Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale

Forty-six bacterial cultures, including one culture collection strain, thirty from the rhizosphere of Alyssum murale and fifteen from Ni-rich soil, were tested for their ability to tolerate arsenate, cadmium, chromium, zinc, mercury, lead, cobalt, copper, and nickel in their growth medium. The resistance patterns, expressed as minimum inhibitory concentrations, for all cultures to the nine different metal ions were surveyed by using the agar dilution method. A large number of the cultures were resistant to Ni (100%), Pb (100%), Zn (100%), Cu (98%), and Co (93%). However, 82, 71, 58 and 47% were sensitive to As, Hg, Cd and Cr(VI), respectively. All cultures had multiple metal-resistant, with heptametal resistance as the major pattern (28.8%). Five of the cultures (about of 11.2% of the total), specifically Arthrobacter rhombi AY509239, Clavibacter xyli AY509235, Microbacterium arabinogalactanolyticum AY509226, Rhizobium mongolense AY509209 and Variovorax paradoxus AY512828 were tolerant to nine different metals. The polymerase chain reaction in combination with DNA sequence analysis was used to investigate the genetic mechanism responsible for the metal resistance in some of these gram-positive and gram-negative bacteria that were, highly resistant to Hg, Zn, Cr and Ni. The czc, chr, ncc and mer genes that are responsible for resistance to Zn, Cr, Ni and Hg, respectively, were shown to be present in these bacteria by using PCR. In the case of, M. arabinogalactanolyticum AY509226 these genes were shown to have high homology to the czcD, chrB, nccA, and mer genes of Ralstonia metallidurans CH34. Therefore, Hg, Zn, Cr and Ni resistance genes are widely distributed in both gram-positive and gram-negative isolates obtained from A. murale rhizosphere and Ni-rich soils.     

Competition between phenanthrene, chrysene, and 2,5-dichlorobiphenyl for high-energy adsorption sites in a sediment

We determined the maximum amounts of added phenanthrene, chrysene, and 2,5-dichlorobiphenyl sorbed onto high-energy adsorption sites in a sediment on bi-solute experiments. The bi-solute pairs were phenanthrene/chrysene and phenanthrene/2,5-dichlorobiphenyl. On the bi-solute sorption experiments, one solute was introduced and equilibrated with sediment prior to addition of the second solute. The values for the maximum amounts adsorbed onto high-energy sites revealed that, after equilibration of the first solute, still some high-energy sites could be occupied by the second solute. Phenanthrene, chrysene, and 2,5-dichlorobiphenyl seem to share about 30% of the accessible high-energy adsorption sites in the sediment employed.     

A synchronized amphibian metamorphosis assay as an improved tool to detect thyroid hormone disturbance by endocrine disruptors and apolar sediment extracts

Amphibian metamorphosis assays are used to evaluate potential effects of endocrine disrupting compounds on the thyroid hormone axis. In this study, Xenopus laevis tadpoles are kept in a solution of 0.2% thiourea (TU) to arrest and synchronise them in their development. The advantage of this synchronized amphibian metamorphosis assays is that synchronised tadpoles are available at any time to start metamorphosis experiments, and experimental groups are much more homogenous at the start of experimental exposure compared with groups selected from an untreated pool of animals. The water volume per animal was kept constant throughout the experimental period to overcome the influence of declining numbers of animals per aquarium due to metamorphosis and mortality on the density dependent development of the remaining tadpoles. Clophen A50 (a technical PCB mixture), the single congener 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and apolar sediment extracts that were previously tested positive in the T-Screen, an in vitro proliferation assay for thyroid hormone disruption, were tested in the Synchronized Amphibian Metamorphosis Assay. Endpoints studied were mortality, malformations, body weight, and percentage of metamorphosed froglets at the end of the 60-day experimental period, percentage of tadpoles in different developmental stages, and developmental stage-dependent awarded penalty points. Dietary exposure to Clophen A50 (0.2-50mg/kg food) resulted in a significant increased percentage of tadpoles that did not pass metamorphosis at concentrations higher than 2mg/kg food. Time until metamorphosis in those animals that were able to metamorphose after the 60-days experimental period was significantly decreased. Dietary exposure to PCB 77, a congener that can be readily metabolised, did not result in significant effects in any exposure group (2-500 microg/kg food). Apolar sediment extracts from two of the three sites that are contaminated with a wide variety of chemicals significantly decreased the percentage of metamorphosed animals and significantly increased the number of tadpoles that remained in early and late metamorphic stages. These effects already occurred when the extracts where diluted more than 1000 times (on an organic carbon base) compared to environmental concentrations. The rank of potency was comparable to results obtained with the T-screen. This suggests the presence of thyroid hormone disrupting compounds in the aquatic environment and possible effects of such compounds on animal development in the wild.     

Androgenic and antiandrogenic activities in water and sediment samples from the river Lambro, Italy, detected by yeast androgen screen and chemical analyses

The river Lambro is the most polluted tributary of the river Po in North Italy and was chosen as a representative water course discharging industrialized areas. Water and sediment samples of the river Lambro were investigated regarding the presence of endocrine disrupting compounds. A combined procedure was used consisting of solid-phase extraction and HPLC based fractionation of samples, of screening for (anti)androgenic activity using the yeast androgen screen (YAS) and of chemical analysis using HPLC-MS/MS and GC-MS. Androgenic and antiandrogenic activities were found in specific fractions of the water and sediment while the total extracts showed antiandrogenic activity only. The chemical analysis of the fractions and total extracts with antiandrogenic activities revealed the presence of compounds with suspected antiandrogenic potency such as bisphenol A, iprodione, nonylphenol, p,p'-DDE and tert-octylphenol but other unknown compounds contributed mainly to the observed antiandrogenic activities. The antiandrogenic load of the river Lambro ranged between 1.34 and 17.1 microM flutamide-equivalents and may pose a risk to aquatic environments. Future screenings for EDC in the environment that have the potential to interfere with reproduction of aquatic organisms should be extended to different modes of actions including (anti)androgenic ones.     

A practical method for quantification of phosphorus- and glycogen-accumulating organism populations in activated sludge systems.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the enrichment of activated sludge with phosphorus-accumulating organisms (PAOs). The presence and proliferation of glycogen-accumulating organisms (GAOs), which compete for substrate with PAOs, may be detrimental for EBPR systems, leading to deterioration and, in extreme cases, failure of the process. Therefore, from both process evaluation and modeling perspectives, the estimation of PAO and GAO populations in activated sludge systems is a relevant issue. A simple method for the quantification of PAO and GAO population fractions in activated sludge systems is presented in this paper. To develop such a method, the activity observed in anaerobic batch tests executed with different PAO/GAO ratios, by mixing highly enriched PAO and GAO cultures, was studied. Strong correlations between PAO/GAO population ratios and biomass activity were observed (R2 > 0.97). This served as a basis for the proposal of a simple and practical method to quantify the PAO and GAO populations in activated sludge systems, based on commonly measured and reliable analytical parameters (i.e., mixed liquor suspended solids, acetate, and orthophosphate) without requiring molecular techniques. This method relies on the estimation of the total active biomass population under anaerobic conditions (PAO plus GAO populations), by measuring the maximum acetate uptake rate in the presence of excess acetate. Later, the PAO and GAO populations present in the activated sludge system can be estimated, by taking into account the PAO/GAO ratio calculated on the basis of the anaerobic phosphorus release-to-acetate consumed ratio. The proposed method was evaluated using activated sludge from municipal wastewater treatment plants. The results from the quantification performed following the proposed method were compared with direct population estimations carried out with fluorescence in situ hybridization analysis (determining Candidatus Accumulibacter Phosphatis as PAO and Candidatus Competibacter Phosphatis as GAO). The method showed to be potentially suitable to estimate the PAO and GAO populations regarding the total PAO-GAO biomass. It could be used, not only to evaluate the performance of EBPR systems, but also in the calibration of potential activated sludge mathematical models, regarding the PAO-GAO coexistence.     

Behavioural inertia places a top marine predator at risk from environmental change in the Benguela upwelling system

In variable environments, organisms are bound to track environmental changes if they are to survive. Most marine mammals and seabirds are colonial, central-place foragers with long-term breeding-site fidelity. When confronted with environmental change, such species are potentially constrained in their ability to respond to these changes. For example, if environmental conditions deteriorate within their limited foraging range, long-lived species favour adult survival and abandon their current breeding effort, which ultimately influences population dynamics. Should poor conditions persist over several seasons, breeding-site fidelity may force animals to continue breeding in low-quality habitats instead of emigrating towards more profitable grounds. We assessed the behavioural response of a site-faithful central-place forager, the Cape gannet Morus capensis, endemic to the Benguela upwelling system, to a rapid shift in the distribution and abundance of its preferred prey, small pelagic shoaling fish. We studied the distribution and the abundance of prey species, and the diet, foraging distribution, foraging effort, energy requirements, and breeding success of gannets at Malgas Island (South Africa) over four consecutive breeding seasons. Facing a rapid depletion of preferred food within their foraging range, Cape gannets initially increased their foraging effort in search of their natural prey. However, as pelagic fish became progressively scarcer, breeding birds resorted to scavenging readily available discards from a nearby demersal fishery. Their chicks cannot survive on such a diet, and during our 4-year study, numbers of breeding birds at the colony decreased by 40% and breeding success of the remaining birds was very low. Such behavioural inflexibility caused numbers of Cape gannets breeding in Namibia to crash by 95% following over-fishing of pelagic fish in the 1970s. In the context of rapid environmental changes, breeding-site fidelity of long-lived species may increase the risk of local or even global extinction, rendering these species particularly vulnerable to global change.     

Robustness of variance and autocorrelation as indicators of critical slowing down

Ecosystems close to a critical threshold lose resilience, in the sense that perturbations can more easily push them into an alternative state. Recently, it has been proposed that such loss of resilience may be detected from elevated autocorrelation and variance in the fluctuations of the state of an ecosystem due to critical slowing down; the underlying generic phenomenon that occurs at critical thresholds. Here we explore the robustness of autocorrelation and variance as indicators of imminent critical transitions. We show both analytically and in simulations that variance may sometimes decrease close to a transition. This can happen when environmental factors fluctuate stochastically and the ecosystem becomes less sensitive to these factors near the threshold, or when critical slowing down reduces the ecosystem's capacity to follow high-frequency fluctuations in the environment. In addition, when available data is limited, variance can be systematically underestimated due to the prevalence of low frequencies close to a transition. By contrast, autocorrelation always increases toward critical transitions in our analyses. To exemplify this point, we provide cases of rising autocorrelation and increasing or decreasing variance in time series prior to past climate transitions.