Rapid Testing of Toxic Chemicals

The measurement of chlorophyll fluorescent signals from photosynthetic enzyme complexes (PEC) has become one of the most powerful indicators for ecophysiologists. PECs have been used for more than three decades in research laboratories to determine the toxicological impact of a vast array of chemicals.1–11 Photosynthesis is the absorption of light energy and its conversion into stable chemical potential in chloroplasts. The major part of the absorbed light energy is used to drive photochemical reactions (redox complexes in an electron transport chain). However, a part of this absorbed light energy is emitted in the form of fluorescence and nonradiative energy dissipation. The presence of electron transport inhibitors (see Figure 1 for inhibition sites) can modify the balance between those energetic processes promoting the dissipation process. Based on chlorophyll fluorescence emission by PECs, the toxicity of a sample is indicated by the modification of fluorescence parameters. This plant bioassay was seen to rapidly evaluate the toxicity of an effluent, thus proving the efficacy of the treatment.

Figure 1 - Representation of PEC with inhibition sites.

This widely accepted technique is the basis of an instrument and consumables, the LuminoTox (Lab-Bell Inc., Québec, Canada).12 The test kit is supplied in two parts: 1) a portable, robust, and compact instrument, and 2) the consumables, the PECs. The PECs have been stabilized following a proprietary technique to ensure a commercially acceptable shelf life. The test takes only 15 min to perform, allowing rapid screening of samples to be selected for further chemical and/or bioassay testing. The system is automated in order to provide on-line monitoring of accidental or intentional toxic discharges in the water supply or other environmental infrastructure.

This paper presents the principle supporting the LuminoTox rapid toxicity screening device. It proposes a simple yet effective method for selecting positive samples as opposed to time-consuming and expensive chemical and/or bioassay testing methods. Results obtained from effluents before and after treatment of industrial wastewater plants (pulp and paper mills and mining industries), municipal wastewater plants, and landfill sites are presented. They demonstrate the system’s ability to assess the efficacy of treatment.

Principle of operation

The LuminoTox measurement uses a fluorescence dissipation process emitted by PSII complexes. The variable fluorescence of chlorophyll a is considered an indicator of electron transport efficiency since it is mostly related to the redox state of the PSII electron acceptor plastoquinone A (QA).5,10

To evaluate the photochemical quantum yield (ϕP)—also called photosynthetic efficiency—by fluorescence measurements, the PECs need to be dark adapted. Two levels of fluorescence are required. The first level (F1) is obtained after the application of a low-intensity light in order to determine the fluorescence of chlorophyll molecules that absorb light in PEC. If this light intensity is too weak to drive the photosynthesis process, the entire PECs will be oxidized or in an “open state.” The second level (F2) is reached following the application of a high-intensity light pulse. The saturating pulse will induce the closure of all enzyme complexes (reduced state).

In the LuminoTox, light intensities have been chosen to provide partial oxidation and reduction of PEC. The photochemical quantum yield (ϕP) is evaluated as follows:3,4

In the LuminoTox, the relative photochemical quantum yield, named the relative efficiency (eff), is evaluated in order to obtain better sensitivity.

F2 (blank) is referred to as the fluorescence value obtained in the control solution.

Materials and method

Apparatus

LuminoTox dedicated fluorometers were used. The test does not work with conventional fluorometers, but specialized fluorescence photosynthetic measurement apparatus may be used. No calibration is required. The LuminoTox apparatus is calibrated upon manufacture.

Reagents

The following reagents were employed:

  • PEC–photosynthetic enzyme complexes
  • Reaction buffer
  • Organic standards, atrazine in water (three concentrations)
  • Inorganic standards, copper in water (three concentrations).

Preparation of PEC

The reaction buffer was added to the PEC and mixed vigorously until all the complexes were totally solubilized. A period of 15 min had to elapse before using the enzymes. The solution was mixed occasionally during the waiting time. It is important to agitate before each measurement.

Procedure

Figure 2 - Test procedure.

A user-friendly method has been developed that can be easily deployed in the field (Lab-Bell). The procedure requires only standard laboratory supplies such as disposable cuvettes and syringes (Figure 2).

Results and discussion

Figure 3 - Results obtained from wastewater samples before and after treatment.

Toxicity control is a major driving force in the follow-up of urban wastewater treatment directives. Most biological treatment plants that accept industrial wastewater experience problems from time to time with toxic substances. This problem is usually due to the presence of toxic chemicals in the incoming waste stream. A fast, reliable test was needed to measure the toxicity of industrial wastewater and to evaluate the efficiency of the treatment. The LuminoTox is not only useful for urban wastewater treatment plants, but also has the potential to help industries eliminate chemicals used or produced. The results shown in Figure 3 are from assays performed in industrial (pulp and paper mills and mining industries) and municipal wastewater treatment plants. In 15 min, IC50 can be evaluated from recommended sample concentrations of 100, 50, 25, 12.5, 6.25, and 3.125%.

The tests demonstrate the ability of the LuminoTox device to provide a rapid screening and monitoring test for toxicity detection of a broad spectra of molecules found in industrial or municipal effluents. It offers a fast, on-site response to the efficacy of a treatment. Standard chemical testing and bioassays require costly equipment and normally allow access to data only after several days. For example, a bioassay on trout takes a minimum of 72 hr of testing. The LuminoTox technology offers a simple yet effective method for selecting positive samples. By slightly increasing the incubation time, it is also possible to increase the test’s sensitivity to detect very low concentrations of certain toxic compounds.

Scientific literature, as well as a wide range of validation tests on different sources of toxic effluents and runoff water samples, have proven the ability of PECs to react to a vast array of chemical elements and molecules.1–11

Another critical application of this technology is water safety. The LuminoTox system is in the process of being automated in order to provide on-line monitoring of accidental or intentional toxic discharges into the water supply or other environmental infrastructures. In light of the ongoing national focus on security, the technology provides utilieffties with the necessary tools to continue to protect public health and meet new challenges.

Warning

A few cautionary words are in order. Because the test is sensitive to pH, light, and temperature, it is very important to treat the blank, samples, and standards in exactly the same manner. The test should be performed at a pH between 6.5 and 7.8 and at temperatures between 4 and 25 °C. Better results are obtained when the blank, samples, and standards are kept in the same conditions, for example, at pH 7.8 at 20 °C.

The consumables, or PECs, should be stored in a freezer. PECs are stable for six months at –20 °C and for two months at 4 °C in the freeze-dried form, as specified by the manufacturer. Once solubilized, the PECs are stable for 24 hr at 4 °C or for 5 hr at 20 °C. The PECs can be frozen once solubilized. It is important to protect the solubilized PECs from light at all times. If exposed to light, regeneration is achieved after 15 min in the dark.

References

  1. Cedeno-Maldonado A, Swader JA, Heath RL. The copper ion as an inhibitor of photosynthetic electron transport in isolated chloroplasts. Plant Physiol 1972; 50:698–701.
  2. de Filippis LF, Hampp R, Ziegler H. The effects of sublethal concentrations of zinc, cadmium and mercury on Euglana. Arch Microbiol 1981; 128:407–11.
  3. Genty B, Briantais JM, Baker NR. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 1989; 990:87–92.
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  5. Light emission by plants and bacteria. In: Govindjee, Amesz J, Fork DJ, eds. Orlando, FL: Academic Press Inc., Harcourt Brace Jovanich, 1986:638.
  6. Koblizek M, Maly J, Masojidek J. A biosensor for the detection of triazine and phenylurea herbicides designed using Photosystem II coupled to a screen-printed electrode. Biotechnol Bioeng 2002; 78(1):110–16.
  7. Laberge D, Chartrand J, Rouillon R, Carpentier R. In vitro phytotoxicity screening test using immobilized spinach thylakoid. Env Toxicol Chem 1999; 18:2851–8.
  8. Laberge D, Rouillon R, Carpentier R. Comparative study of thylakoid membranes sensitivity for herbicide detection after physical or chemical immobilization. Enzyme Microb Technol 2000; 26:332–6.
  9. Schreiber U, Bilger W. Rapid assessment of stress effects on plant leaves by chlorophyll fluorescence measurements. In: Tenhunen JD, ed. Plant response to stress. Berlin, Heidelberg: Springer-Verlag, 1987:27–53.
  10. Krause GH, Weis E. Chlorophyll fluorescence and photosynthesis: the basis. Ann Rev Plant Physiol 1991; 42:313–49.
  11. Boucher N, Carpentier R. Hg2+, Cu2+ and Pb2+ induced changes in Photosystem II photochemical yield and energy storage in isolated thylakoid membranes: a study using simultaneous fluorescence and photoacoustic measurements. Photosynth Res 1999; 59:164–74.
  12. Bellemare F, Boucher N, Lorrain L. Method of testing photosynthetic activities, 2001, WO 2004/046717.

Dr. Boucher, Dr. Lorrain, Ms. Rouette, Ms. Perron, and Dr. Bellemare are with Lab-Bell Inc., 2263 Ave. du Collège, Shawinigan, Québec, Canada; tel.: 819-539-8508, ext. 107; fax: 819-539-8880; e-mail: [email protected]. Ms. Déziel is with Centre National en Electrochimie et Technologies Environnementales (CNETE), and Dr. Tessier is with the Biology Dept., Collège Shawinigan, Québec, Canada.

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