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Biodegradation in water: screening tests

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The test substance ptBP was assessed for being readily biodegradable in a total of three different OECD 301 guideline-based studies. Additionally, ptBP was tested for inherent biodegradability using a Modified MITI test according to the OECD Guidelines for the Testing of Chemicals No. 302C (MITI, 1992). The substance has shown to be readily biodegradable in a DOC Die-Away test (OECD 301A, Huels, 1993) achieving a rate of 98% determined by DOC-removal within 28 days, but failed to pass the 10-day window criterion in the two other studies (OECD 301B CO2 Evolution, OECD 301F Manometric Respirometry), although showing a significant aerobic biodegradation of 60% within 28 days. The modified MITI-test suggested that ptBP could not be considered as inherently biodegradable under the strict terms and conditions of the OECD Guidelines, but this result may be attributable to the inhibitory concentration of ptBP used resulting in a long lag phase under the test conditions. Overall, ptBP did not pass the 10 -days window for biodegradation in the key study, Manometric Respirometry test according to OECD 301 F (Källqvist, 2003), but clearly the substance is readily biodegradable but failing the 10-day window.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable but failing 10-day window

Additional information

The biodegradation of ptBP was tested in a ready biodegradability study according to the DOC Die-Away test and in compliance with the OECD Guidelines for the Testing of Chemicals No. 301A and in accordance with the Principles of Good Laboratory Practices (GLP) (Hüls AG, 1994). The initial test substance concentration was 13 mg/L, corresponding to 10.4 mg DOC/L. The test was performed with unadapted activated sludge from a municipal wastewater treatment plant, and the DOC removal was monitored over a time period of 28 days. The test substance reached a degree of degradation of 98 % (DOC removal), while the reference substance, sodium benzoate (10.7 mg DOC/L), achieved 99% DOC removal after 28 days. The test results were considered valid as the 10-day window criterion was fulfilled for both the test and reference substances. Given these results, ptBP was considered as readily biodegradable. However, it can not be excluded that the inoculum might have been adapted to ptBP, as it was taken from a heavily industrialized area, where industry might have located using ptBP.


The ready biodegradability of ptBP was determined by the Carbon Dioxide Evolution Test Method (OECD Guideline 301B) under the terms of Good Laboratory Practices (GLP) (Matthews, 2013). The test substance ptBP was tested at the two nominal concentrations, 5 and 10 mg C/L. Additionally, toxicity controls and a reference system with sodium benzoate were set up. The test was run for 28 days and CO2 evolution was monitored, expressed as %TO2. The final mean percentage biodegradation was 58.5% at 5 mg C/L and 63.5% at 10 mg C/L respectively. The criterion of ready biodegradability (60 % degradation within a 10-days window, counting from the time the biodegradation passes the 10% level) was not met for ptBP for either concentration tested. By Day 4, 25% TCO2 was achieved for the toxicity control dosed with the test substance at 5 mg C/L and by Day 7 for the toxicity control dosed at 10 mg C/L. Therefore, the test substance can be considered non inhibitory at both concentrations tested.

The aerobic biodegradability of ptBP was evaluated in a ready biodegradability study using a Manometric Respirometry test system according to the OECD Guidelines for the Testing of Chemicals No. 301F, and in compliance with GLP Principles (NIVA, 2003). The test compound in 2 different concentration ranges, 13.8 to 15.1 mg/L and 24.7 to 25.1 mg/L was exposed to microorganiams cultivated in an in-house unadapted activated sludge simulation unit. The biodegradation of ptBP was expressed as the ratio of biochemical oxygen demand (BOD) to the theoretical oxygen demand (ThOD). The test substance reached 60% biodegradation at 15 mg/L ptBP, whereas a significant lower degradation (42%) was obtained at 25 mg/L ptBP after 28 days. In both test ranges a lag-phase was observed before the degradation of the test compound was initiated, and the observed lag-phase was longer at 15 mg/L (16 days) than at 25 mg/L (12 days). This suggests that the lag-phase is likely not related to toxicity but rather to adaptation. The criterion for ready biodegradability (60% degradation within a 10-day window) was not fulfilled for the test compound in this test; however, the reference substance, aniline, met the requirement of 60% degradation within 14 days confirming the viability of the inoculum used. The results of this study indicates that municipal sludge microorganisms require an adaptation period in order to degrade ptBP rapidly


The inherent biodegradability of ptBP was assessed using a Modified MITI test according to the OECD Guidelines for the Testing of Chemicals No. 302C (MITI, 1992). The oxygen consumption (% chemical oxygen demand and ThOD) was monitored in a test system inoculated with 100 mg/L of mixed population of activated sludge and 30 mg/L of ptBP for 28 days. Aniline was used as a reference substance at an initial concentration of 100 mg/L. No biodegradation was observed with ptBP within 28 days, likely due to an inhibitory concentration of ptBP in this study combined with a long lag-phase, while aniline attained 87% degradation within 28 days, confirming the viability of the inoculum used. The total organic carbon (TOC) analysis of ptBP was not possible due to the insoluble nature of the test material. The results of this study suggest that ptBP cannot be considered as inherently biodegradable, under the strict terms and conditions of the OECD Guidelines.