Environmental Pollution 233 (2018) 1113e1124

Contents lists available at ScienceDirect

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

Molecular identification of polymers and anthropogenic particles

extracted from oceanic water and fish stomach e A Raman micro-
spectroscopy study*
Sutapa Ghosal*, Michael Chen, Jeff Wagner, Zhong-Min Wang, Stephen Wall
California Department of Public Health, Environmental Health Laboratory Branch, 850 Marina Bay Parkway, Richmond, CA 94804, USA

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

Article history: Pacific Ocean trawl samples, stomach contents of laboratory-raised fish as well as fish from the sub-
Received 20 March 2017 tropical gyres were analyzed by Raman micro-spectroscopy (RMS) to identify polymer residues and any
Received in revised form detectable persistent organic pollutants (POP). The goal was to access specific molecular information at
26 September 2017
the individual particle level in order to identify polymer debris in the natural environment. The iden-
Accepted 5 October 2017
tification process was aided by a laboratory generated automated fluorescence removal algorithm. Pacific
Available online 13 October 2017
Ocean trawl samples of plastic debris associated with fish collection sites were analyzed to determine the
types of polymers commonly present. Subsequently, stomach contents of fish from these locations were
Keywords:
Microplastic identification
analyzed for ingested polymer debris. Extraction of polymer debris from fish stomach using KOH versus
Microplastic extraction ultrapure water were evaluated to determine the optimal method of extraction. Pulsed ultrasonic
Raman micro-spectroscopy extraction in ultrapure water was determined to be the method of choice for extraction with minimal
Marine litter chemical intrusion. The Pacific Ocean trawl samples yielded primarily polyethylene (PE) and poly-
Fish stomach contents propylene (PP) particles >1 mm, PE being the most prevalent type. Additional microplastic residues
(1 mm - 10 mm) extracted by filtration, included a polystyrene (PS) particle in addition to PE and PP.
Flame retardant, deca-BDE was tentatively identified on some of the PP trawl particles. Polymer residues
were also extracted from the stomachs of Atlantic and Pacific Ocean fish. Two types of polymer related
debris were identified in the Atlantic Ocean fish: (1) polymer fragments and (2) fragments with com-
bined polymer and fatty acid signatures. In terms of polymer fragments, only PE and PP were detected in
the fish stomachs from both locations. A variety of particles were extracted from oceanic fish as potential
plastic pieces based on optical examination. However, subsequent RMS examination identified them as
various non-plastic fragments, highlighting the importance of chemical analysis in distinguishing be-
tween polymer and non-polymer residues.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction effects of physical contamination as documented by wildlife

Plastic contamination of the environment and our food chain
represents an area of growing environmental concern (Teuten et al.,
2009). There is great urgency in understanding the extent and the
potential effects of this contamination (Arthur et al., 2008). For
instance, plastic debris have been detected in broad regions of the
marine environment as well as in marine wildlife, as documented
in several recent publications (Andrady, 2011; Eriksen et al., 2014;
Cozar et al., 2014; Wright et al., 2013). Apart from the obvious
*
This paper has been recommended for acceptance by Eddy Y. Zeng.
* Corresponding author.
E-mail address: sghosal@cdph.ca.gov (S. Ghosal).
entanglement and smothering, plastic debris represent a source of
chemical contamination in the environment (Gall and Thompson,
2015; Koelmans et al., 2016). Polymers can be a direct source of
chemical pollutants such as flame retardants and can concentrate
pollutants present in the environment through adsorption (Bakir
et al., 2014a). Hence, chemical identification of the plastic debris
and any associated chemicals is necessary for understanding the
nature and potential effects of this contamination such as carci-
nogenesis and endocrine disruption (Oehlmann et al., 2009;
Talsness et al., 2009).
Current approaches to polymer identification in environmental
samples involve sample collection and preparation followed by
identification of the sample composition (Rocha-Santos and Duarte,

https://doi.org/10.1016/j.envpol.2017.10.014
0269-7491/© 2017 Elsevier Ltd. All rights reserved.
1114 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124
2015; Hidalgo-Ruz et al., 2012; Qiu et al., 2016; Vandermeersch
et al., 2015). Identification of polymer debris typically relies on one
of the following methods e 1) visual inspection of the sample with
or without the aid of a microscope, 2) chemical identification of the
constituents using spectroscopic methods such as Raman micro-
spectroscopy (RMS) and/or Fourier transform infra-red spectros-
copy (FTIR) (Song et al., 2015; Collard et al., 2015). Plastics debris in
the marine environment are typically subject to biofouling and
degradation which can alter the physical appearance of the poly-
mer surface significantly, thereby complicating the process of visual
identification. Spectroscopic identification offers insights into the
chemical nature of the particles, thereby minimizing potential
misidentification of polymers based on visual inspection alone. The
measured chemical spectrum is typically a composite of all spec-
troscopically active ingredients including the polymer and any
additional constituents. This is relevant since the associated
chemicals can be important sources of contamination in the marine
environment (Wardrop et al., 2016; Rochman et al., 2013). Spec-
troscopic identification not only enables differentiation between
polymer and natural particles with identification of the exact
polymer type, but may also provide insight into the chemistry of
any associated additives.
In recent years, FTIR micro-spectroscopy has dominated the
spectroscopic identification of polymer debris and related items in
the marine environment (Harrison et al., 2012). In contrast, Raman
micro-spectroscopy based assessment of marine polymer residues
has been less extensive. RMS offers spatially resolved composi-
tional information at the micrometer scale, and is suitable for non-
invasive molecular identification/characterization of small plastic
residues in the bio/marine environment (Lenz et al., 2015). It is
typically characterized by minimal interference from water, thus
making it suitable for analyzing biological samples. In addition,
RMS is a nondestructive technique requiring minimal sample
preparation. As such, we have used Raman micro-spectroscopy to
examine polymers and anthropogenically derived chemicals in
laboratory based fish and select samples from the marine
environment.
Raman spectroscopy of biological samples is often accompanied
by a broad auto-fluorescence background. This broad background is
typically of no quantitative interest and tends to overshadow the
less intense Raman peaks, thereby complicating the identification
process. Given the abundance of biological material in ocean
samples, the removal of intrinsic auto-fluorescence background
from individual spectrum was of major concern for the identifica-
tion of potential plastic residues. In this work, we have imple-
mented an automated algorithm to remove the fluorescence
background from RMS spectra to enhance Raman peak identifica-
tions (Lieber and Mahadevan-Jansen, 2003). Extraction of polymer
debris from laboratory based fish is briefly examined here to
demonstrate the optimal method of polymer extraction and the
acquisition of relevant RMS spectra. The extraction procedure is
explored in further detail by Wagner et al. (2016). Subsequently,
marine debris and fish from the marine environment are examined
to assess the applicability of RMS in the identification of plastic and
associated debris in the marine environment.
2. Experimental
2.1. Samples
Four different types of sample were examined in this study to
explore the application of Raman micro-spectroscopy in identifying
polymer debris in the marine environment. These included 1)
reference polymer particles added to the diet of laboratory based
fish, 2) laboratory based fish exposed to microplastic diet, 3) trawl
particles from the North Pacific Ocean, and 4) fish guts from the
North Pacific and South Atlantic Oceans.
Two sets of laboratory-based freshwater Japanese medaka
(Oryzias latipes) were used to develop the extraction method e an
optimal procedure for the extraction of plastics from fish stomach.
The fish were obtained from a laboratory population at the School
of Veterinary Medicine at University of California, Davis (UCD).
Care, maintenance, handling, and sampling of the fish followed
protocols that were in accordance with and approved by the UCD
Animal Care and Use Committee. The fish were exposed to purified
casein fish diet mixed with several common microplastics - poly-
vinyl chloride (PVC), polyethylene (PE), polypropylene (PP), poly-
styrene (PS), polyethylene terephthalate (PET) and a mixture of
anthropogenic fibers from laundry lint. While the concentration of
microplastics added to the diet was not specifically quantified, it
was sufficiently higher than what is typically expected in the nat-
ural environment to ensure intake by the fish and their subsequent
identification.
Surface trawl samples from the North Pacific Ocean were
examined for the presence of polymer debris in oceanic water
(Gassel et al., 2013). The trawl samples were collected for a duration
of 1 h each in a 333 mm mesh net for two days in August 2009 and
were stored frozen in large glass jars. In addition, gastrointestinal
(GI) tracts of oceanic lantern fish from the family Myctophidae were
received from two different locations e South Atlantic Ocean and
North Pacific Ocean. Six of these myctophid fishes were collected in
NovembereDecember 2010 via manta trawl on a sailing trip led by
5Gyres across the South Atlantic Ocean (Rochman et al., 2014).
Another six myctophid fishes were from Project Kaisei, collected via
manta trawl in August 2009 during a research cruise in the North
Pacific Ocean (Gassel et al., 2013). The average length and mass of
the analyzed Pacific fish were 9.2 ± 0.5 cm and 7.5 ± 0.9 g,
respectively (mean and standard deviation). The average length
and mass of the analyzed Atlantic fish were 9.3 ± 1.3 cm and
12 ± 3.9 g, respectively. Contents of the GI tracts were examined for
the presence ingested polymers and any associated primary organic
pollutants (POPs).
2.2. Sample extraction from fish and oceanic particulate debris
Initially, laboratory raised Japanese medaka gut samples were
received as two separate suspensions in e 1) deionized (DI) water
and 2) 10% potassium hydroxide (KOH) solution. For these samples,
the entire GI tract was used for microplastic extraction. GI samples
suspended in DI water were analyzed either as an air-dried drop on
quartz slide or as particles filtered onto a 0.8 mm pore size poly-
carbonate (PC) filter. The KOH suspended sample was neutralized
using 10% nitric acid (HNO3) solution prior to filtration onto a PC
filter, in order to prevent damage to the filter surface from the basic
solution. The neutralized sample was subsequently filtered onto
0.8 mm pore-size PC filters for analyses. The polymer particles used
to spike the diet in this case were 200e2000 mm PET, PE, PP par-
ticles and 50e150 mm PVC particles. A second set of laboratory
medaka suspended in ultrapure water was processed using pulsed
ultrasonic extraction (PUE) to remove ingested particles from the
stomach, which were then filtered onto 10 mm pore-size PC filters
for analysis (Wagner et al., 2016). The PUE extracted solution was
separated into three portions e top, middle and bottom e to
identify any density related distribution of the extracted micro-
plastics in the water column. Each of the three portions was filtered
individually onto a PC filter. In this case, polymer particles of sizes
100e300 mm for PE, PS, PVC, PET and 400e800 mm for PP were
used. The microplastic particles used to spike the diet were also
analyzed separately to determine their size and spectral identifi-
cation. The particles were mounted on double-sided adhesive
 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124
carbon tab on a standard SEM (Secondary Electron Microscope)
stub for analysis.
Two surface trawl samples from the North Pacific Ocean were
analyzed for evidence of plastic contamination in the ocean. These
samples were extracted both as particles >1 mm using a 1 mm
stainless steel sieve and as <1 mm fraction collected on 25 mm,
10 mm pore size PC filter. The manually extracted particles >1 mm
were mounted onto seven SEM stubs with double-sided adhesive
carbon tabs for analysis. There were 2e5 particles per stub for a
total of 30 particles. The <1 mm filtered fraction was analyzed
directly on the PC filter for evidence of polymers.
Ocean-based lantern fish of the family Myctophidae were
analyzed to assess the ingestion of polymer fragments by oceanic
fish. The fish were sampled from two different locations in the
ocean. Gut samples were received individually frozen and were
stored in the freezer prior to analysis. Each gut was dissected for
analysis using a clean stainless-steel scalpel blade and was visually
screened under a stereozoom microscope for ingested polymer
particles. Rigid particles >1 mm in size and resembling polymer
material were set aside in a clean glass Petri dish for further anal-
ysis. The dissected stomach was suspended in ultrapure water in a
clean glass vial for PUE procedure, which had been identified as the
method of choice for the extraction of particles from fish stomach
(Wagner et al., 2016). The PUE extracted stomach contents were
subsequently filtered through a 1 mm screen onto 10 mm PC filters,
thereby screening for both >1 mm particles as well as 1 mme10 mm
fraction. The manually extracted individual particles as well as the
PUE extracted stomach contents were analyzed by RMS. In the case
of 1 mme10 mm PUE fraction extracted on PC filter, a portion of the
filter (~1 mm2) was cut and mounted onto a SEM stub with double-
sided adhesive carbon tab for analysis. Additional details of sam-
pling and extraction methods pertaining to the fish and the trawl
samples analyzed in this study can be found in Wagner et al. (2016)
Special precautions were taken to minimize potential airborne
contamination of the samples. Filtrations as well as drying of
cleaned filtration equipment were performed in a cleanroom rated
at class 2k (less than 2000 particles > 0.5 mm per cubic foot), which
was equipped with particle-free ventilation hoods with HEPA
(high-efficiency particulate air) filtered air curtains. Glassware and
the filtration unit were cleaned in the clean room as well. The
cleanroom was equipped with an optical particle counter (LasAir II,
Particle Measuring Systems) to monitor the airborne particulate
concentration and typically yielded no detectable airborne
particles > 0.5 mm per cubic foot. In addition, laboratory blanks
were periodically analyzed for possible airborne contamination,
which was found to be negligible.
2.3. Optical microscopy
A reflected-light stereozoom microscope (Leica S8APO) with
digital CCD camera (Leica MC170 HD) was used for initial docu-
mentation of the samples prior to RMS analysis. Optical microscopy
was conducted at 6.3X e 80X with a minimum resolution of 10 mm.
2.4. Confocal Raman micro-spectroscopy
Raman spectroscopic measurements were performed using two
micro-Raman setups e 1) Renishaw inVia Raman microscope
(Renishaw Plc., Old Town, Wottonunder-Edge, Gloucestershire,
U.K.), and 2) Senterra Dispersive Raman microscope (Bruker Optics
Inc., Billerica, MA). Both instruments are equipped with near-
infrared 785 nm diode lasers, which were used for these mea-
surements. Samples were excited using 1e100 mW of laser light
focused onto the sample through a 20xe100x objective with signal
acquisition times of 10e60 s per measurement. In case of the
1115
Renishaw system, GRAMS WiRE software package (Galactic In-
dustries Corp., 395 Main St., Salem, NH) was used for instrument
control and data acquisition. The spectral resolution was ~2.5 cm 1.
On the Senterra Raman microscope, all spectra were recorded and
analyzed using OPUS 7.5 software (Opus Software Inc., San Rafael,
CA) with a spectral resolution of ~3e5 cm 1. Wavelength calibra-
tion was performed periodically using neon laser light. A silicon
wafer spectrum was acquired on a daily basis as a quality control
step to check and correct for minor wavelength offsets. Raman
spectra of polymers used in the medaka feeding experiments were
acquired as reference spectra for commercially available polymers.
Raw spectra were processed to eliminate dark noise and con-
tributions from the spectrometer's optical parts. Each spectrum was
baseline corrected using a laboratory built baseline correction
method (see below) and smoothed using a Savitzky-Golay filter. For
spectral identification both a commercial and an in-house library
were used. The spectral library searches were performed using an
automated correlation based search algorithm (GRAMS/AI version
9.00 R2) in order to compare and determine similarities between
the sample and the various library spectra. Within this algorithm
the quality of the spectral match is determined based on the value
of the Hit Quality Index, wherein a value of 0 corresponds to a
perfect match. The final spectral identification was based on indi-
vidual evaluation of each spectrum, wherein the sample spectrum
was identified as a polymer if it contained the characteristic bands
of the suspected polymer. The automated correlation values served
as an additional indicator. In addition to the polymer identification,
each spectrum was also analyzed for additional constituents e.g.
flame retardants.
2.5. Data analyses
As mentioned earlier, Raman spectra of samples associated with
biological material are frequently accompanied by broad auto-
fluorescence backgrounds which tend to complicate the identifi-
cation process. Zhao et al. have proposed an automated algorithm
to remove this broad fluorescence signal, so as to reveal any po-
tential Raman peaks in the processed spectra (Zhao et al., 2007).
The algorithm builds upon the modified multi-polynomial fitting
method (ModPoly) developed by Lieber et al., which iteratively fits
a polynomial function to a spectrum's fluorescence background and
thereby subtracts the auto-fluorescence signal (Lieber and
Mahadevan-Jansen, 2003). A slightly modified in-house MATLAB
GUI implementation of this automated algorithm for broad back-
ground subtraction was adapted in this study in order to remove
the auto-fluorescence background and thereby expose any poten-
tial Raman peaks. In addition, the GUI implementation also
included a smoothing option using a Savitzky-Golay filter to aid the
identification process. The source code for the procedure is avail-
able at https://github.com/michaelstchen/modPolyFit. Fig. 1a
shows the spectra of a manta trawl particle before and after MAT-
LAB GUI processing. Using the processed spectrum, the particle was
identified as PP with additional peaks for phthalocyanine dye.
3. Results
3.1. Extraction and identification of microplastics in laboratory
based Medaka fish
Five polymers - PE, PS, PVC, PET, and PP ewere used as surrogate
microplastic particles to spike the diet of laboratory based medaka
fish. All five polymers have distinct spectral signatures (Fig. 1b)
which were used to identify the microplastic residues. RMS spectra
of virgin microplastic particles and microplastic particle mixed
with fish diet were found to be identical, thereby confirming the
1116 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124
Fig. 1. RMS spectra of microplastic particles. (a) Spectrum of oceanic polymer particle
before and after MATLAB GUI processing. The processed spectrum (black) identifies the
particle as PP (blue). (b) Processed spectrum of known microplastics showing their
unique spectral signatures. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
polymer identity of these particles even when mixed with fish food.
This validated the applicability of using the polymer spectra to
screen for potential microplastics in fish diet. However, prior to
identification, the presence of auto-fluorescence background due to
residual biomaterials on the particles had to be addressed using the
automated background subtraction procedure mentioned
previously.
Fig. 2 presents RMS characterization of medaka fish gut contents
suspended in DI water and analyzed as an air-dried droplet on a
quartz slide. Dispersed particles of varying sizes can be seen on an
optical image of the prepared sample (Fig. 2a). RMS analyses and
subsequent molecular identification of representative particles
identified the presence of PVC and PET microplastics, which
represent only two of the overall plastic types added to the fish diet.
In addition to the microplastics, other identified particles included
plasticizers, cellulose as well as indigo fiber (jeans material) which
was most likely from the lint material added to the food. Some of
the particles were identified as a mixture of cellulose and plasti-
cizer (Fig. 2f). The combined presence of cellulose and plasticizers
may be indicative of the release of plasticizers from the ingested
polymers adsorbing onto other contents in the fish gut.
A portion of the medaka gut suspended in DI water was also
filtered using a PC filter and the filter surface was analyzed for the
evidence of ingested microplastics. Compositions of the identified
particles on the filter were similar to that of the air-dried droplet
sample discussed above. PVC and PET were the only observed
microplastic in this medaka gut.
The contents of a medaka gut digested in 10% KOH solution were
analyzed for comparison. The KOH digestion process is expected to
remove a large portion of the biological residues, thereby making it
easier to extract the microplastic particles. For instance, cellulose is
expected to be digested by the KOH treatment and in fact no cel-
lulose material was detected in the KOH treated sample unlike the
water suspended gut sample discussed previously. Removal of most
of the biological residues through digestion is expected to make it
easier to identify the microplastic particles. Spectral analyses of the
digested and subsequently filtered gut contents revealed the
presence of PVC and PET particles, same as in the previous case.
However, exposure to 10% KOH solution appeared to generate a
significant amount of residual debris which impacted the overall
sample. For instance, Fig. 3aeb shows medaka gut extracted in
water versus 10% KOH solution. The medaka gut extracted in KOH
had attendant residual material which was not present in the water
extracted sample. Furthermore, to determine the impact of KOH on
the polymer particles, virgin PE particles exposed to water or to10%
KOH solution were examined. The particles exposed to 10% KOH
solution appeared degraded with residual material surrounding the
particles, implying possible dissolution of the microplastic surface
(Fig. 3ced). SEM/EDS (Scanning Electron Microscopy/Energy
Dispersive X-Ray Spectroscopy) analyses of microplastics exposed
to KOH treatments of medaka gut revealed fine particulate coatings
and strong potassium peaks (Wagner et al., 2016). Such modifica-
tion of the polymer surface residue is likely to interfere with the
detection of potential POPs on the polymer surface. For both the
medaka guts discussed so far, PVC was the most abundant micro-
plastic identified. It is possible that the predominance of PVC par-
ticles may be indicative of preferential ingestion of these particles
due to their relatively smaller size (50e150 mm) and brighter
appearance compared to the other microplastics (200e2000 mm
sized PET, PE, PP particles).
The final medaka stomach underwent PUE extraction sus-
pended in ultrapure water (Fig. 4aec). Overall, PE, PVC, PS and PET
were among the particles extracted from the processed fish stom-
ach. In this case, RMS identification of the particles was not
complicated by surface residues commonly observed with chemical
extraction. Theoretical densities of the four detected plastics range
from 0.9 to 1.4 g/cm3, therefore suggesting the possibility of density
separation of the of the different plastic types. However, attempted
density separation to distinguish between the extracted micro-
plastics was overall inconclusive. This suggests that the relative
density of the extracted particles did not necessarily match the
expected density of the virgin polymers. The presence of attendant
residual material from the digestive tract on the extracted particles
may help explain the attendant discrepancy. A total of 38 particles
were analyzed, 21 of these were identified as microplastics. The
extracted microplastics were in the size range of 100e300 mm. In
contrast, PP particles which were larger in size (approximately
400e800 mm), were not found in the gut. The remaining 17
analyzed particles were of non-plastic origin and were identified as
cellulose, hydroxyl apatite, aragonite, phosphates, silicates, and
carbonates, apparent components in the fish gut.
 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124 1117

Fig. 2. RMS characterization of water suspended Medaka gut contents air-dried on a quartz slide. (a) Optical image of the glass substrate showing the distribution of particles.
(b)e(e) Processed RMS spectra and optical images of representative identified particles. (f) A multi-component particle showing the presence of cellulose and methyl ricinoleate.
Red and blue spectra correspond to the particle and library match, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

3.2. Characterization of particles from North Pacific Ocean manta
trawl samples
Two trawl samples collected by the Office of Environmental
Health Hazard Assessment (OEHHA) were screened for potential
polymer residues present in the ocean. One of the trawl samples
was manually extracted and the 30 extracted particles mounted on
seven SEM stubs for RMS characterization. The particles ranged in
size from 1 to 5 mm (Fig. 5a) and were identified as 25 PE, 4 PP, 1
particle was a mixture of PE and PP. The identified polymer parti-
cles often included additional ingredients which were identified as
iron oxide (red pigment), phthalocyanine dye, titanium dioxide,
aluminum telenate, and surface associated biofilms. Presence of
biofilm on the polymer particles often resulted in a broad fluores-
cent band around 800-1800 cm 1 region, which overshadowed the
underlying polymer peaks. This is evident in Fig. 5b, where raw
spectrum of a region of the particle with attendant biofilm is
characterized by a broad fluorescent band. This is in direct contrast
to the spectrum of the exposed polymer region, where the polymer
peaks (PE) are evident. Overshadowed polymer peaks can usually
be resolved through background subtraction analysis described in
detail previously.
The second trawl sample was filtered through a 1 mm screen
onto a 10 mm PC filter and the filter surface was screened for plastic
1118 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124

Fig. 3. Impact of KOH extraction. Extraction of Medaka gut in (a) water versus (b) 10% KOH solution. Polyethylene particles exposed to (c) water, (d) 10% KOH solution.

Fig. 4. Extraction of microplastics from Medaka stomach. (a) RMS characterization of particles from Medaka stomach extracted in water. Red and blue spectra correspond to the
particle and library match, respectively. (b) Different particle types identified in the Medaka stomach. (c) Polymer types identified among the three different filtrations of the
Medaka stomach used to examine the effect of polymer buoyancy. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124 1119

Fig. 5. RMS identification of representative plastic particles in OEHHA Pacific Ocean trawl samples mounted on SEM stubs. (a) RMS spectra identifying various trawl particles. (b)
RMS spectra of a trawl particle with biofilm coating. Raw spectrum for the region containing the biofilm (red) does not show distinct polymer related peaks, which become evident
only after background subtraction (lower red spectrum). The polymer was identified as PE. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)

residues. Three pieces of polymers, PP, PE and PS in the size range of
100e300 mm were identified on the filter. The remaining analyzed
particles were identified as various composite minerals.
One of the objectives of RMS analyses was to identify any evi-
dence of POPs including flame retardants that were detectable on
the polymer surfaces. It should be noted that the surface concen-
tration of POPs on polymer particles are expected to be in the parts
per million range. Given the detection limit of RMS analysis is ~1%,
such low concentrations are not anticipated to be readily identifi-
able by RMS. However, for solid flame retardants such as deca-
brominated diphenyl ether (deca-BDE), heterogeneous distribu-
tion of the chemical within the polymer often results in localized
concentrations which are greater than 1% and therefore detectable
by RMS (Ghosal and Wagner, 2013). Inspection of some of the trawl
polymer particle spectra identified potential deca-BDE and anti-
mony trioxide related peaks, thereby suggesting the possible
presence of the flame retardant. However, deca-BDE related peak
intensities were not strong enough for a conclusive confirmation.
3.3. Identification of potential polymer debris ingested by
myctophid fish from subtropical gyres
A selection of six South Atlantic myctophid stomach contents
were analyzed for the evidence of polymer ingestion (Fig. 6). Each
of these gut sample was received individually frozen in optimum
cutting temperature (OCT) media on cork substrate. Two separate
sample preparations - manually extracted particles >1 mm and
1 mme10 mm sized particles filtered onto a PC filter - were
analyzed. For the six processed myctophid stomachs, a total of 103
particles were selected for RMS analysis based on visual identifi-
cation of the particles as potential polymer entities. Of these, three
particles were identified as distinct pieces of plastics in two of the
myctophid stomachs, BB13A and BB14A, as shown in Fig. 6. In case
of myctophid BB13A, two particles on the PC filter were identified
as PP. The particles were approximately 30  5 mm2 and
25  10 mm2 in size and hence identified as microplastic. The two PP
particles were from the top portion of the PUE filtrate and therefore
in agreement with the expected buoyancy of PP. One of these
particles also contained phthalocyanine, a dye for blue color. For
BB14A, a relatively long piece of PE fiber (13  0.2 mm2) was
extracted directly from the dissected stomach prior to PUE pro-
cessing and filtration. The relatively long length of the fiber
appeared to have obstructed the fish stomach as it was virtually
free of additional contents. The fiber also contained phthalocyanine
dye in agreement with its blue color.
Apart from the three distinct polymer particles described above,
six particles identified as co-adsorbed polymer and fatty acid res-
idues were also observed in the PUE filter samples. Identification of
1120 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124

Fig. 6. RMS characterization of particles from Atlantic Ocean Myctophid fish stomach. (a) Summary of Atlantic Ocean Myctophid stomach particle analyses. (b) Optical images and
processed spectra showing three particles identified in the Myctophid stomach as microplastics. Red and blue spectra correspond to the particle and library match, respectively. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the Raman peaks of these composite particles is shown in Fig. 7a.
The peaks pertaining to the fatty acid component were identified as
oleic acid. RMS spectrum of one of these composite particles is
compared to the spectrum of a PE particle from the oceanic trawl
sample, as shown in Fig. 7b. The presence of PE in the composite
particle is confirmed by the spectral match with the trawl PE par-
ticle. Additional peaks in the composite particle spectrum were
identified as oleic acid and the substrate background (carbon). As
such, these particles appeared to be a composite of fatty acids
derived from lipid content in fish stomach and ingested polymer
residues. This observation is consistent with reports that identify
stomach oils as organic solvents solubilizing ingested polymer
particles and associated chemicals, thereby enhancing their
bioavailability (Tanaka et al., 2013, 2015; Kershaw, 2015). These
composite particles were detected in three myctophid stomachs -
four in BB27B, one in BB13A and one in BB18A. The polymer
component of majority of these particles was identified as PS,
except for two particles in BB27B, which were identified as partially
PE.
Several of the extracted particles were anticipated to be of
polymer origin based on optical observations prior to RMS analysis.
However, subsequent RMS examination of these particles identified
them as non-polymer entities; respective spectral matching iden-
tified their actual composition as shown in Fig. 8. This underscores
the difficulty of attempting to identify polymers especially micro-
plastics by optical observations alone, which is often responsible for
the misidentification of biogenic particles as polymers. Further-
more, spectral identification often provides information about
additional ingredients which may be of interest. For instance, one of
the particles extracted from BB27B was identified as a composite of
hydroxyl apatite and propylene glycol ricinoleate, which is a
commercially used skin-conditioning agent. This was also observed
in the case of laboratory based medaka fish which contained par-
ticles with plasticizer signatures.
In addition to the six South Atlantic myctophids, six North Pa-
cific myctophid fishes were analyzed as well. In this case, each gut
 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124 1121

Fig. 7. Characterization of composite microplastic/fatty acid particles. (a) Spectral identification of the microplastic and specific fatty acid in two of the composite particles. Red and
blue spectra correspond to the particle and library match, respectively. (b) RMS spectra comparing a PE particle from OEHHA trawl sample and a composite particle from BB27B
Myctophid stomach along with the substrate background. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

sample was received individually frozen and packaged inside 4. Discussion

aluminum foil, OCT media was not used. In some cases, the
aluminum foil in which the fish gut was frozen was damaged,
resulting in micron scale aluminum foil debris in the fish stomach,
which were readily identifiable and as such were not considered for
results reporting. Each sample was treated to the same polymer
extraction procedure as described for the South Atlantic mycto-
phids. The PUE filter samples for the North Pacific Ocean fish were
analyzed less extensively compared to the South Atlantic Ocean
samples. The polymer particles identified in this case were in the
millimeter size range and were manually extracted directly from
the Pacific Ocean myctophid stomach during dissection, prior to
PUE filtration. Fig. 9 shows the polymer pieces identified in samples
M080 which contained a PE particle and in M143 which had two
particles, PE and PP. In addition, several non-polymer particles
were identified on the PUE filter samples as well.
The work presented here focused primarily on the extraction
and Raman micro-spectroscopy based identification of polymer
residues in ocean water and fish stomach. The extraction of poly-
mers from laboratory based fish stomach was evaluated for water
and KOH based suspensions as part of method development. Water
based PUE extraction was demonstrated to be an effective method
for the extraction of plastic residues from fish stomachs (Wagner
et al., 2016). Stomach sizes of the fish examined in this study
ranged from 2.5 cm (laboratory based medaka) to 9.3 ± 1.3 cm
(oceanic myctophid fish). They were of a reasonable size for water
based PUE extraction. Larger fish species with typically larger
stomach and therefore greater quantities of biomass and lipids in
the stomach, often require the stomach contents to be divided into
multiple portions for PUE based extraction. Additionally, PUE is
often conducted for a longer time period to optimize the extraction/
1122 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124
Fig. 8. RMS identification of non-microplastic particles in fish stomach. Red and blue spectra correspond to the particle and library match, respectively. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
filtration process.
One of the anticipated impacts of plastic ingestion is expected to
be the exposure to POPs that are believed to preferentially adsorb
on the polymer surfaces (Koelmans et al., 2016; Bakir et al., 2014a,
2014b, 2012; Chua et al., 2014). However, there have also been
studies that consider the impact of POPs to be minimal due to a
proposed cleaning mechanism that counteracts their bio-
magnification (Koelmans et al., 2013). In any case, typical the con-
centration of POPs on plastic debris are expected to be in the sub
ng g 1 to mg g 1 range (Teuten et al., 2009). Given the detection
limit of RMS analysis (~1%), most surface adsorbed POPs if present
are not expected to be readily identifiable by RMS in this concen-
tration range. However, to ensure the retention of any potential POP
signatures for possible detection by an applicable technique, it is
necessary to preserve the integrity of the sample by using a mini-
mally invasive extraction procedure for the ingested microplastics.
As such, water based PUE extraction was identified as the method
of choice. KOH based extraction led to modification of the extracted
polymer surface, as such enabling the potential loss of any surface
POPs. The presence of plasticizers identified on some of the parti-
cles, extracted from the laboratory based medaka stomach in water,
are likely indicative of plasticizers that were released from the
ingested plastics and/or laundry lint adsorbing onto other contents
in the fish gut. Hence, these plasticizers represent a form of poly-
mer related residue that the fish are exposed to as a result of plastic
contamination.
The observed size range of polymer particles extracted from the
laboratory based medaka stomach suggest the intake of micro-
plastic <300 mm in size by the fish. There are studies which indicate
visual awareness of particles and often a distinctly preferred
 et al., 2015; Ory et al.,
ingestion of certain particles by the fish (de Sa
2017). However, in this case given that the size distribution of the
ingested polymer particles was not the focus of the study, this
observation was not explored in any detail in the experiments.
Examination of manta trawl samples from the North Pacific
Ocean identified PE and PP as the predominant plastic debris,
which is consistent with the findings in other studies (Cozar et al.,
2014; Hidalgo-Ruz et al., 2012). The densities of virgin PE and PP are
typically 0.94 and 0.85 gm/cm3, respectively, hence less than that of
water (1 gm/cm3). As such, they are expected to be buoyant in the
ocean. This is in agreement with the collection location of the
manta trawl samples, which gathered particles floating near the
ocean surface. Also, as discussed earlier, polymer debris are ex-
pected to be potential carriers of POPs in the marine environment.
The tentative identification of deca-BDE and antimony trioxide on
some of the trawl polymer particles appear to support this view.
However, as stated earlier, the detection of POPs on oceanic poly-
mer residues by RMS is challenging based on their typical
concentrations.
Two types of plastic residues were observed in the stomachs of
some South Atlantic Ocean fish, which were analyzed more
extensively compared to the North Pacific myctophids examined in
this study. These included 1) polymer particles and 2) composite
particles with mixture of polymer and fatty acid residues. Presence
 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124 1123

Fig. 9. RMS characterization of particles from Pacific Ocean Myctophid fish stomach. (a) Summary of analyzed Pacific Ocean Myctophid stomach particle. (b) Optical images and
processed spectra showing three particles identified in the Myctophid stomach as microplastics. Red and blue spectra correspond to the particle and library match, respectively. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

of the composite particles may indicate digestive processing of
consumed plastic fragments whereby the stomach oils serve as
organic solvents solubilizing some of the ingested polymer debris
over time and thereby leading to the formation of these composite
particles. Tanaka et al. have reported the presence of similar plastic-
derived chemicals in tissues of seabirds ingesting marine plastics
(Tanaka et al., 2013, 2015). As such, this may indicate a potential
pathway for the incorporation of ingredients associated with the
polymer into fish tissue.
One of the major advantages of RMS analysis is the ability to
potentially differentiate between polymer and non-polymer ag-
gregates. This is particularly relevant in terms of estimating the
amount of polymer intake by the fish. As this analysis shows, a
number of particles visually identified as polymers were deter-
mined to be non-polymeric residues by RMS analysis (Fig. 8). Some
of the polymer particles identified in oceanic fish were <50 mm in
dimension. This indicates the detection of smaller microplastic
particles by RMS in this study compared to those identified by
Wagner et al. using FTIR, in which case 600 mm was the smallest
microplastic identified (Wagner et al., 2016). This is in agreement
with the work done by Kappler et al., where they show significant
underestimation of microplastics identified by FTIR imaging
compared to Raman imaging, especially in the size range <20 mm
(Kappler et al., 2016). In fact, they were able to identify particles as
small as 5 mm using Raman imaging. However, Cincinelli et al.
report the identification of significantly smaller microplastics
compared to 600 mm, by FTIR (Cincinelli et al., 2017). In addition,
the automated RMS data processing algorithm employed in this
work greatly reduced the potentially negative effects of biomass
fluorescence associated with RMS. However, the time required for
individual RMS analysis (~1 min) is typically longer compared to
FTIR (Kappler et al., 2016).
5. Conclusion
Water based isolation and subsequent screening of resultant
particles by Raman micro-spectroscopy identified polymer con-
taminants in ocean water and representative fish stomach. The
chemical differentiation between polymers versus non-polymer
ingredients accessible through RMS identification highlighted the
1124 S. Ghosal et al. / Environmental Pollution 233 (2018) 1113e1124
importance of chemical speciation versus optical screening alone.
In addition to polymer identification, RMS also identified additional
components associated with the polymer particles, which may in
turn offer insight into additional contaminants introduced by the
plastics residues. Moreover, the detection of composite polymer
and fatty acid particles identified potential pathway for the incor-
poration of ingredients associated with the polymer into fish tissue.
RMS enables identification of particles of size ranges which are
often not easily detectable through other forms of spectroscopy e.g.
FTIR. However, the typically longer spectral acquisition time asso-
ciated with RMS analysis makes the process time intensive.
Acknowledgments
Funding for laboratory work was provided by United States
Environmental Protection Agency Region 9. We thank 5Gyres and
Project Kaisei for making it possible to collect myctophids from the
S. Atlantic and N. Pacific, respectively, and the Aquatic Health
Program at UC Davis for providing Japanese medaka. We also thank
Anna-Marie Cook and Harry Allen of United States Environmental
Protection Agency Region 9 (USEPA/USACE Agreement (W912P97-
15-P-0014), Chelsea Rochman and Swee J. Teh at UC Davis, and
Margy Gassel of CalEPA for their advice on this work.
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