Evaluating the Accumulation of Antioxidant and Macro- and Trace Elements in Vaccinium myrtillus L.

This study was conducted in order to characterise the accumulation ability of Vaccinium myrtillus L for trace elements such as Al, Cd, Cu, Fe, Mn, Pb and Zn and selected macroelements Ca, K, Mg, Na and P. The accumulation of nutrient elements and trace elements (ANE and ATE) and changes in the ecophysiological responses in bilberry in differently polluted areas were compared. The accumulation of the elements in the roots, stems and leaves of bilberry from four sites (in the nearest vicinity of a zinc smelter, a Mining and Metallurgical Plant, a main road with a high traffic volume and an unprotected natural forest community) were measured using optical emission spectrometry with excitation using inductively coupled argon plasma after wet acid digestion. The highest Cd, Cu, Pb and Zn concentrations were found in the V. myrtillus samples that were growing under the influence of the emissions from the zinc smelter. Moreover, the level of the total accumulated trace metals (ATE-17.09 mmolc kg-1) was also highest for the bilberry at this site. However, in the same area, the sum of the accumulated macronutrients (ANE-296.92 mmolc kg-1) was lower than at the other sampling sites. An EF > 2 was found for Cd, Pb, Zn and Mn, which suggests that bilberries may be enriched in these metals. According to the translocation factor, V. myrtillus was an accumulator of Cd, Zn and Mn. An analysis of the ecophysiological responses showed that the greatest concentration of ascorbic acid was found in the leaves of V. myrtillus at the most contaminated site (3.32 mg g-1 fresh weight). There were no significant differences in the total phenols between the contaminated and non-contaminated sites. However, the lowest value of the total phenolic content (490.77 mg g-1 dry weight) was recorded at the site where the highest Fe concentration was detected in the leaves. A significantly positive correlation between the Cd, Pb and Zn concentrations and a strong negative correlation between the Mn concentration and ascorbic acid content in the leaves of bilberry was also observed. The results provide evidence that the ANE method, which is used to interpret the chemical composition of bilberry has made determining the impact of toxic trace metals on the mineral composition of V. myrtillus significantly easier and also that a non-enzymatic antioxidant such as ascorbic acid can be a good biomarker for determining the oxidative stress that is caused by toxic trace metals.

Influence of lead on the activity of soil microorganisms in two Beskidy landscape parks.

The aim of the study was to assess the potential impact of lead on soil metabolism in two landscape parks localized in the Beskid Slaski and Beskid Zywiecki mountains which were affected, among others, by air pollution from the Upper Silesian Industrial Region, the largest industrial zone in Poland. The study was carried out in six locations with different lead levels in the soil environment. Each plot was equipped with four pairs of vacuum ceramic lysimeters to assess the mobility of Pb in the soil. The metabolic activity was assessed by measuring: soil enzyme activity, soil respiration and by studying community-level physiological profiling (CLPP) using Biolog EcoPlates technique. The soil to the examination was collected near the stands with the lysimeters from two soil horizons (A and B layer). The analyses carried out showed that the factors that had the greatest influence on lead mobility were the organic carbon content and the soil pH. The elevated lead level in the topsoil (layer A) could affect the functional biodiversity of soil microorganisms, but low soil pH was a more likely limiting factor. In the subsoil (layer B), lower lead content was found and its probable effect on soil microbial activity was small. In summary, it can be concluded that the assessment of the influence of heavy metals on soil metabolism is not easy, and the Biolog system has proven to be a sensitive tool for assessing the potential impact of heavy metals on the soil environment.

The influence of heavy metals on biological soil quality assessments in the Vaccinium myrtillus L. rhizosphere under different field conditions.

The aim of this study was to determine the influence of heavy metals on biological soil quality assessments in Vaccinium myrtillus L. rhizosphere soil as well as in non-rhizosphere soil from different polluted sites. The presented study was also conducted in order to determine any differences in the soil physicochemical and biological properties between the Vaccinium rhizosphere soil and the non-rhizosphere soil. The content of heavy metals and their potential bioavailability, content of macronutrients, physicochemical soil properties, activity of six soil enzymes and microarthropod communities were determined. Soil organic matter, the levels of C, N and all the studied macronutrients and almost all enzyme activity were significantly higher in the rhizosphere soil than in the non-rhizosphere soil. At the most contaminated site, the content of heavy metals was also higher in the rhizosphere soil, but their bioavailability was lower than in the non-rhizosphere soil. The ß-glucosidase and urease activity in the soil correlated most negatively with the examined metals. The levels of two enzymes were also strongly impacted by the organic matter-the C and N levels and pH. The number of microarthropods as well as the QBS (soil biological quality index) and FEMI (abundance-based fauna index) were higher in the rhizosphere soil. The bilberry rhizosphere soil had stronger correlation coefficient values between the measured parameters than the non-rhizosphere soil, which suggests that rhizosphere soil is more sensitive and could be used in the monitoring and assessment of forest ecosystems. ß-glucosidase and urease were the most sensitive indicators of the adverse impact of Cd, Zn and Pb. The FEMI index seems to be a better indicator than the QBS for identifying differences in soil quality.

Using Plantago major and Plantago lanceolata in environmental pollution research in an urban area of Southern Poland.

The aim of this study was to perform a complex assessment of changes in the elements of an ecosystem that are caused by environmental pollution in industrial and urban biotopes. The study focused on three sites: a park, a road and the site of the metallurgical plant "Pokój" in the city of Ruda Slaska (Southern Poland), which are each under a different level of anthropogenic load. Soil and plant material samples (Plantago major and Plantago lanceolata leaves) were investigated by performing biochemical, ecophysiological and scanning electron microscopy (SEM) analyses. A significant difference was observed in all of the study samples. The content of Pb, Zn and Cd in the soil samples that had been collected at the site of the metallurgical plant exceeded the permitted limits (Cd > 4 mg kg-1, Pb > 100 mg kg-1, Zn > 300 mg kg-1). The content of Fe, Mn, Pb, Cd and Zn in the plant material was much higher in unwashed samples than in washed samples. The concentrations of potentially toxic elements (PTEs) were below the permitted level in the leaves of Plantago lanceolata for Cd (> 5 mg kg-1) and in the leaves of Plantago major for Zn (> 100 mg kg-1). The SEM observations revealed a significant decrease in the stomata pore length (SPL) in the Plantago lanceolata leaves that had been collected at the road site compared with the plants from the park site. The elemental content on the leaf surface was also determined using X-ray microanalysis. The total chlorophyll (Chl) content, ascorbic acid (AA), proline, guaiacol peroxidase (GPX) activity, pH, relative water content (RWC) and air pollution tolerance index (APTI) were evaluated. The APTI for the investigated species ranged from 5.6 to 7.4, which demonstrated that the studied plant species are sensitive to air pollutants.

Antioxidant responses of Triticum aestivum plants to petroleum-derived substances.

Winter common wheat (Triticum aestivum L.) plants were cultivated on petroleum products contaminated soils with and without using biopreparation ZB-01. We determined the impact of soil contamination with petrol, diesel fuel and engine oil on selected antioxidant enzymes and the levels of antioxidants in the leaves of winter wheat. The impact of petroleum products on selected morphological characteristics of the plants, levels of nutrients and heavy metals was also assessed. Winter wheat was relatively resistant to soil contamination with petroleum products, and did not show a significant impact on the morphological characteristics of the plants. The levels of nutrients and heavy metals in the plants depended on the type of pollutant and the analyzed component.‬ Biopreparation ZB-01 generally resulted in an increase in calcium levels in the plants.‬ The winter wheat plants growing in soil contaminated with engine oil were characterized by higher levels of zinc, lead, manganese and cadmium than the control plants.‬ Biopreparation applied to the soil contaminated with petrol resulted in a slight increase in the levels of lead and zinc in the plants.‬ The petroleum products affected the activity of antioxidant enzymes and the levels of antioxidants in the plants.‬ The general markers of soil contaminated with diesel fuel and petrol were POD activity and proline levels. Use of the ZB-01 biopreparation caused an increase in the levels of proline and -SH groups and an increase in the levels of carbon and calcium in the plants and had no effect on the morphological characteristics of plants.‬.

Effect of petroleum-derived substances on life history traits of bird cherry-oat aphid (Rhopalosiphum padi L.) and on the growth and chemical composition of winter wheat.

The aim of the study was to determine the effects of various petroleum-derived substances (PDSs), namely petrol, diesel fuel, and spent engine oil, on life history traits of the bird cherry-oat aphid Rhopalosiphum padi L., and on the growth and chemical composition of its host plant-winter wheat Triticum aestivum L. Each substance was tested separately, using two concentrations (9 and 18 g kg-1). Plants were cultivated in both control and contaminated soils. In early October 2013, soil was contaminated and after 1 week, winter wheat seeds, 'Batuta' cultivar, were sown. In early June 2014, observations of the effect of petroleum-derived substances on traits of three successive generations of aphids were conducted. Aphids were inoculated separately on leaves using cylindrical cages hermetically closed on both sides. Contamination of aphid occurred through its host plant. Results showed that all of the applied petroleum-derived substances have a generally adverse effect on the developmental parameters in aphids, resulting in the decrease of its fecundity, shortening its average life span, and most often lowering of the population intrinsic growth rate. PDSs caused the limitation of growth in wheat plants; whereas, changes in nutrient contents and heavy metals depended on the part of the plant analysed, the substance applied, and on its dose. The negative relationships between the contents of both some macro-elements (Ca, K, P) and heavy metals (Mn, Cd, Cu, and Zn) and the developmental parameters of particular generations of R. padi were observed. The high susceptibility of R. padi to the presence of PDSs in the substrate for the host plant should be emphasised-the clear-cut changes in the life span and fecundity, with relatively small changes in the chemical composition of the plant, constitute an evident indication that the developmental parameters of aphids have the potential for the use as bio-indicator to evaluate the state of the environment contaminated by PDSs.

Bioaccumulation of heavy metals and ecophysiological responses to heavy metal stress in selected populations of Vaccinium myrtillus L. and Vaccinium vitis-idaea L.

The aim of this study was to determine the concentrations of heavy metals (Cd, Pb, Zn, Fe, and Mn) in soil, and their bioavailability and bioaccumulation in Vaccinium myrtillus L. and Vaccinium vitis-idaea L. organs. Analysis also concerned the physiological responses of these plants from three polluted sites (immediate vicinity of a zinc smelter in Miasteczko Slaskie, ArcelorMittal Poland S.A. iron smelter in Dabrowa Górnicza-Losien, and Jaworzno III power plant in Jaworzno) and one pseudo-control site (Pazurek nature reserve in Jaroszowiec Olkuski). All of the sites are situated in the southern parts of Poland in the Slaskie or Malopolskie provinces. The contents of proline, non-protein thiols, glutathione, ascorbic acid, and the activity of superoxide dismutase and guaiacol peroxidase in the leaves of Vaccinium myrtillus L. and Vaccinium vitis-idaea L. were measured. In soil, the highest levels of Cd, Pb, and Zn (HNO3 extracted and CaCl2 extracted) were detected at the Miasteczko Slaskie site. At all sites a several times lower concentration of the examined metals was determined in the fraction of soil extracted with CaCl2. Much higher Cd, Pb, Zn and Fe concentrations were found in V. myrtillus and V. vitis-idaea grown at the most polluted site (located near the zinc smelter) in comparison with cleaner areas; definitely higher bioaccumulation of these metals was found in lingonberry organs. Additionally, we observed a large capability of bilberry to accumulate Mn. Antioxidant response to heavy metal stress also differed between V. myrtillus and V. vitis-idaea. In V. myrtillus we found a positive correlation between the level of non-protein thiols and Cd and Zn concentrations, and also between proline and these metals. In V. vitis-idaea leaves an upward trend in ascorbic acid content and superoxide dismutase activity accompanied an increase in Cd, Pb, and Zn concentrations. At the same time, the increased levels of all tested metals in the leaves of V. vitis-idaea were accompanied by a decreased activity of guaiacol peroxidase. In both species increased Mn accumulation caused a decrease in antioxidant response.

Air pollution tolerance index and heavy metal bioaccumulation in selected plant species from urban biotopes.

This research was carried out on plants Taraxacum officinale, Plantago lanceolata, Betula pendula and Robinia pseudoacacia growing in urban biotopes with different levels of heavy metal contamination in the city of Dabrowa Górnicza (southern Poland). Based on the pollution index, the highest heavy metal contamination was determined in the site 4 (connected with industry emitters) and 6 (high traffic). The metal accumulation index (MAI) values ranged within the biotopes in Dabrowa Górnicza between 7.3 and 20.6 for R. pseudoacacia, 4.71-23.1 for P. lanceolata, 4.68-28.1 for T. officinale and 10.5-27.2 for B. pendula. Increasing tendency in proline content in biotopes connected with high traffic was found in the leaves of investigated plants (except R. pseudoacacia). Similar tendency was observed for ascorbic acid content in the foliage of the plants as well as in T. officinalle in stands connected industrial emission. Non-protein thiols content increased especially in the leaves of R. pseudoacacia in biotopes with high traffic emissions as well as in T. officinale in stands connected with industry. The mean values of APTI (Air Pollution Tolerance Index) within the city of Dabrowa Górnicza for investigated plants were found in the following ascending order P. lanceolata < R. pseudoacacia < B. pendula < T. officinale. Among the investigated plants B. pendula and T. officinale may be postulated as appropriate plants in urban areas with considerable soil and air contamination, especially with heavy metals. The results indicate that species deemed tolerant according to APTI are suitable plants in barriers areas to combat atmospheric pollution.

Effect of petroleum-derived substances on life history traits of black bean aphid (Aphis fabae Scop.) and on the growth and chemical composition of broad bean.

The aim of the study was to determine the effects of various petroleum-derived substances, namely petrol, diesel fuel and spent engine oil, on life history traits and population dynamics of the black bean aphid Aphis fabae Scop. and on growth and chemical composition of its host plant Vicia faba L. Each substance was tested separately, using two concentrations (9 g kg-1 and 18 g kg-1). The experiment was conducted in four replications (four pots with five plants in each pot per treatment). Plants were cultivated in both control and contaminated soils. After six weeks from soil contamination and five weeks from sowing the seeds, observations of the effect of petroleum-derived substances on traits of three successive generations of aphids were conducted. Aphids were inoculated separately on leaves using cylindrical cages hermetically closed on both sides. Contamination of aphid occurred through its host plant. Results showed that all tested substances adversely affected A. fabae life history traits and population dynamics: extension of the prereproductive period, reduction of fecundity and life span, reduction of the population intrinsic growth rate. In broad bean, leaf, roots, and shoot growth was also impaired in most conditions, whereas nutrient and heavy metal content varied according to substances, their concentration, as well as plant part analysed. Results indicate that soil contamination with petroleum-derived substances entails far-reaching changes not only in organisms directly exposed to these pollutants (plants), but also indirectly in herbivores (aphids) and consequently provides information about potential negative effects on further links of the food chain, i.e., for predators and parasitoids.

Accumulation of heavy metals and antioxidant responses in Pinus sylvestris L. needles in polluted and non-polluted sites.

The purpose of this study was to determine the concentrations of heavy metals (cadmium, iron, manganese, lead and zinc) in current-year, 1-year old and 2-year old needles of Pinus sylvestris L. Trees were from three heavily polluted (immediate vicinity of zinc smelter, iron smelter and power plant) and three relatively clean sites (nature reserve, ecologically clean site and unprotected natural forest community) in southern Poland. Analysis also concerned the antioxidant response and contents of protein, proline, total glutathione, non-protein thiols and activity of guaiacol peroxidase (GPX) in the needles. Generally, in pine needles from the polluted sites, the concentrations of the metals were higher and increased with the age of needles, and in most cases, antioxidant responses also were elevated. The highest levels of Cd, Pb and Zn were found in 2-year old pine needles collected near the polluted zinc smelter (respectively: 6.15, 256.49, 393.5 mg kg(-1)), Fe in 2-year old pine needles in the vicinity of the iron smelter (206.82 mg kg(-1)) and Mn in 2-year old needles at the ecologically clean site (180.32 mg kg(-1)). Positive correlations were found between Fe, Mn and Pb and the content of proteins and NPTs, between Cd and non-protein -SH groups, and between Zn and proline levels. The activity of GPX increased under the influence of Mn, while glutathione levels tended to decrease as Mn levels rose. The data obtained show that the levels of protein and non-protein -SH groups may be useful in biological monitoring, and that these ecophysiological parameters seem to be good evidence of elevated oxidative stress caused by heavy metals.

Enzymatic activities and arbuscular mycorrhizal colonization of Plantago lanceolata and Plantago major in a soil root zone under heavy metal stress.

The objectives of the present field study were to examine the soil enzyme activities in the soil root zones of Plantago lanceolata and Plantago major in different heavy metal contaminated stands. Moreover, the investigations concerned the intensity of root endophytic colonization and metal bioaccumulation in roots and shoots. The investigated Plantago species exhibited an excluder strategy, accumulating higher metal content in the roots than in the shoots. The heavy metal accumulation levels found in the two plantain species in this study were comparable to other plants suggested as phytostabilizers; therefore, the selected Plantago species may be applied in the phytostabilization of heavy metal contaminated areas. The lower level of soil enzymes (dehydrogenase, urease, acid, and alkaline phosphatase) as well as the higher bioavailability of metals in the root zone soil of the two plantain species were found in an area affected by smelting activity, where organic matter content in the soil was also the smallest. Mycorrhizal colonization on both species in the contaminated area was similar to colonization in non-contaminated stands. However, the lowest arbuscule occurrence and an absence of dark septate endophytes were found in the area affected by the smelting activity. It corresponded with the lowest plant cover observed in this stand. The assessment of enzyme activity, mycorrhizal colonization, and the chemical and physical properties of soils proved to be sensitive to differences between sites and between Plantago species.

Element accumulation, distribution, and phytoremediation potential in selected metallophytes growing in a contaminated area.

The distribution of elements in three pseudometallophytes species Cardaminopsis arenosa, Plantago lanceolata, and Plantago major, naturally occurring at metalliferous and non-metalliferous sites in southern Poland, was investigated. The accumulation of Al, Cd, Cu, Fe, Mn, Pb, Zn, as well as Ca, P, Na, and K in shoots and roots was measured. The level of the accumulated trace elements (ATE) was visibly higher in C. arenosa and P. lanceolata from metalliferous sites than non-contaminated ones. However, the level of the accumulated nutrient elements (ANE) was visibly higher only in C. arenosa plants. Also, higher potassium share in ANE was found in the shoots of C. arenosa and Plantago species from metalliferous sites than non-contaminated ones. The highest content of Cd, Zn, Pb, Al, Fe, and Mn was found in C. arenosa, which better reflected metal concentrations in the metalliferous and non-metalliferous soil than other plants. In the studied Plantago species, in almost all cases in all sites TF (translocation coefficient) and MR (mobility ratio) were below 1, which indicates they use the excluder strategy. The best accumulation ability was found for C. arenosa. The higher translocation coefficients (TF > 1) for Zn and Cd in C. arenosa shoots make it suitable for phytoextraction from soil, while the lower translocation ratios (TF < 1) for Zn and Cd in Plantago species and also for Pb in C. arenosa make them suitable for phytostabilization. Almost in all cases the plants had enrichment coefficient >2, which suggested that they may act as indicators of the soil metal contamination.

Heavy metal bioaccumulation and antioxidative responses in Cardaminopsis arenosa and Plantago lanceolata leaves from metalliferous and non-metalliferous sites: a field study.

The purpose of this study was to determine the concentrations of heavy metals (cadmium, lead, zinc, copper, iron and manganese) in soil, their bioavailability and bioaccumulation in plants leaves. This study also examined their influences on the antioxidant response of the plants Cardaminopsis arenosa and Plantago lanceolata grown in metal-contaminated and non-contaminated soils. The activities of guaiacol peroxidase and superoxide dismutase and the levels of antioxidants such as glutathione, proline and non-protein thiols were measured. Concentrations of the examined metals were several to thousands of times lower in the potentially bioavailable fraction than in the acid-extracted fraction of the soil. Similar mode of antioxidant responses in plant leaves of metalliferous populations indicates the tolerance of plants towards heavy metals. However POD and GSHt had a particularly strong role in defense reactions, as their increase was the most common reaction to heavy metal contamination.The levels of Zn, Cd and Pb in the leaves of C. arenosa better reflected metal concentrations in the metalliferous and non-metalliferous soil than the determined metal concentrations in P. lanceolata. Bioaccumulated Zn, Cd and Pb concentrations were above or in the ranges mentioned as toxic for plant tissues and therefore the studied plants have potential for use in phytostabilization.

A comparative study of heavy metal accumulation and antioxidant responses in Vaccinium myrtillus L. leaves in polluted and non-polluted areas.

The purpose of this study was to explore a possible relationship between the availability of metals in soil (Cd, Fe, Mn, Pb and Zn) and their concentrations in leaves of Vaccinium myrtillus L. as a species which has been reported to be a successful colonist of acid-and-heavy metal-contaminated soil. Analysis also concerned the antioxidant response of plants from three heavily polluted (immediate vicinity of: zinc smelter, iron smelter and power plant) and three relatively clean sites (nature reserve, ecological site and unprotected natural forest community) in southern Poland. The contents of glutathione, non-protein thiols, protein, proline and activity of guaiacol peroxidase in leaves of bilberry were measured. Generally, the concentrations of metals in the HNO3 and CaCl2 extracants of the soil from the polluted sites were higher. Moreover, the antioxidant responses were also elevated in bilberries in the polluted sites. Significant positive relationships between Cd, Pb and Zn concentrations in soil and in the plants were found. In the leaves of V. myrtillus from the polluted sites, higher concentrations of Cd, Pb and Zn were noted (In Miasteczko Slaskie respectively 6.26, 157.09 and 207.17 mg kg(-1) d.w.). We found a positive correlation between the increase in the NPTs and protein contents as well as the Cd, Pb and Zn concentrations in V. myrtillus. Cd, Pb and Zn also decreased guaiacol peroxidase activity. However, the activity of this enzyme increased under Fe. A decreasing trend in glutathione contents was observed with increasing iron and manganese concentrations in bilberry leaves. Parameters such as protein, non-protein -SH groups and changes in GPX activity seem to be universal, sensitive and correlated well with heavy metal stress.

Accumulation of heavy metals and antioxidant responses in Vicia faba plants grown on monometallic contaminated soil.

The purpose of this study was to explore the effects of soil contamination by selected metals (cadmium, copper, nickel, lead or zinc) on the antioxidant response of Vicia faba plants. The levels of the antioxidants: glutathione, proline, non-protein thiols, as well as guaiacol peroxidase and catalase activities were measured in the upper parts of plants. Additionally, the potential bioavailability of metals in the soil and their concentrations in V. faba plants were compared. Treatment with metal caused the problem of an elevation in its bioavailability in soil and its concentration in leaves and stems. The most serious problems seemed to be metal elevations in soil, especially Zn and Ni as well as in the aerial parts of V. faba plants. The antioxidant responses appeared to be metal specific. The elevation of guaiacol peroxidase activity in leaves and stems as well as the proline in leaves was the only more general reaction to metal exposure. Upon analysis of the effects of soil metal contamination on V. faba plants, we recommend the use of some measurements such as guaiacol peroxidase activity and proline level as useful tools in biological monitoring.

The effects of Aphis fabae infestation on the antioxidant response and heavy metal content in field grown Philadelphus coronarius plants.

The purpose of this study was to explore a possible relationship between the soil availability of metals and their concentrations in various parts of Philadelphuscoronarius plants. Moreover, the possible impact of an aphid infestation on the contamination and antioxidant response of plants from the urban environment of Kraków and the reference rural area of Zagaje Stradowskie (southern Poland) was analyzed. The contents of the glutathione, proline, non-protein -SH groups, antioxidants, and phosphorous and the levels of guaiacol peroxidase and catalase activity in leaves and shoots either infested or not by the aphid Aphis fabae Scop., were measured. The potential bioavailability of metals (Cd; Cu; Ni; Pb; Zn) in the soil and their concentrations in P. coronarius plants originating from both sites were compared. The antioxidant responses were generally elevated in the plants in the polluted area. Such reactions were additionally changed by aphid infestation. Generally, the concentrations of metals in the HNO(3) and CaCl(2) extractants of the soils from two layers at the 0-20 and 20-40 cm depths from the polluted area were higher than in those from the reference area. Such differences were found for nickel and lead (in all examined extractants), zinc (in soil extractants from the layer at 20-40 cm) and cadmium (in HNO(3) extractants). Significant positive relationships between the lead concentrations in the soil and in the plants were found. In the parts of plants from the polluted area, higher concentrations of Pb and Zn (leaves and shoots) and Cd (shoots) were recorded. The shoots and leaves of plants infested with aphids had higher concentrations of Zn but lower Pb. Moreover, their leaves had higher contaminations of Cu and Ni. In conclusion, aphids affected not only the antioxidant response of the plants but also their contamination with metals, especially contamination of the leaves.