Indole

Endpoint summary

Description of key information

Short term toxicity to fish

The short-term toxicity of the test chemical to aquatic fish was predicted using EPI Suite ECOSAR version 1.11. On the basis of effect of test chemical observed in a static system on the mortality of the test organism during the 96 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 85.927 mg/l. Thus, based on the LC50 value, test chemical can be considered as toxic to aquatic fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic and hence, considered to be ‘not classified’ as per CLP classification criteria.

Short term toxicity to aquatic invertebrates

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical (from authoritative database, 2018 and secondary source, 2019). The study was performed under static conditions using Daphnia magna (Water flea) (<24 hours old) as a test organism. Test was carried out under test conditions at 20°C, pH 8.2, dissolved oxygen of>6.5 mg/l and hardness of 250 mg/l as CaCO3, respectively. On the basis of the effect of test chemical on mobility of test organism, the 48 hr median effect concentration (EC50) value was determined to be 1 mg/l. Thus, test chemical can be considered as toxic to aquatic invertebrates. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be ‘not classified’ as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Algal toxicity study was carried out for 96 hrs for assessing the effect of test chemical (from peer reviewed journal, authoritative database and secondary source, 2019)

.Scenedesmus acuminatus(green algae) was used as a test organism.Test culture was kept in continuous culture under a 16:8 h light:dark conditions with two cool white fluorescent tubes (Sylvania, FC40W/CW) as a light source.For obtaining the algal cells in the exponential phase, a static (batch) preculture was inoculated 4 d before the start of a 96-h toxicity experiment.Test chemical concentration used for the study were 0.04, 0.16, 0.63, 2.5, and 10.0 mg/l, respectively. Test chemical concentrations were determined analytically by HPLC. 500 ml of Glass bottle (borosilicate) was used as a test vessel for the study. Test vessel was closed with screw caps containing Teflont inlays. Initial cell density of the test organism used for the study was approx. 1000 cells/ml. Woods Hodle (WH) medium was used for the study. The modifications done in this medium is that, HEPES-buffer & 2-[4-(2-hydroxyethyl)-1-piperazynil]-ethanesulfonic acid was added instead of Tris buffer to the test medium. An aliquot from the preculture was inoculated in each test vessel. After inoculation, the cultures were allowed to acclimate for at least 2 h before the test chemical was added.Test bottles were incubated horizontally on a rolling device at 25 rpm, partly submerged in water at 20°C under conditions of 16:8 hr light: dark with the light source provided by three mercury lamps (Philips HPI-T, 400 W), giving a light intensity of 150µE/m2/sec on the bottles.Two cultures with WH medium and algae but without DMSO were served as a control. One test vessel with WH medium without algae but with the highest test chemical concentration was used as a sterile control in order to determine changes in test chemical concentration due to sorption or chemical (photo-) degradation. All test experiments including the control were performed in duplicates.Each day a sample was taken aseptically from the cultures, starting directly after the addition of algae. Samples (5 ml) were counted in duplicate using a Coulter Counter (Coulter Multisizer; tube diameter, 70 mm) to determine the number and volume of algal cells. At t = 0 and t = 96 h, samples were also taken for the HPLC analysis for the determination of conc. of test chemical.Growth rates were calculated on the basis of both cell number and cell volume. The parameters were plotted versus the 10log of the toxicant concentration and a logistic dose–response model fitted through the data using a nonlinear least-squares method. From this curve the toxicant concentrations giving 10% and 50% inhibition (EC10 and EC50, respectively) of either growth rate or chlorophyll-a content, relative to the controls, were calculated.During the study, the decrease in concentration of test chemical was much higher, being 100% at both 0.16-mg/L treatments and declining to 7% (average) at the 10 mg/L treatments. The strong decrease of the indole concentrations (notably at lower concentrations) might be attributed to transformation by the algal biomass.Test chemical did not show any toxic effect on growth rate & biomass of the test organism upto the highest concentration of 10 mg/l.Based on the effect of test chemical on growth rate and biomass of the test algae, the 96 hrs EC10 value was determined to be > 10 mg/l and on the basis of effect on chlorophyll a of the test organism, the 96 hr EC10 and EC50 value was determined to be 2.43 & 9.42 mg/l, respectively. Thus, test chemical can be considered as toxic to aquatic algae.Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be ‘not classified’ as per the CLP classification criteria.

Toxicity to microorganisms

Toxicity study of micro-organism study was carried out for assessing the effect of the test chemical (from peer reviewed journal (T. Wayne Schultz et. al.; 1980) and secondary source (2019)). Study was performed using Tetrahymena pyriformis as a test organism. Axenic cultures of T. pyriformis were grown in a semi-defined 2% (w/v) proteose peptone medium. Stock solution of conc. (50,000 mg/l) of test chemical was prepared by dissolving the test chemical in ethanol. Erlenmeyer flask of 250 ml volume was used as a test vessel for the study. Test flask containing 50 ml of test chemical-medium solution was inoculated with 0.2 ml of log growth phase culture of test organism. Cultures without test chemical was served as a control. All experiments containing conc. of test chemical were performed in five replicates. 72-hr population density were recorded from a Bausch & Lomb Spectronic 20 spectrophotometer at 540 nm. The reproduction impairment was examined for each compound by plotting the PROBIT value of the percent control absorbance, Y, vs the concentration (mmol/L), X. The best line for these data was fitted by the least-squares method of linear regression and the concentration which inhibits 50% growth (IGC) following 72-hr exposure, 72-hr IGC 50, and 95% confidence intervals were calculated. On the basis of the effect of test chemical growth of the test organism Tetrahymena pyriformis, the 72 hr IC50 value was determined to be 72.633 mg/l (95% C. I. = 51.78 to 101.92 mg/l).

Additional information

Short term toxicity to fish

Predicted data and various experimental studies of the target chemical and supporting weight of evidence studies for its read across analogue were reviewed for short term toxicity to fish endpoint which are summarized as below:

 

In a prediction done using EPI Suite ECOSAR version 1.11, the short-term toxicity of the test chemical to aquatic fish was predicted. On the basis of effect of test chemical observed in a static system on the mortality of the test organism during the 96 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 85.927 mg/l. Thus, based on the LC50 value, test chemical can be considered as toxic to aquatic fishes. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

In a supporting weight of evidence study from review article (1969) and secondary source (2019), short term toxicity to fish study was carried out for 24 hrs. Northern squawfish (Ptychocheilus oregonensis), steelhead (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch) were used as a test organism. The length of these test organisms were about 5 to 10 cm long. Squawfish were seined from the St. Maries River or Santa Creek, a tributary stream, and were either stored in a wooden vat on a tributary of the St. Maries River or brought directly to the campus laboratory for immediate use. The salmonids were flown from the Eagle Creek National Fish Hatchery near Portland, Oregon, to Spokane, Washington. From Spokane, the fish were trucked to Mission Point near St. Maries, Idaho. Later, the salmonids were transported to the campus laboratory. The acclimatization period varies from 3 to 24 hrs, but most fish were conditioned at least overnight. Test fishes were acclimatized at about the temperature of the assay vessel i.e, at 13.88°C, 10°C and 15°C for the respective three test organism. The test animal were starved during acclimatization and transferred to the assay vessels about 2 hrs before the addition of chemical. Test chemical solution was prepared by dissolving the test substance in water or acetone.5 mg/l & 10 mg/l (for northern squawfish (Ptychocheilus oregonensis)) and10 mg/l steelhead (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch). Aseries of insulated, round, stainless steel tubs was used as a test vessel for the study.Aseries of insulated, round, stainless steel tubs (61 cm in diameter x 30 cm deep) were used for water baths. These baths were served by a common refrigerated reservoir through which temperature-controlled water was recirculated. Each tub held four 9.5-liter plastic aquaria. Each aquarium was aerated by a single stone air-breaker.1squawfish and 1 salmonid fish were placed together in one vessel in 4 liters of water, the load being about five grams of fish per liter of solution. Each aquarium was lined with a disposable polyethelene poultry bag (30 cm x 20 cm x 90 cm deep). The bag was closed at the top to prevent fish from escaping. The times at which a fish lost its equilibrium and died were recorded. Equilibrium was defined as lost when a fish was no longer able to remain right-side-up and death was designated when a fish ceased visible movement. All test organisms appeared to be dead after a period of 14 hrs. Thus, on the basis of the effect of test chemical on mortality of the test organism (i.e.,northern squawfish (Ptychocheilus oregonensis), steelhead (Salmo gairdneri) and coho salmon (Oncorhynchus kisutch)was carried out for 24 hrs.), the 14 hr LC100 value was determined to be 10 mg/l.

 

Another short term toxicity test to rainbow trout, bluegill and sea lamprey was carried out for 24 hrs (from review article, 1957 and secondary source). Rainbow trout (Salmo gairdnerii), Bluegill (Lepomis macrochius) and Sea lamprey (Petromyzon marinus) were used as a test organism for the study. Larval lampreys were collected by means of an electric shocker in the Ocqueoc River, Presque Isle County, Michigan, and were held in running water in aquaria and small “races” under conditions which simulated their natural stream habitat. Test fishes (Rainbow trout and Bluegill) were obtained from the stocks of local state and Federal fish hatcheries and were held in large raceways. These specimens were maintained in the best possible physical condition until used in the laboratory. The study was performed under static conditions for 24 hrs. The test chemical concentration used for the study was 5 mg/l. Chemical was weighed in calibrated weighing bottles to the nearest milligram on a Volland Speedigram balance. Five cubic centimeters of the indicated solvent was added to the test chemical. Each concentrated mixture was next added to a predetermined volume of water (as required by actual weight of test chemical and desired conc.) and agitated with a Power-Stir to produce a more dilute solution. Emulsions or suspensions of insoluble compounds were made with the aid of a Waring blender. These prepared solutions, emulsions or suspensions were added to the test containers in which the experimental animals had already been placed. The resultant volume in each test jar varied from 5800 to 6200 cc. The aggregate test animals available, usually six in number, were placed together in a 10-literglass battery jar containing 5 liters of water. The jars were provided with aeration through standard stone air-breakersand were maintained at a constant temperature by immersion in specially constructed constant temperature troughs. Water temperature was maintained within the limits of ± 1.0 °F. Twelve of these test jars (each containing a substance being assayed) were included with one control jar in each trough. Fish and larvae in the control jar were exposed only to the water and physical conditions of the typical test container. Test condition contains dissolved oxygen from 8.6 to 13.7 mg/l and free CO2 from 5.0 to 9.0 ppm. Observations of each test were measured approx. six times, at various intervals, during the 24 hr test period. At each observation, the condition of every test organism was determined and recorded. Chronological histories were thus obtained of any symptoms of illness and the occurrence of death were noted. On the basis of the effect of test chemical on test organism rainbow trout, the 10 hr LOEC value was determined to be 10 mg/l and on the basis of the effect on mortality of the test organism (bluegill and sea lamprey), the 24 hr EC0 value was determined to be 10 mg/l, respectively.

 

In a supporting weight of evidence study, short term toxicity test to fish study was conducted for 24 hrs (from handbook, 2008 and secondary source, 1989). Catastomus columbianus (Bridgelip sucker) and Salmo gairdneri (Steelhead trout) were used as a test organism for the study. Sucker (45-102 mm in length) were was obtained from local drainages and Steelhead trout (33 to 117 mm in length) was obtained from Dwarshak Fish Hatchery (U. S. Fish and Wildlife Service), respectively. The test chemical concentration used for the study was 10 mg/l. Test chemical was dissolved in minimal amounts of water, ethanol or in few cases, acetone before application. Aquarium was used as a test vessel. Biomass loading rate involve 0.6 to 4.0 gram of test fishes/lit of water. Water used in all tests was taken from the Clearwater River in northern Idaho. The study was performed under static conditions with hardness of 0 to 17 mg/l as CaCO3 and pH 7.6 for 24 hrs. Observations recorded the time when aliquots were added to aquaria and noted any changes in the state of the test fish at approx. 1, 2, 3, 4, 6, 8, 12, 16 and 24 hr intervals. On the basis of the effect of test chemical on mortality test organism Salmo gairdneri (Steelhead trout) and Catastomus columbianus (Bridgelip sucker), the 2 hr & 21 hr LC100 value was determined to be 10 mg/l.

 

Additional short term toxicity to fish study from handbook and secondary source was carried out for 24 hrs. Trout (Salmo trutta), bluegill sunfish (Lepomis macrochirus) and goldfish (Carassius auratus)) were used as a test organism. The length of these test organisms were about 4 inches. Test chemical concentration used for the study were 0.01, 0.1, 1 and 5 mg/l, respectively. Test fishes were exposed to different test chemical concentrations in an aquaria of 8 lit capacity containing 5 lit of water. Test vessel was aerated by compressed air released through carborundum spargers. 8 fishes (two of each species) were taken for the study. The study was performed for 24 hr under test conditions at a temperature of 12.8°C, pH of 7.0, dissolved oxygen of 7.5 mg/l, hardness of 300 mg/l, respectively.  The times at which a fish shows obvious distress or died were recorded on plastic cards attached to each aquarium. On the basis of the effect of test chemical on mortality or behaviour of the test organism (i.e., trout (Salmo trutta), bluegill sunfish (Lepomis macrochirus) and goldfish (Carassius auratus)), the 4 hr LC0 and LC100 value was determined to be 1 and 5 mg/l, respectively.

 

For the test chemical, short term fish toxicity was conducted for 96 hrs for assessing the effect of test chemical (Secondary source, 2020). Study was carried out using Oncorhynchus mykiss (Rainbow Trout) of 0.4 g initial weight under static conditions. On the basis of the effect of test chemical on mortality of the test fishes, the 96 hr NOEL and median lethal concentration (LC50) value was determined to be 58.8 and >90.5 mg/l. Thus, based on the LC50 value, test chemical can be considered as toxic to aquatic fishes. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

In a supporting weight of evidence study (2020), short term fish toxicity was conducted for assessing the effect of test chemical. Study was carried out using Pimephales promelas (Fathead Minnow) of 30 days old, 18.6 mm length and 0.089 g initial weight under static conditions. Test was performed at a temperature of 23.9°C, pH of 7.6 and dissolved oxygen of 6.5 mg/l, respectively for 96 hrs. On the basis of the effect of test chemical on mortality of the test fishes, the 96 hr median lethal concentration (LC50) value was determined to be 8.84 mg/l. Thus, based on the LC50 value, test chemical can be considered as toxic to aquatic fishes. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

 

On the basis of the above results, test chemical can be considered as toxic to fish. Since the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Short term toxicity to aquatic invertebrates

Various experimental studies of the target chemical and supporting weight of evidence study for its read across analogue were reviewed for short term toxicity to aquatic invertebrates endpoint which are summarized as below:

 

In an experimental study, an acute immobilisation test from authoritative database (2018) and secondary source (2019) was conducted for 48 hrs for assessing the effect of test chemical. The study was performed under static conditions using Daphnia magna (Water flea) (<24 hours old) as a test organism. Test was carried out under test conditions at 20°C, pH 8.2, dissolved oxygen of >6.5 mg/l and hardness of 250 mg/l as CaCO3, respectively. On the basis of the effect of test chemical on mobility of test organism, the 48 hr median effect concentration (EC50) value was determined to be 1 mg/l. Thus, test chemical can be considered as toxic to aquatic invertebrates. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be ‘not classified’ as per the CLP classification criteria.

 

The short-term toxicity of the test chemical to aquatic invertebrates was predicted using EPI Suite ECOSAR version 1.11. On the basis of effect of test chemical observed in a static system on the mobility of the test organism during the 48 hr exposure duration, the lethal effect concentration (LC50) for the test chemical was estimated to be 49.038 mg/l. Thus, based on the LC50 value, test chemical can be considered as toxic to aquatic invertebrates. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.

 

In a supporting weight of evidence study,short term toxicity to aquatic invertebrate study was conducted for 24 hrs for assessing the effect of test chemical (Secondary source, 2019). The study was performed under static conditions using Artemia franciscana (Brine shrimp) (Nauplli life stage) as a test organism. 96 well microlitre plates was used as a test vessel for the study. Test was carried out under test conditions at 28°C, pH 7.4 to 8.0 and salinity of approx. 35.15 g/l, respectively. DMSO was used as a negative control. All experiments were performed in triplicates. On the effect of test chemical on mortality of test organism, the 24 hr median lethal concentration (LC50) value was determined to be≤ 57.5 mg/l.

 

For the test chemical from secondary source (2020), an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical. The study was performed under static conditions using Daphnia magna (Water flea) (<24 hr old) as a test organism. On the basis of the effect of test chemical on mobility of the test organism Daphnia magna, the 48 NOEL and median effect concentration (EC50) value was determined to be 28.7 mg/l and 57 mg/l (95% C. I. = 46.7 to 73.8 mg/l), respectively. Thus, test chemical was considered as toxic to aquatic invertebrates at environmental relevant concentrations. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic and hence, considered to ‘not classified’ as per the CLP classification criteria.

 

On the basis of the above results, test chemical can be considered as toxic to aquatic invertebrate. Since the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Experimental study and predicted data of the target chemical were reviewed for toxicity to aquatic algae and cyanobacteria endpoint which are summarized as below:

In an experimental study, algal toxicity study was carried out for 96 hrs for assessing the effect of test chemical (from peer reviewed journal, authoritative database and secondary source, 2019). Scenedesmus acuminatus (green algae) was used as a test organism. Test culture was kept in continuous culture under a 16:8 h light:dark conditions with two cool white fluorescent tubes (Sylvania, FC40W/CW) as a light source. For obtaining the algal cells in the exponential phase, a static (batch) preculture was inoculated 4 d before the start of a 96-h toxicity experiment. Test chemical concentration used for the study were 0.04, 0.16, 0.63, 2.5, and 10.0 mg/l, respectively. Test chemical concentrations were determined analytically by HPLC. 500 ml of Glass bottle (borosilicate) was used as a test vessel for the study. Test vessel was closed with screw caps containing Teflont inlays. Initial cell density of the test organism used for the study was approx. 1000 cells/ml. Woods Hodle (WH) medium was used for the study. The modifications done in this medium is that, HEPES-buffer & 2-[4-(2-hydroxyethyl)-1-piperazynil]-ethanesulfonic acid was added instead of Tris buffer to the test medium. An aliquot from the preculture was inoculated in each test vessel. After inoculation, the cultures were allowed to acclimate for at least 2 h before the test chemical was added. Test bottles were incubated horizontally on a rolling device at 25 rpm, partly submerged in water at 20°C under conditions of 16:8 hr light: dark with the light source provided by three mercury lamps (Philips HPI-T, 400 W), giving a light intensity of 150µE/m2/sec on the bottles. Two cultures with WH medium and algae but without DMSO were served as a control. One test vessel with WH medium without algae but with the highest test chemical concentration was used as a sterile control in order to determine changes in test chemical concentration due to sorption or chemical (photo-) degradation. All test experiments including the control were performed in duplicates. Each day a sample was taken aseptically from the cultures, starting directly after the addition of algae. Samples (5 ml) were counted in duplicate using a Coulter Counter (Coulter Multisizer; tube diameter, 70 mm) to determine the number and volume of algal cells. At t = 0 and t = 96 h, samples were also taken for the HPLC analysis for the determination of conc. of test chemical. Growth rates were calculated on the basis of both cell number and cell volume. The parameters were plotted versus the 10log of the toxicant concentration and a logistic dose–response model fitted through the data using a nonlinear least-squares method. From this curve the toxicant concentrations giving 10% and 50% inhibition (EC10 and EC50, respectively) of either growth rate or chlorophyll-a content, relative to the controls, were calculated. During the study, the decrease in concentration of test chemical was much higher, being 100% at both 0.16-mg/L treatments and declining to 7% (average) at the 10 mg/L treatments. The strong decrease of the indole concentrations (notably at lower concentrations) might be attributed to transformation by the algal biomass. Test chemical did not show any toxic effect on growth rate & biomass of the test organism upto the highest concentration of 10 mg/l. Based on the effect of test chemical on growth rate and biomass of the test algae, the 96 hrs EC10 value was determined to be > 10 mg/l and on the basis of effect on chlorophyll a of the test organism, the 96 hr EC10 and EC50 value was determined to be 2.43 & 9.42 mg/l, respectively.

 

Another toxicity of the test chemical to green algae was predicted using EPI Suite ECOSAR version 1.11 (2019). On the basis of effect of test chemical observed in a static system on the growth rate of the test organism during the 96 hr exposure duration, the median effect concentration (EC50) for the test chemical was estimated to be 37.301 mg/l.

 

On the basis of the above results, test chemical can be considered as toxic to aquatic algae. Since the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic algae at environmental relevant concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

Toxicity to microorganisms

Various experimental studies of the target chemical were reviewed for toxicity to microorganisms endpoint which are summarized as below:

 

In an experimental study from peer reviewed journal (T. Wayne Schultz et. al.; 1980) and secondary source (2019), toxicity study of micro-organism study was carried out for assessing the effect of the test chemical. Study was performed using Tetrahymena pyriformis as a test organism. Axenic cultures of T. pyriformis were grown in a semi-defined 2% (w/v) proteose peptone medium. Stock solution of conc. (50,000 mg/l) of test chemical was prepared by dissolving the test chemical in ethanol. Erlenmeyer flask of 250 ml volume was used as a test vessel for the study. Test flask containing 50 ml of test chemical-medium solution was inoculated with 0.2 ml of log growth phase culture of test organism. Cultures without test chemical was served as a control. All experiments containing conc. of test chemical were performed in five replicates. 72-hr population density were recorded from a Bausch & Lomb Spectronic 20 spectrophoto meter at 540 nm. The reproduction impairment was examined for each compound by plotting the PROBIT value of the percent control absorbance, Y, vs the concentration (mmol/L), X. The best line for these data was fitted by the least-squares method of linear regression and the concentration which inhibits 50% growth (IGC) following 72-hr exposure, 72-hr IGC 50, and 95% confidence intervals were calculated. On the basis of the effect of test chemical growth of the test organism Tetrahymena pyriformis, the 72 hr IC50 value was determined to be 72.633 mg/l (95% C. I. = 51.78 to 101.92 mg/l).

 

Another toxicity study of micro-organism study was carried out for assessing the effect of the test chemical at 28°C (T. Wayne Schultz et. al.; 1985 and secondary source, 2019). Study was performed using Tetrahymena pyriformis as a test organism which was grown in 2% (w/v) proteose peptone medium. Each replicate used were freshly prepared stock solutions. Stock solutions were prepared by dissolving the test compounds in reagent-grade dimethyl sulfoxide (DMSO) or sterile distilled water in concentrations of 10, 50, 250 or 500 g/L. Erlenmeyer flask of 250 ml volume was used as a test vessel for the study. Test flask containing 50 ml of test chemical-medium solution was inoculated with test organism. Culture flask without test chemical was used as a control. All experiments were performed in three replicates. Growth inhibition of the test organism was noted after a period of 60 hrs. Statistical analyses were done using Statistical Analysis System (SAS) software. The 60-h IGC5O value (concentration that inhibits 50% of population growth) and 95% confidence limits were determined for each test compound by converting raw absorbance to percent control absorbance prior to probit analysis. On the basis of the effect of test chemical growth of the test organism Tetrahymena pyriformis, the 60 hr IGC50 value was determined to be 70.33 mg/l (95% C. I. = 56.26 to 85.38mg/l).

 

In a supporting study, toxicity study of micro-organism study was carried out for assessing the effect of the test chemical (from handbook, 2009 and Klaus L.E. Kaiser et. al., 1991). Study was performed using Photobacterium phosphoreum, strain NRRL-B-11177 (also referred to as Vibrio fischerii, strain NRRL-B-11177) as a test organism at a temperature of 15°C and pH range 5 to 9. Recommneded reference substance that can be used for the study were Phenol and Sodium pentachlorophenate, respectively. When the test bacterium was exposed to the test chemical, reduction in light output was observed. Thus, based on this effect, the EC50 value during 5, 15 and 30 min exposure period was determined to be 2.436 mg/l, 2.389 mg/l and 2.389 mg/l, respectively.

 

For the test chemical from secondary sources, toxicity study of micro-organism study was carried out for assessing the effect of the test chemical. Study was performed using Tetrahymena pyriformis as a test organism at 28°C. Total 4 concentrations of test chemical were taken for the study. Proteose peptone medium was used as a test medium for the study. Composition of the medium includes 20 g of proteose peptone, 5 g glucose, 1 g yeast extract and 0.03 g EDTA iron salt per litre H2O pH 7.3, respectively. Cultures without test chemical was served as a control. All experiments were performed in triplicates. The cell density was determined with OD 540 nm. On the basis of the effect of test chemical on growth of the test organism Tetrahymena pyriformis, the 60 hr IC50 value was determined to be 72.633 mg/l.

 

On the basis of the above results, the EC50/IC50 value of the test chemical was evaluated to be ranges from 2.389 to 72.633 mg/l, respectively.

On the basis of the available information of aquatic toxicity studies, it can be concluded that the test chemical was considered as toxic to aquatic organisms at environmental relevant concentrations. Since the test chemical is readily biodegradable in water, chemical was considered as non-toxic and hence, considered to be 'not classified' as per the CLP classification criteria.