Plastic Busters MPAs in the lab: HCMR

The Ecotoxicology Laboratory of the Institute of Oceanography, HCMR, investigated the ingestion of microplastics in mussels and fish, the ingestion of marine litter by sea turtles Caretta caretta, the genotoxicity of microplastics in seabirds Calonectris diomedea and the presence of microplastics in the faeces of the Mediterranean monk seal.

  • Microplastics ingestion in mussels and fish
  • Plastic ingestion in sea turtles
  • Genotoxicity investigation in blood samples of seabirds
  • Microplastics in Mediterranean monk seal faeces

 

 

 

 

BIOINDICATORS FacT sheet 

Read more about the sea bird Calonectris diomedea

 

BIOINDICATORS FacT sheet 

Read more about the Mediterranean mussel

Microplastics ingestion in mussels and fish

Mussels and fish were sampled in the National Marine Park of Zakynthos and the coastal area of the island of Zakynthos during two expeditions carried out in 2019. The samples were transported to the Ecotoxicology Laboratory of the Institute of Oceanography, HCMR, in order to detect the amount of the microplastics ingested.

Photos of mussel and fish samples collected during the HCMR expeditions at the National Marine Park of Zakynthos island and coastal area of the island of Zakynthos.

  • In the laboratory, the mussel tissues and fish guts are subjected to digestion of the organic matter by hydrogen peroxide (H2O2 15%, 50 °C).

Figure 2: Digestion process

  • When all organic matter has been removed, distilled water is added and stirred.
    The samples are filtered on a filtering apparatus under airborne contamination free conditions
    (e.g. glove box, laminar flow).

Figure 3: Homogenization process over magnetic agitator and filtration process

  • A stereomicroscope (Olympus SXE) paired with a camera (Infinity) is used to
    detect items resembling plastic on the filters.

Stereoscopic observation.

  • Fourier-Transform Infrared Spectroscopy (FTIR) is used to confirm the polymer composition of the items detected

FTIR reveals the polymer type of the items found.

Plastic ingestion in sea turtles

The Ecotoxicology Laboratory of the Institute of Oceanography, HCMR, recently started studying stranded sea turtles for ingested plastics. Prior to necropsy, various biometric data i.e. curved carapace length, straight carapace length, curved plastron length, curved carapace width, age stage, sex and weight are recorded.
The gastro-intestinal tracts (oesophagus, intestine and stomach) of the sea turtles are individually measured (weight, length, width, fullness) and their content is emptied and washed on top of 3 stacked metallic sieves of different mesh size (5 mm, 1 mm and 300 μm). Each sieve is thoroughly washed and inspected for plastic.
All plastic items are counted, weighed, and categorised by size, shape, colour, and polymer type.

Gastrointestinal analysis of sea turtles.

Plastics found in sea turtles stranded in Greece.

 

Genotoxicity investigation in blood samples of seabirds

In genetics, genotoxicity describes the property of chemical agents that damage the genetic information within a cell, causing mutations, which may lead to cancer. To assay for genotoxic molecules, researchers assay for DNA damage in cells exposed to the toxic substrates.

The micronucleus assay is a technique that allows the evaluation of genotoxicity in living organisms. Using the micronucleus assay, the Plastic Busters MPAs project investigated the genotoxicity in blood samples of living Mediterranean seabird species Calonectris diomedea.

Samples were collected from 26 birds in Strofades islands located at the National Marine Park of Zakynthos. One drop of blood from each bird is smeared on a microscope slide and fixed in ethanol. The slides are then treated with 10% Giemsa stain, that specifically stains the nucleus of the cell (where most of the DNA is located) and allows the observer to study the phenotype of the nucleus.

The observation of the slides is conducted in a microscope using a x100 lens. From each bird, a total of 4,000 cells are observed and the nucleus phenotypes are scored as normal, bi-nucleated, cells with nuclear buds, and cells with micronucleus. The frequency of the micronucleus in cells is then calculated to assess genotoxicity.

Bird red blood cells with stained nuclei to reveal different phenotypes of the nucleus such as a bi-nucleated cell or a micronucleus that can be associated with genotoxicity

Microplastics in Mediterranean monk seal faeces

Faeces of the endangered Mediterranean monk seal (Monachus monachus) were collected at the coastal area of the island of Zakynthos by the NGO “Archipelagos – environment and development”. 

Monk seal faeces collected.

  • The faeces samples were transported in a cool-box to laboratory and were washed through a series of sieves (1 mm, 500 μm, 300 μm). The material collected from the two top sieves was transferred to a vial with 70% ethanol and was left for 24 hours, to remove odour and growth of mould and bacteria. Any macro-litter found was cleaned and dried. The remains at the bottom sieve were transferred to jars and filled up with 10% KOH solution to digest organic matter. Samples were kept for two weeks at lab temperature (over 30°C) or incubated on a hot-plate (~45-50°C).
  • Density separation was applied after sample digestion using two fully saturated solutions: a lower density solution of NaCl and a higher density solution of NaI. This procedure allowed the plastic items in the inorganic and non-digested material to float. Samples were then filtered under a vacuum in the interior of a hermetic plastic pyramid in order to avoid airborne contamination.

Faeces analysed in the HCMR Ecotoxicology laboratory

  • Samples were dried and investigated under an Olympus SXE stereomicroscope with an Infinity camera attached. Anthropogenic litter items were observed and classified (fibres, fragments, beads/micro spheres), measured and photographed. 

Stereoscopic observation of microplastics 

Macro-litter items deriving from fish nets.

  • All anthropogenic litter particles found were investigated using Fourier-Transform Infrared Spectroscopy (FT-IR). 

FTIR reveals the polymer type of the items found