Benzimidazole

Endpoint summary

Description of key information

Short term toxicity to fish:

Short term toxicity study on fish species Salmo gairdreii, Lepomis macrochirus and Petromyzon marinus was carried out for 24 hrs. Test was performed under static condition and fresh water medium (lake water) and pH maintained as 7.5 to 8.2, test temperature 12.77 deg.C and dissolved oxygen as 8.6 to 13.7 ppm. No effects were observed at dose concentration 5 mg/l on the test fish species Salmo gairdreii, Lepomis macrochirus and Petromyzon marinus thus the NOEC value for test chemical was consider to be 5 mg/l for 24 hrs exposure period.

Short term toxicity to aquatic invertebrates:

Experimental study for inhibition of the mobility of daphnids was carried out with the benzimidazole according to OECD Guideline 202.The animals used for the test shall be less than 24 h old and should not be of first brood progeny. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with reconstituted test water. The test substance was tested at the concentrations 0, 7.5, 15, 30, 60 and 120 mg/L. The test was performed under static conditions in a static fresh water system at 20±1°C temperature. Effects on immobilisation were observed for 48 hours. EC50 was calculated using nonlinear regression by the software Prism 4.0. After the experiment, the median effective concentration (EC50) for the test substance, benzimidazole, in Daphnia magna was determined to be 55.5 mg/L on the basis of immobilisation effects. Based on the EC50 value chemical consider to be toxic and classified as aquatic chronic 3 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus it not exist for longer period of time. Thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to aquatic algae and cyanobacteria:

Study was designed to assess the toxic effects of the test compound Benzimidazole on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test solution was prepared in aseptic condition. The test item Benzimidazole 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 30 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. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l, 100mg/l, 200mg/l concentration were used in the study. All the six concentrations were in geometric series spaced by a factor of 2. 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 Benzimidazole to various nominal test concentrations, EC50 was determine to be > 200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

Toxicity to microorganisms:

Inhibitory effects of test chemical Benzimidazole (i.e 1H-benzimidazole) on microorganism species Bacillus spp. was observed using different soil and water samples as inoculum. Test chemical analytically monetarized by thin-layer chromatography. A stock solution (0.276 mg/ml) was prepared in methanol, with an activity of 36 512 dpm/ul). Methanol were used as a vehicle in which test chemical dissolved. Test conducted under the static system for 192 hrs. During experiment the Effect of benzimidazole derivatives on maximum growth of Bacillus strains was observed to be at concentrations 500-1000 mg/l (reported as ug/ml) using tubes shaken in water bath at 30C; Klett-Summerson readings taken at intervals up to 192 h after inoculation. Based on the EC50 value, chemical consider to be nontoxic.

Additional information

Summarized result of toxicity of the chemical Benzimidazole (CAS No. 51 -17 -2) on the growth of fishes, aquatic invertebrates and algae and microorganisms was studied by considering and collecting the data from various databases for target chemical. The studies are as follows:  

 

Short term toxicity to fish:

Based on the experimental data from various database for the target chemical study have been reviewed to determine the mode of action of Benzimidazole (CAS No. 51 -17 -2) on the mortality rate and behavior of fish. The studies are as mentioned below:

In the first key study from handbook or collection of data, short term toxicity study on fish species Salmo gairdreii, Lepomis macrochirus and Petromyzon marinus was carried out for 24 hrs. Test was performed under static condition and fresh water medium (lake water) and pH maintained as 7.5 to 8.2, test temperature 12.77 deg.C and dissolved oxygen as 8.6 to 13.7 ppm. No effects were observed at dose concentration 5 mg/l on the test fish species Salmo gairdreii, Lepomis macrochirus and Petromyzon marinus thus the NOEC value for test chemical was consider to be 5 mg/l for 24 hrs exposure period.

 

First study was supported by another experimental studies. Short term toxicity study on fish species Petromyzon marinus was carried out for 24 hrs. Test was performed under static condition and fresh water medium (lake water) and pH maintained as 7.5 to 8.2, test temperature 12.77 deg.C and dissolved oxygen as 8.6 to 13.7 ppm. No effects were observed at dose concentration 5 mg/l on the test fish species Salmo gairdreii, Lepomis macrochirus and Petromyzon marinus thus for test chemical, the NOEC value was consider to be 5 mg/l for 24 hrs exposure period.

 

Similarly another study was designed to access the toxic effects of the test compound on the Zebra fish (Danio rerio). Test was conducted in compliance with the OECD guideline 203. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 1 g 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. Test conducted at limit test concentration 100 mg/l and Zebra fishes were exposed to this concentration for 96 hours. Test animal was collected from Orange City Aquarium, Sitabuldi, Nagpur, MS (India). 8 fishes exposed to the test concentration. Aeration in test vessels was provided 1 day before the start of experiment. After the exposure of chemical (mortality, visible symptoms, pH, Temperature, dissolved oxygen content) were recorded after 24 hours, 48 hours, 72 hours and 96 hours of the start of the experiment. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for test chemical on Zebra fish (Danio rerio) was determined to be > 100 mg/L by observing the mortality. Thus based on LC50 it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

Thus on the basis of overall studies chemicalBenzimidazole (CAS No. 51 -17 -2)was consider as nontoxic to fishes and not classified as per the CLP classification criteria.

 

Short term toxicity to aquatic invertebrates:

 

Experimental study for inhibition of the mobility of daphnids was carried out with the benzimidazole according to OECD Guideline 202.The animals used for the test shall be less than 24 h old and should not be of first brood progeny. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with reconstituted test water. The test substance was tested at the concentrations 0, 7.5, 15, 30, 60 and 120 mg/L. The test was performed under static conditions in a static fresh water system at 20±1°C temperature. Effects on immobilisation were observed for 48 hours. EC50 was calculated using nonlinear regression by the software Prism 4.0. After the experiment, the median effective concentration (EC50) for the test substance, benzimidazole, in Daphnia magna was determined to be 55.5 mg/L on the basis of immobilisation effects. Based on the EC50 value chemical consider to be toxic and classified as aquatic chronic 3 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus it not exist for longer period of time. Thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to aquatic algae and cyanobacteria:

 

Summarized result for the toxicity of test chemical Benzimidazole (CAS No. 51 -17 -2) on the growth of aquatic algae and cyanobacteria are as follows:

 

In the first experimental report, study was designed to assess the toxic effects of the test compound Benzimidazole on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). The test solution was prepared in aseptic condition. The test item Benzimidazole 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 30 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. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment. 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l, 100mg/l, 200mg/l concentration were used in the study. All the six concentrations were in geometric series spaced by a factor of 2. 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 Benzimidazole to various nominal test concentrations, EC50 was determine to be > 200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

 

Similarly in the second study freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance Benzimidazole to OECD Guideline 201. The stock solution (120 mg/L) was prepared by dissolving white powder in OECD growth medium. The solution was kept in ultrasonic bath for 20 mins. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Conc. of test chemical used for the study was 0, 7.5, 15, 30, 60 and 120 mg/L, respectively. The test was performed under static conditions in a static fresh water system at a temp of 23±2°C. Initial cell density of test organism used was 5x10(3) cells/ml. Determination of cell counting involve the use of microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non-linear regression by the software Prism 4.0. The median effective concentration (ErC50) for the test substance Benzimidazole in Desmodesmus subspicatus was determined to be 26.8 mg/L on the basis of effects on growth rate in a 72 hour study. Based on the ErC50 value, chemical consider to be toxic to the growth of aquatic algae and cyanobacteria and classified as aquatic chronic 3 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus it not exist for longer period of time. Thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

 

Thus based on the above both studies, chemical Benzimidazole (CAS No. 51 -17 -2) was consider to be nontoxic and not classified as per the CLP classification criteria.

 

Toxicity to microorganisms:

Based on the experimental data from various database and peer reviewed journals for the target chemical study have been reviewed to determine the mode of action of test chemical Benzimidazole (CAS No. 51 -17 -2) on the growth rate of microorganisms. The studies are as mentioned below:

 

In the first key study from from peer reviewed journal 1978, inhibitory effects of test chemical Benzimidazole (i.e 1H-benzimidazole) on microorganism species Bacillus spp. was observed using different soil and water samples as inoculum. Test chemical analytically monetarized by thin-layer chromatography. A stock solution (0.276 mg/ml) was prepared in methanol, with an activity of 36 512 dpm/ul). Methanol were used as a vehicle in which test chemical dissolved. Test conducted under the static system for 192 hrs. During experiment the Effect of benzimidazole derivatives on maximum growth of Bacillus strains was observed to be at concentrations 500-1000 mg/l (reported as ug/ml) using tubes shaken in water bath at 30C; Klett-Summerson readings taken at intervals up to 192 h after inoculation. Based on the EC50 value, chemical consider to be nontoxic.

 

First study was supported by another experimental studies. Aim of this study was to determine the effect of test chemical on the population rate of Saccharomyces cerevisiae. Test conducted under the static system for 24 hrs. 25 ml of sterile synthetic media2 were inoculated from a stock culture slant and shaken for 6 to 7 hr at 30 C. After centrifugation and washing, the cells were suspended in fresh sterile media (1.5 X 105cells/ml) and were grown with shaking to a concentration of 1.0 x 107cells/ml (9.5 to 10.5 hrs), at which time aliquots of the culture were transferred into smaller flasks containing the chemical solutions to be tested. After being shaken for 4 hr, aliquots of the culture were removed for mas and count determinations. Culture mass was determined by measurement of turbidity or of dry weights (24 hrs at 105 C). Erlenmeyer flasks were used in the study. After the exposure of test chemical with Saccharomyces cerevisiae for 24 hrs, 50 % cell growth inhibition were observed. The IC50 was determine at 4000 mg/l.

 

Similarly in the third supporting study from secondary source, Study was conducted to evaluate the effect of test chemical on the Proliferation ratio of Tetrahymena pyriformis (Ciliate). Test conducted under the static system for 60 hrs. Based on the inhibition of proliferation rate of test organism Tetrahymena pyriformis (Ciliate) by the chemical exposure for 60 hrs, the EC50 was determine to be 66.34 mg/l.