Abstract

Thirty (30) micrograms of copper was given orally alone or in combination with either zinc or arsenic at two levels (high level, 10 or low levels, 30 micrograms) to pinfish, Lagodon rhomboides in two trials. Each trial consisted of 10 groups (8 fish per group) of pinfish: controls, copper-treated; zinc or arsenic alone at each of the two levels; combinations of copper with zinc or arsenic at low and high level. Each metal produced lesions in the gills; but the response was enhanced when combinations were used. Arsenic produced lesions in the liver and kidneys which were more severe when it was given together with copper. Zinc produced a severe stress which was increased in the presence of copper. The effects of combined doses of these metals on selected clinical values was either synergistic or antagonistic. Toxic effects of mixtures of these metals differ from those of the metals given alone and results could not be predicted.

Introduction

Copper together with the heavy metals zinc and arsenic, has been incorporated into wood preservatives (copper-chrome arsenate), pigments and rodenticides (copper arsenite), fungicides, insecticides, and anti-fouling paints(1). Individually, these metals have many applications. Copper, in the form of cupric bromide, is used as an intensifier in photography while cupric chloride, cupric carbonate, and cupric sulfate are used as additives in animal feeds. Zinc is necessary to make protective coatings for other metals, such as galvanized iron which is commonly used in building. Zinc is also found in rubber products, dry cell batteries, ceramics, linoleum, paints, cosmetics, textiles, glue, fertilizers, and many other goods. Arsenic, a highly toxic compound to most animals, is found in soil sterilants, defoliants, weed killers, and feed additives(1). Arsenical herbicides are employed for the control of weeds and defoliation of cotton plants prior to picking in the south and southeastern Unites States. The Airforce has used the herbicide, dimethylarsenic acid, to prevent forest foliar growth(2). Arsenic is also important in the glass, tanning, and textile industries(1).

Concurrent with the use of these metals is their contribution to environmental pollution. Among the chief industrial pollutants listed for Great Britain are zinc and copper(3). Although there are many products that contain heavy metals, public concern for environmental contamination appears to revolve around industries that produce heavy metals as a waste product. Examples include the smelting of nonferrous ores, such as zinc and copper, and the use of arsenic-rich coal in coal fired plants(4). Copper and zinc are also released directly into the aquatic environment in the form of mining effluent(5,6).

Ultimately, many of these metals reach the estuarine system where they threaten the natural levels of copper, zinc and arsenic. The accumulation of these metals may consequently, result in a massive fish kill as it did in June and July of 1980 at the Indian River and Mosquito Lagoon, in Northeast Florida.

In this incident, approximately one hundred large mature red drum were found dead. Analyses of the stomach contents revealed that ingestion of copper, zinc, and arsenic had occurred prior to death. Elevated levels of sodium, chloride and potassium suggest that copper poisoning was a major factor, although the interaction among the metals may have contributed to the acute effect(7).

The ability of fish to tolerate particular levels of heavy metals in their environment appears to be species related, while factors such as dissolved oxygen, pH, temperature, sediment quality, salinity, and organic and inorganic chemicals may influence this tolerance(8). For example, the common carp, Cyprinus carpio, is more sensitive to zinc than to copper ions, while the reverse is true for the grass carp, Ctenopharyngodon idellus(9). The longfin dace, Agosia chrysogaster, compared to the fathead minnow, Pimephales promelas, is also more sensitive to zinc than to copper(5). In addition to species differences, tolerance to heavy metals differs among individuals with sex, age, length, and stage of development contributing to this variability(6,9,10).

The ultimate sink for heavy metals is the sediment(11,12). Depending upon the chemical form and concentration of these metals, the particle size and agitation of the sediment and the feeding habits and physiological aspects of the aquatic animal, these metals may become available for absorption(11). While metals absorbed in this manner may not produce immediate death of an organism, its growth, reproductive rate, and lifespan may be limited.

When the toxicity of a mixture of metals is more than that predicted by adding the known toxicity of each metal separately, their interaction is considered to be synergistic(3). The interaction of heavy metals may also produce a combination with additive, synergistic or antagonistic toxic effects.

In mammals, sulfate, molybdate, phytate, zinc, cadmium, and iron, all decrease copper absorption, therefore, limiting copper toxicity(1,13). Similarly, zinc toxicity may be alleviated by the addition of copper and iron to the diet. Pigs, for example, fed a potentially toxic dose of 250 ppm copper, showed growth stimulation when 100 ppm of zinc was added to the diet. In cases of copper induced anemia and jaundice, 750 ppm of dietary zinc alleviated these symptoms and returned serum copper, and aspartate transaminase to their normal levels(1). Rats receiving 120 ppm dietary zinc had a lower liver copper content than those that received only 30 ppm zinc(14).

Copper, in the marine environment, interacts with chlorine, pentachlorophenate, cadmium, mercury and zinc to produce synergistic effects in fish. Sodium nitrate and sodium nitrite, however, act antagonistically to reduced copper sulfate toxicity(15,16). Copper and zinc, for example, were implicated in a massive fish kill at the state-run bait station on Sand Island in Honolulu(15). Many experiments have reported the synergistic action of these two metals in fish such as rainbow trout, Salmo gairdneri, atlantic salmon, Salmo salar, mummichog, Fundului--heteroclitus (L.)(17), cyprinid, Agosia chrysogaster, male guppies, Pimephales promelas(5), as well as the eggs of the zebra ciclid, Cichlasoma nigrofasciatum(18). Other reports on fish, such as the blue gill, Lepomis macrochirus(19), juvenile atlantic salmon, Salmo salar(6), and the killifish, Fundulus heteroclitus(20), have shown the interaction of copper and zinc.

Due to the diversity of results, it is important to recognize the experimental conditions to which the fish was exposed. Zinc and copper, for example, have been shown to act synergistically in softwater, but additively in hardwater(3). Specimen species, size, and behavior, as well as water quality, and method of toxicity determination may all influence metal interaction.

The synergistic effects of copper and zinc appear to correlate with the individual toxic properties of these metals at different anatomical locations or sites of activity. Zinc, for example, when added to copper did not affect the degree or extent of copper-induced kidney lesions in the mummichog, although the toxicity of the combination acted synergistically(17).

There is little experimental data available that explores the interaction of arsenic compounds with copper in fish. There is some evidence, however, indicating that arsenite may enhance copper toxicity to some degree(1).

In order to manage the marine environment more efficiently, the full potential of heavy metals and their interaction must be realized. Currently, in marine aquariums, and other aquatic animal facilities, low levels of copper are deliberately added to the water to control protozoans and algae. This implies that a high potential exists for copper interaction with other metals already present in the water. The goal of this study is to determine whether the toxicity of copper and zinc, and copper and arsenic mixtures interact synergistically, additively or antagonistically in the marine Teleost, Lagodon rhomboides, in order to gain a better understanding of the consequences of copper usage and disposal.

Materials and Methods

Using a hook and line, 160 pinfish, Lagodon rhomboides, weighing between 60g and 115g, were collected from a pier located on the intercostal waterway at the University of Florida's Whitney Laboratory. Following capture, all fish were placed in a oxytetracycline hydrochloride (125 mgs/gallon) bath for 1 hour to facilitate recovery after capture. Fish were fed a quantity of shrimp equal to approximately 2% of their body weight daily during the 2-3 week acclimation period.

Metal solutions were prepared as follows: 30 mg/L zinc, 10 mg/L zinc, 30 mg/L arsenic, 10 mg/L arsenic, 30 mg/L copper; 30 mg/L zinc and 30 mg/L copper; 10 mg/L zinc and 30 mg/L copper, 30 mg/L arsenic and 30 mg/L copper; 10 mg/L arsenic and 30 mg/L copper. All dilutions were made using distilled water instead of seawater. This prevented the metals from combining with constituents of the sea water. Sources of copper, zinc, and arsenic were as follows: copper sulfate, zinc chloride, and sodium arsenate. The metal concentration of the solutions were confirmed by analytical testing.

Two trials were run. In each trial, 8 fish were randomly distributed to one of the 10 treatment tanks. All fish were anesthetized with Tricaine Methane Sulfonate (MS-222, Argent Chemical Laboratories, Inc., Redmond, Washington) and 1 ml of the appropriate metal solution was delivered directly to the fishes stomach via a syringe and small diameter tubing. Fish were then placed in a recovery bucket for 45-60 seconds to determine the quantities of metal regurgitated. once a tank was completed, a water sample of the recovery bucket was taken for analysis. The fish were bled from the caudal vein and sacrificed 16 to 18 hours following metal exposure. Liver, gill, heart, brain, kidney, and spleen were collected. Half of each gill and liver were frozen for metal determination. The remaining liverand gill, as well as the other tissues, were fixed in 10% formalin for histological examination. Serumd samples were analyzed for the following: total proteins (PRO), total globulins (G10B), albumin (ALB), albumin to globulin ration (A/G), aspartate aminotransferase (GOT), alanine amino transferase (GPT), alkaline phosphatase (PHOS-ALK), calcium (CA), chloride (Cl), carbon dioxide (CO2), phosphorus (P), potassium (K), sodium (Na), creatinine (CREAT), glucose (GLU), and urea nitrogen (BUN).

Copper and zinc levels were determined using a Perkin Elmer Model 603 air-settling atomic absorption spectrophotometer sensitive to .01 mg/L. Arsenic was determined utilizing a flameless technique, the mercury hydride system (MHS-10) which was sensitive to 0.001 mg/L. Water samples analyzed included those taken before each trial from water supplying the treatment tanks and those from the recovery bucket of each treatment tank. Gill and liver metal concentrations required that samples first be digested by adding 4 ml of concentrated nitric acid to 0.1-1.5 gms of tissue. After 1 hour, test tubes were placed 2 inches deep into 110°C sand. Samples were removed when a volume of 1 ml or less remained, and diluted with 0.2% nitric acid to a final volume of 25 ml.

Stress was monitored using Hemastix to detect hemoglobin in the outer protective mucous layer(21). Tests were made, prior to anesthetization, on several fish from each treatment tank in trial 1 and in all fish in trail 2 by touching the fish with the hemoglobin test strip. All fish in both trials were tested following metal exposure.

Statistical analysis of tissue metal content and serum enzymes, inorganics, organics and proteins was performed utilizing Statistical Analysis System (SAS Institute Box 8000-, Cary, North Carolina). Initially, the experiment x treatment (EXP x TRT) interaction was tested for in a randomized block design (RBD) using the sampling error term: FISH (EXP x TRT)_k(ij)If the EXP x TRT interaction wassignificant, the treatment effects were tested for each experiment separately in a completely randomized design (CRD). If EXP x TRT interaction was not significant, experimentand treatment effects were tested for by using the pooled error term: FISH(EXP x TRT)_k(ij) + EXP x TRT_ij in a RBD. All responses from the RBD in which EXP was not significant were tested for TRT effect in CRD using the pooled error term: EXP(TRT)_i(i) + FISH(EXP x TRT)_k(ij) Pooling the error terms increased the degrees of freedom, making the tests more sensitive.

Duncan's Multiple Range test (DMR) was performed for all responses that were shown to be significant in the CRD's. Responses from the RBD, using the pooled error term, in which both EXP and TRT were significant also was examined using Duncan's Multiple Range Test. In the final analysis of serum constituents and tissue metal content, contrasts were developed in order to test for synergism, antagonism or additivity.

Results

Seawater collected prior to trial 1 contained 0.05 mg/L Cu, 0.08 mg/L Zn and 0.001 mg/L As. Prior to trial 2 these values were 0.04 mg/L Cu, 0.01 mg/L Zn and 0.000 mg/L As. Regurgitation of metal solutions was found to occur. An estimated average value for all treatment groups was 25.3% (+/- 30.7%).

Gross examination showed the body condition of all fish prior to the trials to be good. Following the appropriate metal treatment, many fish were visually hemorrhaging in the tail, dorsal, and caudal fins.

Minor hemorrhaging in 25% of the treatment group was seen in fish exposed to zinc alone at the higher concentration in trial 1. Seven of eight fish treated with 30 micrograms of copper alone had severe hemorrhagic lesions. Copper combined with zinc or arsenic also produced lesions. The most severe of these lesions were found in 50% of the fish treated with copper plus low zinc while 88% of the high zinc and copper group had moderate lesions. Minor lesions were found in fifty percent of the high arsenic with copper group, and 30% in the low arsenic with copper group.

Low zinc and arsenic alone, in trial 2, produced minor lesions in 50% and 25% of the fish, respectively. Copper alone induced moderate lesions in all but one fish which had severe lesions. The combination of low zinc with copper produced moderate lesions in 13% of the fish and severe lesion in 38% of the fish. Both low and high concentrations of arsenic with copper induced minor lesions in 25% of the fish and moderate lesions in 25% of the fish. The gills, liver, kidney, brain, heart and spleen of metal-treated fish were similar in gross appearance to control fish.

Gill sections stained with hematoxylin and eosin were generally characterized by heterophils and lymphocytes in the gill filaments, the bases of the lamellae, and the pharyngeal tissue. With the exception of the copper-treated group in trial 2, control fish had the smallest degree of inflammation while fish treated with copper, zinc, arsenic or metal combination had a greater degree of inflammation.

The degree of histological inflammation seen in gill tissue is summarized. Inflammation was based on the number of heterophils and lymphocytes present within the primary and secondary lamella, degranulation of the heterophils, hypercellularity and congestion of gill filaments.

In trial 1, control fish had some hepatic vacuolar degeneration, but it was primarily confined to the tissue edges. Fish receiving zinc alone in both high and low concentration had livers that were mildly vacuolated throughout the tissue, as compared to just the edges of controls. High and low concentrations of arsenic had a more severe degenerative effect. Vacuolar degeneration occurred in all fish in these two groups. Within the arsenic/copper groups this degeneration was marked, but when copper was added to zinc, degeneration was minimal.

In trial 2 most fish, including controls, had severe hepatic vacuolar degeneration, therefore, comparisons between treatment groups could not be made.

Renal lesions consisted of interstitial congestion and/or hemorrhage. The most severe renal damage was found in fish that were given copper, alone or in combination with arsenic.

The addition of copper to zinc or arsenic altered the majority of mean serum constituent levels. Albumin to globulin ratio (trial 1), potassium (trial 2), aspartate amino transferase (pooled error), globulins (pooled error), and creatinine (pooled error) were the serum constituents that were altered synergistically in fish treated with the arsenic-copper mixture. This metal combination had an antagonistic affect on alanine amino transferase (trial 1), phosphorous (pooled error), sodium (pooled error), and albumin (pooled error). Zinc combined with copper synergistically altered the albumin/globulin ration (trial 1), potassium (trial 2), and glucose (pooled error). No antagonistic results were observed for the zinc mixed with copper treatment. Groups in which copper was given with zinc or arsenic had liver copper levels similar to controls but gill-copper content was reduced in an antagonistic manner in the high dose groups.

Prior to orally dosing the fish in trial 2, the hemastix test for stress indicated that 27.5% of the fish were minimally stressed, 67.5% were slightly stressed, and 5.0% were moderately stressed. A general sampling of fish prior to trial 1 indicated similar results, although all fish were not sampled. Following the period of metal exposure, all fish were slightly to severely stressed. In trial 1, 46.8% of the fish were slightly stressed, 45.6% moderately stressed, and 7.8% severely stressed. Sixty two percent (62.3%) of the fish were slightly stressed, 32.5% moderately stressed, and 7.5% severely stressed in trial 2. All fish that were severely stressed were treated with zinc alone or in combination with copper.

Discussion

The interaction of copper with zinc has been shown to cause an overall synergistic or additive effect in previous experiments(5,6,17,18,19,20). The effects of arsenic alone in marine fish is not considered to be highly toxic(4,22), although the interaction of copper with arsenic has not been studied. The present experiments show that low oral doses (10-30 micrograms) of zinc and arsenic, alone or in combination with copper produce detectable toxic effects. The low doses used in these experiments are believed to approximate exposures that might actually occur. The general increase in stress measured in fish following the experiments may have resulted from handling, anesthesia, intubation and change in environment that occurred during the experimental procedure. Several observations indicate however, that the treatment with metals increased stress. Zinc, and zinc combined with copper were the only treatments that produced severely stressed fish. Gross examination of these fish shown moderate to severe hemorrhaging in the caudal, dorsal, and tail fins. An elevation in serum glucose, which may indicate stress (Benirschke et al., 1978) was found primarily in fish treated with zinc combined with copper, although arsenic alone or in combination with copper also increased glucose levels. The addition of copper to 10 micrograms of zinc had a synergistic effect on glucose levels. Arsenic alone did not significantly increase stress, and gross lesions, when present, were minor. Copper induced severe to moderate gross lesions although the stress test and glucose levels did not confirm this. When arsenic was combined with copper, the glucose levels of these fish dropped from those treated with arsenic alone but the number and severity of gross hemorrhagic lesions increased. This appeared to demonstrate an interaction between copper and arsenic which modified the stress in these fish.

The primary functions of the gills are to meet oxygen demand, and to maintain both an osmotic and electrolyte balance. Any toxic substance that inhibits the ability of the gill to perform these functions may ultimately lead to death. Copper and zinc have been shown to cause lamellar thickening and capillary congestion in fish. While copper(7,24,25) and zinc(26,27) cause disruption of the gills epithelium, only copper is reported to inhibit osmoregulation(7,26). Copper, zinc, and arsenic in these experiments all increased primary and secondary lamellar inflammation and congestion. Distinct microscopic lesions in fish treated with 10 or 30 micrograms of a specific metal or metal combination were difficult to identify. The addition of copper to zinc and arsenic increased gill inflammation to a greater degree in zinc-treated fish. It is not clear why arsenic, and arsenic combined with copper switched from slight inflammation in experiment 1 to most severe inflammation in experiment 2. The observation that K, Na, and P levels were similar to controls, despite the inflammation observed in fish treated with zinc alone, agrees with Skidmore's finding (1970) as osmoregulation is apparently maintained in these zinc-treated fish. Arsenic, however, consistently elevated K and Cl levels while Na, Ca an P levels were similar to controls. Copper elevated K, P, and Na above controls, but only the change in Na was significant. Combining copper with arsenic or zinc increased K levels in an additive manner in the first experiment and in a synergistic manner during the second experiment. Sodium levels in the zinc/copper-treated fish increased overall, but remained lower than copper-treated fish. Arsenic, when combined with copper, acted to produce an antagonistic effect on Na levels and thus, these fish maintained values similar to control fish.

Cardeilhac has hypothesized that copper exposure may elevate K to toxic levels in fish due to cell damage and failure of osmoregulation in the gills and kidney(7,24,25). Absolute serum potassium levels in these experiments were high, but may be exaggerated in terms of magnitude as only small quantities of hemolyzed serum were obtained. The average K level in control fish for example was 7.5 Meq/L which exceeds the normal value of 3.8 Meq/L found by Cardeilhac and Hall(24). A normal value of 8.3 Meq/L has, however, been reported by Holemes and Donaldson for Lagodon rhomboides(28). Sodium, which is fairly stable even with hemolysis, may be a more reliable indicator of the effect that these metals have on electrolyte levels and osmoregulation.

Severe kidney congestion and hemorrhaging were found in fish treated with arsenic combined with copper. This finding seems contrary to the effect produced on Na levels. Copper, arsenic, or a combination of copper and arsenic had the most dramatic affect on renal tissue although lesions were induced by all metals tested. Copper in winter flounder(29), goldfish(30), and marine teleosts(25) induced renal lesions while zinc, alone or in combination with copper, caused little damage to the kidneys(17). Although examination of pinfish exposed to zinc in this experiment revealed a small increase in inflammation compared to controls, the addition of copper did not potentiate this effect. Arsenic, however, induced large hemorrhages and severe congestion in many fish, especially when combined with copper. A significant increase in creatinine levels occurred in a synergistic manner when copper was combined with arsenic. A rise in serum levels implies kidney dysfunction because this compound is actively secreted by the teleost kidney. The present observations correspond well with the excretory properties of these metals. The kidney is the primary route of excretion for arsenic(l), and becomes involved in copper excretion only when internal copper levels are unusually high(31). Zinc excretion was significant by statistical analysis. Fish treated with zinc alone maintained gill-copper content significantly higher than all other treatments while gill-zinc was also slightly elevated. This increase in zinc content may be due to efforts by the teleost to excrete the excess zinc. Arsenic alone also produced relatively high gill-copper content. The addition of copper to both 30 micrograms zinc and 30 micrograms arsenic reduced gill copper content in an antagonistic manner.

Summary

Low levels of copper, zinc, and arsenic all produced intoxication in the pinfish, Lagodon rhomboides. The interaction of copper with zinc of arsenic was shown to have effects on some serum constituents, while other constituents were affected antagonistically or additively. Serum electrolyte levels were altered by copper, and possibly arsenic.

Despite reports that arsenic is relatively non-toxic to fish, these experiments showed it to be highly destructive to the kidneys. Copper also caused renal lesions although not as severe. When these two metals were combined, renal dysfunction was indicated. Arsenic was also associated with hepatic damage which was enhanced when copper was added.

Zinc may have contributed to hepatic damage, and in some fish induced severe stress, especially when combined with copper. Arsenic, singularly and in combination with copper increased stress as well, but to a lesser degree.

The estuarine zone provides shallow habitants that marine finfish such as pinfish, utilize as nurseries(35). Copper, zinc, and arsenic levels may be unusually high in these areas due to boat anti-fouling paints and runoff containing fertilizers and pesticides.

These metals, once absorbed by the fish may ultimately interact to produce a toxic effect (or alter reproductive rate) in a manner not readily attributed to one specific metal.

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