JWBK117-1.4 JWBK117-Quevauviller October 10, 2006 20:11 Char Count= 0 62 Alternative Methods biomarkers, whole-organism tests and biological early warning systems for bio- logical monitoring (Allan et al., 2006). These tools, many being under validation, even if they are commercially available, are actually designed for water bodies mon- itoring and very few for wastewater. However, considering their complementary nature with reference and other alternative methods, there are several new methods for biological monitoring. Further developments will be devoted to direct applica- tion to wastewater quality. Meanwhile, a lot of emerging tools can already be used for discharge toxicity monitoring, such as bioassays and biological early warning systems (see Chapter 5.1). Other emerging tools designed for chemical monitoring are passive samplers, immersed in a stream, for the selective adsorption and con- centration of micropollutants. A recent review (Vrana et al., 2005) has pointed out the huge development of this approach for water quality monitoring. Even if only a few applications exist for wastewater quality monitoring with analysis of polar organic compounds (Alvarez et al., 2005) or trace metals and organic micropollu- tants (Petty et al., 2004), the use of passive samplers appears to be a very promising technique, even if the calibration is difficult as it is strongly dependent on the com- position of water. This is the reason why applications deal with wastewater discharge impact. 1.4.4 COMPARABILITY OF RESULTS The purpose of this section is not to give anexhaustive overview of the tools for qual- ity control and assurance for water quality (the reader will find complete information in Quevauviller, 2002), but rather to stress a simple procedure to check the compa- rability of results between a reference method and an alternative one (candidate for being recognised as an equivalent method). There exist very few standards for the purpose. The French experimental stan- dard (AFNOR XP T90-210, 1999) on the evaluation protocol of a physico-chemical quantitative analysis (for water analysis) regarding a reference method, defines some principles and tools for the comparability of methods. Considering the complexity of the problem, this standard is still experimental, and discussions still exist. How- ever, the principles of this standard have been chosen for the evaluation procedure for comparing two methods intended for the detection or quantification of the same target group or species of microorganisms (ISO 17994, 2004). ISO 17994 provides the mathematical basis for the evaluation of the average relative performance of two (quantitative) methods against chosen criteria of equivalence. Another international standard (ISO 11726, 2004) describes procedures for validating alternative (quan- titative) methods of analysis for coal and coke either directly by comparison with the relevant international standard method or indirectly by comparison with refer- ence materials that have been exhaustively analysed using the relevant international standard method. JWBK117-1.4 JWBK117-Quevauviller October 10, 2006 20:11 Char Count= 0 Comparability of Results 63 Reference method (x) Alternative method (y) Theoretical line y = x Experimental line y = a.x + b 0 Figure 1.4.2 Comparison between reference and alternative methods The principles of comparison are simple and schematically based on two steps: r The first step aims to calculate the analytical characteristics of the two methods (reference and alternative), including the reproducibility for a given value (from a standard solution). A first comparison is carried out on the average values, from a Fisher–Snedecor test. If the test is conclusive (if the two values are not statistically different), the second step can be performed. r Then, the equivalence betweenmethods must be statistically verifiedby plotting the results (Figure 1.4.2) and checking the coordinates of the experimental regression line [comparison of the slope and intercept values which must be not statistically different from, respectively, 1 and 0, values of the theoretical line (y = x)]. For the purpose a Student test is carried out. An exampleisgiven in Table1.4.1,showingtheresultsoftheStudenttestofa compar- ison from realurbanand industrial wastewater (grab samples)for the measurement of total Kjeldahl nitrogen (TKN). Reference and alternative methods are, respectively, standard NF EN 25663 and UV/UV procedure (Roig et al., 1999) for TKN. The re- gression line between the estimated (by the alternative method) and measured values (by the reference method) is: TKN est = 0.96 TKN ref + 0.86 (R 2 = 0.98). The re- sults obtained from the comparison of the slope and intercept values to, respectively, 1 and 0, show that the alternative method can be considered as equivalent. In fact, the scientific decision must be determined by other considerations, such as the improvement of the alternative method if it brings some consistency advantages regarding the reference methods (very cheap, rapid, etc.), and the acceptability of the procedure (Figure 1.4.3). Once the equivalence between methods is confirmed, the validation procedure results given for on-/off-line instruments (permanent measurement) must be com- pleted, taking into account the sampling procedure is different for a laboratory method and a permanent measurement. For example, considering that regulation JWBK117-1.4 JWBK117-Quevauviller October 10, 2006 20:11 Char Count= 0 64 Alternative Methods Table 1.4.1 Results of Student test (95 % confidence interval) for TKN measurement by UV (method described in Roig et al., 1999) Student test Values Slope δ 0.9643 Y intercept γ 0.8609 S δ 0.02 S γ 0.63 Degree of freedom 55 t 0.975 2.01 δ − t 0.975 * S δ 0.92 δ + t 0.975 * S δ 1.0045 δ − t 0.975 * S δ < 1 <δ+ t 0.975 * S δ 0.92 < 1 < 1.0045 γ − t 0.975 * S γ −0.405 γ + t 0.975 * S γ 2.127 γ − t 0.975 * S γ < 0 <γ +t 0.975 * S γ −0.405 < 0 < 2.127 constraints require 24 hcompositesampling before laboratory analysis, the challenge is to obtain equivalent results with this procedure and with permanent measurement. In this case, the results to be compared are the mean values for each measurement during the permanent acquisition, with the reference value of the corresponding composite sample (Thomas and Pouet, 2005). Proposal for alternative method Characterisation of standard and alternative methods Test of comparability (reliability) Proposal for alternative method Comparable? Y N Optimisation of method Validation (method) Abandon Improvement? Y N Use method Seek acceptance Relevance? Y N Validation Figure 1.4.3 Validation procedure of a candidate alternative (equivalent) method (adapted from Bruner et al., 1997) JWBK117-1.4 JWBK117-Quevauviller October 10, 2006 20:11 Char Count= 0 References 65 Finally, the international standards already cited (ISO 17381, 2003; ISO 15839, 2003) should be considered for the general evaluation of ready-to-use test kits methods and on-line systems. Other procedures can also be cited (Battelle, 2002, 2004), including works in progress in the frame of the European project Swift-WFD (www.swift-wfd.com). REFERENCES AFNOR XP T90-210 (1999) Qualit´e de l’eau – Protocole d’´evaluation d’une m´ethode alternative d’analyse physico-chimique quantitative par rapport `a une m´ethode de r´ef´erence. Allan, I.J., Vrana, B., Greenwood, R., Mills, G.A., Roig, B. and Gonzalez, C. (2006) Talanta, 69, 302–322. Alvarez, D.A., Stackelberg, P.E., Petty, J.D., Huckins, J.N., Furlong, E.T., Zaugg, S.D. and Meyer, M.T. (2005) Chemosphere, 61, 610–622. Battelle (2002) Generic verification protocol for long-term deployment of multiparameter water quality probes/sondes. http://www.epa.gov/etv/pdfs/vp/01 vp probes.pdf. Battelle (2004) Generic verification protocol for portable technology for detecting cyanide in water. http://www.epa.gov/etv/pdfs/vp/01 vp cyanide.pdf. Baur`es, E. (2002) La mesure non param´etrique, un nouvel outil pour l’´etude des effluents indus- triels: application aux eaux r´esiduaires d’une raffinerie. PhD thesis, Universit´e Aix Marseille III, France. Bonastre, A., Ors, R., Capella, J.V., Fabra, M.J. and Peris, M. (2005) Trends Anal. Chem., 24(2), 128–137. Bourgeois, W., Burgess, J.E. and Stuetz, R.M. (2001) J. Chem. Technol Biotechnol., 76, 337–348. Bruner, L.H., Carr, G.J., Curren, R.G. and Chamberlain, M. (1997) Comm. Toxicol., 6, 37–51. Castillo, L., El Khorassani, H., Trebuchon, P. and Thomas, O. (1999) Water Sci. Technol., 39(10– 11), 17–23. Dworak, T., Gonzalez, C., Laaser, C. and Interwies, E. (2005) Environ. Sci. Pol., 8, 301–306. European Commission (1991) Council Directive of 21 May 1991 concerning urban wastewater treatment (91/271/EEC). European Commission (2000) Council Directive of 23 October 2000 establishing a framework for Community action in the field of water policy (2000/60/EC). Greenwood, R., Roig, B. and Allan, I.J. (2004) Draft report: operational manual, overview of existing screening methods (available at: http://www.swift-wfd.com). ISO 5664 (1984) Water quality – Determination of ammonium – Distillation and titration method. ISO 6778 (1984) Water quality – Determination of ammonium – Potentiometric method. ISO 7150-1 (1984) Water quality – Determination of ammonium – Part 1: Manual spectrometric method. ISO 7150-2(1986)Water quality –Determinationofammonium– Part 2: Automatedspectrometric method. ISO 11732 (1997) Water quality – Determination of ammonium nitrogen by flow analysis (CFA and FIA) and spectrometric detection. ISO 11348-3 (1998) Water quality – Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteriatest) – Part 3: Method using freeze-dried bacteria. ISO 15839 (2003) Water quality – On line sensors/analysing equipment for water: specifications and performance tests. JWBK117-1.4 JWBK117-Quevauviller October 10, 2006 20:11 Char Count= 0 66 Alternative Methods ISO 17381 (2003) Water quality – Selection and application of ready-to-use test kit methods in water analysis. ISO 11726 (2004) Solid mineral fuels – Guidelines for the validation of alternative methods of analysis. ISO 17994 (2004) Water quality – Criteria for establishing equivalence between microbiological methods. Muret, C., Pouet, M.F., Touraud, E. and Thomas, O. (2000) Water Sci. Technol., 42(5–6), 47–52. Oliveira-Esquerre, K.P., Seborg, D.E., Bruns, R.E. and Mori, M. (2004a) Chem. Engin. J., 104, 73–81. Oliveira-Esquerre, K.P., Seborg, D.E., Mori, M. and Bruns, R.E. (2004b) Chem. Engin. J., 105, 61–69. Petty, J.D., Huckins, J.N., Alvarez, D.A., Brumbaugh, W.G., Cranor, W.L., Gale, R.W., Rastall, A.C., Jones-Lepp, T.L., Leiker, T.J, Rostad, C.E. and Furlong, E.T. (2004) Chemosphere, 54, 695–705. Quevauviller, Ph. (2002) Quality Assurance for Water Analysis. Water Quality Measurements Series. John Wiley & Sons Ltd, Chichester. Roig, B., Gonzalez, C. and Thomas, O. (1999) Anal. Chim. Acta, 389, 267–274. Sperandio, M. and Queinnec, I. (2004) Water Sci. Technol., 49(1), 31–38. Thomas, O. (1995) M´etrologie des eaux r´esiduaires. Tec et Doc: Paris; Cebedoc: Li`ege. Thomas, O. and Constant, D. (2004) Water Sci. Technol., 49(1), 1–8. Thomas, O. and Pouet, M F. (2005) Wastewater quality monitoring: on-line/on-site measurement. In: The Handbook of Environmental Chemistry, 5, part O, Barcelo, D., (Ed.). Springer-Verlag: Berlin, pp. 245–272. Thomas, O., El Khorassani, H., Touraud, E. and Bitar, H. (1999) Talanta, 50, 743–749. Vanrollegem, P.A. and Lee, D.S. (2003) Water Sci. Technol., 47(2), 1–34. Vrana, B., Mills, G.A., Allan, I.J., Dominiak, E., Svensson, K., Knutsson, J., Morrison, G. and Greenwood, R. (2005) Trends Anal. Chem., 24(10), 845–868. JWBK117-1.5 JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 1.5 Biosensors and Biological Monitoring for Assessing Water Quality Carmen Rebollo, Juan Azc´arate and Yolanda Madrid 1.5.1 Introduction 1.5.2 Biosensors 1.5.2.1 Definition and Classification 1.5.2.2 Environmental Applications of Biosensors 1.5.3 Biological Monitoring 1.5.3.1 Microbiological Contamination 1.5.3.2 Algae Monitoring 1.5.4 Future Trends References 1.5.1 INTRODUCTION The implementation of wastewater treatment procedure (WWTP), including sew- erage systems, WWTP and effluent quality control and potential reuse, and the control of environmental impacts on the receiving waters imply the availability of a considerable amount of analytical data in order to facilitate the management of water resources and the decision-making processes. Wastewater Quality Monitoring and Treatment Edited by P. Quevauviller, O. Thomas and A. van der Beken C  2006 John Wiley & Sons, Ltd. ISBN: 0-471-49929-3 JWBK117-1.5 JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 68 Biosensors and Biological Monitoring for Assessing Water Quality These needs are derived basically from the three following points: r Normative requirements. Within the EU environmental policy, the Water Frame- work Directive is likely to cause an important change regarding water quality monitoring. Additionally, other European Directives have been developed, con- cerning protection of water against the harmful effects of particular substances, the quality of water dedicated to different uses and the obligation of wastewater treatment to achieve a degree of performance and effluent quality. This quality control has to be carried out as analytical measurements. r Operation and maintenance needs (O&M). In WWTP and sewerage, analytical data are essential for the monitoring process, detecting changes in the process, fol- lowing the processevolution, betterunderstandingthe process and forperformance evaluation. The monitoring of raw water is also needed as an alarm system to pro- tect biological processes, during water-clean up, which could be easily damaged by uncontrolled industrial discharges. r Research and development (R&D). The increasing use of mathematical models for designing and operation of sewer networks and WWTP demands also lots of raw analytical data in order to validate the model itself for a specific site. For research purposes in the environmental field, to assess the aquatic ecosystem status, etc., analytical data are also important. In order to satisfy these needs on a permanent basis, treatment plant managers, envi- ronmental authorities as well as consumers and polluters require the implementation of rapid and accurate analytical measuring techniques. On-line systems, such as sen- sors, biosensors and other analytical tools in continuous or sequential mode, offer as main advantages faster response, lower cost and easier automatization compared with classical laboratory methodologies. Besides, on-line monitoring provides more detailed information than that obtained from composite samples, because it takes into consideration time-dependent variations. However, on-line methods have limitations. Although they are normally rapid and inexpensive, currently only a narrow range of parameters can be measured automati- cally, satisfying the required quality and sensitivity criteria within a reasonable cost. Thus, it is not always possible to carry out continuous monitoring of the required analytes (direct parameters), and often it is necessary to use indirect parameters, correlated to the former or even global pollution indicators. In addition, accuracy and reliability are often lower than laboratory methods. In most cases, a combination of field analysis, laboratory analysis and on-line monitoring is the best choice. Within the broad range of on-line monitoring devices, special reference should be made to biosensorsconsidering recent advances intechnology and applicationsto the environmental field. A wide range of applications have been described in the litera- ture, both as screening techniques and for the determination of specific compounds. Despite this variations in biosensor-related methods, a common definition could be JWBK117-1.5 JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 Biosensors 69 Table 1.5.1 Main biosensor applications in the monitoring of wastewater systems Area Objective Measured parameter Sewer system Pollution load BOD, biodegradability Industrial discharges Pesticides, phenols, heavy metals, solvents, toxicity Wastewater treatment plants Alarm systems Toxicity Process control BOD, O 2 consumption Environmental monitoring Effluent quality/effluent reuse Microbiological pollution, BOD Aquatic ecosystem evolution Chlorophyll, global chemical parameters ‘an analytical device composed of a biological recognition element directly inter- faced to a signal transducer, which together relate the concentration of an analyte or group of relatedanalytes to a measurable response’ (Allan et al., 2006). The different types of biosensor and the classification criteria will be discussed below. The term biosensor, in a wide sense, could include not only the determination of chemical species but also the determination of biological populations through the changes of chemical or physical properties. This type of on-line technique is referred to within the text as biological monitoring. The main potential applications in which biosensors could offerspecialadvantages are listed in Table 1.5.1. 1.5.2 BIOSENSORS 1.5.2.1 Definition and Classification Despite the wide variation in biosensors and biosensor-related techniques that have been introduced, the widely accepted definition for these devices remained fairly constant. A biosensor can be described as an analytical device composed of a bio- logically active material directly interfaced to a signal transducer. Biosensors for environmental applications have employed a wide variety of bi- ological recognition systems (isolated enzymes, intact bacterial cells, mammalian and plant tissue, antibody and bioreceptor proteins) coupled to a similarly wide range of signal transducers (Allan et al., 2006). In a broad sense, biosensors can be divided into three categories according to the biological recognition mechanism: biocatalytic-, bioaffinity- and microbe-based systems. These biological recognition systems have been linked to electrochemical, optical and acoustic transducers. The biocatalytic-based biosensors for environmental applications are based on the use of enzymes that can act following two operational mechanisms. The first one involves the catalytic transformation of a pollutant (typically from a nondetectable JWBK117-1.5 JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 70 Biosensors and Biological Monitoring for Assessing Water Quality form to a detectable form). The second mechanism involves the detection of pollu- tants that inhibit or mediate the enzyme activity. Bioaffinity-based biosensors for environmental applications depend on the use of antibodies and antigenes to measure a wide variety of substances ranging from complex viruses and micro-organisms to simple pesticide molecules and industrial pollutants. The key reagents in these types of biosensors are antibodies, which are soluble proteins, produced by the immune system in response to infection by foreign substances (called antigens). The fundamental concept behind immunoassays is that antibodies prepared in animals can recognize and bind with relatively affinity and specificity to the anti- gen that stimulated their production. The binding forces involved in the specific interaction between antibodies (Ab) and antigens (Ag) are of a noncovalent, purely physicochemical nature: hydrogen bonds, ionic bonds, hydrophobic bonds and van der Waals interactions. Since these interactions are weaker that the covalent bonds, an effective Ab–Ag interaction requires the presence of a large number of these interactions and a very close fit between the Ab and Ag. Antibodies are glycoproteins produced by lymphocite B cells, usually in conjunc- tion withT-helper cells, as part of the immune system response toforeign substances. Antibodies (also known as immunoglobulins) are found in the globulin fraction of serum and in tissue fluids and they are able to bind in a highly specific manner to foreign molecules. There are five classes of immunoglobulins: IgG, IgM, IgA, IgD and IgE. The predominant immunoglobulin in serum is IgG which has an ap- proximate molecular weight of 160000 Da. All five classes of immumoglobulins share a common basic structure comprised of two light chains and two heavy chains linked by disulfide bonds and noncovalent forces. The antibody molecule usually is represented as a Y-shaped structure. Immunoassays canbe classified ascompetitive and noncompetitive. Becausemost low-molecular-weight organic pollutants in the environment have distinguishing optical or electrochemical characteristics, the detection of stoichiometric binding of these compounds to antibodies is typically accomplished with the use of competitive binding assay formats. Competitive immunosensors rely on the use of an antigen tracer that competes with the analyte for a fixed and limited number of antibody binding sites. As antigen tracer radioisotopes, enzymes, liposomes, fluorophores or chemiluminescent compounds are commonly used. For affinity-based biosensors, this is typically accomplished in several ways. In one method, the antigen tracer competes with analyte for immobilized antibody binding sites. In another format, the antigen is immobilized to the signal transducer while free binding sites on the antibody, which has been previously exposed to the analyte, bind to the surface-immobilized antigen. The third commonly used format requires an indirect competitive assay and relies on the use of an enzyme-labelled antigen tracer. In this format, the assay is completed in two ways. First, the enzyme tracer competes with the analyte for immobilized antibody binding sites. Then, after removal of the unbound tracer, a nondetectable substrate is catalytically converted to a detectable product. JWBK117-1.5 JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 Biosensors 71 Immunosensors are becoming the most popular type of biosensors for environ- mental applications. Micro-organism-based biosensors for environmental monitoring and toxicity as- sessment use devices with sensitivity over a broad spectrum rather than highly specific ones. As the array of contaminants is wide and the threat unknown, the choice of cellular rather than molecular systems is more suitable. Whole cell biosen- sors probably offer the greatest technological changes among the existing alarm systems. In contrast to the previous biosensors, which exploit only one combination, namely, enzyme/substrate or Ag/Ab, microbial biocatalysts are living cells, i.e. complete organisms with multiple biochemical pathways governed by multiplic- ity of enzymes, which thus offer the greatest potential of investigation. Therefore, microbial sensors share the property of presenting a wide spectrum of response to toxicants with vertebrates and invertebrates. These types of biosensors use three mechanisms. For the first mechanism, the pollutant is a respiratory substrate being mainly ap- plied to the measurement of biological oxygen demand (BOD). Another mechanism used for micro-organism-based-biosensors involves the inhibition of respiration by the analyte of interest. Inthis case, these devices might bemost applicable for general toxicity screening or in situations where the toxic compounds are well defined, or where there is a desire to measure total toxicity. Biosensors have also been developed with the use of genetically engineered micro-organisms (GEMs) that recognize and report the presence of specific environmental pollutants. Biological recognition systems have been linked to several types of transduc- ers: electronic, optical and acoustic. Electronic transduction is the most applied in biosensors being classified in potentiometric, amperometric and conductimetric biosensors. The potentiometric transducers are based on the use of ion-selective membranes that make these devices sensitive to various ions, gases and enzyme sys- tems. The enzymatic modification of ion-selective electrodes by covalent binding of the enzymes to the membrane surface is a common procedure for the development of biosensors with high sensibility, stability and fast response. Most potentiometric biosensors fordetection of environmental pollutants have used enzymes thatcatalyse the consumption or production of protons. Amperometric biosensors typically rely on an enzyme system that catalytically converts electrochemically nonactive analytes into products that can be oxidized or reduced at a working electrode. The electrode is maintained at a specific potential and thecurrent produced is linearly proportional tothe nonelectroactive enzyme sub- strate. The enzymes typically used are oxidases, peroxidases and dehydrogenases. Despite efficient electron transfer from redox enzymes with corresponding electron carrier molecules, few redox enzymes can transfer electrons directly to a metal or semiconductor electrode. Several molecular interfaces that enhance electron transfer from redox enzymes on the electrode surface have been developed. Electron medi- ators such as ferrocene and its derivates and Meldona Blue have been successfully applied into enzyme sensors. [...]... the bioluminescence inhibition of Vibrio fisheri have been frequently used, because it is a well-known organism, well introduced and standardized These tests offer rapid, easy handling and cost effective responses, and a large database for many chemicals is available.As standard ISO 11340 protocols exist for this assay, many commercial devices are available Commercial instruments such as Microtox R... the greater the analytical needs Consequently, the potential application of rapid methods in natural water and wastewater monitoring has raised an increasing interest JWBK117-1.5 78 JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 Biosensors and Biological Monitoring for Assessing Water Quality Depending on the principle on which the techniques are based it is possible to differentiate four... within the instrument A standard amount is rehydrated and mixed with the water sample It is widely used in the laboratory (Araujo et al., 2005) Two bioluminescent inhibition assays from Merck, Toxt Alert 10 and Toxt Alert 100, are also based on the inhibition of V fisheri Toxt Alert 100 is a portable device with no temperature control and uses freeze-dried bacterial reagents and Toxt Alert 100 uses liquid–dried... JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 Biosensors and Biological Monitoring for Assessing Water Quality On-line Biochemical Oxygen Demand analysis The estimation of the organic load is a key parameter in conventional wastewater treatment for assessing the environmental effect (oxygen depletion) caused by a wastewater discharge into a receiving aquatic system One of the parameters used... detecting DNA sequences (Cheng et al., 1998) JWBK117-1.5 80 JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 Biosensors and Biological Monitoring for Assessing Water Quality Their applicability to on-line monitoring is still limited due to problems of isolation and processing the micro-organism of interest in order to amplify the selected DNA sequences before the hybridization step The development... promise for use in environmental monitoring Besides the application in biomonitoring of organisms of hygienic/environmental interest and difficult to determine through conventional laboratory procedures, such as viruses, these biosensors, also known as genosensors, can be useful for the detection of chemically induced DNA damage 1.5.3.2 Algae Monitoring Together with pathogen and indicator micro-organisms,... a wide range of biosensors have been developed for water monitoring, most of the work has been performed at research level and relatively few of these devices have been introduced into commercial markets Incorporation of biosensors and, in general, field methods into environmental measurements reduces problems related to sample transportation and time consumption of the analytical measurement JWBK117-1.5... in-situ monitoring of some parameters including global pollution indicators and single compounds or classes of compounds Most of them are dedicated to the determination of BOD, the direct or indirect measurement of toxicity and, less frequently, to specific compounds such as pesticides, phenols, heavy metals, etc JWBK117-1.5 74 JWBK117-Quevauviller October 10, 2006 20:13 Char Count= 0 Biosensors and Biological... reagent and the incubation takes place at controlled temperature (Farr´ et al., 2002) e Other commercial equipment for toxicity measurements are Eclox (Aztec Environmental & Control Ltd) and Aquanox (Randox Laboratories) These use an enhanced chemiluminescent reaction; a free radical reaction for the oxidation of luminol in presence of horse radish peroxidase enzyme using p-iodophenol as an enhancer and. .. indicating the sample toxicity An amperometric biosensor with E coli for the determination of toxicity in textile and tanneries industry wastewater has been reported (Farr´ , 2001) e Chemical substances detection Biosensors for water monitoring cover a broad range of substances Pesticides and chlorinated compounds are a hardly biodegradable group of pollutants for which numerous sensing schemes have . resources and the decision-making processes. Wastewater Quality Monitoring and Treatment Edited by P. Quevauviller, O. Thomas and A. van der Beken C  2006 John Wiley & Sons, Ltd. ISBN: 0 -47 1 -49 929-3 JWBK117-1.5. Li`ege. Thomas, O. and Constant, D. (20 04) Water Sci. Technol., 49 (1), 1–8. Thomas, O. and Pouet, M F. (2005) Wastewater quality monitoring: on-line/on-site measurement. In: The Handbook of Environmental. Springer-Verlag: Berlin, pp. 245 –272. Thomas, O., El Khorassani, H., Touraud, E. and Bitar, H. (1999) Talanta, 50, 743 – 749 . Vanrollegem, P.A. and Lee, D.S. (2003) Water Sci. Technol., 47 (2), 1– 34. Vrana, B.,