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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:
from 2016-07-22 to 2016-11-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Following nominal concentrations were measured with algae: 0 (control), 0.34, 0.58, 0.99, 1.68 and 2.85 mg/L and 2.85 mg/L without algae solution
- Sampling method: The samples were measured after 0, 1, 2 and 3 days.
- Sample storage conditions before analysis: Room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance were directly dissolved in the test medium to prepare the stock solution. The test solutions were performed by diluting the stock solution with the test medium.
- Controls: Blank control performed with test medium and the algal solution in a proportion of 1:1 without the test substance.
- Evidence of undissolved material: no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: algae
- Source: National Chiayi University Freshwater Bioresource Center, in Chiayi City (Taiwan)

ACCLIMATION
- Acclimation period: 2-4 days
- Culturing media and conditions: Same as the test.
- 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 - 24 °C
pH:
The mean of pH value of the control group and each test group ranged between 7.55 - 7.84. The variation in the pH values in the beginning and end of the tests was less than 1.5 units. The validity criteria were met.
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.34, 0.58, 0.99, 1.68 and 2.85 mg/L
- Measured concentrations:
On day 0: 0.332, 0.579, 0.937, 1.640, 2.916 and 2.971 mg/L corresponding to 94.93 - 104.10 % of the nominal concentrations
On day 1: 0.323, 0.541, 0.956, 1.672, 2.901 and 2.939 mg/L corresponding to 93.28 - 102.98 % of the nominal concentrations
On day 2: 0.322, 0.532, 0.954, 1.587, 2.990 and 2.946 mg/L corresponding to 91.72 - 104.77 % of the nominal concentrations
On day 3: 0.319, 0.544, 0.966, 1.620, 2.955 and 2.953 mg/L corresponding to 93.55 - 103.54 % of the nominal concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: 125 mL glass flask
- Volume: 60 mL
- Type of cover: sealed with silicone plugs
- Aeration: No
- Initial cells density: 10 000 cells/mL
- Control end cells density: 528 600 +/- 2.82 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD guideline 201 (2011)
- Culture medium different from test medium: no
- Intervals of water quality measurement: The temperature was measured continuously, and the pH value was measured on day 0 and on day 3.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: 4440-8880 lux

EFFECT PARAMETERS MEASURED: Inhibitory effect of the test substance on the growth of the freshwater green algae species after 0. 24, 48 and 72 hours of exposure in the test media.
- Determination of cell concentrations: The measurements of the cell concentrations were made by cell counting with microscope.
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Range finding study: yes
- Test concentrations: The test concentrations in the range finding test were 0 (control), 0.16, 0.80, 4.00, 20.00 and 100.00 mg/L
- Results used to determine the conditions for the definitive study: Results of the range finding test showed that the percent inhibition in average specific growth rate was determined to be 0.09 - 92.18 % in the concentrations range of 0.16 and 4.00 mg/L (ErC50 between 0.16 – 4 mg/L). The percentage inhibition of yield was determined to be 0.39 – 100.29 % in the concentrations range of 0.16 – 100 mg/L (EyC50 between 0.16 and 100 mg/L).

Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.81 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
69.96 % Test Substance
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.97 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.54 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
69.96 % Test Substance
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.38 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
69.96 % Test Substance
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
100 % Test Substance
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.76 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
69.96 % Test Substance
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
100 % Test Substance
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
69.96 % Test Substance
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
100 % Test Substance
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
69.96 % Test Substance
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
100 % Test Substance
Basis for effect:
biomass
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: not observed
- Unusual cell shape: not observed
- Colour differences: not observed
- Flocculation: not observed
- Adherence to test vessels: not observed
- Aggregation of algal cells: not observed
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Other effects observed: After 72 hours the ErC50 was determined to be 2.81 mg/L and the ErC10 was determined to be 0.54 mg/L. The 72-EyC50 was determined to be 0.76 mg/L and the EyC10 was determined to be < 0.34 mg/L.
Results with reference substance (positive control):
No test with a reference substance were performed.
Reported statistics and error estimates:
No statistics and error estimates were reported.

Table 1. The algal biomass for the definitive test

Group

(mg/L)

Mean of algal biomass (×10 000 cells/mL) ± standard deviation

Day 0

Day 1

Day 2

Day 3

Control group

0.00

1.00 ± 0.00

4.41 ± 0.21

15.61 ± 2.25

52.86 ± 2.82

Test group

2.84

1.00 ± 0.00

2.74 ± 0.13

5.15 ± 0.21

7.08 ± 0.12

1.67

1.00 ± 0.00

2.93 ± 0.16

8.67 ± 0.81

19.90 ± 3.92

0.98

1.00 ± 0.00

3.00 ± 0.97

8.12 ± 1.81

17.21 ± 7.65

0.58

1.00 ± 0.00

4.32 ± 0.29

12.25 ± 2.38

34.72 ± 4.05

0.34

1.00±0.00

4.32 ± 0.15

14.42 ± 1.53

40.70 ± 3.05

Table 2. Theaverage specific growth ratefor the definitive test

Group

(mg/L)

Mean of average specific growth rate (d-1) ± standard deviation

(Coefficient of variation [%])

Day 0-1

Day 1-2

Day 2-3

Day 0-3

Control group

0.00

1.48 ± 0.05

(3.17)

1.26 ± 0.10

(7.88)

1.23 ± 0.10

(7.96)

1.32 ± 0.02

(1.33)

Test group

2.84

1.01 ± 0.05

(4.78)

0.63 ± 0.04

(6.89)

0.32 ± 0.03

(10.51)

0.65 ± 0.01*

(0.83)

1.67

1.07 ± 0.05

(5.06)

1.08 ± 0.12

(10.85)

0.82 ± 0.11

(13.96)

0.99 ± 0.07*

(7.06)

0.98

1.07 ± 0.31

(29.24)

1.01 ± 0.15

(14.64)

0.71 ± 0.21

(29.41)

0.93 ± 0.14*

(14.79)

0.58

1.46 ± 0.07

(4.67)

1.03 ± 0.14

(13.20)

1.05 ± 0.22

(20.50)

1.18 ± 0.04

(3.31)

0.34

1.46 ± 0.03

(2.36)

1.20 ± 0.13

(10.81)

1.06 ± 0.22

(20.89)

1.23 ± 0.03

(2.03)

Table 3.The percent inhibition in average specific growth rate and percent inhibition of yield for the definitive test

Group

(mg/L)

Logarithm of concentration

(mg/L)

Percent inhibition in average specific growth rate (%)

Percent inhibition of yield (%)

2.84

0.45

50.27

88.06

51.09

88.51

50.66

88.28

1.67

0.22

31.03

72.19

21.32

58.21

22.51

60.24

0.98

-0.01

36.84

78.31

17.84

51.75

34.63

76.15

0.58

-0.24

13.72

42.84

7.82

27.24

10.51

34.82

0.34

-0.47

3.28

12.53

7.94

27.61

5.26

19.28

Table 4. ECand 95% confidence interval for the definitive test

Parameter

ErC10a

ErC20

ErC50

EyC10b

EyC20

EyC50

ECX(mg/L)

0.54

0.78

2.81

< 0.34

< 0.34

0.76

95% confidence interval-lower

< 0.34

0.62

2.29

< 0.34

< 0.34

0.65

95% confidence interval-upper

0.64

1.06

> 2.84

< 0.34

0.38

1.06

ErCxwas calculated by percent inhibition in average specific growth rate.

EyCxwas calculated by percent inhibition of yield.

 

Table 5. The pH value for the definitive test

Group

(mg/L)

Mean of pH value ± standard deviation

Day 0

Day 3

Control group

0.00

7.70 ± 0.05

7.84 ± 0.07

Test group

2.84

7.65 ± 0.05

7.71 ± 0.04

1.67

7.55 ± 0.03

7.83 ±0.06

0.98

7.67 ± 0.13

7.74 ± 0.04

0.58

7.63 ± 0.22

7.62 ± 0.09

0.34

7.76 ± 0.09

7.60 ± 0.10

Validity criteria fulfilled:
yes
Conclusions:
The test substance was tested to Pseudokirchneriella subcapitata in a static test system according to OECD 201 (2011). Under the given conditions of this study the 72 h-ErC50 value was determined to be 2.81 mg/L and the 72 h-ErC10 was determined to be 0.54 mg/L (for the test substance with a purity of 69.96 %) and the 72 h-ErC50 value was determined to be 1.97 mg/L and the 72 h-ErC10 was determined to be 0.38 mg/L (for the test substance with a purity of 100 % of the actual ingredient).
Executive summary:

The influence of the test substance on the growth and growth rate of the green algae species to Pseudokirchneriella subcapitata was tested according to OECD 201 (1984) under GLP conditions. The algae were exposed to different concentrations of test material and the growth was calculated after 24, 48, or 72 hours of exposure in the test media. Based on the results of a previous study, the study with the test substance was performed with nominal concentrations of 0 (control), 0.34, 0.58, 0.99, 1.68 and 2.85 mg/L. The nominal concentrations were measured using HPLC Method. The study design included three replicates per test material concentration and control. All nominal concentrations were measured. The measured concentrations were determined to be in a range between 91.72 and 104.77 % of the nominal concentrations. The results are expressed in nominal concentrations. The initial cells density was 10 000 cells/mL. At the end of the test the cells density was determined to be 528 600 cells/mL. In addition the pH values were measured on day 0 and on day 3 of the test. The mean of pH value of the control group and each test group ranged between 7.55 - 7.84. The variation in the pH values in the beginning and end of the tests was less than 1.5 units. The validity criteria were met. No control with reference substance was performed. Under the given conditions of this study the 72 h-ErC50 value was determined to be 2.81 mg/L and the 72 h-ErC10 was determined to be 0.54 mg/L (for the test substance with a purity of 69.96 %) and the 72 h-ErC50 value was determined to be 1.97 mg/L and the 72 h-ErC10 was determined to be 0.38 mg/L (for the test substance with a purity of 100 % of the actual ingredient).

.

Description of key information

The test substance was tested to Pseudokirchneriella subcapitata in a static test system according to OECD 201 (2011). Under the given conditions of this study the 72 h-ErC50 value was determined to be 2.81 mg/L and the 72 h-ErC10 was determined to be 0.54 mg/L (for the test substance with a purity of 69.96 %) and the 72 h-ErC50 value was determined to be 1.97 mg/L and the 72 h-ErC10 was determined to be 0.38 mg/L (for the test substance with a purity of 100 % of the actual ingredient).

Key value for chemical safety assessment

EC50 for freshwater algae:
1.97 mg/L
EC10 or NOEC for freshwater algae:
0.38 mg/L

Additional information

The influence of the test substance on the growth and growth rate of the green algae species to Pseudokirchneriella subcapitata was tested according to OECD 201 (1984) under GLP conditions. The algae were exposed to different concentrations of test material and the growth was calculated after 24, 48, or 72 hours of exposure in the test media. Based on the results of a previous study, the study with the test substance was performed with nominal concentrations of 0 (control), 0.34, 0.58, 0.99, 1.68 and 2.85 mg/L. The nominal concentrations were measured using HPLC Method. The study design included three replicates per test material concentration and control. All nominal concentrations were measured. The measured concentrations were determined to be in a range between 91.72 and 104.77 % of the nominal concentrations. The results are expressed in nominal concentrations. The initial cells density was 10 000 cells/mL. At the end of the test the cells density was determined to be 528 600 cells/mL. In addition the pH values were measured on day 0 and on day 3 of the test. The mean of pH value of the control group and each test group ranged between 7.55 - 7.84. The variation in the pH values in the beginning and end of the tests was less than 1.5 units. The validity criteria were met. No control with reference substance was performed. Under the given conditions of this study the 72 h-ErC50 value was determined to be 2.81 mg/L and the 72 h-ErC10 was determined to be 0.54 mg/L (for the test substance with a purity of 69.96 %) and the 72 h-ErC50 value was determined to be 1.97 mg/L and the 72 h-ErC10 was determined to be 0.38 mg/L (for the test substance with a purity of 100 % of the actual ingredient)

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