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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard test guidelines
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
The test solution was prepared in aseptic condition. The test item was prepared by adding 4 mg of test item in 250ml of BBM to get the final concentration of 16 mg/L. This stock solution was kept for stirring for 20 minutes 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 10 to the power 4 cells/ml. Care was taken to have a homogeneous solution for the experiment.
Test organisms (species):
Chlorella vulgaris
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
22 °C ±2°C.
pH:
No data
Dissolved oxygen:
No data
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
0.5mg/l,1mg/l,2mg/l,4mg/l,8mg/l,16mg/l
Details on test conditions:
Parameter Conditions
1. Study Type: Static
2. Exposure Duration: 72 Hours
3. Temperature: 22 °C ±2°C.
4. Light Quality: White Fluorescent Light
5. Light Intensity: 1500Lux
6. Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
7. RPM Speed: 120 Revolutions per minute
8. Water: Deionized Water
9. Test vessel size: 100ml
10. Test volume: 60ml
11. Dilution water: Bold’s Basal Medium
12. Test concentration: Six test concentration were: 0.5mg/l,1mg/l,2mg/l,4mg/l,8mg/l,16mg/l
13. Replicates per concentration: Three replicates for Control; Two replicates for each test concentration
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
10.58 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Average specific growth rate and percentage inhibition after an interval of 72 hours:

 

CONTROL

0.5 mg/l

1 mg/l

2 mg/l

4 mg/l

8 mg/l

16 mg/l

Average

Specific

Growth rate (μ)

R1

0.231

R1

0.195

R1

0.180

R1

0.130

R1

0.139

R1

0.112

R1

0.112

R2

0.237

R2

0.164

R2

0.164

R2

0.164

R2

0.130

R2

0.148

R2

0.121

R3

0.268

 

Mean of Avg.

Specific

growth rate

0.245

0.180

0.172

0.147

0.135

0.130

0.116

Percentage

Inhibition (%I)

-

26.613

29.695

39.884

45.035

47.041

52.468

 

 pH values at 0 Hours and after 72 Hours of test item exposure to algae

Test vessels and

test concentration

0 Hours

72 Hours

CONTROL

Replicate1

6.77

6.84

Replicate2

6.68

6.88

Replicate3

6.71

6.87

Average

6.72

6.86

 

0.5 mg/l

Replicate1

6.68

7.01

Replicate2

6.73

6.99

1 mg/l

Replicate1

6.71

6.98

Replicate2

6.70

7.00

2 mg/l

Replicate1

6.70

6.99

Replicate2

6.68

6.95

4 mg/l

Replicate1

6.67

6.97

Replicate2

6.63

7.03

8 mg/l

Replicate1

6.70

7.05

Replicate2

6.68

7.10

16 mg/l

Replicate1

6.70

7.12

Replicate2

6.66

7.15

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure of test item to nominal test concentrations of 0.5 mg/L,1 mg/L,2 mg/L,4 mg/L,8 mg/L,16 mg/L, EC50 was determined to be 10.580 mg/l for Chlorella vulgaris graphically and through probit analysis.
Executive summary:

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 0.5 mg/L,1 mg/L,2 mg/L,4 mg/L,8 mg/L,16 mg/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 10.580 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was hazrdous and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

Description of key information

Toxicity to aquatic algae and cyanobacteria:

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 concentrations0.5 mg/L,1 mg/L,2 mg/L,4 mg/L,8 mg/L,16 mg/Lwere 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 10.580 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was hazrdous and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
10.58 mg/L

Additional information

Toxicity to aquatic algae and cyanobacteria:

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 concentrations0.5 mg/L,1 mg/L,2 mg/L,4 mg/L,8 mg/L,16 mg/Lwere 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 10.580 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was hazrdous and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.