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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:
19 January 2018 to 01 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch: 01 Jan 2019
- Purity test date: Not specified

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (5 °C - 30 °C), dark, dry
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 1g/L
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material) N/A

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS: N/A
Analytical monitoring:
yes
Remarks:
Analytical samples were analysed from control and test item concentration 100 mg/L at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions.
Details on sampling:
- Concentrations: 100 mg/L
- Sampling method: Analytical samples were taken at 0 h fresh and 72 h aged
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The necessary amount of test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. Afterwards the solution was clear and transparent. Lower test solutions were prepared by dilution of appropriate solutions with test medium.
- Eluate: N/A
- Differential loading: N/A
- Controls: Yes (six replicates)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): N/A
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: N/A
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
- Age of inoculum (at test initiation): 3 - 4 d
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.

ACCLIMATION
- Acclimation period: 3 - 4 d
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Guideline-recommended exposure duration.
Post exposure observation period:
N/A
Hardness:
Not reported
Test temperature:
22.6 – 23.1 °C
pH:
7.23 - 8.34
Salinity:
N/A
Conductivity:
Not reported
Nominal and measured concentrations:
0.954, 3,05, 9.77, 31.3, 100, 1000 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 100 mL size, containing ~ 50 mL test solution, selaed with aluminium caps
- Aeration: CO2 supply by shaking on a rotating shaker, approximately 105 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 0.5 x 104 cells/mL
- Control end cells density:
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes (AAP-Medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The necessary amount of test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask. Lower test solutions were prepared by dilution of appropriate solutions with test medium.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: Not specified
- Intervals of water quality measurement: pH was recorded at test start and test end. Temperature was measured continuously over the whole test period and recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: With NaOH or HCl, if necessary (to acheive pH 7.5 +/- 0.1)
- Photoperiod: Continuous
- Light intensity and quality: Approx. 4440 - 8880 lux at cell culture level or 60 - 120 µEm-2s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : At 24, 48 and 72 hours from the test initiation, the number of cells in each replicate was determined
- Determination of cell concentrations: Fluorescence detection (Performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0)).
- Chlorophyll measurement: N/A

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: No
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Basis for effect:
growth rate
Remarks on result:
not determinable
Remarks:
No statistically significant inhibitory effects
Duration:
72 h
Dose descriptor:
LOEC
Basis for effect:
other: yield
Remarks on result:
not determinable
Remarks:
No statistically significant inhibitory effects
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopically observed at the end of the test, all cells were considered normal for the control and all test item concentrations.
- Unusual cell shape: None
- Colour differences: None
- Flocculation: None
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Other: N/A
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None

Results with reference substance (positive control):
- Results with reference substance valid?
- EC50: Growth rate: 1.23 mg/L (nominal); Yield: 0.685 mg/L
- Other: The EC50 values calculated in the reference test (performed in a separate study twice a year by the laboratory) were considered to be within an acceptable range therefore it can be considered that the test organism is sensitive.
Reported statistics and error estimates:
After 72 h at termination of the test no statistically significant inhibition of growth rate or yield was observed for test item concentrations up to and including 100 mg/L. Due to an inhibition below 10, 20 and 50 % for growth rate and yield, the calculation of the EC10, 20, 50 was not indicated.
The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-t-test, two-tailed, for growth rate, Jonckheere Terpstra test, two-tailed, for yield).

Validity criteria of the study:

Biomass: Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 54.20, which exceeds the threshold of 16. It corresponds to a growth rate of 1.32937 d-1.

Coefficient of Variation (section by section): The mean coefficient of variation for the section-by-section specific growth rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 20 % and did not exceed 35 %.

Coefficient of Variation (average growth rate): The coefficient of variation of average growth rate in replicate control cultures was 2.6 % and did not exceed 7 % for the whole test period.

Results:

After 72 h at termination of the test no statistically significant inhibition of growth rate or yield was observed for test item concentrations up to and including 100 mg/L

Table 2. Average cell numbers for each sampling time and concentration

Conc.

Average cell numbers [104/mL]

[mg/L]

0 h

24 h

48 h

72 h

Control

0.55

1.61

7.18

29.81

0.954

0.55

2.03

8.01

32.76

3.05

0.55

2.14

9.79

38.78

9.77

0.55

2.31

13.54

58.10

31.3

0.55

2.33

14.55

56.73

100

0.55

1.95

14.51

62.16

Table 3. Percentage inhibition of growth rate

Conc.

% Inhibition of growth rate1)

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

9.5

-1.6

-4.4

-2.2

-7.3

0.3

Control

10.9

4.2

3.4

11.4

8.5

1.2

-18.7

1.6

-0.9

-10.9

-5.4

0.4

Mean

0.0

0.0

0.0

-18.7

-0.5

1.8

0.954

-21.5

-12.0

-7.0

-23.7

0.0

-1.4

Mean

-21.3

-4.2

-2.2

-29.4

-13.7

-9.7

3.05

-27.7

-14.9

-4.3

-21.9

-8.2

-5.8

Mean

-26.3

-12.3

-6.6

-31.4

-27.8

-19.0

9.77

-31.4

-26.1

-15.3

-37.0

-21.0

-16.1

Mean

-33.3

-25.0

-16.8

-30.6

-28.6

-14.2

31.3

-38.6

-27.0

-17.0

-33.9

-27.9

-17.4

Mean

-34.4

-27.8

-16.2

-18.7

-27.7

-17.8

100

-17.7

-28.4

-20.4

-16.3

-27.1

-17.2

Mean

-17.6

-27.7

-18.5

Table 4. Percentage inhibition of yield

Conc.

% Inhibition of yield1)

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

3.8

-16.6

-34.1

-3.8

-21.2

1.8

Control

15.1

10.4

11.8

18.9

23.3

6.9

-17

17.3

9.8

-17

-12.8

3.8

Mean

0.0

0.1

0.0

-34

-0.3

7.4

0.954

-39.6

-37.7

-32.3

-44.3

1.1

-5.3

Mean

-39.3

-12.3

-10.1

-56.6

-44.3

-47.3

3.05

-52.8

-48.9

-18.5

-40.6

-24.2

-26.2

Mean

-50

-39.1

-30.7

-61.3

-110.5

-114.5

9.77

-61.3

-101.1

-84.6

-74.5

-75.3

-91

Mean

-65.7

-95.6

-96.7

-59.4

-115.2

-76.8

31.3

-78.3

-106.2

-98.2

-67

-111.1

-101

Mean

-68.2

-110.8

-92

-34

-109.9

-104.6

100

-32.1

-113.9

-127.2

-29.2

-106.8

-99.9

Mean

-31.8

-110.2

-110.6

Table 5. pH measurements during test

Conc.

t = 0 h

t = 72 h

[mg/L]

Control

7.23

8.12

0.954

7.25

8.10

3.05

7.27

8.05

9.77

7.26

8.05

31.3

7.27

8.13

100

7.28

8.34

Table 6. Temperature during the test

Time

Temperature[°C] min

Temperature[°C] max

[h]

0

23.0

24

22.6

23.1

48

22.6

23.1

72

22.6

23.1

Table 7. Control, growth rates

Growth rates, µ [d-1]

0 h – 24 h

0 h – 48 h

0 h – 72 h

0.97273

1.30075

1.38780

1.09861

1.37480

1.32503

Control

0.95825

1.22658

1.28389

0.95266

1.17215

1.31350

1.27629

1.26036

1.34178

1.19280

1.34969

1.32424

Mean

1.07522

1.28072

1.32937

Std. Dev.

0.13712

0.07634

0.03445

CV [%]

12.8

6.0

2.6

Std.Dev.:Standard deviation

CV: Coefficient of variation

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) at test end. Thus the overall LOEC was not determinable and the overall NOEC was observed to be at 100 mg/L (nominal).
The EC10, EC20 and EC50 for growth rate and yield were considered to be > 100 mg/L (nominal).

Executive summary:

This study was conducted in accordance with the OECD guideline 201; “Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (28 July 2011), in order to determine the effects of the test item on the growth and yield of green alga Pseudokirchneriella subcapitata. Initial target cell densities of 0.5 × 104 cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours. A static test with nominal test item concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L and control was performed. Six replicates were employed for the control and three for each test item concentration. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. Analytical samples were analysed from control and test item concentration 100 mg/L at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions. The measured initial concentration of the test item was 91.2 % of nominal in the highest test item concentration of 100 mg/L. In the aged samples the measured concentrations was 102.6 %.

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any test item concentrations up to the highest concentration tested (100 mg/L, nominal) at test end. No abnormalities in the cells were observed at test end.

The overall LOEC was not determinable and the overall NOEC was observed to be 100 mg/L (nominal). The EC10, EC20 and EC50 for growth rate and yield were considered to be > 100 mg/L (nominal).

Description of key information

72 h ErC50 = > 100 mg/L, 72 h ErC10 = > 100 mg/L; OECD 201; Schuster, K.-A. (2018)

Key value for chemical safety assessment

Additional information

This study was conducted in accordance with the OECD guideline 201; “Freshwater Alga and Cyanobacteria, Growth Inhibition Test" (28 July 2011), in order to determine the effects of the test item on the growth and yield of green alga Pseudokirchneriella subcapitata. Initial target cell densities of 0.5 × 104cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours. A static test with nominal test item concentrations of 100, 31.3, 9.77, 3.05, 0.954 mg/L and control was performed. Six replicates were employed for the control and three for each test item concentration. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution. Analytical samples were analysed from control and test item concentration 100 mg/L at 0 hours (initial value) from fresh test solutions and after 72 hours from aged solutions. The measured initial concentration of the test item was 91.2 % of nominal in the highest test item concentration of 100 mg/L. In the aged samples the measured concentrations was 102.6 %.

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any test item concentrations up to the highest concentration tested (100 mg/L, nominal) at test end. No abnormalities in the cells were observed at test end.

The overall LOEC was not determinable and the overall NOEC was observed to be 100 mg/L (nominal). The EC10, EC20 and EC50 for growth rate and yield were considered to be > 100 mg/L (nominal).