EPA/600/R-12/580 | November 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Sporian Inline Biosensor
System (IBS)
Evaluation Summary
Cartridge
Cartridge Latch
Cartridge Front Side
Cartridge Back Side
Flowch*rn«li
TT
Flow InleUOiitlet
Office of Research and Development
National Homeland Security Research Center
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Notice
The U. S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded and directed the research
described herein under Contracts EP- C-09-041 with Shaw Environmental Incorporated. The EPA
performed the work in collaboration with the US Army Engineering Research and Development
Center (ERDC) Fluorescence Spectrocopy Lab. It has been reviewed by the Agency but does not
necessarily reflect the Agency's views. No official endorsement should be inferred. EPA does not
endorse the purchase or sale of any commercial products or services.
For questions about this report, please contact John Hall of the U.S. Environmental Protection
Agency, National Homeland Security Research Center, 26 West Martin Luther King Drive,
Cincinnati, Ohio, 513-487-2814, hall.john@epa.gov.
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Foreword
Following the events of September 11, 2001, EPA's mission was expanded to address critical needs
related to homeland security. Presidential Directives identify EPA as the primary federal agency
responsible for the country's water supplies and for decontamination following a chemical,
biological, and/or radiological attack.
As part of this expanded mission, the National Homeland Security Research Center (NHSRC) was
established to conduct research and deliver products that improve the capability of EPA to carry out
its homeland security responsibilities. One focus area of this research is the detection of potential
contaminatnts within water systems. The Sporian inline biosensor system is designed for remote
sensing of potable water supplies relevant to civilian and military communities. This research was
performed in collaboaration with the United States Army Corps of Engineers.
NHSRC has made this publication available to assist water system utilities and operators by
providing test data relating for a novel dection technology. This information is intended to move
EPA one step closer to achieving its homeland security goals and its overall mission of protecting
human health and the environment while providing sustainable solutions to our environmental
problems.
Jonathan Herrmann, Director
National Homeland Security Research Center
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Page Left Blank Intentionally
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Contents
Notice ii
Foreword iii
Contents v
List of Figures vi
List of Tables vii
Acronyms and Abbreviations viii
Objective 1
Instrument Overview 1
Instrument Operation 2
Test Protocol 3
Data Collecting and Processing 5
Evalaution Summary and Conclusions 5
Appendix A Sporian Data Plots 7
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List of Figures
Figure 1 IBS side view with cartridge chamber door and latch open 1
Figure 2 Schematic rendering of the MDE 2
Figure 3 Low flow insert 3
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List of Tables
Table 1 Event Summary 4
Table 2 Sporian response data for Table 1 injections 4
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Acronyms and Abbreviations
CPU colony forming unit
CSV comma separated variable
EPA U.S. Environmental Protection Agency
IB S Inline biosensor system
GPH Gallons per Hour
L liter
MDE Molecular detection element
mg milligram
min minute
mL milliliter
mm millimeter
NHSRC National Homeland Security Research Center
PC Personal Computer
S/N Serial Number
Sig/etime Flurosence signal over elapsed time
T&E Test and Evaluation
USAGE United States Army Coprs of engineers
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Objective
To evaluate Inline Biosensor System's (IBS) capability to detect biological contamination
(e.g., B. subtilis, and E. coif) of drinking water in real time and ease of deployment in the
field as a contamination warning device. IBS was designed and manufactured by Sporian
Microsystems, Inc. (Sporian) for the US Army Corps of Engineers (USAGE).
Instrument Overview
The IBS is a flow-through device that is equipped with a proprietary sensor cartridge
designed to detect biological contamination. The IBS sensor cartridge contains a proprietary
molecular detection element (MDE) which is also a fluorescing (emits light) media. When
biological contamination is present, the fluorescing media is released from the MDE and
bound to the contaminant. The rate of change of MDE media is roughly proportional to target
concentration within the environment (how much target interacts with the MDE), and as
such, high target concentrations may result in a very fast response (in the order of minutes)
and depletion of the cartridge. A positive signal response is indicated by a decrease in
fluorescing signal over elapsed time (in microseconds) as the MDE is exposed to the target
biological contaminant within the environment. Due to processing variations, the initial value
will differ from cartridge to cartridge. Over a period of time, the MDE becomes completely
exhausted and needs to be replaced. Figure 1 shows the side view of the Sporian IBS device.
Figure 1. IBS side view with cartridge chamber door and latch open
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Instrument Operation
As shown in Figure 1, the IBS includes a flow valve slide that routes a portion of the total
flow through the inlet and outlet ports through an installed disposable MDE sensor cartridge.
This valve does not affect the main flow from the inlet to the outlet port, but only routes a
portion of the flow through the MDE cartridge. Moving the valve slide to the "Close"
position blocks the flow to and from the cartridge ports. Moving the slide to the "Flow"
position allows water to flow through the cartridge. This allows the user to replace a cartridge
without stopping the main system flow.
The cartridge attached to the IBS includes a glass window coated with the appropriate MDE,
such that when inserted into the IBS, the MDE is positioned in front of the detection optics.
When inserted, the cartridge also forms a channel for flow to the sensing area. Each cartridge
is a one-time use device, meaning that once the positive detection has occurred, the cartridge
has been irreversibly consumed, and needs to be replaced with a fresh cartridge. Figure 2
shows a schematic rendering of the MDE cartridge.
Cartridge Front Side
Cartridge Back Side
MDE Position
Front Optical Window
Flow Channels
Back Window
Flow Inlet/Outlet
Groove
Figure 2. Schematic rendering of the MDE Sensor Cartridge
The IBS was designed for a nominal flow rate of 1,500 Gallons per Hour (GPH) for USAGE,
but will function in the as-shipped configuration at flow rates down to approximately 480
GPH. For flow rates below 480 GPH, inserts are provided (Figure 3) to ensure suitable flow
through the MDE cartridge.
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Figure 3. Low Flow Insert
The low flow inserts were used during the tests conducted at the EPA Test & Evaluation
(T&E) Facility.
Test Protocol
The EPA/Shaw Environmental protocol for contaminant injection for real-time monitoring
typically involves running the instrument for a certain period of time (at least 1-2 hours) to
collect baseline data (representing normal field conditions) and then conducting the injection
event followed by a post injection period to evaluate if the instrument recovers (i.e., the data
are back to the normal baseline levels). This approach is desired by EPA for all online
continuous monitoring equipment from a long-term equipment deployment perspective and it
allows for automated contamination "event detection" using algorithms that are designed to
detect statistically significant changes in water quality from the baseline or "normal"
conditions.
Between August 15, 2011, and September 1, 2011, a total of eight injection events (excluding
overnight and reagent blank runs) were performed for the instrument evaluation. During each
test run, a designated amount of contaminant was mixed in 10-L dechlorinated tap water and
injected into the single-pass pipe loop using a flow-controlled injection pump. Each test run
consisted of the biological contaminant with the growth media and the dechlorinating agent
(sodium thiosulfate) except for the blank runs which contained only the dechlorinating agent.
The water flow rate of the single-pass pipe system was controlled at 23 gallons per minute
(gpm) through the whole experiment. This flow rate is equivalent to one foot per second in
the single- pass pipe. The 10-L contaminant solution was continuously injected into the pipe
for 20 minutes (each event) at the injection port. The travel time from the injection port to the
Sporian IBS is estimated to be roughly 4 minutes. Table 1 is the summary of events
performed for the study. During the biological contaminant injection events, the average
chlorine concentration varied between 1.1 and 1.2 mg/L. Varying amounts of sodium
thiosulfate were injected to neutralize the chlorine: -13 grams for Tests 1, 2, and 3, and 19
grams for the remainder of the test runs. Volumetrically, it is estimated that 4.8 mg/L and
6.95 mg/L of unreacted thiosulfate was injected into the system. Two Sporian units (Serial
Numbers 005 and 007) were tested side by side during this testing.
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Table 1. Event Summary
Date
8/15/2011
8/15/2011
8/15/2011
8/18/2011
8/19/2011
8/19/2011
8/24/201 1
8/25/201 1
9/1/2011
Test
No.
1
2
3
4
Reagent
Blank
5
6
7
8
Start
Time
11:55
14:40
15:40
16:15
10:00
11:01
15:45
14:00
11:00
Stop
Time
12:15
15:00
16:00
16:35
10:20
11:21
16:05
14:20
11:20
Injected Stock
Contaminant
Concentration
Flask #6 E. coli 4.2 x
109CFU/ml
Flask #6 E. coli 4.2 x
109CFU/ml
Flask #6 E. coli 4.2 x
109CFU/ml
Flask #8 E. coli 1.0x
1011 diluted to 1.0x109
Sodium thiosulfate
blank
Flask #8 E. coli 1.0x
1011 diluted to 1.0x109
Flask #8 E. coli 1.0x
1011 undiluted
Bacillus subtilis spores
1.0 x109
Bacillus subtilis spores
1.0 x109
Injected Diluted
Concentration
5 ml = 12,061
CFU/ml
5ml = 12,061
CFU/ml
0.5ml = 1,206
CFU/ml
20 ml = 11, 487
CFU/ml
20 ml = 11, 487
CFU/ml
2ml = 114,881
CFU/ml
2ml = 1,149
CFU/ml
6 ml = 3,446
CFU/ml
Grab
Sample
Sporian Port
119 CFU/ml
110 CFU/ml
12 CFU/ml
190 CFU/ml
194 CFU/ml
1 1 ,000
CFU/ml
2,500
CFU/ml
6,500
CFU/ml
Table 2 Sporian response data for Table 1 injections
Test No.
1
2
3
4
Reagent
Blank
5
6
7
8
Grab Sample
Sporian Port
119 CFU/ml
110 CFU/ml
12 CFU/ml
190 CFU/ml
194 CFU/ml
11,000
CFU/ml
2,500 CFU/ml
6,500 CFU/ml
Unit 005
Signal/Etime
Change
Observed
Yes
Yes
Yes
No
Yes
Yes
No
No
Yes
Unit 005
Estimated Peak
Concentration
100, 000 CFU/ml
100,000 CFU/ml
100,000 CFU/ml
100, 000 CFU/ml
100, 000 CFU/ml
80,000 CFU/ml
30,000 CFU/ml
100,000 CFU/ml
100,000 CFU/ml
Unit 007
Signal/Etime
Change
Observed
No
Yes
Yes
No
No
No
Yes
Yes
Yes
Unit 007
Estimated Peak
Concentration
4000 CFU/ml
100,000 CFU/ml
100,000 CFU/ml
100, 000 CFU/ml
80,000 CFU/ml
0 CFU/ml
80,000 CFU/ml
100,000 CFU/ml
100,000 CFU/ml
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Data Collection and Processing
The IBS instrument is connected to PC/Netbook and utilizes a program named IBIPC that
captures the data and logs the data to files on the PC in a "data" subdirectory. The location of
this directory, by default, is the installation directory of the IBIPC program. Within the data
subdirectory, a file is created for each day of logs. The current day is appended to the file
named "data", and prior days' logs are in files which contain the date, such as "data.2011-08-
15." The log files need to be processed further manually using Sporian- provided software to
extract data into comma separated variable (.CSV) format that can be processed using a
spreadsheet program such as Microsoft Excel.
Subsequent to the completion of the testing, Sporian has also provided a serial cable interface
for enabling direct logging to the NexSens data logger available at the T&E Facility.
Evaluation Summary and Conclusions
The data collected using IBS units 005 and 007 were submitted to Sporian and USAGE for
further evaluation. The key observations from the EPA and Shaw Environmental testing are
summarized below:
1) The IBS unit appears to be capable of detecting both dead and live biological
contaminants since the reported concentrations by the unit were higher than the live
cells determined from plate and culture analysis of the grab sample port. The
estimated concentrations by the IBS units were consistently much higher than the
injected cell densities.
2) The Sporian IBS device is not currently suitable for long-term monitoring as the
cartridge is consumed quickly in continuous service. The overnight tests indicated
that the signal decreases relatively quickly (in about 30 minutes), showing a strong
evidence that chlorine removes the fluorophores from the MDE surface relatively
quickly. The "useful life" of the cartridges should be clearly understood by the end
users in relation to the target concentration of the contaminant.
3) There were numerous test runs where there was no observable deflection in the signal
graph, yet relatively high concentrations of contaminant were reported. This could be
a scaling issue with the graphs, but the correlation between the signal and the reported
concentrations should be better understood to prevent false positives.
4) The units reported significant estimated concentrations of bacteria in response to the
blank test runs indicating that the sodium thiosulfate may be reacting with the
material on the cartridge.
5) In general, the observed signal response to E. coli was stronger than for B. subtilis.
6) The unit is susceptible to condensation and bubbling. EPA/Shaw tested the units at a
much lower flow rate than the design flow rate. Future improvements to the flow cell
may be required for low flow applications. All manufacturer recommendations
should be strictly followed.
7) Inter unit reproducibility was low for the EPA/Shaw testing.
8) All of the EPA approved enzyme based E. coli detection methods require relatively
lengthy incubation periods. This MDE technology if perfected could provide a useful
"real time" rapid screening method for E. coli prior to enzyme based detection.
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Page Left Blank Intentionally
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Appendix A
Sporian Data Plots
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S .2 <£>
> 5 <£
CM
*
01
t—
H
M
j- Overnight t£
rfl u
c|;
03
0
i
T W|Th| F
SB oi
CO CO CO
Figure 1, Timeline of various test events.
Pittr ID tre^ Injectkr on a'isail 1
0.1K-
ai-
BOB-
a ace-
O.D75-
0.07-
Q.D65-
-J--
1
--J-
4
J
3ft5/11 SIKH &1S11 B1S11 I'lSfll B1S11 &1S11 S15I11 B/15'11
1tt35 1C:45 1(155 11:05 11:15 1125 11:36 11:45 11:55
Figure 2. S/N 005 Sig/etime vs. time for period
between set up and first test.
Tes: 1. H«an KIS'JBII a 1 1:55 EOT
atos-
ai-
Q.ffiS-
« a.ce-
a.D7-
O.DK-
r
"t"
H
' 'fv::^ ~
atsm 11:55 8/15/11 lies aisntzis E
15)11 122E S15/11 1235
Figure 4. S,'"N 005 Sig/etime vs. time for injection
#1-E. coh.
Figure 3. S/N 007 Sig/etmie vs. time for period
between set up and fust test.
Tea 1. H«3X3S 915(2011 « 11:55 EOT
6
arm
_i
* .
|
A 0
A A
11:55 8115/111205 S/IS'11 12:15 aiS11 1225 aiS'11 12:35
Figure 5. S/N 005 Estimated concentration vs. time
for injection £ 1 - E. coli.
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1
9
""
Test i. n«*aiaiis'20ii ® itss EOT
11:55 S'l&'n 12:05 3^1&11 1^15 ^'15V11 12:25 S'151
12:35
Test1.r|ectBna'1
Es 5COOD • - -
i:::iiH::::::i::::::
B1S11 11:55 B1S'1112(15 B> 1911 1215
Figiu'e 6. S/N 007 Sig/etmie vs. time for injection Figiire 7. S/N 007 Estimated concentration vs. time
#1 - E. coli. for injection #1 - E. coli.
Test 2. hjeakjn ^t&2D11 (g M:40 EOT
0.1 -
0.095 -
o 0.09 •
E
S 0-OB5 •
» 0.08.
0.075-
0.07-
0.065'
1.06
arisyi
•4:40 &M5m 14:50 S/1&11 15:00 ^15/11 15:10 &15/11 15:20
Tes- 2, Injection 0> 1 SC0 1 1 @ M:40 EOT
!
u
1
(J
1?
1
;
i
• \
y
ffl1&f11 14:40 8/1&11 14:50 8/16/11 1&00 #15m 15:10 an&11 15:20
Fisiu'e 8. S./N 005 Sig/etime vs. time for injection Figure 9. S/N 005 Estimated concentration vs. time
#2 - E. coli. for injection #2 - E. coli.
Test 2, Ejection &1 15/20 11 @ S4:4D' cUT
0105
0.1 •
CL095 -
o 0.09 :
£ 0.065 -
•
® 0.08 •
CL075-
0.07-
0-065-
XL,
ftf15ni 14:40 fln&H 14:50 a/15/n 15:00 & 15/11 15:10 &15/11 15:20
Figure 10. S/N 007 Sig/etime vs. time for injection
#2 - E. coli.
Tesl 2. InjeMioi S11 SCO 11 ® 1+40 EOT
S'15/11 14:40 8V1S/11 14:50 8/15/11 15:00
1&11 15:10 a'15/11 15:20
Figure 11. S/N 007 Estimated concentration vs.
time for injection #2 - E. coli.
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Test 3. Injection &'1S'2011 @ 15:40 EOT
8/15/11 15:40 B/15/11 15:50 6/15/11 16:00 3/15/11 18:10 8/1511 16:20
Figure 12. S/N 005 Sis/etime vs. time for injection
#3-E.cdi.
Test3. hjectksi S 15/2011 @ 15:40 EFJT
0.105
* 0.00
E
tv Q.OB5 •
0.08 •
0.075 •
0.07-
0.065-
8/1&11 t&40 anSII 15:50 8/1&11 16:00 3/1&11 16:10 Q/1S11 18:20
Figiu'e 14. S/N 007 Sig etime vs. time for injection
#3-E.coli.
Test 3. Section fflWO11 @ 15:40 EOT
30000
40000
D
1 15:40 8/15/11 15:50 S'15'11 16:00 8/15/11 18:10 8/1&'t1 16:20
Figure 13. S/N 005 Estimated concentration vs.
time for injection #3 - E. coli.
Test 3. Iqection &15/2011 @ 15:40 EOT
100000
1
Concentration, CFC
fl/l&ll 15:40
a/1&1t 15:50 ffl15/11 16:00 8/15/11 10:10 a/1&11 16:20
Figure 15. S/N 007 Estimated concentration vs.
time for injection #3 - E. coli.
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Test 4, Injection 8/18/2011 @ 18:15 EOT
0.105
0.1-
0.085-
£ 0.085-
51 Q.OB-
0-07-
0.065
a
-------
Test 5. hjecticn B/1ftf2D1 1 @ IftQO EDT
0.105-
0.1 -
1 : v •
5 0.085-
S 0.03 -
0-075 •
0.07-
^**x^^_
10:00 8/19/1110:10 8/19/1110:20 8/19/1110:30
Figure 20. S/N 005 Sig etime vs. time for injection
#5 - sodium thiosulfate.
Test 5. Irvecticn 8n»2D1 1 @ 10:00 EOT
0.1QS-
0.1 -
ROB-
« o.oe-
?
& 0 » -
aore -
0.07-
8/18/11 10:00 e/lft'11 10:10 B'ia'11 10:20 ffl1»11 10:30
Figiu'e 22. S/N 007 Sig/etune vs. time for injection
#5 - sodium thiosulfate.
Test 5. Injection 8/192011 @ 10:00 EOT
NhDO S'lfl/ll 10:10 8/1B,'11 10:20 &iai1 10:30
Figui'e 21. S/N 005 Estimated concentration vs.
Time for injection #5 - sodium tliiosulfate.
Test 5. tijecton 8/1&2011 @ 10:00 EDT
1 60000-
3
2DDQQ-
_*
|
j
. I
,
I
10: DO B/lfli'11 10:10 6/1BM1 "D:2D ft'lSV! 10:30
Figure 23. S/N 007 Estimated concentration vs.
time for injection #5 - sodium tliiosulfate.
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Twte. hj«aior&'1i'2II-l 3 ":01 EOT
91S;'111:C1 8M911 11:11 a'19'1 11:2' ap1911 11:31
Figure 24. S/N 005 Sig etime vs. time for injection
#6 - E. coli.
Test8, Injection &'tft'2D11 @11:01 EOT
I
jj 0.18 - -
1 11:11 &19/11 11:21 8/iaT1 11:31 fflia/11 11:41
Figure 26. S/N 007 Sig/etime vs. time for injection
#6-E. coli.
man 11:01 man 11:11 &piaii< 11:21 a/iapn ;i:3i ana'mt:4i
Figiu'e 25. S/N 005 Estimated concentration vs.
time for injection #6 - E. coli.
TestS, hjecton a/1&'2Q11 @ 11:Dt EUT
3 80000-
1 eoooo-
1 40000 -
0
20000-
s/ian
11:01 &'19-'11 11:11 &'16/11 11:21 an»11 11:31 Bi'1&'1
11:41
Figure 27. S/N 007 Estimated concentration vs.
time for injection #6 - E. coli.
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Test 7. hjectkxi ^2*2011 @ 15:45 ECfT
S2V11 15:45 &24.'11;5:55
1 !8:05 S'24,' 1 1 1 6: 15 S'24j111 18:25
Fiaure 28. S/N 005 Sig/etime vs. time for injection
#7-E. coli.
Test 7, Ejection B'3*2011 6 '5:45 EOT
&24/11 15:45 &24/11i5:55 i24.-11'e05 S24/11 16:15 ffl24/11 16:25
Figure 30. S/N 007 Sig/etime vs. time for injection
#7 - E. coli.
Test 7, k^ec?ian 0/24/20 1 1 @ 15:45 EFJT
s I
20COO - '
o4>
3124/1115:45 3,'24/11 15:55 B/24'Il 16:05 B<24/11 16:15 8/2*1116:25
Figure 29. S/N 005 Estimated concentration vs.
time for injection #7 - E. coli.
Tes! 7. Injection 8(24)2011 @ 15:45 EOT
-:occc
'30COO
E
3 3CCOO
8 4DOOO
o
i'24111c45 8.'24:11 15:55 ^24/1118:05 B/2*11 16:15 a'24.'1 - '6:25
Figure 31. S/N 007 Estimated concentration vs.
time for injection #7 - E. coli.
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Test B, Injection a'2&2011 @ 14:00 EOT
&2&11 14:00 8/25/11 14:10 8/2&11 '4:20 a25'11 14:30 a'25.'tt 14:40
Figm'e 32. S/N 005 Sig/etime vs. time for injection
#8 - B. subtilis.
i
i
W2&1
Test fl. Injector a'2&2Q1 1 @ 14:00 ECT
\ ,
14:00 S2S11 14:10 B/2&'11 14:20 &25'11 14:30 8/25(1
14:4D
Figure 34. S/N 007 Sig etime vs. time for injection
#8 -B. subtilis.
Tes! 8, Injection S/2S2Q11 @ 14:3C EOT
SI'S/11 14:00 &2S11 14:1D &25/11 14:20 BESfll 14:30
Figure 33. S/N 005 Estimated concentration vs.
time for injection #8 - B. subtilis.
Tas: 8. Injeclicn »2&'201 1 fl «.4:OD EOT
100000-
I aCKB-
•
u
| ecoffi-
|
1 40000-
G
20000-
at26/11 1*00 3/2S11 14:10 8/26/1114:20 8/25'H 14:30 8(25/1
14:40
Figure 35. S/N 007 Estimated concentration vs.
time for injection #8 - B. subtilis.
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Test 9. Iriecsicn 8/01/2011 8 appro* 09:25 EOT
PI/11929 E.'1/r S:3f 8/1/116:45 S"/I18:§5
Figure 36. S/N 005 Sig/etime vs. time for injection
#9 - B. subtilis.
Test 6, Injection 9/Qr.'2D1 1 @ approx QQ:25 EDT
|
D.11
/^
|
V
9/1/116:25 9/1/119:35 6.M/11 9:45 6/1/119:55 S/1'11 IttCB
Figm'e 38. S/N 007 Sig/etime vs. time for injection
#9-B. subtilis.
Test a. Injection 9/01/2011 @ approx D9:25 EOT
0/1/11 a25 at/Haas
Figure 37. S/N 005 Estimated concentration vs.
time for injection #9 - B. subtilis.
Test 8. IrtectionanVSDtl @ appnox DS:25 EDT
100030-
I
3 8CODO -
E
L>
| 6C030-
I 4CCO1-
o
o
2DOOO-
8/1/1
1
^
*"t
I--
I
Jv :
9:25 9/1/119:35 9/1/116:45 9/1/116:55 9/1/11
IttOS
Figiu'e 39. S/N 007 Estimated concentration vs.
time for injection #9 - B. subtilis.
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Between test 3 and A
8/17/11 14:00 S>17/11 18:48 8,'17/11 23:36 8/18/114:24 8(18(1'. B:12 8/18/1114:00
Between test 3 and 4
8/17/11 8/17(11 8/17/11 8(17/11 8(17/11 8/17/11 8/17(11 8(17/11 8/17(11
14:00 14:07 14:14 14:21 14:28 14:36 14:43 14:50 14:57
Figure 40. S/N 007 Sig. etiine vs. time for Figure 41. S/N 007 Sig/etime vs. time for
"Ovemiglit Matrix Background" test night of 8/17 "Oveniiglit Matrix Backgi'ound" test night of 8/17
to 8/18/11. to 8/18/11 focusing on signal change in first approx.
30 minutes.
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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