Detection of Herbicide Subclasses by an Optical Multibiosensor Based

Centre de Phytopharmacie, Université de Perpignan, 52. Avenue Paul Alduy, 66860 Perpignan, France. Massive use of herbicides in agriculture over the ...
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Environ. Sci. Technol. 2005, 39, 5378-5384

Detection of Herbicide Subclasses by an Optical Multibiosensor Based on an Array of Photosystem II Mutants M A R I A T E R E S A G I A R D I , * ,† LICIA GUZZELLA,‡ PIERRE EUZET,§ REGIS ROUILLON,§ AND DANIA ESPOSITO† Institute of Crystallography, Department of Molecular Design and Nanotechnology, CNR, Area of Research of Rome, Via Salaria Km 29, 3- Monterotondo Scalo (Rome), Italy, Institute for Environmental Water Analyses, IRSA-CNR, via della Mornera 25, 20047 Brugherio (MI), Italy, and Equipe Biomem, Centre de Phytopharmacie, Universite´ de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan, France

Massive use of herbicides in agriculture over the last few decades has become a serious environmental problem. The residual concentration of these compounds frequently exceeds the maximum admissible concentration in drinking water for human consumption and is a real environmental risk for the aquatic ecosystem. Herbicides inhibiting photosynthesis via targeting photosystem II function still represent the basic means of weed control. A multibiosensor was constructed for detecting herbicides using as biosensing elements photosynthetic preparations coupled to an optical fluorescence transduction system (Giardi et al. EU patent EP1134585, 01830148.1-2204); this paper is about its application in the detection of herbicide subclasses in river water. Photosynthetic material was immobilized on a silicio septum inside a series of flow cells, close to diodes so as to activate photosystem II (PSII) fluorescence. The principle of the detection was based on the fact that herbicides selectively modify PSII fluorescence activity. The multibiosensor has the original feature of being able to distinguish the subclasses of the photosynthetic herbicides by using specific immobilized biomediators isolated from mutated organisms. This setup resulted in a reusable, portable multibiosensor for the detection of herbicide subclasses with a half-life of 54 h for spinach thylakoids and limit of detection of 3 × 10-9 M for herbicides present in river water.

Introduction Photosystem II (PSII) is a light-driven, water-plastoquinone oxidoreductase which catalyzes the most thermodynamically demanding reaction in biology. This highly endergonic reaction splits water into molecular oxygen, protons, and electrons, thereby sustaining an aerobic atmosphere on earth and providing the reducing equivalents necessary to fix carbon dioxide to organic molecules, creating biomass, food, * Corresponding author phone: +39 06 90672704; fax: +39 06 90672630; e-mail: [email protected]. † CNR. ‡ IRSA-CNR. § Universite ´ de Perpignan. 5378

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and fuel (1). PSII with its pigment-protein complexes is embedded in the thylakoid membranes of higher plants, algae, and cyanobacteria, and excitation energy is transferred from the antenna to the core of the PSII complex, where the primary photochemistry takes place (1). The development of biosensors represents a valuable step forward in monitoring pollutant in ecosystems. Biosensors are analytical devices that consist of a biosensing element (enzyme, tissue, living cell) that provides selectivity, and a transducer that transfers the chemical signal to an electrical signal for further processing (2-4). The sensitive core of a biosensor is the biological component on which the peculiarities of the device depend. In our case photosystem II is extremely interesting in the development of a biosensor because of the particular simplicity of the transduction signal, which can be measured immediately without the use of any “marker” or introduction of competitors that would complicate the measuring instruments. The use of PSII in monitoring herbicides has been recommended since 1960 when whole cells of photosynthetic microorganisms were used in laboratories with this goal in mind. However, use of this important biological component on a large scale was limited by its intrinsic instability, which impaired its analytical application. Recently, remarkable progress in the stabilization of the PSII has been achieved, principally due to the fact that it can now be immobilized in several ways (5, 6). The selection of photosynthetic organisms allowed the use of a biomediator, which proved stable over many hours at ambient temperature and under measuring conditions, utilizing a flow cell equipped with a Clark electrode or with printed electrodes (5, 7). This system, however, was complicated because of the volume of equipment involved. Light energy absorbed by chlorophyll molecules in photosystem II is used for the photochemistry of photosynthesis, while energy in excess is dissipated as heat or is re-emitted as longer wavelength red/far-red light energy; this re-emission of light is termed chlorophyll fluorescence. These routes of energy pattern compete with each other in such a way that any increase in the efficiency of one will result in a decrease in the yield of the others. Therefore, by measuring the yield of chlorophyll fluorescence, information about changes in the efficiency of photochemistry and energy dissipation can be obtained (8). The most widely used technique is fluorescence induction, achieved by measuring changes in fluorescence yield when a light is switched on after a dark period; under exciting light, the fluorescence yield rapidly rises and then slowly decreases (8). From an economic point of view, derivatives of urea, triazines, diazines, and phenolic compounds represent classes of highly important compounds in many sectors of the chemical, pharmaceutical, and agricultural industries. They constitute the basic products for the chemical weeding of various crops. They can be applied both in pre- and postemergency crop control, and their mechanism results in the inhibition of electron flow at the level of photosystem II with the consequent interruption of water photolysis and oxygen formation (Hill reaction) and the modification of fluorescence activity as well (9). Wide and prolonged use of certain herbicides, principally triazines, caused selection and consequent diffusion of herbicide-resistant biotypes derived from sensitive infesting species (10). Due to their high toxicity for humans and animals, the indiscriminate use of herbicides results in severe environmental problems; in fact, the problem of pollution due to 10.1021/es040511b CCC: $30.25

 2005 American Chemical Society Published on Web 06/14/2005

herbicides in rivers, lakes, and underground streams is widely known. The European Union, in the “European Water Act of 1980” document, stated that the concentration of herbicides in water must be lower than 0.1 or 0.5 µg/L of any individual or total herbicide class, respectively. Diffuse losses from agriculture fields are a major input source of herbicides in surface waters (11). Therefore, it is obvious that to detect such a low residual concentration extremely sensitive and reliable analytical methods are required. These premises induced us to develop a new multibiosensor able to detect herbicides and herbicide subclasses.

Experimental Section Reagents. Alaclor (2-chloro-2,6-diethyl-N-methoxymethylacetanilide), desethylatrazine (atrazine ) 2-chloro-4-(ethylamino)-6-isopropylamino-1,3,5-triazine), desethylterbutilazine, prometryn (2,4-bis(isopropylamino)-6-methylamino-1,3,5-triazine), and simazine (2-chloro-4,6-bis(ethylamino)-1,3,5-triazine) were purchased from Dr. Ehrenstorfer (Germany) with a certified purity greater than 95%. Tetramethyl-p-benzoquinone (DQ), diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), and atrazine were purchased from Sigma-Aldrich. Mannitol, bovine serum albumin (fraction V), glutaraldehyde, [14C]atrazine, and all other chemicals were purchased from Aldrich. All chemicals were of analytical-reagent grade. Organisms and PSII Membrane Preparations. Spinacia olearacea, Amaranthus retroflexus, Senecio vulgaris, and Solanum Nigrum wild types were cultivated. The plant mutants resistant to herbicide were selected from soil treated with atrazine for 5 years; then resistance to atrazine was determined by radiolabeled herbicide binding as previously described (12). The photosynthetic material was isolated from deveined leaves (12). The leaves were homogenized in a medium containing 300 mM mannitol, 30 mM tetrasodium pyrophosphate, 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.1% bovine serum albumin (BSA) adjusted to pH 7.9. The mixture was filtered through a layer of cheesecloth and centrifuged for 1 min at 500g. The pellet was resuspended in the rinsing medium containing 300 mM mannitol, 25 mM MOPS, and 2 mM EDTA adjusted to pH 7.9 and centrifuged for 2 min at 2500 g. The new pellet was stirred in distilled water for 10 s to obtain thylakoids. The photosynthetic membranes were centrifuged for 2 min at 2000 g, and the pellet was homogenized in 500 mL of the rinsing medium. The amount of photosynthetic material was quantified by measuring the chlorophyll concentration, and the concentration was adjusted to 1 mg mL-1. A 100 g sample of plant material gives a yield of 5 × 104 µg of chlorophyll. Aliquots of 10 µg of chlorophyll were immediately immobilized. Immobilization of Photosynthetic Material. The photosynthetic material was immobilized in an albumin glutaraldehyde matrix (BSA-Glu) (13). All steps were carried out at 4 °C under a weak green light. The procedure consisted in mixing 1.65 mL of a 50 mM sodium phosphate buffer at pH 7.2 or 6.5 (measuring buffer), 0.625 mL of a BSA solution, and 0.5 mL of a 1.5% glutaraldehyde (Glu) solution. The mixture was incubated for 2 min, and then 0.6 mL of a photosynthetic material was added, followed by 3-4 s of agitation using a vortex mixer. Alternatively, the photosynthetic material was treated with the cross-linker CdCl2 (1 mM), in the measuring buffer at pH 7.2 or trapped in 10% gelatin in the measuring buffer. The immobilization mixture was immediately divided into 80 µL aliquots and deposited on a silicio septum filter; with a micropipet tip, the samples were distributed to fit in a circle of 1 cm diameter; this corresponds to the diameter of the measuring chamber in the flow cell. Then, the measuring phosphate buffer was fluxed through the filter of the flow

TABLE 1. GC-MS/MS Conditions for the Analysis of Herbicides in River Water precursor excitation acquisition (m/z) voltage (V) range (m/z) terbuthylazine simazine atrazine DET DEA metolachlor alachlor oxadiazon prometryn

214 201 200 186 172 162 160 175 184

1.00 1.00 1.20 1.00 1.20 1.00 1.00 1.00 1.10

90-214 100-201 90-200 100-186 60-172 70-162 70-160 70-175 100-184

monitored ions (m/z) 104, 132, 17 172, 17 122, 13 145, 16 105, 13 133 132 112, 14 142

cell till it became a green layer. For further measurements the flux of herbicide solution was sent both through and over the biomediator layer. The samples immobilized in the filter were frozen in liquid nitrogen and kept at -80 °C until use. River Samples and Chromatographic Analyses. The stock solutions of herbicides were prepared by dissolving herbicides in 100 mL of a pesticide-free methanol, and the diluted samples were prepared in the phosphate buffer. The four river samples were taken in winter when the probability of finding herbicides is minimal (the Tiber in Rome, the Aqua Marcia in Rome, a small river in Formello, and the Po in Pontelagoscuro, Italy). The Tiber and the Po samples, tested by gas chromatography-mass spectrometry (GC-MS), showed absence of simazine and diuron together with other classical herbicides, whereas atrazine was present at the level of parts per thousand. The Po water was also collected in April 2004 at Pontelagoscuro (FE, Italy). The Po water was filtered through a prefilter (Glasfaser Rundfilter, 142 mm, Schleicher&Schuell, Germany) and a 0.45 µm filter (nitrate cellulose, 142 mm, Sartorius AG, Germany). The filtered water was stored at 4 °C in a 10 L glass bottle prior to analysis for organic compounds. After storage, the first step for the concentration was to allow the water sample to reach ambient room temperature. Filtrated river water was passed through a 200 mg Lichrolut EN commercial cartridge (VWR, Germany) (14). Organic compounds were eluted from the cartridge using 5 mL of acetonitrile/methanol (1:1, v/v), and this process was repeated again a few minutes later. The extract, after being gently concentrated by N2 flux into a Turbovap II (Zymark) apparatus, was retrieved with ethyl acetate, giving a final volume of 1 mL. Herbicides in the water extract were analyzed by using the GC-MS/MS ion-trap technique (see Table 1 for condition analyses). The ThermoFinnigan GC-MS/MS system was equipped with an AI/AS 3000 autosampler, PTV injector, trace GC 2000 gas chromatograph, Polaris Q 1.3.1 spectrometer with CI (, Turbo Pump 230L, and the software Excalibur 1.3.1 acquisition system. The gas chromatographic conditions were PTV injection in a splitless mode, column Varian VF-5ms FS (60 m × 0.25 mm i.d. × 0.25 µm), injected volume 2 µL, carrier gas He (1 mL/min with constant flow), and transfer line 280 °C. The mass spectrometer conditions were EI 70 eV, standard autotune, multipler 1075 V, positive polarity, AGC target 50, temperature source 250 °C, damping gas He (constant flow, 0.3 mL/min). Under these conditions a calibration curve was drawn up, using the precursor and the monitored ions reported in the Results, for the analysis of terbuthylazine, simazine, atrazine, 2-chloro-4-amino-6-tert-butylamino1,3,5-triazine (desethylterbuthylazine, DET), 2-chloro-4amino-6-isopropylamino-1,3,5-triazine (desethylatrazine, DEA), metolachlor (2-chloro-6-ethyl-N-(2-methoxy-1-meVOL. 39, NO. 14, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. (A) A schematic representation of the electron-transfer chain at the level of reaction center II where the herbicide interferes at the level of the QB quinone pocket on the D1 protein of PSII. (B) Fast chlorophyll a fluorescence induction curves as a function of time measured on dark-adapted spinach illuminated at 650 nm with 70 µmol/(m2 s) (graph a) or with 300 µmol/(m2 s) (graph b) and in the presence of herbicide DCMU (graph c). thylethyl)acetotoluidide), alachlor, oxadiazon, and prometryn. An environmental sample of the Po water taken in spring was also prepared: the 1000× extract was dissolved to 300× with Milli-Q water; the herbicide concentrations determined by GC-MS are reported in the Results. Chlorophyll Fluorescence Measurements. Chlorophyll fluorescence emission was measured with the modified plant efficiency analyzer (PEA; Hansatech Instrument Ltd., England), whereas the dynamic measurements were performed using the BioLumi (Biosensor srl, P. Sabina, Via Tivoli (RM), Italy, www.biosensor.it). Illumination was provided by an array of light-emitting diodes (peak wavelength 650 nm) focused with lenses onto the photosynthetic sample. Biosensor Apparatus. The equipment (see Figure 2 in the Results) consists of (1) a black flow cell or a series of flow cells, (2) a peristaltic pump promoting the movement of the herbicide solution or the regenerating buffer in the flow cell, (3) a fluorescence sensor which provides excitation light and detects the fluorescence signal [the 650 nm of excitation light is emitted by a system of crown-arranged light-emitting diodes (LEDs) (focused over the biomediator in the flow cell)], (4) a computer to control the system and to analyze the fluorescence data, (5) an autosampler with timer equipped with (i) a stopped-flow actuator that automatically carries out the fluorescence measurements and (ii) a delayer actuator that allows the fluorescence sensor to carry out the measurement after a prefixed time period, (6) interchangeable series of cylinders in the flow cells containing the biomediators, and (7) containers of the herbicide solutions and regenerating buffers. For a reproducibility in the calculation of the fluorescence area, a special software program was developed by Biosensor srl (www.biosensor.it). This biosensor equipment was assembled in a versatile and easy to use portable structure of about 20 × 20 × 15 cm, thereby lowering both the operating and construction costs (see the scheme in the Results; 15).

Results Fluorescence Parameters. Fluorescence induction kinetics are often used to study photosynthetic activity. The numerical parameters considered are the ratio of the variable fluorescence and the area above the fluorescence curve between F0 and Fm; this parameter is proportional to the pool size of the electron acceptors QA on the reducing side of photosystem II. If the electron transfer from the reaction center of PSII to the quinone pool is blocked, as occurs during the binding of herbicides, the area over the fluorescence curve is 5380

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dramatically reduced (8). Figure 1A shows the electrontransfer PSII chain where herbicide binds the QB pocket, altering the fluorescence activity; a typical pattern of a fluorescence induction curve (called Kausky’s effect) is shown in Figure 1B. The variable fluorescence ratio (Fv/Fm) is proportional to the quantum yield of PSII photochemistry. A decline in Fv/Fm is symptomatic of the effect of various environmental stresses, and such changes indicate a loss of photochemical efficiency. However, an increase in F0 is characteristic of PSII photochemistry inactivation, whereas a decline in Fv may indicate an increase in a nonphotochemical quenching process at or close to the reaction center (8). The presence of diuron (10 mg/L) in the phosphate buffer does not modify Fm but increases F0 by 20 ( 3% and decreases Fv/Fm by 40 ( 5%. The fluorescence area in the presence of the diuron shows the strongest inhibition (95 ( 6%) and for this reason was the selected parameter for the subsequent experiments. The biosensor equipment was composed of the following components: (i) a flow cell with an interchangeable cylinder equipped with a filter septum, where the biomediator is immobilized, (ii) a peristaltic pump promoting the flow of the buffer containing the herbicide through the flow cell and then through the biomediator, and (iii) a fluorimeter which provides the excitation light and detects the fluorescence signal (Figure 2; see the details in the Experimental Section). Biomediator Specificity, Stability, and Immobilization. To improve the stability of the biomediator, we exploited the capacity of the photosynthetic organisms to adapt at a high temperature. These organisms were grown at relatively high temperatures (33-35 °C) or underwent a moderate thermal stress (33-40 °C) for 48 h (12, 16). Moderate heating induces heat stress and chaperone proteins which stabilize the biomediator (12). Furthermore, to optimize the orientation and cohesion of the biomediator molecules, the most important techniques for immobilization of proteins on optical surfaces (covalent bonds, physical adsorption, etc.) were examined as well as protein filming by cross-linking. Experimentally, the technique of PSII immobilization on surfaces by chemical cross-linking with BSA-Glu gives the poorest performance in fluorescence area response but longer stability than immobilization with gelatin or CdCl2 (Table 2). In particular treatment with BSA-Glu, which causes the crosslinking of the -SH groups in the protein, results in a network more compact and resistant to the bleeding activity of the herbicide-containing effluent. Treatment with CdCl2 causes a less effective cross-linking of the protein -SH groups, but surprisingly, it increases both the stability and sensitivity of

FIGURE 2. (A) Setup of the measuring flow system. The apparatus consists of a vessel containing the measuring buffer, herbicide solutions, and a valve for selecting between buffer or sample in the peristaltic pumps, a flow cell with the biomediator, and a fluorescence apparatus where a red LED for excitation and measurement is controlled by a timer. The system is controlled by a computer (15). (B) Flow cell design. It consists of a circular chamber where the biomediator membrane is immobilized using chemicals on a silicio porous disk. The flow cell is connected to the fluorescence sensor. The pollutant can be pumped either through or over the biomediator.

TABLE 2. Recognition Activity of Differently Immobilized Biomediators, Isolated from the Wild Type and Mutants Selected from Soil Treated with Atrazine for 5 Yearsa plant species

types of immobilization in a porous septum

S. oleracea S. oleracea S. oleracea S. vulgaris Senecio mutant A. retroflexus Amaranthus mutant

BSA-Glu gelatin CdCl2 CdCl2 CdCl2 CdCl2 CdCl2

a

half-life (h) pH 7.5 pH 6.5 15.5 7.3 13.6 7.0 5.2 9.6 7.3

54.2 24.3 39.2 24.4 12.3 13.6 21.0

detected herbicide subclasses

range of recognition (M)

urea, diamine, triazine urea, diamine, triazine, phenolic compounds urea, diamine, triazine, phenolic compounds urea, diamine, triazine urea urea, diamine, triazine, phenolic compounds urea, diamine

10-8-10-6 10-8-10-6 10-9-10-6 10-7-10-5 10-7-10-5 10-8-10-5 10-7-10-5

SD in the range (3-9%.

the biomediator to the herbicide. Sensitivity to the herbicide is 10 times greater when immobilized with CdCl2 (Table 2). The pH of the cross-linker buffer strongly influences the halflife of the resulting thylakoid matrix with a half-life of 54 h for the spinach thylakoids at the optimal condition at pH 6.5 (Table 2). Thus, the treated biomediator is further immobilized on a porous septum of a silicio material, a component of the measuring flow cell (see the details in the Experimental Section; 15). To have a specific signal for an individual class of herbicides, the peculiar properties of various PSII preparations were utilized. Various immobilization procedures lead to biomediators with differential activity (Table 2). It is known that the mutation of a single amino acid on the D1 protein of PSII is able to induce resistance to a herbicide; for example, the replacement of a serine with a glycine induces resistance to atrazine but not to other herbicides (16); the biomediator is resistant to the class of triazinic compounds and therefore selective for those classes of herbicides other than triazine (phenolic, ureidic, and diazinic herbicides). The recognition activity of the wild type and mutants was determined for the various immobilization procedures and plant species (S. oleracea, S. vulgaris, A. retroflexus). Two or more biomediators can be used, arranged inside the series of flow cells (Figure 3).

FIGURE 3. Biomediators with differential recognition activity in flow cells connected in series. A pollutant goes either through or over the biomediators. Testing Herbicides in River Water. Surprisingly, the use of real water samples instead of MilliQ water to prepare the buffer caused a PSII activation measured as Hill activity (oxygen evolution). In all tested samples, PSII activation increased by 10-21%. We observed that this increase was in part due to a high concentration of divalent cations in the river water. This is in accordance with the knowledge that PSII activity depends on the presence of charge ionic species (mainly divalent cations) and consequent membrane aggregation (17). The PSII fluorescence activity was also tested using salinity similar to that of seawater ([NaCl] lower than 0.2 M) with a negligible effect on its activity (data not shown). Figure 4 shows the percentage decrease of fluorescence area when the entrapped S. oleracea thylakoids (graphs A-D) VOL. 39, NO. 14, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Percentage decrease of the fluorescence area in the presence of diuron or TBA herbicides (Herb) in Po river water at controlled pH 7.2 with added phosphate at 10 mM, at various molar concentrations and time intervals. After inhibition in the presence of herbicide the biomediator was regenerated (Reg) by fluxing the phosphate buffer, pH 7.2. The figures show typical analysis patterns of five replicates, with SD ) (4-8%.

TABLE 3. The Herbicide Was Fluxed over the Biomediator (S. oleracea Immobilized on Gelatin) for 30 min, and Then for Regeneration, the Phosphate pH 7.2 Buffer Was Fluxed for 20 min

FIGURE 5. Percentage decrease of fluorescence area with various concentrations of TBA. SD in the range (4-8%. or A. retroflexus thylakoids (graphs E-F) are fluxed with a solution of diuron or 2-chloro-4-(ethylamino)-6-tert-butylamino-1,3,5-triazine (terbuthylazine, TBA) herbicides from the Po river water. From the graphs we can deduce the percentage decrease of fluorescence area when the biomediator is treated with herbicide. The regeneration of the biomediator with the 10 mM phosphate buffer, pH 7.2, is possible, and after one biomediator regeneration, the pattern shows approximately a 10% decrease in the herbicide inhibition (compare graphs C and D). However, a funny phenomenon occurs: the higher the concentration of herbicide, the higher the regeneration (compare graphs A and B); at present there is no explanation for such a phenomenon. To determine the response of the fluorescence area to the fluxed herbicide, the analysis was performed over a long time (70 min); however, in the case of a normal analysis 10 min is sufficient. The percentage of area decrease depends on the herbicide concentration in a nonlinear way (Figure 5). This is in accordance with the observation that the interaction of 5382

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herbicide (10-7 M)

area recovery after regeneration (%)

herbicide (10-7 M)

area recovery after regeneration (%)

TBA DET

85 ( 6 10 ( 4

diuron atrazine

93 ( 2 71 ( 4

herbicide with PSII is an equilibrium reaction following the Michaelis-Menten kinetics (17). Since in surface water dealkylated metabolites of triazines are often found (18), we analyzed the behavior of TBA and its metabolite DET. DET is known to be produced by a dealkylation process operated by soil bacteria (19). We observed that the biomediator is irreversibly inhibited by DET at the level of Fv/Fm (Table 3). This behavior may be explained by the fact that DET herbicide has a structure with a free -NH2 group in the ring. Therefore, it is more polar and perhaps less removable from the biomediator than the parental compound. It will probably interfere with the PSII chlorophyll-protein complexes that are involved in the fluorescence emission. On the contrary the PSII electrontransfer activity is affected by DET in a reversible way (data not shown). Testing River Samples. The apparent absence of interfering organic compounds in surface water suggested the utilization of the biosensor for monitoring herbicides in river water. We measured the thylakoid activity in four river samples, taken in winter when the presence of herbicides is less probable: The Tiber river (RM), the Aqua Marcia (RM), the Valle del Sorbo (Formello), and the Po (FE). The waters analyzed by GC-MS showed the absence of simazine, diuron,

TABLE 4. Concentration of Herbicides Found in the Po River in Spring (April) 2004, Detected by GC-MS (See the Experimental Section) herbicide compd

herbicide concn (mg/L)

herbicide compd

herbicide concn (mg/L)

desethylatrazine desethylterbuthylazine atrazine terbuthylazine oxadiazon

0.026 0.027 0.027 0.106 0.007

simazine prometryn alachlor metolachlor sum of herbicides

0.020 0.005 0.006 0.028 0.252

TABLE 5. Analyses of the Po Water Using the Biosensora herbicide concn (M) in Po River

initial activity after Po water addition (%)

recovery after biomediator regeneration (%)

control 10-9 10-8 10-7

100 ( 3 95.8 ( 3 67 ( 4 54 ( 6

nd nd 77.3 ( 5

a

nd ) not determined.

and classical herbicides, while atrazine was present at the level of parts per thousand only in the Tiber and the Po waters. On the contrary, the Po water sampled in April 2004 and analyzed by GC-MS showed the presence of several herbicides as reported in Table 4. The terbuthylazine concentration was close to the maximum admissible concentrations (MACs) for pesticides in drinking water use (equal to 0.1 µg/L). Atrazine, oxadiazon, simazine, metolachlor, alachlor, and prometryn were also detected, but the levels of these herbicides and their metabolites (DEA and DET) were below the MAC value (Table 4). Table 5 represents the data regarding the Po water obtained using the multibiosensor. The data indicate that the analysis by the multibiosensor is possible for the Po water concentrated at least 3 times since the environmental nanomolar concentration is in the error range. The limit of detection was calculated as reported by Koblizek et al. (5, 7).

Discussion Massive use of herbicides in agriculture over the last few decades has become a serious environmental problem. The pollution of soil and water in certain areas represents an important risk factor due to the toxicity or carcinogenicity of some of these compounds. Herbicides inhibiting photosynthesis via targeting PSII function still represent the basic means of weed control. This group consists of several classes of chemicals such as triazines (e.g., atrazine, simazine, cyanazine), diazines, phenylureas (linuron, diuron), or phenols [e.g., ioxynil (4-hydroxy-3,5-diiodobenzonitrile), bromoxynil] (9). Triazine herbicides are used every year in large quantities; for example, in the U.S. about 35 × 106 kg of atrazine, 9 × 106 kg of cyanazine, and 3 × 106 kg of simazine are applied every year (20). This practice frequently leads to soil contamination and subsequent pollution of surface water and groundwater. Triazines are relatively persistent in water and represent the most frequently detected pesticides in groundwater. In the U.S., atrazine contaminates even countries in which it is not used due to its percolation in soil! In the Po river (Italy) in spring, we found atrazine at a concentration of 27 µg/L even though it has not been used since 1989! (18, 21). The residual concentration of these compounds frequently exceeds the maximum admissible concentration in drinking water for human consumption and is a real environmental risk for the aquatic ecosystem (18, 21).

These problems have stimulated research toward the development of sensitive methods and new technology to detect pesticide residues. For this reason, attention has been focused on immunochemical methods for herbicide detection because of their high sensitivity and specificity of detection. However, the disadvantage of the immunological methods is that the antibodies bind specifically to only one compound; moreover, their preparation is difficult. The basic principle shared by all sensors consists of their ability to detect an analyte even at a very low concentration, due to the interaction occurring between a biological/ biochemical system (suitably immobilized) and an analyte. This interaction results in a response of the biological/ biochemical system which is detected (signal transduction) and continuously monitored. Depending on the combined selection of the “biological element-transducer-processing system of the signal”, biosensors differing in detection ability and potential are obtained. Indeed, not all the combinations are viable or advantageous. The selection of the biological element determines the selectivity and sensitivity grade of the biosensor to the analyte, but the coupling mode existing between the reactive biological layer and signal transducer is also essential (2-4). Photoinhibition of photosynthesis, stress acclimation, and effects of air pollution and herbicides (8) are examples of physiological responses in which the fluorescence has been successfully used as an indicator of the physiological state of the photosynthetic apparatus. Measurements of chlorophyll fluorescence have been shown to be a rapid, noninvasive, and reliable method to assess photosynthetic performance in a changing environment. The fluorescence arising from chlorophyll a at room temperature is almost exclusively associated with PSII and hence primarily reflects its state (8). The area parameter calculated over the fluorescence curve shows strong inhibition in the presence of the photosynthetic herbicide, and it was selected for our experiments. PSII thylakoidal preparations, twice immobilized with a cross-linker and in a silicio porous septum, inserted in a flow cell tightly connected to a fluorescence transducer are a good system to test the environmental samples, such as the water of rivers containing TBA and DET (limit of detection 3 × 10-9 M). The extraction of thylakoids is a simple method requiring 15 min, and once the thylakoids are immobilized after 10 min, a huge amount of biomediator is obtained that can withstand being frozen at -20 °C for months (22; see also the Experimental Section). It is possible to distinguish the presence in river water of triazine metabolites with free -NH2 groups (i.e., DET or DEA) by the irreversibility of the PSII inhibition. Hypersensitive mutants have been previously used to increase the sensitivity of the biomediator to herbicides (23). The multibiosensor has the advantage, over previous systems based on the Clark electrode printed electrodes, of being portable and easy to use. Moreover, it has the advantage of providing fluorescence-integrated areas instead of peak measures (24). Furthermore, the multibiosensor has the original feature of being able to distinguish the subclasses of the photosynthetic herbicides by using specific immobilized biomediators isolated from mutated organisms. Such variously immobilized and mutated organisms prove to be resistant to one subclass of photosynthetic herbicides but not to the others; for example, a mutant is resistant to triazines, whereas it is sensitive to urea, with different fluorescence emission. Therefore, a “wild-type” organism is sensitive to triazines, whereas the corresponding mutant is not. It follows that in the first case a response in fluorescence variation associated with a “nonresponse” for the mutated organisms certainly identifies the subclass. VOL. 39, NO. 14, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Acknowledgments This work was supported by the European Community within EC Contract QLK3-CT-2001-01629. M.T.G. thanks Dr. M. Iosa and Dr. P. Giardi for experimental work.

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Received for review September 3, 2004. Revised manuscript received April 21, 2005. Accepted May 2, 2005. ES040511B