Environmental Pollution 272 (2021) 116419

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

A novel approach based on multiple fish species and water column

compartments in assessing vertical microlitter distribution and
composition
Luca Palazzo a, b, *, Stefania Coppa a, Andrea Camedda a, Mariacristina Cocca c,
Francesca De Falco c, Alvise Vianello d, Giorgio Massaro a, Giuseppe Andrea de Lucia a
a
CNR-IAS, National Research Council - Institute of Anthropic Impacts and Sustainability in Marine Environment, Loc. Sa Mardini, 09170, Torregrande, OR,
Italy
b
Department of Ecology and Biology, University of Tuscia, Via S. Camillo de Lellis 44, 01100, Viterbo, VT, Italy
c
Institute for Polymers, Composites and Biomaterials, National Research Council of Italy, Via Campi Flegrei, 34 - 80078, Pozzuoli, NA, Italy
d
Aalborg University, Department of the Built Environment, Thomas Manns Vej 23, 9220, Aalborg, Denmark

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

Article history: The assessment of the distribution and composition of microlitter in the sea is a great challenge. Bio-
Received 23 October 2020 logical indicators can be an irreplaceable tool since they measure microlitter levels in their environments
Received in revised form in a way that is virtually impossible to replicate by direct physical measurements. Furthermore, trends
16 December 2020
can provide policymakers with statistically robust analysis. We looked into the capacity of multiple fish
Accepted 30 December 2020
species to describe the distribution and composition of microlitter vertically across different compart-
Available online 31 December 2020
ments of the water column. A total of 502 individuals from six selected species (Scomber scombrus,
Oblada melanura, Spicara smaris, Boops boops, Merluccius merluccius and Mullus barbatus) were collected
Keywords:
Microplastics
on the western side of Sardinia island and allocated to three compartments: surface, mid-water and
Fish bottom. The species of the surface exhibited a higher frequency of occurrence (41.89%) of microlitter
Compartments ingestion, compared to those of the mid-water and bottom (19.60%; 22.58%). A significant difference in
Mediterranean Sea the average number of ingested microlitter was found between the surface and the bottom compart-
Bioindicators ment. All the microlitter fragments found were analysed through Fourier Transform Infrared Spectros-
copy (FTIR). The comparison of the expected buoyancies of the polymers identified puth faith in the
allocation of the species to the respective compartments. Therefore, considering the Marine Strategy
Framework Directive objective, this approach could be useful in assessing microlitter distribution and
composition vertically across the water column.
© 2021 Elsevier Ltd. All rights reserved.

1. Introduction their size into macroplastics (>25 mm), mesoplastics

(5 < x < 25 mm) and microplastics (<5 mm) (Thompson et al.,
Marine litter is “any persistent, manufactured or processed solid 2004; Arthur et al., 2009). Microplastics are further divided into
material discarded, disposed of or abandoned in the marine and “primary microplastics” (Cole et al., 2011) when they have been
coastal environment” (UNEP, 2009). Various studies have shown purposely manufactured of size less than 5 mm (i.e. microbeads
that it consists primarily of plastics, mainly due to their continu- from cosmetics, hand cleaners and air blast cleaning media; Fendall
ously increasing global production (PlasticsEurope, 2015) and the and Sewell, 2009; Napper et al., 2015) or when they enter the
fact that it is virtually immune to environmental degradation environment already in micrometric size (i.e. microplastics from
(Barnes et al., 2009). Plastics are generally subdivided according to tire wear and tear, from the washing and wearing of synthetic
textiles) (De Falco et al., 2018a, 2020). In contrast, “secondary
microplastics” are the result of a progressive fragmentation once
* Corresponding author. CNR-IAS, National Research Council - Institute of An- introduced into the environment, mainly due to chemical, physical
thropic Impacts and Sustainability in Marine Environment, Loc. Sa Mardini, 09170, and biological action (Andrady, 2011; Browne et al., 2007; Barnes
Torregrande, OR, Italy. et al., 2009). Most of the plastics produced have a lower density
E-mail address: [email protected] (L. Palazzo).

https://doi.org/10.1016/j.envpol.2020.116419
0269-7491/© 2021 Elsevier Ltd. All rights reserved.
L. Palazzo, S. Coppa, A. Camedda et al. Environmental Pollution 272 (2021) 116419

than seawater (PlasticEurope, 2015); thus we would expect to find a addition to giving crucial information on the distribution, compo-
prevalence of floating plastics in marine environments that mix sition and trends, indicators for microplastic ingestion could pro-
with the surface boundary layer (Kukulka et al., 2012). Many studies vide guidance on which species to perform further investigations
have noted a great amount of low-density polymers and their on toxicity (Rochman et al., 2013), chemical transfer (Oliveira et al.,
persistence in surface waters (Ryan et al., 2009; Goldstein et al., 2013; Bakir et al., 2016), biomagnification (Rochman et al., 2013;
2013; Eriksen et al., 2014). Nevertheless, the density of virgin Lusher, 2015) and bioaccumulation (Besseling et al., 2013; Browne
plastics can be modified by a plethora of natural processes once et al., 2013). Microlitter ingestion is currently being assessed in
they are introduced into marine environments. For example, the various organisms ranging from invertebrates to vertebrates
density of polymers that reside for a long time at the surface can be (Wright et al., 2013; Werner et al., 2016). Just recently fish have
altered by solar UV photodegradation reactions, thermal reaction started to be investigated for microlitter ingestion and have been
(thermal oxidation), hydrolysis of the polymer and microbial recognised as potential indicators for specific aquatic compart-
degradation that cause leaching of additives (Andrady, 1996; Barnes ments and/or regions (Galgani et al., 2013; UNEP/MAP SPA/RAC,
et al., 2009; Browne et al., 2010; Derraik, 2002; Thompson et al., 2018; Bray et al., 2019). To date studies on fish have mainly
2004; Kooi et al., 2017) or by biofouling (Harrison et al., 2011; selected representative species of pelagic and dermesal habitats
More
t-Ferguson et al., 2010). Moreover, microplastics density, (Rummel et al., 2016; Guven et al., 2017) and Sardina pilchardus,
together with particle size and shape, can strongly influence the Platichthys flesus, Gadus morhua, Scomber scombrus, Clupea hare-
advection velocity and hence the ability of the particle to reside for ngus are some examples of common species that have been
a long time at different depths in the water column (Ballent et al., investigated. Therefore, it is probable that once appropriate suitable
2012; Enders et al., 2015). sentinel species are selected, fish will start to be mandatorily
The abundance of plastics in marine environments has been monitored (Fossi et al., 2018), also because they could warn of
shown to be inversely related with the particle’s size, and micro- potential threats to human health (Barboza et al., 2018; Wright and
plastics have been found to be ubiquitous (Thompson et al., 2004; Kelly, 2017).
Bergman and Klages, 2012; Galgani et al., 2015). The monitoring of Recent studies on microlitter ingestion in fish species have been
abundance and composition of smaller particles poses quite a lot of trying to understand how to best investigate and interpret the data
challenges since there are a whole variety of sources and pathways in order to help assess microlitter abundance, distribution,
that affect their distribution (Browne, 2015). Moreover, currents composition, fate and impacts. Some studies have compared the
and wind forces make them migrate over long distances and have ingestion of microlitter with fish feeding behaviour or diet (Peters
been observed to accumulate in large convergence zones (Law and Bratton, 2016; Vendel et al., 2017; Mizraji et al., 2017). Others
et al., 2010; More
t-Ferguson et al., 2010; Lebreton et al., 2012). have taken into account the overall habitat use, while most studies
The five gyres (Moore et al., 2001; Davison and Asch, 2011; Eriksen have divided the species into demersal (Avio et al., 2015; Bellas
et al., 2013) are examples of accumulation spots, as is the Medi- et al., 2016; Torre et al., 2016; Güven et al., 2017), mesopelagic
terranean Sea, which is a semi-enclosed basin, with an average (Boerger et al., 2010; Davison and Asch, 2011; Lusher et al., 2016)
concentration of 243,854 plastics/km2 in its surface waters, of and pelagic (Deudero and Alomar, 2015; Romeo et al., 2015).
which 83% are microplastics (Cozar et al., 2015). Different factors Geographical distribution has also been taken into account, for
govern accumulation on the surface and in the sediments, and we example by considering the species proximity to coastal environ-
do not always find accumulation of litter in both compartments ments (Nadal et al., 2016; Neves et al., 2015; Battaglia et al., 2016) or
over the same areas. The sampling tools used to study microlitter by comparing small vs. large scale (Medsealitter project: https://
include manta trawls and bongo nets for the sea surface and mid- medsealitter.interreg-med.eu). Recently, the microlitter frequency
water (Doyle et al., 2011; Eriksen et al., 2013; Colton et al., 1974; of ingestion, among multiple fish species, has been proposed as a
More
t-Ferguson et al., 2010), while Van Veen, Ekman grabs and good proxy to highlight differences between areas (Anastasopulou
various corers have been used to investigate sediment samples et al., 2018; Avio et al., 2020). Although the frequency of ingestion
(Van Cauwenberghe et al., 2013; Vianello et al., 2013; Pagter et al., for multiple fish species seems to be a good parameter to evaluate
2018; Palatinus et al., 2019). It is important to keep in mind that, abundances of microlitter across areas, much remains to be done to
although these can be powerful analytical tools, these instruments assess the different accumulation patterns across compartments of
sample different sections of the water column and microplastics the water column.
concentrations are presented in relation to the area, volume or Thus, the objective of the present study is to evaluate the ca-
length covered. These types of measurements must be considered pacity of multiple indicator fish species to describe the distribution
local and time-dependent (Waldschl€ ager et al., 2020). and composition of microlitter vertically across the water column.
The Marine Strategy Framework Directive (MSFD/2008/56/EC), In order to do so, the species fidelity to three compartments (sur-
which set out the major contaminant issues related to the marine face, mid-water and bottom) was taken into consideration. This
environment and prioritises the topics to be investigated in order to study was conducted with the intention of further supporting the
achieve Good Environmental Status (GES), has made the assess- planned actions for implementing the MSFD.
ment of plastic ingestion in marine species a research priority.
Many species are impacted by marine litter, mainly due to entan- 2. Material and methods
glement or ingestion, and the number reported is constantly
growing (Gall and Thompson, 2015). A few bioindicators for mac- 2.1. Study area
roplastic ingestion have already been adopted and recognised as
invaluable tools to measure the amount of litter in their environ- This study was carried out in Sardinian waters (Western Medi-
ments and trends can provide policymakers with statistically terranean Sea) which are part of the Geographical Sub-Area 11 (GSA
robust analysis (van Franeker, 1985; van Franeker et al., 2011; 11) identified by the FAO’s General Fisheries Commission for the
Matiddi et al., 2017). Moreover, the search for indicators for Mediterranean (GFCM) and in the middle of the Western Medi-
microplastic ingestion is still ongoing and various efforts have been terranean Sea sub-region (MSFD). Fish samples were collected over
made to cover different ecological and biological aspects (Galimany a period of 3 years (2017e2019) from local fisherman and fishing in
et al., 2009; Fossi et al., 2014; Vandermeersch et al., 2015). In first-hand in an area comprised between the Gulf of Oristano
(central west of Sardinia) and the Gulf of Cagliari (south of
2
L. Palazzo, S. Coppa, A. Camedda et al. Environmental Pollution 272 (2021) 116419

Sardinia). The area presents heterogeneous fishing grounds small fish close to the sea bottom during daytime (Alheit and
(Sabatini et al., 2013) and is characterised by a 25 km wide conti- Pitcher, 1995; Buchholz et al., 1995; Carpentieri et al., 2005). We,
nental shelf (De Falco et al., 2015), where the main water mass therefore, collected only juveniles <25 cm in length (Ungaro et al.,
present in the area is the Modified Atlantic Water (MAW). 1993). The red mullet Mullus barbatus is a demersal species that
feeds typically on zoobenthos such as crustaceans, worms and
2.2. Sample collection in relation to the ecology and assignment to molluscs (Mahmoud et al., 2017). All the individuals collected that
compartments presented signs of stomach eversion or of net feeding were dis-
carded. Additional information for each species is presented in
The choice of the species was based on an initial evaluation of Table 1.
the most common and easily available fish species (landings) in the
area. Among these, we chose those species with appropriate spatial 2.3. Laboratory analysis
coverage of the area, and on which we had enough information
related to their ecology and biology. Moreover, importance was All procedures were developed following the indications of the
given to species where microplastics ingestion had been observed “Harmonized protocol for monitoring microplastics in biota”
in the past, Boops boops, for example, has already been proposed by (BASEMAN Project; Bessa et al., 2019). Fish were collected once
Tsangaris et al. (2020) to assess microplastic ingestion in the landed and frozen at
20  C. In the laboratory, each fish was
Mediterranean Sea. Only one species, Oblada melanura, was individually measured (fork length; cm), weighed (second decimal
selected following personal observations and preliminary analysis point; g) and finally dissected on a metal tray in order to extract the
on microplastic ingestion, even if, to our knowledge, this is the first entire gastrointestinal tract (Lusher et al., 2013; Rocha-Santos and
record for this species. The choice of compartments in the water Duarte, 2015; Bessa et al., 2018). At this stage, also the sex (males
column was based on the expected distribution of polymers ac- and females) and the weight (g) of the gastrointestinal tract (GIT)
cording to their density (that would make them float, suspend or were recorded. The extraction of microplastics from biological
sink) and on the fact that, to date, most studies have investigated matrices with H2O2 is one of the most widely employed methods
microplastic distribution and composition in surface and benthic (Renner et al., 2018), it is efficient for the successful extraction of
environments, while the middle of the water column is lacking. most polymers (Hamm, 2018) and is fast and cost-effective (Collard
Literature research was done in order to understand the feeding et al., 2015; Tagg et al., 2017). Therefore, individual GIT were placed
behaviour and deduce when was best to collect the species in order into glass beakers (500 mL) and 15% H2O2 1:20 (w/v) was added in
to be representative of the compartment they used. The mackerel order to digest the organic matter (Nuelle et al., 2014; Mathalon
Scomber scombrus is a pelagic-neritic planktivorous species that and Hill, 2014; Avio et al., 2015), keeping them at room tempera-
was collected during summer when it came closer to the coast. ture (~25e30  C) for maximum five days. If the exothermic reaction
Jansen et al. (2019) showed, by comparing the zooplankton distri- ended before all the organic matter was digested, an extra 1e2 mL
bution and composition, that, during this time of the year, there of 15% H2O2 was topped up. It is important to point out that the
was no evidence that they fed below the mixed layer since they digestion method adopted works well with small GIT tracts, such as
simply shifted between different types of prey as they became in the case of the selected species, which weighed up to 20 g max.
progressively available in the mixed surface layer. The seabream Once the organic material had been removed, the solution was
Oblada melanura is a benthopelagic omnivorous species which filtered onto 100 mm sieve (Giuliani steel sieves). This size of mesh
feeds mainly on copepods (Pallaoro et al., 2003) and was consid- was considered to be an appropriate detection limit for identifying
ered representative of surface coastal environments (Bauchot and microplastics (down to 100 mm) with confidence (Markic et al.,
Hureau, 1986) because of its opportunistic predator behaviour 2018). Moreover, it allowed easy handling, further analysis pro-
(Pallaoro et al., 2003), that takes place at the surface during daytime cessing (optical microscopy, FTIR spectroscopy) and is a meaningful
in the summer season (Pers. Obs.). The bogue Boops boops is a size to allow comparisons with most of the literature. Small aliquots
demersal omnivore fish that feeds on benthic (Crustacea, Mollusca, of digested matter were positioned onto multiple sieves (all of
Anellida, Sipuncula, Plantae) and pelagic preys (Siphonophorae, 100 mm mesh) and covered immediately with a Petri dish in order
Copepoda, eggs) (Derbal and Kara, 2008). This gregarious species to be observed under a stereoscopic microscope (Carl Zeiss Micro-
can be found on the continental shelf in the summer, and it feeds imaging GmbH) equipped with image analysis system (AxioCam
across the water column, generally ascending to the surface at night ERc5s and Zen, 2014 Blue edition software) (Lusher et al., 2013;
(El-maremie and El-mor, 2015). The picarel Spicara smaris is a Murray and Cowie, 2011). While observing at the stereoscopic
pelagic-neritic species and is generally observed in open waters microscope, an item was considered to be a microplastic particle if
feeding on copepods, other crustaceans, fish eggs and larvae during no cellular or organic structure was visible and it was homoge-
summer (Vidalis, 1994; Karachle and Sterglou, 2014) and is there- neously coloured (Hidalgo-Ruz et al., 2012; Primpke et al., 2020).
fore considered a good species to represent the middle of the water Fine-tipped tweezers were used to position the detected micro-
column. The European hake Merluccius merluccius is a demersal plastic particles (>0.1 mm) into individual glass Petri dishes (MSFD-
predator species, and its young typically feed on crustaceans and TSGML, 2013; Lusher et al., 2013; Rocha-Santos and Duarte, 2015).

Table 1
Fish species, compartments, numbers, length, weight and relative abundances of litter in gut contents.

Species Compartment No. fish dissected Average length (cm) (±SE) Average weight (g) (±SE) No. with litter % with litter

Scomber scombrus Surface 65 27.22 ± 1.98 213.13 ± 48.38 32 49.23

Oblada melanura Surface 83 20 ± 3.22 136.82 ± 62.42 30 36.14
Spicara smaris Mid-Water 89 14.19 ± 1.41 29.93 ± 5.28 7 7.87
Boops boops Mid-Water 110 17.78 ± 1.98 54.42 ± 0.91 32 29.09
Merluccius merluccius Bottom 66 22.25 ± 2.01 112.23 ± 21 15 22.73
Mullus barbatus Bottom 89 14.44 ± 1.30 40.31 ± 1.53 20 22.47

Total 502 136 27.29

3
L. Palazzo, S. Coppa, A. Camedda et al. Environmental Pollution 272 (2021) 116419

All suspected microlitter items were photographed, and the thickness with a smooth surface and a shape similar to a long thin
maximum length was measured by means of image analysis. In the cylinder (Cook, 2001; Houck, 2009). On the basis of these charac-
case of fibres that presented bendings, the length was estimated teristics, the morphological features of the observed fibres were
when possible. Colours (white, grey, black, red, orange, pink, pur- used to classify them into synthetic or natural-based fibres,
ple, blue, light blue, green, transparent, multi-colour) and shapes including, in this last case, fibres having a morphology typical of
(circular, angular, spherical, flat, irregular and cylindrical) were natural or artificial fibres.
recorded. Finally, the items were subdivided into typologies, ac-
cording to Hidalgo-Ruz et al. (2012), such as fragment, film, sphere, 2.6. Frequency, rate, and statistical analysis
rope/filament, sponge/foam and fibre. Pellets and microspheres
were grouped under the category “spheres” since the two afore- The particles found in species for each individual fish allowed
mentioned types have rarely been found in the gut of fish. for “frequency of occurrence” (number of fish that ingested
Airborne contamination has been recognised as an important microlitter/total number of fish dissected) and the “encounter rate”
parameter to monitor while performing any study involving syn- (total number of microlitter particles ingested/number of fish
thetic microlitters. Therefore, laboratory atmospheric deposition dissected) to be calculated. Univariate two-way permutational
was monitored to obtain an estimate of the level of potential analysis of variance (Permanova) was used to check for any sig-
airborne contamination. Other precautions were also taken during nificant difference in the average number of microlitter ingested,
dissection, extraction, sorting and visual identification such as: among the compartment and species factors, for all samples (in-
wearing a cotton laboratory coat, cleaning all surfaces and material dividuals). For all the analyses performed, compartment was
with alcohol, covering the samples at all times during analysis and considered a fixed factor, while the species were random and
positioning clean filters while analysing samples to collect any at- nested in compartment. The same analysis was performed by
mospheric microplastics created during laboratory procedures. considering only the fish that presented microlitter ingestion. A
Although we found very few fibres in our blanks, we excluded all multivariate two-way Permanova was used to check for differences
the fibres from the samples when the relative control presented for the typologies identified among compartments and the species
them. in compartments, considering only individuals with microplastics.
Finally, a univariate two-way Permanova was used to test for dif-
2.4. FTIR analysis ferences by considering only fibres. These tests were based on
Euclidean distance for univariate, and Bray Curtis for multivariate
Fragments isolated during the visual examination, by optical and each term is analysed through 9999 random permutations and
microscopy, were analysed by using Fourier Transform Infrared associated with a Monte Carlo test (Anderson et al., 2008).
Spectroscopy (FTIR). In detail, 60 fragments out of the total 70 were
chemically characterised through FTIR while the remaining frag- 3. Results
ments were too small or lost during manipulation. FTIR spectra of
fragments were recorded by means of a PerkinElmer Spectrum A total of 502 individuals (six species) were collected between
Frontier spectrometer (Waltham, MA, USA), equipped with a 2017 and 2019, 148 individuals were assigned to the surface, 199 to
deuterated triclicyne sulphate (DTGS) room temperature detector mid-water and 155 to bottom (Table 1). The overall frequency of
and by collecting the signal with an attenuated total reflectance
accessory (ATR) over the range 4000e650 cm
1, at a resolution of
4 cm
1, and co-adding 4 scans for each sample. The spectra ob-
tained were compared to multiple spectral databases, both com-
mercial (i.e. Hummel Polymer and additives, Aldrich Polymers, and
others) and custom-built (BASEMAN project; siMPle, 2019) (Meyns
et al., 2019; Rist et al., 2020).

2.5. Optical microscopy analysis

Regarding fibres, 43 out of the overall 147, recovered as reported

above, were analysed using a Leica M205 FA light microscope (Leica
Microsystem, Wetzlar, Germany). The morphological features of
fibres allow their discrimination between synthetic and natural or
artificial ones: cotton fibres present convolutions with the typical
twisted ribbon form; wool fibres present cuticular scale patterns;
the artificial fibre rayon is smooth and straight but marked by Fig. 1. Average number of litter particles (±SE) found in species divided into
striations; finally, synthetic fibres present uniform and regular compartments.

Table 2
Species, sex, gastrointestinal tract (GIT) weight, number of litter particles, size and encounter rate for the species analysed.

Species % males % females GIT weight (g) (±SE) No. litter particles Size range (mm) Encounter rate

Scomber scombrus 33.33 66.67 12.40 ± 5.33 69 0.345e19 1.06

Oblada melanura 49.25 50.75 5.56 ± 2.83 77 0.208e7.537 0.93
Spicara smaris 45.95 54.05 1.18 ± 0.71 9 0.175e3.664 0.10
Boops boops 37.27 62.73 2.45 ± 0.91 48 0.257e5.176 0.44
Merluccius merluccius 45 55 5.75 ± 3.09 21 0.396e2.946 0.32
Mullus barbatus 59.46 40.54 2.76 ± 1.53 36 0.102e14.742 0.40

4
 L. Palazzo, S. Coppa, A. Camedda et al.
Table 3
Statistical analysis performed.

Analysis and variables Source of variation df SS MS Pseudo-F p (MC) Pairwise test

Univariate two-way PERMANOVA Compartments 2 5.10*E^10 2.5119*E^10 3.2883 0.0473 Surface s

Bottom
p < 0.01
N of MP considering all individuals Species 3 2.34*E^10 7.812*E^9 3.2561 0.004 S. smaris s B.
boops
p < 0.001
Res 496 1.19*E^12 2.40*E^9
Total 501 1.26*E^12

Transform Fourth root

Univariate two-way PERMANOVA Compartments 2 7.18*E^7 3.5905*E^7 3.0228 0.0601

N of MP considering only individuals with MP Species 3 3.51*E^7 1.169.8*E^7 0.84181 0.5375
Res 131 1.82*E^9 1.39*E^7
Total 136 193*E^9
5

Transform Square root

Multivariate two-way PERMANOVA Compartments 2 994*E^7 4.9697*E^7 0.68169 0.6669 S. scombrus s

O. melanura
p < 0.0001
Typologies considering only individuals with MP Species 3 235*E^8 7.8292*E^7 6.3163 0.0001
Res 131 1.62*E^9 1.24*E^7
Total 136 1.96*E^9

Transform Square root

Univariate two-way PERMANOVA Compartments 2 6.85*E^7 3.43*E^7 0.44146 0.7947 S. scombrus s

O. melanura
p < 0.0001
Fibres considering all individuals Species 3 2.50*E^8 8.34*E^7 6.7346 0.0001
Res 131 1.62*E^9 1.24*E^7
Total 136 1.93*E^9

Environmental Pollution 272 (2021) 116419

Transform Square root
L. Palazzo, S. Coppa, A. Camedda et al. Environmental Pollution 272 (2021) 116419

Fig. 2. Size frequency distribution of microlitter per compartment (surface, mid-water

and bottom).

Fig. 3. Average number of microlitter (±SE) found in species divided into typologies.

occurrence (FO) of microlitter for the six species was 27.29%.

However, if we consider the single compartments, the surface had a
FO of 41.89%, mid-water 19.60% and bottom 22.58%. The highest FO
was found for S. scombrus (49.23%) and the lowest for S. smaris
(7.87%; Table 1). The highest encounter rate was found in the
“surface” compartment where S. scombrus and O. melanura dis-
played 1.06 and 0.93 (Table 2). A significant difference in the
average number of ingested microplastics (considering all in-
dividuals) was found between compartments (p ¼ 0.0473), as well
as for the species contained within compartments (p ¼ 0.004)
(Fig. 1; Table 3). The resultant pairwise test showed a significant
difference between surface and bottom compartment (p ¼ 0.0067)
and among S. smaris and B. boops in the mid-water compartment
(p ¼ 0.0002) (Table 3).
In total 260 litter particles were found in 136 individuals.
Considering compartments, 146 items were found in the surface, 57
Fig. 4. Colour categories found in gastrointestinal tract of fish according to the
in mid-water and 57 in bottom. The number of particles found per compartment. (For interpretation of the references to colour in this figure legend, the
species are shown in Table 2. If we consider only the fish that reader is referred to the Web version of this article.)
ingested microplastics, the average number of microplastics per
specie was: (mean ± SE) 2.16 ± 0.32 S. scombrus, 2.57 ± 0.45
O. melanura, 1.47 ± 0.13 B. boops, 1.28 ± 0.17 S. smaris, 1.4 ± 0.2 and the two most frequent size ranges found were the 1e1.5 and
M. merluccius and 1.8 ± 0.24 M. barbatus. No significant difference 1.5e2 mm ones (Fig. 2). The typologies found, overall, distributed as
was found when comparing these values among compartments follows: 147 fibres (56.53%), 71 fragments (27.31%), 26 filaments
(p ¼ 0.06; Table 3) and the species (p ¼ 0.53; Table 3). The size of (10%), 9 films (3.46%), 4 sponges (1.54%) and 3 spheres (1.15%). In
the litter particles found ranged from a minimum of 102 mm the surface we found: 71 fibres (48.63%), 45 fragments (30.82%), 15
(M. barbatus) to a maximum of 19 mm (S. scombrus; Table 2). Par- filaments (10.27%), 9 films (6.16%), 4 sponges (2.74%) and 3 spheres
ticles were subdivided into size ranges (0.5 bins in size distribution) (1.37). In mid-water we noted: 33 fibres (57.89%), 21 fragments

6
L. Palazzo, S. Coppa, A. Camedda et al. Environmental Pollution 272 (2021) 116419

Fig. 5. FTIR spectra (red line) of some fragments recovered from fish, and reference spectra highlighting the best match used for identification (blue dashed line). (For interpretation
of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

(36.84%), and 3 filaments (5.26%). In contrast, in the bottom we The FTIR analysis allowed the identification of different types of
observed: 43 fibres (75.44%), 8 filaments (14.03%), 5 fragments synthetic polymers such as: polypropylene (PP), polyethylene (PE),
(8.77%), and 1 sphere (1.75%). The average number of typologies polyurethane (PUR), polyester (PES). Some examples are reported
found per species are shown in Fig. 3. No significant difference was in Fig. 5. In addition, other polymeric fragments were detected, but
observed for typology distribution among compartments since their chemical composition was not clearly identified, they
(p ¼ 0.70), while a significant difference was observed between were classified as “others” for the purpose of the article. Morpho-
species within the same compartment (p ¼ 0.0001), specifically logical analysis by optical microscopy allowed the detection of
between S. scombrus and O. melanura (p ¼ 0.0001) in the “surface” synthetic fibres and natural based fibers. These last were mainly
compartment (Fig. 3, Table 3). Considering only fibres, no signifi- constituted by cellulose based fibres, of natural or artificial origin.
cant difference was observed between compartments (p ¼ 0.80; In the surface compartment we found 4 polymers (PP, PE, PUR,
Table 3), while a significant difference was noted for species within PES), 2 polymeric fragments classified as others, 4 synthetic and 1
compartments (p ¼ 0.0001; Table 3). We conducted a pairwise test cellulose based fibres. In mid-water 2 polymers (PP, PE), 1 other, 2
and found that S. scombrus was different from O. melanura synthetic and 7 cellulose based fibres. Finally in the bottom
(p ¼ 0.0001; Table 3). compartment we encountered 1 polymer (PES), 11 synthetic and 18
Overall, the items presented the following morphology per- cellulose based fibres. Polymer fragments, synthetic and cellulose
centages: cylindrical 68.85%, irregular 29.23%, circular 0.77%, based fibres are presented in Fig. 6. From the figure, it is evident
angular 0.77% and spherical 0.38%. In total 12 colour types were that the species from the surface compartment presented a wider
identified (white, grey, black, red, orange, pink, purple, blue, light range of polymers compared to the other two compartments.
blue, green, transparent and multi-colour): 11 in the surface Moreover, by looking into the polymer densities (Enders et al.,
compartment, 7 in mid-water and 8 in bottom (Fig. 4). Of these 2015; Li et al., 2016; Andrady, 2017) of the items found in our fish
S. scombrus presented 10 (all colours apart from grey), O. melanura and comparing them with the sea-water density (1.02e1.07 g/cm3),
10 (all colours apart from grey), S. smaris 3 (blue, green and we noticed that 82% of the items considered were positively
transparent), B. boops 7 (white, black, red, blue, light blue, green buoyant in the surface, 37.5% in mid-water and none in bottom. In
and transparent), M. merluccius 4 (black, blue, green and trans- contrast, 18% at the surface, 62.5% in mid-water and 100% in the
parent) and M. barbatus 8 (white, grey, black, red, blue, light blue, bottom were negatively buoyant.
green and transparent).

7
L. Palazzo, S. Coppa, A. Camedda et al. Environmental Pollution 272 (2021) 116419

Fig. 6. Number of items per polymer types and species analysed through FTIR spectroscopy.

4. Discussion the fraction of fish that ingest microlitter seems to uptake particles
at a rather constant rate, which transcends compartments and
Our results show that by selecting multiple fish species and species.
considering their biology and ecology, we can assign them to Considering all the above, we decided to choose multiple fish
compartments of the water column and therefore assess microlitter species that complement each other (Bonanno and Orlando-
distribution and composition, thereby providing a more detailed Bonaca, 2018) and allocated them according to their prevalent
and comprehensive picture of the potential threats posed to the use of 3 compartments (surface, mid-water, bottom) of the water
marine communities that inhabit different compartments. Micro- column. In order to do so, various factors were taken into account,
litter and particularly microplastics vertically distributes through such as the biology and ecology, as well as the time of year in which
the water column depending on intrinsic factors, such as the den- they were sampled. This has allowed us to create adequate pollu-
sity, size and shape of the particle, which, together, influence its tion indicator fish groups that can be descriptive of the state of the
advection velocity (Ballent et al., 2012; Enders et al., 2015). Sec- different compartments in relation to the pollution caused by
ondly, it is influenced by extrinsic processes like wind-induced microlitter. According to the expected buoyancies (Enders et al.,
turbulence, which can cause, for example, breaking surface waves 2015) of the polymers we found, allow us to trust in the choice of
(Kukulka et al., 2012). The distribution of microplastics throughout species and the subsequent allocation to the respective compart-
the water column has commonly been studied by taking water ments. The reason why we found negatively buoyant particles in
samples, generally pointing to high concentrations in the upper the surface can be explained by the fact that most of these items
layers and an exponential decrease with depth (Goldstein et al., were fibres. Since this typology of microlitter has already been
2013; Reisser et al., 2013; Rios-Fuster et al., 2019). Regarding the proposed empirically to have a slower vertical advection velocity,
spatial distribution of microplastics on the surface, gyres have been therefore contributing to their longer residence time in the upper
recognised as zones of convergence and accumulation (Law et al., layers of the water column (Ballent et al., 2012; Reisser et al., 2013).
2010; Eriksen et al., 2013; Goldstein et al., 2013), as well as closed Our study area is located in the middle of the western side of the
bays, gulfs, watersheds and seas surrounded by densely populated Mediterranean basin, which has been described as among the most
coastlines such as in the case of the Mediterranean Sea (Reisser impacted regions in the world as regards microplastics pollution in
et al., 2013; Collignon et al., 2012). The seafloor also concentrates surface waters (Cozar et al., 2015; Faure et al., 2015; Suaria et al.,
microplastics in great amounts, so much so that it has been 2016) and models have predicted some of the highest concentra-
described as a major sink for microplastics debris (Woodall et al., tions of floating plastics (Lebreton et al., 2012). To our knowledge,
2014). This idea is likely to be true if we consider processes such there are still no studies on microlitter distribution in sediments on
as weathering (Hidalgo-Ruz et al., 2012; Andrady, 2015), biofouling the western side of the island. However, a recent study (Soto-
(Holmstro € m, 1975; Ye and Andrady, 1991), entanglement with Navarro et al., 2020), based on the realistic distribution of marine
planktonic aggregates (Long et al., 2015) and transfer via plankton litter sources, produced a 3D simulation on microplastics distri-
faecal pellets (Cole et al., 2013) that make even the buoyant parti- bution and accumulation (making previsions based on the density:
cles sink to the bottom. As with the surface, also on the seafloor, floating, neutral and sinking), where they found that sinking par-
accumulation happens due to external processes and generally ticles remained very close to where they were released. As a result,
seems to happen when debris becomes entrapped in areas of low this area, having a low population density (1,630,474 ha; ISTAT,
circulation where sediments are already accumulating (Galgani 2019) and modest river outlets, did not present any sinking
et al., 1996; Schlining et al., 2013; Pham et al., 2014). Therefore, microplastics in the model outcome. Moreover, the same model
given this extreme complexity in the distribution of microplastics (referring to neutrally buoyant particles) and de Lucia et al. (2018)
both vertically and horizontally across the water column using measured the distribution of microplastics in levels below the
multiple fish species to monitor compartments of the water column surface and found medium to low values compared to the other
in space and time can be an irreplaceable tool. Another advantage areas they studied. Our multiple fish species divided into com-
presented in using multiple fish species is that the average number partments collected on the western side of the island of Sardinia
of microlitter items ingested per individual is very constant in the seem to confirm these expectations since we found a higher FO in
literature of fish species, generally presenting between 1 and 2 the surface compartment. Moreover, the species from the surface
items/individual (Davison and Asch, 2011; Lusher et al., 2013; Avio compartment (S. scombrus and O. melanura) showed an average
et al., 2015, 2020; Bellas et al., 2016). Even our study showed that number of ingested microplastics, among all individuals, that was

8
L. Palazzo, S. Coppa, A. Camedda et al. Environmental Pollution 272 (2021) 116419

significantly higher compared to the species of the bottom patterns. This study was conducted over an area with a wide con-
compartment (M. merluccius and M. barbatus). Most studies that tinental shelf. Therefore, in the future, it could be interesting to
have sampled the surface of the sea directly, when dividing the investigate if compartments together with multiple fish species can
items into typologies, have decided to exclude fibres because of be a valuable approach to study distribution and composition of
concern about airborne contamination (Suaria et al., 2016; Faure microlitter also in areas beyond the continental shelf, as well as
et al., 2015). This type of contamination can be limited by car- other environments, such as lakes for example.
rying out extraction and analysis in a laboratory as in our case.
Considering all the typologies of debris found and comparing them 5. Conclusions
across the three compartments, they appeared to be distributed
homogeneously throughout the water column, and fibres were the We found microlitter in all the six species and three compart-
dominant type encountered (56.53%), as in many other studies on ments analysed. The surface exhibited the highest values for our
microlitter ingestion in fish (Avio et al., 2015; Bellas et al., 2016; area suggesting that appropriately selected fish species grouped
Peters et al., 2017; Ory et al., 2018; Compa et al., 2018). Browne et al. into compartments can be an extremely important tool, since they
(2011) suggested that washing machine wastewater is the main can continuously measure microlitter levels in their environments
source of fibres released into marine environments. De Falco et al. in a way that is virtually impossible to replicate by direct physical
(2018b) found that release of polyester fabrics, during a simulated measurements. Therefore, these pollution indicator fish groups can
washing test, is about 1,733,000 microfibres per kg of washed potentially give us a better understanding of distribution, charac-
fabric. More recently, De Falco et al. (2019) showed that microfibres terisation, fate and accumulation of microlitter across compart-
released during washing range from 124 to 308 mg/kg of washed ments as well as allow inferences on the possible ecological
fabric, which corresponds to 640,000e1,500,000 microfibres. implications. This approach is well in line with the recent MSFD
Bearing this in mind, it is likely that the Mediterranean Sea, which it objectives and would enable the development of adequate miti-
is a semi-enclosed basin with a slow water exchange (Millot and gation strategies.
Taupier-Letage, 2005), would present very high fibre-based pollu-
tion. Therefore, it is important to consider microfibres when ana- Funding sources
lysing microlitter ingestion in fish, so as to assess their distribution
in different areas, although they probably prevent an accurate This work was supported by the European Commission, with
comparison of the other typologies among compartments. funding from the JPI Oceans project BASEMAN (reference JPIO-
Properties of microlitter such as density and typology seem to CEANS/0001/2015) and INDICIT (DG Env) project (No. 11.0661/
be critical factors to consider when looking for appropriate fish 2016/748064/SUB/ENV.C2) in the framework of which this study
bioindicator species. Therefore, grouping multiple fish species ac- was conducted.
cording to their use of defined compartments may be a good so-
lution to describe the distribution of this type of pollutant and Compliance with ethical standards
could become a valuable approach to incorporate within the MSFD
under Descriptor 10 (properties and quantities of marine litter do The authors declare that they have no conflict of interest.
not cause harm to the coastal and marine environment). Studies so
far have generally analysed the presence of microlitter in the gut of Author statement
fish in relation to their ecology by selecting one species (Tanaka and
Takada, 2016; Alomar et al., 2017; Pellini et al., 2018; Cardozo et al., Luca Palazzo has written the manuscript and conceptualised the
2018; Sbrana et al., 2020) or by subdividing multiple species ac- work, Stefania Coppa, Giorgio Massaro, Andrea Camedda have
cording to habitat use: pelagic, mesopelagic, benthopelagic, collected and analysed data, helped in the artwork and contributed
demersal, benthic (Phillips and Bonner, 2015; Rummel et al., 2016; to revise the manuscript, Maria Cristina Cocca, Francesca De Falco
Güven et al., 2017; Jabeen et al., 2017; Murphy et al., 2017; Lusher and Alvise Vianello have performed the polymer characterisation
et al., 2013; Neves et al., 2015; Bellas et al., 2016). This informa- and have revised the manuscript, Giuseppe Andrea de Lucia has
tion is very useful, and by making appropriate assumptions on the coordinated, conceptualised and helped to write the manuscript.
compartment’s use and the time of year in which they were
sampled, we may be able to evaluate microlitter pollution of Declaration of competing interest
compartments across different areas and further contribute to
providing a more comprehensive mapping of microlitter distribu- The authors declare that they have no conflict of interest.
tion. Moreover, by extending the number of species per compart-
ment, we may be able to increase our accuracy and add other Acknowledgements
valuable information. For example, we observed a significant dif-
ference of typologies ingested in the surface compartment, be- The authors would like to thank Rodney De Souza for checking
tween S. scombrus and O. melanura. This finding, for instance, can be English style and grammar and the anonymous reviewers who
explained by the different scale at which the two species uptake improved the quality of the manuscript.
microlitter from the environment. S. scombrus is a fast-moving
carnivorous predator that shows a prevalence of fibres, while
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