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Environ Monit Assess
Environ Monit Assess
Environmental Monitoring and Assessment
0167-6369
1573-2959
Springer International Publishing Cham

39240383
12996
10.1007/s10661-024-12996-2
Research
Histopathology reveals environmental stress in dusky flounder Syacium papillosum of the Yucatan Peninsula continental shelf
Couoh-Puga Eunice Danilú 13
Chávez-Sánchez María Cristina 2
Vidal-Martínez Víctor Manuel 1
Gold-Bouchot Gerardo 4
Centeno-Chalé Oscar Arturo 1
Aguirre-Macedo M.Leopoldina leopoldina.aguirre@cinvestav.mx

1
1 https://ror.org/009eqmr18 grid.512574.0 Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional/Unidad Mérida, Departamento de Recursos del Mar, Km 6 Carretera Antigua a Progreso, Cordemex, Mérida, 97319 México
2 grid.428474.9 0000 0004 1776 9385 Centro de Investigación en Alimentación y Desarrollo en Acuicultura y Manejo Ambiental (CIAD), Av. Sábalo-Cerritos, Mazatlán, Sinaloa, 82112 México
3 https://ror.org/01tmp8f25 grid.9486.3 0000 0001 2159 0001 Facultad de Química, Unidad de Química en Sisal, Universidad Nacional Autónoma de México, Sisal, Yucatán México
4 https://ror.org/01f5ytq51 grid.264756.4 0000 0004 4687 2082 Oceanography Department and Geochemical and Environmental Research Group, Texas A&M University, College Station, TX USA
6 9 2024
6 9 2024
2024
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8 8 2024
© The Author(s) 2024
2024
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The histological changes in the liver, kidney, spleen, and gills of Syacium papillosum from the continental shelf of the Yucatan Peninsula, Gulf of Mexico, and their statistical associations with environmental conditions and pollutants were assessed in 2010, 2011, and 2012. We evaluated the extension and severity of the lesions through a degree of tissue change (DTC), and, with the sum of the number of lesion types within each of their DTC stages, we determined the histological alteration index (HAI). The liver and kidney were the most affected organs, with HAI values > 100. Fish with the most severe damage were observed on the Campeche Bank and the Caribbean Sea, contrasting with those collected from the northern Yucatan continental shelf. The presence of foci cellular alteration and abundant melanomacrophage centers indicated that these flatfishes were chronically exposed to environmental stress factors. Redundancy analyses showed strong associations between HAI values and hydrocarbon and heavy metal concentrations in muscle. Our results provide evidence for the first time of a differential health condition of the Yucatan shelf through the histopathology shown in S. papillosum, establishing the baseline for future monitoring programs in the region.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s10661-024-12996-2.

Keywords

Yucatan shelf
Anthropogenic pollution
Histological changes
Flatfish
Histological index
issue-copyright-statement© Springer Nature Switzerland AG 2024
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pmcIntroduction

The Gulf of Mexico (GoM) is an important source of marine ecosystems; and the anthropogenic activities developed there have produced chronic inputs of contaminants, such as heavy metals and hydrocarbons (Gold-Bouchot et al., 2023) . The oil extraction and transportation processes in shallow and deep waters throughout the GoM often have resulted in spills of various magnitudes (Pulster et al., 2020). The release of chemical oil products due to intense maritime transportation also represents an important pollution source that can carry out catastrophic effects on biota (Hook et al., 2014; Vidal-Martínez et al., 2019). Additionally, high concentrations of metals, hydrocarbons, and other chemical contaminants from the environment can be accumulated by marine organisms (Bazzi, 2014; Quintanilla-Mena et al., 2019).

Fish are sensitive to toxicants and considered as potential indicators of environmental pollution. Consequently, fish have been proposed as “effect indicators” and “accumulation indicators” because of the variety of ways in which they respond to pollutants such as oil spills, heavy metals, domestic sewage, and agricultural and industrial contamination (Authman, 2015; Tashla et al., 2018). Toxic compounds can be absorbed into the organism’s organs, tissues, and cells, resulting in different degrees of histological lesions, immune responses, and in many cases, harmful diseases (Améndola-Pimenta et al., 2020). In this sense, pathological fish histology can be a helpful tool for evaluating and monitoring aquatic ecosystems (Couoh-Puga et al., 2021; Yancheva et al., 2016). An advantage of histopathological responses is their integrative potential because they reflect an intermediate effect between the biochemical and individual responses. Moreover, different tissue structural changes are considered a response to several environmental threats, thus reflecting the impact of the total environmental mixture of toxic compounds in the respective area (Teh et al., 1997; Gusso-Choueri et al., 2022). Recently, Murawski et al. (2014) and Ali et al. (2014) reported skin lesions in red snapper (Lutjanus campechanus) and splenic responses in sea trout individuals exposed to different hydrocarbons, such as polycyclic aromatic hydrocarbons (PAHs) from the Deepwater Horizon (DWH) oil spill. Despite unexposed trout fish developing sporadic melanomacrophage centers (MMCs), those were significantly greater in number and size in the fish exposed in the GoM.

Histological lesions caused by environmental pollution have been well documented (Khoshnood, 2016; Yancheva et al., 2016). Liver and gill lesions in marine fish, for example, neoplasms, non-neoplastic proliferative lesions, and specific degenerative/necrotic lesions, have been associated with exposure to chemical xenobiotics and are well-established as histological bioindicators in response to pollutants (Adams, 2003; Rojo-Nieto et al., 2014). Moreover, the presence of liver tumors in bottom-dwelling fish has been clearly associated with PAHs in sediment. However, exposure to other chemicals can likely contribute either as an initiator or as a promoter (USEPA et al., 2012). As a response, fish health may reflect a low nutritional status and susceptibility to chemical exposure (Schmitt & Dethloff, 2000). Subsequently, physiological processes can be affected, causing fish death in severe cases (Vázquez-Gómez et al., 2016). In this sense, the physiological responses in fish fitness (estimated as the condition factor (K)) also can be affected. Such responses have been observed as an inverse relationship between the condition factor K and histopathological changes of fishes in environmentally polluted conditions (Javed & Usmani, 2019).

In this context, one of the most significant threats in in the Gulf of Mexico (GoM) in recent years has been the DWH oil spill, which occurred in April 2010. The release of approximately 134 million gallons (4.9 million barrels) of oil at 1600 m depth into the GoM required the addition of 1.84 million gallons of dispersant (Pallardy, 2023; Soto & Botello, 2013), the ecological impact continue under investigation. Although this oil spill was located within 555 km of the Yucatan shelf (YS) (CONAGUA, 2014), there was still concern that its effects could reach the YS. However, the organisms living in the YS could be affected by other environmental problems not yet evaluated, such as pollutants arrived from Cayo Arcas offshore crude oil loading terminal or the heavy maritime traffic of ships that transport petroleum coming from Aruba, Curacao, Maracaibo, and Trinidad and Tobago, all of which use the Yucatan Channel as a frequent route of transportation to North America (Botello, 2005). In addition, tourism activities from the Caribbean zone and the presence of natural hydrocarbon seeps could also contribute to YS pollution (Botello, 2005; Love et al., 2013).

The DWH oil spill established the importance of distinguishing local YS environmental damage sources from their external counterparts. Thus, an environmental baseline of the YS had to be established. This accident gave us an opportunity to start the GoM monitoring program. The National Institute of Ecology and Climatic Change Center (I.N.E.C.C.), together with other national marine and oceanological institutions, implemented a monitoring plan to establish baseline environmental conditions in the southern Gulf of Mexico after the DWH catastrophe. As a component of such baseline establishment, this paper aims to record flatfish (Syacium papillosum) histopathological alterations that may indicate the presence and effects of pollutants on the Yucatan shelf. We hypothesized that severe histological lesions would be present in flatfishes of the sampling sites closest to the oil spill–impacted area and that histological alteration index scores (HAI) would be statistically associated with environmental and contamination variables in sediments and organisms.

Histological biomarkers provide advantages over other environmental stress biomarkers; first, they provide identification of specific target organs and tissues during exposure to potentially toxic pollutants; moreover, they can be used to establish the specific patterns of both acute and chronic deleterious effects of such pollutants on tissues (Au, 2004).

Therefore, the scarce information on the study area about temporal dynamics of xenobiotic exposure in the area after the DWH oil spill and the tissues morphological changes of S. papillosum as a potential bioindicator contributes to the originality and value of this research as future monitoring programs in YS.

Thus, the study aims were (i) to establish quantitative values through a histological index, (ii) to determine whether the histological changes in S. papillosum exhibit differences among subregions from east to west of the YS, and (iii) to determine whether the occurrence of histological damage of S. papillosum is associated with natural physicochemical environmental variables, heavy metals, nutrients, and/or hydrocarbons at the seascape level.

Materials and methods

Study sites and fish sampling

The study area comprised 87 sampling sites on the continental shelf of the Yucatan Peninsula from Isla Arena, Campeche, in the west to Playa del Carmen, Quintana Roo, in the east (Fig. 1). The sampling sites covered the northern shelf of the entire peninsula between 15 and 200 m depth.Fig. 1 Study area. A Sampling transect network for the Gomex oceanographic cruises (GMX) over the Yucatan continental shelf during 2010–2012, A–E transects of the region near the Campeche Bank (Campeche Bank zone); F–K transects of the central zone of the Yucatán shelf (Yucatán zone); L–Q transects of the Mexican Caribbean (Yucatan-Quintana Roo zone). B Sampling sites where fish were collected during the Gomex oceanographic cruises 2010–2012

Three oceanographic cruises (OC) were carried out during consecutive years: Gomex-1 (10–21 September 2010) and Gomex-2 (September 23 to October 3, 2011), during the rainy season, and Gomex-3 (from November 28 to December 2012) during the early north winds season. Once in sampling sites, the fish were collected by net trawling and identified by ichthyologists of the Necton Laboratory (CINVESTAV-IPN, Mérida).

In each OC, a total of 62 environmental variables from water and sediments were measured (hydrocarbons and heavy metals), including bile PAH metabolites in organisms (Supplementary material Table S1). In addition, nutrients and physicochemical water parameters (e.g., oxygen (mg/l), salinity (psu), and temperature (°C)) were measured (Supplementary material Table S1). The physicochemical characteristics and concentrations of hydrocarbons and heavy metals in sediments and organisms were determined at the Marine Geochemistry Laboratory (CINVESTAV-IPN, Mérida) applying standardized and recommended methods (Botello et al., 2015). PAH metabolite concentrations in organisms were provided by the Ecotoxicology Laboratory following standardized methods (Gold-Bouchot et al., 2017).

Immediately after being caught, fish morphometric data were taken. The fish were weighed (g) and measured for total length (cm) and then dissected. The length–weight relationship (LWR) of S. papillosum was estimated using the equation W = αSLβ (Ricker, 1975), where W = total weight (g), SL = standard length (cm), α is the ordinate to the origin, and β is the intercept, also known as the allometry coefficient. Based on the estimations of LWR, we used the condition factor (K) proposed by Bagenal and Tesch (1978) (K = 100 × W/SLβ) to assess the physiological condition of the fish, as well as the effect of pollutant exposure on this physiological biomarker (Couoh-Puga et al., 2021; Snyder et al., 2019).

Histopathological analyses

Each fish was euthanized by brain puncture, to avoid mechanical damage to tissues, mainly in gills, previously reported by anesthetics (Boijink et al., 2017; Couoh-Puga et al., 2021). All organisms were handled under the Guidelines for Care and Manipulation of Laboratory Animals of Cinvestav, the Mexican Official norm NOM-062-ZOO-1999 (http://www.fmvz.unam.mx/fmvz/principal/archivos/062ZOO.PDF) and the Guide for the Care and Use of Laboratory Animals as adapted by the US National Institutes of Health (http://www.aaalac.org/resources/Guide_2011.pdf). Gills, kidney, liver, and spleen tissue sections were fixed immediately in a 10% buffered formalin solution to preserve biological tissues and avoid autolysis or putrefaction.

The tissues were processed following the methodology by Humason (1962) and Luna (1968), dehydrated in an automatic processor (Histokinette), and embedded in paraffin (melting point 56 °C). Afterwards, sections were cut to 5-µm thickness using a microtome and mounted in Entellan® for microscopy. Finally, the slides were stained with hematoxylin and eosin (H&E) for morphological examination under an Olympus BX50 compound microscope (Chávez-Sánchez et al., 2014). Using a Q imagine digital camera of 5-megapixel resolution, photographs of normal and histopathological tissues were taken for the histological baseline comparison. All the material corresponding to this investigation is available for consultation in the Aquatic Pathology Laboratory Histology Section of CINVESTAV-IPN, Mérida.

Histological alterations in tissues and organs were evaluated semi-quantitatively based on two criteria according to Schwaiger et al. (1997) and Simonato et al. (2008): (a) extent of the damage (EOD), where the alterations were classified into three degrees of incidence, and (b) degree of tissue change (DTC) based on the severity of the lesions and the possibility of recovery of tissue and organs (Table 1). Table 1 Evaluation of the histopathological alterations in relation to the extent of the damage (EOD) and the degree of tissue change (DTC) or severity of damage

EOD	The extent of the damage	DTC	Histopathological alterations	
Grade 1	There are no focal alterations	Stage I	Changes that do not damage the organ to such an extent that it cannot repair itself if conditions improve	
Grade 2	Focal changes	Stage II	Changes that are more severe and affect the associated organ or tissue function	
Grade 3	Extensive damage	Stage III	Changes that prevent the organ from repairing itself even if conditions improve	

The histological alteration index (HAI) was calculated with the equation used by Silva and Martinez (2007) and Simonato et al. (2008) and proposed by Poleksic and Mitrovic-Tutundzic (1994).HAI=1·∑I+10·∑II+100·∑III

In this equation, I, II, and III are the histological lesion stages (DTCs). The HAI was calculated for each fish and tissue. The average HAI was ranked into five categories: 0–10, normal organ; 11–20, slightly to moderately damaged organ; 21–50, moderately to heavily damaged organ; 51–100, severely damaged organ; and > 100, irreversibly damaged organ (Améndola-Pimenta et al., 2020; Chávez-Sánchez et al., 2014). The histopathological alterations were compared among the fish of sampling sites of the same cruise and afterward compared among the fish HAI data on different oceanographic cruises.

Data analyses

Normality of both EOD mean values and HAI scores was obtained for each organ for each oceanographic cruise and then assessed with Shapiro–Wilks normality test. If normality was not reached, a nonparametric ANOVA test (Kruskal–Wallis) with a level of significance of p < 0.05 was used to determine potential differences in HAI values among subregions (see below).

Nonmetric multidimensional scaling (NMDS) was performed with all the HAI values of the four organs examined for the fish collected at every sampling site, considering the depth and zone as grouping factors. For these analyses, we used Primer 6 software, based on the Bray–Curtis dissimilarity index, to determine the patterns that are formed concerning HAI with respect to depth (10–49 m, 50–100 m, 101–200 m) and zone (Campeche Bank, Central Yucatan, Caribbean) factors within the YS and for three Gomex cruises from 2010 to 2012. Due to their geographical proximity, all of the individual fish obtained were grouped a priori into subregions: Campeche Bank in the western YS (sampling sites A2, A3, A4, A5, and B6, B7, B8, B9, B10), Central Yucatan (sampling sites F28, F29, G33, G34, G35, H39, H40, I42, I43, I45, and J47, J48), and the Caribbean (sampling sites L58, L59, M63, O71, O72, O73, O74, O75, P77, P78, P79, and P80). Redundancy analyses (RDA) were performed between dependent variables (HAI values) and independent variables (62 variables: physicochemical, nutrient, and contaminant data for sediments and organisms) (Supplementary material Table S1). Monte Carlo permutations were used to determine the significance of the canonical axes based on 4999 permutations. The independent variables were selected by forward stepwise analyses, keeping only the variables that explained the greatest variance (10%) and did not present a variance inflation factor (VIF) higher than four. The significance of the statistics was determined with p < 0.05. RDAs were performed with CANOCO software for Windows 5.0 (Ter Braak & Smilauer, 2012), to these analyses, length, weight, and K were considered covariables.

Results

Water quality

The physicochemical parameters of water and pollutants in sediments and organisms are presented in the Supplemental material (Table S1). Analysis results showed that except for salinity, the physicochemical parameters of water (i.e., oxygen, temperature) were variable concerning to the three OCs (Fig. 2).Fig. 2 Statistical differences in the oxygen and temperature during three Gomex (GMX) oceanographic cruises (OCs). (A) Mean oxygen ± S.D., (B) mean temperature ± S.D., and (C) mean salinity ± S.D. Different letters denote significant differences (p < 0.05). We used Kruskal–Wallis nonparametric one-way ANOVA

Fish sampled

A total of 153 adult flatfish (Syacium papillosum) individuals were captured from 15 sites in 2010, 19 sites in 2011, and 17 sites in 2012 out of 87 sampled sites per year. Fish were captured between 15 and 200 m deep (Fig. 1B and Supplementary material Table S1). Most fish collected during Gomex-1 came from sites at 100 m depth at the eastern and western boundaries of the Yucatan shelf (Supplementary material Table S1: transects A and P, respectively). For Gomex-2 and Gomex-3, it was also possible to collect fish from the F–M transects at 50 to 200 m depth (Fig. 1). The size and weight of collected fishes varied between 20 and 28 cm of total length and from 105 to 270 g of weight. The total length of fishes in Gomex-2 and Gomex-3 presented significant differences compared to Gomex-1, and the weight of the fishes from Gomex-1 and Gomex-2 had significant differences to Gomex-3 (H = 6.97; df = 2; p = 0.0307 and H = 27.92; df = 2; p = 0.0001 to length and weight, respectively) (Fig. 3; Supplementary material Table S1).Fig. 3 Statistical differences in the length and weight of all flatfish for three Gomex (GMX) oceanographic cruises. A Mean total length ± S.D. B Total weight average ± S.D. Different letters denote significant differences (p < 0.05). We used the Kruskal–Wallis nonparametric one-way ANOVA test

Histopathological findings

The external fish examination did not show clinical signs of diseases, injuries, or malformations. Some of the lesions observed were caused by mechanical damage during trawling. Between 8 and 14 types of histopathological alterations were identified in the selected organs (Supplementary material Table S2). The spleen was the organ with the fewest histological lesions, and the liver and kidney were the tissues that showed the most significant histopathologies by number and severity. The lesions prevalence values varied between 3% (gills hypertrophy) and 100% of melanomacrophage centers (MMCs) in the spleen, kidney, and liver. The prevalence values of each lesion were variable among cruises. There was a general reduction of lesion prevalence for the second year concerning to the first, increasing again for the third year (Supplementary material Table S2). However, the histopathologies were focal in most cases.

The spleen presented eight lesions; however, the most prevalent alteration in the three cruises was the presence of MMCs, which in definition contain a varying amount of pigments within vacuoles, these pigments can include both classical melanin, including hemosiderin, and lipofuscins (Agius and Roberts, 2003) (Supplementary material Table S2; Fig. 4). This histological response reached a prevalence higher than 80%, which was maintained for all three OCs.Fig. 4 Histological lesions in spleen tissue during the Gomex 1, 2, and 3 cruises, a normal tissue with focal melanomacrophage centers (↑), 10 × ; b, c splenic tissue with abundant melanomacrophage centers (↑), 10 × and 4 × , respectively. H&E staining

Thirteen histological damages were identified in the gills (Supplementary material Table S2). The prevalence of hypertrophy was 90% in samples from Gomex-1, while in Gomex-2 and Gomex-3, the values decreased to 20% and 3%, respectively. The prevalence of hyperplasia for the three cruises presented values > 89%. The prevalence of inflammation reflected in mucus cells and lamellar fusion tended to decrease through each OCs from 90 to 40%. At the same time, telangiectasia increased its prevalence through every OC (Supplementary material Table S2). The lamellae presented edema, metaplasia, and parasites (mainly protozoa), with 35 to 61% prevalence. The pathological alterations caused by the presence of parasites were usually limited to slight inflammation around the parasite cyst (Fig. 5).Fig. 5 Histological lesions in gills of flatfish Syacium papillosum during oceanographic cruises Gomex 1, 2, and 3. a Healthy gill tissue, 10 × ; b increase in mucus at the branchial apex (metaplasia) (↑), 20 × ; c extensive epithelial lifting in gills with severe lamellar edema (↑), blood congestion (red oval), and mucus cells (Δ), 20 × . H&E staining

MMCs were highly prevalent in the kidneys in each subsequent oceanographic cruise, with 85, 95, and 100%, respectively. However, cellular atrophy and necrosis were the most severe lesions, and they tended to increase through every OC (Supplementary material Table S2; Fig. 6). The rest of the pathologies observed in this organ varied in percentage on all three cruises.Fig. 6 Histological lesions observed in kidney during the Gomex 1, 2, and 3 cruises. a Renal tissue with severe dilation in the glomerular vessels (↑), 40 × ; b renal tissue with abundant MMCs ( ), glomerular atrophy (Δ), and dilation of glomerular blood vessels ( ), 40 × ; c renal tissue with abundant MMCs, 10 × . H&E staining

In the hepatic tissue, we identified 13 lesions where cellular atrophy, fatty degeneration, MMCs, and necrosis were the most prevalent (> 80%) (Supplementary material Table S2, Fig. 7). Additionally, there was an increase in the prevalence of these lesions through the OCs. Injuries such as inflammation, hemorrhage, granulomas, dilation of sinusoids, adenomas, and parasites showed lower prevalence (< 50%). Again, the injuries caused by parasites were focal (slight inflammation).Fig. 7 Histological lesions observed in the liver during the Gomex 1, 2, and 3 cruises. a Normal liver tissue, 40 × ; b congestion of hepatic sinusoids (□), MMCs (○), severe fatty degeneration (*), 40 × ; c liver tissue with abundant MMCs (↑) and extended hyaline droplets (black circle), 40 × . H&E staining

The histological damage, like focal inflammation in the kidney, liver, and spleen and focal telangiectasia in the gills, was slight on most sampling sites. However, in two sampling sites of Gomex-2 (G34 and H40) and Gomex-3 (G35 and H40) (Fig. 1B), the fishes showed severe histological damage such as fatty degeneration in the liver accompanied by cellular atrophy, necrosis, and hypertrophy in the renal epithelial cells, as well as abundant MMCs in the kidney and spleen.

Quantification of histological damage in S. papillosum

Table 1 shows the classification of the histopathological alterations. Table 2 gives the mean values of the extent of the damage (EOD) and histological alteration index (HAI) in organs and tissues for the different lesions observed in the liver, spleen, kidney, and gills of flatfish collected from the Yucatan shelf during the Gomex cruises. The extent of the damage (EOD) of the pathological alterations in the evaluated organs was mainly focal (Grade 2) and generally classified in DTC stage II, except for the neoplastic lesion, severe necrosis, and structural disintegration, which were classified as DTC stage III (Supplementary material Table S2). The highest values of HAI in the liver and kidney are from the coastal zone of the Yucatan shelf, mainly off the coast of Progreso and in front of the upwelling zone, close to the Mexican-Caribbean coast. Table 2 Mean and standard deviation of the extent of damage (EOD) and histological alteration index (HAI) in the liver, spleen, kidney, and gills of the flatfish Syacium papillosum from the continental shelf of the Yucatan Peninsula. These data were obtained during oceanographic cruises Gomex 1, 2, and 3

TISSUE		GOMEX 1
Mean/Std.Dev	GOMEX 2
Mean/Std.Dev	GOMEX 3
Mean/Std.Dev	
LIVER	EOD	1.34 ± 0.27 a	1.29 ± 0.29 a	1.37 ± 0.12 a	
SPLEEN	EOD	1.21 ± 0.36 b	1.13 ± 0.37 a	1.28 ± 0.29 b	
KIDNEY	EOD	1.28 ± 0.46 b	1.25 ± 0.28 a	1.41 ± 0.18 b	
GILLS	EOD	1.56 ± 0.49 c	1.34 ± 0.22 b	1.27 ± 0.23 a	
LIVER	HAI	131 ± 93 c	108 ± 59 c	35 ± 40 b	
SPLEEN	HAI	2 ± 3 b	3 ± 4 a	5 ± 7 b	
KIDNEY	HAI	97 ± 70 c	58 ± 60 b	19 ± 18 b	
GILLS	HAI	30 ± 19 c	7 ± 6 b	4 ± 3 a	
Different letters denote significant differences between values (P <0.05)

The NMDS analyses showed that, although the HAI values of some fish collected in different zones of the Yucatan shelf were high (> 100), they were not associated with depth or sampling zone (Fig. 8A, B). In contrast, Fig. 8C shows differences among oceanographic cruises.Fig. 8 Nonmetric multidimensional scaling (NMDS) of the histological alteration index (HAI) of the dusky flounder Syacium papillosum. A NMDS analysis between HAI values with the depth classification of the Gomex 1, 2, and 3 cruises. B NMDS analysis between HAI values with zone classification of the Gomex 1, 2, and 3 cruises. C NMDS analysis between HAI values and years of Gomex cruises within the Yucatan platform

RDA between HAI and abiotic variables: contaminants, physicochemical parameters, and nutrients

The redundancy analyses (RDA) showed significant and positive associations between the HAI values of the liver, kidney, spleen, and gills (dependent variables) and independent variables such as polycyclic aromatic hydrocarbons (PAHs) and heavy metal concentrations. Although other variables, such as nutrients, water physicochemical variables, fish length, weight, and K as a covariables, were considered, these were not positively associated with the histological index. For Gomex-1, the RDA accounted for 72.6% of the total variance and was highly significant for the first and all canonical axes (F = 6.3473; p value = 0.0226; 4999 permutations). The HAI of the liver and kidney (L_HAI and K_HAI) were positively associated with the total hydrocarbons in muscle (TH (M)) and with aliphatic hydrocarbons in sediments (ALIPH (S)); in contrast, the spleen HAI values (S_HAI) were positively associated with total polycyclic aromatic hydrocarbons in sediment (TPAHs (S)) and with vanadium in muscle (V (M)). For gill HAI (G_HAI), there was a close association with hydroxypyrene bile metabolites (HPY (BM)) (Fig. 9A). For Gomex-2, the RDA accounted for 72.6% of the total variance and was highly significant for the first and all four canonical axes (F = 9.928; p value = 0.0004; 4999 permutations). The HAI for kidney and liver (L_HAI and K_HAI) were positively associated with PAHs of low molecular weight in sediment (LMWPAHs (S)), lead, and barium in muscle (Pb (M) and Ba (M)), while the spleen and gills’ HAI (S_HAI and G_HAI) were positively associated with cadmium in muscle (Cd (M)) (Fig. 9B). For Gomex-3, the RDA accounted for 93.9% of the total variance and was highly significant for second and third canonical axes (F = 2.307; p value = 0.0120; 4999 permutations). The HAI of the liver and spleen (L_HAI and S_HAI) were positively associated with PAHs of high molecular weight (HMWPAHs (L)) and total PAHs in sediment (TPAHs (S)). The kidney HAI (K_HAI) was positively associated with the UCM and nickel in muscle (UCM (M) and Ni (M)), while the gill HAI (G_HAI) was closely associated with low molecular weight PAHs (PAHs LMW (M)), phenanthrene (PHE (BM)), and iron in muscle (Fe (M)) (Fig. 9C).Fig. 9 A–C Redundancy analyses (RDA) of the environmental variables and histological alteration index HAI of Syacium papillosum of the three Gomex cruises (1, 2010; 2, 2011; and 3, 2012). A For Gomex-1, the RDA accounted for 72.6% of the total variance and was highly significant for the first and all canonical axes (F = 6.3473; p value = 0.0226; 4999 permutations). B For Gomex-2, the RDA accounted for 72.6% of the total variance and was highly significant for the first and all four canonical axes (F = 9.928; p value = 0.0004; 4999 permutations). C For Gomex-3, the RDA accounted for 93.9% of the total variance and was highly significant for all four canonical axes (F = 2.307; p value = 0.0120; 4999 permutations). Abbreviations: L_HAI, liver-histological alteration index; S_HAI, spleen-histological alteration index; K_HAI, kidney-histological alteration index; G_HAI, gill-histological alteration index; TH (M), total hydrocarbons (muscle); ALIPH (S), aliphatic hydrocarbons (sediment); TPAHs (S), total polycyclic aromatic hydrocarbons-sediment; BaP(BM), benzo[a]pyrene (bile metabolites); HPY (BM), hydroxypyrene (bile metabolites); NAPH (BM), naphthalene (bile metabolites); HMWPAHs (M), polycyclic aromatic hydrocarbons–high molecular weight (muscle); PHE (BM), phenanthrene (bile metabolites); Ni (M), nickel (muscle); Ba (M), barium (muscle); Cd (M), cadmium (muscle); Pb (M), lead (muscle); V (M), vanadium (muscle); Fe (M), iron (muscle); Pyrene (M), pyrene (muscle); Perylene (L), perylene (liver); LMWPAHs (S), polycyclic aromatic hydrocarbons–low molecular weight (sediment); NH4, ammonia; UCM (M), unresolved complex mixture (muscle); LMWPAHs (M), polycyclic aromatic hydrocarbons–low molecular weight (muscle); HMWPAHs (L), polycyclic aromatic hydrocarbons–high molecular weight (liver)

Discussion

Our original hypothesis that severe histological lesions would be present in S. papillosum at the closest sites to the DWH oil spill, and therefore at the boundary of the Yucatan shelf, was not fulfilled. The distribution of lesions in S. papillosum from the Yucatan shelf showed that these occurred at all sampling sites, being more prevalent and severe in the eastern and western margins of the YS and in the coastal zone in front of Progreso harbor. Regarding the severity of the lesions, the DTC of the collected fish from all Gomex oceanographic cruises showed severe alterations in the liver and kidney (stage III), which can compromise the normal function of these organs. The observed histological damage could be the consequence of chronic exposure to hydrocarbons and heavy metals present in the sediments (Costa et al., 2009; Yancheva et al., 2016) (Fig. 9). Vidal-Martínez et al. (2019) mention that although there are no oil extraction activities in the YS, a possible explanation for the presence of pollutants causing S. papillosum injuries is the proximity of the sampling sites to the oil extraction operations of the Campeche Bank in the western YS and to the loop current carrying these pollutants into the GoM from the Caribbean area in the eastern YS. Moreover, Peters et al. (2021) mention that the eddies formed by the loop current create strong currents that redistribute the water in the surface layers, transporting the pollutants mainly on continental shelves and along the coastal zones of GoM. In addition, according to Love et al. (2013), the study area matches spatially with the presence of hydrocarbon seeps in the Yucatán Peninsula, which could be related to the most severe histological injuries and highest HAI scores in S. papillosum.

Concerning the severity of pathological changes (DTC), most alterations did not hurt the organ structure and function of the liver. Fatty degeneration, MMCs, and necrosis increased their prevalence in fishes of Gomex-2 and Gomex-3. The presence of lesions such as necrosis in liver tissue is focal and does not represent an irreversible change in the liver tissues. These lesions have been associated with biochemical disturbances such as degenerative alterations (granular, vacuolar, hydropic, and fatty degeneration) (Monteiro et al., 2005; Rajeshkumar & Munuswamy, 2011). In addition, we occasionally found foci of cellular alteration (FCA), structural disintegration, and coagulative necrosis, which are pathologies that remain and can extend despite the disappearance of the causal factor. These lesions have been reported previously and linked to PAH exposure (Myers et al., 2003). The extension of these lesions can often affect liver functions, such as the detoxification process and fat metabolism, carbohydrates, and proteins (Straif et al., 2005).

The severity of the histological damages in sampling sites near the coastal area of Yucatan (B6) may be due to strong freshwater discharge from the continent during the rainy season, which contains different compounds, including nutrients such as HCO3, SO4, Cl, Ca, Mg, Na, K, NO2, NO3, and NH4 (Delgado et al., 2010; Herrera-Silveira & Morales-Ojeda, 2010). Additionally, there is evidence of low and high molecular weight PAHs in sediments all along the coastal lagoons of Yucatán, presumably carried from the continent to the coastal region by subterranean currents (Gold-Bouchot et al., 2014). Consequently, fish are exposed to all these compounds in the coastal zone, which could produce the histopathologies found in S. papillosum. On the other hand, histopathological damages observed in the deepest sampling stations (G34, G35, and H40) could be related to additional factors such as pollutants dispersion and frequent maritime traffic (including oil transportation), which often results in frequent small-scale spills, that is one of the main stressors in approximately 50% of the YS (Ocaña et al., 2019).

Furthermore, the RDA analyses suggested associations or causal relationships between the concentrations of hydrocarbons, heavy metals, and the HAI scores of S. papillosum. Despite the positive association between histopathological lesions and the presence of pollutants shown by our analyses, we should not jump to conclusions regarding the source of such pollutants being from the oil industry only. As Vidal-Martínez et al. (2019) point out, the concomitant effect of other environmental variables and other water sources of the discharge into the YS should be considered. To develop a histological lesion, high pollutant concentrations are not always necessary, and low doses are usually sufficient to trigger their development (Améndola-Pimenta et al., 2020). This indicates that localized environmental conditions could lead to similar fish histopathologies elsewhere on the GoM.

In the year of the Deepwater Horizon oil spill, we observed some grade of histological lesions in all fishes from Yucatán shelf stations sampled. However, some sampling stations assessed in A and B transects at western YS where there are not oil activities and are distant from posible crude oil spills can serve as a kind of “baseline/reference,” because they showed minimal or low concentrations of pollutants, such as total polycyclic hydrocarbons, total hydrocarbons, their metabolites, and heavy metals (Supplementary material S1). Although no solid evidence was found of DWH pollutants reaching the YS, the decreasing tendency in prevalence and severity of lesions found in S. papillosum throughout the Gomex cruises is similar to the pattern observed by Murawski et al. (2014). These authors found a similar pattern while sampling considerably near the DWH oil spill. The incidence of skin lesions reported in their 2011 samplings had decreased by 53% when they sampled again by 2012, with the severity of lesions also declining. A likely reason for the decreased prevalence and severity of lesions in Murawski’s research and our own could be the death of the more severely affected fish. It is not yet reasonable to conclude that the histopathologies reported in this paper have been necessarily caused by pollutants originated from the DWH or that the observed decrease in lesion prevalence observed by Murawski and ourselves can be indeed attributed to the death of the most severely affected fish. More precise conclusions on these matters will require additional long-term research.

Conclusions

The prevalence and high HAI values found in the examined organs of flatfish inhabiting the Yucatan platform are evidence of specific areas with poor environmental quality. The histopathologies described in this work also indicate that these fish are chronically exposed to stress sources.

These findings constitute a relevant baseline data set for monitoring the pollutants and their biological effects in the YS. The Yucatan shelf has several anthropogenic and natural stress factors, thus requiring continuous biological and environmental monitoring to understand their effect on the YS communities. Equally important will be experimentally testing the potential relationship between contaminants such as hydrocarbons and heavy metals with histological damage in S. papillosum.

Supplementary Information

Below is the link to the electronic supplementary material.ESM 1 (XLSX 48.1 KB)

Acknowledgements

This study was part of the Ph.D. thesis of E.D.C.P, who thanks CONACYT-Mexico (367616) for providing 4 years of scholarship. Thanks are extended to all the Aquatic Pathology Laboratory of CINVESTAV-IPN Unit Mérida staff, in particular to Gregory Arjona and Francisco Puc Itzá, who carried out the sampling campaigns and the collections of the histological samples; Raúl Sima, who helped in the histological sample processing; and Mr. Ehecatl Vidal Aguirre for his help with the English language. This is a contribution of the Consorcio de Investigación del Golfo de México (CIGoM).

Author contribution

Gerardo Gold Bouchod and Ma. Leopoldina Aguirre Macedo designed the sampling network; Eunice Danilú Couoh Puga, Ma. Leopoldina Aguirre Macedo, and María Cristina Chávez Sánchez worked in the conceptualization of the histological study; Eunice Danilú Couoh Puga and Oscar Arturo Centeno Chalé worked in data curation; Víctor Manuel Vidal Martínez and Ma. Leopoldina Aguirre Macedo were responsible for the funding acquisition; Eunice Danilú Couoh Puga, Víctor Manuel Vidal Martínez, Ma. Leopoldina Aguirre Macedo, and María Cristina Chávez Sánchez worked in general methodology, writing, and editing; and Eunice Danilú Couoh Puga wrote the main manuscript text and prepared all Figs. 1, 2, 3, 4, 5, 6, 7, 8, and 9. All authors reviewed the manuscript.

Funding

Semarnat-INEC project-2010–2012 Monitoreo ambiental en la plataforma norte de la Península de Yucatán. “Oceanographic cruises (Gomex 1, 2, and 3) in response to Deepwater Horizon oil spill.”

Data availability

No extra datasets were generated or analysed during the current study.

Declarations

Ethical approval

All authors have read, understood, and have complied as applicable with the statement on “Ethical responsibilities of Authors.”

Competing interests

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

Adams SM Establishing causality between environmental stressors and effects on aquatic ecosystems Human and Ecological Risk Assessment: An International Journal 2003 9 1 17 35 10.1080/713609850
Adams, S. M. (2003). Establishing causality between environmental stressors and effects on aquatic ecosystems. Human and Ecological Risk Assessment: An International Journal,  9(1), 17–35. 10.1080/71360985010.1080/713609850
Agius, C., & Roberts, R.J. (2003). Melanomacrophage centers and their role in fish pathology. Journal of Fish Diseases, 26(9), 499–509. 10.1046/j.1365-2761.2003.00485.x
Ali AO Hohn C Allen PJ Ford L Dail MB Pruett S Petrie-Hanson L The effects of oil exposure on peripheral blood leukocytes and splenic melano-macrophage centers of Gulf of Mexico fishes Marine Pollution Bulletin 2014 79 87 93 10.1016/j.marpolbul.2013.12.036 24405733
Ali, A. O., Hohn, C., Allen, P. J., Ford, L., Dail, M. B., Pruett, S., & Petrie-Hanson, L. (2014). The effects of oil exposure on peripheral blood leukocytes and splenic melano-macrophage centers of Gulf of Mexico fishes. Marine Pollution Bulletin,  79, 87–93. 10.1016/j.marpolbul.2013.12.03624405733 10.1016/j.marpolbul.2013.12.036
Améndola-Pimenta M Cerqueda-García D Zamora- Briseño JA Couoh-Puga D Montero-Muñoz J Árcega-Cabrera F Ceja-Moreno V Pérez-Vega JA García-Maldonado JQ del Río-García M Zapata-Pérez O Rodríguez-Canul R Toxicity evaluation and microbiota response of the lined sole Achirus lineatus (Chordata: Achiridae) exposed to the light petroleum water accommodated fraction (WAF) Journal of Toxicology and Environmental Health, Part A 2020 10.1080/15287394.2020.1758861
Améndola-Pimenta, M., Cerqueda-García, D., Zamora- Briseño, J. A., Couoh-Puga, D., Montero-Muñoz, J., Árcega-Cabrera, F., Ceja-Moreno, V., Pérez-Vega, J. A., García-Maldonado, J. Q., del Río-García, M., Zapata-Pérez, O., & Rodríguez-Canul, R. (2020). Toxicity evaluation and microbiota response of the lined sole Achirus lineatus (Chordata: Achiridae) exposed to the light petroleum water accommodated fraction (WAF). Journal of Toxicology and Environmental Health, Part A. 10.1080/15287394.2020.175886110.1080/15287394.2020.1758861
Au DWT The application of histo-cytopathological biomarkers in marine pollution monitoring: A review Marine Pollution Bulletin 2004 48 9–10 817 834 10.1016/j.marpolbul.2004.02.032 15111029
Au, D. W. T. (2004). The application of histo-cytopathological biomarkers in marine pollution monitoring: A review. Marine Pollution Bulletin,  48(9–10), 817–834. 10.1016/j.marpolbul.2004.02.03215111029 10.1016/j.marpolbul.2004.02.032
Authman, M. M. (2015). Use of fish as bioindicator of the effects of heavy metals pollution. Journal of Aquaculture Research and Development,  06(04). 10.4172/2155-9546.1000328
Bagenal, T., & Tesch, F. W. (1978). Age and growth. In T. Bagenal (Ed), Methods for assessment of fish production in freshwater waters (pp. 101–136). Blackwell Scientific Publication.
Bazzi AO Heavy metals in seawater, sediments and marine organisms in the Gulf of Chabahar, Oman Sea Journal of Oceanography and Marine Science 2014 5 3 20 29 10.5897/joms2014.0110
Bazzi, A. O. (2014). Heavy metals in seawater, sediments and marine organisms in the Gulf of Chabahar, Oman Sea. Journal of Oceanography and Marine Science, 5(3), 20–29. 10.5897/joms2014.011010.5897/joms2014.0110
Boijink CL Maciel PO Tavares-Dias M Iwashita MKP Morais MS Hide DMV Souza NC Couto MVS Meneses JO Cunha FS Fujimoto RY Anesthesia by sprinkling method in the gills of tambaqui Colossoma macropomum does not influence intensity and morphology of monogeneans Brazilian Journal of Biology 2017 77 1 5 10.1590/1519-6984.15915
Boijink, C. L., Maciel, P. O., Tavares-Dias, M., Iwashita, M. K. P., Morais, M. S., Hide, D. M. V., Souza, N. C., Couto, M. V. S., Meneses, J. O., Cunha, F. S., & Fujimoto, R. Y. (2017). Anesthesia by sprinkling method in the gills of tambaqui Colossoma macropomum does not influence intensity and morphology of monogeneans. Brazilian Journal of Biology, 77, 1–5. 10.1590/1519-6984.1591510.1590/1519-6984.15915
Botello, A. V. (2005). Características, composición y propiedades fisocoquímicas del petróleo, p. 261- 268. In: A. V. Botello, J. Rendón-von Osten, G. Gold-Bouchot y C. Agraz-Hernández (Eds.), Golfo de México contaminación e impacto ambiental: Diagnóstico y tendencias, (2nd ed., pp. 696). Universidad Autónoma de Campeche, Universidad Nacional Autónoma de México, Instituto Nacional de Ecología.
Botello AV Soto LA Ponce-Vélez G Villanueva FS Baseline for PAHs and metals in NW Gulf of Mexico related to the Deepwater Horizon oil spill Estuarine, Coastal and Shelf Science 2015 156 124 133 10.1016/j.ecss.2014.11.010
Botello, A. V., Soto, L. A., Ponce-Vélez, G., & Villanueva, F. S. (2015). Baseline for PAHs and metals in NW Gulf of Mexico related to the Deepwater Horizon oil spill. Estuarine, Coastal and Shelf Science,  156, 124–133. 10.1016/j.ecss.2014.11.01010.1016/j.ecss.2014.11.010
Chávez-Sánchez MC Olvera-Novoa MA Osuna-Durán B Abdo de la Parra I Abad-Rosales SM Martínez-Rodríguez I Ascorbic acid requirement and histopathological changes due to its deficiency in juvenile spotted rose snapper Lutjanus guttatus (Steindachner, 1869) Aquaculture International 2014 22 6 1891 1909 10.1007/s10499-014-9790-6
Chávez-Sánchez, M. C., Olvera-Novoa, M. A., Osuna-Durán, B., Abdo de la Parra, I., Abad-Rosales, S. M., & Martínez-Rodríguez, I. (2014). Ascorbic acid requirement and histopathological changes due to its deficiency in juvenile spotted rose snapper Lutjanus guttatus (Steindachner, 1869). Aquaculture International,  22(6), 1891–1909. 10.1007/s10499-014-9790-610.1007/s10499-014-9790-6
CONAGUA (Comisión Nacional del Agua). (2014). Monitoreo ambiental y evaluación de la calidad del agua en las costas del Golfo de México y mar caribe. Ciudad de México. (CGACC_2014_Monitoreo_Ambiental_CONAGUA), (pp. 1–83).
Costa PM Diniz MS Caeiro S Lobo J Martins M Ferreira AM Caetano M Vale C DelValls TA Costa MH Histological biomarkers in liver and gills of juvenile Solea senegalensis exposed to contaminated estuarine sediments: A weighted indices approach Aquatic Toxicology 2009 92 3 202 212 10.1016/j.aquatox.2008.12.009 19282038
Costa, P. M., Diniz, M. S., Caeiro, S., Lobo, J., Martins, M., Ferreira, A. M., Caetano, M., Vale, C., DelValls, T. A., & Costa, M. H. (2009). Histological biomarkers in liver and gills of juvenile Solea senegalensis exposed to contaminated estuarine sediments: A weighted indices approach. Aquatic Toxicology, 92(3), 202–212. 10.1016/j.aquatox.2008.12.00919282038 10.1016/j.aquatox.2008.12.009
Couoh-Puga ED Vidal-Martínez VM Ceja-Moreno V Árcega-Cabrera F Puch-Hau C Rodríguez-González M-T Aguirre-Macedo ML Histological effects of light crude oil on Sciaenops ocellatus under experimental conditions Bulletin of Environmental Contamination and Toxicology 2021 10.1007/s00128-021-03172-0
Couoh-Puga, E. D., Vidal-Martínez, V. M., Ceja-Moreno, V., Árcega-Cabrera, F., Puch-Hau, C., Rodríguez-González, M.-T., & Aguirre-Macedo, M. L. (2021). Histological effects of light crude oil on Sciaenops ocellatus under experimental conditions. Bulletin of Environmental Contamination and Toxicology. 10.1007/s00128-021-03172-010.1007/s00128-021-03172-0
Delgado C Pacheco J Cabrera A Batllori E Orellana R Bautista F Quality of groundwater for irrigation in tropical karst environment: The case of Yucatán Mexico. Agricultural Water Management 2010 97 10 1423 1433 10.1016/j.agwat.2010.04.006
Delgado, C., Pacheco, J., Cabrera, A., Batllori, E., Orellana, R., & Bautista, F. (2010). Quality of groundwater for irrigation in tropical karst environment: The case of Yucatán. Mexico. Agricultural Water Management, 97(10), 1423–1433. 10.1016/j.agwat.2010.04.00610.1016/j.agwat.2010.04.006
Gold-Bouchot G Arcega-Cabrera F Ceja-Moreno V Dissolved/dispersed polycyclic aromatic hydrocarbon spatial and temporal changes in the Western Gulf of Mexico Frontiers in Marine Science 2023 9 1069315 10.3389/fmars.2022.1069315
Gold-Bouchot, G., Arcega-Cabrera, F., & Ceja-Moreno, V. (2023). Dissolved/dispersed polycyclic aromatic hydrocarbon spatial and temporal changes in the Western Gulf of Mexico. Frontiers in Marine Science,  9, 1069315. 10.3389/fmars.2022.106931510.3389/fmars.2022.1069315
Gold-Bouchot G Ceja-Moreno V Chan-Cocom E Zapata-Perez O Petroleum hydrocarbons, fluorescent aromatic compounds in fish bile and organochlorine pesticides from areas surrounding the spill of the Kab121 well, in the southern Gulf of Mexico: A case study Journal of Environmental Biology 2014 35 1 147 156 24579530
Gold-Bouchot, G., Ceja-Moreno, V., Chan-Cocom, E., & Zapata-Perez, O. (2014). Petroleum hydrocarbons, fluorescent aromatic compounds in fish bile and organochlorine pesticides from areas surrounding the spill of the Kab121 well, in the southern Gulf of Mexico: A case study. Journal of Environmental Biology, 35(1), 147–156.24579530
Gold-Bouchot G Rubio-Piña J Montero-Muñoz J Ramirez-Miss N Echeverría-García A Patiño-Suarez V Puch-Hau CA Zapata-Pérez O Pollutants and biomarker responses in two reef fish species (Haemulon aurolineatum and Ocyurus chrysurus) in the southern Gulf of Mexico Marine Pollution Bulletin 2017 116 2017 249 257 10.1016/j.marpolbul.2016.12.073 28088368
Gold-Bouchot, G., Rubio-Piña, J., Montero-Muñoz, J., Ramirez-Miss, N., Echeverría-García, A., Patiño-Suarez, V., Puch-Hau, C. A., & Zapata-Pérez, O. (2017). Pollutants and biomarker responses in two reef fish species (Haemulon aurolineatum and Ocyurus chrysurus) in the southern Gulf of Mexico. Marine Pollution Bulletin, 116(2017), 249–257. 10.1016/j.marpolbul.2016.12.07328088368 10.1016/j.marpolbul.2016.12.073
Gusso-Choueri PK Choueri RB de Araújo GS Cruz ACF de Oliveira Stremel TR de Campos SX de Souza Abessa DM de Oliveira Ribeiro CA Univariate or multivariate approaches for histopathological biomarkers in the context of environmental quality assessments? Marine Pollution Bulletin 2022 181 113828 10.1016/j.marpolbul.2022.113828 35716493
Gusso-Choueri, P. K., Choueri, R. B., de Araújo, G. S., Cruz, A. C. F., de Oliveira Stremel, T. R., de Campos, S. X., de Souza Abessa, D. M., & de Oliveira Ribeiro, C. A. (2022). Univariate or multivariate approaches for histopathological biomarkers in the context of environmental quality assessments? Marine Pollution Bulletin,  181, 113828. 10.1016/j.marpolbul.2022.11382835716493 10.1016/j.marpolbul.2022.113828
Herrera-Silveira, J. A., & Morales-Ojeda, S. M. (2010). Subtropical Karstic coastal lagoon assessment, Southeast Mexico, the Yucatan peninsula case. In M. J. Kennish & H. W. Paerl (Eds.), Coastal lagoons: Critical habitats of environmental change (pp. 307–333). CRC Press
Hook SE Gallagher EP Batley GE The role of biomarkers in the assessment of aquatic ecosystem health Integrated Environmental Assessment and Management 2014 10 3 327 341 10.1002/ieam.1530 24574147
Hook, S. E., Gallagher, E. P., & Batley, G. E. (2014). The role of biomarkers in the assessment of aquatic ecosystem health. Integrated Environmental Assessment and Management,  10(3), 327–341. 10.1002/ieam.153024574147 10.1002/ieam.1530
Humason, G. L. (1962). Animal tissue techniques (p. 468). Freeman. 10.5962/bhl.title.5890
Javed M Usmani N An overview of the adverse effects of heavy metal contamination on fish health Procedings of the National Academy of Sciences, India, Section B: Biological Sciences 2019 89 389 403 10.1007/s40011-017-0875-7
Javed, M., & Usmani, N. (2019). An overview of the adverse effects of heavy metal contamination on fish health. Procedings of the National Academy of Sciences, India, Section B: Biological Sciences,  89, 389–403. 10.1007/s40011-017-0875-710.1007/s40011-017-0875-7
Khoshnood Z Using biomarkers in ecotoxicology: What and why? Focus on Sciences 2016 2 2 1 2 10.20286/focsci-020237
Khoshnood, Z. (2016). Using biomarkers in ecotoxicology: What and why? Focus on Sciences, 2(2), 1–2.10.20286/focsci-020237
Love M Baldera A Yeung C Robbins C The gulf of Mexico ecosystem: A coastal and marine atlas 2013 Ocean Conservancy, Gulf Restoration Center 162
Love, M., Baldera, A., Yeung, C., & Robbins, C. (2013). The gulf of Mexico ecosystem: A coastal and marine atlas (p. 162). Ocean Conservancy, Gulf Restoration Center.
Luna LG Manual of histologic staining methods of the armed forces institute of pathology 1968 3 McGraw-Hill 258
Luna, L. G. (1968). Manual of histologic staining methods of the armed forces institute of pathology (3rd ed., p. 258). McGraw-Hill.
Monteiro SM Mancera JM Fontaínhas-Fernandes A Sousa M Copper induced alterations of biochemical parameters in the gill and plasma of Oreochromis niloticus Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology 2005 141 375 383 16185936
Monteiro, S. M., Mancera, J. M., Fontaínhas-Fernandes, A., & Sousa, M. (2005). Copper induced alterations of biochemical parameters in the gill and plasma of Oreochromis niloticus. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology,  141, 375–383.16185936
Murawski SA Hogarth WT Peebles EB Barbeiri L Prevalence of external skin lesions and polycyclic aromatic hydrocarbon concentrations in Gulf of Mexico fishes, post deepwater horizon Transactions of the American Fisheries Society 2014 143 4 1084 1097 10.1080/00028487.2014.911205
Murawski, S. A., Hogarth, W. T., Peebles, E. B., & Barbeiri, L. (2014). Prevalence of external skin lesions and polycyclic aromatic hydrocarbon concentrations in Gulf of Mexico fishes, post deepwater horizon. Transactions of the American Fisheries Society, 143(4), 1084–1097. 10.1080/00028487.2014.91120510.1080/00028487.2014.911205
Myers MS Johnson LL Collier TK Establishing the causal relationship between polycyclic aromatic hydrocarbon (PAH) exposure and hepatic neoplasms and neoplasia-related liver lesions in English sole (Pleuronectes vetulus) Human and Ecological Risk Assessment: An International Journal 2003 9 1 67 94 10.1080/713609853
Myers, M. S., Johnson, L. L., & Collier, T. K. (2003). Establishing the causal relationship between polycyclic aromatic hydrocarbon (PAH) exposure and hepatic neoplasms and neoplasia-related liver lesions in English sole (Pleuronectes vetulus). Human and Ecological Risk Assessment: An International Journal, 9(1), 67–94. 10.1080/71360985310.1080/713609853
Ocaña FA Pech D Simões N Hernández-Ávila I Spatial assessment of the vulnerability of benthic communities to multiple stressors in the Yucatan continental shelf, Gulf of Mexico Ocean and Coastal Management 2019 104900 104900 10.1016/j.ocecoaman.2019.104900
Ocaña, F. A., Pech, D., Simões, N., & Hernández-Ávila, I. (2019). Spatial assessment of the vulnerability of benthic communities to multiple stressors in the Yucatan continental shelf, Gulf of Mexico. Ocean and Coastal Management, 104900,. 10.1016/j.ocecoaman.2019.10490010.1016/j.ocecoaman.2019.104900
Pallardy, R. (2023). Derrame de petróleo de deepwater horizon. Enciclopedia Británica. https://www.britannica.com/event/Deepwater-Horizon-oil-spill
Peters, E. M., Zaragoza Álvarez, R. A., Herzka, Sharon Z., & Herguera García, J. C. (Eds.) (2021). Introducción. En S. Z. Herzka, R. A. Zaragoza Álvarez, E. M. Peters y G. Hernández Cárdenas. (Coord. Gral.). Atlas de línea base ambiental del golfo de México. Consorcio de Investigación del Golfo de México
Poleksic, V., & Mitrovic-Tutundzic, V. (1994). Fish gills as a monitor of sublethal and chronic effects of pollution. In R. Müller, & R. Lloyd (Eds.), Sublethal and chronic effects of pollutants on freshwater fish (pp. 339–352). Cambridge Univ
Pulster, E. L., Gracia, A., Snyder, S. M., Deak, K., Fogleson, S., & Murawski, S. A. (2020). Chronic sublethal effects observed in wild caught fish following two major oil spills in the Gulf of Mexico: Deepwater Horizon and Ixtoc 1 (Chap. 24). In S. A. Murawski, C. Ainsworth, S. Gilbert, D. Hollander, C. B. Paris, M. Schlüter, & D. Wetzel (Eds.), Deep oil spills: Facts, fate and effects. Springer International.
Quintanilla-Mena, M., Gold-Bouchot, G., Zapata-Pérez, O., Rubio-Piña, J., Quiroz-Moreno, A., Vidal-Martínez, V. M., Aguirre-Macedo, M. L., & Puch-Hau, C. (2019). Biological responses of shoal flounder (Syacium gunteri) to toxic environmental pollutants from the southern Gulf of Mexico. Environmental Pollution, 113669. 10.1016/j.envpol.2019.113669
Rajeshkumar S Munuswamy N Impact of metals on histopathology and expression of HSP 70 in different tissues of milk fish (Chanos chanos) of Kaattuppalli Island, South East Coast, India Chemosphere 2011 83 4 415 421 10.1016/j.chemosphere.2010.12.086 21257190
Rajeshkumar, S., & Munuswamy, N. (2011). Impact of metals on histopathology and expression of HSP 70 in different tissues of milk fish (Chanos chanos) of Kaattuppalli Island, South East Coast, India. Chemosphere,  83(4), 415–421. 10.1016/j.chemosphere.2010.12.08621257190 10.1016/j.chemosphere.2010.12.086
Ricker WE Computation and interpretation of biological statistics of fish populations Bulletin of the Fisheries Research Board of Canada 1975 191 1 382
Ricker, W. E. (1975). Computation and interpretation of biological statistics of fish populations. Bulletin of the Fisheries Research Board of Canada, 191, 1–382.
Rojo-Nieto E Oliva M Sales D Perales JA Feral finfish, and their relationships with sediments and seawater, as a tool for risk assessment of PAHs in chronically polluted environments Science of the Total Environment 2014 470–471 1030 1039 10.1016/j.scitotenv.2013.10.062
Rojo-Nieto, E., Oliva, M., Sales, D., & Perales, J. A. (2014). Feral finfish, and their relationships with sediments and seawater, as a tool for risk assessment of PAHs in chronically polluted environments. Science of the Total Environment,  470–471, 1030–1039. 10.1016/j.scitotenv.2013.10.06210.1016/j.scitotenv.2013.10.062
Schmitt, C. J., Dethloff, G. M. (2000). Biomonitoring of environmental status and trends (BEST) program: Selected methods for monitoring chemical contaminants and their effects in aquatic ecosystems. U.S. geological survey, biological resources division (p. 81). Information and technology report USGS/BRD-2000--0005.
Schwaiger J Wanke R Adam S Pawert M Hönnen W Triebskorn R The use of histopathological indicators to evaluate contaminant-related stress in fish Journal of Aquatic Ecosystem Stress Recovery 1997 6 75 86 10.1023/A:1008212000208
Schwaiger, J., Wanke, R., Adam, S., Pawert, M., Hönnen, W., & Triebskorn, R. (1997). The use of histopathological indicators to evaluate contaminant-related stress in fish. Journal of Aquatic Ecosystem Stress Recovery, 6, 75–86. 10.1023/A:100821200020810.1023/A:1008212000208
Silva AG Martinez BR Morphological changes in the kidney of a fish living in an urban stream Environmental Toxicology and Pharmacology 2007 23 2 185 192 10.1016/j.etap.2006.08.009 21783756
Silva, A. G., & Martinez, B. R. (2007). Morphological changes in the kidney of a fish living in an urban stream. Environmental Toxicology and Pharmacology, 23(2), 185–192. 10.1016/j.etap.2006.08.00921783756 10.1016/j.etap.2006.08.009
Simonato JD Guedes CLB Martinez CBR Biochemical, physiological, and histological changes in the neotropical fish Prochilodus lineatus exposed to diesel oil Ecotoxicology and Environmental Safety 2008 69 1 112 120 10.1016/j.ecoenv.2007.01.012 17368761
Simonato, J. D., Guedes, C. L. B., & Martinez, C. B. R. (2008). Biochemical, physiological, and histological changes in the neotropical fish Prochilodus lineatus exposed to diesel oil. Ecotoxicology and Environmental Safety,  69(1), 112–120. 10.1016/j.ecoenv.2007.01.01217368761 10.1016/j.ecoenv.2007.01.012
Snyder SM Pulster EL Murawski SA Associations between chronic exposure to polycyclic aromatic hydrocarbons and health indices in Gulf of Mexico tilefish (Lopholatilus chamaeleonticeps) post Deepwater Horizon Environmental Toxicology and Chemistry 2019 1–13 1 10.1002/etc.4583
Snyder, S. M., Pulster, E. L., & Murawski, S. A. (2019). Associations between chronic exposure to polycyclic aromatic hydrocarbons and health indices in Gulf of Mexico tilefish (Lopholatilus chamaeleonticeps) post Deepwater Horizon. Environmental Toxicology and Chemistry, 1–13, 1. 10.1002/etc.458310.1002/etc.4583
Soto LA Botello AV Legal issues and scientific constraints in the environmental assessment of the Deepwater Horizon oil spill in Mexico exclusive economic zone (EEZ) in the Gulf of Mexico International Journal of Geosciences 2013 4 5 39 45 10.4236/ijg.2013.45B007
Soto, L. A., & Botello, A. V. (2013). Legal issues and scientific constraints in the environmental assessment of the Deepwater Horizon oil spill in Mexico exclusive economic zone (EEZ) in the Gulf of Mexico. International Journal of Geosciences, 4(5), 39–45. 10.4236/ijg.2013.45B00710.4236/ijg.2013.45B007
Straif K Baan R Grosse Y Secretan B El Ghissassi F Cogliano V Carcinogenicity of polycyclic aromatic hydrocarbons The Lancet Oncology 2005 6 12 931 932 10.1016/s1470-2045(05)70458-7 16353404
Straif, K., Baan, R., Grosse, Y., Secretan, B., El Ghissassi, F., & Cogliano, V. (2005). Carcinogenicity of polycyclic aromatic hydrocarbons. The Lancet Oncology,  6(12), 931–932. 10.1016/s1470-2045(05)70458-716353404 10.1016/s1470-2045(05)70458-7
Tashla T Žuža M Kenjveš T Prodanović R Soleša D Bursić V Petrović A Pelić LD Bošković J Puvača N Fish as an important bio-indicator of environmental pollution with persistent organic pollutants and heavy metals Journal of Agronomy 2018 1 1 52 56
Tashla, T., Žuža, M., Kenjveš, T., Prodanović, R., Soleša, D., Bursić, V., Petrović, A., Pelić, L. D., Bošković, J., & Puvača, N. (2018). Fish as an important bio-indicator of environmental pollution with persistent organic pollutants and heavy metals. Journal of Agronomy, 1(1), 52–56.
Teh, S. J., Adams, S. M., & Hinton, D. E. (1997). Histopathologic biomarkers in feral freshwater fish populations exposed to different types of contaminant stress. Aquatic Toxicology, 37(1), 51–70. 10.1016/S0166-445X(96)00808-9
Ter Braak, C. J. F., Šmilauer, P. (2012). Canoco reference manual and user’s guide: Software for ordination, version 5 0. Microcomputer Power.
USEPA (U.S. Environmental Protection Agency)., USGS (U.S. Geological Survey)., U.S. Fish and Wildlife Service. (2012). Toxic contaminants in the Chesapeake Bay and its watershed: Extent and severity of occurrence and potential biological effects (pp. 175). USEPA Chesapeake Bay Program Office.
Vázquez-Gómez G Rubio-Lightbourn J Espinosa-Aguirre JJ Mecanismos de acción del receptor de hidrocarburos de arilos en el metabolismo del benzo [a] pireno y el desarrollo de tumores Revista Especializada En Ciencias Químico-Biológicas, TIP 2016 19 1 54 67 10.1016/j.recqb.2016.02.006
Vázquez-Gómez, G., Rubio-Lightbourn, J., & Espinosa-Aguirre, J. J. (2016). Mecanismos de acción del receptor de hidrocarburos de arilos en el metabolismo del benzo [a] pireno y el desarrollo de tumores. Revista Especializada En Ciencias Químico-Biológicas, TIP, 19(1), 54–67. 10.1016/j.recqb.2016.02.00610.1016/j.recqb.2016.02.006
Vidal-Martínez VM Velázquez-Abunader I Centeno-Chalé OA May-Tec AL Soler-Jiménez LC Pech D Mariño-Tapia I Enriquez C Zapata-Pérez O Herrera-Silveira J Hernández-Mena D Herzka SZ Ordoñez-López U Aguirre-Macedo ML Metazoan parasite infracommunities of the dusky flounder Syacium papillosum as bioindicators of environmental conditions in the continental shelf of the Yucatan Peninsula, Mexico Parasites and Vectors 2019 12 277 10.1186/s13071-019-3524-6 31151478
Vidal-Martínez, V. M., Velázquez-Abunader, I., Centeno-Chalé, O. A., May-Tec, A. L., Soler-Jiménez, L. C., Pech, D., Mariño-Tapia, I., Enriquez, C., Zapata-Pérez, O., Herrera-Silveira, J., Hernández-Mena, D., Herzka, S. Z., Ordoñez-López, U., & Aguirre-Macedo, M. L. (2019). Metazoan parasite infracommunities of the dusky flounder Syacium papillosum as bioindicators of environmental conditions in the continental shelf of the Yucatan Peninsula, Mexico. Parasites and Vectors, 12, 277. 10.1186/s13071-019-3524-631151478 10.1186/s13071-019-3524-6
Yancheva V Velcheva I Stoyanova S Georgieva E Histological biomarkers in fish as a tool in ecological risk assessment and monitoring programs: A review Applied Ecology and Environmental Research 2016 14 1 47 75 10.15666/aeer/1401_047075
Yancheva, V., Velcheva, I., Stoyanova, S., & Georgieva, E. (2016). Histological biomarkers in fish as a tool in ecological risk assessment and monitoring programs: A review. Applied Ecology and Environmental Research, 14(1), 47–75. 10.15666/aeer/1401_04707510.15666/aeer/1401_047075
