Metal Concentrations (Mg, Fe, Zn, Cu, Cd, Pb) in the Plasma and Cell Concentrates of Chelonia mydas and Lepidochelys olivacea from Costa Rica

Simple Summary

Sea turtles are widely used as bioindicators for assessing marine pollution, since they have a long lifespan, migrate across large oceanic areas and integrate into the environment over time. Measuring trace elements in their blood provides information regarding both environmental contamination and potential health risks. In this study, magnesium, iron, zinc, copper, cadmium and lead levels were measured in the plasma and red blood cells of green turtles (Chelonia mydas) and olive ridley turtles (Lepidochelys olivacea) from the Caribbean and Pacific coasts of Costa Rica. The blood was analyzed, since it reflects both recent exposure (plasma) and longer-term internal regulation (cell concentrates). The results were compared both between the blood compartments in the two species and with previously published data from other geographic areas and time periods. Essential elements showed different distributions between the plasma and the cell concentrates, confirming their physiological regulation in reptiles. Green turtles had generally higher iron and copper levels, likely related to diet, while olive ridleys showed higher cadmium concentrations, suggesting greater exposure in the Pacific basin. The lead concentrations were low in both species. This study provided baseline blood trace element data for sea turtles in Costa Rica and supported the usefulness of blood analyses for long-term environmental monitoring.

Abstract

Sea turtles are increasingly being used as bioindicators of marine pollution, yet baseline data on trace elements in the blood are still limited. This study quantified magnesium (Mg), iron (Fe), zinc (Zn), copper (Cu), cadmium (Cd) and lead (Pb) in green turtles (Chelonia mydas) (55 plasma samples and 71 cell concentrate samples) and olive ridleys (Lepidochelys olivacea) (101 plasma samples and 65 cell concentrate samples) sampled off the Caribbean (Tortuguero) and Pacific (Ostional) coasts of Costa Rica in 2003–2004. The metals were measured using atomic absorption spectroscopy; whole-blood concentrations were derived from the plasma and the erythrocyte values. The present results were compared with published datasets to evaluate the spatial and temporal patterns of metal exposure over the past two decades. The essential elements showed matrix-specific distributions, with Mg and Cu higher in the plasma, and Fe and Zn higher in the cell concentrates in both species (p < 0.001). C. mydas generally exhibited higher Cu, Fe and Zn levels in the plasma (p < 0.001), whereas L. olivacea showed markedly higher Cd levels (p < 0.001). Overall, the Pb levels were low as compared with many other rookeries worldwide. These data provide one of the earliest, large-sample baselines for trace elements in sea turtle blood in the Eastern Tropical Pacific and Western Caribbean and underscore the value of blood-fraction analysis for long-term ecotoxicological monitoring.

1. Introduction

Sea turtles are considered endangered species as a result of centuries of overexploitation for their meat, eggs and shells, as well as incidental capture; furthermore, sea turtles suffer the harmful effects of the presence of pollutants in the marine environment [1]. Although polychlorinated biphenyls (PCBs), plastics and microplastics represent a significant part of environmental contaminants, metals also pose a major threat to marine ecosystems and living organisms. Therefore, there is a need for bioindicators capable of providing a realistic picture of the presence of pollutants in both the environment and living organisms. Sea turtles are considered excellent environmental bioindicators due to several characteristics common to all species: they have long lifespans, are globally distributed and, in the case of carnivorous species, occupy high trophic levels [2,3,4]. Changes in their health and behavior can reveal important information regarding the state of marine ecosystems, including pollution levels, habitat degradation and the effects of climate change. Consequently, monitoring sea turtle populations can provide valuable data for conservation and ecosystem management. Furthermore, pollutant monitoring in sea turtles may also help in assessing risks to their health, particularly since many species have undergone dramatic demographic declines in recent years [5]. Blood sampling is a simple and minimally invasive method for assessing metal intake in both the extracellular and the intracellular compartments [6,7]. Whole blood can also provide indirect information regarding potential metal accumulation in organs such as the liver, kidneys and adipose tissue [8]. Although plasma and whole blood are the most commonly used samples for assessing the status of essential and non-essential trace elements, there are also advantages in assessing their levels in the cell concentrates in which the levels remain more stable [9]. Erythrocyte concentrations of trace elements are useful for assessing their intracellular concentrations and general homeostasis, making them a more reliable indicator of mid-term nutritional status (also under pathological conditions, such as anemia), or of mid-term exposure in the case of non-essential trace elements.

This study focused on green sea turtles (Chelonia mydas) and olive ridley sea turtles (Lepidochelys olivacea). For the most part, green turtles are found in the tropical and subtropical waters of the Atlantic, Pacific and Indian Oceans. They usually inhabit shallow waters; however, they are also capable of long-distance migrations across open seas. Juveniles (up to three years of age) are carnivorous, feeding on crabs and small invertebrates, while adults shift to an herbivorous diet of algae and marine plants. The females migrate every two to four years to nesting sites, which typically remain constant throughout their lives, as do their foraging grounds [10]. Olive ridley turtles inhabit the tropical waters of the Atlantic, Pacific and Indian Oceans. They are carnivorous, feeding on small crabs, jellyfish, fish and other invertebrates, and they spend most of their lives in the open sea, entering shallow waters only to mate and lay eggs. Owing to the ethical limitations associated with the collection of tissue samples from live animals, studies concerning trace element concentrations in sea turtles have predominantly relied on opportunistic sampling from deceased or stranded individuals [2,10]. Nevertheless, assessing contaminant exposure in living populations is of greater relevance for both toxicological research and sea turtle conservation. Accordingly, there is a pressing need to develop minimally invasive methods for obtaining samples useful to the assessment of trace element exposure in sea turtles [7].

The present study analyzed plasma and blood cell concentrate samples collected from green turtles and olive ridleys in Costa Rica in the early 2000. These samples, which had been preserved in a private biobank, came from two of the country’s most important sea turtle conservation areas: the Ostional Wildlife Refuge and the Tortuguero National Park. Ostional, on the Pacific coast, is a major nesting site for olive ridleys and hosts several “arribadas” each year, whereas Tortuguero, on the Caribbean coast, is one of the most significant nesting sites for green turtles in the Atlantic region. As much as heavy metal pollution varies geographically, the overall risk to each species is determined by evaluating species samples in the same environment, such as the North Caribbean coast for green turtles and the Pacific coast for olive ridley turtles in Costa Rica. After recruiting to neritic waters during the reproductive season, these different species largely occupy similar habitats [11], with turtles foraging in the Northwest Atlantic and the Pacific Ocean. This migratory cycle may expose them to pollutants from a large portion of North and Central America. Moreover, the Caribbean and the Pacific coast of Costa Rica are multi-use coastal zones with a variety of human activities that can cause metal pollution, such as agriculture, untreated sewage discharge, solid waste pollution, marine transport, oil refineries, oil spills and port activities, which have adversely affected sandy beaches, coastal lagoons and coral reefs by increasing sedimentation rates and causing heavy metal contamination of seawater [12,13].

The aims of this study were to (i) quantify the concentration of Mg, Fe, Zn, Cu, Cd and Pb in the plasma and in the blood cell concentrates in green turtles and olive ridleys using samples collected in Costa Rica in the early 2000s, and which had been preserved in a biobank, and (ii) compare these results with published data to evaluate potential changes in metal bioaccumulation in these two species over the past twenty years and to provide a picture of the situation in the early 2000s. To the authors’ knowledge, this is the first study reporting essential and non-essential trace element concentrations in sea turtles sampled at Ostional Wildlife Refuge.

2. Materials and Methods

2.1. Sample Collection

Blood sampling of C. mydas and L. olivacea was carried out in Costa Rica (Figure 1) from June to September in both 2003 and 2004. The green turtles were sampled on the beach of the Tortuguero National Park (10°32′27″, 83°29′ 59″ W–10°21′17″ N, 83°23′29″ W; the North Caribbean coast) at the end of their nesting process, while the olive ridleys were captured and sampled at sea off Ostional (10°00′00″ N, 86°45′50″ W; north Pacific coast). Blood was obtained from the dorsal cervical sinus [13] using manual restraint, 21 G needles and 5 mL sterile plastic syringes (Terumo, Tokyo, Japan). The samples were collected in heparinized tubes and placed on ice. The turtles were marked on the carapace with a yellow marker pen (LACO Industries, Chicago, IL, USA) to avoid re-sampling and were then released. Blood sampling was carried out as part of the sea turtle health monitoring projects carried out by the Tortuguero and Ostional parks (permit 091-2003/2004-OFAU of Costa Rica’s Ministry of Environment and Energy and the National System of Conservation Areas). Only blood samples obtained from turtles which appeared healthy during the external clinical examination (i.e., no visible signs of disease or trauma) were included in this study. The sampling procedures are detailed in Santoro and Meneses [14].

Figure 1.

Map of the sampling locations for the two species of sea turtles, C. mydas and L. olivacea, sampled at the two costal sites of Costa Rica in the Tortuguero National Park (sampling site 1) and in the Ostional Wildlife Refuge (sampling site 2), respectively.

Blood samples were kept refrigerated for 1–2 h until they arrived at the laboratory, where the plasma was separated from the blood by centrifugation at 3000 g for five minutes. It was then transferred into plastic vials and stored at −20 °C until the analysis of the biochemical parameter [14]. The frozen cell concentrate, containing mainly erythrocytes, but also leukocytes and thrombocytes, as well as the remaining plasma were dried by lyophilization to completely remove water from the samples. The lyophilized samples were stored in plastic tubes sealed with parafilm to prevent the sample from absorbing moisture at −20 °C. They were finally sent to the Department of Veterinary Medical Sciences of the University of Bologna, where they were stored at −20 °C until metal analysis.

Seventy-one cell concentrates and 55 plasma samples from female green turtles (the difference was due to the presence of hemolysis in some plasma samples, which made the samples unsuitable for metal analysis due to the passage of the Fe and Zn contained in the cell concentrates into the plasma itself) were samples suitable for additional metal analysis, as were 65 cell concentrates and 101 plasma samples from the olive ridleys (the number of cell concentrate samples is lower than the plasma samples since they were also used for parasitological investigation). Of the L. olivacea samples, it was possible to differentiate 19 plasma samples coming from females and 18 coming from males (Table 1 and Figure 2).

Table 1.

Data regarding the two species studied and the tissues collected.

Species	Sample	n	Males/Females	Eating Habits	Sampling Area
C. mydas	plasma	55	nd	Herbivorous (seagrasses, macroalgae and jellyfish in juveniles)	Caribbean Sea
	cell concentrates	71	nd
L. olivacea	plasma	101	19/18	carnivorous (crustaceans and fish)	Pacific Ocean
	cell concentrates	65	nd

Figure 2.

The dot plot represents the sample size of the plasma and cell concentrates of C. mydas and L. olivacea. Regarding the plasma of L. olivacea, some specimens were identified as male and female.

2.2. Metal Analysis

The metal concentration analysis was carried out in 2020. To avoid contamination, all reagents were handled carefully. Polyethylene disposables were thoroughly washed with 1 N HCl under a fume hood, and disposable gloves were worn during the procedure. All the reagents were of Suprapur grade from Merck (Darmstadt, Germany). The samples (200 mg of dry matrix) were placed in individual acid-washed Teflon jars and were digested in 2 mL 65% HNO₃ and 0.5 mL of 30% H₂O₂ in a microwave oven for 5 min at 250 W, 5 min at 400 W, 5 min at 500 W and 1 min at 600 W. The cooled samples were transferred into 10 mL polyethylene volumetric flasks, diluted with water Suprapur up to 10 mL and directly analyzed using an atomic absorption spectrophotometer with a graphite furnace (Spectra AA, Varian, Palo Alto, CA, USA) equipped with a deuterium lamp background correction for Cd and Pb, and a flame atomic spectrophotometer for Mg, Fe, Zn and Cu (AAnalyst 100, PerkinElmer, Waltham, MA, USA). All the samples were run in batches, which included blanks, and standard reference materials (fish muscle: ERM-BB422); there was no evidence of any contamination in these blanks. All the values for the reference materials were within the certified limits provided by the Community Bureau of Reference—BCR (Brussels).

The detection limits for flame atomic spectroscopy were 0.04 μg/mL for Mg, 0.09 μg/mL for Fe, 0.04 μg/mL for Zn, and 0.01 μg/mL for Cu. When analyses were carried out using an atomic absorption spectrophotometer equipped with a graphite furnace, the detection limits were 0.6 ng/mL for Cd and 1.9 ng/mL for Pb. The metal concentrations in plasma and cell concentrates are expressed in μg/g on a dry weight (d.w.) basis for Mg, Fe, Zn and Cu and in ng/g on a d.w. basis for Cd and Pb.

The concentrations in the whole blood were calculated starting from the metal concentrations in plasma and cell concentrates, considering a hematocrit of 30% [14,15,16] with a weighted mean taken from the two matrices. The trace element concentrations in whole blood expressed as mean ± standard error (SE) in both µg/g d.w. and in µg/g wet weight (w.w.) are also reported in Table S1. When it was necessary to convert data expressed in wet weight from the bibliography, 80% water was used [17].

The Pollution Load Index (PLI) was calculated using the concentration factors (CFs) of each element in the sea turtles’ blood for the total assessment of the degree of contamination in C. mydas, as reported by Morao et al. [18]; when considering the baseline concentrations (C baseline), we used the reference intervals reported by Villa et al [6]. Regarding L. olivacea, reference intervals for trace element concentrations in the blood were not available in any bibliography; therefore, it was not possible to calculate the PLI.

2.3. Statistical Analysis

The central limit theorem and the Saphiro–Wilk test were used to evaluate the normality of the distribution [19,20]. The homogeneity of the variances among the groups, as defined by species, sex and matrix, was evaluated using Levene’s test [21]. When the distribution was normal, a t-test for unpaired samples was used. Conversely, when the distribution was not normal, the Mann–Whitney test was applied [22]. Differences were considered significant, with a p-value < 0.05. Statistical analyses were carried out using R 4.2.1 (R foundation for statistical computing; Vienna, Austria; https://www.R-project.org/ accessed on 4 August 2023). The p values for the different statistical comparisons are reported in Table S2 (nd: not determined).

3. Results

All the metals were present at detectable concentrations in all the samples analyzed. The mean concentrations found in the cell concentrates and plasma of C. mydas and L. olivacea showed considerable variation between matrices and species, as evidenced in Figure 3.

Figure 3.

Concentrations of Mg, Fe, Zn, Cu, Cd and Pb in the plasma and the cell concentrates of specimens of L. olivacea and C. mydas. Values are expressed as mean ± SE in µg/g d.w. for Mg, Fe, Zn and Cu and in ng/g d.w. for Cd and Pb. For each element, the same symbol indicates a significant difference between the species.

Regarding the distribution of elements between the plasma and the cell concentrates, Mg was significantly more concentrated in the plasma than in the cell concentrates in both species (p < 0.001); however, there were no significant differences between the species. Iron was significantly higher in the cell concentrates than in the plasma (p < 0.001), with a higher concentration observed in the plasma of C. mydas than in that of L. olivacea (p < 0.001). Zinc was significantly more concentrated in the cell concentrates than in the plasma (p = 0.003); the Zn content was significantly higher in the cell concentrates of L. olivacea than in those of C. mydas (p < 0.005), while it was more concentrated in the plasma of C. mydas (p < 0.001). The Cu concentration was significantly higher in the plasma and cell concentrates of C. mydas than in those of L. olivacea (p < 0.001). When considering the matrix, the concentration of Cu was significantly higher in the plasma of both species (p < 0.001). Conversely, Cd was present at significantly higher concentrations in the plasma and the cell concentrates of L. olivacea (p < 0.001). Higher concentrations of Cd were observed in the cell concentrates of both species (p < 0.001). Lead did not show significant differences between the species; however, it was significantly more abundant in the plasma than in the cell concentrates (p < 0.001 in L. olivacea, p = 0.013 in C. mydas), exhibiting higher values in L. olivacea.

Considering the differences between the sexes (Table 2 and Table S2), the plasma Fe concentration was significantly higher in the females of L. olivacea (22.82 ± 2.591 µg/g d.w., p < 0.001), whereas Pb was more concentrated in the male plasma of L. olivacea (0.166 ± 0.019 µg/g d.w., p < 0.001).

Table 2.

Concentrations of Mg, Fe, Zn, Cu, Cd and Pb (μg/g d.w.) in the plasma of females and males of L. olivacea. Values are expressed as mean ± SE. The same superscript number in the same column, indicates a significant difference between the sexes for each element.

	Mg	Fe	Zn	Cu	Cd	Pb
Females (n = 19)	725.9 ± 39.83	22.82 ± 2.595 (1)	15.45 ± 3.204	4.445 ± 0.309	0.828 ± 0.079	0.091 ± 0.004 (2)
Males (n = 18)	740.5 ± 23.60	13.14 ± 1.636 (1)	8.241 ± 1.482	4.695 ± 0.420	0.671 ± 0.065	0.166 ± 0.009 (2)

The CF values for each metal are reported in Table 3. Cadmium presented the highest value (5.791 ± 1.062), followed by Cu, Mg, Zn and Pb; on the other hand, Fe exhibited the lowest CF value (0.490 ± 0.054). The overall PLI for C. mydas was 1.301 ± 0.201.

Table 3.

Concentrations of Mg, Fe, Zn, Cu, Cd and Pb (mean ± SE expressed as μg/g d.w.) in C. mydas whole blood, their corresponding CF values and the overall PLI.

	Metal Concentration	Metal CF	PLI
Mg	666.6 ± 12.32	0.490 ± 0.054	1.301 ± 0.201
Fe	553.3 ± 19.63	1.706 ± 0.609
Zn	34.84 ± 2.181	0.745 ± 0.296
Cu	5.401 ± 0.264	2.348 ± 0.618
Cd	0.139 ± 0.008	5.791 ± 1.062
Pb	0.079 ± 0.000	0.580 ± 0.010

4. Discussion

The generation and interpretation of data regarding trace metals in marine organisms are difficult for several reasons. Metal concentrations vary in individuals of the same species depending on sex and age, as well as on the physiological and environmental conditions. In addition, tissues and organs (liver, kidney, blood and shell) of the same organism may accumulate metals in different concentrations; the differences in the bioaccumulation of trace elements have been extensively studied in sea turtles of different species over the years [23,24].

Sea turtles are widely recognized as valuable bioindicators of marine environmental health. Their sensitivity to ecological changes and their broad distribution and presence across multiple trophic levels make them particularly effective for monitoring the health status of ecosystems [2,3,4]. Finally, it is important to note that the present samples were collected almost 20 years ago, providing a snapshot of metal concentrations in sea turtles at a time when such investigations were far less common. To place the present results in context, they were compared with data from several studies carried out in subsequent years which reported trace element concentrations in the blood of C. mydas and L. olivacea from different regions worldwide. This offers an overview of how the situation has evolved from the time of the sampling used in this study to the present. For clarity, a summary table (Table 4) presents trace element concentrations from both the present study and the literature, standardized as mean ± SD in μg/g d.w. The main factors reported to influence the metal content in sea turtles, such as diet, species, sex and habitat [7,23], will therefore be taken into account when discussing the findings in the present study in the following subparagraphs.

Table 4.

Metal concentrations (mean ± SD expressed as μg/g d.w.) in the blood of C. mydas and L. olivacea from various locations worldwide sorted in chronological order of sampling. “NA” not analyzed, “n” number of specimens sampled, “Year” year of sampling.

Source	Species	Locality	Year	Mg	Fe	Zn	Cu	Cd	Pb
This study	C. mydas n = 55	Caribbean Sea, Costa Rica	2003–2004	553.3 ± 158.2	666.6 ± 99.29	34.84 ± 17.57	5.401 ± 2.127	0.139 ± 0.063	0.079 ± 0.006
Labrada-Martagon et al., 2011 [25]	C. mydas n = 42	Eastern Pacific Ocean, California (Punta Abreojos)	2005–2007	0.95 ± 0.35	1717 ± 421	69.6 ± 15.8	NA	0.3 ± 0.01	NA
Van de Merwe et al., 2010 [8]	C. mydas n = 16	Australia	2006–2007	NA	NA	37.20 ± 3.136	4.784 ± 0.093	0.166 ± 0.044	0.104 ± 0.027
Komoroske et al., 2011 [26]	C. mydas n = 30	Eastern Pacific Ocean, California (San Diego Bay)	2007–2009	NA	NA	NA	3.745 ± 1.125	0.066 ± 0.105	6.30 ± 1.11
Ley-Quinonez et al., 2013 [27]	C. mydas agasiizzi n = 12	Northern Pacific Ocean, Mexico	2008	NA	NA	63.58 ± 17.06	1.71 ± 0.73	0.99 ± 0.35	NA
Camacho et al., 2014 [28]	C. mydas n = 21	Central Atlantic Ocean (Cape Verde)	2009–2011	NA	NA	5.2 ± 2.25	1.25 ± 0.6	1.5 ± 0.35	0.35 ± 0.1
McFadden et al., 2014 [29]	C. mydas n = 87	Central Pacific Ocean, Northern Line Islands	2008–2012	533.9 ± 133.7	1190 ± 296	37.5 ± 1.175	2.16 ± 0.88	0.135 ± 0.125	0.09 ± 0.06
Da Silva et al., 2016 [30]	C. mydas (healthy group n = 13)	Southern Atlantic Ocean, Brazil	2011–2012	NA	NA	3.44 ± 0.57	4.64 ± 0.54	0.39 ± 0.05	4.77 ± 0.64
Villa et al., 2017 [6]	C. mydas n = 49	Australia, Queensland, Howick Island Group (baseline)	2014	323.9 ± 35.20	1361 ± 244	46.92 ± 7.97	2.301 ± 0.464	0.024 ± 0.008	0.136 ± 0.080
Villa et al., 2017 [6]	C. mydas n = 40	Australia, Queensland, Cleveland Bay	2014	455 ± 55	1300 ± 335	48 ± 13.5	3.5 ± 1	0.035 ± 0.030	0.1 ± 0.05
Villa et al., 2017 [6]	C. mydas n = 42	Australia, Queensland, Upstart Bay	2014	495 ± 60	1500 ± 425	55 ± 12.5	3 ± 1	0.02 ± 0.01	0.1 ± 0.05
Agostinho et al., 2020 [31]	C. mydas n = 23	Southwestern Atlantic Ocean, Brazil	2018	NA	2005 ± 224	115.6 ± 22.7	8.15 ± 2.14	0.51 ± 0.25	1.15 ± 0.7
Morao et al., 2024 [18]	C. mydas n = 27	Eastern Atlantic (Gulf of Guinea)	2017–2018	NA	2189 ± 869	91.30 ± 57.18	3.79 ± 1.22	0.03 ± 0.06	0.97 ± 0.59
Wilkinson et al.,2023 [32]	C. mydas n = 30	Australia, Queensland, Howick Island Group	2017–2019	365 ± 45	1335 ± 275	44 ± 11	3 ± 0.5	0.05 ± 0.05	0.2 ± 0.1
Wilkinson et al.,2023 [32]	C. mydas n = 35	Australia, Queensland, Cockle Bay	2017–2019	420 ± 55	1000 ± 520	38.5 ± 20.5	3.5 ± 2.5	0.1 ± 0.1	0.4 ± 0.3
Wilkinson et al.,2023 [32]	C. mydas n = 24	Australia, Queensland, Upstart Bay	2017–2019	440 ± 70	1335 ± 500	44.5 ± 14.5	3 ± 1	0.15 ± 0.2	0.15 ± 0.1
This study	L. olivacea n = 65	Northern Pacific Ocean, Costa Rica	2003–2004	594.2 ± 99.36	647.7 ± 101.1	27.70 ± 13.29	3.997 ± 1.059	1.409 ± 0.481	0.102 ± 0.041
Paez-Osuna et al., 2010a [33]	L. olivacea n = 4	Northern Pacific Ocean, Mexico	2005	NA	NA	NA	NA	NA	0.95 ± 0.18
Paez-Osuna et al., 2010b [34]	L. olivacea n = 25	Northern Pacific Ocean, Mexico	2005–2006	NA	NA	58.4 ± 4.7	2.28 ± 1.3	0.45 ± 0.20	NA
Zavala et al., 2014 [35]	L. olivacea n = 19	Northern Pacific Ocean, Mexico	2011	NA	NA	185.6 ± 18.35	5.1 ± 7.35	6.65 ± 1	NA
Cortes-Gomez et al., 2014 [36]	L. olivacea n = 41	Northern Pacific Ocean, Mexico	2012	NA	NA	52.75 ± 18.41	3.05 ± 0.55	0.850 ± 0.400	0.100 ± 0.050
Cortes-Gomez et al., 2018 [37]	L. olivacea n = 20	Northern Pacific Ocean, Mexico	2014	NA	1295 ± 760	40.3 ± 23.5	2.8 ± 1.75	0.65 ± 0.4	0.05 ± 0.05
Andalon et al., 2021 [38]	L. olivacea n = 35	Northern Pacific Ocean, Mexico	2018	NA	NA	47.15 ± 32.81	10.59 ± 4.725	3.095 ± 2.591	0.495 ± 0.095

4.1. Metal Concentrations in C. mydas and L. olivacea

Regarding trace elements, Mg, Fe, Zn and Cu are essential micronutrients present in organisms in concentrations expressible in mg/g, with values ranging from hundreds of micrograms for Mg to a few micrograms for Cu, depending on the tissues. Magnesium concentrations in sea turtles remain poorly investigated, yet improving knowledge in this area would enhance understanding of the risks faced by marine organisms and ecosystems. In this study, the Mg levels were significantly higher in the plasma than in the cell concentrates in both species, while no significant differences were observed between species or between sexes. These results suggested that Mg in the bloodstream is regulated by finely tuned, conserved homeostatic mechanisms essential for maintaining vital physiological functions. Similar results have been reported in C. mydas sampled in the Pacific Ocean between 2008 and 2012 [29], suggesting that the Mg blood concentrations observed herein likely reflect the physiological values. This was in agreement with the findings of Villa et al. [6] and Wilkinson et al. [32], who reported Mg blood concentrations in specimens of C. mydas from Queensland (Australia), collected, respectively, in 2014 and 2017–2019, to be slightly lower, but of the same order of magnitude. We consider the comparison between the data presented in our study and those reported by Villa et al. [7], used as a reference interval, together with the corresponding CF calculation for essential and non-essential trace elements, to be particularly useful, as the analytical techniques used for sample preparation are the same, although the collected specimens of C. mydas were sexually mature in our research and subadult in the group studied by Villa et al. [7]. The CF factor for Mg concentration calculated in C. mydas (1.709 ± 0.60) confirmed that the concentration measured in the present study was quite similar to the value reported as baseline by Villa et al. [6] in animals sampled about ten years later. To the best of the authors’ knowledge, this paper contains the first report of blood Mg concentration in L. olivacea. Iron is an essential element; however, it can be potentially toxic, due to its ability to change its oxidative state [39]. Therefore, it is useful to assess its concentration in different organs and tissues of marine organisms. In sea turtles, dietary habits strongly influence Fe levels. For instance, hepatic Fe concentrations in the herbivorous C. mydas are higher than those reported for Caretta caretta, likely because seagrass beds can accumulate trace metals at levels much higher than those found in fish, mollusks or crabs [23]. The higher plasma Fe concentration in C. mydas, compared to L. olivacea, may reflect its preference for eelgrass, the chloroplast-rich leaves of which contain high levels of Fe, mainly bound to proteins such as ferritin and ferredoxin [40]. However, in the present study, the values of C. mydas (666.6 ± 99.29 μg/g d.w.) were lower than those reported by some authors in the literature. For example, Agostinho et al. [31] reported 2005 ± 224 (Table 4) μg/g d.w. in blood collected from healthy female specimens from Rocas Atoll, a pristine nesting site in a marine biodiversity reserve. Labrada-Martagon et al. [25] reported 1717 ± 421 μg/g d.w. in the blood of both healthy and injured specimens from the eastern Pacific Ocean (California, Punta Abrejos). Conversely, Yipel et al. [41] reported Fe concentrations in the blood of C. mydas from the northeastern Mediterranean Sea of 23.72 ± 5.71 μg/g d.w., while for C. caretta, comparable to L. olivacea because of the same carnivorous diet, they reported values of 62.62 ± 9.75 μg/g d.w., almost ten times lower than the present results. In the study by Yipel et al. [41], the specimens collected were turtles which were stranded, primarily due to traumatic injuries, and it was possible to hypothesize that the values reported by Yipel et al. were due to the pathological states of the animals sampled. Data regarding Zn concentration in the blood remain scarce, despite the importance of monitoring Zn levels in blood and tissues, particularly due to the increasing concern regarding zinc oxide nanoparticles (ZnO NPs), which have been associated with mutagenic and cytotoxic effects in the cell concentrates of Arrau turtles (Podocnemis expansa) [42]. Over the past two decades, the production and use of ZnO NPs have risen significantly, and they are now incorporated into a wide range of commercial products, including toothpaste, cosmetics, sunscreen, textiles, paint and building materials. In sea turtles, Zn is found at the highest concentrations in muscle and adipose tissue [24]. The unusually high levels of Zn in adipose tissue may be explained by its binding to the pigment-related proteins responsible for the green coloration of fat [43,44]. In the present study, the Zn concentration in plasma was significantly higher in C. mydas than in L. olivacea, whereas in the cell concentrates, it was significantly higher in L. olivacea. The higher plasma Zn concentration in C. mydas could be due to diet, since Zn is more prevalent in algae than in small crustaceans. A blood concentration higher than that found in the present study (Table 4) was reported in L. olivacea specimens at La Escobilla beach in Mexico [33,35,36,37]. The different levels could be attributed to the different sampling sites, since Ostional and La Escobilla are 2000 km apart, even if both are located on the eastern Pacific coast. At Boa Vista Isle in Cape Verde, a Zn blood concentration very similar to that found in the present study was reported in 201 specimens of C. caretta [45]; however, the same author reported a lower Zn concentration in the whole blood of 21 C. mydas [28]. Higher Zn blood values in C. mydas were reported by Agostinho et al. in Rocas, a Brazilian atoll in the Atlantic Ocean [31]. Nevertheless, comparison is difficult, even within the same species, due to the limited number of studies, the heterogeneous origin of the samples, and the usually unknown age of the animals sampled. The most probable hypothesis was that the different Zn blood concentrations reported for C. mydas were the result of the different levels of environmental exposure [46]. Copper concentration in the tissues of the different species of sea turtles varies as a result of their different diets. However, it has recently increased in all species due to its presence in antifouling paints used on boat hulls [24]. The concentration of Cu in the blood and tissues of living organisms is finely regulated since this essential trace element is fundamental for Cu-dependent enzymes; however, due to its ability to change oxidation state, it can be harmful and produce reactive oxygen species (ROS) [46]. In this study, a significantly higher Cu concentration was found in the plasma and the cell concentrates of C. mydas than in those of L. olivacea. This was probably due to the different diets of the two species, since Cu is more concentrated in plants and algae than in crabs and crustaceans. As proof of this, a higher Cu concentration has been reported in C. mydas livers and kidneys than in the same tissues of L. olivacea [23]. Copper blood concentrations in specimens of C. mydas and L. olivacea collected in different years from different marine areas and reported in different studies (Table 4) were very similar to those found in the present study [8,26,33,36,47], although with respect to the baseline reported by Villa et al. (2017) [6] for C. mydas, a CF of 2.348 ± 0.618 was calculated. In more recent years, Agostinho et al. [30] found a Cu concentration equal to almost double the one found in this study in the blood of C. mydas from a pristine zone in the Southwestern Atlantic Ocean in Brazil, while in 2021, Andalon et al. reported values for L. olivacea from the northern Pacific Ocean values which were more than twice those found in the present study [38].

Cadmium, due to its structural similarity to Zn, acts as an antagonist, and Cd exposure may cause hepatic and renal dysfunction, neurological and reproductive disorders, as well as neoplasms [48]. Regarding the Cd concentration in the blood of the C. mydas and the L. olivacea considered in the present study, a significantly lower concentration was found in the plasma and the cell concentrates of C. mydas with respect to L. olivacea. It should be noted that the specimens of L. olivacea analyzed in the present study came from the Pacific Ocean, while the C. mydas came from the Atlantic Ocean. Fraga et al. [2] reported a higher Cd concentration in the liver and kidney in the subpopulations of C. mydas inhabiting the Pacific Ocean than in the same organs of the subpopulation inhabiting the Atlantic Ocean. The probable reason lies in the two oceans themselves; the Pacific Ocean is of more ancient origin than the Atlantic, allowing for a greater accumulation of metals in the waters of this ocean. Cadmium and other metals, including Zn, nickel (Ni), Cu, manganese (Mn), but not Pb and aluminum (Al), exhibit this pattern of higher concentration in the deep waters of the Pacific than in the Atlantic [49]. Comparing the blood Cd values in the green turtles from Costa Rica analyzed in the present study with those reported by other authors (Table 4), it was noted that the concentrations were higher in the specimens from the eastern [25] and northern Pacific Oceans [35], but also from the Northern [28] and Southwestern Atlantic Ocean [30,31]. On the other hand, several authors have reported lower values in the blood from C. mydas from Australia [6,32] and from the eastern Atlantic [18]; with reference to the baseline for C. mydas reported by Villa et al. [6], a CF of 5.791 ± 1.062 was therefore calculated. Regarding L. olivacea, since it is a carnivorous species in all the phases of its life, it is prone to accumulate Cd, especially when living in the Pacific Ocean and, in the future, it will be particularly important to continue monitoring Cd levels in the blood and tissues of this species. Cortez Gomez et al. [37] reported carapace asymmetry and a correlation between metal concentration in the blood of L. olivacea from Mexico in specimens found dead as well as a very high Cd concentration in the blood of 35 μg/g d.w. [50]. Moreover, in different olive riddle turtles from the same area, sampled in the same period, Cortes-Gomez [36,37] also reported Cd blood concentrations lower that those reported in this study, while other authors reported higher concentrations [35,38], indicating that Cd levels in organisms could be influenced by several factors, including the clinical conditions and age of the specimens, while there does not appear to be a temporal increase over time.

In the present study, the Pb blood concentration was similar in C. mydas and L. olivacea in both the plasma and the cell concentrates; values for both species were similar to or lower than those reported in previous studies [6,8,29,32,36,37]. The lead plasma levels were significantly higher than those in the cell concentrates, and a significant sex-related difference with male L. olivacea specimens exhibiting higher concentrations than in females was also found. This unexpected result has not previously been reported. Guirlet et al. [51] hypothesized an increase in blood Pb concentration in female L. olivacea during oviposition, due to Ca mobilization. Furthermore, Komoroske et al. [26] reported a very high Pb blood concentration in C. mydas from San Diego Bay, linking this result to high contamination of the bay. Interestingly, an elevated Pb blood concentration was also reported in C. mydas from the South Atlantic Ocean [47] and from the Rocas atoll, a pristine area in the Atlantic Ocean which is far from contamination sources [31]; the authors hypothesized that migratory sea turtles may visit polluted feeding sites, leading to the accumulation of Pb. Similarly, in olive riddle sea turtles from the northern Pacific Ocean in Mexico, Paez-Osuna et al. [33] reported a higher Pb blood concentration. Tropical waters contain more Pb than temperate or cold waters, and in coastal areas more than in offshore regions [52]. This highlights the high risk of Pb exposure for sea turtles. Since the early 2000s, when many countries banned leaded gasoline, Pb contamination has gradually declined [36,53]. However, studies investigating its presence in the wild remain valuable for assessing environmental contamination and evaluating risks to both wildlife and human health. Comparing lead levels across species, regions and trophic levels can reveal patterns of bioaccumulation and highlight areas in which residual contamination persists. Thus, monitoring wildlife also functions as an early warning system for potential human exposure.

4.2. Metal Distributions in Plasma and Cell Concentrates

Measuring the concentration of trace elements in cell concentrates may provide valuable insight in the case of pathological conditions such as anemia or during an inflammatory response. Moreover, trace element concentrations in cell concentrates reflect homeostatic mechanisms by which cells maintain the optimal element levels required for physiological functions, thus making them useful indicators of long-term exposure. Furthermore, an increase in the plasma of a trace element is usually considered to be an index of recent exposure, with variations often associated with oral intake and the type of diet. Previous studies have shown that trace metal concentrations in red blood cells are positively correlated with those in kidney and liver tissues, supporting the use of non-lethal measurements to assess trace element bioaccumulation [8,54]. However, data regarding the concentrations of trace elements in cell concentrates are scarce in the literature and are sometimes estimated indirectly, using blood and plasma concentrations and blood hematocrit. In the present study, the concentrations were determined directly by mineralizing cell concentrates. Of the elements analyzed, Mg, Cu and Pb were significantly more concentrated in the plasma, whereas Zn, Fe and Cd were more concentrated in the cell concentrates in both species. This consistency between the two species reflected the biochemical roles of these elements and represented the general pattern observed in vertebrates. Despite the fact that erythrocytes in reptiles have a higher metabolic demand than those in mammals, due to being nucleated, reptiles contain low levels of Mg in comparison to mammals. Similarly to Mg, Cu concentrations in the plasma were higher than in the cell concentrates; a similar pattern is reported in male athletes [55]. Physical exercise, particularly intense in all swimming animals, including sea turtles, produces a redistribution of Cu in tissues and plasma, triggering plasma Cu for the synthesis of Cu-enzymes, such as cytochrome-c oxidase. The latter catalyzes the final step in the aerobic respiratory chain as well as other Cu proteins, such as ceruloplasmin and superoxide dismutase which have essential anti-oxidant activity, reducing the free radicals formed during physical activity.

The distribution between the plasma and the cell concentrates of the two non-essential elements Cd and Pb showed a different trend, with Pb being more concentrated in the plasma only in L. olivacea and Cd more concentrated in the cell concentrates of both species. Non-essential trace elements primarily accumulate in the cell concentrates, having a lower concentration in the plasma; this reflects the toxicologically active fraction. Regarding Cd, the significantly higher concentration in the cell concentrates could be interpreted as an indicator of long-term low environmental exposure. Conversely, a more severe exposure to Pb in L. olivacea could have caused progressively higher plasma levels as compared to the Pb in the cell concentrates. In human medicine, the plasma-to-erythrocyte concentration ratio was proposed [56] as an index of Pb exposure; a constant ratio of approximately 0.7% could be indicative of a state of equilibrium, whereas an increase in the ratio is expected after a sudden exposure to Pb. In the present study, a ratio of 3.2 was calculated for L. olivacea and of 1.8 for C. mydas, suggesting a probable environmentally higher exposure for Olive Ridley turtles than for green turtles.

Finally, Fe and Zn concentrations in the cell concentrates, particularly in erythrocytes, do not reflect dietary intake, and a nutritional deficiency only leads to a decrease after several months, sometimes after symptoms of deficiency, such as anemia [57,58]. On the other hand, plasma concentration is an index of recent exposure; thus, its variations may be associated with animal diet [59]. In the literature, only Barraza et al. [60] have reported trace element concentrations in the cell concentrates of C. mydas from the eastern Pacific Ocean, California, having values similar to those reported in the present study for Fe and Cu. On the contrary, the Zn, Cd and Pb levels reported by Barraza et al. [60] were higher with respect to the concentrations found in the sea turtles from Costa Rica, suggesting a more severe exposure to these trace elements for specimens inhabiting the marine environment near California.

4.3. Trace Elements of Concern in Sea Turtles

It is important to point out that chronic exposure to heavy metals has been indicated to be one of the possible factors for the development of fibropapillomatosis in sea turtles [30,61]. This tumor disease mainly affects green sea turtles, C. mydas [62,63]. Its exact cause is still unclear; however, it is considered to be multifactorial. A herpesvirus is consistently associated with these tumors [64], while environmental factors such as ultraviolet (UV) radiation [65,66,67], marine toxins [68] and especially pollutants [66,69,70] are thought to contribute to the disease, either by weakening the immune system or by making the virus more aggressive [66,71]. Of the pollutants, metals are increasingly being studied as possible contributors, especially Cu, Cd and Pb. Turtles living in coastal areas close to human activity are often exposed to chemical contaminants [25,35]; metals can promote oxidative stress by generating ROS [72,73]. This condition can damage lipids, proteins and DNA [73,74] and may interfere with immune defenses [75]. In this way, metal exposure can contribute to oxidative stress, immune suppression, viral activation and, finally, tumor development [66,71,76]. Da Silva et al. [30] investigated the relationship between the concentration of blood trace elements and the development of fibropapillomatosis, reporting significantly higher concentrations of Cu, Fe and Pb in the blood of specimens of C. mydas, with severe fibropapilloma lesions with respect to healthy animals. However, the trace element concentrations reported by Da Silva et al. [30] in healthy specimens of C. mydas from the South Atlantic Ocean are similar to those presented in the present study only for Cu, while the Cd and Pb concentrations were three and ten times higher, respectively and Zn was ten times lower. These differences could confirm a low level of non-essential trace element pollution in the aquatic environment of the Caribbean coast of Costa Rica, where the samples in the present study were collected. Regarding the Zn concentration in the blood, it is possible to assume that a decreased concentration, even in clinically asymptomatic subjects, represented a predisposing factor for the onset of skin diseases, as has already been highlighted in numerous species of reptiles [77,78]. It is therefore of paramount importance to continue monitoring essential and non-essential trace element levels in sea turtles, and to correlate them with their health status, evaluating the potential risk of developing fibropapillomatosis.

5. Conclusions

In conclusion, the present study confirmed the usefulness of blood sampling in assessing the environmental contaminants and the health status of the specimens simultaneously, using a sampling technique which minimizes stress to the animals. The Mg and Fe blood concentrations reported herein could be considered a starting point for establishing reference values, given the large number of animals sampled and their good health conditions. The Cd and Pb concentrations were lower than those reported in other studies over the past twenty years, indicating the low level of contamination at the two sampling sites in Costa Rica. Regarding the Cd blood concentration, the differences between the species could be attributed to both the sampling site and diet, while for Fe, Zn and Cu, the differences could be due to diet. Although the data regarding the trace elements presented herein were obtained from samples collected more than two decades ago, they are an important reference for sea turtles as bioindicator organisms in the coastal marine habitats of Costa Rica, where the sources of contaminants to the both the Caribbean and the Pacific coasts are expected to have increased over the past decades due to local coastal development and to the water and sediment loads carried by the rivers. The PLI determined from the present data was lower than the value reported by Morao et al. [18] who obtained their data in more recent years. This suggests a possible increase in the degree of trace element pollution in green turtles over time. A continuous detailed spatial and temporal evaluation of metals in sea turtles and other marine bioindicator organisms is recommended.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ani16040621/s1, Table S1: Metal concentrations (mean ± ES) expressed as μg/g d.w. and as μg/g w.w. in the blood of C. mydas and L. olivacea and Table S2: The statistical test used to calculate p-values.

Author Contributions

Conceptualization, G.A. and M.S.; methodology, G.A. and M.F.; validation, C.R. and G.I.; formal analysis, M.F. and C.R.; investigation, C.R. and G.A.; resources, M.S.; data curation, C.R. and G.A.; writing—original draft preparation, C.R., G.I. and A.L.; writing—review and editing, G.A. and A.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article or Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.