Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl propionate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a guideline study

Justification for type of information:

The acute Daphnia toxicity of Isobornyl propionate is based on read-across from Isobornyl acetate. The documentation is presented in the Aquatic Endpoint summary. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

8.7 mg/L

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

Effect value from the source chemical was recalculated based on Log Kow and molecular weight for the target chemical.

Validity criteria fulfilled:

yes

Remarks:

The read across rationale is documented according to Annex XI criteria.

Conclusions:

Isobornyl propionate has an EC50 for Daphnia of 8.7 mg/l, which value is converted from isobornyl acetate using MW and Log Kow.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jul 2011 to 29 Jul 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Isobornyl propionate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

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.

Version / remarks:

2007

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were collected from one test chamber of each treatment and control group two days prior to the start of the test after conditioning the diluter for approximately four days. Samples of tock solutions were also taken at this time to confirm stock concentrations. Water 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. Two sets of samples were collected at 48 hours. One set was processed immediately for analysis, and the second set was stored refrigerated for possible future analysis. The samples were collected from mid-depth, acidified with two drops of phosphoric acid, placed in glass scintillation vials, and processed immediately for analysis.

Vehicle:

yes

Remarks:

0.1 mL/L dimethylformamide

Details on test 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 220 mg/mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 14, 28, 55 and 110 mg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion, and appeared clear and colorless. Stock solutions were stored refrigerated in glass amber bottles or glass graduated cylinders, and aliquots of each stock were placed in the syringe pumps every other day during the study. The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 15.50 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 test item treatment groups was 0.1 mL/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnids
- Age: Daphnid neonates used in the test were less than 24 hours old
- Source: Wildlife International, Easton, Maryland
- Feeding: 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. The adults were fed prior to test
initiation, but neonates were not fed during the test.

CULTURING CONDITIONS
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 20.6 to 22.1ºC, measured with a hand-held, liquid-in-glass thermometer. The pH of the water ranged from 8.3 to 8.7, measured with a Thermo Orion Model 525 Aplus pH Meter. Dissolved oxygen ranged from 7.9 to 8.9 mg/L (≥88% of saturation), measured with a Thermo Orion Model 850Aplus Dissolved Oxygen Meter or a Thermo Orion Benchtop 3 Star Plus Dissolved Oxygen Meter.

BREEDING
The four adult daphnids used to supply neonates for the test were held for at least 12 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.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

152 mg/L as CaCO3 (at study initiation)

Test temperature:

20.0 - 21.0 °C

pH:

8.0 - 8.1

Dissolved oxygen:

6.0 - 7.9 mg O2/L (A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20°C in freshwater)

Conductivity:

385 µS/cm (at study initiation)

Nominal and measured concentrations:

- Nominal test concentrations: 0 (control), 0 (solvent control), 1.4, 2.8, 5.5, 11 and 22 mg/L
- Mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers were 25-L stainless steel aquaria filled with approximately 22 L of test water. The depth of the test water in a representative chamber was 30.0 cm. Each test chamber contained one test compartment constructed from a glass beaker approximately 6.5 cm in diameter and 12 cm in height, with nylon screen attached to two holes on the sides of the beaker. The depth of the test water in a representative compartment was 9.1 cm.
- Type of flow-through: The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance, a solvent control and a negative control (dilution water) to test chambers. Syringe pumps (Harvard Apparatus, South Natick, 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 and was adjusted to provide approximately 5 volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber was split to deliver test water to two replicate test chambers. The syringe pumps used to deliver stock solutions or solvent to the mixing chambers, and the rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test. The proportion of the test water that was split into each replicate test chamber was checked prior to the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the two replicates. Delivery of test solutions to the test chambers was initiated six days prior to test initiation in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.
- 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

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, Ltd. 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. The well water is characterized as moderately-hard water.
- Total organic carbon: ~2 mg C/L
- Metals: Please see 'Any other information on materials and methods incl. tables' for an overview of the metals analysed in the test medium.
- Pesticides: Please see 'Any other information on materials and methods incl. tables' for an overview of the pesticides and organics analysed in the test medium.
- Chlorine: 3.5 mg/L
- Alkalinity: 176 mg/L as CaCO3 (at study initiation)
- Ca/mg ratio: 2.7
- Culture medium different from test medium: same as test

TEST MEDIUM MEASUREMENTS
- Temperature: The target test temperature during the study was 20 ± 1°C. 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 one negative control test chamber using a Fulscope ER/C Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer.
- Dissolved oxygen and pH: 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.
- Hardness, alkalinity, conductivity, TOC: 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
- Illumination: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight.
- Light intensity: Light intensity at test initiation was 597 lux at the surface of the water of one representative test chamber. Light intensity was measured at the water surface of one representative test chamber at test initiation using a SPER Scientific Model 840006C light meter.
- Photoperiod: 16 hours of light and 8 hours of darkness, controlled by an automatic timer. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting.

EFFECT PARAMETERS MEASURED: immobility and other sub-lethal effects
All organisms were observed periodically to determine the number of immobile organisms in each treatment group. Immobility was defined as a lack of movement by the organism except for minor activity of the appendages. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 3, 24 and 48 hours after test initiation.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: Nominal test concentrations were selected in based on the results of exploratory range finding toxicity data and functional solubility data.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

10.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: The EC50 value was estimated using non-linear interpolation between 5.6 and 12 mg/L

Details on results:

OBSERVATIONS
Daphnia in the negative and solvent control groups generally appeared normal throughout the test. One negative control daphnid and four solvent control daphnia were noted as floating at the 24-hour observations, but were normal in appearance after being gently submerged. All daphnia in the 0.85, 1.2, 2.8 and 5.6 mg/L treatment groups also appeared normal throughout the test, with no immobility or overt signs of toxicity observed. Percent immobility at test termination in the 12 mg/L treatment group was 65%. Floating daphnia were noted in all treatment groups, but were normal in appearance after being gently submerged. In addition to floating, daphnia in the 12 mg/L treatment group were observed to be lethargic.

TEST SOLUTIONS
The test solutions in the test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.

Reported statistics and error estimates:

The immobility data were analyzed using the computer program 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, nonlinear interpolation was used to calculate the 48-hour EC50 value and binominal probability was used to calculate the 95% confidence interval. Due to the method used to calculate the 48-hour EC50 value, the slope of the dose response curve could not be calculated. Since there was <50% immobility at 24 hours, the 24-hour EC50 value, as well as the no-immobility concentration and NOEC, were determined by visual interpretation of the immobility and observation data.

 Table: Cumulative Immobility and Observations

Mean Measured Concentration (mg/L)	Rep.	Daphnia/Replicate	3 Hours		24 Hours		48 Hours		Percent Immobile
			Number Immobile(1)	Observations(2)	Number Immobile(1)	Observations(2)	Number Immobile(1)	Observations(2)
Negative Control	A	10	0	10 AN	0	10 AN	0	10 AN	0
	B	10	0	10 AN	0	1 Q,AN; 9 AN	0	10 AN
Solvent Control	A	10	0	10 AN	0	2 Q,AN; 8 AN	0	10 AN	0
	B	10	0	10 AN	0	2 Q,AN; 8 AN	0	10 AN
0.85	A	10	0	2 Q,AN; 8 AN	0	8 Q,AN; 2 AN	0	2 Q,AN; 8 AN	0
	B	10	0	10 AN	0	6 Q,AN; 4 AN	0	2 Q,AN; 8 AN
1.2	A	10	0	10 AN	0	2 Q,AN; 8 AN	0	2 Q,AN; 8 AN	0
	B	10	0	1 Q,AN; 9 AN	0	5 Q,AN; 5 AN	0	10 AN
2.8	A	10	0	10 AN	0	1 Q,AN; 9 AN	0	10 AN	0
	B	10	0	10 AN	0	3 Q,AN; 7 AN	0	2 Q,AN; 8 AN
5.6	A	10	0	1 Q,AN; 9 AN	0	5 Q,AN; 5 AN	0	10 AN	0
	B	10	0	1 Q,AN; 9 AN	0	5 Q,AN; 5 AN	0	10 AN
12	A	10	0	7 Q,AN; 3 AN	0	2 Q,C; 8 AN	5	2 C; 3 AN	65
	B	10	0	10 Q,AN	0	6 Q,C; 4 AN	8	2 C

1) Cumulative number of immobile daphnids.

2) Observed effects: AN = appear normal; C = lethargy; Q,AN = trapped at water surface but appear normal after gentle submersion; Q,C = trapped at water surface and appear

lethargic after gentle submersion.

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on materials and methods incl tables'.

Conclusions:

The 48-h EC50 is 10.5 mg/L in freshwater invertebrates (D. magna).

Executive summary:

The acute toxicity to aquatic invertebrates was determined in a GLP-compliant study according to OECD TG 202. In this study daphnids (Daphnia magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 0 (solvent control), 1.4, 2.8, 5.5, 11 and 22 mg/L for 48 hours under flow-through conditions. The test item solution was prepared from individual stock solutions of calculated a amount of the test item mixed into HPLC-grade dimethylformamide (DMF) which was used as solvent (0.1 mL/L). Analysis of the test media at 0 and 48 hours showed geometric mean measured test concentrations of <LOQ (control), <LOQ (solvent control), 0.85, 1.2, 2.8, 5.6 and 12 mg/L. The measured test concentrations did not remain remain within ±20% of nominal concentrations throughout the test and hence the results are based on the geometric mean measured test concentrations. Immobilization was recorded after 3, 24 and 48 hours exposure. The water quality parameters were determined to be within the acceptable limits. After 48 hours, no immobility was observed up to the 5.6 mg/L treatment groups. At the highest test concentration treatment (12 mg/L), 65% immobility was observed after 48 hours. Based on these findings, the EC50 value was estimated using non-linear interpolation between 5.6 and 12 mg/L and was determined to be 10.5 mg/L. In this study all OECD validity criteria were met.

Description of key information

Isobornyl propionate

The acute Daphnia toxicity of the Isobornyl propionate is 8.7 mg/l, which is a converted value from Isobornyl acetate based on the difference in Log Kow and molecular weight (Log EC in mmol target= Log ECmmol x log Kow source/Log Kow target)

Isobornyl acetate and acute Daphnia study

Isobornyl acetate's (CAS 125 -12 -2) acute toxicity to aquatic invertebrates was determined in a GLP-compliant study according to OECD TG 202. In this study daphnids (Daphnia magna, 20 per concentration) were exposed to nominal concentrations of 0 (control), 0 (solvent control), 1.4, 2.8, 5.5, 11 and 22 mg/Lfor 48 hours under flow-through conditions.The test item solution was prepared from individual stock solutions of calculated a amount of the test item mixed into HPLC-grade dimethylformamide (DMF) which was used as solvent (0.1 mL/L). Analysis of the test media at 0 and 48 hours showed geometric mean measured test concentrations of <LOQ (control), <LOQ (solvent control), 0.85, 1.2, 2.8, 5.6 and 12 mg/L. The measuredtest concentrations did not remain remain within ±20% of nominal concentrations throughout the testand hence the results are based on the geometric mean measured test concentrations. Immobilization was recorded after 3, 24 and 48 hours exposure. The water quality parameters were determined to be within the acceptable limits. After 48 hours, no immobility was observed up to the 5.6 mg/L treatment groups. At the highest test concentration treatment (12 mg/L), 65% immobility was observed after 48 hours. Based on these findings, the EC50 value was estimated using non-linear interpolation between 5.6 and 12 mg/L and was determined to be 10.5 mg/L. In this study all OECD validity criteria were met.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.7 mg/L