Allyl 3-cyclohexylpropionate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In-life phase of the definitive toxicity test was conducted from January 28 to February 1, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians

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: The samples were collected from mid-depth, two days prior to the start of the test after conditioning the diluter for approximately two days, at the beginning of the test and at 48 and 96 hours (± 1 h). An additional set of samples was taken at 96 hours and was stored for possible future analysis.
- Sample storage conditions before analysis: Samples were placed in glass vials and processed immediately for analysis. Additional samples for possible future analysis were stored refrigerated

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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 15 mg/mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 7.5, 3.8, 1.9 and 0.94 mg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion and appeared clear and colourless. Stock solutions were stored refrigerated in capped glass amber bottles and aliquots of each stock were placed in the syringe pumps every two days during the test. The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 20 μL/min where they were mixed with dilution water delivered at a rate of 200 mL/min 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. Delivery of the test substance to the test chambers was initiated four days prior to the introduction of the test animals to the test water in order to achieve equilibrium of the test substance in the test chambers.
- Chemical name of vehicle: Dimethylformamide (DMF, CAS 68-12-2)
- Concentration of vehicle in test medium: 0.1 mL/L in the solvent control and all treatment groups

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow
- Source: Chesapeake Cultures, Inc. Hayes, Virginia 23072, U.S.A.
- Age at study initiation: Juveniles
- Length at study initiation: The length of the longest fish measured was no more than twice the length of the shortest.

ACCLIMATION
- Acclimation period: The fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test.
- Acclimation conditions: Same as test; during the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 18.5 to 24.0 °C, measured with a liquid-in-glass thermometer. The pH of the water ranged from 8.2 to 8.6, measured with a Thermo Orion Benchtop 4 Star Plus pH/ISE meter. Dissolved oxygen concentrations were ≥ 7.9 mg/L (≥ 91 % of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter.
- Type and amount of food: Brine shrimp nauplii (Artemia sp.) supplied by INVE Aquaculture, Salt Lake City, Utah, U.S.A.
- Feeding frequency: Daily during the holding period, except during periods of fasting prior to testing, the fish were fed. The fish were not fed for at least two days prior to the test or during the test.
- Health during acclimation: During the 2-week period prior to the test the fish showed no signs of disease or stress.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

132 mg/L as CaCO3 in dilution water at day 0 (measurement by titration), mean 141 mg/L as CaCO3 (range 136 to 144) were measured in dilution water during the 4-week period immediately preceding the test

Test temperature:

Water temperatures were within the 22 ± 1 ºC range established for the test.
Temperature was measured in each test chamber at the beginning and end of the test using a liquid-in-glass thermometer. Temperature also was monitored continuously in a negative control test chamber using a Fulscope ER/C Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer.
When 100 % mortality occurred in a test chamber, measurements were discontinued.

pH:

Measurements of pH ranged from 8.1 to 8.2 during the test.
The pH was measured in one replicate test chamber of each treatment and control group at the beginning of the test, at approximately 24-hour intervals during the test and at the end of the test, with measurements typically alternating between replicates in each group at each measurement interval. Measurements of pH were made using a Thermo Orion Model 525Aplus meter.
When 100 % mortality occurred in a test chamber, measurements were discontinued.

Dissolved oxygen:

Dissolved oxygen concentrations remained ≥7.5 mg/L (≥87 % of saturation) throughout the test.
Dissolved oxygen was measured in one replicate test chamber of each treatment and control group at the beginning of the test, at approximately 24-hour intervals during the test 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.
When 100 % mortality occurred in a test chamber, measurements were discontinued.

Nominal and measured concentrations:

Nominal: 0 (Negative Control), 0 (Solvent Control), 0.094, 0.19, 0.38, 0.75 and 1.5 mg/L
Arithmetic Mean Measured: < LOQ, < LOQ, 0.058, 0.11, 0.26, 0.42 and 0.99 mg/L, representing 62, 58, 68, 56 and 66 % of nominal concentrations, respectively
The relatively high volatility of the test substance resulted in analytical arithmetic mean recoveries that were below 70 % in all test concentrations (range in the individual replicates 51 to 71 % of nominal). Therefore the results of the study were based on arithmetic mean measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: The test chambers were indiscriminately placed in a temperature-controlled water bath to maintain the target water temperature throughout the test period.
- Type: Closed (aquaria fitted with Plexiglas covers)
- Material, size, headspace, fill volume: Stainless steel, 25 L, 10 L, 15 L
- Aeration: No
- Type of flow-through: Proportional diluter
- Renewal rate of test solution: Approximately 10 volume additions of test water in each test chamber per day
- No. of organisms per vessel: 10 (impartially assigned to exposure chambers at test initiation)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

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 mg C/L measured once in dilution water during the 4-week period immediately preceding the test
- Metals [mg/L]: below detection limits in dilution water (detailed analytical results given in the original study report)
- Pesticides [µg/L]: below detection limits in dilution water (detailed analytical results given in the original study report)
- Alkalinity: 178 mg/L as CaCO3 in dilution water at day 0 (measurement by titration), mean 178 mg/L as CaCO3 (range 176 to 180) were measured in dilution water during the 4-week period immediately preceding the test
- Ca/Mg ratio: 34.9/13.2 = 2.6/1 (dilution water)
- Conductivity: 400 μS/cm in dilution water at day 0 (measured using an Acorn Series Model CON6 conductivity-temperature meter), mean 342 μS/cm (range 28 to 371) were measured in dilution water during the 4-week period immediately preceding the test

OTHER TEST CONDITIONS
- Photoperiod: 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: Light intensity at test initiation was 505 lux at the surface of the water of one representative test chamber.

EFFECT PARAMETERS MEASURED
Observations of mortality and other signs of toxicity or abnormal behaviour were made approximately 3, 24, 48, 72 and 96 h after test initiation.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.97 to 2.02 (nominal)
- Range finding study: Yes
- Test concentrations (non-GLP range finding study): 0.075, 0.25, 0.83, 2.8 and 9.2 mg/L.
- Results used to determine the conditions for the definitive study: 80 % mortality and signs of toxicity observed among fish in the 0.25 mg/L treatment group and 100 % mortality in the 0.83, 2.8, and 9.2 mg/L test concentrations

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95 % Confidence Interval 0.058 – 0.26 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.058 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

EC0

Effect conc.:

0.058 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

0.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Signs of toxicity observed at the 3-hour observation interval among fish in the 0.99 mg/L treatment group included lethargy and fish lying on the bottom of the test chamber.
- Observations on body length and weight: The average total length of 10 negative control fish measured at the end of the test was 2.9 cm, with a range of 2.7 to 3.1 cm. The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.16 g, with a range of 0.12 to 0.21 g.
- Other biological observations: All fish in the 0.058 mg/L treatment group appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. Percent mortality in the 0.11, 0.26, 0.42, and 0.99 mg/L treatment groups at test termination was 35, 100, 100, and 100 %, respectively. No signs of toxicity were observed in the 0.11, 0.26, and 0.42 mg/L treatment groups prior to the mortality and all surviving fish were normal in appearance at test termination.
- Mortality of control: No.
- Other adverse effects in control: No. All fish in the negative and solvent control groups appeared normal throughout the test, with no mortalities or overt signs of toxicity observed.
- Any observations that might cause a difference between measured and nominal values: During the course of the test, the appearance of the test solutions was observed in the test chambers, as well as in the diluter mixing chambers, where the stock and dilution water were combined prior to delivery to the test chambers. The negative and solvent control solutions and the 0.094, 0.19, 0.38 and 0.75 test solutions appeared clear and colourless, with no evidence of precipitation or oily slicks observed during the test, in the mixing chambers or in the test chambers themselves. Test solutions in the mixing chambers for the 1.5 mg/L treatment group appeared clear and colourless, however, oily slicks were observed in the diluter mixing chambers at both test initiation and termination. Test solutions in the 1.5 mg/L test chambers appeared clear and colourless with no precipitates or oily slicks observed at test initiation and termination.
- Effect concentrations exceeding solubility of substance in test medium: No

Reported statistics and error estimates:

The LC50 (Lethal Concentration 50 %) value was estimated using non-linear interpolation between 0.11 and 0.26 mg/L. The 95 % confidence limit was determined by binomial probability.
The mortality data were analyzed using the computer program of C. E. Stephan (1978). The program was designed to calculate the LC50 value and the 95 % confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation (Finney 1971, Thompson 1947, Stephan 1977). In this study, nonlinear interpolation was used to calculate the 24, 48, 72 and 96-hour LC50 values and binomial probability was used to calculate the 95 % confidence intervals. Due to the method used to calculate the 96-hour LC50 value, the slope of the dose response curve could not be calculated. The no-mortality concentration (LC0) and the no-observed-effect concentration (EC0) were determined by visual interpretation of the mortality and observation data.
- Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London.
- Thompson, W.R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145.
- Stephan, C.E. 1977. “Methods for Calculating an LC50”, Aquatic Toxicology and Hazard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84.
- Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota, U.S.A. Personal communication.

Sublethal observations / clinical signs:

Table 1: Toxicity to fish: mortality data

Treatment [mg test item/L], arithmetic mean measured		No. of fish at start of study	Observation period										Cumulative mean % mortality
			3 h		24 h		48 h		72 h		96 h
			No Dead	% mortality	No Dead	% mortality	No Dead	% mortality	No Dead	% mortality	No Dead	% mortality
Control (dilution water only)	Replicate #1	10	0	0	0	0	0	0	0	0	0	0	0
	Replicate #2	10	0	0	0	0	0	0	0	0	0	0
Solvent control (0.1 mL DMF/L)	Replicate #1	10	0	0	0	0	0	0	0	0	0	0	0
	Replicate #2	10	0	0	0	0	0	0	0	0	0	0
0.058	Replicate #1	10	0	0	0	0	0	0	0	0	0	0	0
	Replicate #2	10	0	0	0	0	0	0	0	0	0	0
0.11	Replicate #1	10	0	0	2	20	3	30	3	30	3	30	35
	Replicate #2	10	0	0	3	30	4	40	4	40	4	40
0.26	Replicate #1	10	0	0	10	100							100
	Replicate #2	10	0	0	10	100
0.42	Replicate #1	10	0	0	10	100							100
	Replicate #2	10	0	0	10	100
0.99	Replicate #1	10	0	0	10	100							100
	Replicate #2	10	0	0	10	100

 

 

Validity criteria fulfilled:

yes

Remarks:

Mortality of the fish in the controls did not exceed 10% by the end of the test, and the dissolved oxygen concentration remained > 60 % of the air-saturation value throughout the test.

Conclusions:

Very toxic to fish, 96-h LC50 = 0.13 mg/L.

Executive summary:

The short-term toxicity of Allyl Cyclohexyl Propionate (CAS 2705-87-5) to fish was investigated in a GLP-compliant study using fathead minnow (Pimephales promelas) according to the OECD TG 203 (1992), OPPTS 850.1075 (1996) and ASTM E729-96 (1996) protocols. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions (Klimisch 1). Following a preliminary range-finding test the fish were exposed, in two groups of ten, to nominal test item concentrations of 0, 0.094, 0.19, 0.38, 0.75 and 1.5 mg/L for a period of 96 h at a temperature of approximately 22 °C under flow-through test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 h after the start of exposure and then daily throughout the test until termination after 96 h. The analysis of the test item at 0, 48 and 96 h showed measured concentrations below 70 % in all test concentrations. The results of the study are therefore based on arithmetic mean measured concentrations of < LOQ, 0.058, 0.11, 0.26, 0.42 and 0.99 mg/L. All fish in the control, the solvent control (DMF at 0.1 mL/L) and in the 0.058 mg/L treatment group appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. Percent mortality in the 0.11, 0.26, 0.42 and 0.99 mg/L treatment groups at test termination was 35, 100, 100, and 100 %, respectively. No signs of toxicity were observed in the 0.11, 0.26 and 0.42 mg/L treatment groups prior to the mortality and all surviving fish were normal in appearance at test termination, while at 0.99 mg/L signs of toxicity were recorded after 3 h and 100 % mortality occurred until 24 h. The LC50 (Lethal Concentration 50 %) value was estimated using non-linear interpolation between 0.11 and 0.26 mg/L. The 95 % confidence limit was determined by binomial probability. Accordingly the acute toxicity of the test item to freshwater fish gave 96-h LC50 of 0.13 mg/L (95 % Confidence Interval 0.058 - 0.26 mg/L) with LC0 and EC0 of 0.058 mg/L and LC100 of 0.26 mg/L. In conclusion the test item was found very toxic (i.e. LC50 < 1 mg/L) to fish and indication for acute aquatic hazard classification is given by the results of this study.

Description of key information

Very toxic to fish

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.13 mg/L

Additional information

The short-term toxicity of Allyl Cyclohexyl Propionate (CAS 2705-87-5) to fish was investigated in a GLP-compliant study using fathead minnow (Pimephales promelas) according to the OECD TG 203 (1992), OPPTS 850.1075 (1996) and ASTM E729-96 (1996) protocols. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions (Klimisch 1). Following a preliminary range-finding test the fish were exposed, in two groups of ten, to nominal test item concentrations of 0, 0.094, 0.19, 0.38, 0.75 and 1.5 mg/L for a period of 96 h at a temperature of approximately 22 °C under flow-through test conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 h after the start of exposure and then daily throughout the test until termination after 96 h. The analysis of the test item at 0, 48 and 96 h showed measured concentrations below 70 % in all test concentrations. The results of the study are therefore based on arithmetic mean measured concentrations of < LOQ, 0.058, 0.11, 0.26, 0.42 and 0.99 mg/L. All fish in the control, the solvent control (DMF at 0.1 mL/L) and in the 0.058 mg/L treatment group appeared normal throughout the test, with no mortalities or overt signs of toxicity observed. Percent mortality in the 0.11, 0.26, 0.42 and 0.99 mg/L treatment groups at test termination was 35, 100, 100, and 100 %, respectively. No signs of toxicity were observed in the 0.11, 0.26 and 0.42 mg/L treatment groups prior to the mortality and all surviving fish were normal in appearance at test termination, while at 0.99 mg/L signs of toxicity were recorded after 3 h and 100 % mortality occurred until 24 h. The LC50 (Lethal Concentration 50 %) value was estimated using non-linear interpolation between 0.11 and 0.26 mg/L. The 95 % confidence limit was determined by binomial probability. Accordingly the acute toxicity of the test item to freshwater fish gave 96-h LC50 of 0.13 mg/L (95 % Confidence Interval 0.058 - 0.26 mg/L) with LC0 and EC0 of 0.058 mg/L and LC100 of 0.26 mg/L. In conclusion the test item was found very toxic (i.e. LC50 < 1 mg/L) to fish and indication for acute aquatic hazard classification is given by the results of this study.