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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:
August 16 2001 to September 14 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline and GLP study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
GLP compliance:
yes
Remarks:
except for the determination of total organic content of mineral medium
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
not applicable
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, domestic, adapted
Details on inoculum:
Type: inoculum was collected from a water treatment plant containing effluent from a predominantly domestic origin.
Reason for this choice: inoculum based on aerated sludge is recommended in OECD and EEC
Collected from: The water treatment plant Emeraude (SIARR) (Petit-Quevilly, France).
Sampling date: 9 August 2001.
Preparation: The inoculum was prepared by initially sieving sewage sludge. The sludge was then centrifuged for 5 minutes, the supernatant was rejected and the pellet was redispersed in the mineral medium. In order to wash out the dissolved organic carbon (DOC) and to lower the carbon organic content, the inoculum was preconditioned for 6 days before use. Air was bubbled through the inoculum during this preconditioning period.

Conditions during preconditioning
During the preconditioning period, the conditions were set as follows:
Temperature: 22°C±2°C. The temperature in the culture room was recorded continuously and records retained.
Aeration: air was bubbled through the inoculum until use.

Mineral medium:
Reconstituted water (DECO and EEC recommended) was prepared using deionized water which contained no more than 5% of the organic carbon content introduced by addition of the test or reference item and analytical grade reagents (see appendix 2 for details of fabrication). The mineral medium is checked periodically for carbon content by Total Organic Carbon analysis (analysis undertaken by the Laboratoire Departemental d'Analyses, Evreux, France).
Duration of test (contact time):
28 d
Initial conc.:
44.9 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Preparation of the test and reference solutions
The test item was weighed on two superposed glass microfibre filters and these filters were placed directly into test vessels containing 3 liters of mineral medium. The quantity added per flask was 44.9 mg for 2,5-dimethyl-2,5-di(2-ethylhexanoyl peroxy)hexane, corresponding to 10.0 mg/L of total organic carbon (TOC). The reference item was placed directly into test vessels containing 3 liters of mineral medium. The quantity added per flask was 102.4 mg for sodium acetate, corresponding to 10.0 mg/L of TOC. When test and reference items were placed into the appropriate test vessels, two superposed glass microfibre filters were also placed into the two blank test vessels and the test vessel containing the reference item.


Test system
Five flasks were used for the test:
two flasks containing the inoculum (inoculum blanks),
two flasks containing the test item (at 10.0 mg/L of TOC) and inoculum (test solutions),
one flask containing the reference item (sodium acetate at 10.0 mgiL of TOC) and inoculum (procedure contro!).

Parallel groups were prepared by adding 2.4 liters of mineral medium to each of the test flasks. Inoculum was added to provide a final concentration of 12.0 mg/L of suspended solids (dry weight) in 3 liters of suspension. The test flasks were aerated with CO2 scrubbed air overnight to purge the system of carbon dioxide and then attached in parallel to a series of three wash bottles filled with 100mL of 0.0125M barium hydroxide solution (to trap any C02 released from the test vessels). The test and reference (sodium acetate) items were added, where appropriate, to the flasks to give a test and reference item concentrations of 10.0 mg of TOC per liter (44.9 mg of 2,5-DIMETHYL-2,5-DI(2-ETHYLHEXANOYLPEROXY)HEXANE and 102.4 mg of sodium acetate). When all the items have been added, the volume of the suspensions was made up to 3 liters. Carbon dioxide-scrubbed air was bubbled through the suspensions at the same rate for all preparations.
Measurements of C02 were made at the following times: days 1, 4, 6, 8, 11, 14, 18, 21, 25 and 29. For each measurement, the first wash bottle nearest to the test flask was disconnected and titrated with 0.05 M HCI, using phenolphthalein as an indicator. The remaining C02 absorber bottles were connected to the test flasks so that the second wash bottle replaced the first one and an extra bottle containing fresh barium hydroxide solution was added to the far end of the series.

For calculation purpose, it was assumed that the volume necessary to titrate untitrated wash bottle would be the same as the volume needed to titrate 100 mL of the Ba(OH)2 stock solution. Each time C02 was analyzed, 100 mL of the barium hydroxide stock solution (used to fill the wash bottles) was titrated with the HCI solution in order to determine the maximum amount of acid required to titrate the wash bottles. Barium hydroxide (from analytical grade Ba(OH)2 crystals) and hydrochloric acid solutions (from 1M analytical grade HCI) were made up as necessary.

On the 28th day, 1 mL of concentrated hydrochloric acid was added to each test flask to stop the biodegradation and test flasks were aerated overnight to drive off the remaining carbon dioxide. A final C02 analysis of all wash bottles was made on the 29th day, this final analysis being representative of biodegradation of the 28th day.


Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
84
Sampling time:
3 d
Remarks on result:
other: Measurements of C02 were made at the following times: days 1,4, 6, 8, 11, 14, 18, 21, 25 & 29.
Interpretation of results:
readily biodegradable, but failing 10-day window
Conclusions:
The 10 day window (the 10 days immediately following the attainment of 10% biodegradation) started on the 6th day. Biodegradation of the test item totalled 54% at the end of this 10 day window (16th day) and 84% at the end of the test.
Executive summary:

Summary

At the request of ATOFINA, Paris-la-Defense, France, the ready biodegradability of the test item 2,5-dimethyl-2,5-di(2-ethylhexanoyl peroxy)hexane was evaluated using a 28-day modified Sturm test according to Commission Directive 92/69/EEC(CA-C.,31st July, 1992), Commission Directive 93/21/EEC (27th April 1993) and OECD guideline No. 301B (l7th July 1992).

In order to classify a test item as readily biodegradable: the biodegradation value of 60% has to be reached in the 10-day window (the 10 days immediately following the attainment of 10% biodegradation) within 28 days according to Commission Directive 93/21/EEC and OECD guideline No. 301B, or the biodegradation must exceed 70% after 28 days according to Commission Directive 93/21/EEC.

Methods

The test item was dispersed in reconstituted water (OECD mineral medium) prepared from deionised water with a conductivity < 10 uS/cm. Five flasks were used to determine the quantity of CO2 evolved by the degradation of the test item:

--2 flasks containing the (inoculum blanks)

--2 flasks containing the test item at 10 mg/L of total organic carbon (TOC) and inoculum (test solutions)

--one flask containing the reference item (sodium acetate at 10 mg/L of TOC) and inoculum (procedure control)

CO2 scrubbed air was bubbled through the flasks for the 28 day test period

Environmental parameters were recorded as

pH: 7.52 to 8.02

temperature: 19oC to 24oC

Results

Validity criteria

All validity criteria were respected

-biodegradation values of test item replicates deviated by less than 20% at the end of the test

-biodegradation in the reference test was at least 60% within 14 days

-the blank value (a measurement of CO2 evolved uniquely from the breakdown of organic matter in the inoculum, mineral medium etc) was <= 70 mg of CO2 evolved per liter of suspension at the end of the test (average of two controls).

Test item biodegradation

The 10 day window (the 10 days immediately following the attainment of 10% biodegradation) started on the 6th day. Biodegradation of the test item totalled 54% at the end of this 10 day window (16th day) and 84% at the end of the test.

Conclusion

Under the experimental conditions, the test item is classified as readily biodegradable in the 28 day modified Sturm test

Description of key information

Results of an OECD 301B test indicated the test substance is readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Test item biodegradation

The 10 day window (the 10 days immediately following the attainment of 10% biodegradation) started on the 6th day. Biodegradation of the test item totaled 54% at the end of this 10 day window (16th day) and 84% at the end of the test.

Conclusion

Under the experimental conditions, the test item is classified as readily biodegradable in the 28 day modified Sturm test