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Bulletin of Environmental Contamination and Toxicology (2018) 101:570–579https://doi.org/10.1007/s00128-018-2464-8
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Histological and Genotoxic Biomarkers in Prochilodus lacustris (Pisces, Prochilodontidae) for Environmental Assessment in a Protected Area in the Northeast of Brazil
Rayssa de Lima Cardoso1 · Raimunda Nonata Fortes Carvalho‑Neta2 · Antonio Carlos Leal de Castro3 · Cássia Fernanda Chagas Ferreira2 · Marcelo Henrique Lopes Silva4 · James Werllen de Jesus Azevedo3 · João Reis Salgado Costa Sobrinho5 · Débora Martins Silva Santos2
Received: 30 April 2018 / Accepted: 4 October 2018 / Published online: 22 October 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018
AbstractThe quality of aquatic environments all around the world is being altered by different human activities that represent direct threat to the ecological system and the aquatic biota. This study aimed to evaluate the occurrence of histological and genotoxic alterations in Prochilodus lacustris as indicators of anthropic impacts in a lacustrine environment in northeast Brazil. The histological alterations were evaluated using the histological alteration index, and the genotoxic alterations were detected using the micronuclei test, at three sampling stations (S1, S2 and S3). The gills presented lesions with three stages of severity, with mild lesions more frequent in the specimens collected at station S1. Mild hepatic tissue lesions were the most frequent type in both areas. Micronucleus analysis showed that station S3 was the most affected. The biological responses observed indicated that the fish are under influence of environmental changes. It is important to highlight that the organisms collected at station S3 had a more compromised health status.
Keywords Liver · Gill · Histopathology · Micronuclei · Biomarkers
Surface water bodies such as lakes, rivers and seas, are receptacles of a complex variety of compounds and sub‑stances that arise from both metabolic activities of organ‑isms and waste produced by anthropic actions, which nor‑mally find their final destination in such environments (He
et al. 2011; Liu et al. 2014; Santana et al. 2018). The variety of compounds and substances includes contaminants and toxic agents that are a direct threat to biota (Chang and Sib‑ley 1993, Ip et al. 2005; Rotter et al. 2011).
* Rayssa de Lima Cardoso [email protected]
Raimunda Nonata Fortes Carvalho‑Neta [email protected]
Antonio Carlos Leal de Castro [email protected]
Cássia Fernanda Chagas Ferreira [email protected]
Marcelo Henrique Lopes Silva [email protected]
James Werllen de Jesus Azevedo [email protected]
João Reis Salgado Costa Sobrinho [email protected]
Débora Martins Silva Santos [email protected]
1 Institute of Science and Technology, São Paulo State University (Unesp), Avenue Three March, 511, Alto da Boa Vista, Sorocaba, São Paulo, Brazil
2 Department of Chemistry and Biology, Postgraduate Program in Aquatic Resources and Fisheries, State University of Maranhão (Uema), University City Paulo VI s/n, Cidade Operária, São Luís, Maranhão, Brazil
3 Department of Oceanography and Limnology, Federal University of Maranhão (Ufma), Avenue of Portugueses, 1966, Vila Bacanga, São Luís, Maranhão, Brazil
4 Postgraduate Program in Biodority and Biotechnology Network of the Legal Amazon (BIONORTE), Department of Biology, Federal University of Maranhão (Ufma), Avenue of Portugueses, 1966, Bacanga, São Luís, Maranhão, Brazil
5 Department of Agricultural Engineering, Soil Chemistry Laboratory, State University of Maranhão (Uema), University City Paulo VI s/n, Cidade Operária, São Luís, Maranhão, Brazil
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Several aquatic organisms can be used as indicators of the changes in habitats. Among the most commonly used taxa, fish are excellent bioindicators, as they assimilate, absorb, accumulate and transfer compounds dissolved into the environment (Minissi et al. 1996; Geiszinger et al. 2009; Bolognesi and Hayashi 2011). Fish respond to environmen‑tal stressors in several ways, and these responses are classi‑fied as biomarkers (Nrc 1987; Peakall 1994; Van Der Oost et al. 2003).
Studies of histological alterations as biomarkers have been widely used in environmental assessments (Bernet et al. 2004; Van Dyk et al. 2012). One of the great advan‑tages of the use of histological biomarkers is the possibil‑ity of examining specific target organs and identifying how the direct and indirect toxic effects are affecting the tissues (Gernhofer et al. 2001). For example, gills are responsible for breathing and osmotic changes in fish species and due to permanent contact with the aquatic environment, this organ may react to stressors present in the environment, conse‑quently presenting alterations in its structure. The liver is an organ responsible for several metabolic functions, including xenobiotic biotransformation, which makes it sensitive to biochemical, physiological and structural alterations (Cama‑rgo and Matinez 2007; Costa et al. 2009; Leonardi et al. 2009; Yasser and Naser 2011).
In addition to histopathology, exposure to chemical sub‑stances in water may also modify genetic material and cause chromosomal mutations in species. The Micronucleus Test (MN) (Rabello‑Gay 1991), associated with the identification of nuclear alterations in fish erythrocytes (Carrasco et al. 1990), therefore represents an important analysis tool.
The increase of these techniques and methodologies of environmental monitoring (Brazil 2005), as well as the use of bioindicator species and their physiological information,
are important tools for the monitoring of aquatic regions and environments with great biological diversity. An example is the northern region of Brazil, which in addition to includ‑ing the largest area of ecological potential on the planet, the Amazon region, there is also an extensive flood plain located in the state of Maranhão, known as Baixada Mara‑nhense, considered by the state government as an Environ‑mental Protected Area and, it is also considered a wetland of international importance under the Ramsar Convention (Matthews 1993).
Despite the importance of the ecosystem services pro‑vided by Baixada Maranhense Environmental Protected Area, little is known about the environmental quality of the region. Thus, the aim of this study was to evaluate the occur‑rence of histological and genotoxic alterations in Prochilo-dus lacustris, endemic neotropical fish, considering a multi‑stressor context of contaminants acting synergistically, in three sites varying in anthropogenic pressure and contamina‑tion level in the Baixada Maranhense Environmental Protec‑tion Area.
Materials and Methods
The study was carried out in the municipality of Conceição do Lago Açu, in the central region of the state of Maranhão, in which Lake Açu is the main water body (Fig. 1).
Specimens were collected during the dry season (August, October and December 2015) and the rainy season (April, May and June 2016). Samplings were performed once a month, at three sampling stations (S1, S2 and S3). Station S1 was the reference station, which is the furthest station from anthropogenic influence; S2 represents the transitional station, in the central portion of the water body; and S3 is the
Fig. 1 Location of the sampling stations of Prochilodus lacustris in Lago Açu, in Baixada Mara‑nhense Environmental Protec‑tion Area, Maranhão, Brasil
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closest station to the municipal area, suffering direct influ‑ence of urban occupation, domestic sewage disposal and in natura discarding of fish and shellfish from commercial fish‑ing on the lake. The distances between the sampling stations and the township are listed in Table 1.
A total of 98 specimens of Prochilodus lacustris were used, of which 55 were collected during the dry season in 2015 and 43 were collected in the rainy season in 2016. All the specimens had their total length (Lt) and total weight (Wt) measured. Moreover, gonadal stage macroscopic analy‑sis (GS) was performed, being classified as GS1 (immature), GS2 (maturing), GS3 (mature) and GS4 (spawned) accord‑ing to the Vazzoler scale (1996), modified by Carvalho‑Neta and Castro (2008). The sex ratio was obtained through the observed frequency of male and female individuals, apply‑ing the Chi‑squared test (χ2) to detect statistically significant differences in the ratios. In this study, the sex ratios were expected to be 1:1.
The sampling procedures were conducted following the national and institutional guidelines (COBEA 2015) for the protection of animal welfare. The protocol was approved by the Ethics Committee of the State University of Maranhão (Nº 15/2016 CRMV‑MA) and complied with the guidelines of the Brazilian College of Animal Experimentation.
To verify the presence of potential genotoxic effects in the local biota was blood sampled through caudal vein puncture with disposable insulin syringes. Consisting of the use of a drop of blood for the preparation of the blood smear on a clean microscope slides, made in duplicate for each animal. Subsequently, the slides were left at room temperature for 24 h for drying according to the methodology described by Nepomuceno et al. (1997). In the laboratory, the red cells were stained with Rosenfeld (Tavares‑Dias and Moraes 2003) for the quantification of 2000 cells per prepared slide examined with a Zeiss microscope (400x and 1000x magni‑fication) (Campana et al. 1999; Palhares and Grisolia 2002). In addition to the micronuclei (MN), nuclear morphological alterations were also classified as indicative of genotoxicity in accordance with Carrasco et al. (1990).
After blood collection, samples the second right gill arch and liver were extracted for histopathological
analysis. The tissues were fixed in 10% formalin for 24 h and then stored in 70% alcohol. During the histological procedure, the gills arcs were decalcified in 10% nitric acid for more 6 h. Then they the livers and gills tissues were dehydrated in increasing concentrations of alcohol, diaphanized in xylol, added to paraffin in sections with a thickness of 5 µm and stained with hematoxylin and eosin (Luna 1968) for microscopic description.
Gills and hepatic histological alterations were evalu‑ated semi‑quantitatively through the Histological Altera‑tion Index (HAI), adapted from Poleksic and Mitrovic‑Tutundzic (1994), based on the severity of each lesion. The alterations were classified into progressive stages of tissue damage: Stage I, which do not impair the func‑tioning of the organ; Stage II, which are more severe and impair the normal functioning of the organ; and stage III, which is extremely severe and irreversible. The HAI value was calculated for each fish using the formula: HAI = (1 × Σ I + 10 × Σ II + 100 × Σ III), where I, II and III cor‑respond to the number of alteration for the stages I, II and III, respectively. The mean HAI value was divided into five categories: 0–10 = normal tissue functioning; 11–20 = mild to moderate tissue alteration; 21–50 = mod‑erate to severe tissue damage; 51–100 = severe tissue alter‑ation; ˃100 = irreparable tissue damage.
Parallel to the fish sample in situ abiotic water variables were measured such as pH, dissolved oxygen, temperature and conductivity using a multiparameter probe Hanna HI 9828. Moreover, turbidity analysis (NTU) was performed using a HANNA HI 93,703 turbidimeter.
During the sampling collections, water samples (3 liters) were also collected for bacteriological analyzes following the methodology recommended by APHA (1998), with the determination of the most probable number of total coli‑forms and Escherichia coli, using chromogenic (ONPG) and fluorogenic (MUG) enzymatic substrate tests.
Water samples were collected to determine the concentra‑tions of Copper, Aluminum, Zinc, Cadmium, Chromium, Lead, Iron, Nickel, Manganese, Mercury and Selenium. Metal determination was performed using a methodology adapted by USEPA 3005A (1992), after reduction of the digested contents, each sample was subsequently subjected to the determination of the concentrations using the plasma optical emission spectrometry method, ICP‑Varian 720‑ES (Tyler 1991), in the Laboratory of Soils of the State Univer‑sity of Maranhão.
The biomarker and physicochemical data were tested for the normality and homogeneity of variances using the Kol‑mogorov–Smirnov and Levene tests. When the assumptions were met, ANOVA and posteriori Tukey test were performed to identify groups that differed among each other. For data that did not meet the assumptions, the Kruskal–Wallis non‑parametric test was applied. Significance was tested to
Table 1 Coordinates and distances between sampling stations
Section Distance (m) Coordinates (UTM)
S1–S2 3849.18 S1–Lat: 513790.288984Long: 9582655.62598S1–S3 7837.31
S2–S3 3403.43 S2–Lat: 511717.525679Long: 9579.650.89142
S1—Township 9589.20 S3–Lat: 511099.927607Long: 9576359.73726S2—Township 4193.63
S3—Township 772.41
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evaluate the monthly/seasonal (dry and rainy) and spatial (collection points) differences.
Results and Discussion
The maximum temperature was 32 °C and the minimum was 29.2 °C during the entire sampling period. The dissolved oxygen level remained above 5.0 mg/L, varying from 5.92 to 9.46 mg/L. The pH was alkaline at all stations with values above 8.0, and electrical conductivity ranged from 154 µS/cm to 326 µS/cm. The concentrations of the chemical ele‑ments Al, Fe, Se and Hg in the samples collected at sta‑tions S2 and S3 were higher than the acceptable limits of the national standards determined by the Conselho Nacional do Meio Ambiente (the National Council for the Environ‑ment) (CONAMA). Pb was found in high concentrations only at station S3, while all other chemical components were either within the range of recommended values or were not detected. The results of bacteriological analysis (total coliforms, thermotolerants and the presence of Escherichia coli), were within the limits established by law. Data on water quality is shown in Table 2.
The parameters temperature, conductivity, pH and dis‑solved oxygen are related to one other and are indicative
of the dynamics of the environments, being indispensable for the evaluation of environmental quality. In the present study, the physicochemical parameters were within the limits established by national legislation (Conama 357/05). The values of conductivity and turbidity are indicative of large amounts of dissolved nutrients and organic and inorganic matter in particulate form, which are very common in lentic environments (Camargo and Valentini 1990; Wetzel 2001).
There were no significant differences (p > 0.05) between the concentrations of heavy metals in the sampling stations, but the water at stations S2 and S3 presented values above the levels established by Brazilian legislation for the ele‑ments Al, Pb, Hg, Fe and Se. Any change in concentrations of heavy metals in the aquatic environment is a concern, since these elements are assimilated by the biota (through food or in solution) and that may cause a high cumulative effect in the trophic chain, reaching the human food level (Pereira et al. 2010; Jabeen et al. 2011; Mert et al. 2014). It is important to highlight the necessity of further studies that analyze the content of chemical elements not only in water samples, but also in the sediment and tissues of organisms.
The Biometric data from Prochilodus lacustris, the fre‑quency of occurrence of the specimens sampled, as well as the result of the Chi‑squared test (χ2), revealed that females predominated significantly throughout the sample period.
Table 2 Mean and standard deviation of physical–chemical and biological parameters monitored in Lago Açu, Baixada Maranhense Environ‑mental Protection Area, Maranhão, Brazil
*Values below the method detection limita All values shown were measured in the months of August, October and December of 2015 for the rainy season, and in the months of May, June and July of 2016 for the dry seasonb Recommended values based on Brazilian legislation (CONAMA) under Resolutions No. 357 of 2005, No. 430 of 2011 and No. 274 of 2000. LD: Detection limit in mg/L
Parameters Dry season Rainy season Rec. values (b) Detection limit (LD)
S1 S2 S3 S1 S2 S3
Temperature (°C) 29.2 ± 2.7 30.3 ± 2.1 32 ± 3.2 30.4 ± 2.5 30.1 ± 1.1 30.5 ± 1.5 28°a 32 °CpH 9.15 ± 0.4 8.26 ± 0.6 9.03 ± 1.2 8.33 ± 1.5 8.72 ± 0.5 8.14 ± 0.3 6–9Dissolved Oxygen (mg/L) 7.58 ± 1.3 9.46 ± 2.3 8.07 ± 2.5 7.09 ± 0.3 6.01 ± 0.7 5.92 ± 1.1 > 5 mg/LTurbidity (NTU) 49 ± 2.6 55 ± 2.9 52 ± 1.8 31 ± 3 39 ± 2.1 30 ± 1.71 ≤ 100 UNTConductivity (ΜS/cm) 319 ± 163.72 326 ± 200.47 311 ± 564.52 155 ± 25.74 154 ± 21.61 163 ± 43.18 –Aluminum (Al) mg/L * 0.25219 * * 0.42765 0.03511 ≤ 0.1 mg/L 0.0083Copper (Cu) mg/L * * * * * 0.01446 ≤ 0.009 mg/L 0.0026Zinc (Zn) mg/L * * * * * * ≤ 0.18 mg/L 0.0020Cadmium (Cd) mg/L * * * * * * ≤ 0.001 mg/L 0.0008Chromium (Cr) mg/L 0.00678 * 0.00433 * * 0.00394 ≤ 0.05 mg/L 0.0014Lead (Pb) mg/L 0.0037 0.0479 0.07758 0.0688 * * ≤ 0.01 mg/L 0.0310Iron (Fe) mg/L 0.14863 0.46650 0.66105 0.19689 0.03734 1.30891 ≤ 0.3 mg/L 0.0061Nickel (Ni) mg/L 0.02240 * 0.00998 * * 0.04332 ≤ 0.025 mg/L 0.0079Manganese (Mn) mg/L 0.00140 0.01537 0.02392 * * 0.42020 ≤ 0.1 mg/L 0.0007Mercury (Hg) mg/L * 0.01214 * 0.00087 0.03196 0.01352 ≤ 0.0002 mg/L 0.00021Selenium (Se) mg/L * 0.07157 0.08957 * * * ≤ 0.01 mg/L 0.0009
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The sex ratio was 4.15 females for each male. The morpho‑metric characteristics showed that the largest specimens of both sexes were captured at S3. The gonadal stage percent‑ages (%GS) indicated that, regarding their sexual stages, the samples were classified as GS1, GS2 and GS3 during the dry season, with a predominance of individuals (75%) identified in the rainy season. The S1 area showed indications of high reproductive activity, with a higher occurrence of mature individuals, while a predominance of immature individuals was found at station S3 (Table 3). There were no occurrences of spawned individuals (GS4).
There were no significant differences between the bio‑metric data and the sexual stages of Prochilodus lacustris specimens between station S1 and the other sampling sta‑tions (S2 and S3). The development of the reproductive tract is a continuous process in all phases of the fish life cycle, but exposure to xenobiotics can lead to delayed developmental stages and gonadal maturation (Adams 1992, 2002), which can explain the number of immature individuals at station S3. It is important to mention that S3 is directly influenced by municipal urban occupation, disposal of domestic sew‑age and “in natura” discharge of fish and shellfish from
commercial fishing on the lake, which, in turn, leads to the production of organic matter, influencing the incidence of young fish that occupy that area for feeding.
The frequency of cytogenetic alterations of the micro‑nucleus type was significantly different (p < 0.05) among P. lacustris specimens collected at station S1 during dry season in relation to the specimens collected at point S3 in both sea‑sons of the year. During the dry season, station S1 presented the lowest frequency (0.01%) of the entire sampling period. During the rainy season, there was a higher frequency of genotoxic biomarkers, but there was no statistical difference between the seasonal periods (p > 0.05).
The nuclear alterations (NA) of the “notched”, “lobed”, “blebbed” and “binucleated” (Carrasco et al. 1990) had a higher occurrence at station S3 during the rainy season. All types of cellular morphological alterations occurred, and the “notched” type was the one that presented the highest amount (Table 4). The presence of micronuclei and other alterations in the nuclear envelope are indicative of muta‑genic action in the analyzed environment.
According to Palhares and Grisolia 2002, fish may pre‑sent variations in the frequency of micronuclei due to their
Table 3 Mean and Standard Deviation (±) of biometric and percentage data of stages of gonadal maturation (%GS1, GS2 and GS3) of males and females of Prochilodus lacustris, collected in Lago Açu, Baixada Maranhense Environmental Protection Area, Maranhão, Brazil
M male; F female, n number of individuals, NI not identified (NI)
Sample station Dry season Rainy season
S1 M (n = 2) F (n = 16)
S2 M (n = 4) F (n = 15)
S3 M (n = 3) F (n = 15)
S1 M (n = 4) F (n = 12)
S2 M (n = 3) F (n = 9)
S3 M (n = 4) F (n = 11)
Total length (Lt) 19.1 ± 0.75 18.6 ± 0.45 20.1 ± 1.36 19.4 ± 1.59 19.5 ± 0.31 22.1 ± 1.27Weight (Wt) 121.02 ± 5.87 124.1 ± 3.67 143.84 ± 6.4 119.46 ± 7.63 128.2 ± 3.88 138.0 ± 2.9Gonadal stage
(%GS)GS1 = ImmatureGS2 = MaturingGS3 = Mature
17.7% (GS1) 38.8% (GS1) 72.2% (GS1) NI (GS1) 75% (GS1) 42.8% (GS1)23.5% (GS2) 22.4% (GS2) 27.8% (GS2) 80% (GS2) 25% (GS2) 28.5% (GS2)58.8% (GS3) 38.8% (GS3) NI (GS3) 20% (GS3) NI (GS3) 28.7% (GS3)
Table 4 Nuclear anormalities and formation of micronucleus in Prochilodus lacustris erythrocytes collected in Lago Açu, Baixada Maranhense Environmental Protection Area, Maranhão—Brazil
NUCLEAR ALTERATIONS
Sample points
Samples ofP. lacustris
Dry periodMICRONUCLEUS NOTCHED LOBED BLEBBED BINUCLEATED
S1 N=18 2a 2S2 N=19 3 4 1S3 N=18 8b 4 1 2
Rainy periodS1 N= 16 3 3S2 N= 12 5 5 2 2 1S3 N= 15 6c 9 1 1
*Different alphabetic superscripts represent indices that are significantly different (p < 0.05)
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food habit, ecological niches, types of pollution the aquatic environment is subject to and seasonal changes. This is very evident in this study, since differences were identified in both spatial and seasonal scales.
Regarding the stations, only S1 showed minimum values of micronucleus frequency (0.01%), which corresponds to the basal frequency of the erythrocytes attributed to fish of the Prochilodus genus, collected in a natural environment and kept in laboratory, as evidenced by Porto et al. (2005) that reported the minimum frequency of 0.01% for Prochilo-dus nigricans; Seriani et al. (2011) reported minimum fre‑quency of 0.2 for Prochilodus argenteu %; Cavalcante et al. (2008) using the species Prochilodus lineatus reported mini‑mum frequency of 0.7% and Santos et al. (2013) established 0.03% for Prochilodus vimboides.
Although a significant difference (p > 0.05) was not iden‑tified on a seasonal scale, there was a greater occurrence of cytogenetic alterations during the rainy season, possibly indicating that the fish remained exposed to a higher con‑centration of chemical agents that accumulated in the water body, since during the rainy period there is a significant increase of sedimentation, nutrients, organic matter, oxygen‑consuming substances, bacteria, chemical compounds, met‑als and toxic agents (Temprano et al. 2005; Righetto et al. 2017). As a result, there may be also a high occurrence of alterations in genetic material (Fenech et al. 2000; Matsu‑moto and Cólus 2000; Pacheco and Hackel 2002).
Regarding gills lesions, most of the individuals collected at station S1 presented only lesions of stage I of severity, and
the lesion hyperplasia and hypertrophy of mucus cells was also found in a low percentage (3.3%) (Table 5). The stage I gills alterations are examples of defense mechanisms that result in increased distance between the external environ‑ment and the blood system, forming a barrier against the entry of contaminants, but may be reversible when environ‑mental conditions are improved (Mallatt 1985; Hinton et al. 1990; Poleksic and Mitrovic‑Tutundzic 1994; Fernandes and Mazon 2003). The station S1 was considered the refer‑ence area, since it was the most distant area of the municipal district, with low anthropogenic action and environmental characteristics still conserved.
The specimens collected at station S2 presented 10 of the 11 types of gills lesions, in other words, it is 90% of all alterations identified in this study. In the dry season the most frequent lesions were vascular congestion, incomplete fusion and complete fusion of several secondary lamellae. During the rainy season, the most frequent lesions were vascular congestion, lamellar epithelium removal, incomplete fusion and complete fusion of several secondary lamellae, complete fusion of all lamellae and aneurysm. Regarding station S3, individuals were found to contain all types of gills lesions, with lesions in stages II and III being frequent, demonstrat‑ing an environmental susceptibility of the species in a spatial scale (Table 5). It was observed the presence of parasite in some specimens, but there was no taxonomic identification.
The identification of lamellar aneurysm (stage III lesion) indicated a collapse of the pilaster cell system, impairing vascular integrity and risk of hemorrhage due to rupture
Table 5 Frequency of occurrence of gill changes in Prochilodus lacustris collected in Lago Açu, Baixada Maranhense Environmental Protection Area, Maranhão, Brazil
S1, S2 and S3 (sampling points); number of individuals (n)*Different alphabetic superscripts represent indices that are significantly different (p < 0.05)
Histopathological changes Frequency (%)
Dry period Rainy period
S1 (n = 18) S2 (n = 19) S3 (n = 18) S1 (n = 16) S2 (n = 12) S3 (n = 15)
Stage I Vascular congestion 11.90 17.20 14.17 14.81 16.67 12.75 Hyperplasia of lamellar epithelium 0.00 3.23 2.50 0.0 10.61 12.75 Lifting of lamellar epithelium 9.52 11.83 14.17 11.11 15.15 14.71 Disorganization of secondary lamella 9.52a 8.60c 2.50b 0.0 7.58 10.78b
Incomplete fusion of several secondary lamellae 11.90 17.20 15.00 14.81 12.12 12.75 Full Fusion of secondary lamellae 7.14 17.20 14.17 3.70a 12.12c 14.71b
Dilation of Venous Sinus 0.0 3.23 2.50 3.70 4.55 2.94 Presence of parasite 0.0 0.0 6.67 0.0 0.0 0.0
Stage II Hypertrophy and hyperplasia of mucus cells 3.3a 8.60 10b 0.0 0.0 3.7 Complete lamellar fusion 0.0 4.30 6.67 0.0 7.58 9.80
Stage III Aneurysm 0.0 6.45 11.67 0.0 9.09a 5.88b
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of the epithelium (Hinton et al. 1990). The Histological Alteration Index (HAI) ranged from 2 to 125, with a mean value of 46.89 in the dry season. During the rainy season, the HAI ranged between 1 and 117, with a mean of 32.39. The biomarkers in the gill tissue of P. lacustris presented alterations ranging from normal level to irreversible damage (Fig. 2). During the dry season, HAI values were signifi‑cantly different (p > 0.05) between the individuals collected at stations S3 and S2 (higher indexes) and those recorded in individual collected at station S1 (lower indexes), unlike the rainy season that did not show significant difference between sampling sites.
Histological alterations were found in hepatic tissue of P. lacustris, such as: nucleus in the periphery of the cell, centers of melanomacrophages, vacuolization, hyperemia and tissue necrosis (Fig. 3). During the entire sample period, lesions of the types vacuolation and melanomac-rophage center, categorized as stage 1 of severity, were the most frequent, and were more expressive at stations S2 and S3 (Table 6). Necrosis, which cause irreversible damage to the hepatic tissue, occurred only at stations S2 and S3. However, no significant difference was observed between the distribution of lesions in a spatial scale (p < 0.05).
Fig. 2 Photomicrographs of gills of Prochilodus lacustris collected at different sampling stations in Lago Açu—MA, Brazil. a Normal gill filament, arrow indicating primary lamellae (PL) and secondary lamellae (SL). b and c Gill histopathology frequent in fish collected in station S1: Lifting of lamellar epithelium (LLE) and Vascular
Congestion (VC); d Gill histopathology frequent in fish collected in station S2: Lamellar fusion (LF) and Lifting of lamellar epithelium (LLE); e and f Gill histopathology frequent in fish collected in sta‑tion S3: Aneurysm (AN) and Presence of parasite (PA). Scale bars: 50 µm, HE staining
Fig. 3 Photomicrographs of liver of Prochilodus lacustris collected in Lago Açu—MA, Brazil. a Normal hepatic tissue. b and c Lesions most frequent in the sampling stations: arrow indicating Melanomac‑
rophage center (MC); c vacuolization of hepatocyte (VA); Hemor‑rhage (HE). Scale bars: 50 µm, HE staining
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The hepatic alterations observed in this study corroborate with a study carried out by Camargo and Martinez (2007) using the species Prochilodus lineatus; where alterations such necrosis, irregular nuclei, nuclear vacuolization, mel‑anomacrophagous centers, among others were found. As the liver is the organ related to the process of detoxifica‑tion and biotransformation (Van Der Oost et al. 2003), the liver lesions found in this tissue are important biomarkers of aquatic contamination (Rodrigues and Fanta 1998).
The increase in vacuolation in hepatocytes is described by Pacheco and Santos (2002) as a sign of a degenerative pro‑cess related to metabolic damage, probably related to expo‑sure to contaminated water. The high incidence of melanoma centers in the liver is related to the increase of phagocytic activity as an immune response, in the elimination, detoxi‑fication or recycling of contaminants (Mondon et al. 2001; Rabitto et al. 2005). The identification of the necrosis‑like lesion in the individuals collected at stations S2 and S3 is an indication that these areas are impacted, as this type of lesion causes functional and structural damages in the liver of the fish (Stentiford et al. 2003). A necrotic tissue has no functionality, so that it may cause cell death in adjacent tis‑sues and consequently, affects higher levels of biological organization (Rabitto et al. 2005).
In conclusion, the biomarkers observed in P. lacustris in the present study indicate that the fish are responding to direct effects caused by environmental changes. Histo‑logical alterations and nuclear modifications proved to be good biomarkers of environmental assessment, as there was a significant distinction between the sampling stations and their respective levels of conservation. There was also seasonal differentiation, with the rainy season presenting a higher incidence of biomarkers, indicating that rain events may cause greater environmental susceptibility for that water body. It is also worth noting that the species Pochilo-dus lacustris was found to be adequate for environmental
studies as it presented responses at molecular and tissue lev‑els, which could be identified, classified and quantified as indicators of environmental quality.
Acknowledgements The authors are grateful to the Biology and Aquatic Environment Study Group (BIOAqua/UEMA), the Animal Morphology Laboratory, the Aquatic Organism Biomarker Labora‑tory (LABOAQ/UEMA), the Fishing and Aquatic Ecology Labora‑tory (LABPEA/UEMA), the Soil Chemistry Laboratory (UEMA), the UEMA (Veterinary Medicine Department) Food and Water Microbiol‑ogy Laboratory and the Oceanography Department of the Universidade Federal do Maranhão (DEOLI/UFMA) for assistance with the labora‑tory analyses.
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Table 6 Frequency of occurrence changes in liver tissue of Prochilodus lacustris collected in Lake Açu, Baixada Maranhense Environmental Protection Area, Maranhão, Brazil
S1, S2 and S3 sampling points, n number of individuals
Histopathological changes Frequency (%)
Dry period Dry period
S1 (n = 18) S2 (n = 19) S3 (n = 18) S1 (n = 16) S2 (n = 12) S3 (n = 15)
Stage I Nucleus in periphery of the cell 13.13 19.76 37.84 22.22 17.86 10.34 Melanomacrophage Center 31.25 35.29 45.95 14.8 25.00 37.93 Vacuolization 25.88 26.35 27.03 48.15 28.57 31.03
Stage II Hyperemia 16.25 23.53 27 14.8 25.0 20.7
Stage III Necrosis 0.0 5.88 5.41 0.00 3.57 3.45
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