Ethyl 2-methylbutyrate

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:

06 August to 19 August 2012

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/l

Vehicle:

yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection 10801, University of Boulevard, Manassas, Virginia, 20110-2209, USA. Subculture maintained at Section - Ecotoxicology, Jai Research Foundation, Valvada - 396 108, Dist. Valsad, Gujarat.

- Date of Receipt (at JRF): May 13, 2010
- Date of Last Subculture: August 11, 2012
- Method of Cultivation: Static condition

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not determined

Test temperature:

21 - 23°C

pH:

7.26 - 7.92

Dissolved oxygen:

not determined

Nominal and measured concentrations:

Test Concentrations: 100.0 mg Ethyl 2-methylbutyrate/L and control [without Ethyl 2-methylbutyrate] and vehicle control [with acetone].

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical flask
- Initial cells density: Initial Mean Number of Cells/mL: 12250

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: See attached report for details of Alga Culture Stock Solution and Medium Preparation
- Intervals of water quality measurement:
- Analysis of Test Concentration: A volume of 50 mL test media were collected in glass bottles for each test concentration. For determination of the stability and a.i. concentration of Ethyl 2-methylbutyrate in test media, sampling was carried out at 0 and 72 h (control, vehicle control, lowest concentration and highest concentration) of sample preparation during range finding study. Samples for a.i. analysis were collected at 0 h during the main study for all the test concentrations. All the samples (range finding and main study) were centrifuged to separate the test media and alga cells before using it for stability and active ingredient analysis, the supernatant was used for stability and active ingredient analysis. Active ingredient concentration in water was determined using the validated analytical method [Gas Chromatograph (Perkin Elmer, Clarus 500), Column: DB-17 (30 m x 0.25 mm; 0.25 µm film thickness), Detector: Flame Ionization Detector (FID), Carrier gas: Nitrogen, Carrier Flow 1.0 mL/minutes, Hydrogen Gas Flow 40 mL/minutes, Air Gas Flow 400 mL/minutes, Injection volume 1.0 µL] (Appendix 4 of attached report).
- Test Culture Maintenance: The cells in the culture flasks were maintained in suspension by agitating the test flasks continuously at 100 ± 2 rpm using an orbital shaker throughout the exposure period. The culture flasks were maintained at temperature of 21°C to 23°C throughout the study. Illumination was provided with florescent lamps emitting between 5566 - 6100 Lux throughout the exposure period. Illumination and temperature were recorded once daily. The pH of the negative control and treated groups was measured at 0 h and 72 h.

OTHER TEST CONDITIONS
- Light intensity and quality: Illumination (Lux) Range 5566 - 6100

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: A volume of 10 mL of the test culture was collected from all replicate flasks (6 replicates from control and 6 replicates from test concentration) at 24, 48 and 72h. The cell concentration of each replicate was determined once at regular intervals of 24h using a haemocytometer and microscope.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study:
- Preliminary Range Finding Study: A preliminary range finding study was conducted with test concentrations of 0 (control without Ethyl 2-methylbutyrate), vehicle control [with acetone], 1.2, 10.0, 25.0, 50.0 and 100.0 mg Ethyl 2-methylbutyrate/L. Two replicates were run for each concentration of the test item and three replicates for the controls. The algal population was assessed at 24, 48 and 72h. The percent inhibitions at the tested concentrations in relation to growth inhibition (EbC50) and growth rate reduction (ErC50) at 0 - 72h for preliminary study are tabulated below:

Nominal concentration Percentage (%) reduction in
(mg Ethyl 2-methylbutyrate/L) Inhibition (EbC) 0-72h Growth rate (ErC) 0-72h
0 (control) - -
Vehicle Control 0.21 -0.16
1.2 0.30 0.00
10.0 0.43 -0.16
25.0 0.36 0.00
50.0 0.69 0.00
100.0 0.94 0.00

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities were observed during the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: No data

- Test Culture Conditions: The pH values ranged from 7.26 - 7.92 (Table 1 and Appendix 1 of the attached report). Temperature was maintained between 21 and 23 °C and illumination values ranged between 5566 and 6100 Lux. Both parameters were within the guidelines limit (Appendix 2 of the attached report).

 

- Algal Cell Count: In the negative control, cell growth in numbers was observed to be more than 77.0 times over 72 h exposure period [as per OECD 201 at least 16 times in control] (Table 2 and Appendix 3 of the attached report). The coefficient of variance of average specific growth rate during the whole test period in replicate control culture was found to be 0.69 percent. The mean coefficient of variation for day 0-1, 1-2 and 2- 3 in control culture was found to be 5.86, 11.11 and 8.70, respectively. Thus coefficient of variation is within the guidelines limit.

 

- Analysis of Test Concentrations: Test media samples were collected in glass bottles for each test concentration for determination of the stability and a.i. concentration of Ethyl 2-methylbutyrate in test media. During range finding study at the start of the test (0 h) 97.46% (lowest concentration) and 97.63% (highest concentration) of the nominal concentrations were found. After 72h test duration 88.37% (lowest concentration) and 89.35% (highest concentration) of the nominal concentrations were found. During main study (0 h) alga were exposed to 98.11% of nominal concentration. All reported results were related to nominal concentration of test item (Appendix 4 of the attached report).

               

- Growth Inhibition, EC50 Values, NOEC and LOEC: As the study was conducted as a limit test the EC50, growth inhibition, growth reduction, NOEC and LOEC of Ethyl 2-methylbutyrate could not be calculated. The Ethyl 2-methylbutyrate does not inhibit the growth of alga (Psudokirchneriella subcapitata) up to the 100 mg/L of alga media.

 

- Validity Criteria of the Study: Cell Density increase in Control Culture: cell increase by the factor of 77.0 within 72 hours, validity criteria was met. Coefficient of Variation of Sectional (Daily) Growth Rate for day 0-1, 1-2 and 2-3 in Control Culture: 5.86. 11.11 and 8.70 thus the validity criterion was met. Coefficient of Variation of Average Growth between Control Replicates: 0.69% and thus the validity criterion was met.

Validity criteria fulfilled:

yes

Conclusions:

As the study was conducted as a limit test the percent growth inhibition and growth reduction of Ethyl 2-methylbutyrate could not be calculated. The Ethyl 2-methylbutyrate does not inhibit the growth of alga (Pseudokirchneriella subcapitata) up to 100 mg/L of alga media.

The results obtained with the positive and negative controls establish the suitability, validity and reliability of the test system, the methods followed and incubation conditions.

Executive summary:

This study was performed to assess the alga (Pseudokirchneriella subcapitata) growth inhibition caused by Ethyl 2-methylbutyrate [Supplied by Toyo Gosei Co., Ltd., Japan]. The method followed was as per the guidelines of OECD No. 201 (March 2006).

 

Exponentially growing cultures of algae (12250 cells/mL) were exposed to limit test concentration of 100mg Ethyl 2-methylbutyrate/L. A negative control (without Ethyl 2-methylbutyrate) and vehicle control (with acetone) were run concurrently. The algal growth was assessed at 24, 48 and 72 h.

 

Test media was analysed for the Ethyl 2-methylbutyrate stability and a.i. concentration to monitor the concentration of test solution at 0 and 72 h during range finding (control, vehicle control, the lowest concentration and highest concentrations) and at 0 h during main study (all the test concentrations). The concentrations selected for range finding and the main study were found to be stable within the guidelines limit.

 

As the study was conducted as a limit test the percent growth inhibition and growth reduction of Ethyl 2- methylbutyrate could not be calculated. The Ethyl 2-methylbutyrate does not inhibit the growth of alga (Pseudokirchneriella subcapitata) up to the concentration of 100 mg/L of alga media.

Description of key information

The concentration at which the substance may affect the growth of algae is >100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Growth Inhibition, EC50 Values, NOEC and LOEC: As the study was conducted as a limit test the EC50, growth inhibition, growth reduction, NOEC and LOEC of Ethyl 2-methylbutyrate could not be calculated. The Ethyl 2-methylbutyrate does not inhibit the growth of alga (Pseudokirchneriella subcapitata) up to the 100 mg/L of alga media.