Phytoprotective influence of bacteria on growth and cadmium accumulation in the aquatic plant Lemna minor

Certain plants are known to accumulate heavy metals, and can be used in remediation of polluted soil or water. Plant-associated bacteria, especially those that are metal tolerant, may enhance the total amount of metal accumulated by the plant, but this process is still unclear. In this study, we investigated metal enhancement vs. exclusion by plants, and the phytoprotective role plant-associated bacteria might provide to plants exposed to heavy metal. We isolated cadmium-tolerant bacteria from the roots of the aquatic plant Lemna minor grown in heavy metal-polluted waters, and tested these isolates for tolerance to cadmium. The efficiency of plants to accumulate heavy metal from their surrounding environment was then tested by comparing L. minor plants grown with added metal tolerant bacteria to plants grown axenically to determine, whether bacteria associated with these plants increase metal accumulation in the plant.Unexpectedly, cadmium tolerance was not seen in all bacterial isolates that had been exposed to cadmium. Axenic plants accumulated slightly more cadmium than plants inoculated with bacterial isolates. Certain isolates promoted root growth, but overall, addition of bacterial strains did not enhance plant cadmium uptake, and in some cases, inhibited cadmium accumulation by plants. This suggests that bacteria serve a phytoprotective role in their relationship with Lemna minor, preventing toxic cadmium from entering plants.     

Haematological status of wintering great tits (Parus major) along a metal pollution gradient

In the long-term biomonitoring of wild populations inhabiting polluted areas, the use of non-destructive biomarkers as markers of condition is very important. We examined the possible effects of metal pollution on the haematological status of adult great tits (Parus major) along a well-established pollution gradient near a non-ferrous smelter in Belgium. We measured blood and feather metal concentrations and assessed the haematological status (amount of red blood cells, haemoglobin concentration, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin) of adult great tits during winter at four study sites. Metal concentrations in blood and feathers indicated that cadmium and lead were the most important metals in the pollution gradient under study. Measurements of haematological parameters revealed that haemoglobin concentration, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin were lower in great tits from the more polluted sites. These parameters were significantly negatively correlated with blood lead concentration. The amount of red blood cells, however, did not significantly differ among study sites. Our results indicate that the haematological status of great tits is negatively affected by metal pollution and may therefore be used as a successful biomarker for monitoring the negative impact of metal exposure in the wild.     

Application of temporal temperature gradient gel electrophoresis for characterisation of fungal endophyte communities of Salix caprea L. in a heavy metal polluted soil

Fungal endophytes can affect the heavy metal uptake of their host plants and increase the tolerance of their host plants to heavy metal stress. Therefore, in the present study, a wide-range screening of the fungal endophyte communities was conducted to determine the fungal distribution and diversity on S. caprea roots on a metal polluted site. Fungal communities were screened using amplification with the 5.8S-ITS2-28S part of the rDNA operon, with the resulting amplicons analysed by temporal temperature gradient gel electrophoresis (TTGE) and sequencing. This technique is reproducible and shows good coverage of ascomycete and basidiomycete taxa, as 68% and 32% of all of the sequences, respectively. No clear shift in fungal ITS-TTGE profiles from S. caprea roots was seen along the secondary succession stages. Ascomycetes dominated the more polluted plots, while there was a greater diversity of basidiomycetes in the less polluted and control plots, suggesting greater tolerance of ascomycetes in comparison with basidiomycete fungi. The high diversity of DSEs was confirmed at the highly metal-enriched locations, with species belonging to the genera Phialophora, Phialocephala and Leptodontidium. Furthermore, the DSE colonisation of S. caprea roots and the frequency of the sequences showing affinity towards DSE genus Phialophora, showed good correspondence with soil Pb, Cd and plant-available P concentrations, possibly indicating that DSEs improve metal tolerance of willows to high heavy metal contamination.     

The effects of Aphis fabae infestation on the antioxidant response and heavy metal content in field grown Philadelphus coronarius plants

The purpose of this study was to explore a possible relationship between the soil availability of metals and their concentrations in various parts of Philadelphuscoronarius plants. Moreover, the possible impact of an aphid infestation on the contamination and antioxidant response of plants from the urban environment of Kraków and the reference rural area of Zagaje Stradowskie (southern Poland) was analyzed. The contents of the glutathione, proline, non-protein − SH groups, antioxidants, and phosphorous and the levels of guaiacol peroxidase and catalase activity in leaves and shoots either infested or not by the aphid Aphis fabae Scop., were measured. The potential bioavailability of metals (Cd; Cu; Ni; Pb; Zn) in the soil and their concentrations in P. coronarius plants originating from both sites were compared.The antioxidant responses were generally elevated in the plants in the polluted area. Such reactions were additionally changed by aphid infestation. Generally, the concentrations of metals in the HNO₃ and CaCl₂ extractants of the soils from two layers at the 0-20 and 20-40 cm depths from the polluted area were higher than in those from the reference area. Such differences were found for nickel and lead (in all examined extractants), zinc (in soil extractants from the layer at 20-40 cm) and cadmium (in HNO₃ extractants). Significant positive relationships between the lead concentrations in the soil and in the plants were found. In the parts of plants from the polluted area, higher concentrations of Pb and Zn (leaves and shoots) and Cd (shoots) were recorded. The shoots and leaves of plants infested with aphids had higher concentrations of Zn but lower Pb. Moreover, their leaves had higher contaminations of Cu and Ni. In conclusion, aphids affected not only the antioxidant response of the plants but also their contamination with metals, especially contamination of the leaves.     

Rhizosphere characteristics of two arsenic hyperaccumulating Pteris ferns

Better understanding of the processes controlling arsenic bioavailability in the rhizosphere is important to enhance plant arsenic accumulation by hyperaccumulators. This greenhouse experiment was conducted to evaluate the chemical characteristics of the rhizosphere of two arsenic hyperaccumulators Pterisvittata and Pterisbiaurita. They were grown for 8 weeks in rhizopots containing arsenic-contaminated soils (153 and 266 mg kg^− 1 arsenic). Bulk and rhizosphere soil samples were analyzed for water-soluble As (WS-As) and P (WS-P), pH, and dissolved organic carbon (DOC). Comparing the two plants, P.vittata was more tolerant to arsenic and more efficient in arsenic accumulation than P.biaurita, with the highest frond arsenic being 3222 and 2397 mg kg^− 1. Arsenic-induced root exudates reduced soil pH (by 0.74-0.92 units) and increased DOC concentrations (2-3 times) in the rhizosphere, resulting in higher WS-P (2.6-3.8 times higher) compared to the bulk soil. Where there was no difference in WS-As between the rhizosphere and bulk soil in soil-153 for both plants, WS-As in the rhizosphere was 20-40% higher than those in bulk soil in soil-266, indicating that the rate of As-solubilization was more rapid than that of plant uptake. The ability to solubilize arsenic via root exudation in the rhizosphere and the ability to accumulate more P under arsenic stress may have contributed to the efficiency of hyperaccumulator plants in arsenic accumulation.     

Metal specific partitioning in a parasite-host assemblage of the cestode Ligula intestinalis and the cyprinid fish Rastrineobola argentea

When evaluating metal accumulation patterns in parasite-host assemblages species specific metal requirements should be taken into account. The aim of the present study was therefore to determine the metal specific partitioning in a parasite-host assemblage of the cestode Ligula intestinalis and the cyprinid fish Rastrineobola argentea and to determine the effect of the parasites on the metal balance of the fish. To this purpose the host-parasite assemblage was analysed for several metals at sites in the coastal zone of Lake Victoria differing in metal contamination. Our results showed that some elements (Ca, Sr, and Mg) reflected the physiological differences of bone formation and ionic balance and pointed to physiological disturbances of infested R. argentea. Other essential metals including Cu and Co were subject of element competition between fish and parasite, while only a micro-element (Cr) and a non-essential metal (Cd) displayed a partitioning with high concentration in the parasite. The present study clearly demonstrated the impact of the large cestodes on their small fish hosts and it is concluded that the partitioning of metals in the assemblage of R. argentea and L. intestinalis is subject to metal specific mechanisms for essential and non-essential elements.     

Metal/metalloid accumulation/remobilization during aquatic litter decomposition in freshwater: a review

The focus of this article is to combine two main areas of research activities in freshwater ecosystems: the effect of inorganic pollutants on freshwater ecosystems and litter decomposition as a fundamental ecological process in streams.The decomposition of plant litter in aquatic systems as a main energy source in running water ecosystems proceeds in three distinct temporal stages of leaching, conditioning and fragmentation. During these stages metals and metalloids may be fixed by litter, its decay products and the associated organisms. The global-scale problem of contaminated freshwater ecosystems by metals and metalloids has led to many investigations on the acute and chronic toxicity of these elements to plants and animals as well as the impact on animal activity under laboratory conditions. Where sorption properties and accumulation/remobilization potential of metals in sediments and attached microorganisms are quite well understood, the combination of both research areas concerning the impact of higher trophic levels on the modification of sediment sorption conditions and the influence of metal/metalloid pollution on decomposition of plant litter mediated by decomposer community, as well as the effect of high metal load during litter decay on organism health under field conditions, has still to be elucidated. So far it was found that microbes and invertebrate shredder (species of the genera Gammarus and Asellus) have a significant influence on metal fixation on litter. Not many studies focus on the impact of other functional groups affecting litter decay (e.g. grazer and collectors) or other main processes in freshwater ecosystems like bioturbation (e.g. Tubifex, Chironomus) on metal fixation/release.     

Does competition for phosphate supply explain the invasion pattern of Elodea species?

Two invasive aquatic plants, Elodea canadensis and Elodea nuttallii, occurred in north-eastern France. In this study, we examine the influence of phosphorus availability in soft water streams to explain the invasion pattern of exotic species (E. nuttallii and E. canadensis) compared to native plants (Callitriche platycarpa, Ranunculus peltatus). Total phosphorus was measured in these four aquatic macrophytes. Sediment total phosphorus and water-soluble reactive phosphorus were also analysed each season in 2001. Phosphorus content in the two invasive species and in R. peltatus was higher than in C. platycarpa. Elodea species are adapted to the seasonal phosphorus fluctuations as well as R. peltatus and exhibited high phosphorus storage ability. The high fluctuation availability of resources in space or/and time favoured the spread of the invasive plants and confirms the theory of invasibility of Davis et al. [2000. Fluctuating resources in plant communities: a general theory of invasibility. J. Ecol. 88, 528-534]. The eutrophication process increases the invasibility of E. nuttallii's, while inducing competition between E. nuttallii and native macrophyte species.     

Arbuscular mycorrhiza alters metal uptake and the physiological response of Coffea arabica seedlings to increasing Zn and Cu concentrations in soil

Studies on mycorrhizal symbiosis effects on metal accumulation and plant tolerance are not common in perennial crops under metal stress. The objective of this study was to evaluate the influence of mycorrhization on coffee seedlings under Cu and Zn stress. Copper (Cu) and zinc (Zn) uptake and some biochemical and physiological traits were studied in thirty-week old Coffea arabica seedlings, in response to the inoculation with arbuscular mycorrhizal fungi (AMF) and to increasing concentrations of Cu or Zn in soil. The experiments were conducted under greenhouse conditions in a 2 × 4 factorial design (inoculation or not with AMF and 0, 50, 150 and 450 mg kg^− 1 Cu or 0, 100, 300 and 900 mg kg^− 1 Zn). Non-mycorrhizal plants maintained a hampered and slow growth even in a soil with appropriate phosphorus (P) levels for this crop. As metal levels increased in soil, a greater proportion of the total absorbed metals were retained by roots. Foliar Cu concentrations increased only in non-mycorrhizal plants, reaching a maximum concentration of 30 mg kg^− 1 at the highest Cu in soil. Mycorrhization prevented the accumulation of Cu in leaves, and mycorrhizal plants showed higher Cu contents in stems, which indicated a differential Cu distribution in AMF-associated or non-associated plants. Zn distribution and concentrations in different plant organs followed a similar pattern independently of mycorrhization. In mycorrhizal plants, only the highest metal concentrations caused a reduction in biomass, leading to significant changes in some biochemical indicators, such as malondialdehyde, proline and amino acid contents in leaves and also in foliar free amino acid composition. Marked differences in these physiological traits were also found due to mycorrhization. In conclusion, AMF protected coffee seedlings against metal toxicity.     

Intra- and inter-annual variation of Cd, Zn, Mn and Cu in foliage of poplars on contaminated soil

The uptake of trace metals in the leaves of fast-growing woody species is a crucial factor in ecological risk assessment and in the evaluation of phytoextraction potentials. In this study, we present a long-term data series of foliar Cd, Zn, Mn and Cu concentrations in poplar (Populus trichocarpa x P. deltoides). Leaves were collected every three weeks from 2001 until 2007 on three sites, (i) a new plantation on an alluvial soil polluted by river sediments, (ii) a new plantation on an unpolluted soil and (iii) a 10-year old plantation on a polluted dredged sediment soil. In addition, tree rings were measured on the alluvial soil in order to better assess growth over the past seven years. Foliar concentrations of Cd, Zn and Mn decreased considerably with time in the new plantation on polluted soil. Concentrations of Zn and Mn decreased in the new plantation on unpolluted soil as well. The older plantation on polluted soil did not show changes in foliar concentrations for Cd, Zn or Mn. Foliar Cu concentrations slightly increased for all sites. Within one growing season, foliar concentrations of Cd, Zn, Cu and Mn increased towards the end of the season. The tree ring data of the poplars on the alluvial soil indicated a strong decrease in growth due to declining tree condition from 2005 onwards, the same year that foliar Cd and Zn concentrations markedly decreased. Lower transpiration rates probably induced a lower uptake of dissolved trace metals. It is concluded that stand health and growth rate have a strong impact on the variation of foliar trace metal concentrations over time.     

Patterns of metal bioaccumulation in two filter-feeding macroinvertebrates: Exposure distribution, inter-species differences and variability across developmental stages

This study focused on the metal bioaccumulation of two aquatic insects (Ephoron virgo and Hydropsyche spp.) in order to evaluate the spatial distribution of metals, the interspecific differences between both filter-feeders and the bioaccumulation dynamics during E. virgo development stages. Hg, Cd, Ni, Cr, As, Pb, Cu, Ti, Zn and Mn were quantified in insects and in suspended particulate matter (SPM) sampled downstream and upstream of a chemical plant, where more than 300,000 t of polluted sediments are deposited. Hg concentrations were one order of magnitude higher downstream of the sediment dump, which showed that the Hg pollution originated in the chemical plant. Cd, Ni, Cr, Pb, Ti, Zn and Mn in invertebrates revealed that metal pollution was present upstream in other parts of the river. Interspecific differences were observed for all metals but Mn; significantly higher concentrations were observed in E. virgo over Hydropsyche exocellata, except for Cd, which showed 10-fold higher values. Hg and Cd increased until E. virgo nymphs reached 11 mm and decreased afterwards in late instars when nymphs were about to emerge. Cr, Pb, Ti and Mn decreased along early instars followed by a steady state in late instars. Similar values were obtained for Cu, As and Zn along all instars. Sexual differences between males and females of E. virgo were observed for Cd, Cu and Mn. Hg and Cd persistence was strong across developmental stages since high concentrations were found in eggs and emerging adults. Because the behavior of different metals varied for the two species and during the developmental stages of E. virgo, care should be taken in the interpretation of insect metal concentrations when analyzing the food chain transfer of metals in river ecosystems.     

Epiphytic bacteria downstream of sewage-works outfalls

Epiphytic microorganisms were investigated in two watercourses which were enriched by sewage-works effluent. These were a disused canal and a small stream in North-East England. Scanning electron microscopy, on the submerged macrophytes Callitriche sp. and Elodea canadensis, from the canal, demonstrated abundant epiphyton which was dominated by rod-shaped bacteria and pennate diatoms, especially Cocconeis sp. An in situ counting technique showed that the density of epiphytic bacteria was greater downstream of the sewage-works outfall on plants from both watercourses. The proportion of metabolically-active epiphytic bacteria, and cell length of epiphytic bacteria, were determined on Callitriche in the small stream, and were greater downstream of the outfall. Thus increase in (1) the population density of epiphytic bacteria, (2) the proportion of cells which were metabolically active, and (3) cell size, combined to increase the potential contribution of epiphytic bacteria to biopurification and downstream recovery. Epiphytic diatoms, in contrast to bacteria, apparently did not increase in abundance downstream of the outfalls.     

Arsenic phytoremediation by Phragmites australis: green technology

The authors report arsenic phytoremediation by the common reed, Phragmites australis (P. australis). The plants for this study were collected from five sampling sites in the Chelpo contaminated area of Khorasan Province in Northeast Iran. P. australis was found to be a hyper‐accumulator plant. Because of the potential for this plant to be used as an agent to remove arsenic from a polluted environment, P. australis has a considerable enrichment factor. Thus, in the P. australis accumulation model following the order of root>rhizome>stem>leaves, the examination of P. australis for the phytoremediation process indicates that this species can cope with arsenic remediation from certain polluted soils.     

Transport rate of arsenic, cadmium, copper and zinc in Potamogeton pectinatus L.: radiotracer experiments with 76As,109,115Cd,64Cu and 65,69mZn

The present study was aimed at obtaining an insight into possible experimental approaches for providing numerical data on both the accumulation of sediment As, Cd, Cu and Zn in the submerged water plant Potamogeton pectinatus L., and the possible corresponding metal flows into the water phase. A hydroculture two-compartment system was used as the experimental set-up, and the selected metals were followed by measurements of their radioisotopes 76As, 109Cd, 115Cd, 64Cu, 65Zn and 69mZn. All experiments were performed in single plant mode. The results stress the extreme importance of leakage tests, which were performed using 99mTcO4-, and which resulted in approximately 30% of all experiments being discarded. Metal flows were shown as very near the metal limits of detection or obscured by and/or numerically very near the occurring leakage phenomena. Bio-concentration factors BCF (fresh wt. fine root basis) were calculated as 100, 10, 10 and 100-500 l/kg for Cu, Zn, Cd and As, respectively. The mobility, expressed as the shoot/root concentration ratio, CR, was obtained as < 10(-4), < 10(-5), 10(-3) and 10(-3) - 10(-2) for Cu, As, Cd and Zn, respectively. Double-labeling experiments showed that the CR values were due to the exclusive root-mediated transport in radiotracer experiments: results for simultaneous applications of 109Cd and 115Cd or 65Zn and 69mZn showed field-simulated CR values of approximately 0.04 and 14, respectively. Single-tracer experiments, using liquid scintillation counting (LSC) with 109Cd and 65Zn, were shown to strongly improve the sensitivities of flow determinations. Under the applied conditions, metal flows could be determined as <5 x 10(-8), <5 X 10(-8), 3.5+/-1.8 x 10(-10) and <8 x 10(-9) mol/h per kg root fresh wt. for Cu, As, Cd and Zn, respectively. Upscaling calculations, assuming plant steady state behavior, indicate that the metal accumulation in the plants may comprise up to 1% of the sediment metal occurrence, that the major part of an accumulated metal is retained in the plant roots, and that plant-mediated metal flow into the water phase (< 0.01% for Cd, Cu and Zn, < 0.1% for As within a growing season) may be regarded as not significantly contributing to the overall process of metal mobilization. It should be noted, however, that the above conclusions should be drawn with care, due to the pilot nature and the short-term duration of the presented experiments.     

A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant

The spread of methicillin-resistant Staphylococcus aureus (MRSA), in which the mecA gene mediates resistance, threatens the treatment of staphylococcal diseases. The aims were to determine the effect of wastewater treatment processes on mecA gene concentrations, and the prevalence of S. aureus and MRSA over time. To achieve this a municipal wastewater treatment plant was investigated for the mecA gene, S. aureus and MRSA, using real-time PCR assays. Water samples were collected monthly for one year, at eight sites in the plant, reflecting different aspects of the treatment process. The mecA gene and S. aureus could be detected throughout the year at all sampling sites. MRSA could also be detected, but mainly in the early treatment steps. The presence of MRSA was verified through cultivation from inlet water. The concentration of the mecA gene varied between months and sampling sites, but no obvious seasonal variation could be determined. The wastewater treatment process reduced the mecA gene concentration in most months. Taken together our results show that the mecA gene, S. aureus and MRSA occur over the year at all sites investigated.     

Survival of manure-borne E. coli in streambed sediment: Effects of temperature and sediment properties

Escherichia coli bacteria are commonly used as indicator organisms to designate of impaired surface waters and to guide the design of management practices to prevent fecal contamination of water. Stream sediments are known to serve as a reservoir and potential source of fecal bacteria (E. coli) for stream water. In agricultural watersheds, substantial numbers of E. coli may reach surface waters, and subsequently be deposited into sediments, along with fecal material in runoff from land-applied manures, grazing lands, or wildlife excreta. The objectives of this work were (a) to test the hypothesis that E. coli survival in streambed sediment in the presence of manure material will be affected by sediment texture and organic carbon content and (b) to evaluate applicability of the exponential die-off equation to the E. coli survival data in the presence of manure material. Experiments were conducted at three temperatures (4 °C, 14 °C, and 24 °C) in flow-through chambers using sediment from three locations at the Beaverdam Creek Tributary in Beltsville, Maryland mixed with dairy manure slurry in the proportion of 1000:1. Indigenous E. coli populations in sediments ranged from ca. 10¹ to 10³ MPN g⁻¹ while approx 10³ manure-borne E. coli MPN g⁻¹ were added. E. coli survived in sediments much longer than in the overlaying water. The exponential inactivation model gave an excellent approximation of data after 6-16 days from the beginning of the experiment. Slower inactivation was observed with the increase in organic carbon content in sediments with identical granulometric composition. The increase in the content of fine particles and organic carbon in sediments led not only to the slower inactivation but also to lower sensitivity of the inactivation to temperature. Streambed sediment properties have to be documented to better evaluate the role of sediments as reservoirs of E. coli that can affect microbiological stream water quality during high flow events.     

Delineation of Pb contamination pathways in two Pectinidae: The variegated scallop Chlamys varia and the king scallop Pecten maximus

Bioaccumulation of Pb was determined in Chlamys varia and Pecten maximus exposed to ²¹⁰Pb via seawater, food and sediment. Both scallops readily concentrated dissolved Pb with whole-body 7-d concentration factors of 250 ± 40 and 170 ± 70, respectively. In both species, more than 70% of Pb taken up from seawater was strongly retained within tissues (biological half-life > 1.5 month) whereas Pb ingested with phytoplankton was poorly assimilated (< 20%). As P. maximus lives buried in the sediment, this exposure pathway was assessed and showed low bioaccumulation efficiency for sediment-bound Pb (transfer factor < 0.015). Despite the poor transfer efficiency of Pb from food and sediment, the use of a global bioaccumulation model indicated that the particulate pathway (food and/or sediment) constituted the major bioaccumulation route of Pb in both scallops. Whatever the exposure pathway, the digestive gland and kidneys always played a major role in Pb accumulation. In scallop tissues, Pb was predominantly associated with the insoluble subcellular fraction, suggesting a low bioavailability of Pb for scallop consumers.     

Utilization of the seagrass Posidonia oceanica to evaluate the spatial dispersion of metal contamination

Metal concentrations have been measured in blades of the endemic Mediterranean seagrass Posidonia oceanica, along transects from three different contaminant point sources (the former asbestos mine of Canari - Corsica, France; the chemical plant of Solvay/Rosignano - Livorno, Italy; and the industrial harbour of Porto-Torres - Sardinia, Italy). The aim of this study was to evaluate the spatial extent of the impact of these sources in terms of metal contamination. The results showed that metal contamination from the former mine of Canari (cobalt - Co, chromium - Cr and nickel - Ni) extends at least 5 km to the north and south. The impact of this mine, which closed in 1965, seems to be lingering still. Mercury (Hg) contamination in the Livorno location was difficult to evaluate due to the presence of others potential sources of mercury in the area (e.g. industrialized city of Livorno, natural cinnabar deposits, intense tectonic activity of the area). At any rate, mercury concentration decreased strongly with distance from the plant. Lead (Pb) contamination at the Porto-Torres harbour was very low and disappeared with distance from the harbour. However, as the Porto-Torres harbour does not appear as a substantial point source of Pb contamination and because of the ubiquitous characteristic of the Pb element, it is difficult to draw any general conclusions concerning this element. The results presented in this study demonstrated the usefulness of the seagrass P. oceanica as a tool for the evaluation of the spatial extent of metal contaminations from point sources and could, therefore, contribute to on-going efforts to manage coastal environments.     

The distribution of cadmium,copper, lead,and zinc in eelgrass (Zostera marina L.)

The distribution of Cd, Cu, Pb, and Zn in eelgrass (Zostera marina L.) was studied at three locations with different heavy metal loads in the Limfjord, Denmark.The eelgrass was fractionated into roots, rhizome, stem, and leaves according to age, and the heavy metal concentrations in each fraction were determined. The distribution patterns of the four heavy metals in eelgrass were independent of the heavy metal loads at the sampling stations. The concentrations of all metals were greater in the roots than in the rhizomes. In the aerial¹ parts two different age-dependent distribution patterns were observed. The concentrations of Cd, Pb, and Zn increased with age while the opposite was true for Cu. The distribution of lead correlated with the distribution of ash content. These age-dependent distribution patterns were maintained throughout the observation period and were most pronounced for Cu and Zn in winter.The heavy metal distribution in eelgrass is discussed in relation to gross morphology, especially age-structure. It is suggested that the accumulation of Cd, Pb, and Zn is due to a slow irreversible uptake or to the existence of more binding sites in old leaves. The distribution of Cu can be explained by translocation within the plant, dilution due to growth or leakage from the older leaves.     

Concentration-dependent response of estrone-degrading bacterial community in activated sludge analyzed by microautoradiography-fluorescence in situ hybridization

Inefficient removal of estrone (E1) in wastewater treatment plants (WWTPs) causes feminizing effects in male aquatic creatures. As E1 is mainly removed by biodegradation, investigation of E1 degradation is important to determine better removal strategies. Using microautoradiography-fluorescence in situ hybridization (MAR-FISH), we demonstrated that the structures of [³H]E1-incorporating bacterial communities were different at different E1 concentrations applied to activated sludge. At 200 μg/L E1, almost all [³H]E1-incorporating cells were associated with either Betaproteobacteria or Gammaproteobacteria (60% and 40% of MAR (+) cells, respectively). The proportion of Betaproteobacteria and Gammaproteobacteria in the total number of [³H]E1-incorporating cells decreased as the concentration of E1 decreased. In contrast, the proportion of Alphaproteobacteria in the total number of [³H]E1-incorporating cells increased as the concentrations of E1 decreased. At the lowest applied concentration (540 ng/L), almost all the [³H]E1-incorporating cells were Alphaproteobacteria (96%). The results of MAR-FISH applied to sludge samples collected from various plant locations and activated sludge processes, and during different seasons also demonstrated the high contribution of Alphaproteobacteria to the entire E1-degrading bacterial community (50.4 ± 11% of the total number of [³H]E1-incorporating cells) at 1 μg/L E1. Since the E1 concentration in domestic wastewater is at sub-μg/L levels, the key E1 degraders in activated sludge of domestic WWTPs are probably be Alphaproteobacteria. All [³H]E1-incorporating Alphaproteobacteria were hybridized with probe ALF968. Few MAR (+) cells were Sphingomonadales. An E1-degrading bacterial community at low E1 concentration appeared to consist of diverse bacterial groups of Alphaproteobacteria. This study suggested that substrate concentration is an essential factor for revealing E1-degrading bacteria in complex communities.