Science of the Total Environment 912 (2024) 168956

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Science of the Total Environment

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

Combined passivators regulate physiological, antioxidant potential and

metals accumulation in potato grown in metals contaminated soil
Hongyang Wang a, 1, Hao Liu b, 1, Junhua Li b, Shengnan Chen a, Qamar uz Zaman c,
Khawar Sultan c, Muzammal Rehman d, Shah Saud e, Khaled El-Kahtany f, Shah Fahad g, **,
Gang Deng b, **, Aie Chen a, h, *
a
Yunnan Key Laboratory of Potato Biology, Yunnan Normal University, Kunming, Yunnan 650500, China
b
School of Agriculture, Yunnan University, Kunming, Yunnan 650504, China
c
Department of Environmental Sciences, The University of Lahore, Lahore 54590, Pakistan
d
Guangxi Key Laboratory of Agro-environment and Agric-products Safety, Key Laboratory of Plant Genetics and Breeding, College of Agriculture, Guangxi University,
Nanning 530004, China
e
College of Life Science, Linyi University, Linyi, Shandong 276000, China
f
Geology and Geophysics Department, College of Science, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia
g
Department of Agronomy, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa 23200, Pakistan
h
Teaching Affairs Department, Yunnan Normal University, Kunming 650500, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Heavy metals immobilization by the

application of passivators is an effective
approach in metal polluted soils.
• Passivators application improved the
soil health by the immobilization of
heavy metals.
• Balanced application of passivators
improved the potato growth and enzy­
matic activities in the stressed plants.
• For safe utilization of metal polluted
soil, combined usage of passivating
agents is recommended for its
remediation.

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

Editor: Baoliang Chen To solve the problem of excessive heavy metals in farmland soil, there is a dire need for research effort to screen
for the soil passivator materials. This study aimed to develop a practical novel approach for improving the potato
Keywords: growth and remedial effectiveness of the metals by optimal combination and dosage of various passivators.
Potato Experimental treatments were comprised of various levels of passivating agents (sepiolite, quicklime and calcium
Passivating agents magnesium phosphate) in individual and combined form. Results showed that application of passivating agents
Quicklime significantly enhanced growth by optimizing photosynthetic attributes, enzymatic antioxidants, and soil health.
Food security

* Correspondence to: A. Chen, Teaching Affairs Department, Yunnan Normal University, Kunming, Yunnan 650500, China.
** Corresponding authors.
E-mail addresses: shah_fahad80@yahoo.com (S. Fahad), denggang1986@ynu.edu.cn (G. Deng), chenaie145@126.com (A. Chen).
1
These authors have contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2023.168956
Received 26 August 2023; Received in revised form 7 November 2023; Accepted 26 November 2023
Available online 1 December 2023
0048-9697/© 2023 Elsevier B.V. All rights reserved.
H. Wang et al.
Antioxidants Balanced application of passivators effectively reduce the bioavailability of metals, curbing their uptake by
Bioaccumulation potato plants. Sole application of all the agents results statistically similar outcomes as compared with combined
form. Additionally, passivators indirectly enhance the activity of essential antioxidant enzymes. Synergistic effect
of all the agents significantly improved the tuber quality by decreasing the accumulation of proline, malon­
dialdehyde content, and bioaccumulation of Cu, Pb, Cd, and As in potato parts. In crux, combined usage of
passivating agents proved to be of better growth, improvement in antioxidative defense system, and better
quality of potato. By mitigating heavy metal contamination, passivators not only enhance crop quality and yield
but also ensure heavy metal-free potatoes that meet stringent food safety standards.
1. Introduction
In addition to being an essential component of the ecosystem, soil is
also a key resource for agricultural production and a vital component of
human survival (Turan, 2020; Pahalvi et al., 2021). Heavy metals can
enter the soil in a number of ways, but the two main methods are
through native and exogenous sources (Lal et al., 2021). Heavy metals in
the soil not only reduce crop quality and yield, but also decrease soil
quality, which is very difficult to degrade (Qayyum et al., 2020; Iqbal
et al., 2023). In the soil-crop system, heavy metals build up in crops,
where they progressively move to the human body through the food
chain (Ali et al., 2020).
Remediation of contaminated soils is necessary for agricultural soils
due to the toxicity, bioaccumulation, and persistence of metals in the
environment (Filote et al., 2021). Several methods, such as digging,
washing, landfilling, immobilizing, and phytoremediation, have been
used to clean up metal-contaminated sites (Turan, 2019; Shabaan et al.,
2021; Jiang et al., 2022). In these circumstances, the passivating ma­
terial’s remedial effect is based on the characteristics of the soil (Turan
et al., 2019). However, in other circumstances, passivation remediation
is based on the kind and level of contamination (Turan, 2022; Gong
et al., 2022). Ameta-analysis of 489 independent observations showed
that using biochar, phosphate, lime, metal oxides, and clay minerals are
very effective in remediation of metal contaminated soil. The most
efficient material for the immobilization of lead was phosphate, while
the most efficient material for fixing metals and metalloids was lime
(Turan, 2021; Titchou et al., 2021). Not only that, but the effect of lime
also depended on the activity of metals in the soil (Wang et al., 2022).
To lessen the bioavailability of different heavy metals in soils by
adsorption, complexation, and precipitation, passivators are extensively
used (Hussain et al., 2020; Zhao et al., 2020). One of the most effective
inorganic passivators, quicklime, can substantially raise soil pH and
passivate the heavy metals in the soil (Huang et al., 2019). However
frequent, excessive usage of lime over an extended period of time may
cause soil particles to bond together, limit soil permeability, and lessen
the diversity of the microbial population (Song et al., 2017; Jing et al.,
2019). Quicklime application in combination with clay minerals like
sepiolite (S) has become quite popular. It has stronger remediation ef­
fects than single quicklime application and prevents soil from becoming
harder as a result of repeated quicklime application (Chen et al., 2019).
Potato tubers are the fourth foodstuff consumed by mankind because
these foods contain carbohydrates (Setiyo et al., 2020). Therefore, these
foods must meet the safety standards set by World Health Organization
(WHO). Food safety standards set by WHO for heavy metal content are
for Fe, Pb, Cd, Cr and Zn <0.4, 0.2, 0.1, 0.1 and 0.35 mg/kg (Zeliha
et al., 2017; Zeng et al., 2018). To produce consumption potatoes that
meet food safety standards, cultivation must be carried out with quality
seeds, and application of environmentally friendly cultivation technol­
ogy (Muktar et al., 2019). Though, there is a research gap concerning the
optimal dosage and combination of passivators for mitigation of metals
stress in potato. To this end, this experimental approach hypothesizes
that sole and synergistic effect of various passivating agents improve the
soil health, potato growth and to minimize metals uptake by reducing
metals bioavailability in the soil. In this novel approach, a pot trail was
performed on multi-metal polluted soil to portray the impacts of various
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Science of the Total Environment 912 (2024) 168956
dosage and combinations of passivating agents on soil health and bio­
accumulation of As, Pb, Cd and Cu in the soil–potato system. The main
objectives of this study were to; 1) determine the most suitable appli­
cation combination and dosages of passivators for maximizing their
effectiveness in improving the potato growth by reducing metal uptake,
2) assess the effectiveness of passivators in immobilizing heavy metals in
contaminated soil, thereby improving the soil’s health and 3) to estab­
lish the potential of passivators as a viable strategy for mitigating the
adverse effects of metal contamination on potato growth and
development.
2. Materials and methods
2.1. Study area, experimental treatments & design
The experimental research work was conducted in the greenhouse at
the Yunnan Normal University, Kunming City, Yunnan Province, during
the period from 23th Jun. to 8th Oct. 2022. The location of the experi­
mental site is 24.867 N and102.859E at an elevation of 1946 mASL
(meters above sea level) having a temperature of 15 ◦ C and an average
annual precipitation of 667 mm. Experimental treatments were: Ck =
control (no-passivating agent), T1 (sepiolite =15 g/kg), T2 (quicklime =
30 g/kg), T3 (calcium magnesium phosphate = 15 g/kg), T4 (sepiolite =
15 g/kg + quicklime = 30 g/kg), T5 (sepiolite = 15 g/kg + calcium
magnesium phosphate = 15 g/kg), T6 (quicklime = 30 g/kg + calcium
magnesium phosphate = 15 g/kg) and T7 (sepiolite = 15 g/kg +
quicklime = 30 g/kg + calcium magnesium phosphate = 15 g/kg).
Sepiolite is primarily composed of hydrated magnesium silicate, which
is a combination of magnesium, silicon, oxygen, and water. Quicklime,
is a chemical compound that is derived from limestone or other calcium-
containing materials through a process called calcination. Calcium
magnesium phosphate contains 12 % P2O5, 20 % CaO, and 3 % MgO. All
reagents were purchased from the company (The Mosaic Company,
USA). The experiment was developed by employing the method of
completely randomized design (CRD) replicated three times.
2.2. Soil sampling and analysis
For the experimental treatments of samples, surface soils were
collected from a contaminated site of a copper mining area in Dong­
Chuan district, Kunming city, Yunnan, China (103.746E, 26.104N), at
the depths of 0 to 30 cm. The initial physio-chemical, elemental, and
heavy metals contents of sampled soil were pH = 8.15, electrical con­
ductivity = 25.8 mS/m, organic matter = 16.2 g/kg, total nitrogen =
996 mg/kg, total phosphorous = 1038 mg/kg, total potassium = 1.79 %,
hydrolytic nitrogen = 53.4 mg/kg, available phosphorus = 33 mg/kg,
quick available potassium = 454 mg/kg, and for metals Cu, As, Cd, Pb,
Hg, Cr, Zn and Ni were 296, 80.2, 9.15, 193, 0.678, 82.2, 247 and 50.9
mg/kg, respectively. According to the risk control standards for agri­
cultural soil pollution, four soil heavy metal content indexes (Cu, As, Cd,
and Pb) exceeded the national soil safety standards.
2.3. Crop management
The potato cultivar Mira, from Yunnan Normal University was
H. Wang et al.
planted in this experimental work. Potato plants were grown in plastic
pots (diameter ~ 40 cm & depth ~ 30 cm) filled with about 10 kg of soil.
In each pot, the soil was homogeneously mixed with the respective
passivators agents according to the treatments. The pots were lined with
plastic sheets to stop any leaching or loss of nutrients and trace elements
during the experiment. All the passivating agent were mixed fully before
sowing. Two weeks before planting the potatoes, seed tubers were
placed in a cardboard box covered with cheesecloth to create airy con­
ditions for germination of buds at the 8 to 10 ◦ C temperature range. The
essential nutrients were added to the soils by application of 20–20–20
NPK (nitrogen–phosphorus–potassium) fertilizer, and the addition of
supplements such as Fe, Mn, Zn, Cu, B, Mo, and EDTA (Plant Products
Co., Ltd., Brampton, Canada) was carried out. Harvesting of full-grown
potatoes took place after 120 days of the plantation. The aboveground
biomass of potato plants was collected and separated into stems, leaves,
and tubers for further investigation.
2.4. Data collection
2.4.1. Morphological attributes
Plants were collected at various growth stages and their roots and
shoots were separated for the measurements of growth variables. Potato
plant height was measured from the soil surface to the top of the plant
employing a meter rod. To quantify the dry weight of plant parts, they
were oven dried (~70 ◦ C) for 48 h.
2.4.2. Photosynthetic attributes
The sample of plant leaves at various growth stages was crushed and
about 5 g was taken in a test tube having 85 % acetone (v/v) for 24 h and
was placed in a dark condition for pigment extraction. Centrifugation of
the sample in test tubes for 10 min at (4000 ×g) at 4 ◦ C to enable the
separation. The supernatant thus obtained was subjected to an analysis
using a spectrophotometer (Halo DB-20/DB-20S, UK) at different
wavelengths of 470, 647, and 664.5 nm for measurements by following
procedures reported by Lichtenthaler (1987) for the content determi­
nation of chlorophyll a & b, and carotenoids. Total chlorophyll contents
were measured by the addition of chlorophyll a and chlorophyll b.
2.4.3. Osmolytes attributes
A plant fresh leaf of about 0.5 g was collected, and ground with a
buffer of 7.2 pH as part of sample preparation. To the powdered sample,
protease inhibitors of a concentration of 1 μM were added with saline
phosphate buffer (PBS) to prepare a homogenous mixture. In 1 L of
deionized water 1.37 mM NaCl, 2 mM KH2PO4, 2.7 mM KCl, and 10 mM
Na2HPO4 were dissolved for the preparation of saline buffer. The pH
values of the buffer used were adjusted by the addition of an HCl. The
solution thus obtained was autoclaved first and then centrifuged
(12,000 ×g) for 5 min for the sample separation of supernatant. Proline
contents were determined as per the protocols described by Maehly and
Chance (1954), while soluble sugars and soluble proteins were deter­
mined by the method described by Giannakoula et al. (2008) and
Bradford assay (Bradford, 1976), respectively.
2.4.4. Biochemical attributes
The supernatant extracted from 1 g leaves with 50 mM phosphate
buffer was centrifuged at (15,000 ×g) for 10 min and used for measuring
the enzyme activity. The activity of peroxidase (POD) was determined
following the methods reported by Velikova et al. (2000). The catalase
activity (CAT) was determined according to procedures described by
Aebi (1974). The superoxide dismutase activity (SOD) was measured
according to the techniques presented by Beauchamp and Fridovich
(1971). The enzyme extract’s ability to inhibit the nitro blue tetrazo­
lium’s photochemical reduction (NBT) was determined at 560 nm. MDA
was assayed by the thiobarbituric acid reaction method (Hodges et al.,
1999).
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Science of the Total Environment 912 (2024) 168956
2.4.5. Soil enzymes and soil chemical attributes
Surface soil samples were collected from a depth of 0 to 15 cm.
Samples were bulked and dried at room temperature conditions (~21
◦
C) and stored in paper bags under dry conditions until further analysis.
By utilizing the water/soil suspension (2:1) the soil pH and electrical
conductivity (EC) were measured by following the protocols given by
Ryan et al. (2001). Measurement of soil organic matter (OM) contents
was carried out according to the method of GB9834-1988, soil available
potassium by the method of GB 9836-1998 by using flame atomic ab­
sorption spectrophotometry, and soil available phosphorus by sodium
hydrogen carbonate solution-Mo-Sb anti spectrophotometric method of
HJ/T 704-2014 following the standards procedures and the protocols
described by the Institute of Soil Science, Chinese Academy of Sciences
(Nanjing, China).
Using a TECAN Infinite M200 spectrophotometer (G10S UV–Vis,
Thermo Fisher, USA) and methylumbelliferone (MUB)-linked model
substrates, the activities of the extracted soil enzymes were estimated
fluorometrically for each rhizosphere soil sample including soil acid
phosphatase activities, urease (Emami et al., 2013), cellulase (Kizilkaya
et al., 2012), sucrase, and soil catalase activities (Allison and Jastrow,
2006), which were measured at the wavelengths of 450 nm for S-CAT
and 365 nm. For the estimation of aryl acylamidase the protocols of
Nakamura et al. (1990) were followed using acetanilide (AAN) as the
substrate. The procedures for β-1,4-glucosidase were adapted from Dick
(2011) measuring the color intensity at wavelengths of 400 nm while
soil dehydrogenase enzymatic activity was measured following the
method reported by Min et al. (2001) and the hydrolyzed nitrogen in the
soil samples were measured according to the protocols of Yunnan San­
biao Agriculture and Forestry Technology Co., Ltd. China.
2.4.6. Tuber quality attributes
Total amylose content of sweet potato starch was determined using
spectrophotometric method after removal of lipids from starch with hot
75 % n-propanol for 7 h in a Soxhlet extractor (Hoover and Ratnayake,
2005). The pure potato amylose (Sigma–Aldrich) and amylopectin from
maize (Sigma–Aldrich) were used to create a standard curve and the
total amylose content of each sample was inferred from this standard
curve. The difference was the considered as the amylopectin content.
Total tuber nitrogen was determined by the Kjeldahl method (Chapman
and Pratt, 1961). Protein contents in tubers was estimated by multi­
plying total nitrogen with a factor 6.25 (AOAC, 1995).
2.4.7. Metals analysis in plant samples
Metal absorption in both soil and plant samples was determined on a
dry-weight basis. By following the wet digestion method, about 2 g
(plant and soil) of dried sample was added to a 10 mL freshly prepared
aqua regia solution of two acids of HNO3 and HCl with a volumetric ratio
of 1:3 and was allowed to digest at 100 ◦ C until it produced a clear so­
lution for further analysis. All the chemicals used in the digestion pro­
cess were of analytical grade quality and were obtained from E. Merck
(Darmstadt, Germany) with a certified purity of better than 99 %. The
solution thus obtained was filtered through the pore size of 42 μm after
cooling the clear solution at room temperature conditions. To bring up
to the required volume of 50 mL for the analytical analysis, high-purity
distilled water was added to the solution. This prepared sample of soil
and plant extract was then used for the determination of dissolved
metals and metalloids concentration by using inductively coupled
plasma mass spectrometry (ICP-MS, ELAN 9000, Perkin-Elmer SCIEX,
USA).
2.4.8. Quality control analysis and assurance
Merck-Germany analytical grade chemicals were utilized for metal
analysis. Using a calibration curve, the atomic absorption spectroscopy
(AAS) instrument was calibrated using a series of standard solutions
from the elemental stock solution. The stock solution was made using
double deionized water. To avoid contamination, high-quality glassware
H. Wang et al. Science of the Total Environment 912 (2024) 168956

(Merck-Germany) was used after washing with diluted HNO3. To
improve the accuracy of the data, the analysis was performed three
times for each sample using the usual reference approach.
2.4.9. Statistical analysis
ANOVA was performed on the dataset to investigate for any statis­
tically significant variations (p ≤ 0.05) in the application of various
passivators treatments. The impact of biochar treatments on the
morphological, biochemical, and enzymatic traits of soil and potato
plants was investigated using linear regression. The correlation between
variables was evaluated using Pearson’s correlation analysis. The sta­
tistical package Statistics 8.01 was used to systematically evaluate the
data. Using Mintab-19 software, principal component analysis was
carried out on the measured variables.
3.2. Biochemical attributes
Various growth stages and passivating treatments significantly (p ≤
0.05) improved the biochemical attributes of the potato crop grown
under contaminated soil. Addition of passivating treatments improved
the soluble sugars (96.70 %, 96.53 %, and 161.74 %) and protein con­
tents (107.03 %, 64.71 % and 65.12 %) at seedling, budding and flow­
ering stage respectively. A linear increase in the whole biochemical
attributes with increase in the addition of passivating agents. The
descending order in terms of biochemical attributes of potato for
passivating treatments were T7 > T6 > T5 > T4 > T3 > T2 > T1 > control
(Fig. 2).
3.3. Enzymatic antioxidant attributes

3. Results Passivating agents’ addition showed significantly effects on the

3.1. Growth and photosynthetic attributes
All the soil applied passivating treatments and growth stages
significantly (p ≤ 0.05) improved the growth and photosynthetic attri­
butes of potato grown under metal contaminated soil (Fig. 1). Maximum
increase in the plant height and photosynthetic attributes at various
growth stages of potato crop was observed when combined form of all
the passivating agents was applied as compared to rest of treatments.
Synergistic effect of all the passivating agents increased the plant height,
total chlorophyll contents and carotenoids contents at seedling stage
(132.80 %, 58.87 % and 252.73 %), budding stage (71.47 %, 114.14 %
and148.95 %), and on flowering stage (72.95 %, 244.91 % and 68.80
%). Sole application of calcium magnesium phosphate proved better
results than passivating agents which are applied in sole form. The
descending order in terms of growth and photosynthetic attributes of
potato for passivating treatments were T7 > T6 > T5 > T4 > T3 > T2 > T1
> control (Fig. 1).
enzymatic attributes at various growth stages of potato crop grown
under contaminated soil (Fig. 2). Maximum decrease in the SOD con­
tents (37.69 %, 47.18 % and 39.84 %), POD contents (32.53 %, 48.97 %
and 46.63 %) and catalase activity (37.44 %, 58.68 %, 31.99 %) at
seedling, budding and flowering stage, respectively, was observed
where combined application of passivating agents was used as compared
to control conditions where no passivating agents was used. The
descending order in terms of enzymatic attributes of potato for passiv­
ating treatments were control > T1 > T2 > T3 > T4 > T5 > T6 > T7.
3.4. Lipid per oxidation and proline contents
Maximum proline contents and lipid per oxidation (MDA contents) in
potato grown under metals contaminated soil at various growth stages
was observed under the control conditions. Metals stress significantly
enhanced the MDA and proline contents potato plants. While, addition
of passivating agents in synergistic form significantly reduced the MDA
contents (43.92 %, 51.02 %, 66.24 %) and proline contents (57.53 %,

Fig. 1. Effect of various soil applied passivating agent’s treatments on growth and photosynthetic attributes of potato grown in contaminated soil. The statistics
illustrate the means with standard deviation (SD) and were replicated three times; the capital letters in the bars indicate significant differences between treatment
means at p ≤ 0.05 using highest significant difference test (HSD). SS = seedling stage; BS = budding stage; FS = flowering stage; ck = control (no-passivating agent);
T1 = sepiolite; T2 = quicklime; T3 = calcium magnesium phosphate; T4 = sepiolite + quicklime; T5 = sepiolite + calcium magnesium phosphate; T6 = quicklime +
calcium magnesium phosphate and T7 = sepiolite + quicklime + calcium magnesium phosphate.

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H. Wang et al. Science of the Total Environment 912 (2024) 168956

Fig. 2. Effect of various soil applied passivating agent’s treatments on enzymatic antioxidants, osmolytes and lipid per oxidation attributes of potato grown in
contaminated soil. The statistics illustrate the means with standard deviation (SD) and were replicated three times; the capital letters in the bars indicate significant
differences between treatment means at p ≤ 0.05 using highest significant difference test (HSD). SS = seedling stage; BS = budding stage; FS = flowering stage; ck =
control (no-passivating agent); T1 = sepiolite; T2 = quicklime; T3 = calcium magnesium phosphate; T4 = sepiolite + quicklime; T5 = sepiolite + calcium magnesium
phosphate; T6 = quicklime + calcium magnesium phosphate and T7 = sepiolite + quicklime + calcium magnesium phosphate.

38.99 % and 47.59 %) at seedling, budding and flowering stage
respectively compared with the control (Fig. 2).
3.5. Dry biomass of various plant tissues
Soil applied passivating treatments significantly (p ≤ 0.05) improved
the dry biomass of leaves, stem and tuber of potato grown under metal
contaminated soil (Fig. 3). Maximum increase in the dry biomass various
plant tissues of potato crop was observed when combined form of all the
passivating agents was applied as compared to rest of treatments.
Maximum leaf dry weight (1.88 g), stem dry weight (1.69 g) and tuber
dry weight (14.28 g) was observed where all the passivating agents were
applied in combined form. Sole application of calcium magnesium
phosphate proved better results than rest of passivating agents which are
applied in sole form (Fig. 3).
compared with the control (Fig. 4).
3.7. Metals analysis in plant parts
According to National Food Safety Standards of China the four
metals were above the permissible limits in the soil and plant tissues.
However, addition of passivating agents significantly reduced the
accumulation of metals in the various parts of the potato crop except for
Cd contents. Under the application of passivators reduced the Pb, Cu, Cd
and As accumulation in the leaf (66.32 %, 50.58 %, 48.11 % and 40.75
%), stem (74.28 %, 59.87 %, 60.20 % and 57.09 %) and tuber (64.78 %,
71.03 %, 73.17 % and 75.37 %) respectively. The descending order in
terms of metals accumulation in various plants parts of potato for
passivating treatments were control > T1 > T2 > T3 > T4 > T5 > T6 > T7
(Fig. 5).

3.6. Tuber quality attributes 3.8. Soil enzymes

Maximum of all the quality attributes of potato tubers was observed
when optimum quantity of all the passivating agents were applied under
metals contaminated soil under the control conditions. Metals stress
significantly affected the quality attributes of potato tubers. While,
addition of passivating agents in synergistic form significantly enhanced
the proteins contents (98.37 %), amylopectin contents (125.10 %),
amylose contents (239.10 %) and revertose contents (198.78 %)
All the soil enzymatic attributes linearly increased by the addition of
synergistic form of all three passivating agents. Maximum activity of soil
aryl-acylamindase (159.95 μg//h/g), soil catalase (144.77 μmol/d/g),
soil alkaline protease (1.54 U/g), soil urease (245.77 U/g), solid-
β-glucosidase (25.25 U/g), soil dehydrogenase (41.65 U/g), soil cellu­
lase (11.44 U/g), soil sucrase (6.43 U/g) and solid-β-glucosidase (25.25
U/g) was observed where application of all the passivating agents was

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H. Wang et al. Science of the Total Environment 912 (2024) 168956

Fig. 3. Effect of various soil applied passivating agent’s treatments on dry biomass of leaf, stem and tuber of potato grown in contaminated soil. The statistics
illustrate the means with standard deviation (SD) and were replicated three times; the capital letters in the bars indicate significant differences between treatment
means at p ≤ 0.05 using highest significant difference test (HSD). ck = control (no-passivating agent); T1 = sepiolite; T2 = quicklime; T3 = calcium magnesium
phosphate; T4 = sepiolite + quicklime; T5 = sepiolite + calcium magnesium phosphate; T6 = quicklime + calcium magnesium phosphate and T7 = sepiolite +
quicklime + calcium magnesium phosphate.

done as compared with before treatment (passivating agents applica­ chlorophyll contents, 0.3286 while the protein contents and POD ac­
tion) (Fig. 6). tivity showed association, 0.0937.

3.9. Correlation analysis 3.11. Principle component analysis

It was observed from the findings of the current study that physio-
biochemical and enzymatic attributes are significantly correlated with
each other (Table 1). All the enzymatic and lipid per oxidation attributes
(MDA) were negatively correlated with the total chlorophyll contents,
carotenoids contents, and soluble sugars. However, plant height, soluble
sugars, and all the proteins contents positively correlated with the rest of
the measured attributes. The MDA showed a non-significant association
with plant height and protein contents. However, soluble sugars and
total chlorophyll contents also showed a non-significant association
with the POD activity.
3.10. Regression analysis
Physio-biochemical and enzymatic attributes parameters were also
tested using regression analysis (Fig. 7). The R2 values of regressions
were: protein contents and soil urease activity, 0.6664; amylopectin
contents and soil sucrase activity, 0.4905; MDA contents and total
The principal component analysis of measured data revealed three
groups of seedlings, budding and flowering stages with the different
passivation agents. A trend from the control sample (no-passivating
agent) to the application of highest number of passivators (sepiolite,
quicklime, calcium magnesium phosphate) is observed (arrow, Fig. 8).
Potato plants have the effective enzymatic antioxidant defense system
that is reflective of activities of catalase (CAT), peroxidase (POD), and
superoxide dismutase (SOD) at the flowering stage especially with the
application of quicklime treatment.
4. Discussion
Mineral nutrients found in soil, such as OM, N, P, and K, that are
crucial for plant growth. The amount of nutrients in soil with heavy
metal contamination has a significant impact on seed germination and
plant establishment (Yu et al., 2016). It is obvious from the findings of
the current study that all the soil applied passivating treatments and

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H. Wang et al. Science of the Total Environment 912 (2024) 168956

Fig. 4. Effect of various soil applied passivating agent’s treatments on tuber quality attributes of potato grown in contaminated soil. The statistics illustrate the means
with standard deviation (SD) and were replicated three times; the capital letters in the bars indicate significant differences between treatment means at p ≤ 0.05 using
highest significant difference test (HSD). ck = control (no-passivating agent); T1 = sepiolite; T2 = quicklime; T3 = calcium magnesium phosphate; T4 = sepiolite +
quicklime; T5 = sepiolite + calcium magnesium phosphate; T6 = quicklime + calcium magnesium phosphate and T7 = sepiolite + quicklime + calcium magne­
sium phosphate.

Fig. 5. Effect of various soil applied passivating agent’s treatments on metals accumulation in various plants of potato grown in contaminated soil. The statistics
illustrate the means with standard deviation (SD) and were replicated three times; the capital letters in the bars indicate significant differences between treatment
means at p ≤ 0.05 using highest significant difference test (HSD). Figure without the lettering represent the non-significant response; ck = control (no-passivating
agent); T1 = sepiolite; T2 = quicklime; T3 = calcium magnesium phosphate; T4 = sepiolite + quicklime; T5 = sepiolite + calcium magnesium phosphate; T6 =
quicklime + calcium magnesium phosphate and T7 = sepiolite + quicklime + calcium magnesium phosphate.

growth stages significantly improved the growth and photosynthetic
attributes of potato grown under metal contaminated soil (Fig. 1).
Agegnehu et al. (2015) and Melo et al. (2016) reported that due to
changes in soil pH, soil amendments result in enhanced plant absorption
of C, N, P, and K as well as increased soil availability of soluble organic
carbon (SOC). As a result, chlorophyll content and plant growth were
enhanced that might be due to the better availability of essential nu­
trients. The calcium supplied by limestone and superphosphate may also
help plants mount defenses because calcium reinforces the cell wall by
cross-linking pectin and takes part in defense signal transduction

7
H. Wang et al. Science of the Total Environment 912 (2024) 168956

Fig. 6. Effect of various soil applied passivating agent’s treatments on metals accumulation in various plants of potato grown in contaminated soil. The statistics
illustrate the means with standard deviation (SD) and were replicated three times; the capital letters in the bars indicate significant differences between treatment
means at p ≤ 0.05 using highest significant difference test (HSD). AT = after treatment; BT = before treatment; ck = control (no-passivating agent); T1 = sepiolite; T2
= quicklime; T3 = calcium magnesium phosphate; T4 = sepiolite + quicklime; T5 = sepiolite + calcium magnesium phosphate; T6 = quicklime + calcium magnesium
phosphate and T7 = sepiolite + quicklime + calcium magnesium phosphate.

Table 1
Correlation matrix of physio-biochemical and growth attributes of potato in response to various rates of soil applied passivating agents under greenhouse conditions
under metal contamination.
Carotenoids CAT MDA PH POD Protein SOD Soluble Sugar

CAT 0.30*
MDA − 0.03NS 0.56**
PH 0.86** 0.57** 0.0023NS
POD 0.36** 0.82** 0.76** 0.48**
Protein 0.86** 0.24* − 0.05NS 0.76** 0.30**
SOD 0.24* 0.87** 0.68** 0.46** 0.92** 0.16NS
Soluble sugar 0.84** 0.30** − 0.26* 0.92** 0.17NS 0.75** 0.16NS
Total chlorophyll 0.69** − 0.16NS − 0.57** 0.61** − 0.27NS 0.67** − 0.32** 0.82**

CAT, catalase activity; SOD, superoxide dismutase activity; POD, peroxidase activity, PH, plant height; MDA, malondialdehyde contents.
**
P ≤ 0.01.
*
P ≤ 0.05.
NS
Non-significant.

(Gordon et al., 2019).
Inorganic passivators contain large amounts of the nutrient Ca. It
contributes to around 0.1–5 % of dry plant biomass and is essential for
plant growth (Cui et al., 2019). Improved growth might be due to role of
calcium regulates membrane permeability and helps in nutrient uptake
and water movement within cells. For crop cell supersession, signal
shift, and nutrient uptake across cell membranes, it serves as the second
messenger in signal transition (Ahmad et al., 2016; Turan et al., 2018a).
In acidic soil, potato yield is frequently limited by a lack of an essential
nutrient (Ca) rather than a lack of other plant nutrients. Soil application
can improve plant stress resistance by increasing PSII efficiency and
increasing antioxidant enzyme activity (e.g., SOD and CAT), and
decreasing MDA content under stress similar observations were also
noticed in the current study (Hamid et al., 2018).
All the enzymatic antioxidants shower a linear decrease by the op­
timum use of passivators. However, under control condition opposite
trend was noticed. The activities of antioxidant enzymes, such as SOD
and CAT, in crops can alleviate oxidative stress and neutralize ROS
toxicity caused by metals (Salam et al., 2019). Elevated heavy metal
concentrations can lead to oxidative stress by promoting the generation
of ROS, which can damage cellular components and disrupt physiolog­
ical processes. Passivators’ ability to curtail metal uptake helps to
maintain the integrity of plant cell membranes, chloroplasts, and other
organelles, preserving their functionality and ensuring optimal photo­
synthesis and metabolic activity. It was previously mentioned that OM
and the main nutrients, such as N and P, are the main components which

8
H. Wang et al. Science of the Total Environment 912 (2024) 168956

Fig. 7. Scatter plots of measured parameters in the soil applied passivating agent’s treatments of potato grown in contaminated soil.

Fig. 8. Biplot of principal component analysis (PCA) showing clusters of measured data and variability accounting for 88.5 % in the potato plants experiments with
different treatments of passivation agents.

alter SOD and CAT activities (Zhu et al., 2016; Zubair et al., 2021). When transport nutrients and water through the xylem, it may also encourage
the crop is under metal stress, essential minerals, particularly K, might the involvement of numerous additional latent enzymes (Shahbaz et al.,
encourage the activity of these enzymes. Similar to how K helps 2019; Naciri et al., 2021). Therefore, with the application of passivating

9
H. Wang et al.
agents having these nutrients increased H2O2 detoxification due to
increased SOD and CAT activity (Mahar et al., 2015; Tauqeer et al.,
2022). Overgeneration of ROS induces production of higher MDA levels
(Al Mahmud et al., 2018). Previous studies have shown that passivators
mediated amendments significantly increased soil pH, alleviated the
toxicity of metals to the photosystem, boosted chlorophyll content, and
improved yields (Gao et al., 2012; Rasool et al., 2022). Passivators, often
in the form of soil amendments interact with heavy metal ions in the soil,
forming stable complexes and reducing their bioavailability for uptake
by plants that might be the possible reason for improved growth.
Addition of synergistic form of all three passivating agents linearly
increased the soil enzymatic attributes. Soil enzymes are produced by
soil microorganisms and are an important part of the soil ecosystem
(Prasad et al., 2021). By regulating microbial enzyme production,
conformational changes in the enzymes themselves brought on by
ionization, and the availability of substrates and enzyme cofactors, soil
pollution impacts the activity of soil enzymes (Puissant et al., 2019).
According to previous studies, several enzyme reactions are extremely
sensitive to variations in soil pH and can only take place within a specific
pH range (Turan et al., 2018b; Pokharel et al., 2020). Our studies
revealed that, in comparison to the soil control treatment, the applica­
tion of passivators dramatically boosted the activities of urease, phos­
phatase, and catalase to various degrees (Fig. 6). After the application of
passivators to the contaminated soil, soil urease activity rose most
noticeably. This might be because quicklime induces a quick rise in pH,
which might encourage soil microbial activity and bacterial abundance.
Other studies have discovered that the use of passivators can boost soil
enzyme activity and increase the nitrogen content and phosphorus
availability in acidic soils (Chang et al., 2007; Xun et al., 2016; Deng
et al., 2019; Lv et al., 2022). The activities of sucrase, phosphatase, and
catalase were all significantly impacted by the combined application of
passivators. Thus, it might have altered the rhizosphere’s habitat and
increased soil fertility to raise soil enzyme activity (Wang et al., 2010).
The addition of quicklime, superphosphate, or plant ash to the replanted
soil raises the soil enzymatic attributes (Zhao et al., 2022).
The adsorption and enrichment of metals by plants is related to the
bioavailability of heavy metals in soils (Khan et al., 2020). A significant
inverse relationship between the metal content of the potato’s above-
and below-ground portions was found in this study. The fall in soil pH
and metal bioavailability following the application of individual and
combined passivators may be the cause of this substantial association
(Wang et al., 2021). Additionally, there was a strong positive correlation
between the metal content of potato above and below ground and the
availability of metals in soils. The recent findings suggest that real metal
uptake by potatoes could be predicted using soil metal availability.
However, the presence of heavy metals in soils could not be used to
forecast rice’s absorption of heavy metals (Chen et al., 2019).
Incorporating passivators into potato cultivation practices not only
enhances enzymatic antioxidant systems but also contributes to the
production of safer, heavy metal-free potatoes for consumption. This
approach aligns with food safety standards and addresses concerns
about heavy metal accumulation in crops, ensuring that potatoes remain
a nutritious and reliable staple food. This finding could be explained by
the fact that quicklime and calcium magnesium phosphate can improve
soil environment, increase soil calcium and phosphorus content, pro­
mote plant growth, reduce ROS damage to chlorophyll molecules,
improve photosynthetic electron transfer and photosynthetic capacity,
lower metal uptake, and improve potato growth.
Passivators employed in the study may not protect against other
environmental elements such as pollution or agro industry-based
chemicals. In some cases, additional coatings (or treatments) may be
needed for the optimum application. Nevertheless, it is suggested that
there is need to optimize passivator formulations, application methods,
and dosage levels, with various slow released coating material to ensure
the efficacy of passivators while considering potential environmental
impacts and sustainable agricultural practices.
10
Science of the Total Environment 912 (2024) 168956
5. Conclusion
Soil applied passivators significantly improved soil health by
increasing soil enzymatic activity and reducing the metals accumulation
in potato tissues. Balanced and optimal combination of passivators
resulted in enhancement of carotenoids, chlorophyll contents and sol­
uble sugars and depicted strong positive correlation with each other and
negative association with MDA contents. Data of current research justify
the growth improving and stress ameliorative role of passivators in
potato under metal contaminated soil. By mitigating heavy metal
contamination, passivators not only enhance potato quality and yield
but also ensure the production of safer, heavy metal-free potatoes that
meet stringent food safety standards. This study provides an effective
and safe technical solution for the remediation of heavy metal
contaminated soil and has positive effects on food safety and soil health.
By effectively mitigating heavy metal contamination, this strategy
contributes significantly to SDG 2 (Zero Hunger) and SDG 1 (No
Poverty) by promoting safe and nutritious food production. However,
continued research efforts are crucial to optimize passivator formula­
tions, application techniques, and dosage levels to strike a balance be­
tween efficacy and environmental sustainability. In the face of
escalating concerns about metal contamination, the innovative use of
passivators offers a promising solution to enhance potato growth and
contribute to the sustenance of global food security.
Funding
This work was partially supported by the National Natural Science
Foundation of China (Grant number 32060499), Yunnan Fundamental
Research Projects (Grant number 202201AT070034 and
202201AT070150), Guangdong Major Project of Basic and Applied
Basic Research (Grant number 2021B0301030004), Open Research
Program (No. YNPKF202201) of Yunnan Key Laboratory of Potato
Biology, Yunnan Normal University and Xingdian Talent Support Plan.
The authors extend their appreciation to the Researchers Supporting
Project number (RSP2023R139) King Saud University, Riyadh, Saud
Arabia.
CRediT authorship contribution statement
Hongyang Wang: Investigation, Methodology, Writing – original
draft, Funding acquisition. Hao Liu: Investigation, Methodology,
Writing – original draft. Junhua Li: Investigation, Methodology.
Shengnan Chen: Formal analysis, Investigation. Qamar uz Zaman:
Data curation, Investigation, Writing – review & editing. Khawar Sul­
tan: Data curation, Investigation, Writing – review & editing. Muzam­
mal Rehman: Data curation, Investigation, Writing – review & editing.
Shah Saud: Data curation, Investigation, Methodology, Writing – re­
view & editing. Khaled El-Kahtany: Data curation, Investigation,
Methodology, Writing – review & editing. Shah Fahad: Data curation,
Investigation, Methodology, Writing – review & editing. Gang Deng:
Supervision, Methodology, Resources. Aie Chen: Supervision, Investi­
gation, Methodology, Conceptualization.
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 will be made available on request.
H. Wang et al. Science of the Total Environment 912 (2024) 168956

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