SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 95 ( 20 0 8 ) 5 1–6 2

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

Aromatic plant production on metal contaminated soils

Valtcho D. Zheljazkov a,⁎, Lyle E. Craker b , Baoshan Xing b , Niels E. Nielsenc , Andrew Wilcoxd
a
Mississippi State, Department of Plant and Soil Sciences and North Mississippi Research and Extension Center, 5421 Highway 145 South,
Verona, MS 38879, USA
b
Department of Plant and Soil Sciences, 12 Stockbridge Hall, University of Massachusetts, Amherst, MA 01003, USA
c
Plant Nutrition and Soil Fertility Lab, Department of Agricultural Sciences, The Royal Veterinary and Agricultural University,
Thorvaldsensvej 40, DK1871, Copenhagen, Denmark
d
Harper Adams University College, Newport, Shropshire, TF10 8NB, UK

AR TIC LE I N FO ABS TR ACT

Article history: Field and container experiments were conducted to assess the feasibility of growing
Received 4 September 2007 aromatic crops in metal contaminated areas and the effect of metals on herbage and oil
Received in revised form productivity. The field experiments were conducted in the vicinities of the Non-Ferrous
17 January 2008 Metals Combine (Zn–Cu smelter) near Plovdiv, Bulgaria using coriander, sage, dill, basil,
Accepted 17 January 2008 hyssop, lemon balm, and chamomile grown at various distances from the smelter. Herbage
Available online 19 March 2008 essential oil yields of basil, chamomile, dill, and sage were reduced when they were grown
closer to the smelter. Metal removal from the site with the harvestable plant parts was as
Keywords: high 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
Aromatic crops for Zn. Sequential extraction of soil demonstrated that metal fractionation was affected by
Essential oil the distance to the smelter. With decreasing distance to the smelter, the transfer factor (TF)
Metal fractionation for Cu and Zn decreased but increased for Cd, while the bioavailability factor (BF) for Cd, Pb,
Trace elements Cu, Mn, and Zn decreased. Scanning electron microscopy and X-ray microanalyses of
contaminated soil verified that most of the Pb, Cd, Mn, Cu, and Zn were in the form of small
(b 1 μm) particles, although there were larger particles (1–5 μm) with high concentrations of
individual metals. This study demonstrated that high concentrations of heavy metals in soil
or growth medium did not result in metal transfer into the essential oil. Of the tested metals,
only Cu at high concentrations may reduce oil content. Our results demonstrated that
aromatic crops may not have significant phytoremediation potential, but growth of these
crops in metal contaminated agricultural soils is a feasible alternative. Aromatic crops can
provide economic return and metal-free final product, the essential oil.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction Kabata-Pendias, 2001; McGrath et al., 2002). Indeed, the pre-

dicted increase in heavy metal contamination of soils over the
Contamination of agricultural soils with toxic heavy metals is a next 30 to 40 years (McGrath et al., 2002) will represent an
major environmental problem that can affect both plant pro- environmental stress that limits land use. Cadmium (Cd), lead
ductivity and safety as food and feed crops (Alloway, 1990; (Pb), copper (Cu), and zinc (Zn) occur as major heavy metal

⁎ Corresponding author. Tel.: +1 662 566 2201; fax: +1 662 566 2257.
E-mail address: vj40@pss.msstate.edu (V.D. Zheljazkov).
Abbreviations: BF, bioavailability factor, the ratio of the metal concentration in the EXCH fraction to the total metal concentration in soil;
CARB, carbonates bound fraction; EDTA, ethylenediaminetetraacetate; EDX, Energy Dispersive X-ray spectrometer; EXCH, exchangeable
fraction of specific element in soil; FeMnOx, iron and manganese bound fraction of metals; OM, organic matter-bound metals; SEM,
scanning electron microscope.

0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2008.01.041
52 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 95 ( 20 0 8 ) 5 1–6 2
contaminants of agricultural soils (Alloway, 1990; Kabata-
Pendias, 2001) and may exert toxic effects on plants at elevated
concentrations (Kabata-Pendias, 2001). For instance, in Bulgaria
large areas of agricultural soils in the vicinity of the Non-
Ferrous Metals Combines near Plovdiv, Kirdzalli, Pirdop, and
Kremikovci, are contaminated with heavy metals, resulting in a
serious environmental problem (Sengalevitch, 1993). While the
soils are fertile, farming in these areas produces crops and
animals contaminated with Cd, Pb, Cu, Zn, and Mn, making
them generally unfit for human or animal consumption.
Cleansing the soil of heavy metals via conventional meth-
odologies is expensive and in most cases not feasible
(Cunningham and Ow, 1996). Phytoremediation (and more
specifically phytoextraction) is a low-cost alternative to the
traditional remediation technologies, such as soil excavation
and other metal extraction technologies (Salt et al., 1998;
Blaylock and Huang, 2000; Schmidt, 2003). Ideally, plants
used for phytoremediation should extract heavy metals from
soils and provide a return on investment, yet not contam-
inate the food or feed. Aromatic crops, used for production of
essential 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 heavy
metals from contaminated soil (Zheljazkov and Nielsen, 1996;
Zheljazkov and Warman, 2003), suggesting the possibility that
such plants could be used in phytoremediation of contaminated
soils. The effects of these metals on growth, essential oil pro-
duction, and metal 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,
largely unknown. Coriander, dill, chamomile, peppermint, basil,
hyssop, lemon balm, and sage are aromatic crops that have been
traditionally 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 in
various non-food industries, such as perfumery, cosmetics, and
aromatherapy. The objectives of this study were to evaluate
the ability of the selected aromatic crops to grow, produce
essential 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 experiments
Coriander ‘Alekseevski’, sage ‘Dessislava’, basil ‘Mesten’ and
‘Trakia’, dill ‘Mesten’, chamomile ‘Lazur’, hyssop ‘Local’, and
lemon balm ‘Melissa 2’ were used in a field study. The plants,
seeded from 1991 to 1994, in the vicinity of the Zn–Cu smelter
near Plovdiv, Bulgaria (situated close to the northeastern
foot of the Rhodopa Mountains in the European continental
climatic zone) were grown in 15 m2 experimental plots at three
sites situated at 0.8, 3, and 9 km (control) from the smelter
in the direction of the prevailing wind using a randomized
complete block design with three replicates. The soil was
alluvial and alluvial-meadow, pH 7.1–7.2, with a humus A
horizon layer 25–28 cm deep, organic matter of 4.2%, a clay
content of 49–52%, sand content of 37–42% and silt content
of 10–11% and CEC of 35–38.8 meq/100 g. Annual rainfall
amounts were 600–650 mm.
The plants were grown using accepted agricultural practices
for the respective crops (Topalov, 1962). Briefly, in late summer
120 kg P2O5 ha− 1 and 100 kg K2O ha− 1 were applied to the growing
area in accordance with soil test results. The land was subse-
quently plowed, disked, and fertilized with N at 80 kg ha− 1 just
before the spring seeding. Coriander and dill were seeded in the
first week of April at 15 kg seed ha− 1 (3–4 cm deep) and 10 kg
seed ha− 1 (2–3 cm deep), respectively. Chamomile was seeded
after disking in the fall at 2.5 kg seed ha− 1 (0–1 cm deep). Basil
and lemon balm seedlings, produced in a greenhouse, were
transplanted to the field on June 1 (in 1992 and in 1993 for basil,
and in 1992 for lemon balm). Garden sage and hyssop seedlings
were started in a nursery and transplanted to the field in
September, 1991. All plants were side-dressed with N at 60 kg
ha− 1 in the last week of June. Heavy metal concentrations were
determined using soil samples (0–20 cm deep) from the
experimental plots taken just before planting. Coriander was
harvested when seeds reached technical maturity, whereas all
other plant species were harvested at the beginning of flower-
ing (the last week of August) (Topalov, 1962). The dry weights of
coriander seed, hyssop inflorescences, and plant shoots of other
species were recorded.
2.1.2. Soilless container experiment
Peppermint, basil, and sage were used in the container study;
the selection was based on their performance in prior field
experiments and their economic value. The peppermint was
grown from rhizome cuttings and basil and sage were started
from 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 by
watering twice a week with 50% strength Hoagland's solution.
The containers with plants were distributed randomly on the
greenhouse benches with a subsequent weekly rotation in
placement.
The concentrations of Cd, Pb, and Cu used to treat plants in
the container study were chosen so that the lowest concen-
tration represented the lowest total critical concentrations for
the respective metal in soil that induces phytotoxicity or a 10%
yield reduction (Kabata-Pendias, 1992), and so that the two
higher concentrations represented the upper limit and 150 to
500% of the critical concentrations for the respective metal in
soil. Hence, the metal treatments consisted of Cd at 2, 6, and
10 mg L− 1, Pb at 50, 100, and 500 mg L− 1, and Cu at 20, 60, and
150 mg L− 1. Metals were added to the plant root environment
by watering the plants with chloride salt solutions (corrected
to neutral pH) of each metal once a week at a rate of 100 mL per
container. Since the chloride salts were corrected for pH, we
assumed no major effect of Cl on plant growth and on the
measured responses. Controls (plants not treated with heavy
metals) were watered without the heavy metal additions. Basil
and peppermint were harvested at 50% flowering to ensure
high 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
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 95 ( 20 0 8 ) 5 1–6 2
distilled for essential oil extraction. The remaining half of the
vegetative tissue was separated into stem and leaf tissue,
dried to a constant weight in a forced air oven at 35 °C, and
weighed.
2.2. Essential oils
Of the harvested plant materials from the field and container
experiments, the sub-sample (one-half of seed, inflores-
cences, or plant shoots) used for oil extraction and analysis
was dried in a shaded, well-aerated location (22 °C) to preserve
essential oil content (Topalov, 1962). The essential oils from all
crops were extracted by steam distillation using a Clevenger-
type apparatus.
2.3. Elemental analyses
The remaining half of the plant material (not used in essential
oil analysis) and the collected soil and perlite samples were
used to determine, respectively, the heavy metal concentra-
tions in the plant tissue and in the growth environment. The
plant tissues used for metal analysis were dried in an oven at
70 °C for 72 h, weighed to determine dry weight, and sub-
sequently ground and sieved through a 1.0 mm screen. For
analyses, the 1 g dry samples of tissue, perlite, and soil were
digested in concentrated HNO3 (Zheljazkov and Nielsen, 1996),
filtered, and made to final volume of 25 mL. It is understood
that the HNO3 digestion of soil samples represent HNO3-
extractable or pseudo-total and not the total concentration of
elements 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 carbonization
using a hot plate and heated in a muffle furnace at 400 °C for
4 h. After cooling, 2 mL of HNO3 were added to the residue of
each sample and the mixture was placed in a heated sand bath
for acid evaporation followed heating in a muffle furnace at
400 °C to produce a colorless acid solution that was brought to
volume with distilled water and 2 mL of HCl.
Fractionation of the heavy metals in soil samples was done
using the sequential extraction procedure of Tessier et al.
(1979), as modified by Luo and Christie (1998), and described in
Zheljazkov and Warman (2003) on 2 g of air-dried, sieved soil
in 50-mL polypropylene centrifuge tubes. Sequential extrac-
tions and fractionation of heavy metals in soils have been
used 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 were
operationally defined as: (1) exchangeable (EXCH), (2) carbo-
nate bound fraction (CARB), (3) iron and manganese bound
fraction (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 was
added to each sample. The samples were shaken for 1 h,
centrifuged at 3000 rpm for 10 min, the supernatant filtered
through No. 40 Whatman filter paper, and the weight of the
sample 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 to
each residue from step 1. Then, the samples were shaken
53
for 5 h at room temperature, centrifuged and filtered as
during step 1; afterwards the weight was recorded.
3. Metals from reducible iron and manganese bound fraction
(FeMnOx): 30 mL of 0.04 M NH2OHHCl in 25% (v/v) CH3COOH
were added to sample residues from step 2. The tubes with
screwed caps were placed for 5 h on a water bath at 96 °C
and 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 were
added to the sample residues from the previous step. The
non-capped tubes with samples were placed on water bath
at 85 °C for 45 min. After that, 4 mL of 30% H2O2 adjusted to
pH 2 with HNO3 were added to the samples, which were put
on a water bath at 85 °C for a further 45 min. This procedure
was 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 deionized
water to 30 mL. The samples were shaken for additional
30 min, centrifuged, and filtered as indicated above.
Heavy metals remaining in soils after extraction for organic
matter-bound fractions were treated as “residual” and their
concentrations were calculated by subtracting the EXCH, CARB,
FeMnOx, and OM fractions from the total metal concentrations.
Heavy metals in tissue and soil digested samples from the field
experiment 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 absorption
spectrometer 5100 (GFAAS) (Zheljazkov and Nielsen, 1996).
Elemental concentrations of plant tissues, growth media, oil
samples, and the water and plant residues from distillation of
container plant material were determined using a Thermo
Jarrell Ash (Franklin, MA), Inductively Coupled Argon Plasma
(ICAP) Spectrometer. Samples from the fractionation were
analyzed on a Varian (Palo Alto, CA) Spectra AA-20 AAS. To
avoid possible interferences of matrices, for each of the
extraction steps as well for the nitric acid digestions, separate
standards were prepared using the same matrices as the
samples from a particular step. Blanks were included with the
digestion of every batch of 20 samples and with the extraction of
each soil fraction. Quality control was ensured by the inclusion
of a barley flour standard, a Certified Reference Material AR2027
(Alpha Resources Inc., Stevensville, MI) and a certified reference
soil SS-2 (SCP Science, Champlain, NY) sample, and calculating
the percent recoveries of the five elements relative to the cer-
tified standard values. The relative elemental concentrations
recovered from the Standard Reference Material SS-2 (contami-
nated soil) using the HNO3 digestion procedure (in % relative to
the elemental concentrations as provided with the certificate of
analyses) were as follows: 89% for Cd, 103% for Pb, 108% for Cu,
93% for Mn, and 96% for Zn. The relative elemental recoveries
from barley flour Standard Reference Material AR2027 were 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 spatial
distribution visualized using a Hitachi (Tokyo, Japan) 3000N
 54
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 of
pollution
SV Herbage yield Essential oil yield Cd Cd Pb Pb Cu Cu Mn Mn Zn Zn
−1 −1
content†† uptake††† content uptake content uptake content uptake content uptake
T ha kg ha

Proximity 0.8 km
Sage 13.5b† 171.1b 13.9a 48.9aA† 182.9a 645.2aA 36.1a 128.0aA 94.6a 331.4aA 202.4a 709.4aA
Basil ‘Trakia’ 17.0b 72.6b 8.4a 28.4aC 154.5a 525.9aB 26.2a 88.8aB 88.2a 300.2aA 186.7a 635.2aA
Basil ‘Mesten’ 16.8b 68.6b 8.4a 28aC 155.3a 520.4aB 25.8a 86.3aB 88.3a 296.0aA 191.8a 641.6aA

SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 95 ( 20 0 8 ) 5 1–6 2
Dill 9.7b 43.8b 9.1a 26.5aC 149.6a 438.7aC 24.1a 70.5aB 77.1a 226.4aB 180.2a 528.7aB
Cham 15.9b 44.5b 11.9a 38.1aBC 149.6a 476.1aBC 26.5a 84.4aB 91.9a 292.5aA 197.0a 627.2cA
Coriander†††† 0.83b 3.8b 8.9a 5.2aD 143.8a 85.4aDE 26.2a 15.6aD 79.5a 47.2aC 145.0a 86.1aC
Lem balm 18.8b 3.9b 10.2a 40.3aB 126.7a 50.1bE 21.1a 83.3bB 81.5a 321.7aA 176.3a 696.1aA
Hyssop†††† 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 km
Sage 17.2a 220.1a 4.2b 18.9bA 57.4b 260.5bA 24.9b 114.2aA 77.7ab 348.4aA 85.4b 383.8bA
Basil ‘Trakia’ 21.1a 90.7a 3.1b 12.9bBC 46.9b 197.8bAB 23.8a 101.0aAB 67.2ab 283.8aAB 73.1b 309.2bA
Basil ‘Mesten’ 19.4a 80.6ab 2.8b 10.9bC 46.5b 180.2bAB 23.5a 91.3aB 67.3ab 261.8aB 72.9b 283.8bA
Dill 12.1a 54.5ab 2.8b 10.2bC 45.5b 165.7bB 20.7b 75.5aC 64.4ab 234.9aB 69.5b 254.1bA
Cham 17.9ab 51.0ab 3.5b 12.6bBC 42.5b 152.0bB 23.6a 84.5aBC 75.2b 268.8aB 75.3b 269.3bA
Coriander 0.93ab 4.2ab 2.4b 1.6aD 39.4b 26.3bC 20.5a 13.7abE 61.5ab 41.0aC 77.2 51.5bB
Lem balm 23.5a 4.9a 2.8b 14.0bB 37.8b 186.5aAB 25.1a 123.8aA 59.8b 295.1aAB 65.7b 324.6bA
Hyssop 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 km
Sage 17.8a 227.9a 0.43c 2.0cA 6.1c 28.6cA 13.8c 65.8bA 62.4b 290.5aA 31.9c 151.4cA
Basil ‘Trakia’ 21.1a 92.7a 0.25c 1.1cAB 4.5c 19.8cA 13.4b 58.9bA 49.4b 216.3bB 32.6c 142.9cA
Basil ‘Mesten’ 20.7a 86.2a 0.24c 0.98cAB 4.5c 18.5cA 12.5b 51.9bA 49.8b 206.3bB 32.6c 133.8cA
Dill 13.1a 58.7a 0.22c 0.91cAB 3.9c 15.9cAB 13.7c 54.6aA 44.3b 176.6bC 29.9c 119.4cA
Cham 20.8a 57.1a 0.27c 1.13cAB 4.2c 17.5cAB 13.5b 54.6bA 44.4c 258.0aAB 31.1c 128.8cA
Coriander 0.96a 4.4a 0.23c 0.16bB 4.1c 0.29bD 11.5b 7.9bB 39.8b 27.4bD 33.9c 23.3cB
Lem balm 21.6a 4.7a 0.26c 1.2cAB 3.5c 15.8cAB 13.5b 61.2bA 35.1c 159.2bC 24.6c 112.0cA
Hyssop 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 represent
significance 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.
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 95 ( 20 0 8 ) 5 1–6 2 55

variable pressure scanning electron microscope (SEM) coupled
to Oxford (Concord, MA) INCA 350 Energy Dispersive X-ray
spectrometer (EDX). The SEM imaging used a backscattered
electron (BSE) detector, a low vacuum (2–50 Pa) and a cooling
stage to stabilize the samples and control charging. The EDX
analyses 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 using
repeated measures analyses (proximity to the source of the
pollution 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 (2
levels), proximity (location) — 3 levels (0.8 km, 3 km and 10 km
from the smelter), species/varieties (8 levels). Differences
among metal treatments and plant responses in the container
experiment were tested for significance using analysis of var-
iance (ANOVA) and the means were separated using Duncan's
New 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 the
smelter were reduced compared with the respective yields at
9 km from the smelter (the control) (Table 1). Yields of sage,
basil, dill, lemon balm, and hyssop at 3 km from the smelter
were higher than respective yields at 0.8 km from the smelter.
Chamomile and coriander yields at 3 km from the smelter
were not different from yields either at 0.8 or 9 km from the
smelter (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 practical
interest, since it allows the estimation of the phytoremedia-
tion potential of these plants. In Table 1, heavy metal uptake
was calculated from metal concentrations in the herbage and
the yield of dry herbage (please note that yields in Table 1 are
fresh herbage yields). Generally, heavy metal concentration in
the experimental plants reflected the metal concentration
in the soils (Tables 1 and 2). Overall, the highest heavy metal
concentrations were measured in plants grown at 0.8 km from
the smelter, lower in plants grown at 3 km from the smelter,
and lowest in the control plants at 9 km from the smelter. At
0.8 km from the smelter, the highest tissue Cd concentrations
were found in sage and the lowest Cd concentrations were in
the two varieties of basil, coriander, and dill. Overall, most Cd
was removed with sage, lemon balm, and chamomile, less
with basil and dill, and least with coriander and hyssop.
Generally, Pb concentrations of sage and hyssop plants were
higher than in other plants. Removal of Pb from the site with
the harvestable biomass was highest with sage plants, lower
with basil, dill, and chamomile, and lowest with coriander,
lemon balm and hyssop. The seven plant species accumulated
similar amounts of Cu and Mn at any given distance from the
smelter. At 0.8 and 3 km from the smelter, more Cu was
removed from site with the harvestable parts of sage, less with
basil, dill, chamomile and lemon balm, and least with hyssop
and coriander. Plants grown at 9 km from the smelter removed
similar amounts of Cu with the harvest. Also, due to the
differences in harvestable yields between plants, more Mn
was removed from the sites with sage, basil, dill, chamomile
and lemon balm, and much less with coriander or hyssop. At
0.8 km from the smelter, lesser amounts of Zn were found in

Table 2 – Fractionation of Cu, Cd, Zn, Zn, Pb, and Mn and in the soils and their pseudo-total concentrations in soils following
HNO3 digestion, bioavailability factor (BF) and transfer factor (TF)
Metal Distance Soil fractions
from the
smelter EXCH CARB FeMnOx OM HNO3 BF TF††
(km) mg kg− 1 % %

Cu 9 (control) 1.6b† 1.8c 0.9c 2.9c 30.7c 5 0.37–0.45

3 2.2ab 8.8b 11.6b 32.4b 115.6b 1.9 0.18–0.23
0.8 2.6a 49.2a 36.5a 122.3a 193.8a 1.3 0.1–0.18
Cd 9 (control) 3.0c 4.6c ud††† ud 4.3c 70 0.05–0.1
3 3.5b 8.5b 3.2b ud 9.9b 35 0.24–0.44
0.8 5.4a 18.5a 6.9a ud 25.1a 22 0.33–0.55
Zn 9 (control) 2.3c 3.2c 9.3c 3.1c 18.3c 13 1.3–1.9
3 3.7ab 29.1b 83.5b 18.8b 113.8b 3.2 0.6–0.8
0.8 6.5a 246.4a 692.1a 208.5a 1092.2a 0.6 0.13–0.18
Pb 9 (control) 14.4b 19.1c 9.3c 10.6c 33.8c 54 0.1–0.19
3 27.7a 115.9b 60.4b 17.8b 220.3b 13 0.17–0.26
0.8 29.1a 892.3a 360.7a 133.7a 1365.2a 2 0.09–0.13
Mn 9 (control) 0.4c 3.4b 20.1b 161.8b 166.4b 0.24 0.19 – 0.38
3 0.5ab 3.8b 107.5a 502.8a 658.1a 0.08 0.09 – 0.12
0.8 0.7a 5.6a 96.2a 511.2a 722.9a 0.09 0.1 – 0.13
†
Separation of means. Data are the means of four replicates. Means with the same letter in a column within a fraction and element are not
significantly different at P ≤ 0.05.
††
TF is different for different crops. The indicated range includes TF for all seven crops.
†††
ud — below the detection limit, undetected amount.
56 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 95 ( 20 0 8 ) 5 1–6 2

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

the harvestable coriander fraction as compared with Zn in the
harvestable 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 were
removed with coriander or hyssop (Table 1).
The concentrations of Cd, Pb, Cu, and Zn in the soils at
0.8 km were about 30 times higher than the respective
concentrations in the control soils (Table 2) and much above
what would be considered phytotoxic concentrations of these
elements in soils (Kabata-Pendias and Pendias, 1992). For
instance, Pb and Zn concentrations in soil at 0.8 km were
approximately 1000 mg kg− 1. Soil metal concentrations at
3 km from the smelter were relatively high, but for most
metals, within the maximum permissible concentrations of
the elements for agricultural soils (Kabata-Pendias, 2001). No
increased concentrations of heavy metals were noted in soil at
9 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
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 95 ( 20 0 8 ) 5 1–6 2 57

fraction, most of the Zn was in FeMnOx fraction, and most of
Pb and Cd were in the CARB fraction (Table 2). The bioavail-
ability factor (BF), the ratio of the metal concentration in the
EXCH fraction to the total metal concentration in soil (Knox
et al., 2000), for Cd, Cu, Zn, Pb, and Mn decreased with
decreasing distance to the smelter. With decreasing distance
to the smelter, the transfer factor (TF), the ratio of the metal
concentration in plant tissue to the total concentration of
metal in the soil (Knox et al., 2000), for Cu and Zn, decreased
but the increased for Cd and was highest at 0.8 km from the
smelter.
SEM/EDX analyses of the soil sampled at 0.8 from the
smelter indicated that Pb, Cd, Mn, Cu and Zn were either
concentrated on certain particles (1–6 μm in diameter), or
smaller than 1 μm and randomly scattered throughout the
sample without forming any significant clusters (Figs. 1 and 2).
This pattern suggested atmospheric deposition of the con-
taminants was the source. Similar results were found by
Adamo et al. (1996), who reported “bright particles” (heavy
metal particles in SEM observations) in Sudbury soil receiving
aerosol contaminants from a smelter for over 100 years. Some
of the particles in our study had predominately Zn and Pb on
the 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 were
in predominately metal particles, as there was a lack of strong
signals from plant nutrients such as P and K. These metal
particles could cause redistribution of metals in the sequential
extraction process as demonstrated previously (Dahlin et al.,
2002).

Fig. 2 – A. BSE detector SEM image of contaminated soil sampled at 0.8 km from the smelter at magnification of 1000×, indicating
four bright particles (S2, S5, S8 and S9) that were analyzed with EDX microanalyzer. Spectrum of the bright particle S2 (B), S5 (C),
S8 (D), and S9 (E).
58 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 95 ( 20 0 8 ) 5 1–6 2

Fig. 2 (continued ).

3.2. Container experiments
Lead at 500 mg L− 1 and Cu at 60 and 150 mg L− 1 reduced
peppermint yields (Table 3) relative to the control not exposed
to heavy metals. At 150 mg L− 1 Cu, peppermint plants had
yellow leaves, stunted growth, and underdeveloped root
systems as compared with control plants. Vegetative yields
of basil were significantly reduced by Cd at 6 and 10 mg L− 1, Pb
at 500 mg L− 1, and Cu at 20 and 60 mg L− 1 as compared with
controls. Cu at 60 and 150 mg L− 1 reduced plant weight and
essential oil content (Tables 3 and 5). Sage biomass yields were
reduced by the applications of Cd, Pb, and Cu at the two
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 95 ( 20 0 8 ) 5 1–6 2 59

Fig. 2 (continued ).

highest rates (Table 3). Overall, plants subjected to 150 mg Cu Higher concentrations of Cu in the growth medium also
L− 1 had foliar chlorosis, underdeveloped (smaller volume) root resulted in some increases of Cu in the plant foliage, most
systems and reduced yields. In the above ground plant parts, notably in basil. The concentrations of Cd, Pb, and Cu in the
Cd accumulations in peppermint were primarily in the leaves, plant residual (steam distilled) material after distillation were
while the Cd accumulations in basil and sage were similar for similar to the respective concentrations of the element in
leaves and stems. The concentrations of Pb in plant shoots plant tissues before the distillation. Cadmium in the distil-
were relatively low as compared with the Pb concentrations of lation water was below the detection limit of 0.06 mg L− 1,
the roots (Table 4). The addition of Cu to the growth medium while Pb and Cu in the distillation water were not different
dramatically increased the accumulations of Cu in the roots from the concentrations of these elements in the control. Trans-
of the three tested species (Table 4), especially in basil roots. fer factor coefficients (TF) were 2 to 14% for Cd, 0.5 to 53% for
Pb, and 0.5 to 14% for Cu, depending on the treatment and
species.
Table 3 – The effects of Cd, Pb, and Cu on plant growth Essential oil contents of the three crops also showed some
Heavy metal Peppermint Basil Sage variation (Table 5). For both peppermint and basil, only the
treatment highest Cu treatment (150 mg L− 1) decreased oil contents
relative to the control. Cadmium, Cu, and Pb in the essential
Mg L− 1 Biomass† yields, g dry weight
oils of basil, sage, and peppermint from all treatments were
Cd 2 147a†† 111a 126ab below the detection limit of ICAP-AES (0.005 mg L− 1 for Cd,
6 138a 80b 109b 0.02 mg L− 1 for Cu, and 0.05 mg L− 1 for Pb). Hence, the actual
10 114ab 48c 104b
concentrations of Cd, Cu, and Pb in the oils were below 0.06 mg
Pb 50 135a 105a 110b
L− 1 Cd, 0.25 mg L− 1 Cu, and 0.63 mg L− 1 Pb.
100 122ab 102ab 128ab
500 91b 20d 98b
Cu 20 124a 77b 151a
60 86b 34cd 138ab 4. Discussion
150 44c 5e 49c
Control††† 130a 113a 146a
4.1. Field experiments
†
Biomass represents all above ground parts of the harvest plant.
††
Means of 4 replicates. Mean separation by Duncan's Multiple Range The total soil concentrations of Cd, Pb, Cu, Mn and Zn at
test. Numbers with the same letter within a treatment and species different distances from the smelter reflect the pattern and
having the same letter are not significantly different, P=0.05. nature of the aerosol pollution around the smelter. Research
†††
Controls were not treated with heavy metals.
has indicated that a number of crops around this particular
60 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 95 ( 20 0 8 ) 5 1–6 2

Table 4 – The effects of heavy metal treatments on Cd, Pb, and Cu accumulation in plant tissues
Heavy Peppermint Basil Sage
metal †† ††
treatment† 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 %
−1
mg Cd kg
Cd
2 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 4
6 199.0b 3.4a 16.0b 18.8b 9 868.0b 129.0b 20.5b 31.3a 4 238.0b 18.5b 22.8b 12.3b 5
10 537.0a 4.2a 21.9a 24.7a 5 1620.0a 201.0a 42.0a 37.9a 2 537.0a 46.5a 40.2a 31.2a 6
0 0.9e bd bd bd – 0.4e 0.9d 0.3c 0.8c – 0.7d 0.7d 0.7d 0.4d –

Pb
50 224.7c 28.5b 108.0c 120.5c 53 3068.0c 59.0b 65.3b 105.7b 3 270.6c 58.3b 61.5b 86.4b 32
100 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 14
500 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 5
0 13.4d 8.8c 7.2d 20.3d – 38.4f 18.1c 19.1c 23.6c – 26.7d 28.0c 22.6c 20.1c –

Cu
20 655.6c 22.3b 21.9b 36.8b 6 931.0c 33.9c 25.8b 48.7b 5 392.7c – 55.9b 56.2a 14
60 1242.2b 37.2a 25.6ab 47.1a 4 3507.9b 62.0b 42.3ab 48.6ab 1 511.5b – 53.4b 62.0a 12
150 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 9
0 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 part
are not significantly different, P≤ 0.05.
††††
MT — metal transfer from roots to shoots in %.

smelter accumulated excessive amounts of heavy metals
(Sengalevitch, 1993). Several earlier reports (Kabata-Pendias
and Pendias, 1992; Zheljazkov and Nielsen, 1996) have dem-
onstrated that high soil concentrations of Cd, Pb, and Cu
would reduce the yields of a variety of crop plants. In this
study, although the yields from crops at 0.8 km from the
smelter were reduced, they were still within the typical yields
for these crops in the region (Topalov, 1962). The above sug-
gested that the tested aromatic crops could remain profitable
even at 0.8 km from the smelter.
Although the concentrations of Cd, Pb, Cu and Zn in soil at
0.8 km were above the critical concentrations in soil for these
elements (Kabata-Pendias and Pendias, 1992), no visual phy-
totoxicity symptoms on crops were observed. However, our
electron microscopy observations of thin sections indicated
alterations in chloroplasts thylakoids in plants grown at
0.8 km from the smelter (data not shown). Such sub-cellular
alterations in morphology may be an indication of the toxic
effects of heavy metals on transport of solutes across the
plasma membrane, and perhaps photosynthesis, and thus
responsible for measured yield reductions. These results are in
agreement with the report of Pietrini et al. (2003), who reported
30% loss of chlorophyll and swelling of thylakoid membranes
in Phragmites australis when exposed to high concentrations of
Cd in solution.
Our results suggested that fractionation of Cu, Cd, Zn, Pb
and Mn in soil was affected by the distance from the smelter.
Results from the metal fractionation study supported the idea
that Cu has a high affinity for organic compounds (Wu et al.,
1999). Cadmium in the OM fraction was below the detection
limit of AAS, supporting the notion for relatively low affinity of
Cd for OM (Kabata-Pendias, 2001; Lim et al., 2002). The largest
Zn fraction in the contaminated and in control soils was
FeMnOx–Zn, also evident from other reports (Luo and Christie,
1998; Shuman, 1999). Evidence exists that Zn has relatively
high 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 the
high affinity of Pb to Mn oxides, Fe and Al hydroxides, clay
minerals, and organic matter (Kabata-Pendias, 2001). Lim et al.
(2002), using a similar fractionation procedure, also observed

Table 5 – Essential oil content (% in dry leaves) in the three plants depending on the treatments
Treatments (mg L− 1 in the applied solution)

Species Control Cd 2 Cd 6 Cd 10 Pb 50 Pb 100 Pb 500 Cu 20 Cu 60 Cu 150

Peppermint 2.5a 2.3a 2.4a 2.1ab 2.6a 2.3a 2.1a 2.7a 2.1ab 1.8b
Basil 1.6a 1.6a 1.6a 1.4a 1.3a 1.2a 1.2a 1.4a 1.1ab 0.9b
Sage 2.1a 2.2a 2.6a 2.0a 2.3a 2.3a 2.1a 2.2a 2.2a 2.0a

Means with the same letter within a crop (a row) are not significantly different at P ≤ 0.05.
 SC IE N CE OF TH E TOTA L E N V I RO N ME N T 3 95 ( 20 0 8 ) 5 1–6 2
that the carbonate fraction contained the greatest amount of
Pb in contaminated clay. Our results were in accordance with
the report of Kabala and Singh (2001) for small organically
bound fractions of Pb in highly contaminated soils.
The BF (Knox et al., 2000) showed that with decreasing
distance to the smelter, the proportion of EXCH forms of Cu,
Cd, Zn, Pb, and Mn relative to the total amounts of these
metals in soil decreased. The concentrations of these metals
in EXCH fractions, however, increased with decreasing dis-
tance to the smelter (for Cd), or decreased in soil at 0.8 km
relative to the control (for Cu, Pb, Zn, and Mn), indicating that
larger 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 (Knox
et al., 2000) indicated increased metal availability with de-
creased distance to the smelter.
4.2. Container experiment
Results from our container experiments demonstrated that
of 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 above
ground biomass of basil, peppermint, and sage in the container
experiment did not reach the high concentrations character-
istic for hyperaccumulators (Brown et al., 1995). Interestingly
however, 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 significant
metal accumulators. Cadmium transfers from roots to shoots
in peppermint, basil, and sage were similar to Cd transfer in
maize grown on two Cd contaminated UK soils (Lombi et al.,
2001). Also, our results indicated that Cd and Pb accumulations
in peppermint, basil, and sage were higher than Cd and Pb in
maize shoots grown on contaminated soils, and comparable to
Cd and Pb in maize shoots after the addition of EDTA (Lombi
et al., 2001).
Traditionally, peppermint, basil, and sage tissue wastes
after distillation are used as feed for sheep (Topalov, 1962).
Results from our container experiment demonstrated that if
peppermint, basil, and sage are grown in highly heavy metal
contaminated medium, Cd, Pb, and Cu may accumulate in
shoots 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 produce
usable final product, metal contaminated distillation wastes
could be composted after mixing with wastes from other low-
metal feedstock.
Overall, our results from field and container experiments
supported the general understanding that Cu and Pb accumu-
late mainly in the roots. However, results from the container
study demonstrated that Cu and Pb may have increased
mobility within plants when grown in substrates with high
concentrations of these metals. Once the metals were taken up,
Cu and Pb tended to accumulate in the roots, while Cd, Mn, and
Zn were easily transported to the shoots (Alloway, 1990; Kabata-
Pendias and Pendias, 1992; Zheljazkov and Warman, 2003).
61
Some studies have noted, however, that leaf tissue Cu equaled
or exceeded root Cu concentration in mustard (Planquart et al.,
1999). Our results indicated significant amounts of Pb and Cu in
above ground plant parts, accumulations that could be due to
continuous aerosol emissions or soil particle entrapment on
above ground plant parts. Overall, the transfers of Pb and Cu
from roots to shoots were greatest at lower concentrations of
these metals in the growth medium and declined with in-
creasing concentrations of Pb and Cu. The accumulations of Cu
in above ground plant parts of peppermint, basil, and sage in the
container experiment were comparable and in some instances
higher than Cu accumulation in Agrostis tenuis grown on Cu
contaminated soil (Thayalakumaran et al., 2003). The transfers
of 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 of
these particular experimental varieties different from other
reports (Planquart et al., 1999), most likely due to genetic dif-
ferences among species (Macnair, 1990).
Our results from field and container experiments indicated
that coriander, dill, chamomile, peppermint, basil, hyssop, lemon
balm and sage were not metal hyperaccumulators (Brown et al.,
1995), and did not have very high phytoremediation potential as
compared with other plants (Brown et al., 1994). Yet the use of
aromatic plants for phytoremediation may have an advantage
over other crop plants in that the harvested foliage is a source of
essential oils, which are the marketable revenue-generating pro-
ducts of aromatic crops. Growing of these aromatic crops in metal
contaminated areas may not introduce heavy metals into the
food chain and may not result in an economic penalty compared
to most other edible crops. In the process of oil extraction by
distillation, heavy metals remain in the extracted plant residues,
limiting the quantities of heavy metals in the commercial oil
product (Zheljazkov and Nielsen, 1996; Scora and Chang, 1997;
Zheljazkov and Warman, 2003). Thus, significant amounts of
heavy metals could be removed from the soil through proper
disposal of the metal contaminated plant residues, while the
metal-free, extracted oils could be safely marketed. High-value
aromatic crops may well be a better alternative for heavy metal
contaminated agricultural soils than the suggested woody
species such as Salix and Betula (Hammer et al., 2003; Rosselli
et al., 2003), or other plants like Sesbania drumondii that have been
shown to hyperaccumulate Pb (Sahi et al., 2002).
Disposal of the metal contaminated plant tissues following
phytoextraction remains a problem in the use of all plant
materials. Several approaches such as composting, incinera-
tion, ashing, pyrolysis, direct disposal and liquid extraction
have been proposed and/or tried (Sas-Nowosielska et al., 2003;
Keller et al., 2005), but if not controlled can still pollute the
environment. Some aromatic plants might demonstrate
significant phytoremediation potential if coupled to other
means for increasing bioavailability and uptake of Cd, Pb, and
Cu, such as chelates (Schmidt, 2003) or biosurfactants
(Mulligan et al., 2001).
Acknowledgements
The research in the vicinity of the Pb–Zn smelter near Plovdiv
was funded by the Bulgarian Ministry of Education. Authors
62 SC IE N CE OF TH E TOTA L E N V IR O N ME N T 3 95 ( 20 0 8 ) 5 1–6 2
thank the Cooperative Farm in Dolni Voden, Plovdiv region for
providing access to contaminated land and logistical support.
The work at the Royal Veterinary and Agricultural University
in Copenhagen, was supported by a visiting scholar research
grant awarded to Dr. Zheljazkov by the Danish Academy of
Sciences. The work at the Nova Scotia Agricultural College was
supported by the Natural Sciences and Engineering Research
Council of Canada (NSERC) Discovery Grant and the Canadian
Foundation 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 to
Dr. Zheljazkov for 12 months as a visiting scholar. This mate-
rial is partially based upon work supported by the Cooperative
State Research, Extension, Education Service, US Department
of Agriculture, and Massachusetts Agricultural Experiment
Station under project No. 729. Publication No. 3259.
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