Tall Oil Fatty Acids, compounds with 2-(2-aminoethoxy) ethanol

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:

20 May 2015 to 16 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution 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:

other: mixed population of activated sewage sludge microorganisms

Details on inoculum:

- Mixed population of activated sewage sludge micro-organisms obtained on 07 September 2015 from the aeration stage of the Severn Trent Water Plc. Sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
- The activated sewage sludge sample was washed twice by settlement and resuspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present.
- The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and used on the day of collection.
- Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper using a Buchner funnel (The Filter paper is rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven).
- Filtration was continued for a further 3 minutes after rinsing the filter three successive time with 10 mL of deionised reverse osmosis water.
- The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to cool before weighing.
- The process was repeated until a constant weight was attained.
- The suspended solids concentration was equal to 2.5 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

MINERAL MEDIUM
- Mineral medium was that recommended in the OECD Guidelines (see Appendix 2, attached).

PRELIMINARY INVESTIGATIONAL WORK
- In order to investigate whether the test item absorbed to filter matrices and/or the activated sewage sludge, samples were taken for Dissolved Organic Carbon (DOC) analysis and, as part of the sample preparation, the samples were filtered or centrifuged to remove the sewage sludge solids.
- The following work was conducted and samples analysed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC-VCPH analyser (see Appendix 3, attached).
- A nominal amount of test item (100 mg) was dispersed in mineral medium (1 L) to give a 100 mg/L stock dispersion.
- Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 μm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter).
- A further nominal amount of test item (100 mg) was dispersed in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 L.
- Two samples were taken for DOC analysis; one after filtration through a 0.45 μm Gelman AcroCap filter (discarding the initial 5 mL to pre-condition the filter) and the other after centrifugation at 4000 g for 15 minutes.
- Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as per the test item samples.

TEST ITEM
-The test item was dispersed directly in mineral medium.
- An nominal amount (300 mg) of test item was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (15 minutes) and the volume adjusted to 1 L to give a 300 mg/L stock dispersion.
- An aliqot (141 mL) of the stock solution was dispersed in inoculated mineral medium.
- The volume was adjusted to 3L to give a final concentration of 14.1 mg/L (equivalent to 10 mg carbon/L).
- The volumetric flask containing the test item was inverted several times to ensure homogenicity.

REFERENCE ITEM
- Sodium benzoate (C6H5COONa) was used as the reference item in 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 5 minutes.
- An aliquot (51.4 mL) of the stock solution was added to the test vessel containing inoculated mineral medium and the volume was adjusted to 3L to give a final test concentration of 17.1 mg/L (equivalent to 10 mg carbon/L).
- The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

TOXICITY CONTROL
- A toxicity control containing the test material and sodium benzoate was prepared in order to assess any toxic effect of the test item on sewage sludge micro-organisms.
- An aliquot of test item (141 mL) of the test item stock solution was dispersed in inoculated mineral medium.
- An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel.
- The volume was adjusted to 3L to give a final concentration of 14.1 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

TEST SYSTEM
- The following were prepared and inoculated in 5L test culture vessels each containing 3L of solution:
(a) Inoculated control, in duplicate, consisting of inoculated mineral medium.
(b) Procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
(c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
(d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
- Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L.
- The test was carried out in a temperature controlled room at 21 to 24 °C in the dark.
- Approximately 24 hours prior to addition of the test and reference items, the vessels were filled with 2400 mL of mineral medium and 36.0 mL of inoculum and aerated overnight.
- The test and reference items were added on day 0 and the pH of all vessels was measured using a Hach HQ40d Flexi handheld meter.
- The pH was adjusted to 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide before the volume in each vessel was adjusted to 3L by the addition of mineral medium which had been purged overnight with CO2-free air.
- The test vessels were sealed and CO2-free air was bubbled through the solution in each vessel at a rate of 30 to 100 mL/min whilst stirring continuously using a magnetic stirrer.
- The CO2-free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb) granules.
- The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH.
- The CO2 absorbing solutions were prepared using purified water.

EVALUATIONS
- The appearance of the test preparations was recorded on Days 0, 6, 13, 20 and 27.

pH MEASUREMENT
- The pH of the test preparations was determined on Days 0 and 28 using a Hach HQ40d Flexi handheld meter or a Hach 160d handheld meter prior to acidification with hydrochloric acid.

Preliminary study:

PRELIMINARY INVESTIGATIONAL WORK
- Results obtained from the samples taken for DOC analysis inidcated that the test item may absorb to filter matrices but did not absorb to activated sewage sludge (see Appendix 3, attached).
- Therefore, for the purposes of the study, the samples taken for DOC analysis were centrifuged to remove the suspended solids present without loss of any test item,

Test performance:

DEFINITIVE TEST
- Inorganic carbon values for the test material, procedure control, toxicity control and inoculum control vessels are shown in Table 1 (attached).
- Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 (attached).
- Biodegradation curves are presented in Figure 1 (attached).
- Total and inorganic carbon values in the culture vessels on Day 0 are given in Table 3 (attached).
- The pH values of the test preparations on Days 0 and 28 are given in Table 4 (attached).
- Observations made on the contents of the test vessels are given in Table 5 (attached).

VALIDATION CRITERIA
- Total CO2 evolution in the inoculum control vessels on Day 28 was 38.78 mg/L and therefore satisfied the validation criterion given in the OECD test guidelines.
- The IC content of the test material suspension in the mineral medium at the start of the test (see Table 3, attached) was below 5 % of the TC content and hence satisfied the validation criterion given in the OECD test guidelines.
- The difference between values for CO2 production at the end of the test for the replicate vessels was < 20 % and hence satisfied the validation criterion given in the OECD test guidelines.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

74

Sampling time:

28 d

Details on results:

BIODEGRADATION
- Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the test guidelines.
- Acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels.
- Any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test item.
- The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels.
- Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.
- The test item attained 74 % biodegradation after 28 days and satisfied the 10-day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301 B.
- The toxicity control attained 77 % biodegradation after 14 days and 108 % biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro-organisms used in the test. Biodegradation values in excess of 100 % were considered to be due to sampling/analytical variation.
- Sodium benzoate attained 80 % biodegradation after 14 days and 77 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The ready biodegradability of the test item was assessed in accordance with OECD Guideline 301B. The test item attained 74 % biodegradation after 28 days and and satisfied the 10 -day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The substance can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301 B.

Executive summary:

GUIDELINE

 

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method was designed to be compatible with OECD Guidelines for Testing of Chemicals (1992) No 301B "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4 -C of Commission Regulation (EC) No 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph m).

 

METHODS

 

The test item at a concentration of 10 mg carbon/L was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures between 21 and 24 °C for 28 days.

 

The biodegradation of the test item was assessed by determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, and a toxicity control were used for validation purposes.

 

RESULTS

 

The test item attained 74 % biodegradation after 28 days and satisfied the 10 -day window validation criterion, whereby 60% biodegradation must be attained within 10 days of the biodegradation exceeding 10%. The test item can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301 B.

 

The toxicity control attained 77 % biodegradation after 14 days and 108 % biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment microorganisms used in the test. Biodegradation values in excess of 100 % were considered to be due to sampling/analytical variation.

 

Sodium benzoate attained 80 % biodegradation after 14 days and 77 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions. The slight decrease in biodegradation between days 14 and 28 was considered to be due to sampling/analytical variation.

 

CONCLUSION

 

The test item attained 74 % biodegradation after 28 days and satisfied the 10 -day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The substance can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301 B.

Description of key information

The ready biodegradability of the test item was assessed in accordance with OECD Guideline 301B.  The test item attained 74 % biodegradation after 28 days and and satisfied the 10 -day window validation criterion, whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. The substance can therefore be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301 B.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

No additional data.