Organophosphates are considered highly toxic to many aquatic species (Qin and Dong, 2004). Additionally, important organs of fish can be affected by organic pesticides such as in the gills (Gill et al., 1988; Guimarães et al., 2007), the liver (Gill et al., 1988; Rodrigues et al., 2001; Fanta et al., 2003) and in the kidney (Gill et al., 1988; Veiga et al., 2002). Although the use of many pesticides is heavily regulated in some parts of the world, studies to assess toxicity of organophosphate insecticides used in Brazil are required to establish safety levels, as some of these are still licensed in this country and more stringent control is needed. Pacu (Piaractus mesopotamicus) is an important farmed fish, natural to the rivers, where agrotoxic products, including trichlorfon, are still discharged by agricultural production systems. Additionally, trichlorfon is one of the most frequently used products against parasites in farmed fish. Bioconcentration of trichlorfon was assessed in pacu (Lopes et al., 2006), but there are no studies on the effects of this organic pesticide in this fish. The present study determined toxic concentrations of trichlorfon to pacu through the LC50/96 h and histopathological alterations to the gills, liver and kidney that are provoked by this organophosphate compound. In the first experiment, a toxicity assay (96-h LC 50) assessed the lethal effects of trichlorfon (O,O–dimethyl–1–hydroxy–2,2,2-trichloroethyl phosphonate, Sigma). One hundred fish were individually weighed (50 ± 10 g) and acclimated for 7 days to experimental conditions, being fed to satiation with a commercial feed (28% crude protein) in 1000 L aquaria with running water at a monitored temperature (26 ± 1°C), pH (7.8 ± 0.1), dissolved oxygen concentration (8.2 ± 0.7 mg L−1) and alkalinity (146 ± 11 mg L−1). The photoperiod was 12L : 12D. Acclimated fish were divided into five groups, conditioned in 100 L aquaria (4 aquaria per treatment, 5 fish per aquarium) and exposed to trichlorfon at 0.0 (control), 0.07, 0.12, 0.2 and 0.34 mg L−1. Acute toxicity bioassays were made in static fish exposure to the insecticide for 96 h; food was not provided. Fish mortality was registered at 24-h intervals. In the second experiment, the effect of low trichlorfon concentrations was assessed through histological analysis of the tissues. Totals of 120 fish were individually weighed (75 ± 15 g), fed as previously described and acclimated for 15 days in 100 L containers with running water, monitored temperature (26.9 ± 0.4°C), pH (7.7 ± 0.1), dissolved oxygen concentration (8.8 ± 0.5 mg L−1) and alkalinity (146 ± 11 mg L−1). Fish were exposed to trichlorfon at 0.0 (control), 0.025, 0.05 and 0.1 mg L−1 (3 aquaria per treatment/10 fish per aquarium; n = 30) during 24 h in a static system while maintaining the oxygen flow. Following exposure, water flow was re-established and fish were sampled at 1, 2, 7 and 15 days after exposure. Anesthetized fish (benzocaine, 1 g L−1) were sacrificed and dissected for removal of the gill, liver and kidney. Tissues were 24-h fixed in Bouin’s solution, embedded in paraffin and stained with hematoxylin/eosin. The LC50–96 h was statistically analyzed using the Trimmed Spearman Karber method (Hamilton et al., 1977). The LC50–96 h for pacu was determined with 0.1906 mg L−1 and the linear equation that represents the concentration-response relation was estimated with: y = 289.14x + 2.2316 (r2 = 0.9955). No control fish died. Concentrations of 0.07, 0.12, 0.2 and 0.34 mg L−1 caused 20%, 40%, 60% and 100% mortality, respectively, during the 96-h exposure. Sensitivity of an organism to any chemical varies with fish size and species (Sprague, 1971). Values of 24-h and 96-h LC for trichlorfon in juvenile yabbies (Cherax destructor) were 0.351–1.167 and 0.018–0.093 mg L−1, respectively (Qin and Dong, 2004). However, Cebrian et al. (1991) reported that the values of 24-h and 96-h LC trichlorfon for adult crayfish Procambarus clarkii were 5.14–0.99 and 0.73–0.28 mg L−1, respectively. Better standardization of bioassays could reduce the unexplained but design-caused resulting variabilities. The exposure of pacu to a 24-h bath of trichlorfon at 0.025, 0.05 and 0.1 mg L−1 showed even at the lowest concentrations that fish presented pathological alterations in the tissues. Control gills exhibited the normal features of pacu gills (Severi et al., 2000) (Fig. 1a). Fish exposed to 0.025 mg of trichlorfon did not present alterations in the branchial architecture, but fish exposed to 0.05 and 0.1 mg showed hyperplasia in the primary and secondary lamellae and a marked swelling of blood sinuses in the secondary lamellae (telangiectasia) (Fig. 1b), some foci of sub-epithelial edema (Fig. 1c,f), lamellar fusion (Fig. 1d) and foci of blood congestion (Fig. 1e) in the experiment. Branchial gills of pacu (HE). (a) Control fish. Primary (P) and secondary lamellae (S). (b) Trichlorfon 0.05 mg L−1 (1 day after exposure). Telangiectasia (t), hyperplasia of secondary lamellae (h). (c) Trichlorfon 0.05 mg L−1 (1 day after exposure). Sub-epithelial edema (e). (d) Trichlorfon 0.1 mg L−1 (1 day after exposure). Lamellar fusion (lf). (e) Trichlorfon 0.1 mg L−1 (15 days after exposure). Blood lamellar congestion (c). (f) Trichlorfon 0.1 mg L−1 (1 day after exposure). Sub-epithelial edema (e) Organic pesticides are described as changing the branchial epithelium (Wendelaar Bonga and Lock, 1992). Pathological changes similar to those observed in pacu were described for tilapia (Oreochromis niloticus) after 4 h of exposure to 0.25 ppm trichlorfon (Guimarães et al., 2007) and for Corydoras paleatus exposed to a sub-lethal concentration (0.58 μl L−1) of methyl parathion (Fanta et al., 2003). Hyperplasia of the gill epithelium and circulatory changes were described in pacu exposed to copper (Tavares-Dias et al., 2002). Changes observed in pacu gills are implicated in damage to the respiratory system and ionic regulation mechanisms controlled by this structure (Evans et al., 2005). Control hepatocytes presented the normal features described to pacu liver (Souza et al., 2001) (Fig. 2a,b). Immediately after exposure to 0.025 mg trichlorfon, no changes were verified in the liver, but at days 2, 7 and 15 thereafter, the hepatocytes lost their normal shape, the nucleus was peripherally located, and cell fusion was verified. Fish exposed to 0.05 mg trichlorfon showed the same alterations verified immediately after exposure, but there were also necrotic hepatic cells with pycnotic nuclei and decreased cytoplasmatic affinity for eosin, in addition to hypertrophied cells. The necrosis foci increased with time, especially those bordering the exocrine pancreas. In fish exposed to 0.1 mg trichlorfon, the liver changes were similar to those described but more severe with the passage of time (Fig. 2c,d,e). The same lesions were described in the liver of Prochilodus lineatus (Rodrigues et al., 2001) exposed to Dipterex 500 (trichlorfon, 0.2 μl L−1) and Corydoras paleatus (Fanta et al., 2003) exposed to methyl parathion (0.58 μl L−1). The histological alterations in pacu livers suggest that the treated fish faced a metabolic crisis caused by serious tissue damage. Liver of pacu (HE). (a) Control fish. Exocrine intra-hepatic pancreas (p), biliar canaliculi (c). (b) Control fish. Cord-like organization of hepatocytes (h). Central lobular vein (v). (c) Trichlorfon 0.025 mg L−1 (1 day after exposure). Necrosis areas (n). (d) Trichlorfon 0.05 mg L−1 (7 days after exposure). Necrosis areas (n). (e) Trichlorfon 0.1 mg L−1 (7 days after exposure). Necrosis areas (n) The kidney tissue in control fish exhibited the structural pattern described in other teleosts (Veiga et al., 2002) (Fig. 3a,b). No changes were verified immediately after exposure to 0.025 mg trichlorfon, but 2 days after exposure to 0.05 mg trichlorfon the kidney showed thickening of the glomerular capsule and an enlargement of the intercapsular space with glomerular atrophy (Fig. 3c). Melano-macrophage centers were seen (Fig. 3d). The same microscopic abnormalities, but more severe, were observed after the first day of exposure to 0.1 mg trichlorfon. Kidney of pacu (HE). (a) Control fish. Kidney organization – Bowman capsule (bc), hematopoietic tissue (ht). (b) Control fish. Urinary. (c) Trichlorfon 0.05 mg L−1 (7 days after exposure). Enlargement of intercapsular space (s). (d) Trichlorfon 0.1 mg L−1 (7 days after exposure). Necrosis areas and parenchyma disorganization (d). Melano-macrophage centers (m) The kidney is a target of toxic chemicals, which can disrupt its functions and cause temporary or permanent damage of the homeostasis (Miller, 2002). The pathological changes observed in treated pacu demonstrated the renal damage provoked by trichlorfon, as also described in other fish (Gill et al., 1988; Veiga et al., 2002). The melano-macrophage centers also confirm the effect of the pollutant on the kidney. These centers act in the sequestration of products of cellular degradation and potentially toxic tissue materials and increase in size or frequency in conditions of environmental stress (Agius and Roberts, 2003). In conclusion, low concentrations of trichlorfon (0.025, 0.05 and 0.1 mg L−1) were toxic to pacu and alternatives to pesticides in pacu farming should be discussed. On the other hand, the results described could be used as a guide by pathologists to compare healthy and damaged tissues.
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Mataqueiro et al. (2008) studied this question.
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