Identification of anthropogenic debris in the stomach and intestines of giant freshwater prawns from the Trang River in southern Thailand

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Ecology

Introduction

The world is well aware of the devastating effect that plastic waste has on the ecosphere. Plastics are a popular material used to make a variety of goods (Plastics Europe, 2018). They are a form of synthetic polymer that has been widely employed due to their light weight, strength, durability, and low cost, as well as their ability to be molded into various shapes and sizes using contemporary manufacturing processes (Bogusz & Oleszczuk, 2017). Thailand is among the top six nations that dump the most plastic into the sea (Jambeck et al., 2015).

Microplastics are microscopic plastic particles that are produced as a byproduct of commercial product manufacturing due to the breakdown of larger plastics by physical, chemical, and biological processes (Arthur, Baker & Bamford, 2009). These processes produce macroplastic (more than 25 mm), mesoplastic (5–25 mm), and microplastic (less than five mm) particles (Desforges et al., 2014), which are derived from primary sources of microplastics such as plastic beads from plastic manufacturers, microbeads in cosmetics, and fishing net fibers, as well as secondary sources of microplastics (GESAMP, 2016). Microplastic pollution in the environment causes microplastics to infiltrate the food chain, where they can directly impact organisms and ecosystems (Lusher, Hollman & Mendoza-Hill, 2017). It can now be detected in a variety of ways. It has been documented that many forms of microplastics have been ingested by zooplankton, shrimp, and animals living in alluvium or mangrove soil (Pradit et al., 2021; Abbasi et al., 2018; Devriese et al., 2015; Moore et al., 2001; Murray & Cowie, 2011). Several studies have shown that consuming too much microplastic-contaminated food on a regular basis increases the likelihood of acquiring allergies (Pironti et al., 2021). Microplastics can obstruct the activity of organs in the body, such as the circulatory system, because they are small enough to enter the bloodstream, causing pain and irritation to internal organs. They can also enter the digestive system where they can cause gastric cancer. The most dangerous effect of microplastics on the body is genetic mutation (Thushari et al., 2017).

South and Southeast Asia, in addition to some parts of the Pacific Islands, are home to giant freshwater prawns (Petcjun & Siriwat, 2016). Giant freshwater prawns are large shrimp that live in freshwater waterways along rivers and canals, and are usually observed in regions where the water is flowing and clean. They are commonly consumed both locally and internationally because the flesh is excellent and has a high nutritional value. Because of their high price, the species is popular among fishermen (Nitiratsuwan et al., 2022). Fertilized female giant freshwater prawns move to the river mouth or brackish water to spawn during the breeding season before moving back to fresh water. The feeding habitats of prawns involve consuming a wide variety of organic material (Sitthi, 2011). Microplastics are abundant in river water and soil, and aquatic species such as prawns may absorb them while feeding. Microplastic consumption has been researched in different shrimp species, such as Paratya australiensis, and were found in 36% of the shrimp, with an average of 0.52 ± 0.55 items/ind (24 ± 31 items/g) (Nan et al., 2020.). The gut of Nephrops norvegicus was investigated, and 83% of the animals analyzed had plastics (mostly filaments) in their stomachs (Murray & Cowie, 2011). There has been little academic research on this topic in Thailand, and there have been no reports of microplastic buildup in the Trang River. This study investigates the presence of anthropogenic waste, such as microplastic-like debris, in the gastrointestinal tracts of giant freshwater prawns.

Materials & Methods

Sample collection and preparation

In September of 2022, a total of 6 kg of giant freshwater prawns (M. rosenbergii) was purchased randomly from coastal fishermen who operate on the Trang River in the Trang province (Fig. 1). To conduct a microplastic analysis, a total of 60 giant freshwater prawns were randomly selected, with 30 males and 30 females. Male and female characteristics of giant freshwater prawns are shown in Fig. 2. The sample size used in this investigation is consistent with previous studies conducted by Cole et al. (2013), Pradit et al. (2020), and Jitkaew et al. (2023). It is important to note that this species is commonly consumed in Thailand. To preserve the giant freshwater prawn samples, they were carefully wrapped in aluminum foil and stored in a freezer at a temperature of −20 °C in preparation for further analysis.

The study area of the Trang River, Trang Province, Thailand.

Figure 1: The study area of the Trang River, Trang Province, Thailand.

Male and female characteristics of giant freshwater prawns.

Figure 2: Male and female characteristics of giant freshwater prawns.

Prevention of microplastic contamination

A blank test was performed using a 250-ml beaker filled with distilled water and placed in a laboratory. After 24 h, the distilled water in the beaker was filtered using filter paper, oven-dried, and examined under a microscope to ensure the absence of microplastics. The experiment took place in a clean room with a fume hood, and no disturbances such as wind were present in the laboratory. The researcher wore gloves, a gown, and a surgical cap throughout the experiment. During the lab analysis, aluminum foil was placed over the glass beaker containing the dissected sample (Pradit et al., 2023). To minimize the impact of exogenous microplastics, no plastic instruments were used on the samples during the experiment (Pan et al., 2021). All materials were cleaned and rinsed with distilled water before use.

Anthropogenic debris identification

The frozen giant freshwater prawn samples (M . rosenbergii) were defrosted at room temperature. Carapace length (CL) and length of abdomen (LA) were measured in centimeters (Fig. 3), and body weight (BW) was measured in grams, in accordance with Food and Agriculture Organization of the United Nations (FAO) guidelines. The description from Dehaut et al. (2016) served as the basis for the sample analysis procedure. The samples’ intestines and stomachs (Fig. 2) were removed using thin forceps, cut into small pieces, and placed in a beaker. The alkaline technique was applied to digest the dissected stomachs and digestive tracts of the samples (Cole et al., 2014; Ding et al., 2018). The samples were then placed in 30 ml of 10% potassium hydroxide (KOH) solution, stirred continuously for 1 min with a stirring rod, covered with aluminum foil to prevent foreign matter contamination from the air, heated to 60 °C for 5 min, and left to degrade for another 12 h at room temperature. The samples were then filtered with a 20-micron filter cloth. The filter cloth (new, made of nylon) was dried in a hot air oven at 50 °C for 5 h. Studies conducted after digestion can benefit from density separation. The primary aim is to separate microplastics from sediment or other inorganic material that was not dissolved during enzymatic or chemical digestion (Stock et al., 2019), and when there is a significant amount of inorganic material present (Lusher et al., 2017). Because there was no organic or inorganic material left (such as sand or chitin) after digestion with KOH (10%) in our investigation, the density separation step was skipped. The method of using alkaline digestion was adapted for the dissolution of the biota of invertebrates and fish and has proven largely efficacious in removing biogenic material (Lusher et al., 2017). In the absence of debris, organic matter, shells, or cartilage, which can prevent the identification of microplastics on the filter, an alkaline digestion was deemed to be efficient (Dehaut et al., 2016).

Carapace length and length of the abdomen of a giant freshwater prawn.

Figure 3: Carapace length and length of the abdomen of a giant freshwater prawn.

The microplastic samples on the filter cloth were carefully counted and their sizes measured. Additionally, their characteristics and color were observed using an Olympus SZ61 three-dimensional viewing system equipped with a light-emitting diode. The microplastics were counted as individual pieces. The size of the microplastics was categorized into four classes: <100 µm; 101–500 µm; 501–1,000 µm; and >1,000 µm. Furthermore, the types of microplastics were classified into two categories: fibers and fragments. Randomly selected microplastics longer than 100 µm were analyzed on a Fourier transform infrared spectrophotometer (FTIR), using the Attenuated Total Reflectance mode to identify their composition; Spectrum Two; Perkin Elmer Spectrum IR version 10.6.2, spotlight 200i; Perkin Elmer, Seer Green, UK. In the study, the wavelength spanned from 4,000 cm 1 to 400 cm1. The acquired spectrum was compared to the standard library spectrum.

Data analysis

Descriptive data on the number, size, color, and shape of microplastics was collected in Microsoft Excel (Office Professional Plus 2019). Data was presented in the form of a mean standard error. The t-test was used to compare the number of microplastics found in the intestines and stomachs of male and female giant freshwater prawns. The relationship between the number of microplastics in the intestines, stomachs, carapace length, length of the abdomen, and body weight between male and female giant freshwater prawns was measured using the Pearson correlation. The significance level was set at p < 0.05.

Results

Abundance of anthropogenic debris in the stomach and intestine of giant freshwater prawns

Sixty giant freshwater prawns (30 females and 30 males) were tested. Each giant freshwater prawn was measured for CL, BW, LA, and SW before the analysis was conducted (Table 1). The number of microplastics in the stomachs and intestines of female and male giant freshwater prawns were 4.87 ± 0.72 items/individual, 1.73 ± 0.36 items/individual, 3.03 ± 0.58 items/individual and 2.70 ± 0.57 items/individual, respectively (Table 2). The number of microplastics in the stomachs of female giant freshwater prawns and male giant freshwater prawns (p = 0.866) and intestines (p = 0.171) was not statistically different.

Table 1:
Carapace length (cm), weight (g), length of abdomen (cm), and stomach (g) in giant freshwater prawns.
Sex Carapace length (cm) Weight (g) Length of abdomen (cm) Stomach (g)
max min mean ± SE max min mean ± SE max min mean ± SE max min mean ±SE
Female (n = 30) 5.40 3.40 4.41 ± 0.08 99.11 28.94 54.83 ± 2.96 10.00 7.00 8.49 ± 0.13 1.28 0.22 0.56 ± 0.04
Male (n = 30) 7.00 3.60 5.15 ± 0.17 175.61 33.06 89.07 ± 8.07 11.40 7.00 9.26 ± 0.19 6.33 0.30 1.39 ± 0.24
DOI: 10.7717/peerj.16082/table-1
Table 2:
Anthropogenic debris abundance in giant freshwater prawns.
Sex Body Microplastics item
Total Microplastics Average item/individual
Female Stomach 146 4.87 ± 0.72
Intestine 52 1.73 ± 0.36
Male Stomach 91 3.03 ± 0.58
Intestine 81 2.70 ± 0.57
DOI: 10.7717/peerj.16082/table-2

Anthropogenic debris size

In the stomachs of female giant freshwater prawns, the most common size of microplastics found was <100 µm whereas in the stomachs of male giant freshwater prawns, the most common size of microplastics found was 100–500 µm. In the intestines of female and male giant freshwater prawns, the most common size of microplastics found was 100–500 µm. See more details in Fig. 4.

Size of anthropogenic debris in female and male giant freshwater prawns.

Figure 4: Size of anthropogenic debris in female and male giant freshwater prawns.

Anthropogenic debris type, color, and polymer

The intestines of male giant freshwater prawns included 91.36% fiber-type microplastics, followed by the stomachs (85.71%) of male giant freshwater prawns and the intestines (69.23%) and stomachs (55.48) of female giant freshwater prawns. Fragment-type microplastics were found in 44.52% of female giant freshwater prawn stomachs, followed by female giant freshwater prawn intestines (30.77%), and male giant freshwater prawn stomachs (14.25%) and intestines (8.64%) (Table 3). The forms and sizes of the microplastics differed.

Table 3:
Anthropogenic debris type and color in female and male giant freshwater prawns.
Category of microplastics Macrobrachium rosenbergii
Female Male
Stomach Intestine Stomach Intestine
Type (%) Fiber 55.48 69.23 85.71 91.36
Fragment 44.52 30.77 14.29 8.64
Color (%) Black 37.67 16.00 27.18 44.44
Blue 57.53 78.00 67.03 50.62
Red 4.79 6.00 8.79 3.70
Yellow 0.00 0.00 0.00 1.23
DOI: 10.7717/peerj.16082/table-3

Blue (61.35%), black (32.70%), red (5.68%), and yellow (0.27%) microplastics were found. Blue was the most prevalent hue discovered in the stomach and intestines of both female and male giant freshwater prawns, followed by black, red, and yellow. Yellow microplastics were not found in the stomachs and intestines of female giant freshwater prawns nor male giant freshwater prawns (Table 3). Cotton (70.37%), rayon (25.93%), and polyvinyl chloride (PVC) (3.70%) were the polymer types found in M. rosenbergii (Fig. 5).

Polymer found in giant freshwater prawns.

Figure 5: Polymer found in giant freshwater prawns.

Correlation between the size of giant freshwater prawns and anthropogenic debris in the stomach and intestines

The study examined the relationship between CL, LA, BW, and stomach weight, and the number of microplastics. The findings revealed a significant association between the number of microplastics and stomach weight in male prawns (R = 0.495; p = 0.005) (Table 4).

Table 4:
The relationship between size of giant freshwater prawns and the amount of anthropogenic debris in stomach (ST) and the intestines (IN).
CL (cm) AL (cm) SW (g) BW (g) ST (items) IN (items)
Macrobrachium rosenbergii (female) CL (cm) 1 .805** .385* .840** .292 −.176
AL (cm) 1 .468** .894** −.062 −.122
SW (g) 1 .321 .109 −.074
BW (g) 1 .222 −.178
ST (items) 1 .083
IN (items) 1
Macrobrachium rosenbergii (male) CL (cm) 1 .912** .371* .932** .029 −.045
AL (cm) 1 .352 .903** .121 −.145
SW (g) 1 .407* .241 .495**
BW (g) 1 .085 −.119
ST (items) 1 .144
IN (items) 1
DOI: 10.7717/peerj.16082/table-4

Discussion

This study confirmed that microplastics were detected in the stomach and intestines of female and male giant freshwater prawns. More microplastics were found in the stomach than in the intestines in both female and male giant freshwater prawns; 4.87 ± 0.72 items/individual, 1.73 ± 0.36 items/individual, 3.03 ± 0.58 items/individual, 2.70 ± 0.57 items/individual, respectively. There was no significant difference in the accumulation of microplastic particles in the stomachs (p = 0.866) and intestines (p = 0.171) of female and male giant freshwater prawns. This is consistent with a study conducted in shrimp, Crangon crangon, that found microplastics were most common in the gastrointestinal tract (Devriese et al., 2015). The number of microplastics found in each type of prawn and shrimp depends on the environment where the prawn samples are selected, such as rivers, aquaculture ponds, lakes, and seas. When comparing the present study with other studies (Table 5), the number of microplastics detected in this study varied by location. The amount of microplastics found in this study is similar to the results obtained by Goh et al. (2022), but less than other studies in Thailand (Jitkaew et al., 2023; Reunura & Prommi, 2022; Pradit et al., 2021). This could be because there are fewer sources of microplastics in the aquatic environment from this study (south-west Thailand) than in the areas from other studies (south-east Thailand). The most common size of microplastics found in this research was 100–500 µm (44.05%), followed by <100 µm (38.11%), >1,000 µm (9.46%), and 501–1,000 µm (8.38%), respectively. The size of the microplastics found is likely related to their toxicity. Smaller microplastics can better absorb hydrophobic materials from a production process or from the environment, resulting in humans being at a greater risk of exposure to toxic chemicals (Lusher, Hollman & Mendoza-Hill, 2017). Microplastics can absorb the additional chemicals (plastic additives) used in the manufacturing process that give plastic products their color and characteristics (Pradit et al., 2020). The results of interactions between selected microplastics and heavy metals strongly support the hypothesis that microplastics can absorb heavy metals and act as a vector for heavy metal ion distribution in the marine ecosystem (Goh et al., 2022). As a result, microplastics appear to be poison transporters for aquatic creatures that consume microplastics. Heavy metals can accumulate in marine creatures, increasing in concentration over time. This concentration provides a record of the availability of metal species in the environment (Rainbow, 2002).

Table 5:
Microplastic abundance in giant freshwater prawns.
Shrimp Location Method Abundance of microplastics Color Shape Size Polymer References
Metapenaeus moyebi (n = 17) Khlong U-Taphao, Songkhla H2O2 30% 14.76 ± 1.98 items/ individual blue, black, other fiber, fragment less than 100 μ m rayon, polyester, PET, PP, Poly (Ethylene:Propylene) (Jitkaew et al., 2023)
Macrobrachium rosenbergii (n = 17) 11.24 ± 1.74 items/individual larger than 1,000 μ m
Litopenaeus vannamei (n = 150) Thailand HCO2K 99% 11.00 ± 4.60 items/ individual black, red, white, blue, yellow, green fiber, fragment, film, spheres 500–1,000 μ m PE, polycaprolactone, polyvinyl alcohol, acrylonitrile butadiene styrene Reunura & Prommi (2022)
Macrobrachium rosenbergii (n = 300) 33.43 ± 19.07 items/ individual (male)
33.31 ± 19.42 items/ individual (female)
Parapenaeopsis hardwickii (n = 18) Songkhla Lake, Southern Thailand KOH 10% 4.11 ± 1.12 pieces/stomach black, red blue, white fiber 500–1,500 μ m rayon, polyester, polyvinyl alcohol, PE, paint Pradit et al. (2021)
Metapenaeus brevicornis (n = 18) 3.78 ± 0.48 pieces/stomach 500–5,000 μ m
Metapenaeus elegans (n = 20) Songkhla Province, Southern Thailand KOH 10% 3.70 ± 1.12 number of MPs/individual black, red blue, gray, transparent fiber 150–3,800 μ m PE Goh et al. (2021)
Fenneropenaeus indicus (n = 20) 3.45 ± 0.04n/individual
Metapenaeus monoceros (n = 60) North Eastern Arabian sea HNO3 69% 7.23 ± 2.63 items/individual 78.48 ± 48.37 MPs/gram of the gut material blue, translucent, black, red fiber, fragment, pellet, film, beads <100 μ m, -greater than 1,000 μ m PE, PP, PA, nylon, PES, PET Gurjar et al. (2021)
Parapeneopsis stylifera (n = 50) 5.36 ± 2.81 items/individual 64.79 ± 24.58 MPs/gram of the gut material
Penaeus indicus (n = 70) 7.40 ± 2.60 items/individual 47.5 ± 38.0 MPs/gram of the gut material
P. monodon / gastrointestinal tract (n = 50) Northern Bay of Bengal H2O2 30% 6.60 ± 2.00 pieces/gram blue, black, transparent, green, red fiber, fragment 250–5,000 μ m rayon, polyamide Hossain et al. (2020)
M . monocerous / gastrointestinal tract (n = 100) 7.80 ± 2.00 pieces/gram blue, black, transparent, green <250–5,000 μ m
Fenneropenaeus indicus (n = 330) coastal waters off Cochin, Kerala, India KOH 10% 0.39 ± 0.60 microplastics/gram red, blue, black, transparent, green fiber, fragment 157–2,785 μ m polyamide, polyester, polyethene, PP Daniel, Ashraf & Thomas (2020)
Paratya australiensis (n = 100) Victoria, Australia NaOH 0.52 ± 0.55 pieces/ individual black, red gray, white blue, green, transparent, yellow fiber, fragment, film, pellet 36–4,668 μ m rayon, polyester, polymide Nan et al. (2020)
Crangon crangon (n = 165) North sea HNO3: HCIO4 4:1 1.23 ± 0.99 items/ individual transparent, translucent, orange, yellow-greenish, purple-blue, pink fiber 200–1,000 μ m Devriese et al. (2015)
Macrobrachium rosenbergii (n = 60) Thailand KOH 10% female; stomach 4.87 ± 0.72 MPs/individual intestine 1.73 ± 0.36 MPs/individual black, red, blue, yellow fiber, fragment cotton, rayon, PVC This study
male; stomach 3.03 ± 0.58 MPs/individual intestine 2.70 ± 0.57 MPs/individual
DOI: 10.7717/peerj.16082/table-5

The most abundant category of microplastics found was fibers, followed by fragments, which is consistent with the findings of several studies (Pradit et al., 2021; Goh et al., 2021; Gurjar et al., 2021; Hossain et al., 2020; Devriese et al., 2015). According to Weinstein, Crocker & Gray (2016), it was reported that polymer fibers can float on water for a long time due to their low density, while fragments with rough surfaces are broken down by natural forces. The fibers found in this study likely originated from floating fibers in river water, and the fiber strands of polymer most likely came from fishing nets and clothing lint (De Witte et al., 2014).

The microplastics found in both male and female giant freshwater prawns were blue, black, and red, while yellow microplastics were only found in the intestines of male giant freshwater prawns. This is similar to the results of previous studies (Jitkaew et al., 2023; Reunura & Prommi, 2022; Pradit et al., 2021; Goh et al., 2021; Gurjar et al., 2021; Nan et al., 2020). It was also found that plastics with a long lifespan and darker colors are more likely to be contaminated with other chemical substances than long-lived lighter-colored plastics (De Witte et al., 2014). Wright, Thompsom & Galloway (2013) reported that living organisms choose to eat plastics that look similar to their regular food, causing them to acquire microplastics in their gastrointestinal tract. According to Sitthi (2011), giant freshwater prawns eat all types of food, both living and nonliving, including fish, seedlings, and other prawns.

In this study, three polymer types were found in giant freshwater prawns. The results reveal that natural polymer cotton was the most abundant, followed by semi-synthetic polymer (rayon) and synthetic polymer (PVC), respectively. The use of detergent in laundering likely results in increased microfibers (Zambrano et al., 2019) which are then suspended and accumulate in bottom sediment or in water currents (Henry, Laitala & Klepp, 2019). This study found polyvinyl chloride (PVC) in the stomachs of male giant freshwater prawns, similar to a study on Litopenaeus vannamei in the Korean Sea (Yoon et al., 2022), which found that the PVC likely came from food packaging and fishing equipment. The study of the correlation between microplastic content and CL, LA, BW, and stomach weight found that there was no correlation between female giant freshwater prawns and microplastic content in the stomach and intestines, while there was a significant correlation between male giant freshwater prawns and intestinal microplastic content and stomach weight at the level of R = 0.495; p = 0.005. This indicates that the high gastric weight of giant freshwater prawns may result in an increase in intestinal microplastic content in proportion to the stomach. CL, LA, and BW were not associated with the number of microplastics in female and male giant freshwater prawns.

It is projected that the problem of plastic waste will worsen due to the excessive use and consumption of single-use plastics (Silva et al., 2021) as well as an increase in the demand for personal protective equipment (PPE) such as masks, and rubber gloves, which will lead to an increase in PPE waste (Okuku et al., 2021). A public awareness campaign aimed at changing people’s attitudes regarding the environment is critical (Sornplang et al., 2022). Diffusion can occur when microplastics are smaller than five mm, causing widespread pollution of the environment. If an organism is exposed to this environment for a prolonged period of time, there is a greater chance that the exposure will have negative effects. These effects could include obstructions in the gastrointestinal tract of organisms, increased mortality rates, decreased ability to reproduce, and inhibition of metabolism. However, depending on the size, shape, and type of contaminated plastic in the environment, as well as the quantity and concentration discovered (Cole et al., 2013; Zhang et al., 2017), other hazardous additive contaminants may be released which could serve as an intermediary to other pollutants, further harming aquatic animals and humans.

Conclusions

In this study, anthropogenic waste was discovered in the stomachs and intestines of giant freshwater prawns (M. rosenbergii). This discovery indicates that microplastic pollution, which is caused by a range of human activities, is harmful because microplastics can enter the food chain. Fibers were the most prevalent category of microplastic found in prawn organs. Blue, black, and red microplastics were identified in the intestines of both male and female giant freshwater prawns, whereas yellow microplastics were found in the intestines of male giant freshwater prawns. Cotton, rayon, and PVC were also discovered in these giant freshwater prawns. Although microplastics are evacuated with waste, some persist in the tissue. Consequently, to reduce plastic pollution in the seas in the future, people need to be informed of the government’s management and act immediately to remedy issues with waste disposal.

Supplemental Information

Raw data of anthropogenic debris in freshwater prawn

DOI: 10.7717/peerj.16082/supp-1

Polymer type found in freshwater prawn

DOI: 10.7717/peerj.16082/supp-2
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