Challenges and Criticality of Analytics for Ecotoxicology Studies

Krithi Shetty B.
Eurofins Advinus Limited, Bangalore

Key analytical requirements for dose formulations to comply with GLP
The dose formulations (test item in the vehicle) prepared for dosing the test systems in toxicology studies are required to be analysed for test concentration verification. The dose formulation preparations need to be checked for their concentration, stability and homogeneity.

Common practice for developing analytical methods
The usual practice is to develop an analytical method covering the concentration range of the dose preparations that are used in the toxicology studies. It is recommended to cover at least 30% lower side of the low dose concentration and at least 10% higher side of the higher dose concentration so as to further cover analytical or preparation variations. After the developed method is validated, this method is used for testing the stability of the preparation. If the preparation is a suspension or dietary, homogeneity is also need to be tested. Based on the stability of the preparation, frequency of preparation of the dose formulations will be decided.


Challenges of analytical methods meant for Ecotoxicology studies
In case of ecotoxicology studies, for example fish toxicity studies, the dosing happens through introducing the test item into the test medium (water, reconstituted water, etc.). Depending on the stability of the test item in the test medium, whether to conduct the study under static conditions or semi-static conditions will be decided. Under semi-static conditions, the test medium is changed, after a period not less than 24 hours, with freshly fortified (with test item) test medium. The frequency of changing the fortified medium under semi-static conditions is decided based on the stability of the test item in water. It is believed that changing test medium at a frequency less than 24 hours can introduce stress on the organisms. Hence, for semi static conditions, the stability requirements will be 24 hours and for static conditions the stability requirements will be up to the end of exposure period (e.g., 96 h, 48 h). There are test items which are not stable in the test medium even for 24 hours. In such cases, the actual analysed concentrations in the test medium at various intervals, as it degrades, till 24 hours will be used for interpretation of the ecotoxicity study results. This means that, it is not enough if the concentration range of the analytical method covers just the initial dose concentrations. The method need to be able to cover concentration of test item present in the test medium, as it degrades during the study, even at the end of the 24-hour period. This means that the Limit of Quantification (LOQ) of the method needs to be several folds lower than the lower dose concentration.
The dose concentrations used in ecotoxicology studies, most of the times, run into ppb and ppt levels. But if the test item is not stable in the test medium for even 24 hours, the concentrations to be covered in the analytical method further get lowered, to be able to analyse the concentrations as the test item degrades in the water over 24-hour period.
Strategies to overcome challenges
To overcome major challenges of analytical methods for ecotoxicology studies, a GLP test facility requires analytical techniques that allow very very low LOQs and a team of experienced analysts to develop and validate methods. Various techniques like, highly sensitive LC-MS/MS and GC-MS/MS are usually used for such analysis, but even these need to be higher-end equipment in order to analyse samples at such lower concentration levels, in spite of concentrating the samples by extracting the test item into volatile solvents or by SPE methods.
The analytical methods for supporting ecotoxicology studies hence become very trying and require enormous analytical expertise to develop methods and to use high-end analytical instrumentation for validation and sample analysis. Further, when working at such low concentration levels, contaminations resulting in ghost peaks or interferences become major problems.
In cases where test item solubility in the water is negligible, the test item is mixed with water thoroughly by physical means (sonication, magnetic stirring, etc.), filtered / centrifuged and the resultant test medium is used in the study. This also makes the analytical method development and validation much more complicated.
Stringent and meticulous efforts are made to solubilize test item using physical and chemical aids (carriers) so that a stable and uniform dispersion of test item is achieved in the test medium. Use of carriers is the last resort for introducing a test item into test medium. In such cases, test item concentrations in test medium are solely dependent on the analysed concentrations.
Overall, the analytical results of the dose preparation analysis become very important part of the ecotoxicology studies and any uncertainty in these analytical results seriously impact the outcome of the entire ecotoxicology studies.
What makes Eurofins Advinus to overcome challenges
Eurofins Advinus is specialized in analytical services for ecotox studies, equipped with well trained and experienced personnel; and top-end analytical equipment like, high-end LC-MS/MS, GC-MS/MS, ICP-MS, etc. Further the analytical sample processing labs for these analyses are well separated from the other routine analytical labs so as to avoid any contamination. Even the glassware used for this purpose are dedicated only for ecotox analytical purpose including a dedicated glassware cleaning area. Analytical expertise at Eurofins Advinus includes developing such methods for analysis at extremely low concentrations for test items containing multiple analytes and also for those unstable in the test medium. Thorough validation of these methods including the matrix effects, qualifier ions in MRM for better specificity, etc., are conducted prior to using them for the study sample analysis, as this become essential for getting reliable analytical results.
Development and validation of analytical methods for ecotoxicology studies are several folds more complex and challenging than those applicable for general animal toxicology studies. Claiming laboratory competence for performing such studies requires not only high-end analytical equipment but also trained people dedicated to generate data for meeting global regulatory requirements.