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.     

Occurrence of Polycyclic Musks in Sewage Sludge and their Behaviour in Soils and Plants. Part 1: Behaviour of Polycyclic Musks in Sewage Sludge of Different Treatment Plants in Summer and Winter (5 pp)

Background, Aim and Scope   Part 1: Behaviour of Polycyclic Musks in Sewage Sludge of Different Treatment Plants in Summer and Winter Part 2: Investigation of Polycyclic Musks in Soils and Plants -  Preamble. In Part 1 of the study, screening tests were performed to investigate the occurrence of PCMs in sewage sludges. For a preliminary risk assessment, further information is needed about their behaviour in the terrestrial environment. Hence, Part 2 examined the adsorption of PCMs to soil, their dissipation and leaching in soil and their uptake by plants. Background, Aim and Scope   Polycyclic Musks (PCMs) enter the environment via the waste water system. Because of their persistence, they can accumulate in different matrices like sewage sludge or biota. By the use of sewage sludge as a fertilizer, PCMs are transferred to agricultural soils. Therefore, in Part 1 of the study, screening tests were performed to investigate the occurrence of PCMs in sewage sludge. For a preliminary risk assessment, further information is needed about their behaviour in the terrestrial environment. Hence, Part 2 of the study examined the adsorption of PCMs to soil, their dissipation and leaching in soil, and their uptake by plants. Materials and Methods: In the screening study, samples of activated sewage sludge were taken both in summer and in winter at 21 treatment plants. In order to get an overview of the contamination situation, sampling covered different types of treatment plants (in rural, urban, industrial areas). Analytical methods for the determination of HHCB, AHTN, ADBI, ATTN, AHDI and ATII in the sludge samples were developed and applied. Results: The analytical screening of PCMs showed their presence in activated and dried sewage sludge samples. HHCB and AHTN represented about 95% of the PCMs investigated. Their concentrations in the activated sludge samples varied between 2.9 and 10.4 mg/kg dry mass (dm) and 1.1 to 4.2 mg/kg dm, respectively. Although different types of sewage treatment plants were investigated, similar PCM levels were found, showing the widespread input of these compounds into domestic waste water. Discussion: PCM concentrations in activated sludge varied widely. The variation drops substantially when concentrations are related to the varying dry mass. In dehydrated sludge, PCM concentrations were up to 24 mg/kg dm for HHCB and up to 6.9 mg/kg dm for AHTN. These high values are comparable to those obtained in other investigations analysing PCMs. If the degradation of organic mass during anaerobic decomposition is included in the evaluation, the figures obtained are comparable to those for activated sludge. Elimination in sewage sludge was higher in summer than in winter. Therefore, the contamination of the sludges in winter reached higher levels compared to the summer. Conclusions: The results show that PCMs are widespread contaminants in sewage sludge. Recommendations and Perspectives: PCM should be considered in a risk assessment as potential contaminants of sewage sludge destined for agricultural use. Due to the high PCM levels in sewage sludge, further investigations into the degradation and elimination behaviour in sewage sludge have to be carried out, including that involving PCM metabolites such as lactone derivatives.     

Saprotrophic fungi transform organic phosphorus from spruce needle litter

Fungal decomposition of and phosphorus transformation from spruce litter needles (Picea abies) were simulated in systems containing litter needles inoculated with individual saprotrophic fungal strains and their mixtures. Fungal strains of Setulipes androsaceus (L.) Antonín, Chalara longipes (Preus) Cooke, Ceuthospora pinastri (Fr.) Höhn., Mollisia minutella (Sacc.) Rehm, Scleroconidioma sphagnicola Tsuneda, Currah & Thormann and an unknown strain NK11 were used as representatives of autochthonous mycoflora. Systems were incubated for 5.5 months in laboratory conditions. Fungal colonization in systems and competition among strains were assessed using the reisolation of fungi from individual needles. After incubation, needles were extracted with NaOH and extracts were analysed using ³¹P nuclear magnetic resonance spectroscopy (NMR). Needle decomposition was determined based on the decrease in C:N ratio. Systems inoculated with the basidiomycete S. androsaceus revealed substantial decrease in C:N ratio (from 25.8 to 11.3) while the effect of ascomycetes on the C:N ratio was negligible. We suppose that tested strains of saprotrophic ascomycetes did not participate substantially in litter decomposition, but were directly involved in phosphorus transformation and together with S. androsaceus could transform orthophosphate monoesters and diesters from spruce litter needles into diphosphates, polyphosphates and phosphonates. These transformations seem to be typical for saprotrophic fungi involved in litter needle decomposition, although the proportion of individual phosphorus forms differed among studied fungal strains. Phosphonate presence in needles after fungal inoculation is of special interest because no previous investigation recorded phosphonate synthesis and accumulation by fungi. Our results confirmed that the ³¹P NMR spectroscopy is an excellent instrumental method for studying transformations of soil organic phosphorus during plant litter decomposition. We suggest that polyphosphate production by S. androsaceus may contribute to the phosphorus cycle in forest ecosystems because this fungus is a frequent litter colonizer that substantially participates in decomposition.     

Immediate Impact of the Flood (Bohemia, August 2002) on Selected Soil Characteristics

In August 2002 extreme floods affected Bohemia a part of the Czech Republic, along Vltava and Labe rivers. After the flood had culminated, soil samples of arable and meadow soil (flooded and control-non-flooded) have been collected in the surroundings of the cities of ?eské Budějovice (CB, South Bohemia, the Vltava river, the upper flood stream) and Ústí nad Labem (UL, the Labe river, the lower flood stream). Different parameters of arable soils have been affected more significantly than those of meadow soils. Soil texture e.g. has been shifted from sandy loam to loam (CB) and clay loam (UL). Organic carbon, total phosphorus and nitrogen contents have been increased as well as the content of some hazardous elements such as arsenic. A shift of soil pH from 6.1 to 4.8 has been noticed in CB and an opposite one from 5.1 to 7.1 was measured in UL. Available phosphorus decreased in CB by 56% while a four-fold increase was measured in UL. Chlorinated organic pesticides level was significantly reduced, but that of polycyclic aromatic hydrocarbons (PAH) increased up to the maximum values of legal limits. The UL arable soil also became heavily contaminated by PAH. The CB arable soil demonstrated high microbial activity, while UL arable soil suffered under stress conditions in this respect. The flooded meadow soils became covered with a layer of drifted soil sediment, which retained enhanced content of the PAH's different hazardous elements, and, in addition, the soil aeration was reduced.     

Statistical Modeling of the Partitioning of Nonylphenol in Soil

Partition coefficients K _P of nonylphenol (NP) in soil were determined for 193 soil samples which differed widely in content of soil organic carbon (SOC), hydrogen activity, clay content, and in the content of dissolved organic carbon (DOC). By means of multiple linear regression analysis (MLR), pedotransfer functions were derived to predict partition coefficients from soil data. SOC and pH affected the sorption, though the latter was in a range significantly below the pK_a of NP. Quality of soil organic matter presumably plays an important but yet not quantified role in sorption of NP. For soil samples with SOC values less than 3 g kg^?1, model prediction became uncertain with this linear approach. We suggest that using only SOC and pH data results in good prediction of NP sorption in soils with SOC higher than 3 g kg^?1. Considering the varying validity of the linear model for different ranges of the most sensitive parameter SOC, a more flexible, nonlinear approach was tested. The application of an artificial neuronal network (ANN) to predict sorption of NP in soils showed a sigmoidal relation between K _P and SOC. The nonlinear ANN approach provided good results compared to the MLR approach and represents an alternative tool for prediction of NP partitioning coefficients.     

Detection of phloridzin in strawberries (Fragaria x ananassa Duch.) by HPLC-PDA-MS/MS and NMR spectroscopy

The phenolic profile of strawberry fruits (Fragaria x ananassa Duch., Rosaceae) was investigated by high-performance liquid chromatography with photodiode array detection. A peak displaying retention time and UV spectral data identical to those of phloridzin (phloretin 2'-O-beta-d-glucoside), a dihydrochalcone glucoside so far considered characteristic of apples, was monitored. For further characterization, crude extracts of strawberries were purified on polyamide, and the target compound was isolated by preparative and analytical HPLC. Structure elucidation was performed on the basis of APCI- and ESI-MS in the negative ion mode as well as by 1D and 2D NMR spectroscopy using authentic phloridzin for comparison. The d-configuration of the sugar moiety was established by HPLC analysis of the corresponding acyclic 1-deoxy-1-(N-acetyl-alpha-methylbenzylamino)alditol acetate. Apart from its chemotaxonomic relevance, this first report on the occurrence of phloridzin in strawberries is of particular interest for the authenticity control of strawberry products such as juices, jams, and fruit preparations since phloridzin has so far been used for the detection of fraudulent admixtures.     

Rapid surface plasmon resonance-based inhibition assay of deoxynivalenol

Deoxynivalenol belongs to a group of highly toxic fungal metabolites produced by Fusarium species that may contaminate food and animal feed, mostly grains. Three different monoclonal mouse anti-deoxynivalenol antibodies were compared for the development of a surface plasmon resonance (SPR)-based immunoassay for the selective and quantitative determination of deoxynivalenol in naturally contaminated matrices. A conjugate of deoxynivalenol with the protein casein was prepared and immobilized on the sensor chip surface. An excess of antibody was added to each test solution before the measurement. The assay was based on the competition for antibody binding between the immobilized deoxynivalenol conjugate on the sensor and the free deoxynivalenol molecules in the test solution. The deoxynivalenol-casein sensor could be reused more than 500 times without significant loss of activity using 6 M guanidine chloride solution for regeneration. The cross-reactivity of the three antibodies in the SPR assay was tested with other trichothecene mycotoxins (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol, HT2-toxin, and T2-toxin). The only sample preparation was extraction with max 80 vol % acetonitrile and 10-fold dilution with the running buffer. The assay had an optimal range between 2.5 and 30 ng/mL deoxynivalenol in the test solution. Most results of the SPR-based assay were in agreement with liquid chromatography/tandem mass spectrometry measurements of naturally contaminated wheat samples.     

Effects of partial drying of an udder quarter in cows with teat injuries

In cows with teat lesions, partial drying-off of the affected mammary quarter is recommended for a period of 3 to 10 days. The other three quarters are normally milked. Partial drying-off reduces the risks of complications due to the daily milking. The slight reduction of milk production, connected with the temporary drying-off a single quarter, is nearly compensated within 4 weeks.     

Antigenic characterization of IBDV field isolates by their reactivity with a panel of monoclonal antibodies

Recently Infectious Bursal Disease Virus isolates have been described in USA displaying an antigenic drift. Many of the new isolates were very virulent for chickens. In several European countries severe outbreaks of Gumboro disease have also been reported from vaccinated and non-vaccinated flocks. Since vaccinated SPF birds were shown to be protected against challenge infection with the new isolates under laboratory conditions, a more detailed investigation of the European isolates is wanted. The similarity between the European and US field situation got us to use a panel of monoclonal antibodies (MCAs) previously applied to characterize US strains for testing European isolates. An antigen capture ELISA has been carried out directly on bursa homogenates of chickens form the field. One European (F52/70) and two US (Var. E and GLS-5) strains have been included as reference viruses. From the results presented here it can be concluded that the European isolates (Netherlands, France, UK, Germany, Jugoslavia and Spain) did not undergo the same antigenic drift as the US strains. A more extensive analysis of the isolates will be done to elucidate their role for disease outbreaks.