1438 Environmental Toxicology and Chemistry, Vol. 20, No. 7, pp. 1438–1449, 2001 q 2001 SETAC Printed in the USA 0730-7268/01 $9.00 1 .00 THE USE OF ACUTE AND CHRONIC BIOASSAYS TO DETERMINE THE ECOLOGICAL RISK AND BIOREMEDIATION EFFICIENCY OF OIL-POLLUTED SOILS CORNELIS A.M. VAN GESTEL,*² J AAP J. VAN DER WAARDE,‡ J.G.M. (ANJA)DERKSEN,§ ELINE E. VAN DER HOEK,\ MARTIN F.X.W. VEUL,# SANDRA BOUWENS,² B EN RUSCH,² R ENE ´ KRONENBURG,²² and GERARD N.M. STOKMAN‡‡ ²Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1087 HV, 1081 HV Amsterdam, The Netherlands ‡Bioclear, PO Box 2262, 9704 CG Groningen, The Netherlands §AquaSense, PO Box 95125, 1090 HC Amsterdam, The Netherlands \KEMA, PO Box 9035, 6800 ET Arnhem, The Netherlands #Witteveen and Bos, PO Box 233, 7400 AE Deventer, The Netherlands ²²Port Management of Amsterdam, Harbour Building, PO Box 19406, 1000 GK Amsterdam, The Netherlands ‡‡Dutch Railway’s Foundation for Soil Remediation (SBNS), PO Box 2809, 3500 GV Utrecht, The Netherlands ( Received 19 July 2000; Accepted 13 November 2000) Abstract—To compare the effectiveness of acute and chronic bioassays for the ecological risk assessment of polluted soils, soil samples from a site with an historical mineral oil contamination (,50–3,300 mg oil/kg dry soil) at the Petroleum Harbour in Amsterdam, The Netherlands, were screened for ecological effects using acute and chronic bioassays. A two-step 0.001 M Ca(NO 3 ) 2 extraction at a final solution-to-soil ratio of 1:1 was used to prepare extracts for the acute bioassays. Acute bioassays (#5 d) applied to the 0.001 M Ca(NO 3 ) 2 extracts from the polluted and reference soils included growth tests with bacteria (Bacillus sp.), algae (Raphidocelis subcapitata), and plants (Lactuca sativa), immobility tests with nematodes (Plectus acuminatus), springtails (Folsomia candida), and cladocerans (Daphnia magna), and the Microtoxt test (Vibrio fischeri). Chronic bioassays (four weeks) performed on the same soil samples included tests with L. sativa, F. candida, and earthworms (Eisenia fetida) and the bait-lamina test (substrate consumption). The acute bioassays on Microtox showed a response that corresponded with the level of oil pollution. All other acute bioassays did not show such a consistent response, probably because pollutant levels were too low to cause acute effects. All chronic bioassays showed sublethal responses according to the contaminant levels (oil and in some soils also metals). This shows that chronic bioassays on soil samples are more sensitive in assessing the toxicity of mineral oil contamination in soil than acute bioassays on soil extracts. A pilot scale bioremediation study on soils taken from the two most polluted sites and a control site showed a rapid decline of oil concentrations to reach a stable level within eight weeks. Acute bioassays applied to the soils, using Microtox, algae, and D. magna, and chronic bioassays, using plants, Collembola, earthworms, and bait-lamina consumption, in all cases showed a rapid reduction of toxicity, which could be attributed to the degradation of light oil fractions. Keywords—Oil pollution Toxicity testing Bioremediation Sublethal endpoints INTRODUCTION Chemical analysis usually is insufficient to provide insight into the potential ecological risk of polluted soils since it does not allow for an integration of the combined effects of the mixture of all chemicals present at a polluted site, including their bioavailability [1,2]. Bioassays do integrate these effects and are therefore recommended for the ecological risk as- sessment of polluted soils [3]. In addition, the use of such biological test methods is recommended for the evaluation of the effectiveness of soil remediation processes [4]. Soil bioassays described in the literature may be classified as acute and sublethal (chronic) tests. Tests using aquatic spe- cies have to be distinguished from methods using real terres- trial organisms [4]. Tests with aquatic organisms are usually performed on extracts of the polluted soils, generally applying short-term exposure periods. The advantages of such methods are the low costs and rapid answers; from an ecological point of view, such acute tests, however, are less relevant. They may however, be used, e.g., to indicate potential effects of chemical pollutants on the filter function of the soil [4]. Long-term bioassay methods, focusing on sublethal endpoints and testing the soil as such, are more relevant from an ecological point * To whom correspondence may be addressed (gestel@bio.vu.nl). of view [5] and may provide information on potential effects on the habitat function of the soil [4]. Such chronic tests are much more expensive and time consuming and have therefore only rarely been applied to polluted and remediated soils. Al- though the difference between acute and chronic tests is de- termined by the lifespan of the test organism, in this study, the border was arbitrarily set at #5 d of exposure. In this way, all tests performed in elutriates were classified as acute bio- assays and all tests using soil substrates as chronic bioassays. When applying acute or sublethal toxicity tests using aquat- ic organisms as a substitute for soil organisms, a suitable ex- traction technique is required. Such a technique should be reproducible and reliable and as much as possible be repre- sentative of the bioavailability of the chemicals in the polluted or remediated soils. In addition, extracting agents should not be harmful for the test organisms to be used. At present, several methods have been described, applying solution-to-soil ratios varying between 1:1 and 100:1, extraction times between 30 min and 48 h, and different methods to separate the extract from the soil. Also, many different extracting agents (water, salt solutions, acids, or chelating agents) have been used (cf., [6]), which may not always be representative of the availability of the pollutants for biota. Extraction procedures for the use