Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

Endpoint summary

Description of key information

Short term toxicity to fish:

Study was conducted to access the effect of test chemical on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).The test substance was soluble in water. Therefore, the stock solution was prepared by dissolving 1 g of the test substance in 1 liters of potable water (passed through reverse osmosis system) with continuous 1 hour stirring for achieving test concentrations of 6.25mg/L,12.5mg/L, 25mg/L,50mg/L, 100mg/L , respectivelyand Zebra FishDanio reriowere exposed to these concentration for 96 hours.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure to test item to nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for test material. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media having concentration 1g/L. Achieving test concentrations of 6.25 mg/L,12.5 mg/L,25 mg/L, 50 mg/L and 100 mg/L, respectively.

The nominal concentration selected for the experiment were 6.25 mg/L,12.5 mg/L,25 mg/L, 50 mg/L and 100 mg/L and test Daphnia magna were exposed to these concentration for 48 hours. The median lethal concentration (EC50) for test material on Daphnia magna in a 48 hours study on the basis of immobilization effect was found to be >100 mg/l.Thus, on the basis of this EC50 value and according to CLP criteria for aquatic classification of the substance, it is concluded that the test substance, does not exhibit short term toxicity to Daphnia.After 48 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be >100 mg/l . Based on the EC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Toxicity to aquatic algae and cynobacteria:

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201.The stock solution 100 mg/l was prepared by dissolving dark red powder in OECD growth medium . Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs.

The median effective concentration (EC50) for the test substance , in algae was determined to be 51.3 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be hazardous to aquatic algae can be classified as aquatic chronic 3 category. The substance is rapidly degradeable in aquatic environment and can be considered to be not classified as per CLP criteria.

Toxicity to microorganism:

Toxicity to micro-organisms study was conducted onStaphylococcus aureusof enterotoxigenic strain ATCC 13565.For the preparation of dye solution, a test portion (approximately 10 mg) was placed in a 10-ml volumetric flask, which was wrapped in aluminum foil, and dissolved by adding aqueous sodium hydroxide (approx. 0.4% NaOH) (1 ml) and water (9 ml).Inoculum was prepared inbrain heart infusion broth (BHI). Cultures were incubated shaking (200 rpm) at 37°C. All the experiments were conducted under conditions of standard room illumination (fluorescent ceiling light, Sylvania Octron 4100K) in tubes containing 3 ml of medium. Growth was analyzed by measuring turbidity (absorbance at a wavelength of 660 to 680 nm) with a spectrophotometer (Spectronic 21D). Experiments were initiated with  ̴0.2 ml of inoculum from a fresh culture grown aerobically at an optical density (OD) of ̴1. Colony counts were measured on BHI plates.

 

For the study,a 300-µl aliquot of dye solution (1 mg/ml) was mixed with 0.7 ml of BHI and then added to a tube of diluted culture. The tubes were aerated vigorously, and the OD was measured every 45 min to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates.

 

Additional information

Short term toxicity to fish:

The short term toxicity to fish for the test material was determined based on the data derived from experimental report which was further supported by data from peer reviewed journal.

The experimental study was conducted to access the effect of test chemical on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test).The test substance was soluble in water. Therefore, the stock solution was prepared by dissolving 1 g of the test substance in 1 liters of potable water (passed through reverse osmosis system) with continuous 1 hour stirring for achieving test concentrations of 6.25mg/L,12.5mg/L, 25mg/L,50mg/L, 100mg/L , respectivelyand Zebra FishDanio reriowere exposed to these concentration for 96 hours.

Bowl aquaria containing 2 liters of potable water (passed through reverse osmosis system) were loaded with 8 fishes. A static procedure was used for the study and it was conducted in compliance with the OECD guideline 203. After 96 hours of exposure to test item to nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria. 

The above experimental data was supported by data in peer reviewed journal In which short term toxicity study to Gambusia Affiniswas carried out for 96 hrs. The concentration of test chemical used for the study was 1, 10, 20, 50, 70, 100, 150 and 200 mg/l, respectively. The fish Gambusia Affinis Baird and Girard, were collected from a pond at Mississippi State University.

 

The test was performed under static conditions for 96 hrs. The fishes were kept in the laboratory for at least 72 hrs for stabilization prior to their use in the test. 20 fishes were then transferred into a 5 gallon glass aquarium after the appropriate amount of dyes was added. The aquarium were illuminated with Cool white Rflorescent lamps giving a surface light intensity of 3800 µW/cm2.Mortality was recorded as a function of illumination time and dye concentration.

 

The criterion for the death of fish was lack of movement immediately after touching with a camel hair brush. The light intensity was measured with an EG & GRphotometer/radiometer, Model 450-1. Abbott’s formula was used to correct for mortality in controls. Probit analysis was used to determine the log-time mortality regression lines and to calculate LC50 values.As no mortality of test organism was observed, the 96 hr LC50 value was found to be > 200 mg/l, respectively.

The above experimental data was further supported by Short term toxicity study to Oryzias latipes was carried out for 48 hrs and survival rate (in %) was determined. Himedaka (Oryzias latipes) of same age (2 cm long and 0.2 in weight) was used for the study. The test fishes were acclimatized for 10 days in tap water.In 1 litre solution of pH 7.0 containing 3,000 mg/l of test substance Eosine, ten fishes were kept in the tank without direct sunlight for 48 hrs and survival rate was determined. Water temp. was 20ᵒC and aeration was provided with bubbler.

 Median Tolerance Limit (TLm) was also determined after 24 and 48 hrs.After observation of dead fish, coloration of gill, fin and mouth by the test substance Eosine was recognized and particularly deep coloration of gill was found. Though internal organs were not colored, the excrement was clearly colored .Significant binding of Eosine to bovine serum albumin was also noted.The TLm value was found to be 2,700 and 2,200 mg/l at 24 and 48 hrs.Based on mortality of test fishes, the LC50 value was found to be 2,700 and 2,200 mg/l at 24 and 48 hrs, respectively and the 48 hr LC100 value was found to be 3,000 mg/l.

In another peer reviewed journal , Short term toxicity study to Oryzias latipes was carried out for 48 hrs and lethal concentration of 50% fish was determined. Himedaka (Oryzias latipes) of same age (2 cm long and 0.2 in weight) was used for the study. The test fishes were acclimatized for 10 days in tap water.Ten fish of Himedaka per one trial were kept in 2 liter of deionized water at 25°C and, after 24 and 48 hrs, lethal concentration of 50% fish was determined.

Median Tolerance Limit (TLm) was determined after 24 and 48 hrs. The TLm value was found to be 1,000 and 620 mg/l at 24 and 48 hrs. Based on mortality of test fishes, the LC50 value was found to be 1,000 and 620 mg/l at 24 and 48 hrs, respectively.

In another journal , In a short-term toxicity to fish study,test material was exposed toOryzias latipes  for 48 hours with or without oxygen. Results shows that number of dead fish increased remarkably by irradiation of test material and all fish died after 7-711 hours. While under the bubbling of oxygen gas, the time of death was delayed and all the fish died after 16-24 hours. Therefore, LC100 was considered to be 3000 mg/l for test material when Oryzias latipes were exposed for 48 hours.

One of the peer reviewed journal short term toxicity study to Oryzias latipes was carried out for 48 hrs and TLm value was determined. Himedaka (Oryzias latipes) of same age (2 cm long and 0.2 in weight) was used for the study. The test fishes were acclimatized for 10 days in tap water.In 1 litre solution of pH 7.0 containing 3,000 mg/l of test substance test material, ten fishes were kept in the tank without direct sunlight for 48 hrs and survival rate was determined. Water temp. was 25ᵒC and aeration was provided with bubbler. Median Tolerance Limit (TLm) was determined after 48 hrs. The TLm value was found to be 1,800 mg/lat 48 hrs. Based on mortality of test fishes, the 48 hr LC50 value was found to be 1,800 mg/l.

Based on the above effect concentrations determined from experimental data and data from all the peer reviewed journals , it can be concluded that test material is not toxic to fish and can be considered to be not classified as per CLP criteria.

Short term toxicity to aquatic invertebrate:

The short term toxicity to aquatic invertebrate for the test material was determined based on the data derived from two experimental reports .

In one report , Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for test material. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 100 mg of the test substance in 100 ml of ADaM’s media having concentration 1g/L. Achieving test concentrations of 6.25 mg/L,12.5 mg/L,25 mg/L, 50 mg/L and 100 mg/L, respectively.

The nominal concentration selected for the experiment were 6.25 mg/L,12.5 mg/L,25 mg/L, 50 mg/L and 100 mg/L and test Daphnia magna were exposed to these concentration for 48 hours. The median lethal concentration (EC50) for test material on Daphnia magna in a 48 hours study on the basis of immobilization effect was found to be >100 mg/l.Thus, on the basis of this EC50 value and according to CLP criteria for aquatic classification of the substance, it is concluded that the test substance, does not exhibit short term toxicity to Daphnia.After 48 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be >100 mg/l . Based on the EC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

In another report Aim of this study was to assess the short term toxicity of test material to aquatic invertebrates daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs.

 The stock solution 100.0 mg/l was prepared by dissolving dark red powder in reconstituted water. The test solutions of required comcentrations were prepared by mixing the stock solution of the test sample in reconstituted water.12 , 20 , 32 , 50 , 80 , 100 mg/lconcentrations were used in the study. Effects on immobilisation were observed for 48 hours. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0.

 The median effective concentration (EC50) for the test substance , in Daphnia magna was determined to be 51.9 mg/L on the basis of mobility inhibition effects in a 48 hour study. Based on the EC50 value, substance is likely to be hazardous to aquatic invertebrate and can be classified as aquatic chronic 3 category .

The substance is rapidly degradeable in aquatic environment and can be considered to be not classified as per CLP criteria.

The effect concentration determined by both the study reports are contradictory to each other since , value of the first report is from recently performed study we would consider that the test material is not classified as per CLP criteria.

Toxicity to aquatic algae and cynobacteria:

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201.The stock solution 100 mg/l was prepared by dissolving dark red powder in OECD growth medium . Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.

With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs.

The median effective concentration (EC50) for the test substance , in algae was determined to be 51.3 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be hazardous to aquatic algae can be classified as aquatic chronic 3 category.

The substance is rapidly degradeable in aquatic environment and can be considered to be not classified as per CLP criteria.

Toxicity to microorganism:

Toxicity of test material was determined based on the data derived from peer reviewed journals ,

In a journal toxicity to micro-organisms study was conducted onStaphylococcus aureusof enterotoxigenic strain ATCC 13565.For the preparation of dye solution, a test portion (approximately 10 mg) was placed in a 10-ml volumetric flask, which was wrapped in aluminum foil, and dissolved by adding aqueous sodium hydroxide (approx. 0.4% NaOH) (1 ml) and water (9 ml).Inoculum was prepared inbrain heart infusion broth (BHI). Cultures were incubated shaking (200 rpm) at 37°C. All the experiments were conducted under conditions of standard room illumination (fluorescent ceiling light, Sylvania Octron 4100K) in tubes containing 3 ml of medium. Growth was analyzed by measuring turbidity (absorbance at a wavelength of 660 to 680 nm) with a spectrophotometer (Spectronic 21D). Experiments were initiated with  ̴0.2 ml of inoculum from a fresh culture grown aerobically at an optical density (OD) of ̴1. Colony counts were measured on BHI plates.

 

For the study,a 300-µl aliquot of dye solution (1 mg/ml) was mixed with 0.7 ml of BHI and then added to a tube of diluted culture. The tubes were aerated vigorously, and the OD was measured every 45 min to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates.

 

During the study period of 250 mins, OD was measured every 45 mins to check culture growth.Experiment was performed in triplicate on separate days to ascertain reproducibility.As the test substance do not have any activity againstS. aureusi.e, no difference was observed between the treated test tubes and the control tubes, the NOEC value was found to be 100 mg/l.

In another study ,toxicity to micro-organisms study was conducted onParamecium caudatumfor 20 mins.The test chemical conc. used for the study was 1000 mg/l (0.1%).The test organismParamecium caudatumwas maintained at22°C on 0.15% dried lettuce infusion and fed withAerobacter aerogenes.Food dye (test chemical) of 0.1% conc. was put in a hollow slide glass, and an equal volume of 0.04 M phosphate buffer, pH 7.0, was added. After 5 to 10Paramecium caudatumwere added, their survival times were measured microscopically. 30 to 40 test organism for each conc. were tested and the mean survival time and the death rate was calculated after 20 mins.

 The survival time was defined as the time required until death was observed for each concentration. Death was assumed to have occurred when there was no movement. The death rate was defined as the percentage of deaths observed during 20 min.

 The mean survival time of test organismParamecium caudatumwas found to be 457 seconds i.e 7.61667 min. Based on mortality, the EC100 value was reported to be 1000 mg/l.

In a data from peer reviewed journal ,toxicity to micro-organisms study was conducted on gram – negative bacteria. Inhibitory activity of test chemical (Eosin Yellowish, Eosin Y)was determined by the traditional agar gel method.The conc. of test chemical used for the study was 0.6918 mg/l (1 µM).

These test organisms were taken from the collection of the Plant Protection Institute, Hungarian Academy of Sciences (Budapest, Hungary).The bacteria were maintained on Nutrient Agar (Oxoid CM3) completed with vitamins pyridoxine.HCl, thiamine. HCl, riboflavine and nicotinamide at 1, 10, 1 and 20 mg/l concentrations. Bacterial suspensions for screening were prepared by washing cells with sterile tap water containing 0.3% peptone from slants of 20 h old cultures grown at (21±1)°C.

 

The culture medium consisted of agar (11 g/l), glucose (5 g/l), peptone (5 g/l), yeast extract (3 g/l), glycerol (2 g/l), Na-β-glycerophosphate.6H2O (0.5 g/l), KH2PO4 (0.5 g/l), Na2HPO4.7H2O (0.5 g/l), KCl (0.25 g/l), MgSO4 (0.15 g/l), CaCl2 (0.15 g/l), FeSO4.5H2O (0.025 g/l), CuSO4.5H2O (0.005 g/l), MnSO4.H2O (0.005 g/l), Ni(NO3)2.6H2O (1 mg/l) and CoCl2.6H2O (1 mg/l), respectively.

 

A layer of 5 mm depth of inoculated medium was dispensed into Petri dishes of 90 mm diameter. Filter paper discs of 5 mm diameter were impregnated with 1µM solution of test compounds and placed centrally on the surface of the agar plate. Growth inhibition zones were measured after 24 h incubation at 21±1°C.After incubation colony diameters were determined and growth inhibition was calculated in percentage of dye free colony growth.Each determination was run in quadruplicate.As no effect on growth of test bacteriaAgrobacterium radiobacter, Escherichia coli, Erwinia atroseptica, Erwinia herbicola, Erwinia uredovoraandPseudomonas phaseolicolawere observed, the NOEC value was found to be0.6918 mg/l.