Background and literature reappraisal. Polycyclic aromatic hydrocarbons ( PAHs ) are a category of hydrocarbons that consist of at least two amalgamate aromatic rings through two or more C atoms. PAHs are one of the major pollutants in urban countries, which can be dispersed in different environments: H2O, dirt and deposits, air, workss and animate beings. Semivolatile organic compounds ( SOCs ) are distributed between the ambiance and the surface of the Earth. If consider that 80 % of land surface of the Earth is covered with flora, which exceeds the surface country of the land on which it is turning by 6-14 times, every bit good as count the fact that works surfaces are covered with tungsten & A ; deg ; x or lipid beds, workss are of import sinks for atmospheric SOCs, playing a important r??le in their one-year cycling. PAHs are airborne contaminations and nowadays in the ambiance in vapor and solid ( particle-bound ) stages. Due to their lipotropic belongingss, PAHs preponderantly divider from the air to the foliage surface, as it mentioned above, which consist of nonionic lipoids and waxes.
The grade to which PAHs accumulate in the leaf depend on several factors:
Physico-chemical belongingss of PAHs. Harmonizing to these belongingss, PAHs can lodge to foliages through a three basic deposition mechanisms: gaseous deposition, particle-bound deposition ( wet and dry ) and w?µt d?µposition of dissolved PAHs. Because of the hydrophobic nature of the PAHs the 3rd deposition manner is of low significance. High molecular weight not volatile PAHs with 5 or more rings tend to divider from the gas stage on atmospheric atoms and so roll up to works surface, whereas low molecular weight volatile PAHs nowadays in the ambiance in a vapour stage and enter works tissues chiefly via unfastened pores by diffusion. Compounds of intermediate volatility with 4 rings are partitioned between gas and particulate stages depending on ambient temperature.
Partition of gaseous PAHs to flick surfaces can be described as sorption procedure between vapour stage and leaf surface, where PAHs are transported from the ambient air to the flora until equilibrium is reached. The octanol-air divider coefficient ( KOA ) is an of import parametric quantity and helps to understand the divider of airborne pollutants to flick surface. KOA is either measured straight or calculated from c??mp??und ‘s air-water divider coefficient and octanol-water divider coefficient ( KOW ) . Therefore, accretion of PAHs can be described with the log of KOA: ( 1 ) log KOA & A ; lt ; ~9 – equilibrium breakdown ; ( 2 ) 9 & A ; lt ; log KOA & A ; lt ; 11 – kinetically limited gaseous deposition ; ( 3 ) KOA & A ; gt ; 11 – atom bound deposition.
Kaupp and McLachlan ( 2000 ) found that 97 % of particle-bound high molecular weight PAHs ( with 5 and more rings ) were attached to atoms with aerodynamic diameters of & A ; lt ; 2.91 µm and merely 1.2 % of them were associated with atoms with & A ; gt ; 8.6 µm aerodynamic diameter. They besides found that with turning atom size PAH burdens invariably decreased.
Second factor which plays function in PAH deposition from the ambiance is belongingss of the roll uping surface. There are great differences in the sum of adsorbed organic pollutants between assorted foliage surfaces which are exposed to the same air concentration. This can be explained by the different physiological characteristics of foliages of different tree species, such as leaf morphology, lipid and epicuticular waxes content, stomata denseness and the presence or absence of hairs, which later lead to a different efficaciousness in retaining airborne pollutants.
Entire leaf country is the most indispensable works characteristic, which affect the interception and accretion rates of PAHs. Leaves contribute to the works surface country much more than roots and branchlets and degree Celsius & A ; deg ; n be used for comparing of accretion rates between tree species. Jouraeva et Al. ( 2002 ) suggested that the mensurating the concentrations of PAHs per leaf country gives an accurate image of the accretion of PAHs instead than normalizing them to per foliage dry weight, & A ; deg ; s the foliages of diff?µrent tree species may change in the & A ; deg ; saddle horse of biomass per lupus erythematosus & A ; deg ; f & A ; deg ; re & A ; deg ; .
Howsam et Al. ( 2000 ) proposed that the pubescent leaves tend to roll up higher sums of PAHs than hairless foliages. Therefore, hairs were found to be effectual at capturing of atoms and forestalling their washing-off. Barthlott and Neinhuis ( 1997 ) showed for the first clip the mutuality between the surface raggedness, caused by epicuticular wax crystalloids, H2O repellence and decreased atom adhesion. Hereby, the rougher the surface, the better the H2O repellence which is accompanied by decreased attachment of contaminant atoms.
The cuticle is the outer uninterrupted bed of the works, which covers the aerial surfaces of the workss. The cuticle covers both ventral and dorsal surfaces of foliages. It limits inactive H2O loss and provides protection against some pathogens and minor mechanical harm. The rates of consumption of non-volatile lipotropic chemicals, which are deposited to flick surfaces and can non come in the works via pores by gaseous diffusion, are determined by cuticle. The works cuticles are composed of the indissoluble biopolymer cutin and waxes of assorted composings. Wax which exists within the cuticle matrix is known as embedded wax or intracuticular wax, whereas wax that covers the cuticle matrix is referred to as epicuticular wax. The cutin was found to represent 40-80 % of the cuticle mass in some species.
There are great differences in the composing and morphology of epicuticular waxes between and within different works species. Baker ( 1974 ) found that epicuticular waxes of Bruxelless sprout ( Brassica oleracea volt-ampere. gemmifera ) can develop different formless and crystalline signifiers, such as tubings, dendrites, fibrils and home bases. It is proposed that there is a strong connexion between morphology and chemical science of epicuticular wax. Therefore, the epicuticular waxes of most grass and Eucalyptus species can organize two different morphology types, simple home base and tubular, harmonizing to the different ruling component of aliphatic constituents, primary intoxicants ( hexacosanol or octacosanol ) and ?-diketones ( hentriacontan-14,16-dione or tritriacontan-12,14-dione ) severally. However, this is non ever the instance, e.g. home bases can besides be correlated with the presence of oleanolic acid in V. common grape vine.
Epicuticular waxes are typically composed of n-alkanes, primary n-alcohols, n-aldehydes and fatty acids, where each category of compounds occur in homologous series with C atoms runing from 20 to 40. In add-on, it was identified that esters of fatty acids ( C16-C34 ) and primary intoxicants ( C20-C36 ) can be formed, giving the compounds with the concatenation length of up to 70 C atoms. It was besides established that n-alkanes, secondary intoxicants, ketones, ?-diketones and their hydroxy- and keto-derivatives show high penchant for uneven C Numberss, whereas even carbon Numberss predominate in primary intoxicants, aldehydes, fatty acids and alkyl esters. The per centum of different single compound categories may change greatly. For illustration, leaf wax of H.vulgare was found to incorporate 89 % of primary intoxicants, 0.2-9.2 % of methane seriess, aldehydes, fatty acids and alkyl esters, whereas harmonizing to Bianchi et Al. ( 1984 ) foliage wax of Zea Mays contained 9-42 % each of above-listed category of compounds.
The sum of epicuticular wax may change widely from 1µg/cm2 to several mgs per square centimeter. It is possible to judge the thickness of the wax bed from its sums presuming that the denseness of wax mixture is comparatively changeless and is about 0.8-1.0 g/cm3. In such instance, 1µg/cm2 of wax would match to 10 nanometers of its bed thickness. It has been estimated for workss with 3-4 µm cuticle thickness the entire sum of epidermal stuff may run from 180 to 1500 kilogram per hectar. This shows that workss play a considerable function in roll uping the lipotropic compounds in the environment. However, workss may merely move as impermanent sinks, as the foliages of deciduous trees are accumulated in the dirt every twelvemonth where it develops a lipotropic site for accretion of pollutants.
Environmental conditions besides influence the accretion rates of PAHs on foliage surfaces. Ambient temperature influences the PAH-vegetation divider procedure. Harmonizing to Simonich and Hites ( 1994 ) in spring and autumn, when the ambient temperature is low gaseous PAHs divider into flora and at high ambient temperatures in summer some PAHs volatalize back to the ambiance.
Leaf surface is surrounded by laminal boundary bed of air. Laminar boundary bed is characterised by about absence of turbulency where the velocity of air current is really undistinguished. The thickness of laminal air depends on raggedness of the leaf surface and velocity of air current of upper air beds. It reduces at high air currents and can be well thickened in unagitated conditions. The atmospheric atoms are trapped by laminal boundary bed and deposited on the foliage surfaces.
Barthlott and Neinhuis ( 1997 ) subjected water-resistant foliages to natural and unreal rains and found that atoms of any size and different chemical nature were about wholly removed from the surface of the foliages until their surface waxy beds were non damaged and they explained it by high kinetic energy of the rain.
PAHs pose a hazard to human wellness owing to their carcinogenic and mutagenic belongingss and therefore it is of import to be able to supervise their concentrations in the environment and present both qualitative and quantitative analysis for these pollutants. Pollution of workss with PAHs is of a turning concern as they may come in a nutrient concatenation get downing with the flora and range worlds. It is besides of import fact that flora Acts of the Apostless as an air filter and play function of a impermanent reservoir for PAHs. Wagrowski and Hites ( 1997 ) estimated that 160 T of PAHs are deposited to workss every twelvemonth and constitutes 4 % of entire emitted PAHs in northeasterly part of the United States.
Another of import point is that tree foliages are widely used as indexs to measure the atmospheric PAH degrees in the urban countries. Such biomonitoring is found to be efficient and cost effectual as it is easier to roll up foliages than air samples.
PAHs are chiefly produced by burning which can be both natural ( e.g. forest fires ) and anthropogenetic. Anthropogenetic beginnings such as burning in vehicles and domestic warming preponderantly contribute to PAH emanations to the environment. PAH concentrations are higher in urban sites than in rural locations due to the intimacy of pollution beginnings such as vehicles.
Purposes and aims
The purpose of the present research undertaking is to find the extent to which leaves accumulate PAHs and look into whether the foliages of different tree species accumulate these pollutants to different extents.
To accomplish the purpose of the undertaking the undermentioned aims were set:
To carry on leaf country and dry weight gravimetric measurings and find the wax contents of the foliages.
To pull out PAHs from foliage samples utilizing supersonic extraction and carry on clean-up processs of the infusions.
To place single PAHs both quantitatively and qualitatively by agencies of gas chromatography/mass spectroscopy ( GC/MS ) .
To compare entire PAH concentrations per foliage countries and/or wax content of foliages of different tree species. Reveal any tendencies in PAH profiles and compare them with those in the literature.
The undertaking aims to find PAHs adsorbed on foliages ( acerate leafs ) of both deciduous tree species and evergreens. The concentrations of PAHs on foliages depending on seasonal fluctuations or over a turning period will non be investigated due to the restricted clip frame. Undertaking will look into the extents to which leaves of different tree species accumulate PAHs by comparing their PAH loads ( a mass per entire foliage surface country and/or wax content ) .
16 USEPA precedence PAHs, every bit good as 2- and 1-methylnaphtalenes, benzo [ J ] fluoranthene, benzo [ vitamin E ] pyrene and perylene were identified and quantified in foliages of 9 different tree species, which included:
Common holly ( Ilex aquifolium )
Saging retem ( Juniperus recurva )
Silver birch ( Betula pendula )
English oak ( Quercus robur )
Rhododendron ( Rhododendron thomsonii )
Common Silver Fir ( Abies alba )
Beech ( Fagus sylvatica )
Sycamore ( Acer pseudoplantanus )
Lime tree ( Tilia europaea )
Study sites and sample aggregation
Two survey sites within the metropolis of Newcastle Upon Tyne were chosen. Back garden of the Drummond edifice of the Newcastle University and Moorbank botanical garden are situated following to the roads loaded with vehicular traffic ( Fig. 3.1 and 3.2 ) . Maps were modified from Ordnance Survey ( 2010 ) .
Figure 3.2 Moorbank botanical garden
Leafs of different tree species were collected in September 2009 and May 2010 from both sites. Sampled countries are shown in Figures 3.1 and 3.2 as shaded circles. The exact locations of sampled trees were recorded utilizing GPS sailing master and facets of the sampled sides of the trees were besides identified ( Table 3.1 ) . All foliages were collected from 1.5-2 m tallness. Sampled tree foliages and acerate leafs were first air-dried for several yearss and so stored in paper envelopes until experiments commenced.
Extraction of PAHs and works waxes
The solvent extraction was non thorough as epicuticular waxes ( non embedded waxes ) and PAHs on which they were adsorbed are of involvement. The analytical process was tested several times prior to chief analyses. There was a possibility that works pigments such as chlorophylls and provitamin As would be co-extracted along with epicuticular waxes and interfere with the analysis. There was besides possibility that wax esters may do extremum overlapping on mass chromatograms. However, trial processs showed that works pigments were retained by chromatographic column and there were no peak overlapping on mass chromatograms in the countries of involvement. PAH degrees on test samples were approximately quantified in order to find the sums of internal and recovery criterions that needed to be added.
Each foliage sample was weighed and appropriate sums of 1,1′-binaphthyl solution in DCM was added as recovery criterion straight to flick samples prior to extraction utilizing micro syringe ( Table 3.3 ) . After 30 proceedingss, when dissolver was evaporated, leaves harmonizing to their sizes were placed into 250 milliliter or 500 milliliter beakers. DCM ( 200-400 milliliter ) was so added to each sample in the beaker. Each beaker was placed into the supersonic bath for 1 minute. 3 back-to-back extractions of each sample were made and infusions were so combined. After that, solvent volume of entire infusion was reduced to ~20 milliliter utilizing rotary evaporator. The figure of foliages which were subjected to extraction every bit good as their weights are given in Table 3.2.
Sample clean-up and hydrometric measurings
First, entire infusions were subjected to filtrating through a class 1 ( 11 µm ) filter paper to extinguish any present atoms and clear up the infusions. After that, dissolver was farther evaporated from the filtered infusions utilizing rotary evaporator to a volume of ~ 5 milliliter. Concentrated infusions were transferred to the 10 milliliter mensurating cylinders and made up to 10 milliliter. A 1/9th aliquot of each sample was taken and transferred into the pre-weighed phials in order to find the sums of foliage waxes. The dissolver was evaporated till waterlessness from aliquots utilizing a watercourse of N gas. It was impossible to vaporize all the dissolver merely by utilizing a watercourse of N gas as the dissolver molecules were trapped within the foliage waxes. To take all the solvent phials were placed in a warm bath ( 30-40 & A ; deg ; C ) . Phials were following weighed until changeless weight and entire sums of extractible waxes were determined ( Table 3.3 ) .
The aliquots incorporating ~60 milligram of extractible waxes or less were concentrated to a volume of ~0.25 milliliter and transferred into the phials with ~0.5 ml aluminum oxide. The staying DCM was allowed to vaporize. The 500 millimeter long – 10 millimeter internal diameter glass columns were foremost plugged with cotton wool and so packed with silicon oxide and aluminum oxide ( 4:1 ) . The aliquots which were adsorbed on aluminum oxides and free of DCM were loaded on top of the jammed columns. PE ( 70 milliliter ) was run through the columns to elute aliphatic hydrocarbons. PAHs incorporating fraction was eluted with DCM: PE ( 1:1 ) dissolvers mix ( 70 milliliter ) . Aliphatic fraction of each sample were concentrated down to a volume of ~3 milliliter and stored in capped phials. PAHs incorporating fractions were concentrated to a volume of ~1 milliliter and transferred to GC phials.
A procedural space was besides prepared by reiterating all the analytical processs but without leaf samples. A mix of criterions was made by adding about equal sums of 1,1′-binaphthyl, p-terphenyl and deuterated PAHs to a GC phial ( Table 3.3 ) .
A mix of deuterated PAHs and p-terphenyl solutions in DCM were added in appropriate sums to each PAH incorporating fraction as internal criterions prior to GC-MS analysis ( Table 3.3 ) . The mix of deuterated criterions included 5 deuterated PAHs: naphthalene-d8, acenaphthene-d10, phenanthrene-d10, chrysene-d12 and perylene-d12.
The aromatic fractions of infusions, the criterions mix and a clean sample were analysed by GC-MS, which was performed on Agilent 6890/7890A GC in split less manner, injector at ( 280 & A ; deg ; C ) linked to a Agilent 5973/5975 MSD ( electron electromotive force 70eV, beginning temperature 230 & A ; deg ; C, quadrupole temperature 150 & A ; deg ; C, multiplier electromotive force 1800V, interface temperature 310 & A ; deg ; C ) . The acquisition was controlled by a HP Compaq computing machine utilizing Chemstation package in selected ion manner ( 30 ions 0.7 hertz 35 MS dwell ) . The sample ( 1µl ) in DCM was injected by an HP7673/7683B autosampler and the split opened after 1 minute. After the solvent extremum had passed the GC temperature programme and informations acquision commenced.
Separation was performed on an Agilent fused silica capillary column ( 30 m -0.25 mm internal diameter ) coated with 0.25 µm dimethyl poly-siloxane ( HP-5 ) stage. The column temperature was programmed from 50-310 & A ; deg ; C at 5 & A ; deg ; C/min and held at concluding temperature for 10 proceedingss with Helium as the bearer gas ( flow rate of 1 ml/min, initial force per unit area of 50 kPa, split at 30 ml/min ) .
Extremums were identified by comparing the elution times of deuterated criterions against matching PAHs every bit good as by recognizable extremum forms, elution order and comparative keeping times of PAHs.
Leaf country measurings
Surface countries of foliages were determined after extraction processs by utilizing AreaS 2.1 freeware ( Samara State Agricultural Academy, 2005 ) . Areas of any complexness can be calculated by comparing of two scanned images where the country of one of them is known. Thus, a scanned image of rectangular with known country of 5×5 cm2 was used to put up the graduated table. The countries of scanned foliages were estimated by the plan automatically. Calculating mistake was no more than 0,001 % . Calculated leaf country was doubled to obtain the entire leaf surface country. Entire surface countries ( S ) of acerate leafs were calculated by mensurating the length ( cubic decimeter ) , diameter ( vitamin D ) and the figure ( N ) of acerate leafs extracted:
The entire surface countries of foliages used in each sample are given in Table 3.4. The images of investigated foliages are given in Appendix 1.
Obtained GC-MS information was processed on Chemstation package ( version E.01.01.335 ) . Peak countries were integrated utilizing RTE planimeter and manual integrating method. Unfortunately, some samples were contaminated with some polar compounds which were retained in the column and caused the shadowing of the extremums in following samples. Contamination may come from either the detergents used in cleansing of the chemical dishes or contaminated dissolvers, aluminum oxide or silicon oxide as the experiments were carried out in a shared research lab.
First, comparative response factor ( RRF ) of internal criterion ( p-terphenyl ) against foster criterion ( 1,1′-binaphthyl ) from the criterions mix sample was calculated:
The chromatogram of criterions mix can be seen from Figure 4.1 ( Section 4 ) . Recovery of 1,1′-binaphthyl was so calculated for each sample as following ( Section 10, Table 10.4 ) :
PAH sums were determined by quantifying each PAH extremum against the appropriate deuterated PAH ( with the same figure of rings ) with the premise that RRF of PAHs is peers to 1 ( Table 3.5 ) as we do non cognize the exact sums of analyte PAHs nowadays in the sample:
, where ten is the analyte PAH.
Estimated weights of the analyte PAHs were so corrected for recovery of foster criterion ( 1,1′-binaphthyl ) presuming that recovery criterion reflects the behavior of the analyte PAHs:
Designation of PAHs
First of all, ion chromatograms of all added criterions were obtained from criterions mix sample. Figure 4.1 represents the combined ion chromatogram which was obtained by come ining the bids in bid line, which is on the chief interface of Chemstation package. The algorithm of bids is given in Appendix 2. As it is known that deuterated PAHs come out merely before their non-deuterated parallels, it was easy possible to find the naphthalene, acenaphthene, phenanthrene, chrysene and perylene ( Figures 4.2-4.5 and 4.8 ) . Figures 4.6 to 4.9 illustrate combined ion chromatograms for all parts of involvement, which were magnified between the peculiar clip periods. Extremums were identified harmonizing to the elution order of the PAHs, recognizable extremum forms and comparative keeping times.
All samples were run in SIM manner and it was impossible to place the extremums utilizing plan library. However, the library could non distinguish and place the extremums within one m/z ratio, e.g. for ion 252 it gives equal per centum of fiting ( 98 % ) for one extremum with benzo [ B ] fluoranthene, benzo [ K ] fluoranthene, benzo [ J ] fluoranthene, benzo [ vitamin E ] pyrene and benzo [ a ] pyrene.
Table 4.1 summarises concentrations of single PAHs and amount of 16 USEPA precedence PAHs in 16 samples ( D1-D7 and M1-M9 ) . The PAHs which were quantified included 16 USEPA precedence PAHs, every bit good as 2- and 1-methylnaphtalenes, benzo [ J ] fluoranthene, benzo [ vitamin E ] pyrene and perylene. The PAH concentrations are normalized to flick surface country and expressed in ng per square decimeter. The natural information is given in Appendix 3.
Phenanthrene and anthracene, every bit good as benzo [ B ] fluoranthene and benzo [ B ] fluoranthene were quantified as a individual extremum due to their hapless declaration.
The analysis of D5, D6 and D7 samples were performed to gauge the preciseness of the analysis. The average concentrations of single PAHs in these replicates are shown in Figure 4.10, with the standard mistakes shown as mistake bars.