Acclimation of Daphnia magna to environmentally realistic copper concentrations

It may be hypothesised that as the bioavailable background concentration of an essential metal increases (within natural limits), the natural tolerance (to the metal) of the acclimated/adapted organisms and communities will increase. In this study the influence of acclimation to different copper concentrations on the sensitivity of the freshwater cladoceran Daphnia magna Straus was investigated. D. magna was acclimated over three generations to environmentally relevant copper concentrations ranging from 0.5 to 100 microg Cu/l (copper activity: 7.18 x 10(-15) to 3700 x 10(-12) M Cu2+). A modified standard test medium was used as culture and test medium. Medium modifications were: reduced hardness (lowered to 180 mg CaCO3/l) and addition of Aldrich humic acid at a concentration of 5 mg DOC/l (instead of EDTA). The effects of acclimation on these organisms were monitored using acute mortality assays and long-term assays in which life table parameters, copper body concentrations and energy reserves were used as test endpoints. Our results showed a two-fold increase in acute copper tolerance with increasing acclimation concentration for second and third generation organisms. Copper acclimation concentrations up to 35 microg Cu/l (80 pM Cu2+) did not affect the net reproduction and the intrinsic growth rate. The energy reserves of the acclimated daphnids revealed an Optimal Concentration range (OCEE) and concentrations between 5 and 12 microg Cu/l (0.5-4.1 pM Cu2+) and 1 and 35 microg Cu/l (0.023-80 pM Cu2+) seemed to be optimal for first and third generation daphnids, respectively. Lower and higher copper concentrations resulted in deficiency and toxicity responses. It was also demonstrated that up to 35 microg Cu/l, third generation daphnids were able to regulate their total copper body concentration. These results clearly indicate that bioavailable background copper concentrations present in culture media have to be considered in the evaluation of toxicity test results, especially when the toxicity data are used for water quality guideline derivation and/or ecological risk assessment for metals.     

A novel piezoelectric immunoagglutination assay technique with antibody-modified liposome

A simple rapid piezoelectric immunoagglutination assay (PEIA) technique with antibody-modified liposome has been developed for direct quantitative detection of human immunoglobulin G (hIgG). This technique is based on specific agglutination of antibody-coated liposome particles in the presence of the corresponding antigen, which can be monitored by the frequency shift of a piezoelectric device. Compared with conventional piezoelectric assays, this liposome-based PEIA does not require the immobilization of antigen or antibody on the quartz crystal surface, making the developed technique especially useful for rapid and renewable immunochemical determination. To alleviate non-specific adsorption of serum proteins, modification of the quartz crystal surface by different protocols and the composition of the assay medium have been investigated. The results indicate that the background interference can be substantially minimized through modifying the quartz crystal surface with a bovine serum albumin (BSA) layer and introducing an appropriate amount of BSA in the assay medium. The effects of the liposome composition, the liposome concentration and the concentration of poly(ethylene glycol) (PEG) in the assay medium, have also been investigated. The frequency responses of the liposome-based PEIA are linearly correlated to hIgG concentration in the range of 0.05-6 microg mL(-1) with a detection limit of 50 ng mL(-1).     

Effects of Photochemical Transformations of Dissolved Organic Matter on Bacterial Metabolism and Diversity in Three Contrasting Coastal Sites in the Northwestern Mediterranean Sea during Summer

The effects of phototransformation of dissolved organic matter (DOM) on bacterial growth, production, respiration, growth efficiency, and diversity were investigated during summer in two lagoons and one oligotrophic coastal water samples from the Northwestern Mediterranean Sea, differing widely in DOM and chromophoric DOM concentrations. Exposure of 0.2-μm filtered waters to full sun radiation for 1 d resulted in small changes in optical properties and concentrations of DOM, and no changes in nitrate, nitrite, and phosphate concentrations. After exposure to sunlight or dark (control) treatments, the water samples were inoculated with the original bacterial community. Phototransformation of DOM had contrasting effects on bacterial production and respiration, depending on the water’s origin, resulting in an increase of bacterial growth efficiency for the oligotrophic coastal water sample (120%) and a decrease for the lagoon waters (20 to 40%) relative to that observed in dark treatments. We also observed that bacterial growth on DOM irradiated by full sun resulted in changes in community structure of total and metabolically active bacterial cells for the three locations studied when compared to the bacteria growing on un-irradiated DOM, and that changes were mainly caused by phototransformation of DOM by UV radiation for the eutrophic lagoon and the oligotrophic coastal water and by photosynthetically active radiation (PAR) for the mesoeutrophic lagoon. These initial results indicate that phototransformation of DOM significantly alters both bacterial metabolism and community structure in surface water for a variety of coastal ecosystems in the Mediterranean Sea. Further studies will be necessary to elucidate a more detailed appreciation of potential temporal and spatial variations of the effects measured.     

The effect of copper on osmoregulation in the freshwater amphipod Gammarus pulex

The influence of copper on osmoregulation in the freshwater amphipod Gammarus pulex was determined from the analysis of water permeability, haemolymph sodium concentration, sodium influx and gill Na(+)/K(+) ATPase and Mg(2+) ATPase activity. Exposure to nominal copper concentrations of 100 microg l(-1) or greater caused a significant reduction in both haemolymph sodium concentration and sodium influx within 4 h. Measurements of water permeability, expressed as the half-time of exchange of body water (t(1/2)), excluded structural gill damage as the cause of this fall in haemolymph sodium. Copper at 10 microg l(-1) or above in the assay solution significantly reduced gill Na(+)/K(+) ATPase activity. In contrast gill Mg(2+) ATPase activity was markedly less affected by copper. These differences in enzyme sensitivity were considered with respect to the potential mechanisms of copper toxicity.     

Growth, lipid class and fatty acid composition in juvenile mud crabs (Rhithropanopeus harrisii ) following larval exposure to Fenoxycarb, insect juvenile hormone analog

This study examines the effects of Fenoxycarb on larval growth, and lipid class and fatty acid composition in first crabs of the mud crab Rhithropanopeus harrisii reared through total larval development in nominal water concentrations from 1 to 100 microg/l. In first crabs of R. harrisii, dry weight (microg) decreased significantly (P < 0.05) from 228.8+/-38.2 microg (n = 9) in the controls to 131.8+/-10.1 microg (n = 4) in animals exposed throughout larval development to 100 microg/l. A significant (P < 0.05) reduction was found between total lipid content in the controls and first crabs reared at concentrations greater than 50 microg/l. In relative terms (% dry weight), different lipid classes predominated in the controls and the various fenoxycarb exposure concentrations. There were no significant (P > 0.05) differences among the treatment groups in phospholipid level, while the triglyceride content was significantly lower in crabs exposed to 10 and 100 microg/l. No significant differences in the percent of free fatty acids were found in crabs exposed to 1-10 microg/l and the controls. Free sterols in crabs exposed to concentrations higher than 10 microg/l were below the detection limit. Control animal fatty acid profiles were dominated by palmitic, stearic, and oleic acid, accounting for 48% of total fatty acids (TFA). The fatty acid composition of crabs exposed to 100 microg/l significantly (P < 0.05) differed from the controls. The results suggest that fenoxycarb has substantial effects on growth, lipid class and fatty acid composition in developing larvae of R. harrisii at water concentrations greater than 10 microg/l.     

The susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae and its resistance under copper sulfate stress.

Addition of copper sulfate (0.1 to 0.4 mg l(-1)) to tryptic soy broth (TSB) had no effect on growth rate of the bacterial pathogen Lactococcus garvieae. Giant freshwater prawns Macrobrachium rosenbergii were injected with L. garvieae (4 x 10(6) colony-forming units [cfu] prawn(-1)) grown in TSB or TSB containing copper sulfate at 0.1, 0.2, 0.3 or 0.4 mg l(-1). After 48 h, the cumulative mortality was significantly (p < 0.05) higher for prawns exposed to L. garvieae grown in 0.4 mg l(-1) copper sulfate than at the lower concentrations examined. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 2 x 10(5) cfu prawn(-1)), then held in water containing copper sulfate. After 8 h the mortality of L. garvieae-exposed prawns held in water containing 0.4 mg l(-1) copper sulfate was significantly higher than prawns held in water containing 0.2 and 0.3 mg l(-1) copper sulfate. At the lower L. garvieae density, cumulative mortality of prawns increased directly with ambient copper sulfate concentrations in the range of 0.2 to 0.4 mg l(-1). All prawns survived a 168 h exposure to 0.1 mg l(-1) copper sulfate. Prawns exposed to different concentrations of copper sulfate were examined for hemocyte density, phenoloxidase activity and respiratory burst. No significant differences in hemocyte density were observed among treatments. In prawns following a 48 h exposure to 0.1 mg l(-1) copper sulfate, phenoloxidase activity was decreased, but respiratory burst was increased. In conclusion, copper sulfate increased the virulence of L. garvieae to M. rosenbergii and modulated its immune system. Copper sulfate at 0.1 mg l(-1) decreased susceptibility of M. rosenbergii to L garvieae infection, whereas at 0.2 mg l(-1) the susceptibility was increased. The generation of superoxide anion by M. rosenbergii exposed to copper sulfate at a concentration higher than 0.2 mg l(-1) was considered to be cytoxic.     

Cooper, cadmium and lead in the fish Cyprinodon dearborni, in the sediments and in the water of two lagoons in Venezuela

The concentration of copper, cadmium and lead in superficial sediment, water and the fish Cyprinodon dearborni was determined in two coastal lagoons of Sucre State, Venezuela. Chacopata lagoon is hyper saline while Los Patos lagoon is hypo saline and receives significant wastewater from Cumaná city. Water, sediment and fish samples were collected in February 1998. In the laboratory, samples underwent acid digestion and were analyzed by atomic absorption spectophotometry. The mean values of the metals in C. dearborni from the Chacopata lagoon were: 159.26 +/- 210.68 microg/g for Cu, 44.71 +/- 45.58 microg/g for Cd, and 9.31 +/- 23.34 microg/g for Pb, while for Los Patos lagoon the mean values were: 64.88 +/- 16.30, 19.48 +/- 5.81 and 22.85 +/- 20.00, respectively. In the water column, the metal concentration ranges were: 2.3-11.6, 3.9-5.4 and 21-32 mg/l for copper, cadmium and lead, respectively. These results suggest that metal levels in sediment, water column and organisms in both lagoons do not differ, except for lead, even though only Los Patos receives waste water.     

An amplified mass piezoelectric immunosensor for Schistosoma japonicum

An ultrasensitive piezoelectric immunosensor using an amplification path based on an insoluble biocatalyzed precipitation product has proposed for Schistosoma japonicum. A mercapto Schistosoma japonicum antigen was self-assembled onto the quartz crystal surface via an Au nanoparticle mediator monolayer to sense the Schistosoma japonicum antibody (SjAb). And the horseradish peroxidase labeled protein A conjugate which was bounded to the SjAb by a "sandwich" format was used as a biocatalyst for the oxidative precipitation of 4-chloro-1-naphthol by H(2)O(2) to yield the insoluble product benzo-4-chlorohexadienone, resulting in an amplified mass sensing of antigen-antibody interaction. The amount of the precipitate accumulated on the quartz crystal is controlled by the antibody concentration. The SjAb can be linearly determined in the range of 10-200 ngml(-1) and the detection limit reaches as low as 5 ngml(-1).     

Operational characteristics of an antibody-immobilized QCM system detecting Salmonella spp

A quartz crystal microbalance (QCM) system detecting Salmonella spp. was developed by an anti-Salmonella antibody immobilization onto one gold surface of a piezoelectric quartz crystal surface with sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP) thiolation. The optimum temperature and pH for the antibody-immobilized sensor were 35 degrees C and 7.2, respectively. The frequency shifts obtained were correlated with the Salmonella concentrations in the range 3.2 x 10(6)-4.8 x 10(8) CFU per ml. The system was quite specific to Salmonella spp. and applicable for repetitive use after a regeneration step employing 1.2 M NaOH. A model sample measurement was done for a market milk spiked with Salmonella typhimurium.     

Series quartz crystal sensor for remote bacteria population monitoring in raw milk via the Internet

A remote monitoring system based on a piezoelectric quartz crystal (SPQC) sensor was developed for the determination of the bacteria population in raw milk. The system employs the Windows XP server operating system, and its programs for data acquisition, display and transmission were developed using the LabVIEW 7.1 programming language. The circuit design consists of a circuit with a piezoelectric quartz crystal (SPQC) and a pair of electrodes. This system can provide dynamic data monitoring on a web-page via the Internet. Immersion of the electrodes in a cell culture with bacteria inoculums resulted in a change of frequency caused by the impedance change due to microbial metabolism and the adherence of bacteria on the surface of the electrodes. The calibration curve of detection times against density of bacteria showed a linear correlation coefficient (R(2) = 0.9165) over the range of 70-10(6) CFU ml(-1). The sensor could acquire sufficient data rapidly (within 4 h) and thus enabled real-time monitoring of bacteria growth via the Internet. This system has potential application in the detection of bacteria concentration of milk at dairy farms.     

Effect of sub-lethal concentrations of aluminium on the filtration activity of the freshwater mussel Anodonta cygnea L. at neutral pH

Significant amounts of aluminium (Al) are commonly present in rivers and lakes, largely in particulate form in neutral waters. Freshwater bivalves, as filter feeders are therefore exposed to both particulate and dissolved metal and are potentially vulnerable to Al. The effect of Al on filtering behaviour of the freshwater mussel Anodonta cygnea L. was investigated during short (1 hour) and long-term (15 days) exposure to environmentally relevant concentrations (250 and 500 microg l(-1)) at neutral pH. Water flow through the outflow siphon was monitored as an indicator of pumping capacity. Short-term (1 hour) exposure to 500 microg l(-1) added Al produced an irreversible decrease in the duration of filtering periods, presumably as an avoidance response to the toxicant. One-hour exposure 250 microg l(-1) Al had no detectable effect. When mussels were exposed to 250 or 500 microg l(-1) added Al for 15 days, siphon activity measured in days 11-15 of exposure was inhibited by 50% and 65%, respectively, compared to pre-exposure levels. Recovery occurred following transfer of mussels to uncontaminated water. Interaction between Al and freshwater bivalves at neutral pH may affect both the performance of the mussels and the chemical speciation of the metal in the natural environment.     

Low filtering rates of <Emphasis Type="Italic">Daphnia magna</Emphasis> in a hypertrophic lake: laboratory and in situ experiments using synthetic microspheres

Zooplankton grazing was measured in Albufera of València (Spain), a shallow turbid hypertrophic lagoon dominated by filamentous bluegreens, during the period of Daphnia magna growth, to evaluate the role of this cladoceran in maintaining a clear water phase which takes place after flushing for rice cultivation practices. We found extremely low ingestion and clearance rates (CR) on latex beads in situ, using a Haney trap suggesting potentially strong inhibition of grazing by the filamentous cyanobacteria, still flourishing in the lagoon. To test the effect of filaments, we undertook laboratory feeding experiments using six different dilutions of the lagoon water to vary filament concentrations, and four different bead concentrations. A highly significant power function between CR and water dilution associated with filament concentrations was found, indicating that the ability of Daphnia to exploit smaller edible algae and thereby control phytoplankton growth would depend on filament concentration levels in the lagoon. From our results only the two more diluted treatments, 9 × 10³ and 18 × 10³ filament ml⁻¹ showed CRs not far from the range of what would be normally expected on the basis of the general relationship of feeding rate as a function of total food concentration. This means that at lower food concentrations, filaments reduce D. magna CR by increasing total food concentration beyond the incipient limiting level, as well as by moderate mechanical interference with the animal’s feeding. However, at lagoon water concentrations above 25% (corresponding to filament concentrations of 75 × 10⁵ filaments ml⁻¹), extremely low CR’s, which did not respond to any food addition, were obtained for D. magna. From these results we can infer that at concentrations beyond the above-mentioned critical level, feeding inhibition by filamentous cyanobacteria is most probably due entirely to mechanical interference. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

Detection and analysis of Bacillus subtilis growth with piezoelectric quartz crystal impedance based on starch hydrolysis

A piezoelectric quartz crystal (PQC) impedance method based on the alpha-amylase-catalyzed hydrolysis of starch present in a culture medium has been developed for in situ monitoring of the whole growth process of Bacillus subtilis and the variation in the activity of alpha-amylase during bacterial growth. An S-shaped response behavior was observed for Deltaf(0), and simultaneously inverse S-shaped responses were found for DeltaR(1) and DeltaL(1). The ratio of DeltaR(1) to Deltaf(0) or DeltaL(1) coincided well with that calculated from Martin's equations reflecting the solution density-viscosity effect, suggesting that the continuing change in liquid loading onto the PQC surface causes significant variation in Deltaf(0), DeltaR(1), and DeltaL(1). Bacterial growth equations were derived from the kinetics of the enzyme-catalyzed hydrolysis of starch, which fit well with the experimental responses of Deltaf(0), DeltaR(1), and DeltaL(1). Kinetic parameters of bacterial growth, including the asymptote (A), the maximum specific growth rate (microm), and the lag time (lambda), were obtained and were in good agreement with those obtained from the pour plate count method. The variation in the activity of alpha-amylase exhibited peak-type behavior with its maximum value at the later stage of the log phase. In addition, the influence of initial bacterial concentration was also investigated.     

Biomass, production and heterotrophic activity of bacterioplankton in the Great Astrolabe Reef lagoon (Fiji)

 Biomass, production and heterotrophic activity of bacterioplankton were determined for two weeks in the Great Astrolabe Reef lagoon, Fiji. Bacterial and Bacterial activities were distributed homogeneously throughout the water column (20 to 40 m deep) and varied little from site to site inside the lagoon. Bacterioplankton biomass and production also varied little over a diel period with coefficients of variation of 9 and 22%, respectively. On average, over the whole study, bacterial abundance was 0.77×10⁹ cells l^-1 and bacterial production averaged 0.36 μg-at. C l^-1 d^-1. Bacterial abundance and production were greater in the lagoon than in oceanic waters. Attachment to particles seems to provide an advantage for bacterioplankton growth because specific growth rates for attached bacterioplankton were, on average, significantly greater than that of the free community. Growth efficiency, determined by correlating the net increase of bacterial biomass and the net decrease of dissolved organic carbon (DOC) in dilution cultures, was very low (average 6.6%). Using carbon growth efficiency and bacterial production rates, heterotrophic activity was estimated to average 5.4 μg-at. C l^-1 d^-1. The turn-over rate of DOC (average 114 μg-at. C l^-1) due to bacterial consumption was estimated to be 0.048 d^-1 during the period of study. Accepted: 25 July 1998     

Effect of long-term silver exposure on survival and ionoregulatory development in rainbow trout (Oncorhynchus mykiss) embryos and larvae,in the presence and absence of added dissolved organic matter

Developing rainbow trout were chronically exposed to silver (as AgNO(3)) from fertilization to swim-up, in moderately hard water (120 mg CaCO(3)(x)l(-1)) in the presence and absence of an additional 12 mg C/L of dissolved organic carbon (DOC, as humic acid, Aldrich). Nominal silver concentrations were 0, 0.1 and 10 microg l(-1) total silver in a flow-through set-up maintained at 12 degrees C. The objectives of the study were to investigate the possible protective effects of DOC on growth, mortality, time to hatch and swim-up, and sublethal ionoregulatory disturbances during chronic exposure to ionic silver. Throughout development, there was a large increase in % daily mortality at 10 microg(x)l(-1) total silver (in the absence of DOC), that was associated with an ionoregulatory disturbance, in particular a 35% reduction in whole body Na(+) just prior to hatch. At nominal 10 microg(x)l(-1) total silver, the presence of additional DOC (reducing dissolved silver to 4.7+/-0.3 microg l(-1)) resulted in a significant reduction in % daily mortality up to hatch, demonstrating a protective effect of DOC. Interestingly, DOC did not appear to mitigate the ionoregulatory disturbance, with the exception of whole body [Cl(-)] on day 44 of exposure. Exposure to 0.1 microg(x)l(-1) total silver (in the absence of DOC) resulted in a statistically significant reduction in growth, and DOC did not prevent an ionoregulatory disturbance [based upon (J(in) Na(+)), whole body Na(+),K(+) ATPase activity and whole body (Na(+))] at this silver concentration relative to controls+DOC. DOC exerted a direct effect on growth and ionoregulatory development that complicates interpretation of the data, however, these data indicate that protective effects of DOC (in the form of Aldrich humic acid) during chronic silver exposure appear to be less than that observed during acute exposure. The ultimate goal of this and future studies is to develop a model that can predict chronic toxicity on a site-specific basis, taking into account protective effects of various ligands present in different waters, as is presently being employed for some metals during acute exposure.     

Capacity of Lemna gibba L. (duckweed) for uranium and arsenic phytoremediation in mine tailing waters

The potential of Lemna gibba L. to clean uranium and arsenic contamination from mine surface waters was investigated in wetlands of two former uranium mines in eastern Germany and in laboratory hydroponic culture. Water and plants were sampled and L gibba growth and yield were monitored in tailing ponds from the field study sites. Contaminant accumulation, growth and yield experiments were conducted in the laboratory using synthetic tailing water. Mean background concentrations of the surface waters were 186.0+/-81.2 microg l(-1) uranium and 47.0+/-21.3 microg l(-1) arsenic in Site one and 293.7+/-121.3 microg l(-1) uranium and 41.37+/-24.7 microg l(-1) arsenic in Site two. The initial concentration of both uranium and arsenic in the culture solutions was 100 microg l(-1). The plant samples were either not leached, leached with deionized H2O or ethylenediaminetetracetic (EDTA). The results revealed high bioaccumulation coefficients for both uranium and arsenic. Uranium and arsenic content of L gibba dry biomass of the field samples were as follows: nonleached samples > deionized H2O leached (insignificant ANOVA p = 0.05) > EDTA leached. The difference in both arsenic and uranium enrichment were significantly high between the nonleached and the other two lead samples tested at ANOVA p > 0.001. Estimated mean L gibba density in surface water was 85,344.8+/-1843.4 fronds m(-2) (approximately 1319.7 g m(-2)). The maximum specific growth rate was 0.47+/-0.2 d(-1), which exceeded reported specific growth rates for L gibba in the literature. Average yield was estimated at 20.2+/-6.7 g m(-2) d(-1), giving approximately 73.6+/-21.4 t ha(-1) y(-1) as the annual yield. The highest accumulations observed were 896.9+/-203.8 mg kg(-1) uranium and 1021.7+/-250.8 mg kg(-1) arsenic dry biomass for a 21-d test period in the laboratory steady-state experiments. The potential extractions from surface waters with L gibba L. were estimated to be 662.7 kg uranium ha(-1) yr(-1) and 751.9 kg arsenic ha(-1) yr(-1) under the above conditions.     

Monitoring and kinetic parameter estimation for the binding process of berberine hydrochloride to bovine serum albumin with piezoelectric quartz crystal impedance analysis

A new method for monitoring, in real time, the drug-binding process to protein with piezoelectric quartz crystal impedance (PQCI) is proposed. The method was used to monitor the binding process of berberine hydrochloride to bovine serum albumin (BSA). BSA was immobilized on the silver electrode surface of a piezoelectric quartz crystal and the optimized experimental conditions were established. The BSA-coated piezoelectric sensor was in contact with berberine solution. The time courses of the resonant frequency and equivalent circuit parameters of the sensor during the protein-drug binding were simultaneously obtained. On the basis of the analysis of the multidimensional information provided by PQCI, it was concluded that the observed frequency decrease was mainly ascribed to the mass increase of the sensor surface resulting from the binding. According to the frequency decrease with time, the kinetics of the binding process were quantitatively studied. A piezoelectric response model for the binding was theoretically derived. Fitting the experimental data to the model, the kinetic parameters, such as the binding and dissociation rate constants (k(1) and k(-1)) and the binding equilibrium constant (K(a)), were determined. The k(1), k(-1), and K(a) values obtained at 25 degrees C were 67.5 (+/-0.1) (mol liter(-1))(-1) s(-1), 1.7 (+/- 0.1) x 10(-3) s(-1), and 3.97 (+/- 0.06) x10(4) (mol liter(-1))(-1), respectively.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.     

Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media with ampicillin. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day(-1)) and yield (60 microg chlorophyll/ml culture) than in pure cultures (0.4 day(-1) and 10 microg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.     

Quantification of tetracycline resistance genes in feedlot lagoons by real-time PCR

A new real-time PCR method is presented that detects and quantifies three tetracycline resistance (Tcr) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tcr gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tcr genes present in seven different cattle feedlot lagoons, to validate the method, and to assess whether resistance gene concentrations correlate with free-tetracycline levels in lagoon waters. The method proved to be sensitive across a wide range of gene concentrations and provided consistent and reproducible results from complex lagoon water samples. The log10 of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r2 = 0.50, P < 0.001; n = 18), with the geometric means of individual resistance concentrations ranging from 4- to 8.3-fold greater in lagoon samples with above-median tetracycline levels (>1.95 microg/liter by enzyme-linked immunosorbent assay techniques) than in below-median lagoon samples. Of the three Tcr genes tested, tet(W) and tet(Q) were more commonly found in lagoon water samples. Successful development of this real-time PCR assay will permit other studies quantifying Tcr gene numbers in environmental and other samples.