Authors are grateful to the French Rescue Centres (Jean-Batiste Senegas), stranding networks (Jacques Sacchi) and veterinarian laboratories (Joanne Belfort) and Jessiaca Martin and Marie Sabatte, Cetacean and sea turtle stranding network of the Valencia Community, including the Marine Zoology Unit of the Cavanilles Institute (University of Valencia) and the Biodiversity Service of the Generalitat Valenciana, the Portuguese Regional Fund for Science and Technology of the Azores (Maria Vale), the Italian Rescue Centres (Stazione Zoologica &#34;Anton Dohrn&#34; Naples and Sardinian CREs) the veterinarian laboratories (IZSLT M. Aleandri Roma; IZSAM G. Caporale Teramo; IZSS G. Pegreffi Otistano;. IZS CReTaM Palermo), members of the INDICIT Advisory Board and the PO for their suggestion, and the Environmental Ministries and Regional Governments of the participant countries for their support.
Two anonymous reviewers for their suggestions and comments.
The present protocol has been performed by INDICIT consortium in the frame of the European DG-ENV project GA No. 11.0661/2016/748064/SUB/ENV.C2.

A series of &#34;basic&#34; and &#34;optional&#34; parameters are proposed to be collected. The basic parameters correspond to the minimum parameters fundamental to accomplish the Criteria D10C3, while the optional parameters allow acquiring more knowledge on sea turtles&#39; behavior/biology.An observation data sheet and a list of material necessary for sampling individuals in the field and analysis of litter in the laboratory are provided here in order to facilitate data recording and statistical analysis by following a standardized table. Marine litter subcategories are chosen according to the shape and type of items. Remains of sea turtle&#39;s food and anything natural that are non-food items (stone, wood, pumice, etc.) are requested for considering thresholds and the animal&#39;s diet. All the experimental activities of this protocol have been conducted on dead turtles according to the law of the involved countries and international rules. All the necropsies must be performed at the authorized centres.
1. Sampling from carcass: fill the observation sheet (Appendix 1 in Supplementary Files 1 and 2)
<ol>
	<li>Fill in the contact details including name, contact (phone, mail) and institution of the observer(s) (data collector).</li>
	<li>Identify the species as follows: Cc (Caretta caretta, Linnaeus 1758); Dc (Dermochelys coriacea, Vandelli 1761); Cm (Chelonia mydas, Linnaeus 1758); Ei (Eretmochelys imbricata, Linnaeus 1766); Lo (Lepidochelys olivacea, Eschscholtz 1829); Lk (Lepidochelys Kempii, Garman 1880); Nd (Natator depressus, Garman 1880).</li>
	<li>Tags: If a tag already exists on the flipper, specify the number (N&#176;. Indicate the presence and number of electronic chips. Otherwise, note NO.</li>
	<li>Specify the animal identification code. For example: &#34;two letters for the country&#34;_&#34;two letters for the location (e.g., region or institution)&#34;_&#34;YY&#34; _ &#34;MM&#34;_&#34;DD&#34;_&#34;chip number&#34;.</li>
	<li>Note the date of discovery (yy/mm/dd).</li>
	<li>Specify the location of discovery which is the recovery area or coordinates in decimal degrees.</li>
	<li>Report the specimen&#39;s body condition level: 1 (Alive), 2 (Fresh -dead recently), 3 (Partially decomposed -internal organs are still in good condition), 4 (Advanced decomposed-skin scales are raised or lost), 5 (Mummified-part of the skeleton or part of the body are missing). See Figure 1.</li>
	<li>Discovery circumstances: Note the circumstances among the four categories: Stranding (animal found on the beach or on the shoreline); By-catch/Fisheries (animal captured actively by fishermen, e.g., ingestion of a hook, trapped in a net, brought back by fishermen, etc.); Found at sea (animal discovered on the sea surface); Dead at the recovery centre (the animal arrived alive, but died during its recovery).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59466/59466fig1.jpg" /> 
Figure 1: Specimen&#39;s body condition level or decomposition status. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/59466fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Sea turtle necropsy: biometrics measurements and extraction of the contents of the gastro-intestinal tract
<ol>
	<li>Arrange for the transport of the animal to the authorized center for necropsy. In case of an extremely decomposed animal, assess the integrity of the digestive tract before disposal at the authorized center. If the necropsy cannot be done immediately after the recovery, freeze the carcass at -20 &#176;C.</li>
	<li>Before the necropsy operation, record the biometric measurements in the specific section of the recovery file. The curved carapace length, notch to tip23, is mandatory; the other measures are optional (e.g., curved carapace width, weight).</li>
	<li>Conduct an external examination of the animal body and report the information in the specific section of the necropsy file. Also inspect the oral cavity for possible presence of foreign material.</li>
	<li>Separate and remove the plastron from the carapace by making an incision along the edge as highlighted by the yellow line (Figure 2a).</li>
	<li>Use a short blade or cut with a horizontal tilt avoiding damaging the interior parts (Figure 2 b - c). The ligament attachment to the pectoral and pelvic girdle must be cut when the plastron is detached from the carapace so that it is easy to access and handle it.</li>
	<li>Expose the gastro-intestinal (GI) tract removing the pectoral muscles and the heart of the turtle (Figure 2d).</li>
	<li>(Optional) Assess the trophic status qualitatively by evaluating the atrophy of pectoral muscles (none-moderate-severe) and fat thickness in the articular cavities and on the coelomic membrane (abundant-normal-low-none).</li>
	<li>Extract the GI and place it on the examination table. Do this with two operators to make the actions easier. While one operator keeps the carcass laying on one side, the other separates the ligaments from the different organs and the membranes from the carapace using small blades or scissors and removes the GI from the animal (Figure 2f).</li>
	<li>Isolate the esophagus, stomach and intestine using plastic clamps. Place these on the esophagus close to the mouth, at the esophageal valve, on the peg and at the cloacae, as close as possible to the anal orifice as indicated by yellow arrows (Figure 2f).</li>
	<li>Record the sex of the animal when possible.</li>
	<li>Separate the esophagus, stomach and intestine definitively by placing a second clamp (corresponding to the cutting point) to avoid spillage of the contents.</li>
	<li>Open the GI section lengthwise using a scissor (or the fingers when possible), and then directly place the material contained into a 1 mm mesh sieve by cleaning the GI walls with running water.</li>
	<li>Take note of each anomaly in the GI (e.g., ulcers, perforations, adhesions, inflammation).</li>
	<li>Inspect the contents in the sieve to eventually detect any tar, oil, or fragile material that must be removed and treated separately.</li>
	<li>Rinse the contents through the sieve in order to remove the liquid portion, mucus and unidentifiable digested matter.</li>
	<li>Repeat the sequence for each GI portion separately.</li>
	<li>Freeze all the material collected by the sieves or store it in jars containing 70% alcohol solution. 
	NOTE: For more details on the anatomy of the sea turtle see also Wyneken (2001)24.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59466/59466fig2.jpg" /> 
Figure 2: Sequence of turtle necropsy. (a) Ventral view of a dead turtle. The yellow line indicates the way to cut in order to separate the plastron from the rest of the turtle. (b,c) Horizontal cuts to prevent affecting the interior organs. (d) Ventral view of the opened turtle. (e) Extraction of the GI tract. (f) View of the entire GI, yellow arrows indicate where clamps must be attached in order to separate the three different GI sections. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/59466fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Data collection and analysis: marine litter classification
<ol>
	<li>Label the sample code and the respective GI section.</li>
	<li>Empty the jars on a 1 mm mesh sieve, gathering all the material.</li>
	<li>Rewash the collected material with water in order to eliminate alcohol and clean the litter.</li>
	<li>Separate marine litter from the organic components or other materials, identifying the category of marine litter by visual analysis, sorting the material on a Petri dish, and sub-diving the collected items into the different categories.</li>
	<li>Fill up the datasheet with collected information.</li>
	<li>Use the stereomicroscope to take a closer look at any unidentifiable materials.</li>
	<li>Dry the marine litter at room temperature or in an oven at 35 &#176;C for 12 h.</li>
	<li>Dry the organic fraction in an oven at 35 &#176;C for 12 h or in a drier.</li>
	<li>Report the number and the dry weight of the different categories of marine litter.</li>
	<li>Report the dry weight of the organic fraction subdivided into food remain(s) and natural non-food remain(s). Total dry mass (weight in grams, accurate to the 2nd decimal place) is the main information used in the monitoring program, followed by the number of items (abundance).</li>
	<li>Record other information such as the color of the items, volume of litter, different incidences of litter in the esophagus, stomach and intestine, and incidence per litter category as this is useful for research and impact analysis. The raw data will provide varied information for each single section of the GI; the total contents of the marine litter within the three parts will be accounted for in the final data.</li>
</ol>

The authors have nothing to disclose.

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&#39;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&#39;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 &#34;marine litter&#34;. 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 studies16,28,29,30,31,32. 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 EcoQO17,25.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59466/59466fig6.jpg" /> 
Figure 6: Fragmentation of single items could occur before ingestion or during the feeding process, producing bias in counting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/59466fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 values16,26,32,33,34. 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 samples16,26. The second scenario is more related to the individual health status and could better answer to the Criteria D10C3: &#34;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&#34;. In fact, impact of ingested plastic items consists most frequently in sub-lethal effects rather than lethal ones28,36,37,38,39. 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 EcoQO17,25 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 environment20,40. 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 &#34;death at sea&#34; turtles washed ashore and are usually used as samples in monitoring activities26,32,38,45. 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&#39;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.

Marine litter is a complex issue to address since it can enter the oceans via multiple sources and forms. Over 80% of the litter that is encountered in marine environments is made up of plastic1. The role of this material from an economic perspective has been increasing in the last 50 years. As a consequence, its production has also increased twentyfold since 1960, reaching 335 million tons in 2016. This value is expected to double over the next 20 years2. Moreover, it has been estimated that around 5 to 13 million tons of plastic end up in the oceans every year (which is equal to 1.5 to 4% of global plastic production)2,3. Plastic movement is influenced by its physical properties (e.g., buoyancy) or environmental variables (e.g., tide and stream), and plastic can be accumulated in all marine compartments4,5. To face the plastic problem, it is important to bear in mind that, as many other environmental issues, it is transboundary and therefore governance solutions are complex to meet6. To better reach this goal we must take into consideration regional and international frameworks, so as to enhance or maintain marine environmental awareness and protection across the globe7. The final objective of the European Union&#39;s Marine Strategy Framework Directive (MSFD) is to achieve a Good Environmental Status (GES) in European waters by 2020, to protect marine biodiversity, and to promote the sustainable use of marine environments. This will be done through 11 qualitative descriptors, of which Descriptor 10 focuses on marine litter and is defined as &#34;Properties and quantities of marine litter do not cause harm to the coastal and marine environments&#34;. Within this descriptor, the New Commission Decision8 decided to add criteria D10C3-&#34;The amount of litter and micro-litter ingested by marine animals is at a level that does not adversely affect the health of the species concerned&#34;-since it was considered to be a relevant criteria in the evaluation of GES. As a result, member states were requested to produce a list of species, to develop methodological standards and define threshold values through regional or sub-regional cooperation.
After the first scientific publication in 18389, on the Storm-Petrel with an ingested candle stick, over 500 marine species have been listed for ingesting marine litter10,11,12,13,14, and sea turtles were among the first taxa recorded to ingest plastic debris15. Given their propensity for ingesting litter, their wide distribution and the large range of habitats used during their life, sea turtles, in particular the loggerhead species Caretta caretta (Linnaeus 1758), was chosen as a potential indicator for the Mediterranean basin16, like the sea bird Fulmarus glacialis (Linnaeus, 1761) for Northern European waters17. Even after five decades of research, discussion of method standardization has been very limited18 and a cohesive approach by the scientific community to quantify plastic ingestion by wildlife is lacking19. Standardized sampling protocols, and analytical detection methods and metrics to assess plastic ingestion by marine biota are needed; a recent paper showed the potential benefits and limitations of using marine species as bio-indicators on plastic pollution20. Following the Matiddi et al. proposal in 201121 to use the loggerhead as bio-indicator, and the Technical Group on Marine Litter Report22, a specific protocol to consider marine litter ingested by sea turtle has been developed and tested by ten partners in seven countries in the Mediterranean Sea and Atlantic Ocean within the European Project INDICIT (GA n&#176;11.0661/2016/748064/SUB/ENV.C2). This protocol provides standardized methodologies for the analysis of marine litter ingested by sea turtles in order to support the new Commission Decision (EU)8, Criteria D10C3, where threshold values are requested. According to the definition provided by COM8, the threshold value is a number or a range that allows to evaluate if the quality level criterion was achieved, therefore helping in assessing the GES. The proposed protocol for assessing the litter ingested by sea turtles, will be useful in gathering data on the composition and abundance of litter and evaluate its impact on marine environments. Moreover, collecting this type of standardized data will help to define threshold values. Here we consider two types of scenarios. The first scenario takes into account the Fulmar litter EcoQO monitoring, which is implemented for the OSPAR area: &#34;There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region, where Y is the average weight of plastic ingested considering all the samples and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one aims at considering a proxy of individual health level: &#34;There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region&#34;, where the weight of ingested plastic is compared with food remaining in each individual.

<table><tbody><tr><td>&lt;strong&gt;For the recovery of the animal and the collection of samples at the discovery site&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Boots</td><td></td><td></td><td></td></tr><tr><td>Bottle/ziploc bags</td><td></td><td></td><td></td></tr><tr><td>Camera</td><td></td><td></td><td></td></tr><tr><td>Cooler</td><td></td><td></td><td></td></tr><tr><td>Cut-resistant gloves</td><td></td><td></td><td></td></tr><tr><td>Garbage bag</td><td></td><td></td><td></td></tr><tr><td>Glasses and protective mask or shield</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Integral protective suit&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Measuring tape</td><td></td><td></td><td></td></tr><tr><td>Observation sheet</td><td></td><td></td><td></td></tr><tr><td>Pen</td><td></td><td></td><td></td></tr><tr><td>Permanent marker</td><td></td><td></td><td></td></tr><tr><td>Rope (to marke-off the zone)</td><td></td><td></td><td></td></tr><tr><td>Transport bins or containers for the turtle</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;For the collection of samples on dead individuals in laboratory and the extraction of the ingested litter from the digestive tract&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;In the laboratory room&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Cold chamber or chest freezers (-20&amp;deg;C) with large storage capacity</td><td></td><td></td><td></td></tr><tr><td>Garbage bags</td><td></td><td></td><td></td></tr><tr><td>Proofer (not mandatory)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;For manipulators&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Boots</td><td></td><td></td><td></td></tr><tr><td>Cut-resistant gloves</td><td></td><td></td><td></td></tr><tr><td>Glasses and protective mask or shield</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Integral protective suit</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;For notes and report&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Camera</td><td></td><td></td><td></td></tr><tr><td>Observation sheet</td><td></td><td></td><td></td></tr><tr><td>Pen</td><td></td><td></td><td></td></tr><tr><td>Permanent marker</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;For biometric measurements&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Measuring tape</td><td></td><td></td><td></td></tr><tr><td>Sliding calliper</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;For the necropsy and the collection of samples&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Clamps (at least 6) and/or kistchen string or plastic cable clamps</td><td></td><td></td><td></td></tr><tr><td>Clips with claws</td><td></td><td></td><td></td></tr><tr><td>Containers for samples (Bottle/zipped bags)</td><td></td><td></td><td></td></tr><tr><td>Metal containers</td><td></td><td></td><td></td></tr><tr><td>Scalpel (possible with interchangeable blade)</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;For the analysis of ingested litter&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Binocular (optional)</td><td></td><td></td><td></td></tr><tr><td>Measuring cylinders (10 ml, 25 ml, 50 ml)</td><td></td><td></td><td></td></tr><tr><td>Measuring decimetre</td><td></td><td></td><td></td></tr><tr><td>Precision balance (0.01 g)</td><td></td><td></td><td></td></tr><tr><td>Sieve with 1 mm mesh</td><td></td><td></td><td></td></tr><tr><td>Sieve with 5 mm mesh (optional &amp;ndash; for the study of the ingested micro-plastics (1-5 mm))</td><td></td><td></td><td></td></tr></tbody></table>

data collection on marine litter ingestion in sea turtles and thresholds for good environmental status

The following protocol is intended to respond to the requirements set by the European Union&#39;s Marine Strategy Framework Directives (MSFD) for the D10C3 Criteria reported in the Commission Decision (EU), related to the amount of litter ingested by marine animals. Standardized methodologies for extracting litter items ingested from dead sea turtles along with guidelines on data analysis are provided. The protocol starts with the collection of dead sea turtles and classification of samples according to the decomposition status. Turtle necropsy must be performed in authorized centers and the protocol described here explains the best procedure for gastrointestinal (GI) tract isolation. The three parts of the GI (esophagus, stomach, intestine) should be separated, opened lengthways and contents filtered using a 1 mm mesh sieve. The article describes the classification and quantification of ingested litter, classifying GI contents into seven different categories of marine litter and two categories of natural remains. The quantity of ingested litter should be reported as total dry mass (weight in grams, with two decimal places) and abundance (number of items). The protocol proposes two possible scenarios to achieve the Good Environmental Status (GES). First: &#34;There should be less than X% of sea turtles having Y g or more plastic in the GI in samples of 50-100 dead turtles from each sub-region&#34;, where Y is the average weight of plastic ingested and X% is the percentage of sea turtles with more weight (in grams) of plastic than Y. The second one, which considers the food remain versus plastic as a proxy of individual health, is: &#34;There should be less than X% of sea turtles having more weight of plastic (in grams) than food remains in the GI in samples of 50-100 dead turtles from each sub-region&#34;.

This protocol, derived from the MSFD guideline22 and has been co-built and improved by more than 50 stakeholders (biologists from rescue centers, stranding networks, veterinarian and research laboratories) from 7 countries across the Mediterranean and the European Atlantic coasts, it proposes a homogenized, feasible and easy evaluation of litter ingestion by sea turtles. The protocol has been tested on loggerhead turtles, and most of the manipulations are also applicable to other sea turtle species. The first important result of this protocol is the description of marine litter items under seven categories according to their visual features (Figure 3). This classification has been derived by the Fulmar EcoQo17,25, and modified as per the authors&#39; experience in sea turtle ecology. The first category, and usually the least abundant one, is industrial plastic (IND PLA) comprised of plastic pellet and granules, usually cylindrical and round shape, but also oval or cubical shapes, rarely found to be ingested by the loggerhead turtle16,26. The second category comprises the remains of sheet-like (USE SHE) materials, such as plastic bags, agricultural sheets or plastic foil. They appear in irregular shapes but are always thin and flexible. The third category includes ropes, filaments, and other threadlike materials such as the remains of ghost fishing gear usually made of nylon (USE THR). The fourth category includes all foamed plastics (USE FOA) such as polystyrene foam or foamed soft rubber. The fifth category includes fragments of hard plastic items (USE FRA). Fragments are highly abundant in the GI contents and they can be found in a variety of different colors. They are derived from broken larger pieces and are usually rigid, with an irregular shapes and sharp crooked edges. Any other plastic items including elastics, dense rubber, balloon pieces, and soft air-gun bullets, are categorized as other user plastic (USE POTH). All the non-plastic marine litter such as cigarette butts, newspapers, rubbish and hard pollutant are included in the last category of litter other than plastic (OTHER) even if they are not easily found in sea turtles. The other two categories not classified as marine litter, are (i) remains of the turtle natural diet (FOO) and (ii) any natural item, not recognized as prey for the sea turtle such as stone, wood or pumice (NFO).
Figure 4&#160;shows an example of representative results on the dry mass of marine litter categories, where sheet-like plastic (USE SHE) was the most abundant class, and plastic bags or parts of them, were the main ingested items. Similar results are shown in Figure 5 in terms of the number of items (abundance). Table 1 shows an example result of litter dry mass analysis in six different areas, which is useful for setting the threshold value according to the requirements from the European Union&#39;s MSFD. These areas should be represented, for example, by countries or sub-region of the Mediterranean basin. The reported average is calculated using all individuals examined, including samples without ingested marine litter. According to our example, area 5 represented the clearest zone of the Mediterranean basin and the data from this area could be used to set the threshold value to be reached. For this area the first scenario could be: &#34;There should be less than 25% of sea turtles having 0.5 g or more plastic in the GI in samples of 50-100 sea turtles&#34;. The second scenario could be: &#34;There should be less than 32% of sea turtles having more plastic grams than food remains (FOO) in the GI in samples of 50-100 sea turtles&#34;.&#160;
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59466/59466fig3v2.jpg" /> 
Figure 3: Examples of marine litter categories established for marine turtle ingestion monitoring. (a) IND PLA, (b) USE SHE, (c) USE THR, (d) USE FOA, (e) USE FRA, (f) USE POTH, (g) OTHER, (h) FOO. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/59466fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59466/59466fig4.jpg" /> 
Figure 4: Example results of weights of marine litter ingested by sea turtles under the various categories. The average weight values are reported in grams of items per individual (&#177; SE). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/59466fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59466/59466fig5.jpg" /> 
Figure 5: Example results for the number of marine litter categories ingested by sea turtles. The average number of items per individual (&#177; SE) are reported. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/59466fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AREA</td>
			<td>Sample size (n)</td>
			<td>Average value &#177;SE for dry weight of ingested plastic (g)</td>
			<td>Percentage of turtles with more plastic than average value (%)</td>
			<td>Percentage of turtles with more plastic than food remain (%)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>100</td>
			<td>1.32&#177;0.03</td>
			<td>27</td>
			<td>64</td>
		</tr>
		<tr>
			<td>2</td>
			<td>100</td>
			<td>1.61&#177;0.01</td>
			<td>28</td>
			<td>67</td>
		</tr>
		<tr>
			<td>3</td>
			<td>100</td>
			<td>1.35&#177;0.02</td>
			<td>26</td>
			<td>62</td>
		</tr>
		<tr>
			<td>4</td>
			<td>95</td>
			<td>0.73&#177;0.02</td>
			<td>34</td>
			<td>40</td>
		</tr>
		<tr>
			<td>5</td>
			<td>65</td>
			<td>0.55&#177;0.03</td>
			<td>25</td>
			<td>32</td>
		</tr>
		<tr>
			<td>6</td>
			<td>50</td>
			<td>0.90&#177;0.04</td>
			<td>44</td>
			<td>54</td>
		</tr>
	</tbody>
</table>
Table 1: Example of results from different areas (e.g., countries, sub-regions, etc.), using dry mass of marine litter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/Table_1.xls.xlsx" target="_blank">Please click here to download this table as an excel file.</a>
Supplementary Files 1.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/Appendix_1.jpg" target="_blank">Please click here to download this file.</a>
Supplementary Files 2.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59466/Appendix_1bis.jpg" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Data Collection on Marine Litter Ingestion in Sea Turtles and Thresholds for Good Environmental Status at JoVE.com

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

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 requirements set by the European Union&#39;s Marine Strategy Framework Directives (MSFD) for the ...

data-collection-on-marine-litter-ingestion-sea-turtles-thresholds-for

Nanyang Technological University

Research

JoVE Journal

Environment

11.9K Views.  Italian National Institute for Environmental Protection and Research (ISPRA). Het volgende protocol is bedoeld om te reageren op het mariene strategiekader van richtlijnvereisten voor de nieuwe criteria. De hoeveelheid zwerfvuil en microstrooisel die door zeedieren wordt ingenomen, is een niveau dat geen nadelige gevolgen heeft voor de gezondheid van de betrokken soort. Het protocol zal nuttig zijn bij het verzamelen van gegevens over de samenstelling en de overvloed aan zwerfvuil en het evalueren van de impact ervan op het mariene milieu.Dit type gestandaardise...

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

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Sampling, Sorting, and Characterizing Microplastics in Aquatic Environments with High Suspended Sediment Loads and Large Floating Debris

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only recently have microplastics in freshwater systems, such as rivers and lakes, been quantified. Microplastic sampling at the surface usually consists of deploying drift nets behind either a stationary or moving boat, which limits the sampling to environments with low levels of suspended sediments and floating or submerged debris. Previous studies that employed drift nets to collect microplastic debris typically used nets with &#8805;300 &#181;m mesh size, allowing plastic debris (particles and fibers) below this size to pass through the net and elude quantification. The protocol detailed here enables: 1) sample collection in environments with high suspended loads and floating or submerged debris and 2) the capture and quantification of microplastic particles and fibers &lt;300 &#181;m. Water samples were collected using a peristaltic pump in low-density polyethylene (PE) containers to be stored before filtering and analysis in the lab. Filtration was done with a custom-made microplastic filtration device containing detachable union joints that housed nylon mesh sieves and mixed cellulose ester membrane filters. Mesh sieves and membrane filters were examined with a stereomicroscope to quantify and separate microplastic particulates and fibers. These materials were then examined using a micro-attenuated total reflectance Fourier transform infrared spectrometer (micro ATR-FTIR) to determine microplastic polymer type. Recovery was measured by spiking samples using blue PE particulates and green nylon fibers; percent recovery was determined to be 100% for particulates and 92% for fibers. This protocol will guide similar studies on microplastics in high velocity rivers with high concentrations of sediment. With simple modifications to the peristaltic pump and filtration device, users can collect and analyze various sample volumes and particulate sizes.

Most microplastic research to date has occurred in marine systems where suspended solid levels are relatively low. Focus is now shifting to freshwater systems, which may feature high sediment loads and floating debris. This protocol addresses collecting and analyzing microplastic samples from aquatic environments that contain high suspended solid loads.

The ubiquitous presence of plastic debris in the ocean is widely recognized by the public, scientific communities, and government agencies. However, only ...

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

A flow-through device for using the biodeposition method to quantify filtration and feeding behavior of bivalve mollusks was modified for shipboard use. A two-dimensional gimbal table built around the device isolates the apparatus from boat motion, thereby allowing the accurate quantification of bivalve filtration variables at offshore shellfish aquaculture sites.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves ...

Assessing Intertidal Populations of the Invasive European Green Crab

Invasive species have caused major disruptions to ecosystems worldwide. The European green crab invaded North America in the 1800s and is considered one of the world&#8217;s 100 worst invaders by the IUCN. Observations of spatiotemporal green crab population dynamics are essential for predicting and managing the ecological and economic impacts of this harmful invasive species. This protocol was developed in an effort to create a standardized method for assessing green crab population dynamics in the rocky intertidal zone of New England and Atlantic Canada. The protocol was designed to be accessible to multiple users including researchers, educators, students, and citizen scientists. Although it was designed for surveying crab populations, this protocol is easy to adapt and could be used for any number of intertidal species. The resulting data collected using this protocol has a wide range of uses, including to inform ecological research, conservation efforts, mitigation strategies, and fishery development, as well as for educational outreach purposes.

Understanding spatiotemporal patterns in green crab population dynamics is essential for predicting and managing the ecological and economic impacts of this harmful invasive species. This protocol was developed in an effort to create a standardized method for assessing green crab populations in the rocky intertidal zone of the Northwest Atlantic.

Invasive species have caused major disruptions to ecosystems worldwide. The European green crab invaded North America in the 1800s and is considered one ...

Ecotoxicological Effects of Microplastics on Bird Embryo Development by Hatching without Eggshell

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues and organs. Ecotoxicological effects of microplastics on the development of bird embryos are not known. The bird egg is a complete development and nutrition system, and the entire embryo development occurs in the eggshell. Therefore, a direct record of bird embryo development under the stress of pollutants such as microplastics is highly limited by the opaque eggshell in traditional hatching. In this study, the effects of microplastics on quail embryo development were visually monitored by hatching without an eggshell. The main steps include the cleaning and disinfection of fertilized eggs, the incubation before exposure, the short-term incubation after exposure, and the sample extraction. The results show that compared with the control group, the wet weight and body length of the microplastics-exposed group displayed a statistical difference and the liver proportion of the whole exposed group significantly increased. Additionally, we evaluated external factors that affect the incubation: temperature, humidity, egg rotation angle, and other conditions. This experimental method provides valuable information on the ecotoxicology of microplastics and a novel way to study the adverse effects of pollutants on the development of embryos.

This paper introduces a method of hatching without using an eggshell for toxicological studies of particle pollutants such as microplastics.

Microplastics are an emerging global pollutant type that poses a great health threat to animals due to their uptake and translocation in animal tissues ...

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

Hellgrammite as a Non-Conventional Biomonitor for Surface Water and Environmental Health Assessment

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of occurrence, and extended period of immaturity, which can last up to one year. Hellgrammites are clearly known to exhibit sensitivity to pollution and habitat structure impacts. Given these unique features, using Corydalus texanus larvae is highly suitable as reliable biomonitoring agents to assess the ecological integrity of aquatic ecosystems. This protocol aims to provide the necessary tools for C. texanus assessment and demonstrate their efficacy through a case study. Research findings have practical implications, indicating that C. texanus larvae exhibit early-warning responses to mining pollution, bioaccumulating high amounts of heavy metals such as Zn, Fe, and Al. The presence or absence of C. texanus populations may serve as a helpful indicator for identifying potential issues related to ecosystem health. The unconventional approach has shown early warnings of pollution in mining-impacted sites, highlighting the need for timely action to protect the environment. Given their unique traits, the use of C. texanus larvae is highly suggested as a reliable non-conventional bioindicator.

This protocol describes the use of biomarkers for the early detection of deleterious impacts in aquatic ecosystems. The biomarkers are closely related to sentinel traits, and their changes aid in detecting early-warning damages.

The larvae of dobsonflies from the genus Corydalus, commonly known as Hellgrammites, are characterized by their notable size, extensive range of ...

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel&rsquo;s ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels Bivalvia: Unionida

Freshwater mussel conservation is dependent on monitoring reproductive patterns and processes of species. This study standardizes a non-lethal protocol for sampling gill contents, characterizing larval development, and providing a digital repository for data collected. This protocol-database package will be an important tool for mussel researchers in recovery of imperiled species.

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater ...

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

Samenvatting

Meer video's verkennen