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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 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:
As chemical is stable
Vehicle:
no
Details on test solutions:
The test solution was prepared in aseptic condition. The test substance tert-butyl-4-methoxyphenol was prepared by adding 10 mg of test substance in 100 ml of BBM to get the final concentration of 100 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. Care was taken to have a homogeneous solution for the experiment.
Test organisms (species):
Chlorella vulgaris
Details on test organisms:
The fresh water green alga Chlorella vulgaris, was used as the test organism. Sterile, unicellular, liquid cultures of algae.
The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated.
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.
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Test temperature:
24 °C ±2°C.
pH:
6.7 to 7.2
Nominal and measured concentrations:
1.5, 2.7, 4.86, 8.748, 15.75 and 28.34 mg/L. All the six concentration were in geometric series spaced by a factor of 1.8.
Details on test conditions:
Light Quality White Fluoroscent Light
Light Intensity 1500Lux
Photoperiod 16 Hour Light Period : 8 Hour Dark Period
RPM Speed 120 Revolutions per minute
Water Deionized Waer
Test vessel size 100ml
Test solution volume 60ml
Dilution water Bold’s Basal Medium
Test concentration Six test concentration were: 1.5, 2.7, 4.86, 8.748, 15.75 and 28.34 mg/L
Replicates per concentration
Three replicates for Control
Two replicates for each test concentration
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculation from equation
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculation from graph
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 in the experimental flask following changes were observed with increase in test substance concentration.
Decrease in cell count
Discoloration of algal cells
Reduced cell size
Fine particles observed inside the cell

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 test concentration

24 Hours

48 Hours

72 Hours

Control

Replicate 1

49.5 x 104

66 x 104

10.5 x 105

Replicate 2

46 x 104

59 x 104

10.55 x 105

Replicate 3

54.5 x 104

66.5 x 104

10.8 x 105

Test chemical

1.5 mg/l

Replicate 1

41.5 x 104

50.5 x 104

47 x 104

Replicate 2

44.5 x 104

48.5 x 104

59.5 x 104

2.7 mg/l

Replicate 1

56 x 104

43 x 104

44 x 104

Replicate 2

48 x 104

35.5 x 104

40 x 104

4.86 mg/l

Replicate 1

39 x 104

40 x 104

28 x 104

Replicate 2

36.5 x 104

43.5 x 104

30 x 104

8.75 mg/l

Replicate 1

32.5 x 104

37 x 104

9 x 104

Replicate 2

44 x 104

37 x 104

10.5 x 104

15.75 mg/l

Replicate 1

39.5 x 104

35.5 x 104

5 x 104

Replicate 2

44.5 x 104

34.5 x 104

8.5 x 104

28.34 mg/l

Replicate 1

46.5 x 104

38.5 x 104

3 x 104

Replicate 2

41 x 104

33 x 104

2.5 x 104

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

 

CONTROL

1.5 mg/l

2.7 mg/l

4.86 mg/l

8.75 mg/l

15.75 mg/l

28.34 mg/l

Average Specific Growth rate (µ )

R1

1.551

R1

1.283

R1

1.261

R1

1.110

R1

0.732

R1

0.536

R1

0.366

 

R2

1.552

R2

1.361

R2

1.229

R2

1.33

R2

0.783

R2

0.713

R2

0.305

 

R3

1.560

 

Mean of Avg. Specific growth rate

1.554

1.322

1.245

1.122

0.758

0.624

0.335

Percentage Inhibition (%I)

_

14.938

19.901

27.829

51.246

59.811

78.403

 

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to test substance to various nominal test concentration, EC50 calculated from equation and graphically through probit analysis was observed to be 9.05 mg/L and 9.33 mg/L respectively.
Executive summary:

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). The test solution was prepared in aseptic condition. The test substance Tert-butyl-4-methoxyphenol was prepared by adding 10 mg of test substance in 100 ml of BBM to get the final concentration of 100 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 104cells/ml. Care was taken to have a homogeneous solution for the experiment. The effect of the test substance on the green algae Chlorella vulgaris culture was observed at nominal test concentration of 1.5, 2.7, 4.86, 8.748, 15.75 and 28.34 mg/L. 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. 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. Decreased in cell count and reduced cell size.  After 72 hours of exposure to test substance to various nominal test concentration, EC50 calculated from equation and graphically through probit analysis was observed to be 9.05 mg/L and 9.33 mg/L respectively.

Description of key information

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). The test solution was prepared in aseptic condition. The test substance Tert-butyl-4-methoxyphenol was prepared by adding 10 mg of test substance in 100 ml of BBM to get the final concentration of 100 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 104cells/ml. Care was taken to have a homogeneous solution for the experiment. The effect of the test substance on the green algae Chlorella vulgaris culture was observed at nominal test concentration of 1.5, 2.7, 4.86, 8.748, 15.75 and 28.34 mg/L. 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. 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. Decreased in cell count and reduced cell size.  After 72 hours of exposure to test substance to various nominal test concentration, EC50 calculated from equation and graphically through probit analysis was observed to be 9.05 mg/L and 9.33 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
9.33 mg/L

Additional information

Based on the various data for the test chemical, studies were reviewed for the toxicity evaluation on the growth of algae and cyanobacteria end point which are summarized as below: 

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). The test solution was prepared in aseptic condition. The test substance Tert-butyl-4-methoxyphenol was prepared by adding 10 mg of test substance in 100 ml of BBM to get the final concentration of 100 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 104cells/ml. Care was taken to have a homogeneous solution for the experiment. The effect of the test substance on the green algae Chlorella vulgaris culture was observed at nominal test concentration of 1.5, 2.7, 4.86, 8.748, 15.75 and 28.34 mg/L. 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. 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. Decreased in cell count and reduced cell size.  After 72 hours of exposure to test substance to various nominal test concentration, EC50 calculated from equation and graphically through probit analysis was observed to be 9.05 mg/L and 9.33 mg/L respectively.

 

Determination of short term toxicity of test chemical on the growth of aquatic algae and cyanobacteria. Test conducted for 72 hrs. Effect were observed on the basis of growth rate inhibition and area under the growth curve. The 72 hour EC50 value of test chemical in green algae was determine to be 1.9 mg/L based on its effect on the growth rate (area under the growth curve) and 5.2 on the basis of growth rate. The NOEC was observed at 0.25 mg/l. Thus based on the EC50 value, chemical consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

Thus based on the above effects, chemical consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.