Analytical dose verification as part of ecotoxicological / toxicological studies

The purpose of analytical dose verification accompanying terrestrial trials is to ensure that the tested organisms (e.g. non-target plants, honey bees, earth worms, collembolen, etc.) were exposed to the correct concentration of the pure active ingredient or formulated products. The dose verification is done in different matrices e.g. the application dilution /spraying solution in case of plant studies, feeding sugar solution in case of honey bee studies or soil in case of soil organisms or plant seedlings. A dedicated team of analytical chemists works together in close collaboration with our biology teams to verify the concentrations of every test substance. Ibacon has also profound experience in working together with external partners who send their samples for dose verification to our laboratories.

Study Design


The challenge for our analytical team is to establish and adapt analytical methods which are suitable for the verification of test concentrations from e.g. several g/L down to the low µg/L range and for each matrix (solvent, sugar solution or soil). We use a wide range of analytical instruments at our disposal that allows us to analyse almost every kind of substance. Our available instruments are:

  • UPLC and HPLC with UV, DAD, or ELSD detection
  • LC MS-MS
  • GC-MS
  • IC (Ion Chromatography)
  • AAS
  • TOC (Total Organic Carbon)

Method Development and Implementation

If a suitable analytical method cannot be provided by the sponsor, our analytical team can develop an appropriate method with the required sensitivity for the concentration range used in the biological study. Method development is carried out under non-GLP conditions. During this stage of method development an appropriate instrument and detection method is chosen (e.g. HPLC with UV detection at a specific wavelength or MS/MS detection using a specific column), along with any necessary sample preparation (e.g. dilution, filtration, centrifugation, extraction etc.).

After initial method development or implementation of a given method often the biological pre-test samples is analysed to obtain information concerning the dosing of the test substance, its stability during the test and during storage of the samples, and the suitability of the analytical method. This information is vital for choosing the appropriate test design and the appropriate analytical method for the main study. At this stage a decision is made if the development of complex sample preparation steps such as enrichment via liquid-liquid extraction or solid phase extraction or derivatisation may be necessary if the limit of quantification of the initial analytical method is not suitable.

Test Set-up

The samples (e.g. stock solutions, soil samples) will be prepared and taken by our biologists and the samples will be analyzed directly on the same day (standby analysis) or they will be stored frozen until analysis. In most cases the method validation will be performed concurrently with the preparation and analysis of the biological test sample. For validation of the analytical method usually at least two fortification levels in the respective test media (e.g. solvent or soil) will be prepared, each level consisting of at least five replicates with concentrations at least 10% higher respectively at least 10% lower than the test sample. The sample preparation (e.g. dilution, filtration, centrifugation, extraction etc.) depends on the properties of the test item, the concentrations and the matrix (e.g. solvent, soil, sugar solution).

Method Verification

The final method will be validated under GLP conditions alongside the analytical measurements of the main biological study samples. The validity of the method will be determined according to the validity criteria set out in the SANCO 3029/99 guidelines. Fortified samples are prepared to check for the precision and accuracy of the method, and various blank samples are used to confirm the absence of analytical contaminants or interferences which could have entered the samples during sampling, sample preparation, or analysis. A calibration curve across the required concentration range is also prepared to confirm the linearity of the instrument response, and to be able to quantify the test substance in the biological samples. These data will be included in the final study report.

Guidelines and Literature

  • SANCO/3029/99 rev.4