Ethyl 2-ethylhexanoate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 June 2016 to 26 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 2-ethylhexanoate

EC Number:

221-043-3

EC Name:

Ethyl 2-ethylhexanoate

Cas Number:

2983-37-1

Molecular formula:

C10H20O2

IUPAC Name:

ethyl 2-ethylhexanoate

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h, t=24 h and t=72 h
Volume 3.0 mL
Storage Samples were stored in a freezer (≤ -15°C) until analysis.

At the end of the exposure period, the replicates with algae were not pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤ -15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of IROTYL tested was a colourless liquid with a purity of 100.0% (by GLC) and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with a loading rate of 100 mg/L applying 3 days of slow magnetic stirring followed by a settlement period of approximately 2 hours. Settlement resulted in a clear and colourless solution with a floating layer of undissolved test item. The fraction containing the Saturated Solution (SS) was collected and used as highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

After preparation, volumes of 120 mL were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms

Test organisms (species):

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

Details on test organisms:

Strain: NIVA CHL 1
Source: In-house laboratory culture.
Stock culture : Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of
1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21°C-23°C

pH:

0h: 7.4-7.7
72h: 7.6-8.0

Nominal and measured concentrations:

Nominal loading rate: 10, 18, 32, 56 and 100% SS prepared at a loading rate of 100 mg/L.
Measured concentration (0h): 1.75, 3.20, 5.86, 11.1, 21.1 mg/L
Measured concentration (24h): 0.632, 1.06, 1.54, 2.05, 2.59 mg/L
Measured concentration (72h): 0.035, 0.067, 0.231, 0.270, 0.404 mg/L
Time Weight Average: 0.45, 0.79, 1.4, 2.1, 3.1 mg/L

Details on test conditions:

-Combined limit/range-finding test
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a SS solution prepared at a loading rate of
100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates per concentration were exposed to 0.10, 1.0 and 10% SS in the combined range-finding test.
• The pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed to verify a normal and healthy appearance.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

-FInal test
Controls: Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration, 6 replicates of the control,1 or 2 extra replicates of each test group for sampling after 24 hours, 1 or 2 replicates of each test concentration without algae.
Test vessels: 120 mL, all-glass, airtight closed containing 120 mL of test solution
Medium: Adjusted M2 (for volatile test items)
Cell density: An initial cell density of 1 x 104 cells/mL
Illumination: Continuously using TLD-lamps with a light intensity within the range of 85 to 91 µE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
Recording of cell densities:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a
spectrophotometer with a cuvette (pathlength =10 mm). Algal medium was used as blank and the extra replicates incubated without algae as background for the treated solutions.

-Acceptability of the test
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 153).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 9.3%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 2.4%).

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 3.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Combined limit/range-finding test
-Mean cell densities, inhibition of growth rate and inhibition of yield
A slight effect on algal growth was observed in the undiluted SS. Unexpectedly, the effects in the dilutions were higher. Based on these results samples taken from 10 and 100% SS were analysed, which showed measured concentrations of 2.0 and 20.5 mg/L at the study start, respectively. This confirmed that dilutions were prepared correctly. These concentrations decreased by approximately 80% during the first 24-hour test period and by more than 95% at the study end. As the observed effects showed no relation to the concentrations that were measured it was decided to continue with a final test testing a range of SS dilutions. All test conditions were maintained within the limits prescribed by the study plan.

Final test
-Measured test item concentrations
The actual test concentrations at the start of the test were 1.8, 3.2, 5.9, 11 and 21 mg/L in samples taken from 10, 18, 32, 56 and 100% SS, respectively. After 24 hours of exposure concentrations had decreased by 60-90% and after 72 hours measured concentrations had decreased by more than 95% below the start concentrations. The measured concentrations in the samples taken from the solution without algae showed a similar decrease. Based on these results, the Time Weight Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations was 0.45, 0.79, 1.4, 2.1 and 3.1 mg/L

-Inhibition of growth rate and inhibition of yield
Growth rates were in the range of the controls at the concentrations from 0.45 to 2.1 mg/L during the 72-hour test period (<4% inhibition), whereas the growth rate of algae exposed to 3.1 mg/L showed a slightly higher inhibition of 9.7%. The inhibition observed at the TWA concentration of 3.1 mg/L was statistically significant.Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the TWA concentration of 3.1 mg/L when compared to the control.

Results with reference substance (positive control):

Pseudokirchneriella subcapitata, strain: NIVA CHL-1. Fresh water algal growth inhibition test with Potassium Dichromate (Project 514940).
Start: 18 July 2016
End : 21 July 2016

Algae were exposed for a period of 72 hours to Potassium Dichromate concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4 cells/mL.

Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.56 mg/L and higher.The EC50 for growth rate inhibition (72h-ERC50) was 1.0 mg/L with a 95% confidence interval ranging from 0.97 to 1.0 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

The study plan, raw data and report of this study are kept in the Charles River Den Bosch archives. The test described above was performed under GLP conditions with a QA-check.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, IROTYL reduced growth rate of this fresh water algae species significantly at a TWA concentration of 3.1 mg/L.
The EC50 for growth rate inhibition (72h-ErC50) were beyond the range tested, i.e. exceeded 3.1 mg/L being the TWA concentration measured in a saturated solution prepared at a loading rate of 100 mg/L.
The 72h-NOEC for growth rate inhibition was 2.1 mg/L.
When based on loading rates, the effect parameters for algal growth rate inhibition all exceeded the highest loading rate tested, being 100 mg/L. Hence, the 72h-ErL10,20,50 based on loading rates exceeded 100 mg/L.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with IROTYL.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of theCommissionRegulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 2016/266 and the OECD series on testing and assessment number 23, 2000.

The batch of IROTYL tested was a colourless liquid with a purity of 100.0% (by GLC) and not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying 3 days of slow magnetic stirring followed by a settlement period of approximately 2 hours. Settlement resulted in a clear and colourless solution with a floating layer of undissolved test item. The fraction containing the Saturated Solution (SS) was collected and used as highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to test groups representing 10, 18, 32, 56 and 100% of a SS prepared at a loading rate of 100 mg IROTYL per litre. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

The actual test concentrations at the start of the test were 1.8, 3.2, 5.9, 11 and 21 mg/L in samples taken from 10, 18, 32, 56 and 100% SS, respectively. After 24 hours of exposure concentrations had decreased by 60-90% and after 72 hours measured concentrations had decreased by more than 95% below the start concentrations. The measured concentration in the samples taken from the solution without algae showed a similar decline. Based on these results, the Time Weight Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations was 0.45, 0.79, 1.4, 2.1 and 3.1 mg/L.The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study, based on TWA concentrations, are summarized in the table below.

	Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	Value	2.1	>3.1	>3.1	>3.1
	lower 95%-cl
	upper 95%-cl

When based on loading rates, the effect parameters for algal growth rate inhibition all exceeded the highest loading rate tested, being 100 mg/L. Hence, the 72h-E_RL_10,20,50based on loading rates exceeded 100 mg/L.