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Introduction
It is well established that microplastic (MP) contamination is affecting ecosystems at a global level (Cox et al., 2019). MPs have been detected in diverse environments, from the deep sea and polar regions to coastal areas across all continents (Blumenröder et al., 2017), as well as in freshwater and terrestrial systems (Horton et al., 2017; Blettler et al., 2018; Dris et al., 2018; Ramachandraiah et al., 2022). The United States Environmental Protection Agency (U.S. Environmental Protection Agency, 2024) defines MPs as plastic particles ranging in size from 5 millimeters (mm) to 1 nanometer (nm). Their widespread distribution is further documented by findings from 2019, which revealed that MPs travel through atmospheric water condensation and are deposited via precipitation, even in remote and uninhabited regions (Allen et al., 2019).
The growing number of publications documenting the presence of MPs in marine organisms is alarming, but similar research on the presence in terrestrial environments is still scarce, although Horton et al., 2017 showed that the amount of plastics released into the terrestrial environment is 4 to 23 times greater than that released into the marine environment. In addition, despite the abundance of terrestrial MPs and their potential to cause deleterious biological effects, there are few studies related to the impact of MPs in terrestrial organisms, although there are already reports of MPs found in milk, meat, feed, blood (Van der Veen et al., 2022), earthworms and chickens for human consumption (Huerta-Lwanga, et al., 2017).
The Central American region is far behind in this regard. Only a few reports on MPs in marine environments have been published (Davidson, 2012; Delvalle de Borrero et al., 2020; Kutralam-Muniasamy et al., 2020; Mazariegos-Ortíz et al., 2020; Fallon & Freeman, 2021; Oldenburg et al., 2021; Aldana Aranda et al., 2022; Orona-Návar et al., 2022), and the lack of relevant literature in the region has been mentioned by authors including Ivar Do Sul & Costa, 2014. The first report on MPs in Costa Rica appeared in 2018, and since then five other reports have been published (Johnson et al., 2018; Bermúdez-Guzmán et al., 2022; Astorga et al., 2022a; Astorga et al., 2022b, Rojas-Jimenez et al., 2022). The information generated so far has primarily focused on marine organisms, highlighting a significant gap in our understanding of Costa Rican terrestrial ecosystems, making it a critical area of study. Studies of MPs in terrestrial animals, especially those consumed by humans, are essential to broaden our knowledge and address this public health concern in the country.
The liver, through which blood from the stomach and intestine passes, has already been determined to be one of the routes through which microplastics are spread throughout the human body (Osman et al., 2023; Kalra et al., 2024). In Costa Rica, bovine and pork liver is widely consumed, with bovine liver typically prepared as a steak and pork liver often made into pâté (Chacón Villalobos, 2021; Madrigal-Meneses & Caravaca-Rodríguez, 2020). This underscores the importance of understanding the potential health implications of contaminants in this widely consumed organ. The objective of the study was therefore to determine the presence of microplastic particles in meat products from Costa Rica that form part of the human diet.
Materials and methods
Samples
For this study, bovine and porcine livers were obtained from butcher shops located in the centers of cities. This implies that they were healthy organs that had successfully passed veterinary inspections at the slaughterhouse. The livers were randomly collected, bought as a whole and not sliced, just as they are customarily sold to the public, and were transported in coolers. A total of 20 bovine livers and 18 porcine livers were processed, obtained from markets in the regions of greatest meat production, to provide a representative sample of both types of liver (Figure 1).
Tissue processing
The first step in preparing the sample analysis was a thorough cleaning of the working areas of the laboratory. To prevent contamination by microplastic particles in the air, work was carried out in a laminar flow chamber, mainly for the preparation of solutions, filtration and sieving. In addition, all glassware used in the process was washed three times, then dried in a laboratory oven and covered with aluminum foil to avoid cross-contamination, following the methodology applied by Astorga et al., 2022b.
For chemical digestion, a sample of 100 grams was taken from the inside of each liver, taking into consideration the central axis of the organ to ensure that the sample allowed part of the hepatic veins and arteries to be seen. To extract the MPs from liver samples, the tissue was chemically digested following the method described by Cole et al (2014), Kühn et al. (2017), and Bessa et al., (2019). A 10% KOH solution was added to the liver tissue sample, ensuring that the liquid covered the sample and did not exceed 50% of the total volume of the Erlenmeyer flask (250 or 500 ml). Next, all Erlenmeyer flasks were covered with aluminum foil and placed in an incubator with orbital shaking at 60 °C and 300 rpm for 24 hours. Once the chemical process was completed, the digested contents were sieved through a 63 μm stainless steel sieve and transferred to a clean glass Petri dish. To remove excess water, the glass Petri dishes were covered with aluminum foil with small holes to allow water evaporation and prevent possible contamination by airborne plastic and placed in an oven at 45 °C for 30 hours (Enders et al., 2020).
As a quality control measure for the experiment, controls were performed with distilled water and KOH without any sample during chemical treatment of the livers, as well as during observation, identification and validation of microplastics (Jabeen et al 2017). Fibers were the most abundant microplastic particles identified in the control samples, and all these plastic microparticles in these samples were excluded from the data analysis.
Identification and validation of microplastic particles
An OPTIKA SZ-ST2 stereoscope with camera (AMSCOPE MU1000) was used for the identification, measurement and photographing of each microplastic particle observed in the processed samples. For data analysis, only plastic particles smaller than 5000 µm were considered (Andrady, 2011), which were classified by type as fibers, fragments, pellets or films, and categorized by color (Hidalgo-Ruz et al., 2012; Qiu et al., 2016; Martin et al., 2017; Enders et al., 2020).
Results
Out of the 20 bovine livers processed, 90% (n= 18) were positive for the presence of MPs. All the particles found were classified as fibers with an average size of 1530 µm (range 230 µm to 3680 µm) and were most frequently (38.10%) blue-green in color. Other colors of fibers detected were black (21.43%), blue (16.67%), purple (7.14%), red (7.14%), white translucent (4.76%), brown (2.38%) and sky blue (2.38%) (Figures 2 and 3). The amount of MPs per gram of liver tissue processed varied from 0.009 to 0.074 with an average of 0.0203 MPs g-1 (Figure 4).
On the other hand, of the 18 processed porcine livers, 83.3% (n = 15) were positive for the presence of MPs. Ninety-five (98.96%) of the particles found were classified as fibers with an average size of 1240 µm (range 220 µm - 4510 µm) and most of them (32.29%) blue-green in color. Other colors of the fibers detected were red (22.92%), blue (15.63%), translucent white (11.46%), black (9.38%), green (5.21%), and purple (3.13%) (Figure 2, 3). The other particle found was classified as a film, red in color, with a size of 2150 μm. The amount of MPs per gram of liver tissue processed varied from 0.013 to 0.2948 with an average of 0.063 MP g-1 (Figure 4).
A summary of these findings, including the percentage of positive samples and MP size ranges for both species, is presented in Table 1.
Table 1 Comparison of microplastic (MP) presence in bovine and porcine livers from Costa Rica.
| Parameter | Bovine Livers | Porcine Livers |
|---|---|---|
| Total samples processed | 20 | 18 |
| Positive samples (%) | 90% (n=18) | 83.3% (n=15) |
| MP classification | 100% fibers | 98.96% fibers, 1.04% film |
| Average MP size (µm) | 1530 µm | 1240 µm |
| MP size range (µm) | 230 - 3680 µm | 220 - 4510 µm |
| Most common MP color (%) | Blue-green (38.10%) | Blue-green (32.29%) |
| Other MP colors (%) | Black (21.43%), blue (16.67%), purple (7.14%), red (7.14%),translucent white (4.76%), brown (2.38%), sky blue (2.38%) | Red (22.92%), blue (15.63%), translucent white (11.46%), black (9.38%), green (5.21%), purple (3.13%) |
| MP per gram of liver (range) | 0.009 - 0.074 MP g⁻¹ | 0.013 - 0.2948 MP g⁻¹ |
| MP per gram of liver (average) | 0.0203 MP g⁻¹ | 0.063 MP g⁻¹ |
Source: data
Figure 3 Microplastic particles found in bovine (A) and porcine (B) livers from the regions of greatest meat production of Costa Rica. Source: data obtained during this investigation.
Discussion
Due to the ubiquity of MPs, living organisms are inevitably taking them into their bodies through ingestion or inhalation (Galloway et al., 2017). Since the objective of the study was to investigate if the Costa Rican human population was consuming MPs in products of animal origin, without attempting to compare the quantity of microplastics between regions or trace the origin of the microplastics, there is no information on the specific food supplied to each of the animals whose livers were sampled. However, in Costa Rica, both dairy and beef cattle are generally fed similar feed ingredients-primarily forage, supplemented with corn, soybean meal, and sugarcane molasses in varying proportions. In specialized dairy systems, the proportion of concentrate in the diet is typically higher compared to beef cattle, whose feed is predominantly pasture-based with minimal supplementation. It is also important to note that feedlot systems, such as those commonly used in the United States for beef cattle fattening, are not used in the country. On the other hand, most pigs are kept housed and have a diet of fattening concentrated feed based mostlyon corn and soybean meal (Instituto Nacional de Estadística y Censos, 2022). The presence of MPs has been documented in salt (Yang et al., 2015; Kim et al., 2018), soybean meal (Walkinshaw et al., 2022), sugarcane (Liebezeit & Liebezeit, 2013) and corn (Haluska, 2020; Garrido Costanzo, 2022; Shi et al., 2023), all of which are ingredients often used in the animal feed industry. Also, MPs of marine origin could be incorporated into foods of terrestrial animals due to the use of derivatives of fish in the manufacture of feed concentrates (Bouwmeester et al., 2015; Walkinshaw et al., 2022). Another possible route of contamination could be drinking water; in a systematic study carried out by Danopoulos et al (2020), it was found that microplastics were nearly ubiquitous and were present in drinking water.
The results of our study show the presence of microplastics (MPs) in liver samples intended for human consumption. Although cross-contamination during processing, from the slaughter of the animals to their subsequent market commercialization in the country, cannot be completely ruled out (Garrido & Costanzo, 2022), this is considered unlikely. Extensive precautions were taken to process the samples carefully, including taking digestion samples from inside the organ and implementing all possible measures to avoid contamination in the laboratory. Regardless of the contamination source, our study sought to determine the presence of microplastics in the product that ultimately reaches the consumer.
In this study, the amount of MPs per gram found in animal livers (bovine: average 0.024 MPs g-1; porcine average 0.074 MPs g-1) is lower than that found in human livers, which in healthy humans were found to have a number of microplastic particles ranging from 0 to 2.2 per gram of tissue (with an average of 1 MP g-1), while in patients with liver disease (cirrhosis) this number increased by more than 8 times (an average of 8.3 MP g-1) (Hovartis et al., 2022).
In our studies, the size of the MPs found was bigger in both bovine (average 1530 µm) and porcine (average 1240 µm) livers than the size of the particles found in cirrhotic human liver tissue, which ranged from 3.0 to 29.5 μm (average 9.8 µm); however, it has not been possible to determine whether the accumulation of MPs is the cause or the consequence of liver disease in human subjects (Hovartis et al., 2022), although they have been reported to damage liver cells, and to alter fat metabolism and enzyme activities in crabs, fish and mice (Lu et al., 2016; Deng et al., 2017; Lu et al., 2018; Chae et al., 2018; Yu et al., 2018).
While the scientific literature has reported translocation of MPs ranging from 1 to 500 μm in size into different tissues (liver, cell membranes, the blood-brain barrier and even the placenta) of several species (Hussain et al., 2001; Vethaak & Leslie, 2016; Triebskorn et al., 2019; Elizalde-Velazquez et al., 2020; Yee, et al., 2021) causing oxidative stress, cell damage and inflammation, some authors have recently challenged whether microplastics are capable of being translocated (Schür et al., 2019). Currently, the disagreement is related to the size of particles that can be taken up by cells and translocated among tissues (Triebskorn et al., 2019).
Haave et al., (2021) carried out a study between 2017 and 2019 on wild animals (mammals, birds and fish), and found MPs in the liver of anchovies. They mentioned that there was a translocation of MPs in the tissues of the animals studied, and that MPs that cross the epithelial tissue of the digestive tract could then be transported through the portal system to the liver (Garrido & Costanzo, 2022).
Based on the criteria for visual identification of microplastics described by Zhang et al., 2020, information regarding the shape, size, and color of the MPs that were encountered in this study provides important information about the types of plastic found, but not about their origin and how the animals may be being contaminated. In the case of shapes, fibers in urban areas have been linked to industrial activities, while flakes are attributed to mechanical and chemical degradation. Colors are due to pigments that are used in the fabrication of plastics - for instance, black plastics have been shown to contain PU, transparent plastics indicate polypropylene, opaque plastics point to LDP, and white plastics indicate polyethylene (Ramachandraiah et al., 2022).
Plastics are generally considered to be inert materials. However, the following have been proposed as possible damages that MPs might cause in the body: vascular embolization, inflammatory responses (associated with cell phagocytosis of particles and increased autoimmune response) (Wright & Kelly, 2017; Yee et al., 2021; Hu et al., 2023), gastrointestinal alterations such as dysbiosis and altered mucus secretion (Garrido & Costanzo, 2022), cytotoxicity due to oxidative stress mechanisms (Schirinzi et al., 2017; Solomando et al., 2020; Urli et al., 2023), liver damage (Garrido & Costanzo, 2022), alterations in energy and lipid metabolism (Deng et al., 2017), and neurotoxicity (Garrido & Costanzo, 2022).
In some plastics, additives (such as bromates as flame retardants, plasticizers such as phthalates, and heat stabilizing metal compounds) may constitute a major fraction of their composition, and the toxicity of a large part of the additives is well known. Some well-documented examples in humans include reproductive toxicity (e.g., bis(2-ethylhexyl) phthalate (DEHP) and bisphenol A (BPA)), carcinogenicity (vinyl chloride and butadiene), and mutagenicity (benzene and phenol) (Campanale et al., 2020). In addition to additives, MPs can release residual monomers and degradation products that have formed on the surface through chemical or photochemical reactions during their stay in the environment (Lucas et al., 2008; Ramachandraiah et al., 2022). Studies of the chemical composition of plastic in field samples are scarce due to the high cost of the tests, but they are necessary to understand the toxicity of these particles.
Finally, consumption of the species studied by humans could increase their consumption of MPs, as other studies have shown. Yee et al., 2021 showed that the human food chain is a major source of MP consumption, based on a review of MP pollution in food and other ingested substances (seafood, sugar, honey, salt, alcohol, bottled water, tap water and air), and we now have evidence of MPs in terrestrial animals which are consumed by humans.
Conclusions
Although the origin of the MPs in the livers studied in this investigation cannot be determined, their presence in these products suggests a high likelihood that the human population consuming them is being exposed to microplastic contamination. This research at the local and regional level has produced several significant findings. Firstly, the identification of microplastics in livers intended for human consumption is crucially important, shedding light on environmental contamination and contributing to public awareness about food safety. These findings do not indicate that consumption of these products should be avoided (this study is not intend to discourage consumption of these products); however, they should be taken into account by healthcare professionals, since the presence of these particles in tissues has been reported to potentially trigger adverse reactions in the digestive and respiratory systems, skin reactions, reproductive issues, and more. Secondly, the findings highlight environmental impacts, emphasizing the interaction between plastic pollution and the food chain, which could encourage initiatives to mitigate plastic contamination and protect local natural resources. Thirdly, the findings may influence policy-making and regulations, leading to stricter measures in waste management and food safety practices, ultimately benefiting public health and the environment at both the local and regional levels. Lastly, this pioneering study opens avenues for further research into microplastic presence and impacts on other organs and food sources, thereby advancing global understanding of this issue.
Acknowledgment
This project (SIA 0514-2019) was supported by the Office of the Vice-Rector for Research through the Institutional Fund for Academic Development (FIDA) at the National University, Costa Rica.
Author contribution statement
All the authors declare that the final version of this paper was read and approved. Authors and CRediT Roles: N. S. B., K. U. N., C. V. M. & A. G. R.: Conceptualization, Methodology & Supervision; K. U. N. & A. G. R.: Data Curation, Formal Analysis; N. S. B., K. U. N., C. V. M. & A. G. R.: Validation, Investigation, Resources, Writing - Original Draft, Writing - Review & Editing, Visualization.
The total contribution percentage for this paper was as follows: N.S.B. 25%, K.U.N. 25%, C.V.M. 25% & A.G.R. 25%.
