Fecal-Indicator Bacteria and Escherichia coli

Pathogen Data Collected Near a Novel Sub-Irrigation 

Water-Treatment System in Lenawee County, 

Michigan, June–November 2007 

By Joseph W. Duris and Stephanie Beeler 

In cooperation with Lenawee County Conservation District

Open File Report 2008–1025

U.S. Department of the Interior 

U.S. Geological Survey 
U.S. Department of the Interior 

DIRK KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia 2008 

For product and ordering information: 

World Wide Web: http://www.usgs.gov/pubprod 

Telephone: 1-888-ASK-USGS 

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and living resources, natural hazards, and the environment: 

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imply endorsement by the U.S. Government 

Athough this report is in the public domain, permission must be secured from the individual 

copyright owners to reproduce any copyrighted material contained within this report. 

Suggested citation: Duris, J.W., and Beeler, Stephanie, 2008 fecal-indicator bacteria and 

Escherichia coli pathogen data collected near a novel sub-irrigation water-treatment system in 

Lenawee County, Michigan, June–November 2007: U.S. Geological Survey Open-File Report 

2008–1025, 13 p. 

ii
Contents 

Abstract................................................................................................................................................................................1 

Introduction .........................................................................................................................................................................1 

Methods ...............................................................................................................................................................................2 

Site Information............................................................................................................................................................2

Sample Collection........................................................................................................................................................2

Bacteriological Indicator Organisms.......................................................................................................................3 

E. coli Gene Analysis...................................................................................................................................................3 

Results ..................................................................................................................................................................................3 

References Cited ................................................................................................................................................................4

Figures 

 1. Map showing sampling locations for fecal-indicator bacteria and E. coli pathogen study, 

Lenawee County, MI, 2007. .................................................................................................................................5 

 2–10. Stacked bar graphs illustrating the fecal coliform bacteria and Escherichia coli

concentrations by date for stations in Lenawee County, Mich., 2007: 

 2. Station 415233084140309 .............................................................................................................................................6 

 3. Station 415224084141108 .............................................................................................................................................6 

 4. Station 415230084142607 .............................................................................................................................................7 

 5. Station 415224084141604 .............................................................................................................................................7 

 6. Station 415227084141405 .............................................................................................................................................8 

 7. Station 415227084141806 .............................................................................................................................................8 

 8. Station 415223084140101 .............................................................................................................................................9 

 9. Station 415230084140602 .............................................................................................................................................9 

10. Station 415233084140603 ...........................................................................................................................................10 

Tables 

 1. U.S. Geological Survey stations sampled in Lenawee County, Michigan .......................................................11 

 2. Sampling dates and conditions................................................................................................................................11 

 3. Microbiological analytes and supplemental information for fecal-indicator-bacteria analysis .................11 

 4. Microbiological data collected in Lenawee County, Michigan.........................................................................12 

iii
Conversion Factors 

Multiply By To obtain 

Length 

inch (in.) 2.54 centimeter (cm) 

foot (ft) 0.3048 meter (m) 

meter (m) 3.281 foot (ft) 

yard (yd) 0.9144 meter (m) 

Volume 

gallon (gal) 3.785 liter (L) 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

°F=(1.8×°C)+32 

Small sample volumes are reported in milliliters (mL) and microliters (μL); very small masses are 

given in nanograms (ng). 

Bacteria concentrations are given in colony-forming units per 100 milliliters (CFU/100 mL). 

iv
Fecal-Indicator Bacteria and Escherichia coli

Pathogen Data Collected Near a Novel Sub-Irrigation 

Water-Treatment System in Lenawee County, 

Michigan, June–November 2007 

By Joseph W. Duris and Stephanie Beeler

Abstract 

The U.S. Geological Survey, in cooperation with the Lenawee County Conservation District 

in Lenawee County, Mich., conducted a sampling effort over a single growing season (June to 

November 2007) to evaluate the microbiological water quality around a novel livestock reservoir 

wetland sub-irrigation system. Samples were collected and analyzed for fecal coliform bacteria, 

Escherichia coli (E. coli) bacteria, and six genes from pathogenic strains of E. coli. 

A total of 73 water-quality samples were collected on nine occasions from June to 

November 2007. These samples were collected within the surface water, shallow ground water, and 

the manure-treatment system near Bakerlads Farm near Clayton in Lenawee County, Mich. Fecal 

coliform bacteria concentrations ranged from 10 to 1.26 million colony forming units per 100 

milliliters (CFU/100 mL). E. coli bacteria concentrations ranged from 8 to 540,000 CFU/100 mL. 

Data from the E. coli pathogen analysis showed that 73 percent of samples contained the eaeA

gene, 1 percent of samples contained the stx2 gene, 37 percent of samples contained the stx1 gene, 

21 percent of samples contained the rfbO157 gene, and 64 percent of samples contained the LTIIa 

gene. 

Introduction 

The management of animal wastes on dairy farms is commonly driven by the need of 

farmers to effectively manage nutrient content in soils where crops are grown, as well as to control 

and manage manure in a way that avoids negative effects on local surface water and ground water. 

Typically, the management of manure in and around a farm is locally controlled by use of 

comprehensive nutrient-management plans that define how manure will be handled on the farm 

under various conditions. To meet the guidelines of these management practices, the Bakerlads 

Farm, working with the U.S. Department of Agriculture and the Lenawee County Conservation 

District, in 2005 installed novel manure-management system called a Livestock Reservoir Wetland 

Sub-Irrigation System (LRWSIS) on the farmstead, which is near Clayton, Mich (fig. 1; all figures 

and tables are at back of report). 

1
The U.S. Geological Survey (USGS), in cooperation with the Lenawee County 

Conservation District (LCCD) in Lenawee County, Mich., sampled nine sites (surface water and 

groud water) over a single growing season, from June to November 2007. The purpose of this study 

was to evaluate the microbiological water quality around the LRWSIS. By studying the populations 

of fecal coliform and E. coli bacteria and by characterizing the types of E. coli pathogens and other 

bacterial markers in manure sources, we can assess how manure-management practices may 

contribute to indicator and pathogen loading in environmental waters. This could lead to a better 

understanding of how and why bacterial indicators and pathogens might be found in other 

environmental settings affected by agriculturally influenced waters. In addition, this work builds on 

a growing body of work by the USGS Michigan Water Science Center related to understanding 

how manure management affects the transport of fecal indicator and pathogenic bacteria. Samples 

were collected and analyzed for fecal coliform bacteria, Escherichia coli (E. coli), and six genes 

from pathogenic strains of E. coli, including E. coli O157:H7. This report summarizes the results of 

this sampling. 

Methods 

Site Information 

The LRWSIS directs dairy barn wash water and runoff from the entire facility into a 

primary settling reservoir. Once settled, the liquid waste is then transferred into an 80,000 gal 

constructed wetland via a 3-in. subsurface PVC pipe that bridges a small unnamed tributary to the 

South Branch of the River Raisin. The liquid waste from the influent pond of the constructed 

wetland is allowed to move via subsurface flow through a packed layer of sand and pea gravel, 

planted with wetland vegetation, into the subsurface irrigation holding pond. This water is then 

pumped directly into a closed subsurface tile irrigation system below a planted cornfield, where it 

supplies water and nutrients to the crop that is planted above. 

Samples for this study were collected from nine locations in and around the Bakerlads Farm 

(table 1 and fig. 1). For flowing water, the tributary to the South Branch of the River Raisin was 

sampled at three locations: upstream from the sub-irrigated field and the LRWSIS, downstream 

from the sub-irrigated field, and downstream from the LRWSIS. For ground water, samples were 

collected from two piezometers within the cropped area of the sub-irrigated field and one 

piezometer 10 m from the bank of the tributary to the South Branch of the River Raisin. For ponded 

water, samples were collected from the primary settling pond, the influent pond of the wetland 

system, and the sub-irrigation holding pond of the wetland system to evaluate potential inputs to the 

ambient water. Samples were collected on nine dates over the 2007 growing season, from June 25 

through November 27, 2007 (table 2). 

Sample Collection 

Samples were collected by the LCCD staff using standard USGS sampling procedures 

documented by the USGS National Field Manual (Myers, 2003). Each sample was evaluated for 

fecal coliform bacteria and E. coli and for six genes from pathogenic E. coli. 

2
Bacteriological Indicator Organisms 

Samples were examined for the presence of fecal-indicator bacteria by means of standard 

membrane filtration and serial dilution methods as indicated in Britton and Greeson (1989), 

whereas E. coli analysis followed protocols of the American Public Health Association and others 

(1998), method 9222D/9222G. Fecal coliform bacteria from the membrane filter with the most 

growth for each site and date were collected and suspended in 1 mL of phosphate buffered saline 

(PBS) with a final concentration of 15 percent glycerol. This fecal coliform stock (FC) was used for 

all subsequent analysis for pathogenic E. coli.

E. coli Gene Analysis 

The FC stocks were analyzed by use of three polymerase chain reaction (PCR) methods. 

Deoxyribonucleic acid (DNA) was extracted from one-tenth of the volume of the FC stock, using 

alkaline polyethylene glycol (APEG) DNA extraction (Chomczynski and Rymaszewski, 2006). A 

volume of 100 μL of FC stock was transferred to a volume of 1,000 μL of APEG reagent and 

incubated at 55°C for 30 minutes, at which point it was stored at 4°C until PCR analysis for E. coli

pathogen genes. 

A volume of 1 μL of DNA representing 1–100 ng of DNA was used to conduct all PCR 

analysis. Samples were analyzed for the gene responsible for the O157 type (rfbO157) as indicated 

by Osek (2003). The gene targeted by this assay is the gene responsible for the production of the 

specific antigen found on the surface of an E. coli O157 cell. 

The samples were also analyzed for the pathogenic E. coli toxin genes eaeA, stx2, and stx1

(Fagan and others, 1999; Sabat and others, 2000). The genes targeted by this assay are those 

required to confirm the presence of enterohemorrhagic E. coli in clinical samples (Nataro and 

Kaper, 1998; Fagan and others 1999). The method was modified to include analysis for a gene 

common to all E. coli, both pathogenic and nonpathogenic, which was used to confirm the presence 

of E. coli (Duris and others, 2003; Fogarty and others, 2005). The LTIIa gene, carried by some 

pathogenic E. coli strains common to bovine sources, was analyzed according to Khatib and others 

(2002) and Chern and others (2004). A summary of all genes and their common functions and 

associations can be found in table 3. All gene assays followed standard protocols (U.S. 

Environmental Protection Agency, 2004). All methods included positive controls and method 

blanks. 

Results 

Water-quality samples were collected on nine occasions from June to November 2007. A 

total of 73 water samples were collected within the surface water, shallow ground water, and 

manure-treatment system near Bakerlads Farm, in Lenawee County, Mich. Fecal coliform bacteria 

concentrations ranged from 10 to 1.26 million CFU/100 mL. E. coli bacteria concentrations ranged 

from 8 to 540,000 CFU/100 mL. Data from the E. coli pathogen analysis showed that 73 percent of 

samples contained the eaeA gene, 1 percent of samples contained the stx2 gene, 37 percent of 

samples contained the stx1 gene, 21 percent of samples contained the rfbO157 gene, and 64 percent 

of samples contained the LTIIa gene. The EC gene that is common to all E. coli was present in 97 

percent of samples. The data from the fecal-indicator bacteria testing at each site are presented in 

table 4 and figures 2–10. Data from the E. coli pathogen analysis are presented in table 4. Those 

samples that had countable E. coli but no detectable EC gene were considered not amplifiable by 

PCR and are noted in table 4. 

3
References Cited 

American Public Health Association, American Water Works Association, and Water Environment 

Federation, 1998, Standard methods for the examination of water and wastewater (20th ed.): 

Washington, D.C., American Public Health Association, p. 9–1 to 9–76. 

Britton, L.J., and Greeson, P.E., eds., 1989, Methods for collection and analysis of aquatic 

biological and microbiological samples: U.S. Geological Survey Techniques of Water-Resources 

Investigations, book 5, chap. A4, p. 37–40. 

Chern E.C., Tsai Y.L., and Olson B.H., 2004, Occurrence of genes associated with enterotoxigenic 

and enterohemorrhagic Escherichia coli in agricultural waste lagoons: Applied and 

Environmental Microbiology, v. 70, no. 1, p. 356–362. 

Chomczynski, Piotr, and Rymaszewski, Michal, 2006, Alkaline polyethylene glycol-based method 

for direct PCR from bacteria, eukaryotic tissue samples, and whole blood: Biotechniques, v. 40, 

p. 454–458. 

Duris, J.W., Haack, S.K., Reeves, H.W., and Kiesler, J.L., Jr., 2003, Pathogenic Escherichia coli 

from agricultural watersheds in Michigan and Indiana, in Proceedings, 2003 Spring Specialty 

Conference, Kansas City, Mo.: American Water Resources Association. 

Fagan, P.K., Hornitzky, M.A., Bettelhein, K.A., and Djordjevic, S.P., 1999, Detection of shiga-like 

toxin (stx1 and stx2), intimin (eaeA), and enterohemorrhagic Escherichia coli (EHEC) hemolysin 

(EHEC hylA) genes in animal feces by multiplex PCR: Applied and Environmental 

Microbiology, v. 65, p. no. 2, p. 868–872. 

Fogarty, L.R., Duris, J.W., and Aichele, S.S., 2005, Preliminary survey of antibiotic-resistant fecal 

indicator bacteria and pathogenic Escherichia coli from river-water samples collected in Oakland 

County, Mich., 2003: U.S. Geological Survey Scientific Investigations Report 2005–5058, 34 p. 

Khatib, L.A., Tsai, Y.L., and Olson, B.H., 2002, A biomarker for the identification of cattle fecal 

pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli: Applied 

Microbiology and Biotechnology, v. 59, p. 97–104. 

Myers, D.N., 2003, Fecal indicator bacteria: U.S. Geological Survey Techniques of WaterResources Investigations, book 9, chap. A7, section 7.1 (3d ed.), accessed January 26, 2005, at 

http://pubs.water.usgs.gov/twri9A7/ 

Nataro, J.P., and Kaper, J.B., 1998, Diarrheagenic Escherichia coli: Clinical Microbiological 

Reviews, v.11, no. 1, p. 142–201. 

Osek, J., 2003, Development of a multiplex PCR approach for the identification of Shiga toxinproducing Escherichia coli strains and their major virulence factor genes: Journal of Applied 

Microbiology, v. 95, no. 6, p. 1217–1225. 

Sabat, G., Rose, P., Hickey, W.J., and Harkin, J.M., 2000, Selective and sensitive method for PCR 

amplification of Escherichia coli 16S rRNA genes in soil: Applied and Environmental 

Microbiology, v. 66, no. 2, p. 844–849. 

U.S. Environmental Protection Agency, 2004, Quality assurance/quality control guidance for 

laboratories performing PCR analysis on environmental samples: Office of Water, 

EPA 815–B–04–001, p 1–57. 

4
5

Figure 1. Sampling locations for fecal indicator bacteria and E. coli pathogen study, Lenawee 

County, Mich., 2007.
Figure 2. Fecal coliform bacteria and Escherichia coli concentrations by date for station 415233084140309 

(D1), in Lenawee County, Mich., 2007. 

Figure 3. Fecal coliform bacteria and Escherichia coli concentrations by date for station 415224084141108 

(D2), in Lenawee County, Mich., 2007.

6
Figure 4. Fecal coliform bacteria and Escherichia coli concentrations by date for station 415230084142607 

(D4), in Lenawee County, Mich., 2007. 

igure 5. Fecal coliform bacteria and Escherichia coli concentrations by date for station 415224084141604 

(W1), in Lenawee County, Mich., 2007. 

F

7
Figure 6. 

Figure 7. 

Fecal coliform bacteria and Escherichia coli concentrations by date for station 415227084141405 

(W2), in Lenawee County, Mich., 2007. 

Fecal coliform bacteria and Escherichia coli concentrations by date for station 415227084141806 

(W3), in Lenawee County, Mich., 2007. 

8
Figure 8. 

Figure 9. Fecal coliform bacteria and Escherichia coli concentrations by date for station 415230084140602 

(P3), in Lenawee County, Mich., 2007. 

Fecal coliform bacteria and Escherichia coli concentrations by date for station 415223084140101 

(P2), in Lenawee County, Mich., 2007. 

9
Figure 10. Fecal coliform bacteria and Escherichia coli concentrations by date for station 415233084140603 

(P4), in Lenawee County, Mich., 2007.

10
Table 1. U.S. Geological Survey stations sampled in Lenawee County, Michigan. 

 [USGS, U.S. Geological Survey; ID, Identification] 

Table 2. Sampling dates and conditions. 

Sample number Sampling date Condition 

1 6/25/2007 4 days prior to treatment system start 

2 7/18/2007 Day 20 of system operation 

3 8/6/2007 Day 39 of system operation 

4 8/20/2007 Day 53 of system operation 

5 9/4/2007 Day 68 of system operation 

6 9/10/2007 Day 74 of system operation 

7 9/18/2007 Day 82 of system operation 

8 10/3/2007 10 days post system shutdown 

9 11/27/2007 37 days after field drained 

Table 3. Microbiological analytes and supplemental information for fecal-indicator-bacteria analysis.

[E. coli, Escherichia coli; NA, Not Applicable] 

Analyte Category Gene 

product Importance 

Fecal coliform Indicator group NA Commonly used group of fecal indicator bacteria. 

E. coli Indicator group NA Commonly used species of fecal indicator bacteria. 

eaeA Gene Intimin 

Product causes tight binding of pathogenic E. coli to 

intestinal cells (Nataro and Kaper, 1998; Fagan and 

others, 1999). 

stx1 Gene Shiga toxin 1 

Product causes intestinal cell death. stx1 is associated 

with bovine sources (Nataro and Kaper, 1998; Fagan 

and others, 1999). 

stx2 Gene Shiga toxin 2 

Product causes intestinal cell death. When found with 

eaeA is associated with human disease (Nataro and 

Kaper, 1998; Fagan and others, 1999). 

EC Gene 16s rRNA Common to all E. coli, pathogenic and nonpathogenic 

(Fogarty and others, 2005). 

rfbO157 Gene O157 surface protein Gene is marker for E. coli O157:H7 (Osek, 2003). 

LTIIa Gene Heat labile toxin 

Product causes diarrheal disease in calves, commonly 

associated with bovine sources (Khatib and others, 

2002; Chern and others, 2004). 

 

USGS 

station 

ID 

USGS 

field 

ID 

Map 

number USGS station name Station type 

415230084142607 D4 1 Tributary to South Branch River Raisin (site D4) near Clayton, MI Surface water 

415224084141108 D2 2 Tributary to South Branch River Raisin (site D2) near Clayton, MI Surface water 

415233084140309 D1 3 Tributary to South Branch River Raisin (site D1) near Clayton, MI Surface water 

415227084141806 W3 4 Unnamed Well W3 near Clayton, MI Ground water 

415227084141405 W2 5 Unnamed Well W2 near Clayton, MI Ground water 

415224084141604 W1 6 Unnamed Well W1 near Clayton, MI Ground water 

415223084140101 P2 7 Unnamed Storage Lagoon P2 near Clayton, MI Place of use 

415230084140602 P3 8 Unnamed Wetland Pond P3 near Clayton, MI Place of use 

415233084140603 P4 9 Unnamed Wetland Pond P4 near Clayton, MI Place of use 

11
Table 4. Microbiological data collected in Lenawee County, Michigan.

[USGS, U.S. Geological Survey; E. coli, Escherichia coli; EC, E. coli 16S ribosomal DNA gene that is common to all 

E. coli; +, gene present in sample; -, gene absent in sample; ns, no sample; na, not amplifiable with polymerase chain 

reaction] 

Colony forming units per 

 100 milliliters water Eschericha coli gene data

 ID Field 

0 D1 6/ + - - + 

Fecal Date E. coli USGS station ID coliforms eaeA stx2 stx1 EC rfb LTIIa

4152330841403 9 25/2007 1,500 700 - + 

41522408414110 D2 6/ + - - + 

0 D4 6 + - - + 

01 P2 6/ - + + 

02 P3 6 - - + 

03 P4 6 - - + 

415224084141604 W1 6/25/2007 ns ns ns ns ns ns ns ns 

7 ns ns ns ns ns ns ns ns 

415227084141806 W3 6/25/2007 ns ns ns ns ns ns ns ns 

0 7/18/2007 28 + - - + - - 

415 4084141 2 10 + - - + - + 

415 0084142 4 7/ ns ns ns ns ns ns ns 

415 3084140 P2 7/ 370,000 + - + + - + 

415 0084140 P3 7/ 140,000 + - + + - + 

415 3084140 P4 7/ 26,000 + - + + + + 

415 4084141 1 7/ ns ns ns ns ns ns ns 

415 7084141 2 7/ ns ns ns ns ns ns ns 

415 7084141 3 7 ns ns ns ns ns ns ns 

415 3084140 1 8/6 200 - - - + - - 

415224084141108 D2 8/6/2007 8,600 5,100 + - + + - + 

415230084142607 D4 8/6/2007 930 370 + - - + - + 

160,000 73,000 + - + + - - 

415233084140603 P4 8 7 13,000 7,000 + - - + - - 

84141 W1 8 360 + - - + - - 

141 8 ns ns 

141 8 07 25,000 

415233084140309 D1 8/20/2007 11,000 

15224084141 D2 8 10,000 

415230084142607 D4 8/20/2007 10,000 + - + + - + 

415223084140101 P2 8/20/2007 630,000 

5230084140 P3 8 40,000 

415233084140603 P4 8/20/2007 49,000 

5224084141 W1 8 970 

415227084141405 W2 8/20/2007 11,000 - + 

415227084141 8/ 8,400 

415233084140 D1 9 330 - + - + 

24084141 D2 9 + 

415230084142607 D4 9/4/2007 1200 

5223084140 P2 9 000 + 

415230084140602 P3 9/4/2007 1,300 - + - + 

8 25/2007 1,100 780 - - 

4152300841426 7 /25/2007 84 64 - - 

4152230841401 25/2007 190,000 160,000 + - + 

4152300841406 /25/2007 240 210 + - - 

4152330841406 /25/2007 10 8 - - + 

415227084141405 W2 6/25/200

41523308414 309 D1 28

22 108 D 7/18/2007 370 3

23 607 D 18/2007 ns 

22 101 18/2007 480,000 

23 602 18/2007 210,000 

23 603 18/2007 31,000 

22 604 W 18/2007 ns 

22 405 W 18/2007 ns 

22 806 W /18/2007 ns 

23 309 D /2007 230 

415223084140101 P2 8/6/2007 340,000 270,000 + - + + - + 

415230084140602 P3 8/6/2007 

/6/200

4152240 604 /6/2007 

07 

160 

415227084

227084

405 W2 /6/20 ns ns ns ns ns ns 

415 806 W3 /6/20 14,000 na na na na na na 

6,300 + - + + - +

7,400 + - + + + + 

8,000 

4 108 /20/2007

430,000 + - + + + + 

41 602 /20/2007 2 140,000 + - + + + + 

35,000 + - - + - + 

300 na na na na na na 

4,500 + - - + 

41 604 /20/2007 

806 W3 

309 

20/2007 

/4/2007 

7,400 - - - + - - 

260 + - 

4152 108 /4/2007 320 200 + - - + - 

1,100 + - - + + + 

150,000 + - + + + 

1,300 - - 

41 101 /4/2007 210,

12
Tabl ntinued

 [US

E. c

reac

C 

e 4. Microbiological data collected in Lenawee County, Michigan.—Co

GS, U.S. Geological Survey; E. coli, Escherichia coli; EC, E. coli 16S ribosomal DNA gene that is common to all 

oli; +, gene present in sample; -, gene absent in sample; ns, no sample; na, not amplifiable with polymerase chain 

tion] 

 olony forming units per 

100 milliliters water Eschericha coli gene data

Fecal USG FieDate 

c

E. c

e t x a

4152 P4 9/ 1,30 - - - + 

oli 

S station ID ld ID oliforms aeA s x2 st 1 EC rfb LTII

33084140603 4/2007 2,100 0 - - 

4152 W1 9 21 + - - + 

4152 W2 9 36,0 17,000 + - - + 

4152 W3 9/ + - - 

4152 D1 9/ 3, 2,00 + - + + 

4152 D2 9/ 2,6 1,500 + - + + 

4152 D4 9/ 6,5 4,100 + - - 

4152 P2 9/ 410,0 300,000 + - + 

4152 P3 9/ 1,1 670 + - - 

4152 P4 9/ 2,7 2,000 - - + + 

4152 W1 9/ 1, 50 + - - + 

4152 W2 9/ 3,2 2,600 - - - 

4152 W3 9 - - - 

4152 D1 9 - - - 

4152 D2 9 + - - 

4152 D4 9 7 530 + - + 

4152 P2 9/ 170,0 160,000 + - + 

4152 P3 9 50 - - - 

4152 P4 9/ 2, 2,00 - - - + 

4152 W1 9/ ns ns ns s s 

4152 W2 9/ ns ns ns ns s s 

4152 W3 9 - - - 

4152 D1 1 1 + - - 

4152 D2 1 3 + - - + 

4152 D4 1 41 + - + + 

4152 P2 1 1,260,0 390,000 + - + 

4152 P3 1 76 + - + 

4152 P4 1 1 60,0 + - - 

4152 W1 1 - - - 

4152 W2 1 6 + - - 

4152 W3 1 + - - 

4152 D1 1 + + + 

4152 D2 1 1, + - + + 

4152 D4 1 2, 1,50 + - + 

4152 P2 1 8 540,0 + - + + 

4152 P3 1 + - - + 

4152 P4 1 7 - - - + 

4152 W1 1 13 - - - + 

4152 W2 1 4 - - - + 

4152 W3 1 - - - 

4152 D1 8/ 3 - - - + 

15223084140101 P2 Rep 8/6/2007 390,000 310,000 + - + + - + 

24084141604 /4/2007 250 0 - +

27084141405 /4/2007 00 - -

27084141806 4/2007 1,800 1,200 + - +

33084140309 10/2007 900 0 - +

24084141108 10/2007 00 + +

30084142607 10/2007 00 + - + 

23084140101 10/2007 00 + + + 

30084140602 10/2007 00 + - - 

33084140603 10/2007 00 - +

24084141604 10/2007 800 0 - - 

27084141405 10/2007 00 + - - 

27084141806 /10/2007 46 42 + - +

33084140309 /18/2007 730 530 + - -

24084141108 /18/2007 1100 770 + - -

30084142607 /18/2007 70 + - + 

23084140101 18/2007 00 + + + 

30084140602 /18/2007 50 + - - 

33084140603 18/2007 100 0 - +

24084141604 18/2007 ns ns n ns n

27084141405 18/2007 ns n ns n

27084141806 /18/2007 14 12 + - +

33084140309 0/3/2007 2,000 ,400 + - -

24084141108 0/3/2007 660 80 - -

30084142607 0/3/2007 570 0 - +

23084140101 0/3/2007 00 + + + 

30084140602 0/3/2007 83,000 ,000 + - + 

33084140603 0/3/2007 40,000 00 + - +

24084141604 0/3/2007 38 20 + - -

27084141405 0/3/2007 2,000 70 + + -

27084141806 0/3/2007 6,800 4,400 + + +

33084140309 1/27/2007 2,500 1,800 + - +

24084141108 1/27/2007 1,900 200 + +

30084142607 1/27/2007 700 0 + + + 

23084140101 1/27/2007 60,000 00 + +

30084140602 1/27/2007 90 70 - +

33084140603 1/27/2007 90 0 - -

24084141604 1/27/2007 300 0 - +

27084141405 1/27/2007 670 00 - +

27084141806 1/27/2007 9,900 4,700 + - -

33084140309 Rep 6/2007 460 00 - -

4

13