Metal homeostasis in Hypogymnia physodes is controlled by lichen substances

The hypothesis was tested that the lichen substances produced by the epiphytic lichen Hypogymnia physodes control the intracellular uptake of divalent transition metals. Incubating lichen thalli with and without their natural content of lichen substances with metal solutions showed that the lichen substances of H. physodes selectively inhibit the uptake of Cu(2+) and Mn(2+), but not of Fe(2+) and Zn(2+). Such behavior is ecologically beneficial, as ambient concentrations of Cu(2+) and Mn(2+) in precipitation and bark are known to limit the abundance of H. physodes, whereas limiting effects of Fe(2+) or Zn(2+) have never been found. This suggests that increasing the Cu(2+) and Mn(2+) tolerance stimulated the evolution of lichen substances in H. physodes. The depsidone physodalic acid is apparently most effective at reducing Cu(2+) and Mn(2+) uptake among the seven lichen substances produced by H. physodes. Probably lichen substances play a general role in the metal homeostasis of lichens.     

Sorption kinetics and leachability of heavy metal from the contaminated soil amended with immobilizing agent (humus soil and hydroxyapatite)

Release of heavy metals onto the soil as a result of agricultural and industrial activities may pose a serious threat to the environment. This study investigated the kinetics of sorption of heavy metals on the non-humus soil amended with (1:3) humus soil and 1% hydroxyapatite used for in situ immobilization and leachability of heavy metals from these soils. For this, a batch equilibrium experiment was performed to evaluate metal sorption in the presence of 0.05 M KNO(3) background electrolyte solutions. The Langmuir isotherms applied for sorption studies showed that the amount of metal sorbed on the amended soil decreased in the order of Pb(2+)>Zn(2+)>Cd(2+). The data suggested the possibility of immobilization of Pb due to sorption process and immobilization of Zn and Cd by other processes like co-precipitation and ion exchange. The sorption kinetics data showed the pseudo-second-order reaction kinetics rather than pseudo-first-order kinetics. Leachability study was performed at various pHs (ranging from 3 to 10). Leachability rate was slowest for the Pb(2+) followed by Zn(2+) and Cd(2+). Out of the metal adsorbed on the soil only 6.1-21.6% of Pb, 7.3-39% of Zn and 9.3-44.3% of Cd leached out from the amended soil.     

Heavy metals effects on proteolytic system in sunflower leaves

Plant proteolytic system includes proteases, mainly localized inside the organelles, and the ubiquitin-proteasome pathway in both, the cytoplasm and the nucleus. It was recently demonstrated that under severe Cd stress sunflower (Helianthus annuus L.) proteasome activity is reduced and this results in accumulation of oxidized proteins. In order to test if under other heavy metal stresses sunflower proteolytic system undergoes similar changes, an hydroponic experiment was carried out. Ten days old sunflower plants were transferred to hydroponic culture solutions devoid (control) or containing 100 microM of AlCl(3), CoCl(2), CuCl(2), CrCl(3), HgCl(2), NiCl(2), PbCl(2) or ZnCl(2) and analyzed for protein oxidative damage and proteolytic activities. After 4 days of metal treatment, only Co(2+), Cu(2+), Hg(2+), and Ni(2+) were found to increase carbonyl groups content. Except for Al(3+) and Zn(2+), all metals tested significantly reduced all proteasome activities (chymotrypsin-like, trypsin-like and PGPH) and acid and neutral proteases activities. The effect on basic proteases was more variable. Abundance of 20S protein after metal treatments was similar to that obtained for control samples. Co(2+), Cu(2+), Hg(2+), Ni(2+), Cr(3+), and Pb(2+) induced accumulation of ubiquitin conjugated proteins. It is concluded that heavy metal effects on proteolytic system cannot be generalized; however, impairment of proteasome functionality and decreased proteases activities seem to be a common feature involved in metal toxicity to plants.     

Changes of metal-induced toxicity by H2O2/NO modulators in Scenedesmus quadricauda (Chlorophyceae)

Effect of nitric oxide donor (sodium nitroprusside, SNP, 500 μM) or hydrogen peroxide scavenger (dithiothreitol, DTT, 500 μM) on cadmium (Cd) or copper (Cu) uptake (150 μM solutions) and toxicity using Scenedesmus quadricauda was studied. Combined treatments (Cd or Cu + DTT or SNP) usually ameliorated metal-induced toxicity at the level of pigments, proteins, and mineral nutrients in comparison with respective metal alone. Viability tests (MTT and TTC) showed the lowest values preferentially in Cu treatments, indicating higher toxicity in comparison with Cd. Cd showed low impact on amino acids while strong Cu-induced depletion was mitigated by DTT and SNP. Amount of ROS and NO showed the most pronounced responses in SNP variants being rather reciprocal than parallel and regulated ascorbate peroxidase activity. Blot gel analyses of hsp70 protein did not reveal extensive changes after given exposure period. Phenols were elevated by DTT alone while all Cu treatments revealed depletion. Total Cu content decreased while total Cd content increased in metal + SNP or metal + DTT. Subsequent experiment using lower Cd, SNP or DTT doses (10 and 100 μM) revealed concentration-dependent impact on Cd uptake. Overall, DTT was found to be more suitable for the amelioration of metal-induced toxicity.     

Toxic effects of low concentrations of Cu on nodulation of cowpea (Vigna unguiculata)

Although Cu is phytotoxic at Cu(2+) activities as low as 1-2 microM, the effect of Cu(2+) on the nodulation of legumes has received little attention. The effect of Cu(2+) on nodulation of cowpea (Vigna unguiculata (L.) Walp. cv. Caloona) was examined in a dilute solution culture system utilising a cation exchange resin to buffer solution Cu(2+). The nodulation process was more sensitive to increasing Cu(2+) activities than both shoot and root growth; whilst a Cu(2+) activity of 1.0 microM corresponded to a 10% reduction in the relative yield of the shoots and roots, a Cu(2+) activity of 0.2 microM corresponded to a 10% reduction in nodulation. This reduction in nodulation with increasing Cu(2+) activity was associated with an inhibition of root hair formation in treatments containing > or =0.77 microM Cu(2+), rather than to a reduction in the size of the Rhizobium population.     

Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions

This paper provides direct evidence that earthworm immune cells, coelomocytes, are exposed to bio-reactive quantities of metals within 3 days after dermal exposure, and that they respond by upregulating metallothionein (MT) and heat shock protein (HSP70, HSP72) expression. Indirect support for the hypothesis that coelomocytes are capable of trafficking metals was also obtained. Coelomocytes were expelled from adult individuals of Eisenia fetida after 3-day exposure either to metal ions (Zn, Cu, Pb, Cd) or to distilled water (controls) via filter papers. The number of coelomocytes was significantly decreased after Cu, Pb, or Cd treatment. Cytospin preparations of coelomocytes were subjected to immunoperoxidase staining with monoclonal antibodies against human heat shock proteins (HSP70 or HSP72), or rabbit polyclonal antibodies raised against metallothionein 2 (w-MT2) of Lumbricus rubellus. Applied antibodies detected the respective proteins of E. fetida and revealed that the expression of HSP70, HSP72 and w-MT2 proteins was either induced or significantly enhanced in coelomocytes from metal-exposed animals. In conclusion, stress protein expression in earthworm coelomocytes may be used as sensitive biomarkers of metal contaminations. Further experimentation is needed for quantitative analysis of kinetics of metal-induced stress protein expression in earthworm coelomocytes.     

Phytotoxicity of short-term exposure to excess zinc or copper in Scots pine seedlings in relation to growth,water status,nutrient balance,and antioxidative activity

The toxic effects of heavy metals pose a significant threat to the productivity and stability of forest ecosystems. Changes in the agrochemical properties of polluted forest soils due to global climate changes can increase the bioavailability of previously immobilized heavy metals. To test this hypothesis, we studied the effects of short-term shock exposure to ZnSO₄ (50, 150, 300 μM) or CuSO₄ (2.5, 5, 10 μM) in hydroculture on 4- to 6-week-old seedlings of Scots pine (Pinus sylvestris L.) with well-developed root systems. The effects of the excess heavy metals on mineral nutrients and the functioning of low-molecular-weight antioxidants and glutathione in protecting plants from oxidative damage were studied. Even short-term exposure to exogenous metals led to their rapid accumulation in the root system and their subsequent transport to aboveground organs. An increase in the 4-hydroxyalkenals content in seedling needles exposed to excess Cu led to an increase in the content of proanthocyanidins and catechins, which act as scavengers of reactive oxygen species. The impact of both metals led to the rapid development of mineral nutrient imbalances in the seedlings, which were most pronounced in the presence of excess Zn. Exposure to excess Zn led to a disruption in the translocation of Fe and a decrease in the Fe content in the needles. The most dramatic consequence of Zn exposure was the development of Mn deficiency in the roots, which was the likely cause of the inhibition of phenolic compound synthesis. A deficiency in phenolic compounds can have serious environmental consequences for pine populations that are at risk of contamination by Zn and Cu salts.

     

Heavy metal toxicity to Lemna minor: studies on the time dependence of growth inhibition and the recovery after exposure

Environmental concentrations of toxic substances are not necessarily constant but fluctuate over time. Periods of intense exposure might be followed by episodes with a relatively low or no exposure, in principle allowing exposed organisms to recover from toxic injury. The growth reproduction assay with the limnic vascular plant Lemna minor allows for convenient studies on the time dependence of the aquatic toxicity of chemicals. Here we report on a study with four priority metals (Zn, Cu, Ni, Cd). Aims of the study were to determine the impact of the exposure duration on the observed toxicities and to determine the potential for recovery. The bioconcentrations of the test metals were recorded during the exposure in order to analyse, whether changes in the internal concentrations are a governing factor for the dynamics of toxicity. After an exposure of 7 days, Cd and Cu showed the highest toxicity to Lemna (EC50's of 1.9 and 9.7 microM respectively), while Ni and Zn had a slightly lower toxicity (EC50's of 56.3 and 46.1 microM respectively). Additionally, Zn showed a severely delayed toxicity and the exposed plants did not recover even 7 days after the exposure had ended. This is in sharp contrast to the other test metals, for which a considerable recovery was observed. These results indicate the necessity to more thoroughly consider the dynamics of toxicity, instead of recording toxic effects only after a constant exposure over a fixed time.     

Sorption studies of heavy metal ions on a novel chelating resin and its application in the stripping of mercury (II)

A chelating resin containing a stable thiol group was synthesised, using polystyrene as the starting material. The resin is stable towards conc. HCl, 0.1M HNO(3) and 0.1M NaOH. The resin shows affinity towards Ag(+), Hg(2+), Bi(3+), Pb(2+), Cu(2+), Zn(2+) and Cd(2+). Extraction of these metal ions as a function of pH, kinetics of exchange and breakthrough capacities is evaluated. The selectivity of the resin for the metal ions is in the order Ag(+) > Hg2+ > Cu2+ > Pb2+ > Cd2+ > Zn2+. The equilibrium constants for exchange and kinetics of exchange are favourable for the recovery of mercury from lean sources. Application of the resin in the stripping of mercury from chlor-alkali plant affluent, and in the enrichment of mercury from seawater, have been investigated. Mercury sorbed resin can be regenerated using 5% thiourea in 0.1M HCl.     

Metal-related oxidative stress in birds

Metals can cause oxidative stress by increasing the formation of reactive oxygen species (ROS), which render antioxidants incapable of defence against growing amounts of free radicals. Metal toxicity is related to their oxidative state and reactivity with other compounds. Our aim is to review the mechanisms on how metals cause oxidative stress and what is known about metal-induced oxidative stress in wildlife. Taking birds as model organisms, we summarize the mechanisms responsible for antioxidant depletion and give a view of how to detect metal-induced oxidative stress in birds by using different biomarkers. The mechanisms producing the harmful effects of oxidative stress are complex with different biomolecular mechanisms associated with ecotoxicological and ecological aspects. The majority of the studies concerning metals and ROS related to oxidative stress have focused on the biomolecular level, but little is known about the effects at the cellular level or at the level of individuals or populations.     

Oxidative stress, liver biotransformation and genotoxic effects induced by copper in Anguilla anguilla L.--the influence of pre-exposure to beta-naphthoflavone

Fish are exposed in the aquatic ecosystems to different classes of pollutants. Polycyclic aromatic hydrocarbons (PAHs) and heavy metals represent two important classes of aquatic contaminants. Thus, one lot of European eels (Anguilla anguilla L.) was pre-exposed during 24 h to 2.7 microM beta-naphthoflavone (BNF; a PAH-like compound), and subsequently exposed during 24 h to 0, 1 and 2.5 microM copper (Cu). Additionally, another lot not pre-exposed to BNF was exposed to the same Cu concentrations. BNF pre-exposure promoted a significant increase in liver ethoxyresorufin O-deethylase (EROD) activity, but did not change the other responses investigated in eels. On the other hand, both Cu concentrations did not modify the liver EROD activity either in eels pre-exposed to BNF or not. Liver total cytochrome P450 was increased in eels exposed to Cu 2.5 microM, being significantly only in eels not pre-exposed to BNF. Free sulfhydryl group content was decreased by 1 and 2.5 microM in eels pre-exposed to BNF or not pre-exposed, being significant at 2.5 microM Cu in eels not pre-exposed compared to its control. Liver total glutathione (TG), reduced glutathione (GSH) and GSH/oxidized glutathione (GSSG) levels were slightly decreased by 1 and 2.5 microM Cu in eels pre-exposed to BNF, whereas a slight tendency to increase was observed in eels not pre-exposed. Thus, liver TG and GSH significantly decreased in 2.5 microM Cu BNF pre-exposed eels compared to eels not pre-exposed to BNF. Liver glutathione reductase and catalase activities were significantly inhibited by 1 and 2.5 microM Cu in eels pre-exposed to BNF, concomitantly with a slight liver glutathione peroxidase tendency to decrease. Lipid peroxidation was significantly increased by 1 microM Cu in eels either pre-exposed or not pre-exposed to BNF. Liver H(2)O(2) was significantly increased by 1 microM Cu in eels pre-exposed to BNF. Liver DNA integrity was significantly decreased by 1 and 2.5 microM Cu in eels pre-exposed to BNF. The oxidative stress and genotoxic effects induced by Cu in eels pre-exposed to BNF revealed that the metal effects are potentiated by previous exposure to BNF.     

浮游球衣菌对Pb2+、Cu2+、Zn2+、Cd2+的吸附性能研究

研究了浮游球衣菌（Sphaerotilus natans）在不同吸附条件下对溶液中Pb^2＋、Cu^2＋、Zn^2＋、Cd^2＋的吸附规律。结果表明,Sphaerotilus natans对这4种重金属离子均有一定的吸附作用,并在20min内达到吸附平衡,pH对吸附过程影响较大,pH为5.5时Sphaerotilus natans对这4种金属离子的吸附效果最好,Sphaerotilus natans对它们的吸附选择性为Pb^2＋〉Cu^2＋〉Zn^2＋〉Cd^2＋,Pb^2＋、Cu^2＋能部分置换出已被菌体吸附的Zn^2＋、Cd^2＋。HCI和EDTA溶液可有效地将金属离子从菌体上解吸下来,解吸后的菌体可重复使用。     

Anguilla anguilla L. oxidative stress biomarkers responses to copper exposure with or without beta-naphthoflavone pre-exposure

The redox cycling of heavy metals as well as their interactions with organic pollutants is a major contributor to the oxidative stress resulting from aquatic pollution. Therefore, in order to evaluate beta-naphthoflavone (BNF), Cu and BNF/Cu-induced oxidative stress with single and subsequent exposures, research was carried out in European eel (Anguilla anguilla L.). Eel gill and kidney oxidative stress biomarker responses such as lipid peroxidation (LPO), glutathione peroxidase (GPX), catalase (CAT), glutathione S-transferase (GST) and total reduced glutathione (GSH) to a single 24 h exposure to two copper concentrations (Cu-1 microM, 2.5 microM) and BNF (2.7 microM) with or without 24 h BNF (2.7 microM) pre-exposure were investigated. Cu exposure alone showed a significant gill GST increase at the lowest concentration and GSH content decrease for the highest concentration. Double BNF exposure in gill demonstrated a significant increase in LPO, CAT, GPX and GST, as well as a decrease in GSH content. However, in sequential BNF/Cu exposures, only the highest Cu concentration exhibited a significant increase in LPO and GSH as well as a decrease in GPX (vs. BNF + CW). In kidney, Cu exposure alone showed a significant CAT and GSH contents decrease for both concentrations, and at highest concentration in GPX; as well as GST increase at the lowest concentration. Double BNF exposure showed a significant increase in LPO and GST. Nevertheless, in sequential BNF/Cu exposures, both concentrations exhibited a significant increase in LPO and decrease in GSH contents. Moreover, LPO was also increased significantly in comparison to BNF+CW and the equivalent Cu exposures without BNF pre-exposure. Concerning GPX, a significant increase was observed at highest Cu concentration. In GST, a significant decrease at the lowest Cu concentration and increase at the highest Cu concentration was observed. Summarizing, a simple copper or BNF exposures have no ability to induce LPO in both gill and kidney. However, double BNF exposure induced LPO in both organs and sequential BNF/Cu exposures potentiated the risk of peroxidative damage occurrence in both organs. BNF/Cu interference on antioxidant responses differs between the studied organs. In gill, antagonistic effects were denoted with probable reflex in terms of peroxidative damage increase. In kidney, BNF pre-exposure prevented CAT and GPX inhibition by copper; though, no advantage of this effect was perceptible as defence against LPO generation. Considering BNF as a surrogate for a PAH and the detected interactions with copper, as well as the likelihood that these effects would be observed in polluted ecosystems, current results demonstrate their relevance to actual ecological exposures contributing to a better knowledge on oxidative stress mechanisms in fish.     

Metal accumulation from contaminated food and its effect on growth of juvenile landsnails Helix engaddensis

Metal accumulation by juvenile landsnails, Helix engaddensis, and its effect on growth rate was studied over a 5-week period of exposure and 2 weeks of recovery. An artificial food contaminated with Cu (4-2500 microg x g(-1)), Cd (50-800 microg x g(-1)), Pb, and Zn (20-12500 microg x g(-1)) was used. During the 7 weeks of the experiment, mortality rates were 20, 27, 30, and 38% among snails fed Cu-, Pb-, Zn-, and Cd-contaminated food, respectively. According to the ability to inhibit growth, metals were found to have the following order: Cd > Zn > Cu = Pb. Inhibitory effects of dietary metals started to be significant from the third week of exposure on. Inhibition of growth by Pb and Cu was found to be reversible, and within the first week of recovery, snails erupted their aestivation and resumed feeding and growth to gain weights similar to those of the control groups. Snails fed Cd- or Zn-contaminated food failed to resume growth during the 2 weeks of recovery. This indicates that in the case of Cu and Pb, growth inhibition was mainly due to starvation due to food rejection and aestivation. On the other hand, growth inhibition caused by Cd and Zn may have been resulting from irreversible toxicity. Therefore, snails were assumed to be sensitive to Cd and Zn but tolerant to Cu and Pb. Accumulation of Cu and Pb was significant only at the highest concentrations. At low and medium concentrations, no signs of accumulation were observed, indicating regulation at these concentrations. Cd and Zn accumulation starts at low concentrations but became significant at medium and high levels indicating accumulation of these metals.     

Toxicity of copper to acetoclastic and hydrogenotrophic activities of methanogens and sulfate reducers in anaerobic sludge

Heavy metals could potentially negatively impact microorganisms in anaerobic sulfate reducing bioreactors. The objective of this is study was to evaluate the inhibitory effect of copper to acetoclastic and hydrogenotrophic activities of methanogens and sulfate reducers in sludge obtained from a full-scale sulfate reducing bioreactor. The 50% inhibiting concentration (50%IC) of Cu(2+) to acetoclastic and hydrogenotrophic methanogens was 20.7 and 8.9 mg l(-1), respectively. The 50%IC of Cu(2+) to acetoclastic sulfate reduction was 32.3 mg l(-1). The hydrogenotrophic sulfate reducers were only inhibited by 27% at the highest concentration of Cu(2+) tested, 200 mg l(-1), indicating a high level of tolerance. The soluble Cu(2+) was observed to decrease rapidly in both the methanogenic and sulfate reducing assays. The highest level of decrease was observed in the hydrogenotrophic sulfate reducing assay which was over 99% in 5h. The results of this study indicate that sulfate reducing biotechnologies would be robust at relatively high inlet concentrations of Cu(2+).     

Behavior of Trifolium repens and Lolium perenne growing in a heavy metal contaminated field: Plant metal concentration and phytotoxicity

The use of a vegetation cover for the management of heavy metal contaminated soils needs prior investigations on the plant species the best sustainable. In this work, behaviors of Trifolium repens and Lolium perenne, growing in a metal-polluted field located near a closed lead smelter, were investigated through Cd, Pb and Zn-plant metal concentrations and their phytotoxicity. In these plant species, metals were preferentially accumulated in roots than in shoots, as follow: Cd>Zn>Pb. Plant exposure to such metals induced oxidative stress in the considered organs as revealed by the variations in malondialdehyde levels and superoxide dismutase activities. These oxidative changes were closely related to metal levels, plant species and organs. Accordingly, L. perenne seemed to be more affected by metal-induced oxidative stress than T. repens. Taken together, these findings allow us to conclude that both the plant species could be suitable for the phytomanagement of metal-polluted soils.     

Effects of pH, organic acids, and inorganic ions on lead desorption from soils

The desorption characteristics of lead in two variable charge soils (one developed from Arenaceous rock (RAR) and the other derived from Quaternary red earths (REQ)) were studied, and the effects of pH value, organic acid, and competitive ions were examined. Desorption of Pb(2+) decreased from nearly 100.0 to 20.0% within pH 1.0-4.0 in both soils, and then the decrease diminished at pH > 4.0. Organic ligands at relatively low concentrations (< or =10(-3) mol L(-1)) slightly inhibited Pb(2+) desorption, but enhanced Pb(2+) desorption at higher concentrations. In this study, citric acid or acetic acid at higher concentrations (>10(-3) mol L(-1)) had the greatest improvement of Pb(2+) desorption, followed by malic acid; and the smallest was oxalic acid. Desorption of the adsorbed Pb(2+) increased greatly with increasing concentrations of added Cu(2+) or Zn(2+). Applied Cu(2+) increased Pb(2+) desorption more than Zn(2+) at the same loading.     

A field method using microcosms to evaluate transfer of Cd,Cu, Ni,Pb and Zn from sewage sludge amended forest soils to Helix aspersa snails

Juvenile Helix aspersa snails exposed in field microcosms were used to assess the transfer of Cd, Cu, Ni, Pb and Zn from forest soils amended with liquid and composted sewage sludge. Zn concentrations and contents were significantly higher in snails exposed to liquid and composted sludge after 5 and 7 weeks of exposure, when compared with control. Trends were less clear for the other metals. Present results show that Zn, among the cocktail of metallic trace elements (MTE) coming from sewage sludge disposal, represents the principal concern for food chain transfer and secondary poisoning risks. The microcosm design used in this experiment was well suited for relatively long-term (about 2 months) active biomonitoring with H. aspersa snails. The snails quickly indicated the variations of MTE concentrations in their immediate environment. Therefore, the present study provides a simple but efficient field tool to evaluate MTE bioavailability and transfer.     

Concentrations and composition of PCB congeners in the air around stored used capacitors containing PCB insulator oil in a suburb of Bangkok, Thailand

Accumulation of Zn, Cu, Pb and Cd was studied in snails fed for 120 days on diets contaminated with each metal separately and with all metals mixed together. The concentrations of Zn in food were in the range 39 to 12 200 mg kg(-1), Cu 9-1640 mg kg(-1), Pb 0.4-12 700 mg kg(-1), and Cd 0.16-146 mg kg(-1) on a dry weight basis. At the highest concentrations of all metals the consumption rates decreased significantly. For the remaining concentrations, Zn and Cu were accumulated in soft tissue in proportion to their concentrations in food. The lowest treatments of Pb and Cd did not cause any increase in soft tissue concentrations of these metals but at average treatments, a clear increase was observed. Copper was accumulated especially efficiently, exceeding concentrations in food throughout the whole range of treatments. Except for the lower end of experimental treatments, Zn was accumulated approximately in direct proportion to its concentration in the diet. Lead was the most efficiently regulated metal, with soft tissue concentrations always substantially lower than in food. Approximately 60% of Zn, 90% of Cu, 43% of Pb and 68% of Cd on average was assimilated from food. The assimilation efficiency of food alone was ca 74%. The concentrations of metals in shells increased significantly with exposure, but (with one exception) the concentrations in shells did not exceed 5% of those found in soft tissue. We argue that snails are more important as agents of food-chain transport of Cu and Cd, than of Zn or Pb. Our results indicate also that snails are not able to deposit significant quantities of metals in their shells, at least during the time scale of our laboratory experiment.