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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:
04 March - 19 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
not specified
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
- Lot No. of test material: 07-170-FIL-2/3
- Appearance: White powder
- Expiration date: May 2010
- Purity: >98.4%
- Storage condition of test material: Ambient
Analytical monitoring:
yes
Details on sampling:
The concentration of test substance was determined in each test solution at 0 and 96 hours by standard analytical methods (not described in the report).
Vehicle:
yes
Remarks:
Dimethyl formamide (DMF)
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test material was determined to be insoluble in water. Therefore, a water-soluble fraction was prepared by adding the required nominal amount of test substance in moderately hard synthetic freshwater and mixing the solution for 20-24 hours. After this mixing time, the undissolved test substance was separated from the solution and only this water-soluble fraction was used for testing.
- Controls: Positive control (3 flasks treated with 10 mg/L zinc chloride) and solvent control (3 flasks containing 0.25 mL DMF in 500 mL of dilution water added to stock solution)
- Chemical name of vehicle: Dimethyl formamide (DMF)
- Concentration of vehicle in test medium: DMF was added as a solvent to the water-soluble fraction at 0.25 mL per 500 mL dilution in water.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Source: StillMeadow, Inc. cultures
- Age of inoculum (at test initiation): 3 - 7 ± 1 days
- Method of cultivation: Three days prior to testing, 1000 mL of autoclaved algal stock culture medium (Woods Hole) was inoculated. This new mass culture was then placed in a ~25°C environmental chamber and maintained on continuous aeration.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
24 ± 2°C (actual temperature 23°C)
pH:
INITIAL pH:
- Control 7.4
- 2.0 mg/L 7.4

TERMINAL pH:
- Control: 8.0 - 8.1
- Test vessels: 8.0 - 8.2
- Positive control: 7.3 - 7.4
- Solvent control: 8.3 - 8.5
Dissolved oxygen:
No data
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal: 0.25, 0.50, 0.75, 1.0 and 2.0 mg/L
Measured: See table 2
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL sterile Erlenmeyer flasks with stoppers and aeration tubing; filled with 100 mL of the appropriate dilution
- Type: stoppered flasks
- Aeration: constant aeration
- Agitation: constant agitation at approximately 100 cycles/minute
- Initial cells density: 10000 cells/mL
- Control end cells density: 368 - 426 x 10^4 cells/mL after 96 hours
- No. of vessels per concentration (replicates): 3 flasks per test concentration
- No. of vessels per control (replicates): 3 flasks
- No. of vessels per vehicle control (replicates): 3 flasks

GROWTH MEDIUM
- Medium used: Algal stock culture medium (Woods Hole) with EDTA up to 0.3 mg/L
- Detailed composition: Not specified

TEST MEDIUM / WATER PARAMETERS
Not specified

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: continuous lighting
- Light intensity and quality: 400 ± 40 ft.c

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Observations of inhibition were made for each test container at 24, 48, 72 and 96 hours following dosing by measuring the mean cell density with a haemocytometer.

TEST CONCENTRATIONS
- Range finding study: There was one flask of test medium for each concentration. The algae were introduced to each concentration at an initial cell density of 10000 cells/mL and at 96 hours the terminal density was measured.
- Test concentrations: 1, 10, 100, 500 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Not specified
Reference substance (positive control):
yes
Remarks:
Zinc chloride (3 flasks treated with 10 mg/L)
Key result
Duration:
96 h
Dose descriptor:
IC50
Effect conc.:
0.24 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
< 0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Control: The growth rate increased by a factor of at least 16 and the 96 hour terminal control increased to at least 3.5x10^6 cells/mL.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes

Table 1: Daily cell counts:

Test concentration (mg/L)

Replicate

Cell density x 104cells/mL

24 hours

48 hours

72 hours

96 hours

Control

A

10

24

76

368

B

9

11

98

402

C

5

22

111

426

0.25

A

8

12

58

175

B

5

18

84

191

C

11

20

95

210

0.50

A

6

8

43

134

B

4

12

45

156

C

5

8

58

117

0.75

A

5

14

51

125

B

6

12

59

152

C

4

12

48

147

1.0

A

4

16

44

119

B

4

14

34

130

C

3

7

40

123

2.0

A

2

6

31

84

B

4

7

35

91

C

3

10

38

76

Positive control

10 mg/L ZnCl2

A

0

0

1

0

B

1

1

0

1

C

0

2

1

1

Solvent control (DMF)

A

9

17

88

379

B

7

28

95

362

C

10

42

106

418

 

 

Table 2: Dose verification

 

Control

mg/L

Positive control

Solvent control

Time point

NA

0.25

0.50

0.75

1.0

2.0

10 mg/L ZnCl2

DMF

Day 0

0.53

0.58

0.57

0.61

0.59

0.68

0.51

0.52

96 hour

0.65

0.56

0.69

0.54

0.58

0.61

0.63

0.59

 

Validity criteria fulfilled:
yes
Remarks:
The 72 hour interim control density and growth rate increased by a factor of at least 16 and the 96-hour terminal control increased to at least 3.5x10^6 cells/mL.
Conclusions:
The test concentration of Formononetin that resulted in 50% inhibition (IC50) was determined to be 0.24 mg/L with 95% confidence limits of 0.22 - 0.38 mg/L. The NOEC was determined to be <0.25 mg/L.

Description of key information

A GLP study was performed in accordance with OECD Guideline 201, to determine the toxic effects of the test substance, Formononetin, on the growth of the freshwater algae Selenastrum capricornutum in a 96 hour test. The test concentration of Formononetin that resulted in 50% inhibition (IC50) was determined to be 0.24 mg/L with 95% confidence limits of 0.22 - 0.38 mg/L. The NOEC was determined to be <0.25 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.24 mg/L

Additional information