Chemosphere 303 (2022) 135162

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

Effects of microplastics on lentil (Lens culinaris) seed germination and

seedling growth
Y. Sanath K. De Silva a, b, Uma Maheswari Rajagopalan c, *, Hirofumi Kadono a, Danyang Li a
a
Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama-shi, Saitama, 338-8570, Japan
b
Department of Mechanical and Manufacturing Engineering, Faculty of Engineering, University of Ruhuna, Hapugala, Galle, 80000, Sri Lanka
c
Department of Mechanical Engineering, Shibaura Institute of Technology, 3-7-5 Toyosu, Koto City, Tokyo, 135-8548, Japan

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

• The effect of polyethylene microplastics

on lentil was investigated using Bio­
speckle Optical Coherence Tomography
(bOCT).
• bOCT is sensitive enough to observe the
adverse effect of PEMPs on seed germi­
nation at an early stage before the
germination.
• A significant reduction of bOCT contrast
was observed after 6 h for all PEMPs
exposure, and the effect was dose-
dependent.
• Physical parameters and antioxidative
enzymes were measured and compared
with bOCT results.

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

Handling Editor: Michael Bank Widespread use of plastics and mishandling has resulted in severe environmental issues affecting seed germi­
nation and seedling growth. This study investigates the effect of polyethylene microplastics (740–4990 nm
Keywords: PEMPs) on lentil (Lens culinaris) seed germination and seedling growth using Biospeckle Optical Coherence
Microplastics Tomography (bOCT), a technique that we successfully demonstrated earlier in visualizing the internal activity of
Optical coherence tomography (OCT)
plants. Lentil seeds were exposed to PEMPs bioassay for seven days with 10, 50, and 100 mg L− 1 concentrations.
Biospeckle
The average speckle contrast was calculated after 0 h, 6 h, 12 h, and 24 h of exposure, and statistically significant
Seed germination
Seedling growth differences were observed just after 6 h of exposure under all the treatments. However, with conventional pa­
rameters, germination viability, germination rate, root and shoot lengths, fresh and dry seedling weights, and
antioxidative enzymes, no significant effect was observed until 2 d of exposure. The results revealed that the
presence of PEMPs significantly reduced the internal activity at the initial stages that could be visualized only by
the use of bOCT, which has never been observed till now. Our results demonstrated for the first time the effect
that microplastics indeed could hinder the internal activity during germination of the seeds, possibly resulting
from the physical blockage of pores leading to stunted growth at later stages.

* Corresponding author.
E-mail addresses: sanath@mme.ruh.ac.lk (Y.S.K. De Silva), uma@shibaura-it.ac.jp (U.M. Rajagopalan), kadono@mail.saitama-u.ac.jp (H. Kadono).

https://doi.org/10.1016/j.chemosphere.2022.135162
Received 16 March 2022; Received in revised form 24 May 2022; Accepted 27 May 2022
Available online 30 May 2022
0045-6535/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Y.S.K. De Silva et al.
1. Introduction
The effects of microplastics on plant growth, seed germination, the
health of animals and humans are getting worldwide attention due to its
widespread usage, and mismanagement. Microplastics (MPs) are plastic
particles with a diameter less than 5 mm are classified as primary or
secondary microplastics based on their formation (Nel and Froneman,
2015). The primary MPs are small plastic debris initially manufactured
with a particle size of less than 5 mm. In general, primary MPs exist in
cosmetics, textiles, scrubbing agents, and toiletries (Boucher and Friot,
2017). Secondary MPs are the most common source of pollution, formed
when the fragmentation of larger plastic particles is fragmented by
natural forces such as photodegradation, biodegradation, thermoox­
idative degradation, mechanical degradation, and physical stress
(Andrady, 2011). They mainly arise from waste incineration and land­
fills (Dris et al., 2016).
Because of overwhelming usage and mismanagement, MPs accu­
mulate at an alarming rate in aquatic and terrestrial environments,
significantly impacting plant growth (De Silva et al., 2021b). It is a
well-known fact that the ocean behaves as the main sink for microplastic
accumulation through various sources such as effluents from wastewater
treatment plants (WWTP) (McCormick et al., 2014), commercial ship­
ping processes (Barnes and Milner, 2005), and atmospheric deposition
(Szewc et al., 2021). Therefore, the aquatic ecosystem is greatly affected
by MPs inhibiting seed germination and aquatic plant growth (Yuan
et al., 2019). Microplastics can be adsorbed onto the root of duckweed
species (Dovidat et al., 2019; Kalčíková et al., 2017) and are largely left
accumulated in mosses (Capozzi et al., 2018), and the mechanical
blockage results in lack of water and nutrient uptake inhibiting the
growth of root and shoot. Reports have shown that uptake of MPs gets
internalized in plants (Bandmann et al., 2012) depending on the root
and xylem characteristics (surface area, volume), plasma membrane
potential, organic and geometric properties of plastic debris (material,
size, and geometry) (Trapp, 2000). Moreover, the growth and photo­
synthesis activity of algae species could be significantly affected by MPs
inducing oxidative stress (Bhattacharya et al., 2010; Zhang et al., 2017;
Mao et al., 2018).
At the beginning, there was less focus on investigating the effect of
MPs on the terrestrial ecosystem. However, recently, due to the rapid
increase of microplastic accumulation by anthropogenic activities and
different environmental sources such as plastic mulching, road dust (Su
et al., 2020), sewage sludge (Gao et al., 2020), atmospheric deposition
(Szewc et al., 2021), and landfilling (Sun et al., 2021) attention has been
shifting toward the terrestrial ecosystem (Feng et al., 2021). Subse­
quently, MPs could pose potential threats to terrestrial biota inhibiting
the growth of plant and soil organisms. Bosker et al. (2019) found that
the vascular plant Lepidium sativum could accumulate MPs on pores
resulting in physical blockage of the seed capsule, thus delaying the
germination and root growth due to inhibition of water and nutrient
uptake.
Polystyrene nanoplastics (PSNPs) were adsorbed and deposited on
the spore surface of Ceratopteris pteridoides while reducing spore size and
germination ratio (Yuan et al., 2019). Under MPs exposure, the seed
germination and the shoot height of Lolium perenne were reduced, and
the root biomass and the shoot to root ratio significantly differed be­
tween treatments (Boots et al., 2019). In wheat (Triticum aestivum L.),
above and below ground sections were impaired during vegetative and
reproductive growth (Triticum aestivum L.) (Qi et al., 2018), where MPs
were taken up and transferred to the stem and leaves through the xylem
(Lian et al., 2019). Moreover, MPs could induce cytotoxic, genotoxic,
and oxidative damages to Vicia faba and Allium cepa, inducing significant
growth reduction and particle internalization in the cellular compart­
ments. The toxic effect was significantly enhanced by nanoplastics, in
which the growth reduction was more significant for the microplastic in
comparison to that of nanoplastics (Jiang et al., 2019; Giorgetti et al.,
2020).
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Chemosphere 303 (2022) 135162
The adverse effect of MPs on soil organisms such as earthworms
(Lwanga et al., 2016) and snails (Song et al., 2019) has been reported by
many studies in which their weight, growth, activity, feeding behavior,
and reproduction were highly affected (Zhu et al., 2018). Furthermore,
earthworms have been recorded transporting MPs from the surface to
the groundwater level, contaminating groundwater and exposing other
soil biotas (Rillig et al., 2017). Recently, the micro and nanoplastics
have been found in edible fruits (apple, pear), vegetables (broccoli,
lettuce, carrot, and potatoes) (Oliveri Conti et al., 2020), and table salt
(Yang et al., 2015), emphasizing the potential threat to human health
due to the transmittance of MPs through the food chain.
Agro-ecosystem, polyethylene (PE) is frequently used as agricultural
material, especially plastic mulching. Therefore, there is a high possi­
bility of accumulation of an excessive amount of PEMPs in the terrestrial
ecosystem, which greatly impacts seed germination. Investigation per­
formed by Pignattelli et al. (2020) on garden cress (L. Sativum) suggested
the acute and chronic toxicity induced under different microplastics
exposure in which the PE showed the highest acute toxicity compared to
other microplastics tested in that study. A significant negative effect of
PE debris on soybean (Glycine max) plant growth and germination
viability was examined (Li et al., 2021a). Moreover, PEMPs can accu­
mulate on the root of hydroponic maize plants and inhibit the water and
nutrient uptake causing the growth reduction (Urbina et al., 2020).
Furthermore, PEMPs have a great tendency to adsorb different organic
pollutants inducing a reduction in leaf development and a 30% reduc­
tion in seed germination in L. sativa, whereas 70% growth reduction was
recorded as the effect of PEMPs alone (Martín et al., 2021).
Lentils are economically substantial legume crops cultivated world­
wide that contain a high amount of protein, fiber, low calories, essential
amino acids, fatty acids, and trace minerals (Alam et al., 2019). In
addition, lentils help in controlling diabetes, boosting metabolism,
preventing cancer, improving digestion, and lowering the risk of car­
diovascular diseases (Danihelová and Šturdík, 2012). Therefore, it be­
comes the most common pulse in day-to-day life, and human
consumption is relatively high, especially in West Asia, East, and North
Africa. In 2018, the estimated global lentil market was 6.3 million tons,
and it is predicted to reach up to 8.4 million tons in 2024 (Asia, 2020).
Moreover, the lentil species have been extensively used to observe the
toxicity of different environmental stressors on seed germination and
plant growth (De Silva et al., 2020, 2021a; Hossain et al., 2020). Many
research studies reported the impact of engineered nanoparticles on the
germination of lentil seeds and seedling growth (Khan et al., 2019;
Siddiqui et al., 2018).
Effects of the external agents on seed germination and the seedling
growth were mainly assessed by conventional measurements such as
germination viability, germination rate, shoot length, root length,
seedling weight, and chemical analysis. However, these are destructive
post-harvesting measurements that require relatively long periods to
obtain reliable results. Consequently, there is an urgent requirement for
futuristic methods that could monitor the impact of an external stressor
on the seed germination at an early stage.
Optical Coherence Tomography (OCT) is a well-known optical
interferometric method that facilitates obtaining a non-invasive struc­
tural image of a biological sample in vivo up to micrometer-scale spatial
resolution with high speed (Huang et al., 1991). A low-coherence light
source is used in OCT, and its applications have been widely spread in
biomedical fields due to non-contact and non-destructive observation
potential (Davis et al., 2019; Rebolleda et al., 2015). Recently, efforts
have been made to use OCT in agriculture, plant biology, and botany
(Wijesinghe et al., 2017; Rateria et al., 2019). Moreover, our group has
proposed the bOCT and has investigated the response of plant leaf to
ozone (Srimal et al., 2013, 2015; Srimal, 2015), change of internal
biological activities in seed germination under different environmental
conditions (Lim et al., 2019), the impact of gibberellic acid (GA3) on
plant leaf (Rajagopalan et al., 2020), micronutrient and toxic effect of
increasing Zn concentration on the seed germination and the seedling
Y.S.K. De Silva et al. Chemosphere 303 (2022) 135162

growth (De Silva et al., 2021a), and impact of Acid Mine Drainage
(AMD) on seed germination (Li et al., 2022).
Many researchers have investigated the impact of MPs on seed
germination and seedling growth and reported different adverse effects
emphasizing the need for more studies to fully understand the effects
(Bosker et al., 2019; Jiang et al., 2019). Nevertheless, there exist a few
limited studies and methods that are able to monitor the internal bio­
logical activity changes under the exposure of MPs. Therefore, this study
investigates the effect of (740–4990 nm) PEMPs on lentil seed germi­
nation and seedling growth by monitoring the changes in internal ac­
tivity using bOCT under different PEMPs concentrations and also
compare with the conventional measures.
2. Materials and methods
2.1. Microplastics particles and characterization
In this study, polyethylene microspheres were used to investigate the
impact of microplastics on lentil seed germination with specified parti­
cle sizes of 740–4990 nm. The polyethylene microspheres were pur­
chased from Cospheric LLC (Santa Barbara, California 93160, USA) in
dry powder form. The small size and pure polyethylene formulation (no

Fig. 1. Schematic representation of fiber-based experimental biospeckle Optical Coherence Tomography system (a). Photograph of practical experimental setup
constructed on optical bench utilizing optical fibers (b). Illumination of seed by laser beam for the scan (c). Time sequence speckle images for calculation of bio­
speckle OCT contrast (d). The system includes an SLD: superluminescent diode, PC: polarization controller, L1–L6: objective and collimating lenses. Incident power
given to the seed sample was around 5 mW, which was well below the damage threshold of seeds.

3
Y.S.K. De Silva et al.
additives, colorants, or solvents) are advantageous for use in micro­
plastics (MPs) to observe the individual effect of microplastics on seed
germination and plant growth. The emulsion solution of 0.05% Tween
80 was made to overcome the hydrophobic characteristics of particles
and achieve proper dispersing ability. Tween is a non-ionic, bio-
compatible surfactant often used for dispersing hydrophobic particles in
aqueous solutions. Different concentrations of 10, 50, and 100 mg L− 1
PEMPs were prepared, and the following protocol was applied to ach­
ieve the high dispersity and uniformity of the emulsion. First, the solu­
tions were vortexed for 1 min, centrifuged for 5 min, and sonicated at 28
◦
C for 10 min. The same protocol was repeated six times until all the
particles were appropriately dispersed and no clusters of PEMPs
appeared in the solutions. The relevant concentrations were selected
based on the concentrations used in similar studies (Jiang et al., 2019;
Guo et al., 2021).
2.2. Plant materials and treatments
Lentil seeds were bought from an organic seed-producing firm
(Greenfield Project Co., Ltd.) and stored in a dry spot until use. Healthy
seeds around an approximate weight of 35 mg, had the surfaces cleaned
and through a disinfection process before the germination to ensure
actual germination ability. The disinfection process was done by
immersing seeds in 2.5% H2 O2 solution for 15 min. After that, seeds
were thoroughly washed thrice and exposed to PEMPs concentrations of
10, 50, and 100 mg L− 1, with distilled water being used as the control.
For the OCT experiments, six seeds of lentils were placed in a Petri dish
and kept inside a growth chamber (Conviron, Controlled Environmental
Ltd, Canada) maintained at an air temperature of 25 ◦ C/20 ◦ C, a light
intensity of 260–350 μmol m− 2 s− 1/0 μmol m− 2 s− 1, and relative hu­
midity of 55–65% following a 12 h/12 h day/night cycle. The bOCT
contrast images were taken after 0, 6, 12, and 24 h of exposure to
PEMPs.
2.3. Optical coherence tomography (OCT) experimental system
Fig. 1 (a) and (b) show the schematic diagram of the OCT system and
practical experimental design developed on the optical bench using
optical fibers used for the image acquisition, respectively. The system
consists of a sample arm, reference arm, custom-built spectrometer, data
processing unit, and low coherent light source (SUPERLUM, SLD-371-
HP3-DBUT-SM-PD, Ireland) of 836.1 nm wavelength and 55.2 nm
bandwidth. In the free space (in the air), the lateral (Δx) and axial (Δz)
resolution of the system was estimated as 22 μm and 6 μm respectively.
According to the following equations,
[ ]
4λ0 f
Δx = , (1)
π d
2 ln 2 λ0 2
Δz = , (2)
π n Δλ
where n is the refractive index of the tissue (n = 1.4) (Seyfried and
Fukshansky, 1983), λ0 is the central wavelength, Δ λ is the bandwidth, f
is the focal length, and d is the beam diameter.
The illuminated light from the source was split into sample and
reference arms using the 2 × 2 50/50 fiber coupler (TW850R5A2-2 × 2
Wideband Fiber Optic Coupler, 850 ± 100 nm, THORLABS, UK) after
passing through a circulator (AC Photonics, Inc. USA). In the sample
arm, the light was focused onto the sample in which the light beam was
laterally scanned by galvano scanning mirror (Model_GVS012, THOR­
LABS, UK) to obtain 3D OCT structural images. The sensitivity roll-off
was around 7.94 dB/mm, and the measured sensitivity was 96 dB at a
depth of 0.24 mm. The lights backscattered from the sample and refer­
ence arm were combined by the coupler and directed to a custom-made
spectrometer where interfering light was collimated by lens L5. The
4
Chemosphere 303 (2022) 135162
spectrometer consists of a grating, line scan camera (L104k-2 k, BASLER,
Germany), and a lens L6 with a 200 mm focal length. After k-λ con­
version, the spectral interference signal was Fourier transformed to get
the depth-resolved reflectivity profile of the sample. At each scanning
position total of a hundred frames of 2048 (x) 512 (z) pixels were
recorded at a rate of 7.4 frames per second while scanning the probing
beam with the Galvano mirror in x-direction using custom made soft­
ware by LabVIEW. Therefore 13.5 s were taken to calculate one bOCT
scan from 100 OCT cross-section images. bOCT images calculations
using MATLAB, and the processing time took around 8 s totaling to 21.5
s to obtain a single bOCT image.
The OCT scans were performed after drying the lentil seeds properly
with paper towels to eliminate the effect of moisture on the seed coat.
For each PEMPs treatment, six replicates were used for the OCT obser­
vations. The bottom surface area close to the meridian of the lentil seeds
has the highest contact with PEMPs solution due to its natural curvature.
Therefore, all the seeds were fixed and adjusted in a proper orientation
where the center of the seeds could be successively scanned to ensure
that OCT images of all the seeds were acquired from the same position. A
photo of the seed illuminated by the probing beam for the scan is shown
in Fig. 1c. The CCD camera used to get this photo has a sensitivity in the
infrared (IR) region.
2.4. Biospeckle phenomena and contrast
The biospeckle technique is a non-destructive and non-invasive
method based on the optical phenomenon, occurring when a biolog­
ical object is subjected to the illumination of coherent laser light.
Because of the interference of backscattered light from the biological
object, a random granular speckle pattern is formed, and that phe­
nomenon is called a biospeckle (Aizu and Asakura, 1996). A static object
has stationary scattering centers that produce granular interference
patterns when illuminated with laser light. Nevertheless, a biological
object has many scattering centers that can change with time; thus the
speckle intensity varies with time. A static speckle pattern could be
observed for a static object in time. Conversely, intensity fluctuation is
observed for a dynamic object such as living biological objects due to
continuous movement of the scattering centers. This phenomenon is due
to the random interference between the scattered lights from cyto­
plasmic streaming, organelle movement, cell growth, cell division, and
biochemical reactions. These dynamic speckle patterns are known as
biospeckle. Useful information regarding the biological and physiolog­
ical activity of biological objects could be extracted from biospeckle, and
it plays a vital role as a bioindicator. Several studies have been per­
formed on the use of biospeckle in monitoring the effect of seed priming
treatments (Singh et al., 2020), disease inspection on seed (Braga et al.,
2005; Rabelo et al., 2011), and as a novel non-destructive approach for
food quality and safety detection (Pandiselvam et al., 2020).
The speckles due to the random interference of multiply scattered
light within the coherence volume appear in OCT as well, even when a
low coherence light source is utilized, and these speckles vary over time.
The time sequence speckle images which are used for the calculation of
biospeckle OCT contrast are illustrated in Fig. 1d.
The temporal variation of the collected OCT structural images was
evaluated in this work using a quantity representing as to biospeckle
contrast (γ), which is defined as the ratio of the standard deviation of the
intensity to the mean value at each pixel along the temporal axis, was
calculated given by,
[ ]1
1 1 ∑
N
{ ( ) }2 2
γ (x, y) = IOCT x, y; tj − < IOCT (x, y) > ,
< IOCT (x, y) > N j=1
(3)
1 ∑N
( )
< IOCT (x, y) > =
N j=1
IOCT x, y; tj
Y.S.K. De Silva et al.
where pixel coordinates are represented by x and y, scan number is
represented by j, and total number of scans is represented by N (N =
100). A larger temporal variation or speckle contrast shows that the
seeds have experienced significant changes throughout time. Conse­
quently, the changes in speckle contrast magnitude could be a useful
measure for predicting the biological activity of seeds under the pres­
ence of external substances.
2.5. Growth parameters analysis
As a comparison to the bOCT results, the effect of different PEMPs
concentrations on traditional metrics such as enzyme activities, seed
germination parameters, root length, shoot length, fresh and dry weight
of seedlings were measured with triplicates for all of the measurements.
2.5.1. Seed germination
Generally, lentil seeds started to germinate after 24 h of exposure to
PEMPs solution. When the radical extension of the seeds reached around
2 mm, they were considered as germinated (Turk et al., 2004). If fungi
grew on the seed coat, it was considered a dead seed and was discarded
from the petri dishes. Seed germination occurs within 48 h or two days.
With PEMPs exposure, the germination got delayed, and we wanted to
test the prolonged effects. Once the seeds germinate, then root formation
happens, and it is no longer a seed. Hence, the experiment lasted after 7
d, and the germination parameters were determined.
Ng at day 2
Germination Viability (GV, %) = X 100%, (4)
Nt
Germination Rate (GR, %) =
Ng at day 7
X 100%, (5)
Nt
where Ng is the number of germinated seeds at particular days of
exposure, and Nt represents the total number of seeds used in the
respective batch.
2.5.2. Growth of seedlings
Seedling growths were recorded after 7 d of PEMPs exposure. Six
seedlings from each sample treated with PEMPs and control samples
were harvested and properly washed five times by distilled water to
remove unnecessary surfactants. After that, seedlings were cut at the
root-shoot junction to measure the shoot and root length with the help of
ImageJ software (NIH, USA).
In order to measure the fresh weight of seedlings, all the separated
roots and shoots were weighed separately using an analytical balance
(AUX 320, UniBloc, Shimadzu, Japan). Thereafter, separated roots and
shoots were kept in the oven (SOFW-450S, AS ONE, Japan) under 105 ◦ C
for 15 min, followed by 70 ◦ C until a constant weight was obtained and
weighed to acquire the dry weight.
2.6. Biochemical indices assay
The antioxidant enzyme activity for reactive oxygen species and
oxidative damages are important conventional measures for the envi­
ronmental stress and determining the detrimental effect of MPs on plant
growth. Therefore, in this study, superoxide dismutase (SOD), catalase
(CAT), hydrogen peroxide (H2O2), and malondialdehyde (MDA) levels
were measured for comparison with bOCT results. The measurements of
enzyme activity, H2O2 concentration, and MDA level were performed
with triplicates.
In the experiment, lentil seedlings were harvested after 7 d of
exposure to different PEMPs concentrations (0, 10, 50, 100 mg L-1) and
weighed after washing the sample properly with distilled water.
Thereafter, samples were homogenated with phosphate buffer (0.01
mol/L, pH = 7.4) and blended in a pre-chilled motor in an ice bath. The
mixture was centrifuged at 3000 rpm and 4 ◦ C for 15 min. The
5
Chemosphere 303 (2022) 135162
supernatants were used to analyze CAT, SOD, H2O2, and MDA content,
and the absorbance was measured using a spectrometer (UVmini-1240
Shimadzu Corporation, Kyoto, Japan). A commercial assay kit was used
to measure the H2O2 (CL-204, National Diagnostics, Atlanta, Georgia)
and SOD (S311-WST, DOJINDO, Japan) content in which CAT and MDA
content were measured using standard procedures (Aebi, 1984; Heath
and Packer, 1968). After the treatments, the absorbance value of H2O2
and SOD were measured at 560 nm and 450 nm, respectively, as
described by the technical manual of each assay kit.
The CAT activity was measured spectrophotometrically (Aebi,
1984). The decrement of the absorbance values was measured for 3 min
with a 1 min interval at 240 nm after the treatments. The MDA contents
were observed according to the thiobarbituric acid test proposed by
Heath and Packer (1968), where the absorbance was recorded at 532
nm, subtracting the absorbance at 600 nm and 450 nm, respectively.
2.7. Statistical analysis
The results of each triplicate sample are presented as the mean ±
standard deviation of the mean. Significant differences between the
treatments and control were determined by one-way analysis of variance
(ANOVA, p < 0.05), followed by a Tukey’s post hoc test using SPSS 16.0.
Origin 9.5 was used to create and modify all the graphs and histograms.
All the OCT image data were acquired using custom-made software of
LabVIEW (13.0.1f5), and the bOCT data analysis of the acquired OCT
data was performed with the help of MATLAB (R2016b) software.
3. Results
3.1. OCT structural images
In this study, the main objective was to observe the change of bio­
logical activity inside the lentil seeds during the germination process
under exposure to PEMPs. Hence, to make sure whether OCT is capable
enough to visualize the changes of internal activity under varying
PEMPs concentrations, OCT images acquired under control were
compared with those obtained after 24 h of exposure to 10, 50, and 100
mg L− 1 PEMPs concentrations as shown in Fig. 2 a-d. However,
noticeable distinguishable changes were not found from OCT structural
images even after 24 h of exposure. With the acquired OCT images ob­
tained after 24 h of exposure to 0, 10, 50, and 100 mg L− 1 MPs con­
centrations, bOCT images were calculated using Equation (3) and
illustrated in Fig. 2 e-h. In the biospeckle images, the blue and red colors
indicate lower and higher contrasts, respectively, as shown in the scale
bar. The bOCT images with higher red color density imply the higher
activity or movement inside the seed. In contrast, a higher blue color
density indicates lower biological activity or lower movement inside the
seed. Interestingly from bOCT images, for all the PEMPs treatments,
significant reductions of the biospeckle contrast could be seen as
compared to that of the control. Further, such difference could not be
seen in the conventional OCT images obtained for different PEMP
concentrations.
3.2. Biospeckle contrast over shorted durations
The bOCT images were found to be even sensitive for shorter imbi­
bition durations. The observations of bOCT following exposure to
different MPs concentrations taken at 0 h, 6 h, 12 h, and 24 h are shown
in Fig. 3. The first row of the figure (a–d) is related to the control samples
at each observation time frame within 24 h. With increasing time, the
natural germination process sets in, and the gradual spread of the red
region, or in other words, the enhancements of internal activity, could
be observed. This is due to the activation of the energy metabolic process
and the expansion of seed size from water absorption during
germination.
On the contrary, for seeds in a medium containing variable amounts
Y.S.K. De Silva et al. Chemosphere 303 (2022) 135162

Fig. 2. First row indicates OCT structural images after 24 h exposure of control (a) and different PEMPs concentration of 10 mg L− 1 (b), 50 mg L− 1 (c), 100 mg L− 1
(d). Second row indicates bOCT contrast images after 24 h exposure of control (e) and different PEMPs concentration of 10 mg L− 1 (f), 50 mg L− 1 (g), 100 mg L− 1 (h).
A clear significant reduction of biospeckle contrast could be seen for all the MPs treatments compared to the control from bOCT images, which is not seen in the
conventional OCT images.

Fig. 3. Comparison of bOCT images of lentil seeds treated with several PEMPs at 0 h, 6 h, 12 h, and 24 h. Control samples (a)–(d); Samples treated with 10 mg L− 1
MPs (e)–(h); Samples treated with 50 mg L− 1 MPs (i)–(l); Samples treated with100 mg L− 1 MPs (m)–(p). A reduction of internal activity could be seen for all PEMPs
treatments, and the effect is more significant when the concentration increases.

PEMPs concentrations, 10 mg L− 1 (e-h), 50 mg L− 1 (i-l), and 100 mg L− 1
(m-p), the spread of red regions was relatively lesser, indicating a
reduction of internal activity. The reduction of the red region was more
pronounced when the concentration was increased, emphasizing the
dose-dependent adverse effects of PEMPs on seed germination. The
mechanism for this internal activity reduction is believed to be that
PEMPs could accumulate near the seed coat and block the pores of the
seed coat, inhibiting the imbibition of water and nutrient uptake (Bosker
et al., 2019; Urbina et al., 2020).
In order to evaluate the effects of changes in the biospeckle contrast
images with increasing concentrations of PEMPs, the contrast images in
Fig. 2 e–h and in Fig. 3 were used to conduct quantitative analysis.
3.2.1. Quantitative analysis
A region of interest (ROI) was first chosen in the biospeckle contrast
image so that the regions do not fall on the surface and also do not lie

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Y.S.K. De Silva et al. Chemosphere 303 (2022) 135162

deep. During the calculations, special attention was devoted to specific
local rectangular sections between the seed coat and the cotyledon,
which were designated as a region of interest (ROI). Six ROIs were
chosen from each bOCT image for this calculation, and the mean of local
contrast with ROIs was determined as average local contrast (ALC). The
grand average of contrast was then obtained by averaging six sets of ALC
over the six seeds under the same treatment condition.
3.2.2. Normalized bOCT contrast
To prevent individual variation of seeds between treatments, the ALC
of each treatment at various time frames was first normalized by those
obtained at 0 h or before the imbibition condition, and the result was
referred to as normalized contrast (NC). The average normalized
contrast (ANC) for each treatment was calculated by averaging the NCs
over six seeds. Fig. 4 shows the comparison of ANC between all of the
treatments. Clear reductions of ANC could be seen for all the PEMPs
treatments where all the reductions are significant (p < 0.05) compared
to that of control at 6 h with the respective reduction rates of 9.6%,
23.3%, and 27.2% for 10, 50, and 100 mg L− 1 MPs concentrations,
respectively. The same tendencies were observed for the reductions of
ANC after 12 h of exposure with the reduction rates of 7.9%, 15.2%, and
19.4% for 10, 50, and 100 mg L− 1 PEMPs concentrations, respectively.
After 24 h of exposure to different PEMPs concentrations, significant
reductions (p < 0.001) of ANC contrast were observed for the 10, 50,
and 100 mg L− 1 MPs concentrations with the reduction rates of 17.5%,
23.7%, and 30.2%, respectively. The reduction is more significant when
the concentration is higher, emphasizing the dose-dependent adverse
effect of microplastics. Interestingly, the significant reductions of ANC
were observed just after 6 h of exposure for all the microplastics con­
centrations highlighting the speed and reliability of the proposed bOCT
method for the observation of the effect on seed germination by external
stress.
3.3. Conventional measurements to compare with bOCT results
To compare the results obtained from bOCT experiments with con­
ventional measurements such as germination viability, germination
rate, shoot length, root length, fresh and dry weights of seedlings, and
enzyme activities were also measured after exposure to PEMPs.
3.3.1. Seed germination indices
The germination viability (GV) and germination rate (GR) are key
indicators used to measure seed vigor and germination ability under the
presence of different environmental stressors. After two days of expo­
sure, the number of germinated seeds of each treatment was recorded to
calculate the germination viability (Equation (4)), and the final germi­
nation rate was calculated after seven days of exposure according to
Equation (5). A significant reduction of germination viability could be
seen for both 50 mg L− 1 (P < 0.01) and 100 mg L− 1 (P < 0.01) PEMPs
concentrations compared to that of control, where the reduction was not
significant for 10 mg L− 1 PEMPs concentration. The same tendency was
observed for the final germination rate, where the effect was significant
for both 50 mg L− 1 (P < 0.05) and 100 mg L− 1 (P < 0.01) concentrations
(Table 1). Therefore, a dose-dependent adverse effect was examined
between PEMPs and lentils, whereas the lowest germination rate was
observed for the PEMPs concentration of 100 mg L− 1.
3.3.2. Growth of lentil seedlings
In this study, averaged root and shoot lengths were also measured
after 7 d of exposure to PEMPs as a conventional parameter to compare
the bOCT results. The root length was inhibited to varying degrees as the
PE concentration increased (Table 1). The inhibition was more signifi­
cant for the higher PEMPs concentration, as shown in Fig. 5a. The
strongest significant inhibition was recorded at a PE concentration of
100 mg L− 1 (P < 0.01). There was a consistent tendency for the inhi­
bition of shoot length, same as the root length and the inhibition was
more significant when the PEMPs concentration increased compared to
the control. Thus, the strongest significant inhibition of shoot length was
recorded for a100 mg L− 1 (P < 0.01) PEMPs concentration.
Fresh weight (FW) and dry weight (DW) of seedlings have often been
utilized as essential indicators of acute toxic effects on plant growth
(Alam et al., 2019; Janas et al., 2010). The effect of increasing PEMPs
concentration on FW and DW of the lentil roots and shoots was illus­
trated in Fig. 5b to compare with those obtained by bOCT results. Sig­
nificant reductions of the FW and DW of the lentil roots was monitored
with respect to those of control conditions. The reductions of root FW
and DW were more significant when the PEMPs concentration increased
highlighting the dose-dependent adverse effect. Almost the same ten­
dency for the reductions of shoot FW and DW was observed where all the
PEMPs treatments showed significant reductions compared to those of
control. The reductions of shoot FW and DW were also more significant
when the PEMPs concentration increased. The highest significant re­
ductions for both root and shoot (FW and DW) were observed for the
highest PEMPs concentration of 100 mg L− 1 (P < 0.01).

Table 1
The effects of four varying PEMPs concentrations (0, 10, 50, and 100 mg L− 1) on
different conventional growth parameters such as germination viability in 2
d old lentil seedlings, seed germination rate, average root length, and shoot
length in 7 d old lentil seedlings. Data reflects mean ± SD (n = 3), * denotes a
significant reduction of growth parameters compared to the control (*P < 0.05,
**P < 0.01).
PEMPs Germination Germination Average Average
concentration viability (GV) rate (GR) (%) root length shoot
(mg L− 1) (%) (mm) length
(mm)

0 80.6 ± 4.81 83.3 ± 8.33 48.6 ± 74.2 ±

2.70 4.52
10 66.7 ± 8.33 77.8 ± 12.73 38.4 ± 50.5 ±
3.4* 1.18**
Fig. 4. Averaged normalized bOCT contrast of lentil seeds subjected to varying 50 50.0 ± 8.3** 58.3 ± 8.33* 28.2 ± 53.2 ±
PEMPs concentrations of 0, 10, 50, and 100 mg L− 1 throughout a 24 h period. 1.75** 4.20**
100 38.9 ± 9.62** 50.0 ± 8.33** 26.1 ± 23.9 ±
Reductions of ANC could be observed for all the PEMPs treatments where
1.89** 1.28**
significant reductions of ANC was monitored just after 6 h of exposure.

7
Y.S.K. De Silva et al. Chemosphere 303 (2022) 135162

Fig. 5. Image of lentil seedling treated by PEMPs for 7 d (a). Effects of varying PEMPs concentrations (0, 10, 50, and 100 mg L− 1) on different conventional growth
parameters of root fresh weight, root dry weight, shoot fresh weight, shoot dry weight in 7 d old lentil seedlings (b). Significant reductions of root length, shoot
length, FW and DW of lentil roots and shoot were monitored with respect to those of the control conditions. The adverse effects were significant when the PEMPs
concentration increased. Data reflect mean ± SD (n = 3), * denotes a significant reduction of growth parameters compared to the control (*P < 0.05, **P < 0.01).

3.4. Enzyme activities and oxidative damage
To characterize the change of antioxidant enzyme activities induced
by the PEMPs and compare those results with those obtained by bOCT
observation, the measured SOD, CAT, H2O2, and MDA activities after 7
d of exposure to different PEMPs treatments are shown in Fig. 6. It
should be noted that to have a reliable change in the enzymes, longer
exposure times are required. The results showed the increment of CAT,
SOD, and H2O2 content when the concentration of PEMPs were
increased. The increments were significant for both 50 mg L− 1 and 100
mg L− 1 compared to those of control. The highest significant increment
was observed for the concentration of 100 mg L− 1. The MDA activities
were also enhanced for all the PEMPs treatments compared to those of
control, and the highest significant increment was recorded for the
concentration of 100 mg L− 1, emphasizing the degree of cell membrane
damage induced by the PEMPs at highest concentrations.

8
Y.S.K. De Silva et al.
Fig. 6. Superoxide dismutase (SOD), catalase (CAT), hydrogen peroxide (H2O2) and malondialdehyde (MDA) content of lentil seedling after 7 d exposure to different
PEMPs concentrations (0, 10, 50, and 100 mg L− 1). Data reflect mean ± SD (n = 3), * denotes a significant reduction of growth parameters compared to the control
(*P < 0.05, **P < 0.01).
4. Discussion
The main objective of this study was to demonstrate the optical
method named biospeckle optical coherence tomography (bOCT) in
investigating the PEMPs effect on the lentil seed germination and to
conduct measurements for comparing bOCT results with conventional
measurements, germination viability, germination rate, shoot length,
root length, seedlings fresh, dry weight, and antioxidative enzyme ac­
tivity. While the conventional measurements rely on lengths, weights for
the effective estimation of the seed germination, bOCT by its principle
rely on the monitoring of the internal activity of the seeds. This in turn
implies, it is possible to visualize the germination process, and this
makes the observations of germination process between stages possible
while waiting for the seed to germinate, usually after 24 h. Thus, in this
study, to observe the acute toxicity effect of PEMPs, bOCT observations
were taken at 0, 6, 12, and 24 h. The bOCT results revealed that sig­
nificant reductions of internal activity for all the PEMPs treatments were
recorded just after 6 h. This demonstrates the potential of the observa­
tion speed by bOCT.
The reduction of internal activity was more significant when the
concentration was increased indicating the possibility of the method to
monitor the dose-dependent adverse effects of the microplastics. During
the germination process, bOCT contrasts were used to measure the
changes in internal activity of seeds, with red and blue indicating high
and low internal activity (Fig. 2). The speckle generated from OCT im­
ages could be utilized to evaluate the change in internal activity of
biological substances in the presence of an external agent (Rajagopalan
et al., 2020). The decrement of internal biological activity was found for
the boiled seed in comparison to that of control by using bOCT (Lim
9
Chemosphere 303 (2022) 135162
et al., 2019). Moreover, in our previous study, bOCT was able to
investigate both positive and negative effects of increased Zn concen­
tration on lentil seed germination at an early stage before the germi­
nation (De Silva et al., 2021a). In several scientific domains, the
conventional OCT images are frequently employed as a strong tool to
visualize the interior structure (Rebolleda et al., 2015), including agri­
culture (Wijesinghe et al., 2017). Nevertheless, it is not able to visualize
the change of internal activity of biological objects as compared to the
bOCT contrast images (Fig. 2).
Rapid increment of ANC could be observed within the first 6 h of
exposure for all the treatments. This is because water diffusion within
the lentil seed reaches a maximum of around 6 h, enhancing internal
activity and then plateaus (Seyhan-Gürtas et al., 2001). During the start
of germination, water will be absorbed to hydrate the seed, and the
metabolic process will be initiated enhancing respiration and protein
synthesis. Following that, stored food in seed will be digested and
translocated to the embryo, resulting in cell division and growth as a
result of seedling development (Bareke, 2018). Therefore, for all the
treatments (without and with PEMPs) enhancement of internal activity
could be examined as a function of time. This results in the alternation of
the dynamic properties of the biospeckle originating from each struc­
ture. These phenomena were used in this investigation to test the in­
fluence of various PEMPs concentrations on lentil seed germination as
an ultra-high-speed measuring technique.
Microplastics can adsorb to the seed surface and root hair in nature
and accumulate on the pores in seed capsule and delay the germination
and root growth while inhibiting imbibition of water and nutrient up­
take through the physical blockage (Bosker et al., 2019). Therefore, the
presence of PEMPs can reduce the germination and seed internal activity
Y.S.K. De Silva et al.
compared to the control (Fig. 4). There exists report where the micro­
plastic accumulation in the seed coat, root, shoot, and root hairs were
investigated using fluorescent microplastics followed by its imaging
with LSM confocal laser microscope (Zeiss, reference and model)
(Bosker et al., 2019). The scanning electron microscope (SEM) (Zhang
et al., 2021), transmission electron microscope (TEM), Fourier trans­
form infrared spectroscopy (FTIR) ( 2021b), and gas chromatography
mass spectrometer (GC-MS) (Park et al., 2021) were also used to observe
the distribution characteristics of microplastics. There are various pros
and cons of these technologies. Specially, TEM, SEM and FTIR all require
destructive inspection and not in vivo. However, confocal can be in vivo
but requires labeling.
In OCT, the changes in reflectivity from structures are mapped.
Therefore, when PEMP and water exist in seed coat, the varying
complexity of the seed tissue structure is enhanced. Their reflectivity
differences would result in the structural OCT image as given in Fig. 2.
The role of refractive index would result in difference reflectivity of the
OCT signal. However, if the PEMPs are bound to the surface without any
movement, then this would result in scattering from the particles within
the coherence volume partially contributing to the OCT reflectivity
signal. As the particles themselves are assumed to be stationary, when
the OCT images were obtained over 13 s and within this duration, the
variation from the particles themselves could be considered as not
contributing to any changes in the bOCT images. Instead, we suggest the
changes would be due to the particles on the surface influencing changes
in the activities within the seed tissue as observed in Figs. 2 and 3.
The bOCT results of the current research corroborate with the study
performed by Bosker et al. (2019) and Li et al. (2021a), where they
confirmed the short-term, transient adverse effect of PEMPs on seed
germination and seedlings growth. Hence, there could be an obvious
inhibitory effect of germination within the first 24 h of exposure to
PEMPs, and the bOCT observations were also taken at 0, 6, 12, and 24 h
after exposure to PEMPs. Interestingly, a significant inhibitory effect of
lentil seed germination was observed just after 6 h of exposure for all the
PEMPs treatments from bOCT which could not be revealed by any of the
existing studies (Guo et al., 2021; Bosker et al., 2019).
According to the existing literature, the contrasting effect of plastic
debris was reported on seed germination and seedling growth. The effect
of plastic residue on seed germination and plant growth depends on
numerous factors such as type of plastic, size, geometry, aging time,
environmental factors, and plant species. However, in this study, the
proposed optical method, bOCT, was able to monitor the influence on
the seed germination and growth with a high sensitivity within a fairly
short period of exposure if the MPs have negative effect on seeds. In
addition, our method could reveal the difference in toxicity of MPs due
to its size and shape etc. In the present study, the seed germination
viability was monitored as a conventional measurement after 2 d of
exposure, and significant inhibitory effects were observed. The same
tendency could be observed for the final seed germination rate calcu­
lated after 7 d of exposure (Table 1). Hence, the effect was not transient
until the whole observation period of 7 d. Moreover, significant re­
ductions of shoot length, root length, fresh and dry weight of seedlings
were also observed after 7 d of exposure. This kind of inhibitory effect of
MPs on seed germination and seedling growth was consistent with
previous results (Guo et al., 2021; Li et al., 2021a, Pflugmacher et al.,
2021).
To compare with bOCT results, the level of conventional parameters
SOD, CAT, H2O2, and MDA were measured after 7 d of exposure to
PEMPs. The production of reactive oxygen species (ROS) maintained a
dynamic balance in plants. When the ROS level exceeds the anti­
oxidative activities of the plants, oxidative damage will occur. In this
study, SOD, CAT, and H2O2 contents were significantly increased when
the concentration of PEMPs increased, emphasizing the adverse effect of
PEMPs on lentil seed germination and seedling growth. Furthermore,
the MDA level was also enhanced for all the PEMPs treatments compared
to that of control, and the highest significant increment was recorded for
10
Chemosphere 303 (2022) 135162
the highest concentration of 100 mg L− 1, highlighting the degree of cell
membrane damage. Those results of enzyme activities and oxidative
damage are primarily consistent with previous results (Guo et al., 2021;
Jiang et al., 2019).
Interestingly, significant influences of MPs on all the conventional
parameters, germination viability, germination rate, enzymatic activity,
shoot length, and root length, as well as FW and DW, were observed
compared to those of the control owing to the inhibition of water and
nutrient through the mechanical blockage of pores in seed capsule and
root hair by microplastics (Bosker et al., 2019; Urbina et al., 2020).
Hence, the growth of the lentil seedling was significantly reduced
compared to control. Consequently, a significant reduction in the fresh
and dry weight of root and shoot could be observed. Moreover, the ef­
fects of microplastics on plant growth were shown for some plant spe­
cies, including lentils. The results imply that the presence of
microplastics reduced plant growth and promoted the enzymatic activ­
ity. Additionally, roots may have suffered physical damage by adsorbing
microplastics. Hence, the presence of microplastics in the ecosystems
could be recognized by the plant as a stressor.
The surface texture and pore structure have an important role in the
penetration of PEMPs. In some research studies, the microstructural
difference between lentils and other food legumes was monitored in
examining the seed coat surface. The seed coats of other legumes appear
relatively smooth, though generally possessing a characteristic pattern
and often being covered with pits and pores or varying amounts of
surface deposits. Lentils, in contrast, possess an uneven seed coat surface
covered with distinctive conical papillae. For lentil seed coats, the
papillae appear mushroom-shaped. However, careful examination re­
veals that the mushroom-shaped papillae are merely conical papillae
with disc-shaped debris attached to their tips (Hughes et al., 1986). The
average pore size of lentils was experimentally observed, and it could be
varied in the range 4–8 μm (Aouaini et al., 2015). In the present study,
the particle size of polyethylene microspheres ranged from 0.744 to
4.990 μm. Therefore, PEMPs could block the pores of seed capsules
while inhibiting the imbibition of water and nutrient. Consequently, the
reduction of internal activity, germination viability, germination rate,
root length, shoot length, fresh and dry weight of seedling could be
observed. The effect is more significant when the concentration of
PEMPs is increased.
A significant effect on germination viability was observed after 2 d of
exposure to PEMPs, whereas all other physiological parameters required
7 d to make a distinguishable impact on lentil seedling growth for
varying PEMPs concentrations. The same effect was examined just after
6 h of exposure to PEMPs using the proposed bOCT technique before the
germination emphasizing the observation speed and the reliability of the
proposed method. Moreover, many research studies describe the adverse
effect of MPs on seed germination and seedling growth with none
available on the observation of the changes within the seed before the
germination under the exposure of microplastics.
5. Conclusions
This research represents the first paper that highlights the reduction
of biological activity by microplastics during seed germination resulting
in the inhibition of germination and seedling growth. There are some
studies that described the different effects of MPs on seed germination.
However, there are no studies that could be able to observe the internal
biological activity of seed under the exposure of MPs before germina­
tion. Here, bOCT was applied to monitor the effect of PEMPs on lentil
seed germination and significant reductions of bOCT contrast were
observed just after 6 h of exposure for all the tested concentrations
emphasizing the dose-dependent adverse effect. The conventional pa­
rameters, germination viability, germination rate, root length, shoot
length, root FW, shoot FW, root DW, shoot DW and oxidative stress
markers, superoxide dismutase (SOD), catalase (CAT), hydrogen
peroxide (H2O2), and malondialdehyde (MDA) concentration were also
Y.S.K. De Silva et al.
measured for comparison. With conventional measurements, similar
effects of PEMPs were observed only after 2 d of exposure in comparison
to just 6 h by current method of bOCT. More studies are required to
understand the mechanism of how speckles could reveal the effect of
MPs on seed germination and seedling growth.
Author contribution statement
Y. Sanath K. De Silva: Data curation, Formal analysis, Investigation,
Methodology, Writing – original draft. Uma Maheswari Rajagopalan:
Conceptualization, Project administration, Supervision, Validation,
Writing – review & editing. Hirofumi Kadono: Conceptualization,
Funding acquisition, Project administration, Supervision, Writing – re­
view & editing. Danyang Li: Data curation.
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.
Acknowledgments
This study was supported by the JSPS Grant-in-Aid for Scientific
Research (B) 19H04289 of the Ministry of Science and Technology,
Japan.
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