Allyl 3-cyclohexylpropionate

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:

2013-01-16 to 2013-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: six test concentrations were prepared form stock solutions.
- Sampling method:
Samples of the test solutions were collected at approximately 0, 24, 48, 72 and 96 hours of exposure to measure concentrations of the test substance. At the start of the test, samples were collected from the individual batches of test solution prepared for each treatment and control group prior to distribution into the test chambers. At 24 and 48 hours of exposure, samples were collected from surrogate biotic replicates included in each treatment and control group. At 72 and 96 hours of exposure, the four biological replicates from each respective treatment and control group sacrificed at that time interval were pooled and then sampled. Samples were collected from additional abiotic replicates included in the highest treatment group at 24, 48, 72 and 96 hours of exposure. All samples were collected in glass vessels and processed immediately for analysis. All samples were acidified with two drops of 10 % phosphoric acid immediately after collection, to stabilize the test substance. Algal cells present in samples collected after inoculation were removed during the extraction process.
- Sample storage conditions before analysis: no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A primary stock solution was prepared by dissolving 3.0002 g of Allyl Cyclohexyl Propionate in 10 mL of N,N-dimethylformamide (DMF) to achieve a nominal concentration of 300 mg/mL. The primary stock was mixed by inversion and appeared clear and colorless after mixing. Five additional stocks solutions were prepared in DMF at nominal concentrations of 3.1, 7.7, 19, 48 and 120 mg/mL by serial dilution. The test solutions were prepared by diluting the required volume of each respective stock solution in freshwater AAP medium with additional sodium bicarbonate added such that each test solution contained the same concentration of solvent, 0.1 mL DMF/L. The test solutions were prepared in 4000 mL glass aspirator bottles, filled to the maximum volume of each bottle, leaving no headspace and were stirred for approximately 15 minutes with a Teflon-coated stirbar and magnetic stirplate. The 0.31 to 12 mg/L test solutions appeared clear and colorless and were otherwise unremarkable after mixing. An oily slick was visible on the surface of the 30 mg/L test solution at test initiation. The negative control solution consisted of freshwater AAP algal medium with additional sodium bicarbonate added. A solvent control solution was prepared by diluting 0.4460 mL of DMF in 4460 mL of freshwater AAP medium with additional sodium bicarbonate added to achieve a nominal concentration of 0.1 mL DMF/L, equivalent to the solvent concentration in all the treatment groups. In addition, test solutions were also prepared approximately 24 hours prior to test initiation to pre-condition the glassware used in this test. The pre-conditioning of glassware was conducted in an attempt to improve day 0 analytical recoveries.
- Controls: negative control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N,N-dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL DMF/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc):
An oily slick was visible on the surface of the 30 mg/L test solution at test initiation. At test termination, there were no visible particulates in any of the test solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater Alga Pseudokirchneriella subcapitata
- Strain: not reported
- Source: Original algal cultures were obtained from the University of Toronto Culture Collection, and had been maintained in culture medium at Wildlife International, Easton, Maryland.
- Age of inoculum at test initiation: Algal cells used in this test were obtained from Wildlife International cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh media four days prior to test initiation. Prior to test initiation, the cell density of P. subcapitata culture 00-1 was determined to be 3.78 x 106 cells/mL using a hemacytometer and microscope. In order to achieve a nominal concentration of approximately 5,000 cells/mL at test initiation 0.397 mL of culture was added to each biotic replicate test vessel.
- Method of cultivation: cultures actively growing in culture medium. The algal cells were cultured and tested in freshwater AAP medium. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified Wildlife International well water.

ACCLIMATION
- Acclimation period: Algal cells for this study were taken from a culture that had been transferred to fresh media four days prior to test initiation.
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: not mentioned

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

assessments of growth after 72 and 96 hours of exposure

Hardness:

not reported

Test temperature:

Day 0: 23.0-23.2 °C; day 1: 23.2-23.3 °C; day 2: 23.1-23.2 °C; day 3: 23.2-24.3 °C; day 4: 24.0-24.3 °C

pH:

Day 0: 7.5-7.9; day 4: 7.2-7.7 (mean measured 4.5-15 mg/L, due to no or low algal growth in these treatments), 10.4-10.5 (mean measured 0.28-1.9 mg/L)

Dissolved oxygen:

not reported

Salinity:

not applicable

Conductivity:

not reported

Nominal and measured concentrations:

nominal: 0.31, 0.77, 1.9, 4.8, 12 and 30 mg/L; measured (day 0): 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L; geometric mean measured (day 4): 0.095, 0.14, 0.37, 0.95, 4.6, 11 and 17 mg/L; study based on measured concentrations of 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L, at Day 0 based on U.S. E.P.A. guidance (U.S. Environmental Protection Agency. 1994. Pesticide Reregistration Rejection Rate Analysis – Ecological Effects. Test Material Decline in Algae and Diatom Tests)

Details on test conditions:

TEST SYSTEM
- Test vessel: gas tight, sterile, glass bottles sealed with glass stoppers
- Type: closed
- Material, size, headspace, fill volume: glass, 300-mL, no head space, 300-mL
- Aeration: No specifici aeration but the test chambers were shaken continuously at 100 rpm.
- Initial cells density: approximately 5,000 cells/mL
- Control end cells density: approx. 623,533 cells/mL
- No. of organisms per vessel: approximately 5,000 cells/mL at test start
- No. of vessels per concentration: 4 for 72-hour assessment, 4 for 96-hour assessment
- No. of vessels per control: 4 for 72-hour assessment, 4 for 96-hour assessment
- No. of vessels per vehicle control: 4 for 72-hour assessment, 4 for 96-hour assessment

GROWTH MEDIUM
- Standard medium used: yes, freshwater AAP medium with additional sodium bicarbonate
- Detailed composition if non-standard medium was used: additional sodium bicarbonate

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: freshwater AAP medium
- Total organic carbon: not relevant as freshwater AAP medium was being used
- Particulate matter: not relevant as freshwater AAP medium was being used
- Metals: all toxic metals analysed were found to be below detection limit (detailed analytical results are given in the original study report)
Mineral composition of test medium:
MgCl2•6H2O: 12.164 mg/L
CaCl2•2H2O: 4.410 mg/L
H3BO3: 0.1855 mg/L
MnCl2•4H2O: 0.4154 mg/L
ZnCl2: 3.27 µg/L
FeCl3•6H2O: 0.1598 mg/L
CoCl2•6H2O: 1.428 µg/L
Na2MoO4•2H2O: 7.26 µg/L
CuCl2•2H2O: 0.012 µg/L
Na2EDTA•2H2O: 0.300 mg/L
NaNO3: 25.50 mg/L
MgSO4•7H2O: 14.70 mg/L
K2HPO4: 1.044 mg/L
NaHCO3: 50.00 mg/L
- Pesticides: all substances analysed were found to be below detection limit (detailed analytical results are given in the original study report)
- Chlorine: not applicable as freshwater AAP medium was being used
- Alkalinity: not measured; estimation based on NaHCO3 concentration of the test medium (50 mg/L) the alkalinity is ca. 0.6 mmol/L
- Ca/Mg ratio: 1:2
- Conductivity: not reported, however detailed mineral compisition of test medium is stated above
- Culture medium different from test medium: Yes. Due to the closed-bottle test design, additional sodium bicarbonate was added to achieve a total concentration of 50 mg NaHCO3/L in the medium to provide a sufficiently supply of CO2 for algal growth.
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5,550 to 6,580 lux

EFFECT PARAMETERS MEASURED: growth rate, area under curve, biomass after 72 and 96 hours
- Determination of cell concentrations: electronic particle counter (Coulter Electronics, Inc.)
- Chlorophyll measurement: no
- Other: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study
- Test concentrations:
nominal 0.09, 0.9 and 9.0 mg/L
measured 55, 37 and 35 % of target (at test start) and less than the limit of quantitation (0.050 mg/L) at test end
- Results used to determine the conditions for the definitive study: -2, -4 and 7 % inhibition of mean cell density after 96 hours of exposure

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI 2.5 to 3.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI 1.1 to 2.2 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95 % CI 0.95 to 4.8 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI 0.95 to 4.8 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI 4.1 to 5.1 mg/L

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI 1.8 to 2.7 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95 % CI 1.9 to 2.8 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CI 2.3 to 3.3 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

< 0.28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: Cells were observed to be adhering to the test vessels in the 0.28, 0.74 and 1.9 mg/L treatment groups and in the negative and solvent controls.
- Aggregation of algal cells: no
- Other: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance not reported

Reported statistics and error estimates:

The 72 and 96-hour EbC50, ErC50 and EyC50 values and their 95 % confidence intervals were calculated, when possible, using nonlinear regression with replicate data (area under the growth curve, growth rate or yield) and day 0 measured test concentrations. Additionally, ErC10 values and their corresponding 95 % confidence intervals were calculated, when possible, using non-linear regression with replicate data (growth rate) and day 0 measured test concentrations.
The negative and solvent control responses for area under the growth curve, growth rate and yield were compared at 72 and 96-hours using a t-test. The responses of the two control groups were determined to be significantly different (p < 0.05) for all three parameters at both 72 and 96 hours. The solvent control responses were reduced relative to the negative control responses and the treatment responses were compared to the negative control response as this provided the most conservative estimates of toxicity and is the appropriate response per U.S. EPA guidance. It was not determined why the negative control replicates performed better than the solvent control replicates over the duration of the test.
The 72 and 96-hour area under the growth curve, yield and growth rate data were evaluated for normality and homogeneity of variance (p = 0.01) using Shapiro-Wilk’s and Levene’s tests, respectively. The 72-hour area under the growth curve, growth rate and yield data and 96-hour yield and growth rate data failed Shapiro-Wilk’s test for normal distribution. The 96-hour growth rate data also failed Levene’s test for homogeneity of variance. Log transformation of the data failed to resolve these issues. The treatment groups were compared to the negative control replicates using Dunnett’s test (p = 0.05) and the Jonckheere-Terpstra trend test (p = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC for each parameter.

Conditions for the Validity of the Test

The mean cell density in the negative control flasks increased by a factor of 102 after three days and 126 after four days, exceeding the growth criteria listed in the study protocol. The coefficient of variation of mean yield and mean average specific growth rates in the negative control replicates at the 0-72 hour interval were 4.9 and 1.0 %, respectively. At the 0-96 hour interval, the coefficient of variation for mean yield and mean average specific growth rate in the negative control replicates were 2.0 and 0.41 %, respectively, achieving the protocol requirement of less than 15 %.

Validity criteria fulfilled:

yes

Conclusions:

The freshwater green alga, Pseudokirchneriella subcapitata, was exposed to six concentrations of Allyl Cyclohexyl Propionate, with day 0 measured concentrations ranging from 0.28 to 15 mg/L. Effects were evaluated based on area under the growth curve, growth rate and yield. The 72-hour EbC50, ErC50 and EyC50 values and their corresponding 95 % confidence intervals, based on area under the growth curve, growth rate and yield, were 2.1 [0.95 to 4.8] mg/L, 3.0 [2.5 to 3.6] mg/L and 2.1 [0.95 to 4.8] mg/L, respectively. The 96-hour EbC50, ErC50 and EyC50 values, based on area under the growth curve, growth rate and yield, were 2.3 [1.9 to 2.8] mg/L, 4.6 [4.1 to 5.1] mg/L and 2.8 [2.3 to 3.3] mg/L, respectively. The 72 and 96-hour NOEC values, based on effects on area under the growth curve, growth rate and yield, were determined to be 0.74 and < 0.28 mg/L, respectively. The 72 and 96-hour EC10 values for growth rate were calculated as 1.6 (1.1 to 2.2) mg/L and 2.2 (1.8 to 2.7) mg/L, respectively.

Executive summary:

In a conclusive, reliable and valid study, the effects of the test item Allyl Cyclohexyl Propionate on the growth of the freshwater green alga Pseudokirchneriella subcapitata were examined after 72 and 76 hours of exposure in a closed test system, according to OECD TG 201, EC No. L383 Method C.3 and OCSPP Number 850.4500. Nominal concentrations were 0.31, 0.77, 1.9, 4.8, 12 and 30 mg/L. The 0.31 to 12 mg/L test solutions appeared clear and colorless and were otherwise unremarkable at test initiation. An oily slick was visible on the surface of the 30 mg/L test solution at test initiation and at test termination. At test termination, there were no visible particulates in any of the test solutions. Measured concentrations of Allyl Cyclohexyl Propionate in samples collected from the 0.31, 0.77, 1.9, 4.8, 12 and 30 mg/L nominal concentrations at exposure initiation were 90, 96, 98, 95, 77 and 50 % of the target nominal concentrations, respectively. Measured concentrations of Allyl Cyclohexyl Propionate in samples collected from the 0.31, 0.77, 1.9, 4.8, 12 and 30 mg/L nominal concentrations at the end of exposure (96 hours) were less than the limit of quantitation in all test solutions except for the 12 and 30 mg/L test solutions. Measured concentrations of Allyl Cyclohexyl Propionate in the 12 and 30 mg/L test solutions at the end of the exposure period were 0.60 and 3.3 mg/L, respectively. Measured concentrations in the abiotic replicates included in the 30 mg/L treatment group were 50, 65, 55, 57 and 63 % of nominal at approximately 0, 24, 48, 72 and 96 hours of exposure, respectively. Measured concentrations of Allyl Cyclohexyl Propionate declined over the 96 hour exposure period indicating that the test substance was not stable under the conditions of the test. Concentrations in the controls were less than the limit of quantitation at each sampling interval. Since the measured concentrations of Allyl Cyclohexyl Propionate declined substantially over the exposure period the results of the study were based on day 0 measured concentrations of 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L, per U.S. E.P.A. guidance (U.S. Environmental Protection Agency. 1994. Pesticide Reregistration Rejection Rate Analysis – Ecological Effects. Test Material Decline in Algae and Diatom Tests). After 72 hours of exposure, inhibition of area under the growth curve in the 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L treatment groups was 36, 2, 44, 99, 100 and 100 %, respectively, relative to the negative control response. Inhibition of growth rate at the 0-72 hour interval in the 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L treatment groups was 10, 0, 13, 82, 98 and 100 %, respectively, relative to the negative control, and inhibition of yield after 72 hours of exposure was 36, 2, 44, 99, 100 and 100 %, respectively, relative to the negative control. Reductions in area under the growth curve, growth rate and yield were statistically significant (Jonckheere-Terpstra trend test; p < 0.05) in the 1.9, 4.5, 9.2 and 15 mg/L treatment groups when compared to the negative control response. The 72-hour NOEC was determined to be 0.74 mg/L, based on statistically significant reductions in area under the growth curve, growth rate and yield after 72 hours of exposure. After 96 hours of exposure, inhibition of area under the growth curve in the 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L treatment groups was 31, 6, 37, 97, 100 and 100 %, respectively, relative to the negative control. Inhibition of growth rate at the 0-96 hour interval in the 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L treatment groups was 3, 4, 3, 55, 85 and 100 %, respectively, relative to the negative control, and inhibition of yield after 96 hours of exposure was 15, 17, 13, 94, 99 and 100 %, respectively, relative to the negative control. Reductions in growth rate were statistically significant (Dunnett’s test; p < 0.05) in the 4.5, 9.2 and 15 mg/L treatment groups when compared to the negative control response. Area under the growth curve was significantly reduced (Jonckheere-Terpstra trend test; p < 0.05) in the 1.9, 4.5, 9.2 and 15 mg/L treatment groups at 96 hours of exposure. Yield was significantly reduced in all treatment groups when compared to the negative control response after 96 hours of exposure (Jonckheere-Terpstra trend test; p < 0.05). Therefore, based on statistical comparisons between treatment and negative control data and evaluation of the dose-response, the 96-hour NOEC was determined to be < 0.28 mg/L, based on reductions in yield after 96 hours of exposure.

Description of key information

The 72-hour EbC50, ErC50 and EyC50 values for growth rate and yield, were 2.1 mg/L, 3.0 mg/L and 2.1 mg/L, respectively. The 72-hour EC10 value for growth rate was determined to be 1.6 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

3 mg/L

EC10 or NOEC for freshwater algae:

1.6 mg/L

Additional information

In a conclusive, reliable and valid study, the effects of the test item Allyl Cyclohexyl Propionate on the growth of the freshwater green alga Pseudokirchneriella subcapitata were examined after 72 and 76 hours of exposure in a closed test system, according to OECD TG 201, EC No. L383 Method C.3 and OCSPP Number 850.4500. Measured concentrations of Allyl Cyclohexyl Propionate declined over the 96 hour exposure period indicating that the test substance was not stable under the conditions of the test. The results of the study were based on day 0 measured concentrations of 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L, as per U.S. E.P.A. guidance for instable substances in algal growth inhibition studies (U.S. Environmental Protection Agency. 1994. Pesticide Re-registration Rejection Rate Analysis – Ecological Effects. Test Material Decline in Algae and Diatom Tests). After 72 hours of exposure, inhibition of area under the growth curve in the 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L treatment groups was 36, 2, 44, 99, 100 and 100 %, respectively, relative to the negative control response. Inhibition of growth rate at the 0-72 hour interval in the 0.28, 0.74, 1.9, 4.5, 9.2 and 15 mg/L treatment groups was 10, 0, 13, 82, 98 and 100 %, respectively, relative to the negative control, and inhibition of yield after 72 hours of exposure was 36, 2, 44, 99, 100 and 100 %, respectively, relative to the negative control. Reductions in area under the growth curve, growth rate and yield were statistically significant (Jonckheere-Terpstra trend test; p < 0.05) in the 1.9, 4.5, 9.2 and 15 mg/L treatment groups when compared to the negative control response. The 72-hour NOEC was determined to be 0.74 mg/L, based on statistically significant reductions in area under the growth curve, growth rate and yield after 72 hours of exposure. The 72 -hour ErC10 value is 1.6 mg/L. The 72-hour EbC50, ErC50 and EyC50 values for growth rate and yield, were 2.1 mg/L, 3.0 mg/L and 2.1 mg/L, respectively.