of vessels per control (replicates): Blank-control (6 replicates), nitrification control and abiotic control
- Concentration of suspended solids in test solution: 1.5 g/L of sludge

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges (Milli-Q)
- Intervals of water quality measurement
pH: At the beginning and at the end of the test period.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): After the 3-hour contact time the oxygen consumption was recorded for a period of 10-11 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x10

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: An average of 28% inhibition was observed at 1000 mg/L

Details on results:

- The combined limit/range-finding test showed 2%, no inhibition and an average of 28% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/l, respectively. The inhibition observed at a loading rate of 1000 mg/l was statistically significant (Two Sample t-Test). The EC50 was above the highest loading rate tested (1000 mg/l).

- There was no oxygen uptake from abiotic processes. The results at 1000 mg/l with a nitrification inhibitor showed 52% heterotrophic inhibition of the respiration rate.

Results with reference substance (positive control):

- The EC50 was 8.9 mg 3,5-Dichlorophenol/L

Reported statistics and error estimates:

- ECx
For m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline no EC50 could be calculated because the test substance proved to be non-toxic (EC50 > a loading rate of 1000 mg/l).

- NOEC estimation
In this present test m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was slightly toxic to waste water (activated sludge) bacteria at all loading rates tested. Therefore, a NOEC could not be determined in the combined limit/range-finding test (Two sample-t-Test α=0.05, Toxstat Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). As discussed with the sponsor no further testing was performed to determine the NOEC.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

- Under the conditions of this present test m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was slightly toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l. As discussed with the sponsor no further testing was performed to determine the NOEC.

- The EC50 exceeded a loading rate of 1000 mg/l.

Executive summary:

The influence of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline on the respiration rate of activated sludge was investigated after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209, 2010. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11 and ISO Standard 8192 (2007).

The batch of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline tested was a clear slightly yellow very viscous liquid and consisted of 100% Mono constituent substance. No correction was made for the purity/composition of the test substance. The test substance was not completely soluble in test medium at the loading rates initially prepared. Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 10 minutes. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for 10-11 minutes.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower concentrations consisted of one replicate. In addition, blank-controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were tested.

The combined limit/range-finding test showed 2%, no inhibition and an average of 28% inhibition of the respiration rate at a loading rate of 10, 100 and 1000 mg/l, respectively. The inhibition observed at a loading rate of 1000 mg/l was statistically significant (Two Sample t-Test : α=0.05). The EC50 was above the highest loading rate tested (1000 mg/l).

There was no oxygen uptake from abiotic processes. The results at 1000 mg/l with a nitrification inhibitor showed 52% heterotrophic inhibition of the respiration rate.

The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

In conclusion, m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was slightly toxic to waste water (activated sludge) bacteria at or below a loading rate of 1000 mg/l. As discussed with the sponsor no further testing was performed to determine the NOEC.

The EC50 exceeded a loading rate of 1000 mg/l.