Geoderma 332 (2018) 180–189

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Geoderma
journal homepage: www.elsevier.com/locate/geoderma

Macrofauna and mesofauna from soil contaminated by oil extraction T

a a a
David García-Segura , Isis Maviel Castillo-Murrieta , Froylán Martínez-Rabelo ,
Antonio Gomez-Anayaa, Jacobo Rodríguez-Camposc, Benito Hernández-Castellanosb,
Silvia M. Contreras-Ramosc, Isabelle Baroisa,⁎
a
Red de Ecología Funcional, Instituto de Ecología A.C. (INECOL), Carretera antigua a Coatepec 351, El Haya, Xalapa 91070, Veracruz, Mexico
b
Facultad de biología, Universidad Veracruzana, Circuito Gonzalo Aguirre Beltrán s/n, Zona Universitaria, 91090 Xalapa, Veracruz, Mexico
c
Unidad de Tecnología Ambiental, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C. (CIATEJ), Av. Normalistas No. 800, Col. Colinas
de la Normal. Guadalajara, A.P. 2-191, Guadalajara, 44270, Jalisco, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: Mexico is an oil producing country; the extraction of oil on land has left many sites with soil contaminated by oil
Bio-indicators spills. The aim of our study was to determine the impact of this contamination on the soil fauna; thus we
Earthworms compared the macro and mesofauna from a non contaminated soil to a moderately and highly polluted soil
Hydrocarbons caused by oil extraction. Total petroleum hydrocarbons (TPH) and soil physicochemical characterization was
Collembola
determined. TPH results showed two areas: highly contaminated (8150 mg TPH/kg) and moderately con-
Soil quality
taminated (1800 mg TPH/kg). The macrofauna abundance was not significantly different between the sites.
Spills
The Hymenoptera, Gastropoda, Isoptera and the earthworms were the most abundant groups. The Gastropoda
population decreased with the increase of TPH concentration while other groups of macrofauna increased their
density (ants, isopteran and earthworms). The mesofauna was significantly more abundant in the moderately
contaminated area (50,500 Ind./m2). The main groups present were the Acari, Collembola and ants. The Acari
orders were present in similar proportions both in the control soil and in the medium and highly contaminated
area, while the Collembola families varied in their proportion in the three areas. The diversity index showed that
the moderately contaminated site was the more diverse both in macro and mesofauna. Many groups of fauna
(earthworms, ants and Isoptera) were positively correlated to some petroleum hydrocarbons (PH), such as
naphthalene. The Gastropoda and Acari were the groups that were most negatively correlated to the different
hydrocarbons. The PCA differentiated significantly three groups, both in the case of macrofauna as well as for
mesofauna. Earthworms were clearly associated with TPH specially the native species Protozapotecia australis.
These results indicate that oil spills could be a source of food for soil organisms after oxygenation and weath-
ering.

1. Introduction
The petrochemical industry is one of the most important activities in
the world due to the great demand for hydrocarbons and their by-
products for other industries. At the same time, oil extraction and as-
sociated activities have negative impacts on the environment, mainly in
the form of spills or waste products, which affect the sea and soil, the
flora and fauna. Mexico is the tenth largest producer of oil worldwide,
with 2267 millions of barrels per day (PEMEX, 2016). All oil activities
have as consequences the spreading of hydrocarbons to the soil. Ac-
cording to the last report, 159 spills in the soil were recorded as hy-
drocarbon pollution in Mexico, with a total of 1162 ha of contaminated
soil in 2015 (PEMEX, 2016). The states with most spills recorded are
Tabasco (40.2%), Veracruz (32.1%) and Chiapas (18.3%) (CNH, 2014).
These spills have received little attention; they are rarely remediated
and they can last for decades before any action is taken (PEMEX, 2015).
Oil hydrocarbons are toxic to organisms particularly to beneficial
soil organisms, such as the meso- and macrofauna: acari, collembola,
potworms, earthworms, etc. (Cébron et al., 2011; Reinecke et al., 2016;
Sverdrup et al., 2001; Van Brummelen et al., 1996). These organisms
have important functions in the maintenance of soil ecosystem services
and they contribute as well in the form of physical engineering by
creating channels and aerating the soil and biochemical engineering by
promoting the decomposition of organic matter in the soil and causing
interactions with fungi and bacteria (Lavelle et al., 2016). In this way,
they are essential for maintaining the functionality of terrestrial

⁎
Corresponding author.
E-mail address: isabelle.barois@inecol.mx (I. Barois).

http://dx.doi.org/10.1016/j.geoderma.2017.06.013
Received 15 November 2016; Received in revised form 1 June 2017; Accepted 14 June 2017
Available online 29 June 2017
0016-7061/ © 2017 Elsevier B.V. All rights reserved.
D. García-Segura et al.
ecosystems. So, the abundance and diversity of soil organisms have
been used as indicators of environmental impact and disturbance due to
natural or anthropogenic activity, such as some microarthropods
(Austruy et al., 2016) and especially collembolas (Ardestani and Van
Gestel, 2014; Gimenes-Rieff et al., 2016). The macrofauna and some of
their groups have also been used as bio-indicators to determine dis-
turbance or contamination (Bedano et al., 2011; Paoletti, 1999;
Rousseau et al., 2013; Ruiz et al., 2011; Velasquez et al., 2007; Wahl
et al., 2012). Earthworms and collembola are sensitive to changes and
have therefore been used for eco-toxicological tests (Römbke et al.,
2006). However, there are few studies concerning meso- and macro-
fauna at contaminated sites or their tolerance to petroleum hydro-
carbons both separately or together. The lower taxonomic resolution of
groups can give a moderate quality of soil faunal function and diversity
with reduced taxonomic effort and cost. Although a full identification of
part of the community at high taxonomic resolution is required espe-
cially when you are looking for bio-indicator groups (Ekschmitt et al.,
2003). Thus, the aim of this study was 1) to determine meso- and
macrofauna groups at a contaminated site due to spills resulting from
oil extraction and to observe their relation with total petroleum hy-
drocarbons (TPH) and specific hydrocarbons (PH) and 2) to observe the
diversity of specific groups like earthworms, acari and collembola
which are groups susceptible to be used as indicators of soil con-
tamination by PH.
2. Materials and methods
2.1. Experimental site and soil sampling
2.1.1. Experimental site
The oil well 98 field Miguel Alemán, is located in the Totonaca
region, near Papantla in the north of the state of Veracruz, Mexico
whose coordinates are 20°27′22.6′′ N and 97°20′8.5′′ W. Its climate is
warm with an average temperature of 24.4 °C, with abundant rain in
summer, its annual average rainfall is 1010 mm and it is 50 m.a.s.l. Its
original vegetation was tropical semi-deciduous forest. The surrounding
vegetation now is secondary forest or grassland and citric plantations;
the contaminated area is partly covered by grass. The main activities in
the surrounding area are the oil industry, agriculture and livestock. In
this region, the tar or “chapopote” as it was called by the pre-Colombian
inhabitants was often found naturally on the soil surface, particularly
during the last century but with intense oil extraction they were less
obvious (Aguilera, 1980).
2.1.2. Soil and macrofauna sampling
In the area of oil well No. 98, where the oil extraction stopped
15 years ago, a contaminated site as well as a surrounding area free of
contamination (which served as a control) was sampled. The con-
taminated site corresponded to the channel, which was used to lead the
oil spill to a small dam that was made to retain it. The non-con-
taminated site (control) was set 100 m apart more or less parallel to the
channel. At both sites a transect of 100 m was established and every ten
meters a monolith (25 × 25 × 30 cm) was excavated and divided into
three strata at a depth of 0–10, 10–20 and 20–30 cm following the
method of the Tropical Soil Biology and Fertility programme (TSBF,
Anderson and Ingram, 1993). A total of ten monoliths were sampled per
site. Each monolith was reviewed manually at different depths and
macrofauna were collected and placed in their respective plastic con-
tainers containing 70% alcohol; with the exception of earthworms
which were kept in containers with formaldehyde at 4%. Afterwards,
the taxonomic groups were identified, counted and weighed in the la-
boratory with the help of taxonomic keys (Dindal, 1990). In general, the
organisms were identified up to the order level for insects and species
level for earthworms (Oligochaeta).
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Geoderma 332 (2018) 180–189
2.1.3. Mesofauna determination
The mesofauna of the soil was sampled next to each of the monoliths
from both transects with a cylinder measuring 5 cm in diameter and
10 cm tall (196.25 cm3) at a depth of 0–10, 10–20 and 20–30 cm. The
samples were transported in plastic bottles in order to protect the or-
ganisms until the mesofauna extraction with a homemade Berlese
funnel (Karyanto et al., 2008). After the extraction, the mesofauna were
counted and identified. Acari were identified to the level of order and
collembola to the family level.
Deeper taxonomic identification in the majority of the groups was
not done because keys, experience, time and money were not available;
the only groups that had a higher taxonomic identification were those
that could be susceptible to be used as indicators.
2.2. Soil characterization
One kilogram of soil was taken at each point from the transects
where the monoliths were made to determine soil characteristics such
as pH (H2O), electrical conductivity (EC), cation exchange capacity
(CEC), available (P-Olsen) and total phosphorous (TP), organic (OC)
and total carbon (TC), total nitrogen (TN), C/N ratio, moisture, field
capacity, bulk density, texture, iron (Fe), manganese (Mn), copper (Cu),
zinc (Zn), nickel (Ni), lead (Pb) and cadmium (Cd). All the techniques
for the physical and chemical parameters were based on the Soil
Science Society of America methods of soil analysis (Sparks et al., 1996)
or according to the standardized Mexican protocols NOM-021-RECNAT-
2000 (SEMARNAT, 2000). Available P was measured with Olsen
method and total P with vanadate-molybdate method. Additionally, a
sub-sample (200 g) from the soil obtained from the contaminated area
in the ten points sampled was taken, stored at 4 °C until its analysis to
measure the content of TPH and individual PH.
2.3. Total petroleum hydrocarbons (TPH) determination
The TPH were determined using gas chromatography with mass
detector (GC-MS) according to the sonication extraction method 3550B
of the Environmental Protect Agency (EPA, 2007) slightly modified.
The extraction of TPH was made from 2 g of dry homogenized soil,
mixed with 2 g of anhydrous NaSO4 and placed into a 20-mL glass tube.
10 mL of dichloromethane (DCM) were added and mixed in a vortex
and after that sonicated in an ultrasonic bath (Brason 5800/5510) at
40 °C for 30 min with vortex agitation every 5 min. After this, the so-
lution was transferred into a 50-mL flask and 10 mL of DCM was added
again to repeat the extraction process twice. The extracted solution was
added to the same flask and left to evaporate up to 3 mL volume and
then passed through a glass column containing 20 g of florisil® (Sigma)
and Na2SO4 (1:1) (Sigma) to clean the extract. The column was con-
ditioned with 25 mL of DCM and samples were eluted with 40 mL of
DCM, filtered using a 45 μm nylon disk filter and concentrated to 1 mL.
The identification and quantification of individual alkanes and
polycyclic aromatic hydrocarbons (PAHs) were measured by gas chro-
matography (GC–MS). The GC (Agilent Technologies model 6890) was
equipped with a capillary column HP S1933
(30 m × 0.25 mm × 0.25 μm). The oven temperature was set at 50 °C
for 1 min, increased until 120 °C at a rate of 25 °C/min after 10 °C/min
until 160 °C, 6 °C/min until 240 °C and 2 °C/min until 315 °C and fi-
nally maintained at 315 °C for 10 min. The carrier gas was high purity
helium at 1.2 mL/min. The injection volume was 1 μL in splitless mode
at 300 °C. The selective mass detector was a quadrupole (Agilent
Technologies Model 5975), with an electronic impact ionisation system
at 70 eV and at 230 °C.
The identification of compounds was achieved with pure standards
and the mass spectra with the NIST5.0/EPA/NIH (version 2.0 d) library
of mass spectra. Standard curves for quantification were obtained by
preparing a solution with a mixture of the different standard com-
pounds of alkanes (Sigma, UST122) and PAH (Sigma, 69281) in
D. García-Segura et al. Geoderma 332 (2018) 180–189

concentrations of 0.5, 1, 5, 10, 25, 50 mg/kg and 1, 10, 100, 250 and
500 mg/kg respectively. The concentrations were calculated with a
linear regression equation from each standard compound, which had an
R2 from 0.989 to 0.9999. TPH data was the sum of all hydrocarbons
found in the extracts. Twenty different hydrocarbons were measured.
All the soil physicochemical parameters and TPH concentration mea-
surements are referred to soil dry weight at 105 °C.
2.4. Data analysis
Ten samples of meso- and macrofauna at 3 depths of the soil were
thus obtained from the uncontaminated and the TPH contaminated
area. The results of the TPH contents indicated that in the contaminated
area there were two average TPH concentrations that were significantly
clustered in two areas (Table 2), thus we treated all data based on 3
sites: unpolluted or control, moderately and highly contaminated with
TPH. The number of replicates in each site was 10 for unpolluted, 6 for
medium and 4 for highly polluted site. To determine whether there
were any significant differences between the 3 sites, the data was
analyzed with a one way ANOVA and the post hoc test was Fisher's, if
the data fitted a normal distribution, and if not with a nonparametric
Kruskal-Wallis analysis. Diversity indices were applied to meso- and
macrofauna data. Pearson's correlations were calculated between fauna
density, different physicochemical variables and TPH of soil; when the
correlation index was above 0.40 a multiple regression was made with
these respective data and simple linear model for specific PH and
abundance of groups. A PCA was carried out with the soil physico-
chemical and TPH variables and meso- and macrofauna data. Data was
analyzed using the Statgraphic Centurion XVII or SAS. The remaining
analyses were performed using Statistica version 10. Statistical sig-
nificance was chosen at a probability of p < 0.05.
3. Results and discussion
3.1. Soil characterization
In general, the soil was neutral or slightly alkaline (pH 7.7–7.8),
moderately rich in organic carbon (2.2–4.4%) and rich in carbonates as
the total carbon was 6.8–11% (Table 1). The organic matter content
was significantly higher in the highly-contaminated soil than in the
other soils; it indicated 40% more carbon than at the other 2 sites. Some
significant differences were found between the 3 sites (Table 1).
The level of total nitrogen was significantly lower in the moderately
contaminated soil than at the other 2 sites (control and highly con-
taminated). The total P content showed no significant difference be-
tween the three soils; however, the P (Olsen) showed a significant in-
crease in the control soil compared to the highly-contaminated soil.
Thus, the C/N ratio was significantly the highest in the highly-con-
taminated soil which proved that the organic matter was less miner-
alized at the highly contaminated area. No significant differences were
found in EC, CEC, and bulk density among the three soils.
The texture was clay loam in the control soil and moderately con-
taminated soil, but there was significantly more clay in the highly-
contaminated soil; which might be the result that the clay had accu-
mulated in the low part of the channel. Field capacity varied from 21 to
40% and, at the time the soil samples were taken, their water content
was 45 to 100%.
The control soil showed significantly lower metal contents (Fe, Cu,
Zn, Ni, Pb, and Cd) than both contaminated soils which in turn showed
a significant increase of 10 times more Zn and 6 to 8 times of Pb. It is
noteworthy that these concentrations are still within the acceptable
limits of contamination for soils (SEMARNAT, 2000; USEPA, 2002).
3.2. TPH contamination
In the study zone of the Miguel Aleman oil field, two contamination
areas were observed along the oil spill, in which the concentration of
TPH was classified as medium (total TPH 1800 mg/kg) and high con-
centration (8150 mg/kg, Table 2). The concentration of Naphthalene,
n-Octadecane, n-Eicosane, Benzo[b]fluoranthene, Tetracosane, Hex-
acosane, Benzo[k]fluoranthene, Benzo[a]pyrene, Octacosane, Tria-
contane, Indenol[1.2.-c,d]pyrene, Dibenzo[a,h]anthracene was sig-
nificantly higher in the soil from the area of high contamination than
the area of moderate contamination (p < 0.05). There were no sig-
nificant differences for Acenaphthylene, n-Tetradecane, Fluorene, n-
Table 2
Hydrocarbon concentration found in medium and high contaminated area in studying site
( ± standard error).

Table 1 Hydrocarbon Carbons Medium High

Average Physicochemical characteristics of soil in control, medium and high con- number/rings mg/kg mg/kg
tamination area ( ± standard error).
1 Naphthalene C10 (2 rings) 61.2 ± 46 a 252.8 ± 79 b
Parameter Control Medium High 2 Acenaphthylene C12 (3 rings) 96.5 ± 50 a 409.4 ± 1 b
3 n-Tetradecane C14 98.9 ± 34 a 214.3 ± 94 a
pH 7.7 ± 0.10 a 7.8 ± 0.06 a 7.8 ± 0.07 a 4 Fluorene C13 (3 rings) 488.2 ± 81 a 1558.4 ± 481 a
Organic matter (%) 5.0 ± 0.33 b 3.8 ± 0.43 b 7.6 ± 0.52 a 5 n-Hexadecane C16 70.0 ± 32 a 270.3 ± 135 a
Organic carbon (%) 2.9 ± 0.19 b 2.2 ± 0.25 b 4.4 ± 0.30 a 6 n-Octadecane C18 55.7 ± 16 a 287.9 ± 108 b
Total carbon (%) 7.0 ± 0.60 b 6.8 ± 0.78 b 11.0 ± 0.96 a 7 Anthracene C14 (3 rings) 47.7 ± 8 a 298.3 ± 5 a
Total nitrogen (%) 0.3 ± 0.01 a 0.2 ± 0.02 b 0.3 ± 0.02 a 8 n-Eicosane C20 61.3 ± 16 a 292.1 ± 112 b
Total P (mg/kg) 582.4 ± 42.08 b 649.5 ± 54.33 ab 754.4 ± 66.54 a 9 Benzo[b] C10 (5 rings) 28.7 ± 7 a 322.1 ± 142 b
P Olsen (mg/kg) 3.4 ± 0.37 b 6.8 ± 2.65 a 13.0 ± 6.51 a fluoranthene
C/N 10.0 ± 0.57 a 12.6 ± 0.74 a 18.2 ± 0.91 b 10 Docosane C22 69.9 ± 39 a 238.2 ± 86 a
EC (mS/cm) 0.10 ± 0.01 b 0.12 ± 0.01 b 0.13 ± 0.03 a 11 Tetracosane C24 37.3 ± 15 a 132.1 ± 33 b
CEC (cmol/kg) 17.5 ± 2.92 a 11.9 ± 1.62 b 16.2 ± 1.70 a 12 Crysene C10 (4 rings) 680.1 ± 127 a 1663.0 ± 1177 a
Moisture (%) 66.5 ± 3.80 b 72.1 ± 4.90 b 91.7 ± 6.00 a 13 Hexacosane C26 28.4 ± 5 a 160.7 ± 85 b
Field capacity (%) 31.9 ± 0.69 b 32.0 ± 0.90 b 37.6 ± 1.10 a 14 Benzo[k] C20 (5 rings) ND a 465.5 ± 500 b
Bulk density (g/m3) 1.2 ± 0.04 b 1.4 ± 0.05 a 1.3 ± 0.06 ab fluoranthene
Clay (%) 38.4 ± 1.57 b 37.3 ± 2.03 b 51.7 ± 2.48 a 15 Benzo[a]pyrene C10 (5 rings) ND a 502.0 ± 596 b
Silt (%) 30.1 ± 1.06 a 31.6 ± 1.37 a 28.3 ± 1.68 a 16 Octacosane C28 30.6 ± 7 a 199.4 ± 87 b
Sand (%) 31.5 ± 2.06 a 31.2 ± 2.66 a 20.0 ± 3.26 b 17 Triacontane C30 41.6 ± 23 a 382.7 ± 361 b
Fe (mg/kg) 10.8 ± 2.46 b 20.4 ± 3.17 a 28.8 ± 2.86 a 18 Indenol[1.2.-c,d] C22 (6 rings) ND a 892.9 ± 12 b
Mn (mg/kg) 2.7 ± 0.23 a 1.8 ± 0.29 a 1.6 ± 0.36 a pyrene
Cu (mg/kg) 0.3 ± 0.24 b 0.9 ± 0.31 ab 1.9 ± 0.38 a 19 Dibenzo[a,h] C10 (5 rings) ND a 872.0 ± 429 b
Zn (mg/kg) 1.1 ± 1.64 b 9.1 ± 2.12 a 9.1 ± 2.56 a anthracene
Ni (mg/kg) 0.3 ± 0.05 b 0.6 ± 0.07 ab 0.8 ± 0.09 a 20 Benzo[g,h,i] C22 (2 rings) 149.5 ± 112 a 1011.7 ± 963 a
Pb (mg/kg) 0.6 ± 0.64 b 4.9 ± 0.83 a 3.3 ± 1.02 a perylene
Cd (mg/kg) 0.081 ± 0.01 b 0.11 ± 0.01 ab 0.14 ± 0.01 a ∑ TPH 1800.5 ± 192 a 8150.1 ± 4045 b

Rows with different letters are significantly different. Rows with different letters are significantly different.

182
D. García-Segura et al.
Hexadecane, Anthracene, Docosane, Crysene, Benzo [g,h,i]perylene
between both soils (Table 2).
This difference in hydrocarbon concentration might be related to
the topography of the site, which induced the lixiviation from the high
part of the channel down to the lowest part (area of high contamina-
tion). This high concentration of poly-aromatic hydrocarbons was re-
lated to the fact that the compounds with more benzene rings can be
concentrated and binding to the soil matrix due to the high amount of
clays and organic matter accumulated (Yang et al., 2010; García-
Delgado et al., 2016), such was found in the lowest part of the channel
in this study. These compounds have been reported to be degraded and
removed slowly by soil organisms because PAH (with > 4 rings ben-
zene) are highly influenced by the soil characteristics, adsorbed by the
clays and organic matter such as was demonstrated by several authors
(Banach-Szott et al., 2015; García-Delgado et al., 2016; Weissenfels
et al., 1992; Yang et al., 2010).
For Erstfeld and Snow-Ashbrooky (1999) a low level of PAH
(polycyclic aromatic hydrocarbons) ranges from 5.28 to 80.46 mg/kg;
soils from a refinery can reach a TPH content of 100 g/kg (Huang et al.,
2005). However, in this case we had many more PAH and only the
naphthalene content was 61 mg/kg in the moderately contaminated
area and 253 mg/kg in the highly-contaminated area. These levels can
be up to20 fold above the one accepted by European countries
(10–160 mg/kg, Carlon, 2007). Nevertheless, the TPH content in the
medium contamination area was below the limits of the Mexican
standard whereas in the highly-contaminated area it was almost twice
as much above (SEMARNAT/SSA, 2012); for the Netherlands, a soil
with > 5000 mg/kg of TPH is qualified as seriously contaminated
(Pinedo et al., 2014), thus our soil from the low part of the channel was
highly contaminated. Besides the concentrations found at this site were
higher than concentrations indicated to represent a risk to animals or
humans (USEPA, 1993).
3.3. Macrofauna
The macrofauna tended to have more individuals at the con-
taminated sites with an average of 900 Ind./m2 (Fig. 1a). This, might be
due to some organisms within the macrofauna using some of the
compounds in a stage of decomposition as a carbon source to satisfy
their nutrition needs. This value is within the average range of mac-
rofauna abundance in a healthy soil (Lavelle personal comment). Re-
garding the vertical distribution > 80% of the organisms were in the
first 20 cm at the three sites, and we noticed that only at the highly-
Fig. 1. Total soil Macrofauna density and biomass with the respective proportion of the taxa in the 3 levels of TPH contamination (vertical lines indicate the standard error).
183
Geoderma 332 (2018) 180–189
polluted site, there were significantly four times more organisms (120
Ind./m2) in the deepest layer (20–30 cm). This could be a behaviour to
avoid the TPH from the surface. Twenty different taxa were found, 13 of
these were present everywhere. The taxa found only at the unpolluted
site were Isopoda, Symphyla and Diplura. This was partly contrasting to
what was observed in the mesofauna, as the two last groups just
mentioned were in all three sites and with what was reported by
Faulkner and Lochmiller (2000). These authors found that the abun-
dance of Isopoda and Hymenoptera in areas contaminated with waste
from oil refineries was higher than that in uncontaminated areas.
Cicacidae and Homoptera were present at the medium con-
taminated site and Thysanoptera and Phasmoidea at the highly-con-
taminated site. The dominant groups were ants, snails and earthworms.
The snails were dominant in the unpolluted site representing 54% of the
total abundance; at the moderately contaminated site, they decreased
to 21% and at the highly contaminated site to 8% of the total. Instead,
the proportion of ants, earthworms and beetles increased in abundance
progressively from the uncontaminated site to the contaminated sites. It
is normal to find snails because the soil is quite rich in inorganic carbon.
The proportion of ants doubled from the control site to contaminated
sites and at the highly-contaminated sites, earthworms doubled and
Coleoptera was six-fold more. There was no significant difference in the
total biomass (Fig. 1b) at all three sites, the control proved to be the
highest due to the presence of snails and earthworms; although the
snails contribute more to the biomass as they have their respective
shells. At the moderately and highly contaminated sites worms pre-
vailed showing > 58% and 71% of the biomass, respectively. The other
groups showing a substantial biomass were Coleoptera (7%) at the
moderately polluted site and Hemiptera (12%) at the highly-con-
taminated site. In the case of earthworms, we identified three species,
as always, Pontoscolex corethrurus was present, there was also Onycho-
chaeta windlei; both of these species are peregrine. Although we found
Protozapotecia australis a native species, which originated in this area
(Cervantes, 2012) and interestingly its abundance was higher than that
of P. corethrurus at the highly-contaminated site. To our knowledge this
is the first report on the presence of P. australis in hydrocarbon-con-
taminated soil and their tolerance to high concentrations
(8150 mg TPH/kg). It is known that native earthworms are very sen-
sitive to soil disturbance and normally P. corethrurus replace the native
species (Fragoso et al., 1999; Marichal et al., 2010). Here, we did not
see this pattern, therefore this suggests that the genus Protozapotecia
and the species australis has evolved and adapted to soils that have
always had “chapopote” or tar at the soil surface with high
D. García-Segura et al.
concentrations of TPH because this genus has originated in this region
of Veracruz (Cervantes, 2012).
Other studies of sites contaminated with hydrocarbons in tropical
areas observed greater abundance of the earthworms; more than double
the abundance in the control soil of our study and the exotic species P.
corethrurus (75%) was dominant versus native species (25%)
(Hernández-Castellanos et al., 2012). This could indicate that external
factors such as the nutritional quality of the soil, its structure and the
level of contamination are crucial factors for different species of
earthworms.
P. corethrurus seems to be able to grow at sites with a high
184
Geoderma 332 (2018) 180–189
Fig. 2. Total soil Mesofauna density and the proportion of the
taxa in the 3 levels of TPH contamination (vertical lines indicate
the standard error).
Fig. 3. Total soil acari density and the proportion of the order in
the 3 levels of TPH contamination (vertical lines indicate the
standard error).
proportion of Benzo[a]pyrene and is able to remove it as Hernández-
Castellanos et al. (2013) showed, although the concentration they used
in their experiment for its removal was 5 times less than the highly-
contaminated site. This supports our idea that P. australis has adapted to
this site and P. corethrurus cannot compete with it.
3.4. Mesofauna
Mesofauna density was significantly higher at the moderately con-
taminated site (50,500 Ind./m2, Fig. 2); it was double that found at the
other two sites. Eighteen taxonomic units were found; the most
D. García-Segura et al. Geoderma 332 (2018) 180–189

Fig. 4. Total soil Collembola density and the proportion of

the families in the 3.

Table 3 dominant groups were acari and collembola. Seven groups were
Simpson, Shannon and Equitability diversity indices of total macrofauna, earthworms, common to the three sites, and nine other groups were found only at
total mesofauna, Collembola and Acari. one of the sites. The groups that were only found at the control site
Macrofauna diversity
were the Hemiptera Thysanoptera and Protura and this it was the site
with highest representation of different groups. Pauropoda and Pseu-
Simpson (1-D) Shannon-H Equitability-J doscorpionida were only found at the moderately contaminated sites.
Control 0.3373 0.7827 0.4023 At the highly-contaminated site there were only 10 groups and no
Medium 0.8032 1.697 0.9473
High 0.6211 1.106 0.798
group was exclusive to it.
Earthworm diversity Among the Acari, the Oribatida was the most represented order at
Control 0.7988 1.815 0.8727 all three sites, followed by the Mesostigmata and the Prostigmata which
Medium 0.7022 1395 0.867 were only present in the non-contaminated site and moderately con-
High 0.4444 0.6365 0.9183
taminated site (Fig. 3). Cébron et al. (2011) found similar results, they
Mesofauna diversity
Control 0.6953 1.594 0.6923 reported an increase in Acarididae abundance with low hydrocarbons
Medium 0.7452 1.505 0.8397 (PAH) contamination.
High 0.6315 1.133 0.8169 In this research, Collembola were present three times more in
Collembola diversity number at the moderately contaminated site (Fig. 4). The families of
Control 0.3755 0.8593 0.4796
Medium 0.6757 1.437 0.7383
Collembola were distributed very differently at each of the three sites.
High 0.2917 0.5661 0.5153 The Odontelidae family at the non-contaminated site was dominant,
Acari diversity and was hardly present at the moderately contaminated site. On the
Control 0.8264 1.973 0.8569 other hand, the Hypogastruridae family was dominant at the con-
Medium 0.7831 1.666 0.9297
taminated sites, and at the control site there were very few (Fig. 4). Our
High 0.6658 1.226 0.8845
results of the mesofauna are similar to those described by Uribe-
Hernández et al. (2010), who also took a sampling in a TPH con-
taminated area in the south of Veracruz, although this area showed a

185
D. García-Segura et al. Geoderma 332 (2018) 180–189

Fig. 5. Biplot of PCA with loadings and scores of macrofauna data in PC1 and PC3. Twenty sampling points were represented according to the 3 levels of TPH contamination: High (black
big circles), medium (grey circles) and not contaminated (NC, white big circles) area. Black small squares represent physicochemical soil parameters, white triangles indicate polycyclic
aromatic hydrocarbon compounds, diamonds indicate alkanes compounds, black small circles are macrofauna taxes, dark grey big circles indicate earthworm species (P < 0.02).

higher proportion of TPH (14,858 mg/kg). In the case of the Collembola
this area was more diverse at the family level but, as in our study, in the
moderately contaminated area there was an important abundance of
Collembola, although the family represented are not the same. For
example, the Hypogastruridae are not present there. Acari are also
shown in this study to be much less preponderant at the contaminated
sites. Probably the soil organisms that are not present in the con-
taminated sites might be affected directly by hydrocarbons because of
their intimate contact with contaminated soil particles and they have
more permeable cuticles (i.e. nematodes or some Apterygota) than
other such as collembola or acari (Blakely et al., 2002), which show
more tolerance to hydrocarbon contamination.
Blakely et al. (2002) also reported that the changes in soil organisms
were an indirect effect of contamination due to changes in soil prop-
erties more than toxicity effects. These changes could be the result of an
increase in the bulk density of the soil that partially determines which
organisms could be present and participate in decomposition by
altering their habitat, interfering with nutrient uptake, inhabitable pore
space, or modify food webs into the soil. In addition, the toxicity is
reduced due to sorption when organic carbon content exceeds 2% and
bioavailability processes in these environments are not possible
(Alexander, 2000). In this study, the soils had > 2% organic carbon and
the clay content in the highly contaminated was > 50%, which sug-
gests that hydrocarbon toxicity was reduced by a sorption process.
3.5. Diversity indices and correlation
Diversity indices (Simpson and Shannon) were generally higher at
the contaminated sites in the case of meso- and macrofauna and col-
lembola. Only the diversity of earthworms and acari was higher at the
non-contaminated sites (Table 3).
Pearson correlation coefficients between TPH compounds and the
abundances of the macrofauna and mesofauna taxa showed many close
correlations mainly with the macrofauna taxa. Some were negative for

186
D. García-Segura et al.
all the compounds such as for snails, others showed a positive corre-
lation with almost all of them such as the earthworms and in the case of
other groups the correlation was in some cases positive and in others
negative, such as Isoptera and Hemiptera. Seven compounds
(Naphthalene, Fluorene, n-Octadecane, n-Eicosane, Benzo[b]fluor-
anthene, Tetracosane Crysene and Octacosane) exhibited a close cor-
relation with > 4 macrofauna taxa (snails, Isoptera, Hemiptera and
earthworms); the correlation coefficients ranged from 0.45 to 0.91. The
native earthworm P. australis was the species that had the highest
coefficient values with the majority of the hydrocarbons. This confirms
what was discussed above, indicating that this species is adapted to live
in the presence of these compounds. On the other hand, snails are
clearly very sensitive to them. The multiple regression made between
the different PH content and the abundance of termites (0.002), ants
(p = 0.012) and earthworms (0.026) was significant, but in the case of
snails it was not, although simple linear regression for the n-
Tetradecane was good (r = 0.82) which reinforced the idea that some
PH are source of energy or toxicity for the soil fauna.
The Pearson's correlation coefficient values between mesofauna and
TPH were in the majority very distant; the only group that showed some
higher values was the ants in the case of seven compounds (0.45–0.55)
where the correlation was positive. The majority of the correlation
values of hydrocarbons with collembola were positive and for Acari
negative, although the values were very distant. These results coincide
with Cotrufo et al. (2014), who found that naphthalene addition to soil
significantly reduced the abundance of mesofauna: oribatid acari
(− 45%), predatory acari (− 52%) and collembola (− 49%).
187
Geoderma 332 (2018) 180–189
Fig. 6. Biplot of PCA with loadings and scores of me-
sofauna data in PC1 and PC3. Twenty sampling points
were represented according to the 3 levels of TPH
contamination: High (black big circles), medium (grey
circles) and not contaminated (NC, white big circles)
area. Black square represent physicochemical soil
parameters, white triangles indicate polycyclic aro-
matic hydrocarbon compounds, diamonds indicate al-
kanes compounds, black small circles are mesofauna
taxes (P < 0.02).
3.6. Principal component analysis
The principal component analysis (PCA) explained a 54.39%
variability of macrofauna data with PC1 and PC3. The positive axis of
PC1 grouped highly contaminated (MC7, MC8 and MC9) sites with TP,
the available P (Olsen), clays, metals, all PAHs, alkanes, total earth-
worms, the native species Z. australis, Coleoptera and termites (Fig. 5).
The negative axis of PC1 separated the sand and earthworms P. core-
thrurus, which was correlated with control site (NC). On the other hand,
the bulk density, pH and silt were located on the positive axis of PC3
and correlated to the medium contamination area. The TN, Mn, OM,
OC, total biomass density, Mollusca, Lepidoptera larva, Coleoptera,
Hymenoptera, Hemiptera and anthracene were grouped on the negative
axis of PC3. These variables were correlated with the non-contaminated
area. All the physical and chemical variables and macrofauna showed a
clear grouping at the 3 sites, which was significant (P < 0.02).
The same PCA analysis was carried out with the physicochemical
variables and mesofauna data (Fig. 6); PC1 and PC3 explain 49.32%
data variability. They showed a clear grouping of each sampled area
according to the level of contamination. All hydrocarbons were grouped
with OM, OC, metals, clay, CIC, EC, TC, available P (Olsen), TP and all
of them correlated to the highly-contaminated area (MC7, MC8, MC9)
on the positive axis of PC1. On the other hand, on the positive axis of
PC3 CIC, silt, Mn, TN, Thysanoptera, Symphyla, Hemiptera, Pauropoda,
Diplopoda, Protura, Larvae, Chilopoda, and Coleoptera were found.
They were correlated with the non-contaminated area (control).
At the moderately contaminated site, some mesofauna groups such
as Araneae, Collembola, Hymenoptera, Pseudoscorpionida, Homoptera,
D. García-Segura et al.
Acari, moisture, pH, bulk density, sand and anthracene were grouped
along the negative axis PC3.
The PCA also highlighted the strong association that earthworms
and P. australis have with the TPH. In the case of the mesofauna the PCA
showed more clearly that the collembola have a positive relationship
with some hydrocarbons at the moderately contaminated site whereas
the Pearson's correlation coefficients were weak. This is similar to what
Erstfeld and Snow-Ashbrooky (1999) suggested: low concentration of
TPH can stimulate the invertebrate community of the soil which re-
inforces our idea that carbon can be a source of food directly for them
or for the bacteria and fungi that are in their food web. It is known that
some bacteria naturally degrade TPH, particularly in the Gulf of Mexico
region (Suidam, 2010). Zavalza-Ramírez, et al. (unpublished data)
isolated some bacteria of the contaminated area, some of them are from
genera that have already been identify to be oil decomposers (Yan et al.,
2013; Das and Chandran, 2011). Besides, warm regions have much
more of a possibility to degrade more rapidly the TPH which probably
happened in our studied area (Das and Chandran, 2011). Thus, at our
site there is probably a natural degradation of the TPH and a trophic
network developed around it; starting from the microorganisms and
facilitated by the meso- and macrofauna, although this degradation is
quite slow as this well has been closed for 15 years. Thus, it is important
to know which organisms are susceptible to degrade TPH and then try
to cultivate them to accelerate the bioremediation of these areas by bio-
augmentation of native species or species found naturally at con-
taminated areas. This has been tested with success by other authors
(Mrozik and Piotrowska-Seget, 2010; Suja et al., 2014; Wu et al., 2016).
4. Conclusions
Mesofauna density was significantly higher in moderately polluted
soil than in the control soil and the macrofauna was more abundant at
the most contaminated areas but it was not significant. Both meso- and
macrofauna were more diverse in moderately contaminated soil as
shown in the diversity indices.
The mesofauna groups were more sensitive to TPH as there was a
strong change of their proportions from non-contaminated soil to
highly-contaminated soil. Although at a family level, the collembola
can be indicators of pollution, thus the Odontelidae may indicate un-
polluted soil and Hypogastruridae contaminated soil.
Several taxa were especially sensitive to some hydrocarbons parti-
cularly to naphthalene and fluorine, negatively in the case of snails and
acari and positively in the case of termites, ants and earthworms spe-
cifically P. australis. The native endogeic earthworm P. australis is as-
sociated with highly contaminated sites. This species has probably
evolved in soils with tar as Veracruz has a lot of tar spills and it is the
area where Protozapotecia originated (Cervantes et al., 2014). Thus, it
could prove a candidate for producing biodegradation of TPH in this
region. It had already been noticed that earthworms can promote the
removal of PH; but most of these studies were performed with epigeic
earthworms and or from temperate regions (Dendooven et al., 2011)
and with the tropical peregrine P. corethrurus (Hernández-Castellanos
et al., 2012, 2013).
These results finally indicate that oil could be a source of food after
oxygenation and weathering and that there is a succession of soil fauna
that shape networks depending on the concentration and composition
of TPH.
Acknowledgments
This research had financial support from CONACYT Mexico through
project 247619. The authors I. M. Castillo-Murrieta and D. García-
Segura are grateful to CONACYT for their undergraduate scholarship
received to carry out this research. To Jose G. Palacios Vargas, Blanca E.
Mejía Recamier, Ricardo Iglesias Mendoza and Gabriela Cervantes
Olvera, thank you for your help in the identification of organisms. We
188
Geoderma 332 (2018) 180–189
are grateful to Imelda Castro Ronzón for TPH analyses and to the
Technicians of the Soil, Water and Plant Laboratory (LABSAP) of
INECOL.
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