# Evidence of niche partitioning among bacteria living on plastics, organic particles and surrounding seawaters

Claire Dussud, Anne-Leïla Meistertzheim, Pascal Conan, Mireille Pujo-Pay, Matthieu George, Pascale Fabre, Jean Coudane, Perry Higgs, Amanda Elineau, Maria-Luiza Pedrotti, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Claire Dussud, Anne-Leïla Meistertzheim, Pascal Conan, Mireille Pujo-Pay, Matthieu George, et al. Evidence of niche partitioning among bacteria living on plastics, organic particles and surrounding seawaters. Environmental Pollution, 2018, 236, pp.807-816. 10.1016/j.envpol.2017.12.027. hal-04186967

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Submitted on 25 Aug 2023

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<figure><img src="image_2.png" /></figure>
Evidence of niche partitioning among bacteria living on
plastics, organic particles and surrounding seawaters

Dussud C¹, Meistertzheim AL¹, Conan P¹, Pujo-Pay M¹, George M², Fabre P², Coudane J³,
Higgs P⁴, Elineau A⁵, Pedrotti ML⁵, Gorsky G⁵, Ghiglione JF¹*

¹Sorbonne Universités, CNRS, UPMC Univ Paris 06, UMR 7621, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique de Banyuls, Banyuls sur mer, France

²Laboratoire Charles Coulomb (L2C), Univ. Montpellier, CNRS, Montpellier, France.

³Institut des Biomolécules Max Mousseron, CNRS UMR5247, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier, BP 14491, F-34093 Montpellier cedex5

⁴Symphony Environmental Ltd, Borehamwood, Hertfordshire WD6 1JD, UK.

⁵Sorbonne Universités, CNRS, UPMC Univ Paris 06, UMR 7093, Laboratoire d'Océanographie de Villefranche, Villefranche sur mer, France

*Corresponding author:

JF Ghiglione, Laboratoire d'Océanographie Microbienne, Observatoire Océanologique de Banyuls, Avenue Fontaulé, F-66650 Banyuls/mer, France. Tel.: +33 (0) 4 68 88 73 16. E-mail address: ghiglione@obs-banyuls.fr

Note : légendes des figures à la fin de la publication.
**Abstract**

The Mediterranean Sea is one of the most plastic-polluted zone and offer a unique opportunity to test the hypothesis of bacterioplankton partitioning between free-living (FL), organic particle-attached (PA) and the recently introduced plastic marine debris (PMD). The large set of samples taken during the *Tara*-Mediterranean expedition revealed for the first time a clear niche partitioning between the three co-occurring communities and marked originality in the PMD fraction. Bacterial counts in PMD presented higher cell enrichment factors than generally observed for PA fraction, when compared to FL bacteria in the surrounding waters. Taxonomic diversity was also higher in the PMD communities, where higher evenness indicated a favorable environment for a very large number of species. *Cyanobacteria* were particularly overrepresented in PMD, together with essential functions for biofilm formation and maturation. The consistent distinction of community structure between the three lifestyles, that exceeded the large-scale geographical variation in the Western Mediterranean basin, support a new trade-off for the maintenance of a large diversity of bacterioplankton in the Oceans. 'Plastic specific bacteria' recovered only on the PMD represented half of the OTUs, thus forming a distinct biota that should be further considered for understanding microbial biodiversity in changing marine ecosystems.

**Keywords:** microbial ecotoxicology, plastisphere, biofouling

**Capsule:** This study is the first description of bacterial abundance and diversity on plastic marine debris (PMD) in the Mediterranean Sea; it describes PMD as a particular biota for marine bacteria compared to particle-attached and free-living fractions in surrounding waters.
**Introduction**

Plastic litter have become the most common form of marine debris and a growing global pollution concern, affecting marine ecosystems and economic activities (Gregory, 2009). The discovery of five large-scale accumulation areas of floating PMD in the subtropical ocean gyres has captured worldwide attention, which often refers to these zones as “great garbage patches” (Cózar *et al.*, 2014; Eriksen *et al.*, 2014). These accumulation areas are dominated by plastic pieces, mainly smaller than 5 mm, commonly referred to as “microplastics” (Andrady, 2011; Law and Thompson, 2014). Recent studies have demonstrated that the Mediterranean Sea can be regarded as another great accumulation region with average concentration of more than 200 000 items km⁻², comparable to subtropical oceans gyres (Lebreton *et al.*, 2012; Pedrotti *et al.*, 2016). The accumulation of floating plastic in the Mediterranean Sea is explained by the high human density pressure together with the hydrodynamics of this semi-enclosed basin with residence time of 70 years (Durrieu de Madron *et al.*, 2011).

Bacterioplankton represent the most abundant organisms in seawater where they are also the main actors of nutrient regeneration and carbon cycling by converting organic matter to biomass, which supplies microbial food webs and transfers carbon and energy to higher trophic levels (Gasol *et al.*, 2008). Dissolution and degradation of 75% of the organic particles sinking to the sea floor revealed the key role played by PA bacteria in the regulation of the carbon storage by the Oceans (Farooq and Malfatti, 2007; Mével *et al.*, 2008). Particulate organic carbon and more precisely “phycospheres” and “detritospheres” represent micro-patches of concentrated substrates that are hot spots for pelagic bacterial processes (Ghiglione *et al.*, 2009). PA bacteria differ from the surrounding FL fraction in many aspects, including cell abundance, morphology, diversity and metabolic activities (Pulido-Villena *et al.*, 2014; Simon *et al.*, 2002). Microplastics may be considered as new particles recently
introduced within the last 60 years in the Oceans, which offer novel habitats for bacteria and
enhance their dispersal over long ways by drifting with currents and winds (Lebreton et al.,
2012). Recently, Zettler et al. (2013) introduced the term “plastisphere” to describe the very
diverse PMD-associated microorganisms, which were found in the seawater sub-surface to
differ from the surrounding FL bacteria. Seafloor plastisphere was also found to be
genetically unique from the FL water-column bacteria and in addition, they were distinct from
the sediment communities in the North Sea (De Tender et al., 2015).

The purpose of this study was first to test the hypothesis of the existence of specific
communities associated to PMD particles compared to the PA bacteria. This question is not
trivial since any surfaces exposed to seawater are rapidly colonized by bacteria and generally
form biofilms (Siboni et al., 2007; Lobelle and Cunliffe, 2011). Because the few studies so far
on floating microplastics collected at Sea only compared the PMD to FL bacteria (Zettler et
al., 2013, 2015), the question of the existence of a ‘plastic-specific’ community in contrast to
the PA fraction is still open. Here, we provide microscopic counts together with a large
dataset of 16S metabarcoding of bacteria living in the PMD, PA and FL fractions collected
during the *Tara*-Mediterranean expedition. We also used a computational approach to predict
the potential functional differences between the three communities. Finally, we investigated
the influence of the location-dependent environmental factors together with the plastic-related
properties as potential drivers of the plastisphere characteristics.
# Materials and methods

## Plastic and surrounding seawater sampling

Samples were taken during the *Tara*-Mediterranean expedition (April-November 2014) aboard the RV *Tara*. PMD were recovered from 32 manta trawls (mesh size 333 µm) carried out in the Western Mediterranean Sea (Figure 1). A subset of these fragments was sorted with sterilized forceps, rinsed with sterile seawater and immediately frozen for further physical, chemical and bacterial diversity analysis. For microscopic observations, PMD were fixed for at least 20 min at room temperature with 2% (v/v) glutaraldehyde (final concentration) before freezing. A conductivity-temperature-fluorescence-depth profiler (CTD – SeaBird SBE 19) was deployed at each of the 32 stations, together with Niskin bottle for surface seawater collection. A fractionation procedure was performed onboard in one step for 2 L seawater sequential filtration through 3 µm-pore size (PA fraction) and 0.2 µm-pore size (FL fraction) polycarbonate filters (47mm diameter, Nucleopore) using a peristaltic pump (pressure <100 mbar) and filters were subsequently frozen until further DNA extraction, as previously described (Ghiglione et al. 1999).

## Physical and chemical characterization of the PMD

The one-dimensional area of the surface, the major length and circularity of the individual PMD were determined using ZooScan system (Hydroptic Ltd., EMBRC France) (Gorsky et al., 2010). Fourier Transform Infrared Spectroscopy (FTIR) analysis was performed on the individual PMD using Spectrum 100 equipped with an ATR attenuated total reflectance (Perkin-Elmer) and compared to references for identification of the nature of the polymer. Carbonyl index values were determined as the ratio of the peak intensity at 1,715 cm⁻¹ to the peak intensity at 1,460 cm⁻¹, as previously described (Selke et al., 2015).
## Microscopic observations

Bacteria were enumerated on 70 plastic pieces sampled at 22 stations (from 1 to 4 pieces per manta, when microscopic observation was possible) by epifluorescence microscopy using an Olympus AX-70 PROVIS after 4', 6-diamidino-2-phenylindole (DAPI) staining. The covering of the surface area of the plastic by bacterial cells was determined using Image J software. Cell enrichment factors were calculated by the ratio between bacterial counts in PMD and in the FL fraction, brought forward to a same volume of 1 mm⁻³ when considering a mean PMD thickness of 100 µm.

For qualitative assessment of biofilm structure, a random collection of 8 samples was chosen for scanning electron microscopy (SEM) using Inspect S50 (FEI, Hillsboro, OR, USA), as described previously (Zettler *et al.*, 2013). Some of the samples were also analyzed by atomic force microscope (AFM) to get more resolved insight of the surface, using a Nanoscope V in dynamic mode (Bruker instruments, Madisson, WI, USA) and standard silicon probes (Bruker, TESP-V2) (Binnig and Quate, 1986).

## DNA extraction and next-generation sequencing

DNA was extracted from 72 PMD and from all the 3 µm- and 0.2 µm-pore size filters from surrounding seawater from each of the 32 stations. Between 2 and 4 plastic fragments were extracted separately at each station to evaluate intra-site variability. DNA extraction followed a classical phenol-chloroform-based protocol (Ghiglione *et al.*, 1999) with an additional sonication step (3 x 5 sec with 30% amplitude, Branson SLPe) for a better disruption of the biofilm. The molecular size and the purity of the DNA extracts were analyzed by agarose gel electrophoresis (1%) and the DNA was quantified by spectrophotometry (GeneQuant II, Pharmacia Biotech). Primers for PCR amplification of the 16S V3-V5 region were 515F-Y and 926R, particularly well-suited for marine samples according to Parada *et al.* (2016), with
Illumina-specific primers and barcodes. Sequencing was performed on an Illumina MiSeq by Research and Testing Laboratories (Lubbock, TX). Raw FASTA files were deposited at GenBank under the accession number SRP102645. Paired raw reads were joined, quality-filtered and assigned to taxa using Qiime pipelines, as previously described (Severin et al., 2016). Operational taxonomic units (OTUs) were defined as clusters sharing 97 % sequence identity. Only Bacteria were treated in this study, due to the small number of Archaea reads. Bacterial sequences were randomly resampled in the OTU file to enable comparison between samples, by normalizing the number of sequences between samples to the sample with the fewest sequences (n=8,541) using MacQiime 1.9.0 (single_rarefaction.py). All further analyses were performed on the randomly re-sampled OTU table.

## Statistical analysis, diversity assessment and predictive metagenome analysis

Alpha diversity was estimated by non-parametric Chao1 species richness estimator using SPADE software; together with Simpson, Shannon and Pielou diversity indexes using PRIMER 6 (PRIMER-E, UK). Differences between FL, PA and PMD richness and diversity indexes were tested using non-parametric Kruskal-Wallis test (R software). Nonmetric multidimensional scaling (NMDS) was used for visualization of beta-diversity based on Bray-Curtis similarities. One-way analysis of similarity was performed on the same distance matrix (ANOSIM, PRIMER 6) to test the significant difference and to partition the variance of the beta-diversity matrix between FL, PA and PMD. We performed a PICRUSt analysis to obtain a predictive functional profile of the bacterial communities, calculated by linking individual species to metabolic functions. The mean of Nearest Sequenced Taxon Index (NSTI) had a score of 0.08 ± 0.03, thus indicating a reliable estimation of the predictions for our samples (Langille et al., 2013). We identified the OTUs or the functions primarily responsible for dissimilarities between pairs of FL, PA and PMD groups using similarity percentage analysis
(SIMPER, PRIMER 6). To investigate the relationships between bacterial community structure and environmental parameters, we used a direct multivariate gradient approach, i.e., a canonical correspondence analysis (CCA) using the software package CANOCO 4.5, as previously described (Berdjeb *et al.*, 2011). Spearman rank pairwise correlations between the environmental variables helped to determine their significance. We used a Monte Carlo permutation full model test (199 permutations) to statistically evaluate the significance of the canonical axes. Explanatory variables were determined first by using a forward-selection procedure (999 permutations) and second, by successive addition of variables until it failed to contribute significantly (p<0.05) to a substantial improvement of the model.

# Results

## PMD characterization and environmental parameters

We visually selected 80 PMD in order to get between 2 to 5 pieces for each of the 32 Manta trawls. The size, shape, color, composition and oxidation state were determined for all pieces (Suppl. Table 1). The pieces showed varied shapes like filaments, pellets or sheets with about 46% transparent, 28% white and 12.5% colored (Figure 1). Zooscan analysis showed that the pieces were on average of 9.3 mm in size (SD=4.7). Ranging from 2.8 to 23.8 mm, most of the pieces did not correspond exactly to the arbitrary limit of microplastics (size <5mm), but were representative of the majority of PMD recovered in all manta nets. The shape of the pieces, as depicted by the contour circularity, ranged between 0.05 and 0.75 (mean=0.39, SD=0.14). FTIR analysis revealed that polyethylene (PE) dominated the composition in the 80 plastic pieces (72.2%), followed by polypropylene (PP) (18.0%), polystyrene (PS) (2.8%) and others, mainly dominated by composite plastics (7.0%). The mean carboxyl index (COi) was on average 15.3% (SD=12.5%; min=1.6% and max=93.2%, both extreme values reached in PE pieces) with more than 8% of PMD presenting higher degree of oxidation (>25 %).
<figure><img src="image_3.png" /></figure>

Bacterial abundance on PMD

Inspection of 72 plastic pieces from 20 manta trawls by epifluorescence microscopy revealed that most of the samples were highly colonized by bacteria (Figure 2). Bacterial counts were on average $4.4 \times 10^4$ cells mm$^{-2}$ (SD = $3.7 \times 10^3$). It corresponded to an average enrichment factor of 888 (SD = 731, min=80 and max=3748) compared to the FL bacteria in surrounding waters, when considering the same volume. Variation in cell density (between $1.1 \times 10^3$ and $1.9 \times 10^5$ cells mm$^{-2}$) could not be statistically correlated to PMD characteristics (surface, shape) or environmental parameters (distance to the coast, temperature, salinity and chlorophyll *a* of the surrounding water). A large variety of morphotype was observed (Figure 2), including coccus-, rod- and spiral-shaped cells with a large number of cells with
prosthecae and long filaments together with cells with pili and flagella. We often observed
Cyanobacteria, such as *Calothrix sp.* frequently embedded in an amorphous mass of
*Pleurocapsa sp.* cells. Often occurring in rows or patches, bacteria were non-uniformly
distributed on the plastic surface, covering between 0 and 3.5% of the surface area of the
plastic. Other eukaryotic organisms were observed on plastics, mainly diatoms and more
randomly dinoflagellates, fungi and bryozoans, but were not further considered in this study.
All plastics showed signs of degradation, including cracks and pitting hosting numerous
organisms, as shown by scanning electron microscopy (SEM) and atomic force microscopy
(AFM) (Figure 2).

<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

Diversity indexes of PMD colonizers compared to FL and PA fractions

Next-generation DNA sequencing resulted in 1,135,953 sequences falling into 1,340
OTUs at the 97% similarity level, after randomly resampling to 8,541 sequences per sample to provide statistical robustness when comparing diversity measures among samples. Each of the FL, PA and PMD fractions were composed of 35.1, 56.0 and 83.6% of the total number of OTUs, respectively. Rarefaction analysis (not shown) suggested that the FL, PA and PMD bacterial diversity approached that of an asymptote. Chao1 estimator showed that the richness of the PA (mean=173, SD= 41.7, n= 24) was significantly higher (Kruskal-Wallis test, p<0.001) than the FL community (mean=125, SD=39.5, n=28), whereas PMD communities presented intermediate values (mean=160, SD=69.5, n=74) non-significantly different from the other two fractions (Figure 3). Pielou index revealed a significantly higher evenness in the PMD community (mean=0.65, SD=0.10) compared to the PA (mean=0.53, SD=0.11) and FL fractions (mean=0.60, SD=0.07) (Kruskal-Wallis test, p<0.001). Consequently, PMD presented a significantly higher Shannon diversity index (mean=3.10, SD=0.47) compared to the PA (mean=2.62, SD= 0.59) and FL fractions (mean=2.71, SD= 0.30) (ANOVA p<0.001).

<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>

**Bacterial community structure in relation to PMD characteristics and environmental parameters**

Non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarities revealed that the global pattern of bacterial diversity was explained in part by the FL, PA and PMD lifestyles (Figure 4). Analysis of similarity (ANOSIM) showed these groups to be highly significant (UniFrac and p test significance, both p<0.001). The overall dissimilarity between
PMD cluster and FL and PA cluster was 69%. FL and PA formed distinct sub-clusters with lower within dissimilarities than in the PMD cluster (41, 52 and 60%, respectively). To evaluate the factors driving the bacterial community structure of the PMD communities, we performed a canonical correspondence analysis (CCA) (Suppl. Figure S1). The best statistical model explained only 11.0% of the total variance (P < 0.05). Variation partitioning indicated that the PMD characteristics (surface, shape and carbonyl index) and the few environmental variables available across all datasets (temperature, salinity and Chl *a*) contributed equally to the total variance explained. No significant relationship was found neither with the polymer type (i.e. PE, PP, PS and others) nor among the geographical sampling sites (inter-site) or within plastic pieces sampled in the same Manta trawl (intra-site).

<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>
Taxonomic difference between FL, PA and PMD fractions

Most of the OTUs in the FL fraction were dominated by Alphaproteobacteria (45.0%, mainly *Pelagibacter* sp.), followed by Cyanobacteria (24.3%, mainly *Synechococcus* sp.), Flavobacteria and Gammaproteobacteria (11.3% and 11.1%, respectively) (Figure 5). OTUs in the PA fraction were characterized by closer percentages between the 3 dominant classes, including Alphaproteobacteria (25.9%, mainly *Erythrobacter* sp.), Gammaproteobacteria (25.0%, mainly *Alteromonas* sp.) and Cyanobacteria (17.9%, mainly *Synechococcus* sp.). The PMD fraction was dominated by Cyanobacteria (40.8%, mainly *Pleurocapsa* sp.) and Alphaproteobacteria (32.2%, mainly *Roseobacter* sp.). Most of the Cyanobacteria belong to Oscillatoriales, Chroococcales, Nostocales and Pleurocapsales orders. *Synechococcus* sp., *Calothrix* sp., *Scytonema* sp. and *Pleurocapsa* sp. were the more represented species.

Venn diagram revealed a large fraction (46.7%, dominated by *Ulvibacter litoralis*, *Rivularia* sp., *Microchaete tenera* and *Shimia marina*) of the total OTUs found only in the PMD fraction. A very small fraction (5.9%, dominated by Marinimicrobia) was found only in FL, whereas specific OTUs found in PA represented 15.3% (dominated by Vibrionaceae). The ubiquitous OTUs found in the three fractions represented 24.6% (Figure 6).

Similarity percentage analysis (SIMPER) revealed 12 individual OTUs contributing to 50% of the dissimilarity between PMD and the surrounding water, including *Pleurocapsa* sp., *Calothrix* sp., *Scytonema* sp. and Oscillatoriales more abundant in the PMD and *Synechococcus* sp., *Pelagibacter* sp., *Alteromonas* sp. more abundant in FL and PA fractions (Figure 7). Comparison between PA and FL showed that five OTUs contributed to 50% of the dissimilarity, including *Pelagibacter* sp. and *Synechococcus* sp. more represented in FL and *Alteromonas* sp. more abundant in PA.
<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Figure 6</figcaption></figure>
<table><caption>Figure 7</caption><thead><tr><th></th><th></th><th>FL</th><th>PA</th><th>PMD</th></tr></thead><tbody><tr><td rowspan="5">Cyanobacteria</td><td>Synechococcus sp.</td><td></td><td>●</td><td>●</td></tr><tr><td>Cyanobacterium sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Pleurocapsa sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Calothrix sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Oscillatoriales</td><td>●</td><td>●</td><td>●</td></tr><tr><td rowspan="2">Alphaproteobacteria</td><td>Scytonema sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Leptolyngbya sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td rowspan="4">Actinobacteria</td><td>Pelagibacter sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Erythrobacter sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Pelagibaca sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Roseobacter sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Bacteroidetes</td><td>Unclassified</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Gammaproteobacteria</td><td>Flavobacteriaceae</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Sphingobacteria</td><td>Alteromonas sp.</td><td>●</td><td>●</td><td>●</td></tr><tr><td>Sphingobacteriales</td><td>Sphingobacteria</td><td>●</td><td>●</td><td>●</td></tr></tbody></table>

## Potential metabolic differences between bacteria in FL, PA and PMD fractions

Analysis of the predicted metabolic functions potentially expressed by the bacterial communities associated to FL, PA and PMD fractions revealed several systematic differences (Figure 8). On KEGG level 2 (high hierarchical level in PICRUSt), membrane transport, cell motility and xenobiotics biodegradation were predicted to be significantly more encoded by PMD compared to FL communities (Kruskal-Wallis rank sum test; p<0.05), while KEGG
terms related to translation, replication and repair and amino acid metabolism were predicted to be more represented in the FL fraction. We further used SIMPER to discriminate the predictive functions at a lower hierarchical level (KEGG level 3) explaining the most differences between the 3 fractions (the 20 most abundant functions contributing to 36.7% difference). General transporters and ABC transporters functions were more detected on PMD and explained from 10.5 to 13.8% of the dissimilarity with FL and PA. Xenobiotic biodegradation and metabolism functions were also more found on PMD. Photosynthesis proteins, cell motility, cellular process and signaling were more represented in both PMD and PA compared to FL.

<table><caption>**Figure 8**</caption><caption>Relative abundance of KEGG terms on PMD</caption><thead><tr><th rowspan="2">Level 2</th><th rowspan="2">Level 3</th><th colspan="2">FL</th><th colspan="2">PMD</th></tr><tr><th>-1%</th><th>0%</th><th>-1%</th><th>0%</th></tr></thead><tbody><tr><td rowspan="3">Membrane transport</td><td>Transporters</td><td></td><td></td><td></td><td></td></tr><tr><td>ABC transporters</td><td></td><td></td><td></td><td></td></tr><tr><td>Secretion system</td><td></td><td></td><td></td><td></td></tr><tr><td>Signal transduction</td><td>Two component system</td><td></td><td></td><td></td><td></td></tr><tr><td>Genet info & process</td><td>Replic., recombin. & repair proteins</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Cell motility</td><td>Bacterial motility proteins</td><td></td><td></td><td></td><td></td></tr><tr><td>Bacterial chemotaxis</td><td></td><td></td><td></td><td></td></tr><tr><td>Cell process & signaling</td><td>Pores ion channels</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">Energy metab</td><td>Nitrogen metab</td><td></td><td></td><td></td><td></td></tr><tr><td>Photosynthesis proteins</td><td></td><td></td><td></td><td></td></tr><tr><td>Oxidative phosphorylation</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">Xenobiotics<br>biodegradation</td><td>Benzoate degradation</td><td></td><td></td><td></td><td></td></tr><tr><td>Chloroalkane and chloroalkene degrad</td><td></td><td></td><td></td><td></td></tr><tr><td>Polycyclic aromatic hydrocarbon degrad</td><td></td><td></td><td></td><td></td></tr><tr><td>Amino acid metab</td><td>Glycine, serine & threonine metab</td><td></td><td></td><td></td><td></td></tr><tr><td>Metab of cofactors & vitam</td><td>Porphyrin & chlorophyll metab</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="2">Replication & repair</td><td>Homologous recombination</td><td></td><td></td><td></td><td></td></tr><tr><td>DNA repair & recombination proteins</td><td></td><td></td><td></td><td></td></tr><tr><td>Nucleotide metab</td><td>Purine metab</td><td></td><td></td><td></td><td></td></tr><tr><td>Translation</td><td>Ribosome</td><td></td><td></td><td></td><td></td></tr></tbody></table>

A closer look at the OTU table for known hydrocarbonoclastic bacteria (77 OTUs including *Glaciecola* sp., *Muricauda* sp., *Pseudoalteromonas* sp., *Hyphomonas* sp., *Phormidium* sp., and *Erythrobacter* sp., which represented generally more than 0.1% of the community for each sample) showed that these OTUs were more present in PMD and PA fractions (7.4 and 7.8%, respectively) compared to FL (4.7%). The most dominant hydrocarbonoclastic OTUs
was *Erythrobacter* sp., which dominated some samples of the PMD (43%) and PA (35%) communities. *Hyphomonas* sp. and *Phormidium* sp. in particular were 10 to 100 times more found in PMD compared to PA and FL, and were observed in almost all the plastic pieces.

We also focused on putative pathogens, based on taxonomic OTU identification. *Vibrio* sp. was generally found in the rare biosphere (<0.1%) in the FL, and sometimes in higher proportion in PMD and PA. Putative pathogen *Vibrio* species such as *V. anguillarum*, *V. harveyi*, *V. pectenicida* and *V. xiamenensis* were found on less than 5 plastic pieces, and neither detected on FL and PA. Other putative pathogens were particularly found on PMD and rather rare in PA and FL, such as the fish pathogen *Tenacibaculum* sp. or the crustacean and invertebrate pathogens *Phormidium* sp. and *Leptolyngbya* sp., which were observed in more than three quarters of plastic pieces and represented 27.7% of the PMD community.

## Discussion

*A high density but patchy distribution of bacteria living on PMD compared to the PA and FL lifestyles.*

We found a very high density of bacteria living on plastics, with a mean of 4.4x10⁴ cells mm⁻². This is in the same order of magnitude as found in seawater biofilms covering glass (Mieszkin *et al.*, 2012), subtidal rocks (Chung *et al.*, 2010) or stainless steel (Devi, 1995). Microplastic concentration ranged between <1 to 36 pieces m⁻³ in the surface layers of the North Western Mediterranean Sea (Pedrotti *et al.*, 2016; Collignon *et al.*, 2012), thus suggesting that microplastics can no longer be considered as a negligible part of the marine particles in this region. Our epifluorescence microscopic observations give the first *in situ* estimation of the bacterial density on PMD in marine waters, even if plastic biofilms have been observed by electron microscopy in a number of studies starting in the 1970s (Carpenter *et al.*, 1972). We found that PMD cell enrichment factor (EF) was up to 4000 and more
generally around 900 compared to FL fraction, which is similar and even higher to EF generally observed for PA bacteria (Simon *et al.*, 2002). The biofilms covered between 0 and 3.5% of the surface area, revealing a rather patchy and non-uniform covering of the PMD. This observation is in accordance with the *in situ* observation of spatial heterogeneity ('patchiness') of mature biofilms in nature, controlled by a complex set of parameters including physical (hydrodynamic fluxes), chemical (nutrients, pH, oxygen) and biological conditions (quorum-sensing, toxic metabolic by-products) (Flemming *et al.*, 2016; Salta *et al.*, 2013).

### *PMD bacteria presented similar richness but higher evenness compared to FL and PA*

We observed that PMD represent favorable environments for a large number of species, with significantly higher evenness compared to FL and PA fractions. These results are in accordance with Zettler et al. (2013), but differed with Mc Cormick et al. (2014), both comparing PMD and FL communities only. Such discrepancy may be due to the low number of samples analyzed in the previous studies (3 and 4 sites with a total of 6 and 4 PMD, respectively) compared to the 32 sites and 80 PMD analyzed here, which make the conclusions more robust. Theoretical studies suggested that spatial isolation (such as the colonization of PMD in seawater in our case) could limit competition, thereby supporting a high richness and a more uniform diversity (Zhou *et al.*, 2002). Further studies are needed to test if this trend may be explained by the superabundant or heterogeneous resources, fluctuating conditions and spatial heterogeneity offered by PMD.

### *Niche partitioning between PMD, FL and PA fractions.*

NMDS results demonstrated niche partitioning in marine bacteria as reflected by the considerable proportion of the variance (69%) in community composition between PMD and
the surrounding FL or PA fractions. Niche partitioning is one of the hypotheses that could
explain the maintenance of the tremendous diversity among marine bacteria, together with
their capacity for dormancy (Baran *et al.*, 2015). The classical barriers known to affect the
pelagic bacterial communities are transitions between marine and freshwater, coast and
offshore or surface and deep waters (Ghiglione *et al.*, 2012). Here, we propose that spatial
isolation provided by the large concentration of PMD offer a distinct strategy to avoid
competition by occupying different physical locations, which support a new trade-off for the
maintenance of a large diversity of bacteria in the Oceans (Sunagawa *et al.*, 2015). Together
with additional metabolic diversification of sympatric microbial populations (i.e., resource
limitation and cross-feeding), the evidence of spatial isolation on PMD give a new example of
bacterial strategy to explain the high diversity of pelagic microbes in the Oceans, analogous to
the “Hutchinson’s paradox of the plankton” for phytoplankton (Hutchinson, 1961).

Transition from one lifestyle to another and niche specificities.

Over the thousands of OTUs identified, a quarter of them were ‘generalists’. It suggests
possible exchanges between the PMD, PA and FL communities rather than considering the
three compartments as non-interacting environments. Attachment and detachment is a
common feature already observed in biofilms and organic particles, with release of cells that
become free, return to the planktonic mode of growth until they encounter another physical
carrier (Stoodley *et al.*, 2002; Ghiglione *et al.*, 2007).

Interestingly, we observed that OTUs only found on PMD represented almost half of the total
OTUs in our samples, thus suggesting a distinct selection strategy and reinforcing the view of
PMD being a particular biota for marine bacteria. We may have hypothesis a high similarity
between OTUs living on PMD and PA, as previously suggested in one study focusing on the
early colonization of polyethylene terephthalate (PET) bottles in seawater (Oberbeckmann et
al. 2016). It was not the case in our study, with more than half of OTUs being distinct between these very different PA and PMD habitats. PA bacteria decompose the support where they live, which is made of detritus-dominated aggregates sinking in the water column (Simon et al., 2002). This is not the case for PMD that presented very low biodegradation rates (Dussud and Ghiglione, 2014). Floating PMD are different habitats, with a longer history of drifting dispersion and being periodically submitted to ultraviolet (UV) radiation, mechanical forces and weathering, as well as to ingestion and release by pelagic filter feeders, and other zooplankton and micronekton (Collignon et al., 2012).

Finally, we observed that ‘FL specialists’ were rather an exception than a rule, since the OTUs found exclusively in a FL state represented less than 6%. Similar tendency was found when comparing only FL and PA communities, using similar metabarcoding approach (Rieck et al., 2015). Our results reinforce the view of Grossart et al. (2010), who underlined that much less attention has been paid to the large diversity of bacteria living on PMD and PA compared to FL, which may influence in an erroneous way emerging concepts in marine microbial ecology.

### Taxonomic identification and predictive functions.

Bacteria living on PMD were dominated by Cyanobacteria and Alphaproteobacteria, whereas FL and PA fractions were in line with the general trends previously observed, i.e. with the FL dominated by Alphaproteobacteria (mainly *Pelagibacter* sp.) and PA dominated by Alphaproteobacteria (mainly *Erythrobacter* sp.) and Gammaproteobacteria (mainly *Alteromonas* sp.) (Crespo et al., 2013; Mohit et al., 2014). Consistent with the typical Cyanobacteria shapes widely observed in our SEM and AFM images, SIMPER analysis revealed that most of the taxonomic groups explaining the difference between the PMD vs. PA and FL belong to this group. It confirmed the specific occurrence of some OTUs in PMD
biofilms, where the presence of light and inorganic carbon released by neighbor microorganisms may be a driving factor for the selection of Cyanobacteria (Stal, 1995). Five genera of Cyanobacteria were particularly more represented in the PMD, including *Leptolyngbya* sp., *Pleurocapsa* sp. and *Rivularia* sp., which were already observed in the plastisphere in the North Pacific gyre (Zettler *et al.*, 2013; Bryant *et al.*, 2016). We also noted the presence of *Scytonema* sp. and *Calothrix* sp. in PMD, which were previously found on biofilms covering rocks or chalky crustaceans (Sivonen *et al.*, 2007; González-Resendiz *et al.*, 2015). Further studies are needed to evaluate the influence of the photosynthetic activities of Cyanobacteria living on PMD on the global pelagic primary production and more generally on their biogeochemical role on the carbon and nitrogen cycles in the Oceans.

In parallel, we used our OTU table for the prediction of microbial metagenomes, as recently proposed by several authors (Langille *et al.*, 2013; Louca *et al.*, 2016). PICRUSt analysis highlighted the dominance of porphyrin and chlorophyll metabolism functions on the PMD compared to FL and PA genomes. Although these observations were based only on predictions made from 16S rRNA gene sequences, this is consistent with the metagenomic data obtained from 12 plastic pieces in the North Pacific Gyre (Bryant *et al.*, 2016). Other prediction of overrepresented functional categories on PMD compared to FL and PA were also plausible: the membrane transport functions and the ABC transporters, as well as the cellular processes and signaling were previously described as essential functions in biofilm formation and maturation (Lasa and Penadés, 2006; Porter *et al.*, 2011; He and Bauer, 2014); the cell motility was described as a prerequisite for the chemotactic behavior and active interactions between bacteria and colonization sites (Simon *et al.*, 2002). Even though the overrepresentation of some functional categories needs to be interpreted with caution, this predictive method provided a good indication for the existence of functional differences in our system.
Evidence of bacterial degradation of hydrocarbon-based PMD remains an unresolved hypothesis in marine waters (Dussud and Ghiglione, 2014). Interestingly, several hydrocarbonoclastic bacteria were overrepresented in the PMD fraction, such as Erythrobacteraceae (e.g. *Erythrobacter* sp., *Croceicoccus naphtovorans*) (Wang *et al.*, 2016) or *Hyphomonas* sp. (Li *et al.*, 2014) found in almost all our plastic pieces. Predicted genomes of bacteria predominantly found in PMD were also notably enriched in xenobiotics biodegradation functions, mainly including hydrocarbon degradation. However, further investigations are needed to evaluate how much these consortia are good candidates for PMD biodegradation, since they may also be selected by hydrocarbon petroleum residues concentrated on their surfaces due to the hydrophobic properties of PMD (Rios *et al.*, 2007).

Interests have been raised about opportunist pathogens dispersal on PMD, such as animal or human pathogenic *Vibrio* sp. (Zettler *et al.*, 2013). The potentially human pathogenic *Vibrio parahaemolyticus* was recently observed on a number of PMD from North/Baltic Sea (Kirstein *et al.*, 2016), but were never detected in our dataset. Other potentially pathogenic *Vibrios* were observed, such as *V. anguillarum*, *V. harveyi*, *V. pectinicida*, *V. xiamenensis*, but they generally belong to the rare biosphere (<0.1%) and they were not related to human diseases. Overall, *Vibrio* sp. was 7 times higher in the PMD compared to FL, but they were not particularly enriched compared to the PA fraction. SIMPER analysis highlighted the overrepresentation of *Tenacibaculum* sp., *Phormidium* sp. and *Leptolyngbya* sp. in the PMD compared to both FL and PA, a few members of these genus being known as fish pathogens (Austin *et al.*, 2012) or brine shrimp and coral black band disease (Rützler and Santavy, 1983; Frazão *et al.*, 2010). However, caution should be taken since metabarcoding approach is not an appropriate method for describing bacterial virulence, and further tests are needed before waving alarmist conclusions on the potential role of PMD as vector for the dispersal of harmful or even human pathogenic species (Kirstein *et al.*, 2016).
## Conclusion

This study successfully evidences the occurrence of a specific and abundant bacterial life on PMD, when compared to the more frequently studied FL and PA fractions in the surrounding waters. Such niche partitioning raised stimulating questions about the functional role of these abundant communities, which are increasing together with the number of PMD in the World Ocean. Based on a large set of samples along the western Mediterranean Sea, our study gives new directions to estimate the ecological role of PMD communities, and in particular the Cyanobacteria group consistently found abundant on PMD that may influence the global carbon and nitrogen biogeochemical cycles in surface Oceans. Computational predictions also gave evidence of specific functions associated to PMD such as hydrocarbon degradation, which needs further studies to evaluate their capability to perform plastic biodegradation in marine waters.

**Acknowledgements.** We thank the commitment of the following institutions, people and sponsors: CNRS, UPMC, LOV, Genoscope/CEA, the Tara Expeditions Foundation and its funders: Agnès B. and Etienne Bourgois, the Veolia Environment Foundation, Lorient Agglomeration, Serge Ferrari, the Foundation Prince Albert II de Monaco, IDEC, the Tara schooner and teams. We are also grateful to the French Ministry of Foreign Affairs for supporting the expedition and to the countries that graciously granted sampling permissions.

We thank Guigui PA, VJS, and Albuquerque N for insightful comments on the manuscript.

This work was also supported by the PLASTICMICRO project founded by CNRS-EC2CO-Microbien.
**Conflict of interest.** All of the reported work is original research, and authors have seen and approved the final version submitted. The material has not been previously published and has not been submitted for publication elsewhere while under consideration. The authors declare no conflict of interest. Likewise, consent is given for publication in *The ISME Journal*, if accepted.

**Supplementary information.** Supplementary information is available at Environmental Pollution website.
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**Figure legends:**

**Figure 1:** Study area and station locations (n=32) in the Western Mediterranean Sea. Upper left: picture of the manta trawls used to collect PMD. At the bottom: light-micrograph of PMD analyzed in this study.

**Figure 2:** A: SEM image showing a plastic colonized by diverse organisms and surface deterioration; B: AFM amplitude error image showing pits and cracks on a very small scale, as well as organisms of different morphologies and size, among which two *Oscillatoria* are pointed (arrows) and a diatom visible on the upper right; C: Epifluorescence microscopy images of biofilms with a “patchy” organization (C-1) and a loaded biofilm with diverse morphotypes (C-2).

**Figure 3:** Alpha-diversity indices (Chao1, Pielou, Shannon, Simpson) of FL, PA and PMD bacteria. The boxplots show the median (heavy horizontal line inside the boxes), the box represents the first and third quartiles and unfilled circles indicate outliers. Lowercase letters (a, b, c) denote statistically different groups ($p \le 0.05$).

**Figure 4:** Nonmetric multidimensional scaling (NMDS) plot showing dissimilarities among FL (open circles), PA (solid circles) and PMD (triangles: red for polyethylene, blue for polypropylene and green for other composition). Samples were grouped according to >30% Bray-Curtis similarity threshold (dotted circles).

**Figure 5:** Histograms showing taxonomic composition at the class level for FL, PA and PMD fractions, as well as for unique and common OTUs between the three fractions (see also Venn
diagram Figure 6). For more clarity, the class representing < 1% are not represented. Numbers of OTUs are in parentheses. (1) Alphaproteobacteria, (2) Cyanophyceae, (3) Gammaproteobacteria, (4) Flavobacteriia, (5) Sphingobacteriia, (6) Proteobacteria (unclas), (7) Actinobacteria, (8) Planctomycetia, (9) Bacteroidetes, (10) Cytophagia, (11) Bacilli, (12) Betaproteobacteria, (13) Opitutae, (14) Deltaproteobacteria, (15) Phycisphaerae, (16) Bacteroidia, (17) Clostridia, (18) Marinimicrobia.

**Figure 6:** Venn diagram showing overlaps between OTUs found in FL, PA and PMD fractions. Numbers inside circles represent the number of shared OTUs. The percentage of unique OTUs found in only one fraction is also indicated. N = number of reads per fraction.

**Figure 7:** Average abundance of the most significant OTUs contributing to 50% (cumulative) of the dissimilarity between FL, PA and PMD fractions based on SIMPER analysis. We indicated OTUs explaining the difference between FL and PA (white bubbles), FL and PMD (dotted bubbles), PA and PMD (stripped bubbles) and both FL/PMD and PA/PMD (black bubbles).

**Figure 8:** KEGG categories predicted by PICRUSt (level 2 and 3) differentially represented in bacterial communities found in PMD, FL and PA fractions (discriminant KEGG terms according to SIMPER analysis). Bars represent the difference of relative abundance of a KEGG term in PMD bacterial communities compared to FL fraction (left graph) or to PA fraction (right graph). Overexpression or underexpression of a KEGG term in PMD compared to FL or PA fractions are indicated by positive and negative percentage, respectively. Abbreviation of KEGG terms: metab, metabolism; info, information; process, processing; vitam, vitamins.
Supplementary files

<figure><img src="image_9.png" /><figcaption>**Supl. Figure S1:** Canonical correspondence analysis of bacterial community structures living on plastics in relation to plastic characteristics (surface, shape and carbonyl index) and environmental variables (temperature, salinity, chlorophyll a, distance to the coast). Arrows point in the direction of increasing values of each variable and the length of the arrows indicates the degree of correlation with the represented axes. The first and second canonical axes explained 38 and 21% of the total variance, respectively. The remaining axes accounted for less than 13% of the total variance each and thus were not considered as significant enough.</figcaption></figure>
<table><caption>Table S1: Contextual and physico-chemical data associated with the samples used in this study. PE: polyethylene, PP: polypropylene, PS: polystyrene, EVA: ethylene-vinyl acetate</caption><thead><tr><th>Manta #</th><th>Study area</th><th>Latitude</th><th>Longitude</th><th>Distance to coast (km)</th><th>Composition</th><th>Shape</th><th>Color</th><th>Major size (mm)</th><th>Surface (mm²)</th><th>Circularity</th><th>Carbonyl Index</th><th>Temperature</th><th>Salinity</th><th>Chi-a</th></tr></thead><tbody><tr><td>M10P1</td><td>French Riviera (Monaco)</td><td>43.71</td><td>7.43</td><td>2.13</td><td>PE</td><td>pellet</td><td>white</td><td>4.92</td><td>15.77</td><td>0.67</td><td>1.61</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M10P2</td><td>French Riviera (Monaco)</td><td>43.71</td><td>7.43</td><td>2.13</td><td>PE</td><td>thick sheet</td><td>green</td><td>8.19</td><td>14.14</td><td>0.30</td><td>6.48</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M10P3</td><td>French Riviera (Monaco)</td><td>43.71</td><td>7.43</td><td>2.13</td><td>PE</td><td>sheet</td><td>white</td><td>11.67</td><td>56.43</td><td>0.19</td><td>9.84</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M15P3</td><td>Ligurian coast (Vintimiglia)</td><td>43.70</td><td>7.80</td><td>12.52</td><td>PE</td><td>sheet</td><td>white</td><td>13.37</td><td>88.43</td><td>0.16</td><td>10.66</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M15P4</td><td>Ligurian coast (Vintimiglia)</td><td>43.70</td><td>7.80</td><td>12.52</td><td>PP</td><td>sheet</td><td>transparent</td><td>17.31</td><td>164.10</td><td>0.46</td><td>8.06</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M19P1</td><td>French Riviera (Nice)</td><td>43.64</td><td>7.21</td><td>0.30</td><td>EVA</td><td>sheet</td><td>transparent</td><td>12.83</td><td>64.05</td><td>0.62</td><td>23.16</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M19P2</td><td>French Riviera (Nice)</td><td>43.64</td><td>7.21</td><td>0.30</td><td>Composite</td><td>sheet</td><td>white</td><td>21.04</td><td>234.02</td><td>0.52</td><td>43.09</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M20P1</td><td>French Riviera (Open sea)</td><td>43.46</td><td>7.54</td><td>28.82</td><td>PE</td><td>sheet</td><td>transparent</td><td>7.33</td><td>32.04</td><td>0.55</td><td>18.81</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M20P4</td><td>French Riviera (Open sea)</td><td>43.46</td><td>7.54</td><td>28.82</td><td>PE</td><td>sheet</td><td>transparent</td><td>5.02</td><td>16.05</td><td>0.75</td><td>23.86</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M20P6</td><td>French Riviera (Open sea)</td><td>43.46</td><td>7.54</td><td>28.82</td><td>PP</td><td>sheet</td><td>transparent</td><td>15.88</td><td>49.10</td><td>0.25</td><td>23.20</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>M32P1</td><td>Gulf of Lions (Portoferraio)</td><td>42.86</td><td>10.18</td><td>5.15</td><td>PE</td><td>sheet</td><td>white</td><td>10.34</td><td>44.17</td><td>0.39</td><td>10.37</td><td>23.94</td><td>37.93</td><td>0.05</td></tr><tr><td>M32P2</td><td>Gulf of Lions (Portoferraio)</td><td>42.86</td><td>10.18</td><td>5.15</td><td>PE</td><td>pellet</td><td>white</td><td>6.28</td><td>22.25</td><td>0.60</td><td>13.49</td><td>23.94</td><td>37.93</td><td>0.05</td></tr><tr><td>M32P5</td><td>Gulf of Lions (Portoferraio)</td><td>42.86</td><td>10.18</td><td>5.15</td><td>PE</td><td>sheet</td><td>transparent</td><td>18.89</td><td>67.52</td><td>0.17</td><td>5.00</td><td>23.94</td><td>37.93</td><td>0.05</td></tr><tr><td>M32P6</td><td>Gulf of Lions (Portoferraio)</td><td>42.86</td><td>10.18</td><td>5.15</td><td>PE</td><td>sheet</td><td>transparent</td><td>6.46</td><td>16.51</td><td>0.38</td><td>8.08</td><td>23.94</td><td>37.93</td><td>0.05</td></tr><tr><td>M38P3</td><td>Gulf of Saint-Florent</td><td>42.82</td><td>9.29</td><td>2.85</td><td>PE</td><td>pellet</td><td>white</td><td>9.35</td><td>27.83</td><td>0.25</td><td>4.01</td><td>22.79</td><td>38.01</td><td>0.03</td></tr><tr><td>M42P1</td><td>Gulf of Saint-Florent (Open sea)</td><td>42.97</td><td>8.94</td><td>32.82</td><td>PE</td><td>sheet</td><td>transparent</td><td>3.41</td><td>8.34</td><td>0.56</td><td>17.39</td><td>23.21</td><td>38.09</td><td>0.04</td></tr><tr><td>M42P2</td><td>Gulf of Saint-Florent (Open sea)</td><td>42.97</td><td>8.94</td><td>32.82</td><td>PE</td><td>thick sheet</td><td>transparent</td><td>3.83</td><td>7.09</td><td>0.55</td><td>17.69</td><td>23.21</td><td>38.09</td><td>0.04</td></tr><tr><td>M42P3</td><td>Gulf of Saint-Florent (Open sea)</td><td>42.97</td><td>8.94</td><td>32.82</td><td>PE</td><td>sheet</td><td>transparent</td><td>7.21</td><td>12.63</td><td>0.31</td><td>9.94</td><td>23.21</td><td>38.09</td><td>0.04</td></tr><tr><td>M47P3</td><td>Tyrrenian Sea (East Corsica)</td><td>42.26</td><td>9.66</td><td>8.59</td><td>PE</td><td>thick sheet</td><td>white</td><td>5.74</td><td>17.00</td><td>0.30</td><td>22.00</td><td>23.57</td><td>37.75</td><td>0.05</td></tr><tr><td>M47P5</td><td>Tyrrenian Sea (East Corsica)</td><td>42.26</td><td>9.66</td><td>8.59</td><td>PE</td><td>sheet</td><td>white</td><td>6.07</td><td>16.57</td><td>0.30</td><td>14.90</td><td>23.57</td><td>37.75</td><td>0.05</td></tr><tr><td>M47P6</td><td>Tyrrenian Sea (East Corsica)</td><td>42.26</td><td>9.66</td><td>8.59</td><td>PE</td><td>sheet</td><td>transparent</td><td>6.26</td><td>17.71</td><td>0.39</td><td>8.15</td><td>23.57</td><td>37.75</td><td>0.05</td></tr><tr><td>M51P2</td><td>Tyrrenian Sea (East Corsica)</td><td>41.95</td><td>9.98</td><td>46.49</td><td>PP</td><td>sheet</td><td>transparent</td><td>5.59</td><td>17.63</td><td>0.62</td><td>8.85</td><td>24.06</td><td>37.87</td><td>0.03</td></tr><tr><td>M51P4</td><td>Tyrrenian Sea (East Corsica)</td><td>41.95</td><td>9.98</td><td>46.49</td><td>PE</td><td>thick sheet</td><td>transparent</td><td>4.50</td><td>10.93</td><td>0.48</td><td>15.38</td><td>24.06</td><td>37.87</td><td>0.03</td></tr><tr><td>M56P1</td><td>Strait of Bonifacio (south Corsica)</td><td>41.37</td><td>9.17</td><td>0.65</td><td>Composite</td><td>sheet</td><td>white</td><td>9.95</td><td>50.25</td><td>0.42</td><td>15.82</td><td>22.27</td><td>38.04</td><td>0.04</td></tr><tr><td>M56P2</td><td>Strait of Bonifacio (south Corsica)</td><td>41.37</td><td>9.17</td><td>0.65</td><td>Composite</td><td>sheet</td><td>transparent</td><td>5.59</td><td>17.51</td><td>0.51</td><td>7.88</td><td>22.27</td><td>38.04</td><td>0.04</td></tr><tr><td>M60P1</td><td>Tyrrenian Sea (East Sardinia)</td><td>41.04</td><td>10.66</td><td>94.45</td><td>PP</td><td>sheet</td><td>transparent</td><td>9.89</td><td>55.72</td><td>0.31</td><td>7.69</td><td>22.88</td><td>38.14</td><td>0.04</td></tr><tr><td>M60P4</td><td>Tyrrenian Sea (East Sardinia)</td><td>41.04</td><td>10.66</td><td>94.45</td><td>PE</td><td>sheet</td><td>transparent</td><td>13.22</td><td>39.25</td><td>0.34</td><td>17.14</td><td>22.88</td><td>38.14</td><td>0.04</td></tr><tr><td>M60P5</td><td>Tyrrenian Sea (North Sicily)</td><td>41.04</td><td>10.66</td><td>94.45</td><td>PE</td><td>sheet</td><td>transparent</td><td>8.21</td><td>17.78</td><td>0.24</td><td>1.92</td><td>22.88</td><td>38.14</td><td>0.04</td></tr><tr><td>M66P1</td><td>Tyrrenian Sea (North Sicily)</td><td>39.00</td><td>12.42</td><td>68.01</td><td>PE</td><td>sheet</td><td>transparent</td><td>5.01</td><td>10.93</td><td>0.54</td><td>16.40</td><td>24.61</td><td>37.63</td><td>0.03</td></tr><tr><td>M66P2</td><td>Tyrrenian Sea (North Sicily)</td><td>39.00</td><td>12.42</td><td>68.01</td><td>PE</td><td>pellet</td><td>transparent</td><td>4.39</td><td>13.68</td><td>0.65</td><td>24.30</td><td>24.61</td><td>37.63</td><td>0.03</td></tr><tr><td>M66P5</td><td>Tyrrenian Sea (North Sicily)</td><td>39.00</td><td>12.42</td><td>68.01</td><td>PE</td><td>pellet</td><td>transparent</td><td>2.84</td><td>4.53</td><td>0.63</td><td>14.73</td><td>24.61</td><td>37.63</td><td>0.03</td></tr><tr><td>M165P1</td><td>Tyrrenian Sea (Palermo)</td><td>38.27</td><td>13.82</td><td>32.52</td><td>PP</td><td>sheet</td><td>transparent</td><td>21.24</td><td>166.11</td><td>0.54</td><td>8.66</td><td>27.56</td><td>37.78</td><td>0.05</td></tr><tr><td>M165P3</td><td>Tyrrenian Sea (Palermo)</td><td>38.27</td><td>13.82</td><td>32.52</td><td>PP</td><td>sheet</td><td>transparent</td><td>23.80</td><td>132.74</td><td>0.38</td><td>12.91</td><td>27.56</td><td>37.78</td><td>0.05</td></tr><tr><td>M165P5</td><td>Tyrrenian Sea (Palermo)</td><td>38.27</td><td>13.82</td><td>32.52</td><td>PE</td><td>sheet</td><td>transparent</td><td>6.23</td><td>7.24</td><td>0.52</td><td>10.12</td><td>27.56</td><td>37.78</td><td>0.05</td></tr><tr><td>M173P1</td><td>Southeast of Algerian Basin</td><td>37.36</td><td>7.15</td><td>30.04</td><td>PE</td><td>sheet</td><td>blue</td><td>14.06</td><td>88.99</td><td>0.36</td><td>9.46</td><td>26.41</td><td>37.36</td><td>0.04</td></tr><tr><td>M173P2</td><td>Southeast of Algerian Basin</td><td>37.36</td><td>7.15</td><td>30.04</td><td>PE</td><td>sheet</td><td>transparent</td><td>17.19</td><td>83.78</td><td>0.32</td><td>7.56</td><td>26.41</td><td>37.36</td><td>0.04</td></tr><tr><td>M173P3</td><td>Southeast of Algerian Basin</td><td>37.36</td><td>7.15</td><td>30.04</td><td>PE</td><td>sheet</td><td>orange</td><td>6.00</td><td>17.44</td><td>0.36</td><td>93.19</td><td>26.41</td><td>37.36</td><td>0.04</td></tr><tr><td>M173P4</td><td>Southeast of Algerian Basin</td><td>37.36</td><td>7.15</td><td>30.04</td><td>PE</td><td>sheet</td><td>transparent</td><td>13.55</td><td>48.53</td><td>0.38</td><td>23.58</td><td>26.41</td><td>37.36</td><td>0.04</td></tr><tr><td>M178P2</td><td>Algerian Basin (Algiers)</td><td>37.73</td><td>3.80</td><td>91.86</td><td>PE</td><td>sheet</td><td>black</td><td>2.97</td><td>5.26</td><td>0.31</td><td>7.32</td><td>25.90</td><td>36.81</td><td>0.08</td></tr><tr><td>M178P3</td><td>Algerian Basin (Algiers)</td><td>37.73</td><td>3.80</td><td>91.86</td><td>PP</td><td>sheet</td><td>transparent</td><td>8.18</td><td>9.14</td><td>0.38</td><td>14.70</td><td>25.90</td><td>36.81</td><td>0.08</td></tr><tr><td>M178P4</td><td>Algerian Basin (Algiers)</td><td>37.73</td><td>3.80</td><td>91.86</td><td>PE</td><td>sheet</td><td>white</td><td>6.79</td><td>23.26</td><td>0.51</td><td>5.85</td><td>25.90</td><td>36.81</td><td>0.08</td></tr><tr><td>M189P1</td><td>Algerian Basin (Balearic Islands)</td><td>39.51</td><td>5.17</td><td>83.34</td><td>PE</td><td>thick sheet</td><td>white</td><td>7.87</td><td>24.94</td><td>0.29</td><td>12.46</td><td>26.37</td><td>37.46</td><td>0.02</td></tr><tr><td>M189P2</td><td>Algerian Basin (Balearic Islands)</td><td>39.51</td><td>5.17</td><td>83.34</td><td>PE</td><td>sheet</td><td>transparent</td><td>13.93</td><td>86.81</td><td>0.20</td><td>7.76</td><td>26.37</td><td>37.46</td><td>0.02</td></tr><tr><td>M189P3</td><td>Algerian Basin (Balearic Islands)</td><td>39.51</td><td>5.17</td><td>83.34</td><td>PE</td><td>sheet</td><td>transparent</td><td>8.24</td><td>17.90</td><td>0.36</td><td>17.48</td><td>26.37</td><td>37.46</td><td>0.02</td></tr><tr><td>M193P1</td><td>Algerian Basin (Balearic Islands)</td><td>40.47</td><td>5.27</td><td>100.01</td><td>PE</td><td>sheet</td><td>yellow</td><td>12.55</td><td>113.84</td><td>0.20</td><td>21.71</td><td>25.83</td><td>37.82</td><td>0.04</td></tr><tr><td>M193P2</td><td>Algerian Basin (Balearic Islands)</td><td>40.47</td><td>5.27</td><td>100.01</td><td>PE</td><td>pellet</td><td>light pink</td><td>10.57</td><td>14.95</td><td>0.30</td><td>14.10</td><td>25.83</td><td>37.82</td><td>0.04</td></tr><tr><td>M193P3</td><td>Algerian Basin (Balearic Islands)</td><td>40.47</td><td>5.27</td><td>100.01</td><td>PE</td><td>sheet</td><td>transparent</td><td>7.21</td><td>29.35</td><td>0.30</td><td>5.23</td><td>25.83</td><td>37.82</td><td>0.04</td></tr><tr><td>M193P4</td><td>Algerian Basin (Balearic Islands)</td><td>40.47</td><td>5.27</td><td>100.01</td><td>PE</td><td>sheet</td><td>blue</td><td>6.99</td><td>10.67</td><td>0.34</td><td>6.67</td><td>25.83</td><td>37.82</td><td>0.04</td></tr><tr><td>M202P1</td><td>Gulf of Lions (Marseille)</td><td>43.26</td><td>5.34</td><td>2.04</td><td>Composite</td><td>sheet</td><td>transparent</td><td>19.13</td><td>118.52</td><td>0.40</td><td>3.19</td><td>22.46</td><td>38.28</td><td>0.04</td></tr><tr><td>M202P2</td><td>Gulf of Lions (Marseille)</td><td>43.26</td><td>5.34</td><td>2.04</td><td>PS</td><td>sheet</td><td>transparent</td><td>7.22</td><td>36.46</td><td>0.46</td><td>22.00</td><td>22.46</td><td>38.28</td><td>0.04</td></tr><tr><td>M202P3</td><td>Gulf of Lions (Marseille)</td><td>43.26</td><td>5.34</td><td>2.04</td><td>PS</td><td>sheet</td><td>transparent</td><td>8.21</td><td>31.88</td><td>0.42</td><td>21.77</td><td>22.46</td><td>38.28</td><td>0.04</td></tr><tr><td>M234P1</td><td>Tyrrenian Sea (Naples)</td><td>40.62</td><td>13.74</td><td>13.89</td><td>PP</td><td>sheet</td><td>white</td><td>7.00</td><td>17.03</td><td>0.45</td><td>12.82</td><td>23.87</td><td>37.90</td><td>0.04</td></tr><tr><td>M234P2</td><td>Tyrrenian Sea (Naples)</td><td>40.62</td><td>13.74</td><td>13.89</td><td>PP</td><td>sheet</td><td>transparent</td><td>10.57</td><td>49.34</td><td>0.60</td><td>11.70</td><td>23.87</td><td>37.90</td><td>0.04</td></tr><tr><td>M238P5</td><td>Tyrrenian Sea (Naples)</td><td>40.71</td><td>14.12</td><td>11.54</td><td>PP</td><td>sheet</td><td>transparent</td><td>9.39</td><td>38.24</td><td>0.48</td><td>11.80</td><td>23.78</td><td>37.95</td><td>0.04</td></tr><tr><td>M238P6</td><td>Tyrrenian Sea (Naples)</td><td>40.71</td><td>14.12</td><td>11.54</td><td>PE</td><td>sheet</td><td>white</td><td>7.43</td><td>28.70</td><td>0.11</td><td>6.48</td><td>23.78</td><td>37.95</td><td>0.04</td></tr><tr><td>M238P7</td><td>Tyrrenian Sea (Naples)</td><td>40.71</td><td>14.12</td><td>11.54</td><td>PE</td><td>sheet</td><td>transparent</td><td>6.03</td><td>20.87</td><td>0.39</td><td>10.98</td><td>23.78</td><td>37.95</td><td>0.04</td></tr><tr><td>M263P1</td><td>Gulf of Lions (Perpignan)</td><td>42.49</td><td>3.45</td><td>25.37</td><td>PE</td><td>sheet</td><td>white</td><td>12.49</td><td>70.84</td><td>0.34</td><td>11.22</td><td>22.26</td><td>38.24</td><td>0.03</td></tr><tr><td>M263P10</td><td>Gulf of Lions (Perpignan)</td><td>42.49</td><td>3.45</td><td>25.37</td><td>PE</td><td>thick sheet</td><td>white</td><td>3.02</td><td>5.05</td><td>0.40</td><td>8.91</td><td>22.26</td><td>38.24</td><td>0.03</td></tr><tr><td>M269P2</td><td>Balearic Sea (Barcelona)</td><td>41.23</td><td>2.13</td><td>6.87</td><td>PE</td><td>pellet</td><td>white</td><td>5.15</td><td>19.29</td><td>0.55</td><td>16.80</td><td>21.56</td><td>38.18</td><td>0.05</td></tr><tr><td>M269P3</td><td>Balearic Sea (Barcelona)</td><td>41.23</td><td>2.13</td><td>6.87</td><td>PE</td><td>sheet</td><td>green</td><td>10.57</td><td>66.04</td><td>0.30</td><td>7.54</td><td>21.56</td><td>38.18</td><td>0.05</td></tr><tr><td>M277P4</td><td>Balearic Sea (Gulf of Valencia)</td><td>39.00</td><td>0.77</td><td>55.73</td><td>PE</td><td>sheet</td><td>transparent</td><td>15.29</td><td>109.20</td><td>0.34</td><td>6.53</td><td>21.67</td><td>38.18</td><td>ND</td></tr><tr><td>M277P6</td><td>Balearic Sea (Gulf of Valencia)</td><td>39.00</td><td>0.77</td><td>55.73</td><td>PE</td><td>sheet</td><td>white</td><td>4.12</td><td>9.86</td><td>0.50</td><td>6.91</td><td>21.67</td><td>38.18</td><td>ND</td></tr><tr><td>M280P2</td><td>Balearic Sea (Costa Blanca)</td><td>38.27</td><td>-0.28</td><td>44.11</td><td>PP</td><td>sheet</td><td>green</td><td>7.31</td><td>17.82</td><td>0.37</td><td>14.07</td><td>23.07</td><td>38.07</td><td>ND</td></tr><tr><td>M280P5</td><td>Balearic Sea (Costa Blanca)</td><td>38.27</td><td>-0.28</td><td>44.11</td><td>PE</td><td>sheet</td><td>white</td><td>8.10</td><td>37.90</td><td>0.40</td><td>11.90</td><td>23.07</td><td>38.07</td><td>ND</td></tr><tr><td>M280P7</td><td>Balearic Sea (Costa Blanca)</td><td>38.27</td><td>-0.28</td><td>44.11</td><td>PE</td><td>sheet</td><td>white</td><td>4.12</td><td>4.78</td><td>0.40</td><td>19.61</td><td>23.07</td><td>38.07</td><td>ND</td></tr><tr><td>M283P4</td><td>Balearic Sea (Cartagena)</td><td>37.48</td><td>0.73</td><td>126.92</td><td>PP</td><td>sheet</td><td>transparent</td><td>9.10</td><td>49.41</td><td>0.24</td><td>18.97</td><td>22.10</td><td>37.92</td><td>ND</td></tr><tr><td>M283P5</td><td>Balearic Sea (Cartagena)</td><td>37.48</td><td>0.73</td><td>126.92</td><td>PE</td><td>pellet</td><td>transparent</td><td>4.05</td><td>10.71</td><td>0.44</td><td>22.25</td><td>22.10</td><td>37.92</td><td>ND</td></tr><tr><td>M283P6</td><td>Balearic Sea (Cartagena)</td><td>37.48</td><td>0.73</td><td>126.92</td><td>PE</td><td>sheet</td><td>transparent</td><td>8.65</td><td>25.85</td><td>0.23</td><td>25.30</td><td>22.10</td><td>37.92</td><td>ND</td></tr><tr><td>M285P1</td><td>Alboran Sea (Strait of Gibraltar)</td><td>36.12</td><td>-5.04</td><td>26.37</td><td>PE</td><td>sheet</td><td>white</td><td>15.05</td><td>127.58</td><td>0.05</td><td>13.92</td><td>18.46</td><td>36.43</td><td>ND</td></tr><tr><td>M285P4</td><td>Alboran Sea (Strait of Gibraltar)</td><td>36.12</td><td>-5.04</td><td>26.37</td><td>PE</td><td>sheet</td><td>transparent</td><td>9.59</td><td>57.38</td><td>0.42</td><td>4.49</td><td>18.46</td><td>36.43</td><td>ND</td></tr><tr><td>M285P5</td><td>Alboran Sea (Strait of Gibraltar)</td><td>36.12</td><td>-5.04</td><td>26.37</td><td>PP</td><td>sheet</td><td>transparent</td><td>9.77</td><td>46.43</td><td>0.45</td><td>4.97</td><td>18.46</td><td>36.43</td><td>ND</td></tr><tr><td>M285P6</td><td>Alboran Sea (Strait of Gibraltar)</td><td>36.12</td><td>-5.04</td><td>26.37</td><td>PE</td><td>thick sheet</td><td>white</td><td>7.70</td><td>22.67</td><td>0.25</td><td>12.80</td><td>18.46</td><td>36.43</td><td>ND</td></tr></tbody></table>