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Ecotoxicological information

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Experimental test result using OECD guideline.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
Storage Conditions
Store in cool place. Keep container tightly closed in a dry and well-ventilated place. Substance is Hygroscopic.
Vehicle:
yes
Details on test solutions:
The test test chemical was prepared by adding 60 mg of test substance in 300 ml of BBM to get the final concentration of 200 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 10E4 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
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 °C ±2°C.
pH:
6.7 to 7.4
Nominal and measured concentrations:
12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200 mg/l
Details on test conditions:
Light Quality White Fluoroscent Light
Light Intensity 1500Lux
Photoperiod 16 Hour Light Period :6 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:
Replicates per concentration
Three replicates for Control
Two replicates for each test concentration
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
75.25 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 experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.

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

39 x 104

41.5 x 104

47 x 104

Replicate 2

28 x 104

46.5 x 104

34 x 104

Replicate 3

34 x 104

42.5 x 104

44 x 104

CAS No. 54-21-7

12.5 mg/l

Replicate 1

27 x 104

17 x 104

19.5 x 104

Replicate 2

27.5 x 104

20.5 x 104

12 x 104

25 mg/l

Replicate 1

10.5 x 104

16 x 104

14.5 x 104

Replicate 2

12.5 x 104

18.5 x 104

15.5 x 104

50 mg/l

Replicate 1

28.5 x 104

11.5 x 104

5.5 x 104

Replicate 2

22 x 104

12.5 x 104

10 x 104

100 mg/l

Replicate 1

22.5 x 104

3.5 x 104

6 x 104

Replicate 2

35 x 104

6 x 104

3.5 x 104

200 mg/l

Replicate 1

20 x 104

22 x 104

2.5 x 104

Replicate 2

24.5 x 104

22 x 104

7 x 104

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

 

CONTROL

12.5 mg/l

25 mg/l

50 mg/l

100 mg/l

200 mg/l

Average Specific Growth rate (µ )

R1

1.283

R1

0.990

R1

0.891

R1

0.568

R1

0.597

R1

0.305

R2

1.175

R2

0.828

R2

0.913

R2

0.767

R2

0.417

R2

0.648

R3

1.261

 

Mean of Avg. Specific growth rate

1.240

0.909

0.902

0.667

0.507

0.477

Percentage Inhibition (%I)

_

26.69

27.26

46.21

59.11

61.53

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

7.3

7.4

Replicate 2

7.2

7.3

Replicate 3

7.4

7.5

CAS No. 54-21-7

12.5 mg/l

Replicate 1

7.3

7.2

Replicate 2

7.0

7.2

25 mg/l

Replicate 1

7.3

7.4

Replicate 2

7.4

7.3

50 mg/l

Replicate 1

7.2

7.2

Replicate 2

7.1

7.1

100 mg/l

Replicate 1

7.0

7.1

Replicate 2

7.1

7.2

200 mg/l

Replicate 1

7.2

7.1

Replicate 2

7.1

7.1

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to test substance to various nominal test concentration, EC50 calculated from probit analysis was observed to be 75.25 mg/L.
Executive summary:

This study was designed to assess the effect of the test substance on the growth of green alga Chlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201 – Alga growth inhibition test”.

The effect of test substance 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 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200 mg/l were prepared using stock solution of the test substance using de-ionized water. 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 test substance. EC50 calculated from probit analysis was observed to be 75.25 mg/L. As the test chemical is readily biodegradable, it cannot be classfied as per CLP classification category.

Description of key information

This study was designed to assess the effect of the test substance on the growth of green algaChlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201 – Alga growth inhibition test”.

The effect of test substance 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 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200 mg/l were prepared using stock solution of the test substance using de-ionized water. 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 test substance. EC50 calculated from probit analysis was observed to be 75.25 mg/L. As the test chemical is readily biodegradable, it cannot be classfied as per CLP classification category.

Key value for chemical safety assessment

EC50 for freshwater algae:
75.25 mg/L

Additional information

Various experimental of test chemical and its read across chemicals have been reviewed to determine the toxicity of target to aquatic algae and their results are summarized below.

 

The first Experimental study performed for the target chemical and it was designed to assess the effect of the test substance on the growth of green algaChlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201 – Alga growth inhibition test”.

The effect of test substance was studied on the growth of fresh water green algaChlorella vulgaris.The study was conducted following OECD guideline 201- Alga growth inhibition test. The test concentration chosen for the study were 12.5 mg/l, 25 mg/l, 50 mg/l, 100 mg/l and 200 mg/l were prepared using stock solution of the test substance using de-ionized water. 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 test substance. EC50 calculated from probit analysis was observed to be 75.25 mg/L. As the test chemical is readily biodegradable, it cannot be classfied as per CLP classification category.

 

Next experimental study was reviewed from handbook in this the green algae were exposed to the test concentration for a period of 14 days to observe average specific growth rate. On the basis of growth rate EC50 value was determined to be 60 mg/L for 3 hrs and > 10 mg/L for 14 days.

 

 Last experimental study was also reviewed from handbook and this study was designed to assess the effect of the test substance on the growth of green algae Haematococcus pluvialis. The green algae were exposed to the test concentration for a period of 4 hr to observe average specific growth rate. On the basis of growth rate EC10 value was determined to be165 mg/L .

 

By considering results of all the studies mentioned above it is concluded that EC50 value of test chemical is in range from 60 mg/L to 165 mg/L. On the basis of this range value and ready biodegradability test chemical is considered to be non toxic and cannot be classified as per CLP regulation.