In the past decades, industrial and scientific communities have developed a complex standardized system (e.g., OECD, ISO, CEN) to evaluate the biodegradability of chemical substances. This system includes for OECD three levels of testing (ready and inherent biodegradability tests, simulation tests). It was adopted by many countries and is completely integrated into European legislation (registration, evaluation, authorization, and restriction of chemicals, REACH). Nevertheless, the different tests have certain deficiencies, and the question arises of how accurately these tests display the situation in the real environment and how the results can be used for predictions. This review will focus on the technical advantages and weaknesses of current tests concerning the technical setup, the inoculum characterization, and its biodegradation potential as well as the use of adequate reference compounds. A special focus of the article will be on combined test systems offering enhanced possibilities to predict biodegradation. The properties of microbial inocula are critically discussed, and a new concept concerning the biodegradation adaptation potential (BAP) of inocula is proposed. Furthermore, a probability model and different in silico QSAR (quantitative structure-activity relationships) models to predict biodegradation from chemical structures are reviewed. Another focus lies on the biodegradation of difficult single compounds and mixtures of chemicals like UVCBs (unknown or variable composition, complex reaction products, or biological materials) which will be an important challenge for the forthcoming decades. Key points: • There are many technical points to be improved in OECD/ISO biodegradation tests • The proper characterization of inocula is a crucial point in biodegradation tests • Combined biodegradation test systems offer extended possibilities for biodegradation tests

Microbial toxicity tests play an important role in various scientific and technical fields including the risk assessment of chemical compounds in the environment. There is a large battery of normalized tests available that have been standardized by ISO (International Organization for Standardization) and OECD (Organization for Economic Co-operation and Development) and which are worldwide accepted and applied. The focus of this review is to provide information on microbial toxicity tests, which are used to elucidate effects in other laboratory tests such as biodegradation tests, and for the prediction of effects in natural and technical aqueous compartments in the environment. The various standardized tests as well as not normalized methods are described and their advantages and disadvantages are discussed. In addition, the sensitivity and usefulness of such tests including a short comparison with other ecotoxicological tests is presented. Moreover, the far-reaching influence of microbial toxicity tests on biodegradation tests is also demonstrated. A new concept of the physiological potential of an inoculum (PPI) consisting of microbial toxicity tests whose results are expressed as a chemical resistance potential (CRP) and the biodegradation adaptation potential (BAP) of an inoculum is described that may be helpful to characterize inocula used for biodegradation tests. Key points: • Microbial toxicity tests standardized by ISO and OECD have large differences in sensitivity and applicability. • Standardized microbial toxicity tests in combination with biodegradability tests open a new way to characterize inocula for biodegradation tests. • Standardized microbial toxicity tests together with ecotoxicity tests can form a very effective toolbox for the characterization of toxic effects of chemicals.

The activated sludge respiration inhibition test and the luminescent bacteria test with Vibrio fischeri are important bacterial test systems for evaluation of the toxicity of chemical compounds. These test systems were further optimized to result in better handling, reliability and sensitivity. Concerning the Vibrio fischeri test, media components such as yeast extract and bivalent cation concentrations like Ca²⁺ and Mg²⁺ were optimized. The cultivation, storage conditions and reactivation process of the stored bacteria were also improved, which enabled simpler handling and led to good reproducibility. Additionally, the respiration inhibition test with a prolonged incubation time was further analyzed using different chlorinated phenols as reference compounds. It could be stated that a longer incubation period significantly improved the sensitivity of the test system.

For a period exceeding five decades, industrial and scientific communities, in conjunction with regulators, have utilized a complexified, standardized system (e.g., OECD, Organisation for Economic Co-operation and Development; ISO, International Organization for Standardization; ASTM, American Society for Testing and Materials; CEN, Comité Européen de Normalisation) for the estimation of biodegradability of organic compounds. This system has been adopted in numerous countries worldwide and has also been integrated into European legislation (REACH, registration, authorisation, and restriction of chemicals). In recent years, a number of deficiencies have been identified in the standardized biodegradation test systems. This comprehensive review sets out the fields in which improvements are necessary to set up the next generation of reliable, standardized biodegradation tests. The main focus of the review is the challenges and modifications needed to test difficult-to-test compounds such as volatile, hydrophobic compounds, UVCBs (unknown or variable composition, complex reaction products or biological materials), water-soluble polymers, and plastics. Recent advances in the characterization of inocula for biodegradation tests are also addressed, which offer a valuable opportunity to enhance the reliability and reproducibility of biodegradation assays. Moreover, the potential for predicting biodegradation in the environment is a subject that is discussed in this text. Key points: • It is essential that the OECD system of biodegradability tests be subjected to a thorough re-examination and further technical development. • It is evident that UVCBs, hydrophobic compounds, and polymers present particular challenges in the context of OECD/ISO biodegradation tests. • It is necessary that inocula for OECD/ISO-based biodegradation tests are characterized in a much more comprehensive manner.