Membrane Dialysis Extraction (MDE): A Novel Approach for Extracting Toxicologically Relevant Hydrophobic Organic Compounds from Soils and Sediments for Assessment in Biotests

Goal, Scope and Background   Organic solvents are routinely used to extract toxicants from polluted soils and sediments prior to chemical analysis or bioassay. Conventional extraction methods often require the use of heated organic solvents, in some cases under high pressure. These conditions can result in loss of volatile compounds from the sample and the degradation of thermally labile target analytes. Moreover, extracts of soils and sediments also frequently contain substantial quantities of organic macromolecules which can act as sorbing phases for target analytes and in doing so interfere with both chemical analysis and bioassays. Membrane dialysis extraction (MDE) is described as a simple, passive extraction method for selectively extracting toxicologically relevant hydrophobic organic compounds (HOCs) from polluted soils and sediments and anaylzed for its applicability in ecotoxicological investigations. Methods   Toxicologically relevant hydrophobic organic compounds were extracted from wet and dry sediments by sealing replicate samples in individual lengths of pre-cleaned low-density polyethylene (LD-PE) tubing and then dialysing in n-hexane. The efficacy of the MDE method for use in ecotoxicological investigations was assessed by testing the concentrated extracts in the neutral red assay for acute cytotoxicity, in the EROD assay for the presence of dioxin-like compounds and in the Danio rerio fish egg assay for embryotoxic and teratogenic effects. Conditions of the sediment sample (with or without water content), dialysis membrane length and duration of dialysis were analyzed with respect to their impact on three endpoints. Results of the MDE investigations were compared to data obtained in samples prepared using conventional Soxhlet extraction. Results and Discussion   The membrane dialysis extraction was found to be at least as efficient as Soxhlet methodology to extract toxicologically relevant HOCs from sediment samples. In most cases, MDE-derived extracts showed a higher toxicological potential than the Soxhlet extracts. Lack of any significant effects in any MDE controls indicated these differences were not caused by contamination of the LD-PE membrane used. The elevated toxicological potential of MDE extracts is most likely the result of enhanced bioavailability of toxic compounds in consequence of lower amounts of organic macromolecules (i.e. sorbing phases) in the MDE extracts. This effect is probably the result of a size-selective restriction by the LD-PE membrane. Conclusion   Membrane dialysis extraction was found to be a simple, efficient and cost-effective method for the extraction of sediment samples. MDE can be used to extract toxicologically relevant hydrophobic organic compounds from both wet and dry sediments without the risk of loosing volatile and thermally labile target analytes. The size-selectivity of the LD-PE membrane also appears to have the capacity to increase the bioavailablity of potential target analytes in the resulting extracts by retaining much of the organic macromolecules present in the sample. Thus, results suggest that MDE may be particularly useful for the extraction of toxicologically relevant hydrophobic organic compounds from soils and sediments for bioassays and other ecotoxicological investigations. Recommendation and Perspective   Further validation of MDE has been initiated and the applicability of the methodology to other sample types will be investigated. Of particular interest is the potential application of MDE to recover hydrophobic target analytes from biological samples such as muscle, other soft tissues and blood.     

Fate of Amoxicillin In Two Water Reclamation Systems

The overall objective of this research was to determine the fate of amoxicillin in two wastewater reclamation systems containing biological and physical/chemical treatment processes. The results of this study indicate that amoxicillin is easily removed in a biological wastewater treatment system (<0.15 and 0.10 mg/L). Further evaluation of the physical/chemical treatment process, which included reverse osmosis, ion exchange, and UV oxidation and disinfection, indicated that amoxicillin will be removed (<0.10 mg/L) should it breakthrough the biological system. The concern of amoxicillin is not its presence in high concentrations in the effluent of a water reclamation system, but low antibiotic concentrations, which are known to encourage antibiotic resistance development.     

Attempts to derive EC50 values for heavy metals from land-applied Cu-, Ni-, and Zn-spiked sewage sludge

We established a field trial to assess the impacts on soil biological properties of application of heavy metal-spiked sewage sludge, with the aim of determining toxicity threshold concentrations of heavy metals in soil. Plots were treated with sludges containing increasing concentrations of Cu, Ni and Zn in order to raise the metal concentrations in the soil by 0-200 mg Cu kg⁻¹, 0-60 mg Ni kg⁻¹ and 0-400 mg Zn kg⁻¹, and were then cultivated and sown in ryegrass-clover pasture and monitored annually for 6 years. All biological properties measured (soil basal respiration, microbial biomass C, and sulphatase enzyme activities), except phosphatase activity, increased in all plots over the duration of the experiment. Consequently, it was only possible to assess effects of heavy metals across time if, each year, all data for each metal were normalised by expressing them as percentages of the activities measured in an un-sludged control plot. When this was done, no significant effects of increasing heavy-metal concentrations on basal respiration, microbial biomass C or respiratory quotient (qCO₂) were observed, although total Cu and soil solution Cu were significantly negatively related to microbial biomass C when it was expressed as a proportion of soil total C. None of the properties measured were affected by increasing Ni concentrations. Phosphatase and sulphatase activities were significantly negatively related to increasing Zn concentrations, but not usually to increasing Cu unless they were expressed as a proportion of total C. A sigmoidal dose-response model was used to calculate EC₂₀ and EC₅₀ values using the normalised data, but generally, the model parameters had very large 95% confidence intervals and/or the fits to the model had small R² values. The factors primarily responsible for confounding these results were site and sample variations not accounted for by the normalisation process and the absence of any data points at metal concentrations beyond the calculated EC₅₀ values. In the few instances where reasonable EC₂₀ values could be calculated, they were relatively consistent across properties, e.g., EC₂₀ for total Zn and phosphatase (330 mg kg⁻¹), total Zn and sulphatase (310 mg kg⁻¹), and EC₂₀ for total Cu and sulphatase (140 mg kg⁻¹) and total Cu and microbial biomass C (140 mg kg⁻¹), when both sulphatase and microbial biomass C were expressed as a proportion of total C. Our results suggest that Cu and Zn at the upper concentrations used in this experiment were possibly having adverse effects on some soil biological properties. However, much higher metal concentrations will be needed to accurately calculate EC₂₀ and EC₅₀ and this may not be easily achievable without many applications of sewage sludge, even if the sludge is spiked with heavy metals.     

Elemental concentrations in fresh snowfall across a regional transect in the northeastern U.S.: Apparent sources and contribution to acidity

Fresh snowfall was collected on the surface of 8 lakes across a 350 km west-east transect from northeastern New York state to the coast of Maine after a single storm. In addition, every snowfall event during the winter of 1993 was collected on a single lake near the center of the transect. Across the transect, midwestern sources appear to dominate Pb and Cd concentrations, while Sb appears to be derived from midwestern sources as well as local and/or industrial East Coast sources. In all samples, the highest Na, Cl and Mg concentrations reflect a marine influence, but at some transect sites roadspray aerosol appears to contribute to Na and Cl concentrations. The regional pattern of Ca, K, Mn and Sr concentrations and Mn/Sr ratios indicate that woodsmoke may be an important winter source of these elements at some sites. In all samples, H⁺ is strongly correlated with NO₃ ^? (R² = 0.97) and mean NO₃ ^?/SO₄ ^2? molar ratios of 6.4 for transect samples, and 4.7 for temporal samples, are higher than mean NO₃ ^?/SO₄ ^2? reported for other precipitation studies in the same region. The contribution of NO₃ ^? to the snowpack greatly exceeds that of SO₄ ^2?, and may be a major source of acidity in aquatic ecosystems during snowmelt.     

Two-dimensional simulation of airflow and carbon dioxide transport over a forested mountain: Part I: Interactions between thermally-forced circulations

Local-scale wind regimes over an idealized two-dimensional (2D) mountain having similar horizontal and vertical scales as Vancouver Island were investigated using a high-resolution mesoscale model. The model-generated flow outputs were then used as ersatz data to assess the impact of limiting assumptions in the eddy-covariance (EC) method as well as the effectiveness of tower flux data time-filtering for the main tower site of the Fluxnet-Canada British Columbia flux station. In this paper, we describe the simulated mesoscale and local-scale flow regimes, and in Part II we describe their use in assessing tower-data analyses of CO₂ fluxes.The numerical model was enhanced to include parameterizations for: tree drag, radiation effects of forest canopies on the surface energy budget, and soil heat conduction. Simulations were performed both over an idealized bare hill and over a forested hill. The simulated flow involved interactions between the land/sea breeze, convective thermals, and mountain circulations under fair-weather conditions.The resulting simulated winds over the forested slope were much weaker than those over a bare slope. The nocturnal drainage flow over the forested slope was separated into sub-canopy and above-canopy regimes due to the temperature inversion at treetop. The strongest downslope flows occurred above the canopy with a minimum velocity occurring in the upper canopy where canopy drag is the strongest. The overturning of large convective eddies during daytime resulted in intermittent downslope flows under the canopy during the day. Wind regimes had a rapid shift from upslope to downslope flow above the canopy just after sunset. Further testing will be made using 3D simulations with higher resolution.     

Nematode community structure as indicator of soil functioning in European grassland soils

This investigation analyses whether soil nematode diversity is correlated with soil functional parameters to serve as bioindicator of soil functioning. The analysis focuses on the interrelations of nematofauna, microflora, and soil nitrogen pools. The sites studied represent six major European grassland types: Northern tundra, atlantic heath, wet grassland, seminatural temperate grassland, East European steppe, and mediterranean garigue. Continental and local climate gradients were combined to a wide and continuous range of microclimate conditions. Nematode richness, as indicated by the number of genera, was highest under temperate conditions and declined towards the climatic extremes. Differences in richness affected all nematode feeding types proportionally. Nematode richness was the only parameter among a range of 15 alternatives tested that exhibited consistent correlations with mass and activity parameters of both nematofauna and microflora in the mineral grassland soils (garigue, wet grassland, seminatural grassland, steppe). In the same soils, the nematode Maturity Index was the best indicator of nitrogen status. We conclude that a high nematode richness can generally be seen as a good indicator of an active nematofauna and microflora in mineral grassland soils, and hence as an indicator of the decomposition function. The prospects of exploiting nematode diversity as an indicator of soil functioning are critically discussed.     

Mineralization of methionine sulphur in soils and forest floor layers

A1-horizon soils and 01, 02 forest floor layers from a mixed mature hardwood forest rapidly converted methionine-S to readily-available (salt-extractable) and less readily-available (acid- and base-extractable) inorganic sulphate (SO^?2₄). It is suggested that this latter conversion represents the incorporation into organic matter of a portion of the (SO^?2₄) released by mineralization. On a dry weight basis, the 02 layer of the forest floor was the most active with respect to both conversions. Moreover, capacities for mineralization and (SO^?2₄) incorporation decreased with increasing sample depth within the mineral horizon. Both conversions were dependent upon temperature and duration of incubation and were absent from samples which had been autoclaved. Sodium azide and the broad-spectrum antibiotic, tetracycline also inhibited each conversion to varying extents depending upon the type of sample incubated with methionine.     

On-line MS/MS monitoring of acrylamide generation in potato- and cereal-based systems

An on-line MS/MS technique was used to study the generation of acrylamide in rye-, wheat-, and potato-based systems during cooking. Acrylamide release to the gas phase was monitored continuously and was correlated with the acrylamide content of samples using a calibration based upon the partition of [1,2,3-(13C3)]acrylamide. On-line results at 180 degrees C were compared with data relating to the same systems obtained through GC-MS analysis. Agreement between the two techniques was notable, both in terms of the temporal profiles of acrylamide generation and when comparing the relative magnitudes of results for potato, wheat, and rye determined by each method. The effects of pH (citric acid) and added amino acids (soy protein hydrolysate) on the generation of acrylamide in hydrated potato flake were modeled at 180 degrees C. It was concluded that a combined treatment of low levels of each additive could result in significant reductions in acrylamide, although the effects of such treatments on sensory properties such as color and flavor remain to be evaluated.     

Meat and ascorbic acid can promote Fe availability from Fe-phytate but not from Fe-tannic acid complexes

This study utilized an in vitro digestion/Caco-2 cell model to determine the levels of ascorbic acid (AA) and "meat factor" needed to promote Fe absorption from Fe complexed with phytic acid (PA) or tannic acid (TA). AA reversed the inhibition of Fe absorption by PA beginning at a molar ratio of 1:20:1 (Fe:PA:AA) but essentially had no effect on the Fe complexed with TA. Fish also reversed the inhibition of Fe uptake by PA but not by TA. TA and fish decreased total Fe solubility. Iron in the presence of PA was highly soluble. AA, but not fish, increased the percentage of soluble Fe as Fe2+ in the presence of both inhibitors. The results indicate that monoferric phytate is a form of Fe that can be available for absorption in the presence of uptake promoters. In contrast, a TA-Fe complex is much less soluble and unavailable in the presence of promoters.     

Patterns in the modern decline of western Australia''s vertebrate fauna: Causes and conservation implications

The conservation status of terrestrial vertebrates occurring on the mainland of Western Australia was assessed. Extinctions and declines are virtually confined to non-flying mammals with mean adult body weights between 35 g and 4200 g. Variation in patterns of attrition within this critical weight range (CWR) can be explained almost entirely by a combination of regional patterns in rainfall and, to a lesser extent, species' habitat and dietary preferences. Similar patterns of mammal attrition were recognisable throughout the continent, except that the CWR was 35 to 5500 g.

Environmental changes since European settlement have emulated an increase in aridity by reducing the environmental productivity available to vertebrates. These include the diversion of environmental resources to humans and introduced species, and a reduction in vegetative cover by exotic herbivores and changed fire regimes. Our analyses support the view that the reduction in available productivity has caused CWR mammals to suffer the greatest attrition because of their limited mobility, but relatively high daily metabolic requirements. The direct elimination of confined populations of mammals by exotic predators has exacerbated this attrition. We derive priorities for the conservation of Australian mammals.