3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid

Endpoint summary

Description of key information

Short-term toxicity to fish :

Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for CAS No. 18472-87-2 on Zebra fish Danio rerio, was determined to be >100 mg/L.

Short term toxicity to aquatic invertebrates :

Aim of this study was to assess the short term toxicity of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2) 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 red powder in reconstituted water. The test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted test water. 0, 0.6, 1.2, 2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 mg/L concentrations 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 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2), in Daphnia magna was determined to be 14.5 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 as per the CLP classification criteria. Since the chemical is readily biodegradable, substance is likely to be not  hazardous to Aquatic organisms at environmentally relevant concentrations and can be considered to be `Not classified`  as per the CLP regulation.

Long-term toxicity to aquatic invertebrates:

In long term toxicity to aquatic invertebrate for Phloxine B in Daphnia pulex when it is exposed for 10 days. Exposure to phloxine B at concentrations of 1 mg/liter or greaterresulted in both a general decline in mean survivorship as well as a significant decline in the mean number offspring per surviving individual. Therefore LOEC was considered to be1mg/l for Phloxine B in Daphnia pulex for 10 days under static condition.

Toxicity to aquatic algae and cyanobacteria :

The median effective concentration (ErC50) for the test substance, 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid, in a freshwater algae Desmodesmus subspicatus was determined to be 7.5 mg/L with 95% confidence limit of 6.9 -8.1 mg/L on the basis of effects on growth rate in a 72 hour study. Since the chemical is readily biodegradable, substance is likely to be not  hazardous to Aquatic organisms at environmentally relevant concentrations and can be considered to be `Not classified`  as per the CLP regulation.

Toxicity to microorganisms :

Minimum inhibitory concentration (MIC) was found to be 25 mg/L  for test chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen- 9-yl) benzoic acid (phloxine B) (CAS No.18472-87-2) exposed to Staphylococcus aureus for 80 min.

Additional information

Short-term toxicity to fish :

Various studies including experimental data following OECD guideline for toxicity to aquatic fish with the target substance 3,4,5,6-tetrachloro-2-(1,4,5,8- tetra bromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS Number: 18472-87-2), were summarized as follows:

 

Study was conducted to access the effect of test chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen -9-yl)benzoic acid on the growth of fish Danio rerio. Test

conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). The test substance was highly soluble in water.In house solubility was found to be 786 g/L. Therefore the stock solution prepared as 1g/1L, with the concentration of 1g/1L, and was kept for 5 min. stirring. After this as it is stock taken for experiment. From this stock solution further test concentrations were prepared for achieving test concentrations of 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L respectively.This test solution was then added to the remaining two liters of water for achieving test concentrations of 100 mg/L and 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 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen -9-yl)benzoic acid to various nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was non hazardous and can be consider to be not classified as per the CLP classification criteria.

 

Moreover, the study was conducted by Pimprikar et al. (Southwest. Entomol.9 (2): 218-222 1984) to determine the toxic effects of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472) to predatory mosquitofish. The toxicity was observed over a period of 4 days. The lethal concentrations (LC50) for the test substance, 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo- 6-hydroxy-3-oxoxanthen-9-yl)benzoic acid, in Gambusia affinis on a mortality effect was observed to be 97.7 mg/L. Also the substance was found to be not rapidly degradable. Thus according to CLP Criteria for aquatic classification of the substance, it is concluded that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6- hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2) exhibits toxicity to aquatic fish in the chronic 3 classification category. Since the chemical is readily biodegradable, substance is likely to be not  hazardous to Aquatic organisms at environmentally relevant concentrations and can be considered to be `Not classified`  as per the CLP regulation.

Futhermore,Short term toxicity study toOryzias latipeswas carried out for 48 hrs and survival rate (in %) & Median Tolerance Limit (TLm) 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 Phloxine B, ten fishes were kept in the tank without direct sunlight for 48 hrs and survival rate was determined. Water tem. was 20ᵒC and aeration was provided with bubbler.Median Tolerance Limit (TLm) was also determined after 24 and 48 hrs.After observation of dead fishes, coloration of gill, fin and mouth by the test substance Phloxine B was recognized and particularly deep coloration of gill was found. Though internal organs were not colored, the excrement was clearly colored. Significant binding of Phloxine B to bovine serum albumin was also noted.The TLm value was found to be 230 and 190 mg/l at 24 and 48 hrs.Based on mortality of all test fishes, the LC100 value was determined to be 230 and 190 mg/l at 24 and 48 hrs respectively. Thus, the survival rate of fish after 48 hrs was determined to be 0%.

 

So, based on the above studies for substance 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS Number: 18472-87-2), the lethal concentration LC50 was found to be in the range of 97.7 mg/L to >100.0 mg/L in a 96 hours of exposure. Although data from peer-reviewed journal suggests that the substance is classified in aquatic chronic 3 but as the data from experimental study has K1 reliability and the test was performed using OECD guidelines which has the value of LC50 as >100 mg/L in a 96 hours of study, indicating that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen -9-yl)benzoic acid (CAS No. 18472-87-2) does not exhibits toxicity to fish criteria of CLP, the same has been considered for the purpose of classification and chemical safety assessment. Additonally, since the chemical is readily biodegradable, substance is likely to be not  hazardous to Aquatic organisms at environmentally relevant concentrations and can be considered to be `Not classified`  as per the CLP regulation.

Short-term toxicity to aquatic invertebrates :

Aim of this study was to assess the short term toxicity of 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2) 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 red powder in reconstituted water. The test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted test water. 0, 0.6, 1.2, 2.5, 5.0, 10.0, 20.0, 40.0 and 80.0 mg/L concentrations 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 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2), in Daphnia magna was determined to be 14.5 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 as per the CLP classification criteria. Since the chemical is readily biodegradable, substance is likely to be not  hazardous to Aquatic organisms at environmentally relevant concentrations and can be considered to be `Not classified`  as per the CLP regulation.

Long-term toxicity to aquatic invertebrates:

In long term toxicity to aquatic invertebrate for Phloxine B in Daphnia pulex when it is exposed for 10 days. Exposure to phloxine B at concentrations of 1 mg/liter or greaterresulted in both a general decline in mean survivorship as well as a significant decline in the mean number offspring per surviving individual. Therefore LOEC was considered to be1mg/l for Phloxine B in Daphnia pulex for 10 days under static condition

Toxicity to aquatic algae and cyanobacteria :

Various experimental studies following OECD guideline for toxicity to aquatic algae and cyanobacteria with the target substance 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2), were summarized as follows:

 

Aim of this study was to evaluate the nature of chemical test chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6- hydroxy-3-oxoxanthen-9-yl)benzoic acid 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 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.Various concentrationes were used.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 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6- hydroxy-3-oxoxanthen-9-yl)benzoic acid, in algae was determined to be 7.5 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 and can be classified as aquatic chronic 2 category as per the CLP classification criteria. Since the chemical is readily biodegradable, substance is likely to be not  hazardous to Aquatic organisms at environmentally relevant concentrations and can be considered to be `Not classified`  as per the CLP regulation.

Moreover,The study was designed to assess the toxic effects of the test compound 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen- 9-yl)benzoic acid (CAS No. 18472-87-2) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance (3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen- 9-yl)benzoic acid (CAS No. 18472-87-2) was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations 0.5mg/l, 1.6mg/l, 5.12mg/l, 16.384mg/l, 52.429mg/l and 167.772mg/l were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution.For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.After 72 hours of exposure to test item 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen- 9-yl)benzoic acid (CAS No. 18472-87-2) to various nominal test concentrations, EC50 was determine to be 166.06 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was non hazardous and can be consider to be not classified as per the CLP classification criteria.

 

So, based on the above experimental studies for target substance 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid (CAS No. 18472-87-2), the median effective concentration (EC50) was found to be in the range of 7.5–166.06 mg/L on the basis of growth rate effect in a 72 hours of exposure. Although data from UERL suggests that the substance is not classified but as the ABITEC (2016) data has K1 reliability and the test was performed using OECD guidelines which has the value of EC50 as 7.5 mg/L in a 72 hours of study and also the substance was found to be not rapidly degradable, indicating that 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3 -oxoxanthen -9-yl)benzoic acid (CAS No. 18472-87-2) exhibits toxicity to aquatic aquatic algae in the chronic 2 classification category, criteria of CLP. Since the chemical is readily biodegradable, substance is likely to be not  hazardous to Aquatic organisms at environmentally relevant concentrations and can be considered to be `Not classified`  as per the CLP regulation.

Toxicity to microorganisms :

Various experimental studies from a peer-reviewed journal for toxicity to microorganism with the target substance3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl)benzoic acid(CAS No. 18472-87-2), were reviewed and are summarized as follows: 

 

80 min toxicity test was conducted by Avraham Rasooly et al. (Antimicrobial Agents And Chemotherapy, Nov. 2002, P. 3650–3653) on bacterial species Staphylococcus aureus to study the toxic effects of chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6- hydroxy-3-oxoxanthen-9-yl)benzoic acid (phloxine B). 90% inhibition was observed after exposure to phloxine B. When dye was added at concentrations of 50 or 100 mg/L, bacterial growth stopped completely and the cell count decreased by 104 and 105, respectively, within 80 min. These values point to a MIC of 25 mg/L. Hence, Minimum inhibitory concentration (MIC) was found to be 25 mg/L for test chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen-9-yl) benzoic acid (phloxine B) (CAS No. 18472-87-2) exposed to Staphylococcus aureus for 80 min.

 

Further, toxicity to micro-organisms test was conducted by Gyula Oros et. al., (Chemosphere, Vol. 52, Pg. no. 185–193, 2003) on 14 different gram – negative bacteria. Inhibitory activity of test chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen- 9-yl) benzoic acid (Phloxine B) (CAS No.18472-87-2) was determined by the traditional agar gel method. Based on growth inhibition of Agrobacterium radiobacter; Agrobacterium tumefaciens; Bradyrhizobium japonicum; Erwinia herbicola; Erwinia uredovora; Pseudomonas fluorescence Midula; Rhizobium trifolii Dangeard [RTR]; Xanthomonas malvacearum (Erw. Smith) Dowson [XMA]; Xanthomonas phaseoli Burkholder [XPH] and X. stewartii (Erw. Smith) Dowson [XST] , the LOEC value was reported to be 0.6918 mg/l.

Since no effect on growth of Escherichia coli; Erwinia atroseptica Dye [EAT]; Pseudomonas phaseolicola ; Pseudomonas syringae were observed, the NOEC value was reported to be 0.6918 mg/l.

 

Moreover in a study by Gyula Oros et. al., (Chemosphere, Vol. 52, Pg. no. 185–193, 2003), toxicity to micro-organisms test was conducted on 8 different gram – positive bacteria. Inhibitory activity of test chemical 3,4,5,6-tetrachloro-2-(1,4,5,8-tetrabromo-6-hydroxy-3-oxoxanthen- 9-yl) benzoic acid (Phloxine B) (CAS No.18472-87-2) was determined by the traditional agar gel method. Based on growth inhibition of gram positive bacteria, the LOEC value was reported to be 0.6918 mg/l.