The following protocol is intended to respond to the Marine Strategy Frame of Directive Requirements for the new criteria. The amount of litter and micro litter ingested by marine animals is at the level that does not adversely effect the health of the species concerned. The protocol will be useful in gathering data on the composition and the abundance of litter and evaluate its impact on marine environment.
This type of standardized data will help to define threshold values. All the experiment activities of this protocol have been conducted on the turtles according to the law of the involved countries and international rules. All necropsies must be performed at your authorized centers.
On the recovery site, recording each observations and possible data on the beaching on the observation sheet. Report the specimen's body condition level.Alive.Fresh. Partially decomposed.
Advanced decomposed.Mummified. Organize the transport of the animal to your authorized center for necropsy. In case of an extremely decomposed animal, assess integrity of the digestive tract before disposal.
If the necropsy cannot be done immediately after the recovery, freeze the carcass at minus 20 Celsius degrees. Before the necropsy operation, record the biometrics measurements in the specific section of the recovery file. The curved carapace length, notch to tip, is mandatory.
The other measures, such as weight or width, are optional. Conduct an external examination of the animal body and report information in the specific section of the necropsy file. Also, the oral cavity must be inspected for possible presence of foreign material.
Separate and remove the plastron from the carapace by performing an incision on the edge as highlighted by the yellow line. Use a short blade or cut with a horizontal tilt avoiding damaging the interior parts. The ligament attachment to the pectoral and pelvic girdle must be cut when plastron is detached from the carapace and it is easy to access and handle it.
Expose the gastrointestinal tracts by removing the pectoral muscles and the heart of the turtle. Extract the GI and place it on the examination surface. Do this with two operators to make the actions easier.
While one of the operators keeps the carcass lying on one side, the other separates the ligaments from the different organs and the membranes from the carapace using small blades or scissors and removing the GI from the animal. Esophagus, stomach and intestines must be isolated using plastic clamps. They are placed on the esophagus close to the mouth, at the esophagal valve, on the bag and at the cloaca, as close as possible to the anal orifice as indicated by yellow arrows.
Open the GI section length ways using the scissors or the fingers when possible. The material contained must be placed directly into a one millimeter mesh sieve, by cleaning the GI walls with running water. Take note of each anomaly in the GI.Inspect the contents in the sieve to eventually detect any tar, oil or particular fragile material that must be removed and treated separately.
Rinse the contents through the sieve in order to remove the liquid portion, mucus and digested unidentifiable matter. Repeat this sequence for each GI portion separately. Freeze all the collected material by the sieves or store it in jars with 70%alcohol solution.
Label the sample code and the respective GI section. Empty the jars on a one millimeter mesh sieve gathering all the material. Rewash the collected material with water in order to eliminate alcohol and clean litter.
Separate marine litter from the organic components or other materials. Identify the category of marine litter by visual analysis, sorting the material on a Petri dish, subdividing the collected items into different categories. Fill the data sheet with collected information.
Use the stereo microscope for any uncertain materials. Dry out the marine litter at room temperature, or in a stove at 35 Celsius degrees for 12 hours. Dry the organic fraction in a stove at 35 Celsius degrees for 12 hours or in a dryer.
Report the number and the dry weight of the different categories of marine litter. Report the dry weight of organic fraction subdivided in food remains and natural no food remains. Total dry mass weight in grams accurate to second decimal is the main information useful for monitoring program followed by number of items.
Other information is color of items, volume of litter, different incidents of litter in esophagus, stomach and intestine. Incidents per litter category are useful for research and impact analysis. The first important result of this protocol is the description of marine litter items into seven categories according to the visual features.
Industrial plastic composed by plastic pellet and granules:IND PLA. Sheet-like items as plastic bags, agricultural sheets or plastic foil:USE SHE. Thread-like material including ropes, filaments or ghost fishing gears:USE THR.
Foam plastics as polyester and foam or foamed soft rubber:USE FOA. Fragments or hard plastic items:USE FRA. Any other plastic items including elastics, dense rubber, balloon pieces:USE POTH.
All the non-plastic marine litter as cigarette butts, newspapers, rubbish and hard pollutants:OTHER. Two categories, not classified as marine litter are remains of the turtle's natural diet:FOO, and any natural item not recognized as prey for the sea turtle as stone, wood, or pumice:NFO. This figure shows an example of representative results of the dry mass of marine litter categories, where sheet-like plastic is the most abundant class.
Big plastic bags were part of the LDMA ingested items. Similar results are showed in this figure considering the number of items. This table shows an example of results of litter dry mass analysis in six different areas useful for setting the threshold value according to the requirements of the European Union's MSFD.
The average reported is calculated using all individuals examined including the samples without ingested marine litter. According to our example, area five represents the clearest zone and data from this area could be used to set the threshold value to be reached using two proposed scenarios. There should be less than 25%of sea turtles having 0.5 grams or more plastic in the GI, in samples of 50 100 sea turtles.
There should be less than 32%of sea turtles having more plastic grams than food remains in the GI, in samples of 50 100 sea turtles. According to the example, area five as the clearest zone, could represent the value to be reached by others. Member states should decide threshold according to the significant reduction of the whole distance from this value.
