Improved methods for quantifying soil invertebrates during ecotoxicological tests: Chill comas and anesthetics.

Soil invertebrate ecotoxicological tests are important when making informed site-management decisions. However, traditional tests are time-consuming and require quantification of high numbers of soil invertebrates burrowed beneath the surface of soil. A commonly used technique to extract invertebrates from the soil is the floatation method. Due to the movement of Collembola, and the presence of small soil particulates and bubbles on the surface of the water, automatic image analysis software may inaccurately quantify the true number of individuals present. Hence, manual counting immediately following extraction, or from images, is still the most effective method utilized for quantifying floated soil invertebrates. This study investigated three novel techniques; the use of an ice-water bath, chest freezer (-12 °C) and ethanol to temporarily immobilize groups of 35 individuals to increase accuracy during the quantification step. Active thermography to aid automatic image analysis was also investigated. Results show that while thermoimaging did not provide a distinct advantage in differentiating soil invertebrates from soil particles, both an ice-water bath and 4.75% ethanol solution were extremely effective at temporarily immobilizing with no apparent ill effects. The outcome of this study will assist future soil invertebrate research by increasing the accuracy of invertebrate quantifications. In addition, as the techniques caused no mortality to the invertebrates, the same individuals remain available for continuous monitoring experiments, repeated exposure, and/or multi-generational studies.