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.     

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.     

Effect of summer throughfall exclusion, summer drought, and winter snow cover on methane fluxes in a temperate forest soil

Soil moisture strongly controls the uptake of atmospheric methane by limiting the diffusion of methane into the soil, resulting in a negative correlation between soil moisture and methane uptake rates under most non-drought conditions. However, little is known about the effect of water stress on methane uptake in temperate forests during severe droughts. We simulated extreme summer droughts by exclusion of 168 mm (2001) and 344 mm (2002) throughfall using three translucent roofs in a mixed deciduous forest at the Harvard Forest, Massachusetts, USA. The treatment significantly increased CH₄ uptake during the first weeks of throughfall exclusion in 2001 and during most of the 2002 treatment period. Low summertime CH₄ uptake rates were found only briefly in both control and exclusion plots during a natural late summer drought, when water contents below 0.15 g cm⁻³ may have caused water stress of methanotrophs in the A horizon. Because these soils are well drained, the exclusion treatment had little effect on A horizon water content between wetting events, and the effect of water stress was smaller and more brief than was the overall treatment effect on methane diffusion. Methane consumption rates were highest in the A horizon and showed a parabolic relationship between gravimetric water content and CH₄ consumption, with maximum rate at 0.23 g H₂O g⁻¹ soil. On average, about 74% of atmospheric CH₄ was consumed in the top 4-5 cm of the mineral soil. By contrast, little or no CH₄ consumption occurred in the O horizon. Snow cover significantly reduced the uptake rate from December to March. Removal of snow enhanced CH₄ uptake by about 700-1000%, resulting in uptake rates similar to those measured during the growing season. Soil temperatures had little effect on CH₄ uptake as long as the mineral soil was not frozen, indicating strong substrate limitation of methanotrophs throughout the year. Our results suggest that the extension of snow periods may affect the annual rate of CH₄ oxidation and that summer droughts may increase the soil CH₄ sink of temperate forest soils.     

Effects of aerobic conditions in the rhizosphere of rice on the dynamics and availability of phosphorus in a flooded soil – A model experiment

The secretion of O₂ by rice roots results in aerobic conditions in the rhizoshere compared to the bulk flooded soil. The effect of this phenomenon on the adsorption/desorption behavior and on the availability of phosphorus (P) in a flooded soil was investigated in a model experiment. An experimental set‐up was developed that imitates both O₂ release and P uptake by the rice root. The results showed that O₂ secretion significantly reduced P adsorption/retention and increased P desorption/release in the “rhizosphere” soil, compared to the anaerobic bulk soil. The P uptake by an anion exchange resin from both unfertilized and P‐amended soil was significantly increased. The results confirm that the O₂ secretion is an important mechanism to enhance P availability and P uptake of rice under flooded conditions, where the “physico‐chemical” availability of P in the anaerobic bulk soil is strongly reduced. The decrease of P availability in the P‐amended flooded bulk soil was mainly associated with the almost complete transformation of the precedingly enriched Al‐P fraction into Fe‐bound P with extremely low desorption/release characteristics during the subsequent flooding.     

Einfluß von Cd,Cu, Ni und Zn auf die Synthese metallothioneinähnlicher Substanzen in Scenedesmus subspicatus

Influence of Cd, Cu, Ni and Zn on the synthesis of metalloproteins by Scenedesmus subspicatus In short term experiments under controlled growth conditions the effect of increasing Cd-, Cu-, Ni-, and Zn-concentrations in the nutrient solution on the amount of biomass, the accumulation of heavy metals and the synthesis of metalloproteins by the unicellular greenalga Scenedesmus subspicatus was studied. Among the 4 elements Cd was taken up at the highest rate. Cu revealed to be the most toxic element according to the biomass production. In the Cd trials a protein fraction could be established, which was not detected in the control and in the Cu-, Ni-, and Zn-trials. The further characterization of the isolated fraction indicates the existence of a Cd-metallothionein in the algae.     

Response of primary plant metabolism to the N-source

Even when plant growth was not visibly affected, ammonium versus nitrate nutrition had distinct effects on some parts of plant metabolism. Barley seedlings growing on 3 mM ammonium rapidly accumulated ammonium up to 20 mM in the roots. In leaves, ammonium accumulation was observed only when the pH of the nutrient medium was very low (pH 4). Yet even under the most extreme conditions there was no indication that plants were suffering from uncoupling of ATP synthesis or from a lack of carbohydrates. Especially dramatic was the response of the organic acid content of pea and barley leaves: it decreased strongly within a few days upon transfer of plants from nitrate to ammonium-media, and this was apparently not due to an inhibition of PEPcarboxylase, which was rather activated under ammonium nutrition. As malate dispappeared from leaves even when pea plants were transferred to an N-free medium, malate degradation was not necessarily connected to increased amino acid synthesis, but eventually to a more rapid decarboxylation by malic enzyme. Also, malate degradation was not a response to ammonium, but rather to (the absence of) nitrate.     

Influence of Soil Type on the Effects of Elevated Atmospheric CO2 and N Deposition on the Water Balance and Growth of a Young Spruce and Beech Forest

Sixteen open-top chambers, each equipped with two non-weighablegravity-drained lysimeter compartments, were used to investigate the impacts of elevated atmospheric carbon dioxide (CO₂) concentration and nitrogen (N) deposition on the water relations and growth of young model forest ecosystems on two different types of soils. The same vegetation of a mixed spruce and beech overstorey and various herbs in the understorey was planted in all treatments on both soils. The soils were repacked on top of a drainage layer. Four combinations of treatments were applied in four replicates each: ambient (370 cm³ m^-3) CO₂ + low (7 kg N ha^-1 a^-1) N deposition, ambient CO₂ + high(70 kg N ha^-1 a^-1) N deposition, elevated (590 cm³ m^-3) CO₂ + low N deposition, and elevated CO₂ + high N deposition. After canopy closure, treatment effects on evapotranspiration and growth during the third year of study were very different for the two soils.On the acidic sandy loam, elevated CO₂ enhanced growth(leaf biomass +21%, roots +27%) at reduced evapotranspiration (–9%). High N deposition increased aboveground growth even more strongly (+50%), but also increased evapotranspiration (+16%). Together, elevated CO₂ and high N had a more than additive fertilizer effect on growth, while their effects on evapotranspirationcompensated. On the calcareous loamy sand, elevated CO₂not only tended to enhance growth (leaf biomass +17%, roots +20%), but also increased evapotranspiration (+5%).On this soil, aboveground growth was stimulated by N only incombination with elevated CO₂, but less than on the acidic soil, while evapotranspiration (–6.5%) and root growth into the subsoil (–54%) were decreased by increased N deposition at both CO₂ concentrations, in contrast to the N treatments on the acidic sandy loam. The influence of the soil on the observed ecosystem responses canbe interpreted in terms of the concept of optimal resource allocation.     

Effects of root exudates and humic substances on weathering kinetics

The effect of complex natural organic ligands on the weathering kinetics of aluminum oxide was investigated in laboratory experiments. A peat-derived humic substance and root exudates obtained from ectomycorrhizal (Picea abies — Hebeloma crustuliniforme) and non-mycorrhizal Norway Spruce trees; and γ-Al₂O₃ were used as a model system. The experimental weathering rates are in accordance with a surface-controlled dissolution mechanism. The effect of the humic material on dissolution rates appears to depend on the degree of protonation of the humic (macro)molecules: we observed dissolution-enhancement or -inhibition at pH 3 and 4, respectively. Ectomycorrhizal exudates proved to be effective weathering agents at pH 4, as opposed to humic material and non-mycorrhizal exudates. Our results suggest that (i) the role of humic materials in mineral weathering and podzolization is different from what is commonly thought, and (ii) mineral weathering rates in the rhizosphere may be higher than in the bulk soil.     

Bedeutung der arbuskulären Mykorrhiza (AM) für die Schwermetalltoleranz von Luzerne (Medicago Sativa L.) und Hafer (Avena sativa L.) auf einem klärschlammgedüngten Boden

Effect of arbuscular mycorrhizal fungi (AMF) on heavy metal tolerance of alfalfa (Medicago sativa L.) and oat (Avena sativa L.) on a sewage-sludge treated soil In pot experiments with a sewage sludge treated soil, the influence of two arbuscular mycorrhizal fungi (AMF) isolates of Glomus sp. (T6 and D13) on plant growth and on the uptake of heavy metals by alfalfa (Medicago sativa L.) and oat (Avena sativa L.) was investigated. Alfalfa showed an increase of biomass with mycorrhizal infection only to a small extent. In oat AMF inoculation increased the growth of both root and shoot by up to 70% and 55% respectively. Mycorrhization raised the P-content and -uptake in alfalfa, but not in oat, in both roots and shoots. Mycorrhizal alfalfa showed lower Zn-, Cd- and Ni-contents and uptake in roots and shoots. The root length was significantly decreased in mycorrhizal alfalfa plants (up to 38%). The translocation of heavy metals into the shoot of mycorrhizal alfalfa was slightly increased. Mycorrhizal infection of oat led to higher concentrations of Zn, Cd and Ni in the root but to less Zn in the shoot. The translocation of heavy metals to the oat shoot was clearely decreased by mycorrhizal colonisation. This may be based on the ability of fungal tissues to complex heavy metals at the cell walls, thus excluding metals from the shoot. This conclusion is supported by the enhanced root length (up to 78%) of mycorrhizal oat plants in this experiment. The mycorrhizal infection seemed to protect plants against heavy metal pollution in soils. It was obvious that different host plants reacted in different ways.