Tall oil, reaction products with ethanolamine

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 January 2015 to 18 February 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:

yes

Remarks:

10-day window not met plus room temperature discrepency (neither deviation affecting validity of study - see discussion below)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

10-day window not met plus room temperature discrepency (neither deviation affecting validity of study - see discussion below)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

yes

Remarks:

10-day window not met plus room temperature discrepency (neither deviation affecting validity of study - see discussion below)

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 19 January 2015 from the aeration stage of Severn Trent Water plc sewage treatment plant at Loughborough, Leicestershire, which treats predominantly domestic sludge.
- 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 using a Buchner funnel through pre-weighed GF/A filter paper rinsed three times with 20 mL deionised reverse osmosis waterprior 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 3.9 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

test mat.

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 SOLUBILITY WORK
- Information provided by the Sponsor indicated that the test item was partially soluble in water.
- Preliminary solubility/dispersibility work was therefore performed in order to determine the most suitable method of preparation (see Appendix 3, attached).

TEST ITEM
- The test item was dispersed directly in mineral medium.
- An amount (41.4 mg) of test item was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm for 15 minutes) prior to dispersal in inoculated mineral medium.
- The volume was adjusted to 3L to give a final concentration of 13.8 mg/L (equivalent to 10 mg carbon/L as recommended in the test guideline).

REFERENCE ITEM
- Sodium benzoate (C6H5COONa) 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 (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 homegeneity 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 sluge micro-organisms.
- An amount of test item (41.4 mg) was dispersed in approximately 400 mL of mineral medium with the aid of high shear mixing (approximately 7500 rpm for 15 minutes) prior to dispersal in inoculated mineral medium.
- An aliquot (51.4 mL) of the sodium benzoate stock solution was also added to the test vessel and the volume adjusted to 3L to give a final concentration of 13.8 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

PREPARATION OF 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 20mg carbon/L to act as a toxicity control (one vessel only).
- Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
- 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 temperatures between 18 and 23 °C in the dark. This slight deviation to the study plan was considered to have had no adverse effect on the study given that all validation criteria were met.
- Approximately 24 hours prior to addition of the test and reference items, the vessels were filled with 2400 mL of mineral medium and 23.1 mL of inoculum and aerated overnight.
- The test and reference items were added on day zero and the pH of all vessels was measured using a Hach HQ160 Flexi handheld meter.
- The volume of all vessels was then 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 de-gassed 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 HQ160 Flexi handheld meter prior to acidification with hydrochloric acid.

Reference substance:

other: sodium benzoate

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 32.43 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.

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 with the exception of procedure control replicate 2.
- The test item attained 74 % biodegradation after 28 days and can therefore be considered to be readily biodegradable.
- The test item failed to meet the 10-day window validation criterion whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals (2006), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individual structures takes place, then the 10-day window should not be applied to interpret the results of the test.
- The toxicity control attained 65 % biodegradation after 14 days and 68 % biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage microorganisms used in the test.

Results with reference substance:

- Sodium benzoate attained 72 % biodegradation after 14 days and 74 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

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 can therefore be considered to be readily biodegradable. The test item failed to meet the 10 -day window validation criterion whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals (2006), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individial structures takes place, then the 10 -day window should not be applied to interpret the results of the test.

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 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 18 to 23 °C for 28 days.

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

RESULTS

The test item attained 74 % biodegradation after 28 days and can therefore be considered to be readily biodegradable.

CONCLUSION

The test item attained 74 % biodegradation after 28 days and can therefore be considered to be readily biodegradable. The test item failed to meet the 10 -day window validation criterion whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals (2006), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individual structures takes place, then the 10 -day window should not be applied to interpret the results of the test.

Description of key information

The test item was investigated using a method compatible with OECD 301B and EU Method C.4-C and attained 74 % biodegradation after 28 days and can therefore be considered to be readily biodegradable. Failure of the test item to meet the 10-day window criterion need not be taken into consideration when interpreting the results because the test item is a complex mixture and a sequential biodegradation of individual structures takes place.

Key value for chemical safety assessment

Biodegradation in water:

readily biodegradable

Additional information

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 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 18 to 23°C for 28 days.

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

RESULTS

The test item attained 74 % biodegradation after 28 days and can therefore be considered to be readily biodegradable.

CONCLUSION

The test item attained 74 % biodegradation after 28 days and can therefore be considered to be readily biodegradable. The test item failed to meet the 10 -day window validation criterion whereby 60 % biodegradation must be attained within 10 days of the biodegradation exceeding 10 %. However, in accordance with the Revised Introduction to the OECD Guidelines for Testing of Chemicals (2006), if testing on a complex mixture is performed, and it is anticipated that a sequential biodegradation of the individual structures takes place, then the 10 -day window should not be applied to interpret the results of the test.