Reaction products of fatty acids, C18 (unsaturated) alkyl and epoxidized fatty esters, with amines polyethylenepoly-, tetraethylenepentamine

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:

Experimental start date: 01 February 2021 and Experimental completion date: 10 August 2021

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:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

A mixed population of sewage treatment micro-organisms was obtained for the definitive test on 12 July 2021 from the final effluent stage of the Severn Trent Water Plc sewage treatment plant at Belper, Derbyshire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The sample of effluent was filtered through coarse filter paper (first approximate 200 mL discarded) and maintained on aeration at measured temperatures of between 20 and 21 °C prior to use.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Initial conc.:

30 mg/L

Based on:

test mat.

Details on study design:

An initial test conducted at a test concentration of 100 mg/L was terminated after 13 days as the toxicity control had attained 0% biodegradation thereby indicating that it would not pass the validation criterion whereby this vessel needs to attain at least 25% biodegradation by
Day 14 of the test for the test item to be considered non-inhibitory.
A second test was conducted which also showed that the toxicity control attained 0% biodegradation after 14 and 28 days which confirmed that the test item was demonstrating an inhibitory effect on the sewage treatment micro-organisms used in the test.
A test concentration of 100 mg/L was initially selected for use in the study following the recommendations of the Test Guidelines.
However, in order to try to minimize any inhibitory effects of the test item, the definitive test was conducted at a reduced test item concentration of 30 mg/L.
The test item was dispersed directly in mineral medium.
A nominal amount of test item (15 mg) was dispersed in mineral medium (350 mL) and subjected to ultrasonic disruption for approximately 15 minutes prior to allowing to cool to temperatures of between approximately 21 and 22 °C and the measurement of the pH values using a Hach HQ30d Flexi handheld meter. Inoculum (5 mL) was then added and the volume adjusted to a final volume of 500 mL with mineral medium to give the test concentration of 30 mg/L.
The inoculum control vessels were prepared in a similar manner without the addition of test item.

Toxicity Control
A toxicity control, containing the test item and aniline, was prepared in order to assess any toxic effects of the test item on the sewage treatment micro-organisms used in the test.
A nominal amount of test item (15 mg) was dispersed in mineral medium (350 mL) and subjected to ultrasonication for approximately 15 minutes prior to allowing to cool to a temperature of approximately 21 °C and the measurement of the pH values using a Hach HQ30d Flexi handheld meter prior to the addition of an aliquot (50 mL) of the 1000 mg/L aniline stock solution (see Section 3.5.3) and measurement of the pH value. The inoculum (5 mL) was then added prior to adjusting to a final volume of 500 mL with mineral medium to give the test concentration of 30 mg test item/L and 100 mg aniline/L.

Reference substance:

aniline

Test performance:

Validation Criteria and Biodegradation
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%.

However, according to the revised introduction to the OECD Guidelines for Testing of Chemicals, Section 3 (OECD, 2006), if a test on a mixture is performed and it is anticipated that a sequential biodegradation of the individual structures is taking place, then the 10-day window should not be applied to interpret the results.

Key result

Value:

0

Sampling time:

28 d

Remarks on result:

other: at 30 mg/l

Details on results:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered as readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F.

Additionally, the toxicity control attained 0% biodegradation after 14 and 28 days, thereby not passing the validation criterion of attaining at least 25% biodegradation by Day 14 of the test for the test item not to be considered to be demonstrating an inhibitory effect. Care should therefore be taken in the interpretation of this result.

Results with reference substance:

Aniline (procedure control) attained 69% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 74% biodegradation was attained.

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F. 

Executive summary:

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.

 

An initial test conducted at a test concentration of 100 mg/L was terminated after 13 days as the toxicity control had attained 0% biodegradation thereby indicating that it would not pass the validation criterion whereby this vessel needs to attain at least 25% biodegradation by Day 14 of the test for the test item to be considered non-inhibitory.

A second test was conducted which also showed that the toxicity control attained 0% biodegradation after 14 and 28 days which confirmed that the test item was demonstrating an inhibitory effect on the sewage treatment micro-organisms used in the test.

Therefore, in order to try to minimize any inhibitory effects of the test item, the definitive test conducted at a reduced test item concentration of 30 mg/L was exposed to sewage treatment micro‑organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between approximately 21 and 22 ºC for 28 days. 

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

 

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F. 

Additionally, the toxicity control attained 0% biodegradation after 14 and 28 days thereby, not passing the validation criterion of attaining at least 25% biodegradation by Day 14 of the test for the test item not to be considered to be demonstrating an inhibitory effect. 

Aniline (procedure control) attained 69% biodegradation after 14 days with greater than 60% degradation being attained in a 10-Day window. After 28 days 74% biodegradation was attained.

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

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 301F. An initial test conducted at a test concentration of 100 mg/L was terminated after 13 days as the toxicity control had attained 0% biodegradation thereby indicating that it would not pass the validation criterion whereby this vessel needs to attain at least 25% biodegradation by Day 14 of the test for the test item to be considered non-inhibitory.

A second test was conducted which also showed that the toxicity control attained 0% biodegradation after 14 and 28 days which confirmed that the test item was demonstrating an inhibitory effect on the sewage treatment micro-organisms used in the test.

Therefore, in order to try to minimize any inhibitory effects of the test item, the definitive test conducted at a reduced test item concentration of 30 mg/L was exposed to sewage treatment micro‑organisms with mineral medium in sealed culture vessels in diffuse light at temperatures of between approximately 21 and 22 ºC for 28 days. 

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

 

The test item attained 0% biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301F. 

Additionally, the toxicity control attained 0% biodegradation after 14 and 28 days thereby, not passing the validation criterion of attaining at least 25% biodegradation by Day 14 of the test for the test item not to be considered to be demonstrating an inhibitory effect.