Ecotoxicology and Environmental Safety 166 (2018) 366–374

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Ecotoxicology and Environmental Safety

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

Land use impact on potentially toxic metals concentration on surface water T

and resistant microorganisms in watersheds
⁎
Luciana Maria Sarana, ,1, Teresa Cristina Tarlé Pissarrab,1, Géssica Aparecida Silveiraa,c,
Milena Tavares Lima Constancioa,c, Wanderley José de Meloa,d, Lúcia Maria Carareto Alvesa,c
a
São Paulo State University (UNESP), School of Agricultural and Veterinarian Sciences, Department of Technology, Jaboticabal, Brazil
b
São Paulo State University (UNESP), School of Agricultural and Veterinarian Sciences, Department of Rural Engineering, Jaboticabal, Brazil
c
Agricultural and Livestock Graduation Program, School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Brazil
d
Brazil University, Descalvado, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Soil and water resources must be conserved and protected. However, the management of production activities
Soil and water resources causes a change in the quality of these natural resources due to accumulation in soil of potentially toxic metals.
Environmental degradation The objective of the present study was to identify the variety and paths of potentially toxic metals (PTMs), such
Agricultural management as cadmium, lead, copper, chromium, nickel and zinc, which are associated spatially and temporally to soil and
Bioremediation
water. We also intended to isolate bacteria resistant to PTMs with important characteristics to be used in
Trace elements
bioremediation processes. Water samples were collected every two months for one year (February–December/
Heavy metals
2014) at eight sites (P1–P8) and the soil samples were collected twice (February and August/2014) from twelve
sites (S1–S12). Results indicated that agricultural land use impacts the environment, increasing the con-
centration of potentially toxic metals, mainly copper, zinc and chromium, in soil and water due to crop man-
agement. Ten bacteria resistant to all the metals studied were isolated, which could be used as tools for bior-
emediation of contaminated soils and water with those metals. The results would positively contribute to land
use policy, and for the development of enhanced agricultural practices.

1. Introduction it is important to know the actual concentration of PTMs in soil and

Land impacts associated with agricultural and urban development
can intensify runoff and increase erosion that can pollute areas down-
stream, and stream banks in river basins (Pacheco et al., 2014; Paul,
2017; Valle Junior et al., 2014, 2015). Due to anthropogenic inputs, the
concentration of potentially toxic metals (Duffus, 2002) has increased
in the environment, and a thorough understanding on the inputs will
enhance our capability to predict land contamination. Singh and Kumar
(2017) and Zhang et al. (2017a) related that potentially toxic metals
have a wide distribution in the environment, mainly in rural and urban
areas (Zhang et al., 2017b).
There are reports in the literature emphasizing that, mainly due to
multiple agricultural, medical, industrial, domestic and technological
applications, the concentration of a potentially toxic metals may exceed
the regulatory standards and lead to harmful toxicity and bioaccumu-
lation (Acosta et al., 2011; Ali et al., 2016; Chapman et al., 2016;
Kadhum et al., 2015; Liu et al., 2016; Mokhtar et al., 2015). Therefore,
water, the agricultural soil managements and other anthropic actions
that contributed for the pollution in other to take providence for the
depollution or to avoid other areas also be contaminated.
Potentially toxic metals like cadmium (Cd), chromium (Cr), cobalt
(Co), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), nickel (Ni) and
zinc (Zn), and are of high concern due to their direct impact on human
health and aquatic ecosystem (Marrugo-Negrete et al., 2017). Thus,
land use and agricultural management practices, which are a source of
additional degradation in watersheds, are particularly distressing.
These metals need to be monitored carefully because their toxicity
depends on several contributing factors and concentrations, and special
consideration should be given to PTMs, which are of particular concern
while implementing land usage policies. According to the U.S. En-
vironmental Protection Agency (U. S. EPA, 1996) and the International
Agency for Research on Cancer, an analysis of their environmental
occurrence must be done to determine the human exposure due to their
carcinogenicity risk (Tchounwou et al., 2012).

⁎
Corresponding author.
E-mail address: lm.saran@unesp.br (L.M. Saran).
1
Shared authorship.

https://doi.org/10.1016/j.ecoenv.2018.09.093
Received 11 May 2018; Received in revised form 12 September 2018; Accepted 21 September 2018
Available online 01 October 2018
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
L.M. Saran et al.
The land uses impact on soil and water and must be studied on sub-
basins to show if the anthropogenic management affect the aspects of
water, geology, geomorphology and soil (Ali et al., 2016; Kelepertzis,
2014; Liu et al., 2016; Valle Junior et al., 2015). Studies have yet to
produce a comprehensive assessment of PTMs pollution associated with
agricultural activities to better understand the contamination sources of
hydrogeology environments.
The ecosystems within watershed streams may be contaminated by
PTMs concentration above their tolerance limits (Tóth et al., 2016;
Singh and Kumar, 2017), compromising the environment (Valle Junior
et al., 2015; Pacheco et al., 2015). In the present study the focus was on
the potential impact of PTMs on natural resources, and the modification
and conversion of land cover in agricultural lands and urban areas by
the interaction of the variables in space and time. Therefore, spatial
assessment of these PTMs should be investigated to evaluate the im-
pacts of those pollutants. How these changes potentially affect the de-
cisions of agribusiness and the consumer was also analyzed.
The objectives of this study were to determine and to understand
how the concentrations of Cd, Pb, Cu, Cr, Ni and Zn linked spatially and
temporally to water by modification of the land uses in watersheds, and
analyze the concentrations of these metals in soil and water. In addi-
tion, a microbiological evaluation of water was performed to determine
the percentage of bacterial isolates resistant to these metals. The new
view on soil and water management is to develop strategies for water
security in relation to PTMs, their toxicity to humans, and their po-
tential to spread pollution. Furthermore, the possibility of using iso-
lated bacteria is being studied in assisting bioremediation processes.
2. Materials and methods
2.1. Studied area
The studied area is located in the Northeast region of the State of
São Paulo, Brazil (see location map in Fig. 1). The central geographical
coordinates are latitude 21°15'22"S and longitude 48°18'58" W Gr and
Fig. 1. Geographic watershed unit on the São Paulo State, Brazil (Jaboticabal Watershed) and the samples points (soil and water).
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Ecotoxicology and Environmental Safety 166 (2018) 366–374
the specific boundaries were defined as the external limits of the wa-
tershed mapping units at Jaboticabal watershed (extent of 76 km2) of
the Córrego Rico Watershed, a watershed of the Mogi-Guaçu River,
located in the 7th Hydrographic Zone of the State of São Paulo and
corresponds to the 9th Water Resources Management Unit (CBH-Mogi-
UGRHI-09, 1999; IBGE, 1971; Pissarra et al., 2004).
The climate, according to Köeppen classification is Aw (Melo et al.,
2017), which is defined as a humid subtropical climate with a dry
winter. The average temperature of the coldest month is below 18 °C
and that of the hottest month above 22 °C. The average annual rainfall
ranges between 1200 and 1500 mm, and the elevation of the Jaboti-
cabal watershed ranges from altitudes of 450 and 650 m.
A dominant geologic feature is formed from the calcareous sand-
stones and sandy limestones of the Bauru Group (Suguio and Barcelos,
1983) and basaltic slopes that border this plateau with the Peripheral
Depression of the Paraná Basin (Penteado and Ranzani, 1971). Mainly,
in the higher and flatter portions of these hills are the Red Oxisols,
whose material was originally basalt and the transition to the lower
part of the hillside (near the bottom of the valleys) to eutrophic Latosols
with clayey texture, originated from the products of the Sandstones’
alteration to Basaltic (Andrioli and Centurion, 1999; Cunha et al.,
2005). The watershed is described on general appearance of the entire
area as undulating landscape. The subbasin hills are gentle topography,
the state of the highest and lowest parts of the area is occupied by
agricultural land uses and the valleys are occupied by rivers. It presents
particular erosion surfaces and this erosive condition and distribution
of soils are highly common in the State (Lepsch, 2010; Pissarra et al.,
2010).
2.2. Water sampling
The water samples were taken from 8 locations that were re-
presentative of the Jaboticabal Watershed Streams Sites’ water source.
In selecting sampling points (P1 to P8) each locality was considered
individually, particularly points uniformly that were proportional to the
L.M. Saran et al.
number of branches and distributed throughout the length of the
drainage net in the Jaboticabal watershed (Fig. 1, also see
Supplementary material Table S1).
The stream sites were compared over space (spatial) and time
(temporal) and with the possibility of identifying local problems such as
cross-connections and contamination from land uses. The water sam-
pling was carried out every two months, during the year 2014, totaling
six collections in the months of February, April, June, August, October
and December. The simple water samples were collected at a depth of
up to 30 cm, adopting the procedures for collecting, storing and pre-
serving water samples as described in the Standard Methods for the
Examination of Water and Wastewater (APHA, 2005).
2.3. Soil sampling
The soil sampling was conducted in the Jaboticabal watershed, an
area of land where all the rainwater drains downward to the
Jaboticabal stream. This area is also called a catchment, because the
water is caught by the surrounding land and then funneled down into
the basin. Simple soil samples were collected at a depth of 0–20 cm. The
soil sampling sites S1–S6 are located on farm field on the subbasin hill,
at three sites uphill and three sites mid-hill, respectively, and S7–S12
are downhill on the riparian forest areas (reforested growth along the
streams). The area is located at the undulating relief, and on mainly
agricultural land use The Brazilian Federal Forest Code has deemed it a
"permanent preservation area", with a range of environmental func-
tions, and landowners are required to respect a specific extension based
on the width of each stream or river. The physical appearance, texture,
color, slope and drainage of the soil was similar throughout the area.
The map of the experimental area was drawn and identifies the
areas where samples were collected. Sampling fields, plot edges and
other non-representative areas were avoided. The stones, roots, and
debris were removed, and the mix subsamples were put directly in the
collection bucket. Soil samples were classified as Eutrophic Red Latosol
(LVef) (Andrioli and Centurion, 1999).
Soil samples were air dried in the shade, gases were exhaled, sieved
to 2 mm and then submitted to the soil laboratory for chemical analysis.
2.4. Chemical analysis
In the water, the determination of the total concentration of Cd, Cr,
Ni, Zn, Cu and Pb was determined by atomic absorption spectroscopy
with air-acetylene flame, in a GBC Avanta atomic absorption spectro-
meter, after nitroperchloric digestion of the samples as described in the
Standard Methods for the Examination of Water and Wastewater
(APHA, 2005).
After the metal was extracted from the soil using EPA 3050B
methodology (U.S. EPA, 1996), Cd, Cr, Ni, Zn, Cu and Pb concentra-
tions were determined using atomic absorption spectroscopy with air-
acetylene flame. In addition, the concentration of extractable metals
was determined by using the method Mehlich I (Jones Junior, 1990).
2.5. Stock solutions of metals for microbiological tests
The solutions of Cd2+, Cu2+, Pb2+, Zn2+, Ni2+, Cr3+ and Cr6+
100 mmol dm−3 were prepared by dissolving the following salts in
deionized water: CdCl2·H2O, CuSO4·5H2O, Pb(CH3COO)2·3H2O,
ZnSO4·7H2O, NiCl2·6H2O, CrCl3·6H2O e K2Cr2O7, respectively. Such
solutions were autoclaved at 121 °C and 1 atm, by 20 min and subse-
quently used in the resistance tests.
2.6. Isolation of microorganisms
The water samples were pooled and homogenized resulting in a
composite sample. The microorganisms were isolated by the serial di-
lution technique and culture in solid medium plates (K2HPO4
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Ecotoxicology and Environmental Safety 166 (2018) 366–374
0.5 g dm−3; MgSO4·7H2O 0.2 g dm−3; NaCl 0.1 g dm−3; yeast extract
1 g dm−3; glicose 10 g dm−3; ágar 9 g dm−3; pH 6,8) plus 10 mL dm−3
cycloheximide.
For metal resistance tests, the isolates were firstly grown in 96-well
plates containing 1.2 mL of nutrient broth (70122 Nutrient Broth,
Sigma-Aldrich), for 72 h, at 30 °C and 150 rpm. The isolates were
transferred to Petri dishes with solid nutrient medium containing Cd2+,
Cu2+, Pb2+, Zn2+, Ni2+, Cr3+ or Cr6+ ions in the concentrations of
1.0–15.0 mmol dm−3. After incubation in a B.O.D type oven at 30 °C for
72 h, the development on the dishes of bacterial monolayer was con-
sidered as a resistance response of the isolate to the metal at the con-
centration used in the medium.
2.7. Sequencing of the 16s rRNA gene
The 16S rRNA gene was sequenced to identify the bacteria resistant
to each metal analyzed. The extraction of genomic DNA from the iso-
lates was performed according to Marmur (1961) and the 16S rRNA
gene was amplified by the polymerase chain reaction (PCR) using
universal primers fD1e rD1 described by Weisburg et al. (1991) and
internal primers 362f (336–362 region), 786f (746–786 region) and
1203f (1179–1203 region) described by Menna et al. (2006). PCR
products were purified using the "Wizzard SV gel and PCR Clean Up
System" Promega kit. Sequencing was performed on ABI 3700 se-
quencer (Applied Biosystems).
Nucleotide sequences were analyzed with the Phred/Phrap/Consed
program package. The fasta sequences obtained were compared with
the data available in GenBank, using the BLAST tool (Basic Local
Alignment Search Tools) proposed by Altschul et al. (1997).
2.8. Statistical analysis and geographic information system
The results of metal concentrations in soil and water were included
in the statistics which described the basic features of the Jaboticabal
watershed, as well as the simple summaries about the sample and
measures. The water statistical analyses were box-and-whisker plot. For
the statistical analysis of the PTMs concentrations on soil was used the
Inverse distance weighting – IDW model. The spatial distribution of the
concentrations and agricultural land uses was calculated by the values
attributed to the unknown points, with the weighted average of the
values available at the points where the soil samples were collected.
The interpolation was determined from the assumption that the nearest
sampling points of the weighted mean of the samples are more similar,
and was made at the geographic information system Arc GIs 10.2.
Statistical analysis software system R 3.3.2 was used to analyze the
data.
3. Results and discussion
3.1. Potentially toxic metals in water
The mean concentrations of the PTMs in Jaboticabal stream system
in eight sampling sites (spatial) and six periods (seasonal) are shown on
Fig. 2. Results of an exploratory investigation of stream waters with
PTMs including Cd, Pb, Cu, Cr, Ni, and Zn contents in the water samples
showed that the metal contamination of the spatial water sample pre-
sented high values concentrations for Cd (0–6.0 µg dm−3), Cr
(0–70.5 µ dm−3), Ni (0–30.8 µ dm−3), Pb (0–27.6 µg dm−3), Zn
(9.5–156.6 µg dm−3) and Cu (0–655.5 µg dm−3) (see Supplementary
material Table S2).
Potentially toxic metals were found throughout the water in all sites
and all of them exceeded the standard limits recommended by the
Brazilian Government for class 1 and 2 of superficial water in basins,
established by the National Environmental Council (CONAMA),
Resolution n. 357, March 17th, 2005 (CONAMA, 2005) (see
Supplementary material Table S3). Within the stream superficial water
L.M. Saran et al. Ecotoxicology and Environmental Safety 166 (2018) 366–374

Fig. 2. (A) Spatial variation of Cd, Cr, Cu, Ni, Pb, and Zn content (µg dm−3) in collected water samples depending on the location (stream sites: P1–P8) in the
Jaboticabal Watershed. (B) Seasonal variation of Cd, Cr, Cu, Ni, Pb, and Zn content (µg dm−3) in the water samples depending on the month of sampling.

class by CONAMA, several fluctuations in the PTMs concentrations
were observed between sites. In view of this, it is noteworthy to high-
light the several hydrological impacts that the sampling sites has had on
water. Furthermore, results from the statistical analysis (Fig. 2) has
shown that changes in the PTMs concentrations were driven majorly
from the changes in season sampling, as in spring (P1 and P2) and in
places of agricultural activities (P6–P8), contributing in the modifica-
tion of the basin hydrological components.
The streams studied belong to the Hydrographic Unit for Water
Resources Management – UGRHI 09-Mogi Guaçu, São Paulo State,
Brazil. According to the normative references published by the
Environmental Sanitation Technology Company of the State of São
Paulo (CETESB, 2008), it is verified that, within the drainage network
of the study area, in all sampling sites, the P1 point is located in an area
classified as class 2, and the other points, classified as class 4 (see
Supplementary material Table S3). This is a strategic and essential
water ordinance for the implementation of the National Environmental
Policy and the National Environmental System. The drainage net of the
class 2 (P1) is considered fresh water that, after conventional treatment,
can be used for domestic supply. This water can also be used as pro-
tection of aquatic communities, recreation of primary contact (water
skiing, swimming and diving), irrigation of vegetables and fruit plants,

369
L.M. Saran et al.
and for the natural and/or intensive (aquaculture) breeding of species
intended for human consumption. However, according to this same
resolution, the fresh water of Class 4 (sampling points P2–P8) can only
be used for navigation and landscape harmony. Therefore, it is not safe
for domestic and irrigation uses.
Seasonal and spatial patterns of PTMs concentrations (Fig. 2) in
streams of the watersheds are one of the important types of information
needed to characterize water quality. An understanding of when trace-
metal concentrations are highest improves the ability to evaluate the
health of hydro resources (Singh and Kumar, 2017; Zhang et al.,
2017a).
The spatial variation of upstream water (Fig. 2) includes the streams
sites P1 and P2 spring sites, in a natural situation where water flows
from the aquifer to the drainage net protected by riparian areas. At P1
and P2 lower values of the PTMs contents can be observed, except by Zn
at all sites. The water from P2 had the lowest values because it is an
area protected by riparian forest and not gave influence of urban areas.
The water flows downstream from three springs and streams located in
rural areas.
The P3 site is in the middle of an urban area and is formed by a
natural waterway that flows through a populated area in Jaboticabal.
The site P4 has influence from an urban area and P5 area is just outside
of urban areas. The sites P6–P8 are in agricultural areas (see
Supplementary material Table S1). The metal contamination of the
spatial water sample showed high value concentrations for Cd and Cu at
P6 (2,65; 230 µg dm−3); P7 (1.82; 170.6 µg dm−3) and P8 (6.02;
160.4 µg dm−3), respectively, and for Pb at 27.6 µg dm−3 at P8 (Fig. 2,
also see Supplementary material Table S3), those were the most
dominant potentially toxic metal in the drainage net.
This shows that land uses surrounded those streams sites played a
significant role in the level of Cd, Cu and Pb in the watersheds. From
this, it is possible to conclude that this stream-water has relatively high
concentrations of those potentially toxic metals, and its effect on the
aquatic systems can be high, resulted from anthropogenic activities
such as agricultural uses (sites P6–P8; see Supplementary material
Table S1) of metals and metal-containing compounds in fertilizers and
sanitary products. Cadmium is a non-essential and highly toxic element,
moreover it has been determined to have more mobility in aquatic
environments than in soil (Bengtsson and Hodges, 2006; Singh and
Kumar, 2017).
A high degree of variability at individual sites (Fig. 2) occurred as a
consequence of changes in water courses on flow, which are the prin-
cipal controls on fluvial morphodynamics from sites and along the year
(Fig. 2). The implication is that estimation techniques of the con-
centration of potentially toxic metal in water can expect to have a high
degree of uncertainty (Valle Junior et al., 2015). The decreases those
metals contents at the stream sites P1 and P2 may be associated with
the riparian zones upstream (P1) and along the shores of water bodies
(P2) that stabilize banks against erosion. The land use of forestry can
contribute to a better quality of water for all living things (National
Research Council, 2002). The riparian areas may control soil erosion on
site, thus reducing sediment in water bodies and through trapping or
filtering other water pollutants by its root distribution. The reduced
concentration (P1 and P2) were quite different from the increases ob-
served for the downstream waters on the others streams sites (P3–P8).
Therefore, the lower concentration of PTMs on P1 and P2 sites is not
clearly understood at this time and warrants further investigation of
water quality in riparian areas and springs protected by native vege-
tation.
The concentration of PTMs were released into the water phase and
increased along the river downstream of the hydrographic basin, and
the high concentrations were observed in agricultural environment
(P6–P8), resulting in the increase of PTMS release from the particulates.
Consequently, the spatial variation of PTMs concentrations in river
water was closely related to agricultural land uses. The dissolved Cd,
Pb, Cu and Zn concentrations in river water were higher than Cr and Ni
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Ecotoxicology and Environmental Safety 166 (2018) 366–374
at most sampling sites (Fig. 2).
Seasonally, at the water streams, variations of Cd concentration
were observed in December, Cu in April and February, and Pb in April
and December (Fig. 2), presumably due to variations quoted for the
PTMs contents of aquatic systems by rainfall in the rainy season (Feb-
ruary – 83 mm; April – 63.3 mm; December – 150.3 mm), and also due
to soil erosion of metal ions due to rainfall intensity. Pb contamination
has received great attention because of its harmful effects on microbiota
(Zahran et al., 2011). In a study carried out in the city of Adrianópolis
(PR), near the banks of the Ribeira River, in most of the collection
points, Pb contents in the water were higher than 10 µg dm−3 (Melo
et al., 2012). Also in April and December, as land agricultural man-
agement happens, higher concentration was observed (Fig. 2).
Rainfall in the watershed resulted in an increase of PTMs in the
stream waters (Fig. 2) presumed by direct discharge of soil from erosive
processes (rainy season), decreasing the water quality due to rainfall.
Agricultural land uses associated with poor land management practices
that lead to erosion can also produce significant concentrations of po-
tentially toxic metals. It has been observed that mainly due to the ap-
plication of crop mixed mineral fertilizer (2% N, 20% P2O5, 20% K2O +
0.05% Cu + 0.1% Zn), the concentration of Cu and Zn was identified as
from agricultural activity developed in the agricultural areas – stream
sites P7–P9. This was considered a major environmental (and water
quality) problem in the studied area.
On agricultural production system, the producer may use a high
amount of fertilizer, both in soil and in leaf spraying, because there is a
great commercial pressure sectors linked to the fertilizers. This practice
has contributed to production costs, soil fertility and plant nutritional
imbalances. In addition, these products may contain relatively high
levels of contaminants and may be pollutants. Excessive levels of these
elements can be extremely toxic and accumulation can lead to serious
diseases and environmental risks (Mills et al., 1985; Marrugo-Negrete
et al., 2017; Zhang et al., 2017a). Thus, it is important to study the
release of residues in soil and sediment, since these elements can be
absorbed by human, animals and plants, causing intoxication in their
food chain.
3.2. Potentially toxic metals in soil
The landform of the Jaboticabal watershed was formed in a natural
process responding to the geomorphology, lithology, soil formation,
vegetation and other forms of life. On the hills of the watershed, the soil
was sampled to better understand the impact of the land uses on the
concentration of PTMs, such as Cd, Pb, Cu, Cr, Ni, and Zn (Fig. 3, also
see Supplementary material Table S4).
The mean concentration of Cr (41.88 mg kg−1), Cd (0.72 mg kg−1),
Zn (92.10 mg kg−1), Cu (19.25 mg kg−1), Ni (14.95 mg kg−1), and Pb
(21.07 mg kg−1) indicated their common source in the watershed hill
(see Supplementary material Table S4). Alexandre (1995) and Fadigas
et al. (2006) also considered the same. These results already indicate
areas with the presence of PTMs in concentrations that require pre-
cautionary thinking, mainly in agricultural lands of the watersheds hills
(Fig. 3).
Given the complex behavior of PTMs in soil and the redistribution in
watershed hills suffered from diverse human disturbances, the excessive
contents of some PTMs in the riparian areas (S11) may be partially
ascribed to soil erosion processes from uphill anthropogenic sources or
naturally in the soil environment from process of soil development by
weathering of bedrock to produce mineral particles at levels that are
regarded as trace. Considering the immobile nature of the riparian soils
(i.e. local weathering products), their enrichment with PTMs must be
attributed to metal adsorption from soluble fractions, due to runoff.
Comparing the data obtained with the guiding values for soils in the
State of São Paulo (see Supplementary material Table S5), defined by
the Environmental Company of the State of São Paulo, Brazil, the total
contents of the PTMs indicate great variability of these elements in the
L.M. Saran et al.
Fig. 3. Concentration of Cd, Pb, Cu, Cr, Ni, and Zn (mg kg−1) in the soil samples on the Jaboticabal watershed hills, extracted: (A) according to EPA 3050B (total
content); (B) with Mehlich I extractor (soluble and exchangeable forms). Graphics in Universal Transverse Mercator (UTM) coordinate system: longitude values with
the x axis and latitude values with the y axis.
studied soils. The guiding values can be seen on CETESB (2016).
The quality reference value (QRV) reflects the mean natural con-
centration of the elements, indicating situations of non-contamination;
the values of prevention (PV) indicate a possible alteration detrimental
to the quality of the soils, being used in a preventive character and,
when exceeded in the ground, the monitoring of the resulting impacts
should be required; the limit of contamination (IV) above which there
are direct or indirect potential risks to human health and, when ex-
ceeded, the area will be classified as contaminated under investigation,
indicating the need for intervention (CETESB, 2005).
The contents of Cr and Cd ranged from 21.24 to 71.88 mg kg−1 and
from 0.19 to 1.03 mg kg−1, respectively. Then, both Cr and Cd contents
are near the prevention value (PV) in some samples. Zinc content
ranged from 8.30 to 367.30 mg kg−1, values higher than the soil quality
reference values (see Supplementary material Table S5). Copper ranged
from 2.75 to 37.85 mg kg−1, Ni ranged from 5.43 to 39.92 mg kg−1 and
Pb ranged from 10.83 to 22.81 mg kg−1 with some sampling point
presenting contents between QRV and PV.
There is no area above the prevention values, but since the con-
centration of Zn is in at least one sample with a higher content
(367.3 mg kg−1) there is a need for further studies in those areas, in
order to subsidize and direct prevention management activities (see
Supplementary material Table S5). Although some contents have ex-
ceeded the soil quality reference values, in general the main values
observed are close to those obtained by and El Azhari et al. (2017) in
Marocco, Liu et al. (2016) in China and Tóth et al. (2016) in European
soil. The data also agrees with those verified by Fadigas et al. (2006),
except for the maximum values for Zn.
The levels of PTMs available to plants extracted from the soluble
and exchangeable fractions with Mehlich I extractor, direct and indirect
affecting human health, presented, in general, low values. Some ele-
ments, in particular Cd, Ni and Zn, had showed relatively high total
content, indicating that only a small part can be considered available to
plants (Fig. 3, also see Supplementary material Table S4).
The results of the analyses of all PTMs for each soil sample were
combined to see, which samples have high concentrations, above
Brazilian reference values (Fadigas et al., 2006) (see Supplementary
material Table S4). The soil points S1–S6 are located in farm field, and
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Ecotoxicology and Environmental Safety 166 (2018) 366–374
S7–S12 are at the riparian forests (reforested areas grown along the
streams).
The alarming figure means that there is an accumulation of PTMs in
soils mainly due to anthropogenic management, throughout agri-
cultural practices due to the use of products containing potentially toxic
metals, especially in land application of mixed mineral fertilizers (2%
N, 20% P2O5, 20% K2O + 0.05% Cu + 0.1% Zn) with Cu and Zn
contents, animal manure and pesticides applied to crop production (Luo
et al., 2009; Shi et al., 2018; Wuana and Okieimen, 2011). This might
be a reason, why some soil samples had potentially high toxic metal
concentrations in sugarcane areas.
Agricultural land is affected mostly higher uphill (S1–S3), due to
land uses associated to soil management practices and application of
crop mixed mineral fertilizer. Although the samples with metal con-
centrations above the reference values are rather high among all of the
samples, their proportion is visibly higher in sugarcane areas and eu-
calyptus plantation. The most critical soil sampling was in the su-
garcane area planted in a conventional tillage. The riparian soil areas
(S7–S9) are less affected by high concentrations of PTMs, but presented
some soil samples with a high content due to local use of fertilizer and
disease control crop management. Those results were also verified by El
Azhari et al. (2017), Singh and Kumar (2017) and Tóth et al. (2016).
Human activities carried out directly on the soil promote input of
PTMs which can contaminant it. Most soils in rural and urban areas
may accumulate one or more the PTMs at high enough rates to cause
risks to the living things in their environment, as also to human health.
3.3. Isolation of microorganisms
A total of 182 bacteria were isolated from water samples and their
resistance to the metal ions Cd2+, Cu2+, Pb2+, Zn2+, Ni2+, Cr3+ and
Cr6+ was evaluated. The percentage of resistant microorganisms at
concentrations of 1–15 mmol dm−3 can be observed in Supplementary
Table S6. Only in the presence of Ni2 + and Zn2 + ions, the isolates
showed capacity to grow at high concentrations, reaching 15 mmol
dm−3. Only 7% of the isolates were resistant to the concentration of
1 mmol Cd2+ dm−3. The maximum concentrations of resistance to
Cu2+, Pb2+, Cr3+ and Cr6+ were between 4 and 5 mmol dm−3. It is
L.M. Saran et al.
Fig. 4. Resistance of the isolates to various metal ion concentrations and their
identification by sequencing the 16S rRNA gene.
observed that the resistance to chromium is related to its oxidation
state, that is, the isolates showed a higher resistance to Cr3 + than to
hexavalent chromium, since 60% of the isolates are sensitive to the
lowest concentration of Cr6 + (1 mmol dm−3).
Ten isolates showed resistance for all tested metals ions (Cu2+,
Cd , Zn2+, Pb2+, Ni2+, Cr3+ and Cr6+) at different concentrations
2+
(Fig. 4). However, the resistance pattern was not equal between any of
the isolates studied. Compared to the others, the isolate 103 showed the
lowest resistance to Zn2+ and Ni2+. Isolates 39, 109 and 144 were the
most resistant to Ni2 +, the last two isolates being the most resistant to
Zn2 + as well.
The 10 isolates with multiple resistances were submitted to classi-
fication by the study of 16S rRNA gene sequencing and comparison in
databases. It can be observed (Fig. 4) that 5 isolates (103, 109, 125, 126
and 138) belonging to the Enterobacteriaceae family could not be
classified by comparison with the sequences of the RDP database,
however by comparison with sequences deposited in the database of
NCBI they could be classified as components of the Enterobacter and
Klebisciela genus. In the Enterobacter genus, isolate 102 was also iden-
tified. Three isolates of Pseudomonas aeruginosa (39, 144 and 173) and a
Bacillus sp. (33 isolates) were also identified in this set of microorgan-
isms.
Potentially toxic metals together with various pollutants can cause
numerous hazards to both human health and the environment even in
low concentrations because they can be accumulated (Nouha et al.,
2016). Potentially toxic metals as Cd, Cr, Cu, Pb and Zn have attracted
lots of attention from researchers because they are widely spread in the
environment. Some of them are necessary for plants in small amount
but turn out to be hazardous in slightly larger quantities than the
372
Ecotoxicology and Environmental Safety 166 (2018) 366–374
required amount (Zhang et al., 2016).
However, metal resistance is a common phenotype in many mi-
croorganisms exposed to metals in their habitat and resistance genes to
biocides and metals are showed to be present in the great majority of
microorganism genomes isolated from different environments (Pal
et al., 2015). Bacterial adaptations to excessive concentrations of metals
have been described in many cultivated species or in complex com-
munities. In this context, microorganisms play an important role in the
maintenance and sustainability of any ecosystem, as they are more
capable of rapid adjustment towards change and deterioration of en-
vironment.
Microorganisms produce diverse metabolic enzymes that can be
employed for the safe removal of contaminants, which can be achieved
either by direct destruction of the chemical or through transformation
of the contaminants to a safer or lesser toxic intermediate (Dash and
Das, 2012). Microbes apply various types of resistance mechanisms in
response to PTMs (Nies, 2003). Several metal resistance mechanisms
have been identified which are responsible for the alteration of normal
cell physiology leading to development of drug resistance in micro-
organisms (Garhwal et al., 2014).
Bacteria have the properties of chelating PTMs. They can remove
them from the contaminated environment by secreting exopoly-
saccharides which has been evident from the reports of a marine bac-
terium Enterobacter cloacae (Iyer et al., 2005). This bacterium has been
reported to chelate up to 65% cadmium, 20% copper, and 8% cobalt at
100 mg L−1 of metal concentration. Due to their molecular versatility,
bacteria and other microorganisms have proved to be useful tools in a
bioremediation of PTMs and other pollutants.
Moreover, bacteria from the genus Pseudomonas and Bacillus are
related to remediation of organic and inorganic mercury (De et al.,
2008; Dash and Das, 2012), or phytoremediation of Ni, Cr and Cd
(Ndeddy Aka and Babalola, 2016).
The long term effect of pollutants has led to the emergence of multi-
metal resistant bacteria (El-Sayed, 2016). Our study showed us 10
isolates described in the scientific literature to be important species of
bacteria related to several processes of potentially toxic metal re-
sistance and bioremediation. These bacteria present multiple re-
sistances to metals, considering that in our soil there is potentially toxic
metals in a concentration that requires precautionary thinking, mainly
in agricultural lands of the watershed's hills, we can suggest the use in
future of these microorganisms as bioremediation of the study area.
Despite some isolates being classified as pathogenic organisms, other
evaluations are necessary.
4. Conclusions
The agricultural activities as a result of land use management by
higher fertilization resulted in an increase of the concentration of PTMs
in soil leading to water loss within the basin, increasing the con-
centration of metals in the water of the drainage network. There have
been very significant changes in soil and water environment, in terms of
water downstream and soil upper hill in watershed due to concentra-
tion of potentially toxic metals. The soil and water samples analyzed in
the Jaboticabal watershed showed increase in some potentially toxic
metals, mainly copper, zinc and chromium, contributing in the mod-
ification of the basin hydrological components and soil. This change is
also responsible for increased the PTMs during the rainy and crop
management season (April), and increased water pollution in the basin
by applying higher values of nutrients by fertilization of crops. It is
therefore important that the government through environmental policy
regulates the concentration of heavy metals in soil, as done in Brazil
with water.
In this polluted environment we have been isolated 10 bacteria
resistant to all metals studied. Bacteria are known as important or-
ganisms for potentially toxic metal bioremediation and then the multi-
resistance characteristic observed in our isolates can be important for
L.M. Saran et al.
their technological application, mainly bioremediation process. The
results will be useful and can contribute to land use policy, for the
development of enhanced agricultural practices and bioremediation
process.
Acknowledgements
The authors would like to thank: the Coordination of Improvement
of Higher Education Personnel (CAPES) for the scholarship; the
National Council for Scientific and Technological Development (CNPq)
for Research funds (Grant 478347/2013-8); the Agricultural and
Livestock Microbiology Graduation Program and the Agronomy (Soil
Science) Graduation Program from School of Agricultural and
Veterinarian Sciences, in São Paulo State University (UNESP). We also
tank Edvaldo Ramos do Nascimento to crop management information
and Msc Renata Cristina Araújo Costa for helping at the geographic
information system and mapping.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2018.09.093.
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