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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
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)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test chemical solution was prepared by dissolving 12.5 mg of test substance in 250 ml of BBM to get the final concentration of 50 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 initialcell 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
- 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
Test temperature:
24°C ± 2°C
pH:
6.5 - 6.8
Nominal and measured concentrations:
Nominal test chemical conc. used for the study were 0, 1.5625, 3.125, 6.25, 12.5, 25 and 50 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: 10000 cells/ml
- No. of organisms per vessel: 10000 cells/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: 1.5625 mg/l, 3.125 mg/l, 6.25 mg/l, 12.5 mg/l, 25 mg/l and 50 mg/l
- 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):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated from equation through probit analysis
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12.59 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Calculated graphically
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 while following changes were observed at the higher concentrations.
• Decrease in cell count
• The substance was both adsorbed and absorbed on the 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) were 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

46.5 x 104

10.95 x 105

13.3 x 105

Replicate 2

71 x 104

10.55 x 105

14.55 x 105

Replicate 3

75 x 104

10.65 x 105

14.45 x 105

Test chemical

1.5625 mg/l

Replicate 1

51 x 104

55.5 x 104

66.5 x 104

Replicate 2

46.5 x 104

52.5 x 104

65 x 104

3.125 mg/l

Replicate 1

56.5 x 104

51 x 104

46 x 104

Replicate 2

56 x 104

35.5 x 104

48.5 x 104

6.25 mg/l

Replicate 1

52 x 104

34 x 104

32 x 104

Replicate 2

51 x 104

34.5 x 104

29.5 x 104

12.5 mg/l

Replicate 1

51 x 104

32 x 104

18.5 x 104

Replicate 2

52.5 x 104

34.5 x 104

23 x 104

25 mg/l

Replicate 1

44 x 104

35.5 x 104

10.5 x 104

Replicate 2

49.5 x 104

33 x 104

11.5 x 104

50 mg/l

Replicate 1

50.5 x 104

30.5 x 104

4.5 x 104

Replicate 2

43.5 x 104

29 x 104

1 x 104

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

 

CONTROL

1.5625 mg/l

3.125 mg/l

6.25 mg/l

12.5 mg/l

25 mg/l

50 mg/l

Average Specific Growth rate (µ )

R1

1.630

R1

1.399

R1

1.276

R1

1.155

R1

0.972

R1

0.783

R1

0.501

 

R2

1.660

R2

1.391

R2

1.293

R2

1.128

R2

1.045

R2

0.814

R2

0

 

R3

1.657

 

Mean of Avg. Specific growth rate

1.649

1.395

1.285

1.141

1.008

0.798

0.250

Percentage Inhibition (%I)

_

15.403

22.086

30.777

38.830

51.558

84.800

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

6.6

7.0

Replicate 2

6.8

7.1

Replicate 3

6.6

7.3

Test chemical

1.5625 mg/l

Replicate 1

6.4

7.2

Replicate 2

6.6

7.1

3.125 mg/l

Replicate 1

6.7

7.4

Replicate 2

6.5

6.9

6.25 mg/l

Replicate 1

6.8

6.6

Replicate 2

6.5

6.8

12.5 mg/l

Replicate 1

6.6

6.6

Replicate 2

6.4

6.7

25 mg/l

Replicate 1

6.5

6.8

Replicate 2

6.4

6.6

50 mg/l

Replicate 1

6.5

6.6

Replicate 2

6.5

6.6

Conclusions:
Based on the growth inhibition of green alga Chlorella vulgaris by the test chemical, the 72 hr median effect concentration (ErC50) calculated from equation and graphically through probit analysis was determined to be 14.05 and 12.59 mg/l.
Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance was prepared by dissolving 12.5 mg of test substance in 250 ml of BBM to get the final concentration of 50 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical ( 0, 1.5625, 3.125, 6.25, 12.5, 25 and 50 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). All the cells appeared healthy, round and green throughout the study duration in the control while changes were observed at the higher concentration which includes, decrease in cell count and substance was both adsorbed and absorbed on the cells. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value calculated from equation and graphically through probit analysis was determined to be 14.05 and 12.59 mg/l. On the basis of the EC50 value, chemical was considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Description of key information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris (Study report, 2015). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance was prepared by dissolving 12.5 mg of test substance in 250 ml of BBM to get the final concentration of 50 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical ( 0, 1.5625, 3.125, 6.25, 12.5, 25 and 50 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). All the cells appeared healthy, round and green throughout the study duration in the control while changes were observed at the higher concentration which includes, decrease in cell count and substance was both adsorbed and absorbed on the cells. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value calculated from equation and graphically through probit analysis was determined to be 14.05 and 12.59 mg/l. On the basis of the EC50 value, chemical was considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
14.05 mg/L

Additional information

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris (Study report, 2015). The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. Bold’s Basal Medium (BBM) composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test substance was prepared by dissolving 12.5 mg of test substance in 250 ml of BBM to get the final concentration of 50 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical ( 0, 1.5625, 3.125, 6.25, 12.5, 25 and 50 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 24±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). All the cells appeared healthy, round and green throughout the study duration in the control while changes were observed at the higher concentration which includes, decrease in cell count and substance was both adsorbed and absorbed on the cells. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value calculated from equation and graphically through probit analysis was determined to be 14.05 and 12.59 mg/l. On the basis of the EC50 value, chemical was considered to be toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.