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AR226-2810
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Study Title
H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricomutum
AUTHORS: BarbraD. Ferrell, B.S. Robert A. Hoke, Ph.D.
STUDY COMPLETED ON: May 20,2003
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
WORK REQUEST NUMBER: ^^^^^B^lM^
STUDY CODE NLMBER:B^B&
Qompany sanitize<!- ^ s nof contain TSCA CB
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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.
Work Completed by:
/D/U^W.. A >Ui^M^
BarbraD.Farcll.B.S.
Associate Scientist
Issued by Study Director;
x^L/^ ^
Robert A. HokeWi.D.
Senior Research Ecotoxicologist and Manager
^>Ml^2^?^ Dale
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H-24750: Influence oh Growth and Growth Rate of the Green Alga Selenastrum capricomutum
STUDY INFORMATION
9th Collective Nomenclature: Synonyms/Codes:
T. H-24750
Submitter's Notebook Numberfs Haskell Number: 24750
CAS Registry Number:
DuPont-12989
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 of the green alga Selenastrum capricornutum^ 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 EC5o, including the 95% confidence intervals (C.I.),13based on nominal
concentrations are as follows:0
Healthy Cell Count
EC5o
NOEC
10.6 mg/L (95% C.I. = 5.8 to 15.5) 1 mg/L
H-24750 exhibited medium concern'1 under 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 EC5o is defined as the "effective concentration" producing a 50% inhibition of growth relative to the
control.
c Slob, W., (2002). Dose-response modeling of 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, ASTMSTP 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 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 Selenastmm capricornutum
DuPont-12989
MATERIALS AND METHODS
A.
Test System
Selenastmm 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.0'21 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 Erienmeyer 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:
Nutrient Flask Volume Solution Volume Volume
Organism___________Medium____(mL)_______(mL)_____Ratio
Selenastrum capricornutum
AAP
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 ofMilli-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 Corningpre-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 of the Green Alga Selenastrum capricornutum
DuPont-12989
2.
Test Culture Preparation
For each blank control and test solution, 2 aliquots (50 mL each) were placed in separate sterilized 250 mL Erienmeyer 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 uL) 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
Illumination
___Population____(lumens/m2 = lux)
10,000 cells/mL
6000 to 10.000
PhotoPeriod (hours)
24
Shaking Speed Temperature
(rpms)____(C)
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 EC5o 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
DuPont-12989
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 of the test solutions at test initiation ranged from 3.20 to 7.47, and at test
termination ranged from 3.33 to 7.86.
CONCLUSIONS
The 72-hour EC^o, including the 95% confidence intervals (C.I.)3 based on nominal test substance concentrations and the NOEC are as follows:^5
Healthy Cell Count
EC5o
NOEC
10.6 mg/L (95% C.L = 5.8 to 15.5) 1 mg/L
H-24750 exhibited medium concern (4) under TSCA and was classified as "harmful" to aquatic organisms (R52/5^under the EU Directive in a 72-hour, acute test using the green alga
Selenastrum capricornutum.
Company Sanitized. Does not contain TSCA CBS
a The EC5o is defined as the "effective concentration" producing a 50% inhibition of growth relative to the
control.
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H-24750: Influence on Growth and Growth Rate
of the Green Alga Selenastrum capricornutum_____________________________DuPont-12989 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 of 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, ASTMSTP 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
DuPont-12989
TABLES
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
DuPont-12989
TABLE 1 pH MEASUREMENTS OF TEST SOLUTIONS
Nominal Concentration
Test Initiation (0 hours)
Test Termination (72 hours)
(mg/L)__________pH______________pH____
Blank Control
7.41
7.74
0.1 1 10
100 1000
7.47 7.42 7.33 6.33 3.20
7.78 7.84 7.86 6.99 3.33
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H-24750: Influence on Growth and Growth Rate
of the Green Alga Selenastrum capricornutum
DuPont-12989
TABLE 2 DATA SUMMARY
Nominal
Day 0 Healthy Cell Count Day 3 Healthy Cell Count
Concentration______(cells/mL)__________(cells/mL)
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
10,000
0
Replicate 2
10,000
0
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastmrn capricornutum
DuPont-12989
FIGURE
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastmm capricornutum
DuPont-12989
FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE
or nulnl 400:
tc
300-
OECO Model 2, Using MARQUARDT Method rORUUKI r011 HOrE.tB14339SC115 fOUUI.AI=CtlI.Conl;2 / 10000
COMCENIBAIIDHS=0 O.I I 10 100 1000
ZOO-
100- I
B-
50 pet iitiintion
-100- i ' ' i ' i ' i i < i ' ' ' i ' i i i i ' ' ' i i ' ' i ' r ' ' ' ' 1 ' ' i ' ' ' i ' ' ' ' i ' ' ' ' r
1DO
200
300
400
50D
SOO
700
BOO
900
1000
110
CDNC
OECO Model 2. Using MARQUARDT Melhod rORUULAI FBOM HOU.AWB14.139SC.115 FOBHULAI.CEII.CoBtm / 10000
COHCCHTIiAIIOIIS^O O.I I 10 100 1000
COHC
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H-24750: Influence brfGrowth and Growth Rate of the Green Alga Selenastnim capricomutum
DuPont-12989
APPENDICES
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum
DiiPont-12989
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 NaHCOs
15.000
Macronutrient Stock #3 K2HP04
1.044
Macronutrient Stock #4 MgS04-7H20
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
2.904"
Ca
1.202
S
1.911
Stock Solutions'
Component
Concentration (mg/L)
Micronutrient Stock
H3B03 MnCl2-4 H20 ZnCl2 CoCl2-6 H20 CuCl2-2H20 Na2Mo04'2 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 (ug/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 capricornutum
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
DuPont-12989
72-HOUR HEALTHY CELL COUNT STATISTICS
MOEC Determination Using StatXact
!K Independent Samples:Jonckhere-Terpstra Test
> KI JT/EX /RO=CONC /CO=COUNT72 All concentrations present
Datafile: C: \AQUA7XCS\hoke^^--------------fctxdata.cy3
JONCKHEERE-TERPSTRA TEST [That the 6 populations are identically distributed] 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
-3.507 } =
=
0.0002 0.0005
Exact p-values: One-sided: Pr { JT*(X) -LE. Pr { JT*(X) -EQ. Two-sided: Pr { |JT*(X)| .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 omitted
Datafile: C: \AQUATICS\hokeBBBBBBBtxdata. cy3
JONCKHEERE-TERPSTRA TEST [That the 5 populations are identically distributed] Statistic based on the 10 observations:
Mean
20.00
Std-dev 5.477
ObservedfJT(x)) 4.000
Standardized(JT*(x)) -2.921
Asymptotic p-value: One-sided: Pr { JT*(X) .LE. Two-sided: 2 * One-sided
-2.921 } =
=
0.0017 0.0035
Exact p-values: One-sided: Pr { JT*(X) -LE. Pr { JT*(X) .EQ. Two-sided: Pr { |JT*(X)| .GE.
-2.921 } = -2.921 } =
2.921 } =
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\hokeM--------|--------Ltxcdya3ta.
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H-24750: Influence on_Growth and Growth Rate of the Green Alga Selenastnim capricomufum
DuPont-12989
JONCKHEERE-TERPSTRA TEST [That the 4 populations are identically distributed] Statistic based on the 8 observations:
Mean
12.00
Std-dev 3.916
Observed(JT(x)) 4.000
Standardized(JT*(x)) -2.043
Asymptotic p-value: One-sided: Pr { JT*(X) -LE. Two-sided: 2 * One-sided
.043 }
=
0 .0205 0 .0411
Exact p-values: One-sided: Pr { JT*(X) ,LE. Pr { JT*(X) .EQ. Two-sided: Pr { |JT*(X)| .GE.
-2.043 } =
-2.043
}
=
2.043 } =
0.0262
0.0139 0.0524
!K Independent Samples:Jonckhere-Terpstra Test
> KI JT/EX /RO=CONC /CO=COUMT72 1000, 100, and 10 mg/L omitted
Datafile: C:\AQUATICS\hoke
stxdata.cy3
JONCKHEERE-TERPSTRA TEST [That the 3 populations are identically
distributed] Statistic based on the 6 observations:
Mean
6.000
Std-dev 2.517
Observed(JT(x)) 4.000
Standardized(JT*(x)) -0.7947
Asymptotic p-value: One-sided: Pr { JT*(X) .LE. Two-sided: 2 * One-sided
-0.7947 } =
=
0.2134 0.4268
Exact p-values: One-sided: Pr { JT*(X) .LE. Pr { JT*(X) .EQ. Two-sided: Pr { |JT*(X)| .GE.
-0.7947 } = -0.7947 } =
0.7947 } =
0.2889 0.1222
0.5778
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H-24750: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricomutum
DuPont-12989
EC50 Estimation Using OECD Model 2
OBSERVED & PREDICTED PERCENT CHANGEFROMCONTROL MEAN
FORMULAl FROM HOK^BIHB--------A
FORMULAl =Ce 1 l_Count72/nffu<^^
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
SSREG
Model 2 Evaluation Measures
SSERR
SSTOTC
SSTOTU
521335.79
5301.12
21827.68
526636.92
note Graph descriptions include the following: formulal: EC50 OECD MODEL 2 FIT BY MARQUARDT,
RSQUARE
.0.75714
cwvwvsm1^^^
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