Abstract: Heavy metal pollution represents a serious problem facing many of the aquatic organisms. Lead is one of the most toxic elements found in many industrial effluents which are metabolized inside bodies and can cause skeletal deformities by impairing developmental processes and bone formation. This study was undertaken to determine the LC50 of Pb-acetate and detect the effect of 1/10 LC50 exposure on fingerlings of Nile Tilapia (Oreochromis niloticus). Modern and accurate diagnostic methods were used; Scanning Electron Microscopy (SEM) of gills, plain X-ray and Energy-dispersive X-ray Spectroscopy (EDX) of the spinal column to explain the elemental analysis. The results indicated that the 96 h LC50 of lead acetate was 146.8 mg L-1. SEM showed a distinct degeneration in gill filaments and pavement cells with increasing of mucus secretion after lead exposure. X-ray revealed slight dorsal spinal curvature. EDX analysis of the spinal column of treated fish showed decrease in calcium and phosphorus weight percents, on the other hand Pb appeared in treated fish but not in control ones. Finally this study can spot a light on the dangerous effects of lead toxicity in developing of fish and impairment of their skeletal ossification which may lead to skeletal deformities.
INTRODUCTION
With the growth of civilization, an increasing number of chemicals are being introduced to our environment. These chemicals are hazardous to all living organisms. The aquatic environment is particularly sensitive and suffered from the toxic effects of contaminants due to industry, urbanization and agriculture activities. Heavy metals pollution has been reported in aquatic organisms by Adham et al. (2002) and Olojo et al. (2005). These pollutants were built up in the food chain and accumulated in the aquatic organisms which were responsible for many adverse effects. Waterborne metals induce disturbances in structure and function of fish organism which led to inhibition of the growth of young stages. Lead has many uses in industry including pipes, paints enamels, glazes, motor industry and others and has not involved in any metabolic processes so it accumulated in the aquatic organism through the food chain causing several effects (Szulkowska-Wojaczek et al., 1992).
Skeletal abnormalities in fishes were widely described and reviewed (Tutman et al., 2000; Jawad and Oktoner, 2007; Eissa et al., 2009). A wide variety of chemicals are known to induce neuromuscular damage that can result in skeletal deformities in fish, in particular lead (Holcombe et al., 1976; Hodson et al., 1980; Bengtsson and Larsson, 1986) Such chemicals can cause skeletal deformities by impairing developmental processes and bone formation. It was suggested that skeletal deformities are useful bio-indicators of pollution (Lemly, 1997; Sun et al., 2009).
Lead was suspected of causing spinal deformities in a wide spectrum of aquatic animals, including fish (Bengtsson and Larsson, 1986) as it produced scoliosis and caudal atrophy and hemorrhaging in the caudal region of fish (Davies et al., 1976). Exposure to low levels of Pb during early developmental stages was found to produce neurobehavioral deficits. These skeletal deformities can impact the normal physiological functions of fish by hindering the ability of fish to eat, reproduce and protect themselves against predators (Eissa et al., 2009). A number of diagnostic methods have been widely employed for detection of fish skeletal deformities; these include histopathology, histochemistry and cell and molecular biological methods, beside the clinical and physical inspection (Martin-Robichaud et al., 2000; Eissa et al., 2009). The method of X-ray can possibly influence the diagnosis and categorization of vertebral deformities (Witten et al., 2009).
The present study was carried out to investigate the effect of exposure to 1/10 LC50 Pb-acetate on the fingerlings of Nile tilapia (Oreochromis niloticus). Influence of Pb on the gill ultra structure was detected by SEM. While the vertebral column and the possible skeletal deformities were examined through plain X-ray and EDX analysis.
MATERIALS AND METHODS
Experimental fish: Fingerlings of Nile tilapia fish Oreochromis niloticus (weight from 10-20 g) were bought from El-Fayoum fish farm and were transported alive in a large plastic bags to Hydrobiology Department, National Research Centre, Dokki, Giza, Egypt. Fish were acclimatized to the laboratory conditions for two weeks before the experiments, during which they fed daily on a fine fish commercial diet.
Determination of 96 h LC50 of lead acetate: To determine the 96 h LC50 of lead acetate (Pb (CH3COO)2.3H2O); a total number of 90 tilapia fingerlings were divided into nine groups (10 per each group); eighty in eight small aquaria (40 L), besides one as control group. The fish were exposed to various concentrations (25, 50, 75, 100, 125, 150, 175 and 200 mg L-1) of lead acetate which previously dissolved in distilled water and added to the aquaria. Fish were exposed to the above concentrations for 96 h. Mortality in each aquarium was recorded daily, then the LC50 value was calculated from the regression line drawn according to the probit method which is a parametric statistical procedure for estimating the LC50 (Finney, 1964). The calculated 96 h LC50 of lead acetate was 146.8 mg L-1.
Effect of 1/10 LC50 of lead acetate on O. niloticus: Total of sixty tilapia fingerlings were divided into two groups, each group was represented by three replicates (10 fish per each aquarium). The first group was the control group while the second group was received a concentration of lead acetate which represents the 1/10 of the 96 h LC50 (14.68 mg L-1). One third of water in each aquarium was renewed every three days, fish feces were siphoned from the aquaria and the Pb concentration was adjusted to maintain its concentration in the treated aquaria. Fish were fed daily as in the accommodation period at rate of 3% of their body weight.
Scanning electron microscopy (SEM) of gills: After one month of exposure to 1/10 LC50 lead acetate; samples of fish were dissected, gills were fixed in 10% formal-saline solution. After rinses and dehydration in ethanol, gills were dried with CO2, mounted onto aluminum stub and coated with gold palladium. These preparations were then examined by Electron microscope; JEOL JXA-840A ELECTRON PROBE MICROANALYZER, JAPAN.
Vertebral analysis
Radiographic plain X-ray examination: At the end of the experiment (one
month), fish were X-ray and radiographs were examined for alterations in vertebral
structures. The X-rays were taken with the fish in dorsoventral and lateral
positions by Toshiba-Varian, TF-6TL6, Utah, USA, with a technique chart utilizing
40-50 kV and 10-20 mA.
Energy-dispersive X-ray spectroscopy (EDX): EDX is an analytical technique used for the elemental analysis of sample. Fish viscera were removed, then muscles were digested using the standard Pepsin-HCl digestion method (Srisawangwong et al., 1997). Fish samples were digested using an acid pepsin solution (1 mL conc. hydrochloric acid, 1 g pepsin, 99 mL 0.85% sodium chloride solution) for 1-2 h at 37°C. The digested material was discarded, bony skeleton become clear, rinsed with saline and kept in room temperature to dry very well. Three points were taken; in the beginning, middle and end of the vertebral columns of treated and control fishes and examined by EDX, INCA X-sight OXFORD Instruments, ENGLAND.
RESULTS
The calculated 96 h LC50 of lead acetate was 146.8 mg L-1. After thirty days of exposure of Oreochromis niloticus to 1/10 LC50 of lead acetate, the treated and control fishes were subjected to SEM, plain X-ray and X-ray spectroscopy (EDX).
SEM of normal non-treated fish: The gills are consists of main filament trunk and are divided into several small filament trunks which are provided by many gill lamella as shown in Fig. 1a and c. The different structures of gills were covered with mucous damping the structural space. The pavement cells were provided with microridges (Fig. 1e).
SEM of lead acetate treated fish: The gill filaments appeared cracked and thin with irregular thickness at their whole lengths due to erosions or failure in bone ossification (Fig. 1b). The gill lamellae are greatly thickened curved and appeared shorter. There were several small notches (n) and bony projections (bp) at the surfaces of each lamella due to bony proliferation changes (Fig. 1d). The lamellar surface is spotted (Fig. 1b, d). Deep dark areas mixed with lobulated areas were observed in the interlamellar space (ir).
| Fig. 1(a-f): | SEM micrographs of Oreochromis niloticus gills after thirty days of exposure to 1/10 LC50 of Pb-acetate, (a, c, e) Control group, (b, d, f) Treated group. a and c showing normal gill filaments (f), Lamellae (l) and the interlamellar region (ir) which was clear. b and d showing a cracked and thin filament with irregular thickness at its whole length, small notches (n) and bony projections (bp) appeared on the lamellae surface and presence of much mucus (m) secretion. e showing the detailed normal ultrastructure of the gill filament with its pavement cells (pc) provided with microridges (mr) while f showing coagulative necrosis of the pavement cells with disappearance of its microridges (arrows) |
The pavement cells (pc) showed coagulative necrosis with disappearance of its architecture together with its microridges (Fig. 1f).
Plain X-ray of normal and lead acetate treated fish: Radiographic examination was shown in Fig. 2. The observed changes in the treated fish included slight dorsal spinal curvature with widening of the intervertebral space.
| Fig. 2(a-c): | Plain X-ray micrograph of control and treated Oreochromis niloticus after thirty days of exposure to Pb-acetate toxicity, (a) Dorsoventral position of normal fish with straight spinal vertebra, (b) Dorsoventral position of treated fish showing slight spinal curvature (arrow) and (c) Lateral position showing marked fusion of dorsal and anal fins in treated fish (arrow) |
Also the radiograph showed marked fusion of dorsal with anal fins. On the other hand, the radiograph of normal non treated fish showed straight spinal vertebrae with normal and proper intervertebral spaces and separation of dorsal spine from anal fin.
Energy-dispersive X-ray spectroscopy (EDX): The determination of elements structure at three different points along the vertebral column of fish showed decrease in weight percent of calcium and phosphorus in head, trunk and tail regions in lead acetate treated fish compared to the control one. Moreover Pb appeared only in the EDX analysis of the treated samples and cannot detect in control samples (Fig. 3, Table 1).
DISCUSSION
Gills play a primary role in osmoregulation in fishes. Studies of teleost gills have described the morphological and functional characteristics of gill epithelial cells. These cells participate in various functions, such as gas exchange, maintenance of blood acid-base balance and ionic regulation (Sturla et al., 2001). While there is a lack of published studies on the cell types involved in these functions, electron microscopy studies have described the ultrastructure of pavement cells and its characteristic indicative of a high metabolic activity and acid-base regulation (Wilson et al., 2000; Carmona et al., 2004). SEM of gills of the lead acetate treated fish presented in the present study revealed impairment and disturbance of bony ossification of gill lamellae and filaments, also the pavement cells showed coagulative necrosis with disappearance of its architecture and microridges, this may be due to the metal toxicity which reduces gill calcium uptake and resulting in changes in gill filament properties; they become flexible (Jezierska et al., 2009).
| Fig. 3(a-f): | Elemental analysis (EDX) of the vertebral column of Oreochromis niloticus after exposure to Pb-acetate toxicity, (a-c) EDX analysis of control fish in head, trunk and tail regions, respectively, (d-f) EDX analysis of treated fish in head, trunk and tail regions, respectively, Arrow shows appearance of Pb in the treated fish |
| Table 1: | Weight percent of Ca, P and Pb elements in different regions of vertebral column of Oreochromis niloticus after exposure to Pb-acetate toxicity |
| nd: Not detected | |
Also the observed spotted lamellar surface may be due to the inflammatory processes preceding the degenerative changes, while the deep dark inter lamellar space with lobulated areas may be due to the organization of the increased mucous mixed with the inflammatory fluid. Similar studies also indicated degeneration of pavement cells of gills of the African freshwater cichlid Oreochromis mossambicus after 35 days of exposure to cadmium (Pratap and Bonga, 1993).
Deformity mechanisms are not yet well understood (Bengtsson, 1979; Al-Harbi, 2001) but in most cases appear to be linked to the disruption of early developmental processes (Longwell et al., 1992). Radiography is faster and simpler to perform than standard histological methods for examining skeletal anatomy and provides considerable detail without requiring the sacrifice of the animal. X-rays can also be used as a rapid survey of known mutants for skeletal abnormalities (Fisher et al., 2003). Minor vertebral changes in fish could be detected radiographically and sonographically, when no observable effects on external morphology were noted. This demonstrates the importance of diagnostic imaging in detecting skeletal deformities in fish. Detected vertebral changes ranged from demineralization, increased density and slight loss of structure to collapse, fusion and change in intervertebral spacing for multiple vertebrae. This impairment could give rise to lordosis, kyphosis or abnormal vertebrae at several locations. The observed curvature in the lead acetate treated fish of the present study may be explained by the effect of lead toxicity on inhibition of enzymatic decomposition of tryptophan; also Pb may cause inhibition of other enzymes due to its affinity to amino, imino and sulfhydryl groups of the enzymatic active centers (Slominska and Jezierska, 2000). A similar effect of lead was observed, since fish exposed to lead had scoliosis (Davies et al., 1976; Holcombe et al., 1976). Many literatures indicate that metal intoxication reduces calcium uptake and bone calcium accumulation which results in the changes in bone properties; they become flexible and easy bend. Metals may also induce disturbances of neuro-muscular transmission which often results in muscular contractures that may lead to skeletal deformities, especially if it is flexible (Jezierska and Witeska, 2001; Jezierska et al., 2009).
Calcium and phosphorus are closely related to the development and maintenance of the skeletal system and the stability of the vertebrae is maintained by a solid phase of calcium phosphate (Lall and Lewis-McCrea, 2007). Concerning the decrease in calcium and phosphorus elements estimated by EDX analysis in the present study, this may explain both the great degenerative changes shown in the ultra structure examination of gills and the spinal alterations examined by X-ray. These results were also in agreement with that of Slominska and Jezierska (2000), who revealed an impairment of skeletal ossification of the larvae of carps after exposure to copper and the skeleton remained cartilaginous. On the other hand the bones were fully calcified in 40 days old in control carps. Only in some fish initial ossification may occurred in the anterior part of the spinal column (Slominska and Jezierska, 2000). This also confirms the high weight percent of these two elements in the head region compared with trunk and tail regions.
CONCLUSION
The present study has succeeded in diagnosis of lead toxicity in fish by modern and accurate methods; SEM and radiographical study. The toxicity by lead revealed disturbance of bone ossification and may be reach to mutation-like tumor in Nile tilapia fish (Oreochromis niloticus).