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

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:

14 June 21019 - 5 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted 28th July, 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

14 February 2017

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
Purity: 99.8%

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

yes

Details on test solutions:

FORMULATION

A stock solution with a nominal concentration of 100.0 mg/L was prepared with direct addition of the test item, mixed into the algal growth medium (OECD Medium) using magnetic stirrer for an hour in a closed system with reduced headspace in order to minimize potential losses due to volatilization. The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test flasks prior to introduction of algae.


Nominal concentration [mg/L] Amount of stock solution (mL) Amount of OECD medium (mL)
100.00 1000.0 -
40.00 240.0 q.s ad 600
16.00 96.0 q.s ad 600
6.40 38.40 q.s ad 600
2.56 15.36 q.s ad 600
q.s ad= quantum sufficient ad (a sufficient quantity to make)


UNTREATED CONTROL

Algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.


REFERENCE CONTROL

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate
satisfactory test conditions. The date of the last study (Study Code: 19/238-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 10 – 13 September 2019.
The 72h ErC 50: 0.82 mg/L, (95 % confidence limits: 0.76 – 0.90 mg/L)
The 72h EbC 50: 0.58 mg/L, (95 % confidence limits: 0.53 – 0.62 mg/L)
The 72h EyC 50: 0.49 mg/L, (95 % confidence limits: 0.46 – 0.54 mg/L)

These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

DILUTION WATER

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionisedwater in order to achieve the final concentrations

Stock soluton 1: Macro nutrients
Substance Final concentration in the prepared growth medium
NH4CL 15.0 mg/L
MgCl2 x 6 H2O 12.0 mg/L
CaCl2 x 2 H2O 18.0 mg/L
MgSO4 x 7 H2O 15.0 mg/L
KH2PO4 1.6 mg/L

Stock solution 2: Iron
Substance Final concentration in the prepared growth medium
FeCL3 x 6 H2O 64.0 µg/L
Na2EDTA x 2H2O 100.0 µg/L

Stock solution 3: Trace elements
Substance Final concentration in the prepared growth medium
H3BO3 185.0 µg/L
MnCl2 x 4 H2O 415.0 µg/L
ZnCl2 3.0 µg/L
CoCl2 x 6 H2O 1.5 µg/L
CuCl2 x 2 H2O 0.01 µg/L
NaMoO4 x 2 H2O 7.0 µg/L

Stock solution 4: Bircarbonate
Substance Final concentration in the prepared growth medium
NaHCO3 50.0 mg/L

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)

Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)

Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Charles River Laboratories Hungary Kft.

Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.

Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae
suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Study design

Water media type:

freshwater

Total exposure duration:

72 h

Remarks on exposure duration:

According to guideline

Post exposure observation period:

No post exposure observation period

Test conditions

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.4 and 22.8 °C measured in the flask and between 22.0 and 23.2 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.10 –8.56 during the experiment.

Nominal and measured concentrations:

Nominal concentrations were tested: 2.56, 6.4, 16.0, 40.0 and 100.0 mg/L
The corresponding initial measured test item concentrations were: 2.66, 6.34, 16.0, 30.9 and 80.3 mg/L.

Details on test conditions:

DESCRIPTION OF THE TEST PROCEDURE

Taking into account that the test item is a highly volatile substance the study was performed in a closed system followed the OECD recommendations. A simple filled closed bottle test with low algal densities and bicarbonate enrichment was performed to minimize the loss of test substance from solution. The link between algal growth, the dissolved CO2 concentration and the pH of the algal medium is crucial for reproducible algal growth inhibition tests. To prevent such limitation of algal growth in a sealed test system the study was performed by the following way (based on Mayer et al., 2000): The algae medium was enriched with 300 mg/L NaHCO3 (to replace the gaseous CO2), the pH was adjusted to 7.0 by addition of HCl, and the resulting CO2 concentration was sufficient to support algal growth in the absence of headspace.

The test was started (0 hours) by inoculation of a biomass of 3 x 103 algal cells per mL test medium. (This slightly lower level did not affect the performance of the test.)
Volumes of 60 mL test solution (already including the appropriate amount of algal suspension) per replicate, in sterile plastic flasks, were continuously shaken by a laboratory orbital shaker. Each test vessel was uniquely identified with at least study code, test group and replicate.

Additional samplings for analysis at 24 hour intervals were used in order to better define loss of the test substance during the exposure period. The test was performed with six replicates per test concentrations and six replicates in the control group. In the test concentrations three replicates were used for observations and three replicates for the 24 and 48-hours analytical measurements.

PRELIMINARY RANGE FINDING TEST

A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group. During the formulation procedure was prepared by the similar method described above.The concentration levels used and results (72 h) of the preliminary range-finding test are summarised in the following table.

Results of the Preliminary Range-Finding Test

Nominal concentrations [mg/L] Untreated control 0.1 1 10 100
Average of cell number at 72 hours (x 10^4cell/mL) 52 50.5 47.5 31.5 1
Inhibition of growth rate (Iµ) at 72 hours (%) - 0.6 1.8 9.9 78.1


TEST ITEM CONCENTRATIONS IN THE DEFINITIVE TEST

Because significant inhibition was observed at the highest concentration level (100.0 mg/L) during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 2.5 and one control were tested in the main experiment. The following nominal concentrations were tested: 2.56, 6.4, 16.0, 40.0 and 100.0 mg/L. Due to rapid degradation of the test item in the test media the analytically measured concentrations could not be measured 48 hours start of the test, the biological results are based on the initial measured test item concentrations. The corresponding initial measured test item concentrations were: 2.66, 6.34, 16.0, 30.9 and 80.3 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

42.99 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.34 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

cell number

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.79 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

biomass

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.66 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.66 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.66 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.34 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.34 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

cell number

Effect concentrations 9

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.34 mg/L

Nominal / measured:

meas. (initial)

Basis for effect:

biomass

Details on results:

VALIDITY

The cell density in the control cultures increased by the factor of 51.67 within three days. The mean coefficient of variation for section-by-section specific growth rates (days 0-1;1-2; 2-3) in the control cultures was 5.01 %. The coefficient of variation of average specific growth rates during the whole test period
(day 0-3) in the control cultures was 1.01 %. All validity criteria were met; therefore, the study can be 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 of the test and 24-hour intervals thereafter during the experiment. Due to rapid degradation of the test item in the test media the analytically measured concentrations decreased significantly within 24 hours during the experiment; then, they could not be measured and were below of the Limit of Quantification 48 hours after the start of test.The corresponding initial measured geometric mean test item concentrations were: 2.66, 6.34, 16.0, 30.9 and 80.3 mg/L. The biological results are related to the initial measured test item concentrations

Any other information on results incl. tables

Calculation of exposure concentrations

Nominal Concentration (mg/L)	Measured Concentrations (mg/L)
	0h/start	24h	48h	72h/end
Control	n.d	*	*	n.d
2.56	2.66	bql	bql	bql
6.4	6.34	3.37	bql	bql
16	16	7.3	bql	bql
40	30.9	bqlx	bqlx	bql
100	80.3	41.3	bqlx	bql

n.d. – not detected *No samples were taken.

bql= below of the quantification limit - (LOQ)/10 (LOQ: Limit of Quantification) = bqlx

Growth Rates (µ) and Percentage Inhibition of µ during the Test Period

Concentration		Growth rate (µ) and % inhibition of µ
Nomial [mg/L]	Initial measured [mg/L]	0-24 h		0-48 h		0-72h
		µ	%	µ	%	µ	%
Control	-	0.0959	0.0	0.0752	0.0	0.0715	0.0
2.56	2.66	0.0903	5.9	0.0749	0.4	0.0712	0.4
6.4	6.34	0.079	17.6	0.0690	8.2	0.0672*	6.0
16	16	0.0694*	27.6	0.0635*	15.6	0.0644*	9.9
40	30.9	0.0598*	37.7	0.0452*	40.0	0.0466*+	34.9
100	80.3	0.0502*	47.7	0.0251*	66.6	0.0167*	76.6

* : statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

+ : at this value the rounding of the EXCEL and TOXSTAT software was different. The table contains

the values calculated with EXCEL.

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration		Area under the Growth Curves (a) and Percentage Inhibition of A
Nominal [mg/L]	Initial measured [mg/L]	0-24 h		0-48 h		0-72 h
		A	%	A	%	A	%
Control	-	32.4	0.0	195.2	0.0	942.0	0.0
2.56	2.66	28.4	12.3	185.2	5.1	918.0	2.5
6.4	6.34	20.4*	37.0	137.2*	29.7	686*	27.2
16	16	16.4*	49.4	105.2*	46.1	546*	42.0
40	30.9	12.4*	61.7	53.2*	72.7	182*	80.7
100	80.3	8.4*	74.1	25.2*	87.1	42*	95.5

* : statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration		Yeild (Y) and % inhibition of Y
Nominal [mg/L]	Initial Measured [mg/L]	0-72 h
		Y	%
Control	-	51.4	0
2.56	2.66	50.4	1.9
6.4	6.34	37.7*	26.6
16	16	30.7*	40.2
40	30.9	8.4*	83.7
100	80.3	0.7*	98.6

* : statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of Reaction mass of 2-methylbutyl butyrate and pentyl butyrate test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control
group at the initial test concentration range of 6.34 – 80.3 mg/L (measured), therefore the NOEC was determined as 2.66 mg/L (measured) and LOEC was
determined as 6.34 mg/L (measured). The biological results are summarised in the following table:

Influence of Reaction mass of 2-methylbutyl butyrate and pentyl butyrate on the Growth of Pseudokirchneriella subcapitata

Parameter(0-72h) Growth rate (r) [mg/L] Yield (y) [mg/L] Biomass (b) [mg/L]
Calculation based on initial measured test item concentrations
EC50 42.99 14.34 14.79
95% conf. limits 36.69-50.37 12.63-16.28 12.91-16.95
NOEC 2.66 2.66 2.66
LOEC 6.34 6.34 6.34

Executive summary:

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period

of 72 hours. As significant toxic response was observed at the examined concentration levels during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.5) and one control were tested in the main experiment. 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.Test concentrations were analytically determined at the start of the test and 24-hour intervals thereafter during the experiment. Due to rapid degradation of the test item in the test media the analytically measured concentrations could not be measured 48 hours after the start of the test, the biological results are based on the initial measured test item concentrations (more details are given in 4.2.). The initial measured test item concentrations were: 2.66, 6.34, 16.0, 30.9 and 80.3 mg/L.

The test design included three replicates at each test concentration and six replicates for the untreated controls.Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group at the initial test concentration range of 6.34 – 80.3 mg/L (measured), therefore the NOEC was determined as 2.66 mg/L (measured) and LOEC was determined as 6.34 mg/L (measured).

The results indicate that the registered substance is classified as aquatic chronic category 3 in accordance with the Classification, Labelling and Packaging (CLP) regulation (1272/2008)