Environmental Pollution 316 (2023) 120548

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Microplastic in northern anchovies (Engraulis mordax) and common murres

(Uria aalge) from the Monterey Bay, California USA - Insights into
prevalence, composition, and estrogenic activity☆
Sami Michishita a, Corinne Gibble b, Christopher Tubbs c, Rachel Felton c, Jenessa Gjeltema d,
Jackelyn Lang d, Myra Finkelstein a, *
a
Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA, 95064, USA
b
California Department of Fish and Wildlife, Office of Spill Prevention and Response, Marine Wildlife Veterinary Care and Research Center, 151 McAllister Way, Santa
Cruz, CA, 95060, USA
c
Conservation Science Wildlife Health, San Diego Zoo Wildlife Alliance, 15600 San Pasqual Valley Road, Escondido, CA, 92027, USA
d
Department of Medicine and Epidemiology and the Karen C. Drayer Wildlife Health Center, University of California Davis School of Veterinary Medicine, One Shields
Drive, Davis, CA, 95616, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Microplastic (particle size <5 mm) is considered an emerging threat to the marine environment, yet data are
Microplastic limited for coastal ecosystems. To provide information related to microplastic in a coastal system, we used
Raman spectroscopy alkaline tissue digestion and Raman spectroscopy to quantify the prevalence and composition (e.g. fiber, frag­
Monterey Bay
ment, foam, etc.) of anthropogenic microparticles in the digestive tracts of northern anchovies (Engraulis mordax,
Xenoestrogen
Estrogen
anchovy, n = 24), and common murres (Uria aalge, murre, n = 19) from the Monterey Bay, California USA. We
Common murre also determined microplastic prevalence and composition in seawater (n = 12 17-h sampling periods repre­
Northern anchovy senting ~46,000 L sampled) from two Monterey Bay intake systems (Moss Landing, CA and Santa Cruz, CA USA).
Microparticles recovered from murre digestive tracts were assessed for estrogenic activity using an in-vitro es­
trogen receptor activation assay. Suspected anthropogenic microparticles based on visual characteristics were
recovered from all sample types with ~2 particles per 1000 L from the seawater sampling periods, 58% prev­
alence in anchovies, and 100% prevalence in murres. Across samples of seawater, anchovies, and murres, the
most abundant microparticle type found were fibers (78%), followed by fragments (13%), foam (6%), film (2%),
and beads (1%). Raman spectroscopy identified 57% of microparticles (excluding dye-prominent and unknown)
as plastic (synthetic, semi-synthetic, or blends). Almost one quarter (23%) of the murre digestive tracts contained
microparticles that exhibited estrogenic activity. Our study describes the widespread occurrence, composition,
and potential estrogenic activity of microplastic in the Monterey Bay and provides important information to aid
in the understanding of microplastic contamination in coastal systems.

1. Introduction
Plastic pollution is widespread throughout the marine environment
with over 700 marine species documented with ingested plastic (Azou­
lay et al., 2019; Geyer et al., 2017; Kühn and Van Franeker, 2020).
Indeed, unless prevention measures are implemented, by 2050, the
ocean is predicted to have more plastic than fish by weight (Dąbrowska
et al., 2021), and 99% of seabird species are expected to be affected by
plastic ingestion or entanglement (Wilcox et al., 2015). While
macroplastic (size >5 mm) ingestion by marine species has been widely
documented (Kühn and van Franeker, 2020; Provencher et al., 2017),
information on microplastic (size <5 mm) ingestion and risk for adverse
effects is less understood, due in part to the logistical challenges of
quantifying ingested microplastic compared to macroplastic (Pro­
vencher et al., 2020).
Common methods to quantify ingested macroplastic include nec­
ropsy and collection of plastic identifiable by the naked eye (Donnel­
ly-Greenan et al., 2014; Provencher et al., 2017). In contrast, to

☆
This paper has been recommended for acceptance by Professor Christian Sonne.
* Corresponding author.
E-mail address: myraf@ucsc.edu (M. Finkelstein).

https://doi.org/10.1016/j.envpol.2022.120548
Received 28 June 2022; Received in revised form 8 October 2022; Accepted 28 October 2022
Available online 4 November 2022
0269-7491/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
S. Michishita et al.
document ingested microplastic, methods typically require digestion of
the full digestive tract and collection of particles under a microscope
(Provencher et al., 2019). Standardized procedures to quantify ingested
microplastics have been recently proposed (Lusher and
Hernandez-Milian, 2018), including the use of chemical analysis for
definitive identification of microparticles as plastic (Provencher et al.,
2017). For example, spectroscopic polymer identification methods like
Fourier-transform infrared (FT-IR) and Raman spectroscopy are quan­
titative techniques to analyze particle composition and can improve the
understanding of abundance, sources, and toxicity of microplastics in
environmental samples (Schmid et al., 2021).
In terms of toxicity associated with plastic pollution, plastic can
adsorb persistent organic pollutants (e.g., polychlorinated biphenyls)
(Tanaka et al., 2013; Teuten et al., 2009) and thus is considered a
pathway for exposure to toxic chemicals in marine systems (Tanaka
et al., 2019). However, the plastic matrix is composed of numerous
potentially toxic compounds including the structural plastic polymer (e.
g., polyester, polystyrene) and additive chemicals (e.g., plasticizers,
flame retardants) that can leach into tissues when ingested (Koelmans
et al., 2014; Tanaka et al., 2015). One concern is that many
plastic-associated chemicals are xenoestrogenic and thus able to mimic
estrogen by binding to nuclear estrogen receptors and disrupt physio­
logical functions (Jenssen, 2006; Wang et al., 2021; Yang et al., 2011).
Nonetheless, little is known about the estrogenic activity of microplastic
ingested by marine species (Provencher et al., 2020).
The overall goal of our study was to provide information about the
prevalence, composition, and estrogenic activity of microplastic in the
Monterey Bay (California, USA), a highly valuable economic and pro­
ductive ecosystem (National Ocean Service, n.d.). As seabirds are
well-established indicators of marine plastic pollution (Van Franeker
and Law, 2015), we used common murres (Uria aalge, murre), an
abundant resident seabird of the Monterey Bay (Roth et al., 2008), to
investigate the prevalence, composition, and estrogenic activity of
ingested microplastic. To examine a probable pathway for microplastic
exposure to murres, we assessed microplastic prevalence and composi­
tion ingested by northern anchovies (Engraulis mordax, anchovy), a
Monterey Bay resident filter feeder and main component of the murre’s
diet (Roth et al., 2008). We also evaluated the prevalence and compo­
sition of microplastic in seawater samples from two locations in the
Monterey Bay. Specifically, we quantified the prevalence and compo­
sition of anthropogenic microparticles in 1) seawater samples, 2)
digestive tracts of anchovies, and 3) digestive tracts of murres. A sub-set
of recovered microparticles were analyzed by Raman spectroscopy to
determine what proportion of collected microparticles were micro­
plastic as well as the plastic’s specific chemical composition and poly­
mer type. To investigate the potential toxicity of ingested microplastic,
we measured the estrogenic activity of microparticles recovered from
murre digestive tracts with an in-vitro estrogen-receptor activation
assay. Taken together, our study provides important information about
the prevalence, composition, and potential estrogenic activity of
microplastic in a coastal system.
2. Materials and methods
2.1. Sample collection
Samples were collected within the Monterey Bay, located in coastal
central California, USA (Fig. 1) and along a portion of the Pacific
Flyway, an important migration thoroughfare for a multitude of bird
species (National Ocean Service, n.d.). The Monterey Bay is a well-mixed
oceanographic system due to strong upwelling and horizontal circula­
tions of the California Current System (Tseng et al., 2005).
2.1.1. Seawater sampling periods
Microparticles were collected from seawater intake systems at the
University of California, Santa Cruz Long Marine Laboratory (Santa
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Cruz, CA, USA) (August 2020: 4 sampling periods,1 May 2021: 2 sam­
pling periods, July 2021: 2 sampling periods) and Moss Landing Marine
Laboratories (Moss Landing, CA, USA) (July 2021: 4 sampling periods)
following methods modified from Mason et al. (2016). Access points
were located after the intake system’s initial filtration for sand: Long
Marine Laboratory’s vertical filter size was 0.7 cm and Moss Landing
Marine Laboratories’ mesh size was 0.02 cm. At each sampling period (n
= 12), seawater gently flowed through two stacked steel sieves, where a
500 μm-mesh sieve was on top a 150 μm-mesh sieve for 17 h overnight.
The time duration to fill a 1-L glass beaker was measured in triplicate
before and after each 17 h sampling period and used to estimate the
volume of seawater sampled. The average of the six readings (sec/L) was
used to calculate flow rate (L/sec) and estimate the total volume (L)
sampled. Sieves were covered with a bucket to protect from weather and
reduce atmospheric contamination, as even sea spray can contain
microplastic (Brahney et al., 2021). Sieves were retrieved in the morning
and covered with aluminum foil until processing.
2.1.2. Anchovy
Whole anchovies (n = 24) were generously provided by Ocean2T­
able, a sustainable Community Supported Fishery group based in Santa
Cruz, CA, USA. Anchovies were caught within a 100-mile radius from
Monterey, California by local fishing partners (Fig. 1) and stored at
− 20 ◦ C until processing.
2.1.3. Murre
Samples of whole digestive tracts (n = 19) were provided by Cali­
fornia Department of Fish and Wildlife (CDFW) Office of Spill Preven­
tion and Response (OSPR) Seabird Health Program at the Marine
Wildlife Veterinary Care and Research Center (MWVCRC) in Santa Cruz,
California USA. Freshly deceased murre carcasses were collected be­
tween July 2019 to November 2020 for the CDFW-OSPR Seabird Health
Program by USFWS BeachCOMBERS (Beach Coastal Ocean Mammal and
Bird Education and Research Surveys) Program volunteers, or the So­
ciety for the Prevention of Cruelty to Animals for Monterey Bay County
(MSPCA) Wildlife Rescue and Rehabilitation Center (Fig. 1). Murres
acquired from the MSPCA were dead on arrival and did not undergo
treatment prior to death. Murres were examined via systematic necropsy
by CDFW-OSPR MWVCRC Seabird Health Program personnel, and the
digestive tract was removed following described methods (Van Fra­
neker, 2004; Van Franeker and Meijboom, 2002). Digestive tracts were
stored in aluminum foil and frozen at − 20 ◦ C until processing.
2.2. Contamination control
Sample integrity and prevention of atmospheric contamination are
crucial to monitor when studying microplastics (Lusher et al., 2014;
Tanaka and Takada, 2016). Working areas were cleaned prior to use;
tools and equipment were triple rinsed with Milli-Q water and visually
checked under a dissecting microscope for particles before use. Lab coats
and gloves were worn and easily-shedding clothes (e.g. fleece, sweaters,
etc.) were avoided. Amber glass vials were baked in a muffle furnace at
450 ◦ C prior to use (i.e., kilned). A triple-rinsed 100 mL-glass beaker
filled with Milli-Q water (procedural blank) was placed at the working
station to monitor atmospheric contamination. Care was taken to
identify microparticle contamination during each sample processing
procedure. Filtration and chemical digestion processes were completed
in laminar hoods and in a class 100 clean room, respectively.
1
August 2020 samples were not included in analysis of particle/L due to the
sieves overflowing and our inability to determine the number of microparticles
recovered per L. August 2020 samples were included in the physical (particle
type) and chemical (polymer identification) composition analyses.
S. Michishita et al.
Fig. 1. Map of sample collection locations. A) From most northern to most southern sample collection. B) Magnified view of Monterey Bay region. Open square:
common murre collection sites, open triangle: seawater sampling sites, open dashed circle: northern anchovies were collected within boundary lines.
2.3. Sample processing and microparticle physical composition
Visual selection of anthropogenic microparticles from seawater
sampling periods, anchovy digestive tracts, and murre digestive tracts
followed guidelines by Lusher and Hernandez-Milian (2018) and con­
ducted using a dissecting microscope (American Optical Company
Model 570, total magnification 40X). Anthropogenic microparticles
were categorized as: fiber, fragment, foam, film, or bead (Lusher and
Hernandez-Milian, 2018). Unless verified as plastic by Raman spec­
troscopy, we refer to recovered anthropogenic microparticles as
‘microparticles’.
2.3.1. Seawater sampling periods
Milli-Q water was used to separate sediment, algae, and other natural
materials such as micro-invertebrates and shell fragments from
anthropogenic particles from each seawater sampling period. Using a
dissecting scope, anthropogenic microparticles were recovered from
sieves with clean forceps and stored in sterile amber glass vials.
2.3.2. Anchovy and murre
Digestive tracts were processed following Rochman et al. (2015)
with minor modifications. Specifically, in a class 100 clean room,
digestive tracts were placed in triple rinsed glass beakers (anchovies) or
500 mL kilned amber glass jars (murres) with 15 mL (anchovies) or three
times the volume (murres) of 20% potassium hydroxide (Sigma-Aldrich,
St. Louis, MO), a concentration that is not expected to alter plastic
polymer composition (Munno et al., 2017). Digestive tract samples were
loosely capped for 2–3 weeks at room temperature. Although heat is
often used in the alkaline tissue digestion process, we excluded this step
to reduce potential chemical leaching from the particles (Carlin et al.,
2020; Provencher et al., 2019). After the organic material was digested,
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Environmental Pollution 316 (2023) 120548
samples were filtered through a vacuum filtration apparatus with a 10
μm-pore size polycarbonate filter (MilliporeSigma™ Isopore™ Mem­
brane Filters). Using a dissecting scope, anthropogenic microparticles
were removed from the filter and stored in sterile amber glass vials using
clean forceps.
2.4. Microparticle chemical composition
A subset of microparticles (~20%) recovered from seawater samples
(n = 29/110 particles), anchovies (n = 20/31 particles), and murres (n
= 62/115 particles) were analyzed using Raman spectroscopy in the
Health Effects of Anthropogenic Litter Lab at the University of California
Davis School of Veterinary Medicine (Davis, CA USA) for chemical
identification and classification.
Analyses were performed using the LabSpec 6 software suite and an
XploRA™ PLUS Raman confocal microspectroscope, equipped with 785
nm and 532 nm monochromatic lasers, a cooled charge-coupled device
detector (1024 × 256 pixels), and an automated stage. Calibration of the
unit was performed using zero-order correction of the spectral peak at
520.7 cm− 1 of a reference silicon wafer for each grating and laser
combination prior to analysis. Targeted point analysis of microparticles
was performed using optimized acquisition parameters (laser wave­
length, laser power, grating, and acquisition time) for each micropar­
ticle to maximize spectral quality.
After acquisition, the spectrum for each target microparticle was
2
Six microparticles (one fragment, one foam, one film, and three fibers)
recovered from common murres were analyzed by Raman spectroscopy due to
logistical constraints. Dark-colored microparticles were intentionally excluded
to maximize the potential for polymer identification.
S. Michishita et al. Environmental Pollution 316 (2023) 120548

baseline corrected, matched to Raman spectral libraries of known 3. Results

polymers (including Wiley KnowItAll, SLoPP, and SLoPP-E) with visual
confirmation of key peak matches for identification. Each microparticle 3.1. Contamination control
was classified into one of the following polymer groups: natural, syn­
thetic, semi-synthetic, blends, dye-prominent, and unknown. Semi- Samples were not blank-corrected for atmospheric contamination as
synthetic polymers are derived from natural materials, but undergo 97% (34/35) of the procedural blanks were particle-free. One proce­
chemical processing (e.g. cellulose acetate and rayon). Blends referred dural blank from a seawater sampling period (LML04) contained a fiber
to mixtures of both synthetic and natural polymers, such as cotton that is that did not match the color, type, texture, or shape of the microparticles
coated or combined with polyester. One limitation to Raman spectros­ found in sample LML04. Four additional fibers were observed after
copy is that the spectrum of some dark dyes can mask the spectrum of initial sample processing (transfer of microparticles from vial to Petri
the underlying polymer, which make definitive identification difficult or dish) and thus not reported as results (Table S5).
ambiguous; in this case microparticles were classified as dye-prominent
(Schmid et al., 2021; Zhu et al., 2019). Microparticles that could not be 3.2. Microparticle prevalence
positively matched to known polymer spectra were classified as
unknown. 3.2.1. Seawater sampling periods
67 microparticles were recovered from approximately 39,000 L (n =
8 sample periods3), with a mean of 8 ± 1 S.E. microparticles per sam­
2.5. Estrogenic activity of microparticles from murres (n = 13 digestive
pling period or a mean of 1.9 ± 0.4 S.E. microparticles per 1000 L
tracts)
sampled (Table S1). The mean microparticles per 1000 L sampled from
Long Marine Laboratory and Moss Landing Marine Laboratories were
2.5.1. Incubation
not significantly different (1.8 ± 0.3 S.E. and 2.0 ± 0.8 S.E., respec­
Microparticles from murre digestive tracts were transferred to kilned
tively, Wilcoxon test, p = 0.69).
amber glass vials and placed under UV light in a laminar flow hood for
30 min to kill bacteria. We used two slightly different methods for
3.2.2. Northern anchovy and common murre
microparticle incubation for leaching of plastic-associated chemicals: 1)
At least one microparticle was recovered from 14 of the anchovy
Particles were incubated in 1.5 mL of 100% ethanol (EtOH, Fisher Sci­
digestive tracts (58%). Out of the digestive tracts with microparticles
entific, Pittsburgh, PA) for 7 days at 38 ◦ C in a temperature controlled
recovered, the abundance was 2.2 ± 0.5 per anchovy (mean ± S.E.,
shaker (New Brunswick Scientific Co., Inc. Series 25) at 100 rpm (n = 9;
median = 1, range 1–7, Fig. 2). Our results were consistent with the
Murre1-9), or 2) particles were incubated in 1.5 mL 100% EtOH over­
prevalence (30%, p = 0.26, Fisher’s exact test) and mean abundance (1,
night for 16 h in an orbital shaker at room temperature (n = 4; Murre16-
p = 0.18, Wilcox test) of particles found in anchovies caught offshore in
19). Samples were incubated in sets of three or four with an EtOH
Central California in 2014 (Rochman et al., 2015).
control blank (Coffin et al., 2019; Yang et al., 2011). Vials were tightly
At least one microparticle was recovered from 100% of murre
sealed with Parafilm and Teflon tape. Sample volume and incubator
digestive tracts (n = 19). The abundance was 6.1 ± 1.0 per bird (mean
temperature were checked daily (for 7-day incubation). After incuba­
± S.E., median = 5, range 1–17, Fig. 2). Murres had significantly higher
tion, microparticles were separated from the leachates and placed on
particle prevalence (p = 0.001, Fisher’s exact test right-tailed) and
double-sided tape adhered to a labeled transparent plastic film in a clean
abundance (p = 0.00012, Wilcoxon 2-way test) than anchovies (Fig. 2).
Petri Dish for analysis by Raman spectroscopy. Leachates (500 μL) were
dried down and concentrated under forced air in glass tubes before
3.3. Microparticle physical composition
resuspending in 50 μL of EtOH (concentration 10X).

Particle size was classified by measuring each particle’s longest

2.5.2. Estrogen receptor alpha (ERα) activation assay
dimension. Microparticles (seawater: n = 29, anchovy: n = 20) analyzed
Microparticle leachates were assessed for estrogenic activity using a
with Raman spectroscopy ranged from 119 μm to 4545 μm with a mean
human estrogen receptor alpha (ERα) activation assay previously
of 1383 μm ± 143 μm (S.E.). Fibers were the smallest and longest mi­
described in Felton et al. (2015, 2020) with minor modifications. Briefly,
croparticles recovered (Table S4). Due to logistical constraints, size was
human embryonic kidney cells (HEK293) were co-transfected with the
not measured for microparticles recovered from murres.
human estrogen receptor 1 gene (hESR1) in a pcDNA3.1+ expression
Fibers were the most common particle type recovered from the
vector (www.cdna.org), pCMX- β-galactosidase (β-gal) and pGL2-3xERE
seawater sampling periods, anchovies, and murres (80%, 71%, and 77%,
luciferase reporter plasmids (Addgene plasmid 11,354)(Hall and
respectively, Fig. 3). Seawater: 110 microparticles were recovered from
McDonnell, 1999). After 24 h cells were treated, in triplicate, with an
approximately 46,000 L (12 sampling periods4) (Fiber: 88, fragment: 11,
endogenous estrogen 17β-estradiol (E2: 10− 12-10− 7 M; Steraloids,
foam: 8, film: 3). Anchovy: 31 microparticles were recovered from 14
Newport, RI), BPA (10− 5 M; Sigma-Aldrich, St. Louis, MO), a vehicle
anchovies (Fiber: 22, fragment: 4, foam: 2, bead: 2, film: 1). Murre: 115
treatment of 0.1% DMSO, 0.1% EtOH control, or microparticle leachate.
microparticles were recovered from 19 murres (Fiber: 89, fragment: 20,
After the 24 h final incubation, cells were lysed to measure luciferase
foam: 5, film: 1). No beads were recovered from seawater sampling
and β-gal activity (Felton et al., 2020, 2015). Luciferase activity was
periods or murre samples.
standardized to β-gal activity, and the fold activation was measured by
normalizing to a vehicle (DMSO or EtOH). Final results were normalized
3.4. Microparticle chemical composition
to maximum E2 activation as determined by Graph Pad Prism Software
(San Diego, CA) (Felton et al., 2020, 2015).
Fifty-five microparticles from seawater sampling periods (n = 29
particles), anchovy digestive tracts (n = 20 particles), and murre
2.6. Statistical analysis digestive tracts (n = 6 particles) were analyzed with Raman spectros­
copy and classified as natural, synthetic, semi-synthetic, blends, dye-
Statistical analyses (Wilcoxon test and Fisher’s exact test) were prominent, and unknown polymers (Fig. 4). 33% of analyzed
performed with JMP Pro 16 and RStudio version (1.3.1093) to assess
differences in microparticle prevalence and abundance between the two
seawater intake locations as well as between anchovy and murre 3
Excludes August 2020 samples (n = 4, Refer to Methods).
digestive tract samples. Significance was determined by P-values < 0.05. 4
Includes August 2020 samples (Refer to Methods).

4
S. Michishita et al. Environmental Pollution 316 (2023) 120548

Fig. 2. Microparticle prevalence and abundance in anchovies and murres. A) Prevalence of microparticles in common murres (murre) was higher compared to
northern anchovies (anchovy; p = 0.001, Fisher’s exact test right-tailed). At least one microparticle was found in 57% of anchovies (n = 24) and 100% of murres (n =
19). B). Murres (n = 19) had higher mean abundance of ingested microparticles than anchovies (n = 14; p = 0.00012, Wilcoxon 2-Way Test). Tukey’s boxplot
comparing quantity of ingested microparticles found in the digestive tracts of anchovies and murres. Ends of the box represents lower (Q1) and upper (Q3) quartiles,
with median line bolded. Open triangle represents mean. Also refer to Tables S2, S3.

Fig. 3. Physical composition of microparticles

recovered from seawater sampling periods, an­
chovies, and murres. A total of 256 particles were
recovered from all samples: 110 particles from
~45,000 L (12 seawater sampling periods), 31 par­
ticles from 14 northern anchovies (anchovy) and 115
particles from 19 common murres (murre). Fibers
were the most dominant particle type followed by
fragments, foam, and film. Beads were observed in
one anchovy sample. Also refer to Tables S2 and S3.

microparticles were dye-prominent, 27% were natural polymers, 25% microparticles that could be identified (excluding dye-prominent and
were synthetic polymers, and 11% were partially synthetic polymers unknown) were plastic. No animal-based polymers (fur/hair, shells,
(semi-synthetic and blends) (Table S4) (Figure S2). 57% of bones, etc.) were detected, validating the visual processing protocol to

5
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6

Fig. 4. Chemical composition of microparticles recovered from seawater samples, anchovies, and murres. A subset of recovered microparticles (n = 55) was analyzed by Raman spectroscopy for chemical composition

Environmental Pollution 316 (2023) 120548

identification: 29 particles from seawater sampling periods, 20 particles from northern anchovies (anchovy), and 6 non-randomly selected particles from murres (refer to methods). A) Distribution of categorized polymer
bins based on chemical identification of microparticles. B-E) Selected Raman spectra: red line is spectrum of microparticle; black line is spectrum of matched polymer from spectral libraries (Wiley KnowItAll, SLoPP, and
SLoPP-E). Y-axis is intensity and x-axis is Raman effect wavelength (cm− 1). B) Natural fiber from seawater identified as cotton (cellulose). C) Synthetic foam from a murre identified as polystyrene. D) Synthetic fiber
from an anchovy identified as polyethylene terephthalate. E) Dye-prominent fiber from seawater. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of
this article.)
S. Michishita et al.
recover anthropogenic particles (Lusher and Hernandez-Milian, 2018).
3.4.1. ERα activation assay: murre (n = 13)
Leachates of microparticles recovered from three murre digestive
tracts (Murre3, 4, 9) activated at 27%, 32%, and 31% of maximal E2
activation, respectively, which were 2.9-, 3.5-, 3.3-fold over their
respective EtOH controls (Fig. 5). Murre5 activated at 14% of maximal
E2 activation, which was 1.5-fold over its EtOH control. Leachates from
the overnight incubation (Murre16-19) did not exhibit estrogenic ac­
tivity relative to the EtOH control (Fig. S1).
4. Discussion
We found a similar composition of anthropogenic microparticles in
the digestive tracts of anchovies and murres as well as the seawater
sampling periods (Fig. 3, Table S2; Table S3) from the Monterey Bay,
California USA. Although we cannot conclude that microplastics
recovered from murres were due to trophic-level transfer (i.e., ingestion
of anchovies), findings of similar spectra of synthetic fibers in anchovies
and murres support a potential trophic transfer exposure pathway
(Provencher et al., 2018a). Other studies have shown evidence for tro­
phic transfer of microplastic, as well as of plastic-associated chemicals
(Bang et al., 2021; Tanaka et al., 2013), to apex predators (e.g., grey
seals, Halichoerus grypus) (Nelms et al., 2018) and humans (Van Cau­
wenberghe and Janssen, 2014). Thus, the trophic transfer of micro­
plastics in the Monterey Bay seems plausible.
4.1. Microparticle prevalence in seawater, anchovy, and murre
4.1.1. Seawater sampling periods
We recovered microparticles from all seawater sampling periods and
found no significant difference in mean number of microparticles per
1000 L between our two seawater collection sites, despite the 35-fold
difference in intake filter sizes (Long Marine Laboratory: 0.7 cm; Moss
Landing Marine Laboratories: 0.02 cm). We found fewer median mi­
croparticles per liter (median = 0.002 particles/L) compared to samples
collected at 5 m depth nearshore and offshore of Monterey Bay [median
Fig. 5. Estrogenic activity measured from microparticles recovered from murre
digestive tracts. Three common murre particle leachates (Murre3, 4, 9) (▾)
stimulated 27%–32% maximal activation of human estrogen receptor alpha
(ERα) relative to activation from 17β estradiol (E2). Murre5 stimulated 14%
maximal activation of E2. Data represent mean ± S.E. of fold activation relative
to maximal E2 activation. E2: endogenous estrogen, BPA: bisphenol-A (positive
control), EtOH blank: average of three ethanol incubation blanks. Also refer to
Figure S1 and Table S2.
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Environmental Pollution 316 (2023) 120548
of 2.9 particles/L (Choy et al., 2019)], a similar depth as the intake to the
systems we used. While there are many factors such as abiotic conditions
to consider, we suspect our lower median of particles/L is due in part to
our upper size limit from operational boundaries of the seawater intake
systems’ sand filters. Indeed, the Moss Landing site’s intake system
limits recovery of microparticles above ~2 mm in size. One of the many
obstacles in synthesizing microplastic data is the ability to compare
studies due to operational limitations. Similar to others (Miller et al.,
2021; Provencher et al., 2017), we recommend the adoption of stan­
dardized methods and reporting to aid in the assessment of spatial and
temporal trends of microplastic in marine systems.
4.1.2. Anchovy
In the Monterey Bay, anchovies are ecologically valuable to pisci­
vores such as brown pelicans, endangered marbled murrelets, and
common murres (MacCall et al., 2016). For murres, anchovies comprise
the third highest biomass of the adult diet and the highest biomass of the
chick diet (Koehn et al., 2016; Roth et al., 2008). In terms of economic
value, anchovies contributed $1 million in average revenue in the US
West coast between 2004 and 2013 and continue to be recreationally
and commercially fished in the Monterey Bay (Koehn et al., 2016). Thus,
due to the high ecological and economic importance of anchovies in the
Monterey Bay, our finding that almost 60% of anchovies sampled con­
tained microparticles, of which 50% were identified as plastic
(Table S4), is concerning as well as consistent with data from anchovies
collected in 2014 (Rochman et al., 2015).
4.1.3. Murre
Over 200 seabird species have been reported to ingest macroplastic,
primarily following methods with visual and tactile cues for particle
selection using a minimum of 1 mm mesh sieves (Bond et al., 2013; Kühn
and van Franeker, 2020) as opposed to the alkaline tissue digestion and
smaller pore size filters (e.g. 10 μm) recommended for microplastic
quantification (Rochman et al., 2017). Previous studies in murres
following macroplastic ingestion protocols resulted in a cumulative 6%
macroparticle prevalence (Kühn and van Franeker, 2020) compared to
our findings of 100% microparticle prevalence. The higher prevalence of
plastic ingestion using micro-versus macro-quantification was also
found in flesh-footed shearwaters (Ardenna carneipes) with a prevalence
of 50% [n = 638, 8 studies (Kühn and van Franeker, 2020)] using
macroplastic ingestion protocols compared to 92% (n = 57) using
microplastic digestion protocols (Lavers et al., 2019). Our results,
similar to the results for flesh-footed shearwaters (Kühn and van Fra­
neker, 2020, Lavers et al., 2019), indicate that quantifying plastic
ingestion in seabirds using macroplastic detection methods might sub­
stantially underestimate plastic exposure. We encourage the use of tissue
digestion of the entire digestive tract to quantify plastic (macro- and
micro-) ingestion rates in seabirds.
4.2. Microparticle physical and chemical composition
Consistent with other marine microplastic studies (Mason et al.,
2016; Provencher et al., 2018b; Rochman et al., 2015), fibers were the
most common particle type recovered (seawater sampling periods: 80%,
anchovy: 71%, murre: 77%, Fig. 4). Of the 25 fibers analyzed using
Raman spectroscopy (excluding dye-prominent), ~50% were either
fully or partially composed of synthetic polymers and out of these ~70%
were polyester (Fig. 4, Table S4). The high proportion of fibers found in
our samples is not surprising as the textile industry is considered a large
contributor of plastic fibers to the environment with ~40 million tons of
synthetic fibers, of which polyester is the most common, produced
annually (Carr, 2017). Further, a single polyester clothing item can
release over 1900 fibers from one wash in a laundry machine and
wastewater treatment plant effluent is commonly discharged to marine
systems (Browne et al., 2011; McIlwraith et al., 2019; Vassilenko et al.,
2021). Laundry machine filter attachments have proven effective at
S. Michishita et al.
reducing the outflow of fibers in wastewater by 87% (McIlwraith et al.,
2019), illustrating how simple preventative actions can help decrease
the amount of microplastic entering the marine environment.
We emphasize the recommendation by other studies to use Raman
(or FT-IR) spectroscopy to identify particle composition when con­
ducting microplastic research (Miller et al., 2021). While there are some
limitations to Raman and FT-IR spectroscopy (cost, detectable particle
size, dye interference), visual categorization is a less accurate method of
plastic identification (Araujo et al., 2018; Schmid et al., 2021). Identi­
fication of specific polymers can provide important information with
respect to the relative abundances and possible sources of microplastic
in the environment (Choy et al., 2019).
4.3. Estrogenic activity of microparticles ingested by murres
To our knowledge, we are the first to investigate the potential es­
trogenic activity of anthropogenic microparticles recovered from
seabird digestive tracts. The number and composition of microparticles
recovered from the murre digestive tracts did not appear to correspond
to activation of the in vitro estrogen receptor (ERα) (Table S2). For
example, three out of 13 samples (Murre3, 4, 9) exhibiting the highest
levels of ERα activation (27%–32% max activation relative to endoge­
nous E2) did not have the highest observed number of particles (Fig. 5,
Table S2). Murre1 had the highest number of microparticles (n = 16),
but exhibited low estrogenic activity, while Murre9 had only two mi­
croparticles but exhibited high estrogenic activity (Fig. 5, Table S2).
Noteworthy is that one foam particle from Murre3 with estrogenic ac­
tivity was identified as polystyrene (Fig. 4C), a known xenoestrogen
(Coffin et al., 2019; Hwang et al., 2020; Yang et al., 2011). Our detection
of estrogenic activity from microparticles ingested by murres is con­
cerning as even low doses of exposure to xenoestrogens can lead to
adverse effects (Wang et al., 2021).
5. Conclusion
Our findings demonstrate the widespread occurrence of microplastic
in the Monterey Bay with ~60% of anchovies and 100% of murres
having anthropogenic microparticles in their digestive tract. Using
Raman spectroscopy, we determined that ~60% of microparticles were
plastic. The similar composition (e.g. proportion of fibers, foam, etc.) of
microparticles in the digestive tracts of anchovies and murres suggest a
potential trophic transfer of microparticles in the Monterey Bay. We also
show that microparticles from ~25% of the murre digestive tracts
exhibited estrogenic activity, illustrating the potential harm of micro­
plastic ingestion. Overall, our study underscores the concern of micro­
plastic pollution in coastal systems. We emphasize the importance of
monitoring microplastic prevalence, composition, and potential adverse
effects to assess risk and inform waste reduction practices.
Author statement
We will provide additional underlying data or methods not listed in
the main text or supplementary materials upon request.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data not provided in the Supplementary Information will be made
available on request.
8
Environmental Pollution 316 (2023) 120548
Acknowledgments
We thank Dr. Chelsea Rochman at University of Toronto for sharing
tissue digestion protocols and comments on a manuscript draft. We also
thank Colin Carney, assistant specialist at the UCSC Stable Isotopes
Laboratory, Daniel Gossard at Moss Landing Marine, and Brooke Doblar,
a UCSC undergraduate student, for field and laboratory assistance. We
thank Julianna M. Ramirez for assisting Sami Michishita in coding in
RStudio. We would also like to thank BeachCOMBERS volunteers, staff
at the MSPCA Wildlife Rescue and Rehabilitation Center for carcass
collection and coordination, and Katherine Greenwald for necropsy
support at CDFW-OSPR MWVCRC. Funding: This work was supported by
the Friends of Seymour Center for the Student Research and Education
Award (2019), UC Santa Cruz Re-entry student scholarship (2020 &
2021), and NorCal Society of Environmental Toxicology and Chemistry
Research Scholarship Award (2021).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.envpol.2022.120548.
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