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Environmental fate & pathways

Biodegradation in water: screening tests

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Description of key information

OECD 301B_enhanced study: The test substance may be classified as readily biodegradable
OECD 301B_modified_35 days: 64 % degradation - biodegradable and not persistent in the environment
Read-across substance_Phosphoric acid, mono- and di-(C8-C10) ester, compds. with C12 - 14 amine_OECD 301B_28 days: 53 % degradation - biodegradable and not persistent in the environment

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

Two experimental results about biodegradability in water are available for the target substance (Reaction products of diphosphorus pentaoxide and alcohol C7-9-iso, C8 rich, salted with 2-ethylhexylamine) as well as one for the read-across substance (Phosphoric acid, mono- and di-(C8 -C10) ester, compds. with C12 - 14 amine). For the detailed justification of the read-across procedure, please refer to the corresponding statement by Chemservice S.A. (2013d).

The biodegradability of the target substance was investigated two times according to OECD Guideline 301B with different enhancements / modifications.

In the first experiment, inoculated mineral medium (mixture of activated sludge and soil) is dosed with a known amount of test substance as the nominal sole source of organic carbon and aerated with CO2-free air (Schäfer and Matthews, 2009). The CO2 produced from the mineralization of organic carbon within the test chambers is displaced by the flow of CO2-free air and trapped as K2CO3 in KOH trapping solution. The amount of CO2 produced by the test substance (corrected for that evolved by the blank inoculum) is expressed as a percentage of the theoretical amount of CO2 (TCO2) that could have been produced if complete biodegradation of the test substance occurred. Enhancements from Guidance on information requirements and chemical safety assessment Chapter R.7b: Endpoint specific guidance May 2008 were employed over conventional protocols to optimize demonstration of biodegradability. These enhancements included the use of large incubation vessels, the use of dispersal powders, the use of inoculum derived from both activated sludge and soil and maximizing loading rates for inocula total suspended solids. The test volume of 3 L and the total suspended solids level of 30 mg/L in the test system were consistent with 301B study guidelines. Overall, the study conditions remained inside the boundaries for the results to be considered for readily biodegradability. The experiment contained a blank control group, a reference group and three treatment groups. Each group contained three replicate test chambers. The blank control was used to measure the background CO2 production of the inoculum and was not dosed with a carbon source. The reference chambers were dosed with sodium benzoate, a substance known to be biodegradable, at a concentration of 10 mg C/L. Each treatment group test chamber was used to evaluate a test substance at 10 mg C/L. The results indicate that the activated sludge inoculum was active by degrading the reference substance approximately 70.8 %. The temperature range recorded during the test was 20.3 to 22.3 °C and was within the protocol specified range throughout the test. The results of the standard plate count and TSS measurement performed on the inoculum were 1.14 x 10E5 CFU/mL and 1773 mg/L, respectively. The control chambers evolved an average of 110.9 milligrams of CO2 over the test period. This value has been corrected for the amount of CO2 in the trapping solution since potassium hydroxide solution, even when freshly prepared, contains carbonates. The amount of CO2 evolved by the control chambers did not exceed the 40 mg/L (120 mg total) value considered the acceptable limit for CO2 evolution tests. The test results indicate that the test substance may be classified as readily biodegradable since the conditions established for Ready Biodegradability Tests were fulfilled and the administration of the test substance on an inert support is an acceptable route of administration of poorly water-soluble chemicals. The final mean percent biodegradation was 93.9 % and the pass level of 60 % TCO2 being achieved within 10 days of reaching 10 % TCO2 without the use of pre-adapted inoculum. In addition, the suspended solids concentration used did not exceed the 30 mg/L limit stated in the guidelines for tests of ready biodegradability.

The second experiment with the target substance was performed with following modifications: The test duration was increased to 35 days (instead of 28 days), the test volume employed was increased from 3 L to 4 L and another modification was the increase of the biomass (50 mg suspended solids (ss) /L instead of 30 mg ss/L) (Clarke, 2009). A composite of microbial inoculum, derived from soil and a wastewater treatment facility was used as test species. During the study, samples are taken for Dissolved Oxygen Carbon (DOC) analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge soils. The initial test substance concentration was 10 mg Carbon/L. Sodium benzoate was used as standard material. Also a control vessel was included. All preparations were prepared and evaluated in duplicate. The test was carried out in a temperature controlled room at approximately 21 °C, in darkness. CO2 analysis was carried out on Days 0, 2, 6, 8, 10, 14, 21, 24, 28, 31, 35, and 36 with the first absorber vessel and on Days 0 and 36 with the second absorber vessel, respectively. Each analysis was carried out in triplicate. The pH of the test preparations was determined on Day 35, prior to acidification with hydrochloric acid. Preliminary investigational work indicated that the test substance did not absorb to filter matrices or to activated sewage sludge. The test substance attained 61 % degradation after 28 days and 64 % after 36 days. Thus, the test substance can be considered as biodegradable and not persistent in the environment. The suitability of the inoculum and test conditions was confirmed by sodium benzoate, which attained 62 % degradation after 14 and 28 days and 74 % degradation after 36 days.

The biodegradability of the read-across substance (Phosphoric acid, mono- and di-(C8 -C10) ester, compds. with C12 -14 amine) was also investigated according to OECD Guideline 301B / EU Method C4.-C (Mead, 1999a). The study was conducted under certificated GLP compliance and last for 28 days exposure duration. A mixed population of activated sewage sludge from an aeration stage of a sewage treatment plant, predominantly treating domestic sewage, was used as inoculum. Sodium benzoate served as reference substance, in order to confirm that the inoculum as well as the test procedure is suitable. Furthermore a blank and a toxicity control were included. The study was carried out in a temperature controlled room of 21 °C in darkness. CO2 analysis was performed on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28, and 29 (first absorber vessel). The second absorber vessel was sampled on Days 0 and 29. Analysis of Dissolved Organic Carbon (DOC) was not carried out due to the insoluble nature of the test substance in water.The total CO2 evolution in the control vessels on day 28 was 34.2 mg/L (= 102.6 mg/L) and therefore satisfied the validation criterion given in the OECD Guideline.The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels. These increases are considered to be due to CO2 present in solution being driven off by the addition of hydrochloric acid on Day 28 and resulted in an increase in the percentage degradation value for the test material from 53 % on Day 28 to 56 % on Day 29.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 toxicity control attained 49 % degradation after 28 days, confirming that the test substance was not toxic to the sewage treatment microorganisms used. The reference substance attained 94 % degradation at the end of the experiment. The test material attained 53 % degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline 301B.