Pedosphere 24(2): 280284, 2014

ISSN 1002-0160/CN 32-1315/P

c 2014 Soil Science Society of China

Published by Elsevier B.V. and Science Press

Ecotoxicological Tools for Landfarming Soil Evaluation

in a Petrochemical Complex Area1
1,2

1 , K. A. DE ALMEIDA1 , P. R. M. BAISCH2
F. M. R. DA SILVA JUNIOR
, P. F. SILVA1 , F. S. GUIMARAES
1,3
and A. L. MUCCILLO-BAISCH
1 Laborat
orio de Ensaios Farmacol
ogicos e Toxicol
ogicos, Instituto de Ci
encias Biol
ogicas, Universidade Federal do Rio Grande do
Sul (FURG), Av. It
alia, km 8, Campus Carreiros, CEP 96201-900, Rio Grande-RS (Brazil)
2 Laborat
orio de Oceanograa Geol
ogica, Instituto de Oceanograa, Universidade Federal do Rio Grande do Sul (FURG), Av. It
alia,
km 8, Campus Carreiros, CEP 96201-900, Rio Grande-RS (Brazil)
3 Programa de P
os graduac
ao em Ci
encias Fisiol
ogicas, Fisiologia Animal Comparada, Instituto de Ci
encias Biol
ogicas, Universidade
Federal do Rio Grande do Sul (FURG), Av. It
alia, km 8, Campus Carreiros, CEP 96201-900, Rio Grande-RS (Brazil)

(Received July 28, 2013; revised January 9, 2014)

ABSTRACT
The demand for simple and rapid bioassays in ecotoxicological evaluations is of paramount importance in order to speed up
environmental monitoring programs. In this study we performed bioassays with lettuce seeds and two species of terrestrial isopods
(Armadillidium vulgare and Porcellio dilatatus) for the ecotoxicological assessment of a landfarming soil from a petrochemical complex
area. The solubilized content of test soil demonstrated a concentration-response type toxic eect on seed germination rate, and a delay
on germination, but showed toxic eect on seedlings wet weight only at the highest concentration. Toxic eects were also observed
in mortality rate and avoidance behavior of the two woodlice species. These results demonstrated the sensitiveness of the organisms
studied, and highlighted the possibility to use these bioassays in environmental monitoring programs in areas contaminated with fossil
fuels.
Key Words:

Armadillidium vulgare, lettuce, Porcellio dilatatus, terrestrial woodlice

Citation: Da Silva J
unior, F. M. R., Silva, P. F., Guimar
aes, F. S., De Almeida, K. A., Baisch, P. R. M. and Muccillo-Baisch, A. L.
2014. Ecotoxicological tools for landfarming soil evaluation in a petrochemical complex area. Pedosphere. 24(2): 280284.

The use of bioassays to detect alterations caused

by toxic agents has become an important complement
to the simple chemical analysis. Ideal bioassays must
have some particular properties, such as being robust,
standardized, cheap and simple. They must also attempt to include dierent ecological/biological functions, to have a clear endpoint and to allow comparisons between dierent sites, besides responding to environmental stress and changes in such stress (Paton
et al., 2005).
Ecotoxicological evaluations on soils have been
performed mainly with aqueous fraction and using
aquatic organisms. Studies with terrestrial species have
been focused on plants and invertebrates (earthworms
and springtails) (Sverdrup et al., 2003; Paton et al.,
2005). These organisms are chosen based on the sensitiveness of their response. Such choice of organisms
is normally related to the benets of the soil and to
ecosystem functions such as water storage, decomposition and nutrient cycling (Doran and Zeiss, 2000).
1 Supported

In this study, we performed bioassays with a vegetable species (Lactuca sativa, lettuce) and with two
species of terrestrial invertebrates which are important to the decomposition process in the soil (Armadillidium vulgare and Porcellio dilatatus, two common Isopoda species). Germination, growth and dry or
wet biomass are the parameters used in phytotoxicity
evaluation of contaminated soils, which can be analyzed by planting the seeds directly in the soil or using
aqueous fractions for watering the seeds (Wang and
Freemark, 1995; Gong et al., 1999; Henner et al., 1999;
Plaza et al., 2005; Smith et al., 2006). Lettuce is one of
the most used vegetables in phytotoxicity assessment
(Robidoux et al., 2004; Eom et al., 2007; Mart et al.,
2007; Valerio et al., 2007).
Terrestrial invertebrates have become important
organisms for monitoring polluted sites because they
play important roles in decomposing organic matter
and recycling nutrients. Among such invertebrates, terrestrial woodlice are particularly interesting because

by Brazil National Petroleum Agency and Petrobras (No. PRH-ANP/MME/MCT 27).

author. E-mail: f.m.r.silvajunior@gmail.com.

2 Corresponding

ECOTOXICOLOGICAL TOOLS FOR SOIL EVALUATION

281

they are detritivore and accumulate heavy metals.

Also, it is a group of organisms composed of species
that can be very resistant or even highly sensitive
(Cortet et al., 2000). Mortality, growth and reproduction rates and also their avoidance behavior have been
the most important endpoints used in ecotoxicological studies with woodlice (Paoletti and Hassall, 1999;
J
ansch et al., 2005; Loureiro et al., 2005).
The objectives of the present study were to investigate the responses of dierent terrestrial organisms
to a soil from a petrochemical area treated by landfarming with the lettuce and woodlice bioassays and
to indicate the recovery status of the site.

llary column (60 m 0.25 mm 0.25 m). Electron

impact mass spectra were obtained at 1 keV of ionization energy. Helium was used as the carrier gas at
a ow of 1 mL min1 . Temperature was programmed
from 120 to 220 C at 5 C min1 , followed by a 10

C min1 increasing rate until it reached 280 C. The

interface temperature was 280 C.
Copper and zinc in the soil samples were analyzed by ame atomic absorption spectrophotometry

(AASPerkin-Elmer 800, Uberlingen,

Germany), while
electrochemical atomization mode with Zeeman correction was used in chromium, nickel, lead and arsenic
analysis in the soil samples.

MATERIALS AND METHODS

Organisms test

Study area and measurements of polycyclic aromatic

hydrocarbons and heavy metals
The soil was collected from a waste treatment
system SICECORS in an area under landfarming bioremediation process at Polo Petroqumico do Sul, Triunfo, Rio Grande do Sul, Brazil, and was supplied by
Companhia Petroqumica do Sul (COPESUL). This
soil was at initial treatment stage (less than one year),
and was contaminated with many types of pollutants,
including polycyclic aromatic hydrocarbons (PAHs)
and heavy metals, listed in Table I. The landfarming
soil had sand 65.4%, silt 13.7%, and clay 20.9%. A
control articial soil with the same granulometry was
prepared to mix with landfarming soil.
PAH analysis was performed using gas chromatography coupled to mass spectrometry (GCMS) by
injecting 1 L aliquot of the extracts, with a split/
splitless injector (1:50) and an HP-5 fused silica capi-

Lettuce seeds from TopSeed Garden, Petropolis,

Brazil, were submerged in diluted hypochlorite (10 g
L1 ), and rinsed in tap water before use. All lettuce
experiments were initialized on the same day and they
were performed with the seeds from the same batch.
The woodlice A. vulgare and P. dilatatus were collected in areas of organic farming and maintained in laboratory conditions (temperature 28 C, in the dark,
and frequently moistened using water sprinklers). Only
antenna-bearing adults of undistinguished sex were selected for the tests. No gravid females were used. The
animals were kept in the laboratory in earthworm humus and were fed with jambul tree leaves (Syzygium
jambolanum).
Acute toxicity test with lettuce
The soil was solubilized at six dierent concentrations (0, 5, 15, 50, 150 and 500 mg L1 ) using mineral
water as solvent. The stock solution (500 g L1 ) was

TABLE I
Contents of polycyclic aromatic hydrocarbons (PAHs) and heavy metals in the landfarming soil from a petrochemical industrial
complex area
PAH

Concentration

Naphthalene
2-Methyl naphthalene
1-Methyl naphthalene
2,6-Dimethyl naphthalene
1,7-Dimethyl naphthalene
Biphenyl
Acenaphthylene
Acenaphthene
Fluorene
Dibenzothiophene
Phenanthrene
Anthracene
Fluoranthene
Pyrene

g kg1 dry soil

62.60
71.87
70.56
83.77
< 1.66
115.48
5 442.47
118.56
1 056.52
21.04
3 422.89
3 630.22
1 540.49
1 299.72

PAH

Concentration

Benzo(a)anthracene
Chrysene
Benzo(b)uoranthene
Benzo(k)uoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Perylene
Indeno(1,2,3-cd)pyrene
Dibenzo(a,h)anthracene
Benzo(g,h,i)perylene
Total PAHs
23 rings PAHs
46 rings PAHs

g kg1 dry soil

2 940.08
5 348.95
930.58
205.93
860.07
838.45
226.40
359.68
188.00
343.62
29 177.97
14 095.99
13 541.49

Metal

Concentration

Nickel
Zinc
Lead
Copper
Chromium
Arsenic

mg kg1 dry soil

19.49
97.78
24.37
21.23
27.95
3.61


F. M. R. DA SILVA JUNIOR
et al.

282

prepared in a soil:solvent proportion of 1:2 (w:v) according to Da Silva J

unior et al. (2009) and it was
mechanically shaken for 24 h. All concentrations used
in the bioassays were obtained from this stock solution.
Solutions of 0.02 mol L1 CuSO4 were used as a positive control. Every concentration was tested in 5 replicates using 25 lettuce seeds distributed in a Petri plate
containing a paper lter, to which was added 3 mL of
the corresponding concentration solution. At the end
of 5-d exposure, the rate of germination, daily germination, and wet weight of the plants were recorded.
Acute toxicity test with woodlice
The test-soil was diluted with the control-soil to
ve concentrations of 0, 30, 100, 300 and 1 000 g
kg1 . Fifty grams of soil were placed into jars (8 cm
height, 7 cm diameter). Every concentration was tested
in 6 replicates with 5 animals each. The animals were
exposed for 14 d with access to food (jambul tree
leaves), at a temperature of 28 C, in the dark and in
conditions of high humidity. At the end of the exposure
period, the number of dead organisms was counted.
Avoidance behavior response test
This test was divided in two sections: group and
individual. The animals were put in plastic boxes (18
cm 13 cm 12 cm) divided in two compartments
with the same dimensions, one containing the test-soil
and the other the control-soil. For the group avoidance
test ten animals of each species were put in each box in
ve replicates. The individual avoidance test was made
with a single animal in each box in 10 replicates due to
the fact that woodlice present an aggregative behavior
(Loureiro et al., 2009). The animals were kept in the
boxes for 48 h, without food, at a temperature of 28

C, in the dark and in conditions of high humidity. After the exposure period, a wall was put in the center of
the box, and the number of animals in both test- and
control-soil was counted.
Data analysis
Parametric data in wet weight of seedling as well in
daily and nal germination in dierent treatments were
analyzed by Dunnetts test. The median eective concentration (EC50) of the assay with lettuce seeds and
the median lethal concentration (LC50) of the assay
with terrestrial isopods were obtained using the CurveExpert 1.4 software (Daniel Hyams, Hixson, USA)
(Meng et al., 2010, Silva et al., 2012).
For the analysis of group and individual avoidance
behavior tests, the Guideline for Earthworm Avoidance

Test (ISO 17512) was used, in which the habitat function of the soil is considered limited when more than
80% of specimens are found in the control-soil. Statistical analysis was conducted using STATISTICA 8.0.
RESULTS
Acute toxicity test with lettuce
The EC50 value for lettuce seeds after 5 d was 309
mg L1 of aqueous extract of soil. The germination
rate of the positive control was null after 5 d of experiment (data not shown). The increase in concentration
of landfarming soil caused a diminished germination
rate and a delay on lettuce seed germination. Seedling
wet weight decreased only at 500 mg L1 , the highest
concentration tested (Table II).
TABLE II
Cumulative average percentages (n = 5) of germinated seeds exposed for 5 d and wet weights of 5-d-old seedlings under dierent
concentrations of solubilized landfarming soil, using mineral water as solvent, collected from a petrochemical industrial complex
area
Soil
Seed germination
concentration
1d
2d
3d
mg
0
5
15
50
150
500

L1
70.4
59.2
48.8
49.6
41.6*
13.6*

76.8
66.4*
60.0*
60.0*
50.4*
26.4*

%
84.0
69.6
68.8
63.2
53.6*
28.0*

Wet
weight
4d

5d

85.6
78.4
78.4
69.6*
60.0*
36.8*

88.0
85.6
85.6
73.6
61.6*
40.8*

g plant1
0.170
0.173
0.170
0.183
0.174
0.086*

*Signicant at P < 0.05 when compared to the control (0 mg

L1 ) within each column.

Acute toxicity test with woodlice

The experiments with the two woodlice species was
conducted with a mixture of landfarming soil and control soil at dierent concentrations. The LC50 values
for the two species were 74% (A. vulgare) and 27% (P.
dilatatus), respectively. The landfarming soil (concentration 100%) caused a mortality rate of 70% for A.
vulgare and 100% for P. dilatatus.
Avoidance behavior response test
There was no mortality during the avoidance behavior response test. Both woodlice species escaped
from the tested landfarming soil, in which less than
20% of the animals were found in the contaminated
part in the group test, whereas 20% of the animals
were found in the contaminated portion in the individual test. The results of these tests were similar for
both species tested (Fig. 1).

ECOTOXICOLOGICAL TOOLS FOR SOIL EVALUATION

Fig. 1 Percentages of the exposed test organisms Armadillidium vulgare and Porcellio dilatatus in the landfarming soil from
a petrochemical industrial complex (test soil). The dash line
(20%) indicates the habitat function limit. Vertical bars represent the standard deviations of the means (n = 50).

DISCUSSION
Several studies have demonstrated the need for using dierent organisms to evaluate environmental risks
or remediation processes (Eom et al., 2007; Leitgib et
al., 2007; Lors et al., 2010). In this study an acute toxicity test using lettuce seeds and an avoidance behavior
test using two species of terrestrial woodlice were conducted to evaluate the bioremediation quality of a soil
contaminated with PAHs from a petrochemical complex area in the South of Brazil.
The evaluation of soil quality using phytotoxicity is a common strategy (Keddy et al., 1995; Wang
and Freemark, 1995), and bioassays with lettuce seeds
are commonly used for the evaluation of contaminated sites (USEPA, 1989). Bioassays with plants have
demonstrated sensitiveness while measuring the eciency of bioremediation processes (Hamdi et al., 2006,
2007). Plaza et al. (2005) studied 6 higher plants as
bioremediation indicators of a soil contaminated by oil.
They found that L. sativa growth was a more sensitive
toxicity indicator than germination itself. Eom et al.
(2007) also highlighted the low sensitivity of lettuce
seed germination rates as a phytotoxicity parameter,
but they indicated that dry and wet weights are valuable parameters, mainly the latter.
In our study the seedling wet weight was ecient to
demonstrate phytotoxicity of the studied soil, but only
at the highest concentration tested (500 mg L1 ). On
the other hand, germination after 5 d of incubation
was aected even at low concentration of the studied
soil ( 100 g kg1 ).
The strategy of evaluating daily germination increased the sensitiveness of the bioassay with lettuce
seeds. For instance, after only 1 d of incubation, concentrations of the landfarming soil above 30 g kg1

283

were enough to reduce germination. Such evaluation

has already demonstrated to be an invaluable tool in
studies of soils with moderate contamination (unpublished data). Thus, in the present study it shows to be
a useful tool for the assessment of the process of remediation.
Similarly to the lettuce seed bioassay, bioassays
performed with the two terrestrial woodlice species
demonstrated sensitiveness while detecting lethal and
sub-lethal toxic eects. Both species of woodlice studied (A. vulgare and P. dilatatus) are found worldwide and can live in aggregation (Loyola e Silva and
Coraiola, 1999). From the morphological point of view,
they are part of two distinct groups: A. vulgare is a
roller and P. dilatatus is a runner. In the avoidance
behavior test the responses of the two species were similar (escaping from the landfarming soil) and the soil
provoked a high rate of mortality in both species after 14 d of exposure, which indicates an early stage of
bioremediation of the soil. Lethal and sub-lethal parameters were demonstrated to be useful for determining soil toxicity, in disagreement to the proposition
of Geissen et al. (2008), who defended the use of sublethal parameters to estimate the eciency of restoration techniques.
Even though terrestrial woodlice have been targeted in various studies of toxicity related to metals
(K
ohler and Eckwert, 1997; Jones and Hopkin, 1998;
Odendaal and Reinecke, 2003; Godet et al., 2011) and
pesticides (Ribeiro et al., 2001; Jansch et al., 2005),
the use of these animals in studies of organic compounds derived from petroleum is restricted to bioaccumulation via feeding (Van Brummelen et al., 1996). In
our study, these organisms have demonstrated to be
sensitive enough to indicate the presence of toxic
compounds in the soil, among which are metals and
petroleum derived-hydrocarbons, and to reveal the
early stage of remediation in the soil studied.
CONCLUSIONS
The bioassays (lettuce seed germination, woodlice
mortality and woodlice avoidance behavior) used in
this study highlighted the sensibility of the test organisms for the assessment of soils contaminated by
fossil fuels. They are suitable for environmental monitoring programs that aim at following-up bioremediation processes and environmental restoration.
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