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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 August 2015 to 16 September 2015
Reliability:
1 (reliable without restriction)
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 micro-organisms
Details on inoculum:
- Mixed population of activated sewage sludge micro-organisms obtained on 17 August 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
- 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 3.6 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10 other: mg carbon/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 insoluble in water.
- Preliminary solubility/dispensibility work was therefore performed in order to determine the most suitable method of preparation (see Appendix 3, attached).

TEST MATERIAL
-The test item was dispersed directly in mineral medium.
- An amount (37.8 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 12.6 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.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation (Exception with regard to GLP and has been reflected in the GLP compliance statement).

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 in mineral medium.
- 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 amount of test item (37.8 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 12.6 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).
- 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 23 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 25.0 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 and adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution 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 prior to acidification with hydrochloric acid.
Reference substance:
benzoic acid, sodium salt
Preliminary study:
DEFINITIVE TEST
- Inorganic carbon values for the test item, 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).
Test performance:
VALIDATION CRITERIA
- Total CO2 evolution in the inoculum control vessels on Day 28 was 36.46 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:
53
Sampling time:
28 d
Remarks on result:
other: Did not satisfy criteria for rapid biodegradability (> 60% within 28 days)
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 a decrease in all replicate vessels with the exception of inoculum control R1 and test vessel R1 (and was considered to be due to sampling/analytical variation).
- 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 53 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD guideline No. 310B.
- The toxicity control attained 63 % 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 63 % biodegradation after 14 days and 84 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
no
Interpretation of results:
not inherently biodegradable
Conclusions:
The test item attained 53 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guidelines No. 301B.
Executive summary:

GUIDELINE

A study was performed to assess the 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 23 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, together with a toxicity control were used for validation purposes.

RESULTS

The test item attained 53 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

The test item attained 53 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable (OECD Guideline No. 301B).

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

GUIDELINE

A study was performed to assess the 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 23 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, together with a toxicity control were used for validation purposes.

RESULTS

The test item attained 53 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.