Document em50xqXEaQjwR4DX64dM279m9
AR226-2810
DuPont-12989
TRADE SECRET
Study Title H-24750: Influence on Growth and Growth Rate of the Green
Alga Selenastrum capricomutum
A u th o rs: Barbra D. Ferrell, B.S. Robert A. Hoke, Ph.D.
Study C om pleted o n : May 20,2003 P erform ing La bo r a to ry : E.I. du Pont de Nemours and Company
Haskell Laboratoiy for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050
W ork Request N umber: Study Code Number:
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricomutum
DuPont-12989
CERTIFICATION
I, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study.
W ork Completed by:
jG laM
Barbra ]D. Ferrell, B.S.
Associate Scientist
3 ? Abiti
Date
Issued by Study Director:
Robert A. HokerPh.D. Senior Research Ecotoxicologist and Manager
o20 /V/ JVy oJ e o 3 fifee
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H-24750: Influence oh Growth and Growth Rate of the Green Alga Selenastrum capricomutum
% STUDY INFORMATION 9th Collective Nomenclatur Svnonvms/Corie
DuPont-12989
Submitter's Notebook Nnmhcrfs
Haskell Number: 24750
CAS Registry Number
Purity
Known Impurities
Physical Characteristics: White Solid
Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.
Study Initiated/Completed: April 15, 2003 / (see report cover page)
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum_____________
DuPont-12989
SUMMARY
A study was conducted to determine the effect of H-24750 on the growth and growth rate o f the green alga Selenastrum capricomutum.a The algae were exposed to nominal concentrations of 0.1, 1, 10, 100, and 1000 mg H-24750/Liter of nutrient medium (ppm).
The algae 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 (also referred to as cell density) relative to the blank (culture medium) control for the 72-hour (day 3) interval of the test.
The 72-hour ECso, including the 95% confidence intervals (C.I.),bbased on nominal concentrations are as follows:0
H ealthy Cell C ount
EC50
10.6 mg/L (95% C.I. = 5.8 to 15.5)
NOEC
1 mg/L
H-24750 exhibited medium concemdunder TSCA and was classified as "harmful" to aquatic organisms (R52)e under the EU Directive in a 72-hour, acute test using the green alga Selenastrum capricornutum.
a Also known as Pseudokirchneriella subcapitata. b The EC50 is defined as the "effective concentration" producing a 50% inhibition of growth relative to the
control. c Slob, W., (2002). Dose-response modeling o f continuous endpoints. Toxicological Science, V66; pp 298-312. d Smrchek, J., Clements, R., Morcock, R., and Rabert, W. (1993). Assessing ecological hazard under TSCA:
methods and evaluation of data. Environmental Toxicology and Risk Assessment, A ST M ST P 1179. (W.G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia. e Official Journal o f the European Communities, Volume 36, 4 May 1993, pp. 68, section 5.2.1.
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
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MATERIALS AND METHODS
A. Test System
Selenastrum capricornutum, a freshwater, unicellular, non-motile, green alga, was used in this study. The original culture source was the Department of Botany - Culture Collection of Algae The University of Texas at Austin - Austin, Texas 78713-7640. The culture method for Selenastrum capricornutum was based on published literature.(1,2) Prior to the study, Selenastrum capricornutum 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) synthetic algal-assay procedure (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.
B. Test Design
The test design used for the Selenastrum capricornutum study is described below:
Organism Selenastrum capricornutum
Nutrient Medium
AAP
Flask Volume Solution Volume Volume
(mL)
(mL)
Ratio
250 50 5:1
C. AAP Nutrient Medium Preparation for Selenastrum capricornutum (Appendix A)
AAP nutrient medium0) 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 of the medium required for the study.
The medium pH was adjusted to approximately 7.5. The medium was filter-sterilized using Coming pre-sterilized filtration systems each with a 0.22 pm cellulose acetate filter.
1. Test Solution Preparation
Five nominal test concentrations (0.1, 1, 10, 100, and 1000 mg H-24750/L AAP nutrient medium) were used in this study. Aliquots of the filter-sterilized AAP nutrient medium were used for the blank (normal culture medium) control solution.
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H-24750: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
2. Test Culture Preparation
DuPont-12989
For each blank control and test solution, 2 aliquots (50 mL each) were placed in separate sterilized 250 mL Erlenmeyer flasks fitted with sterilized foam stoppers. To achieve the desired nominal concentration of approximately 10,000 Selenastrum capricornutum cells/mL at test initiation, an approximate 0.475 mL (= 475 pL) aliquot of algal inoculum from a logarithmically growing stock culture was aseptically transferred to each flask.
D. Experimental Design and Test Conditions
The test flasks were placed on a shaker table in an environmental chamber in a non-systematic design and were re-positioned each working day. Air temperature in the chamber was recorded continuously with a continuous temperature recorder. Each blank control and test concentration was tested as 2 replicates. The algae were incubated without test medium renewal and cell counts were made after 72 hours. Illumination was supplied by cool-white fluorescent tubes. The target environmental parameters are described in the following table.
Initial Population
10,000 cells/mL
Photo Shaking
Illumination Period Speed Temperature
(lumens/m2= lux) (hours) (rpms)
(C)
6000 to 10,000
24
100
24 2
E. Selenastrum capricornutum Growth Measurement
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 72 hours from study initiation. The counts were conducted using a hemacytometer and a compound microscope. An aliquot of each sample was loaded into each side of the hemacytometer and 8 grids from each side were selected for counting. All cells located in the 16 grids 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. Observations and counts of healthy and unhealthy cells (e.g., deformed, scenescent, stunted) were recorded in the study records. The statistical calculation of the EC50 was based on mean healthy cell counts and nominal test substance concentrations.
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
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RESULTS
Based on visual observations, the blank control solution and the 0.1, 1, 10, and 100 mg/L test solutions were clear and had no color before addition of algal cells. The 1000 mg/L test solution was clear and had undissolved particles of test material and a surface film. After the addition of algal cells, the test and blank control flasks were placed on a shaker table in a chamber with a temperature of approximately 25C. The algae were incubated for 72 hours without medium renewal. Illumination was supplied by cool-white fluorescent tubes. The mean light intensity in the chamber was 7564 lumens/m2 (= lux). The shaking speed was 96 revolutions per minute (rpm). The pH measurement of the culture medium was approximately 7.5 at study start. The pH measurements o f the test solutions at test initiation ranged from 3.20 to 7.47, and at test termination ranged from 3.33 to 7.86.
C O N C L U SIO N S
The 72-hour EC50, including the 95% confidence intervals (C.I.)a based on nominal test substance concentrations and the NOEC are as follows:(3)
H ealthy Cell C ount
EC50
10.6 mg/L (95% C.I. = 5.8 to 15.5)
NOEC
1 mg/L
H-24750 exhibited medium concern (4) under TSCA and was classified as "harmful" to aquatic organisms (R52) under the EU Directive in a 72-hour, acute test using the green alga Selenastrum capricornutum.
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a The EC50 is defined as the "effective concentration" producing a 50% inhibition o f growth relative to the control.
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
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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. Slob, W., (2002). Dose-response modeling of continuous endpoints. Toxicological Science,
V66;pp 298-312.
'
4. Smrchek, J., Clements, R., Morcock, R., and Rabert, W. (1993). Assessing ecological hazard under TSCA: methods and evaluation of data. Environmental Toxicology and Risk Assessment, A ST M STP 1179. (W.G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia.
5. Official Journal of the European Communities, Volume 36, 4 May 1993, pp. 68, section 5.2.1.
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
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TABLES
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
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TABLE 1 pH MEASUREMENTS OF TEST SOLUTIONS
Nominal Concentration
(mg/L)
Test Initiation (0 hours) pH
Test Termination (72 hours) pH
Blank Control
7.41
7.74
0.1 7.47 1 7.42 10 7.33 100 6.33 1000 3.20
7.78 7.84 7.86 6.99 3.33
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H-24750: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
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% TABLE 2
DATA SUMMARY
N om inal
Day 0 H ealthy Cell C ount Day 3 Healthy Cell Count
C o n cen tra tio n
(cells/m L )
(cells/m L )
Blank Control
Replicate 1 Replicate 2
10,000 10,000
3,140,000 2,580,000
0.1 mg/L
Replicate 1 Replicate 2
10,000 10,000
3,080,000 3,420,000
1 mg/L
Replicate 1 Replicate 2
10,000 10,000
2,540,000 2,600,000
10 mg/L
Replicate 1 Replicate 2
10,000 10,000
1,460,000 1,760,000
100 mg/L
Replicate 1 Replicate 2
10,000 10,000
10,000 0
1000 mg/L
Replicate 1 Replicate 2
10,000 10,000
0 0
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H-24750: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
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FIGURE
9
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H-24750: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
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FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE
OCEOrCoNDrCROERMHulmIoTURdLAieAlTIr2I'Da,CoKUeSuel=inI0h.gCo0oMu.o1A.naRl wIQ12Ur1Ai0o/RjD1o10T0s00cM0j10e0itsh0o0d
O EC D M odel 2. Uslna MARQUARDT Method r O R I I U L A I r R0 U KOKE:. AWR 1 4 3 3 9 S C 3 I 5 r O R H U L A I - C e I I _ C o u n I 72 / 10000
C O NC E N TRA T10NS = 0 0 . 1 I 10 100 1 0 0 0
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H-24750: Influence brf Growth and Growth Rate of the Green Alga Selenastrum capricornutum
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APPENDICES
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H-24750: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum
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APPENDIX A AAP Nutrient Medium Constituents
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum________________
DuPont-12989
AAP NUTRIENT MEDIUM CONSTITUENTS
Stock Solutions3
Component
Concentration (g/L)
Macronutrient Stock #1
NaN03
25.500
Macronutrient Stock #2
NaHC03
15.000
Macronutrient Stock #3
K2H P04
1.044
Macronutrient Stock #4
M gS04*7H20
14.700
Macronutrient Stock #5
MgCl2*6H20
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 Solutions8
Component
Concentration (mg/L)
Micronutrient Stock
H 3B O 3
MnCl2*4 H20 ZnCl2 CoC12*6 H20 CuC12*2H20 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 2. b Includes magnesium from both magnesium sulfate and magnesium chloride.
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum caDricornutum -- --------------------------------------- ------------------- ---------.----------------------------------------------------------DuPont-12989
APPENDIX B 72-Hour Healthy Cell Count Statistics
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricomutum
NOEC Determination Using StatXact
!K Independent Samples:Jonckhere-Terpstra Test > KI JT/EX /RO=CONC /CO=COUNT72 All concentrations present
Datafile: C:\AQUATICS\hoki
txdata.cy3
JONCKHEERE-TERPSTRA TEST distributed]
[That
the 6 populations are
identically
Statistic based on the 12 observations:
Mean 30.00
Std-dev 7 -129
Observed(JT(x)) 5.000
Standardized(JT*(x)) -3.507
Asymptotic p-value: One-sided: Pr { JT*(X) .LE. Two-sided: 2 * One-sided
0.0002 0.0005
Exact p-values:
One-sided: Pr { JT*(X) Pr { JT*(X)
Two-sided: Pr { |JT*(X)|
.LE. .EQ. .GE.
-3.507 } = -3.507 } =
3.507 } =
0.0001 0.0000 0.0001
!K Independent Samples:Jonckhere-Terpstra Test > KI JT/EX /RO=CONC /CO=COUNT72 1000 mg/L omittec
Datafile: C:\AQUATICS\hokJ
txdata.cy3
JONCKHEERE-TERPSTRA TEST distributed]
[Thlt the 5 populations are
identically
Statistic based on the 10 observations:
Mean 20.00
Std-dev 5-477
Observed(JT(x)) 4.000
Asymptotic p-value:
One-sided: Pr { JT*(X) .LE. Two-sided: 2 * One-sided
-2.921 }
Exact p-values:
One-sided: Pr { JT*(X) Pr { JT*(X)
Two-sided: Pr {|JT*(X)|
.LE. .EQ. .GE.
-2.921 } -2.921 }
2.921 ]
Standardized(JT*(x)) -2.921
0.0017 0.0035
0.0012 0.0007 0.0024
!K Independent Samples:Jonckhere-Terpstra Test > KI JT/EX /RO=CONC /CO=COUNT72 1000 and 100 mg/L omitted
Datafile: C:\AQUATICS\hoki
txdata.cy3
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H-24750: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum
JONCKHEERE-TERPSTRA TEST distributed]
[That the 4 populations are
identically
Statistic based on the 8 observations:
Mean 12.00
Std-dev 3.916
Asymptotic p-value: One-sided: Pr { JT*(X) .LE. Two-sided: 2 * One-sided
Observed(JT(x) ) 4.000
-2.043 }
Exact p-values:
One-sided: Pr Pr
Two-sided: Pr
{ JT*(X)
{ JT*(X) { |JT*(X)|
.LE.
.EQ. .GE.
-2.043 } -2.043 }
2.043 }
Standardized!J T * (x)) -2.043
0.0205 0.0411
0.0262 0.0139 0.0524
'K Independent Samples:Jonckhere-Terpstra Test > KI JT/EX /RO=CONC /CO=COUNT72 1000, 100, and ll
mg/L omitted
Datafile: C:\AQUATICS\hokei
txdata.cy3
JONCKHEERE-TERPSTRA TEST distributed]
[That
the 3 populations
are identically
Statistic based on the 6 observations:
Mean 6.000
Std-dev 2.517
Asymptotic p-value: One-sided: Pr { JT*(X) .LE. Two-sided: 2 * One-sided
Observed(JT(x)) 4.000
-0.7947 }
Exact p-values: One-sided: Pr { JT*(X) .LE. Pr { JT*(X) .EQ. Two-sided: Pr { JJT*(X) | .GE.
-0.7947 } -0.7947 }
0.7947 }
Standardized(JT*(x)) -0.7947
0.2134 0.4268
0.2889 0.1222 0.5778
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H-24750: Influence on Growth and Growth Rate cifthe Green Alga Selenastrum capricomutum
DuPont-12989
EC50 Estimation Using OECD Model 2
OBSERVED & PREDICTED PERCENT1 OANGE FROM CONTI FORMULAI FROM HOK:
FORMULAI=Ce 1l_Coun?72
Obs
CONC N
MEAN
PRED
PCTCHNG
PREDPCTCHNG
1 0.0 2 286.0 297.537
2 0.1 2 325.0 295.603
3 1.0 2 257.0 278.759
4 10.0 2 161.0 155.034
5
100.0
2
0.5
0.439
6
1000.0
2
0.0
0.000
0
14 -10 -44 -100 -100
0 -1
-6
-48
-100
-100
EC50 using Method=MARQUARDT, OECD Model 2 Covariance Matrix and Standard Error of Parameter Estimates
_NAME
yO EC50
SE
EC50 yO
-8.778 102.717
6.06778 -8.77808
2.4633 10.1350
Model 2 Parameter Estimates, EC50 and 95% Confidence Bounds
yO EC50 LB
UB
297.537
10.6328
5.80478
15.4609
Model 2 Evaluation Measures
SSREG
SSERR
SSTOTC
SSTOTU
521335.79
5301.12
21827.68
526636.92
note
Graph descriptions include the following: formulai: EC50 OECD MODEL 2 FIT BY MARQUARDT,
RSQUARE 0.75714
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