Hexyl propionate

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:

01 June 2018 to 06 July 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 Propionate
Appearance: Clear colourless liquid
Purity/Composition: 99.54% (assumed 100% for testing)
Test item storage: At room temperature protected from light

Analytical monitoring:

yes

Details on sampling:

Hexyl Propionate Solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L.
Frequency at t=0 h, t=24 h and t=72 h.
Volume 6.0 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.

Vehicle:

no

Details on test solutions:

The batch of Hexyl Propionate tested was a clear colourless liquid with a purity of 99.54% 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 54-69 minutes. 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.


Control(s): Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration,
6 replicates of the control,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.

Test organisms (species):

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

Details on test organisms:

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 20 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
\HEPES buffer 6 mmol/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22-23 °C

pH:

8.1 ± 0.2

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

1.0, 3.2, 10, 10*, 32, 100 % Saturated solution
Measured concentrations in mg/L at t=0h: 0.752, 2.33, 7.25, 7.56,19.0, 38.0
Measured concentrations in mg/L at t=24h: 0.025**, 0.025**, 0.013, 0.018, 1.38, 1.15
Measured concentrations in mg/L at t=72h: 0.025**, 0.025**,0.025**, 0.025**,0.025**, 0.025**
*Without algae.
**Estimated as half of the LOQ (LOQ = 0.05 mg/L).

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/l LR

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/l LR

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Combined Limit/Range-Finding Test:

The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively.

A dose-related inhibition of algal growth rate and yield was found at the two highest test concentrations at the end of the test, resulting in an inhibition of 28 and 75% at the highest test concentration, respectively.

Based on these results, samples taken from solutions containing 1.0 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 0.26 and 20 mg/L, respectively. During the exposure period, the concentrations decreased significantly and were below reliable detectability at the end of the test (see also Table 2 of the appended Analytical Report).

At the end of the test, algae exposed to the highest test concentration were observed to form flocs, while algal cells exposed to the 10% of SS solution were observed to stick to the walls of the test vessel at the surface of the test solution. Subsequently, these test solutions were observed under the microscope. The algal cells showed a healthy and normal appearance when compared to the control.

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

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 measured concentrations at the start of the test were 0.75, 2.3, 7.6, 19 and 38 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. During the exposure period, the concentrations decreased significantly and were below reliable detectability at the end of the test. The concentrations measured in the samples taken from solutions with algae were comparable to the concentrations measured in the corresponding samples without algae.

A limit of detection could not be determined with sufficient reliability. Instead the Limit of Quantification (LOQ) as determined during the analytical method development (Charles River Den Bosch project 20152292) was used to estimate concentrations in samples where concentrations were detected at levels similar to the blank Quality Control (QC) samples. It should be noted that small responses were recorded in the control samples. These were, however, most likely caused by the analytical system.

Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated and used to determine the effect parameters (see Table 4).

Mean Cell Densities:
Figure 1 shows growth curves at different TWA concentrations of Hexyl Propionate. 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 in Table 7 (see Appendix 1 for the individual values). Statistical analysis of the data is shown in Appendix 2 and Appendix 3.

Growth rates were in the range of the control at the three lowest test concentrations. At the two highest test concentrations, inhibition of growth rates increased with increasing concentration of Hexyl Propionate resulting in 20% inhibition the highest test concentration. Statistically significant inhibition of growth rates was found at the two highest test concentrations. However, growth rate inhibition was considered to be biologically not relevant at 1.8 mg/L, where the observed inhibition was below 10%.

Inhibition of yield increased with increasing concentration of Hexyl Propionate from 0.097 mg/L upwards resulting in 64% inhibition at the highest test concentration. Statistically significant inhibition of yield was found at the two highest test concentrations.

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

Determination of Effect Concentrations:
Table 8 shows the effect parameters based on TWA concentrations, see also Appendix 4.

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 1
Mean Cell Densities (x10⁴ Cells/mL) during the Combined Limit/Range-Finding Test

Time (h)	Hexyl Propionate;%SS prep. at 100 mg/L
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	7.7	n.a.	n.a.	5.4
48	47.6	n.a.	n.a.	27.5
72	238.5	246.0	147.1	61.1

n.a.: not applicable

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

Hexyl Propionate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.825	0.0075	6
1.0	1.835	0.0136	3	-0.56
10	1.662	0.0433	3	8.9
100	1.308	0.2346	6	28

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

Hexyl Propionate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	237.5	5.39	6
1.0	245.0	9.97	3	-3.1
10	146.1	18.55	3	38
100	60.1	37.36	6	75

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

Hexyl Propionate %SS prep. at 100 mg/L	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
1.0	0.752	0.025^2	0.025^2	0.062
3.2	2.33	0.025^2	0.025^2	0.097
10	7.56	0.013	0.025^2	0.12
10^1	7.25	0.018	0.025^2	0.13
32	19	1.38	0.025^2	1.8
100	38.0	1.15	0.025^2	2.3

¹Without algae.;²Estimated as half of the LOQ (LOQ = 0.05 mg/L).

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

Hexyl Propionate TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.763	0.0165	6
0.062	1.766	0.0189	3	-0.17
0.097	1.763	0.0156	3	0.035
0.12	1.746	0.0341	3	1.0
1.8	1.720	0.0165	3	2.5^#
2.3	1.417	0.0786	3	20*

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

Table 6
Growth Rate and Percentage Inhibition at Different Time Intervals

 

Hexyl Propionate TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.822		1.882		1.587
0.062	3	2.030	-11	1.743	7.4	1.526	3.9
0.097	3	2.043	-12	1.695	9.9	1.551	2.3
0.12	3	1.886	-3.6	1.744	7.3	1.607	-1.2
1.8	3	1.863	-2.3	1.765	6.2	1.532	3.5
2.3	3	1.164	36	1.534	18	1.553	2.2

Table 7
Yield and Percentage Inhibition for the Total Test Period

Hexyl Propionate TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	197.6	9.87	6
0.062	199.4	11.20	3	-0.89
0.097	197.2	9.27	3	0.22
0.12	187.8	18.85	3	5.0
1.8	173.3	8.56	3	12*
2.3	70.5	15.83	3	64*

* Effect was statistically significant.

Table 8
Effect Parameters

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	0.12	1.8	2.1	2.3^1	>2.3
	lower 95%-cl			2.0	2.2
	upper 95%-cl			2.2	2.4
Yield	Value	0.12	0.12	1.8	1.9	2.2
	lower 95%-cl			1.6	1.8	2.1
	upper 95%-cl			1.9	2.0	2.2

cl – confidence limit;* Based on statistical significance.;^#Based on biological relevance.;
¹Determined by extrapolation.

Table 9
pH Levels Recorded during the Final Test

Hexyl Propionate TWA conc. (mg/L)	pH
	t=0h	t=72h
Control	8.2	8.5
0.062	8.1	8.4
0.097	8.1	8.4
0.12	8.0	8.4
1.8	8.0	8.3
2.3	8.0	8.2

 

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Hexyl Propionate inhibited growth rate and yield of this fresh water algae species significantly at TWA concentrations of 1.8 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested, i.e. exceeded a TWA concentration of 2.3 mg/L being considered to represent the maximum solubility of the test item in test medium over 72 hours of exposure.

The 72h-EC50 for yield inhibition (EYC50) was 2.2 mg/L with a 95% confidence interval ranging from 2.1 to 2.2 mg/L.
The 72h-NOEC for growth rate inhibition was 0.12 mg/L based on statistical significance.
The 72h-NOEC for growth rate inhibition was 1.8 mg/L based on biological relevance.
The 72h-NOEC for yield inhibition was 0.12 mg/L based on statistical significance as well as on biological relevance.

Executive summary:

The objectiveofthe study was to evaluate Hexyl Propionate 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, EC₂₀ and 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 Propionate tested was a clear colourless liquid with a purity of 99.54% and not completely soluble in test medium at the loading rate initially prepared.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 to solutions containing 1.0, 3.2, 10, 32 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 measured concentrationsat the start of the testwere 0.75, 2.3, 7.6, 19 and 38 mg/L in solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively.During the exposure period, the concentrations decreased significantly and were below reliable detectability at the end of the test.Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated to be 0.062, 0.097, 0.12, 1.8 and 2.3 mg/L, respectively, and used to determine the effect parameters.

Growth rates and yield were in the range of the control at the three lowest test concentrations. At the two highest test concentrations, inhibition of growth rates increased with increasing concentration of Hexyl Propionate resulting in 20 and 64% inhibition the highest test concentration, respectively.

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	0.12	1.8	2.1	2.3^1	>2.3
	lower 95%-cl			2.0	2.2
	upper 95%-cl			2.2	2.4
Yield	Value	0.12	0.12	1.8	1.9	2.2
	lower 95%-cl			1.6	1.8	2.1
	upper 95%-cl			1.9	2.0	2.2

cl – confidence limit; * Based on statistical significance.;^#Based on biological relevance.;
¹Determined by extrapolation.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Hexyl Propionate inhibited growth rate and yield of this fresh water algae species significantly at TWA concentrations of 1.8 mg/L and higher.

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Additional information

72 hour EC50 >2.3 mg/l (100% saturated solution).

The substance is not classified for CLP as the effect levels are above the limit of water solubility (0.0631 mg/l), the registered substance is readily biodegradable, and the Log Pow is < 4 (3.9).