Portable Contamination Biomonitors

PCB-TOX & PCB-TOX-SPOT

Session I - Basics

Agenda

Recap- bioassays, toxicity, bioluminescence-based assays Key applications Product line overview & history EPA/ETV verification

PCB-TOX (ToxScreen3) - Key applicationsBenefits/features/functionSensitivityKit components & luminometerrs

PCB-TOX-SPOT - Key applicationsBenefits/features/functionSensitivityKit components & luminometer

Summary

Use of Bioassays

A bioassay can be defined as a biological assay performed to measure the effects of a substance on a living organism.

A toxicity bioassay may be run as a screening test (or qualitative), where the toxicity of a sample is compared to that of a control water. The screening tests indicate whether toxicity is present in the sample.

A toxicity bioassay may be run as a definitive test (or semi-quantitative), where several portions of the sample are diluted with varying amounts of the control water and their results compared to the control water. The definitive tests indicate the amount of toxicity presented by the sample.

Additionally, the results of a toxicity bioassay may be measured as either an acute or chronic response.

Bioassays Applications

Mapping to identify toxicity/concentration hotspots

Investigate cause/effect relationships

Detection of “active” or toxic compounds or mixtures not identified by classical analysis

Selection of samples for further/more expensive analysis

Mapping after pollution incidents/accidents

Benefits of Using Bioassays

The use of bioassays provides a holistic approach that allows the toxicity evaluation of the total integrated effect of all constituent components, including toxicants and confounding variables, in a given complex sample matrix. The net assessment is the combined interactive evaluation of additive, antagonistic and synergistic effects of all sample components.

As bioassays directly allow measurement of the potential environmental effects of complex sample matrices, their use for pollution monitoring and control in regulatory framework is becoming increasingly important.

“While there are several different organisms that can be used to monitor for toxicity (including bacteria, invertebrates, and fish), bacteria-based bio-sensors are ideal for use as early warning screening tools for drinking water security because bacteria usually respond to toxics in a matter of minutes”. [EPA - Biological Sensors for Toxicity-Water and Wastewater Security Product Guide]

General Features of a Bioassay Based on Luminous Bacteria

The freeze-dried preparations of luminous bacteria are stable for long periods.

Millions of cells can be introduced to a small water sample, increasing the number of test organisms and reducing the effect of biological variability.

Chemical toxicants that effect cell’s metabolism result in rapid decay of luminescence.

Luminescence may be easily measured by readily available luminometers. High correlation exists between the luminescence test and bioassays that

apply higher organisms, such as fish.

Decreased luminescence

pesticides

Chlorinated hydrocarbons

Heavy metalsdetergents

gasolinepetrol oil

herbicides

Membrane function Protein & lipid synthesis

ATP generationElectron transport

Cell respiration

Luminescence Level Reflects Degree of Toxicity

Product Line Overview & History

ToxScreen1 was first presented in 2001.Verified by the ETV in 2003.

An improved version - ToxScreen2 - was presented in 2003. Verified by the USA-

EPA/ETV in 2006.

Additional improvements were implemented in the latest version - ToxScreen3 -

presented in 2008. Renamed PCB-TOX(ToxScreen3) in 2009.

A dedicated field test kit based on the ToxScreen technology, named, PCB-TOX-

SPOT, was introduced in 2009.

PCB-TOX & PCB-TOX-SPOT are part of CheckLight’s new AquaVerity - a

comprehensive solution to drinking water safety and security.

USA-EPA/ETV Verification Studies

USA-EPA/ETV Verification Studies on Deliberate Contamination of Drinking Water

Sources included Sixteen different rapid toxicity test systems were tested for-

> Sensitivity

> Rapidity

> Field use

> User friendliness

> Sample throughput

Any final solution to the rapid detection problem will likely include a

combination of sensing technologies including bioassays and more

conventional chemical sensors all tailored to the characteristics of individual

water supplies.

An effective bioassay will indicate the presence of something that needs looking

into. But it won't replace qualified hand sampling and further testing.

ETV Conclusions

Out of the 16 different test systems evaluated in 2003 and 2006,

ToxScreen was on the top of the list is terms of -

Sensitivity

Field use

High throughput

User friendliness

ETV/EPA Conclusions

SummarySummary

Rapid toxicity tests are a vital part of a desired Early Warning System for

detecting drinking water contamination.

CheckLight’s ToxScreen test was found to be one of the best rapid toxicity

tests for drinking water.

The test is rapid, sensitive to a broad spectrum of chemical contaminants,

reliable, and user-friendly.

PCB-TOX (ToxScreen3)- Applications

Rapid lab monitoring tool for early detection of chemical toxicity due to spills, accidents, or sabotage in drinking water.

The test can be used to test different water sources - raw waters (including ground water, surface water, recycled water and effluents) and finished drinking water.

It can be used throughout the distribution network to determine dangerous changes in drinking water quality.

It can be used to determine the relative toxicity of inlet and outlet of sewage treatment plants.

PCB-TOX (ToxScreen3) - Benefits

Provides early warning of dangerous water contamination events due to spills,

accidents, sabotage or terrorism.

Ideal for routine monitoring of changes in water quality in surface and ground

water sources.

Able to detect active toxic compounds / mixtures, not identified by classical

analysis.

PCB-TOX (ToxScreen3)- Features

Sensitive - sub-ppm detection levels; for most of the tested chemical agents the test is up to hundreds of times more sensitive than other available bioluminescence-based tests.

Fast - results are obtained in 15 minutes.

Discriminatory - a unique buffer set (Pro-Metal & Pro-Organic) enables

preliminary discrimination between organic toxicants and cationic heavy metals.

Flexible – different testing protocols can be used (quantitative/qualitative)

User-friendly - minimal lab experience is necessary.

Cost effective - extended usability of suspended reagent (up to 5 days). High

sample throughput. Microplate testing format option.

PCB-TOX (ToxScreen3)- Function

Various lab testing protocols are recommended (detailed in the user guide) to enable either:

Rapid screening of multiple samples for qualitative determination of toxicity

Semi-quantitative measurements to determine degree of toxicity of discrete samples.

Low cost per test encourages frequent testing for rapid response to changing conditions in water quality.

ToxScreen3 Sensitivity - 1

Sensitivity as tested in Pro-Metal Buffer

IC50 – the minimal concentration of the toxic agent that results in 50% inhibition of luminescence.

LDL - the minimal concentration of the toxic agent that results in 20% inhibition of luminescence

NF – not found

ToxicantIC50 (mg/L)LDL (mg/L)

ToxScreen3MicrotoxTMToxScreen3MicrotoxTM

Arsenate (5)0.091.500.015NF

Cadmium(2)0.241.40.0820

Lead(2)0.18110.060.6

Selenium(2)0.15NF0.0333

ToxScreen3 Sensitivity - 2

Sensitivity as tested in Pro-Organic Buffer

ToxicantIC50 (mg/L)LDL (mg/L)

ToxScreen3MicrotoxTMToxScreen3MicrotoxTM

2,4,5-T0.1352.50.05NF

DDT0.0570.01NF

2,4-Dintrophenol0.210.60.05NF

KCN0.458.40.10.73

Arsenic(3)6NF27.8

2,4-D0.15.70.04NF

Chromium(6)0.07310.0315.3

PCB-TOX (ToxScreen3)- Key References & Case Studies

The test kit has been routinely utilized by the North Carolina DWQ Aquatic Toxicology Unit for assessing watershed toxicity.

The test kit has been used by various US-based water utilities to determine potential contamination to their water sources during high terrorist alert periods.

The test kit has been routinely used by research groups in Japan to assess leachate toxicity.

The test kit has been used in order to monitor the toxicity in the tidal estuaries in Europe, as part of the WFD monitoring requirements.

Kits & Accessories Components

PCB-TOX (ToxScreen3):

Freeze dried bacteria box

Assay Buffers box

Empty tubes

Portable luminometer

PCB-TOX- SPOT:

Freeze dried bacteria boxAssay Buffers boxEmpty tubes

Portable low-cost luminometer

Pipettor & tipsTimerPortable incubatorCarrying case

PCB-TOX (ToxScreen3) -Kit components & Luminometer

The kit holds: 5 vials of freeze dried bacteria 50 ml of concentrated Pro-Metal &

Pro-Organic buffers (each) 5ml storage Buffer 1ml concentrated positive controls

(metal & organic, each) 500 empty test tubes

The luminometer is a portable photomultiplier-based instrument.

Sensitivity - 1fmol ATP Data storage- 2000 points Dynamic range- 106

Interface- RS232 Weight- 2kg net

PCB-TOX-SPOT -Kit components & Luminometer

The kit holds: 25 vials of freeze dried bacteria 20 ml of concentrated Pro-Metal & Pro-

Organic buffers, each. 23 ml Hydration Buffer 200 empty tubes 2.5 ml of concentrated positive controls

(metal & organic, each)

The luminometer is a portable photodiode- based instrument.

Weight- 250gr net

Dynamic range - 105

Data storage- 2000 points

Interface- USB

LUM-KK-01

PCB-TOX-SPOT -Accessories

The starter kit is supplied with the following accessories-

Carrying case (11x40x50cm)

Pipettor (0.1-1ml)

425 pipettor tips plus storage box

Two boxes to hold the empty test tubes

Timer

Incubator block (30°C) with a car cigarette plug connector

PCB-TOX-SPOT -Key Applications & Benefits

Designed for field operation, the test provides early warning of dangerous water

contamination in emergency situations due to spills, accidents, sabotage or

terrorism.

PCB-TOX-SPOT enables rapid on site screening to determine changes in water

quality in raw and treated drinking water sources and remote locations throughout

the distribution network.

The test can be used by emergency response teams, water companies, health

and environmental supervising & monitoring authorities, municipal water system

authorities, military units, etc, for: Mapping after pollution incidents / accidents Selection of samples for further/more expensive analysis Mapping to identify toxicity / concentration hotspots

PCB-TOX-SPOT -Features

Sensitive - sub-ppm detection levels; for most of the tested chemical agents the test is up to hundreds of times more sensitive than other available bioluminescence-based tests.

Fast - results are obtained in 15 minutes.

Discriminatory - a unique buffer set (Pro-Metal & Pro-Organic) enables preliminary discrimination between organic toxicants and cationic heavy metals.

User-friendly & Robust - minimal skill and training is necessary. Can be used anywhere.

Cost effective – saves valuable time and money by minimizing shipment of samples to distant labs.

Summary - 1

FeaturePCB-TOX (ToxScreen3)PCB-TOX-SPOT

Tests per freeze dried bacteria vial

482

Preparation time (pre-incubation of hydrated bacteria)

3 hrs pre-incubation once a week

10 min

Life span of hydrated bacteria

One weekOne hour

Optimal assay temperature

30°C

(Operational: 18°C - 35°C)

Nature of generated dataQualitative & semi-quantitative

Qualitative

Comparative sensitivityOrganics - very high

Metals - very high

Summary - 2

FeaturePCB-TOX (ToxScreen3)PCB-TOX-SPOT

Data analysisDownload data from luminometer to PC. Transfer data to programmable excel module for automatic calculation of toxicity

Recommended for Routine lab testing Screening large numbers of samples Determination of relative toxicity

On site testing in emergency situations