ETV Joint Verification Statement Hidex Oy Bio Tox


THE ENVIRONMENTAL TECHNOLOGY VERIFICATION

PROGRAM ^

ETV

SEPA

ETV Joint Verification Statement

Batteiie

U.S. Environmental Protection Agency	Puttjng Tcchnohgy To Work

TECHNOLOGY TYPE: RAPID TOXICITY TESTING SYSTEM
APPLICATION:	DETECTING TOXICITY IN DRINKING WATER

TECHNOLOGY NAME: BioTox™

COMPANY:	Hidex Oy

ADDRESS:	Mustionkatu 2	PHONE: +358 2 275 0557

FIN-20750 Turku, Finland FAX: +358 2 241 0075
WEB SITE:	www.hidex.com

E-MAIL:	risto.juvonen@hidex.com

The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology Verification (ETV)
Program to facilitate the deployment of innovative or improved environmental technologies through performance
verification and dissemination of information. The goal of the ETV Program is to further environmental protection
by accelerating the acceptance and use of improved and cost-effective technologies. ETV seeks to achieve this goal
by providing high-quality, peer-reviewed data on technology performance to those involved in the design,
distribution, financing, permitting, purchase, and use of environmental technologies.

ETV works in partnership with recognized standards and testing organizations, with stakeholder groups
(consisting of buyers, vendor organizations, and permitters), and with individual technology developers. The
program evaluates the performance of innovative technologies by developing test plans that are responsive to the
needs of stakeholders, conducting field or laboratory tests (as appropriate), collecting and analyzing data, and pre-
paring peer-reviewed reports. All evaluations are conducted in accordance with rigorous quality assurance (QA)
protocols to ensure that data of known and adequate quality are generated and that the results are defensible.

The Advanced Monitoring Systems (AMS) Center, one of seven technology areas under ETV, is operated by
Batteiie in cooperation with EPA's National Exposure Research Laboratory. The AMS Center has recently
evaluated the performance of rapid toxicity testing systems used to detect toxicity in drinking water. This
verification statement provides a summary of the test results for the BioTox™ testing system.

VERIFICATION TEST DESCRIPTION

Rapid toxicity technologies use bacteria, enzymes, or small crustaceans that produce light or use oxygen at a steady
rate in the absence of toxic contaminants. Toxic contaminants in drinking water are indicated by a change in the
color or intensity of light or by a change in the rate of oxygen use. As part of this verification test, which took place
between July 14 and August 22, 2003, various contaminants were added to separate drinking water samples and

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analyzed by BioTox™. Response to interfering compounds in clean drinking water also was evaluated.
Dechlorinated drinking water samples from Columbus, Ohio, (DDW) were fortified with contaminants at
concentrations ranging from lethal levels to levels 1,000 times less than the lethal dose and analyzed. Endpoint and
precision, toxicity threshold for each contaminant, false positive/negative responses, ease of use, and sample
throughput were evaluated.

Inhibition results (endpoints) from four replicates of each contaminant at each concentration level were evaluated
to assess the ability of the BioTox™ to detect toxicity at various concentrations of contaminants, as well as to
measure the precision of the BioTox™ results. The response of BioTox™ to compounds used during the water
treatment process (interfering compounds) was evaluated by analyzing separate aliquots of DDW fortified with
each potential interferent at approximately one-half of the concentration limit recommended by the EPA's National
Secondary Drinking Water Regulations guidance. For analysis of by-products of the chlorination process, unspiked
DDW was analyzed because Columbus, Ohio, uses chlorination as its disinfectant procedure. For the analysis of
by-products of the chloramination process, a separate drinking water sample from St. Petersburg, Florida, which
uses chloramination as its disinfection process, was obtained. The samples were analyzed after residual chlorine
was removed using sodium thiosulfate. Sample throughput was measured based on the number of samples
analyzed per hour. Ease of use and reliability were determined based on documented observations of the operators
and the verification test coordinator.

Quality control samples included method blank samples, which consisted of American Society for Testing and
Materials (ASTM) Type II deionized (DI) water; positive control samples fortified with zinc sulfate; and negative
control samples, which consisted of the unspiked DDW. EPA QA staff also performed a technical systems audit
while testing was being conducted.

QA oversight of verification testing was provided by Battelle and EPA. Battelle QA staff conducted a technical
systems audit, a performance evaluation audit, and a data quality audit of 10% of the test data. EPA QA staff also
performed a technical systems audit while testing was being conducted.

TECHNOLOGY DESCRIPTION

The following description of BioTox™ was provided by the vendor and was not subjected to verification in this
test.

BioTox™ luminescent toxicity screening uses the Triathler™ luminometer, together with the freeze-dried
BioTox™ reagent, to determine the inhibitory effect of water-soluble samples, including suspensions of solid
samples. The BioTox™ reagent contains naturally luminescent Vibrio fischeri, which produce luciferase as a part
of their metabolic pathway. Luciferase catalyzes the oxidation of a long-chain aldehyde and coenzyme, flavin
mono-nucleotide. Substances affecting any part of the metabolic pathway of the bacteria directly affect the amount
of light they emit. Toxic compounds interfere with this metabolic process, resulting in a reduction of light
emission. To determine the toxicity of a sample, changes in light output are measured with the Triathler™
luminometer.

Sample dilutions and a control sample (2% sodium chloride) are pipetted into test tubes (500 microliters [|iL]
each), and the Triathler™ injector is filled with the V. fischeri reagent. The tube containing the control sample is
placed in the Triathler™ luminometer, and 500 |iL of the reagent are measured and injected. The measurement is
taken after 5 seconds. The tube is set aside, and the same procedure is repeated for each sample. After a 30-minute
reaction time, the tubes are shaken, and end-point readings from the control and each sample are measured. The
inhibition of each sample dilution is calculated.

To determine whether a contaminant caused detectable inhibition, the inhibition exhibited by drinking water
spiked with a contaminant was compared to the inhibition exhibited by the unspiked drinking water. Four
replicates of each spiked sample were analyzed. A result was considered positive if the inhibition of the water

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sample spiked with a contaminant plus or minus the standard deviation of four replicates did not include the
inhibition of the unspiked drinking water.

The BioTox™ kit, which provides for 144 measurements, contains six vials of freeze-dried V. fischeri reagent, six
vials of reagent diluent (12.5 milliliters [mL] each), and one 50-mL bottle of concentrated sample diluent. Reagent
injection and data acquisition can be performed by a computer connected to the Triathler™ luminometer. The
dimensions of the Triathler™ luminometer are 10 inches by 10 inches by 6 inches, and it weighs approximately
10 pounds. It only can be operated on 110-volt alternating current electricity. The BioTox™ kit costs $128, the
Triathler™ injector costs $1,950, and the luminometer with liquid scintillation counter costs $6,950.

VERIFICATION OF PERFORMANCE

Endpoint and Precision/Toxicity Threshold: The table below presents BioTox™ percent inhibition data and
range of standard deviations for the contaminants and potential interferences that were tested. The toxicity
thresholds also are shown for each contaminant tested.

Parameter

Compound

Lethal
Dose (LD)
Cone.
(mg/L)

Average Inhibitions at Concentrations
Relative to the LD Concentration (%)

Range of
Standard
Deviations (%)

Toxicity
Thresh.

(mg/L)

LD/10

LD/100

LD/1,000

Contaminants in
DDW

Aldicarb

280

-1

-10

3-12

ND(a)

Colchicine

240

-8

-15

-27

11-27

Cyanide

250

-1

2-16

Dicrotophos

1,400

4-10

Thallium
sulfate

2,400

-4

6-16

Botulinum

toxin(b)

0.30

5-8

Ricin(c)

-5

6-10

Soman

0.068(d)

-1

2-3

0.22

2-9

Potential
interferences in
DDW

Interference

Cone.
(mg/L)

Average Inhibitions at a
Single Concentration (%)

Standard
Deviation (%)

Aluminum

0.36

Copper

0.65

Iron

0.069

Manganese

0.26

Zinc

3.5

131 ND = Not detectable.

lbl Lethal dose solution also contained 3 mg/L phosphate and 1 mg/L sodium chloride.

lcl Lethal dose solution also contained 3 mg/L phosphate, 26 mg/L sodium chloride, and 2 mg/L sodium azide.
ldl Due to the degradation of soman in water, the stock solution confirmation analysis confirmed that the concentration of the lethal
dose was 23% of the expected concentration of 0.30 mg/L.

False Positive/Negative Responses: Slightly exaggerated inhibitions (false positive responses) may result if
BioTox™ is used to analyze chloraminated water, which produced 13% ± 2% inhibitions, with respect to ASTM
Type IIDI water. Inhibition greater than the negative control was not detected for lethal doses of aldicarb,
colchicine, dicrotophos, botulinum toxin, ricin, soman, and VX; and, therefore, these results were considered false
negative. Inhibition was -49% ±33% for water from the system treated by chlorination, resulting in a risk of false
negative responses when using ASTM Type II DI water as the control sample.

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Field Portability: A single concentration of cyanide was analyzed in the field and in the laboratory. The inhibition
of 2.5 milligrams per liter of cyanide in the field was 57% ± 4%, and in the laboratory it was 10% ± 9%.
Practically, the operation did not seem much different. However, the Triathler™ is not equipped for use with
batteries, so electricity was required. A field-portable case may be purchased. A flat, sturdy surface is needed to
operate BioTox™ because a beaker of bacteria must be connected to the injector.

Other Performance Factors: Although determining how to operate the BioTox™/Triathler™ was difficult
without an instruction manual and required significan t intervention from the vendor, it was easy to use once the
correct procedure was determined. Although the operators had scientific backgrounds, based on the observations
of the verification test coordinator, an operator with little technical training would probably be able to analyze
samples successfully once provided with adequate guidance in the form of contact with the vendor or an improved
instruction manual. Sample throughput was 50 samples per hour.

Original signed by Gabor J. Kovacs 11/13/03
Gabor J. Kovacs Date

Vice President
Environmental Sector
Battelle

Original signed by Timothy E. Oppelt 12/1/03

Timothy E. Oppelt Date

Director

National Homeland Security Research Center
U.S. Environmental Protection Agency

NOTICE: ETV verifications are based on an evaluation of technology performance under specific, predetermined
criteria and the appropriate quality assurance procedures. EPA and Battelle make no expressed or implied
warranties as to the performance of the technology and do not certify that a technology will always operate as
verified. The end user is solely responsible for complying with any and all applicable federal, state, and local
requirements. Mention of commercial product names does not imply endorsement.

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