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104105Materials and methodsGrowth of plant materialChara corallina is a freshwater alga with single giant internodal cells which can be typically8cm long.The internodal cells used in the study were collected from established cultures growing in indoor tanks.Inter-nodal cells were cut from neighbouring cells,washed in distilled water and bathed in artificial pond wa-ter(APW:0.2m M K2SO4,1.0m M NaCl,1.0m M CaSO4,5.0m M Mes-NaOH,pH6.0)prior to experi-mental treatment.Internodes were transferred to APW solutions containing either0.5m M or1m M NH4+(as (NH4+)2SO4)for a minimum of two days before the measurements of intracellular NH4+.Chara sap analysisSingle Chara internodal cells were transferred from the nutrient solution to a glass microscope slide and one end was cut using a scalpel allowing the sap to exude onto the slide.A known volume(3–9µL)of vacuolar sap was collected using a Microcap(Drum-mond Scientific Co.,Broomall,PA)and immediately frozen in liquid nitrogen for storage.Stored samples were defrosted and analysed for NH4+using a Dionex Model DX500HPLC,fitted with an Ionpac Fast Cation1column(Dionex,Camberley,UK)and detected by non-suppressed conductivity.NH4+selective electrode measurementsDouble-barrelled micropipettes were prepared and sil-anized as described previously(Miller and Zhen, 1991).The composition of the ammonium sensor cocktail was10%(w/w)nonactin(ammonium iono-phore I,Fluka09877),1%potassium tetrakis(4-chlorophenyl)borate and89%dibutyl sebacate.Fi-nally,20%high molecular weight polyvinyl chloride was added to this cocktail and the mixture dissolved in5volumes of tetrahydrofuran(THF).All chem-icals were of analytical grade and were purchased from Fluka,except THF(Sigma).After a minimum of48h at room temperature in silica-dried air,the ammonium-selective membrane had gelled in the tip and each barrel was backfilled with electrolyte solu-tion.The ammonium-selective barrel was backfilled with100m M NH4Cl using a29-G needle attached to a1mL disposable syringe.The reference barrel was backfilled with200m M NaCl using a Microfil needle (World Precision Instruments(WPI),Sarasota,USA).The calibration and intracellular measurements were made using a high-impedance differential electrometer (model FD223,WPI)which was connected to a PC as described previously(Walker et al.,1995).The circuit was completed with a bath electrode(Flexref,WPI) which was placed in the APW bathing the Chara cells.The NH4+barrel was calibrated using solutions containing a constant background activity of K+(a K) of72m M and buffered at pH6.0.For intracellular recordings,Chara internodal cells were clamped in a 5mL perspex chamber and perfused with APW solu-tion.Microelectrodes were calibrated for NH4+before and after intracellular recordings and the combined calibration data were used to produce a curve from which the intracellular a NH4was calculated. ResultsIn the presence of background cytosolic a K(Walker et al.,1996),the electrodes can detect NH4+activ-ities in the m M range(Figure1A).Calibrating these microelectrodes in this background gave a mean slope ±sd of55.6±5.0mV(sample size,n=47)per ten fold change in concentration and a detection limit of9.4m M(IUPAC,1994).However,when several calib-ration points are obtained around the detection limit, the practical use of the electrodes can be extended to around1m M by using the non-linear portion of the calibration curve(see Figure1A).In Chara cells growing in APW containing0.5m M NH4+,the electrode measurements were almost at the limit of detection(Figure1B).The electrodes were more successful when cells were incubated in APW supplemented with1m M NH4+.Intracellular meas-urements of a NH4in Chara cells were in the range 6–39m M and these measurements could be separ-ated into two populations(Figure1B).However,the membrane potential measurements obtained with each a NH4value did not separate into two populations(data not shown).For cells placed in the higher NH4+con-centration,HPLC analysis of whole cell sap samples gave a mean value±sd for NH4+concentration of 25.1±1.3(n=4)m M.DiscussionWe have developed an ion-selective microelectrode, based on the sensor nonactin,that allows the direct measurement of intracellular a NH4in the millimolar106range.In the cytosol of plant cells,these electrodes can measure intracellular a NH4down to1m e of the electrodes on cells of Chara incubated in APW supplemented with1m M NH4+,gave two populations of measurements(Figure1B),with means of7.3and 30.8m M.It is probable that these represent the cytosol and vacuole,respectively.In Chara,the vacuole oc-cupies90–96%of the intracellular volume and the cytoplasm4–10%(see references in Miller and Zhen, 1991).This means that the whole-cell sap samples must be dominated by the vacuolar a NH4and thus the HPLC analysis will measure vacuolar paring the results of the sap sample analysis with the electrode measurements suggests that the higher population in Figure1B must represent vacu-olar impalements.Therefore,the cytosolic electrode measurements are the lower population with values around7m M.Further evidence for this assignment of these two populations comes from previous work measuring vacuolar concentrations of NH4in Chara growing in similar conditions which showed a mean vacuolar concentration of31m M(Ryan and Walker, 1994).Using14C-labelled methylammonium as an analogue for NH4+,these authors calculated the cyto-plasmic concentration to be18m M,but suggested that further compartmentation into acidic organelles may give2mm in the actual cytosol(Ryan and Walker, 1994).Another advantage of the electrode technique is that the measurement directly reports cytosolic a NH4 and so does not require such assumptions to be made.These are thefirst intracellular measurements of NH4+using NH4+-selective microelectrodes and they demonstrate the feasibility of the method.The dis-tribution of NH4+in Chara cells reveals a4-fold accumulation of ammonium in the vacuole compared to the cytosol.The future use of triple-barrelled mi-croelectrodes incorporating a pH-selective barrel will allow the unequivocal measurement of vacuolar and cytosolic NH4+activities.In these electrodes,the pH barrel allows identification of the compartment in which the electrode tip is located(Walker et al., 1995).Measurements of compartmental pH are also important as the equilibrium between NH3and NH4+ will be determined by this parameter.Finally,even though cytosolic K+activity is regulated in higher plants(Walker et al.,1996),it may be important to check with K+-selective microelectrodes that the cytosolic K+activity in Chara cells does not change when NH4+is added to the APW. AcknowledgementsWe wish to thank John Andralojc and David Carden for their help with the HPLC analysis.This work was funded by EU grant number BIO4-CT97-2310.IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK. ReferencesHenriksen G H,Bloom A J and Spanswick R M1990Measure-ment of netfluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes.Plant Physiol.93,271–280.IUPAC1994Recommendations for the nomenclature of ion-selective electrodes(IUPAC recommendations1994).Pure& Appl.Chem.66,2528–2536.Kronzucker H J,Siddiqi M Y and Glass A D M1995Compartment-ation andflux characteristics of ammonium in spruce.Planta196, 691–698.Miller A J and Zhen R-G.1991Measurement of intracellular nitrate concentrations in Chara using nitrate-selective microelectrodes.Planta184,47–52.Ryan P R and Walker N A1994The regulation of ammonia uptake in Chara australis.J Exp.Bot.45,1057–1067.Scholer R P and Simon W1970Antibiotika-Membranelektrode zur selektiven Erfassung von Ammoniumionenaktivitäten.Chimia 24,372–374.Tyerman S,Whitehead L F and Day D A1995A channel-like trans-porter for NH4+on the symbiotic interface of N2-fixing plants.Nature378,629–632.Walker D J,Smith S J and Miller A J1995Simultaneous meas-urement of intracellular pH and K+or NO3−in barley root cells using triple-barreled,ion-selective microelectrodes.Plant Physiol.108,743–751.Walker D J,Leigh R A and Miller A J1996Potassium homeostasis in vacuolate plant A93,10510–10514.Wang M Y,Siddiqi M Y,Ruth T J and Glass A D M1993 Ammonium uptake by rice roots.I.Fluxes and subcellular distribution of13NH4+.Plant Physiol.103,1249–1258.。

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ASSESSMENT OF THE HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION IN THE BAIX LLOBREGAT(CATALONIA,NE SPAIN)DANUTA ZIMAKOWSKA-GNOI´NSKA1,JAUME BECH2and FRANCISCO J.TOBIAS21International Centre of Ecology PAS,Dziekanów Le´s ny n.Warsaw,05-092Łomianki,Poland 2Catedra de Pedologia,Facultat de Biologia,Universitat de Barcelona,Spain(Received21July1998;accepted13January1999)Abstract.A main goal of investigations is to determine could a soil respiration be an indicator of the soil pollution.In this case a measured level of the soil oxygen of its pollution.It also means that the pollution reduces biological processes soil samples were taken from polluted and non-polluted places in the Baix(Catalonia, NE Spain).Soil samples were taken from the top of soil(0–5cm)without a litter.Soil analysis were done,determining percentage shares of coarse fragments,coarse sand,fine sand,coarse silt,fine silt, clay,CaCO3,organic matter as well as water pH and conductivity CE(1:5[mS cm−1]).Also were determined(in mg kg−1)quantities of heavy metals,as Fe,Al,Mn,Zn,Cr,Ni,V,Cu,Cd,Pb.The soil respiration was investigated in temperatures15and30◦C and with controlled humidity.The respiration in30◦C is number of times greater then in15◦C both for polluted and non-polluted soils.Particularly high coefficients of correlation between the soil respiration and soil pollution in polluted soils were obtained for Pb:r=0.75in15◦C and r=0.98in30◦C;for Ba:0.90and0.57;for V:0.99and0.81.In non-polluted soils highest correlation coefficients are for Pb:r=0.70in15◦C; Fe:0.60and0.72;Al:0.68and0.64;Mn:0.51and0.66;Ba:0.63and0.61;Cr:0.94and0.70;Ni: 0.64and0.65;Cu:0.69and0.48;as well as V:0.62in15◦C;and Cd:0.69in15◦C.This way the soil respiration could be a good indicator of the soil pollution.Keywords:biological activity of soil,constant-pressure volumetric respirometer,heavy metals,respirom-etry methods,soil,soil degradation1.IntroductionThe aim of this study is to try correlating the effect of metal pollution and the respiration of soil in two areas–one polluted and a neighbour non-polluted area.An unfavourable influence of heavy metals on soil environment is well known. Many autors indicate their particularly destructive influence on soil microflora (Strojan,1975;Freedman and Hutchinson,1980).An activity of soil microbial population is also influenced by heavy metals as well as an intensity of biological processes in edaphon(Nordgren et al.,1983,1985,1986).Scientists are looking for relatively sensitive indicator of degradating processesin soil environment.An intensity of biological processes is generally estimated by quantity of ejected carbon dioxide(Witkamp,1969;Edwards,1974;Elmholt,1992;Environmental Monitoring and Assessment61:301–313,2000.©2000Kluwer Academic Publishers.Printed in the Netherlands.302 D.ZIMAKOWSKA-GNOI´NSKA ET AL.Gildon et al.,1993;Nakadai et al.,1993).This indicator covers both metabolisms –aerobic and anaerobic.Investigation of oxygen respiration could give more sen-sitive and explicit indication.Investigations of Fischer(1996)show that measured changes of the carbon dioxide ejection are less sensitive to changes of temperature and humidity of the soil as well as to changes of the soil pollution.Probably,the carbon dioxide ejection is a result of two simultanous processes–aerobic and anaerobic.The oxygen intake is connected only with aerobic process.In presented paper a respirometry method was applied to oxygen respiration measurements of polluted and non-polluted soils from the Barcelona area.Results have to show an influence of pollution on biological activity of soils. These results are also influenced by other physical and biological characteristics of investigated soil sample.The main objective of said study is to prove that res-piration of soil depends of soil pollution and moreover indicates the extent of the pollution harmfulness on soil biological processes.2.Area of Studies,Climatic Conditions and VegetationStudied areas are located in the Baix Llobregat,about20km SW of Barcelona and inside of the metropolitan area of the city.For purposes of this paper two areas were taken into account.Thefirst one,named‘non-polluted area’(Figure1),belongs to mountainous side of Gavá,Viladecans and Torelles de Llobregat.This area can be considered as non-polluted area because it is forest zone with relatively low anthropogenic impact.The second one,named‘polluted area’(Figure2),is located SW of Gaváand Viladecans and represents degradated part of metropolitan area.The climate of area is typically Mediterranean with mean annual temperature of 16◦C and mean annual rainfall(mostly in spring and in autum)of650mm.Spring and autumn precipitation are135and240mm,respectively;summer precipitation is only105mm.The summer is hot and dry with mean temperature over20◦C from June to September.The mean January temperature is9±10◦C.According to the Koppen climatic classification,it is‘Csa’;pedoclimic regimes are xeric(SMR) and thermic(STR).A main vegetation in the non-polluted area are low‘garriga’scrubs,Quercus coccifera,Pistacia lentiscus,Olea europea,Chamaerops humilis,Rhamnus ly-cioides,Rosmarinus officinalis,Erica multiflora,Ampelodesma mauritanica,Cer-atonia siliqua,Fumana laevipes,frequently accompanied by Pinus halepensis.In degradated area are herbes such as Hyparrhenia hirta,Sonchus tenerrimus, Brachypodium ramosum,Brachypodium phoenicoides,Foeniculum vulgare,Pso-ralea bituminosa,Inula viscosa etc.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION303Figure1.The sampling points of the non polluted area are indicated.Number2shows the location of the polluted area.304 D.ZIMAKOWSKA-GNOI´NSKA ET AL.Figure2.Sampling points in the polluted area:enlarged view(A:composting plant,B:used cars dump).3.Material and Methods3.1.M ATERIALWere studied22topsoil samples(0–5cm,without litter).Eleven samples were taken from non-polluted area(Figure1)and other eleven from polluted area(Fig-ure2).Those from polluted area were taken from three transects–two near a composting plant and one near an used car dump.3.2.A NALYTICAL METHODSSoil samples were air-dried and passed through a2mm sieve to obtain a‘fine earth’(fraction less then2mm).Sieving has also to eliminate plant roots and other plant parts.In this‘fine earth’shares of coarse fractions(>0.2mm)andfine fractions (<0.2mm)were determined for sands and for silts.In the‘fine earth’the organic carbon content was determined using the Walkley-Black dichromate acid oxidation method.Soil pH values were determined for soil solution suspensions in distilled water and in the1N KCl.This determination was done potentiometrically with a glass electrode.Calcium carbonate contents were determined using the acid neutralization method.Particle-size distribution was determined using the pipette method and by sieving sand fractions.Conduc-HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION305 timetry was determined in1:5soil to water extract.Total nitrogen contents were determined using the Carlo Erba1500elemental analyzer.The‘fine earth’was grounded in a tungsten carbide mill,dried at105◦C and then used to determine metal contents.Soil samples(3g)were extracted with28ml of aqua regia(HNO3to HCl as1:3)during two hours at130◦C after a predigestion of16hr at a room temperature.Metal concentrations were measured infiltered extract,using the Polyscan61E spectrometer.3.3.S OIL RESPIRATIONSoil respirations were determined in laboratory because in this case experimental conditions are more stabile than in the case offield measurements.Soil samples were taken from the top of soil(0–5cm)without a litter,trans-ported to laboratory and stored there in a cooler in temperature3◦C.Soil samples were sieved to eliminate plant roots,as recommends Anderson(1982).Soil respirations were measured using constant pressure volumetric respirime-ters,modified by Klekowski(1975).Respirometers were placed in water ther-mostate with temperature controlled with±0.1◦C precision.Experiments were done in two temperatures:15and30◦C.In respirometric vessels were placed30g samples of soil.Samples were moist-ened up to60%of humidity adding sufficient quantity of boiled cold water.Moist-ened samples were adapted during12hr to the temperature of experiments.Mea-surements of oxygen consumption were done for the next3hr.After respiration and after drying in temperature55◦C a dry weight of sample was weighted.An humidity of sample was determined from the ratio of dry weight to wet weight of sample.A share of organic matter in the soil sample was determined by combustion in a muffle oven in temperature450◦C.Results obtained when using this method of determination are more applicable to biological processes in the investigated soil.The oxygen consumption for a sample30g of the wet soil is expressed in mm3O2·h−1and per gram of the dry soil or per gram of the organic matter in mm3O2·h−1·g−1.Mean values and standard deviations were calculated for all experiments.The aim of this preliminary study is tofind simple correlations between heavy metal pollution and soil respiration in two areas:polluted and non-polluted.4.Results and DiscussionThe increase of antropogenic heavy metals in soils is significant on outskirts of industrial zones like the Metropolitan Area of Barcelona.In Table I are compared physico-chemical characteristics of sampled soils.In Table II are shown shares of heavy metals in the same soil samples.In polluted area in the Llobregat delta plane306 D.ZIMAKOWSKA-GNOI´NSKA ET AL.TABLE IPhysico-chemical data of soil samplesNo.Coarse Coarse Fine Coarse Fine Clay Organic pH N CaCO3CE1:5 fragments sand sand silt silt matter H2O(%)(%)(%)(%)(%)(%)(%)(%)(%)(mS cm−1)1 3.6254.7122.53 4.407.5010.85 3.827.290.1025.580.23029.8922.0220.0215.6622.4819.83 1.887.340.0923.400.5843 3.5735.6010.0234.430.0019.97 3.597.120.2417.850.764419.6142.8611.719.8225.4410.17 2.907.230.1116.650.804565.1635.8815.677.0721.3120.078.037.260.7218.82 2.368619.7144.8812.267.1317.4018.32 4.167.540.2115.200.240722.2430.8216.2012.6319.8520.51 6.097.530.3118.820.303833.5746.4212.287.8919.2514.169.177.450.1529.680.347937.3634.7014.5412.8719.6218.27 4.277.270.1623.890.27710 6.3318.4110.758.1739.3823.29 6.557.300.2831.130.551 1119.8217.988.817.5640.3925.279.637.210.4329.440.653 1232.1617.55 6.5612.9338.7724.19 3.707.260.197.850.517 1352.0615.00 6.3214.1342.1122.44 3.137.360.17 2.500.103 1465.5522.52 6.3911.0339.5920.4714.537.200.60 3.450.213 1511.1340.3441.50 4.44 6.277.45 6.097.060.23 2.610.104 1646.5816.07 4.63 5.3137.6936.307.58 6.470.49 4.040.135 1715.7014.128.7210.7931.7334.64 4.967.180.1847.740.157 1813.667.8169.649.18 5.787.59 3.13 6.920.09 1.550.089 1913.527.4238.6516.4918.8118.62 6.557.370.2411.430.204 2019.6115.0245.407.9013.5918.09 6.217.480.1811.540.231 2140.2826.0916.2610.1124.5023.0412.827.070.38 5.590.319 2250.5514.94 5.447.4931.4340.718.157.300.22 6.880.183main sources of pollution are composting plant and the used car dump.The non-polluted area is nearby hilly forest area.Point16placed in this area is an anomaly –its soil contains natural high share of metals.These tables confirm that the polluted area is really a polluted one and concen-tration of heavy metals grows when approaching to the plant and car dump.In paper Bech et al.(1994)were presented correlations between physico-chemical characteristics and concentrations of heavy metals.For example,conductivity(CE 1:5)positively correlates with concentration of heavy metals in the contaminated area,but not in noncontaminated area.Mean conductivity in polluted area is0.65mS cm−1.In extremely polluted point No.5conductivity is even2.37mS cm−1.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION307TABLE IIShares of heavy metals in soil samplesNo.%Fe%Al Mn Zn Cr Ni V Cu Cd Pb(ppm)1 1.56 1.35445.86197.0120.6616.1297.5871.930.71166.762206 2.24455.38122.1327.1920.69100.2833.040.4462.723 2.88 2.57575.72355.8050.0938.01128.49131.18 1.84241.304 3.12 2.34497.67898.4344.5628.79114.91196.33 4.46761.21513.64 2.961395.843770.36705.91130.17128.121093.7411.911970.656 1.98 1.78 4.38166.6324.0821.0298.9035.390.7465.067 3.08 2.24549.01537.1039.9335.21102.25112.85 1.30205.738 3.54 1.94610.201573.7155.3463.7383.16216.29 1.88458.669 2.32 2.36501.02391.9932.2024.13117.3858.310.76149.1710 2.22 2.48512.89199.0627.7622.89104.3745.200.4963.2911 2.15 2.48488.32247.5227.8922.96111.1629.140.9648.7212 4.11 3.10813.0877.6432.8437.7593.1025.200.3739.0813 4.60 3.68705.9484.1942.0544.21106.0327.270.3041.9714 3.47 2.79978.56214.3343.0638.25100.4033.090.5885.50 150.710.72103.4347.4511.608.7765.98 4.720.3028.04 1610.77 4.3313584.39111.4556.72111.15169.70129.82 1.2974.45 17 1.80 2.77426.8292.7127.9319.30117.8411.480.4740.94 180.870.98522.3323.2019.438.8879.07 3.980.1220.2619 1.79 2.20805.0652.0323.2519.9587.4015.550.3027.0020 1.68 1.851153.5742.6521.5915.1498.0310.020.3026.8621 3.64 1.74786.78128.3721.5036.3890.3358.600.6977.7122 4.31 4.16256.9178.3341.0442.74116.7225.17 1.0850.99In polluted area is observed considerable concentration of Fe,Mn,Zn,Be,Cr, Ni,V,Cu,Cd and Pb.In non-polluted area concentration of heavy metals is much lower and mean conductivity is only0.20mS cm−1.Share of organic matter decreases with increased concentration of heavy metals and increased salinization of soils.In the non-polluted area,natural Fe,Al,Zn,Cr,Ni,V,Cd and Pb show correla-tions with size fractions(negative correlations withfine sand and positive withfine silt and clay).In both areas metal contents generally correlate with each other:–in the non-polluted area these correlations are due to similar pedogonic processes and also to similar parent materials.308 D.ZIMAKOWSKA-GNOI´NSKA ET AL.Figure3.Oxygen consumptions of soil samples from contaminated areas:=15◦C, =30◦C.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION309Figure4.Oxygen consumptions of soil samples from contaminated areas:=15◦C, =30◦C.310 D.ZIMAKOWSKA-GNOI´NSKA ET AL.–in the polluted area these correlations are due to contaminant processes which lead to a proportional increase of metal concentrations.In Figure3are presented oxygen consumptions of soil samples from contaminated areas and in Figure4oxygen consumptions of soil samples from noncontaminated areas.In both cases results are given for two temperatures of experiment:15and 30◦C.Was observed generally lower oxygen consumption in soil samples from con-taminated areas in comparison with oxygen consumption in soil samples from noncontaminated areas.In the extremely contaminated point No.5oxygen con-sumption has the lowest value.In this point concentration of heavy metals is con-siderably higher then in other points and also there is an extremal salinization (conductivity2.37mS cm−1compared with mean values:0.65in contaminated area and0.20in noncontaminated).In contaminated area a share of the organic matter is significantly lower,a mean value for all points in this area is only9.18%,when in noncontaminated area a mean value is12.77%.It could be a result of unfavourable influence of heavy metals and their salts on biological processes in edaphon,mostly on soil microfauna and soil microbial populations.In Table III are presented correlations between measured soil respirations and components of soil samples from contaminated area.In Table IV are presented correlations between measured soil respirations and components of soil samples from noncontaminated area.Particularly high coefficients of correlation between the soil respiration and soil pollution in polluted soils were obtained for Pb:r=0.75in15◦and r=0.98in 30◦C,for Ba:0.90and0.57;for V:0.99and0.81.In non-polluted soils highest correlation coefficients are for Pb:r=0.70in15◦C;Fe:0.60and0.72;Al:0.68 and0.64;Mn:0.51and0.66;Ba:0.63and0.61;Cr:0.94and0.70;Ni:0.64and 0.65;Cu:0.69and0.48;V:0.62in15◦C;and Cd:0.69in15◦C.Obtained results attest that measurements of soil respirations using volumetric respirometry could be used for estimations and comparations of soil ecological conditions as well as biological activity of soils.5.Conclusions1.Obtained results of experiments and analysis confirm that some investigatedsoils in the Baix Llobregat near Barcelona are contaminated by heavy metals.Concentration of pollutants increases in vinicity of a composting plant and a car dump.2.In contaminated soils conductivities are three times higher than in noncontam-inated soils.Soil conductivity depends in high degree on salinization of soils by dissociated heavy metal salts.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION311TABLE IIICorrelations between measured soil respirationand components of this soil from contaminatedarea15◦C30◦CSoil humidity–0.326∗–0.471∗0.328∗∗0.144∗∗Organic matter–0.543∗–0.385∗0.084∗∗0.243∗∗CaCO3–0.544∗–0.334∗0.083∗∗0.315∗∗CE–0.493∗–0.528∗0.123∗∗0.095∗∗Fe–0.445∗–0.382∗0.170∗∗0.247∗∗Al–0.432∗–0.341∗0.185∗∗0.305∗∗Mn–0.532∗–0.442∗0.092∗∗0.173∗∗Sr–0.546∗–0.446∗0.082∗∗0.169∗∗Zn–0.476∗–0.304∗0.139∗∗0.364∗∗Ba0.044∗0.193∗0.897∗∗0.570∗∗Cr–0.418∗–0.399∗0.201∗∗0.224∗∗Ni–0.517∗–0.368∗0.104∗∗0.266∗∗V–0.003∗–0.084∗0.992∗∗0.806∗∗Cu–0.431∗–0.359∗0.186∗∗0.278∗∗Cd–0.352∗–0.295∗0.288∗∗0.379∗∗Pb–0.109∗0.010∗0.749∗∗0.976∗∗∗:r=Correlation Coefficient.∗∗:Significance level p<0.05.312 D.ZIMAKOWSKA-GNOI´NSKA ET AL.TABLE IVCorrelations between measured soil respirationand components of this soil from noncontami-nated area15◦C30◦CSoil humidity–0.325∗–0.569∗0.330∗∗0.068∗∗Organic matter–0.515∗–0.708∗0.105∗∗0.015∗∗CaCO3–0.655∗–0.515∗0.029∗∗0.105∗∗CE0.313∗–0.192∗0.349∗∗0.572∗∗Fe0.179∗–0.122∗0.599∗∗0.721∗∗Al–0.141∗–0.161∗0.679∗∗0.637∗∗Mn0.221∗–0.152∗0.513∗∗0.657∗∗Sr–0.595∗–0.606∗0.053∗∗0.048∗∗Zn–0.138∗–0.542∗0.685∗∗0.085∗∗Ba0.162∗–0.175∗0.633∗∗0.607∗∗Cr0.025∗–0.132∗0.941∗∗0.699∗∗Ni0.159∗–0.153∗0.641∗∗0.653∗∗V–0.170∗–0.337∗0.617∗∗0.311∗∗Cu0.135∗–0.238∗0.692∗∗0.482∗∗Cd–0.137∗–0.474∗0.690∗∗0.141∗∗Pb–0.133∗0.492∗0.697∗∗0.124∗∗∗:r=Correlation Coefficient.∗∗:Significance level p<0.05.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION313 3.In contaminated area a share of the organic matter is significantly lower(9.18%)then in noncontaminated area(12.77%).It could be a result of unfavourable influence of heavy metals and their salts on biological processes in edaphon, mostly on soil microfauna and soil microbial populations.4.Oxygen consumptions are considerably lower in soil samples from contami-nated areas in comparation with noncontaminated areas.5.V olumetric respirometry could be used for estimations and comparations ofecological condition and biological activity of soils.ReferencesAnderson,J.P.E.:1982,‘Soil Respiration’,in:Page,A.L.,Miller,R.H.and Keeney,D.R.(eds.),Methods of Soil Analysis:2Chemical and Microbiological Properties,Am.Soc.Agron., Madison,Wisconsin,pp.467–476.Bech,J.,Tobias,F.J.and Zimakowska-Gnoi´n ska,D.:1994,‘Comparison of Heavy Metals in pol-luted and unpolluted soils at the NE edge of the Garraf Massif(Catalonia,Spain)’,Abstracts of the15th International Congress of Soil Science,Acapulco,Mexico,152–153.Edwards,N.T.:1974,‘A moving chamber design for measuring soil respiration rates’,OIKOS25, 97–101.Elmholt,S.:1992,‘Effect of Propiconazole on Substrate Amended Soil Respiration Following Laboratory and Field Respiration’,Pestic.Sci.34,139–146.Fischer,Z.:1996,‘Ecological equilibrium disturbances in the soil exposed to simulated acid rain.Part VI’,Environ.Monit.Assess.41,61–65.Freedman,B.and Hutchinson,T.C.:1980,‘Effect of smelter pollutants on forest litter decomposition near a nickel-copper smelter at Sudbury,Ontario’,Can.J.Bot.58,1722–1736.Freedman,B.and Hutchinson,T.C.:1980,‘Pollutant inputs from the atmosphere and accumulation in soils and vegetation near a nickel-copper smelter at Sudbury,Ontario’,Can.J.Bot.58,108–132.Gildon,A.and Rimmer,D.L.:1993,‘Soil respiration on reclaimed coal-mine spoil’,Biol.Fertile Soils16,41–44.Klekowski,R.Z.:1975,‘Constant Pressure V olumetric Microrespirometer for Terrestrial Inver-tebrates’in W.Grodzi´n ski,R.Z.Klekowski and A.Duncan(eds.),Methods for Ecological Bioenergetics,IBP Handbook Blackwell Sci.Publ.,Oxford-London-Edinburgh-Melbourne,24, pp.212–225.Nakadai,T.,Koizumi,H.,Usami,Y.,Satoh,M.and Oikawa,T.:1993,‘Examination of the method for measuring soil respiration in cultivated land:Effect of carbon dioxide concentration on soil respiration’,Ecological Research8,65–71.Nordgren,A.,Baath,E.and Soderstrom,B.:1983,‘Microfungi and microbial activity along a heavy metal gradient’,Appl.Environ.Microbiol.46,1829–1837.Nordgren,A.,Baath,E.and Soderstrom,B.:1985,‘Soil microfungi in an area polluted by heavy metals’,Can.J.Bot.63,448–455.Nordgren,A.,Kauri,T.,Baath,E.and Soderstrom,B.:1983,‘Soil microbial activity,mycelial lengths and physiological groups of bacteria in a heavy metal polluted area’,Environ.Poll.(Serie A)41, 89–100.Strojan,C.L.:1978,‘Forest litter decomposition in the vicinity of a zinc smelter’,Oecologia(Berl.) 32,203–212.Witkamp,M.:1969,‘Cycles of temperature and carbon dioxide evolution from litter and soil’, Ecology50,922–924.。

Plant and Soil222:119–137,2000.©2000Kluwer Academic Publishers.Printed in the Netherlands.119Foliar free polyamine and inorganic ion content in relation to soil and soil solution chemistry in two fertilized forest stands at the Harvard Forest, MassachusettsRakesh Minocha1,∗,Stephanie Long1,Alison H.Magill2,John Aber2and William H. McDowell31USDA Forest Service,Northeastern Research Station,P.O.Box640,Durham,NH03824,USA;2Complex Systems Research Center,University of New Hampshire,Durham,NH03824,USA and3Department of Natural Resources, University of New Hampshire,Durham,NH03824,USAReceived24September1999.Accepted in revised form29February2000Key words:ammonium nitrate,calcium,Harvard Forest,magnesium,nitrate leaching,polyaminesAbstractPolyamines(putrescine,spermidine,and spermine)are low molecular weight,open-chained,organic polycations which are found in all organisms and have been linked with stress responses in plants.The objectives of our study were to investigate the effects of chronic N additions to pine and hardwood stands at Harvard Forest,Petersham, MA on foliar polyamine and inorganic ion contents as well as soil and soil solution chemistry.Four treatment plots were established within each stand in1988:control,low N(50kg N ha−1yr−1as NH4NO3),low N+sulfur(74kg S ha−1yr−1as Na2SO4),and high N(150kg N ha−1yr−1as NH4NO3).All samples were analyzed for inorganic elements;foliage samples were also analyzed for polyamines and total N.In the pine stand putrescine and total N levels in the foliage were significantly higher for all N treatments as compared to the control plot.Total N content was positively correlated with polyamines in the needles(P≤0.05).Both putrescine and N contents were also negatively correlated with most exchangeable cations and total elements in organic soil horizons and positively correlated with Ca and Mg in the soil solution(P≤0.05).In the hardwood stand,putrescine and total N levels in the foliage were significantly higher for the high N treatment only as compared to the control plot.Here also, total foliar N content was positively correlated with polyamines(P≤0.05).Unlike the case with the pine stand,in the hardwood stand foliar polyamines and N were significantly and negatively correlated with foliar total Ca,Mg, and Mn(P≤0.05).Additional significant(P≤0.05)relationships in hardwoods included:negative correlations between foliar polyamines and N content to exchangeable K and P and total P in the organic soil horizon;and positive correlations between foliar polyamines and N content to Mg in soil solution.With few exceptions,low N +S treatment had effects similar to the ones observed with low N alone for both stands.The changes observed in the pine stand for polyamine metabolism,N uptake,and element leaching from the soil into the soil solution in all treatment plots provide additional evidence that the pine stand is more nitrogen saturated than the hardwood stand. These results also indicate that the long-term addition of N to these stands has species specific and/or site specific effects that may in part be explained by the different land use histories of the two stands.Abbreviations:Perchloric acid(PCA),Polyamines(PAs),Zero tension lysimeter(ZTL),hardwoods(HW)IntroductionThere is increasing concern about the potential ad-verse effects of elevated rates of N deposition on water ∗FAX No:6038687604.E-mail:rminocha@ quality and the health of forest ecosystems(Aber et al.,1989,1998;Rasmussen and Wright,1998).This concern stems from the fact that in1990the United States(US)Clean Air Act targeted a50%reduction in S deposition but only a10%reduction in N depos-120ition(McNulty et al.,1996).Although most temperate forests are N limited,continuous deposition of N from the atmosphere can move them towards nitrogen sat-uration.Nitrogen saturation has been defined as the availability of ammonium and nitrate in excess of total combined plant and microbial nutritional demand (Aber et al.,1989).It is important to understand how chronic additions of N to ecosystems change the struc-ture and function of forest ecosystems(Asner et al., 1997;Jefferies and Maron,1997).Long-term elevated N deposition typically leads to an increase in the concentration of total foliar N, with or without similar changes in the important base elements such as Ca,Mg and K(Aber et al.,1995; Magill et al.,1997,1999;Rasmussen and Wright, 1998;van Dijk and Roelofs,1988).This increase in leaf N content also leads to significant shifts in the internal partitioning of N within the leaf.For example in conifers,N deposition significantly increases leaf N present in the form of free amino acids such as arginine (Ericsson et al.,1993,1995;Näsholm et al.,1997). Little is known about N partitioning for hardwoods under these wlor(1992)suggested that these changes in N partitioning are probably not re-lated to leaf function.This idea,however,has yet to be experimentally tested in terms of whether the alter-ations in N partitioning due to long-term N deposition actually have a positive or a negative effect on photo-synthetic capacity and biomass production.A possible decoupling of the relationship between foliar N and photosynthetic rate may occur under these conditions.The aliphatic polyamines(putrescine,spermidine, and spermine)play an important role in the growth and development of all living organisms.They are meta-bolically derived from the amino acids arginine and ornithine,and at cellular pH they carry a net positive charge(Cohen,1998).Abiotic stress conditions such as low pH,high SO2,high salinity,osmotic shock, nutrient stress,low temperature(Flores,1991and ref-erences therein)and high Al(Minocha et al.,1992, 1996)all result in an increase in cellular putrescine levels.Polyamines show a reverse proportionality to cellular elements such as Ca,Mg,Mn,and K in re-sponse to Al treatment in periwinkle(Catharanthus roseus)and red spruce(Picea rubens)cells(Minocha et al.1992,1996;Zhou et al.,1995).A key distinction between the polyamines(organic cations)and the inor-ganic elements is that,even if the latter(e.g.Ca2+and Mg2+)undergo recompartmentalization in response to external stimuli,their cellular levels are derived mainly from uptake across the biological membranes and this uptake ultimately depends upon their availab-ility in the soil or soil solution.In contrast,polyamines are synthesized within the cell,permitting adjustment of their cellular concentrations to meet physiological needs.Also,cellular polyamine levels can be regu-lated by conjugation,degradation,and sequestration via enzymatic means(Minocha et al.,1996).Thus polyamine synthesis may play an important role in the survival of plants under stress(Galston,1989).Recent work examining the concentration of polyamines in plant foliage has been aimed at using foliar polyam-ine concentrations as indicators of stress(Dohmen et al.,1990;Minocha et al.,1997;Santerre et al.,1990). In the case of mature red spruce trees,an increase in foliar putrescine concentration was associated with a decrease in foliar and soil Ca and Mg concentrations and an increase in the Oa horizon soil Al or Al:Ca ratios(Minocha et al.,1997).Structurally,polyamines are composed of carbon, hydrogen,and nitrogen.Therefore,we suspect that their levels may also change in response to chronic N additions,thus affecting the internal N partitioning within the leaf,a situation similar to that observed with free amino acids in conifers.The objective of this study was to determine the effects of chronic additions of N on:(1)foliar polyamines(a proposed stress in-dicator)and inorganic ions;(2)soil and soil solution inorganic ion chemistry,especially Al mobilization; and(3)the relationship between polyamines and soil chemistry in pine and hardwood stands.Materials and methodsStudy sitesThe study plots are located at Harvard Forest,Peter-sham,MA(42◦30 N,72◦10 W).This site is a part of the National Science Foundation’s Long-Term Eco-logical Research(LTER)program.These plots are a part of the ongoing study on chronic nitrogen addi-tions since1988(Aber et al.,1993;Magill et al.,1997, 1999).An even-aged red pine(P.resinosa)stand,74 yr old,and an adjacent mixed hardwood stand,approx. 55yr old,were chosen for this study.The hardwood stand is dominated by black oak(Quercus vetulina Lam.)and red oak(Q.Rubra Michx.f.,respectively) with significant amounts of black birch(Belutinaa lenta L.),red maple(Acer rubrum L.),and American beech(Fagus grandifolia Ehrh.).Most of the currently forested area at this site was in cultivation or pas-tureland during the mid-1800’s(Foster,1992).The121dominant soil types are stony sandy loams classified as Typic Dystrochrepts.Mean annual temperature ranges from19◦C in July to–12◦C in January and mean total annual precipitation is112cm.The estimated nitrogen deposition to the forest is about6kg ha−1yr−1wet and2kg ha−1yr−1dry(Aber et al.,1993;Magill et al.,1997;Ollinger et al.,1993).TreatmentsFour treatment plots were established within each stand:control,low N,low N+sulfur(N+S),and high N.Each plot measured30×30m(0.09ha)and was divided into36subplots(each5×5m).Fertilizer additions of NH4NO3and Na2SO4began in1988as six equal applications over the growing season.The fertilizer was weighed,mixed with20L of water (equivalent to0.002cm rainfall)and applied using a backpack sprayer.Two passes were made across each plot to ensure an even distribution of fertilizer.As described in Magill et al.(1997),a partial ap-plication was made in year1(1988).The total amount of fertilizer applied was38kg N ha−1yr−1to the low nitrogen treatment and the nitrogen portion of the N+S treatment,113kg N ha−1yr−1to the high nitrogen treatment,and74kg(S)ha−1yr−1to the N+S plots. Applications for all following years were at the rate of 50kg N ha−1yr−1to the low and N+S plots and150 kg N ha−1yr−1to the high N plots.Sulfur additions remained the same as used for year one.Collection and analyses of needle samplesFoliage samples were collected during thefirst week of August each year from mid to upper canopy branches of dominant or co-dominant trees using a shotgun.Early August was chosen as the sampling time because the trees were still physiologically very active at this time,compared to early or late summer. At each sampling time,current-year needle samples were collected from20different red pine trees in the pine stand and leaves from10different trees of black or red oak and red maple each were collected from the hardwood plots.A sub-sample was taken from each in-dividual tree collection for analyses of polyamine and exchangeable inorganic elements.The remaining pine needle samples from20different trees in each plot were pooled into5composite samples of4trees per sample for total inorganic elements and N analyses. Similarly,the remaining samples from10–12trees per species collected from each plot in the hardwood stand were pooled into4composite samples.Red and black oak were treated as a single species in all collections. Total elements and nitrogen analysesThe composite samples were dried at70◦C for48 h and ground using a Wiley mill with a1mm mesh screen.The ground samples were dried overnight at 70◦C and analyzed for N content using near-infrared (NIR)spectroscopy(Bolster et al.,1996;McLel-lan et al.,1991).These samples were also used for extraction of total inorganic ion content by a modi-fication of the method of Isaac and Johnson(1976) as described in Minocha and Shortle(1993).The ex-tracts were analyzed for total Ca,Mg,Mn,K,Al, and P content using a Beckman Spectrospan V ARL DCP(Direct Current Plasma Emission Spectrometer, Beckman Instruments,Inc.,Fullerton,CA)using the Environmental Protection Agency’s method number 66-AE0029(1986).Analysis of polyamines and exchangeable inorganic elementsThe fresh foliage samples were placed in individual pre-weighed microfuge tubes containing1ml of5% perchloric acid(PCA).The tubes were kept on ice dur-ing transportation to the laboratory and then stored at –20◦C until they were processed.The samples were weighed,frozen and thawed(3X),and centrifuged at 13,000rpm in a microfuge for10min.Details of the freeze-thawing extraction procedure are described in Minocha et al.(1994).The freeze-thawing method was also chosen for the extraction of exchangeable fraction of inorganic ions.This method extracted a consistent fraction of the total acid extractable inor-ganic ions from foliage of various species of mature trees and the quantity of this fraction varied for each ion type and tree species(Table1and Minocha et al.,1994).The moisture content data for individual exchangeable ion samples was not collected.There-fore,for comparison of total and exchangeable ions on weight basis,an average moisture content of53%, 62%,and61%has been used for this site from data collected in1992for pine(n=19),oak(n=33),and maple(n=45),respectively to convert fresh weight to dry weight.Briefly,the samples for both inorganic ions as well as for polyamines were frozen at–20◦C and thawed at room temperature,repeating the process two more times.Duration of the freezing step could vary from4122parison of effects of nitrogen treatments on total and exchangeable inorganic ion levels in the foliage of pine,oak and maple trees.Data has been averaged over three year period(1995–1997).Data for exchangeable ions are mean±se of n=60for pine stand and n=30for hardwoods.Data for total ions are mean±se of n=15for pine stand and n=12for hardwoods.The numbers in parenthesis indicate%of total ions extracted as exchangeable.The moisture content data for individual exchangeable ion samples was not collected.Therefore,for comparison of total and exchangeable ions on weight basis,an average moisture content of53%,62%,and61%has been used for this site from data collected in1992for pine(n=19),oak(n=33),and maple(n=45),respectively to convert FW to DWTreatment Ion Inorganic ions Pine(µmol g−1DW)Oak(µmol g−1DW)Maple(µmol g−1DW)Control Ca Total62.5±4.8121.5±5.9156.0±6.8Low N72.3±5.1113.5±8.1137.0±6.7High N60.0±5.571.8±6.2116.4±6.1Control Exchangeable11.4±0.6(18.3%)102.1±5.9(84.0%)79.2±4.7(50.8%)Low N11.9±0.7(16.5%)87.1±4.7(76.7%)69.7±2.3(50.9%)High N9.9±0.9(16.5%)57.3±5.0(79.9%)59.2±3.4(50.9%)Control Mg Total32.4±1.562.8±2.164.7±3.3Low N35.5±1.363.1±2.359.9±1.3High N35.4±1.354.1±3.355.7±2.9Control Exchangeable13.6±0.5(41.9%)56.1±3.4(89.3%)43.7±2.3(67.5%)Low N14.7±0.4(41.3%)58.0±3.4(91.9%)43.5±1.4(72.7%)High N13.5±0.4(38.1%)51.0±3.0(94.2%)38.5±1.9(69.1%)Control Mn Total16.7±1.141.6±2.337.4±2.3Low N22.4±2.127.3±2.023.6±0.9High N15.8±1.615.3±0.922.5±1.9Control Exchangeable 3.6±0.2(21.3%)50.8±3.5(122.1%)∗21.1±1.5(56.6%)Low N 3.9±0.3(17.3%)33.4±2.4(122.4%)∗14.5±1.0(61.6%)High N 3.2±0.4(20.5%)17.4±1.5(113.4%)∗11.1±0.9(49.5%)Control K Total120.1±6.7218.7±9.5184.6±11.8Low N125.5±7.2265.7±12.0171.6±10.5High N135.3±7.4202.2±9.4176.0±9.7Control Exchangeable72.8±3.8(60.6%)158.0±6.8(72.3%)132.2±6.0(71.6%)Low N79.6±3.6(63.4%)165.1±7.2(62.2%)138.8±5.9(80.9%)High N84.0±3.2(62.1%)157.5±6.7(77.9%)147.0±5.8(83.5%)∗The calculation of greater than100%extraction for Mn in the case of oak was possibly caused by slight overestimation of the mean moisture content for1995–1997.h to a few days.Samples were allowed to thaw com-pletely(approximate time1–1.5h)before refreezing. After freeze-thawing,the samples were centrifuged at 13,000×g.This supernatant was used directly for free polyamine analysis without further dilution and for inorganic-ion analysis after proper dilution with distilled,deionized water(final concentration of PCA 0.01or0.02N)by the procedures described below. The diluted fractions were analyzed for inorganic ion content with a Beckman Spectrospan V ARL DCP as described above.For quantitation of polyamines, heptanediamine was added as an internal standard to aliquots of the above extracts prior to dansylation. Fifty or one hundredµL of the extract were dansylated according to the procedure described in Minocha et al.(1990).Dansylated polyamines were separated by reversed phase HPLC(Perkin-Elmer Corp.,Norwalk,CT)using a gradient of acetonitrile and heptane-sulfonate,and quantified by afluorescence detector (Minocha et al.,1990).Collection and analyses of soil and soil solution samplesThree sets of two adjacent soil cores(<30cm apart) were taken to a depth of10cm in the mineral soil in each of the three designated subplots(nine samples per plot).Cores were split into organic(Oe+Oa)and min-eral horizons(top10cm)and placed in gas-permeable polyethylene bags.The soils were air-dried,sieved (<2mm size),and stored at room temperature prior to analyses.Before analyses,the mineral soil and organic soil samples were oven-dried at105◦C and70◦C, respectively.123Exchangeable inorganic elements were determined from a sub-fraction of the above samples using a modi-fication of the procedure of Taylor(1987).Briefly, either6g of mineral soil or1g of organic soil was added to30ml of extraction solution(0.05N HCl and 0.025N H2SO4)and placed on a gyratory shaker at90 rpm for15min.The extract wasfiltered with a glass fiber syringefilter(Gelman A/E,Gelman Sciences, Ann Abror,MI)and stored at4◦C until quantitation by DCP.Prior to digestion for total elemental analysis,a sub-sample of air-dried and sieved soil was processed for1min in a Shatter Box Laboratory Mill(Spex Industries,Inc.,Edison,NJ)to powder the sample. Microwave digestion(Hallett and Hornbeck,1997) was used for obtaining inorganic elements.Briefly, for mineral soils,0.1g sample was digested with5 ml of concentrated HNO3,2ml of concentrated HCl, 2ml offluoroboric acid(HBF4)and2ml of H2O2. For organic soils,0.1g sample was digested the same way with acids but without the presence of H2O2.The following microwave programs were used.Given in pairs are:Time(min)–Power(Watts).For mineral soil, 1–250,1–0,5–250,5–400,5–500,and5–600.For organic soil,1–250,1–0,5–250,5–400,1–0,5–500, 1–0,5–600,1–0,and2.5–650.Total running time was 27.5min.The details on installation of zero tension lysi-meters(ZTL)and soil solution sample collection are described in Currie et al.(1996)and McDowell et al. (1998).Briefly,5polyethylene ZTL’s were installed per plot except for N+S plots.Solutions were collected after major rain events and all5samples per plot were pooled prior to analyses.Over the course of three years (1995–1997),the collections were made approx.50 times from each plot.Samples were transported on ice to the laboratory andfiltered through pre-combusted Whatman GF/F glassfiberfilters(Whatman Inc., Clifton,NJ)within36h of collection before freezing. These solutions were analyzed for inorganic elements using DCP as described earlier.Statistical methodsLinear regression analyses were performed to es-tablish the strength and significance of relationships between two different variables(n=12except for soil samples where n=8)using Excel5.0for Windows(Mi-crosoft Corporation,Roselle,IL).Data for each vari-able(e.g.,foliar or soil Ca,Mg and Al)were analyzed as a series of one-way analysis of variance(ANOV A)to determine whether statistically significant differ-ences occurred between control and treatment plots for each individual variable.When F values for one-way ANOV A were significant,differences in treatment means were tested by Tukey’s multiple comparis-ons test.ANOV A and Tukey’s tests were performed with Systat for Windows,version7.01(SYSTAT Inc., Evanston,Il)and a probability level of0.05was used for tests unless specified otherwise.ResultsWith few exceptions,the low N+S treatment showed effects similar to the ones observed for low N treat-ment alone for both stands.For this reason,results for low N+S will not be discussed separately.Also,in foliage exchangeable ions always represented a con-sistent fraction of the total ions.Even though the quantity of this fraction varied for each ion type and tree species,nitrogen treatments had similar effects on both exchangeable and total Ca,Mg,Mn,and K levels in each case for all three species(Table1).Thus due to this similarity of trends between exchangeable and total ions,only total inorganic ion data are used for further comparison of foliar results with soil and soil solution data.Foliar polyamines and N contentPine:There was a significant increase in the level of putrescine in the needles of trees growing on all three treatment plots as compared to the control plot (Figure1A–D).A small but statistically significant increase was also observed in spermidine levels in response to high N treatment.Spermine,which was a relatively small proportion of free polyamines in red pine,increased significantly in response to low and high N additions.In spite of year to year vari-ations in the total amounts of polyamines(possibly due to different growth conditions resulting from vari-able weather patterns),similar trends were observed for each of the three years of data collection. Hardwoods:Putrescine levels in oak leaves were three-to four-fold higher in response to high N treat-ment for each of the three years of this study(Fig-ure2A–D).For other treatments no significant change was observed.The level of spermidine and spermine did not change significantly in response to low and high N treatments.While there were annual variations124Figure1.The effect of chronic N additions on foliar polyamines in pine.Data presented for each treatment are mean±SE of n=20for A–C. Figure1D presents cumulative data for three years(n=60).The asterisks denote the significant differences from control.in the total amount of polyamines,the trends were the same for each year.High N treatment caused a significant increase in putrescine levels in maple leaves(Figure3A–D)for two of the three years.Spermidine and spermine levels were also present in quantities comparable to putres-cine in maple leaves.For1995,a parallel increase in spermidine was also observed(Figure3A).No change in spermine content was observed for any of the three years.As with pine and oak,year to year variation in the three polyamines was also observed in maple.The total N content of pine needles and maple leaves increased in response to all N treatments(Fig-ure4A and4C).The total N content in oak foliage also rose significantly but only in response to the high N treatment(Figure4B).The changes in N were always positively and significantly correlated with changes in foliar polyamines for all three species(Figure4A–C). Foliar inorganic elementsPine:No significant changes in total foliar Ca,Mg, Mn,and K were observed in response to chronic N additions to the soil.However,there was a decrease in total Al concentration in response to all N treatments and an increase in total P with high N treatment only (Figure5).Hardwoods:Total Ca and Mn levels showed a sig-nificant decrease in response to high N treatment in both maple and oak leaves(Figure6).Changes in total Mn were also significant for low N treatment in both species.The only other statistically significant change observed was a decrease in P with all N treat-ments in maple.Mg showed a statistically insignificant decrease with high N addition for both species. Foliar Al:Ca and Mg:N ratiosA significant decrease in foliar total Mg:N and Al:Ca ratios was observed with all N treatments in case of pine(Figure7A–B).In contrast,an increase in the Al:Ca ratio(though statistically insignificant for maple)accompanied by a decrease in total Mg:N ra-tio was observed in response to high N treatment for maple and oak(Figure7C–F).Inorganic elements in the organic horizon of soil Pine:A significant decrease in exchangeable Ca, Mg,Mn,and K in the organic soil was observed in125 Figure2.The effect of chronic N additions on foliar polyamines in oak.Data presented for each treatment are mean±SE of n=10for A–B and n=5for C.Figure2D presents cumulative data for three years(n=25).The asterisks denote the significant differences from control.response to all N treatments.There was an increase in exchangeable P in response to the low N treatment only(Figure8:Pine).However,there was no signi-ficant effect of these treatments on exchangeable Al levels.Whereas total Ca decreased in response to high N treatment only,total Mn and P decreased in response to all N treatments with no significant change in K in the organic soil.A slight but statistically insignific-ant decrease in the level of total Mg and Al with N treatments was also observed(Figure9:Pine). Hardwoods:N additions alone had no significant effect on exchangeable or total Ca levels of the or-ganic horizon.However,exchangeable Mg,K,and P concentrations were significantly lower in high N plots with Mn being low in all N treatment plots(Fig-ure8:HW).Total Mg,Mn,and P levels decreased in response to all N treatments in this soil horizon (Figure9:HW).Inorganic elements in the mineral horizon of soil Pine:Whereas changes in the exchangeable Ca,Mg, Mn,and K did not show any specific and statistically significant patterns,P increased significantly in rela-tion to all N additions in the mineral soil.Both low and high N treatments decreased the exchangeable Al con-centrations significantly in this soil layer(Figure10: Pine).Hardwoods:The exchangeable ion chemistry of the mineral horizon showed decreases in the levels of some inorganic elements,but the changes were not statistically significant except for Al in the high N plot (Figure10:HW).Inorganic elements in zero tension lysimeter(ZTL) solutionPine:A significant increase was observed in the levels of all inorganic elements tested in response to high N addition.Levels of P and Al were high even for low N treatment(Figure11:Pine). Hardwoods:There was typically a small increase in the levels of most elements in the ZTL solution samples in response to N treatments,but the only sig-nificant increase that was observed was in Al levels in response to high N treatment(Figure11:HW).126Figure3.The effect of chronic N additions on foliar polyamines in maple.Data presented for each treatment are mean±SE of n=10for A–B with one exception and n=4for C.Figure3D presents cumulative data for three years(n=24).The asterisks denote the significant differences from control.Relationship between foliar and soil chemistry Pine:Putrescine levels did not correlate with most inorganic elements in the pine needles.However,pu-trescine was negatively correlated with all but P of the exchangeable and all of the total inorganic ions in the organic soil horizon(Table2).There was no correlation between putrescine or total polyamines and exchangeable elements in the mineral soil except for P and Al.In contrast to the results for organic soil hori-zon,there was a positive correlation between Ca,Mg, and Al in soil solution and foliar putrescine as well as total polyamines(Table2).N content in the pine needles was positively correl-ated with foliar Mg and P only.There was also a strong negative correlation between foliar N and exchange-able Ca,Mg,Mn,and K in the organic soil horizon. Similar to the situation with total polyamines,foliar N showed a significant negative correlation only with total Mn and P in the organic soil horizon.N content, however,was positively correlated with all elements in the soil solution(Table2).Hardwoods:Unlike the situation with pine,putres-cine and total PAs in oak and maple leaves showed a strong negative correlation with foliar Ca,Mg,Mn, and P.In the organic soil horizon,putrescine,total polyamines as well as N were significantly and in-versely related with exchangeable K and total P in both oak and maple.In the mineral soil,putrescine in oak was negatively correlated only with Al but in maple it had a negative correlation with several elements.Fo-liar putrescine and N were also positively correlated with soil solution Mg and/or Al.For more details on individual correlations refer to Table2.DiscussionBiochemical changes that occur in response to expos-ure to a particular stress can be measured before any visible symptoms appear at the level of the organism and may even be used as early indicators of change(s) in the vitality of trees within a stand(Baur et al.,1998; Minocha et al.,1997).Among the physiological and molecular responses of plants to high N deposition, is the increase in cellular content of leaf N.Nitrogen has been shown to be stored in the leaf in the form of nitrate and/or specific free amino acids such as ar-ginine(Aber et al.,1995;Ericsson et al.,1993,1995;。

政治学主要英文期刊简介1、刊名称American Political Science Review, with PS: Political Science & Politics.ISSN: 0003-0554创刊年:1906出版期次4/yr. 开本:21.5x15.5 页数:300 目录价:154/USD出版机构American Political Science Association,USA 出版地:美国译名简介《美国政治科学评论》附《政治科学与政治学》美国政治科学学会会刊。

刊载政治理论、政治学、比较政治学、国际政治学等方面的文章、札记和书评。

附刊《政治科学与政治学》，报道该学会的研究动态和会议消息，并刊载学位论文。

SSCI来源刊，影响因子2.4482、报刊名称Comparative Politics. 创刊年:1968ISSN:0010-4159出版期次4/yr. 开本:21.5x15.5 页数:90 目录价:69/USD出版机构City University of New York, Political Science Program,USA 出版地:美国译名简介《比较政治学》刊载美国和其它国家的政治、社会、经济与宗教的研究和比较研究方面的文章与评论。

SSCI来源刊，影响因子1.0833、报刊名称Capitalism, Nature, Socialism. 创刊年:1989ISSN:1045-5752出版期次4/yr. 开本:21.5x15.5 页数:144 目录价:99/USD出版机构Guilford Publications Inc.,USA 出版地:美国译名简介《资本主义、自然、社会主义》由加利福尼亚大学社会学与经济学教授James O’Connor担任主编的探讨和论述社会主义生态学的国际性刊物。

内容涉及政治学、经济学、社会学和环境科学等。

PROQUEST有全文4、报刊名称Public Administration. 创刊年:1922ISSN:0033-3298出版期次4/yr. 开本:21.5x15.5 页数:124 目录价:324/GBP出版机构Blackwell Publishers Ltd.,UK 出版地:英国译名简介《公共行政》刊载公共行政管理和政策制定以及国际行政管理和比较研究等方面的文章。

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▪David Marius Bressoud David Marius Bressoud的主页。

David Marius编写了一系列的大学课本，在这些课本中，他使用物理和数学的历史来激发学生对多变量微积分学，数论以及实际分析的学习兴趣。

三、数学词表▪1991 Mathematics Subject Classification (MSC) - Chris Eilbeck; Heriot-Watt University, Edinburgh 爱丁堡Heriot-Watt大学Chris Eilbeck编著的1991数学主题分类表。

这是1991数学主题分类表（Mathematics Subject Classification）的超文本版本。