Appunti completi di Marine Ecotoxicology

MARINE ECOTOXICOLOGY

What ecotoxicology is about?

• Traditional and legacy pollutants: origin, fate, distribution

• Effects of on marine organisms and bioindicators

• Mechanisms of action and biomarkers

• New contaminants of emerging concern

• From molecular effect to ecological risk assessment

Traditional chemical pollutants in the marine environment are for instance metals, PAHs, pesticides, PCBs,

dioxins, etc. We have advanced knowledge on the origin, distribution and biological effects of these

pollutants, while we have to understand more about others.

Annual loads of traditional chemicals in the Mediterranean:

• 120.000 Tons of oils and 60.000 Tons of detergents

• 85.000 Tons of metals (Cu, Pb, Hg, Cd)

• 1.960.000 Tons of crude oils

• Uncertain loads for pesticides and organochlorines

• 200.000 Tons of volatile organic compounds (VOC), 47 Tons of carcinogenic aromatic, 55 Kg of TCDD/F

Many anthropogenic activities are responsible for the emission of these pollutants: industrial areas, petro-

chemical, industrial accidents and principal boat routes.

At global level more than 1.500 new compounds are produced every year and more than 100.000 synthetic

compounds (that don’t exist in nature) are classified in mid-80s. At least 11.000 compounds are produced in

amounts which can represent an environmental hazard: the risk can be a mix of their ecotoxicological impact

and their amount. For several compounds toxicological effects are poorly known. European Chemicals

regulation was adopted in December 2006 and is called REACH (Registration, Evaluation, Authorisation and

Restriction of Chemicals). Long term effects and chemical mixtures are difficult to evaluate and there is the

necessity to integrate chemical data with biological evaluations: the actual problem is that we are not exposed

to only one pollutant, but a mix of them and we don’t know what’s the effect of the mixture and it is not

necessary the sum of their singular effects. Acute ecotoxicological effects are easier to study, but it takes

several years to understand the long-term effects of a compound and to predict in advance the possible

negative effects of a new synthetized compound.

How many chemicals in the environment? More than 100,000 commercial chemical substances were recorded

in 1981 (European Inventory) with more than 1,500 new chemicals added every year.

• 500 chemicals with extensive characterization for exposure and toxicological hazard

• 10.000 chemicals with sufficient characterization for exposure and toxicological hazard

• 20.000 chemicals with limited characterization for exposure and toxicological hazard

• 70.000 chemicals with poor/absent characterization for exposure and toxicological hazard

Environmental pollution is linked with human health: tumors, cardiopathies, ischemic failures, respiratory

problems can be due to the exposition to some toxic compounds. At European level, every year 10% of deaths

are due to pollution, and this percentage is higher in more vulnerable countries. At global level, in 2015

pollution caused 9 mil premature death (16 % of the total), 15 folds higher than deaths caused by wars and

violence acts. Contrast to pollution is also a fight for equity and equality: more harmful and evident

repercussions act on the most vulnerable groups, socio-economic effects are worst in populations living in less

favorable conditions and in low-income countries, 92% of deaths appear to be linked to pollution.

Pollution is considered one of the top 5 direct drivers of biodiversity loss, but there is the lack of extensive

toxicological data, most of toxicological data are from few species (often freshwater species). There are many

gaps of knowledge on long-term effects, interactions among cell pathways, interactions in chemical mixtures

(synergic effect of the pollutant mixture) and cross-species extrapolations.

The importance of monitoring emerging pollutants integrating biological effects with chemical levels:

• Chemical hazard assessment is typically performed on a single-basis compound

• Organisms are exposed simultaneously to several chemicals

• Mixture effects: synergistic, additive, antagonistic relationships

• Integrated assessment: effect-based methodologies

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To assess pollutants effects, we use bioindicator organisms, from plankton to top predators. Example of them

are sea urchins, mussels, polar bears, etc. Today we use many approaches to study negative effects, from

morphological to intra-cellular analysis, molecular and genomic approaches, etc. Bioinformatic is an important

step of the process and a new powerful tool, but we need to understand the biological and ecological

meaning of the machine output.

There is a new way to think about ecotoxicology. The common paradigm was the exposure-response

continuum.

The new paradigm is the activation of toxicity pathways.

Emerging pollutants in the marine environment are for example pharmaceuticals, endocrine disruptors,

microplastics and nanoparticles and algal metabolites and toxins. Metals we saw before are defined by a

chemical definition, while these new pollutants like endocrine disruptors and pharmaceuticals are described

by their effects. Today the world is warming and the oceans are more acid, but if these effects occur in a

polluted area consequences can be even worse, because we found in a multiple stressor system; here the

effect of a pollutant can be different thanks to the different chemical environment.

Overview on more common traditional pollutants of the marine environment

Crude oil and Polycyclic Aromatic Hydrocarbons (PAHs): crude oil is a mixture of aliphatic and polycyclic

aromatic hydrocarbons (PAHs) which have a different significance in terms of environmental risk. We normally

associate the presence of pollutant with human activities, but hydrocarbons are naturally produced. The

pollution is related to extraction, transport, transformation, use (combustion processes) of them, which are

anthropogenic activities. We use to say that pollution has no limits or boundaries, but it is not true for every

pollutant: for example hydrocarbons have a limited-medium atmospheric transport (around some tens of km).

there is a different bioaccumulation-biotransformation rate of PAHs in invertebrates and vertebrates.

Vertebrate can expel hydrocarbons through fecal pellets. Invertebrates do not have a mechanism of expulsion

of hydrocarbons, so they tend to bio-accumulate them. So, we expect to find higher concentration of them on

mussels than in fishes for example, but that do not mean that fishes are not exposed. Another important

aspect is that there is no biomagnification along food webs: it means that top predators do not have higher

concentration than lower trophic levels, and that’s because vertebrate can expel them as we just saw. That’s

why hydrocarbons do not have a wide dispersion in marine habitats.

The main sources of hydrocarbons are not marine, but they come

from urban environments through rivers, urban and industrial

runoff, combustion processes, urban pollution (washout,

discharges, fires and atmospheric fallout), direct discharges,

maritime traffic and harbors, loss from ships (accidental or

systematic off-shore exploitation and activities). The toxicity of a

compound can rise if it is activated and usually the activation

means the transformation in radical: hydrocarbons are subject of

photo-activation promoted by light or energy transmitted by fire

during combustion. Even if many oil spills events seem to be

disastrous, if correctly threated, we can be totally recover the

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environment: oil spill is not an irreversible event. Depending on the amount of the release, a part of

hydrocarbons naturally evaporates: if the oil spill reaches the coast before the evaporation occurs it will have

more disastrous effects then the oil which hits the coast days or weeks after the spill. Hydrocarbons are

lipophilic, so they do not dissolve in marine water: the problem is that marine water is not a pure substance,

and hydrocarbons are very effective in binding lipidic compounds. An environment hit by an oil spill usually

can be fully recovered in less than 10 years.

Other pollutants are halogenated hydrocarbons: they are only synthetic compounds added with a halogen,

which usually was a chlorine. They are not present in nature, but they are only produced by humans. They are

largely used in industrial applications (PCBs), agriculture (pesticides, herbicides, insecticides) or secondary

products in synthetic and combustion processes (dioxins). Dioxin is for instance produced during accidental

fires in plastic industries and that’s why we are so worried when it happens. Most pesticides are now banned,

and they have not an acute toxicity. They have atmospheric transport, or through river, rural urban and

industrial runoff, direct discharges. They are grouped in Persistent Organic Pollutant (POPs): it means that

they tend to be accumulated in the environment, with chemical and biological implications: for example, they

have bio magnification though the food web. Bioaccumulation is the accumulation of a contaminant or toxin

in or on an organism from all sources (e.g., food, water, air), not depending on their trophic level and it occurs

in polluted areas. Biomagnification is the increase in concentration of toxin as it passes through successive

levels of the food web, because it is not absorbed or expelled; biomagnification can occur far from the

polluted site. Thanks to biomagnification, top predators will have extremely higher concentrations of toxic

compounds than organisms belonging to lower trophic levels. The diffusion routes of «persistent organic

pollutants» (POPs) depends on atmospheric currents, food webs, sea currents and rivers. In addition of that,

we can import food coming from other places in the world and so we can change the natural routes of

pollutant. Human can act as top predators when we eat tuna for example (a top predator which is subject to

bio magnification), but we can also eat mussels and so we can act also as a lower trophic level organism: for

this reason, we are exposed to different levels of pollutants.

The grasshopper effect is responsible for transport and biomagnification in the Artic and other cold

environments as mountains: the global circulation transports POPs to these environments, where the cold

temperatures do not allow a high evaporation rate and that’s why they tend to accumulate in these

environment (condensation is higher than evaporation there). POPs deposit on ice and when it melts, they are

released into water: it happens when the phytoplankton blooms occur, enhancing the biomagnification due to

the high pollution exposition of the lower trophic levels like phytoplankton. The indiscriminate use and

environmental persistence of these pollutants (POPs) makes their control particularly important. For example,

in some populations the presence of DDTs, PCBs and dioxins in breast milk was discovered.

Modern mechanism-based toxicology increased public awareness of environmental toxicology. “The “control

of nature” is a phrase conceived in arrogance, born of the Neanderthal age of biology and the convenience of

man” (Rachel Carson Silent Spring, 1962).

Examples of notorious halogenated hydrocarbons:

• DDT and derivates: wildly used in the ’50 and ’60, most used pesticide for mosquitos’

control and pest control in agricultural crops. After more than a decade of massive

use, some bird populations and sea lions exhibited sharp decline, with several cases

of death, nervous system damage, and reproductive failure… which were then associated to DDT

accumulation. As a result, DDT was banned in the US in 1972.

• Vinyl chloride (PVC)

• Paraquat (herbicide)

• 2,3,7,8-tetrachlorodibenzodioxin (potent carcinogenic)

• Polychlorinated biphenyls (PCBs)

After DDT is applied, some DDT volatizes, some remains on the plant, and some washes off the plant into the

soil, eventually making its way to a stream, river, or lake. The DDT that remains on the leaves of plants may be

ingested by primary consumers, such as insects and rodents. DDT that has washed into a waterbody, remains

in the sediment, or is consumed by bottom-feeding organisms or absorbed by fish gills and skin. In the body of

an animal, DDT is metabolized to DDE which is fat-soluble and is stored in fatty tissues. Its progressive

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accumulation causes: reproductive failure (in birds-eggshell thinning), immune system problems, nervous

system damage, death. High levels of DDE cause the female ospreys to lay eggs with thin eggshells. The DDE is

an inhibitor of the enzyme responsible for the control of the Ca present in the shell. Thin eggshells have a

greater chance of breaking, leading to embryo death. With high levels of DDE, female ospreys can also lay

eggs that contain high enough concentration of DDE to prevent embryo development. Below 3 μg/g DDE has

no effect on reproduction, above 16 μg/g brings to failure. DDT is still used in some countries for insect

control on crops. In countries where mosquitoes carry malaria, DDT is currently used for mosquito control.

After the banning of DDT other pesticides has been used: more than 2000 pesticides on the market with > 500

different chemical compounds. >80% of the soil tested in EU has pesticide residues, 45% of which contained

Glyphosate/AMPA (max content: >2 mg/kg). Obviously, the chemicals released in the soli will enter in the

cycle of water/food web through they will be spread everywhere.

Dioxins and furans

Dioxins are the most toxic chemical molecule that can be released in the environment. In the ‘60/’70 dioxins

become “popular” for their first time during their deliberate use in Agent Orange in Vietnam, as a defoliant,

and the effects are still visible today. In the ’76 there has been an industrial accident with release of dioxin at

Seveso (northern Italy), during production of 2,4,5 Trichlorophenol, in which still nowadays the cancer

incident is higher than in the rest of Italy. One year later Dioxins has been detected in ash of urban

incinerators, and lead in ‘77-’85 to technical modifications on incinerators to remove dioxins from ash

(combustion at higher temperatures): they did not solve the problem. In the ’80-’90: dioxins are produced

exclusively as unavoidable by-products during numerous processes of production, utilization and disposal of

compounds containing chlorine such as: DDT, Herbicides, Defoliants, Anti-mold for wood, PVC. Excluding

acute, accidental events, the main route of exposure to dioxins is represented by food (those rich in animal

fats). Since the 90's a better control of the release of dioxins from the incineration systems begins. Accidental

production of dioxins is caused by waste combustion (uncontrolled temperatures), accidental fires and

atmospheric emission.

Dioxins are mostly accumulated through the diet and are concentrated in animal fat, one liter of cow's milk

can make the same dose of dioxin breathed by the cow in eight months (Connett and Webster,1987). Dioxins

accumulate stably in the fats of the human body. Man has no chance of getting rid of it, but the woman does

it when she has a baby: maternal dioxin can pass to the fetus and then to the newborn through the placenta

and the mother's milk. Dioxins act as fat-soluble hormones and destroy numerous hormone systems: male

and female sex hormones, insulin, gastrin, and glucocorticoids. Examples of chronic and acute toxicity effects:

• Damage to the immune system.

• Damage to the reproductive system.

• Carcinogenic effects.

• Early acute effects (such as Chloracne).

The dioxins have a typical chronic toxicity effect, unless one is exposed to massive

amounts of dioxins at the same time. It is made of two aromatic rings, linked by two O

atoms and with a variable amount of Cl, for a total of 75 type of dioxins: 2,3,7,8-

tetraCDD. Furans are basically the same but with only one O linking the two aromatic rings and

are a total of 135 molecules: 2,3,7,8-tetraCDF. Minimal differences in the chemical structure of

molecules, profoundly affect the reactivity and biological toxicity of dioxins. No biological

effects of 2,3,6,8TCDF at concentrations more than 100 folds higher than those observed to be biologically

reactive for 2,3,7,8-TCDF. The molecule does not provoke any adverse reaction: it depends on the interaction

with the genetic expressions: if it’s accumulated in the cell, recognized by the receptor of the cell (interaction

in a specific receptor, that is activated), once into the nucleus it interacts with specific region of the DNA that

modulate the expression of various genes. All the effects that we saw are modulated because something

wrong is happening at the expression level, this is why if this interaction is blocked the dioxin is not reactive

and do not cause any harm. The main factor making a dioxin toxic is its affinity with the specific receptor. The

position of the Cl atom interferes with the affinity with the AHR receptor (max affinity with the Cl present in

positions 2,3,7,8). Even if we have a high number of type of dioxins and furans, at the end only a few have a

specific affinity with this receptor and are 7 dioxins and 10 furans, they all have a planal shape, caused by the

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presence of Cl in positions 2, 3, 7 and 8. If the molecules are brominated instead of chlorinated the toxicity

does not change much: dioxin-like has a biological, not chemical meaning (affinity with AhR). The brominated

ones are flame retardants, that we are in contact with each day. For many years these have been largely used

and are today banned

Polychlorinated biphenyls: biphenyl structure, very simple: two aromatic rings. Polychlorinated

biphenyls, PCBs = a family of compounds in which chlorine atoms are substituted for hydrogen

at 1 to 10 positions of the BIPHENYL structure. Largely used in industrial processes, in

refrigeration liquids, highly toxic and nowadays banned. Depending on the position of the Cl it

changes its shape and therefore its effects/toxicity dioxins-like biphenyls, different molecule.

Levels of toxicity

So many chemical forms with so different toxicological potential require simplification when

expressing data: TEF, Toxicity Equivalent Factor, to normalize the data in biological terms.

• 2,3,7,8-TCDD (the most toxic) has a TEF equal to 1

• Other dioxins or furans have a lower T