Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status

This protocol allows evaluation of the total abundance of marine litter, and identification of the main litter categories ingested by sea turtles. It is less expensive compared to other monitoring programs with sea activities because sea turtles could be collected after stranding on the beach or be recovered by fishermen. The identification of marine litter categories is easy and rapid as the lower limit on item size is 1 mm. A limitation of the protocol is the use of sea turtle considering that all 7 species of marine turtles are listed on Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora²⁷; therefore, only authorized personnel can handle live and dead animals or parts of them. Turtle management and recovery should be reported and coordinated with the corresponding authorities. Sanitary precautions must be taken when handling dead or alive wild animals to minimize risks of zoonosis. This protocol has been tested on loggerhead species but it is applicable to all the seven turtle species. Data analysis should be carried out separately for each species. The specimen's body conditions as considered under five levels from alive to mummified turtles. Level 1 (Alive) is considered for a more detailed classification of the specimen's body condition in case the turtle died at the rescue center after the recovery. The protocol is applicable to dead individuals from levels 2 to 4, but also on individuals that died after recovery (circumstances: dead at the recovery center). Levels 2 and 3 are adequate for the protocol while level 4 allows to measure biometrics data and assessing the presence/absence of ingested litter for the evaluation of frequency of occurrence (FO%), and the percentage of turtles with ingested marine litter on the entire sample. Individuals of level 5, where generally the gastrointestinal content has been lost cannot be considered for the collection and quantification of litter ingestion. Taking photos of the animal before handling, could provide additional information on the sample as the probable cause of death or main injuries and entanglement. It is important to include a scale bar on the pictures. Even if often sea turtles had fishing hooks in their GI, data do not have to be included in the analysis because fishing hooks on which longline victims are actively caught are not considered as "marine litter". Hook presence should be recorded in the notes. Collection of data should be performed separately in each part of the GI (esophagus, stomach, intestine), in order to evaluate the degree of tolerance to marine litter ingestion considering GI blockage or the capability to eliminate it through defecation, as demonstrated in previous studies^{16,28,29,30,31,32}. A critical step of the protocol could be found in the collection of the number of items. Multiple pieces could be derived from fragmentation of the same object inside the GI or as a consequence of separate ingestion. Subjective interpretation of a single item or multiple separate pieces could correspond to a potential bias in recording number (Figure 6). For this reason, threshold values have been elaborated using only ingested marine litter mass data, like the Fulmar EcoQO^17,25.

Figure 6: Fragmentation of single items could occur before ingestion or during the feeding process, producing bias in counting. Please click here to view a larger version of this figure.

The protocol requires the categorization of different plastic items according to their shapes (USE SHE, USE THR). This sub-division is useful to identify the source of marine litter with a list of items according to their abundance. It aides policy makers in their programs of measures, providing rapid evidence of their efficiency in targeting items by evaluating their strength. For example, the ban of plastic bags in the markets should correspond to a reduction of USE SHE category ingested (Figure 4, Figure 5) in sea turtle samples collected in the future. The application of this protocol will allow EU member states to answer to the MSFD requirements, evaluating their own baselines and defining the threshold values at which GES is achieved. Thresholds should be determined in pristine or next to pristine areas. Due to the ubiquity of plastic in the marine environment, a pristine area does not exist. According to the example data (Table 1), area 5, was the clearest zone and could represent the value (Y) to be reached for the Mediterranean basin. Member states should decide thresholds according to the significant reduction of their own distance from this value. According to a recent review¹⁸, marine litter ingestion units should be normalized to the size of the turtle, especially if the goal is to compare different age classes. Nevertheless, a relationship between the mass of ingested litter and turtles size has been detected by different authors with positive, negative or zero values^{16,26,32,33,34}. Our protocol does not include animal size in the first scenario, but it could be possible to estimate the body burden, evaluating mass of the turtle using curved carapace length (CCL)³⁵ and use the ratio of weight of plastic weight of turtle instead of only grams of ingested plastic (Y). In any case we suggest verifying any possible significant differences before merging oceanic stage turtles with neritic ones or early juveniles with adults, in order to better stratify the samples^16,26. The second scenario is more related to the individual health status and could better answer to the Criteria D10C3: "The amount of litter and microlitter ingested by marine animals is at a level that does not adversely affect the health of the species concerned". In fact, impact of ingested plastic items consists most frequently in sub-lethal effects rather than lethal ones^{28,36,37,38,39}. We also rarely found an occlusion or a perforation due to plastic ingestion, which could cause the death of the turtles. Sub-lethal effects are not easy to be detected and to be distinguished from impacts due to other pollutants⁴⁰. Dietary dilution or assimilation of contaminants happens when marine litter is inside the GI of the turtle⁴¹. Thus the sample with more grams of plastic than food remains could indicate an animal in a very bad health condition. In order to remain in line with the Fulmar EcoQO^17,25 used by the northern European countries, both scenarios consider plastic weight instead of marine litter weight.

Finally it is important to clarify the differences between (i) analyzing the ingestion of plastic in sea turtles as indicator of impact on population with consequences for population conservation and (ii) analyzing the ingestion of plastic in sea turtles as bio-indicator of impact on the coastal and marine environment^20,40. To understand the implications of this impact on conservation of turtles population, more information is needed and better data stratification is necessary⁴². By confronting the opinion of 35 specialists from 13 nations, who are experts in sea turtle biology and conservation, it is clear that sea turtles have been widely studied across the years, although it is still necessary to investigate the interactions with human activities and therefore assess the population status and potential threats⁴³.

This means that a single protocol cannot be considered as exhaustive for all the thematic and more studies are necessary to understand the impact of plastic at population level.

Even tough plastic could be considered to cause a low level of damage to sea turtles, with respect to by-catch or habitat destruction, its reduction has been challenging in the last few years and quick methods of measurement must be elaborated. There is a controversy in the use of stranded turtles for monitoring purposes because, according to some authors they are not representative of the whole population⁴⁰, while others have declared that stranded turtles do not represent a bias of marine litter ingestion rates in the background population⁴⁴. Moreover, in many countries there is not a well-organized stranding network or system linking rescue centers to fishermen and there is a lack of information on by-catch and post release mortality by fisheries. Hence, stranded samples cannot always be considered as sick turtles without normal feeding behavior for a time period before dying and reaching the beach; many of them are "death at sea" turtles washed ashore and are usually used as samples in monitoring activities^26,32,38,45. We believe that stranded samples are useful in providing information on the level of marine litter abundance in the environment and we suggest excluding only turtles with completely empty gastrointestinal tract from this analysis as they could be sick from a long time before death. The use of this protocol would enable evaluation of environmental status and marine litter availability for marine organisms. It could also be helpful in improving our knowledge on turtle behavior. The significance of the method with respect to the MSFD TS-ML guidelines²², is due to the harmonization in seven countries and the number of samples on which it has been tested (n = 700). Specimen's body condition level has been defined and marine litter ingested categories have been reduced according to the preliminary results. Moreover, this is the first time representative results have been shown and connected to the GES thresholds.

The protocol is an efficient tool for researchers to understand the impact of plastic on the marine environment, globally or at a local scale, and for comparing standardized data with neighboring countries. This result could not be reached before, due to the discrepancies in data among different countries, preventing any spatial comparison.