Marine environments are naturally exposed to terrestrial and aerial inputs of rock weathered trace metals and human activities (Fattorini, Alonso-Hernandez, et al., 2004; Fattorini, Notti and Regoli, 2006).

Marine organisms are able to bioaccumulate some of these elements (Fattorini, Alonso-Hernandez, et al., 2004; Fattorini, Notti and Regoli, 2006).

Many metals have essential functionality for the health of organisms. Some are only toxic at high concentrations whereas others are highly toxic even at minimal concentrations (Tchounwou et al., 2012; Giangrande et al., 2017).

Arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb) and mercury (Hg) are considered the five most dangerous metals for public health due to their key role in (oxidative) stress within body cells and the inactivation of enzymes. They can cause damage to DNA and changes to enzymes which can result in cell mutation or cell death (Gebel, 2001; Fattorini and Regoli, 2004; Tchounwou et al., 2012).

Metals can be taken up by marine organisms through water or diet; diet represents the major uptake pathway (Giangrande et al., 2017).

Some polychaete species have shown to hyperaccumulate compounds within their tissues as a suggested anti-predatory defence mechanism (Fattorini and Regoli, 2004). These hyperaccumulation phenomena lead to very high levels in specific tissues, concentrations usually found in various marine organisms after human accidents; for example the acute arsenic contamination that occurred in 2001 in Cienfuegos Bay, Cuba (Fattorini, Alonso-Hernandez, et al., 2004).

Polychaetes are a highly diverse annelid class (segmented worms) with more than 16,000 described species inhabiting all marine environments (Díaz-Castañeda and Reish, 2009; World Register of Marine Species, 2020). .

Some species live in the pelagic (water column), others prefer sedentary lifestyles; feeding on sediment surfaces or simply burrowing or constructing tubes through calcareous secretion or sand grain cementation (Díaz-Castañeda and Reish, 2009).

The Sabella spallanzanii (Gmelin, 1791) of the Sabellidae clade for example are living on hard surfaces where they can attach and extend their mouth appendages from their protective mucous-mud composed tubes for filter-feeding and respiration (Fattorini et al., 2005; Di Carlo et al., 2017).

These animals have the extraordinary ability to survive in a wide range of environmental conditions, even some of the most extremes in the marine environment (Pearson and Rosenberg, 1978; Díaz-Castañeda and Reish, 2009):

• Oxygen minimum zones (OMZ)

• Hydrothermal vents

Polychaete species serve as a food source for various marine predators and are an important pioneer species arriving early at disturbed sediments that were defaunated (no animals present) or polluted (Fattorini and Regoli, 2004; Díaz-Castañeda and Reish, 2009).

Polychaetes have certain tools, like the antioxidants, to counteract damage on a cellular level and deal with higher levels of toxic metal species incorporated in their bodies.

Changes in water temperature and ocean acidification due to climate change might pose a risk to those species capable of hyperaccumulation. Trace metal bioavailability increases under low pH levels and can make inorganic arsenic more available in seawater and sediments (Ricevuto et al., 2016).

Under such conditions, a significant drop of antioxidant enzymes was found and a higher presence of inorganic arsenic and MMA (instead of DMA) measured in Sabella spallanzanii. This indicates that with increased oxidative stressors due to ocean acidification the methylation mechanism is impaired.

This could have repercussions on the animals ability to synthesise methylated arsenic species, bioaccumulate them in the branchial crown and reducing its ability to discourage predators with distasteful tissues.

The aspect of unfavourable conditions caused by ocean acidification could partly explain the total absence of Sabella spallanzanii in environments characterised by a natural increase in CO₂ like the Mediterranean vent systems (Ricevuto et al., 2016).

With climate change antioxidants are likely needed to combat reacitve oxygen species (ROS) instead of converting toxic metals into a less-toxic state (Bocchetti et al., 2004; Ricevuto et al., 2016). This could impair the overall fitness (ability to survive and reproduce) of the animals as well as to lower their antipredatory defence mechanisms.

• Bocchetti, R. et al. (2004) ‘Trace Metal Concentrations and Susceptibility to Oxidative Stress in the Polychaete Sabella spallanzanii (Gmelin) (Sabellidae): Potential Role of Antioxidants in Revealing Stressful Environmental Conditions in the Mediterranean’, Archives of Environmental Contamination and Toxicology, 46(3), pp. 353–361.
  doi: 10.1007/s00244-003-2300-x.

• Di Carlo, M. et al. (2017) ‘Trace elements and arsenic speciation in tissues of tube dwelling polychaetes from hydrothermal vent ecosystems (East Pacific Rise): An ecological role as antipredatory strategy?’, Marine Environmental Research, 132, pp. 1–13.
  doi: 10.1016/j.marenvres.2017.10.003.

• Díaz-Castañeda, V. and Reish, D. J. (2009) ‘Polychaetes in Environmental Studies’. In Annelids in Modern Biology. ed. by Shain, D. S. Hoboken: Wiley-Blackwell, pp. 203–227.
  doi: 10.1002/9780470455203.ch11.

• Domingo, J. L. (1996) ‘Vanadium: A review of the reproductive and developmental toxicity’, Reproductive Toxicology, 10(3), pp. 175–182.
  doi: 10.1016/0890-6238(96)00019-6.

• European Council (2000) Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy. Brussels: European Council.

• Fattorini, D., Alonso-Hernandez, C. M., et al. (2004) ‘Chemical speciation of arsenic in different marine organisms: Importance in monitoring studies’, Marine Environmental Research, 58(2–5), pp. 845–850.
  doi: 10.1016/j.marenvres.2004.03.103.

• Fattorini, D., Bocchetti, R., et al. (2004) ‘Total content and chemical speciation of arsenic in the polychaete Sabella spallanzanii’, Marine Environmental Research, 58(2–5), pp. 839–843.
  doi: 10.1016/j.marenvres.2004.03.102.

• Fattorini, D. et al. (2005) ‘Levels and chemical speciation of arsenic in polychaetes: A review’, Marine Ecology, 26(3–4), pp. 255–264.
  doi: 10.1111/j.1439-0485.2005.00057.x.

• Fattorini, D. et al. (2010) ‘Hyperaccumulation of vanadium in the Antarctic polychaete Perkinsiana littoralis as a natural chemical defense against predation’, Environmental Science and Pollution Research, 17(1), pp. 220–228.
  doi: 10.1007/s11356-009-0243-0.

• Fattorini, D., Notti, A. and Regoli, F. (2006) ‘Characterization of arsenic content in marine organisms from temperate, tropical, and polar environments’, Chemistry and Ecology, 22(5), pp. 405–414.
  doi: 10.1080/02757540600917328.

• Fattorini, D. and Regoli, F. (2004) ‘Arsenic speciation in tissues of the Mediterranean polychaete Sabella spallanzanii’, Environmental Toxicology and Chemistry, 23(8), pp. 1881–1887.
  doi: 10.1897/03-562.

• Fattorini, D. and Regoli, F. (2012) ‘Hyper-Accumulation of Vanadium in Polychaetes’. In Vanadium: Biochemical and Molecular Biological Approaches. ed. by Michibata, H. Dordrecht: Springer Science + Business Media B.V., pp. 73–92.
  doi: 10.1007/978-94-007-0913-3.

• Gebel, T. W. (2001) ‘Genotoxicity of arsenical compounds’, International Journal of Hygiene and Environmental Health, 203(3), pp. 249–262.
  doi: 10.1078/S1438-4639(04)70036-X.

• Geiszinger, A. E., Goessler, W. and Francesconi, K. A. (2002a) ‘Biotransformation of arsenate to the tetramethylarsonium ion in the marine polychaetes Nereis diversicolor and Nereis virens’, Environmental Science and Technology, 36(13), pp. 2905–2910.
  doi: 10.1021/es015808d.

• Geiszinger, A. E., Goessler, W. and Francesconi, K. A. (2002b) ‘The marine polychaete Arenicola marina: Its unusual arsenic compound pattern and its uptake of arsenate from seawater’, Marine Environmental Research, 53(1), pp. 37–50.
  doi: 10.1016/S0141-1136(01)00106-4.

• Giangrande, A. et al. (2017) ‘Heavy metals in five Sabellidae species (Annelida, Polychaeta): ecological implications’, Environmental Science and Pollution Research. Environmental Science and Pollution Research, 24(4), pp. 3759–3768.
  doi: 10.1007/s11356-016-8089-8.

• Gibbs, P. E. et al. (1983) ‘Tharyx marioni (polychaeta): A remarkable accumulator of arsenic’, Journal of the Marine Biological Association of the United Kingdom, 63(2), pp. 313–325.
  doi: 10.1017/S0025315400070703.

• Irgolic, K. J. (1992) ‘Arsenic’. In Techniques and Instrumentation in Analytical Chemistry. Hazardous Metals in the Environment. ed. by Stoeppler, M. Amsterdam: Elsevier B.V., pp. 287–350.
  doi: 10.1016/S0167-9244(08)70110-0.

• Notti, A. et al. (2007) ‘Bioaccumulation and biotransformation of arsenic in the Mediterranean polychaete Sabella spallanzanii: Experimental observations’, Environmental Toxicology and Chemistry, 26(6), pp. 1186–1191.
  doi: 10.1897/06-362R.1.

• Pearson, T. H. and Rosenberg, R. (1978) ‘Macrobenthic succession in relation to organic enrichment and pollution of the marine environment.’, Oceanography and Marine Biology: An Annual Review, 16, pp. 229–331.

• Ricevuto, E. et al. (2016) ‘Arsenic speciation and susceptibility to oxidative stress in the fanworm Sabella spallanzanii (Gmelin) (Annelida, Sabellidae) under naturally acidified conditions: An in situ transplant experiment in a Mediterranean CO2 vent system’, Science of the Total Environment, 544, pp. 765–773.
  doi: 10.1016/j.scitotenv.2015.11.154.

• Stabili, L. et al. (2006) ‘Sabella spallanzanii filter-feeding on bacterial community: Ecological implications and applications’, Marine Environmental Research, 61(1), pp. 74–92.
  doi: 10.1016/j.marenvres.2005.06.001.

• Tchounwou, P. B. et al. (2012) ‘Heavy Metals Toxicity and the environment’. In Molecular, Clinical and Environmental Toxicology. ed. by Luch, A. Basel: Springer, pp. 133–164.
  doi: 10.1007/978-3-7643-8340-4.

• The Comission of the European Communities (2006) Comission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs, Official Journal of the European Union. Brussels: European Comission

• Venkataraman, B. V and Sudha, S. (2005) ‘Vanadium toxicity ’, Asian J. Exp Sci, 19(2), pp. 127–134.

• World Register of Marine Species (2020) Polychaeta [online] Available from
  <https://www.marinespecies.org/aphia.php?p=taxdetails&id=883> [23 April 2020].

Mytilus mussels are characterised by two identical halves not having real teeth, a small anterior adductor mussel, a mantle forming a closed exhalant siphon and lameillibranched gills (lungs) (Bayne, 2009; Delahaut, 2012).

These animals are living a sedentary lifestyle after a short larvae stage freely swimming in open water (Dame, 1996; Bayne, 2009).

Sexual maturity is normally reached within the first year of their life (Bayne, 2009; Azpeitia et al., 2017). Under natural conditions the reproduction development of gonads occurs during winter months when food is generally scarce and spawning is kicked off in spring when food availability (phytoplanktonic bloom) is high (Bayne, 2009; Delahaut, 2012). The onset of a spawning event is a combination of biotic and abiotic factors such as nutrient availability, hormone presence, temperature and salinity (Bayne, 2009; Delahaut, 2012).

Larvae settling occurs under forming so called byssal threads, composed of thanned proteins that stick to any hard surface (Bayne, 2009). After settlement, the mussels start filter feeding on the water column for phytoplankton (Dame, 1996; Bayne, 2009). Filter feeding requires a constant water flow, thus, settlement occurs preferably in areas with sufficient water motion; where the tides are recognisable and in estuarine environments (transition zone between rivers and the sea) (Dame, 1996; Delahaut, 2012).

Pumping and filtration are a combination of three types of cilia (hairlike structure) working together on the gill filaments to maintain the filter feeding process (Bayne, 2009):

• Lateral cilia are moving the inhalant water flow,

• Laterofrontal cilia retain potential food particles and

• Frontal cilia transport particles in mucus trains towards the labial palps (mouth), where particles are sorted based on being suitable for digestion (Bayne, 2009).

Food particles are mainly composed of organic material, unicellular algae and bacteria (Delahaut, 2012). Particles are either transported to the mouth or being rejected as pseudofeces (Dame, 1996).

Mytilidae have an open cardiovascular system, where the haemolymph (blood) is getting oxygenated at the gills (lungs) and pumped by the heart into the haemocoel (body cavity) (Bayne, 2009; Delahaut, 2012). The bivalves possess a non-specific immune system (innate immunity) which manifests itself as a cellular and a humeral response (antibodies) (Delahaut, 2012).

In general, Mytilus sp. can filter up to 1-2 litres per hour and this makes these animals a sensitive indicator of environmental changes; due to their open circulatory system these organisms are constantly exposed to contaminants (Bocchetti and Regoli, 2006; Fattorini et al., 2008; Bayne, 2009; Hu et al., 2014).

Mussels tend to accumulate (harmful) compounds easily which result in the creation of reactive oxygen species (ROS) and DNA fragmentation reducing the animal’s overall fitness depending on exposure intensity and timescale (Regoli et al., 2004).

In the marine environment harmful components range from heavy metals to organic pollutants which includes polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), dioxins, herbicides and the effect of PAHs in combination with sunlight radiation (Pisanelli et al., 2009).

These compounds can either be introduced into the marine environment through natural pathways like riverine inputs or natural oil seepage or through human activities like smelting, extraction and shipping (Bocchetti et al., 2008; Gorbi et al., 2008; Benedetti et al., 2014). Often additional human activities such as dredging for seafood or building activities reintroduces previously sedimented compounds back into the marine environment (Bocchetti et al., 2008).

In mussels such components interact by direct or indirect mechanisms. These can range from

• Inhibition of enzymes

• Creation of oxyradicals (containing reactive oxygen atoms)

• The reduction of antioxidative defences

These effects can result in DNA strand breaks and other cellular damages like targeting the lysosomal system (cell organelles) (Bocchetti et al., 2008; Pisanelli et al., 2009).

PAHs metabolites are able to form stable adducts to DNA, causing instability and strand breaks (Regoli et al., 2004).

Types of analyses that can be carried out depends on the kind of contaminant of interest (Regoli et al., 2004; Bocchetti et al., 2008; Fattorini et al., 2008; Gorbi et al., 2008; Pisanelli et al., 2009):

• Gas-chromatography with flame ionization, high performance liquid chromatography (HPLC) with fluorometric detection and atomic adsorption spectrophotometry are used to determine the bioaccumulation of compounds in the organism’s tissues.

• The total oxyradical scavenging capacity (TOSC) is used to quantify the overall oxyradical scavenging capacity of an organism at a certain point in time and is carried out by measuring various cellular antioxidants by spectrophotometry.

• And the Comet assay is used to measure DNA strand breakage.

Abiotic fluctuations in temperature, organic matter, nutrient availability, water mass circulation or upwelling, riverine runoffs as well as biotic factors like the species specific gametogenesis (reproduction cycles) are interfering factors (Fattorini et al., 2008).:

• With changes in salinity most bivalves close their shells and this isolation can allow isosmotic intracellular regulation to begin. Changes in salinity can lead to suppression of physiological filtering rates (Dame, 1996).

• Basic levels of trace elements can vary depending on the geographical location due to site specific environmental features (Fattorini et al., 2008).
  For example, the runoff of the Po river south of Venice, respectively the lack of it in 2003, resulted in little nutrient input, higher water temperatures and an upwelling event at the Portonovo sampling location in the Marche region affecting the mussels behaviour (Fattorini et al., 2008).

• Inter-annual variability and seasonal fluctuations of trace metals in mussel tissue occur (Fattorini et al., 2008). The accumulation of trace metals during winter before the spring tissue growth was demonstrated by the high levels of metallothionein measured (Gorbi et al., 2008). As such natural variability of responses to ROS have to be carefully examined when monitoring genotoxicity (Pisanelli et al., 2009).

• Different responses may occur while monitoring.
  For example, the antioxidant glutathione S-transferase levels stayed almost constant for the first two weeks when exposed to ROS and then gradually declined (Regoli et al., 2004). The same results derived from resuspension of PAHs after dredging the port of Piombino, where caged mussels showed a reduced ability to counterbalance ROS after time (Regoli et al., 2004; Bocchetti et al., 2008).

Complex interactions of numerous molecular-cellular alterations occur constantly (Fattorini et al., 2008). It is useful to apply a multimarker approach when examining mechanisms that cause biological disorder (Regoli et al., 2004).

Overall, the use of caged mussels has been demonstrated as a useful strategy for biomonitoring marine pollution (Gorbi et al., 2008).
The transplantation of caged mussels next to the Costa Concordia shipwreck by Regoli et al. (2014) was a very good example of how mussels can be used to assess any leakage of high- or low molecular hydrocarbons, which might not be detectable on the surface.

• Azpeitia, K. et al. (2017) ‘Variability of the reproductive cycle in estuarine and coastal populations of the mussel Mytilus galloprovincialis Lmk. from the SE Bay of Biscay (Basque Country)’, International Aquatic Research. Springer Berlin Heidelberg, 9(4), pp. 329–350.
  doi: 10.1007/s40071-017-0180-3.

• Bayne, B. L. (2009) Marine mussels: their ecology and physiology. Cambridge: Cambridge University Press, pp. 506.

• Benedetti, M. et al. (2014) ‘Environmental hazards from natural hydrocarbons seepage: Integrated classification of risk from sediment chemistry, bioavailability and biomarkers responses in sentinel species’, Environmental Pollution, 185, pp. 116–126.
  doi: 10.1016/j.envpol.2013.10.023.

• Bocchetti, R. et al. (2008) ‘Contaminant accumulation and biomarker responses in caged mussels, Mytilus galloprovincialis, to evaluate bioavailability and toxicological effects of remobilized chemicals during dredging and disposal operations in harbour areas’, Aquatic Toxicology, 89(4), pp. 257–266.
  doi: 10.1016/j.aquatox.2008.07.011.

• Bocchetti, R. and Regoli, F. (2006) ‘Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea’, Chemosphere, 65(6), pp. 913–921.
  doi: 10.1016/j.chemosphere.2006.03.049.

• Curtis, T. M., Williamson, R. and Depledge, M. H. (2000) ‘Simultaneous, long-term monitoring of valve and cardiac activity in the blue mussel Mytilus edulis exposed to copper’, Marine Biology, 136(5), pp. 837–846.
  doi: 10.1007/s002270000297.

• Dame, R. F. (1996) Ecology of marine bivalves: an ecosystem approach. London: CRC Press, pp. 284.

• Delahaut, V. (2012) Development of a challenge test for the blue mussel, Mytilus edulis. [MSc Aquaculture] [online]. Ghent University. Available from <https://lib.ugent.be/fulltxt/RUG01/001/894/276/RUG01-001894276_2012_0001_AC.pdf> [12 May 2020].

• Etiope, G. et al. (2014) ‘A thermogenic hydrocarbon seep in shallow Adriatic Sea (Italy): Gas origin, sediment contamination and benthic foraminifera’, Marine and Petroleum Geology. Elsevier Ltd, 57, pp. 283–293.
  doi: 10.1016/j.marpetgeo.2014.06.006.

• Fattorini, D. et al. (2008) ‘Seasonal, spatial and inter-annual variations of trace metals in mussels from the Adriatic sea: A regional gradient for arsenic and implications for monitoring the impact of off-shore activities’, Chemosphere, 72(10), pp. 1524–1533.
  doi: 10.1016/j.chemosphere.2008.04.071.

• Glasby, T. M. (1997) ‘Analysing data from post-impact studies using asymmetrical analyses of variance: A case study of epibiota on marinas’, Australian Journal of Ecology, 22, pp. 448–459.
  doi: 10.1111/j.1442-9993.1997.tb00696.x.

• Gorbi, S. et al. (2008) ‘An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic sea’, Marine Environmental Research, 65(1), pp. 34–49.
  doi: 10.1016/j.marenvres.2007.07.006.

• Hu, W. et al. (2014) ‘Toxicity of copper oxide nanoparticles in the blue mussel, Mytilus edulis: A redox proteomic investigation’, Chemosphere, 108, pp. 289–299.
  doi: 10.1016/j.chemosphere.2014.01.054.

• Pisanelli, B. et al. (2009) ‘Seasonal and inter-annual variability of DNA integrity in mussels Mytilus galloprovincialis: A possible role for natural fluctuations of trace metal concentrations and oxidative biomarkers’, Chemosphere. Elsevier Ltd, 77(11), pp. 1551–1557.
  doi: 10.1016/j.chemosphere.2009.09.048.

• Regoli, F. et al. (2004) ‘Time-course variations of oxyradical metabolism, DNA integrity and lysosomal stability in mussels, Mytilus galloprovincialis, during a field translocation experiment’, Aquatic Toxicology, 68(2), pp. 167–178.
  doi: 10.1016/j.aquatox.2004.03.011.

• Regoli, F. et al. (2014) ‘A multidisciplinary weight of evidence approach for environmental risk assessment at the Costa Concordia wreck: Integrative indices from Mussel Watch’, Marine Environmental Research. Elsevier Ltd, 96, pp. 92–104.
  doi: 10.1016/j.marenvres.2013.09.016.

• Smith, E. P. (2002) ‘BACI Design’. In Encyclopedia of Environmetrics. ed. by El-Shaarawi, A. H. and Piegorsch, W. W. Chichester: John Wiley & Sons, Ltd., pp. 141–148.
  doi: 10.1002/9781118445112.stat07659.

• Thompson, I. S. et al. (1997) ‘Effects of low level chlorination on the recruitment , behaviour and shell growth of Mytilus edulis Linnaeus in power station cooling water’, Scientia Marina, 61, pp. 77–85.

• Underwood, A. J. (1992) ‘Beyond BACI: the detection of environmental impacts on populations in the real, but variable, world’, Exp. Mar. Biol. Ecol., 161, pp. 145–178.

• Underwood, A. J. (1993) ‘The mechanics of spatially replicated sampling programmes to detect environmental impacts in a variable world’, Australian Journal of Ecology, 18, pp. 99–116.

• Underwood, A. J. (1996) Enviromental design and analysis in marine enviromental sampling. Paris: UNESCO.

Plastic pollution of the oceans has come into focus of science and the public awareness in recent years (Avio, Gorbi and Regoli, 2017).

Roughly 300 million tons of plastics are produced each year of which 10 - 20% end up in oceans. They're found in coastal waters to the hadal zone (deepest regions) and from the tropics to the polar regions.

Current estimates approximate a minimum of 5.25 trillion particles having a weight of 268,940 tons that are currently floating in our oceans (Avio, Gorbi and Regoli, 2017).

Land sources are representing over 80% of the global oceanic microplastic pollution (Wright, Thompson and Galloway, 2013; Jambeck et al., 2015; Avio, Gorbi and Regoli, 2017; Magni et al., 2019).

Major pathways are mismanaged wastes and waste water treatment plants (WWTPs) derived sources in industrialised countries (Jambeck et al., 2015; Magni et al., 2019).

Owing to the lightweight and durable nature, plastics became the prevailing litter in all marine waters (Wright, Thompson and Galloway, 2013). Especially microplastics (<5mm in size) have become the omnipresent marine debris originating either from primary (manufactured) or secondary (fragmentation) sources (Wright, Thompson and Galloway, 2013; Avio, Gorbi and Regoli, 2015).

The most frequent hydrocarbon polymers found in the marine environment are

• Polyethylene (PE)

• Polypropylene (PP)

• Polystyrene (PS)

• Polyvinylchloride (PVC)

• Polyamide (PA)

• Polyethylene terephthalate (PET)

• Polyvinyl alcohol (PVA)

PP, PE and PCV account for 64% of total plastic production (Wright, Thompson and Galloway, 2013).

Due to their hydrophobic (water repellant) properties and large surface to volume ratio they can absorb various organic pollutants readily, showing contamination levels up to six orders of magnitude compared to surrounding levels in marine waters (Wright, Thompson and Galloway, 2013; Avio, Gorbi and Regoli, 2015, 2017). The surface to volume ratio further provides an area for microbial colonisation and are a possible extensive form of transport for these communities (Zettler, Mincer and Amaral-Zettler, 2013; De Tender et al., 2015; Avio, Gorbi and Regoli, 2017).

Depending on density, some polymers float (PE and PP) in the water column whereas others sink (PCV) and sedimentation of polymer particles is accelerated through biofouling (accumulation of organisms) and colonisation (Avio, Gorbi and Regoli, 2017).

Given the constant fragmentation of plastic particles, their concentrations are increasing continuously with decreasing size and are made bioavailable to many different marine organisms (Wright, Thompson and Galloway, 2013; Zettler, Mincer and Amaral-Zettler, 2013; De Tender et al., 2015; Avio, Gorbi and Regoli, 2017).

Ingestion through filter feeding, scavenging and predation is likely to happen and as such, accumulate throughout the marine food web (Wright, Thompson and Galloway, 2013; Avio, Gorbi and Regoli, 2017).
For example, microplastics were found in the gut cavity and digestive tubules of the mussel Mytilus edulis or in the gastrointernal tract and liver of the mullet Mugil cephalus (Wright, Thompson and Galloway, 2013; Avio, Gorbi and Regoli, 2017). Ingested microplastics can even be incorporated in cells through endocytosis and thus, even translocated between tissues (Pittura et al., 2018).

The frequency of ingested microplastics was found to be higher in benthic (seafloor) species when compared to pelagic (open sea) species (Avio, Gorbi and Regoli, 2015) indicating a homogenous distribution of microplastics in sediments which in turn is having repercussions for benthic species feeding above or within sediments. Ingestion can lead to blockages of the digestive tract producing possible satiation, starvation, abrasions and physical deterioration causing an overall reduction in fitness (ability to survive) (Wright, Thompson and Galloway, 2013).

The effects of additives are of additional concern. Plastic additives are used to improve certain properties: heat resistance or elasticity. Additive compounds like phthalates, bisphenol A, alkylphenols, polybrominated diphenyl esters act as endocrine (hormone messangers) disruptors in biota (Avio, Gorbi and Regoli, 2017).

Of further concern are the plastics property to adsorb organic and inorganic pollutants (Avio, Gorbi and Regoli, 2017; Pittura et al., 2018.

PE, PP, PS and PCV bind:

• DDT

• Polycyclic aromatic hydrocarbons (PAHs)

• Hexachlorocyclohexanes

• Chlorinated benzenes

• Polychlorinated biphenyls (PCBs)

• Organo-halogenated pesticides

• Nonylphenols

• Dioxins

Pittura et al. (2018) could show in their study with Mytilus galloprovincialis that the combination of physical stress and PAHs jeopardised the organism’s fitness. Adsorbed organic pollutants were transmitted from microplastic particles to the tissues, having damaging effects on cellular and molecular levels (Pittura et al., 2018).

Halo-organic pollutants are difficult to break down due to their chemical structure and bonding of aromatic hydrocarbons with halogens, and therefore remain a persistent polluting factor in the environment.

Also metals have shown to bind to weathered microplastics, possibly due to increased polarity of the microparticles (Avio, Gorbi and Regoli, 2017).

Elevated temperatures intensify toxic impacts of metals in marine organisms (Res, Sokolova and Lannig, 2008; Hawkins and Sokolova, 2017). This could be partially explained by higher uptake rates to cope with a higher energy demand (Res, Sokolova and Lannig, 2008; Nardi et al., 2017). A growing energy demand leads to increased ventilation and or feeding rates. Both are resulting in higher exposure to metal compounds (soluble or organic) (Res, Sokolova and Lannig, 2008).

Metal detoxification under higher temperatures lead to an enhanced cell maintenance. An enlarged energy demand is needed to counterbalance the maintenance and detoxification by increasing protective and repair mechanisms (Res, Sokolova and Lannig, 2008).

Metal speciation (distribution) is predicted to change in ocean acidification with an expected decrease in pH from currently 8.1 to 7.7 by 2100 and could free vast amounts of metals.(Millero and Woosley, 2009).
For example, divalent or trivalent metals such as copper [Cu(II)] or iron {Fe(III)] are likely to be freed from their complexes with carbonates (CO₃^2-) and hydroxides (OH^-) as these (anionic) complexes are thought to decrease by 77% and 82% respectively (Millero and Woosley, 2009; Ivanina, Hawkins and Sokolova, 2016).

• Copper:

An essential micronutrient, it engages as cofactor in enzymes which play important roles in aerobic metabolism (Ivanina, Hawkins and Sokolova, 2016). In high concentrations however, free copper is toxic for most marine organisms due to the metals pro-oxidant characteristics (Millero and Woosley, 2009; Ivanina, Hawkins and Sokolova, 2016).
Metals that are forming carbonate complexes like copper, are bound to be most strongly affected by pH changes: Cu²⁺ is showing the highest increase of all metals (30%) (Millero and Woosley, 2009). This effect might even be accelerated in estuarine systems that are mixing with seawater. River waters tend to have a pH of 6 and mixing with seawater of low pH (7.4) could have drastic effects on biogeochemical processes and Cu²⁺ toxicity (Millero and Woosley, 2009; Ivanina, Hawkins and Sokolova, 2016; Hawkins and Sokolova, 2017).

Copper accumulation under hypercapnia (high carbon dioxide levels) of 800 µatm CO₂ increased drastically in haemolymph (blood) and haemocytes (haemolymph cell) of two different orders of bivalves, although accumulation levels displayed a strong species-specific pattern (Hawkins and Sokolova, 2017). On the other hand, increased copper in haemolymph might act as an inhibitor of pathogens as exposure sensitised the bivalves against bacteria (Ivanina, Hawkins and Sokolova, 2016).

Free ionic copper could also have potential harmful effects on primary producers yet a lower pH could also free more dissolved iron which in turn stimulates primary productivity (Millero and Woosley, 2009).

• Iron

Under a decrease in pH to 7.4 iron [Fe(III)] solubility is bound to increase by 40% to current levels. This could have a potentially enormous impact on primary productivity as it is one of the most important micronutrients (Millero and Woosley, 2009).

Both, iron [Fe(II)] and copper [Cu(II)], may help breaking up oxyradicals in surface waters, where iron is oxidised to Fe(III) and copper reduced to Cu⁺.

Additionally, a decrease in pH to 7.4 has shown to increase Fe(II)'s half-life in seawater from 1 to 24 minutes, making dissolved iron more available for primary producers (Millero and Woosley, 2009).

The pressure on all species caused by temperature alone is worrisome (Benedetti et al., 2016). Thermal stress is not only going to amplify toxic effects of pollutants, with rising temperatures metabolic rates are to increase and as such the ingestion and accumulation of the here described pollutants (Múgica, Izagirre and Marigómez, 2015). With further acceleration of ocean acidification, the overall stress is about to increase even more. As a result, marine organisms will be exposed to a mixture of multiple stressors and their synergistic effects (Mezzelani, Gorbi and Regoli, 2018; Nardi et al., 2018a).

Yet, different stressors are also species specific. Biological and physical factors like metabolic rates, seasonal fluctuations or pollutant bioavailability have different effects on different species. This can influence individual responses differently (Hawkins and Sokolova, 2017). For example, Mytilus galloprovincialis have a higher antioxidant production during winter temperatures (Nardi et al., 2018a). Or carbon dioxide induced oxidative stress (hypercapnia) negatively influences metal detoxification in oysters but not in clams (Hawkins and Sokolova, 2017).

Overall, the adverse effects of the polluting factors described here have effects on immune responses and (larval) development. Reactions range from histological (body tissue) alterations, inflammation and ecotoxicological disturbances at cellular, biochemical and molecular levels. On the physiology level the respirate system, nutrition uptake and the organisms growth are handicapped (Pittura et al., 2018).

It is not difficult to imagine that a combination of physical stressors in combination with chemical stressors are reducing species survivability. And, although a species might be able to buffer one stressor easily, too many stressor at once could turn out to be fatal.

In order to reduce human introduced stressors like pharmaceuticals and microplastics it is of crucial importance that our wastewater treatment is scaled up to a tertiary stage, where the effective filtering of these compounds and products can be successfully achieved (Jambeck et al., 2015; Mezzelani, Gorbi and Regoli, 2018; Gunnarsson et al., 2019; Magni et al., 2019).

Also, it is of equal importance that the public is informed, attention is drawn and awareness is created in order to push forward for changes that require political decisions (Mezzelani, Gorbi and Regoli, 2018; Gunnarsson et al., 2019).

It is of relevance to tackle these problems at different levels, be it technological or political.

• Avio, C. G., Gorbi, S. and Regoli, F. (2015) ‘Experimental development of a new protocol for extraction and characterization of microplastics in fish tissues: First observations in commercial species from Adriatic Sea’, Marine Environmental Research, 111, pp. 18–26.
  doi: 10.1016/j.marenvres.2015.06.014.

• Avio, C. G., Gorbi, S. and Regoli, F. (2017) ‘Plastics and microplastics in the oceans: From emerging pollutants to emerged threat’, Marine Environmental Research. Elsevier Ltd, 128, pp. 2–11.
  doi: 10.1016/j.marenvres.2016.05.012.

• Benedetti, M. et al. (2016) ‘Oxidative responsiveness to multiple stressors in the key Antarctic species, Adamussium colbecki: Interactions between temperature acidification and cadmium exposure’, Marine Environmental Research 121, pp. 20–30.
  doi: 10.1016/j.marenvres.2016.03.011.

• Gunnarsson, L. et al. (2019) ‘Pharmacology beyond the patient – The environmental risks of human drugs’, Environment International, 129(May), pp. 320–332.
  doi: 10.1016/j.envint.2019.04.075.

• Hawkins, C. A. and Sokolova, I. M. (2017) ‘Effects of elevated CO2 levels on subcellular distribution of trace metals (Cd and Cu) in marine bivalves’, Aquatic Toxicology, 192(August), pp. 251–264.
  doi: 10.1016/j.aquatox.2017.09.028.

• Ivanina, A. V, Hawkins, C. and Sokolova, I. M. (2016) ‘Interactive effects of copper exposure and environmental hypercapnia on immune functions of marine bivalves Crassostrea virginica and Mercenaria mercenaria’, Fish and Shellfish Immunology, 49, pp. 54–65.
  doi: 10.1016/j.fsi.2015.12.011.

• Izagirre, U. et al. (2014) ‘Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis’, Aquatic Toxicology, 149, pp. 145–156.
  doi: 10.1016/j.aquatox.2014.01.013.

• Jambeck, J. R. et al. (2015) ‘Plastic waste inputs from land into the ocean’, Science, 347(January), pp. 768–771.
  doi: 10.1126/science.1260352 VIRAL.

• Magni, S. et al. (2019) ‘The fate of microplastics in an Italian Wastewater Treatment Plant’, Science of the Total Environment, 652, pp. 602–610.
  doi.: 10.1016/j.scitotenv.2018.10.269

• Mezzelani, M., Gorbi, S. and Regoli, F. (2018) ‘Pharmaceuticals in the aquatic environments: Evidence of emerged threat and future challenges for marine organisms’, Marine Environmental Research, 140, pp. 41–60.
  doi: 10.1016/j.marenvres.2018.05.001.

• Millero, F. J. and Woosley, R. J. (2009) ‘Effect of Ocean Acidification on the Speciation of Metals in Seawater’, Oceanography, 22(4), p. 15.
  doi: 10.5670/oceanog.2009.98.

• Múgica, M., Izagirre, U. and Marigómez, I. (2015) ‘Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels’, Aquatic Toxicology, 164, pp. 99–107.
  doi: 10.1016/j.aquatox.2015.04.020.

• Nardi, A. et al. (2017) ‘Chemosphere Indirect effects of climate changes on cadmium bioavailability and biological effects in the Mediterranean mussel Mytilus galloprovincialis’, Chemosphere, 169, pp. 493–502.
  doi: 10.1016/j.chemosphere.2016.11.093.

• Nardi, A. et al. (2018a) ‘Effects of ocean warming and acidification on accumulation and cellular responsiveness to cadmium in mussels Mytilus galloprovincialis: Importance of the seasonal status’, Aquatic toxicology, 204, pp. 171–179.
  doi: 10.1016/j.aquatox.2018.09.009.

• Nardi, A. et al. (2018b) ‘Oxidative and interactive challenge of cadmium and ocean acidification on the smooth scallop Flexopecten glaber’, Aquatic Toxicology, 96, pp. 53–60.
  doi: 10.1016/j.aquatox.2018.01.008.

• Pittura, L. et al. (2018) ‘Microplastics as Vehicles of Environmental PAHs to Marine Organisms: Combined Chemical and Physical Hazards to the Mediterranean Mussels, Mytilus galloprovincialis’, Frontiers in Marine Science, 5(103), p. 15.
  doi: 10.3389/fmars.2018.00103.

• Res, C., Sokolova, I. M. and Lannig, G. (2008) ‘Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change’, Clim Res, 37, pp. 181–201.
  doi: 10.3354/cr00764.

• De Tender, C. A. et al. (2015) ‘Bacterial Community Profiling of Plastic Litter in the Belgian Part of the North Sea’, Environmental Science and Technology, 49(16), pp. 9629–9638.
  doi: 10.1021/acs.est.5b01093.

• Wright, S. L., Thompson, R. C. and Galloway, T. S. (2013) ‘The physical impacts of microplastics on marine organisms: A review’, Environmental Pollution, pp. 1–10.
  doi: 10.1016/j.envpol.2013.02.031.

• Zettler, E. R., Mincer, T. J. and Amaral-Zettler, L. A. (2013) ‘Life in the “Plastisphere”: microbial communities on plastic marine debris’, Environ. Sci. Technol., 47, pp. 7137–7146.
  doi: 10.1021/es401288x