Microplastics in eviscerated flesh and excised organs of dried fish

Abstract

There is a paucity of information about the occurrence of microplastics (MPs) in edible fish tissues. Here, we investigated the potential presence of MPs in the excised organs (viscera and gills) and eviscerated flesh (whole fish excluding the viscera and gills) of four commonly consumed dried fish species (n = 30 per species). The MP chemical composition was then determined using micro-Raman spectroscopy and elemental analysis with energy-dispersive X-ray spectroscopy (EDX). Out of 61 isolated particles, 59.0% were plastic polymers, 21.3% were pigment particles, 6.55% were non-plastic items (i.e. cellulose or actinolite), while 13.1% remained unidentified. The level of heavy metals on MPs or pigment particles were below the detection limit. Surprisingly, in two species, the eviscerated flesh contained higher MP loads than the excised organs, which highlights that evisceration does not necessarily eliminate the risk of MP intake by consumers. Future studies are encouraged to quantify anthropogenic particle loads in edible fish tissues.

Full text

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Scientific RepoRts | 7: 5473 | DOI:10.1038/s41598-017-05828-6
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Microplastics in eviscerated esh
and excised organs of dried sh
Ali Karami1, Abolfazl Golieskardi1, Yu Bin Ho1, Vincent Larat3 & Babak Salamatinia2
There is a paucity of information about the occurrence of microplastics (MPs) in edible sh tissues. Here,
we investigated the potential presence of MPs in the excised organs (viscera and gills) and eviscerated
esh (whole sh excluding the viscera and gills) of four commonly consumed dried sh species (n = 30
per species). The MP chemical composition was then determined using micro-Raman spectroscopy and
elemental analysis with energy-dispersive X-ray spectroscopy (EDX). Out of 61 isolated particles, 59.0%
were plastic polymers, 21.3% were pigment particles, 6.55% were non-plastic items (i.e. cellulose or
actinolite), while 13.1% remained unidentied. The level of heavy metals on MPs or pigment particles
were below the detection limit. Surprisingly, in two species, the eviscerated esh contained higher MP
loads than the excised organs, which highlights that evisceration does not necessarily eliminate the risk
of MP intake by consumers. Future studies are encouraged to quantify anthropogenic particle loads in
edible sh tissues.
Worldwide plastic production was estimated to reach 322 million metric tons in 20151 whereby 5 to 13 million
metric tons were reported to be disposed into the marine environment annually2. e plastics dumped in the
environment may never completely degrade3 but instead fragment into smaller particles called microplastics
(MPs), sized between 1 and 1000 µm4. e widespread distribution of MPs in aquatic bodies [e.g.,5, 6] has sub-
sequently contaminated a diverse range of aquatic biota including those sold for human consumption such as
sh7 and mussels8. erefore, seafood products could be a major route of human exposure to MPs. For example,
it was estimated that top European shellsh consumers might take up to 11,000 MPs per annum8. Microplastics
were suggested to exert their harmful eects by providing a medium to facilitate the transport of other toxic
compounds such as heavy metals9 and persistent organic pollutants (POPs)10 to the body of organisms. Upon
ingestion, these chemicals may be released and cause toxicity11.
Dried sh are considered low-cost protein sources in many developing countries12. e purpose of the drying
process is to create a desirable avor and texture and/or to increase the shelf life by reducing the moisture con-
tent13. So far, nothing is known about the occurrence of MPs in dried sh that are intended for direct human con-
sumption. Dried sh are oen processed without any cleaning process, and although evisceration prior to drying
helps to reduce bacterial contamination in sh14, this is not practical to many small sh species such as anchovies.
Subsequently, this could potentially increase the chance of anthropogenic particle exposure to consumers. In
this study, we investigated the potential presence of anthropogenic particles (MP and pigment particles) in the
eviscerated esh and excised organs of Indian mackerel (Rastrelliger kanagurta), spotty-face anchovy (Stolephorus
waitei), greenback mullet (Chelon subviridis), and belanger’s croaker (Johnius belangerii). ese species were cho-
sen since they are oen caught from the coastal waters of many Asian countries as well as in some other parts of
the world15–18.
In aquatic organisms, the gills are the rst organ exposed to anthropogenic particles during respiration19, 20,
which increases the possibility that these particles can become stuck among the gill laments. For example, fol-
lowing the exposure to high-density polyethylene (HDPE) fragments, these particles became trapped on the gills
of the blue mussel (Mytilus edulis)21. Laboratory studies have later shown that MPs were able to be translocated
into other tissues of sh22, 23. Most eld studies on sh have investigated the occurrence of MPs in the gastrointes-
tinal tract [e.g.,24–26] but little is known about their presence in their edible tissues.
Here, we investigated MP (0.001–1 mm), mesoplastic (1–10 mm), and macroplastic (>10 mm) loads and mor-
phology (fragments, lms, laments, beads, and foams27) in the viscera and gills (hereaer are called excised
1Laboratory of Aquatic Toxicology, Department of Environmental and Occupational Health, Faculty of Medicine and
Health Sciences, Universiti Putra Malaysia, 43400, Selangor, Malaysia. 2Discipline of Chemical Engineering, School of
Engineering, Monash University Malaysia, 47500, Selangor, Malaysia. 3HORIBA Jobin Yvon S.A.S., 231, rue de Lille,
59650, Villeneuve d’Ascq, France. Correspondence and requests for materials should be addressed to A.K. (email:
alikaramiv@gmail.com)
Received: 10 March 2017
Accepted: 5 June 2017
Published: xx xx xxxx
OPEN

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organs) and eviscerated esh of 4 dried sh species. All the isolated particles were initially sampled based on their
similar density and morphology to MPs and then analyzed for their chemical composition using micro-Raman
spectroscopy. Finally, to investigate if the extracted MPs contained hazardous inorganic substances, we further
assessed the atomic composition of MP particles using eld emission scanning electron microscopy (FESEM)
equipped with an energy-dispersive X-ray spectroscopy (EDX). e results of this study will help to understand if
removing viscera and gills could mitigate the intake of anthropogenic particles by consumers.
Results
No particles were found in the procedural blanks. A total of 61 MP-like particles were isolated from the four
dried sh species. As depicted by Fig.1a, 36 particles (59.0%) were conrmed as MPs (i.e. particles conrmed
as plastic polymer or plastic polymer plus pigment), 13 particles (21.3%) as pigments (i.e. particles conrmed as
pigment), 4 particles (6.55%) were non-plastic items (i.e. cellulose or actinolite), and 8 particles (13.1%) remained
unidentied. e most abundant plastic polymers were polypropylene (PP, 47.2%) followed by polyethylene (PE,
41.6%), polystyrene (PS, 5.56%), polyethylene terephthalate (PET, 2.77%), and nylon-6 (NY6, 2.77%) (Fig.1b).
Particles identied as pigments were phthalocyanine (84.6%) and hostasol green (15.3%) (Fig.1c). Figure2 shows
the Raman spectra of a PE particle containing phthalocyanine. Figure3a–f are the microscopic images of some of
the isolated particles. With regards to morphology, the predominant type of anthropogenic particles were frag-
ments (85.7%) followed by lms (10.0%), and laments (4.08%) (Fig.4). No beads or foams were found among
the samples.
Between 0 and 3 pigments and MP particles were isolated from each individual sh. Figure5a and b, present
the frequency histograms of pigment and MP particle numbers across the tested species, respectively. Figure6a
and b are stacked bar charts of the number of extracted pigments and MP particles, respectively, isolated from
the excised organs or the eviscerated esh of each dried sh species. Surprisingly, 29 MPs and 9 pigment particles
Figure 1. Particle compositions. (a) Pie chart of the percentage and chemical composition of the extracted
particles from dried sh samples and their corresponding proportion of (b) plastic polymers, and (c) pigments.
Figure 2. Raman spectrum of a particle identied as polyethylene + phthalocyanine and spectra of the
reference materials.

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were isolated from the eviscerated esh while 7 MPs and 4 pigment particles were from the excised organs. e
abundance of anthropogenic particles per species ranged from 2 in S. waitei to 24 in C. subviridis. In C. subviridis
and J. belangerii, Mann–Whitney U tests showed that the number of MPs in the eviscerated esh was signicantly
higher than excised organs (Z = −2.43 and Z = −2.21, respectively; p < 0.05). No signicant dierences were,
however, indicated in the number of pigment particles between the eviscerated esh and excised organs. In R.
kanagurta and S. waitei, the number of pigment particles or MP particles was comparable (p > 0.05) between the
eviscerated esh and excised organs.
ere was a signicant dierence in the number of MPs (Kruskal-Wallis, H = 15.7, df = 3, p < 0.01) isolated
from eviscerated esh among the dried sh species. Dunn’s multiple comparison tests found higher MP loads in
the eviscerated esh of C. subviridis and J. belangerii than R. kanagurta and S. waitei. However, the number of
pigment or MP particles in the excised organs was comparable among the sh species. Similarly, no signicant
dierence, however, was noticed in the load of pigment particles extracted from eviscerated esh among the
species. Among the 5 identied plastic polymers, the prevalence of PP (Kruskal-Wallis, H = 8.56, df = 3, p < 0.05)
Figure 3. Isolated particles from dried sh. ese particles were identied using micro-Raman spectroscopy as
(a) Phthalocyanine, (b) Polypropylene + Phthalocyanine, (c) Polyethylene terephthalate, (d) Polyethylene, (e)
Hostasol green, and (f) Actinolite.

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and PE (Kruskal-Wallis, H = 9.14, df = 3, p < 0.05) were signicantly higher in C. subviridis and J. belangerii than
R. kanagur ta and S. waitei while the concentration of other plastic polymers were comparable among all the tested
species. Elemental analysis of the particles showed that all the anthropogenic particles contained carbon (C) and
oxygen (O) while a few had nitrogen (N), chlorine (Cl) and sodium (Na) as well (Supplementary Information
Fig.1).
Discussion
e ability of MPs to translocate from the digestive systems into other tissues of aquatic organisms22, 23, 28 has
raised concerns about the safety of seafood products. Most of the studies on wild sh have assessed MP loads in
the digestive tract while little has been done to investigate the presence of MPs in the edible sh tissues.
e absence of particles in the procedural blanks ensured the reliability of contamination prevention proce-
dure employed by this study. Approximately one-h (21.3%) of the recovered particles were identied as pig-
ments (phthalocyanine or hostasol green) owing to the strong Raman spectra of these additives. Recent studies
have indicated that additives could complicate the identication of the chemicals within the samples [e.g.,29].
ese synthetic pigments are extensively employed during the manufacturing of dierent materials, including
plastics30–33. Initially, we suspected that these particles could be dyes. However, this hypothesis was rejected since
none of the isolated particles shared the main characteristics associated with dyes (i.e. brittleness34). Some earlier
studies inferred that pigment particles were plastics [e.g.,35] while others only suspected these particles to be
Figure 4. Pie chart of the morphology of isolated anthropogenic particles.
Figure 5. Frequency histograms of pigment and microplastic particles in the whole body (eviscerated
esh + excised organs) of tested dried sh species. Frequency histogram of (a) pigment and (b) microplastic
particles in the whole body of Chelon subviridis, Johnius belangerii, Rastrelliger kanagurta, and Stolephorus
waitei.

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plastics8, 33. Similar to the approach followed by the latter studies, we ensured that the pigment particles had an
anthropogenic origin, but we could not conrm if they were in fact plastic polymers. One particle isolated from
the excised organs of R. kanagurta was identied as actinolite. Actinolite is one of the 6 naturally occurring min-
eral silicate bers that are referred to as asbestos, which has a wide application such as in textile, plastics, roong,
electrical insulation36–38.
Micro-Raman spectroscopy was unable to identify 13.1% of the isolated particles. e spectra of the samples
collected from the eld oen diers with the spectra of pure materials, possibly due to the degradation pro-
cess39. All the isolated particles in this study were sampled according to their similar density (i.e. having density
<1.5 g/cm3) and morphology with MPs. erefore, the unidentied particles are suspected to be MPs. e high
occurrence of fragments in the sh could indicate their dominance in Malaysian coastal environments, which is
consistent with sh caught in other regions of the world23, 40. Coastal areas have oen been reported to be con-
taminated with MPs41, which is due to their vicinity to anthropogenic activities along the coast. e dominance
of fragments in the tested sh in this study could reect their prevalence in the water and sediments of Malaysian
marine ecosystems. Consistent with our ndings, Barasarathi et al.42 showed the dominance of fragments in the
soils of a Malaysian mangrove forest. e absence of bead or foam microparticles in the eviscerated esh or the
excised organs of the tested sh could reect their negligible prevalence in the natural environments.
Polypropylene and PE were the major recovered plastic polymers in the tested species, which is consistent
with their massive production load and demand by various industries1. Consequently, this can lead to their
broad distribution in the marine environment43, 44. Also, the lower density of PP (0.90–0.91 g/cm3) and PE (0.91–
0.96 g/cm3) than seawater would cause them to oat on the water surface. Over time, biofouling by micro- and
macro-organisms have been suggested as a potential mechanism that could cause positively buoyant plastics to
become less buoyant45 and, therefore, led to a more homogeneous distribution throughout the water column.
Surprisingly, in C. subviridis and J. belangerii, the MP load was signicantly higher in the eviscerated esh than
excised organs. Initially, it was hypothesized that the sh might have been contaminated during handling on the
shing vessels or during the salting and drying processes. Interestingly, our recent study have shown the occur-
rence of MPs in most of the tested sea and lake salts produced in dierent countries46. However, this hypothesis
was rejected because the sh were gutted in the laboratory aer rinsing the body surface with water and ethanol
(see Contamination control). Alternatively, the particles found in the eviscerated esh could have been translo-
cated from the alimentary tract. Several laboratory-based studies on sh have shown the translocation of MPs
from the digestive system into other organs. For example, PE and PS particles (sizes: 200–600 µm) translocated
from the stomach to the liver of athead grey mullet (Mugil cephalus)23. In another study on zebrash (Danio
rerio), waterborne exposure to PS microspheres resulted in their accumulation in the gills and liver22. An earlier
study in rats demonstrated the translocation of PS particles from the gut into the lymph47. Uptake through the
layer of Peyer’s Patches from M-cells located within the small intestinal lymphoid tissue is a common route for
the absorption of nano- and micro-particles48. M-cells are the dierentiated epithelial cells with the ability to
transcytosize macromolecules and particles49. Other mechanisms such as persorption might have been involved
in the translocation of particles across the gastrointestinal mucosa50. e higher load of MPs in the eviscerated
esh of C. subviridis and J. belangerii highlights that evisceration does not fully eliminate the risk of MP uptake
by consumers. Moreover, this study showed that quantifying MPs in the viscera may not truly represent their
Figure 6. Stacked bar chart of the isolated particles from the excised organs or the eviscerated esh. (a) e
prevalence (n) of plastic polymer and (b) pigment particles; n = 30.

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concentrations in the entire organism. Future studies are urged to assess MP loads, in not only the digestive tract,
but also in the edible tissues of the sh. is strategy should better reect the risks associated with the consump-
tion of the sh caught from the natural environment.
In 2014, a total of 17 million tons of dried, smoked, or salted sh were produced for human consumption51.
ere is insucient data, however, regarding the global consumption of dried sh. e species employed in this
study were caught from Malaysian waters. us, consumers in neighboring countries could be exposed to the
similar MP loads. Based on a report on the consumption of sh and sh products in the Asia Pacic region, the
annual dried sh consumption in Bangladesh is 370 g/capita52. Considering the average sh weight (Table1) and
the number of anthropogenic particles (pigment + MP) per individual sh (between 0 and 3), consumers of S.
waitei are expected to ingest between 0 to 246 MPs per annum. Similarly, consumers of C. subviridis (containing
between 0 and 3 anthropogenic particles per sh), J. belangerii (containing between 0 and 3 anthropogenic parti-
cles per sh), or R. kanagurta (0 and 1 MP particles per sh) could ingest 0–68, 0–44, and 0–6 anthropogenic par-
ticles per annum, respectively. However, the majority of the tested sh in this study did not contain MP (Fig.5b).
erefore, it is less likely that an individual would ingest the suggested maximum number of MPs per annum.
Previous studies have shown that MPs adsorb POPs from the surrounding environment53 or contain additives
that were incorporated into them during the manufacturing process54. Subsequently, these chemicals may desorb
from the particles into the body of organisms upon ingestion55. However, recent studies have shown the intake of
POPs by aquatic organisms from water and food exceeded the potential transfer of POPs from ingested MPs56, 57.
According to the results of this study, the undetectable level of toxic heavy metals on the isolated particles does
not support their potential toxicity and the mechanisms whereby MPs cause toxicity are still unclear. erefore,
despite the potential for a maximum of 246 anthropogenic particles to be ingested by a human per year, we cannot
evaluate the health risks associated with the consumption of dried sh at this moment. e increase in plastics
disposal2 coupled with their continuous fragmentation58, is expected to increase MPs concentrations over time.
As such, it will become increasingly important to regularly assess MP loads in seafood products, including dried
sh.
Given the fact that dried sh are oen consumed as a whole, they may be responsible for the translocation
of a signicant amount of MPs into the body of consumers. Higher MP loads in the edible tissues than excised
organs of two dried sh species indicates that removing gills and viscera does not necessarily reduce MP uptake
by consumers of dried sh. e results of this study underscores the importance of assessing edible sh tissues for
MP presence and to better understand the ability of MPs to translocate into other organs.
Methods
Materials and chemicals. Packed dried C. subviridis, J. belangerii, R. kanagurta, and S. waitei were pur-
chased from local markets in Malaysia (Table1). Sodium iodide (NaI), potassium hydroxide (KOH), and ethanol
95% were purchased from R&M Chemicals (UK). Solutions of NaI (4.4 M) and KOH (10% w/v) were prepared
by dissolving the powder/pellet in ultrapure deionized water. e GF/D microber lter membranes (pore
size 2.7 μm) and lter membranes No. 540 (hardened ashless, pore size 8 μm) were supplied by Whatman. e
149 μm-pore size lter membranes were purchased from Spectrum Laboratories (USA).
Sample preparation. Each sh was placed on pre-cleaned aluminum foil, and the total length and weight
was recorded. An equal number of sh per pack (3–6 packs per species) was used to provide a total number of 30
sh for each species (n = 30). e gill arches were carefully removed by cutting through the bone at the top and
bottom where the gills joined the head. e viscera was removed by cutting the sh beginning at the vent and con-
tinuing to the throat. Gills and viscera (excised organs) were placed together into a 250 mL DURAN laboratory
glass bottle sealed with a premium cap and pouring ring (Schott, Germany). e eviscerated esh (sh without
gills and viscera) was placed into a separate 250 mL laboratory bottle and were subjected to digestion.
Microplastic isolation. Microplastic isolation from the eviscerated esh and excised organs of dried sh
were done according to the method of Karami et al.59. Briey, 200 mL of KOH (10% w/v) was added to each bot-
tle containing either excised organs, or the eviscerated esh, and were incubated at 40 °C for 72 h. e digestate
was then vacuum ltered through a 149 μm lter membrane. To separate the high-density particles (i.e. bone
fragments and scales), the lter membrane was soaked in 10–15 mL of 4.4 M NaI (density: 1.5 g/mL), sonicated
at 50 Hz for 5 min and agitated on an orbital shaker at 200 rpm for 5 min. Eventually, the solution was centrifuged
at 500 × g for 2 min, and the supernatant containing MPs was vacuum ltered through another lter membrane
(pore size 8 μm). is process was repeated once more to ensure complete isolation of MPs.
Visual identication. Microscopical examination of the lter membranes was performed using a Motic
SMZ-140 stereomicroscope (Motic, China). Particles resembling MPs were sampled based on their morphological
Common name Species Total weight (g) Total length (cm)
Greenback mullet Chelon subviridi s 16.3 ± 1.812 12.7 ± 0.4460
Belanger’s croaker Johnius belangerii 25.2 ± 0.381 13.6 ± 0.2160
Indian mackerel Rastrelliger kanagurta 58.5 ± 3.755 18.6 ± 0.2687
Spotty-face anchovy Stolephorus waitei 1.50 ± 0.1568 6.66 ± 0.4440
Table 1. Average total weight and length ± SD of the dried sh used in this study. Number of sh examined per
species (n) = 30.

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characteristics, such as color and shape, as explained by Karami et al.46 Fragments (irregular shape with une-
ven surface), bers/laments (thin and elongated), beads (spherical and ovoid), lms (thin plane of appy), or
foams (lightweight and highly porous). Selected particles were photographed using a camera (AxioCam, ERc 5S,
Germany) coupled with a microscope.
Raman spectroscopy and FESEM-EDX analyses. Particles were analyzed over a range of 150 to
3000 cm−1 using a Raman spectrometer (Horiba LabRam HR Evolution) equipped with a Single Mode Open
Beam Laser Diode (Innovative Photonic Solutions) operating at a wavelength of 785 nm coupled with a
charge-coupled device detector (Horiba Synapse). Before the library search, to reduce noise and enhance the
spectrum quality without losing subtle spectral information, each spectrum passed through a baseline correction
and denoising procedure (Labspec 6, Horiba Scientic). Pre-processed spectra were then evaluated and compared
to the following spectral libraries: Raman polymers and monomers from Bio-Rad Sadtler and Raman Forensic
from Horiba using the KnowItAll soware from Bio-Rad. e Correlation algorithm (KnowItAll, Bio-Rad) was
used to evaluate each query spectrum to the spectra of the databases. e Hit Quality Index (HQI) was used to
rank the results of the spectral search. To assess the inorganic composition of isolated MPs, all particles identied
as plastic polymers were examined using a FESEM (Hitachi Ultra-high resolution SU8010) operating at 5 keV and
equipped with an Oxford-Horiba Inca XMax50 energy-dispersive X-ray (EDX; Oxford Instruments Analytical,
High Wycombe, England). e detection limit of the machine was around 1000 pg/µg for most of the heavy
metals.
Contamination control. To minimize contamination, cotton lab coat, nitrile gloves were worn at all times.
All the liquids (deionized water, ethanol, KOH, and NaI) were ltered through a GF/D microber lter mem-
brane (pore size 2.7 μm). e glassware and instruments, such as dissecting scissor and forceps, were washed once
with dishwashing liquid, rinsed with deionized water, and nally with ethanol. To remove any potential particles
attached to the sh body surface, the outer part of the sh was rinsed twice with ultrapure deionized water and
once with ethanol. e work surface was pre-cleaned with 70% ethanol every time before dissection. e entire
procedure was carried out in a horizontal laminar ow cabinet (AHC-4A1-ESCO) to avoid potential contamina-
tion with airborne MPs. To monitor and correct potential contamination, one procedural blank with 10% KOH
was tested simultaneously during the isolation procedure, and another procedural blank containing NaI solution
was tested simultaneously during the density separation process.
Statistics. Shapiro–Wilk test was used to assess normality of the data. Upon data transformations, normal
distribution was not achieved. erefore, the Mann–Whitney U test was used to compare pigment or MP particle
loads between the eviscerated esh and excised organs of each sh species. A Kruskal–Wallis test (non-parametric
one-way analysis of variance) was employed to compare the number of extracted pigments or MP particles in the
excised organs or eviscerated esh among the tested species. Also, the concentration of each polymer (PP, PE,
PS, NY6, PET) was compared among the species with a Kruskal–Wallis test. e analysis was followed by using
Dunn’s multiple comparison tests if a signicant dierence (P < 0.05) was obtained. Statistical analyses were per-
formed using SPSS (IBM SPSS Statistics V. 24).
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Author Contributions
A.K. designed and supervised the research, interpreted the results, and wrote the manuscript; A.G. performed
the extraction experiment and co-wrote the manuscript; A.K., Y.B.H., V.L., and B.S. performed micro-Raman
spectroscopy analysis. All authors discussed the results.
Additional Information
Supplementary information accompanies this paper at doi:10.1038/s41598-017-05828-6
Competing Interests: e authors declare that they have no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.

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