J Soils Sediments (2016) 16:785–800

DOI 10.1007/s11368-015-1306-0

SOILS, SEC 1 • SOIL ORGANIC MATTER DYNAMICS AND NUTRIENT CYCLING • RESEARCH ARTICLE

Phytotoxicity of natural soils using physiological and biochemical

endpoints reveals confounding factors: can a weight of evidence
tackle uncertainty?
Sara C. Antunes 1,2 & Bruno B. Castro 2,3 & Maria Celeste Dias 4 & José Moutinho-Pereira 5 &
Carlos M. Correia 5 & Maria T. Claro 3 & Ana Gavina 1,6 & Conceição Santos 1,7 &
Fernando Gonçalves 2,3 & Glória Pinto 2,3

Received: 13 April 2015 / Accepted: 16 November 2015 / Published online: 21 November 2015
# Springer-Verlag Berlin Heidelberg 2015

Abstract Organisation for Economic Co-operation and Development

Purpose Standard assays for phytotoxicity provide a reduc-
tionist view on the performance of plants under toxic stress.
To address two of the most prominent issues in plant toxicity
studies, our aims were (1) to assess how well physiological
and biochemical parameters complement standard toxicolog-
ical endpoints when testing natural soils and (2) to assess the
suitability of three commonly used control soils as compara-
tive references.
Material and methods We compared the performance of Zea
mays and Helianthus annuus in three control soils (artificial
Responsible editor: Caixian Tang
* Sara C. Antunes
scantunes@fc.up.pt; gpinto@ua.pt
1
Departamento de Biologia da Faculdade de Ciências da Universidade
do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal
2
Centro de Estudos do Ambiente e do Mar (CESAM), Universidade
de Aveiro, Campus Universitário de Santiago,
3810-193 Aveiro, Portugal
3
Departamento de Biologia, Universidade de Aveiro, Campus
Universitário de Santiago, 3810-193 Aveiro, Portugal
4
Centro de Ecologia Funcional (CEF), Departamento de Ciências da
Vida, Faculdade de Ciências e Tecnologia da Universidade de
Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal
5
Centro de Investigação e de Tecnologias Agroambientais e
Biológicas (CITAB), Department Biologia e Ambiente,
Universidade de Trás-os-Montes e Alto Douro, 5000-801 Vila
Real, Portugal
6
Centro Interdisciplinar de Investigação Marinha e Ambiental
(CIIMAR), Rua dos Bragas n° 289, 4050-123 Porto, Portugal
7
GreenUP, CITAB-UP, Faculdade de Ciências da Universidade do
Porto, Rua do Campo Alegre, 4769-007 Porto, Portugal
(OECD) soil, standard LUFA 2.2 soil, and turf-perlite) against
three natural soils representing a gradient of contamination
(from a deactivated uranium mine). Standard endpoints (emer-
gence and biomass) were estimated, along with pigment con-
tent, photosynthetic parameters, cellular injury, and proline
content.
Results and discussion The toxicological profile of natural
soils was highly dependent on the control soil used as refer-
ence; also, plant physiological performance was influenced by
the soils’ properties. We discuss the need to interpret and
combine multiple lines of evidence as a way to increase the
degree of confidence one classifies soils based on their
ecotoxicity, and this is where the integration of physiological
and biochemical parameters bring added value.
Conclusions When facing large variability in soil characteris-
tics, it is best to collect and integrate as much information
possible to strengthen conclusions about phytotoxicity of nat-
ural soils. Obviously, this refutes reductionist views and
places the final conclusion in the hands of expert judgment.
Keywords Natural and standard soils . Physiological
performance . Plant bioassays . Standard protocols
1 Introduction
Human activities, such as industry, agriculture, and mining
exploitation, can harm the environment in numerous ways,
causing disturbances in ecosystems and consequent loss of
biodiversity and sustainability (Ernst and Peterson 1994).
The soil compartment is crucial for land-based life, and
(Abrahams 2002; O’Halloran 2006) it is highly vulnerable to
contaminant accumulation. Some authors suggest that
786
concentrations of contaminants in soil are higher than in any
other environmental component (O’Halloran 2006). For ex-
ample, the widespread accumulation of metals presents a risk
for primary and secondary consumers and, ultimately, humans
(Nedelkoska and Doran 2000). Although some metals are
essential or beneficial micronutrients for microorganisms,
plants, and animals, high concentrations have strong toxic
effects over the biota, and metals may become an environmen-
tal threat after a certain threshold (Nedelkoska and Doran
2000; Munzuroglu and Geckil 2002). This has been studied
thoroughly in deactivated mining areas, due to the deposition
of mine tailing and sludge (Wong 2003; Antunes et al. 2011;
Pereira et al. 2014).
In order to assess environmental impacts from metals and
other pollutants, ecological risk assessment (ERA) is an in-
creasingly important part of the decision-making process for
managing contaminated soils, and its application to mine ex-
ploitation zones has been largely documented (Weeks et al.
2004; Weeks and Comber 2005; Antunes et al. 2008a; Pereira
et al. 2008). Ecological risk assessment requires an integrated
appraisal of distinct lines of evidence and information flows,
as suggested, for example, in the triad approach (Rutgers et al.
2000). In this approach, an important role is played by bioas-
says, because chemical analyses alone are not adequate to
assess the potential ecological impact of contaminated soil
(Breure and Peijnenburg 2003; Banks and Schultz 2005).
Bioassays are experiments where organisms are exposed to
chemicals or environmental samples, and their response is
observed under standardized conditions (van Straalen 2002).
For example, plant toxicity assays (ISO 1995) are particularly
relevant when phytotoxic contaminants are present in soil,
such as metals, since plants are naturally exposed to these
stressors in their habitat (Munzuroglu and Geckil 2002).
Standard protocols for plant toxicity assays have been suc-
cessfully implemented, yet they still provide a somewhat reduc-
tionist view: protocols require the determination of emergence
and plant growth (biomass) in early stages of development
(OECD 1984; ISO 1995; OECD 2006). Emergence was found
to be less sensitive or even insensitive to metal toxicity (e.g.,
Cd) and is not considered by several authors (e.g., An et al.
2004; Wang and Zhou 2005) as a good endpoint to assess metal
toxicity in soils. Classical parameters such as these do not give
a complete idea of the physiological performance of plants
under toxic stress. Higher plants, being sessile organisms, have
developed tolerance against several stresses (Hirayma and
Shinozaky 2010), displaying an array of defenses to environ-
mental challenges (e.g., proline, an amino acid which plays a
highly beneficial role in plants exposed to various stress condi-
tions; Hayat et al. 2012). Various other physiological modifica-
tions and injuries have been documented with different degrees
of impact in the photosynthetic process, as well as in plant
growth and metabolism. This includes stomatal and non-
stomatal limitations to photosynthesis, photosynthetic
J Soils Sediments (2016) 16:785–800
pigments, sugar contents, and biochemical injury (Ali et al.
2004; Yu et al. 2007; Monteiro et al. 2009; Gill and Tuteja
2010). Gavina et al. (2013) found that photosynthetic and cell
injury parameters provided increased discriminatory power in
plant performance relatively to emergence and biomass esti-
mates. Unlike for terrestrial and aquatic animals, where bio-
markers have been used extensively in the field, more research
is necessary before plant biomarkers can be used in environ-
mental surveys and regulation (Ernst and Peterson 1994;
Lagadic et al. 1994; Brain and Cedergreen 2009). Such markers
are considered to be early warning signs, with biochemical
injury occurring at lower levels of contamination than individ-
ual endpoints (Peakall 1992; Sanchez-Hernandez 2011); see
Brain and Cedergreen (2009) rationale for aquatic plants.
Another important issue when assessing the phytotoxic po-
tential of soils is that noxious effects are inferred by compar-
ing plant performance of test or spiked soils relatively to a
reference or control soil(s). Therefore, the discriminatory
power is dependent on the choice of control or reference
soil—Gavina et al. (2013) used Organisation for Economic
Co-operation and Development (OECD) as the control soil
plus an additional local reference. In this sense, ecotoxicolog-
ical assays lack full standardization of a proper control soil,
although numerous attempts have been made to provide syn-
thetic and natural alternatives for a multipurpose and adequate
substrate for both plants and edaphic invertebrates (e.g.,
Gawlik et al. 2003; Hofman et al. 2009; Caetano et al.
2012). Also, other studies have shown that soil properties
add uncertainty to ecotoxicological assessments (Amorim
et al. 2005; OECD 2006; Römbke et al. 2006; Hofman et al.
2009; Chelinho et al. 2011), which is especially important in
plants (see differences in TBT toxicity between natural and
control soils in Römbke et al. 2006), as they physically depend
on the soil substrate. So, the biological interpretation of plant-
soil interactions (both physical and chemical) strongly de-
pends on experimental design procedures and this is, in part,
the approach explored in this manuscript.
Bearing in mind this background information, the aim of
this study was twofold: (1) to assess how well physiological
and biochemical parameters complement standard toxicolog-
ical endpoints when testing natural soils and (2) to assess the
suitability of three commonly used control soils as compara-
tive references in terms of plant performance. The first objec-
tive (use of physiological measures) is especially important to
provide additional information in ecotoxicological assess-
ments. The second objective relates to the use of proper
control/reference soils, in order to reduce uncertainties in eco-
toxicological assessments. This is especially relevant in stud-
ies with natural soils, as these provide an additional challenge
given the heterogeneity of the substrates’ properties. While
physiological endpoints may help teasing out contaminant-
induced changes from natural variation, they may also be
themselves subjected to confounding factors (Römbke et al.
J Soils Sediments (2016) 16:785–800
2006). Our experiment aims at clarifying this, by using natural
soils that have been thoroughly characterized in terms of their
chemical composition and ecotoxicity.
2 Material and methods
2.1 Natural and artificial soils
The natural soils used in this study were from a deactivated
uranium mine in the area of Cunha Baixa, Mangualde
(Portugal). This was considered one of the mining areas of
major concern due to the in situ acid leaching of pore ore
performed in the past, which still contributes for the produc-
tion of acid mine drainage. This mine was classified as deserv-
ing priority intervention, after environmental impact studies
revealed water and soil contamination due the dispersion and
accumulation of chemical elements originating from the min-
ing area (Antunes et al. 2007; Antunes et al. 2008a; Pereira
et al. 2008; Marques et al. 2009; Pereira et al. 2009). The small
borough of Cunha Baixa is particularly close to the mine, and
inhabitants use the local soil for subsistence farming and use
subterranean water for agriculture, domestic, and animal
drinking purposes (Lourenço et al. 2013). Three natural soils
from the deactivated mining area were selected as test soils.
Two of these soils were characterized as being potentially
toxic (TA and TE) based on previous chemical and ecotoxico-
logical screening (Antunes et al. 2008a, 2008b, 2011; Pereira
et al. 2008; 2009). According to these studies, an addi-
tional soil (TI) was selected displaying a lower level of
environmental risk.
Three standard soils were used as negative controls: artifi-
cial OECD substrate (10:20:70 mixture of sphagnum peat
(turf), kaolinite clay, and quartz sand; OECD 2004); standard
LUFA 2.2 (Agricultural Research Centre, Speyer, Germany),
a natural soil; and a mixture of turf-perlite (80:20). They will
be referred to as CO, CL, and CT throughout the manuscript.
Soils CO and CL are commonly used as reference or control
soils, as well as standard substrates for edaphic organism cul-
turing (Antunes et al. 2008a, 2008b). Soil CT is an example
usually used in plant physiology studies and plant perfor-
mance evaluation (e.g., Correia et al. 2014) by our re-
search team. A rapid soil characterization was conducted
for the six soils, by measuring soil pH, conductivity, and
organic matter (OM), according to standard protocols
(FAOUN 1984; SPAC 2000).
2.2 Biological material
The species used in this study were Zea mays L. (maize) and
Helianthus annuus L. (sunflower), both proposed as test spe-
cies by ISO (1995). These species belong to two different
classes (monocotyledon and dicotyledonous, respectively)
787
and may have different strategies to cope with soil contami-
nants. Moreover, standardized protocols usually select a min-
imum of two species, comprising at least one species from
each of the two groups mentioned (ISO 1995; OECD 2004).
Seeds were purchased in a local supplier and visually
inspected before using; damaged seeds were discarded.
2.3 Experimental procedure
Simple test chambers were used in the experiments, consisting
of polystyrene cups with 55 mm of diameter and 125 mm of
height, whose base was perforated to allow proper drainage.
Cups were filled with 75 g of each standard or natural soil
(<2 mm fraction). Germination and growth assays were per-
formed following standard procedures described in ISO
11269-2 guideline (ISO 1995). Fifteen replicates for each
plant species×soil combination were used. Soil moisture
was adjusted to 80 % water-holding capacity (ISO 2005) with
distilled water, prior to adding the seeds. Seeds of both species
(in separate) were individually added to each cup and gently
covered with ca. 1 cm of corresponding soil. To minimize
potential differences among soils in terms of plant growth
due to the test soils’ nutrient content, half-strength (0.8 g l−1)
Hoagland’s solution (Sigma-Aldrich, Germany) was added
every 2 days to provide adequate and equal nourishment. A
half-strength solution was used as a precaution, because this
nutritive mixture contains trace metals and chelating agents
that could alter soil contaminant bioavailability; we found this
to be an acceptable trade-off to minimize potentially large
differences in the mineral and organic profile of the test soils,
which could exacerbate differences among test soils in terms
of plant performance. Test chambers were checked daily for
seed germination and soil moisture and were maintained at
constant temperature (22±2 °C), photoperiod (16 hL:8 hD)
and light intensity (ca. 200 μmol m−2 s−1), in a climatic growth
chamber (Model Microclima 1000, ASL-Snijders,
The Netherlands). Assay duration depended on plant perfor-
mance in control soils (see 2.4 Seed emergence and plant
biomass section).
2.4 Seed emergence and plant biomass
Seed emergence (%) and time-to-emergence (days) were
quantified in each replicate test vessel; time-to-emergence
was calculated solely with data from the test vessels where
seeds emerged. Total plant biomass (g) was determined
14 days after 50 % of the seeds in any of the control soils
emerged (following ISO 1995), by harvesting plants (root,
stem, and leaves) and determining dry weight (DW) after
16 h at 70 °C. Plant biomass was determined in six individual
replicates, while six others were harvested for the quantifica-
tion of several physiological parameters (see below).
Whenever emergence was insufficient (<80 %), the number
788
of replicates for biomass determinations was sacrificed (i.e., n
<6). For H. annuus in soil TA, no plants were available for
biomass determination.
2.5 Pigment quantification (chlorophylls and carotenoids)
Pigments were extracted from leaf samples in cold acetone/
Tris 50 mM pH 7.8 buffer solution (80:20, v:v) and centri-
fuged at 28,000g during 5 min, according to Sims and
Gamon (2002). The absorbance at 470, 537, 647, and
663 nm was determined with a Thermo Fisher Scientific spec-
trophotometer (Genesys 10-uv S). The contents of chlorophyll
a (Chl a), chlorophyll b (Chl b), and carotenoids were calcu-
lated using the formulae of Sims and Gamon (2002).
2.6 Chlorophyll a fluorescence and gas exchange
parameters
Chlorophyll a fluorescence parameters were determined in
situ on the adaxial side in full expanded leaves, with a pulse-
amplitude-modulated fluorimeter (FMS 2, Hansatech
Instruments, Norfolk, England). Maximum quantum efficien-
cy of photosystem II (PSII) was calculated as F v /
Fm =(Fm−F0)/Fm by measuring the fluorescence signal from
a dark-adapted leaf when all reaction centers are open using a
low-intensity pulsed measuring light source (F0) and during a
pulse saturating light (0.7 s pulse of 15,000 μmol photons
m−2s−1 of white light) when all reaction centers are closed
(Fm). Leaves were dark adapted for 30 min using dark-
adapting leaf clips for these measurements. Following Fv/Fm
estimation, after a 20-s exposure to actinic light
(1500 μmol m−2 s−1), light-adapted steady-state fluorescence
yield (Fs) was averaged over 2.5 s, followed by exposure to
saturating light (15,000 μmol m−2s−1) for 0.7 s to establish F′
m. The sample was then shaded for 5 s with a far-red light
source to determine F′0. From these measurements, the effi-
ciency of electron transport as a measure of the quantum ef-
fective efficiency of PSII (ΦPSII =ΔF/F′m =(F′m−Fs)/F′m) was
calculated, according to van Kooten and Snel (1990).
In situ leaf gas exchange measurements (net CO2 assimila-
tion rate, A; transpiration rate, E; and the ratio between inter-
cellular and ambient CO2 concentration, Ci/Ca) were per-
formed using a portable infrared gas analyzer (LCpro+,
ADC, Hoddesdon, UK), operating in open mode under growth
chamber conditions. The stomatal conductance (gs) was auto-
matically calculated according to von Caemmerer and
Farquhar (1981). Measurements were always performed in
the youngest fully developed leaf, between 10:00 and 12:00
a.m. (6–8 h after the beginning of the light period), at growth
temperature (24± 2 °C) conditions and atmospheric CO2
concentration.
J Soils Sediments (2016) 16:785–800
2.7 Cellular injury: lipid peroxidation and electrolyte
leakage
Lipid peroxidation was estimated by determining the
malondialdehyde (MDA) contents in the leaves according to
Elkahoui et al. (2005). Leaf samples (0.5 g per replicate) were
homogenized in 5 ml of 0.1 % trichloroacetic acid (TCA). The
homogenate was centrifuged at 10,000g for 5 min at 4 °C. An
aliquot of 0.3 ml of supernatant was mixed with 1.2 ml of
0.5 % thiobarbituric acid (TBA) prepared in 20 % TCA, and
incubated at 95 °C for 30 min. After stopping the reaction in
an ice bath for 5 min, samples were centrifuged at 10,000g for
10 min at 25 °C. The supernatant absorbance at 532 nm was
then measured using a Thermo Fisher Scientific spectropho-
tometer (Genesys 10-uv S). After subtracting the non-specific
absorbance at 600 nm, MDA concentration was determined
using the extinction coefficient 0.155 μM−1 cm−1 (Elkahoui
et al. 2005), following the equation:
MDA equivalent ¼ ðA532 −A600 Þ=0:155
For electrolyte leakage assessment, leaf samples were
washed with deionized water to remove surface-adhered elec-
trolytes according to Lutts et al. (1996). One leaf from each
replicate was weighed, washed with Milli Q water, and then
placed in falcon tubes filled with Milli Q water. The
mass:volume ratio was the same in all tubes. The tubes were
shaken mechanically for 12 h in an orbital shaker (100 rpm) at
23 °C, after which the electrical conductivity of the bathing
solution (Lt) was determined. Samples were then autoclaved at
120 °C for 20 min, and a final conductivity reading (L0) was
obtained upon equilibration at room temperature. Electrolyte
leakage was defined as Lt/L0 and expressed as percent.
2.8 Proline content
Proline was measured as described by Bates et al. (1973).
Frozen leaf samples (0.1 g) were homogenized in 1.5 ml of
3 % sulphosalicylic acid and centrifuged (4000g). Then, 2 ml
of glacial acetic acid and 2 ml of ninhydrin acid were added to
100 μl of the extract and incubated for 1 h at 100 °C. After
cooling in ice, 1 ml of toluene was added. The chromophore
phase containing toluene was measured at 520 nm. The
amount of proline was determined from a standard curve
(R2 =0.99).
2.9 Statistical analysis
One-way analyses of variance (ANOVA) were used to deter-
mine significant differences between natural and standard
soils, for plant biomass and physiological and biochemical
endpoints. The normal procedure in ecotoxicological assess-
ments is to compare putatively contaminated soils with a
J Soils Sediments (2016) 16:785–800
standard control or reference soil (OECD 1984, 2004). To
assess whether different control soils provide different out-
comes using the latter approach, we conducted separate
ANOVAs for comparing the three natural soils with each
one of the standard soils (i.e., three ANOVAs per endpoint×
plant species=78 separate ANOVAs). These are clearly a se-
ries of tests of multiple related hypotheses, which suffer from
loss of control of the significance level and loss of power
(Benjamini and Hochberg 1995; Quinn and Keough 2002).
Because Bonferroni-type adjustments are very conservative
and inappropriate for such a large number of multiple com-
parisons (Benjamini and Hochberg 1995; Pike 2011), we
corrected for multiplicity by adjusting p values with the con-
servative one-stage procedure based on the control of false
discovery rate (Benjamini and Hochberg 1995), using the
spreadsheet provided by Pike (2011). In the cases where the
ANOVA was significant, we tested differences between the
control soil and natural soils with a Dunnett test (Quinn and
Keough 2002).
Non-parametric correlation analysis (Spearman rs, Quinn
and Keough 2002) was used to explore relationships between
mean values of physical and chemical soil characteristics and
mean values of measured endpoints in plants (a total of six
data points). The use of this nonparametric method allowed a
conservative approach, thus avoiding the caveats of the large
variability and non-normality of the data. All statistical anal-
yses used a 0.05 significance level. Statistical software
Minitab (v16) and SPSS (v17) were used.
3 Results
3.1 Soil characteristics and standard endpoints of plant
performance
The standard soils differed greatly, namely in their organic
matter and mineral content (indirectly measured as
conductivity, see Table 1). Soil CT was, by far, the richest
substrate for plants because of its high content in turf
(80 %). On the other hand, soil CL—the only non-artificial
control soil—had the lowest organic matter content among the
standard soils. The test soils from the mining area were very
heterogeneous, ranging from very low (TA) to moderate levels
(TI) of organic matter content. These natural soils were also
more acidic than the control soils (Table 1). High metal con-
centrations were found in all test soils, and the most important
are shown in Table 1.
Seeds from both species had variable performance in the
tested soils (Table 2). In control soils, H. annuus and Z. mays
plantlets emerged in average after 5–6 days or 3–4 days, re-
spectively. Soil CO promoted 100 % seed germination in both
species, while the lowest performance among control soils
was achieved in soil CL, particularly for H. annuus (only
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67 % emergence, which is below the recommended 80 %
emergence for control soils—see Section 2). In the test soils,
Z. mays seeds performed equally well—or even better (soil
TI)—when compared to controls (100 % emergence after 3–
4 days). This was not the case of H. annuus, which was more
sensitive than Z. mays (Table 2). Emergence of H. annuus was
suboptimal in all test soils, and it was particularly low in soil
TA. Also, its seeds took longer to germinate in this soil (up to
1–2 more days than in control soils); in soils TE and TI, time to
emergence was similar to control soils.
Plant growth was very different among the control soils,
with very high biomass accumulation of both species recorded
in soil T, relatively to soil CO and CL. Consequently, the ability
to discriminate differences between control and test soils was
highly dependent on the choice of control soil (Fig. 1 and
Table 3). When soil CT was used as the control benchmark,
plants from both species underperformed in all test soils.
When soil CO was used as the control benchmark, Z. mays
biomass was significantly lower in soil TA; unfortunately, no
comparison is available for H. annuus in soil TA, because of
insufficient number of individuals for assessing biomass.
When soil CL was used as the control benchmark, no signif-
icant differences in plant performance were observed.
3.2 Photosynthetic pigment contents
The response profile of the plants to the test and control soils
was similar within the same species, but completely different
between Z. mays and H. annuus (Fig. 2 and Table 3). In the
former species, plants grown on soil TA had higher levels of
chlorophylls and carotenoids, which were in line with the
level of pigments on control soils CO and CL. Plants grown
on soils TE and TI had lower levels of pigments, but these were
in line with the observed pigment content of control soil CT.
For H. annuus, plants on soil TI achieved the highest chloro-
phyll and carotenoid contents, which were significantly higher
than those of the plants grown on the control soils CT and CO.
Plants grown on soils TA and TE had a pigment content similar
to control soils CT and CO, but significantly lower than soil
CL. This lack of consistency between species and the highly
variable response of control soils suggest that other factors
may be involved, some of which may be species specific
and related with morphological, physiological, and biochem-
ical features of the two groups of plants included in this study
(H. annuus: C3 photosynthetic pathway and Z. mays: C4 pho-
tosynthetic pathway).
3.3 Chlorophyll a fluorescence and gas exchange
No significant differences (Table 3) were observed in the Fv/
Fm of plants grown in the test soils, independently of the
control soil used, for both species (data not shown). The other
photosynthetic related endpoints (quantum effective
790 J Soils Sediments (2016) 16:785–800

Table 1 Mean (± standard error) physical and chemical parameters of control (CT, CL, and CO) and test soils (TA, TE, and TI; from a deactivated mining
area)

pH (H2O) Conductivity (μS/cm) Organic matter (%) Pseudo-total metal (mg/Kg soil)

CT 6.42±0.04 1080±80.8 54.2±0.80

CL 5.97±0.13 333±32.4 2.88±0.12
CO 6.77±0.08 97.7±4.54 7.43±0.99
TAa 5.28±0.04 9.63±0.12 0.66±0.57 U=13.4; Al=545; Fe=617
TEa 5.84±0.11 61.4±2.19 6.32±0.15 U=524; Al=9007; Fe=4747
TIa 4.81±0.04 20.7±0.66 11.5±0.16 U=16.4; Al=3963; Fe=2650
a
From Pereira et al. (2008)

efficiency of PSII, ΦPSII, and gas exchange) were elevated in not with CL. Similarly to Z. mays, the lowest values of Ci/Ca
control soil CT, except for Ci/Ca ratio (see below). This was ratio were found in soil I, which were significantly lower to
particularly noticeable in Z. mays, where in most cases, the control soils CO and CT.
performance of plants in the test soils (TA, TE, TI) was consis-
tently lower than control soil CT (Table 3, Fig. 3). Still, of all 3.4 Lipid peroxidation, cell membrane damage,
the tested soils, performance was less suboptimal in soil CI, and proline content
namely in transpiration and net CO2 assimilation rate (espe-
cially the latter). Using CL as the benchmark, results also point For Z. mays, some consistent patterns were observed (Table 3,
to a lower performance in soils TA and TE, relatively to TI—no Fig. 4). All test soils had electrolyte leakage levels similar to
significant differences between CL and TI for transpiration control soils CO and CL, but lower than control soil CT. Soil TI
rate, net CO2 assimilation rate, and stomatal conductance pa- had significantly higher lipid peroxidation relatively to all
rameters. Plant photosynthetic performance in soil CO was controls; similarly, soil TE had significantly higher proline
comparable to that of test soils (almost no significant differ- content. This response profile for H. annuus was not as
ences were found between soil CO and soils TA, TE, and TI), straightforward, particularly because of the very high levels
with the exception of ΦPSII, where test soils had significantly of lipid peroxidation and proline in the control soil CL, which
higher values. The Ci/Ca ratio had an opposite response pat- were significantly higher to all of the test soils. Soils TA and
tern, being significantly higher in soils TA (compared to soil TE had significantly higher lipid peroxidation levels than con-
CL and CT) and TE (compared to CT). trol soils CT and CO, while soils TA and TI had significantly
Regarding H. annuus, its response pattern is less consistent higher proline content than control soil CT.
(Table 3, Fig. 3). Overall, there are significantly lower values
of ΦPSII and net CO2 assimilation rate in all test soils, com- 3.5 Associations between plant physiological performance
pared to any control (except for ΦPSII in soil CO). On the and soil features
contrary, no differences were found among soils for transpira-
tion rate. Soil TE had extremely high levels of stomatal con- Plant biomass (at the end of the experiments) was not corre-
ductance, which were in line with control soils CO and CT, but lated with any physiological or biochemical parameters in
Z. mays and H. annuus (Spearman correlation, p>0.05). On
the contrary, significant associations were found between
Table 2 Emergence and mean (±standard error) time-to-emergence of
H. annuus and Z. mays in control (CT, CL, and CO) and test soils (TA, TE,
emergence and net CO2 assimilation rate (rs =0.89, p=0.018)
and TI; from a deactivated mining area) and MDA content (rs =−0.83, p=0.042), for H. annuus, but
none for Z. mays.
Emergence (%) Time-to-emergence (days) Significant correlations were found between soil prop-
H.annuus Z.mays H. annuus Z. mays erties (pH organic matter content and soil conductivity)
and plant physiological status. In H. annuus, plant bio-
CT 86.7 100 5.2±0.4 3.0±0.0 mass was positively correlated with organic matter con-
CL 66.7 93.3 5.5±1.2 3.1±0.5 tent (rs = 0.90, p= 0.037). Also, electrolyte leakage and
CO 100.0 100 5.9±1.0 4.1±1.8 net CO2 assimilation rate were significantly correlated
TA 46.7 100 7.1±3.8 3.9±1.1 with soil conductivity (rs =−0.83, p=0.042; rs =0.89, p=
TE 60.0 100 5.0±0.5 3.3±0.9 0.019), while pigments were negatively associated with
TI 73.3 100 5.0±1.3 2.9±0.3 soil pH (rs =−0.83, p=0.042). In Z. mays, biomass was
also strongly associated with soil conductivity (rs =0.82,
J Soils Sediments (2016) 16:785–800
1.2
Z. mays
1.0
0.8
g.DW -1
0.6 * * *
0.4
*
0.2
0.0
TA TE TI
Soils
Fig. 1 Total biomass of Z. mays and H. annuus exposed to natural soils
from a deactivated mining area (TA,TE, TI). For comparative purposes,
reference lines (dashed) represent average biomass in standard soils (CT,
CO, CL). Bars represent mean ± SD. Asterisks stand for significant
p=0.046) and organic matter content (rs =0.82, p=0.046).
Net CO2 assimilation rate (rs =0.83, p=0.042), leaf Ci/Ca
(rs = −0.83, p = 0.042), and MDA (rs = −0.89, p = 0.019)
were correlated with soil conductivity.
4 Discussion
One of the purposes of this study was to assess how
physiological and biochemical parameters of plant
performance complement standard toxicological
endpoints when testing natural soils. Gavina et al.
(2013) found that photosynthetic and cell injury parame-
ters provided increased discriminatory power in plant
physiological performance relatively to emergence and
biomass estimates. However, this discriminatory power
is dependent on the control or reference soil used
(Gavina et al. 2013 used OECD as the control soil and
a local reference). In the present study, we showed that
the toxicological profile of natural soils from a
deactivated mining area was highly dependent on the
control/reference soil used (part of the second objective
of this study). Also, plant physiological performance in
control soils was very variable, and some confounding
factors have arisen. Here, differences in nutrient levels
among soils were minimized by adding a nutritive solu-
tion; still, other variables (e.g., pH, texture, organic mat-
ter) seem to play an important role, especially in a later
stage of development (post-plantlet). During this study,
we were challenged by the influence that soil properties
exert in plant physiological status and growth. In fact,
soil combines various properties that interact with each
other, directly or indirectly influencing the overall plant
status in a positive or negative manner. Dealing with
such uncertainty is a challenge in phytotoxicity studies,
and we address this below taking into account the two
791
Total Biomass H. annuus
CT
* *
CT
CO CO
CL
CL
TA TE TI
Soils
differences (Dunnett test) between natural soils and the corresponding
standard soil, when placed next to respective reference line; multiple
asterisks for the same natural soil mean that it is significantly different
than more than one standard soil
objectives initially proposed and our specific case study
(soils from deactivated mining area).
Plant performance varied between test species: H. annuus
was apparently more sensitive than Z. mays to soil characteris-
tics, displaying suboptimal emergence in all test soils (<75 %)
and also in control soil CL. This was also reflected in differ-
ences in the physiological and biochemical profiles of both
species. Overall, literature suggests that dicotyledonous (such
as H. annuus) are more sensitive to stress than monocotyledon-
ous (Gong et al. 1999; Shao et al. 2013), but this maybe a
premature generalization (Sunahara et al. 2000). Piršelová
et al. (2011) reported that soybean (dicotyledonous) was more
tolerant to metals and showed more pronounced defense re-
sponses than maize. Once again, the differences between stud-
ies are highly influenced by experimental procedures, the type
of metal, and the doses tested. Still, interspecific differences are
natural, hence the need for using more than one species.
4.1 Plant physiology meets ecotoxicology
As expected, plant performance was variable among soils
and this variation strongly depended on the measured
endpoint. If only standard endpoints are considered,
two soils stand out: soil CT, which promoted high yield
in terms of biomass of both species, and soil TA, which
led to slight underperformance of Z. mays (in terms of
biomass) and severe underperformance of H. annuus (in
terms of emergence). The superior performance of plants
in soil CT is a likely consequence of its distinctly higher
organic matter (OM) content. On the contrary, soil TA
had the lowest amount of OM. Together, these two facts
may partially explain the correlations observed between
plant biomass and OM (and also conductivity). However,
the lower performance of H. annuus (see emergence,
Table 2) in soil TA cannot be explained solely by its
low organic content, and results suggest this soil is
 792

Table 3 Summary of one-way ANOVA of biomass and physiological/biochemical endpoints measured in Z. mays and H. annuus exposed to natural (TA, TE, and TI) and standard (CT, CL, and CO) soils

TA, TE, TI vs. CT TA, TE, TI vs. CO TA, TE, TI vs. CL

F MS df adj-p F MS df adj-p F MS df adj-p

Zea mays Chl a+b 6.532 0.344 3, 11 <0.001 12.230 0.867 3, 12 0.001 10.212 0.706 3, 11 0.003
Carotenoids 5.107 0.034 3, 12 0.024 5.230 0.034 3, 13 0.021 3.302 0.032 3, 12 0.076
Fv/Fm 0.868 1.9E−4 3, 19 0.507 0.912 1.9E−4 3, 19 0.492 0.797 1.7E−4 3, 19 0.538
ΦPSII 22.76 0.008 3, 15 <0.001 4.859 1.7E−3 3, 15 0.022 3.771 1.2E−3 3, 15 0.047
E (transpiration rate) 10.40 0.169 3, 14 0.002 2.689 0.050 3, 14 0.112 11.72 0.151 3, 14 0.001
A (net CO2 assimilation rate) 14.36 19.12 3, 16 <0.001 10.75 5.455 3, 16 0.001 12.79 8.508 3, 16 <0.001
gs (stomatal conductance) 34.41 1008 3, 13 <0.001 3.437 166.5 3, 14 0.062 14.15 468.0 3, 14 <0.001
Ci/Ca 9.013 0.091 3, 15 0.002 3.781 0.027 3, 15 0.047 6.372 0.046 3, 15 0.010
Lipid peroxidation 26.23 30.55 3, 9 <0.001 24.23 23.37 3, 10 <0.001 26.68 32.38 3, 11 <0.001
Electrolyte leakage 17.68 62.47 3, 13 <0.001 0.099 0.319 3, 12 0.959 0.742 2.385 3, 12 0.569
Proline 25.84 0.010 3, 8 <0.001 17.09 0.009 3, 8 0.002 17.75 0.008 3, 8 0.002
Biomass 11.45 0.179 3, 19 <0.001 3.553 0.016 3, 19 0.047 1.802 0.008 3, 19 0.224
Helianthus annuus Chl a+b 5.065 0.204 3, 13 0.023 12.04 0.346 3, 14 0.001 6.547 0.220 3, 14 0.010
Carotenoids 5.266 0.034 3, 14 0.020 11.68 0.046 3, 14 0.001 6.231 0.029 3, 13 0.013
Fv/Fm 1.235 1.0E−4 3, 17 0.365 1.142 8.9E−5 3, 17 0.396 1.391 1.4E−4 3, 17 0.326
ΦPSII 32.33 0.026 3, 14 <0.001 9.393 5.3E−3 3, 14 0.002 29.49 0.017 3, 15 <0.001
E (transpiration rate) 1.598 0.434 3, 17 0.272 0.482 0.110 3, 16 0.718 0.225 0.063 3, 17 0.889
A (net CO2 assimilation rate) 36.38 27.81 3, 17 <0.001 10.28 8.618 3, 17 0.001 4.864 3.204 3, 17 0.020
gS (stomatal conductance) 161.2 5.5E4 3, 10 <0.001 207.7 6.3E4 3.12 <0.001 19.49 4.1E4 3, 12 <0.001
Ci/Ca 10.90 0.085 3, 12 0.002 15.66 0.103 3, 14 <0.001 6.717 0.078 3, 14 0.009
Lipid peroxidation 23.01 7.317 3, 16 <0.001 24.82 7.398 3, 16 <0.001 24.26 13.46 3, 14 <0.001
Electrolyte leakage 1.317 21.13 3, 14 0.348 2.567 14.82 3, 14 0.123 2.469 20.97 3, 14 0.132
Proline 5.515 0.006 3, 12 0.020 1.441 2.2E−3 3, 12 0.326 7.741 0.014 3, 10 0.010
Biomass 19.27 0.716 2, 8 0.002 1.394 0.031 2, 8 0.347 2.178 0.033 2, 7 0.224

For each endpoint, three ANOVAs were conducted, so that natural soils were separately compared to each standard control soil. For each ANOVA, we present F statistic (F), mean squares (MS), degrees of
freedom (df), and probability level—adjusted for multiple comparisons (adj-p), by controlling false discovery rate (Pike 2011)
J Soils Sediments (2016) 16:785–800
J Soils Sediments (2016) 16:785–800 793

Fig. 2 Pigment content (Chl a+b Z. mays Chlorophyll a+b H. annuus

2.0
and carotenoids) of Z. mays and CO
H. annuus exposed to natural ** ** CL CL
soils from a deactivated mining 1.5
* *

mol.gFW -1
area (TA,TE, TI). For comparative CT
purposes, reference lines (dashed) CT *
represent average pigment 1.0 * CO
content in standard soils (CT, CO,
CL). Bars represent mean±SD. 0.5
Asterisks stand for significant
differences (Dunnett test)
between natural soils and the 0.0
corresponding standard soil, Z. mays Carotenoids H. annuus
0.8
when placed next to respective
reference line; multiple asterisks
CL
for the same natural soil mean that 0.6
* *

mol.gFW -1
CO
it is significantly different than
more than one standard soil CL CT
0.4
* *
CT * CO

0.2

0.0
TA TE TI TA TE TI
Soils Soils

potentially hazardous. Concern about this soil had al-
ready been reported in several studies in terms of soil
metal concentrations (U, Al, Fe, Mn; Pereira et al.
2008), toxicological screening with animals (Antunes
et al. 2008a; 2008b), and community structure and func-
tion (Antunes et al. 2011, 2013).
In order to get a finer resolution in terms of plant performance
in the tested soils, additional measures complemented the stan-
dard endpoints. Plant physiological and biochemical parameters
such as photosynthetic pigments, gas exchange parameters,
chlorophyll fluorescence, cell injury, lipid peroxidation, and pro-
line are commonly be used to determine plant response to stress
conditions (Piršelová et al. 2011; Correia et al. 2014; Nunes
et al. 2014). This was the underlying reason for the choice of
the specific parameters used in this study, which were expected
to confirm the information provided by standard endpoints, thus
validating it, while allowing the detection of stress responses or
specific pathways that could be affected. Few correlations be-
tween physiological/biochemical parameters and standard end-
points were observed, suggesting that these responses are not
necessarily coupled with plant biomass or seed emergence.
Therefore, a more detailed analysis is in order.
Photosynthetic pigments content was highly variable
among soils and species. Plants grown in soil T I (for
H. annuus) and TA (for Z. mays) had the highest chlorophyll
and carotenoid content. The profile of both species was
completely opposite, both in terms of the test soils and the
relative position of the control soils. These species-specific
and contradictory results suggest that factors associated to soil
features (e.g., pH, soil nutrients) may play a role in the
observed differences in pigments content among soils, not
necessarily suggesting phytotoxicity. Indeed, other works
from our team have shown that pigments, for example, can
respond to natural stressors (Santos 2004; Correia et al. 2014).
Therefore, pigments do not seem to be a good marker for
assessing the toxicity of natural soils.
Photosynthetic related indicators were also variable, but
some patterns emerge. In general, plants performed better in
control soils than in test soils. Diminished net CO2 assimila-
tion rate and augmented Ci/Ca ratio were observed in soil TA
and, partially, TE. While the lower performance of plants in TA
confirms the information provided by standard endpoints (see
above), some uncertainty is associated to soil TE, as revealed
by the unexplainable profiles in terms of stomatal conductance
and Ci/Ca in H. annuus and by the low values in transpiration
and net CO2 assimilation rate in Z. mays. Previous chemical
and ecotoxicological studies did not observe evidences of po-
tential toxicity in this particular site (see Pereira et al. 2008;
Antunes et al. 2008a; 2011; 2013), nor did biomass and
emergence.
Cell injury parameters revealed large variations, show-
ing that natural variability can drastically affect these
parameters. In Z. mays, soil TI caused high levels of lipid
peroxidation, contrarily to the pattern found in
H. annuus. Proline levels were significantly higher in
soil TE for Z. mays but not for H. annuus. As for pig-
ment data, contrasting results do not aid in our ability to
detect putative phytotoxicity of test soils. Again, control
soils behaved sometimes very differently; plants from
soil CL showed high levels of lipid peroxidation and
794 J Soils Sediments (2016) 16:785–800

Fig. 3 Chlorophyll a Z. mays  PSII - Quantum yield of photosystemII H. annuus

fluorescence and gas exchange 0.5

parameters of Z. mays and CT

H. annuus exposed to natural 0.4 * * * CL
soils from a deactivated mining * * *
area (TA,TE, TI). For comparative 0.3 CO
CT *
purposes, reference lines (dashed) * * *
represent average of the 0.2
CL
corresponding parameter in *
0.1
standard soils (CT, CO, CL). Bars * * * CO
represent mean±SD. Asterisks
stand for significant differences 0.0
(Dunnett test) between natural Z. mays E - transpiration rate H. annuus
4
soils and the corresponding
CT
standard soil, when placed next to CO
respective reference line; multiple 3
CL

mmol.m-2s-1
asterisks for the same natural soil
mean that it is significantly
2
different than more than one
standard soil
CT
1
** ** CL
CO
0
Z. mays A - net CO2 assimilation rate H. annuus
12
* * * CT
10 CO
* * *
* * *
-2 -1

8
mol.m s

CL
CT
6 * * *
CL
4 * *
* CO
2

0
Z. mays gS - stomatal condutance H. annuus

300 CO
* *
* * *
mmol.m-2s-1

CT
200
CL
*
100
CT
** ** * CL

CO
0

Z. mays Ci/Ca - internal [CO2]/environment[CO2] H. annuus

1.0

CO
0.8 *
CO * CT
*
0.6 * CL
CL

* * CT
0.4

0.2

0.0
TA TE TI TA TE TI
Soils Soils
J Soils Sediments (2016) 16:785–800 795

proline in H. annuus, which suggests a stress response.
Unlike the other physiological parameters, cell injury da-
ta did not clearly discriminate soil TA as a potential most
noxious test soil, with the exception of H. annuus data
on lipid peroxidation.
4.2 Standard control soils as benchmarks of plant
performance
Regardless of the use of standard (biomass, emergence) or
physiological endpoints, much of the conclusions regarding
phytotoxicity depended greatly on the comparative reference
used, i.e., the control soil(s)—see Figs. 1, 2, 3, and 4. Because
of high variability among control soils, our results can be
interpreted either as augmented or diminished plant perfor-
mance, depending on the control soil used (Table 4). This
produces considerable uncertainty, whether one considers
standard or physiological responses. Large differences among
plant performance in control soils were probably attributable
to soil features. This was the case of soil CT, which has very
high organic matter content, and where plants consistently
overperformed in terms of biomass, quantum yield, and pho-
tosynthetic assimilation rate. This is not surprising, as organic
matter is rapidly mineralized in the soil matrix, transferring
nutrients to the plant and thus promoting growth (Seiter and
Horwat 2004). However, this soil also promoted high electro-
lyte leakage in Z. mays, which is an unexpected sign of dis-
tress. Of course, the overall excellent performance of plants in
CT increases the chances of overestimating toxicity, when it is
used as a comparative reference (i.e., control). This was prob-
ably the case for biomass, quantum yield, and net assimilation
rate (see Figs. 1 and 3).
On the opposite end, soil CL caused plant underperfor-
mance; indeed, this was the only control soil not to fulfill the
validation criterion (<80 % emergence for H. annuus). Plants
grown in this soil displayed lower biomass (for both species),
net assimilation rate, and stomatal conductance (for H. annuus
only) than the other controls, and they even underperformed
plants in some of the test soils (see e.g., biomass results,
Fig. 1). Moreover, soil CL caused elevated lipid peroxidation
and proline content, suggesting some degree of cell injury.
Being a natural soil, unlike CT and CO (artificial or formulated

Fig. 4 Cellular injury parameters Z. mays Electrolyte leakage H. annuus

(electrolyte leakage, MDA 30
concentration and proline
25 CO
content) in leaves of Z. mays and
H. annuus exposed to natural
mg.gFW -1

20 CT, CL
soils from a deactivated mining
area (TA, TE, TI). For comparative 15 CT
purposes, reference lines (dashed)
10
* * *
represent average of the CL
corresponding parameter in 5
standard soils (CT, CO, CL). Bars CO
represent mean±SD. Asterisks 0
stand for significant differences Z. mays Lipid Peroxidation H. annuus
16
M MDA equivalent.gFW -1

(Dunnett test) between natural

14
soils and the corresponding
standard soil, when placed next to 12
respective reference line; multiple 10
CO
asterisks for the same natural soil
mean that it is significantly
8 * CL CL

different than more than one 6

* * * *
* CT
standard soil 4
CT
2 * * CO
0 * *
Z. mays Proline H. annuus
0.30
CL
0.25
* * *
mol.gFW -1

0.20

0.15
CO
0.10 CL
* CT
* CT
0.05 * *
* CO
0.00
TA TE TI TA TE TI
Soils Soils
796 J Soils Sediments (2016) 16:785–800

Table 4 Summary table of significant effects observed in each parameter, using three different control soils as a comparative reference (see
Section 4.2)

Symbols and stand for a corresponding significant decrease or increase of the parameter relatively to each control soil. Underlined symbols ( or
) represent changes in parameters that are considered deleterious
ND no data
a
A deleterious effect was considered to occur when a decrease in gS was accompanied by an increase in Ci/Ca, suggesting underperformance in terms of
CO2 metabolism

soils), one cannot exclude the presence of natural contami-
nants, despite its widespread use in soil toxicity studies
(Hund-Rinke et al. 2003; Römbke et al. 2006; Antunes et al.
2008a; Frankenbach et al. 2014; Gomes et al. 2014;
Scheffczyk et al. 2014) and the claims of its adequateness as
a reference benchmark (Kalsch et al. 2006). Indeed, a few
metals have been quantified in samples of this soil, and there
is evidence they are bioavailable (Antunes et al. 2008b), al-
though the observed levels do not seem to deserve concern.
Irrespective of the reasons, evidence suggests soil CL is a poor
control, as CT seems to be Btoo good.^ Note that the use of a
single control or reference soil would have provided
completely opposite results: if only soil CT or CL had been
used, all test soils could be considered potentially hazardous
or environmentally safe, respectively, when considering stan-
dard endpoints.
Plant performance in soil CO was intermediate between
soils CT and CL. The only apparent negative finding in this
soil was the low quantum yield, which was significantly lower
than all test soils in Z. mays. Other than that, this soil
J Soils Sediments (2016) 16:785–800
performed better than or equally well as test soils, despite
having low OM content (see Table 1). When used as the ref-
erence soil, it discriminated plants grown in soil TA for having
lower emergence (in H. annuus), lower biomass, lower net
assimilation rate, and higher lipid peroxidation (in
H. annuus). These are consistent signs of plant
underperformance, and they are partially corroborated when
using soil CT as benchmark (see also Table 4). Evidence of
damage in soils TE and TI (higher proline content and lipid
peroxidation, respectively, in Z. mays) were also supported,
when using both soils as benchmark. Thus, consistent trends
emerge when natural soils are compared with both CO and CT.
Adding all together, results suggest that CL is not an ade-
quate control soil in this case, while CO and CT promoted
good plant performance, although the use of CT should be
restricted to studies wishing to determine optimal plant per-
formance (acknowledging it may overestimate phytotoxicity).
Although the use of natural reference soils is suggested by
various authors (Kalsch et al. 2006; Römbke et al. 2006;
Pereira et al. 2009; Chelinho et al. 2011; Frankenbach et al.
2014), this only makes sense if these are local natural refer-
ences, with the same background levels of contaminants and
geological features. The use of a local soil from another area
(such as CL), even if fully characterized physically (texture,
etc.) and chemically (metals, pesticides, etc.), should be
avoided. This idea is partially corroborated by OECD guide-
lines (2006), which advise against the use of natural soils. The
use of formulated control soils seems more adequate, although
necessarily more artificial. Because of that, a combination of
several control/reference artificial soils (as in this study) may
provide a more balanced approach; the use of a single all-
purpose control soil is unrealistic.
4.3 An open gate to phytotoxicity studies
A weight-of-evidence approach suggests that soils from the
mining area are potentially hazardous, particularly soil TA.
This is supported both by standard parameters and physiolog-
ical endpoints and after comparing this soil to the most suit-
able controls (CO and CT)—Table 4. Additionally, physiolog-
ical endpoints raised some uncertainty regarding soils TE and
TI, which deserves further research. The physiological condi-
tion of plants is associated with many abiotic variables, espe-
cially those that depend on soil physicochemical properties
(Morgan and Connolly 2013). The properties of soil affect
plant root vigor, length, density, and uptake of water and nu-
trients, among others. Therefore, the inclusion of physiologi-
cal parameters brings added value and potential discriminato-
ry power to phytotoxicity bioassays, as shown here. However,
this study also shows that species-specific responses to con-
founding factors may add some uncertainty in the assessment
of noxious soil samples (see pigment profiles, Fig. 2). To
properly tackle the potential influence of confounding factors
797
on the plants’ physiological profile, a multiplicity of combi-
nations between factors would be required (e.g., pH correc-
tion, nutrient addition, particle-size manipulation); this is vir-
tually impossible in terms of experimental design, taking into
account the necessary multifactorial framework, especially
when studying natural soils, as in the case of this study.
Therefore, in our opinion, there is a need to interpret and
combine multiple lines of evidence (not restricted to standard
ecotoxicological parameters) as a way to increase the degree
of confidence with which one classifies soils based on their
ecotoxicity. With this approach, there is more information
available to distinguish noise (sporadic fluctuations) and con-
sistent trends (among species and measured parameters), with
the overall physiological profile confirming or rejecting the
conclusions derived from the standard endpoints (see the
integration of information in Table 4). This brings added value
for reducing uncertainty in the assessment of phytotoxicity of
contaminated soils.
Taking into account the correlations observed between
plant performance and soil organic matter and conductivity,
it is desirable that the reference soils allow proper control of
these variables. Indeed, there were major differences in terms
of organic matter and conductivity between soils CT and CO,
partly explaining the differences in plant performance of the
plants grown in two soils (see above). Given the natural var-
iability (see Table 1) and importance (see biomass data, Fig. 1)
of organic matter, a suitable solution would be the use of
control soils differing in the amount of organic matter, thus
mimicking the natural variability of the natural soils under
test. For soils CO and CT, this would be as easy as adjusting
the amount of turf/peat added to the formulation (see example
in Spurgeon and Hopkin 1996). The same would be valid for
texture (particle size) and other simple variables, provid-
ed that the number of potential control soils does not
turn the experimental design unfeasible. There is no
doubt that a proper benchmark is pivotal for reaching a
conclusion about the phytotoxic potential of natural soils.
Again, using several control/reference soils and combin-
ing multiple lines of evidence seems to be the way to go
(Antunes et al. 2011), as it is likely that, with so many
confounding factors, we will never completely erase the
existing uncertainty degree in phytotoxicity assessments
of natural, contaminated soils.
5 Conclusions
Our results clearly demonstrate the need for a weight-of-
evidence approach, where all the different types of data,
comprehending different species, are integrated to generate a
weighted conclusion. In our case, data suggests some degree
of concern of the tested soils, albeit some noise and, even
more important, a strong dependence on the choice of control
798
soil. This study case illustrates the need to overcome con-
founding factors and control variability, using a flexible ap-
proach that places the final conclusion in the hands of expert
judgment. In our opinion, no single species is better than the
next one, no control soil performs better than the other, nor a
single endpoint or group of endpoints provides better resolu-
tion than the others. On the contrary, available data should be
integrated and analyzed in a way resembling a systems ap-
proach (Keurentjes et al. 2011).
Acknowledgments This work was supported by European Funds
through COMPETE and by National Funds through the Portuguese Sci-
ence Foundation (FCT) within project PEst-C/MAR/LA0017/2013 and
UID/AMB/50017/2013. Maria Celeste Dias and Glória Pinto were sup-
ported by FCT by means of a post-doctoral grant (SFRH/BPD/100865/
2014 and SFRH/BPD/101669/2014, respectively).
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