2,6,8-trimethylnonan-4-ol

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 September - 3 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 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S. EPA - TSCA 40 CFR 797.1500 (1992)

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 Analysis
The bulk dose solutions were sampled for analytical confirmation on day 0 of the study immediately following preparation. For the control AAM, 1.56 and 3.13 mg/L solutions, a 10-mL aliquot was extracted with 2-mL iso-octane (5-fold enrichment). For the 6.25, 12.5,25.0, 50.0, and 100 mg/L dose levels, a 2-mL aliquot was extracted with 2-rnL isooctane. The extractions were achieved by shaking the biphasic mixtures on a flat-bed
shaker for 30 minutes. The phases were allowed to separate with centrifuging, as needed. The organic phase was then transferred to autosampler vials for analysis by gas chromatography with a flame ionization detector (GC/FID).

On day 4, the three test solutions containing algae at each dose level were pooled to provide one composite algae-containing sample per dose level for analytical confirmation. The test solutions at each dose level containing no algae were sampled separately. A 15-mL aliquot was collected from the control AAM, 1.56 and 3.13 mg/L solutions and a 5-mL aliquot was collected from the 6.25, 12.5,25.0, 50.0, and 100 mg/L solutions. The samples were centrifuged for 10 minutes at 2000 rpm in order to sediment the algae. Aliquots (10-mL from control AAM, 1.56 and 3.13 mgL; 2-mL from 6.25, 12.5,25.0, 50.0, and 100 mg/L) of the supernatant were extracted with 2-mL iso-octane by shaking on a flat-bed shaker for 30 minutes. The phases were allowed to separate with centrifuging, as needed. The organic phase was then transferred to autosampler vials for analysis by GC/FID.

Method Precision and Homogeneity
To assess analytical method precision and solution homogeneity, three additional samples were taken on day 0 from bulk dose solutions at nominal concentrations of 1.56 and 100 mg/L. These additional samples were prepared for analysis as described above and analyzed along with the other day 0 samples.

Extraction Efficiency of Trimethyl Nonanol from Algal Assay Media
The recovery (extraction efficiency) of trimethyl nonanol from AAM by solvent partitioning with iso-octane was determined by fortifying AAM with trimethyl nonanol at concentrations representative of the expected range of dose solutions and then conducting the extraction as described in "Sample Collection/Extraction and Analysis." Triplicate spikes of trimethyl nonanol in AAM were prepared on both analysis days at target concentrations of 0.972,9.63, and 96.3 mgIL AAM. This assessment of extraction efficiency yielded average recovery values of 99.6% for day 0 and 96.1 % for day 4, which were used to adjust the analyzed concentrations of the test solutions for method recovery on each analysis day, as needed.

Standard PreparationIDetector Calibration
Primary stock solutions of trimethyl nonanol were prepared in acetone at nominal concentrations of 9729 and 973 mg trimethyl nonanol/L on day 0 of the study. The stock solutions were stored at approximately 8°C and used on both analysis days. Analytical standards were prepared in iso-octane over a concentration range extending from 0.486 to 145 mg trimethyl nonanola, which encompassed the expected sample concentration range (after accounting for appropriate sample enrichment during the sample preparation). Standards were analyzed with each set of samples to define the detector response.

Response factors calculated for each of the individual analytical standards were averaged to yield an overall mean response factor and standard deviation for each daily set of analyses. The measured concentrations (mg trimethyl nonanol/L AAM) of the test solutions were then calculated as follows:

Response Factor (RF) = Standard Trimethyl Nonanol Concentration/Peak Height

Sample Concentration (mg/L) = (RF * Peak Height * Dilution Factor)/(Spike Recovery)

To provide some measure of the quality (applicability) of the daily calibration factors (mean response factors), a relative standard deviation was calculated for each daily set of analyses by dividing the standard deviation of the individual response factors by the mean response factor. The relative standard deviation values derived from the two calibrations did not exceed 8% for trimethyl nonanol.

Test solutions

Vehicle:

no

Details on test solutions:

The probe test was conducted between 04 September and 08 September 2003 using four nominal trimethyl nonanol concentrations of 0.800,4.00,20.0, and 100 mg/L, plus a medium control. Due to the low solubility of trimethyl nonanol in AAM, it was decided to prepare the 100 mg/L test solution by direct addition of trimethyl nonanol to AAM in a separatory funnel. This bulk solution was mixed vigorously and then allowed to settle for
one hour to allow the biphasic mixture (top layer of insoluble test material versus bottom layer of AAM containing solubilized test material) to separate. After one hour, the bottom (soluble test material layer) of the solution was decanted and used as the highdose level. The remaining test solutions were prepared as serial dilutions of the 100 mg/L test solution. Percent inhibition compared to controls was -13,20, 35, and 96% for the nominal 0.800,4.00, 20.0, and 100 mg/L test levels, respectively (negative percent inhibition values indicate stimulation of growth). Based on this, the empirically determined 4-day EC50 value was between 20.0 and 100 mg trimethyl nonanon. The information derived from this test was used to set the range of concentrations for the definitive test.

As a result, the definitive test concentrations were defined as 0 (control), 1.56, 3.13, 6.25, 12.5,25.0, 50.0, and 100 trimethyl nonanol/L AAM. The test was conducted under static exposure conditions with test vessels (250-mL Erlenmeyer flasks, four replicate vessels per dose) containing AAM dosed at the target concentrations.

Test solutions for the definitive test were prepared in the same manner as the probe test. A 100 mg trimethyl nonanol/L solution was prepared via direct addition of trimethyl nonanol to AAM in a separatory funnel. Since the test material was a liquid, the weight of the test material added to the AAM 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). This was then adjusted for purity of the compound (95.7%). Based on this, a 0.1275-mL aliquot of trimethyl nonanol was added to 1000 mL in a 2-L separatory funnel. The funnel was stoppered and shaken vigorously to maximize dissolution of the test material in AAM. After shaking, the solution was allowed to settle for approximately one hour to allow the biphasic mixture (top layer of insoluble test material versus bottom layer of AAM containing solubilized test material) to separate. The bottom (soluble test material layer) of the solution was then decanted and used as the high dose solution and
to make subsequent serial dilutions for the remaining test levels. 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 AAM. The flask was stoppered and the solution mixed thoroughly. Subsequent bulk dose solutions (25.0, 12.5,6.25,3.13, and 1.56 mgL) were similarly prepared as serial dilutions of the next highest dose solution. The control dose solution was AAM with no test material added.

Test organisms

Test organisms (species):

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

Details on test organisms:

Axenic samples of Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) were received on 13 February 2003 from the University of Toronto Culture Collection at the University of Toronto, Toronto, Ontario, Canada. Stock cultures of this organism were maintained aseptically by weekly transfer into sterile AAM.

The algal inoculum was prepared from a 3-day old stock culture of Pseudokirchneriella subcapitata.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No data

Test conditions

Hardness:

No data

Test temperature:

24.7 - 24.8C

pH:

Day 0 range - 7.0 to 7.1
Day 4 with algae range - 8.9 to 9.8
Day 4 without algae range - 7.0 to 7.1

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

See Table below

Details on test conditions:

Exposure Phase
The definitive test was conducted from 29 September to 03 October 2003. Four replicate test vessels were prepared per test concentration, each containing 100 mL of test solution. Three replicates at each test concentration and the control group were inoculated with approximately 10,000 cells/mL. Inoculations were made after all the replicate test vessels at each test concentration were poured. The fourth replicate at each test concentration
and the control group was not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the interference of the test material and to monitor pH and concentration of the test material without the algal biomass. The exposure phase was carried out aseptically under static conditions for 4 days (approximately 96 hours). The replicate test flasks were placed in a walk-in environmental chamber (Lab-Line Environmental Chamber, Lab-Line Inc., Melrose, Illinois) according to a computer-generated randomization scheme. The replicate test flasks were randomized daily after sampling for cell counts. The incubator was thermostated at 24 +/- 2C with continuous light at approximately 8000 +/- 1600 lux.

Physical Analysis
At test initiation, a pH measurement was taken from a sample of the bulk preparation of each test concentration and the control. At test termination, a final pH measurement was taken from a pooled sample of the replicates with algae at each test concentration and the control and from each blank replicate. The incubator temperature was continuously monitored with a Fischer minimum/maximum thermometer probe placed in a representative vessel within the incubator. The light intensity was monitored daily at positions corresponding to the test flasks in the incubator with a VWR Dual Display Light Meter.

Density DeterminationsIObservations
Algal cell densities of the initial inoculum and test cultures were determined by electron particle counting using a Coulter Multisizer 3. Total cell counts were determined at approximately 24,48,72, and 96 hours. Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.6 pm to a higher threshold equivalent spherical diameter of approximately 8.7 pm. Three separate cell count readings were made per replicate. The readings for the blank replicates were used to correct for background in
daily calculations. Since there was no test material effect on the blanks, the mean of all the blanks was calculated and used to adjust the cell counts. The adjusted cell counts were converted to cells x 10000/mL (cell density) for statistical analysis and reporting.

In addition, at test termination morphological observations were done on a composite sample of the three inoculated replicates at each test concentration. The cells were observed under a microscope (Olympus@ BHB-DO System Microscope; 20x or 40x objective lens; WF10x eyepiece; 1.25Dual Observation Deck) using a Bright Line Hemacytometer Counting Chamber

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.63 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Remarks on result:

other: 95% CI 2.21 - 7.04 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.252 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: cell density

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Effect Concentration is the approximate value, empirically estimated. Calculated values were >10.0 mg/L, but 53% effect was noted at 10.0 mg/L.

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.503 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.54 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 0.968 - 6.68 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.252 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

6.09 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Cell Density

Remarks on result:

other: 95% CI 4.17 - 8.01 mg/L

Effect concentrations 8

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.252 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Cell Density

Effect concentrations 9

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 10

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.869 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 11

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

3.55 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI 0.915 - 13.8 mg/L

Effect concentrations 12

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.252 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Chemical Analysis
Results from the day 0 analysis yielded percent of target values for the bulk dose solutions ranging from 20.0 to 30.6%, with an average percent of target of 27.7 +/- 3.61%. The low recoveries are likely due to the limited solubility of trimethyl nonanol in water. Trimethyl nonanol was not present at quantifiable concentrations in any of the day 4 test solutions (with or without algae). Mean measured concentrations were calculated for all dose levels by averaging the day 0 bulk dose solution concentrations and the day 4 exposure solution concentrations. However, since the day 4 measured concentrations were all less than the lowest level quantified (LLQ) of 0.0972 mg trimethyl nonanol/L AAM, a value of 0.0486 mg/L (equivalent to 1/2 LLQ) was used in the calculations as a conservative estimate of day 4 exposure concentrations. The mean measured concentrations ranged from 10.0 to 16.2 percent of target, with an overall study average mean measured value across all dose levels (n=7) of 14.3 +/- 2.10 percent of target.

None of the analyses of the AAM controls exhibited a peak eluting at the retention time of trimethyl nonanol at concentrations exceeding the lowest level quantified of 0.0972 mg/L AAM.

Method Precision and Solution Homogeneity
The variability associated with the analytical method, as well as solution homogeneity, was assessed on day 0 of the study. Four replicate samples were collected from day 0 bulk dose solutions at nominal concentrations of 1.56 and 100 mg trimethyl nonanol/L AAM. Four repeated measurements (4 samples x 1 injectionlsample) resulted in percent RSD values of 1.47 and 7.05% in the low and high samples, respectively.

Linearity
The GC/FID instrumentation exhibited a linear response over the concentration range extending from 0.486 to 145 mg trimethyl nonanolll iso-octane.

Lowest Level Ouantified
The lowest level quantified was set at 0.0972 mg trimethyl nonanol/L AAM, based on the concentration of the lowest standard analyzed multiplied by the lowest dilution factor.

Test Conditions
Temperatures during the exposure period ranged from 24.7C to 24.8C. The mean (+/- standard deviation) light intensity was 7645 +/- 528 lux, with a range of 6450 - 8600 lux. The pH values ranged from 7.0 to 7.1 at test initiation, from 8.9 to 9.8 in replicates with algae at test termination, and from 7.0 to 7.1 in blank replicates without algae.

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

Mean cell densities at 72 hours were 173.3, 161.8, 137.6, 129.6, 106.7, 104.5, 53.49, and 11.26 x 10(4) cells/mL for the control, 0.252, 0.503, 0.869, 1.86, 3.76,6.97, and 10.0 mg/L test levels, respectively. Response relative to the controls ranged from 7% to 94% inhibition of growth. The 72-hour calculated EC25 and EC50 values (95% confidence intervals) for cell density were 1.63 (< 0.252-4.04) and 4.63 (2.21-7.04) mg/L , respectively. Based on the Dunnett's test, the 72-hour cell density was significantly less than the controls at test levels >/- 0.503 mg/L; therefore, the 72-hour NOEC value for cell density was determined to be 0.252 mg/L.

Mean cell densities at 96 hours were 476.7, 449.3, 424.7, 406.2, 356.6, 349.1, 237.7, and 75.32 x 10(4) cells/ml for the control, 0.252, 0.503, 0.869, 1.86, 3.76, 6.97, and 10.0 mg/L test levels, respectively. Response relative to the controls ranged from 6% to 84% inhibition of growth. The 96-hour calculated EC25 and EC50 values (95% confidence intervals) for cell density were 2.73 (0.839-4.62) and 6.09 (4.17-8.01) mg/L, respectively. Based on the Dunnett's test, the 96-hour cell density was significantly less than the controls at test levels >/- 0.503 mg/L ; therefore, the 96-hour NOEC value for cell density was determined to be 0.252 mg/L.

Mean specific growth rates at 72 hours were 1.718, 1.694, 1.641, 1.619, 1.555, 1.549, 1.32 1, and 0.805 day(-1) for the control, 0.252, 0.503, 0.869, 1.86, 3.76, 6.97, and 10.0 mg/L test levels, respectively. Response relative to the controls ranged from 1% to 53% inhibition of growth rate. Based on the estimation model used, the 72-hour calculated ErC50 value (95% confidence intervals) for specific growth rate was > 10.0 mg/L, the highest level tested. However, since there was a 53% effect at the10.0 mg/L test level (the highest test level tested), an approximate 72-hour ErC50 value of 10.0 mg/L can be empirically determined. Based on the Dunnett's test, the 72-hour specific growth rate was significantly less than the controls at test levels >/- 0.869 mg/L; therefore, the 72-hour NOEC value for specific growth rate was determined to be 0.503 mg/L.

Mean specific growth rates at 96 hours were 1.542, 1.527, 1.513, 1.501, 1.469, 1.463, 1.365, and 1.079 day(-1) for the control, 0.252, 0.503, 0.869, 1.86, 3.76, 6.97, and 10.0 mg/L test levels, respectively. Response relative to the controls ranged from 1 % to 30% inhibition of growth rate. The 96-hour calculated ErC50 value for specific growth rate was > 10.0 mg/L, the highest level tested. Based on the Dunnett's test, the 96-hour specific growth rate was significantly less than the controls at test levels >/- 1.86 mg/L; therefore, the 96-hour NOEC value for specific growth rate was determined to be 0.869 mg/L .

Mean biomass area values at 72 hours were 3170, 3038, 2613, 2403, 1937, 1798, 896, and 187 for the control, 0.252, 0.503, 0.869, 1.86, 3.76, 6.97, and 10.0 mg/L test levels, respectively. Response relative to the controls ranged from 4% to 94% inhibition of biomass area. The 72-hour calculated EbC50 value (95% confidence intervals) for biomass area was 2.54 (0.968-6.68) mg/L. Based on the Dunnett's test, the 72-hour biomass area was significantly less than the controls at test levels >/- 0.503 mg/L ; therefore, the 72-hour NOEC value for biomass area was determined to be 0.252 mg/L .

Mean biomass area values at 96 hours were 10945, 10348, 9336, 8809, 7473, 7216, 4366, and 1201 for the control, 0.252,0.503, 0.869, 1.86, 3.76, 6.97, and 10.0 mg/L test levels, respectively. Response relative to the controls ranged from 5% to 89% inhibition of biomass area. The 96-hour calculated EbC50 value (95% confidence intervals) for biomass area was 3.55 (0.915-13.8) mg/L . Based on the Dunnett's test, the 96-hour biomass area
was significantly less than the controls at test levels 2 0.503 mg/L; therefore, the 96-hour NOEC value for biomass area was determined to be 0.252 mg/L.

Microscopic evaluation of cells at each test concentration and the control revealed no abnormal observations

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

The 72-hour results, based on mean measured trimethyl nonanol concentrations, were as follows:
The 72-hour EC25, EC50, and NOEC values for cell density were 1.63, 4.63, and 0.252 mg/L, respectively.
The 72-hour ErC50 and NOEC values for growth rate (day(-1)) were approximately 10.0 and 0.503 mg/L, respectively.
The 72-hour EbC50 and NOEC values for biomass area (area under the growth curve) were 2.54 and 0.252 mg/L, respectively.

The 96-hour results, based on mean measured trimethyl nonanol concentrations, were as follows:
The 96-hour EC25, EC50, and NOEC values for cell density were 2.73, 6.09, and 0.252 mg/L, respectively.
The 96-hour ErC50 and NOEC values for growth rate (day(-1)) were > 10.0 and 0.869 mg/L, respectively.
The 96-hour EbC50 and NOEC values for biomass area (area under the growth curve) were 3.55 and 0.252 mg/L, respectively.

For error estimates see results above

Any other information on results incl. tables

Table. Mean Cell Density (x 10⁴ cells/ml) of Pseudokirchneriella subcapitata after

24, 48, 72, and 96 Hours of Exposure to Trimethyl Nonanol

Trimethyl Nonanol Concentration (mg/L)		Mean Cell Density (x 10^4 cells/mL) + S.D.Exposure Hour
Nominal	Mean Measured	24	48	72	% Inhib.^1	96	% Inhib.
Control	<LLQ^2	6.844 + 1.002	41.13 + 3.01	173.3 + 12.3	---^3	476.7 + 15.0	---
1.56	0.252	6.415 + 0.388	41.77 + 1.91	161.8 + 16.0	7	449.3 + 17.8	6
3.13	0.503	5.882 + 0.252	36.72 + 5.50	137.6 + 2.6*	21	424.7 + 13.9*	11
6.25	0.869	5.542 + 0.185	32.27 + 1.26	129.6 + 20.4*	25	406.2 + 36.1*	15
12.5	1.86	4.670 + 0.364	25.20 + 2.79	106.7 + 12.8*	38	356.6 + 17.5*	25
25.0	3.76	3.941 + 0.521	21.24 + 1.81	104.5 + 8.6*	40	349.1 + 25.3*	27
50.0	6.97	2.175 + 0.155	10.90 + 2.82	53.49 + 11.72*	69	237.7 + 39.6*	50
100	10.0	1.865 + 0.508	2.778 + 0.434	11.26 + 1.59*	94	75.32 + 9.35*	84

¹ % Inhib. = Percent inhibition from control.

² Less than the lowest level quantified (0.0972 mg trimethyl nonanol/L).

³---- = not applicable, % inhibitions are based on control values.

*: Significant difference from control; p < 0.05, one tailed Dunnett's t-test on raw data.

Table. Mean Specific Growth Rate (day^-1) of Pseudokirchneriella subcapitata after 72

and 96 Hours of Exposure to Trimethyl Nonanol

Trimethyl Nonanol Concentration (mg/L)		72 -hour Specific		96 -hour Specific
Nominal	Mean Measured	Growth Rate (day^-1)	% Inhibition^1	Growth Rate (day^-1)	% Inhibition
Control	<LLQ^2	1.718 + 0.023	---^3	1.542 + 0.008	---
1.56	0.252	1.694 + 0.032	1	1.527 + 0.010	1
3.13	0.503	1.641 + 0.006	4	1.513 + 0.008	2
6.25	0.869	1.619 + 0.052*	6	1.501 + 0.023	3
12.5	1.86	1.555 + 0.040*	9	1.469 + 0.012*	5
25.0	3.76	1.549 + 0.027*	10	1.463 + 0.018*	5
50.0	6.97	1.321 + 0.079*	23	1.365 + 0.041*	11
100	10.0	0.805 + 0.049*	53	1.079 + 0.030*	30

¹ % Inhibition = Percent inhibition from control.

² Less than the lowest level quantified (0.0972 mg trimethyl nonanow).

³---- = not applicable, % inhibitions are based on control values.

*: Significant difference from control; p < 0.05, one tailed Dunnett's t-test on raw data.

Table. Mean Biomass (Area Under the Growth Curve) of Pseudokirchneriella

subcapitata after 72 and 96 Hours of Exposure to Trimethyl Nonanol

Trimethyl Nonanol Concentration (mg/L)		72 -hour Biomass		96 -hour Biomass
Nominal	Mean Measured	(area under the growth curve)	% Inhibition^1	(area under the growth curve)	% Inhibition
Control	<LLQ^2	3170+162	---^3	10945+325	---
1.56	0.252	3038+224	4	10348+431	5
3.13	0.503	2613+102*	18	9336+174*	15
6.25	0.869	2403+240*	24	8809+887*	20
12.5	1.86	1937+159*	39	7473+455*	32
25.0	3.76	1798+131*	43	7216 +442*	34
50.0	6.97	896+208*	72	4366+780*	60
100	10.0	187+18*	94	1201 +1193*	89

¹% Inhibition = Percent inhibition from control.

² Less than the lowest level quantified (0.0972 mg trimethyl nonanol/L).

³---- = not applicable, % inhibitions are based on control values.

*: Significant difference from control; p < 0.05, one tailed Dunnett's t-test on raw data.

Table Summary Results of Trimethyl Nonanol Algae Study

Endpoint:

Mean Measured Trimethyl Nonanol Concentration (mg/L)

72 -Hour Cell Density:
EC25	1.63 (<0.252 -4.04)^a
EC50	4.63 (2.21 - 7.04)
NOEC	0.252
72-Hour Specific Growth Rate:
ErC50	10.0^b
NOEC	0.503
72 -Hour Biomass Area:
EbC50	2.54 (0.968 - 6.68)
NOEC	0.252
96 -Hour Cell Density:
EC25	2.73 (0.839 - 4.62)
EC50	6.09 (4.17 - 8.01)
NOEC	0.252
96 -Hour Specific Growth Rate:
ErC50	>10.0
NOEC	0.869
96 -Hour Biomass Area:
EbC50	3.55 (0.915 - 13.8)
NOEC	0.252

^a 95% Confidence Interval.

^b Approximate value, empirically estimated; calculated values were >10.0 mg/L, but 53% effect was noted at 10.0 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the 72-hour results, mean measured trimethyl nonanol concentrations were, the 72 hour EC25, EC50 and NOEC values for cell density were 1.63, 4.63, and 0.252 mg/L respectively. The 72 hour ErC50 and NOEC values for growth rate (per day) were approximately 10.0 and 0.503 mg/L, respectively. The 72 hour EbC50 and NOEC values for biomass area (area under the growth curve) were 2.54 and 0.252 mg/L, respectively.
The 96 hour results, based on mean measured trimethyl nonanol concentrations were, the 96 hour EC25, EC50 and NOEC values for cell density were 2.73, 6.09 and 0.252 mg/L respectively. The 96 hour ErC50 and NOEC values for growth rate(per day) were >10.0 and 0.869 mg/L, respectively. The 96 hour EbC50 and NOEC values for biomass area ( area under the growth curve)were 3.55 and 0.252 mg/L, respectively.

Executive summary:

TEST SUBSTANCE: Trimethyl Nonanol, Batch No. RC1355T3D4, a transparent pale yellow liquid, reported to have a measured purity of 95.7%.

DEFINITIVE TEST DATES: 29 September to 03 October 2003

TEST ORGANISM: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricomutum), inoculum - three days since

previous transfer. Source - In-house culture.

TEST CONDITIONS: 96-hour duration. Temperatures during the exposure period ranged from 24.7^oC to 24.8^oC. The mean (+ standard deviation) light intensity was 7645 + 528 lux, with a range of 6450 - 8600 lux. The pH values ranged from 7.0 to 7.1 at test

initiation, from 8.9 to 9.8 in replicates with algae at test termination, and from 7.0 to 7.1 in blank replicates without algae. Shaking rate was set at 100 rpm

DILUTION WATER: The growth and test medium used was algal assay medium (AAM) designated for the EPA algal assay bottle test.

NOMINAL TEST CONCENTRATIONS: Seven trimethyl nonanol exposure concentrations of 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg/L AAM, and a medium control group were tested.

MEAN MEASURED CONCENTRATIONS: Analytical reesidues at test initiation were 0.455, 0.957, 1.69, 3.68, 7.48, 13.9, and 20.0 mg trimethyl nonanol/L for the nominal concentrations of 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg trimethyl nonanol/L, respectively. Recoveries on day 0 ranged from 20.0 to 30.6% of nominal. Recovery in the control was less than 0.0972 mg trimethyl nonanol/L, the lowest level quantified (LLQ). Analytical residues at test termination (day 4) were less than the LLQ at all test concentrations and the control. Based on this, mean measured concentrations were calculated for each test concentration using day 0 recoveries and one half the day 4 LLQ (0.0486 mg trimethyl nonanolL). Mean measured concentrations were 0.252, 0.503, 0.869, 1.86, 3.76, 6.97, and 10.0 mg trimethyl nonanol/L for the nominal concentrations of 1.56, 3.13, 6.25, 12.5, 25.0, 50.0, and 100 mg

trimethyl nonanol/L, respectively. The mean measured concentrations were used for statistical analysis of the biological data.

EFFECT CRITERIA: Inhibition of growth (cell density) at 72 and 96 hours (EC25 and EC50), inhibition of growth rate (day(-1)) for the 0- to 72-hour and 0- to 96-hour intervals (ErC50), and inhibition of biomass area (area under the control growth curve) at 72 and 96 hours (EbC50), relative to the controls.

Results

Endpoint:

Mean Measured Trimethyl Nonanol Concentration (mg/L)

72 -Hour Cell Density:
EC25	1.63 (<0.252 -4.04)^a
EC50	4.63 (2.21 - 7.04)
NOEC	0.252
72-Hour Specific Growth Rate:
ErC50	10.0^b
NOEC	0.503
72 -Hour Biomass Area:
EbC50	2.54 (0.968 - 6.68)
NOEC	0.252
96 -Hour Cell Density:
EC25	2.73 (0.839 - 4.62)
EC50	6.09 (4.17 - 8.01)
NOEC	0.252
96 -Hour Specific Growth Rate:
ErC50	>10.0
NOEC	0.869
96 -Hour Biomass Area:
EbC50	3.55 (0.915 - 13.8)
NOEC	0.252

^a95% Confidence Interval.

^bApproximate value, empirically estimated; calculated values were >10.0 mg/L, but 53% effect was noted at 10.0 mg/L.