Ecotoxicology and Environmental Safety 246 (2022) 114179

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Ecotoxicology and Environmental Safety

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

Environmental conditions and plant physiology modulate Cu phytotoxicity

in field-contaminated soils
Pedro Mondaca a, Patricio Valenzuela b, Waldo Quiroz c, Monika Valdenegro a,
Sebastián Abades d, Juan L. Celis-Diez a, e, *
a
Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Chile
b
Departamento de Sistemas y Recursos Naturales, Universidad Politécnica de Madrid, Madrid, Spain
c
Instituto de Química, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
d
GEMA Center for Genomics, Ecology & Environment, Facultad de Ciencias, Ingeniería y Tecnología, Universidad Mayor, Santiago, Chile
e
Instituto de Ecología y Biodiversidad, Chile

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

Keywords: Foliar Cu concentration has been widely used as a biomarker of plant growth in phytotoxicity bioassays. This
Copper relation has helped find plant processes altered by Cu in dose-response experiments (a bivariate approach).
Contamination However, when plants are grown in field conditions, their responses can vary in function of multiple variables,
Ionomics
such as the environment, plant physiology, and other elements in plant (plant ionome). These sources of vari­
Structural equation modeling
ability are commonly unreported, which could limit bioassays’ utility. Thus, the present study aimed to assess
and integrate the mentioned sources of variability on Cu phytotoxicity. Lettuce was used as plant model. Lettuces
were grown in growth chambers with contrasting light and air humidity conditions and on two different field-
contaminated soils (sandy and loamy soils). Results showed that environmental conditions significantly
affected foliar Cu and plant growth, but this effect differed in the two studied soils. Foliar Cu was not a good
biomarker of plant growth. In contrast, integrating the potential phytotoxicity effect with the plant’s nutritional
status allowed a better understanding of plant growth. We remarked on using a structural equation modeling
approach (SEM) to integrate plant physiology and plant ionome as moderators of plant growth. Results showed
that plant growth was primarily related to plant nutritional status rather than Cu phytotoxicity. Also, the foliar
Cu concentration would affect plant nutritional status due to photosynthesis-related plant processes and cation
balance. Finally, this research invites to state and include sources of variability when assessing phytotoxicity.
This way, it is possible to advance toward understanding complex linked processes occurring in field conditions.

1. Introduction
Copper (Cu) is an essential micronutrient in plants, but high Cu
concentrations can affect their growth (Yruela, 2005). Traditionally,
dose-response models have allowed understanding and assessing the
potential toxicity of Cu in a particular media (solution, substrate, or soil)
on plants. These models have worked with increasing doses of Cu,
leaving other conditions in ceteris paribus. However, several conditions
could moderate the relation between Cu exposure and the plant
response, such as environmental conditions, plant physiology, and other
nutrients and metals. Thus, a multivariable approach could improve the
current knowledge of Cu phytotoxicity.
Cu phytotoxicity has been associated with increased leaf Cu
concentration (i.e., foliar Cu). Thereby foliar Cu has been used as a
biomarker of Cu phytotoxicity in bioassays (e.g., Cook et al., 1997). An
advantage of foliar Cu as a biomarker is the specificity for a plant species
and the theoretical independence of soil properties and origin. This
biomarker is based on Cu association with several plant functions, pri­
marily related to photosynthesis and enzymatic processes (Broadley
et al., 2012; Pilon et al., 2006). However, these physiological processes
are also influenced by environmental conditions such as light intensity
(Poorter et al., 2019) and air relative humidity (Xu et al., 2020).
Therefore, if the environmental conditions alter plant processes, the
relation between leaf Cu concentration and plant growth may change
too. Light intensity and air relative humidity also have a direct effect on
Cu assimilation and Cu uptake, respectively (Jiao et al., 2022; Yruela,

* Corresponding author at: Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Chile.
E-mail address: juan.celis@pucv.cl (J.L. Celis-Diez).

https://doi.org/10.1016/j.ecoenv.2022.114179
Received 19 February 2022; Received in revised form 30 September 2022; Accepted 9 October 2022
Available online 13 October 2022
0147-6513/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
P. Mondaca et al.
2005). Moreover, these climate variables gain particular interest in
future climate change scenarios since they could lead to different eco­
toxicological responses in plants to what is currently known.
Although environmental conditions are crucial for bioassays in
controlled conditions (Poorter et al., 2012), this information is scarcely
reported (e.g., Hamels et al., 2014; Mondaca et al., 2017; Wang et al.,
2017). Even in standardized methods such as the ISO (2005) for chronic
toxicity of higher plants, the relative humidity is not established, while
light intensity is considered as lux despite photosynthetic photon flux
density (PPFD) being more appropriate (Poorter et al., 2012). Therefore,
when phytotoxicity thresholds are reported, caution should be taken
about whether the thresholds represent general environmental condi­
tions or are only valid for a specific experiment. Especially when envi­
ronmental conditions are not reported, results might not be
reproducible, and they could not be contrasted with other experimental
conditions.
Another concern is the assumed dose-response relationship in bio­
assays which challenges their utility. Such assumptions deal with leaf Cu
as an independent variable while other variables, like other leaf chem­
ical elements, are in ceteris paribus. This approach has been successful
for plant bioassays on hydroponic or substrates culture conditions.
However, the trace element of concern is not the only chemical element
varying in plant bioassays on field-contaminated soils (McBride et al.,
2009; Mondaca et al., 2017), which could affect the validity of the
dose-response approach. For example, using a dose-response model,
Verdejo et al. (2015) aimed to find effective concentrations (ECx) for
lettuces grown in field-contaminated soils. They found a decrease of
10% and 25% of shoot length (EC10 and EC25) with 11 and 16 mg kg− 1 of
shoot Cu. However, they obtained wide and overlapping 95% confi­
dence intervals (7–16, and 13–20 mg kg− 1 of shoot Cu, for EC10 and
EC25, respectively). Such variability in the results was explained by the
significant effect of nutrients on the growth of lettuces. Nevertheless,
they reported a second biomarker (available soil phosphorus) instead of
integrating it with leaf Cu.
A dose-response approach to assessing Cu phytotoxicity bioassays on
field-contaminated soils also denies the principle that a whole’s com­
ponents are inherently related (Parent et al., 2013a). For example, it is
known that Cu involves a complex network of metal trafficking path­
ways that regulate its homeostasis (Yruela, 2005). Also, several enzy­
matic processes are in function of Cu and other elements, especially
those bivalent cationic elements like Zn2+, Ca2+, and Mg2+ (Broadley
et al., 2012). Therefore, other nutrients can interact with Cu alleviating
or increasing its toxicity (Kader et al., 2018; Le et al., 2013).
A novel approach to studying the complex response of plants is
ionomics, which is the study of the mineral nutrient and trace element
composition of an organism (Salt et al., 2008). Due to the fundamentals
of ionomics, multivariate data analysis are required. In this sense,
principal component analysis (hereafter PCA) has allowed to find re­
lations among nutrients and trace elements (Campos et al., 2021; Huang
and Salt, 2016). Similarly, PCA has been helpful to define nutrient
balances instead analyze elements separately (von Wettberg et al.,
2018). While PCA is a first-generation multivariate method, this analysis
relies on correlative information that can limit the study’s findings. In
contrast, structural equation modeling (hereafter SEM) approaches
(so-called second-generation multivariate methods) bring researchers
closer to causal understanding (Eisenhauer et al., 2015). Thus, multi­
variate methods would better to understand the assumed Cu phytotox­
icity processes.
To go deeper on Cu phytotoxicity using field-contaminated soils, in
this study we proposed the hypothesized that both leaf Cu concentration
(biomarker) and the plant dry weight (DW) (endpoint) would be altered
by the environmental conditions. Thus, the first objective of this study
was to measure the dry weight of lettuces as model plant and the Cu
concentration in lettuce leaves under two relative humidity levels and
three light intensity levels. Then we questioned if foliar Cu is enough as a
biomarker of plant growth or if considering other plant responses is
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Ecotoxicology and Environmental Safety 246 (2022) 114179
necessary? Thus, the second objective was to assess the relationship
between DW and foliar Cu by bivariate statistics and that relation in­
tegrated with the leaf ionome and plant physiological status by first- and
second-generation multivariate data analyses. Finally, we added a final
consideration by comparing the results obtained in the current experi­
ment and a similar research experience but performed in field
conditions.
2. Material and methods
2.1. Soil recollection and characterization
We studied two soils collected from two contrasting areas in Medi­
terranean central Chile near Valparaíso city (Fig. S1). The first area is the
Puchuncaví Valley, where the Ventanas smelter is located and is char­
acterized by a coastal area with predominantly sandy soils. The second
area is in Catemu, where the Chagres smelter is located. It is near the
Aconcagua River and has predominantly loamy soils. Both study areas
were selected based on previous knowledge (Aguilar et al., 2018;
Alekseev et al., 2018; Lizardi et al., 2020; Mondaca et al., 2022). We
collected 40 kg of agricultural topsoil from each study area. Considering
the importance of soil texture, as clay has a higher sorption capacity
than coarser particles (Adriano, 2001b), we labeled the soils surround­
ing the Ventanas smelter as sandy soil and those surrounding the Cha­
gres smelter as loamy soils.
Collected soils samples were dried, sieved (4 mm) and mixed for 10
min. Then, three soil subsamples of each soil were sent to the laboratory
for physicochemical analyses. Soil texture was analyzed by the hy­
drometer method (Sheldrick and Wang, 1993). Soil pH was measured in
a 1:2.5 w:v solution with deionized water. Electrical conductivity was
measured in a saturated paste extract, and soil organic matter content
was determined by the potassium dichromate method (Sparks et al.,
1996).
Nutrients were determined according to routine methods (Sadzawka
et al., 2015). Available N (the sum of N–NO3– and N–NH+ 4 ) was
extracted by 2 mol L− 1 KCl and determined by titration following NH3
distillation. Available P was determined by the Olsen method (extraction
with 0.5 mol L− 1 NaHCO3 at pH 8.5) and measured colorimetrically with
the molybdenum blue method. Extraction with 1 mol L− 1 CH3COONH4
at pH 7.0 was used to determine available K by atomic emission spec­
trophotometry and available Ca and Mg by atomic absorption spec­
troscopy (AAS) after adding lanthanum.
To determine total Cu, Zn, and As, the samples were digested in
boiling nitric acid followed by perchloric acid addition (Maxwell, 1968).
While their soluble concentrations were determined in a 0.1 M KNO3
extract (Stuckey et al., 2008). Total and soluble concentrations of Cu,
Zn, and As were determined by AAS.
Quality was assured by analyzing duplicate ISE-973 and ISE-859
certified reference samples obtained from Wageningen University,
Netherlands. The obtained results for the standard reference materials
were within 10% of the certified values. Quality control was assured by
introducing standards and blanks.
2.2. Design of the experiment
The field-contaminated soils were used to perform a phytotoxicity
trail under different environmental conditions. Two environmental pa­
rameters were tested: Air humidity and light intensity. Air humidity was
tested at two levels of relative humidity (RH), low humidity (LH) and
high humidity (HH) with about 30% and 65% of RH, respectively. Light
intensity was tested at three light intensity levels defined by the PPFD.
The three levels were low light (LL), medium light (ML), and strong light
(SL), which represent ranges of 200–300, 400–500, and 600–700 PPFD,
respectively. The experimental design was a 3 × 2 factorial (with
interaction) design resulting in 6 treatments: LH-LL, LH-ML, LH-SL, HH-
LL, HH-ML, and HH-SL. For each treatment, we prepared mini plant
P. Mondaca et al.
growth chambers (hereafter MPGC) of 3 m2. Each MPGC was set at 25 ◦ C
and a 16/8 h light/dark photoperiod. Moreover, we used fans to ho­
mogenize the environmental conditions inside of each MPGC.
2.3. Bioassay of plant development
Lettuces (Lactuca sativa L.) var “Victoriosa” were grown at each
MPGC. We considered five potted lettuces (replicates) from each studied
soil; thus, we grew 10 potted lettuces per MPGC and 60 in total. Pots (1
L) used in this experiment were filled with either 800 g of the loamy soil
or 700 g of the sandy soil, being volumetrically similar. Soils were
moistened to 60% of field capacity, and aggregates were formed with a
knife at next day.
Initially, 4 seeds of lettuces were sown in each pot. After one week of
seed emergence, we left just one lettuce per pot. The selected plants were
daily watered to maintain 60% of field capacity. After every watering,
plants were randomly placed in the corresponding MPGC. One week
before lettuce harvesting, plant physiological measurements were made.
The leaf senescence was measured with the CL-01 instrument (Han­
santech Instrument Ltd, UK). This instrument indicates photosynthetic
activity related to leaf nitrogen concentrations, determining indirect
chlorophyll content (Chl) from dual 620 nm and 940 nm wavelength
optical absorbance levels ranging from 0 to 2000 Chl units. On the other
hand, the stomatal conductance (gs) level was evaluated with the SC-1
Leaf Porometer (METER Group, Inc. USA). Leaf porometer measured
leaf transpiration, where low gs values are water stress indicators.
Finally, the selected lettuces were harvested once they reached harvest
maturity (~ 50 days after sown).
2.4. Lettuce samples preparation and laboratory analyses
Once the lettuces were harvested, they were carefully washed in the
following sequence: tap water, 0.1 M HCl, distilled water, 0.05 M EDTA,
distilled water, and distilled water again (Steubing, 1982). HCl and
EDTA were used to assure soil particles remotion from lettuces leaves.
Then, the samples were put into paper bags and dried in an oven at 70 ◦ C
for 72 h. Later, the samples were weighed, ground, sieved through
18-mesh (aperture size 1 mm), and homogenized.
We measured the concentration of N, P, K, Ca, Mg, Cu, Zn, and As in
lettuces. For N determination, samples (0.5 g) were digested with sul­
furic acid and salicylic acid (Temminghoff and Houba, 2004), distilled,
and titrated (Kalra, 1998). For P, K, Ca, Mg, Cu, and Zn, samples (1 g)
were calcinated at 500 ◦ C, dissolved with HCl 2 M and filtered (Kalra,
1998). K and P concentrations were measured in the filtrates using flame
photometry and nitro-vanado-molybdate colorimetry, respectively.
Concentrations of Ca, Mg, Cu, and Zn in the filtrates were determined by
atomic absorption spectroscopy (Flame–AAS). For As, samples (0.5 g)
were digested in boiling nitric acid, followed by perchloric acid
(Maxwell, 1968). A Teflon stopper with a 30-cm-long glass reflux tube
was used (adapted from Verlinden, 1982) to prevent As volatilization
during the digestion process. Then, As was determined by hydride
generation–atomic fluorescence spectrometry (HG–AFS) under experi­
mental conditions detailed in Vargas et al. (2015).
Quality was assured by analyzing in duplicate certified reference
IPE-907 (Spinach) and IPE-951 (Aubergine (leaf + fruit)) samples ob­
tained from Wageningen University, Netherlands. The obtained results
for the standard reference materials were within 10% of the certified
values. Quality control was assured by introducing standards and blanks
every 20 samples.
2.5. Data analyses
Data analyses were performed using the statistical software R v4.1.2
(R Core Team, 2021) on RStudio (v2022.02.3; RStudio Team, 2022).
Soil physicochemical characteristics of the sandy and loamy soils were
compared using an analysis of variance (ANOVA). The effect of light
3
Ecotoxicology and Environmental Safety 246 (2022) 114179
intensity and relative humidity on plant responses were analyzed with
two-way ANOVA of generalized linear models (GLMs) for gamma or
log-normal distributions (α = 0.05). Scatterplot and Pearson correlations
were performed as bivariate data analyses.
As multivariate analysis, PCA was performed by using the eight
elemental concentrations through the “FactoMineR” and “factoextra”
packages, and the princomp function from the R software (R Core Team,
2021) with all of the default settings (Baxter et al., 2008). Then, element
balance was built from the results of PCA. Specifically, elements with
loading > 0.5 were considered in the numerator when positive or in the
denominator when negative. Also, all elements were standardized with
the corresponding atomic weights. Elements considered in the leaf
ionome and the created variables by PCA were used to explain the dry
weight of lettuces using stepwise multiple regression analyses, verifying
a normal distribution and homogeneity of residuals (Kutner et al.,
2004). Multiple regression analyses were performed with Minitab 17
(Minitab, 2010).
2.6. Structural equation modeling (SEM) approach for ecotoxicological
analysis
Partial least squares path modeling (PLS-PM) was used to integrate
other variables more than foliar Cu to explain plant growth. As an SEM
approach, PLS-PM allows testing models to represent causal hypotheses,
including mediators and indirect effects (Grace et al., 2015). PLS-PM
estimates path coefficients of latent variables with their measured in­
dicators (inner models) and computes the loadings among the latent
variables (outer model) by an iterative process. Thus, PLS-PM provides a
practical summary of how the predictors systematically explain the set
of dependent variables (Sanchez, 2013).
We aimed to relate the multiple relations of plant physiological
status, nutrients, stress, and dry weight as a final response. In the hy­
pothetical model, we assessed the plant ionome as a biomarker of plant
growth. The plant ionome was sectioned in (1) the plant stressors: Cu
and As, and in (2) the remaining nutrients altered by the stressors. In the
nutrients group, Zn was excluded since it was highly correlated to Cu but
without the potential of causing phytotoxicity (according to the findings
in Section 3.4). In addition, the plant ionome was modeled in function of
the plant physiological status (according to the findings in Section 3.1).
Unidimensionality, determination coefficients of latent variables, and
redundancy were assessed to check the quality of the model. Further­
more, the model was assessed using the Goodness of Fit (GoF) statistic, a
measure of the overall prediction performance (Henseler and Sarstedt,
2012; Sanchez, 2013).
3. Results and discussion
3.1. Studied soils
Table 1 shows the main difference between the two studied soils.
Sandy soil was slightly acidic, while loamy soil was slightly basic. Loamy
soil showed higher Cu and As content than the sandy soil, yet both soils
have Cu concentrations with potential phytotoxicity (Mondaca et al.,
2017). Despite the lower total concentrations of Cu and As in the sandy
soil, it showed higher Cu and As solubility. This result is explained by the
lower content of clay and lower pH (Abbasi et al., 2021; McBride et al.,
1997), but higher soil organic matter content (SOM) in the sandy soil
than in the loamy soil. Despite some literature showing that SOM de­
creases Cu solubility, we have seen the contrary behavior in circum­
neutral soils as in this study (details in Mondaca et al., 2015).
As shown in Table 1, the studied soils differ in soil texture, nutrient
content, and trace element concentration. These differences would
condition plant responses. Thus, results of lettuces grown on the two
studied soils should be understood as contrasting cases.
P. Mondaca et al. Ecotoxicology and Environmental Safety 246 (2022) 114179

Table 1 concentration in lettuces under LH conditions.

Soil physicochemical properties of both studied soils (n = 3). Results of the stomatal conductance showed a similar pattern (Fig
Soil parameter Unit Average ± SD S2a,b) to the results of dry weight. We inferred that our experimental
conditions led to water stress on lettuces, but HH-ML would reduce such
Loamy soil Sandy soil
stress. Indeed, HH-ML reached stomatal conductance over
Clay % 20.33 ± 0.94 a 8.67 ± 0.47 b 100 mmol m− 2 s− 1, which was reported as the optimal status by Kim
pH – 8.12 ± 0.06 a 6.49 ± 0.08 b
Electrical conductivity dS m− 1
1.73 ± 0.12 a 1.24 ± 0.03 b
et al. (2004). We also found that the higher the light intensity, the lower
Soil organic matter % 3.76 ± 0.08 a 4.39 ± 0.01 a chlorophyll concentration in lettuces grown on the sandy soils. How­
Macronutrients ever, this relation is not clear on lettuces grown on the loamy soils.
Available N mg kg− 1 10.08 ± 0.98 b 54.53 ± 1.24 a Chlorophyll content showed a similar pattern to that found for leaf total
Available P mg kg− 1 40.37 ± 0.50 b 206.61 ± 5.31 a
1 Cu under the experimental condition of the study. This result would be
Exchangeable K cmol+ kg− 0.32 ± 0.01 b 4.01 ± 0.03 a
Exchangeable Ca cmol+ kg− 1
37.88 ± 0.15 a 18.58 ± 0.71 b explained by the importance of Cu on plastocyanin formation (Pilon
Exchangeable Mg cmol+ kg− 1
6.23 ± 0.10 b 7.28 ± 0.33 a et al., 2006).
Trace elements Finally, our results found that environmental conditions: light in­
1
Total Cu mg kg− 609.58 ± 9.11 a 244.64 ± 3.36 b tensity and air relative humidity, can alter leaf Cu concentration, DW,
1
Total Zn mg kg− 122.40 ± 9.73 b 168.12 ± 2.8 a
Total As mg kg− 1
29.55 ± 1.03 a 20.70 ± 0.60 b
and plant physiological responses. Thus, studies must report the envi­
Soluble trace elements ronmental conditions through which the experiments have been
Soluble Cu mg L− 1
0.36 ± 0.008 b 1.1 ± 0.030 a performed.
1
Soluble Zn mg L− UDL* 0.18 ± 0.0034
1
Soluble As mg L− 0.0063 ± 0.001 b 0.025 ± 0.0034 a
3.3. Analisys of the plant growth and the leaf ionome by bivariate statics
Different letters in the same row indicate significant differences between the two
studied soils (p ≤ 0.05). *UDL= Under detection limit (0.028 mg L-1 for Zn).
We found a poor correlation between plant growth and foliar Cu
(Fig. 1). Hence, foliar Cu concentration was not a good biomarker of
3.2. Effect of light intensity and air relative humidity on lettuce dry weight plant phytotoxicity (Fig. 2). This was not surprising as we found that the
and foliar Cu environmental condition altered both, dry weight and foliar Cu (Fig. 1).
This finding reaffirms the importance of reporting environmental con­
Light intensity and air relative humidity altered dry weight (DW) and ditions since the foliar Cu concentration as a biomarker of plant growth
foliar Cu, as shown by two-way ANOVA results (Fig. 1; Table S1). DW of would have limited robustness.
lettuces reached a maximum average value at ML treatments, especially Besides leaf Cu concentration, correlations between the concentra­
under HH conditions (Fig. 1a, b). On the other hand, light and air tion of other elements in leaves with DW showed other potential bio­
relative humidity were also significant factors altering the leaf total Cu markers of plant growth (Fig. S3). DW of lettuces grown on sandy soils
concentration. Specially, Fig. 1c, d shows a much higher Cu were correlated with: Plant N, Ca, Cu, Zn (negative relations), and As

Fig. 1. Boxplot of plant responses and results of two-way ANOVA for light and humidity factors. Notes inside each box are the significant factors for the corre­
sponding models (P-value < 0.05): L x H = light and humidity interaction; L + H = light and humidity but not the interaction; and H = only humidity is significant
(details in Table S1).

4
P. Mondaca et al.
Fig. 2. Scatterplot between plant dry weight and the plant Cu concentration grouped by the studied site. P-values and R2 are for loamy soils and sandy soils are
placed in the upper and lower part of the box, respectively. Density plots are also shown for each variable.
(positive). While for lettuces grown on loamy soils, only plant P was
significant (positive). This latter relation is attributed to a potential
deficit of P due to the high nutrient-demanding of agricultural plants like
lettuces, as found in the previous research of Verdejo et al. (2015). In
addition, this result could be explained by the higher As availability in
the loamy soil (Table 1). Specifically, arsenate can compete with phos­
phate for the plant uptake sites (Moreno-Jiménez et al., 2012).
We expected a positive relation of DW with the plant Zn concen­
tration, as Zn competes with Cu for the same adsorption sites (Tiecher
et al., 2016). Even, It has been proposed to add Zn to moderate Cu in
plants (Upadhyay and Panda, 2010). However, Zn was negatively
correlated with DW. On the other hand, foliar concentration of Zn was
strongly correlated with Cu (r = 0.93; p < 0.01). These results could be
explained since Cu and Zn are required in the chloroplast as cofactors of
superoxide dismutase (SODs) (Palmer and Guerinot, 2009) involved in
the detoxification of O2- generated in photosynthesis process (Elstner
and Osswald, 2011). In this sense, Wang et al. (2004) stated that the
induction of a particular group of enzymes, such as SOD, plays an
essential role in the cellular defense strategy against oxidative stress
caused by metals. Therefore, future studies of plant ionome should
include enzymatic analyses to assess this inference.
The positive relation between DW and Ca could be associated with a
Ca deprived by Cu. Ca is readily replaced by other cations from its
binding sites at the exterior surface of the plasma membrane; thus, Ca
requirement increases with increasing external concentrations of heavy
metals (Hawkesford et al., 2012; Wallace et al., 1966). Similarly, toxicity
tests have shown that Cu toxicity decreased with increasing Ca2+ or
Mg2+ concentrations (Wang et al., 2017). Thus, Ca potential deficit or
alleviating effects would be significant only in lettuces grown on sandy
soil since Ca is poorly available in sandy and acidic soils (Adcock et al.,
2001). Indeed, the studied sandy soil showed significantly lower Ca
availability and higher Cu solubility than the loamy soil (Table 1).
The negative relation of DW and plant N in the sandy soil can be
5
Ecotoxicology and Environmental Safety 246 (2022) 114179
explained by N affecting Cu mobility within the plant (Broadley et al.,
2012). Besides, N is the major constituent of the chlorophyll molecule
(Gholizadeh et al., 2017; Hawkesford et al., 2012), and the treatments
with the highest chlorophyll concentration in lettuces showed low plant
DW (Fig. 1; Fig. S2). In contrast, the positive correlation found for DW
and plant As, could be explained by the inhibitory effect of As on
chlorophyll molecules (Azizur Rahman et al., 2007). Alternatively, a
potential explanation is that As can stimulate root growth (Adriano,
2001a), which could partly mitigate the affliction of roots by copper.
Alternatively, As could reflect the DW, that is, the higher DW, the higher
As accumulation. This potential explanation is supported by the low soil
As solubility (Table 1). Therefore, the potential As and P competition
inferred for the loamy soil would not occur in the sandy soil.
3.4. Analisys of the plant growth and the leaf ionome by first generation
multivariate methods
Apart from discussing whether foliar Cu is significant or not, our
previous results open the challenge of disentangling what is behind the
error variance. Thus, they invite to increase scientific knowledge by
integrating related variables on Cu phytotoxicity. Accordingly, we used
PCA on the plant ionome of lettuces grown in both soils (Fig. 3). PCA
showed three PCs with eigenvalue > 1, explaining 76.2% of the total
variance. These latent variables (LVs) were represented as follows: PC-
1 = P, K (positives), Ca and Zn (negatives); PC-2 = Cu, and Zn (posi­
tives), Mg (negative); and PC-3 = As (positive), and, N and Mg
(negatives).
Considering the abovementioned biochemical functions of elements
in plants, we can describe the PCs as follows: PC-1 (36.8% variance
explained) containing Ca and P, which would be affected by Cu and As
competition, respectively. PC-2 (21.7%) with Cu, Zn and Mg, possibly
defined by the formation of enzymes Cu-Zn SOD and the Mg’s alleviating
Cu-induced oxidative stress (Juang et al., 2021). Finally, PC-3 (17.7%)
P. Mondaca et al.
Fig. 3. Principal component analysis (PCA) of the plant ionome.
with As, N, and Mg are related to chlorophyll as explained in the pre­
vious section.
The previous relations among leaf elements could establish plant
responses by multivariable shoot ionomics signatures (Baxter et al.,
2008). Therefore, we performed linear regression models with the po­
tential ionomic biomarkers (Table 2). A stepwise regression model
including foliar Cu and other elements in leaves allowed to establish a
regression model with a high determination coefficient (Model A).
However, this regression fails in multicollinearity as shown in Fig. S4.
Model B showed a simplified model excluding multicollinearity. In this
model we can see that the potential biomarkers of DW in plants would be
Cu as negative, and P and Mg as positive. However, Cu and Mg in this
model were at the margin of statistical significance (p = 0.060 and
0.053, respectively). Alternatively, we built a regression model synthe­
sizing the number of variables by element’s balance (Model C). This
model slightly decreased the determination coefficient, yet it was more
parsimonious than Model B, and all the variables met the significance
level (α = 0.05).
Table 2 shows the need to consider other elements determining plant
growth as the single stressor is not a suitable biomarker. Thus, dose-
response models traditionally used in bioassays would not be suitable
for plants grown in field-contaminated soils. Specifically, the DW of
plants showed that other elements also affect the plant response. In fact,
we found in model C that dry weight is mainly related to other nutrients
than to Cu. Such finding can also suggest that the potential effect of the
stressor (Cu in this case) is not only by toxicity but also by plant elements
balance.
Table 2
Determination coefficients (R2) and P-values for multiple regression models
between dry weight (DW) and the lettuce foliar ionome.
Model Stepwise multiple regression R2
A DW = 0.81 + 0.0056 Cu (0.010) - 0.084 Zn (<0.001) + 0.0016 P 74.41
(0.006) + 0.000035 K (0.102) - 0.000134 Ca (0.099) + 0.000936
Mg (0.009)
B DW = − 3.63 to 0.0019 Cu (0.060) + 0.0027 P (<0.001) 48.45
+ 0.00076 Mg (0.053)
C DW = 2.82 + 0.085 (P * K/Ca*Zn) (<0.001) - 23.97 (Cu*Zn/Mg) 41.51
(0.003)
P-values (in parentheses) for each variable in the regression models are shown.
Model A: Alpha to enter = 0.15 in stepwise multiple regression. Model B: Similar
to Model A but excluding multicollinearity. Model C: Using nutrient ratios ac­
cording PCA.
6
Ecotoxicology and Environmental Safety 246 (2022) 114179
3.5. Analisys of Cu phytotoxicity by a second-generation multivariate
method. Using a SEM approach
Cu phytotoxicity in field-contaminated soils was assessed using PLS-
PM. This method is based on a Structural Equation Modeling (SEM)
approach allowing to establish a network of interactions (Fig. 4). In this
analysis, we aimed to establish the relation of the plant ionome and the
DW, while the plant physiological status moderates the plant ionome.
The model show that DW was mainly explained by the plant macronu­
trient latent variable (loading = 0.70), which is affected by plant Cu
(loading = - 0.24). On the other hand, we found a negative direct effect
of Cu on plant growth (loading = - 0.29). Therefore, we integrated the
plant basal nutritional status with the potential stressor.
The quality of the model was assessed with the redundancy index
(mean redundancy for dry weight= 0.66), which showed a high ability
of independent latent variables to explain variation in the plant dry
weight. On the other hand, the measure of the overall prediction per­
formance was assessed using the Goodness of Fit statistic (GoF=0.48).
We found that plant physiological status would explain better the
plant basal nutrition status but not foliar Cu. These results contrast with
the findings using the bivariate statistic of Section 3.2. Therefore, we
inferred that the environmental conditions used in this experiment
mainly altered the plant basal nutritional status, which varied the effect
of Cu on plant growth. This type of interaction shows the weakness of
dose-response models since they cannot integrate more than two vari­
ables. Bivariate analyses are helpful for isolated phenomena but can not
address more complex issues. In the same context, nutrient balances
have been proposed as an alternative to traditional approaches like the
“Law of minimum” illustrated by Liebig’s barrel (Parent et al., 2013a,
2013b). However, our findings argue that using SEM to analyze plant
ionome could be a better approach. Specifically, the SEM approach
proved logical interactions while discerning among direct, indirect, and
no significant effects.
3.6. Outlook of using multivariate approaches in ecotoxicological
experiments
The results of the multivariate approaches indicates the importance
of considering multiple variables to interpret phytotoxicity. If only the
stressor and the endpoints are measured, we suggest that results of
bioassays should be considered for those local specific cases, that is,
specific environmental conditions and soil characteristics. Conse­
quently, this information should be clarified properly.
P. Mondaca et al.
Fig. 4. Diagram of the Partial Least Squares Path Model (PLS-PM). Observed variables are represented in rectangular form, while the latent variables are represented
in elliptical form. Numbers represent the loadings (the correlations between a composite and its observed variables), and the path coefficients calculated after 999
bootstraps. Dashed and blank arrows represent the indirect and no significant effect (α = 0.05), respectively. The Goodness of Fit was 0.48.
Alternatively, we suggest integrating multiple variables by novel
data analysis methods as performed here. For that purpose, caution
should be taken in experimental designs, since by increasing the number
of variables considered, a increasing number of replicates is recom­
mended. According to our results, if a similar or higher number of var­
iables are added, a n > 5 is suggested. On the other hand, the number of
samples can be lower if the variability on plant growth is reduced (e.g.
using field-contaminated soils, but with constant environmental condi­
tions). We also want to encourage similar experiments to train models by
novel informatics analyses, especially those where linearity is not
needed.
3.7. Final consideration: comparison of lettuce ionome in growth chamber
vs field-conditions
Representativeness of field conditions is often desired in bioassays.
An effort to represent these field conditions is using field-contaminated
soils in bioassays instead of artificially-spiked soils (Mondaca et al.,
2017). However, environmental conditions should also be considered,
as shown in Table S3. Specifically, we found drastic variations in the
plant Cu and As concentrations. HH-LM showed the most similar
magnitude among all the treatments than those obtained in field con­
ditions (Mondaca et al., 2022). However, in field conditions, the Cu
concentration was lower but the As higher. Consequently, other factors,
such as those affecting roots, such as watering, soil physic conditioning,
and the soil microbiome, should be assessed in future studies.
4. Conclusion
We found that plant growth, leaf Cu concentration, plant physiology,
and leaf ionome were modified by the environmental conditions: light
intensity and air relative humidity. These changes rarely follow a linear
pattern. Hence, caution should be taken when results such as phyto­
toxicity thresholds or the efficacy of a soil amendment are extrapolated
to universal conditions. In this context, we suggest that light intensity
and air relative humidity shall always be reported.
Foliar Cu concentration showed poor performance as a biomarker of
plant growth. In contrast, other elements showed better determination
7
Ecotoxicology and Environmental Safety 246 (2022) 114179
coefficients. The first-generation multivariate analysis allowed inte­
grating the different elements of the foliar ionome by building nutrient
and trace element balances. On the other hand, the Structural Equation
Modeling (SEM) approach go further by including physiological re­
sponses, and discerning the effect of Cu affecting plant growth by
phytotoxicity consequences and the indirect effect when Cu alters plant
basal nutritional status. Thus, the multivariate analysis allowed finding
plant ionomic signatures, which can be calibrated by using big data. In
another way, the results of bioassays should be considered only for the
specific conditions under which they were performed.
Finally, we found that lettuces grown in high air relative humidity
and medium light intensity showed the closest concentration of As and
Cu to those found in an experiment with similar soils but in field con­
ditions. Yet, results still differ. Thus, we encourage future studies to
analyze other properties, especially those related to root growth and
uptake.
Funding
ANID (Chile) FONDEF ID17AL0056 supported this research. P.M.
was funded by the ANID doctoral scholarship grant 21201569. P.V. was
funded by the ANID postdoctoral scholarship grant 74200084. W.Q. was
funded by FONDECYT 1191041. S.A. was funded by FONDECYT
1170995. J.L.C.-D. was funded by PUCV 039.431/2020 “Nú cleo de
Soluciones de Base Natural” and by ANID ACE210006 to the Instituto de
Ecologiá y Biodiversidad (IEB-Chile). ANID/PIA/ACT192027 funded P.
M., S.A., and J.L.C.-D.
CRediT authorship contribution statement
Pedro Mondaca, Sebastián Abades, Juan L. Celis-Diez: Concep­
tualization. Pedro Mondaca, Waldo Quiroz, Sebastián Abades:
Methodology. Pedro Mondaca, Patricio Valenzuela: Investigation.
Waldo Quiroz, Monika Valdenegro, Juan L. Celis-Diez: Resources.
Pedro Mondaca: Writing − original draft preparation. Sebastián
Abades, Juan L. Celis-Diez: Writing − review & editing. All authors
have read and agreed to the published version of the manuscript.
P. Mondaca et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data and R codes supporting the analyses are available from the
GitHub Repository (https://github.com/pemori/ligh-humidity-lettuce).
Acknowledgments
P.M. wants to acknowledge Fenisa Osses, Dayana Olivares, and
Yasna Peña for their support in laboratory analyses. We thank Alexander
Neaman for funding acquisition and discussion of experimental design.
J.L.C.-D. is an associate researcher at the Centro de Acción Climática-
PUCV.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2022.114179.
References
Abbasi, S., et al., 2021. The influence of long-term ageing on arsenic ecotoxicity in soil.
J. Hazard. Mater. 407, 124819–124825. https://doi.org/10.1016/j.
jhazmat.2020.124819.
Adcock, K.G., et al., 2001. Calcium deficiency of wheat grown in acidic sandy soil from
Southwestern Australia. J. Plant Nutr. 24, 1217–1227. https://doi.org/10.1081/
PLN-100106977.
Adriano, D.C., Arsenic. In: D. C. Adriano, (Ed.), Trace Elements in Terrestrial
Environments: Biogeochemistry, Bioavailability, and Risks of Metals. Springer New
York, New York, USA, {C}2001a{C}, pp. 219–261.
Adriano, D.C., Copper. In: D. C. Adriano, (Ed.), Trace Elements in Terrestrial
Environments: Biogeochemistry, Bioavailability, and Risks of Metals. Springer New
York, New York, USA, {C}2001b{C}, pp. 499–546.
Aguilar, M., et al., 2018. Comparison of exposure to trace elements through vegetable
consumption between a mining area and an agricultural area in central Chile.
Environ. Sci. Pollut. Res. 25, 19114–19121. https://doi.org/10.1007/s11356-018-
2116-x.
Alekseev, I., et al., 2018. Assessment of potential health risk due to consumption of
vegetables grown near a copper smelter in central Chile. Taurida Herald Agrar. Sci 2,
9–14. https://doi.org/10.25637/TVAN.2018.02.01.
Azizur Rahman, M., et al., 2007. Effect of arsenic on photosynthesis, growth and yield of
five widely cultivated rice (Oryza sativa L.) varieties in Bangladesh. Chemosphere
67, 1072–1079. https://doi.org/10.1016/j.chemosphere.2006.11.061.
Baxter, I.R., et al., 2008. The leaf ionome as a multivariable system to detect a plant’s
physiological status. Proc. Natl. Acad. Sci. USA 105, 12081–12086. https://doi.org/
10.1073/pnas.0804175105.
Broadley, M., et al., 2012. Function of nutrients: Micronutrients. In: Marschner, P. (Ed.),
Mineral Nutrition of Higher Plants. Elsevier, pp. 191–248.
Campos, A., et al., 2021. 1,135 ionomes reveal the global pattern of leaf and seed mineral
nutrient and trace element diversity in Arabidopsis thaliana. Plant J. 106, 536–554.
https://doi.org/10.1111/tpj.15177.
Cook, C.M., et al., 1997. Effects of copper on the growth, photosynthesis and nutrient
concentrations of Phaseolus plants. Photosynthetica 34, 179–193. https://doi.org/
10.1023/a:1006832321946.
R. Core Team, 2021, R: A language and environment for statistical computing. R
Foundation for Statistical Computing, Vienna, Austria.
Eisenhauer, N., et al., 2015. From patterns to causal understanding: Structural equation
modeling (SEM) in soil ecology. Pedobiologia 58, 65–72. https://doi.org/10.1016/j.
pedobi.2015.03.002.
Elstner, E.F., Osswald, W., 2011. Mechanisms of oxygen activation during plant stress.
Proc. R. Soc. B 102, 131–154. https://doi.org/10.1017/s0269727000014068.
Gholizadeh, A., et al., 2017. Leaf chlorophyll and nitrogen dynamics and their
relationship to lowland rice yield for site-specific paddy management. Inf. Process.
Agric. 4, 259–268. https://doi.org/10.1016/j.inpa.2017.08.002.
Grace, J.B., et al., 2015. Structural equation modelling: Building and evaluating causal
models. In: Fox, G.A., et al. (Eds.), Ecological Statistics: Contemporary Theory and
Application. Oxford University Press, New York, NY, United States of America.
Hamels, F., et al., 2014. Phytotoxicity of trace metals in spiked and field-contaminated
soils: Linking soil-extractable metals with toxicity. Environ. Toxicol. Chem. 33,
2479–2487. https://doi.org/10.1002/etc.2693.
Hawkesford, M., et al., 2012. Functions of macronutrients. In: Marschner, P. (Ed.),
Mineral Nutrition of Higher Plants. Elsevier, pp. 135–189.
8
Ecotoxicology and Environmental Safety 246 (2022) 114179
Henseler, J., Sarstedt, M., 2012. Goodness-of-fit indices for partial least squares path
modeling. Comput. Stat. 28, 565–580. https://doi.org/10.1007/s00180-012-0317-1.
Huang, X.Y., Salt, D.E., 2016. Plant ionomics: from elemental profiling to environmental
adaptation. Mol. Plant 9, 787–797. https://doi.org/10.1016/j.molp.2016.05.003.
ISO 22030, Soil quality — Biological methods — Chronic toxicity in higher plants.
Geneva, Switzerland, 2005.
Jiao, X., et al., 2022. Effects of rising VPD on the nutrient uptake, water status and
photosynthetic system of tomato plants at different nitrogen applications under low
temperature. Sci. Hortic. 304. https://doi.org/10.1016/j.scienta.2022.111335.
Juang, K.W., et al., 2021. Modeling Alleviative Effects of Ca, Mg, and K on Cu-Induced
Oxidative Stress in Grapevine Roots Grown Hydroponically. Molecules 26. https://
doi.org/10.3390/molecules26175356.
Kader, M., et al., 2018. Copper interactions on arsenic bioavailability and phytotoxicity
in soil. Ecotoxicol. Environ. Saf. 148, 738–746. https://doi.org/10.1016/j.
ecoenv.2017.11.025.
Kalra, Y.P., 1998. Handbook of Reference Methods for Plant Analysis. CRC Press, Boca
Raton, FL, USA.
Kim, H.H., et al., 2004. Stomatal conductance of lettuce grown under or exposed to
different light qualities. Ann. Bot. 94, 691–697. https://doi.org/10.1093/aob/
mch192.
Kutner, M., et al., 2004, Applied Linear Regression Models. McGraw-Hill Education,
Boston.
Le, T.T.Y., et al., 2013. Modelling metal–metal interactions and metal toxicity to lettuce
Lactuca sativa following mixture exposure (Cu2+–Zn2+ and Cu2+–Ag+). Environ.
Pollut. 176, 185–192. https://doi.org/10.1016/j.envpol.2013.01.017.
Lizardi, N., et al., 2020. Human health risk assessment from the consumption of
vegetables grown near a copper smelter in central Chile. J. Soil Sci. Plant Nutr. 20,
1472–1479. https://doi.org/10.1007/s42729-020-00226-w.
Maxwell, J.A., 1968. Rock and Mineral Analysis. Interscience Publishers, New York.
McBride, M., et al., 1997. Solubility control of Cu, Zn, Cd and Pb in contaminated soils.
Eur. J. Soil Sci. 48, 337–346. https://doi.org/10.1111/j.1365-2389.1997.tb00554.x.
McBride, M.B., et al., 2009. A comparison of tests for extractable copper and zinc in
metal-spiked and field-contaminated soil. Soil Sci. 174, 439–444. https://doi.org/
10.1097/SS.0b013e3181b66856.
Minitab, I., 2010, Minitab 17 Statistical Software. State College, PA, USA.
Mondaca, P., et al., 2015. Solubility, partitioning, and activity of copper-contaminated
soils in a semiarid region. J. Soil Sci. Plant Nutr. 178, 452–459. https://doi.org/
10.1002/jpln.201400349.
Mondaca, P., et al., 2017. Advances on the determination of thresholds of Cu
phytotoxicity in field-contaminated soils in central Chile. Environ. Pollut. 223,
146–152. https://doi.org/10.1016/j.envpol.2016.12.076.
Mondaca, P., et al., 2022. Remediation of agricultural soils with long-term contamination
of arsenic and copper in two chilean mediterranean areas. Agronomy 12. https://doi.
org/10.3390/agronomy12010221.
Moreno-Jiménez, E., et al., 2012. The fate of arsenic in soil-plant systems. In:
Whitacre, D.M. (Ed.), Reviews of Environmental Contamination and Toxicology.
Springer, New York, USA, pp. 1–37.
Palmer, C.M., Guerinot, M.L., 2009. Facing the challenges of Cu, Fe and Zn homeostasis
in plants. Nat. Chem. Biol. 5, 333–340. https://doi.org/10.1038/nchembio.166.
Parent, S.E., et al., 2013a. The plant ionome revisited by the nutrient balance concept.
Front Plant Sci. 4, 39. https://doi.org/10.3389/fpls.2013.00039.
Parent, S.E., et al., 2013b. Plant ionome diagnosis using sound balances: case study with
mango (Mangifera indica. Front. Plant Sci. 4, 449. https://doi.org/10.3389/
fpls.2013.00449.
Pilon, M., et al., 2006. Copper cofactor delivery in plant cells. Curr. Opin. Plant Biol. 9,
256–263. https://doi.org/10.1016/j.pbi.2006.03.007.
Poorter, H., et al., 2012. The art of growing plants for experimental purposes: a practical
guide for the plant biologist. Funct. Plant Biol. 39. https://doi.org/10.1071/
fp12028.
Poorter, H., et al., 2019. A meta-analysis of plant responses to light intensity for 70 traits
ranging from molecules to whole plant performance. N. Phytol. 223, 1073–1105.
https://doi.org/10.1111/nph.15754.
Sadzawka, A., et al., 2015, Métodos de análisis de lodos y suelos. Sociedad Chilena de la
Ciencia del Suelo, Universidad de Concepción, Chillán, Chile.
Salt, D.E., et al., 2008. Ionomics and the study of the plant ionome. Annu. Rev. Plant Biol.
59, 709–733. https://doi.org/10.1146/annurev.arplant.59.032607.092942.
Sanchez, G., P.L.S. Path Modeling with R. Berkeley, USA, 2013.
Sheldrick, B.H., Wang, C., 1993. Particle size distribution. In: Carter, M. (Ed.), Soil
Sampling and Methods of Analysis. Canadian Society of Soil Science. Lewis
Publishers, Boca Raton, FL, USA, pp. 499–511.
Sparks, D.L., et al., 1996. Methods of Soil Analysis. Part III: Chemical Methods. Soil
Science Society of America. American Society of Agronomy,, Madison, WI, USA.
Steubing, L., 1982. Problems of bioindication and the necessity of standardization. In:
Steubing, L., Jäger, H. (Eds.), Monitoring Air Pollutants by Plants. Dr. Junk.
Publishers, The Netherlands, pp. 19–27.
Stuckey, J.W., et al., 2008. Highly Charged Swelling Mica Reduces Free and Extractable
Cu Levels in Cu-Contaminated Soils. Environ. Sci. Technol. 42, 9197–9202. https://
doi.org/10.1021/es801799s.
R.Studio Team, 2022, RStudio: Integrated Development Environment for R. RStudio,
PBC, Boston, MA, USA.
Temminghoff, E.J.M., Houba, V.J.G., 2004, Plant Analysis Procedures. Kluwer Academic
Publishers.
Tiecher, T.L., et al., 2016. Effects of zinc addition to a copper-contaminated vineyard soil
on sorption of Zn by soil and plant physiological responses. Ecotoxicol. Environ. Saf.
129, 109–119. https://doi.org/10.1016/j.ecoenv.2016.03.016.
P. Mondaca et al.
Upadhyay, R., Panda, S.K., 2010. Zinc reduces copper toxicity induced oxidative stress by
promoting antioxidant defense in freshly grown aquatic duckweed Spirodela
polyrhiza L. J. Hazard. Mater. 175, 1081–1084. https://doi.org/10.1016/j.
jhazmat.2009.10.016.
Vargas, C., et al., 2015. Stability of arsenic during soil treatment and storage. JCCHEMS
60, 3045–3048. https://doi.org/10.4067/s0717-97072015000300015.
Verdejo, J., et al., 2015. Thresholds of copper toxicity to lettuce in field-collected
agricultural soils exposed to copper mining activities in Chile. J. Soil Sci. Plant Nutr.
15, 11–24. https://doi.org/10.4067/s0718-95162016005000011.
Verlinden, M., 1982. On the acid decomposition of human blood and plasma for the
determination of selenium. Talanta 29, 875–882. https://doi.org/10.1016/0039-
9140(82)80260-9.
von Wettberg, E.J.B., et al., 2018. Ecology and genomics of an important crop wild
relative as a prelude to agricultural innovation. Nat. Commun. 9, 649. https://doi.
org/10.1038/s41467-018-02867-z.
Ecotoxicology and Environmental Safety 246 (2022) 114179
Wallace, A., et al., 1966. Calcium requirements of higher plants. Nature 209, 634.
https://doi.org/10.1038/209634a0.
Wang, H., et al., 2004. Responses of antioxidative enzymes to accumulation of copper in
a copper hyperaccumulator of Commoelina communis. Arch. Environ. Contam.
Toxicol. 47, 185–192. https://doi.org/10.1007/s00244-004-2222-2.
Wang, X., et al., 2017. Extended biotic ligand model for predicting combined Cu–Zn
toxicity to wheat (Triticum aestivum L.): Incorporating the effects of concentration
ratio, major cations and pH. Environ. Pollut. 230, 210–217. https://doi.org/
10.1016/j.envpol.2017.06.037.
Xu, C., et al., 2020. High humidity alleviates photosynthetic inhibition and oxidative
damage of tomato seedlings under heat stress. Photosynthetica 58, 146–155. https://
doi.org/10.32615/ps.2019.168.
Yruela, I., 2005. Copper in plants. Braz. J. Plant Physiol. 17, 145–156. https://doi.org/
10.1590/s1677-04202005000100012.