3-methylbutyl butyrate

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:

2017-04-10 to 2017-04-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analytically verified via GC-MS after 0 (start of exposure) and 72 hours (end of the exposure).
- Sampling method: Separate replicates for each test item concentration and control for the test item analysis at the beginning of the exposure were prepared with algae. The samples for the test item analysis after 72 hours were prepared with algae and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until the start of the analysis, if necessary. Prepared samples were stored in the autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 50.0 mg test item/L was freshly prepared with dilution water and stirred for 3 hours at room temperature, 1100 rpm.
- Controls: Six replicates (without test item) were incubated under the same conditions as the test concentrations.
- 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 freshwater algae
- Strain: SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day.


ACCLIMATION
- Acclimation period: 72 h
- Culturing media and conditions (same as test or not): The culturing conditions were not the same as the test conditions (the light intensity in the test was higher: approximately 4440 to 8880 lux)
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

mean 22.8 °C (range 22.0 - 23.5 °C)

pH:

test start: 7.53 - 7.55
test end: 7.57 - 8.96

Nominal and measured concentrations:

nominal: 0.0949, 0.300, 0.949, 3.00, 9.49 and 30.0 mg/L
geometric mean measured: 0.0149, 0.140, 0.356, 0.736, 2.50 and 22.1 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile headspace flasks, volume: 59 mL, with aluminium tops with PTFE seals.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Filled without headspace.
- Aeration: no
- Initial cells density: Nominal: approximately 5 x 10^3 - 10^4 cells/mL; Actual: 6839 cells/mL
- Control end cells density: 497365 cells /mL (72 h)
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes, threefold concentrated AAP medium according to the guidelines

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD 201.
- Culture medium different from test medium: Yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24 hours/day light.
- Light intensity and quality: 4440 to 8880 lux, corresponding to 60 to 120 µE*m^-2*s^-1 (nominal)

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: via Chlorophyll a-fluorescence
- Other: Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.13 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.:

4.68 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

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 at test start were in the range of 93 to 100% of the nominal values and between < LOQ and 57% of the nominal values after 72 hours.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the reference item toxicity is in the valid range
- ErC50: with headspace: 0.435 mg/L; without headspace: 0.811 mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-value with confidence intervals of yield inhibition after 72 hours was calculated by sigmoidal dose-response regression.
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield. The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01 for growth rate and yield.
Levene’s test on variance homogeneity was done with a significance level of 0.01 for growth rate and yield.
Monotonicity was done by trend analysis by contrasts (significance level 0.05) for growth rate and yield.
William’s test on significant differences was done with a significance level of 0.05 for growth rate and yield.

Table 1: Measured concentrations and percentage of the nominal concentrations of Isoamyl butyrate.

Sampling date	0 h		72 h
Nominal concentration of the test item (mg/L)	Meas. conc. (mg/L)	%	Meas. conc. (mg/L)	%	Geometric mean measured concentration (mg/L)
30.0	28.4	95	17.2	57	22.1
9.49	9.44	99	0.662	7	2.50
3.00	2.84	95	0.191	6	0.736
0.949	0.922	97	0.138	14	0.356
0.300	0.301	100	0.0654	22	0.140
0.0949	0.0883	93	< LOQ^1		0.0149
Control	< LOQ		< LOQ		< LOQ

 

Meas. conc.= Measured concentration of the test item, dilution and enrichment factor taken into account

% = Percentage of the nominal concentration of the test item

LOQ = Limit of quantification (0.005 mg test item/L)

¹= ½ LOQ taken into account for calculation of the geometric mean measured concentration

Table 2: Evaluation after 72 hours

Geometric mean measured test item concentration [mg/L]	Replicate No.	Growth rate [d-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
22.1	1	-0.270	100	-3798	100
	2	-0.135	100	-2282	100
	3	-0.205	100	-3140	100
	mean	(+)     -0.203	100	(+)        -3073	100
2.50	1	1.37	4	405175	17
	2	1.40	2	451303	8
	3	1.32	7	356340	27
	mean	(+)         1.36	4	(+)     404273	18
0.736	1	1.43	0	489649	0
	2	1.42	0	479905	2
	3	1.42	1	471888	4
	mean	(-)         1.42	0	(-)      480481	2
0.356	1	1.43	0	497900	-2
	2	1.38	3	421853	14
	3	1.34	6	369741	25
	mean	(-)         1.38	3	(-)      429831	12
0.140	1	1.41	1	461852	6
	2	1.44	-1	507380	-3
	3	1.38	3	422936	14
	mean	(-)         1.41	1	(-)      464056	5
0.0149	1	1.36	5	399411	19
	2	1.38	3	427325	13
	3	1.33	7	362806	26
	mean	(-)         1.36	5	(-)      396514	19
Control	1	1.41		461383
	2	1.44		503313
	3	1.47		562418
	4	1.45		516685
	5	1.41		457375
	6	1.40		441984
	mean	1.43		490526

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test substance to the green freshwater algae Pseudokirchneriella subcapitata was determined according to OECD 201 and GLP. The results are as follows:
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 4.68 mg/L and 3.84 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 3.13 mg/L and 1.23 mg/L, respectively.

Description of key information

ErC10 (72 h): 3.13 mg/L (Pseudokirchneriella subcapitata; OECD 201)

ErC50 (72 h): 4.68 mg/L (Pseudokirchneriella subcapitata; OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

4.68 mg/L

EC10 or NOEC for freshwater algae:

3.13 mg/L

Additional information

The toxicity of the test substance to the green freshwater algae Pseudokirchneriella subcapitata was determined according to OECD 201 and GLP. The algae were exposed to 0.0949, 0.300, 0.949, 3.00, 9.49 and 30.0 mg/L, equivalent to 0.0149, 0.140, 0.356, 0.736, 2.50 and 22.1 mg/L geometric mean measured concentrations, of the test substance for 72 hours. The concentrations and the control were analytically verified via GC-MS after 0 and 72 hours. The respective effect values were ErC50 = 4.68 mg/L, ErC10 = 3.13 mg/L, EyC50 = 3.84 mg/L and EyC10 = 1.23 mg/L (based on geometric mean measured concentrations).