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) - Benefits/features/function Sensitivity Kit components & luminometer rs PCB-TOX-SPOT - 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 Luminescence Level Reflects Degree of Toxicity Membrane function Protein & lipid synthesis ATP generation Electron transport Cell respiration Chlorinated hydrocarbons pesticides gasoline petrol oil herbicides Heavy metals detergents Decreased luminescence

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

ETV Conclusions 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/EPA 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

Summary 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 LDL (mg/L) IC50 (mg/L) Toxicant MicrotoxTM ToxScreen3 NF 0.015 1.50 0.09 Arsenate (5) 20 0.08 41.4 0.2 Cadmium(2) 0.6 0.06 11 0.18 Lead(2) 33 0.03 0.15 Selenium(2) 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

ToxScreen3 Sensitivity - 2 Sensitivity as tested in Pro-Organic Buffer LDL (mg/L) IC50 (mg/L) Toxicant MicrotoxTM ToxScreen3 NF 0.05 52.5 0.13 2,4,5-T 0.01 7 DDT 10.6 0.2 2,4-Dintrophenol 0.73 0.1 8.4 0.45 KCN 7.8 2 6 Arsenic(3) 0.04 5.7 2,4-D 15.3 0.03 31 0.07 Chromium(6)

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 box Assay Buffers box Empty tubes Portable low-cost luminometer Pipettor & tips Timer Portable incubator Carrying 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 PCB-TOX-SPOT PCB-TOX (ToxScreen3) Feature 2 48 Tests per freeze dried bacteria vial 10 min 3 hrs pre-incubation once a week Preparation time (pre-incubation of hydrated bacteria) One hour One week Life span of hydrated bacteria 30°C (Operational: 18°C - 35°C) Optimal assay temperature Qualitative Qualitative & semi-quantitative Nature of generated data Organics - very high Metals - very high Comparative sensitivity

Summary - 2 PCB-TOX-SPOT PCB-TOX (ToxScreen3) Feature Download data from luminometer to PC. Transfer data to programmable excel module for automatic calculation of toxicity Data analysis On site testing in emergency situations Routine lab testing Screening large numbers of samples Determination of relative toxicity Recommended for