The impact of extraction methodologies on the toxicity of sediments in the zebrafish (Danio rerio) embryo test

Purpose

Traditionally, methods for sediment extractions are characterised using chemical analyses. However, in order to evaluate sediment extracts with regard to biological effects and, thus, bioaccessibility, extraction methods have to be compared to effect data obtained from experiments with in situ exposure scenarios, i.e., sediment contact tests. This study compares four extraction methods and sediment contact test data from a previous project with respect to predictive power in the fish embryo test with zebrafish (Danio rerio).

Materials and methods

A natural and an artificial sediment spiked with a mixture of six organic pollutants (2,4-dinitrophenol, diuron, fluoranthene, nonylphenol, parathion and pentachlorophenol) were extracted using (a) membrane dialysis extraction (MDE), (b) a Soxhlet procedure, (c) hydroxypropyl-??-cyclodextrin (HPCD) or (d) Tenax^®-TA. Whereas the former two are regarded being exhaustive with respect to non-covalently bound contaminants, the latter two are considered to predict bioaccessibility. Resulting extracts were tested in the fish embryo assay with D. rerio for embryotoxic and teratogenic potential.

Results and discussion

Mortalities caused by organic extracts from Soxhlet extraction and MDE were high. However, HPCD extracts turned out to be at least as effective as extracts obtained with these two methods. One possible reason might be short ageing time of the spiked sediments. Only Tenax^®-TA extracts gave results comparable to the sediment contact assay for natural sediment, but revealed low reproducibility. Significant differences between natural and artificial sediment were found for extracts obtained with techniques using native (i.e., non-freeze-dried) sediments, i.e., HPCD and Tenax^®-TA. In contrast, MDE and Soxhlet extracts used freeze-dried sediment and did not differentiate between natural and artificial sediment. Therefore, freeze-drying has likely altered and equalised sediment properties that influence accessibility, such as composition of bacterial communities and organic matter quality.

Conclusions

Four extraction methods were successfully characterised with respect to their stringency and predictiveness for bioaccessibility. MDE was confirmed as an alternative to Soxhlet extraction. High mortalities induced by HPCD extracts underline the need to include ageing into consideration when assessing sediments. Although Tenax^®-TA may basically be used to predict bioaccessibility in the fish embryo test, the high variability observed warrants further investigation of the relation between effect and extractability. Apparently, freeze-drying can severely affect sediment properties, potentially eliminating individual properties of natural sediments.     

Comparison of different exhaustive and biomimetic extraction techniques for chemical and biological analysis of polycyclic aromatic compounds in river sediments

Purpose

The importance of the bioaccessible fractions of particle-bound contaminants is a key feature for the assessment of their likely risks to the aquatic environment. The present study investigated the extractability and potential toxicity of polycyclic aromatic hydrocarbons (PAHs) in river sediments. This study combined chemical with bioanalytical tools to establish a more in-depth insight into the relationship between the extractability of sediment contaminants, the chemodynamic properties of each extraction approach, and resulting toxicity.

Materials and methods

Sediment samples were treated with different extraction methods, namely Soxhlet extraction with acetone (SOX), membrane dialysis extraction (MDE) with n-hexane, ultrasonic extraction with acetone (USE), and extraction with (2-hydroxpropyl)-??-cyclodextrin (HBCD). The extracts were analyzed for PAHs using gas chromatography/mass spectrometry and tested using the neutral red retention assay and the ethoxyresorufin-O-deethylase (EROD) induction assay with the permanent fish cell line RTL-W1.

Results and discussion

The SOX and MDE approaches were comparable in their extraction power regarding PAHs and their cytotoxicity. However, the EROD activity differed between SOX and MDE, possibly due to retardation effects of the polyethylene membrane used in MDE. Thus, macromolecular components of the sediment were not extracted and therefore did not contribute to toxicity. HBCD extraction provided 3.4?% of the total PAH content in the sediments and might be an appropriate approach to predict the bioaccessible fraction. USE showed an extraction power between the HBCD approach and the MDE as well as SOX and MDE method. Hence, USE is neither appropriate for exhaustive extraction nor for biomimetic extraction.

Conclusions

MDE and SOX have been approved for the exhaustive extraction of PAHs from sediments for a worst case assessment of effect potentials. The influence of the low-density polyethylene membrane and the experimental conditions on the diffusion of compounds with broad physical?Cchemical properties, and the establishment of models such as quantitative-structure relationships for the prediction of diffusion behavior of a broad range of compounds require further assessment. HBCD was confirmed as a method providing a certain, putatively bioaccessible, fraction. Mechanistic research is required regarding desorption and uptake kinetics of particularly bound compounds with different physical?Cchemical properties in the solid phase?Cwater?CHBCD system to get more insight in the underlying partitioning processes.     

Extraction of polycyclic aromatic hydrocarbons from spiked soil

A homogenization method was evaluated for extracting polycyclic aromatic hydrocarbons (PAHs) from soils. Fifteen PAHs were spiked and recovered from 2 soils at concentrations ranging from 1 to 1000 micrograms/g, using the homogenization method and a Soxhlet extraction method. Each extraction method performed well in removing the 15 PAHs from both soils over a broad range of concentrations. In general, Soxhlet extraction yielded slightly but significantly (P less than 0.05) higher recoveries than did the homogenization method. The homogenization method, however, was easy to use, and the extraction step turnaround time was less than 15 min/sample. The method should be suitable for other applications requiring the extraction of hydrophobic organic compounds from soils.     

Differentiation between bioavailable and total hazard potential of sediment-induced DNA fragmentation as measured by the comet assay with Zebrafish embryos

Goals, Scope and Background  While water quality strongly improved over decades in the Rhine River, sediments still reflect elapsed contaminations of organic pollutants and heavy metals. In comparing genotoxic effects induced by both sediment extracts and whole sediments, a ratio of bioavailable toxicity and total extractable toxicity is obtained. Since contaminated sites whose contaminants are toxic and as well bioavailable present an elevated risk to the ecosystem, such ratios may be used as a warning signal to identify sites of primary concern. Methods  Accordingly, two different exposure scenarios were compared to reveal the genotoxic potential of 18 sediment samples derived from 9 sample sites along the River Rhine. For assessment of effects on genome integrity, DNA fragmentation was measured using the comet assay with primary cells isolated from zebrafish embryos previously exposed to either organic sediment extracts or freeze-dried sediments at sublethal concentrations. Additionally, chemical data were used to determine responsible pollutants and correlate them with biological effects. Results  Whereas 17 out of 18 sediment extracts caused significant DNA damage to the embryo cells, only 4 native sediments showed a genotoxic potential. Thus, under field-like exposure conditions, a major part of potentially genotoxic compounds seem to remain particle-bound and ineffective, as shown for whole sediment exposure. Conversely, the organic extracts seem to contain enriched concentrations even of hardly soluble substances. Hence, organic extracts may be used as a screening tool to address potentially polluted sites, even though the relevance of these results for the field situation may be questionable. Investigations on native sediments determined few sites with bioavailable and therefore ecologically most relevant genotoxic sediment compounds. Discussion  However, these results may underestimate the total hazard potential of sample sites with hardly bioavailable substances. Chemical data revealed a variety of anthropogenic pollutants, ranging from PAHs to heavy metals. Nevertheless, chemical data on the measured priority pollutants did not fully explain the pollution pattern of the bioassays but clearly determined substances of concern (e.g., HCB, heavy metals) in particular sample sites. Conclusions  There is a striking advantage in assessing the genotoxicity by means of different exposure scenarios that focus on either bioavailable or extractable fractions, as the combination of the results allows obtaining information on specific properties of the genotoxicants and their bioavailability. An additional correlation with chemical data should be required to identify priority pollutants, as long as the responsible contaminant is known a priori. As many studies revealed inherent failures of such a correlation, an effect-driven analysis of pollutants is recommended as a promising tool to identify even non-priority pollutants by means of their ecotoxicological effectiveness.     

Redistribution of organic pollutants in river sediments and alluvial soils related to major floods

Background, Aims, and Scope  More frequent occurrence of stronger floods in Europe as well as in other parts of the world in recent years raises major concern about the material damages, but also an important issue of contamination of the affected areas through flooding. The effects of major floods on levels and distribution of contamination with hydrophobic organic pollutants were examined from the continuous set of data for floodplain soils and sediments from a model industrial area in the Czech Republic where a 100-year flood occurred in 1997. The goal of this study was to evaluate the risk related to contamination associated with such extensive natural events and characterize the spatial and temporal distribution and dynamics of pollutants related to a major flooding shortly after the floods and also in the time period several years after floods. Methods  Sediments and alluvial soils from fourteen sites each were repeatedly sampled during the period from 1996 until 2005. The sampling sites represented five regions. Collected top-layer sediment and soil samples were characterized and analyzed for hydrophobic organic pollutants PCBs, OCPs and HCB using GC-ECD and PAHs using a GC-MS instrument. Spatial and temporal differences as well as the relative distribution of the pollutants were examined in detail by statistical analysis including multivariate methods with special emphases placed on the changes related to floods. Results  The organic pollutants levels in both alluvial soils and sediments exceeded the safe environmental limits at numerous sites. Pollutants concentrations and relative distribution as well as organic carbon content in both sediment and floodplain soils were significantly affected by the flooding, which resulted in a decrease of all studied contaminants in sediments and significant rise of the PAH pollution in the flooded soils. There was a unique and highly conserved PAH pattern in soils regardless of the floods and greater changes in PAH pattern in sediments related to floods. The relative distribution of individual PAHs reflected a combustion generated PAH profile. PAH levels in the river sediments rose again at the sites with continuous sources several years after floods. Discussion  The results showed different dynamics of PAHs and PCBs during the floods when PAHs were redistributed from the sediments to alluvial soils while PCBs have been washed out of the study regions. The data reveal longer contamination memory and consistent contamination pattern in soils, whereas sediments showed more dynamic changes responding strongly to the actual situation. The stable PAH pattern within the regions also indicates that a relative amount of all compounds is comparable across the samples and, thus, that the sources at different sites have similar character. Conclusions  Sediments have the potential to function as a secondary source of contamination for the aquatic ecosystem, but also for the floodplain soils and other flooded areas. The floods served as a vector of PAHs contamination from sediments to soils. The reloading of river sediments in time, namely with PAHs, due to present sources increases their risk as a potential source in the next bigger flood event both to the downstream river basin and affected alluvial soils. Recommendations and Perspectives  The results stress the importance of including the floodplain soil contamination in the risk assessment focused on flood effects. Floodplain soils have stable long-term environmental memory related to contamination levels, pattern and distribution, whereby they can provide relevant information on the overall contamination of the area. The sediments will continue to serve as a potential source of contaminants and alluvial soils as the catchment media reflecting the major flood events, especially until effective measures are taken to limit contamination sources. ESS-Submission Editor: Dr. Henner Hollert (Hollert@uni-heidelberg.de) This article is openly accessible!     

Hazard identification of contaminated sites—ranking potential toxicity of organic sediment extracts in crustacean and fish

Background, aim, and scope  It is well known that contaminated sediments represent a potential long-term source of pollutants to the aquatic environment. To protect human and ecosystem health, it is becoming common to remediate contaminated sites. However, the great cost associated with, e.g., dredging in combination with the large numbers of contaminated sites makes it crucial to pinpoint those sites that are in greatest need of remediation. In most European countries, this prioritization process has almost exclusively been based on chemical analyses of known substances; only seldom toxicity data has been considered. The main objective of the current study was therefore to develop a tool for hazard identification of sediment by ranking potential toxicity of organic sediment extracts in a crustacean and a fish. A secondary objective was to investigate the difference in potential toxicity between compounds with different polarities. Materials and methods  Early life stages of the crustacean Nitocra spinipes and the fish Oncorhynchus mykiss, which represent organisms from different trophic levels (primary and secondary consumer) and with different routes of exposure (i.e., ingestion through food, diffusive uptake, and maternal transfer), were exposed to hexane and acetone fractions (semi-polar compounds) of sediment from five locations, ranging from heavily to low contaminated. Preliminary tests showed that the extracts were non-bioavailable to the crustacean when exposed via water, and the extracts were therefore loaded on silica gel. Rainbow trout embryos were exposed using nano-injection technique. Results and discussion  Clear concentration–response relationships of both mortality and larval development were observed in all tests with N. spinipes. Also for rainbow trout, the observed effects (e.g., abnormality, hemorrhage, asymmetric yolk sac) followed a dose-related pattern. Interestingly, our results indicate that some of the locations contained toxic semi-polar compounds, which are normally not considered in risk assessment of sediment since they are focused on compounds isolated in the hexane fraction. Conclusions  The ranking of the five sediments followed the expected pattern of potential toxicity in both organisms, i.e., sediments with known pollution history caused major effects while reference sediments caused minor effects in the two test systems. Silica gel turned out to be an excellent carrier for exposure of N. spinipes to very hydrophobic and otherwise non-bioavailable sediment extracts. Recommendations and perspectives  Since both test systems demonstrated that a substantial part of the potential toxicity was caused by semi-polar compounds in the acetone fractions, this study enlightens our poor understanding of which compounds are causing adverse effects in environmental samples. Therefore, by investigating potential toxicity (i.e., hazard identification) as a first screening step in prioritizing processes, these implications could be avoided. For proper sediment risk assessment, we however recommend whole sediment toxicity tests to be used for selected sites at following tiers.     

Amino acids and amino sugars extracted by EUF from a sandy soil incubated with green manure,bacterial biomass or cellulose

The extraction of soils by the electro-ultrafiltration (EUF) method yields organic N which has been used as an index for mineralisable N in soils. This EUF extractable organic fraction contains a mixture of various N compounds not yet completely identified. It has been proposed that the amino N compounds are more indicative for the potentially mineralisable N in soils than the total organic N extracted (Mengel et al., 1999). An amendment of soils with easily mineralisable organic matter may, therefore, alter the amino N concentrations of the organic N extracted. Our determination of the amino N compounds aimed to prove this hypothesis. The principle of our experiment was to mix soil with green manure, bacterial biomass and cellulose, respectively, and to incubate the treated soil aerobically for 80 days at 20°C in the laboratory. Control treatments without organic amendment were also incubated. Soil samples were taken several times during the incubation period and analysed for the inorganic N (NO₃⁻-N and NH₄⁺-N) and for the EUF extractable organic N. Amino acids and amino sugars were determined in the hydrolysed EUF extracts. The concentrations of amino acids and amino sugars in the organic N extracted varied with time and differed between the treatments. Glutamic acid has been found to be the most relevant amino acid in the EUF extracts and was particularly indicative for the existence of mineralisable green manure in the soil. Glucosamine was the most relevant amino sugar in the EUF extracts and this amino sugar appears to be indicative for the easily mineralisable relics of microbial cells in the soil.     

Fractionation of dissolved organic carbon in soil water: Effects of extraction and storage methods

Abstract

We measured the concentration and composition (sensu Leenheer, 1981) of dissolved organic carbon (DOC) in lysimeter solutions from the forest floor of a spruce stand in Maine and in laboratory extracts of organic (Oa horizon) and mineral soils collected from various forests in Maine, New Hampshire, and Vermont. All soils were acid Spodosols developed from glacial till. The effects of different storage, extraction and filtration methods were compared. Extracts from Oa horizons stored fresh at 3°C contained a larger fraction of hydrophobic neutrals than lysimeter forest floor solutions (31 and 4% of DOC in stored and lysimeter solutions, respectively), whereas extracts from Oa horizons which had been extracted, incubated at 10–15°C, and extracted again had DOC compositions similar to that in lysimeter solutions. Mechanical vacuum and batch extractions of Oa horizons yielded DOC similar in concentration and composition if the extracts were filtered through glass fiber filters. Nylon membrane filters, however, removed more hydrophobic acids from batch extracts. Dissolved organic carbon extracted from frozen, air‐dry, and oven‐dry Oa and Bh horizons was relatively rich in hydrophilic bases and neutrals and was similar to that released after chloroform fumigation, indicating that common soil‐storage methods disrupt microbial biomass.     

Extraction and characterization of water–repellent materials from Australian soils

Organic materials responsible for water–repellency in some Australian soils were extracted with an amphiphilic mixture of iso -propanol/15.7 m ammonia (7:3, v:v) in a Soxhlet apparatus, after which the water–repellent soils were rendered wettable. The successful extraction by an organic solvent system indicates that the bulk of hydrophobicity in these soils is not covalently linked to the surface of the sand. The extracted materials restored hydrophobicity on acid washed sands or ignited sands at levels comparable to the original soils.
Spectroscopic and chromatographic examination of the extracted materials indicated that both free and esterified long–chain, 16–32 carbon atom, fatty acids were present with a bimodal distribution showing maxima at C₁₆ and C₂₂. The ¹³C–NMR and infrared spectra of the most hydrophobic extract suggest that hydrophobicity is caused by molecules with extensive polymethylene chains. Calculations with model compounds indicate that at least a close packed monolayer is required before measurable hydrophobicity can be detected with the molarity of ethanol droplet penetration test.     

Comparison of methods for the exhaustive extraction of hypericins, flavonoids, and hyperforin from Hypericum perforatum L

Renewed interest in plant-derived drugs has led to an increased need for efficient extraction methods. Hypericum perforatum L. contains several groups of bioactive compounds with noteworthy pharmacological activities. Direct sonication of H. perforatum was investigated and compared with conventional maceration, indirect sonication, Soxhlet extraction, and accelerated solvent extraction (ASE). Highly selective liquid chromatography/tandem mass spectrometry analysis showed that the content of six investigated active compounds (hypericin, pseudohypericin, hyperoside, rutin, quercitrin, and hyperforin) in extracts obtained by direct sonication was significantly higher than in extracts obtained by the other methods. The active compound contents increased on increasing the ultrasonic power from 40 to 60 W when using direct sonication. Conventional maceration gave the lowest amount of analyzed active compounds. Soxhlet extraction gave better results than ASE or indirect sonication.     

Properties of and heavy metal complexation by aqueous humic extracts

Aqueous extracts of terrestrial and underwater soils were prepared by Soxhlet extractions at temperatures between 100 and 37°C (under reduced pressure). From the law of mass action, stability constants were derived for the complexation of Cd, Cu, and Pb by these extracts. The activity of the heavy metals was determined polarographically, whereas the concentration of acid functional groups was used for ligand activity. Visible spectra, electrical conductivity, organic matter contents, and complexation constants depended on extraction temperature as well as on soil properties. In most cases, complex stability increased with increasing pH of the polarographic medium, but sometimes maximum stability was found around pH 7.     

Comparison among soil Series and extraction methods for the analysis of trifluralin

Accurate analytical procedures are needed to improve understanding of the fate and transport of trifluralin, a chemical widely used as a herbicide. Analytical determination of trifluralin is challenging due to its hydrophobic, yet volatile, character and its tendency to degrade into numerous metabolites. In this research, efficient analytical methods for fortified and field-incurred soils were developed for simultaneous quantitation of trifluralin, I [2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine, CAS Registry No. 1582-09-8; CAS Registry No. have been provided by the author], a trifluralin metabolite, II [2,6-dinitro-N-propyl-4-(trifluoromethyl)benzenamine, CAS Registry No. 2077-99-8], and a related trifluoromethyldinitroaniline isomer of trifluralin, III [2,4-dinitro-N,N-dipropyl-6-(trifluoromethyl)benzenamine, CAS Registry No. 23106-20-9]. Extractions of trifluralin (0.5 and 2.5 microg/g) from silt loam, sandy loam, and silty clay soils were compared. A method was developed for the supercritical fluid extraction of trifluralin from soil using modified supercritical carbon dioxide, and the effects of cosolvent, pressure, and flow rate on recovery were evaluated. Supercritical fluid extraction was compared to liquid vortex extraction and automated Soxhlet (soxtec) extraction. Solid-phase extraction was examined for purifying soil extracts. Protocols were developed for analysis of extracts by gas and/or liquid chromatography. Immunoassay was investigated but proven to be impractical for this analysis. Soil properties and extraction methods were observed to affect the level of coextracted background interferences. Trifluralin exhibited concentration-dependent recovery regardless of soil series or extraction method.     

Organic compounds of different extractability in total solvent extracts from soils of contrasting water repellency

Previous studies examining organic compounds that may cause water‐repellent behaviour of soils have typically focussed on analysing only the lipophilic fraction of extracted material. This study aimed to provide a more comprehensive examination by applying single‐ and sequential‐accelerated solvent extraction (ASE), separation and analysis by GC/MS of the total solvent extracts of three soils taken from under eucalypt vegetation with different degrees of water repellency. Water repellency increased in all the soils after extraction with DCM/MeOH (95:5), but was eliminated with iso‐propanol/ammonia (95:5). Quantities of major lipid compound classes varied between solvents and soils. Iso‐propanol/ammonia (95:5) solvent released saccharides, glycerol, aromatic acids and other polar organic compounds, which were more abundant in fractionated extracts from the single extraction and the third step sequential ASE extraction, than in the extracts from the DCM/MeOH ASE solvent. Dominant compounds extracted from all soils were long‐chain alkanols (>C₂₂), palmitic acid, C₂₉ alkane, β‐sitosterol, terpenes, terpenoids and other polar compounds. The soil with the lowest repellency lacked >C₁₈ fatty acids and had the lowest concentrations of alkanols (C_26,C₂₈ and C₃₀) and alkanes (C₂₉, C₃₁), but a greater abundance of more complex polar compounds than the more repellent soils. We therefore speculate that the above compounds play an important role in determining the water repellency of the soils tested. The results suggest that one‐stage and sequential ASE extractions with iso‐propanol/ammonia and subsequent fractionation of extracts are a useful approach in providing a comprehensive assessment of the potential compounds involved in causing soil water repellency.     

A Novel Particle Contact Assay with the Yeast Saccharomyces cerevisiae (8 pp)

Goal, Scope and Background   Numerous xenobiotics released into surface waters are transferred to suspended particulate matter and finally attached to sediments. Aquatic organisms may be exposed to them by direct particle feeding, by physical contact with contaminated surfaces as an exposure route, and by the uptake of dissolved contaminants after equilibration via the free water phase. In order to assess potential sediment toxicity, each of these exposure routes has to be addressed. This paper presents a newly developed particle contact assay that uses the fermentation performance of a specific Saccharomyces cerevisiae strain for the assessment of toxic effects in sediments. The test procedure is based on the characteristic feature of growing yeast cells to attach to sediment particles, which are also relevant for the accumulation of contaminants. The physical contact with lipophilic contaminants mirrors an exposition pathway for the direct uptake into the cells. In order to quantitatively characterize the toxic effects of particle attached pollutants on the fermentation performance, unpolluted native reference sediment was spiked with representatives for widely distributed anthropogenic contaminants. Methods   Saccharomyces cerevisiae was established as sensitive eukaryotic microorganism for the ecotoxicological assessment of particle attached anthropogenic contaminants in freshwater sediments. For this purpose, yeast cells were cultivated in sediment samples and the resulting fermentation performance was continuously measured. Sediments artifically spiked with HCB, PCB, g-HCH, DDT, and benzo(a)pyrene and solutions of each contaminant were comparatively investigated by means of their adverse effects on yeast fermentation performance. Additionally, four native river sediments characterized by increasing levels of pollution were assessed by the yeast particle contact assay, and simultaneously by standard aquatic tests with algae, daphniae, and luminescent bacteria using pore water and elutriates. Results of the bioassays were related to specific sediment contamination with respect to metals and organic priority pollutants. Results and Discussion   In sediments spiked with PCB and benzo(a)pyrene fermentation, performance was affected extensively below concentrations inhibiting fermentation in contaminant solutions. This suggests a high efficiency of the exposure route by physical contact. The fermentation performance was only slightly affected by single lipophilic pollutants, whereas mixtures of individually spiked sediments caused critically reduced fermentation performance suggesting additive synergistic effects. Native river sediments modestly to critically polluted by hazardous organic compounds lead to a slightly to dangerously reduced fermentation performance in the yeast contact assay. These inhibitory effects were much less pronounced in the standard bioassays conducted with algae, daphniae and luminescent bacteria, applying pore waters and elutriates as sample matrices. Using pore water, inhibition was measured only in the most polluted sediment, elutriates lead to a slight inhibition of the algal growth in the undiluted sample only. These results indicate an improved sensitivity of the yeast particle contact assay compared to the standard assays, due to uptake and physical cell contact as additional routes of exposure. Conclusion   The yeast particle contact assay is a valuable tool for the assessment of ecotoxicological potential in freshwater sediments. Since the assay addresses physical contact as an exposure route, it indicates bioavailability of lipophilic compounds in sediments. Outlook   The sensitive indication of bioavailable contaminants associated to sediment particles by the newly developed yeast particle contact assay recommends it as a complementary microbial bioassay in a test battery for assessing major pathways of contaminants in whole sediments.     

A new sediment contact assay to assess particle-bound pollutants using zebrafish (<Emphasis Type="Italic">danio rerio</Emphasis>) embryos

Goal, Scope and Background. Based on a bioassay battery covering only primary producers and consumers as well as degraders, the potential ecological hazard of sediments to vertebrates cannot be estimated comprehensively. Therefore, there is an urgent need to develop and standardize integrated vertebrate-based test systems for sediment investigation strategies. Whereas vertebratebased in vitro systems have frequently been used for the investigation of aqueous samples, there is a significant lack of whole sediment assays. Thus, the purpose of the present study was: (1) to develop a rapid and reliable, but comprehensive method to investigate native sediments and particulate matters without preceding extraction procedures; (2) to compare the hazard potential of solid phase sediments to the effects of corresponding pore waters and organic extracts in order to characterize the bioavailability of the particle-bound pollutants; and (3) to relatively evaluate the embryotoxic effects of sediments from the catchment areas of the rivers Rhine, Neckar and Danube. Methods (or Main Features).  To investigate the toxicity of sediment samples on vertebrates, the standard embryo toxicity test with the zebrafish (Danio rerio; Hamilton-Buchanan 1922) according to DIN 38415-6 was modified with respect to exposure scheme and toxicological endpoints. Sediments from the catchment area of the Neckar River were assessed using pore waters, acetonic extracts and native sediments in order to get inside into the potential bioavailability of particle-bound pollutants. A comprehensive test protocol for the investigation of native sediments in the embryo toxicity test with the zebrafish is presented. Results and Discussion.  The fish embryo assay with Danio rerio can be carried out with both aqueous and organic sediment extracts as well as native (whole, solid phase) sediment samples. Elongation of exposure time from 48 to up to 196 h significantly increased the mortality. Using the fish egg assay with native sediments, a broad range of embryotoxic effects could be elucidated, including clear-cut dose-response curves for the embryotoxic effects of contaminated sediments; in contrast, absence of embryotoxic effects could be demonstrated even for the highest test concentrations of unpolluted sediments. With native sediments, embryotoxicity was clearly higher than with corresponding pore waters, thus corroborating the view that — at least for fish eggs — the bioavailability of particle-bound lipophilic substances in native sediments is higher than generally assumed. The relative ranking of sediment toxicity was identical using both native sediments and sediment extracts, EC₂₀ values of the latter, however, being eight time lower higher than with the native sediments. A comparison of the embryo toxic effects of samples from the Neckar area with locations along the Rhine and Danube rivers elucidated a broad range of results, thus indicating different levels of contamination. Conclusions.  A modified protocol of the zebrafish embryo test allows the assessment of sediment toxicity in both aqueous extracts and native sediments. The isolated investigation of pore waters may result in a clear-cut underestimation of the bioavailability of lipophilic particle-bound substances (as determined by native sediments). Recommendations and Perspectives.  The zebrafish embryo test with native (whole, solid phase) sediments appears very promising for the evaluation of the bioavailable fraction of lipophilic particle-bound substances and can therefore be recommended for the evaluation of vertebrate toxicity in tiered sediment test strategies and dredging directives such as the HABAB-WSV. Whereas acetone extracts may be tested as a rough estimation of embryotoxicity, native sediment samples will provide a more comprehensive and realistic insight into the bioavailable hazard potential     

Characterization and optimization of the preparation procedure for solution P-31 NMR analysis of organic phosphorus in sediments

Purpose

The single-step sodium hydroxide?Cethylenediaminetetraacetic acid (NaOH?CEDTA) extraction is currently the most common preparation technique for the measurement of organic phosphorus (P) composition in sediments using solution ³¹P nuclear magnetic resonance (NMR). In this study, detailed investigations were conducted to evaluate the performance of this technique, with an objective of finding an optimal procedure for the measurement of sediment organic P.

Materials and methods

A single-step extraction with NaOH?CEDTA was investigated on two types of sediment, i.e., Fe/Al-rich sediment and calcareous sediment. The influence of different sediment preparation methods, NaOH?CEDTA compositions, solid:solution ratios, extraction time, pre-concentration techniques, and NMR sample frozen storage time on P extraction and solution ³¹P NMR analysis were investigated.

Results and discussion

Both air- and freeze-drying increased organic P extraction for the calcareous sediment. An extraction time of 16?h was sufficient for quantitative recovery of extracted organic P for both Fe/Al-rich and calcareous sediments. The use of a 1:8 solid:solution ratio achieved stronger NMR signals and greater P diversity than the use of a 1:20 ratio. Extraction of the two sediments with 0.25 NaOH?C50?mM EDTA favored ³¹P NMR detection by reducing the relaxation times required and the risk of organic P degradation compared to the use of stronger NaOH?CEDTA solutions. Rotary evaporation was a better technique for pre-concentration of the NaOH?CEDTA extracts than freeze drying. The concentrated extracts could be preserved by freezing (?20?°C) for at least 2?months.

Conclusions

An optimized procedure was developed based on these investigations, including freeze-drying of fresh sediments, extraction with 0.25?M NaOH?C50?mM EDTA for 16?h using a solid:solution ratio of 1:8, pre-concentration of the extract to the level of ??10 times of its original concentration using rotary evaporation, and storage of the NMR sample at ?20?°C until ³¹P NMR analysis.     

土壤/沉积物中天然有机质对疏水性有机污染物的吸附作用

通过对土壤/沉积物中天然有机质的组成分析,阐述了天然有机物在吸附疏水性有机污染物过程中所起的重要作用,其中包括可溶性有机质的增溶作用和不溶性有机质的吸附作用。本文着重探讨了天然有机质结构的异质性对吸附作用的影响,导致吸附等温线的非线性,从而从土壤/沉积物有机质的微观结构上进一步解释了非线性吸附机理。     

Does biochar interfere with standard methods for determining soil microbial biomass and phenotypic community structure?

Due to its high sorption affinity for organic compounds, biochar may interfere with extraction procedures involving such compounds used for microbially-related assays commonly applied to soils. Here we assessed the impact of two biochars (derived from pine bark and produced at 300 and 600 °C) at three concentrations (0, 12.5, and 50 g kg⁻¹) in three distinct arable soils with contrasting textural classes (loamy sand, sandy loam, and clay) on the determination of soil microbial biomass C by fumigation–extraction, fungal biomass by ergosterol analysis, and microbial community structure as defined by phospholipid fatty acid (PLFA) profiling. Biochar did not affect the apparent concentration of soil microbial biomass C and had no significant impact on apparent PLFA profiles. By contrast, the apparent extraction efficiency of ergosterol was affected dependent on soil type, biochar production temperature, and biochar concentration. Nonetheless, ergosterol contents of biochar-amended soils can be accurately estimated by correcting for reduced recovery using an ergosterol spike.     

Source relationships between streambank soils and streambed sediments in a mercury-contaminated stream

Purpose

In contaminated streams, understanding the role of streambank and streambed source contributions is essential to developing robust remedial solutions. However, identifying relationships can be difficult because of the lack of identifying signatures in source and receptor pools. East Fork Poplar Creek (EFPC) in Oak Ridge, TN, USA received historical industrial releases of mercury that contaminated streambank soils and sediments. Here, we determined relationships between the contaminated streambank soils and sand-sized streambed sediments.

Materials and methods

Field surveys revealed the spatial trends of the concentrations of inorganic total mercury (Hg) and methyl mercury (MeHg), Hg lability as inferred by sequential extraction, particle size distribution, and total organic carbon. Statistical tests were applied to determine relationships between streambank soil and streambed sediment properties.

Results and discussion

Concentrations of Hg in streambank soils in the upper reaches averaged 206 mg kg^?1 (all as dry weight) (n?=?457), and 13 mg kg^?1 in lower reaches (n?=?321), while sand-sized streambed sediments were approximately 16 mg kg^?1 (n?=?57). Two areas of much higher Hg and MeHg concentrations in streambank soils were identified and related to localized higher Hg concentrations in the streambed sediments; however, most of the streambank soils have similar Hg concentrations to the streambed sediments. The molar ratio of Hg to organic carbon, correlation between MeHg and Hg, and particle size distributions suggested similarity between the streambank soils and the fine sand-sized fraction (125–250 μm) collected from the streambed sediments. Mercury in the fine sand-sized streambed sediments, however, was more labile than Hg in the streambank soils, suggesting an in-stream environment that altered the geochemistry of sediment-bound Hg.

Conclusions

This study revealed major source areas of Hg in streambank soils, identified possible depositional locations in streambed sediments, and highlighted potential differences in the stability of Hg bound to streambank soils and sediments. This work will guide future remedial decision making in EFPC and will aid other researchers in identifying source–sink linkages in contaminated fluvial systems.