Keith N. White

Metal transport in a stream polluted by acid mine drainage—The Afon Goch, Anglesey, UK

Sampling of the Afon Goch over a 14-month period revealed maximum dissolved Fe, Al, Mn, Cu and Zn concentrations of 259, 167, 49, 60 and 42 mg dm(-3), respectively, and pH as low as 2.3, making it one of the most metal- and acid-contaminated streams in the UK. The river produces particulates by precipitation of ferrihydrite, due to the entry of near-neutral tributary waters, under all discharge conditions. Consequently, metal transport in this stream is dominated by processes different from those in less contaminated streams. The stream acts as a sink for contaminants, except under high discharge, when accumulated metals are flushed from the system. The implications of these observations for the monitoring and management of streams polluted by acid mine drainage are discussed.

CLONING AND CHARACTERIZATION OF METALLOTHIONEIN CDNAS IN THE MUSSEL MYTILUS EDULIS L. DIGESTIVE GLAND

Metallothioneins are small metal-binding proteins found in all species of animals and are transcriptionally-induced by heavy metal ions, oxidative stresses, and inflammation. In the blue sea mussel, Mytilus edulis, several apparent subtypes of each isoform have been purified and biochemically sequenced. To determine whether the high number of metallothionein forms present in M. edulis were specific to the digestive gland, and to understand how these proteins evolved, we cloned five variants of metallothionein from M. edulis. MT10 and MT20 isoform fragments were amplified by PCR, and used as radiolabelled probes to screen digestive gland cDNA libraries. The MT10 transcripts were 321-353 nucleotides long and the MT20 transcripts, 513-555 nucleotides. Previously identified primary structures of MT10 subtypes were confirmed and, in addition, a novel subtype was identified. Expression of MT10 and MT20 isoforms shown by clonal representation and Northern blot analysis indicated that the MT10 message was more prevalent than the MT20 message. Only the MT20 II transcript could be identified among the MT20 clones. The high degree of untranslated region similarity between each isoform indicates that these additional forms are recent gene duplication events in the Mytilus lineage. Exposure of 0.4 mg l-1 of cadmium to the mussels resulted in a marked increase in both mRNAs suggesting that the MT20 isoform represents a primarily inducible metallothionein not highly expressed under basal conditions.

Bioaccumulation of aluminium in the freshwater snail Lymnaea stagnalis at neutral pH

This study examined the accumulation of aluminium (Al) by the freshwater snail Lymnaea stagnalis at neutral pH, when most Al would be predicted to be in an insoluble form (Al(OH)(3)). Snails were exposed to a range of Al concentrations (38-285 microg l(-1)) for 30 days, followed by 20 days in clean water. Aluminium was measured using atomic absorption spectroscopy. Significant accumulation of Al occurred in the whole soft tissues, gut, digestive gland and kidney at the latest by day 10. High concentration factors were observed, ranging from 4.5 x 10(3) in the whole soft tissues to 6.3 x 10(4) in the kidney, corresponding to actual concentrations of 800 to 7500 microg g(-1), respectively. Proportionality between environmental (water) and tissue concentrations of Al was observed in the gut but not in the other tissues. Following transfer to clean water, rapid loss of Al from the whole soft tissues and gut was seen over the first 10 days. Loss of Al from the digestive gland was much less as a proportion of the total, with approximately 90% of the Al remaining in the tissue. In contrast, significant loss of Al from the kidney occurred between days 20 and 30, even in the continued presence of Al; little further loss occurred following transfer to clean water. Aluminium is clearly available to the snail at neutral pH, the most likely route of entry being the gut. This could facilitate entry of the metal into the food chain. The possible roles of the digestive gland and kidney in the handling of Al are discussed.

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

Abstract 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.

Temporal changes of 210Po in temperate coastal waters

The temporal variation of Polonium-210 (210Po) was examined in coastal sea water, the mussel Mytilus edulis, the winkle Littorina littorea and green alga Ulva lactuca in order to investigate the entry of 210Po into the marine food chain. More than 99% of 210Po in the water column occurred in the particulate phase. Dissolved 210Po concentrations peaked during the spring phytoplankton bloom and it is suggested this is related to preferential scavenging of 210Po by the increased numbers of bacteria, viruses and small dissolved particulates. Changes in L. littorea 210Po specific activity are thought not to be related to food, but to a drop in body weight following spawning. Much of the 210Po accumulated by M. edulis was located in the digestive gland. The specific activity of 210Po in the digestive gland of M. edulis was shown to be strongly correlated with changes in sea water suspended particulate specific activity. Examination of other trace metal (Ag, Al, As, Ca, Cd, Cr, Co, Cu, Fe, Hg, K, Mg, Mn, Na, Ni, Sb, Se, Sn and Zn) variations in the digestive gland revealed that class B and borderline metals had a strong positive correlation with 210Po. On-going work is investigating whether the accumulation and loss of 210Po is affected by the presence of metallothioneins.

Functional and structural rearrangements of salivary gland polytene chromosomes of Chironomus riparius Mg. (Diptera,Chironomidae) in response to freshly neutralized aluminium

Although recent work has shown that environmentally relevant concentrations of freshly neutralized aluminium (AI) are bioavailable and toxic to freshwater invertebrates, the genotoxicity of Al has not been examined. Here we show that freshly neutralized Al affects structure and function of the salivary gland polytene chromosomes of the ubiquitous chironomid larva Chironomus riparius over three generations. Exposure to 500 microg l-1 added Al for 24-25 days resulted in a significantly higher frequency of numerous somatic aberrations, while no structural aberrations were found in F1 controls and few in the second and third generation. Aberrations also included deletions of sections of chromosome G of C. riparius larvae as well as deletions of one or more Balbiani rings. Changes in functional activity included decreased activity of the Balbiani rings (BR), and an increase in the number of decondensed centromeres. The activity of the nucleolar organizer (NOR) significantly decreased in F1 chironomids exposed to Al, while in the F2 and F3 generations the NOR showed normal (high) activity. First generation chironomids were generally more susceptible to Al although no clear evidence of tolerance was apparent over three generations. The possible use of alterations in chironomid polytene chromosomes as biomarkers of trace metal pollution is discussed.

Copper adsorption kinetics of cultured algal cells and freshwater phytoplankton with emphasis on cell surface characteristics

Comparative studies were carried out on the adsorption of copper by a range of laboratory-cultured algae and freshwater phytoplankton samples. The level of surface mucilage associated with the cultured algae ranged from high (Anabaena spiroides, Eudorina elegans) to moderate (Anabaena cylindrica, Microcystis aeruginosa) to complete absence (Chlorella vulgaris, Asterionella formosa, Aulacoseira varians, Ceratium hirundinella). With laboratory cultures, the rapid uptake, EDTA release and quantitative similarity between living and dead (glutaraldehyde-fixed) algae were consistent with physical binding of Cu at the cell surface. The higher Cu adsorption per unit surface area and longer adsorption time of mucilaginous algae in the time-course study, and the relatively high level of Cu bound to mucilage found by X-ray microanalysis suggest that mucilage played an important role in metal binding. For all species examined, Cu adsorption kinetics (external Cu concentrations 1 to 1000 mg L−1) showed a clear fit to the Freundlich, but not the Langmuir isotherm, indicating a monolayer adsorption model with heterogenous binding sites. The Freundlich adsorption capacity constant (Kf) was higher in mucilaginous (3.96–12.62) compared to nonmucilaginous (0.36–3.63) species, but binding intensity (Freundlich constant 1/n) did not differ between the two cell types. The results suggest that mucilaginous algal species may have potential as biosorbents for treatment of industrial effluents containing heavy metals. Investigation of the Cu adsorption behavior of four mixed phytoplankton samples also revealed a good fit to the Freundlich, but not the Langmuir, isotherm. Freundlich constants were in the range 2.3–3.2 for samples dominated by Chlorophyta, Bacillariophyta and Cyanophyta, but recorded a value of 7.4 in the sample dominated by Dinophyta. Comparison with data from laboratory monocultures suggested that the adsorption kinetics of mixed environmental phytoplankton samples cannot be predicted simply in terms of the major algal species.

Aluminum-dependent regulation of intracellular silicon in the aquatic invertebrate Lymnaea stagnalis

Silicon is essential for some plants, diatoms, and sponges but, in higher animals, its endogenous regulation has not been demonstrated. Silicate ions may be natural ligands for aluminum and here we show that, in the freshwater snail (Lymnaea stagnalis), intracellular silicon seems specifically up-regulated in response to sublethal aluminum exposure. X-ray microanalysis showed that exposure of snails to low levels of aluminum led to its accumulation in lysosomal granules, accompanied by marked up-regulation of silicon. Increased lysosomal levels of silicon were a specific response to aluminum because cadmium and zinc had no such effect. Furthermore, intra-lysosomal sulfur from metallothionein and other sulfur-containing ligands was increased after exposure to cadmium and zinc but not aluminum. To ensure that these findings indicated a specific in vivo response, and not ex vivo formation of hydroxy-aluminosilicates (HAS) from added aluminum (555 μg/liter) and water-borne silicon (43 μg/liter), two further studies were undertaken. In a ligand competition assay the lability of aluminum (527 μg/liter) was completely unaffected by the presence of silicon (46 μg/liter), suggesting the absence of HAS. In addition, exogenous silicon (6.5 mg/liter), added to the water column to promote formation of HAS, caused a decrease in lysosomal aluminum accumulation, showing that uptake of HAS would not explain the loading of aluminum into lysosomal granules. These findings, and arguments on the stability, lability, and kinetics of aluminum–silicate interactions, suggest that a silicon-specific mechanism exists for the in vivo detoxification of aluminum, which provides regulatory evidence of silicon in a multicellular organism.

Occurrence of cell-associated mucilage and soluble extracellular polysaccharides in Rostherne Mere and their possible significance

The cell-associated mucilage and soluble extracellular polysaccharides (EPS) were investigated in a eutrophic freshwater lake (Rostherne Mere, Cheshire, U.K.) over up to 2 year annual cycles. Five particular lake algae (Anabaena spiroides Klebahn, Anabaena flos-aquae Brébisson ex Bornet & Flahault,Anabaena circinalis Rabenhorst, Microcystis aeruginosa Kützing emend. Elenkin and Eudorina elegans Ehrenberg) were found to be the major contributors to cell-associated mucilage, particularly M. aeruginosa. Calculation of the total amount of cell-associated mucilage in the phytoplankton samples showed that it occupied 0.0001–0.007% (the latter during a bloom of Microcystis) of lake water volume within the epilimnion. Seasonal changes in the total volume of associated mucilage reflected the succession of mucilage-producing algal species in Rostherne Mere, which was closely correlated with the physico-chemical (temperature, oxygen, pH, nutrients) and biological (Secchi depth, phytoplankton) parameters within the lake. High levels of cell-associated mucilage present in the lake may have potential for binding metals or other ions in the aquatic environment. Colourimetric determination of the concentration of soluble EPS revealed concentrations of between 2.5 and 60 mg l−1, with peak levels during the bacillariophyceaen bloom and late clear water phase. The second phase did not appear to relate directly to changes in algal population, and may result from bacterial activity, algal lysis or zooplankton activity. As soluble EPS forms a major component of the total amount of dissolved carbon in lakes, the study of the soluble EPS is important to understand the carbon cycle in freshwaters. No direct correlation occurred between algal-associated mucilage and soluble EPS over a single annual cycle.

Rearrangements in the salivary gland chromosomes of Chironomus riparius Mg. (Diptera, Chironomidae) following exposure to lead

Abstract The effect of lead (Pb) nitrate on the structural and functional organization of the polytene chromosomes of Chironomus riparius larvae was examined. No specimens with standard polytene chromosomes were found in the material treated with different concentrations of Pb ions. The polytene chromosomes of all individuals exposed to Pb showed various somatic chromosome rearrangements (heterozygous inversions, deletions, duplications and deficiencies), which were not detected in the studied controls. Deletions in chromosome G occurred in a high frequency resulting in the formation of so called “pompons”. The activity of Balbiani rings (BRs) and nucleolar organizer (NOR) significantly decreased with increasing the concentrations of Pb ions. The number of BRc/BRb showing little or no activity (-/-) increased following exposure of the F2 generation to low and high concentrations of Pb ions. Sections of the polytenechromosomes where somatic aberrations were concentrated are considered as “weak” sites. It was shown that in these sites were distributed repetitive DNA elements (Alu and Hinf) that might be activated by stress agents and generated many chromosome rearrangements. It is evident that lead nitrate has a marked genotoxic effect on the salivary polytene chromosomes of Chironomus riparius.