Determination of organochlorine and organophosphorus pesticide residues in eggs using a solid phase extraction cleanup

A multiresidue solid phase extraction (SPE) method for the isolation and subsequent gas chromatographic determination of nonpolar organochlorine and polar organophosphorus pesticide residues in eggs is described. The method uses an acetonitrile extraction followed by an SPE cleanup using graphitized carbon black and aminopropyl SPE columns. Organophosphorus pesticides are determined by gas chromatography with flame photometric detection. After further cleanup of the extract using Florisil SPE columns, organochlorine pesticides are determined by gas chromatography with electron capture detection. Studies were performed using eggs containing both fortified and incurred pesticide residues. The average recoveries were 86-108% for 8 fortified organochlorine pesticide residues and 61-149% for 28 fortified organophosphorus pesticide residues.     

Comparison of thermal and chemical stability of Cu–humic complexes

Purpose

The purpose of this study was to investigate relationships between chemical and thermal stabilities of Cu–humic complexes. The study of the chemical stability was based on pedological methods used for the determination of the bond strength of metal ions in soils by chemical leaching agents. The samples with various contents of the Cu(II) ions and their bond strength were put to the thermal analysis in order to correlate their thermo-oxidative behavior with their stability determined by leaching.

Materials and methods

The humic acid was extracted from the South-Moravian lignite by standard alkaline extraction. The humic sample was used in two different forms: as the solid powder and as the hydrogel prepared by the acidic precipitation of humate. Six various concentrations of copper(II) solutions were used for the complexation of the humic powder and the hydrogel, in order to study the influence of their initial concentration on both the determined stabilities of the prepared complexes. Their chemical stability was assessed in terms of the Cu(II) ions release from the humic acid structure into two different extraction agents (MgCl₂ and HCl solutions). Their thermo-oxidative behavior was investigated employing the thermogravimetry.

Results and discussion

The complexation capacity of the humic hydrogel was higher in comparison with the humic powder. The amounts extractable from the Cu–humic complexes by the used leaching agents are higher for the humic powder, which shows on the lower chemical stability. The thermal degradation of the prepared complexes proceeds in several steps and this character remains also after the removal of the mobile and the ion-exchangeable fractions by the MgCl₂. The elimination of these fractions as well as the extraction of the strongly bound Cu(II) ions shift the thermal degradation to higher temperatures. The incombustible residue increases with the Cu(II) content in the complexes except for the samples extracted by the HCl.

Conclusions

The form of humic sample used for the preparation of the Cu–humic complexes influences both the chemical stability and the thermal one. The main reason is probably a better accessibility of the functional groups in the humic gel, which enables forming stronger binding copper(II) ions. The results showed that the thermal and chemical stabilities are closely related, which corresponds with the shift of the thermal degradation to higher temperatures after removing the less stable fractions from the humic complexes.     

Advanced solid phase extraction using molecularly imprinted polymers for the determination of quercetin in red wine

Solid phase extraction (SPE) based on molecularly imprinted polymers (MIPs) is a novel approach for sample preparation and preconcentration, gaining increased interest in the fields of environmental, clinical, and food analysis. The first application combining MIPs with SPE for advanced beverage analysis is reported. MIPs for the flavonoid quercetin have been generated, using quercetin as a template molecule in a self-assembly approach and yielding imprinting of 1% of the used template. The MIP achieved a capacity of 0.4 g quercetin per gram polymer and a recovery rate of 98.2%. The application of these synthetic receptors as SPE material for the selective extraction and preconcentration of quercetin from synthetic and red wine samples was investigated. Red wine samples from a French Merlot were directly applied onto the SPE cartridge. The collected fractions were analyzed by high-pressure liquid chromatography. For verification of the obtained results, a similarly prepared nonimprinted polymer and a classical octadecyl silane reversed-phase cartridge were applied as the SPE matrix during control experiments. The MIP enabled the selective extraction of quercetin from a complex matrix, such as red wine, spiked with 8.8 mg per liter quercetin, demonstrating the potential of molecularly imprinted solid phase extraction for rapid, selective, and cost-effective sample pretreatment.     

Chemically Modified Silica Gel with N-(2-aminoethyl)-Salicylaldimine for Simultaneous Solid Phase Extraction and Preconcentration of Cu(II), Ni(II), Cd(II) and Zn(II) in waters

In this study, N-(2-aminoethyl)salicylaldimine bonded silica gel was synthesized and characterized using Fourier transform infrared and C, H, N elemental analysis. The analytical conditions such as the pH and volume of the solution, flow rates of the sample solution and the type of eluent to achieve the simultaneous preconcentration of Cu(II), Ni(II), Cd(II) and Zn(II) were optimised using the modified silica gel loaded column using a solid phase extraction technique. Samples (50?C500?ml) containing metal ions at optimal pH of 8 were passed through the column filled with the modified silica gel at 7?ml min^?1 and then elution was achieved using 5?ml of 0.25?M HCl. The concentrations of metal ions in the eluates were determined using flame atomic absorption spectrometry (FAAS). The effects of matrix ions were also studied and none of the major ions interfered to the proposed method. The accuracy of the developed method was validated using a certified reference water sample (Ontario Lake water, NWTMDA-54.4). The method was successfully applied to the analysis of various natural water samples. The adsorption capacities of the modified silica gel for Cu(II), Ni(II), Cd(II) and Zn(II) ions were determined and found to be 0.332, 0.261, 0.130 and 0.375?mmol g^?1, respectively.     

Influence of humate-solute interactions on aqueous heavy metal ion levels

The extent to which heavy metal ions (Cu, Pb, Zn, Cd) are removed from aqueous solution by humic acid suspensions has been found to vary with solution pH, concentration of competing cations, nature of the organic material, and the complexing power of any ligands present. The amount adsorbed in acid media increased with pH until the threshold value required for partial dissolution of the solid, and formation of soluble metal humates, was exceeded The adsorption maximum pH, and the apparent capacity at lower values, varied with the substrate used and cation being sorbed. The affinity order sequence, as derived from adsorption isotherm studies, was Pb > Cu > Cd > Zn ? Ca > Mg. The retention of metal ion by the solid was reduced in the presence of ligands, with zero uptake occurring when the soluble complexes formed had a greater effective stability than those resulting from humic acid-metal ion interactions. With environmental systems having a high organic content, the humic acid component can play a dominant role in determining the final distribution of metal ions, and for predictive purposes, investigation of the effect of pH on uptake is more informative than measurement of adsorption isotherm parameters, since the observed trends reflect differences in functional group properties, relative affinities and solubility effects. The curves have minima which fall within the pH spreads encountered in natural systems, and small pH changes can cause significant variations in solution levels.     

Selective solid-phase extraction of Sudan I in chilli sauce by single-hole hollow molecularly imprinted polymers

A new single-hole hollow molecularly imprinted polymer (SHH-MIP) was prepared by multistep seed swelling polymerization using Sudan I as template molecule and successfully applied to selective solid-phase extraction (SPE) of Sudan dyes in chilli sauce samples. The polymers possessed high specific surface area obtained by nitrogen adsorption and good thermal stability without decomposition lower than 380 °C by thermogravimetry analysis. Much higher binding capacity was exhibited than with irregular solid MIP prepared by bulk polymerization, because most of the binding sites were located in the surface of the polymers, facilitating template removal and mass transfer. Accordingly, the SHH-MIP was employed as SPE adsorbent for chilli sauce analysis and offered high recoveries for Sudan I in the range of 87.5-103.4% with the precision of 1.94-5.33% at three spiked levels. The SHH-MIP with high selectivity and high stability was demonstrated to be potentially applicable for high selective preconcentration and determination of trace Sudan dyes in complicated samples.     

Kinetic, Isotherm and Thermodynamic Analysis on Adsorption of Cr(VI) Ions from Aqueous Solutions by Synthesis and Characterization of Magnetic-Poly(divinylbenzene-vinylimidazole) Microbeads

The magnetic-poly(divinylbenzene-1-vinylimidazole) [m-poly(DVB-VIM)] microbeads (average diameter 53-212?μm) were synthesized and characterized; their use as adsorbent in removal of Cr(VI) ions from aqueous solutions was investigated. The m-poly(DVB-VIM) microbeads were prepared by copolymerizing of divinylbenzene (DVB) with 1-vinylimidazole (VIM). The m-poly(DVB-VIM) microbeads were characterized by N(2) adsorption/desorption isotherms, ESR, elemental analysis, scanning electron microscope (SEM) and swelling studies. At fixed solid/solution ratio the various factors affecting adsorption of Cr(VI) ions from aqueous solutions such as pH, initial concentration, contact time and temperature were analyzed. Langmuir, Freundlich and Dubinin-Radushkvich isotherms were used as the model adsorption equilibrium data. Langmuir isotherm model was the most adequate. The pseudo-first-order, pseudo-second-order, Ritch-second-order and intraparticle diffusion models were used to describe the adsorption kinetics. The apparent activation energy was found to be 5.024?kJ?mol(-1), which is characteristic of a chemically controlled reaction. The experimental data fitted to pseudo-second-order kinetic. The study of temperature effect was quantified by calculating various thermodynamic parameters such as Gibbs free energy, enthalpy and entropy changes. The thermodynamic parameters obtained indicated the endothermic nature of adsorption of Cr(VI) ions. Morever, after the use in adsorption, the m-poly(DVB-VIM) microbeads with paramagnetic property were separeted via the applied magnetic force. The magnetic beads could be desorbed up to about 97% by treating with 1.0?M NaOH. These features make the m-poly(DVB-VIM) microbeads a potential candidate for support of Cr(VI) ions removal under magnetic field.     

Concentration of Polycyclic Aromatic Hydrocarbons and Base Cations in the Air and Drinking Water of the Silesian Rehabilitation Centre,Poland

Investigations were carried out to assess air and potable waterpollution with selected highly bioactive organic compounds,including polycyclic aromatic hydrocarbons (PAHs), in theenvironment of the Silesian Rehabilitation Centre, Repty ?l?skie, Poland. The results were compared with those forother towns of the region.Solid phase extraction (SPE) and capillary gas chromatography(CGC) with flame ionization detector (FID) and mass spectrometer(MS) were used for a qualitative-quantitative analysis. Moreover, water concentration of calcium, magnesium ions andnitrite ions were measured by spectrophotometric methods. Thequality of the Centre environment air and water found to besatisfactory and better than that in the other sampling sites which were studied for all the parameters.

     

Gas chromatographic-mass spectrometric method for the analysis of dimethomorph fungicide in dried hops

An analytical method for the determination of dimethomorph [(E,Z)-4-[3-(4-chlorophenyl)-3-(3,4-dimethoxyphenyl)acryloyl]morpholine] residues in dried hops was developed utilizing liquid-liquid partitioning, automated gel permeation chromatography (GPC), Florisil and aminopropyl solid phase extraction (SPE) column cleanups, and gas chromatography (GC) with mass selective detection (MSD). Method validation recoveries from dried hops ranged from 79 to 103% over four levels of fortification (0.1, 1.0, 5.0, and 20 ppm). Control and dimethomorph-treated hop samples collected from three field sites had residue levels of <0.10 and 4.06-17.32 ppm, respectively. The method was validated to the limit of quantitation at 0.10 ppm. The limit of detection for this method was 0.045 ppm.     

Interactive Sorption of Metal Ions and Humic Acids onto Mineral Particles

The sorption of metal ions (Pb2+, Zn2+ and Cu2+) and soil humic acids (HA) from aqueous solutions onto mineral particles (sand, calcite and clay) was investigated using a batch equilibrium system. The sorption reactions in two- component systems (heavy metals-mineral particles and humic acids- mineral particles), as well as interactions in three-component system (heavy metals-humic acids-mineral particles) were examined. Results showed that the presence of humic acids, dissolved or bound onto mineral surfaces, considerably influenced the fixation of heavy metals. The various effects, depending on mineral type, humic concentration and specific metal-ion, were observed in three- component system. Sorption of Cu2+-ions on all minerals studied rapidly increased as the concentration of dissolved HA increased. The amount of Pb2+-ions sorbed on sand slightly decreased, while on kaolin increased between 15 and 20%. Sorption of Zn2+-ions on all minerals studied decreased at pH 4. At pH 5.5 the sorption of Zn2+-ions onto calcite decreased, while on kaolin and sand increased as a function of the humic acid concentration giving the curve with maximum at c(HA) = 2.5 mmol C L-1. At pH 6.5 sorption onto kaolin and sand increased. This effect occurs as a result of the conditional stability constant of Zn-HA complexes increasing at higher pH which in turn promotes the chelation of Zn2+-ions to mineral- bound humic substances. The enhanced sorption of metal ions from the aqueous phase in three-component systems is not only the result of mineral sorption of free metals but also the result of chelation with HA sorbed on the mineral surface.     

Applications of the Continuous Flow Stirred Cell (CFSC) technique to adsorption of zinc, cadmium and mercury on humic acids

We discuss the sorption of Zn (II), Cd (II) and Hg (II) by H-saturated humic substances using the continuous flow stirred cell technique. Sorption isotherms were determined at two different flow rates and for each of two different humic preparations using radioisotopes of the above metals.

Sorption of mercury using two different anions (NO₃⁻ or Cl⁻) revealed very different behaviour indicating the importance of the anionic species in such studies. In general the adsorption isotherms determined for these metal ion-humic interactions were similar to those predicted from the Langmuir equation.

The data indicate two distinct mechanisms for the adsorption of the metal ions by the humic preparations. At the low metal concentrations used in all the CFSC measurements, sorption occurs without the release of a significant quantity of H⁺ ions, presumably by chelate formation. At higher metal concentrations interactions also involve the release of H⁺ ions from carboxyl-type exchange sites and another model may be required.     

Adsorptionsisothermen als Regelgrößen beim Transport von Schwermetallen in Böden

Adsorption isotherms as regulators controlling heavy metal transport in soils The adsorption and desorption of Pb²⁺ and Cd²⁺ from equilibrium solutions with heavy metal contents up to 5000 μg/1 were determined in bulk experiments for soil samples from an acid Braunerde developed in loess loam, taken from the humic surface layer of the mineral soil (0–10 cm) and from the subsoil fairly free from organic matter (30–40 cm). Pb and Cd in solutions were determined by flameless atomic absorption spectroscopy. Pb was more strongly retained in the solid phase than Cd, and higher amounts of heavy metals were retained in the humic surface soil than in the subsoil free of organic matter. In the case of Pb adsorption/desorption showed slight hysteresis in the subsoil. The quantity/intensity (Q/I) relationships found in the experiments could be described by the Freundlich equation. The Q/I-relationships were substituted in the general transport equation. With a simulation model the transport of Pb and Cd through the soil with vertical water flow was calculated by the use of the Continuous Simulation Programming Language (CSMP). Two different cases were considered: a small, continuous increase in the heavy metal input of the soil surface, and a high, instantaneous peak input. Simulation of the transport and distribution mechanisms induced by the inputs over a period of 10 years showed strong retention of lead in the surface layer and consequently a strong damping of the concentration peak in the soil solution. In contrast, cadmium is distributed more quickly over the whole profile, yet the concentration peak in the solution phase is, too, damped considerably in the surface layer by temporary retention in the solid phase. The results of the simulation runs are in accordance with the situation in real soils where often strong accumulation of Pb is found in the top soil, while Cd is accumulated only slightly.     

Estimating Sorption Affinities of Heavy Metals on Humic Acid and Silica using a Constant Capacitance Model

The adsorption of heavy metals [cadmium (Cd ²⁺), cobalt (Co²⁺), nickel (Ni²⁺), zinc (Zn²⁺), and lead (Pb)] and calcium (Ca²⁺) on humic acid and silica were investigated to understand the adsorptive selectivities of heavy metals on the constituents of soil. The experiments for the adsorption of Cd and Pb were carried out in a 0.1 mol L^?1 (M) sodium nitrate (NaNO₃) background solution, whereas those for the other metals were done in a 0.1 M sodium chloride (NaCl) solution. The adsorptive affinities of the metal ions on the humic acid and silica were ranked by the intrinsic surface complexation constants [K _m ¹(int)] that were calculated approximately from the adsorptive data using a constant capacitance model. The log [K _m ¹(int)] values of the metals were in the order Zn²⁺(?2.29) > Cd²⁺(?2.41) > Co²⁺(?2.74) > Ni²⁺(?2.92) ?> Ca²⁺ (?3.33) for the humic acid and Zn²⁺(?4.23) > Cd²⁺(?4.49) > Ni²⁺(?4.51) ? Co²⁺ (?5.99) > Ca²⁺(?6.37) for silica.     

KINETICS OF ION EXCHANGE IN SOIL ORGANIC MATTER

The rate of ion exchange of humic substances (humic acid and peat in the hydrogen form) with lead ions was investigated. As in processes occurring in nature, the lead solution was added in all our experiments slowly and continuously at a constant feed rate to the well-stirred water-suspended humic substances. Control experiments were also performed with a well-defined synthetic ion- exchange resin (carboxylic acid type). The uptake of divalent lead ions by the humic substance and by the ion-exchange resin, which is coupled with the release of an equivalent amount of hydrogen ions, proceeds as a function of time according to a characteristic sigmoidal curve. During the initial phase, the rate of ion exchange increases continuously with time, even though the humic substances become more saturated with lead ions. Theoretical calculations, based on a film diffusion process as the rate-determining step, show that this behaviour should indeed be observed if any solution, containing exchangeable counter ions, is added continuously to an ion exchanger suspended in a solvent. The theory predicts further, in agreement with the experimental results, that in the initial phase of the ion-exchange reaction, the uptake of the counter ions is proportional to the square of the time. If experiments are performed with different rates of addition of the counter ions, the time required to exchange a certain amount of counter ions should be proportional to α^-1/2, where α is the rate at which the ions are added. This prediction is also in accordance with the experimental results.     

The effect of pH on the retention of Cu,Pb, Zu and Cd by clay-humic acid mixtures

The amount of metal ion sorbed by the solids increased with increasing pH over the range 3 to 6, and with mixtures of clay-cellulose or illite-humic acid the uptake corresponded to the direct addition of individual substrate adsorption values. When the humic acid samples were admixed with Na⁺-form kaolinite or montmorillonite, there was some reduction in adsorption, attributable to component interactions, and this effect was most marked in the presence of Cu and Zn ions. In alkaline media there was competition between the ability of the organic material to form soluble metal humates and the tendency of the clays to strongly retain the sparingly soluble metal hydroxy species formed at pH > 6. In most systems studied retention by the solid phase predominated. In the presence of tannic acid there were distinct pH regions in which the four metal ions formed compounds of limited solubility, with the pH for maximum precipitation ranging from 4.5 (Cu) to 7 (Cd). The introduction of clay suspensions increased the amount precipitated/sorbed in these pH regions. The amount of Cu, Pb, Zn or Cd retained by mixed suspensions varied markedly with pH, nature of the clay and the chemical nature of organic components.     

An Experimental and Modelling Study of Cu2+ Binding on Humic Acids at Various Solution Conditions. Application of the NICA-Donnan Model

Humic substances are characterized by a strong binding capacity for both metals and organic pollutants, affecting their mobility and bioavailability. The understanding of the mechanisms of proton and metal binding to humic substances is of fundamental importance in geochemical modelling and prediction of cation speciation in the environment. This work reports results on copper binding on humic acids obtained through a thorough experimental and modelling approach. Two humic acids, a reference purified peat humic acid isolated by the International Humic Substances Society (IHSS) and a humic acid from a Greek soil, were experimentally studied at various pH values (4, 6 and 8), humic acid concentrations (ranging from 20 to 200 mg?L^?1) and ionic strength (0.1 and 0.01 M NaNO₃). The binding of copper to humic acids was determined over wide ranges of copper ion concentrations using a copper ion selective electrode. The copper binding isotherms obtained at different conditions have shown that copper binding is dependent on the pH and ionic strength of the solution and on the concentration of both humic acids. Copper binding experimental data were fitted to non-ideal competitive adsorption NICA-Donnan model and the model parameter values were calculated. Both Cu²⁺ and CuOH⁺ species binding to humic acid with different binding affinities were considered. Two sets of the NICA-Donnan parameters have been calculated: one for humic acid concentrations of ??100 mg?L^?1and one for humic acid concentration of 20 mg?L^?1. The meaning of the parameters values for each concentration level is also discussed.     

Iron oxidation in different types of groundwater of Western Siberia

Background, aim, and scope  The groundwaters of Western Siberia contain high concentrations of iron, manganese, silicon, ammonium, and, in several cases, hydrogen sulfide, carbonic acids, and dissolved organic substances. Generally, the groundwaters of Western Siberia can be divided into two major types: one type with a relatively low concentration of humic substances and high hardness (water of A type) and a second type with a relatively low hardness and high concentration of humic substances (water of B type). For drinking water production, the waters of A type are mostly treated in the classical way by aeration followed by sand bed filtration. The waters of B type often show problems when treated for iron removal. A part of iron practically does not form the flocs or particles suitable for filtration or sedimentation. The aim of this work was to determine the oxidizability of Fe(II), to characterize the iron colloids, and to investigate the complexation of the iron ions with humic substances and the coagulation of the iron colloids in the presence of dissolved organic matter. Materials and methods  Water samples of the A and B types were taken from bore holes in Western Siberia (A type: in Tomsk and Tomsk region, B type: in Beliy Yar and Kargasok). Depth of sampling was about 200 m below surface. The oxidation of the groundwater samples by air oxygen and ozone was done in a bubble reactor consisting of a glass cylinder with a gas-inlet tube. To produce ozone, a compact ozone generator developed by Tomsk Polytechnic University was used. For the characterization of the colloids in the water of B type, the particle size distribution and the zeta potential were measured. To investigate the formation of complexes between iron and humic substances in the water of B type, size exclusion chromatography was used. The coagulation behavior of iron in the presence of dissolved organic substances was investigated at different pH values. The agglomerates were detected by measuring the optical density using a UV-Vis spectrometer. Results  Ozone showed, as expected, a faster oxidation of Fe(II) than air oxygen. The rate constants for Fe(II) oxidation were not much different for the waters of A and B types when the same oxidation process was used. However, the removal of iron after oxidation and filtration was higher in the water of A type than in the water of B type. No evidence for the formation of soluble complexes between iron and humic substances were found. In the water of A type, the coagulation process started at pH = 4.5 and accelerated with increasing pH value. In the water of B type, the coagulation of colloids occurred only at pH = 11 and higher. Discussion  The oxidation experiments indicated no major effect of dissolved organic carbon concentration on the kinetics of Fe(II) oxidation. In contrast to this, the humic substances showed a significant influence on the aggregation behavior of the iron hydroxide colloids. Due to the sorption of humic substances on the iron hydroxide colloids, they were highly stable in the pH range between 4.5 and 10. The particle size measurements confirmed the presence of small colloids in the water of B type. In contrast to this, the iron hydroxide colloids aggregated rapidly at pH = 11. Conclusions  The results showed a great influence of humic substances on the iron removal from groundwaters of Western Siberia with high organic content. The sorption of humic substances on the iron colloids does not obviously allow their coagulation and formation of flocs suitable for filtration or sedimentation. Recommendations and perspectives  By treatment of groundwaters containing high amounts of humic substances, some problems with the removal of iron are likely to occur. To increase the effectiveness of iron removal, the surface coating and pH-dependent charge effects should be taken into account by the selection and optimization of water treatment processes. The iron colloids coated by humic substances should be separated from the water phase by membrane filtration or by flocculation followed by filtration through different solid materials.     

Analysis of chlorothalonil and three degradates in sediment and soil

A method has been developed for the simultaneous extraction of chlorothalonil and three of its degradates (4-hydroxy-2,5,6-trichloroisophthalonitrile, 1-carbamoyl-3-cyano-4-hydroxy-2,5,6-trichlorobenzene, and 1,3-dicarbamoyl-2,4,5,6-tetrachlorobenzene) from soils and sediments; the compounds were extracted using sonication with acetone and isolation of the parent compound and matrix interferences from the degradates by solid phase extraction (SPE). The chlorothalonil fraction underwent further coextracted matrix interference removal with Florisil. The degradates were derivatized with N, O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS). All compounds were analyzed by gas chromatography-mass spectrometry (GC-MS). Recoveries on a spiked (20 and 200 microg kg (-1)) sediment ranged from 80% to 91% with calculated limits of detection of 1-5 microg kg (-1) dry weight sediment. An additional 20 sediment samples were collected in watersheds from the Southeastern United States where chlorothalonil is used widely on peanuts and other crops. None of the target compounds were detected. Laboratory fortified recoveries of chlorothalonil and its degradates in these environmental sediment samples ranged from 75% to 89%.     

长期使用含铜杀真菌剂对苹果园土壤中自由态铜离子、固相铜形态分级以及微生物活性的影响

Soil samples were collected from apple orchards 5,15,20,30,and 45 years old,and one adjacent forest soil was used as reference to investigate the free Cu2+ion activity in soil solution and the soil Cu fractionation in the solid phase following long-term application of copper fungicide,Bordeaux mixture,in apple orchards and to investigate the relationships among soil free Cu2+ions,Cu fractionation and soil microbial parameters.The total Cu concentration in the orchard soils varied from 21.8 to 141 mg kg-1,increasing with the orchard age,and the value for the reference soil was 12.5 mg kg-1.The free Cu2+ion concentrations in the soil solutions extracted by 0.01 mol L-1 KNO3 ranged from 3.13×10-8(reference)to 4.08×10-6 mol L-1(45 years-old orchard).The concentration of Cu complexed in the fulvic fraction increased with orchard age from 5.16 to 52.5 mg kg-1.This was also the case for other soil Cu fractions except the residual one.The residual soil Cu remained practically constant,ranging from 4.28 to 5.66 mg kg-1,suggesting that anthropogenic soil Cu mainly existed in the more labile active fractions.Regression analyses revealed that both the free Cu2+ions in the soil solution and the humic acid-complexed Cu fraction in the solid phase were strongly related with soil microbial parameters.