the interactions among the heavy metals in soils and in

Research ArticleThe Interactions among the Heavy Metals in Soils and in Weedsand Their Antioxidant Capacity under the Mining Activities inThai Nguyen Province Vietnam

Ngoc-Lien Nguyen1 Cam-Tu Vu 2 Hien-Minh To1 Hoang-Nam Pham 1

Hai-Dang Nguyen 3 Tien-Dat Nguyen 4 and Kieu-Oanh Nguyen Thi 1

1Department of Life Sciences University of Science and Technology of Hanoi Vietnam Academy of Science and Technology18-Hoang Quoc Viet Cau Giay Hanoi Vietnam2Department of Water-Environment-Oceanography University of Science and Technology of HanoiVietnam Academy of Science and Technology 18-Hoang Quoc Viet Cau Giay Hanoi Vietnam3University of Science and Technology of Hanoi Vietnam Academy of Science and Technology 18-Hoang Quoc Viet Cau GiayHanoi Vietnam4Center for Research and Technology Transfer Vietnam Academy of Science and Technology 18-Hoang Quoc Viet Cau GiayHanoi Vietnam

Correspondence should be addressed to Kieu-Oanh Nguyen i nguyenthikieuoanh0711gmailcom

Received 20 August 2020 Accepted 15 October 2020 Published 31 October 2020

Academic Editor Agnieszka Saeid

Copyright copy 2020 Ngoc-Lien Nguyen et al is is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

In this study the relationship between heavy metals (HMs) concentrations in soils and several weeds including Cyclosorusparasiticus Dicranopteris linearis Pityrogramma calomelanos and Pteris vittata in three mining sites (Cam Gia (ai Nguyencity) Tan Long (Dong Hy district) and Hauong (Dai Tu district)) in ai Nguyen province Vietnam have been investigatede levels of HMs varied among soil origins and showed the contaminations of As Cu and Pb in soil samples collected in DongHy and Dai Tu districts In addition the HM distribution and cocontamination phenomena in different soils significantly affectedthe HM residues and transportation abilities into different species as well as tissues Moreover based on the analysis of bio-accumulation factor (BF) and translocation factor (TF) C parasiticus and D linearis were found potentially for phytoextractionby roots while P calomelanos and P vittatawere suitable for hyperaccumulation in shoots and leaves Consequently the strongestantioxidant property by 11-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion (SRSA) radical scavenging assays weredemonstrated in the methanol root extracts of C parasiticus and P vittata respectively In conclusion the correlation among HMin soils and tissues with antioxidant property allows us to hypothesize that the presence of these elements can enhance theantioxidant activity of plant extracts suggesting to apply the weeds in phytoremediation as well as in phytomedicine

1 Introduction

Heavy-metal (HM) pollution is raising alarms since they cancause massive development in industrialization and miningespecially in developing countries Although HM explora-tions bring many economic benefits the serious pollution byexceeding HMs in the soil and water occurs as a matter ofobsolete technology or the lack of systematic mining processand preliminary treatment In the world up to 80 of

industrial polluted sites which cause health-related threatsand HM pollutants such as As Pb Cd Zn are listed in thetop 12 red-flag contaminants [1] In Northern Vietnamseveral mining areas inai Nguyen province are famous fortheir rich mineral resources of Fe Ti Zn Cu Pb ferrousand nonferrous ores e studies of soil analysis in theseregions reportedly contained several HMs contaminatedsites in the scenario of the foreign firmrsquos accelerating ex-ploitation and outdated manual mining technology [2]

HindawiJournal of ChemistryVolume 2020 Article ID 8010376 12 pageshttpsdoiorg10115520208010376

Analysis of soil samples in the Pb-Zn ore Dong Hy aiNguyen showed concentrations of the elements Pb Zn Cdand As that exceeded many times the permissible standards[3 4] In Dai Tu district mining activities at local craft havecreated a significant amount of waste tailings and waste rockResults of several waste rock samples have shown that theaverage As levels reached 5000mgkg Other HM contents inthe samples are also very high (Cu 1260mgkg Pb 105mgkg Cd 05mgkg Se 17mgkg) which indicates that there iscocontamination of several HMs in a site [5] Since Ascontaminated sites have been proved to simultaneously existwith high levels of Cu Cr Cd Pb and Zn [1 6] the ex-ceeding HMs distributed to agricultural soil (paddy tea andforest fields) surrounding tin and tungsten mines in Dai Tudistrict present at up to 1000mgkg for As 300mgkg forCu and abnormal high level of other HMs including Cd ZnPb Mn Other soil analysis in the mining sites of NorthernVietnam also shows cocontamination of Pb As Zn Cd Cuwith the factors of 314 2990 280 28 11 times respectivelyhigher the level of Vietnamese standard for mining soil(QCVN 03-MT 2015BTNMT 2015) [7 8]

Excess of HMs in the soil can cause phytotoxic effects inplants in several ways including the excessive production offree radicals and reactive oxygen species (ROS) ROS beinghighly unstable could play a dual role (i) damage cellularcomponents and (ii) act as an important secondary mes-senger for inducing plant defense systems which affect plantgrowth and biomass [9] To counteract this damage plantsare equipped with enzymatic and nonenzymatic defensemechanisms Several plants are known as hyper-accumulators since they are capable of growing in soils withvery high concentrations of metals absorbing these metalsthrough their roots and concentrating extremely high levelsof metals in their tissues [10 11] ese hyperaccumulatorspossess several antioxidative defense systems to scavengetoxic free radicals in order to protect themselves from theoxidative stress that is caused by HMs

Interestingly many hyperaccumulators belong to pte-ridophytes that are important plants from a phylogeneticand evolutionary perspective [12] Botanically these tem-perate-thriving plants were wild and many of them wereweeds a thousand years ago As these plants have survivedsince Paleozoic times pteridophytes have survived moredisruptive changes in the environment than any othervascular plants is can be explained as the reason whythese ferns were considered as hyperaccumulators C par-asiticus D linearis P calomelanos and P vittata are fourferns that dominate inmining sites inai Nguyen provinceVietnam in which the two latter weeds P calomelanos andP vittata have been reported as strong arsenic hyper-accumulators [6 13ndash15]

In order to explore the relationship between heavy-metalconcentrations in the metalliferous soils on the heavy-metaluptake capabilities of the corresponding local severaldominant ferns including C parasiticus D linearis Pcalomelanos and P vittata collected in the three mining sitesin ai Nguyen province Vietnam were evaluated for theirantioxidant activity via 11-diphenyl-2-picrylhydrazyl(DPPH) and superoxide anion (SRSA) radical scavenging

assays e screening results can be a good start for furtherstudies of the antioxidants which can be applied in the healthindustry Consequently the investigation of HMs contam-inated levels can be useful for applying some methods inorder to reduce pollution andor protect the environmentWe also try to find out the relationship between antioxidantactivity of plants and HMs levels in soil in order to incor-porate the environment purpose and natural sourcesvalorization

2 Materials and Methods

21 Chemicals Hydrochloric acid HCl (365ndash38) nitricacid HNO3 (63) sulfuric acid H2SO4 (98) HClO4DPPH methanol (MeOH HPLC grade) ascorbic acid(gt99) catechin and xanthine were purchased from SigmaAldrich (Taufkirchen Germany) in analytical grade Ul-trapure water (UPW) was prepared using a Millipore (Milli-Q) purification system (Millipore GmbH SchwalbachGermany 182MΩmiddotcm TOClt 2 μgL)

22 Soil and Plant Sampling Four dominant plant specieswere found at three mine sites that were described in Table S1C parasiticus (-elypteridaceae) were collected at the Campusof ai Nguyen Iron and Steel Corporation Cam Gia Wardai Nguyen City ai Nguyen province Two other weedsD linearis (Gleicheniaceae) and P calomelanos (Adiantaceae)were collected in Ha uong commune Dai Tu district aiNguyen province P vittata (Pteridaceae) was collected inHich village Tan Long commune Dong Hy district aiNguyen province Vietnam All plant samples were identifiedby Dr Nguyene Cuong Institute of Ecology and BiologicalResources VietnamAcademy of Science and Technologyevoucher of specimens was deposited in the Department of LifeSciences University of Science and Technology of Hanoi ecorresponding soil samples were dug in the rhizospheric area(up to 20 cm depth from the topsoil and 10 cm diameter fromplant position) of the collected plants All plant and soilsamples were collected under dry weather conditions March9th 2015 stored in polyethylene bags labeled appropriately

For sample preparation each plant was divided into 3parts leaves shoots and roots All plant tissues were washedby tap water to remove all dust and soil residues and thenrinsed by UPW All parts were cut down into smaller partsand dried in the oven at 55degC for 48 h to achieve the stableweight of samples Similarly soil samples were also preparedin the same manner of drying in the oven at 105degC for 24 huntil constant weight after removing all the moisture enplant and soil samples were crushed by using the clean anddry mortar followed by screening steps using 2mm and018mm sieves respectively e prepared samples werefinally stored at minus80degC until analysis

23 Atomic Absorption Spectrometry (AAS) Technique Todetermine the Pb Cu Cd and Zn concentrations in soilsamples 15mL HCl 37 and 5mL HNO 3 65 were addedto 2 g of dried and ground soil samples which were containedin 50mL baker e mixtures were digested on a hot plate at

2 Journal of Chemistry

95degC until getting the transparent solutions and then let toroom temperature and prepared for analysis Especially forAs analysis 5mL HNO 3 65 was added to 2 g of preparedsoil samples and then the mixture was treated in a mi-crowave oven for 40min e sample was cooled down toroom temperature After the treatment all five HMs in soilsamples were formed in the dissolved solution and filteredthrough 045 μm cellulose acetate membrane (WhatmanEngland) and ready for analysis

In order to determine the HM concentration in theplants each 05 g of each tissue powder was digested by amixture including 3mL concentrated H2SO4 98 05mLHClO4 and 10mL of concentrated HNO3 e mixture wasthen heated in the oven at temperature between 150 andminus200degC for at least 120mine operation was repeated untilthe digested solutions became clear and homogenous enthe obtained solution was normed up to 25mL in theErlenmeyer flask All the final sample solutions were shakenwell at 180 rpm on IKAreg C-MAG HS 4 magnetic stirrer(Cologne Germany) for 20min filtered by 045 μm celluloseacetate membrane to remove all the particulars and thendiluted to 25mL for measuring Cu Pb Zn Cd concen-tration For As concentration measurement 15mL of thesolution on the surface was made up with 10mL UPW to getfinally 25mL for measuring

All measurements were done in triplicate by theAtomic Absorption Spectrometry method proposed byAPHA (1995) based on the Varian 280FS and AA280Z(Australia) e results were reported by the averagevalues with relative standard deviation ( RSD) lowerthan 10

24 DPPH Scavenging Assay In order to evaluate the an-tioxidant activity of plant tissues approximately 50 g of eachtissue powder was extracted with MeOH solution(100mLtimes 3 times) at room temperature in an ultrasonicbath Skymen JP-008 (Guangdong China) e combinedextracts were concentrated in Buchi Rotavapor (FlawilSwitzerland) to obtain crude extracts

e DPPH radical scavenging capacity was evaluatedaccording to the previously described method [16] Brieflytwo concentrations of 100 μgmL and 500 μgmL of crudeextracts dissolved in dimethyl sulfoxide (5 μl) were addedto 195 μl DPPH solution (150mM) in 96-well plates emixed solution was incubated at room temperature for30min en the absorbance of the reaction mixture wasmeasured at 517 nm on a microplate reader iMarktrade(BioRad California USA) Ascorbic acid was used as apositive control (SC 913 at 100 μgmL)e differences inabsorbance between the test sample and the blank (DPPHin MeOH) was calculated and expressed as () scavengingcapacity

SC() (Ab minus As)

Ab1113888 1113889 times 100 (1)

where As is the absorbance of the test sample and Ab is theabsorbance of the blank Samples with SC values above 50were considered to be active

25 Superoxide Anion Radical Scavenging Assay e su-peroxide anion scavenging activity was measured based onthe describedmethod [17] In this experiment crude extractswere prepared at 2 concentrations as 100 μgmL and 500 μgmL in DMSO e standard reaction mixture in a finalvolume of 1mL contained 10 μL of sample solution 490 μLof xanthine (1mM in potassium phosphate buffer 01M pH78) and 490 μL of NBT (04mM in potassium phosphatebuffer 01M pH 78) e reaction was initiated by theaddition of 10 μL of xanthine oxidase Incubation at roomtemperature for 20minutes and the samplesrsquo absorbance wasread at 560 nm by using microplate reader iMarktrade (BioRadCalifornia USA) Catechin was used as standard control(positive sample) and the blank well without sample wasused as a negative sample All experiments were performedin triplicate and the reported results are the averages of threeindependent measurements

Superoxide radical scavenging activity (SRSA) is cal-culated by the following equation

SC() (Ac minus As)(Ac minus Ab)

1113888 1113889 times 100 (2)

Ac is the absorbance of the positive sample As is theabsorbance of the test sample and Ab is the absorbance ofthe negative sample Samples with SC values above 50 wereconsidered to be active

26 Statistical Analysis Statistics were performed in Rversion 353 [18] Data was normalized by taking theirlogarithm base 10 In order to discriminate the accumulationability of different species the principal component analysis(PCA) was done by PCA and dudipca function in Facto-MineR factoextra 107 and ade4 R-package [19 20] Toevaluate the effect of environmental factors (HM in soils) onthe accumulation of HM in the plants a function envfit invegan 25ndash6 R-package was used [21] e correlation be-tween the HM concentrations in soil and plants with an-tioxidant activities was calculated and visualized byPerformance Analytics R-package [22]

3 Results and Discussion

31HMsLevel inSoil Five HMs As Cd Zn Pb and Cu werequantified in 25 soil samples collected at the same positionswith 25 plant samples including 5 samples of C parasiticus(in ai Nguyen city) 6 samples for each type of D linearisand P calomelanos plants (in Dai Tu district) and 8 samplesof P vittata (in Dong Hy district) According to eachsampling station the sample abbreviation was used inter-changeably for the respective soil and plant samples

Table 1 shows the highest As Zn Pb and Cd concen-trations in P vittata correspondent soil while the mostconcentration of Cu was found in the soil growing Cparasiticus plant According to the Vietnam National reg-ulations on the allowable limits of HMs in the soils [7] wherethe maximum allowable limits of As Zn Pb Cu and Cd inindustrial soils are 25 300 300 300 and 10mgkg re-spectively It was investigated that soil samples collected in

Journal of Chemistry 3

Dong Hy district (P vittata corresponding soil) were con-taminated by both As and Pb whereas soils from Dai Tu wascontaminated with Cu Soils from Cam Gia ai Nguyencity witnessed the Pb pollution Significantly there were noobservations of Zn and Cd contaminations in all soil samplescollected from different sampling sites

It is found that the distribution of HMs in soils wasfollowing the different soil origins and plant bio-accumulation andor absorption [23] To understand morethis phenomenon the principal component analysis (PCA)revealed the difference among the HM levels of three lo-cations where 25 soil samples were collected by dis-tinguishing P vittata D linearis P calomelanos and Cparasiticus zones (Figure 1) e first two axes of the PCAexplained 664 of the variance e ordination arrowrepresents the dominance of As and Zn in the soil samplescollected in the rhizosphere of P vittata in Dong Hydistrict More Pb and Cd than other HMs were distributedin soils sampled in Dong Hy district and ai Nguyen citywhile Cu significantly existed in soils collected in Dai Tudistrict ese distributions of HMs in soils have thuscorresponded to their detected concentration in differentsoils (Table 1) In addition the arrow direction of vectorswhich represented the distribution tendency of HMsaccording to soil samples were also indicated for thecocontamination relationship between HMs in the soils Cuvectors tend to contradict the vectors of other metalsindicating that Cu has less ability to coaccumulate con-currently with other metals in the soil is was identifiedmore clearly based on the Spearman index (Figure S1)when the indices representing the correlation between Cuand other metals were always very low (from minus076 tominus036) is phenomenon can be explained by highersolubility and leachability of Cu at the ideal soil pH of60ndash65 compared to other HMs [24] In contrast Cdappeared to have a stronger correlation in soil accumu-lation with other metals except Cu while the lowestconcentrations of Cd were often found in all soil samples(Table 1) Consequently the Spearman indices indicatingthe cocontamination between Cd and other HMs followingthe order Zn (076)gtAs (059) gt Pb (037) is could bepartly clarified for the highest concentrations of Zn As Pband Cd were found in Dong Hy soil as previous investi-gation (Table 1) Also Pb had significant cocontaminationwith As and Zn in soil following Spearman indices of 039and 051 respectively confirming for high abilities of ZnAs and Pb accumulations in soils corresponding to Pvittata plant e analysis results therefore presented thesignificant effects of cocontaminations between soil originsand the HM residences in those soils

32HMsLevels inPlantTissues e statistical description ofHM concentration (in mgkg) in 75 plant tissue samples wassummarized by the average concentration with standarddeviation For each HM the bioaccumulation factor (BF)was calculated by the ratio between the HM concentration intissue and those in corresponding soil which is described asthe ability of plants for elemental accumulation from thesubstrate e efficiency of phytoremediation can bequantified by calculating the translocation factor (TF) isindex expresses the capacity of a plant to store the HMs in itsupper part which is defined as the ratio of metal concen-tration in the upper part to that in the roots [25]

e total concentration of selected metals as well as BFand TF indices in plant roots shoots and leaves is shown inTable 2 In plant roots the highest As Pb and Zn con-centrations were found in P vittata whereas the most Cuconcentration was measured in P calomelanos e largeamounts of As Pb Zn and Cu found in plant roots of Pvittata and P calomelanos could be explained by the highlevel of these metal concentrations detected in the soilswhere corresponding plants were cultivated (Table 1) Onthe other side there was no significant difference in Cdconcentration in the roots of 4 plants is correlated to thelow level of Cd found in all corresponding soils (Table 1) Onaverage the HM concentrations in the roots were muchhigher than those in the leaves and shoots implying thatfewer HMs were translocated into leaves and shoots for allfour species

In plant shoots the As concentration ranged from119plusmn 09mgkg (in C parasiticus) to 331plusmn 753mgkg (in Pvittata) e total concentration (mgkg) of Cu Pb and Znin plant shoots varied significantly with the highest valuesbeing 591plusmn 153 (in P calomelanos) 654plusmn 183 (in P vit-tata) and 542plusmn 222 (in P vittata) respectively Similarlyin the case of metals accumulation in leaves higher metalsconcentrations of As Cu Pb and Zn were found in plantleaves of P vittata and P calomelanos On the other sideaccording to different plant types a similar distributiontendency of measured Cd concentrations was found in plantroots and shoots and the lower metals concentrations wereoften found in all tissues of C parasiticus and D lineariscompared to other ones It could be seen that the distri-bution of metal concentration in plant tissues varied greatlyamong plant species and had a significant correlation withHM content in soils from different sampling sites

Moreover the bioaccumulation factor (BF) indicates theability of metals stored in plant tissues compared to soil [26]For all plant tissues the BF values often lower than 10 forCd Cu Pb and Zn In contrast the BF values of As whichwere extremely high in all tissues of almost all plants except

Table 1 e average of five HMs (As Zn Pb Cd and Cu) concentrations (in mgkg) of soil samples corresponding to 4 plant species

Correspondence As Zn Pb Cd CuC parasiticus 931plusmn 421 108plusmn 319 307plusmn 138 198plusmn 070 171plusmn 100D linearis 254plusmn 045 841plusmn 194 206plusmn 566 081plusmn 082 453plusmn 313P calomelanos 292plusmn 098 643plusmn 154 239plusmn 669 038plusmn 016 338plusmn 102P vittata 539plusmn 139 265plusmn 111 450plusmn 248 234plusmn 146 112plusmn 806


C parasiticus with BFAs lower than 1 is indicated thehigher moving ability of As from the external environmentinto plant tissues through the soil exposure route comparedto the other 3 metals It is also found that P vittata sampledat Dong Hy possessed a higher metal bioaccumulation factor

(up to 4025) than other plants indicating its hyper-accumulating ability ere are a number of reports indi-cating that P vittata is an As hyperaccumulator [27 28]capable of translocating As and storing it in the shoots[29 30]

Similarly most shoot and root tissues of all almost plantshad TF values lower than 1 except TFAs (in C parasiticus)and TFCd (in C parasiticus and D linearis plant) valueswhich are much higher than 1 In more detail the maximumvalues of TFCd and TFAs found in shoots of C parasiticusplants were 184 and 451 respectively Furthermore theTFmetals indices in the shoots were often higher than that inleaves of C parasiticus and D linearis plants whereas higherTF metals indices were found in leaves of P calomelanos andP vittata High root to shoot translocation of these metals inC parasiticus andD linearis indicated vital characteristics tobe used in phytoextraction of these metals in shoot [31 32]On the other side the results in TF and BF values of Pcalomelanos and P vittata were considered suitable forphytoextraction which generally requires translocation ofmetals easily harvestable leaves tissue to remove the metalcontamination from the soil without destroying soil struc-ture and fertility [33 34] In fact plant species were judged asbeing suited for phytoremediation based on several criteriaincluding wide distribution high above-ground biomasshigh bioaccumulation factors (hyperaccumulators) shortlife cycles and high propagation rates [35] erefore itcould be concluded that P vittata and P calomelanos can beused in phytoremediation in mine sites of ai Nguyenprovince as they were considered as native plants thatmeet all criteria for a hyperaccumulator

33 Species-Tissue Discrimination of HMs Levels in PlantsIn order to see whether there are differences in the con-centration of HMs level (ie variables) in samples (ieindividuals) PCA biplots where both individuals and var-iables are graphed on the same plot were constructed In thefirst two PCs more than 60 of the variance in the data isexplained for all PCA plots In accordance with the PCAbiplot (Figure 2(a)) HM levels in 75 plant tissues weregrouped in a different pattern based on species e dis-crimination of these patterns indicates that four speciesshow a different capacity in uptaking 5 mentioned HMsePCA biplots containing only data of HM levels in shoots orleaves showed better separation between four species(Figures 2(c) and 2(d)) while that of roots separated well 3patterns corresponding to speciesD linearis P calomelanosand P vittata (Figure 2(b)) is indicates that there is acontribution to the difference in accumulation ability amongtissues on the discrimination of 75 samples based on speciesis result would be useful for understanding the differenttranslocation factors of 4 ferns calculated in Table 2 Inaddition taking into account the variable vector in a PCAbiplot it can be seen that Cu was accumulated with a higherlevel in all tissue of P calomelanos which confirms again theprevious finding of the Cu accumulation ability of this fern[13 36 37] In the current study P calomelanos does notshow the arsenic accumulation capability whereas many

As

Zn

Pb

Cd

Cu

minus2

0

2

4

minus25 00 25PC1 (447)

PC2

(21

7)

Correspondence

C parasiticus

D linearis

P calomelanos

P vittata

Figure 1 PCA of the concentration of HMs (As Cd Cu Pb andZn) in 25 soil samples corresponding to 25 plants of 4 species (Cparasiticus D linearis P calomelanos and P vittata) Each pointdisplays a sample each vector displays the level of an HM Soilsamples are colored and shaped according to different specieswhere they were collected


studies have reported that this fern can accumulate morethan 2000mgkg in their tissues [12 38] even better than Pvittata [39] However the arsenic accumulation behavior ofthese two ferns changed according to field conditions iesubtropical or tropical highly or lightly contaminated soilsdrought and other weeds under field conditions [40] Otherstudies also reported the ability of P calomelanos in As-Pbcoaccumulating [15 41] In this study the poor performanceof P calomelanos in HM accumulation at Hauong Dai Tumay be due to low concentration of As Pb Cu in this sitecompared to Tan Long Dong Hy where P vittata werecollected It can also be highlighted from Figure 3 that AsPb Zn were accumulated with the highest levels in P vittatais finding is consistent with the previous research con-clusion that P vittata is an arsenic hyperaccumulator [27]useful for the remediation of As polluted sites cocontami-nated with Zn [11] Pb [15 41] and Cd [6]

34 Relationship between the HMs Levels in Soils and PlantsTo evaluate whether the HMs levels in soils have impact onthe difference in accumulation activity of plant tissuesenvironmental factors (ie HMs levels in soils) were fittedonto the ordination represented by the principal componentanalysis As a result the significant correlation between HMslevels in soils and samples similarities of the dataset for 75samples and the subsets of roots shoots and leaves with p

values below 005 are reported in Table 3 For all 75 samplesthe concentration of two elements Zn and Cu in soils showeda significant correlation (r2 042 p valuelt 0001 andr2 040 p valuelt 0001 respectively) Furthermore con-cerning the impact of HMs in soils on the sample similaritiesin only one tissue these two above-mentioned variablespresented more important correlations (r2 057 andr2 065 in roots r2 059 and r2 055 in shoots r2 071and r2 059 in leaves respectively with all p valuelt 0001)is suggested that the concentration of Zn and Cu in soils ismost responsible for the discrimination based on speciesbetween all samples as well as roots shoots and leaves econcentration of three remaining HMs (As Cd Pb) was lesscontributed to the variation in HMs accumulation of 4weeds (r2lt 04 except the levels of Cd and Pb in shoots)

e correlation between the HMs levels in soils and intissues was calculated by the Spearman method to reveal therelationship among these variables As can be seen fromFigure 4 Pb level in tissues correlated very strongly with Asconcentration in tissues (r 086 p valuelt 0001) whichindicates there is a coaccumulation of Pb and As of theseferns To the best our knowledge it is the first time these twoelements were found to be proportionally uptaken in planttissues Moreover Zn concentration in tissues correlatedmoderately with Zn level in soils (r 043 p valuelt 0001)indicating the Zn accumulation capacity of 4 ferns does notdepend very much on the fern species

35 Antioxidant Properties of Plant Extracts e radicalscavenging capacity of the crude plant extracts was deter-mined from the reduction in the optical absorbance due tothe scavenging of DPPH and SRSA radicals activity Ourstudy indicated that the roots possessed a high antioxidantactivity while shoots and leaves expressed the lower DPPHscavenging effects for all four species (Figure 3) At 500 μgmL of concentration most of the samples were considered tohave DPPH radical scavenging activity At a concentrationof 100 μgmL roots of C parasiticus presented the highestDPPH scavenging activity with the inhibition of radicalsgeneration value 881plusmn 261 at the concentration 100 μgmL (Table 4) To date there is almost no research about theantioxidant of C parasiticus except a study on the chemicalcomposition of this fern that reveals the presence of somecoumarin-chalcone hybrids in this weed [42] ConcerningSRSA scavenging activity only the roots of P vittata wereconsidered to show an inhibitory effect on the O2-genera-tion (SC of 544plusmn 581) In this study two differentapproaches were applied to measure the scavenging capacityagainst stable nonbiological radicals and against specificROSRNS Two levels of screening were applied to seewhether the antioxidant properties of these weeds can beexplained by different mechanisms related to enzymatic(SRSA assay) and nonenzymatic (DPPH assay) [43] Fromthe applied point of view this property opens the way tobenefit these ferns as phytosource of natural antioxidantsthat could be used in the chemical catalyst food

Table 2 e average concentration (plusmnSD) of five HMs in plant tissues (root shoot and leave) from 4 species C parasiticus D linearis Pcalomelanos and P vittata and corresponding bioaccumulation factor (BF) and translocation factor (TF)

Plantspecies

Planttissue

As Cd Cu Pb ZnConc BF TF Conc BF TF Conc BF TF Conc BF TF Conc BF TF

Cparasiticus

Root 119plusmn 177 014 mdash 058plusmn 049 029 mdash 197plusmn 592 012 mdash 434plusmn 148 001 mdash 121plusmn 736 112 mdashShoot 119plusmn 090 021 451 066plusmn 030 036 184 131plusmn 111 008 088 279plusmn 164 001 128 280plusmn 130 027 110Leave 135plusmn 158 018 443 077plusmn 030 045 837 152plusmn 880 009 081 551plusmn 195 002 061 110plusmn 507 110 028

D linearisRoot 122plusmn 322 500 mdash 019plusmn 009 047 mdash 758plusmn 530 047 mdash 373plusmn 209 021 mdash 355plusmn 108 045 mdashShoot 183plusmn 144 803 032 008plusmn 005 024 761 411plusmn 222 024 038 885plusmn 449 005 047 221plusmn 107 029 099Leave 334plusmn 305 149 057 032plusmn 025 082 044 284plusmn 142 017 059 154plusmn 622 009 025 437plusmn 315 057 059

Pcalomelanos


P vittataRoot 1732plusmn 2991 403 mdash 027plusmn 026 013 mdash 598plusmn 130 106 mdash 244plusmn 607 088 mdash 188plusmn 305 035 mdashShoot 331plusmn 753 762 020 013plusmn 006 008 084 509plusmn 237 096 084 654plusmn 183 019 028 542plusmn 222 023 065Leave 438plusmn 176 995 109 033plusmn 028 018 149 613plusmn 34 117 103 141plusmn 515 047 059 267plusmn 125 113 318


Pb

Cd

Cu

Zn

As

minus25

00

25

50

minus2 0 2 4 6PC1 (446)

PC2

(22

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(a)

Pb

Cd

Cu

Zn

As

minus50

minus25

00

25

minus2 0 2PC1 (381)

PC2

(21

9)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(b)

Species

Pb

Cd

Cu

Zn

As

minus2

0

2

4

minus25 00 25 50PC1 (509)

PC2

(28

3)

C parasiticus

D linearis

P calomelanos

P vittata

(c)

Pb

Cd

Cu

Zn

As

minus25

00

25

minus2 0 2 4PC1 (56)

PC2

(23

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(d)

Figure 2 Principal component analysis of HM levels in 75 samples (3a) in 25 roots (3b) in 25 shoots (3c) and in 25 leaves (3d) from fourspecies C parasiticusD linearis P calomelanos and P vittata Samples are colored and shaped according to different species e length ofthe HM vector shows how high the concentration of these HMs levels is in the samples where this vector is directed toward (a) All tissues(b) roots (c) shoots and (d) leaves


Species

C parasiticus

D linearis

P calomelanos

P vittata

25

50

75

Leave Root Shoot

DPP

H 1

00 (micro

gm

l)

(a)

Species

C parasiticus

D linearis

P calomelanos

P vittata

40

60

80

Leave Root ShootD

PPH

500

(microg

ml)

(b)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

10

20

30

40

Leave Root Shoot

SRSA

100

(microg

ml)

(c)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

20

40

60

Leave Root Shoot

SRSA

500

(microg

ml)

(d)

Figure 3 Boxplots of the DPPH and SRSA scavenging inhibition of leaves roots and shoots from 4 studied ferns


Pb (t)

minus10

00

10

20

minus10

12

minus10

00

10

20

minus10

12

3

0 1 2 3

minus10 05 20

Cd (s)

046

Zn (t)

minus1 1 3

minus1 0 1 2

076

043

Zn (s)

086

041

As (t)

minus05 10 25

minus10 05 20

032

DPPH 100

046

044

070

DPPH 500

minus25minus10 05

minus1 1 2 3

066

043

062

x

SRSA 100

01

23

070

minus10

12

34

033

038

minus05

05

15

25

067

minus25

minus10

00

10

078

minus1 1 2

minus10

12SRSA 500

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast lowastlowastlowast

lowastlowastlowastlowastlowastlowast lowastlowastlowast

Figure 4 e significant pair to pair correlation of nine variables including As Pb Zn concentration in tissue Zn concentration on soilDPPH scavenging activity at 100 μgmL and 500 μgmL SRSA scavenging activity at 100 μgmL and 500 μgmL e distribution of eachvariable is shown on the diagonal On the bottom of the diagonal the bivariate scatter plots with a fitted line are displayed On the top of thediagonal the value of the correlation plus the significance level is denoted as stars Each significance level is associated with a symbol lowastlowast lowastp

valuelt 0001 lowastlowastp valuelt 001 and lowastp value lt005 e Pearson method was used to compute the correlation coefficient

Table 3e correlation coefficient of 5 environmental factors on the HMs in plant tissues roots shoots and leaves was calculated by envfitfunction in vegan R-package

All tissue n 75 Root n 25 Shoot n 25 Leave n 25r2 p r2 p r2 p r2 p

Cu 040 plt 0001 065 plt 0001 055 plt 0001 059 plt 0001Zn 042 plt 0001 057 plt 0001 059 plt 0001 071 plt 0001As 018 0002 003 072 037 0008 034 0009Cd 030 plt 0001 039 0007 044 0005 035 0009Pb 010 0036 013 024 045 0003 012 0244


nutraceuticals cosmetic and pharmaceutical industriesthereby restricting the use of synthetic antioxidants

36 Relationship betweenHMLevel in Soil and in Plant Tissueand -eir Antioxidant Activity In order to investigatewhether HMs in soils and HMs in tissues can affect theantioxidant activity of plants we analyzed the relationshipbetween the concentrations of five metals in soil and plantsamples and the percentages of DPPH and SRSA scavengingof 25 plants growing in these soils Pair to pair correlationsamong 14 variables were calculated by the Spearmanmethod e correlation coefficient was calculated as thestatistical measurement of how strong a linear relationshipof the paired data is Taking into consideration the signif-icant correlations (p valuelt 001) and important correla-tions that were contributed by antioxidant activities thesimplified pairwise correlations between 5 variables (Pb intissues Cd in soils Zn in tissues Zn in soils and As intissues) and 4 antioxidant variables (SC DPPH at 100 and500 μgmL SC SRSA at 100 and 500 μgmL) were shown inFigure 4 Noticeably the concentration of As in tissues wasproportional to both SRSA scavenging activity at 100 and500 μgmL (r 062 and r 067 respectively plt 0001) butnot correlated significantly with SC DPPH is findinghas been consistent with the previous study of Cao et al2004 which showed that at low levels of As exposure(0ndash20mgkg) enzymatic antioxidants (which may con-tribute to SRSA activity) play a more important role in Asdetoxification than nonenzymatic antioxidants (which maycontribute to DPPH activity) in P vittata [44] By otherresearch P calomelanos also presented a remarkable changein amino acid levels in As-treated plants including thoserelated to the biosynthesis of enzymatic antioxidants of thisweed [9] Similarly it was also observed that Pb concen-tration in tissues correlated significantly and strongly to SCof SRSA at two studied concentrations (r 066 p val-uelt 0001 and r 070 p valuelt 0001 respectively) whiledid not correlate to SC of DPPHis allows hypothesizingthat the excess of Pb accumulation significantly enhancedthe synthesis of some enzymatic antioxidants in plants thusinduced the antioxidant activity of plants

4 Conclusion

is study firstly showed the investigation of HM concen-trations in soils that had a significant variation according tomining soils and corresponding plant types As accumulatedat high levels in soils and plant tissues (shoot roots leaves)Cd had a strong cocontamination relationship with othermetals As Zn Pb according to high Spearman values (from037 to 076) Cu contaminated in Dai Tu district and had alow ability to cocontaminated with four other HMs For allweeds with BF and TF values almost lower than 1 Cu ZnPb and Cd had limited ability to uptake in plant tissues Inaddition the distribution or discrimination of HM followedthe different species tissues and soil sampling regionsIndeed it is found that the roots of P vittata of C parasiticuspossess the antioxidant activity via DPPH and SRSA radicalscavenging assay respectively erefore the correlationbetween antioxidant activity of plants and HMs content hasopened a prospect in the identification of hyperaccumulatorspecies with high potential in pharmaceutical as well asphytoremediation research

Data Availability

e data used to support the findings of this study are in-cluded within the article

Conflicts of Interest

e authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

is research was funded by the Vietnam National Foun-dation for Science and Technology Development(NAFOSTED) under grant no 10602ndash201730 e corre-sponding authors would like to thank Dr Tran i anhTam Department of Life Sciences University of Science andTechnology of Hanoi Vietnam Academy of Science andTechnology for her advice in the statistical analysis part eauthors also gratefully acknowledge Laboratoire Mixte

Table 4 DPPH scavenging capacity (SC) of the plant extracts

Plant species Plant tissues DPPH SC at 100 μgmL DPPH SC at 500 μgmL SRSA SC at 100 μgmL SRSA SC at 500 μgmL

C parasiticusRoots 881plusmn 261 879plusmn 196 965plusmn 538 146plusmn 501Shoots 292plusmn 734 804plusmn 139 783plusmn 103 123plusmn 953Leaves 377plusmn 101 779plusmn 610 116plusmn 000 648plusmn 111

D linearisRoots 615plusmn 261 811plusmn 979 594plusmn 321 170plusmn 746Shoots 645plusmn 289 766plusmn 111 544plusmn 305 128plusmn 505Leaves 318plusmn 160 660plusmn 151 399plusmn 174 101plusmn 090

P calomelanosRoots 445plusmn 242 692plusmn 210 263plusmn 251 360plusmn 482Shoots 268plusmn 129 650plusmn 202 105plusmn 641 188plusmn 118Leaves 173plusmn 504 473plusmn 822 114plusmn 541 224plusmn 839

P vittataRoots 417plusmn 200 835plusmn 953 299plusmn 913 544plusmn 581Shoots 310plusmn 114 619plusmn 163 108plusmn 281 303plusmn 611Leaves 462plusmn 118 893plusmn 063 149plusmn 604 243plusmn 567

Ascorbic acid mdash 913 mdash mdash mdashCatechin mdash mdash mdash 487 mdash


International ldquoDrug Resistance in South East Asiardquo (LMI-DRISA) for supporting the repair fee of the microplatereader in an urgent situation

Supplementary Materials

Table S1 description of sampling sites Figure S1 theSpearman indices indicate the correlation of heavy metalsrsquococontamination in soils (Supplementary Materials)

References

[1] L Sun X Liao X Yan G Zhu and D Ma ldquoEvaluation ofheavy metal and polycyclic aromatic hydrocarbons accu-mulation in plants from typical industrial sites potentialcandidate in phytoremediation for co-contaminationrdquo En-vironmental Science and Pollution Research vol 21pp 12494ndash12504 2014

[2] N K Chu V K Nguyen D B Nguyen et al ldquoArsenic andheavy metal concentrations in agricultural soils around tinand tungsten mines in the dai tu district n Vietnamrdquo WaterAir Soil and Pollution vol 197 no 1ndash4 pp 75ndash89 2009

[3] V B Nguyen D Q Nguyen M Q Vu and Q T Le ldquoeheavy metal distribution in the soil and water of Son DuongSn minerdquo Journal of Earth Science vol 22 pp 134ndash139 2000

[4] A Nguyen ldquoe soil pollution associated with mining invietnamrdquo in Proceedings of Contaminated Agricultural LandManagement and Remediation Workshop Hanoi VietnamDecember 2005

[5] B T Anh D D Kim T V Tua N T Kien and D T AnhldquoPhytoremediation potential of indigenous plants from thainguyen province Vietnamrdquo Journal of Environmental Biol-ogy vol 32 no 2 pp 257ndash262 2011

[6] X Xiao T Chen Z An et al ldquoPotential of Pteris vittata L forphytoremediation of sites co-contaminated with cadmiumand arsenic the tolerance and accumulationrdquo Journal ofEnvironmental Sciences vol 20 no 1 pp 62ndash67 2008

[7] Vietnam Ministry of Natural Resources and EnvironmentQCVN 03-MT2015BTNMTmdashNational Technical Regulationon the Allowable Limits of Heavy Metals in the Soils VietnamMinistry of Natural Resources and Environment HanoiVietnam in Vietnamese 2015

[8] T H Nguyen M Sakakibara S Sano and T N Mai ldquoUptakeof metals andmetalloids by plants growing in a lead-zincminearea Northern Vietnamrdquo Journal of Hazardous Materialsvol 86 no 2 pp 1384ndash1391 2011

[9] N V Campos T O Araujo S Arcanjo-Silva L Freitas-SilvaA A Azevedo and A Nunes-Nesi ldquoArsenic hyper-accumulation induces metabolic reprogramming in Pity-rogramma calomelanosto reduce oxidative stressrdquo PhysiologiaPlantarum vol 157 no 2 pp 135ndash146 2016

[10] H Sarma ldquoMetal hyperaccumulation in plants a review fo-cusing on phytoremediation technologyrdquo Journal of Envi-ronmental Science and Technology vol 4 no 2 pp 118ndash1382011

[11] Z-Z An Z-C Huang M Lei X-Y Liao Y-M Zheng andT-B Chen ldquoZinc tolerance and accumulation in Pteris vittataL and its potential for phytoremediation of Znminus and As-contaminated soilrdquo Chemosphere vol 62 no 5 pp 796ndash8022006

[12] T K A Bui D K Dang T K Nguyen N M NguyenQ T Nguyen and H C Nguyen ldquoPhytoremediation of heavymetal polluted soil and water in Vietnamrdquo Journal of Viet-namese Environment vol 6 no 1 pp 47ndash51 2014

[13] Z C Win L J L Diaz T R Perez and K NakasakildquoPhytoremediation of heavy metal contaminated wastes fromsmall-scale gold mining using Pityrogramma calomelanosrdquoE3S Web of Conferences vol 148 2020

[14] H B Wang Z H Ye W S Shu W C Li M H Wong andC Y Lan ldquoArsenic uptake and accumulation in fern speciesgrowing at arsenic-contaminated sites of southern China fieldsurveysrdquo International Journal of Phytoremediation vol 8no 1 pp 1ndash11 2006

[15] P Soongsombat M Kruatrachue R ChaiyaratP Pokethitiyook and C Ngernsansaruay ldquoLead tolerance andaccumulation Inpteris Vittata and Pityrogramma calomelanosand their potential for phytoremediation of lead-contami-nated soilrdquo International Journal of Phytoremediation vol 11no 4 pp 396ndash412 2009

[16] T T Huong N X Cuong L H Tram et al ldquoA new pre-nylated aurone from Artocarpus Altilisrdquo Journal of AsianNatural Products Research vol 14 no 9 pp 923ndash928 2012

[17] J-H Lee and J-S Park ldquoAntioxidant activities of solvent-extracted fractions from Kummerowia Striata (thunb)schindlrdquo Indian Journal of Science and Technology vol 8no 1 pp 28ndash31 2015

[18] R Core Team R A Language and Environment for StatisticalComputing R Foundation for Statistical Computing ViennaAustria 2018 httpswwwR-projectorg

[19] S Le J Josse and F Husson ldquoFacto mine R an R package formultivariate analysisrdquo Journal of Statistical Software vol 25no 1 pp 1ndash18 2008

[20] A Kassambara and M Fabian ldquoFactoextra extract and vi-sualize the results of multivariate data analysesrdquo 2020 httpsCRANR-projectorgpackage=factoextra

[21] J Oksanen F G Blanchet F Michael et al ldquoVegan com-munity ecology package R package version 24-3rdquo 2017httpsCRANR-projectorgpackagevegan

[22] G P Brian C Pete B Kris B Ross U Joshua and Z EricldquoPerformanceanalytics econometric tools for performanceand risk analysisrdquo R Package Version vol 107 no 1 p 35412014

[23] J Luo W He X Xing J Wu X W Sophie Gu andX W S Gu ldquoe variation of metal fractions and potentialenvironmental risk in phytoremediating multiple metalpolluted soils using Noccaea Caerulescens assisted by LEDlightsrdquo Chemosphere vol 227 pp 462ndash469 2019

[24] C E Martınez and H L Motto ldquoSolubility of lead zinc andcopper added to mineral soilsrdquo Environmental Pollutionvol 107 p 1 2000

[25] R Malik S Husain and I Nazir ldquoHeavy metal contaminationand accumulation in soil and wild plant species from in-dustrial area of islamabad Pakistanrdquo Journal of Botanyvol 42 pp 291ndash301 2010

[26] G Arumugam R Rajendran A Ganesan and R SethuldquoBioaccumulation and translocation of heavy metals inmangrove rhizosphere sediments to tissues of Avicenia ma-rina-a field study from tropical mangrove forestrdquo Environ-mental Nanotechnology Monitoring amp Management vol 10pp 272ndash279 2018

[27] L Q Ma K M Komar C Tu W Zhang and Y Cai ldquoA fernthat hyperaccumulates arsenicrdquo Nature vol 409 no 6820p 579 2001

[28] T Kennelley C Wei Z Huang Q Huang Q Lu and Z FanldquoArsenic hyperaccumulator Pteris vittata L and its arsenic ac-cumulationrdquo Chinese Science Bulletin vol 47 no 11pp 902ndash905 2002


[29] S P McGrath F J Zhao and E Lombi ldquoPlant and rhizo-sphere processes involved in phytoremediation of metal-contaminated soilsrdquo Plant and Soil vol 232 no 1pp 207ndash214 2001

[30] L Ozturk S Karanlik F Ozkutlu I Cakmak andL V Kochian ldquoShoot biomass and zinccadmium uptake forhyperaccumulator and non accumulator thlaspi species inresponse to growth on a zinc-deficient calcareous soilrdquo PlantScience vol 164 pp 1095ndash101 2003

[31] M Ghosh and S Singh ldquoA review on phytoremediation ofheavy metals and utilization of its byproductsrdquo AppliedEcology and Environmental Research vol 3 no 1 2018

[32] J L Dıez P Kidd and C M Monterroso ldquoA biogeochemicalstudy of the tras-os-montes region (NE portugal) possiblespecies for plant-based soil remediation technologiesrdquo -eScience of the Total Environment vol 354 no 2 pp 265ndash2772006

[33] M Ghosh and S Singh ldquoA review on phytoremediation ofheavy metals and utilization of its byproductsrdquo AppliedEcology and Environmental Research vol 3 pp 1ndash18 2005

[34] J Yoon and X Cao Q Zhou ldquoAccumulation of Pb Cu andZn in native plants growing on a contaminated Florida siterdquoScience of -e Total Environment vol 368 no 2 pp 456ndash4642006

[35] A Ma G Arun S Vinothkumar and R Rajaram ldquoBio-accumulation and translocation efficacy of heavy metals byRhizophora mucronata from tropical mangrove ecosystemsoutheast coast of Indiardquo Ecohydrology amp Hydrobiologyvol 19 no 1 pp 66ndash74 201

[36] F Perlatti T O Ferreira R E Romero M C G Costa andX L Otero ldquoCopper accumulation and changes in soilphysical-chemical properties promoted by native plants in anabandoned mine site in northeastern Brazil implications forrestoration of mine sitesrdquo Ecological Engineering vol 82pp 103ndash111 2015

[37] D Dahilan ldquoAssessment of copper in Pityrogramma calo-melanosrdquo Philippine Journal of Science vol 146 no 3pp 331ndash338 2017

[38] K Francesconi P Visoottiviseth W Sridokchan andW Goessler ldquoArsenic species in an arsenic hyper-accumulating fern Pityrogramma calomelanos a potentialphytoremediator of arsenic-contaminated soilsrdquo Science of-e Total Environment vol 284 no 1 pp 27ndash35 2002

[39] P Visoottiviseth K Francesconi and W Sridokchan ldquoepotential of thai indigenous plant species for the phytor-emediation of arsenic contaminated landrdquo EnvironmentalPollution vol 118 no 3 pp 453ndash461 2002

[40] N K Niazi B Singh L Van Zwieten and A G KachenkoldquoPhytoremediation of an arsenic-contaminated site usingPteris vittata L and Pityrogramma calomelanos var austro-americana a long-term studyrdquo Environmental Science andPollution Research vol 19 no 8 pp 3506ndash3515 2012

[41] A L Salido K L Hasty J-M Lim and D J ButcherldquoPhytoremediation of arsenic and lead in contaminated soilusing Chinese brake ferns (Pteris vittata) and Indian mustard(Brassica juncea)rdquo International Journal of Phytoremediationvol 5 no 2 pp 89ndash103 2003

[42] H Wei J Ruan and X Zhang ldquoCoumarin-chalcone hybridspromising agents with diverse pharmacological propertiesrdquoRoyal Society of Chemistry Advances vol 6 pp 10846ndash108602016

[43] L M Magalhatildees M A Segundo S Reis and J L F C LimaldquoMethodological aspects about in vitro evaluation of

antioxidant propertiesrdquo Analytica Chimica Acta vol 613pp 1ndash19 2008

[44] X Cao L Q Ma and C Tu ldquoAntioxidative responses toarsenic in the arsenic-hyperaccumulator Chinese brake fern(Pteris vittata L)rdquo Environmental Pollution vol 128pp 317ndash325 2004


Analysis of soil samples in the Pb-Zn ore Dong Hy aiNguyen showed concentrations of the elements Pb Zn Cdand As that exceeded many times the permissible standards[3 4] In Dai Tu district mining activities at local craft havecreated a significant amount of waste tailings and waste rockResults of several waste rock samples have shown that theaverage As levels reached 5000mgkg Other HM contents inthe samples are also very high (Cu 1260mgkg Pb 105mgkg Cd 05mgkg Se 17mgkg) which indicates that there iscocontamination of several HMs in a site [5] Since Ascontaminated sites have been proved to simultaneously existwith high levels of Cu Cr Cd Pb and Zn [1 6] the ex-ceeding HMs distributed to agricultural soil (paddy tea andforest fields) surrounding tin and tungsten mines in Dai Tudistrict present at up to 1000mgkg for As 300mgkg forCu and abnormal high level of other HMs including Cd ZnPb Mn Other soil analysis in the mining sites of NorthernVietnam also shows cocontamination of Pb As Zn Cd Cuwith the factors of 314 2990 280 28 11 times respectivelyhigher the level of Vietnamese standard for mining soil(QCVN 03-MT 2015BTNMT 2015) [7 8]

Excess of HMs in the soil can cause phytotoxic effects inplants in several ways including the excessive production offree radicals and reactive oxygen species (ROS) ROS beinghighly unstable could play a dual role (i) damage cellularcomponents and (ii) act as an important secondary mes-senger for inducing plant defense systems which affect plantgrowth and biomass [9] To counteract this damage plantsare equipped with enzymatic and nonenzymatic defensemechanisms Several plants are known as hyper-accumulators since they are capable of growing in soils withvery high concentrations of metals absorbing these metalsthrough their roots and concentrating extremely high levelsof metals in their tissues [10 11] ese hyperaccumulatorspossess several antioxidative defense systems to scavengetoxic free radicals in order to protect themselves from theoxidative stress that is caused by HMs

Interestingly many hyperaccumulators belong to pte-ridophytes that are important plants from a phylogeneticand evolutionary perspective [12] Botanically these tem-perate-thriving plants were wild and many of them wereweeds a thousand years ago As these plants have survivedsince Paleozoic times pteridophytes have survived moredisruptive changes in the environment than any othervascular plants is can be explained as the reason whythese ferns were considered as hyperaccumulators C par-asiticus D linearis P calomelanos and P vittata are fourferns that dominate inmining sites inai Nguyen provinceVietnam in which the two latter weeds P calomelanos andP vittata have been reported as strong arsenic hyper-accumulators [6 13ndash15]

In order to explore the relationship between heavy-metalconcentrations in the metalliferous soils on the heavy-metaluptake capabilities of the corresponding local severaldominant ferns including C parasiticus D linearis Pcalomelanos and P vittata collected in the three mining sitesin ai Nguyen province Vietnam were evaluated for theirantioxidant activity via 11-diphenyl-2-picrylhydrazyl(DPPH) and superoxide anion (SRSA) radical scavenging

assays e screening results can be a good start for furtherstudies of the antioxidants which can be applied in the healthindustry Consequently the investigation of HMs contam-inated levels can be useful for applying some methods inorder to reduce pollution andor protect the environmentWe also try to find out the relationship between antioxidantactivity of plants and HMs levels in soil in order to incor-porate the environment purpose and natural sourcesvalorization

2 Materials and Methods

21 Chemicals Hydrochloric acid HCl (365ndash38) nitricacid HNO3 (63) sulfuric acid H2SO4 (98) HClO4DPPH methanol (MeOH HPLC grade) ascorbic acid(gt99) catechin and xanthine were purchased from SigmaAldrich (Taufkirchen Germany) in analytical grade Ul-trapure water (UPW) was prepared using a Millipore (Milli-Q) purification system (Millipore GmbH SchwalbachGermany 182MΩmiddotcm TOClt 2 μgL)

22 Soil and Plant Sampling Four dominant plant specieswere found at three mine sites that were described in Table S1C parasiticus (-elypteridaceae) were collected at the Campusof ai Nguyen Iron and Steel Corporation Cam Gia Wardai Nguyen City ai Nguyen province Two other weedsD linearis (Gleicheniaceae) and P calomelanos (Adiantaceae)were collected in Ha uong commune Dai Tu district aiNguyen province P vittata (Pteridaceae) was collected inHich village Tan Long commune Dong Hy district aiNguyen province Vietnam All plant samples were identifiedby Dr Nguyene Cuong Institute of Ecology and BiologicalResources VietnamAcademy of Science and Technologyevoucher of specimens was deposited in the Department of LifeSciences University of Science and Technology of Hanoi ecorresponding soil samples were dug in the rhizospheric area(up to 20 cm depth from the topsoil and 10 cm diameter fromplant position) of the collected plants All plant and soilsamples were collected under dry weather conditions March9th 2015 stored in polyethylene bags labeled appropriately

For sample preparation each plant was divided into 3parts leaves shoots and roots All plant tissues were washedby tap water to remove all dust and soil residues and thenrinsed by UPW All parts were cut down into smaller partsand dried in the oven at 55degC for 48 h to achieve the stableweight of samples Similarly soil samples were also preparedin the same manner of drying in the oven at 105degC for 24 huntil constant weight after removing all the moisture enplant and soil samples were crushed by using the clean anddry mortar followed by screening steps using 2mm and018mm sieves respectively e prepared samples werefinally stored at minus80degC until analysis

23 Atomic Absorption Spectrometry (AAS) Technique Todetermine the Pb Cu Cd and Zn concentrations in soilsamples 15mL HCl 37 and 5mL HNO 3 65 were addedto 2 g of dried and ground soil samples which were containedin 50mL baker e mixtures were digested on a hot plate at







SC() (Ab minus As)

Ab1113888 1113889 times 100 (1)





1113888 1113889 times 100 (2)




















As

Zn

Pb

Cd

Cu

minus2

0

2

4

minus25 00 25PC1 (447)

PC2

(21

7)

Correspondence

C parasiticus

D linearis

P calomelanos

P vittata









Plantspecies

Planttissue


Cparasiticus



Pcalomelanos




Pb

Cd

Cu

Zn

As

minus25

00

25

50

minus2 0 2 4 6PC1 (446)

PC2

(22

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(a)

Pb

Cd

Cu

Zn

As

minus50

minus25

00

25

minus2 0 2PC1 (381)

PC2

(21

9)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(b)

Species

Pb

Cd

Cu

Zn

As

minus2

0

2

4

minus25 00 25 50PC1 (509)

PC2

(28

3)

C parasiticus

D linearis

P calomelanos

P vittata

(c)

Pb

Cd

Cu

Zn

As

minus25

00

25

minus2 0 2 4PC1 (56)

PC2

(23

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(d)



Species

C parasiticus

D linearis

P calomelanos

P vittata

25

50

75

Leave Root Shoot

DPP

H 1

00 (micro

gm

l)

(a)

Species

C parasiticus

D linearis

P calomelanos

P vittata

40

60

80

Leave Root ShootD

PPH

500

(microg

ml)

(b)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

10

20

30

40

Leave Root Shoot

SRSA

100

(microg

ml)

(c)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

20

40

60

Leave Root Shoot

SRSA

500

(microg

ml)

(d)



Pb (t)

minus10

00

10

20

minus10

12

minus10

00

10

20

minus10

12

3

0 1 2 3

minus10 05 20

Cd (s)

046

Zn (t)

minus1 1 3

minus1 0 1 2

076

043

Zn (s)

086

041

As (t)

minus05 10 25

minus10 05 20

032

DPPH 100

046

044

070

DPPH 500

minus25minus10 05

minus1 1 2 3

066

043

062

x

SRSA 100

01

23

070

minus10

12

34

033

038

minus05

05

15

25

067

minus25

minus10

00

10

078

minus1 1 2

minus10

12SRSA 500

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast











4 Conclusion


Data Availability




Acknowledgments













References





















































SC() (Ab minus As)

Ab1113888 1113889 times 100 (1)





1113888 1113889 times 100 (2)




















As

Zn

Pb

Cd

Cu

minus2

0

2

4

minus25 00 25PC1 (447)

PC2

(21

7)

Correspondence

C parasiticus

D linearis

P calomelanos

P vittata









Plantspecies

Planttissue


Cparasiticus



Pcalomelanos




Pb

Cd

Cu

Zn

As

minus25

00

25

50

minus2 0 2 4 6PC1 (446)

PC2

(22

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(a)

Pb

Cd

Cu

Zn

As

minus50

minus25

00

25

minus2 0 2PC1 (381)

PC2

(21

9)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(b)

Species

Pb

Cd

Cu

Zn

As

minus2

0

2

4

minus25 00 25 50PC1 (509)

PC2

(28

3)

C parasiticus

D linearis

P calomelanos

P vittata

(c)

Pb

Cd

Cu

Zn

As

minus25

00

25

minus2 0 2 4PC1 (56)

PC2

(23

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(d)



Species

C parasiticus

D linearis

P calomelanos

P vittata

25

50

75

Leave Root Shoot

DPP

H 1

00 (micro

gm

l)

(a)

Species

C parasiticus

D linearis

P calomelanos

P vittata

40

60

80

Leave Root ShootD

PPH

500

(microg

ml)

(b)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

10

20

30

40

Leave Root Shoot

SRSA

100

(microg

ml)

(c)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

20

40

60

Leave Root Shoot

SRSA

500

(microg

ml)

(d)



Pb (t)

minus10

00

10

20

minus10

12

minus10

00

10

20

minus10

12

3

0 1 2 3

minus10 05 20

Cd (s)

046

Zn (t)

minus1 1 3

minus1 0 1 2

076

043

Zn (s)

086

041

As (t)

minus05 10 25

minus10 05 20

032

DPPH 100

046

044

070

DPPH 500

minus25minus10 05

minus1 1 2 3

066

043

062

x

SRSA 100

01

23

070

minus10

12

34

033

038

minus05

05

15

25

067

minus25

minus10

00

10

078

minus1 1 2

minus10

12SRSA 500

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast











4 Conclusion


Data Availability




Acknowledgments













References





























































As

Zn

Pb

Cd

Cu

minus2

0

2

4

minus25 00 25PC1 (447)

PC2

(21

7)

Correspondence

C parasiticus

D linearis

P calomelanos

P vittata









Plantspecies

Planttissue


Cparasiticus



Pcalomelanos




Pb

Cd

Cu

Zn

As

minus25

00

25

50

minus2 0 2 4 6PC1 (446)

PC2

(22

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(a)

Pb

Cd

Cu

Zn

As

minus50

minus25

00

25

minus2 0 2PC1 (381)

PC2

(21

9)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(b)

Species

Pb

Cd

Cu

Zn

As

minus2

0

2

4

minus25 00 25 50PC1 (509)

PC2

(28

3)

C parasiticus

D linearis

P calomelanos

P vittata

(c)

Pb

Cd

Cu

Zn

As

minus25

00

25

minus2 0 2 4PC1 (56)

PC2

(23

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(d)



Species

C parasiticus

D linearis

P calomelanos

P vittata

25

50

75

Leave Root Shoot

DPP

H 1

00 (micro

gm

l)

(a)

Species

C parasiticus

D linearis

P calomelanos

P vittata

40

60

80

Leave Root ShootD

PPH

500

(microg

ml)

(b)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

10

20

30

40

Leave Root Shoot

SRSA

100

(microg

ml)

(c)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

20

40

60

Leave Root Shoot

SRSA

500

(microg

ml)

(d)



Pb (t)

minus10

00

10

20

minus10

12

minus10

00

10

20

minus10

12

3

0 1 2 3

minus10 05 20

Cd (s)

046

Zn (t)

minus1 1 3

minus1 0 1 2

076

043

Zn (s)

086

041

As (t)

minus05 10 25

minus10 05 20

032

DPPH 100

046

044

070

DPPH 500

minus25minus10 05

minus1 1 2 3

066

043

062

x

SRSA 100

01

23

070

minus10

12

34

033

038

minus05

05

15

25

067

minus25

minus10

00

10

078

minus1 1 2

minus10

12SRSA 500

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast











4 Conclusion


Data Availability




Acknowledgments













References






















































Plantspecies

Planttissue


Cparasiticus



Pcalomelanos




Pb

Cd

Cu

Zn

As

minus25

00

25

50

minus2 0 2 4 6PC1 (446)

PC2

(22

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(a)

Pb

Cd

Cu

Zn

As

minus50

minus25

00

25

minus2 0 2PC1 (381)

PC2

(21

9)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(b)

Species

Pb

Cd

Cu

Zn

As

minus2

0

2

4

minus25 00 25 50PC1 (509)

PC2

(28

3)

C parasiticus

D linearis

P calomelanos

P vittata

(c)

Pb

Cd

Cu

Zn

As

minus25

00

25

minus2 0 2 4PC1 (56)

PC2

(23

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(d)



Species

C parasiticus

D linearis

P calomelanos

P vittata

25

50

75

Leave Root Shoot

DPP

H 1

00 (micro

gm

l)

(a)

Species

C parasiticus

D linearis

P calomelanos

P vittata

40

60

80

Leave Root ShootD

PPH

500

(microg

ml)

(b)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

10

20

30

40

Leave Root Shoot

SRSA

100

(microg

ml)

(c)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

20

40

60

Leave Root Shoot

SRSA

500

(microg

ml)

(d)



Pb (t)

minus10

00

10

20

minus10

12

minus10

00

10

20

minus10

12

3

0 1 2 3

minus10 05 20

Cd (s)

046

Zn (t)

minus1 1 3

minus1 0 1 2

076

043

Zn (s)

086

041

As (t)

minus05 10 25

minus10 05 20

032

DPPH 100

046

044

070

DPPH 500

minus25minus10 05

minus1 1 2 3

066

043

062

x

SRSA 100

01

23

070

minus10

12

34

033

038

minus05

05

15

25

067

minus25

minus10

00

10

078

minus1 1 2

minus10

12SRSA 500

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast











4 Conclusion


Data Availability




Acknowledgments













References
















































Pb

Cd

Cu

Zn

As

minus25

00

25

50

minus2 0 2 4 6PC1 (446)

PC2

(22

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(a)

Pb

Cd

Cu

Zn

As

minus50

minus25

00

25

minus2 0 2PC1 (381)

PC2

(21

9)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(b)

Species

Pb

Cd

Cu

Zn

As

minus2

0

2

4

minus25 00 25 50PC1 (509)

PC2

(28

3)

C parasiticus

D linearis

P calomelanos

P vittata

(c)

Pb

Cd

Cu

Zn

As

minus25

00

25

minus2 0 2 4PC1 (56)

PC2

(23

)

Species

C parasiticus

D linearis

P calomelanos

P vittata

(d)



Species

C parasiticus

D linearis

P calomelanos

P vittata

25

50

75

Leave Root Shoot

DPP

H 1

00 (micro

gm

l)

(a)

Species

C parasiticus

D linearis

P calomelanos

P vittata

40

60

80

Leave Root ShootD

PPH

500

(microg

ml)

(b)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

10

20

30

40

Leave Root Shoot

SRSA

100

(microg

ml)

(c)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

20

40

60

Leave Root Shoot

SRSA

500

(microg

ml)

(d)



Pb (t)

minus10

00

10

20

minus10

12

minus10

00

10

20

minus10

12

3

0 1 2 3

minus10 05 20

Cd (s)

046

Zn (t)

minus1 1 3

minus1 0 1 2

076

043

Zn (s)

086

041

As (t)

minus05 10 25

minus10 05 20

032

DPPH 100

046

044

070

DPPH 500

minus25minus10 05

minus1 1 2 3

066

043

062

x

SRSA 100

01

23

070

minus10

12

34

033

038

minus05

05

15

25

067

minus25

minus10

00

10

078

minus1 1 2

minus10

12SRSA 500

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast











4 Conclusion


Data Availability




Acknowledgments













References
















































Species

C parasiticus

D linearis

P calomelanos

P vittata

25

50

75

Leave Root Shoot

DPP

H 1

00 (micro

gm

l)

(a)

Species

C parasiticus

D linearis

P calomelanos

P vittata

40

60

80

Leave Root ShootD

PPH

500

(microg

ml)

(b)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

10

20

30

40

Leave Root Shoot

SRSA

100

(microg

ml)

(c)

Species

C parasiticus

D linearis

P calomelanos

P vittata

0

20

40

60

Leave Root Shoot

SRSA

500

(microg

ml)

(d)



Pb (t)

minus10

00

10

20

minus10

12

minus10

00

10

20

minus10

12

3

0 1 2 3

minus10 05 20

Cd (s)

046

Zn (t)

minus1 1 3

minus1 0 1 2

076

043

Zn (s)

086

041

As (t)

minus05 10 25

minus10 05 20

032

DPPH 100

046

044

070

DPPH 500

minus25minus10 05

minus1 1 2 3

066

043

062

x

SRSA 100

01

23

070

minus10

12

34

033

038

minus05

05

15

25

067

minus25

minus10

00

10

078

minus1 1 2

minus10

12SRSA 500

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast











4 Conclusion


Data Availability




Acknowledgments













References
















































Pb (t)

minus10

00

10

20

minus10

12

minus10

00

10

20

minus10

12

3

0 1 2 3

minus10 05 20

Cd (s)

046

Zn (t)

minus1 1 3

minus1 0 1 2

076

043

Zn (s)

086

041

As (t)

minus05 10 25

minus10 05 20

032

DPPH 100

046

044

070

DPPH 500

minus25minus10 05

minus1 1 2 3

066

043

062

x

SRSA 100

01

23

070

minus10

12

34

033

038

minus05

05

15

25

067

minus25

minus10

00

10

078

minus1 1 2

minus10

12SRSA 500

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast











4 Conclusion


Data Availability




Acknowledgments













References


















































4 Conclusion


Data Availability




Acknowledgments













References



















































References
















































the interactions among the heavy metals in soils and in

Documents