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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
Study period:
22/05/2017 - 25/05/2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result performed using standard test guidelines
Qualifier:
equivalent or similar 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
Vehicle:
yes
Details on test solutions:
The test solution was prepared in aseptic condition. The test item was prepared by adding 4 mg of test item in 250 ml of BBM to get the final concentration of 16 mg/L. This stock solution was kept for stirring for 20 minutes to obtain a homogenous solution for the experiment. The test concentrations 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. To have a better growth and visibility of cells, the initial of the culture was kept 1 X 104cells/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 system (organism). Sterile, unicellular, suspension cultures of algae were obtained from National Environmental Engineering Research Institute (NEERI), Nagpur and maintained at Unique Ecotox Research Laboratory, Nagpur. 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
Post exposure observation period:
24, 48, 72 hrs
Test temperature:
22 °C ±2°C.
pH:
6.7 to 7.4
Nominal and measured concentrations:
0.5mg/l,1mg/l,2mg/l,4mg/l,8mg/l,16mg/l- All the six concentration were in geometric series spaced by a factor of 2.
Details on test conditions:
1. Study Type: Static
2. Exposure Duration: 72 Hours
3. Temperature: 22 °C ±2°C.
4. Light Quality: White Fluorescent Light
5. Light Intensity: 1500Lux
6. Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
7. RPM Speed: 120 Revolutions per minute
8. Water: Deionized Water
9. Test vessel size: 100ml
10. Test volume: 60ml
11. Dilution water: Bold’s Basal Medium
12. Test concentration: Six test concentration were:0.5mg/l,1mg/l,2mg/l,4mg/l,8mg/l,16mg/l
13. Replicates per concentration: Three replicates for Control, Two replicates for each test concentration
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.249 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
Details on results:
The microscopic observations were also noted in each of the test vessel. All the cells appeared healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 16 mg/l. EC50 was found to be 9.249 mg/l graphically through probit analysis.

Average specific growth rate and percentage inhibition after an interval of 72 hours:

 

CONTROL

0.5mg/l

1 mg/l

2 mg/l

4 mg/l

8 mg/l

16 mg/l

Average

Specific

Growth rate (μ)

R1

0.231

R1

0.188

R1

0.262

R1

0.210

R1

0.291

R1

0.262

R1

0.256

R2

0.237

R2

0.172

R2

0.172

R2

0.237

R2

0.164

R2

0.217

R2

0.231

R3

0.268

 

Mean of Avg.

Specific

growth rate

0.245

0.180

0.217

0.224

0.228

0.240

0.243

Percentage

Inhibition (%I)

-

26.613

11.375

8.869

7.110

2.323

0.799

 

 pH values at 0 Hours and after 72 Hours of test item exposure to algae

Test vessels and

test concentration

0 Hours

72 Hours

CONTROL

Replicate1

6.77

6.84

Replicate2

6.68

6.88

Replicate3

6.71

6.87

Average

6.72

6.86

 

0.5 mg/l

Replicate1

6.63

7.68

Replicate2

6.67

7.98

1 mg/l

Replicate1

6.70

7.26

Replicate2

6.71

7.21

2 mg/l

Replicate1

6.71

7.22

Replicate2

6.78

7.19

4 mg/l

Replicate1

6.77

7.17

Replicate2

6.74

7.14

8 mg/l

Replicate1

6.67

7.05

Replicate2

6.72

7.04

16 mg/l

Replicate1

6.68

7.02

Replicate2

6.68

7.01

 

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to test chemical to nominal test concentrations of 0.5mg/L,1mg/L,2mg/L,4mg/L,8mg/L and 16mg/L, EC50 was determined to be 9.249 mg/l graphically and through probit analysis.
Executive summary:

The effect of test chemical was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.5mg/L,1mg/L,2mg/L,4mg/L,8mg/L,16mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 9.249 mg/L.

This value indicates that the test chemical is toxic to aquatic algae and hence can be classified as Aquatic chronic toxic category 2 as per the CLP regulation.

Description of key information

The effect of test chemical was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.5mg/L,1mg/L,2mg/L,4mg/L,8mg/L,16mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 9.249 mg/L.

This value indicates that the test chemical is toxic to aquatic algae and hence can be classified as Aquatic chronic toxic category 2 as per the CLP regulation.

Key value for chemical safety assessment

EC50 for freshwater algae:
9.456 mg/L

Additional information

In the following information from experimental studies report and different peer reviewed journal on toxicity to aquatic algae and cyanobacteria for target chemical is summarized:

The effect of test chemical was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 0.5mg/L,1mg/L,2mg/L,4mg/L,8mg/L,16mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 9.249 mg/L.This value indicates that the test chemical is toxic to aquatic algae and hence can be classified as Aquatic chronic toxic category 2 as per the CLP regulation.

Toxicity to algae test was performed on Pseudokirchneriella subcapitata for test chemical for 7 days. Basic ASM was used as a test medium with test conditions as 20 degree C and at pH 7.Based on population changes effect, the No-observable-effect-concentration (i.e.NOEC) value of test chemical to Pseudokirchneriella subcapitata was determined to be 1 mg/l.

Toxicity to algae test was performed on Pseudokirchneriella subcapitata for test chemical for 7 days. Basic ASM was used as a test medium with test conditions as 20 degree C and at pH 7.Based on population changes effect, the Lowest-observable-effect-concentration (i.e.LOEC) value of test chemical to Pseudokirchneriella subcapitata was determined to be 10 mg/l.

The main objective of this study was to understand the effect of test chemical on the growth of fresh water algae, Desmodesmus subspicatus. The test was performed, in accordance with the OECD 201 guideline. A range of concentration was selected (2.5, 5, 10, 20, and 40 mg/L ) in a geometric sequence of 2, In parallel to the test chemical, control was also run simultaneously in triplicates.The study was conducted in the static exposure conditions, for the period of 3 days (72h). After the exposure period the growth rate was calculated, to evaluate the inhibition caused by the test chemical. Based on the obtained results, the EC50 was calculated to be 20.5 mg/L. As per the new CLP classification criteria the test chemical can be classified into aquatic chronic 3 category.

Even though varied results for toxicity to aquatic algae and cyanobacteria were available but, based on the maximum notifiers in ECHA and the effect concentration from first experimental study report and data from peer reviewed journal,we can consider that the test chemical is toxic and can be classified as aquatic chronic 2.

 

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