Chemosphere 158 (2016) 56e65

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Impact of cement dust pollution on Cedrela fissilis Vell. (Meliaceae): A

potential bioindicator species
Advanio Ina
cio Siqueira-Silva a, Eduardo Gusma
~o Pereira b, Luzia Valentina Modolo a,

Pires Lemos-Filho a, Elder Antonio Sousa Paiva a, *
Jose
a
Departamento de Bota ^nica, Instituto de Ci^
gicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, MG, Brazil
encias Biolo
b
Instituto de Ci^
gicas e da Saúde, Universidade Federal de Viçosa, Campus Florestal, 35690-000, Florestal, MG, Brazil
encias Biolo

h i g h l i g h t s

A potential bioindicator species to cement dust pollution is proposed.

Exposure to cement dust causes leaf chlorosis and necrosis in Cedrela fissilis.

Cement dust induces leaf Fe deficiency, fall in gs and Fv/Fm in sensitive species.

Antioxidant enzyme activities reduces in highest alkaline dust concentration.

a r t i c l e i n f o a b s t r a c t

Article history: Considering the impacts caused to vegetation in the vicinity of cement factories, the aim of this study
Received 16 September 2015 was to evaluate the impacts of cement dust on the structural organization and physiological/biochemical
Received in revised form traits of Cedrela fissilis leaflets, a woody species native to tropical America. Plants were exposed to 2.5 or
7 April 2016
5 mg cm2 cement dust applied to the leaf surface, the soil or leaf surface and simultaneously. Leaves of
Accepted 17 May 2016
shoot-treated plants exhibited chlorosis, marginal and inter veins necrosis, diminished thickness,
epidermal cells less turgid, cellular collapse, obstructed stomata, senescence, rolling and some abscission.
Handling Editor: Frederic Leusch In few cases, individual death was recorded. Cement dust-treated plants also presented decreased
amount of photosynthetic pigments and iron (Fe) and increase in calcium (Ca) levels. The cement crust
Keywords: formed in leaves surface blocked from 30 to 50% of the incoming light and reduced the stomatal
Pollution conductance and the potential quantum yield of photosystem II. Control or soil-treated plants did not
Stress exhibit morphophysiological changes throughout the experiment. The activity of superoxide dismutase,
Plant-environment interaction catalase and ascorbate peroxidase increased in leaves of plants upon treatment with 2.5 mg cm2 cement
Cement
dust, independent of the site application. Overall, these results indicate that C. fissilis is highly sensitive to
cement dust at the initial stage of development.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction impacted by the limestone extraction and processing.

The Dry Forest is among the most threatened ecosystems in the
world (Nassar et al., 2008; Melo Júnior et al., 2015), especially due
to industrial exploitation of limestone (Silva and Scariot, 2004;
Felfili et al., 2007; Duarte et al., 2015), the main raw material of
cement. In general, cement factories are built nearby limestone
outcrops to reduce transportation costs. However, sometimes these
areas are also near remnant Dry Forest, which can be negatively
* Corresponding author.
E-mail address: epaiva@icb.ufmg.br (E.A.S. Paiva).
The Dry Forest is a unique ecosystem surrounded by diversity
and endemism (Pennington et al., 2006). However, the lack of
knowledge regarding its flora (Silva and Scariot, 2004; Arruda et al.,
2013; Melo Júnior et al., 2015) and the various plant species syno-
nyms worldwide (Pennington et al., 2006) have contributed to the
destruction of such ecosystem. Cedrela fissilis Vell. (Meliaceae) is a
native woody plant of tropical America that occurs in the limestone
outcrops of the Dry Forest. It is a deciduous species with par-
ipinnate, compound leaves and pubescent leaflets and is widely
distributed in South America and Costa Rica (Pennington et al.,
1981). Over the years C. fissilis woods have been commercially

http://dx.doi.org/10.1016/j.chemosphere.2016.05.047
0045-6535/© 2016 Elsevier Ltd. All rights reserved.
 A.I. Siqueira-Silva et al. / Chemosphere 158 (2016) 56e65
exploited (Garcia et al., 2011). Considering its occurrence in areas of
cement industries activities, it is likely that this plant species is at
risk of extinction.
In addition to the limestone extraction by industries, cement
processing is a major source of particulate material (Grantz et al.,
2003; Rodrigues and Joekes, 2011). This particulate material of
variable chemical composition, consists of particles of 10 mm
aerodynamic diameter or lower (Grantz et al., 2003). Also known as
cement dust, such material may affect plant growth and develop-
ment by blocking the incident solar radiation and causing abrasion,
leaf heating, stomatal pore obstruction and decreasing in photo-
synthetic rate (Farmer, 1993; Hirano et al., 1995; Grantz et al., 2003;
Pereira et al., 2009; Maletsika et al., 2015). Direct effects of partic-
ulate material uptaken via stomata or cuticle fissures (Lau and Luk,
2001; Eichert and Fern
andez, 2012; Rocha et al., 2014) include
accumulation in leaf tissues of both essential and non-essential
mineral elements (Lau and Luk, 2001). Alkaline particulate mate-
rial can also affect the activity of antioxidant enzymes such as su-
peroxide dismutase, catalase and peroxidase, among others to
varied extents, according to the plant species (Erdal and Demirtas,
2010; Dziri and Hosni, 2012).
Great amount of particulate material is released to the atmo-
sphere during cement production chain, from limestone extraction
to product packaging and shipping (Abdul-Wahab, 2006; Paoli
et al., 2014). Calcium (Ca) is the primary component of cement
dust (Zhao et al., 2008), an alkaline material that can contain Zn, Cu,
Fe, and heavy metals (Cd, Pb, Cr etc) as well (Mandre and Ots, 2012;
Mandre, 2014; Hua et al., 2016). Even tough Ca is an essential
nutrient for plants, its cytosolic concentration must be maintained
at certain levels, otherwise it can be phytotoxic (White and
Broadley, 2003; Hawkesford et al., 2012).
Exposure to cement dust has been shown to be detrimental to
plants (Paal et al., 2013; Paal and Degtjarenko, 2015). A great
amount of Ca was found on leaves surface of Pinus halepensis Mill.
individuals near a cement factory, which, in turn, led to crust for-
mation, epicuticular waxes erosions and stomata obliteration (Ba ci

c Plants and/or soil received two applications of cement dust: one
et al., 1999). Other effects included decrease in leaves chlorophyll
content (Nanos and Ilias, 2007; Maletsika et al., 2015) and plant
vegetative growth and reproduction, cell turgor losses, chlorosis,
necrosis, leaf senescence etc (Farmer, 1993).
Many woody species are sensitive, to different extents, to soil
and leaf alkalization due to contact with alkaline particulate ma-
terial (Farmer, 1993; Mandre et al., 2012a; Mandre and Lukjanova,
2011). According to Mandre et al. (2012b), plants nearby a cement
industry, subjected to low amounts of particulate material via wind
may benefit from it due to increment of soil fertility. Based on these
findings, we hypothesize that particulate cement material is toxic
to C. fissilis plants, which makes this plant species not suitable for
revegetation programs in areas impacted by cement factories ac-
tivities. Then, the objectives of this study were to evaluate the
degree of sensitivity of C. fissilis to cement dust by evaluating the
morphophysiological and biochemical responses of individual
plants subjected to two concentrations of particulate cement
material.
2. Materials and methods
2.1. Cultivation of plants
Seeds of C. fissilis were collected from mother plants cultivated
on the campus of the Universidade Federal de Minas Gerais, Belo
Horizonte, Brazil (19 51047.300 S, 43 580 00.200 W). A voucher spec-
imen was deposited in the BHCB Herbarium, UFMG, MG, under the
number 49,391. Seeds were planted in plastic trays
(440  300  90 mm), containing a mixture of eutrophic soil
57
(saturation base index higher than 50%) and organic compost (1:1).
Forty-five day-old plants were then transferred to polypropylene
pots (5 L; 1 plant/pot) containing the same soil mixture used for
germination, and grown for six months for use in future treatment
applications.
The plants were cultivated throughout the experiment in a
protected greenhouse of the Departamento de Bota ^nica, Uni-
versidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG,
Brazil (19 520 09.300 S, 43 580 0000 W). The soil was irrigated every
2 day at 90% field capacity.
2.2. Application of cement dust
Treatments consisted of exposing, or not, plants to cement at the
concentrations of 2.5 or 5 mg cm2 applied to the leaf surface (Leaf
Treatment), to the soil (Soil Treatment) or simultaneously to the
leaf surface and the soil (Leaf/Soil Treatment). The experimental
design was completely randomized with four replicates for each
factorial combination (3  3) of three concentrations of cement and
three different sites of cement dust application.
Previous analyses were performed with individuals of different
plant species at the immediate vicinity of cement factories to
determine the minimal amount of cement dust to be used in the
greenhouse experiments. The action of natural agents, particularly
wind and moisture, made difficult to assure that amounts of
cement dust as high as 5 mg cm2 were effectively in contact with
plant leaves.
Dust was applied manually for the treatments that received
alkaline dust only to the soil (2.5 or 5 mg cm2 Soil) to prevent
contamination of plants shoot. For the other treatments (2.5 or
5 mg cm2 Leaf or Leaf/Soil), a chamber was used according to
Hirano et al. (1995) to simulate the natural deposition of solid
particulate material. Plants in which leaves were treated solely with
cement dust had the soil was covered with a plastic lid and poly-
ethylene film to prevent cross contamination.
when plants were seven month-old and another one 22 days after
the first application. The replenishment of cement after 22 days
was necessary to cover any leaflets grown after the first application,
as well as to replenish any possible losses on the previously dusted
leaves. The same experimental conditions and concentrations of
particulate material were used for both applications. The nominal
concentrations of the particulate cement material were estimated
by gravimetry from Petri dishes placed inside the chamber.
Applications of cement dust, performed between 9 a.m. and 12
p.m. under natural light conditions, were carried out to until
completely covering leaves and/or soil surfaces. The aerial parts of
the plants and the soil were manually sprayed with deionized
water after each application of the particulate material and every
five days thereafter to simulate the natural conditions of the
impacted area and to ensure the adherence of the pollutants to the
applied surfaces. This procedure is known to potentiate the toxic
effects of cement dust to plants (Maletsika et al., 2015).
The plants were distributed among two shelves positioned
70 cm from the ground and completely covered with white nylon
net (Mesh 14, thread 0.30 mm) and above by a transparent thin film
of polypropylene with maximum irradiance of 1291 mmol m2 s1.
The experiment lasted 41 days from the first cement dust
application as injuries were clearly observed in plants.
2.3. Quantification of particulate material deposited on leaf
surfaces
At the end of the experiment, the amount of cement deposited
on leaf surfaces was determined using the methodology proposed
58 A.I. Siqueira-Silva et al. / Chemosphere 158 (2016) 56e65

by Kuki et al. (2008). Five leaflets from the second node were
randomly harvested from treated and untreated plants Leaflet area
was measured using the software AxioVision 4.9.1 (Carl Zeiss
Microlmaging GmbH, Jena, Deutschland).
2.4. Nutrient content in leaves and chemical features of soil and
cement
contrasted with 0.2% ruthenium red solution (Chamberlain, 1932),
and mounted in Entellan®. Photographic documentation was done
using a photomicroscope (model BX41 TF, Olympus Optical, Tokyo,
Japan) equipped with an image-capture camera (model SC 30,
Olympus Soft Imaging Solutions GmBH, Munster, Germany).
2.6. Electron microscopy

At the end of the experiment all leaves were washed in running
water to remove cement deposited on surface. Leaves were then
dried at 75  C until they reached a constant weight and processed
in a Willye grinder (model TE648, Tecnal Equipamentos para Lab-
orato
rio Ltda, Sa
~o Paulo). Then, 0.5 g of ground plant material was
digested in 10 mL of nitric perchloric acid solution (3:1), following
the methodology proposed by Tedesco et al. (1995). The chemical
feature of cement was also determined from samples digested with
a nitric perchloric acid solution. Soil fertility analysis of all treat-
ments was determined for a composed sample following according
to Defelipo and Ribeiro (1981) and Embrapa (1997).
The levels of Zn, Fe, Mn, Cu, Ca and Mg were determined using
an Atomic Absorption Spectrometer (model SpectrAA-20, Varian,
Victoria, Australia) while K content was determined by flame
photometry (model B 462, Micronal, Sa ~o Paulo, Brazil). Quantifi-
cation of total N was done essentially as described by Malavolta
et al. (1997), and the levels of B, P and S were determined using a
digital spectrophotometer (model E 225D, Celm, Sa ~o Paulo Brazil).
The soil and cement pH values were determined in water (1:2.5;
soil/water and particulate material/water, respectively) using a
digital-microprocessor pH meter (model AJX 511, Micronal, S~ ao
Paulo, Brazil).
2.5. Light microscopy
At the end of the experiment, apparently healthy, totally
expanded second-node leaflet samples (5th leaflets pair from the
basal portion of the leaf blade) grown during the treatments were
fixed in Karnovsky solution (Karnovsky, 1965) for 24 h after being
washed abundantly in deionized water to remove cement dust
from leaf surface. Subsequently, the samples were dehydrated in an
ethanol series and embedded in Leica historesin (Leica Micro-
systems Inc., Heidelberg, Germany), according to the manufac-
turer’s recommendations. Transverse sections (5 mm) were
obtained with a rotary microtome, stained with 0.05% toluidine
blue in a 0.1 M phosphate buffer pH 6.8 (O’Brien et al., 1964),
At the end of the simulation experiment, apparently healthy,
totally expanded second-node leaflet samples (6th leaflets pair
from the basal portion of the leaf blade) grown during the treat-
ments were used for ultrastructural analysis. Unwashed Leaf frag-
ments were fixed in Karnovsky solution for 24 h, dehydrated in an
ethanol series and mechanically dried with CO2 (model CPD 030,
Bal-Tec, Liechtenstein) to critical point. Afterwards leaf fragments
were placed on an aluminum surface and metalized with gold
(model MED 020, Bal-Tec, Liechtenstein) for further analysis using a
scanning electron microscope (Quanta model 200, FEI Company,
Eindhoven, Netherlands), a 15e20 kV.
For transmission electron microscopy analysis, samples were
washed in deionized water to remove the cement deposited on the
leaf surface, fixed in Karnovsky solution for 24 h, post-fixed in 1%
osmium tetroxide (0.1 M phosphate buffer, pH 7.2) and processed
according to standard methods (Roland, 1978). Ultrathin sections
were contrasted with uranyl acetate and lead citrate and examined
in a transmission electron microscope (model Tecnai G2-12 Spirit
BioTWIN, FEI Company, Eindhoven, Netherlands), at 120 kV.
2.7. Determination of photosynthetic parameters
Apparently healthy, fully expanded fifth-node leaflets (5th
leaflets pair from the basal portion of the leaf blade) treated with
cement dust from the first day of experiment were used to assess
photosynthetic parameters in response to the stress. Stomatal
conductance (gs) was determined in intact leaflets of apparently
healthy plants between 10 a.m. and 11 a.m. every ten days using a
porometer (model AP4, Delta-T Devices Ltd, Cambridge, UK).
Leaf surfaces were gently cleaned with a soft-bristle brush 2 h
prior to chlorophyll a fluorescence analysis. The maximum quan-
tum yield of photosystem II (Fv/Fm) was determined between 11
a.m. and 12 p.m. in leaflets submitted to 41 days of treatment, after
60 min of dark adaptation, using a portable pulse-modulated
fluorometer (model MINI-PAM, Walz, Effeltrich, Germany).
After 41 days since the beginning of plant treatments, leaflets

Table 1
Levels of mineral elements in the dry matter of leaflets of Cedrela fissilis after 41 days of exposure to different concentrations of cement applied on the leaf surface (Leaf); on the
soil (Soil) or simultaneously on the leaf surface and the soil (Leaf/Soil).

Concentration Site N K Ca Mg P S Fe B Mn Zn Cu

(mg cm2) (g kg1) (mg kg1)

0 13.4 ± 0.8 Aa 12.0 ± 1.2 14.8 ± 1.3 1.5 ± 0.3 5.2 ± 0.5 2.9 ± 0.2 251.5 ± 27.0 Aa 67.2 ± 4.9 13.3 ± 1.2 17.5 ± 1.2 4.3 ± 0.2 Aa

2.5 Leaf 12.6 ± 1.8 Aa 10.8 ± 1.6 22.5 ± 2.3 2.1 ± 0.3 6.4 ± 0.7 2.9 ± 0.7 101.7 ± 17.3 Bb 65.3 ± 3.9 27.0 ± 4.7 20.7 ± 1.2 4.0 ± 0.0 Aa
Soil 15.5 ± 1.3 Aa 12.1 ± 0.8 22.7 ± 1.1 2.5 ± 0.3 6.2 ± 0.5 3.9 ± 0.6 239.0 ± 34.8 Aa 74.7 ± 6.9 18.5 ± 2.3 23.3 ± 1.3 3.3 ± 0.5 Aab
Leaf/Soil 12.8 ± 1.0 Aa 10.5 ± 0.3 20.5 ± 1.8 1.8 ± 0.2 5.4 ± 0.7 2.5 ± 0.3 83.3 ± 12.2 Bb 51.2 ± 3.0 18.8 ± 2.5 18.8 ± 1.5 1.5 ± 0.3 Bc

5 Leaf 8.7 ± 0.7 Bb 10.3 ± 1.1 20.3 ± 0.8 1.8 ± 0.3 4.9 ± 0.3 1.9 ± 0.2 123.8 ± 18.1 Bb 53.4 ± 1.7 32.8 ± 5.1 16.3 ± 0.5 2.0 ± 0.4 Ab
Soil 15.5 ± 0.4 Aa 10.4 ± 0.8 20.0 ± 1.8 2.5 ± 0.4 6.2 ± 0.4 3.3 ± 0.1 242.8 ± 8.5 Aa 61.1 ± 4.8 15.8 ± 2.5 20.0 ± 1.3 2.3 ± 0.5 Ab
Leaf/Soil 11.2 ± 1.0 Ba 12.3 ± 1.4 18.6 ± 1.4 1.8 ± 0.1 5.1 ± 0.4 2.6 ± 0.2 89.5 ± 10.1 Bb 56.6 ± 4.3 22.3 ± 2.9 15.5 ± 0.9 2.5 ± 0.3 Ab

Analysis of variance
Concentration n.s. n.s. * * n.s. n.s. * * * * *
Site * n.s. n.s. n.s. n.s. * * n.s. * * n.s.
Concentration  Site * n.s. n.s. n.s. n.s. n.s. * n.s. n.s. n.s. *

Means followed by the same letter in a column do not differ according to Newman-Keuls test at 5% probability. Given a concentration, capital letters compare locations; given a
location lowercase letters compare concentrations. (*) indicates a significant interaction between concentration and application location of the cement dust. Means ± standard
error (n ¼ 4), except for the treatment of 2.5 mg cm2, Leaf (n ¼ 3). (n.s.) ¼ not significant.
 A.I. Siqueira-Silva et al. / Chemosphere 158 (2016) 56e65 59

were harvested and washed in deionized water for evaluation of
the levels of chlorophyll a (Chl a), chlorophyll b (Chl b) and carot-
enoids (Barros et al., 2012). Three leaf discs (0.5 cm2) from each
plant were weighed, immersed in 80% acetone. Supernatants were
analyzed in a spectrophotometer (model Genesys 10 UV, Thermo
Spectronic, Rochester, New York, USA) at 663, 646 and 470 nm for
quantification of chloroplastidial pigments according to
Lichtenthaler and Wellburn (1983).
At the end of the experiment, the degree of blockage of photo-
synthetic active photon flux density (PPFD) by the cement dust was
evaluated in gently cleaned leaflets with or without particulate
deposition (2.5 or 5 mg cm2 of cement). The PPFD was measured
using a quantum sensor (model LI-189, LI-COR, Lincoln, NE, USA)
according to Lemos-Filho and Paiva (2006).
2.8. Activity of enzymes of the antioxidant system
Washed and apparently healthy, fully expanded fifth-node
leaflets (6th leaflets pair from the basal portion of the leaf blade)
were harvested at the end of the experiment for the enzymatic

Fig. 1. Leaflets of Cedrela fissilis (AeI) of the control treatment (A, F and H) and treated with cement (BeE, G and I), after 41 days of deposition on the leaf surface (B, C, E and I), on
the soil (D) or simultaneously on the leaf surface and the soil (G), in the concentrations of 2.5 (B, D, G and I) and 5 mg cm2 (C and E). A: Asymptomatic control. B and C: Injuries on
the leaf blade characterized by necrosis (N) and chlorosis. D: Intact leaf blade. E: Necrotic leaf blade with alteration in the pattern of differentiation of the tissues. F: Intact vein. G:
Vein necrosis. H: Intact leaf margin. I: Leaf margin necrosis. Legend: (AdE) adaxial surface of leaflet, (AbE) abaxial surface of leaflet, (PP) palisade parenchyma, (SP) spongy pa-
renchyma, (S) stomata, (Cr) prismatic crystal, (X) xylem, (P) phloem. Bars: 30 mm (AeC); 100 mm (DeI).
60 A.I. Siqueira-Silva et al. / Chemosphere 158 (2016) 56e65

assays. p-nitro blue tetrazolium (NBT) by 50%.

Leaf samples (0.2 g) were ground in a mortar in the presence of
liquid nitrogen and 20 mg of polyvinylpolypyrrolidone. Total pro-
tein was extracted with 700 mL of sodium phosphate buffer (pH 6.8)
0.05 M containing 100 mM EDTA and a protease inhibitor cocktail
(Sigma). The homogenates were centrifuged at 10,000g for
15 min at 4  C and the supernatants used to determine the activity
of superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC
1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11). Enzymatic
activity was express as a function of the total protein content
determined following the method of Bradford (1976).
The SOD activity was measured according to Del Longo et al.
(1993), using 100 mM EDTA. One unit of SOD activity was defined
as the amount of enzyme necessary to inhibit the photoreduction of
The CAT activity was determined according to Havir and McHale
(1987), using sodium phosphate buffer (pH 6.8) instead. The APX
activity was evaluated as described by Nakano and Asada (1981),
with modifications. Reaction medium contained sodium phosphate
(pH 6.0), 1 mM ascorbic acid, 2 mM hydrogen peroxide and plant
extract. The CAT and APX activities were determined by the change
in absorbance at 240 and 290 nm due to the reduction of H2O2 and
oxidation of ascorbic acid, respectively.
2.9. Statistical analysis
The experiment followed a completely randomized design with
four replicates for each factorial combination (3  3) of the three

Fig. 2. Micromorphology of adaxial leaflet surface (A and B) and abaxial leaflet surface (C and D), and ultrastructural aspects of the mesophyll cells of Cedrela fissilis (EeG). Control
treatment (A, C and E). Treatment with cement (B, D, F and G), after 41 days after the deposition on the leaf surface (B, D, F and G), in the concentrations of 2.5 (D and F) and
5 mg cm2 (B and G). A: Turgid epidermal cells and intact tectorial trichomes (T). B: Epidermal cells with loss of turgidity. C: Intact stomata (S). D: Obliterated stomata and stomatal
ledges with altered appearance (ellipse). EeG: Intact plastids (Pl) without disorganization of the membrane system. Legend: (Nu) nucleus, (/) starch grains, (*) plastoglobules.
Bars: 50 mm (A and B); 10 mm (C and D); 1 mm (EeG).
 A.I. Siqueira-Silva et al. / Chemosphere 158 (2016) 56e65 61

cement concentrations and three sites of dust application. To model
each of the dependent variables from the factors of concentration,
site and the interaction of concentration/site, we used the gener-
alized linear model (GLM) followed by the post-hoc Newman-Keuls
test at 5% probability using the software Statistica 7.0 (StatSoft Inc.
2004). The regressions were carried out with the program Sigma
Plot 11.0, Systat Software, Inc., California, USA.
3. Results
3.1. Quantification of the particulate cement material on leaf
surface
At the end of the experiment, the concentrations of the partic-
ulate cement material on the leaf surface were 3.6 and 4.4 mg cm2,
in relation to the nominal concentrations of 2.5 and 5 mg cm2,
respectively.
3.2. Quantification of nutrients in the leaves and chemical
composition of the soil and cement
The pH of cement dust was found to be 13.2 when re-suspended
in water. The Ca (43%) was the major constituent, although Mg
(2.5%), Fe (1.0%), S (0.7%), K (0.6%), Mn (0.3%) and P (0.07%), among
others, were also present.
Cedrela fissilis plants were initially grown on a eutrophic soil
(base saturation index  50%; Embrapa, 2009) at pH in the range
from 5.6 to 7.4 (See Supplementary data, Table S1). Such soil
exhibited high iron concentration (49.5 mg dm3). At the experi-
mental conditions tested, the imposed treatments did not affect the
soil pH or nutrient concentrations (See Supplementary data,
Table S1).
Exposure to cement dust caused an alteration of the mineral
composition of leaflets, except for the elements K and P whose
content remained unchanged (Table 1). Leaf treated with the
highest dust concentration showed lower N levels in comparison to
control ones. The B and Cu levels were also lower than that of
control plants independent of the application site. There was
reduction in the Fe level in leaves (Leaf and Leaf/Soil treatments),
independent of cement dust concentration (Table 1).
Exposure to the pollutant, however, promoted an increase in the
Ca and Mg levels in leaves regardless of the site of cement appli-
cation. The dust also promoted an increase in Mn content in leaves,
especially for plants under Leaf treatment. When applied to the soil
(Soil treatment), the cement dust caused an increase in S and Zn
levels in leaves relative to other treatments (Table 1).
3.3. Morphoanatomical analysis
Injuries were observed in leaves of C. fissilis plants two weeks
after the first application of cement dust on Leaf or Leaf/Soil
(Fig. 1AeC). The symptoms started with chlorosis followed by
marginal and intervein necrosis on apical portion and winding of
leaf blade towards the adaxial face (Fig. 1B and C). Senescence event
and leaf abscission were observed in cement dust-treated plants
(Leaf ones) 25 days post treatment that, in turn, resulted in the
death of some individuals. The leaflets of control (Fig. 1A) and Soil-
treated plants remained asymptomatic until the end of the exper-
iments. C. fissilis has dorsiventral and hypostomatic leaves with
heterogeneous mesophyll formed by a single layer of palisade pa-
renchyma and three to five layers of spongy parenchyma (Fig. 1D, F
and H).
After exposure to cement, leaf blades of plants from the Leaf or
Leaf/Soil treatments exhibited necrosis of epidermal and mesophyll
cells (Fig. 1E, G and I) regardless of the concentration tested.
Alteration in the pattern of tissues differentiation with cells
collapse were likely responsible for the occurrence of thinner leaf
blade in C. fissilis (Fig. 1E, G and I). No structural changes were
observed among leaflets of the Soil treatment (Fig. 1D) at both
concentrations used.
Ultrastructural analysis indicated that the leaf surface and the
stomata of the control plants remained intact (Fig. 2A and C),
differently from the observed for dust-treated plants from Leaf
treatment (Fig. 2B and D). Plants from Leaf treatment exhibited less
turgid epidermal cells (Fig. 2B), stomata with obliterated pores and
distorted leadges (Fig. 2D). Such events were absent in control
plants (Fig. 2A and C). Plastids of C. fissilis presented intact thyla-
koids and apparently uniform stroma (Fig. 2EeG). In contrast to
control plants, plastids of dust-treated plants tended to accumulate
plastoglobules instead of starch grains.
3.4. Physiological analysis
The deposition of cement dust on the leaflets of C. fissilis (Leaf
treatment) caused a reduction in the levels of chlorophyll a, chlo-
rophyll b and total chlorophyll, when compared to plants from the
other treatments (Table 2). A significant negative influence by dust
concentration and location of application, without an interaction

Table 2
Levels of photosynthetic pigments (chlorophyll a e Chl a, chlorophyll b e Chl b, carotenoids e Car and total chlorophyll e TChl) and potential quantum yield of photosystem II
(Fv/Fm) of leaflets of Cedrela fissilis at the end of the experiment with deposition of cement.

Concentration Site Chl a Chl b Car TChl Chl a/b Fv/Fm

(mg cm2) (mg g1 FM)

0 0.53 ± 0.04 0.43 ± 0.04 0.73 ± 0.07 0.96 ± 0.07 1.26 ± 0.03 0.80 ± 0.01

2.5 Leaf 0.39 ± 0.18 0.31 ± 0.14 0.50 ± 0.19 0.70 ± 0.31 1.09 ± 0.16 0.71 ± 0.05
Soil 0.52 ± 0.08 0.43 ± 0.07 0.66 ± 0.09 0.95 ± 0.15 1.22 ± 0.03 0.81 ± 0.01
Leaf/Soil 0.55 ± 0.03 0.49 ± 0.04 0.71 ± 0.03 1.04 ± 0.07 1.11 ± 0.02 0.78 ± 0.01

5 Leaf 0.19 ± 0.13 0.18 ± 0.10 0.32 ± 0.10 0.37 ± 0.24 0.85 ± 0.16 0.72 ± 0.04
Soil 0.55 ± 0.03 0.48 ± 0.03 0.69 ± 0.04 1.04 ± 0.06 1.15 ± 0.01 0.79 ± 0.02
Leaf/Soil 0.48 ± 0.03 0.43 ± 0.03 0.62 ± 0.07 0.91 ± 0.06 1.14 ± 0.06 0.73 ± 0.03

Analysis of variance
Concentration n.s. n.s. * n.s. * *
Site * * * * * *
Concentration  Site n.s. n.s. n.s. n.s. n.s. n.s.

Means followed by the same letter in a column do not differ according to Newman-Keuls test at 5% probability. Given a concentration, capital letters compare locations; given a
location lowercase letters compare concentrations. (*) indicates a significant interaction between concentration and application location of the cement dust. Means ± standard
error (n ¼ 4), except for the treatments of 2.5 and 5 mg cm2 Leaf, (n ¼ 3). (n.s.) ¼ not significant.
62 A.I. Siqueira-Silva et al. / Chemosphere 158 (2016) 56e65
among these factors, was observed on the level of carotenoids and
the ratio of chlorophyll a/b (Table 2). Both concentrations of cement
caused a decrease in the chlorophyll a/b ratio in leaflets. A 10%
decrease in the potential quantum yield (Fv/Fm) of photosystem II
(PSII) in leaflets of Leaf and Leaf/Soil treatments was caused by the
maximum tested concentration of cement dust in comparison to
control plants (Table 2). The particulate material did not affect PSII
performance when applied to soil surface (Soil treatment),
regardless of the concentration tested.
Stomatal closure was observed in the first 10 days of treatment
(Leaf and Leaf/Soil treatments), as evidenced by the reduction in
Fig. 3. Effect of cement dust on stomatal conductance (AeC) of the leaflets of Cedrela
fissilis. Plants of seven months of age treated or not with cement dust of 2.5 or
5 mg cm2 applied on the leaf surface (A); on the soil (B) or simultaneously on the leaf
surface and the soil (C), for a period of 41 days. The points in AeC represent the
mean ± standard error (n ¼ 4, except for the Leaf treatments of 2.5 and 5 mg cm2
after 30 and 41 days, respectively, for which n ¼ 3).
the stomatal conductance (gs) of the leaflets compared to those of
the control plants (Fig. 3AeC). A sharp decrease in gs values was
recorded after 30 days of all treatments, except for the 2.5 mg cm2
Soil treatment, in relation to control (Fig. 3AeC).
A crust was formed on leaf surface of dust-treated plants at 2.5
or 5 mg cm2, which caused the blockade of incoming light
(PPFD  800 mmol m2 s1) by 30 and 50%, respectively (Fig. 4).
Indeed, removal of dust from leaf surface allowed for the reestab-
lishment of light transmission to normal levels as it occurs for
control plants.
3.5. Effect of cement dust on the activity of antioxidant enzymes
The activities of superoxide dismutase (SOD), catalase (CAT) and
ascorbate peroxidase (APX) were 3.3-, 2.4- and 2.7-fold higher in
leaflets of plants subjected to the 2.5 mg cm2 cement treatment
than that of control ones, regardless of the site of dust application
(Table 3). Higher concentration of cement dust, however, led to
SOD, CAT and APX activities similar to those found in leaves of
untreated plants (Table 3).
4. Discussion
Application of cement dust on C. fissilis leaf surface led to
senescence and premature leaf abscission. The effect of cement on
C. fissilis was observed to be dependent upon contact, and there is
no indication of systemic action, especially with regard to structural
damage.
The decrease in the leaf blade thickness was a result of leaf
tissue collapse. Such event was also observed in Pinus sylvestris L.
(Lukjanova and Mandre, 2010; Mandre and Lukjanova, 2011) and
Populus tremula x Populus tremuloides Michx. (Mandre et al., 2012a)
in response to cement dust. The chlorosis in the leaflets exposed to
2.5 or 5 mg cm2 cement dust (Leaf and Leaf/Soil treatments) was
due to the blockade of light caused by cement crust formation and
iron depletion in leaves; the latter is essential for chlorophyll
biosynthesis (Broadley et al., 2012). The biosynthesis of chlorophyll
and carotenoids was slowed down in Datura innoxia Mill. plants
cultivated at different distances from a cement factories exhibited
(Salama et al., 2011). The depletion of Fe in leaves upon cement dust
Fig. 4. Effect of cement dust on photon flux density in photosynthetic active leaflets of
Cedrela fissilis, after 41 days of deposition on the leaf surface at the concentrations of
2.5 or 5 mg cm2. The points represent the mean ± standard error (n ¼ 4). Regression
equations of the treatments (R2 ¼ 0.99): y ¼ 0.0840x  1.4736 (control);
y ¼ 0.0514x  2.7580 (2.5 mg cm2); and y ¼ 0.0408x  2.2058 (5 mg cm2).
 A.I. Siqueira-Silva et al. / Chemosphere 158 (2016) 56e65
Table 3
Effect of cement dust on the activity of enzymes of the antioxidant system of leaflets of Cedrela fissilis after 41 days of exposure.
Concentration SOD
(mg cm2) (U min1 mg1 prot.)
0 33 ± 2 b
2.5 107 ± 27 a
5 90 ± 29 ab
Analysis of variance
Concentration * *
Site n.s.
Concentration  Site n.s.
Means followed by the same letter in a column do not differ according to Newman-Keuls test. (*) indicates a level of significance of p  0.05. (n.s.) ¼ not significant.
Means ± standard error (n ¼ 4 for concentration of 0 mg cm2 and n ¼ 11 for the concentrations of 2.5 and 5 mg cm2, with the data of a particular site being grouped within
each concentration, they do not represent statistically significant differences). Superoxide dismutase e SOD, catalase e CAT and ascorbate peroxidase e APX.
treatment was severe reaching values that are typical of most plant
species under iron nutritional deficiency as determined by Broadley
et al. (2012). Plants growing on soils with pH greater than 7 usually
exhibit Fe deficiency (George et al., 2012) due to its low solubility
under alkali conditions (Mandre and Lukjanova, 2011; Broadley
et al., 2012). Considering that the soil pH was not affected by the
treatments and leaves of control or soil-treated plants did not
exhibit Fe depletion, the lower content of Fe in plant leaves of the
other treatments may be a result of impairment of iron trans-
location to shoots via transpiration by the formation of cement
crust on leaves. This is supported by the fact that the Fe amount
remaining in the soil of treated plants was higher than that of soil
containing control plants and that stomatal conductance of treated
leaves lower compared to control ones. Iron content was also found
to be low in five species (Anchusa leptophylla Roem. & Schult., Se-
necio vernalis Waldst. & Kit., Crambe orientalis L., Euphorbia ori-
entalis L. and Convolvulus sepium L.) growing in to the vicinity of a
cement factory, compared to the control group growing far away
from the factory (Mutlu et al., 2013). Likewise, Eichert and
Ferna
ndez (2012) claimed that the cement dust alkalinity and the
thick cement crust formed on leaves surface contribute to the
decrease of nutrient solubility and absorption by leaves (Eichert
and Ferna
ndez, 2012).
Leaves with low Fe levels, as those of C. fissilis plants grown
under 2.5 or 5 mg cm2 cement dust (Leaf and Leaf/Soil treat-
ments), exhibited lower photosynthetic rate and higher chance to
be photoinhibited and under photooxidation. The lower levels of
photosynthetic pigments in leaves of such treatments can be
attributed to the lower efficiency in capturing and using light (Fv/
Fm). Lower potential quantum yield (Fv/Fm) and gs values were
also previously reported for Clusia hilariana Schltdl. exposed to Fe
particulate material (Pereira et al., 2009). Although Fe is pivotal for
the plants redox system (Broadley et al., 2012), its depletion in C.
fissilis leaves upon cement dust treatment did not affect the activity
of antioxidant enzymes (CAT, SOD and APX) herein investigated.
The increase in the activity of antioxidant enzymes in leaves
under 2.5 mg cm2 cement dust was another indicative that C.
fissilis were under stress. Abiotic stresses are known to contribute
to an increased production of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) in plant cells. The stimulation of
CAT, APX and SOD activities is one of the ways to control the
increased ROS levels and therefore to prevent cell damage. Increase
in antioxidant enzyme activities has been observed in response to
stress in many other plant species (Hung et al., 2005; Zhang et al.,
2007; Horta et al., 2014).
Specifically, increased SOD, CAT and peroxidase activities have
been reported for P. halepensis grown in the vicinity of cement
factories (Dziri and Hosni, 2012). In response to cement dust, ac-
tivities of APX and SOD but not CAT increased in Arabidopsis
63
CAT APX
(mmol min1 mg1 prot.) (mmol min1 mg1 prot.)
650 ± 26 b 86 ± 5 b
1.556 ± 319 a 229 ± 53 a
1.312 ± 354 ab 171 ± 43 ab
*
n.s. n.s.
n.s. n.s.
thaliana (Abu-Romman and Alzubi, 2015). As for C. fissilis, object of
our study, the increment in SOD, APX and CAT activities certainly
was not enough to confer plant tolerance to 2.5 mg cm2 cement
dust as attested by the alteration of some phenotypic features
namely loss of turgidity of the epidermal cells obliteration of sto-
matal pores, stomata deformation. Indeed, SOD, CAT and APX ac-
tivities in plants treated with higher amount of cement dust
(5 mg cm2) were not significantly different from those of control
plants, suggesting that C. fissilis failed to cope with the stress caused
by cement dust. On the other hand, aquatic plants grown near a
cement factory presented lower activity of SOD, CAT and peroxi-
dase, in comparison to control plants, indicating the negative effect
of cement dust on antioxidant enzymes (Erdal and Demirtas, 2010).
In general, plants submitted to cement dust are asymptomatic
or do not present visible symptoms, which can mask the actual
effect of the pollutant (Mandre and Lukjanova, 2011). In situations
like this, and even in symptomatic cases such as that observed for C.
fissilis, the integrated use of morphophysiological and biochemical
variables is critical for the diagnosis and prognosis of injuries
caused by pollutants (Sant’Anna-Santos et al., 2012). The moni-
toring of these plant traits in ex situ studies has been proven to be
valuable to predict the degree of phytotoxicity exhibited by cement
dust on a particular species.
In areas with fully active cement factories, the daily deposition
of cement dust can reduce or eliminate most sensitive species, such
as C. fissilis and favor the most tolerant and/or generalist species. In
fact, decreased plant diversity was observed in regions around
cement factory activity, a condition that was attributed to the
presence of cement dust (Mutlu et al., 2009). In a short period of
shoot exposure to cement dust and prior to any changes in soil pH
and chemical composition, there may be leaf damage in C. fissilis, a
main threat to plant survival in these environments.
5. Conclusions
Cement dust should be considered an atmospheric phytotoxic
pollutant that severely affect C. fissilis, a species here determined to
be highly sensitive to such abiotic stress. The C. fissilis exposure to
alkaline dust causes morphoanatomical (leaf chlorosis and necrosis,
leaf thickness alteration, cell turgor loss, stomatal pore obliteration
and cellular collapse), physiological (decreased photosynthetic
pigments, leaflets nutritional alteration, lower leaf Fe content,
decreased gs and Fv/Fm and early leaves senescence and abscission)
and biochemical (increased CAT, SOD and APX activities) alterations
in their leaflets. The harmful effects of cement dust appear prior to
possible changes in the soil pH and chemical composition, mainly
due to the direct contact of leaves with the stressing agent. The
morphophysiological and biochemical alterations in leaves of C.
fissilis in response to cement dust indicate that this species is not
64 A.I. Siqueira-Silva et al. / Chemosphere 158 (2016) 56e65
suitable for revegetation of areas near cement factories, which also
represent a threat to the survival of the plants that remain in these
areas.
Acknowledgments
The authors thank Conselho Nacional de Desenvolvimento
Científico e Tecnolo
gico (CNPq) for financial support (473261/2010-
3), PhD scholarship to AI Siqueira-Silva and research grants to EAS
Paiva, JP Lemos-Filho and LV Modolo. We thank Josiane A.T. Oliveira
for helping with the experiments and plant harvesting. Raquel O.
Faria kindly provided assistance with the enzymatic analyses. The
Centro de Microscopia da Universidade Federal de Minas Gerais
(http://www.microscopia.ufmg.br) provided infrastructure and
technical support for the ultrastructural analyses. We also thank
two anonymous reviewers for their contributions to the improve-
ment of the manuscript.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.chemosphere.2016.05.047.
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