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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:
02 July - 22 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
yes
Remarks:
The applied dose could not be analytically verified successfully.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Test material: Harpin fermentation extract (Harpin-αβ extract)
- Batch No. of test material: R134-1
- Appearance: Liquid/ tan, light brown
- Active ingredient content: 0.401% w/w
- Storage conditions: On dry ice (≤ -70 °C), dark, dry
Analytical monitoring:
yes
Details on sampling:
Analytical samples were taken and analysed in the main test from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- 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. Lower test solutions were prepared by dilution of the stock solution with test medium and appeared clear. Algae were added to each solution individually resulting in target cell densities of 0.5 × 104 cells per mL in each solution.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The solution appeared clear and transparent.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae)
- Strain: SAG 61.81
- Source: MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- 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. Stock cultureswere ordered regularly from the commercial supplier.

ACCLIMATION
- Acclimation period: 3 to 4 days prior to start of test
- Culturing media and conditions: Same as test
- Any deformed or abnormal cells observed: None specified
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Test temperature:
22.7 - 23.3°C
pH:
Control during the test: 7.17 - 7.97
Test concentrations during the test: 7.22 - 8.24
Nominal and measured concentrations:
Nominal test item concentrations: 0.954, 3.05, 9.77, 31.3 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks with aluminium caps
- Type: closed
- Material, size, headspace, fill volume: 100 mL Erlenmeyer flasks with aluminium caps each containing ~ 50 mL test solution
- Aeration: By continuous agitation
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 0.5 × 10^4 cells/mL
- Control end cells density: 24.67 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3 replicates for each test concentration
- No. of vessels per control (replicates): 6 replicates and one additional replicate without algae

GROWTH MEDIUM
- Standard medium used: yes (AAP-Medium, according to Annex 3 of OECD 201)
- Detailed composition if non-standard medium was used: Not applicable

OTHER TEST CONDITIONS
- pH of the control: 7.17 - 7.97
- Adjustment of pH: The pH was adjusted with NaOH and HCL to 7.5 ± 0.1.
- Photoperiod: Continuous illumination
- Light intensity and quality: 88.1 µEm-2s-1 at cell culture level (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At 24, 48 and 72 hours from the test initiation, the number of cells in each replicate was determined by fluorescence detection.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
- Test concentrations: Nominal test item concentrations: 0.0100, 0.100, 1.00, 10 and 100 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (tested in a separate study twice a year to confirm the sensitivity of the test organisms against compounds with known effects under the test conditions).
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
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
Biological results: After 72 h at termination of the test no concentration response relation was observed for the inhibition of growth rate and yield. The inhibition of growth rate peaked in -23.0 % at a nominal test item concentration of 100 mg/L and the inhibition of yield peaked in -148.2 % at a nominal test item concentration of 100 mg/L.
The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item concentration of 100 mg/L.

Analytical results: The initial measured content of TOC in the test item concentrations was below LOD (LOD = 0.5 mg/L) and 0.6131 mg/L. In the aged samples the measured TOC was between 0.9025 mg/L and 2.750 mg/L. The measured TOC concentrations might result from the test medium, since the TOC content of the solution of 100 mg/L in deionized water was determined to be below LOD. Furthermore, the measured concentrations of TOC were of similar values in the test item concentration as corresponding results from the control media measured values, in both the fresh and aged analysed samples.
Due to the properties and the composition of the test item no analytical dose verification was possible with commonly applied analytical methods. Due to correct routinely handling of the test item and the test item solutions, a correct application of the test item is assumed thus toxicological endpoints were evaluated using the nominal of the test item.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes
- EC50: The EC50 value for growth rate was determined to be 1.60 mg/L (nominal). The EC50 value for yield was 0.990 mg/L (nominal). The EC50 value for biomass integral was 1.06 mg/L (nominal).
- Other: Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 0.800 mg/L (nominal) and above. Significant inhibitory effects were determined for biomass integral at 0.320 mg/L (nominal) and above. The overall NOEC was therefore determined to be 0.320 mg/L (nominal), the corresponding LOEC was set at 0.800 mg/L (nominal).

Table1:      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

0.51

1.30

6.28

24.67

0.954

0.51

1.79

9.44

38.30

3.05

0.51

1.26

8.06

35.60

9.77

0.51

1.80

10.06

45.28

31.3

0.51

1.94

12.89

58.23

100

0.51

2.28

14.42

60.47

 

Table2:         Percentage inhibition of growth rate

Conc.

% Inhibition of growth rate

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

Control

0.0

0.0

0.0

0.954

-28.0

-16.1

-10.9

3.05

6.1

-8.9

-8.7

9.77

-34.0

-18.0

-14.7

31.3

-42.7

-28.7

-22.0

100

-60.2

-33.3

-23.0

 

 

Table3:         Percentage inhibition of yield

Conc.

% Inhibitionof yield

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

Control

0.0

0.0

0.0

0.954

-62.0

-54.8

-56.4

3.05

5.1

-30.8

-45.2

9.77

-63.3

-65.5

-85.3

31.3

-81.0

-114.6

-138.9

100

-124.1

-141.1

-148.2

 

Table 4:       Determined concentration ofTOC

Test item nominal [mg/L]

Harpin-αβnominal [mg/L]

Sampling

TOC [µg/L]

% Standard Deviation

Control

0

0 h fresh

<LOD (384.4)

2.60

24 h aged

902.5

2.39

48 h aged

2120

1.88

72 h aged

933.7

0.49

0.954

0.004

0 h fresh

<LOD (301.6)

9.28

24 h aged

1380

0.49

48 h aged

2830

0.43

72 h aged

2460

0.98

3.05

0.0122

0 h fresh

613.1

3.02

24 h aged

1020

1.83

48 h aged

1460

0.31

72 h aged

2020

0.91

9.77

0.0391

0 h fresh

<LOD (418.1)

1.66

24 h aged

2150

1.24

48 h aged

2400

0.46

72 h aged

2610

1.27

31.3

0.125

0 h fresh

506.7

1.90

24 h aged

2540

0.49

48 h aged

2100

0.13

72 h aged

2350

1.09

100

0.400

0 h fresh

<LOD (460.6)

1.48

24 h aged

1560

0.09

48 h aged

2310

1.74

72 h aged

2750

0.92

Limit of Detection (LOD) = 0.5 mg/L TOC

Validity criteria fulfilled:
yes
Remarks:
Control cell numbers, were found to increase by a factor of 48.37. Coefficient of Variation (section by section) in the control cultures was 29 %. Coefficient of Variation (average growth rate) in replicate control cultures was 2.2 %.
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 set at 100 mg/L (nominal).
The EC10-, EC20- and EC50-value for growth rate and yield were considered to be > 100 mg/L (nominal).

Description of key information

A GLP study was performed in accordance with OECD Guideline 201 to assess the toxicity of the test material, Harpin fermentation extract, to the single cell green alga Pseudokirchneriella subcapitata Hindák.

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 set at 100 mg/L (nominal).

The EC50-value for growth rate and yield was considered to be > 100 mg/L (nominal).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information