Disodium succinate

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 test solution was prepared by dissolving 400 mg of the test substance in 4 liters of potable water (passed through reverse osmosis system) with continuous stirring for achieving test concentrations of 100 mg/L,respectively.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 toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

Daphnia magna Acute Immobilization Test according to OECD Guideline 203 was conducted for test material. The nominal concentration selected for the experiment were 100mg/L and test Daphnia magna were exposed to these concentration for 48 hours. 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. Achieving test concentrations of 1 g/L, respectively. The median lethal concentration (EC50) for disodium butanedioate 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 substance, test material does not exhibit short term toxicity to Daphnia. EC50 (48 hours) Experimental = >100 mg/L.

Long term toxicity to aquatic invertebrate:

Long term toxicity to aquatic invertebrate of the test chemical was performed according toOECD Guideline 211 (Daphnia magna Reproduction Test)to evaluate its effect on the reproduction potential of Daphnia magna for the period of 21 days .The EC50 and No Observed Effect Concentration (NOEC) values for the est substance to Daphnia magna is found to be >100 mg/L. ased on the above effect concentration it can be considered that test material is not classified as per CLP criteria.

Toxicity to aquatic algae and cyanobacteria:

The study was designed to assess the toxic effects of the test compound 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 item  was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 00 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit.

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.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 100.99 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Toxicity to microorganism:

Effect of test material was evaluated on microorganisms based on the data of target chemical from peer reviewed journal.

Klebsiella pneumonieH12 is a polysaccharide flocculant that is involved to mediate plant-microbe interaction. In the given study, , two- way regulation of polysaccharide-mediated interactions betweenA. pneumoniasandRaphanus sativuswas studied using test material. Polysaccharide productivity and flocculation activity assayed using kaolin, polysaccharide and test material was studied. In an assay to study polysaccharide productivity, Strain H12 was cultured for 4 days at 28°C in 250-ml Erlenmeyer flasks containing  30 ml  of  YPE  medium. Production of the polysaccharide was measured by the dry weight after ethanol  precipitation   of  the  culture supernatant. Polysaccharide production was promoted at low concentration of upto 10mM but the productivity decreased from 20mM ( 3241.04 mg/L) dose range. The EC for the test material is thus found to be 20mM ( 3241.04 mg/L).

Additional information

Short term toxicity to fish:

Toxicity of test material was evaluated on fish based on study report of the test material and data from authoritative database.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 test solution was prepared by dissolving 400 mg of the test substance in 4 liters of potable water (passed through reverse osmosis system) with continuous stirring for achieving test concentrations of 100 mg/L,respectively.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 toxic and can be consider to be not classified as per the CLP classification criteria.

In an authoritative data base , the short term toxicity test on fish was evaluated according to OECD guideline 203 . The LC50 value of the test substance to fish is found to be >100 mg/L. This value indicates that the substance is not likely to exhibit short term toxicity to fish.Based on the LC50, it can be consider that the chemical was toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

Toxicity of test material was evaluated for aquatic invertebrate based on study report of the test material and data from authoritative database.

Daphnia sp., Acute Immobilization Test according to OECD Guideline 203 was conducted for test material. The nominal concentration selected for the experiment were 100mg/L and test Daphnia magna were exposed to these concentration for 48 hours. 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. Achieving test concentrations of 1 g/L, respectively. The median lethal concentration (EC50) for disodium butanedioate 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 substance, test material does not exhibit short term toxicity to Daphnia. EC50 (48 hours) Experimental = >100 mg/L.

The above study report was further supported by data fromauthoritative database.The EC50 value of the test substance was evalauted according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) .Test material to Daphnia magna is found to be >1000 mg/L. This value indicates that the substance is not likely to exhibit short term toxicity to aquatic invertebrate.

Based on the above data it can be considered that the test material is not toxic to aquatic invertebrate and can not be classified as per CLP criteria.

Long term toxicity to aquatic invertebrate:

Long term toxicity to aquatic invertebrate of the test chemical was performed according toOECD Guideline 211 (Daphnia magna Reproduction Test)to evaluate its effect on the reproduction potential of Daphnia magna for the period of 21 days .The EC50 and No Observed Effect Concentration (NOEC) values for the est substance to Daphnia magna is found to be >100 mg/L. ased on the above effect concentration it can be considered that test material is not classified as per CLP criteria.

Toxicity to aquatic algae and cyanobacteria:

Toxicity of test material was evaluated for aquatic algae and cyanobacteria based on study report of the test material and data from authoritative database.

The study was designed to assess the toxic effects of the test compound 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 item  was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 00 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit.

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.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 100.99 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

In another study from authoritative databasethe toxicity to aquatic algae and cynobacteria was performed according toOECD Guideline 201 (Alga, Growth Inhibition Test)for the test chemical.The EC50 and NOEC values for the test substance is found to be >1000 mg/L based on both growth rate and AUG parameters. This value indicates that the substance is not likely to exhibit short term toxicity to aquatic algae and cyanobacteria.

Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Toxicity to microorganism:

Effect of test material was evaluated on microorganisms based on the data of target chemical from peer reviewed journal.

Klebsiella pneumonieH12 is a polysaccharide flocculant that is involved to mediate plant-microbe interaction. In the given study, , two- way regulation of polysaccharide-mediated interactions betweenA. pneumoniasandRaphanus sativuswas studied using test material. Polysaccharide productivity and flocculation activity assayed using kaolin, polysaccharide and test material was studied. In an assay to study polysaccharide productivity, Strain H12 was cultured for 4 days at 28°C in 250-ml Erlenmeyer flasks containing  30 ml  of  YPE  medium. Production of the polysaccharide was measured by the dry weight after ethanol  precipitation   of  the  culture supernatant. Polysaccharide production was promoted at low concentration of upto 10mM but the productivity decreased from 20mM ( 3241.04 mg/L) dose range. The EC for the test material is thus found to be 20mM ( 3241.04 mg/L).

In another study ,Klebsiella pneumonieH12 is a bacteria and polysaccharide flocculant that is involved to mediate plant-microbe interaction. In the given study, two- way regulation of polysaccharide-mediated interactions betweenA. pneumoniasandRaphanus sativuswas studied using sodium succinate. Polysaccharide productivity and flocculation activity assayed using kaolin, polysaccharide and title comp was studied. In the flocculation assay -in a test tube, 9 ml of a kaolin clay suspension (5.5 g /l) was mixed with 1 ml of polysaccharide solution, 100 µl of or- ganic acid solution, and 100 µl CaCl2.2H2O solution in this sequence with a Vortex mixer for 5 sec. After 5 min, OD_550c of the supernatant was measured. A control experiment was done in the absence of the polysaccharide and the OD_550cwas measured. Flocculation of bacterial cells by polysaccharide was inhibited at a concentration of 0.3mM (48.61 mg/l) by the test compound .The EC for the test material Sodium succinate is thus found to be 0.3mM (48.61 mg/l).