Technology & EWS Basics CCB-TOX Tutorial Sections 1-3 Technology & EWS Basics

Overall Agenda Section 1: Company information Section 2: Technology basics Section 3: Early Warning Systems basis Section 4: CCB Overview Section 5: CCB Operations Section 6: CCB Installation Section 7: CCB Maintenance Section 8: CCB “Hands-On” session.

Section 1 Section 1: Company Information Background Product lines Product range

Company Background Vendor of water quality monitoring technologies & products. Headquarters are in Israel. Operates internationally. Private company, founded in 2001 by Dr. Nirit Ulitzur - a biology expert. Equity investment by Whitewater in 2008 – an Israeli investment group focusing on water technologies. Israel’s Chief Scientist Office supports CheckLight’s R&D. Scientific research is headed by Prof. (Emeritus) Shimon Ulitzur (Technion Institute), a leading scientist in the field of microbial luminescence.

Product Lines Portable Contamination Biomonitors - Test kits (bacteria & reagents) and Luminometers Continuous Contamination Biomonitors - On-Line monitors (hardware), reagents cartridges, software, accessories & options.

Product Range

Section 2 Section 2: Technology Basics Definitions Toxicity Use of Bioassays Chemical analysis vs. Toxicity testing Bioassays benefits Bioluminescent Bacteria

Definitions A toxicity test can be considered a bioassay that allows measurement of damage. It is a measure of the degree to which a substance can elicit a deleterious effect (including death) in a given organism. Acute, Sub-Acute Immediate or almost immediate adverse health effects from exposure to substance (for water contaminants, usually within a day) Chronic, Sub-Chronic Adverse health effects resulting from long-term or repeated (chronic, >10% of lifespan) exposure to a substance over a period of time Can occur at low levels that have no ACUTE effects Chronic health effects can be as severe as acute effects, but take much longer to manifest Lethal, Sub-Lethal Causes death immediately or over a short period of time Sub-lethal is not quite lethal; less than lethal

Toxicity Measures Some toxicity measurements are more applicable than others in assessing the concentration at which a contaminant will have acute or immediate impacts, while others will have more chronic, long-term impacts. Assessing acute or immediate impacts of contaminant: Lethal Dose 50 (LD50), Infectious Dose 50 (ID50), or Lethal Concentration 50 (LC50) No Observed Adverse Effect Level (NOAEL) Lowest Observed Adverse Effect Level (LOAEL) Assessing chronic, or long-term impacts of contaminant: Maximum Contaminant Level (MCL) Maximum Contaminant Level Goal (MCLG) Basic tenet of toxicology: “Dosis facit venenum “ - The dose makes the poison (Paracelus)

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.

Chemical Analysis and Toxicity Testing Toxicity testing is not and never will be a substitute for chemical analysis. The traditional approach to environmental assessment based on chemical analysis fails to provide an adequate interpretation of toxicity to biota in the ecosystem in the context of bioavailability. An environmental toxicant can be defined as a substance that, in a given concentration and chemical form, challenges the organisms of the ecosystem and causes adverse or toxic effects. This definition includes an element of chemical characterization, toxicity testing and eco-assessment. This triad of techniques, which can be used alone or in combination, forms a particular approach that is often employed in the environmental management of pollutants.

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

Luminescent bacteria strains used for toxicity testing Vibrio fischeri Photobacterium leiognathi Strain name Sea water Origin 15-16°C 18-35°C Optimal temperature for light emission MicrotoxTM LumisToxTM BioFixTM TOX-SCREEN, TOX-SPOT CCB-TOX Toxicity test utilized in Low High Stability to changing environmental conditions

Section 3 Section 3: Early Warning Systems Basics What is CCB-TOX & key benefits Key uses and users Vulnerability, sources and effects of contaminations EWS structure, function & criteria EWS design EWS – the tiered approach Bioassays & technology selection Other considerations & response AquaVerity solution and application CL CCB value proposition EWS solutions comparison

What is the CCB-TOX ? Automatic water toxicity biomonitor Continuously monitors chemical contamination events. On-Line - sends alerts in real-time. Key element of an Early Warning System. CCB-TOX

CCB - key benefits Significantly reduces the threats associated with accidental and intentional chemical contamination of water: Spills, accidents, dumping Equipment malfunction Natural disasters Sabotage & terror Acute & chronic exposure Illness & death Direct & indirect costs Liability suits

CCB - key uses Drinking water monitoring: protecting public health by sensing changes in water quality at reservoirs, intake, during & after treatment, throughout the distribution network, protected zones, etc. Environmental monitoring: protecting the environment by sensing changes in water quality along rivers, lakes & natural reservoirs, as well as near potential effluent discharge areas. Water treatment monitoring: improving treatment processes effectiveness by sensing water quality changes at intake, and during treatment processes, enabling process modifications decisions & quality assurance.

CCB -potential users Water companies: raw water suppliers, drinking water utilities, municipal water suppliers, water treatment plants. Authorities: environmental supervising & monitoring, river basin monitoring, health supervising, municipal water systems. Secured facilities: industrial zones & parks, hospitals, governmental, military. Industrial companies: water intake & discharge for bottlers, food, pharma and other water related processing companies. Recycle & re-use systems.

Vulnerability & Sensitivity of Water Sources Surface water > Runoff > Ground water infiltration Ground water > Infiltration from the surface > Injection of contaminants > Naturally occurring substances

Health effects caused by contaminated source water Acute health effects mainly by - > viruses > pathogenic bacteria > parasites, particularly protozoa and cysts > algal microtoxins Chronic health effects mainly by - > volatile organic chemicals (VOCs) > inorganic chemicals (IOCs) > synthetic organic chemicals (SOCs)

Vulnerability Within the Distribution System Backpressure can cause backflow to occur when a potable system is connected to a non-potable supply operating under a higher pressure than the distribution system by means of a pump, boiler, elevation difference, air or steam pressure, or other means. Backflow is any unwanted flow of used or non-potable water, or other substances from any domestic, industrial, or institutional piping system back into the potable water distribution system. Cross-connections and backflow represent a significant public health risk (US EPA, 2000b) by allowing chemical and biological contaminants into the potable water supply (a conclusion of the Microbial/Disinfection Byproducts Federal Advisory Committee (M/DBP FACA)). A wide number and range of chemical and biological contaminants have been reported to enter the distribution system through cross-connections and backflow. Pesticides, sewage, antifreeze, coolants, and detergents were the most frequent types of contaminants reported.

Sources of Contaminants with Acute and Chronic Health Effects Industrial activities Animal feeding operations Agriculture runoff Septic systems and cesspools Chronic: Industrial & commercial activities Landfills & surface impoundments Urban uses

Early Warning System (EWS) Structure & Function An effective EWS is an integrated system for deploying the monitoring technology, analyzing and interpreting the results, and utilizing the results to make decisions that protect public health. An ideal contamination warning system that monitors toxic events in water should have the following features: Rapid results Sensitive Wide detection spectrum Reliable Continuous operations Fit for field testing User-friendly Affordable

EWS - Core Criteria Currently, an EWS with all of these features does not exist. However, there are some technologies that can be used to build an EWS that can meet certain core criteria: provide rapid response screen for a number of contaminants while maintaining sufficient sensitivity perform as automated systems that allow for remote monitoring Any monitoring system that does not meet these minimum criteria should not be considered an effective EWS.

EWS Design Considerations There are many issues and water system characteristics that need to be considered when designing an EWS: Planning and Communication System Characterization Target Contaminants

Planning and Communication The objectives of the program should be defined clearly, and a plan should be developed for the- > Interpretation > Use > Reporting of monitoring results. The plan should be developed in coordination with - > The water utility > Local and state health departments > Emergency response units > Law enforcement agencies > Local political leadership

System Characterization The system should be characterized with respect to - > Access points > Flow and demand patterns > Pressure zones If not already available, a hydraulic model should be constructed. System vulnerabilities should be identified and characterized, preferably through a formal vulnerability assessment.

Target Contaminants Even the most complex array of monitoring equipment cannot detect the entire spectrum of agents that could pose a threat to public health via contaminated water. Thus, the design of an EWS should focus on contaminants that are thought to pose the most serious threat. Many factors may go into this assessment, including: Concentration of a particular contaminant that is necessary to cause harm Availability and accessibility of a contaminant Persistence and stability of a contaminant in an aqueous environment Difficulty associated with detecting a contaminant in the water

EWS - The Tiered Approach A balance between the need for screening function of the system (i.e., the ability to detect a wide range of contaminants) and the need for specificity (i.e., the ability to positively identify and quantify a specific contaminant) can be achieved through tiered monitoring. First tier - continuous, real-time screen for a range of contaminants utilizing a broad-based screening technology such as assays designed to detect changes in toxicity. Second tier - a positive result from the first stage would trigger the second stage of confirmatory analysis using more specific and sensitive techniques. A positive result from the confirmatory analysis would trigger a response action.

Observed Water Quality Change Tiered Response Model Observed Water Quality Change (determined by broad-based continuous screening) Increasing: Certainty Response Cost Automated Sample Collection Confirmation Bioassay If positive Chemical Analysis If positive Public Health Regulatory or Remedial Action

Broad-Based Continuous Screening A major problem in the development of EWS quality monitoring systems is that there are an almost unlimited number of potential contaminants that could threaten a water asset. While many products have been developed that monitor for specific contaminants or specific types of contaminants, it is impractical to design a system that can detect every potential threat to water quality. One approach is to use biological organisms as living "sentinels" that will warn operators of contamination. Sophisticated continuous and automatic biomonitors are now available that detect and alert whenever a notable change occurs in the behavior of the sensing organisms (such as, bacteria, fish, algae, mussels, daphnia).

Bioassays - Applications & Benefits Mapping to identify toxicity/concentration hotspots Selection of samples for further/more expensive analysis Mapping after pollution incidents/accidents “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] The Luminescent bacteria provided by CheckLight offer the unique advantage of both automatic and hand held testing capabilities.

EWS Technology Selection Performance of the chosen field deployable monitoring technology must meet the data quality objectives of the monitoring program that were defined during the design of the EWS and include: > Specificity > Sensitivity > Accuracy > Precision > Recovery > False positives/negatives rates

Alarm Levels For the alarms to be triggered at the appropriate levels, one must identify the concentrations at which the agents pose a threat to human health. The basis for setting alarm levels will depend on the capability of the EWS employed. The alarm should be triggered by a combination of events, not a single detection, which may be a false positive.

Sensor Location and Density The location and density of sensors in an EWS is dictated by the results of the system characterization, vulnerability assessment, threat analysis, and usage considerations. Proper characterization of the distribution system, including usage patterns, and the location of critical system nodes (e.g., hospitals, law enforcement and emergency response agencies, government facilities, etc.) is necessary to design an effective monitoring network. However, even if sensors can be optimally located within a distribution system, there may not be sufficient time to prevent exposure of a portion of the public to the contaminated water. At best, monitoring conducted within the distribution system will provide time to limit exposure, isolate the contaminated water, and initiate mitigation/ remediation actions.

Data Management, Interpretation, and Reduction One of the challenges of a continuous, real- time monitoring system is management of the large amounts of data that are generated. Use of data acquisition software and a central data management center is critical. The data management system should be capable of performing some level of data analysis and trending in order to assess whether or not an alarm level has been exceeded and minimize the rate of false alarms. At a minimum, the system should notify operators, public health agencies, and/or emergency response officials. In some cases, it may be appropriate to program the data management system to initiate preliminary response actions, such as closing valves or collecting additional samples. However, these initial responses should be considered simple precautionary measures, and public officials should make judgments regarding decisive response actions. Adopted in part from: Safeguarding The Security Of Public Water Supplies Using Early Warning Systems: A Brief Review .J Hasan et al. Journal Of Contemporary Water Research And Education Issue 129, Pages 27-33, October 2004.

Response The possible responses when an EWS triggers an alarm may include- Modification to the drinking water system (e.g., shutdown, addition of disinfectants, etc.) Notification (e.g., boil water advisory) either to the general public or to target communities or subpopulations Additional data gathering or monitoring Follow-up surveillance and epidemiologic studies No action, or some combination of these The type of response will be dependent on the nature of both the threat to and the nature of the drinking water system, including the population it serves.

The ETV-Verified ToxScreen Technology Serves as the Basis for the AquaVerity The Comprehensive Solution for Water Utilities to Ensure Drinking Water Safety and Quality

AquaVerity Components Continuous Contamination Biomonitor Portable Contamination Biomonitor Control & Analysis Software package Solution Implementation Service package

Application Key part of a comprehensive Early Warning Solution: Effective coverage of drinking water systems. Located at various stations throughout the water distribution system, Coupled with Portable Contamination Biomonitors. Pinpoint contamination boundaries & trace contamination sources. Seamless integration with other monitors / sensors and customer management systems (i.e. SCADA).

AquaVerity Comprehensive, Early Warning Biomonitoring System to ensure water safety and quality. Continuously detects contamination events and issues real-time alerts. Significantly reduces the threats & risks associated with water contamination. Composed of hardware, software and consumables. Includes both continuous (on-line) and portable equipment.

AquaVerity - Tiered Response Observe water quality change - broad, continuous. Increasing: Certainty, Response. Cost CCB-TOX Automated Sample Collection Confirmation Bioassay. TOX-SPOT / TOX-SCREEN Chemical Analysis. Public Healthy / Remedial Action

AquaVerity xxx

CheckLight’s Value Proposition Functional Benefits: Early detection of contamination in drinking water Enabling to pinpoint location & boundaries of contamination sources Reducing direct & indirect costs of illnesses & deaths Preventing widespread illness and severe symptomps. Saving lives. Reducing liability Emotional Benefits: Providing a sense of safety & security Reducing perceived risk of malpractice/liability

CCB - Continuous Contamination Biomonitor For deployment in monitoring stations positioned at strategic locations Includes various monitoring models & refill reagent kits (for detecting chemical & biological contaminants) Easily integrated with other systems Suspicious samples are captured by an automatic sampler for further analysis Easy installation, operation and maintenance No need for adjustments due to changing environmental conditions Remotely operated & controlled Requires minimal operator intervention

How does the AquaVerity solution compare to competitive approaches on the market?

EWS Matrix (1)- Detection & Warning Capabilities CheckLight’s AquaVerity Other Biomonitors Multi-parameter Factor One million luminescent bacteria 5-20 live organisms 5-6 sensors Method Wide range of contaminants, including unknown types. 5-6 parameters Detection spectrum Yes No Determines toxicity Discrimination between organic & heavy metals contaminants High Medium (depends on the parameter) Detection sensitivity Yes, by using the portable detectors Partial Contamination boundaries assessment 49

EWS Matrix (2)- Implementation CheckLight’s AquaVerity Other Biomonitors Multi-parameter Factor Very simple to install & maintain. Unattended operation. Complex & requires on- going human supervision Complex due to variability of sensors used Installation & maintenance Minimal adjustment needed. Complex Adjustment to changing water environment Wide distribution possible due to simple installation, minimal training & maintenance. Limited distribution due to complexity & costs Limited to wide distribution. Depends on parameters mix & complexity Effective coverage in large water networks Effort is minimal - reagents replacement once a month. Complex - Requires skilled personnel & special training Medium – requires skills & training On-going usage Very high. Includes built-in control mechanism. Low to Medium. Depends on biota used & environment. conditions Medium. Depends on parameters used. Overall reliability Upgrade refill kits with enhanced detection capabilities Unknown unknown Future Enhancements 50

EWS Matrix (3) - Cost Effectiveness CheckLight’s AquaVerity Other Biomonitors Multi-parameter Factor Medium Medium to very high Low to very high Depending on chosen parameters Initial capital investment Low due to minimal intervention High due to the required human supervision Medium due to complexity of sensors’ arrays and baseline build up On-going costs Low to medium High Medium to high Total cost of ownership 51

Positioning Sensitive to a broad range of contamination sources Reliable Cost-effective Easy to operate Customer oriented

CheckLight Ltd. P.O. Box 72, Qiryat Tiv-on 36000, Israel Tel: 972 4 9930530 Fax: 972 4 9533176 info@checklight.biz www.checklight.biz