2,6,8-trimethylnonan-4-ol

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 September-16 October 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 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

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, 1, (spent and renewed test solutions) and 2 of the study using gas chromatography with a flame-ionization detector (GC/FID). Aliquots (10 mL from the control ALDW, 3.13, 6.25, and 12.5 mg/L dose levels and 2 mL from the 25.0, 50.0, and 100 mg/L dose levels) were collected from the test solutions (bulk dose solutions and ALDW control on day 0, replicate spent test solutions as well as renewed dose solutions on day 1 and replicate spent test solutions on day 2). The samples were extracted with 2 mL iso-octane by shaking on a flatbed shaker set on low speed for 30 minutes. The biphasic mixtures were allowed to separate with centrifuging as needed. The organic phase (top) was transferred to autosampler vials for analysis by GC/FID.

Method Precision/Homogeneity
To assess analytical method precision and solution homogeneity, three additional samples were taken on day 0 from the 3.13 and 100 mg trimethyl nonanol/L ALDW bulk dose solutions. These additional samples were collected, extracted, and analyzed along with the other day 0 samples as described above.

Extraction Efficiency of TMN from Adjusted Laboratory Dilution Water
The recovery (extraction efficiency) of trimethyl nonanol from ALDW by solventpartitioning with iso-octane was determined by fortifying ALDW 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 three concentrations of trimethyl nonanol in ALDW were prepared at target concentrations of 3.06, 12.2, and 97.3 mg/L on day 0 and 0.614, 9.14, and 24.5 mg/L on days 1 and 2. This assessment of extraction efficiency yielded average recovery values of 103% for day 0, 105% for day 1, and 103% for day 2, which were used to adjust the analyzed concentrations of the test solutions for method recovery on each analysis day.

Test solutions

Vehicle:

no

Details on test solutions:

For the definitive test, bulk solutions were prepared in ALDW at nominal exposure concentrations of 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L, at test initiation and renewed at approximately 24 hours. These bulk dose solutions, in addition to control test solution (ALDW with no test material added and referred to as water control) were apportioned (~ 200 mL) between two replicate test vessels per dose level.

Dose Solution Preparation
Bulk dose solutions were prepared at test initiation and renewed at approximately 24 hours at nominal (target) concentrations of 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L. Due to the limited solubility of the test material in aqueous solution (observed as an oily film on the test solution surface) and under these test conditions (ALDW @ ~ 20°C), the 100 mg/L bulk test solution was prepared using a separatory funnel.

Since the test material was a liquid, the weight of the test material added to the ALDW 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, 0.255 mL of TMN was added to 2L of ALDW in a 2-L separatory funnel. The solution was shaken vigorously and then allowed to settle for ~ 1 hour to enable the biphasic mixture (top layer of insoluble test material versus bottom layer of ALDW containing solubilized test material) to completely separate. Following the 1-hour settling time, the bottom (soluble test material layer) of the solution was decanted into a clean 2-L
volumetric flask. 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 500 mL of the 100-mg/L bulk dose solution to a 1-L volumetric flask and filling it to volume with ALDW. The flask was stoppered and the solution mixed thoroughly. Subsequent bulk dose solutions (25.0, 12.5, 6.25, and 3.13 mg/L) were similarly prepared as serial dilutions of the next highest test solution.

These bulk dose solutions, in addition to control test solution (ALDW with no test material added and referred to as water control) were apportioned (~ 200 mL) between two replicate test vessels per dose level prior to the addition of the test organisms. The process was repeated on day 1 (~ 24-hours) and the test organisms transferred, via a pipette, from the old (spent) solutions to clean vessels containing freshly prepared solutions of the same respective nominal test concentrations.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test organism was the freshwater daphnid, Daphnia magna, from in-house cultures initially obtained from Aquatic Biosystems, Fort Collins, Colorado. Daphnia instars less than 24-hours old from a laboratory-reared culture was used as the test organism. This species is widely accepted and recommended for toxicity testing. Rearing conditions were as follows: illumination (cool-white fluorescent) 2050 ± 350 lux; 16-hour light/8-hour dark photoperiod; temperature 20 ± 2°C. Daphnia were fed a mixed diet of Selenastrum capricornutum (algae) and YCT (yeast-ceraphyll trout) trout chow five times weekly. The day before instars were needed for testing, stock tanks with Daphnia greater than 14-days old which have had at least four broods were removed from the incubator. The instars were separated from adults by gently lifting the screened insert from the 2-L stock tank, releasing instars through the nylon mesh screen while retaining the adult Daphnia. The screened insert, with adult Daphnia, were then placed in another stock tank that contained daphnid water. The original solution with instars was poured through a metal sieve into another stock tank. The instars collected on the sieve were discarded, and the original solution was poured back into the initial stock tank. The corresponding screened insert holding adult Daphnia was then put back in place. This procedure was repeated the day the study was set to cull < 24-hour old instars for use in the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No data

Test conditions

Hardness:

Control Water (ALDW) Hardness (mg/L CaCO3): 167 and 181 on 14 and 15 Oct 2003, respectively.
100 mg/L Bulk Solution, Nominal Hardness (mg/L CaCO3): 178 and 182 on 14 and 15 Oct 2003, respectively.

Test temperature:

20.1-20.9 (20.4 ± 0.2) Measured from the bulk dose solutions and the individual test vessels.
19.4-20.2 (Continuous temperature monitoring range from surrogate vessel.)

pH:

7.5-7.6 (7.5 ± 0.1)

Dissolved oxygen:

8.3-9.9 (9.0 ± 0.4) (Percent oxygen saturation averaged 100% and remained >/- 93% throughout the exposure, based on a theoretical value of 8.9 mg/L.)

Salinity:

No data

Nominal and measured concentrations:

See Table below.

Details on test conditions:

Preliminary Study
A 48-hour static-renewal (renewal of test solutions on day 1) probe study was conducted between 16 September and 18 September 2003 with one replicate of ten Daphnia per dose level exposed to nominal test concentrations of 0 (water control), 25.0, 50.0, and 100 mg trimethyl nonanol/L. The test material was not soluble at the 100 mg/L dose level as indicated by the presence of an oily film on the surface of the bulk test solution. Therefore,
the bulk solution was mixed vigorously and then sat undisturbed for one hour to allow the biphasic mixture (top layer of insoluble test material versus bottom layer of ALDW containing solubilized test material) to separate. After one hour, the bottom (soluble test material layer) of the solution was decanted and used as the high-dose level and for the preparation of the lower dose levels via subsequent serial dilutions. The process was repeated on day 1 (~ 24-hours) and the test organisms transferred, via a pipette, from the old (spent) solutions to clean vessels containing freshly prepared solutions of the same respective nominal test concentrations.

No immobility was observed in any of the dose levels following 24-hours of exposure. Following 48-hours of exposure, immobility observed in the 100% (10/10) of the Daphnia at both the 100 and 50.0 mg/L dose levels, and in 40% (4/10) of the Daphnia at the 25.0 mg/L dose level. No immobility was observed in the water control during the study. Based on these preliminary study results, the 48-hour EC50 was estimated to fall between the nominal 25.0 and 50.0 mg/L dose levels.

Analytical chemistry verification of trimethyl nonanol concentrations in the test solutions during the probe indicated that the test material was not very soluble or stable under these test conditions (in ALDW at ~ 20°C). Day 0 and 1 bulk treatment solutions measured approximately 15% of nominal concentrations indicating limited solubility of the test material in aqueous solutions and declined to approximately 9% and below after 24-hours in the spent solutions indicating test material instability.

The information derived from this preliminary test was used to set the range of concentrations for the definitive test and supported the decision to conduct the definitive study under static-renewal conditions.

Definitive Study
The definitive study was conducted between 14 October and 16 October 2003. Based on the results from the preliminary study, twenty Daphnia (two replicates of ten Daphnia per dose level) were exposed to nominal test concentrations of 0 (water control), 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L, over a 48-hour static-renewal (renewal of test solutions at ~ 24-hours) exposure period. Daphnia were impartially added to each test vessel within 30 minutes of initial test solution sampling.

Daphnia were observed for immobility (inability to swim within 15 seconds after gentle agitation of the test container) at 24 and 48 hours of exposure. Dissolved oxygen, pH, and temperature data were recorded from each bulk dose solution at test initiation and approximately 24-hours and from all replicate test vessels (spent test solutions) at approximately 24-hours and test termination. Water temperature was continuously monitored from one surrogate vessel throughout the study; test temperature was maintained at 20.0 ± 1°C. Water quality parameters such as alkalinity, hardness, residual chlorine, and conductivity were measured from the day 0 and day 1 (~ 24-hour) control water and the bulk dose solution of the highest exposure level with surviving organisms. Daphnia were not fed during the test.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

6.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 5.07–7.63 mg/L

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.44 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI 3.79–5.21 mg/L

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.02 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Chemical Analysis
Daily residual values (bulk dose solutions for days 0 & 1; average of two replicate spent test solutions for days 1 & 2) are calculated along with daily percent of target values (daily value divided by the target value). Results from the day 0 analysis yielded percent of target values for the bulk dose solutions ranging from 20.5 to 26.1%. Results from the day 1 analysis of spent test solutions showed a decline in analyte concentrations, with trimethyl nonanol levels ranging from 3.12 to 14.0% of target after 24 hours. The renewed dose solutions on day 1 had percent of target comparable to the day 0 bulk dose solutions, with levels ranging from 16.1 to 22.5%. Results from the day 2 analysis of spent test solutions showed percent of target levels ranging from 5.80 to 11.4% for all dose levels. A study average and percent of target was also calculated for each dose level by averaging the day 0 bulk dose solution, day 1 spent test solution, day 1 renewed test solution and day 2 spent test solution daily values. Study average percent of target values ranged from 11.7 to 17.4%. The overall average percent of target and standard deviation values calculated across all dose levels (n=6) was 15.4 ± 2.04%. The low measured concentrations from the day 0 and 1 bulk dose solutions are due to the limited solubility of trimethyl nonanol in water (ALDW) and the declining measured concentrations from the day 1 and 2 spent solutions are likely due to the instability of trimethyl nonanol in ALDW.

None of the analyses of the ALDW controls exhibited a peak eluting at the retention time monitored for trimethyl nonanol at concentrations exceeding the lowest level quantified of 0.0982 mg analyte/L ALDW, which was the concentration of trimethyl nonanol in the lowest standard analyzed times the lowest dilution factor.

Method Precision/Homogeneity
The variability associated with the analytical method as well as solution homogeneity was assessed for trimethyl nonanol. Four replicate samples were collected from the bulk dose solutions at nominal concentrations of 3.13 and 100 mg trimethyl nonanol/L on day 0. Four repeated measurements (4 samples x 1 injection/sample) yielded a percent RSD value of 5.58% and 6.34% for the low and high levels, respectively (data not shown).

Lowest Level Quantified/Limit of Detection
The lowest level quantified (LLQ) was set at 0.0982 mg trimethyl nonanol/L ALDW. The LLQ value was based on the concentration of analyte in the lowest standard analyzed times the lowest dilution factor.

Test Conditions
Dissolved oxygen levels ranged from 8.3-9.9 (9.0 ± 0.4), averaged 100% of saturation and remained greater than or equal to 93% over the 48-hour exposure period. Temperatures measured from the bulk test solutions and the individual test vessels ranged from 20.1-20.9 °C (20.4 ± 0.2°C); the temperature continuously measured from a surrogate vessel ranged from 19.4-20.2°C and maintained within 20.0 ± 1°C throughout the study. The pH ranged from 7.5-7.6 (7.5 ± 0.1) and the light intensity ranged from 1774-2090 lux (1916 ± 111 lux).

Water quality parameters such as alkalinity, hardness, residual chlorine, and conductivity were measured from the freshly prepared bulk dose solution of the water control and the nominal 100-mg/L dose level on days 0 and 1. Hardness ranged from 167-182 mg/L (as CaCO3), alkalinity ranged from 38-41 mg/L (as CaCO3), and conductivity from 422-434 mmhos/cm. Residual chlorine was < 10 ppb (instrument’s limit of detection) in all solutions measured.

Biological Data
All biological results are expressed in terms of mean measured concentrations of trimethyl nonanol.

Daphnia were observed at 24 and 48 hours of exposure and the primary endpoint of interest in this acute study was immobility. Immobility was observed in 80% (16/20), 75% (15/20), and 20% (4/20) of the Daphnia at the 11.7, 8.41, and 2.17 mg/L dose levels, respectively, following 24 hours of exposure. One of the four immobile Daphnia immobile at the 2.17 mg/L dose level was observed dead and stuck to the side of the test vessel above the test solution surface. It is not certain whether this individual organism died as a result of the chemical exposure or from getting stuck to the vessel during Daphnia loading or test vessel placement. Therefore, to remain conservative, this immobile (dead) Daphnia was included in the statistical evaluation of the 24- and 48-hour data. Following 48-hours of exposure, immobility was observed in 100% (20/20), 100% (20/20), 10% (2/20), and 25% (5/20) of the Daphnia at the 11.7, 8.41, 3.73, and 2.17 mg/L dose levels, respectively. No immobility was observed in the 1.02 mg/L and below dose levels or in the water control during the conduct of the study.

The resulting acute toxicity values for trimethyl nonanol with Daphnia magna were statistically evaluated, where applicable, using mean measured concentrations and are listed below:
· The 24-hour EC50 value was 6.22 mg/L, with a 95% confidence interval of 5.07–7.63 mg/L; the Spearman-Karber trim was 20%.
· The 48-hour EC50 value was 4.44 mg/L, with a 95% confidence interval of 3.79–5.21 mg/L; the Spearman-Karber trim was 0%.
· The 48-hour NOEC was 1.02 mg/L and was determined based on biological interpretation of the data and the highest exposure level exhibiting no Daphnia immobility.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

The resulting acute toxicity values for trimethyl nonanol with Daphnia magna were statistically evaluated, where applicable, using mean measured concentrations and are listed below:
· The 24-hour EC50 value was 6.22 mg/L, with a 95% confidence interval of 5.07–7.63 mg/L; the Spearman-Karber trim was 20%.
· The 48-hour EC50 value was 4.44 mg/L, with a 95% confidence interval of 3.79–5.21 mg/L; the Spearman-Karber trim was 0%.

Any other information on results incl. tables

Table. Summary of Biological Observations

Trimethyl Nonanol Concentrations (mg/L)		Observations
Mean analyzed	Nominal	24 -hour	48 -hour
<LLQ^a	Water Control	20N	20N
0.480	3.13	20N	20N
1.02	6.25	20N	20N
2.17	12.5	16N, 4I	15N, 5I
3.73	25.0	20N	18N, 2I
8.41	50.0	5N, 15I	20I
11.7	100	4N, 16I	20I
EC50^b (mg/L)95% Confidence Limits (mg/L)		6.22(5.07 - 7.63)	4.44(3.79 -5.21)
Spearman-Karber Trim^b (%)		20	0
48 -hour NOEC^c (mg/L)			1.02

N = Normal, I = Immobile

^a <LLQ = Less than the lowest level quantified of 0.0982 mg trimethyl nonanol/L-ALDW.

^bThe U.S. EPA Trimmed Spearman-Karber Program, Version 1.5, was used to calculate the EC50 value, confidence limits, and corresponding percent trim values. Values determined using mean measured trimethyl nonanol concentrations.

^cDetermined based on biological interpretation of the data and the highest exposure level exhibiting no Daphnia immobility.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of 24 and 48 hours of immobility (inability to swim within 15 seconds after gentle agitation of the test container), the 24 hour EC50 value was 6.22mg/L, with a 95% confidence interval of 5.07-7.63 mg/L; the Spearman Karber trim was 20%. The 48 hour EC50 value was 4.44mg/L, with a 95% confidence interval of 3.79-5.21 mg/L; the Spearman Karber trim was 0%. The 48 hour NOEC was 1.02 mg/L and was determined based on biological interpretation of the data and the highest exposure level exhibiting no Daphnia immobility.

Executive summary:

The purpose of this study was to estimate the acute toxicity of trimethyl nonanol to the daphnid (Daphnia magna), over a 48-hour static-renewal exposure period. These data were used to calculate the 24- and 48-hour EC50 values (the concentrations estimated to immobilize 50 percent of the Daphnia after 24 and 48 hours of exposure, respectively) and a 48-hour no-observed-effect concentration or NOEC.

The study was conducted with two groups of ten Daphnia exposed to nominal test concentrations of 0 (water control), 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L, over a 48-hour exposure period. The concentration of trimethyl nonanol in the test solutions was confirmed by collecting samples on days 0 and 1 (from bulk solutions) and days 1 and 2 (from spent solutions of each replicate test vessel). The collected samples were analyzed by gas chromatography/flame ionization detection (GC/FID). Overall, the average percent recovery of nominal dose concentrations ranged from 11.7 to 17.4%. The overall average percent recovery of nominal and standard deviation values for the entire study (n = 6) was 15.4 ± 2.04%. The low percent recoveries of nominal dose concentrations observed were mainly due to the limited solubility of trimethyl nonanol in aqueous solution. The resulting mean measured trimethyl nonanol concentrations for the study were less than the lowest level quantified of 0.0982 mg/L for the water control and 0.480, 1.02, 2.17, 3.73, 8.41, and 11.7 mg/L for the treatment solutions.

Observations were made at 24 and 48 hours for immobility (inability to swim within 15 seconds after gentle agitation of the test container). These data were statistically evaluated, where applicable, using mean measured trimethyl nonanol concentrations. The resulting values are listed below:

• The 24-hour EC50 value was 6.22 mg/L, with a 95% confidence interval of 5.07–7.63 mg/L; the Spearman-Karber trim was 20%.

• The 48-hour EC50 value was 4.44 mg/L, with a 95% confidence interval of 3.79–5.21 mg/L; the Spearman-Karber trim was 0%.

• The 48-hour NOEC was 1.02 mg/L and was determined based on biological interpretation of the data and the highest exposure level exhibiting no Daphnia immobility.