3-methylbutyl isovalerate

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:

2015-02-02 to 2015-02-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified via GC-FID after 0 hours (start of exposure) and 72 hours (end of exposure).
- Sampling method: Separate replicates for the test item analysis at the beginning of exposure were prepared without algae. For the test item analysis after 72 hours, separate replicates were prepared with algae at the beginning of exposure and incubated under test conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For preparation of the test concentrations the density was taken into account. Appropriate volumes of the test item were pipetted directly into measuring flasks, containing the respective volume of the dilution water. After dispersion treatment the prepared test solutions were transferred to the test vessels, which were tightly closed, without headspace. The test solutions were stirred with approximately 1100 rpm for 30 minutes at room temperature.
- Controls: Six replicates (without test item) were incubated under the same conditions as the test vessels
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG),
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE*m^-2*s^-1

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L (dilution water)

Test temperature:

22-23 °C

pH:

7.90-9.34

Nominal and measured concentrations:

nominal: 0 (control), 0.0632, 0.200, 0.632, 2.00, 6.32, 20.0 mg/L
measured (geometric mean, 72 h): 0 (control), 0.0549, 0.171, 0.452, 1.60, 4.62, 8.50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks (volume 119 mL) with aluminium tops with PTFE seals
- Type (delete if not applicable): closed
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL; current: 6089 cells/mL
- Control end cells density: 889503 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Ca/mg ratio: 1.5

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: 60 - 120 µE*m^-2*s^-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Microscopic evaluation of the cells at the start and the end was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence od algae to the test containers and agglutination of algae cells
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the test item concentration of 48.0 mg/L

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1^1/10
- Range finding study: yes
- Test concentrations: 0 (control), 0.48, 4.80, 48.0 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- The measured concentrations of the test item in the fresh media (0 h) were in the range of 70 to 84 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 23 to 90 % of the nominal values of all concentrations. Therefore, all effect values given are based on the geometric mean measured test item concentrations of the test item.

Results with reference substance (positive control):

- Results with reference substance valid? yes, the reference item toxicity is in the valid range following test facility SOPs.
- ErC50: with headspace: 0.749 mg/L; without headspace: 0.774 mg/L

Table 1: Analytical measurements

Nominal test item concentration     [mg/L]	test item 0 h		test item 72 h		Geometric mean measured concentration
	Meas.conc. [mg/L]	[%]	Meas.conc. [mg/L]	[%]	[mg/L]
20.0	15.9	79 (1)	4.56	23	8.50
6.32	4.48	71 (1)	4.77	75	4.62
2.00	1.65	82 (1)	1.56	78	1.60
0.632	0.442	70 (1)	0.463	73	4.52
0.200	0.166	83	0.175	87	0.171
0.0632	0.0530	84	0.0568	90	0.0549
Control	< LOQ		< LOQ

Meas. Conc = Measured concentrations of the test item (dilution factor taken into account)

% = Percent of the nominal concentration of the test item

LOQ = Limit of Quantification (5 µg/L test item)

(1) = mean value of 3 measured concentrations, two replicates reanalyzed on 2015-02-04

 

 

Table 2: Cell densities and algae evaluation

Geometric mean measured test item concentration [mg/L]	Cell density mean [cells/mL]   0 hours	24 hours	48 hours	72 hours	Growth rate mean [d^-1]	Inhibition of growth rate mean [%]	Yield mean [cells/mL]	Inhibition of yield mean[%]
8.50	6089	10187	7003	5973	(+) -0.01	100	(+) -116	100
4.62	6089	26802	116605	527513	(+) 1.49	10	(+) 521424	41
1.60	6089	33718	197179	732855	(-) 1.60	4	(-) 726766	18
0.452	6089	40068	252995	838762	(-) 1.64	1	(-) 832673	6
0.171	6089	38648	261439	862855	(-) 1.65	0	(-) 856766	3
0.0549	6089	36615	232210	774034	(-) 1.61	3	(-) 767945	13
Control	6089	37537	299206	889503	1.66		883414

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item on Pseudokirchneriella subcapitata was tested in a GLP study according to OECD 201. The determined effect levels for growth inhibition were ErC10 4.6 mg/L and ErC50 5.47 mg/L based on geometric mean measured concentrations.

Description of key information

The toxicity of the test item on Pseudokirchneriella subcapitata was tested in a GLP study according to OECD 201. The determined effect levels for growth inhibition were ErC10 4.6 mg/L and ErC50 5.47 mg/L based on mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.47 mg/L

EC10 or NOEC for freshwater algae:

4.6 mg/L

Additional information

The toxicity of the test item on Pseudokirchneriella subcapitata was tested in a GLP study according to OECD 201. Algae were exposed to 0 (control), 0.0632, 0.20, 0.632, 2.00, 6.32 and 20 mg/L of the test substance, equivalent to geometric mean measured concentrations of 0 (control), 0.0549, 0.171, 0.452, 1.60, 4.62 and 8.50 mg/L. Concentrations were analytically verified with GC-FID. After 72h the growth inhibition effect levels were ErC10 4.6 mg/L and ErC50 5.47 mg/L, and corresponding yield inhibition effect levels were EyC10 3.6 mg/L and EyC50 4.75 mg/L based on mean measured concentrations. The NOEC was determined at 1.6 mg/L.