2,6,8-trimethylnonan-4-ol

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 November to 14 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1400 (Fish Acute Toxicity Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,6,8-trimethylnonan-4-ol

EC Number:

204-606-8

EC Name:

2,6,8-trimethylnonan-4-ol

Cas Number:

123-17-1

Molecular formula:

C12H26O

IUPAC Name:

2,6,8-trimethylnonan-4-ol

Details on test material:

- Name of test material (as cited in study report): Trimethyl Nonanol
- Molecular weight (if other than submission substance): 186 g/mole
- Physical state: Transparent pale yellow liquid
- Analytical purity: 95.7%
- Lot/batch No.: RC1355T3D4

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sample Collection/Extraction and Chemical Analysis
The test solutions were analyzed on days 0 (from the freshly prepared solutions) 1, 2, and 3 (from both renewed and spent solutions) and 4 (spent solutions only) of the study using gas chromatography with a flame-ionization detector (GC/FID). Aliquots were collected (40 mL from the control LDW, 6.25 and 12.5 mg/L solutions; 10 mL from the 25.0, 50.0 and 100 mg/L solutions) from the day 1, 2, 3 and 4 test solutions (two replicates per dose level including the controls). The samples were extracted with 2 mL iso-octane by shaking on a flatbed shaker for 30 minutes (low speed). The organic extract was transferred to autosampler vials for analysis by GC/FID.

Method Precision and Homogeneity
To assess the variability associated with the analytical method as well as solution homogeneity, three additional samples were collected on day 0 from representative 6.25 and 100 mg/L test solutions (replicates A). These additional samples were prepared for analysis along with the other day 0 samples as described above.

Extraction Efficiency of Trimethyl Nonanol from Laboratory Dilution Water (LDW)
The recovery (extraction efficiency) of trimethyl nonanol from LDW by solvent partitioning with iso-octane was determined by fortifying LDW with trimethyl nonanol at concentrations representative of the range of dose solutions and then conducting the extraction as described in “Sample Collection/Extraction and Chemical Analysis.” Triplicate spikes at of trimethyl nonanol in LDW were prepared on days 0 and 1 at target concentrations of 0.0527, 1.05, and 10.5 mg/L LDW. However, the 10.5 mg/L spike was outside the standard range. Therefore, triplicate spikes of trimethyl nonanol in LDW were prepared on days 2, 3, and 4 at target concentrations of 0.0527, 1.05, and 4.21 mg/L LDW. The assessment of extraction efficiency yielded average recovery values of 97.1% for day 0, 98.6% for day 1, 96.8% for day 2, 92.2% for day 3, and 95.6% for day 4, which were used to adjust the analyzed concentrations of the test solutions for method recovery on each analysis day.

Test solutions

Details on test solutions:

For the definitive test, bulk solutions were prepared in LDW at nominal exposure concentrations of 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L, at test initiation and renewed daily throughout the study. These bulk dose solutions, in addition to control test solution (LDW with no test material added and referred to as water control) were apportioned (~ 10 L) between two replicate test vessels per dose level.

Test Solution Preparation
Bulk dose solutions were prepared at test initiation and renewed daily at nominal (target) concentrations of 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L. Since the test material was a liquid, the weight of the test material added to the LDW was calculated on a weight-to-volume basis and was then converted to a volume using the specific gravity of the test material (0.8193 g/mL) and adjusted for the test material purity of 95.7%.

Based on this, 2.93 mL of trimethyl nonanol was added to 23 L of LDW in a 20-L glass carboy in duplicate. Due to the limited solubility of the test material in LDW (observed as an oily film on the test solution surface), the solutions were stirred via a large magnetic stirrer and stir bar and then allowed to settle for ~ 1 hour to enable the biphasic mixture (top layer of insoluble test material versus bottom layer of LDW containing solubilized
test material) to completely separate. Following the 1-hour settling time, the bottom layers (soluble test material layer) from each solution were decanted and pooled into a clean 45-L glass carboy. This solution became the highest dose level bulk solution (100 mg/L, nominal). The 50.0-mg/L bulk dose solution was prepared by adding 10 L of the 100-mg/L bulk dose solution to 10 L of LDW in a 20-L glass carboy. The solution
was then mixed thoroughly. Subsequent bulk dose solutions (25.0, 12.5, and 6.25 mg/L) were similarly prepared as serial dilutions of the next highest test solution. All test solutions were prepared with LDW (which was aerated overnight) at room temperature to maximize the solubility of the test material in LDW.

These bulk dose solutions, in addition to control test solution (LDW with no test material added and referred to as water control) were apportioned (~ 10 L) between two replicate test vessels per dose level. The vessels were then placed in at static water trough (water bath) and allowed to reach test temperature prior to analytical chemistry sampling and the addition of the test organisms. The process was repeated daily and the test organisms transferred, via the screened insert as described in “Test Vessels”, from the old (spent) solutions to clean vessels containing freshly prepared solutions of the same respective nominal test concentrations.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test organism was the rainbow trout, Oncorhynchus mykiss Walbaum (Lot # RT100703). This species is widely accepted and recommended for toxicity testing. The fish were obtained as juveniles from Thomas Fish Company, Anderson, California. Upon arrival, the juveniles were visually inspected and placed into a holding tank (~ 230 L). All fish were held on a 16-hour light/8-hour dark transitional photoperiod and observed for at least 14 days before testing. During holding, the fish received a standard diet (Aquatic Diet Number 1 Lot #218452, Harlan-Teklad, Madison, Wisconsin) at least once daily. The test lot used for the definitive study was acclimated to 15.0 ± 2°C for at least 7 days prior to test initiation and held without food for at least 48 hours before testing. Temperature changes did not exceed ± 3°C in 72 hours pre-test, and mortality did not exceed five percent of the population in the 48 hours before testing. Fish averaging 4.2 ± 0.2 cm in total length and 0.576 ± 0.096 g in mass (post-exposure) were used as test organisms. The length of the largest fish was no more than twice that of the shortest fish.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96

Remarks on exposure duration:

h

Post exposure observation period:

No data.

Test conditions

Hardness:

Control Water (LDW) Hardness (mg/L CaCO3): ranged from 110 - 114
100 mg/L Test Solution, Nominal Hardness (mg/L CaCO3): 110 - 116

Test temperature:

14.4-15.9 (15.4 ± 0.3) Measured from the individual test vessels.
14.4-15.8 (Continuous temperature monitoring range from surrogate vessel.)

pH:

7.1-7.9 (7.4 ± 0.3)

Dissolved oxygen:

7.3-8.8 (8.0 ± 0.5) (Percent oxygen saturation averaged 82% and remained ³ 74% throughout the exposure, based on a theoretical value of 9.8 mg/L.)

Salinity:

No data

Nominal and measured concentrations:

See Table below.

Details on test conditions:

Preliminary Study
No 96-hour preliminary trout study with trimethyl nonanol was conducted. Information obtained from a 96-hour static-renewal (daily renewal of test solutions) acute test conducted with rainbow trout exposed to isobutyl heptyl ketone (IBHK) was used to determine the test concentrations and test design for the definitive study with trimethyl nonanol. IBHK is a chemical with similar chemical/physical properties to trimethyl nonanol. Therefore, it was believed that trimethyl nonanol would have similar water solubility and stability and would result in a similar dose response. In the IBHK study, rainbow trout were exposed to the water-soluble portion (also referred to as water accommodated fraction) of a nominal 100 mg/L test solution and subsequent dilutions prepared in LDW. The resulting 96-hour LC50 value was 1.24 mg/L and represented the maximum solubility of IBHK in this test medium (LDW) using the test solution preparation procedure employed.

It was evident that the solubility of trimethyl nonanol in aqueous solution was limited. Analytical chemistry results from a related study with Daphnia magna exposed to trimethyl nonanol at 20°C supported that the test material had limited solubility (< 20 mg/L) in aqueous solution. In the Daphnia study, the water-soluble portion (also referred to as water accommodated fraction) of the nominal 100 mg/L test solution prepared in daphnid water resulted in measured trimethyl nonanol concentrations of approximately 16 mg/L. In addition, it was determined and confirmed during the Daphnia testing that trimethyl nonanol was also not stable in aqueous solution. We anticipated that the trimethyl nonanol would have a lower solubility in testing with trout due to the water temperature of ~ 15°C.

The information acquired from the above testing was used to set the range of concentrations for the definitive test. Due to the limited solubility of the test material and the lack of a significant dose response in the IBHK test, the definitive test was conducted with the highest dose level equal to the highest water solublility of trimethyl nonanol attainable in LDW using the test solution preparation procedure employed. In addition, since it was determined that trimethyl nonanol was not stable in aqueous solution, the test was conducted as a static-renewal test with the daily renewal of test solutions in the attempt to maintain exposure concentrations during the study.

Definitive Study
The definitive study was conducted between 10 November and 14 November 2003. Based on results from preliminary testing, ten fish (two replicates of five fish per dose level) were exposed to nominal test concentrations of 0 (water control), 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L, over a 96-hour static-renewal (daily renewal of test solutions) exposure period. Fish were impartially added to each test vessel within 30 minutes of initial test solution sampling.

The fish were observed after 24, 48, 72, and 96 hours of exposure for mortality (no response to touching of the caudal peduncle and no opercular movement) and sublethal effects. Dead fish were removed when observed. Any behavioral effects were noted and reported, e.g., swimming at the surface, complete or partial loss of body equilibrium, lethargy, hyperactivity, and erratic movement. Any gross pathological conditions were
also noted and reported; e.g., exophthalmia (bulging eyes), ascites (fluid accumulation in the abdomen), hemorrhage (discharge of blood), excess mucus, sloughing of epidermis, and melanosis (increased amount of dark pigmentation).

Dissolved oxygen, pH, and temperature data were recorded in each test vessel on days 0 (from the freshly prepared solutions) 1, 2, and 3 (from both renewed and spent solutions) and 4 (spent solutions only). Water quality parameters such as alkalinity, hardness, residual chlorine, and conductivity were measured on days 0 through 3 from one freshly prepared control and highest exposure level solution (highest level with surviving test organisms). Water temperature was continuously monitored from one test vessel throughout the study; test temperature was maintained at 15.0 ± 2°C. Loading of the test vessels did not exceed 1.0-g fish per liter of test solution. This loading rate resulted in dissolved oxygen levels that were equal to or greater than 60% of saturation throughout the exposure period; aeration was not required. Fish were not fed during the test. Terminal body weight (nearest 1 mg) and total length (nearest 1 mm) measurements were recorded for all surviving fish. Fish were euthanatized with tricaine methanesulfonate prior to the length and weight measurements.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 2.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.239 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

behaviour

Details on results:

Chemical Analysis
Average exposure values (average of two replicates per dose level) are calculated along with the corresponding percent of target values (average value divided by the target value). Results from the analyses of renewed test solutions (days 0, 1, 2 and 3) yielded measured concentrations that ranged from 1.59 (day 3) to 3.94 (day 2) percent of target. The analyses of spent test solutions (days 1, 2, 3 and 4) showed a general decline in concentration over the 24-hour exposure period with measured concentrations ranging from 0.968 (day 1) to 2.74 (day 3) percent of target. A study average and percent of target was also calculated for each dose level by averaging the measured values obtained throughout the course of the study. Study average percent of target values ranged from 1.83 to 2.30%. The overall average percent of target and standard deviation values for the entire study (n = 5) was 2.03 ± 0.195%. The low measured concentrations from the freshly prepared (renewed) solutions are due to the limited solubility of trimethyl nonanol in water. The declining measured concentrations of trimethyl nonanol from the spent solutions are likely due to the instability of this chemical in aqueous solution.

None of the analyses of the LDW controls exhibited a peak eluting at the retention time of trimethyl nonanol at concentrations exceeding the lowest level quantified of 0.0281 mg/L LDW, which was equivalent to the concentration of the lowest standard analyzed multiplied by the lowest dilution factor.

Method Precision and Homogeneity
The variability associated with the analytical method as well as solution homogeneity was assessed on day 0 of the study by collecting four replicate samples from representative test solutions at nominal concentrations of 6.25 and 100 mg/L. Four repeated measurements (4 samples x 1 injection/sample) of the low and high concentration test solutions yielded percent RSD values of 3.11 and 1.16%, respectively (data not shown).

Lowest Level Quantified/Limit of Detection
The lowest level quantified (LLQ) was set at 0.0281 mg trimethyl nonanol/L LDW. The LLQ value was based on the concentration of analyte in the lowest standard analyzed multiplied by the lowest dilution factor.

Test Conditions
Dissolved oxygen levels ranged from 7.3–8.8 (8.0 ± 0.5), averaged 82% of saturation and remained greater than or equal to 74% over the 96-hour exposure period. Temperature in the vessels ranged from 14.4–15.9°C (15.4 ± 0.3°C); the temperature continuously measured from a surrogate vessel ranged from 14.4–15.8°C and maintained within 15 ± 2°C throughout the study. The pH ranged from 7.1–7.9 (7.4 ± 0.3).

Water quality parameters such as alkalinity, hardness, residual chlorine, and conductivity were measured on days 0 through 3 from one freshly prepared control and highest exposure level solution (highest level with surviving test organisms). Alkalinity ranged from 36–43 mg/L (as CaCO3), hardness ranged from 110–116 mg/L (as CaCO3), and conductivity ranged from 164–170 mmhos/cm. Residual chlorine was < 10 ppb (instrument’s limit of detection) in all solutions measured.

Biological Data
The post-exposure lengths (total) and weights of all surviving fish used in this study were measured and recorded. The mean (+/- standard deviation) length and weight of the water control fish was 4.1 ± 0.2 cm and 0.550 ± 0.117 g, respectively. The mean length and weight of all surviving trimethyl nonanol exposed fish was 4.2 ± 0.2 cm and 0.581 ± 0.092 g, respectively, indicating that fish used in this study were of a similar size class. Pooled length and weight means of all surviving fish combined was 4.2 ± 0.2 cm and 0.576 ± 0.096 g, respectively. The resulting average biological loading rate, calculated as: [(mean fish weight * five fish per vessel)/10.0 L of solution in each vessel], was 0.3-g fish/L of test.

Fish were observed and any mortality and sublethal effects recorded at 24, 48, 72, and 96 hours of exposure. Sublethal effects were observed down through the 0.491 mg/L measured dose level during the conduct of this 96-hour exposure study and included lethargy, partial loss of equilibrium and complete loss of equilibrium. No fish mortality was observed at or below the highest measured concentration tested of 2.17 mg/L or in the water control during the conduct of this study. The resulting acute toxicity values for trimethyl nonanol with rainbow trout are listed below:
· The 24-, 48-, 72-, and 96-hour LC50 values were empirically determined to be greater than 2.17 mg/L, which was the highest measured concentration tested. This concentration represents the maximum solubility of trimethyl nonanol in this test medium (LDW) using the test solution preparation procedure employed.
· The 96-hour NOEC was 0.239 mg/L and was determined based on biological interpretation of the data and the highest exposure level exhibiting no fish mortality and/or sublethal effects.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

No statistical analysis programs were used and the LC50 values empirically determined.

The 24-, 48-, 72-, and 96-hour LC50 values were empirically determined to be greater than 2.17 mg/L, which was the highest measured concentration tested. This concentration represents the maximum solubility of trimethyl nonanol in this test medium (LDW) using the test solution preparation procedure employed.
The 96-hour NOEC was 0.239 mg/L and was determined based on biological interpretation of the data and the highest exposure level exhibiting no fish mortality and/or sublethal effects.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table. Summary of Biological Observations

Trimethyl Nonanol Concentrations (mg/L		Observations
Mean Analyzed	Nominal	24 -hour	48 -hour	72 -hour	96 -hour
<LLQ^a	Water Control	10N	10N	10N	10N
0.144	6.25	10N	10N	10N	10N
0.239	12.5	10N	10N	10N	10N
0.491	25.0	10N	10N	10N	9N, 1L
0.916	50.0	10N	10N	10N	3N, 7L
2.17	100	7N, 2L, 1PE	9PE, 1CE	10PE	9PE,1L
LC50^b (mg/L)		>2.17	>2.17	>2.17	>2.17
95% Confidence Limits^b (mg/L)		ND^d	ND	ND	ND
96 -hour NOEC^c(mg/L)					0.239

N = normal, PE/CE = partial/complete loss of equilibrium, L = lethargy

^a < LLQ = Less than the lowest level quantified of 0.0281mg trimethyl nonanol/L LDW.

^b The 24-, 48-, 72-, and 96-hour LC50 values were empirically determined to be greater than 2.17 mg/L, which was the highest measured concentration tested. This concentration represents the maximum solubility of trimethyl nonanol in this test medium (LDW) using the test solution preparation procedure employed.

^c Based on biological interpretation of the data and the highest exposure level exhibiting no fish mortality and/or sublethal effects.

^d ND = Not determined.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 24-, 48-, 72-, and 96-hour LC50 values were empirically determined to be greater than 2.17 mg/L, which was the highest measured concentration tested. This concentration represents the maximum solubility of trimethyl nonanol in this test medium (LDW) using the test solution preparation procedure employed. The 96-hour NOEC was 0.239 mg/L and was determined based on biological interpretation of the data and the highest exposure level exhibiting no fish mortality and/or sublethal effects.

Executive summary:

The purpose of this study was to estimate the acute toxicity of trimethyl nonanol to the rainbow trout (Oncorhynchus mykiss), over a 96-hour static-renewal (daily renewal of test solutions) exposure period. These data were used to calculate 24-, 48-, 72-, and 96-hour LC50 values (the concentration that is lethal to 50 percent of the test population during a defined period) and a 96-hour no-observed-effect concentration or NOEC.

The study was conducted with two groups of five fish exposed to nominal test concentrations of 0 (water control), 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L, over a 96-hour exposure period. The concentration of trimethyl nonanol in the test solutions was confirmed by collecting samples on days 0 (from the freshly prepared solutions) 1, 2, and 3 (from both renewed and spent solutions) and 4 (spent solutions only) of the study. The collected samples were analyzed by gas chromatography/flame ionization detection (GC/FID) and external standard quantitation. Overall, the average percent recovery of nominal dose concentrations ranged from 1.83 to 2.30%. The overall average percent recovery of nominal and standard deviation values for the entire study (n = 5) was 2.03 ± 0.195%. The low percent recoveries of nominal dose concentrations observed were mainly due to the limited solubility of trimethyl nonanol in aqueous solutions. The resulting mean measured trimethyl nonanol concentrations for the study were less than the lowest level quantified of 0.0281 mg/L for the water control and 0.144, 0.239, 0.491, 0.916, and 2.17 mg/L for the treatment solutions.

Fish were observed and any mortality and sublethal effects recorded at 24, 48, 72, and 96 hours of exposure. Sublethal effects were observed down through the 0.491 mg/L measured dose level during the conduct of this 96 -hour exposure study and included lethargy, partial loss of equilibrium and complete loss of equilibrium. No fish mortality was observed at or below the highest measured concentration tested of 2.17 mg/L or in the water control during the conduct of this study. The resulting acute toxicity values for trimethyl nonanol with rainbow trout are listed below:

• The 24-, 48-, 72-, and 96-hour LC50 values were empirically determined to be greater than 2.17 mg/L, which was the highest measured concentration tested. This concentration represents the maximum solubility of trimethyl nonanol in this test medium (LDW) using the test solution preparation procedure employed.

• The 96-hour NOEC was 0.239 mg/L and was determined based on biological interpretation of the data and the highest exposure level exhibiting no fish mortality and/or sublethal effects.