The loggerhead sea turtle, Caretta caretta (C. caretta), is the most abundant sea turtle species living and reproducing in the Mediterranean Sea (Pagano et al., 2019; Esposito et al., 2020; Oriá et al., 2020). This species is classified as vulnerable to endangered in the International Union for Conservation of Nature’s Red List of Threatened Species (https://www.iucnredlist.org ). Factors threatening C. caretta extinction are mainly linked to human activities (www.iucn.org): coastal areas anthropisation, high level of anthropisation in the coastal areas, marine traffic, sea macroscopic pollution (ingestion of plastic and other debris), chemical pollution, fisheries (entanglement in longlines, trawls and gill nets as well as ingestion of hooks and lines) (Tomas et al., 2001; Camiñas et al., 2006; Báez et al., 2010; Echwikhi et al., 2012; Blasi and Mattei, 2017; Caracappa et al., 2018; Pagano et al., 2019).

Chemical pollution, caused by human and industrial activities (use of fertilisers and pesticides, plastic, oil, and gas production), determines the release in the marine environment of large quantities of nonessential toxic metals, such as arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) (Valavanidis and Vlachogianni, 2010; Varol, 2011; Esposito et al., 2020; Lambiase et al., 2021b), which are accumulated by sea turtles mainly through the ingestion of contaminated prey, but also through marine sediment and seawater (Storelli et al., 2011; Bucchia et al., 2015; Cammilleri et al., 2017; Lambiase et al., 2021a). The accumulation of these chemicals, as already reported for other marine pollutants (Gonzalez-Fernandez and Cuesta, 2022), could weaken the immune system of sea turtles (Chaousis et al., 2023), making them more vulnerable to bacterial or viral infections (Fiorito et al., 2019; Fiorito et al., 2021). In this regard, they are susceptible to an alphaherpesvirus belonging to the Herpesviridae family, named chelonid herpesvirus 5 (ChHV5). The virus can infect several species of aquatic turtles and is considered the causative agent of the formation and development of fibropapillomas (Aguirre et al., 1994; Quackenbush et al., 1998). Even though fibropapillomatosis (FP) mostly affects green sea turtles (Chelonia mydas), all marine turtles can contract the disease (Lawrance et al., 2018). FP is characterised by the presence of cutaneous lesions growing on the external soft tissue of the animal body, on the carapace, plastron, and cornea (Jones et al., 2016). The tumours, although not malignant, can hinder crucial functions (Rodenbusch et al., 2014), like the animal’s ability to see, breathe, swim, dive, forage, and avoid predators (Milton and Lutz, 2003; Perrault et al., 2021), thus indirectly causing its death. The lesions can also be observed (with reported prevalence varying from 17% to 41%, Work et al., 2004) in visceral organs; they mainly occur in the lungs and kidneys, but also, to a lesser extent, in the heart, gastrointestinal tract, and liver (Herbst and Klein, 1995; Alfaro-Nunez et al., 2016; Jones et al., 2016). ChHV5 has been consistently associated with FP, with fibropapillomas containing high concentrations of ChHV5 DNA (Alfaro-Nunez et al., 2016). However, recent PCR-based studies have detected high levels of ChHV5 DNA also in apparently healthy tissues without fibropapillomas (Page-Karjian et al., 2012), demonstrating that large proportions of asymptomatic sea turtles can also be virus carriers (Alfaro-Nunez et al., 2016) and suggesting the probable ability of ChHV5 to latently persist in their organism (Alfaro-Nunez et al., 2014, Alfaro-Nunez, 2016). In this regard, it is conceivable that, if particular conditions compromise the immune system of animals, like critical water temperature, coinfection with other pathogens, or the presence of pollutants (Jones et al., 2016; Lawrance et al., 2018; Chaousis et al., 2023), the virus could be reactivated and trigger the onset of a disease, not necessarily FP.

The present study was carried out (from 2015 to 2020) on specimens of sea turtles stranded along the coasts of Southern Italy overlooking the Mediterranean Sea. Animals underwent viral, bacterial, histological, and chemical investigations with the aim of carrying out a comprehensive analysis of the causes of turtle stranding and, more generally, to trace a picture of the anthropogenic and opportunistic factors involved in the animal health status and in the endangerment of the species.

In the last few years, marine animal stranding has become frequent, and loggerhead sea turtles are involved in this phenomenon. Indeed, in our study, during the 6 years of observation, 195 sea turtles have been found dead along the coasts of the Campania and Calabria regions. Potential health risks for this species are mainly caused by increased anthropogenic pressure on the marine ecosystem (Monteiro et al., 2021), due to marine traffic, sea pollution, both macro- and microscopic, fishing activities (Pagano et al., 2019), and infectious diseases (Monteiro et al., 2021).

Results from our study have confirmed that anthropogenic factors are involved, to a different extent, in a large rate of turtle stranding/death, as already reported by other authors (Casale et al., 2010). Overall, our findings indicate that fishing activities are among the most important threats to sea turtles in our basin, in agreement with existing literature (Tomas et al., 2008; Casale et al., 2010; Caracappa et al., 2018). Necropsies have revealed that 117 sea turtles out of 195 (60%) suffered from anthropic impact. Indeed, we detected internal and/or external injuries caused by collision with boats, entanglement in fishing gears, and marine litter ingestion. Marine litter, a growing problem worldwide, impacts all marine organisms in different ways. Sea turtles usually ingest plastic debris and other litter, mistaking them for food (Campani et al., 2013). We found a high percentage (38.5%) of animals with objects in their digestive tract, similar to other studies (Casale et al., 2008; Lazar and Gračan, 2011), carried out on C. caretta in the Mediterranean Sea (37.9% and 48.1%, respectively), but lower than the rate reported by other authors (71.0% and 75.9%) in the same basin (respectively Tomás et al., 2002; Campani et al., 2013). Ingestion of plastic objects was found in 31/195 (15.9%) of the sea turtles analysed, but marine plastic ingestion has never been associated with direct mortality. However, it may cause sublethal effects, such as gastrointestinal lacerations, blockage, reduced feeding, and absorption of toxic compounds, as well as death from complete intestinal occlusion (Kühn et al., 2015; Matiddi et al., 2017; Sala et al., 2021).

Observing the injuries and the internal/external lesions has helped to clearly address the specific cause of mortality in 53 animals. In the other 64 sea turtles, the death could be ascribed to a mix of anthropic effects. Of the remaining 54 animals, 24 have been excluded from this analysis because they were mummified, and 30 have not been visibly damaged by anthropic factors, but various hypotheses about mortality were developed; for example, a lethal infection can be difficult to diagnose or die from embolism and/or drowning due to prolonged apnoea caused by fishing by-catch (trawling), which may not leave any visible traces on stranded carcasses (Casale et al., 2010).

In this study, a complete absence of both internal and external fibropapillomas in the animals tested was observed. The analysis included a substantial number of turtles (195) and covered an extended period of observation (6 years). Thus, so far, the Mediterranean Sea seems not to be affected by FP, a neoplastic disease whose development is likely linked to the presence of ChHV5 (James et al., 2021; Loganathan et al., 2021; Perrault et al., 2021), as well as too polluted and eutrophicated habitats (Dujon et al., 2021; Monteiro et al., 2021). Our results were quite surprising, considering that this disease is panzootic in green turtles (Williams et al., 1994; Jones et al., 2016), has been described in marine turtles worldwide (Alfaro-Nunez et al., 2014; Espinoza et al., 2020), and appears to be increasing in some parts of the world (Loganathan et al., 2021). It has been established that FP prevalence is related to marine pollution (Loganathan et al., 2021). In this regard, the Mediterranean Sea, a semi-closed sea mainly surrounded by industrialised countries, is considered a contaminated ecosystem (Gomez-Ramirez et al., 2020). Hence, the complete absence of FP in turtles from the Mediterranean Basin seems to indicate that other factors than pollution are involved in FP formation.

For the first time, according to our knowledge, the presence of ChHV5 was detected in 21 specimens of C. caretta living in the Mediterranean Sea. The detection of the virus without FP association did not surprise us since the occurrence of this alphaherpesvirus in sea turtles without tumours is well documented (Alfaro-Nunez et al., 2014; Alfaro-Nunez et al., 2016). The prevalence of ChHV5 observed in this study (10.8%) agrees with previous researchers who reported a percentage of viral positivity of 11.5% in healthy turtles belonging to the same species (Alfaro-Nunez et al., 2014). Furthermore, in 19 of the 21 positive C. caretta, beyond FP, the presence of the virus could not be associated with any specific pathological condition of the animal.

As already supposed by other authors (Page-Karjian et al., 2017), our results suggest the presence of persistent subclinical and latent infection due to ChHV5 in apparently healthy sea turtles. Indeed, the virus is characterised by the common feature of establishing long latency to escape immune system detection, in line with other herpesviruses (Alfaro-Nunez et al., 2016; Oriá et al., 2020; Monteiro et al., 2021; Piret and Boivin, 2021; Esposito et al., 2022a). The presence of ChHV5 in all the organs analysed (heart, kidney, brain, spleen, liver) agrees with data reported by other authors (Page-Karjian et al., 2017), who have identified the virus in kidney, heart, and nerve from green turtles not affected by FP. In addition, the simultaneous identification of the virus in multiple anatomical sites of some turtles may be due to the capacity of alphaherpesviruses to infect a wide variety of host cell types (Page-Karjian et al., 2017). According to age, the rates of ChHV5 positivity in the large juvenile (12.5%) and adult (9.5%) groups were similar, while a lower prevalence was revealed in the early juvenile group (4.2%). These results could be explained by the age-dependent difference in habits of sea turtles, as they spend the first period of life in the open seas, while in their juvenile or adult phase, they live in coastal areas, not deep waters (Pagano et al., 2019), and are probably more exposed to viral contamination.

In chelonians (turtles and tortoises), herpetic infections have been related to different pathological states, including proliferative and ulcerative stomatitis, respiratory infections, dermatitis, conjunctivitis, hepatitis, and dermatitis (Jacobson et al., 1986; Pettan-Brewer et al., 1996; Hervas et al., 2002; Johnson et al., 2005). In C. caretta, herpesviruses have been associated not only with FP but also with oral, respiratory, cutaneous, and genital lesions (Stacy et al., 2008). Interestingly, our results showed in two animals real-time PCR positivity for ChHV5 (in the lungs), a clinical frame of bronchopneumonia observable both macroscopically and histologically ( Figures 6 , 7 ). Our histopathological findings are consistent with studies describing, in chelonians, herpesvirus infections associated with heterophilic bronchitis and pneumonia, with the presence of heterophilic inflammation and sloughed epithelial cells in the airways (Origgi et al., 2004; Stacy et al., 2008; Marschang et al., 2009; Marenzoni et al., 2018). Indeed, to date, ChHV5 has been associated, apart from FP, only with ocular pathological manifestations (Oriá et al., 2020), while its involvement in respiratory disorders has never been reported in the literature. Similarly, heterophilic inflammation in the lung could be due to any insult, and it is not pathognomonic for ChHV5. Nevertheless, even though no other pathogen associable with respiratory disease has been found in the lungs of these two specimens of C. caretta, the hypothetical implication of ChHV5 in bronchopneumonia does not fulfil all the criteria necessary to claim a causal relationship between a pathogen and a disease (Fredricks and Relman, 1996).

An important aspect of this research is the analysis of the levels of trace metals in various organs of the turtles. Climate changes and the consequent geochemical differences considerably affect the quality, temperatures, and biochemical parameters of seawater in Southern Europe Seas (https://ec.europa.eu/clima/climate-change; Mitchell et al., 2011; Iordache et al., 2022). The large-scale (6 years; 94 animals) survey provides a very clear overall picture of the current situation regarding trace element levels in loggerhead turtles from Southern Italy’s coasts. Thus, it makes it easier to evaluate accumulated concentrations as well as to compare the levels of time revealed among different surveys in these areas. Overall, a relevant time-dependent accumulation increase of As levels was detected in liver and muscle tissues (Storelli and Marcotrigiano, 2000; Esposito et al., 2020; Canzanella et al., 2021). In particular, the concentrations of As in the muscle were higher than in the kidney and liver. Metalloid levels, such as As, in the liver tend to decrease more than in other tissues, because of a detoxification process that occurs in the liver before going into other tissues (kidney and muscle) (da Silva et al., 2014; Sulato et al., 2022), as detected in the liver of C. mydas (Sulato et al., 2022). The levels of Hg, which are similar to Cd, appeared similar to or lower than those detected in other studies (Maffucci et al., 2005; Storelli et al., 2005; Esposito et al., 2020; Gomez-Ramirez et al., 2020; Canzanella et al., 2021) and were instead higher in the liver and kidney than in the muscle. Whereas the highest levels of Pb and Cd were found in the kidney, followed by the liver and muscle. A similar pattern of bioaccumulation was previously found in turtles stranded along the southwestern coasts of the Tyrrhenian Sea (Esposito et al., 2020; Canzanella et al., 2021). These findings suggest that the accumulation of each metal is tissue-dependent. Interestingly, the levels of As in the liver and kidney of turtles from Calabria were considerably higher than those from the Campania region. Herein, the values of As in loggerhead turtles were highest in the muscle, followed by the kidney and liver. This trend, probably due to their feeding habit rich in molluscs (generally containing high levels of this metalloid), was already described by us (Esposito et al., 2020; Canzanella et al., 2021; Lambiase et al., 2021b). It is well known indeed that the main route of turtles’ exposure to marine environment contaminants occurs through food, consequently, the levels of metals found in the organs depend on the specific dietary habits of the loggerhead turtles, which is directly correlated to the levels of contamination of the marine environment (Esposito et al., 2020; Canzanella et al., 2021; Lambiase et al., 2021a). In this regard, about one-fifth of the foraging areas in the Mediterranean resulted contaminated; therefore, they represent a potential risk of exposure for turtles (Almpanidou et al., 2022). Although a lowering in Pb levels was observed in our samples, likely due to reduced use of lead in gasoline in European Countries since early 1980 (https://www.eea.europa.eu), regrettably, climate warming affects both saltwater and freshwater, provoking metal toxic level perturbation (LeMonte et al., 2017; Iordache et al., 2022). The concentration of As in the Calabria region is affected by multiple hydrogeochemical processes, which determine variable levels in groundwater (Apollaro et al., 2022). In addition, sea level rise also induces arsenic release from contaminated coastal soils (LeMonte et al., 2017). Another plausible explanation for such high levels of As contamination in the loggerheads of the Calabria coast may be connected with harbours, ship traffic, and the oil and gas industry. Indeed, other studies have pointed out that high levels of As are closely related to industrial contamination, including the oil and gas industry in the case of the green sea turtles found stranded in Southeastern Brazil (Bruno et al., 2021). Important information, which could explain the higher contamination of turtles from Calabria than the animals from Campania, is described by Garofalo et al. (2013) in a study on the genetic characterisation of central Mediterranean stocks of the loggerhead turtles using mitochondrial and nuclear markers. Garofalo et al. (2013), using MSA analysis, found out that the Calabrian colony represented the only considerable contribution to the Tyrrhenian stock. Furthermore, it has been reported that loggerhead turtles are able to be active even in colder waters, and this could increase their exposure to environmental contaminants (Garofalo et al., 2013). Overall, higher concentrations of As were detected in the liver and kidney of turtles from Calabria. Moreover, in turtles from the Calabria region, males had almost significantly higher Cd levels in the liver than females. Probably, this variation could be explained by metabolic differences between males and females (Maffucci et al., 2005) and by their feeding habits. No significant differences between the sexes (p > 0.05) were found for Pb and Hg. Age-dependent bioaccumulation of Pb and As was detected in the liver and muscle, respectively. Indeed, an adult group from the Campania region showed Pb (L) and As (M) values significantly higher than the large juvenile group. Moreover, Pb in the muscle of adults from the Campania region was remarkably higher than those of early juvenile turtles from the Calabria region. The levels of Pb (L) in adults from the Campania region were significantly higher than those in early juvenile and large juvenile groups from Calabria. Moreover, a significant site-dependent accumulation of As was detected in the liver of both early juvenile and large juvenile turtles from the Calabria region vs. large juvenile turtles from Campania. This difference may be explained as reported above. The phenomenon of age-dependent bioaccumulation, as well as the size, may explain these differences (Adel et al., 2017). The relationship between the accumulation of the contaminants and the size of the turtles was studied by performing statistical tests such as Spearman’s rank correlation. For the statistical study, the contamination levels determined in the liver, kidney, and muscle were correlated with the BW, SCL, and SCW morphometric parameters of the turtles. The Spearman’s rank test showed a statistically significant positive correlation (p < 0.01) between the levels of As found in the kidneys of Campania turtles and their SCL, indicating that the accumulation of this metal is size-dependent ( Supplementary Table S1 ). The increase in metal concentrations with size was also shown by a linear regression model ( Supplementary Figure S1 ), in accordance with the results reported in other studies on the bioaccumulation of environmental contaminants in turtles (Lazar et al., 2011; Lambiase et al., 2021a). Considering that the size of the turtles can be related to their age, it is possible to assume that the older turtles had a higher body burden of metals due to longer exposure to contaminants (Lambiase et al., 2021a). The increase in the metal accumulation with size could also be explained by considering the feeding behaviour of the turtles, which depends on their developmental stage. In fact, during the oceanic stage (CCL < 40 cm), turtle feeding consists mostly of planktonic organisms, while in the neritic stage (CCL > 40 cm), their diet becomes mainly constituted by benthic organisms (Lazar et al., 2011; Nicolau et al., 2017; Lambiase et al., 2021a). Genetic characterisation of Mediterranean loggerhead turtles C. caretta indicates that the distribution of juveniles from different rookeries may not be homogeneous among the major Mediterranean foraging grounds because of a complex pattern of surface currents in the Mediterranean (Clusa et al., 2014; Garofalo et al., 2013; Caracappa et al., 2016). These findings might have possible implications for the burden of contaminants found in this population, indicating a special concern. For example, abnormalities in the number of scutes have already been found by Caracappa et al. (2016) through an epigenetic mechanism probably caused by the environmental context during embryonic growth. Despite the data reported in the literature as well as the results shown herein on the bioaccumulation of the metals in the turtles from the Campania region, which highlight the correlation between the contamination levels found in the animal tissues and their size, the statistical analysis did not show any significant correlation between the As, Pb, Cd, and Hg concentrations in the turtles from the Calabria region and their morphometric parameters ( Supplementary Table S2 ). Moreover, the statistical analysis showed strong positive correlations (p < 0.001) between the Pb, As, and Hg levels found in the tissues of the turtles from both regions, indicating that there is a strong correlation between the bioaccumulation of contaminants in the different organs, despite the fact that it is tissue-dependent ( Supplementary Table S1 ).

In the frame of anthropogenic threats, it is important to consider that other environmental contaminants, such as organic pollutants including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and poly-chlorinated biphenyls (PCBs), may also cause harmful effects in turtles (Lambiase et al., 2021b; Esposito et al., 2022b). Some chemicals may reduce the immune function of marine animals and increase susceptibility to microbes (Fiorito et al., 2019; Fiorito et al., 2021). Particularly, a recent study using nontargeted proteomics has revealed altered immune responses in geographically distinct populations of green sea turtles (Chelonia mydas) from the Queensland coast (Chaousis et al., 2023). Here, to establish a relationship between viruses and chemicals, we have examined this hypothetical correlation. Nevertheless, the statistical analysis did not show significant correlations between the presence of the virus and the metal concentrations. The lack of relationships among chemical and viral parameters was probably due to the too-small number of virus-positive tissues, which did not allow us to perform an accurate statistical analysis.

In conclusion, this study provided valuable information on various aspects related to loggerhead sea turtles living in the Mediterranean Sea, with a focus on the factors involved in the endangerment of the species. We identified for the first time the presence of chelonid herpesvirus 5 in C. caretta living in this ecosystem. We found relatively high levels of lead, mercury, arsenic, and cadmium in the organs of the sea turtles investigated, even though no correlation was found with animal health status, including viral infection. One-fifth of the sea turtles analysed presented injuries caused by collision with boats or caused by entanglement in nets and lines, and more than one-half of these injuries were probably fatal. In some cases, ingested debris such as plastic, filament line, and fishhooks were involved in the animals’ death. For some of the investigated turtles, the cause of death remained unknown and was considered probably due to a mix of not-determined factors. Moreover, in the last few years, maybe due to climate warming allowing the turtles to find new habitats previously too cold for embryos incubation, a notable increase in nesting activity of loggerhead turtles has been detected in the Western Mediterranean (Hochscheid et al., 2022). In 2021, from the Italian coasts, 244 nests were reported. Specifically, both Campania and Calabria coasts (53 and 77 nesting sites, respectively) have been chosen for turtle spawning (Pietroluongo et al., 2023). However, these beaches are characterised by a high flow of tourism that may threaten eggs and hatchlings, as well as the development of new rookeries. Hence, further studies are needed to monitor and identify risk parameters that might involve C. caretta, with the aim of implementing conservation measures to protect the species from extinction.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

Ethical review and approval was not required for this study as our investigation was performed on stranded/dead turtles. Consequently, no ethical declarations were requested.

FF, ME, SL, and MA delineated the study. FS, DI, FF, FN, ME, SL, GF, and MA collected the data. FS, DI, FF, FN, ME, EE, CC, SL, BU, GL, and MA processed and analysed the data. FS, DI, FF, FN, ME, SL, BU, GL, EC, GF, and MA contributed resources and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.