3-[[3-(dimethylamino)propyl]amino]propiononitrile

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 27 June 2016 to 25 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

Referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

Deviations:

no

Principles of method if other than guideline:

Not required - method to guideline

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item:
Identification: 3-[[3-(Dimethylamino)propyl]amino]propanenitrile (TK2710)
Physical state/Appearance: clear colorless liquid
Batch: AAE1131000
Purity: 98.7%
Expiry Date: 27 July 2018
Storage Conditions: room temperature in the dark

Reference Item:
Identification: Aniline
Physical state/Appearance: yellow/orange liquid
Batch: 1479758
Purity: 99.99%
Expiry Date: 29 January 2017
Storage Conditions: room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, adapted

Remarks:

filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at temperatures of between 20 and 22 ºC prior to use.

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained on 22 June 2016 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration in a temperature controlled room at temperatures of between 20 and 22 ºC prior to use. This was a deviation to the study plan which states that the inoculum is maintained on aeration prior to use at test temperature which was 22 ± 1 ºC. This deviation was considered to have had no effect on the integrity of the study given that all validation criterion were met.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

The mineral medium used in this study was that recommended in the OECD Guidelines.

Test Item Preparation
The test item was dissolved directly in mineral medium.
A nominal amount of test item (500 mg) was dissolved in mineral medium and the volume adjusted to 500 mL to give a 1000 mg/L stock solution. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and inoculum (5 mL) to give the final test concentration of 100 mg/L. The volumetric flasks containing the stock solution and the test concentration were inverted several times to ensure homogeneity.
A test concentration of 100 mg/L was selected for use in the study following the recommendations of the Test Guideline.

Reference Item Preparation
A reference item, aniline (C6H5NH2), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium with the aid of ultrasonication for approximately 10 minutes. An aliquot (50 mL) of this stock solution was diluted with mineral medium (445 mL) and the inoculum (5 mL), to give the test concentration of 100 mg/L. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effect of the test item on the sewage treatment micro-organisms used in the test.
An aliquot (50 mL) of the 1000 mg/L test item stock solution (see Section 3.5.1) and an aliquot (50 mL) of the 1000 mg/L aniline stock solution was diluted with mineral medium (395 mL) and inoculum (5 mL) to give the test concentration of 100 mg test item/L and 100 mg aniline/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 500 mL bottles:
a) Three replicate bottles containing inoculated mineral medium to act as the inoculum control.
b) Two replicate bottles containing inoculated mineral medium and the reference item, aniline, at a concentration of 100 mg/L.
c) Three replicate bottles containing inoculated mineral medium and the test item at a concentration of 100 mg/L.
d) Two replicate bottles containing inoculated mineral medium, the reference item, aniline, at a concentration of 100 mg/L and the test item at a concentration of
100 mg/L to act as toxicity control vessels.
All vessels were inoculated with the prepared inoculum at a rate of 1% v/v.
On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter prior to the addition of the inoculum and the volume in all the vessels being adjusted to 500 mL by the addition of mineral medium.
In order to confirm that the aniline and test item stock solutions were prepared correctly, a diluted, 100 mg/L stock solution (in reverse osmosis water) was also sampled for DOC analysis.
All inoculum control, test item, procedure control and toxicity control vessels were placed in a CES Multi-Channel Aerobic Respirometer.
The system consists of a sample flask sealed by a sensor head/CO2 trap immersed in a temperature controlled water bath. The samples were stirred for the duration of the test with a magnetically coupled stirrer.
As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into the ethanolamine solution (50% v/v) causing a net reduction in gas pressure within the sample flask (see Figure 1). The pressure reduction triggers the electrolytic process, generating oxygen and restoring the pressure in the sample flask. The magnitude of the electrolyzing current and the duration of the current is proportional to the amount of oxygen supplied to the micro-organisms. The data generated from the respirometer’s own battery backed memory was collected on the hard disk drive of a non-dedicated computer.
The test was conducted in diffuse light at a temperature of between 22 and 24 ºC. The temperature of the water bath where the test vessels were incubated was recorded as being approximately 24 °C on Days 23, 24, 25, 26 and 27 of the study. Therefore this was a deviation to the study plan which states that the study will be conducted at a temperature of between 20 to 24 °C with a maximum deviation of ± 1°C during the test.
This deviation was considered to have had no effect on the integrity of the study given that all validation criterion were met.
On Day 28, two inoculum control, one procedure control, two test item and one toxicity control vessel were sampled for pH analysis.
The remaining vessels which were not sampled were discarded and are not reported. Additional replicate vessels were prepared and incubated in order that in the event of a leak in the test system a replicate vessel could be discarded without jeopardizing the integrity of the test.

Assessments
Physico-chemical Measurements
The temperature of the water bath was recorded daily.

pH Measurements
In order to confirm whether the pH of the test preparations changed, the pH was measured using a Hach HQ40d Flexi handheld meter on Days 0 and 28.

Validation Criteria
The BOD of the inoculated mineral medium (control) is normally 20 to 30 mg O2/L and should not exceed 60 mg O2/L after 28 days. Values higher than 60 mg O2/L require critical examination of the data and the experimental technique.
If the pH of the inoculated test vessels after 28 days is outside the range 6.0 to 8.5 and the biodegradation rate of the test item is less than 60%, the test should be repeated at a lower test concentration.
The test is considered valid if the difference between extremes of replicate BOD values at the time the plateau is reached, at the end of the test or at the end of the 10-day window, as appropriate, is less than 20%.
The toxicity control (test item and aniline) should attain ≥ 25% biodegradation by Day 14 for the test item to be considered as non-inhibitory.
The percentage biodegradation of aniline calculated from oxygen consumption values must be 60% (in a 10-Day window) after 14 days.
Test items giving a measured BOD value which is ≥ 60% of the ThOD value (within 28 days) are regarded as readily biodegradable. This level must be reached within 10 days of the biodegradation rate exceeding 10%.

Reference substance:

aniline

Preliminary study:

Not required

Test performance:

Validation Criteria
The mean BOD of the inoculated mineral medium (control) was 27.20 mg O2/L after 28 days and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The pH of the inoculated test item vessels on Day 28 ranged from 7.8 to 7.9 and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between extremes of replicate BOD values at the end of the test was less than 20% and therefore satisfied the validation criterion given in the OECD Test Guidelines.

Key result

Parameter:

% degradation (DOC removal)

Value:

7

Sampling time:

28 d

Details on results:

The test item attained 7% biodegradation after 28 days, calculated from the oxygen consumption values, and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.
The toxicity control attained 34% biodegradation after 14 days and 41% biodegradation after 28 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the test.

Results with reference substance:

Aniline (procedure control) attained 64% biodegradation after 14 days and 71% biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 7% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Executive summary:

Introduction                  

The study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301F, “Ready Biodegradability; Manometric Respirometry Test” referenced as method C.4-D of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (q)).

Methods                  

The test item at a concentration of 100 mg/L was exposed to sewage treatment micro-organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between 22 and 24 ºC for 28 days. 

The biodegradation of the test item was assessed by the measurement of daily oxygen consumption values on Days 0 to 28. Control solutions with inoculum and the reference item, aniline, together with a toxicity control were used for validation purposes.

Results                  

The test item attained 7% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Description of key information

The biodegradability of Aradur 1019 was determined according to OECD test guideline 301F "Ready biodegradability; Manometric Respirometry Test".

The study concludes that Aradur 1019 is not readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed