As part of the research programme "Processes for the bioremediation of soils" sponsored by the German Federal Ministry for Education and Research (BMBF), a chronic plant test is developed as a contribution to an ecotoxicological test battery for soil quality assessment. The plant test, together with other bioassays currently under development within the BMBF joint project, are considered to be versatile tools to identify hazards associated with contaminated soils as well to verify the success of remediation of these soils. Results from bioassays with soils can improve the understanding of the effects of known substances in a variety of soils as well as of unknown chemicals (e.g. transformation products) or mixtures of chemicals and therefore complement soil quality assessment based solely on analysis of known chemicals in soils. Both techniques, in conjunction, can assure a safe assessment of the risks associated with contaminated soil and thus allow the ecological and economic value of land to be estimated.

Under the German law for the protection of soils and its test guidelines, biotests are not yet mandatory. Decisions are made on the basis of trigger concentrations of single chemicals. As a prerequisite for the acceptance of biotests, they have to be applicable to different types of soil and to a variety of contaminants. Like others, the chronic plant test is based on test procedures for the assessment of effects of single chemicals in defined media (e.g. ISO-standard ISO/DIS 11269-2 or OECD guideline 208). Therefore, research is still needed to recognise the effects of experimental handling factors on the measured endpoints like seedling emergence, plant biomass, flowering and seed production. Furthermore, it is important to differentiate between effects caused by soil contaminants and those caused by other properties of the test soils like nutrient concentrations or texture.

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Test species	Brassica rapa (rapid cycling variant CrGC) Avena sativa (oat)
Control soil	Standard field soil (LUFA sp. 2.2) or OECD artificial soil. If available, a reference soil is collected near the site of contamination.
Soil preparation	Sieving (5 mm), storage in refrigerator until testing (max. 10 days). Mixing with control soils at five concentrations (plus control). Soil mixtures are filled loosely in the test vessels (400 g)
Test vessels	Polystyrene (approx. height x length x width 9.5 x 10.5 x 7 cm)
Sowing	10 seeds are placed in each vessel at a depth of 1 cm.
Watering	With deionised water by glass fiber wicks. Soils take up water until the maximum water holding capacity is reached.
Incubation	At 22 ? 3°C. Illumination of 12000 lx for 16 h/d.
Test duration	35 - 42 days (Brassica rapa), 49 - 56 days (Avena sativa)
Harvest	Within the first week, reduction to 8 plants per vessel. Harvest of 4 plants each after 14 days and of the remaining plants at the end of the test.
Variants (tested in a subset of experiments each)	Handling tests with Brassica nigra, two variants each of Brassica rapa and Avena sativa Daily manual watering (60% of water holding capacity) Use of different wicks (1, 3, 11 mm in diameter) Addition of nutrients by solution or by initial mixing into soils Plant density Sowing depth (5 - 20 mm) Seed weight (A. sativa only) Method of pollination (B. rapa only)
Endpoints	Plant height and weight, no. of inflorescences (oat) and seed pods

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Up to now (end of 1998) soils from a variety of sites contaminated with either PAHs (polyaromatic hydrocarbons), or MO (mineral oil) or TNT (2,4,6-trinitrotoluene) and a few remediated soils have been tested. By the end of 1999, when the project will end, additional soils will be examined after remediation.

A guidance textbook will be prepared towards summer 1999 by DECHEMA (Principal Coordinators of the BMBF Joint Research Project "Processes for the bioremediation of soils") comprising methods for soil remediation as well as for the ecotoxicological bioassays. By that time, a critical examination of the chronic plant test data will be needed to establish a reasonable scheme for the assessment of soil quality and remediation success on the basis of this new laboratory test. An initial guideline proposal will be drafted by the end of 1999.

Validation of the test procedure and measures for quality assurance, e.g. by comparing results between laboratories and expanding the number of contaminated soils studied to include more contaminants and soil types, will depend on further financial support.

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Dr. Joerg Roembke
j-roembke@ect.de
++6145 956411

The financial support of the Federal Ministry of Education, Science, Research and Technology is gratefully acknowledged.
(Project Identification No.: 1491077)

Kalsch W. and Römbke J. Zur chronischen Wirkung von TNT auf die Stoppelrübe Brassica rapa im Labortest. In: Oehlmann, J. & Markert, B. (1999): Ökotoxikologie - Ökosystemare Ansätze und Methoden, Urban & Fischer, New York, Stuttgart, Jena [in press].

Kula C. and Römbke J. [1998] Evaluation of soil ecotoxicity tests with functional endpoints for the risk assessment of plant protection products. Environ. Sci. & Pollut. Res, 5: 55-60.

Römbke J., Bauer C., Brodesser J., Brodsky J., Danneberg G., Heimann D., Renner I. und Schallnaß H.-J. [1995] Grundlagen für die Beurteilung des ökotoxikologischen Gefährdungspotentials von Altstoffen im Medium Boden. UBA-Texte 53/95, German Federal Environmental Agency (Umweltbundesamt), Berlin (editor), ISSN 0722-186X.

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Financial Support: BMBF (Federal Ministry for Education and Research)

Principal Coordinator "Processes for the bioremediation of soils": DECHEMA

Coordination "Ecotoxicological test batteries": Technical University of Berlin

Source for rapid cycling Brassicas: Crucifer Genetics Cooperation at the University of Madison, Wisconsin

Technical forum on soil remediation: Bioremediation discussion group

Scientific opinion on research needs: CARACAS (Concerted Action on Risk Assessment for Contaminated Sites in the European Union)

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