BioVir Laboratories, Inc.

Transcription

Page 1 of 17
685 Stone Road, Unit 6 ! Benicia, CA 94510 ! (707) 747-5906 !1-800-GIARDIA ! FAX (707) 747-1751 ! WEB: www.biovir.com
TEST REPORT
Project Title
CamelBak All Clear Microbiological UV Water Purifier
Project No.
110957
Sponsor:
CamelBak Products, LLC.
2000 S. Mc Dowell Blvd. Suite 200
Petaluma, CA 94594
C/O:
Chris Blain, Jeremy Galten, Jon Wambold
Date:
August 9, 2011
From:
R.C. Cooper, V.P.
Introduction:
BioVir Laboratories Inc. was contracted by CamelBak Products, LLC. to perform a
series of microbiological purifier tests on their All Clear Microbiological UV Water Purifier which
employs ultraviolet light as the sanitizing agent. The protocol was designed in the spirit of the
EPA "Guide Standard" (EPA Guide Standard and Protocol For Testing Microbiological Water
Purifiers (1987).) The guidelines for ultraviolet light units was the model for this evaluation. The
All Clear UV Water Purifier bottle is designed to treat “clear” water. When turbid or colored
water is to be treated the user is instructed to filter the water prior to the UV treatment using a
filter recommended by CamelBak. A detailed protocol for this evaluation is contained in
Attachment A.
In summary, the protocol calls for testing three 750 mL All Clear Bottles fitted with a
screw cap which incorporates a battery (rechargeable) operated germicidal UV light source
(see Attachment B.) Each UV lamp had been cycled (on and off) approximately 15,000 times
by Camelbak prior to receipt at BioVir (According to CamelBak 15,000 cycles exceeds the
alarm point of 10,000 cycles at which point the production unit will stop functioning.) The units
were challenged with test water containing known concentrations of the Bacterium Raoultella
terrigena, the two enteric viruses: poliovirus and rotavirus and the protozoan parasite
Cryptosporidium parvum. Each test unit was challenged a total of 12 times half of which were
performed with the microorganisms suspended in EPA Guideline Test Water GTW1 and half
in EPA Test Water GTW 4. This test schedule is illustrated in Table 1.
Page 2 of 17
Table 1. Schedule of Test Challenges - CamelBak All Clear UV Bottle
Week
Test
Water
Challenge
Microbe(s)
Unit #1
Unit #2
Unit #3
One
GTW1
Bacteria & Virus
3
3
3
Cryptosporidium
3
3
3
Bacteria & Virus
3
3
3
Cryptosporidium
3
3
3
Two
GTW4
The bacteria and virus challenge were run separately from the Cryptosporidium challenge as
the presence of bacteria and viruses interfere with the cell culture assay for viable
Cryptosporidium oocysts.
Test Water:
The water quality parameters for the two test waters used in the microbial challenges are
shown in Table 2.
Table 2. Challenge Water1 Quality Parameters
Challenge in Test Water GTW1
Challenge in Test Water GTW4
Component
Bacteria &
Virus
Cryptosporidium
Bacteria &
Virus
Cryptosporidium
Chlorine (mg/L)
Non-detect
Non-detect
Non-detect
Non-detect
pH
7.2
7.4
8.2
8.3
TOC (mg/L)3
0.5
2.0
13.9
13.8
NTU2
0.1
0.1
39
33
Temp oC
24
20
5
5
TDS (mg/L)
203
177
1420
1370
1
Initial Source: De-chlorinated City of Benicia Tap Water. 2Adjusted prior to Humic Acid (TOC)
addition, 3 TOC source Sigma-Aldrich Humic Acid Lot # 21520BB
Microorganisms:
The bacterium R. terrigena (ATCC 33257) culture was prepared, as per the EPA Guide
Standard, by overnight growth in Tryptic Soy Broth (TSB.); harvested by centrifugation at 2,600
X g, re-suspended in phosphate buffer, re-centrifuged, re-suspended in buffer and filtered
through a sterile 5µm Acrodisc® filter. The assays were performed by membrane filtration and
growing colonies on mFC agar incubated at 35oC. The bacteria were added to the challenge
test water to reach a concentration in the range of 1x107colony forming units (Cfu) per 100 mL.
The polio and rota viruses were harvested from infected BVBGMK cell cultures. The
assays were performed by plaque assay using BGMK cells. The viruses were added to the test
water to result in a concentration range of 1x107 Plaque forming units (Pfu) per L.
The Cryptosporidium parvum oocysts were obtained from Bunch Grass Farms and
arrived at an oocyst count of 1x107 oocysts per mL. Direct microscopic examination and
Page 3 of 17
enumeration was performed as described in the EPA 1622/23 method. Cryptosporidium
infectivity assays were conducted following the method of Slifko et al, 1999 employing growth in
cell culture as evidence by the presence of fluorescence foci using epifluorescence microscopy.
Exposure Procedure:
The test water was prepared in 10L volumes and adjusted as necessary to meet the
water quality parameters set forth in the Guideline (see Table 1.) The 10L were inoculated with
the appropriate amount of challenge microorganisms and the entire volume continuously mixed
using a magnetic stirrer. The exposure procedure followed the CamelBak instructions.
Test Water GTW1 Challenge:
The All Clear Bottle was filled to the 750 mL mark with seeded water and closed with the
UV cap and the UV light activated. During the exposure period the contents were mixed by
alternating the system from the upright to the inverted position in a 3ft arc with an inversion
frequency of about one second. The exposure period of one minute is programed into the UV
cap. At the end of the exposure period the contents of the bottle were poured into a sterile one
liter bottle and refrigerated prior to assay.
Test Water GTW4 Challenge:
In this challenge the test water was filtered through a hand pumped MSR® Miniworks®
EX Filter into a one liter sample bottle and then filtered a second time, using the same filter into
the All Clear Bottle. When the bottle was filled it was closed with the UV cap and exposed to
the UV light for two successive one minute periods. The mixing regimen was the same as that
described for the Test Water GTW1 exposure. At the end of the second exposure period the
contents of the bottle were transferred to a sample bottle and refrigerated prior to assay.
Results:
Detailed results of the microbiological challenges are shown in Tables 3 through 6. A
summary of these data is shown in Table 7.
Table 3.CamelBak All Clear Purifier Results - R. terrigena Challenge
Test Water GTW1
Sample #
1
Cfu/100 mL
Log reduction
Test Water GTW4
Cfu/100 mL
Log reduction
Influent
1.1 x 107
0.0
4.2 x 107
0.0
1A1
4.3 X 100
6.4
<1
>7.6
1B
0
6.7 X 10
6.2
<1
>7.6
1C
1.6 X 101
5.8
<1
>7.6
2A
6.0 X 100
6.3
<1
>7.6
2B
7.7 X 100
6.2
<1
>7.6
2C
1
3.8 X 10
5.5
<1
>7.6
3A
3.0 X 101
5.6
<1
>7.6
3B
6.0 X 100
6.3
<1
>7.6
3C
1.1 X 101
6.0
<1
>7.6
The numerals = test unit # and the letters = challenge iteration.
Page 4 of 17
The reduction in the concentration of Challenge viruses are shown in Tables 4.
Table 4. CamelBak All Clear Purifier Results - Polio and Rota Virus Challenge
Test Water GTW1
Sample #
1
Test Water GTW4
Pfu/ mL
Log reduction
Pfu/mL
Log reduction
Influent
1.2 x 105
0.0
2.8 x 104
0.0
1A1
<1
>5.1
<1
>4.5
1B
<1
>5.1
<1
>4.5
1C
<1
>5.1
<1
>4.5
2A
<1
>5.1
<1
>4.5
2B
<1
>5.1
<1
>4.5
0
2C
<1
>5.1
4.2 x 10
3.8
3A
<1
>5.1
<1
>4.5
3B
<1
>5.1
<1
>4.5
3C
<1
>5.1
3.1 x 100
4.0
The numerals = test unit # and the letters = challenge iteration.
The reduction in the viability and/or concentration of Challenge Cryptosporidium are
shown in Tables 5 and 6. The initial challenge performed in GTW1 water gave anomalus
fluorescent foci results. Because of this the challenge was repeated and the results shown in Table 5
are those of the repeat challenge. The water quality parameters of the GTW1 used in the repeat
challenge are shown in Table 2.
Table 5. Results of Cryptosporidium Viability Assay In Test W ater GTW 1
Sample
Concentration of Oocyst Distributed in Four Wells per Concentraton
Number Wells containing Florescent Foci
105
104
103
102
Influent
4
4
4
4
1A
0
0
0
0
1B
0
0
0
0
1C
0
0
0
0
2A*
0
0
0
0
2B
0
0
0
0
2C
0
0
0
0
3A
0
0
0
0
3B
0
0
0
0
3C
0
0
0
0
5
*The number of oocysts in this influent sample were reported to be 1.3 X10 and did not
allow for the distribution of 105 oocysts across 4 wells.
The influent results indicate that when 105 oocysts are applied to the wells all 4 were
Page 5 of 17
positive and when the same number are applied in the treated samples none were positive.
The inference is that a 5 Log or greater reduction in viable oocysts occurred. The one exception
as noted in Table 5 was in Sample 2A in which the inference is a reduction of 4 logs or greater.
In the GTW4 Cryptosporidium challenge no oocysts were seen in the concentrated volume of
the 750 mL treated samples due to the pre-filtration steps. The influent samples were at the
expected level. In this instance the resuts are stated as actual oocyst microscopic direct counts.
Table 6 CamelBak All Clear Purifier Results -Cryptosporidium parvum Challenge In
GTW 4
1
Sample #
Oocysts / 750 mL
Log reduction
Influent
3.3 x 106
0.0
1A1
<1
>6.5
1B
<1
>6.5
1C
<1
>6.5
2A
<1
>6.5
2B
<1
>6.5
2C
<1
>6.5
3A
<1
>6.5
3B
<1
>6.5
3C
<1
>6.5
The numerals = test unit # and the letters = challenge iteration
As shown in Table 7 all the values are above the Guideline minimum log reduction values
of 5 for bacteria, 3 for viruses and 2.5 for cryptosporidium oocysts.
Table 7. Geom etric Average Reduction Of Challenge Microorganism s in the
Two Test W aters And In Total
Test W ater
R. terrigena
Polio and Rota virus
Cryptosporidium
GTW 1
6
>5
$5
GTW 2
>8
$4
>7
GTW 1 & 2
$7
$4
$6
In averaging the log reduction, greater than (>) values are treated as the
whole num ber. The log reduction values are rounded to one significant
figure.
F:\WP\COOPER\BV\Camelbak\Purifier\Final Report.wpd
Page 6 of 17
ATTACHMENT A
CamelBak All Clear UV Water Purifier Protocol
Page 7 of 17
Project No. 110957
CONFIDENTIAL INFORMATION
DOCUMENT NO.:
Page 1 of
Final
TITLE: CamelBak All Clear UV Water Purifier Purifier Test Protocol
DATE: June 27, 2011
PREPARED FOR:
PREPARED BY:
CamelBak Products, LLC.
In cooperation with CamelBak Products LLC
7
Page 8 of 17
Project No. 110957
1.
Page 2 of
7
Introduction: The goal of this protocol is to define the conditions under which
the CamelBak “All Clear UV Water Purifier bottle will be evaluated to ascertain if
it meets water purifier criteria. The protocol will follow the spirit of the EPA
"Guide Standard" (EPA Guide Standard and Protocol For Testing Microbiological
Water Purifiers (1987). The guide lines for ultra violet light units will be the
model for this evaluation. The UV Water Purifier bottle is designed to treat
“clear” water. When turbid or colored water is to be treated the user is instructed
to filter the water prior to the UV treatment using a filter supplied with the unit by
CamelBak.
2.
Biologics/Media/Reagents
a.
Challenge Microorganisms:
i.
Bacterium: Raoultella terrigena (ATCC 33257)
ii.
Animal Virus
iii.
b.
c.
(1)
Poliovirus type 1 (ATCC VR -59)
(2)
Rotavirus SA11 (ATCC VR-899)
Cryptosporidium parvum oocysts
Media
i.
TSB - Tryptic soy broth (Difco)
ii.
mFC agar (Difco)
iii.
MEM/L-15 medium (Hyclone)
iv.
BV-BGMK (BioVir)
Reagents
i.
Sodium Hydroxide 5N
ii.
Hydrochloric Acid 5N
iii.
Phosphate buffed saline (PBS), pH 7.4
iv.
Ten percent sterile solution of sodium thiosulfate
v.
De-chlorinated Benicia City tap water
vi.
Fine Test Dust, Nominal 0-5µm (Powder Technologies, Inc.
Brunsville, MN.)
BIO VIR LABO RATO RIES, IN C .
F:\WP\COOPER\BV\Camelbak\Purifier\Purifier Protoccol.6..15.11.wpd
Page 9 of 17
Project No. 110957
3.
4.
vii.
Sea Salts (Sigma Chemical)
viii.
Humic Acid (Aldrich Chemical)
Page 3 of
7
Equipment
a.
CamelBak All Clear UV Water Purifier bottle units
b.
Sterile screw cap bottles
c.
Sterile disposable petri dishes
d.
Dilution blanks containing PBS or TSB
e.
pH meter
f.
Turbidity meter
g.
HACH total chlorine test kit
h.
Conductivity meter
i.
UV / Visible spectrophotometer
j.
Vortex mixer
k.
Magnetic stirrers
l.
Carboys
m.
Water Bath
n.
Sterile pipettes
o.
Stop watch
p.
35 °C Incubators
q.
Steam autoclave
r.
Inoculating loops and needles
Procedure
a.
Challenge Seed
i.
Raoultella terrigena will be grown and maintained as per EPA
Guide Standard Protocol. The challenge seed will contain .1X107
Colony forming units (Cfu) per 100 mL.
ii.
Polio and Rotavirus will be produced and maintained using the BVBGMK cell line. The challenge seed will be a mixture of equal parts
of the two viruses at a concentration of .1X107 plaque forming
units (Pfu) units per Liter.
Page 10 of 17
Project No. 110957
iii.
Page 4 of
7
Purified (gradient centrifugation) viable Cryptosporidium parvum
oocysts are obtained either from Bunch Grass Farms, Deary, Idaho
or from the University of Arizona. Before they are used their
viability status is confirmed by the excystation method according to
NSF/ANSI Std 53. The challenge seed will contain .1 x 106 oocysts
per Liter.
b.
Bioassays
i.
Raoultella terrigena concentration in the test water will be
determined by the membrane filtration method using mFC agar
incubated at 35oC.
ii.
Virus bioassays will be done by the plaque method using BGMK
cells. The tissue culture medium will be MEM/15-L Medium.
iii.
Enumeration of viable Cryptosporidium oocysts is conducted using
the tissue culture method of Slifko et al. (2000.)
iv.
Assay dilution series will be in a range required to determine if the
required log reduction has been achieved.
c.
d.
Test Units:
i.
Three All Clear UV Water Purifier bottle treatment units
ii.
Operated as per CamelBak instructions.
Test Waters
i.
The test water will be similar to that described in the EPA Purifier
Guidelines.
ii.
These waters have the following qualities:
(1)
The source water will be City of Benicia de-chlorinated tap
water. The water will be de-chlorinated by carbon filtration
followed, if necessary, by the addition of the minimum
amount of sterile sodium thiosulfate to insure total chlorine
removal as shown by chlorine residual test (HACH test kit).
The water will be adjusted as necessary to meet the water
quality requirements shown in Table 1.
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Project No. 110957
(2)
Page 5 of
7
The EPA Guide lines call for two types of test water, as
described in Table 1 below:
Table 1. Test Water Quality
Water Type
Residual
pH
TOC mg/L
NTU
TDS mg/L
Temp oC
Chlorine
mg/L
#1
0
6.5 - 8.5
0.1 - 5.0
0.1 - 5.0
50 - 500
20±5
#4
0
6.5 - 8.5
$10
$30
1350 -1650
4±1
(3)
TOC will be adjusted by the addition of humic acid (Aldrich)
(4)
NTU will be adjusted using specified AC fine test dust
(Nominal 0-80 µm from Powder Technologies, Inc.
Brunsville, MN). TDS will be adjusted using sea salts
(Sigma.)
(5)
e.
Water temperature (test water #4 )will be reduced to 4±1oC.
Test schedule
i.
The test schedule is shown in Table 2.
Table 2. Number of Test Challenges To Be Conducted - CamelBak UV Bottle
Week
One
Two
ii.
Test
Challenge
Water
Microbe(s)
GTW1
GTW4
Unit #1
Unit #2
Unit #3
Bacteria & Virus
3
3
3
Cryptosporidium
3
3
3
Bacteria & Virus
3
3
3
Cryptosporidium
3
3
\
Three test units will be challenged
(1)
Each test unit will be challenged 12 times
(a)
Six times with test water #1
(i)
Three times with R. terrigena and Polio / Rota
virus;
Page 12 of 17
Project No. 110957
Page 6 of
(ii)
(b)
7
Three times with Cryptosporidium oocysts
Six times with test water #4
(i)
Three times with R. terrigena and Polio/Rota
virus;
(ii)
(2)
Three times with Cryptosporidium oocysts
As per CamelBak’s direction, when test water #4 is used the
water will be filtered twice through filters (supplied by
CamelBak) prior to the UV exposure. Each test unit
challenge will employ a new pre-filter.
iii.
The test exposures will be conducted as per the CamelBak
instructions.
iv.
The bacteria and virus challenge will be run separately from the
Cryptosporidium challenge. In the former instance the entire
volume of treated water (750 mL) will be collected in a sterile
Nalgene bottle (or equivalent) and sub sampled for bacteria and
viruses assay.
v.
The Cryptosporidium challenge will be run separately and the entire
750 mL sample used for the assay.
f.
The challenges in each of the test waters will be initiated at the beginning
of each week . The All Clear UV Water Purifier test units will be sanitized
at the end of the test period.
5.
Quality Control
a.
BioVir Laboratories, Inc. is a NELAP and California ELAP accredited
laboratory.
b.
Positive and negative controls will be included for all
microbiological media including the cell culture system for
Polio/ Rotaviruses and Cryptosporidium oocysts.
Page 13 of 17
Project No. 110957
6.
Page 7 of
7
Data Reporting
a.
Concentrations of bacteria will be reported as colony forming units (Cfu)
per 100 mL of untreated challenge or product water.
b.
Virus challenge results will be reported as Plaque forming units (Pfu) per
assay volume.
c.
Viable cryptosporidium oocysts will be reported as MPN per assay
volume.
d.
The log10 reduction in challenge microorganisms will be determined by
subtracting the log10 concentration of the test microbe in the product water
from the log10 concentration of microbe in the influent to the Units under
test.
Approvals:
Date June 27 2011
Robert C. Cooper, Ph.D.
Vice President
CamelBak Products, LLC. ______________________
Date __________
Name:
Title:
Page 14 of 17
Page 15 of 17
ATTACHMENT B
CamelBak All Clear UV Water Purifier Photos
Page 16 of 17
Figure 1 All Clear Bottle With Cap
Figure 2 All Clear Bottle with UV On
(Taken in the dark)
Page 17 of 17
Figure 4 Aspects of Bottle Caps
Figure 5 All Clear Bottle With Filter Unit
Attached

BioVir Laboratories, Inc.

Transcription

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