Delayed mortality of bycaught turtles due to sequelae of near-drowning, metabolic disturbance, and gas embolism upon release17 is suspected to be high. To date, relatively few studies with small sample sizes have been conducted on GE in trawled sea turtles17,20,21. The present study provides the largest data set to date for sea turtles affected by GE, including a diverse collection of variables and clinical data. Approximately 20% (40/204) of GE-affected turtles in this study died, while 80% recovered and were returned to the sea in 1-13 days without specific therapy. About half (21/204) of the GE-affected animals arrived already dead at the STC while the other half (19/204) died during hospitalization within 48-72 hours post-capture, although in severe cases, they died in the first 6 to 8 hours. In previous studies on lower numbers of turtles accidentally caught by trawls and gillnet, the mortality caused by GE was between 41 and 30%17,21. These turtles died during hospitalization in a time comparable to that observed in our study. In a recent study20 12 of 28 (43%) animals died on-board fishing vessels and 3 of 15 (20%) turtles released with satellite tags died within 6 days. About 90% turtles with GE alive upon arrival at STC recovered from the disease in a time ranging from 1 and 13 days without any supportive drug therapy. They were only dry-docked or kept in tanks with low water level in a warm and quiet room. Animals were discharged only when complete resolution of clinical and radiographic signs and normal blood gases were assessed.
According to several studies14,15, direct mortality due to trawling depends on tow duration and hence to the submergence time. We found a statistically significant correlation between presence of GE and duration and depth of fishing nets, size, weight and body temperature of the turtles. Intense loggerhead turtle interactions with trawl nets have previously been described in Adriatic Sea23,24,25,26. This area is characterized by shallow waters (<100 m) and rich benthic communities where turtles in the demersal stage spend the winter13,23,24,25,26,27. Mediterranean loggerhead sea turtles increase time of submergence and rest on the bottom during the coldest periods of the year23,27. Bottom trawling mostly interferes with the demersal stage of the loggerheads that are more likely to assemble in shallow water in order to feed on abundant benthic and epibenthic prey24,25. Turtles in the present study were trawled mostly in the winter with juveniles comprising over 60% of the bycaught animals. After April in the southern Adriatic Sea, turtles accidentally caught by trawling are almost never observed. The highest rate of bycatch in Valencian coast of Spain occurred between November and March, when most GE cases were encountered17.
The majority of the observed turtles (80%) presented with good body condition and normal fat stores. We found peripheral edema in 95% of the turtles and prolapse of the cloaca in 15%, with edema of the cloacal mucosa presumably due to mechanical obstruction of vasculature by bubbles. These anomalies took 3-7 days to resolve. In humans with GE, bubbles may cause platelet and leucocyte aggregation, cytokine release, and activation of the complement, kinin, fibrinolytic and coagulation cascades. The subsequent acute inflammatory response results in increased capillary permeability, edema and haemoconcentration28.
Respiratory rates did not differ significantly between turtle that survived (median 0.4, range 0.2–0.6 breaths/min) and those that died (median 0.4, range 0.2–0.5 breaths/ min), though respiration was slower in all the GE turtles than reported respiratory rates of loggerhead sea turtles after 30 min on-board recovery following capture by trawl net (trawl T0, median 5.2, range 1.0–10 breaths/min). Respiratory rates were similar to captive swimming unrestrained subadult loggerheads at 22–25 °C (0.34 breaths per min)29.
Almost all the turtles (10/12) in a lethargic/comatose state and with progressive neurological symptoms and the three turtles with blood coming from the larynx and out the oral cavity died within 8-24 hours. We found a statistically significant correlation between mortality and neurological deficit, comatose state, hind limb retraction under the carapace, and lower heart rate. Seven turtles (4%) with GE showed retraction of the hind limbs under the carapace. We must consider that the hind limb retraction under the carapace is also a frequent sign of severe stress in sea turtles and it is considered a normal protective rear flipper clasp reflex30.
We did not find a correlation between mortality and duration and depth of fishing nets, animal size, sex, weight, body temperature, respiratory rate and peripheral edema. Unlike what has been reported recently20 where PCV was significantly lower in turtles that died from GE, we found no statistically significant PCV alteration in the turtles that died. However, in about one third turtles analyzed (71/204) we found that PCV was slightly above normal ranges, without any statistical correlations with mortality. In humans, severe GE can be accompanied by hemoconcentration resulting from increased vascular permeability mediated by endothelial damage.
On radiographic examination, 65/204 (32%) animals with radiographic signs of systemic GE also showed signs of drowning and 48% of these turtles died. Conversely, of the 278 trawled turtles without GE, only 8 (3%) showed radiographic signs of drowning and none of these died. Very interesting is that in the multivariable analysis drowning is not a risk factor for death but the only risk factors are the number of the CAS and the presence of GE in the left atrium and sinus venosus/right atrium. According to these data it can be deduced that trawled sea turtles rarely undergo drowning if not also suffering from GE and that mortality is closely associated with the presence of drowning contextually to GE. Specifically, we can therefore hypothesize that the animals probably first undergo severe GE involving the heart and only than drown. In a recent study, GE entails a worse prognosis if it occurs concurrently with water aspiration20. In our opinion therefore trawling turtles essentially die for severe undiagnosed GE and water aspiration associated with forced submergence is not the leading cause of death.
In the present study radiographic examination proved to be an excellent diagnostic tool to evaluate GE in sea turtles. Full-body radiographs in the DV projection is easy to perform and allows effective diagnostic evaluation because it is possible to observe all the defined CAS affected by the accumulation of gas. The DV projection is helpful for identifying pathological radiopacity of the lungs as in drowning, but the cranial-caudal and LL projections were more useful to view the lungs without the overlap of celomic soft tissues. The LL projection is useful for the evaluation of the renal CAS (external iliac and renal portal vessels), which however are evident also in the dorsal-ventral projection, and severe accumulation of gas in the heart. As with the liver, the presence of gas in the external iliac and renal portal vessels is not significant for the prognosis because gas in these vessels is present in almost all GE cases (94% and 90% respectively).
Compared to ultrasound, the radiographic examination allows a better over-view of all the CAS with less stress for the animal. In our experience, because 12/14 CAS was a statistically significant risk factor associated with mortality, it is important to be able to observe all the CAS through the radiographic examination. The advantage of ultrasound is that sea turtles can be examined on-board fishing vessels, but in animals larger than 30 cm length (CCL), many areas, such as the heart or major vessels, cannot be examined20. In the present study high risk of mortality was identified just for these CAS (left and right atrium, pulmonary vessels, sinus venosus, and major vessels) and the median CCL of the turtles was 64 cm. Therefore, in most cases radiographic examination is essential for a complete evaluation of the lungs and the CAS and then to define the severity of the disease. Ultrasonography for animals larger than 30 cm in length is useful as a screening to evaluate if they suffer from GE because the renal vessels are involved in more than 90% of the cases and are always easily identifiable17,20. During the radiographic follow-up of the turtles with GE we observed that gas in the renal vessels is also the last to disappear, so ultrasonography can be useful to monitor resolution of GE.
One of the goals of the study reported here was to document venous blood gas and acid-base values for loggerhead turtles with GE 5-7 hours after trawling and to investigate differences in these values between turtles that survived and those that died. In turtles with GE the blood circulation is dramatically slowed or even stopped due to the gas emboli disseminated along the cardiovascular system, and peripheral tissue O2 extraction should be considerable.
In the present study pH, pCO2, tCO2, HCO3, SO2 and potassium concentration were found to be relevant in the clinical assessment of turtles affected by GE. However, the multivariate analysis showed that only pH was an independent risk factor for death. Turtles that died had significant acidosis compared with turtles that survived.
Considering the high amount of gas present in the cardiovascular system of turtles affected by GE, pathologically this disease could be compared to the massive systemic air macroembolism of humans with a rare but catastrophic and largely underdiagnosed, undertreated, and underreported disease31. The brain and heart are the end organs most vulnerable to these ischemic events, which can lead to irreversible sequela or death. The air emboli cause pathological changes by two mechanisms: a reduction in perfusion distal to the obstruction and an inflammatory response32. These processes lead to vasogenic edema and worsen ischemia of the end organs33. Hyperbaric oxygen (HBO) is the main therapy for massive gas embolism from any cause because may not only decrease the size of air bubbles but also provide an adequate supply of oxygen to the ischemic tissue but such a treatment can be performed only whit specialized equipment34. There is no evidence that treatment with corticosteroids, anticoagulants, or lidocaine is related to a beneficial outcome in massive gas embolism32.
It is well known that in sea turtle during diving, apnea a right-to- left-shunt occurs. Besides intracardiac shunts, pulmonary-to-systemic shunting has been described between the pulmonary arteries and veins suggesting control of perfusion at several levels35,36. Recently multiple physiological artero-venous anastomoses were detected between pulmonary arteries and veins on high resolution Multidetector Computed Tomography images37,38.
With systemic GE in turtles, the gas is present throughout the venous and arterial circulation because the gas may pass from the heart or arteriovenous anastomoses from the venous to the arterial compartment. It is plausible that the significant amount of gas found throughout the venous and arterial circulation and in the heart causes a slowing or even an arrest of the blood circulation. Nevertheless, “only” 20% of the turtles in the present study died and many animals with severe disease spontaneously eliminated the gas bubbles and recovered.
Freshwater turtles survive weeks of anoxia at low temperatures without suffering from oxidative damage upon reoxygenation. A recent study showed that ATP/ADP pools and low succinate accumulation likely protect turtle hearts from anoxia/ reoxygenation injury39. Sea turtles continue to survive long after there is a total absence of oxygen in blood and lung40. Loggerheads also maintained electrical activity of the heart for over 1 hour of total anoxia41 in very sharp contrast to mammals. Turtle brain ATP levels are maintained for at least 2 hours of anoxia42 while depletion occurs within minutes in mammals.
During extended dives all tissues may become anaerobic and large changes in blood pH and pCO2 are tolerated43. The major adaptation that allows turtles to endure total anoxia for many hours is the special ability of the brain to function in the absence of 02.
Loggerhead turtles can rest on the ocean bottom for over 7 hours in cold temperatures27,44 but if turtles are struggling in nets, heart rate will increase from vigorous activity, which will result in rapid oxygen depletion. Higher heart rate will increase pulmonary blood flow, which may contribute to increased nitrogen absorption, potentially resulting in the development of gas emboli45. However, the exact etiopathogenetic mechanism of this systemic macroembolism in sea turtles has not yet been clarified. In the present study the range of withdrawal time of fishing gear was from 13 minute/depth of 20 m up to a maximum of 20 minutes/depth of 140 m and we cannot ascribe that this rate of ascent definitely causes decompression sickness in sea turtles. Moreover, this ascent rate did not differ between animals that developed GE and those that did not.
The turtles with GE, those that arrived alive at the STC and were then dry-docked or kept in tanks with low water level in a room at approximately 25-30 °C without any pharmacological treatment had a mortality of about 10%. Theoretically, gas embolism in turtles must be treated in a hyperbaric chamber as soon as possible but studies on large numbers of animals must be carried out to understand if it is promptly effective on turtles with very severe GE, destined to die within a few hours.
There has been still limited study and with small sample size of turtles for post-release mortality of marine turtles following capture by trawl fisheries20,46,47. Parga et al. (2020) described that on 15 animals that survived and were released with sPAT tags three (20%) died during the first 6 days and the outcome of a fourth animal was unknown. The immediate release of turtles into the sea can only be an option if there is no rescue center available to refer the turtles to. On board the fishermen cannot stage the disease and understand its severity. From the statistical data of our study only turtles in a comatose state are obviously serious, but turtles without pathognomonic clinical signs can also be severe and RX staging is always required. All the animals in our study underwent radiographic checks every 48 hours with progressive improvement of the gas inside the vessels and clinical improvement. In no animal have we seen a worsening in quantity of gas in the following days.
In light of the results achieved in this research, it is strongly recommended to transport the turtles as soon as possible to rehabilitation or rescue centers, rather than releasing them directly into the sea, even after a resting phase; in fact, turtles that apparently appear to be in good condition may actually have a GE in progress. Waiting to understand how to stem the phenomenon during fishing, collaboration with the fishermen is essential because it is important that the sea turtles are immediately transport to the rescue centers in order to be housed in the tanks in case of GE so as to control and to reduce their mortality.
The adoption of conservation actions has become a strategic issue in the Mediterranean, where the commercial fishing appears to be the main driver of mortality for marine turtles12,26,48. In bottom trawling the most effective solution to reduce GE phenomena is to limit the residence time of turtles inside the net, once incidentally captured. This can be achieved by introducing technological improvements in fishing gear, the so-called Bycatch Reducer Devices (BRDs) as the Turtle Excluder Device (TED) that seems to be the most effective. In the last 6 years, the effect of a new prototype of TED, a flexible grid, on the catching efficiency and performance of commercial bottom trawl has been tested in several bottom trawlers in the Adriatic Sea49,50. Easy storage and handling compared with previous devices tested in this area51 make the flexible TED a practical and valuable solution to reduce turtle bycatch in coastal Mediterranean demersal multispecies fisheries. Because of their effectiveness, which has mainly been demonstrated in prawn trawl fisheries, TEDs have become mandatory in several countries, out of the Mediterranean. Unfortunately, despite BRDs being an increasingly urgent need, they are not accepted in any Mediterranean country, because conservation aims are often hampered by competing political and economic factors52.
In conclusion, besides to applying the BRDs and outreach programs, the measures to reduce the impact of bycatch consist of raising fishers’ awareness and in training them in the best practices in sea turtle recovery after capture. We deem that the conservation of sea turtles is mainly a technical and political challenge, because all these measures require significant investments but are capable of improving the conservation prospects of these endangered species16. Such efforts would so be designed to reach the UN Sustainable Development Goals of 2030 Agenda, particularly the targets of Goal 1453, and in the context of the European Union Green Deal and the recent Recovery and Resilience Facility54.
Currently, the synergy between fishermen, rescue and rehabilitation centers is important to reduce mortality from systemic GE in sea turtle, because 80% of all the affected animals will have a good chance of survive and be released, if kept in shallow water or dry for at most 2 weeks needed to recover from systemic macroembolism.