Does nitrate co-pollution affect biological responses of an aquatic plant to two common herbicides?

Aquatic systems in agricultural landscapes are subjected to multiple stressors, among them pesticide and nitrate run-off, but effects of both together have rarely been studied. We investigated possible stress-specific and interaction effects using the new OECD test organism, Myriophyllum spicatum, a widespread aquatic plant. In a fully factorial design, we used two widely applied herbicides, isoproturon and mesosulfuron-methyl, in concentration-response curves at two nitrate levels (219.63 and 878.52mg N-NO3). We applied different endpoints reflecting plant performance such as growth, pigment content, content in phenolic compounds, and plant stoichiometry. Relative growth rates based on length (RGR-L) were affected strongly by both herbicides, while effects on relative growth rate based on dry weight (RGR-DW) were apparent for isoproturon but hardly visible for mesosulfuron-methyl due to an increase in dry matter content. The higher nitrate level further reduced growth rates, specifically with mesosulfuron-methyl. Effects were visible between 50 and 500μgL(-1) for isoproturon and 0.5-5μgL(-1) for mesosulfuron-methyl, with some differences between endpoints. The two herbicides had opposite effects on chlorophyll, carotenoid and nitrogen contents in plants, with values increasing with increasing concentrations of isoproturon and decreasing for mesosulfuron-methyl. Herbicides and nitrate level exhibited distinct effects on the content in phenolic compounds, with higher nitrate levels reducing total phenolic compounds in controls and with isoproturon, but not with mesosulfuron-methyl. Increasing concentrations of mesosulfuron-methyl lead to a decline of total phenolic compounds, while isoproturon had little effect. Contents of carbon, nitrogen and phosphorus changed depending on the stressor combination. We observed higher phosphorus levels in plants exposed to certain concentrations of herbicides, potentially indicating a metabolic response. The C:N molar ratio decreased strongly with isoproturon and increased with mesosulfuron-methyl. The C:P and N:P ratios did not vary for most herbicide concentrations, indicating homeostasis. Nitrate level had no effect on the C:N ratio, but the N:P ratio increased in high nitrate level treatments, indicating that the former is more strictly regulated by the plant than the latter. We conclude that the multi-stress impacts caused to aquatic primary producers by herbicides and nitrate enrichment, as often observed in agricultural run-off, not only affected growth and pigment content, but also structural traits (dry matter content) and other physiological traits (elemental stoichiometry, phenolic compounds). Changes in those might have indirect effects on biotic interactions and elemental cycles. We suggest considering some of these endpoints in future studies in environmental risk assessment for agricultural run-off.