Allyl 3-cyclohexylpropionate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-28 to 2013-02-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

US EPA, May 7, 2009

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples of test water collected from each treatment and control group
- Sampling method: Stock solutions were collected and analyzed for test substance at the beginning of the test to ensure test solutions were being delivered correctly to the test chambers. In addition, samples were collected from one test chamber of each treatment and control group at both six and four days prior to the start of the test after conditioning the diluter for approximately two and four days, respectively. Samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test and at 48 hours (± 1 hour) to measure concentrations of the test substance. The test solution samples were collected from mid-depth, placed in glass vials with 2 drops of 10 % phosphoric acid, and processed immediately for analysis. Finally, an additional set of samples was collected at termination (day 2) and stored refrigerated for possible future analysis.
- Sample storage conditions before analysis: refrigerator

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Flow-trough method with stock solutions. Individual stock solutions were prepared for each of the five concentrations tested. A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 200 mg/mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 13, 25, 50, and 100 mg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion and appeared clear and colorless. Two sets of stock solutions were prepared, one at the start of flows, and the second on day 4 prior to initiation of the test. Stock solutions were stored refrigerated in glass amber bottles and aliquots of each stock were placed in the syringe pumps every two days during the study. The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 15.5 μL/minute where they were mixed with dilution water delivered at a rate of 155 mL/minute to achieve the desired test concentrations. The negative control received dilution. water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all Allyl Cyclohexyl Propionate (CAS# 2705-87-5) treatment groups was 0.1 mL/L.
- Eluate: not appicable
- Differential loading: not applicable
- Controls: control and solvent control
- Chemical name of vehicle: dimethylformamide (DMF)
- Concentration of vehicle in test medium: 0.1 mL/L
- Evidence of undissolved material: All test solutions in the test chambers appeared clear and colorless with no precipitates observed at test initiation and termination. The negative and solvent control test solutions in the mixing chambers appeared clear and colorless at test initiation and termination. At test initiation and termination, the test solutions in the mixing chambers for the 1.3, 2.5, 5.0, 10 and 20 mg/L nominal test concentrations, appeared clear and colorless with an oily slick observed on the surface of the test solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: not reported
- Source: cultures maintained by Wildlife International, Easton, Maryland
- Age at study initiation: neonates (less than 24 hours old)

ACCLIMATION
- Acclimation period: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.5 to 20.4ºC. The pH of the water ranged from 8.1 to 8.6. Dissolved oxygen concentrations remained above 7.8 mg/L (more than 86% of saturation).
The six adult daphnids used to supply neonates for the test were held for at least 20 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 10 daphnids. Each group of daphnids then was transferred to the test compartment in an indiscriminately assigned test chamber to initiate the test. All transfers were made below the water surface using wide-bore pipettes.
- Acclimation conditions: same as test
- Type and amount of food: The adults were fed prior to test initiation, but neonates were not fed during the test.
- Feeding frequency: Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media and trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata and a supplemental vitamin stock.
- Health during acclimation (any mortality observed): no observations on health problems

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Six adults used to supply neonates for the test were held for at least 20 days before test start and produced at least three young per adult per day over the 7-day period prior to the test. Application of test item 8 days before introduction of neonates.

Hardness:

142 mg/L as CaCO3

Test temperature:

0 hours: 19.8-19.9 °C
48 hours: 19.9-20.0 °C

pH:

0 hours: 8.1
24 hours: 8.1
48 hours: 8.0-8.1

Dissolved oxygen:

0 hours: 8.0-8.5 mg/L (a dissolved oxygen concentration of 5.4 mg/L represents 60 % saturation at 20 °C in freshwater)
24 hours: 7.0-8.1
48 hours: 7.2-8.1

Salinity:

not applicable

Nominal and measured concentrations:

pretest: nominal 0.075, 0.25, 0.83, 2.8 and 9.2 mg/L; definitive test: nominal 1.3, 2.5, 5.0, 10 and 20 mg/L; definitive test: measured 0.86, 1.5, 2.4, 5.4 and 11 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers were aquaria with one test compartment within
- Type: open
- Material, size, headspace, fill volume: 25-L stainless steel aquaria with 22 L of test water and 3 L of headspace, one test compartment approximately 6.5 cm in diameter and 12 cm in height, with nylon screen attached to two holes on the sides of the beaker and within the test water volume
- Aeration: not reported but oxygen levels above the validity limit throughout the study period.
- Type of flow-through (e.g. peristaltic or proportional diluter): Syringe pumps (Harvard Apparatus, Massachusetts) were used to deliver test substance stock solutions or solvent to impartially assigned mixing chambers where the stocks or solvent were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters.
- Renewal rate of test solution: approximately 5 volume additions of test water in each test chamber per day.
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2
- Biomass loading rate: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- Total organic carbon: 1.376 mg C/L
- Particulate matter: not applicable
- Metals: 29 metals and measured concentrations (mg/L) reported. All toxic metals below limit of detection.
- Pesticides: 54 pesticides and measured concentrations (µg/L) reported. All below limit of detection.
- Chlorine: not applicable
- Alkalinity: 180 mg/L as CaCO3
- Ca/Mg ratio: not reported; based on analytical results for dilution water the Ca/Mg ratio is ca. 1:0.6
- Conductivity: 316 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen and pH were measured in one replicate test chamber of each treatment and control group at the beginning of the test, at approximately 24 hours, and at the end of the test, with measurements typically alternating between replicates in each group at each measurement interval. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion Model 525Aplus meter. When 100 % immobility occurred in a test chamber, measurements of temperature, dissolved oxygen and pH were taken in that test chamber and then discontinued. Hardness, alkalinity, specific conductance and total organic carbon (TOC) in the dilution water at the beginning of the test were measured. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using an Acorn Series Model CON6 conductivity-temperature meter. TOC was measured using a Shimadzu Model TOC-VCSH total organic carbon analyzer based on procedures in Standard Methods for the Examination of Water and Wastewater.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006C light meter.
- Light intensity: at test initiation387 lux at the surface of the water of one representative test chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: performed
- Test concentrations: 0.075, 0.25, 0.83, 2.8 and 9.2 mg/L
- Results used to determine the conditions for the definitive study: 40 % immobility and signs of toxicity observed among daphnia in the 9.2 mg/L treatment group after 48 hours, and signs of toxicity observed in the 0.83 and 2.8 mg/L test concentrations.

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 3.2-4.5 mg/L

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.86 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks:

lethargy

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

7.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 5.4-11 mg/L

Details on results:

- Behavioural abnormalities: lethargic daphnids were observed in the 1.5 and 2.4 mg/L treatment groups at the 48 hour observation interval, and at all intervals for the 5.4 mg/L treatment group.
- Observations on body length and weight: not applicable
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: 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 immobility data were analyzed using the computer program of C. E. Stephan (1977). The program was designed to calculate the EC50 value and the 95 % confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation. In this study, probit analysis was used to calculate the 48-hour EC50 value and the 95 % confidence interval, and binomial analysis with non-linear interpolation was used to calculate the 24-hour EC50 value and the 95 % confidence interval. The no-immobility concentration and NOEC, were determined by visual interpretation of the immobility and observation data.

Table 1 Cumulative Immobility and Observations

Hours after introduction			5 hours					24 hours					48 hours
Mean Measured Concen-tration (mg/L)	Repl.	No. of Daphnia per replicate	immobile	normal	lethargic	trapped at water surface and normal after gentle submersion	trapped at water surface and lethargic after gentle submersion	immobile	normal	lethargic	trapped at water surface and normal after gentle submersion	trapped at water surface and lethargic after gentle submersion	immobile	normal	lethargic	trapped at water surface and normal after gentle submersion	trapped at water surface and lethargic after gentle submersion
Negaitive control	A	10	0	10	0	0	0	0	10	0	0	0	0	10	0	0	0
	B	10	0	10	0	0	0	0	9	0	1	0	0	10	0	0	0
Solvent control	A	10	0	10	0	0	0	0	10	0	0	0	0	10	0	0	0
	B	10	0	10	0	0	0	0	9	0	1	0	0	10	0	0	0
0.86	A	10	0	6	0	4	0	0	7	0	3	0	0	10	0	0	0
	B	10	0	4	0	6	0	0	8	0	2	0	0	10	0	0	0
1.5	A	10	0	8	0	2	0	0	7	0	3	0	0	4	0	4	2
	B	10	0	6	0	4	0	0	4	0	6	0	0	0	2	5	3
2.4	A	10	0	4	0	6	0	0	2	0	8	0	0	0	3	0	7
	B	10	0	6	0	4	0	0	1	0	9	0	2	0	1	0	7
5.4	A	10	0	0	0	0	10	0	0	7	0	3	9	0	0	0	1
	B	10	0	0	2	0	8	0	0	10	0	0	8	0	0	0	2
11	A	10	10	0	0	0	0	10	0	0	0	0	10	0	0	0	0
	B	10	10	0	0	0	0	10	0	0	0	0	10	0	0	0	0

 

Validity criteria fulfilled:

yes

Conclusions:

The cladoceran, Daphnia magna, was exposed for 48 hours under flow-through conditions to five mean measured concentrations of Allyl Cyclohexyl Propionate (CAS# 2705-87-5) ranging from 0.86 to 11 mg/L. The 48-hour EC50 value was 3.8 mg/L, with a 95 % confidence interval of 3.2 to
4.5 mg/L. The no-immobility concentration was 1.5 mg/L and the NOEC was 0.86 mg/L.

Executive summary:

In a valid, conclusive and reliable study, the acute toxicity of the test item Allyl Cyclohexyl Propionate (CAS# 2705-87-5) on Daphnia magna was tested according to OECD TG 202, OPPTS 850.1010 and ASTM Standard E729-96 under GLP. Nominal concentrations selected for use in this study were 1.3, 2.5, 5.0, 10 and 20 mg/L. Measured concentrations of the samples ranged from 45.9 to 66.7 % of nominal. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 0.86, 1.5, 2.4, 5.4 and 11 mg/L, representing 66, 60, 48, 54 and 55% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations. A single floating daphnid was observed in both the negative and solvent control groups, but both appeared normal after gentle submersion. All other daphnia in the negative and solvent control groups appeared normal throughout the test with no immobility or signs of toxicity. Percent immobility at test termination in the 0.86, 1.5, 2.4, 5.4, and 11 mg/L mean measured concentration treatment groups was 0, 0, 10, 85, 100 %, respectively. Floating daphnids were also observed in the 0.86 mg/L treatment group, but all appeared normal after gentle submersion. No immobility or overt signs of toxicity were observed at the 0.86 mg/L test concentration. In addition to floating, lethargic daphnids were also observed in the 1.5 and 2.4 mg/L treatment groups at the 48 hour observation interval, and at all intervals for the 5.4 mg/L treatment group. The no-immobility concentration was 1.5 mg/L and the NOEC was 0.86 mg/L. EC50 values at 48 hours were determined to be 3.8 mg/L, with a 95 % confidence interval of 3.2 to 4.5 mg/L.

Description of key information

Toxic to aquatic invertebrates (EC50(48h) = 3.8 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

3.8 mg/L

Additional information

In a valid, conclusive and reliable study, the acute toxicity of the test item Allyl Cyclohexyl Propionate (CAS# 2705-87-5) on Daphnia magna was tested according to OECD TG 202, OPPTS 850.1010 and ASTM Standard E729-96 under GLP. Nominal concentrations selected for use in this study were 1.3, 2.5, 5.0, 10 and 20 mg/L. The results of the study were based on the mean measured concentrations of 0.86, 1.5, 2.4, 5.4 and 11 mg/L, representing 66, 60, 48, 54 and 55% of nominal concentrations, respectively. A single floating daphnid was observed in both the negative and solvent control groups, but both appeared normal after gentle submersion. All other daphnia in the negative and solvent control groups appeared normal throughout the test with no immobility or signs of toxicity. Percent immobility at test termination in the 0.86, 1.5, 2.4, 5.4, and 11 mg/L mean measured concentration treatment groups was 0, 0, 10, 85, 100 %, respectively. Floating daphnids were also observed in the 0.86 mg/L treatment group, but all appeared normal after gentle submersion. No immobility or overt signs of toxicity were observed at the 0.86 mg/L test concentration. In addition to floating, lethargic daphnids were also observed in the 1.5 and 2.4 mg/L treatment groups at the 48 hour observation interval, and at all intervals for the 5.4 mg/L treatment group. The no-immobility concentration was 1.5 mg/L and the NOEC was 0.86 mg/L. EC50 values at 48 hours were determined to be 3.8 mg/L, with a 95 % confidence interval of 3.2 to 4.5 mg/L.