Segmentation of Renal Thyroid Follicle Colloid in Common Carp: Insights into Perfluorooctanoic Acid-Induced Morphometric Alterations.

Perfluorooctanoic acid (PFOA) is a globally prevalent contaminant of concern recognised for its persistence and detrimental effects on both wildlife and humans. While PFOA has been established as a disruptor of thyroid function, limited data exist regarding its impact on thyroid morphology. The kidney of the common carp (Cyprinus carpio) harbours numerous thyroid follicles, rendering it a valuable biomarker organ for investigating PFOA-induced thyroid alterations. Renal tissue slides, stained with the Alcian blue/PAS method, were examined from carp in three experimental groups: unexposed, exposed to 200 ng L-1, and exposed to 2 mg L-1 of PFOA over 56 days. Thyroid follicle colloids were segmented, and related morphometric parameters, including perimeter, area, and shape descriptors, were obtained. Statistical analyses revealed significant reductions in thyroid follicle colloid perimeter and area in the 200 ng L-1 PFOA group compared to the unexposed and 2 mg L-1 PFOA groups. Additionally, the fish exposed to PFOA exhibited a significantly higher follicle count compared to the unexposed fish. These findings collectively suggest that PFOA induces thyroid folliculogenesis, emphasising its impact on thyroid morphology even at an environmentally relevant concentration (200 ng L-1).

Teleost innate immunity, an intricate game between immune cells and parasites of fish organs: who wins, who loses.

Fish, comprising over 27,000 species, represent the oldest vertebrate group and possess both innate and adaptive immune systems. The susceptibility of most wild fish to parasitic infections and related diseases is well-established. Among all vertebrates, the digestive tract creates a remarkably favorable and nutrient-rich environment, which, in turn, renders it susceptible to microparasites and macroparasites. Consequently, metazoan parasites emerge as important disease agents, impacting both wild and farmed fish and resulting in substantial economic losses. Given their status as pathogenic organisms, these parasites warrant considerable attention. Helminths, a general term encompassing worms, constitute one of the most important groups of metazoan parasites in fish. This group includes various species of platyhelminthes (digeneans, cestodes), nematodes, and acanthocephalans. In addition, myxozoans, microscopic metazoan endoparasites, are found in water-dwelling invertebrates and vertebrate hosts. It is worth noting that several innate immune cells within the fish alimentary canal and certain visceral organs (e.g., liver, spleen, and gonads) play active roles in the immune response against parasites. These immune cells include macrophages, neutrophils, rodlet cells, and mast cells also known as eosinophilic granular cells. At the site of intestinal infection, helminths often impact mucous cells number and alter mucus composition. This paper presents an overview of the state of the art on the occurrence and characteristics of innate immune cells in the digestive tract and other visceral organs in different fish-parasite systems. The data, coming especially from studies employed immunohistochemical, histopathological, and ultrastructural analyses, provide evidence supporting the involvement of teleost innate immune cells in modulating inflammatory responses to metazoan and protozoan parasitic infections.

Perfluorooctanoic Acid Promotes Recruitment and Exocytosis of Rodlet Cells in the Renal Hematopoietic Tissue of Common Carp.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous environmental contaminants, with perfluorooctanoic acid (PFOA) being a prominent member. PFOA poses a risk to aquatic ecosystems and human health due to its presence in water, environmental persistence, and bioaccumulation. Since rodlet cells (RCs) have emerged as potential biomarkers for chemical stressors, this study aimed to investigate the effects of sub-chronic PFOA exposure on RCs in the renal hematopoietic tissue of common carp. Three groups of fish were used: an unexposed control group and two groups exposed to environmentally relevant (200 ng L-1) and elevated (2 mg L-1) PFOA concentrations. Light and transmission electron microscopy were employed to assess RCs' distribution patterns and exocytosis, while biometry quantified RCs in the hematopoietic tissue. The results showed that, even at environmentally relevant concentrations, PFOA significantly influenced RCs' distribution patterns, leading to increased occurrence and cluster formation, as well as heightened exocytosis activity. This research highlights PFOA's immunotoxicity in fish and suggests the potential of RCs as sentinel cells in the immunological response to environmental contaminants. These findings enhance our understanding of PFAS toxicity and emphasise the importance of monitoring their impact on fish as representative vertebrates and reliable animal models.

Ultrastructural Alterations of the Glomerular Filtration Barrier in Fish Experimentally Exposed to Perfluorooctanoic Acid.

Per- and polyfluoroalkyl substances can be referred to as the most critical group of contaminants of emerging concern. They can accumulate in high concentration in the kidney and are known to potentially affect its function. Nonetheless, there is a lack of knowledge about their morphopathological effect on the glomerular filtration barrier. Since previous research suggests perfluorooctanoic acid (PFOA) induces glomerular protein leakage, the glomerular filtration barrier of 30 carp from the same parental stock (10 unexposed; 10 exposed to 200 ng L-1 of PFOA; and 10 exposed to 2 mg L-1 of PFOA for 56 days) was screened for possible PFOA-induced ultrastructural lesions in order to shed light on the related pathophysiology. PFOA exposure affected the glomerular filtration barrier in carp experimentally exposed to 2 mg L-1, showing ultrastructural alterations compatible with glomerulonephrosis: podocyte effacement, reduction of filtration slits and filtration slit diaphragms, basement membrane disarrangement, and occurrence of proteinaceous material in the urinary space. The results of the present research confirm the glomerular origin of the PFOA-induced protein leakage and can contribute to the mechanistic comprehension of PFOA's impact on renal function and to the assessment of the exposure effect of environmental pollutants on animals and humans, according to the One Health approach.

Ecology and effects of metazoan parasites of fish in transitional waters.

Given the abundance, heterogeneity and ubiquity of parasitic organisms, understanding how they influence biodiversity, evolution, health and ecosystem functionality is crucial, especially currently when anthropogenic pressures are altering host­parasite balances. This review describes the features, roles and impacts of metazoan parasites of fish occurring in transitional waters (TW). These aquatic ecosystems are highly productive and widespread around the globe and represent most favourable theatres for parasitism given the availability of hosts (invertebrates, fishes and birds) and an increased probability of parasite transmission, especially of those having complex life cycles. Fascinating examples of how parasitism can influence different hierarchical levels of biological systems, from host individuals and populations to entire aquatic communities, through effects on food webs come from this kind of ecosystem. Edible fish of commercial value found in TW can harbour some parasite species, significantly reducing host health, marketability and food safety, with possible economic and public health consequences. Many TW are historically exploited by humans as sources of relevant ecosystem services, including fisheries and aquaculture, and they are highly vulnerable ecosystems. Alteration of TW can be revealed through the study of parasite communities, contributing, as bioindicators, for assessing environmental changes, health and restoration. Fish parasites can provide much information about TW, but this potential appears to be not fully exploited. More studies are necessary to quantify the ecological, economic and medical impacts fish parasites can have on these important ecosystems.

Perfluorooctanoic Acid Affects Thyroid Follicles in Common Carp (Cyprinus carpio).

Carp kidney is comprised of nephrons, hemopoietic tissue, and also hormonally-active thyroid follicles. Given this anatomical trait, it has been used to assess the thyroid disrupting potential of perfluorooctanoic acid (PFOA), a widespread and feared per- poly-fluoroalkyl substance and a persistent organic pollutant capable of interfering with the endocrine system in animals and humans. The occurrence and morphology of thyroid follicles in kidneys of carp experimentally exposed to 200 ng L-1 or 2 mg L-1 waterborne PFOA for 56 days were studied. The abundance of thyroid follicles was significantly higher and vesiculation increased in exposed fish as compared to controls. The number of vesiculated follicles/total number of follicles was positively correlated with PFOA blood concentration in fish exposed to the highest dose (2 mg L-1). The structure and ultrastructure of thyroid follicles were affected by PFOA also at the lower, environmentally relevant, concentration (200 ng L-1). Increased cellular projections, enhanced colloid endocytosis, rough endoplasmic reticulum enlargement and fragmentation and cytoplasm vacuolation were the main features displayed by PFOA-exposed carp. These results show that PFOA affects the occurrence and status of follicles and suggest the utility of fish kidney as a multipurpose biomarker organ in environmental pathology research, according to the One Health approach.

Effect of waterborne exposure to perfluorooctanoic acid on nephron and renal hemopoietic tissue of common carp Cyprinus carpio.

Per- and poly-fluoroalkyl substances (PFAS) are synthetic contaminants of global concern for environmental and public health. Perfluorooctanoic acid (PFOA) is an important PFAS, and considerable attention has been paid to its hepatotoxicity and reproductive and developmental impact, while potential nephrotoxic effects are largely ignored, especially in fish. This study documents the structural and ultrastructural effects on kidney of common carp Cyprinus carpio exposed to waterborne PFOA at an environmentally relevant concentration of 200 ng L-1 and at 2 mg L-1. Dilation of the glomeruli capillary bed, increased vesiculation in the proximal tubular segment, compromised mitochondria, apical blebbing, and sloughing of collecting duct cells occurred in exposed fish, primarily at 2 mg L-1. Perfluorooctanoic acid exposure resulted in higher numbers of rodlet cells (RC), putative immune cells exclusive to fish, mainly in the renal interstitium, than seen in controls, increased association with cells of myeloid lineage and modifications to ultrastructure. No differences in other cells of innate immunity were observed. Despite the absence of severe histological lesions, PFOA was shown to affect both nephron and hemopoietic interstitium at high concentration, raising concern of the impact on renal and immune function in fish. The response of RCs to PFOA concentration of 200 ng L-1 suggests a potential role as a biomarker of PFOA exposure.

Survival of metazoan parasites in fish: Putting into context the protective immune responses of teleost fish.

Defence mechanisms of fish can be divided into specific and non-specific that act in concert and are often interdependent. Most fish in both wild and cultured populations are vulnerable to metazoan parasites. Endoparasitic helminths include several species of digeneans, cestodes, nematodes, and acanthocephalans. Although they may occur in large numbers, helminth infections rarely result in fish mortality. Conversely, some ectoparasites cause mass mortality in farmed fish. Given the importance of fish innate immunity, this review addresses non-specific defence mechanisms of fish against metazoan parasites, with emphasis on granulocyte responses involving mast cells, neutrophils, macrophages, rodlet cells, and mucous cells. Metazoan parasites are important disease agents that affect wild and farmed fish and can induce high economic loss and, as pathogen organisms, deserve considerable attention. The paper will provide our light and transmission electron microscopy data on metazoan parasites-fish innate immune and neuroendocrine systems. Insights about the structure and functions of the cell types listed above and a brief account of the effects and harms of each metazoan taxon to specific fish apparati/organs will be presented.

Perfluorooctanoic acid-induced cellular and subcellular alterations in fish hepatocytes.

Liver perfluorooctanoic acid (PFOA) pathophysiology and related morphofunction disturbances were studied in common carp at the cellular and subcellular level and with box-counting fractal analysis of ultrathin sections to assess the effect of PFOA exposure on hepatocyte structure complexity and heterogeneity. Three experimental groups were investigated: unexposed; low exposure (200 ng L-1 PFOA); high exposure (2 mg L-1 PFOA). PFOA-exposed cells showed differences from controls at both tested concentrations, manifested mainly as cloudy swelling and reversible vacuolar degeneration. Subcellular modifications primarily involved mitochondria and secondarily endoplasmic reticulum, with evidence of increased subcellular turnover. The alterations were consistent with oxidative stress related pathophysiology. Fractal analysis discriminated exposed from unexposed fish and low from high PFOA exposure based on lacunarity and fractal dimension, respectively. The absence of irreversible organelle alterations and apoptosis/necrosis, along with the increase of cellular complexity, led to the conclusion that the patterns observed represented an adaptive recovery response.

Temporal dynamics of species associations in the parasite community of European eels, Anguilla anguilla, from a coastal lagoon.

The resilience of biological communities is of central importance in ecology, but is difficult to investigate in nature. Parasite communities in individual hosts provide good model systems, as they allow a level of replication usually not possible with free-living communities. Here, using temporal data (2005-2017) on the communities of endohelminth parasites in European eels, Anguilla anguilla, from brackish-water lagoons in Italy, we test the resilience of interspecific associations to changes in the abundance of some parasite species and the disappearance of others. While most parasite species displayed changes in abundance over time, three trematodes that were present in the early years, two of which at high abundance, completely disappeared from the parasite community by the end of the study period. Possibly other host species required for the completion of their life cycles have declined in abundance, perhaps due to environmental changes. However, despite these marked changes to the overall community, pairwise correlations in abundance among the three most common parasite species (all trematodes) were stable over time and remained mostly unaffected by what happened to other species. We explore possible reasons for these resilient species associations within a temporally unstable parasite community inhabiting a mostly stable host population.

Perfluorooctanoic Acid Exposure Assessment on Common Carp Liver through Image and Ultrastructural Investigation.

Perfluorooctanoic acid (PFOA) poses particular concern as an emerging pollutant in both surface and ground waters. Fish, as a natural inhabitant of these waters and being highly representative of vertebrates, represents an ideal animal model to assess the toxic effects of PFOA. Hereby, liver microscopic texture was comparatively evaluated in individuals of common carp subchronically exposed to PFOA using grayscale differential box counting, a fractal analysis method. Furthermore, liver cytoplasmic glycogen areas and ultrastructure were also evaluated and compared to the image analysis findings. Redundancy Analysis was performed to assess, in summary, how much the variation of fractal dimension and lacunarity was explained by the concentration of PFOA in liver, the mass of liver and the number of proliferating cell nuclear antigen (PCNA)-immunoreactive nuclei. Treatment group ordination was better determined by fractal dimension than lacunarity. Interestingly, a significant complexity increase was associated with the modification of liver microscopic texture due to PFOA exposure. This complexity increase was related to "cloudy swelling", possibly representing a primarily adaptive strategy against PFOA challenge, rather than a slight, reversible form of degeneration as traditionally proposed. The occurrence of endoplasmic reticulum stress, unfolded protein reaction and hormetic response was proposed and discussed.

Description of epithelial granular cell in catshark spiral intestine: Immunohistochemistry and ultrastructure.

We evaluated the histology of the spiral intestine of the blackmouth catshark (Galeus melastomus), a small shark distributed in the eastern Atlantic and Mediterranean Sea basin. Entire digestive tracts of 10 G. melastomus were studied using histochemical, immunohistochemical, and ultrastructural methods. Our studies identified a unique, large granular cell type in the intestinal epithelium. Transmission electron microscopy showed that the epithelial granular cell type made intimate contact, by means of junctional complexes, with adjacent epithelial and mucous cells. Several histochemical staining methods showed that the cytoplasmic granules were strongly eosinophilic. Immunostaining of intestinal sections revealed immunoreactivity of the granular cell to tumor necrosis factor-α (TNF-α) antibody. However, no reactivity to inducible-nitric oxide synthase (i-NOS), interleukin-6 (IL-6), interleukin IL-1ß, lysozyme, serotonin 5-HT antibodies was detected.

Pike intestinal reaction to Acanthocephalus lucii (Acanthocephala): immunohistochemical and ultrastructural surveys.

BACKGROUND: The Northern pike, Esox lucius, is a large, long-lived, top-predator fish species and occupies a broad range of aquatic environments. This species is on its way to becoming an important model organism and has the potential to contribute new knowledge and a better understanding of ecology and evolutionary biology. Very few studies have been done on the intestinal pathology of pike infected with helminths. The present study details the first Italian record of adult Acanthocephalus lucii reported in the intestine of E. lucius. RESULTS: A total of 22 pike from Lake Piediluco (Central Italy) were examined, of which 16 (72.7%) were infected with A. lucii. The most affected areas of gastrointestinal tract were the medium and distal intestine. The intensity of infection ranged from 1 to 18 parasites per host. Acanthocephalus lucii penetrated mucosal and submucosal layers which had a high number of mast cells (MCs) with an intense degranulation. The cellular elements involved in the immune response within the intestine of pike were assessed by ultrastructural techniques and immunohistochemistry using antibodies against met-enkephalin, immunoglobulin E (IgE)-like receptor (FCεRIγ), histamine, interleukin-6, interleukin-1ß, substance P, lysozyme, serotonin, inducible-nitric oxide synthase (i-NOS), tumor necrosis factor-α (TNF-α) and the antimicrobial peptide piscidin 3 (P3). In intestines of the pike, several MCs were immunopositive to 9 out of the 11 aforementioned antibodies and infected fish had a higher number of positive MCs when compared to uninfected fish. CONCLUSIONS: Pike intestinal tissue response to A. lucii was documented. Numerous MCs were seen throughout the mucosa and submucosal layers. In infected and uninfected intestines of pike, MCs were the dominant immune cell type encountered; they are the most common granulocyte type involved in several fish-helminth systems. Immunopositivity of MCs to 9 out of 11 antibodies is of great interest and these cells could play an important key role in the host response to an enteric helminth. This is the first report of A. lucii in an Italian population of E. lucius and the first account on positivity of MCs to piscidin 3 and histamine in a non-perciform fish.

Pigmented macrophages and related aggregates in the spleen of european sea bass dosed with heavy metals: Ultrastructure and explorative morphometric analysis.

The ultrastructure and morphometrics of pigmented macrophages (PMs) were assessed in the spleen of European sea bass experimentally dosed with Cd and Hg. PMs occurred either as solitary cells or as variably structured aggregations, defined as macrophage aggregates (MAs). Light microscopy revealed a high degree of morphological heterogeneity amongst MAs of all experimental groups. At the ultrastructural level, MAs showed a heterogeneous pigment content that was not influenced by the treatment. Cytoplasm rarefaction/vacuolation and euchromatic nuclei, were observed in PMs of dosed fish. Undosed and Cd-dosed samples differ significantly with regard to the following morphometric features: the Minor axis of the best fitting ellipse, Aspect Ratio, and Roundness. In Cd-dosed fish, MAs showed reduced size and complexity. Lacunarity showed significant differences between undosed and both Cd and Hg-dosed samples. These results suggest that heavy metals, and especially Cd, may influence the dynamics of PM aggregation/disaggregation. Variability in splenic MAs was observed both by light and electron microscopy. However, only the morphometric techniques adequately and objectively described the phenomenon, allowing a quantitative/statistical comparison of morphology among experimental groups. These morphometric analyses could be usefully applied in toxicological and ecotoxicological, as well as morpho-functional studies.

Environmental doses of perfluorooctanoic acid change the expression of genes in target tissues of common carp.

We aimed to evaluate the effects of environmental doses of perfluorooctanoic acid (PFOA) on bioconcentration and gene expression in common carp (Cyprinus carpio). Adult male and female carp were exposed to environmental (200 ng/L) and experimental (2 mg/L) doses of PFOA for 56 d. Carp exposed to 200 ng/L had levels of PFOA below the level of detection in all tissue samples analyzed, whereas variable concentrations were measurable in various tissues from carp exposed to 2 mg/L. The expression level of the glutathione S-transferase (GST) gene, coding for a detoxifying enzyme, increased in a PFOA dose-dependent manner in liver tissues from 200 ng/L to 2 mg/L exposure (p < 0.05). The expression levels of CYP19A gene, coding for the enzyme that converts testosterone into estrogen, were altered in gonadal tissues from male and female carp exposed to either 200 ng/L or 2 mg/L; expression increased in male gonads and decreased in female gonads. Unexpectedly, the expression levels of CYP19A in male and female gonads from carp exposed to 200 ng/L or 2 mg/L were similar (p > 0.05). Therefore, even though environmental doses of PFOA did not accumulate in tissues of the common carp, they did affect the gene expression levels of GST in the liver and CYP19A in the gonads. These observations raise concerns that exposure to environmental doses of PFOA may affect gene expression in animals and possibly in humans, with important health consequences. Environ Toxicol Chem 2018;37:942-948. © 2017 SETAC.

Histological and ultrastructural study of Myxobolus mugchelo (Parenzan, 1966) with initial histopathology survey of the Liza ramada host intestine.

This paper provides histological and ultrastructural data on Myxobolus mugchelo (Myxozoa), a parasite of the intestinal wall of thinlip mullet Liza ramada from the Comacchio lagoons (Northern Adriatic Sea, Italy). About 80% of the examined mullets were infected with this myxozoan. In histological sections, the plasmodia appeared rounded to ovoid and spindle shaped, measured 181.61 ± 97 × 122.79 ± 53.16 µm (mean ± standard deviation, SD) in size. Early sporogenic plasmodia were located primarily in the muscle layer where no pronounced inflammatory response was detected. Plasmodia containing mature spores were situated closer to mucosal folds and were encircled by numerous immune cells, especially mast cells. Mature spores were generally oval and 6.06 ± 0.40 × 3.48 ± 0.92 µm in size. The spores showed symmetrical and smooth valves. The anterior end of the spores contained two equal pyriform polar capsules measuring 2.19 ± 0.47 × 1.59 ± 0.29 µm. Inside the polar capsules, an isofilar polar filament displayed 5-6 coils perpendicular or slightly oblique to the longitudinal axis. The wall of the polar capsule was filled with a hyaline substance contrasting with the very dense internal matrix. The mucus layer surrounded the spore surface. A thorough comparison with the Myxobolus species infecting intestine of fish is provided. This is the first report on occurrence of this myxozoan species in L. ramada population of the North Adriatic Sea.

Nematode infection in liver of the fish Gymnotus inaequilabiatus (Gymnotiformes: Gymnotidae) from the Pantanal Region in Brazil: pathobiology and inflammatory response.

BACKGROUND: A survey on endoparasitic helminths from freshwater fishes in the Pantanal Region (Mato Grosso do Sul, Brazil) revealed the occurrence of third-larval stage of the nematode Brevimulticaecum sp. (Heterocheilidae) in most organs of Gymnotus inaequilabiatus (Gymnotidae) also known by the local name tuvira. The aim of the present study was to examine Brevimulticaecum sp.-infected tuvira liver at the ultrastructural level and clarify the nature of granulomas and the cellular elements involved in the immune response to nematode larvae. METHODS: Thirty-eight adult specimens of tuvira from Porto Morrinho, were acquired in January and March 2016. Infected and uninfected liver tissues were fixed and prepared for histological and ultrastructure investigations. RESULTS: The prevalence of infection of tuvira liver by the nematode larvae was 95 %, with an intensity of infection ranging from 4 to 343 larvae (mean ± SD: 55.31 ± 73.94 larvae per liver). In livers with high numbers of nematode larvae, almost entire hepatic tissue was occupied by the parasites. Hepatocytes showed slight to mild degenerative changes and accumulation of pigments. Parasite larvae were surrounded by round to oval granulomas, the result of focal host tissue response to the infection. Each granuloma was typically formed by three concentric layers: an outer layer of fibrous connective tissue with thin elongated fibroblasts; a middle layer of mast cells entrapped in a thin fibroblast-connective mesh; and an inner layer of densely packed epithelioid cells, displaying numerous desmosomes between each other. Numerous macrophage aggregates occurred in the granulomas and in the parenchyma. CONCLUSIONS: Our results in tuvira showed that the larvae were efficiently sequestered within the granulomas, most of the inflammatory components were confined within the thickness of the granuloma, and the parenchyma was relatively free of immune cells and without fibrosis. Presumably this focal encapsulation of the parasites permits uninfected portions of liver to maintain its functions and allows the survival of the host.

Multivariate approach to gill pathology in European sea bass after experimental exposure to cadmium and terbuthylazine.

The combined use of guided quantitative expert analysis and of multivariate exploratory data analysis is reported as a robust, sensitive and sufficiently specific approach to study European sea bass gill secondary lamellar pathology after exposure to incremental doses of cadmium and terbuthylazine up to 48h. The following elementary pathological findings were considered: "epithelial lifting", "epithelial shrinkage", "epithelial swelling", "pillar cells coarctation", "pillar cells detachment", "channels fusion", "chloride cells swelling", and "chloride cells invasion". The relative spatial extension was determined according to exposure class and data were analyzed by means of canonical correspondence analysis (CCA), linear discriminant analysis (LDA) and canonical variates analysis (CVA). Histologically and ultrastructurally, cellular shrinkage/coarctation prevailed in cadmium exposed lamellae, whereas cellular swelling and epithelial lifting were predominant in terbuthylazine exposed lamellae compared to unexposed fish. Both CCA and CVA permit a good graphical data grouping according to exposure classes by means of the convex hull minimum polygons. This also reveals exposure dose and time gradients in CCA plot. Accordingly, epithelial swelling and epithelial shrinkage were comparatively associated to higher exposure time, whereas epithelial shrinkage and pillar cells coarctation were comparatively associated to higher exposure dose. LDA with only "epithelial shrinkage", "epithelial swelling" and "pillar cells coarctation" in the model classified correctly 87.5% of the cross-validated cases. A possible pathogenetic relationship between the discriminant elementary lesions and the toxic mode of action at the cellular level of both cadmium and terbuthylazine is also discussed.

Common carp Cyprinus carpio responses to sub-chronic exposure to perfluorooctanoic acid.

Perfluorooctanoic acid (PFOA) is an important and diffuse perfluorinated alkylated substance, but knowledge of the toxicological effects of this endocrine disrupter in fish is limited. Adult common carp Cyprinus carpio, L. were exposed to 200 ng/l (a concentration reported in impacted aquatic ecosystems) and 2 mg/l PFOA solutions in a flow-through system for 56 days to determine tissue accumulation and histological alterations of the primary target organs. PFOA was extracted from blood, gill, liver, muscle, kidney, gonad, and brain by an ion-pairing liquid extraction procedure and quantified using high performance liquid chromatography with electrospray ionization tandem mass spectrometry. The limit of detection (LOD) was 0.4 ng/g wet weight (ww). PFOA was not detectable in unexposed fish or in fish exposed to 200 ng/l, but was >LOD in most samples of carp exposed to 2 mg/l. Mean PFOA concentration ranged from 0.5 to 65 ng/g ww, depending on the tissue, with highest levels in the blood and liver. There were no significant differences in condition factor, hepato-somatic index, or gonado-somatic index among the fish of the three groups. Histological, histochemical, and immunohistochemical staining was performed on sections of liver and gonad. Occurrence of atretic oocytes and a paucity of spermatozoa were documented in carp treated with 2 mg/l PFOA. Exposed fish did not show gross hepatic anomalies, but there was enhancement of hepatocytes in proliferation (positive to anti-PCNA antibody) compared to controls.

Local connected fractal dimension analysis in gill of fish experimentally exposed to toxicants.

An operator-neutral method was implemented to objectively assess European seabass, Dicentrarchus labrax (Linnaeus, 1758) gill pathology after experimental exposure to cadmium (Cd) and terbuthylazine (TBA) for 24 and 48h. An algorithm-derived local connected fractal dimension (LCFD) frequency measure was used in this comparative analysis. Canonical variates (CVA) and linear discriminant analysis (LDA) were used to evaluate the discrimination power of the method among exposure classes (unexposed, Cd exposed, TBA exposed). Misclassification, sensitivity and specificity, both with original and cross-validated cases, were determined. LCFDs frequencies enhanced the differences among classes which were visually selected after their means, respective variances and the differences between Cd and TBA exposed means, with respect to unexposed mean, were analyzed by scatter plots. Selected frequencies were then scanned by means of LDA, stepwise analysis, and Mahalanobis distance to detect the most discriminative frequencies out of ten originally selected. Discrimination resulted in 91.7% of cross-validated cases correctly classified (22 out of 24 total cases), with sensitivity and specificity, respectively, of 95.5% (1 false negative with respect to 21 really positive cases) and 75% (1 false positive with respect to 3 really negative cases). CVA with convex hull polygons ensured prompt, visually intuitive discrimination among exposure classes and graphically supported the false positive case. The combined use of semithin sections, which enhanced the visual evaluation of the overall lamellar structure; of LCFD analysis, which objectively detected local variation in complexity, without the possible bias connected to human personnel; and of CVA/LDA, could be an objective, sensitive and specific approach to study fish gill lamellar pathology. Furthermore this approach enabled discrimination with sufficient confidence between exposure classes or pathological states and avoided misdiagnosis.