(Z)-3-hexenyl salicylate

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:

8th June 2009 to 19th June 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/l) of test material in culture medium for a period of 24 hours prior to removing any undissolved test material present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test material.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the control (replicates R1-R6 pooled) and each test group (replicates R1-R3 pooled)
- Sampling method: Samples were taken at 0 and 72 hours for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 hours and stored at approximately -20 °C for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Vehicle:

no

Details on test solutions:

PRE-STUDY MEDIA PREPARATION TRIAL
Pre-study solubility work indicated that the test substance was practically insoluble in water using traditional methods of preparation. A test concentration of 1 mg/L was obtained using a preliminary solution in dimethylformamide. An estimate of the water solubility for the test substance using EPIWIN (Version 3.20 2007) indicated that the solubility was approximately 9.5 mg/L. The test substance was categorised as being a “difficult substance”. A media preparation trial was conducted in order to determine the solubility of the test substance under test conditions.

- Saturated solution preparation: 550 mg of test substance was dispersed, in duplicate, in 11 litres of culture medium with the aid of a propeller stirring at approximately 1500 rpm at a temperature of approximately 21 °C for periods of 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
* Centrifugation at 10000 g for 30 minutes
* Centrifugation at 40000 g for 30 minutes
* Filtration through a 0.2 µm Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter).
* Filtration through a 0.2 µm Sartorius Sartopore filter (approximately 2 litres discarded in order to pre-condition the filter).

- Solvent spike preparation: 50 mg of test substance was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 50 mg/10 mL solvent stock solution. An aliquot (1000 µL) of this 50 mg/10 mL solvent stock solution was dispersed in 10 litres of culture medium with the aid of magnetic stirring at approximately 21 °C for approximately 10 minutes to give the required test concentration of 0.50 mg/L prior to taking samples for chemical analysis after the following pre-treatments:
* Untreated
* Centrifugation at 10000 g for 30 minutes
* Centrifugation at 40000 g for 30 minutes
* Filtration through a 0.2 µm Gelman Acrocap filter (approximately 100 mL discarded in order to pre-condition the filter)
* Filtration through a 0.2 µm Gelman Acrocap filter (approximately 500 mL discarded in order to pre-condition the filter)
The remainder of the 0.50 mg/L test concentration was returned to the magnetic stirrer and stirred for a further 48 hours with samples being taken for analysis after both 24 and 48 hours stirring.

The result from the pre-study media preparation trial conducted indicated that a dissolved test substance concentration of approximately 4.8 mg/L could be obtained using a saturated solution method of preparation.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances): Definitive Test
- Method: Due to the low aqueous solubility and high purity of the test substance, the test concentrations used in the definitive test were prepared by diluting (with culture medium) a saturated solution prepared from an initial test substance dispersion at a concentration of 50 mg/L. 550 mg of test substance was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After 24 hours the stirring was stopped and any undiluted test substance was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution with a 0-hour measured concentration of 3.6 mg/L. A series of dilutions was made from this saturated solution to give stock solutions of 1.5, 0.74, 0.35 and 0.15 mg/L. An 1 litre aliquot of each of the stock solutions was separately inoculated with 4.5 mL algal suspensions to give the 0-Hour measured test concentrations of 0.15, 0.35, 0.74, 1.5 and 3.6 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test substance in the test solutions were verified by chemical analysis at 0 and 72 hours.

- Controls: The control group was maintained under identical conditions but not exposed to the test substance.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, UK

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

pH 7.1-7.2 at 0 hours and pH 7.5-7.6 at 72 hours

Nominal and measured concentrations:

Nominal concentrations: 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L
Measured concentrations (0-hour): 0.15, 0.35, 0.74, 1.5 and 3.6 mg/L
Measured concentrations (Geometric mean): 0.036, 0.055, 0.079, 0.3 and 0.58 mg/L
Results are based on 0-hour measured concentrations.

Details on test conditions:

TEST SYSTEM
- Type: Closed, plugged with a polyurethane foam bung
- Test vessel material, size, headspace, fill volume: 250 mL glass conical flasks containing 100 mL test solution
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 8.91 x 10^5 cells per mL.
- Control end cells density: 2.25 x 10^5
- Initial cell density: Approximately 4 x 10^3
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Detailed composition:
NaNO3 25.5 mg/L
MgCl2.6H20 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H20 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
Na2SeO3.5H2O 0.000010 mg/L
The culture medium was prepared using reverse osmosis purified deionised water (Elga optima 15+ Elga Purelab Option R15 BP) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl.
- Temperature: 24 ± 1 °C
- Photoperiod: Constant illumination
- Light intensity and quality: 7000 lux provided by warm lighting (380-730 nm)
- Agitation: Constantly shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x2

- Range finding study: Desmodesmus subspicatus cells were exposed to 0.048, 0.48 and 4.8 mg/L for a period of 72 hours. 550 mg of test substance was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at approximately 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test substance was removed by filtration through a 0.2 µm Sartortius Sartopore filter (first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution with a nominal concentration of 4.8 mg/L. A series of dilutions was made from this saturated solution to give further stock solutions of 0.48 and 0.048 mg/L. A 250 ml aliquot of each stock solution was separately inoculated with 3.7 mL algal suspension to give the required test concentrations of 0.048, 0.48 and 4.8 mg/L. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test solution and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The control group was maintained under identical conditions but not exposed to the test substance. At the start of the range-finding test, a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated at 24 ± 1ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380-730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined.

The results showed no effect on growth at the nominal test concentrations of 0.048 and 0.48 mg/L. However, growth was observed to be reduced at 4.8 mg/L.

- Test concentrations: Nominal 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results based on the 0-Hour measured test concentrations:

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results based on the 0-Hour measured test concentrations:

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.61 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI 0.51-0.73 mg/L

Remarks:

results based on the 0-Hour measured test concentrations:

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: results based on the 0-Hour measured test concentrations:

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: results based on the 0-Hour measured test concentrations:

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI 0.25-0.30 mg/L

Remarks:

results based on the 0-Hour measured test concentrations:

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results based on the 0-Hour measured test concentrations:

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After 72 hours all control and 0.15 mg/L test cultures were observed to be pale green dispersions. The 0.35 mg/L test cultures were observed to be extremely pale green dispersions whilst the 0.74, 1.5 and 3.6 mg/L test cultures were observed to be clear colourless solutions.
- Other: Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.15 to 3.6 mg/L. Analysis of the test preparations at 72 hours showed a significant decline in measured test concentrations in the range of less than the limit of quantitation (LOQ). As the preliminary stability analyses indicated that the test substance was stable in culture medium, this decline was attributed to the absorption of the test substance to the algal cells. Whilst the preliminary recovery analyses conducted in the presence of algal cells indicated that no immediate absorption occurred, this does not preclude long-term adsorption over the test period. The geometric mean measured test concentrations were calculated. In cases where the measured concentrations was less than the LOQ of the analytical method a value half of the LOQ was used to calculate the geometric mean measured concentration. As the test substance was known to be stable in aqueous test medium and the decline observed attributed to adsorption to algal cells it was considered that the algal cells present were exposed to equivalent 0-hour measured concentrations of the test substance throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? The results from the positive control with potassium dichromate were within the normal ranges.
- EC50: ErC50 (0-72 h) : 0.79 mg/L
- Other: EyC50 (0-72 h): 0.30 mg/L (95 % CI 0.27-0.34 mg/L)
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant difference between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999-2001) Under the conditions of the study, the 72 hour growth rate EC50 of the test substance with Desmodesmus subspicatus was determined to be 0.61 mg/L. The 72 hour yield EC50 was determined to be 0.28 mg/L. The NOEC for both effects was determined to be 0.15 mg/L and the LOEC 0.35 mg/L.

Summary of results

Response variable	EC50 (mg/L)	95% C.I. (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Growth rate	0.61	0.51-0.73	0.15	0.35
Yield	0.28	0.25-0.30	0.15	0.35

Geometric mean measured test concentrations

0 hour mean test concentrations (mg/L)	Geometric mean measured test concetration (mg/L)	Expressed as a percentage of the 0 hour measured test concentration
0.15	0.036	24
0.35	0.055	16
0.74	0.079	11
1.5	0.30	20
3.6	0.58	16

Table 1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/l)		Cell Densities*(cells per ml)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.47E+03	1.74E+06
	R2	4.34E+03	1.23E+06	-	-
	Mean	4.41E+03	1.49E+06
0.048	R1	4.41E+03	2.05E+06
	R2	4.62E+03	1.51E+06	[2]	[20]
	Mean	4.51E+03	1.78E+06
0.48	R1	4.23E+03	1.16E+06
	R2	4.45E+03	1.61E+06	1	7
	Mean	4.34E+03	1.38E+06
4.8	R1	4.55E+03	2.08E+05
	R2	4.36E+03	2.56E+05	32	85
	Mean	4.46E+03	2.32E+05

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

Table 2              Cell Densities and pH Values in the DefinitiveTest

0-Hour Measured Test Concentration (mg/l)		pH	Cell Densities*(cells per ml)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	7.2	4.53E+03	1.19E+04	3.50E+04	2.08E+05	7.6
	R2	7.2	3.91E+03	1.16E+04	3.46E+04	1.95E+05	7.5
	R3	7.2	4.18E+03	1.12E+04	3.73E+04	2.19E+05	7.6
	R4	7.1	3.78E+03	1.28E+04	3.16E+04	2.22E+05	7.6
	R5	7.1	3.98E+03	1.12E+04	3.44E+04	2.35E+05	7.6
	R6	7.1	3.82E+03	1.13E+04	3.43E+04	2.68E+05	7.5
	Mean		4.04E+03	1.17E+04	3.45E+04	2.25E+05
0.15	R1	7.1	4.11E+03	1.07E+04	6.28E+04	2.53E+05	7.5
	R2	7.1	3.95E+03	1.30E+04	4.99E+04	2.84E+05	7.5
	R3	7.1	3.74E+03	1.10E+04	6.12E+04	2.64E+05	7.5
	Mean		3.93E+03	1.16E+04	5.79E+04	2.67E+05
0.35	R1	7.1	4.44E+03	7.82E+03	2.75E+04	5.69E+04	7.3
	R2	7.0	4.06E+03	6.70E+03	2.10E+04	5.37E+04	7.3
	R3	7.1	4.10E+03	6.82E+03	3.04E+04	5.72E+04	7.3
	Mean		4.20E+03	7.11E+03	2.63E+04	5.59E+04
0.74	R1	7.1	4.09E+03	6.38E+03	9.44E+03	2.42E+04	7.2
	R2	7.0	4.30E+03	6.65E+03	7.88E+03	2.59E+04	7.2
	R3	7.0	4.14E+03	6.66E+03	7.93E+03	2.32E+04	7.2
	Mean		4.18E+03	6.56E+03	8.42E+03	2.44E+04
1.5	R1	7.1	3.96E+03	6.66E+03	7.71E+03	7.50E+03	7.1
	R2	7.0	3.86E+03	6.26E+03	7.68E+03	8.00E+03	7.1
	R3	7.1	4.51E+03	6.41E+03	9.23E+03	9.00E+03	7.1
	Mean		4.11E+03	6.44E+03	8.21E+03	8.17E+03
3.6	R1	7.0	4.35E+03	2.95E+03	3.01E+03	3.59E+03	7.1
	R2	7.0	4.33E+03	2.54E+03	3.32E+03	3.42E+03	7.1
	R3	7.0	4.28E+03	3.42E+03	3.42E+03	4.29E+03	7.1
	Mean		4.32E+03	2.97E+03	3.25E+03	3.77E+03

*Cell densities represent thean number of cells per ml calculated from thean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Table 3              Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/ml/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.045	0.045	0.074
	R2	0.044	0.046	0.072
	R3	0.043	0.050	0.074
	R4	0.049	0.038	0.081
	R5	0.043	0.047	0.080
	R6	0.043	0.046	0.086
	Mean	0.045	0.045	0.078

R₁- R₆= Replicates 1 to 6

Table 4              Inhibition of Growth Rate and Yield in the Definitive Test

0-Hour Measured Test Concentration (mg/l)		Growth Rate (cells/ml/hour)		Yield (cells/ml)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.055		2.04E+05
	R2	0.054		1.91E+05
	R3	0.056		2.15E+05
	R4	0.056	-	2.18E+05	-
	R5	0.057		2.31E+05
	R6	0.058		2.64E+05
	Mean	0.056		2.20E+05
	SD	0.001		2.54E+04
0.15	R1	0.058	[4]	2.49E+05
	R2	0.059	[5]	2.80E+05
	R3	0.058	[4]	2.60E+05
	Mean	0.058	[4]	2.63E+05	[19]
	SD	0.001		1.58E+04
0.35	R1	0.037	34	5.24E+04
	R2	0.036	36	4.96E+04
	R3	0.037	34	5.31E+04
	Mean	0.037	35	5.17E+04	77
	SD	0.001		1.85E+03
0.74	R1	0.025	55	2.01E+04
	R2	0.026	54	2.16E+04
	R3	0.024	57	1.91E+04
	Mean	0.025	55	2.03E+04	91
	SD	0.001		1.28E+03
1.5	R1	0.009	84	3.54E+03
	R2	0.010	82	4.14E+03
	R3	0.011	80	4.49E+03
	Mean	0.010	82	4.06E+03	98
	SD	0.001		4.78E+02
3.6	R1	-0.001	102	-7.60E+02
	R2	-0.002	104	-9.12E+02
	R3	0.001	98	8.00E+00
	Mean	-0.001	101	-5.55E+02	100
	SD	0.002		4.93E+02

*In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated

R₁– R₆= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 72 hour growth rate EC50 of hexentyl-3-cis salicylate with Desmodesmus subspicatus was determined to be 0.61 mg/L. The 72 hour yield EC50 was determined to be 0.28 mg/L. The NOEC for both effects was determined to be 0.15 mg/L and the LOEC 0.35 mg/L.

Executive summary:

In a GLP compliant study conducted in line with OECD 201 and EU Method C.2, the effect of the test material hexenyl-3-cis salicylate on the growth of Desmodesmus subspicatus was determined. The 72 hour growth rate EC50 of the test material was determined to be 0.61 mg/L, the 72 hour yield EC50 was determined to be 0.28 mg/L, the NOEC for both effects was determined to be 0.15 mg/L and the LOEC 0.35 mg/L.

Description of key information

Acute algae 72 hour EC50 0.61 mg/L (growth); 72 hour NOEC 0.15 mg/L (growth and yield); study conducted in accordance with OECD 201, EU C.3; Vyrenhoef & Mullee (2010)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.61 mg/L

EC10 or NOEC for freshwater algae:

0.15 mg/L

Additional information

In a GLP compliant acute study conducted in line with standardised guidelines, the effect of hexenyl-3-cis salicylate on Desmodesmus subspicatus was determined. The 72 hour growth rate EC50 was determined to be 0.61 mg/L, the 72 hour yield EC50 was determined to be 0.28 mg/L, the NOEC for both effects was determined to be 0.15 mg/L and the LOEC to be 0.35 mg/L.

Analysis of the test preparations at 72 hours showed a significant decline in measured test concentrations. As the preliminary stability analyses indicated that the test substance was stable in culture medium, this decline was attributed to the absorption of the test substance to the algal cells. As the test material was known to be stable in aqueous test medium and the decline observed attributed to adsorption to algal cells it may be considered that the algal cells present were exposed to equivalent 0-Hour measured concentrations of the test material throughout the test period.