Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Biodegradation in water: screening tests

Description of key information

During 10 days aerobic experiment, Food Red 2 was decomposed about  20% in 10 days
From the oxygen uptake by Warburg’s manometer, the low activity of the sludge to dye was obtained.
Based on the results obtained from the aerobic degradation assay and Warburg method, it can be concluded that Amaranth is not readily biodegradable under aerobic conditions

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Additional information

From the available experimental data and prediction data for the target chemical, the information is summarized as below:

 

The aim of the study (biochemical decomposition of coal-tar dyes i.- biochemical decomposition and identification of decomposed products, 1978) was to estimate the microbial decomposition of Amaranth dye by sludge under aerobic conditions. Return activated sludge was obtained from the municipal sewage treatment plant, Nakahama, Osaka. Synthetic sewage preparation: Glucose ,peptone and potassium dihydrogen phosphate, 30g each, were dissolved in 1 liter water and the pH was adjusted to pH 7.0 with sodium hydroxide Seeded Dilution water: To 1 liter, 10 ml of supernatant of sludge was added. Aerobic biodegradation assay: To 750ml of sludge (MLSS ca, 3,000 ppm) 250 ml of O.03 M dye solution was added, and bubbled with air sufficiently at 20°C. 5ml sample was taken out once a day. After sampling 5ml of synthetic sewage was added to the mixture. Each sample was filtered through filter paper and diluted twenty times prior to the spectrophotometric measurement at the absorption maximum within the visible range. The decrease of dyes concentration was expressed in terms of percent to the initial absorption. The experiment was carried out for 10 days. Oxygen uptake of sludge [Warburg Method]- 2.0 ml of sludge, 0.2 ml of 1000 ppm dye solution, and 0.2 ml of 20% potassium hydroxide were pipetted into the vessel, the side arm and central well, respectively. The sludge and the dye solution were mixed and the vessel was shaken at 25°C. The oxygen uptake was measured. The oxygen uptake by sludge alone was subtracted from those by dyes addition. During 10 days aerobic experiment, Food Red 2 was decomposed about 20% in 10 days From the oxygen uptake by Warburg’s manometer, the low activity of the sludge to dye was obtained. Based on the results obtained from the aerobic degradation assay and Warburg method, it can be concluded that Amaranth dye is not readily biodegradable under aerobic conditions

 

 

Anaerobic decomposition of dyes using return activated sludge was determined(biochemical decomposition of coal-tar dyes i.- biochemical decomposition and identification of decomposed products, 1978). Return activated sludge was obtained from the municipal sewage treatment plant, Nakahama, Osaka. The return sludge was acclimated to the synthetic sewage for a week or longer, and it was used for the aerobic and anaerobic decomposition experiments. Synthetic sewage preparation: Glucose ,peptone and potassium dihydrogen phosphate, 30g each, were dissolved in 1 liter water and the pH was adjusted to pH 7.0 with sodium hydroxide To 750 ml of sludge (MLSS ca, 3000 ppm), 250 ml of 0.03 M dye solution was added and standard in room with stirring by a magnetic stirrer, 5 ml of the solution was taken out once a day for measurement. After sampling 5ml of synthetic sewage was added to the mixture.After every sampling, nitrogen gas was introduced into the solution and the vessels were closed tightly to shut the air.Each sample was filtered through filter paper and diluted twenty times prior to the spectrophotometric measurement at the absorption maximum within the visible range. The decrease of dyes concentration was expressed in terms of percent to the initial absorption.Sewage acclimated sludge was used for the experiment, because the digested sewage gives too large blank values and interfere to spectra absorption.The reaction mixture was filtered and concentrated up to 25 ml under reduced pressure at 50°C. The concentrates were subjected to subsequent separation and identification.

 

Amaranth dye decomposed more rapidly, under the anaerobic conditions than the aerobic complete fading took place within 3-6 days in the solutions of azo dyes.The fading could be attributed to the partial reduction of azo compound at the double bond in anaerobic circumstance. The new maximum absorption was at 250, 240 and 245 nm for Food Red 2. After anaerobic decomposition for 10 days, each reaction mixture was concentrated and analyzed. The decomposed products of the dye by sludge under anaerobic conditions were determined quantitatively by high performance liquid chromatography. Quantitative determination of biochemical decomposition products of Amaranth dye by anaerobic sludge showed that napthionic acid was one of the primary products of degradation. The amounts of sulfanilic or naphthionic acids formed by decomposition during the 10 days accounted for more than 50% of the initial contents of the Amaranth dye on a molar basis. Amaranth dye decomposed about 60% in 3 days under anaerobic conditions. Hence it was concluded that decomposition of Amaranth dye took place easily in anaerobic conditions than in aerobic conditions. Amaranth dye is readily biodegradable under anaerobic conditions.

 

COD and BOD values from biochemical decomposition of coal-tar dyes i - biochemical decomposition and identification of decomposed products , 1978; of the 10mg/l dye solution were determined according to the procedure described in the JIS K0102. Determination of BOD – Dye solution (10, 20, and 40 ppm) were prepared with the seeded dilution water and kept at 20°C (Japanese Industrial Standards Committee, 1971). The dissolved oxygen contents were measured by a dissolved oxygen meter, because of the coloration of the solutions. The COD, BOD and absorbance at 245 and 545 nm (representing ultraviolet and the visible absorbance) was studied. Amaranth dye showed small BOD and COD values less than 100 ppm at concentration of 100mg-dye/l. The general limit of BOD and COD for effluent by the Law is regulated to be below 160ppm (daily average 120 ppm) In BOD experiment, the dissolved oxygen contents on the 5^thday were essentially the same to the initial values. From the results of BOD, COD experiments, it was concluded that Amaranth dye is not readily biodegradable under aerobic conditions.

 

Reduction of seven azo dyes (amaranth, Ponceau SX, Allura Red, Sunset Yellow, tartrazine, Orange II, and methyl orange) was carried out (Reduction of Azo Dyes by Intestinal Anaerobes, 1978) by cell suspensions of predominant intestinal anaerobes. The organisms studied were obtained from the Anaerobe Laboratory, Virginia Polytechnic Institute (VPI), Blacksburg, except Fusobacterium sp. 2, which was obtained from Wadsworth Hospital Center, University of California, Los Angeles. A round-bottom flask containing 500 ml of brain heart infusion medium was inoculated with 1 ml of a pure culture grown in a chopped-meat broth and incubated at 37°C for 17 to 19 h. After incubation, the cells were centrifuged at 20,000 x g for 20 min and anaerobically washed once with 0.4 M potassium phosphate buffer (pH 7.4). The bacterial pellets were suspended in 25 to 40 ml of the same phosphate buffer and immediately used for azo reduction. One milliliter of dye (2 µmol/ml), 2 ml of cell suspension, and 2 ml of 0.4 M phosphate buffer (pH 7.4) with or without glucose and electron carriers were placed in roll tube (18 by 142 mm). The various electron carriers were prepared anaerobically at 0.025 mmol/ml. Each reagent was added anaerobically under nitrogen, using a VPI apparatus (12), and the reaction tubes were closed with rubber stoppers. Incubation was generally carried out at 37°C for 1 h, but the results mentioned were obtained with 2.5-h incubation. A zero-time control was also run for each dye. The azo reductase is expressed as micromoles of azo dye disappearing per hour per 100 mg (dry weight) of cells. At the end of the incubation period, a 2-ml sample of the reaction mixture was added to 2 ml of 6% trichloroacetic acid and centrifuged at 29,000 x g for 20 min, and the clear supernatants were used for spectrophotometric measurements. The final dye concentration was within the reading range of the spectrophotometer (optical density, about 1.0). The absorption maximum of Amaranth was 520 nm.

A standard curve for Amaranth was prepared by dissolving the dye in phosphate buffer and adding an equal volume of 6% trichloroacetic acid before spectrophotometric measurement. The blank was a solution consisting of equal volumes of the above 0.4 M phosphate buffer and 6% trichloroacetic acid.

The results show that all of the anaerobes tested reduce Amaranth. It indicates that reduction of azo compounds can be achieved by anaerobes rather than aerobes. Amaranth dye was determined to be readily biodegradable under anaerobic conditions by anaerobic bacteria.

 

From predicted data from EPI suit, the screening test inherent to the biodegradability of the substance was calculated using the software BIOWIN v4.10. The test chemical is estimated to be not readily biodegradable.

 

On the basis of available information for the target as well prediction data for substance, the test substance cannot be consideredbiodegradable in nature, though two study indicates that the substance is biodegradable in nature. On the basis of the experimental and predicted data, the substance is considered as the not biodegradation in nature.