1. Portable Contamination Biomonitors PCB-TOX & PCB-TOX-SPOT
Session I - Basics

2. 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

3. 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.

4. 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

5. 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]

6. 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.

7. 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

8. Product Line Overview & History
ToxScreen1 was first presented in 2001.Verified by the ETV in 2003.
An improved version - ToxScreen2 - was presented in Verified by the USA-EPA/ETV in 2006.
Additional improvements were implemented in the latest version - ToxScreen3 - presented in 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.

9. 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

10. 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.

11. 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

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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

18. 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)

19. 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.

20. 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

21. 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 points
Dynamic range- 106
Interface- RS232
Weight- 2kg net

22. 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 points
Interface- USB
LUM-KK-01

23. 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

24. 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

25. 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.

26. 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

27. 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