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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
for read across
Justification for type of information:
Data for the target chemical is summarized based on the structurally similar read across chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
WoE report is based on two toxicity study of aquatic algae for the test chemical :
The effect of test substance was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga growth inhibition test.
GLP compliance:
no
Analytical monitoring:
no
Details on test solutions:
2.The test substance test material was prepared by adding 75 mg of test substance in 150 ml of BBM to get the final concentration of 500 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml.

3.The test substance , was prepared by adding 106.25 mg of test substance in 250 ml of BBM to get the final concentration of 425 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above stock solution, under aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml. Care was taken to have a homogeneous solution for the experiment.

4.The test substance was prepared by adding 60 µl of test substance in 300 ml of BBM to get the final concentration of 200 mg/L. The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10e4 cells/ml. Care was taken to have a homogeneous solution for the experiment.
Test organisms (species):
Chlorella vulgaris
Details on test organisms:
The fresh water green alga Chlorella vulgaris, was used as the test organism. Sterile, unicellular, liquid cultures of algae.
The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated.
The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Test temperature:
24 °C ±2°C.
pH:
6.7 to 7.4
Nominal and measured concentrations:
2) 6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200 All the six concentration were in geometric series spaced by a factor of 2.

3) 4.32mg/l, 10.88 mg/l, 27.2mg/l, 68mg/l, 170mg/l and 425mg/l All the six concentration were in geometric series spaced by a factor of 2.

4)6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/l nominal concentration were used in the study. All the six concentrations were in geometric series spaced by a factor of 2.
Details on test conditions:
Light Quality White Fluoroscent Light
Light Intensity: 1500Lux
Photoperiod 16 Hour Light Period : 8 Hour Dark Period
RPM Speed 120 Revolutions per minute
Water Deionized Waer
Test vessel size 100ml
Test solution volume 60ml
Dilution water :Bold’s Basal Medium
Test concentration Six test concentration were:
Replicates per concentration
Three replicates for Control
Two replicates for each test concentration
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
220.8 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated from equation through probit analysis
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
138.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated from equation through probit analysis
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated from equation through probit analysis
Validity criteria fulfilled:
not specified
Conclusions:
After 72 hours of exposure to test material to various nominal test concentration, EC50 calculated from equation through probit analysis was found to be in the range of 138.96 to 220 mg/L.
Executive summary:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae of the test chemical .The studies are as mentioned below:

For a read across substance , the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

The test substance test material was prepared by adding 75 mg of test substance in 150 ml of BBM to get the final concentration of 500 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml.

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/l were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item ( ) to various nominal test concentrations, EC50 was determine to be 220.89 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic for aquatic algae and can be consider to be not classified as per the CLP classification criteria.

For another read across , the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized.The test substance , was prepared by adding 106.25 mg of test substance in 250 ml of BBM to get the final concentration of 425 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above stock solution, under aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml. Care was taken to have a homogeneous solution for the experiment.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 138.96 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was non-toxic and can be consider to be not classified as per the CLP classification criteria.

Data from read across substance was used to further support the above data , the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance 1-phenylethan-1 ol was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations 6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/l were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201. After 72 hours of exposure to test item 1-phenylethan-1 ol to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis.

Based on the EC50 values , it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Description of key information

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae of the test chemical .The studies are as mentioned below:

For a read across substance ,the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

The test substance test material was prepared by adding 75 mg of test substance in 150 ml of BBM to get the final concentration of 500 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml.

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/l were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item ( ) to various nominal test concentrations, EC50 was determine to be 220.89 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic for aquatic algae and can be consider to be not classified as per the CLP classification criteria.

For another read across , the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized.The test substance , was prepared by adding 106.25 mg of test substance in 250 ml of BBM to get the final concentration of 425 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above stock solution, under aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml. Care was taken to have a homogeneous solution for the experiment.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 138.96 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was non-toxic and can be consider to be not classified as per the CLP classification criteria.

Data from read across substance was used to further support the above data , the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations 6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/l were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201. After 72 hours of exposure to test to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis.

Based on the EC50 values , it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
220 mg/L

Additional information

Toxicity to aquatic algae and cyanobacteria:

Data available for the structurally and functionally similar read across chemicals has been reviewed to determine the toxicity of aquatic algae of the test chemical .The studies are as mentioned below:

For a read across substance ,the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

The test substance test material was prepared by adding 75 mg of test substance in 150 ml of BBM to get the final concentration of 500 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above prepared stock solution under aseptic conditions. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml.

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/l were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item ( ) to various nominal test concentrations, EC50 was determine to be 220.89 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic for aquatic algae and can be consider to be not classified as per the CLP classification criteria.

For another read across , the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized.The test substance , was prepared by adding 106.25 mg of test substance in 250 ml of BBM to get the final concentration of 425 mg/L. The sock solution was ultrasonically agitated for 30 minutes to obtain a homogenous solution for the experiment. The remaining test solutions were prepared by dilution from the above stock solution, under aseptic condition. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104 cells/ml. Care was taken to have a homogeneous solution for the experiment.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item to various nominal test concentrations, EC50 was determine to be 138.96 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was non-toxic and can be consider to be not classified as per the CLP classification criteria.

Data from read across substance was used to further support the above data , the study was designed to assess the toxic effects of the test compound on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test). Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring/ sonication for 0 minutes to obtain a homogenous solution for the experiment. The test concentrations 6.25 mg/l, 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200mg/l were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution. For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined. Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate. As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201. After 72 hours of exposure to test to various nominal test concentrations, EC50 was determine to be >200 mg/l graphically and through probit analysis.

Based on the EC50 values , it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.