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56
Soil & Water Res., 8, 2013 (2): 56–62
Assessment of a Soil with Moderate Level
of Contamination using Lettuce Seed Assay
and Terrestrial Isopods Assimilation Assay
F M R DA SILVA JÚNIOR1, E M GARCIA1,
P R M BAISCH 2, N MIRLEAN 2
and A L MUCCILLO-BAISCH 1
1Laboratório de Ensaios Farmacológicos e Toxicológicos, Instituto de Ciências Biológicas
and 2Laboratório de Oceanografia Geológica, Instituto de Oceanografia,
Universidade Federal do Rio Grande do Sul – FURG, Rio Grande, Brasil
Abstract
D S J F.M.R., G E.M., B R.M., M N., M-B A.L. (2013): Assessment of
a soil with moderate level of contamination using lettuce seed assay and terrestrial isopods assimilation assay.
Soil & Water Res., 8: 56–62.
Lettuce (Lactuca sativa) seeds play a significant role in toxicity tests of isolated chemicals, pesticides, and envi-
ronmental samples. Commonly, the main variables under study are the rate of seed germination and root elon-
gation at the end of five days of exposure. Another organisms used in environmental assessment of soil quality
are terrestrial isopods. The parameter evaluated in this assay is usually mortality rate. In this study, we suggest
to use the daily number of germinated seeds and wet weight of plants, and feeding measurements (consumption
rate, assimilation rate, assimilation efficiency and growth rate) in woodlice (Armadillidium vulgare and Porcellio
dilatatus) to detect toxicity of moderately contaminated soil samples. The lettuce seed assay proved to be more
efficient in the tested conditions, however, we do not reject the use of feeding parameters in terrestrial isopods
in toxicological screening of contaminated soils.
Keywords: Lactuca sativa; short-term bioassays; soil contamination; woodlice
The use of bioassays for detecting changes caused
by toxic agents has become increasingly common
in diagnostic and monitoring studies. For the use
in environmental evaluation, it is necessary to
reunite some features like: to be standardized,
simple, low cost, to have a defined endpoint, and
be sensitive enough to distinguish differences
among sites (P et al. 2005).
Few studies have been conducted for evaluation
of soil toxicity. Most of these studies use plants
and evaluate endpoint germination and growth,
while there are also assessments of survival and
reproduction of terrestrial invertebrates (E et al.
2007). Some of these bioassays are ruled by specific
standards (USEPA 1989) and recommended in the
battery for assessment of soil quality (K et
al. 1995; W & F 1995).
Bioassays with plants have been used for evalu-
ation of phytotoxicity of soils contaminated by
various substances (G et al. 1999; P et
57
Soil & Water Res., 8, 2013 (2): 56–62
al. 2005; S et al. 2006; M et al. 2007;
V et al. 2007). However, parameters such
as germination (K et al. 1995; S et
al. 2003) and growth (R et al. 2004) have
been under debate on whether being really sensi-
tive endpoints in phytotoxic evaluation.
H et al. (2005) suggested the use of
terrestrial isopods (woodlice) as a tool of eco-
toxicological evaluation for contaminated sites.
Terrestrial isopods are an important member of
the invertebrate community of the soil, they are
also easy to capture in the field and maintain in
laboratory. Among soil invertebrates, woodlice
play an important role in decomposition processes
through the fragmentation of litter by their feeding
activity (Z et al. 2003).
This study aimed to investigate the ecotoxi-
cological status of a soil with moderate levels of
contamination using two approaches: (I) daily
counting of germinated seeds of lettuce and fresh
weight of plants after five days of experiment and
(II) mortality (21 days) and feeding parameters in
woodlice on the 22nd day of experiment.
MATERIAL AND METHODS
Soil samples. Soil samples were collected from
two distinct sites in Rio Grande municipality, satate
of Rio Grande do Sul, southern Brazil: (i)soil of
an industrial-urban area, under the influence of
fertilizer industries via atmosphere and petroleum
refining industry (contaminated), and (ii)soil of
forested area on the Federal University of Rio
Grande (Universidade Federal do Rio Grande –
FURG) campus (control).
The study area (industrial-urban area of the city
of Rio Grande) is located in the southern part of
the coastal plain of Rio Grande do Sul State, Bra-
zil. This area is of Holocene age, and was formed
by sediment deposition resulting from marine
regression. The soils are recent and evolved over
udic moisture regime and thermic soil tempera-
ture regime controlled by the humid subtropical
climate. These soils have sparse vegetation, mainly
represented by grasses.
These soils (of the urban and industrial area
of the city of Rio Grande) are composed mainly
of sand of aeolian and marine origins and they
have very low concentrations of organic matter,
nutrients and clay minerals. These soils have no
horizons with different colour or texture, are not
saturated with water, and are classified as Typic
Quartzipsamments (C et al. 1995)
Collection and storage of surface soil. At each
collection site, surface sampling of soil was carried
out at a depth of 20 cm, using a plastic shovel. Stones
and plant material were removed. In the field, the
material was placed in plastic bags and transported
into the laboratory. In the laboratory, the soil was
separated and stored at –20 °C for biological assays
and at room temperature for metal analysis.
Soil (solubilized) extraction. Soil samples were
shaken (116 rpm) at room temperature for 24h with
mineral water (soil to solvent, 1:2 g/ml) (DS
J et al. 2009).
Acute toxicity test with lettuce (Lactuca sativa).
In order to evaluate acute toxicity in lettuce seeds,
contaminated and control soils were solubilized in
five different concentrations (5, 15, 50, 150, and
500 mg/l of solvent). Negative control employed
only the solvent and positive control a 0.02M
CuSO4 solution. Each concentration was tested
in four replicates, using 25 seeds distributed in
Petri dishes containing filter paper and 3 ml of
solvent (only at the beginning) was added in the
respective concentrations. No more water was
added to the plants during assay.
In a second experiment, contaminated soil was
mixed with control soil in six concentrations
(0, 1, 3, 10, 30, and 100%) and 25 seeds were placed
directly on the soil (30 g). Plates were watered
with 3 ml of mineral water. This experiment was
performed in triplicate.
After five days of exposure, the germination rate
(classic endpoint) was assessed, in addition to the
daily germination rate and wet weight of plants at
the end of the experiment (new variables).
Feeding measurements in woodlice. The isopods
used in these experiments (Porcellio dilatatus and
Armadillidium vulgare) were obtained from labora-
tory cultures, composed of animals collected from
a compost pill and brought to the laboratory where
they were fed ad libitum with tree leaves of jambol
(Syzygium jambolanum) and maintained at 28°C
with a 16:8 h (light:dark) photoperiod for 3 months
before use in experiments. Only adult animals with
antennae were selected for the tests and the sexes
were not distinguished. Humidity was maintained
by regular spraying with distilled water and the
ventilation in the boxes was ensured by periodic
openings on the box cover (R et al. 2001).
The animals were kept in the control and contami-
nated soil for 21 days in Petri plates. Each soil was
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Soil & Water Res., 8, 2013 (2): 56–62
tested in six replicates (plates), with five animals
in each plate. At day 20, animals were left without
food to empty their gut. At day 21, the isopods
were removed from the soil and the number of
dead organisms was counted. The living organisms
were weighed and placed on Petri plates with filter
paper and food (jambol). The food offered to the
animals was weighed. The faeces of the woodlice
and the remaining leaf material were removed and
also weighed (wet weight) (R et al. 2001).
Besides the mortality rate after 21 days, the pa-
rameters measured were similar to those employed
by R et al. (2001): consumption rate (mg
food consumed mg/animal/day), assimilation rate
(mg food assimilated mg/animal/day), assimila-
tion efficiency (percentage of assimilated food in
relation to the consumed food), and growth rate
(biomass gain/average initial weight). Assimilation
and consumption were evaluated after weighing
the pooled food, animals, faeces and the remaining
food of each Petri dish. The sum was then divided
by the number of living animals.
Metal quantification in the extracts. Copper
and zinc in the soil and soil extract samples were
analysed by flame atomic absorption spectropho-
tometry (AAS Perkin-Elmer 800, Perkin-Elmer,
Shelton, USA), while electrochemical atomization
mode with Zeeman correction was used in chro-
mium, nickel, lead, arsenic and cadmium analysis
in soil and soil extract samples. Maximal value of
relative standard deviation for the analysis of 3
replicates of an individual sample was less than 4%.
Data analysis. Results were expressed as mean
± standard deviation. For comparison of means,
analysis of variance (ANOVA) was carried out
whenever assumptions were met. Duncan’s test
was applied for comparison among groups, at
5% statistical significance using Assistat 7.6 beta.
RESULTS
Acute toxicity test with lettuce (Lactuca sativa)
Table 1 shows the germination of lettuce seeds
treated with aqueous solubilized soils of the control
and contaminated soils at different concentra-
tions. The commonly used endpoint to evaluate
toxicity in lettuce seeds has been the inhibition
of germination after 5 days of exposure. However,
this variable did not show sensitivity to reveal the
toxic effects of contaminated soil. After 5 days, the
germination rate did not differ between the control
and any concentrations of the contaminated soil.
Table 1. Average number of germinated seeds and wet weight of seedlings exposed for five days to different con-
centrations of solubilized (aqueous extract) from two soil samples (control and contaminated)
Day Concentration (mg/kg)
0 5 15 50 150 500 positive control
Control soil
123.25 24.25 21.25 24.25 23.25 23.25 0
224.12 24.25 23.75 24.75 24.50 24.75 0
324.25 24.25 24.00 24.75 24.50 25.00 0
424.25 24.25 24.25 24.75 24.50 25.00 0
524.25 24.25 24.25 24.75 24.50 25.00 0
Wet weight 0.3160 0.2862 0.27497 0.2761 0.2857 0.2728 0
Contaminated soil
123.25 23.75 23.75 23.00 22.50 20.25 0
224.12 24.25 24.25 24.25 24.25 23.75 0
324.25 24.25 24.50 24.50 24.25 23.75 0
424.25 24.25 24.50 24.50 24.50 23.75 0
524.25 24.25 24.50 24.50 24.50 23.75 0
Humid weight 0.3160 0.2946 0.2841 0.2655 0.2622 0.2524 0
Data in bold refer to results that are significantly different from the control
59
Soil & Water Res., 8, 2013 (2): 56–62
The strategy to investigate the germination
daily until the 5th day revealed significant differ-
ences between the highest tested concentration
of contaminated soil and the control on day 1 of
observation. The control soil showed no toxicity
at all tested concentrations (Table 1).
Another endpoint used – the fresh weight of
plants after five days of exposure – was also sen-
sitive to highlight the toxic effects of the con-
taminated soil. The fresh weight was reduced at
the highest concentrations of contaminated soil,
whereas there were no changes at different con-
centrations of control soil (Table 1).
In the experiments with lettuce seeds exposed
directly on the ground, the germination delay on
the first day of exposure was more pronounced
than the results obtained with the seeds exposed
to soluble fraction, while by the end of five days
the germination rate did not differ among any of
soil concentrations. However, the wet weight did
not differ among the different concentrations
or between any concentration and the control
(Table 2).
Feeding measurements in woodlice
Mortality rate was between 10 and 20% for both
woodlice species in the control and contaminated
soil (Table 3). Feeding parameters and growth in
Table 2. Average number of germinated seeds and wet weight of seedlings exposed for five days to different con-
centrations of a mixture of control and contaminated urban soil
Day Concentration (%)
0 1 3 10 30 100
120.33 20.67 18.67 15.33 15.67 13.67
222.33 22.33 21.33 22.00 23.33 22.67
323.67 23.33 2267 24.67 24.33 24.00
423.67 25.00 22.67 25.00 24.67 24.00
524.00 25.00 23.67 25.00 24.67 24.67
Humid weight 0.4608 0.4637 0.4808 0.4647 0.4839 0.4558
Data in bold refer to results that are significantly different from the control
Table 3. Mortality and feeding parameters of A. vulgare and P. dilatatus cultured for 21 days in control and con-
taminated urban soil
Parameter Control soil Contaminated soil
A. vulgare
Mortality (%) 10 20
Consumption rate 0.28 ± 0.12 0.31 ± 0.12
Assimilation rate 0.20 ± 0.15 0.23 ± 0.11
Assimilation efficiency (%) 64.22 ± 28.19 73.99 ± 19.03
Growth rate (%) 12.04 ± 6.74 17.02 ± 11.06
P. dilatatus
Mortality (%) 20 20
Consumption rate 0.39 ± 0.09 0.65* ± 0.23
Assimilation rate 0.29 ± 0.07 0.52* ± 0.19
Assimilation efficiency (%) 76.01 ± 13.83 81.67 ± 13.09
Growth rate (%) 7.23 ± 11.51 30.36* ± 11.22
Results are expressed as the mean of the parameter and standard deviation; *indicate significant differences between
control and contaminated soil
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Soil & Water Res., 8, 2013 (2): 56–62
A. vulgare were not significantly different between
the control and contaminated soils, while some
parameters in P. dilatatus were statistically distinct
between both soils. However, the contaminated
soil improves feeding measurements in P. dilatatus
in the following parameters: consumption rate,
assimilation rate and growth rate (Table 3).
Metal quantification
Among the metallic elements analysed, the
concentration of Cr, Cu, Pb and Zn in contami-
nated soil was above prevention levels laid down
by Brazilian legislation, but no evaluated metal
exceeded the investigation levels for residential
soils (CONAMA 2009), characterizing this soil as
moderately contaminated. In the control soil, only
Cr had levels above the level of prevention, while
none of the elements exceeded the investigation
levels for residential soils (Table 4).
On the other hand, the Cr, Ni and Pb levels in the
aqueous extract of contaminated soil were above the
Brazilians limits allowed for groundwater while in the
control soil extract none of the elements exceeded
the Brazilians levels (CONAMA 2009) (Table 4).
DISCUSSION
This study evaluated the toxicity of a soil located
in an urban-industrial zone, where the major input
of contaminants from industries originates from
atmospheric dispersion. Soil contamination under
these conditions requires a sensitive endpoint for
visualization of environmental damage, especially
in cases where the element concentrations are at
levels permitted by legislation. For this study, bio-
assays with three species were used for assessing
toxicity: acute toxicity bioassay with lettuce seeds
and toxicity tests with woodlice (Armadillidium
vulgare and Porcellio dilatatus).
In the bioassay with lettuce seeds, the number
of seeds germinated after five days of exposure is
commonly used as a classical endpoint. If only this
endpoint was taken into account, the soil shows
no toxicity for the experimental model, neither for
the solubilized test nor seeds exposed directly to
soil. However, when other endpoints were added
(daily number of germinated seeds and fresh weight
of plants after five days of exposure) toxicity was
observed in the sample of contaminated soil and
absence of toxicity in the control soil sample, which
confirmed the sensitivity of these other endpoints.
Studies evaluating the phytotoxicity of soils have
been recommended for assessment of soil quality
(K et al. 1995; W & F 1995).
However, some studies have shown the low sensitivity
of seed germination to predict phytotoxicity of soil
samples (S et al. 2003; S et al. 2006),
including the toxicity test with lettuce seeds (E et
al. 2007). R et al. (2004) and M et al.
(2007) used parameters related to growth, and they
also found low sensitivity of the assay with lettuce to
predict toxicity of soil samples. ese results under-
line the importance of finding endpoints sensitive
in bioassay with lettuce, including soils with low or
moderate levels of contamination.
Table 4. Metal concentration in soil samples and soil aqueous extracts
As Cd Cr Cu Ni Pb Zn
Soil samples (mg/kg)
Control soil 2.28 0.558 95.50 31.64 19.66 36.70 102.45
Contaminated soil 8.71 0.902 185.83 88.91 39.75 179.03 504.56
Soil prevention levels115 1.3 75 60 30 72 300
Soil investigation levels1
(residential soil) 55 8300 400 100 300 1000
Soil extract samples (µg/l)
Control soil 0.367 –28.30 55.71 12.79 8.075 77.92
Contaminated soil 2.529 0.87 50.59 91.68 36.93 58.20 101.50
Brazilian standards
to groundwater110 5 50 2000 20 10 1050
1CONAMA (2009)
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Soil & Water Res., 8, 2013 (2): 56–62
Our strategy to evaluate the toxicity of the solu-
bilized soil and the effect of the seeds directly on
the soil was useful, considering that each form of
exposure showed different sensitivity to the two
alternative endpoints: assessment of the solubilized
presented wet weight as an important endpoint, while
seeds deposited directly on the ground revealed a
pronounced delay in germination at the highest
concentrations of contaminated soil. is delay in
germination was previously reported by V et
al. (2007) working with soluble elements of soil. In
this study, the soluble fraction also induced germina-
tion delay, but it was more pronounced in the study
with the seeds deposited directly on the ground.
Regarding the sensitivity of the wet weight as
endpoint for evaluation of toxicity of soil sam-
ples, E et al. (2007) found that wet weight was
a more sensitive parameter than dry weight to
investigate soil toxicity in lettuce grown directly
on the ground. In this study, the wet weight of
plants growing directly on the ground showed no
differences between the control and the highest
concentrations of contaminated soil. However, the
fresh weight was a sensitive endpoint for plants
grown in the solubilized treatment, decreasing
weight as the concentration of contaminated soil
on soluble increased. Phytotoxicity of soil under the
influence of atmospheric dispersion of pollutants
investigated by bioassay with lettuce seeds could
be related to concentrations of metallic elements
found in soil and its extracts
Regarding the soil invertebrates, O and
R (2004) commented that little attention was
paid to the toxic effects of environmental mixtures
on non-target organisms and that this strategy in-
volved more realistic models for the understanding of
environmental toxicity. For both woodlouse species,
there was no difference between mortality rates from
animals exposed to control and contaminated soil.
The feeding-related parameters have been re-
ported as a promising tool for the investigation of
sub-lethal toxicity in terrestrial isopods exposed to
metals (D & H 1995; D 1997).
In the present study, these parameters were not af-
fected by metals present in the urban soil under the
influence of atmospheric dispersion of pollutants.
The short exposure period (21 days) could be one
reason for this absence of toxic effect in woodlice
(R et al. 2001). Another explanation also ap-
proached by R et al. (2001) is a narrow line
between lethal and sub-lethal effects in woodlice.
This can be possible because the woodlice can trap
certain metals in granules in their internal organs
(D 1992; P & H 1999), not
suffering the toxic effects of the metals. The con-
taminated urban soil may not contain the toxic
concentrations of metals for the species studied,
or metals in the mixture have antagonistic effects.
Bioassays used in the present study showed dis-
tinct responses for assessment of an urban soil with
moderate level of contamination. Some researches
affirm to be unsatisfactory to use a single ecotoxicity
test for soil quality assessment, revelling a dispro-
portionate effect on different organisms (P
et al. 2005; L et al. 2010). Our results reaffirm
the view of using different organisms to investigate
the soil quality, mainly in soil with moderate or low
levels of contamination. Even at moderate concen-
trations, the mixture of contaminants in the soil
seems to exert phytotoxic action in lettuce seeds
while not displaying toxicity to woodlice. Although
the concentration of metals in contaminated soil
and its extract appears to contribute to phytotox-
icity, we cannot discard the negative contribution
of organics, especially petroleum hydrocarbons
arising industrial and vehicle emissions.
CONCLUSION
The alternative parameters analysed in the lettuce
seed bioassay proved to be sufficiently sensitive
for evaluation of acute toxicity of soil extracts with
moderate level of contamination. On the other
hand, the terrestrial isopod assimilation assay
did not show any differences between both soils
evaluated. However, this bioassay can be tested
in other contamination conditions.
Acknowledgements. e authors thank Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior, CAPES, for
the Doctorate (F.M.R. S J) and Master’s (E. M.
G) scholarships.
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Received for publication June 4, 2012
Accepted after corrections November 21, 2012
Corresponding author:
F M R S J, Universidade Federal do Rio Grande do Sul – FURG, Instituto de Ciências
Biológicas, Laboratório de Ensaios Farmacológicos e Toxicológicos, Campus Carreiros, CEP 96203-900, Rio Grande, Brasil
e-mail: f.m.r.silvajunior@gmail.com
