Josiane Rodrigues Rocha da Silva1 
, Camila Oliveira de Andrade1, Fernanda Losi Alves de Almeida2 and Carlos Alexandre Fernandes1,3
PDF: EN XML: EN | Supplementary: S1 S2 S3 S4 S5 | Cite this article
Abstract
Corantes reativos, comumente utilizados na indústria têxtil, têm mostrado potencial para causar danos a organismos aquáticos. Diante da falta de estudos abrangentes sobre os efeitos dos corantes reativos, nosso estudo teve como objetivo avaliar o impacto do corante reativo Novacron® Bold Deep Navy nas brânquias do peixe Astyanax lacustris. Especificamente, buscamos determinar a toxicidade do corante antes e após o tratamento utilizando um sistema de wetlands construídos de fluxo vertical (WCFV). Durante o experimento, espécimes de A. lacustris foram expostos a concentrações subletais (10, 20, 30 e 40 mg L-1) do corante Novacron® Bold Deep Navy e ao corante tratado pelo WCFV por 96 horas. Nossos resultados indicaram que o corante causou danos significativos nas brânquias, incluindo fusão das lamelas secundárias, hiperplasia e hipertrofia das células epiteliais lamelares, além de telangiectasia e aneurisma, em comparação com o grupo controle (p < 0,05) em todas as concentrações. As imagens de Microscopia Eletrônica de Varredura (MEV) revelaram o desaparecimento das microdigitações à medida que a concentração do corante aumentava. O sistema WCFV demonstrou eficiente biorremediação do corante, conforme evidenciado pela redução dos danos nas brânquias e análises físico-químicas favoráveis. Esses resultados destacam o risco potencial do corante para organismos aquáticos, ao mesmo tempo em que mostram a eficácia do sistema WCFV na remoção do corante.
Palavras-chave: Biorremediação, Corantes reativos, Peixes, Toxicidade, Wetlands.
Introduction
The textile industry is one of the oldest sectors in the world. Despite being an important economic activity, its production has contributed to environmental pollution. This is due to its high water usage and various toxic chemical products discarded as effluents with high polluting potential (Kumar, Gunasundari, 2017). An estimated 10 to 15% of synthetic dyes used in the textile industry are lost during the dyeing process, and as much as 20% are directly released into various environmental components (Yanto, Tachibana, 2014). Textile dye waste is highly toxic, and recent studies have highlighted dyes’ carcinogenic and mutagenic properties (Karthick et al., 2018). In humans, they can also cause allergic reactions (Huessin et al., 2019), respiratory issues (Leme et al., 2015), kidney diseases, dermatitis, dementia, and diarrhea, among other health concerns, which can be transmitted through contaminated water (Lin et al., 2022). Additionally, apart from the loss of aquatic biodiversity, persistent dyes can accumulate in the fatty tissues of aquatic animals, mainly fish (Pande et al., 2019). Several studies show the bioaccumulation of heavy metals, which are present in textile dyes (Aldoghachi et al., 2016; Sanyal et al., 2017; Mehra, Chadha, 2020). When consumed by humans, this leads to the bioaccumulation of substances in the body (Pande et al., 2019).
Synthetic dyes utilized in various industries encompass diverse organic compounds with differing origins, chemical and physical properties, and application processes (Paz et al., 2017). Consequently, numerous studies have investigated the effects of these xenobiotics on various facets of fish (Silva et al., 2023). However, the rapid expansion of man-made substances necessitates increased research into the cyto/genotoxicity and other metabolic disturbances caused by these compounds (Silva et al., 2024). This is crucial for understanding organisms’ responses to contamination, evaluating the impact on cells, tissues, and organs, and predicting potential consequences for species and ecosystems (Puvaneswari et al., 2006).
Despite being widely used in the textile industry, Novacron® Bold Deep Navy, a reactive dye with a blue hue, has not been the subject of specific studies. This lack of research is significant, as reactive dyes are classified as harmful substances for aquatic organisms and may cause long-term adverse environmental effects (European Commission and European Parliament, 2008).
Fish, particularly Astyanax lacustris (Lütken, 1875) specimens, play a crucial role as biological models in ecotoxicological studies (Bu, 2012; Silva et al., 2023; Andrade et al., 2024). These fish have shown a high sensitivity to various chemical contaminants present in polluted waters and are commonly exposed to mutagens or carcinogens, which can lead to changes in their DNA structure (Tincani et al., 2019). Gills play a crucial role in respiration, excretion, and osmoregulation, comprising approximately 50% of the surface area of fish. They are primary targets for toxicants (Singh, 2014; Sumi, Chitra, 2017). Due to their susceptibility, analyzing histological biomarkers from gills provides valuable insights into the environmental damage inflicted by toxicants such as textile dyes, aiding in the development of more effective monitoring strategies. Biotechnological methods have been suggested to mitigate the adverse effects of chemical pollutants in aquatic environments, primarily through their removal. Constructed Wetlands (CWs) have garnered attention as a comprehensive and viable option for phytoremediation. Their effectiveness lies in the combination of physical (filtration and sedimentation), chemical (adsorption and precipitation), and biological (biodegradation and phytoremediation) processes, making them suitable for pretreating effluents before their discharge into water bodies (Hassan et al., 2021; Kiflay et al., 2021). CWs are essentially shallow artificial lagoons or channels with a filtering bed, hosting aquatic plants that aid in reducing various types of contaminants in the effluent (Dotro et al., 2017).
Given the urgent need for research in this area, the present study aims to evaluate the histopathological effects of the textile dye on A. lacustris before and after treatment through the vertical flow constructed wetland system (VFCW) using gill histological biomarkers.
Material and methods
Textile dye. The commercial Reactive dye, Novacron® Bold Deep Navy (Huntsman International LLC Textile Effects located at 3400 Westinghouse Blvd, Charlotte, NC 28273, USA. LOT: 0071443800 No CAS: 68259–02–9 /130201–57–9 / 72214–18–7 / 77447–41–8) was obtained from a local source and used directly for experimental purpose. A stock solution was prepared by dissolving accurately weighed dye in distilled water to the concentration of 100 mg L-1. It was stored it at 4 °C temperature. The experimental concentrations were obtained by diluting the stock solution in accurate proportions with dechlorinated water.
The dye was utilized in different concentrations for each group, with group 1 serving as a negative control with dechlorinated water. Group 2 had a concentration of 10 mg L-1, group 3 at 20 mg L-1, group 4 at 30 mg L-1, group 5 at 40 mg L-1, and group 6 at 40 mg L-1, which was treated through the VFCW system using phytoremediation. The concentrations chosen for this study were carefully tested and selected based on previous research on other textile dyes that have demonstrated cyto/genotoxic changes at these levels (Anlinker, 1977; Zhang et al., 2012; Parmar, Barot, 2016; Parmar, Shah, 2019; Yaseen, Scholz, 2019).
Construction of experimental built wetland treatment units with the vertical flow (VFCW). The making of the VFCW units followed some suggestions from the manual by Sezerino et al. (2018), with adaptations according to Silva et al. (2024). This study made them from two cylindrical high-density polyethylene (HDPE) containers (0.80 m in height X 0.55 m in diameter). The filter mass of the first stage was composed of 0.2 m of crushed stone (25–50 mm), 0.15 m of crushed stone (19–25 mm), and 0.45 m of crushed stone (7–9.5 mm) (gravel). The second stage consisted of 0.2 m of gravel (19–25 mm), 0.15 m of gravel (7–9.5 mm), and 0.45 m of sand (1.2–2 mm). The adduction system consisted of 25 mm diameter polyvinyl chloride pipes and connections. The effluent was drained through an adductor system with perforations of 8.0 mm in diameter distributed along its entire extremity. The drainage pipe was positioned horizontally at the bottom of the bed, extending across the whole diameter of the units. The system contains a faucet for outputting the treated effluent at the bottom of the reservoir, installed 10 cm from the bottom.
The beds were populated with Typha domingensis at 16 plants per square meter density. The propagules were collected manually in a naturally flooded area located on a rural property in the municipality of Cianorte at the beginning of December 2021. The collection was carried out so that the rhizomes were preserved, carefully transported, and transplanted in the experimental units. The residence time of the effluent in the treatment system was 96 h, 48 h in the first stage, and 48 h in the second stage.
Physical-chemical analysis of the effluent. After treatment, the raw and treated Novacron® Bold Deep Navy dye were stored in a thermal box, kept at ± 5 ºC, and taken to the laboratory for physical-chemical characterization. The analyses were conducted in the Departamento de Engenharia Civil, Laboratório de Qualidade da Água e Controle da Poluição – Saneamento Ambiental da Universidade Estadual de Maringá (UEM). Six physicochemical parameters of this textile effluent – pH, apparent color, BOD, COD, total suspended solids, and electrical conductivity were determined using standard methods (Rice et al., 2012).
Experimental design. Adult individuals (males and females) of A. lacustris, with a mean weight of 4±5 g and length of 6±8 cm, were obtained from a local breeding facility. The fish were acclimatized in aquaria with dechlorinated water at room temperature, constant aeration, a natural photoperiod (12:12 h light/dark cycle), and fed with specific small fish feed once daily (Basic Alcon® Fish Food, Camboriú/SC, Brazil) for 10 days in the Sectorial Vivarium for Fish Keeping and Experimentation at the Universidade Estadual de Maringá (UEM) in Maringá, Paraná State, Brazil. Voucher specimens were deposited in the Fish Collection of the Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (NUPELIA), Universidade Estadual de Maringá, municipality of Maringá, Paraná State, Brazil, as Astyanax lacustris (NUP 25442).
Six groups/ conditions were tested, with four fish per group, in total, 30 fish were used in the study. The number of individuals per aquarium was established according to the body mass/water volume ratio, which should not exceed 0.5–2 g L-1 (CONCEA Guide, annex I, fish). In the control group (Group 1), the fish were maintained in dechlorinated water. In the other five experimental groups (Group 2 to 6), the fish were exposed to the Novacron® Bold Deep Navy dye at concentrations of 10, 20, 30, 40 mg L-1 and 40 mg L-1 treated by phytoremediation through the VFCW system dissolved in 10 L of water for 96 h for each concentration, corresponding to an acute test. In all cases, the total aquarium volume was 10 L. All samples were coded and analyzed under blind conditions.
Histological analysis. After four days of exposure, the fish were carefully removed from the aquarium one at a time and bathed in an anesthetic solution of clove oil, as per Inoue et al. (2005). The animal was only manipulated after it failed to respond to physical stimulus, they were euthanized by cranial perforation, which denotes brain death (following CONCEA Normative Resolution No 37/2018).
Immediately after euthanasia, their gills were excised, rinsed in a 0.9% saline solution, and subsequently fixed in Bouin’s aqueous solution for 12 h (Behmer et al., 1976). Following this, the gills were stored in 70% alcohol. For histological processing, the specimens were dehydrated using a sequential increase of alcohol concentrations (80%, 90%, and 100%), clarified with xylene, and embedded in paraffin. Semi-serial cross-sections with a thickness of 5 μm were prepared using a LEICA rotary microtome at the Laboratório de Histotecnologia Animal do Departamento de Ciências Morfológicas of the Universidade Estadual de Maringá. The slides were stained using the Hematoxylin-Eosin (H.E.) and Periodic Acid-Schiff (PAS) methods (Behmer et al., 1976). An optical microscope (Olympus CX31RBSFA) was employed to analyze the observed changes.
Scanning electron microscopy (SEM). Three individuals were randomly selected from each of the Novacron® Bold Deep Navy dye samples to analyze their gills using scanning electron microscopy. The gills were carefully dissected and then fixed in Bouin solution (composed of 7.5% picric acid, 2.5% formaldehyde, and 0.5% acetic acid) for duration of 24 h at room temperature. Following this, the samples underwent a series of dehydration steps with alcohol (7.5%, 15%, 30%, 50%, 70%, 90%, and 100%) and were subsequently critically dried in a LEICA CPD030 drier. They were then coated with gold using a Shimadzu IC-50 metalizer. Analyses were conducted using a Quanta 250-Fei scanning microscope at the Centro de Microscopia do Complexo de Centros de Apoio à Pesquisa (COMCAP) at the Universidade Estadual de Maringá, Paraná, Brazil (Gigliolli et al., 2015). The sample height was set at 10 mm from the detector, with an accelerating voltage of 15.00 KV and a spot size of 3.0. The microscope interface was managed via the xT Microscope program. To eliminate potential bias, the operator was blinded to the treatment groups, and the resulting images were randomized prior to final evaluation. Qualitative analyses of gill alterations were performed, focusing on capturing microridge images from the primary lamellae to ensure consistency in the tissue location during image capture.
Quantitative analysis of gill changes. For morphological analysis and changes in the gills, 30 random fields per animal were evaluated under an optical microscope with a total magnification of 40x (Olympus CX31RBSFA) according to the semiquantitative method proposed by Schwaiger et al. (1997). Graduated Histological Change Index (HAI) scale, depending on the severity of the lesions, as described by Mallatt (1985) with adaptation: Damage 0: no histological changes; Damage 1: small changes; Damage 2: moderate and specific changes; Damage 3: moderate and extensive changes; Damage 4: severe, extensive and irreparable changes, due to secondary lamellar fusion, hypertrophy and hyperplasia of lamellar epithelial cells. For telangiectasia and aneurysm, only qualitative analyses of gills were performed, in which 30 random fields per animal were evaluated under an optical microscope at 40x total magnification (Olympus CX31RBSFA), and the quantity per field was counted.
Statistical analysis. Statistical analyses of the collected data were conducted to assess normality using the Kolmogorov-Smirnov test. Levene’s test was used to assess the homogeneity of variances between the experimental groups. The results indicated that the variances between the groups were comprehensive (p > 0.05), allowing the application of ANOVA for data analysis. A one-way analysis of variance (ANOVA) was performed, accompanied by Tukey’s post-test. A significance level of 5% was set, and the results were reported as mean ± standard error.
For the analysis of the frequency distributions of damage in the alterations of secondary lamellar fusion, hyperplasia, and hypertrophy, between the experimental groups and the control group, two statistical tests were performed: the Chi-square test and the Fisher’s exact test (p > 0.05).
Results
This study examined the histology of Astyanax lacustris gills exposed to the textile dye Novacron® Bold Deep Navy, revealing various gill abnormalities such as lamellar fusion, epithelial cells hyperplasia, and hypertrophy, which were quantitatively evaluated. Aneurysms and telangiectasia were found to be significantly different from the control across all concentrations and the treated effluent, with a dose-dependent frequency of lesions.
Histological analyses revealed normal gills (Fig. 1A), with telangiectasia (Fig. 1B), aneurysm (Fig. 1C), secondary lamellar fusion (Fig. 1D), lamellar epithelium hyperplasia (Fig. 1E), and hypertrophy (Fig. 1F). The normal gills presented the expected structure, with well-defined and organized lamellae, without signs of damage or abnormalities. However, several other pathological changes were observed in samples exposed to the dye, varying in severity and quantity as dye concentrations increased (Fig. 2). The scanning electron microscope (SEM) revealed the disappearance of microdigitations as the dye concentration increased (Fig. 3).
FIGURE 1 | Photomicrograph of Astyanax lacustris gills exposed to different concentrations of Novacron® Bold Deep Navy textile dye and stained with hematoxylin and eosin (HE). A. Normal lamellar, B. Telangiectasia, C. Aneurysm, D. Secondary lamellar fusion, E. Hyperplasia and F. Hypertrophy. The arrows indicate the changes in the gills.
FIGURE 2| Proportional distribution of damage (0, 1, 2, 3, and 4) in the gills of Astyanax lacustris at different concentrations of the textile dye Novacron® Bold Deep Navy in each group: A. Secondary lamellar fusion, B. Hyperplasia, and C. Hypertrophy.
FIGURE 3| Photomicrograph of Astyanax lacustris gills exposed to different concentrations of Novacron® Bold Deep Navy textile dye in scanning electron microscopy. A. Control, B. Treated by phytoremediation through the VFCW system, C. 10 mg L-1, D. 20 mg L-1, E. 30 mg L-1 and F. 40 mg L-1. It is worth noting the reduction in microdigitations (arrows) with increasing doses. Scale bar = 10 µm.
FIGURE 4| Frequency mean of aneurysm (A) and telangiectasia (B) in Astyanax lacustris gills in different concentrations of Novacron® Bold Deep Navy textile dye.
Secondary lamellar fusion, for example, in the control group showed only 11 mild changes, while the group tested at a concentration of 40 mg L-1 showed 128 severe and extensive and/or irreparable changes (Tab. S1). In lamellar epithelium hyperplasia, the control group showed only 19 mild changes, while the group tested at a concentration of 40 mg L-1 showed 101 severe and extensive and/or irreparable changes (Tab. S2); similarly, in epithelial cells hypertrophy, the control group showed only 15 mild changes, while the group tested at a concentration of 40 mg L-1 showed 64 severe and extensive and/or irreparable changes (Tab. S3).
With the exception of the hypertrophy alteration, for which there was no significant difference between the control and treated group, in the other alterations, all concentrations presented significant differences when compared to the control group (p < 0.05) (Fig. 2). The results for changes in telangiectasia (Tab. S4) and aneurysm (Tab. S5) were also dose-dependent. On the other hand, a significant reduction in changes was observed in the group treated by the VFCW system, both quantitatively and qualitatively. However, the statistical analysis for telangiectasia and aneurysm, comparing the control with the other concentrations of the dye and the treated dye, resulted in significant differences (p < 0.05) (Fig. 4).
The results of the physical-chemical analysis of the textile dye are summarized in Tab. 1. Particularly, there was a substantial 99.79% decrease in Biochemical Oxygen Demand (BOD), which signifies the oxygen required for the breakdown of biodegradable organic matter. Additionally, the Chemical Oxygen Demand (COD), reflecting both biodegradable organic matter and that produced by chemical processes, showed a notable reduction of 99.76%. There has been a decrease in conductivity by 98.55% and a 61% reduction in apparent color. This finding is critical because the color of the effluent is directly linked to the presence of organic substances and can have a negative impact on the receiving water bodies. Furthermore, there has been a decrease in total nitrogen by 94%, turbidity by 99.75%, suspended solids by 93.5%, and total dissolved solids by 34%, indicating a reduction in salts and other dissolved components in the treated effluent.
TABLE 1 | Values of physicochemical parameters before and after treatment of textile dye treated by vertical flow constructed wetland system (VFCW).
Assay | Raw textile dye | VFCW-treated textile dye |
Electric conductivity (μS/cm) | 15500 | 224 |
Apparent color (uH) | 286.9 | 111.6 |
Biochemical oxygen demand (BOD) (mg L-1) | 6800 | 14 |
Chemical oxygen demand (COD) (mg L-1) | 20100 | 48 |
pH | 7.0 | 7.93 |
Total dissolved solids (mg L-1) | 155 | 102 |
Total phosphorus (mg L-1) | 0.09 | 0.08 |
Total Nitrogen (mg L-1) | 182 | <10 |
Suspended solids (mg L-1) | 158 | 1.0 |
Turbidity (uT) | 328 | 0.80 |
Discussion
Histopathological changes in gills can serve as biomarkers in fish, considering that fish gills are crucial for respiration, excretion, gas exchange, and osmoregulation, making them an important indicator of population health and reflective of the overall well-being of an aquatic ecosystem in the biomonitoring process (Sumi, Chitra, 2017; Hasan et al., 2022).
Increased size of branchial epithelium, lamellar fusion, and hyperplasia are primary indicators of fish pathology (Thophon et al., 2003; Carvalho et al., 2020; Pramanik, Biswas, 2024). These changes are part of the fish’s natural defense mechanism to prevent the entry of contaminants, creating a barrier between the blood and the aquatic environment, ultimately reducing the efficiency of gill gas exchange (Hesni et al., 2011; Maurya et al., 2019). Hyperplasia and hypertrophy of lamellar epithelial cells can typically be reversed upon removal of the contaminant from the water (Nilsson et al., 2012). However, continuous exposure can lead to irreversible stages where restoration is no longer possible.
Aneurysms and telangiectasia may arise from increased blood flow in the branchial lamellae, causing the rupture of pillar cells and vascular structures (Ahmed et al., 2013). Aneurysms represent a severe form of vascular damage characterized by abnormal widening or ballooning of blood vessels, which can result in bleeding and significant compromise to the body’s health (Azadbakht et al., 2019; Hasan et al., 2022). Telangiectasia involves the dilation of capillaries in the branchial lamellae, leading to visibly enlarged blood vessels (Strzyżewska-Worotyńska et al., 2017).
The study conducted by Shahid et al. (2021) on Ictalurus punctatus, as well as the research by Sharma et al. (2022) on Channa punctatus and Kaur, Dua (2015) on Labeo rohita, all observed similar results when the fish were exposed to different types of polluted effluents. The lesions found in the gills are believed to be the result of exposure to a combination of substances, rather than a specific contaminant, a view supported by Virgens et al. (2015) and Barbieri et al. (2022).
The scanning electron microscopy (SEM) revealed that the microdigitations of the gill epithelial cells gradually diminished as the dosage of the contaminant increased. These structures, resembling fingerprints and commonly present on the epithelial cells located on the primary lamellae of gill (Eiras-Stofella et al., 2001), seemingly contribute to the structural support of the branchial epithelium and enhance cell surface area, which in turn facilitates substance absorption (Lam et al., 2015). While their specific function is not yet established, they are believed to be crucial to the physiology of healthy organisms. Research on various xenobiotics has shown alterations in gill microdigitations (Ba-Omar et al., 2011; Barbieri et al., 2022).
The effectiveness of treatment using Constructed Wetlands (CW) has been highlighted in various research studies. For instance, a study by Watharkar et al. (2015) demonstrated that textile effluent treated by CW with bacteria immobilized in Etheostoma olmstedi fish maintained the integrity of the lamellae without causing any toxic effects on the gills of the fish. Similarly, Rane et al. (2015) examined sulfonated remazol red dye and textile effluents and found that histological observations of the gills of fish exposed to untreated effluents showed various distortions, whereas those exposed to effluents treated by phytoremediation in constructed wetlands with the macrophyte Alternanthera philoxeroides Griseb did not exhibit significant deformities. These studies, along with our own, confirm the efficacy of constructed wetlands in treating and decolorizing dyes from the textile industry.
Upon analyzing all the changes in gill health and emphasizing the importance of a thriving ecosystem, the reduction of environmental damage observed in the group treated with dye by the CW is noteworthy. The results of the physicochemical analyses, a substantial improvement in the quality of the textile dye was observed. It is important to highlight that, in addition to the removal of organic matter, the effective treatment of chemical pollutants, such as heavy metals, commonly found in industrial effluents, is of utmost importance. Several studies demonstrate the effectiveness of the treatment in eliminating both organic and chemical pollutants present in the effluent (Andrade et al., 2024; Silva et al., 2024). These findings emphasize the need to mitigate the negative impacts of effluents on aquatic ecosystems, highlighting that the use of systems such as constructed wetlands can be decisive in the removal of these pollutants. Regulations, such as the UN Sustainable Development Goals (SDGs), specifically Goal 6 (Clean Water and Sanitation), underscore the importance of effective wastewater treatment systems to protect water resources and aquatic life globally.
Similar results have been reported in other studies, such as the work by Hussein, Scholz (2017), which focused on a wetland constructed with vertical flow (WCVF) for treating Acid Blue 113 and Basic Red 46 dyes and demonstrated a COD removal rate ranging between 50% and 94% over different hydraulic retention periods (48 h and 96 h). Furthermore, according to the study conducted by Shehzadi et al. (2014), the Typha domingensis plant was used WCVF and exhibited the potential to degrade textile effluent. The inoculation of endophytic bacteria further improved the degradation of the textile effluent, leading to reduced BOD, COD, and apparent color values compared to uninoculated plants.
The findings of this study indicate that the dye has a harmful impact on aquatic organisms, affecting the well-being and respiratory function of exposed fish. The biomarkers and bioindicator utilized proved to be effective for environmental biomonitoring. The Wastewater Treatment Plant’s process yielded positive results in terms of the effluent’s physical-chemical parameters and the reduction of toxicity observed in the gill histology of the specimens. However, further research is needed to investigate various types of treatment systems that can be employed to assess their efficacy in the presence of different flora, microorganisms, and pollutants.
Acknowledgments
We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), through the Dean of the Departamento de Pós-Graduação e Pesquisa of the Universidade Estadual de Maringá (UEM-PPG), for the master’s scholarship granted to JRRS.
References
Ahmed MdK, Habibullah-Al-Mamun Md, Parvin E, Akter MS, Khan MS. Arsenic induced toxicity and histopathological changes in gill and liver tissue of freshwater fish, tilapia (Oreochromis mossambicus). Exp Toxicol Pathol. 2013; 65(6):903–09. https://doi.org/10.1016/j.etp.2013.01.003
Aldoghachi MAJ, Rahman M, Yusoff IB, Azirum MS. Bioaccumulation and histopathological changes induced by toxicity of mercury (HgCl2) to tilapia fish Oreochromis niloticus. Sains Malays. 2016; 45(1):119–12.
Andrade CO, Silva JRR, Barbieri PA, Borin-Carvalho LA, Portela-Castro ALB, Fernandes CA. The effect of acute exposure of yellowtail tetra fish Astyanax lacustris (Lütken, 1875) to the glyphosate-based herbicide Templo®. Mutat Res Genet Toxicol Environ Mutagen. 2024; 897:503771. https://doi.org/10.1016/j.mrgentox.2024.503771
Azadbakht F, Shirali S, Ronagh MT, Zamani I. Assessment of gill pathological responses in yellowfin sea bream (Acanthopagrus Latus) under Aeromonas Hydrophila exposure. Arch Razi Inst. 2019; 74(1):83–89. https://doi.org/10.22092/ari.2017.114702.1139
Ba-Omar TA, Al-Jardani S, Victor R. Effects of pesticide temephos on the gills of Aphanius dispar (Pisces: Cyprinodontidae). Tissue Cell. 2011; 43(1):29–38. https://doi.org/10.1016/j.tice.2010.11.002
Barbieri PA, Mari-Ribeiro IP, Lupepsa L, Gigliolli AAS, Paupitz BR, Melo RF et al. Metformin-induced alterations in gills of the freshwater fish Astyanax lacustris (Lütken, 1875) detected by histological and scanning electron microscopy. Ecotoxicology. 2022; 31(8):1205–16. https://doi.org/10.1007/s10646-022-02580-0
Behmer OA, Tolosa EMC, Neto AGF. Manual de técnicas para histologia normal e patológica. Vol. 1. 1st ed. São Paulo: EDART Livraria Editora LTDA; 1976.
Bu A. Fish ecogenotoxicology: an emerging science, an emerging tool for environmental monitoring and risk assessment. J Environ Anal Toxicol. 2012; 3(1):100065. https://doi.org/10.4172/2161-0525.1000165
Carvalho TLAB, Nascimento AA, Gonçalves CFDS, Santos MAJ, Sales A. Assessing the histological changes in fish gills as environmental bioindicators in Paraty and Sepetiba bays in Rio de Janeiro, Brazil. Lat Am J Aquat Res. 2020; 48(4):590–601. https://doi.org/10.3856/vol48-issue4-fulltext-2351
Dotro G, Molle P, Nivala J. Biological wastewater treatment series. Vol. 7. London: Treatment Wetlands; 2017.
Eiras-Stofella DR, Charvet-Almeida P, Fanta E, Casagrande Vianna AC. Surface ultrastructure of the gills of the mullets Mugil curema, M. liza and M. platanus (Mugilidae, Pisces). J Morphol. 2001; 247(2):122–33. https://doi.org/10.1002/1097-4687(200102)247:2<122::AID-JMOR1007>3.0.CO;2-5
European Commission and European Parliament. Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures [Internet]. OJEU; 2008. Available from: https://eur-lex.europa.eu/eli/reg/2008/1272/oj
Gigliolli AAS, Lapenta AS, Ruvolo-Takasusuki MCC, Abrahão J, Conte H. Morpho-functional characterization and esterase patterns of the midgut of Tribolium castaneum, Herbst, 1797 (Coleoptera: Tenebrionidae) parasitized by Gregarina cuneata (Apicomplexa: Eugregarinidae). Micron. 2015; 76:68–78. https://doi.org/10.1016/j.micron.2015.04.008
Hasan J, Ferdous SR, Rabiya SBA, Hossain MF, Hasan AM, Shahjahan M. Histopathological responses and recovery in gills and liver of Nile tilapia (Oreochromis niloticus) exposed to diesel oil. Toxicol Rep. 2022; 9:1863–68. https://doi.org/10.1016/j.toxrep.2022.10.005
Hassan I, Chowdhury SR, Prihartato PK, Razzak SA. Wastewater treatment using constructed wetland: current trends and future potential. Processes. 2021; 9(11):1917. https://doi.org/10.3390/pr9111917
Hesni MA, Savari A, Mortazavi MS, Sohrab AD. Gill histopathological changes in milkfish (Chanos chanos) exposed to acute toxicity of diesel oil. World Appl Sci J. 2011; 14(10):1487–92.
Huessin H, Mensah RK, Brown RS. Diagnosis and management of oral allergy syndrome, the itchy tongue allergic reaction. Compend Contin Educ Dent. 2019; 40(8):502–05.
Hussein A, Scholz M. Dye wastewater treatment by vertical-flow constructed wetlands. Ecol Eng. 2017; 101:28–38. https://doi.org/10.1016/j.ecoleng.2017.01.016
Inoue LAKA, Afonso LOB, Iwama GK, Moraes G. Effects of clove oil on the stress response of matrinxã (Brycon cephalus) subjected to transport. Acta Amazon. 2005; 35(2):289–95. https://doi.org/10.1590/S0044-59672005000200018
Karthick K, Namasivayam C, Pragasan LA. Kinetics and isotherm studies on acid dye adsorption using thermal and chemical activated Jatropha husk carbons. Environ Prog Sustain Energy. 2018; 37(2):719–32. https://doi.org/10.1002/ep.12745
Kaur R, Dua A. 96 h LC50, behavioural alterations and histopathological effects due to wastewater toxicity in a freshwater fish Channa punctatus. Environ Sci Pollut Res. 2015; 22(7):5100–10. https://doi.org/10.1007/s11356-014-3710-1
Kiflay E, Selemani J, Njau K. Integrated constructed wetlands treating industrial wastewater from seed production. Water Pract Technol. 2021; 16(2):504–15. https://doi.org/10.2166/wpt.2021.008
Kumar PS, Gunasundari E. Sustainable wet processing – an alternative source for detoxifying supply chain in textiles. In: Muthu S, editor. Textile science and clothing technology. Singapore: Springer; 2017. p.37–60. https://doi.org/10.1007/978-981-10-4876-0_2
Maurya PK, Malik DS, Kumar YK, Gupta N, Kumar S. Haematological and histological changes in fish Heteropneustes fossilis exposed to pesticides from industrial waste water. Hum Ecol Risk Assess. 2019; 25(5):1251–78. https://doi.org/10.1080/10807039.2018.1482736
Lam P-y, Mangos S, Green JM, Reiser J, Huttenlocher A. In vivo imaging and characterization of actin microridges. PLoS ONE. 2015; 10(1):e0115639. https://doi.org/10.1371/journal.pone.0115639
Leme DM, Primo FL, Gobo GG, Costa CRV, Tedesco AC, Oliveira DP. Genotoxicity assessment of reactive and disperse textile dyes using human dermal equivalent (3D cell culture system). J Toxicol Environ Health A. 2015; 78(7):466–80. https://doi.org/10.1080/15287394.2014.999296
Lin L, Yang H, Xu X. Effects of water pollution on human health and disease heterogeneity: a review. Front Environ Sci. 2022; 10:880246. https://doi.org/10.3389/fenvs.2022.880246
Mallatt J. Fish gill structural changes induced by toxicants and other irritants: a statistical review. Can J Fish Aquat Sci. 1985; 42(4):630–48. https://doi.org/10.1139/f85-083
Mehra S, Chadha P. Bioaccumulation and toxicity of 2-naphthalene sulfonate: an intermediate compound used in the textile industry. Toxicol Res (Camb). 2020; 9(2):127–36. https://doi.org/10.1093/toxres/tfaa008
Nilsson GE, Dymowska A, Stecyk JAW. New insights into the plasticity of gill structure. Respir Physiol Neurobiol. 2012; 184(3):214–22. https://doi.org/10.1016/j.resp.2012.07.012
Pande V, Pandey SC, Joshi T, Sati D, Gangola S, Kumar S et al. Biodegradation of toxic dyes: a comparative study of enzyme action in a microbial system. In: Smart Bioremediation Technologies. Academic Press; 2019. p.255–87. https://doi.org/10.1016/B978-0-12-818307-6.00014-7
Parmar A, Barot Jk. Determination of genotoxic effect of azo dye CI RR 120 on fish Catla catla. Biotechnol Res. 2016; 2(2):77–80.
Parmar A, Shah A. Cytogenotoxicity of azo dye Reactive Red 120 (RR120) on fish Catla catla. Environ Exp Biol. 2019; 17(3):151–55.
Paz A, Carballo J, Pérez MJ, Domínguez JM. Biological treatment of model dyes and textile wastewaters. Chemosphere. 2017; 181:168–77. https://doi.org/10.1016/j.chemosphere.2017.04.046
Pramanik S, Biswas JK. Histopathological fingerprints and biochemical changes as multi-stress biomarkers in fish confronting concurrent pollution and parasitization. IScience. 2024; 27(12):111432. https://doi.org/10.1016/j.isci.2024.111432
Puvaneswari N, Muthukrishnan J, Gunasekaran P. Toxicity assessment and microbial degradation of azo dyes. Indian J Exp Biol. 2006; 44(8):618–26.
Rane NR, Chandanshive VV, Watharkar AD, Khandare RV, Patil TS, Pawar PK et al. Phytoremediation of sulfonated remazol red dye and textile effluents by Alternanthera philoxeroides: an anatomical, enzymatic and pilot scale study. Water Res. 2015; 83:271–81. https://doi.org/10.1016/j.watres.2015.06.046
Rice EW, Baird RB, Eaton AD, Lesceri LS. Standard methods for the examination of water and wastewater. 22nd ed. Denver, CO: American Water Works Association; 2012.
Sanyal T, Kaviraj A, Saha S. Toxicity and bioaccumulation of chromium in some freshwater fish. Hum Ecol Risk Assess. 2017; 23(7):1655–67. https://doi.org/10.1080/10807039.2017.1336425
Schwaiger J, Wanke R, Adam S, Pawert M, Honnen W, Triebskorn R. The use of histopathological indicators to evaluate contaminant-related stress in fish. J Aquat Ecosyst Stress Recovery. 1997; 6(1):75–86. https://doi.org/10.1023/A:1008212000208
Sezerino PH, Rousso BZ, Pelissari C, Santos MO, Freitas MN, Fechine VY et al. Wetlands construídos aplicados no tratamento de esgoto sanitário: recomendações para implantação e boas práticas de operação e manutenção. Tubarão: Fundação Nacional da Saúde – Funasa; 2018.
Shahid S, Sultana T, Sultana S, Hussain B, Irfan M, Al-Ghanim KA et al. Histopathological alterations in gills, liver, kidney and muscles of Ictalurus punctatus collected from pollutes areas of River. Braz J Biol. 2021; 81(3):814–21. https://doi.org/10.1590/1519-6984.234266
Sharma K, Sharma P, Dhiman SK, Chadha P, Saini HS. Biochemical, genotoxic, histological and ultrastructural effects on liver and gills of fresh water fish Channa punctatus exposed to textile industry intermediate 2 ABS. Chemosphere. 2022; 287:132103. https://doi.org/10.1016/j.chemosphere.2021.132103
Shehzadi M, Afzal M, Khan MU, Islam E, Mobin A, Anwar S et al. Enhanced degradation of textile effluent in constructed wetland system using Typha domingensis and textile effluent-degrading endophytic bacteria. Water Res. 2014; 58:152–59. https://doi.org/10.1016/j.watres.2014.03.064
Silva JRR, Andrade CO, Ribeiro AC, Macruz PD, Bergamasco R, Fernandes CA. Cyto and genotoxicity induced by acute exposure to Novacron® Bold Deep Navy dye on Astyanax lacustris can be reduced after treatment through a vertical flow constructed wetland system. Environ Sci Pollut Res. 2024; 31(59):66630–42. https://doi.org/10.1007/s11356-024-35687-4
Silva JRR, Gregorio A, Portela-Castro AL de B, Fernandes CA. Genotoxicity and cytotoxicity of textile production effluents, before and after Bacillus subitilis bioremediation, in Astyanax lacustris (Pisces, Characidae). Mutat Res Genet Toxicol Environ Mutagen. 2023; 886:503588. https://doi.org/10.1016/j.mrgentox.2023.503588
Singh RN. Effects of dimethoate (EC 30%) on gill morphology, oxygen consumption and serum electrolyte levels of common carp, Cyprinus carpio (Linn). Int J Sci Res Environ Sci. 2014; 2(6):192–98. https://doi.org/10.12983/ijsres-2014-p0192-0198
Strzyżewska-Worotyńska E, Szarek J, Babińska I, Gulda D. Gills as morphological biomarkers in extensive and intensive rainbow trout (Oncorhynchus mykiss, Walbaum 1792) production technologies. Environ Monit Assess. 2017; 189(12):611. https://doi.org/10.1007/s10661-017-6278-7
Sumi N, Chitra KC. Histopathological alterations in gill, liver and muscle tissues of the freshwater fish, Pseudetroplus maculatus exposed to fullerene C60. Int J Fish Aquat Stud. 2017; 5(3):604–08.
Thophon S, Kruatrachue M, Upatham ES, Pokethitiyook P, Sahaphong S, Jaritkhuan S. Histopathological alterations of white seabass, Lates calcarifer, in acute and subchronic cadmium exposure. Environ Pollut. 2003; 121(3):307–20. https://doi.org/10.1016/S0269-7491(02)00270-1
Tincani FH, Santos GS, Azevedo ACB, Marques AEML, Pereira LS, Castellano GC et al. Climbing the taxonomic ladder: could a genus be used as bioindicator? The ecotoxicological relationship between biomarkers of Astyanax altiparanae, Astyanax bifasciatus and Astyanax ribeirae. Ecol Indic. 2019; 106:105474. https://doi.org/10.1016/j.ecolind.2019.105474
Virgens AC, Castro RL, Cruz ZMA. Alterações histológicas em brânquias de Orechromis niloticus (Tilapia-Do-Nilo) expostas o acefato, difenoconazol e sulfluramida. Natureza Online. 2015; 13(1):27–31.
Watharkar AD, Khandare RV, Waghmare PR, Jagadale AD, Govindwar SP, Jadhav JP. Treatment of textile effluent in a developed phytoreactor with immobilized bacterial augmentation and subsequent toxicity studies on Etheostoma olmstedi fish. J Hazard Mater. 2015; 283:698–704. https://doi.org/10.1016/j.jhazmat.2014.10.019
Yanto DHY, Tachibana S. Enhanced biodegradation of asphalt in the presence of Tween surfactants, Mn2+ and H2O2 by Pestalotiopsis sp. in liquid medium and soil. Chemosphere. 2014; 103:105–13. https://doi.org/10.1016/j.chemosphere.2013.11.044
Yaseen DA, Scholz M. Textile dye wastewater characteristics and constituents of synthetic effluents: a critical review. Int J Environ Sci Technol. 2019; 16(2):1193–226. https://doi.org/10.1007/s13762-018-2130-z
Zhang W, Liu W, Zhang J, Zhao H, Zhang Y, Quan X et al. Characterisation of acute toxicity, genotoxicity and oxidative stress posed by textile effluent on zebrafish. J Environ Sci. 2012; 24(11):2019–27. https://doi.org/10.1016/S1001-0742(11)61030-9
Authors
Josiane Rodrigues Rocha da Silva1 , Camila Oliveira de Andrade1, Fernanda Losi Alves de Almeida2 and Carlos Alexandre Fernandes1,3
[1] Programa de Pós-Graduação em Biotecnologia Ambiental, Departamento de Biotecnologia, Genética e Biologia Celular, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil. (JRRS) josianerrs@hotmail.com (corresponding author), (COA) pg404535@uem.br, (CAF) cafernandes@uem.br.
[2] Departamento de Ciências Morfológicas, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil. (FLAA) flaalmeida@uem.br.
[3] Núcleo de Pesquisa em Limnologia, Ictiologia e Aquicultura (NUPELIA), Centro de Ciências Biológicas, Universidade Estadual de Maringá, Av. Colombo, 5790, 87020-900 Maringá, PR, Brazil.
Authors’ Contribution 
Josiane Rodrigues Rocha da Silva: Data curation, Formal analysis, Methodology, Visualization, Writing-original draft, Writing-review and editing.
Camila Oliveira de Andrade: Methodology, Visualization, Writing-review and editing.
Fernanda Losi Alves de Almeida: Data curation, Formal analysis, Visualization, Writing-review and editing.
Carlos Alexandre Fernandes: Conceptualization, Project administration, Supervision, Visualization, Writing-original draft, Writing-review and editing.
Ethical Statement
The Ethics Committee approved all procedures for using Animals in Research (CEUA – UEM), license number 3359040723.
Competing Interests
The author declares no competing interests.
How to cite this article
Silva JRR, Andrade CO, Almeida FLA, Fernandes CA. Histopathological changes in the gills of Astyanax lacustris specimens exposed to a reactive textile dye, before and after treatment with the constructed wetland system. Neotrop Ichthyol. 2025; 23(2):e250002. https://doi.org/10.1590/1982-0224-2025-0002
Copyright
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Distributed under
Creative Commons CC-BY 4.0
© 2025 The Authors.
Diversity and Distributions Published by SBI
Accepted May 19, 2025
Submitted January 8, 2025
Epub August 04. 2025