In the recent review of the control of marketing surfactants used in detergents, the EU decided to increase the severity of the testing procedure by using the criterion of ultimate biodegradability (mineralization) rather than primary biodegradation (removal of the parent molecule) to ensure that possible harmful organic metabolites do not reach the environment. The relatively new ISO headspace CO2 test, considered to be an improvement on the OECD 301B (Sturm CO2) test was chosen. The method was subjected to a ring test by 11 laboratories using one of each of four classes of surfactants plus a poorly degradable reference surfactant; all laboratories satisfactorily applied the method. The necessary addition of silica gel to the medium containing the cationic surfactant, known as a class to be more inhibitory than other classes, was confirmed as a technique for avoiding inhibition of the inoculum. The biodegradability of the surfactants was in general agreement with results reported in the literature and the often reported variable values of % inorganic carbon (IC) produced of the theoretical was found. The anionic and cationic surfactants were readily biodegradable (%IC>60), the non-ionic surfactant was well below the pass value, while the amphoteric was borderline. The IC production by the blank controls, one of the validity criteria, was about 0.3 mgC/100 ml test medium, equivalent to 3 mgC/l, as recommended in the ISO text. Mild conditions of pre-exposure of the inoculum to the test surfactant did not produce consistent worthwhile effects on either the percentage biodegradation or on its variability.

Current test guidelines for assessing 'inherent' (potential) biodegradability were designed for water-soluble, organic compounds of low volatility and are unsuitable for most oil products. It was against this background, that CONCAWE (the oil companies' European organisation for environment, health and safety) formed a task force to develop a standard test protocol for assessing the 'inherent' biodegradability of oil products.

Toxicity AssessmentVolume 1, Issue 4 p. 515-524 Technical Method Section Testing the toxicity of chemicals by the inhibition of respiration of activated sludge† H. A. Painter, H. A. Painter Water Research Centre, Henley Road, Medmenham, PO Box 16, Marlow, Bucks, SL7 2HD, EnglandSearch for more papers by this author H. A. Painter, H. A. Painter Water Research Centre, Henley Road, Medmenham, PO Box 16, Marlow, Bucks, SL7 2HD, EnglandSearch for more papers by this author First published: Autumn (Fall) 1986 https://doi.org/10.1002/tox.2540010410Citations: 1 † This method is presented by permission of the Director, Water Research Centre, Medmenham Laboratory AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume1, Issue4Autumn (Fall) 1986Pages 515-524 RelatedInformation

Out of fourteen chemicals tested, two - hexamethylenetetramine and sodium benzene sulphinate - were degraded in screening tests (>70% DOC removal, >60% ThOD exerted) but not in activated sludge simulation tests (<25% DOC removal). It is suggested from data in the literature that these essentially non-biodegradable chemicals undergo hydrolysis or autoxidation in the 28-day screening tests to form biodegradable products, which are not produced in sufficient quantity in the 3-hour simulation test. The need is stressed for taking account of the stability of chemicals in water when assessing biodegradability in the environment.

Assessment of the toxicity of chemicals to the respiration of activated sludge microorganisms is a relatively simple and reproducible method when applied to a standard population of bacteria. However, activated sludge is, by nature, a variable commodity, and varying results are often reported. Within‐plant variations can occur from day to day as a result of shifts in the bacterial population, probably caused by changes in the strength of components of the sewage feed. Between‐plant variations can be considerably greater. Factors such as differing operating conditions and presence of industrial waste waters in the sewage can result in differing species composition of the sludge, which, in turn, may account for differing biooxidation rates of components of the waste water and thus different respiration rates. Sludge from different sources, and/or grown under different conditions, may also vary in response to inhibitors, because of varying degrees of reaction of some inhibitors with non‐living sludge components. Therefore, in practice, it has been usual to consider EC 50 values from the inhibition of respiration of activated sludge test in terms of order of magnitude. Variations in EC 50 results of the ‘ISO’ inhibition of respiration test have been quantified for a number of chemicals, both within and between batches of sludge, and with sludges from different sources. The coefficient of variation of EC 50 results within batches of sludge was found to be approximately 9%. Between batch and between source variations were 28–76% depending on the test chemical.

The various ways in which organic chemicals can be changed by the action of micro-organisms are reviewed. All organic compounds are capable of releasing sufficient energy for growth on degradation, but the necessary enzyme systems are often not available for the conversion of xenobiotic compounds to mineral products. Microbial enzymes having broad substrate specificity are capable of relatively slowly effecting single, simple structural changes, or a series of changes in xenobiotic chemicals, resulting in transformation products which are sufficiently recalcitrant for them to persist in the environment. Three broad classes of reaction are identified — transformation, cleavage and conjugation — and examples of many types of reaction are quoted. Difficulties in predicting events likely to occur in the environment from results of tests with monocultures with single or multi-substrates are discussed. A large body of data on transformations are available and it is suggested that a register be set up and maintained recording the known reactions for application to environmental problems.

A field trial has been performed to measure the biodegradability of two typical alcohol ethoxylate nonionic surfactants, “Dobanol” 45-7 and “Dobanol” 45-11, by dosing them to biological filters in a mixture with domestic sewage. Influent levels were 10 and 25 mg l−1 of each surfactant and 96–98% degradation was achieved within a temperature range of 5–10°C. The surfactants had no adverse effects on the filters in terms of the usual sanitary parameters (BOD, COD, TOC and ammoniacal nitrogen). Tests on the effluents indicated no residual acute lethal toxicity to rainbow trout Salmo gairdneri).

Interest in biodegradability grew as a necessity when problems were created by the use and subsequent discharge of chemicals which did not degrade but persisted in the environment. Probably the best known example is the introduction of the first synthetic detergents in the 1950’s. These detergents, now described as “hard,” were only partially removed in sewage treatment and passed through treatment works, largely unchanged, to rivers. It was common, at least in the UK, to see huge banks of foam created by aeration in activated sludge plants and also at weirs and other sites of turbulence on polluted rivers. The problem, caused by the inability of aquatic bacteria to degrade sufficiently rapidly the highly branched chain alkyl benzene sulphonates, was satisfactorily solved by replacement by the manufacturers of the “hard” type with a biodegradable (“soft”) product in which the degree of branching had been considerably reduced.

A collaborative exercise involving twelve laboratories was organized for the European Economic Community to check the validity of an enclosed respirometric method for assessing the ready biodegradability of test chemicals, including insoluble substances. The method, based partly on the OECD-Japanese Ministry of International Trade and Industry (MITI) I method (301C) and partly on the U.K. method, allowed the use of a less restrictive, natural inoculum and a wider variety of respirometers than the original MITI I method. Eight compounds of a wide range of degradative behavior were tested over an incubation period of 28 days. The agreement between participating laboratories in the lag period before biodegradation started and in the proportions of theoretical oxygen uptake achieved was at least as good as in other ring tests; it is proposed that the method be accepted. Differences in behavior of pentaerythritol reported here and in the literature are examined and suggestions for future study are discussed.

Legislation controlling the use of new chemicals and concern for environmental protection have resulted in a surge of interest in tests for toxicity to bacteria. Currently used tests for inhibition of respiration, nitrification and growth of sewage bacteria are normally short-term and deliberately use unacclimatised microorganisms. This ‘failsafe’ design is open to criticism, in that safety margins are normally high, taking no account of acclimatisation which may reduce toxicity for continuously discharged chemicals.

A mathematical model, based on a modification of the Michaelis-Menten and Monod equations describing bacterial growth, has been used to predict the course of removal of the organic substrates in the modified OECD and other screening tests. A range of initial concentrations of bacteria in the inoculum has been selected, using published data on the total bacterial count in secondary sewage effluents and activated sludge, to simulate the wide range of volumes of these materials used in practice. The arbitrary definition of ready biodegradability adopted by the OECD, that is, 5 to the equivalent of 70% DOC removal in not more than 10 days in a total incubation period of 28 days, is expressed in terms of inoculum size and kinetic constants. Modifications are proposed to the screening test with the object of making it more useful and realistic in predicting whether or not a chemical will be removed in sewage treatment.

The specific growth rates of nitrifying organisms were determined under non-equilibrium conditions in laboratory-scale activated sludge plants at three temperatures and six pH values. The highest growth rate for the first stage (to nitrite) was 0.61 d−1; no nitrification was observed over a 7 week period at pH 6.0 at any temperature, indicating a μ value of <0.06 d−1. Fewer data were obtained for the second stage (to nitrate); in all but one of ten comparisons the specific growth rate was lower (by an average of 16%) than for the first stage. The effects of temperature and pH value were not as expected probably because of greater effects resulting from the differences in composition and strength of the sewages used.