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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:
key study
Study period:
02 November 2019 – 03 March 2020
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.0 (control), 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L
- Sampling method: samples (plus retain samples) were taken from a randomly chosen replicate per treatment group at 0 hour and 72 hours in order to determine the concentration of test item during the test. A volume of 10 mL of sample solutions were collected from all test concentration in duplicates and diluted in the corresponding quantity of methanol before being analyzed.
- Sample storage conditions before analysis: All samples were analyzed on the same day of collection from the respective test vessels.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was freshly prepared prior to exposure, by weighing 100 mg of test item in a beaker and diluted in 100 mL of OECD alga medium. After complete soluble of test item by mixing well using a glass rod, the test item formulation was transferred into a measuring cylinder and the beaker was rinsed with OECD alga medium and transferred again to the measuring cylinder. Rinsing process was repeated until the complete transfer of test contents. A homogeneous distribution of the test item within the stock solution was achieved by stirring using magnetic stirrer shortly before preparation of the test vessels. The required test concentrations were prepared by adding appropriate amount of stock solutions and made up to required volume using OECD alga medium.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection.

ACCLIMATION
- Acclimation period: 3 days (pre-culture).
- Culturing media and conditions (same as test or not): Inoculum was pre-cultured (in a flask containing 100 mL of culture medium) during 3 days prior to the test. The biomass increased by factor of 9.0 (on day 2) and 21.2 (on day 3) for the dose range finding study and by factor of 9.6 (on day 2) and 22.0 (on day 3) for the main study.
- Any deformed or abnormal cells observed: Not reported.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
22.2 to 22.6ºC
pH:
7.99 to 8.18 (0 h) and 8.00 to 8.12 (72 h)
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L.
Measured concentrations for 0, 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L (initial time, T0h): -, 0.57, 1.42, 3.52, 9.03, 21.66 and 52.36 mg/L.
Measured concentrations for 0, 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L (final time, T72h): -, 0.55, 1.31, 3.50, 8.28, 20.37 and 51.42 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: erlenmeyer flask
- Material, size, headspace, fill volume: glass, 250 mL capacity, 100 mL fill volume.
- Aeration: Test vessels were covered by air permeable stoppers.
- Initial cells density: 5528 cells/mL
- Control end cells density: 933167 cells/mL (average)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD medium as per OECD TG 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the OECD medium was prepared as per the OECD TG 201.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The temperature (culture medium) and light intensity (parallel to the level of culture medium was recorded at four random locations using a Lux meter) was monitored daily during pre-culture and exposure. The pH of the test concentrations was measured at the beginning (0 hours) and at the end (72 hours) of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Constant illumination
- Light intensity and quality: 8185 to 8923 lux with white fluorescent lamp. The maximum value recorded was slightly outside of the recommended range stated in the OECD TG 201 (4440-8880 lux). LIght intensity applied during test was near higher value of the menioned range because the test item is a coloured substance which can absorb light and limit growth of alga and then it could result in growth inhibition which would have been difficult to distighish from inherent toxicity.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Counting chamber: Samples collected were loaded on haemocytometer and the cells were counted manually using microscope.
Cell density is obtained by calculating the average cell count [average cell counts calculated by summating the number of cells in all the four large corner squares (squares 1 to 4) and dividing the sum by 4] and multiplied with chamber conversion factor (CCF). Then the following formula was used for calculating cell density in mL:
Cell Density (cells/mL)= Average cell count × 10000 (CCF) × Dilution factor

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric factor of 2.5
- Justification for using less concentrations than requested by guideline: Not applicable; 6 concentrations used for the main test.

- Range finding study
- Test concentrations: 0 (control), 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L.
- Results used to determine the conditions for the definitive study: At the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, the average inhibition growth rate was calculated to be 0.13, 1.24, 5.15, 20.75, 100 and 100% respectively.
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol 97% )
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
27.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval = [19.16 – 40.78]
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
8.15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval = [6.47 – 10.27]
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval = [3.23 – 5.67]
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence interval = [4.08 – 7.69]
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
2.26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence interval = [1.53 – 3.34]
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence interval = [0.86 – 2.30]
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Determined by SPSS statistical procedure for multiple sample comparison using individual replicate values of the specific growth rate.
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Determined by SPSS statistical procedure for multiple sample comparison using individual replicate values of the specific growth rate.
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Not reported.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported.

Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50 (0-72h) = 1.12 mg/L (0.95 – 1.33)
Reported statistics and error estimates:
The 72 hours EC10, EC20 and EC50 values were calculated for growth rate and yield using the Probit Finneys method. Probit regression line was drawn for assessed parameters by plotting the Probit inhibition values against the corresponding log concentration. Student’s t-test was performed for response variable to determine NOEC and LOEC.

Table 1: Mean cell density after exposure during main study

Group

Test Conc.
 (mg/L)

R

Mean Cell Density (104Cells/mL)

Observation Periods

24 h

48 h

72 h

G1

0.0

R1

4.95

28.60

92.95

R2

5.50

28.05

95.70

R3

4.95

28.60

91.85

R4

5.50

28.05

92.95

R5

5.23

28.88

94.60

R6

5.23

29.70

91.85

G2

0.5

R1

4.95

30.53

92.95

R2

5.23

29.15

93.23

R3

4.95

30.80

91.58

G3

1.3

R1

4.40

23.65

78.65

R2

4.68

24.75

78.38

R3

4.68

25.03

78.65

G4

3.2

R1

3.85

18.98

66.55

R2

4.68

19.53

67.65

R3

4.40

20.35

67.93

G5

8.0

R1

2.75

15.68

43.45

R2

2.20

17.05

44.83

R3

2.48

16.50

44.28

G6

20.0

R1

1.38

11.55

9.35

R2

1.65

11.00

9.63

R3

1.38

10.73

9.08

G7

50.0

R1

0.00

0.00

0.00

R2

0.00

0.00

0.00

R3

0.00

0.00

0.00

Table 2: Average specific growth rate during main study

Group

Test Conc.
(mg/L)

R

Growth Rate

Mean Growth
Rate

Observation Intervals (Days)

0 to 1

1 to 2

2 to 3

0 to 3

G1

0

1

2.19

1.75

1.18

1.71

1.71

2

2.30

1.63

1.23

1.72

3

2.19

1.75

1.17

1.70

4

2.30

1.63

1.20

1.71

5

2.25

1.71

1.19

1.71

6

2.25

1.74

1.13

1.70

Mean

2.25

1.70

1.18

1.71

±SD

0.05

0.06

0.03

0.01

CV

2.10

3.46

2.77

0.32

G2

0.5

1

2.19

1.82

1.11

1.71

1.71

2

2.25

1.72

1.16

1.71

3

2.19

1.83

1.09

1.70

Mean

2.21

1.79

1.12

1.71

±SD

0.03

0.06

0.04

0.00

CV

1.41

3.39

3.32

0.19

G3

1.3

1

2.07

1.68

1.20

1.65

*1.65

2

2.14

1.67

1.15

1.65

3

2.14

1.68

1.15

1.65

Mean

2.11

1.68

1.17

1.65

±SD

0.03

0.01

0.03

0.00

CV

1.65

0.47

2.63

0.04

G4

3.2

1

1.94

1.60

1.25

1.60

*1.60

2

2.14

1.43

1.24

1.60

3

2.07

1.53

1.21

1.60

Mean

2.05

1.52

1.23

1.60

±SD

0.10

0.08

0.03

0.00

CV

4.85

5.50

2.09

0.23

G5

8.0

1

1.60

1.74

1.02

1.45

*1.46

2

1.38

2.05

0.97

1.47

3

1.50

1.90

0.99

1.46

Mean

1.49

1.90

0.99

1.46

±SD

0.11

0.15

0.03

0.01

CV

7.47

8.11

2.69

0.36

G6

20.0

1

0.91

2.13

-0.21

0.94

*0.94

2

1.09

1.90

-0.13

0.95

3

0.91

2.05

-0.17

0.93

Mean

0.97

2.03

-0.17

0.94

±SD

0.11

0.12

0.04

0.01

CV

10.83

5.82

-22.87

1.04

G7

50.0

1

0.0

0.0

0.0

0.0

0.0

2

0.0

0.0

0.0

0.0

3

0.0

0.0

0.0

0.0

Mean

0.0

0.0

0.0

0.0

±SD

0.0

0.0

0.0

0.0

CV

0.0

0.0

0.0

0.0

±SD: Standard deviation; CV: Coefficient of Variation;*: Statistical Significance of >0.005.

Table 3: Yield of alga during main study

Group

Test Conc.
(mg/L)

R

Cell density (Cells/mL)

Yield
(72 hours)

0 h

72 h

G1

0.0

1

5528

929500

923973

2

5528

957000

951473

3

5528

918500

912973

4

5528

929500

923973

5

5528

946000

940473

6

5528

918500

912973

Mean

933167

927639

±SD

15426

15426

G2

0.5

1

5528

929500

923973

2

5528

932250

926723

3

5528

915750

910223

Mean

925833

920306

±SD

8840

8840

G3

1.3

1

5528

786500

780973

2

5528

783750

778223

3

5528

786500

780973

Mean

785583

780056

±SD

1588

1588

G4

3.2

1

5528

665500

659973

2

5528

676500

670973

3

5528

679250

673723

Mean

673750

668223

±SD

7276

7276

G5

8.0

1

5528

434500

428973

2

5528

448250

442723

3

5528

442750

437223

Mean

441833

436306

±SD

6921

6921

G6

20.0

1

5528

93500

87973

2

5528

96250

90723

3

5528

90750

85223

Mean

93500

87973

±SD

2750

2750

G7

50.0

1

5528

0

-5528

2

5528

0

-5528

3

5528

0

-5528

Mean

0

0

±SD

0

0

±SD: Standard deviation

Table 4: Percent inhibition in yield, cell density and growth rate during main study

Group

Test Conc. (mg/L)

% inhibition in Yield

% inhibition in Cell Density

% inhibition in Growth Rate

G1

0

-

-

-

G2

0.5

0.79

0.79

0.15

G3

1.3

15.91

15.82

3.36

G4

3.2

27.97

27.80

6.35

G5

8.0

52.97

52.65

14.58

G6

20.0

90.52

89.98

44.86

G7

50.0

100.00

100.00

100.00

Table 5: Acceptance criteria for control during main study

0 – 72 Hours Mean Coefficient of Variation of Section-By-Section Average Specific Growth Rate

Daily average growth rate

±SD

% CV out of the daily growth rates

1.71

0.51

29.75

1.72

0.54

31.47

1.70

0.51

30.19

1.71

0.55

32.43

1.71

0.53

30.91

1.70

0.56

32.82

Mean % of CV 0 – 72 hours

31.26

Biomass increase during the whole test period

Initial biomass (cells/mL)

 Cell density on day 3 (cells/mL)

Biomass increase Factor

5528

929500

168.16

5528

957000

173.13

5528

918500

166.17

5528

929500

168.16

5528

946000

171.14

5528

918500

166.17

Mean

933166.67

168.82

±SD: Standard deviation

Table 6: Summary of test concentration analysis during the 72 hour alga growth inhibition test during main study with violet LCC6D dried

 

0 Hour New Solution

72 hour Aged Solution

Group

Nominal concentration

(mg/L)

 Conc. obtained                        

 (mg/L)

Recovery

(%)

Nominal concentration

(mg/L)

 Conc. obtained                        

 (mg/L)

Recovery

(%)

G1

0.0

-

-

0.0

-

-

G2

0.5

0.57

113.00

0.5

0.55

109.00

G3

1.3

1.42

108.85

1.3

1.31

100.39

G4

3.2

3.52

109.85

3.2

3.50

109.22

G5

8.0

9.03

112.82

8.0

8.28

103.51

G6

20.0

21.66

108.30

20.0

20.37

101.83

G7

50.0

52.36

104.71

50.0

51.42

102.83

Table 7: Temperature and light intensity during pre-culture and exposure - main study

Day

Temperature of the surrogate (°C)

Light intensity (Lux)

Revolutions per minute (rpm)

1

2

3

4

During Acclimatisation (pre-culture) & Exposure

0

22.5

8215

8326

8254

8357

100

1

22.4

8354

8337

8257

8421

100

2

22.3

8562

8389

8454

8356

100

3

22.3

8457

8185

8256

8347

100

4

22.5

8554

8621

8529

8923

100

5

22.6

8726

8524

8611

8715

100

6

22.2

8531

8429

8357

8562

100

Mean

22.4

8486

8402

8388

8526

-

±SD

0.1

164.2

141.9

145.8

221.8

-

Min

22.2

8185

Max

22.6

8923

Table 8: pH of test medium during main study

Group 

Test Conc. (mg/L)

pH

0 hours

72 hours

R1

R2

R3

R4

R5

R6

R1

R2

R3

R4

R5

R6

G1

0

8.11

8.09

8.13

7.99

8.03

8.05

8.10

8.08

8.12

8.00

8.02

8.01

G2

0.5

8.12

8.11

8.13

-

-

-

8.11

8.10

8.12

-

-

-

G3

1.3

8.07

8.12

8.09

-

-

-

8.05

8.10

8.06

-

-

-

G4

3.2

8.12

8.18

8.14

-

-

-

8.08

8.12

8.11

-

-

-

G5

8.0

8.13

8.11

8.09

-

-

-

8.10

8.09

8.06

-

-

-

G6

20.0

8.07

8.06

8.03

-

-

-

8.03

8.02

8.01

-

-

-

G7

50.0

8.05

8.01

8.01

-

-

-

8.04

8.00

8.03

-

-

-

 

Min

7.99

8.00

 

Max

8.18

8.12

Validity criteria fulfilled:
yes
Remarks:
(increase of biomass in control during 72h>16 fold; coefficient of variation of the mean specific growth rate among replicates in control (t0-t72)<7%; the mean of the replicate coefficients of variation in section-by-section growth rate in control < 35%)
Conclusions:
In an Alga Growth Inhibition Test with Pseudokirchneriella subcapitata green algae, the growth rate inhibition effect concentration (72 h-ErC50) of the test substance was found to be 27.96 mg/L.

Executive summary:

An Algae Growth Inhibition Test was performed with Pseudokirchneriella subcapitata green algae on the test substance over a period of 72 h in static conditions according to OECD Guideline 201, following GLP. OECD medium was used for algal culturing. In the previous solubility study the test item was found soluble in OECD medium and thus no vehicle was needed. In a preliminary range finding study the average growth inhibition rate were 0.13, 1.24, 5.15, 20.75, 100 and 100% at the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively. Based on these results, the definitive test was conducted with nominal test item concentrations of 0 (control), 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L (factor of 2.5). Test medium alone and 3,5-dichlorophenol 97% were used as negative and positive controls respectively. Three replicates were performed for each concentration of the test item, along with six replicates for the negative control group. A validated analytical method based on LC-MS/MS was used to monitor the concentration and stability of the active ingredient in the test solution. The test item was considered as stable in the testing conditions after 72 hours of exposure as the measured concentrations in samples were maintained within ± 20 per cent of the nominal concentrations. All validity criteria were fulfilled. The percent inhibitions in average specific growth rate were 0.15, 3.36, 6.35, 14.58, 44.86 and 100.0% and the percent inhibitions in yield were 0.79, 15.91, 27.97, 52.97, 90.52 and 100.0% at the tested concentrations of 0.5, 1.3, 3.2, 8.0, 20.0 and 50.0 mg/L respectively, when compared to the negative control. Based on these results, the growth rate inhibition effect concentrations were determined to be 72 h-ErC10 = 4.28 mg/L, 72 h-ErC20 = 8.15 mg/L and 72 h-ErC50 = 27.96 mg/L, and the yield inhibition effect concentrations were 72h-EyC10 = 1.41 mg/L, 72h-EyC20 = 2.26 mg/L and 72h-EyC50 = 5.60 mg/L. The 72 hours No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) were calculated to be 0.5 mg/L and 1.3 mg/L respectively.

Description of key information

Key study. Test method according to OECD 201, GLP study. The growth rate inhibition effect concentrations to Pseudokirchneriella subcapitata green algae were determined to be 72 h-ErC50 = 27.96 mg/L and 72 h-ErC10 = 4.28 mg/L. The NOEC and LOEC were calculated to be 0.5 mg/L and 1.3 mg/L respectively for the 72 h-study.

Key value for chemical safety assessment

EC50 for freshwater algae:
27.96 mg/L
EC10 or NOEC for freshwater algae:
4.28 mg/L

Additional information