3-aminophenol

Endpoint summary

Description of key information

Short term toxicity to fish:

Study was conducted to assess the effect of test chemical 3-aminophenol on the growth of fish Danio rerio. Test conducted according to OECD Guideline 203 (Fish, Acute Toxicity Test). The nominal concentration selected for the experiment were 68.30 mg/L, 75.13 mg/L, 82.64 mg/L, 90.90 mg/L & 100 mg/L and Zebra Fish Danio rerio were exposed to these concentration for 96 hours. The test substance was soluble in water. Therefore, the test solution was prepared by dissolving 683.0 mg, 751.3 mg, 826.4 mg, 909.9 mg & 1 gm of the test substance in 10 liters of potable water (passed through reverse osmosis system) with continuous stirring for achieving test concentrations of 68.30 mg/L, 75.13 mg/L, 82.64 mg/L, 90.90 mg/L & 100 mg/L, respectively. Bowl aquaria containing 10 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. Based on nominal concentrations, experimental median lethal Concentrations [LC-50 (96 h)] for 3-aminophenol on Zebra Fish Danio rerio was determine to be > 100 mg/l. Based on the LC50, it can be consider that the chemical 3-aminophenol was nontoxic and can be consider to be not classified as per the CLP classification criteria.

Long term toxicity to fish:

The test item was tested according to OECD 204 guideline, prolonged toxicity test in fish, and GLP. The test was conducted in flow-through conditions on the orange killifish (Oryzias latipes) during 21 days. The tested concentrations were 6.25, 12.5, 25.0, 50.0 and 100 mg/L. Analytical monitoring showed that measured concentrations were in the range of 89.7% to 105% of the nominal concentrations. Mortality, growth rate, other toxic symptoms and behaviour were evaluated. Non-Observed Effect Concentration for 21 days (NOEC) = 25.0 mg/L (based on nominal values). The highest concentration of the concentration groups at which no dead individual was observed was 50 mg/L, the highest concentration at which no toxic symptom was observed was 25.0 mg/L, the highest concentration at which no abnormality in food consumption status was found was 25.0 mg/L, and the highest concentration at which no significant difference was suggested compared with control group in the growth rate of test organisms was 50 mg/L. LC50 at day 7, 14, and 21 were all > 100 mg/L (nominal concentrations). All the validity criteria were met.

Short term toxicity to aquatic invertebrates:

The Acute Toxicity of the test item to Daphnia magna was determined in a Semi-Static 48-hour Immobilisation Test, according to OECD 202 guideline and following GLP. This study encompassed 6 treatment groups (5 dose rates of the test item, control) each containing 20 individuals. The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. 1.0, 0.45, 0.21, 0.093 and 0.042 mg test item/L, corresponding to geometric mean measured concentrations of 0.99, 0.387 and 0.164 mg test item/L (the two lowest concentrations were below LOQ) and a control were tested. Water temperature: 20 to 21 °C; pH value: 7.7 to 7.9; dissolved oxygen concentration: 8.1 to 9.3 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 810 - 1080 lux; and thus were within the ranges requested by guideline OECD 202. Additionnally all the validity criteria are fulfilled according the oecd 202 guideline. After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 0.164 mg test item/L. At the concentration of 0.387 mg test item/L, 16 animals were immobile and 20 animals were immobile at the concentration of 0.99 mg test item/L. The quantification of the test item was performed using liquid chromatography (HPLC-UV). In the both lowest test concentrations of nominal 0.042 and 0.093 mg test item/L all measured values are below the limit of quantification of the analytical method. In the freshly prepared test media 90% of the nominal test concentration was found (average of the nominal test concentrations of 0.21, 0.45 and 1.0 mg test item/L). In the 24 hours old test media 85% of the nominal value (average of the nominal test concentrations of 0.21, 0.45 and 1.0 mg test item/L) was determined. During the test the daphnids were exposed to a mean of 88% of nominal (average of the nominal test concentrations of 0.21, 0.45 and 1.0 mg test item/L). All reported biological endpoints refer to mean measured concentrations. For the concentration levels of 0.042 and 0.093 mg test item/L, which were below the limit of quantification (LOQ), 50% of the LOQ (= 0.01 mg test item/L) were introduced for the statisitical evaluation of the biological endpoints (as recommended in OECD No.23). Based on the effect concentration it was consider that the chemical was toxic to the mobility of daphnia magna and consider to be classified as aquatic acute 1 as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates:

21d-LC50 = 0.131 mg/L (95% confident interval: 0.112 – 0.157 mg/L)

21d-EC50% Reproduction Inhibition = 0.116 mg/L (95% confident interval: 0.0991 – 0.137 mg/L)

21d-NOECreproduction = 0.050 mg/L

21d-LOECreproduction = 0.10 mg/L

All of the above concentrations are the values based on nominal values

Toxicity to aquatic algae and cyanobacteria:

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test, according to OECD 201 guideline and EC method C.3, and GLP. Exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. This study encompassed 8 treatment groups (7 dose rates of the test item 100, 32, 10, 3.2, 1.0, 0.32 and 0.1 mg test item/L and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement. At the start of the test 104% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 100% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 102% of nominal. Therefore, all reported results refer to nominal concentrations. The 72-hour EyC50 was calculated to be 41.0 mg test item/L and the 72-hour ErC50 value was calculated to be 67.7 mg test item/L. The 72-hour NOEyC was determined to be 10.0 mg test item/L and the associated 72-hour LOEyC was 32 mg test item/L. The 72-hour NOErC was determined to be 10.0 mg test item/L and the associated 72-hour LOErC was 32 mg test item/L. All the validity criteria were fulfilled.

Toxicity to microorganisms: 

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to the OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted July 22, 2010. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. Concentrations of 10, 32, 100, 320 and 1000 mg test item/L and concentrations of 3.2, 10 and 32 mg 3,5-Dichlorophenol/L and six inoculum controls were used to determine the inhibition on total respiration and heterotrophic respiration (without nitrification) in a dose-response study. In comparison to the inoculum controls the total respiration rates of the activated sludge were moderately inhibited by 27.3% and 41.3% at the test concentrations of 10 and 32 mg/L. For the concentrations of 100 mg/L and 320 mg/L the inhibition rates were 46.5% and 42.8%. The highest test concentration of 1000 mg/L resulted in an inhibition of 48.7%. Concentrations exceeding 1000 mg/L nominal were not tested. In comparison to the inoculum controls the heterotrophic respiration (respiration without nitrification) was not inhibited at concentrations of 10 mg/L and 32 mg/L. For the test item concentration of 100 mg/L and 320 mg/L the inhibition was 4.7% and 7.3%. At the concentration of 1000 mg/L the respiration rate was moderately inhibited by 18.7%. Test item has a strong inhibiting effect on nitrifying bacteria. At a concentration of 10 mg/L the inhibition was 65.3%. For the concentrations of 32 and 100 mg/L the inhibition rates were 97.5% and 98.3%. For 320 and 1000 mg/L the inhibition rates were 86.8% and 86.0%. The 3-hour EC50 for total respiration was calculated to be 961.9 mg/L. For heterotrophic respiration the 3-hour EC10 was 406.4 mg/L. For the oxygen uptake due to nitrification the 3-hour EC50 could not be calculated. The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration, heterotrophic respiration and for the oxygen uptake due to nitrification was in the range recommended by the test guidelines and confirms the suitability of the activated sludge used.

Additional information

Summarized result of toxicity of the chemical 3-aminophenol 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 of3-aminophenolon the mortality rate and behavior of fish. The studies are as mentioned below:

 

In the first key study from experimental report 2014 toxicity was measured. Study was conducted to assess 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 683.0 mg, 751.3 mg, 826.4 mg, 909.9 mg & 1 gm of the test substance in 10 liters of potable water (passed through reverse osmosis system) with continuous stirring for achieving test concentrations of 68.30 mg/L, 75.13 mg/L, 82.64 mg/L, 90.90 mg/L & 100 mg/L, respectively 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 > 100 mg/l and LC0 was observed at 100 mg/l. Based on the LC50, it can be consider that the chemical was nontoxic and can be consider to be not classified as per the CLP classification criteria.

 

First key study was supported by the second study from authoritative database 2018. Short term toxicity study of test chemical were carried out on the mortality of Oryzias latipes. Test conducted in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test). Chemical analytically monitorized by HPLC at the start of exposure, before renewal of test solution. Test performed under the semi-static system for 96 hrs. 300, 200, 133, 88.9, 59.3 mg/L (geometric ratio 1.5), control concentration is used. 10 Oryzias latipes per vessel incorporated and conducted in 2 replicates. After 96 hrs effect were calculated on thew basis of mortality of test organism. The lethal concentration (LC50) value of 3-aminophenol in fish (Oryzias latipes) in a 96 hr study was determine to be 121 mg/L.

 

Similar the primary purpose of the present study (from peer reviewed journal, 2004) was to investigate the effect of test chemical on the growth of fishes. The eggs of Zebrafish were transferred into wells of a 24-well multiplate (1 egg per well) after differentiation. 20 wells were prepared with 2 mL test solution each, 4 wells were filled with dilution water and served as internal controls. The tests were replicated 3 times consecutively and 7 concentrations were tested. The tests were only considered valid when at least 90% of the embryos in the control were viable after 72hr. the eggs were incubated at 26°C and embryonic development was observed with an inverted microscope and recorded daily. 20 wells were prepared with 2 mL test solution each, 4 wells were filled with dilution water and served as internal controls. Eggs of the fish were obtained according to the method prescribed by OECD. The eggs were collected in 30 min and washed with tap water. A suitable number of eggs were transferred into prepared chemical concentrations withour regard for fertilisation. The lethal concentration (LC50) value of test chemical in eggs of fish (Danio rerio) in a 72 hr study was determine to be 22.34 mg/L. This value indicates that chemical will exhibit aquatic toxicity to fish.Thus classified in aquatic chronic category 3 based on the CLP criteria for classification of the substance.

 

As the maximum experimental studies performed according to the OECD guidelines supports the nontoxic classifications of test chemical thus on basis chemical 3-aminophenol was consider as nontoxic to fishes and not classified as per the CLP classification criteria.

 

Long term toxicity to fish:

The test item was tested according to OECD 204 guideline, prolonged toxicity test in fish, and GLP. The test was conducted in flow-through conditions on the orange killifish (Oryzias latipes) during 21 days. The tested concentrations were 6.25, 12.5, 25.0, 50.0 and 100 mg/L. Analytical monitoring showed that measured concentrations were in the range of 89.7% to 105% of the nominal concentrations. Mortality, growth rate, other toxic symptoms and behaviour were evaluated. Non-Observed Effect Concentration for 21 days (NOEC) = 25.0 mg/L (based on nominal values). The highest concentration of the concentration groups at which no dead individual was observed was 50 mg/L, the highest concentration at which no toxic symptom was observed was 25.0 mg/L, the highest concentration at which no abnormality in food consumption status was found was 25.0 mg/L, and the highest concentration at which no significant difference was suggested compared with control group in the growth rate of test organisms was 50 mg/L. LC50 at day 7, 14, and 21 were all > 100 mg/L (nominal concentrations). All the validity criteria were met.

 

Short term toxicity to aquatic invertebrates:

Based on the experimental data from various database for the target chemical study have been reviewed to determine the mode of action of 3-aminophenol on the mobility rate and behavior of aquatic invertebrates. The studies are as mentioned below:

 

In the first key study from experimental report 2015. The Acute Toxicity of the test item to Daphnia magna was determined in a Semi-Static 48-hour Immobilisation Test, according to OECD 202 guideline and following GLP. This study encompassed 6 treatment groups (5 dose rates of the test item, control) each containing 20 individuals. The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. 1.0, 0.45, 0.21, 0.093 and 0.042 mg test item/L, corresponding to geometric mean measured concentrations of 0.99, 0.387 and 0.164 mg test item/L (the two lowest concentrations were below LOQ) and a control were tested. Water temperature: 20 to 21 °C; pH value: 7.7 to 7.9; dissolved oxygen concentration: 8.1 to 9.3 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 810 - 1080 lux; and thus were within the ranges requested by guideline OECD 202. Additionnally all the validity criteria are fulfilled according the oecd 202 guideline. After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item concentration of 0.164 mg test item/L. At the concentration of 0.387 mg test item/L, 16 animals were immobile and 20 animals were immobile at the concentration of 0.99 mg test item/L. The quantification of the test item was performed using liquid chromatography (HPLC-UV). In the both lowest test concentrations of nominal 0.042 and 0.093 mg test item/L all measured values are below the limit of quantification of the analytical method. In the freshly prepared test media 90% of the nominal test concentration was found (average of the nominal test concentrations of 0.21, 0.45 and 1.0 mg test item/L). In the 24 hours old test media 85% of the nominal value (average of the nominal test concentrations of 0.21, 0.45 and 1.0 mg test item/L) was determined. During the test the daphnids were exposed to a mean of 88% of nominal (average of the nominal test concentrations of 0.21, 0.45 and 1.0 mg test item/L). All reported biological endpoints refer to mean measured concentrations. For the concentration levels of 0.042 and 0.093 mg test item/L, which were below the limit of quantification (LOQ), 50% of the LOQ (= 0.01 mg test item/L) were introduced for the statisitical evaluation of the biological endpoints (as recommended in OECD No.23). Based on the effect concentration it was consider that the chemical was toxic to the mobility of daphnia magna and consider to be classified as aquatic acute 1 as per the CLP classification criteria.

 

First key study was supported by the second study from authoritative database 2018. Acute Immobilization effect of test chemical to Daphnia magna was studied in accordance with the OECD Guideline 202 (Daphnia sp., Acute Immobilisation on 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 m-Aminophenol, the EC50 for 48hrs of exposure was 0.447 mg/l. Based on the EC50, it can be concluded that the chemical was toxic and be consider to be classified in aquatic acute category 1.

 

 

Similarly damaging effects of environmental chemicals (from peer reviewed journal 1989) in the Daphnia test as a basis for evaluation of the environment hazard in aquatic systems was measured. The effect of potential pollutants on the ability of Daphnia magna Straus to swim was carried out in accordance with the DIN 38412, Part II. Test conducted under the static system. From the solutions with the pollutants, dilution series were prepared using "dilution water". The concentration steps of the test solution were selected so as to give 3--4 EC values in a range between EC 0 and EC 100. Effect measured on the 6-24 hours old daphnia magna Straus which were not feed during the time of testing. 50 ml beakers and two 50 ml beakers with ground-glass stoppers were used. Loading amounted to one test animal per 2 ml test medium as ten 6-24 h old daphnids were placed in each test and control vessel, i.e. 20 animals per concentration step. Based on the mobility of daphnia magna by the exposure of chemical 3-aminophenol, in the interval of 24hrs and 48 hrs effect were measured. After 24 and 48 hrs effect (EC50) was observed at 2.2 mg/l and 1.1 mg/l. based on the EC50, chemical was consider as toxic to aquatic environment and classified as aquatic chronic 2 as per the CLP classification criteria.

 

As the maximum experimental studies performed according to the OECD guidelines supports the toxic and hazardous nature of test chemical thus on basis chemical 3-aminophenol was consider as toxic to aquatic invertebrates and classified as aquatic acute 1 as per the CLP classification criteria.

 

Long term toxicity to aquatic invertebrates:

 

The purpose of the study was to determine the long term effect over 21 days of the test item on Daphnia magna, according to the OECD 202 guideline (1984) and following GLP. Forty daphnids (10 animals per replicate, 4 replicates), were used per concentration level and control. At test start the daphnids were ared 24 hours old or less. The study was carried out under flow-through conditions. Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the first appearance of living juveniles, occurrence of resting eggs, adult mortality, size and conditions of parental animals. Nominal concentrations of the test item were as follows: 0.40 – 0.20 – 0.10 – 0.050 – 0.0250 mg/L. The test item concentrations were analytically verified via HPLC (High Performance Liquid Chromatography) on days 0, 3, 7, 14 and 21. The measured values of test substance concentration during exposure period were 0.0202 – 0.454 mg/L (nominal value 0.0250 – 0.40 mg/L), which were in the range 74.1 – 120% of the nominal concentrations. The environmental conditions were within the acceptable limits. The validity criteria of the OECD 202 test guideline were met. According to the effect chemical was consider as toxic and classified as aquatic acute 1/ chronic 1 category as per the CLP classification criteria.

21d-LC50 = 0.131 mg/L (95% confident interval: 0.112 – 0.157 mg/L)

21d-EC50% Reproduction Inhibition = 0.116 mg/L (95% confident interval: 0.0991 – 0.137 mg/L)

21d-NOECreproduction = 0.050 mg/L

21d-LOECreproduction = 0.10 mg/L

All of the above concentrations are the values based on nominal values.

 

 

Long term toxicity study of test chemical were reviewed by test conducting according to OECD Guideline 202 (Daphnia sp., Acute Immobilisation Test and Reproduction Test). Test conducted under the flow-through method. Chemical analytically monitorized by HPLC with the frequency of 0th, 3rd, 7th, 14th, 21st day. 40 per concentration (10 per vessel) daphnia were incorporated. The effective concentration (EC50) value of test chemical based on the effect on reproduction in Daphnia magna in a 21 day study was determine to be 0.116 mg/L and the NOEC value was observed to be 0.05 mg/L. Thus based on the long term effects, it was concluded that the chemical was toxic and classified as aquatic chronic 2 category as per the CLP classification criteria.

 

Thus chemical consider to be toxic

 

Toxicity to aquatic algae and cyanobacteria:

 

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 on the growth rate, biomass and behavioral changes of aquatic algae and cyanobacteria. The studies are as mentioned below:

 

In the first key study from experimental report 2015. The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test, according to OECD 201 guideline and EC method C.3, and GLP. Exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. This study encompassed 8 treatment groups (7 dose rates of the test item 100, 32, 10, 3.2, 1.0, 0.32 and 0.1 mg test item/L and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrically measurement. At the start of the test 104% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 100% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 102% of nominal. Therefore, all reported results refer to nominal concentrations. The 72-hour EyC50 was calculated to be 41.0 mg test item/L and the 72-hour ErC50 value was calculated to be 67.7 mg test item/L. The 72-hour NOEyC was determined to be 10.0 mg test item/L and the associated 72-hour LOEyC was 32 mg test item/L. The 72-hour NOErC was determined to be 10.0 mg test item/L and the associated 72-hour LOErC was 32 mg test item/L. All the validity criteria were fulfilled.

 

First key study was supported by the second study from authoritative database 2018. Short term toxicity to green algae study was carried out for 72 hrs. The study was based on the effects of the test compound on green algae in a static fresh water system. Based on effect on growth rate of the test organism green algae, the 72 hr EC50 and NOEC value was determined to be 160 and 25 mg/l, and on the basis of biomass and area under the growth curve, the 72 hr EC50 and NOEC value was determined to be 62 and 25 mg/l, respectively. Thus, based on the EC50 value (62 mg/l), it can be concluded that the substance 3 -aminophenol can be considered as toxic to aquatic organisms and thus can be considered to be classified as aquatic chronic 3 as per the CLP classification criteria.

 

Based on the overall studies for the toxicity of test chemical on the growth of aquatic algae, chemical was consider as toxic and classified as aquatic chronic 3 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 3-aminophenol on the growth rate, and mortality of microorganisms. The studies are as mentioned below:

 

In the first key study from experimental report 2015. The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions according to the OECD Guideline for Testing of Chemicals, No. 209: "Activated Sludge, Respiration Inhibition Test", adopted July 22, 2010. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours. Concentrations of 10, 32, 100, 320 and 1000 mg test item/L and concentrations of 3.2, 10 and 32 mg 3,5-Dichlorophenol/L and six inoculum controls were used to determine the inhibition on total respiration and heterotrophic respiration (without nitrification) in a dose-response study. In comparison to the inoculum controls the total respiration rates of the activated sludge were moderately inhibited by 27.3% and 41.3% at the test concentrations of 10 and 32 mg/L. For the concentrations of 100 mg/L and 320 mg/L the inhibition rates were 46.5% and 42.8%. The highest test concentration of 1000 mg/L resulted in an inhibition of 48.7%. Concentrations exceeding 1000 mg/L nominal were not tested. In comparison to the inoculum controls the heterotrophic respiration (respiration without nitrification) was not inhibited at concentrations of 10 mg/L and 32 mg/L. For the test item concentration of 100 mg/L and 320 mg/L the inhibition was 4.7% and 7.3%. At the concentration of 1000 mg/L the respiration rate was moderately inhibited by 18.7%. Test item has a strong inhibiting effect on nitrifying bacteria. At a concentration of 10 mg/L the inhibition was 65.3%. For the concentrations of 32 and 100 mg/L the inhibition rates were 97.5% and 98.3%. For 320 and 1000 mg/L the inhibition rates were 86.8% and 86.0%. The 3-hour EC50 for total respiration was calculated to be 961.9 mg/L. For heterotrophic respiration the 3-hour EC10 was 406.4 mg/L. For the oxygen uptake due to nitrification the 3-hour EC50 could not be calculated. The positive control 3,5-Dichlorophenol was tested in the same way as the test item. The 3-hour EC50 for total respiration, heterotrophic respiration and for the oxygen uptake due to nitrification was in the range recommended by the test guidelines and confirms the suitability of the activated sludge used.

 

 

Similarly study from peer reviewed journal 1994, toxicity study was conducted to determine the effect of test chemical on the growth of microorganisms. Test conducted under static system in which Escherichia coli exposed for 12 hrs with the test chemical. After the 12 hhrs of exposure the Effective concentration (EC50) value of test chemical to Escherichia coli was determine to be 4054.5 mg/l on the basis of growth rate inhibition.

 

 

Similar study was conducted to determine the rate of toxicological effect of test chemical on the strain of microorganisms E.coli ATCC 11775 for 45 minutes. The strain of E.coli used was purchased from the American Type Culture Collection. Stock cultures were maintained on agar slants at room temperature. For culture broth dextrose-salts-casaminoacids-(vitamin free)-medium was used. From agar cultures the culture broth was inoculated and allowed to grow for 12 to 16 h at 37 °C. Test cultures were prepared by dilution with culture medium (104 cells/ml) and 150 ml portions were transferred to 1 liter Erlenmeyer culture flasks. After 0.5 h the toxicant was added. Samples of the incubated cultures (37 °C) were taken every 45 minutes and the number of bacteria was determined with a Coulter Counter. Therefor samples were diluted with particle free saline-(0.85%)-formaldehyde-(2%)-solutions to obtain counts of 1000 to 10000 organisms. The Coulter Counter Model ZB was equipped with a 0.030 mm orifice obtaining counts for 0.050 ml. After the exposure of test chemical for 45 minutes effect on the bacterial population were count. Based on the effect the I50 was determine to be 4070.176 mg/l.

 

 

Above key study was supported by the second study from authoritative database 2018. Toxicological study was conducted to determine the nature and effect of test chemical when comes in contact with the Tetrahymena Thermophile. Test performed for the total exposure period of 48 hrs. Based on the effect observed on the Population growth rate inhibition of Tetrahymena thermophile (Ciliate Protozoa) during the exposure period of 48 hrs, the EC50 was determine to be 2.55 mg/l. Based on the effect observed on the microorganisms, chemical was consider as toxic.

 

Similarly in the fifth study from authoritative database 2018 toxicity was measured. Toxicological study was conducted to determine the nature and effect of test chemical when comes in contact with the Tetrahymena Thermophile. Test performed for the total exposure period of 48 hrs. Based on the effect observed on the Population growth rate inhibition of Tetrahymena thermophile (Ciliate Protozoa) during the exposure period of 48 hrs, the EC50 was determine to be 2.9 mg/l. Based on the effect observed on the microorganisms, chemical was consider as toxic.

 

Sixth study from secondary sources supports the previous 2 and 3rd study. Aim of this study was to investigate the effect of test chemical on the growth and population effect of Tetrahymena Thermophile by providing the incubation period of 2 days. Based on the effect observed on the Population growth rate inhibition of Tetrahymena thermophile (Ciliate Protozoa) during the exposure period of 48 hrs, the EC10 was determine to be 0.7 mg/l and EC20 was observed in the range of 1.25mg/l – 1.5 mg/l. Based on the effect observed on the microorganisms, chemical was consider as toxic.