OECD 216/217: Nitrogen and Carbon Transformation Test

Soil microorganisms are very important for the breakdown and transformation of organic matter through mineralization. Long-term interference with the biochemical processes could potentially affect the nutrient cycling thus altering the functionality of the soil. The impact of chemicals on the soil microbial community needs to be assessed if products are applied to soil or if an exposure of soil is likely. For agrochemicals, the effect of two test item concentrations on nitrogen transformation is required. If non-agrochemicals are tested, a dose-response study might be necessary.

Study Design


A sandy soil according to the OECD specifications is used. The soil is freshly collected e.g. from an untreated field site near ibacon. After sampling the soil is air dried, sieved and pre-incubated in ventilated boxes at room temperature with frequently moistening.

Test concentrations

For agrochemicals and medicinal products usually two test item concentrations are tested. The test item concentrations are based on the single application field rate and up to ten times that concentration (depending on the number of applications per season) or on the predicted environmental concentration (PEC) and a multiple of it. For biocides and industrial chemicals under REACH, usually five concentrations are tested in order to receive an effect concentration (ECx).

Course of the test

The test item is applied onto soil either as a solution in water or applied onto quartz sand. Three replicates are used for the control and the test item concentrations. For nitrogen transformation studies, the soil is mixed with powdered Lucerne meal to supply the microorganisms with a source of required carbon to nitrogen ratio. As a reference item, a control is tested in parallel. After application to the soil, the samples are incubated in a controlled environmental room in the dark. Soil moisture is adjusted and maintained during the study. These conditions allow rapid microbial metabolism. Samples of the incubated soils are taken after application (day 0) and after 7, 14 and 28 days, respectively. At each sampling date the dry weight, nitrate content and / or short-term respiration is determined. The results of the nitrogen transformation (nitrate levels) and the short-term respiration measurements are statistically analyzed for comparison of the control group with the treatment groups. If, after four weeks, the results for the control sample and the test item treated samples differ for more than 25 %, the test is prolonged up to 100 days.

A test with a toxic reference substance is performed at least once a year to proof sensitivity of the test design.

Measurement of nitrogen transformation

The soil samples collected at sampling dates are extracted with a suitable extraction solvent (e.g. KCl) by shaking and centrifugation afterwards. The extracts were analysed for nitrate-N (and nitrite-N and ammonium-N if necessary) by means of Continuous Flow Analysis and comparision with known standards.

Measurement of carbon transformation

The collected soil samples are analysed for glucose induced respiration rates. An appropiate amount of glucose is mixed with the samples and incubated in closed vessels. To calculate oxygen consumption and carbon dioxide release, the pressure decrease in the reaction vessels is measured up to 24 consecutive hours.


The soil respiration of the treated samples and the nitrate-N content of test item treatments are compared with the untreated control. Furthermore, incremental (between two sucessive sampling dates) or cumulative (between sampling date and day 0) nitrate rates are calculated. The nitrate-N rates and nitrate content and the soil respiration of the test item treated samples should be lower than 25% compared to the control at the end of the test. The endpoints for a dose-response study are the calculated ECx values.

Guidelines and literature

  • OECD-Guideline for the Testing of Chemicals, Soil Microorganisms: Nitrogen Transformation Test, Guideline 216, January 21, 2000.
  • OECD-Guideline for the Testing of Chemicals, Soil Microorganisms: Carbon Transformation Test, Guideline 217, January 21, 2000.