BS ISO 21285:2019 pdf download – Soil quality — Inhibition of reproduction of the soil mite (Hypoaspis aculeifer) by soil contaminants
7.1.2? Range-finding? test? (preliminary? test) A preliminary test to find the range of mixture ratio affecting predatory mites is optional, e.g. 0 %, 1 %, 5 %, 25 %, 50 %, 75 %, 100 % soil, or of the test substance, e.g. 0 mg/kg, 1 mg/kg, 10 mg/kg, 100 mg/kg and 1 000 mg/kg [the concentrations being expressed in milligrams of test substance per kilogram of dried control soil (see 5.2.2) and a control using 10 mites per container]. The preliminary test is conducted without replication. The duration of the range finding test is 14 d (exposure time), followed by an extraction time of two days. After a total 16 d, mortality of the adult mites and the number of juveniles is determined. Based on the results of the range finding test, the ER 50 /EC 50 is roughly determined by calculating the geometric mean of those two concentrations showing 0 % and 100 % mortality. The concentration/dilution range in the final test should preferably be chosen so that it includes concentrations at which juvenile numbers are affected while survival of the maternal generation is not. This, however, may not be possible for substances that cause lethal and sub-lethal effects at similar concentrations. When no effects are observed, even at 100 % contaminated soil or at concentrations of 1 000 mg test substance/kg standard soil (dry mass), the definitive test can be designed as a limit test.
7.1.3 Definitive test
The design of the definitive test depends on the test objectives. Typically, the habitat properties of samples of a field-collected soil are characterized by comparison of the biological effects found in the soil to be tested with those found in a reference soil, or if not available or not appropriate due to toxicity or atypical physicochemical characteristics, in a standard soil. Results for the standard soil assist in distinguishing contaminant effects from non-contaminant effects caused by soil physicochemical properties. Regardless of whether a reference soil or standard soil is used for the statistical comparisons, the results from standard soil shall be used to judge the validity and acceptability of the test  . The duration of the definitive test is 14 d (exposure time), followed by an extraction time of two days. After a total 16 d, mortality of the adult mites and the number of juveniles is determined. If for characterization purposes a test design including dilution series is required, three designs are possible (the concentrations shall be spaced by a factor not exceeding 2):
— For the NOER/NOEC approach, at least five concentrations or test mixtures in a geometric series should be used. Four replicates for each treatment plus eight controls are recommended.
— For the ER x /EC x approach, 12 concentrations or test mixtures should be used. Two replicates for each concentration plus six controls are recommended. The spacing factor can be variable; smaller at low concentrations, larger at high concentrations.
— For the mixed approach, 6 to 8 concentrations or test mixtures in a geometric series should be used. Four replicates for each treatment plus eight controls are recommended.
This mixed approach allows a NOER/NOEC as well as an ER x /EC x evaluation. To facilitate checking of the pH and humidity of the test sample, use of additional containers for each concentration and for the control is recommended. Each test container (replicate) is filled with 20 g dry mass of the test sample. To ensure easy migration of mites the substrate in the test container should not be compressed. 7.1.4 Limit test If no effects are observed at the highest concentration in the range-finding test (i.e. 1 000 mg/kg or 100 %), the reproduction test can be performed as a limit test, using a test concentration of 1 000 mg/kg or undiluted soil. A limit test will provide the opportunity to demonstrate that the NOEC/NOER or the EC 10/ ER 10 for reproduction is greater than the limit concentration while minimising the number of mites used in the test. Eight replicates should be used for both the treated soil and the control.