Chemosphere 262 (2021) 127810

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Review

Copper bioavailability, uptake, toxicity and tolerance in plants: A

comprehensive review
Vinod Kumar a, *, Shevita Pandita b, Gagan Preet Singh Sidhu c, Anket Sharma d,
Kanika Khanna e, Parminder Kaur f, Aditi Shreeya Bali g, Raj Setia h
a
Department of Botany, Government Degree College, Ramban, Jammu, 182144, India
b
Department of Botany, University of Jammu, Jammu and Kashmir, India
c
Centre for Applied Biology in Environment Sciences, Kurukshetra University, Kurukshetra, 136119, India
d
State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, 311300, China
e
Independent Researcher, House No.282, Lane no. 3, Friends Colony, Opposite DAV College, Jalandhar, 144008, Punjab, India
f
Independent Researcher, House No. 472, Ward No. 8, Dhariwal, Gurdaspur, 143519, Punjab, India
g
Department of Botany, Dyal Singh College, Karnal, Haryana, 132001, India
h
Punjab Remote Sensing Centre, Ludhiana, India

h i g h l i g h t s

Biogeochemical behaviour of Cu in soil-plant-human system was pre
cised.

Cu speciation and soil microbes oversee its biogeochemical behaviour in soil-plant system.

Cu incites copious harmful impacts to biochemical processes.

Plants endure Cu toxicity through various detoxification mechanisms.

a r t i c l e i n f o a b s t r a c t

Article history: Copper (Cu) is an essential element for humans and plants when present in lesser amount, while in
Received 21 May 2020 excessive amounts it exerts detrimental effects. There subsists a narrow difference amid the indis-
Received in revised form pensable, positive and detrimental concentration of Cu in living system, which substantially alters with
14 July 2020
Cu speciation, and form of living organisms. Consequently, it is vital to monitor its bioavailability,
Accepted 21 July 2020
Available online 1 August 2020
speciation, exposure levels and routes in the living organisms. The ingestion of Cu-laced food crops is the
key source of this heavy metal toxicity in humans. Hence, it is necessary to appraise the biogeochemical
Handling Editor: Lena Q. Ma behaviour of Cu in soil-plant system with esteem to their quantity and speciation. On the basis of existing
research, this appraisal traces a probable connexion midst: Cu levels, sources, chemistry, speciation and
Keywords: bioavailability in the soil. Besides, the functions of protein transporters in soil-plant Cu transport, and the
Copper detrimental effect of Cu on morphological, physiological and nutrient uptake in plants has also been
Bioavailability discussed in the current manuscript. Mechanisms related to detoxification strategies like antioxidative
Hyperaccumulation response and generation of glutathione and phytochelatins to combat Cu-induced toxicity in plants is
Speciation
discussed as well. We also delimits the Cu accretion in food crops and allied health perils from soils
Phytotoxicity
encompassing less or high Cu quantity. Finally, an overview of various techniques involved in the
Phytoremediation
reclamation and restoration of Cu-contaminated soils has been provided.
© 2020 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Copper in the environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

* Corresponding author.
E-mail address: vinodverma507@gmail.com (V. Kumar).

https://doi.org/10.1016/j.chemosphere.2020.127810
0045-6535/© 2020 Elsevier Ltd. All rights reserved.
2 V. Kumar et al. / Chemosphere 262 (2021) 127810

2.1. Occurrence, uses and sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2. Chemistry of copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3. Threshold intake limits of copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.4. Copper toxicity in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.5. Natural/background copper concentration in the soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Copper in food plants in relation to soil-plant-human transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4. Cu speciation and bioavailability in soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.1. Impact of speciation on Cu bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4.2. Effect of soil chemical properties on Cu bioavailability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.3. Effect of soil pH on copper bioavailability and speciation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4.4. Effect of soil microbes on copper bioavailability and speciation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5. Copper acquisition and translocation in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.1. Copper uptake by plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.1.1. P-type ATPase copper transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
5.1.2. COPT copper transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.1.3. ZIP family transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.1.4. NRAMP family transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.2. Copper translocation in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5.3. Copper sequestration in plant (roots/shoots) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6. Beneficial effects of copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7. Copper toxicity and tolerance in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7.1. Morphological parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7.2. Physiological parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
7.3. Nutrient uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
8. Copper detoxification and tolerance mechanisms in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
8.1. Antioxidative response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
8.2. Glutathione production in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
8.3. Phytochelatin production in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
9. Phytoremediation of copper . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
10. Management of copper-contaminated soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
11. Concluding remarks and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

1. Introduction et al., 2020a). In food crops the recommended value of Cu is

30 mg kg1 (Sharma et al., 2018). Additionally, Cu excess in plants
Copper (Cu) is the vital micronutrient for the plant growth and fosters oxidative stress by generating reactive oxygen species (ROS)
generally 5e30 mg kg1 Cu is considered as satisfactory in plant which are detrimental for the plants and these are scavenged by
tissues (Wuana and Okieimen, 2011). Copper shortage/excess has superoxide dismutase and peroxidase, which plays imperative
detrimental impact on the human health. The prescribed dose- functioning in decreasing Cu toxicity in the plants (Huang et al.,
response curve of Cu in humans is U-shaped (Stern et al., 2007). 2020). Oxidative stress through lipid peroxidation results in
The deficiency of Cu leads to severe disorders like anaemia and disruption of numerous metabolic pathways and causes harm to
neutropenia (Oliver, 1997), while it’s excessive level leads to liver essential macromolecules (Juang et al., 2019). Uptake and transport
and Alzheimer disorders, and may also cause nervous breakdown of Cu in plants depend predominately on Cu level and growth en-
(Uriu-Adams and Keen, 2005). The content of Cu depends on virons (Saleem et al., 2020b). Nevertheless, their concentration in
complex interfaces between parent materials, physio-chemical cells prerequisite to be kept at low level as high Cu persuade
characteristics and additions due to agronomic practices or in- amendments in DNA, photosynthesis, cell membrane integrity,
dustry. For example, Cu content reduces with high pH, soil organic enzyme activity and respiration leading to growth reduction and
carbon and clay content (de Vries et al., 2013). Globally, the normal imperilled existence of plants (Nair and Chung, 2015). The decline
values of Cu in uncontaminated soils varied from 2 to 109 mg kg1 in plant biomass is the most usual indication in plants under high
(Baize, 1994). The key anthropogenic sources of Cu include mining, Cu stress (Nazir et al., 2019).
refinery, fossil fuel combustion, waste incineration, traffic, fertil- The biogeochemical cycling of Cu in soil-plant-human system is
izers, soil amendments etc. (Brunetto et al., 2019). The mining of regarded as the base of Cu cycling in living organisms. However, the
Cu-containing ores and industrial activities are responsible for Cu concentration in plants parts is allied with the soil Cu concen-
enhancing Cu level in the environment. Kumar et al. (2019a) sug- tration (Fig. 1). Consequently, it is vital to observe Cu concentration
gested 20 mg kg1 permissible limit for Cu in soil samples. In in soil and its transfer to plants and humans system. Most of the
natural topsoil the background level of Cu reported is 5 mg kg1 studies revealed that Cu2þ and Cuþ are the most abundant forms of
(Ferreira et al., 2018). Cu for plant uptake, and their mobility is reliant on pH, organic
Copper also plays a key function in CO2 assimilation and ATP matter etc. (Amery et al., 2008). Now it is recognized that transfer of
production (Marques et al., 2018). It is the main constituent of metal from soil to root/shoot/grains and finally to humans/animals
diverse proteins like plastocyanin of photosynthetic system and is regulated by speciation of metal and its numerous detoxification/
cytochrome oxidase of electron transport chain (Zeng et al., 2019). tolerance approaches operative inside the plants (Pourrut et al.,
Cu concentration in plants beyond critical limits affects the plant 2011; Khalid et al., 2017). Consequently, in addition to appraising
growth, promotes leaf chlorosis and causes cytotoxicity (Saleem Cu toxicity or essentiality in living organisms and threshold levels
 V. Kumar et al. / Chemosphere 262 (2021) 127810
in routine diet, it is extremely imperative to explicate all the ap-
proaches/mechanisms influencing soil-plant transfer (Rafiq et al.,
2018). The current review provides the comprehensive state-of-
the-art about the Cu status in the environment, bioavailability
and speciation, toxicity, uptake, hyperaccumulation and detoxifi-
cation mechanisms in plants. Threshold levels and relation to soil-
plant-human transfer will also be appraised.
2. Copper in the environment
2.1. Occurrence, uses and sources
Copper is an essential micronutrient with atomic number 29
and atomic weight 63.55. It is a d-block transition metal with
electronic configuration [Ar] 3 d10 4s1 and belongs to group XI and
period IV of the periodic table. Cu finds its role in various metabolic
and physiological processes in plants and animals. It is an essential
component of many enzymes such as cytochrome oxidase, nitrate
reductase etc. At room temperature its density is more than
5 g cm3 (i.e., 8.96 g cm3), so it is classified as a heavy metal.
Various characteristics of soil like pH, type of soil, organic matter
content, cation exchange capacity and soil texture affects Cu con-
centration and its distribution in soil (Wu et al., 2010a, 2010b). The
normal concentration of Cu in soil ranges from 2.0 to 100 mg kg1
(Willard, 1979). In United States, Cu is present in the soil at the
concentration range of 5e70 mg kg1 (ATSDR, 2017).
Copper sulphate (CuSO4) is the most common naturally occur-
ring compound of Cu. It is used in preservation of wood, in agri-
culture as fungicides and in leather industry. Cu is widely used as a
building material, constituent of many alloys like sterling silver,
cupronickel and constantan that are used in jewellery, coins and in
making gauge for instruments. Moreover, Cu finds its usage in
Fig. 1. Biogeochemical cycle of Cu in environment (soil-plant-human). (a) Different sources responsible for copper in the environment, (b) copper transporter like COPT, HMA6/8,
ZIP family, Nramp are responsible for Cu uptake and transport in the plants, (c) reduction of Cu(II) to Cu(I) by ferric reductase oxidase, (d) detrimental impact of Cu(II) on plant
morphology and physiology, (e) Cu(II) induces geneotoxicty by breaking DNA strands and oxidative dna damage, and (f) impact of Cu on humans by causing Wilson’s disease.
3
dental products and cosmetics. It has many applications owing to
its distinctive electricity conducting properties, which also makes it
challenging to replace. It may become more important for the
people due to probable upsurge of copper-intensive low carbon
energy and electrification of transport technologies (Schipper et al.,
2018). The growing requirement causes evils and can contribute to
environs problems. For instance, reducing ore grades consequence
in greater energy demands for the similar quantity of copper
extraction, so growing the greenhouse gas releases (Memary et al.,
2012; UNEP, 2013).
Natural sources of Cu in soil include parent soil material,
weathering of rocks and soils, volcanoes, forest fires and many
disturbances in soil. High concentration of Cu is carried by wind in
the form of dust or copper particulate matter from copper in-
dustries and gets settled in the soil. Sewage sludge from treatment
plants is another source of Cu in soil. Beside natural sources, large
amount of Cu is added in the soil from various anthropogenic
sources like mining, smelting, industrial and agricultural waste and
agrochemicals (Wu et al., 2010a, 2010b). Rapid industrialization
and urbanization have increased Cu levels in soil profile and sedi-
ments. Application of inorganic fertilizers and manure in agricul-
ture adds high content of Cu in soil (Dogra et al., 2020). Fig. 2
depicts the Global production and distribution of Cu in mine tail-
ings of various countries (USGS, 2016).
2.2. Chemistry of copper
Copper is a soft, malleable, ductile metal with melting point
1084.62  C and boiling point 2562  C. It reacts with atmospheric
oxygen to form copper oxide; however, in moist air Cu slowly forms
copper carbonate (Martınez and Motto, 2000). Cu readily gets
dissolves in ammonia to form cupric ammonium complex ion in the
4 V. Kumar et al. / Chemosphere 262 (2021) 127810
presence of air (Zhang and Richardson, 2000). Cu exists in two
oxidation states viz. þ1 (Cu (I), cuprous ion) and þ2 (Cu (II), cupric
ion (Burdett and Sevov, 1995). The most common oxidation state of
copper is Cu (II) or cupric ion that form co-ordination bond with six
water molecules in solution. The complexes or compounds of Cu (II)
are blue or green in colour. Cu exists as free ions, exchangeable,
organic, precipitated and residual forms in the soil (Shuman and
Hargrove, 1985). Cu has high affinity for organic matter, leached


relatively less into the soil, and thus accumulates in the soil (Sauve
et al., 1997). Continuous accumulation of Cu in the agricultural soils
increases phytotoxicity and poses havoc to food safety (Wu et al.,
2010a, 2010b).
Typically Cu encompassing enzymes like cytochrome c oxidase,
NADH dehydrogenase-2, and tyrosinases reside in the cytoplasmic
membrane or periplasm, while Cu-chaperone proteins like Atx1
and Ccs identified in yeast. Atx1 intermingles directly with Ccc2, a
P-type Cu-transporting ATPase like to that occurred in bacterial
cells and pumps Cu into the lumen of the secretory compartment
where it is fused with ligands of secreted Cu metalloproteins. Atx1
is structurally analogous to the cytoplasmic Cu binding domains of
Ccc2, giving alike protein surfaces to facilitate Cu allocation amid
two proteins (Festa and Thiele, 2011). The Ctr family of importers
have distinguishing methionine (met) rich amino termini which
bind with extracellular Cuþ and augmenting the local level of Cu.
Moreover, there is MxxxM motif within the second of its three
transmembrane domains, with the met probably effective in Cuþ
binding during translocation athwart the membrane (Puig et al.,
2007). Aspartic acid, histidine, glutamic acid and cysteine are the
strong amino acid residues of Cu and these are position conserva-
tion of amino acid residues in a good display of protein-metal ion
binding (Cao et al., 2017). Wu et al., 2010a, 2010b reported in their
study that cysteine side chains are vital Cuþ ligands and these metal
ion-ligand interactions have a strong impact on the disintegration
chemistry of Cuþ ions.
2.3. Threshold intake limits of copper
Copper induces positive effect at sufficient quantity, while have
adverse impact at high quantity. So, diverse national and interna-
tional health organizations have suggested its minimum, suitable
or maximum intake quantity through food or its supplements. The
lesser threshold ingestion is the lowest appraisal of ultimatum from
the normal distribution curve, and is commonly enumerated based
on average necessities minus twice its SD (Strand et al., 2017). This
can encounter the demand of 2.5% of the individuals in the in-
habitants (Strand et al., 2017). Tolerable upper intake level is the
highest amount of total chronic daily intake of metal from all
sources arbitrated to be implausible to pose a peril of detrimental
health impacts to humans. The average Cu intakes through food in
Fig. 2. Global production of Copper in mines.
adults of European countries varied from 1 to 2.3 mg/day for males
and 0.9e1.8 mg/day for females (Van Dokkum, 1995). The intake of
Cu in adults from omnivore diet ranged from 1 to 1.5 mg/day, while
in vegetarian diet Cu intake varied from 2.1 to 3.9 mg/day (Gibson,
1994). The Finnish and Swedish legislations for soil contamination
are the utmost cited recommendations and threshold standard for
Cu (MEF, 2007). As recommended by the Government Verdict on
the appraisal of Soil Contamination and Remediation Needs 214/
2007 (MEF, 2007), Cu threshold value is 100 mg kg1 and the
guideline value is 150 mg kg1. The threshold is the level which
requires further appraisal in the site, whereas guideline value
represents the ecological or health peril (To
th et al., 2016). The
threshold of 100 mg kg1 is recommended to indicate Cu unpol-
luted soils (Sharma et al., 2020). 5e30 mg kg1 is considered as
ideal range of Cu in croplands (Adrees et al., 2015). Table 1 shows
the outline of upper levels of Cu in adults set by various authorities.
Table 2 depicts US and European Union oral dietary reference
values for copper.
2.4. Copper toxicity in humans
Copper is an essential nutrient that is an important constituent
of various metalloenzymes responsible for carbohydrate meta-
bolism, haemoglobin formation and various other metabolic ac-
tivities. Due to anthropogenic activities, the concentration of Cu has
increased many-folds in the environment and is hazardous to living
biota especially humans. Elevated Cu concentration leads to various
hepatic and neurodegenerative disorder due to disturbed homeo-
stasis of Cu in body (Gaetke et al., 2014). Cu dust from smelters and
refineries is inhaled by workers or population growing nearby that
cause severe health effects such as anaemia, damaged liver and
kidney (ATSDR, 2017). Moreover, in Wilson’s disease, an autosomal
recessive genetic disorder, exposure to higher Cu level alters the
structure and function of liver and brain due to accumulation of Cu
in these organs (Wilson and Pyatt, 2007). Cu toxicity in humans
might be due to the increased oxidative stress combined with
pronounced disturbances in the endocrine system (Uriu-Adams
and Keen, 2005). According to the report of World Health
Organisation (2017), excessive consumption of Cu in beverages
and drinking water resulted in nausea, vomiting and diarrhoea in
humans. Winge and Mehra (1990) documented that early stage Cu
poisoning causes weakness, lethargy and anorexia in humans and
later on it damages the gastrointestinal tract and causes necrosis in
the kidney (Barceloux and Barceloux, 1999). Desai and Kaler (2008)
pointed that Cu excess in human body results in mitochondrial
damage, breaks in DNA, brain injury and various other neurological
disorders. Finally we can accomplish that the excess level of Cu is
harmful for the health of human beings.
2.5. Natural/background copper concentration in the soil
Copper exist naturally in the soil environs with mean value of
about 30e35 mg kg2 dry soil, while in the earth’s crust the average
value of Cu concentration is 60 mg kg2 (Kupiec et al., 2019). In soil,
it is bounded to various fractions owing to cation exchange, pre-
cipitation or adsorption, and however the stability depends upon
pH (Young, 2013). The background level of Cu varies from 2 to
50 mg kg2, and it depends mainly on geology of soil (Oorts, 2012).
Their background content in parent rocky materials and soils is
related with organic matter and texture. Soils comprising high
percentage of clay and organic matter usually have greater back-
ground concentration of Cu. Copper has two stable isotopes such as
63
Cu and 65Cu with twenty seven radioisotopes. The utmost stable
radioisotope is 67Cu, whereas least stable radioisotope is 54Cu with
half-life of 61.83 h and 75 ns respectively. Unstable Cu isotopes with
 V. Kumar et al. / Chemosphere 262 (2021) 127810 5

Table 1
Summary of upper levels in adults set by numerous authorities.

UL (mg/day) NOAEL (mg/day) LOAEL (mg/day) UF References

10 10 30 1 IOM (2001)
5 10 30 2 SCF (2003)
10 16 10  10 (rats) EVM (2003)
5 10 1 (human) NNR (2012)

Where UL (tolerable upper intake level); NOAEL (no observed adverse effect level); LOAEL (lowest observed adverse effect level) and UF (uncertainty factor).

Table 2
US and European oral dietary reference values for Cu.

Group and age Recommended oral dietary intake (mg Tolerable upper intake level (mg Recommended oral dietary intake (mg Cu/ Body weight
Cu/day) Cu/day) kg BW/day) (kg)

Infant (0e12 months)1 0.20e0.22 e 0.020e0.022 10

Infant (7e11 months)2 0.4 e 0.04 10
Children (1e8 years)1 0.34e0.44 1e3 0.034e0.044 10
Children (9e13 years)1 0.70 5 0.02 37.5
Children (1 to <3 years)2 0.7 e 0.07 10
Children (3 to <10 years)2 1 e 0.01 10
Boys, girls (10 to <18 years)2 1.3, 1.1 e 0.02 53.9
Adolescents (14e18 years)1 0.89 8 0.02 53.9
Adults, both genders (19e70þ 0.90 10 0.013 70
years)1
Men, women2 1.6, 1.3 e 0.02 70
Women, pregnant lactating (14 1.0, 1.3 8e10 0.014e0.019 70
e50 years)1
Adult, pregnant and lactating 1.5 e 0.02 70
women2
IOM (2001)1; EFSA (2015)2

atomic masses less than 63 undergo bþ decay, whereas isotopes
with atomic masses greater than 65 undergo b decay. 64Cu decays
both by bþ and b (Audi et al., 2003). The influence of soil parent
material and texture also exhibits in diverse Cu content through
main soil types. Soil types like vertisols, histosols and spodosols
have greatest Cu amount (Oorts, 2012). The background concen-
tration of copper in parent materials and soils are given in Table 3.
crops (Rai et al., 2019). Health hazard indices are used to appraise
the health perils results from the ingestion of food crops contam-
inated with heavy metals (e.g. Cu). If value of Cu for hazard quotient
were found above one in food crops, it signifies detrimental influ-
ence on the human health (Kumar et al., 2019a, b). Finally, we can
conclude that if hazard quotient value of Cu was found above one,
then it poses harmful impact on the human beings, and if found less
than one, then it poses less harm to the human health.

3. Copper in food plants in relation to soil-plant-human

transfer 4. Cu speciation and bioavailability in soil

Certain natural and man-made activities like deposition of solid/
liquid wastes, agricultural practices, bedrock, urban or industrial
wastes spoil the land by addition of heavy metals like Cu (Wilson
and Pyatt, 2007). Deposition of high doses of metals on the land
contaminates the soil and food crops cultivated on these soils, en-
ters into the living organism through their consumption. Due to
aerial load, vegetables are the most visible food among food system,
in the environmental pollution (Lokeshappa et al., 2012). Vegeta-
bles uptake heavy metals like Cu and stored them in their edible as
well as non-edible parts in excessive amounts, hence leads to
health problems in both humans and other animals via consump-
tion of these vegetables (Maleki and Zarasvand, 2008). The heavy
metals like Cu in soil can accrue in food crops and are transported to
other media via the food chain. The bioaccumulation factor (BCF) of
heavy metals in soil-food crops system, mainly in foremost staple
crops like wheat and rice grains are well studied (Wang et al., 2017).
Table 4 enlisted the copper contamination in various food crops.
The consumption of vegetables contaminated with metals leads
to severe health complications like gastrointestinal cancer, mental
growth retardation and malnutrition (Dickin et al., 2016; El-Kady
and Abdel-Wahhab, 2018). The soil-plant transfer factor of metals
is a vital measure to appraise the health issues throughout the
globe (Woldetsadik et al., 2017). Furthermore, the human health
perils are nearly allied with the intake of metal-contaminated food
Speciation is the identification and quantification of the diverse,
definite species, types or stages in which a metal arises, whereas
bioavailability is the portion of total quantity of metal accessible to
an organism for uptake/adsorption across its cellular membrane.
The speciation and bioavailability inclusively appraises the physi-
ological and detrimental impact of metal on the biological systems
(Cui et al., 2020).
4.1. Impact of speciation on Cu bioavailability
Copper has been known to be the vital mineral nutrient for soil
organisms at optimum concentrations (Eriksen et al., 2001). It is
primarily as well as secondarily found in different minerals (Young,
2013). Although, they are necessary in trace levels for plants as a
micro-nutrient, but they can be accumulated in plants at higher
toxic levels due to anthropogenic sources (Uriu-Adams and Keen,
2005). Cu is associated with many key enzymes and various tran-
scription factors that are important in maintaining cellular integ-
rity, signalling processes, cell proliferation, oxidation/reduction
reactions and defence pathways (Kardos et al., 2018). It is the
speciation that can determine the levels of copper in environment,
whether they are biologically available or unavailable or in toxic or
non-toxic range (Montenegro et al., 2015). Moreover, there is a
direct correlation between Cu levels and its bioavailability in soils,
6 V. Kumar et al. / Chemosphere 262 (2021) 127810

Table 3
Background concentration of copper in parent materials and soils.

Cu (mg/kg)

Average Median Range References

Earth’s crust 60 e e Sparks (2002)

Igneous rocks e e 30 to 160 Nagajyoti et al. (2010)
Basic igneous rocks e e 4 to 30
Acid igneous rocks
Sedimentary rocks e e 18 to 120 Nagajyoti et al. (2010)
Shales and clays e e 20 to 200
Black shales
USA, agricultural soils 29.6 18.5 0.6 to 495 Holmgren et al. (1993)
Indian agricultural soils 48.70 e 0.98 to 218 Kumar et al. (2019a)
Indian roadside soils 42.25 e 0.22 to 202.24 Kumar et al. (2019a)
Indian industrial soils 72.49 e 5.4 to 193 Kumar et al. (2019a)
China soils 23 Chen et al. (1991)
Europe, natural topsoil (XRF) 0.8 to 256 13 17.3 Salminen (2005)
Europe, natural topsoil (AR) 1.0 to 239 12 16.4 Salminen (2005)
Europe, natural subsoil (XRF) 0.9 to 125 13.9 17.2 Salminen (2005)
Europe, natural subsoil (AR) 1 to 118 13 16.5 Salminen (2005)

Table 4
Copper contamination from varied sources in food crops.

Food crops Country Sources of heavy metal Concentration of Copper recorded in food crops References
contaminants affecting food (mg/kg*)
chains

Brassica sp., Chenopodium sp., leafy India Sewage seepage 1.7e12.9* Rattan et al. (2005)
and root vegetables, grains
Grain, maize green cabbage, China Sewage seepage 0.16e0.85* Khan et al. (2008)
Radish. turnip, spinach, cauliflower,
and lettuce
Brassica sp., food grains, and leafy China Both sewage and industrial 0.15e0.86* Liu et al. (2005)
vegetables leftover
Rice Australia Arsenic- and metal polluted 1.0e9.4* Rahman et al.
groundwater (2014)
French beans, beetroot and kale Australia Urban stormwater 0.016e0.66* Tom et al. (2014)
Spinach India Sewage wastewater 0.09* Chary et al. (2008)
Radish China Ineffectively treated 0.34* Song et al. (2009)
wastewater
Industrially processed food stuffs United States of America Industries/food processing 0.01e6.44a Gonzalez-Martin
(USA), Spain, Portugal, industries/modern et al. (2018)
Belgium, England, and pesticides based agriculture
Chile
Potato/other foodstuffs Egypt Ineffectively treated 0.83* El-Kady and Abdel-
wastewater Wahhab (2018)
Potato China Ineffectively treated urban 1.03* Song et al. (2009)
wastewater
Radish India Diverse pollution sources 5.96* Arora et al. (2008)
Cauliflower China Urban wastewater 0.6* Song et al. (2009)
Amaranthus India e 1.4 mg kg1 Chary et al. (2008)
Food crops (wheat grains, mustard India e Wheat grains (0.32e21.50), Sharma et al. (2018)
grains, rice and maize grains) Mustard grains (1.56. e 17.98), rice grains (19.30
e167.41), and maize grains (8.62e83.84)*
a
ppm

indicating its direct influence on bulk speciation and ecotoxicology
(Cuypers et al., 2013). Copper can form complexes with both
organic and inorganic ligands (iron and manganese oxides) through
cation exchange, precipitation, biosorption or adsorption in order
to reduce the magnitude of free metal ions than entire metal con-
taminants (Oorts et al., 2012; Cui et al., 2017). The process of Cu
binding to various different fractions is mainly dependent of pH in
order to maintain their stability (Young, 2013). However, the free
metal ions are more reactive than the complexed forms, thereby
found to be mostly available in their biological forms (Cui et al.,
2019). In general, Cu is present predominantly in soils in the form
of Cu (II) in natural organic forms or in association between organic
and inorganic forms, or to Fe oxides (Fang et al., 2009). Cu also
binds to inorganic forms via clay such as montmorillonite kaolinite,
illite, ashes, Fe oxyhydroxides and carbonates (Lu et al., 2017). Soil is
a dynamic environment for Cu speciation, different physicochem-
ical and biochemical parameters affecting the metal equilibrium
are altered due to change in pH, organic matter concentration, ionic
levels etc. thereby significantly affecting the Cu distribution be-
tween different phases (Nielsen et al., 2015). Even though,
bioavailability is dependent on plant or animal species but in most
cases it is found that the labile forms like soluble and exchangeable
forms Cu (II) are readily available in comparison to stable forms or
precipitated ones (Montenegro et al., 2015).
Numerous studies have been explored that have quantified the
adsorption and bioavailability of Cu in soils of different types (Soler-
Rovira et al., 2010). It has been earlier revealed that the bioavail-
ability of Cu is directly proportional to the Cu speciation in soils,
 V. Kumar et al. / Chemosphere 262 (2021) 127810
therefore the determination of Cu forms is more relevant rather
than the total measurement of all the metal ions present within the
soil (Bourgeault et al., 2013; Nielsen et al., 2015). Among all, the
sequential selective extraction process has been projected to
measure speciation of sorbed Cu particles among different soil
fractions (Hass and Fine, 2010). These methods are dependent on
treatment of samples sequentially with different chemicals/re-
actants in order to increase their reactivity to selectively remove or
dissolve the sorbed species adsorbed with the solid phase
(component) followed by the desorbing using specific adsorbate
(Montenegro et al., 2015). These steps are used to remove the
pollutants associated with the soil particles and based upon the
nature and soil composition, it is impossible to prevent that the
smallest fraction of analyte separated by reactant might be bound
to highly stable soil particles (Peng et al., 2016). Consequently,
various steps involved in sequential selective extraction cannot be
counted as quantitative, however, sequential selective extraction
are otherwise useful in studying the speciation of Cu in soil.
4.2. Effect of soil chemical properties on Cu bioavailability
Even though, Cu levels in soil range between 3 and 100 mg kg2,
only 1e20% is bioavailable, whereas maximum is bound as organic
complexes (Adrees et al., 2015). Cu in soil is found in solid as well as
liquid phase. In case of solid phase, Cu being water soluble form
complexes with secondary minerals (clays and metal oxides/oxy-
hydroxides), organic materials and primary minerals (silicates,
carbonates and phosphates) (Giacalone et al., 2005). The avail-
ability of Cu in the soil is dependent on number of factors such as
soil type and its morphological and chemical properties (Zhao et al.,
2018). Furthermore, Cu retention time in soil is also dependent
upon the particle size, soil aggregates and many other soil factors
(Wang et al., 2015). In addition, Cu availability is also regulated by
total Cu concentration, organic matter, pH, moisture and cation
exchange capacity of soil (Wu et al., 2010a, 2010b). On the other
hand, the Cu (II) concentration in soil is increased when the soil pH
falls down followed by the reduction in the adsorption capacity of
Cu (Cui et al., 2020). Furthermore, Cu bioavailability is more in-
clined by rhizospheric pH compared to soil pH in Cu-contaminated
soils (Chaignon et al., 2009). Also, the dissolved organic matter
along with rhizospheric pH alters Cu dynamic speciation more than
inorganic material present in soil (Bravin et al., 2012). However,
plant roots have been known to plays a crucial role on Cu
bioavailability but more or less it also depends on the species of the
plant and concentration of Cu present in soil (Yruela, 2009).
Various studies revealed the influence of Cu bioavailability on
physical, chemical and biological properties occurring at soil-root
interface in rhizospheric region. The different properties such as
soil type, pH, organic matter, clay particles, soil moisture, temper-
ature, permeability, retention and presence of different metal ions
and their oxides in soils have been known to influence Cu
bioavailability in soils (Hinsinger et al., 2009). It is well known from
previous studies that soil organic matter having higher negative
charge and cation exchange capacity have a huge impact on
elemental distribution, especially Cu in soil profile (Zhang et al.,
2020). Moreover, the binding of Cu to kaolinite was also triggered
by alginic and humic acid (Beiyuan et al., 2017). Several studies
illustrated the retardation of Cu ions in ultisols and oxisols were
positively regulated by organic matter, pH, clay and Fe oxides (De
Matos et al., 2001). Cu has been mainly entered into the soils
through different fungicides and known to be however, sorbed to
organic matter occurring between soil aggregates and Fe oxides
(Antoniadis et al., 2017). The binding of Cu (II) to dissolved organic
matter in soils is also an important aspect in regulating the
chemical speciation, bioavailability and toxicity of Cu (II) (Fulda
7
et al., 2013). In addition, the behaviour and availability of Cu ions
also varies between the different soil types. The metal ion have
been affecting different soils such as oxisols, ultisols and alfisols
and oxisols were found to reach the critical point load to the earliest
(Medas et al., 2015). The higher absorption coefficient of clay par-
ticles with metal ions particularly Cu are mainly attributed to more
surface negative charge intensity (Rieuwerts, 2007). The bioavail-
ability of Cu is very much dependent on soil properties as suggested
through their associations with different types of metal oxides,
organic matter and clay particles. Furthermore, the contamination
of soils with Cu has also been influenced with the type of soils, their
pH along with the higher concentrations of Al and Fe (Zhang et al.,
2018a). Finally we can accomplish that soil properties mainly soil
pH and organic carbon has great impact on the bioavailability of Cu.
4.3. Effect of soil pH on copper bioavailability and speciation
pH has a significant role in effecting the biosorption/sorption of
different metal ions existing in soil (Rieuwerts, 2007; Rutkowska
et al., 2013). Gao et al. (2019) see the impact of pH, organic mat-
ter and moisture contents of copper oxide nanoparticles and
observed that soil pH and organic matter have great influence on
their dissolution behaviour, while moisture content has insignifi-
cant impact on the dissolution behaviour. Lower pH is shown to
reduce the sorption while increasing in the bioavailability and
mobility of different metal ions and vice-versa. Relatively, very few
studies have been reported with this aspect and further this area
needs to be explored. For instance, a study depicted that Cu ions
when added to 21 different types of samples in A, B and C horizons
within United states region showed variation in exchangeable Cu
ions with respect to pH in all the three horizons (A, B, C) (King,
1988). This variation in the pH is attributed to soil properties
such as soil texture, particle size etc. Moreover, a study of Appel and
Ma (2002) demonstrated that in ultisol and oxisol varieties of soil
showed higher metal adsorption with increased soil pH. According
to their findings, the point of zero net charge for ultisol and oxisol
were 2.3 and 3.5, while oxisol retained the trace metal amounts
below point zero net charge revealing the inner sphere adsorption
or complex formation towards permanent charge sites. However,
distribution coefficient (Kd) values for metal ions have also been
known to get effected between 4.4 and 7.3 pH range in lateritic soil
(Twining et al., 2004). The metals were considerably noticed to get
mobilise under lower pH range and higher oxidising state of soils
(Gnandi and Tobschall, 2002). One more aspect showed that ni-
trogen fertilization has contributed towards decreased soil pH, due
to nitrification of NHþ 4 ions that further maximised the levels of Cu
(II) concentration in soils. The application of long-term farmyard
manure also induced the levels of organic matter in soil thereby
enhancing the levels of metal-organic complexes mainly Cu in soil
solution (Cances et al., 2003).
Very few reports on least effect of pH on metal sorption in
tropical soils have also been investigated. The studies showing the
influence of soil characteristics on cation adsorption in different
soils have also been explored, where least correlation of Cu with pH
have been reported (Gomes et al., 2001). Similar to this study, Cu
concentration in 57 different types of Indian soils including oxisols
and ultisols have been found to be correlated with clay particles
present in soil irrespective of the pH or other soil properties (Katyal
and Sharma, 1991). Another study realized by De Matos et al.
(2001), investigated that reduction in the concentration of Cu in
ultisols and oxisols were significant and positively allied with
cation exchange capacity and Ca2þ content in the soils and no
correlation was existed with soil pH. Although, the reduction was
significantly elevated when the soils were limed that can be
attributed to precipitation at maximum pH value. Therefore, we can
8 V. Kumar et al. / Chemosphere 262 (2021) 127810
emphasise that soil pH may or may not necessarily support the
greater sorption capacity of different metal ions (particularly Cu), so
the sorption abilities may vary with the specific region and soil type
and environmental conditions.
4.4. Effect of soil microbes on copper bioavailability and speciation
Copper bioavailability and speciation has led to impending risk
to soil and its fertility along with the microbe-mediated soil pro-
cesses such as mineral cycling, making it an essential aspect to
measure Cu toxicity in soil (Cao et al., 2020). In this dilemma, there
are very few alternatives left for Cu fungicides, but the system
mainly relies on beneficial micro-organisms that form associations
at plant-soil interfaces. It is reported that Cu toxicity causes
antagonistic effect on soil microbiota, declining the biomass, car-
bon levels, respiration rate, microbe activities and alterations in the
metabolic quotient and structural community of microbes (Zhang
et al., 2016; Li et al., 2015). But on the contrary, there are many
microbes that have well developed resistance mechanisms against
heavy metal stresses and are able to multiply and proliferate under
higher Cu concentrations (Naik and Dubey, 2013) possibly due to
their capability to mobilise, convert or sequester the metal ions
affecting their mobilities (Keiblinger et al., 2018). Moreover, they
can also affect metal speciation, bioavailability through effective
production of organic matter and oxidation/reduction reactions
(Cui et al., 2019).
Studies were found in which Bacillus subtilis DBM strain isolated
from paddy fields reduced the bioavailability of Cu from soil
through biosorption, bioaccumulation or enzymatic transformation
of Cu ions into stable forms (Bai et al., 2017). The interactions of
microbes in soil and heavy metals are very complex, in which
nearly 90% of total soil biomass constituted of mineral nutrients
that add to the inorganic constituent of the soil (Bai et al., 2019). It
has been noticed that 90% of the soil microbes form complexes with
the minerals and organic matter and the adsorption of metal ions
with these complexes enable to understand the role of immobili-
sation (Nannipieri et al., 2017). The microbes contain diverse
functional sites on their surfaces such as carboxyl, amide, amine,
hydroxyl, exopolysaccharides (EPS), proteins, nuclic acids, poly-
saccharides to bind and form complexes through hydrogen bonds
(Fang et al., 2012; Shehata et al., 2019).
Recently, there is wide range of studies associated with the
microbe-mediated transformation of Cu in rhizospheric soils. As
the studies associated with Cu accumulator have been widespread,
the potential of Elsholtzia splendens as a Cu hyperaccumulator and
Trifolium repens as a non-hyper accumulator have been conducted
in association with soil microbial activities and their composition
(Wang et al., 2008). The Cu levels in shoots of Elsholtzia splendens
were noticed to be 2.1e2.4 times more than that of Trifolium repens
under the influence of different Cu concentrations. The total soil
microbiome composition and phosphatase enzyme activity in the
rhizospheric zone was reported to be maximum in Elsholtzia
splendens than that of Trifolium repens. It was further verified using
PCR-denaturing gradient gel electrophoresis (PCR-DGGE) finger-
print examination that assessed that after the addition of Cu
(200e500 mg kg1) in soil with Elsholtzia splenden, the number of
bands was considerably lowered. In comparison to non-
contaminated soils, microbes belonging to genera Firmicutes was
present only in Elsholtzia splendens rhizosphere of Cu-
contaminated soils and a very few species such as Deinococci-
Thermus was observed, indicating that Cu pollution altered the
microbial communities in the rhizosphere (Shi et al., 2018). How-
ever, the spatial arrangement of microbial communities were
studied in different soils through real time PCR by studying the
gene expression levels of flagellin (fliC) and chemotaxis gene (cheA)
and their expression levels were found to be altered by Cu toxicity
in different zones of soil (Yuan et al., 2014). Elsholtzia splendens as a
hyperaccumulator governs microbial activity and bacterial
composition in the rhizospheric zone under Cu toxicity. Further-
more, Cu-resistant bacteria Pseudomonas putida CZ1 isolated from
Elsholtzia splendens rhizosphere showed higher MIC (minimum
inhibitory concentration) in the presence of Cu (3 mmol l1) and
was able to remove 87.2% Cu with biosorption capacities
(24.2 mg l1) (Chen et al., 2005). The optimum pH for removal of Cu
(II) was 4.5e5.0 and under these conditions Cu (II) biosorption was
enhanced in the dose dependent manner. About 50% of Cu was
taken up bacterium, while the rest of it was passively facilitated
into the bacterium. In addition, desorption efficiency of Cu (II) was
72% in case of living cells via intracellular accumulation (Chen et al.,
2005). The binding ability of Cu by bacterium was favoured by
chemical modifications of soil biomass as studied through infrared
and X-ray absorption analysis ((Kumari et al., 2017; Lin et al., 2020).
Finally, we can reveal that microbial communities residing within
the rhizosphere plays an essential role in the regulation of Cu
motilities in the soil ecosystem. Table 5 showed the microbial
induced copper tolerance in plants.
5. Copper acquisition and translocation in plants
Plants uptake metal ions from the soil matrix through epidermal
root cells and then transfer to its center through parenchyma,
endodermis and xylem. This one way uptake of transition metals is
supported by various heavy metal transporters which works in co-
ordination with other metal transporter molecules (sequesters/
chelators) for proper uptake and translocation of minerals in all the
tissue during plant metamorphogenesis. Fig. 3 showed the uptake
and sequestration of Cu in plant cells through different transporters
and Cu chaperons.
5.1. Copper uptake by plants
The acquisition of Cu by the roots is similar to the mechanism
involved for the uptake of Fe and suggests that its uptake from roots
involves reductive Cu (I) uptake mechanisms at root cell surface
from Cu (II) (Printz et al., 2016). In Arabidopsis SPL7 mutants, the
activity of Cu (II) chelate reductase was up regulated at the root
membrane under Cu deficit conditions which was mediated by
SPL7. Moreover, it was found that the enzyme Cu (II) chelate
reductase was encoded by ferric reductase oxidase (FRO4/5) which
regulates the reduction of cupric ions at the roots surface (Bernal
et al., 2012). The uptake of Cu involves various heavy metal trans-
porters as mentioned below:
5.1.1. P-type ATPase copper transporters
The P-type heavy metal transporters are involved in the trans-
membrane movement of range of toxic metals, i.e., Cu2þ, Zn2þ, Cd2þ
and Pb2þ (Williams et al., 2000). They belong to the family of P-type
transporters that uses ATP as an energy source to pump the various
charged molecules across the cell membranes. These heavy metal
transporters are of two types namely 1 A ATPase and 1 B ATPase
(involved in Kþ transport) (Palmgren and Nissen, 2011). The gene
encoding the transporters further divide 1 B ATPase into two
classes, i.e., monovalent cation transporters (Ag, Cu) and divalent
cation transporters (Cd, Co, Pb, Zn) (Grønberg et al., 2016). It was
reported by Voskoboinik and Camakaris (2002) that the substrate
molecule for 1 B P-type transporters is Cu (I) and not Cu (II) as
showed by biochemical analysis of membrane vesicles. The type 1 B
family consists of eight members as found from the genome of
Arabidopsis and Oryza sativa (Gupta and Lutsenko, 2009), and have
been named as HMA1 to HMA8. However, three of these HMA6,
 V. Kumar et al. / Chemosphere 262 (2021) 127810 9

Table 5
Microbial induced Cu tolerance in plants.

Microorganism Plant Cd treatment Time Effect on plant traits References

/Medium span

Paenibacillus mucilaginosus (plant Medicago Pre-Cu -contaminated soil, soil 60 days Co-inoculation increased the nutrient concentration (C, N) and Ju et al.
growth promoting bacteria), sativa enzymatic activities (urease (55.6%), saccharase (29.5%), and b- (2020)
Sinorhizobium meliloti (rhizobia) glucosidase (31.4%)) of soil, hence promoted the plant growth by
decreasing Cu accumulation (48.6%) in shoots compared to the
uninoculated plants.
Stenotrophomonas maltophilia Saxifraga 150, 300 mM, petridish 5 weeks Plant inoculation with both bacteria and fungus enhanced the Ryszka
(bacteria), Mortierella sp. (fungus) stellaris (in vitro) growth, survival and photosynthetic performance and tolerance et al.
of plants compared to the uninoculated plants. (2019)
Sinorhizobium meliloti Medicago 50, 100, 200, 300, and 60 days Inoculation regulated the gene expression involved in Chen et al.
sativa 400 mg kg1, 100 g mixture of antioxidant response, phytochelatin synthesis and ascorbate- (2018)
vermiculite and perlite (2:1) glutathione cycle in plants. In inoculated plants copper uptake
was significantly increased in roots than shoots, decrease
transfer coefficient and promote phytostabilisation.
Commelina communis Elsholtzia Pre-contaminated Cu mine 2 Bacterial community composition and richness increased Yang et al.
splendens spoil months enhanced the plant growth as well as exudation of (2016)
phytochemicals in soil.
Arbuscular micorrhizal fungi Elsholtzia 125 mg l1, hydroponics 2 weeks AMF inoculation enhanced the germination rate, index, fresh Li et al.
splendens weight of the hypocotyl and radicle of the seedlings grown under (2017)
Cu stressed conditions.
Glomus coronatum Tagetes 50, 100 mM, hydroponics 7 days AMF inoculated plants alleviated noxious effects of Cu on root Zhou et al.
patula system activity, photosynthesis rate and mineral nutrient (2017)
accumulation. Additionally, the intraradical hyphae at root
epidermis immobilise large amount of Cu and restricted its
translocation to the aerial plant parts,
Funneliformis mosseae, Capsicum 2, 4 and 8 mM, soil 120 days Plant colonisation with AMF alleviated the deleterious effects of Ruscitti
Rhizophagus intraradices annuum Cu in pepper plants and enabled them to withstand Cu induced et al.
toxicity. (2017)
Rhizophagus clarus Mucuna 60 mg kg1, soil 45 days Phosphorus fertilization and mycorrhization favoured the plant Ferreira
cinereum growth, altered chlorophyll a fluorescence parameters and et al.
modulate oxidative stress (SOD, POD) enzyme activities by (2018)
reducing the stress induced by available Cu.
Glomus mosseae Dysosma 200, and 400 mg kg1, soil 120 days AMF inoculation enhanced plant biomass, root podophyllotoxin Luo et al.
versipellis concentration and decreases Cu absorption in roots, thus (2020)
alleviated Cu toxicity and activate key components crucial for
improving the productivity and stress tolerance in plants
Rhizophagus irregularis Phragmites 1 and 5 mg l1, hydroponics 21 days AMF inoculation improved the growth and physiological activity, Wu et al.
australis up-regulated the expression of transmembrane protein (2020)
epigment complexes CP43 (photosystem II) and FNR
(ferredoxin-NADP þ oxidoreductase related to photosynthetic
electron transport) and enhanced Cu tolerance in plants.

Fig. 3. Uptake and sequestration of Cu in plant cells through different transporters and Cu chaperons.
10 V. Kumar et al. / Chemosphere 262 (2021) 127810
HMA7 and HMA8 were previously identified as PAA1, RAN1 and
PAA2, respectively (Catty et al., 2011). From them, four HMA
transporters HMA5, HMA6, HMA7, and HMA8 are closely related to
1 B sub-family. HMA7 (RAN1) regulates the signalling of ethylene
by interplaying with trans-cyclooctene, responsible for the role of
Cu in ethylene-signalling pathway. The trans-cyclooctene is an
ethylene antagonist and it was identified that ethylene receptors
are Cu-dependent proteins (Li et al., 2017). HMA7 mediated the
transfer of Cu to secretory pathway for the synthesis of ethylene
receptors (Woeste and Kieber, 2000; Huang et al., 2016). HMA6
(PAA1) is responsible for the transfer of Cu to chloroplast which
provides the cofactor for the enzyme Cu/Zn SOD in the stroma
(Zhang et al., 2018b). HMA5 and HMA8 have the similar sequence to
HMA7 and HMA6, respectively and their roles are not defined
much. However, HMA8 (PAA2) which is similar to PacS transporters
of cyanobacteria might be involved in the transfer of Cu across
thylakoid membrane. OsHMA4 gene identified by Huang et al.
(2016) in rice plants which is a type of P-type ATPase, which con-
trols Cu accretion in rice grains. OsHMA4 functions to confiscate Cu
in the root vacuoles, reducing their accretion in grains and the al-
terations in grain Cu accrual is associated with a single amino acid
replacement that leads to diverse OsHMA4 transportation action.
5.1.2. COPT copper transporters
The major family of proteins involved in Cu transportation in
reduced form is COPT protein transporters which has sequence
similarity with eukaryotic Cu transports. It consists of five mem-
bers, i.e., COPT1, COPT2, COPT3, COPT4 and COPT5 has been iden-
tified in Arabidopsis thaliana. The constituents of this family
comprises of three transmembrane domains (TMDs) which has a
His-and/or Met-rich N-rich boxes, a standard motif attached with
TMD2 and TMD3. However, the COPT6 family has Cys/His group
facing the cytoplasm at the C-terminal (Puig, 2014). It was observed
that under Cu deficit conditions increased expression of COPT 1,
COPT 2 and COPT 6 was induced. The role of Cu in regulating the
expression of COPT 3 and COPT 5 has not been observed yet. The
role of COPT4 which is short of Met residues needed for the
movement of Cu (I) has been explored (Sanz et al., 2019). The gene
expression analysis of COPT family demonstrated the induction of
these protein transporters for Cu uptake which is not only depen-
ded on the cation availability but also on the plant type and the
tissue controlling its transportation (Kavitha et al., 2015).
The COPT1 transporters is mainly responsible for Cu uptake
which is having high affinity for Cu (I) and is primarily located on
the plasma membrane of plant cells particularly root tips. When
there is Cu limited conditions, plant uses the SPL7 mechanism and
upregulates the expression of COPT1 resulting in high acquisition of
Cu from the growing matrix (Carrio
-Seguí et al., 2019). The ab-
sorption of Cu in plants can generate hydroxyl radical (OH.) through
Fenton and Haber-Weiss reactions. The radical can also bind to the
plasma membrane non-selective cationic channel (NSCC) which
opens the channels for Ca2þ leading to enhanced root growth
(Rodrigo-Moreno et al., 2013). The other members of COPT family,
COPT2 also showed upregulation of its expression during decreased
Cu availability in a SPL mediated system (Perea-García et al., 2013).
The COPT2 gene is mainly expressed in the root epidermal cell,
roots hairs but not in the meristematic zones of roots and also in the
chlorophyll containing tissues hence the uptake of Cu by COPT2 is
restricted and the transfer takes place through secondary pathway
(Puig, 2014). Additionally, the second class of this family, i.e., COPT3
and COPT5 by a single Met- and His-terminus and an intermediate
rate of Cu affinity and transportation and it may have a secretive
pathway for indirect transport of Cu (Garcia-Molina et al., 2013).
Escaray et al. (2019) identified four COPT genes that encode
members of the Lotus copper transporter family which functions in
Cu uptake. Finally we can accomplish that COPT transporters play
imperative role in transport of Cu especially Cu(I).
5.1.3. ZIP family transporters
The uptake of Cu is also regulated by the expression of ZIP family
transporters (zinc iron regulated protein transporters). The avail-
ability of Cu ions in the growing medium decides their transport
through ZIP2 and ZIP4 transporters (Kavitha et al., 2015). It was
found that in Arabidopsis thaliana there was upregulation of ZIP2
and ZIP4 under Cu deficit conditions and down-regulation in
excessive Cu availability (Milner et al., 2013). However, over-
expression of ZIP4 was observed under enhanced Cu ion concen-
tration (Wu et al., 2013). Liu et al. (2019a) in their study reported
the OsZIP1 function in restraining surplus Cu accretion in rice. The
results of their study inferred that less and perpetual expression of
OsZIP1 in endoplasmic reticulum should be needed to retain
OsZIP1 at least level to confirm appropriate distribution of Cu
within the cells and precludes the harm of vital metals. But under
conditions of excessive metals, the OsZIP1 expression is
upregulated.
5.1.4. NRAMP family transporters
The Natural resistance associated macrophage protein (NRAMP)
transporters are used to relocation various ions such as Fe, Ni, Mn,
Zn and Cu in diverse kingdoms (Jain et al., 2018). The plant proteins
aid in the relocation of toxic metal ions from roots and shoots
athwart cell membrane and vacuolar membrane (Kra €mer et al.,
2007). NRAMP family of metal ion transporters plays role in var-
ied organisms from bacteria to human beings. NRAMP1 plays role
in metal transport across the phagosomal membrane of macro-
phage and imperfect NRAMP1 leads sensitivity to various intra-
cellular pathogens. NRAMP2 or DCT1 transport metal ions acts at
the plasma membrane of cells of duodenum and peripheral tissues,
and malfunctioning NRAMP2 leads to anemia. The proton gradient
is the driving force for metal-ion transport (Nevo and Nelson,
2006). Data on three rice NRAMPs such as OsNramp1, OsNramp2
and OsNramp3discovered their existence in varied parts and a
foremost function in transferring metal ions (Cailliatte et al., 2009).
Finally, we can conclude that these transporters play significant
role in transport of metal ions.
5.2. Copper translocation in plants
Copper ions taken up by plant roots can be transferred in the
form of Cu(I) or Cu(II) but generally it is done in the form of Cu-
complexes (Lang et al., 2017). The xylem vessels act as major
source for root to shoot transfer of Cu. The xylem tissue responsible
for this action is studied in mutants of Oryza sativa having P1B-type
ATPase OsHMA5 transporter showed better translocation of Cu from
roots to shoots (Deng et al., 2013). The transporter OsHMA5 is
located in the pericycle region of vascular tissues in the well-
developed roots and the translocation is observed in both vegeta-
tive and reproductive stage (Deng et al., 2013). The re-oxidation of
Cu (I) to Cu(II) was observed in Lycopersicon esculentum in aerial
transfer as Cu(II) forms better complexes favourable for long dis-
tance transporters (Ryan et al., 2013). Another amino acid nic-
otianamine (NA), synthesized by NA synthases (NAS) also has vital
role in mineral translocation (Hofmann, 2012). The NA has ability to
bind to varied type of transition metals, i.e., Mn, Fe, Co, Zn, Ni and
Cu and in the xylem vessels. Na and histone showed maximum
affinity to bind with the Cu defining their role in the transfer of Cu
in long distance translocation (Curie et al., 2009; Tamez et al., 2019).
Various transporters like P-type ATPase, ZIP, NRAMP and COPT are
involved in the transport of Cu.
 V. Kumar et al. / Chemosphere 262 (2021) 127810
5.3. Copper sequestration in plant (roots/shoots)
Plants have evolved different strategies to cope with the free
absorbed metal ions in the cytosol. Plants either inhibit the uptake
of the cations or can develop certain tolerance mechanisms by
converting the reactive ions into stable complexes (Kr~amer, 2005).
To inhibit the formation of reactive oxygen species, Cu is taken up
by the plants and is sequestered as metal chelating complex in the
vacuoles to reduce Cu toxicity. Cu chaperons, the family of receptor
proteins, are cytosolic low-molecular-weight receptors and
responsible for cross membrane transfer of metal ions within the
cells. Cu chaperons join with the specific receptors of Cu ions and
deliver it to the active sites of Cu dependent enzymes therefore
preventing the Cu interaction with other intracellular apparatus
(Markossian and Kurganov, 2003). In plants, three major type of Cu
chaperons are characterized i.e. CCH, COX17 and CCS at various
levels. The CCH and ATX1 (for antioxidant Protein1) control the
movement of Cu to Cu-carrying ATPases (Shin et al., 2012). The
upregulation of ATX1 has been observed in regulating plant growth
under unfavorable Cu conditions by mediating the Cu homeostasis
by sequestering Cu through interaction with MxCxxC domain (Shin
and Yeh, 2012). The expression of CCH was observed in the stems of
tomato (Lycopersicon esculentum; LeCCH) infected with fungal
infection. The plant CCH expression depends upon the status of
plant oxidative stress and aging. The CCH binds with the Cu (I) and
directs interacts with the P-type ATPases for Cu transportation
(Grønberg et al., 2016). The transport of Cu to Cu-carrying ATPase
(i.e. PAA1) is controlled by plastid chaperone which is a plant
specific Cu chaperone (PCH1). The transport of Cu to PAA is medi-
ated through stromal CCS which is responsible for delivering
cytosolic Cu (CuC) to stromal Cu (CSD). This also simultaneously
interact with PAA2 in Arabidopsis which controls the transport of
CuC to important cuproroteins essential CSD (Blaby-Haas et al.,
2014). The AtCOX17 gene isolated from Arabidopsis plant having
genome similarity with COX17. The COX17 is a Cu transporter and
helps in mitochondrial cross membrane mediation of Cu (Printz
et al., 2016). The AtCOX17 supplied Cu to mitochondria is used for
cytochrome oxidase complex and cytosolic enzymes such as Cu/Zn
superoxide dismutase.
6. Beneficial effects of copper
The mineral nutrients are prerequisite to the plants in order to
sustain normal growth and development, mainly from the soil
nonetheless from the foliar applications. At cellular level, Cu plays
significant function in cell wall metabolism, signalling to the
transcription protein trafficking apparatus, oxidative phosphory-
lation, iron armament and biogenesis of molybdenum cofactor
(Puig et al., 2007; Yruela, 2009). The Cu is needed for normal
growth and development, and when not available plant develops
precise deficiency symptoms, which mainly influences the young
leaves and reproductive organs (Yruela, 2009). Cu partakes in
numerous biological roles being a component of various enzymes
and proteins. However, a small escalation in Cu concentration in
soils persuades phytotoxic impacts on plants. Being a redox active
metal, it helps in free radical generation mainly reactive oxygen
species such as superoxide radicals, hydroxyl radicals and hydrogen
peroxide through Haber-Weiss and Fenton reactions (Yadav et al.,
2018). Cu is one of the seven micronutrients necessary for the
growth and mainly 5e30 mg kg1 is considered as satisfactory in
crop tissues (Sharma et al., 2019). Lastly, we can reveal that low
level of Cu has positive effect, whereas when present in excess it
exerts negative impact.
11
7. Copper toxicity and tolerance in plants
Copper is a necessary co-factor of various proteins (Cambrolle
et al., 2015). As trace element, an optimal quantity of Cu is
required to ensure cellular roles, but in excessive quantity it in-
duces harmful impact on the primary production and survival of
plants (Printz et al., 2016). Cu is a vital metal take part in many
metabolic processes of plants like undue acquisition of Cu by the
roots can rigorously compromise plant growth and productivity by
persuading alterations in root system design, nutritional inequities,
oxidative stress and accretion of reactive oxygen species (De Conti
et al., 2019; Marastoni et al., 2019). Ryegrass plants cultivated in
intercropping systems with plants like vine signify an auspicious
approach to regulate Cu toxicity in vineyard soils (De Conti et al.,
2020). Decline in plant biomass, reduction of root growth, chlo-
rosis, bronzing and necrosis are the common symptoms associated
with excessive Cu owed to enhanced generation of reactive oxygen
species and detrimental interfaces at cellular level (Lange et al.,
2017). Table 6 showed the toxicity and tolerance mechanisms of
Cu in plants. Fig. 4 exhibited the general physiological and
biochemical mechanism in plants under Cu stress.
7.1. Morphological parameters
Excess Cu concentrations have significant effects on plant
growth. Cu toxicity effects largely root growth and morphology
since it tends to accumulate in the root tissue and can be trans-
ferred to the shoots (Yang et al., 2015). The general visual symptoms
of Cu toxicity, apart from root growth inhibition, include interveinal
foliar chlorosis, wilted leaves, necrotic leaf tips and root abnor-
malities (Chen et al., 2015; Jung et al., 2015). More precisely excess
Cu levels are damaging to plant roots, with symptoms ranging from
disruption of the root cuticle and reduced root hair proliferation to
severe deformation of root structure (Sheldon and Menzies, 2005).
Depending on plant species, concentrations higher than
20e30 mg g1 may cause toxicity, while concentrations of
1e5 mg g1 are essential for plant growth (Marschner, 1995; Khatun
et al., 2008). The effects on root growth and form are significant
often before any effect on above-ground growth (Minnich et al.,
1987). However, at low concentrations, Cu is a plant micro-
nutrient and the visible symptoms may be less obvious, or
detectable, although reduction of crop yield and biomass may
persist (Marschner, 1995; Yruela, 2005). Hossain et al. (2020)
studied the morphological parameters under Cu stress in lentil
plants and observed that high treatment of 3.0 mM CuSO4. 5H2O
reduced the root, shoot, seedling growth and biomass of the plants.
Lalau et al. (2015) in their studies appraised the impact of CuO-
nanoparticles on Landoltia punctata plant and inferences indi-
cated that morphology was affected more substantially than
growth rate. Marques et al. (2018) in another study pointed the
toxic effect of Cu on Hymenaea courbaril plants. From their results, it
was obtained that under 200 mg kg1 treatment, Cu preferred the
growth and root morphology of this plant, while under
800 mg kg1, it has detrimental impact on the growth as well as
root morphology of this plant. Underneath micronutrient environs,
Cu has positive influence on root growth of Hymenaea courbaril as
per Imada et al. (2008). Further, the root surface area is mostly
associated with nutrient absorption, and enhancement in root
volume leads to better capability in nutrient absorption and
eventually has positive effect on the plant advancement. The
decline in root volume under Cu treatment indicates the significant
reduction in root biomass, which is directly allied with cell division
(Cai et al., 2014; Bochicchio et al., 2015). Batool et al. (2015) high-
lighted that apparent decline in root growth also linked with
decrease in cell division, leading to enhancement in cell wall
12 V. Kumar et al. / Chemosphere 262 (2021) 127810

Table 6
Copper toxicity and tolerance in plants.

Plant species Copper concentration Growth Time Span Effect on plant traits Tolerance factors in plants References
medium

Betula papyrifera 9.16, 1312, 3936 mg kg1 Soil 8 days Cu treatment for 8 days
imposed severe damage to the
plants. Highest Cu application
killed all the plants within 4
days
Hymenaea 100, 200, 400, 800 mg kg1 Soil 6 months At 800 mg kg1, Cu induced
courbaril toxicity with a dramatic
decrease in photosynthetic
machinery, biochemical status
and alterations in root and leaf
anatomy
Brassica juncea 0.25, 0.50, 0.75 mM Soil 60 days Highest Cu regime enhanced
superoxide anion and hydrogen
peroxide content up to 31.71%
and 68.29%, respectively.
Generation of reactive oxygen
species impeded parameters
related to gas exchange and
photosynthetic machinery.
Vitis vinifera, 0.2, 5, 25, 50 mM Hydroponics 14 days A reduction in shoot biomass
Vitis berlanderi and shoot-to-root ratio at
highest Cu regime was
observed.
Lolium perenne 0.2, 5.0, 25, 50 mM Hydroponics 14 days Impaired growth of the plants
with a decrease in root-shoot
biomass, leaf size and area.
Peltophorum 100, 200, 400 mg kg1 Soil 150 days Cu excess altered morphology
dubium of the root system, induced a
reduction in growth, biomass,
photosynthesis and
transpiration rate.
Vitis labrusca 60 mg kg1 Soil 90 days Cu regimes posed pessimistic
effects on plant growth,
pigment concentration,
nutrient content and
antioxidant response (SOD) of
the plants
Pisum sativum 200 mM Hydroponics 6 days Reduced root and shoot dry
biomass by 30% and 37%,
respectively, disrupt
nicotineamide redox status and
ultimately caused cell death.
Egeria densa 100 mM Hydroponics 4 days Combination of Cu þ urea
(5 mM) adversely affected the
photosynthetic machinery,
oxidative status and activities of
urease and catalase enzymes.
Vitis vinifera Available 122 mg kg1 Soil 12 months Decreased root length, biomass,
surface area, chlorophyll
content, stomatal conductance
and carboxylation efficiency of
the leaves under Cu stress.
Malus hupehensis 105, 161, 533 mg kg1 Soil 10 months Under highest Cu treatment,
Ralls cultivar accumulated 13%
more Cu in fibrous roots and
also exhibited higher
bioaccumulation factor values
compared to tap root and
lateral roots
Expression of single recessive Theriault and
gene induced retention of toxic Nkongolo (2016)
Cu ions in the roots in resistant
genotypes.
At 800 mg kg1, maximum Cu Marques et al. (2018)
content was retained inside the
roots and only 4.45% Cu was
translocated to upper aerial
parts, thus enabled plants to
withstand Cu toxicity.
Supplementation of Yadav et al. (2016)
castasterone in the growth
medium triggered antioxidant
machinery and enhanced
polyphenol accumulation in
plant tissues to increase
tolerance towards Cu toxicity.
In an intercropping system Marastoni et al. (2019)
there prevailed a competing
environment for nutrient
availability, the plants were
affected less due to Cu toxicity
due to the increased secretion
of flavnoids, flavonols and total
phenolic compounds.
Fe fertilization affect Cu De Conti et al. (2020)
availability in the rhizosphere
by decreasing succinate and
fumarate concentration in the
roots.
Cu accumulated more in roots, Marques et al. (2019)
minimises toxicity on
photosynthetic system and
promoted Cu tolerance
mechanisms in plants
Rhizophagus clarus (AMF) Brunetto et al. (2019)
inoculation along with
phosphorus fertilization
enhanced the quantum yield of
PSII, protects the
photosynthetic apparatus of the
plants by reducing Cu
translocation from roots to the
shoots.
Supplementation of gibberellin, Ben Massoud
auxin, citric acid and calcium et al. (2019)
promoted plant tolerance by
reducing Cu acclimatization
through hindering Cu
mobilization and by regulating
nicotinamide redox
homeostasis in plants.
Ascorbate peroxidase (APX) and Maleva et al. (2016)
glutathione peroxidase (GPX)
activity was enhanced to
promote plant tolerance and to
combat lethal effect of
combined Cu þ urea (5 mM)
treatment.
Soil supplemented with Trentin et al. (2019)
limestone enhanced the
development and growth,
hence proved an efficient
strategy to limitise Cu
availability and phytotoxicity.
Formation of Cu precipitates Wang et al. (2016)
with oxalates, phosphates and
its deposition in the cell wall
and vacuole of fibrous roots of
Ralls and Fuji cultivars is
tolerance strategy adopted by
the plants to overcome Cu
stress.
 V. Kumar et al. / Chemosphere 262 (2021) 127810 13

Table 6 (continued )

Plant species Copper concentration Growth Time Span Effect on plant traits Tolerance factors in plants References
medium

Betula papyrifera 9.16, 1312, 3936 mg kg1 Soil (growth 8 days After 2 days, a severe damage to Upregulation and over- Djeukam et al. (2016)
chamber) the plant growth was observed expression of multi-drug
at highest Cu level. However, allresistance associated protein
the plants died after 4 days of (MRP4) and metallothioneins
Cu treatment. (MT2B) genes in leaves at
intermediate Cu
(1312 mg kg1) treatment is the
tolerance strategy acquired by
plants to cope Cu toxicity.
Gossypium 50, 75, 100 mM MS media 21 days Hydrogen peroxide and In root tissue of Ersan-92 Kurt-Gür et al. (2018)
hirsutum malondialdehyde levels in cultivar formate dehydrogenase
tissues of Ersan-92 cultivar (FDH) activity was increased by
were increased significantly 6.35-fold and metallothionein
with the elevated Cu regimes. (GhMT3a) transcript level was
dramatically enhanced to
combat Cu stress in plants.
Hordeum 2000 mg kg1 Soil 45 days Disrupted cell wall integrity High Cu concentrations were Minkina et al. (2020)
sativum and deposition of electron- acclimatised in root
1
dense material in the (459.5 mg kg ), stem
cytoplasmic membranes of the (79.4 mg kg1) and leaf
roots. In leaves and stem parts, (66.3 mg kg1) part of the
Cu stress induced alterations in plants
the structures of endoplasmic
reticulum, peroxisomes,
chloroplasts and mitochondria.
Lens culinaris 1000 mg kg1 Soil 103 days Cu excess in soils induced a Copper-resistant bacteria Islam et al. (2016)
significant reduction in nutrient Providencia vermicola confer Cd
content (nitrogen and tolerance in plants by
phosphorus) with a parallel influencing a noticeable
increase in hydrogen peroxide increase in growth, chlorophyll
and malondialdehyde content content, root nodulation and
in plant tissues. antioxidative response.
Medicago sativa 25 and 75 mg l1 Petri dishes 6 days A dramatic reduction in Nano Cu(OH)2 at 25 mg l1 Cota-Ruiz et al. (2018)
nutrient content (Ca, Mg, Zn, P, overexpressed Cu/Zn
Mn and K) along with nitric superoxide gene, while ionic
oxide level, concurring with treatment at 75 mg l1
decreased root length. upregulated 10-fold increase in
metallothionein transcripts.
Cucumis sativus 80 mmol l1 Hydroponics 2 weeks Cu excess induced chlorosis, Melatonin enhanced the levels Cao et al. (2019)
decreased biomass and growth of glutathione and
of the plants phytochelatins to chelate
excessive Cu ions, promoted
cell wall trapping of Cu ions in
vacuoles and cell wall, thus
incremented Cd tolerance in
plants
Cucumis sativus 10 mM Hydroponics 5 days Decreased biomass, molar ratio Silicon addition in the growth Bosni
c et al. (2019)
of micronutrients (Fe:Cu, Zn:Cu medium enhanced the
and Mn:Cu) and significantly accumulation of nicotianamine
affect nicotianamine and histidine in the leaves,
accumulation in leaves of the promoted the chelation of Cu
plants. ions to alleviate Cu induced
toxicity in plants.
Nicotiana 35 mM Hydroponics 21 days Stunted growth, affect the Silicon supplementation Flora et al. (2019)
tabacum chlorophyll pigment drastically reduced Cu uptake by roots due
along with decreased to decreased expression of
photosynthetic efficiency of the copper transporter 1 (COPT1)
plants. gene and overexpression of
ethylene (ET) biosynthetic gene
mitigate Cu stress in plants.
Avena strigosa 50 mg kg1 Soil 30 days Affected the growth, Increased concentration of lime Ambrosini et al. (2017)
development, internal mineral (0.5, 1.0 g kg1) enhanced the
distribution, chlorophyll accumulation of micronutrients
concentration with enhanced like (N, P, K, Ca and Fe) in both
concentration of starch and roots and shoots but
carbohydrates. ameliorated Cu toxicity in
plants
Brassica pekinensis 5 and 10 mM Vermiculite 10 days Cu regimes cause stuted plant Upregulation of Aghajanzadeh et al. (2019)
growth, reduced biomass and CYP79B2 and CYP83B1 genes
nitrofen content in leaves was and MYB51, a transcription
reduced dramatically at 10 factor involved in the
10 mM Cu2þ regime. biosynthesis of indolic
glucosinolates in roots to cope
Cu toxicity.
14 V. Kumar et al. / Chemosphere 262 (2021) 127810
thickness of roots under heavy metal stress. Interveinal foliar
chlorosis is a common early symptom of Cu toxicity (Reckova et al.,
2019), while chlorosis has also been observed in higher plants, e.g.
Eucalyptus eximia (yellow bloodwood) and Casuarina distyla (she-
oak) (Mitchell et al., 1988). Cu toxicity can also be associated with a
purpling of foliage (Mostofa and Fujita, 2013) but this is not
apparent in all species (Sinha et al., 2015). The decline of leaf area
under Cu environs may be attributed to lignin accretion in xylem
and ultimately leads to thickening and hardening of cell wall, hence
pose pessimistic effects on cell development and leaf enlargement
by decreasing its elasticity (Garcia et al., 2018). Under Cu toxicity no
necrosis was observed in Zea mays and Rhodes grass (Reckova et al.,
2019; Sheldon and Menzies, 2005). From all these studies we can
conclude that treatment of Cu reduces the morphological param-
eters like root length, shoot length and leaf area etc. Further,
Interveinal foliar chlorosis, necrosis, purpling of foliage and chlo-
rosis was also found under Cu stress.
7.2. Physiological parameters
Copper can affect the biosynthesis of the photosynthetic ma-
chinery, by altering the pigment and protein composition of
photosynthetic membranes. Excess Cu disturbs chloroplast and
thylakoid membrane composition, triggers oxidative stress in plant
cells, and diminishes content of photosynthetic pigments, electron
carriers and subsequently photosynthetic electron transport
(Maksymiec, 1997; Vassilev et al., 2003; Gonzalez-Mendoza et al.,
2013). Experiments conducted by Küpper et al. (2009) showed
that Cu induced inhibition of photosynthesis mainly by affecting
the photosystem II (PSII) reaction center in Crassula helmsii plants.
Cu-induced chlorosis can result from the inhibition of pigment
Fig. 4. Overview of physiological and biochemical mechanisms of copper pollution in plants.
accumulation and retardation of chlorophyll integration into pho-
tosystems. A dramatic decline in photosynthetic gas exchange and
chlorophyll fluorescence parameters has been detected in Avicennia
germinans leaves (Gonzalez-Mendoza et al., 2013). Excess Cu can
cause oxidative stress in plants and subsequently increase the
antioxidant responses due to increased production of highly toxic
oxygen free radicals. Hossain et al. (2020) studied the photosyn-
thetic pigment such as chlorophyll a, b and carotenoids in lentil
plants and observed that high treatment of 3.0 mM CuSO4. 5H2O)
decreased their content. However, Cu toxicity was reduced with
application of acetate along with Cu, which helps in mitigating the
Cu stress. These inferences may be allied with changing the
photosynthetic metabolism of leaves accountable for ATP and
NADPH for root growth, which indicates in morphological varia-
tions in the root system (Batool et al., 2015). The decline in
photosynthetic rates and PSII quantum yield, and the escalation in
initial and maximium fluorescence under Cu treatment in soils are
allied to the decrease in chlorophyll concentrations, which medi-
cates deficit in photosynthetic machinery and enhanced the pho-
toinhibition of PSII (Cambrolle
et al., 2015). Nevertheless, low
chlorophyll content might be concerned with the reduction in iron
and phosphorus levels in shoots, which also decreased under
enhanced Cu regimes (Ambrosini et al., 2017). The detrimental in-
fluence of Cu in the soil disturbs the pigment composition of
thylakoid membrane and so causes reduction of electron transport
and PSII activities (Xu et al., 2013). Consequently, excessive Cu ac-
crues in plant tissues may disrupt the chloroplast structure and
therefore causes alterations in the constituents of thylakoid
membrane (Rehman et al., 2019a). Lastly, we can accomplish that
under Cu stress chlorophyll a, b, carotenoids, photosynthetic gas
exchange, photosystem II and PSII quantum yield are affected by Cu
 V. Kumar et al. / Chemosphere 262 (2021) 127810
treatment.
7.3. Nutrient uptake
Heavy metals inhibit the absorption and uptake of nutrients,
disturb their composition and induce phytotoxicity by affecting
nutrient metabolism in the plants (Chibuike and Obiora, 2014).
With the increase in Cu level there was decline in the quantity of
phosphorus (roots and leaves) and potassium (roots, stems and
leaves), signifying that excessive Cu caused the impairment in
nutrient uptake (Ambrosini et al., 2018). The lesser nutrient uptake
is a distinctive indicator of Cu toxicity, meanwhile the membrane
permeability and transporters role are adversely influenced
(Cambrolle
et al., 2015). The degree of nutrient disproportion is
based upon plant species, Cu level in the rhizosphere, treatment
time and growth environs (Adrees et al., 2015). The phosphorus
level decline in leaves and roots is related with the production of
insoluble metal-phosphate complex in the roots. In earlier studies,
Guimara ~es et al. (2016), discovered this complex, while signifying
the impact of phosphate in declining Cu uptake from roots to
shoots, so the complex halts Cu and phosphorus, decreasing their
uptake to the shoots. Under excessive Cu stress, various reports in
young grapevines also observed decline in nutrient uptake. For
instance Melo et al. (2008), Toselli et al. (2009) and de Oliveira et al.
(2015), reported reduction in accretion of nitrogen, potassium,
calcium, magnesium and iron in Sangiovese’ plants and Red
Niagara’ grapevines. Shams et al. (2019) studied the mineral
nutrient accumulation in Lactuca sativa under Cu stress and their
findings showed that under highest 400 mM treatment of CuSO4 the
nitrogen, potassium, phosphorus, magnesium, sodium, zinc and
boron accumulation is declined significantly in roots, whereas iron,
silicon, calcium and chloride remains unaltered in the leaves under
200 and 400 mM treatment of CuSO4. Zeng et al. (2019) also studied
the mineral uptake in Sugarcane Seedlings under Cu stress and
their findings revealed that under concentration of 200 mmol/l
Cu2þ, the Zn, Fe, Ca, Mg and Mn levels were enhanced in shoots,
whereas Zn and Mg reduced in roots. Li et al. (2019) also appraised
nutrient accretion in citrus seedlings under Cu stress and their
results indicated that iron, boron, nitrogen, phosphorus, potassium,
calcium, magnesium and sulphur contents were declined with Cu
stress. The complex dynamics of nutrient uptake and transport
under Cu stress might be due to competition between Cu and nu-
trients for transporters, alterations in gene expression participated
in nutrient uptake at transcriptional or post-transcriptional level
and variations in plasma membrane permeability (Andres-Borderia
et al., 2017; Roy et al., 2017; Zhang et al., 2017). The varied level of
nutrients in plants under Cu environs may be elucidated by diverse
tolerance approaches espoused by several plants (Yruela, 2009).
From these studies we can conclude that excessive Cu leads to the
impairment in nutrient uptake.
8. Copper detoxification and tolerance mechanisms in plants
Copper is considered as an important element, which plays vital
role in various physiological activities of plants like photosynthesis,
respiration, regulation of antioxidative system, electron transport
chain, nitrogen and protein metabolism, hormonal signalling and
also in the growth and development of plants. Apart from this, it is
having significant role in cellular metabolism and as a structural
and metabolic components of proteins and enzymes
(Chandrasekhar and Ray, 2017). Though, Cu is recounted as one of
the most toxic heavy metal (Rehman et al., 2019a). When plants are
exposed to very high concentrations of Cu, it leads to disturbances
in physiological functioning of plants including retardation in the
growth (Chen et al., 2015). Damage at molecular level is caused in
15
plants due to the oxidative burst produced by the formation of free
radicals and ROS in extreme doses of Cu (Habiba et al., 2015). Cu
stress leads to the oxidative burst by causing oxidation of almost
every organic components of the cells including proteins, nucleic
acids etc., and also stimulates the process of lipid peroxidation and
hence disturbs carbohydrate metabolism (Marques et al., 2018).
Certain antioxidative defensive system including both enzymatic
and non-enzymatic strategies like proline, glutathione, SOD, APOX,
GPX, ascorbic acid, phenols, tocopherol, phytochelatins etc., can
protect the plants from the ROS generated due to Cu toxicity (Liu
et al., 2018).
8.1. Antioxidative response
On exposure of plants to Cu distress, the antioxidative defence
response of plants get boost up due to ROS production and accu-
mulation. According to the reports, high concentration of Cu acti-
vates the defensive genes, which further boost up the antioxidative
enzymes like CAT, SOD, APOX, GPX etc., for the removal of free
radicals and combating the adverse effects of stress (Adrees et al.,
2015). SOD dismutase superoxide ions and convert them into ox-
ygen and hydrogen peroxide. CAT activity promotes detoxification
of H2O2 produced by SOD (Sidhu et al., 2020). The antioxidant en-
zymes are considered as the moderator of oxidative damage, hence
provide protection to the biomolecules against Cu induced stress
(Chandrasekhar and Ray, 2017). Saleem et al. (2020a) while work-
ing on Linum usitatissimum under the application of Cu, and their
inferences indicated that SOD and POD activities were enhanced
with increasing concentrations of Cu in the soil and utmost
enhancement was recorded at 600 mg kg1 of Cu in contrast with
no treatment of Cu. Saleem et al. (2020b) in another study on Linum
usitatissimum under Cu treatment, reported there was enhance-
ment in the SOD and POD activities in roots and leaves of Linum
usitatissimum which take part in scavenging of ROS. Liu et al. (2018)
in another study indicated that toxic level of Cu for 24 h caused
enhancement in the activities of SOD, CAT and APX in maize plant.
Further, they inferred that Cu caused oxidative stress which triggers
defence antioxidant enzymes through MAPK pathway in maize
plant. In another study, SOD, POD and APX activities were increased
significantly at 5 mg L1 concentration of Cu, whereas CAT activity
was reduced under high treatment of Cu in Salvinia natans (Liu
et al., 2019b). Rehman et al. (2019b) while working on Boehmeria
nivea under Cu treatment observed that SOD and POD activities
were enhanced with treatment of 200 mg kg1 Cu, but with
consequent enhancement in the Cu dose, i.e., 300 mg kg1 and
400 mg kg1 the SOD and POD activities were significantly reduced.
Contreras et al. (2018) in their findings on Colobanthus quitensis
under Cu exposure revealed that SOD, POD and CAT activities were
enhanced under Cu exposure, while APX exhibits reduction in their
activity under Cu treatment. Younis et al. (2018) exhibited the
enhancement in SOD and CAT activities under low dose of Cu, but
their activities declined under high level of Cu in Phaseolus vulgaris.
Furthermore, GR and APX activities showed significant enhance-
ment under Cu stress.
8.2. Glutathione production in plants
Under long term exposure of stress conditions, plants produced
antioxidant defense system to combat the reactive oxygen species
accrual like glutathione (Miteva et al., 2010). In Scenedesmus biju-
gatus cells, activities of enzymes involved in the glutathione
metabolism and level of GSH (glutathione reduced) content were
studied under copper stress, where formation of metal-binding
peptides were also investigated. With enhancing Cu concentra-
tion, drastic decrease in GSH was found in the cells in dose
16 V. Kumar et al. / Chemosphere 262 (2021) 127810
dependent manner. Similarly, protein thiol content was found to
enhance with Cu stress, whereas non-protein thiol content was
inhibited. Cu toxicity caused enhancement in gamma-
glutamylcysteine synthetase, GSH S-transferase and GSH-
peroxidase activities and reduction in GSSG-reductase activity.
These findings revealed that Cu leads to alteration in equilibrium
between formation and consumption of GSH moreover due to its
antioxidant functions or by helping as a precursor in the production
of phytochelatins (Chu et al., 2013). Rodríguez-Rojas et al. (2019)
appraised the function of mitogen-activated protein kinases
(MAPK) path in detoxification responses induced by Cu (10 mM) in
Ulva compressa and their inferences revealed that stoppage of more
than one MAPK at 6 days of Cu treatment leads to decline in total
glutathione, while in the end it did not occur when complete MAPK
was blocked. The higher Cu detoxification is interceded by gluta-
thione in Ulva compressa. The intracellular augmentation in GSH
levels might participate in Cu accrual and detoxification. Lastly, it
was inferred that exudation of GSH to the extracellular medium in
green seaweed, signifying its function complexing external surplus
of bio accessible Cu (Navarrete et al., 2019).
8.3. Phytochelatin production in plants
Phytochelatins (PCs) are the utmost vital type of metal chelators
which counters the detrimental impact of heavy metals stress.
These are synthesized in the cytosol with the help of phytochelatin
synthase (PCS) under heavy metal toxicity (Sharma et al., 2016).
Toxic effects of heavy metals like Cu can be ameliorated by different
defensive mechanisms. Plants internal tolerance to Cu metal stress
may provide resistance against metal toxicity. Under the stress
conditions, chelation of metals occurs due to the production of GSH,
amino acids, metallothioneins (heavy metal binding ligand) (Yadav,
2010). Sequestration of Cu metals occur with the help of PCs which
further leads to the activation of antioxidative defensive system and
glyoxalases which ameliorates the ROS produced by Cu metal
(Conte and Carroll, 2013). PCs are produced by GSH with the help of
PC synthase enzyme, which is a constitutive enzyme that is needed
for the post-translational amendment to execute as an active
enzyme (Mellado et al., 2012). In order to maintain balance be-
tween the synthesis of PCs and their formation might be essential
in counteracting Cu stress. There are abundant reports regarding
the production of PC-copper complexes and their sequestration in
the vacuole (Jozefczak et al., 2012). For instance, Song et al. (2014)
in their studies provides inferences for the transport of Cu linked to
PCs in the vacuole through ABCC-type transporters. Moreover,
findings of Flores-Ca
ceres et al. (2015) suggested that there was no
formation of PCs under Cu treatment in Medicago sativa, signifying
less significance of PCs in detoxification of Cu ions. Chen et al.
(2018) in another on Medicago sativa under Cu stress reported
that Cu influenced PC-biosynthesis-coupled gene expression and
stopped the PC-Cu production. Pochodylo and Aristilde (2017) in-
ferences represented the impact of Cu-PC structure on the Cu-
binding thermodynamics and PC conformation, mutually these
are vital to appraise the intracellular function of PCs in arbitrating
algal response to toxic Cu treatment. The PCs (PC2-PC5) was arbi-
trated as defensive mechanisms in Colobanthus quitensis under Cu
exposure (Contreras et al., 2018). The PCS1 gene expression was
assessed in Azolla pinnata and Azolla filiculoides at varied doses of
Cu, and maximum PCS1 gene expression was observed in Azolla
pinnata and their expression was enhanced at 500 mM dose of Cu
after 24 h, but significant decline and again enhancement after
72 h. The Azolla species were genetically varied and the mecha-
nisms take part in phytoremediation was prospective to differ and
so, the gene expression was varied (Talebi et al., 2019). Finally, we
can reveal that PCs are produced under different levels of Cu, plant
species and their genetic make-up.
9. Phytoremediation of copper
Phytoremediation is an evolving approach which uses plants to
decontaminate the polluted soil/water (Bonanno et al., 2017). There
are many plants which have phytoremediation potential and are
best accrual of heavy metals like copper (Kumar et al., 2019c). The
tolerance, accrual and transport of metals to the aerial tissues of
plants are vital features which are taken into consideration during
species choice in phytoremediation strategy (Fan et al., 2011).
Moreover, metal endurance is also an indispensable prerequisite for
metal accrual and so effective execution of phytoremediation
(Hussain et al., 2017). Firstly, the limit of Cu hyperaccumulation was
fixed to 1000 mg g1 but later on modified to 300 mg g1 due to least
amount of evidence for its accumulations in plants (Baker and
Brooks, 1989; Kra ~mer, 2010; Van der Ent et al., 2013). Lu et al.
(2018) conducted a study to remediate the Cu pollution by using
aquatic plants such as Sagittaria sagittifolia, Arundina graminifolia,
Juncus effusus, Eichhornia crassipes, Pistia stratiotes, Echinodorus
major, Acorus calamus and Nymphaea tetragona. Among the ana-
lysed aquatic plants Eichhornia crassipes and Pistia stratiotes are the
superlative capability for bioaccumulation, whereas Acorus
calamus, Arundina graminifolia and Nymphaea tetragona exhibited
good potential for remediation of Cu pollution. Covre et al. (2020) in
another study checked the phytoremediation potential of Khaya
ivorensis and Cedrela fissilis in increasing dose of Cu, i.e., 60, 200,
400, and 600 mg kg1, and both these plants showed good potential
for accrual of Cu. Further, the translocation factor was recorded low,
indicating restraining and accruing Cu in the roots, signifying their
phytostabilization ability. The less heavy metal transfer is dis-
tinguishing feature of many tropical plants signifying their ability
for phytostabilization of Cu (Souza et al., 2012; Meyer et al., 2016;
Asensio et al., 2018). Several authors like Nirola et al. (2015); Silva
et al. (2015) and Kang et al. (2018) reported Cu-tolerant tree spe-
cies in their work. Chu et al. (2020) in their study on coal gangue
and Vetiveria zizanioides for phytostabilization of Cu in Cu-mine
tailing and their results inferred that treatment of coal gangue
reduced the DTPA-extractable content of Cu in amended tailings.
Furthermore, they reported that with the treatment of coal gangue
transformed the exchangeable and carbonate portions of Cu into
unavailable forms of organic matter portions, hence decreasing Cu
concentration in mobile fractions of the amended tailings. Finally,
their inferences revealed that coal gangue assisted phytostabiliza-
tion of Cu in Cu-mine tailings. Ju et al. (2020) in another study also
reported the phytoremediation ability of alfalfa in Cu-polluted soils.
Khalid et al. (2020) conducted study using Alternanthera bettzicki-
ana plant and applied varied doses of Cu 2.5, 5, 7.5 and 10 mM. The
results indicated that Cu level and accretion enhanced with their
increasing doses in different plant parts and maximum accumula-
tion takes place in shoots and roots, indicating the capability of this
plant to remediate Cu-polluted regions. Table 7 presents the plants
involved in remediation of Cu-contaminated mediums.
10. Management of copper-contaminated soils
Due to twofold function (vital and harmful) of Cu, both the
deficiency as well as toxicity is a worldwide apprehension. In Cu-
deficient soils, usage of Cu fertilizers is the superlative potential
approach. The application of Cu-amended fertilizers is usual in
nation’s deficient of Cu in the soils (Taylor et al., 2020). In Europe,
about 19% of the arable land is deficient for bioavailable Cu
(Shorrocks and Alloway, 1985). The pollution of soils and ground-
water by metals mainly Cu is a prime apprehension at many
polluted regions, most of which are encompassed in the National
 V. Kumar et al. / Chemosphere 262 (2021) 127810 17

Table 7
Plants involved in remediation of Cu-contaminated mediums.

Plant species Copper concentration/ Time span Cu accumulation Phytoremediation References

Medium traits
Roots Shoots

Copaifera 60, 100, 500 mg kg1, 60 days Accumulated and stabilised Cu Translocate high Cu Plant species Asensio et al.
langsdorffi, soil (more than 300 mg kg1) in concentrations in the accumulated more (2018)
Myracrodruon roots with very high aerial parts with high than 300 mg kg1
urundeuva, bioaccumulation factor. translocation factor for Cu in roots and
Cecropia sp. Cu. shoots and showed
potential to
phytostabilise and
phytoextract Cu
from contaminated
soils.
Elsholtzia 632 mg kg1, Pre- 26th April to mid- For three years Pennisetum Cu-tolerant plant Addition of Xu et al. (2019)
splendens, contaminated soil December for 3 sinese cumulatively Elsholtzia splendens hydroxyapatite
Pennisetum consecutive years accumulated total Cu up to the exhibited high Cu enhanced the soil
sinese (2013, 2014, 2015) level of 8.67  103 g h m2. absorption of enzymatic activity
202 mg kg1. and also the effect
of Cu toxicity on
plant biomass was
reduced, enabled
the plants to
remediate Cu-
contaminated soils
Pistia stratiotes 5, 10, 15 mg l1, 32 days At 5 mg l1, roots accumulated At 5 mg l1, shoots Pistia stratiotes Kumar et al. (2019)
hydroponics Cu upto 1.39 mg kg1 accumulated Cu upto achieved maximum
1.61 mg kg1 with growth at 5 mg l1
translocation factor and can be
more than 1. employed for
remediation of Cu.
Limnocharis flava 30 ppm, hydroponics 5 days, Highest rate of Cu absorption e Plants exhibited Alikasturi et al.
was 39.3%, while the absorption high efficacy in (2019)
efficiency of Cu in surface and absorbing Cu
mineral water was 31.5% and contaminant.
23.6%, respectively.
Brassica nigra 100, 150, and 120 days Highest Cu accumulation and e Plants exhibited Lothe et al. (2016)
200 mg kg1, soil its retention were found in root ~42% Cu removal
part of the plants. efficiency from the
soil
Corchorus 100, 200, 300, 30, 60, 90, 120 days Maximum Cu concentration After 120 days of After 120 days, the Saleem et al.
capsularis 400 mg kg1, soil was accumulated in roots growth, Cu was plants exhibited (2020c)
(470 mg kg1) at the early accumulated upto the high
growth stage (30 days) level of 534 mg kg1 in bioaccumulation
the leaves. factor and
translocation factor
values more than
the critical limits
set for
hyperaccumulator
plants.
Dendrocalamus 40, 80,120 mM, 7, 14, 21 days After 7 days, roots accumulated During the first 14 days, Roots acclimatised Chua et al. (2019)
asper hydroponics highest Cu levels at 120 mM Cu the concentration of Cu highest Cu
treatment. The in aerial parts increased concentration
bioconcentration after 21 days progressively but a indicating the
of growth at 40, 80 and 120 mM decline was noticed phytostabilisation
was 41.86, 50.57 and 29.63, thereafter. potential of the
respectively plant species.
Potamogeton 1, 10, 100, 1000 mM, 96 h Roots accumulated more than The bioconcentration Bioaccumulation Costa et al. (2018)
pectinatus hydroponics 2800 mg Cu/g fresh weight at factor values of Cu was factor values were
highest Cu concentration in the 0.04 and 0.03 for stem on the lower side
medium and leaves at 1000 mM but the plant can be
concentration used as an effective
biomonitor of
copper-
contaminated
areas.
Linum 2153 mg kg1, pre- 60 days Plant roots accumulated e The plant species Saleem et al.
usitatissimum contaminated soil maximum Cu upto the displayed good (2020a)
concentration of 36 mg kg1 potential to revoke
grown under highest Cu Cu ranging from 22
treatment (1:0). to 27% from Cu-
contaminated soil
Eichhornia crassipes 2, 4, 6, 8 mg l1, 30 days Roots accumulated 86 mg kg1 Leaves accumulated Concentration of Cu Tabinda et al.
hydroponics Cu at highest treatment 50 mg kg1 Cu at in the medium (2020)
8 mg l1 Cu treatment. decreased with the
(continued on next page)
18 V. Kumar et al. / Chemosphere 262 (2021) 127810

Table 7 (continued )

Plant species Copper concentration/ Time span Cu accumulation Phytoremediation References

Medium traits
Roots Shoots

increase in time
period and
maximum Cu was
retained in the
roots.
Pistia stratiotes 2, 4, 6, 8 mg l1, 30 days Roots acclimatised 96 mg kg1 Leaves acclimatised No effect was Tabinda et al.
hydroponics Cu 70 mg kg1 Cu shown on plant (2020)
growth and
biomass and the
plants exhibited
bioaccumulation
factor greater than
1 for Cu.
Helianthus annuus 10,000 ppm, soil 4 weeks e Leaves and stem Plants efficiently Forte and Mutiti
accumulated 783 and uptake Cu from soil (2017)
99 ppm Cu and translocate it to
concentration, the aerial parts to
respectively. show effective
phytoremediation
potential of the
plants.
Schinus molle 11,151 mg kg1 pre- 1 year Bioconcentration values for Cu Plants exhibited high Combination of Lam et al. (2017)
contaminated Cu mine were less than 1 for all the translocation factor AMF, compost and
tailings, soil treatments values, but the values CaCO3 enhanced
were less than 1 to the uptake
consider them potential of the
hyperaccumulators plants but retain
high Cu
concentration
inside the roots,
hence
phytostabilise Cu
from contaminated
soils.
Canavalia 100 mg kg1, soil 45 days Mycorrhization at low e Vermicompost Santana et al.
ensiformis vermicompost dose (10 and addition at low (2018)
20 mg P kg1) enhanced Cu dose (10 and
accumulation in the roots by 20 mg P kg1) and
106% and 97%, relative to non- inoculation of
inoculated plants. mycorrhizal fungi
(Rhizophagus
clarus) enhanced
the
phytostabilisation
potential of Cu-
contaminated
sandy soils
Leersia hexandra 80, 130, 180, 230, 330, 60 days At highest Cu treatment, roots Cu translocated to the Fertilization and 3- Lin et al. (2019)
430, 480 mg kg1, soil accumulated 5501.7 mg kg1 shoots upto 755.4 mg indole-acetic acid
kg1at 480 mg kg1Cu supplementation
treatment. increased the tissue
dry weight and Cu
extraction by 52.3%,
thus significantly
enhanced the Cu
phytoextraction
efficiency upto
204.6%.
Solanum nigrum 50, 100, 150, 4 weeks The extent of Cu acclimatised in e Cu treatment Abdel-Wahab et al.
200 mg l1, culture the callus of plants was ~20.9 induced alterations (2019)
media and 40.4-fold at 150 and in the functional
200 mg l-1 Cu levels with groups and
respect to the controlled plants. modulates the
action of phenols,
lignins and proteins
in chelating Cu,
hence confer
tolerance to
withstand Cu
toxicity.
Amorpha fruticosa 400 mgkg1 pre- 150 days Bioconcentration factor and e Plant showed Shi et al. (2016)
contaminated Cu mine translocation factor values were highest tolerance
tailings, soil less than 1, maximum Cu was and normal
phytostabilised in the roots biomass
production.
 V. Kumar et al. / Chemosphere 262 (2021) 127810 19

Table 7 (continued )

Plant species Copper concentration/ Time span Cu accumulation Phytoremediation References

Medium traits
Roots Shoots

Myracrodruon 125, 1525, 3050 mg Cu 90 days At highest Cu treatment, roots Stem and leaves The plant species Asensio et al.
urundeuva kg1, soil accumulated upto 2600 mg Cu accumulated upto 3241 showed the (2019)
kg1 dry weight and 12.4 mg Cu kg1 potential to
dry weight at highest stabilise and
Cu dose phytoextract Cu
from Cu-
contaminated soils
Cedrela fissilis 125, 1525, 90 days At highest Cu treatment, roots Stem and leaves The plants Asensio et al.
3050 mg kg1, soil accumulated upto 5800 mg Cu accumulated upto 5008 accumulated high (2019)
kg1 dry weight and 52.2 mg Cu kg1 Cu concentration in
dry weight at shoots (more than
1
3050 mg kg Cu 300 mg kg-1) and
treatment. even higher in roots
and strongly
exhibit the
potential to reclaim
Cu-polluted soils.
Thymus Pre-contaminated Cu e Roots accumulated e High Ghazaryan et al.
1
kotschyanus mine tailings, soil ~775 mg kg Cu and the plant bioconcentration (2019)
species exhibited high and translocation
phytostabilisation potential for factor values for Cu
Cu-contaminated soils made this plant
species a valuable
resource for
restoring Cu
polluted
ecosystems.

Priorities List (NPL) (Mulligan et al., 2001a). Due to allied detri-
mental impacts, various severe rules have been made to reduce the
amount of harmful metals in the environs. Nevertheless, clean-up
of metal polluted regions is a challenging and expensive process
(Lo and Yang, 1999). China is the foremost Cu generating country
where 600,000 tons of Cu was mined per year from 1607 mines,
and affecting the soil health. So, it is vital to reinstate Cu-
contaminated soils for agricultural development and ultimately
for human health (Lin et al., 2019). Xu and Zhao (2005) applied
Poly(amidoamine) Dendrimers in their studies for remediation of
Cu-contaminated sites. The findings of their study indicated that
90% Cu initially sorbed was remediate by 66 bed volumes of 0.10%
(w/w) of a production of 4.5 dendrimer with carboxylate terminal
groups with pH (6.0). Based upon same dose, dendrimers of less
generation remediate more Cu, while reducing pH increased Cu
removal for all dendrimers confirmed. Di Palma and Medici (2002)
in another study removed Cu in polluted soil by flushing with a
0.1 M aqueous solution of ethylenediaminetetraacetic acid sodium
salt. A maximum of 60% removal of Cu was perceived. Liu et al.
(2019c) applied potassium lignosulfonate (KLS) for removal of Cu
in polluted soils. KLS content of 8%, pH 5.24 and 6 h duration, the
elimination ratios for the stimulated and mining area soils in the set
investigation were 73.42% and 55.20% for Cu. The inferences of this
study revealed that KLS remediate Cu from soil, although increase
nutrient echelons in the soil. In another study, Lin et al. (2019) used
Leersia hexandra Swartz to mitigate the pollution of Cu in
contaminated areas, and their results inferred that the shoots
accrue maximum Cu (755.4 mg kg1), signifying the phytor-
emediation potential of this plant to mitigate Cu-contaminated
soils. Xu et al. (2019) also reported in their work that Elsholtzia
splendens and Sedum plumbizincicola are the accumulators of Cu.
Amid the extraction strategies established to reduce metals from
the polluted soils, phytoextraction via tolerant and accreting plants
like Alyssum lesbiacum, Ambrosia artemisiifilia and Brassica juncea,
appeared to be an auspicious in-situ process to de-contaminate the
Cu levels (Raskin et al., 1997; Padmavathiamma and Li, 2007).
Though, this method needs long duration to effectively mitigate the
pollutants from soil and only effective strategy to reduce the mobile
metals into the contaminated regions (Wang et al., 2018). Another
technique, electrokinetic processes are also applied for the miti-
gation purposes and it comprises of low intensity electric current
between cathode and anode introduced into the polluted site. Due
to applied electric current, the ions and charged particles are
migrated to anode or cathode based upon their charges. This
technique is also applied for the elimination of Cu and other metals
from the contaminated regions (Mulligan et al., 2001b). Finally we
can recommend that if soil is deficient with Cu then we can apply
Cu encompassing fertilizers, and if Cu level is more in the soil then
it is managed by using phytoremediation plant species, electroki-
netic, potassium lignosulfonate and poly(amidoamine) dendrimers.
11. Concluding remarks and future perspectives
This review elucidates the biogeochemical behaviour of Cu in
soil plant with respect to human health. Cu exists in diverse
oxidation states; however the utmost ample form is Cu2þ. The
mobility, soil-plant relocation, accretion in plant tissues, phyto-
toxicity and detoxification of Cu varies substantially according to
speciation. The soil physiochemical characteristics like pH, organic
matter and microbial activity have pronounced impact on Cu
speciation and biogeochemical behaviour of Cu. Copper accretion
through roots takes place by specific and non-specific transporters
like Nramp, COPT, Zip family, etc. Copper in Cu2þ form is converted
into Cuþ inside the plant roots. Excess of Cu is toxic for plants, but,
at low concentrations, Cu positively regulates the plant growth. At
lower levels, Cu shields plant from diverse kinds of abiotic stress
environs. Copper protects the plants from harmful effect of reactive
oxygen species at low levels, while at higher levels, it act as pro-
oxidant and augment ROS generation, leads to lipid peroxidation
and protein oxidation. Moreover, the positive or negative impact of
Cu is mainly dependent on the plant species, speciation and its total
concentration in the growth medium. Ingestion of food crops
comprising Cu is the prime route of Cu revelation to living organ-
isms, and depends on soil Cu level and speciation. Based on the
20 V. Kumar et al. / Chemosphere 262 (2021) 127810
literature used in the review, following point’s needs to be
sightseen:
In what way diverse plant species have dissimilar Cu detoxifi-
cation and tolerance strategies are not properly explicated. Also, the
threshold lethal quantity of Cu according to their chemical form is
not unblemished.
More detailed research is needed to improve knowledge about
mobility of Cu inside the plants, leading to increased transferral of
Cu to shoot tissues.
Copper showed both positive as well as negative impacts in
plants, however the strategies, and ideal, vital and harmful level of
Cu in soil are not well recognized for diverse plant species and soil
types. Many workers have scrutinized the antioxidative activities of
Cu, but only fewer studies have appraised the pro-oxidative im-
pacts of Cu.
Copper also plays imperative function in health of human be-
ings, however fewer nations throughout the world have established
Cu status in soil inhabitants to appraise Cu adequacy/paucity. There
is prerequisite to made more worker mainly in developing nations
to appraise nationwide Cu status in soils and human beings, which
is further assembled to worldwide level.
Declaration of competing interest
All authors declared that there is no conflict of interest.
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