Science of the Total Environment 819 (2022) 152965

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Science of the Total Environment

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

Detection of plastic particles in marine sponges by a combined infrared

micro-spectroscopy and pyrolysis-gas chromatography-mass
spectrometry approach
Francesco Saliu a, Greta Biale b, Clarissa Raguso a, Jacopo La Nasa b, , Ilaria Degano b,c, Davide Seveso a,d,
⁎
⁎
Paolo Galli a,d, Marina Lasagni a, Francesca Modugno b,c,
a
Earth and Environmental Science Department, University of Milano Bicocca, Piazza della Scienza 1, 20126 Milano, Italy
b
Department of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 13, Pisa, Italy
c
Center for Instrument Sharing of the University of Pisa (CISUP), University of Pisa, Italy
d
MaRHE Center (Marine Research and High Education Center), Magoodhoo Island Faafu Atoll, Maldives

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

• We developed an approach based on py-

rolysis and infrared spectroscopy to study
microplastics in marine sponges.
• We performed both qualitative and quan-
titative determination of micro/nano plas-
tics.
• We tested the new protocol to study Mal-
divian marine sponges.

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

Article history: Plastic pollution threatens the marine environment, especially due to the adverse effects caused by micro and nano par-
Received 6 October 2021 ticles interacting with the marine biota. In order to provide reliable data regarding micro and nanoplastic contamina-
Received in revised form 21 December 2021 tion and the related impacts, efficient analytical solutions are needed. We developed a new analysis workflow that uses
Accepted 4 January 2022
marine sponges to monitor plastic pollution by characterizing the plastic particles accumulated in their tissue. Speci-
Available online 10 January 2022
mens of cf. Haliclona (Haplosclerida) were sampled in the Maldivian archipelago. The aim was to optimize the method
Edito: Yolanda Picó and to carry out a pilot study of the contamination of the related reef habitat. Particles were isolated, size fractioned,
counted and submitted to morphological and chemical characterization. The constituting polymer was identified by
Keywords: infrared microspectroscopy for particles >25 μm, and by pyrolysis coupled with gas chromatography mass spectrom-
Microplastics etry for those <25 μm. Method recoveries were between 87 and 83% and limits of quantitation (LOQs) were between
Nanoplastics 6.6 and 30.2 ng/g. Analyses showed that 70% of the sponges presented plastic contamination, with an average of 1.2
Pollution particles/g tissue for the 25–150 μm size range, and a total plastic concentration of up to 4.8 μg/g in the 0.2–25 μm size
Coral reefs range, with polyolefin being the most represented polymer in both size ranges. Overall, the study demonstrated the
Bioindicator
reliability of the proposed analytical workflow and of the use of sponges as biosamplers for plastic particles.
© 2022 Elsevier B.V. All rights reserved.

⁎ Corresponding authors at: Department of Chemistry and Industrial Chemistry, University of Pisa, Italy.
E-mail addresses: [email protected] (J. La Nasa), [email protected] (F. Modugno).

http://dx.doi.org/10.1016/j.scitotenv.2022.152965
0048-9697/© 2022 Elsevier B.V. All rights reserved.
F. Saliu et al. Science of the Total Environment 819 (2022) 152965

1. Introduction can process over 24 L of water per hour (Batista et al., 2014). Due to this
high filtration rate, sponges can accumulate high levels of foreign particles
The escalating production of plastic worldwide and the related and show a high concentration of metals and/or organic pollutants
mismanaged disposal of plastic waste is a matter of great concern (Bennett et al., 2018; Carballo et al., 1996), as they are susceptible to
(Andrady, 2011). In fact, today plastic is one of the most represented frac- water pollution (Gentric et al., 2016; Perez et al., 2003; Rizzi et al., 2020;
tions in marine litter. However, the scientific literature has mainly focused Roveta et al., 2021; Srikanth and Rao, 2016).
on the risks posed to the marine environment by the presence of plastic par- Sponges have thus already been proposed as a sentinel of the health
ticles down to a 5 mm size range (microplastics, MPs). These particles status of marine ecosystems, including MPs (de Mestre et al., 2012; Rao
mainly originate from the photo-oxidative degradation of larger plastic de- et al., 2007; Rao et al., 2008). In addition, a recent study (Girard et al.,
bris or are directly dumped from primary sources into aquatic systems (Gall 2021) found that sponges had taken up anthropic material, including
and Thompson, 2015). polystyrene particles. Interestingly, the authors also observed that the
Microplastics can threaten marine life by direct physical interaction, number of particles taken up by the sponges was not related to the
i.e., blocking of the digestive tract after ingestion (Wright et al., 2013), by chemical composition of the particles. They hypothesized that the
acting as a vector for persistent organic pollutants (Lamb et al., 2018), main driver in determining the differences in particle accumulation in
and by leaching toxic substances present as additives or produced due to marine sponges was the fluctuation in particle concentration and com-
degradation processes (Biale et al., 2021; Koelmans et al., 2013; Teuten position in the nearby water.
et al., 2007). Moreover, finer fractions within a 1–1000 nm size range, We thus aimed to investigate the feasibility of using marine sponges as a
called nanoplastics (NPs) (Gigault et al., 2018), may exhibit intrinsic toxic- “tool” for sampling plastic particles. Consequently, we tested an integrated
ity correlated with their capacity to overcome biological barriers (da Costa analytical protocol exploiting the respective advantages of microspectroscopy
et al., 2016; Gonçalves and Bebianno, 2021; Wright et al., 2013). and Py-GC–MS in providing the visualization and counts of particles larger
One of the main current challenges in tackling MP and NP pollution is than 25 μm and mass quantification of the smaller size range present in the
the lack of standardized and universally accepted analytical procedures sponges.
(Löder and Gerdts, 2015; Primpke et al., 2020; Sridhar et al., 2022). Several The study was focused on optimizing a sample preparation workflow
methods have been described in the scientific literature to approach MPs to isolate the plastic particles incorporated in the tissues of marine
and NPs analysis in different matrices (biota included), which differ in sponges and onto the evaluation of the related efficiency. We validated
particle isolation and detection (Saliu et al., 2020b; Tirkey and Upadhyay, the procedure and assessed whether a spatial variability of this pollution
2021). Two instrumental approaches are generally used for detecting parti- could be observed in a real case study by applying the procedure to de-
cles: micro spectroscopy (Käppler et al., 2016) and thermoanalytical tech- termine the plastic contamination in sponge samples collected in the
niques (La Nasa et al., 2020; Peñalver et al., 2020). Maldivian atoll reef. Lastly, we investigated a possible correlation be-
Micro spectroscopy is based on visualizing the particles after isolating tween the presence in the sponge tissue of different sized plastic parti-
them on a filter by physical separation (flotation and filtration) and the si- cles and plastic associated contaminants (phthalates) to verify the
multaneous collection of the chemical signature with either Fourier trans- adoption of associated plastic contaminants as a possible proxy of plas-
formed infrared (Löder et al., 2015) or Raman (Anger et al., 2018) tic contamination.
spectroscopy. This approach enables both the morphological information
(color, size, and shape of the particles) and chemical identification of indi- 2. Materials and methods
vidual plastic items to be obtained. The main drawbacks are the size limita-
tion imposed by the physical interaction of the radiation with the selected 2.1. Chemicals and reference materials
particles and the difficulty in providing mass concentration data (Löder
and Gerdts, 2015). The organic solvents hexane, dichloromethane (DCM) and acetone were
The second analytical approach based on thermoanalytical tech- purchased from Merck (Darmstadt, Germany). Potassium hydroxide (KOH)
niques is gaining momentum in MP and NP studies. It is based on analyt- in pellet form was purchased from Sigma-Aldrich (221473–2.5KG). Ultra-
ical pyrolysis coupled with gas chromatography and mass spectrometry pure water (18.2 MΩ cm−1) was obtained from a Milli-Q filtration unit
(Py-GC–MS), which is already widely used to obtain the molecular char- (Merck Millipore, MA, USA). A total of six different polymers, selected by
acterization of synthetic polymers in material sciences. With this ap- considering the most common petroleum-based plastics reported to occur
proach, the processed sample is introduced into the instrument, where in the marine environment, were used to assess and optimize the efficiency
the high molecular weight components are fragmented during pyroly- of the solvent extraction method, to optimize the Py-GC–MS conditions, to
sis. The pool of obtained fragments is separated, identified, and quanti- assess micro-infrared spectra acquisition and the library matches: polysty-
fied by gas chromatography–mass spectrometry. A pyrolysis fingerprint rene (PS), polyvinylchloride (PVC), polycarbonate (PC), polyethylene
is thus provided in the form of a GC–MS chromatogram (Tsuge et al., (PE), polyethylene terephthalate (PET), and polypropylene (PP). Further in-
2011). This method does not provide any information on the size, formation regarding the reference materials is available in Section S.1 of the
shape, color, and number of the analyzed particles. However it can Supporting Information.
also be used as a quantification tool by targeting specific pyrolysis prod-
ucts. When Py-GC–MS is used, quantitative information is obtained in
mass, with very low instrumental limits of detection and quantification. 2.2. Sample collection for method validation
This thus provides accurate information on the presence of NPs, which
is not possible with spectroscopy (Fischer and Scholz-Böttcher, 2019; A large sponge (72 g) specimen was collected in July 2019 in the water
Goedecke et al., 2020; Käppler et al., 2018; La Nasa et al., 2021; of the Portofino Marine Protected Area (Camogli, North Italy, 44° 18′ 40′′
Okoffo et al., 2020; Picó and Barceló, 2020; Ribeiro et al., 2021). N, 9° 12′ 47′′ E) for use as a reference matrix for method development
Considering the limitations of these two approaches, we developed and and evaluation. Based on pre-surveys, a single sampling site was selected
validated an integrated analytical protocol exploiting the respective advan- at a depth of 10 m. The sample was wrapped in aluminum foil and frozen
tages of spectroscopy and analytical pyrolysis. In addition, we tested the use at −20 °C until subsequent analysis.
of marine sponges as plastic particle biosamplers.
Sponges constitute abundant phyla, ubiquitous in all marine habitats (de 2.3. Microplastic isolation workflow
Goeij et al., 2013; Perez et al., 2003; Webster et al., 2013). They are long-
living, sessile and active filter-feeding invertebrates, capable of processing Fig. 2 shows the optimized analytical workflow, including the prelimi-
large quantities of water. It has been estimated that one gram of sponges nary determination of phthalic acid esters by SPME-LC/MS following a

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F. Saliu et al. Science of the Total Environment 819 (2022) 152965

previously described method (Saliu et al., 2020a). Regarding the provided by Perkin Elmer was used to confirm the assignment of the parti-
microplastic analysis, the workflow consisted of the following: cles by comparison with the reference spectra library (Saliu et al., 2018).
Positive identification with the reference library was assigned for matches
Step 1 Alkaline digestion: aliquots of 2.5 g of the selected marine sponges >80%. An example of the results obtained is provided in Fig. S1.
were weighed on an analytical balance (Gibertini® E50S) and added
with a 25 mL solution of 10% KOH. The mixture was then submitted 2.6. Py-GC-MS
to 24 h digestion at 60 °C in an Erlenmeyer flask sealed with aluminum
foil, under agitation (150 rpm). The accomplishment of complete diges- Analyses were performed with an EGA/PY-3030D® micro-furnace
tion was evaluated according to a previously published procedure pyrolizer (Frontier Laboratories Ltd., Japan) coupled to a Mass Selective
(Prata et al., 2019). Detector 5977B (Agilent Technologies, USA) using parameters adapted
Step 2 Density separation: after digestion, samples were cooled, filtered from La Nasa et al. (La Nasa et al., 2021). Briefly, samples obtained after
Step 5 of the workflow were pyrolyzed in single-shot mode at 600 °C for
onto glass fiber filters and added to a brine solution of ZnCl2 density
0.2 min. The dried extracts were injected with a split ratio of 20:1, and
(1.6 g cm−3) according to Quinn et al. (Quinn et al., 2017). The mixture
the GC injector was set at 280 °C. Additional details on the Py-GC-MS con-
was vigorously shaken and then left to stand and covered with a watch
ditions are available in Section S.3 of the Supporting Information, while the
glass. This procedure aimed to induce plastic particle floatation and sep- full instrumental conditions are reported in Biale et al. (2021). Calibration
arate the plastic particles from any denser inorganic particles associated curves were performed based on the integration of peak areas of the EIC
with the sponge tissue (i.e., sponge spicules and foreign mineral parti- (extracted ion chromatograms) of specific pyrolysis markers for each
cles taken up by the sponge). The complete settling of the denser parti- polymer according to the literature (Dierkes et al., 2019; Fischer et al.,
cles after mixture agitation took 20 min. The supernatant containing the 2019; Fischer and Scholz-Böttcher, 2019; Gomiero et al., 2019; Okoffo
floating plastic particles was then collected and filtrated. et al., 2020; Ribeiro et al., 2020). In detail, for PS: styrene dimer; PVC: hy-
Step 3 Isolation of larger microplastics (>25 μm): the supernatant from the drochloric acid; PC: bisphenol; PP: 2,4-dimethyl-1-heptene; PE: α,ω-alkenes
density separation step was filtered using a vacuum glass apparatus C15-C25.
onto stainless steel filters in order to isolate the larger microparticles
2.7. Determination of phthalic acid esters by SPME-LC/MS
(25 μm mesh size. Paco GMBH, German). The filters were examined
by μ-FTIR. In addition to plastic particles, the amount of plastic associated contam-
Step 4 Neutralization of the solution and isolation of the smaller microplastics inants, specifically of phthalic acid esters (PAEs), was evaluated in all the
(<25 μm): the filtrated solution from Step 3 was neutralized using HCl sampled sponges to assess any correlation between PAEs and the plastic
2 M solution added dropwise. Neutral pH was checked using litmus particle concentration (Saliu et al., 2020a). Analyses were carried out by
paper. The neutralized solution was filtered onto a glass microfiber fil- the SPME-LC/MS method described elsewhere (Saliu et al., 2020b). A sam-
ter to isolate the smaller microparticles (Whatman GF/D 0.2 μm) pling of the target compounds was carried out using BioSPME LC Tips,
using a dedicated vacuum glass apparatus. made of C18 functionalized silica particles (3 μm diameter) coated
Step 5 Pressurized Solvent Extraction (PSE): glass fiber filters collected (45 μm thickness) onto a 200 μm metal fiber and embedded with a biocom-
patible binder (Sigma Aldrich-Merck, Milano, Italy), which were inserted
after the second filtration (Step 4) were submitted to pressurized solvent
into a portion of each sponge for 40 min exposure in order to extract the
extraction (PSE). The conditions of PSE are detailed in Section 2.4. The
analytes. The analytes were then eluted from the fiber using methanol. Fi-
extracts obtained were dried and transferred into the pyrolysis cups for
nally, the extracts were directly injected into the LC-MS system for the
Py-GC–MS analysis. quantitative determination of 24 PAEs, by applying the selected mass tran-
sition and chromatographic conditions reported in Saliu et al. (2020b).
2.4. PSE condition
2.8. Quality assurance and quality control (QA/QC)
PSE was performed by employing a Speed Extractor® E-914 apparatus
provided by Buchi (Buchi Labortechnik AG, Flawil, Switzerland). The in- To limit background contamination and ensure data quality control
strumental conditions were previously tested on a glass bead matrix (Stile from sampling to extraction and analysis, precautions described in the liter-
et al., 2021) to verify recoveries and background contamination. They ature were adopted. The use of plastic labware was extremely limited.
were then adapted and validated for the extraction of plastic particles Glassware was washed with high purity dichloromethane (picograde
from the marine sponge tissues (Fuller and Gautam, 2016; Okoffo et al., from Promochem), heated at 350 °C overnight and rinsed before handling
2020). Briefly, filters were placed into 40 mL stainless steel extraction with the same ultrapure solvents adopted for the procedural steps (ultra-
cells, sealed with glass fiber filters, and submitted to a pre-extraction pure methanol or hexane). Whatman GF/D filter and stainless-steel filters
cycle with methanol at 100 °C, and three consecutive extraction steps in di- were individually wrapped with aluminum foil and heated at 450 °C for
chloromethane at 190 °C. Section S.2 and Table S1 of the Supporting Infor- 4 h in a muffle furnace (Fischer and Scholz-Böttcher, 2017). Sample manip-
mation provide additional details on the PSE procedure and conditions. ulation and vial preparation were performed in a clean air flow cabinet. Fil-
ters were stored in Petri dishes (P5481 Sigma-Aldrich) and covered in
2.5. Micro-FTIR analysis aluminum foil to prevent any additional contamination prior to the analy-
sis.
The presence of plastic particles ≥25 μm was verified by analyzing the Before each extraction, PSE cells were washed with acetone and soni-
entire surface of the stainless filters obtained after Step 3 of the workflow by cated three times for 30 min, followed by a DCM wash (Stile et al., 2021).
collecting three spectra for each detected particle. A Spotlight® 200i FT-IR In addition, all the deactivated steel cups used for Py-GC-MS were cleaned
(Perkin Elmer) equipped with liquid nitrogen cooled mercury cadmium tel- with a butane blow torch at 1400 °C in order to remove all the possible or-
luride (MCT) single detector was used to collect the spectra in point mode ganic contaminations before use.
according to a previously described procedure (Saliu et al., 2021): a total The Py-GC-MS system was cleaned in between analyses by performing
of 32 co-added scans were collected by operating in total reflection onto a two subsequent fast runs. The first run was performed using a clean
25 × 25 μm window by applying a wave-number range of empty cup containing 2 μL of the silylating agent hexamethyldisilazane
4000–400 cm−1 and a resolution of 4 cm−1. Background spectra were col- (HMDS) to remove any polar, low-volatility contaminants from the GC-
lected prior to each acquisition. COMPARE™ spectral comparison algorithm MS system. The second run was performed on the same empty cup without

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F. Saliu et al. Science of the Total Environment 819 (2022) 152965

HMDS to remove any excess derivatizing agent from the system. The lack of suitable sampling sites and organisms. Fig. 1 shows the study area and
consistent airborne microplastic contamination and the cleanliness of the the morphological appearance of the marine organisms under study. Spec-
laboratory and μ-FTIR equipment were checked by analyzing filters left in imens of the marine sponge cf. Haliclona (Haplosclerida) were collected in
the laboratory and in the μ-FTIR stage for 8 h and 1 h, respectively. the water surrounding two Maldivian islands, namely Magoodhoo (Faafu
Atoll, 3°4′49.08”N, 72°57′57.19″E) and Thudufushi (South Ari Atoll, 3°78′
2.9. Method validation 62” N, 72°73′10″ E). Table 1 reports a detailed list of the samples.
Magoodhoo is an inhabited island (~ 900 inhabitants) that measures
Validation parameters were calculated according to ICH guidelines 900 × 450 m, located in the south-eastern part of the atoll rim, about
(https://www.ich.org/, 2021). Tests to establish recoveries and accuracy 140 km south of the capital Malè, and its reef is approximately 2.9 km
were carried out on pre-spiked sponge tissue, since certificated materials long and 1.55 km wide. Thudufushi is a circular shaped resort island that
are not commercially available for this specific biological matrix. measures 3.5 km in length, including coralline reefs (land area of approxi-
Procedural blanks were carried out by submitting pre-washed glass mately 0.046 km2). Both reefs exhibit the features of typical low-energy
beads to the workflow used for the sponges to determine the average reefs, with a luxuriant growth of coral and gentle slopes on all sides
level of background contamination. In addition, the procedural blanks (Montalbetti et al., 2019; Montano et al., 2020; Montano et al., 2012).
were used to evaluate the limits of quantitation of the two analytical On each island, sponges were sampled from pristine and healthy reef
methods. areas characterized by no signs of mortality, bleaching, algal overgrowth,
For the μ-FTIR procedural blank analysis, the filters obtained after Step 3 disease, sedimentation, or any other anthropogenic stressors, and from im-
of the workflow were used. Since the particles studied were relatively large pacted reefs showing signs of degradation and mortality and anthropogenic
and the filter's entire surface was visually inspected, defining the minimum pressures. Two sites were selected in the waters surrounding Magoodhoo.
sample size and confidence interval was unnecessary (Schwaferts et al., Site 1 was characterized by a healthy coral reef and located at a greater dis-
2021). Specifically, only particles ≥25 μm were identified, considering tance from the island than Site 2 (~250 m far from the island). Site 2 was
that the lateral resolution of micro-FTIR spectroscopy is diffraction limited characterized by a degraded and almost totally dead coral reef near the
(i.e. 10 μm at 1000 cm−1). Therefore, the limit of quantitation was deter- island's solid waste landfill (~30 m far from the landfill) (Fig. 1). Two
mined starting with blanks count by applying the 10-Sigma method sites were also selected on Thudufushi based on their distance from the
(Schwaferts et al., 2021). sewage effluent outfall located at a depth ranging between 30 and 35 m.
The residue after Step 5 was analyzed for the Py-GC-MS procedural These consisted of Site 3, a sewage pipe outfall site situated on the eastern
blank analysis and the detection and quantitation limits were calculated. side of the island, and Site 4, a healthy coral reef located along with the
Specifically, LOQs were calculated by considering peak areas and the re- same reef system at approximately 200 m from the pipe (Fig. 1).
lated standard deviation in the procedural blank chromatogram (LOQ = In all these sites, different sponge fragments (from 2 to 8 g) were col-
mean + 6 SD). LODs were calculated by considering the slope of the curves lected using a knife and by snorkeling or SCUBA diving, depending on
of the mixed-matched calibration and residual standard variation, as indi- the depth (details reported in Table 1). Each sponge individual was sampled
cated in the ICH guidelines (https://www.ich.org/, 2021). once. To increase representativeness, different sponge individuals were
sampled at each site. All the fragments were wrapped in aluminum foil
2.10. Sample collection for pilot study and frozen at −20 °C in the MaRHE Center laboratory until subsequent
analyses.
Sampling for the pilot study was performed in coral reefs in the
Maldives in November 2019, using the Marine Research and High Educa- 2.11. Statistical analysis
tion (MaRHE) Center as a logistics base (https://marhe.unimib.it/),
which is located on Magoodhoo Island, Faafu Atoll, the Republic of The obtained data, expressed as mean ± standard error (SE), were sub-
Maldives. Pre-surveys were conducted by snorkeling and diving to select mitted to statistical analysis to assess possible significant differences

Fig. 1. Left: Map of the study area reporting the two islands in the Republic of Maldives selected for the sampling activities: Magoodhoo located in Faafu Atoll and Thudufushi
located in Ari Atoll. The sampling sites of sponges in each island are also indicated (see Table 1 for additional information). In Magoodhoo the arrow indicates the solid waste
landfill. Scale bars: 100 m.; Right: Marine sponge cf. Haliclona (Haplosclerida).

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Table 1
Sponge samples analyzed for microplastic determination and description of the related sampling points.
ID Weight (g) Island Typology of island Site Coral reef environment Lat N Long E

S1 2.64 Magoodhoo Inhabited 1 Healthy 3° 04′ 58′′ 72° 57′ 58′′

S2 3.66 Magoodhoo Inhabited 1 Healthy 3° 04′ 58′′ 72° 57′ 58′′
S3 2.48 Magoodhoo Inhabited 2 Degraded 3° 04′ 33′′ 72° 57′ 53′′
S4 2.07 Thudufushi Resort 3 Degraded 3° 47′ 04” 72° 43′ 54”
S5 3.32 Thudufushi Resort 3 Degraded 3° 47′ 04” 72° 43′ 54”
S6 7.90 Thudufushi Resort 4 Healthy 3° 47′ 11” 72° 43′ 56”
S7 5.78 Thudufushi Resort 4 Healthy 3° 47′ 11” 72° 43′ 56”

(p < 0.05), using SPSS v. 27 (IBM, New York). Since data were not normally subtracting the average amounts observed in the six non-spiked samples
distributed, the nonparametric Kruskal-Wallis H test was used to investi- (in all cases below 1% of the spiked amount). All aliquots were processed,
gate variations in the concentration of microplastics (particles/g and ng/g and the amount recovered on the filters after the filtration steps was evalu-
tissue) among the different exposures and sample sites. The Spearman coef- ated. The stainless steel filters obtained after Step 3 were weighed and in-
ficient was measured to assess possible correlations between the concentra- vestigated by infrared microscope to check recoveries as particles counts
tion of plastic particles and the concentration of phthalic acid esters (PAEs), and to assess possible background contamination. The glass fiber filters ob-
and in particular of DEHP, detected in the sponge tissues. tained after Step 4 were weighed and submitted to PSE.
The liquid extracts were reduced in volume using a Syncore apparatus
3. Results (Buchi Labortechnik AG, Flawil, Switzerland). The reduced extracts were
then transferred into pre-weighed vials and completely dried using a gentle
3.1. Analytical performance of the procedure stream of nitrogen at 40 °C. The weight of the dried residues was used to de-
termine the mass recovery for Step 5 (0.02 mg of precision using a Gibertini
The recovery of Steps 3, 4, and 5 and the efficiency in removing matrix analytical balance). A 95% recovery was found for the largest particles
interferences of the proposed analytical procedure were evaluated using a (50–250 μm, SD 5%), while for the smallest particles (<25 μm, SD 10%)
pre-spiked amount of reference sponge tissue digestate since certified mate- the recovery was 94% (Table 3).
rials are not commercially available for this specific biological matrix To evaluate the matrix effect of the constituents of the sponge tissue on
(Martins et al., 2011). Py-GC–MS analysis and the efficiency of the established analytical
Specifically, the digestate obtained after alkaline treatment (Step 1) of workflow in the removal of interference, single shot pyrolysis was prelimi-
the analytical workflow (Fig. 2) of 72 g of Portofino sponge was divided narily performed on a raw subsample of the Portofino sponge tissue, with-
into 12 aliquots. Six aliquots of the digestate were spiked with reference out any pretreatment. The profile obtained is reported in Fig. S2, and
plastic particles (25 mg at 50–250 μm size range and 25 mg of plastic par- features the pyrolysis markers characteristic of a protein material, i.e. pyr-
ticles <25 μm size, each). The other six aliquots were analyzed without role derivatives, toluene derivatives, styrene, and diketopiperazines. How-
spiking to evaluate the background amount of microplastics contained in ever, none of the signals deriving from the pyrolysis of the sponge tissue
the sponge matrix and use this value to calculate the recoveries. Since the except for styrene were detected in the procedural blanks.
Portofino sponge samples might have contained inherent plastic contami- Matrix effect was also evaluated for the Py-GC–MS analysis by drawing
nation, the calculations of the polymer recoveries were corrected by matrix-matched calibration curves for polystyrene (PS), polyvinyl chloride

Marine sponge SPME-LC/MS of phthalic

(Demospongiae) acid esters

Alkaline digeson
1 KOH 60° C, 24 h

Density separaon
2 ZnCl2 25%

μFTIR analysis of MPs Filtraon of the surpernatant

isolated onto the filters Stainless steel filters
3
(25 μm)

Neutralizaon and filtraon

Glass microfiber
4 Time--> 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

filters (0.2 μm)

Pressurized solvent
Py-GC-MS analysis
extracon (PSE)
5 of the dried extracts
of the filters

Fig. 2. Scheme of the optimized analytical workflow, including the non-destructive SPME-LC/MS analysis of phthalic acid esters.

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Table 2 Table 4
Characteristic pyrolysis products with their m/z signals used for the GC–MS identi- Results of the analysis by SPME-LC/MS of the phthalic acid esters, of the plastic par-
fication and construction of matrix-matched calibration curves of the different poly- ticles determination in the >25 μm fraction identified by μ-FTIR (after Step 3 of the
mers. The m/z signal reported in bold was integrated to obtain the peak areas of the workflow) and of the quantitation of the polymers in the 0.2–25 μm fraction ex-
corresponding specie. The figures of merit of the procedure are also reported, tracted by PSE and identified and quantified by Py-GC–MS (after Step 5 of the
namely the limits of detection (LOD) and quantitation (LOQ), and r2 of the calibra- workflow).
tion in the 0.05–10 μg/g range. Island Magoodhoo Thudufushi
Polymers Selected pyrolysis Retention m/z LOD LOQ r2
Sample n. S1 S2 S3 S4 S5 S6 S7
products time (min) signals (ng/g) (ng/g)
SPME-LC/MS (ng/g) DEHP 8.6 – – 15 – 15 –
PVC HCl 2.5 36, 38 4.8 9.4 0.9915
PS – – – 0.4 – – –
PP 2,4-dimethyl-1-heptene 8.8 70, 126 9.8 13.8 0.9994
PVC – – – – – – –
PS 2,4-diphenyl-1-butene 16.2 91, 208 6.6 9.3 0.9905
PC – – – – – – –
(dimer)
PP – 0.1 0.4 0.4 0.4 – –
PC bisphenol 18.5 213 3.0 6.6 0.9866 μ-FTIR (particles/g)
PE 0.8 1.6 – – 1.2 – –
PE α,ω-alkenes C15-C25 19–25 82 25.1 30.2 0.9972
PL 0.4 0.4 – 0.8 – – –
(average of the areasa)
PA – – – 0.4 0.4 – –
a
11 peaks were integrated for quantifying PE22. TOTAL 1.2 2.1 0.0 1.2 1.6 – –
PS 252 – 121 190 – 63 –
PVC 4.7 129 4.7 40.0 139 67 100
(PVC), polycarbonate (PC), polypropylene (PP), and polyethylene (PE). The PC 59 – – – – – –
Py-GC/MS (ng/g)
entire sponge specimen was subjected to extraction, and increasing aliquots PP 2457 – 964 2370 1166 578 –
of solid standard polymers were added to five aliquots of the extract after PE 15 – 573 2245 2087 275 389
Step 5 of the workflow and analyzed to construct the matrix-matched cali- TOTAL 2866 129 1673 4853 3406 985 506

bration. The curves showed good linearity within a 0.05–10 μg/g

range with r2 between 0.9866 and 0.9972. LODs and LOQs were calcu-
lated between 3.0 and 25.1 ng/g and 6.6 and 30.2 ng/g, respectively 3.3. Quantification of the plastic particles in Maldivian sponges by μ-FTIR
(Table 2).
The μ-FTIR analysis of the filters obtained after Step 3 of the workflow,
3.2. Quantification of the plastic particles in Maldivian sponges by Py-GC-MS and related to the particles >25 μm in the sponge tissue, revealed plastics in
most of the analyzed specimens, with a maximum of 2.1 particles/g of
Table 4 reports the results of the quantitative Py-GC-MS analysis of the sponge in sample S2 (Table 4). Among the particles >25 μm, the largest
seven Maldivian sponges. The analyses entailed the quantitation of plastic fraction consisted of polyolefins (PE and PP), representing more than
polymers in filtrates obtained in Step 5 of the workflow, which comprise 75% of the total number of particles detected (Supporting information,
the finer microplastics in the 0.2–25 μm size range that the spectroscopic Fig. S4). In addition, polyamides (PA), polystyrene (PS) and polyester
approach cannot detect. The pyrogram of sample S5 is shown in Fig. S3 (PL) were also detected in relatively lower abundances.
as an example. Other particles detected in the samples consisted mainly of carbonates
The analysis of three replicates showed that polyolefins (PE and PP) retained during the floatation process and probably originating from the
were the most represented polymers in most of the samples (>75% in six sponge spicules. Concerning the color, three main colours were detected
samples), with an average concentration of 1076 ± 328 ng/g for PP and (Supporting information, Fig. S5): transparent/grey was the most repre-
797 ± 361 ng/g for PE. Considering the total amount of plastic per gram, sented (68%), followed by brown (27%) and black (5%). Concerning the
the most polluted samples were S4 and S5 located on the resort island shapes, the observed particles were mainly fragments, foams, and granules,
near the sewage pipe with the total detected plastic values of up to with some filaments (Supporting Information, Fig. S6). The size of the par-
4852 ng/g. On the other hand, the least polluted sample was S2, from the ticles was between 57 μm and 278 μm. No larger particles (>500 μm) were
Magodhoo island at the check-dive point, with a total plastic concentration observed. The average concentration of microplastics >25 μm in the sponge
of 129 ng/g. Interestingly, this sample was the only one with PVC predom- tissues, as detected by inspecting the filters after Step 3 of the workflow, was
inance among the detected polymers. The 0.2–25 μm range fraction ana- 0.9 ± 0.3 particles/g. Specifically, the sponges sampled on Magoodhoo is-
lyzed after Step 5 of the workflow of sample S1 was the only one land showed an average of 1 ± 0.58 particles/g, whereas the sponges from
containing a significant amount of PC, quantified based on the pyrolysis Thudufushi island showed an average of 1 ± 0.71 particles/g. The highest
marker, bisphenol. Note that as it is a common plasticizer, bisphenol concentration of microplastics was found in a sample collected near the
may be present in environmental samples; in this case, it would be pres- sewage pipe in Thudufushi, with a concentration of 2.5 particles/g. One
ent in its free molecular form; thus it will not be retained on the filter sponge collected near the rubbish dump and another collected from the
surface. control sites showed no microplastics.

Table 3
Percentage mass recovery and standard deviation obtained for the 6 not spiked and spiked aliquots of sponge tissue digestate, after Steps 3, 4 and 5 of the workflow (mass
recovered /mass submitted to extraction ×100); * In the case of unspiked samples the values in percentage are theoretical and used only to express the contribution of
the background contamination in the calculation.
Recovery Sample description Particles 250–50 μm Particles < 25 μm

Recovery SD Recovery SD
(%) (%) (%) (%)

Recovery onto stainless steel filter (recovery of Step 3) Spiked with 25 mg of plastmix at 50–250 μm size range and 95 5.4 0.8 1.2
25 mg of plastmix <25 μm size
Recovery onto glass fiber filter before SPE (recovery of Step 4) Spiked with 25 mg of plastmix at 50–250 μm size range and 0.8 1.3 93 7.8
25 mg of plastmix <25 μm size
Recovery in the SPE extract (recovery of Step 5) Spiked with 25 mg of plastmix at 50–250 μm size range and 0.3 0.7 94 9.5
25 mg of plastmix <25 μm size

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F. Saliu et al. Science of the Total Environment 819 (2022) 152965

3.4. Non-destructive determination by SPME-LC/MS of phthalic acid esters different sites (Fig. 3). Considering the total amount of plastic detected by
(PAEs) in the Maldivian marine sponges Py-GC–MS, no significant differences were found between the different
sites and the different islands sampled. However, the concentration of poly-
SPME-LC/MS analysis showed that of the 23 investigated PAEs, Di(2- ethylene was found to be higher in the samples collected from sites with a
ethylhexyl)phthalate (DEHP) was the only one detected above LOQs. Di degraded coral reef environment (S3, S4, S5) (Fig. 3).
(2-ethylhexyl)phthalate was found in three of the seven samples (Table 4)
and specifically in those from Magoodhoo (S1), and Thudufushi (S4 and 4. Discussion
S6), with an average concentration of 8.6 ± 2.3 and 15.2 ± 3.0 ng/g
respectively. Biological matrices need rigorous sample preparation to obtain a stable
and sensitive determination of microplastics by Py-GC-MS, mainly because
3.5. Comparison of sampling sites some of their biochemical components may be co-extracted and cause ma-
trix interference (Fischer and Scholz-Böttcher, 2019).
The whole data set was subjected to statistical analysis to assess possible Preliminary sample preparation is also needed for microspectroscopy
correlations, as summarized in Fig. 3. Analysis of variance was carried out analysis since organic layers on the filter surface along with the presence
by considering the average and standard deviation of the detected concen- of a large number of non-plastic particles deriving from the matrix
trations measured in each specimens from the related subgroup. Overall, no (e.g. minerals, ingested planktonic organisms) may prevent plastic par-
significant correlations were found between DEHP and plastic polymer con- ticle identification either because they may be covered or the large num-
centrations or considering the total plastic concentration and the concentra- ber of foreign items may significantly slow down the point-by-point
tion of each plastic polymer. In addition, no correlation was observed even identification during the filter survey.
for PVC, a polymer that is known to be generally formulated with high The results of the analytical optimization reported in Section 3.1
levels of phthalates as additives (Saliu et al., 2020b). showed that our proposed method efficiently removes the matrix interfer-
Variance analysis (one-way ANOVA, p > 0.05) of the data obtained by μ- ence that may have a detrimental effect on both the microspectroscopy
FTIR – considering both the polymers individually and in total – did not and Py-GC–MS polymer identification and quantification. In addition, the
highlight any significant differences between the two islands or the results show that the method provides reliable isolation and determination

Fig. 3. (A) Concentration (ng/g of sponge) of DEHP, (B) levels of the different plastic polymers (particles size, μ-FTIR: > 25 μm) and (C) concentration (ng/g of sponge) of the
different plastic polymers (Py-GC/MS: 0.2–25 μm) found in the Maldivian sponges collected in Magoodhoo and Thudufushi islands and in areas with healthy or degraded
coral reef environment. All the values (ng or n° particles) are expressed in relation to the sponge dry weight (g). Data are expressed as mean ± SE (standard error of the
mean). Asterisk: a significant difference is detected by 1-way ANOVA in the levels of PE between sites with a different status of coral reef environment (healthy/degraded).

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F. Saliu et al. Science of the Total Environment 819 (2022) 152965

of the plastic particles in marine sponge tissue with good sensitivity and re- Another possible mechanism of transporting plastics to the seafloor is
peatability. It is worth noting that since the determinations were carried out through the marine food chain. Small particles may be ingested by zoo-
on the particles isolated by filtration, our workflow enables us to rule out plankton and excreted as fecal pellets, which may speed up particle sedi-
any possible contamination from plasticizers such as bisphenol, and thus mentation (Choy et al., 2019).
provides a confident determination of the strict polymeric origin of the sig- Lastly, the colloidal interaction of the plastic particles with the marine
nals detected by Py-GC–MS. The protocol was also improved by the SPME- snow or the turbidity currents may act as an active force pump (Woodall
LC/MS analysis of phthalic acid esters to establish them as a possible proxy et al., 2014). This could thus explain the heterogeneous distribution of
of plastic contamination. the various plastic polymers observed in our sponge samples, with positive
The identification and characterization by microspectroscopy of the buoyant polyolefins being the most represented plastic polymers in both
larger particles unambiguously highlight the ingestion of plastic items the size range fractions examined. Interestingly, no harmful buoyant poly-
by the sponge, with helpful information regarding their morphological mers, such as PVC nor PC, were detected in any sample in the form of par-
appearance and the level of photooxidative degradation (i.e. by measur- ticles >25 μm. Since these polymers are widely employed for several
ing the carbonyl index in polyolefins). In addition, Py-GC–MS provided applications, they were also expected to be detected as marine pollutants
the identification and quantification of the polymers present in the plas- in the Maldivian environment.
tic particle fraction, which is not available for visualization by micros- The survey results showed no statistically significant differences
copy (< 1 μm). between the two islands or the different sites surveyed. However,
Our results on the Maldivian sponges highlighted that these marine in- local factors may play a key role in determining variations in the abun-
vertebrates are heavily affected by microplastic contamination since more dance of plastic materials. Nonetheless, the most degraded sites in
than half of the analyzed specimens showed plastic particles with an aver- terms of coral reef environments were characterized by the highest
age concentration of 1.0 ± 0.5 microplastics/g. number of polyethylene particles in the sponge samples, and thus a
Previous studies on filter feeder marine invertebrates used as cause-effect relationship cannot be ruled out and needs further inves-
bioindicators indicate similar levels of incidence and contamination levels tigation.
(Van Cauwenberghe et al., 2015). For the brittle star, Amphiura filiformis An analysis of a much larger set of samples is necessary to assess
and the polychaete Sabella pavonina, Bour et al. (2018) reported the pres- whether microplastic contamination is ubiquitous in sponges from the
ence of plastic particles in 20% and 25% of the analyzed individuals, re- Maldivian archipelago and to evaluate statistical differences in the sponge
spectively. Vered et al. (2019) reported a higher incidence for individual populations.
ascidians, with most specimens collected in the Red Sea showing average We believe that sponges are widely distributed in benthic organisms
levels of 1.37 ± 1.29 particles per individual (n = 35). Cho et al. (2021) that are resilient to environmental stressors. They can be used as a sentinel
reported a mean concentration of 0.33 ± 0.23 microplastics/g in oysters of plastic contamination before the pollution reaches levels that may endan-
and 0.43 ± 0.32 microplastics/g in clams. ger more sensitive species with irreversible effects on the invaluable natural
To the best of our knowledge, there is still a lack of knowledge regarding marine heritage.
the mechanism of transferring the plastic particles into the marine sponge We have demonstrated that sponges may incorporate plastic parti-
tissues, and no studies have reported rates either for passive or active inclu- cles and have highlighted the differences between different sampling
sion. In fact, this information is obtainable through artificial exposure ex- areas. Since for some marine invertebrates the ingestion of plastic parti-
periments that have still not been carried out. In order to discuss the cles has been reported to be the result of a possible chemoreceptive mis-
evidence of the plastic particle found in the marine sponges reported identification (Procter et al., 2019), it is important to also investigate
here, it is important to first consider that marine sponges are diploblastic or- this mechanism for marine sponges in order to highlight possible excre-
ganisms (Becerro, 2008). Therefore they are likely to be exposed to tion and/or detoxification mechanisms active after ingestion. Future
microplastic penetration by diffusion. However, active filtration is expected studies should also investigate the presence of molecular stress markers
to play significant role in incorporating plastic particles. It is known that in the sponge tissues, which would provide information on the physio-
food particles enter sponges by porocytes, are trapped by choanocytes, logical health conditions of sponges, finding specific molecular patterns
retained in vacuoles and submitted to digestive processes (Hammel and that could discriminate the impact of plastic particles from other envi-
Nickel, 2014). A similar process may likely occur with plastic particles, ronmental stressors.
which cannot be digested. The phagocytosis mechanism, carried out by
the exopinacoderm in non-spiculate sponges (Cerrano et al., 2007) and ca- 5. Conclusions
pable of promoting the incorporation of particles up to 2 mm in diameter, is
not considered here since cf. Haliclona is a spiculate sponge, and we found A micro spectroscopy and Py-GC-MS combined approach was opti-
no particles larger than 230 μm. mized for detecting plastic particles in marine sponges, which was then ap-
The evidence that plastic particles may reach a benthic organism plied to survey plastic contamination in the Maldivian reef. Analyses
merits careful consideration in terms of marine pollution. Plastic con- showed the prevalence of polyolefin plastic polymers and the contamina-
tamination is in fact widely described for the sea surface and within a tion levels between the different sites surveyed. Overall, the study demon-
neustonic habitat (Fossi et al., 2012; Moore et al., 2001), whereas evi- strates the suitability of sponges as benthic organisms for monitoring
dence that microplastics may reach the seafloor has only recently been plastic contamination in marine environments.
presented (Sanchez-Vidal et al., 2018). The sedimentation of plastic
items that are not buoyant is to a certain degree expected, however plas- CRediT authorship contribution statement
tic degradation and fragmentation in anoxic conditions occur more
slowly in relation to sea surface (Bagaev et al., 2021; Chubarenko FS: conceptualization, data curation, investigation, methodology, vali-
et al., 2016). dation, visualization, writing (original draft); GB: formal analysis, data
On the other hand, the sedimentation of plastic items that are less dense curation, investigation, methodology, validation, writing (original draft);
than seawater is generally explained by the fact that floating particles are CR: formal analysis, data curation, investigation, methodology, validation;
subjected to long range transport, and may eventually impact sediments JLN: formal analysis, data curation, investigation, methodology, validation,
in coastal areas (Chubarenko et al., 2016). Due to prolonged exposure to writing (original draft); ID: conceptualization, data curation, visualization,
weathering conditions and mechanical stress, fragmentation occurs and writing (original draft), supervision; DS: conceptualization, visualization,
micro and nano particles are formed (Efimova et al., 2018). In addition, writing (original draft); PG: financing, supervision; ML: financing, supervi-
due to photo-oxidative degradation and/or biofouling, these particles sion; FM: conceptualization, data curation, visualization, writing (original
may increase in density and sink to the seafloor (Egger et al., 2020). draft), financing, supervision.

8
F. Saliu et al. Science of the Total Environment 819 (2022) 152965

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“FAR 2019”. leen whales exposed to the threat of microplastics? A case study of the Mediterranean fin
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ests or personal relationships that could have appeared to influence the ments and organic pollutants in marine sponges from the South Brittany coast, France.
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bioindicators for microparticulate pollutants? Environ. Pollut. 268 (Part A), 115851.
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Evaluation of thermoanalytical methods equipped with evolved gas analysis for the de-
versity of Pisa (CISUP) for providing the Py-GC-MS instrumentation. We tection of microplastic in environmental samples. J. Anal. Appl. Pyrolysis 152.
would like to thank Inga Denhert and Sara Vencato for their valuable help de Goeij, J.M., van Oevelen, D., Vermeij, M.J.A., Osinga, R., Middelburg, J.J., de Goeij,
in the sampling. A.F.P.M., et al., 2013. Surviving in a marine desert: the sponge loop retains resources
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iments of an urban fjord in south West Norway using a thermal degradation method.
Chemosphere 227, 705–714.
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Gonçalves, J.M., Bebianno, M.J., 2021. Nanoplastics impact on marine biota: a review. Envi-
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