Barnacle analysis as a microplastic

pollution bioindicator on the East Coast

of Surabaya
Miftakhul Sefti Raufanda1 , Aunurohim Aunurohim2 and
Romanus Edy Prabowo1
1
Faculty of Biology, Universitas Jenderal Soedirman, Banyumas, Indonesia
2
Department of Biology, Institut Teknologi Sepuluh Nopember, Surabaya, Indonesia

ABSTRACT
Background. Plastic pollution is a significant issue on the East Coast of Surabaya,
emphasizing the need to develop microplastic monitoring programs. Barnacles became
one of the potential microplastic bioindicator species on the East Coast of Surabaya.
This study aimed to characterize the visual and polymers of microplastics found in
barnacles and assess their potential as a bioindicator species for microplastic pollution
on the East Coast of Surabaya.
Methods. Microplastic polymer analysis was performed using ATR-FTIR.
Results. A total of 196 microplastic particles were found in barnacles, water, and
sediment. The size of microplastics in barnacles, water, and sediment varied, with the
size in barnacles dominated by class 1 (1–10 µm), in water by class 2 (10–50 µm), and
in sediments by class 3 (50–100 µm). Fragments dominated the shape of microplastics
in barnacles, while water and sediment were dominated by fiber. The microplastic
color in barnacles, water, and sediment was dominated by blue, and the microplastic
polymer composition on barnacles, water, and sediments was dominated by cellophane
(36%). Amphibalanus amphitrite was found to be predominant and identified as a
potential microplastic bioindicator because it is a cosmopolitan species. Its population
was found to correlate positively with cellophane (CP) accumulation. The Pearson’s
correlation test between barnacle length and microplastic length at a = 0.05 was inversely
Submitted 15 September 2023
Accepted 20 May 2024 proportional to r = −0.411 (p < 0.05), categorized as a strong enough correlation.
Published 24 June 2024 These findings are essential in developing monitoring programs and mitigating the
Corresponding author impact of microplastics on the marine environment.
Aunurohim Aunurohim,
aunurohim@its.ac.id
Subjects Ecology, Marine Biology, Ecotoxicology, Environmental Impacts, Biological
Academic editor
Haider Mahmood Oceanography
Keywords Barnacle, Bioindicator, Microplastics, Monitoring, Marine debris, Pollution,
Additional Information and Amphibalanus amphitrite, Plastic, Cellophane, FTIR-ATR
Declarations can be found on
page 17
DOI 10.7717/peerj.17548 INTRODUCTION
Copyright The issue of plastic pollution in marine waters has gained significant attention from the
2024 Raufanda et al.
scientific community and the public due to its potential dangers. About 10% of plastic
Distributed under produced ends up in the oceans, where it accumulates and does not decompose (Saeed et al.,
Creative Commons CC-BY 4.0
2020). Indonesia is considered the second-largest plastic waste contributor globally, with an
OPEN ACCESS estimated 0.48−1.29 million metric tons of plastic waste in the sea per year, following China

How to cite this article Raufanda MS, Aunurohim A, Prabowo RE. 2024. Barnacle analysis as a microplastic pollution bioindicator on
the East Coast of Surabaya. PeerJ 12:e17548 http://doi.org/10.7717/peerj.17548
 (Jambeck et al., 2015; Kurniadi & Hizasalasi, 2017). The breakdown of plastic into smaller
fragments occurs when exposed to ultraviolet radiation from the sun, surface waves, and
turbulence of water currents (Li et al., 2019; Saeed et al., 2020). These small fragments with
dimensions less than 5 mm are known as microplastics. Microplastics can be categorized
into two classifications—primary and secondary microplastics. The main categories of
microplastics include molded plastic powders, surface blast cleaning scrubbers, industrial
plastic nanoparticles, and microbeads commonly present in cosmetic items. Moreover,
spherical or cylindrical virgin resin pellets are widely used in plastic production processes.
Secondary microplastics are generated by the breakdown or fragmentation of bigger plastic
trash (Foo et al., 2022). Microplastics can present a significant danger to ecosystems and
marine species due to their widespread distribution and the possibility of being consumed
by marine organisms (Li et al., 2019).
There are several ways aquatic organisms can be exposed to plastic materials, including
entanglement, ingestion, and interaction. Entanglement refers to the entrapment of
the organism, including ghost fishing (Law, 2017). Ingestion of plastic debris could be
intentional, accidental, or indirect (via ingesting organisms that have ingested the plastic).
It has been observed in various animals, from planktonic invertebrates to large aquatic
mammals (Al-Thawadi, 2020). The negative impacts of microplastics on aquatic animals
have been identified to suppress growth performance, hinder reproductive functions,
potentially induce neurotoxicity, depress feeding and foraging activity, alter oxidative
stress, destroy metabolic responses (Jimoh et al., 2023), and alternatively increase mortality
rates among aquatic organisms with an accumulation as low as 184 µg/L (Li et al., 2021).
Microplastics accumulated in the neural system are also known to interfere with the defense
mechanisms of aquatic animals, thereby acting as highly high-risk stressors by decreasing
phagocytic activity (Mallik et al., 2021) and disrupting the lysosomal membrane (Sharifinia
et al., 2020). The impacts of microplastic pollution extend to human health, the economy,
tourism, and beach aesthetics (Joesidawati, 2018). Furthermore, microplastics can act as
vectors for metal pollutants. Therefore, it is urgent to develop microplastic monitoring
programs for marine ecosystems’ different components, such as water, sediment, and biota
(Xu et al., 2020; Welden, 2020).
A monitoring program for environmental quality using bioindicators is crucial to
assess the toxic effects on an organism. The bioindicator species selected for such a
program should have a wide distribution, be abundant and tolerant to environmental
conditions, and be easily sampled (Xu et al., 2020). Macrozoobenthos are advantageous as
bioindicators due to their sedentary habitat and their sensitivity to changes in the ecological
conditions of aquatic environments (Sidik, Dewiyanti & Octavina, 2016). There are two
compartments from which benthic species can directly take up microplastics, depending on
the animal’s feeding behavior: the sediment and the water column. Epifaunal filter feeders,
for example, will uptake microplastics suspended in the water above the sediment, while
infaunal deposit feeders will uptake microplastics within the sediment. Also, microplastics
deposited on the sediment can be resuspended by mechanical forces and become available
to the water column and its turbulent processes at different scales (Pinheiro, Sul & Costa,
2020). Because organisms inhabiting and feeding in benthic habitats are at the base of food

Raufanda et al. (2024), PeerJ, DOI 10.7717/peerj.17548 2/22

 webs, it is crucial to understand how microplastics interact with and affect them (Wright,
Thompson & Galloway, 2013). The main form of interaction is the uptake and assimilation
of particles in the digestive system (i.e., ingestion). However, benthic species also interact
with microplastics in other ways, which could affect the movement of these pollutants
within benthic environments. They may bury, mobilize, and even act as stepping-stones
for the transmission of microplastics to other environmental compartments such as the
nekton (Pinheiro, Sul & Costa, 2020).
The macrozoobenthos community consists of five groups: mollusca, polychaeta,
crustaceans, echinoderms, and other groups of several small taxons such as Sipunculidae
and Pogonophora (Ndale, Restu & Wijayanti, 2021). Barnacles (Crustaceans: Cirripedia)
are a group of crustaceans that dominate certain zones of rocky coastal areas and are
considered excellent biomonitoring organisms due to their widespread and abundant
distribution around the world, easy collectibility, sessile adult life, and relative tolerance to
contaminants (Vaezzadeh et al., 2021). Barnacles can potentially serve as bioindicators for
microplastics because they are sessile and can accumulate microplastics from surrounding
waters, thus aiding in identifying microplastic sources (Xu et al., 2020). According to
the research (Scotti et al., 2023), provide an assessment of the efficiency of the biofouler
Lepas (Lepas) anatifera Linnaeus, 1758 in capturing microplastics and microfiber particles
floating in the water column. In this context, pelagic gooseneck barnacles are collected at
fixed moorings in the Capo Milazzo Marine Protected Area (MPA). Fibers and fragments
were found in the digestive tract of 30% of the 120 specimens collected. The ingested debris
was mainly fibers (85.9%) of synthetic (30.6%) and natural (11.7%) origin, with lengths
ranging between 1 and two mm (33.3%) and transparent (47.2%) (Scotti et al., 2023).
Another investigation conducted in Hong Kong examined the presence of microplastics in
four species of barnacles over 30 locations. The study found that the median quantity of
microplastics varied from 0 to 8.63 particles per gram of wet weight, or 0 to 1.9 individual
particles per barnacle. Among the several types of microplastics, fibers were found to be
the most prevalent (Xu et al., 2020).
Surabaya produces significant plastic waste, estimated at 400 tons, which could result
in significant environmental problems if not adequately managed (Ni’am et al., 2019).
The East Coast of Surabaya is particularly susceptible to plastic pollution due to human
activities in the coastal areas, fisheries, and tourism, as well as the discharge of plastic waste
from rivers (Ni’am et al., 2019). The research area is located in the Suramadu Bridge Area.
The Suramadu Bridge crosses the Madura Strait. Kenjeran Beach is a coastal area that
directly faces the Madura Strait. On land, it is mainly characterized by tourism activities,
fishermen’s houses, and mangrove ecosystems. The waters of Kenjeran Beach are regularly
used for fishing and marine tourism activities (Cordova, Purwiyanto & Suteja, 2019). A
study conducted by Kurniawan & Imron (2019) revealed that a quantity of visible plastic
debris (VPD) gathered along the seashore of the Madura Strait in both the dry and wet
seasons. Polyethylene terephthalate accounted for the majority of plastic types distributed,
reaching a share of 59.77% during the wet season. During the dry season, low-density
polyethylene was the primary plastic component collected in all sampling points, making up
73.13% of the collected VPD composition. To address this pressing issue, a comprehensive

Raufanda et al. (2024), PeerJ, DOI 10.7717/peerj.17548 3/22

 study is necessary to understand the characteristics and composition of microplastics in
this region. Furthermore, identifying effective biomonitoring organisms for microplastic
pollution is critical. Barnacles have shown promise as bioindicator species for microplastics
since they are sessile and can accumulate plastic particles from surrounding waters, making
microplastic sources identifiable (Xu et al., 2020). Therefore, this study aims to explore
the potential of barnacles as a bioindicator species for microplastic pollutants and to
characterize the visual and polymers of microplastics in barnacles in the East Coast Waters
of Surabaya. The findings of this study will contribute to the development of monitoring
programs and help mitigate the impact of microplastics in the marine environment.

MATERIALS & METHODS

Experimental design
Barnacle sampling was carried out in 2021 at the Suramadu Bridge Area on the East
Coast of Surabaya, with coordinate points of 112.7799857 BT and −7.1905940 LS (Fig. 1).
The sampling location was chosen because abundant barnacle species were found and
fishing activities under bridges could be one of the causes of microplastics. The analysis
of microplastic size, shape, and color on the barnacle samples was performed at the
Ecology Laboratory, Department of Biology, Faculty of Science and Data Analytics, Institut
Teknologi Sepuluh Nopember. The analysis of the microplastic polymer composition
in barnacle, water, and sediment samples was collected at the Material Characterization
Laboratory, Department of Materials Engineering and Metallurgy, Faculty of Industrial
Technology and Systems Engineering, Institut Teknologi Sepuluh Nopember.

Barnacle, water, and sediment sample collection

Fifty individual barnacle samples were collected from the research site in March 2021. The
barnacles were removed from a pole of the Suramadu Bridge in the intertidal zone and
placed in sealed glass bottles to ensure the durability of ingested microplastics. The bottles
were stored in a cool box (Xu et al., 2020). The identification of barnacle samples is based
on morphological characteristics of the hard part of the shell and shell cover plate, using
Darwin’s identification book from 1851 and 1954, as well as Chan, Prabowo & Lee (2009).
The barnacles obtained at the research site were identified as the Amphibalanus amphitrite.
The barnacles underwent morphometric length, width, and tissue weight measurements.
The barnacles used as research samples were in the size range of 0.4−0.7 cm in length and
0.2−0.6 cm in width. Water sampling was conducted by pulling a plankton net for 10 min
at 2 knots, with a net size of 80 µm. The sample was taken horizontally at the surface at the
study site, and the net was rinsed using water from the direction of the mouth of the net
towards the cod end. The cod end was then released slowly, and the water sample in the cod
end was rinsed using 70% ethanol to keep the water samples in good condition. All water
samples were collected in sample bottles, sealed tightly, and placed in a cool box (Prata
et al., 2019; Zhang et al., 2020; Viršek et al., 2016). The sediment samples were collected
using an Ekman bottom grab (Model LEG-150 PROTIRTA standard size, sample area 232
cm2 ) from the top 5 cm depth. Samples of 400 grams were collected at the study site, and
the coordinates were marked using GPS. The sediment samples were placed in previously

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 Figure 1 Sampling location on Suramadu Bridge Pillar, East Coast of Surabaya (adapted from Google
Maps, 2023). Map imagery credit: ©2023 TerraMetrics.
Full-size DOI: 10.7717/peerj.17548/fig-1

labeled plastic bags and stored in a cool box for further analysis in the laboratory (Wang &
Wang, 2018; Prata et al., 2019).

Sample preparation for microplastic analysis

To prevent air contamination, the trial table is sterilized with 75% ethanol before
measurements of the length and width of the barnacle shell are taken using a funnel
term. The wet weight of the barnacle soft tissue is then separated from the body using
forceps and measured to the nearest 0.01 g. Five barnacles were put together as one
replicated and placed in a 100 ml beaker glass. Next, 60 ml of KOH 10% (Rochman et
al., 2015) is added to each replicate, and the solution is incubated at 40 ◦ C for 48 h. The
solution is then filtered through Whatman paper no. 41 (pore size 20 µm) and placed in
a petri dish for further analysis (Xu et al., 2020). For water, samples are transferred into
a test tube and dried in an oven at 80 ◦ C for 24 h. To degrade organic materials, 2 ml of
30% H2 O2 is added to each sample (Masura et al., 2015). The remaining water samples
containing solid matter are mixed with 30% H2 O2 at a volume ratio of 1:1 and left to stand
for 24 h. The sample is closed using aluminum foil and left for 24 h before incubation in a
water bath at 80 ◦ C until the solution is clear. The clear solution is then dried on Whatman
filter paper no. 41 using a vacuum pump (Gewert et al., 2017). For microplastic analysis in
sediment samples, sediment samples are dried at 74 ◦ C in an oven for 24 h. Then, 200 g
of sediment is mixed with 600 ml of concentrated NaCl in a beaker glass and stirred for
2 min before being left for 1 h until the sediment settles. This process is repeated twice, and
the supernatant containing microplastic particles is carefully transferred to another beaker
glass (Cordova, Hadi & Prayudha, 2018). The supernatant is filtered through Whatman
filter paper no. 41 using a vacuum pump, and the filter paper is placed in a petri dish for
further analysis (Xu et al., 2020).

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 Analysis of visual characteristics of microplastics
Visual analysis of microplastics is based on their morphological characteristics, including
shape, size, and color. All microplastic particles were observed, photographed, and marked
under an Olympus® SZ61 stereo microscope with the Optilab Advance 2.2 tool using
Optilab Viewer software for visual analysis. The Optilab Advance 2.2 cameras, installed
with ocular lenses on stereo microscopes (USB Digital Microscope 1600X Zoom Magnifier
Monocular Lens), are connected to laptops for real-time observation. The Raster Image
software is used to measure microplastics based on the most extended microplastic scales.
Microplastic particles were categorized into four size classes: class 1 (1–10 µm); class 2
(10–50 µm); class 3 (50–100 µm); and class 4 (>100 µm) (Shen et al., 2021). Microplastic
color analysis can vary greatly, leading to subjectivity in visual recognition. Therefore, the
standard size and color sorting system (SCS System) is used to categorize plastic pieces
effectively based on size and shape. The SCS system classifies microplastic shapes into five
types: fiber, film, fragments, pellets, and foam (Crawford & Quinn, 2017).

Analysis of the polymers composition of microplastics

The chemical composition of microplastics was analyzed using Attenuated Total Reflection
Fourier Transform Infrared Spectroscopy (ATR-FTIR) Thermo Scientific Nicolet 1S10. The
spectra were investigated and subtracted from the baseline in the scan range of 400–4000
cm−1 at a resolution of two cm−1 . ATR-FTIR allows the analysis of large-sized microplastics
(>500 µm) and also enables the direct analysis of microplastic particles with irregular shapes
without preparation (Wang & Wang, 2018). There were ten replications of the barnacle
samples; we only used the replications with the highest number of microplastics, namely
replications 1, 2, 4, 8, and 10, for FTIR testing. Next, we also tested water and sediment
samples. Of 155 particles suspected of microplastics in barnacles, 97 were observed for
polymer analysis. For water and sediment samples, 17 particles and 24 particles suspected
of being microplastics were observed for polymer analysis. Using the instrument’s software
(OMNIC Picta v1.7, Thermo Fisher Scientific Inc., US), the examined particle spectra
were compared to a number of built-in reference spectra libraries. The libraries that were
utilized were the HR Polymer Additives and Plasticizers (PAP), HR Hummel Polymer and
Additives (HPA), Hummel Polymer Sample Library (HPSL), HR Aldrich Polymers (AP),
and HR Spectra Polymers and Plasticizers by ATR–corrected (SPPATRc). Matches against
the ATR corrected ATR libraries were preferred for determining the polymer identification
(Copyright, 2008 Thermo Fisher Scientific Inc. for Nicolet FT-IR, Thermo Fisher Scientific
Inc., Waltham, MA, USA). Additives, plasticizers, and comonomers such as acrylonitrile
and propylene glycol that are used to create polymers have been counted as microplastics
(Carrillo-Barragán et al., 2024).

Quality control
To ensure quality control, all materials were washed with distilled water and ethanol
to eliminate any cross-contamination. Additionally, the work surface was disinfected
using ethanol. In addition, distilled water and NaCl solutions used for MPs separation
in sediment samples were also analyzed for contamination and identified as being free of

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 MPs. The results revealed that both solutions were found to be devoid of any microplastics.
Furthermore, the KOH solutions utilized for barnacle extraction and the H2 O2 solutions
for water extraction were examined for contamination and determined to be devoid of
microplastics. Analysis showed that neither of the solutions contained any microplastics.
Before usage, the filters were examined under a microscope to verify their absence of
microplastic particles. The samples were consistently handled using latex gloves and
stainless-steel tweezers (Flores-Ocampo & Armstrong-Altrin, 2023). Upon acquiring the
microplastics, they were appropriately preserved in sealed glass petri dishes. To avoid
contamination from clothing, the researcher stood at the edge or a distance from the
sample while keeping and collecting it, wearing only cotton coats. Cotton laboratory coats
were also always worn to avoid microplastic fibers from clothes in the laboratory air. In
order to reduce the accumulation of microplastics in the laboratory, we aimed to examine
the samples in a controlled setting with little air circulation (such as closed doors and
windows and limited human activity within the laboratory) (Shruti et al., 2022).

Data analysis
The data were analyzed to determine if there were significant differences between
microplastics in barnacles, water, and sediment. Before the test, the data were analyzed
normally using the Shapiro–Wilk test. When the data is distributed normally, the Pearson
Correlation test is performed. All statistical tests were conducted at a significant level of
5% (a = 0.05). Pearson correlation of barnacle species was used to analyze the correlation
between the size of microplastics and the size of barnacles. All statistical analysis was done
using SPSS (Armonk, NY, USA).

RESULTS
Barnacles on the East Coast of Surabaya
Only one barnacle species, A. amphitrite, was found at the research site because this species
has a significantly higher resistance to disease. The hard shells of A. amphitrite protect the
soft parts of the barnacle against predators and adverse conditions. The relatively higher
resistance to physical stress may give A. amphitrite a competitive advantage over other
more fragile species (Encarnação & Calado, 2018). A. amphitrite is found living attached to
rigid natural substrates in the bridge pillars (see Fig. 2). The vertical density of barnacles
found in this study is thought to be related to their harmful phototactic properties, which
means that barnacles tend to stay away from light, resulting in a higher population of
barnacles in the water column than at the water’s surface (Nasution & Mudzni, 2016).
The morphology of A. amphitrite is characterized by a six-sided shell that appears to be
pursed or condensed. The body’s exterior is smooth and white with a vertical purple
pattern, and there are no horizontal striations (see Fig. 3).

Microplastic in barnacles, water, and sediment in the East Coast

of Surabaya
This study found that all barnacles, water, and sediment samples contained microplastics.
Microplastics were found in the barnacle samples, with 155 particles and an average of 15.5

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 Figure 2 Appearance of pillars showing substrate attachment of barnacles.
Full-size DOI: 10.7717/peerj.17548/fig-2

Figure 3 Morphology of Amphibalanus amphitrite found at the site (magnification of 8×).

Full-size DOI: 10.7717/peerj.17548/fig-3

± 5 particles per replication. The average abundance of microplastics in barnacles was 5.37
particles per gram (see Table 1).
Water and sediment samples contained microplastics, with as many as 17 and 24
particles, respectively. The total abundance of microplastics in the waters off the East
Coast of Surabaya was 0.38 particles/m3 , while the total abundance in sediments was 60
particles/kg.

Microplastic size
The most commonly found size class of microplastics was class 1, with 79 particles
identified (78 in barnacles, 1 in water). Class 2 had 61 particles (45 in barnacles, nine

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 Table 1 The number of microplastics found in barnacles (Amphibalanus amphitrite), water, and sedi-
ments in the East Coast of Surabaya.

Number of Microplastic
microplastics abundance
(particles)
Barnacles 155 5.37 particles/g
Water 17 0.38 particles/m3
Sediments 24 60 particles/kg
Total 196

Figure 4 Microplastic size in barnacles, water, and sediments in East Coast of Surabaya.
Full-size DOI: 10.7717/peerj.17548/fig-4

in water, and seven in sediment), class 4 had 35 particles (21 in barnacles, six in water,
and eight in sediment), and class 3 had 21 particles (11 in barnacles, one in water, and
nine in sediment) (See Fig. 4). The correlation test results between barnacle length and
microplastic length showed a negative correlation value or were inversely proportional to
r = −0.411 (p < 0.05), categorized as having a strong enough correlation.

Microplastic shapes
Visual observations of microplastics in barnacles, water, and sediments along the East
Coast of Surabaya revealed the presence of three types of microplastics: fragments, fibers,
and pellets. In barnacles, fragments (104 particles), fibers (47 particles), and pellets (four
particles) were the dominant shapes of microplastics. In water and sediments, most

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 Figure 5 Percentage of microplastic shapes in barnacles, water, and sediments found on the East Coast
of Surabaya.
Full-size DOI: 10.7717/peerj.17548/fig-5

microplastics were in the shape of fibers (10 particles and 18 particles), followed by
fragments (seven particles and six particles), with no pellets found (See Fig. 5).

Microplastic color
This study identified six different colors of microplastics: blue, red, yellow, black, brown,
and green. In the barnacle samples, blue was the dominant color (69.7%), followed by
brown (12.9%), black (10.9%), red (5.1%), and yellow (1.3%). In the water samples,
blue was also the dominant color (41.2%), followed by brown (23.5%), red (11.8%),
black (11.8%), and green (11.8%). Sediment samples were primarily blue (91.6%), with

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 Figure 6 Percentage of microplastic color in barnacles, water, and sediments found on the East Coast
of Surabaya.
Full-size DOI: 10.7717/peerj.17548/fig-6

smaller amounts of red (4.2%) and black (4.2%). Figure 6 provides a detailed graph of the
percentage and color of microplastics.

Microplastic polymer types

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) was
used to analyze barnacle, water, and sediment samples for microplastic particles. The
ATR-FTIR analysis of barnacle samples revealed the presence of four different microplastic
polymers consisting of cellophane, poly (1,3-oxathiolane), hydroxyethylcellulose, and hexa
methylol melamine. Meanwhile, cellophane, Pullulan P2000, Pullulan P800, and polyester
with kaolin filler were found for polymer analysis of water samples. No plastic polymers
were found in the sediment samples. Cellophane accumulation in this study was 36%,
followed by poly (1,3-oxathiolane) at 29%. Figure 7 provides a spectrum of microplastic
that found in barnacle and sediment.

DISCUSSION
Microplastic in barnacles, water, and sediment in the East Coast of
Surabaya
This is likely due to the pollution of the East Coast of Surabaya by various types of garbage,
mainly plastic waste. Plastic waste dominates about one-third of the debris found in the
waters (Anjarwati et al., 2017). Microplastics are small particles, typically micrometers in
size, that result from the breakdown of larger plastic materials and fibers released during
laundry and cleaning (Anjarwati et al., 2017). This assertion is supported by the condition
of the research site, where plastic waste is observed floating on the water’s surface, and a
significant amount of plastic waste is present on the coast (see Fig. 8).

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 Figure 7 Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) spectra
and match degrees of selected microplastics. (A) Cellophane in barnacles and (B) cellophane in water.
Full-size DOI: 10.7717/peerj.17548/fig-7

Barnacles, being filter feeders, catch food by sticking out their cirri to capture food
particles (Xu et al., 2020). Previous studies have shown that adult barnacles can digest
microplastics (Browne et al., 2008). Other field studies conducted in three different sites in
Thailand have found 0.23−0.43 particles per gram of tissue in Amphibalanus amphitrite
(Thushari et al., 2017). Microplastics originate from the fragmentation of larger plastics
transported by river run-offs, tides, and wind are sourced from the ocean, including
fishing gear, aquaculture equipment, and clothing fibers from household waste. The high
concentration of microplastics is also attributed to the passing ships that significantly
contribute to microplastic pollution (Ayuningtyas, 2019). The amount of microplastics
found at the bottom of the sediment was greater than on the water’s surface, which is
affected by factors such as gravity, currents, waves, and density. Microplastics settle in
sediment when the water density is lower than that of microplastics (Laksono, Suprijanto &
Ridlo, 2021). Microplastics with lower densities in the water will float in the water column,
while those with higher densities will sink and settle in the sediment (Victoria, 2017).
Regarding the abundance of microplastics in the water, this study found levels of 0.38
particles/m3 . Natural factors that affect the abundance of microplastics in the water include
currents and waves, as the magnitude of waves can cause stirring and lift debris from the

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 Figure 8 The condition of the research site. (A) appears to be a plastic bottle floating under the Sura-
madu bridge and (B) pieces of crepe plastic on Kenjeran Beach.
Full-size DOI: 10.7717/peerj.17548/fig-8

bottom of the water to accumulate on the surface (Muchlissin et al., 2020). The movement
of waste particles occurs due to the deflection of currents and waves in the ocean, with
the weight and amount of microplastics determining their trajectory. Lighter microplastics
are carried away by currents and tides (Febriani, Amin & Fauzi, 2020). Anthropogenic
activities around the waters are another factor causing microplastic pollution. For instance,
microplastics can originate from domestic activities such as washing clothes or from
agricultural activities where wastewater from farms is often untreated and becomes a
source of pollutants that empties into the ocean (Permatasari & Radityaningrum, 2020).

Microplastic size, shapes, and color

The size range of microplastics is significant because it determines their potential impact
on biota and ecosystems (Nor & Obbard, 2014). Microplastics below 40 µm have the same
size range as most microplankton and nanoplankton and have a greater potential to be
digested by various organisms. Generally, the size limit of microplastics is operationally
defined by sampling and analytical methods (Zhang et al., 2020). A negative correlation
can be interpreted as the larger the barnacle, the smaller the microplastics. This is thought
to occur due to the process of thickening food in barnacles. Larger barnacles can be
selective about food to recognize the presence of microplastics. However, this argument is
inconsistent with the research of Xu et al. (2020), which stated that the size of microplastics
has a very weak correlation with barnacle size for species A. amphitrite: r = 20.117,
p = 0.485, n = 38.
This study indicates that fragments are the most common shape of microplastics found
in barnacles (67%). Fragments are often textured and dense and likely originate from the

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 fragmentation of plastic packaging, including drink bottles, plastic bags, and PVC pipes
(Victoria, 2017). Plastic waste can also influence the prevalence of fragmentary shapes
due to tourist and household activities, such as plastic bottles, mica packaging, and other
objects with high plastic content (Azizah, Ridlo & Suryono, 2020). In water and sediment,
the dominant shape of microplastics was fiber (58% and 75%). The long, colored fibers
found in marine habitats may come from domestic waste, including synthetic cloth, fishing
boat waste, and fishing gear such as fishing nets and lines (Ayuningtyas, 2019). Fishing
activity is a significant contributor to fiber microplastics, as most fishing nets are made
of fiber (Victoria, 2017; Azizah, Ridlo & Suryono, 2020). The study’s findings suggest that
the dominant shape of microplastic particles (fragments and fibers) likely originates from
secondary sources, such as plastic waste that has degraded in the marine environment
over time (Nor & Obbard, 2014). Pellets (2.5%) are another shape of microplastic found in
barnacles, which are round-shaped and primarily derived from plastic factories, cleaning
and beauty products, and resin powders (Victoria, 2017). Pellets are often produced directly
by raw material manufacturers making plastic products, including those used to shape
larger plastic products, and micro-scale products, such as industrial plastics and medical
devices, including those used in pharmaceutical applications. The results of this study
support the findings of Viršek et al. (2016), which state that the most commonly found
microplastic particles in water ecosystems are fragments from larger plastic waste and
fibers from textile fibers. The shapes of microplastics discovered in this study are consistent
with those reported by Xu et al. (2020), who found microplastic fibers (95.7%), fragments
(3.4%), and pellets (0.8%) in barnacles. This study revealed the absence of other forms
of microplastics apart from fibers and fragments in the water samples. This is caused by
the use of techniques carried out by researchers, namely by carrying out a drying process
on water samples at a temperature of 80 degrees. This was confirmed by Munno et al.
(2018), who confirmed that the choice of chemical digestion technique to remove natural
organic matter and isolate microplastic particles, as well as the specific conditions under
which the method is applied, can have a significant impact on the recovery of certain
types of microplastics particle. The investigation revealed that heating conditions impacted
two of the five categories of microbeads present in the personal care products evaluated.
Beads melt when the solution’s temperature, resulting from the application of heat or an
exothermic oxidation process, exceeds 60 ◦ C (Munno et al., 2018).
Some studies suggest that certain microplastics, such as fibers, are commonly colored
red and blue and primarily originate from domestic water waste, such as laundry water,
from residential areas and water waste management plants. If microplastics are still highly
concentrated in color, it suggests that they have not undergone significant discoloration
(Dekiff et al., 2014). A yellowish color indicates prolonged exposure to the sea and oxidation.
Chocolate color is caused by the oxidation of polymers in microplastics that have been
exposed to sunlight for an extended period. The black color of microplastics suggests they
are composed of polystyrene (PS) or polypropylene (PP) and may also contain pollutants
such as polycyclic aromatic hydrocarbons (PAHs) and absorbed polychlorinated biphenyls
(PCBs) (Azizah, Ridlo & Suryono, 2020; Laksono, Suprijanto & Ridlo, 2021).

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 Microplastic polymer types
However, no microplastic polymers were detected in sediment samples using FTIR analysis,
likely due to the challenges associated with analyzing irregularly shaped fragments, which is
usually a refractive error and interference (Dris et al., 2018). Other studies have shown that
the degradation can cause changes in the FTIR spectrum, interfering with the identification
process (Jung et al., 2018). The dominance of cellophane (CP) in this study is consistent
with previous findings by Xu et al. (2020), who identified CP as one of the three dominant
polymers in barnacles, with polyethylene terephthalate (PET) and polypropylene (PP).
Ding et al. (2018) also reported the common presence of CP and PP in marine bivalves.
Cellophane was the most commonly identified microplastic fibers and fragments in this
study. Although marketed as ‘‘biodegradable’’, the low biodegradability of regenerated
cellulose, such as cellophane (36%), suggests that biodegradable plastics are a ‘‘false
solution’’ to marine waste. Cellophane is extensively used in food packaging and as a release
agent in fiberglass rubber production. The dominance of cellophane as a microplastic type
has also been reported at several sampling points in the United Kingdom estuarine complex
and salt lakes in China (Zhang et al., 2019). Poly (1-3-oxathiolane) (29%) was found in
this study, as well as in zooplankton, according to Sun et al. (2018). The prevalence of
fiber and cellulose in commercial species highlights their limited ability to recognize and
avoid microplastics during consumption (Wu, Zhang & Xiong, 2018). Rayon, a regenerated
cellulose commonly found in personal hygiene products and textiles, is likely a primary
source of cellulose in marine environments (Acharya et al., 2021). Hydroxyethyl cellulose
(7%) is a water-soluble, non-ionic polymer used in various industrial fields, such as color
thickening, textile finishing, and thickening in cement mortars (Abdel-Halim, 2014). Hexa
methylol melamine (7%) contains resins used in the production of coatings and plastics
for cans, wires, and cars (Dsikowitzky & Schwarzbauer, 2015). Pullulan P200 (7%) and
Pullulan P800 (4%) have high potential in thin filmmaking, are transparent, odorless,
tasteless, and consumable, and are widely used as packaging materials and to produce
dental strips (Kraśniewska, Pobiega & Gniewosz, 2019).

Barnacles as potential microplastic bioindicators

Barnacle crustaceans, known for their high tolerance to environmental pressures, are
commonly used in coastal areas for monitoring marine pollution (Scotti et al., 2023).
Barnacles feed by extending their cirri to trap brine shrimps, copepods, and phytoplankton
from the water column (Xu et al., 2020). Due to their feeding efficiency, surface position
in the water column, and opportunistic behavior, these organisms are susceptible to
consuming microplastics. As a result, they can be regarded as appropriate species for
evaluating the presence and dispersion of microplastics and fibers in the surrounding
waters (Scotti et al., 2023). The previous study by Goldstein & Goodwin (2013) reported
that 385 individuals of gooseneck barnacles (Lepas spp.) collected from the North Pacific
Subtropical Gyre (NPSG) had plastic particles present in their gastrointestinal tract, ranging
from one plastic particle to a maximum of 30 particles. Results by Thushari et al. (2017)
show that barnacles are highly susceptible to microplastic accumulation showing the
highest micro debris density compared to other filter feeders. Barnacles use a filter-feeding

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 mechanism by extending their spines outward to create water currents. During their
feeding, water with suspended particles, food materials, and plastic waste enters the body
cavity without selection and accumulates in their bodies.
The Amphibalanus amphitrite of barnacles found in this study holds promise as a
potential microplastic bioindicator due to its cosmopolitan distribution and abundance
in diverse marine habitats worldwide. A. amphitrite primarily engages in regular and
rapid planktivory, with the addition of simultaneous maxilliped microfiltration. During
normal beat, water flow through the mantle cavity is intensified to enhance the filtration
of microscopic food. On the other hand, during a fast beat, there is a higher probability of
capturing particulate food through the large cirri (Pasternak & Achituv, 2007). A previous
study by Xu et al. (2020) supports this, noting the presence of A. amphitrite in various
habitats, including mudflats, piers, and dams. The high abundance of microplastics
found in barnacles compared to surrounding sediments and seawater (5.37 particles/g
vs. 0.06 particles/g and 0.38 particles/m3 , respectively) indicates that barnacles can track
microplastic exposure in the environment. Furthermore, the smaller size of A. amphitrite
reduces the efficiency of microplastic expenditure from the digestive tract, which can result
in higher microplastic content in the habitat. This study also revealed similarities in the
characteristics of microplastics found in barnacles, water, and sediment, including the
dominance of fragment and fiber shapes of microplastics, as well as the blue color of the
particles. The analysis of microplastic polymers found in barnacles and the environment
also showed a high percentage of cellophane (36%), which is consistent with the results
of the previous study by Xu et al. (2020) that found cellophane (57.69%), polyethylene
terephthalate (PET, 11.54%), and polypropylene (PP, 9.62%). To strengthen the findings of
this study, it is crucial to increase the sample size and replicate measurements across various
environmental compartments. This would provide a more comprehensive understanding
of microplastic distribution and accumulation patterns. Furthermore, incorporating
multiple bioindicator species can validate observed correlations, and conducting long-term
monitoring studies can reveal temporal trends in microplastic concentrations, enhancing
the assessment of microplastic pollution in marine ecosystems.

CONCLUSIONS
This study identified 196 microplastic particles in Amphibalanus amphitrite, water, and
sediments. Barnacles had the highest concentration of microplastics (155 particles),
followed by sediment (24 particles) and water (17 particles). The size, shape, and color
of the microplastics varied across the samples, with barnacles dominated by class 1 size
(1–10 µm), fragments, and blue color. Water and sediment were dominated by class 2
and 3 sizes (10–50 µm and 50–100 µm, respectively) and fiber shapes, with blue being
the most common color. Cellophane was the most dominant chemical composition
found in all samples. Based on the findings, Amphibalanus amphitrite shows promise as
a potential bioindicator for microplastics, particularly the cellophane type. Although we
did not calculate correlations due to the lack of sample replication across environmental
compartments, our observations suggest a linear relationship between its population and
the accumulation of this specific type of microplastic.

Raufanda et al. (2024), PeerJ, DOI 10.7717/peerj.17548 16/22

 ACKNOWLEDGEMENTS
We thank the lecturers from the Faculty of Biology at Universitas Jenderal Soedirman for
knowledge sharing regarding this research.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
Miftakhul Sefti Raufanda received a scholarship from the Indonesia Endowment Fund for
Education (LPDP), under the supervision of the Ministry of Finance of the Republic of
Indonesia. LPDP also supports facilitating the publication of this article. This work was
supported by the Department of Biology FSAD ITS and the Directorate of Research and
Community Service while facilitated the research activities through Research Contract No.
1882/PKS/2022 Batch 2. The external funders had a role in the decision to publish. The
funders had no role in study design, data collection and analysis, or preparation of the
manuscript.

Grant Disclosures
The following grant information was disclosed by the authors:
the supervision of the Ministry of Finance of the Republic of Indonesia.
Indonesia Endowment Fund for Education (LPDP) under the supervision of the Ministry
of Finance of the Republic of Indonesia.
The Department of Biology FSAD ITS and the Directorate of Research and Community
Service: 1882/PKS/2022.

Competing Interests
The authors declare there are no competing interests.

Author Contributions
• Miftakhul Sefti Raufanda conceived and designed the experiments, performed the
experiments, analyzed the data, prepared figures and/or tables, authored or reviewed
drafts of the article, and approved the final draft.
• Aunurohim Aunurohim conceived and designed the experiments, analyzed the data,
authored or reviewed drafts of the article, and approved the final draft.
• Romanus Edy Prabowo conceived and designed the experiments, analyzed the data,
authored or reviewed drafts of the article, and approved the final draft.

Data Availability
The following information was supplied regarding data availability:
The raw data is available in the Supplemental File.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.17548#supplemental-information.

Raufanda et al. (2024), PeerJ, DOI 10.7717/peerj.17548 17/22

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