Application of a new sediment contact test with <Emphasis Type="Italic">Myriophyllum aquaticum</Emphasis> and of the aquatic <Emphasis Type="Italic">Lemna</Emphasis> test to assess the sediment quality of Lake Skadar

Goal, Scope and Background  Situated in the transboundary belt between Montenegro and Albania, Lake Skadar is the largest freshwater reservoir in Southeastern Europe. Because of the wide range of endemic, rare or endangered plant and animal species it supports, Lake Skadar and its extensive adjacent wetlands are internationally recognised as a site of significance and importance (Ramsar site). Within the last 10 to 20 years, Lake Skadar was exposed to intensive pollution. For the assessment of the ecotoxic load of the sediments sampled in Lake Skadar, a triad approach was recently applied. Overall, a complex spectrum of ecotoxic loads was elucidated. The aim of the present study was to use plant-based bioassays for assessing the sediment quality of Lake Skadar in order to facilitate and complement the triad test battery. The newly developed sediment contact test with Myriophyllum aquaticum and the aquatic growth inhibition test with Lemna minor were applied to native sediments and pore water, respectively, allowing the investigation of different toxicity-effects caused by particle-bound pollutants as well as pollutants in the interstitial water. This investigation is the first application of the novel sediment contact test with Myriophyllum aquaticum to lake sediments. Methods  Sediment samples were taken from nine selected sites at Lake Skadar and investigated by the sediment contact assay with Myriophyllum aquaticum. The pore water was extracted from these sediment samples to be analysed in the aquatic growth inhibition test with Lemna minor. The results of the sediment contact tests were compared with each other and with those of the aquatic growth inhibition test. Results and Discussion  Both applied macrophyte biotests revealed distinct changes in the growth behaviour of the two macrophytes subsequent to the exposure to the investigated natural sediments of Lake Skadar. The Myriophyllum sediment contact test revealed significant toxicity in the sediment samples from Radus and Kamenik, whereas the aquatic Lemna test showed inhibition effects for the samples from Sterbeq, Plavnica and Kamice. Data obtained with the newly developed Danio rerio contact test and the Arthrobacter globiformis contact test confirmed the Myriophyllum results. Analyses of the heavy metal content in the sediments revealed low or moderate contamination levels. Correlation analyses between the content of heavy metals in the sediments and growth inhibition of Myriophyllum aquaticum showed a significant correlation between Cr concentrations and growth inhibition. Comparable findings are available for a German river system. In contrast, no significant correlation between inhibition rates and concentration of metals could be observed with Lemna minor. Conclusions  It was shown that the newly developed sediment contact test with Myriophyllum aquaticum is applicable to lake sediments. In both the sediment contact test with Myriophyllum aquaticum on whole sediments and the aquatic growth inhibition test with Lemna minor on pore water, plant growth was influenced by the natural sediments and its components. Therefore, both test systems were found to be suitable for the detection of phytotoxic effects upon exposure to sediments. Myriophyllum aquaticum as test organism of the contact test grows directly in the sediment without an additional water-layer. Thus, it is able to detect toxicity caused by particle-bound phytotoxic substances as well as pore water-related contamination, while the floating Lemna minor can only detect effects emanating from pore water. Significant differences of the results were observed between these two test systems and, accordingly, the two different exposure scenarios. Hence, none of the tests can replace the other one and, as a consequence, both should be included into a test battery for the assessment of sediment toxicity. Recommendations and Perspectives  Both plant assays were shown to be reliable tools for the evaluation of the eco-toxicological risk potentials of pore water and solid-phase sediment. They should become a complement to the standardised test battery generally used for comprehensive hazard assessment. ESS-Submission Editor: Dr. Ulrike Kammann (ulrike.kammann@ifo.bfa-fisch.de)     

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     

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.     

Integrating the fish embryo toxicity test as triad element for sediment toxicity assessment based on the Water Framework Directive approach

Purpose  

The objective of this study was to complement analyses according to the European Union Water Framework Directive (WFD) with a sediment toxicity analysis as part of an integrated river assessment. To this end, Hessian water courses were analyzed using the sediment quality triad concept according to Chapman with chemical analyses, in situ effect evaluations, and ecotoxicological assessments. For the ecotoxicological assessment (fish embryo toxicity test with Danio rerio), a new evaluation scheme was developed, the fish teratogenicity index (FTI), that allows for a classification of sediments into ecological quality classes compliant to the WFD.     

Ecotoxicity of Alternative De-icers

Background  De-icers are essential to wintertime road maintenance in Nordic countries. The use of road salts (NaCl, CaCl₂ and MgCl₂) is having significant effects on groundwater. There has been a growing concern of environmental effects resulting from de-icing. Regulatory bodies have tried to find de-icers which have less harmful impacts on soil and groundwater. Alternative new organic de-icers are e.g. potassium formiate, potassium acetate, and calcium magnesium acetate. Objectives  At present, the decision finding for soil and water quality assessment is mainly based on the results of chemical analyses. However, ecotoxicological characterization will provide more useful information for determining environmental effects of de-icers. Methods  Six different de-icers, NaCl, CaCl₂, MgCl₂, potassium formicate KFo, potassium acetate KAc and calcium magnesium acetate CMA were studied with different bioassays. These bioassays were two plant tests (onion Allium cepa root elongation test and duckweed Lemna growth inhibition test), an enzyme inhibition test (reverse electron transport test, RET) and a microbial test (luminescent bacteria Vibrio fischeri, BioTox test). Results and Discussion  Most of the responses of the de-icers tested were toxic to test organisms. CMA was unsuitable for RET and BioTox tests because it was not possible to analyze turbid samples with these bioassays. On the whole, organic chemicals were more toxic than the inorganic salts. According to the Lemna and Allium cepa tests KFo was the most toxic and the toxicity ranking of the de-icers was KFo > KAc > CMA > CaCl₂ > NaCl > MgCl_2. The sensitivity ranking for tests in decreasing order was Lemna > Allium cepa > RET > BioTox. The plant tests proved to be relevant, easy, cost-effective and sensitive screening methods of de-icers: Lemna test was 92 times more sensitive and Allium test 70 times more sensitive than BioTox to KFo. Conclusion and Outlook  The tested de-icers had significant effects on plant growth, microbial luminescence and enzymatic activity. Toxicity could hardly be predicted from the chemical concentrations of these de-icers. In summary, these results demonstrate a need for including toxicity measurements in the evaluation of technologies used in soil treatment and illustrate the potential value of such measurements for ensuring that no or minimal environmental contamination would arise from de-icing process.     

Chemical and Microbiological <Emphasis Type="Italic">in situ</Emphasis> Characterization of Benthic Communities in Sediments with Different Contamination Levels

Background  River sediments are natural habitats of complex bacterial and fungal communities and therefore play a decisive role in the mineralization process of organic matter in freshwater systems. By means of comparative temporal and spatial analyses of microbial communities, the in situ impact of anthropogenically generated pollutants on these biofilm associations can be assessed and discriminated from seasonal variations. Aim  The aim was the adaptation of hybridization with fluorescently labelled rRNA-targeted oligonucleotides (FISH) for the in situ characterization of the structural and functional diversity of native microbial communities in complex lotic sediments. The impact of qualitatively and quantitatively different water pollutants on the microbial diversity, metabolic potential, and relative abundance of characteristic bacterial groups was assessed by oligonucleotide probes on different phylogenetic levels. In particular, sulfate reducing bacteria (SRB) were investigated to evaluate their potential applicability as microbial biomonitors in sediments. Methods  Sediment samples from the German lowland rivers Elbe and Oder were investigated over 12 months with regard to physico-chemical parameters and the composition of the attached microbial communities. Mechanical treatment including ultrasonification and sagitation under aerobic conditions combined with the use of pyrophosphate ensured the equal dispersion of fixed microbial cells within the sediment samples. The optimized whole-sediment FISH-technique was combined with an improved cell extraction procedure and applied, due to the specific grain size fraction distribution, at the different sampling sites. Resultsand discussion  Up to 85.6% of the total bacterial cell counts as determined by DAPI (4’, 6-diamidino-2-phenylindole) staining could be successfully monitored by the eubacterial oligonucleotide probe set EUB338, EUB338-II and EUB338-III, simultaneously indicating a high proportion of Eubacteria and the high metabolic potential of the bacterial community. Desulfobacteriaceae could be detected by the specific probe SRB385Db in various relative percentages ranging from 2.4 to 16.0% of the total bacterial cell counts. The total number of bacteria and the metabolic potential of sediment related bacteria were barely affected by the different pollution pattern of the sampling sites. Conclusions  The pre-treatment step as conducted by cell extraction as well as the FISH hybridization procedure was successfully optimized to the specific conditions present within freshwater sediments. Beside seasonal variations, particularly occurring at hydrologically influenced sites, sampling sites with different pol lution levels could be successfully distinguished by the relative abundance of Desulfobacteriaceae used as microbial indicator organisms. Outlook  The integration of ongoing insights into pollution induced changes of natural bacterial consortia should result in a system of ecotoxicological classes representing the different ecological status of riverine systems. Physiological directed methods like Community Level Physiological Profiling (CLPP) or Pollution Induced Community Tolerance (PICT), and structural techniques as FISH or microarrays should be used to investigate the influence of harmful substances on the biodiversity in natural microbial sediment communities.     

Ecotoxicological evaluation of Harbour sediments using marine organisms from different trophic levels

Background, Aim and Scope  The toxicity of contaminated sediments should be evaluated considering the direct exposure of laboratory organisms to whole sediments and the indirect exposure to elutriates or extracts (Tay et al. 1992, Byrne and Halloran 1999, Nendza 2002). The alga Dunaliella tertiolecta is indicated for the use in toxicity bioassays because it is highly sensitive to several xenobiotics. Harpacticoid copepods have been already used for toxicity testing and Tigriopus fulvus is a promising Mediterranean target-species in ecotoxicology (Todaro et al. 2001, Faraponova et al. 2003, Pane et al. 2005a). In this study, the toxicity of sediments collected in harbour sites of the Northeastern Adriatic Sea was evaluated by growth inhibition test with free living and alginate-immobilized Dunaliella tertiolecta and acute toxicity test with nauplii and adult Tigriopus fulvus with the aim of pointing out the importance to utilize model organisms from different trophic levels in sediment ecotoxicology. Methodology  Elutriates and whole sediments were tested on free living and immobilized (Pane et al. 1998) algal cells, and on laboratory reared copepods. Free-living D. tertiolecta were exposed to diluted elutriates in a static, multi-well plate system. Naalginate immobilized D. tertiolecta were placed in polystyrene inserts fitted with polyester mesh bottoms and exposed to a thin layer (2 mm) of whole sediments in multi-well plates (EPS 1992, Pane and Bertino 1999). Toxicity tests with copepods were carried out on Tigriopus fulvus nauplii (elutriates) and adults (whole sediments and elutriates). Same-aged nauplii useful for toxicity tests were obtained by egg sac detaching and consequent hatching stimulation (Pane et al. 2006). Newborn nauplii (I–II stage) were exposed to elutriates in multi-well plates provided with polystyrene inserts. Adult T. fulvus maintained in polystyrene inserts fitted with polyester mesh bottoms were placed in contact with a thin layer (2 mm) of whole sediment placed on multi-well plate bottoms. All end-points were evaluated after 96 h. Results  In general, the effects increased with the increasing of elutriate concentration up to 50%; the stimulation or inhibition of algal growth was statistically significant in comparison to the control. The inhibiting elutriates induced EC₅₀ variations of algal growth ranging from 66.9% to 74.3%. The mortality of T. fulvus nauplii was always < 25% after treatment with 100% elutriates and < 10% after treatment with 50% dilution; no effect was shown up with 25% dilution; therefore LC₅₀ was not calculable. The effect of elutriates was negligible on adult copepods and LC₅₀ values were never calculable; percent mortality always resulted in < 10% after treatment with whole sediments. Discussion  Both experimental systems gave substantially similar results after exposition to whole sediments and elutriates. During the experiment with algal cells, the immobilization in Na-alginate and the employment of inserts which allowed the contact of organisms with sediments and their easy counting were particularly useful. Likewise, the employment of inserts of adequate mesh size in the tests with copepods allowed the contact of organisms with the sediment and made organism handling and counting easy, as well as the evaluation of mortality. The methodology here described and the utilization of the proposed test-species could have an importance also considering that the current trend in ecotoxicological research is towards finding the most appropriate organism for specific areas of concern by using indigenous species (Mariani et al. 2006) and towards the major significance of chronic and reproductive end-points. Conclusions  Based on the above results, it can be stated that the bioassay with Dunaliella tertiolecta could be a good estimation tool for the ecotoxicological assessment of marine sediments. The immobilization of algae in Na-alginate was seen to be useful to evaluate the toxicity of whole sediments; the employment of polystyrene inserts allowed an improvement of the procedures. T. fulvus nauplii and adults, as other harpacticoids such as Tigriopus japonicus (Yoon et al. 2006), satisfy the basic criteria for the employment of a standard species in marine bioassays. To date only pelagic Acartia tonsa are utilized in the standardized procedure to evaluate the risk assessment of chemicals or wastewaters (ISO 1999). As, on the contrary, the exposure of copepods to solid-phase contaminants it is not yet standardized, the employment of polystyrene inserts improved the procedures for T. fulvus too. So, the rapidity and the possibility to solve practical problems could be the main attractive features of this technique (Pane et al. 2005a) when applied to whole sediments. Recommendations and Perspectives  The methodology here developed being also applicable to long term and reproduction tests should be recommended because it provides relevant information in comparison with other frequently applied, standardized biotests with crustaceans (ISO 1999). The procedure has been shown to be easily applicable to selected marine organisms. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Optimisation of the Solid-Contact Test with Arthrobacter globiformis (7 pp)

Background, Aim and Scope   A number of biotests are available for the characterisation of solid matters such as soil or sediment. Among these, bacterial biotests using single test species often analyse the toxicity of water-soluble contaminants in aquatic extracts, but there is also a need for a fast and inexpensive bacterial solid-contact test. In this study, a solid contact test with added bacteria (Arthrobacter globiformis) was optimised (through miniaturization) for the development of a test kit with conserved bacteria. As in other tests, the results can be influenced by natural soil factors, often masking anthropogenic impacts. For this reason, a further goal of this study was the investigation of the influence of natural soil characteristics on the result of the solid contact test. The project is part of the joint research project 'Optimization of ecotoxicological test methods for routine use' (abbreviated as ERNTE-Forschungsvorhaben: 0330305). Materials and Methods: This method is based on an existing German standard (DIN 38412 L 48) using Arthrobacter globiformi for testing whole soils and sediments. The test principle is the measurement of the dehydrogenase activity of the test organism A. globiformis after an incubation time of two hours with the solid material. To attain the miniaturization in microplates, dye measurement was changed from spectrophotometrical determination of the substrate resazurine to the fluorimetric measurement of the product resorufin. A second step towards optimisation was the use of freeze-dried bacteria. Seven selected uncontaminated soils were tested in order to determine the influence of natural soil characteristics on the results of the solid contact test with A. globiformis. Freshly spiked and polluted field soils were analysed in order to obtain information about the sensitivity of the test. Results: It is possible to perform the contact test in microplates. The fluorimetric dye measurement can be carried out in the presence of the solid material, so the work-intensive step of centrifugation and filtration is no longer necessary. The measurement in the optimised contact test is based on the kinetics of the enzyme reaction. The investigation showed that conserved bacteria have the same activity and sensitivity as cultivated bacteria. Discussion: The study of the uncontaminated soils demonstrated the influence of various soil characteristics on the results of the solid contact test. This information is the basis for the selection of the control and reference soils and is crucial for setting the threshold value in toxicity testing. The investigation of freshly spiked and contaminated soils showed a different sensitivity dependent on the kind of the contamination. Conclusions: The solid contact test was successfully optimised using microplates, whereas now less than six hours are necessary for the analysis. The optimised test is rapid and sensitive, requiring small samples and no stock culture of the bacteria A. globiformis if using freeze-dried bacteria. In this study, the effect of natural soil factors such as pH-value was shown. This information is used to define the threshold value for toxicity. Therefore, the optimised contact test can be used for an efficient assessment of soil or soil substrates. Further studies will clear up if this optimisation is also valid for aquatic sediments and waste. Recommendations and Perspectives: Due to its short analysis time, the test is suitable for screening different kinds of solid matter and can be used for on-site analysis. - The optimised contact test with freeze-dried bacteria as part of a battery of tests is appropriate for the assessment of contaminated soils, sediments and waste.     

Comparisons of terrestrial and aquatic bioassays for oil-contaminated soil toxicity

Background  Petroleum products are widely used in various sections of industry and they are one of the most abundant sources of environmental contamination. These products are classified by their physico-chemical properties such as boiling point, density and viscosity. Oil contamination in the environment is primarily evaluated by measuring the chemical concentrations of petroleum products in the solid or water phase. The results of chemical analyses do not correspond directly with the harmful environmental effects of petroleum products on the soil flora and fauna, because the interactions between oil compounds and the production of their methabolites in soil are not measured in chemical assessments. These kinds of effects of complex chemical mixtures in soil can be estimated by bioassays. Therefore, ecotoxicological tests are important for estimating soil quality in the risk assessment of oil-contaminated soil sites. Objectives  The objective of this study was to examine the oil-contaminated soil site of a closed petrol station with both chemical and ecotoxicological methods. The goals of this study were to compare the sensitivity of the terrestrial and aquatic bioassays and to compare the toxicity responses of aquatic bioassays determined from three different extraction procedures. In addition, our aim was to characterise a cost-effective battery of bioassays that could be applied to a comparison of oil-contaminated soils. It was in our interest to investigate oil-contaminated soil with oil concentrations of 2500–12000 mg/kg and to find out the possible differences between terrestrial and aquatic toxicity tests. Methods  Six soil samples from a closed petrol station were examined for toxicity with terrestrial and aquatic tests. Terrestrial tests includedEnchytraeus albidus survival and reproduction assays and seed germination assays using wheat, cress, lettuce, and red clover seeds and growth inhibition assays of onions. The toxicities of the water-extractable fractions of the soil samples obtained from three different extractions were tested with aquatic bioassays based on plants (onion and duckweed growth inhibition tests), microbes (luminescent bacteria test), and enzyme inhibition (reverse electron transport test, RET). Chemical analyses of the solid samples were carried out simultaneously. Results. Oil concentrations ranged from 2500 to 12000 mg/kg, BTEX varied from 300 to 2800 mg/kg, and fuel additives: MTBE and TAME from 0.0 1 to 260 mg/kg. Only the sample contain-ing 12000 mg/kg oil had a significant toxic impact on all test organisms. Soil samples with oil concentrations 2500–6200 mg/ kg had no or only slight adverse effects on the test organisms with one exception, theE. albidus reproduction test. TheE. albidus survival and reproduction tests were the most sensitive bioassays of the terrestrial tests, and the luminescent bacteria test of the aquatic tests.     

The impact of storm water runoff on a small urban stream

Background, aim, and scope  In urban areas, storm water runoff often transports various pollutants, some of which settle and form sediments. In order to have the comprehensive view of the ecological state of storm water runoff recipients, both water and sediments of the stream must be assessed. In the Baltic Sea Area, the Water Framework Directive & HELCOM Recommendations aim to prevent or minimise pollution caused by harmful substances arising from storm water runoff, in order to promote the ecological restoration of the Baltic Sea—one of the most vulnerable seas. The aim of the study was to investigate the toxicity of bottom sediments of a small storm water runoff recipient focusing on the potential impact of successive discharges of urban storm water. Some storm water runoff quality parameters and the toxicity of bottom sediments of recipients was studied in this research. Materials and methods  During 9 years, at four discharge points, minimum four grab samples per year at each discharge point were taken for chemical characterisation. General parameters (pH, SS, BOD₇, COD_Cr and TPH) in liquid phase samples were analysed according to standard methods. Annual limit values were taken from the Lithuanian EPA requirements for the management of storm water runoff with a focus on prevention and control of contamination. Eleven composite samples of stream bottom sediments, each consisting of ten sub-samples, were collected in 2006. Toxicity screening from sediments was performed using the plant Lepidium sativum according to modified I. Magone’s methodology (Magone I, Bioindication of phytotoxicity of transport emission. In: Kachalova O-L, Zinatne (eds) Bioindication of toxicity of transport emissions in the impact of highway emissions on natural environment. Riga, pp 108–116, 1989). The level of toxic impact of Lepidium sativum (compared to control) was assessed according to the modified method of Wang (Rev Environ Contam Toxicol 126:88–127, 1992). Results  The mean pH of urban storm water runoff does not vary much from neutral, but range values are quite different, from 4.0 up to 8.7. The highest concentration of SS reached 800 mg L⁻¹, TPH—2.4 mg L⁻¹, BOD₇—300 mg O₂ L⁻¹ and COD_Cr—1,400 mg L⁻¹. The SS was above the limit in 64% of total amount of grab samples, TPH—37%, BOD₇—41% and COD_Cr—55%. The toxicity analysis of the bottom sediments showed varying toxicity of bottom sediments along the stream. From nine analysed samples of bottom sediments, 30% had weak toxicity, 30% medium and 30% strong toxicity on the test organism plant L. sativum. There was one single sample with no toxic effects, so that the results showed that urban storm water has an unacceptable environmental impact on recipients. It was also indicated that storm water runoff discharge alone is not the potential source of toxicity of bottom sediments. The litter demonstrated a weak toxicity of bottom sediments as well. Discussion  Most local authorities do not consider storm water runoff discharges to be a matter of great concern because they believe that surface runoff arising from rainfall is still relatively clean. The study showed that the current method of monitoring storm water runoff quality by chemical analyses is not the best tool for environmental impact assessment and must be combined with toxicity tests of bottom sediments of recipients. Recommendations and perspectives  To avoid the environmental impact of storm water runoff more attention should be paid to the development and implementation of storm water runoff pollution prevention measures. The study implies that future research concerning the relationships between storm water runoff deposit characteristics and biological activities must be developed to evaluate the contamination potential of stream sediment deposits for local aquatic ecosystems. Further studies should be developed to characterise the activities of the microbial community of storm water runoff sediments, and to monitor bioremediation in situ.     

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.     

Importance of dose metrics for lethal and sublethal sediment metal toxicity in the oligochaete worm Lumbriculus variegatus

Background, Aims, and Scope  There is an increasing demand for controlled toxicity tests to predict biological effects related to sediment metal contamination. In this context, questions of metal-specific factors, sensitivity of toxicity endpoints, and variability in exposure duration arise. In addition, the choice of the dose metrics for responses is equally important and is related to the applicability of the concept of critical body residue (CBR) in exposure assessments, as well as being the main focus of this study. Methods  Experiments were conducted to assess toxicity of Cd, Cr, Cu and Pb to the oligochaete worm Lumbriculus variegatus with the aim of determining CBRs for two response metrics. Mortality and feeding activity of worms exposed to sediment-spiked metals were used as end-points in connection with residue analyses from both the organisms and the surrounding media. Results  LC50 values were 0.3, 1.4, 5.2, and 6.7 mg/L (from 4.7 μmol/L to 128.0 μmol/L), and the order of toxicity, from most toxic to least toxic, was Cu > Cd > Pb>Cr. By relating toxicity to body residue, variability in toxicity among the metals decreased and the order of toxicity was altered. The highest lethal residue value was obtained for Cu (10.8 mmol/kg) and the lowest was obtained for Cd (2.3 mmol/kg). In the 10-d sublethal test, both time and metal exposure were an important source of variation in the feeding activity of worms. The significant treatment effects were observed from worms exposed to Cd or Pb, with the controls yielding the highest feeding rate. However, quantitative changes in the measured endpoint did not correlate with the exposure concentrations or body residues, which remained an order of magnitude lower than in the acute exposures. Discussion  Both response metrics were able to detect a toxic effect of the metals. However, the ranking of metal toxicity was dependant on the choice of the dose metric used. An attempt to form a causal mortality-mediated link between tissue residues and metal toxicity was successful in water-only exposures. The results also indicated that egestion rate was a sensitive toxicity end point for predicting the effects of sediment contamination. Conclusions  By relating the biological response with the tissue metal residues, toxicity data was comparable to both environmental media as well as different response metrics and time scales. The results also revealed the importance of metal toxicity ranking on a molar basis and, furthermore, a direct link to the CBR concept was established. Recommendations and Perspectives  There is a growing demand for methods to assess the effects of contaminated sediments to benthic fauna and whole aquatic ecosystems. Such information is needed for sediment quality guidelines that are currently being developed in many countries and remediation processes. The use of body residues as a dose metric in metal toxicity studies may help to overcome difficulties related to bioavailability issues commonly faced in sediment toxicity studies. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Flumequine Uptake and the Aquatic Duckweed,Lemna minor L.

Water, Air, & Soil Pollution - Phytotoxicity of Flumequine (F) on the aquatic duckweed, Lemna minor L., and plant drug uptake were evaluated by a simple ecotoxicological test. Flumequine, at...     

Contaminated sediments as a potential source of Zn, Pb, and Cd for a river system in the historical metalliferous ore mining and smelting industry area of South Poland

Background, aim, and scope  Elevated levels of heavy metals in the aquatic and soil systems can be caused by the weathering of mineralized rocks. This enrichment is often considerably enlarged by historical and current mining and smelting activities. In Poland, the most contaminated river systems are those in the Silesia region. The metalliferous ore mining and smelting industries have been the main sources of heavy metal pollutions over the last 100–170 years. The previous and present studies have shown very high concentrations of heavy metals in the bottom sediments of the Mala Panew River, the most polluted tributary of the Oder River. The main objective of this work was to study temporary changes of selected metal (Zn, Pb, and Cd) concentrations in upper layer of bottom sediments at the measuring point near the outlet of the Mala Panew River into the Oder River, and to determine the vertical distribution of the metals in the sediment cores from the most polluted middle part of this river. The mobility of the metals and their potential bioavailability were assessed based on metal partitioning in the sediments and metal concentrations in pore waters. The presented data were compared with metal concentrations in aquatic sediments from similar historical mining and smelting sites in Poland and other countries. Methods  The upper layer of bottom sediment samples from the same Mala Panew River measuring point were collected six times in the period 1997–2005, while five sediment cores were collected once from the middle course of Mala Panew River in 2006. Abiotic parameters such as pH and Eh have been determined in situ. Metal contents were determined in the <20 and <63 μm size fractions of sediments after digestion in a microwave oven with aqua regia or concentrated nitric acid. Metal mobility was assessed in the selected sediment cores by the chemical forms of metals (sequential extraction method) and their concentrations in pore waters were investigated. Results  The concentrations of Cd, Pb, and Zn in the upper layer of sediments varied, depending on both the season and the year of sampling. Their mean concentrations (from six samplings) are [mg/kg]: Zn 1,846, Pb 229 and Cd 73. The metal concentrations in the sediment cores varied with the depth in the range of [mg/kg]: 0.18–559 for Cd, 26.2–3,309 for Pb and 126–11,153 for Zn, although the highest accumulations generally could be observed in the deeper layers. The most mobile metal fractions, i.e., exchangeable, carbonate and easily reducible fractions, are typical of Zn and Cd. Cadmium was found to be the most mobile metal and its relative contribution ranges from 84 to 96%, while in the case of Zn it ranged from 45 to 94%. Lead is mainly associated with the moderately reducible fraction (30–60%). Relative contributions of metal chemical forms slightly vary with the depth in the sediment profile. The results obtained for the pore water samples show very high concentrations of the metals studied, especially in the case of Cd (31–960 μg/dm³) and Zn (300–4,400 μg/dm³). Discussion  Accumulation of Cd, Pb, and Zn in the upper layer of the bottom sediments and in the sediment core samples from the Mala Panew River is very high, considerably exceeding the local geochemical background. High contributions of mobile Cd and Zn and the toxicity of cadmium can cause environmental risk. Our measurements also suggest that mobile metals can migrate into groundwater, whereas the groundwater itself can leach some chemicals from river sediments, because of a relatively high water table in the study area, especially during rainfall periods. Comparison of the results obtained with the literature data from the last decade shows that the concentrations of Cd and Zn in the sediments from the Mala Panew River are the highest among other submersed sediments in Poland and other regions (e.g., the Mulde River, Germany). Conclusions  The Mala Panew River is one of the most polluted rivers when compared with similar historical mining and smelting areas in Poland and elsewhere. The sediments studied are strongly polluted with the metals analyzed. In the upper layer of the bottom sediments there has been no reduction of Zn and Cd amounts over the last decade, which could suggests a long-term migration and a secondary contamination. Considerably higher accumulations of metals in overbank sediment cores and in the deeper core section could result from strong contamination in previous decades and translocation of Cd and Zn (secondary pollutants). The relatively high concentrations of the two metals in pore waters support these findings. Cadmium is crucial in the environmental risk assessment because of its high mobility and toxicity. These data are important for water/sediment management in the transboundary Oder River catchment, situated in Poland, Germany and the Czech Republic. Recommendations and perspectives  It is important to assess mobility phase and pore water in the contaminated historical aquatic sediments. Such studies may help explain the changes, which take place in the sediment layers as well as at the water–sediment interface. Obtained results should be used for the risk assessment of the historical contaminated sediments at the local river-basin scale. The treatment of contaminated sediments, e.g., dragging activity, should be considered as very important in management strategies in order to avoid remobilization of metals.     

厌氧发酵牛粪对浮萍生长和养分吸收的影响

养分从牛粪到农田的循环利用是养牛场可持续发展、资源利用和环境保护的重点研究内容。利用奶牛场废水种植水生植物被认为是一种有效的废水处理及养分循环的方法。本文研究了3种浮萍[少根紫萍0128（Landoltia punctata 0128）、膨胀浮萍7589（Lemna gibba 7589）和小浮萍9517（Lemna minuta 9517）]在厌氧发酵过的奶牛场废水中种植时的养分吸收和生物质变化。在28 d的测试期间,种植在稀释比例为1：18的厌氧发酵过的牛奶场废水中的少根紫萍01283获得最高的总氮吸收率（11.6%±1.6%）,种植在稀释比例为1：27厌氧发酵过的牛奶场废水中的少根紫萍0128获得最高的总磷吸收率（15.4%±4.4%）;相应地少根紫萍鲜重的增长率分别为0.11 g·d^-1和0.17 g·d^-1。3种浮萍中,少根紫萍最具有吸收牛奶场废水氮、磷并获得较高生物质的潜力。     

生态毒理学工具在评价石油化工厂区土地耕作适宜性中的运用

The demand for simple and rapid bioassays in ecotoxicological evaluations is of paramount importance in order to speed up environmental monitoring programs. In this study we performed bioassays with lettuce seeds and two species of terrestrial isopods （Armadillidium vulgare and Porcellio dilatatus） for the ecotoxicological assessment of a landfarming soil from a petrochemical complex area. The solubilized content of test soil demonstrated a concentration-response type toxic effect on seed germination rate, and a delay on germination, but showed toxic effect on seedlings wet weight only at the highest concentration. Toxic effects were also observed in mortality rate and avoidance behavior of the two woodlice species. These results demonstrated the sensitiveness of the organisms studied, and highlighted the possibility to use these bioassays in environmental monitoring programs in areas contaminated with fossil fuels.     

Acute and chronic isopod testing using tropical Porcellionides pruinosus and three model pesticides

An approach for testing acute and chronic ecotoxicological effects of pesticides on isopods in the laboratory is presented. Laboratory cultures of tropical Porcellionides pruinosus were used. The pesticides Benomyl, Carbendazim and lambda-Cyhalothrin served as model chemicals. The toxicity tests were done with five to six concentrations and one untreated control in four replicates containing 10 individuals per vessel. The test was performed at 28 °C, in permanent darkness and it lasted 14 days. Each chemical was mixed in 250 g DW per vessel of OECD artificial, LUFA 2.2 standard, tropical artificial or tropical natural soil in acute tests. Chronic tests were performed using 150 g DW OECD artificial or LUFA 2.2 standard soil. Both adult mortality and number of juveniles showed a high sensitivity of P. pruinosus towards lambda-Cyhalothrin while Carbendazim and Benomyl had a very low and no toxicity, respectively. Further research and optimization of the chronic test design are required.     

A combined hydraulic and toxicological approach to assess re-suspended sediments during simulated flood events—part II: an interdisciplinary experimental methodology

Purpose

Flood events are expected to increase both in intensity and frequency due to climate change in the near future. From an environmental toxicology perspective, there is concern that such flood events could lead to the remobilization of contaminated sediment layers in rivers. The aim of this pilot study was to establish a novel and interdisciplinary framework combining methods of hydrodynamic engineering and ecotoxicological assessment to enable investigation of the potential risks associated with such remobilization events.

Materials and methods

Formulated sediment was prepared according to OECD guideline 218 and spiked with a mixture of four polycyclic aromatic hydrocarbons (phenanthrene, chrysene, pyrene, benzo[a]pyrene) at concentrations of 3.3?C8.3?mg?kg^?1 dry weight. Rainbow trout (Oncorhynchus mykiss) were exposed as test animals to re-suspended sediments in three out of five experiments. The experiments were carried out in an annular flume designed to investigate transport behaviour of fine-grained sediments. Several physicochemical (e.g. pH) and sedimentological (e.g. turbidity) parameters were measured to characterise environmental conditions and erosion behaviour of sediments. Furthermore, exposure concentrations were measured by means of an in vitro assay (7-ethoxyresorufin-o-deethylase (EROD), RTL-W1 cell assay) and chemical analysis.

Results and discussion

Preparation and spiking of large amounts of formulated sediments were feasible but not practical. Successful spiking could be confirmed by the bioanalytical methods with the spiked sediments showing significantly elevated EROD induction compared to control sediments. Conditions within the annular flume remained stable throughout all experiments and were adequate to support rainbow trout. Flood events were successfully simulated, resulting in re-suspension of formulated sediment. Different erosion behaviours of sediments during the simulated flood events were observed and could be associated with changes in microbial composition of sediments due to differences in storage conditions. Therefore, maintaining constant storage conditions of formulated sediments is crucial to enable consistency and comparability among erosion experiments.

Conclusions

This study clearly demonstrated the feasibility of a combined hydro-toxicological approach in support of the investigation of the potential ecotoxicological relevance of sediment re-suspension events. However, based on the results presented here, it is recommended to include additional physicochemical parameters, such as redox potential and conductivity, and to extend the experimental setup to natural sediments and different aquatic organisms. Future studies will use natural sediments containing representative microbial communities and extracellular polymeric substances to enable extrapolation from the annular flume to conditions in natural flowing waters.     

Sediment Toxicity Assessment: Rationale for effect classes (5 pp)

-  Dedicated to Prof. Dr. Ulrich Förstner on his 65th birthdayBackground, Aims and Scope   The main challenge in the application of biotests for hazard assessment of sediments is a transparent interpretation, especially if combinations of biotests are applied and an integrated assessment of all results is wanted. For decision making, assigning results of biotesting into different classes that indicate increasing hazards can be a useful tool. In this paper we suggest an approach on how to set up a site-independent classification system for sediment toxicity tests.Methods   About 250 sediment samples were collected from two rivers and evaluated. The bioassays applied are used in a standardized procedure for all samples over years. The test battery include toxicological endpoints like nematode growth and reproduction, algae growth, bacteria activity, and luminescence inhibition.Results and Discussion   The classification system described emphasizes the following steps: the assessment of test-specific response spans for each applied biotest and the subsequent interpretation of inhibition values in terms of toxicity, estimation of accuracy and uncertainty of different test systems, and the use of cluster- and k-means analyses in order to identify occurring pattern within a large biotest data base, followed by a ranking. The outcome of the procedure were 5 effect classes with an increasing potential of hazard. Conclusion   The effect classes represent the result of a logical procedure of deduction. The procedure is transparent giving an increased degree of certainty. The site independent ranking of 5 classes could be used for sediment monitoring and facilitate fast interpretation. Recommendation and Perspective   A large database comprising differently contaminated sediments is the precondition for setting up effect classes, because it is essential to know the range of effects and the variability of the test methods. The proposed procedure could then provide the basic rules for designing an expert system.