Addition of organic acids to acid mine drainage polluted wetland sediment leads to microbial community structure and functional changes and improved water quality.

Acid mine drainage (AMD) is a serious environmental problem worldwide that requires efficient and sustainable remediation technologies including the use of biological mechanisms. A key challenge for AMD bioremediation is to provide optimal conditions for microbial-mediated immobilisation of trace metals. Although organic carbon and oxygen can enhance treatment efficiency, the effect on microbial communities is unclear. In this study, surface sediments from a natural wetland with proven efficiency for AMD bioremediation were artificially exposed to oxygen (by aeration) and/or organic carbon (in the form of mixed organic acids) and incubated under laboratory conditions. In addition to measuring changes in water chemistry, a metagenomics approach was used to determine changes in sediment bacterial, archaeal and fungal community structure, and functional gene abundance. The addition of organic carbon produced major changes in the abundance of microorganisms related to iron and sulfur metabolism (including Geobacter and Pelobacter) and increased levels of particulate metals via sulfate reduction. Aeration resulted in an increase in Sideroxydans abundance but no significant changes in metal chemistry were observed. The study concludes that the utilisation of organic carbon by microorganisms is more important for achieving efficient AMD treatment than the availability of oxygen, yet the combination of oxygen with organic carbon addition did not inhibit the improvements to water quality.

Prolonged phenanthrene exposure reduces cardiac function but fails to mount a significant oxidative stress response in the signal crayfish (Pacifastacus leniusculus).

Crustaceans are important ecosystem bio-indicators but their response to pollutants such as polyaromatic hydrocarbons (PAHs) remains understudied, particularly in freshwater habitats. Here we investigated the effect of phenanthrene (at 0.5, 1.0 and 1.5 mg L-1), a 3-ringed PAH associated with petroleum-based aquatic pollution on survival, in vivo and in situ cardiac performance, the oxidative stress response and the tissue burden in the signal crayfish (Pacifastacus leniusculus). Non-invasive sensors were used to monitor heart rate during exposure. Phenanthrene reduced maximum attainable heart rate in the latter half (days 8-15) of the exposure period but had no impact on routine heart rate. At the end of the 15-day exposure period, the electrical activity of the semi-isolated in situ crayfish heart was assessed and significant prolongation of the QT interval of the electrocardiogram was observed. Enzyme pathways associated with oxidative stress (superoxide dismutase and total oxyradical scavenging capacity) were also assessed after 15 days of phenanthrene exposure in gill, hepatopancreas and skeletal muscle; the results suggest limited induction of protective antioxidant pathways. Lastly, we report that 15 days exposure caused a dose-dependent increase in phenanthrene in hepatopancreas and heart tissues which was associated with reduced survivability. To our knowledge, this study is the first to provide such a thorough understanding of the impact of phenanthrene on a crustacean.

Understanding the cardiac toxicity of the anthropogenic pollutant phenanthrene on the freshwater indicator species, the brown trout (Salmo trutta): From whole heart to cardiomyocytes.

Freshwater systems are faced with a myriad of stressors including geomorphological alterations, nutrient overloading and pollution. Previous studies in marine fish showed polyaromatic hydrocarbons (PAHs) to be cardiotoxic. However, the cardiotoxicity of anthropogenic pollutants in freshwater fishes is unclear and has not been examined across multiple levels of cardiac organization. Here we investigated the effect of phenanthrene (Phe), a pervasive anthropogenic pollutant on a sentinel freshwater species, the brown trout (Salmo trutta). We first examined the electrical activity of the whole heart and found prolongation (∼8.6%) of the QT interval (time between ventricular depolarization and repolarization) of the electrocardiogram (ECG) and prolongation (∼13.2%) of the monophasic action potential duration (MAPD) following ascending doses of Phe. At the tissue level, Phe significantly reduced trabecular force generation by ∼24% at concentration 15 µM and above, suggesting Phe reduces cellular calcium cycling. This finding was supported by florescent microscopy showing a reduction (∼39%) in the intracellular calcium transient amplitude following Phe exposure in isolated brown trout ventricular myocytes. Single-cell electrophysiology was used to reveal the mechanism underlying contractile and electrical dysfunction following Phe exposure. A Phe-dependent reduction (∼38%) in the L-type Ca2+ current accounts, at least in part, for the lowered Ca2+ transient and force production. Prolongation of the MAPD and QT interval was explained by a reduction (∼70%) in the repolarising delayed rectifier K+ current following Phe exposure. Taken together, our study shows a direct impact of Phe across multiple levels of cardiac organization in a key freshwater salmonid.

Macroalgae as spatial and temporal bioindicators of coastal metal pollution following remediation and diversion of acid mine drainage.

Acid mine drainage (AMD) is a significant contributor of metal pollution leading to ecosystem damage. Bioindicator organisms such as intertidal brown macroalgae have an important role in quantifying the risks of metal bioaccumulation in coastal locations exposed to AMD contamination. Measurement of As, Cd, Cu, Fe, Pb, and Zn accumulation was performed in Fucus serratus, Fucus vesiculosus and Ascophyllum nodosum sampled from two marine locations near to an abandoned Cu mine in Anglesey, Wales, UK. Transect samples were taken from a coastal location (Amlwch) that has seen a substantial increase in AMD contamination over 15 years, in comparison to a nearby estuarine location (Dulas Estuary leading to Dulas Bay) with a historic legacy of pollution. These were compared with samples from the same sites taken 30 years earlier. Some of the Dulas macroalgae samples had Cd, Cu and Zn concentrations that were above background but in general indicated a non-polluted estuary in comparison to substantial pollution over previous decades. In contrast, Fucus samples collected from directly below an AMD outflow at Amlwch showed extremely elevated metal bioaccumulation (>250 mg Fe g-1, >6 mg Cu g-1, >2 mg Zn g-1, >190 µg As g-1) and evidence of macroalgae toxicity, indicating severe pollution at this site. However, the pollution dispersed within 200 m of the outflow source. This study has demonstrated the efficiency of three brown macroalgae species as indicators for metal bioavailability at high spatial resolution and over time.

The association of microbial activity with Fe, S and trace element distribution in sediment cores within a natural wetland polluted by acid mine drainage.

Natural recovery and remediation of acid mine drainage (AMD) reduces the generation of acidity and transport of trace elements in the runoff. A natural wetland that receives and remediates AMD from an abandoned copper mine at Parys Mountain (Anglesey, UK) was investigated for better understanding of the remediation mechanisms. Water column concentrations of dissolved Fe and S species, trace metal (loid)s and acidity decreased markedly as the mine drainage stream passed through the wetland. The metal (loid)s were removed from the water column by deposition into the sediment. Fe typically accumulated to higher concentrations in the surface layers of sediment while S and trace metal (loid)s were deposited at higher concentration within deeper (20-50 cm) sediments. High resolution X-ray fluorescence scans of sediment cores taken at three sites along the wetland indicates co-immobilization of Zn, Cu and S with sediment depth as each element showed a similar core profile. To examine the role of bacteria in sediment elemental deposition, marker genes for Fe and S metabolism were quantified. Increased expression of marker genes for S and Fe oxidation was detected at the same location within the middle of the wetland where significant decrease in SO42- and Fe2+ was observed and where generation of particulate Fe occurs. This suggests that the distribution and speciation of Fe and S that mediates the immobilization and deposition of trace elements within the natural wetland sediments is mediated in part by bacterial activity.

Metabolic adaptation of a Chlamydomonas acidophila strain isolated from acid mine drainage ponds with low eukaryotic diversity.

The diversity and biological characteristics of eukaryotic communities within acid mine drainage (AMD) sites is less well studied than for prokaryotic communities. Furthermore, for many eukaryotic extremophiles the potential mechanisms of adaptation are unclear. This study describes an evaluation of eight highly acidic (pH 1.6-3.1) and one moderately acidic (pH 5.6) metal-rich acid mine drainage ponds at a disused copper mine. The severity of AMD pollution on eukaryote biodiversity was examined, and while the most species-rich site was less acidic, biodiversity did not only correlate with pH but also with the concentration of dissolved and particulate metals. Acid-tolerant microalgae were present in all ponds, including the species Chlamydomonas acidophila, abundance of which was high in one very metal-rich and highly acidic (pH 1.6) pond, which had a particularly high PO4-P concentration. The C. acidophila strain named PM01 had a broad-range pH tolerance and tolerance to high concentrations of Cd, Cu and Zn, with bioaccumulation of these metals within the cell. Comparison of metal tolerance between the isolated strain and other C. acidophila strains previously isolated from different acidic environments found that the new strain exhibited much higher Cu tolerance, suggesting adaptation by C. acidophila PM01 to excess Cu. An analysis of the metabolic profile of the strains in response to increasing concentrations of Cu suggests that this tolerance by PM01 is in part due to metabolic adaptation and changes in protein content and secondary structure.

Microbial Community Shifts in Response to Acid Mine Drainage Pollution Within a Natural Wetland Ecosystem.

Natural wetlands are known to play an important role in pollutant remediation, such as remediating acid mine drainage (AMD) from abandoned mine sites. However, many aspects of the microbiological mechanisms underlying AMD remediation within wetlands are poorly understood, including the role and composition of associated microbial communities. We have utilized an AMD-polluted river-wetland system to perform rRNA sequence analysis of microbial communities that play a role in biogeochemical activities that are linked to water quality improvement. Next-generation sequencing of bacterial 16S rRNA gene amplicons from river and wetland sediment samples identified variation in bacterial community structure and diversity on the basis of dissolved and particulate metal concentrations, sediment metal concentrations and other water chemistry parameters (pH and conductivity), and wetland plant presence. Metabolic reconstruction analysis allowed prediction of relative abundance of microbial metabolic pathways and revealed differences between samples that cluster on the basis of the severity of AMD pollution. Global metabolic activity was predicted to be significantly higher in unpolluted and wetland sediments in contrast to polluted river sediments, indicating a metabolic stress response to AMD pollution. This is one of the first studies to explore microbial community structure dynamics within a natural wetland exposed to AMD and our findings indicate that wetland ecosystems play critical roles in maintaining diversity and metabolic structure of sediment microbial communities subject to high levels of acidity and metal pollution. Moreover, these microbial communities are predicted to be important for the remediation action of the wetland.

Bioaccumulation of silver nanoparticles into Daphnia magna from a freshwater algal diet and the impact of phosphate availability.

This study assessed the bioavailability, toxicity, and transfer of silver nanoparticles (AgNPs) in comparison with AgNO3 in two model food chain organisms: the alga Chlamydomonas reinhardtii and the grazing crustacean Daphnia magna. The effects of phosphate, a potential Ag(+)-binding ligand and a determinant of phytoplankton productivity, were evaluated. Nano Ag derived from coated AgNPs and AgNO3 was accumulated at similar concentrations into microalgae during high phosphate treatment, but AgNO3 accumulation was increased by low phosphate availability. After feeding on Ag-containing algae, D. magna equally accumulated AgNO3 and nano-derived Ag. There were significant reductions in feeding when D. magna were fed Ag-contaminated algae, with the AgNO3, low phosphate-exposed cells being ingested the least. Nutritional quality characteristics including fatty acid and trace nutrient content were similar in all algal samples, indicating that feeding reduction is specifically due to the presence of Ag, with AgNO3 being more toxic than nano Ag.

Natural wetlands are efficient at providing long-term metal remediation of freshwater systems polluted by acid mine drainage.

This study describes the first long-term (14-year) evaluation of the efficacy of an established (>100 years) natural wetland to remediate highly acidic mine drainage (AMD). Although natural wetlands are highly valued for their biodiversity, this study demonstrates that they also provide important ecosystem service functions through their ability to consistently and reliably improve water quality by mitigating AMD. The Afon Goch river flows from Parys Mountain copper mine via a natural wetland, and was the major source of Zn and Cu contamination to the Irish Sea. Prior to 2003 the wetland received severe acidic metal contamination and retained a large proportion of the contamination (55, 64, and 37% in dissolved Fe, Zn, and Cu) leading to a greatly reduced metal flow to the Irish Sea. Reduced wetland loadings midway through the sampling period led to a reduction of metals by 83-94% and a pH increase from 2.7 to 5.5, resulting in long-term improvements in the downstream benthic invertebrate community. High root metal accumulation by the dominant wetland plant species and the association of acidophilic bacteria in the wetland rhizosphere indicate that multiple interacting processes provide an efficient and self-sustaining system to remediate AMD.

Aluminium exposure disrupts elemental homeostasis in Caenorhabditis elegans.

Aluminium (Al) is highly abundant in the environment and can elicit a variety of toxic responses in biological systems. Here we characterize the effects of Al on Caenorhabditis elegans by identifying phenotypic abnormalities and disruption in whole-body metal homeostasis (metallostasis) following Al exposure in food. Widespread changes to the elemental content of adult nematodes were observed when chronically exposed to Al from the first larval stage (L1). Specifically, we saw increased barium, chromium, copper and iron content, and a reduction in calcium levels. Lifespan was decreased in worms exposed to low levels of Al, but unexpectedly increased when the Al concentration reached higher levels (4.8 mM). This bi-phasic phenotype was only observed when Al exposure occurred during development, as lifespan was unaffected by Al exposure during adulthood. Lower levels of Al slowed C. elegans developmental progression, and reduced hermaphrodite self-fertility and adult body size. Significant developmental delay was observed even when Al exposure was restricted to embryogenesis. Similar changes in Al have been noted in association with Al toxicity in humans and other mammals, suggesting that C. elegans may be of use as a model for understanding the mechanisms of Al toxicity in mammalian systems.

Toxicity of aluminium in natural waters controlled by type rather than quantity of natural organic matter.

Extension of the conditions under which Al toxicity is tested is required. Environmentally representative preparation of waters is used in investigating roles of alginate (AA) and humic acids (HA) in partitioning of Al (0.5 mg L(-1)), subsequent uptake and accumulation by and toxicity to Lymnaea stagnalis. HA and AA did not alter precipitation of Al(OH)3, but altered subsequent behaviour of Al. High (40 mg L(-1)) HA concentrations, and to a lesser extent AA, prevented settling and availability for benthic grazing but made deposited Al more likely to be ingested. HA detoxified but AA increased toxicity relative to Al alone. Low concentration (4 mg L(-1)) AA and HA do not change partitioning but increase uptake; they both detoxify, but AA less than HA. The study shows OC:Al ratio is critical in predicting Al behaviour in natural waters, also uptake is mediated by snail behaviour, not solely a function of concentration and form of Al. Therefore, predicting Al behaviour will be subject to errors in determining relevant water composition and response of biota to the new speciation. However, with respect to toxicity, rather than other aspects of Al behaviour, different ratios of HA and Al are insignificant compared to whether AA is present rather than HA.

Trophic transfer of aluminium through an aquatic grazer-omnivore food chain.

The potential for trophic transfer of aluminium (Al) was investigated using a grazing detritivore, the freshwater snail Lymnaea stagnalis, and a predator, the signal crayfish Pacifastacus leniusculus. Snails were exposed to either aqueous Al (500 microg l(-1)) in the presence or absence of an inorganic ligand (phosphate (+P); 500 microg l(-1)) for 30 days, or kept as unexposed controls. Subcellular partitioning of Al in the snail tissues was characterised using ultracentrifugation. Al content in the soft tissues and the subcellular fractions was measured using inductively coupled plasma atomic emission spectroscopy. Exposed and control snails were fed to individually housed crayfish (n=6 per group) over 40 days. Water samples, uneaten snail tissue and faeces were collected throughout the experiment in order to assess the fate of Al. Behavioural toxicity to the crayfish was assessed at four time points, and tissue accumulation of Al in soft tissues was measured following a 2-day depuration period. Snails exposed to Al+P accumulated more Al per snail than those exposed to Al only (291 microg vs 206 microg), and also contained a higher proportion of detoxified Al (in inorganic granules and associated with heat stable proteins) (39% vs 26%). There were no significant differences in behavioural activity between the different groups of crayfish at any time point. Crayfish fed snails exposed to only Al accumulated significant levels of Al in their total soft tissues, compared to controls; crayfish fed Al+P-exposed snails did not, even though concentrations of Al in these snails were higher. The highest concentrations of Al were found in the green gland in both crayfish feeding groups, and the gut and hepatopancreas in crayfish fed Al only exposed snails; all of these were significantly higher than in crayfish fed control snails. There was no significant accumulation of Al in the gills or flexor muscle in any group. At least 17% of trophically available Al in the snail tissues was accumulated by the crayfish. This proportion was similar in both feeding groups but, as the proportion of trophically available Al in the snails exposed to Al+P was lower, this led to lower accumulation in the Al+P crayfish feeding group. This study indicates that in comparison to vertebrates, aquatic invertebrates accumulate a higher proportion of Al via oral ingestion but it does not accumulate in tissues that may pose a threat to human consumers.

The suitability of gallium as a substitute for aluminum in tracing experiments.

Aluminum is a toxic metal whose complex aquatic chemistry, mechanisms of toxicity and trophic transfer are not fully understood. The only isotope of Al suitable for tracing experiments in organisms-(26)Al-is a rare, costly radioisotope with a low emission energy, making its use difficult. Gallium shares a similar chemistry with Al and was therefore investigated as a potential substitute for Al for use in aquatic organisms. The freshwater snail, Lymnaea stagnalis was exposed to either Al or Ga (0.0135 mM) under identical conditions for up to 40 days. Behavioural toxicity, metal accumulation in the tissues, and sub-cellular partitioning of the metals were determined. Al was more toxic than Ga and accumulated to significantly higher levels in the soft tissues (P < 0.05). The proportion of Al in the digestive gland (DG; detoxificatory organ) relative to other tissues was significantly lower than that of Ga (P < 0.05) from day 14 onwards. There were also differences in the proportions of Al and Ga associated with heat stable proteins (HSPs) in the digestive gland, with significantly more HSP present in the DGs of snails exposed to Al, but significantly less Al than Ga associated with the HSP per unit mass protein present. From this evidence, we conclude that Ga may be of limited use as a tracer for Al in animal systems.

Tissue accumulation of aluminium is not a predictor of toxicity in the freshwater snail, Lymnaea stagnalis.

The amount of toxic metal accumulated by an organism is often taken as an indicator of potential toxicity. We investigated this relationship in the freshwater snail, Lymnaea stagnalis, exposed to 500 microg l(-1) Al over 30 days, either alone or in the presence of phosphate (500 microg l(-1) P) or a fulvic acid surrogate (FAS; 10 mg l(-1) C). Behavioural activity was assessed and tissue accumulation of Al quantified. Lability of Al within the water column was a good predictor of toxicity. FAS increased both Al lability and behavioural dysfunction, whereas phosphate reduced Al lability, and completely abolished Al-induced behavioural toxicity. Tissue accumulation of Al was not linked to toxicity. Higher levels of Al were accumulated in snails exposed to Al + P, compared to those exposed to Al alone, whereas FAS reduced Al accumulation. These findings demonstrate that the degree of tissue accumulation of a metal can be independent of toxicity.

Oxidation state and size of Fe controlled by organic matter in natural waters.

In a well mixed-stream, in which the iron/organic carbon (OC) ratio varied from 0.333 to 0.05 with sampling point and discharge, 40-70% of the Fe load was found to be present as lightly bound Fe(II). In laboratory simulations of streamwater, after 24 h of aeration at pH 6.5, and with an Fe/OC concentration ratio of 0.417, 97% of Fe(II) was converted to Fe(III) (hydr)oxides, while at a ratio of 0.083, 87% of Fe(ll) remained unoxidized. The particle size distribution of Fe contained < 0.2 microm fractions only when OC was present and comparison of Fe and OC size distributions suggested that there was more than one mechanism by which colloidal Fe was produced. At high Fe/ OC ratios, < 0.2 microm fractions may be predominantly Fe(III) (hydr)oxides stabilized by OC, but at low ratios, they must consist of otherwise soluble Fe(ll) attached to < 0.2 microm OC. The recognition in the field of the consequences of processes demonstrated in the laboratory suggests that OC may be a predominant control of both size and oxidation state of Fe in many natural waters.

Avoidance of aluminum toxicity in freshwater snails involves intracellular silicon-aluminum biointeraction.

Silicon (Si) ameliorates aluminum (Al) toxicity to a range of organisms, but in almost all cases this is due to ex vivo Si-Al interactions forming inert hydroxyaluminosilicates (HAS). We hypothesized a Si-specific intracellular mechanism for Al detoxification in aquatic snails, involving regulation of orthosilicic acid [Si(OH)4]. However, the possibility of ex vivo formation and uptake of soluble HAS could not be ruled out Here we provide unequivocal evidence for Si-Al interaction in vivo, including their intracellular colocalization. In snails preloaded with Si(0H)4, behavioral toxicity in response to subsequent exposure to Al was abolished. Similarly, recovery from Al-induced toxicity was faster when Si(OH)4 was provided, together with rapid loss of Al from the major detoxificatory organ (digestive gland). Temporal separation of Al and Si exposure excluded the possibility of their interaction ex vivo. Elemental mapping using analytical transmission electron microscopy revealed nanometre-scale colocalization of Si and Al within excretory granules in the digestive gland, consistent with recruitment of Si(OH)4, followed by high-affinity Al binding to form particles similarto allophane, an amorphous HAS. Given the environmental abundance of both elements, we anticipate this to be a widespread phenomenon, providing a cellular defense against the profoundly toxic Al(III) ion.

Influence of aqueous aluminium on the immune system of the freshwater crayfish Pacifasticus leniusculus.

Little is known of the effects of aluminium (Al) on invertebrate immunity despite the ubiquitous nature of the metal and its toxicity to aquatic organisms. Here we examine the effect of Al at neutral pH on the immune system of the freshwater crayfish Pacifasticus leniusculus. Heat-killed bacteria were injected at intervals into the haemolymph of crayfish continuously exposed to a sub-lethal concentration (500 microg l(-1)) of Al over 40 days. Circulating haemocyte and bacterial numbers were monitored for 16 days post-injection at each time interval. In the absence of bacterial challenge, the number of circulating haemocytes increased in Al-exposed crayfish compared to unexposed controls. Aluminium exposure initially reduced the crayfish's ability to clear bacteria from the circulation and decreased the rate of recovery in haemocyte numbers following bacterial challenge. These effects on bacterial and haemocyte numbers were abolished after prolonged exposure (>10 days) to Al, indicating adaptation to the metal. Aqueous Al impairs gill function in P. leniusculus by inducing hypersecretion of mucus and we suggest that the decrease in immunocompetence is due to haemolymph hypoxia. We conclude that exposure to episodic pulses of aqueous Al over the short term (<10 days) increases the risk of infection in the crayfish by impairing the ability of haemocytes to recognise and/or remove bacteria from the circulation.

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis.

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high-M(r) glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high-M(r) material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.

Effect of orthosilicic acid on the accumulation of trace metals by the pond snail Lymnaea stagnalis.

Silicon (Si) has a marked affinity for aluminium (Al(III)), but not other trace metals such as cadmium (Cd(II)) and zinc (Zn(II)). Exogenous orthosilicic acid (Si(OH)(4)) ameliorates the toxicity of Al(III) to the pond snail Lymnaea stagnalis, but its mechanism of action is unclear. Here, studies were conducted to ascertain whether interaction between orthosilicic acid and Al(III) occurs in the water column to prevent Al(III) uptake, or in the tissues to reduce the toxicity of accumulated metal. Silicon did not reduce the accumulation of Al(III) by the digestive gland (the main "sink" for trace metals in L. stagnalis) following exposure of the snail for 30 days to 500 microg l(-1) added Al(III) and 13-fold molar excess of orthosilicic acid. However, Si concentrations correlated well with Al(III) levels in the digestive gland (R(2)=0.77), giving a ratio of 2.5:1 (Al(III):Si). Exposure to Zn(II) or Cd(II) and 13-fold molar excess of orthosilicic acid did not prevent uptake of these metals, or result in a correlation between metal and Si concentrations of the snail digestive gland. These data show that aquated orthosilicic acid does not prevent Al(III) accumulation by L. stagnalis. However, following exposure, the ratio of Al(III) to Si in the digestive gland is suggestive of the early formation of hydroxyaluminosilicates, probably proto-imogolites (2-3:1 Al(III):Si). Whether hydroxyaluminates are formed ex vivo in the water column and taken up by snails into the digestive gland, or formed in situ within the digestive gland remains to be established. Either way, orthosilicic acid clearly prevents the in vivo toxicity of Al(III) rather than reducing its uptake. Silicon appears to have an important role in the handling Al(III) by the pond snail which may also have wider relevance in understanding the role of Si in ameliorating Al(III) toxicity.

Interaction of mucus with freshly neutralised aluminium in freshwater.

This study examined the interaction of mollusc trail mucus, and its biochemical constituents, with environmentally relevant concentrations of freshly neutralised aluminium (Al) in freshwater. Upon neutralisation Al starts to polymerise. In the presence of mucus the metal is rapidly localised into the hydrated mucus gel resulting in a likely reduction of its overall degree of polymerisation. A simple Al binding assay identified large-M(r) glycoconjugates as major Al-complexing molecules in mucus. Subsequent isolation and purification of these mucus glycoconjugates showed the metal readily bound to the carbohydrate portion and, in particular, to acidic components such as those containing carboxyl functionality. It is suggested gel-forming extracellular glycoconjugates play a crucial role in preventing the diffusion of Al into biological systems and thus serve to maintain metabolic homeostasis.