Marine Ecotoxicology, F. Regoli

Prof Francesco Regoli Marine Ecotoxicology

Traditional chemical pollutants in the marine environment

Classes of pollutants, origin, distribution, biological effects → important.

The ecotoxicological approach plays a role in understanding the pollutants, based on the use of organisms as biological bioindicators. Bioaccumulation → integrated biological effects: analyzing biological markers to know the toxicity and better predict the risk of pollutants.

Bioindicator organisms

From plankton to top predators, new models and new approaches have been developed in the last few years. Which is the best organism? It depends on what we want to study.

• Old models: used to study molecular and biological processes linked to ecotoxicology, but we developed a lot of new models and kits.
• We can use cell cultures, isolated organs or tissues, molecular approaches using gene markers…
• New analytical technologies: genomics, proteomics, metabolomics (only in the last few decades), engineered tissue-based screenings, virtual tissues systems.
• New bioinformatics and elaboration procedures: a network architecture for canonical transcriptomes. It produces thousands of data that require powerful computers for the analysis.

From all these new techniques, we have a new way to think about ecotoxicology: the common paradigm of the exposure-response continuum (a stress of specific intensity and duration should enhance a correlated response) has been rejected:

• There aren't necessarily variations of the normal biological activity in the organism.
• The organism might have detoxification pathways that, at low doses, fight sources of contamination, while at higher concentrations the pollutant might show some impacts.
• Even if the pollutant enhances immediate effects on the organism, it doesn’t mean that it would have a physiological impact on the organism.

A biological effect of a pollutant can occur at different levels. Effects at the cellular level might not have a physiological impact on the organism. This level can be monitored as a prognostic value, since it can be an early warning signal that anticipates further damage at higher biological levels. If contamination reaches high intensities or long exposures, defenses at the cellular level may not be able to compensate or adapt to the stress condition: the cell may be damaged and impacts of the contamination could appear also at higher levels.

Chemical pollutants in the marine environment

Traditional:

• Trace metals and organometallics
• Polycyclic aromatic hydrocarbons (PAHs = oil spills)
• Halogenated hydrocarbons (dioxins, pesticides)
• Nitroaromatic and heterocyclic
• Organophosphates and estrogenic chemicals

New emerging compounds:

• Microplastics
• Nanoparticles
• Pharmaceuticals
• Endocrine disruptors (long-term chronic effects)
• Toxins and metabolites (algal > natural!)

Annual loads in the Mediterranean:

• 120,000 tons of oils and 60,000 tons of detergents
• 85,000 tons of heavy metals (Cu, Pb, Hg, Cd)
• 1,960,000 tons of crude oils (15,000 tons from a power plant in Lebanon bombed in 2006)
• Uncertain loads of pesticides and organochlorine
• 200,000 tons of volatile organic compounds (VOC), 47 tons of carcinogenic aromatic, 55kg of TCDD/F (dioxins).

Anthropogenic sources

• Industrial areas
• Petrochemical sites
• Principal routes for navigation that lead to industrial accidents and oil spills – the Mediterranean is a closed basin, with slow turnover of water; all the chemicals have the possibility to exert their toxicological effect.
• Ships traffic is huge > spills/collisions.

In recent years, at a global scale:

• More than 15,000 new compounds are produced every year.
• More than 100,000 synthetic compounds have been classified in the mid-80s.
• At least 11,000 compounds are produced in amounts which can represent an environmental hazard.
• Toxicological effects are still poorly known.

EU chemicals regulation adopted in December 2006, REACH (registration, evaluation, authorization, and restriction of chemicals – this approach doesn’t include ecotoxicological effects; it’s very generic and it’s based on the measure of acute toxicological response, which is totally different from chronic toxicity).

Long-term effects and chemical mixtures are still difficult to evaluate; organisms are exposed to all types of chemicals, and the toxicological effect of mixtures can be totally different from those of the individual chemicals → difficult to study, assessing environmental pollution by performing just one chemical analysis is not feasible. We also have to consider a biological approach to have an idea of how organisms respond. Compounds with low toxicity can also be dangerous if they’re present in high amounts: the concentrations of the contaminants should be carefully measured.

Most of the time, the effects of chemicals combine with other stressors, such as high temperatures, acidification, eutrophication… Biological pathways can be sensitive to some classes of chemical compounds, but there is not a specific response for each chemical, and most of the time, they interact with other physical or chemical stressors (pH, temperature…).

More common pollutants in the marine environment

1. Crude oil and polycyclic aromatic hydrocarbons (PAHs)
2. Halogenated hydrocarbons
3. Trace metals

Crude oil (PAHs)

Crude oil is a mixture of aliphatic and polycyclic aromatic hydrocarbons, which have different relevance in terms of environmental risk. They are different molecules, originated in different ways, which are related to the extraction area (e.g., crude oil Iran, crude oil Azerbaijan…). Crude oil is formed by carbon, and the energy derives from the H-bond of the carbons. When dealing with oil spills, two completely different portions should be considered:

• Aliphatic = linear, branched, cyclic, saturated or unsaturated chains, but no aromatic rings.
• Aromatic = presence of one aromatic ring with at least 2 double bonds important for the toxicology of the molecule.

Ecotoxicological relevance of aromatic hydrocarbons

• Natural origin and pollution – it is related to the extraction, transport, transformation, and use. Anthropogenic impact is highly different from natural bubbles developing from natural oil spills from the bottom. A similar natural condition can be observed for trace metals.
• Limited medium atmospheric transport for PAHs – this is true for some chemicals and not for others, it depends. This factor is important for monitoring. PAHs are moved due to water circulation, food webs, partly atmospheric circulation: the way of transport is responsible for the global scale diffusion of certain contaminants.
• Different bioaccumulation/biotransformation rates of PAHs in invertebrates and vertebrates (e.g., fishes have a cellular system that transforms this molecule and excretes the PAHs from the tissue, although it bioactivates carcinogenic events).
• They’re not biomagnified along food webs: pollutants are assumed from the food web and each organism can bioaccumulate it itself.

Sources and environmental fate of PAHs in the marine environment

• Rivers runoff
• Urban and industrial areas runoff
• Combustion processes, urban pollution (washout, discharges, fires, and atm fallout)
• Direct discharges
• Maritime traffic and harbors
• Loss from ships (accidental or systematic)
• Offshore exploitation and activities, with implications for both biota and the sediments.

The fate of oil spills

The fate of oil released in the sea is very complex because it’s influenced by physical, chemical, and biological processes involving both water and atmosphere and the biota. Some of these processes occur simultaneously and others at different moments.

• Oil slick sea surface – the oil can remain at the surface and spread, depending on the amount that is discharged, its molecular capacity (it tends to be in a monomolecular phase), currents, and winds.
• Water-in-oil emulsion – the water enters the oil, producing a dark viscous mousse. When the emulsion loses water, tar balls can be formed.
• Tar balls – they usually go to the seafloor and become part of the sediments (depending on the viscosity of the oil).
• Volatile compounds – these fractions of the oil spills can evaporate; among them, there are some inflammable aromatic hydrocarbons that are carcinogenic and highly toxic.
• Coasts – effects on the beaches are acute if the oil reaches them immediately; if it arrives after some weeks, after some of the more toxic compounds have evaporated, the long-term effects are less severe.
• Adsorption – being mostly hydrophobic, oil tends to adhere to particulate matter, either organic or inorganic, and be transported along the water column or in the sediments. Most of the free chemicals transit in seawater, and they’re not appropriate for analyses; sediments and organisms are much more reliable to assess the concentration of pollutants in the sea.
• Biodegradation – many bacteria and microbes, in general, are able to attack and degrade at least a portion of the hydrocarbons.

An oil spill could take from weeks to several months or years to undergo all these transformations and processes.

Operational measures to remove oil slick

1. Containment and recovery – mechanical and manual operations to remove hydrocarbons from the sea or the coasts and limit its spreading; if the oil changes viscosity, it also changes its chemical and toxicological effects. We can use a Skimmer, a system that creates water circulation with a pump that filters the oil; it can only be applied in the early phases of an oil spill, before the oil becomes thicker. Local fishermen with small boats can also make a great contribution by fragmenting the oil slick; the operations must be organized since they present some difficulties related to maintenance, recovery, storage, and treatment of oil onboard. Manual collection is another option that proved valid, especially in difficult areas. A chemical waste chain can be created, going from the coasts near the collection site, to the storage site, to temporary storage systems, and then to treatment systems.
2. Adsorbent products – they are disposed of on adsorbing and floating barriers, which should prevent the diffusion of the oil.
3. Dispersant products – they are chemical compounds which, in contact with oil slick, promote its spreading and emulsion in small droplets, which are easily dispersed. These molecules, however, are still toxic and can cause damage to benthic habitats; moreover, there are still some concerns in relation to the time for spreading and some other technical problems. They should be used before the oil gains viscosity.

Chemical dispersion

• Suspension of finer oil droplets in the water column
• Reducing/avoiding the amounts of pollutant on shorelines
• Enhancing oil biodegradation
• Limitations on viscous and/or weathered oils
• Temporarily increases the local toxicity.

The possibility for dispersion should be linked to the volume of seawater available for the dilution process: in other words, the amount of oil which can be safely dispersed is related to the volume of available water, the renewal of water, all conditions needed to get a rapid decrease of the dispersed oil concentration to safe levels. Different countries have different regulations for the use of chemical dispersants. In general, the current policies for dispersants include:

• Applying them mainly offshore
• Restrictions in coastal areas (geographic limits).

For example, in Italy, dispersants can be considered only as an extreme solution, and we can only use the compounds which have been previously tested and approved through:

• Effectiveness and stability tests
• Biodegradability test (based on the Arabic light, liquid oil, but not representative for all the oils)
• Bioaccumulation tests (based on partition coefficients: a theoretical calculation, not considering the physiological aspects)
• Ecotoxicological bioassays → but they test the effects of a compound alone and not when interacting with oil; these two combined together could have increased toxicity.

An experimental design for the approval of a dispersant should be more or less like this:

• Control sample
• Oil sample
• Dispersant sample
• Oil + dispersant sample, with a ratio of 1:10.

Bioindicator organisms

• Mytilus galloprovincialis (whole tissues, digestive gland, gills, hemolymph)
• Vibrio fischeri (bioluminescence)
• Phaeodactylum tricornutum (growth inhibition)
• Crustaceans (mortality)
• Paracentrotus lividus (larval development)
• Crassostrea gigas (larval development)

2 days of incubation plus 21-35 days of recovery; sampling after 2, 7, 14, 21, 35 days.

Ecotoxicological bioassays concerning

• Bioaccumulation (HYPs and PAHs)
• Genotoxic damage
• Immunological parameters
• Lipid peroxidation
• Neurotoxicity biomarkers
• Oxidative stress biomarkers.

The bioavailability of PAHs is investigated, in terms of:

• Total PAHs
• Low molecular weight PAHs
• High molecular weight PAHs (carcinogenic)
• Total aliphatic compounds.

The results clearly show that not only the mixture oil + dispersant is more toxic than the individual compounds, but also that on most bioindicator organisms the dispersant alone already shows higher toxicity than the very same PAHs it’s meant to eliminate! Moreover, high toxicity of the mixture can still be observed after 3-5 weeks of recovery.

Discobiol project

It is an ongoing project aiming to assess the toxicity of dispersed oil in different environmental conditions and on various bioindicator organisms, with the final objective to determine if the use of dispersant is beneficial or not. Some early results show that:

• The chemical dispersion impact is always higher than that of mechanical dispersion.
• The presence of mineral particles tends to reduce the effects of chemical dispersion on fish.
• Even at strong exposure levels, the effects of chemical dispersion seem to be limited and temporary, usually with dissipation after 2 weeks recovery.

Further investigations are planned on the effects of dispersed oil on larval stages and in other climates; the effects of dispersants, in fact, change depending on the sea temperature.

Responses to oil spills in the Arctic environment

This area is not a pristine environment anymore; on the contrary, it is recently interested by oil extraction and transportation. Moreover, climate change is particularly hitting these environments, causing ice thickness reduction; the more reduced the ice thickness and extension, the more exploitation of the Arctic seafloor is possible. Geologically speaking, the hydrocarbon deposits in the Arctic are among the dirtiest in the world and extracting them could generate 3-4 times more CO₂ than normal extraction!

Development of countermeasures for oil spill in the ice

Ice interference, cold temperature, isolated locations make it much harder to fight oil spills in Arctic environments; traditional tools such as skimmers have to be adapted. We can also accelerate the natural recovery of the environment by the promotion of Oil Mineral Aggregate (OMA) formation: interaction of micron-sized mineral particles with droplets of bulk oil, to form solids-stabilized, oil-in-water emulsions.

Clay-oil flocculation – some laboratory studies with oiled sediments from the Exxon Valdez spill (Alaska) revealed that micron-sized mineral fines, seawater, and weathered oil interact to form clay-oil flocs. Clay-oil flocculation was responsible for the natural cleaning of sheltered shorelines in Prince Williams Sound observed a year after the spill.

In 2008, a Canadian vessel tested the effectiveness of fighting oil spills using OMA formation in a field campaign near the Saint Lawrence estuary, using a controlled oil spill: analyses were performed both in field and in the lab.

In-situ instruments

• UW camera
• Laser scattering and transiometer
• Turbidometer.

Water samples were collected every 30 minutes for 2 hours, at different depths (surface, 1 m, 5 m, 10 m), and analyzed through:

• Gc-FID analysis of TPH distribution
• Epifluorescent microscopy
• UV-fluorometer analysis.

The aim was to assess the biodegradation processes and monitor the persistence of oil at cold temperatures. Results:

• Oil spilled on ice-covered waters may be effectively dispersed by promotion of OMA formation.
• The propeller wash on ice-breakers can provide sufficient mixing energy to facilitate OMA formation.
• Verification of significant oil biodegradation under ambient low temperature conditions.
• Ongoing wave tank and numerical model studies are focused on the influence of factors such as mixing energy, type of mineral fines, chemical dispersants, and toxicity issues.

Halogenated hydrocarbons

• Only synthetic compounds having at least one halogenated atom
• Largely used in industries (PCBs), agriculture (pesticides, herbicides, insecticides), secondary products in synthetic and combustion processes (dioxins)
• Interested by atmospheric transport, rivers, rural urban and industrial runoff, direct discharges
• Persistent organic pollutants
• Biomagnified along food webs (e.g., DDT, HCB).

They’re defined as persistent for their presence but also for their biological effects; persistent pollutants typically aren’t acutely toxic molecules.

Notorious halogenated hydrocarbons

• DDT and derivates
• Vinyl chloride (PVC)
• Tetrachlorodibenzo-dioxin (highly carcinogenic)
• Polychlorinated Biphenyls (PCBs).

Diffusion of persistent organic pollutants can be mediated by:

• Atmospheric currents
• Food webs
• Sea currents
• Rivers.

The indiscriminate use and environmental persistence of these pollutants make their control particularly important. Halogenated hydrocarbons are ubiquitous; they can be found in the atmosphere, but also in the oceans, the soil, and in the ice Arctic. There, molecules don’t evaporate, so they tend to accumulate; this offers the possibility to measure the concentrations effectively.