Contamination sites pose significant environmental hazards for terrestrial and aquatic ecosystems. They are important sources of pollution and may result in ecotoxicological effects. At severely contaminated sites acute effects occur, but the core problem lies in possible long-term chronic effects. Ecotoxicological effects occur at all levels of the biological organization, from the molecular to the ecosystem level. Not only certain organisms may be affected, but the ecosystems as a whole in its function and structure. Contaminants at large contaminated sites often share critical properties such as toxicity, high environmental persistence, often high mobility prone to contamination of groundwater, and high lipophilicity resulting in bioaccumulation in food webs. Contaminants present at polluted sites occur as mixtures; therefore, interactions between individual compounds may be of importance. The bioavailability is a key factor responsible for ecotoxicological effects of contaminants: only the bioavailable fraction induces ecotoxicological effects, as shown for organotin compounds. Organotins belong to the most toxic pollutants known so far for aquatic life. Widespread contamination of harbor sediments occurs globally due to the ongoing use of organotins in antifouling paints on large ships. In lake sediments, tributyl- and triphenyltin are very persistent and bioavailable to biota even after a long time. The bioavailability of organotins is dependent on the pH and the content of organic matter. Organotins accumulate in sediments, but remobilization occurs during disturbance and dredging. A key question in dealing with contaminated sites is whether, and to what extent ecotoxicity occurs. Usually, established OECD tests and whole effluent toxicity tests are performed for an ecotoxicological evaluation and for risk assessment. However, these assays are often expensive, laborious and sometimes not sensitive enough. As a consequence, we have used rapid and inexpensive in vitro systems such as fish cell lines for the evaluation of sediments and landfill leachates, which were contaminated by polycyclic aromatic hydrocarbons (PAHs). The determination of cytotoxicity as a measure for acute toxicity, and the induction of cytochrome P4501A (CYP1A) as a biomarker of exposure and effects were found to be important measures, which can be used for hazard and risk assessment. We have developed a concept for the ecotoxicological evaluation of PAH contamination based on induction equivalents, which can be applied for aquatic and terrestrial ecosystems. One of the key question and present gaps, however, includes the long-term chronic ecotoxicological effects of single compounds and mixtures on soil and aquatic biota at contaminated sites. This should be addressed in the future.