3-nitrobenzoic acid

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 stock solution was prepared by dissolving 2 g of the test substance in 2 liters of potable water (passed through reverse osmosis system) with continuous 1 hour stirring. After stirring the stock was analytically detected and the solubility was found 937.84 mg/L.By considering this value calculations done for following concentrations 6.25mg/L,12.5mg/L,25mg/L,50mg/L,100mg/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 all nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

Determination of the inhibition of the mobility of daphnids was carried out with the substance . Test was performed according to OECD Guideline 202.

The test substance was tested at the concentrations 0, 6.2, 12.4, 25, 50, 100, 200 mg/l. The stock solution 200 mg/l was prepared by dissolving white powder in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water. Effects on immobilisation were observed for 48 hours. The median effective concentration (EC50) for the test substance, 3-Nitrobenzoic Acid in Daphnia magna was determined to be 71.3 mg/L for immobilisation effects. Tested substance is an acid and that is why the higher concentration the lower Ph. The optimum pH for daphnids is 6.0-6.9. The inhibition of the mobility of daphnids could be influenced by pH at the concentration 200 mg/l and that is why EC50 was calculated also for results without the concentration 200 mg/l with low pH. Adjustment of pH would change substance properties. The EC50 without 200 mg/l concentration of test chemical was 96.7 with 95% CI was 68.6-136.4 mg/l.

Toxicity to aquatic algae and cyanobacteria:

Freshwater algal growth inhibition test was carried out on Desmodesmus subspicatus with the substance for total exposure period of 72 hrs. Test conducted according to OECD Guideline 201. The stock solution 100 mg/l was prepared by dissolving white powder in OECD growth medium. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture and tested at the concentrations 0, 10, 18, 32, 58, 100  mg/L. Effects on the growth rate of the organism were studied by using positive reference substance Potassium dichromate (K2Cr2O7).

The median effective concentration (EC50) for Potassium dichromate in Desmodesmus subspicatus was determined to be 0.77 mg/L. The median effective concentration (EC50) for the test substance, in Desmodesmus subspicatus was determined to be 106.4 mg/L with 95% CI was 98.6 - 114.9 mg/l.

This value indicates that the substance test substance is likely to be non-hazardous to aquatic algae and cannot be classified as toxic and not classified as per the CLP criteria.

Toxicity to microorganism:

In different studies, the given test chemical, has been investigated for toxicity to microorganisms to a greater or lesser extent. The studies are summarized as below –

 

In the first key studyevaluation of toxicity of test chemical on Staphylococcus aureus toxicity to micro-organisms test was carried for 24 h under static condition.An overnight culture of each organism S. aureus was prepared. The 0.1 ml of organism was taken into 9.9 ml of sterile distilled water (SDW) to give 10 ml of 1:100 (10 ) dilution. The stock was maintained on nutrient agar slant and sub-cultured in nutrient broth for incubation at 37 °C prior to each antimicrobial testing. Inoculation of the test organisms on nutrient agar-prepared plates was achieved by flaming a wire loop on a spirit lamp, cooling the wire loop (air cooling) and fetching the test organisms.The discs were prepared using a Grade No. 1 Whatman filter paper. One hundred discs were obtained by punching and putting in vial bottles and sterilizing in an oven at 150 °C for 15 min. Thereafter the cups (9 mm diameter) were aseptically bored into the solid nutrient agar using a sterile cork-borer. The test solution of Methyl salicylate was introduced .The plates were left at room temperature for 2 h, allowed to diffuse into the medium, turned upside-down and thereafter incubated at 37 °C for 24 h in an incubator. The minimum inhibition concentration (MIC) of test chemical on Staphylococcus aureus was observed to be 200mg/l.

First study was supported by the second study from peer reviewed journal. To evaluate antibacterial activity of  test chemical on Activated sludge toxicity to micro-organisms test was carried out according to the OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test. The test was carried for 3 h under static condition. Test chemicalwas used as a test material to evaluate toxicity to microorganisms as per OECD guideline 209. Activated sludge was used as the test culture at pH 7.5 for 3 h The EC50 value on the basis of respiration inhibition test was observed to be >1000 mg/l.

Thus based on the above effects chemical toxicity value ranges from 200 to > 1000 mg/l.

Additional 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 stock solution was prepared by dissolving 2 g of the test substance in 2 liters of potable water (passed through reverse osmosis system) with continuous 1 hour stirring. After stirring the stock was analytically detected and the solubility was found 937.84 mg/L.By considering this value calculations done for following concentrations 6.25mg/L,12.5mg/L,25mg/L,50mg/L,100mg/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 all nominal test concentrations, LC50 was determine to be >100 mg/l . Based on the LC50, it can be consider that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

Determination of the inhibition of the mobility of daphnids was carried out with the substance . Test was performed according to OECD Guideline 202.

The test substance was tested at the concentrations 0, 6.2, 12.4, 25, 50, 100, 200 mg/l. The stock solution 200 mg/l was prepared by dissolving white powder in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water. Effects on immobilisation were observed for 48 hours. The median effective concentration (EC50) for the test substance, 3-Nitrobenzoic Acid in Daphnia magna was determined to be 71.3 mg/L for immobilisation effects. Tested substance is an acid and that is why the higher concentration the lower Ph. The optimum pH for daphnids is 6.0-6.9. The inhibition of the mobility of daphnids could be influenced by pH at the concentration 200 mg/l and that is why EC50 was calculated also for results without the concentration 200 mg/l with low pH. Adjustment of pH would change substance properties. The EC50 without 200 mg/l concentration of test chemical was 96.7 with 95% CI was 68.6-136.4 mg/l.

Toxicity to aquatic algae and cyanobacteria:

The effect of test material was evaluated for aquatic algae and cyanobacteria based on results of two experimental report as described below:

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 was prepared in aseptic condition. The test item  was prepared by adding 50mg of test item in 250ml of BBM to get the final concentration of 200mg/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 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 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

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 335.805 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.

The above report was supported by another eperimental study which suggests , that 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 was prepared in aseptic condition. The test item  was prepared by adding 50mg of test item in 250ml of BBM to get the final concentration of 200mg/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 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 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

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, EC10 value was determined to be 9.05±5.93 graphically and through probit analysis.

Toxicity to microorganism:

In different studies, the given test chemical, has been investigated for toxicity to microorganisms to a greater or lesser extent. The studies are summarized as below –

 

In the first key studyevaluation of toxicity of test chemical on Staphylococcus aureus toxicity to micro-organisms test was carried for 24 h under static condition.An overnight culture of each organism S. aureus was prepared. The 0.1 ml of organism was taken into 9.9 ml of sterile distilled water (SDW) to give 10 ml of 1:100 (10 ) dilution. The stock was maintained on nutrient agar slant and sub-cultured in nutrient broth for incubation at 37 °C prior to each antimicrobial testing. Inoculation of the test organisms on nutrient agar-prepared plates was achieved by flaming a wire loop on a spirit lamp, cooling the wire loop (air cooling) and fetching the test organisms.The discs were prepared using a Grade No. 1 Whatman filter paper. One hundred discs were obtained by punching and putting in vial bottles and sterilizing in an oven at 150 °C for 15 min. Thereafter the cups (9 mm diameter) were aseptically bored into the solid nutrient agar using a sterile cork-borer. The test solution of Methyl salicylate was introduced .The plates were left at room temperature for 2 h, allowed to diffuse into the medium, turned upside-down and thereafter incubated at 37 °C for 24 h in an incubator. The minimum inhibition concentration (MIC) of test chemical on Staphylococcus aureus was observed to be 200mg/l.

First study was supported by the second study from peer reviewed journal. To evaluate antibacterial activity of  test chemical on Activated sludge toxicity to micro-organisms test was carried out according to the OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test. The test was carried for 3 h under static condition. Test chemicalwas used as a test material to evaluate toxicity to microorganisms as per OECD guideline 209. Activated sludge was used as the test culture at pH 7.5 for 3 h The EC50 value on the basis of respiration inhibition test was observed to be >1000 mg/l.

Thus based on the above effects chemical toxicity value ranges from 200 to > 1000 mg/l.