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Biodegradation in water: screening tests

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Biodegradation in water ability was predicted for the test substance D & C Orange No. 5 (CAS no. 596 -03 -2) using the SSS QSAR prediction model (SSS QSAR Prediction Model, 2016).The ready biodegradability of the test substance D & C Orange No.5 was estimated as 9.8% in 28 days by using BOD parameter. This result indicates that D & C Orange no. 5 was estimated to be not readily biodegradable in water.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Various predicted data for the test compound D & C Orange No. 5(CAS No. 596-03-2)and the study for its read across substance were reviewed for the biodegradation end point which are summarized as below:

Biodegradation in water ability was predicted for the test substance D & C Orange No. 5 (CAS no. 596 -03 -2) using the SSS QSAR prediction model (SSS QSAR Prediction Model, 2016).The ready biodegradability of the test substance D & C Orange No.5 was estimated as 9.8% in 28 days by using BOD parameter. This result indicates that D & C Orange no. 5 was estimated to be not readily biodegradable in water.

 

Estimation Programs Interface Suite (EPI suite, 2016) was run to predict the biodegradation potential of the test compound D & C Orange No. 5 (CAS no. 596 -03 -2). The screening tests ready biodegradability of the test substance was calculated using the software BIOWIN v4.10. The results indicatethatD & C Orange No. 5is not expected to be readily biodegradable in nature.

A combination of two biodegradation tests (Closed Bottle test and Manometric Respiratory test, OECDs 301 D, F) was carried out to investigate the biodegradability of the Uranine (Lukasz Gutowski et. al; 2015). Closed Bottle Test was performed according to the guidelines of the Organization for Economic Co-operation and Development OECD 301D. This test is characterized by low bacteria density (102–105 colony forming units (CFUs)/mL), low nutrient content, and constant temperature (20 ± 1 °C) and it was kept in the dark. The average biodegradation value after 28 days for Uranine monitored by measurement of the oxygen concentration was 7.6. Based on the results obtained, Uranine is expected to be not readily biodegradable.

A combination of two biodegradation tests (Closed Bottle test and Manometric Respiratory test, OECDs 301 D, F) was carried out to investigate the biodegradability of the Uranine (Lukasz Gutowski et. al; 2015). The Manometric Respiratory tests works with higher bacterial density (5–10×106CFUs/mL) and diversity as the Closed Bottle test thus increasing the probability for biodegradation. This test was performed according to the OECD guidelines (OECD, 1992) in the dark at room temperature (20 ± 1 °C) under gentle stirring. CO2 production as the parameter of the endpoint biodegradation was measured indirectly by the OxiTop OC110-system. The concentration of standard solution for uranine was 16.7 mg/L, corresponding to the theoretical oxygen demand ThOD of 30 mg/L. Inoculum was derived from the municipal sewage treatment plant (Lüneburg, Germany).Aliquots (measuring) of 80ml of inoculum were added to 1 L of mineral medium. The test consisted of four different series: (i) a blank series (containing only the mineral medium and inoculum), (ii) quality control (containing readily biodegradable sodium acetate as the only relevant carbon source apart from the inoculum), (iii) a test series (containing the target compound) and (iv) toxicity control (containing target compound and sodium acetate as carbon source). The amount of sodium acetate for each series corresponded to ThOD of 5 mg/L. A compound is qualified as “ready biodegradable” when 60% of ThOD expressed as percentage of oxygen consumption is consumed within a period of 10 days after the oxygen uptake reached 10% of ThOD. Samples from the beginning (day 0) and the end of the test (day 28) were collected and stored at−20 °C until analysis with HPLC-FLD and LC–M/MS. No toxic effects on bacteria were observed in the toxicity control as well as no degradation was observed in the sterile control. The measurements with HPLC-FLD confirmed that no elimination of uranine occurred during the Manometric Respiratory Test. Only 0.1% degradation of test substance Uranine was observed. The reason for the negative values in Manometric Respiratory test might be interpreted as high degradation in the blank control and should be considered could be due to some background in the blanks and can be considered as 0% degradation of the test substance.Based on the results obtained in Manometric Respiratory test method, Uranine is expected to be not readily biodegradable.

 

Aerobic degradation of fluorescein sodium was studied by a modification of the OECD Guideline 302B used as the static test method (U. Pagga and O. Brown, 1986). In the test condition, test chemical was exposed with concentration of 100 mg/l in an activated sludge of 0.5g/l dry material upto 42 days.feeding of the inoculums carried out each week at 100 mg/l yeast extract.The parameter followed for biodegradation was DOC elimination. Based on study results sodium fluorescein i.e, only 11% degradation by DOC removal after 42 days and thus the substance cannot be considered as inherently biodegradable.

Biodegradation screening test was conducted for 28 days (4 weeks) for evaluating the percentage biodegradability of the test substance (J-CHECK, 2016). The purity of test substance by HPLC was determined to be 92.1% and water solubility value is ≥ 300 g/l at 20ᵒC (Flask method). By analytical methods, the recovery ratio was found to be- (Water + test substance) system: 98.4 % and (Activated sludge + test substance) system:99.0 %.Concentration of inoculum i.e, sludge is 30 mg/l and initial test substance conc. used in the study is 100 mg/l. The percentage degradation of test substance is found to be 0% by O2 consumption and by TOC removal whereas only 1% degradation was observed by HPLC. Thus, the substance Fluorescein sodium is non-biodegradable in water.

28-days Manometric respirometry test following the OECD guideline 301Fto determine the ready biodegradability of the test item Disodium 2-(2, 4, 5, 7-tetraiodo-6-oxido-3oxoxanthene-9-yl) benzoate (CAS No. 16423-68-0). The test system included control, test item and reference item[UERL study report, Sustainability Support Services (Europe) AB (Report no. 16423-68-0/01/2015/RBD), 2016]. The concentration of test and reference item (Sodium Benzoate) chosen for both the study was 100mg/L, while that of inoculum was 10ml/l. ThOD (Theoretical oxygen demand) of test and reference item was determined by calculation. % Degradation was calculated using the values of BOD and ThOD for test item and reference item. The BOD28value of Disodium 2-(2, 4, 5, 7-tetraiodo-6-oxido-3oxoxanthene-9-yl) benzoate (CAS No. 16423-68-0) was observed to be 0.058 mgO2/mg. ThOD was calculated as 0.709 mgO2/mg. Accordingly, the % degradation of the test item after 28 days of incubation at 20 ± 1°C according to manometric respirometry test was found to be 8.18%.Based on the results, the test item, under the test conditions, was found to be non-biodegradable at 20 ± 1°C over a period of 28 days.

 

On the basis of above results for target and read across substance, it can be concluded that the test substance D & C Orange No. 5 can be expected to be not readily biodegradable in nature.