Environ. Sci. Technol. 2001, 35, 2423-2427
Identification of the Isoflavonoid Genistein in Bleached Kraft Mill Effluent YIANNIS KIPARISSIS, RICHARD HUGHES, AND CHRIS METCALFE* Water Quality Centre, Trent University, Peterborough, Ontario, K9J 7B8 Canada THOMAS TERNES ESWE Institute for Water Research and Technology, Wiesbaden, Germany
Plants synthesize many phytochemicals, including flavonoids, which may be present in the heartwood of trees used in the pulp and paper industry. Extracts were prepared from wood pulp and mill effluent collected from a bleached kraft mill in Ontario, Canada, and these extracts were subfractionated by LH-20 gel filtration chromatography and analyzed by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and LC-ESI-tandem mass spectrometry (LC-ESI-MS-MS). Initial LC-MS analysis in negative ion mode was conducted by monitoring ions corresponding to the deprotonated molecular ions of a range of flavonoid compounds. The presence of chromatographic peaks generated by selected ion monitoring (SIM) in samples of both mill effluent and wood pulp encouraged further analysis. Of the compounds highlighted by LC-ESIMS-SIM analysis, the isoflavonoid genistein was positively identified by LC-ESI-MS-MS. Genistein was quantified at a concentration of 30.0 µg/kg in air-dried wood pulp and concentrations of 13.1 µg/L and 10.5 µg/L in untreated and treated (final) effluent, respectively. Genistein is a known endocrine disruptor substance and, therefore, could contribute to the alterations in sex steroid levels and reduced reproductive capacity observed in fish captured near the discharges of pulp mills.
Many sublethal biochemical and physiological responses have been observed in populations of fish collected near pulp mills, including induction of hepatic cytochrome P-450dependent monooxygenases, changes to serum steroid levels, alterations in secondary sex characteristics, and reproductive dysfunction (6-10). Several studies have shown that these responses may be induced in fish through exposure to phytochemicals originating from the wood pulp, including chemicals in black liquor (11), alkyl-substituted polynuclear aromatic hydrocarbons (12), phytosterols (13-15), stilbenes (15, 16) and juvabiones (17). It is possible that flavonoids may also contribute to the biological responses observed in fish near pulp mill discharges. There have been no previous studies directed at identifying flavonoid compounds in the effluents of pulp mills, even though these compounds have been detected in tree heartwood (1, 2). Final pulp mill effluents are complex mixtures of numerous chemicals and their composition depends on various parameters, including the pulping process, wood species, bleaching technology, and wastewater treatment (18). Thus, research directed at isolating and identifying the toxic or bioactive chemicals in these effluents is a challenging task. The objective of this study was to analyze effluents and wood pulp from a bleached kraft mill to determine whether flavonoid compounds are present in these complex mixtures. Samples of wood pulp after oxygen delignification as well as mill effluents collected prior to treatment and after treatment were analyzed by LC-MS and LC-MS-MS techniques to identify and quantify flavonoid compounds.
Materials and Methods
Introduction Plants synthesize many secondary metabolites such as terpenoids, alkaloids, lignans, tannins, coumarins, and flavonoids for protection against pathogens and herbivores. In the heartwood and sapwood of trees, the presence of these phytochemicals makes the wood disease-resistant (1). Compounds from the flavonoid group of phytochemicals, which includes both the flavones and isoflavones (Figure 1) have been identified in significant quantities in the heartwood of tree species that are used for manufacturing wood pulp (1, 2). The presence of flavonoid compounds in wood pulp may have environmental significance since several flavonoid compounds, especially the ones lacking sugar moieties (i.e. aglycones), are known to be biologically active (3). For instance, flavonoids affect reproduction in mammals by acting upon the pituitary-gonadal axis, either as competitors for steroid receptor sites (4) or by inhibiting aromatase (5). * Corresponding author phone: (705)748-1272; fax: (705)748-1587; e-mail:
[email protected]. 10.1021/es001679+ CCC: $20.00 Published on Web 05/05/2001
FIGURE 1. General structure of flavone and isoflavone compounds, showing sites of hydroxy-substitution.
2001 American Chemical Society
Chemicals. Flavone (2-phenyl-4H-1-benzopyran-4-one), chrysin (5,7-dixydroxyflavone), apigenin (3′,5,7-trihydroxyflavone), kaempferol (3,3′,5,7-tetrahydroxyflavone), quercetin (3,3′,4′,5,7-pentahydroxyflavone), naringenin (4′,5,7-trihydroxyflavanone), (+)-catechin, flavanone, flavonol (3-hydroxyflavone), 6-hydroxyflavone, 7-hydroxyflavone, transchalcone, and galangin (3,5,7-trihydroxyflavone) were purchased from Sigma-Aldrich, Inc., Toronto, ON, Canada. Genistein (4′,5,7-trihydroxyisoflavone, 4′-hydroxyflavone and daidzein (4′,7- dihydroxyisoflavone) were purchased from Apin Chemicals Ltd, Oxfordshire, England. Sample Collection. Wood pulp and pulp mill effluent were collected in August, 1998 from a pulp mill located in northern Ontario, Canada. Wood pulp was obtained from the softwood pulping line immediately after oxygen delignification. The pulp mill effluent, which originated from both the softwood and hardwood pulping lines, was collected in 4 L glass bottles from points in the mill either (a) prior to its biological treatment in the secondary wastewater treatment VOL. 35, NO. 12, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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facility (i.e. before treatment) or (b) after treatment just prior to its discharge into receiving waters (i.e. after treatment). Upon arrival at Trent University, the samples were immediately stored in the dark at 4° C. All samples were extracted within 4-7 days of sample collection. Preparation of Extracts. Effluent samples and procedural blanks of distilled water were extracted on a column of Amberlite XAD-7 resin (BDH, Toronto, ON, Canada), using the methods described by Metcalfe et al. (19). The effluent sample was filtered with 1.5 µm glass fiber filters (Whatman, 934-AH), which were previously extracted in a Soxhlet apparatus with hexane. A 2 L aliquot of the filtrate was acidified to pH 2 with 16 mol/L HCl and then passed through a glass chromatography column (35 cm × 2 cm i.d.) packed with XAD-7 resin. Prior to extraction, XAD resin was sequentially extracted in a Soxhlet apparatus with hexane (1.5 h), acetone (1.5 h), and methanol (2 h) and stored in excess methanol. Glass wool (hexane washed) was added to the bottom of the column, and resin was added to within 3 cm of the top of the column. Another glass wool plug was placed on the top of the column prior to extraction of samples. The column was initially eluted with 100 mL of methanol, followed with 100 mL of HPLC-grade water, and then the effluent sample was passed through the column at a rate of 25 mL/minute. The column was allowed to run dry, and any residual effluent was forced out by air pressure. After removal of the top plug, 100 mL of methanol was added to the column and mixed thoroughly with the resin. After 10 min, the methanol was eluted from the column and passed through sodium sulfate (hexane washed) into a 500 mL boiling flask. Finally, the volume of the extract was reduced on a rotary evaporator to approximately 20 mL. A sample of air-dried softwood pulp (100 g) was placed in five thimbles (solvent washed), and each subsample was extracted with 100 mL of methanol in a Soxhlet apparatus for 3 h. The methanol extracts from all flasks were pooled in a 1000 mL boiling flask, and the volume was reduced in a rotary evaporator to a volume of 20 mL. The extracts of wood pulp, effluents, and procedural blanks were subfractionated by gel filtration chromatography with Sephadex LH-20 (BDH, Toronto, ON, Canada) using methods based on those described by Johnston et al. (20). LH-20 resin (40 g dry weight) was soaked overnight in excess methanol and then packed into a glass chromatography column (2.5 × 33 cm i.d.). The extracts were applied to the column and eluted with methanol at a rate of 4-5 mL/min. Three eluent fractions of 0-150 mL (fraction 1), 151-260 mL (fraction 2), and 261-400 mL (fraction 3) were collected, the volumes were reduced in a rotary evaporator to 15 mL and stored at 4 °C until further analysis. The dihydroxy- and pentahydroxy-flavonoids, chrysin and quercetin, respectively, were used as the model standards to determine the elution patterns in the above fractionation process. Chrysin and quercetin were eluted in the second and third fractions, respectively, and percent recoveries of these analytes were 93% and 88%, respectively. Analysis. Extracts were initially analyzed by liquid chromatography-electrospray ionization-mass spectrometry in selected ion mode (LC-ESI-MS-SIM) using a Hewlett-Packard 1100 Model LC-MSD. The HPLC was equipped with a 3 µm Phenomenex (Torrance, CA) Luna C18 column, 150 × 4.6 mm. There was gradient elution of analytes with a binary mobile phase consisting of solvent A of 0.5% acetic acid in HPLC-grade water and solvent B of HPLC-grade methanol. The gradient solvent program at a constant flow rate of 670 µL/min consisted of 100% solvent A at t ) 0, followed by immediate linear addition of solvent B to a concentration of 70% solvent B by t ) 3 min, followed by a hold time of 10 min. Solvent A was then added so that by t ) 11 min, there was 0% solvent B. The column was flushed with 100% solvent 2424
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A until t ) 15 min. The electrospray ionization source was operated at 350 °C in negative ion mode with nitrogen drying and nebulizer gas. The compounds eluting from the column were analyzed for putative flavonoids by monitoring at massto-charge ratios corresponding to the deprotonated molecular ions for seven flavonoid compounds in an analytical standard; specifically, chrysin (m/z 253), genistein, apigenin and galangin (m/z 269), kaempferol (m/z 285), catechin (m/z 289), and quercetin (m/z 301). The limits of quantitation (LOQ), determined from analysis of procedural blanks according to the method of Keith et al. (21), were between 100 and 200 ng/mL for the flavonoids in the standard. To confirm the presence of genistein in samples, extracts were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) using a Micromass “Quattro” LC-Tandem Mass Spectrometer. This system consisted of a Waters 600 quaternary HPLC pump and Waters 717 autosampler and separation was performed on a Phenomenex Luna C18 column (5 µm, 150 × 4.6 mm). Analytes were eluted with an isocratic mobile phase consisting of 65% methanol:35% formic acid (20% solution) at a flow rate of 0.3 mL/min. The Z-spray electrospray ionization source was operated in negative ion mode at a temperature of 300 °C and nitrogen was used as a desolvation gas and nebulizer gas. In MS-MS experiments, UHP argon was used to generate collision induced dissociation (CID). MS-MS analysis was conducted using product ion scans. Genistein was quantified in samples by LC-MS-MS using a PE-Sciex API 365 Tandem Mass Spectrometer. The PESciex system consisted of a PE 200 Series quaternary HPLC pump and autosampler, and separation was performed on a Lichrosphere 100 RP-18 endcapped (5 µm) stationary phase (Merck, Darmstadt, Germany) packed in a Merck EcoCart column (125 × 3 mm). The binary gradient solvent program at a constant flow rate of 300 µL/min consisted of a 55:45 solution (v/v) of 0.1% acetic acid in HPLC-grade acetonitrile, and the content of acetonitrile was increased linearly to a 20:80 solution (v/v) of 0.1% acetic acid in acetonitrile. The electrospray ionization source was operated in positive ion mode at a temperature of 475 °C, and nitrogen was used as a desolvation gas and nebulizer gas. MS-MS analysis of genistein was conducted using multiple reaction monitoring (MRM) of the precursor ion, m/z 271, and the product ion, m/z 153, which are the major ions generated by product ion scans (Figure 2). In MS-MS, analysis, UHP nitrogen was used to generate collision induced dissociation (CID). A calibration curve with an external standard of genistein was used to quantify concentrations of the analyte in effluent and pulp samples. The limit of quantitation was 50 ng/L, as calculated by analyzing samples of wastewater spiked with genistein at concentrations between 10 ng/L to 1 µg/L.
Results and Discussion When extracts from wood pulp and treated and untreated mill effluent were analyzed by LC-ESI-MS (negative ion mode), several peaks were detected by monitoring ions corresponding to the deprotonated molecular ions of the seven flavonoid compounds in the analytical standard (i.e. chrysin, genistein, apigenin, galangin, kaempferol, catechin, and quercetin). Total ion chromatograms of the different subfractions of the extracts were compared to identify any peaks that consistently appeared in all extracts. Of the major peaks detected in all extracts, only one eluted at a retention time corresponding to a flavonoid compound in the analytical standard: genistein (Figure 3). Subsequent analysis of the extracts by LC-ESI-MS-MS using the Micromass system confirmed identification of genistein by analysis of product ion spectra (Figure 4).
FIGURE 2. Collision induced dissociation (CID) mass spectrum of genistein generated by LC-MS-MS analysis in positive ion mode (PE-Sciex API 365).
FIGURE 3. Total ion chromatograms (m/z ) 253, 269, 271, 285, 289, 301) generated by LC-ESI-MS in negative ion mode analysis (HewlettPackard Model 1100) of extracts prepared from LH-20 fraction 2 of extracts prepared from (A) wood pulp, (B) mill effluent after treatment, and (C) a procedural blank. The compound identified as genistein was subsequently confirmed by LC-MS-MS. The retention time of the unknown compound did not correspond to any of the flavonoid compounds in the analytical standard. Genistein concentrations were determined by LC-MSMS-MRM analysis of the various subfractions of the extracts. These data indicated that this isoflavone compound was present in air-dried wood pulp at a concentration of 30.0 µg/kg and was present in the mill effluent before treatment at 13.1 µg/L and after treatment at 10.5 µg/L (Table 1). The majority of the genistein in the extract from wood pulp was present in fractions 2 and 3 (Table 1). However, in extracts from the effluent samples, genistein appeared in significant quantities in all three subfractions (Table 1). Although Sephadex LH-20 gel filtration chromatography efficiently fractionated quercetin and chrysin in preliminary trials of the method, the more complex matrix of the extracts prepared from effluent appeared to cause channeling in the LH-20 column that interfered with the efficiency of the subfractionation process. However, gel filtration appeared to remove some other components of the effluent that interfere with analysis of raw extracts and so was considered a useful step for sample cleanup. Further work is required to optimize the procedures for extracting and isolating
flavonoids from complex environmental samples such as pulp mill effluents. These findings indicate that the isoflavone, genistein is present in hardwood pulp and in mill effluent. Since genistein was present in the bleached pulp, it appears that this compound persisted through the “ODcEoDnD” bleaching process (i.e. elemental oxygen, molecular chlorine with chlorine dioxide, caustic extraction with oxygen, chlorine dioxide with neutralization). In addition, the presence of genistein in both the untreated and treated (final) effluent indicates that this compound persisted through the wastewater treatment process, which consisted of a settling basin (primary treatment) and an aerated stabilization basin with 7-d hydraulic retention (secondary treatment). However, the genistein in final effluent could also have originated from washing losses during earlier pulping stages. Genistein has been identified in a range of plant species, including clover (22), alfalfa (23), and soybeans (24). This is the first study to identify genistein or any other flavonoid compound in a pulp mill effluent. This finding is of VOL. 35, NO. 12, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Collision induced dissociation (CID) mass spectra generated by LC-MS-MS analysis in negative ion mode (i.e. Micromass Quattro) of the peak monitored at m/z 269 that was tentatively identified as genistein in (A) extract prepared from mill effluent after treatment (fraction 2), (B) extract prepared from mill effluent before treatment (fraction 2), and (C) a standard of genistein.
TABLE 1. Concentrations of Genistein Quantified by LC-MS-MS-MRM in Extracts of Wood Pulp and in Untreated and Treated Mill Effluent Collected from a Bleached Kraft Pulp Mill in Ontario, Canada in August, 1998a genistein concentration sample
units
fr. 1
fr. 2
fr. 3
total
pulp effluent before treatment effluent after treatment
µg/kg µg/L µg/L
4.7 2.5 4.0
18.3 6.3 2.1
7.0 4.3 4.0
30.0 13.1 10.1
a Extracts were subfractionated into fractions 1, 2, and 3 by LH-20 gel filtration chromatography and the fractions analyzed separately.
environmental significance because genistein is an endrocrine-disrupting substance that binds to the estrogen receptor and mimics the action of endogenous sex steroids (25-27). Thus, genistein may be one of the phytochemicals in pulp mill effluents that is responsible for the biological effects observed in fish near pulp mills, such as alterations in serum steroid levels, effects on secondary sex characteristics, and reproductive dysfunction (6-10). However, other chemicals in the heartwood of trees used to make pulp may contribute to biological effects in fish near pulp mills. Some bioactive compounds such as stilbenes (15, 16) are formed through biosynthetic pathways that are similar to those for flavonoids (28). Phytochemicals such as flavonoids, stilbenes, and various terpenoids that are accumulated in relatively large amounts in heartwood often compete for common enzymes, substrates and cofactors, but the state of knowledge of these systems is presently inadequate to understand how plants control these competing pathways (28). As mentioned previously, several flavonoid compounds were included in the analytical standard used to initially search for flavonoids in extracts by LC-MS-SIM. However, aside from genistein, none of the flavonoid compounds in the analytical standard were identified in extracts, including quercetin, kaempferol, naringenin, chrysin, apigenin, galangin, and catechin. All of these flavonoids have been reported in the heartwood and bark of trees. For instance, quercetin and kaempferol are the most abundant aglycone flavonoids in a number of trees, such as, Douglas fir and 2426
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Larix species (2). The change in color of wood chips during storage at pulp mills is attributed to the formation of flavonoids from their flavanonol precursors by aerial oxidation (29). Catechin is the monomer flavonoid that comprises the condensed tannins in chestnut wood and in Eucalyptus species (1). Fang et al. (30) extracted 9.4 g of flavonoids from 1.1 kg of the heartwood of a pine species (Pinus morrisonicola) native to Taiwan, including relatively large quantities of chrysin (7.2 g), apigenin (14.5 mg), and galangin (17.4 mg). That these compounds were not identified in this study may be explained either by their absence from the tree species used in the pulp mill (e.g. jackpine, spruce, birch, maple, and poplar) or by their inability to persist through the pulping, bleaching, and effluent treatment processes at the mill. In any event, further studies of effluents from other pulp mills should not be confined to analysis of genistein, as other flavonoid compounds may occur in effluents. Methylsubstituted flavonoids have been identified as important classes of flavonoid glycosides in some plant species (31). Work continues to identify other flavonoid compounds in pulp mill effluents and other environmental samples (32). In conclusion, LC-MS and LC-MS-MS analysis of wood pulp and effluents (before and after treatment) of a bleached kraft pulp mill in Ontario, Canada indicated that the isoflavonoid compound genistein was present at µg/kg concentrations in air-dried wood pulp and µg/L concentrations in effluent samples. Similar concentrations of genistein in untreated and treated (final) effluent are consistent with persistence of genistein through the effluent treatment process. Genistein is a known endocrine disruptor substance and, therefore, could contribute to the alterations in sex steroid levels and reduced reproductive capacity observed in fish captured near the discharges of pulp mills. More work is required to identify whether flavonoids are present in the effluents from other pulp mills and whether these compounds are present in receiving waters near mill discharges.
Acknowledgments This work was financially supported by grants to Metcalfe from the Natural Sciences and Engineering Research Council (NSERC) of Canada and from the Canadian Network of Toxicology Centres and a grant to Ternes from the international office of the Ministry of Education and Research
(BMBF), Germany within the bilateral Germany/Canada cooperation program.
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Received for review September 19, 2000. Revised manuscript received March 19, 2001. Accepted March 23, 2001. ES001679+
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