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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:
01-27 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 201 without any major deviation
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
pH level in the control varied more than 1.5 units at the end of the test
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for analysis were taken from the control and all test concentrations.
- Sampling method: Samples were taken at the start (t = 0 h) and the end of the test (t = 72 h). Approximately 1 mL were taken for each sample and analysed by UHPLC-MS/MS.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis or analysed directly after sampling.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared with a nominal concentration of 200 mg/L for the final test. A 200 mg (nominal) of the test item (actual weight: 200.72 mg) were carefully added to 1 L of test water in a sealed glass vessel of 1 L capacity containing a magnetic stirring bar. The mixing was performed during 24 h in the dark. Following mixing, the contents of the vessel were allowed to stand undisturbed for 1 h at room temperature. Then, the stock solution (200 mg/L nominal) was diluted with test water and a fixed amount of inoculum to obtain the required test concentrations in the flasks and a cell density of 1 x 10^4 cells/mL/vessel.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: Pseudokirchneriella subcapitata, CCAP 278/4
- Source (laboratory, culture collection): Museum National d'Histoire Naturelle, Paris, bred in the Laboratory under standardised conditions according to the test guidelines

ACCLIMATION
- Acclimation period: 2-4 days
- Culturing media and conditions (same as test): Same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
No data
Test temperature:
22.4-22.9 °C
pH:
7.73-10.22
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0.5, 1.3, 3.0, 7.2, 17.4, 41.7 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass flasks (100 mL) sealed with a fritted glass stopper
- Type: Closed
- Shaking: During incubation, the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 10000 cells/mL
- Control end cells density: 539000 ± 76000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per control (replicates): Six
- Moreover, a replicate of each test concentration without algae was prepared in order to assess potential bioaccumulation and/or adsorption effects of the test item by P. subcapitata during the test period and to determine maintenance of actual concentrations.

GROWTH MEDIUM
- Standard medium used: Yes; original medium of OECD TG 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Original medium of OECD TG 201, prepared using sterilised water
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was measured continuously in the growth chamber, over the study period and pH was recorded at the beginning and at the end of the test in one vessel per concentration and the control.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 6403-7258 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.4
- Range finding study
- Test concentrations: 0, 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: In the range-finding test after 72 h, no significant effect on algal growth was observed at the nominal exposure concentrations of 0.1 mg/L while a slight effect was observed at 1.0 mg/L. Significant algal growth inhibition was found at the concentration of 10 mg/L. Concerning the concentration of 100 mg/L results showed 100 % algal growth inhibition. Based on these results, test solutions used in the definitive test were prepared to obtain the following nominal concentrations: 0.5, 1.3, 3.0, 7.2, 17.4, 41.7 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (non-concurrent)
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
9.54 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL = 7.68-11.85
Key result
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
2.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL = 1.55-2.91
Details on results:
- Exponential growth in the control (for algal test): Yes
- ErC10 (72 h) = 3.48 (1.87-4.78) mg/L (nominal)
- ErC20 (72 h) = 4.91 (3.14-6.31) mg/L (nominal); EyC20 (72 h) = 0.46 (0.22-0.72) mg/L (nominal)
- ErC50 (72 h) = 9.54 (7.68-11.85) mg/L (nominal); EyC50 (72 h) = 2.16 (1.55-2.91) mg/L (nominal)
- Refer tables 6.1.5/2 and 6.1.5/3 for details
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Historical data (dated: May 2012): ErC50 (72 h): 0.91 mg/L
Reported statistics and error estimates:
- Effective concentrations were determined using the software ToxRat® Professional.

Table 6.1.5/2: Yield (Y) and its inhibition relative to control (%)

Treatment [mg/L]

0-24 h

0-48 h

0-72 h

Y

% I

Y

% I

Y

% I

Control

3.400

0

27.667

0

52.933

0

0.5

2.867

15.7

25.400

8.2

35.800

32.4

1.3

1.933

43.1

18.467

33.3

36.867

30.4

3

0.067

98.0

14.733

46.7

21.800

58.8

7.2

0.600

82.4

11.933

56.9

19.267

63.6

17.4

-0.333

109.8

1.667

94.0

1.000

98.1

41.7

-0.467

113.7

0.333

98.8

-0.067

100.1

100

-0.467

113.7

-0.200

100.7

-0.200

100.4

 

Table 6.1.5/3: Growth rate (G) and its inhibition relative to control (%)

Treatment [mg/L]

0-24 h

0-48 h

0-72 h

G

% I

G

% I

G

% I

Control

1.458

0

1.662

0

1.326

0

0.5

1.344

7.8

1.627

2.1

1.202

9.4

1.3

1.062

27.1

1.482

10.8

1.209

8.8

3

0.008

99.5

1.375

17.2

1.041

21.5

7.2

0.239

83.6

1.271

23.5

0.997

24.9

17.4

-3.219

320.8

0.475

71.4

0.181

86.3

41.7

-3.219

320.8

0.109

93.5

-0.029

102.2

100

-3.315

327.4

-0.160

109.6

-0.074

105.6

Validity criteria fulfilled:
yes
Remarks:
control cultures (0-72 h): cell concentration increased by 54 folds; mean CV for section by section specific growth rate was 18 %; CV for average specific growth rate was 3.6 %
Conclusions:
The ErC50 and EyC50 at 72 h for nerol to green algae (Pseudokirchneriella subcapitata) based on nominal concentration were determined to be 9.54 (7.68-11.85) and 2.16 (1.55-2.91) mg/L, respectively.
Executive summary:

An algal toxicity study was performed in accordance with OECD guideline 201 and GLP. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to nerol at nominal concentrations of 0, 0.5, 1.3, 3.0, 7.2, 17.4, 41.7 and 100 mg/L (three replicate flasks per test concentration and six replicates for control) for 72 h, under constant illumination and shaking at a temperature of 22 ± 1 °C. Measurement of biomass and growth inhibition was done by determination of cell densities at 24, 48 and 72 h using a counting chamber.

After 72 h, cell concentration in the control culture increased by 54 folds; mean CV for section by section specific growth rate was 18 and CV for average specific growth rate was 3.6 %. Hence, it met the validity criteria for the control vessels.

The concentrations of the test item (with and without algae) were determined by chemical analyses at start and at the 72-h incubation period. Chemical analyses revealed test item losses in the presence of algae mainly at low-level concentrations, suggesting metabolism and/or adsorption effects of the test item by P. subcapitata, since test item concentrations remained relatively stable without algae during the test period (within ± 20 % of the nominal).

Therefore, the ErC50 and EyC50 at 72 h for nerol to green algae (Pseudokirchneriella subcapitata) based on nominal concentration were determined to be 9.54 (7.68-11.85) and 2.16 (1.55-2.91) mg/L, respectively.

Description of key information

The substance exhibits an ErC50 for freshwater algae of 7.45 mg/L and an EC10 of 2.58 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
7.45 mg/L
EC10 or NOEC for freshwater algae:
2.58 mg/L

Additional information

One reliable study is available for the registered substance. The toxicity to aquatic algae was studied according to OECD TG No. 201 and Test Method C.3 from Commission Regulation (EC) No. 440/2008. Algae cultures (Pseudokirchneriella subcapitata) were exposed to substance at nominal test concentrations of 0.5, 1.3, 3.0, 7.2, 17.4, 41.7 and 100 mg/L over a test period of 72 hours (static test). Concentrations were analytically verified at the start and daily until the end of the test. Measured concentrations deviated more than 20% of the initial concentrations (at low concentration). Thus the endpoints were based on measured concentration (averaged exposure concentration). Based on the average measured exposure concentrations in the biotic test and under the experimental conditions, the 72-hour EC50 for growth rate was determined to 7.45 mg/L and EC10 to 2.58 mg/L.