Forest Canopy Can Efficiently Filter Trace Metals from Deposited Precipitation in a Sub-Alpine Spruce Plantation

Trace metals can enter some natural regions with low human disturbance from atmospheric circulation, but little information is available regarding how the canopy can retained trace metals. Therefore, a representative sub-alpine spruce plantation was selected to investigate the net throughfall fluxes of eight trace metals (Fe, Mn, Cu, Zn, Al, Pb, Cd and Cr) of closed canopy and gap-edge canopy from August 2015 to July 2016. Over a one-year observational period, the annual fluxes of Al, Zn, Fe, Mn, Cu, Cd, Cr and Pb were 7.29 kg·ha-1, 2.30 kg·ha-1, 7.02 kg·ha-1, 0.16 kg·ha-1, 0.19 kg·ha-1, 0.06 kg·ha-1, 0.56 kg·ha-1 and 0.24 kg·ha-1, respectively, in the deposited precipitation. The annual net throughfall fluxes of these trace metals were 1.73 kg·ha-1, 0.9 kg·ha-1, 1.68kg·ha-1, -0.032 kg·ha-1, 0.04 kg·ha-1, 0.018 kg·ha-1, 0.093 kg·ha-1 and 0.087kg·ha-1, respectively, in the gap-edge canopy and -1.6 kg·ha-1, 1.13 kg·ha-1, 1.65 kg·ha-1, -0.10 kg·ha-1, 0.05 kg·ha-1, 0.03 kg·ha-1, 0.26 kg·ha-1 and 0.15 kg·ha-1, respectively, in the closed canopy. The closed canopy displayed a greater filter effect on the trace metals from precipitation than did the gap-edge canopy in the sub-alpine forest. In the rainy season, the net filtering ratio of trace metals ranged from -66%-89% in the closed canopy and from -52% to 25% in the gap-edge canopy. However, the net filtering ratio of all trace metals was greater than 50% in the closed canopy in the snowy season. Therefore, the results suggested that the most trace metals moving through the forest canopy are taken up rather than by rainfall leaching; moreover, the closed canopy can efficiently take up trace metals in the snowy season.


Introduction
It is well known that trace metals originate mainly from metal refining, fossil fuel combustion, automotive exhaust and other human activities [1].Increasing studies have documented that trace metals mainly exist as particles in the atmosphere and can enter some natural regions with low human disturbance from atmospheric circulation [1,2].The input of trace metals via atmospheric deposition is a large source of contamination for plants, soil and water and a large number of continuous trace metal inputs would have a lasting negative impact on the biogeochemical cycle in an ecosystem [3].Forest ecosystems can often be called ecologic filters that can efficiently decrease atmospheric pollutants and improve air quality [4,5], however, the efficiency of this filter is often controlled by the precipitation and canopy characteristics.
Trace element is rather loosely used in current literature to designate the elements which occur in small concentrations in natural biologic systems [6].Trace metals are introduced into terrestrial ecosystems in two forms; specifically, they may be dissolved in rain and snow (i.e., wet deposition), or they may enter directly as particulate deposition (i.e., dry deposition) [7].When precipitation passes through the forest canopy, the precipitation altered by the wash-off of some particles on the canopy and that are deposited in the dry periods or by ion exchange, i.e., uptake or leaching [8].Finally, some trace elements (e.g., Pb or Zn) [9,10] are taken up by canopy.In addition, Pb, Cd and Cr are classified as non-essential trace elements; however, these elements can be highly toxic and can inhibit growth or cause organismal death [11].Additionally, Fe, Mn, Cu, Zn and Al are essential trace elements that participate in plant physiological and biochemical processes, but excessive amounts of these elements can also be toxic to plants [6].
In forest ecosystems, canopy gaps are created by dead and fallen trees and by intermediate cuttings, which are the primary modes of forest disturbance and regeneration [12,13].The gap-edge canopy differs substantially from the interior forest zones.First, the gap-edge canopy and the closed canopy represent two different forest canopy conditions, and the coverage of the gap-edge canopy area is less than that of the closed canopy.Therefore, the interception of precipitation will influence the trace metal contents received from the precipitation.Second, the structure of the canopy influences the ability of the canopy to capture suspended particles and suggestion that more trace metals were interception by canopy.Due to the obstruction of the wind profile, which causes local advection and turbulent exchanges, the edge canopy can catch more atmospheric deposition than can the closed canopy [9].In addition, several studies have focused on the effect of a closed canopy on trace metals from atmospheric deposition [4,14], but these studies have neglected to address the effect of the gap-edge canopy layer on trace metals; thus, the gap-edge canopy should be considered [15].Therefore, we hypothesis that the fluxes of trace metals in the gap-edge canopy were higher than those in the closed canopy, but the filtration effect on the trace metals in the gap-edge canopy was lower than that in the closed canopy.
Constituting an important freshwater conservation area in the Yangtze River basin, subalpine forests in Southwest China play important roles not only in regulating the regional climate, conservation biodiversity, but also in holding freshwater, and conserving water and soil [16].Since the 1950s, more than 400,000 hectares of pure dragon spruce plantations have replaced natural coniferous forests on the Tibetan Plateau, and these plantations are harvested by large-scale industrial logging operations.Meanwhile, the forest canopy in plantation has single canopy level rather than natural forest has complex canopy level so the spruce plantation could have not effective in the interception of atmospheric deposition than natural deposition.
The migration and transformation of trace metals occurs through the two external inputs of wet and dry deposition into forest ecosystems, and these processes affect the pollution of trace metals in other parts of the ecosystem.This quantification is necessary given the important biogeochemical role of certain trace metals, either as some essential trace metals such as Cu and Zn et al. or as non-essential trace metals (e.g., Pb, Cd and Cr) in spruce plantation forest ecosystems [9].Before reaching the soil surface, the chemical composition of precipitation can be modified by the vegetation.The measurement of throughfall is commonly used to describe and quantify the load of atmospheric pollutants in forest ecosystems [17,18], because the content of pollutants in throughfall is changed after rainfall through the forest canopy [9].Therefore, we measured the trace metals in the throughfall in this area to (1) observe the patterns of annual trace metal concentrations and fluxes from the deposited precipitation and (2) estimate the filter effect of trace metal fluxes between the gap-edge canopy and the closed canopy in a sub-alpine spruce plantation.Knowledge of these processes can enhance the knowledge of the forest canopy trace metal filter effect and evaluate the main processes that control metal behavior after interaction with the forest canopy; Thus, these results can provide the distribution effect of the filtration of trace metals from precipitation in different forest canopy patterns and provide more information for researches on water quality in the upper reaches of the Yangtze River.

Site description
The experiment site is located at the Long-term Research Station of Alpine Forest Ecosystems, Bipenggou Nature Reserve (102-53′-102-57′E, 31-14′-31-19′ N; 2458-4619 m a.s.l.), Li County, Sichuan, southwestern China; specifically, the site is situated on the eastern edge of the Tibetan Plateau and in the upper Yangtze River [19].The mean annual air temperature is 2~4°C, and the maximum and minimum temperatures are 23.7°C and -18.1°C, respectively.The mean annual precipitation ranges from 801 mm to 850 mm, with most rainfall occurring between May and August; furthermore, snowfall mainly occurs from October to April of the following year.The amount of snowfall was approximately 138.56 mm.The canopy forest vegetation is dominated by Picea asperata with some understory shrubs (e.g., Salix paraplesia and Rhododendron spp.) and grasses (e.g., Berberis diaphana, Sorbus rufopilosa, and Deyeuxia scabrescens).The expanded gap (the canopy gap plus the area that expands to the bases of the surrounding canopy trees) covers 23% of the experimental site [20].

Experimental design
Three plots with similar topographical and environmental features were selected in a typical spruce forest gap (the area: 100 m 2 ) along a gradient from the gap-edge to the closed canopy (the closed canopy area was 20 m×20 m) at 3000 m a.s.l.The mean tree age was approximately 60 a.The average diameter at breast height (DBH) and the average tree height was 19.53 ± 1.99cm and 7.63 ± 0.45 m, respectively, in the experimental plots.In addition, we selected an open area (20 m×20 m) that was approximately 50 m from the edge of the spruce plantation forest, and this area was selected as the non-forest site to collect precipitation.

Precipitation observation and water sampling
Precipitation: the rainfall was sampled in the non-forest site using 5 homemade continuous rain gauges (surface collection area of 0.64 m 2 ).
Snowfall: 5 cone-shaped collectors (top diameter of 100 cm, bottle diameter about of 20 cm) made of PVC and gridding cloth were used to observe and sample snowfall in the open site and were established 1 m above the floor, and each collector was drained into a polyethylene (PE) bucket, the snowfall was straight drop into the polyethylene bucket, that won't be exposed for a long time outside and decrease the snowfall evaporation.
Throughfall in the rainy season: the throughfall was recorded using 5 PVC rectangular grooves (surface collection area of 400 cm × 16 cm) that were arranged beneath the closed canopy and the gap-edge canopy in each plot; additionally, 5 grooves were established 1 m above the floor to avoid ground splash effects.Furthermore, the gutters were also established at a 5° horizontal angle to promote drainage, and the lower end of each gutter was equipped with a plastic bucket.
Throughfall in the snowy season: 5 cone-shaped collectors that were similar to the snowfall collectors were distributed beneath the closed canopy and the gap-edge canopy in each plot, and each collector was drained into a PE bucket.

Chemical analyses
Water samples were collected immediately after each rainfall event during the rainy season from August 2015 to July 2016.And we collected the snowfall samples once each month from November 2015 to April 2016 because the snowfall was heavy, and the field conditions were difficult and placed them in clean polyethylene bottles.The samples were quickly transported to the laboratory, where they were filtered using qualitative filter paper with diameter of 12.5 cm.The filtered samples were adjusted to a pH of 1~2 with high-purity grade (GR) nitric acid.The concentrations of tracer metals (i.e., Fe, Mn, Cu, Zn, Al, Pb, Cd and Cr) were determined using inductively coupled plasma optical emission spectrometry (Agilent 5000).

Calculations
The fluxes of precipitation and throughfall was calculated by formula (1) as follow: [21]  =  ×  100 where Fluxj is the deposition flux of the solute j in different forms of water, VWMj is the weighted concentration (mg L -1 ) of the solute j in different forms of water, Vj is the water is the water of different forms (mm), and 100 is the unit conversion factor.

Statistical analysis
All statistical analyses were carried out using IBM SPSS Statistics 20.0.The Univariate analysis was used to compare the concentrations, fluxes and net throughfall fluxes of trace metals at different canopy and season.The statistical tests were considered significant at the P<0.05 level.

The annual variation of trace metal concentrations in precipitation and throughfall
After precipitation passes through the canopy, the concentrations of trace metals increase and decrease at different levels in the closed canopy and gap-edge canopy.In the rainy season, the throughfall concentrations of the essential trace metals of Fe, Mn and Cu in the closed canopy and the gap-edge canopy were higher than those in the precipitation (Figure 1); furthermore, and the concentration of Mn was 2.63-fold and 1.68-fold higher in the closed canopy and gap-edge canopy than in the precipitation.In addition, the concentrations of Fe, Mn and Cu in the closed canopy were higher than those in the gap-edge canopy.Only Cr increased in the gap-edge canopy among the non-essential trace metals (Figure 2).In the snowy season, the throughfall concentrations of all trace metals in the closed canopy and gap-edge canopy were lower than those in the precipitation, and the concentrations of trace metals in the closed canopy were higher than those in the gap-edge canopy (except for Al and Pb).Furthermore, there were no significant differences in the trace metal concentrations between the gap-edge canopy and the closed canopy and the season has significant differences in trace metal concentration in table 2 (P<0.05).

The annual variation of trace metal fluxes in precipitation and throughfall
The annual fluxes of Al, Zn, Fe, Mn, Cu, Cd, Cr and Pb were 7.29 kg•ha -1 , 2.30 kg•ha -1 , 7.02 kg•ha -1 , 0.16 kg•ha -1 , 0.19 kg•ha -1 , 0.06 kg•ha -1 , 0.56 kg•ha -1 and 0.24 kg•ha -1 , respectively.And shown the rainy season higher than snowy season (figure 3,4).The input of all trace metals from the precipitation, gap-edge canopy and closed canopy was 1.15 kg•ha -1 , 0.29 kg•ha -1 and 0.30 kg•ha -1 , respectively, in the snowy season and 16.68 kg•ha -2 , 13.02 kg•ha -2 and 15.96 kg•ha -2 , respectively, in the rainy season.Additionally, the maximum values of all trace metal fluxes in the precipitation, closed canopy and gap-edge canopy were observed for Fe and Al in both seasons.In the snowy season, the throughfall fluxes of all trace metals in the closed canopy and gap-edge canopy were lower than those in the precipitation.Furthermore, there were no significant differences in trace metal fluxes between the gap-edge canopy and the closed canopy, and the season has significant differences in trace metal fluxes in table 2 (P<0.05).

Discussion
Before reaching the soil surface, the chemical composition of precipitation can be modified by vegetation [22], due to the interaction between precipitation and the canopy can exchange with ions and the concentrations of the elements were changed [23].The concentration changes in snowy season and rainy season have obvious seasonal characteristics (figure 1,2), because the effect of freezing and thawing, the cell membrane of plant leaves is damaged, and the change of water content in tissues affects the exchange ability between cells and ions [24,25,26], and the variation of season has a significant effect on trace metals (table 2).The enrichment extent is relevant to the solute characteristics [27], the higher concentrations of trace metals in precipitation are from terrigenous particles, such as Al and Fe, and the Sonja which supported our findings [28].thus, higher concentrations of Al and Fe among the trace metals result in higher fluxes in precipitation.Compared with non-forest, the trace metal concentration of Al was lower in the gap-edge canopy and the closed canopy during both seasons, but the concentration of Fe was higher in the gap-edge canopy and closed canopy in the rainy season.And the concentrations of Al and Fe account for 42.5% and 40.8% of the non-forest input in the rainy season, and 37.6% and 38.0%, in the snowy season.
The brief interaction between vegetation and precipitation creates high spatial variability of metal deposition from throughfall, which is very important for elemental cycling in forest ecosystems [29,30].The concentration of trace metals in precipitation were higher than those in throughfall due to snowfall has higher evaporation and the open field effect higher solar radiation in winter.However, the canopy has no significant effect on fluxes of trace metals.Consistent our hypothesis, the results indicated that the annual fluxes of most trace metals (e.g., Zn, Cd, Cr, Cu and Pb) in the gap-edge canopy were higher than those in the closed canopy.First, due to the proximity of the edge affects the wind speed and enhances the air turbulence, increasing the dry deposition velocities via inflow and advection processes and the coniferous foliage surface has a particular capacity to trap the dry deposition [31,32].Then, the precipitation can wash off more of these metal particles from the gap-edge canopy, and the throughfall deposition of Zn, Cd, Cr, Cu and Pb at the gap-edge canopy is significantly enhanced relative to that in the closed canopy in our study.The throughfall deposition of Zn, Cd, Cr, Cu and Pb in gap-edge canopy was 1.20-, 0.14-, 1.57-, 1.05-and 2.68-fold higher than those in closed canopy.The gaps have higher air temperatures and solar radiation than do the closed forests, and evaporation on the leaves is another critical contributing factor that is responsible for the increase in metal concentrations in throughfall [33 34] So would increase concentration of Cd, Cr and Pb in the gap-edge canopy were 1.31-, 1.43-and 1.43-fold higher than those in the closed canopy, respectively.
The net throughfall input is the combined result of leaching and uptake in the canopy [10] and also relative with the vegetation the element of Fe and Mn in pine-oak forest and the oak forest was leaching, but in our study, the filter was often the result of trace metals leaching (expect for Mn), which indicates that the tracer metals were filtered (e.g., adsorbed or retained) by the canopy, and the Mn in closed canopy was 1.11-and 2.38 fold higher than the pine-oak forest and oak forest [35].In addition, Zn, Fe, Cu, Cd, Cr and Pb were filtered by the gap-edge canopy and the closed canopy, and the net filtering ratio of these metals in the closed canopy was higher than that in the gap-edge canopy, and the net filtering ratio of Zn was 30.25% and 25.00% in two evergreen oak stands in Spain was lower than the Zn in our study [9].In addition, the net filtering ratio was 36% of Al was filtered in the gap-edge canopy, and 49% was leached in the closed canopy during the rainy season.However, the net filtering ratio of all trace metals was over than 50% in the snowy season.There are two mechanisms in which foliar structures filter metals: (1) through the absorption and internalization of the cuticle and (2) the penetration of metals through the stomatal pore [36].The stomatal openings and cuticle expansion allow a high level of metal penetration from the atmosphere [37].The canopy retention of Zn and Cd has been reported in other studies [38,39], and some studies also found canopy uptake of Zn, Cd, Cu and Pb [40].Non-essential trace metals, such as Pb [41,42], Cd [43] and Cr [44], can also enter plant leaves via foliar transfer, and these metals can penetrate cuticles and finally accumulate in leaf tissues, and the canopy filtered the Pb and Cd was over than 80% in mid-subtropical forest which was higher than gap edge canopy and closed canopy [45].Among the essential trace metals, Mn presented the highest level of leaching in both canopies in the rainy season, and this result is widely attributed in the literature to canopy leaching [10,38,46,47,48,49,50].Additionally, the enrichment factor of Mn demonstrated that there was a higher enrichment in throughfall, and this phenomenon was also found in Gandois' research [51], who attributed the results to internal cycling [38].

Conclusion
The forest canopy can be regarded as a self-regulating system that filters certain trace metals from deposited precipitation.The annual fluxes of trace metal in precipitation was 17.83 kg•ha -1 , and the rainy season account for 93.55%, and the trace metals in precipitation were filtered by the closed canopy and gap-edge canopy by 4.3% and 21.94%, respectively.Meanwhile, the snowy season, account for 6.45% of precipitation, and the trace metals in precipitation were filtered by the closed canopy and gap-edge canopy by 73.95% and 75.11%, respectively.For essential trace metals, the closed canopy leached 28.06% of Al and 71.10% of Mn, while the closed canopy filtered 47.14%, 20.43%, 16.66%, 52.55%, 46.35% and 53.32% of the Zn, Fe, Cu.Pb, Cd and Cr, respectively.The gap-edge canopy leached 25.99% of Mn, and it filtered 20.58%, 35.93%, -20.50%, 25.99%, 31.10%,15.30% and 22.44% of Al, Zn, Fe, Zn, Pb, Cd and Cr, respectively.However, all trace metals demonstrated a high net filtering ratio in the gap-edge canopy and closed canopy in the snowy season.These results which provided us a clearer idea to the filtration effect of subalpine forest on trace metals from precipitation deposition and help protect water quality in the upper reaches of the Yangtze River.

Figure 1 .Figure 2 .
Figure 1.The concentrations of essential-trace metals in bulk precipitation and throughfall: BP: bulk precipitation.GE: gap-edge canopy.CC: closed canopy.The bar graphs with error bars are the means with 95% confidence intervals.

PreprintsFigure 3 .Figure 4 .
Figure 3.The fluxes of essential trace metals in bulk precipitation and throughfall: BP: bulk precipitation.GE: gap-edge canopy.CC: closed canopy.The bar graphs with error bars are the means with 95% confidence intervals.

Table 1
The net throughfall fluxes (NTF)and net throughfall ratio (NTR) and standard deviation (in brackets) of trace metals in closed canopy and gap edge canopy during rainy and snowy season.

Table 2
The result (F value) of Univariate analysis on the effect of canopy and season on concentration and fluxes of trace metals in throughfall *:P < 0.05; **:P<0.01.Preprints (www.