Ethyl 2-methylbutyrate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 August to 22 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A volume of 25 ml test media was collected in glass bottles for each test concentration.
- Sample storage conditions before analysis: No data

Vehicle:

yes

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: The master culture of Daphnia magna was originally procured from the Department of Zoology, University of Pune, Pune, India and is regularly subcultured at Ecotoxicology Laboratory of Jai Research Foundation. The daphnids were cultured and maintained in reconstituted water medium.
- Feeding during test: They were fed with unicellular alga, Pseudokirchneriella subcapitata (formally known as Selenastrum capricornutum) during culturing and experiment.
- Food type: They were fed with unicellular alga, Pseudokirchneriella subcapitata (formally known as Selenastrum capricornutum) during culturing and experiment.

ACCLIMATION
- Healthy adult daphnids were acclimated under the same conditions, as specified for the main study. First instar daphnids (≤ 24 h old) were separated and used for the study.

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

21 d

Post exposure observation period:

- Observations were carried out daily to obtain information about the number of offspring produced in each replicate during the exposure period of 21 days. The number of live and dead offspring produced during the course of the experiment were recorded and removed from the glass beakers. The adults were observed for mortality, if any, throughout the duration of the experiment on a daily basis.
- Temperature, dissolved oxygen and pH of the test media were measured initially (0 h) and after every 48 h by using a dissolved oxygen meter and pH meter before every medium renewal. Total hardness of the test media was measured prior to every medium renewal.
- The whole experiment was conducted in a specially designed low temperature water bath preset to a water temperature of 19.8°C. Uniform temperature in the water chamber of the water bath was obtained by maintaining a slow circulation of water by a motor. The beakers containing the test solutions with daphnids were immersed in the water bath. The beakers were held in position by special clamps provided within the chamber of the water bath.
- A photoperiod of 16 hours light and 8 hours dark was maintained using an automatic timer and lighting was provided by using fluorescent tubes. Light intensity was measured during exposure period.

Hardness:

Total hardness of test media ranged between 204 and 216 mg/L as CaCO3m

Test temperature:

Temperature of test media ranged between 20.0 and 22.1°C

pH:

pH values of test media ranged between 7.30 and 8.06

Dissolved oxygen:

Dissolved oxygen values of test media ranged between 7.29 and 9.98 mg/L

Salinity:

N/A

Nominal and measured concentrations:

Acute study
- Preliminary study: control [without Ethyl 2-methylbutyrate], vehicle control [with acetone], 1.2, 10.0, 25.0, 50.0 and 100.0 mg of Ethyl 2-methylbutyrate/L
- Main study: Based on the results of the preliminary study the main study was restricted to a limit study. The limit study was conducted with single test concentration of 100 mg Ethyl 2-methylbutyrate/L

Reproduction test
- Preliminary study: control [without Ethyl 2-methylbutyrate], vehicle control [with acetone], 1.2, 5.0, 10.0, 50.0 and 100 mg Ethyl 2-methylbutyrate/L
- Main study: control [without Ethyl 2-methylbutyrate], vehicle control [with acetone], 1.3, 3.6, 9.6, 25.9 and 70.0 mg Ethyl 2-methylbutyrate/L were selected for the main study.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker of 600 mL capacity
- Material, size, headspace, fill volume: Each replicate comprising of one daphnid, the volume of test media used was 125 mL
- No. of organisms per vessel (for Reproduction main test): 10 daphnids per group. Each group consisted of ten replicates, 1 daphnid per replicate

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water: Reconstituted water was used as the test media. It was prepared by adding different chemicals to distilled water. The detailed procedure is recorded in Appendix 5 of the attached report.
- Intervals of water quality measurement:
- Temperature, dissolved oxygen and pH of the test media were measured initially (0 h) and after every 48 h by using a dissolved oxygen meter and pH meter before every medium renewal. Total hardness of the test media was measured prior to every medium renewal.
- Determination of Total Viable Count: A Volume of 25 mL test media was collected in glass bottles for each test concentration. For determination of the stability and a.i. analysis concentration of Ethyl 2-methylbutyrate in test media sampling was carried out on days 0 (0 h), 2nd (48 h), 10th (0 h), 12th (48 h), 18th (0 h) and 20th (48 h) from control, vehicle control, lowest concentration and highest concentrations of sample preparation during the range finding study and on day 0 (0 h) from all the test concentrations during the main study. Active ingredient concentration in test media 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 µL] (Appendix 4 of the attached report).

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: 1030 and 1130 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): NOEC, LOEC, 21d EC50

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study:
- Acute Study: A preliminary range finding study was conducted with 70 daphnids, divided into seven groups of 10 daphnids per group. The concentrations selected for the preliminary screening were control [without Ethyl 2-methylbutyrate], vehicle control [with acetone], 1.2, 10.0, 25.0, 50.0 and 100.0 mg of Ethyl 2-methylbutyrate/L. The percent immobility observed were 0, 0, 0, 0, 0, 0 and 0% at the test concentrations of control [without Ethyl 2-methylbutyrate], vehicle control [with acetone], 1.2, 10.0, 25.0, 50.0 and 100.0 mg/L, respectively. Based on the results of the preliminary study the main study was restricted to a limit study. The limit study was conducted with single test concentration of 100 mg Ethyl 2-methylbutyrate/L. On the base of above results, the preliminary range finding study for reproduction study was conducted.

- Preliminary Range Finding Study (Reproduction): A preliminary range finding study for reproduction was conducted with concentrations of control [without Ethyl 2-methylbutyrate], vehicle control [with acetone], 1.2, 5.0, 10.0, 50.0 and 100 mg Ethyl 2-methylbutyrate/L. No adult mortality was observed at any of the test concentrations of control [without Ethyl 2-methylbutyrate], vehicle control [with acetone], 1.2, 5.0, 10.0, 50.0 and 100 mg Ethyl 2-methylbutyrate/L. The total number of live offspring produced per group were 446, 438, 382, 314, 299, 296 and 166 at the test concentrations of 0 (control), vehicle control (acetone), 1.2, 5.0, 10.0, 50.0 and 100 mg Ethyl 2-methylbutyrate/L.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Basis for effect:

other: reproductive output

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.6 mg/L

Basis for effect:

other: reproductive output

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

22.53 mg/L

Basis for effect:

other: reproductive output

Remarks on result:

other: 95% fiducial limits between 5.48 and 92.67 mg/L

Reported statistics and error estimates:

The EC50 values for reproductive output was calculated (Tables 5 and Figure 1 of the attached report) using the Probit analysis method (Finney, 1971). The No Observed Effective Concentration (NOEC) of Ethyl 2-methylbutyrate tested at which no statistically significant difference in reproductive output was observed was 1.3 mg/L, as compared with vehicle control. The Lowest Observed Effective Concentration (LOEC) tested at which statistically significant difference in reproduction output was observed was 3.6 mg/L, as compared with vehicle control. The total numbers of offspring per group were recorded and analysed statistically. There was a significant decrease (p ≤ 0.01) in the production of offspring during exposure period at all of the test concentrations except the lowest (1.3 mg Ethyl 2-methylbutyrate /L) as compared with the vehicle control group (Table 1 of the attached report). The 21 day EC50 for reproductive output in Daphnia magna with Ethyl 2-methylbutyrate was 22.53 mg/L with the 95% fiducial limits between 5.48 and 92.67 mg/L. The regression equation established [Probit mortality (y) vs log concentration (mg/L) of Ethyl 2-methylbutyrate (x)] was y = 0.99 + 0.92x.

- Reproductive Output and Mortality: The cumulative number of offspring produced in control, vehicle control and at the test concentrations of 1.3, 3.6, 9.6, 25.9 and 70.0 mg Ethyl 2-methylbutyrate/L was 786, 783, 751, 433, 404, 372 and 347, respectively. No dead offspring were observed in any of the test concentrations throughout the exposure. There was no adult mortality observed in any of the test concentrations (Table 1 of the attached report).

The mean numbers of offspring survivor per adult in the control and vehicle control groups were 78.60 and 78.30 at end of the experiment.

- Live Birth Index: The live birth index for each group was calculated on the basis of total number of live offspring produced in each group following 21 day exposure period. The live birth index was found to be 100% at all of the test concentrations of 0 (control), vehicle control (acetone), 1.3, 3.6, 9.6, 25.9 and 70.0 mg Ethyl 2-methylbutyrate/L (Table 1 of the attached report).

- Survival Index of Offspring: Day-wise survival index was calculated for each group starting from the day (day 8) of first appearance of offspring to the day of completion (day 21) of the experiment. The survival indices for the control and the treated groups were 100% (Table 3 of the attached report)

 

                                     No. of live offspring on day  

Survival Index = --------------------------------------- x 100

                        Total No. of offspring on day

- Water Quality Parameters: Ethyl 2-methylbutyrate did not alter any of the selected water quality parameters (temperature, dissolved oxygen, total hardness and pH) of the test media (Table 4, Appendix 1 of the attached report). Temperature and dissolved oxygen values of test media were ranged between 20.0 and 22.1°C and 7.29 and 9.98 mg/L, respectively. The pH values of test media were ranged between 7.30 and 8.06. Total hardness of test media were ranged between 204 and 216 mg/L as CaCO₃. Light intensity were ranged between 1030 and 1130 lux. All the parameters were found to be within the limits of the guideline.

 

- Analysis of Test Concentration: A Volume of 25 mL test media were collected in glass bottles for each test concentration. For determination of the stability and a.i. analysis concentration of Ethyl 2-methylbutyrate in test media was carried out during the range finding study at the start of the test day 0 (0 h) 97.46 (lowest concentration) and 98.09 (highest concentration), 2^nd (48 h) 95.93 (lowest concentration) and 96.60 (highest concentration), 10^th (0 h) 92.03 (lowest concentration) and 92.77 (highest concentration), 12^th (48 h) 88.39 (lowest concentration) and 90.21 (highest concentration), 18^th (0 h) 85.82 (lowest concentration) and 86.33 (highest concentration) and 20^th (48 h) 80.82 (lowest concentration) and 81.99 (highest concentration). During the main study (0 h) daphnids were exposed to 96.98% of nominal concentration, all reported results were related to nominal concentration of the test item. The test concentrations selected during the range finding and main studies were within the guideline limit.

- Validity Criteria of the Study: Mortality of the parent animals should not exceed 20% in the control (blank) at the end of the test. Mean number of live offspring produced per parent animal surviving at the end of the test should be ≥ 60 in the control.

- Validity Criteria of the Study: The mortality of the parent animals was 0% in the control (blank) at the end of the test thus the validity criteria was met. The mean numbers of offspring survivor per adult in the control and vehicle control groups were 78.60 and 78.30 at end of the experiment.

Validity criteria fulfilled:

yes

Conclusions:

- The No Observed Effective Concentration (NOEC) of Ethyl 2-methylbutyrate tested at which no statistically significant difference in reproductive output was observed was 1.3 mg/L, as compared with vehicle control.
- The Lowest Observed Effective Concentration (LOEC) tested at which statistically significant difference in reproduction output was observed was 3.6 mg/L, as compared with vehicle control.
- The total numbers of offspring per group were recorded and analysed statistically. There was a significant decrease (p ≤ 0.01) in the production of offspring during exposure period at all of the test concentrations except the lowest (1.3 mg Ethyl 2-methylbutyrate/L) as compared with the vehicle control group.
- The 21 day EC50 for reproductive output in Daphnia magna with Ethyl 2-methylbutyrate was 22.53 mg/L with the 95% fiducial limits between 5.48 and 92.67 mg/L. The regression equation established [Probit mortality (y) vs log concentration (mg/L) of Ethyl 2-methylbutyrate (x)] was y = 0.99 + 0.92x.

Executive summary:

This study was conducted to assess the reproductive output of Daphnia magna exposed to different concentrations of Ethyl 2-methylbutyrate [Supplied by Toyo Gosei Co., Ltd., Japan] over a period of 21 days.The method followed was as per the guidelines of OECD No. 211 (2008).

              

A preliminary range finding study was conducted with 35 daphnids, divided into seven groups, each comprising one daphnid per replicate and five replicates per group. The concentrations selected for the preliminary screening were 0 (control), vehicle control (acetone), 1.2, 5.0, 10.0, 50.0 and 100.0 mg Ethyl 2-methylbutyrate /L.

 

In the main study, five groups of daphnids, each comprising one daphnid per replicate and ten replicates per group were exposed to concentrations of 1.3, 3.6, 9.6, 25.9 and 70.0 mg Ethyl 2-methylbutyrate/L and observed for a period of 21 days. A concurrent control (without Ethyl 2-methylbutyrate) and vehicle control (acetone) were run simultaneously and observed for the same period. A semi-static test procedure was followed with the test medium being changed at regular intervals of 48 h.

 

Water quality parameters, viz., temperature, pH, dissolved oxygen and total hardness of the test medium were measured initially and at the time of every test medium renewal. The whole experiment was conducted in a specially designed low temperature water bath, having temperature and photoperiod control facilities. All the parameters were found to be within the limits of the guideline.

 

Daphnids were fed with live algal (Pseudokirchneriella subcapitata) feed at the rate of 1.0 mL per daphnid at the time of every test medium renewal.The algal concentration were 4.02 x 10⁶ cells/mL (0.14 mg Carbon/mL) during the main study.

 

Observations were made at intervals of 24 h, from day 1 to day 21 for number of live and dead offspring produced per replicate per group and adult mortality. Live birth index and survival index of offspring for each group starting from the day of appearance of first brood to the day of termination were calculated.

 

The total number of offspring produced during the 21 days exposure period were 786, 783, 751, 433, 404, 372 and 347 at the test concentrations of 0 (control), vehicle control (acetone) , 1.3, 3.6, 9.6, 25.9 and 70.0 mg Ethyl 2-methylbutyrate/L, respectively. No adult mortality or sign of toxicity was observed at any of the groups.

 

The total numbers of offspring per group were recorded and analysed statistically. There was a significant decrease (p ≤ 0.01) in the production of offspring during exposure period at all of the test concentrations except the lowest concentration (1.3 mg Ethyl 2-methylbutyrate /L) as compared with the vehicle control group.

 

The mean numbers of offspring survivor per adult in the control and vehicle control groups were 78.60 and 78.30 at end of the experiment.

 

The No Observed Effective Concentration (NOEC) of Ethyl 2-methylbutyrate tested at which no statistically significant difference in reproductive output was observed was 1.3 mg/L, as compared with vehicle control.

The Lowest Observed Effective Concentration (LOEC) tested at which statistically significant difference in reproduction output was observed was 3.6 mg/L, as compared with vehicle control.

 

The 21 day EC₅₀ for reproductive output in Daphnia magna with Ethyl 2-methylbutyrate was 22.53 mg/L with the 95% fiducial limits between 5.48 and 92.67 mg/L. The regression equation established [Probit mortality (y) vs log concentration (mg/L) of Ethyl 2-methylbutyrate (x)] was y = 0.99 + 0.92x.

Description of key information

The NOEC for EMB was determined to be 1.3 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.3 mg/L

Additional information

- The No Observed Effective Concentration (NOEC) of Ethyl 2-methylbutyrate tested at which no statistically significant difference in reproductive output was observed was 1.3 mg/L, as compared with vehicle control.

- The Lowest Observed Effective Concentration (LOEC) tested at which statistically significant difference in reproduction output was observed was 3.6 mg/L, as compared with vehicle control. The total numbers of offspring per group were recorded and analysed statistically. There was a significant decrease (p ≤ 0.01) in the production of offspring during exposure period at all of the test concentrations except the lowest (1.3 mg Ethyl 2-methylbutyrate/L) as compared with the vehicle control group.

- The 21 day EC50 for reproductive output in Daphnia magna with Ethyl 2-methylbutyrate was 22.53 mg/L with the 95% fiducial limits between 5.48 and 92.67 mg/L. The regression equation established [Probit mortality (y) vs log concentration (mg/L) of Ethyl 2-methylbutyrate (x)] was y = 0.99 + 0.92x.