Methyl valerate

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:

2002

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

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

As a parameter for the cell density, the chlorophyll fluorescence in the test and control solutions were determined at 0, 24, 48 and 72 hours of incubation

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM:
- Source: "Sammlung von Algenkulturen, Universität Goettingen" (Collection of Algae Cultures, University Goettingen, Germany)
- Maintenance and Acclimatisation: kept on slant agar containing a nutrient solution under standardized conditions (10-15 °C, diffuse light [light/dark rhythm 12 hr/12 hrs); this culture was dissolved in a liquid nutrient solution and subsequently transferred approx. once weekly
- Preparation of pre-cultures: The algae for the pre-culture were taken from a stock up to seven days old, approx. 72 hours before the start of the incubation in the test; kept in nutrient solution according to the guideline, starting with an approximate cell density of 10,000 cells/mL, counted with the Coulter Counter
- Incubation conditions: incubated for 3 days under continuous light in an incubator shaker apparatus; the temperature was between 21 and 24 °C
- Test cultures: After the incubation period the cell concentration of the algae reached a sufficient density (approx. 380,000 cells/mL), and the pre-culture was diluted to approx.100,000 cells/mL with nutrient solution, ready to be used as inoculum

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

22 - 24 °C

pH:

at the end of the test: 8.8 - 9.6

Dissolved oxygen:

No data

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

1.25, 2,5, 5, 12.5, 25 and 100 mg/L (nominal concentration)
No verification of the test concentration was made due to the good water solubility and the assumed stability of the test substance.

Details on test conditions:

I. Preparation of the stock solution, test solutions and addition inoculum:
- For the stock solution 63 mg of test item were diluted in 500 mL demineralized water under constant stirring for approx. 1 hour at room temperature
- The resulting solution with a concentration of 100 mg/L (including dilution by nutrient solution and inoculum) was used for the highest test concentration
- The pH-value (7.6) was measured in the stock solution
- The highest stock solution was further diluted with demineralized water by an electronically controlled syringe resulting to 1:5, 1:10, 1:25, 1:50, and 1:100.
- Further, a control prepared with demineralized water was set up
- 2.5 mL of nutrient solution were added to the 20 mL test substance solutions; finally, 2.5 mL of the inoculum were added, all by electronically controlled syringing
- Each test concentration was set up in triplicate, the control without test compound was incubated in six paralleis

II. Incubation conditions
- Stirring time: approx. 72 hours
- Incubating apparatus: Abimed AlgenTest XT, with continuous light (at approximately 6000-8000 lux) and controlled temperature (water bath)

III. pH and temperature measurements:
- After exposure the pH was measured in two control and one test vessel of each concentration
- The temperature in the water bath was continuously monitored by a minimax thermometer

IV. Fluorescence determination:
- As a parameter for the cell density, the chlorophyll fluorescence in the test and control solutions were determined at 0, 24, 48 and 72 hours of incubation
- All measurements were made by means of a fluorescence photometer at a wavelength of 320 and 800 nm for agitation and measurement, respectively
- Sampling volume and sampling time were electronically controlled

V. Microscopic examination:
- The algae were microscopically examined at the end of exposure
- The algae of one test vessel of each test concentration group including the control were examined with regard to cell abnormalities

VI. Calculation of results and statistical analysis
- The values for the average area below the growth curves, specific growth rate, and the percentage inhibition were calculated by the computer program Abimed "AlgenTest XT 2.21". The program calculates the respective parameters according to the mathematical formulas given in the OECD guideline
- The EC50-values as well as the 95% confidence limits were calculated by the above referenced computer program on the basis of a probit analysis

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

61.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The growth inhibition (biomass and growth rate) showed no clear concentration dependent pattern. Already at 24 hours, the inhibition of biomass growth varied between 38% at the lowest concentration and 6% at the second highest concentration. At 48 and 72 hours this range and distribution was similar. The inhibition of growth rate showed a comparable pattern, in that at the lowest concentration had much higher inhibition rates at all time points than at the second highest concentration. At the highest concentration (100 mg/L) there was the maximum inhibition of both, biomass and growth rate. Since there was no concentration related pattern of inhibition up to the second highest concentration, the observed inhibition of growth was not considered substance related but a spontaneous reaction. The growth inhibition at the highest concentration, in contrast, is considered to be a substance related effect, since it exceeded the inhibition at all other concentrations and the microscopical examination revealed clear signs of adverse effects on algal cells.
As the guideline defines the inhibition of growth as the endpoint for algae study EC 50 > 100 mg/L is taken as key value.

Inhibition of biomass and the growth rate (%) of Desmodesmus subspicatus after 72 hours exposure to methylvalerat

		Inhibition in percent
Dilution of methylvalerat	Nominal concentration of methylvalerat (mg/L)	Biomass (integral)	Growth rate
0	0.0 (control)	--	--
1:100	1.25	35.1	13.2
1:50	2.5	20.9	2.2
1:25	5	14.7	4.4
1:10	12.5	22.9	10.2
1:5	25	-7.4	-6.3
1:1.25	100	63.5	37.2

Validity criteria fulfilled:

yes

Remarks:

> 16-fold increase of algae cells after 72 hours in the controls

Conclusions:

After 72 hours of exposure methyl valerate had an inhibitory effect at the highest concentration (100 mg/L) on the growth of Desmodesmus subspicatus. The EbC5O (biomass) was 61.5 mg/L [95% confidence limits not calculated] and the ErC50 (growth rate) was higher than 100 mg/L. The results are relating to nominal concentrations.

Executive summary:

The purpose of this study was to determine the effects of methyl valerate on the growth of the green algae Desmodesmus subspicatus. The study was conducted in agreement with the test guideline OECD no. 201. Methyl valerate was incubated in an aqueous solution including nutrients with an algae population of Desmodesmus subspicatus for a test duration of approximately 72 hours in an electronically controlled dosing and incubation apparatus. The nutrient solution was made up of mainly nitrate, phosphates and some trace elements. For the preparation of the test solutions 125 mg/L methyl valerate was dissolved in water and stirred for approximately 1 hour. The resulting solution served as the highest concentration, which was a 1:1.25 dilution by adding nutrient solution and inoculum. It was further diluted 1:5, 1:10, 1:25, 1:50, 1:100 with demineralized water. Additionally, a control solution was prepared with demineralized water without test material. The algae were exposed to each concentration in triplicate. Six vessels were prepared for the control. The algae were incubated under standard conditions (continous light, controlled temperature ). A chemical analysis was not performed since the test compound was assumed to be weIl soluble and stable in an aqueous solution. As a parameter for the growth of the algae population, the fluorescence of the algal cells was measured with a fluorescence-photometer. The increase of biomass and the growth rate were calculated on the basis of the fluorescence. The calculated biomass and growth rate of each concentration were compared to those of the controls, and the inhibition was calculated. The biomass and growth rate was inhibited at several concentrations, however, not in a clear concentration related manner. Only at 100 mg/L there was an obvious inhibition of growth resulting in high biomass and growth rate reduction. This was considered to be a compound related effect. The EbC5O (biomass) was 61.5 mg/L [95% confidence limits not calculated], the ErC50 (growth rate) was higher than 100 mg/L both on the basis of nominal concentrations. As the guideline defines the inhibition of growth as the endpoint for algae study EC 50 > 100 mg/L is taken as key value. This toxicity study is classified as acceptable and satisfies the guideline requirements for the Algae study.

Description of key information

After 72 hours of exposure methyl valerate had an inhibitory effect at the highest concentration (100 mg/L) on the growth of Desmodesmus subspicatus. The ErC50 (growth rate) was higher than 100 mg/L. The result relates to nominal concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

"Should read: > 100 mg/L"

The EbC5O (biomass) determined in the study was 61.5 mg/L [95% confidence limits not calculated] relating to nominal concentrations.