Environmental Research 213 (2022) 113711

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Environmental Research
journal homepage: www.elsevier.com/locate/envres

Green synthesis of gold nanoparticles using Gracilaria crassa leaf extract

and their ecotoxicological potential: Issues to be considered
Chinnaperumal Kamaraj a, **, Sengodan Karthi b, Appadurai Daniel Reegan c,
Govindasamy Balasubramani d, Govindaraju Ramkumar b, Kandaswamy Kalaivani e,
A. Abduz Zahir f, Paramasivam Deepak g, Sengottayan Senthil-Nathan b,
Md Mostafizur Rahman h, i, Abu Reza Md Towfiqul Islam j, Guilherme Malafaia k, l, m, *
a
Interdisciplinary Institute of Indian System of Medicine (IIISM), Directorate of Research and Virtual Education, SRM Institute of Science and Technology (SRMIST),
Kattankulathur, 603203, Tamil Nadu, India
b
Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Centre for Excellence in Environmental Sciences, Manonmaniam Sundaranar University,
Alwarkurichi, 627 412, Tirunelveli, Tamil Nadu, India
c
National Center for Disease Control, Bengaluru Branch, No:08, NTI Campus, Bellary Road, Bengaluru, 560 003, Karnataka, India
d
Division of Research & Innovation, Department of Biotechnology, Saveetha School of Engineering, Saveetha Institute of Medical and Technical Sciences (SIMATS),
Thandalam, Chennai, 602105, Tamil Nadu, India
e
Post Graduate and Research Centre, Department of Zoology, Sri Parasakthi College for Women, Courtrallam, 627 802, Tirunelveli, Tamil Nadu, India
f
Unit of Nanotechnology and Bioactive Natural Products, Post Graduate and Research Department of Zoology, C. Abdul Hakeem College (Autonomous), Melvisharam,
632 509, Vellore District, Tamil Nadu, India
g
Department of Biotechnology, Dr. N.G.P. Arts and Science College, Dr.N.G.P. - Kalapatti Road, Coimbatore, 641048, Tamil Nadu, India
h
Department of Environmental Sciences, Jahangirnagar University, Dhaka, 1342, Bangladesh
i
Laboratory of Environmental Health and Ecotoxicology, Department of Environmental Sciences, Jahangirnagar University, Dhaka, 1342, Bangladesh
j
Department of Disaster Management, Begum Rokeya University, Rangpur, 5400, Bangladesh
k
Post-Graduation Program in Conservation of Cerrado Natural Resources, Goiano Federal Institute, Urutaí, GO, Brazil
l
Post-Graduation Program in Ecology, Conservation, and Biodiversity, Federal University of Uberlândia, Uberlândia, MG, Brazil
m
Post-Graduation Programa in Biotechnology and Biodiversity, Federal University of Goiás, Goiânia, GO, Brazil

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

Keywords: The use of vegetal species for gold nanoparticles (AuNPs) biosynthesis can constitute an alternative to replacing
Green nanoparticles the extensive use of several hazardous chemicals commonly used during NPs synthesis and, therefore, can reduce
Gracilaria crassa biological impacts induced by the release of these products into the natural environment. However, the “green
AuNPs
nanoparticles” and/or “eco-friendly nanoparticles” label does not ensure that biosynthesized NPs are harmless to
Anopheles stephensi
Ecotoxicology
non-target organisms. Thus, we aimed to synthesize AuNPs from seaweed Gracilaria crassa aqueous extract
through an eco-friendly, fast, one-pot synthetic route. The formation of spherical, stable, polycrystalline NPs with
a diameter of 32.0 nm ± 4.0 nm (mean ±SEM) was demonstrated by UV–vis spectroscopy, field emission
scanning electron microscopy, and high-resolution transmission electron microscopy, energy-dispersive X-ray
and X-ray diffraction measurement, and Fourier-transform infrared spectroscopy analysis. In addition, different
phytocomponents were identified in the biosynthesized AuNPs, using Gas Chromatography-Mass Spectrometry
(GC-MS). However, both G. crassa aqueous extract and the biosynthesized AuNPs showed high ecotoxicity in
Anopheles stephensi larvae exposed to different concentrations. Therefore, our study supports the potential of
seaweed G. crassa as a raw material source for AuNPs biosynthesis while also shedding light on its ecotoxico­
logical potential, which necessitates consideration of its risk to aquatic biota.

* Corresponding author. Laboratory of Toxicology Applied to the Environment, Goiano Federal Institute, Urutaí Campus, Rodovia Geraldo Silva Nascimento, 2,5
km, Zona Rural, Urutaí, GO, CEP, 75790-000, Brazil.
** Corresponding author.
E-mail addresses: kamarajc@srmist.edu.in (C. Kamaraj), karthientomology@gmail.com (S. Karthi), danielreegan85@gmail.com (A.D. Reegan), balabio62@gmail.
com (G. Balasubramani), ayvidram@gmail.com (G. Ramkumar), senkalai14@gmail.com (K. Kalaivani), abduzzahir1984@gmail.com (A.A. Zahir), deepaksiva1252@
gmail.com (P. Deepak), senthil@msuniv.ac.in (S. Senthil-Nathan), towfiq_dm@brur.ac.bd (A.R. Md Towfiqul Islam), guilhermeifgoiano@gmail.com (G. Malafaia).

https://doi.org/10.1016/j.envres.2022.113711
Received 6 April 2022; Received in revised form 2 June 2022; Accepted 14 June 2022
Available online 18 June 2022
0013-9351/© 2022 Elsevier Inc. All rights reserved.
C. Kamaraj et al.
1. Introduction
Over the last two decades, nanotechnology has emerged as a favor­
able field of interdisciplinary exploration due to its immense application
across a diverse field of science. Currently, in nano-enabled currencies,
personal care, medications, food, and agricultural items, thousands of
tons of metallic nanoparticles (MNPs) are being generated and used
(Chandrakala et al., 2022). The reaction compounds and the methods
used in the industrial manufacture of MNPs are widely agreed not to be
environmentally friendly (Peralta-Videa et al., 2016). Green synthesis
has been suggested as an alternative to minimizing the use of toxic
compounds and harsh reaction conditions in the production of MNPs
(Peralta-Videa et al., 2016; Hano and Abbasi, 2022). The green synthesis
produces high yield with low-cost, environmentally sustainable MNPs
using plants, seaweeds, and microorganisms (Salem &Fouda, 2021). As
discussed by Zhang et al. (2020), these organisms or their extracts are
used for the green synthesis of MNPs through bio-reduction of metallic
particles leading to the synthesis of NPs, whose biosynthesized MNPs
have a range of applications in numerous fields of human interest.
For example, gold nanoparticles (AuNPs) are exceptionally versatile
bionanotechnological materials that can exhibit different size and
shape-dependent properties and various physicochemical properties
such as surface plasmon resonance, redox behavior, electromagnetic
conductivity, etc. (Sayed-Pathan et al., 2022). Such NPs can be boosted
by aquatic and terrestrial plant extracts, bacteria, and fungi and have
transformed the medical sector because of their extensive use in drug
delivery (Hong et al., 2022; Chandrakala et al., 2022), imaging (Luo
et al., 2021; D’Acunto et al., 2021; Yim et al., 2021), and medical
therapy (Mahhengam et al., 2022; Essa et al., 2022). Furthermore,
AuNPs are recognized as possible candidates in many other areas,
including chemical sensing (Chang et al., 2019; Hu et al., 2021; Nguyen
et al., 2022) and catalyzation (Ciriminna et al., 2016). This is mainly due
to the intrinsic features of AuNPs, such as optical, electronic, physico­
chemical, and surface plasmon resonance (SPR); which can be altered by
changing the characterizations of particles such as shape, size, aspect
ratio, or environment; ease of synthesis and functionalization properties
have resulted in various applications in different fields of biomedicine
such as sensing, targeted drug delivery, imaging, photothermal and
photodynamic therapy as well as the modulation of two or three ap­
plications [see Elahi et al. (2018) and references therein].
The biogenesis of AuNPs via seaweed extracts has excellent
biocompatibility and non-toxicity, low risk for clinical research, and the
ease of reducing gold salt; the technique is simple, one-step approach
and productive for large-scale processing (Rotimi et al., 2019). Algae are
photoautotrophic and oxygenic in natural environments and can bio­
accumulate heavy metals. Such distinctive characteristics and their
abundance as a raw material have attracted many researchers to look for
a cleaner method for MNPs synthesis [see Mahmood Ansari et al. (2021)
and references therein]. For the last three decades, new and effective
drugs of natural origin have been studied in marine macro and micro­
algae because they have high levels of hidden bioactive principles.
Numerous secondary metabolites, such as saponins, phenols, flavonoids,
carbohydrates, alkaloids, steroids, etc., are used in a wide range of in­
dustrial and biotechnological applications (Mahmood Ansari et al.,
2021). According to Ponnuchamy and Jacob (2016), compared to other
species of macroalgae, brown macroalgae have been exploited more in
the synthesis of MNPs since the cell wall of their cells contains a variety
of mucilaginous polysaccharides and functional groups that aid in the
absorption of metals. It has already been shown, for example, that the
synthesis of AuNPs from marine brown macroalgae Sargassum muticum
(Namvar et al., 2015), S. polycystin (Sivaraj et al., 2015), S. crassifolium
(Ouano et al., 2018), S. cichorioides (González-Ballesteros et al., 2021),
Turbinariaornata (Deepak et al., 2018) and Padina tetrastromatica
(Princy& Gopinath, 2018; Kumar et al., 2021) provide fruitful alterna­
tives to chemical methods conventional. Other macro- and microalgae
species already used in the synthesis of AuNPs include Stoechospermum
2
Environmental Research 213 (2022) 113711
marginatum, S. myriocystum, Turbinaria conoides, T. ornata, Spirulina
platensis, Corallina officinalis, Cladosiphonokamuranus, Laminaria
japonica, Ecklonia cava, Gracilaria verrucose, among others [see Roy et al.
(2013) and references therein].
Using these species for AuNPs biosynthesis can constitute an alter­
native to replacing the extensive use of several hazardous chemicals
commonly used during NPs synthesis and, therefore, can reduce bio­
logical impacts induced by the release of these products into the natural
environment. However, the “green nanoparticles” and/or “eco-friend
nanoparticles” label does not ensure that biosynthesized AuNPs are
harmless to non-target organisms. Although macro-and microalgae is
promising in synthesizing these particles (Mahmood Ansari et al., 2021),
previous studies have already warned about their (eco)toxicological
potential. According to Roy et al. (2013), “green nanoparticles” possess
the same dimensions as biomolecules such as proteins, DNA, and en­
zymes, to facilitate easy entry into living cells and the bloodstream from
where they can translocate into other organs. Furthermore, like tradi­
tionally synthesized NPs, these “green nanoparticles” can remain in
suspension in aquatic environments for more extended periods,
exposing living organisms to enhance the duration of toxicity. Previous
studies have reported that different “green nanoparticles” can cause
oxidative stress (which affects proteins/enzymes functioning in repair of
damage, cell membrane breakage, leakage of cellular components, and
inflammatory reactions) and genotoxicity (which leads to DNA damage,
mutation, and chromosomal aberration) [see Rana et al. (2020) and
references therein]. However, there is still no consensus among the
different studies. Therefore, depending on the raw material used in the
biosynthesis of NPs and the characteristics of the nanomaterials, toxicity
may be higher, lower, or non-existent. Therefore, this constitutes a
knowledge gap that needs to be filled to contribute to the biosafety
assessment of “green NPs” and predict their potential ecological impact
if disposed of (accidently or voluntarily) in natural environments.
Thus, we aimed to synthesize AuNPs from seaweed Gracilaria crassa
aqueous extract in the present study. As discussed by Baghel et al.
(2014), G. crassa (well-known red algae) presents grows abundantly
along the Mandapam coast (Tamil Nadu, India) and has potential sec­
ondary metabolites (Almeida et al., 2011). In addition, the species has
high biotechnological potential, having already been used in the
biosynthesis of nanoparticles for different purposes (e.g., Lavakumar
et al., 2015). Therefore, these aspects justify the choice of G. crassa in
this study. After synthesizing AuNPs from seaweed G. crassa aqueous
extract, we evaluated their toxicological potential, using Anopheles ste­
phensi larvae as model systems (in vivo). Our study provides new insight
into the use of G. crassa as a source of raw material for the biosynthesis of
AuNPs, but at the same time, it sheds light on its danger to non-target
organisms.
2. Material and methods
2.1. Preparation of seaweed biomasses for nanoparticle synthesis
The brown seaweed G. crassa was collected from the Mandapam
coast (Lat. 9◦ 28′ N; Long. 79◦ 12′ E) of Ramanathapuram District (Tamil
Nadu, India), similarly to Baghel et al. (2014). The samples were
exhaustively washed in seawater at the sampling site to remove epi­
phytes and debris attached to the fronds and then brought to the labo­
ratory in an icebox. After complete drying, the seaweed material was
ground to a fine powder using an electrical blender [as described in
Sangeetha (2021)] and stored in a brown bottle, protected from expo­
sure to sunlight until further use.
2.2. Preparation of aqueous broth and green synthesis of AuNPs
One gram of seaweed (G. crassa) fine powder was mixed with 100 mL
of deionized water, boiled 60 ◦ C for 30 min, cooled, and filtered through
filter paper (pore size: 11 μm, Whatman® 1001-325). From this, 30 mL
C. Kamaraj et al.
of G. crassa aqueous broth was supplemented with 70 mL of HAuCl4
solution (at 1 mM, pH 7.2), and the mixture was placed in an orbital
shaker at room temperature for the bio-reduction of Au+3 to Au0. The
dark violet color formed after adding HAuCl4 was characterized in the
range of 200–700 nm using a spectrophotometer operated at a resolu­
tion of 1 nm (Cyberlab UV-100). The bio-reduction of Au0 was moni­
tored periodically by measuring the absorbance of the solutions. The
AuNPs obtained from the reaction mixture were purified by repeated
centrifugation at 4000 rpm for 10 min, then dispersed the pellet thrice in
deionized water to remove the water-soluble biomolecules such as
proteins and secondary metabolites. The water-suspended NPs were
dried at 30 ◦ C overnight and then kept under a vacuum for 24 h to dry
the NPs as per Ramkumar et al. (2017).
2.3. Chemical components of G. crassa
To identify the phytocomponents present in the G. crassa extract,
samples were analyzed using Gas Chromatography-Mass Spectrometry
(GC-MS), like the study by Balasubramani et al. (2015). Such analysis
was made by injecting 1 μL of sample on a 5MS column of the GC-MS
model (PerkinElmer, Clarus 500, Waltham, Massachusetts), and Heli­
um was used as the mobile phase. The qualitative and quantitative an­
alyses of the samples were carried out using a CP 3800 Saturn 2200
GC-MS system. The following standard GC-MS operating procedure was
followed: initial program temperature was 80 ◦ C, then it was gradually
increased to 350 ◦ C at the rate of 3 C/min, Ion temperature: 200 ◦ C, and
◦
scan range: 20–500 AMU (Atomic Mass Unit). The peaks representing
mass to charge ratio characteristics of the solvent fractions were
compared with those in the mass spectrum library of the corresponding
organic compounds.
2.4. Characterization of biosynthesized AuNPs
2.4.1. UV–visible spectroscopy
The bio-reduction of seaweed aqueous extract into AuNPs after
adding HAuCl4 was monitored periodically using a UV–visible spectro­
photometer (Cyber Lab UV-100, Millbury, MA) in the range of 200–800
nm at regular intervals, similar to the studies by Wahab et al. (2018) and
Li et al. (2014).
2.4.2. Field emission scanning electron microscopy and high-resolution
transmission electron microscopy analyses
The field emission scanning electron microscopy (FESEM) analysis
(Zeiss Sigma FE-SEM, Germany) was executed by dropping a very small
amount of the thin films of the sample onto a carbon-coated copper grid.
The extra solution was removed with blotting paper, the grids were
dried for 5 min, and the specimen was observed under a mercury lamp.
The high-resolution transmission electron microscopy (HRTEM) surface
morphology of AuNPs was examined under the JEOL JEM 2100 high-
resolution transmission electron microscope at an accelerating poten­
tial of 0.1 mV.
2.4.3. Energy-dispersive X-ray (EDX) and X-ray diffraction (XRD)
measurement
The elemental composition of the biosynthesized AuNPs was
analyzed using the Energy-dispersive X-ray detection instrument (EDX)
similar to Fakhari et al. (2019) and Eltaweil et al. (2022) (Oxford INCA
400, United Kingdom). According to Shukla and Iravani (2018), this
technique gives a comprehensive sample mapping by analyzing
near-surface elements and estimating the elemental proportion at
different positions. Furthermore, the AuNPs formation was assessed by
an X-ray diffractometer (Rigaku Ultima IV, Japan), which constitutes a
non-destructive technique that provides detailed information about the
crystallographic structure, chemical composition, and physical proper­
ties of materials (Bandyopadhyay et al., 2016). The instrument was
operated at a voltage of 40 kV and a current of 30 mA with Cu kα
3
Environmental Research 213 (2022) 113711
radiation in a θ–2θ configuration. Based on the width of the XRD peaks,
the crystallite domain size of the AuNPs was calculated using Scherer’s
formula, also adopted by Dipankar and Murugan (2012) and Eltaweil
et al. (2022): D = 0.94 λ/βCosθ, where “D” is the average crystallite
domain size perpendicular to the reflecting planes, “λ” is the wave­
length, “β” is the full width at half maximum (FWHM), and “θ” is the
diffraction angle. To eliminate additional instrumental broadening, the
FWHM was corrected, using the FWHM from a large-grained Si sample:
βcorrected = FWHM2sample− FWHM2Si)1/2. The modified formula is valid
only when the crystallite size is smaller than 100 nm (Boulc’h et al.,
2001).
2.4.4. Fourier-transform infrared spectroscopy (FT-IR) analysis
Fourier-transform infrared spectroscopy (FT-IR) analyses were per­
formed to find the roles of the micro and macromolecules present in the
seaweed extract in the synthesis and the stabilization of the AuNPs. For
this, we adopted the procedures described in Manjunath et al. (2017),
with some modifications. Briefly, the biosynthesized AuNPs (100 mL))
residual solution was centrifuged at 5000 rpm for 10 min, and the
resulting pellet was redispersed in 10 mL of sterile distilled water. This
step aimed to remove any free biomass residue that was not the capping
ligand of the nanoparticles. Subsequently, the purified suspension was
freeze-dried to obtain a dried powder. About 0.2–0.5 mg of the dried
sample material was pressed with 300 mg dried KBr to get a pellet. The
background spectrum was recorded with a pellet containing 300 mg
KBr.
2.5. Assessment of toxicological potential
To evaluate the possible toxic effect of biosynthesized AuNPs in an in
vivo system, we used Anopheles stephensi larvae. Although A. stephensi is
the most important malaria vector in Asia (Sinka et al., 2011; Khan et al.,
2021), which causes a huge further medical and financial burden, its
ecological importance is unquestionable and cannot be neglected (Fang,
2010). Furthermore, it is becoming a model Anopheline species for
physiological, genetics, and toxicological studies, mainly due to its easy
reproduction in the laboratory environment and sensitivity to various
chemical compounds (Mead and Tu, 2008; Abinaya et al., 2018; Good­
arzi et al., 2019; Sharma et al., 2022).
The eggs A. stephensi were collected from water reservoirs in Salem
district (Tamil Nadu, India) using an “O” type brush, similarmente à
Kumar et al. (2012). These eggs were brought to the laboratory and
transferred to 18 cm × 13 cm x 4 cm enamel trays containing 500 mL of
water for hatching, having been kept under controlled conditions (28 ±
2 ◦ C, 70–80% relative humidity; a light-dark cycle of 14 h of light fol­
lowed by 10 h of dark). A. stephensi larvae were fed daily with 5 g of
ground dog biscuits (Pedigree, USA) and hydrolyzed yeast (Sigma-Al­
drich, Germany) in a 3:1 ratio, according to Kamaraj et al. (2012). Newly
emerged IV instar larvae were collected and used in the experiments.
Then, the larvicidal activity was assessed by following the WHO
(1996) procedure with some modifications and per Kamaraj et al. (2012)
methods. Briefly, the bioassay test was carried out in separate groups of
20 larvae/each (I, II, III, and IV instars) in 249 mL of water and 1.0 mL of
the G. crassa extract (nominal concentrations: 31.25, 62.5, 125, 250, and
500 ppm) and biosynthesized AuNPs (at the same concentrations as
above). The control was set up with distilled water. Larval food (0.5 mg)
was provided for each concentration tested. After 24 h of exposure, the
dead larvae were counted, and the percentage of mortality was reported
from the average of five replicates. Furthermore, at the end of the
experiment, the dead larvae were kept on a glass slide, observed external
morphology using a light microscope (10x magnification), and photo­
graphed using a digital camera. Deformities were designated according
to their similarities with reports from previous studies (Yu et al., 2015;
Mahyoub et al., 2016; Ragavendran et al., 2017; Chantawee and
&Soonwera, 2018; Murgia et al., 2022), involving anophelines (e.g.,
damage to the anal papillae, body distortion, alteration in body color,
C. Kamaraj et al. Environmental Research 213 (2022) 113711

damage to the digestive tract, modifications in the respiratory tubes, biosynthesized AuNPs, using Probit analysis.
detachment of bristles and cuticles, among others).
3. Results and discussion
2.6. Statistical analysis
3.1. Phytochemical composition of G. crassa extract and characterization
of the biosynthesized AuNPs
Data were analyzed using GraphPad Prism Software Version 9.0)
software for all analyzes, and significance levels were set at Type I error
Initially, to determine the phytocomponents present in the G. crassa
(p) values lower than 0.05. Initially, all data obtained were evaluated
extract, samples were analyzed via GC-MS. The GCMS chromatogram of
regarding the assumptions for using parametric models. For this, we
G. crassa extract showed 11 peaks identified after comparing the mass
used the Shapiro-Wilk test to assess the distribution of residual data, and
spectra and NIST libraries, indicating the presence of 11 phytocompo­
the Bartlett test was used to assess the homogeneity of variances. The
nents. Their chemical structure and active principles with their retention
two-way ANOVA (with Tukey post-test) was used to evaluate the effects
time, molecular weight, and molecular and structural formula are pre­
of the factors “larval stage” (four levels: I, II, III, and IV instars) and
sented in Table 1. We show that some phytocomponents in the G. crassa
“concentration” (five levels: 31.25, 62.5, 125, 250, and 500 ppm) and
extract have also been identified in other plant species. This is the case
their possible interactions on the mortality index of A. stephensi larvae
with naringin, 5-O-caffeoyl shikimic acid, phytol, and epiceanothic acid.
exposed to both the aqueous extract of G. crassa and the biosynthesized
The naringin (4′ ,5,7-trihydroxy flavanone 7-rhamnoglucoside) and its
AuNPs. In addition, correlation analyses were performed, followed by
metabolite naringenin are ubiquitously distributed in plant foods,
regression analysis. For a comparative evaluation of the influence of the
especially in grapefruit other related citrus species (Jagetia et al., 2003;
compounds added to the exposure media on the mortality rate of
Yilma et al., 2013). Although this phytocomponent is currently listed in
A. stephensi larvae (for each larval stage), we performed a two-way
the register of flavoring substances, allowing its use in food without
ANOVA (with Tukey post-test), considering the “exposure” factors.
restriction, its toxicological effects are still uncertain. While Kim et al.
(two levels: aqueous extract of G. crassa and biosynthesized AuNPs) and
(1998) reported that naringenin showed toxicity in the human hepa­
concentrations (five levels: 31.25, 62.5, 125, 250, and 500 ppm). The 24
toma cell line the HepG2, Macacus’ rhesus monkey kidney cell line
h lethal concentrations (LC50) were obtained by plotting the mortality
MA-104, and human lung cancer cell line A549; Li et al. (2014) found
index against the concentrations of the aqueous extract of G. crassa and

Table 1
Important compounds identified in the GC-MS analysis of G. crassa extract.
Compound name RT (min) MW Molecular formula Structure

Naringin 2.96 433 C21H21O10

5-O-caffeoylshikimic acid 13.94 335 C16H15O8

Eriodictyolhexoside 19.44 449 C21H21O11

Phytol 19.99 296 C20H40O

-
Trichloroacetic acid, 6-ethyl-3-octyl ester 20.99 302 C12H21O2Cl3

Phenyl-D-Mannoheptuloside 23.32 286 C13H18O7

Heptanoicacid, docosylester 26.46 438 C29H58O2

Epiceanothic acid 26.82 486 C30H46O5

Luteolin-4′ -O-rhamnosyl(1 → 2)glycoside 27.69 593 C27H29O15
3-Isopropyltricyclo[4.3.1.1(2,5)]Undec-3-En-10-Ol 28.48 206 C14H22O

5-Diethylsilanyloxy-4-ethyl-2 phenyl-3a,4,7,7a-tetrahydro-isoindole-1,3-dione 29.94 385 C22H31NO3Si

4
C. Kamaraj et al. Environmental Research 213 (2022) 113711

that no-observed-adverse-effect-level (NOAEL) of naringin in rats is

greater than 1250 mg/kg/day when administered orally for six
consecutive months.
The 5-O-Caffeoylshikimic is a compound of the Palmae family
(Harborne et al., 1974) and has currently been widely used in the pro­
duction of Tamiflu® or other antiviral drugs (Singh et al., 2020).
However, few plant sources of these acids have been described (e.g.,
Vaccinium corymbosum (Wan et al., 2012; Sheridan et al., 2012), Elaeis
Guineense (Gan et al., 2010), Solanum somalense (Chideh et al., 2014),
Vaccinium dunalianum (Gao et al., 2022), and Gaultheria spp. (Mier­
es-Castro et al., 2022). Therefore, our findings also reveal that the
seaweed G. crassa may constitute a promising source of this phyto­
component. However, the toxicity of this compound has also not been
widely evaluated. In Fukuoka (1982), for example, isolated 5-O-Caf­
feoylshikimic was isolated from bracken fern (Pteridium aquilinum) is a
principal constituent of its acutely toxic fraction, which causes depres­
sion of leucocytes and thrombocytes in calves, as well as an antihista­
mine effect in vitro, but had no hematuric effect in guinea pigs. On the
other hand, previous studies have shown several properties of phytol,
acyclic diterpene alcohol found in the essential oils of some aromatic
plants (e.g., Cleome serrata (McNeil et al., 2012), Lantana radula (Passos
et al., 2012), Adhatoda vasica (Saha and Bandyopadhyay, 2020);
Myzuspersicae(Gliszczyńska et al., 2021)], including antibacterial ac­
tivity (Lee et al., 2016; Saha and Bandyopadhyay, 2020; Nazemi, 2022),
anticonvulsant (Costa et al., 2012), antispasmodic (Pongprayoon et al.,
1992), antihyperglycemic (Upadhyay et al., 2022), antitumor (Alencar
Fig. 1. (A) Bio-reduction of seaweed aqueous extract into Au nanoparticle after
et al., 2018; De-Alencar et al., 2019), among others [see Islam et al.
adding HAuCl4 and(B)UV–Vis spectra of aqueous leaf extracts solution and
(2018)) and references therein]. The epiceanothic acid is a naturally
synthesized AuNPs Gracilaria crassa showing the surface plasmon resonance at
occurring ceanothane-type pentacyclic triterpenes isolated from the
547 nm.
seeds of traditional Chinese medicine [e.g., Ziziphus jujube (Li et al.,
2005) and has been previously evaluated as an anti-HIV-1 agent (Zhang
index of 1.3328 and viscosity of 0.8878 (cP) and scattering intensity was
et al., 2011). However, it should be noted that although these phyto­
found to be 31,499 (cps).
components have presented promising pharmacological and medicinal
The EDX profile of the AuNPs showed strong Au signals at 1.6 and
properties, their effects on non-target organisms are unknown are those
2.2 keV (Fig. 3A). An additional two peaks corresponding to carbon and
of other compounds identified in our study [e.g., trichloroacetic acid,
oxygen were observed. The carbon and oxygen peaks may be attributed
6-ethyl-3-octyl ester, phenyl-D-Mannoheptuloside, and heptanoic acid,
to the biomolecules present in the extract. Our EDX profile observations
docosyl ester (Table 1)], about which we know little.
agree with the previously reported results in the literature (Deepak
About biosynthesized AuNPs, our analyses revealed different mate­
et al., 2018). Furthermore, the other weak elemental signals are prob­
rial characteristics. Through the UV-VIS spectrophotometric analysis,
ably due to the X-ray emission of constituents from proteins, enzymes,
we observed that the reduction of HAuCl4 was indicated by the color
and other metabolites from the G. crassa extract. These constituents
changes of G. crassa extract, as shown in Fig. 1A. The reaction was rapid
often surround AgNP with a thin layer of capping organic material from
as the yellowish-brown color of the G. crassa extracts turned into ruby
the plant broth, and this enhances the AgNP stability in the solution for
red color within a few minutes, indicating formation of Au-NPs. Fig. 1B
several weeks after synthesis (Dinesh et al., 2015).
displays the UV–vis spectra of AuNPs as a function of reaction time and
The structure, size, and crystallinity of the biosynthesized AuNPs are
the progress of absorbance bands at 547 nm. Even though the scanning
shown in Fig. 3. The HRTEM photomicrographs (Fig. 3B–F) showed that
wavelength was between 200 and 800 nm, no other peaks were observed
an aqueous extract of G. crassa could synthesize triangles and spherical
besides the one at 547 nm. It indicates the formation of anisotropic
AuNPs after 24 h of reaction. The majority of spherical AuNPs were
(spherical and triangular) AuNPs using an aqueous extract of G. crassa,
around range from 18.7 to 93.7 nm. Also, the bright hexagonal spot in
the reducing agent. The UV–vis spectra did not show any significant
the selected area electron diffraction (SAED) pattern (Fig. 3G) from one
changes in λmax after 24 h, indicating the stable particle size and size
of the gold nano triangles in Fig. 3C–E reveals that the biosynthesized
distribution. These results confirm the previously reported studies on the
AuNPs are single crystalline. The hexagonal nature of the diffraction
seaweed extract of T. ornata with HAuCl4 transmuted color rapidly from
spots is a clear indication that the triangular gold nanoprisms are highly
yellowish-brown to ruby red, indicating the formation of AuNPs due to
oriented, with the top normal to the electron beam. The spots could be
the localized SPR (Deepak et al., 2018).
indexed based on gold’s face-centered cubic (FCC) structure. Similar
The FESEM provided further insight into the morphology and size
findings have been recorded in the terrestrial plants of Callistemon cit­
details of the biosynthesized NPs. The micrograph observation showed a
rinus-mediated AuNPs (Rotimi et al., 2019).
high density of AuNPs, which are uniformly distributed on the surface of
Regarding the XRD study, we observed that reflection peaks (Fig. 4A)
the cells, and the formation of polydispersed NPs, with mostly spherical
are distinctly exhibited at 2θ = 38.27o, 44.54o, 64.53o, and 76.44o were
and some polygonal shapes (Fig. 2A–B), similarly to what was reported
indexed with the planes (111), (200), (220), and (311) for the FCC gold,
by Kumar et al. (2012). The average diameter of the biosynthesized
as per the ICSD no.98-005-3763 (Jiang et al., 2014). The Bragg re­
Au-NPs was 32.0 nm ± 4.0 nm (mean ±SEM) (Fig. 2). The cumulative
flections (111) were intense relative to the (220) and (311) reflections.
result part from the analysis liberated was shown to be 65.1 nm as the
This feature indicates that the nanocrystals are mainly oriented along
average diameter of all bigger biosynthesized AuNPs. The polydispersity
(111), and it agreed with the SAED results. The unidentified broad peak
index value of the same Au-NPs released by the same machine was
at 20–30o is due to the low crystallinity of the organic material from the
0.419 at a measurement condition when the temperature is maintained
aqueous extract. The XRD pattern observed in this study (Fig. 4A) was
25 ◦ C inside the water diluents of the colloidal gold with a refractive

5
C. Kamaraj et al.
Fig. 2. (A–B) Electron microscopy images of biosynthesized AuNPs using the aqueous extract of Gracilaria crassa and information about its size.
Fig. 3. (A)Energy dispersive x-ray (EDX) spectrum showing the typical chem­
ical composition of the biosynthesized AuNPs using the aqueous extract of
Gracilaria crassa. (B–F) High-resolution transmission electron microscopy
(HRTEM) photomicrograph and (G) SAED pattern show biosynthesized AuNPs.
consistent with previous reports on the seaweed-mediated synthesis of
Sargassum muticum (Madhiyazhagan et al., 2015). Rotimi et al. (2019)
reported that diffraction peaks at 2θ values of 44.4o, 64.5o, and 77.5o,
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Environmental Research 213 (2022) 113711
Fig. 4. (A) X-ray diffraction (XRD) and (B) Fourier-transform infrared spec­
troscopy (FT-IR) pattern analysis of the biosynthesized AuNPs using the
aqueous extract of Gracilaria crassa.
which correspond to the (111), (200), and (220) facets of the
face-centered cubic crystal structure, confirm the successful synthesis
and the crystalline nature of the material.
Furthermore, we use FT-IR measurements to find the role of the
micro and macromolecules present in the seaweed extract in the syn­
thesis and the stabilization of the AuNPs (Fig. 4B and Table 2). The FTIR
spectra of the biosynthesized AuNPs showed a strong peak at 3307
cm− 1, which is characteristic of the O–H group of the phenolic and
alcoholic molecules. The NPs were stabilized by the hydrophilic
C. Kamaraj et al. Environmental Research 213 (2022) 113711

Table 2 in capping and stabilizing the biosynthesized AuNPs. The variations in

FT-IR shows the functional group involved biosynthesis of gold nanoparticles the absorption bands of the AuNPs may result from the bio-reduction
using the aqueous extract of Gracilaria crassa. process catalyzed by the phenolic compounds in the form of enzyme
Absorption peak (cm− 1) Functional groups Compound class reductase present in the G. crassa extract.
3420.62 O–H stretch, H–bonded Alcohols, phenols
2928.70 O–H stretch Carboxylic acids
2855.85 C–H stretch Alkanes 3.2. Assessment of toxicological potential
1649.81 –C– stretch
–C– Alkenes
1556.79 N–O asymmetric stretch Nitro compounds After biosynthesis and characterization of AuNPs, we evaluated their
1413.26 C–C stretch (in–ring) Aromatics
possible toxicity, considering that few studies assessed the toxicological
1332.69 N–O symmetric stretch Nitro compounds
1231.78 C–H wag (–CH2X) Alkyl halides potential of newly biosynthesized NPs in concomitance with the pro­
1143.44 C–N stretch Aliphatic amines cesses necessary for their manufacture. According to Sharma et al.
1080.45 C–N stretch Aliphatic amines (2022), the biosynthesis of metallic NPs via algae provides a greener
1028.91 C–N stretch Aliphatic amines route with no toxicity levels. However, this may not be valid for all
998.48 = C–H bend Alkenes
866.67 N–H wag 1◦ , 2◦ amines
biosynthesized AuNPs, and its toxicological potential may be dependent
827.81 C–H “oop” Aromatics on the evaluated model system. Therefore, assessing the (eco)toxico­
571.26 C–Cl stretch Alkyl halides logical potential of the new biosynthesized NPs is essential to provide
535.34 C–Br stretch Alkyl halides information about their biosafety, especially concerning non-target or­
ganisms, and may direct their use to activities or sectors that do not
impact the biota.
flavonoids present in the natural products. The absorption band at 2364
Thus, we compared in our study the possible toxicity of aqueous
cm− 1 corresponds to the O–H stretching of the carboxylic acids. The
extract of G. crassa vs. toxicity induced by biosynthesized NPs against
peak in the region of 1650 cm− 1 is due to the combination of the amide-I
A. stephensi larvae at different stages of development (I to IV instars). In
bond of proteins and the secondary stretching vibrations in the sec­
the bioassay involving the aqueous extract of G. crassa, we observed the
ondary structure of the proteins. This study indicates the role of protein
effect of the interaction between the factors “larval stage” and

Fig. 5. (A) Mortality index, (B) heatmap of Pearson correlation coefficient matrix, and (C) relationship between the mortality rate of Anopheles stephensi larvae and the
concentrations of aqueous extract of Gracilaria crassa tested (in ppm). Under “A,” the bars indicate the mean +SD. The data were submitted to two-way ANOVA, with
Tukey’s post-test at 5% probability. Distinct letters or symbols indicate significant differences between the mortality rates of each larval stage exposed to the different
concentrations of aqueous extract of G. crassa. Distinct lowercase letters positioned at the base of the bars indicate differences between the larval stages at each tested
concentration. The summaries of the statistical analyses [two-way ANOVA (in “A") and linear regression (in “C")] are presented at the top of the figures..

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C. Kamaraj et al. Environmental Research 213 (2022) 113711

“concentration” (Fig. 5A). However, the “concentration” factor
accounted for 86.49% of the total variance (F-value = 513.3; p<0.001)
and was therefore considered an extremely significant effect. In addi­
tion, we observed positive and statistically significant correlations be­
tween the variables “concentration (ppm)" vs. “mortality (%)" in all
larval stages evaluated (Fig. 5B), and the regression model best fitting
the data was the simple linear regression model (Fig. 5C). On the other
hand, the 1st instar larvae were more sensitive than the other stages
when exposed to 62.5, 125, 250, and 500 ppm (Fig. 5A). No mortality
was found in the control group.
In the bioassay involving the biosynthesized AuNPs, we observed a
significant effect of the factors “larval stage” and “concentration”
(alone), with no interaction between the factors (Fig. 6A). Similarly, to
the results obtained for the aqueous extract of G. crassa, the factor
“concentration” was responsible for more than 98% of the total variance
(F-value = 733.0; p<0.001) and, therefore, was also considered highly
significant in the evaluation of the toxicity of biosynthesized AuNPs.
However, although we observed significant correlations between the
variables investigated (in all larval stages) (Fig. 6B) and linear increase
in the toxicity of AuNPs as their concentrations were increased (Fig. 6C),
the mortality rate of larvae (I, II, III and IV instars) did not differ within
each concentration tested (Fig. 6A). In addition, we observed that the
mortality rate of i-instar larvae did not vary among the different con­
centrations of biosynthesized AuNPs vs. aqueous extract of G. crassa
(Fig. 7A). However, the larvae of II, III, and IV instars were more sen­
sitive to biosynthesized AuNPs (i.e., they presented a higher mortality
rate) compared to the aqueous extract of G. crassa when exposed to
concentrations of 62.5, 125, 250, and 500 ppm (Fig. 7B–D, respectively).
Regarding probit analysis, Table 3 presents the statistical summary
for the determination of LC50 of the aqueous extract of G. crassa and
biosynthesized AuNPs against A. stephensi larvae (I to IV instars). The
concentration-response curves are presented in Fig. 8. Generally, the
mortality rates were associated with different morphological abnor­
malities, the frequency of which was similar among the groups exposed
to the aqueous extract of G. crassa and biosynthesized AuNPs. In both
groups, treatments induced toxic effects on many body regions
(including the thorax, abdomen, and anal gills), emphasizing loss of
external hairs, crumbled epithelial layer of the outer cuticle, and
shrinkage of the larvae (Fig. 9).
In general, these results confirm that exposure to aqueous extract of
G. crassa and biosynthesized AuNPs induces significant toxicological
effects in A. stephensi larvae. However, the lowest LC50 values observed,
especially in larvae of II, III, and IV instars exposed to biosynthesized
AuNPs, compared to the aqueous extract of G. crassa, suggest a more
significant effect on the survival of the animals. On average, the LC50
values of biosynthesized AuNPs obtained for these stages were 49%
lower than those obtained for larvae exposed to aqueous extract of
G. crassa, which is in line with other reports on the biosynthesized

Fig. 6. (A) Mortality index, (B) heatmap of Pearson correlation matrix, and (C) relationship between the mortality rate of Anopheles stephensi larvae and biosynthesis
concentrations of gold nanoparticles using the aqueous extract of Gracilaria crassa. Under “A,” the bars indicate the mean +SD. The data were submitted to two-way
ANOVA, with Tukey’s post-test at 5% probability. Distinct letters or symbols indicate significant differences between the mortality rates of each larval stage exposed
to the different concentrations of aqueous extract of G. crassa. Distinct lowercase letters positioned at the base of the bars indicate differences between the larval
stages at each tested concentration. The summaries of the statistical analyses [two-way ANOVA (in “A") and linear regression (in “C")] are presented at the top of the
figures. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

8
C. Kamaraj et al. Environmental Research 213 (2022) 113711

Fig. 7. Mortality index of Anopheles stephensi larvae [(A) I instar, (B) II instar, (C) III instar, and (D) IV instar] exposed to the aqueous extract of Gracilaria crassa and
to biosynthesized AuNPs. Bars indicate the mean +SD. The data were submitted to two-way ANOVA, with Tukey’s post-test at 5% probability. Statistical summaries
are displayed at the top of the charts. Distinct lowercase letters indicate differences between groups exposed to each type of exposure concentration. Asterisks
indicate significant differences between groups exposed to biosynthesized AuNPs vs. aqueous extract of Gracilaria crassa. 31.25, 62.5, 125, 250, and 500 ppm refer to
the concentrations to which A. stephensi larvae were exposed.

Table 3
Lethal concentration 50% mortality (LC50) calculated (via probit analysis) of aqueous extract of Gracilaria crassa and gold biosynthesized nanoparticle from G. crassa
leaf extract against the larvicidal activity of Anopheles stephensi larvae (I to IV instars).
Larval stage Exposure Best-fit values 95% CI (profile likelihood Goodness of fit

LogLC50 LC50(ppm) LogLC50 LC50 (ppm) Degrees of freedom R square

I instar AE G. crassa 1.851 70.96 1.798 to 1.902 62.79 to 79.86 16 0.9784

II instar AE G. crassa 2.158 143.9 2.118 to 2.198 131.2 to 157.8 16 0.9834
III instar AE G. crassa 2.186 153.6 2.144 to 2.229 139.4 to 169.4 16 0.9819
IV instar AE G. crassa 2.151 141.5 2.115 to 2.186 130.4 to 153.6 16 0.9895
I instar Bio AuNPs 1.817 65.55 1.774 to 1.858 59.45 to 72.06 16 0.9858
II instar Bio AuNPs 1.841 69.34 1.793 to 1.888 62.02 to 77.26 16 0.9815
III instar Bio AuNPs 1.869 73.98 1.828 to 1.909 67.32 to 81.12 16 0.9864
IV instar Bio AuNPs 1.902 79.81 1.865 to 1.938 73.31 to 86.77 16 0.9885

AuNPs that showed the high toxicity of these NPs against many mos­
quito larvae (Lallawmawma et al., 2015; Sundararajan and Kumari,
2017; Deepak et al., 2018; Alhag et al., 2021), which have been sug­
gested that different phytocomponents present in plant extracts play an
essential role in the larvicidal potential of biosynthesized NPs, which
may, as demonstrated by Oliveira et al. (2016), interfere in the func­
tioning of distinct biochemical and physiological processes that hurt the
survival and growth of the mosquito larvae. Similar mechanisms may
have been dominant for the toxicological potential of biosynthesized
AuNPs in our study, especially when considering the prior knowledge of
the toxicity of some phytocomponents identified in G. crassa, such as
naringin, 5-O-caffeoyl shikimic acid, phytol, and epiceanothic acid
(Table 1). However, the most significant effect of biosynthesized AuNPs
is likely related to the size of NPs that make them more accessible and
easier to penetrate or expose to larval tissue with high efficiency in
killing the larvae.
On the other hand, our data demonstrate that the toxicity of bio­
synthesized AuNPs in our study presents toxicological potential distinct
from other AuNPs synthesized from different biological materials. Alhag
et al. (2021), for example, identified that the LC50 (24 h) of bio­
synthesized AuNPs from Acalypha fruticosa leaf extracts against the third
instar of C. pipiens was 30,248 ppm. In Sundararajan and Kumari (2017),
the LC50 (24 h) of synthesized AuNPs from Artemisia vulgaris leaf extract
and essential oil against Aedes aegypti (III instar) were 62.47 ppm and
111.15 ppm, respectively. On the other hand, when A. stephensi larvae
(IV instar) were exposed to biosynthesized AuNP from Turbinariaornata,
Deepak et al. (2018) identified an LC50 (24 h) of 12.79 ppm, which was
approximately 6.5 times lower than that reported for biosynthesized
AuNPs using Jasminum nervosum leaf extract against Culex quinque­
fasciatus larvae (III instar) (LC50 (24 h) = 82.62 ppm) (Lallawmawma
et al., 2015). The AuNPs synthesized from Anthocephalus cadamba
extract were highly toxic to C. quinquefasciatus larvae (I instar: 0.612
ppm; II urge: 1291 ppm; III instar: 3526 ppm; IV instar: 5631 ppm)
(Jeyalalitha et al., 2013). The different LC50 (24 h) observed for the
different AuNPs are explained by the chemical and biological nature of
the biological materials used as raw material, size, and format of NPs, as
well as the animal models (and stages of larval development) that were
used as test organisms. On the one hand, these studies (as well as ours)
point to the potential use of AuNPs in the control of disease-transmitting
mosquitoes; on the other hand, they shed light on the (eco)toxicological
danger of these NPs, if present in aquatic environments. In this sense, the
ecological role of mosquitoes [including their environmental services (e.

9
C. Kamaraj et al.
Fig. 8. Mortality curve, estimated by the concentration-response (Probit) for Anopheles stephensi larvae [(A) I instar, (B) II instar, (C) III instar, and (D) IV instar] in
response to different concentration de aqueous extract of Gracilaria crassa and gold biosynthesized nanoparticle from G. crassa leaf extract. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
g., pollination and release of nutrients that happen when their offspring
feed on organic waste) and their importance in maintaining food chains]
cannot be overlooked (Fang, 2010). Therefore, knowing the impact of
new substances, compounds, or (nano)materials on the survival of these
animals is a crucial step for the conservation of species and considering
the risks inherent in the possible disposal of these new products in the
sweetening ecosystems.
Finally, it is necessary to consider that our study is not exhaustive
and that further investigations should be carried out to expand our
knowledge about the diversity of applications of biosynthesized AuNPs,
as well as their (eco)toxicological potential. Although our study points
to the dangerousness of AuNPs for the experimental model used, other
organisms at different trophic levels should also be evaluated. Further­
more, evaluations involving concentrations of AuNPs on a micro-and
nanoscale (μg/L and/or ng/L), which simulate their possible presence
in the freshened ecosystems, will be necessary for a more realistic
assessment of the impact of these new nanomaterials on the biodiversity
of aquatic ecosystems. It is equally important to evaluate the mecha­
nisms intrinsic to the toxicity of AuNPs in the animal model studied and
in other organisms. In this case, biochemical, physiological, genotoxic,
mutagenic, molecular, and histopathological biomarkers can help
elucidate how biosynthesized AuNPs can drastically alter individuals’
fitness. On the other hand, our study is the first to support, in the same
work, the feasibility of the synthesis of AuNPs from the brown seaweed
G. crassa, as well as aspects of its (eco)toxicological potential, contrib­
uting to the initial evaluation of its environmental/ecological safety.
This aspect has not been the focus of many studies of a similar nature.
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Environmental Research 213 (2022) 113711
4. Conclusion
In conclusion, our study demonstrated the feasibility of using
seaweed Gracilaria crassa aqueous extract for the biosynthesis of AuNPs,
which can be part of the list of raw materials available for use in
different sectors, with the advantage of being produced from natural
sources. However, the in vivo assays performed in our study demon­
strate that caution is necessary regarding the extensive use of bio­
synthesized AuNPs given the high toxicity of the evaluated model
organisms (A. stephensi larvae), confirming that the “green nanoparticles”
and/or “eco-friend nanoparticles” label does not ensure that bio­
synthesized AuNPs are harmless to non-target organisms. In this sense, it
is strongly recommended that further studies be conducted to expand
our knowledge about the environmental/ecological biosafety of bio­
synthesized AuNPs, combining nanotechnological development with
environmental/ecological issues. Studies that anticipate the evaluation
of the (eco)toxicological effects of newly synthesized materials are
essential for directing strategies for their sustainable use.
Author contribution statements
Chinnaperumal Kamaraj: study conception and design, data collec­
tion, analysis, and interpretation of results, and draft manuscript prep­
aration, Sengodan Karthi: study conception and design, data collection,
analysis, and interpretation of results, and draft manuscript preparation,
Appadurai Daniel Reegan: study conception and design, data collection,
analysis, and interpretation of results, and draft manuscript preparation,
Govindhasamy Balasubramani: study conception and design, data
collection, analysis, and interpretation of results, and draft manuscript
C. Kamaraj et al.
Fig. 9. (A) Anatomy schematic design of a mosquito larva. (B) Representative image of Anopheles stephensi larvae not exposed to treatments (“control” group) and
(C–F) of some abnormalities identified in A. stephensi larvae exposed to aqueous extract of Gracilaria crassa and gold biosynthesized nanoparticle from G. crassa leaf
extract. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
preparation, Govindaraju Ramkumar: study conception and design, data
collection, analysis, and interpretation of results, and draft manuscript
preparation, Kandaswamy Kalaivani: study conception and design, data
collection, analysis, and interpretation of results, and draft manuscript
preparation, A. Abduz Zahir: study conception and design, data collec­
tion, analysis, and interpretation of results, and draft manuscript prep­
aration, Paramasivam Deepak: study conception and design, data
collection, analysis, and interpretation of results, and draft manuscript
preparation, Sengottayan Senthil-Nathan: study conception and design,
data collection, analysis, and interpretation of results, and draft manu­
script preparation, Md. Mostafizur Rahman: analysis, and interpretation
of results, and drafted manuscript preparation, Abu Reza Md Towfiqul
Islam: analysis, and interpretation of results, and drafted manuscript
preparation, Guilherme Malafaia: analysis, and interpretation of results,
and drafted manuscript preparation, All authors reviewed the results
and approved the final version of the manuscript.
Ethical aspects
The ethical standards for animal experimentation performed all
experimental procedures and meticulous efforts were made to ensure
that the animals suffered as little as possible and reduce external sources
of stress, pain, and discomfort. The current study has not exceeded the
number of animals needed to produce reliable scientific data. This
article does not refer to any study with human participants performed by
any authors.
11
Environmental Research 213 (2022) 113711
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We are grateful to the Sophisticated Test and Instrumentation Centre
(STIC) situated at Cochin University of Science and Technology, Cochin
682 022, Kerala, India, for providing the facilities to carry out the
characterization studies, such as XRD, FTIR, and SEM-EDX analyses. CK
acknowledges the Department of Science & Technology, Science and
Engineering Research Board (SERB), New Delhi, India, for the grant of
the National Post-Doctoral Fellowship (PDF/2016/000496). Further­
more, the authors are grateful to the Brazilian National Research Council
(CNPq) (Brazilian research agency) and Instituto Federal Goiano (proc.
n. 23219.000875.2022-75) for the financial support. Malafaia G. holds a
productivity scholarship from CNPq (proc. n. 307743/2018-7).
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