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-AcuteImmediate 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

pesticides

Chlorinated hydrocarbons

Heavy metalsdetergents

gasolinepetrol oil

herbicides

Membrane function Protein & lipid synthesis

ATP generationElectron transport

Cell respiration

Luminescence Level Reflects Degree of Toxicity

Luminescent bacteria strains used for toxicity testing

Strain namePhotobacterium leiognathi

Vibrio fischeri

OriginSea water

Optimal temperature for light emission

18-35°C15-16°C

Toxicity test utilized in

TOX-SCREEN,

TOX-SPOT

CCB-TOX

MicrotoxTM

LumisToxTM

BioFixTM

Stability to changing environmental conditions

HighLow

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

Acute: Industrial activities Animal feeding operations Agriculture runoff Septic systems and cesspools

Chronic: Industrial & commercial activities Agriculture runoff 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.

Tiered Response Model

Increasing:

Certainty

Response

Cost

Observed Water Quality Change

)determined by broad-based continuous screening(

Automated Sample Collection

Confirmation Bioassay

Chemical Analysis

If positive

Public Health Regulatory or Remedial Action

If positive

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

TOX-SPOT/

TOX-SCREEN

CCB-TOXObserve water quality change -

broad, continuous.

Automated Sample Collection

Confirmation Bioassay.

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

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

CCB - Continuous Contamination Biomonitor

How does the AquaVerity solution

compare to competitive approaches on

the market?

EWS Matrix (1)- Detection & Warning Capabilities

FactorMulti-parameterOther Biomonitors

CheckLight’sAquaVerity

Method5-6 sensors5-20 live organismsOne million luminescent bacteria

Detection spectrum5-6 parametersWide range of contaminants, including unknown types.

Determines toxicityNoYesYes

Discrimination between organic & heavy metals contaminants

NoNoYes

Detection sensitivity

Medium (depends on the parameter)

HighHigh

Contaminationboundaries assessment

PartialNoYes, by using the portable detectors

EWS Matrix (2)- Implementation

FactorMulti-parameterOther BiomonitorsCheckLight’sAquaVerity

Installation & maintenance

Complex due to variability of sensors used

Complex & requires on- going human supervision

Very simple to install & maintain. Unattended operation.

Adjustment to changing water environment

ComplexComplexMinimal adjustment needed.

Effective coverage in large water networks

Limited to wide distribution. Depends on parameters mix & complexity

Limited distribution due to complexity & costs

Wide distribution possible due to simple installation, minimal training & maintenance.

On-going usageMedium – requires skills & training

Complex - Requires skilled personnel & special training

Effort is minimal - reagents replacement once a month.

Overall reliabilityMedium. Depends on parameters used.

Low to Medium. Depends on biota used & environment. conditions

Very high. Includes built-in control mechanism.

Future EnhancementsunknownUnknownUpgrade refill kits with enhanced detection capabilities

EWS Matrix (3) - Cost Effectiveness

FactorMulti-parameterOther BiomonitorsCheckLight’sAquaVerity

Initial capital investment

Low to very highDepending on chosen parameters

Medium to very highMedium

On-going costsMedium due to complexity of sensors’ arrays and baseline build up

High due to the required human supervision

Low due to minimal intervention

Total cost of ownership

Medium to highHighLow to medium

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