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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 using standard OECD test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
no
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
The test solution was prepared by dissolving 200 mg of test chemical in 200 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined. The final solubility value obtained after analytical detection is 895.54 mg/l. To have a better growth and visibility of cells, the initial cell count of the culture was kept 10,000 cells/ml. Test chemical concentrations used for the study were 0, 6.25, 12.5, 25, 50 and 100 mg/L, respectively.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Weight: 2 - 3 μm
- Source: Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium and maintained in
Laboratory.
- Method of cultivation: OECD medium

ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was OECD medium. 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 OECD medium.
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
21 to 24 ± 2°C
Nominal and measured concentrations:
Test chemical concentrations used for the study were 0, 6.25, 12.5, 25, 50 and 100 mg/L, respectively.
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, OECD medium was used as a test medium in the study.

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(3000-4000 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were measured using microscope.
- 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: 0, 6.25, 12.5, 25, 50 and 100 mg/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 22 ° 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):
yes
Remarks:
Potassium dichromate (K2Cr2O7) was used as a reference substance.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
77.75 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
Results with reference substance (positive control):
- Results with reference substance valid
- EC50: 0.868 mg/l
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: Assessment of dose range concentrations

Sr. no.

Concentrations (mg/l)

Wavelength (nm)

Absorbance

Temperature (°C)

1

blank

610

0.0001

25°C

2

5

610

0.08

25°C

3

10

610

0.17

25°C

4

15

610

0.24

25°C

5

20

610

0.32

25°C

6

25

610

0.40

25°C

7

30

610

0.48

25°C

8

35

610

0.56

25°C

9

40

610

0.64

25°C

10

45

610

0.71

25°C

11

50

610

0.81

25°C

 

The absorbance and concentrations were recorded at 610 nm.

Table: Concentration after analytical Determination

Sr. No

Concentrations (mg/L)

Absorbance

(mean) (0 hour)

Analytical

Concentrations (0 hour)

Absorbance

(mean) (48 hour)

Analytical

Concentrations (48 hour)

1

blank

0.0001

0.0063

0.0000

0.0004

2

6.25

0.10

6.24

0.10

6.27

3

12.5

0.20

12.65

0.19

12.25

4

25

0.40

25.56

0.39

24.65

5

50

0.79

49.14

0.76

47.34

6

100

0.32

100.85

0.31

96.1

 

Table: pH and Temperature

Test

Concentration(mg/L)

 

 

Experimental

Flasks

pH

 

 

Temperature °C

0 Hours

72 hours

0 Hours

72 hours

control

R1

7.7

7.4

25

25

control

R2

7.8

7.4

25

25

control

R3

7.8

7.5

25

25

6.25

R1

7.8

7.5

25

25

6.25

R2

7.8

7.5

25

25

6.25

R3

8.0

7.5

25

25

12.5

R1

8.0

7.8

25

25

12.5

R2

7.9

7.7

25

25

12.5

R3

7.8

7.6

25

25

25

R1

7.9

7.6

25

25

25

R2

7.8

7.6

25

25

25

R3

7.9

7.7

25

25

50

R1

7.9

7.6

25

25

50

R2

8.0

7.7

25

25

50

R3

7.9

7.8

25

25

100

R1

7.9

7.8

25

25

100

R2

7.8

7.7

25

25

100

R3

7.9

7.6

25

25

 

Table: Cell count and percent inhibition

Experimental Flasks

and Test

Concentration(mg/L)

0 Hr

Cell

Count

24 Hr

Cell

Count

48 Hr

Cell Count

72 Hr

Cell Count

Avg Specific

Growth Rate

(μ)

Mean Avg

Specific Growth

Rate (μ)

Percent

Inhibition(%)

control

10000

30000

85000

260000

1.09

1.09

 

control

10000

35000

100000

265000

1.09

control

10000

30000

90000

260000

1.09

6.25 (R1)

10000

15000

80000

225000

1.04

0.99

9.17

6.25 (R2)

10000

20000

80000

185000

0.97

6.25 (R3)

10000

25000

80000

180000

0.96

12.5 (R1)

10000

25000

70000

160000

0.92

0.91

16.51

12.5 (R2)

10000

20000

65000

150000

0.90

12.5 (R3)

10000

15000

75000

145000

0.89

25 (R1)

10000

20000

75000

80000

0.69

0.69

36.70

25 (R2)

10000

20000

75000

75000

0.67

25 (R3)

10000

20000

80000

85000

0.71

50 (R1)

10000

20000

60000

75000

0.67

0.65

40.37

50 (R2)

10000

20000

65000

70000

0.65

50 (R3)

10000

15000

65000

75000

0.62

100 (R1)

10000

20000

45000

60000

0.60

0.52

52.29

100 (R2)

10000

15000

40000

40000

0.46

100 (R3)

10000

15000

45000

45000

0.50

 

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test chemical to freshwater green algae Pseudokirchneriella subcapitata was determined according to OECD guideline 201 In this study, the test chemical was found to inhibit the growth of the green algae after 72 hrs with the following effect value. The 72 hr median effect concentration (ErC50) value was determined to be 77.75 mg/l (calculated from equation through probit analysis).
Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to the principles of the OECD guideline no. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical (0, 6.25, 12.5, 25, 50 and 100 mg/l) (factor 2) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 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. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the mean coefficient of variation of for section-by-section specific growth rate in the control cultures was not exceeded 35% (i.e., reported as 5.84%) and the mean coefficient of variation of average specific growth rates in the replicate control culture was not exceeded 7% in test with Pseudokirchneriella subcapitata (i.e., reported as 0.34%), respectively. Thus, all validity criteria of the test guideline were fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 77.75 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as 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 Pseudokirchneriella subcapitata (Experimental study report, 2020). The test was performed in accordance to the principles of the OECD guideline no. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical (0, 6.25, 12.5, 25, 50 and 100 mg/l) (factor 2) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 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. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the mean coefficient of variation of for section-by-section specific growth rate in the control cultures was not exceeded 35% (i.e., reported as 5.84%) and the mean coefficient of variation of average specific growth rates in the replicate control culture was not exceeded 7% in test with Pseudokirchneriella subcapitata (i.e., reported as 0.34%), respectively. Thus, all validity criteria of the test guideline were fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 77.75 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as 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:
77.75 mg/L

Additional information

Experimental study of the test chemical and supporting weight of evidence study for its structurally and functionally similar read across chemical were reviewed for toxicity to aquatic algae end point which are summarized as below:

 

In an experimental study from study report (2020), a freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to the principles of the OECD guideline no. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The test solution was prepared by dissolving 200 mg of test chemical in 200 mL of OECD medium to get the final concentration of 1000 mg/L, which was then analytically determined by UV-VIS spectrophotometer. Green algae were exposed to nominal concentration of test chemical (0, 6.25, 12.5, 25, 50 and 100 mg/l) (factor 2) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 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. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16, the mean coefficient of variation of for section-by-section specific growth rate in the control cultures was not exceeded 35% (i.e., reported as 5.84%) and the mean coefficient of variation of average specific growth rates in the replicate control culture was not exceeded 7% in test with Pseudokirchneriella subcapitata (i.e., reported as 0.34%), respectively. Thus, all validity criteria of the test guideline were fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 77.75 mg/l (calculated from equation through probit analysis).

 

Another algal growth inhibition test was conducted for 96 hrs for assessing the growth inhibition potential of test chemical on green algae (from handbook, 2008 and secondary sources). This test was performed by following the OECD 201 guideline (Alga, Growth Inhibition Test) under static condition for 96 hrs. Desmodesmus subspicatus (former scientific name: Scenedesmus subspicatus) was used as the test organism. The algae was cultured in a nutrient solution prepared according to OECD Guideline 201. Initial cell density of the culture used was 10,000 cells/ml of nutrient solution. The cells were taken from a pre-culture, which was set up 3 days prior to the test. On the basis of preliminary study, series of sequential dilutions of test chemical are prepared in a test medium. 400 mg test material was suspended up to 100 ml with test medium. Nominal test chemical concentrations used for the study were 0, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mg/l, respectively. Due to precipitation, test chemical concentrations were not verified analytically. Green algae were exposed to various nominal concentration of test chemical in 50 ml Erlenmeyer flask containing 30 ml of algal suspension were set up per each concentration of test chemical. These flasks were stoppered with cotton wool plugs. Test vessel were placed in shaking incubator for 96 hrs at a room at a temperature of 21°C, pH 7.5 with a continuous uniform illumination of 800 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 only the OECD medium) was also included in the test. Samples (2 to 5 ml) of algae were taken after 24, 48, 72 and 96 hours of incubation and the cell count was noted with the help of a microscope. The pH of one solution per concentration (including the control) was also measured at 96 hours. As per the OECD guideline No. 201 – Alga growth inhibition test, the drift in the pH did not increase by > 1.5 units after 96 hrs as compared to 0 hrs in the control vessel. Algal growth inhibition was determined from the growth curves (biomass integral). The EC50 values with confidence limits were estimated by logit analysis whereas the NOEC and LOEC values were determined statistically using the Dunnett’s test. On the basis of effect on biomass of the test organism Desmodesmus subspicatus, the 96 hrs NOEC, LOEC, EbC50 value was determined to be 25 mg/l, 50 mg/l and 41.1 mg/l, respectively.

 

On the basis of the above results, it can be concluded that the test chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.