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Biodegradation in water

A number of studies for the test compoundSodium fluorescein(CAS no 518-47-8) were reviewed for the biodegradation end point which are summarized as below:

 

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 (102–105colony 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 found to be 7.6%.

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 also performed according to the OECD guidelines 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. 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. Based on the results obtained in Closed Bottle test and Manometric Respiratory test method, Uranine is expected to be not readily biodegradable.

 

Aerobic biodegradation study (U. Pagga and O. Brown, 1986) of fluorescein sodium was studied by a modification of the OECD Guideline 302B used as the static test method.In the test experiment, 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 of test substance 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 (J-CHECK, 2016) was conducted for 28 days (4 weeks) for evaluating the percentage biodegradability of the testsubstance. The purity of test substance by HPLC was found to be 92.1% and water solubility value is ≥ 300 g/l at 20ᵒC (Flask method). GLP compliance is followed for biodegradation study. By analytical methods, the recovery ratio was found to be-

(Water + test substance) system: 98.4 %

(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 isexpected to benot readily biodegradable.

 

 

On the basis of above results of various studies for target substance, it can be concluded that the substance can be considered as non-biodegradable in nature.

Biodegradation in water and sediment

A number of studies and predicted data for the test compoundSodium fluorescein(CAS no 518-47-8) were reviewed for the biodegradation end point which are summarized as below:

 

Estimation Programs Interface (EPI) Suite (2016) prediction model was run to predict the half-life in water and sediment for the test compound disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoate (CAS no. 518 -37 -8). Half-life of disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoatein water is estimated to be 37.5 days (900 hrs.) while in sediment it is 337.5 days (8100 hrs). Based on these half-life values of disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoate, it is concluded that the chemical is not persistent in water and persistent in sediment.

 

The Biodegradation study involve the use of two OECDs (301D and 301F) tests and a screening water sediment test (WST) to investigate the biodegradability of Uranine. An artificial matrix was introduced to achieve higher reproducibility and stability of the test system. All components of the artificial medium (sediment, inoculum, mineral medium) were standardized and based on OECD guidelines (Lukasz Gutowski et. al; 2015).

The sediment was placed in the test vessels (230 g wet weight) with a water layer above containing mineral medium and inoculum. Separately, in vessels conditioned for ‘sterile control’ contained no inoculum and addition of sodium azide in sediment and water. These vessels were acclimated for 7 days under the test conditions. Conditioning allows stabilization of important parameters e.g. pH, redox potential, and adaptation of bacteria and their growth on the sediment. During the conditioning the pressure development and BOD were measured to monitor the processes inside the sediment.

Briefly, the water sediment test (WST) consisted of five different series (details can be found in: blank, quality control, test, toxicity control and sterile control; each run in three parallels. Each of the series was placed in glass bottles (1 L) equipped with two septum sealed bottle nozzles. With water phase (500 mL) and artificial sediment (230 g) volumetric ratio was 1:5. The aniline (used as quality control) and test substance concentrations were prepared in a way that they corresponded to 40 mg/ L of theoretical oxygen demand (ThOD). The nominal concentrations were 17.2 and 24.4 mg/L for aniline and uranine, respectively. To obtain abiotic conditions in the sterile control, sodium azide was added in a concentration of 400 mg/ L in water phase and 800 mg/kg in sediment. All assays were incubated in the dark at 20 °C in closed vessels. Test duration was 28 days as in related OECD tests. The water phase in the bottles was gently stirred to improve water exchange between water and sediment without disturbing the sediment. During the experiment, pressure change inside the vessels was monitored by pressure sensors.A substance was considered to be toxic if measured toxicity control was lower than 25%, which corresponded to less than 50% degradation of aniline. If the measured toxicity control was lower than calculated, a substance was assumed to have inhibitive or toxic impact on the inoculum. The primary elimination was monitored by means of HPLC-FLD (Prominence series Shimadzu, Duisburg, Germany). Sample injection volume was 5 μL and the oven temperature was settled at 30 °C, flow rate was 1.0mL/min. Retention time for UR was 6.0min. The total time of chromatographic run was 16 min. The excitation and detection wavelengths were set to 476 and 515 nm, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) for UR were 1.0μg/l and 3.0μg/l respectively.

During the Water Sediment screening Tests [WST], the inoculum was of sufficient activity (‘quality control’ 79 ± 5% biodegradation). The biodegradability of Uranine (UR) was slightly higher and reached 28 ± 16% compared with 0% in Manometric Respirometric Tests [MRT]. The explanation of slightly higher degradation rates in WST in comparison to MRT could be the higher bacterial diversity of the inoculum used for this test, which in fact was a mixture of bacterial cultures from several natural water bodies and secondary effluent from sewage treatment plant. UR was not toxic to the inoculums as biodegradation in ‘toxicity control’ reached 53 ± 7%. At the end of WST the HPLC-FLD analysis showed elimination of 2.4 mg/l (11.7%) of initial UR concentration from the water phase. This might be a result of partial sorption to the sediment particles or due to the bacterial metabolism. In the WST, 93% of sediment mixture consisted of quartz sand, while the rest was clay and peat. Hence sorption cannot be excluded. However, the low degree of mineralization was observed indicating that the UR was not fully degraded but transformed into TP’s [Transformation Products]. TPs were assumed to be possibly mono- and di-hydroxylated derivatives of UR. The hydroxylation could take place in any of the UR aromatic rings. The fragmentation patterns confirmed that TP1a,b and TP2 are hydroxylated products whereas TP3a,b belong to the carboxylated compounds. However, due to many possible addition sites to the aromatic ring of UR, it is difficult to know the exact position of either the hydroxyl or the carboxyl groups.The study demonstrated that Uranine was not readily biodegradable in nature.

Thus, based on the above experimental data, it can be concluded that the substance is not readily biodegradable and on the basis of the estimation model, it is evident that the substance is not persistent in water but persistent in sediment compartment.

 

Biodegradation in soil

Based on EPI prediction Level III Fugacity Model (EPI suite, 2016), the estimated half life period ofSodium fluoresceinin soil was obtained to be 75 days. Based on this half life value of sodium fluorescein, it is concluded that the chemical is not persistent in the soil environment.