Ecotoxicology and Environmental Safety 118 (2015) 149–157

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Ryegrass cv. Lema and guava cv. Paluma biomonitoring suitability for
estimating nutritional contamination risks under seasonal climate in
Southeastern Brazil
Patricia Bulbovas n, Carla Z.S. Camargo, Marisa Domingos
Instituto de Botânica, Caixa Postal 68041, São Paulo 04045-972, SP, Brazil

ar t ic l e i nf o a b s t r a c t

Article history: The risks posed by nutrient deposition due to air pollution on ecosystems and their respective services to
Received 8 January 2015 human beings can be appropriately estimated by bioindicator plants when they are well acclimated to
Received in revised form the study region environmental conditions. This assumption encouraged us to comparatively evaluate
17 April 2015
the accumulation potential of ryegrass cv. Lema and guava cv. Paluma macro and micronutrients. We also
Accepted 20 April 2015
Available online 4 May 2015
indicated the most appropriate species for biomonitoring nutrient contamination risks in tropical areas
of Southeastern Brazil, which are characterized by marked dry and wet seasons and complex mixtures of
Keywords: air pollutants from different sources (industries, vehicle traffic and agriculture). The study was conducted
Air pollution in 14 sites with different neighboring land uses, within the Metropolitan Region of Campinas, central-
Nutrient enrichment
eastern region of São Paulo State. The exposure experiments with ryegrass and guava were consecutively
Lolium multiflorum
repeated 40 (28 days each) and 12 (84 days each) times, respectively, from Oct/2010 to Sept/2013. Macro
Psidium guajava
Biomonitoring and micronutrients were analyzed and background concentrations and enrichment ratios (ER) were
Contamination risk estimated to classify the contamination risk within the study region. Significantly higher ER suggested
that ryegrass were the most appropriate accumulator species for N, S, Mg, Fe, Mn, Cu and Zn deposition
and guava for K, Ca, P and B deposition. Based on these biomonitoring adjustments, we concluded that
the nutrient deposition was spatially homogeneous in the study area, but clear seasonality in the con-
tamination risk by nutritional inputs was evidenced. Significantly higher contamination risk by S, Fe, K
and B occurred during the dry season and enhanced contamination risk by Mn, Cu and Zn were high-
lighted during the wet season. Distinctly high contamination risk was estimated for S, Fe and Mn in
several exposure experiments.
& 2015 Elsevier Inc. All rights reserved.

1. Introduction acidification. Disproportional ratios between nitrogen and phos-

phorus or mobile cations, for instance, are commonly reported in
The industrial production, fuel combustion by light and heavy native plant species growing in forest ecosystems affected by an-
vehicles, energy production and fertilizer and herbicide applica- thropogenic N deposition (van den Berg and Ashmore, 2008;
tions in agriculture in major urbanized centers (such as me- Huang et al., 2012).
tropolitan regions) are currently among the most important The risks posed by anthropogenic nutrient deposition on nat-
sources of anthropogenic nutrient deposition (Lehndorff and ural or agricultural ecosystems due to air pollution can be esti-
Schwark, 2010; Sawidis et al., 2011; Boian and Andrade, 2012). mated by bioindicator plants, which are able to react in a pre-
Several pollutants may deposit on natural and agricultural eco- dictable and quantifiable way to environmental disturbances by
systems and occur at toxic levels in their different compartments, changing their chemical composition or vital functions (Arndt and
Schweizer, 1991; Fräinzle, 2003; Markert et al., 2003; Abril et al.,
even if they are essential macro and micronutrients (e.g. nitrogen,
2014). However, we may assume that the validity of risk prediction
sulfur, iron, copper, zinc) (Lehndorff and Schwark, 2010). In such
using bioindicator plant species will depend on their acclimation
condition, reduced biomass production and disturbances in plant
level to the regional natural conditions.
physiology and biochemistry may be expected due to both direct
Ryegrass (Lolium multiflorum Lam. ssp. italicum Beck cv. Lema)
and indirect effects induced by nutritional imbalances and has been well acclimated and appropriately used as a bioaccu-
mulator of trace metals, sulfur, fluorine, and organic pollutants
n
Corresponding author. Fax: þ 55 11 50733678. since the early 1970s in temperate regions. It has shown high
E-mail address: pbulbovas@hotmail.com (P. Bulbovas). tolerance against most air pollutants, without showing any visible

http://dx.doi.org/10.1016/j.ecoenv.2015.04.024
0147-6513/& 2015 Elsevier Inc. All rights reserved.
150 P. Bulbovas et al. / Ecotoxicology and Environmental Safety 118 (2015) 149–157

injury due to environmental pollution levels (VDI, 2003; Klumpp

et al., 2009). Some studies using ryegrass cv. Lema also highlighted
its usefulness as a bioaccumulator in tropical regions (Klumpp and
Klumpp, 1994; Klumpp et al., 1996; Domingos et al., 1998; Sandrin
et al., 2008; Rinaldi et al., 2012; Nakazato et al., 2015).
Wild guava (Psidium guajava) also seemed to be efficient as an
accumulator plant of nitrogen, sulfur, fluorine and some few heavy
metals, mostly micronutrients, in a tropical environment (Moraes
et al., 2002). In addition, some studies revealed that guava cv.
Paluma is also interesting for biomonitoring toxic elements in
tropical regions (Perry et al., 2010; Nakazato, 2014).
However, the satisfactory biomonitor properties of both species
in tropical regions were only detected in areas with high water
availability and affected by high levels of industrial air pollution,
fact that raised the following question: which of them is more
appropriate for biomonitoring anthropogenic nutrient deposition
in tropical areas characterized by marked dry and wet seasons and
generally by complex mixtures of air pollutants emitted by dif-
ferent sources, such as those found in the Metropolitan Region of
Campinas (Southeast, Brazil)? In order to answer this question, the
present study aimed at (1) comparing the accumulation potential
of macro and micronutrients shown by ryegrass cv. Lema and
guava cv. Paluma, indicating the most appropriate species for
biomonitoring risks to natural and agricultural ecosystems asso-
ciated with nutrient deposition in an area typically affected by
alternate dry and wet seasons. (2) Verifying whether the risk
posed by nutrient deposition varies among sites and seasons in the
study region based on leaf accumulation in both cultivars.

Fig. 1. Total monthly rainfall and monthly averages of global radiation, relative
2. Material and methods humidity, temperature, particulate material, nitrogen dioxide and sulfur dioxide in
MRC from October/2010 to September/2013.

2.1. Study area

2.2. Plant cultivation and field exposure
The Metropolitan Region of Campinas (MRC), which is com-
posed of 20 municipalities and located in the central-eastern re- The following procedures were repeatedly performed to pro-
gion of São Paulo State, Brazil (Map, Supplementary material), was duce similar lots of plants in all field experiments. Ryegrass cv.
selected to be subjected to the field experiments because (a) It is Lema seeds (0.8 g per pot) were germinated and cultivated in
the second most important economical center of São Paulo State, plastic pots (1 L) containing a mixture of standardized substrate
which is characterized by different land uses, among cities, high- (Tropstrato Hortaliça HT) and vermiculite (3:1 v/v). During culti-
ways, industries and extensive agricultural lands mainly devoted vation, the plants (approximately 20 grown in each pot) were
to sugarcane, orange and ornamental plantations (CETESB, 2013); weekly excised to a height of 4 cm above the substrate and ferti-
(b) These activities emit considerable amounts of air pollutants lized with macronutrient solution recommended by Epstein
(Tresmondi and Tomaz, 2004), which may potentially cause nu- (1975) (40 cm3 per pot), according to the protocol established by
tritional imbalances in crops and native plants growing in the VDI (2003). Guava cv. Paluma saplings, with approximately 30 cm
Atlantic Semideciduous Forest remnants that still exist in MRC; height and 12 leaves produced from the rooting of semi-herbac-
(c) In addition to local emissions, the MRC is also affected by siz- eous cuttings were taken from a specialized Brazilian producer.
able emissions from the Metropolitan Region of São Paulo (MRSP), Fifteen days before the beginning of each field experiment, the
as a result of the predominantly southerly and southwesterly saplings were transplanted into plastic pots (3.0 L) with the same
winds (Boian and Andrade, 2012); (d) Finally, more than three standardized substrate used for the ryegrass (one sapling per pot).
million people live close to these air, water and soil pollution They were fertilized with 100 ml of a solution containing NPK
sources in MRC, with consequent loss of ecosystem service quality. (20:20:20) and insects and mites were controlled with tiametoxan
The biomonitoring using Lolium multiflorum Lam. ssp. italicum 25% (Syngenta) applications.
Beck cv. Lema (ryegrass) and Psidium guajava cv. Paluma (guava) The plants of both species were kept inside a greenhouse under
was performed in fourteen sites within the MRC, which were ca- charcoal-filtered air and ideal climatic growth conditions
tegorized according to their major neighboring land uses: I1, I2 throughout the cultivation process. They were continuously wa-
and I3 were located near an industrial pole; I/A was placed near tered by nylon strings inserted into the bottom of the pots at one
the industrial pole and agricultural crops; A1, A2, A3, A4, A5, A6 end and immersed in water reservoirs at the other.
and A7 were predominantly surrounded by agricultural crops, After growth, three pots of ryegrass cv. Lema plants were ex-
mainly sugarcane plantation; A/U was located near agricultural posed in each of the fourteen sites for 28 days, following the
crops and an urban area; and U1 and U2 were close to urban areas methods suggested by VDI (2003). Ten potted saplings of guava cv.
(Map, supplementary material). The last one was chosen due to its Paluma were exposed in the same sites on racks 1 m high from the
proximity to an automatic air quality and weather conditions soil surface for 84 days, following well-succeeded methods de-
monitoring station. The sites A1, A4, A5, A6, A7 and A/U were close scribed by Moraes et al. (2002). These exposure experiments with
to the last Atlantic Semideciduous Forest remnants, a subtype of ryegrass cv. Lema cultures and guava cv. Paluma plants were
the Atlantic forest domain in Southeastern Brazil. consecutively repeated 35 and 12 times, respectively, during the
 P. Bulbovas et al. / Ecotoxicology and Environmental Safety 118 (2015) 149–157 151

same experimental period (from October/2010 to September/ enrichment dataset and the nutritional preferences of each accu-
2013), thus minimizing eventual biases due to unequal exposure mulator species, were estimated as previously described. As ex-
times. pected, bve was approximately equal to one in all nutrients. As-
At the end of each exposure experiment, the expanded leaves suming that the higher the ER values above bve, the higher the
from each plant were collected. The leaf samples were oven-dried contamination risk, a scale was suggested to classify the regional
at 60 °C, milled in an agate mill and stored in polypropylene vials contamination risk by each element during the experimental
in order to determine macro and micronutrients. period following the method adopted by Klumpp et al. (2006),
with necessary adaptations. This procedure allowed classifying the
2.3. Chemical analyses contamination risk in all sites and during all the exposure ex-
periments in the following four degrees: I – non-contamination
The N concentration was determined in aliquots of dried and risk (enrichment ratio of a nutrient r bve); II – low contamination
milled leaf samples, after digestion with a solution mixture con- risk (enrichments higher than bve and lower than bve plus three
taining 30% hydrogen peroxide, lithium sulfate, selenium powder times the standard deviation); III – high contamination risk (en-
and sulfuric acid in a digester block that was gradually heated up richments higher than bve plus three times the standard deviation
to 350 °C. The nitrogen concentration was then measured by the and lower bve plus six times the standard deviation); and IV –
Kjeldajhl method (Sarruge and Haag, 1974). Other aliquots of dried distinctly high contamination risk (enrichments Zbve plus six
and milled leaf samples were digested in nitric acid at room times the standard deviation).
temperature for 12 h and then gradually heated up to 160 °C. After A Principal Component Analysis (PCA) was performed with ER
partial evaporation, perchloric acid was added and samples were dataset of both accumulator species, transformed by ranging,
heated up to 210 °C. The resulting extract was diluted with deio- aiming to summarize the total variability of the data and to
nized water and the K, Ca, Mg, B, Cu, Fe, Mn and Zn concentrations highlight the suitability of each species for biomonitoring seasonal
were determined by atomic absorption spectrophotometry. P and and spatial contamination risks posed by anthropogenic nutrient
S were also determined in the same extracts by colorimetric and deposition due to air pollution in the study region, which is
turbidimetric methods, respectively (Malavolta et al., 1997). The characterized by marked dry and wet seasons.
accuracy and precision of the analyses were checked by estab- In addition, aiming to detect eventual nutritional imbalances,
lishing concentrations of the same elements in analytical and concentration ratios between nitrogen and phosphorus (N/P) or
methodological blanks and standard reference material. sulfur (N/S) were calculated only for ryegrass cv Lema, since the N
content in its leaves exceeded the bve in greater proportion than
2.4. Monitoring abiotic conditions that found in guava ‘Paluma’ leaves (see more details in Section 3).
Non-parametric analyses identified significant differences in
Data regarding global radiation, relative humidity and tem- the nutritional enrichments, N/P and N/S among exposure sites
perature as well as concentrations of PM10, NO2 and SO2, which (Kruskal–Wallis One Way Analysis of Variance on Ranks) and wet
characterize the nutrient inputs in the MRC, were obtained from and dry seasons (Mann–Whitney Rank Sum Test), also based on
an automatic monitoring station located at the U2 site (available in data from different exposure experiments.
www.cetesb.sp.gov.br/ar/). Rainfall data and global radiation were All the results (nutrient concentrations, enrichment ratios, N/P
supplied by a meteorological station located next to I2 site, in the and N/S) are presented as box plots. Each plot shows the 25th to
city of Paulínia. 75th percentiles of the original datasets (rectangles), the medians
(horizontal line dividing the rectangles), the error bars and the
2.5. Data evaluation and statistics outlier values (●). The results from each exposure experiment
were individually included in such type of graphic presentation,
First, background values (bv) of each nutrient analyzed in leaf allowing showing all the data variability during the long experi-
samples of both species were estimated, following the method mental period and the application of non-parametric statistics to
described by VDI (2003) and employed by Klumpp et al. (2009). check overall tendencies.
The mean concentration (x1) of each element and respective
standard deviation (s1) were calculated for the original dataset
obtained in all exposure experiments for each species. After that, 3. Results and discussion
single values exceeding a threshold (tv) defined as the mean value
plus 1.96 times the standard deviation (tv ¼x1 þ1.96 * s1) were 3.1. Abiotic conditions during the experimental period
removed from the mentioned dataset and a new mean value and
standard deviation were then calculated. This procedure was re- Environmental parameters varied characteristically among wet
peated until no single concentration exceeded the threshold value. and dry seasons, in all three years of biomonitoring, resembling
The arithmetic mean plus standard deviation of the final adjusted the yearly seasonal pattern previously described in the region by
dataset was considered the background value (bv). After that, an CETESB (2013) and Alvares et al. (2014). Higher values of meteor-
enrichment ratio (ER) was calculated by dividing the single con- ological variables were measured during the wet seasons (spring
centration of each element that composed the original dataset by and summer), notably rainfall, which significantly contributes to
the respective bv. define the climatic typology in the MRC ((Fig. 1). The climate in the
Significant differences between the enrichment ratio of each region is predominantly Cwa according to Koeppen’s classification
nutrient in ryegrass cv. Lema and guava cv. Paluma leaves were (humid subtropical zone with dry winter and hot summer) (Al-
identified by non-parametric Mann–Whitney Rank Sum Test, vares et al., 2014). Monthly total rainfall and mean temperature
based on the enrichment ratio calculated in the different exposure generally reach 200 mm and 24 °C, respectively, during the wet
experiments. These comparisons allowed identifying which nu- season (from October to March) and drop down to 30 mm and
trients were more strongly accumulated in the leaves of each 20 °C during the dry season (from April to September),
biomonitor species, allowing performing the next step of data respectively.
treatment that focused on the evaluation of spatial and seasonal In opposition to the meteorological variables, monthly mean
variations in the nutritional enrichments within the study region. values of nitrogen and sulfur dioxide and particulate material that
Thus, new background values (bve), now based on the may carry different macro and micronutrients were
152 P. Bulbovas et al. / Ecotoxicology and Environmental Safety 118 (2015) 149–157

Cu * Cu
Zn Zn
B B
Mn * Mn
Fe * Fe
Mg * Mg
P P
S S
Ca Ca
N *N
K K

0,0001 0,001 0,01 0,1 1 10 100 1000 0 2 4 6 8

element concentration (g Kg -1) enrichment

Cu Cu
Zn Zn
B B
*
Mn Mn
Fe Fe
Mg Mg
P
* P
S S
Ca Ca
*
N N
K
* K

0,0001 0,001 0,01 0,1 1 10 100 1000

0 2 4 6 8
element concentration (g Kg-1) enrichment
Fig. 2. Box plot representation of the concentration ranges and respective enrichment ratios relative to background values (red symbols) in leaf samples of ryegrass cv. Lema
(A) and guava cv. Paluma (B) throughout all the experimental period. Values greater than one (indicated by the red line) denote enrichment. n indicates significant higher
enrichment ratio of a nutrient in one accumulator species in comparison to that estimated in the other species (p o0.05, Mann–Whitney Rank Sum Test). (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)

distinguishably higher during the dry seasons (Fig. 1). Maximum cv. Lema ranged from 0.001 g kg  1 of Cu to 2.13 g kg  1 of Fe and in
mean values of 63 μg NO2 m  3, 30 μg SO2 m  3 and 94 μg PM10 guava cv. Paluma from 0.001 g kg  1 of Cu to 1.3 g kg  1 of Fe
m  3 were recorded during the dry season. By contrast, lower (Fig. 2). The background concentrations of N, P, K, Mg, Cu and Fe
concentrations were measured during the wet season (45 μg NO2 tended to be higher in ryegrass cv. Lema than in guava cv. Paluma,
m  3, 24 μg SO2 m  3 and 63 μg PM10 m  3). In fact, the con- whereas the background concentrations of Ca, B and Mn found in
centration of air pollutants in the MRC is directly influenced by the guava cv. Paluma were higher than in ryegrass cv. Lema (Fig. 2).
predominant weather conditions, in addition to the distribution Nakazato (2014) worked around a petrochemical industry in Cu-
and intensity of pollutant emissions and topography. As a rule, the batão region (SE Brazil) and found higher background values of N
highest concentrations of pollutants occur, except ozone, from and K in ryegrass cv. Lema and of K and Zn in guava cv. Paluma
April to September (dry season), due to the higher occurrence of than those estimated in the current study.
thermal inversions at low atmosphere levels, high percentage of In addition, the concentrations of most of the nutrients mea-
slight winds and low rainfall (Boian and Andrade, 2012; CETESB, sured in several single samples of both ryegrass cv. Lema and
2013). guava cv. Paluma were above the respective background values,
indicating that these samples were enriched by these nutrients
3.2. Comparing nutrient accumulation potential of ryegrass cv. Lema (Fig. 2), similarly to the results found by Nakazato (2014). There-
and guava cv. Paluma fore, the estimate of enrichment ratios in relation to background
concentrations would give a more reliable overview of the bio-
The concentrations and the background values in ryegrass cv. monitor plants accumulation capacity and their suitability for
Lema and guava cv. Paluma decreased similarly according to the biomonitoring purposes than of the proper leaf concentrations, as
following order of magnitude: K4N 4Ca 4S4 P4Mg 4Fe4 discussed in other biomonitoring studies (Domingos et al., 1998;
Mn 4B 4Zn 4Cu (Fig. 2). The minimum and maximum con- Moraes et al., 2002; Perry et al., 2010). In fact, ER 41 was fre-
centrations of macronutrients in ryegrass cv. Lema ranged from quently estimated during the experimental period for all nutrients
0.5 g kg  1 of Ca to 131.0 kg  1 of K, respectively, and in guava cv. in leaf samples of both bioacumulator species (Fig. 2).
Paluma from 1.2 kg  1 of Mg and S to 76.5 g kg  1 of K. The mini- Ryegrass cv. Lema plants showed significantly higher enrich-
mum and maximum concentrations of micronutrients in ryegrass ments of N (median ERN ¼0.92), Mg (median ERMg ¼1.03), Fe
 P. Bulbovas et al. / Ecotoxicology and Environmental Safety 118 (2015) 149–157 153

Axis 2 (20%)
Sites
Lw Industrial
Lw Lw
Industrial/Agriculture
0,6 Agriculture
Lw Lw
Lw Agriculture/Urban
Lw Lw Lw Urban
Lw
Lw
Lw Lw Pd
N Pd Pw Pw
Lw Pd Pd
0,2 Pd
Pw Mn
Pd Pd Pd Pd
Pd Pd Axis 1 (30%)

-0,8 -0,4 Pd 0,0 0,4 0,8

P
Ld Pd Pw
Pd Pw Pw
Ld -0,2 K
Fe Pw Pw Pw
Ld Ld B
Ld Pw Pw
Ld Mg Pw
Ld Pw Pw
Ld Ld
Ld Ld Ld Ld S
-0,6

Ld

-1,0

Axis 3 (17%)
1,0

Ld

0,6
Lw
Mg Lw
Cu Lw Mn
Lw Lw Pw
Lw Ld
Ld Lw Pw
Lw Lw Lw Pw
Lw 0,2 Pw Pw
Ld Ld Lw Lw Pw Pw
Ld Pd Ca Pw Pw
Ld Ld N Lw Axis 1 (30%)
Ld
-0,8 Ld Fe -0,4 0,0 0,4 K P 0,8 Pw
Pw
Ld
Pd Pd
Ld -0,2
Ld
Pd Pd
Ld Pd Pd
Pd Pd
Pd Pw
Pd Pw
Pd Pd Pw
Pd
-0,6

K N Ca S P Mg Fe Mn B Zn Cu
PC 1 0.78 -0.47 0.44 -0.28 0.87 -0.23 -0.72 0.49 -0.06 -0.11 -0.22
PC 2 0.40 0.49 -0.19 -0.81 -0.32 -0.56 -0.46 0.39 -0.47 0.24 0.31
PC 3 -0.11 -0.09 -0.11 0.41 -0.18 0.71 -0.17 0.67 0.11 0.40 0.67
Fig. 3. Principal Components Analysis (PCA) summarizing the nutritional enrichment ratios in both accumulator species. The table shows the correlation coefficients of each
variable with the most explicative principal components (PC 1 to 3). Abbreviations: L – sampling unities of ryegrass cv. Lema; P – sampling unities of guava ‘Paluma’; d – dry
season; w – wet season. K – potassium; N – nitrogen; Ca – calcium; S – sulfur; P – phosphorus; Mg – magnesium, Fe – iron; Mn – manganese; B – boron; Zn – zinc; Cu – copper.

(median ERFe ¼1.21), Mn (median ERMn ¼1.06) and Cu (median to certain elements (Fig. 3). The analysis showed that 67% of the
ERCu ¼0.91) than guava cv. Paluma plants (median ERN ¼0.90, variability of data from both ryegrass cv. Lema and guava cv. Pa-
ERMg ¼0.87, ERFe ¼ 1.04, ERMn ¼0.98, ERCu ¼0.90). On the other luma were summarized in the first 3 axes (Fig. 3). Sampling units
hand, the last species presented higher ability to accumulate K of ryegrass cv. Lema exposed during the wet season were more
(median ERK ¼0.93), Ca (median ERCa ¼ 0.89), P (ERP ¼1.06) and B related to high concentration of N (positive side of axis 2), Mg and
(ERB ¼0.81) over the background levels than ryegrass cv. Lema Cu (positive side of axis 3), and sampling units of plants exposed in
(median ERK ¼ 0.88, ERCa ¼ 0.88, ERP ¼0.84, ERB ¼0.76). No sig- the dry season were more related to high concentration of Fe
nificant differences were found between the capacities of both (negative side of axis 1) and S (negative side of axis 2) (Fig. 3).
accumulator species to enlarge S and Zn concentrations in the Sampling units of guava plants exposed during the wet season
leaves over the respective bv (Fig. 2). Nakazato (2014) also found were associated to higher values of K and P (positive side of axis
higher enrichments of Mg, Fe, Cu, S and Zn in ryegrass cv. Lema 1). Guava cv. Paluma exposed during the dry season showed low
and of B, Fe, N and P in guava cv. Paluma (Fig. 2). The author as- correlation with all elements (Fig. 3).
sociated N, S, P, Fe, B and Zn to oil refinery emissions and Mg to sea
salt that comes from the Atlantic Sea. 3.3. Spatial and seasonal nutritional enrichments in the study region
The Principal Component Analysis (PCA) performed with en- based on leaf accumulation
richment values highlighted the differences between species ex-
posed in different seasons and allowed seeing the species affinity According to the data treatment planning, the evaluation of
154 P. Bulbovas et al. / Ecotoxicology and Environmental Safety 118 (2015) 149–157

I II III IV I II III IV I II III IV I II III IV

Dry *Dry Dry *Dry
U2 U2 U2 U2
U1 U1 U1 U1
A/U A/U A/U A/U
A7 A7 A7 A7
A6 A6 A6 A6
A5 A5 A5 A5
A4 A4 A4 A4
A3 A3 A3 A3
A2 A2 A2 A2
A1 A1 A1 A1
I/A I/A I/A I/A
I3 I3 I3 I3
I2 I2 I2 I2
I1

sites
sites

I1 I1 I1

sites

sites
U2 U2 U2 U2
U1 U1 U1 U1
A/U A/U A/U A/U
A7 A7 A7 A7
A6 A6 A6 A6
A5 A5 A5 A5
A4 A4 A4 A4
A3 A3 A3 A3
A2 A2 A2 A2
A1 A1 A1 A1
I/A I/A I/A I/A
I3 I3 I3 I3
I2 I2 I2 I2
I1 I1 I1 I1
Wet Wet Wet Wet
0 1 2 3
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 5 6 7
nitrogen sulfur magnesium iron

I II III IV I II III IV I II III IV

Dry U2
Dry Dry
U2 U2
U1 U1 U1
A/U A/U A/U
A7 A7 A7
A6 A6 A6
A5 A5 A5
A4 A4 A4
A3 A3 A3
A2 A2 A2
A1 A1 A1
I/A I/A I/A
I3 I3 I3
I2 I2 I2
sites

sites
sites

I1 I1 I1
U2 U2 U2
U1 U1 U1
A/U A/U A/U
A7 A7 A7
A6 A6 A6
A5 A5 A5
A4 A4 A4
A3 A3 A3
A2 A2 A2
A1 A1 A1
I/A I/A I/A
I3 I3 I3
I2 I2 I2
I1 I1
*Wet I1
*Wet *Wet
0 1 2 3 4 5 6 0 1 2 3 4 0 1 2 3 4 5
manganese copper zinc

Fig. 4. Boxplot representation of enrichment ratios in leaf samples of ryegrass cv. Lema exposed in different sites in MRC during wet and dry seasons. I1, I2, and I3 –
industrial areas; I/A – industrial and agricultural areas; A1, A2, A3, A4, A5, A6 and A7 – agricultural areas; A/U – agricultural and urban areas; U1 and U2 – urban areas. n
indicates significant higher enrichment ratios in one season in comparison to the other (p o0.05; Mann–Whitney Rank Sum Test). Contamination risk classification: I. Non-
contamination; II. Low contamination; III. Elevated contamination; IV. Distinctly elevated contamination.

spatial and seasonal variations in the nutritional enrichments in were evidenced by both significant differences between dry and
the study region (Figs. 4–6) was based on the results of the wet seasons and distinct degrees of contamination risk estimated
mentioned statistical comparisons between cultivars. Although during the whole experimental period (Figs. 4 and 5), thus re-
significant differences were not proved between both accumulator inforcing tendencies shown by PCA analysis (Fig. 3). Significant
plants for S and Zn, the ER dataset of both elements in the ryegrass enhanced enrichment of S and Fe (in ryegrass leaf samples) and of
cv. Lema cultures was employed to compare sites and seasons due K and B (in guava leaf samples) were estimated during the dry
to two reasons: (a) maximum single ER for S and Zn were calcu- seasons. We may assume that these higher ER were consequences
lated in ryegrass plants leaf samples (ER 44.0); (b) the ryegrass cv. of a greater supply of these elements to the plants during the dry
Lema is indicated by VDI (2003) as a standardized biomonitor of S seasons, when atmospheric concentrations of SO2 and particulate
and metals. material containing nutrients, such as S, Fe, K and B, were also
First, it is noteworthy that similar nutritional enrichment ratios observed (Fig. 1). One probable source of Fe to ryegrass in the MRC
were estimated in all sites throughout the biomonitoring period, during the dry seasons was the resuspension of oxisol soils that
whether ryegrass cv. Lema or guava cv. Paluma leaf samples were are rich in such element according to Lopes et al. (2015), due to the
analyzed or not (Figs. 4 and 5). This non-spatiality may be ex- traffic in unpaved roads commonly observed in rural areas.
plained by the daily wind circulation. Although the yearly wind Moreover, crop fertilization with compounds containing S, K and B
prevailing direction (Map, supplementary material) in the MRC is in their composition and vinasse applications in sugarcane, with
southeast, the wind direction changes typically during the day: high K concentration (Christofoletti et al., 2013) generally occur at
(a) the predominant wind directions are SSE and SE in early the end of the dry seasons, contributing to nutritional inputs and
morning and evening; (b) SSE, SE and NE in the morning and possibly imbalances to the bioaccumulator species and, by ana-
(c) NNE, SSE, N, SSW and S in the afternoon. Then, prevailing logy, to the forest remnants that still exist in the study region.
winds stay between east and south during a single day (Tresmondi Therefore, these agricultural practices may pose a contamination
and Tomaz, 2004; Boian and Andrade, 2012). This typical daily risk to be routinely monitored in the region.
wind behavior might have dispersed the air pollutants to all ex- The ryegrass also revealed to be an appropriate accumulator
posure sites, causing a uniform plant exposure to air pollutants. In plant of S and Fe in the study region by analyzing ER ranges.
addition, the regional flat topography does not offer barriers Median values of S enrichments largely reached the low con-
against the pollution dispersion (Boian and Andrade, 2012), fa- tamination risk (included in class II) and many individual enrich-
voring the mixture of pollutants in the study area. ment values were classified in the III and IV degrees (high or
In contrast, seasonal variations in the nutritional enrichment distinctly high contamination) in most sites. Most of the Fe
 P. Bulbovas et al. / Ecotoxicology and Environmental Safety 118 (2015) 149–157 155

I II III IV I II III IV

U2
*Dry Dry
U2
U1 U1
A/U A/U
A7 A7
A6 A6
A5 A5
A4 A4
A3 A3
A2 A2
A1 A1
I/A I/A
I3 I3
I2 I2
I1
sites

I1

sites
U2 U2
U1 U1
A/U A/U
A7 A7
A6 A6
A5 A5
A4 A4
A3 A3
A2 A2
A1 A1
I/A I/A
I3 I3
I2 I2
I1 I1
Wet Wet
0 1 2 3 0 1 2
potassium calcium
I II III IV I II III IV

U2
Dry *Dry
U2
U1 U1
A/U A/U
A7 A7
A6 A6
A5 A5
A4 A4
A3 A3
A2 A2
A1 A1
I/A I/A
I3 I3
I2 I2
I1 I1
sites
sites

U2 U2
U1 U1
A/U A/U
A7 A7
A6 A6
A5 A5
A4 A4
A3 A3
A2 A2
A1 A1
I/A I/A
I3 I3
I2 I2
I1 I1
Wet Wet

0 1 2 3 4 5 0 1 2 3
phosphorus boron
Fig. 5. Boxplot representation of enrichment ratios in leaf samples of guava cv. Paluma in different sites in MRC during wet and dry seasons. I1, I2, and I3 – industrial areas; I/
A – industrial and agricultural areas; A1, A2, A3, A4, A5, A6 and A7 – agricultural areas; A/U – agricultural and urban areas; U1 and U2 – urban areas. n indicates significant
higher enrichment ratios in one season in comparison to the other (p o 0.05; Mann–Whitney Rank Sum Test). Contamination risk classification: I. Non-contamination; II. Low
contamination; III. Elevated contamination; IV. Distinctly elevated contamination.

enrichment median values were included in the high contamina- containing these elements from the petrochemical industries and
tion class (III), and a large amount of enrichment values reached traffic of vehicles (Conti et al., 2009; Lehndorff and Schwark, 2010;
distinctly high contamination risk (degree IV), such as in the urban Calvo et al., 2013; Nakazato et al., 2015).
site U2, located in Paulínia city (Fig. 4). The ryegrass plants ex- However, K enrichment in guava leaves suggested absence of
posed in this urban site also tended to accumulate Zn and Cu contamination risk (degree I) even during the dry seasons. As for
during the dry season, indicating distinctly high contamination in B, despite the fact that most ER were included in the first
some exposure experiments (Fig. 4). These tendencies observed in class, some single values were classified in the second degree
U2 site may be associated with emissions of particulate matter (low contamination risk), rarely reaching higher potential
156 P. Bulbovas et al. / Ecotoxicology and Environmental Safety 118 (2015) 149–157

Dry Dry
U2 U2
U1 U1
A/U A/U
A7 A7
A6 A6
A5 A5
A4 A4
A3 A3
A2 A2
A1 A1
I/A I/A
I3 I3
I2 I2
I1

sites
I1
sites

U2 U2
U1 U1
A/U A/U
A7 A7
A6 A6
A5 A5
A4 A4
A3 A3
A2 A2
A1 A1
I/A I/A
I3 I3
I2 I2
I1 I1
* Wet * Wet

0 5 10 15 20 25 0 2 4 6 8 10

N/S N/P
Fig. 6. Boxplot representation of the N/S and N/P ratios in leaf samples of ryegrass cv. Lema exposed in different sites in MRC during wet and dry seasons. I1, I2, and I3 –
industrial areas; I/A – industrial and agricultural areas; A1, A2, A3, A4, A5, A6 and A7 – agricultural areas; A/U – agricultural and urban areas; U1 and U2 – urban areas. n
indicate significant difference between wet and dry seasons (p o 0.05, Mann–Whitney Rank Sum Test).

contamination level in the U2 urban site (elevated contamination terms of the amount of water consumed by Brachiaria brizantha
or distinctly elevated contamination risks, respectively) (Fig. 5). plants. They reasoned that the sulfur required by plants, although
Mn, Cu and Zn were proportionally more concentrated than the small, is closely related to the nitrogen absorption and the meta-
respective background values and N/S and N/P ratios were higher bolism in plants. It explains why high ratios between nitrogen and
in ryegrass exposed during the wet season (Figs. 4 and 6). Mn was sulfur were found during the wet seasons (Fig. 6), periods when
the most enriched element in ryegrass plants during the wet higher water availability and lower SO2 concentration were ob-
season. Most median ER were classified in the second con- served (Fig. 1). High N/P ratios were consequences of high N
tamination degree and a considerable number of ryegrass samples concentrations and/or low P levels in ryegrass cv. Lema exposed in
accumulated more than twice Mn when compared to the back- the field during the wet seasons (Fig. 6). Huang et al. (2012) stated
ground concentration, indicating high to distinctly high con- that several species have leaf contents of N and P positively cor-
tamination risks in all MRC sites (III and IV degrees, respectively). related in a highly significant level, in order not to limit their
The dataset of Cu and Zn enrichments were mainly classified be- productivity. However, this association was not proved in the
tween degrees I and II; only few enrichment values were included present study, as shown by the PCA analysis (Fig. 3). The P and N
in the 3rd and 4th contamination risk classes (Fig. 4). Although vectors positioned on the opposite sides of axis 1 indicated that
Mn, Cu and Zn are essential and non-toxic in low concentrations to the stoichiometric balance between both elements was changed
plants, their levels may increase in a polluted environment due to by increases in the atmospheric deposition of N compounds pos-
atmospheric deposition derived from vehicular traffic (Conti et al., sibly of anthropogenic origin. In fact, depending on the intrinsic
2009; Klumpp et al., 2009; Guzmán-Morales et al., 2011), also features of species, the high N availability in the environment does
increasing the possibility of toxic effects on living organisms. not ensure the high accumulation of other elements (Güsewell,
Therefore, Mn toxic effects might be more probable than those of 2004), including P. Thus, the N/P ratio in leaves was also a mea-
Cu and Zn in the MRC. According to Klumpp et al. (2009), the surement of the potential limitation of P not only to bioindicator
maximum Cu and Zn values measured in ryegrass plants exposed plants, but also to crops and native plants of forest ecosystems in
in the MRC (0.064 g kg  1 and 0.158 g kg  1, respectively) were far the MRC, according to Huang et al. (2012).
below the recommended limits for animal feed, contrasting with
other biomonitoring studies. For example, high concentrations of
Cu and Zn and other traffic-related elements were observed in 4. Conclusions
ryegrass cv. Lema after exposure in Spanish cities (Klumpp et al.,
2009). It was also observed in Tillandsia capillaries exposed in The results led us to the following conclusions: (a) Significantly
Cordoba, Argentina (Abril et al., 2014). higher enrichment ratios (ER) pointed that ryegrass cv. Lema was
The ryegrass plants tended to accumulate more nitrogen during the most appropriate accumulator species for biomonitoring the
the wet season, an expected result if we consider that water plays contamination risk associated with N, S, Mg, Fe, Mn, Cu and Zn
an important role in the nitrogen uptake by plants, and thus in the deposition from several emission sources in the MRC, such as
absorption of other nutrients (Artur et al., 2014). These authors vehicular traffic, petrochemical industries and agricultural prac-
found significant interaction between nitrogen and sulfur rates in tices; (b) Significantly higher enrichment ratios also revealed that
 P. Bulbovas et al. / Ecotoxicology and Environmental Safety 118 (2015) 149–157 157

the guava plants were the most appropriate accumulator species Christofoletti, C.A., Escher, J.P., Correia, J.E., Marinho, J.F.U., Fontanetti, C.S., 2013. Su-
garcane vinasse: environmental implications of its use. Waste Manag. 33,
for biomonitoring K, Ca, P and B deposition originated from crop
2752–2761.
fertilization procedures, such as the vinasse application in ex- Conti, M.E., Pino, A., Botrè, F., Bocca, B., Alimonti, A., 2009. Lichen Usnea barbata as
tensive sugarcane plantations, which is rich in K; among other biomonitor of airborne elements deposition in the Province of Tierra del Fuego
(southern Patagonia, Argentina). Ecotoxicol. Environ. Saf. 72, 1082–1089.
nutrients; (c) Based on the mentioned biomonitoring capacities of Domingos, M., Klumpp, A., Klumpp, G., 1998. Air pollution impact on the Atlantic Forest
both accumulator species, we may state that the nutrient de- at the Cubatão region, SP, Brazil. Cienc. Cult. 50, 230–236.
Epstein, E., 1975. Nutrição Mineral das Plantas. Princípios e Perspectivas. Editora da
position associated with air pollution is spatially homogeneous in Universidade de São Paulo/Livros Técnicos e Científicos, Editora S.A., Rio de Janeiro.
the entire study area; (d) In contrast, clear seasonality in the Fräinzle, O., 2003. Bioindicators and environmental stress assessment. In: Markert, B.A.,
contamination risk by nutritional inputs were evidenced by both et al. (Eds.), Bioindicators and Biomonitors – Principles, Concepts and Applications.
Elsevier Science Ltd., Oxford, pp. 41–84.
accumulator cultivars, either in response to strong climatic sea- Güsewell, S., 2004. N:P ratios in terrestrial plants: variation and functional significance.
sonality that influence air pollution dispersion or to specific agri- New Phytol. 164, 243–266.
Guzmán-Morales, J., Morton-Bermea, O., Hernández-Álvarez, E., Rodríguez-Salazar, M.T.,
cultural practices; significantly higher contamination risk by S, Fe García-Arreola, M.E., Tapia-Cruz, V., 2011. Assessment of atmospheric metal pollu-
(indicated by ryegrass plants), K and B (indicated by guava plants) tion in the urban area of Mexico city, using Ficus benjamina as biomonitor. Bull.
Environ. Contam. Toxicol. 86, 495–500.
occurred during the dry season; enhanced contamination risk in-
Huang, W.J., Zhou, G.Y., Liu, J.X., 2012. Nitrogen and phosphorus status and their influ-
dicated by higher ER of Mn, Cu and Zn, as well as of N/S and N/P ence on aboveground production under increasing nitrogen deposition in three
ratios in ryegrass cv. Lema leaf samples were highlighted during successional forests. Acta Oecol. 44, 20–27.
Klumpp, A., Ansel, W., Klumpp, G., Vergne, P., Sifakis, N., Sanz, M.J., Rasmussen, S., Ro-
the wet season. Poulsen, H., Ribas, A., Peñuelas, J., Kambezidis, H., He, S., Garrec, J.P., Calatayud, V.,
2006. Ozone pollution and ozone biomonitoring in European cities Part II. Ozone-
induced plant injury and its relationship with descriptors of ozone pollution. Atmos.
Environ. 40, 7437–7448.
Acknowledgments Klumpp, A., Ansel, W., Klumpp, G., Breuer, J., Vergne, P., Sanz, M.J., Rasmussen, S., Ro-
Poulsen, H., Ribas, A.A., Peñuelas, J., He, S., Garrec, J.P., Calatayud, V., 2009. Airborne
trace element pollution in 11 European cities assessed by exposure of standardised
The authors thank Programa SISBIOTA-BRASIL (proc. CNPQ ryegrass cultures. Atmos. Environ. 43, 329–339.
563335/2010; proc. FAPESP 2010/52319-2) for financial support. Klumpp, A., Domingos, M., Klumpp, G., 1996. Assessment of the vegetation risk by
fluoride emissions from fertiliser industries at Cubatão, Brazil. Sci. Total Environ. 92,
The Refinaria de Paulínia (REPLAN), Usina Ester, Fazenda São José, 219–228.
Sítio Americana, Cooperativa Agropecuária Holambra, Haras Patente, Klumpp, A., Klumpp, G., 1994. Plants as bioindicators of air pollution at the serra do mar
near the industrial complex of Cubatão, Brazil. Environ. Pollut. 85, 109–116.
sítio Cosmópolis, Áreas de Relevante Interesse Ecológico “Mata da Lehndorff, E., Schwark, L., 2010. Biomonitoring of air quality in the Cologne Conurbation
Santa Genebra”, Prefeitura de Paulínia, Companhia de Saneamento using pine needles as a passive sampler e Part III: Major and trace elements. Atmos.
Environ. 44, 2822–2829.
Básico do Estado de São Paulo (SABESP) for permitting the plant
Lopes, M.I.M.S., Santos, A.R., Camargo, C.Z.S., Bulbovas, P., Giampaoli, P., Domingos, M.,
exposure in their own areas; Norddeutsche Pflanzenzucht Hans- 2015. Soil chemical and physical status in semideciduous Atlantic Forests affected
Georg Lembke KG (NPZ-Lembke) for donating seeds of ryegrass cv. by atmospheric deposition in central-eastern of São Paulo State, Brazil. iForest. http:
//dx.doi.org/
Lema Coordenação Especial para Restauração de Áreas Degradadas- 10.3832/ifor1258-007, in press.
CERAD and Paulo R.T. Ortiz for designing Map (supplementary Malavolta, E., Vitti, G.C., Oliveira, S.A., 1997. Avaliação doe studo nutricional das plantas:
princípios e aplicações. Associação Brasiliera Para Pesquis da Potassa e do Fosfato,
material); Amariles C. de Souza, Andressa R. dos Santos, Cristiane Piraciaba – SP.
Aguiar Silva, Francisco R. da Silva, Leonardo K. Fujita, Patricia Markert, B.A., Breure, A.M., Zechmeister, H.G., 2003. Definitions, strategies and principles
Giampaoli, Solange E. Brandão, and Valdenice S. Amorim, for for bioindication/biomonitoring of the environment. In: Markert, B.A., et al. (Eds.),
Bioindicators and Biomonitors – Principles, Concepts and Applications. Elsevier
technical assistance. Science Ltd., Oxford, pp. 3–39.
Moraes, R.M., Klumpp, A., Furlan, C.M., Klumpp, G., Domingos, M., Rinaldi, M.C.S., Mod-
esto, I.F., 2002. Tropical fruit trees as bioindicators of industrial air pollution in
southeast Brazil. Environ. Int. 28, 367–374.
Appendix A. Supplementary material Nakazato, R.K., 2014. Caracterização de riscos à Floresta Atlântica associados à con-
taminação atmosférica por elementos tóxicos, no entorno de uma refinaria de pet-
róleo, em Cubatão/São Paulo, com plantas acumuladoras [Thesis]. Instituto de Bo-
Supplementary data associated with this article can be found in tânica da Secretaria de Estado do Meio Ambiente Available from the World Wide
the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.04. Web. 〈http://www.ambiente.sp.gov.br/pgibt/dissertacoesteses/〉.
Nakazato, R.K., Rinaldi, M.C.S., Domingos, M., 2015. Will technological modernization for
024. power generation at an oil refinery diminish the risks from air pollution to the
Atlantic Rainforest in Cubatão, SE Brazil? Environ. Pollut. 196, 489–496.
Perry, C.T., Divan Jr., A.M., Rodriguez, M.T.R., Atz, V.L., 2010. Psidium guajava as a bioac-
cumulato rof nickel around an oil refinery, southern Brazil. Ecotoxicol. Environ. Saf.
References 73, 647–654.
Rinaldi, M.C.S., Domingos, M., Dias, A.P.L., Esposito, J.B.N., Pagliuso, J.D., 2012. Leaves of
Lolium multiflorum ‘Lema and tropical tree species as biomonitors of polycyclic
Abril, G.A., Wannaz, E.D., Mateos, A.C., Pignata, M.L., 2014. Biomonitoring of airborne aromatic hydrocarbons. Ecotoxicol. Environ. Saf. 79, 139–147.
particulate matter emitted from a cement plant and comparison with dispersion Sandrin, C.Z., Figueiredo-Ribeiro, R.C.L., Carvalho, M.A.M., Delitti, W.B.C., Domingos, M.,
modelling results. Atmos. Environ. 82, 154–163. 2008. Sub-tropical urban environment affecting content and composition of non-
Alvares, C.A., Stape, J.L., Sentelhas, P.C., Gonçalves, J.L.M., Sparovek, G., 2014. Köppen’s structural carbohydrates of Lolium multiflorum ssp. italicum cv. Lema. Environ. Pollut.
climate classification map for Brazil. Meteorol. Z. 22, 711–728. 156, 915–921.
Arndt, U., Schweizer, B., 1991. The use of bioindicators for environmental monitoring in Sarruge, J.R., Haag, H.P., 1974. Análises químicas em plantas. ESALQ, Piracicaba 56 pp..
tropical and subtropical countries. In: Ellenberg, et al. (Eds.), Biological Monitoring. Sawidis, T., Breusteb, J., Mitrovic, M., Pavlovic, P., Tsigaridas, K., 2011. Trees as bioindicator
Signals from the Environment. Vieweg, Eschborn, pp. 199–298. of heavy metal pollution in three European cities. Environ. Pollut. 159, 3560–3570.
Artur, A.G., Garcez, T.B., Monteiro, F.A., 2014. Water use efficiency of marandu palisade Tresmondi, A.C.C.L., Tomaz, E., 2004. Air pollution and influence of sources on Paulínia
grass as affected by nitrogen and sulphur rates. Rev. Cienc. Agron. 45, 10–17. (Brazil) and surroundings. Int. J. Environ. Pollut. 22, 490–505.
Boian, C., Andrade, M.F., 2012. Characterization of ozone transport among Metropolitan van den Berg, L., Ashmore, M., 2008. Nitrogen. In: Jørgensen, S.E., Fath, B.D. (Eds.), En-
regions. Rev. Bras. Meteorol. 27, 229–242. cyclopedia of Ecology. Academic Press, Oxford, pp. 2518–2526.
Calvo, A.I., Alves, C., Castro, A., Pont, V., Vicente, A.M., Fraile, R., 2013. Research on aerosol VDI – Verein Deutscher Ingenieure, 2003. Biological measuring techniques for the de-
sources and chemical composition: past, current and emerging issues. Atmos. Res. termination and evaluation of effects of air pollutants on plants (bioindication).
120–121, 1–28. Method of Standardised Grass Exposure. VDI-Guideline 3957/2 (draft). In: VDI/DIN
CETESB – Companhia de Tecnologia de Saneamento Ambiental, São Paulo, 2013. Relatório Handbuch Reinhaltung der Luft, vol. 1a. Beuth Verlag, Berlin.
de qualidade do ar no estado de São Paulo 2012. Série Relatórios. Secretaria de Es-
tado de Meio Ambiente, São Paulo. Available from the World Wide Web 〈http://
www.cetesb.sp.gov.br/ar/〉.