Three weeks after the final experimental harvest (3/2013), ryegrass was grown again (height ca. 10 cm) and the pots on Table B (biochar application 20 g/L) and D (80 g/L) were treated with glyphosate (Roundup® Bio; Monsanto, Copenhagen, Denmark) mixed with tap water (15:1000). Glyphosate was sprayed according to the GEP protocol (Good Experimental Practice) used by Agrifood Research Finland (MTT), adopted from the EEC Directive 93/71/EEC. Before spraying, the pots were taken out of the greenhouse and glyphosate was spread using a 2 m wide sprayer at 50 cm height. The glyphosate dose per pot was calculated by placing 16 filter-papers with similar surface-area as the test pots between the pots during glyphosate spraying. The glyphosate dose per surface area was calculated from the mass difference of the filter-paper before and after spraying. The RoundUp dose was 0.11 g/pot comprising 1.5% glyphosate, i.e. 1650 μg/pot, corresponding glyphosate dose of 1300 g/ha. Grasses in the glyphosate treated pots withered and died between 3 and 21 d after spraying. After spraying, the pots were taken back to the greenhouse and returned to their original positions. The pots were kept moist and the temperature at 17:15 °C day:night (16:8 h). After 4 weeks the pots were irrigated with 500–850 ml of 18–20 °C tap water to mimic heavy rain. The water was applied gradually during a period of 1.5 h (average) so that 500 ± 25 ml of the leachate per pot was collected for the analysis of glyphosate, AMPA and solid material. The water leachate was collected, quantified and stored in a freezer (−18 °C) until analyses. Solid material was separated from the melted leachates by centrifuging (15 min, 3500 rpm × G). Glyphosate and AMPA concentrations in the liquid part of the melted leachates were measured using the UHPLC-MS-MS-method (2.23 Internal method of MTT Research Finland, LOQ 0.02 μg/l)