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.     

Contribution of anecic earthworms to biopore formation during cultivation of perennial ley crops

Large sized biopores (diameter >2 mm) in the subsoil can be created by tap roots, which leave voids after their decay, or by the burrowing activity of anecic earthworms which may benefit from the temporary lack in tillage in perennial cropping systems. However, the interactions between root growth and earthworm activity in the process of biopore formation during perennial ley cropping are not well understood. The aim of this field study was to quantify the development of the abundance of the anecic earthworm Lumbricus terrestris and the biopore density during the cultivation of lucerne (Medicago sativa L.), chicory (Cichorium intybus L.) and tall fescue (Festuca arundinacea Schreb.) grown for either one, two or three years. An increased abundance of L. terrestris was already recorded after two years of continuous ley when compared with one year cultivation. The ley crop species had only minor influence on the abundance of L. terrestris. Biopore densities of both diameter classes under study (2–5 mm and >5 mm) were not significantly affected by the duration of ley cropping. In contrast, biopore densities were influenced by ley crop species. More biopores of diameter class 2–5 mm were recorded after chicory than after fescue. Lucerne cropping resulted in intermediate biopore density. Additionally, in an incubation experiment under field conditions, we quantified whether L. terrestris preferentially created new biopores or colonized abandoned, previously existing ones. After three weeks of incubation, one third of the adult individuals incubated in the experimental area created new biopores at 0.4 m soil depth. A similar percentage of individuals colonized previously existing biopores, partially widening the lumen of smaller sized biopores. The remaining individuals remained in the topsoil. Sub-adult individuals rarely formed new pores. Half of the introduced sub-adults remained in the topsoil. We conclude that in crop rotations new biopores can be generated during perennial ley cropping by taproot systems of ley crops, but that a two to three- year period without tillage is not sufficient for populations of anecic earthworms to make a marked contribution to biopore density in the subsoil. The relevance of anecic earthworms for altering physical and chemical properties of biopores during ley cropping still needs further investigation.     

The importance of the soil skeleton for plant-available nutrients in sites of the Southern Black Forest,Germany

The skeletal fraction of forest soils in the Southern Black Forest has a cation exchange capacity (CEC) within the same order of magnitude as fine earth. As the base saturation in the skeleton is much higher than in the fine earth, stones provide a physiologically more favourable environment for nutrient uptake by plants. The fact that 70% of the skeleton grains showed adherent rhizomorphae and/or fine roots suggests that this potential is widely used by nutrient-adsorbing tissues. Therefore the ecological importance of this additional and, until now, unconsidered ion pool available to plants is probably higher than what is reflected by simple ion quantities. The effective CEC of the coarse fraction in forest sites with crystalline bedrock is reasonably predictable considering the water vapour adsorption of the 105°C dried skeleton at a suction of pF 4.6 (vapour pressure equilibrium with an oversaturated K₂SO₄ solution). The content of exchangeable base cations in the coarse fraction can be estimated using the content of exchangeable Mg in the fine earth. These stochastic relations are causally plausible. The regressions are a first approach to identify pedo-transfer functions predicting the skeletons nutrient potential with the aid of easily measurable site parameters.     

Seroprevalence of anaplasmosis among cattle in Switzerland in 1998 and 2003: no evidence of an emerging disease

Anaplasma marginale infection in Europe has been limited to the Mediterranean and eastern countries, to Austria and to very sporadic cases in Switzerland. There are no reports of its occurrence in the countries north of Switzerland. A severe outbreak of anaplasmosis in August 2002 in a cattle farm in the canton Grisons, Switzerland, north of the Alps, with more than 300 cattle that had to be culled, came unexpected and gave reason to hypothesize presence of an increased yet undetected prevalence of A. marginale in Switzerland. Randomly selected bovine serum samples collected in 1998 and 2003 were tested using a competitive inhibitory ELISA (cELISA) to test the hypothesis. Our validation of the diagnostic sensitivity and specificity of this test, done in the outbreak herd, yielded 99.2 and 83.3%, respectively, probably underestimating the true specificity. The true seroprevalence of anaplasmosis in Swiss cattle determined by cELISA was likely to be zero with upper 95% confidence limits of 2.49% in the canton Grisons and 1.17% in the rest of Switzerland, respectively, in 1998. For 2003, these estimates were even lower. There was no significant difference in apparent prevalences between 1998 and 2003. In search of a possible reservoir, three chamoises out of 46 free ranging wild ruminants from the Swiss National Park, Grisons, tested positive in the cELISA. This reaction is in accordance with A. marginale or a cross reacting agent such as Anaplasma ovis. From our results we conclude that the hypothesis of an increased prevalence of anaplasmosis in cattle in Switzerland must be rejected.     

Correction to: “Current-IPM-Fit”: a new proposal for enhanced efficacy labelling of plant protection products

Journal of Plant Diseases and Protection - In the original publication, old version of Table 1 was included by mistake. The correct version of Table 1 is given here.     

Priming effects in soil size fractions of a podzol Bs horizon after addition of fructose and alanine

It is well known that the addition of easily available substrates to soils can affect microbial activity and thus the mineralization of soil organic carbon (SOC). Up to now, little is known about the processes leading to these priming effects and which fractions of organic matter (OM) are affected. The objectives of this study were to determine if SOC associated with isolated soil size fractions showed different susceptibility to priming effects, whether these pools are easily depleted, or whether the amount of substrate addition affects the extent of priming effects. In an incubation experiment, the effect of the uniformly ¹⁴C‐labeled substrates fructose and alanine on the mineralization of the SOC of a Bs horizon of a Haplic Podzol was investigated. The soil sample was fractionated into the three soil size fractions sand, silt, and clay by a mild sonication followed by sieving and sedimentation. Additionally, nonfractionated soil of the horizon was included in the experiment. Every soil sample received four substrate additions repeated at weekly intervals with 3.325 μg substrate‐C (mg SOC)^–1 and a final addition of 13.3 μg substrate‐C (mg SOC)^–1 after 4 weeks. The respiration was determined hourly and ¹⁴CO₂ was analyzed every 2, 4, and 7 d after the respective substrate addition. After 56 d, between 42% and 58% of the added substrates had been mineralized. Both substrates strongly increased the mineralization of the OM in all fractions (positive priming effects). The priming effects were always higher after the addition of the high substrate dose than during the first 4 weeks when four small doses were added. In general, the priming effects increased with decreasing particle size. Alanine generally caused higher priming effects than fructose in the soil size fractions (up to 280% vs. 231%, respectively). This indicates that alanine serves not only as an energy substrate but also as a N source and, thus, also promotes microbial growth. The strong priming effects in the silt and clay fraction (133% and 125% with fructose, 172% and 168% with alanine) showed, that not only the labile pool of OM is affected, but also a more stable pool characterized by higher ¹⁴C ages. We assume that the stability of the OM in these fractions is not only due to recalcitrance or to interactions with the minerals, but that it may also be caused by a substrate limitation of the degrading microorganisms.