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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:
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 chemical 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:
As the chemical was stable
Vehicle:
no
Details on test solutions:
The test solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 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 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
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
24, 48, 72 hrs
Test temperature:
24 ° C ± 2°C
pH:
6.5 - 7
Nominal and measured concentrations:
2 mg/l, 4.4 mg/l, 9.68 mg/l, 21.296 mg/l, 46.8512 mg/l and 103.07 mg/l nominal concentrations were used in the study. 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 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.
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
50.26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Effect were also observed at the concentration 50.12 mg/l.
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 following changes were observed with increase in test substance concentration.
• Decrease in cell count
• Discoloration of algal cells
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

75.5 x 104

97.5 x 104

12.4 x 105

Replicate 2

80.5 x 104

10.7 x 105

14.4 x 105

Replicate 3

87.5 x 104

95 x 104

14.7 x 105

Test chemical

2 mg/l

Replicate 1

71 x 104

98.5 x 104

74 x 104

Replicate 2

70 x 104

93 x 104

69.5 x 104

4.4 mg/l

Replicate 1

64.5 x 104

98 x 104

62.5 x 104

Replicate 2

61 x 104

35.5 x 104

61 x 104

9.68 mg/l

Replicate 1

79 x 104

95 x 104

48.5 x 104

Replicate 2

73.5 x 104

91.5 x 104

50.5 x 104

21.296 mg/l

Replicate 1

59 x 104

91 x 104

28 x 104

Replicate 2

58.5 x 104

91.5 x 104

36 x 104

46.8512 mg/l

Replicate 1

51.5 x 104

77.5 x 104

11 x 104

Replicate 2

51 x 104

79 x 104

11.5 x 104

103.07 mg/l

Replicate 1

50.5 x 104

77.5 x 104

5 x 104

Replicate 2

48.5 x 104

64.5 x 104

5.5 x 104

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

 

CONTROL

2 mg/l

4.4 mg/l

9.68 mg/l

21.296 mg/l

46.8512 mg/l

103.07 mg/l

Average Specific Growth rate (µ )

R1

1.606

R1

1.434

R1

1.378

R1

1.293

R1

1.110

R1

0.799

R1

0.536

 

R2

1.658

R2

1.413

R2

1.370

R2

1.307

R2

1.194

R2

0.814

R2

0.568

 

R3

1.663

 

Mean of Avg. Specific growth rate

1.643

1.424

1.374

1.300

1.152

0.806

0.552

Percentage Inhibition (%I)

_

13.317

16.354

20.842

29.848

50.901

66.381

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

6.9

7.0

Replicate 2

7.1

6.9

Replicate 3

7.0

7.0

Test chemical

2 mg/l

Replicate 1

6.8

6.9

Replicate 2

6.8

6.8

4.4 mg/l

Replicate 1

6.5

6.6

Replicate 2

6.7

6.6

9.68 mg/l

Replicate 1

6.7

6.5

Replicate 2

6.5

6.6

21.296 mg/l

Replicate 1

6.5

6.5

Replicate 2

6.5

6.5

46.8512 mg/l

Replicate 1

6.4

6.5

Replicate 2

6.5

6.5

103.07 mg/l

Replicate 1

6.6

6.5

Replicate 2

6.5

6.4

Validity criteria fulfilled:
yes
Conclusions:
Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determine to be 50.26mg/L and 50.12mg/L calculated from equation and graphically 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 solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 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 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 of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test 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). 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 solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 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 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 of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26mg/L and 50.12mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test chemical was toxic and can be consider to be classified as aquatic chronic 3 as per the CLP classification criteria.

But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
50.26 mg/L

Additional information

Based on the various experimental data for the test chemical study have been reviewed to determine the toxic nature of test chemical on the growth of aquatic algae and cyanobacteria. The studies are as mentioned 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). 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 solution was prepared in aseptic condition. The test substance was prepared by adding 25.767 µl of test substance in 250 ml of BBM to get the final concentration of 103.07 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 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 of test organism with test chemical to various nominal test concentrations, EC50 was determine to be 50.26 mg/L and 50.12 mg/L graphically and through probit analysis. Based on the EC50, it can be concluded that the test 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 the second study from peer reviewed journal. Toxicity of test chemical was accessed on the growth of green algae Microcystis aeruginosa. Experiments were carried out in 250mL conical flasks containing 94.9mL CT liquid medium, and 100L of test solutions in dimethyl sulfoxide (DMSO). Prior to these experiments, 0.2% (v/v) DMSO had no obvious effect on the growth and photosynthetic processes of M. aeruginosa NIES-843. Five milliliters of exponential-phase cultures of M. aeruginosa NIES-843 (in CT medium) were added to the conical flasks in order to reach a final volume of 100 mL. The final test concentrations were set as 0, 6.67, 10, 15, 22.5, 33.75 mg/L, respectively, and the initial cellular concentrations of M. aeruginosa NIES-843 were 6.5×100000 cells/mL. Each treatment was replicated three times. All treatments were cultured under the same conditions as mentioned above. The increase in M. aeruginosa cell number was calculated after 48 h using a hemocytometer. Chl a, and carotenoid content were determined. Cultures were sampled 48 h after inoculation to measure content of photosynthetic pigments. The EC50 of test chemical on the growth of M. aeruginosa NIES-843 was shown to be 21.23±1.87 mg/L. Results based on transcript expression of genes, polyphasic Chl a fluorescence transients and ultrastructural examinations through TEM indicated that the reaction centre of PS II and electron transport at the acceptor side of PS II are the targets responsible for the toxicity of test chemical on the PS II of M. aeruginosa NIES-843. Based on the EC50 value, chemical consider to be toxic and consider to be classified as per the CLP classification criteria.

 

Similarly in the third study short term toxicity study of test chemical was performed on the growth of aquatic algae Scenedesmum subspicatus. Test conducted under the static system for total exposure period of 96 hrs, effect after 72 hrs were also observed. After the exposure of test chemical for 96 hrs effect were measured on the basis of growth rate inhibition and biomass of Scenedesmum subspicatus. On the basis of biomass and growth rate for 72 hrs, the EC50 was determine as 20.9 and 22.9 mg/l, respectively. After the exposure period of 96 hrs the effect were observed at 12.2 and 21.9 mg/l.

 

Thus based on the above experimental data it was concluded that the chemical was toxic and consider to be classified as aquatic chronic category 3 as per the CLP classification criteria. But as the chemical was readily biodegradable in water, thus on that basis chemical consider to be nontoxic and not classified as per the CLP classification criteria.