Standardised bioassays

In recent years there has been increasing interest in the use of primary and established animal and human cell lines in which mycotoxin toxicity is determined on morphological criteria and end-points like protein or DNA synthesis. The development of immortalised human cell lines is now being actively pursued to serve as standardised bioassays for the detection of food toxins including mycotoxins (Lewis et al., 1998). 

The above example illustrates the inherent problems that can arise in the use of standardised protocols for assessing chemicals naturally occurring in the food chain. Had work on comparative metabolism and pharmacokinetics been undertaken before any animal bioassay work, it could have given more useful information. The extrapolation of effects obtained in high-dose animal studies to a large number of people exposed to a low dose is not the most effective use of resources. Nor are such experiments consistent with biological reality. There are few chemicals that would not cause illness or death if the daily intake was increased some 100-1000 fold as is the situation in many... 

For standardised instrumental analytical methods, i.e. biomarkers, biosensors and bioassays, there are well-established standard protocols on the national level, e.g. under Association Francaise de Normalisation (AFNOR), British Standard Institute (BSI), DIN (German Organisation for Standardisation), etc., and all those standards are formed by ISO-Working Groups and by validation studies into ISO - and CEN - Standards. Normal accredited and well-qualified laboratories should be able to perform the monitoring. 

The ISO protocol for the biochemical response EROD (ISO 23893-2/AWI) as a recent example of a bioanalytical (biomarker) [49,50] method standardised under ISO for fish needs harmonisation with the other test systems and between the laboratories (users) before implementation. Use of biomarkers (biochemical responses) in multi-arrays for environmental monitoring according to Hansen et al. [50] is complementary to chemical analysis since they can alert for the presence of ecotoxic compounds. Bringing into the WFD, the effect-related approaches concerning bioassays and biomarkers are only relevant in the context of the QN of environmental relevant substances and the good chemical status. But it is rather difficult to transfer the monitored biochemical responses or biomarkers into an operational effect-related standard. They serve as the basis for environmental protection against hazardous substances. In relation to... 

Biological assay (bioassay) is the process by which the activity of a substance (identified or unidentified) is measured on living material e.g. contraction of bronchial, uterine or vascular muscle. It is used only when chemical or physical methods are not practicable as in the case of a mixture of active substances, or of an incompletely purified preparation, or where no chemical method has been developed. The activity of a preparation is expressed relative to that of a standard preparation of the same substance. Biological standardisation is a specialised form of bioassay. It involves matching of material of unknown potency with an International or National Standard with the objective of providing a preparation for use in therapeutics and research. The results are expressed as units of a substance rather than its weight, e.g. insulin, vaccines. 

Abbondanzi F, Cachada A, Campisi T, et al. (2003) Optimisation of a microbial bioassay for contaminated soil monitoring Bacterial inoculum standardisation and comparison with Microtox((R)) assay. Chemosphere 53(8) 889-897. 

For all these tests, interpretation and use would be easier if data on baseline responses were available. Currently, the quantity and quality of baseline data vary between tests. In the cases of standardised tests, such as the earthworm reproduction bioassay, performance criteria exist that can be used to discern if an observed effect is significant. For other assays, information of this type is not available. One area that any work to establish baselines should focus on is the interplay between soil factors, ecotypes and measured responses. This will give baselines for variation within the tests and will help to derive performance criteria that can be used as part of an algorithm to discern the status of a given location. 

A strategy for the application of biological tests (partly standardised) and a useful concept for the evaluation of contaminated soils using bioassay data have been established by a DECHEMA working group [21]. Under certain circumstances these tests may be used also to characterise wastes. 

A selection of tests that may be employed for the investigation of soil pollution is given in 10.5 Bioassays. The listing comprises frequently used bioassays. Many of the tests described have been standardised by agencies or associations... 

Schantz, E.J. et al. Purified poison for bioassay standardisation./. A oc. Off. Anal. Chem.Al, 160, 1958. McCulloch, A.W. et al. Zinc from oyster tissue as causative factor in mouse deaths in official bioassay for paraytic shellfish poison. J. AOAC, 72,384, 1989. 

Figure 4.3 Influence of the amount of water used for periodic watering on plant growth in bioassays with standardised substrate alone and with 2% added starch. Adapted from J.I. Fritz in Ecotoxicity of Biogenic Materials During and After their Biodegradation, University of Natural Resources and Life Sciences, Wien, Austria,...

Aquatic bioassays made with elutriates from polymeric materials are possible, although limited to water-soluble components. The elutrition procedure should be designed properly to simulate the conditions of the natural environment of the application. Examples could be the continuous elutrition in aquatic environments or a periodic exposure at times of rainfall in terrestrial environments. However, neither a standardised nor an otherwise validated method is currently available for such investigations. 

Figure 4.3 Influence of the amount of water used for the periodical watering on the plant growth in bioassays with standardised substrate alone and with 2% starch...

Algal species which are representative for a local ecosystem can be isolated as well and used for bioassays. Some basic properties should be determined and included in the final report. Local species have the advantage of being adapted to the environment under investigation and are further not inhibited by an unbalanced relationship of minerals or by the geogenic presence of some heavy metals. Kusel-Fetzmann [69] recommends that such local factors should be determined and reported anj ay and that bioassays from real samples using standardised... 

It is obvious that bioassay procedures have to be standardised as to tester strains, metabolic activation systems, and quantitation procedures. In addition, future workers in this field should be aware of some of the more important research concerning isolation and recovery of microorganisms from sub-lethal stresses. The effects of chemical stresses on recovery of microorganisms has not received the attention it deserves. For further insight the reader is referred to papers by Rosenkranz and Amsden et a/. , which clearly show that growth conditions have a marked effect on the behaviour of microorganisms in a variety of assay media. The problems of enumeration and recovery of injured organisms should not be overlooked in these systems. 

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