Artigo Científico

1. RESEARCH ARTICLE
Lung injury caused by exposure to the gaseous fraction of exhaust
from biomass combustion (cashew nut shells): a mice model
Daniel Silveira Serra1
& Karla Camila Lima de Souza2
& Soujanya Talapala Naidu2
& Jéssica Rocha de Lima3
&
Fladimir de Lima Gondim2
& Maria Diana Moreira Gomes2
& Rinaldo dos Santos Araújo3
&
Mona Lisa Moura de Oliveira1
& Francisco Sales Ávila Cavalcante1
Received: 4 July 2019 /Accepted: 29 December 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Currently, to reduce the use of nonrenewable energy sources in energy matrices, some industries have already incorporated
biomass as a source of energy for their processes. Additionally, filters are used in an attempt to retain the particulate matter present
in exhaust gases. In this work, the emission gases of a cashew nut shell (CNS) combustion reactor and the deleterious effects on
the respiratory system of mice exposed to gaseous fraction present in CNS emissions (GF-CNS) are analyzed. The system for
CNS combustion is composed of a cylindrical stainless steel burner, and exhaust gases generated by CNS combustion were
directed through a chimney to a system containing two glass fiber filters to retain all the PM present in the CNS exhaust and,
posteriorly, were directed to a mice exposure chamber. The results show changes in the variables of respiratory system mechanics
(G, H, CST, IC, and PV loop area) in oxidative stress (SOD, CAT, and NO2
−
), as well as in the histopathological analysis and lung
morphometry (alveolar collapse, PMN cells, mean alveolar diameter, and BCI). Through our results, it has been demonstrated
that even with the use of filters by industries for particulate material retention, special attention should still be given to the gaseous
fraction that is released into the environment.
Keywords Pollution . Biomass . Cashew nut shells . Combustion . Pulmonary toxicology
Introduction
Due to harmful effects on the environment and on public
health, there is a worldwide endeavor to reduce the use of
nonrenewable energy sources, especially those originating
from fossils, such as petroleum and its by-products.
Government organizations are joining efforts to this end, as
well as to increase the use of cleaner energy matrices that are
also renewable, such as biomass.
Brazil is a country of continental dimensions and is one
of the largest agricultural producers in the world, character-
istics that place it third in terms of capacity to produce
energy from renewable resources, and first in energy pro-
duction capacity from renewable solid biofuels and waste
(IRENA 2018).
Among the varying renewable resources used for power
generation in Brazil, we highlight the use of biomass. The
term biomass describes all organic matter that, when burned,
decomposed, or recycled, directly and/or indirectly generates
some form of mechanical, thermal, or electrical energy. Thus,
agricultural waste, animal waste, human waste, organic mu-
nicipal waste, and vegetable waste can be processed for
energy.
Some biomass residues already contribute to the growth of
alternative energy production in the industrial sector, such as
coffee grounds (Park et al. 2018), rice husks (Weldekidan et al.
2018), sugarcane bagasse (Jayaraman et al. 2018), Eucalyptus
(Pighinelli et al. 2018), Pinus (Oliveira et al. 2018), and cash-
ew nut shells (CNS) (Mgaya et al. 2019), the latter being the
subject of analysis in this study.
Responsible editor: Philippe Garrigues
* Daniel Silveira Serra
daniel.silveira@uece.br
1
Science and Technology Center, State University of Ceará, Av. Dr.
Silas Munguba, 1700, Fortaleza, CE 60714-903, Brazil
2
Institute of Biomedical Sciences, State University of Ceará,
Fortaleza, CE, Brazil
3
Department of Chemistry and Environment, Federal Institute of
Ceará, Fortaleza, CE, Brazil
Environmental Science and Pollution Research
https://doi.org/10.1007/s11356-019-07576-8

2. One of the problems faced by cashew nut processing in-
dustries has been the final destination of residues such as
CNS. An alternative solution employed by the production
sector has been to incorporate this biomass during cashew
processing and use it in boilers to generate energy
(Figueiredo 2009). However, CNS combustion for the pur-
pose of energy production promotes the release of some pol-
lutants (such as CO2, SO2, and NOx) that, once dispersed in
the atmosphere, can cause harmful effects on the environment
and on human health.
In a previous study, mice were exposed to a concentration
of 30 μg/kg of total suspended particulate matter (TSP) or
particulate matter below 4 μm (PM4.0) from CNS combustion
exhaust gases; it was observed that a single exposure to this
material was sufficient to yield harmful effects on the respira-
tory system, such as increased airway resistance, bronchial
hyperresponsiveness, and decreased lung compliance, as well
as redox imbalance (Josino et al. 2017).
Studies associate inhalation of pollutants from biomass
combustion to cardiovascular disease, stroke, and adverse ef-
fects on neonatal development (Silva et al. 2015). Such expo-
sure may also potentiate the immune system’s response to
allergens, through increased epithelial permeability, recruit-
ment and activation of inflammatory cells, and oxidative stress
in the airways (Nagato 2007).
In addition to the increased use of biomass in the industrial
sector, around 50% of the world’s population and 90% of
households in rural areas use this residue as the primary source
of energy for cooking or heating (Kodgule and Salvi 2012).
Approximately 80% of households in China, India, and Africa
use biomass as fuel, while in rural areas of Latin America, this
ratio is between 30 and 70% (Salvi and Barnes 2009). Around
three million people in the world are exposed to smoke from
biomass burning, and due to sociocultural reasons, women
and children are the most vulnerable groups (Silva et al.
2015).
In an attempt to reduce harm to the environment and to
human health caused by exposure to pollutants from combus-
tion processes, some industries currently use centrifugal sep-
arators (cyclones and multicyclones), manhole filters, or iner-
tial chambers (Malvisi 2010), as well as filters made from
various materials such as melt-blown fibers, spun-bonded fi-
bers, and glass fiber (Uppal et al. 2013; Zhang et al. 2017), to
separate the particulate matter (PM) present in exhaust gases.
However, there are few studies that analyze the health effects
of exposure to only the gaseous fraction (after removal of the
PM) from biomass exhaust generated by these industries.
In view of the above, there is an urgent need to evaluate the
health impacts of exposure to the gaseous fraction of biomass
combustion emissions. Due to the increasing use of biomass
for energy recovery, in the present work, we used CNS for
combustion. We analyzed the whole process of thermal energy
generation and gas emission of a CNS combustion reactor, as
well as the deleterious effects on the respiratory systems of
mice exposed to the gaseous fraction present in CNS emis-
sions (GF-CNS). To conduct this research, we performed anal-
yses of respiratory system mechanics, oxidative stress, pulmo-
nary parenchyma histopathology and morphometry, and the
chemical composition of the GF-CNS.
Materials and methods
CNS analysis
The CNS used in the present study were provided by the
Amêndoas do Brasil Company (Fortaleza, Ceará, Brazil). In
order to analyze the properties of the shells used in the com-
bustion process, moisture, volatile materials, ash, and fixed
carbon contents were determined.
To establish the moisture content, crucibles were previous-
ly dried in an oven (QUIMIS®
) at 105 °C for approximately
20 min, to eliminate any moisture content present in the por-
celain. The crucibles were then transferred to a desiccator to
cool and were subsequently weighed with an analytical bal-
ance (Shimadzu®
). Approximately 1.0 g of CNS were then
placed in each crucible, where they remained until the residues
reached constant mass. The crucibles were weighed with the
CNS after 30 min, 1 h, 1 h and 30 min, and 2 h in the oven.
The moisture content was determined according to the fol-
lowing equation (Brazilian Association of Technical
Standards 1986) (NBR 8112):
TU ¼
m0−mi
m0
·100 ð1Þ
where TU = moisture content (%), m0 = initial sample mass
(g), and mi = final sample mass (g).
To determine the volatile materials content, approximately
1.0 g of moisture-free CNS were placed in porcelain crucibles,
which were then placed in a muffle (FDG3P-S) previously heat-
ed to 900 ± 10 °C for 7 min. The crucibles were then removed
from the muffle and placed in a desiccator to cool. Once cooled,
they were weighed to determine the final mass. The content of
volatile materials was determined according to the equation:
MV ¼
m2−m3
m
·100 ð2Þ
where MV = content of volatile materials (%), m2 = initial mass
of the crucible with residue sample (g), m3 = final mass of the
crucible with residue sample (g), and m = mass of the residue
sample (g).
The ash content was determined using crucibles containing
approximately 1.0 g of moisture-free CNS placed in a
preheated furnace at 700 ± 10 °C and allowed to burn
completely. Afterwards, the crucibles were removed from
Environ Sci Pollut Res

3. the muffle and placed in a desiccator to cool, and then
weighed to determine the final mass.
The ash content was determined according to the following
equation:
CZ ¼
m1−m0
m
·100 ð3Þ
where CZ = ash content (%), m0 = crucible mass (g), m1 =
mass of the crucible with residue sample (g), and m = mass of
the residue sample (g).
To establish the fixed carbon content, the following equa-
tion was used as an indirect measurement:
CF ¼ 100− CZ þ MVð Þ ð4Þ
where CF = fixed carbon content (%), CZ = ash content (%),
and MV = content of volatile materials (%).
Animals
All animals received humane care, and the experiments com-
plied with the following guidelines: ARRIVE; the National
Institutes of Health Guide for the Care and Use of
Laboratory Animals (NIH Publications No. 8023, revised
1978); and regulations issued by the National Council for
Controlling Animal Experimentation, Ministry of Science,
Technology and Innovation (CONCEA/MCTI), Brazil.
Female balb/c mice (7–8 weeks of age), with a body mass of
25 ± 5 g and water and food ad libitum, were used in this
study. Mice were housed in plastic cages under controlled
environmental conditions. All animal use and care procedures
were previously approved by the Animal Ethics Committee of
the State University of Ceará.
Exposure to the gaseous fraction of CNS combustion
Mice exposure to GF-CNS was performed during 3 h each
day, 5 days a week, for 6 weeks, an adaptation of the protocol
proposed by Tesfaigzi et al. (2002), used to investigate the
subchronic effects of exposure to gas from wood smoke, in
rats.
Thirty animals were randomly divided into two groups.
In the first group (n = 15), the mice were placed in the test
chamber and exposed to ambient air during the previously
reported period (CTRL group). In the second group (n =
15), the mice were placed in the chamber and exposed to
the GF-CNS combustion during the previously reported
period (CNS group). All analyses were performed 24 h
after the final exposure.
All animals were preconditioned in the exposure envi-
ronment (test chamber) during 3 h per day, 5 days per
week, for 1 week (week 0). This period was used to adapt
the animals to the environmental conditions of the
experiment and to reduce stress. Initially, there was expo-
sure of animals from the CTRL group during the period
described above and then exposure of the animals from
the CNS group. We used different exposure times to avoid
probable contamination of CTRL group animals by GF-
CNS used in the CNS group exposure.
CNS combustion and exposure chamber
The system for CNS combustion and exposure of animals to
GF-CNS is shown in Fig. 1. CNS (400 g per h) were first
placed in a cylindrical stainless steel burner (Fig. 1 A).
Initial combustion ignition was then performed by supplying
liquefied petroleum gas (LPG—Fig. 1 B) with a flow rate of
1.2 L/min and ambient air from a compressor (Fig. 1 C), with
a flow rate of 25 L/min. The CNS combustion process was
monitored using thermocouples (Fig. 1 D) and flow transduc-
ers (Fig. 1 E) connected to a data acquisition system
(FieldLogger®
—Fig. 1 F), for analysis of the reactor’s internal
temperature; all information was then transferred to a note-
book (Fig. 1 G).
Exhaust gases generated by CNS combustion were directed
through a chimney (Fig. 1 H), to a reservoir containing a
porous aluminum oxide (Al2O3)-based medium with a diam-
eter of 13 mm (Fig. 1 I), in order to retain the tar, moisture, and
part of the cashew nut liquid (LCN) generated during the
process. The system was fed with a suction pump
(AirChek®
XR5000—Fig. 1 J), using a flow rate of 1 L/min.
In order to retain all the PM present in the CNS exhaust, the
gases flowed through a system containing two glass fiber fil-
ters (Fig. 1 K) and were directed to an exposure chamber (Fig.
1 L).
The temperature inside the reactor (base, middle, and top)
was monitored throughout the combustion process, with the
use of thermocouples (Fig. 1 D). We also analyzed the pollut-
ants generated by CNS combustion, using an electrochemical
analyzer (GreenLine 8000®
) placed directly on top of the
chimney.
To ensure that only GF-CNS was present in the animal
exposure chamber, with no contamination with PM, a particle
counter (DT 9881 CEM) was placed inside the chamber in
order to ascertain that no solid particles were present. The
interior of the chamber contained 2 hoods and was divided
into 15 individual capsules for animal containment, for uni-
form subject distribution and to guarantee homogeneous
exposure.
Gases inside the chamber were monitored during the whole
animal exposure period using an electrochemical analyzer
(GreenLine 8000®
). Carbon monoxide (CO) concentration
within the chamber during exposure of the CNS group was
monitored and maintained at mean values of 10 ppm, to avoid
animal intoxication (Serra et al. 2017).
Environ Sci Pollut Res

4. Identification and quantification of polycyclic
aromatic hydrocarbons and carbonyl compounds
For chemical analyses of polycyclic aromatic hydrocarbons
(PAHs) and carbonyl compounds (CCs) present in GF-CNS,
Anasorb 100/50 (SKC®
) tubes filled with 150 mg of coconut-
shell–activated carbon were placed after the system containing
the two glass fiber filters (Fig. 1 K). Extraction of aromatic
derivatives was carried out by placing 150 mg of adsorbent
coal in contact with 1 mL of 99.9% carbon disulfide, under
magnetic stirring in a 2-mL vial for 30 min, as described in
Method 1501 (NIOH 2003). At the end of the contact time, the
solvent was filtered through a PTFE membrane (22 mm,
0.45 μm). The extract was collected in a 2-mL clean vial
and taken for analysis by liquid chromatography.
PAH analysis was achieved through high-performance liq-
uid chromatography, according to application note 20572
adapted from EPA 610 (Thermo Scientific 2012), using a
Varian ProStar HPLC with diode arrangement detector,
Hypersil green PAH column (25 cm × 4.6 mm × 5 μm), with
a wavelength of 225 nm, and mobile phase composed of
CH3CN/H2O at a flow rate of 1.25 mL/min under elution
gradient. Individual concentrations of the 16 PAHs were esti-
mated by the external standard method, by using a PAH cal-
ibration mix (10 μg mL−1
in acetonitrile) acquired from
Sulpeco (Belleforte, PA, USA).
The carbonyl compounds (aldehydes and ketones) were
collected and analyzed according to the EPA method TO-
11A. LpDNPH S10 cartridges (Supelco Analytical), contain-
ing 350 mg of high-purity silica gel coated with DNPH, were
used in in situ adsorption and derivatization of the carbonyl
compounds. CC elution was accomplished by passing
acetonitrile through the cartridges, with percolation occurring
by gravity. The procedure was performed with 1 mL of ace-
tonitrile divided into two equal portions of 500 μL each.
Compounds eluted in acetonitrile were collected in 1.5 mL
vials.
Analysis of CC was performed using HPLC-DAD (Varian
Prostar) with a Hypersil C18 column (250 mm × 4.6 mm ×
5 μm), at a wavelength of 360 nm and injection volume of
40 μL. The mobile phase was composed of CH3CN/H2O at a
flow rate of 0.75 mL/min under gradient schedule. The anal-
yses were performed immediately after the collections. The
analytical standard (T011/IP-6A Aldehyde/Ketone-DNPH
Mix—Supelco) was used in the quantification by the external
standard method.
Pulmonary function analysis
Respiratory system mechanics
Animals were anesthetized 24 h after the end of the exposure
period, by intraperitoneal (i.p.) injection of ketamine:xylazine
(80 mg/kg:10 mg/kg), and then tracheostomized. The animals
were intubated with a 14-gauge cannula (Eastern Medikit,
Delhi, India) that was then connected to a computer-
controlled ventilator for small animals (Scireq-flexiVent®
,
Montreal, QC, Canada). The animals were ventilated with
the following baseline settings: respiratory frequency of 90
breaths/min, tidal volume of 10 mL/kg, pressure limit of
30 cmH2O, and positive end-expiratory pressure (PEEP) of
3 cmH2O. Mice were then paralyzed with pancuronium bro-
mide (0.5 mL/kg, i.p., Cristália, Brazil).
Fig. 1 Combustion system and
exposure chamber. A—Biomass
combustion reactor; B—LPG;
C—air compressor; D—
thermocouples; E—flow trans-
ducers; F—data acquisition sys-
tem (FieldLogger); G—notebook;
H—chimney; I—dehumidifying
system; J—suction pump; K—
PM retention filters; L—exposure
chamber
Environ Sci Pollut Res

5. Initially, we standardized the mechanical history of the
respiratory system with two deep insufflations (DI, 6-s
long, peak pressure 30 cmH2O), followed by ventilation
during 5 min with baseline settings. Soon after, the im-
pedance of the respiratory system (Zrs) was measured
using the forced oscillation technique (Hantos et al.
1992), during 12 sequential 30-s sampling intervals, for
a total of 6 min (Bates 2009).
The experimental Zrs was fitted to the constant phase model
as previously described (Hirai et al. 1999):
Zrs ¼ RN þ I 2πfð Þi þ
G−Hi
2πfð Þα ð5Þ
α ¼
2
π
tan−1 H
G
ð6Þ
where RN is the Newtonian resistance, which represents the
central airways resistance; i ¼
ffiffiffiffiffiffi
−1
p
; f is the frequency (Hz); I
represents airway inertance; and G and H are, respectively, the
dissipative and elastic properties of lung tissue (Hantos et al.
1992).
Thereafter, starting at the functional residual capacity
(FRC) defined by the PEEP, the flexiVent delivered 7 in-
spiratory pressure steps for a total pressure of 30 cmH2O,
followed by 7 expiratory steps, pausing at each step for
1 s. Plateau pressure (P) was recorded for each step and
correlated to the total volume (V) delivered, to produce a
quasi-static PV (pressure–volume) curve. Static compli-
ance (CST) was calculated as the slope of the curve
(Salazar and Knowles 1964). Two quasi-static PV curves
were obtained to measure CST, an estimate of inspiratory
capacity (IC), and the PV loop area. Another forced oscil-
lation ensued to determine respiratory system mechanics.
Methacholine challenge
Immediately after measurements of respiratory system
mechanics, two DIs (deep insufflations) were performed,
followed by 5 min of ventilation with baseline settings.
Airway smooth muscle hyperresponsiveness was evaluat-
ed by inhalation of methacholine (MCh) (Sigma-Aldrich,
St. Louis, MI, USA) delivered by aerosol produced by an
ultrasonic nebulizer (Inalasonic, NS, São Paulo, Brazil)
coupled to the inspiratory line of the ventilator. To
achieve this, 4 mL of MCh solution (30 mg/mL) was
added to the nebulizer container.
Nebulization was carried out during 30 s under mechanical
ventilation (Xue et al. 2008), and the average amount of MCh
solution delivered to the animal was 1.2 mg/kg. After nebuli-
zation, forced oscillation was repeated (30-s sequential inter-
vals for 6 min), followed by two DIs and another forced os-
cillation for data gathering.
Histological study
Immediately after determination of respiratory system me-
chanics, the rib cage was opened and heparin (1000 IU) was
injected in the right ventricle of the heart. The trachea was
clamped at end-expiration, and the abdominal aorta and vena
cava were sectioned, yielding a massive hemorrhage that
quickly euthanized the animals. The lungs were perfused with
saline and then removed en bloc. The right lung was isolated,
frozen in liquid nitrogen, and stored for biochemistry analysis;
the left lung was kept at functional residual capacity and fixed
in Millonig’s formaldehyde (100 mL HCHO, 900 mL H2O,
18.6 g NaH2PO4, 4.2 g NaOH). Slides containing left lung
sections were stained with hematoxylin and eosin (HE) and
examined by optical microscopy according to their qualitative
and quantitative aspects. An investigator, blinded to the origin
of the coded material, examined the samples microscopically.
Quantitative analysis was performed using an integrated
eyepiece with a coherent system, consisting of a 100-point
and 50-line grid coupled to a conventional light microscope.
The fraction area of collapsed alveoli or normal pulmonary
areas and the amount of polymorphonuclear (PMN) cells and
pulmonary tissue were determined by the point-counting tech-
nique (Weibel 1990). The air-space enlargement was quanti-
fied by the mean linear intercept length of the distal air spaces
(Lm) in 30 randomly chosen fields of tissue sections per group
(Knudsen et al. 2010).
Cellularity was assessed at × 1000 magnification across
10–15 random noncoincident microscopic fields for each an-
imal. Morphometric analyses and determination of the
bronchoconstriction index (BCI) were performed at × 400
magnification. BCI was determined in 10 noncoincident mi-
croscopic fields per animal, by counting the number of points
in the airway lumen (NP) and intercepts through the airway
wall (NI), using a reticulum and applying the equation:
BCI ¼ NI=
ffiffiffiffiffiffiffi
NP
p
. Only airways in which the long diameter
did not exceed the short diameter by more than 20% were
accepted for measurement (Sakae et al. 1994).
Biochemical analyses
The right lungs of each group were homogenized in 5 mM
Tris-HCl buffer (pH 7.4), containing 0.9% NaCl (w/v),
0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 1 μg/
mL aprotinin, followed by centrifugation at 750×g for
10 min at 4 °C. The supernatants were stored at − 80 °C for
the biochemical analyses (catalase and superoxide dismutase
activity, nitrite levels). The total protein in the samples was
determined by the Bradford method (Bradford 1976).
Catalase (CAT) activity was measured at 240 nm
(Evolution 60S UV-Visible Spectrophotometer, Madison,
WI, USA), using the rate of decrease in hydrogen peroxide
Environ Sci Pollut Res

6. concentration, and was expressed as CAT equivalents (U/mg
protein) (Aebi 1984).
Superoxide dismutase activity (SOD) was measured from
the pulmonary homogenate of the two groups (different vol-
umes 5, 10, and 15 μL) diluted in glycine buffer (3.75 mg/mL
amino acetic acid + 200 mL distilled water, pH = 10.2), cata-
lase (2.4 mg/mL in distilled water), and adrenaline (60 mM).
A kinetic curve of this enzyme was plotted every 10 s, during
3 min, for each sample. The color change in the mixture was
detected by spectrophotometry (UV-Vis-Evolution 60S
Spectrophotometer), with absorbance at 480 nm. SOD values
were corrected for the protein value of each sample (U SOD/
mg protein) (Bannister and Calabrese 1987).
Detection of nitrite levels is an indirect way of measuring
nitric oxide; this was achieved through the Griess reaction
(Green et al. 1982). In this method, the nitrite is first subjected
to reaction with sulfanilamide in an acid medium to form an
intermediate compound, diazonium salts. These salts are then
added to N-naphthylethylenediamine, to form a purple-
colored stable azo compound. Aliquots of 100 μL lung ho-
mogenate were plated in duplicate and subjected to reaction
with 50 μL of 1% sulfanilamide solution in 2.5% phosphoric
a c i d , d u r i n g 1 0 m i n . T h e n , 5 0 μ L o f 0 . 1 %
naphthylethylenediamine solution in 2.5% phosphoric acid
was added to the mixture. The formation of the purple com-
pound could be observed and read immediately at a wave-
length of 540 nm (VersaMax™ ELISA Microplate Reader).
The resulting value was expressed in mMol/mg protein.
Statistical analysis
Results are presented as mean ± SD, where n represents the
number of samples. Data normal distribution and variance
homogeneity were tested with the Kolmogorov–Smirnov
(with Lilliefors’s correction) and the Levene median tests,
respectively. If both conditions were satisfied, Student’s t test
was used. If any condition was refused, the Mann–Whitney
nonparametric test was used instead. A difference was consid-
ered significant if p 0.05.
Results
CNS analysis
The results of the chemical analyses of the CNS used in the
study show the values of moisture (9.31 ± 0.14), ash (1.16 ±
0.37), volatile (78.12 ± 0.14), and fixed carbon (20.71 ± 0.38).
Knowledge of these properties is decisive for planning and
execution of an efficient combustion process, providing en-
hanced energy utilization of the biomass.
Thermal analysis and determination of gas
concentrations in the combustion reactor
and exposure chamber
Temperatures inside the CNS combustion reactor (base,
middle, and top) were monitored during the whole pro-
cess. In addition, the temperatures and the concentrations
of O2, CO, CO2, SO2, and NOx present in the combustion
exhaust were measured directly within the chimney.
Figure 2 shows the average temperature inside the com-
bustion reactor (Fig. 2a) and the average temperatures
inside the combustion reactor and the chimney with re-
spect to O2 percentage (Fig. 2b), CO concentration (Fig.
2c), CO2 percentage (Fig. 2d), SO2 concentration (Fig.
2e), and NOx concentration (Fig. 2f).
We also monitored temperature and gas concentrations
inside the chamber during the animal exposure period.
Animals of the CTRL group were subjected to an envi-
ronment O2 fraction of 21.1 ± 0.2%, with no CO, NOx, or
SO2 present, and an average temperature of 25.89 ±
0.27 °C. The animals of the CNS group were exposed to
an environment with an O2 fraction of 21.0 ± 0.1%, CO
concentration of 10.1 ± 0.4 ppm, NOx concentration of
1.0 ppm, SO2 concentration of 1.1 ± 0.3 ppm, and an av-
erage temperature of 27.35 ± 0.73 °C.
Polycyclic aromatic hydrocarbons and carbonyl
compounds
The results for the concentrations of CCs and PAHs
pertaining to the gaseous fraction of the CNS exhaust
are shown in Table 1. Analysis of the CCs identified the
presence of some compounds that are potentially harmful
to human health, such as acetaldehyde, acrolein, and
formaldehyde. Special attention should be given to the
presence of potentially carcinogenic PAHs, such as chrys-
ene, pyrene, benzo[a]pyrene (BaP), and benzo[a]-
anthracene (IARC 2013).
Temporal evolution of subjects’ body mass
The mean weekly body mass gain for the CTRL and CNS
groups during the adaptation period (week 0), as well as
during the 6 weeks of exposure (weeks 1 to 6) to either
ambient air (CTRL group) or GF-CNS (CNS group), was
monitored. The results showed that subjects of the CTRL
group experienced an average increase of 3.01 g in body
mass, whereas the animals of the CNS group gained an
average of 0.29 g throughout the total 7 weeks of the
experiment (1 week of adaptation and 6 weeks of
exposure).
Environ Sci Pollut Res

7. Pulmonary function analysis
Respiratory system mechanics
The results pertaining to the analysis of the respiratory system
mechanics after exposure of mice to ambient air (CTRL
group) or the CNS combustion exhaust gaseous fraction
(CNS group) are shown in Table 2. Notably, there were sig-
nificant changes in the variables obtained by the forced oscil-
lation technique (G and H), with exception of the RN, in the
CNS group, when compared to the CTRL group. Similar re-
sults were obtained by analyzing the PV curve variables (CST,
IC, and PV loop area), in which the data displayed significant
changes in the CNS group, as compared to the CTRL group.
Methacholine challenge
Figure 3 shows the variation in ΔRN values after administra-
tion of MCh (30 mg/mL) during 30 s, in both the CTRL and
CNS groups. The results show a significant increase in only a
few points within the CNS group, when compared to the
CTRL group, thus not completely demonstrating airway
hyperresponsiveness (AHR).
Histological study
Figure 4 shows photomicrographs of the pulmonary paren-
chyma for the CTRL and CNS groups. Analysis of this mate-
rial reveals the presence of areas of atelectasis (circles), thick-
ened septa (gray arrows), cellular infiltration (black arrows),
and airways (stars) in the parenchyma of the animals exposed
to ambient air (CTRL group) or to GF-CNS (CNS group).
Table 3 displays the results of the pulmonary parenchymal
morphometry studies, showing that the fraction area of col-
lapsed alveoli, the number of polymorphonuclear cells
(PMN), the mean linear intercept lengths of the distal air
spaces (Lm), and the bronchoconstriction index (BCI) were
Fig. 2 Data on temperature and concentration of pollutants during the
CNS combustion process. a Temperatures inside the combustion reactor.
b Relation between temperatures (reactor and chimney) and O2. c
Relation between temperatures (reactor and chimney) and CO. d
Relation between temperatures (reactor and chimney) and CO2. e
Relation between temperatures (reactor and chimney) and SO2. f
Relation between temperatures (reactor and chimney) and NOx
Environ Sci Pollut Res

8. all increased. In general, these results may suggest pulmonary
inflammation in the animals of the CNS group.
Biochemical analysis
In order to evaluate the imbalance between oxidants and anti-
oxidants suffered by the lung after exposure to ambient air
(CTRL group) or GF-CNS (CNS group), biochemical tests
were carried out to measure the activity of the antioxidant
enzymes SOD and CAT and to quantify nitrite (NO2
−
). The
analyses showed statistically significant changes in the quan-
tities of all three biochemical markers in the lung parenchyma
samples of the CNS group, when compared to the CTRL
group (Table 3).
Discussion
CNS analysis
Chemical analyses of the CNS used in the study were per-
formed. Knowledge of the characteristics of the plant material
used for combustion is of fundamental importance for efficient
combustion tests.
The moisture content of the plant material has a direct
influence on combustion, mainly due to thermodynamics in-
volving the processes inside the reactor. Different plant mate-
rials used as biomass fuel have different moisture contents,
such as rice husk (10.61%), soybean residue (6.30%), and
sugarcane bagasse (8.20%) (Ekinci 2011). The CNS that we
used in the present study had a mean moisture content of
9.31%, close to that of similar biomasses.
The ash content determines the mass of the existing inor-
ganic solid residue (Arantes 2009). Values found in similar
plant materials are approximately 11.30% for rice husk
(Diniz 2005) and 17.10% for sugarcane bagasse (Demirbas
2004). These values are considered elevated, as proportions
below 7% are desirable, in order to avoid compromising the
combustion process (Pereira and Seye 2014). We found mean
values of 1.16% for the CNS, within the desirable range.
Regarding the volatile content, similar vegetable materials
used for energy generation presented values of 64.10% for
rice hulls, 80.00% for soybean residues, and 80.42% for sug-
arcane bagasse (Arantes 2009). Such values are close to that
found in our study for CNS, 78.12%. The volatile material
content determines ease of ignition, flame stability, and com-
bustion rate. A high volatile content facilitates ignition and
combustion.
The fixed carbon content is related to the yield of the com-
bustion process; the lower its value, the higher the yield (Lima
et al. 2007). Values of 18.32% were found in corn bagasse
(Demirbas 2004) and 79.92% in coal (Neves et al. 2011). In
the present study, we found the mean value of 20.71% for
CNS, which is higher than the content found for corn bagasse,
but much lower than that of coal.
Thermal indices and gas concentrations
in the combustion reactor and exposure chamber
The whole process of heat generation inside the combustion
reactor and the chimney, as well as the concentrations of O2,
CO, CO2, SO2, and NOx, present in the CNS exhaust gases, is
shown in Fig. 2. Temperature fluctuations throughout the 3 h
of exposure (Fig. 2a) were close to 45, 90, and 135 min, in the
three different regions of the reactor (base, middle, and top);
this was due to the addition of 400 g of CNS at 45 min inter-
vals. Along the interior of the combustion reactor, temperature
peaks of 699.96 °C were observed in the base, 724.95 °C in
the middle, and 739.53 °C in the top. In the exhaust gases of
the chimney, we found peaks of 191.28 °C.
Oxygen concentrations in the exhaust gases did not
change significantly, with small negative variations ac-
cording to the temperature decay inside the reactor (Fig.
2b). In relation to the concentrations of CO (Fig. 2c), SO2
Table 1 Composition of gaseous phase of CNS combustion
Aldehydes and ketones (μg/m3
) PAHs (μg/m3
)
Formaldehyde 0.430 Phenanthrene 0.013
Acetaldehyde 1.306 Fluoranthene 0.009
Acrolein 0.173 Benzo[a]anthracene 0.026
Propionaldehyde 0.104 Criseno 0.880
Crotonaldehyde 0.051 Pyrene 0.033
Butyraldehyde 0.095 Benzo[a]pyrene 0.170
Benzaldehyde 0.021 Benzo[b]fluoranthene 0.010
Isovaleraldehyde/valeraldehyde 0.038
o,m,p-Tolualdehyde 0.010
2,5-Dimethylbenzaldehyde 0.020
Distribution of aromatic polycyclic hydrocarbons (PAHs), aldehydes, and ketones contained in gaseous fraction from the combustion exhaustion of CNS
Environ Sci Pollut Res

9. (Fig. 2e), and NOx (Fig. 2f) and the percentage of CO2
(Fig. 2d), more significant variations were observed as a
function of internal temperature variations within the re-
actor. Here, we observed peaks of 18,900 ppm CO,
21 ppm SO2, 286 ppm NOx, and 1.6% CO2.
Various harmful effects are caused by the release of these
gases into the atmosphere. According to the World Health
Organization, chronic exposure to small concentrations of
CO can generate deleterious effects that go beyond the hyp-
oxia observed in acute exposure, such as endothelial inflam-
mation and immune activation (WHO 2010). Increased atmo-
spheric concentration of CO2 accentuates the greenhouse ef-
fect, leading to global warming (Lam et al. 2012). SO2 is a
precursor for acid rain formation (Radmann et al. 2011). In
relation to NOx, oxidation of nitrogen monoxide (NO) can
lead to the formation of nitrogen dioxide (NO2), a pollutant
that causes serious respiratory diseases (Castro et al. 2013). In
addition, SO2 and NOx are responsible for blocking sunlight
and forming photochemical smog (Sun et al. 2015).
Polycyclic aromatic hydrocarbons and carbonyl
compounds
Analyses of PAHs performed from GF-CNS samples showed
a more significant presence of chrysene and BaP (Table 1).
According to the International Agency for Research on
Cancer (IARC 2013), there is sufficient evidence to classify
both substances as carcinogenic. BaP is considered to be a
complete carcinogen, as it both initiates and promotes carci-
nogenesis. Due to this characteristic, BaP has been used in
several studies as a model of carcinogenesis in the lungs
(Paul et al. 2011; Ravichandran et al. 2011), stomach (Goyal
et al. 2010), and skin (Shimizu et al. 2000). Exposure to BaP-
containing HPAs by any route of administration has been
shown to lead to an increased risk of lung cancer and other
tumors in humans (Luttrell and Thomas 2007).
Regarding CCs, chronic exposure to these compounds is
associated with various adverse health effects, such as respi-
ratory and ocular tract irritation, headaches, nausea, and diz-
ziness (Sousa 2011). These compounds are considered to be
both carcinogenic and mutagenic, due to their easy absorption
by the airways (Barro et al. 2009). Our results demonstrated
significantly elevated levels of acetaldehyde, formaldehyde,
and acrolein (Table 1).
Besides being mostly present in vehicle emissions, acetal-
dehyde and formaldehyde are also generated by industrial
processes (Gaffney et al. 1997). Acetaldehyde represents a
risk for severe health impacts, mainly in the respiratory sys-
tem, such as throat and respiratory tract irritation (US EPA
1999). Short-term exposure to formaldehyde can provoke
symptoms such as eye and airway irritation, tearing, sneezing,
coughing, nausea, and dyspnea, which can lead to death.
Table 2 Differences between lung function parameters
Measure Group Value Student’s t
Forced oscillation technique Airway resistance (RN) (cmH2O s/mL) CTRL 0.149 ± 0.037 p = 0.115
CNS 0.174 ± 0.021
Tissue damping (G) (cmH2O/mL) CTRL 4.18 ± 0.47 p 0.001*
CNS 5.32 ± 0.81
Tissue elasticity (H) (cmH2O/mL) CTRL 17.65 ± 3.51 p = 0.005*
CNS 22.98 ± 4.90
PV curve Static compliance (CST) (mL/cmH2O) CTRL 0.111 ± 0.015 p 0.001*
CNS 0.085 ± 0.070
Inspiratory capacity (IC) (mL) CTRL 1.00 ± 0.12 p 0.001*
CNS 0.78 ± 0.11
PV loop area (mL) CTRL 2.12 ± 0.40 p 0.014*
CNS 2.69 ± 0.66
Data are presented as mean ± standard deviation. p 0.05 was considered statistically significant
*Values significantly different from the CTRL group
Fig. 3 Changes in ΔRN after nebulization of methacholine. White circle =
animals exposed to ambient air (CTRL group). Black squares = animals
exposed to the gaseous fraction from the combustion exhaustion of CNS
(CNS group). DI = deep inflation. * Values significantly different from
the CTRL group
Environ Sci Pollut Res

10. Long-term exposure to formaldehyde results in airway and
eye irritation, as well as inflammatory and hyperplastic chang-
es of the nasal mucosa (Kotzias et al. 2005). Acrolein is a
common environmental pollutant that induces airway toxicity
(Liu et al. 2018). According to the US EPA, the reference
concentration for acrolein is 2.0 × 10−5
mg/m3
, and the
DL50 is a result of exposure to 66 ppm for 6 h (US EPA 2003).
Temporal evolution of subjects’ mass
Animal body mass was verified continuously during the 7-
week study period. Our results demonstrate increased mass
among the animals of the CTRL group, a characteristic not
observed in the CNS group. The mean mass increase in the
CTRL group was 3.01 g, whereas the animals in the CNS
group gained only 0.29 g. This phenomenon can be explained
through analysis of the temperature to which the subjects of
the different groups were exposed, as the animals of the CTRL
group were submitted to a mean temperature of 25.89 ±
0.27 °C, while those of the CNS group were exposed to an
average temperature of 27.35 ± 0.73 °C.
Studies report that ambient temperature influences animal
metabolic processes (Damy et al. 2010) and that the mouse is
particularly susceptible to changes in environmental
conditions (Chorilli et al. 2007). Small fluctuations in temper-
ature (2 to 3 °C) may cause changes in its physiology
(Johnson-Delaney 1996). A study of the influence of temper-
ature and air flow on the food and water consumption of
Wistar rats showed that increasing temperature led the animals
to consume less food and water, due to a lower caloric con-
sumption being necessary to maintain their metabolism
(Martinewski et al. 2008).
Pulmonary function analysis
Although the literature contains some works in which animals
were exposed to various pollutants generated by the combus-
tion of some types of biomass, we found no studies exposing
animals to only the gaseous fraction of combustion exhaust,
such as GF-CNS.
An inhalation study in rats using firewood, with smoke
concentrations between 1 and 10 mg/m3
for a period of 4 to
12 weeks, showed as main result the development of chronic
inflammation, laryngeal squamous cell metaplasia, alveolar
septum thickening, and an increased number of mononuclear
cells in groups submitted to 30 and 90 days of inhalation
(Tesfaigzi et al. 2002). In another study, repeated and
prolonged exposure to ambient particles produced by either
Fig. 4 Photomicrographs of the
pulmonary parenchyma stained
with HE representative of the
animals of the CTRL and CNS
groups in × 200 magnification.
Gray arrow = alveolar septal
thickening; black arrow = cellular
infiltrate; circle = areas of
atelectasis; star = airway
Table 3 Morphometric (alveolar collapse, PMN cells, mean alveolar diameter, and BCI) and lung antioxidant defense system (SOD, CAT, NO2
−
)
analysis between the CNS and control groups
Groups Alveolar
collapse (%)
PMN cells
(×10−3
/μm2
)
Mean alveolar
diameter (μm)
BCI SOD
(U/g)
CAT
(mcat/g)
NO2
−
(nmol/mg)
CTRL 5.96 ± 1.61 16.10 ± 3.10 47.19 ± 4.98 2.32 ± 0.32 4.17 ± 2.05 57.56 ± 13.81 9.99 ± 2.94
CNS 24.97 ± 4.72*
33.49 ± 4.17*
33.13 ± 4.58*
2.63 ± 0.35 1.50 ± 0.60*
46.69 ± 5.90*
21.56 ± 8.25*
Values are mean ± SD of CTRL and CNS groups. The data were collected in 10 matched fields per mice
PMN, polymorphonuclear; BCI, bronchoconstriction index
*Different from the CTRL group (Student’s t test, p 0.05)
Environ Sci Pollut Res

11. traffic or sugarcane combustion was seen to induce significant
impairment in pulmonary mechanics and lung histology in
mice, as well as a higher influx of macrophages into the lung
(Mazzoli-Rocha et al. 2014).
Regarding our results referring to respiratory system me-
chanics, data collected from the constant phase model (RN,
G ∧ H) show statistically significant variations in tissue resis-
tance (G) and tissue elastance (H), but not in RN (Table 2). The
RN represents an estimate of the total resistance of the central
airways (Bates 2009), and our results suggest that exposure to
GF-CNS did not cause significant damage to airway smooth
muscle. This finding can be corroborated with the BCI result
(Table 3), which represents a quantitative measure of the in-
ternal geometry of the airways, and with the photomicro-
graphs (Fig. 4), which show a representative airway of the
CNS group with preservation of its internal area.
In a study with rats exposed to smoke from wood combus-
tion for 70 days, reduced lung compliance, increased CRF,
and lung parenchyma inflammation were observed
(Tesfaigzi et al. 2005). However, no significant lesions were
seen in the airways, results that corroborate those of our study.
Changes in the pulmonary parenchyma can be observed
through analysis of G and H (Bates 2009). Inflammatory pro-
cesses within the pulmonary parenchyma may lead to an in-
crease in these two variables. This inflammation can be con-
firmed by photomicrography, showing altered alveolar septum
morphology and cellular infiltrates (Fig. 4), as well as an in-
crease in PMN cells observed in the animals from the CNS
group (Table 3).
In addition, the increased G and H within the CNS group
may also be related to stiffening of the pulmonary parenchyma
caused by tissue remodeling. This is due to the increase in
reactive oxygen species (ROS) within the lung parenchyma of
these animals, which can be confirmed by the decreased SOD
and CAT seen in this group (Table 3). This results in the stim-
ulation of alveolar macrophages and endothelial and epithelial
cells, which leads to the release of inflammatory cytokines that
act on fibroblasts. In turn, these cells migrate to the areas of
lesion, where they are stimulated to secrete collagen and other
matrix proteins, in an attempt to promote tissue repair.
However, deposition of collagen and elastin fibers can also lead
to formation of scar tissue (tissue remodeling), contributing to
pulmonary parenchyma stiffening (Loivos 2013).
Regarding the parameters obtained from the quasi-static
PV curve, the static compliance (CST) indirectly measures
the degree of lung tissue distensibility, and estimate of inspi-
ratory capacity (IC) quantifies the volume of air received by
the lungs up to a pressure of 30 cmH2O, while PV loop area
provides an estimate of the amount of atelectasis (air-space
closure) that existed before the PV loop maneuver (West
2012). An explanation similar to that for the alterations seen
in (G and H) can be used to justify the decreased CST.
However, the decrease in IC and the increase in the PV loop
area can be explained by an increase in atelectasis, observed
through photomicrograph analysis (Fig. 4—circle) and by the
increased percentage of collapsed alveoli (Table 3).
Still with regard to the respiratory mechanics analysis, the
methacholine challenge was performed to evaluate airway re-
sponsiveness (Fig. 3). Few points were found with a statisti-
cally significant difference during the analysis, demonstrating
an absence of airway smooth muscle hyperresponsiveness in
the CNS group. In spite of this, the initial inflammatory pro-
cess may explain the observed increasing trend in the CNS
group values when compared to the CTRL group, as previous
studies associate airway hyperresponsiveness with the pres-
ence of an inflammatory process (Serra et al. 2017; Wang
et al. 2008).
In relation to the biochemical analyses for oxidative stress,
activity of the antioxidant enzymes SOD and CAT and the
NO2
−
quantification were analyzed in order to verify any al-
terations in the pulmonary redox balance, in response to ex-
posure to GF-CNS (Table 3).
Increased production of free radicals often causes an in-
crease in antioxidant enzyme levels (Escobar et al. 1996).
However, there are other studies that demonstrate that high
rates of free radicals can trigger enzymatic inactivation, with
reduction of antioxidant activity, thus causing oxidative dam-
age (Kwon et al. 2000).
Our results demonstrate a statistically significant reduction
in SOD and CAT values in lung tissue samples from the CNS
group, when compared to the CTRL group (Table 3). A hy-
pothesis for this result can be associated to the presence of BaP
in the gas fraction of CNS combustion (Table 1). An earlier
study reported a decrease in SOD and CAT enzymes after oral
administration of BaP in rats; according to the authors, BaP-
induced damage may cause increased ROS and, consequently,
oxidative stress due to ineffective protection afforded by the
endogenous antioxidant system (Saunders et al. 2006).
We also observed an increase in NO2
−
(Table 3), which was
analyzed to determine the formation of NO•
. One possible
explanation for this result may be the reduction of SOD, where
its decrease may lead to an increase in O2
•–
. The O2
•–
probably
had reacted with NO•
present in the pulmonary environment,
culminating in the generation of NO2
−
and ONOO−
; the latter
is known to cause reduction and/or inactivation of SOD and
CAT. It is also important to note that ONOO−
is able to react
with several molecules, including proteins, lipids, carbohy-
drates, and nucleic acids, thus damaging them (Tutka et al.
2007).
Another hypothesis for the increased NO2
−
can be associ-
ated to the inflammatory process in the pulmonary parenchy-
ma, as seen in the photomicrograph data (Fig. 4—CNS group)
and by the increased number of PMN cells (Table 3), since
NO•
can also be formed from the synthesis of recruited inflam-
matory cells, such as macrophages and neutrophils (Dias-
Junior et al. 2008).
Environ Sci Pollut Res

12. Conclusion
The results of our study demonstrated and quantified the dif-
ferent types of harm to the respiratory system caused by ex-
posure to GF-CNS. This exposure caused significant changes
in lung mechanics, particularly at the tissue level, accompa-
nied by histological and inflammatory changes, and redox
imbalance in the lungs of mice. GF-CNS has been shown to
present chemical components that are potentially harmful to
health. Thus, we proved that, even with the care that industries
are taking when using particulate retention filters, special at-
tention should still be given to the gaseous fraction of exhaust
from the combustion of substrates such as biomass.
Author contributions DS and FSA: conception and design of the study;
DS, KLC, SN, JR, FL, MDM, RS, and MLM: data collection and anal-
yses; DS and FSA: wrote the manuscript. All the authors edited and
approved the final version of the manuscript.
Funding information The authors are highly thankful to CAPES –
Brazilian Federal Agency for Support and Evaluation of Graduate
Education for the financial support through scholarship.
Compliance with ethical standards
All animals received humane care, and the experiments complied with the
following guidelines: ARRIVE; the National Institutes of Health Guide
for the Care and Use of Laboratory Animals (NIH Publications No. 8023,
revised 1978); and regulations issued by the National Council for
Controlling Animal Experimentation, Ministry of Science, Technology
and Innovation (CONCEA/MCTI), Brazil. All animal use and care pro-
cedures were previously approved by the Animal Ethics Committee of the
State University of Ceará.
Conflict of interest The authors declare that they have no conflict of
interest.
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