2,3-xylenol

Endpoint summary

Description of key information

Short term toxicity to fish

Short term toxicity to fish study was carried out for 96 hrs. Gadus morrhua (eggs and larvae) was used as a test organism. Test stock solutions (10 mg/l) was analysed by fluorometric (FL) analysis. Test fishes were exposed to test chemical in beaker. Test beakers were covered with aluminum foil. It contains 100 ml filtered seawater mixed with the test substance. Each test vessel contains 50 fish eggs, or 20 larvae. Test organisms were observed on  1 ,2 and 4 the day. All deviations from the control development were registered as effects: Death, pathology, inhibition of cleavage and differentiation, and pigment defects. The 96 hour EC50 values were plotted for test chemical. On the basis of the effect on mortality of the test organism Gadus morhua L (egg and larva), the 96 hr EC50 value was determined to be 13 mg/l.

Short term toxicity to aquatic invertebrate

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of M7 media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 990.12 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 1.79 mg/L,  3.04 mg/L, 5.17 mg/L,8.79 mg/L and 14.9 mg/L, respectively was from the saturated test concentration. Study was performed using 20 daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be 6.32 mg/L (4.22-11386). Thus, based on the EC50 value, chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 250mg of test chemical in 250ml of OECD media, to get at concentratino of 1000 mg/l , which was then filtered and the final saturated stock solution obtained was 989.8 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 7000 -8000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 57.5 mg/l (calculated from equation through probit analysis).

Toxicity to microorganisms

Toxicity test was performed with Laboratory-prepared freeze-dried photobacterial reagent (Photobacterium phosphoreum strain FEI 162095) registered in the Finnish Environment Institute. The freeze-dried bacteria were reconstituted by adding 12.5-ml 2% NaCl into the reagent bottle and incubating it at +4°C for 20 min before use. The concentration of test chemical in the test (mg/L) which caused a 50% reduction in light after exposure of was measured after an exposure period of 15 min. The EC50 value of test chemical was determined to be 28 mg/l for Photobacterium phosphoreum bacteria.

Additional information

Short term toxicity to fish

Various experimental studies of the test chemical were reviewed for toxicity to fish end point which are summarized as below:

 

In an experimental study from peer reviewed journal (Inger-Britt Falk-Petersen et. al., 1985), short term toxicity to fish study was carried out for 96 hrs. Gadus morrhua (eggs and larvae) was used as a test organism. Test stock solutions (10 mg/l) was analysed by fluorometric (FL) analysis. Test fishes were exposed to test chemical in beaker. Test beakers were covered with aluminum foil. It contains 100 ml filtered seawater mixed with the test substance. Each test vessel contains 50 fish eggs, or 20 larvae. Test organisms were observed on 1 ,2 and 4 the day. All deviations from the control development were registered as effects: Death, pathology, inhibition of cleavage and differentiation, and pigment defects. The 96 hour EC50 values were plotted for test chemical. On the basis of the effect on mortality of the test organism Gadus morhua L (egg and larva), the 96 hr EC50 value was determined to be 13 mg/l.

 

Another short term toxicity to fish study was carried for assessing the effect of test chemical (from publication and secondary source). The study was performed under static conditions for 24 hrs at a temperature of 22°C, pH 7.8 to 8.8 and hardness of 150 mg/L CaCO3, respectively. On the basis of the effect on mortality of the test organism, the 24 hr LC50 value was determined to be 10 to 35 mg/l.

 

In a supporting weight of evidence study, Estimated 96 hrs LC50 value of test chemical on Fathead minnow was determined by three different models i.e, Battery, Leadscope and SciQSAR used within Danish QSAR database. Based on mortality, the estimated 96 hrs LC50 value was determined to be 20.21 mg/l.

 

On the basis of the above results, it can be concluded that the test chemical was considered to be toxic to aquatic fishes and hence, considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

Short term toxicity to aquatic invertebrate

Experimental studies of the test chemical and various supporting weight of evidence studies for its structurally similar read across chemical were reviewed for short term toxicity to aquatic invertebrate end point which are summarized as below:

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of M7 media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 990.12 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 1.79 mg/L,  3.04 mg/L, 5.17 mg/L,8.79 mg/L and 14.9 mg/L, respectively was from the saturated test concentration. Study was performed using 20 daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 20°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be 6.32 mg/L (4.22-11386). Thus, based on the EC50 value, chemical was considered as toxic to aquatic invertebrates and hence, considered to be classified in 'aquatic chronic category 2' as per CLP classification criteria.

 

In an experimental study from study report, an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on aquatic invertebrates. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp., Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 100.0 mg/l was prepared by dissolving dark orange powder in reconstituted water. The test solutions of required concentrations were prepared by mixing the stock solution of the test sample in reconstituted water. Study was performed using 5 organisms per vessel/replicates in a static system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also run simultaneously during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. The 24 hr EC50 value of reference substance was determined to be 0.76 mg/l. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 6.2 mg/l (95% C. I. = 3.5 to 11.2 mg/l).

 

In a supporting weight of evidence study (A. Kahru et. al., 2000), short term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical. The bioassays were performed according to the standard operational procedures of the Toxkits (Daphtoxkit FTM magna). Daphtokit FTM magna follows the standard protocol as specified in the OECD TG 202 and ISO test method respecitvely. Test chemical was dissolved in deionized water. Daphnia magna (Water flea) neonates was used as a test organism. Test organism was fed with Spirulina powder. Test daphnids were exposed to test chemcial using standardized microplate test containers constructed of biologically inert materials ensure uniform exposure conditions as specified in Daphtoxkit FTM magna was used as a test vessel during the study. Study was performed under static conditions in dark for 48 hrs at 20°C. Based on the effect on mobility of the test organism, the 48 hr LC50 value was determined to be 11.2 mg/l.

 

For the test chemical, short term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical (HSDB). Study was performed using Daphnia magna as a test organism under static conditions for 48 hrs. Based on the effect on mobility of the test organism, the 48 hr LC50 value was determined to be 16 mg/l.

 

Another short term toxicity to aquatic invertebrate study was carried out for assessing the effect of test chemical (from James Devillers, 1988 and secondary source). The study was performed following the AFNOR test method. Acetone (volume not exceeded 0.1 ml/l) was used as a dispersant solvent. Test chemical was diluted with AFNOR reconstituted hard water (pH = 7.8 to 8.2, hardness = 200 mg/l as CaCO3) for toxicity tests. Daphnia magna Straus 1820 (water flea) of < 72 h old obtained from IRCHA Laboratory was used as a test organism. Test daphnids have been cultured parthenogenetically in the Pasteur Institute Laboratory. Daphnids were cultured in 10 l tanks, with aerated hardwater (7.5 + 0.4mg/l as Ca; 5.2 + 0.3 mg/l as Mg; pH = 7). The breeding room was maintained at a regulated temperature of 22 + I°C under a photoperiod of 16 h daylight/8 h darkness. During breeding, test organism was fed with a diet of Chlorella vulgaris (1000000 cells/daphnia/h). Preliminary tests were performed in order to determine the range of concentrations at which the definitive test should be carried out. A range of geometrical concentrations were chosen so as to include chiefly the concentrations which had given 0, 10, 90, and 100% immobilization during the preliminary tests. Test daphnids (5 daphnids/vessel) were exposed to different test chemical concentrations (i.e., 0.1, 0.35, 1, 3.5, 10, 35, 100 and 350 mg/l arrange in single geometric series) in a test tube. Test vessel was covered with plastic stoppers and placed in dark conditions for 24 hrs. No aeration was provided in the test vessel during the study. During the course of the experiments, test daphnids were not fed. Control tubes (reconstituted water and dispersent-solvent) were included in the tests. Potassium dichromate was used as a reference substance during the study. All experiments were performed in triplicates. After an exposure period of 24 hr, observations of test organisms were carried out. Daphnids which were unable to swim within 15 s after stimulation by gentle agitation of the water were considered to be immobilized, even if they could still move their antennae. On the other hand; dissolved oxygen, pH, and temperature were measured at the end of the toxicity tests. The statistics, i.e., percentages of immobilization (between 10 and 90% on the basis of total number of Daphnia per concentration) were recorded and plotted as a function of concentration on log-probit paper. The points obtained were fitted to a straight line from which the IC50 was read as the abscissa of the point corresponding to 50% immobilization. The EC50 value of the reference substance was determined to be in the between range of 0.9 to 1.5 mg/l. On the basis of the effect of test chemical on mobiity of the test daphnids, the 24 hr IC50 was determined to be 13.56 mg/l with 95% C. I. of 12.338 to 14.659 mg/l.

 

In an additional study from publication (2000), short term toxicity to Thamnocephalus platyurus study was carried out for assessing the effect of test chemical. The bioassays were performed according to the standard operational procedures of the Toxkits (Thamnotoxkit FTM). Test chemical was dissolved in deionized water. Thamnocephalus platyurus larvae were exposed to test chemcial in test plate and incubated under static conditions in dark for 24 hrs at 25°C. Based on the effect on mortality of the test organism, the 24 hr LC50 value was determined to be 6 mg/l.

 

In a supporting weight of evidence study, short term toxicity to aquatic invertebrate study was carried out for 48 hrs for assessing the effect of test chemical (from peer reviewed journal and secondary sources). Ceriodaphnia dubia (Water flea) of <12 h old was used as a test organism. Test organism were acclimatized under the test conditions as reported in the main study. Each test organism was fed daily with 100 µl of yeast-trout chow -Cerophyl mixture and 50 µl of a Selenastrum capricornutum suspension. Stock solutions of the test chemical was prepared by dissolving toxicant directly into culture medium. Stock solutions were diluted to desired concentrations (4 test concentrations and 1 control) with culture medium. Test chemical concentrations were analyzed by using appropriate analytical technique atleast once during the test. Concentrations were analyzed by reverse-phase HPLC using a Water Resolve C18 column. Detection was performed on either a Hitachi F1000 fluorescence detector or a Waters 490 variable wavelength UV-Vis detector, depending on the spectral characteristics of the organic chemical. Concentration values were determined by computerized peak integration and comparison to known standards. Thus, 4 total test chemical concentrations were taken for the study. Test daphnids (10 test organism) were exposed to different concentrations of test chemical in 30 ml polystyrene cups for 48 hrs. Test conditions involve a 16:8 Light:dark photoperiod with a cool white fluorescent lamps at an intensity of 28 lux, a temperature of 25 ± 1°C, hardness of 57.07 ± 4.14 mg/l as CaCO3, pH 8.18 ± 0.04 and alkalinity of 81.00 ± 4.22 mg/l as CaCO3, respectively. All experiments were performed in a replicate. For every 24 hr over a 48 hr exposure period, mortality of the test organism was noted. 48 hr LC50 value were estimated using TOXCALC, an IBM microcomputer adaptation of a program, which calculates LC50 estimates utilizing binomial probabilities, moving averages and/or probit analysis. Survival of control animals in acute test was ≥90%. On the basis of the effect of test chemical on mortality fo the test daphnids, the 48 hr LC50 value was determined to be 3.1 mg/l.

 

For the test chemical, an acute toxicity study was conducted on aquatic invertebrates for 48 hrs for assessing the effect of test chemical (from Gerald A. LeBlanc, 1980). The study was performed using the U. S. EPA method. Daphnia magna (Water flea) of < 24 hr old obtained from laboratory stocks cultured at EG&G, Bionomics was used as a test organism. Water used to culture the organisms was deionized reconstituted well water having a total hardness of 72 + 6 mg/L as CaCO3 and a pH of 7.0 + 0.2, respectively. A stock solution of the chemical was prepared in distilled water and used to provide the desired concentrations for testing. Further, test chemical was added to 500 ml of diluted water in 2 lit jar to prepared test solution. At the initiation of test, the dissolved oxygen concentration of diluent water was > 60% of saturation. Test daphnids were exposed to test chemical in 250 ml beaker. Five daphnids were randomly placed in each 150 mL test solution within 30 minutes of the solution preparation. Test vessel was incubated for a period of 48 hrs at a temperature of 22 ± 1°C and under test conditions such as hardness of water as 72 mg/L as CaCO3, 6.7 to 8.1 and dissolved oxygen of 6.5 to 9.1 mg/l, respectively. All test experiments were performed in triplicates. A negative and positive control was run simultaneously during the study. Negative control vessel consists of the same dilution water, test conditions, and test organisms, but containing no test chemical or co-solvent. On the other hand, positive control consists of same dilution water, conditions, and a number of test organisms as in the negative control and containing the highest concentration of the co-solvent present in any test vessel. The tests were also conducted in unreplicated 500 mL solutions containing 15 daphnids if dividing the solution into triplicate test vessels presented a risk of the loss of the  test substance through volatilization or if vapors of the substance posed a high health risk to the investigators. During these tests, the dissolved oxygen concentration, pH and temperature of test solutions were measured at the initiation and termination of the toxicity tests in the high, middle and low test concentrations and controls. Dissolved oxygen concentration and temperature were measured with a YSI Model 54BP dissolved oxygen meter and combination oxygen-temperature probe. The pH's were measured with an Instrumentation Laboratory pH meter. No correction was made for control mortality. At 24 and 48 hr of an exposure period, observations of the test populations and any mortalities were recorded. The LC50's and 95% confidence limits were calculated utilizing a moving average angle method when possible. With the moving average angle method, the nominal test concentrations were transformed to logarithms and the corresponding percentage mortalities to angles. Each group of three successive angles was then averaged and the LC50 was estimated by linear interpolation between the successive concentrations whose average angles bracketed 45 °C. When test data did not meet the moving average angle method requirements, the LC50's were estimated by probit analysis by converting the concentrations to logarithms and percentage mortalities to probits and then calculating a least squares linear regression analysis. Finally, if the data did not permit a probit analysis, then a binomial probability analysis was performed on these data. Calculations were performed with a Hewlett-Packard Model 9815A calculator programmed to scan the data base. Mortality among water flea control populations never exceeded 10% in the test. On the basis of the effect of test chemical on mortality fo the test daphnids, the 24 and 48 hr LC50 value was determined to be 8.3 mg/l (95% C. I. = 5.9 to 11 mg/l) and 2.1 mg/l (95% C. I. = 1.8 to 2.5 mg/l), respectively.

On the basis of the above results, it can be concluded that the test chemical was considered to be toxic to aquatic invertebrates and hence, considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.

Toxicity to aquatic algae and cyanobacteria

Experimental studies of the test chemical and various supporting weight of evidence studies for its structurally similar read across chemical were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

Short term toxicity to Algae:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 250mg of test chemical in 250ml of OECD media, to get at concentratino of 1000 mg/l , which was then filtered and the final saturated stock solution obtained was 989.8 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 7000 -8000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 57.5 mg/l (calculated from equation through probit analysis).

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 250mg of test chemical in 250ml of OECD media, to get at concentratino of 1000 mg/l , which was then filtered and the final saturated stock solution obtained was 989.8 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 7000 -8000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 57.5 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria. the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% (i.e., reported as 12.66%) and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10% (i.e., reported as 5.27%), thus, fulfilling the validity of the criteria. All the cells appeared healthy, round and green throughout the study duration in the control and no significant changes were observed up to the concentration of 16 mg/l. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value was determined to be 2.726 mg/l.

 

In a supporting weight of evidence study, toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 100 mg/l was prepared by dissolving test chemical in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 10, 18, 32, 58 and 100 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 1.7% (in control). The 24 hr EC50 value of the reference substance was determined to be 0.77 mg/l. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr EC50 value was determined to be 92.3 mg/l (95% C. I. 73.1 to 116.5 mg/l) (nominal concentration).

 

Another toxicity to aquatic algae study was carried out for assessing the effect of test chemical (from A. Kahru et. al., 2000). The bioassays were performed according to the standard operational procedures of the Toxkits (Algaltoxkit FTM). Algaltoxkit FTM follows the standard protocol as specified in the OECD TG 201 and ISO test method respecitvely. Test chemical was dissolved in deionized water. Selenastrum capricornutum green algae) was used as a test organism. The initial algal culture was prepared from the immobilized algal beads and the deimmobilized cells were pregrown in the sterile growth medium (25°C, 6000 - 8000 lux). Before the experiment the culture was diluted to OD670 = 0.001 (corresponds to approx. 10000 cells/ml) and cultivated further for 4 days. Study was performed under static conditions for 72 hrs at 25°C under a test condition with light intensity of 6000 to 8000 lux. The inhibition of the growth rate of algae Selenastrum capricornutum was measured. On the basis of the effect of test chemical on growth rate of the test algae, the 72 hr EC50 was determined to be 50 mg/l.

 

For the test chemical from secondary source (2012), toxicity to aquatic algae study was carried out for assessing the effect of test chemical. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) ATCC22662 obtained from American Type Culture Collection was used as a test organism. 200mg of the test substance was dissolved with dilution water to produce a stock solution of 1000 mg/L . Test solution was prepared in each test vessel by mixing the appropriate volume of the stock solution with dilution water.  Test chemical concentrations were verified analytically by HPLC-UV instrument. For the analytical determnations, samples were diluted with pure water. Thus, test chemical concentrations used for the study were  0, 5.6, 10, 18, 32, 56 and 100 mg/l (nominal concentrations) and 0, 5.41, 9.64, 17.5, 31.1, 54.2 and 96.8 mg/l (measured conc.), respectively. Test algae was exposed with the test chemical in an 300 ml erlenmeyer flask with silicon rubber plug (air permeability). Standard OECD medium was used as a test medium for the study. Test organism was exposed for a period of 72 hr and at test conditions of 23°C temperature, pH 8.8-9.1 (at the start of exposure) and 8.1-10.4 at the end of exposure under a continuous photoperiod with light intensity of 4000-5000 Lx. Dilution water was used for the control group. All experiment was performed in triplicates. Algal cells were counted with a particle counter at 24, 48, and 72 h. Microscopic observation were done at 72 h. The pH of the test solution was measured at the start, and the end of the exposure. The temperature in the incubator and light intensity were measured once a day during the exposure. Potassium dichromate was used as a reference substance. The 72 hr EbC50 value of the reference substance potassium dichromate was determined to be 0.65 mgl. On the basis of the effect of test chemical on growth rate of the test algae, the 72 hr NOEC and EC50 value was determined to be 32 and >100 mg/l (nominal conc.). On the basis of effect on biomass, the 72 hr NOEC and EC50 value was determined to be 31.1 and 55.6 mg/l (measured (arithmetic mean) conc.), respectively.

 

In a supporting weight of evidence study from authoritative database, toxicity to aquatic algae study was carried out for assessing the effect of test chemical. The study was performed following the principles of OECD Guideline 201 (Alga, Growth Inhibition Test) under static conditions. Based on the effect of test chemical on growth rate of the test algae, the 72 hr NOEC and EC50 value was determined to be 1.8 and 9.7 mg/l. On the basis of effect on AUG, the 72 hr NOEC and EC50 value was determined to be 2.0 and 6.7 mg/l, respectively.

 

On the basis of the above results, it can be concluded that the test chemical was considered to be toxic to aquatic algae and hence, considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.

Toxicity to microorganisms

Various experimental studies of the test chemical were reviewed for toxicity to micro-organisms end point which are summarized as below:

 

In an experimental study from peer reviewed journal (2000), toxicity test was performed with Laboratory-prepared freeze-dried photobacterial reagent (Photobacterium phosphoreum strain FEI 162095) registered in the Finnish Environment Institute. The freeze-dried bacteria were reconstituted by adding 12.5-ml 2% NaCl into the reagent bottle and incubating it at +4°C for 20 min before use. The concentration of test chemical in the test (mg/L) which caused a 50% reduction in light after exposure of was measured after an exposure period of 15 min. The EC50 value of test chemical was determined to be 28 mg/l for Photobacterium phosphoreum bacteria.

 

In a supporting weight of evidence study, mirotox test was performed with Vibrio fischeri to assess the toxicity of test chemical. MicrotoxTM Reagent (Vibrio fischeri NRRL-B 11177). (AZUR Environmental, Carlsbad, California, USA). The freeze-dried reagent was reconstituted and bacteria cultivated, harvested, and frozen in cryoprotectant as described in paper (Kahru et al., 1996). The thawed bacterial suspension was used for the toxicity tests. The amount of viable bacteria in the case of all photo bacterial tests was ~10⁶ bacteria per ml test solution. The concentration of test chemical in the test (mg/L) which caused a 50% reduction in light was measured after exposure of test chemical for 15 min. The EC50 value of test chemical was considered to be 5.3 mg/l in Vibrio fischeri NRRL-B 11177.

 

Biotox test was performed with Vibrio fisheri to find toxicity of test chemical. Vibrio fisheri 1500 Reagent (Vibrio fischeri NRRL-B11177) from the BioTox test (Labsystems, Helsinki, Finland). The freeze-dried reagent was reconstituted with 12.5-ml 2% NaCl, incubated at +4°C for 20 min and, prior to the assay, further diluted five-fold with 2% NaCl. The concentration of test chemical in the test (mg/L) which caused a 50% reduction in light was measured after exposure of test chemical for 15 min. The EC50 value of test chemical was considered to be 5.2 mg/l in Vibrio fischeri NRRL-B 11177.

 

For the test chemical, toxicity study of micro-organism study to the test chemical was conducted using Photobacterium phosphoreum, strain NRRL-B-11177 (also referred to as Vibrio fischerii, strain NRRL-B-11177) (Klaus L.E. Kaiser et. al., 1991). The study was performed at a temperature of 15°C and pH range 5 to 9, respectively. Recommneded reference substance that can be used for the study were Phenol and Sodium pentachlorophenate, respectively. Test bacteriumPhotobacterium phosphoreum, strain NRRL-B-11177 was exposed to the test chemical, reduction in light output was observed. Thus, based on this effect, the EC50 value during 30 min exposure period was determined to be 3.36 mg/l.

 

On the basis of the experimental studies, the EC50 value of the test chemical on test organism was determined to be ranges from 3.36 to 28 mg/l.

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 and hence, considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.