Effect of Chronic Sublethal Exposure of Major Heavy Metals on Filtration Rate, Sex Ratio, and Gonad Development of a Bivalve Species

The chronic toxic effects of major heavy metals including copper (Cu), zinc (Zn), lead (Pb), and cadmium (Cd) on the filtration rate (FR), sex ratio, and gonad development of immature blood clams, Tegillarca granosa, were investigated. The FRs were significantly inhibited by Cu, Pb and Cd, with rates generally decreasing with both increasing metal concentrations and exposure time. EC₅₀ values for FR after 28 days of exposure were 12.9, 12.7 and 14.4 μg/L for Cu, Pb and Cd, respectively. Zn exposure had no effect on FR. Sex ratios were significantly altered from controls in favor of an increased proportion of males at metal concentrations of ≥14.2, ≥86 and ≥110 μg/L for Cu, Pb and Cd, respectively; and at ≥1.68 mg/L for Zn. The gonado-somatic index was significantly reduced in clams at all metal exposures, except for the lowest concentration of Cu (7.1 μg/L).     

Comparison of different biological methods for the assessment of ecotoxicological risks

To test the effects of heavy metals in river water, we compared the sensitivity of seven different biological test methods, using bacteria (Vibrio fischeri), human FL cells, protozoans (Paramecium spp.), nematodes (Rhabditis oxycerca), aquatic plants (Lemna minor), and fishes (Leuciscus idus melanotus). As test substance we used a representative mixture of 3.0 microg/l As, 2.5 microg/l Pb, 0.8 microg/l Cd, 1.7 microg/l Cr, 3.9 microg/l Cu, 6.7 microg/l Ni, 0.4 microg/l Hg, and 23.0 microg/l Zn, imitating the detectable heavy metal contamination of the Odra estuary (NE Germany/NW Poland). This mixture was defined as normal concentration (NC). Most sensitive was the test with L. minor (exposure time 10 d). The plants already showed phytotoxic effects at the heavy metal concentration found in the Odra estuary. Test systems with human cells, protozoans, and nematodes (exposure time 1-7 d) reacted at concentrations 85-100 times above the NC. Fish toxicity (exposure time 2 d) occurred from 130-fold concentration upwards. In contrast, the standard test carried out with luminescent bacteria (exposure time 30 min) was affected only at > 1000-fold concentrations. This test is therefore not suitable as an early warning system to detect ecological risks. Even the NC of heavy metals measured in the Odra estuary inhibits cells and the growth of aquatic plants. Of the single heavy metals tested, copper produced the strongest effects. Therefore, the reduction of heavy metal emissions, especially of copper-which can amplify the toxicity of other heavy metals-should be a task of highest priority.     

Concurrent exposure to lead,manganese, and cadmium and their distribution to various brain regions,liver, kidney,and testis of growing rats

Growing rats were exposed to 5 mg/L Pb,ad libitum in drinking water, and administered low or high doses of Mn and Cd intraperitoneal (i.p.) for 30 days. Some groups of animals were also administered combinations of Pb + Mn and Pb + Cd in an identical manner. Analysis of Pb, Mn, and Cd in tissue samples showed the expected dose-dependent accumulation when the metal was administered singly. However, combined treatment produced different types of metal shift in different tissues. Enhanced accumulation of all three metals in the brain, Mn in liver, Pb in kidney and Cd in testis and kidney after combined exposure may make target organs vulnerable to the toxic effects of metals, even when encountered at low concentrations. Further, the decreased levels of blood Pb after combined treatment with Cd or Mn suggests that the significance of blood Pb level as a diagnostic aid for Pb toxicity in coexposed conditions may not be of much value. Changes in the metallic distribution within the tissues after coexposure may be the result of a competition between the administered metals for common binding sites.     

Cellular localization of lead using an antibody-based detection system and enzyme activity changes in the gills and digestive gland of the blue mussel Mytilus edulis

Marine organisms are continuously exposed to heavy metals in their environment. Bivalve mollusks such as the blue mussel Mytilus edulis accumulate high levels of heavy metals effecting cellular homeostasis and functions. Lead (Pb) exposure (2.5 mg/L of lead (II) nitrate for 10 d) and depuration (10 d in clean seawater) experiments were conducted to study its intracellular fate in the gills and digestive gland of M. edulis. For this purpose, an antibody-based detection method for ultrastructural localization and a subcellular fractionation approach for chemical analysis of Pb were used. In addition, effects of Pb on enzyme activities involved in oxyradical scavenging, such as the conjugative enzyme glutathione-S-transferase and the antioxidative enzyme catalase, were determined. The ultrastructural studies showed that Pb was mainly detected in lysosomes of gill epithelial cells and digestive cells. Lead was also detected in cell nuclei and granular hemocytes. Higher metal concentrations were measured by chemical analysis in subcellular fractions of the gills compared to those of the digestive gland. Increased activities of glutathione-S-transferase were found in gills after exposure and remained elevated during the depuration period, whereas glutathione-S-transferase activity remained unaffected in the digestive gland. Catalase activities showed no changes after Pb exposure, neither in the gills nor in the digestive gland. We conclude that gill cells are major sites of uptake and accumulation for dissolved Pb and are involved in sequestration and detoxification of this metal in M. edulis.     

Dietary Strategies for the Treatment of Cadmium and Lead Toxicity

Cadmium (Cd) and lead (Pb) are toxic heavy metals that cause adverse health effects in humans and animals. Chelation therapy, the conventional treatment for heavy metal toxicity, is reported to have a number of safety and efficacy issues. Recent studies have shown that dietary supplements play important roles in protecting against Cd and Pb toxicity. This paper reviews the evidence for protective effects of essential metals, vitamins, edible plants, phytochemicals, probiotics and other dietary supplements against Cd and Pb toxicity and describes the proposed possible mechanisms. Based on these findings, dietary strategies are recommended for people at risk of Cd and Pb exposure. The application of these strategies is advantageous for both the prevention and alleviation of Cd and Pb toxicity, as such supplements can be added easily and affordably to the daily diet and are expected to have very few side effects compared to the chelation therapy.     

Sublethal toxicity of trace metals to larvae of the blacklip abalone, Haliotis rubra

The availability of literature regarding sublethal and chronic toxicity of heavy metals to early life stages of marine species is restricted to a few species of invertebrate mollusks. The early life stage of abalone, an important gastropod both environmentally and commercially, has been involved in limited research investigating the effects of heavy metal toxicity. Fertilized eggs of Haliotis rubra were exposed to a range of dissolved nominal concentrations of cadmium, copper, iron, lead, mercury, and zinc in individual solutions for 48 h. After 48 h of exposure, the test was completed by recording survival success and morphological abnormalities of veliger larvae in each heavy metal treatment. The mean 48-h median effective concentrations affecting normal morphological development of veliger larvae determined in this test shows a decreasing order of toxicity of copper (7 mg/L), mercury (21 mg/L), zinc (35 mg/L), iron (4102 mg/L), cadmium (4515 mg/L), and lead (5111 mg/L).     

Accumulation and depuration of metals by duckweed (Lemna perpusilla)

Duckweed (Lemna perpusilla), inhabiting a heavy coal ash, secondary retaining basin from a coal-fired power plant, was the most abundant macrophyte sampled (occupying ～95% of the surface area) during the summer-fall period of 1979. Lemna in the basin accumulated the most abundant heavy and soft metals (Cd, Cu, Fe, Mn, Zn, Cr, Pb, Ni) affiliated with heavy ash to a greater extent than found in water or coal ash sediment. After a 14-day holding period under laboratory conditions of low metal influence, some duckweed metal concentrations declined to levels similar to those found in the ash basin sediments (Pb, Cr, Ni, and Cu), while other tissue concentrations (Cd, Zn, Mn, and Fe) remained higher. During the 10-day laboratory exposure bioassay (concentrations of 0.5, 1, 10, 25, and 100 times those measured in the basin for all metals) after the depuration period, high duckweed mortality was evident within 2 to 10 days at concentrations of 10× and higher, which were coupled with excessive pH reduction (3.6-2.6). Significant accumulation relative to conditions in the ash basin occurred at ≥10× concentration (≥0.50 and 0.10 mg/liter, respectively) for Pb and Cr and at the 1× level (0.10 mg/liter) for Ni. Depuration concentrations returned to control levels within 8 days. Cu and Cd showed similar high increases (～1200 and 100 mg/liter, respectively) in bioconcentration during the 0.5 to 1.0 × concentrations but failed to be significantly depurated during the 8-day recovery period. Exposures to 0.10 mg/liter Zn and 0.05 mg/liter Mn failed to show depuration to levels similar to the control concentrations. The capacity of duckweed to accumulate potentially toxic heavy metals in coal-ash-retaining systems may have an important role upon the displacement of these elements on a seasonal basis. Aquatic elemental releases of duckweed may be minimal during the growing season due to the bioconcentration capability but can be maximal in the fall during the process of natural mortality and the resulting depuration process into the receiving drainage system.     

Gender and age differences in mixed metal exposure and urinary excretion

Background

Little is known about the variation in exposure to toxic metals by age and gender and other potential modifying factors. We evaluated age and gender differences by measurements of metal/element concentrations in urine in a rural population in Matlab, Bangladesh, in three age groups: 8–12 (N=238), 14–15 (N=107) and 30–88 (N=710) years of age, living in an area with no point sources of metal exposure but where elevated water arsenic concentrations are prevalent.

Results

We found marked differences in urine concentrations of metals and trace elements by gender, age, tobacco use, socioeconomic and nutritional status. Besides a clearly elevated urinary arsenic concentration in all age groups (medians 63–85 μg As/L), and despite the low degree of contamination from industries and traffic, the urine concentrations of toxic metals such as cadmium and lead were clearly elevated, especially in children (median 0.31 μg Cd/L and 2.9 μg Pb/L, respectively). In general, women had higher urinary concentrations of toxic metals, especially Cd (median 0.81 μg/L) compared to men (0.66 μg/L) and U (median 10 ng/L in women, compared to 6.4 ng/L in men), while men had higher urinary concentrations of the basic and essential elements Ca (69 mg/L in men, 30–50 years, compared to 52 mg/L in women), Mg (58 mg/L in men compared to 50 mg/L in women), Zn (182 μg/L in men compared to 117 μg/L in women) and Se (9.9 μg/L in men compared to 8.7 μg/L in women). Manganese was consistently higher in females than in males in all age groups, suggesting a biological difference between females and males in Mn metabolism. Increasing socioeconomic status decreased the toxic metal exposure significantly in children and especially in men. Poor iron status was detected in 17% of children, adolescents and women, but only in 6% of men. Also zinc deficiency was more prevalent in females than in males.

Conclusions

Women and children seemed to be more at risk for toxic metal exposure than men and at the same time more vulnerable to micronutrient deficiency. Higher concentrations of the toxic metals in urine in women are likely to reflect an increased gastrointestinal absorption of these metals at micronutrient deficiency, such as low body iron stores and Zn deficiency. Higher urinary concentrations of the essential elements in men likely reflect a better nutritional status. There is a need for information on exposure, lifestyle and socioeconomic factors, stratified by gender and age, for the purpose of conducting balanced risk assessment and management that considers such differences.     

Winter third- to fourth-instar larvae of Chironomus plumosus as bioassay tools for assessment of acute toxicity of metals and their binary combinations

The ecotoxicological effect expressed as mortality of four metal ions (Cd, Cu, Zn, Al) and their associations on winter third- to fourth-instar larvae of Chironomus plumosus was determined. The effect of individual metals was introduced as acute toxicological effect and expressed as LC(50) and LC95 values with 95% intervals of confidence. On the basis of the LC50 values the toxicity of metals after 96 h treatment was ranked Cu>Cd>Zn>Al. Copper was at least 100 times more toxic than other metals tested. When the acute toxic effect of metal pairs was observed, in general, deleterious effects were directly proportional to metal concentrations. The toxicity of metals in combinations was different from that of individual metals, because of either antagonism or synergism. From the results obtained it can be concluded that when in metal pairs in which the original metal (the metal for which the interaction was determined) is at the lower concentration (Cd 10 mg x L(-1), Zn 25 mg x L(-1), Al 25 mg x L(-1), Cu 0.1 mg x L(-1)), the prevailing interaction is synergism (mortality was higher for metal combinations than for individual metals). Except for an overadditive effect (synergism), additivity was also confirmed in some cases (Al25+Cd10; Al25+Cd25; Al25+Cu0.1; Cu0.1+Cd10; Cu0.1+Cd25; Cu0.1+Al50). Synergism, in combinations in which the original metal is at the higher concentration (Cd 25 mg x L(-1), Zn 50 mg x L(-1), Al 50 mg x L(-1), Cu 1.0 mg x L(-1)), was observed only for the pairs Zn50+Al25 and Zn50+Cu1.0. Reciprocal additivity was observed after 96 h of treatment only for the combination Zn50+Al50. For all other binary combinations in which the original metal was at the higher concentration, an antagonistic effect was confirmed.     

Factors influencing variability and potential uptake routes of heavy metals in black kites exposed to emissions from a solid-waste incinerator

Exposure to heavy metals from emissions of the solid-waste incinerator (SWI) of Madrid (Spain) was assessed by determining the relationships between the distance from black kite (Milvus migrans) nests to the SWI and metal concentrations in nestling blood and failed eggs. Lead levels increased as the distance to the SWI decreased, while no significant relationship was found for the remaining elements. Analysis of frequency distributions showed that Pb was normally distributed, suggesting a generalized exposure to this contaminant. Variability in metal levels within and between nests indicated a differential nest exposure to contaminant metals with sibling chicks showing similar metal levels. Correlations between essential and contaminant metals suggested detoxifying mechanisms mediated by essential metals. The contribution of metal residues in blood coming from deposition in eggs was minimal compared with residues coming from posthatch exposure. Exposure to particulate volatile Pb in the form of dust from fly ash emitted to the air by the SWI is suggested to be the main route of uptake via inhalation, while both dietary and respiratory routes may contribute to Cd uptake. The increasing human population living close to the SWI may be increasingly at risk from exposure to air-borne contaminants depending on the emission standards from the SWI.     

Redox and Essential Metal Status in The Brain of Wistar Rats Acutely Exposed To a Cadmium and Lead Mixture

Most Pb and Cd neurotoxicity studies investigate exposure to either of the toxic metals alone, while data on co-exposure are scarce. The aim of our study was to fill that gap by investigating acute combined effects of Pb and Cd on redox and essential metal status in the brain of Wistar rats. Animals were randomised in four groups of six to eight rats, which received 15 or 30 mg/kg of Cd, 150 mg/kg of Pb, or 150 mg/kg of Pb + 15 mg/kg of Cd by gavage. The fifth, control, group received distilled water only. Co-treatment with Pb and Cd induced significant increase in malondialdehyde (MDA) and thiobarbituric acid-reactive substances (TBARS) compared to control and groups receiving either metal alone. This is of special importance, as MDA presence in the brain has been implicated in many neurodegenerative disorders. The groups did not significantly differ in Zn, Cu, Mn, and Fe brain levels. Our findings highlight the importance of metal mixture studies. Neurotoxicity assessments of single chemicals do not provide a real insight into exposure to mixtures in real life. Further research should look into interactions between these metals to reveal complex molecular mechanisms of their neurotoxicity.     

Continuous Monitoring of Folsomia candida (Insecta: Collembola) in a Metal Exposure Test

Current recommended ecotoxicological tests with the parthenogenetic springtail Folsomia candida using standard OECD soil do not allow for continuous monitoring during the exposure period. Effects of chemicals cannot be determined until the end of the experiment (typically after 4 weeks), since the animals stay below the soil surface. In this study, F. candida were maintained on a plaster of Paris/graphite substrate for 7 weeks and were supplied with an aqueous suspension of yeast contaminated with Cd, Cu, Pb, and Zn as nitrate salts. Growth rate, time to first batch of eggs, quantity of food consumed, and the presence of graphite in the gut (a sign of avoidance of yeast) were all affected by metal contaminated diets. The relative toxicities of Cd:Cu:Pb:Zn in the yeast were 1.0:1.07:12.0:4.3, respectively (on a weight basis) with Cd being the most toxic. Internal body concentrations increased, and the concentration factor (metal concentration in F. candida/metal concentration in yeast) decreased with increasing metal exposure. In general, metals are much less toxic when added to the food of F. candida than when incorporated into soil in standard tests. It is suggested that Collembola have a greater tolerance of metals in the diet since they avoid contaminated food, and are able to excrete assimilated metals at moulting via exfoliation of the midgut epithelium where the elements are retained as part of a storage–detoxification system. The methodology described in this article allows effects on growth to be observed as early as 7 days after the beginning of the experiment.     

Application of classic epidemiological studies and proteomics in research of occupational and environmental exposure to lead,cadmium and arsenic

The metals/metaloids lead (Pb), cadmium (Cd), and arsenic (As) are among the leading toxic agents in the environment. They represent an important source of dangerous exposure in humans, particularly in industry workers. The most serious consequences of exposure to those heavy metals are cancers and cardiovascular diseases. In studies of human exposure to Pb, Cd, and As alone and in a mixture markers signaling the subtle, early lesions that occur in the human body are still lacking. It is appropriate to develop high throughput techniques and use non-invasive materials. The techniques currently used in classic epidemiological studies allow selection of single markers of changes of exposure to chronic low dose levels of metal/metaloids mixtures. However, application of proteomic techniques in such studies may allow the selection of new biomarkers of environmental and occupational exposure to heavy metals characterized by high sensitivity and specificity. As presented in our review paper, proteomic techniques in combination with appropriate statistical methods allow the selection of potential markers to estimate the changes in the human body when exposed to those heavy metals.     

Continuous monitoring of Folsomia candida (Insecta: Collembola) in a metal exposure test

Current recommended ecotoxicological tests with the parthenogenetic springtail Folsomia candida using standard OECD soil do not allow for continuous monitoring during the exposure period. Effects of chemicals cannot be determined until the end of the experiment (typically after 4 weeks), since the animals stay below the soil surface. In this study, F. candida were maintained on a plaster of Paris/graphite substrate for 7 weeks and were supplied with an aqueous suspension of yeast contaminated with Cd, Cu, Pb, and Zn as nitrate salts. Growth rate, time to first batch of eggs, quantity of food consumed, and the presence of graphite in the gut (a sign of avoidance of yeast) were all affected by metal contaminated diets. The relative toxicities of Cd:Cu:Pb:Zn in the yeast were 1.0:1.07:12.0:4.3, respectively (on a weight basis) with Cd being the most toxic. Internal body concentrations increased, and the concentration factor (metal concentration in F. candida/metal concentration in yeast) decreased with increasing metal exposure. In general, metals are much less toxic when added to the food of F. candida than when incorporated into soil in standard tests. It is suggested that Collembola have a greater tolerance of metals in the diet since they avoid contaminated food, and are able to excrete assimilated metals at moulting via exfoliation of the midgut epithelium where the elements are retained as part of a storage--detoxification system. The methodology described in this article allows effects on growth to be observed as early as 7 days after the beginning of the experiment.     

Heavy Metal Distribution in Tissues and Eggs of Chinese Alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. Concentrations of heavy metals (As, Fe, Mn, Cu, Pb, Cd, Cr, Zn, and Hg) were examined in the tissues of Chinese alligators to elucidate the background distribution of these metals in the alligator body. Generally, within the body compartments, metal concentrations were high in liver, kidney, and heart, and low in pancreas and gonad. Study of heavy metal levels in the feces and eggs of Chinese alligator suggested that Chinese alligators could reduce body burden of toxic substances by excreting them to feces and/or sequestrating them into eggs to a lesser extent. In addition, to test whether eggshell or egg membrane could be used as surrogates to measure heavy metal load in egg contents, the correlation of metal concentrations between three egg compartments was determined. Of the nine elements analyzed, concentrations of iron, copper, and zinc in the shell membrane were highly correlated with the levels in egg contents, whereas no metal was significantly correlated between eggshell and egg contents. This suggested that the shell membrane could be a useful bioindicator for Fe, Cu, and Zn contaminations in the eggs of Chinese alligator. In a comparison of metal contents in the eggs of individuals from the Anhui captive population, the wild population in Anhui Province, and those of the Changxing captive population, higher Cu, Zn, and Cd levels and a lower Pb level were found in the Changxing individuals, indicative of specific pollutants in different areas. In addition, the majority of metal elements in the muscles of Chinese alligators and American alligators are in the same ranges. As a result of the data found in the eggs of the two alligator species, the Chinese alligators may be exposed to a higher level of metal pollutants. The study provided measurement of the heavy metal distribution in the endangered Chinese alligator for the first time and could serve as the background for the monitoring of possible heavy metal contaminations in the alligator habitats.     

Accumulation of Heavy Metals in Different Body Tissues of Atlantic Salmon,Salmo salar L., Exposed to a Model Mixture (Cu,Zn, Ni,Cr, Pb,Cd) and Singly to Nickel,Chromium, and Lead

One-year-old Atlantic salmon smolts were exposed for 2 weeks either to six priority heavy metal mixture or to Ni, Pb, and Cr singly at a concentration corresponding to Lithuanian inland water standards: Cu – 0.01, Zn – 0.1, Ni – 0.01, Cr – 0.01, Cd – 0.005 and Pb – 0.005 mg/L, respectively under semi-static conditions. The presence of metal mixture in the water only partly (by 50 %) affected the accumulation of single metals in body tissues (muscle, gills, liver and kidneys) probably due to the synergistic interactions among metals. Although metal concentrations increased in most cases, only Pb exceeded recommended level for human consumption (0.2 Pb mg/L) by 1.1-fold to 2.1-fold.     

Feeding and Growth Responses of the Snail <Emphasis Type="Italic">Theba pisana</Emphasis> to Dietary Metal Exposure

The effects of dietary exposure to copper (Cu), lead (Pb), and zinc (Zn) on feeding activities, growth response, and mortality of Theba pisana snails were studied in 5-week feeding tests. Snails were fed on an artificial diet containing the following Cu, Pb, or Zn concentrations: 0, 50, 100, 500, 1000, 5000, 10,000, and 15,000 μg/g dry food. At the end of 5 weeks, the food consumption rate was decreased with increasinges in both metal concentrations and time of exposure. The estimated concentrations of metals that reduces food consumption to 50% (EC₅₀) after 5 weeks were 56, 118, and 18 μg/g dry food for Cu, Pb, and Zn, respectively. All tested metals in the diet were found to inhibit growth of the snails in a dose-dependent manner. The toxic effect on growth of the tested metals could be arranged in the order Cu > Zn > Pb. The cumulative percentage mortality among snails fed a Cu- or Zn-contaminated diet was 73.3% and the respective value for a Pb-contaminated diet was 13.3%. There was a positive correlation between growth coefficient and food consumption for all tested metals.     

Chronic accumulation of cadmium in the edible tissues of Oreochromis aureus (Steindachner): Modification by mercury and lead

The influence of mercury and lead on cadmium accumulation was investigated by exposing the fish Oreochromis aureus, or Blue Tilapia, to two heavy metals simultaneously. The chronic accumulation profile of cadmium in the liver, brain, gill filaments, intestine, caudal muscle, spleen, trunk kidney, and gonads was determined for exposure to cadmium alone and with lead or mercury. O. aureus was exposed to cadmium alone at 0.05 and 0.10 mg/L, and mixtures of 0.05 mg/L cadmium with 0.05 mg/L mercury or lead (0.05 and 0.50 mg/L). Little research has previously been carried out on the long-term interaction between these heavy metals and their effects on tissue accumulation of heavy metals. In a food fish such as O. aureus, a knowledge of toxic metal accumulation patterns is of great importance. The highest levels of cadmium were consistently accumulated by the kidney, and the presence of other mercury or lead did not change this trend. The spleen, intestine, and liver also accumulated high levels of cadmium, while the caudal muscle and brain accumulated the lowest levels of cadmium. Separating the viscera fom the muscle would therefore reduce the cadmium burden of fishmeal.     

Heavy metal burdens in patches of asphyxiated swamp areas within the Qua Iboe estuary mangrove ecosystem

This study examined the burden of Zn, Cu, Ni, Pb, Cr and V in patches of asphyxiated mangrove swamp areas within Qua Iboe Estuary mangrove ecosystem by sediments and surface water analysis; in order to establish natural background levels of these metals and to assess anthropogenic influences on them. The analysis shows that the mean concentrations (mg/kg, dw) of Zn, Cu, Ni, Pb, Cr and V in sediments from asphyxiated and healthy mangrove ecosystems of Qua Iboe vary from 36.3-179.4, 29.2-43.2, 3.6-37.4, 39.6-93.8, 0.15-0.53 and 2.9-9.3, with the former exhibiting higher metal accumulating potential. Although heavy metal concentrations in surface water of the asphyxiated swamp were low, their accumulative effect is significant. The concentrations of Cu and Pb in surface water of this ecosystem exceeded the water quality criteria while Ni and Cr were under the maximum concentration for drinking water quality and protection of aquatic life. The values of pollution load index (PLI), which are generally greater than unity, show that the sediments and the surface water from the asphyxiated mangrove ecosystem were polluted with heavy metals, thus suggesting anthropogenic activities as a possible source of these metals. The mean concentrations of Zn, Ni and Pb exceeded the effects range-low (ERL), indicating that there may be some ecotoxicological risk to organisms living in asphyxiated mangrove sediments.     

Heavy Metals Inhibit Limb Regeneration in Horseshoe Crab Larvae

We studied the effects of heavy metals on the regeneration of walking legs in horseshoe crabs (Limulus polyphemus). The second walking leg was amputated in embryos (stage 20 and 21) and first instar (trilobite) larvae, and the length and morphology of the regenerated appendage was observed after molting to the second instar stage. Regeneration following continuous exposure to TBT (0.001–100 mg/L), mercury (0.001–100 mg/L), cadmium (0.01–100 mg/L), chromium (0.1–100 mg/L), lead (0.1–100 mg/L), and copper (1–100 mg/L) was measured relative to regeneration in seawater. Although regeneration was incomplete in controls, treatment with heavy metals led to smaller and/or malformed legs. The impacts of heavy metals on survival, molting, and regeneration of horseshoe crab larvae were ranked as follows: organotin > Hg > Cd > Cr > Zn > Pb ≧ Cu. Cu and Pb did not inhibit regeneration, even at 100 mg/L. TBT, Hg, Cd, Cr, and Zn inhibited the regeneration of appendages, although first instar larvae successfully molted into second instars even after treatment. Regeneration was comparable to seawater controls in less than 2.5 mg/L Zn. In 5.0 and 10.0 mg/L Zn, regeneration was inhibited and the length of regenerated appendages remained shorter in all second instars. Larvae treated with 10 mg/L Zn for 1-week intervals during the molt cycle showed similar patterns of regeneration. The regeneration of claws was not all or none, and formation of the claw was proportional to the length of regenerated appendages. Limb regeneration in horseshoe crab larvae may be a useful model system for the study of pollutant impacts. Received: 17 July 1997/Accepted: 24 February 1998