Hexyl butyrate

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:

19 May 2018 to 25 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures

Version / remarks:

OECD series on testing and assessment number 23, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Hexyl Butyrate
Appearance: Clear colourless liquid
Purity/Composition: 99.26%
Test item storage: At room temperature protected from light
Test Facility test item number: 209412/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name): Hexyl butanoate
CAS number: 2639-63-6
EC number: 220-136-6
Volatile: Not indicated
Solubility in water: 20.3 mg/L at 20°C
Stability in water: Stable

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 6).

Frequency at t=0 h, t=24 h and t=72 h.
Volume 12 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 32% of the SS 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 12 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.

Vehicle:

no

Details on test solutions:

The batch of Hexyl Butyrate tested was a clear colourless liquid with a purity of 99.26% 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 a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. The obtained mixture was allowed to settle for a period of three hours. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

Any residual volumes were discarded.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21-24°C ±1 °C

pH:

6-9, not varying by more than 1.5 units

Nominal and measured concentrations:

Nominal: Solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L.
Measured mean:TWA (time weighted average) 0.015, 0.019, 0.10, 0.41, 0.67 mg/L

Details on test conditions:

Test System:
Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.


Fresh Water Algae 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.

Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 2 0 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

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 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

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 saturated 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 solutions containing 1.0 and 10% of the SS prepared at a loading rate of 100 mg/L in the combined range-finding test.
• 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.
• pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.


Test Procedure and Conditions:
Test duration: 72 hours
Test type: Static
Test vessels: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
Test Medium: M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity of 99 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.



Measurements and Recordings:
pH: At the beginning and at the end of the test, for all test concentrations and the control
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells : At the end of the final test, microscopic observations were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

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 immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Statistical significance

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Biological relevance

Details on results:

See below ("Any other information on results incl. tables").

Reported statistics and error estimates:

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Results

Combined Limit/Range-Finding Test

The mean cell densities measured during the combined limit/range-finding test are presented inTable 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively. No inhibition of algal growth rate or yield was found at the two lowest test concentrations. An inhibition of 17 and 57% was observed for growth rate and yield, respectively, at the highest test concentration.

Based on these results, samples taken from solutions containing 10 and 100% of the SS prepared at a loading rate of 100 mg/L were analysed. The measured concentrations at the start of the test were 2.7 and 27 mg/L, respectively. During the exposure period, the concentrations decreased significantly and were at the level of the carry-over effect of the analytical method at the end of the test (see also Table 1 and Table 2 of the appended Analytical Report).

All test conditions were maintained within the limits prescribed by the study plan.

Table 1
Mean Cell Densities (x10⁴Cells/mL) during the Combined Limit/Range-Finding Test

Time (h)	Hexyl Butyrate;%SS prep. at 100 mg/L
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	5.3	n.a.	n.a.	2.5
48	32.6	n.a.	n.a.	14.2
72	173.2	158.8	173.7	74.8

n.a.: not applicable

Table 2
Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test

Hexyl Butyrate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.718	0.0167	6
1.0	1.689	0.0173	3	1.7
10	1.718	0.0299	3	0.0
100	1.427	0.0987	6	17

Table 3
Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test

Hexyl Butyrate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	172.2	8.59	6
1.0	157.8	8.18	3	8.4
10	172.7	15.48	3	-0.3
100	73.8	21.01	6	57

Final Test

Measured Test Item Concentrations       

The results of analysis of the samples taken during the final test are described in Table 3 of the appended Analytical Report.

Samples taken from all test concentrations and the control were analysed. The concentrations measuredat the start of the test were 1.0, 1.8, 2.7, 5.7 and 9.5 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations decreased below reliable quantifiability, i.e. the detected concentrations were probably caused by carry-over during the chemical analysis of the samples. The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae.

Based on these results, the Time Weighted Average (TWA) concentrations were calculated and used to determine the effect parameters (seeTable 4). To account for concentrations falling into the range of carry-over levels, the respective concentrations were estimated as half of the detection limit as a worst-case assumption.

It should be noted that the concentration measured for the undiluted SS at the start of the final test was lower than the concentration measured at the start of the combined limit range-finding test. The water solubility of the test item was determined to be 20 mg/L. The concentration at the start of the combined limit range-finding test was above this value and it is likely that the solution was oversaturated.

Table 4
Time Weighted Average vs. Percentage of the Saturated Solution

Hexyl Butyrate %SS prep. at 100 mg/L	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
10	1.01	0.0017^2	0.0017^2	0.015
18	1.76	0.0017^2	0.0017^2	0.019
32	2.73	0.028	0.0017^2	0.10
32^1	3.03	0.209	0.0017^2	0.28
56	5.65	0.25	0.0017^2	0.41
100	9.52	0.40	0.0017^2	0.67

¹Without algae.;²Estimated as half of the LOD (LOD = 0.0033 mg/L).

Mean Cell Densities       

Figure 1 shows growth curves at different concentrations of Hexyl Butyrate. The individual and group mean cell densities measured at 24h intervals are given in Table 10.

Inhibition of Growth Rate and Inhibition of Yield  

Table 5 shows group mean growth rates and the percentages of growth rate inhibition (total test period), whereas Table 6 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized inTable 7(see Appendix 1 for the individual values). Statistical analysis of the data is shown inAppendix 2 and Appendix 3.

Growth rates were in the range of the controls at the three lowest test concentrations. Inhibition of growth rates increased with increasing concentration at the two highest test concentrations, resulting in 4.9 and 8.8% inhibition, respectively, at the end of the test. Statistically significant inhibition of growth rates was found at average concentrations of 0.41 mg/L and higher. However, growth rate inhibition was considered to be biologically not relevant at 0.41 and 0.67 mg/L, where the observed inhibition was below 10%.

Yield was in the range of the controls at the two lowest test concentrations. Inhibition of yield increased with increasing concentration at the three highest test concentrations, resulting in 37% inhibition at the highest test concentration at the end of the test. Statistically significant inhibition of growth rates was found at average concentrations of 0.10 mg/L and higher. However, yield inhibition was considered to be biologically not relevant at 0.10 mg/L, where the observed inhibition was below 10%.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to highest test concentration when compared to the control.

Table 5
Growth Rate and Percentage Inhibition for the Total Test Period

Hexyl Butyrate TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.753	0.0186	6
0.015	1.736	0.0190	3	0.99
0.019	1.738	0.0048	3	0.86
0.10	1.724	0.0241	3	1.7
0.41	1.668	0.0220	3	4.9^#
0.67	1.600	0.0446	3	8.8^#

^#Effect was statistically significant, but biologically not relevant (<10%).

Table 6
Growth Rate and Percentage Inhibition at Different Time Intervals

Hexyl Butyrate TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.903		1.778		1.579
0.015	3	1.888	0.78	1.638	7.9	1.683	-6.5
0.019	3	1.849	2.8	1.988	-12	1.378	13
0.10	3	1.874	1.5	1.729	2.8	1.570	0.60
0.41	3	1.677	12	1.648	7.3	1.679	-6.3
0.67	3	1.488	22	1.798	-1.1	1.514	4.2

 

Table 7
Yield and Percentage Inhibition for the Total Test Period

Hexyl Butyrate TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	191.8	10.72	6
0.015	182.0	10.49	3	5.1
0.019	183.1	2.67	3	4.5
0.10	175.7	12.80	3	8.4^#
0.41	148.3	9.66	3	23*
0.67	121.2	16.43	3	37*

* Effect was statistically significant;^#Effect was statistically significant, but biologically not relevant (<10%).

Determination of Effect Concentrations 

Table 8 shows the effect parameters based on TWA concentrations, see also Appendix 4.

Table 8
Effect Parameters

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	0.10	0.67	>0.67	>0.67	>0.67
	lower 95%-cl
	upper 95%-cl
Yield	Value	0.019	0.10	0.10	0.27	>0.67
	lower 95%-cl			0.039	0.17
	upper 95%-cl			0.16	0.35

cl – confidence limit; * based on statistical significance;^#based on biological relevance.

Experimental Conditions    

Table 9 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 units).

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21‑24°C, constant within ±1°C).

Table 9
pH Levels Recorded during the Final Test

Hexyl Butyrate TWA conc. (mg/L)	pH
	t=0h	t=72h
Control	8.2	8.4
0.015	8.2	8.4
0.019	8.2	8.4
0.10	8.2	8.3
0.41	8.2	8.2
0.67	8.1	8.1

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the concentrations of Hexyl Butyrate tested.

The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested, i.e. exceeded a TWA concentration of 0.67 mg/L being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L during the test period of 72 hours.

The 72h-EC50 for yield inhibition (EYC50) was beyond the range tested, i.e. exceeded a TWA concentration of 0.67 mg/L being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L during the test period of 72 hours.

The 72h-NOEC for growth rate inhibition was 0.10 mg/L based on statistical significance.

The 72h-NOEC for growth rate inhibition was 0.67 mg/L based on biological relevance.

The 72h-NOEC for yield inhibition was 0.019 mg/L based on statistical significance.

The 72h-NOEC for yield inhibition was 0.10 mg/L based on biological relevance.

Executive summary:

The objectiveofthe study was to evaluate Hexyl Butyrate for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀and EC₅₀ for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Hexyl Butyrate tested was a clear colourless liquid with a purity of 99.26% 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.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

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

Samples taken from all test concentrations and the control were analysed. The concentrations measuredat the start of the testwere 1.0, 1.8, 2.7, 5.7 and 9.5 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations decreased below reliable quantifiability, i.e. the detected concentrations were probably caused by carry-over during the chemical analysis of the samples. Based on these results, the Time Weighted Average (TWA) concentrations were calculated to be 0.015, 0.019, 0.10, 0.41 and 0.67 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS. These were used to determine the effect parameters.

Growth rates were in the range of the controls at the three lowest test concentrations. Inhibition of growth rates increased with increasing concentration at the two highest test concentrations, resulting in 4.9 and 8.8% inhibition, respectively, at the end of the test.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	0.10	0.67	>0.67	>0.67	>0.67
	lower 95%-cl
	upper 95%-cl
Yield	Value	0.019	0.10	0.10	0.27	>0.67
	lower 95%-cl			0.039	0.17
	upper 95%-cl			0.16	0.35

cl – confidence limit; * based on statistical significance;^#based on biological relevance.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata(formerly known as Pseudokirchneriella subcapitata), no biologically relevant inhibition of growth rate was recorded at any of the concentrations of Hexyl Butyrate tested.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Additional information

In this guideline (OECD 201) study, the test material (EC220-136-6) was determined to not be classified for environmental toxicity. The LD50 for growth rate was determined to be >0.67 mg/l.