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Short term toxicity to fish :

The acute toxicity of industrial organic chemicals including test chemical to the fathead minnow was determined for 96 hrs.

Concentrations were determined by analytical methods utilizing direct aqueous injection (Knuth and Hoglund, 1984) and solvent extraction gas chromatographic (GC) techniques, and high pressure liquid chromatographic (HPLC) analyses. GC analyses were performed on a Hewlett-Packard model 5730A gas chromatograph equipped with flame ionization and electron capture detectors.

Tests were conducted in diluters with continuous-flow water delivery and toxicant introduction systems similar in principle to those described by Benoit et al.(1982) and Anderson and Weber (1975). Each of two diluters for binary tests contained a control, and four treatment levels in duplicate at each of seven mixture ratios were distributed between the diluters. This gave a total of 28 treatments in duplicate plus two controls. The seven ratios used to define the binary isobole diagrams were 5:0, 4:1, 2:1, 1:1, 1:2, 1:4, and 0:5. The diluter for equitoxic multiple chemical tests had one control and five single treatment levels. The toxicant concentrations followed a geometric series (0.8 dilution factor) for all tests. The dilution water was fed from a main head tank to a similar tank over each diluter. The water in the head tanks was vigorously aerated to remove excess dissolved gases. The toxicant solutions for binary tests were delivered by FMI metering pumps (Fluid Metering,

Inc., Oyster Bay, NY) from separate stock bottles or chemical 'saturators'(Gingerich et al., 1979) into a chamber which was designed to dilute each stock solution independently before combining them in mixtures. The toxicant concentrations in each test treatment were thus controlled separately.

Test chambers were illuminated with wide spectrum fluorescent bulbs (Durotest 'Vitalite') for 16 h daily. This included a 30-rain gradual brightening and dimming period with incandescent light to simulate dawn and dusk (Drummond and Dawson, 1970). The light intensity at the test water surface ranged from 22 to 38 lumens/sq ft.

95% confidence intervals were computed by the Trimmed Spearman-Karber Method (Hamilton et al., 1977) or a log-probit method (Stephan, 1977).

From experimental result the the lethal concentration in 96 hrs exposure period was observed to be 45.9 mg/l with 95% confidence limit (43.5-48.4).Thus based on the result it is concluded that the test substance 2-Octanone is toxic to fisn and thus classified in aquatic chronic 3 as per the CLP criteria.

Short-term toxicity to aquatic invertebrates:

Determination of the inhibition of the mobility of daphnids was carried out with the substance according to OECD Guideline 202.The test substance was tested at the concentrations3.8, 7.5, 15, 30, 60 and 120 mg/L. Effects on immobilisation were observed for 48 hours.The stock solution (120 mg/L) was prepared by dissolving clear yellow coloured iquid in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.The median effective concentration (EC50) for the test substance, 2 -Octanone, in Daphnia magna was determined to be 59.6 mg/L for immobilisation effects.This value indicates that the test substance is likely to be hazardous to aquatic invertebrates , hence it can be classified as Aquatic Chronic category 3 as per the CLP criteria.

Toxicity to aquatic algae and cyanobacteria:

Aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201.The solution (100 mg/L) was prepared by dissolving clear yellow coloured liquid in OECD growth medium.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. Effect on the growth of algae was determine after an exposure period of 72 hrs.

The median effective concentration (EC50) for the test substance in algae was determined to be >100 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as as per the CLP classification criteria.

Toxicity to microorganisms:

The microtox test was conducted on Photobacterium phosphoreum to determine the EC50 value of chemical exposed with duration of 5 min to 15 min.Analytical analyses done by Gas chromatography/mass spectrometry. and statistical analysis for EC50 determination used Student t-test. During experimental result the EC50 value was found to be 8.910 mg/l (69.5±9.2µM), 9.616 mg/L (75±11µM); 10 mg/L (78±11µM) when exposed to Photobacterium phosphoreum with test chemical 2-octanone in microtox test.

Additional information

Short-term toxicity to fish:

Five results including experimental studies and modelling database were reviewed for short term fish toxicity, from data having Klimish rating 2 and 4 considering key and supporting, which is summarised as followed:

First experimental study (Aquatic Toxicology, 6 (1985) 307-322) suggest the acute toxicity of industrial organic chemicals including test chemical to the fathead minnow was determined for 96 hrs. Concentrations were determined by analytical methods utilizing direct aqueous injection (Knuth and Hoglund, 1984) and solvent extraction gas chromatographic (GC) techniques, and high pressure liquid chromatographic (HPLC) analyses. GC analyses were performed on a Hewlett-Packard model 5730A gas chromatograph equipped with flame ionization and electron capture detectors.Tests were conducted in diluters with continuous-flow water delivery and toxicant introduction systems similar in principle to those described by Benoit et al.(1982) and Anderson and Weber (1975). Each of two diluters for binary tests contained a control, and four treatment levels in duplicate at each of seven mixture ratios were distributed between the diluters. This gave a total of 28 treatments in duplicate plus two controls. The seven ratios used to define the binary isobole diagrams were 5:0, 4:1, 2:1, 1:1, 1:2, 1:4, and 0:5. The diluter for equitoxic multiple chemical tests had one control and five single treatment levels. The toxicant concentrations followed a geometric series (0.8 dilution factor) for all tests. The dilution water was fed from a main head tank to a similar tank over each diluter. The water in the head tanks was vigorously aerated to remove excess dissolved gases. The toxicant solutions for binary tests were delivered by FMI metering pumps (Fluid Metering, Inc., Oyster Bay, NY) from separate stock bottles or chemical 'saturators'(Gingerich et al., 1979) into a chamber which was designed to dilute each stock solution independently before combining them in mixtures. The toxicant concentrations in each test treatment were thus controlled separately. Test chambers were illuminated with wide spectrum fluorescent bulbs (Durotest 'Vitalite') for 16 h daily. This included a 30-rain gradual brightening and dimming period with incandescent light to simulate dawn and dusk (Drummond and Dawson, 1970). The light intensity at the test water surface ranged from 22 to 38 lumens/sq ft. 95% confidence intervals were computed by the Trimmed Spearman-Karber Method (Hamilton et al., 1977) or a log-probit method (Stephan, 1977).From experimental result the the lethal concentration in 96 hrs exposure period was observed to be 45.9 mg/l with 95% confidence limit (43.5-48.4).Thus based on the result it is concluded that the test substance is toxic to fish and thus classified in aquatic chronic 3 as per the CLP criteria.

Supporting study from peer reviewed journal (Environmental Toxicology and Chemistry, Vol. 16, No. 5, pp. 948–967, 1997)indicate Short term toxicity study to Fathead Minnow (Pimephales promelas) was carried out for 96 hrs. The objective of this study was to develop procedures that relate modes of acute toxic action in the fathead minnow (Pimephales promelas) to chemical structures and properties. An empirically derived database for diverse chemical structures of acute toxicity and corresponding modes of toxic action was developed through joint toxic action studies, the establishment of toxicodynamic profiles, and behavioral and dose–response interpretation of 96-h LC50 tests. Chemicals in the fathead minnow database were evaluated through analyses of dose–response relationships and behavioral responses associated with 96-h LC50 bioassays. Based on the amount of available information for a test compound, a level of confidence was assigned to a mode of action determination.28 - 30 d old juveline test organisms were used for the study. The test was performed under flowthrough condition with temp. of 25 ± 1°C. Test was conducted using Lake Superior water at 25±1°C.Aqueous toxicant concentrations were measured in tests with quality assurance criteria requiring 80% agreement between duplicate samples and 90 to 110% spike recovery. Flow-through exposures were conducted using cycling proportional, modified Benoit, or electronic diluters. Tests conducted which were conducted on the Benoit and electronic diluters did not have replicate tank exposures.Median lethal concentration (LC50) was calculated using the Trimmed Spearman–Karber Method, with 95% confidence intervals being calculated when possible. The change of LC50 values over time (LC50 ratio) and the ratio of measured 96-h LC50 values to those predicted from a baseline narcosis (narcosis I) QSAR were used as supportive data for assessing potential modes of action. Each mode of action classification was assigned a level of confidence based on the type of data used when making the assessment; confidence levels were high (Level A using FATS and joint toxic action data), moderate (Level B using behavior syndrome, LC50 ratios, Te values, and structural similarity to Level A compounds), and low (Level C using behavioural comments, concentration/response slope, and structural similarity within a chemical class). Thus, the mode of action for the test chemical was Narcosis I: Level A confidence. Based on mortality, the 96 hr LC50 value for Fathead Minnow (Pimephales promelas) was found to be 36 mg/l.

Another peer reviewed study for target suggests the acute toxicity test has been performed on fish with exposure duration of 96 hrs. The animals were exposed by test chemical  with dose range to 35 to 37 mg/L.Under the test condition LC50 value was found to be 36 mg/L when exposed to fish for 96 hrs by test chemical 2-octanone.Thus based on the lethal concentration it is concluded that the test substance 2-octanone is toxic to fish and thus classified in aquatic chronic 3 as per the CLP criteria.( Center for Lake Superior Environmental ,University of Wisconsin-Superior, Superior, WI:414 p.)

And study report GSBL – Joint Substance Data Pool of the German Federal Government and the German Federal States indicate based on mortality, the 96 hrs LC50 value for Pimephales promelas was found to be > 36 mg/l for test chemical .

Overall all available studies give assistance as the test substance showed the adverse effects on the basis of mortality effects on aquatic fish(Pimephales promelas) thus considering the test substance for the aquatic classification in Chronic Category 3 as per the CLP criteria.

Short-term toxicity to aquatic invertebrates:

Determination of the inhibition of the mobility of daphnids was carried out with the substance according to OECD Guideline 202.The test substance was tested at the concentrations3.8, 7.5, 15, 30, 60 and 120 mg/L. Effects on immobilisation were observed for 48 hours.The stock solution (120 mg/L) was prepared by dissolving clear yellow coloured iquid in reconstituted water. Test solutions of required concentrationas were prepared by mixing the stock solution of the test sample with reconstituted test water.The median effective concentration (EC50) for the test substance, 2 -Octanone, in Daphnia magna was determined to be 59.6 mg/L for immobilisation effects.This value indicates that the test substance is likely to be hazardous to aquatic invertebrates , hence it can be classified as Aquatic Chronic category 3 as per the CLP criteria.

Toxicity to aquatic algae and cyanobacteria:

Various experimental study for the target compound were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental key study,aim of this study was to evaluate the nature of chemical test chemical when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). Test was conducted according to the OECD guideline 201.The solution (100 mg/L) was prepared by dissolving clear yellow coloured liquid in OECD growth medium.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. Effect on the growth of algae was determine after an exposure period of 72 hrs.

The median effective concentration (EC50) for the test substance in algae was determined to be >100 mg/L on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be non-hazardous to aquatic algae and cannot be classified as as per the CLP classification criteria.

While in supporting study, short term toxicity to Pseudokirchneriella subcapitata study was carried out for 48 hrs . The test chemical used for the study was 99% pure. The conc. of test chemical 2-Octanone(CAS no. 111-13-7)used for the study was in the range of 10.3 – 49.4 mg/l. Stock solutions of test chemical was prepared in foil-wrapped glass containers. The test organism used for the study is Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum, UTEX 1648). Before starting the experiment, stock solution was freshly prepared and its concentration was analyzed using a HPLC analyzer. The alga Pseudokirchneriella subcapitata(formerly known as Selenastrum capricornutum, UTEX 1648) was grown in a 4-Ltransparent chemostat incubator. The growth medium wascontinuously supplied by a variable-speed pump. Air agitation was used to achieve adequate mixing. The chemostatreactors were placed in a constant-temperature room at24±1°C. Light intensity was set at 65mEm-2s-1(±10%).Growth medium composition is basically the same as that described by the EPA bottle technique. However, NaNO3, K2HPO4, and EDTA contents were reduced to 12.75, 0.52 mg/L, and 30mg/L, respectively. The dilution rate (D) for the chemostat was set at 0.25/day. Toxicity testing was conducted after the algal incubator has reached the steady state by transferring adequate amounts of algal suspension, dilution water (with growth medium), and test chemical into 300-mL BOD bottles. The BOD bottles were completely filled up with no headspace left. Water seal was provided to ensure a closed test environment. The bottles were then placed on an orbital shaker operated at 100 rpm. Temperature and light intensity used for the study was 24 ± 1°C and 65 µEm-2s-1(± 10%). After 48 hrs of test duration, the EC50 value was calculated based on the dissolved oxygen production and algal growth rate.

In the toxicity experiment, the dilution water was stripped by nitrogen gas to reduce the dissolved oxygen level. In addition, the N2 gas contained 0.5% carbon dioxide as an extra carbon source. The DO level at the beginning of the test was approximately 1–3 mg/L. The initial inoculated cell density in the test vessel was 15,000 cells/mL

Algal cell density was measured by electronic particle counting (Coulter Multisizer). Probit analyses were applied to obtain the concentration–response relationships and EC50 values. Based on the dissolved oxygen production by organism and growth rate of test organism Pseudokirchneriella subcapitata, the EC50 value was found to be 32.9 mg/l and 41.2 mg/l, respectively.

 

In a supporting study from peer reviewed journal , short term toxicity to Pseudokirchneriella subcapitata study was carried out for 48 hrs. The test organism used for the study is Pseudokirchneriella subcapitata, UTEX 1648. Before commencing the experiment, stock solution was freshly prepared and its concentration was analyzed using a HPLC (HPLC; 2996 Photodiode Array Detector; Waters, Milford, MA, USA). Algal inoculum was withdrawn from a chemostat operated under steady state and transferred into 300 ml, biochemical oxygen demand (BOD) test bottles together with dilution water (with growth medium) and test chemical. The BOD bottles were completely filled, with no headspace left. A water seal was provided to ensure a close-test environment. The BOD bottles were then placed on an orbital shaker operated at 100 rpm. Temperature and light intensity were kept at 24 ± 1°C and 65 µEm-2s-1 (±10%), respectively. After 48 hrs of test duration, the EC50 value was calculated algal growth rate. The inhibition rate on the net increase of algal cell density was calculated. Probit analysis was used for determining the EC50 values. The population density of the algae was determined using an electronic particle counter. Based on the cell density of test organism Pseudokirchneriella subcapitata, the EC50 value was found to be 32.20 mg/l.

 

Another short term toxicity to Pseudokirchneriella subcapitata study was carried out for 48 hrs . The test organism used for the study is Pseudokirchneriella subcapitata. The test chemical (2-Octanone) concentrations used in the study are in the form of nominal concentrations. Toxicity tests were conducted using the 300-ml biochemical oxygen demand (BOD) test bottles, with no headspace left. A water seal was provided to ensure a closed-test environment. All tests were conducted in triplicate with test duration of 48 h. Three different endpoints were used to analyze the toxic effects of various organic compounds: dissolved oxygen (DO) production, algal growth rate (GR), and the net production of algal cell density (final cell density-initial cell density, biopopulation). One-tail Dunnett’s procedure was applied for the estimation of NOEC and LOEC values at 5% level of significance. NOEC was defined as the toxicant concentration which caused no significant difference compared to the test controls, with respect to all test endpoints (i.e., DO production, growth rate, and biopopulation). The EC10 value was determined using the best-fit-model approach. Regression analyses were performed by using MINITAB (Ver14.2, MINITAB, StateCollege, PA, USA) to establish prediction models for NOEC and EC10. In addition to this, leave- one-out cross-validation was carried out to test the significance of each prediction model. Based on various effects such as biopopulation and dissolved oxygen production of test substance 2-octanone on Pseudokirchneriella subcapitata, the 48 hrs NOEC, LOEC, EC10 and EC50 value was found to be 10.3, 24.7, 21.3 and 32.2 mg/l, respectively.

 

Thus, based on the reported results for target chemical (study report and from peer reviewed journals), it can be observed that the EC50 value is found to be between 32.2 - > 100 mg/L. Although the data from peer reviewed journals indicate the substance to be low toxic to aquatic algae but considering the reliability and authenticity of the study report, the test substance is considered to be Not classified as per the CLP criteria.

Toxicity to microorganisms:

Four studies including experimental studies and modelling database were reviewed for toxicity to microorganisms for test substance , having Klimish rating 2 considering key and supporting, is summarised as followed:

First peer reviewed study suggest The microtox test was conducted on Photobacterium phosphoreum to determine the EC50 value of chemical exposed with duration of 5 min to 25 min.Analytical analyses done by Gas chromatography/mass spectrometry. and statistical analysis for EC50 determination used Student t-test. During experimental result the EC50 value was found to be 8.910mg/l (69.5±9.2µM), 9.616 mg/L (75±11µM); 10 mg/L (78±11µM) when exposed to Photobacterium phosphoreum with test chemical 2-octanone in microtox test.

Another one from SAR and QSAR in Enuironmental Research, journal indicate the toxicity to micro-organisms study was conducted onTetrahymena pyriformisfor 48 hrs. Stock solutions of test chemical was prepared in dimethyl sulfoxide (DMSO) at conc. of 2.5, 5, 10, 25 or 50 mg/l. The volume of stock solution added to each test flask did not exceed 0.35 ml, an amount that does not alter Tetrahymena population growth.Test chemical was tested in a range-finder prior to testing in duplicate for three additional replicates. Each replicate was set up using freshly prepared stock solutions.After 48 hrs of exposure duration of test organism to test chemical 2-octanone,population density was measured spectrophotometrically at 540 nm. The 50 percent growth inhibitory concentration, IGC50, was determined using Probit Analysis of Statistical Analysis System (SAS) software with Yas the absorbency normalized as percentage of control and X as the toxicant concentration in parts per million.Based on growth inhibition of test organism, the IGC50 value was found to be 61.41 mg/l.

And from Microtox study for target from different journal suggest in microtox test which is conducted on Photobacterium phosphoreum to determine the EC50 value of chemical exposed with duration of 5 min. The EC50 value was found to be 17.7 mg/L when exposed to Photobacterium phosphoreum with test chemical in microtox test.

Last study from one another peer reviewed journal Toxicology Methods, indicate toxicity to micro-organisms study was conducted on Tetrahymena pyriformis strain GL for 72 hrs. The assay was conducted in a buffered medium under static conditions.Stock solutions of test chemical was prepared either in solvent DMSO or in distilled water.The solvent DMSO has low toxicity to Tetrahymena,low volatility, and high ability to dissolve organic chemicals. Concentration not greater than 0.75% DMSO (350 µL per 50 mL of medium) are used. This conc. was shown to have no effect on Tetrahymena population growth. Standard stocks are prepared on a milligram per liter basis. When using distilled water for prepare stock solutions, extra care must be taken to maintain sterility. tests were conducted in a 250 mL Erlenmeyer flask containing 50 mL of sterile, semidefined proteose – peptone – based medium and five different conc. of test substance. Then the flasks were inoculated with log-growth-phase culture of Tetrahymena pyriformis of initial cell density of approx. 2500 cells/ml and incubated for about 40 hrs at27 ± 1ᵒC with pH 7.4. After incubation, growth inhibition was measured spectrophotometrically or by electronic particle counting and 50% effect levels are determined at 72 hrs. The nutritional requirement of test organism was met by a solution of proteaose-peptone, yeast extract, glucose, and Fe-EDTA. For test bacterial strain, the optimum temp. for growth was in between 27ᵒC and 35ᵒC and pH range is 5.0 – 8.6, with the optimum pH being 7.5, respectively. Control test vessel contains the test medium inoculated with the test organism with no addition of test chemical and blank was also prepared for the study. The 50% inhibitory growth concentration in mg/l (IGC50) and the 95% fiducial interval are determined for each test compound. The IGC50 was calculated with the probit procedure. Thus, based on growth inhibition of test organism, the IGC50 value was found to be 181.1 and 90.7 mg/l, respectively.

Overall all experimental studies indicate that the EC50 values for 5 min in the range 8.91 mg/l to 17.7 mg/l, for 15 min. -9.616 mg/l and for 25 min-10 mg/l respectively.IGC50 for 72 hrs.was found to be 90.7 mg/l-181.1 mg/l and for 48 hrs it become 61.41 mg/l thus based on the available results it is concluded that test substance may have toxicity concern to micro organisms in acute exposure period.