Biodegradable chelate enhances the phytoextraction of copper by Oenothera picensis grown in copper-contaminated acid soils

Oenothera picensis plants (Fragrant Evening Primrose) grow in the acid soils contaminated by copper smelting in the coastal region of central Chile. We evaluated the effects of the biodegradable chelate MGDA (methylglycinediacetic acid) on copper extraction by O. picensis and on leaching of copper through the soil profile, using an ex situ experiment with soil columns of varying heights. MGDA was applied in four rates: 0 (control), 2, 6 and 10 mmol plant⁻¹. MGDA application significantly increased biomass production and foliar concentration, permitting an effective increase in copper extraction, from 0.09 mg plant⁻¹ in the control, to 1.3 mg plant⁻¹ in the 6 and 10 mmol plant⁻¹ treatments. With 10 mmol plant⁻¹ rate of MGDA, the copper concentration in the leachate from the 30 cm columns was 20 times higher than in the control. For the 60 cm columns, copper concentration was 2 times higher than the control. It can be concluded that at increased soil depths, copper leaching would be minimal and that MGDA applications at the studied rates would not pose a high risk for leaching into groundwater. It can thus be stated that applications of MGDA are an effective and environmentally safe way to improve copper extraction by O. picensis in these soils.     

Partitioning of metals in a degraded acid sulfate soil landscape: influence of tidal re-inundation

The oxidation and acidification of sulfidic soil materials results in the re-partitioning of metals, generally to more mobile forms. In this study, we examine the partitioning of Fe, Cr, Cu, Mn, Ni and Zn in the acidified surface soil (0-0.1 m) and the unoxidised sub-soil materials (1.3-1.5 m) of an acid sulfate soil landscape. Metal partitioning at this acidic site was then compared to an adjacent site that was previously acidified, but has since been remediated by tidal re-inundation. Differences in metal partitioning were determined using an optimised six-step sequential extraction procedure which targets the “labile”, “acid-soluble”, “organic”, “crystalline oxide”, “pyritic” and “residual” fractions. The surficial soil materials of the acidic site had experienced considerable losses of Cr, Cu, Mn and Ni compared to the underlying parent material due to oxidation and acidification, yet only minor losses of Fe and Zn. In general, the metals most depleted from the acidified surface soil materials exhibited the greatest sequestration in the surface soil materials of the tidally remediated site. An exception to this was iron, which accumulated to highly elevated concentrations in the surficial soil materials of the tidally remediated site. The “acid-soluble”, “organic” and “pyritic” fractions displayed the greatest increase in metals following tidal remediation. This study demonstrates that prolonged tidal re-inundation of severely acidified acid sulfate soil landscapes leads to the immobilisation of trace metals through the surficial accumulation of iron oxides, organic material and pyrite.     

Histopathological effects of copper and lithium in the ramshorn snail, Marisa cornuarietis (Gastropoda, Prosobranchia)

The aim of this study was to determine and quantify effects of copper and lithium in tissues of early juveniles of the ramshorn snail, Marisa cornuarietis. For this purpose, hatchlings of M. cornuarietis were exposed for 7 days to a range of five different sublethal concentrations of copper (5, 10, 25, 50, and 75 μg Cu²⁺ L⁻¹) and lithium (50, 100, 200, 1000, and 5000 μg Li⁺ L⁻¹). Both metals changed the tissue structure of epidermis, hepatopancreas, and gills, varying between slight and strong reactions, depending on the copper and lithium concentration. The histopathological changes included alterations in epithelial and mucous cells of the epidermis, swelling of hepatopancreatic digestive cells, alterations in the number of basophilic cells, abnormal apices of digestive cells, irregularly shaped cilia and changes in the amount of mucus in the gills. The most sensible organ in M. cornuarietis indicating Cu or Li pollution is the hepatopancreas (LOECs were 10 μg Cu²⁺ L⁻¹, or 200 μg Li⁺ L⁻¹). In epidermis, mantle and gills relevant effects occurred with higher LOECs (50 μg Cu²⁺ L⁻¹, or 1000 μg Li⁺ L⁻¹). Base on LOECs, our results indicated that histopathological endpoints are high sensitivity to copper and lithium compared to endpoints for embryonic developmental toxicity.     

Comparing the sensitivity of soil invertebrates to pesticides with that of Eisenia fetida

The sole routine testing of the standard earthworm Eisenia fetida for the terrestrial risk assessment of pesticides has been under much debate since other soil invertebrates may be more sensitive than this standard test species. However, the very low availability of laboratory toxicity data for taxa other than E. fetida has greatly hampered sensitivity comparisons. In the present study, the relative tolerance (T_rel) approach was used to enable comparing toxicity thresholds obtained from the US-EPA ECOTOX database, for main terrestrial taxonomic groups and pesticidal types of action (insecticides, fungicides, herbicides, and other) separately. Analyses confirmed previously reported lower and higher sensitivity of collembolans to fungicides and insecticides, respectively. However, various other discrepancies in susceptibility relative to E. fetida were encountered as indicated by species sensitivity distributions and/or calculated 95% confidence intervals of T_rel values. Arachnids and isopods were found to be more sensitive to insecticides, and nematodes to fungicides, as compared to E. fetida. Implications of study findings for the terrestrial risk assessment of pesticides are discussed.     

Trade-offs between elimination and detoxification in rainbow trout and common bivalve molluscs exposed to metal stressors

The purpose of this paper was to examine trade-offs between elimination and detoxification in rainbow trout and three common bivalve molluscs (clam, oyster, and scallop) exposed to cadmium (Cd), copper (Cu), and zinc (Zn) based on recent reported experimental data. We incorporated metal influx threshold with subcellular partitioning to estimate rate constants of detoxification (k_d) and elimination (k₂). We found that the relationships between k₂ and k_d were negative for rainbow trout and positive for bivalve molluscs. However, the relationships between k_d and % metal in metabolically detoxified pool were found positive for rainbow trout and negative for bivalve molluscs. Our results also indicated that rainbow trout had higher accumulation (∼60-90%) in metabolically active pool when exposed to essential metals of Cu and Zn and had only 10-50% accumulation in response to non-essential metal of Cd. Based on a cluster analysis, this study indicated that similarity of physiological regulations among study species was found between Cd and Zn. Our study suggested that detoxification can be predicted by an elimination-detoxification scheme with the known elimination rate constant. We concluded that quantification of trade-offs between subcellular partitioning and detoxification provides valuable insights into the ecotoxicology of aquatic organisms and enhances our understanding of the subcellular biology of trace metals.     

Mobility of Cd and Cu in formulated sediments coated with iron hydroxides and/or humic acids: a DGT and DGT-PROFS modeling approach

The diffusive gradients technique in thin films (DGT) was used to investigate the kinetic resupply of Cd and Cu to pore water from the solid phase. For the sake of simplification, experiments were performed using formulated sediments that differed in the presence or absence of humic acids (HA) and/or of iron hydroxides (i.e., goethite and ferrihydrite). The effects of the time after the contamination of the solid phase (aging effect) on formulated sediments that were coated with goethite and HA and spiked with Cd were also evaluated. Kinetic DGT results were interpreted using the newly developed, multi-compartmental model DGT-PROFS.Due to Cu humate formation, the addition of HA slightly increased the Cu concentration in the pore water independent of the effect of the iron hydroxide coating on the formulated sediments and slightly decreased that of Cd. The impact of 8-190 d of aging resulted in a significant decrease in the Cd concentration of the pore water over an increasing incubation time.Modeling our results with DGT-PROFS led to the following conclusions concerning the impact of HA and iron hydroxides on Cd and Cu availability. First, in the presence of HA and absence of iron hydroxides, Cd is associated mainly with weak sites, while Cu is bound to strong sites. Similarly, in the presence of both iron hydroxides and HA, Cu appeared to be more heavily associated with the strong sites than did Cd. When the incubation time increased from 8 to 190 d, a proportion of Cd initially adsorbed onto weak sites transferred to the strong sites, suggesting that the adsorption of Cd on sediments is controlled partially by slow kinetic processes.     

Dry and heat stress affects H2S production of Salmonella on selective plating media

The pH of Salmonella pre-enrichment media can become acidic (pH 4.0–5.0) when feeds/ingredients are incubated for 24?h. Salmonella in feed that have been stressed by heat and desiccation exhibit different pH tolerances than non-stressed cultures. Acidic conditions can result in cell injury/death and affect biochemical pathways. In this study, eight serotypes of Salmonella were grown in sterile meat and bone meal that was subjected to desiccation and heat stress. Cultures of non-stressed and stressed isolates were subsequently exposed to acidic pH from 4.0 to 7.0 in 0.5?pH increments (3 replicates/pH increment) in citrate buffer. At 6 and 24?h, serial dilutions were plated in duplicate on XLT-4 (xylose lysine tergitol-4) agar. Four serotypes showed an impaired ability to decarboxylate lysine on XLT-4. This inability to decarboxylate lysine was dependent on isolate, stress status, and incubation time. When the isolates’ ability to decarboxylate lysine was examined using biochemical tests, cultures were found to be able to decarboxylate lysine with the exception of S. Infantis. This suggests that XLT-4 contains a biochemical stressor(s) which affects the rate of decarboxylation by these Salmonella. These results suggest that acidic conditions may influence the detection and confirmation of Salmonella in feed.     

Evolution of tolerance to PCBs and susceptibility to a bacterial pathogen (Vibrio harveyi) in Atlantic killifish (Fundulus heteroclitus) from New Bedford (MA, USA) harbor

A population of the non-migratory estuarine fish Fundulus heteroclitus (Atlantic killifish) resident to New Bedford (NB), Massachusetts, USA, an urban harbor highly contaminated with polychlorinated biphenyls (PCBs), demonstrates recently evolved tolerance to some aspects of PCB toxicity. PCB toxicology, ecological theory, and some precedence supported expectations of increased susceptibility to pathogens in NB killifish. However, laboratory bacterial challenges of the marine pathogen Vibrio harveyi to wild fish throughout the reproductive season and to their mature laboratory-raised progeny demonstrated comparable survival by NB and reference killifish, and improved survival by NB males. These results are inconsistent with hypothesized trade-offs of adaptation, and suggest that evolved tolerance in NB killifish may include mechanisms that minimize the immunosuppressive effects of PCBs. Compensatory strategies of populations persisting in highly contaminated environments provide a unique perspective for understanding the long-term ecological effects of toxic chemicals.     

Integral assessment of estrogenic potentials in sediment-associated samples

Background, aim, and scope  The enzyme-linked receptor assay (ELRA) detects estrogenic and anti-estrogenic effects at the molecular level of receptor binding and is a useful tool for the integrative assessment of ecotoxicological potentials caused by hormonally active agents (HAA) and endocrine disrupting compounds (EDC). The main advantage of the ELRA is its high sample throughput and its robustness against cytotoxicity and microbial contamination. After a methodological adaptation to salinity of the ELRA, according to the first part of this study, which increased its salinity tolerance and sensitivity for 17-β-estradiol, the optimised ELRA was used to investigate 13 native sediments characterised by different levels of salinity and chemical contamination. The applicability of the ELRA for routine analysis in environmental assessment was evaluated. Salinity is often a critical factor for bioassays in ecotoxicological sediment assessment. Therefore, salinity of the samples was additionally adjusted to different levels to characterise its influence on elution and binding processes of receptor-binding substances. Materials and methods  The ELRA was carried out with the human estrogen receptor α (ER) in a 96-well microplate format using the experimental setup known from the competitive immunoassay based on ligand–protein interaction. It is an important improvement that a physiologically relevant receptor was used as a linking protein instead of an antibody. The microplates were coated with a 17-β-estradiol-BSA conjugate, and dilution series of estradiol and of native sediment samples were added and incubated with the ER. After a washing step, a biotinylated mouse anti-ER antibody was added to each well. Receptor binding to estradiol, agonistic and antagonistic receptor binding, were determined by a streptavidin-POD-biotin complex with subsequent measurement of the peroxidase activity at the wavelength of 450 nm using a commercial ELISA multiplate reader. The sediment elutriates and pore water samples of sediments were tested in a dilution series to evaluate at which dilution step the receptor-binding potential ends. In the elution process (see Section 2.1 to 2.2), a method was developed to adjust the salinity to the levels of the reference testings, which offers an appropriate option to adjust the salinity in both directions. Statistical evaluation was made with a combination of the Mann–Whitney U test and the pT-method. Results  This part of the study characterised the environmental factor ‘salinity’ for prospective applications of the ELRA. Using reference substances such as 17-β-estradiol, the ELRA showed sigmoid concentration-effect relations over a broad range from 0.05 μg/l to 100 μg/l under physiological conditions. After methodological optimisation, both sensitivity and tolerance of the assay against salinity could be significantly raised, and the ELRA became applicable under salinity conditions up to concentrations of 20.5‰. The mean relative inter-test error (n = 3) was around 11% with reference substances and below 5% for single sediments elutriates in three replicates each. For sediment testings, the pore water and different salinity-adjusted elutriates of 13 sediments were used. A clear differentiation of the receptor-binding potential could be reached by application of the pT-method. Thereby, pT-values from one to six could be assigned to the sediments, and the deviation caused by the different salinity conditions was one pT-value. The mean standard deviation in the salinity adaptation procedure of the elutriates was below 5%. Discussion  Although the ELRA has already been used for assessments of wastewater, sludge and soil, its applicability for samples to different salinity levels has not been investigated so far. Even if the ELRA is not as sensitive as the E-screen or the YES-assay, with regard to reference substances like 17-β-estradiol, it is a very useful tool for pre-screening, because it is able to integrate both estrogenic as well as anti-estrogenic receptor-binding effects. According to the results of sediment testing, and given the integrative power to detect different directions of effects, the ELRA shows sufficient sensitivity and salinity tolerance to discriminate receptor-binding potentials in environmental samples. Conclusions  The optimised ELRA assay is a fast, cost-effective, reliable and highly reproducible tool that can be used for high-throughput screening in a microplate format in detecting both estrogenic and anti-estrogenic effects. Additionally, the ELRA is robust against microbial contaminations, and is not susceptible towards cytotoxic interferences like the common cell-culture methods. The general applicability and sufficient sensitivity of the ELRA was shown in freshwater environments. Marine and brackish samples can be measured up to salinity levels of 20.5‰. Recommendations and perspectives  In view of the proven sensitivity, functionality and the fastness of the ELRA, it is recommendable to standardise the test method. At the moment, no adequate in vitro test procedure exists which is standardised to DIN or ISO levels. The E-screen and the yeast estrogen/androgen screens (YES/YAS) sometimes underlie strong cytotoxic effects, as reported in the first part of this study. Further development of an ELRA assay using human androgen receptors appears to be very promising to gain information about androgenic and anti-androgenic effects, too. This would offer a possibility to use the ELRA as a fast and reliable pre-screening tool for the detection of endocrine potentials, thus minimising time and cost-expensive animal experiments.     

Correlation of foliar MT2b expression with Cd and Zn concentrations in hybrid aspen (Populus tremula × tremuloides) grown in contaminated soil

Metal uptake and its effect on foliar metallothionein 2b (MT2b) mRNA levels were studied in hybrid aspen (Populus tremula × tremuloides) in field conditions. The trees were planted on a site contaminated with several metals, including cadmium (mean 5.1 mg kg⁻¹), chromium (80 mg kg⁻¹), copper (180 mg kg⁻¹), nickel (81 mg kg⁻¹), vanadium (240 mg kg⁻¹) and zinc (520 mg kg⁻¹). Of the ten trace elements analyzed, only Cd and Zn accumulated in the leaves with maximal foliar concentrations of 35 and 2400 mg kg⁻¹ (dry weight), respectively. There was a strong correlation between Cd and Zn concentrations and bioaccumulation factors (concentration in plant/soil) in the leaves, branches and roots, suggesting similar transport mechanisms for these two metals. The levels of MT2b correlated with Cd and Zn concentrations in the leaves, demonstrating that increased MT2b expression is one of the responses of hybrid aspen to chronic metal exposure.