Environ Sci Pollut Res DOI 10.1007/s11356-015-5890-8 BIOMONITORING OF ATMOSPHERIC POLLUTION: POSSIBILITIES AND FUTURE CHALLENGES Heavy metal and polycyclic aromatic hydrocarbon concentrations in Quercus ilex L. leaves fit an a priori subdivision in site typologies based on human management Flavia De Nicola 1 & Daniela Baldantoni 2 & Giulia Maisto 3 & Anna Alfani 2 Received: 29 June 2015 / Accepted: 27 November 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract Concentrations of four heavy metals (HMs) (Cd, Cr, Fe, Pb) and four polycyclic aromatic hydrocarbons ( PA H s ) ( f l u o r a n t h e n e , p h e n a n t h r e n e , c h r y s e n e , benzo[a]pyrene) in Quercus ilex L. leaves collected at the Campania Region (Southern Italy) in previous air biomonitoring studies were employed to (1) test the correspondence with an a priori site subdivision (remote, periurban, and urban) and (2) evaluate long temporal trends of HM (approximately 20 years) and PAH (approximately 10 years) air contaminations. Overall, Q. ilex leaf HM and PAH concentrations resulted along the gradient: remote < periurban < urban sites, reflecting the a priori subdivision based on human management. Over a long time, although a clear decrease of leaf Pb, chrysene, fluoranthene, and phenanthrene concentrations occurred at the urban sites, a high contamination level persists. Keywords Holm oak . Inorganic and organic pollutants . Long-term biomonitoring . Air contamination gradients . Campania Region (Southern Italy) Responsible editor: Constantini Samara * Daniela Baldantoni dbaldantoni@unisa.it 1 Dip. Scienze e Tecnologie, Università degli Studi del Sannio, via Port’Arsa 11, 82100 Benevento, Italy 2 Dip. Chimica e Biologia BAdolfo Zambelli^, Università degli Studi di Salerno, via Giovanni Paolo II 132, 84084 Fisciano, SA, Italy 3 Dip. Biologia, Università degli Studi di Napoli Federico II, via Cinthia, 80126 Naples, Italy Introduction Recently, some heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs), widely recognized as carcinogenic and teratogenic pollutants (IARC 2013). have been considered as causes of many human diseases. Therefore, the BDirective on ambient air quality and cleaner air for Europe^ (Directive 2008/ 50/EC) states that air HM and PAH concentrations must be routinely monitored. These pollutants are emitted in the air by various mobile and stationary sources (motor vehicles, domestic heating, power plants) that are very abundant in urban or industrial areas, and may also move to remote areas. Anyway, air pollutant emissions in remote areas (biological activities, fires, or pedogenetic alterations) are not negligible. Inhalation and ingestion of contaminated food are among the main intake ways of these pollutants by humans (Ravindra et al. 2008a). Due to the high costs for installation and maintenance, the monitoring stations can be usually placed only at a few critical sites of the cities. Thus, the deriving data can represent a local situation and cannot be extended to wider areas. To bypass these inconveniences (i.e., costs and area representation), living organisms can be effectively used to monitor air quality. Besides, living organisms, accumulating air pollutants during their exposure time, can be also used to assess air quality either at a brief or long term (Alfani et al. 1996, 2000, 2005; De Nicola et al. 2005; Aničić et al. 2011). In the last decades, as biomonitoring experienced great interest, the Directive 2004/107/EC (arsenic, cadmium, mercury, nickel, and polycyclic aromatic hydrocarbons in ambient air) also recommends, in addition to mandatory measurements, the use of bioindicators to assess contamination patterns at a regional scale. In this frame, many higher plants can be effectively used as biomonitors of air quality as their morphology, physiology, and ecology are better known than in lower plants (Wittig 1993) and as leaf age and exposure time can be easily recognized (Bargagli Environ Sci Pollut Res et al. 1998). Leaves can accumulate air gaseous and particulate pollutants by stomata and/or by interception, impaction, or sedimentation on leaf surface, and leaf morphological characteristics (i.e., surface area, presence of tricomes, chemistry of cuticular waxes) play an important role in particulate pollutants adsorption (Wittig 1993; Song et al. 2015). Despite soil can contribute to leaf pollutant concentrations, some HMs are accumulated in the roots and scarcely translocated to the aboveground plant portion (Domínguez et al. 2011) whereas PAHs are negligibly absorbed by roots (Simonich and Hites 1995). The aim of this paper was to test the correspondence between an a priori subdivision of sites of Campania Region (Southern Italy) in three typologies (remote, periurban, and urban) on the basis of human management and the concentrations of HMs (Cd, Cr, Fe, and Pb) and PAHs (benzo[a]pyrene, chrysene, fluoranthene, and phenanthrene) in leaves of Quercus ilex L., a typical Mediterranean tree, widely employed as biomonitors (Alfani et al. 2000; De Nicola et al. 2005, 2011). In addition, this paper aimed to evaluate, through the leaf analyses, temporal trends of the inorganic and organic pollutants over a long period (approximately 20 years for HMs and 10 years for PAHs). Materials and methods Background The data reported in this paper come from the analyses of Q. ilex leaves sampled and processed in previous studies, Fig. 1 Remote (diamonds), periurban (triangles), and urban (circles) sites of Campania Region (Italy) where HMs (white), PAHs (gray), or both (black) were investigated (9 remote, 8 periurban, and 26 urban sites for HMs; 4 remote, 6 periurban, and 18 urban sites for PAHs) according to standardized procedures. These studies were performed in order to respond to relevant and different subjects about biomonitoring and here synthetically reported: (1) the possibility to use Q. ilex leaves as biomonitors of air quality through the evaluation of HM and PAH concentrations; (2) the correspondence between leaf pollutant accumulation and leaf time exposure; (3) the leaf uptake of air pollutants and their accumulation in the tissues or on the surface of leaves; and (4) the relationships between leaf and soil concerning these two classes of pollutants. Considered the great number of observations (43 sites for HMs and 28 sites for PAHs) and the long data series (1989–2009 for HMs and 1998–2009 for PAHs), the authors propose to use all the previously obtained data to respond to the aims of this paper. Sampling sites and sample collection The employed sampling sites of the Campania Region (Southern Italy) were grouped, basing on the human management, into three site typologies: remote (9 sites for HMs and 4 for PAHs), periurban (8 and 6 sites for HMs and PAHs, respectively), and urban (26 sites for HMs and 18 for PAHs) (Fig. 1). The study area is characterized by a Mediterranean climate, with warm and dry summers and cold and rainy winters (a climate diagram of the area is reported in De Nicola et al. 2013). At each site, 4–8 Q. ilex trees were chosen to perform the leaf samplings. Small branches located 2–4 m above the ground and from the outer part of the canopies were cut by pruning shears. In order to Environ Sci Pollut Res 0.3 HMs 0.2 Pb −20 0 0.1 20 40 80 0.0 MDS2 60 Fe 100 Cd 120 −0.1 Cr 140 160 −0.2 180 200 −0.3 220 −0.4 −0.2 0.0 0.2 0.4 MDS1 Fig. 2 Non-metric multidimensional scaling (NMDS) biplot of HMs in Q. ilex leaves from remote (diamonds), periurban (triangles), and urban (circles) sites of Campania Region (Italy). The temporal gradient (gray lines) and the confidence ellipses (α=0.05) for remote (dotted), periurban (dashed), and urban (solid) sites are also shown obtain a homogeneous sample, a large number of 1-year old leaves was collected by hand, taking into account that the leaf bud break mainly occurs each year in May (De Lillis and Fontanella 1992). The samplings were carried out minimizing the contact with the leaf surface. The unwashed leaves were differently treated to measure the HM and PAH concentrations. HM and PAH determinations For HM analyses, leaves were oven dried at 75 °C until constant weight and pulverized with agate ball mills, using a Fritsh Pulverisette or a Retsch PM4. Subsequently, the powder was used to prepare three replicates. The samples (250 mg) were mineralized with the addition of 4 ml 65 % HNO3 and 2 ml 50 % HF in a microwave oven system (Milestone, Ethos) and diluted to a final volume of 50 ml, as reported in Baldantoni et al. (2009). Sample mineralization was obtained through the following steps: 250 W for 2 min, 0 W for 2 min, 250 W for 5 min, 400 W for 5 min, 0 W for 2 min, and 500 W for 5 min. The metal concentrations were detected using Varian (AA20) and PerkinElmer (AAnalyst 100) atomic absorption spectrometers, via graphite furnace (Cd, Cr, and Pb) or flame (Fe). Multipoint linear calibration curves were performed for each HM; when outside the linear range, the samples were adequately diluted. In order to ascertain the accuracy of the employed method and the right quantification of the investigated HMs, a concurrent analysis of reference materials was carried out (Olive leaves BCR62 and Pine needles NIST1575a), obtaining percentage recoveries of 80–86 % for Pb, 94–98 % for Cr, 98–100 % for Fe, and 105–110 % for Cd. The precision of the method, calculated as relative standard deviation (n=9), was 2 % for Pb, 5 % for Cr and Fe, and 9 % for Cd. For PAH analyses, fresh leaves (5 g) were extracted by three consecutive sonications (Misonix, XL2020 sonicator), each in 100 ml of a mixture of dichloromethane and acetone (1:1 = v/v). Subsequently, the extracts were reduced in volume (De Nicola et al. 2005) and the concentrations of fluoranthene (Flt), phenanthrene (Phen), chrysene (Crys), and benzo[a]pyrene (B[a]P) were detected by gas chromatography coupled to mass spectrometry detector (HP 5890/5971). The GC-MS conditions were described in De Nicola et al. (2005). To quantify the PAHs, multipoint calibration curves were performed using standard mixtures. To evaluate the extraction efficiency, labeled PAHs (phenanthrene-d 10 , chrysene-d 12 , and perylene-d 12) at known concentrations, were added to each sample before the extraction. The percent recovery of labeled PAHs, approximately of 70 % for each, was used to correct the quantification of the investigated PAHs. The precision of the method, calculated as relative standard deviation (n=6), ranged from 4 % for Phen to 12 % for B[a]P. For each leaf sample, the PAH analyses were carried out in triplicates. Data analysis The overall differences in leaf HM and PAH concentrations among site typologies and among sampling times were evaluated using two-way multivariate analysis of variance (MANOVA) and non-metric multidimensional scaling (NMDS). The MANOVA models, with the HMs or the PAHs as dependent variables and the site typologies and sampling times as fixed factors, were based on the Pillai’s statistic. Upon the NMDS HM and PAH ordinations, based on the Euclidean distance and on two axes, the confidence ellipses (for α = 0.05) for the three site typologies, as well as the temporal fields evaluated through cubic splines, w ere superimposed. The MANOVAs were followed by ANOVA models for each dependent variable, using the site typologies and sampling time as fixed factors. The Tukey HSD post hoc test was then employed to evaluate differences among each pair of site typologies. Homoscedasticity and normality of the residuals were assessed using the Breuch-Pagan and the Kolmogorov-Smirnov tests, respectively. All the analyses were performed using the R 3.1.1 programming environment (R Core Team 2014) with functions from the Bstats^, Bvegan^, Bmgcv^, Bnortest^, and Blmtest^ packages. Environ Sci Pollut Res 0,3 Cd ( g/g d.w.) Fig. 3 HM concentrations (mean values±standard errors of the means) measured in Q. ilex leaves collected from 1989 to 2009 in remote, periurban, and urban sites of Campania Region (Italy) remote periurban urban 0,2 0,1 0,0 6 Cr ( g/g d.w.) 5 4 3 2 1 Fe (mg/g d.w.) 0 1,8 1,2 0,6 0,0 55 Pb ( g/g d.w.) 44 33 22 11 Results Heavy metals Leaf metal concentrations widely varied among the sites, and the ranges were 0.001–0.693 μg g−1 dry weight (d.w.) for Cd, Ma y2 00 8 Ma y2 00 9 Se M pte a y Ja mbe 200 nu r 2 1 ary 00 Ma 20 1 y 2 02 00 2 tob er 19 98 Oc 19 96 Ma r ch Ma rch 19 89 0 0.03–10.03 μg g−1 d.w. for Cr, 0.1–4.5 mg g-1 d.w. for Fe, and 0.01–147.86 μg g−1 d.w. for Pb. The MANOVA test, considering the leaf concentrations of all HMs, highlighted significant differences among the site typologies (P<0.001) and among the time samplings (P<0.001). In particular, the NMDS analysis Environ Sci Pollut Res 0.2 PAHs 0.1 40 60 Crys 30 0.0 Flt 100 50 −0.1 130 Phen 60 12 0 MDS2 50 B[a]P −0.2 50 110 70 90 80 −0.4 −0.3 50 highlighted a site distribution with a partial overlapping between confidence ellipses of urban and periurban sites and a better separation of the confidence ellipse of remote sites (Fig. 2). Moreover, Pb showed higher concentrations in leaves of the urban sites and differentiated them from the leaves of the remote sites, which were characterized by high Cd concentrations (Fig. 2). Finally, leaf Pb concentrations decreased over the time (Fig. 2), with a reduction in urban and periurban sites, respectively, from 43.09 and 5.16 μg g−1 d.w., at the beginning of the monitoring, to 1.32 and 0.35 μg g−1 d.w. at the end (Fig. 3); on the other hand, the other leaf HM concentrations showed unclear temporal trends (Figs. 2 and 3). The ANOVA tests showed differences of leaf Cd, Cr, Fe, and Pb concentrations among the site typologies (P<0.01 for Pb, P<0.001 for the other HMs) and among the time samplings (P<0.01 for Cd and Fe, P<0.001 for Cr and Pb). In detail, HM concentrations in the leaves from urban sites were higher than those measured in the leaves from periurban (P<0.05 for Pb, P<0.001 for Cd, Cr, and Fe) and remote sites (P <0.01 for Cd and Pb, P<0.001 for Cr and Fe). −0.4 −0.2 0.0 0.2 0.4 MDS1 Fig. 4 Non-metric multidimensional scaling (NMDS) biplot of PAHs in Q. ilex leaves from remote (diamonds), periurban (triangles), and urban (circles) sites of Campania Region (Italy). The temporal gradient (gray lines) and the confidence ellipses (α=0.05) for remote (dotted), periurban (dashed), and urban (solid) sites are also shown Polycyclic aromatic hydrocarbons Discussion The MANOVA test, carried out on leaf PAH concentrations, highlighted significant differences among the site typologies (P < 0.01) and among the time samplings (P < 0.001). Among the sites, leaf Phen concentrations ranged between 14.5 and 1172.1 ng g−1 d.w., Flt concentrations between 12.5 and 1734.8 ng g−1 d.w., Crys concentrations between 0.1 and 710.5 ng g−1 d.w. and B[a]P concentrations between 0.1 and 136.7 ng g−1 d.w. The NMDS analysis showed a site distribution with a partial overlapping among confidence ellipses of urban, periurban, and remote sites, and a B[a]P increase over the time (Fig. 4). In particular, the ANOVA tests showed statistically (at least P<0.01) significant differences in leaf PAH concentrations among the site typologies and the Tukey HSD post hoc tests highlighted higher concentrations in the urban area with respect to the periurban (P < 0.05 for Flt and Phen; P < 0.01 for Crys) and the remote (P<0.05 for B[a]P; P<0.01 for Flt and Phen; P<0.001 for Crys) ones. Regarding the temporal trend, a decrease in leaf concentrations was observed for Crys, Flt, and Phen, mainly at the urban sites, in 2008 and 2009 when B[a]P concentrations increased (Fig. 5). Statistically significant differences among the sampling times were found for leaf Phen (P<0.01) and B[a]P (P<0.001) concentrations, with the highest concentrations in winter. The variations in leaf concentrations of the investigated HMs and PAHs among the sites highlighted a wide heterogeneity of Campania Region, regarding the air pollution. Moreover, the human activities, characterizing the sites in different typologies (remote, periurban, and urban), play an important role in leaf pollutant accumulation. For both kinds of pollutants, a clear separation between leaves of urban and remote sites was observed. Anyway, an overall consideration highlighted a different behavior of HMs and PAHs in leaf accumulation at the periurban sites. In fact, at these sites, leaf HM concentrations were more similar to those observed for the urban sites than for the remote ones, whereas, leaf PAH concentrations at the periurban sites were more similar to those observed for the remote than the urban sites. In Campania Region, leaf mean concentrations at the urban sites were 15.20 μg g−1 d.w. for Pb and 0.137 μg g−1 d.w. for Cd, exceeding of one order of magnitude the fingerprint values for Q. ilex leaves, equal to 1.05 and 0.04 μg g−1 d.w., respectively (Bargagli et al. 1998). Anyway, whereas Pb contamination exclusively interested the urban area, being the mean values equal to 0.30 and 2.25 μg g−1 d.w., respectively, in the leaves collected at the remote and periurban sites, Cd contamination interested also many of the remote sites, being the mean value for this site typology equal to 0.056 μg g−1 d.w. Since it is widely recognized that Pb and Cd are, respectively, markers of vehicular traffic (Monaci et al. 2000; Salvagio Manta et al. 2002) and industrial activity (Celo and Environ Sci Pollut Res Dabek-Zlotorzynska 2010), these emission sources would seem to be mainly responsible for air contamination in the Campania Region. However, these metals would seem to be linked to different air particulate sizes: mostly coarse for Pb and fine for Cd (Ny and Lee 2011; Gonzáles-Castanedo et al. 2014). For this reason, notwithstanding Cd emissions in the atmosphere of the remote areas are low (Pacyna 1987). the B[a]P (ng/g d.w.) 150 remote periurban urban 100 50 Crys (ng/g d.w.) 0 350 280 210 140 70 Phen (ng/g d.w.) 0 600 450 300 150 0 800 600 400 20 08 Ma y2 00 9 Ma y 20 05 Ma r ch 0 Se M pte ay m Ja ber 2001 nu ar 200 May 20 1 y 2 02 00 2 200 Ju ly 1 99 8 Flt (ng/g d.w.) Fig. 5 PAH concentrations (mean values±standard errors of the means) measured in Q. ilex leaves collected from 1998 to 2009 in remote, periurban, and urban sites of Campania Region (Italy) high presence of Cd in Q. ilex leaves at these sites could be due to the transport of fine particulate from the most contaminated urban or industrial sites. As Pb, also Cr and Fe, metallic pollutants emitted by motor vehicles (Monaci et al. 2000) showed the highest concentrations in the Q. ilex leaves collected at the urban sites, although their concentrations did not exceed the chemical fingerprint (Bargagli et al. 1998). Environ Sci Pollut Res Among the detected PAHs, B[a]P appeared the less abundant in the urban air as its mean value in Q. ilex leaves was 37.8 ng g−1 d.w. as compared to the mean values of 257.0, 331.2, and 190.9 ng g−1 d.w., respectively, of Phen, Flt, and Crys. Anyway, as the mean values at the urban sites were approximately 1.8- and 2.1-fold higher than those found, respectively, for the periurban and remote sites, B[a]P accumulated mostly in the urban leaves. The mean values of Phen, Flt, and Crys for the urban sites were, respectively, 2.9-, 4.9-, and 4.6-fold higher than those for the periurban sites, and 6.8-, 10.6-, and 11.4-fold higher than those for the remote sites. Although B[a]P was lower than the other investigated PAHs, its leaf concentration fell in the range reported for leaves of the same species collected in other urban sites (Orecchio 2007). whereas Phen, Flt, and Crys resulted to one order of magnitude higher. B[a]P is dominantly emitted from light-duty gasoline vehicles and it is marked for industrial stacks, together with other 4- and 5-ring PAHs (Ravindra et al. 2008a). B[a]P is the only PAH for which a target value in air was established (1 ng m−3 as annual mean, Directive 2004/107/CE), and it is used as marker to assess the toxicity of a PAH mixture. Phen is usually found in high level in motor vehicle emissions and, together with Crys and B[a]P, in steel industry emissions. Phen is also emitted at high levels with Flt in incineration and oil combustion, and the two PAHs are indicated as identifying diesel emissions (Ravindra et al. 2006; Ravindra et al. 2008a). For each site typology, and overall for the urban sites, a seasonal trend in PAH leaf concentrations, with higher winter values, was observed according to the scientific literature that reports for winter major emission sources and lower temperature favoring the condensation/sorption of PAHs on air particles (Ravindra et al. 2008b). Although the evidence of spatial gradients for either HMs or PAHs (remote < periurban < urban sites), in the leaves collected at the urban sites an overall decrease of Pb, Crys, Flt, and Phen concentrations over the time (1989–2009 for HMs and 1998–2009 for PAHs) occurred. Pb decrease in Q. ilex leaves (Alfani et al. 2000; De Nicola et al. 2015) is expected as a Pb reduction in the air occurred since 1986 (Directive 1985/210/EEC). when unleaded fuels were introduced. The lack of temporal variations for Cr and Fe, mainly emitted by vehicular exhausts (Amato et al. 2011) in the urban area, suggests that over the long period, the intensity of the traffic is still high and that the quality of the exhausts changed only for Pb. However, during the investigated time period, the improved technology of cars (Alves et al. 2015) likely permits a decrease of traffic-related PAHs (i.e., Flt and Phen) at the urban sites where the traffic flow is the main source of PAHs. The overall decrease, over the time, of many investigated pollutants in Q. ilex leaves might be also due to management directives aiming to monitor the air quality (Directive 2004/ 107/EC). in order to not exceed the pollutant threshold values (i.e., especially for those that are considered causes of human diseases), and to improve the whole environmental quality. Conclusions The spatial analysis of Q. ilex leaves highlighted the following gradient of HM and PAH concentrations: remote < periurban < urban sites. Thus reflected the a priori subdivision based on human management of the investigated areas of the Campania Region. The temporal analysis highlighted differences over a long time in the contamination degree, as leaf pollutant concentrations remained low and almost constant at the remote sites, and high and severe at the urban sites, notwithstanding the clear decrease of Pb, Crys, Flt, and Phen. Finally, either spatial or temporal dynamics of HM and PAH concentrations further validated Q. ilex leaves as good monitors of air quality. Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests. References Alfani A, Bartoli G, Rutigliano FA, Maisto G, Virzo De Santo A (1996) Trace metal biomonitoring in the soil and the leaves of Quercus ilex in the urban area of Naples. 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