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Aromatic plant production on metal contaminated soils

Valtcho D. Zheljazkova,⁎, Lyle E. Crakerb, Baoshan Xingb, Niels E. Nielsenc, Andrew Wilcoxd

aMississippi State, Department of Plant and Soil Sciences and North Mississippi Research and Extension Center, 5421 Highway 145 South,Verona, MS 38879, USAbDepartment of Plant and Soil Sciences, 12 Stockbridge Hall, University of Massachusetts, Amherst, MA 01003, USAcPlant Nutrition and Soil Fertility Lab, Department of Agricultural Sciences, The Royal Veterinary and Agricultural University,Thorvaldsensvej 40, DK1871, Copenhagen, DenmarkdHarper Adams University College, Newport, Shropshire, TF10 8NB, UK

A R T I C L E I N F O

⁎ Corresponding author. Tel.: +1 662 566 2201E-mail address: vj40@pss.msstate.edu (V.DAbbreviations: BF, bioavailability factor, the

CARB, carbonates bound fraction; EDTA, ethfraction of specific element in soil; FeMnOxscanning electron microscope.

0048-9697/$ – see front matter © 2008 Elsevidoi:10.1016/j.scitotenv.2008.01.041

A B S T R A C T

Article history:Received 4 September 2007Received in revised form17 January 2008Accepted 17 January 2008Available online 19 March 2008

Field and container experiments were conducted to assess the feasibility of growingaromatic crops in metal contaminated areas and the effect of metals on herbage and oilproductivity. The field experiments were conducted in the vicinities of the Non-FerrousMetals Combine (Zn–Cu smelter) near Plovdiv, Bulgaria using coriander, sage, dill, basil,hyssop, lemon balm, and chamomile grown at various distances from the smelter. Herbageessential oil yields of basil, chamomile, dill, and sage were reduced when they were growncloser to the smelter. Metal removal from the site with the harvestable plant parts was ashigh as 180 g ha−1 for Cd, 660 g ha−1 for Pb, 180 g ha−1 for Cu, 350 g ha−1 for Mn, and 205 g ha−1

for Zn. Sequential extraction of soil demonstrated that metal fractionation was affected bythe distance to the smelter. With decreasing distance to the smelter, the transfer factor (TF)for Cu and Zn decreased but increased for Cd, while the bioavailability factor (BF) for Cd, Pb,Cu, Mn, and Zn decreased. Scanning electron microscopy and X-ray microanalyses ofcontaminated soil verified that most of the Pb, Cd, Mn, Cu, and Zn were in the form of small(b1 μm) particles, although there were larger particles (1–5 μm) with high concentrations ofindividual metals. This study demonstrated that high concentrations of heavymetals in soilor growthmedium did not result inmetal transfer into the essential oil. Of the testedmetals,only Cu at high concentrations may reduce oil content. Our results demonstrated thataromatic crops may not have significant phytoremediation potential, but growth of thesecrops in metal contaminated agricultural soils is a feasible alternative. Aromatic crops canprovide economic return and metal-free final product, the essential oil.

© 2008 Elsevier B.V. All rights reserved.

Keywords:

Aromatic cropsEssential oilMetal fractionationTrace elements

1. Introduction

Contamination of agricultural soils with toxic heavymetals is amajor environmental problem that can affect both plant pro-ductivity and safety as food and feed crops (Alloway, 1990;

; fax: +1 662 566 2257.. Zheljazkov).ratio of themetal concen

ylenediaminetetraacetate, iron and manganese bo

er B.V. All rights reserved

Kabata-Pendias, 2001; McGrath et al., 2002). Indeed, the pre-dicted increase in heavy metal contamination of soils over thenext 30 to 40 years (McGrath et al., 2002) will represent anenvironmental stress that limits land use. Cadmium (Cd), lead(Pb), copper (Cu), and zinc (Zn) occur as major heavy metal

tration in the EXCH fraction to the totalmetal concentration in soil;; EDX, Energy Dispersive X-ray spectrometer; EXCH, exchangeableund fraction of metals; OM, organic matter-bound metals; SEM,

.

52 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 3 9 5 ( 2 0 0 8 ) 5 1 – 6 2

contaminants of agricultural soils (Alloway, 1990; Kabata-Pendias, 2001) andmay exert toxic effects on plants at elevatedconcentrations (Kabata-Pendias, 2001). For instance, in Bulgarialarge areas of agricultural soils in the vicinity of the Non-Ferrous Metals Combines near Plovdiv, Kirdzalli, Pirdop, andKremikovci, are contaminatedwith heavymetals, resulting in aserious environmental problem (Sengalevitch, 1993). While thesoils are fertile, farming in these areas produces crops andanimals contaminated with Cd, Pb, Cu, Zn, and Mn, makingthem generally unfit for human or animal consumption.

Cleansing the soil of heavymetals via conventional meth-odologies is expensive and in most cases not feasible(Cunningham and Ow, 1996). Phytoremediation (and morespecifically phytoextraction) is a low-cost alternative to thetraditional remediation technologies, such as soil excavationand other metal extraction technologies (Salt et al., 1998;Blaylock and Huang, 2000; Schmidt, 2003). Ideally, plantsused for phytoremediation should extract heavy metals fromsoils and provide a return on investment, yet not contam-inate the food or feed. Aromatic crops, used for production ofessential oils as opposed to food or feed, may be suitable alter-native crops in heavy metal contaminated agricultural soils.Some aromatic plants appear capable of accumulating heavymetals from contaminated soil (Zheljazkov and Nielsen, 1996;Zheljazkov and Warman, 2003), suggesting the possibility thatsuchplants could be used inphytoremediationof contaminatedsoils. The effects of these metals on growth, essential oil pro-duction, andmetal accumulation of most commercially impor-tant essential oil producing aromatic crops, such as coriander(Coriandrum sativum L.), dill (Anethum graveolens L.), chamomile(Chamomilla recutita (L.) K.), peppermint (Mentha x piperita L.), basil(Ocimum basilicum L.), hyssop (Hyssopus officinalis L.), lemon balm(Melissa officinalis L.), and sage (Salvia officinalis L.) are, however,largelyunknown.Coriander, dill, chamomile, peppermint, basil,hyssop, lemonbalm, andsagearearomatic crops thathavebeentraditionally grown as cash crops in Europe (Topalov, 1962), US,and recently in Canada. Essential oils are low-volume high-value products that are widely used as aromatic agents invarious non-food industries, such as perfumery, cosmetics, andaromatherapy. The objectives of this study were to evaluatethe ability of the selected aromatic crops to grow, produceessential oil, and accumulate Cd, Pb, Cu, and Zn in an en-vironment heavily polluted with heavy metals.

2. Materials and methods

2.1. Plant and growth conditions

2.1.1. Field experimentsCoriander ‘Alekseevski’, sage ‘Dessislava’, basil ‘Mesten’ and‘Trakia’, dill ‘Mesten’, chamomile ‘Lazur’, hyssop ‘Local’, andlemon balm ‘Melissa 2’ were used in a field study. The plants,seeded from 1991 to 1994, in the vicinity of the Zn–Cu smelternear Plovdiv, Bulgaria (situated close to the northeasternfoot of the Rhodopa Mountains in the European continentalclimatic zone) were grown in 15m2 experimental plots at threesites situated at 0.8, 3, and 9 km (control) from the smelterin the direction of the prevailing wind using a randomizedcomplete block design with three replicates. The soil was

alluvial and alluvial-meadow, pH 7.1–7.2, with a humus Ahorizon layer 25–28 cm deep, organic matter of 4.2%, a claycontent of 49–52%, sand content of 37–42% and silt contentof 10–11% and CEC of 35–38.8 meq/100 g. Annual rainfallamounts were 600–650 mm.

The plants were grown using accepted agricultural practicesfor the respective crops (Topalov, 1962). Briefly, in late summer120 kg P2O5 ha−1 and 100 kgK2Oha−1were applied to the growingarea in accordance with soil test results. The land was subse-quently plowed, disked, and fertilized with N at 80 kg ha−1 justbefore the spring seeding. Coriander and dill were seeded in thefirst week of April at 15 kg seed ha−1 (3–4 cm deep) and 10 kgseed ha−1 (2–3 cm deep), respectively. Chamomile was seededafter disking in the fall at 2.5 kg seed ha−1 (0–1 cm deep). Basiland lemon balm seedlings, produced in a greenhouse, weretransplanted to the field on June 1 (in 1992 and in 1993 for basil,and in 1992 for lemon balm). Garden sage and hyssop seedlingswere started in a nursery and transplanted to the field inSeptember, 1991. All plants were side-dressed with N at 60 kgha−1 in the last week of June. Heavy metal concentrations weredetermined using soil samples (0–20 cm deep) from theexperimental plots taken just before planting. Coriander washarvested when seeds reached technical maturity, whereas allother plant species were harvested at the beginning of flower-ing (the last week of August) (Topalov, 1962). The dry weights ofcoriander seed, hyssop inflorescences, and plant shoots of otherspecies were recorded.

2.1.2. Soilless container experimentPeppermint, basil, and sage were used in the container study;the selection was based on their performance in prior fieldexperiments and their economic value. The peppermint wasgrown from rhizome cuttings and basil and sage were startedfrom seeds. The plants were grown and maintained (3 plants/container) in plastic containers (20 cm deep) filled with perlite.After seeding, all containers were supplied with nutrients bywatering twice a week with 50% strength Hoagland's solution.The containers with plants were distributed randomly on thegreenhouse benches with a subsequent weekly rotation inplacement.

The concentrations of Cd, Pb, and Cu used to treat plants inthe container study were chosen so that the lowest concen-tration represented the lowest total critical concentrations forthe respectivemetal in soil that induces phytotoxicity or a 10%yield reduction (Kabata-Pendias, 1992), and so that the twohigher concentrations represented the upper limit and 150 to500% of the critical concentrations for the respective metal insoil. Hence, the metal treatments consisted of Cd at 2, 6, and10 mg L−1, Pb at 50, 100, and 500 mg L−1, and Cu at 20, 60, and150 mg L−1. Metals were added to the plant root environmentby watering the plants with chloride salt solutions (correctedto neutral pH) of eachmetal once aweek at a rate of 100mL percontainer. Since the chloride salts were corrected for pH, weassumed no major effect of Cl on plant growth and on themeasured responses. Controls (plants not treated with heavymetals) werewateredwithout the heavymetal additions. Basiland peppermint were harvested at 50% flowering to ensurehigh content and quality of the essential oil. Sage was har-vested after 14 weeks when the plants had substantial veg-etative growth. Half of the aboveground shoots were dried and

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distilled for essential oil extraction. The remaining half of thevegetative tissue was separated into stem and leaf tissue,dried to a constant weight in a forced air oven at 35 °C, andweighed.

2.2. Essential oils

Of the harvested plant materials from the field and containerexperiments, the sub-sample (one-half of seed, inflores-cences, or plant shoots) used for oil extraction and analysiswas dried in a shaded, well-aerated location (22 °C) to preserveessential oil content (Topalov, 1962). The essential oils from allcrops were extracted by steam distillation using a Clevenger-type apparatus.

2.3. Elemental analyses

The remaining half of the plant material (not used in essentialoil analysis) and the collected soil and perlite samples wereused to determine, respectively, the heavy metal concentra-tions in the plant tissue and in the growth environment. Theplant tissues used for metal analysis were dried in an oven at70 °C for 72 h, weighed to determine dry weight, and sub-sequently ground and sieved through a 1.0 mm screen. Foranalyses, the 1 g dry samples of tissue, perlite, and soil weredigested in concentrated HNO3 (Zheljazkov and Nielsen, 1996),filtered, and made to final volume of 25 mL. It is understoodthat the HNO3 digestion of soil samples represent HNO3-extractable or pseudo-total and not the total concentration ofelements in soil, because concentrated HNO3 cannot comple-tely dissolve all soil matrices (Alloway, 1990). Portions (2.5 mL)of the essential oil samples were dry ashed by carbonizationusing a hot plate and heated in a muffle furnace at 400 °C for4 h. After cooling, 2 mL of HNO3 were added to the residue ofeach sample and themixturewas placed in a heated sand bathfor acid evaporation followed heating in a muffle furnace at400 °C to produce a colorless acid solution that was brought tovolume with distilled water and 2 mL of HCl.

Fractionation of the heavymetals in soil samples was doneusing the sequential extraction procedure of Tessier et al.(1979), as modified by Luo and Christie (1998), and described inZheljazkov and Warman (2003) on 2 g of air-dried, sieved soilin 50-mL polypropylene centrifuge tubes. Sequential extrac-tions and fractionation of heavy metals in soils have beenused to predict metal mobility in soil and their phytoavail-ability to plants (Alloway, 1990; Kabata-Pendias and Pendias,2001; Tessier et al., 1979). The chemical fractions wereoperationally defined as: (1) exchangeable (EXCH), (2) carbo-nate bound fraction (CARB), (3) iron and manganese boundfraction (FeMnOx), (4) organic bound metals (OM), and (5)residual. The chemical fractions were extracted as follows:

1. Exchangeable (EXCH): 16 mL of 1 M Mg(NO3)2 at pH 7.0 wasadded to each sample. The samples were shaken for 1 h,centrifuged at 3000 rpm for 10min, the supernatant filteredthroughNo. 40Whatman filter paper, and the weight of thesample and the tube was recorded.

2. Carbonate bound fraction (CARB): 16 mL of 1 M CH3-

COOHNa adjusted to pH 5.0 with CH3COOH was added toeach residue from step 1. Then, the samples were shaken

for 5 h at room temperature, centrifuged and filtered asduring step 1; afterwards the weight was recorded.

3. Metals from reducible iron and manganese bound fraction(FeMnOx): 30mL of 0.04MNH2OHHCl in 25% (v/v) CH3COOHwere added to sample residues from step 2. The tubes withscrewed caps were placed for 5 h on a water bath at 96 °Cand agitated occasionally. Then the tubes were centri-fuged, filtered, and weighed as indicated above.

4. Organic matter-bound metals (OM): 6 mL of 0.02 M HNO3

and 4 mL of 30% H2O2 adjusted to pH 2.0 with HNO3 wereadded to the sample residues from the previous step. Thenon-capped tubes with samples were placed on water bathat 85 °C for 45 min. After that, 4 mL of 30% H2O2 adjusted topH 2with HNO3were added to the samples, whichwere puton awater bath at 85 °C for a further 45min. This procedurewas repeated three more times for a total period of 5 h.After cooling, 8 mL of 3.2 M CH3COONH4 in 20% (v/v) HNO3

were added and the samples were diluted with deionizedwater to 30 mL. The samples were shaken for additional30 min, centrifuged, and filtered as indicated above.

Heavy metals remaining in soils after extraction for organicmatter-bound fractions were treated as “residual” and theirconcentrations were calculated by subtracting the EXCH, CARB,FeMnOx, and OM fractions from the totalmetal concentrations.Heavy metals in tissue and soil digested samples from the fieldexperiment were measured on a Perkin-Elmer (Perkin-Elmer,Norwalk, CT) Model 5000 atomic absorption spectrophotometer(AAS), whereas metals in the essential oil samples were mea-sured on a Perkin-Elmer Graphite Furnace atomic absorptionspectrometer 5100 (GFAAS) (Zheljazkov and Nielsen, 1996).Elemental concentrations of plant tissues, growth media, oilsamples, and the water and plant residues from distillation ofcontainer plant material were determined using a ThermoJarrell Ash (Franklin, MA), Inductively Coupled Argon Plasma(ICAP) Spectrometer. Samples from the fractionation wereanalyzed on a Varian (Palo Alto, CA) Spectra AA-20 AAS. Toavoid possible interferences of matrices, for each of theextraction steps as well for the nitric acid digestions, separatestandards were prepared using the same matrices as thesamples from a particular step. Blanks were included with thedigestion of every batchof 20 samples andwith the extractionofeach soil fraction. Quality control was ensured by the inclusionof a barley flour standard, a Certified ReferenceMaterial AR2027(Alpha Resources Inc., Stevensville, MI) and a certified referencesoil SS-2 (SCP Science, Champlain, NY) sample, and calculatingthe percent recoveries of the five elements relative to the cer-tified standard values. The relative elemental concentrationsrecovered from the Standard ReferenceMaterial SS-2 (contami-nated soil) using the HNO3 digestion procedure (in % relative tothe elemental concentrations as providedwith the certificate ofanalyses) were as follows: 89% for Cd, 103% for Pb, 108% for Cu,93% for Mn, and 96% for Zn. The relative elemental recoveriesfrom barley flour Standard ReferenceMaterial AR2027were 92%for Cu, 88% for Mn, and 91% for Zn.

2.4. Electron microscopy and X-ray microanalyses

The metal contamination of soil was assessed and the spatialdistribution visualized using a Hitachi (Tokyo, Japan) 3000N

Table 1 –Mean values of plant responses (fresh herbage yield, essential oil yields, tissue Cd, Pb, Cu, Mn, and Zn content and uptake) depending on proximity to the source ofpollution

SV Herbage yield Essential oil yield Cdcontent††

Cduptake†††

Pbcontent

Pbuptake

Cucontent

Cuuptake

Mncontent

Mnuptake

Zncontent

Znuptake

T ha−1 kg ha−1

Proximity 0.8 kmSage 13.5b† 171.1b 13.9a 48.9aA† 182.9a 645.2aA 36.1a 128.0aA 94.6a 331.4aA 202.4a 709.4aABasil ‘Trakia’ 17.0b 72.6b 8.4a 28.4aC 154.5a 525.9aB 26.2a 88.8aB 88.2a 300.2aA 186.7a 635.2aABasil ‘Mesten’ 16.8b 68.6b 8.4a 28aC 155.3a 520.4aB 25.8a 86.3aB 88.3a 296.0aA 191.8a 641.6aADill 9.7b 43.8b 9.1a 26.5aC 149.6a 438.7aC 24.1a 70.5aB 77.1a 226.4aB 180.2a 528.7aBCham 15.9b 44.5b 11.9a 38.1aBC 149.6a 476.1aBC 26.5a 84.4aB 91.9a 292.5aA 197.0a 627.2cACoriander†††† 0.83b 3.8b 8.9a 5.2aD 143.8a 85.4aDE 26.2a 15.6aD 79.5a 47.2aC 145.0a 86.1aCLem balm 18.8b 3.9b 10.2a 40.3aB 126.7a 50.1bE 21.1a 83.3bB 81.5a 321.7aA 176.3a 696.1aAHyssop†††† 3.7b 30.1b 12.6a 11.2aD 175.2a 156aD 30.5a 27.1aC 72.8a 65.0aC 175.8a 157.0aC

Prox 3 kmSage 17.2a 220.1a 4.2b 18.9bA 57.4b 260.5bA 24.9b 114.2aA 77.7ab 348.4aA 85.4b 383.8bABasil ‘Trakia’ 21.1a 90.7a 3.1b 12.9bBC 46.9b 197.8bAB 23.8a 101.0aAB 67.2ab 283.8aAB 73.1b 309.2bABasil ‘Mesten’ 19.4a 80.6ab 2.8b 10.9bC 46.5b 180.2bAB 23.5a 91.3aB 67.3ab 261.8aB 72.9b 283.8bADill 12.1a 54.5ab 2.8b 10.2bC 45.5b 165.7bB 20.7b 75.5aC 64.4ab 234.9aB 69.5b 254.1bACham 17.9ab 51.0ab 3.5b 12.6bBC 42.5b 152.0bB 23.6a 84.5aBC 75.2b 268.8aB 75.3b 269.3bACoriander 0.93ab 4.2ab 2.4b 1.6aD 39.4b 26.3bC 20.5a 13.7abE 61.5ab 41.0aC 77.2 51.5bBLem balm 23.5a 4.9a 2.8b 14.0bB 37.8b 186.5aAB 25.1a 123.8aA 59.8b 295.1aAB 65.7b 324.6bAHyssop 4.4a 34.9a 4.3b 4.6bD 48.2b 51.0bC 26.7a 28.3aD 55.8a 59.0aC 88.2b 93.3bB

Prox 9 kmSage 17.8a 227.9a 0.43c 2.0cA 6.1c 28.6cA 13.8c 65.8bA 62.4b 290.5aA 31.9c 151.4cABasil ‘Trakia’ 21.1a 92.7a 0.25c 1.1cAB 4.5c 19.8cA 13.4b 58.9bA 49.4b 216.3bB 32.6c 142.9cABasil ‘Mesten’ 20.7a 86.2a 0.24c 0.98cAB 4.5c 18.5cA 12.5b 51.9bA 49.8b 206.3bB 32.6c 133.8cADill 13.1a 58.7a 0.22c 0.91cAB 3.9c 15.9cAB 13.7c 54.6aA 44.3b 176.6bC 29.9c 119.4cACham 20.8a 57.1a 0.27c 1.13cAB 4.2c 17.5cAB 13.5b 54.6bA 44.4c 258.0aAB 31.1c 128.8cACoriander 0.96a 4.4a 0.23c 0.16bB 4.1c 0.29bD 11.5b 7.9bB 39.8b 27.4bD 33.9c 23.3cBLem balm 21.6a 4.7a 0.26c 1.2cAB 3.5c 15.8cAB 13.5b 61.2bA 35.1c 159.2bC 24.6c 112.0cAHyssop 4.3a 32.9ab 0.35c 0.26cB 6.3c 6.5cC 12.7b 13bB 32.2b 33.4bD 25.2c 25.9cB

†Means with the same letter are not significantly different at P≤0.05. Lower case letters represent significance between locations (distance) for a species or cultivar. Upper case letters representsignificance between crop species or cultivars within one location.††Concentration, in mg kg−1.†††Uptake, in g ha−1.††††Coriander yields are seed yields. Hyssop yields are yields from inflorescences.

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variable pressure scanning electronmicroscope (SEM) coupledto Oxford (Concord, MA) INCA 350 Energy Dispersive X-rayspectrometer (EDX). The SEM imaging used a backscatteredelectron (BSE) detector, a low vacuum (2–50 Pa) and a coolingstage to stabilize the samples and control charging. The EDXanalyses were done on unpolished samples at working dis-tance of 15 mm, and accelerating voltages of 10–30 kV.

2.5. Statistical analyses

Data collected from field experiments were analyzed usingrepeated measures analyses (proximity to the source of thepollution was repeated) (proc mixed option in SAS (SAS, 2000).The model was: Yijk=µ+βj+τ+τβj+γk+γτ+βγjk+τβγijk, where βj =whole plot (proximity to the smelter), τ = block effect (year), τβj =interaction,γk= subplot effect (species), year—blocking factor (2levels), proximity (location) — 3 levels (0.8 km, 3 km and 10 kmfrom the smelter), species/varieties (8 levels). Differencesamong metal treatments and plant responses in the containerexperiment were tested for significance using analysis of var-iance (ANOVA) and the means were separated using Duncan'sNew Multiple Range Test (P≤0.05).

3. Results

3.1. Field experiments

Fresh yields of all 8 crops and varieties at 0.8 km from thesmelter were reduced compared with the respective yields at9 km from the smelter (the control) (Table 1). Yields of sage,basil, dill, lemon balm, and hyssop at 3 km from the smelterwere higher than respective yields at 0.8 km from the smelter.Chamomile and coriander yields at 3 km from the smelter

Table 2 – Fractionation of Cu, Cd, Zn, Zn, Pb, andMn and in the sHNO3 digestion, bioavailability factor (BF) and transfer factor (T

Metal Distancefrom thesmelter(km)

EXCH CARB FeMn

mg k

Cu 9 (control) 1.6b† 1.8c 0.9c3 2.2ab 8.8b 11.6b0.8 2.6a 49.2a 36.5a

Cd 9 (control) 3.0c 4.6c ud†††

3 3.5b 8.5b 3.2b0.8 5.4a 18.5a 6.9a

Zn 9 (control) 2.3c 3.2c 9.3c3 3.7ab 29.1b 83.5b0.8 6.5a 246.4a 692.1a

Pb 9 (control) 14.4b 19.1c 9.3c3 27.7a 115.9b 60.4b0.8 29.1a 892.3a 360.7a

Mn 9 (control) 0.4c 3.4b 20.1b3 0.5ab 3.8b 107.5a0.8 0.7a 5.6a 96.2a

†Separation of means. Data are the means of four replicates. Means withsignificantly different at P≤0.05.††TF is different for different crops. The indicated range includes TF for a†††ud — below the detection limit, undetected amount.

were not different from yields either at 0.8 or 9 km from thesmelter (control). Despite the yield reduction, no visible phy-totoxicity symptoms were observed on any of the crops.

Heavy metal uptake by the crop is of a great practicalinterest, since it allows the estimation of the phytoremedia-tion potential of these plants. In Table 1, heavy metal uptakewas calculated from metal concentrations in the herbage andthe yield of dry herbage (please note that yields in Table 1 arefresh herbage yields). Generally, heavy metal concentration inthe experimental plants reflected the metal concentrationin the soils (Tables 1 and 2). Overall, the highest heavy metalconcentrations weremeasured in plants grown at 0.8 km fromthe smelter, lower in plants grown at 3 km from the smelter,and lowest in the control plants at 9 km from the smelter. At0.8 km from the smelter, the highest tissue Cd concentrationswere found in sage and the lowest Cd concentrations were inthe two varieties of basil, coriander, and dill. Overall, most Cdwas removed with sage, lemon balm, and chamomile, lesswith basil and dill, and least with coriander and hyssop.Generally, Pb concentrations of sage and hyssop plants werehigher than in other plants. Removal of Pb from the site withthe harvestable biomass was highest with sage plants, lowerwith basil, dill, and chamomile, and lowest with coriander,lemon balm and hyssop. The seven plant species accumulatedsimilar amounts of Cu and Mn at any given distance from thesmelter. At 0.8 and 3 km from the smelter, more Cu wasremoved from site with the harvestable parts of sage, less withbasil, dill, chamomile and lemon balm, and least with hyssopand coriander. Plants grown at 9 km from the smelter removedsimilar amounts of Cu with the harvest. Also, due to thedifferences in harvestable yields between plants, more Mnwas removed from the sites with sage, basil, dill, chamomileand lemon balm, and much less with coriander or hyssop. At0.8 km from the smelter, lesser amounts of Zn were found in

oils and their pseudo-total concentrations in soils followingF)

Soil fractions

Ox OM HNO3 BF TF††

g−1 % %

2.9c 30.7c 5 0.37–0.4532.4b 115.6b 1.9 0.18–0.23122.3a 193.8a 1.3 0.1–0.18ud 4.3c 70 0.05–0.1ud 9.9b 35 0.24–0.44ud 25.1a 22 0.33–0.553.1c 18.3c 13 1.3–1.918.8b 113.8b 3.2 0.6–0.8208.5a 1092.2a 0.6 0.13–0.1810.6c 33.8c 54 0.1–0.1917.8b 220.3b 13 0.17–0.26133.7a 1365.2a 2 0.09–0.13161.8b 166.4b 0.24 0.19 – 0.38502.8a 658.1a 0.08 0.09 – 0.12511.2a 722.9a 0.09 0.1 – 0.13

the same letter in a column within a fraction and element are not

ll seven crops.

Fig. 1 –Backscattered electron (BSE) SEM and EDX analyses of contaminated soil taken at 0.8 km from the smelter. BSE image of arandom soil sample and X-ray dot mapping of Fe (B), Mn (C), Zn (D), Cu (E), and Pb (F).

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the harvestable coriander fraction as compared with Zn in theharvestable fraction of other species. Among the test sites,greater amounts of Zn were removed with sage, basil, dill,chamomile and lemon balm, and lesser amounts wereremoved with coriander or hyssop (Table 1).

The concentrations of Cd, Pb, Cu, and Zn in the soils at0.8 km were about 30 times higher than the respectiveconcentrations in the control soils (Table 2) and much abovewhat would be considered phytotoxic concentrations of these

elements in soils (Kabata-Pendias and Pendias, 1992). Forinstance, Pb and Zn concentrations in soil at 0.8 km wereapproximately 1000 mg kg−1. Soil metal concentrations at3 km from the smelter were relatively high, but for mostmetals, within the maximum permissible concentrations ofthe elements for agricultural soils (Kabata-Pendias, 2001). Noincreased concentrations of heavymetals were noted in soil at9 km from the smelter. Fractionation of the soil Cu, Cd, Zn, Pb,and Mn revealed that most of the Cu and Mn were in the OM

57S C I E N C E O F T H E T O T A L E N V I R O N M E N T 3 9 5 ( 2 0 0 8 ) 5 1 – 6 2

fraction, most of the Zn was in FeMnOx fraction, and most ofPb and Cd were in the CARB fraction (Table 2). The bioavail-ability factor (BF), the ratio of the metal concentration in theEXCH fraction to the total metal concentration in soil (Knoxet al., 2000), for Cd, Cu, Zn, Pb, and Mn decreased withdecreasing distance to the smelter. With decreasing distanceto the smelter, the transfer factor (TF), the ratio of the metalconcentration in plant tissue to the total concentration ofmetal in the soil (Knox et al., 2000), for Cu and Zn, decreasedbut the increased for Cd and was highest at 0.8 km from thesmelter.

SEM/EDX analyses of the soil sampled at 0.8 from thesmelter indicated that Pb, Cd, Mn, Cu and Zn were eitherconcentrated on certain particles (1–6 μm in diameter), orsmaller than 1 μm and randomly scattered throughout the

Fig. 2 –A. BSE detector SEM image of contaminated soil sampled afour bright particles (S2, S5, S8 and S9) that were analyzed with EDS8 (D), and S9 (E).

samplewithout forming any significant clusters (Figs. 1 and 2).This pattern suggested atmospheric deposition of the con-taminants was the source. Similar results were found byAdamo et al. (1996), who reported “bright particles” (heavymetal particles in SEM observations) in Sudbury soil receivingaerosol contaminants from a smelter for over 100 years. Someof the particles in our study had predominately Zn and Pb onthe surface (Fig. 2B), some others had predominately Fe and Pb(Fig. 2C and E), others had predominately Pb (Fig. 2D). The SEM/EDX results revealed that significant amounts of metals werein predominately metal particles, as there was a lack of strongsignals from plant nutrients such as P and K. These metalparticles could cause redistribution ofmetals in the sequentialextraction process as demonstrated previously (Dahlin et al.,2002).

t 0.8 km from the smelter atmagnification of 1000×, indicatingXmicroanalyzer. Spectrum of the bright particle S2 (B), S5 (C),

Fig. 2 (continued ).

58 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 3 9 5 ( 2 0 0 8 ) 5 1 – 6 2

3.2. Container experiments

Lead at 500 mg L−1 and Cu at 60 and 150 mg L−1 reducedpeppermint yields (Table 3) relative to the control not exposedto heavy metals. At 150 mg L−1 Cu, peppermint plants hadyellow leaves, stunted growth, and underdeveloped root

systems as compared with control plants. Vegetative yieldsof basil were significantly reduced by Cd at 6 and 10mg L−1, Pbat 500 mg L−1, and Cu at 20 and 60 mg L−1 as compared withcontrols. Cu at 60 and 150 mg L−1 reduced plant weight andessential oil content (Tables 3 and 5). Sage biomass yields werereduced by the applications of Cd, Pb, and Cu at the two

Fig. 2 (continued ).

59S C I E N C E O F T H E T O T A L E N V I R O N M E N T 3 9 5 ( 2 0 0 8 ) 5 1 – 6 2

highest rates (Table 3). Overall, plants subjected to 150 mg CuL−1 had foliar chlorosis, underdeveloped (smaller volume) rootsystems and reduced yields. In the above ground plant parts,Cd accumulations in peppermint were primarily in the leaves,while the Cd accumulations in basil and sage were similar forleaves and stems. The concentrations of Pb in plant shootswere relatively low as compared with the Pb concentrations ofthe roots (Table 4). The addition of Cu to the growth mediumdramatically increased the accumulations of Cu in the rootsof the three tested species (Table 4), especially in basil roots.

Table 3 – The effects of Cd, Pb, and Cu on plant growth

Heavy metaltreatment

Peppermint Basil Sage

Mg L−1 Biomass† yields, g dry weight

Cd 2 147a†† 111a 126ab6 138a 80b 109b

10 114ab 48c 104bPb 50 135a 105a 110b

100 122ab 102ab 128ab500 91b 20d 98b

Cu 20 124a 77b 151a60 86b 34cd 138ab

150 44c 5e 49cControl††† 130a 113a 146a

†Biomass represents all above ground parts of the harvest plant.††Means of 4 replicates. Mean separation by Duncan's Multiple Rangetest. Numbers with the same letter within a treatment and specieshaving the same letter are not significantly different, P=0.05.†††Controls were not treated with heavy metals.

Higher concentrations of Cu in the growth medium alsoresulted in some increases of Cu in the plant foliage, mostnotably in basil. The concentrations of Cd, Pb, and Cu in theplant residual (steam distilled) material after distillation weresimilar to the respective concentrations of the element inplant tissues before the distillation. Cadmium in the distil-lation water was below the detection limit of 0.06 mg L−1,while Pb and Cu in the distillation water were not differentfrom theconcentrationsof these elements in the control. Trans-fer factor coefficients (TF) were 2 to 14% for Cd, 0.5 to 53% forPb, and 0.5 to 14% for Cu, depending on the treatment andspecies.

Essential oil contents of the three crops also showed somevariation (Table 5). For both peppermint and basil, only thehighest Cu treatment (150 mg L−1) decreased oil contentsrelative to the control. Cadmium, Cu, and Pb in the essentialoils of basil, sage, and peppermint from all treatments werebelow the detection limit of ICAP-AES (0.005 mg L−1 for Cd,0.02 mg L−1 for Cu, and 0.05 mg L−1 for Pb). Hence, the actualconcentrations of Cd, Cu, and Pb in the oils were below 0.06mgL−1 Cd, 0.25 mg L−1 Cu, and 0.63 mg L−1 Pb.

4. Discussion

4.1. Field experiments

The total soil concentrations of Cd, Pb, Cu, Mn and Zn atdifferent distances from the smelter reflect the pattern andnature of the aerosol pollution around the smelter. Researchhas indicated that a number of crops around this particular

Table 4 – The effects of heavy metal treatments on Cd, Pb, and Cu accumulation in plant tissues

Heavymetaltreatment†

Peppermint Basil Sage

Roots Stems Leaves Waste†† MT Roots Stems Leaves Waste†† MT Roots Stems Leaves Waste†† MT††††

mg L−1 mg metal kg−1 dry weight % mg metal kg−1 dry weight % mg metal kg−1 dry weight %

mg Cd kg−1

Cd2 45.0c††† 1.1b 4.2c 6.3c 14 277.0c 21.4c 13.6b 7.4b 3 134.0c 5.0c 6.0c 5.2c 46 199.0b 3.4a 16.0b 18.8b 9 868.0b 129.0b 20.5b 31.3a 4 238.0b 18.5b 22.8b 12.3b 510 537.0a 4.2a 21.9a 24.7a 5 1620.0a 201.0a 42.0a 37.9a 2 537.0a 46.5a 40.2a 31.2a 60 0.9e bd bd bd – 0.4e 0.9d 0.3c 0.8c – 0.7d 0.7d 0.7d 0.4d –

Pb50 224.7c 28.5b 108.0c 120.5c 53 3068.0c 59.0b 65.3b 105.7b 3 270.6c 58.3b 61.5b 86.4b 32100 1369.0b 29.0b 147.0b 177.0b 13 5515.0b 88.7b 86.0b 85.1b 2 997.0.b 81.1b 75.3a 143.3a 14500 8465.0a 81.3a 278.0a 447.8.a 5 31558.0a 248.0a 123.2a 140.1a 0.5 2902.1a 95.2a 109.4a 136.2a 50 13.4d 8.8c 7.2d 20.3d – 38.4f 18.1c 19.1c 23.6c – 26.7d 28.0c 22.6c 20.1c –

Cu20 655.6c 22.3b 21.9b 36.8b 6 931.0c 33.9c 25.8b 48.7b 5 392.7c – 55.9b 56.2a 1460 1242.2b 37.2a 25.6ab 47.1a 4 3507.9b 62.0b 42.3ab 48.6ab 1 511.5b – 53.4b 62.0a 12150 5047.a 36.5a 31.8a 55.3a 1 11478.8a 84.7a 63.4a 58.2a 0.5 764.6a – 65.8a 65.2a 90 30.3d 15.1c 9.0c 11.0c – 29.4e 34.6c 15.1c 19.5c – 28.4d – 43.6bc 27.7b –

†Indicated heavy metal treatments were applied to plants growing in perlite filled pots.††Wastes are the plant residues after extraction of the essential oil.†††Means of 4 replicates, mean separation by Duncan's Multiple Range test, numbers followed by the same letter within a species and a plant partare not significantly different, P≤0.05.††††MT — metal transfer from roots to shoots in %.

60 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 3 9 5 ( 2 0 0 8 ) 5 1 – 6 2

smelter accumulated excessive amounts of heavy metals(Sengalevitch, 1993). Several earlier reports (Kabata-Pendiasand Pendias, 1992; Zheljazkov and Nielsen, 1996) have dem-onstrated that high soil concentrations of Cd, Pb, and Cuwould reduce the yields of a variety of crop plants. In thisstudy, although the yields from crops at 0.8 km from thesmelter were reduced, they were still within the typical yieldsfor these crops in the region (Topalov, 1962). The above sug-gested that the tested aromatic crops could remain profitableeven at 0.8 km from the smelter.

Although the concentrations of Cd, Pb, Cu and Zn in soil at0.8 km were above the critical concentrations in soil for theseelements (Kabata-Pendias and Pendias, 1992), no visual phy-totoxicity symptoms on crops were observed. However, ourelectron microscopy observations of thin sections indicatedalterations in chloroplasts thylakoids in plants grown at0.8 km from the smelter (data not shown). Such sub-cellularalterations in morphology may be an indication of the toxiceffects of heavy metals on transport of solutes across theplasma membrane, and perhaps photosynthesis, and thusresponsible formeasured yield reductions. These results are in

Table 5 – Essential oil content (% in dry leaves) in the three plan

Treatments (mg L−1 in

Species Control Cd 2 Cd 6 Cd 10 Pb 5

Peppermint 2.5a 2.3a 2.4a 2.1ab 2.6aBasil 1.6a 1.6a 1.6a 1.4a 1.3aSage 2.1a 2.2a 2.6a 2.0a 2.3a

Means with the same letter within a crop (a row) are not significantly dif

agreementwith the report of Pietrini et al. (2003), who reported30% loss of chlorophyll and swelling of thylakoid membranesin Phragmites australiswhen exposed to high concentrations ofCd in solution.

Our results suggested that fractionation of Cu, Cd, Zn, Pband Mn in soil was affected by the distance from the smelter.Results from the metal fractionation study supported the ideathat Cu has a high affinity for organic compounds (Wu et al.,1999). Cadmium in the OM fraction was below the detectionlimit of AAS, supporting the notion for relatively low affinity ofCd for OM (Kabata-Pendias, 2001; Lim et al., 2002). The largestZn fraction in the contaminated and in control soils wasFeMnOx–Zn, also evident from other reports (Luo and Christie,1998; Shuman, 1999). Evidence exists that Zn has relativelyhigh affinity for sorption on the surfaces of Fe and Mn oxides,especially with an increase of soil pH (Luo and Christie, 1998;Kabala and Singh, 2001). Our results also supported the gen-eral understanding of very low mobility of Pb in soil and thehigh affinity of Pb to Mn oxides, Fe and Al hydroxides, clayminerals, and organicmatter (Kabata-Pendias, 2001). Lim et al.(2002), using a similar fractionation procedure, also observed

ts depending on the treatments

the applied solution)

0 Pb 100 Pb 500 Cu 20 Cu 60 Cu 150

2.3a 2.1a 2.7a 2.1ab 1.8b1.2a 1.2a 1.4a 1.1ab 0.9b2.3a 2.1a 2.2a 2.2a 2.0a

ferent at P≤0.05.

61S C I E N C E O F T H E T O T A L E N V I R O N M E N T 3 9 5 ( 2 0 0 8 ) 5 1 – 6 2

that the carbonate fraction contained the greatest amount ofPb in contaminated clay. Our results were in accordance withthe report of Kabala and Singh (2001) for small organicallybound fractions of Pb in highly contaminated soils.

The BF (Knox et al., 2000) showed that with decreasingdistance to the smelter, the proportion of EXCH forms of Cu,Cd, Zn, Pb, and Mn relative to the total amounts of thesemetals in soil decreased. The concentrations of these metalsin EXCH fractions, however, increased with decreasing dis-tance to the smelter (for Cd), or decreased in soil at 0.8 kmrelative to the control (for Cu, Pb, Zn, and Mn), indicating thatlarger amounts of metals in soil at 0.8 km were in easily ac-cessible forms to plants relative to the control. Despite differ-ences between the elements, overall, variation in the TF (Knoxet al., 2000) indicated increased metal availability with de-creased distance to the smelter.

4.2. Container experiment

Results from our container experiments demonstrated thatof the three heavy metals, only Cu (at elevated concentra-tions) caused a reduction in essential oil content and yields(in peppermint and basil). Cadmium concentrations of abovegroundbiomass of basil, peppermint, and sage in the containerexperiment did not reach the high concentrations character-istic for hyperaccumulators (Brown et al., 1995). Interestinglyhowever, tissue Cd in peppermint, basil and sage in our experi-ment was similar to Cd shoot concentrations in hydroponi-cally grown Brassica juncea (Indian mustard), B. rapa (turnip),B. napus (colza), Festuca rubra (red fescue), and Thlaspi caer-ulescens (Ebbs et al., 1997), which are considered significantmetal accumulators. Cadmium transfers from roots to shootsin peppermint, basil, and sage were similar to Cd transfer inmaize grown on two Cd contaminated UK soils (Lombi et al.,2001). Also, our results indicated that Cd and Pb accumulationsin peppermint, basil, and sage were higher than Cd and Pb inmaize shoots grownon contaminated soils, and comparable toCd and Pb in maize shoots after the addition of EDTA (Lombiet al., 2001).

Traditionally, peppermint, basil, and sage tissue wastesafter distillation are used as feed for sheep (Topalov, 1962).Results from our container experiment demonstrated that ifpeppermint, basil, and sage are grown in highly heavy metalcontaminated medium, Cd, Pb, and Cu may accumulate inshoots and wastes from distillation above the maximum per-missible concentrations for these elements in animal feed(NRC, 1985), making these waste products unsuitable as ani-mal feed. To reduce the amount of Cd, Pb, and Cu and produceusable final product, metal contaminated distillation wastescould be composted after mixing with wastes from other low-metal feedstock.

Overall, our results from field and container experimentssupported the general understanding that Cu and Pb accumu-late mainly in the roots. However, results from the containerstudy demonstrated that Cu and Pb may have increasedmobility within plants when grown in substrates with highconcentrations of thesemetals. Once themetals were taken up,Cu and Pb tended to accumulate in the roots, while Cd, Mn, andZnwere easily transported to the shoots (Alloway, 1990; Kabata-Pendias and Pendias, 1992; Zheljazkov and Warman, 2003).

Some studies have noted, however, that leaf tissue Cu equaledor exceeded root Cu concentration in mustard (Planquart et al.,1999). Our results indicated significant amounts of Pb and Cu inabove ground plant parts, accumulations that could be due tocontinuous aerosol emissions or soil particle entrapment onabove ground plant parts. Overall, the transfers of Pb and Cufrom roots to shoots were greatest at lower concentrations ofthese metals in the growth medium and declined with in-creasing concentrations of Pb and Cu. The accumulations of Cuinabove groundplantparts of peppermint, basil, and sage in thecontainer experiment were comparable and in some instanceshigher than Cu accumulation in Agrostis tenuis grown on Cucontaminated soil (Thayalakumaran et al., 2003). The transfersof Cd, Pb, and Cu from roots to shoots were species and con-centration dependent. The sequence of trace element concen-trations observed in different plant organs reflects specificity ofthese particular experimental varieties different from otherreports (Planquart et al., 1999), most likely due to genetic dif-ferences among species (Macnair, 1990).

Our results from field and container experiments indicatedthat coriander,dill, chamomile, peppermint, basil, hyssop, lemonbalm and sage were not metal hyperaccumulators (Brown et al.,1995), and did not have very high phytoremediation potential ascompared with other plants (Brown et al., 1994). Yet the use ofaromatic plants for phytoremediation may have an advantageover other crop plants in that the harvested foliage is a source ofessential oils, which are themarketable revenue-generating pro-ductsofaromaticcrops.Growingof thesearomatic crops inmetalcontaminated areas may not introduce heavy metals into thefood chain andmay not result in an economic penalty comparedto most other edible crops. In the process of oil extraction bydistillation, heavymetals remain in the extracted plant residues,limiting the quantities of heavy metals in the commercial oilproduct (Zheljazkov and Nielsen, 1996; Scora and Chang, 1997;Zheljazkov and Warman, 2003). Thus, significant amounts ofheavy metals could be removed from the soil through properdisposal of the metal contaminated plant residues, while themetal-free, extracted oils could be safely marketed. High-valuearomatic crops may well be a better alternative for heavy metalcontaminated agricultural soils than the suggested woodyspecies such as Salix and Betula (Hammer et al., 2003; Rosselliet al., 2003), or other plants like Sesbania drumondii that have beenshown to hyperaccumulate Pb (Sahi et al., 2002).

Disposal of the metal contaminated plant tissues followingphytoextraction remains a problem in the use of all plantmaterials. Several approaches such as composting, incinera-tion, ashing, pyrolysis, direct disposal and liquid extractionhave been proposed and/or tried (Sas-Nowosielska et al., 2003;Keller et al., 2005), but if not controlled can still pollute theenvironment. Some aromatic plants might demonstratesignificant phytoremediation potential if coupled to othermeans for increasing bioavailability and uptake of Cd, Pb, andCu, such as chelates (Schmidt, 2003) or biosurfactants(Mulligan et al., 2001).

Acknowledgements

The research in the vicinity of the Pb–Zn smelter near Plovdivwas funded by the Bulgarian Ministry of Education. Authors

62 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 3 9 5 ( 2 0 0 8 ) 5 1 – 6 2

thank the Cooperative Farm in Dolni Voden, Plovdiv region forproviding access to contaminated land and logistical support.The work at the Royal Veterinary and Agricultural Universityin Copenhagen, was supported by a visiting scholar researchgrant awarded to Dr. Zheljazkov by the Danish Academy ofSciences. Thework at the Nova Scotia Agricultural Collegewassupported by the Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) Discovery Grant and the CanadianFoundation for Innovation (CFI) Grant awarded to Dr. V.Zheljazkov (Jeliazkov). The research at the University of Mas-sachusetts was supported by a Fulbright grant awarded toDr. Zheljazkov for 12 months as a visiting scholar. This mate-rial is partially based upon work supported by the CooperativeState Research, Extension, Education Service, US Departmentof Agriculture, and Massachusetts Agricultural ExperimentStation under project No. 729. Publication No. 3259.

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