Reaction mass of pentyl ester butanoic acid and 2-methylbutyl ester butanoic acid

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 June 2019 - 05 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

(Adopted: 13 April 2004) OECD: Paris.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pentyl butyrate

EC Number:

208-739-2

EC Name:

Pentyl butyrate

Cas Number:

540-18-1

Molecular formula:

C9H18O2

IUPAC Name:

pentyl butyrate

Constituent 2

Reference substance name:

2-methylbutyl butyrate

EC Number:

256-973-9

EC Name:

2-methylbutyl butyrate

Cas Number:

51115-64-1

Molecular formula:

C9H18O2

IUPAC Name:

2-methylbutyl butyrate

Test material form:

liquid

Specific details on test material used for the study:

Name: Reaction mass of 2-methylbutyl butyrate and pentyl butyrate
EC Number: 908-712-1
Description: Clear colorless to pale yellow liquid
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.
Purity: 99.8%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test concentrations were analytically determined at the start and at the end of the renewal periods.

Test solutions

Vehicle:

yes

Remarks:

Reconstituted water (ISO medium, according to OECD 202)

Details on test solutions:

FORMULATION
A stock solution with a concentration of 100.0 mg/L (nominal) was prepared with direct addition of the test item, mixed into the test medium (ISO Medium) and stirred for approximately an hour using magnetic stirrer in a closed system with reduced headspace in order to minimize potential losses due to volatilization. The test solutions were prepared by appropriate diluting of this stock solution just before introduction of the Daphnia (start of the treatments). Control containers were treated exactly the same as test item containers.

Table 1: Preparation of test solutions from stock solution
Nominal concentration Amount of stock solution Amount of ISO Medium
[mg/L] (mL) (mL)

100.0 1000.0 ---
40.0 160.0 q.s. ad 400
16.0 64.0 q.s. ad 400
6.4 25.6 q.s. ad 400
2.56 10.24 q.s. ad 400
q.s. ad = quantum sufficient ad (a sufficient quantity to make)


UNTREATED CONTROL
The dilution water (ISO-medium) was used without addition of the test item.

REFERENCE CONTROL
For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test
conditions.

DILUTION WATER
Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.
The composition of the reconstituted water and the chemicals used are given in Appendix 5.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species and strain: Daphnia magna
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Breeding: The Daphnia are bred in Ecotoxicological Laboratory of Charles River Laboratories Hungary Kft. The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 20 animals in test group and control group respectively, divided into 4 replicates (5 animals / replicate)
Age of the animals: They were less than 24 h old at the beginning of the test
Acclimatization: There was no acclimatization because the water used was similar to the culture water.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

According to guideline

Post exposure observation period:

No post-exposure observation period

Test conditions

Hardness:

The reconstituted water (ISO medium) had a total hardness of 247 mg/L (as CaCO3)

Test temperature:

The water temperature was measured at the start and at the end of the renewal periods in one test vessel at the control. The test temperature was in the range of 20.6– 20.9 °C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 20.5 and 21.1 °C.

pH:

The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the renewal periods in one test vessel at all concentration levels and was in the range of 7.32 – 7.57.

Dissolved oxygen:

The dissolved oxygen concentration was measured in one test vessel at all concentration levels at the start and at the end of the renewal periods and was in the range of 6.7 – 8.6 mg/L.

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

The nominal concentrations of test item used in the main experiment were: 2.56, 6.4, 16.0, 40.0 and 100.0 mg/L.
The corresponding measured geometric mean test item concentrations were: 1.91, 5.61, 14.34, 32.95 and 90.66 mg/L.

Details on test conditions:

Light: The test was carried out in 16-hour light and 8-hour dark cycle.
Twenty animals, divided into four groups (in Erlenmeyer flasks with stopper) of five animals each (at least 5 mL test solution/animal) were used at the test concentrations and for the control group under semi-static conditions.
The test was conducted in a closed system with minimized headspace in the test vessels under semi-static conditions. The frequency of the water renewal periods was 24 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

20.85 mg/L

Nominal / measured:

meas. (initial)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

13.85 mg/L

Nominal / measured:

meas. (initial)

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

32.95 mg/L

Nominal / measured:

meas. (initial)

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

meas. (initial)

Effect concentrations 5

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

14.34 mg/L

Nominal / measured:

meas. (initial)

Details on results:

VALIDITY

There was no immobilization in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L.
All validity criteria were within acceptable limits and therefore the study is considered as valid.

CONCENTRATIONS OF THE TEST ITEM

The following nominal concentrations were tested: 2.56, 6.4, 16.0, 40.0 and 100.0 mg/L.

Test concentrations were analytically determined at the start and at the end of the renewal periods. The nominal concentration of 2.56 mg/L could not be analytically determined at the end of the second renewal period, so the measured concentration was below the Limit of Quantification (LOQ). In order to calculate mean value, where measured concentration was detected, but not quantified, concentration should be taken as the half of the Limit of
Quantification (LOQ = 2.0 mg/L) according to OECD 23; paragraph 3.3.

The corresponding measured geometric mean test item concentrations were: 1.91, 5.61,14.34, 32.95 and 90.66 mg/L.
As the measured concentrations deviated more than 20 per cent from the nominal in more cases, biological results are related to the measured geometric mean test item concentrations.

Any other information on results incl. tables

Calculation of exposure concentrations

Nominal concentration(mg/l)	Measured concentrations(mg/l)1st renewal period		Measured concentrations(mg/l)2nd renewal period		Geometric mean
	start	end	start	end	mg/l
Control	n.d	n.d	n.d	n.d	-
2.56	2.08	1.0*	3.10	2.06	1.91
6.4	5.32	5.98	7.01	4.52	5.61
16	13.9	16.9	15.8	11.4	14.34
40	29.9	40.6	34.2	28.4	32.95
100	78.2	105.1	-	-	90.66

Number and percentage of immobilised animals

			Immobillised animals
Concentration		Number of treated animals	24 hours		48 hours
Nominal[mg/L]	Measured[mg/L]		number	percent	number	percent
Control	-	20	0	0	0	0
2.56	1.91	20	0	0	1	5
6.4	5.61	20	0	0	1	5
16	14.34	20	4	20	7*	35
40	32.95	20	17*	85	20*	100
100	90.66	20	20*	100	20*	100
*: Statistically significantly different compared to the control values (Dunnett's Test;α=0.05)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Acute toxicity of Reaction mass of 2-methylbutyl butyrate and pentyl butyrate was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Reaction mass of 2-methylbutyl butyrate and pentyl butyrate were the following:

The 24h EC50 value: 20.85 mg/L (measured) (95 % conf. limits: 19.09 – 22.78 mg/L)
The 48h EC50 value: 13.84 mg/L (measured) (95 % conf. limits: 12.28 – 15.60 mg/L)
The 48h EC100 value: 32.95 mg/L (measured)
The 48h No-Observed Effect Concentration (NOEC): 6.4 mg/L (measured)
The 48h Lowest Observed Effect Concentration (LOEC): 14.34 mg/L (measured)

Executive summary:

Acute toxicity of Reaction mass of 2-methylbutyl butyrate and pentyl butyrate on Daphnia magna was assessed in an acute immobilisation test, over an exposure period of 48 hours in a semi-static test system in a closed system.Because significant immobility was observed at the higher examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.5) and one control group were tested in the definitive test under semi-static conditions in a closed system.

The nominal concentrations of test item used in the main experiment were: 2.56, 6.4,16.0, 40.0 and 100.0 mg/L. The test concentrations were analytically determined at the start and at the end of the renewal periods. The corresponding measured geometric mean test item concentrations were: 1.91, 5.61, 14.34, 32.95 and 90.66 mg/L. As the measured concentrations deviated more than 20 per cent from the nominal in more cases, biological results are based on the measured geometric mean test item concentrations.

Twenty animals, divided into four groups (in Erlenmeyer flasks with stopper) of five animals each were used at the test concentrations and for the control as well.The 24 and 48 hours EC50 values of the test item were calculated using Probit analysis by TOXSTAT software. The 48h EC100 values of the test item were determined directly from the raw data.

For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software. All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Reaction mass of 2-methylbutyl butyrate and pentyl butyrate were the following:

The 24h EC50 value: 20.85 mg/L (measured) (95 % conf. limits: 19.09 – 22.78 mg/L)

The 48h EC50 value: 13.84 mg/L (measured) (95 % conf. limits: 12.28 – 15.60 mg/L)

The 48h EC100 value: 32.95 mg/L (measured)

The 48h No-Observed Effect Concentration (NOEC): 6.4 mg/L (measured)

The 48h Lowest Observed Effect Concentration (LOEC): 14.34 mg/L (measured)

The results indicate that the registered substance do not meet the criteria for acute aquatic toxicity in accordance with the Classification, Labelling and Packaging (CLP) regulation (1272/2008)