Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status
May 18th, 2019
The protocol focuses on the collection of sea turtle samples, describing all the steps from the animal recovery and necropsy to the classification and quantification of ingested marine litter. Moreover, the representative results show how to use the collected data to elaborate the possible thresholds for Good Environmental Status.
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.
Overview
This protocol outlines the collection and analysis of sea turtle samples to assess the impact of ingested marine litter on their health. It details the steps from animal recovery and necropsy to the classification and quantification of the litter.
Key Study Components
Area of Science
- Marine Biology
- Environmental Science
- Ecotoxicology
Background
- The ingestion of marine litter by sea turtles poses potential health risks.
- Standardized data collection is essential for evaluating environmental impacts.
- The protocol adheres to international regulations for animal handling.
- Data gathered will help establish threshold values for marine litter.
Purpose of Study
- To collect data on the composition and abundance of marine litter ingested by sea turtles.
- To evaluate the impact of this litter on marine ecosystems.
- To define threshold values for Good Environmental Status.
Methods Used
- Recovery and necropsy of sea turtles following legal guidelines.
- Recording observations and biometrics during the recovery process.
- Dissection and examination of gastrointestinal tracts for ingested materials.
- Classification and quantification of marine litter using visual analysis.
Main Results
- Identification of marine litter items categorized into seven types.
- Data on the dry mass and number of litter items collected from turtles.
- Insights into the prevalence of different litter types in the marine environment.
- Establishment of baseline data for future monitoring and research.
Conclusions
- The protocol provides a comprehensive method for assessing marine litter ingestion.
- Findings contribute to understanding the ecological impact of marine debris.
- Results can inform policy and conservation efforts aimed at marine health.
Frequently Asked Questions
Executive Industry Relevance
This protocol provides a standardized framework for quantifying marine litter ingestion in sea turtles, supporting environmental risk assessment and regulatory compliance under the EU Marine Strategy Framework Directive. By establishing measurable thresholds for plastic burden relative to biological health indicators, it enables data-driven decision-making in ecological monitoring programs. The methodology offers translational value for assessing anthropogenic stressors in marine ecosystems, with relevance to predictive modeling and risk de-risking in environmental toxicology and conservation science.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of marine litter as a potential environmental toxin through standardized quantification of ingested plastic mass and abundance.
- Operational Value: Provides a reproducible necropsy and tissue processing workflow for isolating gastrointestinal tracts and filtering ingested debris using a 1 mm mesh sieve.
Screening & Assay Development
- Scientific Value: Supports development of standardized assays for classifying and quantifying seven marine litter categories and two natural remains categories via visual analysis and dry mass measurement.
- Operational Value: Ensures assay readiness through standardized drying protocols (35°C for 12 hours) and reporting of litter as total dry mass (grams, two decimal places) and item count.
Translational & Preclinical Research
- Scientific Value: Facilitates disease-relevant system modeling by linking plastic burden to food remains as a proxy for individual health status in sea turtles.
- Operational Value: Enables cross-subregional comparison using samples of 50–100 dead turtles to establish threshold values for Good Environmental Status.
Pipeline & Workflow Integration
The method integrates into environmental toxicology workflows from sample collection through necropsy, tissue processing, litter classification, and quantitative reporting to support threshold setting and risk assessment.
- Discovery Biology: Supports hypothesis testing regarding marine litter ingestion as a stressor by enabling standardized recovery, biometrics recording, and external examination for foreign material.
- Screening: Ensures assay reproducibility through standardized GI tract isolation, lengthwise opening, and sieving of contents to remove mucus and digested matter.
- Analytics: Generates quantitative outputs including dry mass of litter categories (plastic, non-plastic) and organic fractions (food remains, natural non-food) for comparative analysis across sub-regions.
- Translational Research: Connects litter burden to health proxies by comparing plastic weight to food remains in the GI tract, supporting risk-adjusted advancement decisions in environmental monitoring.
- Enterprise Reuse: Establishes a reusable capability for longitudinal monitoring programs, allowing member states to set region-specific thresholds based on baseline data from reference zones.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in assessing marine litter impact through standardized classification of seven litter types (e.g., industrial plastic, sheet-like, thread-like, foam, fragments, other plastics, other debris) and two natural categories.
- Operational Value: Standardization and reproducibility via mandated necropsy in authorized centers, biometrics recording (curved carapace length), and protocol-driven tissue handling (freezing at -20°C or alcohol storage).
- Strategic Value: Informs go/no-go thresholds for environmental status: <25% of turtles with ≥0.5g plastic GI burden or <32% with plastic exceeding food remains weight in 50–100 turtle samples.
- Portfolio Impact: Enables risk-adjusted prioritization of monitoring efforts by identifying sub-regions approaching or exceeding GES thresholds based on litter dry mass and abundance data.
Implementation Considerations
- Requires expertise in marine vertebrate necropsy, gastrointestinal tract dissection, and visual litter classification under stereo microscopy for uncertain materials.
- Dependent on authorized necropsy centers, access to 1 mm mesh sieves, drying equipment (stove or dryer at 35°C), and alcohol or freezing storage for sample preservation.
- Necessitates cross-team standardization in data sheet completion, litter categorization (IND PLA, USE SHE, USE THR, USE FOA, USE FRA, USE POTH, OTHER, FOO, NFO), and reporting of anomalies in GI tract contents.
- Adaptation considerations include handling varying decomposition states (fresh, partially decomposed, advanced decomposed, mummified) and assessing digestive tract integrity in severely degraded specimens.
- Practical limitations include the need to treat tar, oil, or fragile materials separately during sieving and the reliance on visual analysis for litter categorization without chemical confirmation.
