TREATABILITY, TOXICITY AND BIODEGRADABILITY TEST METHODS
Authors: KILROY A. , GRAY N. F. 
Published: January 2008

1. This review confirms that treatability and biodegradability test methods have been cited extensively in the literature. It is clear that the method selected depends on the specific objectives of the test, i.e. the determination of whether a substance is toxic, biodegradable or treatable. Factors that have to be considered when selecting the test methods are the cost of performing the test, the time and resources involved, and the accuracy required. It often appears that more extensive simulation studies are required after initial screening tests have been performed.

2. Many of the enzyme and bacterial growth tests which have been developed for monitoring or screening of toxicants and their persistence in water and wastewaters have been reviewed. Most of these tests are rapid, inexpensive, and reproducible. Most of the biochemical and microcalorimetric approaches, although promising, are still in their infancy as regards toxicity testing. Therefore, biological testing still appears to be most suitable for routine assessment.

3. Micro‐organisms are particularly suitable for use in toxicity testing of chemicals as they are inexpensive to culture, have rapid growth rates, and usually provide reproducible results (Vaishnav & Korthals, 1990). Many bioassays have been developed to evaluate the toxicity and treatability of municipal and industrial effluents. Numerous single species tests have been recommended by several authors (Dutka et al., 1983; Beaubien et al. 1986). Such approaches are mainly based on the belief that, by selecting the most sensitive species and by using appropriate factors to allow for variability not included in the test, the highest levels of biological organization will be adequately protected. Single species tests are now quite well established, and when properly used, are easy to analyse and quantify. However, it has been pointed out (Levin, 1984) that the results obtained from single species tests cannot easily be applied to natural field conditions because the test organisms are extensively laboratory acclimated; also the test conditions provide for optimized growth and survival, a situation unlikely to be found in the field. Moreover, a fundamental problem with this approach is that it assumes that the ecosystem is a collection of single species exposed to toxicants under constant conditions (Cairns, 1982).

4. Multi‐species toxicity tests, that is the use of mixed cultures or communities of micro‐organisms for a testing protocol, are found to be generally much less sensitive than single species tests (Dutka & Kwan, 1984). It has been claimed that the use of multi‐species tests give more realistic results than single species tests, and that single species adaptation and replacement is a common phenomenon in natural systems (Cairns, 1982).

5. Chemical persistence is the other major area of testing involving micro‐organisms. Many biodegradation procedures have been developed (UK Standing Committee of Analysts, 1981), but surprisingly little interlaboratory validation of test methods has occurred. Those studies which have attempted validation of test methods have generally found good agreement using either non‐biodegradable or readily degradable chemicals. Poor agreement has been reported when testing partially degradable or problematic chemicals, especially when non‐acclimated inocula were used (Gerike & Fischer, 1979). Most biodegradation test methods have been criticized as the nonspecific analytical techniques employed require the use of relatively high substrate concentrations (Gledhill, 1987). Chemical degradation may be proportional to concentration, may not occur below certain threshold concentrations, may proceed via alternate metabolic pathways, or may occur more rapidly via alternate metabolic pathways. Studies by Alexander (1985) indicate the persistence of chemicals at realistic environmental concentrations may be different from that at higher concentrations. More realistic test systems which simulate actual ecosystems are needed for studies of microbial effects and persistence. Test systems should incorporate realistic chemical concentrations, natural microbial communities, and as much of the physical structure of the environment as practical. They should also be adapted to routine laboratory use. Most biodegradation studies look at the disappearance of parent material or the formation of metabolic products such as CO2. Because of the relative high variation and lack of reproducibility of results of die‐away tests, and as CO2 may be a poor method of measuring toxicity because of the slow conversion of organic carbon to CO, both of these approaches have serious limitations when examination of typical environmental chemicals is desired. They also become too laborious, expensive or impractical to be useful in routine test procedures.

6. Respiration is a universally applicable parameter for assessing toxicity to aerobic bacteria. Inhibitory effects on respiration are rapid and can be measured with simple, inexpensive equipment. This method offers some advantages over others in that it requires little attention and also simulates quite closely the conditions found at a wastewater treatment plant. Among the disadvantages are the high concentration of test compound required. In the presence of a readily biodegradable substrate, bacteria respire rapidly, but when the substrate is exhausted the respiration rate falls rapidly to an endogenous level in which the cells are using stored substrate and other expendable cell constituents. If substrate is reintroduced after a long period of endogenous respiration, a lag period may be observed before active growth resumes. It has been found that bacteria are less susceptible to inhibitors when respiring in the endogenous phase, and the effects on growth and cell division cannot be ascertained on cells in this state. Thus in order to maximize sensitivity, the bacteria should be provided with a readily oxidizable substrate so that they are exogenously respiring, growing, and dividing when exposed to the potential inhibitor. Generally, a mixture of substrates similar to those on which the culture would grow naturally is suitable, for example synthetic sewage is commonly used as a growth medium for bacteria from sewage treatment plants.

7. Activated sludge is variable in populations of bacteria and varying results are often reported for inhibition of respiration studies. 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 in the influent. Between plant variations can be greater. 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 (Kilroy & Gray, 1992b). Therefore, in practice, it has been usual to consider EC50 values from the inhibition of respiration of activated sludge tests in terms of order of magnitude.

8. Normal short‐term tests, including inhibition of respiration of activated sludge, are deliberately carried out using unacclimated micro‐organisms. This design is aimed to be as conservative as possible for the purpose of predicting effects in the environment, but may be unnecessarily stringent for the purposes of investigating the treatability of a chemical by activated sludge as no account is taken of the possible reduction in toxicity by acclimation of the micro‐organisms to the chemical when it is continuously discharged to the wastewater treatment plant. Unlike chemical determinations where all the reagents can be clearly specified, the microbial inoculum or reagent used in biodegradability testing can rarely be fully characterized and is usually only vaguely specified (e.g. as activated sludge, sewage effluent). The BOD method is not labour intensive, but it does require a 5‐day test period. While it does not allow a quick assessment of toxicity, it spans a number of generations of bacteria and is therefore more likely to identify effects on growth and cell division than short‐term tests lasting for a few hours. If respiration is slow, the test can be extended (to 20 d) to allow for acclimation of the bacteria to the toxic chemical. Recovery of the respiration in the BOD test has been observed in the presence of some initially inhibitory chemicals by many workers (Busch, 1982; Trudgill et al., 1971).

9. Although useful, short‐term tests are limiting as experimental conditions differ considerably from those in full‐scale plants. The tests are batch processes in which the progress of a biochemical reaction is investigated over a relatively short time period, from 30 min in respiration inhibition studies to 5 d in BOD toxicity testing. The mixing characteristics therefore exhibit completely plug‐flow dispersion. In contrast, the dispersion in full‐scale continuous‐flow plants approaches either completely mixed or dispersed plug‐flow character. Substances described as non‐toxic and degradable in short‐term tests can exhibit other deleterious effects on full‐scale operation. In short‐term tests the biodegradation of a substance does not require that micro‐organisms should be in any particular physical state, whereas the efficient operation of full‐scale activated sludge plants requires that the flocculation of biomass occurs in order that separation of solids from the final effluent is achieved. Some substances can cause deflocculation without inhibiting respiratory activity. Other substances are responsible for the proliferation of filamentous micro‐organisms resulting in sludge bulking. For these reasons simulation tests should be carried out in order to determine the treatability of chemical substances in activated sludge plants.