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DuPont-11954
TRADE SECRET
Study Title H-24768: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
Test Guidelines: OECD Guideline for the Testing of Chemicals Section 2: Effects on Biotic Systems, Number 201 (1984)
Commission Directive 92/69/EEC EEC Method C3 (1992)
Author: Terry Lee Sloman, B.S.
Study Completed on: October 31,2003
Performing Laboratory: E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan Number 3850).
The test substance was characterized by the sponsor prior to the initiation o f this study. Although the characterization was not performed under Good Laboratory Practice Standards, the characterization was done by an ISO 9000 certified laboratory, and the accuracy o f the data is considered sufficient for the purposes of this study.
Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.
Study Director:
iJLvw terryLee Sloman, B.S. Associate Scientist
3>l-QcT~- 2.Q03. Date
Applicant / Sponsor:_________________________________________ __________
DuPont Representative
Date
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
QUALITY ASSURANCE STATEMENT
Haskell Sample Number(s): 24768
Dates of Inspections: Protocol: July 10, 2003 Conduct: July 23, 2003
Records, Reports: October 8, 9, 2003
Dates Findings Reported to: Study Director: October 13, 2003 Management: October 13, 30, 2003
DuPont-11954
Reported by:
Staff Quality Assurance Auditor
Date
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
CERTIFICATION
We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study.
Analytical Evaluation by:
( Bogdan Szostek, Fh.D. Senior Research Chemist
Date
Statistical Analysis Reported by:
(4L S ' * ' - '
__________
' John W. Green, Ph.D.,Ph.D.
Principal Research Statistician
Z c r o J ~ 2- o o z Date
Issued by Study Director: ' l j U w
q
Terry Lee Slamati, B.S. Associate Scientist
M - i X T ' - U )<?3 Date
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
TABLE OF CONTENTS
Page
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT...................................2
QUALITY ASSURANCE STATEMENT......................................................................................3
CERTIFICATION............................................................................ .................................................. 4
LIST OF TABLES.......................... ................................................. .................................................. 6
LIST OF FIGURES..........................................................................
6
LIST OF APPENDICES..................................................................................................................... 6
STUDY INFORMATION...................................................................................................................7
STUDY PERSONNEL......................................................................................................................... 8
SUMMARY........................................................................................................................................... 9
INTRODUCTION...............................................................................
10
MATERIALS AND METHODS.....................................................
10
A. Test Guidelines.... ...................................................................................
10
B. Test Substance................................. ........................... .............. ,............. ............................. 10
C. Test System..................................................................................................
10
D. Test D esign.... ...............................
11
E. Test Preparation..........................
11
F. Experimental Design................................................................................................................ 12
G. Test Solutions........... .............
12
H. Test Conditions - Definitive and Recovery T ests..................................................................12
I. Selenastrum capricomutum Growth Measurement......................... ....................................13*
J. Statistical Analyses......................................................................
13
K. Analysis of Test Solutions - Definitive Test...........................
14
RESULTS AND DISCUSSION........................................................................................................18
A. Environmental Conditions - Definitive and Recovery T ests................................................ 18
B. Test Substance Stability and Analytical Verification............................................................18
C. Selenastrum capricomutum Growth.............................................................
19
CONCLUSIONS.................................................................................................................................20
RECORDS AND SAMPLE STORAGE...........................................
20
REFERENCES......................................................................................
21
TABLES............................................................................................................................................... 22
FIGURES........................................................................................
31
^ APPENDICES.....................................................................................................................................44
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
LIST OF TABLES
Page
1. pH MEASUREMENTS OF TEST SOLUTIONS........................................................................................................23 2. CHAMBER LIGHT INTENSITY, SHAKING SPEED, AND TEMPERATURE JRANGE.................................23 3. MEASURED CONCENTRATIONS OF H-24768 IN TEST SOLUTIONS........................................................... 24 4. HEALTHY CELL COUNT DATA SUMMARY - DEFINITIVE TEST................................................................ 27 5. AREA UNDER THE GROWTH CURVE DATA SUMMARY - DEFINITIVE TEST....................................... 28 6. GROWTH RATE DATA SUMMARY - DEFINITIVE TEST .................................. .........................................29 7. 72-HOUR (DAY 3) STATISTICAL ANALYSES SUMMARY - DEFINITIVE TEST....................................... 30
LIST OF FIGURES
Page
2. REPRESENTATIVE CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION.......................... 35 3. REPRESENTATIVE CHROMATOGRAM OF THE BLANK CONTROL SOLUTON.......................................37 4. REPRESENTATIVE CHROMATOGRAM OF A TEST CONCENTRATION SOLUTION............................... 39 5. 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE FOR THE DEFINITIVE TEST................ 41 6. 72-HOUR AREA UNDER THE GROWTH CURVE DOSE-RESPONSE CURVE FOR THE
DEFINITIVE TEST..........................................................................................................................................................42 7. 72-HOUR GROWTH RATE DOSE-RESPONSE CURVE FOR THE DEFINITIVE TEST............................... 43
LIST OF APPENDICES
Page
A. A AAP NUTRIENT MEDIUM CONSTITUENTS....................................................................................................45 B. 72-HOUR HEALTHY CELL COUNT STATISTICS FOR THE DEFINITIVE TEST........................................ 47 C. 72-HOUR AREA UNDER THE GROWTH CURVE STATISTICS FOR THE DEFINITIVE TEST............... 52 D. 72-HOUR GROWTH RATE STATISTICS FOR THE DEFINITIVE TEST.........................................................56
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
STUDY INFORMATION
DuPont-11954
Haskell Number: 24768 CAS Registry Number:
Composition:
Purity: Known Impurities:
Physical Characteristics: Tan to light brown paste Stability: The test substance stability was evaluated based on analytical measurements conducted during the study. Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.
Study Initiated/Completed: July 3, 2003 / (see report cover page) Experimental Initiated/Completed: July 22, 2003 / July 25, 2003
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
STUDY PERSONNEL Study Director: Teny Lee Sloman, B.S.
Management: Robert A. Hoke, Ph.D.
Analytical Chemist: Bogdan Szostek, Ph.D. Management: S. Mark Kennedy, Ph.D.
Statistical Analysis: John W. Green, Ph.D., Ph.D. Management: Janice L. Connell, M.S., B.A., C.I.H.
Ecotoxicology Report Preparation: Wanda F. Dinbokowitz Management: Nancy S. Selzer, M.S.
D u P o n t-11954
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
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SUMMARY
A study was conducted to determine the effect o f H-24768 on the growth and growth rate o f the green alga Selenastrum capricornutum.s The algae were exposed to nominal concentrations of 6.25,12.5,25, 50, and 100 mg H-24768/liter o f nutrient medium (ppm).
For the definitive (dose-response) test, the organisms were exposed for 72 hours (3 days) without test medium renewal. The effect was expressed as percent inhibition in growth based on healthy cell count (cell density), area under the growth curve, and growth rate relative to the blank (normal culture medium) control for the 72-hour (day 3) interval o f the test.
The 72-hour results,15based on nominal concentrations are as follows:
Healthy Cell Count EC50 NOECc
> 100 mg H-24768/L 25 mg H-24768/L
Area Under the Growth Curve
EC50
> 100mgH-24768/L
NOECc
25 mg H-24768/L
Growth Rate EC50 NOECc
>100 mg H-24768/L 25 mg H-24768/L
None o f the definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore, a recovery test was not performed.
a Also known as Pseudokirchneriella subcapitata, b The EC50 is defined as the "effective concentration" producing a 50% inhibition o f growth relative to the
control. The NOEC is defined as the highest concentration o f test substance that has no significant effect on the measured parameter relative to the control, c As determined by the Jonckheere trend test.
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
INTRODUCTION
This study was conducted to determine the effect o f H-24768 on the growth and growth rate o f the green alga Selenastrum capricomutum.a
MATERIALS AND METHODS
A. Test Guidelines The study design complies with the following test guidelines:
Organisation for Economic Co-Operation and Development (OECD) (1984). 201 Effects on Biotic Systems. Guidelinefo r the Testing o f Chemicals.
European Economic Communities (EEC) (1992). Directive 92/69/EEC Annex V, Part C.3, Algal Inhibition Test. Methodsfo r the Determination o f Toxicity.
B. Test Substance The test substance, H-24768, was supplied by the sponsor as a tan to light brown paste.
C. Test System Selenastrum capricomutum, a freshwater, unicellular, non-motile, green alga, was used in this study.
1. Original Culture Source The original culture source was the Department o f Botany - Culture Collection o f Algae - The University of Texas at Austin - Austin, Texas 78713-7640.
2. Culture Maintenance The culture method for Selenastrum capricomutum was based on published literature/ 1'2-1 Prior to the study, Selenastrum capricomutum cultures were maintained under the same environmental conditions used in the study. The organisms were cultured in sterilized 250 mL Erlenmeyer flasks containing approximately 50 mL of filtered (= filter-sterilized) AAP nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with foam stoppers to permit gas exchange.
a Also known as Pseudokirchneriella subcapitata.
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
D. Test Design
DuPont-11954
The test design used for the Selenastrum capricornutum definitive and recovery tests is described below:
Organism
Nutrient Flask Volume Solution Volume Volume
Medium
(mL)
(mL)
Ratio
Selenastrum capricornutum AAP
250
50 5:1
E. Test Preparation (Appendix B)
1. AAP Nutrient Medium Preparation for Selenastrum capricornutum
AAP nutrient medium(1) was prepared by adding 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution to approximately 800 mL of Milli-Q (deionized) water, with mixing after each addition. The volume of the medium was brought to 1 liter with additional Milli-Q water. Appropriate proportions (1 mL of each stock solution for each 1 liter of medium) were used to prepare the larger volumes o f the medium required for the definitive and recovery tests.
The medium pH was adjusted to 7.52 with 0. IN sodium hydroxide. For both the definitive and recovery tests, the medium was filter-sterilized using Coming pre-sterilized filtration systems each with a 0.22 pm cellulose acetate filter. The containers with the resulting filter-sterilized AAP nutrient medium for use in the definitive and recovery tests were stored in the refrigerator in the dark at approximately 4C and acclimated to room temperature prior to use. Any remaining unused medium was properly stored.
2. Test Solution Preparation
Prior to the initiation o f the definitive test, a range-finding test was conducted to determine the appropriate concentrations to be used in the definitive test.
A working stock solution was prepared by dissolving the test substance in 1000 mL filtersterilized AAP nutrient medium for a nominal concentration o f 100 mg H-24768/L (= 100 ppm). Aliquots of the filter-sterilized AAP nutrient medium were used for the blank (normal culture medium) control solution. Test solutions were prepared using aliquots o f the nominal 100 mg/L working stock solution and diluting with filter-sterilized AAP nutrient medium to make nominal concentrations of 6.25, 12.5,25, and 50 mg H-24768/L. Aliquots o f the nominal 100 mg/L working stock solution were used for the 100 mg H-24768/L test concentration solution and the abiotic (stability) control solution.
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
3. Test Culture Preparation
For each definitive control and test solution, 3 aliquots (50 mL each) were placed in separate sterilized 250 mL Erlenmeyer flasks fitted with sterilized foam stoppers. Each flask, excluding the abiotic control, was randomly assigned a number to eliminate bias while counting. To achieve the desired nominal concentration o f approximately 10,000 Selenastrum capricornutum cells/mL at test initiation, an approximate 0.340 mL (= 340 pL) aliquot of algal inoculum from a logarithmically growing stock culture was aseptically transferred to each flask, except the abiotic control.
F. Experimental Design
1. Definitive Test
The organisms were exposed for 72 hours (3 days), without test medium renewal. Test flasks were arranged on a lighted shaker table in a chamber in a non-systematic design and were re positioned each working day. Each blank control, test concentration, and abiotic control was tested as 3 replicates. Cell counts were made approximately 24, 48, and 72 hours after the definitive test initiation (0-hour or day 0).
2. Recovery Test
Since none of the definitive test concentration exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control, a recovery test was not necessary.
G. Test Solutions Solutions used in the definitive test were as follows: Blank Control AAP nutrient medium containing no H-24768
Treatment
nominal 6.25, 12.5,25, 50, and 100 mg H-24768/liter nutrient medium
Abiotic Control nominal 100 mg H-24768/liter nutrient medium; (no Selenastrum capricornutum)
H. Test Conditions - Definitive and Recovery Tests
The control and test flasks were placed in a chamber (Hotpack model 06084) and air temperature in the chamber was recorded continuously with a continuous temperature recorder (Dickson Model SL445C7). The algae were incubated for 72 hours without test medium renewal for the definitive test. Illumination was supplied by cool-white fluorescent tubes. The target environmental parameters are described in the following table.
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H-24768: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
DuPont-11954
Photo- Shaking
Initial
Illumination Period Speed Temperature
Test
Population
(Iumens/m2= lux) (hours) (rpms)
(C)
Definitive 10,000 cells/mL
6000 to 10,000
24
100
24 2
Recovery
variable; based on definitive test, termination-day counts from selected test concentrations
6000 to 10,000
24
100
24 2
I. Selenastrum capricornutum Growth Measurement
1. Definitive Test
Selenastrum capricornutum growth measurement was determined by visually counting the number of cells taken from an approximate 0.2 mL sample from each flask at approximately 24, 48, and 72 hours after the definitive test initiation. The counts were conducted using a hemacytometer and a compound microscope. An aliquot of each sample was loaded into the 2 grid areas of the hemacytometer and 8 squares from each grid area (16 total squares) were selected for counting. All cells located in the 16 squares were counted and recorded as healthy or unhealthy. The total number of cells counted was multiplied by 10,000 to determine the number of cells per milliliter. Cells outside these 16 squares were not counted nor included in the total number. Observations and counts of healthy and unhealthy cells (e.g., deformed, scenescent, stunted) were recorded in the study records. Most counts were made at approximately the same time each day. Statistical calculations of the EC50 and NOEC were based on mean healthy cell counts and nominal concentrations.
J. Statistical Analyses
The mean healthy cell count, area under the growth curve, and growth rate at the 72-hour interval for each test concentration were expressed relative to the blank control. All calculations were based on the nominal concentrations.
The area under the growth curves for each interval were calculated according to the following formula:
A = [0.5 x A x (Ai - No)] + [0.5 x (t2 - 1\) x (A2 + A, - 2A0)] + ...+ [0.5 x (rn- fn.i) x (An+ N n.i - 2A0)]
where t\, t2, ta.1, and tn are times of observation measured from the initiation of the test and No, N h An.1, and An are the initial and subsequent healthy cell counts (cells/mL) corresponding to the observation times.
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornuium
DuPont-11954
The growth rate (p) for each test concentration and for the controls were calculated according to the following formula:
= l n ( N , / N 0) H tn
where tn is the time of observation measured from the initiation of the test and N0and Nnare the initial and subsequent healthy cell counts (cells/mL) corresponding to the observation time.
The percent growth inhibition, % I, was calculated according to the following formula:
%I =
x 100
where C is equal to the mean healthy cell count, area under the growth curve, or growth rate for the blank control at a selected sampling interval and T is the mean healthy cell count, area under the growth curve, or growth rate for each test concentration at a selected sampling interval. Negative values of inhibition indicate stimulation of growth.
The means and standard errors of the healthy cell counts, areas under the growth curve, and growth rate for each test concentration and the blank control were calculated using standard procedures.1
The healthy cell counts, areas under the growth curve, and growth rates were used to calculate the appropriate EC5ovalues. This "effective concentration" was defined as the concentration producing a 50% inhibition of growth relative to the blank control. The appropriate EC50values and associated 95% confidence limits were determined by weighted least-squares non-linear regression o f the log of the test concentration against the measured parameter. The NOEC, defined as the highest concentration of test substance that had no significant effect on the measured parameter relative to the blank control, was determined from a trend test (Jonckheere- . Terpstra) applied in a step-down manner. Such a procedure assumed a monotone dose response. All tests of significance were at a = 0.05.(4)
K. Analysis of Test Solutions - Definitive Test
1. Sample Collection
The concentrations of H-24768 in the working stock (nominal 100 mg H-24768/L), blank control, and test concentration (nominal 6.25, 12.5, 25, and 50 mg H-24768/L) solutions were verified at test initiation (day 0). An aliquot from each o f the blank control and test concentration solutions was taken prior to the addition of Selenastrum capricornuium. The pH was determined for each of the working stock, blank control, and test concentration solutions.
The concentrations o f H-24768 in the blank control, test concentration (nominal 6.25, 12.5, 25, 50, and 100 mg H-24768/L), and abiotic control (nominal 100 mg H-24768/L) solutions were verified at test termination (day 3). These samples were prepared by pooling all 3 replicates for
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
each of the control and test concentration solutions and taking an aliquot from each of the pooled samples. The pH was determined for each of these day 3 samples.
The concentrations o f H-24768 in each o f the test solutions were determined by high performance liquid chromatography/mass spectrometry (HPLC/MS).
2. Sample Treatment
An aliquot from the blank control, working stock, and test concentrations were sampled at the study start (day 0) and submitted for analysis. An aliquot from the pooled replicates of the blank control, test concentrations, and abiotic control were sampled on day 3 and submitted for analysis. Back-up solutions also were provided for each sample. All samples were analyzed on the day of receipt. The day 3 exposure samples were centrifuged in the IEC clinical centrifuge for 30 minutes in order to remove algae. Aliquots o f all test solutions were transferred to autosampler vials for analysis by LC/MS. All samples were diluted 1:1 with acetonitrile (5 mL of sample added to 5 mL acetonitrile) at the time of sampling. Additionally, the following samples were diluted with 50/50% acetonitrile/AAP nutrient medium before analysis: 6.25 mg/L (diluted 1:6.25), 12.5 mg/L (diluted 1: 12.5), 25 mg/L (diluted 1:25), 50 mg/L (diluted 1:50), 100 mg/L (diluted 1:100).
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
3. Instrumentation and Conditions
DuPont-11954
HPLC Instrument: MS Instrument:
Model 2795, Waters ZQ, Micromass
LC Parameters: Column: Mobile Phase:
Luna 3u C18 (2), 20 mm x 2mm
A - 25 mM ammonium acetate in water
B - acetonitrile
C - 25 mM ammonium acetate in acetonitrile
Gradient:
Time (min)
% A %B %c Flow (mL/min)
0
40 0
60
0.3
5 0 0 100 0.3
6.5 0 0 100 0.3
6.51
0 100 0
0.3
8.5
0 100 0
0.3
8.51
40 0
60
0.3
12
40 0
60
0.3
Column Temperature: Injection Volume: Column Switch:
30C 35 pL 1.25 and 7 min
MS Parameters: Ionization mode: Capillary voltage: Cone voltage: Source Temperature: Desolvation Temperature: Data Acquisition Function:
Electrospray, positive ions
3.5 kV
30 V
120C
500C
_
SIR of 690.5, 734.5, 778.6, 822.6,
Lz; u -/ mm
JS IR o f 790.5, 834.5, 878.6, 922.7,
96A7nFz^0-7min.
of 890.6, 934.6, 978.7,
1022.7, 1066.7 m/z; 0-7 min
4. Quantitation
-25436 and FFZ4768 chemically represent! lo f the following structure|CF,-iCF,h-CH,-CIL-0-fCH,-CH-,-QL-H. wherq] | The major components o f the mixture are derived for three Five of th ^ n o s t intense individt
/ere selected for each of the j^ ^ ^ jF o r e a c h of the|^^H considered! [five o f the most in te n s e n lH ^ B r e p r e s e n te c ^ ^ ^ ^ K i The!
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
ionized in positive ion mode electrospray predominately as the ammonium ion adducts, when the
mobile phase contains significant concentration of ammonium acetate. The quantitation was
based on the total signal obtained for five s e l c c t e d f 9 H H H | H H J | Q Separate
calibration curves wgre made for each group o f ^ H ^ ^ H H u T h e content o f each o f the
s e l e c t e l p ^ ^ H t t i the test substance was not known. The total concentration o f the test substance (either H^?5436 or H-24768) was assigned to each group o f f l H H B d m t were
monitored.
t*
A primary stock solution of the reference compound, H-2543oH | ^ ^ ^ ^ H | [ | [ ^ H [ was made in 10 mL of acetonitrile. Calibration standards were prejfjfred on each analysis day by dilution with 50/50 % acetonitrile/AAP nutrient medium at concentrations ranging from 0.103 to 1.03 mg/L. Duplicate injections of calibration solutions were made and peak heights were determined by electronic integration.
The calibration curves were generated by regression analysis of the peak height data obtained from chromatographic analysis of the calibration solutions. Data for test solutions was fitted to the calibration curve to determine the concentrations o f H-24768. The limit of detection (LOD) and limit of quantitation (LOQ) were determined by calculating the average noise level in chromatograms of blank control samples and comparing them to the signal of a calibration standard o f known concentration. Two chromatograms were examined for noise-related peaks near the retention time o f the analyte. The LOD was calculated as 3 times the concentration equivalent of the mean noise level. The LOQ was calculated as 10 times the concentration equivalent of the mean noise level.
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastmm capricornutum
D u P o n t-1 1954
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RESULTS AND DISCUSSION
A. Environmental Conditions - Definitive and Recovery Tests (Tables 1-2)
For the definitive test, the pH measurements of the test solutions ranged from 7.49 to 7.68 at the 0-hour and from 7.23 to 7.59 at the 72-hour interval.
For the definitive test, light intensity for the areas used in the chamber ranged from 6250 to 7440 lumens/m2(= lux). The mean light intensity was 6800 lumens/m2.
For the definitive test, the shaking speed was 98 revolutions per min (rpm).
For the definitive test, the temperature from the digital readout on the temperature recorder ranged from 24.7 to 24.8C for the duration o f the study.
B, Test Substance Stability and Analytical Verification (Table 3, Figures 1-4)
1. * Chromatographic Results
eluted as a well-resolved peak with a retention time of approximately
2.0 minutes.
luted as a well-resolved peak with a retention time of
approximately 3.6 minutes.YfcHMMHHHVMHwluted as a well-resolved peak with a retention
time o f approximately 5.0 minutes. Figure 1 shows representative calibration curves obtained for
Chromatograms of
typical standards are shown in Figure 2. Figures 3 and 4 show chromatograms o f a blank control
solution and test concentration solution, respectively.
The LOD and LOQ foi& ^H H M B B Q H fl^w ered etermined to be 0,005 mg/L and 0.016 mg/L of H-25436, respectively. The LOD and LOQ fo r ^ H B tiH ttH R H N |w e re determined to be 0.002 mg/L and 0.007 mg/L of H-25436, respectively. The LOD and LOQ f o i j j M H M Q
rere determined to be 0.019 mg/L and 0.062 mg/L ofH-25436, respectively.
2. Test Solution Results
The mean, measured concentration
the day 0 working stock
(= nominal 100 mg H-24768/L test concentration, = abiotic control) was 110 mg/L. This
^.represents 110% recovery of the active ingredient. The mean, measured concentration o f j ^ j
[ j S H f l H M H f r 1the day 0 working stock (=nominal 100 mg H-24768/L test concentration,
= abiotic controlf was 95 mg/L. This represents 95% recovery of the active ingredient. The
mean, measured concentration
the day 0 working stock (= nominal
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum. capricornutum
DuPont-11954
100 mg H-24768/L test concentration, ;abiotic control) was 99 mg/L. This represents 99% recovery o f the active ingredient.
The mean, measured concentrations
the day 0 nominal 6.25, 12.5, 25,
and 50 mg/L test concentration solutions were 6.3, 12, 26, and 52 mg/L, respectively. This
represents 101, 96, 104, and 104% recovery of the active ingredient, respectively. The mean,
measured concentrations o f H H B ^ H f l H f l R n the day 0 nominal 6.25, 12.5, 25, and
50 mg/L test concentration somiKm^vere 5.4, 11, 23, and 47 mg/L, respectively. This represents
86, 88, 92, and 94%o recovery of the active ingredient, respectively. The mean, measured
concentrations
the day 0 nominal6.25, 12.5, 25, and 50 mg/L test
concentration solutions were 5.3, 11, 24, and 49 mg/L, respectively. This represents 85, 88, 96,
and 98% recovery o f the active ingredient, respectively. These data indicated the stock and test
concentration solutions were prepared at the desired nominal concentrations. After 3 days, the
mean, measured concentrations
nom^na^ 6.25, 12.5,25, 50, and
100 mg/L test concentrations, and abiotic control solutions were 4.3, 8.9, 18, 35, and 73 mg/L
and 75 mg/L, respectively. This represents 69, 71, 72, 70, 73, and 75% recovery o f the active ^ _
AJngredient, respectively. The blank control solutions contained no detectable concentrations o f
| 0n both day 0 and day 3. After 3 days, the mean, measured concentrations of
[in the nominal 6.25, 12.5, 25, 50, and 100 mg/L test concentrations, and
abiotic control solutions were 3.2, 7.1, 16, 32, and 68 mg/L and 69 mg/L, respectively. This
represents 51, 57, 64, 64, 68, and 69% recovery o f the active ingredient, respectively. The blank
control solutions contained no detectable concentrations otJjpM IH H H M B BB ^on both day 0
and day 3. After 3 days, the mean, measured concentrations
the
nominal 6.25, 12.5, 25, 50, and 100 mg/L test concentrations, and abiotic control solutions were
2.9, 7.1,16, 32, and 72 mg/L and 74 mg/L, respectively. This represents 46, 57, 64, 64, 72, and
74% recovery o f the active ingredient, respectively. The blank control solutions contained no
detectable concentrations
both day 0 and day 3.
The stability o f H-24768 is difficult to assess since it is composed!
Recovery o f
the abiotic control on day 3 based on
measured concentration were 68, 73,_and 75% o f day 0 measured concentrations. These results
suggest marginal stability of thesej p H P H g p v e r the 3 day study period using 70% of day 0
sured concentrations as the benchmark for stability on day 3. Recoveries o f t h e f | | p A
jon day 3 in test solutions containing Selenastrum capricornutum
ranged from 65-74% o f day 0 measured values with the exception of the day 3 recoveries for the
___ |in the nominal 6.25 mg/L H-24768 test
"concentration.
C. Selenastrum capricornutum Growth (Tables 4-8, Figures 5-7, Appendices C-E)
1. Definitive Test
The organisms were exposed for 72 hours without test medium renewal. The effects were expressed in terms o f percent inhibition in growth based on healthy cell count, area under the
i?m pa"y * " * * * * * eortato TSCA OB,
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
growth curve, and growth rate relative to the blank control for the 72-hour interval o f the test based on nominal test concentrations. The 72-hour results are as follows:
Healthy Cell Count EC5o NOEC
> 100mgH-24768/L 25 mg H-24768/L
Area Under the Growth Curve
EC5o
> 100mgH-24768/L
NOEC
25 mg H-24768/L
Growth Rate EC50 NOEC
> 100mgH-24768/L 25 mg H-24768/L
The reductions in healthy cell count, area under the growth curve, and growth rate indicate a dose-dependent response with increasing concentrations of the test substance.
2. Recovery Test
None of the definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore, a recovery test was not performed.
CONCLUSIONS
The reductions in healthy cell count, area under the growth curve, and growth for Selenastrum capricornutum at 72 hours (3 days) indicate a dose-dependent response for increasing concentrations of the test substance, H-24768. However, the EC50 is greater than 100 mg/L based on nominal test concentrations.
RECORDS AND SAMPLE STORAGE
Laboratory-specific or site-specific raw data, such as personnel files and equipment records will be retained by the facility where the work was done.
Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, the DuPont Crop Protection Archives, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. Characterization data (percent purity, composition, and known impurities) will be stored at Regional Analytical Services (RAS), Jackson Laboratories, Deepwater, New Jersey.
C mpany Sanf*ed. Does not contain TSCA c m
-20-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
REFERENCES
1. Miller, W.E.; Greene, J.C.; Shiroyama, T. The Selenastrum capricornutum Printz Algal Assay Bottle Test; U.S. Environmental Protection Agency; U.S. Government Printing Office: Washington, DC, 1978; EPA-600/9-78-018.
2. American Society for Testing and Materials (ASTM). (1990). "Standard Guide for Conducting Static 96-h Toxicity Tests with Microalgae" in ASTM Annual Book o f Standards, E1218-90. Vol. 11.04, Philadelphia, PA.
3. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition. The Iowa State University Press, Ames.
4. Tamhane, A.C., Dunnett, C.W., Green J.W., and Wetherington, J.D. (2001). Multiple test procedures for identifying maximum safe dose. JAm er. Statist. Assoc. 96, 835-843.
5. U.S. Environmental Protection Agency. (1996). Ecological Effects Test Guidelines. OPPTS 850.1000. Special Considerations for Conducting Aquatic Laboratory Studies, Public Draft. EPA 712-C-96-113.
" PanySanmzed. Doss not contain TSCA CBI
-21 -
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
TABLES
(Company Sanitized, Does not contain TSCA CBi
H-24768: Influence on Growth and Growth Rate o f the Green Alga Seienastrum capricornutum
DuPont-11954
TABLE 1
pH MEASUREMENTS OF TEST SOLUTIONS
Nominal H-24768 Concentration
Exposure Initiated: 22-Jul-2003
0-Hour (Day 0)
pH
Exposure Ended: 25-JuI-2003
72-Hour (Day 3)
Blank Control
7.49
7.49
6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L
7.51 7.68 7.60 7.54 7.59
7.56 7.59 7.59 7.58 7.52
Abiotic Control (100 mg/L)
7.59
7.23
TABLE 2
CHAMBER LIGHT INTENSITY, SHAKING SPEED, AND TEMPERATURE RANGE
Mean Light Intensity
at Test Initiation
Oscillations Temperature
Test (lumens/m2= lux)
(rpms)
(C)
Definitive
6800
98 24.7 to 24.8
Company Sanitized. Does not contain TSCA CBI
-23 -
)
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
)
)
DuPont-11954
TABLE 3
MEASURED CONCENTRATIONS OF H-24768 IN TEST SOLUTIONS
Sample Description3
Nominal
Concentration (mg/L)
Measured
oncentratiitomnj Mean, Measured
Concentration
Day 0
Day 3
(mg/L)c
Blank Control
0
NDe
ND
-
Working Stock (100 mg/L)
100
110 NSf
-
6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L
6.25
6.3 4.3
5.3
12.5
12 8.9
10.5
25
26 18
22.0
650
52 35
43.5
100
no 73
91.5
Abiotic Control (100 mg/L)
100
110s 75
92.5
% Recovery*1
-
-
85 84 88 87 92
93
a Defined in terms oCH-24768 mg/liter nutrient medium, b H-24768 w a s H B )u r e by analysis, c Mean, measured concentration calculated as (day 0 + day 3)/2. d Calculated as nominal/mean, measured concentration. e ND stands for not detected. The limit o f detection was calculated as 0.005 mg/L. f NS stands for not submitted. g No separate analysis was conducted since the working stock, the day 0 nominal 100 mg/L test concentration, and the day 0 abiotic control were
aliquots o f the same solution. Therefore, the measured concentration obtained for the working stock can be used for the 100 mg/L test concentration and the abiotic control.
jPgmpany Sanitized. Does not contain TSf'.* <'1*i
-24-
)
H-24768: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
)
DuPont-11954
TABLE 3 (Continued)
MEASURED CONCENTRATIONS OF H-24768 IN TEST SOLUTIONS
El
Sample Description3
Nominal (mg/L)
Measure
[Concentration
TA
Day 0
Day 3
Mean, Measured Concentration (mg/L)c
Blank Control
0
NDe
ND
-
Working Stock (100 mg/L)
100
95 NSf
-
6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L
6.25
5.4 3.2
4.3
12.5
11 7.1
9.1
25
23 16
19.5
50
47 32
39.5
100
95g 68
81.5
Abiotic Control (100 mg/L)
100
95g 69
82
% Recovery11
-
-
69 73 78 79 82
82
a Defined in terms oOJ-24768 mg/liter nutrient medium, b H-24768 w a s^ M f^ u r e by analysis. c Mean, measured concentration calculated as (day 0 + day 3)/2. d Calculated as nominal/mean, measured concentration. e ND stands for not detected. The limit o f detection was calculated as 0.002 mg/L. f NS stands for not submitted. g No separate analysis was conducted since the working stock, the day 0 nominal 100 mg/L test concentration, and the day 0 abiotic control were
aliquots o f the same solution. Therefore, the measured concentration obtained for the working stock can be used for the 100 mg/L test concentration and the abiotic control.
Company Sanitized. Does not contain TSCA CBI
-25-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
TABLE 3 (Continued)
MEASURED CONCENTRATIONS OF H-24768 IN TEST SOLUTIONS
Sample Description8
Nominal
_ M easured^^^^Hf \
Concentration Mean, Measured
Concentration1*
;/L) Concentration
(mg/L)
Day 0
Day 3
(mg/L)c
Blank Control
0
NDe
ND
-
Working Stock (100 mg/L)
100
99 NSf
-
6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L
6.25
5.3 2.9
4.1
12.5
11 7.1
9.1
25
24 16
20
50
49 32
40.5
100
99e 72
85.5
Abiotic Control (100 mg/L)
100
99e 74
86.5
% Recovery11
-
-
66 73 80 81 86
87
a Defined in terms o fH -24768 mg/liter nutrient medium, b H-24768 w asaH H p u re by analysis. c Mean, measured concentration calculated as (day 0 + day 3)/2. d Calculated as nominal/mean, measured concentration. e ND stands for not detected. The limit o f detection was calculated as 0.019 mg/L. f NS stands for not submitted. g No separate analysis was conducted since the working stock, the day 0 nominal 100 mg/L test concentration, and the day 0 abiotic control were
aliquots of the same solution. Therefore, the measured concentration obtained for the working stock can be used for the 100 mg/L test concentration and the abiotic control.
Company Sanitized. Does not contain TSCA CBf
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H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
TABLE 4 HEALTHY CELL COUNT DATA SUMMARY - DEFINITIVE TEST
Exposure Initiated:
Exposure Ended:
Nominal
_______________ Day 0; 22 July 2003_______________________________ Day 3: 25 July 2003
H-24768
__________________________ Healthy Cell Count (ceils/mL) by Exposure Period___________
Concentration______Rep.__________ 0-Hour_________________ 24-Hour________________ 48-Hour_________________72-Hour
1
10,000
50,000
290,000
2,360,000
Blank Control
2
10,000
30,000
270,000
2,590,000
3
10,000
140,000
600,000
2,160,000
Mean
10,000
73,333
386,667
2,370,000
Standard Error
0.00
33,829.64
106,822.80
124,230.97
Std. Dev.
0.00
58,594.65
185,022.52
215,174.35
Variance
0.00E+00
3.43E+09
3.42E+10
4.63E+10
1
10,000
100,000
630,000
2,520,000
6.25 mg/L
2
10,000
60,000
340,000
2,030,000
3
10,000
20,000
470,000
2,180,000
Mean
10,000
60,000
480,000
2,243,333
Standard Error
0.00
23,094.01
83,864.97
144,952.10
Std. Dev.
0.00
40,000.00
145,258.39
251,064.40
Variance
O.OOE+OO
1.60E+09
2.11E+10
6.30E+10
% Inhibition
0.00
18.18
-24.14
5.34
1
10,000
120,000
500,000
2,940,000
12.5 mg/L
2
10,000
20,000
340,000
2,310,000
3
10,000
70,000
150,000
2,860,000
Mean
10,000
70,000
330,000
2,703,333
Standard Error
0.00
28,867.51
101,159.94
198,017.96
Std. Dev.
0.00
50,000.00
175,214.15
342,977.16
Variance
O.OOE+OO
2.50E+09
3.07E+10
1.18E+11
% Inhibition
0.00
4.55
14.66
-14.06
1
10,000
120,000
310,000
1,870,000
25 mg/L
2
10,000
50,000
330,000
1,630,000
3
10,000
40,000
470,000
1,490,000
Mean
10,000
70,000
370,000
1.663,333
Standard Error
0.00
25,166.11
50,332.23
110,955.45
Std. Dev.
0.00
43,588.99
87,177.98
192,180.47
Variance
O.OOE+OO
1.90E+09
7.60E+09
3.69E+10
% Inhibition
0.00 4.55 4.31 29.82
1
10,000
50,000
340,000
1,630,000
50 mg/L
2
10,000
20,000
110,000
1,640,000
3
10,000
80,000
470,000
1,520,000
Mean
10,000
50,000
306,667
1,596,667
Standard Error
0.00
17,320.51
105,251.02
38,441.88
Std. Dev.
0.00
30,000.00
182,300.12
66,583.28
Variance
0.00E+00
9.00E+08
3.32E+10
4.43E+09
% Inhibition
0.00 31.82 20.69 32.63
1
10,000
10,000
320,000
1,410,000
100 mg/L
2
10,000
40,000
150,000
1,400,000
3
10,000
80,000
170,000
1,370,000
Mean
10,000
43,333
213,333
1,393,333
Standard Error
0.00 20,275.88 53,644.92
12,018.50
Std. Dev.
0.00 35,118.85 92,915.73 20,816.66
Variance
O.OOE+OO
1.23E+09
8.63E+09
4.33E+08
% Inhibition
0.00 40.91 44.83 41.21
Company Sanitized. Does not contain TSCA cb
- 27-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
TABLE 5
AREA UNDER THE GROWTH CURVE DATA SUMMARY - DEFINITIVE TEST
Nominal H-24768 Concentration
Blank Control
Mean Standard Error
Std. Dev. Variance
6.25 mg/L
Mean Standard Error
Std. Dev. Variance % Inhibition
12.5 mg/L
Mean Standard Error
Std. Dev. 'Variance % Inhibition
25 mg/L
Mean Standard Error
Std. Dev. Variance % Inhibition
50 mg/L
Mean Standard Error
Std. Dev. Variance % Inhibition
100 mg/L
Mean Standard Error
Std. Dev. Variance % Inhibition
Exposure Initiated:
Exposure Ended:
DayO: 22 July 2003______________________________________ Day 3: 25 July 2003
Area Under the Growth Curve Based on
____________________Healthy Cell Count (cells/mL) by Exposure Period
Rep. 0-24 Hour
0-48 Hour
0-72 Hour
1
480,000
4,320,000
35,880,000
2
240,000
3,600,000
37,680,000
3
1,560,000
10,200,000
43,080,000
760,000
6,040,000
38,880,000
405,955.66
2,090,358.82
2,163,330.77
703,135.83
3,620,607.68
3,746,998.80
4.94E+11
1.31E+13
1.40E+13
1
1,080,000
9,600,000
47,160,000
2
600,000
5,160,000
33,360,000
3
120,000
5,760,000
37,320,000
600,000
6,840,000
39,280,000
277,128.13
1,390,827.09
4,102,487.05
480,000.00 2.30E+11
2,408;983.19 5.80E+12
7,105,716.01 5.05E+13
21.05
-13.25
-1.03
1
1,320,000
8,520,000
49,560,000
2
120,000
4,200,000
35,760,000
3
720,000
3,120,000
39,000.000
720,000
5,280,000
41,440,000
346,410.16
1,649,727.25
4,166,341.32
600,000.00
2,857,411.42
7,216,314.85
3.60E+11
8.16E+12
5.21E+13
5.26 12.58
-6.58
1
1,320,000
6,240,000
32,160,000
2
480,000
4,800,000
28,080,000
3
360,000
6,240,000
29,520,000
720,000
5,760,000
29,920,000
301,993.38
480,000.00
1,194,654.76
523,067.87
831,384.39
2,069,202.75
2.74E+11
6.91E+11
4.28E+12
5.26 4.64
23.05
1
480,000
4,920,000
28,320,000
2
120,000
1,440,000
22,200,000
3
840,000
7,200,000
30,840,000
480,000
4,520,000
27,120,000
207,846.10
1,674,753.71
2,565,307.00
360,000.00
2,900,758.52
4,443,242.06
1.30E+11
8.4E+12
1.97E+13
36.84
25.17
30.25
1
0 3,720,000
24,240,000
2
360,000
2,400,000
20,760,000
3
840,000
3,600,000
21,840,000
400,000
3,240,000
22,280,000
243,310.50
421,426.15
1,028,396.81
421,426.15
729,931.50
1,781,235.53
1.78E+11
5.33E+11
3.17E+12
|
47.37
46.36
42.70
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- 28-
/ " "N
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
TABLE 6
GROWTH RATE DATA SUMMARY - DEFINITIVE TEST
Nominal H-24768 Concentration
Blank Control
Mean Standard Error
Std. Dev. Variance
6.25 mg/L
Mean Standard Error
Std. Dev. Variance % Inhibition
12.5 mg/L
Mean Standard Error
Std. Dev. Variance % inhibition
25 mg/L
Mean Standard Error
Std. Dev. Variance % Inhibition
50 mg/L
Mean Standard Error
Std. Dev. Variance % Inhibition
100 mg/L
Mean Standard Error
Std. Dev. Variance % Inhibition
Exposure Initiated:
Exposure Ended:
DavO: 22 July 2003________________ ______________________ Day 3: 25 July 2003
Growth Rate Based on
Healthy Cell Count (cells/mL) by Exposure Period__________ ____
Rep.
0-24 Hour
0-48 Hour
0-72 Hour
1
0.0671
0.0702
0.0759
2
0.0458
0.0687
0.0772
3
0.1100
0.0853
0.0747
0.0743
0.0747
0.0759
0.0189
0.0053
0.0007
0.0327
0.0092
0.0013
1.07E-03
8.43E-05
1.56E-06
1
0.0959
0.0863
0.0768
2
0.0747
0.0735
0.0738
3
0.0289
0.0802
0.0748
0.0665
0.0800
0.0751
0.0198
0.0037
0.0009
0.0342
0.0064
0.0015
1.17E-03
4.10E-05
2.33E-06
10.50
-7.05
1.05
1
0.1035
0.0815
0.0789
2
0.0289
0.0735
0.0756
3
0.0811
0.0564
0.0786
0.0712
0.0705
0.0777
0.0221
0.0074
0.0011
0.0383
0.0128
0.0018
1.47E-03 4.22
1.64E-04 5.71
3.33E-06 -2.33
1
0.1035
0.0715
0.0727
2
0.0671
0.0728
0.0707
3
0.0578
0.0802
0.0695
0.0761
0.0748
0.0710
0.0139
0.0027
0.0009
0.0242
0.0047
0.0016
5.83E-04
2.20E-05
2.61E-06
-2.47
-0.13
6.54
1
0.0671
0.0735
0.0707
2
0.0289
0.0500
0.0708
3
0.0866
0.0802
0.0698
0.0609
0.0679
0.0704
0.0169
0.0092
0.0003
0.0294
0.0159
0.0006
8.61E-04
2.52E-04
3.03E-07
18.08
9.14
7.24
i
0.0000
0.0722
0.0687
2
0.0578
0.0564
0.0686
3
0.0866
0.0590
0.0683
0.0481
0.0625
0.0685
0.0255
0.0049
0.0001
0.0441
0.0085
0.0002
1.94E-03
7.18E-05
4.33E-08
35.22
16.32
9.75
Company Sanitized. Does not contain TSCA OBJ -29-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
TABLE 7
72-HOUR (DAY 3) STATISTICAL ANALYSES SUMMARY - DEFINITIVE TEST
Nominal H-24768 Concentration
Mean Standard Response Error N
Test Concentration Response (mg/L)
Level
Value
Healthy Cell Count
Blank Control 6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L
2,370,000 2,243,333 2,703,333 1,663,333 1,596,667 1,393,333
124,231 144,952 198,018 110,955 38,442
12,019
3 3 3 3 3 3
e c 50 NOEC
> 100 25
Area Under the Growth Curve
Blank Control 6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L
38,880,000 39,280,000 41,440,000 29,920,000 27,120,000 22,280,000
2,163,331 4,102,487 4,166,341 1,194,655 2,565,307 1,028,397
3 3 3 3 3 3
e c 50 NOEC
> 100 25
Growth Rate
Blank Control 6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L
0.0759 0.0751 0.0777 0.0710 0.0704 0.0685
0.0007 0.0009 0.0011 0.0009 0.0003 0.0001
3 3 3 3 3 3
e c 50 NOEC
> 100 25
Company Sanifized. Does nof contain TSCA CBI -30-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
FIGURES
Gpmpany Sanitized. Does not contain TSCA
-31 -
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
FIGURE 1
REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVES
A B C
Compound nami Coefficient of Determination: RA2 = 0.999195 Calibration curve: -3.31776e+006 * xA2 + 1.7707e+007 * x + 154110
Company Sanitized. Does not contain TSCA CB$ -32-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
FIGURE 1 (Continued)
REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVES
A B C
Compound name Coefficient of Determination: RA2 = 0.998645
ComWr*
Does not contain TSCA r *
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastmm capricornutum
DuPont-11954
FIGURE 1 (Continued)
REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVES FORH-25436
Al b| c
Compound n a m e f ^ H m ^ P r l Coefficient of Determination: RA2 = 0.998745 Calibration curve: -223095 * xA2 + 4.51527e+006 * x + 16289.8
Company Sanitized. Does not contain TSCA CBI
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
FIGURE 2
REPRESENTATIVE CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION
072203b1
10C 1.97 370114.91 1958138
3
F1:SIR of 5 channels,ES+ TIC
2.079e+006
%-
llute at a retention time o f approximately 2.0 minutes. Calibration standard solution contains T-25436 at a concentration o f 0.103 mg/L.
072203b11
100-1
3.55 285804.84 1263437
F2:SIRof5 channels,ES+ TIC
1.291e+006
%-
1.00 2.00 3.00 4.00 5.00 6.00
min
lute at a retention time of approximately 3.6 minutes. Calibration standard solution contains at a concentration o f 0.103 mg/L
.Company Sanitized. Does not contain Tsna n m
-35 -
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
FIGURE 2 (Continued) REPRESENTATIVE CHROMATOGRAM OF A
CALIBRATION STANDARD SOLUTION
DuPont-11954
blute at a retention time of approximately 5.0 minutes. Calibration standard solution contains H-25436 at a concentration o f 0.103 mg/L.
Company Sanitized. Does not contain TSCA -36-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenasfrum capricomutum
DuPont-11954
FIGURE 3 REPRESENTATIVE CHROMATOGRAM OF THE BLANK CONTROL SOLUTON
072203b10
F1:SIR of 5 channels,ES+ TIC
1.00 2.00 3.00 4.00 5.00 6.00
& rould elute at a retention time o f approximately 2.0 minutes.
jCgmpany Sanitized. Does not contain tsca
-37 -
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastmm capricornutum
DuPont-11954
FIGURE 3 (Continued) REPRESENTATIVE CHROMATOGRAM OF THE BLANK CONTROL SOLUTON
ould elute at a retention time o f approximately 5.0 minutes.
pmpany Samlbed. Does not coniata TSCA CBI
-38-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
FIGURE 4 REPRESENTATIVE CHROMATOGRAM OF A TEST CONCENTRATION SOLUTION
_______________ plute at a retention time of approximately 2.0 minutes. Test solution sample contains H-24768 "at a nominal concentration o f 6.25 mg/L (diluted 1:12.5 before analysis).
072203b27 IOOt
1099472.38 5018560
F2:SIR of 5 channels,ES+ TIC
5.062e+006
To
1.00 2.00 3.00 4.00 5.00 6.00 rr- mm
elute at a retention time o f approximately 3.6 minutes. Test solution sample contains H-24768 'at a nominal concentration o f 6.25 mg/L (diluted 1:12.5 before analysis).
-39-
Pnrpanysannteed. Does con,8i,, TSCA a a
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastntm capricomutum
D u P o n t- 11954
FIGURE 4 (Continued) REPRESENTATIVE CHROMATOGRAM OF A TEST CONCENTRATION SOLUTION
at a retention time of approximately 5.0 minutes. Test solution sample contains H24768 at a nominal concentration o f 6.25 mg/L (diluted 1:12.5 before analysis).
Company Sanitized. Does not contain TSCA CBf
- 40-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
FIGURE 5
72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE FOR THE DEFINITIVE TEST
Bruce & Veisleeg Probit type Regression, Using M A R Q U A R D T Method RESPONSE : C O R B U U I from D A T A S E T : S 10UAN . AHR I 3593 FORMULA I = Cou n I / 10000 CONCENTRATIONS 6 . 2 5 12.5 25 50 100
Bruce & Versteeg Probit Type Rgression, Using M A R Q U A R D T M ethod RESPONSE : FORMULA! trois ATASET : SLOMAN. AWR13593 FORMULAI-Counl / i 0000 CO NC E NT R A TIO N S^ 6.25 12.5 25 50 100
Rate m p a n y Sanitized. Does nof contain TSCA c m
-41 -
/**W*>N.
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
FIGURE 6
72-HOUR AREA UNDER THE GROWTH CURVE DOSE-RESPONSE CURVE FOR THE DEFINITIVE TEST
Brace & Veisteeg Probtt Type Regression. Using M A R Q U A R D T Method RESPONSE : E0RUULA2 iron H A U S E T : 5 LORAN. ANRI 3593 F O R U U U S U tso / 100008 CONCENTRAT I0IIS =0 6.2 5 12.5 25 50 100
B ruce & V e rsle e g P ro bit Type R e gre ssio n , U sin g M A R Q U A R D T M ethod R E SPO NS E: F0HMULA2 from D ATASET: S LO MAN. AWR13593 F0RMU LA2=A r eo / 100000 C O N C E N T R A T I O N S ^ 6.2 5 12 . 5 25 5 0 100
Rote CompanySanitized. Does not contain TSCA CBl
-42-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
FIGURE 7 72-HOUR GROWTH RATE DOSE-RESPONSE CURVE FOR THE DEFINITIVE TEST
Bruce & Versteeg ProbR Type Regression, U sing M A R Q U A R D T Method RESPONSE: G.RATE Iron DATASET: SLOU AN.AWR13593 CONCERT RAT I ORS= 0 6 . 2 5 ! 2 . 5 25 50 100
Bruce & Versteeg Pro bit Type R e gre ssio n, U sin g M A R Q U A R D T M ethod R E S P O N S E : G . R A T E I r o n D A T A S E T : S L O W AN . AVIR 1 3 5 9 3 C O N C E N T R A T I O N S ^ 6,2 5 12.5 25 50 100
Role
jppmpany Sanitized. Does not eonf' **'*'
-43 -
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
APPENDICES
Company SanFffzed. pes not contain TSCA ca r
-44-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
APPENDIX A AAP Nutrient Medium Constituents
Company Sanitized. Does no! oonfain TSCA CB!
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricorrmtum
DuPont-11954
AAP NUTRIENT MEDIUM CONSTITUENTS
Stock Solutions3
Component
Concentration (g/L)
Macronutrient Stock #1
NaN03
25.500
Macronutrient Stock #2
NaHCOa
15.000
Macronutrient Stock #3
k 2h p o 4
1.044
Macronutrient Stock #4
M gS04*7H20
14.700
Macronutrient Stock #5
MgCl2'6 H 20
12.164
Macronutrient Stock #6
CaCl2*2H20
4.410
Final Prepared Medium Component Concentration (mg/L)
Na 11.001 N 4.200
C 2.143 K 0.469
P 0.186 Mg 2.904b
Ca 1.202 S 1.911
Stock Solutions3
Component
Concentration (mg/L)
Micronutrient Stock H3BO3 MnCl2*4 H20 ZnCl2 C oC12*6 H20
C u C12* 2 H 20
Na2M o04*2 H20 FeCl3*6 H20 Na2EDTA*2 H20
185.520 415.610 3.271 1.428 0.012 7.260 160.000 300.000
Final Prepared Medium Component Concentration (pg/L)
B 32.460 Mn 115.374 Zn 1.570 Co 0.354 Cu 0.004 Mo 2.878 Fe 33.051
a As reported in Reference 1
b Includes magnesium from both magnesium sulfate and magnesium chloride.
______ -- ^ S m i l z s . P9g8 not nnritain
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
APPENDIX B 72-Hour Healthy Cell Count Statistics for the Definitive Test
-Company Sanitized. Does noloonlaln r s e .
-47-
/`""v
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
72-HOUR HEALTHY CELL COUNT STATISTICS FOR THE DEFINITIVE TEST
STATISTICAL ANALYSIS LIST FILE
REPORT CREATED ON 17 :19 THURSDAY--. 31JUL03 __ ECOTOX MEASUREMENTS FROM DATASET!M M f ]
GROUP STATISTICS FOR Count BY DOSE
dose dosevai COUNT MEAN mg/L
MEDIAN
STD_DEV
STD_ERR
1 0 3 2370000.00 2 6.25 3 2243333.33 3 12.5 3 2703333.33 4 25 3 1663333.33 5 50 3 1596666.67 6 100 3 1393333.33
2360000 2180000 2860000 1630000 1630000 1400000
215174.35 251064.40 342977.16 192180.47
66583.28 20816.66
124230.97 144952.10 198017.96 110955.45
38441.88 12018.50
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 THURSDAY 31JUL03
SHAPIRO-WILK TEST OF NORMALITY OF Count AQUATIC SELENASTRUM: FULL DATA
STD
SKEW
KURT
SW_STAT P_VALUE SIGNIF
177819.03
-0.32849
-0.034462
0.95919
0.58599
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 THURSDAY 31JUL03
LEVENE TEST FOR Count - FULL Model ANALYSIS OF VARIANCE ON FULL DATA SET
Effect DF
LEVENE P_VALUE SIGNIF
DOSE
5 0.67167 0.65269
NOTE
The data was found to be normally distributed with equal variances.
An analysis of variance will be performed.
STATISTICAL ANALYSIS LIST FILE
REPORT CREATED ON 17:19 THURSDAY 31JUL03
ANALYSIS OF SELENASTRUM - FULL DATA
DOSES 0, 6.25, 12.5, 25, 50,, 100 mg/L
Obs Class Levels
Values
1 dose
6 123456
1! oEt
OVERALL F-TESTS FOR ANOVA Count FROM FULL DATA Using Weight WEIGHT and Variance Ratio=0.
Num Den Obs Effect DF DF FValue ProbF
1 dose
5 12 17.88 <0001
-48Company Sanflfeed. Does nof oonlalh TSCa
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
D u P o n t- 11954
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 THURSDAY 31JUL03
ANALYSIS OF SELENASTRUM - FULL DATA LSMEANS - DOSE MEANS ADJUSTED FOR THE EFFECTS OF SELENASTRUM Count FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0.33
Obs LEVEL
LSMEAN
SE DDF
10 2 6.25 3 12.5 4 25 5 50 6 100
2370000.00 2243333.33 2703333.33 1663333.33 1596666.67 1393333.33
122194.44 122194.44 122194.44 122194.44 122194.44 122194.44
12 12 12 12 12 12
ESTIMATED DOSE EFFECTS & DUNNETT FOR Decreasing ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL
Count FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0.33
Estimate
DOSE TREND DOSE QUAD DOSE 2-1 DOSE 3-1 DOSE 4-1 DOSE 5-1 DOSE 6-1
SIGNIF
** *
** ** **
Dunnett 1-sided p-value
0.00001 0.04616 0.91420 0.25156 0.00613 0.00315 0.00046
Test Group Mean
. 2243333.33 2703333.33 1663333.33 1596666.67 1393333.33
N
. 3 3 3 3 3
ESTIMATED DOSE EFFECTS & DUNNETT FOR Decreasing ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL
Count FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0.33
Check for Number of Reps in count Maximum Number of Reps in Any Treatment Group or Control Maximum Number of Reps per Group is 3 Exact Permutation Methods Recommended
Analysis o f M M H M g D a t a Test for TREND IN EITHER DIRECTION in Response=count ANALYSIS USING ALPHA=0.05 FULL DATA
KEY _JT_ IS JONCKHEERE STATISTIC Z_JT IS STANDARDIZED JONCKHEERE STATISTIC XPR_JT IS P-VALUE FOR UPWARD TREND XPL_JT IS P-VALUE FOR DOWNWARD TREND P-VALUES ARE FOR TIE-CORRECTED TEST WITH CONTINUITY CORRECTION FACTOR SIGNIF RESULTS ARE FOR D ALTERNATIVE HYPOTHESIS
Company Sanitized. Does not oontain TSCA CBt -49-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
' Jonckheere Trend Test on Dose 0 + Lowest 5 Doses thru 100 MG/L
___JT____
Z_JT
XPL_JT
31 -3.466358 0.0001346
XPR_JT SIGNIF DOSE
. **
6
Jonckheere Trend Test on Dose 0 + Lowest 4 Doses thru 50 MG/L
___JT____
Z_JT
XPL_JT
29.5 -2.281172- 0.0114348
XPR_JT SIGNIF DOSE
. **
5
Jonckheere Trend Test on Dose 0 + Lowest 3 Doses thru 25 MG/L
___JT____
Z_JT
24 -1.279204
XPL_JT 0.1166342
XPR_JT SIGNIF DOSE .4
NOTE Jonckheere test results are included in the summary table. Group means should be examined to check for lack-of-fit to a linear trend before trend test results are accepted.
Spnipany Sanitized. Does not contain TSCA CBf
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
EC50 for Cell Count (Note: Formularceli Count/100000)
OBSERVED Sc PREDICTED PERCENT CHANGE FROM CONTROL MEAN RESPONSE: FORMULAl from DATASET:^J|
Obs
CONC N
MEAN
mg/L
PRED
PCTCHNG PREDPCTC
1 0.00 3 237.000 245.544 2 6.25 3 224.333 233.638 3 12.50 3 270.333 220.312 4 25.00 3 166.333 198.514 5 50.00 3 159.667 167.920 6 100.00 3 139.333 131.071
0 -5 14 -30 -33 -41
0 -5 -10 -19 -32 -47
EC50 using Method=MARQUARDT, Bruce & Versteeg Weighted Probit Covariance Matrix and Standard Error of Parameter Estimates
_NAME_
logecx
gamma
yO SE
logecx
ygoamma
0.08794 0.05019 -2.08530
0.05019 0.40994 7.92425
-2.085 7.924
355.372
0.2965 0.6403 18.8513
Model Parameter Es'timates, EC50 and 95% Confidence Bounds
logecx
gamma
yo
LB
UB EC50
4.75450
1.76051
245.544
64.9268
207.625
116.105
Model Evaluation Measures
SSREG
SSERR
SSTOTC
SSTOTU RSQUARE
3591
78.6661
237.796
3669.67
0.66919
note Graph descriptions include the following: formulai: EC50 BVP FIT BY MARQUARDT,
DuPont-11954
jCpmpanjf Sanitized. Does not contain TSCA CBt
-51 -
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
APPENDIX C 72-Hour Area Under the Growth Curve Statistics for the Definitive Test
Company Sanitized. Does no! oontain TSCA CBf
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-31954
72-HOUR AREA UNDER THE GROWTH CURVE STATISTICS FOR THE DEFINITIVE TEST
STATISTICAL ANALYSIS LIST FILE ECOTOX MEASUREMENTS FROM DATASET GROUP STATISTICS FOR Area BY DOS
dose dosevai mg/L
COUNT MEAN
MEDIAN
STD_DEV
STD_ERR
1 0 3 38880000 37680000 3746998.80 2163330.77 2 6.25 3 39280000 37320000 7105716.01 4102487.05 3 12.5 3 41440000 39000000 7216314.85 4166341.32 4 25 3 29920000 29520000 2069202.75 1194654.76 5 50 3 27120000 28320000 4443242.06 2565307.00 6 100 3 22280000 21840000 1781235.53 1028396.81
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 THURSDAY 31JUL03 SHAPIRO-WILK TEST OF NORMALITY OF Area
OBS STD
SKEW
KURT
SW_STAT P_VALUE SIGNIF
18 4113152.51 0.57085 -0.17048 0.94614 0.36764
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 THURSDAY 31JUL03 LEVENE TEST FOR Area - FULL Model ANALYSIS OF VARIANCE ON FULL DATA SET
Effect DF
LEVENE P_VALUE SIGNIF
DOSE
5 0.55480 0.73255
NOTE The data was found to be normally distributed with equal variances. An analysis of variance will be performed.
REPORT CREATED ON 17:19 THURSDAY 31JUL03 ANALYSIS OF SELENASTRUM - FULL DATA DOSES 0, 6.25, 12.5, 25, 50, 100 mg/L ECOTOX OF AQUATIC SELENASTRUM
Obs Class Levels
Values
1 dose
6 123456
OVERALL F-TESTS FOR ANOVA Area FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0
Num Den Obs Effect DF DF FValue ProbF
1 dose
5 12
7.61 0.0020
Company Sanitized. Does no! oontain TSCA CB
-53 -
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
LSMEANS - DOSE MEANS ADJUSTED FOR THE EFFECTS OF SELENASTRUM Area FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0.33
Obs LEVEL 10 2 6.25 3 12.5 4 25 5 50 6 100
LSMEAN 38880000 39280000 41440000 29920000 27120000 22280000
SE 2826493.71 2826493.71 2826493.71 2826493.71 2826493.71 2826493.71
DDF 12 12 12 12 12 12
ESTIMATED DOSE EFFECTS & DUNNETT FOR Decreasing ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL Area FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0.33
Estimate
SIGNIF
Dunnett 1-sided p-value
Test Group Mean
N
DOSE TREND ** 0.00013
DOSE QUAD
0.10086
,
DOSE 2-1
0.99999 39280000 3
DOSE 3-1
0.94736 41440000 3
DOSE 4-1
0.15377 29920000 3
DOSE 5-1
* 0.04655 27120000 3
DOSE 6-1
** 0.00549 22280000 3
ESTIMATED DOSE EFFECTS & DUNNETT FOR Decreasing ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL Area FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0.33
KEY _JT_ IS JONCKHEERE STATISTIC Z_JT IS STANDARDIZED JONCKHEERE STATISTIC XPR_JT IS P-VALUE FOR UPWARD TREND XPL_JT IS P-VALUE FOR DOWNWARD TREND P-VALUES ARE FOR TIE-CORRECTED TEST WITH CONTINUITY CORRECTION FACTOR SIGNIF RESULTS ARE FOR Decreasing ALTERNATIVE HYPOTHESIS
Jonckheere Trend Test on Dose 0 + Lowest 5 Doses thru 100 MG/L
JT
Z_JT
XPL_JT
28 -3.733532 0.0000343
XPR_JT SIGNIF DOSE ** 6
Jonckheere Trend Test on Dose 0 + Lowest 4 Doses thru 50 MG/L
JT
Z_JT
XPL_JT
25.5 -2.734396 0.0027539
XPR_JT SIGNIF DOSE
- **
5
Jonckheere Trend Test on Dose 0 + Lowest 3 Doses thru 25 MG/L
JT
Z_JT
XPL_JT
XPR iJT SIGNIF DOSE
21 -1.705606 0.0514529
4
NOTE
Jonckheere test results are included in the summary table.
Group means should be examined to check for lack-of-fit
to a linear trend before trend test results are accepted.
GomwmirSimBta,,,. Do.S ,,o!oor.(rn w r . r x
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
EC50 for AREA (UC)
OBSERVED Sc PREDICTED PERCENT CHANGE FROM CONTROL MEAN RESPONSE: FORMULA2 from DATASET:]
Obs
CONC N
MEAN
PRED
PCTCHNG PREDPCTCHNG
mg/L
1 0.00 3 388.8 404.834 2 6.25 3 392.8 389.304 3 12.50 3 414.4 368.726 4 25.00 3 299.2 332.613 5 50.00 3 271.2 279.532 6 100.00 3 222.8 214.191
0 1 7 -23 -30 -43
0 -4 -9 -18 -31 -47
EC50 using Method=MARQUARDT, Bruce & Versteeg Weighted Probit Covariance Matrix and Standard Error of Parameter Estimates
_NAME_
logecx
gamma
yO SE
logecx gamma yO
0.06314 0.03519 -2.56309
0.0352 0.3039 10.1754
-2.563 10.175 781.952
0.2513 0.5513 27.9634
Model Parameter Estimates, EC50 and 95% Confidence Bounds
logecx
gamma
yo
LB
UB EC50
4.72438
1.63377
404.834
68.8452
184.362
112.661
Model Evaluation Measures
SSREG
SSERR
SSTOTC
SSTOTU RSQUARE
5967.60 111.608 390.035 6079.21 0.71385
note Graph descriptions include the following: formula2: EC50 BVP FIT BY MARQUARDT,
Company Sanitized. Does not oonfain TSCA cb
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
APPENDIX D 72-Hour Growth Rate Statistics for the Definitive Test
jQompany Sanitized. Does not contain T sca cqi
-56-
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
DuPont-11954
72-HOUR GROWTH RATE STATISTICS FOR THE DEFINITIVE TEST
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 TOURSDAY^31JUL03_ ECOTOX MEASUREMENTS FROKTDATASETl GROUP STATISTICS FOR G Rate BY DOSE
dose dosevai mg/L
COUNT MEAN MEDIAN
STD_DEV
STD_ERR
1 0 3 0.075933 0.0759 .001250333 .000721880 2 6.25 3 0.075133 0.0748 .001527525 .000881917 3 12.5 3 0.077700 0.0786 .001824829 .001053565 4 25 3 0.070967 0.0707 .001616581 .000933333 5 50 3 0.070433 0.0707 .000550757 .000317980 6 100 3 0.068533 0.0686 .000208167 .000120185
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 THURSDAY 31JUL03 SHAPIRO-WILK TEST OF NORMALITY OF G_Rate AQUATIC SELENASTRUM: FULL DATA
OBS
STD SKEW
KURT
SW_STAT P_VALUE
18 .001094729 -0.13041 -0.58919 0.96847 0.76851
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 THURSDAY 31JUL03 LEVENE TEST FOR G_Rate - FULL Model ANALYSIS OF VARIANCE ON FULL DATA SET
Effect DF
LEVENE P_VALUE SIGNIF
DOSE
5 0.55036 0.73563
NOTE The data was found to be normally distributed with equal variances. An analysis of variance will be performed.
STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 17:19 THURSDAY 31JUL03 ANALYSIS OF SELENASTRUM - FULL DATA DOSES 0, 6.25, 12.5, 25, 50, 100 mg/L ECOTOX OF AQUATIC SELENASTRUM
Obs Class Levels
Values
1 dose
6 123456
OVERALL F-TESTS FOR ANOVA G_Rate FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0.33
Num Den Obs Effect DF DF FValue ProbF
1 dose
5 12 23.27 <.0001
.Company Sanitized, Poes not------
H-24768: influence on Growth and Growth Rate o f the Green Alga Selenastnim capricornutum
DuPont-11954
LSMEANS - DOSE MEANS ADJUSTED FOR THE EFFECTS OF SELENASTRUM G_Rate FROM FULL DATA Using Weight=NO WEIGHT and Variance
Obs LEVEL 10 2 6.25 3 12.5 4 25 5 50 6 100
LSMEAN 0.075933 0.075133 0.077700 0.070967 0.070433 0.068533
SE .000752280 .000752280 .000752280 .000752280 .000752280 .000752280
DDF 12 12 12 12 12 12
ESTIMATED DOSE EFFECTS & DUNNETT FOR Decreasing ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL G_Rate FROM FULL DATA Using Weight=NO WEIGHT and Variance Ratio=0.33
Estimate
DOSE TREND DOSE QUAD DOSE 2-1 DOSE 3-1 DOSE 4-1 DOSE 5-1 DOSE 6-1
SIGNIF *'k
k
kk kk kk
Dunnett 1-sided p-value
0.00000 0.02339 0.90629 0.37087 0.00227 0.00099 0.00007
Test Group Mean
. 0.075133 0.077700 0.070967 0.070433 0.068533
N
. 3 3 3 3 3
ESTIMATED DOSE EFFECTS & DUNNETT FOR Decreasing ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL
KEY _JT_ IS JONCKHEERE STATISTIC Z_JT IS STANDARDIZED JONCKHEERE STATISTIC XPR_JT IS P-VALUE FOR UPWARD TREND XPL_JT IS P-VALUE FOR DOWNWARD TREND P-VALUES ARE FOR TIE-CORRECTED TEST WITH CONTINUITY CORRECTION SIGNIF RESULTS ARE FOR D ALTERNATIVE HYPOTHESIS
Jonckheere Trend Test on Dose 0 + Lowest 5 Doses thru 100 MG/L
___JT____
Z_JT
XPL_JT
31 -3.466368 0.0001346
XPR_JT SIGNIF DOSE . ** 6
Jonckheere Trend Test on Dose 0 + Lowest 4 Doses thru 50 MG/L
___JT____
Z,,JT
XPL_JT
29.5 -2.281172 0.0114348
XPR_JT SIGNIF DOSE . ** 5
Jonckheere Trend Test on Dose 0 + Lowest 3 Doses thru 25 MG/L
___JT____
Z_JT
XPL_JT XPR_JT SIGNIF DOSE
24 -1.279204 0.1166342
.
4
NOTE
Jonckheere test results are included in the summary table.
Group means should be examined to check for lack-of-fit
to a linear trend before trend test results are accepted.
.Company Sanitized. Does not oontain TSCA CBt
H-24768: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
DuPont-11954
EC50 for Growth Rate
OBSERVED & PREDICTED PERCENT CHANGE FROM CONTROL MEAN RESPONSE: G RATE from DATASET:1
Obs CONC N mg/L
MEAN
PRED
PCTCHNG PREDPCTCHNG
1 0.00 3 0.07593333 0.076499
2 6.25 3 0.07513333 0.075457
3 12.50 3
0.0777 0.074588
4 25.00 3 0.07096667 0.073179
5 50.00 3 0.07043333 0.071030
6 100.00 3 0.06853333 0.067947
0 -1
2 -7 -7 -10
0 -1 -2 -4 -7 -11
EC50 using Method=MARQUARDT, Bruce & Versteeg Weighted Probit Covariance Matrix and Standard Error of Parameter Estimates
_NAME_
logecx
gamma
yO SE
logecx gamma yO
1.39520 1.11491 0.00074
1.11491 0.93955 0.00077
.000743038 .000774690 .000001339
1.18119 0.96930 0.00116
Model Parameter Estimates, EC50 and 95% Confidence Bounds
logecx
gamma
yO
LB UB EC50
8.01017
2\79779
0.076499
297.391
30494.21
3011.43
Model Evaluation Measures
SSREG
SSERR
SSTOTC SSTOTU
RSQUARE
1.3161 .000910377 .003008721 1.31701 0.69742
95% FIELLER CONFIDENCE BOUNDS ON TARGETED PERCENT EFFECT CONC
note Graph descriptions include the following: G_Rate: EC50 BVP FIT BY MARQUARDT,
.Company Sanitized. Does not oontain TSCA Bf -59-
