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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 algae 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:

72 h

Dose descriptor:

EC50

Effect conc.:

> 18.7 mg/L

Basis for effect:

growth rate

Remarks on result:

other: The converted value based on MW and log Kow is used

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.9 mg/L

Basis for effect:

growth rate

Remarks on result:

other: 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 information is documented according to Annex XI criteria

Conclusions:

Isobornyl propionate has an EC50 growth rate of 18.7 and a NOEC of 9.9 mg/l using converted values from Isobornyl acetate including conversion with MW and log Kow.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jul 2011 to 1 Aug 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for Isobornyl propionate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

March 2006

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Qualifier:

according to guideline

Guideline:

other: ASTM Standard Guide E1218-04: Standard Guide for Conducting Static Toxicity Tests with Microalgae

Version / remarks:

2004

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance. At test initiation samples were collected for each treatment and control group prior to distribution into test chambers. At test termination, the biological replicates from each respective treatment and control group were pooled and then sampled. All samples were collected in glass vials, acidified with two drops of 10% phosphoric acid and processed immediately for analysis.

Vehicle:

yes

Remarks:

0.1 mL/L dimethylformamide

Details on test solutions:

A primary stock solution was prepared by dissolving 2.6316 g of test item in 10 mL of N,N-dimethylformamide (DMF) to achieve a nominal concentration of 250 mg a.i./mL. The primary stock was mixed by inversion and was sonicated for approximately one minute. The 250 mg a.i./L primary stock appeared clear and colorless after mixing. The 250 mg a.i./mL primary stock was serially diluted with DMF to prepare four additional stocks at target nominal concentrations of 15.63, 31.25, 62.50 and 125 mg a.i./mL. Each test solution was prepared by diluting 50 μL of each respective stock in 500 mL of freshwater AAP algal medium. The test solutions were prepared using this method to ensure that the solvent concentration in each treatment group was equivalent. All test solutions were mixed by inversion, in addition, the 13 and 25 mg a.i./L test solutions were sonicated for 10 minutes. The 1.6, 3.1, 6.3 and 13 mg a.i./L test solutions appeared clear and colorless and were otherwise unremarkable after mixing. The 25 mg a.i./L test solution appeared clear with a visible oily slick present at the surface. The negative control solution consisted of freshwater AAP algal medium without test substance. The solvent control solution contained 0.1mL DMF/L, which was equivalent to the solvent concentration in all of the treatment groups.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source: University of Toronto Culture Collection and maintained in culture medium at Wildlife International, Easton, Maryland
- Method of cultivation:

ACCLIMATION
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24.0 - 25.0 °C

pH:

7.4 - 7.8

Nominal and measured concentrations:

- Nominal test concentrations: 0 (control), 0 (solvent control), 1.6, 3.1, 6.3, 13 and 25 mg/L
- Initial measured concentrations: were all less than the limit of quantitation (LOQ), 0.8 mg a.i./L. A detailed overview of the analytical results is provided in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: Test chambers were sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers and contained 100 mL of test or control medium. The test flasks were indiscriminately positioned daily on a mechanical shaker in an environmental chamber designed to maintain the desired test temperature throughout the test. The test flasks were shaken continuously at 100 rpm.
- Type: closed
- Initial cells density: Prior to test initiation, an inoculum of the algal cells was prepared in freshwater (AAP) algal medium at a concentration of approximately 5.0 X 105 cells/mL. The concentration of algal cells was verified using a hemacytometer and microscope, and 1.0 mL of the inoculum was added to each test chamber to achieve a nominal concentration of approximately 5,000 cells/mL at test initiation.
- Control end cells density: 8.79E+05 cells/mL in the negative control
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- No. of vessels per vehicle control: 6

GROWTH MEDIUM
- Standard medium used: yes, Freshwater AAP Algal Medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algal cells were cultured and tested in Freshwater AAP Algal Medium. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified Wildlife International well water. The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to purified well water.
- Culture medium different from test medium: same as test

TEST MEDIUM MEASUREMENTS
- Temperature: Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC. The temperature of a container of water adjacent to the test chambers in the environmental chamber was recorded twice daily during the test using a liquid-in-glass thermometer. In addition, temperature in the environmental chamber was measured continuously using a minimum-maximum thermometer.
- pH: The pH of the medium in each treatment and control group was measured at test initiation and exposure termination using a Thermo Orion Model 525Aplus pH meter, or equivalent. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and control group. At exposure termination, pH was measured in pooled samples of test solution collected from each of the replicates of each treatment and control group.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was adjusted to 7.5 using 0.1 N sodium hydroxide and 10% hydrochloric acid, and the medium was then sterilized by filtration (0.22 µm) prior to use.
- Illumination: The algae were held under continuous cool-white fluorescent lighting throughout the test.
- Light intensity and quality: 5540 - 6330 lux. Light intensity was measured at test solution level at five locations surrounding the test flasks at test initiation using a SPER Scientific Inc. 840006C light meter.


EFFECT PARAMETERS MEASURED: algal cell densities, morphology
- Determination of cell concentrations: Test medium samples were collected from each replicate of the treatment and control group for the determination of algal cell densities. Samples were collected at approximately 24-hour intervals during the 72-hour exposure and were held for a maximum of two days under refrigerated conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were performed using an electronic particle counter (Coulter Electronics, Inc.). Prior to conducting cell counts, the linearity of the instrument response was determined at settings previously established for Pseudokirchneriella subcapitata. A primary counting standard containing P. subcapitata cells was prepared, the density was verified using a hemacytometer and a microscope, and the standard was subsequently diluted to provide a series of seven counting standards for the determination of instrument linearity.
- Morphology: Samples of test solution were collected from each of the replicates per treatment and control group at the end of the test. These samples were pooled within their respective treatments, and subsamples were removed and examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Cells in the replicate test chambers also were assessed for aggregations or flocculations of cells, and adherence of the cells to the test chamber.

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 the solubility of the test substance.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 24 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

OBSERVATIONS
After 72 hours of exposure, inhibition of cell density in the 1.3, 2.8, 5.9, 12 and 24 mg a.i./L treatment groups was 11, 0, 4, 8 and 27%, respectively, relative to the pooled control replicates. Inhibition of yield in the 1.3, 2.8, 5.9, 12 and 24 mg a.i./L treatment groups was 11, 0, 4, 8 and 27%, respectively, relative to the pooled control replicates, and inhibition of growth rate at the 0-72 hour interval was 2, 0, 0, 1 and 6%, respectively, relative to the pooled control replicates. An overview of the results is provided in 'Any other information on results incl. tables'.

MORPHOLOGY
After 72 hours of exposure, there were no signs or noticeable aggregation, flocculation, or adherence to the test chambers in the negative control or in any treatment groups. P. subcapitata cells in the treatment levels appeared normal when compared to cells present in the negative control.

TEST MEDIUM
Nominal concentrations selected for use in this study were 1.6, 3.1, 6.3, 13 and 25 mg a.i./L. The 1.6, 3.1, 6.3 and 13 mg a.i./L test solutions appeared clear and colorless and were otherwise unremarkable at test initiation. There was a visible oily slick on the surface of the 25 mg a.i./L test solution at test initiation. At test termination, there were no visible particulates or surface-slicks in any of the test chambers.

Reported statistics and error estimates:

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

Table: Mean Cell Density, Mean Yield and Percent Inhibition

Day 0 Measured Test Concentration (mg a.i./L)	24 Hours		48 Hours		72 Hours		72-Hour Yield(3)
	Mean Cell Density(1) (Cells/mL)	Percent Inhibition(1,2)	Mean Cell Density(1) (Cells/mL)	Percent Inhibition (1,2)	Mean Cell Density(1) (Cells/mL)	Percent Inhibition (1,2)	Mean Yield(1,2) (Cells/mL)	Percent Inhibition(1,2)
Negative Control	16,465	--	130,383	--	878,996	--	873,996	--
Solvent Control	13,445	--	120,801	--	790,290	--	785,290	--
Pooled Control	14,955	--	125,592	--	834,643	--	829,643	--
1.3	14,234	5	118,345	6	743,077	11	738,077	11
2.8	12,594	16	125,010	0	833,664	0	828,664	0
5.9	9,955	33	128,929	-3	805,152	4	800,152	4
12	11,933	20	117,974	6	766,755	8	761,755	8
24	8,995	40	83,169	34	613,320*	27	608,320*	27

1) Calculations were performed using SAS Version 8.2. Manual calculations may differ slightly.

2) Percent inhibition was calculated relative to the pooled control mean (n=6).

3) Yield was calculated at 72 hours as the final biomass (cell density) in the exposure period minus the initial biomass (5,000 cells/mL).

* Statistically significant difference (Dunnett’s test, p<0.05) from the pooled control mean. Determined at 72 hours only, per study protocol.

 

 Table: Mean Growth Rate (per Hour) and Percent Inhibition

Day 0 Measured Test Concentration (mg a.i./L)	0-24 Hours		0-48 Hours		0-72 Hours
	Mean Growth Rate (per day)(1)	Percent Inhibition(1,2)	Mean Growth Rate (per day)(1)	Percent Inhibition(1,2)	Mean Growth Rate (per day)(1)	Percent Inhibition(1,2)
Negative Control	0.0496	--	0.0677	--	0.0717	--
Solvent Control	0.0411	--	0.0663	--	0.0701	--
Pooled Control	0.0453	--	0.067	--	0.0709	--
1.3	0.0436	4	0.0658	2	0.0695	2
2.8	0.0383	15	0.067	0	0.071	0
5.9	0.0287	37	0.0677	-1	0.0706	0
12	0.0361	20	0.0658	2	0.0699	1
24	0.0225	50	0.0585	13	0.0667*	6

1) Calculations were performed using SAS Version 8.2. Manual calculations may differ slightly.

2) Percent inhibition was calculated relative to the pooled control mean (n=6).

* Statistically significant difference (Dunnett’s test, p<0.05) from the pooled control mean. Determined at 0-72 hours only, per study protocol.

EFFECT VALUES

Measured concentrations of the test item in samples collected from all test concentrations at exposure initiation ranged from 80 to 97% of the target nominal concentrations. Measured concentrations of the test item in samples collected at the end of exposure (72 hours) were all less than the limit of quantitation (LOQ), 0.8 mg a.i./L. Extrapolated values are reported, and are considered to be accurate even though the analytical recoveries were outside of the range of the standard curve. Concentrations in the controls were <LOQ at each sampling interval. Results of the study were based on Day 0 measured test concentrations.

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The 72-h ErC50 and 72-h NOErC are 24 mg/L and 12 mg/L, respectively, in green algae (Pseudokirchneriella subcapitata).

Executive summary:

The toxicity towards freshwater algae was determined in a GLP-compliant study according to OECD TG 201. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 0 (solvent control), 1.6, 3.1, 6.3, 13 and 25 mg/L for 72 hours under static conditions. Test concentrations were analytically verified at t=0h (start) and t=72h (end). The analytical results showed that all test concentrations at exposure initiation ranged from 80 to 97% of the target nominal concentrations. Measured concentrations of the test item in samples collected at the end of exposure were all less than the LOQ, 0.8 mg a.i./L. Therefore, all results are based on the initial measured concentrations, also because the algae toxicity was highest after 24 hours, indicating that the parent was the key toxicant. At the respective nominal concentrations the measured concentrations were determined to be <LOQ (control), <LOQ (solvent control), 1.3, 2.8, 5.9, 12 and 24 mg/L. At the start of the test and after 24, 48 and 72 hours exposure duration, cell densities were determined and the yield growth rates of the treaments were calculated. At the end of the test the algae were inspected microscopically for abnormalities. After 72 hours of exposure, inhibition of cell density in the 1.3, 2.8, 5.9, 12 and 24 mg a.i./L treatment groups was 11, 0, 4, 8 and 27%, respectively, relative to the pooled control replicates. Inhibition of yield in the 1.3, 2.8, 5.9, 12 and 24 mg a.i./L treatment groups was 11, 0, 4, 8 and 27%, respectively, relative to the pooled control replicates, and inhibition of growth rate at the 0-72 hour interval was 2, 0, 0, 1 and 6%, respectively, relative to the pooled control replicates. No abnormal cells were observed. Based on these findings, the 72-h EC50 values for growth rate and yield were both determined at 24 mg/L. The 72-h NOEC for growth rate and yield were both determined at 12 mg/L.

Description of key information

Isobornyl propionate

The acute and long-term Algae toxicity of the Isobornyl propionate is > 18.7 and 9.9 mg/l, respectively. These are converted values 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 its algae toxicity

The toxicity towards freshwater algae was determined in a GLP-compliant study according to OECD TG 201. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of0 (control), 0 (solvent control), 1.6, 3.1, 6.3, 13 and 25 mg/Lfor 72 hours under static conditions. Test concentrations were analytically verified at t=0h (start) and t=72h (end).The analytical results showed thatall test concentrations at exposure initiation ranged from 80 to 97% of the target nominal concentrations. Measured concentrations of the test item in samples collected at the end of exposure were all less than the LOQ, 0.8 mg a.i./L. Therefore, all results are based on the initial measured concentrations, also because the algae toxicity was highest after 24 hours, indicating that the parent was the key toxicant. At the respective nominal concentrations the measured concentrations were determined to be <LOQ (control), <LOQ (solvent control), 1.3, 2.8, 5.9, 12 and 24 mg/L. At the start of the test and after 24, 48 and 72 hours exposure duration, cell densities were determined and the yield growth rates of the treaments were calculated. At the end of the testthe algae were inspected microscopically for abnormalities.After 72 hours of exposure, inhibition of cell density in the 1.3, 2.8, 5.9, 12 and 24 mg a.i./L treatment groups was 11, 0, 4, 8 and 27%, respectively, relative to the pooled control replicates. Inhibition of yield in the 1.3, 2.8, 5.9, 12 and 24 mg a.i./L treatment groups was 11, 0, 4, 8 and 27%, respectively, relative to the pooled control replicates, and inhibition of growth rate at the 0-72 hour interval was 2, 0, 0, 1 and 6%, respectively, relative to the pooled control replicates.No abnormal cells were observed.Based on these findings, the 72-h EC50 values for growth rate and yield were both determined at 24 mg/L. The 72-h NOEC for growth rate and yield were both determined at 12 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

18.7 mg/L

EC10 or NOEC for freshwater algae:

9.9 mg/L