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

Endpoint summary

Description of key information

Short term toxicity to fish:

Study was conducted to assess the effect of test chemical on the mortality of fishes. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). Test conducted on Danio rerio (previous name: Brachydanio rerio). The stock solution was prepared by dissolving 1 gm of the test substance in 1 liters of potable water (passed through reverse osmosis system). This stock solution was used for preparing test concentrations of 6.25 mg/L, 12.5 mg/L, and 25 mg/L, 50 mg/L & 100 mg/L, respectively and Zebra Fish Danio rerio were exposed to these concentration for 96 hours. 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 of test chemical to various nominal test concentrations, LC50 was calculated and the mortality was used as a key parameter for the determination of exact effect on fishes. Based on nominal concentrations, experimental median lethal Concentrations [LC-50] of test chemical on Zebra Fish (Denio rario) after exposing for 96 hours was determined to be > 100 mg/L, as no mortality were observed at 100 mg/l. Thus, it can be consider that the chemical was nonhazardous and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates:

The study was conducted on two different species i.e. Daphnia magna and Artemia salina.After exposure period of 48 hours, effects were calculated. The median effective concentration (EC50 value) of the test substance on Daphnia magna was determined to be 8.1 mg/L on the basis of mobiity inhibition effects in a 48 hour study. Whereas on the basis of mortality of test organism Artemia Salina due to the test chemical exposure for 48 hours, the LC50 and LC100 value was observed to be > 87.9842 mg/ and 879.842 mg/l.

Toxicity to algae and cyanobacteria:

Based on the growth rate inhibition of algae Desmodesmus subspicatus and Chlorella vulgaris, due to the test chemical exposure for 72 hours, the EC50 value was determine to be 34.1 mg/L and > 200 mg/l. As the toxicity were observed on the growth of algae Desmodesmus subspicatus after exposure period of test chemical for 72 hours, chemical consider to be toxic and can be consider to be classified in aquatic chronic category 3 as per the CLP classification criteria.

Toxicity to microorganisms:

Toxicity to micro-organisms study was conducted on Staphylococcus aureus of enterotoxigenic strain ATCC 13565. High-pressure liquid chromatography grade were used for analytical measurements. Inoculum was prepared in brain 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. OD was measured every 45 mins to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates. Experiment was performed in triplicate on separate days to ascertain reproducibility. As the test substance do not have any activity against S. aureus i.e, no difference and no growth rate inhibition was observed between the treated test tubes and the control tubes, the NOEC value was observed to be 100 mg/l.

Additional information

Short term toxicity to fish:

Summarized result from the various experimental sources for the determination of effect of test chemical on the mortality of fishes are as mentioned below:

 

Study was conducted to assess the effect of test chemical on the mortality of fishes. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). Test conducted on Danio rerio (previous name: Brachydanio rerio). The stock solution was prepared by dissolving 1 gm of the test substance in 1 liters of potable water (passed through reverse osmosis system). This stock solution was used for preparing test concentrations of 6.25 mg/L, 12.5 mg/L, and 25 mg/L, 50 mg/L & 100 mg/L, respectively and Zebra Fish Danio rerio were exposed to these concentration for 96 hours. 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 of test chemical to various nominal test concentrations, LC50 was calculated and the mortality was used as a key parameter for the determination of exact effect on fishes. Based on nominal concentrations, experimental median lethal Concentrations [LC-50] of test chemical on Zebra Fish (Denio rario) after exposing for 96 hours was determined to be > 100 mg/L, as no mortality were observed at 100 mg/l. Thus, it can be consider that the chemical was nonhazardous and can be consider to be not classified as per the CLP classification criteria.

 

Above study further supported by the second study from peer reviewed journal. Principle of this study was to determine the effect of test chemical on the mortality rate of fishes. Gambusia affinis was used as a test organism and the study performed under the static system for 96 hours. Different test concentrations 1, 10,20,50,70,100,150 and 200 mg/l respectively were used. Fishes were collected from the pound and kept in the laboratory for at least 72 hr for the stabilization prior to their use in the tests. Fishes were starved for 24hr before test. 20 fishes were transferred to the 5 gallon glass aquaria were illuminated with fluorescent lamps giving surface light intensity of 3800 µw/cm2. Probit analysis was used to determine the effect of test chemical, whereas mortality were used as a key parameter for the LC50 calculation. After the exposure of test chemical for 96 hrs with fishes Gambusia affinis, the LC50 value was determine to be > 200 mg/l on the basis of mortality observations.

 

Short term toxicity test was performed on Himedaka (Oryzias latipes). All fishes acclimated for 10 days in tap water before experiment. In lab, 1 liter solution having pH 7 containing 3,000 mg/liter of test chemical to be tested. 10 tested fish were kept in the tank without direct sunlight for 48 hrs and survival rate was determined. It was observed that at the 3000 mg/liter concentration, the survival rate of fish was observed to be 0 % and thus the LC100 was consider to be at 3000 mg/l.

Similarly an acute study was conducted to determine the effect of test chemical on the fishes. Test conducted in accordance with the Japanese Industrial Standards Committee. Himedaka commonly known as Oryzias latipes of same age i.e. 2 cm long and 0.2 gram 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. Based on the mortality of fishes Oryzias latipes due to the test chemical exposure for 24 and 48 hours, the LC50 value was determine to be 710 and 340 mg/l respectively.

 

The fifth study from peer reviewed journal was conducted on Oryzias latipes was carried out for 48 hrs and TLm value was determined. Himedaka (Oryzias latipes) of same age i.e. 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 liter solution of pH 7.0 containing test substance, ten fishes were added in the tank without direct sunlight for 48 hrs and survival rate was determined. Water temperature was 25°C and aeration was provided with bubbler. Median Tolerance Limit (TLm) was determined after 48 hrs. Based on the mortality of test fishes, the 48 hrs LC50 value was determine to be 900 mg/l.

 

Thus, on the basis of above all studies and effects observations on fishes, test chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

 

Short term toxicity to aquatic invertebrates:

Summarized result from the various experimental sources for the determination of effect of test chemical on the aquatic invertebrates are as mentioned below:

An acute immobilisation test was conducted for 48 hrs for assessing the short-term toxicity of test chemical to aquatic invertebrate. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna was used as a test organism. The stock solution 200.0 mg/L was prepared in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted test water. 0, 2.5, 5, 10, 20, 40, 80 and 100 mg/L, respectively nominal concentrations were used in the study. 5 daphnids per concentration were used in the study. Effects on immobilisation were observed for 48 hours and conducted under the static system. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Observations including immobility, pH, Temperature, dissolved oxygen content were recorded in the interval of 24 and 48 hours. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Based on the immobilisation of Daphnia magna due to the exposure of test chemical for 48 hours, the EC50 value was determine to be 8.1 mg/l. EC50 value indicates that the substance is likely to be hazardous to aquatic invertebrates and can be classified in aquatic chronic 2 category as per the CLP criteria.

 

Above study further supported by the next study from peer reviewed journal. Short term toxicity study to Artemia Salina was carried out 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 a desiccator 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 phototactic 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 determine to be 87.9842 mg/ and 879.842 mg/l.

 

On the basis of effects observation on the standard species Daphnia magna at the concentration of 8.1 mg/l, thus, test chemical consider to be toxic and classified in aquatic chronic category 2 as per the CLP classification criteria.

 

Toxicity to algae and cyanobacteria:

Summarized result from the various experimental sources for the determination of effect of test chemical on the growth of algae are as mentioned below:

 

An acute study was conducted to determine the effect of test chemical on the growth of green algae when exposed for 72 hours. Test conducted according to the OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as a test organism. The stock solution 100 mg/L was prepared 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 nominal concentrations 0, 8, 15, 29, 55 and 100 mg/L, respectively were used in the study. Test conducted under the static system. 50 ml Glass vessel filled with 15 ml of test solution were used in the study, in which 5x10e3 cells/ml were added. Test performed using 3 replicates. Temperature maintained at 23±2°C and pH value 8 changed to 7.1 in sample, whereas for control 8.2 changes to 7.6 during test. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration ErC50 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 green algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical for 72 hours, the ErC50 value was determined to be 34.1 mg/l with 95 % CI of 21.4 to 54.2 mg/l. Thus, based on the ErC50 value, test chemical considered to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Above study further supported by the second study from experimental source. The principle of this study was to determine the effect of test substance on the growth of fresh water green algae. The study was conducted following OECD guideline 201- Alga growth inhibition test. Chlorella vulgaris was used as test organism.The test solution was prepared by adding 70mg of test chemical in 350 ml of BBM to get the final concentration of 200 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10e4cells/ml.The test concentrations chosen for the study 6.25mg/l, 12.5mg/l, 25mg/l, 50 mg/l, 100mg/l and 200 mg/l were prepared using stock solution of the test substance using de-ionized water and the study conducted under the static system. 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. The test is considered to be valid as met all the standard parameters. The green algae was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of test substance. EC50 calculated through probit analysis was observed to be > 200 mg/L. Thus based on the effects observed on the growth rate of algae, chemical consider to be nontoxic and not classified as per the CLP classification criteria. 

 

As the toxicity were observed on the growth of algae Desmodesmus subspicatus after exposure period of test chemical for 72 hours, chemical consider to be toxic and can be consider to be classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Toxicity to microorganisms:

Summarized result from the various experimental sources for the determination of effect of test chemical on the growth of microorganisms are as mentioned below:

 

Toxicity to micro-organisms study was conducted on Staphylococcus aureus of enterotoxigenic strain ATCC 13565. High-pressure liquid chromatography grade were used for analytical measurements. Inoculum was prepared in brain 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. OD was measured every 45 mins to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates. Experiment was performed in triplicate on separate days to ascertain reproducibility. As the test substance do not have any activity against S. aureus i.e, no difference and no growth rate inhibition was observed between the treated test tubes and the control tubes, the NOEC value was observed to be 100 mg/l.

 

Above study further supported by the supported data from peer reviewed journal. Toxicity study of test chemical on micro-organisms was conducted on 10 streptococcal strains. The assay was performed using paper-disk agar diffusion method under aerobic and anaerobic conditions. The test chemical conc. used for the study was 5000 and 10,000 mg/l (0.5 and 1%) respectively. 10 Streptococcal strains were used for the study. The Streptococcal strains include AHT, BHT, CHT, HHT, HS-10, GF-71, GS-5, E-49, FA-1 and PK-1. Tryptic soy agar enriched with 1% sucrose was used as a medium. The assay was performed using paper-disk agar diffusion method in which the paper disk charged with 0.08 ml of aqueous solution of test chemical was placed on the surface of agar plates inoculated with the respective cultures. The plate was incubated for 16 to 30 hrs at 37°C and the presence of zone of growth inhibition around the paper disks was measured. One set of plates was incubated aerobically; the second set was incubated in anaerobic jars which were filled with a gaseous mixture of 95% nitrogen and 5% carbon dioxide. Based on growth inhibition of test organism Streptococcal strains AHT, BHT, HS-10, GF-71, FA-1 and PK-1 under both aerobic and anaerobic condition, the MIC value was observed to be 5,000 mg/l. Similar effects were observed under aerobic condition for Streptococcal strains GS-5 and E-49, based on which the MIC value was observed to be 5,000 mg/l. Under aerobic condition, traces of inhibition in growth of test organism Streptococcal strain HHT was observed, the LOEC value was determine to be 10,000 mg/l. As growth inhibition of test organism Streptococcal strain GS-5 was observed under anaerobic condition, the MIC value was found to be 10,000 mg/l. Also No effects on growth inhibition of test organism Streptococcal strains HHT and E-49 under anaerobic conditions was observed, the NOEC value was observed to be 10,000 mg/l. Similar effects were observed under both aerobic and anaerobic condition for Streptococcal strains CHT, the NOEC value was determine to be 10,000 mg/l.

 

Similarly in the third study, toxicity to micro-organisms was conducted on 8 different organisms of yeast. The assay was performed using paper-disk agar diffusion method under aerobic and anaerobic conditions. The test chemical conc. used for the study was 5000 and 10,000 mg/l (0.5 and 1%) respectively. 8 different yeasts were used for the study. These includes Hansenula wingei ATCC 14256 and ATCC 14355, Candida albicans ATCC 752 and ATCC 11651, Saccharomyces cerevisiae ATCC 4098, Saccharomyces fragilis ATCC 8635, Saccharomyces carlsbergensis ATCC 9080 and S. carlsbergensis SRI-206. All yeasts cultures were cultivated on a medium which contained 0.7% yeast extract, 0.5 KH11PO4, 1.5% agar, and 1.0% sucrose. The organism was autoclaved separately and added aseptically to all media. The assay was performed using paper-disk agar diffusion method in which the paper disk charged with 0.08 ml of aqueous solution of test chemical and was placed on the surface of agar plates inoculated with the respective cultures. The plate was incubated for 24 hrs at 25°C and the presence of zone of growth inhibition around the paper disks was measured. One set of plates was incubated aerobically; the second set was incubated in anaerobic jars which were filled with a gaseous mixture of 95% nitrogen and 5% carbon dioxide. Based on growth inhibition of test organism under aerobic condition by the test chemical, the MIC value was observed to be at 5000 mg/l for organism Candida albicans ATCC 752, Candida albicans ATCC 11651, Saccharomyces fragilis ATCC 8635, Saccharomyces carlsbergensis ATCC 9080 and Saccharomyces carlsbergensis SRI-206. As no effects on growth of test organism was observed under anaerobic condition, the NOEC value was consider to be 10, 000 mg/l for test organism Candida albicans ATCC 752, Candida albicans ATCC 11651, Saccharomyces fragilis ATCC 8635, Saccharomyces carlsbergensis ATCC 9080 and Saccharomyces carlsbergensis SRI-206. Similar effects were observed for test organism Hansenula wingei ATCC 14256, Hansenula wingei ATCC 14355 and Saccharomyces cerevisiae ATCC 4098 under both aerobic and anaerobic condition, the NOEC value was determine to be 10,000 mg/l.

 

Similar study was conducted to determine the effect of test chemical on the growth of Paramecium caudate (ciliated) after providing exposure period of 20 min. Test conducted using 1000 mg/l concentration. In this study, test chemical was put in a hollow slide glass and an equal volume of 0.04 M phosphate buffer, pH 7.0, was added. After that 5 to 10 Paramecium caudatum were added, their survival times were measured microscopically. 30 to 40 Paramecium caudate PC for 0.1% concentration were tested by the same method, and the mean survival time and the death rate were calculated. The death rate was defined as the percentage of deaths observed during 20 min. After the exposure of test chemical with Paramecium caudate (ciliated), mortality were observed after 4 minutes i.e. LC100 value was observed to be at concentration of 1000 mg/l.

 

Toxicity to micro-organisms study was conducted on 12 gram – negative bacteria. The assay was performed using paper-disk agar diffusion method under aerobic and anaerobic conditions. The test chemical concentration used for the study was 5000 and 10,000 mg/l (0.5 and 1%) respectively. 12 gram – negative bacteria as a test organism was used for the study. These includes, Escherichia coli ATCC 9637, E. coli ATCC 9723, E. coli ATCC 11303, Proteus vulgaris ATCC 9484, Salmonella typhimurium ATCC 7823, S. typhosa ATCC 9993, Pseudomonas aeruginosa ATCC 12055, P. aeruginosa ATCC 8709, P. aeruginosa ATCC 10145, Shigella boydii ATCC 9905, S. flexneri ATCC 9582 and Aerobacter aerogenes SRI* - 160. Tryptic soy agar enriched with 1% sucrose was used as a medium. The assay was performed using paper-disk agar diffusion method in which the paper disk charged with 0.08 ml of aqueous solution of test chemical was placed on the surface of agar plates inoculated with the respective cultures. The plate was incubated for 16 to 30 hrs at 37°C and the presence of zone of growth inhibition around the paper disks was measured. One set of plates was incubated aerobically; the second set was incubated in anaerobic jars which were filled with a gaseous mixture of 95% nitrogen and 5% carbon dioxide. As no inhibition on the growth of test organisms was observed due to the test chemical exposure for 30 hours, the NOEC value was observed to be 10,000 mg/l.

 

Thus, based on the above all studies from various sources, no effects were observed on the microorganisms, and thus the chemical can be consider to be nontoxic.

 

As the study was conducted on different species and organisms including fishes, invertebrates and algae and the toxicity and effects ratio were vary from species to species. But the chemical consider to be sensitive for the invertebrates and thus on that basis, chemical consider to be toxic and classified in aquatic chronic category 2 as per the CLP classification criteria.