Allyl 3-cyclohexylpropionate

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-3-14 to 2019-4-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples of test water collected from each treatment and control group at the beginning of the test, at weekly intervals during the test and at test termination
- Sampling method: Water samples and backup samples were collected from alternating replicate test chambers of each treatment and control group on Days 0, 7, 12, 21, 28 and 33 (test termination) to determine concentrations of the test substance in the test chambers, while the backup samples were stored refrigerated for possible analysis if needed. Additional water samples were also collected from the mixing chambers and delivery line, prior to deliver to the test chambers, on Days 0, 12 and 33 of the test. All water samples were collected at mid-depth in the test chambers or directly from the mixing chambers and placed in glass French square bottles or glass scintillation vials.
- Sample storage conditions before analysis: stored in a refrigerator.

Vehicle:

yes

Remarks:

0.1 mL DMF/L

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Stock solutions were prepared six times during the test. At each preparation, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 3.0 mg/mL. Proportional dilutions of the primary stock were made in DMF to prepare additional stock solutions at nominal concentrations of 0.037, 0.11, 0.33 and 1.0 mg/mL.
- Eluate: freshwater
- Controls: dilution water without test item (negative control) and dilution water with HPLC grade dimethylformamide (solvent control)
- Chemical name of vehicle: N,N-Dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): The solvent control was prepared by injecting HPLC-grade DMF into the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all treatment groups was 0.1 mL/L.
- Evidence of undissolved material: No
- Other relevant information: A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (HPLC grade dimethylformamide) control, and a negative (dilution water) control.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow,
- Strain: Pimephales promelas
- Source: Eurofins EAG Agroscience, LLC, Easton, Maryland
- Age at study initiation: < 24 hours Embryos

POST-HATCH FEEDING
- Food type: live brine shrimp nauplii (Artemia sp.)
- Frequency: three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Embryos collected for use in the test were from 17 individual spawns
- Subsequent handling of eggs: examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development (gastrula to neurula)

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

33 d

Hardness:

124-148 mg/L as CaCO3

Test temperature:

22.96-25.84 °C

pH:

8.0 (7.9-8.0)

Dissolved oxygen:

>= 89% (7.3 mg/L)

Salinity:

174-182 mg/L as CaCO3

Conductivity:

318-356 μS/cm

Nominal and measured concentrations:

Nominal concentration: 3.7, 11, 33, 100 and 300 μg/L; measured concentrations:1.9, 6.3, 15, 59 and 180 μg/L (mean)

Details on test conditions:

TEST SYSTEM
- Embryo cups: glass cylinders approximately 50 mm in diameter with 425 μm nylon screen mesh attached to the bottom with silicone sealant
- Test vessel: glass aquaria, 7 L, filled with 6 L of test solution
- Type of flow-through: proportional diluter
- Renewal rate of test solution: stock solution at a rate of 34.0 µL/min, dilution water at a rate of 340 mL/minute
- No. of fertilized eggs/embryos per vessel: 20 embryos
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4
- Biomass loading rate: 0.17 g of fish per liter

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: obtained from a well approximately 40 meters deep located on the Eurofins-Easton site. 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

- Metals: Magnesium, 23.2 mg/L; Potassium, 6.64 mg/L; Sodium, 17.1 mg/L; Others < LOQs
- Pesticides: < LOQs
- Chlorine: 3.9 mg/L
- Alkalinity: 174 mg/L as CaCO3
- Intervals of water quality measurement: 4-week period

Reference substance (positive control):

no

Duration:

5 d

Dose descriptor:

EC10

Remarks:

Hatching Phase

Effect conc.:

> 180 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

28 d

Dose descriptor:

NOEC

Remarks:

Post-Hatch Phase

Effect conc.:

59 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: survial and growth

Duration:

28 d

Dose descriptor:

LOEC

Remarks:

Post-Hatch Phase

Effect conc.:

180 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival and growth

Key result

Duration:

28 d

Dose descriptor:

EC10

Remarks:

Post-Hatch Phase

Effect conc.:

64 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Post-Hatch Larval Survival

Remarks:

Post-Hatch Larval Survival

Duration:

28 d

Dose descriptor:

EC10

Remarks:

Post-Hatch Phase

Effect conc.:

66 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Overall Larval Survival

Duration:

28 d

Dose descriptor:

EC10

Remarks:

Post-Hatch Phase

Effect conc.:

105 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

28 d

Dose descriptor:

EC10

Remarks:

Post-Hatch Phase

Effect conc.:

71 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

wet weight

Duration:

28 d

Dose descriptor:

EC10

Remarks:

Post-Hatch Phase

Effect conc.:

85 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

dry weight

Details on results:

See following tables (Any other information on results incl. tables)

Reported statistics and error estimates:

Controls: t-test, α = 0.05
Treatment groups: Hatching success, post hatch survival and overall survival data were considered to be discrete-variable data, while mean time to hatch and growth data were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (α = 0.05) from the pooled control. All continuous-variable data (mean time to hatch and growth) were evaluated for normality using Shapiro-Wilk’s test, and for homogeneity of variance using Levene’s test (α = 0.01). Since the mean time to hatch and wet and dry weight data passed the assumptions of normality and homogeneity of variance, analysis of variance (ANOVA) was used to determine whether or not statistically significant differences existed among the experimental groups (α = 0.05). Those treatments that were significantly different from the control means were identified using Dunnett’s test (α = 0.05) (5, 6). When the length data failed the assumption of homogeneity of variance and log transformation did not correct the condition, a non-parametric trend test (Jonckheere-Terpstra step down trend test, α = 0.05) was used to determine the t treatments that were significantly different from the pooled control.

Percent Inhibition from the Pooled Control:

Mean Measured concentration [µg/L]	Percent Hatching Success	% Inhibition From Pooled Control	Mean Time to Hatch [day]	Percent Post-Hatch Survival	% Inhition From Pooled Control	Percent Overall Survival	% Inhibition From Pooled Control
Negative Control	99	‒	4.2	91	‒	90	‒
Solvent Control	99	‒	4.3	91	‒	90	‒
Pooled Control	99	‒	4.2	91	‒	90	‒
1.9	99	0.0	4.3	90	1.1	89	1.1
6.3	100	-1.0	4.3	90	1.1	90	0.0
15	100	-1.0	4.4	89	2.2	89	1.1
59	100	-1.0	4.2	84	7.7	84	6.7
180	100	-1.0	4.2	15	84	15	83
Note: Stimulation or a greater response in the test substance treatment than the control is reported as negative % inhibition.

Mean Measured concentration [µg/L]	Mean Total Length [mm]	% Inhibition From Pooled Control	Mean Wet Weight [mg]	% Inhibition From Pooled Control	Mean Dry Weight [mg]	% Inhibition From Pooled Control
Negative Control	24.2	‒	109.3	‒	23.4	‒
Solvent Control	23.8	‒	105.2	‒	21.2	‒
Pooled Control	24	‒	107.3	‒	22.3	‒
1.9	23.8	0.8	103.1	3.9	21.7	2.7
6.3	23.8	0.8	110.4	-2.9	21.9	1.8
15	23.9	0.4	104.8	2.3	22.3	0.0
59	23.4	2.5	100.2	6.6	21.5	3.6
180	17.5	27.0	50.9	33	9.89	56
Note: Stimulation or a greater response in the test substance treatment than the control is reported as negative % inhibition.

Validity criteria fulfilled:

yes

Conclusions:

Based on the most sensitive endpoints, survival and growth, the LC10, NOEC and LOEC were determined to 64 μg/L, 59 μg/L and180 μg/L, respectively.

Executive summary:

The early life-stage toxicity of test item to fish was conducted with Fathead minnows (Pimephales promelas) under flow-through conditions over a period of 33 days in accordance with OECD 210, EPA OCSPP 850.1400 and GLP. Based on the results from an exploratory range-finding toxicity data, five nominal concentrations, 3.7, 11, 33, 100, and 300 µg/Lwith a spacing factor of 3 were tested. A negative (dilution water) control and a solvent control (0.1 mL/L HPLC-grade dimethylformamide) were performed in parallel. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos < 24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. The effects of test item on time to hatch, hatching success, growth, and survival were observed. The concentrations maintained from one test chamber of each treatment and control group were analyzed via GC-MS measurement. The mean measured concentrations were 1.9, 6.3, 15, 59 and 180 μg/L, representing 51, 57, 45, 59 and 60% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations. Based on the most sensitive endpoints, survival and growth, the NOEC and LOEC were determined to 59 μg/L and 180 μg/L, respectively. A value of EC10 based on the effect of post-hatch larval survival was used as the key value for chemical risk assessment.

Description of key information

28-day EC10 = 64 µg/L (geom. mean measured)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

64 µg/L

Additional information

An experimental study on the early life-stage toxicity of test item to fish is available, which was conducted with Fathead minnows (Pimephales promelas) under flow-through conditions over a period of 33 days in accordance with OECD 210, EPA OCSPP 850.1400 and GLP. Based on the results from an exploratory range-finding toxicity data, five nominal concentrations, 3.7, 11, 33, 100, and 300 µg/Lwith a spacing factor of 3 were tested. A negative (dilution water) control and a solvent control (0.1 mL/L HPLC-grade dimethylformamide) were performed in parallel. Four replicate test chambers were maintained in each treatment and control group, with one incubation cup in each test chamber. Each incubation cup contained 20 embryos, resulting in a total of 80 embryos per treatment. At test initiation, embryos < 24 hours old were impartially distributed to incubation cups and exposed to test solution in the test chambers. After a 5-day embryo hatching period, the larvae were released into the test chambers, where exposure continued during a 28-day post-hatch juvenile growth period. The effects of test item on time to hatch, hatching success, growth, and survival were observed. The concentrations maintained from one test chamber of each treatment and control group were analyzed via GC-MS measurement. The mean measured concentrations were 1.9, 6.3, 15, 59 and 180 μg/L, representing 51, 57, 45, 59 and 60% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations. Based on the most sensitive endpoints, survival and growth, the NOEC and LOEC were determined to 59 μg/L and 180 μg/L, respectively. A value of EC10 = 64 µg/L (geom. mean measured) based on the effect of post-hatch larval survival was used as the key value for chemical risk assessment.