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<p>See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/378524714</p><p>Nondestructive Evaluation of Metal Bioaccumulation and Biochemical</p><p>Biomarkers in Blood of Broad-Snouted Caiman (Caiman latirostris) from</p><p>Northeastern Brasil</p><p>Article  in  Environmental Toxicology and Chemistry · February 2024</p><p>DOI: 10.1002/etc.5823</p><p>CITATIONS</p><p>0</p><p>READS</p><p>71</p><p>7 authors, including:</p><p>Rayssa Lima dos Santos</p><p>Universidade Federal Rural de Pernambuco</p><p>16 PUBLICATIONS   21 CITATIONS</p><p>SEE PROFILE</p><p>Célio Freire Mariz Jr</p><p>Federal University of Pernambuco</p><p>11 PUBLICATIONS   61 CITATIONS</p><p>SEE PROFILE</p><p>Paulo Braga Mascarenhas-Junior</p><p>Florida International University</p><p>28 PUBLICATIONS   61 CITATIONS</p><p>SEE PROFILE</p><p>Rafael Sa Leitao Barboza</p><p>Universidade Federal Rural de Pernambuco</p><p>24 PUBLICATIONS   46 CITATIONS</p><p>SEE PROFILE</p><p>All 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© 2024 SETAC</p><p>Environmental Toxicology and Chemistry—Volume 00, Number 00—pp. 1–18, 2024</p><p>Received: 22 May 2023 | Revised: 2 July 2023 | Accepted: 4 January 2024 1</p><p>Environmental Toxicology</p><p>Nondestructive Evaluation of Metal Bioaccumulation</p><p>and Biochemical Biomarkers in Blood of Broad‐Snouted</p><p>Caiman (Caiman latirostris) from Northeastern Brasil</p><p>Rayssa Lima dos Santos,a,b,c,* Célio Freire Mariz Jr.,a,c Paulo Braga Mascarenhas‐Júnior,a,b Rafael Sá Leitão Barboza,b,d</p><p>Ednilza Maranhão dos Santos,d Jozélia Maria de Sousa Correia,d and Paulo Sérgio Martins de Carvalhoc</p><p>aPrograma de Pós‐Graduação em Biologia Animal, Universidade Federal de Pernambuco, Recife, Brasil</p><p>bLaboratório Interdisciplinar de Anfibios e Répteis, Universidade Federal de Pernambuco, Recife, Brasil</p><p>cLaboratório de Ecotoxicologia Aquática, Universidade Federal de Pernambuco, Recife, Brasil</p><p>dPrograma de Pós‐Graduação em Biodiversidade, Universidade Federal Rural de Pernambuco, Recife, Brasil</p><p>Abstract: Studies on the bioaccumulation and toxicity of contaminants in Crocodylians are scarce. We evaluated alterations</p><p>in concentrations of the nondestructive biomarkers butyrylcholinesterase (BChE), glutathione‐S‐transferase (GST), superoxide</p><p>dismutase (SOD), and reduced glutathione (GSH), together with bioaccumulation of the metals iron (Fe), copper (Cu), zinc</p><p>(Zn), manganese (Mn), chronium (Cr), aluminium (Al), and lead (Pb) in Caiman latirostris captured in Tapacurá Reservoir (TR;</p><p>São Lourenço da Mata, Pernambuco, Brasil), in urbanized areas of Pernambuco State (UA; Brasil) and from the AME Brasil</p><p>caiman farm (AF; Marechal Deodoro, Alagoas, Brasil); the latter was used as a potential reference with low levels of</p><p>contamination. For metal analysis, 500 µL of blood was digested in 65% HNO3 and 30% H2O2. The samples were analyzed by</p><p>inductively coupled plasma–optical emission spectrometry. For analysis of biomarkers, an aliquot of blood was centrifuged to</p><p>obtain plasma in which biochemical assays were performed. Blood concentrations of metals analyzed</p><p>status,</p><p>and the presence of specific oxidative stimuli can influence the</p><p>relationship between these enzymes.</p><p>Metals exhibit intricate relationships in both environmental</p><p>settings and biological systems, often yielding synergistic,</p><p>potentiating, additive, inhibiting, or antagonistic effects</p><p>(Nordberg et al., 2007). The scarcity of studies reporting in-</p><p>terelementary correlations in whole blood from crocodilians</p><p>poses challenges in providing a comprehensive discussion of</p><p>the data pertinent to these results. Dos Santos, de Sousa</p><p>Correia, Paim, et al. (2021) observed relationships in the blood</p><p>of C. latirostris captured in the TR. They reported positive</p><p>correlations in concentrations between Cr and Al (r= 0.52),</p><p>similar to the findings in our study. Cortés‐Gómez et al. (2014)</p><p>observed positive correlations between Zn and Cu in the blood</p><p>of L. olivacea captured in Escobilla Beach, Oaxaca, Mexico</p><p>(p= 0.000), and Camacho et al. (2013) reported strong positive</p><p>and negative correlations among 11 elements (Cu, Mn, Pb, Zn,</p><p>Cd, Ni, Cr, As, Al, Hg, and Se) in the blood of loggerhead turtle</p><p>(Caretta caretta) in Cape Verde, West Africa. Similar to the</p><p>findings for C. latirostris, Camacho et al. (2013) identified re-</p><p>lationships between Cu and Zn (0.422), Cr (0.326), and Al</p><p>(0.187; p</p><p>draft;</p><p>Writing—review and editing.</p><p>Data Availability Statement—The data supporting the findings</p><p>of our study are available on request from the corresponding</p><p>author, Rayssa Lima dos Santos (rayssa.limas@ufpe.br or</p><p>rayssalimasantos00@gmail.com).</p><p>REFERENCES</p><p>Araújo Freitas, J. B., Cabral, J. J. dS. P., Luiz, A., Paiva, R., Albuquerque,</p><p>T. B. V., & Nascimento Silva, N. B. (2018). 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See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons L</p><p>icense</p><p>View publication stats</p><p>https://doi.org/10.1016/j.chemosphere.2017.04.102</p><p>https://doi.org/10.1016/j.ecoenv.2019.110057</p><p>https://doi.org/10.1016/j.ecoenv.2019.110057</p><p>http://www2.cprh.pe.gov.br/wp-content/uploads/2021/02/quali_agua_2019.pdf</p><p>http://www2.cprh.pe.gov.br/wp-content/uploads/2021/02/quali_agua_2019.pdf</p><p>https://doi.org/10.1007/s13280-019-01159-0</p><p>https://doi.org/10.1007/s00128-009-9866-6</p><p>https://doi.org/10.1016/j.cbpc.2015.08.003</p><p>https://doi.org/10.1016/j.cbpc.2015.08.003</p><p>https://doi.org/10.1016/j.envpol.2022.119685</p><p>https://doi.org/10.1016/j.envpol.2022.119685</p><p>https://doi.org/10.1016/j.scitotenv.2021.145829</p><p>https://doi.org/10.1002/etc.5620220209</p><p>https://doi.org/10.1016/j.envpol.2004.05.008</p><p>https://doi.org/10.1016/j.envpol.2004.05.008</p><p>https://doi.org/10.1016/j.envpol.2018.07.004</p><p>https://doi.org/10.1016/0269-7491(87)90173-4</p><p>https://doi.org/10.1201/9780203647295.ch16</p><p>https://doi.org/10.1201/9780203647295.ch16</p><p>https://ttu-ir.tdl.org/server/api/core/bitstreams/cf66aa61-7ade-4573-9a5d-36bbaea919ae/content</p><p>https://ttu-ir.tdl.org/server/api/core/bitstreams/cf66aa61-7ade-4573-9a5d-36bbaea919ae/content</p><p>https://doi.org/10.1155/2020/1913853</p><p>https://doi.org/10.4025/actasciagron.v41i1.42620</p><p>https://doi.org/10.1016/j.envpol.2007.06.009</p><p>https://doi.org/10.1016/j.envpol.2007.06.009</p><p>https://doi.org/10.1088/0967-3334/28/4/R01</p><p>https://doi.org/10.1088/0967-3334/28/4/R01</p><p>https://doi.org/10.1016/j.ygcen.2008.02.011</p><p>https://doi.org/10.1016/S0016-6480(03)00199-0</p><p>https://doi.org/10.3390/ijms22157820</p><p>https://doi.org/10.1016/S0300-483X(00)00392-9</p><p>https://doi.org/10.1016/S0300-483X(00)00392-9</p><p>https://doi.org/10.1201/EBK1420064162-c3</p><p>https://doi.org/10.1016/S1382-6689(02)00126-6</p><p>https://doi.org/10.1016/S1382-6689(02)00126-6</p><p>https://doi.org/10.1016/0378-4274(84)90179-6</p><p>https://doi.org/10.1126/science.abe9090</p><p>https://doi.org/10.1126/science.abe9090</p><p>https://doi.org/10.1007/s10646-016-1652-8</p><p>https://doi.org/10.1007/s10646-016-1652-8</p><p>https://doi.org/10.1071/WR9840201</p><p>https://doi.org/10.1071/WR9840201</p><p>https://www.researchgate.net/publication/378524714</p><p>Nondestructive Evaluation of Metal Bioaccumulation and Biochemical Biomarkers in Blood of Broad-Snouted Caiman (Caiman latirostris) from Northeastern Brasil</p><p>INTRODUCTION</p><p>METHODS</p><p>Study areas</p><p>Animal sampling procedures</p><p>Analyses of metals</p><p>Analyses of biochemical biomarkers</p><p>Statistical analyses</p><p>RESULTS</p><p>Biometric parameters</p><p>Metals in blood of C. latirostris</p><p>Biochemical biomarkers in C. latirostris</p><p>Correlation analysis</p><p>DISCUSSION</p><p>CONCLUSIONS</p><p>Acknowledgments</p><p>Disclaimer</p><p>Author Contributions Statement</p><p>Data Availability Statement</p><p>REFERENCES</p><p>in animals from AF</p><p>were lower compared with TR and UA, confirming that animals from the caiman farm could be used as references with low</p><p>levels of contamination. Iron, Cu, Mn, Al, and Pb exceeded toxic levels for other vertebrates in animals from TR and UA.</p><p>Butyrylcholinesterase activity showed significant reduction in adults from UA and TR compared with AF. An increase in the</p><p>activity of GST and GSH, in adults of TR and UA in relation to AF, was verified. Superoxide dismutase activity showed a</p><p>significant reduction in adults of TR in relation to AF, and the concentrations of Cu and Mn were negatively correlated with</p><p>SOD activity. Animals from UA and TR showed greater concentrations of the analyzed metals compared with reference</p><p>animals, and changes in biomarkers were seen, confirming the potential of these nondestructive chemical and biological</p><p>parameters in blood of C. latirostris for biomonitoring of pollution. Environ Toxicol Chem 2024;00:1–18. © 2024 SETAC</p><p>Keywords: Biomarkers; Blood; Crocodylians; Ecotoxicology; Metals</p><p>INTRODUCTION</p><p>Freshwater aquatic ecosystems are the main destinations of</p><p>several potentially toxic organic and inorganic chemical com-</p><p>pounds used in urban, domestic, industrial, and agricultural</p><p>activities (Wang et al., 2021). The intensification in the use of</p><p>these contaminants with consequent chronic exposure and</p><p>bioaccumulation by organisms can cause sublethal toxicity,</p><p>which has been quantified in laboratory and field studies. Many</p><p>reptile species are under threat from factors such as climate</p><p>change, habitat loss, and hunt for subsistence or illegal trade.</p><p>In addition, exposure to anthropogenic contaminants repre-</p><p>sents a relevant factor for the conservation of reptiles, a group</p><p>for which studies on the bioaccumulation and effects of con-</p><p>taminants are relatively scarce (Barraza et al., 2021). Reptiles</p><p>can be directly exposed to contaminants through several</p><p>routes, including ingestion of contaminated food and water,</p><p>ingestion of soil, inhalation, maternal transfer to eggs/young,</p><p>dermal exposure, and absorption of contaminants through the</p><p>surface of eggs (Poletta et al., 2017; Smith et al., 2007).</p><p>Crocodilians are long‐lived, top predatory carnivores that</p><p>inhabit tropical and subtropical locations throughout the world,</p><p>characteristics that favor their use as model organisms in</p><p>* Address correspondence to rayssa.limas@ufpe.br and</p><p>rayssalimasantos00@gmail.com</p><p>Published online in Wiley Online Library</p><p>(wileyonlinelibrary.com).</p><p>DOI: 10.1002/etc.5823</p><p>mailto:rayssa.limas@ufpe.br</p><p>mailto:rayssalimasantos00@gmail.com</p><p>http://crossmark.crossref.org/dialog/?doi=10.1002%2Fetc.5823&domain=pdf&date_stamp=2024-02-27</p><p>ecotoxicological studies to evaluate both bioaccumulation and</p><p>toxic effects caused by contaminants (Campbell, 2003; Todd</p><p>et al., 2010). The toxic effects caused by exposure of crocodi-</p><p>lians to persistent organic pollutants, pesticides, and endocrine</p><p>disruptions have been related to alteration of sex steroids, al-</p><p>tered sexual development, developmental abnormalities, and</p><p>sexual reversal (Barraza et al., 2021).</p><p>The main route for contaminant absorption in reptiles is</p><p>through the pulmonary and gastrointestinal routes, resulting</p><p>from the ingestion of both food and materials rich in metals</p><p>that are accidentally ingested, such as stones, soil, and sedi-</p><p>ment. At the molecular level, tissue distribution occurs via</p><p>nonselective transport, such as through metallothioneins that</p><p>transport zinc (Zn), cobalt (Co), copper (Cu), silver (Ag), cad-</p><p>mium (Cd), mercury (Hg), and albumins (Zn, Cd; Grillitsch &</p><p>Schiesari, 2010). Metallothioneins can also sequester and de-</p><p>toxify nonessential metals (Samuel et al., 2021). At the pop-</p><p>ulation level, the main route of metal transfer is via the food</p><p>chain (Camus et al., 1998; Hammerton et al., 2003; Lance</p><p>et al., 2006), where biomagnification occurs for arsenic (As), Cd,</p><p>Hg, lead (Pb), selenium (Se), and tin (Sn), mainly in their fat‐</p><p>soluble organic forms (Grillitsch & Schiesari, 2010).</p><p>Some studies on the effects of age using body length as a</p><p>proxy for bioaccumulation of Hg in crocodilians have shown</p><p>that larger animals have higher concentrations in tissues than</p><p>smaller ones, such as in muscle, liver, and blood of the caiman</p><p>Melanosuchus niger in the Amazon (Eggins et al., 2015;</p><p>Schneider et al., 2013). However, middle‐aged Alligator mis-</p><p>sissippiensis exhibited higher Hg concentrations in whole</p><p>blood than younger and older animals at the same study site,</p><p>contrary to bioaccumulation predictions (Lawson et al., 2020).</p><p>Sex, in turn, seems to be a factor that directly influences Hg</p><p>concentrations in American alligators, with females losing up to</p><p>2.5% of their body mass during the reproductive period, which</p><p>reflects a loss of 1.6% to 5.7% of the total body load of Hg/year</p><p>(Schneider et al., 2013). During the nesting season, blood Hg</p><p>concentrations in female A. mississippiensis predicted egg</p><p>yolk Hg concentration, indicating maternal transfer (Nilsen</p><p>et al., 2020). Length and sex did not influence hepatic con-</p><p>centrations of the metals As, Cd, Co, chromium (Cr), Hg, nickel</p><p>(Ni), Pb, and Se in A. mississippiensis (Campbell et al., 2010).</p><p>Studies suggest that reptiles are resistant to metal in-</p><p>toxication, but with some elements, such as Cd, Pb, and Hg</p><p>potentially more toxic than others. However, more empirical</p><p>research, particularly laboratory‐based studies, is needed to</p><p>assess this potential resistance and the degree to which it may</p><p>attenuate adverse effects of chronic exposure to low metal</p><p>concentrations (Grillitsch & Schiesari, 2010). Although few ex-</p><p>perimental studies have evaluated the effects of metals on</p><p>crocodilians, correlations have been found between Hg and</p><p>the neuroendocrine response to stress, as well as the rela-</p><p>tionship of Cd and Zn to oxidative damage and antioxidant</p><p>activity (Romero‐Calderón et al., 2022). Changes were also</p><p>observed in the metallothionein biomarker in Morelet's</p><p>crocodile (Crocodylus moreletii) blood, which was positively</p><p>correlated with anthropic environments and with high con-</p><p>centrations of Cd (Buenfil‐Rojas et al., 2022).</p><p>The broad‐snouted caiman (Caiman latirostris) is widely</p><p>distributed in South America, from the Northeast and across</p><p>the Southeast and South of Brasil to Uruguay, northeast</p><p>Argentina, southern Paraguay, and southwest to Bolivia</p><p>(Verdade & Piña, 2007). Caiman latirostris has been used as a</p><p>bioindicator of environmental contamination using chemical</p><p>(Dos Santos, de Sousa Correia, & dos Santos, 2021), bio-</p><p>chemical (Burella et al., 2018), genotoxic (López González</p><p>et al., 2013; Poletta et al., 2011), histological (Stoker</p><p>et al., 2008), and endocrine (Stoker et al., 2003) blood pa-</p><p>rameters. Blood samples offer a highly valuable approach</p><p>for assessing the accumulation and biological consequences</p><p>of contaminants in vertebrates, while avoiding destructive</p><p>procedures (Fossi et al., 1998; Grillitsch & Schiesari, 2010).</p><p>However, ecotoxicological monitoring based on chemical</p><p>measurements of internal concentrations of metals combined</p><p>with biochemical biomarkers in nondestructive blood samples</p><p>from free‐living broad‐snouted caimans residing in natural en-</p><p>vironments has not yet been documented in the literature.</p><p>Inhibition of the enzyme butyrylcholinesterase (BChE) in blood</p><p>plasma has been used as a relatively specific biomarker that in-</p><p>dicates exposure to organophosphate and carbamates in-</p><p>secticides in lizards (Sánchez‐Hernández et al., 2004), freshwater</p><p>turtles, C. latirostris (Attademo et al., 2012), and birds (Grosset</p><p>et al., 2014). The Phase 2 biotransformation enzyme glutathione‐</p><p>S‐transferase (GST) has been used in the monitoring of exposure</p><p>to pollutants in the Nile crocodile (Crocodylus niloticus; Arukwe</p><p>et al., 2015). Cellular antioxidant defenses that attempt to</p><p>counteract the deleterious effects of reactive oxygen species</p><p>(ROS) include antioxidant molecules such as glutathione and</p><p>enzymes such as superoxide dismutase (SOD) and catalase.</p><p>These biomarkers have been quantified in the blood</p><p>of C. latir-</p><p>ostris exposed under controlled conditions to agrochemicals</p><p>used in soybean cultivation (Poletta et al., 2016).</p><p>Tapacurá Reservoir was formed from damming the Tapacurá</p><p>River, located in the rural area of São Lourenço da Mata City,</p><p>Brasil. The area is occupied by local communities and house-</p><p>holds that practice subsistence or commercial activities such as</p><p>polyculture, livestock farming, sugar cane plantation, and flour</p><p>factories (Aprile & Bouvy, 2008). Organic and inorganic pollu-</p><p>tants are deposited in the reservoir from upstream cities along</p><p>the course of the Tapacurá River (De Araújo & Nunes, 2003;</p><p>Mascarenhas‐Junior et al., 2021). Within the highly urbanized</p><p>metropolitan region of Pernambuco's capital, Recife, broad‐</p><p>snouted caiman are also found and rescued by the state en-</p><p>vironmental agency (Mascarenhas‐Junior et al., 2021) from</p><p>water bodies within the drainage basins of rivers polluted by</p><p>domestic sewage and industrial activities (Pernambuco State</p><p>Environmental Agency, 2019).</p><p>Given this context, the objective of our study was to use</p><p>nondestructive blood samples from C. latirostris residing in the</p><p>rural Tapacurá Reservoir and in urbanized water bodies of</p><p>the Recife metropolitan region, as well as from a caiman farm, as</p><p>potential baseline controls, to evaluate bioaccumulation of the</p><p>metals aluminum (Al), Cr, Cu, iron (Fe), manganese (Mn), Zn, Cd,</p><p>Pb, and Ni and concentrations of the biochemical biomarkers</p><p>BChE, GST, SOD, and glutathione (GSH). The influence of</p><p>2 Environmental Toxicology and Chemistry, 2024;00:1–18—dos Santos et al.</p><p>© 2024 SETAC wileyonlinelibrary.com/ETC</p><p>15528618, 0, D</p><p>ow</p><p>nloaded from</p><p>https://setac.onlinelibrary.w</p><p>iley.com</p><p>/doi/10.1002/etc.5823 by Florida International U</p><p>niversity, W</p><p>iley O</p><p>nline L</p><p>ibrary on [27/02/2024]. See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons L</p><p>icense</p><p>caiman sex and age on biomarker response to metal exposure</p><p>was also evaluated.</p><p>METHODS</p><p>Study areas</p><p>Caiman latirostris were captured between the years 2019</p><p>and 2021 in three areas representing different contaminant</p><p>exposure situations: the rural area of Tapacurá Reservoir (TR;</p><p>São Lourenço da Mata, Pernambuco, Brasil), urbanized areas</p><p>within the Recife (Brasil) metropolitan region (UA), and the AME</p><p>Brasil caiman farm (AF; Marechal Deodoro, Alagoas, Brasil), the</p><p>latter being a possible basal reference with lower levels of</p><p>contamination (Figure 1).</p><p>Tapacurá Reservoir was built in the 1970s for public water</p><p>supply and flood control along the course of the Tapacurá</p><p>River. Its drainage area of 470.5 km2, surface area of 9.5 km2,</p><p>and volume of 94,200m3 contribute more than 36% of the</p><p>water consumed by the 2 million people inhabiting Recife</p><p>Metropolitan Region (de Andrade et al., 2009). Currently, land</p><p>use in the Tapacurá River basin includes agriculture</p><p>(390.94 km2), pastures (46.6 km2), urbanized areas (15.46 km2),</p><p>and 12.42 km2 forest. This mosaic of activities with inadequate</p><p>soil management caused by human actions is potentially</p><p>causing environmental impacts related to increased erosion</p><p>and contamination by agrochemicals (Aprile & Bouvy, 2008).</p><p>The Pernambuco State Wild Animal Rehabilitation and</p><p>Screening Center (CETAS) receives wild fauna resulting from in-</p><p>spections, rescues, or voluntary deliveries. The organization</p><p>identifies, evaluates the health of, rehabilitates, and reintroduces</p><p>animals to proper habitats. It is common for C. latirostris to arrive</p><p>at CETAS from rescue operations in urban areas within the state</p><p>of Pernambuco, especially during the rainy season, from May to</p><p>August (Mascarenhas‐Junior et al., 2021). Rescued animals were</p><p>evaluated for their health, and blood samples were taken</p><p>through a partnership between CETAS and the Laboratório In-</p><p>terdisciplinar de Anfíbios e Répteis. Caimans were rescued</p><p>from urbanized areas (UA) within the cities shown in Figure 1.</p><p>Camaragibe, Recife, São Lourenço da Mata, and Vitória de Santo</p><p>Antão are located along the Capibaribe River Basin. Degradation</p><p>caused by human development and sugarcane agricultural ac-</p><p>tivities is clearly evident along the Capibaribe River, where</p><p>farming and urbanization caused a loss of diversity in fish fauna</p><p>(Collier et al., 2019) and increased toxicity to fish (Alves</p><p>et al., 2021).</p><p>The company AME Brasil breeds caimans in a farming</p><p>system, a method by which adult animals are kept in captivity</p><p>to reproduce, eggs are incubated, and young are raised until</p><p>adulthood. The farm works exclusively with C. latirostris for the</p><p>wholesale trade of hides and other inedible byproducts of</p><p>animal origin; it has been on the market since 2015 in Marechal</p><p>Deodoro City, Alagoas State, with a current herd of approx-</p><p>imately 1600 animals (248 breeding reproducers and 1352</p><p>animals from 1 to 5 years old). The water used for the enclo-</p><p>sures, greenhouses, and nurseries comes from the artesian well</p><p>located on the property, authorized and inspected by the</p><p>Alagoas State Environmental and Water Resources Agency.</p><p>Caimans are fed chickens slaughtered from farms of the</p><p>Salamba Company located near AME.</p><p>Animal sampling procedures</p><p>The location and visual identification of caimans was carried</p><p>out with the aid of a motorboat, using spotlight headlamps at</p><p>TR. Animals from urbanized areas were typically visually located</p><p>during the day, and animals from the AME Brasil farm were</p><p>sampled within their enclosures. Caiman were captured using a</p><p>steel cable loop fixed to a telescopic pole with a range of 4m.</p><p>Physical restraint was performed with ropes and adhesive</p><p>tapes, immobilizing the mandibles and the fore and hind legs</p><p>(Bayliss, 1987). Sex was determined by cloacal examination of</p><p>genitalia (Webb et al., 1984). The size of the animals was</p><p>measured with the aid of a tape measure, caliper, and weight</p><p>scales from 1 to 100 kg. Caiman age was estimated based on</p><p>the measurement of the snout–vent length (SVL) proposed by</p><p>Leiva et al. (2019). From each animal, a blood sample was</p><p>collected from the postoccipital sinus using a sterile needle and</p><p>syringe (Myburgh et al., 2014).</p><p>The samples were carefully homogenized by inversion in</p><p>VACUETTE® tubes containing the anticoagulant heparin.</p><p>Whole blood samples were preserved for metal analysis, and a</p><p>1‐mL aliquot was centrifuged at 3600 rpm for 15min to obtain</p><p>blood plasma. Samples were immediately frozen in a –20 °C</p><p>freezer and transported to a –80 °C freezer within 1 day, where</p><p>they remained stored until analysis.</p><p>Analyses of metals</p><p>Whole blood samples were divided into two replicates of</p><p>0.5mL, and wet digestion was performed in two steps: in Step</p><p>1, 2mL of 65% nitric acid (ultrapure HNO3) was added in a</p><p>microwave oven (CEM MARS Xpress) at 400W and 75% power,</p><p>with a heating ramp of 10min until reaching a temperature of</p><p>105 °C, which was maintained for 20min. Step 2 took place</p><p>after the samples from the microwave were removed for</p><p>cooling; 1mL of 35% hydrogen peroxide (H2O2) was added,</p><p>and the samples were again subjected to a 5‐min ramp until</p><p>reaching 105 °C, where they remained for 10min. After</p><p>cooling, the samples were transferred to 15‐mL Falcon tubes</p><p>and centrifuged at a speed of 3000 rpm for 15min, adapting</p><p>the methodology proposed by Loh et al. (2016). Finally, the</p><p>samples were filtered with 2‐µm filter paper to remove any</p><p>suspended solids and diluted with ultrapure deionized water to</p><p>a final volume of 10mL.</p><p>Two replicate samples were analyzed for Pb, Cd, Cu, Cr,</p><p>Ni, Fe, Al, and Mn in an inductively coupled plasma–optical</p><p>emission spectrometer (ICP–OES; model 5100; Agilent</p><p>Technologies) with the axial view. Specifications of the in-</p><p>strumental parameters used in the determination of the ele-</p><p>ments were: radiofrequency power (1.2 kW), nebulization flow</p><p>(0.7 L min–1), auxiliary flow (1 L min–1), plasma flow (12 L</p><p>min–1), and sample capture time (10 s). The following emis-</p><p>sion lines (nm) in the axial plasma view were monitored: Al</p><p>Metal and biochemical biomarkers in Caiman latirostris—Environmental Toxicology and Chemistry, 2024;00:1–18 3</p><p>wileyonlinelibrary.com/ETC © 2024 SETAC</p><p>15528618, 0, D</p><p>ow</p><p>nloaded from</p><p>https://setac.onlinelibrary.w</p><p>iley.com</p><p>/doi/10.1002/etc.5823 by Florida International U</p><p>niversity, W</p><p>iley O</p><p>nline L</p><p>ibrary on [27/02/2024]. See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons L</p><p>icense</p><p>FIGURE 1: Sampling areas for the populations of Caiman latirostris from the rural area of the Tapacurá Reservoir (TR; São Lourenço da Mata,</p><p>Pernambuco State) from different urbanized areas of Pernambuco (UA), and from the AME Brasil caiman farm (AF; Marechal Deodoro, Alagoas</p><p>State, 35W51ʹ01ʹʹ, 9S46ʹ02ʹʹ).</p><p>4 Environmental Toxicology and Chemistry, 2024;00:1–18—dos Santos et al.</p><p>© 2024 SETAC wileyonlinelibrary.com/ETC</p><p>15528618, 0, D</p><p>ow</p><p>nloaded from</p><p>https://setac.onlinelibrary.w</p><p>iley.com</p><p>/doi/10.1002/etc.5823 by Florida International U</p><p>niversity, W</p><p>iley O</p><p>nline L</p><p>ibrary on [27/02/2024]. See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons L</p><p>icense</p><p>396.152, Cd 226.502, Cr 267.716, Cu 327.395, Fe 259.940,</p><p>Mn 259.372, Ni 231.604, Pb 220.353, and Zn 213.857. Ana-</p><p>lytical curves were prepared using standard multi‐element</p><p>solutions (Merck Certipur®) diluted in 65% v/v ultrapure</p><p>HNO3 to avoid adsorption or precipitation of the analytes in</p><p>the containers. Curves were prepared at concentrations of 0,</p><p>0.1, 0.5, 1, 2, and 3 µgmL–1 for further analysis and calibration</p><p>of the ICP–OES.</p><p>Limits of detection (LOD) and quantification (LOQ) for each</p><p>element were determined according to the Brasilian National</p><p>Institute of Metrology, Standardization, and Industrial Quality</p><p>(2020) equations:</p><p>= /s bLOD 3.3</p><p>where s is the standard deviation of 10 blank samples, and</p><p>b is the slope of the analytical curve.</p><p>= /s bLOQ 10</p><p>where s is the standard deviation of the blank response, and</p><p>b is the slope of the analytical curve.</p><p>The LOD and LOQ for each element were determined as</p><p>(µg mL–1) as follows: Al (0.04, 0.11), Cd (0.0001, 0.001), Cr</p><p>(0.0002, 0.0007), Cu (0.001, 0.004), Fe (0.005, 0.14), Mn (0.001,</p><p>0.003), Ni (0.005, 0.015), Pb (0.008, 0.024), and Zn (0.02, 0.07).</p><p>After ICP–OES analysis, the concentration of metals in the</p><p>samples were expressed in µg mL–1 based on the analytical</p><p>curves, and transformed to µg mL blood–1, considering the</p><p>total mass of each metal present in the analyzed solution after</p><p>digestion, and the initial volume of digested blood equal to</p><p>0.5mL. Analytical curves and respective coefficients of</p><p>determination were all above 0.99, and are provided in the</p><p>Supporting Information.</p><p>Ratios between the blood concentrations of each metal</p><p>quantified in animals from TR or UA relative to AF (defined as</p><p>reference animals) were calculated to analyze rates of blood</p><p>concentrations increase (ratio >1) or decrease (ratio 0.05 for the</p><p>factors sex, age, and interactions). Blood Mn and Fe were</p><p>significantly higher at UA than at TR and AF, for both sexes</p><p>(Figure 2D and E).</p><p>Significant differences were found for the age class factor for</p><p>the metals Al (two‐way ANOVA, pAl>Cu>Zn>Mn></p><p>Cr>Pb), UA (Fe>Al>Cu>Zn>Mn>Pb>Ni>Cr),</p><p>and AF (Fe>Al></p><p>Cu>Zn>Mn>Cr>Pb).</p><p>The highest ratios between the blood concentrations of the</p><p>metals Fe, Al, Cu, Zn, Mn, Pb, and Cr quantified in animals from</p><p>TR or UA relative to AF were 18, 10, 4, 11, 8, 83, and 4, re-</p><p>spectively (Table 1). Cadmium was not detected in any of the</p><p>analyzed samples (Table 1).</p><p>Biochemical biomarkers in C. latirostris</p><p>Butyrylcholinesterase. Butyrylcholinesterase activity in</p><p>plasma of adult animals from UA and TR and subadults from TR</p><p>was lower compared with animals from AF (two‐way ANOVA,</p><p>p 0.05 for the factors location and</p><p>sex; Figure 4B).</p><p>Glutathione S‐transferase. Glutathione S‐transferase activity</p><p>of males and females from TR was higher compared with ani-</p><p>mals from AF (two‐way ANOVA, p 0.05 for the factors location and sex; Figure 7B).</p><p>Correlation analysis</p><p>Significant inverse correlations were observed between Cu</p><p>and SOD (r= –0.32) and Mn and SOD (r= –0.32). A significant</p><p>positive correlation (r= 0.73) was verified between GST and</p><p>SOD. Significant positive correlations were observed between</p><p>the essential metals Al, Cr, Cu, Fe, Mn, and Zn, but non-</p><p>essential Pb did not correlate with any other metal (Table 2).</p><p>DISCUSSION</p><p>Concentrations of the essential metals Fe, Cu, Zn, and Mn,</p><p>and the nonessentials Cr, Al, and Pb in blood of caimans from</p><p>FIGURE 3: (A–F) Blood metal concentrations (mean and standard error) of Al, Cr, Cu, Fe, Mn, and Zn in Caiman latirostris sampled from the rural</p><p>area of Tapacurá Reservoir (TR), from different urbanized areas of Pernambuco (UA), and from AME Brasil caiman farm (AF), as a function of the</p><p>factors location and age. Different letters (a, b, and c) indicate statistically significant differences between sampling locations (two‐way analysis of</p><p>variance, pLOD (%) Mean SD Ratio to AFa No. >LOD (%) Mean SD No. >LOD (%)</p><p>Fe 879.42 782.93 9.8 18 94 1678.11 665.34 18.7 33 100 89.90 91.79 35 100</p><p>Al 18.04 18.64 8.2 18 94 22.90 5.41 10.4 33 100 2.20 0.97 35 100</p><p>Cu 5.36 2.69 3.8 18 28 6.34 3.02 4.5 33 100 1.41 4.73 35 29</p><p>Zn 1.45 0.79 9.7 18 11 1.69 0.66 11.3 33 100 0.15 0.09 35 100</p><p>Mn 0.47 0.33 4.7 18 33 0.85 0.62 8.5 33 6 0.10 0.11 35 37</p><p>Pb 0.16 0.02 16.0 18 11 0.83 1.29 83.0 33 70 0.01 0.00 35 6</p><p>Ni — — — 18 — 0.43 0.05 — 33 9 — — 35 —</p><p>Cr 0.19 0.10 3.8 18 44 0.22 0.15 4.4 33 12 0.05 0.10 35 3</p><p>Cd — — — 18 0 — — 33 0 — — 35 0</p><p>aMean concentration in TR or UA divided by mean concentration in AF.</p><p>LOD = limit of detection; No. = number of samples analyzed.</p><p>Metal and biochemical biomarkers in Caiman latirostris—Environmental Toxicology and Chemistry, 2024;00:1–18 7</p><p>wileyonlinelibrary.com/ETC © 2024 SETAC</p><p>15528618, 0, D</p><p>ow</p><p>nloaded from</p><p>https://setac.onlinelibrary.w</p><p>iley.com</p><p>/doi/10.1002/etc.5823 by Florida International U</p><p>niversity, W</p><p>iley O</p><p>nline L</p><p>ibrary on [27/02/2024]. See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons L</p><p>icense</p><p>AF were significantly lower than concentrations in free‐living</p><p>animals from TR and UA, confirming the potential use of these</p><p>farmed animals as a reasonable basal reference for exposure to</p><p>these contaminants. Significant contamination of aquatic sedi-</p><p>ments by the metals Fe, Cu, Zn, Mn, Cr, and Pb was verified at</p><p>a site close to the area where animals were captured at TR,</p><p>corroborating the presence of potential sources of these ele-</p><p>ments to the reservoir (Aprile & Bouvy, 2008).</p><p>Among analyzed metals, Fe reached the highest blood</p><p>concentrations, probably due to the large concentration of Fe‐</p><p>rich hemoglobin in blood. Mean blood Fe levels in four</p><p>crocodylian species from French Guiana ranged from 285 to</p><p>424 μgmL–1, and this small variation would suggest that Fe</p><p>regulation would be similar between species, because it is</p><p>important for hemoglobin function (Lemaire et al., 2022).</p><p>However, the results from our study indicate a wider range of</p><p>blood Fe levels, from a lower mean of 89 μgmL–1 in caimans</p><p>from AF and reaching 879 and 1678 μgmL–1 in animals from TR</p><p>and UA, respectively (Table 3). Animals from UA displayed</p><p>blood Fe approximately four to five times higher than the range</p><p>of concentrations reported for other species previously studied</p><p>(Humphries et al., 2022; Lemaire et al., 2022). Excess iron</p><p>FIGURE 4: Butyrylcholinesterase (BChE) activity in plasma of Caiman latirostris sampled from the rural area of Tapacurá Reservoir (TR), from</p><p>different urbanized areas of Pernambuco (UA), and from AME Brasil caiman farm (AF), as a function of age (adults and subadults; A) and sex (males</p><p>and females; B). Different letters (a, b, and c) indicate statistically significant differences between locations (Tukey's test, p</p><p>significant differences between locations (Tukey's test, p</p><p>0.000 0.306 0.000 0.638 0.357 0.415 0.569</p><p>Cu 0.65 0.60 0.05 0.68 −0.21 0.13 −0.11 −0.36</p><p>0.000 0.000 0.863 0.000 0.133 0.370 0.448 0.016</p><p>Fe 0.90 0.02 0.84 0.11 −0.02 −0.03 −0.10</p><p>0.000 0.949 0.000 0.369 0.891 0.832 0.445</p><p>Mn 0.30 0.82 −0.09 −0.13 −0.05 −0.32</p><p>0.329 0.000 0.540 0.370 0.721 0.036</p><p>Pb 0.21 −0.25 0.43 −0.40 −0.57</p><p>0.469 0.491 0.260 0.264 0.120</p><p>Zn −0.08 0.05 0.07 −0.25</p><p>0.519 0.665 0.578 0.050</p><p>BChE −0.10 −0.06 0.04</p><p>0.411 0.597 0.742</p><p>GSH −0.19 −0.19</p><p>0.113 0.139</p><p>GST 0.73</p><p>0.000</p><p>Numbers in bold indicate significant correlation coefficients and associated p values.</p><p>BChE = butyrilcholinesterase; GSH = total glutathione; GST = glutathione S‐transferase; SOD = superoxide dismutase.</p><p>10 Environmental Toxicology and Chemistry, 2024;00:1–18—dos Santos et al.</p><p>© 2024 SETAC wileyonlinelibrary.com/ETC</p><p>15528618, 0, D</p><p>ow</p><p>nloaded from</p><p>https://setac.onlinelibrary.w</p><p>iley.com</p><p>/doi/10.1002/etc.5823 by Florida International U</p><p>niversity, W</p><p>iley O</p><p>nline L</p><p>ibrary on [27/02/2024]. See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons 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><p>ns</p><p>is</p><p>0.</p><p>37</p><p>–1</p><p>.2</p><p>2</p><p>0.</p><p>78</p><p>‐</p><p>Fl</p><p>or</p><p>id</p><p>a,</p><p>U</p><p>SA</p><p>N</p><p>ils</p><p>en</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>01</p><p>7)</p><p>A</p><p>.</p><p>m</p><p>is</p><p>si</p><p>ss</p><p>ip</p><p>ie</p><p>ns</p><p>is</p><p>0.</p><p>28</p><p>6–</p><p>1.</p><p>22</p><p>5</p><p>0.</p><p>82</p><p>0.</p><p>23</p><p>B</p><p>ea</p><p>r</p><p>Is</p><p>la</p><p>nd</p><p>,</p><p>SC</p><p>,</p><p>U</p><p>SA</p><p>B</p><p>as</p><p>al</p><p>re</p><p>fe</p><p>re</p><p>nc</p><p>e</p><p>N</p><p>ils</p><p>en</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>01</p><p>9)</p><p>A</p><p>.</p><p>m</p><p>is</p><p>si</p><p>ss</p><p>ip</p><p>ie</p><p>ns</p><p>is</p><p>0.</p><p>86</p><p>2–</p><p>1.</p><p>50</p><p>1</p><p>1.</p><p>20</p><p>0.</p><p>21</p><p>K</p><p>is</p><p>si</p><p>m</p><p>m</p><p>ee</p><p>,</p><p>FL</p><p>,</p><p>U</p><p>SA</p><p>N</p><p>ils</p><p>en</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>01</p><p>9)</p><p>C</p><p>.</p><p>la</p><p>tir</p><p>os</p><p>tr</p><p>is</p><p>0.</p><p>30</p><p>–2</p><p>.6</p><p>2</p><p>1.</p><p>45</p><p>0.</p><p>79</p><p>TR</p><p>Pr</p><p>es</p><p>en</p><p>t</p><p>st</p><p>ud</p><p>y</p><p>C</p><p>.</p><p>la</p><p>tir</p><p>os</p><p>tr</p><p>is</p><p>0.</p><p>94</p><p>–3</p><p>.6</p><p>2</p><p>1.</p><p>69</p><p>0.</p><p>66</p><p>U</p><p>A</p><p>Pr</p><p>es</p><p>en</p><p>t</p><p>st</p><p>ud</p><p>y</p><p>M</p><p>.</p><p>ni</p><p>g</p><p>er</p><p>4.</p><p>87</p><p>–8</p><p>.0</p><p>4</p><p>6.</p><p>27</p><p>0.</p><p>99</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>C</p><p>.</p><p>cr</p><p>oc</p><p>od</p><p>ilu</p><p>s</p><p>5.</p><p>16</p><p>–1</p><p>4.</p><p>25</p><p>8.</p><p>54</p><p>2.</p><p>37</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>P.</p><p>p</p><p>al</p><p>p</p><p>eb</p><p>ro</p><p>su</p><p>s</p><p>8.</p><p>20</p><p>–1</p><p>9.</p><p>15</p><p>11</p><p>.8</p><p>6</p><p>4.</p><p>41</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>P.</p><p>tr</p><p>ig</p><p>on</p><p>at</p><p>us</p><p>7.</p><p>10</p><p>–4</p><p>9.</p><p>86</p><p>18</p><p>.6</p><p>7</p><p>14</p><p>.5</p><p>3</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>M</p><p>n</p><p>M</p><p>.</p><p>ni</p><p>g</p><p>er</p><p>0.</p><p>01</p><p>–0</p><p>.0</p><p>8</p><p>0.</p><p>03</p><p>0.</p><p>02</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>P.</p><p>p</p><p>al</p><p>p</p><p>eb</p><p>ro</p><p>su</p><p>s</p><p>0.</p><p>01</p><p>–0</p><p>.0</p><p>6</p><p>0.</p><p>04</p><p>0.</p><p>02</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>C</p><p>.</p><p>cr</p><p>oc</p><p>od</p><p>ilu</p><p>s</p><p>0.</p><p>03</p><p>–0</p><p>.1</p><p>4</p><p>0.</p><p>06</p><p>0.</p><p>03</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>P.</p><p>tr</p><p>ig</p><p>on</p><p>at</p><p>us</p><p>0.</p><p>02</p><p>–0</p><p>.3</p><p>5</p><p>0.</p><p>09</p><p>0.</p><p>07</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>C</p><p>.</p><p>la</p><p>tir</p><p>os</p><p>tr</p><p>is</p><p>0.</p><p>00</p><p>–0</p><p>.4</p><p>4</p><p>0.</p><p>10</p><p>0.</p><p>11</p><p>A</p><p>F</p><p>Pr</p><p>es</p><p>en</p><p>t</p><p>st</p><p>ud</p><p>y</p><p>(C</p><p>on</p><p>tin</p><p>ue</p><p>d</p><p>)</p><p>Metal and biochemical biomarkers in Caiman latirostris—Environmental Toxicology and Chemistry, 2024;00:1–18 11</p><p>wileyonlinelibrary.com/ETC © 2024 SETAC</p><p>15528618, 0, D</p><p>ow</p><p>nloaded from</p><p>https://setac.onlinelibrary.w</p><p>iley.com</p><p>/doi/10.1002/etc.5823 by Florida International U</p><p>niversity, W</p><p>iley O</p><p>nline L</p><p>ibrary on [27/02/2024]. See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons L</p><p>icense</p><p>toxicity in animals including neurological, reproductive, and</p><p>developmental effects. Normal Mn blood levels range from</p><p>0.02 µgmL–1 in dogs to 0.09 µgmL–1 in cattle, but several</p><p>mechanisms of Mn toxic action are not properly understood,</p><p>and blood Mn concentrations do not correlate with neuro-</p><p>toxicity symptoms (Milatovic & Gupta, 2018). Blood Mn in an-</p><p>imals from TR and UA exceeded these normal Mn ranges,</p><p>similar to the results noted for blood Cu, and higher Cu and Mn</p><p>blood concentrations were inversely correlated with decreases</p><p>in SOD activity (Table 2). Similarly, a decrease in kidney SOD</p><p>activity associated with the presence of metals (As, Cd, Cu, Ni,</p><p>Pb, Se, and Zn) was observed in sea turtles (Lepidochelys oli-</p><p>vacea; Cortés‐Gómez, Morcillo, et al., 2018).</p><p>Aluminum is a nonessential metal, and its toxic effects in</p><p>rodents are low compared with Cu, Zn, and Hg (Scheu-</p><p>hammer, 1987), although effects on reptiles are not well</p><p>documented (Nilsen et al., 2017). Nilsen et al. (2019) detected</p><p>blood Al averages between 0.03 and 0.04 µgmL–1 in A. mis-</p><p>sissippiensis in Florida, concentrations almost 400 times lower</p><p>than those found in our study for C. latirostris from TR</p><p>(18 µgmL–1) and UA (22.9 µgmL–1), and still 40 times lower</p><p>compared with animals from our reference area AF</p><p>(2.2 µgmL–1). Aluminum was detected</p><p>in serum of Geoffroy's</p><p>freshwater turtle from the Capibaribe River, at concentrations</p><p>ranging from 1.2 to 2.1 µgmL–1 (Da Fonseca et al., 2023),</p><p>within the range we found in caimans from AF. In humans,</p><p>blood Al concentrations of 0.46 and 0.16 µgmL–1 were found in</p><p>patients with symptoms of severe bone disease and neuro-</p><p>toxicity, respectively (Coulson & Hughes, 2022). The toxic ef-</p><p>fects of Al are the result of its pro‐oxidant activity, which results</p><p>in oxidative stress through the attack of free radicals on cellular</p><p>proteins and lipids (Exley, 2013). There is no information</p><p>available concerning the sensitivity of reptiles to Al accumu-</p><p>lation in blood, and the concentrations observed in our study</p><p>suggest that significant Al toxicity could be occurring in C.</p><p>latirostris, especially those from TR and UA.</p><p>Lead is a nonessential metal capable of altering and mod-</p><p>ifying physiological and biochemical systems (Pain et al., 2019).</p><p>Lead accumulation in blood of crocodilians has been the subject</p><p>of several studies. Lead exposure and accumulation was first</p><p>associated with ingestion of Pb fishing weights in a wild pop-</p><p>ulation of C. niloticus at Lake St Lucia, South Africa, and high</p><p>blood Pb levels with a mean of 0.98 µgmL–1 were observed in</p><p>males (Table 3), including one individual with the highest con-</p><p>centration of 9.6 µgmL–1. However, no evident gross signs of</p><p>(Warner et al., 2016). In a subsequent study in Lake St Lucia,</p><p>Humphries et al. (2022) detected blood Pb concentrations</p><p>ranging from 0.08 to 13.1 µgmL–1, a mean of 3.7 µgmL–1 for</p><p>males (Table 3), and found higher concentrations above</p><p>6 µgmL–1 associated with anemia and tooth loss, deemed as</p><p>possible clinical signs of long‐term environmental exposure to</p><p>Pb. Camus et al. (1998) detected high blood Pb with a mean of</p><p>0.80 µgmL–1 in A. mississippiensis fed with prey meat con-</p><p>taminated by lead bullets, and concentrations higher than</p><p>0.5 µgmL–1 were considered diagnostic of lead poisoning. In the</p><p>present study, mean blood Pb varied from 0.01 µgmL–1 in ani-</p><p>mals from AF to 0.16 and 0.83 µgmL–1 in animals from TR andTA</p><p>B</p><p>LE</p><p>3:</p><p>(C</p><p>on</p><p>tin</p><p>ue</p><p>d</p><p>)</p><p>El</p><p>em</p><p>en</p><p>t</p><p>Sp</p><p>ec</p><p>ie</p><p>s</p><p>Ra</p><p>ng</p><p>e</p><p>(m</p><p>in</p><p>–m</p><p>ax</p><p>)</p><p>M</p><p>ea</p><p>n</p><p>SD</p><p>Lo</p><p>ca</p><p>lit</p><p>y</p><p>Re</p><p>fe</p><p>re</p><p>nc</p><p>ea</p><p>Re</p><p>fe</p><p>re</p><p>nc</p><p>es</p><p>C</p><p>.</p><p>la</p><p>tir</p><p>os</p><p>tr</p><p>is</p><p>0.</p><p>00</p><p>–1</p><p>.0</p><p>5</p><p>0.</p><p>47</p><p>0.</p><p>33</p><p>TR</p><p>Pr</p><p>es</p><p>en</p><p>t</p><p>st</p><p>ud</p><p>y</p><p>C</p><p>.</p><p>la</p><p>tir</p><p>os</p><p>tr</p><p>is</p><p>0.</p><p>09</p><p>–2</p><p>.2</p><p>2</p><p>0.</p><p>85</p><p>0.</p><p>62</p><p>U</p><p>A</p><p>Pr</p><p>es</p><p>en</p><p>t</p><p>st</p><p>ud</p><p>y</p><p>Pb</p><p>C</p><p>.</p><p>la</p><p>tir</p><p>os</p><p>tr</p><p>is</p><p>—</p><p>0.</p><p>01</p><p>0</p><p>0.</p><p>00</p><p>4</p><p>A</p><p>F</p><p>Pr</p><p>es</p><p>en</p><p>t</p><p>st</p><p>ud</p><p>y</p><p>M</p><p>.</p><p>ni</p><p>g</p><p>er</p><p>0.</p><p>00</p><p>–0</p><p>.1</p><p>1</p><p>0.</p><p>01</p><p>5</p><p>0.</p><p>02</p><p>3</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>C</p><p>.</p><p>cr</p><p>oc</p><p>od</p><p>ilu</p><p>s</p><p>0.</p><p>00</p><p>–0</p><p>.1</p><p>0</p><p>0.</p><p>03</p><p>3</p><p>0.</p><p>02</p><p>2</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>A</p><p>.</p><p>m</p><p>is</p><p>si</p><p>ss</p><p>ip</p><p>ie</p><p>ns</p><p>is</p><p>0.</p><p>03</p><p>–0</p><p>.0</p><p>4</p><p>0.</p><p>03</p><p>5.</p><p>77</p><p>Lo</p><p>ui</p><p>si</p><p>an</p><p>a</p><p>al</p><p>lig</p><p>at</p><p>or</p><p>fa</p><p>rm</p><p>,</p><p>U</p><p>SA</p><p>B</p><p>as</p><p>al</p><p>re</p><p>fe</p><p>re</p><p>nc</p><p>e</p><p>C</p><p>am</p><p>us</p><p>et</p><p>al</p><p>.</p><p>(1</p><p>99</p><p>8)</p><p>A</p><p>.</p><p>m</p><p>is</p><p>si</p><p>ss</p><p>ip</p><p>ie</p><p>ns</p><p>is</p><p>0.</p><p>03</p><p>–4</p><p>.4</p><p>7</p><p>0.</p><p>12</p><p>7</p><p>–</p><p>Fl</p><p>or</p><p>id</p><p>a,</p><p>U</p><p>SA</p><p>N</p><p>ils</p><p>en</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>01</p><p>7)</p><p>A</p><p>.</p><p>m</p><p>is</p><p>si</p><p>ss</p><p>ip</p><p>ie</p><p>ns</p><p>is</p><p>0.</p><p>01</p><p>2–</p><p>0.</p><p>26</p><p>6</p><p>0.</p><p>07</p><p>6</p><p>0.</p><p>98</p><p>3</p><p>B</p><p>ea</p><p>r</p><p>Is</p><p>la</p><p>nd</p><p>,</p><p>SC</p><p>,</p><p>U</p><p>SA</p><p>B</p><p>as</p><p>al</p><p>re</p><p>fe</p><p>re</p><p>nc</p><p>e</p><p>N</p><p>ils</p><p>en</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>01</p><p>9)</p><p>A</p><p>.</p><p>m</p><p>is</p><p>si</p><p>ss</p><p>ip</p><p>ie</p><p>ns</p><p>is</p><p>0.</p><p>00</p><p>5–</p><p>0.</p><p>58</p><p>7</p><p>0.</p><p>08</p><p>0</p><p>0.</p><p>16</p><p>8</p><p>K</p><p>is</p><p>si</p><p>m</p><p>m</p><p>ee</p><p>,</p><p>FL</p><p>,</p><p>U</p><p>SA</p><p>N</p><p>ils</p><p>en</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>01</p><p>9)</p><p>C</p><p>.</p><p>la</p><p>tir</p><p>os</p><p>tr</p><p>is</p><p>0.</p><p>14</p><p>–0</p><p>.1</p><p>8</p><p>0.</p><p>16</p><p>0</p><p>0.</p><p>02</p><p>0</p><p>TR</p><p>Pr</p><p>es</p><p>en</p><p>t</p><p>st</p><p>ud</p><p>y</p><p>P.</p><p>tr</p><p>ig</p><p>on</p><p>at</p><p>us</p><p>0.</p><p>02</p><p>–0</p><p>.7</p><p>3</p><p>0.</p><p>15</p><p>7</p><p>0.</p><p>20</p><p>9</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>C</p><p>.</p><p>ni</p><p>lo</p><p>tic</p><p>us</p><p>0.</p><p>22</p><p>7–</p><p>0.</p><p>25</p><p>7</p><p>0.</p><p>24</p><p>6</p><p>0.</p><p>01</p><p>7</p><p>So</p><p>ut</p><p>h</p><p>A</p><p>fr</p><p>ic</p><p>a</p><p>B</p><p>as</p><p>al</p><p>re</p><p>fe</p><p>re</p><p>nc</p><p>e,</p><p>ca</p><p>p</p><p>tiv</p><p>e</p><p>H</p><p>um</p><p>p</p><p>hr</p><p>ie</p><p>s</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>C</p><p>.</p><p>ni</p><p>lo</p><p>tic</p><p>us</p><p>0.</p><p>08</p><p>6–</p><p>0.</p><p>74</p><p>2</p><p>0.</p><p>26</p><p>6</p><p>0.</p><p>23</p><p>0</p><p>So</p><p>ut</p><p>h</p><p>A</p><p>fr</p><p>ic</p><p>a</p><p>Fe</p><p>m</p><p>al</p><p>es</p><p>H</p><p>um</p><p>p</p><p>hr</p><p>ie</p><p>s</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>P.</p><p>p</p><p>al</p><p>p</p><p>eb</p><p>ro</p><p>su</p><p>s</p><p>0.</p><p>01</p><p>–0</p><p>.7</p><p>7</p><p>0.</p><p>28</p><p>5</p><p>0.</p><p>30</p><p>2</p><p>Fr</p><p>en</p><p>ch</p><p>G</p><p>ui</p><p>an</p><p>a</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>b</p><p>A</p><p>.</p><p>m</p><p>is</p><p>si</p><p>ss</p><p>ip</p><p>ie</p><p>ns</p><p>is</p><p>0.</p><p>43</p><p>–2</p><p>.8</p><p>0</p><p>0.</p><p>80</p><p>2</p><p>0.</p><p>77</p><p>0</p><p>Lo</p><p>ui</p><p>si</p><p>an</p><p>a</p><p>al</p><p>lig</p><p>at</p><p>or</p><p>fa</p><p>rm</p><p>,</p><p>U</p><p>SA</p><p>H</p><p>ig</p><p>h</p><p>d</p><p>os</p><p>e</p><p>C</p><p>am</p><p>us</p><p>et</p><p>al</p><p>.</p><p>(1</p><p>99</p><p>8)</p><p>C</p><p>.</p><p>la</p><p>tir</p><p>os</p><p>tr</p><p>is</p><p>0.</p><p>07</p><p>–4</p><p>.3</p><p>4</p><p>0.</p><p>83</p><p>0</p><p>1.</p><p>29</p><p>0</p><p>U</p><p>A</p><p>Pr</p><p>es</p><p>en</p><p>t</p><p>st</p><p>ud</p><p>y</p><p>C</p><p>.</p><p>ni</p><p>lo</p><p>tic</p><p>us</p><p>0.</p><p>03</p><p>–9</p><p>.6</p><p>0.</p><p>98</p><p>1</p><p>2.</p><p>17</p><p>0</p><p>So</p><p>ut</p><p>h</p><p>A</p><p>fr</p><p>ic</p><p>a</p><p>H</p><p>ig</p><p>h</p><p>d</p><p>os</p><p>e</p><p>in</p><p>m</p><p>al</p><p>es</p><p>W</p><p>ar</p><p>ne</p><p>r</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>01</p><p>6)</p><p>C</p><p>.</p><p>ni</p><p>lo</p><p>tic</p><p>us</p><p>0.</p><p>08</p><p>–1</p><p>3.</p><p>1</p><p>3.</p><p>78</p><p>0</p><p>4.</p><p>69</p><p>0</p><p>So</p><p>ut</p><p>h</p><p>A</p><p>fr</p><p>ic</p><p>a</p><p>H</p><p>ig</p><p>h</p><p>d</p><p>os</p><p>e</p><p>in</p><p>m</p><p>al</p><p>es</p><p>H</p><p>um</p><p>p</p><p>hr</p><p>ie</p><p>s</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>a B</p><p>as</p><p>al</p><p>re</p><p>fe</p><p>re</p><p>nc</p><p>e</p><p>d</p><p>os</p><p>es</p><p>m</p><p>ea</p><p>su</p><p>re</p><p>d</p><p>in</p><p>b</p><p>lo</p><p>od</p><p>fr</p><p>om</p><p>an</p><p>im</p><p>al</p><p>s</p><p>in</p><p>le</p><p>ss</p><p>co</p><p>nt</p><p>am</p><p>in</p><p>at</p><p>ed</p><p>ar</p><p>ea</p><p>s,</p><p>an</p><p>d</p><p>“</p><p>hi</p><p>g</p><p>h</p><p>d</p><p>os</p><p>es</p><p>”</p><p>or</p><p>p</p><p>os</p><p>iti</p><p>ve</p><p>re</p><p>fe</p><p>re</p><p>nc</p><p>e</p><p>va</p><p>lu</p><p>es</p><p>m</p><p>ea</p><p>su</p><p>re</p><p>d</p><p>in</p><p>st</p><p>ud</p><p>ie</p><p>d</p><p>an</p><p>im</p><p>al</p><p>s.</p><p>b</p><p>D</p><p>at</p><p>a</p><p>w</p><p>er</p><p>e</p><p>tr</p><p>an</p><p>sf</p><p>or</p><p>m</p><p>ed</p><p>to</p><p>w</p><p>et</p><p>w</p><p>ei</p><p>g</p><p>ht</p><p>of</p><p>b</p><p>lo</p><p>od</p><p>b</p><p>as</p><p>ed</p><p>on</p><p>b</p><p>lo</p><p>od</p><p>w</p><p>at</p><p>er</p><p>co</p><p>nt</p><p>en</p><p>t</p><p>fo</p><p>r</p><p>th</p><p>e</p><p>sp</p><p>ec</p><p>ie</p><p>s</p><p>fr</p><p>om</p><p>Le</p><p>m</p><p>ai</p><p>re</p><p>et</p><p>al</p><p>.</p><p>(2</p><p>02</p><p>2)</p><p>.</p><p>Tr</p><p>an</p><p>sf</p><p>or</p><p>m</p><p>at</p><p>io</p><p>ns</p><p>as</p><p>su</p><p>m</p><p>ed</p><p>a</p><p>b</p><p>lo</p><p>od</p><p>d</p><p>en</p><p>si</p><p>ty</p><p>of</p><p>1</p><p>(1</p><p>g</p><p>w</p><p>et</p><p>b</p><p>lo</p><p>od</p><p>=</p><p>1</p><p>m</p><p>L</p><p>w</p><p>et</p><p>b</p><p>lo</p><p>od</p><p>).</p><p>12 Environmental Toxicology and Chemistry, 2024;00:1–18—dos Santos et al.</p><p>© 2024 SETAC wileyonlinelibrary.com/ETC</p><p>15528618, 0, D</p><p>ow</p><p>nloaded from</p><p>https://setac.onlinelibrary.w</p><p>iley.com</p><p>/doi/10.1002/etc.5823 by Florida International U</p><p>niversity, W</p><p>iley O</p><p>nline L</p><p>ibrary on [27/02/2024]. See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons L</p><p>icense</p><p>UA, respectively, 18 and 83 times above concentrations from</p><p>the local reference site AF (Table 1). Plasma Pb in turtles</p><p>P. geoffroanus sampled from the Capibaribe River, within similar</p><p>locations included in our UA, ranged from 0.73 to 0.81 µgmL–1,</p><p>similar to the mean concentration detected in our study at UA</p><p>(Da Fonseca et al., 2023). High concentrations of Pb in serum of</p><p>1.15 µgmL–1 were also detected in P. geoffroanus from the Pi-</p><p>racicaba River (São Paulo, Brasil), and possible sources include</p><p>illegal industrial sewage (Piña et al., 2009). A case of Pb poi-</p><p>soning in the snapping turtle Chelydra serpentina after ingestion</p><p>of fishing lead sinker was reported; the blood Pb had reached</p><p>3.6 µgmL–1 (Borkowski, 1997). Basal concentrations from AF are</p><p>within the range reported for control animals by Camus et al.</p><p>(1998; Table 3). The highest mean Pb blood of 0.83 µgmL–1</p><p>detected in animals from UA is similar to the reported high doses</p><p>in A. mississippiensis in Louisiana (Camus et al., 1998) and lower</p><p>than blood Pb concentrations in male C. niloticus from South</p><p>Africa (Humphries et al., 2022; Warner et al., 2016), where toxic</p><p>effects were considered likely (Table 3). For birds, Pb levels of</p><p>intoxication have varied from subclinical (0.5 µgmL–1) to severe</p><p>clinical intoxication (1 µgmL–1; Pain et al., 2019) and for mam-</p><p>mals, intoxications have occurred from 0.6 to 0.8 µgmL–1</p><p>(Ma, 2011). Blood Pb concentrations for caiman captured in the</p><p>urbanized areas of our study are higher than these stipulated for</p><p>mammals and within the range of severe intoxication in birds. By</p><p>exposing caiman to Pb gelatin capsules at various doses and</p><p>collecting blood samples to analyze Pb concentrations and the</p><p>enzyme delta‐aminolevulinic acid dehydratase (ALAD), Lance</p><p>et al. (2008) observed that at the lowest dose tested (0.25 g kg –1</p><p>body wt) after 2 weeks there was a depression in ALAD activity of</p><p>approximately 90% in whole blood. A similar result was observed</p><p>in blood from the land tortoise Testudo graeca in which ALAD</p><p>activity was decreased by 30% when the Pb concentration in the</p><p>blood was 0.17 µgmL–1 (Martínez‐López et al., 2010). Therefore,</p><p>additional studies exploring ALAD as a biomarker of Pb toxicity,</p><p>especially in animals from UA, will be important to further char-</p><p>acterize risks (Lance et al., 2008).</p><p>Glutathione‐S‐transferase is a Phase 2 biotransformation</p><p>enzyme that facilitates the excretion of toxicants after its</p><p>conjugation with GSH, and as a result of this conjugation a</p><p>water‐soluble compound is formed that can be readily ex-</p><p>creted (Timbrell, 2000). Glutathione‐S‐transferase can be in-</p><p>duced by polycyclic aromatic hydrocarbons in fish (Santana</p><p>et al., 2018), although contradictory results involving</p><p>either</p><p>induction, no change, or inhibition have been reported in the</p><p>field (Schlenk et al., 2008). There is consistent evidence that</p><p>metals inhibit GST at toxic concentrations in animals in-</p><p>cluding invertebrates, fish, and mammals (Dobritzsch</p><p>et al., 2020).</p><p>Liver GST activity in A. mississippiensis from organochlorine‐</p><p>contaminated areas of Lake Apopka, Florida (USA) and from</p><p>Merrit Island, Florida (USA), which is contaminated by metals,</p><p>was inhibited in relation to animals from reference sites</p><p>(Gunderson et al., 2016). Glutathione‐S‐transferase did not</p><p>differ in adult C. latirostris from the different analyzed areas,</p><p>and an induction of GST was verified in male and female sub-</p><p>adult caimans only from TR compared with animals from AF,</p><p>suggesting an increase in energy expenditure with the bio-</p><p>transformation of contaminants in these individuals.</p><p>Poletta et al. (2016) characterized GSH in blood of captive</p><p>C. latirostris from Argentina, and found large cellular reservoirs</p><p>of GSH that can function as a nonenzymatic antioxidant, as well</p><p>as high antioxidant enzyme activities to deal with oxidative</p><p>stress. In the present study, the concentration of GSH in adult</p><p>animals from UA and TR was higher compared with farmed</p><p>animals (AF), suggesting that higher blood metal concen-</p><p>trations would be involved. However, no correlations were</p><p>observed between any metal and GSH (Table 2). In captive</p><p>C. moreletii captured in Mexico, a negative relationship was</p><p>found between Cd concentrations and GSH in the liver</p><p>(Romero‐Calderón et al., 2022). Captive male C. niloticus</p><p>exhibited significantly higher GSH compared with females</p><p>(Arukwe et al., 2015), differing from our results; we found that</p><p>both males and females exhibited similar blood GSH concen-</p><p>trations, confirming the tendency toward lower GSH in animals</p><p>from AF that was just mentioned (Figure 6B).</p><p>Blood plasma BChE activity has been proposed as a bio-</p><p>marker of exposure to carbamate and organophosphate in-</p><p>secticides in reptiles (Sánchez‐Hernández, 2003), including</p><p>C. latirostris (Attademo et al., 2012). In the present study, the</p><p>mean basal BChE activity of 0.22 μmol TNBmin–1mL plasma–1 in</p><p>C. latirostris from AF is within the basal range of activities of</p><p>0.33 and 0.65 μmol TNBmin–1mL plasma–1 in C. latirostris</p><p>(Attademo et al., 2012) and A. mississippiensis (Schmidt, 2003),</p><p>respectively. Higher basal BChE activities of 2.85 and</p><p>4.15 μmolmin–1mL–1 plasma were observed in the lizards</p><p>Tupinambis merianae (Basso et al., 2012) and Gallotia</p><p>galloti (Sánchez‐Hernández et al., 2004), respectively. Butyr-</p><p>ylcholinesterase is thought to play a protective role by seques-</p><p>tering organophosphates and carbamates and decreasing</p><p>potential brain AChE inhibition and neurotoxicity (Sánchez‐</p><p>Hernández et al., 2004). Thus it has been suggested that the</p><p>lower BChE activity in C. latirostris may be related to a lower</p><p>tolerance to organophosphate and carbamate toxicity</p><p>(Attademo et al., 2012; Basso et al., 2012; Sánchez‐Hernández</p><p>et al., 2004). Butyrylcholinesterase activity was inhibited by 58%</p><p>and 59% in adult caiman from UA (0.11± 0.07 μmolmin−1</p><p>mL−1 plasma) and TR (0.10± 0.02 μmolmin−1mL−1 plasma), re-</p><p>spectively, and by 27% in subadults from TR (0.14± 0.04 μmol</p><p>min−1mL−1 plasma) relative to control caimans from AF</p><p>(0.21± 0.08 μmolmin−1mL−1 plasma; Figure 4). These results</p><p>suggest that exposure of these animals to cholinesterase in-</p><p>hibitor insecticides such as carbamates and/or organo-</p><p>phosphates is occurring both at TR and UA. Although there is no</p><p>chemical information available on the presence of carbamate</p><p>and organophosphate insecticides in the Capibaribe River basin</p><p>or in other urbanized areas, the drainage basin of the Tapacurá</p><p>River that forms Tapacurá Reservoir is influenced by the depo-</p><p>sition of agrochemicals derived from the sugarcane industry,</p><p>which include the nematicides represented by the carbamate</p><p>carbofuran and the organophosphate terbufos (de Moura & de</p><p>Macedo, 2005). In addition, the contribution to TR from diffuse</p><p>sources is significant and includes illegal dumps and landfills, as</p><p>well as drainage from agricultural activities that cover 88% of the</p><p>Metal and biochemical biomarkers in Caiman latirostris—Environmental Toxicology and Chemistry, 2024;00:1–18 13</p><p>wileyonlinelibrary.com/ETC © 2024 SETAC</p><p>15528618, 0, D</p><p>ow</p><p>nloaded from</p><p>https://setac.onlinelibrary.w</p><p>iley.com</p><p>/doi/10.1002/etc.5823 by Florida International U</p><p>niversity, W</p><p>iley O</p><p>nline L</p><p>ibrary on [27/02/2024]. See the T</p><p>erm</p><p>s and C</p><p>onditions (https://onlinelibrary.w</p><p>iley.com</p><p>/term</p><p>s-and-conditions) on W</p><p>iley O</p><p>nline L</p><p>ibrary for rules of use; O</p><p>A</p><p>articles are governed by the applicable C</p><p>reative C</p><p>om</p><p>m</p><p>ons L</p><p>icense</p><p>basin area and include polyculture (35%), livestock (30%), and</p><p>sugarcane cultivation (12%; Gunkel et al., 2003).</p><p>Superoxide dismutase is considered the most important</p><p>antioxidant enzyme (Miller, 2012), and SOD activity ranges</p><p>quantified in our study were similar to those established in</p><p>blood of captive C. latirostris in Argentina (Poletta et al., 2016).</p><p>Superoxide dismutase inhibition was observed in adult caimans</p><p>from TR, potentially related to exposure to the detected higher</p><p>blood concentrations of metals and possibly other con-</p><p>taminants not measured.</p><p>The Spearman correlation coefficients revealed negative</p><p>associations between Cu and Mn concentrations and the</p><p>activities of SOD. Superoxide dismutase is a crucial anti-</p><p>oxidant enzyme for defense against oxidative stress, cata-</p><p>lyzing the transformation of superoxide (ROS) into hydrogen</p><p>peroxide and oxygen. From the multiple forms of SOD, we</p><p>highlight cuprozine‐SOD and manganese‐SOD (SOD2), with</p><p>the former containing Cu ions and the latter Mn ions, serving</p><p>as essential cofactors for optimal SOD activity. Both Cu and</p><p>Mn directly participate in the catalytic mechanism of the en-</p><p>zyme, facilitating the conversion of free radicals into less toxic</p><p>products, ensuring efficient neutralization (Harris, 1992). De-</p><p>spite the lack of documented studies investigating the rela-</p><p>tionship between the SOD enzyme and metals in</p><p>crocodilians, observations in other reptiles such as Olive</p><p>Ridley sea turtle (L. olivacea) revealed reduction in kidney</p><p>SOD activity associated with the presence of several metals,</p><p>including As, Cd, Cu, Ni, Pb, Se, and Zn (Cortés‐Gómez,</p><p>Morcillo, et al., 2018). Similarly, metal concentrations of Cd,</p><p>Sr, Fe, Zn, Ni, Si, Se, and Mg in the blood of green turtles</p><p>(Chelonia mydas) captured in Mexico have indicated both</p><p>positive and negative correlations with the levels of trace</p><p>elements (Labrada‐Martagón et al., 2011). Despite the limited</p><p>understanding of the pathogenic effects of metallic elements</p><p>on crocodilians, especially those lacking a recognized bio-</p><p>logical function, there are documented instances suggesting</p><p>that prolonged exposure can trigger the generation of free</p><p>radical species, leading to the onset of oxidative stress</p><p>(Asmat et al., 2016; Flora et al., 2008; Gupta et al., 2015;</p><p>Sharma et al., 2020).</p><p>The positive correlation between GST and SOD found in our</p><p>study may indicate an effective coordination between these</p><p>antioxidant enzymes in combating oxidative stress (Tim-</p><p>brell, 2000). This can be observed when the body is experi-</p><p>encing a significant increase in the production of free radicals</p><p>and needs a more robust antioxidant response. A positive</p><p>correlation suggests conditions where there is a greater anti-</p><p>oxidant demand and both enzymes are being activated and</p><p>working together to neutralize free radicals; however, there is a</p><p>lack of studies that evaluate the impact of variables on the</p><p>activity levels of biomarkers related to oxidative stress in a</p><p>multicentric manner (Somogyi et al., 2007), particularly in</p><p>crocodilians. This cooperation between GST and SOD is crucial</p><p>for maintaining redox balance in cells and preventing oxidative</p><p>damage (Storey, 1996; Van der Oost et al., 2003), but it is</p><p>important to note that correlations may vary in different con-</p><p>texts and conditions. Factors such as tissue type, health</p>