Barnacle analysis as a microplastic pollution bioindicator on the East Coast of Surabaya

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 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% H₂O₂ is added to each sample (Masura et al., 2015). The remaining water samples containing solid matter are mixed with 30% H₂O₂ 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).

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 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 H₂O₂ 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).

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 ± 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 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 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 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.

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).

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 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 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).

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 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.