Toxicity of Copper in Buttersh (Poronotus triacanthus): Tissues Accumulation and Ultrastructural Changes

Wannee Jiraungkoorskul, Somphong Sahaphong, Niwat Kangwanrangsan Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

Received 19 August 2005; revised 12 October 2006; accepted 4 November 2006 ABSTRACT: The present study investigated the accumulation of copper in various tissues and ultrastructural alterations in buttersh, Poronotus triacanthus. In acute toxicity test, buttersh were exposed to 250 g/L copper for 24 h. In subacute toxicity test, sh were exposed to 25 g/L copper for 7 days and then returned to normal water for 48 h. The levels of copper accumulation in the tissues were determined by using an atomic absorption spectrometer. After the 7 day exposure, the highest level of copper was found in the liver, followed by kidney, gills, and muscle tissues (3.64, 0.62, 0.59, and 0.34 g/L, respectively). The recovery group has shown some reduction in copper level in these tissues when compared with those of the 7 day exposure group (3.09, 0.34, 0.30, and 0.27 g/L, respectively). In gills, the major changes such as lament cell proliferation, increase in intercellular spaces, epithelial lifting, and thickening of the lament and lamellar epithelium were observed. In liver, the major changes such as swollen mitochondria, fragmented in rough endoplasmic reticulum, increases in number and size of lysosomes and lipid droplets. Inltration of leukocytes, increasing hepatocyte size with pyknotic nuclei, and presence of vacuoles were also observed. In kidney, the changes included alterations of the rst proximal tubules, as well as vacuolization of the cytoplasm, proliferation of lysosomes and mitochondria, dilation of endoplasmic reticulum, and nally, cell necrosis. The transmission electron microscopic analysis revealed that the 7 day exposure group had more severe effect in tissue alterations than the 24 h exposure group. Tissue regeneration was also observed in the 48 h recovery group. # 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 92100, 2007. Keywords: copper; buttersh; Poronotus triacanthus; ultrastructure

INTRODUCTION
The aquatic environment has suffered due to the growing number of xenobiotics such as heavy metal and pesticide. Copper (Cu) is a trace metal, classied as an essential element for most living organisms but, in high concentrations, it can be a toxic pollutant. Cu is used to control fungal diseases in vineyard plants in France, South Africa (Schlotfeldt, 1992), and orange orchard in Thailand. High concenCorrespondence to: W. Jiraungkoorskul; e-mail: tewjr@mahidol.ac.th Contract grant sponsors: Thailand Research Fund (TRF) and the Commission on Higher Education (the New Researchers Grant); Faculty of Science, Mahidol University. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.20238
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trations of Cu were detected in some aquatic ecosystems collecting vineyard runoff water (GERBE, 1996). High concentrations, as high as 0.6 mg/L Cu, were detected in orange orchard runoff in Prae province, Thailand (Duangduen et al., 1999). The toxic effect of Cu is related to its capacity for catalyzing oxidative reactions, leading to the production of reactive oxygen species (Lopes et al., 2001). These highly reactive compounds may also induce tissue alterations and physiological derangement in sh (Varanka et al., 2001). Many different types of biomarkers are in use nowadays, ranging from biochemical and cellular biomarkers to physiological indicators and ecosystem monitors. They have to be more sensitive, less variable, and often easier to measure. When the concentration of pollutants is high enough, changes occur within an entire organ or specic parts of it.

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Changes which alter the cells and tissues of an organism and dont result in death can be viewed under light microscope or electron microscope. Histopathology has received increasing interest as an endpoint because histopathological changes are often the result of the integration of a large number of interactive physiological processes (van der Oost et al., 2003). Ultrastructure of tissues and organs is altered when levels of the contaminant are still at low levels, therefore histopathological assays provide a valuable screening method before sever damage occurs. The application of environmental toxicology studies among nonmammalian vertebrates is rapidly expanding, and, for aquatic systems, sh have become indicators for the evaluation of the effects of noxious compounds. Buttersh (Poronotus triacanthus) is a commercialized freshwater sh that is not only widely known around the world, but it is also one of the most popular sh in Thailand. At the present time, this species is the rst Southeast Asia sh investigated in relation to copper exposure. It can provide a good model to study responses and possible adaptations of local sh populations to aquatic pollutants. Fish possess different defensive mechanisms to counteract the impact of toxicants. The objectives of this study were to determine the toxicity of copper in buttersh (P. triacanthus), including tissue accumulation and ultrastructural alterations under experimental conditions.

of the initial body weight per day. Copper (II) sulfate pentahydrate (CuSO4 5H2O, Cat. No. 102790, Merck, Germany) was directly diluted in water to obtain the desired exposure concentrations.

Acute Toxicity Tests

The acute toxicity tests were performed according to the US EPA procedure for the static nonrenewal technique (US EPA, 2002) as described in the previous study (Jiraungkoorskul et al., 2002). Fish were not fed 48 h before starting and 96 h during the experiment. Preliminary screening was carried out to determine the appropriate concentration range for testing the chemical. The test consisted of a control and at least ve concentrations of Cu, three replicates per group, with ten sh in each replicate. At the beginning of the tests and after every 24 h, the symptoms and the number of dead sh were recorded. The results of the median lethality concentration (LC50) at 24, 48, 72, and 96 h were computed using the SPSS probit analysis computer program (Finney, 1971).

Experimental Design
The 96 h LC50 value of buttersh exposed to Cu was determined in our laboratory as 502.95 g/L (Fig. 1). In this study, sh were randomly divided into three groups, with three replicates performed for each group. Group I (n 18), the control group; group II (n 18), sh were exposed to 250 g/L Cu (50% 96 h LC50) for 24 h; and group III (n 18), sh were exposed to 25 g/L Cu (5% 96 h LC50) for 7 days and then returned to normal water for 48 h. All glass ow-through aquaria (50 50 120 cm3) with continuous aeration were lled with 200 L of dechlorinated tap water whose physicochemical characteristics were the same as those described previously. At different times (after 24 h and 7 days exposure, and after 48 h of recovery), two sh of each group were anesthetized with 0.2 g/L MS-222 (tricaine methan sulfonate), weighed, and measured. The organs were removed and prepared for accumulation and ultrasturctural studies. The relative weight of the organ was expressed as a percentage of the body weight.

MATERIALS AND METHODS Animals

Buttersh (P. triacanthus), 1520 g in body weight and 8 10 cm in total length, were purchased from a commercial hatchery in Bangkok, Thailand. Tap water was ltered with activated charcoal (Aquapur, thysen, FRG) to eliminate chemical contamination. The physicochemical characteristics of water were measured daily, according to the experimental procedures described in Standard Methods for the Examination of Water and Wastewater (APHA, 2005). Conductivity was measured with Hanna instruments Model 3 DiST WP (Hanna Instruments, Rhode Island, USA). The pH was measured with a Cyberscan 510 (Eutech Instruments, Illinois, USA) and the temperature was measured with a glass mercury thermometer. A 16 h light and 8 h dark photo-period was maintained. Acclimatization to laboratory conditions for 7 days was done using dechlorinated tap water that had the following physicochemical characteristics: temperature 26.0 6 2.08C, pH 6.87.3, total hardness 5070 mg/L (as CaCO3), alkalinity 6265 mg/L, and conductivity 185210 mhos/cm. Chlorine residue and ammonia were below detection limits. Fish were fed twice a day with 37%-protein commercial sh food (Charoen Pokphand Group, Bangkok, Thailand). The quantity of food was 2%

Copper Accumulation Studies

The dry weights of dissected tissues were determined after being kept at 1058C for 48 h. Dried tissues were digested with 3.0 mL of a mixture of HNO3 and HCIO4 in a 2:1 ratio (Muramoto, 1983). Acid digestion was carried out at 1208C for 3 h. After dilution with distilled water, Cu concentrations of tissues were determined using a GBC 932 ame atomic absorption spectrophotometer.

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Atomic Absorption Analysis

The levels of copper accumulation in various tissues of P. triacanthus are shown in Table I. In the exposure groups, the highest levels were measured in the liver, followed by the kidney, gills, and muscle tissues. They were increased signicantly when compared with those of the control group (3.64, 0.62, 0.59, and 0.34 g/L in the 7 day exposure group, respectively; and 3.09, 0.34, 0.30, and 0.27 g/ L in the 48 h recovery group, respectively). In the 48 h recovery group signicant elimination was detected in the kidney and gills when compare with those of the 7 day exposure group.

Histopathological Studies Gills

Fig. 1. The probit transformed responses of buttersh exposed to LC50 value of copper.

Specimen Preparation for Transmission Electron Microscopic Study

Small pieces of tissue were xed in 4% glutaraldehyde phosphate buffer (0.1 mol/L, pH 7.4) at 48C for 24 h and postxed in 1% osmium tetroxide for 1 h. They were dehydrated through a graded series of alcohol, cleared in propylene oxide, and embedded in polyethylene beam capsule containing Epon 812 resin. Ultrathin sections were cut on an ultramicrotome, stained with uranyl acetate and lead citrate. They were examined by a Hitachi H-7000 transmission electron microscope at 75 kV (Humason, 1972).

Statistical Analysis
All statistical analyses were performed using the SPSS 12.0 for Windows software (SPSS, Chicago, IL, USA). They were expressed as means 6 SD. A two-way analysis of variance was used to determine the signicance of copper accumulation. The least-signicant difference (LSD) was used for determination of signicant differences at P 0.05.

Control Group. Investigation of the gill structure at the ultrastructural level showed signicant differences between control and treated groups. There was no evidence of ultrastructural alterations in the gills of control sh throughout the course of this experiment; therefore, the following description was representative of all time periods sampled. Briey, each gill consisted of numerous primary laments and secondary lamellae, the relationship between both of them was seen at low magnication. The secondary lamellae were implanted in parallel rows on either side of the primary lament. In the electron micrograph, the gill was composed of two distinct epithelial surfaces, the lament and lamellar epithelia. The lament epithelium was multilayered and consisted of various cell types such as mucous goblet cells and chloride cells. The lamellae consisted of a network of pillar cells alternating with the blood spaces formed. They were separated from the aqueous environment by two epithelial layersthe inner serosal epithelial layer and the outer mucosal epithelial layer. The apical surfaces of mucosal cells were characterized by complex system of microridges [Fig. 2(A)]. After 24 h exposure, the initial alteration tended to appear in the mucosal cells of the lamellar epithelium. Occasionally, these cells appeared swollen and exhibited
TABLE I. Copper level (lg/g dry weight) in the tissues of buttersh (P. triacanthus) Control Group (n 6) 0.04 6 0.01 0.03 6 0.01 0.04 6 0.01 0.06 6 0.00 7 Day Exposure Group (n 6)a 3.64 6 0.53 0.62 6 0.22 0.59 6 0.13 0.30 6 0.12 48 h Recovery Group (n 6)a 3.09 6 2.05 0.34 6 0.17a,b 0.30 6 0.09a,b 0.27 6 0.29

Tissue

RESULTS Acute Toxicity Tests

From the probit transformed responses curve (Fig. 1) of buttersh exposed to 24, 48, 72, and 96 h of Cu, the values of LC50 were log of 3.03, 2.86, 2.80, and 2.70 or 1077.94, 724.12, 629.53,and 502.95 g/L, respectively (Fig. 1).

Liver Kidney Gills Muscle

a The mean difference was signicant when compared with the control group (P 0.05). b The mean difference was signicant when compared with the 7 day exposure group (P 5).

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Fig. 2. Transmission electron micrographs of the gills of buttersh, P. triacanthus. (A) In control group, gills composed of the stratied epithelium of the primary lament and the secondary lamellar. These cells included mucous cells (mc), choride cells (cc), microridges (mr), and pillar cells () with blood sinuses (bs) (bar 2 m). (B) In the 250 g/L copper exposure 24 h group, the mucosal cell () of the secondary lamellar epithelium appeared swollen and had small microridges (mr) (bar 2 m). (C) In the 25 g/L copper exposure 7 day group, the mucosal cell () of the secondary lamellar epithelium appeared swollen. Note that the microridges (mr) were irregularly formed and absent from many cells. They were found the necrotic cells (nc) and myelinoid bodies in the lament (bar 2 m). (D) In 48 h recovery group, the primary lament and the secondary lamellar showing similar to that of the control. Note small microridges (mr) and the normal appearance of the mucous cell (mc) (bar 2 m).

an increased thickness. The microridges were irregularly formed and absent from many cells. The intercellular spaces between the mucosal and serosal epithelial layers increased markedly. The lament epithelium exhibited an

increased thickness [Fig. 2(B)]. After 7 days exposure, the mucosal cells were severe swollen and the microridges were absent. The lamellar epithelium generally contained higher densities of myelinoid bodies characterized by their

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onionoid whorls of membranous material, and the intercellular spaces had increased in both size and number compared to that observed following the 24 h exposure group. The lament epithelium had been presented necrotic cells showing cytoplasmic disorganization and containing electron dense granular material of undened morphology [Fig. 2(C)]. After 48 h recovery, there was no evidence of ultrastructural alterations in the lament epithelium except the occurrence of few intercellular spaces. The mucosal cells were still swollen and had small microridges on the cell surface [Fig. 2(D)].

Liver
Control Group. The nucleus was large, round or oval in shape and enclosed by the nuclear envelope that consisted of two layers of membrane. Furthermore, the nucleus contained heterochromatin that was randomly distributed in the nucleoplasm. The cytoplasm had a well-developed rough endoplasmic reticulum, mitochondria, and Golgi complex. The rough endoplasmic reticulum was arranged near the nucleus in 56 parallel arrays, and ribosomes were attached to the membranes while the SER was scarce. Mitochondria appeared as spherical or elongated proles with numerous cristae. Lysosomes were few and smaller in size; their distribution was restricted to the peripheral area of the cell [Fig. 3(A)]. After 24 h exposure, the most conspicuous perturbation was encountered within the lipid inclusions. Hepatocytes exhibited many large electron dense lipid droplets. The nuclear outline was irregular. Condensation of the nuclear chromatin was found. The ribosomes were detached from the surface of the RER. There was a random distribution of ribosomes throughout the cytoplasm. Mitochondria were increased and some showed swelling [Fig. 3(B)]. Any of the observation described for the 7 day exposure groups could be veried in the 24 h exposure groups but they displayed more pronounced damages. Hepatocytes exhibited many large electron dense lipid droplets, mitochondrial swelling with loss of the matrix and cristae. Chromatin material was clumped within the nucleus. A large number of vacuoles and lysosomes were observed [Fig. 3(C)]. After 48 h recovery period, hepatocytes exhibited the normal appearance compared to that of control group. Some mitochondria were still swollen [Fig. 3(D)].

be long and were orientated perpendicular to the basal lamina [Fig. 4(A)]. After 24 h exposure, the apical vacuoles increased in diameter, some mitochondria were contracted and showed a considerable variability in size and shape [Fig. 4(B)]. After 7 days exposure, the nucleus displayed irregular outlines with condensed heterochromatin. There were dilations of RER cisternae. In addition, lysosomes were increased in number and no longer restricted to the apical cell portions. There was an increase in the size of mitochondria and proliferation of atypical mitochondrial prole [Fig. 4(C)]. After the 48 h recovery period, the rst proximal tubular epithelial cells exhibited the normal appearance compared to that of control group [Fig. 4(D)].

DISCUSSION
The problem in determining 96 h LC50 for sh is that the lethal concentrations will vary depending upon the species, sex, and age of the sh and the physiochemical characteristic of water including, pH, hardness, conductivity and temperature (Howarth and Sprague, 1978). Therefore 96 h LC50 values were obtained from studies performed in our laboratory where experimental conditions were as similar to those used in the present study as possible. The accumulation of heavy metals in the tissues of aquatic organisms may cause various physiological defects and mortality (Karakoc, 1999). Copper accumulation in all tissues of sh increased with increasing concentrations of the metal and with increasing exposure period (Cogun and Kargin, 2004). In this study, the highest Cu accumulation occurred in the liver, followed by the kidney, gills, and muscles, respectively. Our ndings were in agreement with the previous studies in Tilapia nilotica (Karakoc, 1999) and Oreochromis niloticus (Cogun and Kargin, 2004). In addition to the gill which is exposed and which accumulates copper when water-born copper concentrations are elevated (Grosell et al., 2003), the gastrointestinal tract is also in direct contact with copper as a result of drinking. Studies carried out in various sh species have shown that liver and kidney are the main sites of accumulation and storage (Capelli and Minganti, 1987; Cogun and Kargin, 2004). The slightly elevated Cu accumulations were observed at 1 day after onset of Cu exposure and it continuously increased through 7 days exposure. They had returned to normal or were only slightly elevated during the 48 h recovery period when compared to those of the control group. The elimination routes of metals from sh are generally through gills, bile, urine, and skin (Heath, 1987). In the present study, the kidney and gills showed signicant decrease in the level of copper when compared with those of the 7 day exposure group. The environmental copper load was adequate to cause signicant structural changes in the sh tissues (Heerden

Kidney
Control Group. Ultrastructural investigations were restricted to the cells of the proximal segments of the nephronic tubules. The rst proximal segment was lined by low columnar cell having long and slender microvilli of brush border, apical vacuoles below the brush border, and a welldeveloped lysosome system. The rough endoplasmic reticulum was found around the nucleus. Mitochondria tended to

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Fig. 3. Transmission electron micrographs of the liver of buttersh, P. triacanthus. (A) In control group, each hepatocyte has a normal central round nucleus (N) and a small amount of heterochromatin, rough endoplasmic reticulum (RER), and mitochondria (M) (bar 1 m). (B) In the 250 g/L copper exposure 24 h group, hepatocytes showing large lipid droplets(L) and lysomes (ly). Some mitochondria (M) are swollen. Chromatin materials are clumped within the nucleus (N) (bar 1 m). (C) In the 25 g/L copper exposure 7 day group, hepatocytes showing the reduction in number and length of rough endoplasmic reticulum (RER) cisternae. Mitochondrial (M) swelling with loss of matrix and cristae. A large number of lysomes (ly) have been observed. Note chromatin materials are clumped within the nucleus (N) (bar 1 m). (D) In 48 h recovery group, hepatocytes showing the regular outline of nucleus (N), large lipid droplets (L). Mitochondria (m) are similar to that of the control. Note a few numbers of lysosomes (ly) (bar 1 m).

et al., 2004). This electron microscopic study, after 24 h Cu exposure, showed that the initial alteration of gills appeared in the mucosal cells of the lamellar epithelium. The loss of

microridges and appearance of intercellular spaces were observed. Additionally, the thickening of the primary lamellar epithelium, edema, lifting and fusion of secondary

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Fig. 4. Transmission electron micrographs of the kidney of buttersh, P. triacanthus. (A) In control group, the epithelial cells of proximal segment showing apical vacuoles (av) and lysosome (ly) underneath a high brush border (BB). Nucleus (N) is surrounded by small stacks of rough endoplasmic reticulum (RER) interspersed with mitochondria (M) (bar 1 m). (B) In the 250 g/L copper exposure 24 h group, the nucleus (N) shows irregular outline of nuclear membrane. There are dilations of rough endoplasmic reticulum (RER), proliferation of lysosomes (ly), increase in size of apical vacuoles (av). M mitochondria, BB brush border (bar 1 m). (C) In the 25 g/L copper exposure 7 day group, the nucleus (N) shows irregular outline of nuclear membrane and condensed heterochromatin. The cytoplasm shows dilations of rough endoplasmic reticulum (RER), condensation of mitochondria (M) (bar 1 m). (D) In 48 h recovery group, the epithelial cells of proximal segment showing similar to that of the control. N, nucleus; M, mitochondria; BB, brush border (bar 1 m).

lamellae were observed. After 7 days Cu exposure, lesions on the laments and lamellae were similar to those of 24 h Cu exposure, but more severe and were largely reversed by the end of the 48 h recovery period. Similar results were reported in subacute toxicity tests of sublethal Cu concen-

trations on Prochilodus scrofa (Mazon et al., 2002). Lamellar epithelial lifting, hyperplasia, and lamellar fusion are nonspecic responses that are known to be induced by many gill tissue irritants (Mallatt, 1985). However, focal points of cellular hypertrophy and necrosis followed by

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epithelial rupture reect the direct deleterious effects of heavy metals in sh gills (Mallatt, 1985; Sola et al., 1995). All these lesions may impair respiratory function. Lifting or hyperplasia of epithelium results in an increase in the diffusion distance, thus affecting exchange of gases. Microridges increase the surface area of cells in contact with the external environment. They are generally considered important structures for mucus retention on gill epithelia (Lewis, 1979) and are perhaps as indicative of cell activity (Goss et al., 1994). In this study, the fusion of lamellae and the loss of microridges may be caused by the decrease in the total respiratory area of the gills, resulting in decreased oxygen-uptake capacity of sh gills. Fish fail to get adequate oxygen for total metabolic activities. In the liver, after 24 h Cu exposure, the most conspicuous alteration was encountered within the RER; they displayed progressive reduction and fragmentation. Mitochondria were swollen and there were increases in number and size of lysosomes and lipid droplets. Inltration of leukocytes, increasing hepatocyte size with pyknotic nuclei, and presence of vacuoles were also observed. After 7 days Cu exposure, lesions in the hepatocyte were similar to those of 24 h Cu exposure, but more severe and were largely reversed by the end of the 48 h recovery period. Similar results were reported in subacute toxicity tests of sublethal Cu concentrations on Channa punctatus (Khangarot, 1992). The lesions include increase in the numbers of lysosomes and electron dense bodies, dilation of the rough endoplasmic reticulum, loss of normal material density, cristae, or outer or inner membranes with mitochondrial swelling. A highly dilated endoplasmic reticulum and shortened and reduced mitochondria were the prominent features of acute Cu toxicity. Nuclei of the necrotic cells showed marked clumping of chromatin with the aggregation of interchromatin material at the center of the nucleus. The changes consisted of extensive proliferation of the smooth endoplasmic reticulum and dilation of the rough endoplasmic reticulum, suggesting an active detoxication attempt by the liver. In the kidney, after 24 h Cu exposure, the ultrastructural alterations consisted of the degeneration of the nuclear membrane, mitochondrial contraction and/or swelling, and accumulation of large electron-dense particles. Lysosome and apical vacuoles increased in number and size. Finally, the necrosis appeared in some cells. After 7 days Cu exposure, lesions in the renal tubular were similar to those of 24 h Cu exposure, but more severe and were largely reversed by the end of the 48 h recovery period. Similar results were reported in subacute toxicity tests of Cu concentrations on Coregonus lavaretus L. (Moiseenko and Kudryavtseva, 2001). The lesions showed small cytoplasmic vacuoles and nuclear deformation in the epithelium of the rst segments of the proximal tubule. Because the excretion of divalent ions is a major function of the renal tubular epithelium, pollution with heavy metals or pesticides would be highly likely to affect these cells.

The toxic effect of Cu is related to its capacity for catalyzing oxidative reactions, leading to the production of reactive oxygen species (Lopes et al., 2001). These highly reactive compounds may also induce tissue alterations and physiological derangement in sh (Varanka et al., 2001). Fish possess different defensive mechanisms to counteract the impact of toxicants. One is the immune system, where the kidney is the one of the major organs of the sh immune system. The otherwise, the gills, liver, and also kidney are as the organs implicated in the metabolism and excretion of toxicants. From our results, it is possible to conclude that tissue accumulation and ultrastructural alterations in buttersh (Poronotus triacanthus) can be used as markers of environmental pollution and can be monitored by transmission electron microscopy.
We wish to thank the Center of Nanoimaging, Faculty of Science, Mahidol University for providing the transmission electron micrographs.

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Environmental Toxicology DOI 10.1002/tox