We predicted that killing live vegetation will have clear impacts on the abundance of soil fauna, litter mass loss and soil mineral N availability. This prediction was fully confirmed in terms of ecosystem functioning: litter mass loss and resin NH 4 capture were lower and resin NO 3 capture higher in H than W plots. In soil fauna, the effects were more mixed with enchytraeids having no response and the response of nematodes depending on the soil layer and study year. Immediately after the first treatment session in June 2016, elevated numbers of nematodes, and especially bacterivorous nematodes, were found in the upper layer of H plots, but throughout the second study year, the effect of hoeing on nematode numbers was clearly negative in both soil layers. Our second prediction was that the effects of killing plants with a Roundup treatment before hoeing would not significantly differ from the effects of hoeing alone. This prediction was confirmed for the most part: animal abundances, litter mass loss and soil mineral N availability showed no differences between H and RH plots except for the number of total nematodes, which was lower in the upper soil layer in RH than H plots in the June 2016 sampling. Another indication of Roundup effects that we observed was a decreased mass loss of plant litter sprayed with Roundup in 2017. Taken together, our results suggest that recognizing the effects of killing plants as such is essential when testing direct herbicide effects on the structure and functioning of soil communities. These effects can be extensive and need to be carefully disentangled from direct herbicide effects. Secondly, our results suggest that treating live plants or plant litter with Roundup may not have major effects on the structure and functioning of soil decomposer communities.

Due to the high functional redundancy in species rich soil communities43, relations between soil biodiversity and ecosystem functioning typically depend more on the structural and functional diversity of soil organisms than on their species richness or other taxonomic parameters19. In our study, we therefore focused on the trophic groups of soil micro- and mesofauna rather than on their taxonomy. We used nematodes and enchytraeids as study organisms because their abundances are known to quickly respond to disturbances36,37,38,39, soil nutrient mineralization is controlled by these animals15,16,17,18 and changes in the abundance of bacterial- and fungal-feeding nematodes can be used as surrogates of changes in bacterial and fungal growth40,41,42. We expected that the different trophic groups would respond to hoeing as it affects the basal resource supply of soil food webs by killing live roots, terminating root carbon release and increasing dead plant material. Indeed, of the nematodes, the abundance and relative proportion of bacterial feeders was increased and the relative proportion of fungal feeders decreased by hoeing in the June 2016 sampling, suggesting that bacterial growth in particular was enhanced by greater plant litter supply in H plots. However, this positive effect was already absent in the October 2016 sampling and in 2017, the total number of nematodes was 75% lower in H than W plots, which illustrates the dependence of nematode abundance on continuous primary production. None of the trophic groups nor the community composition responded to the Roundup treatment in 2016, i.e. the abundances or relative proportions of trophic groups did not differ between H and RH plots, whereas the total number of nematodes was lower in RH than H plots in the June 2016 sampling. This effect is hard to explain as none of the trophic groups had a parallel pattern, and as this effect did not take place in any other sampling, its importance appears limited. Overall then, the effects of Roundup on nematode abundances can be considered negligible. We neither found effects of Roundup on the biomass of enchytraeids. While this was in line with our prediction, we expected that enchytraeids would respond to hoeing. Their numbers have been shown to respond to the availability of decomposing plant material31 and changes in soil density39,44, which both were likely affected by hoeing. It might be, however, that the effects of hoeing on the physical and nutritional conditions of enchytraeids did not significantly differ from those caused by the earlier tilling of the experimental field.

There is limited earlier evidence of glyphosate effects on soil nematodes, and no evidence of effects on enchytraeids, but Zhao et al.45. recently published a meta-analysis of nematode responses to different herbicides. Their analysis suggests that the abundance of bacterial-feeding, plant parasitic and omnivorous nematodes increase in herbicide treated soils, while those of fungivorous and predatory nematodes decrease. However, it is not clear whether the studies in the meta-analysis had distinguished the direct herbicide effects from the indirect effects mediated through reduced root carbon flow and increased dead plant material. Moreover, only one of the studies focused on glyphosate effects. In that three-year study, Liphadzi et al.46 showed that the total nematode abundance and the proportions of trophic groups in conventionally tilled and no-till soybean and corn fields in Kansas, USA, did not under glyphosate application (1.12 kg ha−1 applied once or twice during the growth period) differ from those observed under application of other herbicides. To our knowledge, this study is the only field experiment of glyphosate effects on soil nematodes and since then, only one short-term laboratory experiment of the effects of single glyphosate application on the nematodes of Australian banana plantation soil has been published47. In this study, no significant effects of glyphosate application on the total number of nematodes or nematode trophic groups were found. Our results are in line with these findings and suggest that major glyphosate effects on soil nematodes are neither likely in soils under cold climatic conditions. We did not examine the whole decomposer food web, and it is possible that other groups of organisms such as protozoa and arthropods are more sensitive to Roundup than nematodes and enchytraeids. However, considering that our target organisms covered all trophic groups of soil animals and included microbial feeders and their predators, major changes within the decomposer food web should have become visible through competitive and predatory interactions. It also appears that our sampling protocol was efficient enough to notice changes in nematode and enchytraeid abundances as manifested by the observed differences between soil layers, seasons and the W and H plots.

To examine the effect of Roundup on soil ecosystem services supplied by decomposer organisms, we measured plant litter mass loss and soil availability of mineral N. As we hypothesized, destroying live vegetation affected litter mass loss. The lower mass loss in H than W plots can be explained by litter being on average drier and microbial activity therefore lower on the exposed soil surface than among dense vegetation. The higher soil NO 3 − availability in H than W plots was in turn most likely a consequence of two processes: first, there was more dead plant material (i.e. organic N) available for microbial consumption and N mineralization in H than W plots, and secondly, plants did not exploit NO 3 − in H plots. In 2017, more NH 4 + was found in W than H plots. This was unexpected since plants utilize both mineral forms of N and their concentrations in soil should therefore be lower under active vegetation than in bare soil. The explanation is therefore likely related to differences in the activity of nitrification bacteria between W and H plots. We did not measure soil temperature, but most likely the soil was on average warmer in open, hoed plots than in shaded weed plots and the warmer soil may have raised the nitrification rate (i.e. the microbial oxidation of NH 4 + to NO 3 −) in the hoed plots. Overall, in contrast to these clear effects of plant absence on soil functioning, no differences in functioning were detected between H and RH plots. This suggests that killing weeds using Roundup, even when applying the highest allowable dose, may not have impacts on soil functioning.

The only indication of direct Roundup effects on soil functioning that we found was a 20% decrease in the mass loss of litter sprayed with Roundup in 2017. This finding suggests that in places where plant remains are subjected to herbicide application after crop plant harvest, herbicide effects on decomposers may also be transmitted by plant litter. The effect was most likely not due to Roundup-induced changes in litter C/N-ratio48 as the slightly increased N concentration and reduced C/N –ratio should have enhanced rather than reduced microbial growth. Hoesel et al.49 recently found no effects of Roundup application on decomposition using a tea bag method, but no studies have to date examined the decomposition rate of glyphosate treated plant litter. Most of the studies that have tested the impact of Roundup on soil micro-organisms indicate negligible or minor effects on microbial community structure when the herbicide is applied in recommended doses50,51,52,53. Increased microbial activity has also been reported, which indicates that microbes can utilize Roundup as a source of carbon, nitrogen or phosphorus52,54,55,56. In our study, the litter was only accessible to soil microbes and micro- and mesofauna, not to macrofauna such as earthworms, whose straw incorporation activity has been reported to decrease after glyphosate spraying29,48. The reduced litter mass loss we observed therefore needs to be due to changes in the activity of microbes and/or the micro- and mesofauna. The reason for decreased litter degradation in 2017, but not in 2016, could be related to higher precipitation and higher overall microbial activity in summer 2017, but this idea is not supported by equal mean litter mass loss in 2016 and 2017. All in all, considering that reduced litter mass loss was observed in one year only and that the effect was statistically not highly significant (thus having relatively high possibility of being coincidental), further field-scale tests of the effects of subjecting plant litter to Roundup are warranted. It is also important to note that glyphosate was in our study applied as a formulated product, Roundup Gold, which contains a surfactant, etheralkylamine ethoxylate (EtO-EA: CAS 68478-96-6). There is evidence that EtO-EA is toxic to human cells57,58, but we are not aware of any studies that would have tested its effects on soil microbes. Nevertheless, when carrying out further tests of glyphosate risks, this is a viewpoint that might be worth of more attention.