Science of the Total Environment 778 (2021) 146395

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Critical review on microplastics in fecal matter: Research progress,

analytical methods and future outlook
Fermín Pérez-Guevara a,b, Gurusamy Kutralam-Muniasamy a,⁎, V.C. Shruti c,⁎
a
Department of Biotechnology and Bioengineering, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Ciudad de México, Mexico
b
Nanoscience & Nanotechnology Program, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Ciudad de México, Mexico
c
Instituto Politécnico Nacional (IPN), Centro Mexicano para la Producción más Limpia (CMP+L), Av. Acueducto s/n, Col. Barrio la Laguna Ticomán, Del Gustavo A. Madero, C.P. 07340,
México City, Mexico

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• First review on microplastics contami-

nation in fecal matter.
• Digestion is the most commonly used
approach for microplastic separation.
• Fiber and fragment (>100 μm) shaped
microplastics were prevalent in fecal
samples.
• Owing to significant methodological
variance, standardization is required.

a r t i c l e i n f o a b s t r a c t

Article history: There has been an ever-increasing attention over years for investigating microplastics in feces of lower to higher
Received 4 January 2021 trophic organisms from diverse environments. Focusing on the standardization of methodologies for reliable
Received in revised form 20 February 2021 generation and comparison of data is one of the important aspects in microplastic area. This first review, compris-
Accepted 7 March 2021
ing 20 studies in total, critically summarizes and compares the methodological approaches for the determination
Available online 11 March 2021
of microplastics in feces as well outlines the levels and characteristics of microplastics detected in feces world-
Editor: Damia Barcelo wide. Contaminations and QA/QC measures are also discussed. Despite variations among the approaches, most
studies (n = 12) described herein rely on the digestion processes involving H2O2 (n = 7) and KOH (n = 6) for
the separation of microplastics, whereas very few included wet sieving (n = 5), density separation using NaCl
Keywords: (n = 3) and NaI (n = 1) and enzymatic digestion (n = 2). Microscopical sorting and spectroscopic methods
Human stool such as infrared and Raman were combined for identification and characterization of microplastics. The detected
Microplastics microplastics varied by size, shape, color and polymer types and the differences in reporting units of microplastic
Plastic pollution abundance make comparison across studies difficult. Taking advantage of the current knowledge, our review
Seal
identified analytical challenges and suggested appropriate methods on research into microplastic contamination
Standardization
in feces. This work will serve as a valuable information of available analytical methods for examining
microplastics in feces and will stimulate further research to advance our understanding of microplastics
from feces.
© 2021 Elsevier B.V. All rights reserved.

⁎ Corresponding authors.
E-mail addresses: mgurusamy@cinvestav.mx (G. Kutralam-Muniasamy), shrutifrnd@gmail.com (V.C. Shruti).

https://doi.org/10.1016/j.scitotenv.2021.146395
0048-9697/© 2021 Elsevier B.V. All rights reserved.
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti Science of the Total Environment 778 (2021) 146395

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Literature retrieval and data collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Overview of methodological approaches for microplastics determination in feces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.1. Sample collection, volume and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.2. Sample pre-treatment and microplastics extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3.3. Identification, chemical characterization and quantification of microplastics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4. Contamination prevention and QA/QC measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5. Analytical challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
6. Current knowledge of microplastics in feces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6.1. Abundance of microplastics in feces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
6.2. Characteristics of microplastics in feces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7. Practices and recommendations for future studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Declaration of competing interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1. Introduction
Microplastics are emerging environmental pollutants that originate
either from the disintegration of larger plastics via physical and biolog-
ical degradation or from the applications of personal care products
which contain microplastics that are primarily manufactured in specific
smaller sizes (Koelmans and Pahl, 2019; Guerranti et al., 2019). The oc-
currence of microplastics has been noted in all of Earth's major ecosys-
tems, including marine (Andrady, 2011), freshwater (Li et al., 2020),
estuary (Han et al., 2020) and atmosphere (Chen et al., 2020). The
slower degradation rate of microplastics in aquatic environments in-
creases their persistence in the environment and their bioavailability
to a wide range of organisms (Nelms et al., 2018; Caruso, 2019). There
is currently numerous evidence of microplastic ingestion from low to
high trophic organisms, including fish, shrimp, turtles, bivalves, zoo-
plankton and whales (Abbasi et al., 2018; Hermabessiere et al., 2019;
Duncan et al., 2019; Zhang et al., 2020). The risks associated with the in-
gestion/retention of microplastics in organisms include substantial
threats on growth and reproduction of organisms (De Sá et al., 2018).
The ingested microplastics can be transferred to another organism via
prey ingestion and thereby, gaining entry into the food web.
Microplastics have been shown to act as a carrier of heavy metals and
organic pollutants to environments and to organisms after ingestion
(Caruso, 2019). While the uptake of microplastics by biota is becoming
apparent, evidence from laboratory experiments started appearing as
well for the egestion of microplastics in feces or solid biowastes
(Jinhui et al., 2019; Moreschi et al., 2020). Many organisms utilize a nat-
ural process of expulsion of digestible and non-digestible material for
eliminating the microplastics from their internal system in feces. Even
mussels have shown to eliminate microplastics as pseudofeces
(Woods et al., 2018). Inspired by these interesting findings, in recent
years, a number of studies have examined excreta from low to high tro-
phic organisms for understanding the extent of microplastics contami-
nation in feces at ambient conditions and brought important
information on concentration and characteristics of microplastics de-
tected in feces (Zhao et al., 2018; Beriot et al., 2020; Moore et al.,
2020; Provencher et al., 2018). Remarkably, Schwabl et al. (2019) pub-
lished a comprehensive study demonstrating the presence of
microplastics belonging to 9 different polymer types in human stool.
Based on these contributions, it can be said that the phenomenon of
microplastics expulsion is common across trophic organisms. At pres-
ent, very little is known about the real-world behavioral implications
of the microplastics in feces from being released into environment.
However, it has been speculated that the frequency of occurrence
and the number of plastic pieces packed in fecal matter may play a
significant role when evaluating the potential harm of marine plastic
litter.
Despite the huge amount of effort and promising advances, one of the
main issues related to microplastics research is the quantification and
identification of microplastics and contaminations during this process.
The constant increase in literature along with the variability in methodol-
ogy that mediate particle separation, quantification and identification can
lead to erroneous comparability of data and conclusions. That is because
of the extensive development of analytical and characterization methods
for providing fundamental information (e.g., concentration and polymer)
on microplastics. Additionally, it is worth highlighting that the availabil-
ity of completely different processes may not always be very compatible
and will require systematic testing with different environment samples.
This fundamental problem has driven the need for a standardization of
methodologies and aroused great interest among researchers to combine
the available data by enabling the path towards a feasible harmonization
of different approaches and scientific communications. Importantly, due
to the emerging interesting in this topic, many significant and exciting re-
views have acquired knowledge of the detection and identification of
microplastics in sample matrices like water, sediment, biota and food
(Prata et al., 2019; Kutralam-Muniasamy et al., 2020; Fok et al., 2020;
Shruti et al., 2021), taking a first step towards standardization of method-
ologies. Although there is no standardized definition of methods for a
wide range of environmental samples, most researchers concur that the
development of appropriate preparation methods and advanced charac-
terization techniques is the premise for efficient generation and repro-
duction of reliable results. Since the investigations on microplastics in
feces are gaining great importance globally, it is crucial to synthesize, an-
alyze and understand the variety and variability of available analytical
methods for separation, quantification and characterization of
microplastics in feces reported in literature. However, to date, there is
no published review focusing on this topic and remains unexplored.
Hence, this first review intends to give a comprehensive overview of
the previous research on microplastics in feces to address and fill the im-
portant gap in microplastic area. First, the currently used methods involv-
ing separation, quantification and characterization for assessing the
microplastics in fecal samples collected from aquatic and terrestrial envi-
ronments are briefly discussed. Next, we highlight the occurrences, dis-
tribution, and characteristics of detected microplastics in feces as
reported in previous research. Then, we suggest analytical challenges
and appropriate methods that are adaptable for effective determination
of microplastics contamination in feces. Finally, to inspire more research,
future challenges and opportunities in the field of microplastics in feces
are presented.
2. Literature retrieval and data collection
A search by using combination of terms, “microplastics”, “feces”,
“pseudofeces”, “fecal matter”, “egestion”, “scat” and “excretion” was

2
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti
conducted in several databases such as ISI Web of Science, PubMed, Sci-
ence Direct, and Google Scholar on 15th December 2020 and thus all lit-
erature published from 2015 till 15th December 2020 was screened. The
initial search yielded a total of 1285 papers. Of these, we selected 20 pa-
pers directly related to microplastics in feces according to the following
criteria: 1) the paper was an original research study rather than a re-
view, 2) only field-based studies were included, excluding the labora-
tory exposure experiments, and 3) the conference proceedings, book
chapters and articles not published in the English language were also
excluded in this review. Of the selected studies, 2 studies focused on ag-
ricultural farm creatures (i.e., chicken, earthworm and sheep), 1 study
on pet animals (i.e., cat and dog), 2 studies on human, 2 studies on mi-
gratory birds, 2 studies on ducklings and 11 studies on marine organ-
isms (i.e., whale and seals). From the selected studies, we retrieved
and summarized the information regarding sample collection and num-
ber of samples (Section 3.1), pre-treatment and microplastics extraction
(Section 3.2), identification and quantification methods (Section 3.3),
contamination prevention and QA/QC measures (Section 4), and
microplastic concentration (Section 6) and characteristics (i.e., size,
shape, color and polymer type) (Section 6) in Table 1–3. Majority of
the publications into microplastics and feces were from North America
(n = 8) and Europe (n = 7), followed by Asia (n = 2), South America
(n = 2), Antarctica (n = 1) and Africa (n = 1). If the methodology
data was not completely presented in the paper, we asked for the orig-
inal information from the corresponding authors.
3. Overview of methodological approaches for microplastics deter-
mination in feces
In practice, the determination of microplastics in feces consists of
three main steps (1) sample collection and storage, (2) sample
pre-treatment and microplastics extraction and (3) identification and
characterization of microplastics. Fig. 1 gives a schematic overview of
the applied different methods in previous research for investigating
microplastics in feces.
3.1. Sample collection, volume and storage
The description of sample collection was mentioned in 13 out of 20
studies and there was a great variation in the collection of samples
across studies. Samples were collected using a variety of objects includ-
ing, metal spatula, metal spoon, plastic scoop, crow bill and by hand
after wearing nitrile gloves. Among the studies reported, sample collec-
tion by metal spatula (n = 4) and hand (n = 3) were highly practiced,
followed by using crow bill (n = 2), plastic scoop (n = 1) and brown
glass bottle (n = 1). It is of great importance to register whether the
feces samples collected were fresh or aged during sampling. There is
not any relevant information in most studies reviewed. However, in
the studies on penguin, seals and birds, authors collected fecal samples
immediately after their defecation (Gil-Delgado et al., 2017; Perez-
Venegas et al., 2018; Reynolds and Ryan, 2018; Masiá et al., 2019; Le
Guen et al., 2020). Moreover, the possible measures associated with
the choice of cohort especially with humans are critical while collecting
the stool samples. Based on Schwabl et al. (2019), participants should
be generally healthy and excluded those with diet followed for medical
reasons. Furthermore, persons diagnosed with diseases (e.g., ulcerative
colitis or Crohn's disease, cancer, organ transplant, HIV), pregnant
women and those involved in clinical trials, consuming drug affecting
stool frequency and consistency (e.g., loperamide or lactulose) and alco-
hol within previous 2–4 week should not be considered. Accordingly,
they selected a number of volunteers of 3 men and 5 women aged 33
to 65 years and recorded food intake for 6 to 7 days before sampling
(Schwabl et al., 2019). The samples were asked to send to the laboratory
from the individuals, while in another study fecal samples of cats and
dogs were provided by individual owners (Zhang et al., 2019). One
study collected fecal samples from seals grown in sanctuary and not
3
Science of the Total Environment 778 (2021) 146395
exposed to anthropogenic litter items (Nelms et al., 2018). Sample col-
lection time period (e.g., season) is one of the important factors to be
considered while collecting fecal samples in environment as the species
and habits of organisms may differ between different seasons, which in
turn may have a certain significance for the source of microplastics in
feces. Taking this into account, for example, Beriot et al. (2020) collected
the fecal samples from herds of sheep in the farms just after the winter
harvest during which plastic mulch is used to cover the soil, suspecting
that it could be one of the major sources of microplastics in the farmland
where the sheep herd graze. After sampling, fecal samples were stored
into one of the following items such as aluminum foil, glass tubes, plas-
tic bag, Whirl-pak polyethylene bag, glass bottle and paper bag. The
fecal samples were immediately kept frozen (−20 °C) in laboratory to
prevent further/any organic and plastic decomposition until being proc-
essed for microplastic separation. The amount of sample analyzed was
not often reported in the literature related to the microplastic investiga-
tions on feces. Only in 7 out of 20 studies, the data for the sample vol-
ume taken for microplastic extraction than that of the volume of total
sample collected in the field was mentioned. The sample volume varied
greatly between 0.1 and 380 g (Table 1). Different to other studies,
Moore et al. (2020) processed a total of 100 mL sample. More impor-
tantly, the quantity of samples was different between studies for fecal
samples of same organism. For instance, Yan et al. (2020) took 5 g of
chicken feces for microplastic separation analysis, whereas Lwanga
et al. (2017) analyzed only 0.7 g which is approximately 10 times of
magnitude lesser to the latter study. There have been some speculations
that the interpretation of the results related to microplastic abundance
is likely to be affected by volume of fecal sample analyzed and heterog-
enous distribution of microplastics in the samples. Additionally, it is im-
portant to note that the number of samples collected and analyzed
greatly differ between studies reviewed. The greatest number of sam-
ples noted were 283 for duckling feces and the least number of samples
being 2 for chicken feces.
3.2. Sample pre-treatment and microplastics extraction
Knowledge about the feces or scats of the organism of interest is vital
to design high-performance microplastic extraction approaches. That is
because, fecal consistency and chemical content vary considerably
among/between organisms with regard to nutritional diet, body weight
and health conditions. For example, fresh feces of humans contain
around 75% water and the remaining solid fraction is organic solids
which mainly consist of 25–54% bacterial biomass, 2–25% protein or ni-
trogenous matter, 25% carbohydrate or undigested plant matter and
2–15% fat (Rose et al., 2015). There is also possibility at times to find un-
digested food materials such as seeds, nuts and corn, mainly because of
their high fiber content (Rose et al., 2015; Karu et al., 2018). Further-
more, they may also be enriched with calcium and phosphate associated
particles. The high solid particulate matter content along with other or-
ganic impurities pose interferences during microplastic separation and
identification. Thus, attempts to reduce the organic content are the
first critical step in the microplastic separation from fecal samples.
As seen in Fig. 1, majority of the studies have had their own approach
and performed a certain number of processing steps to enhance
microplastic extraction from fecal samples. Based on the previous re-
search results, the fecal samples were either oven dried, frozen or disag-
gregated in water to obtain aqueous solution before processing for
microplastics separation. Irrespective of the origin of feces, it is noted
that the current organic content removal is performed by means of se-
ries of reactions/pre-treatments that mostly include oxidizing agents,
alkali digestion, density separation and/or a combination of these proce-
dures. Among the studies reviewed, 14 performed organic content re-
moval experiments, 5 studies did not process their fecal samples for
microplastic separation and only one study processed as required for
HPLC MS/MS analysis (refer Table 1). Only in 5 out of 20 studies, sieving
was the first step, in which, the mesh sizes vary greatly between 40 μm
 Table 1
Summary of employed approaches for investigating microplastics in fecal samples from previous research.

Location Organism Sample Sampling Sample size Sample Pre-treatment Density Filter type and Identification Spectral References
collection period equipment volume separation filter pore size methods analysis
(g) (μm) software

Japan, UK, Russia, Human NR Duran 8; 3 men 7 - Shaker at 150 rpm for 12 h – Whatman FTIR-ATR Perkin Elmer Schwabl et al.,
Netherlands, Borosilicate and 5 - 30% H2O2 at 25 °C for two weeks room temperature Anodisc; 0.2 library 2019
Italy, Poland, glass bottle women - Saponified with 0.05 M NaOH, room temperature
Finland and - Biological fibers were dissolved in Imidazolium salt
Austria
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti

Murcia, SE Spain Sheep 2018 NR 8–18 5 30 mL distilled water; Two centrifugation 150 rpm – Whatman No. Visual – Beriot et al.,
samples for 30 min, 3000 rpm for 10 min 42; 2.5 inspection and 2020
each herd heat technique
(n = 5)
Albany, New Dog January–March Directly using 37 0.1 - Ground and sieved through a 2.4 mm sieve and – – HPLC-MS/MS – Zhang et al.,
York, USA 2019 PP container feces fortified with 500 ng of D4-TPA and 200 ng of 2019
13C12-BPA and 0.5 g of KOH (0.1 g for feces) pellets
Cat 41 were taken in a 100 mL round-bottom flask, which
contained 20 mL of 1-pentanol
- Concentration in an orbital shaker at 180 rpm for
5 min followed by centrifugation at 3000 rpm
SE Mexico Chicken NR NR 2 0.7 10 mL of demineralized water was added to – NR Visual – Lwanga et al.,

4
facilitate the floatation of microplastics inspection and 2017
Earthworm - 0.5 heat technique
Nanjing, China Chicken NR NR 5 5 - Fenton's reagent: 30% H2O2 and iron catalyst – Cellulose ester Visual InVia-Reflex Yan et al.,
solution (1:2.5) for less than 5 h at 40 °C membrane inspection and 2020
Human 10 1–5 - 65% HNO3 added to the filters and incubated 50 °C filters; 1 Raman
water-bath for 30 min PTFE filter; 1
- Ultrasonic treatment (100 KHz) for 10–15 min
Eastern Beaufort Whales Summer of 2017 NR 2 100 mL 10% KOH for two weeks at room temperature – Polycarbonate; Visual KnowItAll Moore et al.,
Sea, Canada and 2018 20 inspection and Biorad 2020
FTIR-ATR
Bird island, South Penguin 2009 Collected by 80 NR 100 mL of 10% KOH for 24 h at 40 °C; filtered and – Glass Visual Spectral Bessa et al.,
Georgia, Signy hand using further processed with 10% H2O2 for 24 h microfiber inspection and library 2019
island and nitrile gloves filter; 1.2 micro-FTIR database
South Orkney
island
South Georgia, Penguin 2016–2017 Clean metal 47 NR Dried and powdered; 40 mL prefiltered 10% KOH 100 mL of Glass Visual Opus7.5 Le Guen et al.,
USA spatula solution for 24 h at 50 °C prefiltered microfiber inspection and software 2020
NaCl solution filter; 1.2 micro-FTIR-ATR (Bruker),
was added JPI-OCEANS
and repeated project
twice BASEMEN
Central Spain Ducklings 2013–2015 Clean metal 139 NR - Disaggregated each feces in water – – Visual – Gil-Delgado
spatula identification et al., 2017
Freshwater Duck 2013–2014 NR 283 NR - Washed through a set of stacked sieves with – 1 mm, 250 μm Examined – Reynolds and
ecosystem, distilled water and 63 μm under a Ryan, 2018
South Africa mesh sizes binocular
microscope
Zeluán Beach, Migratory January–April NR 10 NR - 20 mL of 30% H2O2 for 24 h and filtered 100 mL of 0.45 Visual NR Masiá et al.,
Spain bids 2019 prefiltered identification 2019
NaCl solution and FTIR
Science of the Total Environment 778 (2021) 146395
 Labrador Sea Seabird July 2015 NR 30 NR - Intestinal-cloacal segments treated with 30% H2O2 – Nitex mesh Visual – Provencher
for 24 h sieve; 100 identification et al., 2018
-Later heated to 75 °C for 8 h using an oven and then
filtered.
Cape Cod, Fur seals Jan 2016- March Collected 32 harbor NR - Washed into the pre-cleaned stack of metal – mesh sizes: Visual Aldrich™ Hudak and
Massachesetts, 2017 using cylindrical graduated sieves 2000, 1000 and identification Polymers FTIR Sette, 2019
USA pre-cleaned 129 Gy 500 and FTIR
white plastic
slotted scoop
or wooden
sterilized
tongue
depressors
Western Fur seals NR Metallic 42 10-12 g - One part was wet sieved and in other part of – -3, 1 and 5 mm Visual Self-generated Garcia-Garin
Antarctica shovel 35-380 g sample 20% KOH was added -Fiberglass identification polymer et al., 2020
filter 1.2 and μFTIR library
St. Paul island, St. Seal July–October Collected 44 NR - Washed using two drops of Dawn Ultra Original NaCl for 24 h Stainless steel Visual Spectral Donohue et al.,
Miguel island 2015 manually dishwashing liquid soap and 75 mL water was added mesh: 500 and identification database 2019
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti

and Bogoslof using medical to it, homogenized and filtered. 250 and FTIR Perkin Elmer
island, USA grade nitrile - decant was filtered again. Nytex mesh;
gloves - sieved samples were treated with 30% H2O2 at 330
75 °C
Northern South Decemeber Pre-cleaned 205 NR - 20% KOH digestion – GF/F filter; 0.7 Visual – Perez-Venegas
Patagonia American 2015-March crow bills identification et al., 2018
fur seals 2016
Perú and Chile Eared Seal NR NR 51 NR - 20% KOH digestion – GF/F filter; 0.7 Visual NR Perez-Venegas
Coasts identification et al., 2020
and micro-FTIR
United Kingdom Seal NR Metal scraping 31 NR - Wet sieving – 2000, 1000, Visual Agilent FTIR Nelms et al.,
instrument - Enzymatic digestion: 3 g sample 15 mL of 500, 200 and identification Spectral 2018

5
homogenizing solution (400 mL Tris-HCI buffer, 40 and FTIR Library Poly
120 mL ethyl-enediaminetetraacetic acid, 30 mL −8
NaCl,100 mL Sodium Dodecyl Sulphate, 350 mL
Milli-Q water was added to per gram of dried scat.
- Proteinase-K was added to each gram of dried scat
and incubated for up to24 h at 50
°C
- 3 mL 5 M sodium perchlorate was added per gram
of dried scat and samples shaken at 20
°C for >30 min
Skomer Island, Grey Seal NR Metal spatula 15 NR -Enzymatic digestion: 3 g sample 15 mL of – NR Visual Spectrum™ Nelms et al.,
Wales homogenizing solution (400 mL Tris-HCI buffer, identification PerkinElmer 2019
120 mL ethyl-enediaminetetraacetic acid, 30 mL and FTIR-ATR
NaCl,100 mL Sodium Dodecyl Sulphate, 350 mL
Milli-Q water was added to per gram of dried scat.
- Proteinase-K was added to each gram of dried scat
and incubated for up to24 h at 50
°C
- 3 mL 5 M sodium perchlorate was added per gram
of dried scat and samples shaken at 20
°C for>30 min
Avery point, USA Mussels April-Sepember, NR 16 NR - 30 mL of 30% H2O2 at room temperature for 20 mL of PC filters; 0.8 Visual BioRad and Zhao et al.,
2016 pseudofeces 36-48 h prefiltered NaI inspection, Bruker 2018
for 24 h Raman and database
37 feces FTIR-ATR

NR: not reported; FTIR-ATR: Fourier transform infrared spectroscopy-attenuated total reflection; HPLC-MS = High performance liquid chromatography mass spectrometry.
Science of the Total Environment 778 (2021) 146395
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti
Fig. 1. Schematic overview of methods used in previous studies for the analysis of microplastics in fecal samples.
and 5 mm. Most studies (n = 14) described herein rely on the acid and
alkali digestion processes for the efficient removal of organic content
and other impurities. The digestion involving H2O2 was carried out in
7 studies, with 10% (n = 1) and 30% (n = 6) concentrations, whereas
alkaline oxidizing agents such as KOH (n = 6) and NaOH (n = 1)
were used as well. Moreover, the concentration of KOH was between
10% and 20% in the studies reviewed. More notably, Bessa et al. (2019)
combined oxidizing agent and alkaline digestion, in which the authors
primarily digested organic content using 10% KOH and further proc-
essed with 10% H2O2. In contrast, Yan et al. (2020) carried out two
steps of digestion involving Fenton's reagent (30% H2O2 and iron cata-
lyst generally a ferrous (II) ion catalyst) and 65% HNO3. Schwabl et al.
(2019) treated fatty stool samples with 0.05 M NaOH and also
employed imidazolium salt to dissolve biological fibers. In addition to
the differences in concentrations of digestion solution, we observed
great disparities in the reaction time and the temperature across the
studies. These parameters could have significant impact on the effi-
ciency of organic matter digestion and subsequently, microplastic ex-
traction. In the studies reviewed, the temperature range was between
25 °C and 75 °C, whereas the reaction time extended between 30 min
and 14 days depending on the type of fecal sample and chemicals
used in digestion step. There has been a small number of papers that
have performed density separation alongside using NaCl (n = 3) and
NaI (n = 1) as extraction liquids for microplastic extraction. Two studies
on seal fecal samples have performed an additional step of extracting
microplastics with enzymes such as Proteinase K at 50 °C for 24 h.
Few authors have also proposed alternative methods to estimate rele-
vant concentrations of microplastics in fecal samples. Lwanga et al.
(2017) and Beriot et al. (2020) disaggregated fecal samples in water
followed by centrifugation to obtain the liquid solution containing
microplastics. In contrast, several studies took directly the disaggre-
gated fecal samples after sieving for microplastic separation and identi-
fication (Hudak and Sette, 2019; Reynolds and Ryan, 2018; Gil-Delgado
et al., 2017).
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Science of the Total Environment 778 (2021) 146395
A variety of filter membranes including, polycarbonate, glass
fiber, stainless steel mesh, cellulose ester membrane and
polytetrafluoroethylene from sizes ranging from 0.2 μm to 2 mm have
been utilized in the studies reviewed (Supplementary Material Table S1).
Once after the completion of dissolution of organic impurities in fecal sam-
ples, the filtration was carried out to separate the microplastics employing
the supernatant fluid obtained from density separation or digestion
solutions. Afterwards, the filter membranes were placed in glass Petri
dishes and dried at room temperature or in an oven, stored until further
inspection.
3.3. Identification, chemical characterization and quantification of
microplastics
After separation, the immediate step in microplastic area is to ob-
serve, identify and classify the suspected microplastic particles based
on size, shape, color and polymer type. As shown in Fig. 1, microscopical
sorting or visual inspection of microplastics is the primary step in ma-
jority of the studies reviewed. Visual inspection of filter membranes
under optical or stereomicroscope greatly depends on the shape, color
and texture of the microplastics. With human vision as the only detec-
tion method while sorting microscopically, the counting errors may
occur which are largely due to person-to-person perceptual differences.
As the visual interpretation is user-dependent, the individuals must be
trained well enough to achieve microscopical analysis. Moreover, a
number of techniques namely heat-rod technique, hardness tests are
available in literature that can be performed in parallel to microscopical
inspection for enhancing the identification process of microplastics
(Eriksen et al., 2014; Masura et al., 2015). Lwanga et al. (2017) and
Beriot et al. (2020) identified the plastic particles and distinguished
them from other particles by burning suspected particles between 120
and 130 °C for a time period of 10s. Accordingly, researchers were
able to differentiate organic matter from microplastics ensuring a qual-
ity data for quantitative measurement.
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti
On the other hand, determining the physio-chemical characteristics
of microplastics is of pivotal importance. Spectroscopic techniques such
as including, attenuated total reflection Fourier transform infrared (ATR
- FTIR), micro-FTIR or Raman spectroscopy are important and com-
monly practiced in previous research for the characterization of
microplastics to obtain chemical information on them. It is widely
been accepted that the quantification of microplastics through chemical
identification is effective for the evaluation of the relative amount of
microplastics from the viewpoint of the minimization of visual/human
errors. In order to obtain a quality data, it would also be preferred to
use a combination of spectroscopic techniques. Micro-FTIR is well-
suited for particles >10 μm, whereas with Raman spectroscopy, one
can able to identify particles smaller than 1 μm (Käppler et al., 2016;
Xu et al., 2019). Out of 20 studies, 13 studies utilized a combination of
microscopy as well as spectroscopic approaches to ensure the quality
of quantification and one study used both ATR-FTIR and Raman tech-
niques for polymer identification. FTIR techniques including ATR-FTIR
and micro-FTIR were employed in majority of the studies (n = 11),
whereas Raman spectroscopy was applied only in 2 studies. The number
of potential microplastic samples tested for polymer identification var-
ied between studies reviewed and only one study did not mention the
amount of microplastics examined for polymer types (Hudak and
Sette, 2019). For instance, a small number of papers have performed
chemical identification of all particles found on the surface of the filter
(Zhao et al., 2018; Nelms et al., 2018; Bessa et al., 2019; Moore et al.,
2020; Le Guen et al., 2020). Schwabl et al. (2019) examined only parti-
cles >50 μm and Perez-Venegas et al. (2020) randomly selected
microplastics for chemical identification. Masiá et al. (2019) investi-
gated less than 10 particles, whereas Nelms et al. (2019) examined 17
potential microplastics for polymer types. Further, high performance
liquid chromatography mass-spectrometry was also applied to identify
microplastics (Zhang et al., 2020).
Another major concern in microplastic area comes from the
reporting units of microplastic abundance in literature. Any inconsis-
tency in the reporting units of microplastic abundance often pose diffi-
culties to generate a feasible comparison of results between studies.
Accordingly, the geographic distribution and comparison of the level
of microplastic contamination cannot be achieved. Similar to the find-
ings from recent reviews on other environmental matrices (Prata
et al., 2019; Kutralam-Muniasamy et al., 2020; Fok et al., 2020; Shruti
et al., 2021), there is a great variation in the reporting units for
microplastics in fecal samples which include 10 g−1 (n = 1), ng/g
(n = 1), particles kg−1 (n = 1), particles g−1 (n = 2), items ind−1
(n = 1), particles (n = 2), microfiber g−1 (n = 1), sample−1 (n = 2),
pieces dw. g−1 (n = 1), microplastic particles (n = 1), fibers and frag-
ments (n = 1), items g−1 (n = 2), particles scat−1 (n = 1) and
microplastics scat−1 (n = 1). Moreover, we noted that some studies
have reported the microplastic abundance as a mean for the fecal sam-
ples examined, whereas others reported statistics only for the fecal sam-
ples detected as positives.
4. Contamination prevention and QA/QC measures
Strict control measures are mandatory to be followed during sam-
pling and laboratory analysis in microplastics research to prevent sec-
ondary contamination from various sources like airborne, lab coat,
gloves and equipment used in the laboratory. In 20 studies reviewed,
3 studies did not describe any contamination protocol and thus, we con-
sider them as not reported (Table 2). Care must be taken right from the
first step of the study i.e., sample collection to avoid potential contami-
nation. For instance, it is advisable for sampling team to wear natural
fiber clothing wherever possible or clothing of same fabric. During
fecal sample collection, it is essential to prevent its contact from collec-
tion bags, gloves or with any plastic items such as synthetic fleece cloth-
ing. Hence, the use of clean glass bottles in the place of objects related to
plastics is highly recommended. Furthermore, care should be taken to
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Science of the Total Environment 778 (2021) 146395
minimize the extraneous contamination from underlining substrate
soil or sediment while collecting the fecal samples from the ground. Be-
fore each sample collection, the sampling device (metal scoop, spatula)
must be cleaned/rinsed with pre-filtered ethanol to prevent sample
cross-contamination. In addition, it is optimal to store the collected
fecal samples in sterile paper bags or aluminum foil. In the reviewed lit-
erature, only 5 studies carried out some/many of the above-mentioned
field contamination prevention measures (Gil-Delgado et al., 2017;
Donohue et al., 2019; Hudak and Sette, 2019; Le Guen et al., 2020;
Moore et al., 2020). Another important step involves the use of field
and laboratory blank controls and analyze them alongside the samples
for obtaining credible quantification of microplastics. Only one study
collected the substrate sample (i.e., soil underneath the fecal matter)
and treated as field blank to explore the potential contamination of
fecal samples with any microplastics present in substrate (Donohue
et al., 2019). Laboratory and procedural blanks were reported in 14
out of 20 studies which mainly included damp filter paper (n = 3), air
blank (n = 3), pre-filtered Milli-Q water (n = 6), tap water (n = 1)
and blank without sample (n = 1). Additionally, several measures
need to be adopted in laboratory to minimize the secondary contamina-
tion and 13 out of 20 studies reported them in detail. The most common
methods used were to clean surfaces with 70% ethanol (n = 2), pre-
clean all the glassware and equipment prior to use (e.g., ethanol, Milli-
Q water; n = 12), pre-filter all the solutions before analysis (n = 2),
pre-screening of the sieves and filter paper (n = 6), carrying out analy-
sis under laminar flow hood with restricted access (n = 9) and HEPA air
filter in laboratory (n = 2) and finally, wearing cotton lab coat and ni-
trile gloves (n = 8) were regularly followed. Keeping samples and filters
covered with aluminum foil (n = 6) was also considered appropriate. In
the studies where microplastic particles were observed in the blanks,
the results were subtracted accordingly from the obtained results.
Moreover, the studies that did not mention any contamination proto-
cols would result in under/over-estimation of microplastics and thus,
we advocate all the researchers to carry out strict contamination pre-
vention measures during their field and laboratory activities.
5. Analytical challenges
(1) A known issue for example is the discrepancies in the methods of
investigation applied so far including, mesh sizes of sieves and fil-
ters, the quantity of sample, number of samples and reporting
data. Furthermore, the detection limit of microplastics quantifica-
tion set by the filter membrane (i.e., pore or mesh size) and by the
authors (during identification and characterization) can contrib-
ute difficulties to establish comparison of microplastic concentra-
tions between studies and are completely laboratory dependent.
The successful comparison of the results depends greatly on the
uniformity of the methodological approaches. Thus, the need for
the standardization of the methods and units is clear in order to
obtain more reliable and comparable data in future studies.
(2) For field-based observations, owing to the extraneous
microplastic contamination from the surrounding environments,
it is important that fecal samples should be taken immediately
after its deposition and the fecal samples that were already
found in the environment must be avoided.
(3) The amount of sample analyzed seems completely arbitrary or
random and not fully described mostly in previous research. If pos-
sible, future research can divide an individual fecal sample (e.g., in
case of humans, dog etc.,) into two or three portions and try to pro-
cess them to obtain microplastic data. Doing these measurements
are important and the information can shed light on accurate de-
scriptions of the heterogenous distribution of microplastics within
a fecal sample. Additionally, it can provide paths to understand
better the microplastic concentration in fecal samples.
(4) On one hand, performing replicates (based on biologically
distinct samples from an individual of interest) are crucial for in-
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti Science of the Total Environment 778 (2021) 146395

Table 2
Contamination prevention measures practiced in previous research for investigating microplastics in fecal samples.

References Blanks Secondary contamination from Contamination prevention measures

blanks

Schwabl et al., Laboratory: 1 procedural blank (prefiltered Milli-Q No contamination in blanks - All the utensils were rinsed with Milli-Q water
2019 water; 0.22 μm) without stool sample - Antibacterial solution AQUA KEM BLUE
- All the solutions were filtered 50 μm metal sieve prior to use
Beriot et al., Laboratory: 6 blanks without fecal samples NR NR
2020
Zhang et al., Laboratory: procedural blanks for every 15–20 NR - Feces samples were lyophilized to avoid extraneous
2019 samples contamination
- Round-bottom flasks were muffled at 450 °C for 12 h and
rinsed with acetone and methanol prior to use
- Surface layer of feces was removed before microplastics
separation
Lwanga et al., NR NR - feces samples were lyophilized to avoid extraneous
2017 contamination
Yan et al., Recovery rates detection using PE (150 μm) PS NR - Stored in 1 and 2 L brown glass container
2020 (250 μm) and PVC (75 μm) - Feces samples were lyophilized to avoid extraneous
contamination
- Cotton lab coat and gloves were used
- All the equipment was washed thrice with Milli-Q water
- Filters were tested with Raman spectra prior to use
Moore et al., Damp filter paper and procedural blanks NR - Feces samples were lyophilized to avoid extraneous
2020 contamination
- HEPA filter used to control airflow
- Tyvex suits, non-shedding rubber shoes and nitrile gloves
- Tools and glassware were rinsed three times with prefiltered
distilled water
Bessa et al., 3 blanks using Milli-Q water NR - Samples were stored in sterile plastic bags
2019 - Feces samples were lyophilized to avoid extraneous
contamination
- All solutions and reagents were filtered using 1.2 μm
- All utensils were rinsed using ultrapure water
- Closed and restricted laboratory access
- Cotton lab coat and nitrile gloves
Le Guen et al., 17 procedural blanks using Milli-Q water 59 microfibers were found in 17 - After each sample collection, the metal spatula was rinsed with
2020 procedural blanks and subtracted prefiltered ethanol
- Feces samples were stored in 2 mL Eppendorf tubes containing
prefiltered 80% ethanol solution and kept frozen
- Clean glass microfiber filter was left open next to the samples
for the entire processes
- Cotton lab coat was used
- All utensils were rinsed using ultrapure water
Gil-Delgado NR NR - Samples were stored in paper bags
et al., 2017 - Feces samples were lyophilized to avoid extraneous
contamination
Reynolds and NR NR - Stored in individual vials
Ryan, 2018
Masiá et al., NR NR - Stored in glass bottles
2019
Provencher 10 blank samples with tap water 1 or 2 per sample - Cotton lab coat and nitrile gloves
et al., 2018 - All utensils were rinsed using filtered water
- All filters, beakers, and petri dishes were washed and
pre-inspected for microfiber contamination
Hudak and NR NR - Stored in sterile plastic collection bags or aluminum foil and
Sette, 2019 frozen.
- The field scoop was cleaned before each sample collection to
prevent cross-contamination.
- Sieves were soaked in soap and hot water, cleaned, dried,
and inspected visually for any remaining material.
Garcia-Garin - 1 Procedural blank for every 10 samples 3 blue fibers - Stored in aluminum foil and frozen
et al., 2020 - Cotton lab coat
- All glass beakers were rinsed with purified water
Donohue et al., - 2 control samples from study area - No contamination from control - Stored into sterile, polyethylene “Whirl-Pak” 207 mL sample
2019 - Laboratory and procedural blanks samples of study area collection bags and frozen.
- Procedural blank: 22 fibers - Prevented contact of collection bags and gloves with plastic
- Laboratory control (filter air): 49 items, such as synthetic fleece clothing during sampling.
fibers - Cotton lab coat, nitrile gloves were used
- Used materials were carefully washed and dried with low-lint
wipes
Perez-Venegas - One blank filter every ten samples 2 microfibers were observed and - Stored in ashed aluminum foil envelope and frozen
et al., 2018 subtracted
Perez-Venegas - One blank filter every ten samples NR - Stored in ashed aluminum foil envelope and frozen
et al., 2020
Nelms et al., -Damp filter paper NR - Sample collection pots were thoroughly rinsed with Milli-Q.
2018 -Procedural blank using Milli-Q water - Cotton lab coat and gloves were worn.
- All work surfaces were wiped down with70% ethanol

8
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti
Table 2 (continued)
References Blanks Secondary contamination from
blanks
Nelms et al., -Damp filter paper NR
2019 -Procedural blank using Milli-Q water
Zhao et al., Procedural blanks (triplicate) performed using NR
2018 Milli-Q water and H2O2
NR: not reported.
depth understanding of microplastics distribution in feces. How-
ever, the collection of a number of replicate samples per individual
under relatively environmental conditions is not always possible
to achieve. This is likely due to the difficulties associated with
fecal collection from same individual (say seal or bird) which
often requires substantial investment of resources and time to fol-
low the presence and movement of an individual in certain areas
of geographic interest until its next defecation. On the other
hand, technical replicates are difficult to perform in case of organ-
isms that produce relatively little amounts of feces in the environ-
ment (e.g., small birds).
(5) In large part, the concentrations of digestion and density separa-
tion protocol solutions, temperature and incubation time need to
be optimized and standardized.
(6) The direct observation from the disaggregated feces samples
through microscope is simple; however, its widespread use is lim-
ited by the following factors: On one hand, microplastics can still
bound in/onto the solid disaggregated materials and cannot be
counted. On other hand, feces enriched with calcium and phos-
phate particles could be counted as microplastics leading to erro-
neous data and results. As there is no data for the level of non-
microplastic particles found/discarded using heat techniques in
previous studies, we cannot assume the proportion of these parti-
cles in the fecal samples analyzed. Additional challenges may arise
under the brown background, when dealing with microplastic
particles of colorless-to-colored or highly transparent.
(7) If possible, future research can try using alternatives to NaCl solu-
tion like ZnCl2 and NaI for density separation protocols as they are
proven effective in recovery of both low-density plastics
(e.g., polypropylene (PP) and polyethylene (PE)) and high-
density plastics (e.g., polyvinylchloride (PVC), polyamide (PA),
polystyrene (PS) and polyurethane (PU)).
(8) It may be better to include damp filters, open jars or soil sample
underneath the feces as field control during fecal sampling, for
evaluating the extent of contamination arising from the
environment.
6. Current knowledge of microplastics in feces
6.1. Abundance of microplastics in feces
Table 3 shows the recent research developments on abundance and
characteristics of microplastics in feces collected from different organ-
isms of distinct environments. Of 20 studies reviewed, 18 studies have
demonstrated the occurrence of microplastics in fecal samples, only
one study found no presence of microplastics (Garcia-Garin et al.,
2020) and one study did not report the level of microplastics (Yan
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Science of the Total Environment 778 (2021) 146395
Contamination prevention measures
-All equipment was thoroughly rinsed with Milli-Q water
-Inspection of sieves and filter paper under microscope
- Stored in sterile centrifuge tubes
- Sample collection pots were thoroughly rinsed with Milli-Q.
- Cotton lab coat and gloves were worn.
- All work surfaces were wiped down with70% ethanol
- All equipment was thoroughly rinsed with Milli-Q water
- Inspection of sieves and filter paper under microscope
- All the solutions were prefiltered with Glassfiber filter
Whatman GF/F; 0.7 μm prior to use
- Filters were checked for any extraneous contamination of
microplastics
- Utensils were rinsed thoroughly with Milli-Q water
- Analyses conducted under laminar flow hood equipped with
0.2 μm HEPA filter
- Feces samples stored in glass tube at 4 °C
et al., 2020). Majority of studies reported microplastics in feces of higher
trophic level organisms like whale, seal and penguin in marine environ-
ments. Moore et al. (2020) studied the fecal samples of beluga whales
(Delphinapterus leucas) and observed a range of 0–2 microplastic parti-
cles in the two feces sample from Eastern Beaufort Sea. Likewise, King
penguins (Aptenodytes patagonicus) foraging from South Georgia
contained an average of 21.9 ± 5.8 microfibers g−1 in 36 out of 47
fecal samples collected (Le Guen et al., 2020). Bessa et al. (2019) studied
the gentoo penguin (Pygoscelis papua) from the Antarctic region and ob-
served a mean microplastic abundance of 0.23 ± 0.53 items
individual−1 scat in the fecal samples. 7 out of 20 studies focused on
seals collected mainly from USA (n = 2), Latin America (n = 2), UK
(n = 2) and Antarctic (n = 1) regions. The reported microplastic con-
centrations in seal fecal samples from USA was 4 microplastic particles
(Donohue et al., 2019; Hudak and Sette, 2018), 2 to 15 items g−1 in
Latin America (Perez-Venegas et al., 2018, 2020), 0–5 particles scat−1
in UK (Nelms et al., 2018, 2019) and 0 in Antarctic region (Garcia-
Garin et al., 2020). Only one field-based evidence exists for lower tro-
phic organisms like Blue mussels (Mytilus edulis) which exhibited an
abundance of 3 and 7 microplastic particles in its feces and pseudofeces,
respectively (Zhao et al., 2018). Apart from marine organisms,
microplastics in fecal samples of agricultural farmland animals like
chicken (n = 2; 129.8 ± 82.3 particles g−1), earthworm (n = 1;
14.8 ± 28.8 particles g−1) and sheep (n = 1; 997 ± 971 particles
kg−1) were also investigated (Lwanga et al., 2017; Beriot et al., 2020;
Yan et al., 2020). Also, stool samples of domestic animals like cats and
dogs were analyzed and the range of microplastics were polyethylene
terephthalate (PET): <2300–340,000 ng/g; polycarbonate (PC):
<32–13,000 ng/g and PET: 7700–190,000 ng/g; PC: <32–26,000 ng/g,
respectively (Zhang et al., 2019). Schwabl et al. (2019) brought an im-
portant breakthrough by examining human feces for the presence of
microplastics and identified that all 8 stool samples collected were
tested positive for microplastics with a median of 20 microplastics (50
to 500 μm in size) per 10 g of human stool.
Nonetheless, several aspects about microplastics in feces including
microplastic transformations, the transport between ecosystems
(ingested in one environment and defecated in another by migratory
organisms) and accumulations are relatively little known. For example,
Masiá et al. (2019) analyzed 10 depositions from migratory organisms
and found a total of 114 MP particles with the number of microplastic
varied between 3 and 33 per deposition (average 12.7, standard devia-
tion 9.1). Overall, the results of these studies provided new insights into
the degree of microplastic contamination in feces collected from diverse
environments. However, there is a limited knowledge on how seasonal
changes influence the prevalence of microplastics in feces. Since the
abundance and distribution of organisms including migratory species,
zooplankton, fishes etc. may vary based on locality and season, it is
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti Science of the Total Environment 778 (2021) 146395

Table 3
Summary of occurrence and characteristics of detected microplastics in fecal samples as reported in previous studies.

Location Organism Positive Prevalencea Microplastics Size (μm) Polymer type Sources References
samples (%) abundance and
shape

Japan, UK, Russia, Human 8 (8); 3 100 20 pieces per 10 g; 50–500 PP, PET, PE, PS, PC, PA, PVC, Sea food, water from Schwabl et al.,
Netherlands, men and 5 Fragment and film PU and POM plastic bottles, food 2019
Italy, Poland, women wrapped in plastic
Finland and and cosmetic products
Austria
Murcia, SE Spain Sheep 8–18 – 997 ± 971 particles – – Agricultural mulch Beriot et al.,
samples kg−1 films, manure, 2020
each herd fertilizer, plastic bags
(n = 5)
Albany, New York, Dog 37 (37) 100 PET: – PET and PC Dog foods Zhang et al.,
USA 7700–190,000 ng/g 2019
PC: <
32–26,000 ng/g
Cat 41 (41) 100 PET: – PET and PC Cat foods
<2300–340,000 ng/g
PC:
<32–13,000 ng/g
SE Mexico Chicken 2 (2) 100 129.8 ± 82.3 10 - > 50 – Agricultural mulch Lwanga et al.,
particles g−1 films, manure, 2017
fertilizer, plastic bags
Earthworm – – 14.8 ± 28.8 particles – Direct ingestion of soil
g−1
Nanjing, China Chicken 5 (10) 50 – 1 Nylon and PET Agricultural mulch Yan et al.,
films, manure, 2020
fertilizer, plastic bags
Human 10 – – 1 PVB and PBT Contaminated air,
drinking water and
food
Eastern Beaufort Whales 2 (2) 100 0–2 particles; – – Ingestion of prey Moore et al.,
Sea, Canada Fragment and fiber species 2020
Bird island, South Penguin 16 (80) 20 0.23 ± 0.53 items 118–4954 Polyester, rayon, PE, PP, Ingestion of prey Bessa et al.,
Georgia, Signy feces ind−1; microfiber, polyacrylate and species 2019
island and South fiber, fragment and polyacrylonitrile
Orkney island film
South Georgia, Penguin 36 (47) 77 21.9 ± 5.8 186–9280 Natural cellulosic material, Ingestion of prey Le Guen et al.,
USA microfiber g−1; polyester, nylon and rayon species 2020
microfiber
Central Spain Ducklings 64 (139) 46 -; Fragment and 500–1200 – User plastics in Gil-Delgado
thread agricultural landscape et al., 2017
and prey ingestion
Freshwater Duck 14 (283) 5 1.53 ± 0.64 – – Availability of plastics Reynolds and
ecosystem, sample−1; Fiber in freshwater Ryan, 2018
South Africa environment
Zeluán Beach, Migratory 10 (10) 100 114 particles; 3–33 – PVC Ingestion of prey Masiá et al.,
Spain birds sample−1; Fiber and species 2019
fragment
Labrador Sea Seabird 14 (30) 47 12.79 pieces dw. g−1; – – Ingestion of prey Provencher
Fulmars Fragment, fiber, film, species et al., 2018
foam and rubber
Cape Cod, Fur seals 2 (32) 6 4 microplastic >500 Alkyd resin, cellophane, Ingestion of prey Hudak and
Massachesetts, harbor particles EPDM and rubber species Sette, 2019
USA
2 (129) 1.5
grey
Western Fur seals 0 (42) 0 0 microplastics – – – Garcia-Garin
Antarctica et al., 2020
St. Paul island, St. Seal 44 (44) 100 398 fragments <1000–>10,000 – Ingestion of prey Donohue et al.,
Miguel island 186 fibers species or direct 2019
and Bogoslof consumption
island, USA
Perú and Chile Otariids; 44 (205) 21 2–15 items g−1; Cotton, PET, nylon and Ingestion of prey Perez-Venegas
Coasts Eared Seal Fiber and fragment cellulose species et al., 2020
Northern South 34 (51) 67 2.7 to 13.35 items >100 – Ingestion of prey Perez-Venegas
Patagonia American g−1; Fiber species et al., 2018
fur seals
United Kingdom Seal 15 (31) 48 0–4 particles scat−1; 1500–2000 Ethylene propylene, PP, PE, Ingestion of prey Nelms et al.,
Fiber and fragments PA, Styrene butadiene species or direct 2018
rubber, PU, neoprene and consumption
polyamide kevlar
Skomer Island, Grey seals 8 (15) 53 1–5 microplastics Fragment: Nylon, low density PE, PET Ingestion of prey Nelms et al.,
United Kingdom scat−1; Fiber and 300–5500 and PE species or direct 2019
fragment Fiber: 150–400 consumption
Avery point, USA Mussels 16 3 particles; Fiber and 780.9 ± 196.6 Polyester, synthetic Direct consumption Zhao et al.,

10
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti
Table 3 (continued)
Location Organism Positive Prevalencea Microplastics
samples (%) abundance and
shape
pseudofeces fragment
37 feces 7 particle; Fiber and
fragment
PP – polypropylene; PET – polyethylene terephthalate; PE – polyethylene; PS – polystyrene; PC – polycarbonate; PA – polyamide; PVC – polyvinylchloride; PU – polyurethane; POM –
polyoxymethylene; PVB- poly (vinyl alcohol-co-vinyl butyral); PBT- polybutylene terephthalate; POM – polyoxymethylene; EPDM - ethylene propylene diene monomer.
a
Prevalence = positive samples/no. of samples ∗ 100.
important to get information on the abundance of egested microplastics
with size distribution and polymer composition in different seasons.
Thereby, determining seasonal changes in the amount and characteris-
tics of microplastics in feces will contribute to a better understanding of
their influencing factors and potential risks in the environment.
More often than not, a deeper understanding of the results of
microplastic abundance, under a variety of reporting units, between
studies, is, at best, difficult. In this context, we calculated the percentage
of prevalence for microplastics considering the number of samples ana-
lyzed and the number of positive samples (Table 3). By this percentage
of prevalence, we can be able to analyze and compare the extent of
microplastic contamination in fecal samples between studies. In 6 out
of 20 studies, 100% prevalence of microplastics in feces were witnessed,
while, more than 50% occurred in 4 studies and the least prevalence of
1.5–6% were displayed in 2 studies. Thereby, from the observed results
with 30% studies exhibiting 100% prevalence of microplastics in samples
collected, suggest that the exposure of organisms from diverse environ-
ments to microplastic pollution is growing and demands scientific at-
tention. One of the potential source of microplastics in feces have been
identified as plastic contaminated prey ingestion i.e., trophic transfer
and the prevalence also depends upon the capacity of prey to retain
the microplastics. Another important route of ingestion may result
from its presence in the foraging grounds and surrounding environ-
ment. Moreover, the extent of exposure of an organism to microplastic
contaminated environment plays a vital role in its uptake and egestion.
For example, in almost pristine marine region of Western Antarctica, the
fecal samples of fur seals presented none microplastic abundance indic-
ative of possible absence of plastic pollution in the environment or that
the incidences of microplastic ingestion by fur seals are very less and
negligible (Garcia-Garin et al., 2020). On contrary, humans are being
constantly being exposed to higher levels of microplastic contamination
via drinking water, air, packed foods etc. and that is probably the reason
for displaying 100% prevalence of microplastics in the stool samples an-
alyzed (Schwabl et al., 2019).
6.2. Characteristics of microplastics in feces
In general, ingestion of microplastics at ambient conditions can be se-
lective depending on the species and animals may uptake plastics differ-
entially depending on the plastic's color, shape and size. Understanding
these characteristics could provide clues into the type of microplastics
ending in the environment through feces. The color, size, shape and poly-
mer characteristics of detected microplastics in recent studies are sum-
marized in Table 3. The lack of detailed information and numerical
representation (e.g., percentage) on size, shape and color of
microplastics extracted from fecal samples make the complete under-
standing of the distribution and morphological characteristics difficult
in the studies reviewed. 11 out of 20 studies examined the color of
microplastics extracted from the feces; a large variety of colors including
white, black, blue, red, green and purple etc. have been recorded in liter-
ature. It is important to highlight here that the diversity of colors ob-
served in the microplastics from fecal samples are more similar to
those observed in different environmental media (e.g., water, sediment
and air). The observed shape of detected microplastics was majorly
fiber (n = 13) and fragment (n = 11) with very less studies reporting
Science of the Total Environment 778 (2021) 146395
Size (μm) Polymer type Sources References
394 ± 167.8 cellulose, nylon, PE, 2018
synthetic cotton
film (n = 3), microfiber (n = 2) and foam (n = 1) which suggest that
fibers and fragments could be considered more bioavailable via ingestion
by organisms. From the available literature, only one field-based study
analyzed the shape of microplastics in the fecal precursor sample and
in the gut contents (Provencher et al., 2018). The authors observed sig-
nificant differences in the detected microplastics between fecal precur-
sor and gut contents. Microfibers, pellet, film, foam and rubber were
detected in fecal sample suggesting their passage through the gastroin-
testinal tract without being retained in the stomach. Whereas only frag-
ments were found retained predominantly in the gut contents. The
predominance of fiber and fragment shape microplastics in feces across
diverse trophic levels indicates that they are mainly secondary
microplastics (fragment, fiber, film and foam) rather than primary
microplastics (pellets) which could be formed from the disintegration
and fragmentation of larger plastic items. In addition, the formation of
secondary microplastics (fiber and fragment) can also occur within the
organisms´ internal system (i.e., gastro-intestinal tract or gizzard). For in-
stance, organisms like seabirds, snail, chicken and earthworm are known
to ingest plastics from surrounding environment and are thought to
break down retained plastics into smaller ones in their internal system
(Lwanga et al., 2018; Song et al., 2020). Thus, laboratory experiments fo-
cusing on how the gastro-intestinal tract and digestive fluids of biota in-
fluences the shape of microplastics in feces are needed. Nevertheless, the
excretion of retained microplastics over time by organism has been
rarely discussed or reported in microplastics research to date.
With respect to size, larger size microplastics (100–10,000 μm)
accounted for major proportions in the fecal samples analyzed and re-
ported in 13 out of 20 studies. On the other hand, Lwanga et al. (2017)
reported the presence of smaller sized microplastics in chicken feces col-
lected from traditional Mayan home gardens in Southeast Mexico. In this
study, microplastics encountered in chicken gizzards were > 5 mm,
while in feces were between 0.1 and 1 mm and the authors assumed
that smaller size microplastics excreted in feces originated from the
transformation of macroplastics to microplastics during the passage
through the digestive canal. From the evidence discussed in this review,
it is evident that the shape and size of microplastics packed in feces elim-
inated by organisms also greatly depends on the process that undergoes
in the internal system (e.g., gut, gizzard). Polymer type is another impor-
tant criterion that could aid in identifying the source of microplastics.
Only, 13 out of 20 studies performed spectroscopic analysis (FTIR and
Raman) to identify the polymer type extracted from fecal samples of di-
verse organisms. The most common polymer types identified in feces in-
cluded PE, PP and PET (Table 3). These polymer types are highly
expected to be observed as they are widely used in short-life cycle prod-
ucts used in day-to-day life and accounted for nearly 58% of global plastic
production in 2018 (PlasticsEurope, 2018). Other high dense polymers
such as PA, nylon, polyester, PS, PVC and PU were also reported in
some of the studies reviewed. Diverse polymer types recorded in the
fecal samples suggests their prime intake from surrounding environ-
ment as well transfer via prey ingestion.
7. Practices and recommendations for future studies
Based on the available literature, on one hand, there is a lack of stud-
ies that compare the efficiency of methods for extracting microplastics
11
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti
from feces across different species, with the exception of one study in
the total of 20 studies reviewed. Thus, increased knowledge on this
topic can lead to better optimization of the current approaches, which
in itself can serve as a valuable information for further research and sci-
entific professionals will benefit from this to standardize the methodol-
ogies. On the other hand, remarkably, the recent experiences on feces
materials of different species have been more successful. More
supportively, the current approaches showed some similarities in
terms of sample preparation, detection and characterization of
microplastics for different kinds of fecal samples of marine and terres-
trial organisms. It is promising that the available approaches can be eas-
ily adjusted and quickly adapted to a wide range of fecal samples that
are not investigated in literature. From the practical perspective, a pro-
cedural workflow is given in Fig. 2 considering the current analytical
methods for the determination of microplastics in feces. This workflow
includes and combines the selected analytical steps for improved sam-
ple preparation and microplastic identification. Upon sampling, the
immediate storage by freezing or lyophilization of collected fecal
samples is appropriate. It is advisable to not use the surface layer of
the feces collected from terrestrial environments for microplastic ex-
traction. The removal of organic content and other impurities is
the most straightforward route to analysis. The consistency of selection
of pretreatment method is expected and as a starting point for standard-
ization. However, the optimized conditions and concentrations of re-
agent solution are still an issue for these reactions. As stated earlier,
the optimization requires further investigation and must consider by
comparing different fecal samples of origin. The field and laboratory ac-
tivities must consider strict contamination measures as described ear-
lier in this study (i.e., Section 4) and as summarized in Fig. 2. The
combination of visual inspection and spectroscopic techniques is an ad-
vantageous strategy for microplastic identification and quantification.
None of the existing studies have employed fluorescent labels such as
Nile Red to quantify microplastics. Compared to current methods, Nile
Red staining approach has the advantage of tracing small microplastics
or even nanoplastics and is particularly helpful when quantifying
microplastics under microscope. Therefore, it is highly recommended
to include staining approaches in future studies. Other methods
Fig. 2. A schematic representation of the proposed workflow for determination of microplastics in the fecal samples from diverse species. FTIR = Fourier transform infrared spectroscopy;
GC–MS = gas chromatography mass spectrometry; HPLC-MS = High performance liquid chromatography mass spectrometry; Dye tagging = e.g. Nile red.
12
Science of the Total Environment 778 (2021) 146395
including mass spectrometry and gas chromatography techniques
such as pyrolysis-GC–MS and TED-GC–MS could also be explored for
subsequent identification and quantification of microplastics. Finally, it
is necessary representing numerically (e.g., percentage) the morpholog-
ical characteristics (e.g., size, shape) to advance and understand better
the knowledge of the detected microplastics in feces.
8. Conclusion
Microplastics expulsion in fecal matter is ubiquitous and an emerg-
ing concern with unknown consequences in the environment. In this
paper, we reviewed the analytical methods for determining
microplastics in feces across diverse trophic organisms and have sum-
marized occurrences and characteristics of current literature on
microplastic contamination in feces. The increased awareness together
with the better detection methods have brought important scientific ev-
idence and provided novel insights into microplastics in feces. However,
the discrepancy between the current approaches suffers greatly from
critical operational limitations and are often criticized for susceptibility
to reduce the comparability of the results between studies. In order to
advance the field and make data comparable, the need for standardiza-
tion of methodological approaches is evident and a big challenge to con-
front in future.
In addition, there remain opportunities that need to be addressed for
further advancing our scientific understanding of microplastics in feces.
(1) A lack of literature examples dealing with the freshwater organ-
isms and that of other domestic animals like cow and donkey has been
noticed and needs to be addressed. (2) In future studies, the extended
collection of data on the types of plastic particles ingested and egested
by organisms is of pivotal importance and may provide path to ade-
quately assess internal exposure, toxic effects, and trophic transfer of
microplastics. (3) There is a limited information regarding the contribu-
tion of feces or scats for the release of microplastics into environment
and in any risk assessment study, exposure concentration would be a
critical input. As the microplastic research on feces is in its infancy,
more studies should be conducted to estimate how much microplastics
is being released from feces into the environment. (4) It is necessary to
F. Pérez-Guevara, G. Kutralam-Muniasamy and V.C. Shruti
identify and evaluate the presence of toxic additives or other contami-
nants associated to microplastics. (5) When microplastics are packed
in feces, there remains possibility for microplastics to have interactions
with fecal components like microbiota, metals and others. Thus, it is im-
portant conducting investigations to have an overview on the interac-
tions between microplastics and fecal associated chemical and
microbial components.
Regardless of promising efforts and progress made in the last few
years, microplastics contamination in feces in the field of microplastic
research is still emerging. With the scarcity in global studies, the data
on the extent of anthropogenic contamination of microplastics through
feces is much limited to assess their environmental consequences. We
believe that researchers and students will benefit from the methods
presented in this review and will lead to future improvements and
rapid advances in the coming years.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2021.146395.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
VCS thanks CONACYT project no. 274276 “Fase I De La Remediación
de Áreas Contaminadas Con Hidrocarburos En La Refinería Gral. Lázaro
Cárdenas” for Postdoctoral fellowship.
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