Hexyl hexanoate

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:

15 Jun 2018 - 8 Oct 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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item information
Identification: Hexyl Caproate
Appearance: Clear colourless liquid
Purity/Composition: 99.29%
Test item storage: At room temperature

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 10).
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 First and Second Full Test:
Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Third Full Test:
At t=0 h, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling. At t=24 h and 72 h, samples were stored in a freezer (≤-15°C) for possible analysis.
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 6.0 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.

Vehicle:

no

Details on test solutions:

Preliminary Data
The water solubility at 20°C was determined to be 0.951 mg/L, using the slow stirring flask method (Test Facility Study No. 20152316).
After the combined limit/range-finding test, the hydrolytic stability of the test item in test medium was investigated in Test Facility Study No. 20152328. To this end, five saturated solutions were prepared in test medium at a loading rate of 100 mg/L and stirred for 1, 3, 6, 24 and 48 hours, respectively, before analysis. The concentrations measured in M2 medium ranged between 1.0 and 2.8 mg/L after one to 48 hours of stirring (detailed results are archived in the raw data of Test Facility Study No. 20152328). It was thus concluded that the test item was hydrolytically stable enough to perform a full test with an unchanged protocol for preparing the test solutions.

Preparation of Test Solutions
The batch of Hexyl Caproate tested was a clear colourless liquid with a purity of 99.29% 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.

Combined Limit/Range-Finding Test, First and Second Full Test
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 2.5 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. In the combined limit/range-finding test, all test solutions were observed to be clear and colorless at the end of the preparation procedure. In the first and second full test, the highest three test concentrations were increasingly hazy, while the lower test concentrations were clear and colourless.

Third Full Test
Preparation of test solutions started with a loading rate of 100 mg/L applying a one-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 2.5 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. The two highest test concentrations were slightly hazy, while the lower three 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):

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

Details on test organisms:

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 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
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

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:

21-24 °C

pH:

8.0-8.4

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56, and 100% SS

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 Saturated Solution (SS) 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.

Full Tests
Test Concentrations
Hexyl Caproate: Solutions containing 10, 18, 32, 56 and 100% of the SS, prepared at a loading rate of 100 mg/L.
Control: Test medium without test item or other additives.
Replicates: 6 replicates of the control, 3 replicates of each test concentration, 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 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 (see paragraph 4.7.)
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 98 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 each full test, microscopic observations were performed on an intermediate test concentration (first full test: 32% SS, second full test: 56% SS) and the corresponding 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):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

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:

NOEC

Effect conc.:

10 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 100 mg/l LR

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. No inhibition of algal growth rate or yield was found in solutions containing 1.0 and 10% of the SS prepared at a loading rate of 100 mg/L. At the highest test concentration, growth rate and yield were inhibited by 15 and 50%, respectively, at the end of the test.
Based on these results, samples taken from the 10 and 100% SS test groups were analysed. The measured concentrations at the start of the test were 0.18 and 2.1 mg/L, respectively. At the end of the exposure period, the concentrations had decreased to 0.32 3.9% of the initial concentrations (see also Table 2 of the appended Analytical Report).
During the chemical analysis of the samples, problems were encountered with the preparation of the Quality Control (QC) samples. These did not yield adequate recovery levels despite repeated preparation of the according standards. It was hypothesized that the test item may not be sufficiently stable in test medium and thus affected the recoveries during the analysis. It was subsequently decided, in consultation with the Sponsor, to investigate the hydrolytic stability of the test item under Test Facility Study No. 20152328. Based on the obtained results, it was eventually concluded that the test item was hydrolytically stable enough to perform a full test with an unchanged protocol for preparing the test solutions (detailed results are archived in the raw data). The cause for the previous problems with the analytical QC samples could not be identified.
All test conditions were maintained within the limits prescribed by the study plan.

First Full Test
Measured Test Item Concentrations
No samples taken during the first full test were analysed. The first full test was performed at the time, when the analytical results of the combined limit/range-finding test became available and the considerations regarding the hydrolytic stability of the test item emerged. By the time that the hydrolysis test was performed, the samples taken during the first full test were almost two months old, causing uncertainties regarding the integrity of the samples. As a result, it was decided to repeat the full test with a timely analysis of the samples.

Mean Cell Densities
Figure 2 shows growth curves at different concentrations of Hexyl Caproate during the first full test. The individual and group mean cell densities measured at 24h intervals are given in Table 13.

Inhibition of Growth Rate and Inhibition of Yield
Table 4 shows group mean growth rates and the percentages of growth rate inhibition (total test period), whereas Table 5 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 6 (see Appendix 1 for the individual values). Statistical analysis of the data is shown in Appendix 3 and Appendix 4.
Inhibition of growth rates and yield increased with increasing concentration of Hexyl Caproate from 18% SS upwards resulting in 45 and 93% inhibition, respectively, in the undiluted SS. Statistically significant inhibition of growth rates and yield was found at test concentrations of 18% SS and higher. However, growth rate inhibition was considered to be biologically not relevant at 18% SS, 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 32% SS when compared to the control.

Experimental Conditions
Table 7 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).

Second Full Test
Measured Test Item Concentrations
The results of analysis of the samples taken during the second full test are described in Table 3 and Table 4 of the appended Analytical Report.
Samples taken from all test concentrations and the control were analysed. No concentrations could be detected in any of the analysed samples. The reserve samples were subsequently analysed with the same result, except for one sample taken at the end of the test. However, the determined concentration for this sample was close to the limit of detection and the quantification of the detected response was based on extrapolation of the calibration curve.
It was subsequently decided to repeat the test with analysis of the samples on the day of sampling. It was furthermore decided to shorten the period of stirring the test item with the test medium before collecting the saturated solution. These measures were expected to yield measured concentrations at the level of the previous analyses. The hydrolysis test had shown that the maximum concentration of the test item in M2 medium was reached after 24 hours of stirring. Additionally, the samples of the hydrolysis test had been analysed on the day of sampling without storage in a freezer.

Mean Cell Densities
Figure 2 shows growth curves at different concentrations of Hexyl Caproate during the second full test. The individual and group mean cell densities measured at 24h intervals are given in Table 17.

Inhibition of Growth Rate and Inhibition of Yield
Table 8 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 9 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 10 (see Appendix 1 for the individual values). Statistical analysis of the data is shown in Appendix 5 and Appendix 6.
Inhibition of growth rates and yield increased with increasing concentration of Hexyl Caproate from 18% SS upwards resulting in 43 and 91% inhibition, respectively, in the undiluted SS. Statistically significant inhibition of growth rates and yield was found at test concentrations of 18% and higher. However, growth rate inhibition was considered to be biologically not relevant at 18 and 32% SS, 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 56% SS when compared to the control.

Experimental Conditions
Table 11 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 at 22°C. Temperature remained within the limits prescribed by the study plan (21 24°C, constant within ±1°C).

Third Full Test
The third full test did not meet the validity criteria and the results were accordingly rejected. As such, the biological results will not be reported. It should, however, be noted that no concentrations could be detected in the samples taken at the start of the test. Subsequently, no further samples were analysed as the adjusted test strategy did not appear to have delivered the expected results.
All test conditions were maintained within the limits prescribed by the study plan.

GENERAL DISCUSSION
The analysis of samples taken during the full tests showed no measurable concentrations at the start of the second and third full test, which was in contrast to the results of the combined limit/range-finding test and a hydrolysis test. Shortening the time of preparing the test solutions in the third full test in combination with analysis of the corresponding samples on the day of sampling did not change this result. It could not be identified why no test item could be detected in the full tests although the preparation of the test solutions was the same in the combined limit/range-finding and the second full test, and although the preparation of test solutions proved to be reproducible in the hydrolysis test with M2 and adjusted ISO medium applying different stirring times.
Considering the clear dose-response of algal growth inhibition in the first and second full tests and that the observed effects were comparable, it was concluded that the algae had been exposed to a dissolved fraction of the test item that was not captured by the analytical method.
After discussion with the Sponsor, it was decided to not perform additional tests changing the protocol for preparing test solutions further by, e.g., applying a spiking approach with solvent-based stock solutions. Instead, it was decided to base the effect parameters on the percentage of the saturated solutions, which was prepared at a loading rate of 100 mg/L and which had during the initial study phases shown measured concentrations of approximately 2 mg/L. This was clearly above the determined water solubility of 0.951 mg/L.
It was decided to use the results of the first full test for the determination of the effect parameters as those proved to provide the most conservative values and thus complied the strongest with the principles of worst-case assumptions.

Determination of Effect Concentrations
Table 12 shows the effect parameters based on percentages of the saturated solution prepared at a loading rate of 100 mg/L, see also Appendix 7.

Results with reference substance (positive control):

The objective of the study was to evaluate Potassium dichromate for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) (strain: NIVA CHL-1) during an exposure period of 72 hours and, if possible, to determine the EC50 for inhibition of both growth rate and yield (Charles River Den Bosch Study Number 20161585).
Start of first exposure: 02 Jul 2018
Completion last exposure: 05 Jul 2018
The study procedures described in this report were based on the OECD guideline No. 201, Adopted March 23, 2006; Annex 5 corrected 28 July 2011 and ISO Standard 8692, Third edition, February 2012.
This reference test was carried out to check the sensitivity of the test system used by Charles River Den Bosch to Potassium dichromate (Merck, Art. 1.04864, Batch K44879664).
Algae were exposed for a period of 72 hours to K2Cr2O7 (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 104 cells/ml.
Potassium dichromate inhibited growth rate and yield of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.30 mg/L with a 95% confidence interval ranging from 0.29 to 0.31 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L.
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.

Reported statistics and error estimates:

See details on results

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

Time (h)	Hexyl Caproate;%SS prep. at 100 mg/L
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	8.3	n.d.	n.d.	4.7
48	51.9	n.d.	n.d.	21.5
72	231.1	232.6	250.9	115.5

n.d.: not determined

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

Hexyl Caproate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.814	0.0204	6
1.0	1.816	0.0241	3	-0.12
10	1.842	0.0106	3	-1.5
100	1.541	0.1866	6	15

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

Hexyl Caproate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	230.1	14.52	6
1.0	231.6	16.55	3	-0.65
10	249.9	7.90	3	-8.6
100	114.5	63.68	6	50

Table 4. Growth Rate and Percentage Inhibition for the Total Test Period (First Full Test)

Hexyl Caproate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.832	0.0554	6
10	1.803	0.0381	3	1.6
18	1.786	0.0426	3	2.5^#
32	1.589	0.0337	3	13*
56	1.337	0.0360	3	27*
100	1.007	0.0357	3	45*

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

Table 5. Growth Rate and Percentage Inhibition at Different Time Intervals (First Full Test)

Hexyl Caproate %SS prep. at 100 mg/L	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.765		1.899		1.579
10	3	1.865	-5.7	1.741	8.3	1.558	1.3
18	3	1.899	-7.6	1.674	12	1.495	5.3
32	3	1.786	-1.2	1.392	27	1.155	27
56	3	1.508	15	1.166	39	0.334	79
100	3	0.994	44	1.021	46	0.678	57

Table 6. Yield and Percentage Inhibition for the Total Test Period (First Full Test)

Hexyl Caproate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	188.572	13.1853	6
10	174.005	6.4170	3	7.7
18	157.902	3.6493	3	16*
32	75.358	5.9192	3	60*
56	19.283	1.4158	3	90*
100	13.807	1.2939	3	93*

* Effect was statistically significant.

Table 7. pH Levels Recorded during the First Full Test

Hexyl Caproate %SS prep. at 100 mg/L	pH
	t=0h	t=72h
Control	8.2	8.4
10	8.2	8.4
18	8.1	8.4
32	8.1	8.2
56	8.0	8.2
100	8.0	8.2

Table 8. Growth Rate and Percentage Inhibition for the Total Test Period (Second Full Test)

Hexyl Caproate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.790	0.0125	6
10	1.792	0.0246	3	-0.097
18	1.748	0.0079	3	2.4^#
32	1.696	0.0142	3	5.3^#
56	1.409	0.0392	3	21*
100	1.011	0.0387	3	43*

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

Table 9. Growth Rate and Percentage Inhibition at Different Time Intervals (Second Full Test)

Hexyl Caproate %SS prep. at 100 mg/L	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.938		1.857		1.576
10	3	1.753	9.5	1.987	-7.0	1.635	-3.7
18	3	1.886	2.7	1.690	9.0	1.668	-5.9
32	3	1.903	1.8	1.689	9.0	1.495	5.1
56	3	1.784	8.0	1.402	25	1.042	34
100	3	1.207	38	0.911	51	0.917	42

Table 10. Yield and Percentage Inhibition for the Total Test Period (Second Full Test)

Hexyl Caproate %SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	214.084	8.1010	6
10	215.479	16.2922	3	-0.65
18	188.406	4.4495	3	12*
32	161.080	6.8757	3	25*
56	67.833	7.9506	3	68*
100	19.884	2.5025	3	91*

* Effect was statistically significant.

Table 11. pH Levels Recorded during the Second Full Test

Hexyl Caproate %SS prep. at 100 mg/L	pH
	t=0h	t=72h
Control	8.3	8.2
10	8.2	8.2
18	8.2	8.2
32	8.1	8.2
56	8.1	8.2
100	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 Caproate inhibited growth rate and yield of this fresh water algae species significantly in test solutions containing 18% and more of a saturated solution prepared at a loading rate of 100 mg/L.
The 72h-EC50 for growth rate inhibition (ERC50) was beyond the range tested, i.e. exceeded the concentration achieved in a test solution prepared at a loading rate of 100 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was equivalent to a concentration achieved in a test solution containing 29% of a saturated solution prepared at a loading rate of 100 mg/L with a 95% confidence interval ranging from 28 to 29% of the SS.
The 72h-NOEC for growth rate inhibition was equivalent to a concentration achieved in a test solution containing 10% of the SS prepared at a loading rate of 100 mg/L based on statistical significance and equivalent to a concentration achieved in a test solution containing 18% of the SS based on biological relevance.
The 72h-NOEC for yield inhibition was equivalent to a concentration achieved in a test solution containing 10% of the SS prepared at a loading rate of 100 mg/L based on statistical significance and biological relevance.

Executive summary:

The objectiveofthe study was to evaluate Hexyl Caproate 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 Caproate tested was a clear colourless liquid with a purity of 99.29% 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.

Two full tests were performed based on the results of a preceding combined limit/range-finding test. For each full test, six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions 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 10⁴ cells/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.

No concentrations could be detected in the samples taken during the test. It was decided to base the effect parameters on percentage of the saturated solution.

Inhibition of growth rates and yield increased with increasing concentration of Hexyl Caproate from 18% SS upwards. Both tests yielded comparable results with algal growth being slightly stronger inhibited in the first test. Growth rate and yield were inhibited by 45 and 93%, respectively, in the undiluted SS. Assuming a worst case scenario, the results of the first full test were used to determine the effect parameters.

The studies 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 (%SS prep. at 100 mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	10	18	20	34	>100
	lower 95%-cl			19	33
	upper 95%-cl			21	35
Yield	Value	10	10	14	18	29
	lower 95%-cl			14	18	28
	upper 95%-cl			15	19	29

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), Hexyl Caproate inhibited growth rate and yield of this fresh water algae species significantly in test solutions containing 18% and more of a saturated solution prepared at a loading rate of 100 mg/L. The 72h-EC₅₀ for growth rate inhibition (E_RC₅₀) was beyond the range tested, i.e. exceeded the concentration achieved in a test solution prepared at a loading rate of 100 mg/L. The 72h-NOEC for growth rate inhibition was equivalent to a concentration achieved in a test solution containing 10% of the SS prepared at a loading rate of 100 mg/L based on statistical significance and equivalent to a concentration achieved in a test solution containing 18% of the SS based on biological relevance.

Description of key information

Study conducted to internationally recognised testing guideline with GLP certification.

Additional information

EC50 >100% saturated solution (100 mg/l loading rate)