Plastic Busters MPAs in the lab: UNISI

THE JOURNEY OF A SKIN BIOPSY: FROM THE SEA TO THE LAB TO DEFINE THE IMPACT OF MARINE LITTER ON ENDANGERED SPECIES

 

UNIVERSITÀ DEGLI STUDI DI SIENA

How can a 5 mm plastic debris (microplastics) affect filter feeder baleen whales that are up to 20-meters long?
How can a few kilograms of plastic debris affect Sperm whales that weight more than 40 tons? What are the interactions between charismatic megafauna and micro- and macro-plastics?

Questions like these receive their answer in the lab, through the work of scientists such as those of the Plastic Busters MPAs team, as they investigate marine litter impact and their related toxicological effects. Let’s see how this goes…..

After days at sea, collecting superficial microplastics with manta trawl, observing and surveying floating marine litter and wild animals in the waters of marine project areas, and collecting skin biopsies from dolphins and whales (Figure 1), the action moves to the lab.

 
FROM THE SEA TO THE LAB TO DEFINE THE IMPACT OF MARINE LITTER ON:
- MEDITERRANEAN CETACEANS
- MEDITERRANEAN SEA TURTLES

 

BIOINDICATORS FacT sheet 

Read more about the Sperm whale 

 

BIOINDICATORS FacT sheet 

Read more about the Fin whale 

 

BIOINDICATORS FacT sheet 

Read more about the Sea Turtle Caretta Caretta 

FROM THE SEA TO THE LAB TO DEFINE THE IMPACT OF MARINE LITTER ON :

MEDITERRANEAN CETACEANS

First of all, the precious biopsies get divided in different sub-samples - each characterized by a different and specific purpose - and stored in liquid nitrogen already on the boat, to be ready to be analyzed once back in the lab (Figure 4).

Figure 4: Storing biopsies in liquid nitrogen on the boat right after the sampling

In the lab, each tissue takes centre stage given its capacity to store different and important information thus becoming an important diagnostic tool (Figure 5). A part of the blubber, for instance, is used to quantify the concentrations of Persistent Bioaccumulative and Toxic compounds and plastic additives by using chromatography and mass-spectrometry techniques. 

Figure 5: skin sample sub-samples

Meanwhile, a few grams of the skin help our scientists evaluate the toxicological effects caused by plastic debris ingestion in cetaceans through innovative -omics techniques and many other biological analyses (from DNA to protein expressions).

As a result, data extracted from small amounts of tissues collected from these living organisms - which are obtained by investigating the alterations of the smallest molecules constituting the mammalian cells – ends up telling us the extent to which both small and large plastic particles can affect the marine mammal health status and, thus, the state of biodiversity in Mediterranean MPAs.

Let’s see the lab workflow to obtain these data

Once in the lab, the samples are retrieved from the liquid nitrogen to be safely stored in ultrafreezer at -80°C or directly processed (Figure 6).

Part of the samples (skin part) is homogenized to extract proteins, RNA and DNA for the different analysis.
To analyse the alteration at the DNA and RNA level, the samples are processed so that the tissues are disrupted and the molecules of interest are extract from the cells and nucleus using buffers and centrifugation steps (see Figure 7); the end results of this process is a solution containing the molecules at the basis of the life: DNA and RNA. 

Figure 6: Samples retrieved from the liquid nitrogen and ready to be analysed in the lab     Figure 7: Centrifugation steps to isolate DNA and RNA from the cetacean tissues

Figure 8: Phases of the reactions to analyze RNA from cetaceans

Some of the procedures require the use of enzymes and specific buffers for the nucleic acids (RNA and DNA) to be available to be processed. This will allow obtaining from a small amount of tissue a considerable amount of DNA and RNA to be analyzed by means of quantitative Real-Time PCR (polymerase chain reaction – see Figure 8); in other words, a very small amount of the tissue collected at sea from free-ranging organisms can be used to assess the effects of plastics on this species. All these procedures need to be performed with high accuracy and precision. To prevent degradation and any form of contamination they are performed on ice and under biological hoods using sterile material and equipment (Figure 9). 

Figure 9: phases of the analysis of the RNA expression using the quantitative Real Time PCR

Once the reaction is ready, it is incubated in a thermal cycler at different temperature and using florescent dyes to account for the number of copies of DNA and RNA present in the samples.

After a few hours, the results are ready to be analyzed and a bunch of the alteration of some genes can be correlated to the exposure to plastic and plastic additives to diagnose the health status of the cetaceans.

Meanwhile, the chemical analysis of Persistent Bioaccumulative and Toxic compounds and plastic additives by means of chromatography and mass-spectrometry techniques is carried out on the blubber part of the biopsies, since lipophilic compounds are likely to be accumulated in the fat tissues.

The chemical compounds are extracted from the blubber tissue by first disrupting it by several passages using specific solvents (different according to the compounds to be analysed) and temperatures and purified in columns, in order to obtain different purified liquid fraction containing the toxic compounds to be analysed using chromatography and mass-spectrometry techniques (see Figure 10-12).

 

Figure 10: phases of the extraction and injection in the Gas chromatography/mass spectrometry instrument for the analysis of toxic compounds (Plastic additives) accumulated in the cetacean tissues

Figure 11: vials containing the solution extracted from each sample and ready to be injected in the GC/MS 

Figure 12: analysis of the results of the GC/MS data

At the end of each run, data is displayed as chromatograms and qualitatively and quantitively analyzed to understand which and how much of the toxic compounds have been accumulated in the cetacean tissue and correlate these levels to the biological responses obtained for the same organisms.

FROM THE SEA TO RESCUE CENTRE AND UP TO THE LAB: DEFINING THE IMPACT OF MARINE LITTER ON

MEDITERRANEAN SEA TURTLES

Sea turtles are long lived reptiles inserted in the IUCN Red List because they are considered endangered species. How can marine litter affect these species distributed in the Mediterranean Sea?

In the laboratories, the UNISI team performs analyses that focus on the evaluation of the presence and effects of macro and micro-litter in the excreta of sea turtles, following the protocol developed within the Plastic Busters MPAs ‘Toolkit for monitoring marine litter and its impact on biodiversity in Med MPAs’. The protocol is also used to evaluate the toxicological effects associated with the presence of marine litter and related contaminants using a set of diagnostic and prognostic methodologies, by biomarkers.

Certain contaminant compounds can interact with DNA forming adducts, and/or reactive oxygen species (ROS) capable of inducing single or double DNA strand breaks. These breaks can be evaluated by different methodologies, and in this perspective, micronucleus (MN) assay and ENA assay can constitute suitable complementary indicators.

The ENA (Erythrocytic Nuclear Abnormalities) assay is a technique that permits to investigate the micronuclei and other abnormalities that are considered irreparable lesions, representing later and less transient alterations in living organisms. Using this test, the Plastic Busters MPAs team evaluates the genotoxicity in blood samples of hospitalized sea turtles.

The analysis of samples collected from sea turtles hospitalized in different rescue centers located along the coast of the Pelagos Sanctuary starts with the preparation of two blood smears per sample, for which slides were fixed and stained with Diff-Quick (Figure 13).

Figure 13: Slides of blood sea turtles fixed and stained with Diff-Quick in the UNISI Ecotoxicology laboratory.

The stained slides are analyzed under a light microscope at a final magnification of 1000× and for each studied specimen of sea turtle, 1000 mature erythrocytes are scored (Figure 14). 

The erythrocytic nuclear abnormalities are scored into one of the following categories: micronuclei, lobed nuclei, segmented nuclei and kidney-shaped nuclei (see Figure 15). The results are expressed as the ENA frequency, the mean value (‰) of each abnormalities and the sum of all the lesions observed.

For the analysis of micro-plastics in the faeces of sea turtles, samples are collected from hospitalized specimens in the Pelagos Sanctuary (see Figure 16) and transported in a cool-box to laboratory where they were lyophilized in an Edwards freeze drier. 

Plastics are isolated from the faeces (Figure 17), observed under a stereomicroscope and then classified (fibres, fragments, beads/micro spheres), measured and photographed

The same faeces samples used for the observation of plastic are also for the evaluation of concentrations of uroporphyrin, coproporphyrin and protoporphyrins with a fluorimetric method.

Porphyrins are tetrapyrrolic pigments, widespread in nature. They are intermediate metabolites of heme biosynthesis (protoporphyrin) or oxidative by products of the intermediate metabolites porphyrinogens (coproporphyrin and uroporphyrin). Environmental contaminants (such as PAHs, heavy metals, OCs) can alter heme biosynthesis, so altering the profile of porphyrins.

Porphyrins are extracted with acid solvents (Figure 18).

The samples are then placed in a micro-cuvette and measured using a spectrophotofluorimeter, due to their characteristic of fluorescent compounds. The analysis s of porphyrins in the faeces of sea turtle could allow to understand whether marine litter ingestion can alter their profile and cause negative effects of the health of this species (Figure 19).