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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:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
This 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).
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test solution was prepared in aseptic condition. As the test substance has a very low water solubility value, a stock WAF (Water Accommodated Fraction) solution was prepared by adding 100 mg of test substance in 1 Litre of BBM with a constant mechanical stirring for 48 hours. The remaining test solutions were prepared by dilution from the stock WAF. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 cells/ml. Care was taken to have a homogeneous solution for the experiment.
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
Total exposure duration:
72 h
Post exposure observation period:
24, 48, 72 hrs
Test temperature:
24 °C ±2°C.
Nominal and measured concentrations:
2.5mg/l , 3.75m/l, 5.62mg/l, 8.43mg/l, 12.65mg/l and 18.98 mg/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: 2.5mg/l , 3.75m/l, 5.62mg/l, 8.43mg/l, 12.65mg/l and 18.98mg/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 24° 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.
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
20.42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Other details not known
Duration:
72 h
Dose descriptor:
other: EL30
Effect conc.:
7.943 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Other details not known
Details on results:
The microscopic observations were also noted in each of the experimental flasks. While in the control flasks, all the cells appeared healthy, round and green throughout the test duration, the cells in all the test loading rates appeared to be deformed and shrinked.
Reported statistics and error estimates:
To obtain a quantitative concentration-response relationship by regression analysis, a linearising transformation of the response data into probit was performed. Using the same, effective loading rates (EL) were determined. ELs are considered as equivalent to standard effective concentrations (EC) for sparingly soluble or complex substances.

Test item loading rates: The effect of the test substance on the green algaeChlorella vulgaris culture was observed at nominal loading rates of 2.5mg/l, 3.75mg/l, 5.62mg/l, 8.43mg/l, 12.65mg/l and 18.98mg/l. All the six loading rates were in geometric series spaced by a factor of 1.5.

The microscopic observations were also noted in each of the experimental flasks. While in the control flasks, all the cells appeared healthy, round and green throughout the test duration, the cells in all the test loading rates appeared to be deformed and shrinked.

The deformity in the shape of the cells was clearly observed after 48 hours of test duration wherein the cell count had also decreased. At the end of the test, when the cells of test vessels were compared to that of control, change in the morphology and density of the cells could be observed clearly.

 

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

Experimental Flasks and Loading Rates

24 Hours

48 Hours

72 Hours

Control

Replicate 1

75 x104

90 x104

103.5 x104

Replicate 2

77 x104

96 x104

109.5 x104

Replicate 3

70.5 x104

84.5 x104

103 x104

Test chemical

2.5 mg/l

Replicate 1

74 x104

63.5 x104

55.5 x104

Replicate 2

53 x104

69.5 x104

58.5 x104

3.75 mg/l

Replicate 1

35.5 x104

71 x104

51 x104

Replicate 2

32.5 x104

69 x104

45.5 x104

5.62mg/l

Replicate 1

20 x104

67 x104

37 x104

Replicate 2

18 x104

67.5 x104

33.5 x104

8.43mg/l

Replicate 1

31 x104

61 x104

24.5 x104

Replicate 2

26 x104

64.5 x104

24 x104

12.65mg/l

Replicate 1

31 x104

23.5 x104

20 x104

Replicate 2

32.5 x104

26 x104

19.5 x104

18.98mg/l

Replicate 1

25 x104

31 x104

10 x104

Replicate 2

24 x104

30 x104

10 x104

 

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

 

CONTROL

2.5mg/l

3.75mg/l

5.62mg/l

8.43mg/l

12.65mg/l

18.98mg/l

Average Specific Growth rate (µ )

R1

1.546

R1

1.338

R1

1.310

R1

1.203

R1

1.066

R1

0.998

R1

0.767

 

R2

1.565

R2

1.356

R2

1.272

R2

1.170

R2

1.059

R2

0.990

R2

0.767

 

R3

1.544

 

Mean of Avg. Specific growth rate

1.552

1.347

1.291

1.187

1.062

0.994

0.767

Percentage Inhibition (%I)

 _

13.185

16.792

23.525

31.532

35.940

50.553

 

 

 

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

 

Experimental Flasks and Loading Rates

0 Hours

72 Hours

CONTROL

Replicate 1

7.3

7.5

Replicate 2

7.3

7.5

Replicate 3

7.1

7.2

Test chemical

2.5mg/l

Replicate 1

7.2

7.6

Replicate 2

7.0

7.6

3.75mg/l

Replicate 1

7.2

7.5

Replicate 2

7.2

7.5

5.62mg/l

Replicate 1

7.2

7.6

Replicate 2

7.18

7.6

8.43mg/l

Replicate 1

7.3

7.8

Replicate 2

7.14

7.6

12.65mg/l

Replicate 1

7.2

7.7

Replicate 2

7.15

7.7

18.98mg/l

Replicate 1

7.3

7.8

Replicate 2

7.18

7.7

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure with test chemical to various nominal test loading rates, EL50 value for Chlorella vulgaris was observed to be 20.4213 mg/l and EL30 was observed to be at 7.943 mg/l when calculated 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).

The test solution was prepared in aseptic condition. As the test substance has a very low water solubility value, a stock WAF (Water Accommodated Fraction) solution was prepared by adding 100 mg of test substance in 1 Litre of BBM with a constant mechanical stirring for 48 hours. The remaining test solutions were prepared by dilution from the stock WAF. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 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.

After 72 hours of exposure to test item to various nominal test concentrations, EL50 was determine to be 20.4213 mg/l graphically and and EL30 was observed to be at 7.943 mg/l when calculated through probit analysis. Based on the EL50, it can be concluded that the chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

Description of key information

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 solution was prepared in aseptic condition. As the test substance has a very low water solubility value, a stock WAF (Water Accommodated Fraction) solution was prepared by adding 100 mg of test substance in 1 Litre of BBM with a constant mechanical stirring for 48 hours. The remaining test solutions were prepared by dilution from the stock WAF. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 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.

After 72 hours of exposure to test item to various nominal test concentrations, EL50 was determine to be 20.4213 mg/l graphically and and EL30 was observed to be at 7.943 mg/l when calculated through probit analysis. Based on the EL50, it can be concluded that the chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
20.421 mg/L

Additional information

Summarized result for the toxicity of test chemical on the growth of algae were mention below:

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 solution was prepared in aseptic condition. As the test substance has a very low water solubility value, a stock WAF (Water Accommodated Fraction) solution was prepared by adding 100 mg of test substance in 1 Litre of BBM with a constant mechanical stirring for 48 hours. The remaining test solutions were prepared by dilution from the stock WAF. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 10000 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. After 72 hours of exposure to test item to various nominal test concentrations, EL50 was determine to be 20.4213 mg/l graphically and and EL30 was observed to be at 7.943 mg/l when calculated through probit analysis. Based on the EL50, it can be concluded that the chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

Above study was supported by second study from authoritative database. Study was conducted to determine the effect of test chemical on the growth of algae. The no observed effect concentration (NOEC) value of test chemical in aquatic algae in a 72 hr study on the basis of growth rate effect and areas under the growth curves was determine to be 3.2 and 1.8 mg/L respectively. Also the EC50 value was reported to be 9.7 mg/L.

Thus based on the above both study, it was concluded that the chemical consider to be classified as toxic and in aquatic chronic 3 category.