Standards Development

Scope

This guidance document aims at assisting project leaders within ISO/TC 190/SC 4 and ISO/TC 147/SC 5 in designing and organizing trials for validation of biotests. The validation activities during the different steps of the standardization process are described. This guidance document comprises the overall data evaluation and subsequent validation study conclusion.

An interlaboratory validation program supporting the standardization of a new test method typically involves three major steps:

1) Demonstration of Readiness for Testing: This 1st step is an opportunity for the Lead Laboratory to ensure that recommended instrumentation is in place and that test organism cultures have been established using health criteria targets;

2) Demonstration of Laboratory Capability and Transferability of the Biotest: This 2nd step would primarily consist of interlaboratory testing with a focus on these participating laboratories achieving successful control performance and successful completion of a preliminary testing round with a reference toxicant added either to a solid matrix or to a liquid medium according to the aim of the interlaboratory validation study. This demonstration of ability to conduct the testing standard is proven by fulfilling the validity criteria (e.g. variability of controls expressed as the coefficient of variation for the number of juveniles in a reproduction test) qualifies the laboratory for the final method validation step.

3) Method Validation: The 3rd step involves only laboratories who have demonstrated the expertise in conducting the ISO standard under development. In case of validation of an ecotoxicological or microbiological testing method, one or two rounds of interlaboratory Ring testing using a contaminated environmental sample (or samples) would be conducted and the results from each round would be used to calculate the within- and between-laboratory variability of the ISO testing standard as a demonstration of method precision (Annex A). If repeated performance of the biotest is feasible, repeatability is determined. Obtained results are used for confirming or adjusting the validity criteria.

In the course of the validation study, representative samples should be selected according to the intended scope of the standard (e.g. contaminated soils, amended soils, soils after remediation, waste materials, wastewaters, eluates, surface water, groundwater, sediments and extracted samples).

Following the successful completion of these interlaboratory validation steps, the ISO draft standard is to be revised to include a summary of the inter-lab testing results and is now considered to be ready for the final round of voting (i.e. DIS or FDIS vote).

Purpose

Standards prepared by ISO TC-147 and ISO TC-190 are used as reference test methods by governments, agencies working in the area of environment protection, research institutes and testing laboratories to evaluate the quality of water and soil samples, to determine risks resulting from present pollutants or to reveal harmful effects of various non-contaminant conditions (e.g. acidification, salinization, organic matter content, etc.) on water and soil biota. These testing standards are often a component of environmental monitoring programs or studies aimed at generating data for site-specific ecological risk assessments (e.g. TRIAD analyses including chemical, ecotoxicological and ecological data). Test results are often used for the enforcement of various environmental regulations, permits and control orders. In such legal situations, it is vital that the associated uncertainties in the tests' results be known. A test procedure can only be published as an ISO standard when fully validated. The key parameter of validation is precision expressed as reproducibility (inter-laboratory variability) and repeatability (intra-laboratory variability) which are estimated by means of an inter-laboratory ring test. Although there are some general principles which have to be followed during the validation process of any analytical method, biotests require a specific approach which differentiate them from physical and chemical test method validation studies. The most important aspects of biotest validation which have to be considered are:

- differences in culturing and handling of living organisms which can have an influence on the testing outcomes (i.e., health of test organisms);

- longer-term nature of some biological tests (e.g., reproduction inhibition test) which makes it more difficult and costly to repeat experiments;

- data resulting are often non-normal in their distribution which could require the expertise of a statistician to evaluate difficult data sets; and

- the effect of individual stressors on test organisms cannot be estimated directly through testing of a complex contaminated effluent or surface soil but the total toxic potential of a sample can be estimated by comparing the treatments to the results of a suitable negative control.

This proposed standard describes the process and considerations for performing of method validation studies during the development of ISO standards within TC 190/Subcommittee 4 (Soil Quality – Biological Characterization) and TC 147/Subcommittee 5 (Water Quality – Biological Methods). This document will give special attention to the factors listed above which may have a significant impact on estimated reproducibility and/or repeatability if not considered in a new work item. The intention of this standard is to provide a harmonized approach for method validation of all biotests in estimation of soil and water quality in the ISO TC 147 and TC 190 work program.