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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 substance
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 and cyanobacteria for the test chemical :
2.Aim of this study was to evaluate the nature of chemical when comes in contact with the test organism. Test was conducted according to the OECD guideline 201.
3.This study was designed to assess the effect of test material on the growth of green alga Chlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201- Alga, growth inhibition test”.
4.Short term toxicity to Chlorella pyrenoidosa (green algae) study was carried out for 72 hrs.Details on sampling
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Details on test solutions:
2.The stock solution 200 mg/l was prepared by dissolving light grey powder 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.

3.This study was designed to assess the effect of test material on the growth of green alga Chlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201- Alga, growth inhibition test”.

4.Short term toxicity to Chlorella pyrenoidosa (green algae) study was carried out for 72 hrs.Details on sampling
- Concentrations: Five conc. levels of test chemical were used for the tests (100, 500, 1000, 1500 and 2000 mg/l, respectively).
- Sampling method: No data available
- Sample storage conditions before analysis: No data available

Test organisms (species):
other: 2.Desmodesmus subspicatus 3.Chlorella vulgaris 4.Chlorella pyrenoidosa
Details on test organisms:
2.Desmodesmus subspicatus
TEST ORGANISM
- Common name: green algae
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Institute of botany of the ASCR
- Initial biomass concentration: 5x10(3) cells /ml
- Method of cultivation: No data available

ACCLIMATION - No data available

3.- Strain: No data
- Source (laboratory, culture collection): National Environmental Engineering Research Institute (NEERI), Nagpur
- Age of inoculum (at test initiation): 1x10E4 cell/ml
- Method of cultivation: no data

ACCLIMATION
- Acclimation period:no data
- Culturing media and conditions (same as test or not):no data
- Any deformed or abnormal cells observed: no

4.- Common name: Green algae
- Strain: Emerson strain of bacteria free, experimentally reproducible cultures of Chlorella pyrenoidosa was used as a test organism.
- Method of cultivation: A test tubes was used in the screening and final tests. These test tubes contained 15 ml of inorganic culture medium, a predetermined amount of test chemical and 5 ml of algal culture. The tubes were incubated for 72 hrs and chlorophyll content of the algal suspensions was measured every 24 hrs. Control tubes containing no test chemical was also used in the experiment.

Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Hardness:

3) 22 ° C ± 2°C.
4) 25 ± 1°C
pH:
2) the sample at concentration 120.0 mg/l: pH = 7.1 changed to pH=7.3 during the test,
control: pH = 8.2 changed to pH = 7.4 during the test
Nominal and measured concentrations:
2) 2.2 , 11 , 25 , 55 , 120 mg/l
3) 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l, 100mg/l, 200mg/l
4)100, 500, 1000, 1500 and 2000 mg/l
Details on test conditions:
2) TEST SYSTEM
- Test vessel: 15 ml Glass vessel
- Sample volume: 50 ml
- Initial cells density: 5x10(3) cells/ml
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 6000-8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: microscope with counting chamber Cyrus I or electronic particle counter.

3) TEST SYSTEM
- Test vessel: All the tests were carried out in 100mL conical flasks which were carefully autoclaved and sterilized.
- Type : No data available
- Material, size, headspace, fill volume: 60ml
- Aeration: No data available
- Type of flow-through (e.g. peristaltic or proportional diluter): No data available
- Renewal rate of test solution (frequency/flow rate): No data available
- Initial cells density: 13.625 x10E4 cells/mL
- Control end cells density: No data available
- No. of organisms per vessel: No data available
- No. of vessels per concentration (replicates): Two replicates
- No. of vessels per control (replicates): Three replicates
- No. of vessels per vehicle control (replicates): No data available

GROWTH MEDIUM
- Standard medium used: Yes
- The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrien ts, 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.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: White Fluorescent Light (1500Lux)
- RPM speed: 120 Revolutions per minute

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell counting: Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer.
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.
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.
- Chlorophyll measurement: No
- Other: EC50 value was determined.

4)TEST SYSTEM
- Test vessel: Test tubes
- Initial cells density: 1 gm/l dry weight


TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Knop's solution, including the Hutner-EDTA microelement addition, was used as the culture medium.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes, pH of culture medium was adjusted to 7.0 using KOH before use.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Yes
- Chlorophyll measurement: Effect of the test substance on the algae was evaluated by measuring the changes in chlorophyll content of the algal suspensions every 24 hr.


TEST CONCENTRATIONS
- Range finding study: Yes, screening tests were performed to establish the threshold and algicidic concentrations.
- Test concentrations: Five conc. levels of test chemical were used for the tests (100, 500, 1000, 1500 and 2000 mg/l, respectively).

Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
129 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CI 108.4 - 154.3 mg/l
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: No substantial toxicity of test substance was observed at the mentioned effect conc. value.
Validity criteria fulfilled:
not specified
Conclusions:
The test chemical is not likely to be toxic to aquatic algae and cyanobacteria atleast in the concentration range of 129 - 1000 mg/l.
Executive summary:

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

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).The stock solution 200 mg/l was prepared by dissolving light grey powder 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, 2.2 , 11 , 25 , 55 , 120 mg/lconcentration were used. 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 non linear 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 129.4 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 can not be classified as per the CLP classification criteria.

Data from another study report for 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).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized.The test solution was prepared in aseptic condition. The test item  was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours to obtain a homogenous solution for the experiment. The test concentrations 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. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 10E4cells/ml.

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 >200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Above data was further supported by data from peer reviewed journal for read across substance,short term toxicityto Chlorella pyrenoidosa(green algae) study was carried out for 72 hrs. Emerson strain of bacteria free, experimentally reproducible cultures ofChlorella pyrenoidosawas used as a test organism. The procedure involve the use of test tubes in both the screening and final tests. These test tubes contained 15 ml of inorganic culture medium, a predetermined amount of test chemical and 5 ml of algal culture. The tubes were incubated for 72 hrs andchlorophyll content of the algal suspensions was measured every 24 hrs. For chlorophyll measurement, the chlorophyll pigment was extracted with hot methanol in two separate extractions. An algal suspension, 2.5 ml, was removed from the test tube, centrifuged, washed with distilled water, and recentrifuged in preparation of chlorophyll analysis. After discarding the supernatant, the deposited cell material was coagulated by placing the cells in a boiling water bath for about 40 sec. About 2.5 ml of methanol were used in each extraction. Finally, the chlorophyll solution was diluted to a total volume of 10 ml with an acetone-water mixture (80 per cent by volume). A Beckman Spectrophotometer, Model DB, was used to measure the chlorophyll content according to MACKIN~v (1941) and ARNON (1949). For this a wavelength of 652 m/z was used because different proportions of chlorophyll a and b least affect the results at this wavelength. Control tubes containing no test chemical was also used in the experiment. Knop's solution, including the Hutner-EDTA microelement addition, was used as the culture medium.pH of culture medium was adjusted to 7.0 using KOH before use. The test organism was maintained under steady-state conditions, provided a chlorophyll content of 38 mg/l. Environmental control was rigidly maintained. The temp. of water bath was 25 ± 1°C. The test apparatus consisted of a constant-temperature water bath, a light source containing four 200W fluorescent lamps with attached aluminum reflectors, a gas manifold to supply an air-CO2 mixture to each test tube, and a rack to hold the test tubes. A stream of 5 % CO2 in air gas mixture was supplied to culture medium in order to provide the inorganic carbon source and also to keep the algal ceils in suspension. Based on destruction of chlorophyll of test organism by test chemical Amino-1-phenol-4-sulfonic acid, the LOEC value was found to be1500 mg/l and as no toxic effect at 1000 mg/l was observed, the NOEC value was found to be 1000 mg/l.Thus, based on this value, it can be concluded that the substancecan be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

Description of key information

Toxicity to aquatic algae and cyanobacteria:

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

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).The stock solution 200 mg/l was prepared by dissolving light grey powder 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, 2.2 , 11 , 25 , 55 , 120 mg/lconcentration were used. 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 non linear 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 129.4 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 can not be classified as per the CLP classification criteria.

Data from another study report for 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).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized.The test solution was prepared in aseptic condition. The test item  was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours to obtain a homogenous solution for the experiment. The test concentrations 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. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 10E4cells/ml.

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 >200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Above data was further supported by data from peer reviewed journal for read across substance,short term toxicityto Chlorella pyrenoidosa(green algae) study was carried out for 72 hrs. Emerson strain of bacteria free, experimentally reproducible cultures ofChlorella pyrenoidosawas used as a test organism. The procedure involve the use of test tubes in both the screening and final tests. These test tubes contained 15 ml of inorganic culture medium, a predetermined amount of test chemical and 5 ml of algal culture. The tubes were incubated for 72 hrs andchlorophyll content of the algal suspensions was measured every 24 hrs. For chlorophyll measurement, the chlorophyll pigment was extracted with hot methanol in two separate extractions. An algal suspension, 2.5 ml, was removed from the test tube, centrifuged, washed with distilled water, and recentrifuged in preparation of chlorophyll analysis. After discarding the supernatant, the deposited cell material was coagulated by placing the cells in a boiling water bath for about 40 sec. About 2.5 ml of methanol were used in each extraction. Finally, the chlorophyll solution was diluted to a total volume of 10 ml with an acetone-water mixture (80 per cent by volume). A Beckman Spectrophotometer, Model DB, was used to measure the chlorophyll content according to MACKIN~v (1941) and ARNON (1949). For this a wavelength of 652 m/z was used because different proportions of chlorophyll a and b least affect the results at this wavelength. Control tubes containing no test chemical was also used in the experiment. Knop's solution, including the Hutner-EDTA microelement addition, was used as the culture medium.pH of culture medium was adjusted to 7.0 using KOH before use. The test organism was maintained under steady-state conditions, provided a chlorophyll content of 38 mg/l. Environmental control was rigidly maintained. The temp. of water bath was 25 ± 1°C. The test apparatus consisted of a constant-temperature water bath, a light source containing four 200W fluorescent lamps with attached aluminum reflectors, a gas manifold to supply an air-CO2 mixture to each test tube, and a rack to hold the test tubes. A stream of 5 % CO2 in air gas mixture was supplied to culture medium in order to provide the inorganic carbon source and also to keep the algal ceils in suspension. Based on destruction of chlorophyll of test organism by test chemical Amino-1-phenol-4-sulfonic acid, the LOEC value was found to be1500 mg/l and as no toxic effect at 1000 mg/l was observed, the NOEC value was found to be 1000 mg/l.Thus, based on this value, it can be concluded that the substancecan be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
200 mg/L

Additional information

Toxicity to aquatic algae and cyanobacteria:

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

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).The stock solution 200 mg/l was prepared by dissolving light grey powder 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, 2.2 , 11 , 25 , 55 , 120 mg/lconcentration were used. 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 non linear 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 129.4 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 can not be classified as per the CLP classification criteria.

Data from another study report for 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).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized.The test solution was prepared in aseptic condition. The test item  was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 24 hours to obtain a homogenous solution for the experiment. The test concentrations 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. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 10E4cells/ml.

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 >200 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was not toxic and can be consider to be not classified as per the CLP classification criteria.

Above data was further supported by data from peer reviewed journal for read across substance,short term toxicityto Chlorella pyrenoidosa(green algae) study was carried out for 72 hrs. Emerson strain of bacteria free, experimentally reproducible cultures ofChlorella pyrenoidosawas used as a test organism. The procedure involve the use of test tubes in both the screening and final tests. These test tubes contained 15 ml of inorganic culture medium, a predetermined amount of test chemical and 5 ml of algal culture. The tubes were incubated for 72 hrs andchlorophyll content of the algal suspensions was measured every 24 hrs. For chlorophyll measurement, the chlorophyll pigment was extracted with hot methanol in two separate extractions. An algal suspension, 2.5 ml, was removed from the test tube, centrifuged, washed with distilled water, and recentrifuged in preparation of chlorophyll analysis. After discarding the supernatant, the deposited cell material was coagulated by placing the cells in a boiling water bath for about 40 sec. About 2.5 ml of methanol were used in each extraction. Finally, the chlorophyll solution was diluted to a total volume of 10 ml with an acetone-water mixture (80 per cent by volume). A Beckman Spectrophotometer, Model DB, was used to measure the chlorophyll content according to MACKIN~v (1941) and ARNON (1949). For this a wavelength of 652 m/z was used because different proportions of chlorophyll a and b least affect the results at this wavelength. Control tubes containing no test chemical was also used in the experiment. Knop's solution, including the Hutner-EDTA microelement addition, was used as the culture medium.pH of culture medium was adjusted to 7.0 using KOH before use. The test organism was maintained under steady-state conditions, provided a chlorophyll content of 38 mg/l. Environmental control was rigidly maintained. The temp. of water bath was 25 ± 1°C. The test apparatus consisted of a constant-temperature water bath, a light source containing four 200W fluorescent lamps with attached aluminum reflectors, a gas manifold to supply an air-CO2 mixture to each test tube, and a rack to hold the test tubes. A stream of 5 % CO2 in air gas mixture was supplied to culture medium in order to provide the inorganic carbon source and also to keep the algal ceils in suspension. Based on destruction of chlorophyll of test organism by test chemical Amino-1-phenol-4-sulfonic acid, the LOEC value was found to be1500 mg/l and as no toxic effect at 1000 mg/l was observed, the NOEC value was found to be 1000 mg/l.Thus, based on this value, it can be concluded that the substancecan be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP criteria.