A Chronic Plant Test for the Assessment of Contaminated Soils. Part 2: Testing of contaminated soils (10 pp)

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PREAMBLE According to the German Federal Soil Protection Act (BBodSchG 1998), the habitat function of soils must be protected. Despite the fact that in the Federal Soil Protection Ordinance (BBodSchV 1999) it has not been established how this goal can be reached reliably, it is clear that such a biological function can only adequately be assessed using biological test methods. This is especially true when a soil is contaminated by a mixture of often unknown chemicals. In such a case the use of chemical analysis aiming at a small range of known substances is not sufficient and must therefore be supplemented by biological methods. For this reason, several standardised test methods are available (e.g. using earthworms, collembolans or plants; Römbke and Knacker 2003; ISO 2003). Since acute tests are usually not sensitive enough for the assessment of potentially contaminated soils (e.g. Hund-Rinke et al. 2002), chronic tests like the earthworm reproduction tests (ISO 1998) are recommended for this purpose.

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A chronic plant test for the determination of phytotoxicity was missing until quite recently. The term phytotoxicity is understood here as the capacity of a compound or a contaminated soil to cause temporary or long-lasting damage to plants (EPPO 1997). Therefore, the German Ministry for Education and Research sponsored a project (1997 – 1999) in which – based on existing standardised methods – such a chronic plant laboratory test was developed and partly validated (Kalsch and Römbke 2000). The new test can be used for the evaluation of single chemicals (see Part 1 of this mini-series) as well as for the assessment of contaminated or remediated soils (see Part 2 of this mini-series).

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ABSTRACT Background and Scope. A new chronic plant test system which is based on experiences with various acute plant tests (e.g. published by OECD or ISO) and existing North American Plant-Life-Cycle Bioassays was standardised in a project sponsored by the German government. Characteristic properties of the test system, which can be performed either with Brassica rapa (turnip rape) or Avena sativa (oat), are described in Part I of this mini-series.

Methods

This new test was used to assess the effects of natural soil samples contaminated with TNT (2,4,6-Trinitrotoluene) or PAHs (poly-aromatic hydrocarbons). The soils were tested after taken from the field as well as after being remediated. Different control and reference soils were used to evaluate the test results. In addition, they were compared with the results of tests in which either TNT or Pyrene was spiked to field and standard soils (see Part I of this mini-series).

Results

All contaminated soils showed clear effects in the Chronic Plant Test (usually B. rapa was more sensitive than A. sativa). LUFA 2.2 standard soil and OECD artificial soil are well-suited as control and mixture substrates, while reference soils collected at uncontaminated sites were several times phytotoxic. In most of the latter cases, soil properties could be identified as the main cause of these effects (e.g. the pH value). While in general the sensitivity of the reproduction and biomass endpoints did not differ much, it is recommended to measure different endpoints (i.e. biomass and reproduction) due to the different mode-of-action of contaminants. In the case of TNT a good agreement between the results of single chemical tests and tests with TNT-contaminated soils was found (due to the minimal effects of Pyrene the same statement is not possible for PAHs).

Outlook

The results of this work have been used to prepare a draft test guideline, which has recently been standardised by the International Organisation for Standardisation (ISO). Based on these experiences, recommendations are given how this chronic test system could be used as part of a test battery for the assessment of contaminated soils.     

Influence of Soil Organic Carbon on Acute and Chronic Toxicity of Plant Protection Products to Poecilus cupreus (Coleoptera, Carabidae) Larvae (4 pp)

Background, Aims and Scope   Variability of results from terrestrial ecotoxicological tests with arthropods demonstrate the importance of understanding the impact of soil composition has on the bioavailability of pollutants. Beside other soil parameters, the organic C content is known to be relevant to bioavailability. The aim of the present paper was to detect lethal and sublethal effects of plant protection products on soil-dwelling larvae of the carabid beetle Poecilus cupreus under different concentrations of soil organic C. The study was based on a familiar laboratory test method. Methods   Larvae of the laboratory-reared carabid beetle P. cupreus were exposed to dimethoate in a standardised soil (Lufa 2.1), modified to four different concentrations of organic C between 0.5 to 2.8% by admixing peat. Dimethoate was applied to the soil surface at rates ranging between 40 and 250 g a.i./ha. Some larvae were exposed to an application of water to act as a control. One larva was released per test tube, with at least 20 larvae per treatment group. Effects on mortality and developmental time of the larvae and on the hatching weight of adult beetles were recorded. Dimethoate was selected as the test item because some dose-response data for different soils were already available and because the reproducibility of test results with dimethoate had already been shown in a previous ring test. Results and Discussion   With increasing concentration of organic C in the soil substrate, mortality decreased at all tested rates of dimethoate. Consequently, LC50 values increased from 39 to 107 g a.i./ha with increasing organic C content from 0.5 to 2.8%. The results suggest higher bioavailability of dimethoate with decreasing content of organic C. The replication and magnitude of sublethal effects (hatching weight and developmental time) was insufficient to detect a clear interaction between dimethoate and organic C. Conclusion   The results of the experiment reveal that the concentration of organic C in the soil substrate is influential on the effects and, by association, the toxicity of dimethoate to larvae of P. cupreus. For the interpretation of terrestrial ectotoxicological tests with arthropods, the composition of the substrate has to be taken into account. Recommendations and Outlook   For reliable and reproducible test results, a well-defined and thoroughly controlled composition of the test soil is highly recommended. Test results should be used for risk assessments only after having taken the soil composition, especially the organic C content, into account.     

Occurrence of Polycyclic Musks in Sewage Sludge and their Behaviour in Soils and Plants. Part 2: Investigation of Polycyclic Musks in Soils and Plants (9 pp)

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Part 2: Investigation of Polycyclic Musks in Soils and PlantsPart 1: Behaviour of Polycyclic Musks in Sewage Sludge of Different Treatment Plants in Summer and Winter

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Preamble. In Part 1 of the study, screening tests were performed to investigate the occurrence of PCM 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 in plants.

Goal, Scope and Background

Polycyclic Musks (PCMs) enter the terrestrial environment via the use of sewage sludge as fertilizer. After entry into soil, they can be taken up by plants or move into the groundwater body. However, until now no overall risk assessment for polycyclic musks is available. For a preliminary risk assessment further information is needed about their behaviour in the soil environment. Therefore, Part 2 of the study (Part 1, see J Soils Sediments, OnlineFirst ) examined the adsorption of PCMs to soil, their dissipation and leaching in soil and their uptake by plants.

Methods

Analytical methods for the determination of HHCB and AHTN in soil and plant samples were developed and applied. The adsorption/desorption studies were performed according to OECD guideline 106 and draft guideline 121 using three soils. Dissipation (aerobic degradation) was examined according to BBA guideline 4-1 in three soils over a test period of 37 weeks under controlled conditions. Leaching experiments in soil columns were performed using columns of 14 cm Ø and 30 cm filling height for a test period of 48 hours. Finally the uptake of PCMs by lettuce and carrots was studied in laboratory and outdoor experiments.

Results

The adsorption/desorption studies resulted in Koc values between 4200 and 7900 for HHCB and between 4800 and 13600 for AHTN showing strong sorption to the soils investigated. The dissipation of the PCMs occurred very slowly with elimination rates after 37 weeks of approx. 50% and 25% for HHCB and AHTN, respectively. The leaching experiments showed leaching rates of < 0.001% for HHCB and AHTN during a test period of 48 hours. The slight leaching is presumably due to ‘preferential flow'. The transfer factors (ratio of concentrations in the plant to concentrations in the soil ) for HHCB in lettuce and carrot leaves determined in plant uptake experiments were as low as 0.003. For HHCB in carrot roots however high transfer factors of 0.095 for a humic and 0.48 for a loamy soil were obtained. The high uptake may be caused by partitioning of the HHCB into the essential oil cells of the carrot roots.

Discussion

The polycyclic musk compounds HHCB and AHTN showed high adsorption to soil. A desorption phenomenon occurred after three desorption steps. High adsorption influences the slow degradation kinetic. Comparable investigations into the degradation behaviour show the same results, whereas other authors observed a faster degradation. We assume a dependency on microbial consortia, which can vary as a function of the substrate and adaptation of the microbial population. Leaching tests were carried out in our study, but not by other studies. The results reflect the adsorption into the soil matrix.

Conclusions

The results show that PCMs are widespread contaminants in sewage sludge and should be considered in a risk assessment as potential contaminants of sewage sludge destined for agricultural use. When applied to soil they may remain in the upper soil layers due to their high sorption, low degradability and low leaching behaviour. Uptake by some plants like carrot roots may be relevant.

Recommendations and Perspective

. This study examined the adsorption of PCMs to soil, their dissipation and leaching in soil and their uptake into plants. For a qualitative risk assessment more data on adsorption /desorption, and degradation in soils under different soil conditions needs to be generated. Also, further studies have to be carried out to gain a better understanding of plant uptake of PCMs, especially HHCB and AHTN.

     

Technical Recommendations for the Update of the ISO Earthworm Field Test Guideline (ISO 11268-3) (5 pp)

Background, Aim and Scope   Background, Aims and Scope. The earthworm field test firstly was developed by the German Federal Biological Research Centre for Agriculture and Forestry (BBA) for testing of pesticides and later internationally standardised by the International Organization for Standardization (ISO) as a tool for characterizing soil quality. It is mainly used for the assessment of effects of pesticides on earthworms under field conditions. Growing experience has shown that the practical performance of the test can be improved. Main Features: In two meetings organised by the Federal Biological Research Centre for Agriculture and Forestry (Braunschweig, 2002) and later the German Federal Agency for Consumer Protection and Food Safety (Lille, 2005) an ad-hoc working group of experts from various countries and institutions discussed the practical performance and recommendations that should be taken into account in a revised draft following the current ISO guideline after the proposed revision has been approved by voting in the periodical review. Results: The outcome of the discussions and the background of the recommendations of the ad-hoc working group are briefly summarised in this paper. These are mainly methodological details in order to improve the test performance (e.g. by decreasing variability or increasing practicability). Discussion: These recommendations are proposed to be considered for the next update of the current ISO guideline. They should be seen as technical amendments still waiting for their approval by standardisation organisations. Conclusions: Although this method focuses on the assessment of effects of pesticides on earthworms, the recommendations will also be valuable for testing contaminated soils. The recommendations presented may also be a first step for splitting the current guideline into two fields of application, one for testing of chemicals and pesticides (i.e. within the scope of OECD) and another one for testing soil quality (i.e. within the scope of ISO). Perspectives: Nevertheless, there is still a major need for guidance concerning the interpretation of effects determined in such complex field tests.     

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.     

A laboratory test protocol to evaluate effects of plant protection products on mortality and reproduction of the predatory mite<Emphasis Type="Italic">hypoaspis aculeifer</Emphasis> canestrini (acari: laelapidae) in standard soil

We provide a test protocol to evaluate the effects of plant protection products on mortality and reproductive performance ofHypoaspis aculeifer (Acari: Laelapidae). The test design is in compliance with standard characteristics of extended laboratory studies as formulated by international expert panels dealing with non-target arthropod testing schemes. Potential effects on survival rates are evaluated by assessing mortality after an exposure period of 14 days that starts with protonymphs. The reproductive performance of surviving mites is studied by following the egg production of individually isolated females over a 7-day period. The hatching success of these eggs is assessed subsequently. Using several years’ data obtained from water control treatments, we propose criteria to evaluate the validity of individual experiments.     

Comparability of and Alternatives to Leaching Tests for the Assessment of the Emission of Inorganic Soil Contamination (11 pp)

Goal, Scope and Background

The suitability of leaching tests for the assessment of soil water concentrations is a controversial issue. The aim of this paper is to review and critically discuss the applicability and comparability of leaching tests in the scope of groundwater risk assessment of inorganic contamination and to discuss soil water sampling methods as alternative.

Main Features

First, the theoretical background of leaching tests and the main leaching controlling parameters (pH, redox, ionic strength, reaction kinetics, surface, and speciation) are discussed. Experience with common batch leaching tests (the German DEV S4 test (S4), saturation soil extraction (SSE), ammonia nitrate extraction (ANE), and pHstatic tests) are compiled and an emphasis is set on the comparability of the results of batch leaching tests. Additionally, the comparability between batch tests and column tests is discussed and comparison studies are reviewed. As a feasible alternative, soil water sampling strategies (the suction cup method and centrifugation) are outlined.

Results and Conclusions

For an expedient application of leaching tests, it is crucial to identify the main release controlling parameters, which can be both site and contaminant specific. Possible controlling parameters are, for example, pH, redox conditions, specific surface area of the investigated material, liquid to solid-ratio, ionic strength, cation exchange capacity, DOC, speciation, temperature and contact time. It depends on the release process of the contaminant in the investigated material, which parameters are influencing the release predominantly. The following questions have to be answered: Is the release process availability controlled or solubility controlled and are there kinetic limitations? Suction cups are particularly useful for long-term monitoring. In contrast, centrifugation is a fast and simple method to sample soil water also at larger and heterogeneous sites. SSE or similar batch tests for coarse material are suitable if the soil water cannot be investigated directly. Contaminant concentrations of the SSE fit best to contaminant concentrations in soil water compared to other leaching tests. Concentrations measured with S4 tests are usually significantly lower than in real soil water due to the unrealistically high liquid to solid-ratio. The ANE is used for the evaluation of plant uptake. It does not represent realistic soil water concentrations. Cationic contaminants are usually significantly higher concentrated in ANE eluates. pHstatic tests provide an evaluation of the pH dependency of contaminant release and buffer capacity of the investigated material. It provides indications to release processes and often explains deviations in the results of different leaching tests. Previous practical experience and fundamental research studies show that a conversion of leaching test results, as is proposed by the (already withdrawn) DIN V 19735, is impossible. Correlations of results from different methods, regarding different kinds of materials and different sites, are not significant. This is due to different leaching processes.

Recommendation

. For short-term risk assessment, soil water should be sampled and investigated directly by suction cups (for monitoring purposes) or centrifugation (for inventory purposes). If this is not possible, the SSE or analogous batch tests for coarse material with a narrow L/S ratio should be applied. A suggestion could be a modified saturation soil extraction where a soil column is saturated and the eluate is removed by suction at the bottom of the column. With this method, an unsaturated column test could follow in the same vessel. In order to assess the long-term leaching behavior, total contents of the investigated material have to be taken into account additionally. Furthermore, it is essential to understand the dominant physical and chemical release processes and to figure out the main leaching controlling parameters. Therefore, the following methods are recommended: pHstatic tests at different pH values provide an insight to leaching processes and possible future leaching scenarios. Batch tests similar to the S4 procedure with different L/S ratios are useful to find out whether the release process is solubility or availability controlled. Additionally, this method allows the determination of maximum solubility and maximum availability, respectively. Furthermore, unsaturated column tests provide an insight into leaching processes and releasable amounts even though they require great experimental effort. Other leaching tests like S4 or ANE are not suitable for the estimation of soil water concentrations.     

Effects-based Assays in the Earthworm Aporrectodea caliginosa: Their Utilisation for Evaluation of Contaminated Sites before and after Remediation (8 pp)

Goal, Scope and Background   In a preliminary ecological risk assessment, potential adverse effects of contaminants are often evaluated by measuring chemical residues and comparing these with regulatory guidelines. However limitations with this approach with regards to establishing actual effects have resulted in the increasing usage of sublethal effects-based assays, including biomarkers, to evaluate the hazard posed by contaminants in the environment. In this study a number of effects-based endpoints in the earthworm Aporrectodea caliginosa were evaluated to determine their comparative sensitivity for assessment of adverse effects of soil contaminated with petroleum hydrocarbons. Methods   Adult and juvenile earthworms were exposed for 4 weeks to sublethal concentrations of soil collected before and after remediation of a petroleum-contaminated site. A suite of endpoints were measured in these earthworms, including mortality, fecundity, growth, and juvenile maturation, and two less traditional endpoints, the biomarker, the neutral red retention assay (NRRA) and an avoidance behaviour test. Results and Discussion   Cocoon viability in this species is not a reliable parameter to measure, due to low viability in controls and a high coefficient of variation. Growth in adult earthworms was a more sensitive parameter than cocoon production. Maturation and growth of juveniles have been proposed as more sensitive endpoints than adult cocoon production and growth respectively. This was not apparent in the growth parameters, but maturation of juveniles did appear to be more sensitive than cocoon production by adults. The NRRA was a more sensitive parameter than cocoon production, and the NRRA and growth were both affected at the lowest concentration tested. The NRRA response appeared to be more sensitive than growth, but NRRT was only evaluated in one soil only, while the other parameters were assessed in two soils. However, the NRRA has previously been found to be more sensitive than growth after exposure to a number of contaminants. The avoidance behaviour assay exhibited similar sensitivity to growth and fecundity and could therefore be useful as a simple pre-screening test. Conclusion   The chronic endpoints, growth, cocoon production, and juvenile maturation parameters, were all sensitive endpoints for detecting exposure to the petroleum-hydrocarbon-contaminated soil. The NRRA was the most sensitive of the endpoints assessed and could be used as an early-warning indicator to predict adverse impacts. Avoidance behaviour could be used as a simple pre-screening test to evaluate contaminated soils prior to more extensive and invasive testing. Recommendations and Perspective   Measuring chemical concentrations in environmental samples is not always useful, as the toxicological impacts of exposure to these concentrations are not always discernible. However, the use of effects-based endpoints, either in situ or in the laboratory using laboratory-reared earthworms, can account for the bioavailability of chemicals in the soil, and can therefore provide information on the toxicological impacts of exposure. The assays tested in this research were sensitive indicators of exposure, and therefore can be used to determine potential ecological risks at contaminated sites and to monitor the progress of remediation at these sites.     

Quantifying the fine scale (1km × 1km) exposure and effects of ozone. Part 1. Methodology and application for effects on forests

Surface ozone concentrations show very large local variability. This, in rural areas, is largely a consequence of the degree to which the surface air is decoupled from the bulk of the atmosphere by nocturnal and winter stability effects and of the aerodynamic roughness and topography of the countryside. Procedures have been developed to simulate these effects and have been used with O₃ data from a regional monitoring network to quantify exposure of vegetation to mean and peak O₃ concentrations. The index of exposure to potentially phytotoxic O₃ concentrations is the AOT40 (accumulated exposure over a threshold of 40 ppb) expressed in ppb.h. The accumulated exposure of crop and forest surfaces to ozone is shown to increase approximately linearly with altitude and along a gradient from 3000 ppb.h in north west Scotland to 7000 ppb.h in the south of England. The area of UK forest which exceeds the critical level for ozone effects (10⁴ ppb.h above 40 ppb) totals 2.0 × 10⁴ km² and represents 47% of afforested land. The areas affected include most of southern and central England and Wales and large plantation forests of Northumberland and the Scottish borders.     

Evaluation of a Multi-species Test System for Assessing Acute and Chronic Toxicity of Sediments and Water to Aquatic Invertebrates: Effects of Pentachlorophenol on Daphnia magna and Chironomus prasinus (6 pp)

Background, Aims and Scope   Single-species toxicity tests are currently used as main tools for assessing effects in ecological risk assessments. Nevertheless, in higher-tier evaluations, alternative ecotoxicological methods can be required. Alternative methods should cover more realistic exposure conditions and/or additional species or endpoints. Water/sediment systems can be considered for covering additional exposure routes. A typical feature of water/sediment systems is the modification of exposure levels with time. The simultaneous exposure of different species within the same system can cover differences in sensitivity and relevance of exposure routes. The aim of this study was to develop a water/sediment test that could detect relevant effects on two selected species and explore the relevance of sediment and waterborne exposure routes. This alternative method may provide relevant information facilitating the design of microcosm and mesocosm studies.Methods   A multi-species water/sediment system was designed using two invertebrates: Chironomus prasinus a benthic detritivore invertebrate and Daphnia magna a pelagic filtering invertebrate was designed. Seven static water/sediments systems were built up: two controls and five treatments where sediments were spiked with NaPCP at nominal concentrations of 1.25, 2.5, 5, 10 and 20 mg/kg, respectively. Each system contained caged and free (unrestrained) Daphnia magna organisms to consider differences in the exposure route (through sediment and/or water column) and Chironomus prasinus organisms. Fate and partitioning of PCP within the water/sediment compartments were assessed. Acute (lethality) and chronic effects (reproduction) for Daphnia magna were monitored after 48 h and 16 days of exposure, respectively. Adult emergence and oviposition success of Chironomus prasinus were monitored at the end of test (16 days).Results and Discussion   Mortality and reproduction inhibition of Daphnia magna occurred at the two higher doses, corresponding to maximum measured PCP water concentrations of 1.95 and 0.746 mg/l, respectively. Chironomus prasinus was less sensitive to PCP than Daphnia magna. Concentrations inhibiting reproduction and provoking almost 100% adult mortality in D. magna only reduced slightly the emergence and reproduction of chironomids. The experimental design focussed on suitability of the system for assessing simultaneously assessing effects on D. magna and C. prasinus instead of calculations of toxicity endpoints for PCP. The results indicate that this experimental design can be useful to the field of sediment/aquatic toxicity testing, particularly through the comparison with single- species results. Assessment of relative contribution of two exposure routes for Daphnia magna and complementary chronic endpoints derived from two different species can be achieved. Another advantage is that differences in reproduction strategies of selected species (sexual vs. parthenogenetic) should permit one to detect mechanisms of action associated to with endocrine disrupters.Conclusion   The feasibility of a two-species test built-up in an artificial sediment/water system has been demonstrated. This experimental design represents a cost/effective test which can assess effects through water and sediment pathways and incorporates several relevant chronic endpoints in a single assay. Recommendation and Outlook   By building on the experience gained, improvements for further test designs are discussed. Test validation with a set of model chemicals will contribute to address the effectiveness of the test developed as comparing compared to single- species tests.     

Bioassay-directed Fractionation of Organic Extracts of Marine Surface Sediments from the North and Baltic Sea - Part II: Results of the biotest battery and development of a biotest index (8 pp)

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Part I: Determination and identification of organic pollutants Part II: Results of the biotest battery and development of a biotest index

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Preamble. This series of two papers presents the results of an interdisciplinary research project (ISIS) dealing with bioassay-directed fractionation of marine sediment extracts. Part I presents the extraction and fractionation procedure as well as the results of chemical analysis, including non-target analysis of sediments. Part II describes the results of the biotest battery in relation to chemicals possibly causing parts of the observed effects. A biotest index is used to compare the toxicities of the samples.

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AUTHORS / AFFILIATIONS Ninja Reineke (3), Werner Wosniok (4), Dirk Danischewski (1), Heinrich Hühnerfuss (3), Angelika Kinder (5), Arne Sierts-Herrmann (5), Norbert Theobald (2), Hans-Heinrich Vahl (6), Michael Vobach (1), Johannes Westendorf (6) and Hans Steinhart (5).

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(1) Federal Research Centre for Fisheries, Institute for Fishery Ecology, Palmaille 9, 22767 Hamburg, Germany (2) Federal Maritime and Hydrographic Agency, Bernhard-Nochtstr. 78, 20359 Hamburg, Germany (3) University of Hamburg, Institute for Organic Chemistry, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany (4) University of Bremen, Institute of Statistics, Bibliothekstr. 1, 28334 Bremen, Germany (5) University of Hamburg, Institute for Food Chemistry, Grindelallee 117, 20146 Hamburg, Germany (6) University of Hamburg, University Hospital Hamburg-Eppendorf, Department for Toxicology, Vogt-Kölln-Str. 30, 22527 Hamburg, Germany (7) Eurofins Wiertz-Eggert-Jörissen, Stenzelring 14b, 21107 Hamburg, Germany

Goal, Scope and Background

The ecological relevance of contaminants in mixtures is difficult to assess, because of possible interactions and due to lacking toxicity data for many substances present in environmental samples. Marine sediment extracts, which contain a mixture of environmental contaminants in low concentrations, were the object of this study. The extracts were investigated with a set of different biotests in order to identify the compound or the substance class responsible for the toxicity. For this goal, a combination of biotests, biotest-directed fractionation and chemical analysis has been applied. Further on, a strategy for the development of a biotest index to describe the toxicity of the fractions without a prior ranking of the test results is proposed. This article (Part II) focuses on the biological results of the approach.

Methods

The toxicological potential of organic extracts of sediments from the North Sea and the Baltic Sea was analyzed in a bioassay-directed fractionation procedure with a set of biotests: luciferase reporter gene assays on hormone receptor and Ah receptor, arabinose resistance test, fish embryo test (Danio rerio), comet assay, acetylcholinesterase inhibition test, heat-shock protein 70 induction, oxidative stress and luminescence inhibition test (Vibrio fischeri). The test results provided the basis for the calculation of a biotest index by factor analysis to compare the toxicity of the samples and fractions.

Results and Discussion

Results of 11 biotests on different fractionation levels of the samples were described and discussed with regard to the occurrence of contaminants and their toxic potentials. Polychlorinated biphenyls, polycyclic aromatic hydrocarbons, quinones, brominated indoles and brominated phenols were in the focus of interest. A biotest index was constructed to compare the toxic responses in the samples and to group the biotest results.

Conclusion

The procedure presented in this study is well suited for bioassay-directed fractionation of marine sediment extracts. However, in relatively low contaminated samples, high enrichment factors and sufficient fractionation is necessary to allow identification of low concentrations of contaminants which is required to link effects and possible causes. In the present case, the relation between substances and effects was difficult to uncover due to relatively low concentrations of pollutants compared to the biogenic matrix and to the remaining complexity of the fractions. The results, with respect to the brominated phenols and indoles in the samples, highlight the successful use of bioassay directed fractionation in the case of high concentrations and high toxicity.

Recommendation and Outlook

In general, it has been shown that a marine risk assessment requires focusing on the input of diffuse sources and taking into account the fact of mixture toxicity. Effects resulting from biogenic substances will make the assessment of the influence of anthropogenic substances even more difficult.     

Quantifying the fine scale (1km × 1km) exposure,dose and effects of ozone: Part 2 estimating yield losses for agricultural crops

In Britain wheat is an important crop accounting for 41% of the total cereal production. In this study ozone concentrations for 1989 estimated as described in Part 1 of the paper are integrated with the estimated wheat distribution to derive a detailed estimate of the impact of ozone on wheat yields at a fine spatial scale (1km × 1km). These data provide estimates for calculating regional and national yield losses. The methodology can be applied to other crop species. Recent research on a range of crops has established relationships between the economic yield loss for certain crops, including wheat, and ozone exposure. Exposure is described as the accumulated exposure above a threshold experienced during the daylight hours (AOT). Critical AOT values are derived from yield exposure relationships which show linear reductions of yield loss with increasing ozone concentrations. This study has made use of land cover data from remotely sensed imagery at 25m resolution and nationally collected agricultural statistics for counties. These data were combined using an areal interpolation technique to provide more spatially articulate estimates of the location and intensity of wheat production. The results demonstrate the economic importance of ozone as a pollutant. Wheat yield losses attributed to ozone vary between different parts of the country but, for years when ozone levels are high, yield losses are likely to be significant in some areas.     

Sediment Properties for Assessing the Erosion Risk of Contaminated Riverine Sites. An approach to evaluate sediment properties and their covariance patterns over depth in relation to erosion resistance. First investigations in natural sediments (11 pp)

Background, Aim and Scope   Riverine sediments store large quantities of hazardous contaminants, remaining a 'legacy of the past' world-wide. Natural events such as floods may cause the resuspension of polluted sediments and accordingly, the former immobilized contaminants might become bioavailable and toxic again. Hence, a comprehensive erosion risk assessment of contaminated sites is of crucial importance. The present study aimed to implement 'master-variables' for a reliable, easy-to-manage and economically more viable determination of stability in cohesive sediments. Materials and Methods: A wide range of physico-chemical (bulk density, water content, particle size, mineral composition, cation exchange capacity / CEC, total organic matter / TOC, liquid and plastic limits of a soil) and biological (macrofauna abundances, microalgal biomass and species composition, bacterial cell numbers, EPS fractions such as carbohydrates and proteins) properties were determined simultaneously over depth spanning the zone between 0-35 cm. The data were related to sediment stability, determined as the 'critical shear stress for mass erosion' in the SETEG (Stroemungskanal zur Ermittlung der tiefenabhaengigen Erosionsstabilitaet von Gewaessersedimenten) - flume. The investigations were done on natural sediments, thereby covering vertical (over depth), spatial (different study sites) and temporal (different seasons) aspects to ensure the transferability of the data. Here, first data originating from three contaminated reservoirs in the lock-regulated River Neckar / Germany are presented. Results: Comparison of the rather low critical shear stress values (resisting force of sediment, determined in SETEG) with the possibly occurring natural bottom shear stresses (attacking force, calculated for different hydraulic scenarios) at the three reservoirs indicated a severe risk of sediment erosion even under moderate hydraulic conditions and was not restricted to the surface. Critical shear stress was characterised by the following sediment properties of depth, grain size, CEC (Cation Exchange Capacity) and concentrations of TOC (Total Organic Carbon), proteins as well as carbohydrates (water- and resin-extractable). Firstly, biological stabilisation by extracellular polymeric substances (EPS) could be shown for riverine sediments, even over depth. Secondly, erosion resistance was determined by the inter-particles forces, an interplay of the biologically produced compounds constituting active surfaces and the binding capacity as well as charge densities of the sediments. The combined influence of sedimentological and biological properties on sediment stability over depth was assessed by PCA (Principal Component Analysis). Discussion: Hence, a better correlation coefficient between sediment stability and the master variables could be achieved (Main component II: Polymeric substances, R = 0.7, Main component III: Grain size, TOC, CEC, R = 0.9) compared to single correlations. Conclusions: The present paper revealed the combined influence of physico-chemical and biological properties on sediment stability over depth by simultaneous investigation and statistical evaluation. It can be shown, that inter-particle forces, determined by particles size classes, CEC, TOC and polymeric substances such as proteins and carbohydrates, affected sediment stability most. Thereby, the impact of biogenic sediment mediation on riverine sediment stabilisation became evident, even over depth, where mostly sedimentological parameters were considered as important before. Recommendations and Perspectives: The importance of a comprehensive risk assessment of contaminated riverine sites was again highlighted in the present study by the comparison of natural occurring bottom shear stresses with the determined sediment erosion resistance. If a realistic risk assessment is to be derived, the stabilizing potential of micro-organisms needs to be taken into account and the covariance patterns of biological and physico-chemical sediment properties have to be addressed.