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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., (© 2024. The Author(s).)

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., (© 2023. The Author(s).)

The microbial elimination of nitrogen by combined nitrification and denitrification is a well-known technology in wastewater treatment processes. Nitrification in wastewater treatment plants is a sensitive microbiological process, which can be disturbed by toxic substances. Two laboratory methods for nitrification inhibition (ISO standard 9509 and a modification of the respiration inhibition test ISO 8192) were investigated with selected substances and compared with results from sewage model plants. For the prediction and prevention of interferences, the simple but reliable laboratory test methods proved to be very suitable. The tests in laboratory plants allowed a much more comprehensive evaluation of nitrification processes and showed their dependence on biodegradation and adaptation processes. The inhibition of nitrification depended on the mode of application and the biodegradability of the potential inhibitory compound and was much less severe with biodegradable substances than with poorly degradable ones.

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO(2)) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO(2) method with existing CO(2) evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation--i.e., CO(2) evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)--in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO(2) and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.

The activated sludge respiration inhibition test is an important bacterial test system for the determination of bacterial toxicity of chemical compounds. The exposure period recommended by OECD 209 and ISO 8192 protocols is 30 and 180 min. A modified version of the test was developed which allowed a prolonged incubation period of 27 h to enhance the possibilities of the test system. The test system with the prolonged incubation time was evaluated by the recommended reference compound 3.5-dichlorophenol and showed an EC50 of 6.3 mgl(-1) with a coefficient of variation of 12.7%. Furthermore, the use of an open test system was evaluated showing a comparable toxicity but a higher coefficient of variation than the closed test system. A storage of activated sludge for several days accompanied by a daily feed with OECD nutrient solution should be avoided, for it can cause a decreased sensitivity of the inoculum. Different statistical fit procedures were tested indicating that Weibull fit procedures were superior for extended data sets covering a wide range of concentrations whereas Gamma and Probit fits were appropriate for smaller data sets mainly restricted to the linear part of the dose response curve.

Biodegradation studies of organic compounds in the aquatic environment gain important information for the final fate of chemicals in the environment. A decisive role play tests for ready biodegradability (OECD 301) and in this context, the respirometric test (OECD 301F). Two different respirometric systems (Oxitop and Sapromat) were compared and in two of ten cases (diethylene glycol and 2-ethylhexylacrylate) differences were observed indicating that the test systems are not always equivalent. For 2-ethylhexylacrylate and cyclohexanone we could not state differences in the extent of biodegradation with a municipal and industrial inoculum whereas for cyclohexanone the degradation rate was faster with a municipal inoculum. Allylthiourea (ATU) proved to be an effective inhibitor of nitrification processes and did not affect the heterotrophic biodegradation activity. Modelling of biodegradation processes could be successfully performed with a first-order and a modified logistic plot., (Copyright 2002 Elsevier Science Ltd.)

Results of an international ring-test of two laboratory methods are presented for investigating the biodegradability of organic polymeric test materials in aquatic test systems based on respirometry and the evolution of carbon dioxide. These methods are developed further from the well-known standardized biodegradation tests ISO 9408 (1999) and ISO 9439 (1999), which have been successfully used for many years. The most important improvements are the extension of the test period up to six months, the increase of the buffer capacity and nutrient supply of the inorganic medium, an optimization of the inoculation, and optionally, the possibility of a carbon balance. A ring test, organized by the International Biodeterioration Research Group (IBRG), was run using a poly(,-caprolactone)-starch blend and an aliphatic-aromatic co-polyester as test materials and a microcrystalline cellulose powder as a reference material. The test results and the experience gained by the participants showed that the methods are suitable and practicable. The test methods have been meanwhile established as standards ISO 14851 (1999) and ISO 14852 (1999).

In order to classify packaging materials as capable of organic recovery according to the packaging regulation of the European Union, the intended materials must be tested for their compostability. An important prerequisite is the determination of biodegradability by standardized test methods. Details of the intended tests are not comprehensible without further knowledge of the problems of biodegradation testing. The test methods and the limit values used to evaluate the biodegradation results are presented and discussed against the background of established test methods and limit values for chemicals. Furthermore the problem of ecotoxicity tests in connection with compost quality is discussed and an overview of European test laboratories is given.

Laboratory test methods are used by industry laboratories to determine biodegradability, an important parameter for the evaluation of the ecological behaviour of substances. Biodegradability has a key role due to the simple fact that a degradable substance will cause no long term risk in the environment. The great variety of biodegradation processes in the natural environment and in technical plants for treating waste water and solid wastes gave rise to a rather large number of test methods based on different test principles. To guarantee the acceptance of the test results by authorities and customers internationally standardized methods (ISO, OECD) and established quality criteria (GLP, EN 45,000, ISO 9000) are used.

The objective of this publication is to present a new dynamic aerobic biodegradation test method simulating a river. A laboratory cascade test system and standardized batch shake flask tests were used for biodegradation studies with the non-volatile and non-sorbing model compounds 2,4-dinitrophenol, naphthalene-1-sulphonic acid and sulphanilic acid. To be closer to the often very low concentrations of substances in the environment the concentrations of the compounds used were standard test concentrations and lower. 14C labelled compounds were measured at 50 micrograms/l, capillary electrophoresis at 5000 micrograms/l and the removal of dissolved organic carbon at 50000 micrograms/l. The test results obtained confirmed the known ultimate biodegradability of the test compounds and showed that biodegradation degrees, rates and degradation durations depended on the test systems, the concentrations of test compounds and the inocula. The river model is a suitable simulation test for natural dynamic surface waters which can be used to perform biodegradability studies at low test concentrations if adequate analytical tools, preferably radioactive-labelled substances, are available.

A bacterial toxicity test method using the determination of growth inhibition of sewage bacteria within an incubation period of 6 hours will be standardized at ISO. A ring test was performed with 24 participants using 3.5-dichlorophenol and potassium cyanide as test substances. The analysis of the test results showed that the method is very accurate and appropriate for determining the bacterial toxicity of chemical compounds. Due to the results obtained 3.5-dichlorophenol was proposed as a reference substance.