Phenol, mono-C24-28 (even)-sec-alkyl derivs, ortho- and para-

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:

01 November 2016 to 30 November 2016

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:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

INOCULUM
- A mixed population of activated sewage sludge micro-organisms was obtained on 31 October 2016 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

PREPARATION OF INOCULUM
- The activated sewage sludge sample was washed twice by settlement and re-suspension 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 (50 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper (rinsed three times with 20 mL deionised reverse osmosis water prior to drying in an oven) using a Buchner funnel. Filtration was continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was dried in an oven at approximately 105 °C for at least 1 hour and allowed to cool before weighing. The process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.7 g/L prior to use.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

other: Carbon

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

MEDIUM
- The mineral medium used in this study was that recommended in the OECD guidelines (see Annex 2, attached).

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

TEST ITEM PREPARATION
- The test item was dispersed directly in mineral medium.
- An amount of test item (35.7 mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (approximately 15 minutes) prior to dispersal in inoculated mineral medium. The volume was adjusted to 3 L to give a final concentration of 11.9 mg/L, equivalent to 10 mg carbon/L.
- A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

REFERENCE ITEM PREPARATION
- A 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 this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 L 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 item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
- An amount of test item (35.7mg) was dispersed in approximately 400 mL of mineral medium with the aid of ultrasonication (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 3 L to give a final concentration of 11.9 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

PREPARATION OF THE TEST SYSTEM
- Test preparations were prepared and inoculated in 5 L test culture vessels each containing 3 L of solution.
(a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The 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).
- 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 of between 22 and 24 °C, in darkness.
- On Day 0 the test and reference items were added and the pH of all vessels measured using a Hach HQ40d Flexi handheld meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 L by the addition of mineral medium which had been purged overnight with CO2 free air.
- The test vessels were sealed and CO2-free air bubbled through the solution at a rate of 30 to 100 mL/min per vessel and stirred continuously by 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.

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

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

Reference substance:

benzoic acid, sodium salt

Remarks:

Batch SLBM8408V; Purity > 99.5 %; Expiry date 11 May 2017; Room temperature storage conditions over silica gel

Key result

Parameter:

% degradation (CO2 evolution)

Value:

48

Sampling time:

28 d

Details on results:

DEFINITIVE TEST
- Inorganic carbon values for the test item, procedure control, toxicity control and inoculum control vessels at each analysis occasion are given in Table 1 (attached).
- Percentage biodegradation values of the test and reference items and the toxicity control are given in Table 2 (attached)
- The 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 35 are given in Table 4 (attached).
- Observations made on the contents of the test vessels are given in Table 5 (attached).

VALIDATION CRITERIA
- The total C02 evolution in the inoculum control vessels on Day 28 was 36.33 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
- The IC content of the test item 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 the 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.

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. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore 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 inoculum control replicate 1, procedure control replicate 1 and 2. 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 48 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
- The toxicity control attained 54% biodegradation after 14 days and 77 % 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.

Results with reference substance:

- Sodium benzoate attained 78 % 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:

under test conditions no biodegradation observed

Conclusions:

The test item was determined to be not readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B

Executive summary:

GUIDELINE

A study was performed to assess ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the 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 of between 22 and 24 °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 48 % biodegradation after 28 days.

 

CONCLUSION

The test item was determined to be not readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

The test item attained 48 % biodegradation after 28 days and was determined to be not readily biodegradable (OECD 301 B, EU Method C.4 -C and OCSPP 835.3110).

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

GUIDELINE

A study was performed to assess ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301 B, "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 of between 22 and 24 °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 48 % biodegradation after 28 days.

 

CONCLUSION

The test item was determined to be not readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.