Effects of extremely low-frequency electromagnetic fields (ELF-EMF) exposure on B6C3F1 mice

Objective

Long-term exposure study was conducted to investigate the effects of extremely low-frequency electromagnetic field on the tumor promotion process and fertility.

Methods

Ten pregnant C57BL/6NCrj mice were exposed to 50 Hz field 500 mG for 1 week (12 h per day), and 24 male and 42 female B6C3F1mice born from them were further exposed up to 15.5 months. As a control group, 10 pregnant mice were bred without exposure, and 30 produced male and 32 female mice were observed without exposure for the same period.

Results

Mean body weights of exposed groups of male and female mice were decreased significantly than those of the control groups. In exposed mice, there was no increased incidence of liver and lung tumor. In female mice, the incidence of chronic myeloid leukemia [3/42 (7 %)] in the exposed group was significantly greater than in the control group. The size of seminiferous tubules in the EMF exposed groups were significantly less than the control group.

Conclusions

These data support the hypothesis that long-term exposure of 50 Hz magnetic fields is a significant risk factor for neoplastic development and fertility in mice.     

Effects of a 4.7 T static magnetic field on fetal development in ICR mice

In order to determine the effects of a 4.7 T static magnetic field (SMF) on fetal development in mice, we evaluated fetal teratogenesis and endochondral ossification following exposure in utero. Pregnant ICR mice were exposed to a 4.7 T SMF from day 7.5 to 9.5 of gestation in a whole-body dose, and sacrificed on day 18.5 of gestation. We examined the incidence of prenatal death, external malformations and fetal skeletal malformations. There were no significant differences observed in the incidence of prenatal death and/or malformations between SMF-exposed mice and control mice. Further, we evaluated the immunoreactivity for the vascular endothelial growth factor (VEGF), which is implicated in angiogenesis and osteogenesis, in the sternum of fetal mice following magnetic exposure. Our studies also indicated that on day 16.5 of gestation following SMF exposure, the immunoreactivity for VEGF was increased compared to unexposed controls. However, it was decreased in the exposed group compared to the control group on day 18.5 of gestation. DNA and proteoglycan (PG) synthesis were also measured in rabbit costal growth plate chondrocytes in vitro. No significant differences were observed in DNA synthesis between the SMF exposed chondrocytes and the control chondrocytes; however, PG synthesis in SMF exposed chondrocytes increased compared to the controls. Based on these results, we suggest that while SMF exposure promoted the endochondral ossification of chondrocytes, it did not induce any harmful effects on fetal development in ICR mice.     

Raised concentration of serum bile acids following occupational exposure to halogenated solvents, 1,1,2-trichloro-1,2,2-trifluoroethane and trichloroethylene

Objectives: The objectives of this study were␣threefold. First, to examine the hepatic effects of occupational exposure to 1,1,2-trichloro-1,2,2-trifluoroethane (FC 113) using conventional and newer tests (serum bile acids) of hepatobiliary function. Second, to assess the effects of altered work practices that included a reduced exposure to a different halogenated solvent (trichloroethylene) on the same parameters of liver function; and finally, to gather further data to support or refute the contention that serum bile acid (SBA) levels could provide a sensitive biological marker of exposure to these solvents. Design: Two groups of workers (control and exposed) in an Australian steel industry participated in the study. The exposed group (n = 5–6) comprised individuals who had either exposure to FC␣113 (68.2 ± 12.6 ppm) or trichloroethylene (8.9 ± 3.1 ppm) during the application of these solvents in a cleaning procedure, whereas the control group (n = 7–11) was composed of non-solvent-exposed office workers in the same company. The initial investigation involved exposure to FC 113 while a follow-up study was undertaken after changes in work practices were made including replacement of FC 113 with trichloroethylene (TRI). Methods: Standard liver function tests and individual serum bile acids (ISBA) were measured before and after exposure to solvents and simultaneously in the control subjects by enzymatic methods and high performance liquid chromatography (HPLC), respectively. Results: Statistical analysis of the data showed a significant increase in the concentration of total serum bile acids (TSBA), some of the subgroups of SBA, and a few of the ISBA in workers after a period of exposure to FC 113. After TRI replaced FC 113 together with other changes in work practices to give substantial reduction in exposure to solvent, a repeat study also found elevated SBA after the cleaning procedure but to a lesser extent. No other indications of adverse liver effects, as measured by conventional parameters of hepatobiliary function, were detected. Conclusion: Exposure to FC 113 was clearly associated with a significant rise in SBA levels, which are sensitive indicators of liver function. This finding is consistent with, and provides further support for, our previous investigations on chlorinated aliphatic hydrocarbon solvents which showed that SBA levels are a sensitive biological marker of exposure to these solvents. Changes in work practices including replacement of FC 113 resulted in a reduced effect on SBA, consistent with lower exposures. Received: 22 October 1996 / Accepted: 21 February 1997     

PBPK modeling/Monte Carlo simulation of methylene chloride kinetic changes in mice in relation to age and acute, subchronic, and chronic inhalation exposure.

During a 2-year chronic inhalation study on methylene chloride (2000 or 0 ppm; 6 hr/day, 5 days/week), gas-uptake pharmacokinetic studies and tissue partition coefficient determinations were conducted on female B6C3F1, mice after 1 day, 1 month, 1 year, and 2 years of exposure. Using physiologically based pharmacokinetic (PBPK) modeling coupled with Monte Carlo simulation and bootstrap resampling for data analyses, a significant induction in the mixed function oxidase (MFO) rate constant (Vmaxc) was observed at the 1-day and 1-month exposure points when compared to concurrent control mice while decreases in glutathione S-transferase (GST) rate constant (Kfc) were observed in the 1-day and 1-month exposed mice. Within exposure groups, the apparent Vmaxc maintained significant increases in the 1-month and 2-year control groups. Although the same initial increase exists in the exposed group, the 2-year Vmaxc is significantly smaller than the 1-month group (p < 0.001). Within group differences in median Kfc values show a significant decrease in both 1-month and 2-year groups among control and exposed mice (p < 0.001). Although no changes in methylene chloride solubility as a result of prior exposure were observed in blood, muscle, liver, or lung, a marginal decrease in the fat:air partition coefficient was found in the exposed mice at p = 0.053. Age related solubility differences were found in muscle:air, liver:air, lung:air, and fat:air partition coefficients at p < 0.001, while the solubility of methylene chloride in blood was not affected by age (p = 0.461). As a result of this study, we conclude that age and prior exposure to methylene chloride can produce notable changes in disposition and metabolism and may represent important factors in the interpretation for toxicologic data and its application to risk assessment.     

No inhibition of γ‐glutamyl transpeptidase‐positive foci by vitamin e with or without phenobarbital

The effect of vitamin E on γ‐glutamyl transpepti‐dase‐positive foci, with or without phenobarbital, was investigated. Groups of six female Sprague‐Dawley rats were initiated with diethylnitrosamine (15 mg/kg) at 24 hours of age. After weaning, they were fed diets with 10% (wt/wt) fish oil; the diets contained 0, 5,000, or 15,000 ppm vitamin E supplementation with or without phenobarbital (500 ppm) for six months. Phenobarbital significantly increased liver weight and liver weight as a percentage of body weight ( p < 0.05), suggesting a liver hypertrophic effect of phenobarbital. Phenobarbital significantly decreased hepatic phospholipid arachidonate, eicosapentaenoate, and docosa‐hexaenoate (p < 0.05) this may indicate that phenobarbital stimulates phospholipase A₂ activity and results in the increased release of polyunsaturated fatty acids from phos‐pholipids and the decrease of hepatic phospholipid polyunsaturated‐to‐saturated fat ratio. In rats fed phenobarbital, hepatic vitamin E content was lower than in rats fed no phenobarbital; this suggests that phenobarbital causes oxidative stress or induces enzymes that metabolize the vitamin. Phenobarbital exposure significantly increased hepatic prostaglandin F_2a and glutathione S‐transferase activity (p < 0.05). Vitamin E did not influence hepatic γ‐glutamyl trans‐peptidase‐positive foci area and number with or without phenobarbital, and phenobarbital showed a strong promoting action on enzyme‐altered hepatic foci.     

Changes of some serum parameters and amino acids content in rats after chronic sublethal doses of dimethoate

An intraperitoneal repeated dose of dimethoate (O,O-dimethyl S-[N-methylcarbamoyl methyl] phosphorodithioate) was injected to male Wistar rats for 8 successive days. Body weight and liver weight decreased significantly while liver/body weight ratio and mg protein/g liver remained unchanged. Twenty serum amino acids were assayed in blood samples collected from hepatic vein, portal vein, and aorta. Most of the measured amino acids showed a tendency of decrease compared to control. The decrease was significant only for taurine, serine, threonine, methionine, tyrosine, histidine, and arginine but not for the rest of the amino acids measured. Glucose, 3-hydroxybutyrate, urea, ammonia, and lactate were measured in the same serum samples. These serum parameters showed a tendency of decrease (except urea) of them from control but the decrease was not significant. Present address: Laboratory of Environmental Toxicology, Mario-Negri Institute, Via Eritrea 62, Milan 20157, Italy     

Effects of whole body exposure to extremely low frequency electromagnetic fields (ELF-EMF) on serum and liver lipid levels,in the rat

Backgound  

The effects of extremely low-frequency electromagnetic fields (ELF-EMF) on the blood serum and liver lipid concentrations of male Wistar rats were assessed.     

Effect of electromagnetic field exposure on chemically induced differentiation of friend erythroleukemia cells

Whether exposure of humans to extremely low frequency electromagnetic fields (ELF-EMF) can cause cancer is controversial and therefore needs further research. We used a Friend erythroleukemia cell line that can be chemically induced to differentiate to determine whether ELF-EMF could alter proliferation and differentiation in these cells in a manner similar to that of a chemical tumor promoter. Exposure of this cell line to 60 Hz ELF-EMF resulted in a dose dependent inhibition of differentiation, with maximal inhibition peaking at 40% and 40 mG (4 microT). ELF-EMF at 10 mG (1.0 microT) and 25 mG (2.5 microT) inhibited differentiation at 0 and 20%, respectively. ELF-EMF at 1.0 (100) and 10.0 G (1,000 microT) stimulated cell proliferation 50% above the sham-treated cells. The activity of telomerase, a marker of undifferentiated cells, decreased 100[times] when the cells were induced to differentiate under sham conditions, but when the cells were exposed to 0.5 G (50 microT) there was only a 10[times] decrease. In summary, ELF-EMF can partially block the differentiation of Friend erythroleukemia cells, and this results in a larger population of cells remaining in the undifferentiated, proliferative state, which is similar to the published results of Friend erythroleukemia cells treated with chemical-tumor promoters.     

全氟辛烷磺酸对小鼠脾细胞NO分泌水平影响

目的观察短期、高剂量全氟辛烷磺酸(PFOS)对C57BL/6雄性小鼠一氧化氮(NO)分泌水平影响。方法选择雄性C57BL/6小鼠24只,PFOS经口灌胃染毒,剂量分别为5,20,40 mg/kg。每天1次,染毒7 d后,制备脾脏淋巴细胞悬液,细胞培养48 h后收取上清液,检测NO含量。结果20,40 mg/kg PFOS染毒组小鼠体重明显下降(P0.05);20 mg/kg PFOS染毒组小鼠脾脏和胸腺指数分别为(0.36±0.04)和(0.21±0.02),明显低于对照组(P0.05)。40 mg/kg PFOS染毒组NO分泌水平为(0.66±0.14)μmol/L,明显低于对照组(P0.05)。结论高剂量PFOS暴露可降低小鼠脾淋巴细胞NO分泌水平,抑制小鼠巨噬细胞的活化。     

The Impact on Reproduction of an Orally Administered Mixture of Selected PCBs in Zebrafish (Danio rerio)

Zebrafish (Danio rerio) were orally exposed to a mixture of 20 PCBs in three different dose levels (0.008, 0.08, and 0.4 μg of each congener per gram of freeze-dried chironomids). Generally, the PCBs accumulated in a dose-related manner. After 13 weeks of exposure body, liver, and ovary weights, as well as the liver and ovary somatic index, were significantly lower in exposed groups. In addition, the PCB mixture was an effective inducer of hepatic EROD activity. The reproduction study performed with exposed females and unexposed males after 9 weeks revealed that median survival time for larvae was only 7.7 days in the high-dose group as compared with 14 days in controls. Furthermore, egg production was reduced in all three groups exposed. No differences in hatching frequency or median hatching time were recorded. Histologically, females in both the intermediate and high-dose groups contained a reduced number of mature oocytes. The present study demonstrates that the potency of the mixture of selected PCBs induces hepatic EROD activity and has a clearly negative effect on zebrafish reproduction. Received: 7 May 1997/Accepted: 29 November 1997     

Acute effects of 1-octyl-3-methylimidazolium bromide ionic liquid on the antioxidant enzyme system of mouse liver

The effects of acute exposure of intraperitoneal injection of aqueous 1-octyl-3-methylimidazolium bromide on the antioxidant enzymes and lipid peroxidation of the treated mouse liver were investigated in the present paper. Thirty mice were treated with 17.9 (0.5 median lethal dose, LD₅₀) and 35.7 mg/kg (1 LD₅₀) of the ionic liquid or saline solution by intraperitoneal injection for 10 h. Significant increase in hepatosomatic index and decrease in liver protein content were found in 1 LD₅₀ group. No statistically significant change was observed in the activity of superoxide dismutase in both treated groups compared to the control group. In the case of catalase activity, the decrease was recorded only at dose of 1 LD₅₀. As for the activity of glutathione peroxidase, increase in 0.5 LD₅₀ group while significant decrease at dose of 1 LD₅₀ was noted. There was an increase in the activity of glutathione-S-transferase in these two treated groups in comparison to the control. However, no effect on the level of lipid peroxidation was found in the treated groups. These results showed that the acute exposure of aqueous ionic liquid could cause damage to mouse, leading to antioxidant responses in livers of the treated mice.     

PBDE-209致小鼠肝脏病理学改变及其氧化应激机制研究

目的 通过对小鼠进行十溴联苯醚(decabrominated diphenyl ether,PBDE-209)染毒,研究该物质对小鼠肝脏的毒性作用及其可能的氧化应激机制.方法 将24只小鼠随机分3组,即生理盐水组、PBDE-209低剂量组和高剂量组,每组8只,将小鼠按10ml/kg体重剂量经口灌胃染毒连续6周,观察各组小鼠肝脏组织形态学和超微结构改变,并测定小鼠各项生化指标.结果 染毒组小鼠血清谷丙转氨酶和谷草转氨酶水平均升高,与生理盐水组比较,差异有统计学意义.高、低剂量组小鼠肝脏组织丙二醛含量均高于生理盐水组,差异有统计学意义;高剂量组小鼠肝脏谷胱甘肽含量低于生理盐水组和低剂量染毒组,差异有统计学意义;高、低剂量组总超氧化物歧化酶活性低于生理盐水组,差异有统计学意义.光镜下观察染毒组小鼠肝脏出现肝细胞浊肿,胞浆内有细小脂质空泡,肝索排列发生改变,部分肝细胞模糊,空泡化显著;电镜下观察染毒组小鼠肝脏出现染色质浓缩聚合,线粒体膨胀,内质网增大.结论 PBDE-209可以引起肝脏组织脂质过氧化指标和组织形态学以及超微结构的改变.     

Herbicide Formulation with Glyphosate Affects Growth, Acetylcholinesterase Activity, and Metabolic and Hematological Parameters in Piava (Leporinus obtusidens)

The teleost fish Leporinus obtusidens (piava) was exposed to different concentrations of Roundup, a commercial herbicide formulation containing glyphosate (0, 1, or 5 mg L⁻¹), for 90 days. Acetylcholinesterase (AChE) activity was verified in brain and muscle. Hepatic and muscular metabolic parameters as well as some hematological parameters were determined. The results showed that brain AChE activity was significantly decreased in fish exposed to 5 mg L⁻¹ Roundup, whereas muscular AChE activity was not altered. Both Roundup concentrations significantly decreased liver glycogen without altering the muscle glycogen content. Hepatic glucose levels were reduced only in fish exposed to 5 mg L⁻¹ Roundup. Lactate levels in the liver and muscle significantly increased in fish exposed to both Roundup concentrations. Hepatic protein content remained constant at 1 mg L⁻¹ but increased at 5 mg L⁻¹ Roundup. In the muscle however, protein content decreased with increasing exposure concentration. The herbicide exposure produced a decrease in hematological parameters at both concentrations tested. The majority of observed effects occur at environmental relevant concentrations, and in summary, the results show that Roundup affects brain AChE activity as well as metabolic and hematologic parameters of piavas. Thus, we can suggest that long-term exposure to Roundup causes metabolic disruption in Leporinus obtusidens.     

Hepatic content of free sulfhydryl compounds in animals exposed to vinyl acetate

Summary The content of free non-protein thiols (–SH) was investigated in the livers of guinea pigs, rats and mice after intraperitoneal injection of vinyl acetate (VA). A rapid change of the hepatic -SH level was found in guinea pigs after injection of 500 mg/kg VA. This resulted in a 50% decrease in –SH content, In mice the decrease was slower and amounted to only 23% four hours after injection of 300 mg/kg VA. Rats responded to a single dose of 450 mg/kg VA with only a 10% reduction of the –SH content of the liver. An approximately 20% decrease was observed after chronic intermittent exposure (5 h d^–1 for 6 months) to 10, 100 or 500 mg/m³ VA.Supported by the Programme MZ-IX, 1.13     

Effects of chromium on humoral and cell-mediated immune responses and host resistance to disease in a freshwater catfish, Saccobranchus fossilis (Bloch).

The effects of subtoxic levels of Cr on humoral and cell-mediated immune responses, blood parameters, susceptibility to bacterial (Aeromonas hydrophila) infection, and macrophage activity in the freshwater air-breathing Asian catfish, Saccobranchus fossilis, during a 28-day exposure were examined by a static bioassay test procedure. At 0.1, 1.0, and 3.2 mg / liter Cr, dose-dependent Cr accumulation in kidney, liver, and spleen was observed at the end of the experiment. Chromium exposure caused a significant change in spleen to body weight ratio. Fish exposed to Cr concentrations had lower antibody titer values, reduced numbers of splenic and kidney plaque-forming cells, and higher counts of splenic lymphocytes but reduced counts of kidney cells when compared with the control group. At 0.1, 1.0, and 3.2 mg /liter Cr, dose-dependent decreases in red blood cell counts, hemoglobin content, and packed cell volume were observed. Differential leukocyte counts revealed that Cr exposure caused a significant decrease in large and small lymphocytes, whereas neutrophils and thrombocytes increased. Effects of Cr exposure to mitogen (Con A) on proliferation of splenic and pronephric lymphocytes suggests a decrease in mitogenic response. The eye-allograft rejection time, as a parameter of cell-mediated immunity, was statistically increased at 1.0 and 3.2 mg/liter Cr. Fish exposed to Cr for 28 days exhibited higher susceptibility to A. hydrophila infection than control fish. The results suggest that Cr exposure reduced the resistance of catfish to bacterial infections. The phagocytic activity of splenic and pronephros macrophages was examined in vitro and found to be significantly decreased.     

Activities of liver and lung cytochrome P450-dependent monooxygenases and antioxidant enzymes in laboratory and wild Norway rats exposed to reference and contaminated soils

Laboratory and wild Norway rats were exposed in the laboratory to an uncontaminated soil and to a soil from a site contaminated with petrochemical waste. Activities of microsomal lung and liver cytochrome P450-dependent monooxygenases, including 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD) and 7-benzoxyresorufin O-debenzylase (BROD) were measured at selected times during the course of the study. The highest degree of induction of hepatic EROD (7-fold) was shown after 3 days of exposure to the contaminated soil. However, two months later, the EROD activity declined to fourfold increase over the control. The PROD and BROD activities displayed a similar time course of induction, but the degree of induction was lower. The induction of hepatic monooxygenase activities was observed in both laboratory and wild rats. Lung monooxygenase EROD was highly induced (up to 28-fold) after 3 days of exposure, and the activity remained elevated throughout the two-month experiment. BROD and PROD activities were not induced. The activities of three antioxidant enzymes, namely superoxide dismutase, glutathione peroxidase (Se- and non-Se-dependent) and catalase also were measured in lung and liver cytosol, but no significant changes were observed after two months of exposure to contaminated soil.     

Hepatic Metallothionein and Glutathione-S-Transferase Responses in Two Populations of Rice Frogs, <Emphasis Type="Italic">Fejervarya limnocharis</Emphasis>, Naturally Exposed to Different Environmental Cadmium Levels

Glutathione-S-Transferase (GST) and metallothionein are important biomarker endpoints in studying the effect of Cd exposure. The purpose of this research was to study the correlation between hepatic GST and metallothionein with hepatic Cd in wild Fejervarya limnocharis exposed to environmental Cd. Results showed that frogs from contaminated sites had significantly higher hepatic metallothionein (3.58 mg/kg wet weight) and GST activity (0.259 μmol/min/mg total protein) than those from the reference site (2.36 mg/kg wet weight and 0.157 μmol/min/mg total protein respectively). There was a significantly positive correlation between hepatic Cd and GST activity (r = 0.802, p = 0.009) but not between hepatic Cd and metallothionein (r = 0.548, p = 0.139). The results concluded that while frogs from the contaminated site had higher GST and metallothionein, only GST showed significant positive correlation with hepatic Cd levels, indicating that hepatic GST activity may be used as a biomarker endpoint.     

Exposure to a Commercial Glyphosate Formulation (Roundup<Superscript>®</Superscript>) Alters Normal Gill and Liver Histology and Affects Male Sexual Activity of <Emphasis Type="Italic">Jenynsia multidentata</Emphasis> (Anablepidae,Cyprinodontiformes)

Roundup is the most popular commercial glyphosate formulation applied in the cultivation of genetically modified glyphosate-resistant crops. The aim of this study was to evaluate the histological lesions of the neotropical native fish, Jenynsia multidentata, in response to acute and subchronic exposure to Roundup and to determine if subchronic exposure to the herbicide causes changes in male sexual activity of individuals exposed to a sublethal concentration (0.5 mg/l) for 7 and 28 days. The estimated 96-h LC₅₀ was 19.02 mg/l for both male and female fish. Gill and liver histological lesions were evaluated through histopathological indices allowing quantification of the histological damages in fish exposed to different concentrations of the herbicide. Roundup induced different histological alterations in a concentration-dependent manner. In subchronic-exposure tests, Roundup also altered normal histology of the studied organs and caused a significant decrease in the number of copulations and mating success in male fish exposed to the herbicide. It is expected that in natural environments contaminated with Roundup, both general health condition and reproductive success of J. multidenatata could be seriously affected.     

A biomonitoring study of genotoxic risk to workers of transformers and distribution line stations

A cytogenetic monitoring study was carried out on a group of workers from transformer and distribution line stations in the Bursa province of Turkey, to investigate the genotoxic risk of occupational exposure to extremely low frequency electric (ELF) and magnetic fields (EMF). Cytogenetic analysis, namely chromosomal aberrations (CAs) and micronucleus (MN) tests were performed on a strictly selected group of 55 workers and compared to 17 controls. CA and MN frequencies in electrical workers appeared significantly higher than in controls (p < 0.001, 0.05, respectively). The frequency of CA in exposed groups were significantly enhanced with the years of exposure (p < 0.01). The effect of smoking on the level of CA and MN was not significant in the control and exposure groups. The results of this study demonstrated that a significant induction of cytogenetic damage in peripheral lymphocytes of workers engaged to occupational exposure to ELMF in electric transformer and distribution stations.     

Combined Effects of Different Food Rations and Sublethal Copper Exposure on Growth and Energy Metabolism in Common Carp

Common carp (Cyprinus carpio) were fed two different rations, 0.5% body weight (low ration; LR) and 5% body weight (high ration; HR), throughout acclimation, sublethal (64 μg/L) Cu exposure for 28 days, and a subsequent 2-week recovery period. Growth, liver water content, and liver energy stores were assessed during this period. Growth rates were elevated in HR fish compared to LR fish, as was the hepatic lipid content. This was associated with a higher water content in the livers of LR fish. Liver glycogen levels were similar in both feeding treatments and remained relatively stable during Cu exposure. Cu exposure caused a significant reduction in growth in both HR and LR fish. Reduction in growth coincided with significant changes in liver and blood composition. Liver lipid levels dropped significantly during the first days of the exposure in both feeding treatments, and the difference between LR and HR lipid levels disappeared during Cu exposure. During the first week of Cu exposure, the difference in liver water content disappeared as well, with a significant reduction of the water levels in the LR fish. A rise of hepatic protein was observed after 2 and 4 weeks of Cu exposure in the LR carp and after 4 weeks of exposure in the HR fish. Cu exposure led to pronounced increases in plasma ammonia concentrations in HR carp. Growth and energy stores recuperated during the 2-week recovery period in both feeding treatments. The observed changes during Cu exposure are probably related to physiological disturbances like hypoxia and stress, as well as an inhibition of ammonia excretion.