7-hydroxy-3-(4-methoxyphenyl)-4-benzopyrone

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

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

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

not specified

Qualifier:

according to guideline

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 10^4cells/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