Brazilian Journal of Animal and Environmental Research 4070 ISSN: 2595-573X
Exposição à amônia e alterações de pH desencadeiam danos branquiais e mortalidade em peixes tetras da Amazônia
Ammonia exposure and pH alterations trigger gill damage and mortality in Amazonian tetras fish
DOI: 10.34188/bjaerv4n3-098
Recebimento dos originais: 04/03/2021 Aceitação para publicação: 30/06/2021
Wallice Paxiúba Duncan Doutor em Ecologia e Recursos Naturais pela Universidade Federal de São Carlos Universidade Federal do Amazonas, Laboratório de Morfologia Funcional Endereço: Av. Rodrigo Octávio, 6200, Coroado I, 69080-900. Manaus, AM, Brasil E-mail: [email protected]
RESUMO O objetivo deste estudo foi avaliar os efeitos da toxicidade aguda pela amônia e alterações de pH na estrutura branquial dos peixes tetras, Paracheirodon axelrodi e Paracheirodon simulans. Estas espécies são importantes caracídeos neotropicais usados como peixes ornamentais. Os danos branquiais foram analisados e quantificados. Como esperado, o cardinal (P. axelrodi) foi a espécie menos tolerantes à toxicidade da amônia e à água ácida. Por outro lado, o neon (P. simulans) foi a espécie mais resistente à amônia e menos tolerante à água alcalina. Hiperplasia epitelial, descolamento do epitélio lamelar e fusão das lamelas foram mais frequentemente observadas nas brânquias de P. simulans expostos aos estressores (amônia e alterações de pH) do que nas brânquias de P. axelrodi. Tais lesões são inespecíficas, porém podem reduzir a reabsorção de amônia, bem como proteger o epitélio branquial de danos ainda mais graves causados pelas alterações de pH. Por outro lado, alterações na estrutura dos vasos sanguíneos, tais como aneurismas, telangiectasias e necrose focal das células pilares foram comumente observadas no cardinal. A necrose dos componentes branquiais está relacionada às taxas de mortalidade dos peixes. Embora os caracídeos pertençam ao mesmo gênero e ocorram na mesma bacia hidrográfica, tais espécies apresentam respostas morfológicas diferentes aos mesmos agentes estressores.
Palavras-chave: efeito da amônia, alterações branquiais, caracídeos neotropicais, peixes ornamentais, alterações de pH
ABSTRACT The goal of this study was to evaluate the effects of acute ammonia toxicity and pH changes on the gill structure of tetras fish, Paracheirodon axelrodi and Paracheirodon simulans, two important neotropical characins used as ornamental fish. Fish mortality was recorded, and damage to the gills was examined and quantified. As expected, cardinal tetras (P. axelrodi) were less tolerant to ammonia toxicity and acidic water. Conversely, green neon tetras (P. simulans) were more resistant to acute ammonia levels and less tolerant to alkaline water. Epithelial hyperplasia, lamellar lifting and lamellar fusion were more frequently observed in the gills of the green neon tetras that were exposed to stressors (ammonia toxicity and pH alterations) than in the gills of cardinal tetras. These non-specific lesions may reduce ammonia uptake, as well as protect the gill epithelium from severe damage caused by pH alterations. In contrast, blood vessel alterations, such as aneurysms and telangiectasis, and focal necrosis of the pillar cells were commonly observed in the cardinal tetras. Necrosis in the gill components was related to the mortality rates of these tetra fish. Although these
Brazilian Journal of Animal and Environmental Research, Curitiba, v.4, n.3, p. 4070-4084 jul./set. 2021.
Brazilian Journal of Animal and Environmental Research 4071 ISSN: 2595-573X characins belong to the same genus and live in the same hydrological system, they present different morphological responses to the same stressors.
Keywords: ammonia effects, gill alterations, neotropical characins, ornamental fish, pH alterations
1 INTRODUCTION Cardinal tetras (Paracheirodon axelrodi Schultz, 1956) and green neon tetras (Paracheirodon simulans Géry, 1963) are found in the black water that extends throughout the middle to upper Rio Negro and Orinoco basin (WALKER, 2004). In general, they are sympatric and syntopic, but they differ in their preferred microhabitats and are rarely found together in same environment (MARSHALL et al., 2011). Both species are widely exploited as ornamental fish. P. axelrodi and P. simulans represent 64.6 % and 7.5%, respectively, of total export volume between 2006 and 2015 from the Amazonas state, Brazil. In this period, more than 100 million individuals were exported (TRIBUZY-NETO et al., 2020). Detrimental water conditions, such as a concomitant increase in the pH of water and ammonia, produce higher concentrations of the more toxic un-ionized form of ammonia (MIRON et al., 2008), which may contribute to high mortality during transport and shipment. In the case of cardinal tetra, the mortality rates can be as high as 70 % during the capture and transport due to the poor water quality (WAICHMAN et al., 2001). However, the sensitivity to ammonia appears to be species-specific, since the cardinal tetras appear to be less tolerant than the green neon tetras (SOUZA-BASTOS et al., 2017). The gills are particularly sensitive to changes in the aquatic environment (EVANS et al., 2005). Structural damage, such as epithelial lifting, lamellar fusion and haemorrhage, can lead to alterations in those physiological processes (LEASE et al., 2003), such as, reduce respiratory effectiveness and decrease swimming performance (MIRON ET AL., 2008). Furthermore, toxic substances and extreme changes in pH tend to promote excessive mucus production due to the proliferation of mucous cells (PAULINO et al., 2014). However, the proliferation of mucous and pavement cells results in an increase in the blood-water barrier (EVANS et al., 2005). With this background, it is possible that extreme pH conditions and high ammonia concentrations will result in severe gill damage, contributing to the high mortality of tetra fish during management in fisheries and aquariums.
In this study, the experimental design of the LC50-96h as reported by OLIVEIRA et al. (2008) was replicated. The goal was to investigate the acute effects of pH stress and ammonia concentrations on the gill morphology of two ornamental fish. In addition, a correlation and/or a
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Brazilian Journal of Animal and Environmental Research 4072 ISSN: 2595-573X causal relationship was established between stress-induced alterations and mortality rates in these fishes.
2 MATERIALS AND METHODS 2.1 ANIMALS AND MAINTENANCE Cardinal tetras (0.24 ± 0.02 g and 2.74 ± 0.11 cm) and green neon tetras (0.12 ± 0.03 g and 2.05 ± 0.16 cm) collected in the middle sector of the Rio Negro (00º40’S/62º58’W), were purchased from a local aquarium supply store. Fish were kept in a 1,000 L holding tank with local well water for two weeks, and the following conditions were maintained: a stabilized temperature (29.2 ± 1.1 oC), dissolved oxygen 5.4 ± 0.8 mg L-1, conductivity 24.5 ± 2.7 µS cm-1, pH 5.5 ± 1.3 and natural photoperiod. Fish were fed every morning with commercial feed (38 % crude protein). During this period, the ammonia concentrations ranged from 0.05 to 0.10 mM, and feeding was stopped 24 h prior to the beginning of the experiments. All in vivo procedures were approved by Ethical Committee of Animal Experimentation (CEUA/UFAM) protocol Nº 014/2015 (in accordance with the guidelines of CONCEA, National Council of Control of Animal Experimentation).
2.2 EXPERIMENTAL DESIGN In order to evaluate the effects of pH alterations and ammonia concentrations on the gill morphology of cardinal and green neon tetras, the experimental conditions of LC50-96 h that were reported by OLIVEIRA et al. (2008) have been replicated. The fish were placed in tanks (40 L) and kept for 48 hours in tap water (pH 6.4 ± 0.7; temperature 29.4 ± 0.5; electrical conductivity 12.1 ± 0.4 µS/cm; dissolved oxygen 4.7 mg/L, and ammonia was not detected) before the beginning of the experiments. For ammonia experiments, three groups of ten fish (with three replicates for each treatment) were exposed to two different levels (1.54 ± 0.06 and 2.75 ± 0.17 mmoles/L of total ammonia) and tap water without ammonia was used as a control group. The pH monitoring of the ammonia experiments ranged from 6.7 ± 0.2 and 7.3 ± 0.4 in the tests with 1.54 and 2.75 mmoles/L of ammonia, respectively. For study with extreme pH conditions, three different levels of acidic water (with three replicates for each treatment) were used: pH 2.9 ± 0.2 (min – max, 2.5 - 3.2), 4.3 ± 0.4 (3.8 - 4.5) and 6.0 ± 0.3 (5.8 - 6.2), as a control group. While three different levels of alkaline water were tested: pH 7.2 ± 0.5 (6.9 - 7.5), 7.8 ± 0.3 (7.5 - 7.9) and 8.8 ± 0.4 (8.6 - 9.0). The ammonia levels, acidic and alkaline pH were adjusted as reported by OLIVEIRA et al. (2008). briefly, adjustment to acidic pH was performed with HCl, while alkaline pH was performed with Tris and
NaOH. Tests with ammonia were done with a NH4Cl solution. In all experiments, solutions were slowly added every 1-h until reaching the desired value. In addition, the water was partially renewed
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Brazilian Journal of Animal and Environmental Research 4073 ISSN: 2595-573X daily by 30% of the tank volume. The water quality was analysed twice a day, before the partial renewal of the tank water. In the ammonia experiments, the physical and chemical variables of the water were recorded as the fallowing: temperature (29.5 ± 1.2 oC), dissolved oxygen (5.3 ± 1.1 mg/L), and conductivity (92.4 ± 33.7 µS/cm). While in the pH experiments, these variables were: temperature (30.2 ± 0.8 oC), dissolved oxygen (4.1 ± 2.4 mg/L), and conductivity (70.8 ± 52.1 µS/cm). In all cases, monitoring was recorded using a Consort C535 multiparameter analyser + (CONSORT bvba). Ammonia concentrations were assayed as total ammonia levels (NH3 + NH4 ) using Nessler methods. Animals that lost their equilibrium and died due to the acute toxicity of the ammonia were immediately necropsied, and haemorrhages were observed in the gills.
2.3 TISSUE PROCESSING At the end of the exposure period (96 h), the fish were euthanized with a high dose of anaesthetic 0.1 % benzocaine. The gills were removed and fixed in phosphate-buffered containing 2.5 % glutaraldehyde. Gill arches were washed and then dehydrated through a graded series of ethanol baths. The right sides of the gills were embedded in 2-hydroxyethyl methacrylate resin. To quantify the histological changes, thin sections (3 µm) of tissue were cut using a tungsten knife mounted on a semi-rotary microtome (CUT 5062, Slee Medical GmbH). Slides for quantitative measurements were stained with 0.12 % toluidine blue. Some slides were stained with periodic acid- Schiff reagent (PAS) and Alcian blue at pH 2.5 (AB) to quantify the mucous cells with neutral and acidic mucosubstances, respectively. Changes in cell morphology, such as karyorrhexis, karyolysis, and pyknosis were also recorded
2.4 GILL MORPHOMETRIC ANALYSIS The gill components (epithelial lamellar/filament and blood space) were evaluated in random selected fields of the histological sections. The density of volume of the gill components (undamaged and damaged) was analysed using a stereological technique. The tissue elements were quantified from digital images using a square lattice grid of points superimposed by the free software STEPanizer Stereology Tool version 1 (www.stepanizer.com). The thickness of the water-blood barrier was estimated using the test line of a semi-circular grid (Merz’s grid) and analysed using IMAGEJ 1.45s software (www.imagej.nih.gov/ij/).
2.5 STATISTICAL ANALYSIS The data are expressed as mean values ± standard deviation (SD). In the experiments, ammonia and pH data are expressed as mean ± SD (minimum - maximum). The volume density
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Brazilian Journal of Animal and Environmental Research 4074 ISSN: 2595-573X values are proportions that required arcsine-square root transformation before analysis (ZAR, 2010). The uniformity of the data was tested using the Kolmogorov-Smirnov test. The relationship between mortality rate and histological changes was examined using Pearson's correlation analysis. Stereological and morphometric measurements were analysed by parametric one-way ANOVA followed by Tukey’s test (significance level was set at α < 0.05). The data were analysed using the Sigma Plot package version 11.0 (Systat software Inc.).
3 RESULTS The green neon tetras were slightly more sensitive to extreme alkaline water than the cardinal tetras. In green neon tetras, 100 % mortality occurred when pH 8.8 was reached, while in cardinal tetras 70 % of mortality was observed at pH 8.8 (Figure 1A). In contrast, the mortality rate reached 100% in the cardinal tetras exposed to pH 2.9; while, at this pH (2.9), 78 % of green neon tetras died. In both species, the gills were necropsied and exhibited typical haemorrhages, high mucous content and focal necrosis. In green neon tetras, the high mortality was also positively associated with the focal necrosis of the gill epithelium (r2 = 0.82; p = 0.011; Figure 1B). Conversely, this relationship was not observed in cardinal tetras.
Figure 1. Variations in the mortality rate (A) and focal necrosis (B) of the gill components in cardinal tetras and green neon tetras exposed to different pH values.
As expected, the cardinal tetras were more sensitive to high ammonia concentrations than the green neon tetras. At an ammonia level of 2.75 mM, 80.3 ± 3.5 % of the cardinal tetras died over 96 hours, whereas only 22.6 ± 1.9 % of the green neon tetras died. Ammonia was toxic to the cardinal tetras even at low concentrations (1.54 mM), and 24.3 ± 2.1 % of the fish in this group died.
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Brazilian Journal of Animal and Environmental Research 4075 ISSN: 2595-573X
Meanwhile, no cardinal tetras died in this concentration. The gills of the control fish appeared normal in both species. The density of volume of the gill components with histological alterations in the green neon and cardinal tetras exposed to the pH challenges are detailed in the Tables 1 and 2, respectively. Although some of the fish were healthy, evidence (< 0.7 %) of epithelial hyperplasia on the lamellae and gill filament was still observed. Complete fusion of all lamellae was also observed. In the healthy cardinal tetras, hyperplasia occurred more frequently (0.7 % of total tissue alterations) in the lamellar epithelium, while in the green neon tetras, it occurred in the filament epithelium (0.7 %).
Table 1. Gill damage (as volume density, %) in Paracheirodon axelrodi (cardinal tetra) exposed to the pH challenges. Different letters on the same line indicate significant differences (p < 0.05) according to the (Tukey test). Gill alterations Acidic pH Alkaline pH 2.9 4.3 6.0 7.2 7.8 8.8 Hyperplasia of lamellar epithelium 5.8 ± 0.4a 0.4 ± 0.1b 0.7 ± 0.2b 0.5 ± 0.2b 4.6 ± 1.1a 3.7 ± 1.4a Hyperplasia of filament epithelium 0.0a 5.7 ± 1.6b 0.3 ± 0.2c 1.8 ± 1.0d 7.1 ± 5.2b 5.4 ± 1.1b Epithelial lifting of lamellae 2.0 ± 0.5a 0.0b 0.0b 0.0b 14.4 ± 8.4c 7.1 ± 2.7d Dilation of the marginal channel 0.0a 1.3 ± 1.0b 0.0a 0.2 ± 0.1a 7.7 ± 2.3c 3.6 ± 1.3d Complete fusion of all lamellae 0.0a 3.4 ± 2.1b 0.1 ± 0.1a 0.0a 4.3 ± 2.1b 11.4 ± 2.9c Lamellar aneurysm 1.7 ± 0.6a 0.9 ± 0.4a 0.0b 0.2 ± 0.1b 9.5 ± 2.2c 2.5 ± 1.2a Undamaged lamellae 3.4 ± 0.8a 52.2 ± 5.0b 73.5 ± 9.8c 52.4 ± 9.1b 19.8 ± 2.5d 4.4 ± 1.9a Undamaged filament 16.5 ± 6.7a 22.5 ± 3.9b 24.8 ± 6.4b 45.4 ± 7.8c 16.7 ± 8.4a 18.7 ± 5.4a
At extremely low pH levels (e.g., 2.9), hyperplasia due to pavement cell proliferation on the filament epithelia and chloride cell proliferation on the lamellae was observed. Epithelial lifting of lamellae, which resulted in the complete fusion of all lamellae, was most frequent in the gills of the green neon tetras (10.5 %), while this was not observed in cardinal tetras. Conversely, focal necrosis was most common in the cardinal tetras (70 %) than in the green neon tetras (12.3 %). The neon tetras exposed to slightly alkaline water (pH 7.8) often had multiple lesions, such as hyperplasia (on the lamellae and filament epithelia), epithelial lifting of lamellae, dilation of the marginal channel and fusions between adjacent lamellae. However, these alterations were most frequently observed in the cardinal tetras. Unlike the cardinal tetras, the green neon tetras had severe blood alterations (such as lamellar aneurysms) and focal necrosis. Moreover, all fish in the highly alkalized water (pH 8.8) died during the experiments. The green neon tetras exhibited focal necrosis in almost 80 % of their gill components, whereas the cardinal tetras exhibited focal necrosis in 43.3 % of their gill components.
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Brazilian Journal of Animal and Environmental Research 4076 ISSN: 2595-573X
Table 2. Gill damage (as volume density, %) in Paracheirodon simulans (green neon tetra) exposed to pH alterations. Different letters on the same line of data indicate significant differences (Tukey test, p < 0.05). Gill alterations Acidic pH Alkaline pH 2.9 4.3 6.0 7.2 7.8 8.8 Hyperplasia of lamellar epithelium 12.1 ± 1.4a 0.1 ± 0.1b 0.0b 1.1 ± 0.5c 1.2 ± 0.2c 2.3 ± 0.9d Hyperplasia of filament epithelium 8.7 ± 2.3a 0.0b 0.7 ± 0.4c 0.9 ± 0.7c 4.9 ± 1.4d 1.7 ± 0.6c Epithelial lifting of lamellae 9.2 ± 0.7a 0.2 ± 0.1b 0.0b 0.3 ± 0.3b 1.5 ± 0.5c 0.8 ± 0.4c Dilation of the marginal channel 5.1 ± 0.4a 4.5 ± 2.7a 0.0b 0.4 ± 0.2b 2.8 ± 1.1c 1.0 ± 0.4d Complete fusion of all lamellae 10.5 ± 0.3a 2.4 ± 1.8b 0.3 ± 0.2c 0.5 ± 0.4c 3.9 ± 1.3b 1.2 ± 0.7d Lamellar aneurysm 2.2 ± 1.4a 2.9 ± 0.9a 0.0b 3.0 ± 1.1a 11.9 ± 2.9c 4.3 ± 1.5d Undamaged lamellae 9.2 ± 7.0a 60.5 ± 9.8b 59.2 ± .5b 45.9 ± 8.7b 10.3 ± 2.8a 3.4 ± 1.2c Undamaged filament 31.4 ± 8.7a 27.6 ± 5.7a 39.2 ± 5.2b 48.5 ± 7.8b 22.8 ± 8.6a 7.3 ± 2.1c
Acute ammonia concentrations induced severe gill alterations in both tetra fishes (Table 3). The most severe types of damage were extensive focal necrosis, complete fusion of all lamellae, aneurysms, hyperplasia of chloride cells and lamellar fusion. Ammonia also induced cell proliferation in the gill epithelium. Nevertheless, focal necrosis was more common in the cardinal tetras than in the green neon tetras. Some representative histological alterations are showed in the figure 2. Necrotic cells, both in the gill epithelium and those in support (pillar cells), exhibit morphological changes such as karyorrhexis, and karyolysis.
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Figure 2. Sagittal sections of the gill tissues showing normal aspects of the lamellae of the cardinal (a) and green neon tetras (b) maintained in tap water (pH 6.0 without ammonia). Representative histological changes in the gills (arrowheads), such as chloride cell proliferation in cardinal tetras (c), epithelial lifting in green neon tetras (d), hemorrhages in cardinal tetras (e), complete fusion of lamellae in green neon tetras (f), lamellar aneurysms (g) and focal necrosis and partial lamellar fusion in green neon tetras (h). Scale bar = 10 µm). Sections were stained with toluidine blue 0.25 %.
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Table 3. Gill damage (as volume density, %) in the gill alterations of Paracheirodon axelrodi and Paracheirodon simulans after 96 h at different levels of ammonia exposure. Different letters are significantly different according to one-way ANOVA (Turkey’s test, p < 0.05). P. axelrodi P. simulans Histopathologies (total ammonia, mM) (total ammonia, mM) 0 1.54 2.75 0 1.54 2.75 Hyperplasia of lamellar epithelium 0.3 ± 0.2a 0.9 ± 0.2b 1.9 ± 0.5c 0.0a 12.8 ± 0.5b 3.2 ± 1.1c Hyperplasia of filament epithelium 0.2 ± 0.1a 1.0 ± 0.6b 1.8 ± 1.2b 0.5 ± 0.3a 13.0 ± 0.5b 4.7 ± 0.7c Epithelial lifting of lamellae 0.0a 2.5 ± 1.7b 3.3 ± 1.7b 0.0a 6.6 ± 1.2b 13.3 ± 1.8c Dilation of the marginal channel 0.4 ± 0.2a 12.1 ± 0.1b 9.2 ± 0.4c 0.4 ± 0.3a 6.5 ± 1.5d 7.1 ± 1.3cd Complete fusion of all 0.0a 3.6 ± 0.6b 3.5 ± 0.7b 0.0a 8.8 ± 1.6b 6.5 ± 1.7c lamellae Lamellar aneurysm 0.0a 15.3 ± 3.8b 8.1 ± 2.4c 0.0a 0.6 ± 0.4b 1.7 ± 0.8c Necrosis 0.0a 23.2 ± 2.2b 44.8 ± 3.6c 0.0a 9.3 ± 2.8b 19.5 ± 3.8c Undamaged lamellae 48.3 ± 3.9a 17.4 ± 1.2b 7.5 ± 1.4c 46.9 ± 8.4a 22.1 ± 7.3b 14.6 ± 1.6c Undamaged filament 51.5 ± 5.2a 22.8 ± 9.8b 18.7 ± 3.6b 51.9 ± 5.8a 20.2 ± 2.7b 29.3 ± 6.5b
Ammonia toxicity and extreme pH conditions also affected the water-blood distance in the neon tetras (Figure 3). The water-blood barrier became thicker (p < 0.05) in highly acidified or alkalinized water. At a very low pH, the diffusion barrier increased to 1.45 ± 0.26 µm and 1.34 ± 0.09 µm in the cardinal and green neon tetras, respectively. Moreover, at the highest pH, the water- blood distance also increased to 1.42 ± 0.13 µm and 1.63 ± 0.35 µm in the cardinal and green neon tetras, respectively. Similarly, the water-blood distance tended to increase significantly with the ammonia concentration in both tetra fish.
Figure 3. Effect of pH alterations (A) and total ammonia concentrations (B) on the water-blood distance barrier (2/3 of the harmonic mean) of cardinal tetras (P. axelrodi) and green neon tetras (P. simulans). Data are presented as the means ± SD. Different letters over bars indicate significant difference among treatments (Tukey test, p < 0.05).
Both stressors (ammonia exposure and pH alterations) induced mucous cell (MC) proliferation in the leading and trailing edges, as well as in the tips of the gill filaments. In both fish
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Brazilian Journal of Animal and Environmental Research 4079 ISSN: 2595-573X species, the MCs contained neutral and/or acidic mucosubstances, which were stained with periodic acid-reagent of Schiff (PAS) and Alcian blue, respectively. Exposure to alkaline pH levels (>pH 7.8) increased the number of MCs containing neutral mucosubstances in the gills of cardinal tetras (p < 0.05), whereas more MCs with acidic glycoproteins (p < 0.0%) were observed in the green neon tetras. In contrast, very acidic water ( Figure 4. Mucous cell density in the gills of cardinal, P. axelrodi (A) and green neon tetras, P. simulans (B) after acute exposure to pH. Bars with different letters are significantly different according to one-way ANOVA (Turkey test, p < 0.05). Figure 4. Mucous cell density in the gills of cardinal, P. axelrodi (A) and green neon tetras, P. simulans (B) after acute exposure to pH. Bars with different letters are significantly different according to one-way ANOVA (Turkey test, p < 0.05). Brazilian Journal of Animal and Environmental Research, Curitiba, v.4, n.3, p. 4070-4084 jul./set. 2021. Brazilian Journal of Animal and Environmental Research 4080 ISSN: 2595-573X 4 DISCUSSION A previous study has described the acute lethal effects of pH alterations and ammonia toxicity on cardinal tetras (OLIVEIRA et al., 2008). The LC50-96 h for cardinal tetras was estimated to be pH 2.9 in acidified water, pH 8.8 in alkalinized water and 2.7 mM for ammonia toxicity. More recently, SOUZA-BASTOS et al. (2017) studied 11 Amazonian fish species and concluded that cardinal tetras were more sensitive to ammonia toxicity than green neon tetras. Similarly, I observed the same results in both species. However, I herein provide a histopathological approach to explain the mortality caused by acute pH variations and ammonia levels. Furthermore, no comparable study of pH challenges has been carried out to date using green neon tetras, which are a congeneric species of cardinal tetras and are of equal importance to the ornamental fish market. Extreme pH challenges (acidic or alkaline) and high ammonia concentrations cause severe gill damage in tetra fish. However, these two characins exhibited different morphological responses to the same stressor. For example, green neon tetras submitted to alkaline pH (>8.8) has a greater degree of gill injuries compared to cardinal tetras; while at acid pH (<2.9), these damages are more intense in cardinal than in green neon tetras. This may potentially explain the different sensitivities, as the histological alterations were clearly associated with mortality rates in both fish species. The types and amounts of damage observed in both tetra species indicates that the fish were directly affected by extreme pH levels and ammonia concentrations. This partially explains the relationship between focal necrosis and mortality rates in tetra fish, as these injuries may reduce gill function. Several studies have reported gill lesions as a result of exposure to very acidic water (DAYE and GARSIDE, 1976), extremely alkaline pH (LEASE et al., 2003) and elevated ammonia concentrations (MIRON et al., 2008). Elevated pH and ammonia levels induce hyperplasia and hypertrophy of mucous cells (MCs) on the gill lamellae of larval Lost River Suckers, Deltistes luxatus Cope, 1879 (LEASE et al., 2003). However, MC proliferation increases mucus secretions and helps to protect the gill epithelium from chemical injury (PAULINO et al., 2014). In green neon tetras, the increase in the number of MCs with neutral mucosubstances (instead of acidic mucins) in the gill suggest a response to prevent the collapse of gill lamellae, since these cell types produce low-viscosity glycoproteins (FIERTAK and KILARSKI, 2002; RAMOS et al., 2018). Similar results were observed in the cardinal tetras subjected to elevated pH levels (>pH 7.8). Numerous chloride cells (CCs) were also observed on the filaments (interlamellar space) and lamellar epithelium of tetras fish. The proliferation of CCs and pavement cells on the lamellar epithelium was evident in both species under pH challenges. Hyperplasia of the lamellar epithelium may result in an increase in the water-blood distance (CAMARGO and MARTINEZ, 2007). It is Brazilian Journal of Animal and Environmental Research, Curitiba, v.4, n.3, p. 4070-4084 jul./set. 2021. Brazilian Journal of Animal and Environmental Research 4081 ISSN: 2595-573X possible that these alterations reduced the gas-exchange capacity of the gills. The increase in respiratory epithelium thickness and the associated thickening of the mucus layer due to excessive secretions from MCs may result in mortality due to hypoxia (EVANS et al., 2005). Moreover, this can be potentially hazardous to the cardinal tetras, since this species is more sensitive to hypoxia than the green neon tetras (CAMPOS et al., 2017; SOUZA-BASTOS et al., 2017). Extreme pH conditions also cause extensive epithelial lifting, lamellae fusion and aneurysms. In highly acidic water, these lesions were more common in green neon tetras. However, more deleterious damage, such as necrosis, was observed in the cardinal tetras. Epithelial hypertrophy and hyperplasia can lead to complete lamellar fusion, thereby reducing the respiratory surface in neotropical fish (PAULINO et al., 2014). Oedema with epithelial lifting is an inflammatory process that is initiated as a defence mechanism to reduce the gill surface area of green neon tetras due to stress at extreme pH levels ( Brazilian Journal of Animal and Environmental Research, Curitiba, v.4, n.3, p. 4070-4084 jul./set. 2021. Brazilian Journal of Animal and Environmental Research 4082 ISSN: 2595-573X progressed under acute ammonia intoxication and resulted in epithelial rupture and haemorrhages (MIRON et al., 2008). In fact, the moribund fish showed signs of haemorrhage in their gills. The tetra fish that were exposed to high ammonia levels exhibited focal necrosis in the pillar cells. These cells that support the blood space in the lamellae showed karyorrhexis. Additionally, the chloride cells that proliferated on the lamellae epithelium exhibited karyolysis and nuclear shrinkage (pyknosis) in the erythrocytes. All these alterations were more severe in the cardinal tetras, which may explain the high mortality rate of this species when exposed to high ammonia concentrations. 5 CONCLUSIONS Overall, this study demonstrates that extreme pH conditions and high ammonia levels cause severe damage to the gill structures and increase water-blood diffusion barrier. The gills of cardinal tetras exposed to ammonia had 2-fold more necrotic tissue than the gills of the green neon tetras. These histological alterations explain the different mortality rates reported in these tetra fish. The present study aimed to reduce mortality and increase the understanding of the effects of common stressors that occur during the harvest, holding and transport of tetra fish for ornamental purposes. Brazilian Journal of Animal and Environmental Research, Curitiba, v.4, n.3, p. 4070-4084 jul./set. 2021. Brazilian Journal of Animal and Environmental Research 4083 ISSN: 2595-573X REFERENCES CAMARGO, M.M. & MARTINEZ, C.B.R. 2007. Histopathology of gills, kidney and liver of a Neotropical fish caged in an urban stream. Neotropical ichthyology, 5(3): 327-336. https://doi.org/10.1590/S1679-62252007000300013. CAMPOS, D.F.; JESUS, T F.; KOCHHANN, D.; HEINRICHS-CALDAS, W.; COELHO, M.M &; ALMEIDA-VAL, V.M.F. 2017. 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