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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 according to the guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
This study was designed to access 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).
GLP compliance:
no
Analytical monitoring:
no
Remarks:
Stable in recommended storage conditions
Vehicle:
no
Details on test solutions:
The test substance , was prepared by adding 100 mg of test substance in 250 ml of BBM to get the final concentration of 400 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 104 cells/ml.
Test organisms (species):
Chlorella vulgaris
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Source (laboratory, culture collection): National Environmental Engineering Research Institute (NEERI), Nagpur (Laboratory)
- Method of cultivation: Bold’s Basal Medium(BBM)


ACCLIMATION
- Culturing media and conditions (same as test or not): 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
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
24, 48, 72 hrs
Test temperature:
22 °C±2°C
Nominal and measured concentrations:
12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l, 200 mg/l and 400mg/l All the six concentration were in geometric series spaced by a factor of 2.
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: 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.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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 data
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Test concentrations: Six test concentration were: 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l and 200mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Mortality of test organisms


Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 22 ° C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
other: EC30
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated graphically
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 400 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: through probit analysis
Details on results:
The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.
Reported statistics and error estimates:
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) was determined.

Table 1: Showing the average cell count using Haemocytometer of the test vessels at an equal interval of 24hrs, 48hrs and 72hrs

Experimental Flasks and test concentration

24 Hours

48 Hours

72 Hours

Control

Replicate 1

88.5 x 104

74.5 x 104

14 x 105

Replicate 2

85 x 104

75 x 104

13.8 x 105

Replicate 3

89.5 x 104

87.5 x 104

12.95 x 105

12.5 mg/l

Replicate 1

60 x 104

57.5 x 104

84.5 x 104

Replicate 2

57.5 x 104

58.5 x 104

10.2 x 105

25 mg/l

Replicate 1

58.5 x 104

96.5 x 104

10.2 x 105

Replicate 2

62.5 x 104

97 x 104

99 x 104

50 mg/l

Replicate 1

23 x 104

40 x 104

87 x 104

Replicate 2

25 x 104

43 x 104

88 x 104

100 mg/l

Replicate 1

35 x 104

33.5 x 104

36 x 104

Replicate 2

36.5 x 104

37.5 x 104

45 x 104

200 mg/l

Replicate 1

21 x 104

41 x 104

26.5 x 104

Replicate 2

24 x 104

43.5 x 104

30 x 104

400 mg/l

Replicate 1

15.5 x 104

39.5 x 104

22 x 104

Replicate 2

19.5 x 104

40 x 104

27.5 x 104

Table 2 : Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

 

CONTROL

12.5 mg/l

25 mg/l

50 mg/l

100 mg/l

200 mg/l

400 mg/l

Average Specific Growth rate (µ )

R1

1.647

R1

1.478

R1

1.541

R1

1.488

R1

1.194

R1

1.092

R1

1.030

 

R2

1.606

R2

1.541

R2

1.531

R2

1.492

R2

1.268

R2

1.133

R2

1.104

 

R3

1.621

 

Mean of Avg. Specific growth rate

1.625

1.510

1.536

1.490

1.231

1.113

1.067

Percentage Inhibition (%I)

_

7.063

5.438

8.278

24.206

31.507

34.308

Table 3: Depicting pH values at test initiation (0 Hours) and test termination ( 72 Hours)

Experimental Flasks and test concentration

0 Hours

72 Hours

CONTROL

Replicate 1

7.7

7.4

Replicate 2

7.7

7.5

Replicate 3

7.7

7.4

CAS No. 14852-17-6

12.5 mg/l

Replicate 1

7.6

6.3

Replicate 2

7.6

6.2

25 mg/l

Replicate 1

7.6

6.2

Replicate 2

7.6

6.1

50 mg/l

Replicate 1

7.6

6.0

Replicate 2

7.6

6.0

100 mg/l

Replicate 1

7.7

6.1

Replicate 2

7.7

7.9

200 mg/l

Replicate 1

7.7

7.9

Replicate 2

7.7

7.9

400 mg/l

Replicate 1

7.7

7.8

Replicate 2

7.7

7.8

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to test material to various nominal test concentration, EC50 calculated from equation through probit analysis and EC30 calculated graphically was found to be >400 mg/L and 200 mg/L respectively.
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). The test substance was prepared by adding 100 mg of test substance in 250 ml of BBM to get the final concentration of 400 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 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 concentrations12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l, 200 mg/l and 400mg/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 material to various nominal test concentration, EC50 calculated from equation through probit analysis and EC30 calculated graphically was found to be >400 mg/L and 200 mg/L respectively. Based on the EC50, it can be concluded that the chemical was non-hazardous and can be consider to be not classified 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). The test substance was prepared by adding 100 mg of test substance in 250 ml of BBM to get the final concentration of 400 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 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 concentrations12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l, 200 mg/l and 400mg/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 material to various nominal test concentration, EC50 calculated from equation through probit analysis and EC30 calculated graphically was found to be >400 mg/L and 200 mg/L respectively. Based on the EC50, it can be concluded that the chemical was non-hazardous and can be consider to be not classified as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for marine water algae:
400 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). The test substance was prepared by adding 100 mg of test substance in 250 ml of BBM to get the final concentration of 400 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 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 concentrations12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l, 200 mg/l and 400mg/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 material to various nominal test concentration, EC50 calculated from equation through probit analysis and EC30 calculated graphically was found to be >400 mg/L and 200 mg/L respectively. Based on the EC50, it can be concluded that the chemical was non-hazardous and can be consider to be not classified as per the CLP classification criteria.