TOXI-SCREENING KIT

Microbiotest for ultra-rapid on-site

toxicity screening of water

STANDARD OPERATIONAL

PROCEDURE

TABLE OF CONTENTS Introduction to the Toxi-Screening microbiotest 2 - Origin 2

- Biological background of the assay 2

- Principle 2

- Scope 3

- Assets of the Toxi-Screening microbiotest 3

- Features 3

- Shelf life 4

- Sensitivity and detection threshold 4

- Specificity 4

Contents of the Toxi-Screening kit 5 Contents of the Luminescence Measurement Case 6 Test procedure 8 - Transfer of the materials to the Luminescence Measurement Case 8

- Rehydration of the freeze-dried bacteria 8

- Sampling 10

- Transfer of the luminescent bacteria in the sample tubes

and the control tubes 10

- Measurement at t0 10

- Measurement at t30 12

- Data evaluation 14

- Results sheet 15

INTRODUCTION TO THE TOXI-SCREENING

MICROBIOTEST

ORIGIN

The development of the Toxi-Screening Kit has been instigated by the worldwide demand for “field tests” for very rapid screening of the toxic hazard of contaminated waters. The research performed in the company MicroBioTests Inc. eventually led to the elaboration of this practical, user-friendly and low cost “field microbiotest”, which can be performed “anytime and anywhere” with the aid of a small low cost portable luminometer. The portable luminometer also allows to perform concurrently in the field a “Bacterial Contamination Screening” microbiotests to evaluate the degree of contamination of suspect water samples by bacteria and biological residues.

BIOLOGICAL BACKGROUND OF THE ASSAY

The toxicity screening microbiotest is a simplified “field modification” of bioassays which measure the decrease in luminescence in specific (mostly marine) bacteria producing bioluminescence as a by-product of their cellular respiration. The amount of light produced by the bacteria is proportional to the intensity of cellular respiration and is measured in the luminometer in “Relative Light Units” (RLU).

PRINCIPLE

Inhibition of the bacterial respiration under toxic stress automatically leads to decrease of the bioluminescence. The decrease of the luminescence in the water sample is measured after a short exposure time (30 minutes) and is compared to the decrease in luminescence in a (non-toxic) control water.

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The magnitude of the decrease in luminescence in the analysed water versus that in the control water is an indication for the degree of toxicity of the water sample.

SCOPE

The Toxi-Screening microbiotest is suited for a variety of analyses such as : toxicity screening of surface waters, ground waters and leachates of solid wastes, routine toxicity screening of drinking waters, water contamination emergencies and in situ follow up of the efficiency of various kinds of detoxification treatments of aquatic and terrestrial environments

ASSETS OF THE TOXI-SCREENING MICROBIOTEST

1. The Toxi-Screening test is a “single shot” bioassay on non-diluted waters without preparation of dilution series.

2. Conventional bacterial bioluminescence inhibition tests are mostly “laboratory based” and require expensive equipment and materials.

3. The Toxi-Screening microbiotest in turn can be performed anytime and anywhere in the field with the small box containing the reagents and the small lightweight Luminescence Measurement Case.

3. The manipulations are extremely simple and the whole procedure only takes one hour.

4. The tests can be performed at ambient temperature within the range 15 °C to 25 °C.

6. Toxi-Screening microbiotests can be applied to freshwaters as well as to estuarine and marine waters.

FEATURES

The Toxi-Screening Kit is composed of one box containing 10 vials of freeze-dried Vibrio fischeri luminescent bacteria, and of 10 boxes containing tubes and reagents, each allowing 2 assays on (one or two) water samples. The ”Luminescence Measurements Case” contains the portable luminometer and various items for the preparation of the tests. The Luminescence Measurements Case also contains the materials for performance of Bacterial Contamination Screening tests.

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SHELF LIFE

The vials with the freeze-dried bacteria must be stored deep frozen at -18°C prior to use. The other reagents can be stored at ambient temperature. If stored properly the bacteria and the reagents have a shelf life of 6 months to 1 year.

SENSITIVITY AND DETECTION THRESHOLD

The change in bioluminescence upon toxic stress is “chemical dependent” and the sensitivity of the assay hence ranges from very high (a few ppb) for many toxicants to much less (ppm) for others. The sensitivity of the assay is furthermore also dependent of the chemical composition of the water, i.e. the amount of dissolved salts. As a general rule it appears that the higher the conductivity and the hardness of the water, the lower the sensitivity of the assay for toxic chemicals. It is therefore strongly advised to make in parallel with the Toxi-Screening test a measurement of the conductivity and/or the hardness of the analysed water samples. Both former facts must therefore always be taken into consideration for a meaningful evaluation of the findings !

SPECIFICITY

Like any other bioassay the Toxi-Screening microbiotest does not give any information about the “chemical nature” of the toxicant(s) present in the analysed water. Yet this microbiotest is a very simple and practical tool to rapidly determine the presence of toxic compound(s) in water samples in field conditions, from which decisions can immediately be drawn about the suitability of the concerned water for specific uses, and/or the need for treatment.

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CONTENTS OF THE TOXI-SCREENING KIT Box with freeze-dried bacteria One small cardboard box containing 10 vials of freeze-dried Vibrio fischeri bioluminescent bacteria. Boxes with tubes and reagents Ten small cardboard boxes containing tubes already prepared with reagents and 3 finntips for transfer of the reagents and the water samples. Results sheets 10 sheets for scoring of the bioluminescence data of 2 water samples. Standard Operational Procedure manual A detailed brochure with all the instructions and illustrations for performance of the microbiotest. Bench protocol An abbreviated version of the Standard Operational Procedure manual. Specification sheet A sheet indicating the batch number of the vials with freeze-dried bacteria and of the tubes with reagents.

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CONTENTS OF THE LUMINESCENCE

MEASUREMENT CASE

(see Figure 1) The Luminescence Measurements Case contains the portable luminometer and a number of specific materials for performance of the Toxi-Screening microbiotests. The case also contains various items for performance of “Bacterial Contamination Screening” microbiotests. 1. Portable luminometer Small and lightweight luminometer (20 x 8 x 5 cm; 0.3 kg) with digital display, for direct scoring of the bioluminescence in Relative Light Units (RLU). The luminometer is a Lumitester PD-10 instrument from Kikkoman Corporation, and is provided with 2 AA (or R6) alkaline batteries, for use in the field. The luminometer can be connected to the electrical mains with an AC connector and to a printer or a computer with an RS-232C connector. 2. Holes for the sample tubes and the reagent tube The holes in the foam insert layer of the Luminescence Measurement Case allow to put and keep the tubes in a vertical position during the manipulations. 3. Syringe A 10 ml synthetic syringe with Luer fitting (only needed for Bacterial Contamination Screening tests). 4. Box with membrane filters A small box containing small membrane filters (only needed for Bacterial Contamination Screening tests). 5. Mini-membrane filter holder A two-piece holder in inert plastic with Luer fitting (only needed for Bacterial Contamination Screening tests).

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Figure 1 : Contents of the Luminescence Measurement Case 6. Pincette A pair of plastic tweezers (only needed for Bacterial Contamination Screening tests). 7. Cleaning stick A long metal stick provided at the tip with a small piece of foam, to clean the measurement compartment of the luminometer in case of spilling of the reagent. 8. Holders of dipstick units Two plastic holders to fit to the tubes with the control water and the water sample for the bioluminescence measurements in the luminometer. 9. Finpipette 1000 µl A Finpipette for the transfer of 1 ml Osmotic Adjustment medium to the vial with freeze-dried bacteria, and for the transfer of 1 ml water sample into the corresponding tube. 10. Finpipette 200 µl A Finpipette for the transfer of 0.2 ml hydrated bacteria into the tubes with control water and sample water. 11. Timer A small timer with digital display to set the 30 minutes hydration time of the freeze-dried bacteria and the 30 minutes incubation time of the test.

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TEST PROCEDURE Toxi-Screening tests can be performed in the field at any selected time. One vial of freeze-dried bacteria and one box with tubes and finntips allow for toxicity screening of two water samples. The analyses must, however, be performed within a few hours after rehydration of the freeze-dried bacteria.

TRANSFER OF THE MATERIALS TO THE LUMINESCENT

MEASUREMENT CASE

On arrival in the field, open one box “Unit for 2 water samples” and place all the items in the holes located in the upper left corner of the Luminescence Measurement Case (Figure 2) : a) the Control 1 tube and Sample 1 tube next to each other in the 2 holes

on the top b) the Control 2 tube and Sample 2 tube in the 2 holes just below the

former holes c) the tube with “Osmotic Adjustment Medium” in the left hole in the 3rd

row. d) the 3 finntips in the little holes just underneath the 3rd row. Put the vial with freeze-dried bacteria in the (last and largest) hole located to the right of the tube with Osmotic Adjustment Medium.

REHYDRATION OF THE FREEZE-DRIED BACTERIA

1. Put one 1 ml finntip on the fitting of the 1 ml Finnpipette. 2. With the aid of the Finnpipette, transfer the total contents of the

Osmotic Adjustment Medium tube into the vial with bacteria (Figure 3). 3. Close the vial with bacteria, shake the contents thoroughly and put the

vial back into its place in the Luminescence Measurement Case. 4. Set the timer to 30 minutes.

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Figure 2

Figure 3

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SAMPLING

N.B. : An analysis can be made either on two separate water samples, or

alternatively the assay can be performed “in duplicate” on only one water sample to check the precision of the first measurement.

1. Put the second 1 ml finntip on the fitting of the 1 ml Finnpipette. 2. Take a 1 ml water sample with the Finnpipette and transfer it into the

Sample 1 tube (Figure 4). 3. Take again a 1 ml water sample (either from the same water, or from a

different water - in the latter case after rinsing the finntip) and transfer it into the Sample 2 tube.

N.B. : The Sample tubes already contain a (very small) volume of liquid to

adjust the osmotic pressure of the water sample to the appropriate value for the (marine) Vibrio fischeri bacteria.

TRANSFER OF THE LUMINESCENT BACTERIA IN THE SAMPLE

TUBES AND THE CONTROL TUBES

1. Put the 200 µl finntip on the fitting of the 200 µl Finnpipette. 2. After 30 min rehydration, shake the vial with the bacteria and transfer

200 µl of the bacterial suspension in the two Sample tubes and the two Control tubes (Figure 5).

N.B. : The Control tubes already contain the right volume of non-toxic

water already adjusted to the appropriate osmotic pressure for the bacteria.

3. Close the tubes, shake the contents thoroughly and put the tubes back

into their respective holes in the Luminescence Measurement Case. 4. Set the timer again to 30 minutes. MEASUREMENT AT TIME t0

1. Take the luminometer and switch it on by pushing the “Power” button.

Wait till the instrument has calibrated itself by a 10 seconds countdown, visible on the display.

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Figure 4

Figure 5

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2. Remove the caps from Sample 1 and Control 1 tubes and adjust each tube tightly to one of the 2 holders included in the Luminescence Measurement Case (Figure 6).

3. Open the lid of the luminometer and insert Control tube 1 in its holder

(Figure 7). 4. Close the luminometer (Figure 8). N.B.: The luminometer must always be kept vertically when containing a

tube in its holder ! 5. Push the “Enter” button of the luminometer and wait for the 10 seconds

countdown prior to see the luminescence score on the display (Figure 9).

6. Score the luminescence (in RLU) on the Results Sheet. 7. Take the tube and the holder out of the luminometer. 8. Separate the tube from the holder, put the cap back on the tube and

put the tube back into its original place in the Luminescence Measurement Case.

N.B.: When a tube is kept for more than 30 seconds in the luminometer,

the instrument will give beep signals indicating that the tube must be removed !

9. Repeat the former operation with Sample tube 1 and score the result. 10. Repeat the same operations with Control tube 2 and Sample tube 2.

MEASUREMENT AT TIME t30

After 30 minutes exposure of the luminescent bacteria to the 2 control waters and the 2 water samples, repeat all the operations indicated above for the measurements at t0, for a second series of measurements at t30 minutes. All the tubes must be shaken again prior to the measurements.

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Figure 6 Figure 7

Figure 8 Figure 9

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DATA EVALUATION

During the 30 minutes exposure time, the intensity of the bacterial luminescence will normally decrease, so the t30 RLU figure will be lower than the t0 figure. In case of toxic stress, the decrease in luminescence in the tubes with the water sample will be more pronounced than that in the control water. The degree of toxic stress can be calculated as the ratio of the magnitude of the luminescence decrease in the water sample, versus that in the control water. The percentage toxicity of the water sample can be calculated with the formula :

% toxicity = (RLU at t0 – RLU at t30) sample x 10 (RLU at t0 – RLU at t30) control

It should be emphasized, as already indicated above, that both the chemical nature of the toxicant(s) and the interfering influences due to the (different) chemical composition of the analysed water versus that of the control will determine the detection threshold and the magnitude of the toxic signal. The former two features of the Toxi-Screening microbiotest must therefore always be taken into consideration for the interpretation of the results. As a general rule of thumb, samples with a toxicity percentage of 20-25% clearly signal the presence of toxic compound(s) which should trigger further attention.

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TOXI-SCREENING KIT

RESULTS SHEET

All bioluminescence scorings are expressed in Relative Light Units (RLU)

Name of operator : ………………………………………………….

Date of test performance : ………………………………………….

SAMPLE 1

Sampling site : ………………………………………………………

RLU at t0 RLU at t30

Control

Sample

A = Luminescence decrease in control

= (RLU at t0 – RLU at t30)

B = Luminescence decrease in sample

= (RLU at t0 – RLU at t30)

% toxicity = (B/A) x 10

SAMPLE 2

Sampling site : ………………………………………………………

RLU at t0 RLU at t30

Control

Sample

A = Luminescence decrease in control

= (RLU at t0 – RLU at t30)

B = Luminescence decrease in sample

= (RLU at t0 – RLU at t30)

% toxicity = (B/A) x 10

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MANUFACTURED BY :

MicroBioTests Inc.

Kleimoer 15 9030 Mariakerke (Gent)

Belgium www.microbiotests.be