Butyl 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:

From 2017-03-27 to 2017-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L, and a control
- Sampling method: Duplicate samples from the freshly prepared test media of all test concentrations and from the control were taken at the start, after 24h, 48h and at the end of the test (72h).
- Sample storage conditions before analysis: All samples were extracted directly after sampling. The extracts of the samples were stored in a fridge (4 ± 4°C) immediately after extraction, and were kept stored until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 100.3 mg test item into 1003 mL test water by intense stirring for 10 minutes and sterilized via membrane filtration. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
- Test concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L, and a control
- Control: Test water was used without addition of the test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: KORSHIKOV, SAG 61.81
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
- Source: see study report for details

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

21.9 – 23.6 °C
measured daily in an Erlenmeyer flask

pH:

5.9 at test start and 6.0 to 6.2 at test end

Nominal and measured concentrations:

Nominal concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L, and a control
Measured concentrations: 57.5, 17.1, 5.68, 1.84 and 0.602 mg test item/L, and a control

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilized Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: at least 50 mL volume containing as much test medium as possible
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, As recommended in OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Mean light intensity: 6633 lux (range: 5900 to 7300 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometric measurement

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 30.7-43.8 mg/L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

27.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

23.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 16.5-32.4 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

21 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

18.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Microscopic Examination:The shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: most recent test: yield 0.549 mg/L (95% CI: 0.49-0.615), growth rate 1.12 mg/L (95% CI: 1.06-1.17)

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50, the corresponding EC20 and EC10 values and where possible their 95%-confidence limits were calculated by Probit analysis.
For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield) and Bonferroni-Welch t-test (growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Algal densities during the test period of 72h

geometric mean test conc (mg/L)	Mean algal density (10,000/mL)
	24h	48h	72h
Control	1.632	5.221	34.718
0.602	2.929	8.311	42.731
1.84	3.124	6.299	39.713
5.68	2.475	6.802	36.407
17.1	3.578	5.58	33.317
57.5	2.216	0.749	0.821

Growth rates μ and percentage inhibition of μ during the test period

 

Mean measured concentrations (mg test item/L)	Growth ratesμ (1/day) and % inhibition of μ
	0-24hr		0-48hr		0-72hr
	μ	%	μ	%	μ	%
Control	1.144	-	1.164	-	1.410	-
0.602	1.758	-53.6	1.402	-20.5	1.482	-5.1
1.84	1.792	-56.6	1.257	-8.0	1.458	-3.4
5.68	1.595	-39.4	1.287	-10.6	1.422	-0.8
17.1	1.959	-71.2	1.190	-2.2	1.397	0.9
57.5	1.486	-29.9	0.183	84.2*	0.150	89.4*

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control (tested with Williams t-test (24 and 48h) and Bonferroni- Welch t-test (72h),α= 0.05, one-sided)

Summary of analytical results:

nominal concentration (mg/L)	measured concentration (mg/L)
	0h	24h	48h	72h
1	0.65	0.61	0.57	0.58
3.2	2.00	1.86	1.72	1.79
10	6.27	6.18	5.13	5.15
32	19.02	18.47	15.83	15.07
100	64.49	59.26	52.54	53.90

Validity criteria fulfilled:

yes

Conclusions:

The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 27.4 mg test item/L, and the 72-hour ErC50 value was calculated to be 36.7 mg test item/L. The 72-hour NOEC for both, growth rate and yield, was determined to be 17.1 mg test item/L.

Executive summary:

The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test according to OECD Guideline 201.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

The quantification of the test item in the test samples was performed using gas chromatography with flame ionization detector.

At the start of the test 63% of the nominal test concentrations were found (average of all test concentrations). After 24, 48 and 72 hours test duration, 55% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 57% of nominal. Therefore, all reported results refer to mean measured concentrations.

The 72-hour EyC50 was calculated to be 27.4 mg test item/L, and the 72-hour ErC50 value was calculated to be 36.7 mg test item/L. The 72-hour NOEC for bot, growth rate and yield, was determined to be 17.1 mg test item/L.

Description of key information

The 72 -hour ErC50 value was determined to be 36.7 mg test item/L for Pseudokirchneriella subcapitata.

The 72 -hour ErC10 value was determined to be 23.1 mg test item/L for Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:

36.7 mg/L

EC10 or NOEC for freshwater algae:

23.1 mg/L

Additional information

For this endpoint only one test is available. The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test according to OECD Guideline 201.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

The quantification of the test item in the test samples was performed using gas chromatography with flame ionization detector.

At the start of the test 63% of the nominal test concentrations were found (average of all test concentrations). After 24, 48 and 72 hours test duration, 55% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 57% of nominal. Therefore, all reported results refer to mean measured concentrations.

The 72 -hour ErC50, ErC10 and NOErC value were calculated to be 36.7, 23.1 and 17.1 mg test item/L, respectively.

The 72-hour EyC50, EyC10 and NOEyC were calculated to be 27.4 and 17.1 mg test item/L, respectively.