2-amino-4-nitrophenol

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 62.5 mg, 125 mg, 250 mg, 500 mg & 1 g of the test substance in 10 liters of potable water passed through reverse osmosis system with continuous stirring 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 three 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 to various nominal test concentrations, LC50 was determine to be >50 mg/l LC100 was observed at the concentration 100 mg/l . Based on the LC50, it can be consider that the chemical was toxic to fish and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

The evaluation of acute lethal toxicity of test material on Daphnia pulex was performed , test organism was obtained from Plant ToxicologyDepartment, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, TaiwanBefore the experiment, the test organisms were acclimatized in aquaria for 2 weeks. Twenty daphnia, within 24 hr after hatching, were introduced in each 250-mL beaker containing100 mL of test chemical solution with different concentrations. Six different concentrations, 0, 50, 100, 200, 500, and 1,000 mg/l . Mortality of daphnia was observed after an incubation period of 4 h. Sub lethal concentration (LC50) values were calculated by the trimmed Spearman Karber method.The median lethal concentration LC50 was observed to be 0.039 mg/l. Based on the above effect concentration it can considered that test material is highly toxic to aquatic invertebrates.Based on the EC50, it can be concluded that the test chemical was toxic and be consider to be classified in aquatic acute category 1.

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 substance was prepared by adding 75 mg of test substance in 150 ml of BBM to get the final concentration of 500 mg/L. The whole system was warmed for 5 minutes at 30° C and kept for 30 minutes stirring to get the uniform dissolution. The remaining test solutions were prepared by dilution from the above stock solution. The test solutions were prepared in aseptic condition. 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 90.98 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was toxic to aquatic algae and can be consider to be as aquatic chronic 3 as per the CLP classification criteria.

Toxicity to microorganism:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical .The studies are as mentioned below:

Acute toxicity of test material was evaluated for microorganisms, the test material showed no effect on Pseudomonas putida when tested for 16 h at concentration 7 mg/l.

In another study , the effect of test material was evaluated on microorganism Photobacterium phosphoreum for 30 min inhibition of bioluminance test  the EC50 was obseved to be 29 mg/l. The test chemical  is  likely to be toxic to microorganism atleast in the concentration  range of 7 - 29 mg/l

Additional information

Short term toxicity to fish:

The toxicity of test material for fish was evaluated using data from lab report along with a publication.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 62.5 mg, 125 mg, 250 mg, 500 mg & 1 g of the test substance in 10 liters of potable water passed through reverse osmosis system with continuous stirring 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 three 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 to various nominal test concentrations, LC50 was determine to be >50 mg/l LC100 was observed at the concentration 100 mg/l . Based on the LC50, it can be consider that the chemical was toxic to fish and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

In a peer reviewed journal ,short term toxicity to fish was evaluated for the test chemical for 48 h . Carp(Ciprinus carpio) and was provided by Lu-Kan Fish Culture Station, Taiwan Fisheries Research Institute, Taichung, Taiwan. Before the experiment, the test organisms were acclimatized in aquaria for 2 weeks under conditions similar to those under which the tests was performed. Four carp , 2 to 6 cm in size, were introduced to each 10-L beaker containing5 L of different concentrations of test chemical . Five different concentrations 0.1 , 0.5 , 1 , 2 and 4 mg/l were prepared. Sub-lethal concentration (LC50) values were calculated by the trimmed Spearman} act concentration EC50 of test material on fish when observed after 48 h was noted to be 0.453 mg/l. Based on the above effect concentration it can be concluded that the test material is highly toxic to fish and can be classified as aquatic acute 1.

As we have two studies which are classifying test material in two diffrent categories , hence we will consider effect concentration obtained from lab report as it has higher relaibility then that of data from publication .Based on the LC50, it can be consider that the chemical was toxic to fish and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate:

Toxicity to aquatic invertebrate for the test material was described based on the data in peer reviewed journal and data from structurally similar read across substances.The evaluation of acute lethal toxicity of test material on Daphnia pulex was performed , test organism was obtained from Plant ToxicologyDepartment, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, TaiwanBefore the experiment, the test organisms were acclimatized in aquaria for 2 weeks. Twenty daphnia, within 24 hr after hatching, were introduced in each 250-mL beaker containing100 mL of test chemical solution with different concentrations. Six different concentrations, 0, 50, 100, 200, 500, and 1,000 mg/l . Mortality of daphnia was observed after an incubation period of 4 h. Sub lethal concentration (LC50) values were calculated by the trimmed Spearman Karber method.The median lethal concentration LC50 was observed to be 0.039 mg/l. Based on the above effect concentration it can considered that test material is highly toxic to aquatic invertebrates.

Another data for structurally similar read across substance was used to support the above data ,The evaluation of acute lethal toxicity of test material on Daphnia pulex was performed , test organism was obtained from Plant ToxicologyDepartment, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, TaiwanBefore the experiment, the test organisms were acclimatized in aquaria for 2 weeks. Twenty daphnia, within 24 hr after hatching, were introduced in each 250-mL beaker containing100 mL of test chemical solution with different concentrations. Six different concentrations, 0, 50, 100, 200, 500, and 1,000 mg/l . Mortality of daphnia was observed after an incubation period of 4 h. Sub lethal concentration (LC50) values were calculated by the trimmed Spearman Karber method.The median lethal concentration LC50 was observed to be 0.087 mg/l. Based on the above effect concentration it can considered that test material is highly toxic to aquatic invertebrates.

The above study was further supported by data of another structurally similar read across substance from an authoritative database, acute Immobilization effect of test material to Daphnia magna was studied in accordance with the OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test and Reproduction Test). Test conducted in the semi-static system for the total exposure period of 48hrs. Sample analyzed by the HPLC with the frequency of analysis at the start of exposure, before renewal of test solution. Toxicity measured at the nominal concentration 10, 4.0, 1.60, 0.640, 0.256, 0.102 mg/L (geometric ratio 2.5), control. 100 mL per vessel test solution volume was added, on 5 daphnia magna per vessel (20 per concentration) toxicity was checked. Results after 24-hour exposure at 50 % Effective concentration (EC50) for immobilization was 1.79 mg/L (95 % CL: 1.28 - 2.57 mg/L). After 48-hour exposure No observed effect concentration (NOEC) for immobilization was 0.256 mg/L, and Lowest concentration causing 100 % immobility at 1.60 mg/L. Based on the immobility of daphnia magna due to the exposure of chemical , the EC50 for 48hrs of exposure was 0.447 mg/l.

Based on the EC50, it can be concluded that the test chemical was toxic and be consider to be classified in aquatic acute category 1.

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 substance was prepared by adding 75 mg of test substance in 150 ml of BBM to get the final concentration of 500 mg/L. The whole system was warmed for 5 minutes at 30° C and kept for 30 minutes stirring to get the uniform dissolution. The remaining test solutions were prepared by dilution from the above stock solution. The test solutions were prepared in aseptic condition. 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 90.98 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was toxic to aquatic algae and can be consider to be as aquatic chronic 3 as per the CLP classification criteria.

Toxicity to microorganism:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of microorganism of the test chemical .The studies are as mentioned below:

Acute toxicity of test material was evaluated for microorganisms, the test material showed no effect on Pseudomonas putida when tested for 16 h at concentration 7 mg/l.

In another study , the effect of test material was evaluated on microorganism Photobacterium phosphoreum for 30 min inhibition of bioluminance test  the EC50 was obseved to be 29 mg/l. The test chemical  is  likely to be toxic to microorganism atleast in the concentration  range of 7 - 29 mg/l