The effect of food rations on tissue-specific copper accumulation patterns of sublethal waterborne exposure in Cyprinus carpio

Common carp (Cyprinus carpio) were fed to two different food rations, 0.5% body weight (low ration [LR]) and 5% body weight (high ration [HR]), and were exposed to sublethal (1 microM) copper levels for 28 d in softened Antwerp (Belgium) city tap water (Ca2+, 79.3 mg/L; Mg2+, 7.4 mg/L; Na+, 27.8 mg/L; pH 7.5-8.0). Copper accumulations in the liver, gills, kidney, anterior intestine, posterior intestine, and muscle were determined. Copper accumulation in the gills, liver, and kidney of LR fish was significantly higher than in HR fish. The only time copper uptake in HR fish was significantly higher than in LR fish was in the posterior intestine after two weeks of exposure. No difference was found between the two rations in the anterior intestine. Copper accumulation in the liver of both feeding treatments occurred in a time-dependent manner and did not reach steady state in any treatment. On the contrary, copper concentration in the gills reached a steady state for both HR and LR fish within the first week of exposure. No copper accumulation was found in muscle tissues of either treatment. Copper concentration dropped to control levels in all tissues, except liver tissue, two weeks after the exposure ended. Our studies indicated that copper uptake was influenced by the food ration in carp. The difference in copper accumulation probably is related to the amount of dietary NaCl and different rates of metallothionein synthesis. Low food availability provides less Na+ influx and leads to increased brachial uptake of Na+ and copper. In addition, it has been shown that starved animals show increased levels of metallothionein, possibly causing higher copper accumulation.     

Differential metallothionein induction patterns in fed and starved carp (Cyprinus carpio) during waterborne copper exposure

Starved and fed carp (Cyprinus carpio) were exposed to sublethal waterborne copper exposure (1 microM) for 28 d in softened Antwerp, Belgium, city tap water. Copper accumulation in liver and gill tissues was determined, and changes in branchial Na+/K+-adenosine 5'-triphosphatase (ATPase) activity and metallothionein (MT) induction in gill and liver tissues were investigated following 28-d copper exposure. Gill Na+/K+-ATPase activity in exposed fish, both starved and fed, was at its lowest values after 3 d of exposure, after which it slowly recovered to preexposure values. No significant differences in branchial Na+/K+-ATPase activity were found between starved and fed fish. Copper accumulation in the liver and gills of the exposed starved carp was significantly higher than that in the exposed fed carp. The highest MT induction was found in liver tissues. Different patterns of MT induction were observed in the starved and fed carp during copper exposure. Before exposure, MT concentrations in the livers of the starved fish were significantly higher than those in the fed ones. Copper exposure significantly increased MT concentration in the liver of the fed fish, but no changes occurred in the starved fish. In contrast, copper exposure increased MT concentrations in the gills of the starved fish during the first week of exposure, whereas only a slight increase in MT concentration in the gills of the fed fish was observed. When taking into account the role of feeding status in MT induction, hepatic MT was a more relevant indicator for long-term monitoring of copper pollution in carp, but gill MT provided useful information regarding short-term copper toxicity.     

Effects of Sublethal Copper Exposure on Copper Accumulation, Food Consumption, Growth, Energy Stores, and Nucleic Acid Content in Common Carp

Juvenile common carp were exposed for 28 days to three different sublethal copper concentrations (0.20 μM, 0.55 μM, and 0.80 μM). Food consumption was monitored on a daily basis during the exposure period, while growth, copper accumulation, energy stores, and nucleic acid contents were assessed weekly. Copper exposure to 0.80 μM affected both growth and feeding behavior in common carp. At 0.55 μM, growth was affected despite normal food consumption. Even at the lowest copper concentration (0.20 μM), metabolic demand for the fish increased, challenging the carp with an increased demand for food. Copper accumulation mainly occurred in the liver, reaching an equilibrium between uptake and excretion after 1 month of exposure. Substantial biochemical changes were observed at the two highest copper exposure concentrations, but the correlation between growth rate and RNA:DNA ratio was poor considering the substantial differences in growth rate. The use of the RNA:DNA ratio as a sensitive biomarker is questioned. Received: 27 November 1996/Accepted: 12 May 1997     

Histological changes and copper accumulation in liver and gills of the Senegales sole, Solea senegalensis.

Senegales sole, Solea senegalensis, adult specimens were subjected to sublethal copper exposure (100 microg SO(4)Cu/L) for 7 days (contamination period) and afterward kept for 4 days without copper exposure (depuration period). In contaminated and detoxified specimens, the hepatocytes indicated a great number of lipid droplets compared to the control specimens. Some ultrastructural alterations such as partial disruption of the microvilli and endothelial lining of the sinusoids were observed in the hepatocytes. Lifting and swelling of the lamellar epithelium were common lesions in S. senegalensis gills due to the copper exposure. On the other hand, in contaminated and detoxified specimens, gills demonstrated separation of respiratory epithelium; chloride and pavement cells were also altered. Copper levels in gills, and especially in liver, increased during sublethal copper exposure (up to 7 days). During the detoxification period, Cu concentrations in gills and liver experienced variable changes. The results of the study indicate that histology is a successful tool capable of revealing sensitively and selectively even the sublethal effects of heavy metals on the environment and aquatic biota.     

Effects of Sublethal Copper Concentrations on Gills of White Shrimp (Litopenaeus vannamei, Boone 1931)

The objective of this study was to measure the copper (Cu) concentration in gills of juveniles Litopenaeus vannamei after exposure to Cu at sublethal concentrations, and to evaluate its effect upon the structure of gill tissue. The Cu concentration in gills of control shrimp was 0.075 mg/kg. Copper concentrations increased significantly by 147 %, 180 % and 205 % in gills of shrimp exposed to 0.675, 1.325 and 2.010 mg Cu/L, respectively. After exposure to 0.675 mg Cu/L for 15 days, gill tissue hyperplasia was observed, with a narrowing of the hemolymphatic lacunae. Necrosis and loss of hemolymphatic lacunae were observed at exposures of 1.325 and 2.010 mg Cu/L.     

3,4-dichloroaniline-induced oxidative stress in liver of crucian carp (Carassius auratus)

3,4-dichloroaniline (DCA) and its analogs are widely used as chemical intermediates in the synthesis of herbicides, azo dyes, and pharmaceuticals. They bring danger to growth, development, and propagation of aquatic organisms. The purpose of this study was to show DCA-induced oxidative stress response in liver of crucian carp (Carassius auratus). Superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO), and NO synthase (NOS) in liver were measured in crucian carp after exposure to DCA solution (0.2, 0.4, and 0.8 mg/L, respectively) for 15 days and compared with the control. DCA significantly induced SOD activity and enhanced MDA concentration in liver of crucian carp. Compared with the control, GSH and NO concentrations decreased and NOS activity was inhibited in crucian carp liver 15 days after DCA treatment. The data suggested that DCA-induced free radical generation and antioxidant depletion, and caused oxidative stress and lipid peroxidation in liver of crucian carp.     

Morphological and metabolic changes in common carp, Cyprinus carpio, during short-term copper exposure: interactions between Cu2+ and plasma cortisol elevation

The effects of increased endogenous cortisol levels were compared with those of sublethal copper exposure in the freshwater common carp, Cyprinus carpio. Fish were exposed to either increased levels of endogenous cortisol (200 ng/ml) or sublethal copper (1.9 microM) alone or were pretreated by elevating plasma cortisol levels prior to copper exposure to assess whether interactions between both treatments occurred. Effects induced by increased cortisol levels included increased Na+/K(+)-adenosine triphosphate (ATPase) activity and increased plasma Na+ and plasma osmolarity, while copper exposure induced anaerobic metabolism, gill damage, decreasing Na+/K(+)-ATPase activity, decreasing plasma ion levels, and blood thickening. Pretreatment of copper-exposed fish with cortisol partially protected these fish by reducing the copper-induced decrease in Na+/K(+)-ATPase activity. Overall, the results obtained in this study argue against a major role for cortisol as an intermediate for the toxic effects of copper.     

Toxic Responses and Catalase Activity ofLemna minorL. Exposed to Folpet, Copper, and Their Combination

Toxicity of copper and folpet—two fungicides widely used on grape—was evaluated onLemna minorL., a sensitive aquatic weed regularly used for (eco)toxicological studies. Toxicity assessments were based on inhibition of growth and chlorophyll content ofL. minorcultures after 7 days. IC₁₀, IC₅₀, and IC₉₀were determined for both compounds alone and were respectively, 0.03, 0.16, and 0.95 mg liter⁻¹for copper and 1.20, 7.50, and >40 mg liter⁻¹for folpet. When both compounds were combined, the response ofL. minordepended on the initial folpet concentration. Indeed, a slight synergy was observed for 5 mg liter⁻¹folpet, while at folpet concentrations of 20 to 35 mg liter⁻¹, the two fungicides were antagonists. The antagonism was positively correlated with folpet concentration. Antagonism between Cu and folpet could not be explained by a reduced bioavailability of Cu since concentration of free copper in the mixture did not depend on the presence of folpet. One physiological defense response elicited by copper in plants is an increase in catalase activity. Copper and folpet stimulated catalase activity and changes in the activity of the enzyme could not account for the synergy but possibly for the antagonism. Nevertheless, catalase activity increased significantly after a 24-h exposure to 25 μg liter⁻¹of copper. The use of this property as a rapid and sensitive biomarker to monitor the toxicity of xenobiotics alone or in combination and of environmental water is discussed.     

Effects of sublethal concentrations of atrazine and nitrate on metamorphosis of the African clawed frog

Tadpoles of the African clawed frog (Xenopus laevis) were exposed to sublethal concentrations of atrazine (0, 40, and 320 microg/L) and nitrate (0, 37, and 292 mg/L) from feeding stage to metamorphosis. A 3 x 3 factorial design was used to identify both single and interactive effects. At metamorphosis, tadpole weight, snout-vent length (SVL), and hematocrit were determined. Mean mortality was greater in tanks receiving 320 microg/L atrazine; nitrate had no effect on mortality. Significant differences for all mean traits at metamorphosis occurred among atrazine treatments; higher atrazine exposure increased time to metamorphosis and decreased weight, SVL, and hematocrit. Nitrate treatments were not significantly different. Significant interaction tests between atrazine and nitrate occurred for weight and SVL at metamorphosis; the specific type of interaction varied among treatments. Assuming an additive mixture model, at low atrazine (40 microg/L), the addition of 37 mg/L nitrate produced SVL values less than expected (a synergistic effect) while the addition of 292 mg/L nitrate yielded SVL values greater than expected (an antagonistic effect). A similar response was noted for tadpoles in the 320-microg/L atrazine treatments. These results indicate that environmentally realistic concentrations of atrazine exert a negative impact on amphibian metamorphosis. Also, this study suggests that mixtures of agricultural chemicals, even if sublethal, may exert negative and not necessarily consistent mixture effects.     

Phototactic behavior of Daphnia and the continuous monitoring of water quality: interference of fish kairomones and food quality

We carried out a laboratory study to evaluate the sensitivity of phototactic behavior of Daphnia magna to sublethal concentrations of pentachlorophenol (PCP) and copper. More specifically, we determined whether the sensitivity of a D. magna clone to those pollutants is influenced by food quality and the presence of fish kairomones. Test animals were fed either unicellular green algae (Scenedesmus acutus) or fresh baker's yeast (Saccharomyces cerevisiae) and were cultured in the presence or absence of fish kairomones. Four concentrations of PCP (0.4, 0.8, 1.2, and 1.6 mg/L PCP) and one concentration of copper (0.02 mg/L Cu2+) in International Standards Organisation (ISO, Geneva, Switzerland) standard medium were applied in the experiments. Animals were exposed for 3 h to the pollutants prior to the experiments. In the absence of fish kairomones, a strong negative relationship between the phototactic index and nominal sublethal concentrations of PCP was found in animals fed either algae or yeast. The sensitivity of the Daphnia clone to sublethal concentrations of PCP was, however, less clear cut in animals fed yeast than in animals fed algae. The detection limit was 0.4 mg/L PCP with algae as food but was as high as 1.2 mg/L PCP when yeast was used as food. The ability to detect sublethal concentrations of copper and PCP using phototactic behavior was lost when the clones were cultured in the presence of fish kairomones. At a concentration of 0.02 mg/L Cu2+ and in the absence of fish kairomones, the D. magna clone tested became significantly less positively phototactic than in the control treatment regardless of the quality of the food used to culture the test animals. These results suggest that assays using the phototactic behavior of Daphnia to monitor water quality should use genetically stable (clonal) material, positively phototactic, and insensitive to the presence of fish kairomones.     

Copper and zinc absorption in the rat: mechanism of mutual antagonism

The influence that copper and zinc exert on each other's absorption was studied by using the isolated, vascularly perfused rat-intestine system. In the first series of experiments, rats were fed for 1 wk one of nine diets, with different copper and zinc concentrations representing low, adequate and high dietary metal intakes. Copper concentrations were 1, 6 and 36 mg/kg diet and zinc concentrations were 5, 30 and 180 mg/kg. The small intestine was perfused with M199 tissue culture medium containing 6 mg/L copper and 30 mg/L zinc. Neither metal was found to significantly alter the other's absorption. High dietary zinc increased metallothionein-bound copper but did not change the intracellular copper concentration. In the second series of experiments, the dietary copper and zinc concentrations were held at 6 mg/kg and 30 mg/kg, respectively, while the metal concentrations in the luminal perfusate were changed (from 1 to 36 mg/L and from 5 to 180 mg/L for copper and zinc, respectively). The higher copper concentrations in the perfusate increased zinc accumulation in mucosal cells and decreased the zinc transferred to the portal perfusate at the highest luminal zinc concentration. These data indicate that a competition and/or inhibition of a pathway for zinc out of the mucosal cell occurs at high luminal copper concentrations. High luminal zinc concentrations in the perfusate decreased the copper concentration in the mucosal cell cytosol and the amount transferred to the portal effluent. These results taken together indicate that a competition and/or inhibition of copper or zinc intake into intestinal cells occurs when the luminal concentration of the respective congener is very high.     

Aquatic toxicity of nitrogen mustard to Ceriodaphina dubia, Daphnia magna, and Pimephales promelas

Investigation of toxicity of mustard compounds to aquatic organisms has been limited although their effects on terrestrial mammal species have been well studied. In this study, the 48-h LC50 values of nitrogen mustard (HN2) are reported for two aquatic invertebrate species (Daphnia magna and Ceriodaphnia dubia) and for one fish species (Pimephales promelas). Mean LC50 values to C. dubia, D. magna, and P. promela were 1.12, 2.52, and 98.86 mg/L, respectively. C. dubia was the species most sensitive to HN2. Seven-day lethal and sublethal tests with P. promelas and C. dubia were also conducted. In chronic tests, fathead minnow growth was significantly reduced by 2.50 mg/L HN2, while C. dubia reproduction was significantly affected by 7.81 mug/L HN2. These adverse effects on aquatic organisms caused by lower-level concentrations of HN2 indicate that a possible aquatic ecosystem disaster could occur either after a chemical spill or during chemical warfare.     

Adverse effects of chronic copper exposure in larval northern leopard frogs (Rana pipiens)

Northern leopard frogs (Rana pipiens) were exposed to environmentally relevant concentrations of copper in water (control and 5, 25, and 100 microg/L, as CuSO4) in a static renewal system for 154 d from posthatch stage to metamorphosis. After 10 d of copper exposure (from Gosner stages 19-25), survival of tadpoles was not significantly different between treatments (averages range from 96.7 to 99.3%), but a significantly higher incidence of deformities (p < 0.05) was observed in the 100-microg/L treatment. Tadpole growth rate was decreased in the 25- and 100-microg/L treatments relative to controls. Tadpole survival, swimming performance, percent metamorphosis, time to metamorphosis, and survival during and time required for tail resorption were also negatively affected in the 100-microg/L treatment. Copper concentrations in tadpole tissues ranged from 46.5 to 769.5 mg/ kg dry mass and were positively related to copper concentrations in the water. In conclusion, copper was shown to affect development, growth, survival, and behavior in larval northern leopard frogs at ecologically relevant concentrations. The existing U.S. Environmental Protection Agency freshwater-quality chronic criterion for copper appears to be protective for larval Rana pipiens.     

Effects of Elevated Nitrate Concentration on Mortality, Growth, and Egestion Rates of Gammarus pseudolimnaeus Amphipods

There is relatively little known about nitrate toxicity in aquatic invertebrates, especially sublethal effects of elevated nitrate. We exposed the amphipod Gammarus pseudolimnaeus to elevated nitrate concentrations (up to 128 mg NO₃-N/L) in a laboratory experiment and measured mortality, growth rates, egestion rates, molting, and C:N ratio. Nitrate concentration did not affect mortality, egestion rate, molting, and C:N ratio. Amphipod growth decreased slightly with increasing nitrate concentration based on the results of a linear regression, but a one-way ANOVA suggested that mean growth rates were not different among nitrate treatments. We suggest that additional research is needed on the sublethal effects of elevated nitrate concentration on aquatic invertebrates, particularly for those taxa that have demonstrated sensitivity to other chemical stressors.     

Effects of sublethal sodium dichromate exposure in freshwater on the salinity tolerance and serum osmolality of juvenile coho salmon,Oncorhynchus kisutch,in seawater

To simulate a potential field situation in which seawardmigrating juvenile coho salmon,Oncorhynchus kisutch, pass through Crpolluted freshwater (FW) before being exposed to seawater (SW), the effects of sublethal FW exposure to Cr (as sodium dichromate) on salinity tolerance and serum osmolality after transfer to SW were investigated. SW survival was significantly decreased in salmon exposed in FW to 0.23 mg Cr/L for four weeks or to 0.5 mg Cr/L for two weeks and transferred to 20 or 30 parts per thousand (%) salinity, respectively. Serum osmolality, measured after two weeks of FW exposure to 0.0 or 0.5 mg Cr/L and after transfer to 20 or 30% SW, was significantly higher in Cr-exposed fish from one-half to two days after SW transfer but not at the end of the FW exposure period or at seven days after SW transfer. These effects in SW occurred at Cr exposure concentrations which had no effect on survival or serum osmolality in FW. It is suggested that Cr acted non-specifically on osmoregulatory epithelia to cause excessive dehydration in SW, and that measurement of salinity tolerance and serum osmolality may be sensitive and ecologically realistic indicators of sublethal toxicity in aquatic animals.     

Copper concentrations in tap water and possible effects on infant's health--results of a study in Lower Saxony,Germany

Copper in drinking water has been associated with non-Indian childhood cirrhosis (NICC), a form of liver cirrhosis in childhood. This epidemiological study examines the exposure of infants to increased copper concentrations through drinking water from public water supplies in southern Lower Saxony, Germany, and whether this dietary copper intake causes liver damage in early childhood. In total, water samples from 1674 households with infants were tested for copper. The mean copper concentration was 0.18 mg/L in the 1619 collected stagnation samples and 0.11 mg/L in the 1660 random daytime samples. There were notable regional differences in copper values. In 10.3% of all sampled households a copper value of 0.5 mg/L or more was found. These families were requested to additionally collect 2 composite samples. An increased level of copper in the drinking water with copper concentrations at or above 0.8 mg/L in the composite samples and a defined minimum ingestion of tap water was followed by a recommendation of a pediatric examination. Fourteen infants were examined by a pediatrician and of these 11 received a blood serum analysis. None of the examined infants showed any signs of liver malfunction. From the results of the study, no indication of a hazard due to copper pipes connected to public water supplies could be found.     

Aluminum-induced oxidative stress and neurotoxicity in grass carp (Cyprinidae--Ctenopharingodon idella)

Aluminum is used in a large number of anthropogenic processes, leading to aquatic ecosystems pollution. Diverse studies show that in mammals this metal may produce oxidative stress, is neurotoxic, and is involved in the development of neurodegenerative disorders, such as Alzhaimer's and Parkinson's diseases. Nevertheless, there are only few studies with respect to Al-induced neurotoxicity on aquatic fauna, particularly on fishes of economical interest, such as the grass carp (Ctenopharingodon idella). This study evaluates Al-induced toxicity on the grass carp C. idella. Specimens were exposed to the maximum concentration allowed in order to protect aquatic life (0.1 mg L?1), for 12, 24, 48, 72 and 96 h. After the exposure time, lipid peroxidation degree, superoxide dismutase and catalase activity, as well as dopamine, adrenaline and noradrenaline levels were evaluated. Al concentration in organisms and water was also measured, in order to determine the bioconcentration factor. Results show that Al bioconcentrates in grass carp inducing oxidative stress (increment of 300 and 455 percent on lipid peroxidation degree and SOD activity, and decrement of 49 percent on CAT activity) and neurotoxicity (increment of 55 and 155 percent on dopamine and adrenaline levels and decrement of 93 percent on noradrenaline level).     

Extent of thermal processing of infant formula affects copper status in infant rhesus monkeys

BACKGROUND: Infant rhesus monkeys are excellent models in which to study the effect of infant formulas on trace element absorption and status. Infants fed powdered formula from birth exhibit normal growth and have blood variables similar to those of breast-fed infants. OBJECTIVES: The objectives were to evaluate the effects of feeding ready-to-feed (RTF) formulas exposed to different heat treatments to infant monkeys, and, for one of these formulas, to compare the effect of fortification with 2 iron concentrations. DESIGN: From birth to age 5 mo, infant monkeys (n = 6/group) were fed one of the following formulas exclusively: 1) 12 mg Fe/L processed in cans (RTF-12), 2) formula in glass bottles with 12 mg Fe/L and manufactured by an ultrahigh-temperature (UHT) process (UHT-12), or 3) formula manufactured by a standard thermal process (STP), containing either 8 (STP-8) or 12 (STP-12) mg Fe/L. All formulas had similar copper concentrations (0.6 mg Cu/L). Anthropometric measures and venous blood samples were taken monthly. RESULTS: Weight and length gain did not differ among groups; however, the STP-12 group weighed less than the UHT-12 group at ages 2, 4, and 5 mo. Hemoglobin values were significantly lower in the RTF-12 group than in all other groups at ages 4 and 5 mo and serum ferritin was lower in the RTF-12 group than in the STP-12 group at age 5 mo. Copper status was lower in STP-12 infants than in STP-8 infants. There was a progressive and significant decline in plasma copper, ceruloplasmin, and Cu/Zn superoxide dismutase activity in infants fed canned formula (RTF-12). Furthermore, coat color changed from normal brown to silver. These outcomes suggest that the canned formula induced copper deficiency in infant monkeys. CONCLUSIONS: Excessive heat treatment of formula can have a pronounced negative effect on copper status. High iron concentrations did not improve iron status but may adversely affect copper status.     

Effect of Glyphosate on the Development of Pseudosuccinea columella Snails

Glyphosate (Roundup) is one of the most commonly used broad-spectrum herbicides with little to no hazard to animals, man, or the environment. Due to its widespread use, there is continuous contamination of the environment in both soil and water with this herbicide. There is a paucity of long-term exposure studies with sublethal concentrations of glyphosate on aquatic snails. This study was developed to determine the effects of sublethal concentrations of glyphosate on development and survival of Pseudosuccinea columella (intermediate snail host of Fasciola hepatica). This was assessed by continuously exposing three successive generations of snails to varying concentrations (0.1–10 mg/L) of glyphosate. Glyphosate had little effect on the first- and second-generation snails. However, third-generation snail embryos exposed to 1.0 mg/L glyphosate developed much faster than other embryos exposed to 0.1 mg/L, 10 mg/L, and 0 mg/L (control). Hatching was inhibited at 10 mg/L and inhibited slightly at 0.1 mg/L. The egg-laying capacity was increased in snails exposed to 0.1 and 10 mg/L. Abnormalities and polyembryony were observed in snails exposed to 0.1 and 10 mg/L. These results indicate that glyphosate does affect snail reproduction and development. This, in turn, could possibly have an effect on the population dynamics of F. hepatica, which could result in increased infections in animals, including man. Received: 15 November 1996/Accepted: 15 July 1997     

The gastrointestinal tract and acute effects of copper in drinking water and beverages

Copper is an essential element for all living beings. Exposure to copper results almost exclusively from the ingestion of food and water. Generally, potable water contains low levels of copper, but high concentrations of this mineral have been found in water from private wells or when water or beverages with low pH have been conducted through copper piping. Some authors have associated acute gastrointestinal symptoms (diarrhea, abdominal pain, nausea, and vomiting) with elevated levels of copper in water or beverages, but without excluding other confounding factors that could produce such symptoms. Recently, various controlled studies have demonstrated that a concentration of 2 mg Cu/L of potable water does not produce an increase in gastrointestinal symptoms in infants, and that in women, only concentrations greater than 3 mg Cu/L increase the number of episodes of nausea, vomiting, and abdominal pain, but not diarrhea. This critical analysis of scientific publications verifies the WHO provisional level for copper in drinking water (2 mg/L) as safe for human health.