4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one

Endpoint summary

Description of key information

Toxicity to aquatic algae and cyanobacteria:

Aim of this study was to evaluate the nature of chemical test chemical 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one 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 100.0 g/l was prepared by dissolving pink powder in OECD growth medium. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at 0, 6, 12, 25, 50 and 100 mg/l oncentration. 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. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical, the ErC50 was determine to be 50.9 mg/l. Based on the ErC50 value, it was concluded that the substance 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one

is likely to be hazardous to aquatic algae and can be be classified as aquatic chronic 3 category as per the CLP criteria.

Short-term toxicity to aquatic invertebrate:

Various predicted studies of the test compound 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one (CAS number: 596-03-2) and read across chemical to aquatic invertebrates were reviewed from reliable sources.

48 hrs aquatic toxicity studies (SSS QSAR prediction model, 2016) were conducted to assess toxic effects of the 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one (CAS number: 596-03-2) and the results were predicted. The study was based on the effects of the test compound on the Daphnia magna in a static fresh water system. The predicted data suggests the effective concentration (EC50) for the 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one (CAS number: 596-03-2) was estimated to be 17 mg/l on the basis of mobility.

 

The toxicity of dye, fluorescein sodium salt, to Daphnia pulex was assessed (W.K. Walthall, J.D. Stark, 1999) using acute level toxicological test. The test organisms were obtained from cultures at or beyond the third filial (F3) generation. The final concentrations used in the generation of the acute mortality regression are 0, 150, 250, 400, 600 and 800 mg/liter. No control mortality occurred in the acute toxicity experiments. For each concentration tested, 25 ml of test solution was transferred into a 30-ml plastic cup and five neonates were transferred into the test container using a disposable glass pipette. Each concentration was replicated a minimum of four times. The testing regime was 48 h static, nonrenewal. The LC₅₀for Fluorescein sodium was found to be 337(278 ± 403) mg/liter for Daphnia pulex neonates on the basis of the results obtained from mortality and the response predicted by regression analysis.

According to the CLP regulations the test chemical was considered as not classified for short term toxicity to aquatic invertebrates.

 

Short term toxicity study was conducted to determine the aquatic toxicity of sodium fluorescein (Acute Toxicity of Sodium Fluorescein to Ashy Pebble snails Fluminicola fuscus, 2011) .The dose concentration used are 0.2; 20.0; 50.0 71.0 100.0 141.0 and 200.0 mg/L. The snail behavior was observed every 15 minutes during the first hour of exposure, and then every hour for the next 4 h. At the end of a 24h exposure, we poured the test system into small sieve to recover all snails, and then rinsed the snail three times with clean, aged laboratory water. The snails are then placed s into small cups with aged water, assessed mortality immediately, then again at 24 and 48 h. Each beaker is inspected with the aid of a dissecting microscope and watched for snail movement, and touched individual snails that were not active with a probe to elicit movement or tactile response. Each trial was replicated three times over two weeks for a total of 9 beakers tested at each concentration. Probit model and normal distribution were used for estimating the probability of mortality. The median lethal concentration was found to be 377 mg/L. Our model had decreasing precision as probability of mortality increased, because we never achieved high mortality in trials, even at the highest tested concentrations.

According to the CLP regulation the test chemical was considered as not classified for short term toxicity to aquatic invertebrates.

 

Ecological assessments included both acute and chronic effects for the 3 life forms: fish, Cladocera (water flea, daphnia), and algae (An assessment of the potential adverse properties of fluorescent tracer dye used for groundwater tracing, 1995). The concern levels identified by use of the SAR technique are expressed ad HIGH, MODERATE, LOW. These values are toxicity values, not exposure values. SAR results are generally regarded by EPA as being reliable. Dyes with aquatic toxicity values below 1mg/l received a HIGH concern rating, those with toxicity values ranging from 1-100mg/l received a MODERATE concern rating and those with toxicity values greater than 100mg/l received a LOW concern rating. The Median Lethal Concentration which is estimated to be lethal to 50% of Cladocera (water flea) in 48 hours exposure to the test chemical (LC50) was found to be 165mg/l. Since the LC50 for the test chemical was determined to be 165 mg/l, it falls in the low concern category in the aquatic toxicity ratings. Thus, based on the result according to the CLP regulation it was considered that the test chemical was not classified to short term aquatic invertebrates.

Short term toxicity study to Artemia Salina was carried out in read across erythrosine sodium (16423-68-0) (Ascreening method for the toxicity of food dyes using artemia salina larvae, 1977) for 24-48 hrs. The test chemical concentration used for the study was 879.842 mg/l and 87.9842 mg/l, respectively. A. salina eggs (encysted dried gastrulae) were commercially obtained, and were stored at -20°C; Eggs used in experiments were washed and stored at room temperature in desiccators over anhydrous granular CaCl₂. Larvae were obtained by incubating eggs in petri dishes containing muslin-filtered sea water at 30°C for 24 hours. The larvae were separated from shells, dead larvae and unhatched eggs by their photo tactic movements toward a light source.  Food dyes of various concentrations were placed in a petri dish, and sea water containing 20 to 30 larva e was added. After this was incubated at 30°Cfor 24 hours and 48 hours, larvae surviving were measured by direct count. The same method was tested from 5 to 6 times for each concentration, and the death rate was calculated. The study was performed under static conditions for 24 – 48 hrs at 30°C. Death was assumed to have occurred when there was no movement. The death rate was defined as the average of the percentage of deaths observed for 24 hours and 48 hours. Based on death rate or mortality of test organism, the 48 hr > LC50 and LC100 value was found to be 87.9842 mg/ and 879.842 mg/l. Erythrosine sodium was considered as not classified according to CLP regulation

On the basis of results for toxicity to aquatic invertebrates from target chemical and read across studies, it can be considered that the 4', 5’-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one (596-03-2) is not likely to be toxic to aquatic invertebrates at environmentally relevant concentrations and can be considered as not classified as per the criteria of CLP regulation.

Additional information

Toxicity to aquatic algae and cyanobacteria:

Aim of this study was to evaluate the nature of chemical test chemical 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one 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 100.0 g/l was prepared by dissolving pink powder in OECD growth medium. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at 0, 6, 12, 25, 50 and 100 mg/l oncentration. 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. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical, the ErC50 was determine to be 50.9 mg/l. Based on the ErC50 value, it was concluded that the substance 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one

is likely to be hazardous to aquatic algae and can be be classified as aquatic chronic 3 category as per the CLP criteria.

Short-term toxicity to aquatic invertebrate:

Various predicted studies of the test compound 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one (CAS number: 596-03-2) and read across chemical to aquatic invertebrates were reviewed from reliable sources.

48 hrs aquatic toxicity studies (SSS QSAR prediction model, 2016) were conducted to assess toxic effects of the 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one (CAS number: 596-03-2) and the results were predicted. The study was based on the effects of the test compound on the Daphnia magna in a static fresh water system. The predicted data suggests the effective concentration (EC50) for the 4',5'-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one (CAS number: 596-03-2) was estimated to be 17 mg/l on the basis of mobility.

 

The toxicity of dye, fluorescein sodium salt, to Daphnia pulex was assessed (W.K. Walthall, J.D. Stark, 1999) using acute level toxicological test. The test organisms were obtained from cultures at or beyond the third filial (F3) generation. The final concentrations used in the generation of the acute mortality regression are 0, 150, 250, 400, 600 and 800 mg/liter. No control mortality occurred in the acute toxicity experiments. For each concentration tested, 25 ml of test solution was transferred into a 30-ml plastic cup and five neonates were transferred into the test container using a disposable glass pipette. Each concentration was replicated a minimum of four times. The testing regime was 48 h static, nonrenewal. The LC₅₀for Fluorescein sodium was found to be 337(278 ± 403) mg/liter for Daphnia pulex neonates on the basis of the results obtained from mortality and the response predicted by regression analysis.

According to the CLP regulations the test chemical was considered as not classified for short term toxicity to aquatic invertebrates.

 

Short term toxicity study was conducted to determine the aquatic toxicity of sodium fluorescein (Acute Toxicity of Sodium Fluorescein to Ashy Pebble snails Fluminicola fuscus, 2011) .The dose concentration used are 0.2; 20.0; 50.0 71.0 100.0 141.0 and 200.0 mg/L. The snail behavior was observed every 15 minutes during the first hour of exposure, and then every hour for the next 4 h. At the end of a 24h exposure, we poured the test system into small sieve to recover all snails, and then rinsed the snail three times with clean, aged laboratory water. The snails are then placed s into small cups with aged water, assessed mortality immediately, then again at 24 and 48 h. Each beaker is inspected with the aid of a dissecting microscope and watched for snail movement, and touched individual snails that were not active with a probe to elicit movement or tactile response. Each trial was replicated three times over two weeks for a total of 9 beakers tested at each concentration. Probit model and normal distribution were used for estimating the probability of mortality. The median lethal concentration was found to be 377 mg/L. Our model had decreasing precision as probability of mortality increased, because we never achieved high mortality in trials, even at the highest tested concentrations.

According to the CLP regulation the test chemical was considered as not classified for short term toxicity to aquatic invertebrates.

 

Ecological assessments included both acute and chronic effects for the 3 life forms: fish, Cladocera (water flea, daphnia), and algae (An assessment of the potential adverse properties of fluorescent tracer dye used for groundwater tracing, 1995). The concern levels identified by use of the SAR technique are expressed ad HIGH, MODERATE, LOW. These values are toxicity values, not exposure values. SAR results are generally regarded by EPA as being reliable. Dyes with aquatic toxicity values below 1mg/l received a HIGH concern rating, those with toxicity values ranging from 1-100mg/l received a MODERATE concern rating and those with toxicity values greater than 100mg/l received a LOW concern rating. The Median Lethal Concentration which is estimated to be lethal to 50% of Cladocera (water flea) in 48 hours exposure to the test chemical (LC50) was found to be 165mg/l. Since the LC50 for the test chemical was determined to be 165 mg/l, it falls in the low concern category in the aquatic toxicity ratings. Thus, based on the result according to the CLP regulation it was considered that the test chemical was not classified to short term aquatic invertebrates.

Short term toxicity study to Artemia Salina was carried out in read across erythrosine sodium (16423-68-0) (Ascreening method for the toxicity of food dyes using artemia salina larvae, 1977) for 24-48 hrs. The test chemical concentration used for the study was 879.842 mg/l and 87.9842 mg/l, respectively. A. salina eggs (encysted dried gastrulae) were commercially obtained, and were stored at -20°C; Eggs used in experiments were washed and stored at room temperature in desiccators over anhydrous granular CaCl₂. Larvae were obtained by incubating eggs in petri dishes containing muslin-filtered sea water at 30°C for 24 hours. The larvae were separated from shells, dead larvae and unhatched eggs by their photo tactic movements toward a light source.  Food dyes of various concentrations were placed in a petri dish, and sea water containing 20 to 30 larva e was added. After this was incubated at 30°Cfor 24 hours and 48 hours, larvae surviving were measured by direct count. The same method was tested from 5 to 6 times for each concentration, and the death rate was calculated. The study was performed under static conditions for 24 – 48 hrs at 30°C. Death was assumed to have occurred when there was no movement. The death rate was defined as the average of the percentage of deaths observed for 24 hours and 48 hours. Based on death rate or mortality of test organism, the 48 hr > LC50 and LC100 value was found to be 87.9842 mg/ and 879.842 mg/l. Erythrosine sodium was considered as not classified according to CLP regulation

On the basis of results for toxicity to aquatic invertebrates from target chemical and read across studies, it can be considered that the 4', 5’-dibromo-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one (596-03-2) is not likely to be toxic to aquatic invertebrates at environmentally relevant concentrations and can be considered as not classified as per the criteria of CLP regulation.