Evidence of common cadmium and copper uptake routes in zebrafish Danio rerio.

Cadmium and copper accumulations in gills of zebrafish were measured during a 48 h exposure to 0.025 µM 106Cd and 0.05 or 0.5 µM 65Cu as a single metal or their mixtures. The gill transcript levels of genes involved in the transport of Cu (CTR1 and ATP7A), Na (NHE-2), Ca (ECaC), divalent metals (DMT1), and Zn (ZIP8) were also compared between treatments at 24 and 48 h. Cd uptake was significantly suppressed in the presence of Cu, indicating interaction between Cu and Cd at uptake sites, but Cu uptake was unaffected by Cd. The decrease in Cd accumulation rates in the presence of Cu was associated with an increase in transcript abundance of ECaC at 24 h and DMT1 at 48 h and a decrease in Zip8 transcript levels, all known as routes for Cd uptake. Fish exposed to 0.5 µM 65Cu show an increase in gill ATP7a transcript abundance, suggesting that Cu is removed from the gill and is transferred to other organs for detoxification. A reduction in gill CTR1 transcript abundance was observed during the Cu-Cd exposure; this may be a regulatory mechanism to reduce Cu loading if Cu is entering the gills by other uptake routes, such as ECaC and DMT1.

Global transcriptome profiling reveals molecular mechanisms of metal tolerance in a chronically exposed wild population of brown trout.

Worldwide, a number of viable populations of fish are found in environments heavily contaminated with metals, including brown trout (Salmo trutta) inhabiting the River Hayle in South-West of England. This population is chronically exposed to a water-borne mixture of metals, including copper and zinc, at concentrations lethal to naïve fish. We aimed to investigate the molecular mechanisms employed by the River Hayle brown trout to tolerate high metal concentrations. To achieve this, we combined tissue metal analysis with whole-transcriptome profiling using RNA-seq on an Illumina platform. Metal concentrations in the Hayle trout, compared to fish from a relatively unimpacted river, were significantly increased in the gills, liver and kidney (63-, 34- and 19-fold respectively), but not the gut. This confirms that these fish can tolerate considerable metal accumulation, highlighting the importance of these tissues in metal uptake (gill), storage and detoxification (liver, kidney). We sequenced, assembled and annotated the brown trout transcriptome using a de novo approach. Subsequent gene expression analysis identified 998 differentially expressed transcripts and functional analysis revealed that metal- and ion-homeostasis pathways are likely to be the most important mechanisms contributing to the metal tolerance exhibited by this population.

Visualising fat reserves in an insect: A method using X-ray micro-computerised tomography of the Common Wasp (Vespula vulgaris).

The Common Wasp, Vespula vulgaris (Hymenoptera: Vespidae), has an annual nest cycle with new colonies initiated by over-wintered queens. Survival of adult queen wasps through winter dormancy is enabled through the deposition of substantial quantities of triglycerides in fat bodies. Worker (and male) wasps lack these fat reserves. By comparing micro-CT scans of workers, pre-hibernation queens and post-hibernation queens, we demonstrate that it is possible to semi-quantitatively measure fat reserves using arbitrary X-ray attenuation ranges. Venom in the venom gland of the queen wasps, has a significantly lower X-ray attenuation value than the triglyceride-rich fat bodies. This may be due to its content of low molecular weight volatile pheromones in addition to its other known constituents. We also demonstrate the utility of micro-CT for visualising a range of physiological and anatomical features of insects. This non-destructive method for measuring fat reserves can be used on appropriately preserved or freshly collected insect specimens.

The tracheal system of the Common Wasp (Vespula vulgaris) - A micro-CT study.

X-ray micro-CT has been used to study the tracheal system of Pre and Post hibernation Queen wasps (Vespula vulgaris) and their workers. We have compared our findings in wasps with Snodgrass's description of the tracheal system of the honeybee as characterised by anatomical dissection. Our images, whilst broadly similar, identify the tracheal system as being considerably more complex than previously suggested. One of the 30 wasps imaged had a markedly different, previously undescribed tracheal system. Since completing this study, a large micro-CT study from the American Museum of Natural History (AMNH) has been published. This used different software (Slicer) and analysed 16bit digital data. We have compared our methods with that described in the AMNH publication, adopted their suggested nomenclature and have made recommendations for future studies.

Analysis of the rainbow trout solute carrier 11 family reveals iron import < or = pH 7.4 and a functional isoform lacking transmembrane domains 11 and 12.

A Xenopus oocyte heterologous expression system was used to characterise iron transport properties of two members of the solute carrier 11 (slc11) protein family isolated from rainbow trout gills. One cDNA clone differed from the trout Slc11alpha containing an additional 52bp in the exon between transmembrane domains (TM) 10 and 11. The 52bp contained a stop codon, resulting in a novel isoform lacking the last two TM (termed slc11gamma). Slc11gamma and another isoform slc11beta, import Fe(2+) at external pHs < or = to 7.4. Trout slc11beta Fe(2+) import was more sensitive to inhibition by divalent metals. The novel vertebrate slc11gamma isoform functions without TM11 and 12.

The effects of dietary iron concentration on gastrointestinal and branchial assimilation of both iron and cadmium in zebrafish (Danio rerio).

Zebrafish (Danio rerio) were fed either a diet containing 33mgFekg(-1) (low) or 95mgFekg(-1) (normal) for 10 weeks, after which short-term Cd and Fe uptake by the gastrointestinal tract and gill was assessed. Carcass metal content and transcript levels of the iron importer, Divalent Metal Transporter 1 (DMT1) and an iron exporter, ferroportin1, in both the gastrointestinal tract and gill were also measured. Fish fed the low Fe diet accumulated 13 times more Cd into their livers via the gastrointestinal tract than those fed the normal Fe diet. However, no significant increase in liver Fe accumulation was measured. Concomitantly, when exposed to 48nmolCdL(-1) fish fed the low Fe diet exhibited a approximately 4-fold increase in Cd accumulation on the gill and in the liver, compared to those fed a normal diet. In addition, fish fed the low Fe diet also significantly accumulated more Fe on the gill (nine-fold increase) and into the carcass (four-fold increase) when exposed to 96nmolFeL(-1), compared to fish fed a normal diet. Surprisingly, carcass Fe, Ca and Mg concentrations were increased in fish fed the low Fe diet, which suggests that Fe body levels may not be a good indicator of whether a fish is more or less susceptible to increased non-essential metal accumulation via an Fe uptake pathway. However, significantly elevated transcript levels of DMT1 and ferroportin1 (2.7- and 3.8-fold induction, respectively) were seen in the gastrointestinal tract, and DMT1 in the gills (1.8-fold induction) of zebrafish fed a low Fe diet. The correlation between Cd uptake and DMT1 expression suggests that one route of uptake of Cd, either from the diet or from the water, could be via DMT1.

11-deoxycorticosterone is a potent agonist of the rainbow trout (Oncorhynchus mykiss) mineralocorticoid receptor.

The teleost fish are thought to lack the mineralocorticoid hormone aldosterone but possess mineralocorticoid receptor (MR) homologs. Here we describe the characterization of two rainbow trout (Oncorhynchus mykiss) MRs, called rtMRa and rtMRb. The open reading frame of rtMRa cDNA encoded a protein of 1041 amino acids. The rtMRb predicted protein sequence is similar, differing in only 10 amino acids in the nonconserved A/B domain and lacking a three-amino acid insertion between the two zinc fingers of the C domain. Expression of rtMR mRNA (sum of both forms), measured in juvenile trout by real-time RT-PCR, shows that the transcripts are ubiquitous. Expression was significantly higher in brain than the other tissues studied (eye, trunk kidney, head kidney, gut, gills, liver, spleen, ovary, heart, white muscle, skin). Hormonal stimulation of receptor transactivation activity was studied in COS-7 cells transiently cotransfected with receptor cDNA and a mouse mammary tumor virus-luciferase reporter. The mineralocorticoids 11-deoxycorticosterone and aldosterone were more potent enhancers of rtMRa transcriptional activity (EC50 = 1.6 +/- 0.5 x 10(-10) and 1.1 +/- 0.4 x 10(-10) M, respectively) than the glucocorticoids cortisol and 11-deoxycortisol (EC50 = 1.1 +/- 0.3 x 10(-9) and 3.7 +/- 1.9 x 10(-9) M, respectively). A similar response was observed in transactivation assays with rtMRb. These results are discussed in the view of reported circulating levels of corticosteroids in trout.

Toxicity and the fractional distribution of trace metals accumulated from contaminated sediments by the clam Scrobicularia plana exposed in the laboratory and the field.

The relationship between the subcellular distribution of accumulated toxic metals into five operational fractions (subsequently combined into presumed detoxified and non-detoxified components) and toxicity in the clam Scrobicularia plana was investigated under different laboratory exposures. Clams were exposed to metal contaminated media (water and diet) and analysed for the partitioning of accumulated As, Cu and Zn into subcellular fractions. In general, metallothionein-like proteins, metal-rich granules and cellular debris in different proportions acted as main storage sites of accumulated metals in the clam soft tissues for these three metals. No significant differences were noted in the accumulation rates of As, Cu and Zn of groups of individuals with or without apparent signs of toxicity after up to 30 days of exposure to naturally contaminated sediment mixtures. There was, however, an increased proportional accumulation of Cu in the non-detoxified fraction with increased Cu accumulation rate in the clams, suggesting that the Cu uptake rate from contaminated sediments exceeded the combined rates of elimination and detoxification of Cu, with the subsequent likelihood for toxic effects in the clams.

Evidence for two distinct functional glucocorticoid receptors in teleost fish.

Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7.3 kb transcript was observed in various tIssues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K(d)=5.05+/-0.45 nM; GR2 K(d)=3.04+/-0.79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10(-)(6 )M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor. Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC(50)=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC(50)=46+/-12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.

The effects of cyanobacteria and the cyanobacterial toxin microcystin-LR on Ca2+ transport and Na+/K+-ATPase in tilapia gills

The effects of cytotoxic substances from cyanobacteria on ionic transport processes in tilapia (Oreochromis mossambicus) were examined. Inhibitory effects on ionic transport including whole-body Ca2+ fluxes and P-type ATPases of the gill were found. The compounds tested were (1) purified microcystin-LR (MC-LR), a heptapeptide hepatotoxin produced by the cyanobacterium Microcystis aeruginosa, (2) extracts from M. aeruginosa strain PCC 7820, a strain producing MC-LR and other microcystin variants, and (3) extracts of M. aeruginosa CYA 43, a strain producing toxins including small quantities of MC-LR. Whole-body Ca2+ influx was inhibited by a 24 h exposure to extracts of M. aeruginosa CYA 43 and 7820, but not by exposure to an equivalent amount (90 mg l-1) of purified MC-LR. Shorter exposure times (4 h) were ineffective. Fish exposed to extracts from M. aeruginosa CYA 43 showed significant plasma hypocalcaemia. Both strains of M. aeruginosa inhibited Ca2+ uptake by basolateral plasma membrane vesicles (BLMVs), endoplasmic reticulum (ER) and mitochondria, as well as BLMV K+-dependent p-nitrophenol phosphatase (pNPPase) activity. The hydrophobic fractions of the cyanobacterial extracts were the most potent, inhibiting BLMV, ER and mitochondrial Ca2+ uptake by up to 99 %, but they were less inhibitory of BLMV K+-dependent pNPPase activity. Purified MC-LR was without effect on these preparations. In conclusion, cytotoxic substances from cyanobacteria have the potential to disrupt normal physiological processes dependent upon Ca2+ transport processes in tilapia gills.

A mineralocorticoid-like receptor in the rainbow trout, Oncorhynchus mykiss: cloning and characterization of its steroid binding domain.

Using reverse transcriptase polymerase chain reaction (PCR) (RT-PCR) with degenerate primers followed by 3' rapid amplification of cDNA ends PCR (3'Race-PCR) we have isolated a new fish steroid receptor cDNA sequence of 1806 bp from rainbow trout (Oncorhynchus mykiss) testis. This sequence has clear homology with various mineralocorticoid receptor cDNA sequences (rat, human, African toad: 68-70% amino acid identity), and encompasses the second part of DNA binding domain (C domain), the whole hinge region (D domain) and the steroid binding domain (E domain) plus 726 bp of 3'untranslated sequence. COS-1 cells transfected with a pCMV5 expression vector containing the whole E domain (pCMV5-rtMR) showed high affinity binding for cortisol (K(a) = 0.53+/-0.03 nM, K(d) = 1.9 nM) in the cytosol, which could not be detected in untransfected cells. Aldosterone displaced (3)H-cortisol binding, though was less effective by than unlabeled cortisol (P<0.05). Competition experiments with other steroids gave the following hierarchy for the displacement of the (3) dexamethasone, whereas 17, 20beta-dihydroxy-4-pregnen-3-one and 17,20beta,21beta-trihydroxy-4 pregnen-3-one (two fish specific progestins) did not show any specific binding. These results strongly suggest that this cDNA sequence encodes a rainbow trout mineralocorticoid-like receptor, and represent the first description of such a receptor in teleost fish where aldosterone, the classic mineralocorticoid, is believed to be absent.

Assessment of ram sperm membrane integrity following different thawing procedures.

Semen from five 2.5-yr-old rams selected for use in an AI program was collected over 3 consecutive days using an artificial vagina. The semen was diluted with a skim milk extender containing 7% glycerol (v/v), packed in French mini-straws (approx. 100 mill/straw), and frozen in a programmable freezer. Three freezing operations were carried out per ram. Three straws per freezing operation were subjected to the following thawing procedures: 1) 70 degrees C, 5 sec; 2) 50 degrees C, 9 sec and 3) 35 degrees C, 12 sec. Post-thaw sperm motility was subjectively assessed using a phase contrast microscope; while the combined fluorochromes carboxyfluorescein diacetate and propidium iodide (CFDA/PI), the hypo-osmotic swelling test (HOS) and the presence of normal apical ridges (NAR's) were used to determine the degree of sperm membrane integrity. Significant differences between thawing treatments were found for post-thaw motility (P < .05) and membrane integrity (P < 0.01), and variation among rams was statistically significant. Post-thaw sperm motility as well as the percentage of spermatozoa showing intact membranes were significantly higher (P < 0.01) for straws thawed at 70 degrees C than for those thawed at 35 degrees C (67.0 +/- 1.1 and 63.0 +/- 1.1%, and 50.5 +/- 1.5 and 41.7 +/- 1.5%, respectively). However, no corresponding statistically significant difference could be found for these parameters when 70 degrees C and 50 degrees C thawing were compared. It was concluded that sperm can be thawed at 50 degrees C for 9 sec instead of 70 degrees C for 5 sec without further reducing sperm motility or membrane integrity. This lower thawing temperature would facilitate the widespread use of frozen/thawed ram semen under farm conditions in Sweden.

Biodynamic modelling of the bioaccumulation of trace metals (Ag, As and Zn) by an infaunal estuarine invertebrate, the clam Scrobicularia plana.

Biodynamic modelling was used to investigate the uptake and accumulation of three trace metals (Ag, As, Zn) by the deposit feeding estuarine bivalve mollusc Scrobicularia plana. Radioactive labelling techniques were used to quantify the rates of trace metal uptake (and subsequent elimination) from water and sediment diet. The uptake rate constant from solution (±SE) was greatest for Ag (3.954±0.375 l g(-1) d(-1)) followed by As (0.807±0.129 l g(-1) d(-1)) and Zn (0.103±0.016 l g(-1) d(-1)). Assimilation efficiencies from ingested sediment were 40.2±1.3% (Ag), 31.7±1.0% (Zn) and 25.3±0.9% (As). Efflux rate constants after exposure to metals in the solution or sediment fell in the range of 0.014-0.060 d(-1). By incorporating these physiological parameters into biodynamic models, our results showed that dissolved metal is the predominant source of accumulated Ag, As and Zn in S. plana, accounting for 66-99%, 50-97% and 52-98% of total accumulation of Ag, As and Zn, respectively, under different field exposure conditions. In general, model-predicted steady state concentrations of Ag, As and Zn matched well with those observed in clams collected in SW England estuaries. Our findings highlight the potential of biodynamic modelling to predict Ag, As and Zn accumulation in S. plana, taking into account specific dissolved and sediment concentrations of the metals at a particular field site, together with local water and sediment geochemistries.

Cadmium bound to metal rich granules and exoskeleton from Gammarus pulex causes increased gut lipid peroxidation in zebrafish following single dietary exposure.

There has been a growing interest in establishing how the sub-cellular distribution of metals in macro-invertebrate prey affects metal trophic bioavailability and toxicity. In this study, the crustacean Gammarus pulex was exposed to 300mugCdl(-1) spiked with (109)Cd for 13 days, from which the two principal metal containing sub-cellular fractions, the metallothionein-like protein (MTLP) and the metal rich granule and exoskeleton (MRG+exo) were isolated. These fractions were produced at equal metal content, incorporated into gelatin and fed to zebrafish as a single meal; assimilation efficiency (AE), carcass and gut tissue metal concentrations and gut lipid peroxidative damage measured as malondialdehyde (MDA) were assessed. The AE of cadmium bound to the MTLP fraction was 32.1+/-5.6% which was significantly greater than the AE of MRG+exo bound Cd, 13.0+/-2.1% (p<0.05). Of the metal retained by the fish at 72h post-feeding, 94% of MTLP-Cd had been incorporated into the carcass, whereas a significant proportion (46%) of MRG+exo-Cd, although assimilated, appeared to remain associated with intestinal tissue. However, this did not translate into a gut tissue concentration difference with 6.8+/-1.2ngCdg(-1) in fish fed MTLP-Cd compared to 9.5+/-1.4ngCdg(-1) in fish fed MRG+exo fraction. Both feeds led to significantly increased MDA levels compared to the control group (gelatin only feed), but MRG+exo feed caused significantly more oxidative damage than the MTLP feed (p<0.01). Thus, MTLP-Cd is more bioavailable than the cadmium bound to granules and exoskeleton, but it was the latter fraction, largely considered as having limited bioavailability, that appeared to exert a greater localised oxidative injury to the digestive tract of zebrafish.

Effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations.

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish (Opsanus beta).

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.

Bicarbonate secretion plays a role in chloride and water absorption of the European flounder intestine.

Experiments performed on isolated intestinal segments from the marine teleost fish, the European flounder (Platichthys flesus), revealed that the intestinal epithelium is capable of secondary active HCO3(-) secretion in the order of 0.2-0.3 micromol x cm(-2) x h(-1) against apparent electrochemical gradient. The HCO3(-) secretion occurs via anion exchange, is dependent on mucosal Cl(-), results in very high mucosal HCO3(-) concentrations, and contributes significantly to Cl(-) and fluid absorption. This present study was conducted under in vivo-like conditions, with mucosal saline resembling intestinal fluids in vivo. These conditions result in a transepithelial potential of -16.2 mV (serosal side negative), which is very different from the -2.2 mV observed under symmetrical conditions. Under these conditions, we found a significant part of the HCO3(-) secretion is fueled by endogenous epithelial CO2 hydration mediated by carbonic anhydrase because acetazolamide (10(-4) M) was found to inhibit HCO3(-) secretion and removal of serosal CO(2) was found not to influence HCO3(-) secretion. Reversal of the epithelial electrochemical gradient for Cl(-) (removal of serosal Cl(-)) and elevation of serosal HCO3(-) resulted in enhanced HCO3(-) secretion and enhanced Cl(-) and fluid absorption. Cl(-) absorption via an anion exchange system appears to partly drive fluid absorption across the intestine in the absence of net Na(+) absorption.

Mechanism of branchial apical silver uptake by rainbow trout is via the proton-coupled Na(+) channel.

The branchial uptake mechanism of the nonessential heavy metal silver from very dilute media by the gills of freshwater rainbow trout was investigated. At concentrations >36 nM AgNO(3), silver rapidly entered the gills, reaching a peak at 1 h, after which time there was a steady decline in gill silver concentration and a resulting increase in body silver accumulation. Below 36 nM AgNO(3), there was only a very gradual increase in gill and body silver concentration over the 48-h exposure period. Increasing water sodium concentration ([Na(+)]; 0.05 to 21 mM) significantly reduced silver uptake, although, in contrast, increasing ambient [Ca(2+)] or [K(+)] up to 10 mM did not reduce silver uptake. Kinetic analysis of silver uptake at varying [Na(+)] showed a significant decrease in maximal silver transport capacity (173 +/- 34 pmol. g(-1). h(-1) at 0.1 mM [Na(+)] compared with 35 +/- 9 at 13 mM [Na(+)]) and only a slight decrease in the affinity for silver transport (K(m); 55 +/- 27 nM at 0.1 mM [Na(+)] compared with 91 +/- 47 nM at 13 mM [Na(+)]). Phenamil (a specific blocker of Na(+) channels), at a concentration of 100 microM, blocked Na(+) uptake by 78% of control values (58% after washout), and bafilomycin A(1) (a specific blocker of V-type ATPase), at a concentration of 2 microM, inhibited Na(+) uptake by 57% of control values, demonstrating the presence of a proton-coupled Na(+) channel in the apical membrane of the gills. Phenamil (after washout) and bafilomycin A(1) also blocked silver uptake by 62 and 79% of control values, respectively, indicating that Ag(+) is able to enter the apical membrane via the proton-coupled Na(+) channel.

Derivation of a toxicity-based model to predict how water chemistry influences silver toxicity to invertebrates.

The effect of altering water chemistry on acute silver toxicity to three invertebrate species, two Daphnids, Daphnia magna and Daphnia pulex, as well as an amphipod Gammarus pulex was assessed. In addition, the physiological basis of Ag(I) toxicity to G. pulex was examined. Daphnia magna and D. pulex were more sensitive than G. pulex and 48 h LC(50) values in synthetic ion poor water were 0.47, 0.65 and 2.1 microg Ag(I) l(-1), respectively. Increasing water [Cl(-)] reduced Ag(I) toxicity in all species, and increasing water [Ca(2+)] from 50 to 1,500 microM reduced Ag(I) toxicity in G. pulex. Whole body Na(+) content, but not K(+) or Ca(2+) was significantly reduced in G. pulex exposed to 6 microg Ag(I) l(-1) for 24 h, but there was no inhibition of whole body Na(+)/K(+)-ATPase activity. Both increasing water [Cl(-)] and [Ca(2+)] reduced this Ag(I)-induced Na(+) loss. For D. magna, the presence of 10 mg l(-1) humic acid or 0.5 microM 3-mercaptoproprionic acid (3-MPA) increased the 48 h LC(50) values by 5.9 and 58.5-fold, respectively, and for D. pulex the presence of 1 microM thiosulfate increased the 48 h LC(50) value by four-fold. The D. magna toxicity data generated from this study were used to derive a Daphnia biotic ligand model (BLM). Analysis of the measured LC(50) values vs. the predicted LC(50) values for toxicity data from the present and published results where water Cl(-), Ca(2+), Na(+) or humic acid were varied showed that 91% of the measured toxicity data fell within a factor of two of the predicted LC(50) values. However, the daphnid BLM could not accurately predict G. pulex toxicity. Additionally, the Daphnia BLM was under-protective in the presence of the organic thiols 3-MPA or thiosulphate and predicted an increase in the LC(50) value of 114- and 74-fold, respectively. The Daphnia toxicity based BLM derived from the present data set is successful in predicting Daphnia toxicity in laboratory data sets in the absence of sulfur containing compounds, but shows its limitations when applied to waters containing organic thiols or thiosulphate.