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Description of key information

Short term toxicity to fish:

Acute toxicity of test chemical on fish Oryzias latipes were studied under the static system. Static system were used for the study. Fish acute toxicity was tested using Japanese medaka (Oryzias latipes) purchased from the National Institute for Environmental Studies and acclimated in the laboratory of the University of Tokushima at least for 3 months. The acute tests were conducted in conformity to the OECD guideline for testing chemicals no. 203. Duplicates of five juvenile fish were prepared in a 100 ml beaker for at least five concentrations and blanks (and solvent blanks for TCC). The test was conducted at 25 °C with a photoperiod of 16 h light and 8 h dark. The solution was replaced at 48 h and the concentrations of the tested antimicrobials were analyzed using the HPLC. The DO and pH of the solution were checked at 0, 48, and 96 h. The number of dead individuals was counted at 96 h and the median lethal concentration (LC50) was determined on the basis of geometrical mean of the measured concentrations of the tested antimicrobials using probit or logit conversion with Ecotox-Statics version 2.6d. The Lethal effect concentration (LC50) value of test chemical in fish in a 96 hr study on the basis of mortality effect was observed to be 0.085 mg/L. Thus based on the LC50 value, chemical consider to be toxic and classified as aquatic acute 1 category as per the CLP classification criteria.

Long term toxicity to fish:

35 days long term fish toxicity study was conducted to test the effects of test chemical on Pimephales promelas. The No observed effect concentration (NOEC) value of test chemical in 35 day study in Pimephales promelas was observed to be 0.005 mg/L on basis of effect on Hatchability of eggs, growth, and survival.

Short term toxicity to aquatic invertebrate:

Aim of this first study was to assess the short term toxicity of test chemical to aquatic invertebrate daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs. The stock solution 1 g/l was prepared by dissolving white powder in acetone. Test solutions of required concentration as were prepared by mixing the stock solution of the test sample with reconstituted test water. 0, 0, 0.0025, 0.0050, 0.0100, 0.0200, 0.0400 mg/l concentrations were used in the study. Effects on immobilisation were observed for 48 hours. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. The median effective concentration (EC50) for the test substance, in Daphnia magna was determined to be 0.0055 mg/L on the basis of immobility of daphnia magna in a 48 hour study. Based on the EC50 value, substance is likely to be hazardous to aquatic invertebrate and can be classified in aquatic acute 1 category as per the CLP classification criteria.

Long term toxicity to aquatic invertebrates:

Long term toxicity of test chemical were studied and determine on the reproduction of daphnia magna. The LOEC was observed to be 0.0047 mg/L for 21 days based on the effect on reproduction.

Toxicity to algae and cyanobacteria:

Objective of this study was to observe the action of test chemical when it exposed with the Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) for 72 hrs. Test conducted in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test). The stock solution 1 g/l was prepared by dissolving white powder in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test conducted on various nominal concentration of test chemical is 0, 0.0025, 0.0050, 0.0100, 0.0200, 0.0400, 0.0800, 0.1600 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration ErC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical, the ErC50 was determine to be 0.0344 mg/l. Thus on the basis of ErC50 value, chemical concluded to be toxic and can be consider to be classified in aquatic acute category 1 as per the CLP classification criteria.

Toxicity to microorganisms:

The MIC values were determined by the microbroth dilution assay according to the National Committee for Clinical Laboratory Standards methods for antimicrobial susceptibility tests for aerobically growing bacteria (Approved Standard M7-A2, NCCLS, Villanova, PA, 1990).

Approximately 6.5 3 108 cells of strain RN4220 (25) were plated on to brain-heart infusion agar medium (Becton-Dickinson) containing 0.5 mg/ml test material. The plates were incubated at 37 °C with periodic monitoring. After 48 h, resistant clones were picked and their phenotype confirmed by regrowth on the original selective concentration of test material.

Three strains of Staphylococcus aureus namely RN4220 (wild-type) , RN4220/pTri-1 , GR664 (fabIG23S) and three starins of E.coli

W3110 (wild-type) , W3110/pFabI , RJH108 (fabIG93V) . The wild-type FabI proteins from either S. aureus or E. coli were expressed using plasmids pTri-1 or pFabI, respectively, to determine if resistance was conferred by the presence of multiple copies of the fabI genes.

The minimum ihhibition concentration (MIC) was observed in the range of 0.25 to 2 mg/l in case of Staphylococcus aureus and for E.Coli MIC was observed in concentration range of 0.5 - 35 mg/l.

Additional information

Summarized result for the toxicity of test chemical on the growth and mortality of aquatic life’s including fish, invertebrates, algae and microorganism were studied and are as mention below:

Short term toxicity to fish:

Based on the short term toxicity study available for the test chemical, studies has were reviewed to determine the toxic nature of test chemical on the mortality of fish. The studies are as mentioned below:

Acute toxicity of test chemical on fish Oryzias latipes were studied under the static system. Static system were used for the study. Fish acute toxicity was tested using Japanese medaka (Oryzias latipes) purchased from the National Institute for Environmental Studies and acclimated in the laboratory of the University of Tokushima at least for 3 months. The acute tests were conducted in conformity to the OECD guideline for testing chemicals no. 203. Duplicates of five juvenile fish were prepared in a 100 ml beaker for at least five concentrations and blanks (and solvent blanks for TCC). The test was conducted at 25 °C with a photoperiod of 16 h light and 8 h dark. The solution was replaced at 48 h and the concentrations of the tested antimicrobials were analyzed using the HPLC. The DO and pH of the solution were checked at 0, 48, and 96 h. The number of dead individuals was counted at 96 h and the median lethal concentration (LC50) was determined on the basis of geometrical mean of the measured concentrations of the tested antimicrobials using probit or logit conversion with Ecotox-Statics version 2.6d. The Lethal effect concentration (LC50) value of test chemical in fish in a 96 hr study on the basis of mortality effect was observed to be 0.085 mg/L. Thus based on the LC50 value, chemical consider to be toxic and classified as aquatic acute 1 category as per the CLP classification criteria.

 

First study was supported by the second and third study from peer reviewed journal. The exposed adult fathead minnows (Pimephales promelas) for 48 hours to test chemical were studied. Chemicals were added to the conditioned water after water exchanges (100% static renewal). The second day of exposure, fish were removed from the tanks and placed in beakers that were filled with the tank’s water and with air stones. Then, tanks were emptied, and filled with new conditioned water. Chemicals were added to the conditioned water and fish were placed again in the tanks. This was done in order to maintain constant chemical concentrations in the media during the 48 hours of exposure. Three water samples were collected from each tank in pre-rinsed 40 mL amber glass vials with Teflon septa. The first and second water samples (day 0 and day 1) were taken 5 min after water exchange in order to verify that our stock solution delivered to the renewed tanks provided for similar initial conditions for each 24 h period. The last water sample (day 2, no water exchange) was taken 5 min before fish were removed to estimate the degradation rate using a firstorder decay. All water samples were stored at 4C for later chemical analyses by the USEPA Region 8 Laboratory. Samples arrived at the USEPA within 72 h and upon arrival were stored at -20°C until they were extracted at 96 h. The No Observed effect concentration (NOEC) value of test chemical in fish in a 48 hr study on the basis of mortality effect was determine to be 0.0014 mg/L.

 

 

 

96 hr short term toxicity study was conducted to determine the lethal effect of test chemical in Oncorhynchus mykiss. The Lethal effect concentration (LC50) value of test chemical in Oncorhynchus mykiss in a 96 hr study on the basis of mortality effect was determine to be 0.18 mg/L. Thus based on the LC50 value, chemical consider to be toxic and consider to be classified as aquatic acute 1 as per the CLP classification criteria.

 

In the fourth study from authoritative database short term toxicity of test chemical were studied on fish Ictalurus punctatus. Fish exposed with the chemical for 48 hrs and performed under the static system. Based on the mortality of test fish Ictalurus punctatus by the chemical after the exposure period of 48 hrs, the LC50 was determine to be 0.14 mg/l. Thus based on the LC50 value, it is concluded that the substance will exhibit toxicity to fish and classified in aquatic acute 1 category.

 

 

Similar study was conducted to determine the effect of test chemical on fish Pimephales promelas. After the exposure period of 96 hr study of test chemical on Pimephales promelas, no effect were observed (NOEC) at 0.054 mg/l on basis of effect on behavior of fish.

 

Thus based on the overall studies, chemical consider to be toxic and classified in aquatic acute 1 category as per the CLP classification criteria.

 

Long term toxicity to fish:

Various long term studies available for the test chemical were reviewed to determine the toxic nature of test chemical on the mortality of fish. The studies are as mentioned below:

35 days long term fish toxicity study was conducted to test the effects of test chemical on Pimephales promelas. The No observed effect concentration (NOEC) value of test chemical in 35 day study in Pimephales promelas was observed to be 0.005 mg/L on basis of effect on Hatchability of eggs, growth, and survival.

First study was supported by the second study. Long term fish toxicity study was conducted to test the effects of test chemical on danio rerio larvae in 9 days. At least three replicates of 15 embryos were placed in 50 ml beaker for five concentrations. The solutions were replaced every 48 h (i.e. a semi-static system) and the concentration of the selected antimicrobials was monitored using the HPLC as presented above. The DO and pH were also monitored at the replacement of the solutions. The percentage hatching and the survival rate of the larvae were evaluated until the egg yolk had disappeared. The No observed effect concentration (NOEC) value of test chemical in 9 day study in Danio rerio larvae was observed to be 0.024 mg/L on basis of effect on hatching and the survival of larvae.

 

Aim of this study was to evaluate the effect of test chemical on mortality of Pimephales promelas. Test conducted under the flow-through system. The Low  observed effect level (LOEL) value of test chemical in 30 day study in Pimephales promelas on basis of effect on mortality was determine to be 0.01 mg/L.

 

Similar 35 days long term fish toxicity study was conducted to test the effects of test chemical on pimpheles promelas. No treatment related effects were observed at the used concentrations. The No observed effect concentration (NOEC) of test chemical in 35 day study in Pimephales promelas on basis of effect on egg hatchability or growth of the fry was observed to be 0.005 mg/L.

 

Thus based on the above results, chemical consider to be toxic and classified in aquatic chronic category 2 as per the CLP classification criteria.

 

Short term toxicity to aquatic invertebrate:

Summarized result for the evaluation of toxicity of test chemical on the mobility of aquatic invertebrate were studied and mention as below:

 

Aim of this first study was to assess the short term toxicity of test chemical to aquatic invertebrate daphnia magna. Study was performed according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) in a static system for the total exposure period of 48 hrs. The stock solution 1 g/l was prepared by dissolving white powder in acetone. Test solutions of required concentration as were prepared by mixing the stock solution of the test sample with reconstituted test water. 0, 0, 0.0025, 0.0050, 0.0100, 0.0200, 0.0400 mg/l concentrations were used in the study. Effects on immobilisation were observed for 48 hours. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. The median effective concentration (EC50) for the test substance, in Daphnia magna was determined to be 0.0055 mg/L on the basis of immobility of daphnia magna in a 48 hour study. Based on the EC50 value, substance is likely to be hazardous to aquatic invertebrate and can be classified in aquatic acute 1 category as per the CLP classification criteria.

 

Similar acute toxicity of test chemical on Daphnia magna were studied for 48 hrs. Test conducted under the static system. 3 months old daphnia magna were used. The effective concentration (EC50) value of test chemical on (Daphnia Magna) in a 48 hr study based on mortality was estimated to be 0.01 mg/L. Thus based on the EC50 value, chemical consider to be toxic and classified as aquatic acute 1 as per the CLP classification criteria.

 

Study was conducted to determine the effect of test chemical on the developmental changes on embryos of the hard clam Mercenaria mercenaria for 48 hrs. Test conducted under the static system. The EC50 value of test chemical on clam Mercenaria mercenaria in a 48 hr study based on developmental changes was observed to be 0.032 mg/L. Thus on the basis of EC50 value, chemical consider to be toxic and classified as aquatic acute 1 as per the CLP classification criteria.

 

Above studies was supported by this study from authoritative database. Aim of this study was to evaluate the effect of test chemical on the mobility of Ceriodaphnia dubia. Test conducted under the static system on less than 24 hrs old Ceriodaphnia dubia for 48 hrs. The effective concentration (EC50) value of test chemical on Ceriodaphnia dubia in a 48 hr study based on Intoxication was determine to be 0.0031 mg/L. Thus chemical consider to be toxic and classified as aquatic acute category 1 category as per the CLP classification criteria.

 

Short term toxicity of test chemical on Crustaceans were studied. The LC50 value of chemical on (Daphnia Magna) in a 48 hr study based on mortality was observed to be 0.011 mg/L.

 

In the sixth study toxicity of test chemical on invertebrate. After the exposure period of 48 hrs of chemical with daphnia magna, mortality were observed and calculated. The LC50 value of test chemical on Daphnia Magna in a 48 hr study based on mortality was determine to be 0.0077mg/L.

 

Thus based on the overall studies from experimental study, peer reviewed journal. Authoritative database and secondary source, it was concluded that the chemical was toxic and classified as aquatic acute category 1 as per the CLP classification.

 

 

Long term toxicity to aquatic invertebrates:

Based on the various long term studies available for the test chemical, studies were reviewed to determine the toxic nature of test chemical on the mobility and reproduction of aquatic invertebrates. The studies are as mentioned below:

 

In the first long term toxicity of test chemical were studied and determine on the reproduction of daphnia magna. The LOEC was observed to be 0.0047 mg/L for 21 days based on the effect on reproduction.

 

Above study was supported by the below study from peer reviewed journal. 8 days long term toxicity studies were conducted to test the effect of chemical on ceriodaphnia .Five concentrations and blanks were prepared with dechlorinated tap water for each antimicrobial test, and the survival and the number of neonates was counted for eight days, when most of the Ceriodaphnia had finished delivering three broods. The solutions were replaced every 48 h (i.e. a semi-static system) and the concentration before and after the replacement was analyzed using the HPLC. Only the numbers of first three broods were counted and compared with the blanks. Multiple comparisons were conducted to determine NOEC values as presented above for the algal test. After the exposure period of 8 days of chemical with Ceriodaphnia, no effect were observed on the survival rate and reproduction rate of Ceriodaphnia at the concentration 0.0019 mg/l. Thus based on the NOEC value chemical consider to be toxic and classified as aquatic chronic 1 as per the CLP classification criteria.

 

Similar long term toxicity of test chemical were studied on Americamysis bahia. Test conducted under the flow- through system for 28 days. The effective concentration (EC50) value of test chemical in Americamysis bahia(Opossum Shrimp) in a 28 days study based on growth was observed to be > 0.001 mg/L. Thus considering the value from CLP Criteria for aquatic classification of the substance, it is concluded that test chemical could exhibit short term toxicity to aquatic invertebrate (Americamysis bahia) and classified in Aquatic chronic category 1.

 

 

Aim of this study was to determine the effect of test chemical on the aquatic invertebrates. Test conducted under the flow-through system in which effect of chemical were tested by observing the effect of chemical on mortality and survival rate of daphnia magna. The Low observed effect concentration (LOEC) of test chemical on Daphnia magna in 21 days study on the basis of effect on mortality was determine to be 0.05 mg/L.

 

In the fifth study from secondary source, 21 days long term toxicity studies were conducted to test the effect of test chemical on daphnia magna. The NOEC was observed to be 0.0029 mg/L for 21 days based on the effect on reproduction.

Thus based on the overall studies from peer reviewed journal, authoritative database and secondary sources, chemical consider to be toxic and classified as aquatic chronic 1 as per the CLP classification criteria.

 

 

Toxicity to algae and cyanobacteria:

Various studies were reviewed to determine the toxic nature of test chemical on the growth of aquatic algae and cyanobacteria. The studies are as mentioned below:

 

Objective of this study was to observe the action of test chemical when it exposed with the Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) for 72 hrs. Test conducted in accordance with OECD Guideline 201 (Alga, Growth Inhibition Test). The stock solution 1 g/l was prepared by dissolving white powder in acetone. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test conducted on various nominal concentration of test chemical is 0, 0.0025, 0.0050, 0.0100, 0.0200, 0.0400, 0.0800, 0.1600 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration ErC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of chemical, the ErC50 was determine to be 0.0344 mg/l. Thus on the basis of ErC50 value, chemical concluded to be toxic and can be consider to be classified in aquatic acute category 1 as per the CLP classification criteria.

 

First study was supported by the second study from peer reviewed journal. The present study was based on the effect of test chemical on the growth of the freshwater algae Pseudokirchneriella subcapitata. Test conducted in accordance with the OECD guideline 201 with some modification. 1000 mg/l stock solution of test chemical was prepared in methanol. The freshwater unicellular green alga P. subcapitata Hinda´k was obtained from the Algae Culture Collection. Test conducted under the static system for 72 hrs. Five to six concentrations of each test substance in a geometric series (common ratio = 2) were used and performed in triplicates. Yield of algal cells was calculated indirectly by measuring the optical density of the cell suspension at a wavelength of 442 nm using a Helios Alpha spectrophotometer. The effects of test chemical on the growth of the green alga P. subcapitata over 72-h were determined. The Low observed effect concentration (LOEC) of test chemical in Pseudokirchneriella subcapitata in 72hr study on the basis of growth was observed to be 0.01 mg/l. Whereas, the NOEC of test chemical in Pseudokirchneriella subcapitata in 72hr study on the basis of growth was determine to be < 0.01 mg/l. Also, inhibitory concentration (IC50) in 72hr on the basis of growth was observed 0.017 mg/l. Thus chemical consider to be toxic and classified in aquatic acute category 1.

 

 

Similar acute toxicity of test chemical on aquatic green algae were determine under the static system. The effects of test chemical on the growth of the green alga (Pseudokirchneriella subcapitata) over 72-h were determined. The effective concentration (EC50) of test chemical on Pseudokirchneriella subcapitata in 72hr study on the basis of growth rate inhibition was determine to be 0.029 mg/l. Thus based on the EC50 value, chemical consider to be toxic and classified as aquatic acute 1 as per the CLP classification criteria.

 

Thus based on the above all studies from various sources, chemical consider to be toxic and classified as aquatic acute category 1 as per the CLP classification criteria.

 

Toxicity to microorganisms:

The MIC values were determined by the microbroth dilution assay according to the National Committee for Clinical Laboratory Standards methods for antimicrobial susceptibility tests for aerobically growing bacteria (Approved Standard M7-A2, NCCLS, Villanova, PA, 1990).

Approximately 6.5 3 108 cells of strain RN4220 (25) were plated on to brain-heart infusion agar medium (Becton-Dickinson) containing 0.5 mg/ml test material. The plates were incubated at 37 °C with periodic monitoring. After 48 h, resistant clones were picked and their phenotype confirmed by regrowth on the original selective concentration of test material.

Three strains of Staphylococcus aureus namely RN4220 (wild-type) , RN4220/pTri-1 , GR664 (fabIG23S) and three starins of E.coli

W3110 (wild-type) , W3110/pFabI , RJH108 (fabIG93V) . The wild-type FabI proteins from either S. aureus or E. coli were expressed using plasmids pTri-1 or pFabI, respectively, to determine if resistance was conferred by the presence of multiple copies of the fabI genes.

The minimum ihhibition concentration (MIC) was observed in the range of 0.25 to 2 mg/l in case of Staphylococcus aureus and for E.Coli MIC was observed in concentration range of 0.5 - 35 mg/l.

In another secondary data for test chemical the effect of test chemical on the domestic sewage by providing the exposure period of 16 days. Domestic sewage was obtain ocean County South Treatment Plant Test. Anaerobic Digester Inhibition test was performed. After the exposure period of 16 days, effect of test chemical on the domestic sewage were determine. The NOEC was determine to be at 100 mg/l.