Mass spectrometry-based metabolomics unravel the transfer of bioactive compounds between rye and neighbouring plants.

Translocation of metabolites between different plant species provides important hints in understanding the fate of bioactive root exudates. In the present study, targeted and untargeted mass spectrometry-based metabolomics was applied to elucidate the transfer of bioactive compounds between rye and several crops and weed species. Our results demonstrated that benzoxazinoids (BXs) synthesized by rye were taken up by roots of neighbouring plant species and translocated into their shoots. Furthermore, we showed that roots of rye plants took up compounds originating from neighbouring plants. Among the compounds taken up by rye roots, wogonin was detected in the rye shoot, which indicated a root-to-shoot translocation of this compound. Elucidating the transfer of bioactive compounds between plants is essential for understanding plant-plant interactions, developing natural pesticides and understanding their modes of action.

De novo transcriptome assembly analysis of weed Apera spica-venti from seven tissues and growth stages.

BACKGROUND: Loose silky bentgrass (Apera spica-venti) is an important weed in Europe with a recent increase in herbicide resistance cases. The lack of genetic information about this noxious weed limits its biological understanding such as growth, reproduction, genetic variation, molecular ecology and metabolic herbicide resistance. This study produced a reference transcriptome for A. spica-venti from different tissues (leaf, root, stem) and various growth stages (seed at phenological stages 05, 07, 08, 09). The de novo assembly was performed on individual and combined dataset followed by functional annotations. Individual transcripts and gene families involved in metabolic based herbicide resistance were identified. RESULTS: Eight separate transcriptome assemblies were performed and compared. The combined transcriptome assembly consists of 83,349 contigs with an N50 and average contig length of 762 and 658 bp, respectively. This dataset contains 74,724 transcripts consisting of total 54,846,111 bp. Among them 94% had a homologue to UniProtKB, 73% retrieved a GO mapping, and 50% were functionally annotated. Compared with other grass species, A. spica-venti has 26% proteins in common to Brachypodium distachyon, and 41% to Lolium spp. Glycosyltransferases had the highest number of transcripts in each tissue followed by the cytochrome P450s. The GSTF1 and CYP89A2 transcripts were recovered from the majority of tissues and aligned at a maximum of 66 and 30% to proven herbicide resistant allele from Alopecurus myosuroides and Lolium rigidum, respectively. CONCLUSIONS: De novo transcriptome assembly enabled the generation of the first reference transcriptome of A. spica-venti. This can serve as stepping stone for understanding the metabolic herbicide resistance as well as the general biology of this problematic weed. Furthermore, this large-scale sequence data is a valuable scientific resource for comparative transcriptome analysis for Poaceae grasses.

Integrated weed management systems with herbicide-tolerant crops in the European Union: lessons learnt from home and abroad.

Conventionally bred (CHT) and genetically modified herbicide-tolerant (GMHT) crops have changed weed management practices and made an important contribution to the global production of some commodity crops. However, a concern is that farm management practices associated with the cultivation of herbicide-tolerant (HT) crops further deplete farmland biodiversity and accelerate the evolution of herbicide-resistant (HR) weeds. Diversification in crop systems and weed management practices can enhance farmland biodiversity, and reduce the risk of weeds evolving herbicide resistance. Therefore, HT crops are most effective and sustainable as a component of an integrated weed management (IWM) system. IWM advocates the use of multiple effective strategies or tactics to manage weed populations in a manner that is economically and environmentally sound. In practice, however, the potential benefits of IWM with HT crops are seldom realized because a wide range of technical and socio-economic factors hamper the transition to IWM. Here, we discuss the major factors that limit the integration of HT crops and their associated farm management practices in IWM systems. Based on the experience gained in countries where CHT or GMHT crops are widely grown and the increased familiarity with their management, we propose five actions to facilitate the integration of HT crops in IWM systems within the European Union.

Toward a Reduced Reliance on Conventional Pesticides in European Agriculture.

Whether modern agriculture without conventional pesticides will be possible or not is a matter of debate. The debate is meaningful within the context of rising health and environmental awareness on one hand, and the global challenge of feeding a steadily growing human population on the other. Conventional pesticide use has come under pressure in many countries, and some European Union (EU) Member States have adopted policies for risk reduction following Directive 2009/128/EC, the sustainable use of pesticides. Highly diverse crop production systems across Europe, having varied geographic and climatic conditions, increase the complexity of European crop protection. The economic competitiveness of European agriculture is challenged by the current legislation, which banned the use of many previously authorized pesticides that are still available and applied in other parts of the world. This challenge could place EU agricultural production at a disadvantage, so EU farmers are seeking help from the research community to foster and support integrated pest management (IPM). Ensuring stable crop yields and quality while reducing the reliance on pesticides is a challenge facing the farming community is today. Considering this, we focus on several diverse situations in European agriculture in general and in European crop protection in particular. We emphasize that the marked biophysical and socio-economic differences across Europe have led to a situation where a meaningful reduction in pesticide use can hardly be achieved. Nevertheless, improvements and/or adoption of the knowledge and technologies of IPM can still achieve large gains in pesticide reduction. In this overview, the current pest problems and their integrated management are discussed in the context of specific geographic regions of Europe, with a particular emphasis on reduced pesticide use. We conclude that there are opportunities for reduction in many parts of Europe without significant losses in crop yields.

The effect of glyphosate on the growth and competitive effect of perennial grass species in semi-natural grasslands.

Biodiversity within European semi-natural biotopes in agro-ecosystem is declining, and herbicide drift from neighbouring fields is considered as an important factor for the decline. The aim of the present study was to investigate whether the growth and competitive interactions in a model system of two perennial grass species, Festuca ovina and Agrostis capillaris, are affected by sub-lethal doses of glyphosate in field margins. In a glasshouse experiment with ample nitrogen, the interspecific competitive interactions were found to be significantly affected by glyphosate; the competitive effect of F. ovina on A. capillaris increased and the competitive effect of A. capillaris on F. ovina decreased with increasing doses of glyphosate. Furthermore, the importance of interspecific competition increased with the glyphosate dose. The results of the study of competitive interactions are in agreement with the observed plant community dynamics at the field site where F. ovina was found to be more dominant in plots treated with a relatively high dose of glyphosate. Importantly, the effects of glyphosate on the plant community dynamics critically depended on the effect of glyphosate on the plant competitive interactions. The study concludes that the current practice in the environmental risk assessment of non-target effects of herbicides, where single species are tested in the greenhouse, may be inadequate for assessing the effect of herbicides in semi-natural plant communities. The presented methods can be used for assessing the importance of competitive interactions for the sensitivity of non-target plants to herbicides in risk assessment.

Integrated LC-MS and GC-MS-Based Metabolomics Reveal the Effects of Plant Competition on the Rye Metabolome.

Plants compete with their neighbors about the limited resources available to them. Plants under induced stress resulting from competition may alter their metabolome to increase their resilience or enhance their defense mechanisms. In the present study, rye (Secale cereale) plants were cocultivated with different densities (3, 12, and 18 plants per pot) of Austrian pea (Pisum sativum subsp. arvense), hairy vetch (Vicia villosa), and Alexandrian clover (Trifolium alexandrinum L.) to elucidate the changes in the rye metabolome in response to the different levels of competition. Global metabolic profiling by liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS), liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) was performed on rye plants, and the acquired data were analyzed using uni- and multivariate statistics. Targeted analysis showed that a high level of competition reduced the concentration of aglycone benzoxazinoids (BXs) and increased glycoside BXs in rye roots. Untargeted metabolomics analysis indicated an increase in the rye root content of the allelopathic compounds 4-hydroxybenzoic acid and uracil in response to competition. Untargeted analysis of rye shoots revealed that the plant competition increased the d-pyroglutamic acid, which is an elicitor of reactive oxygen species (ROS). Our results have enhanced the knowledge of the biochemical response of plant species to cocultivation.

Perspective: common errors in dose-response analysis and how to avoid them.

Dose-response experiments are conducted to determine the toxicity of chemicals on organisms. The relationship between dose and response is described by different statistical models. The four-parameter log-logistic model is widely used in pesticide sciences to derive biologically relevant parameters such as ED50 and resistance index (RI). However, there are some common errors associated with the calculation of ED50 and RI that can lead to erroneous conclusions. Here we discuss five common errors and propose guidance to avoid them. We suggest (i) all response curves must be fitted simultaneously to allow for proper comparison of parameters across curves, (ii) in the case of nonparallel curves absolute ED50 must be used instead of relative ED50 , (iii) standard errors or confidence intervals of the parameters must be reported, (iv) the e parameter in asymmetrical models is not equal to ED50 and hence absolute ED50 must be estimated, and (v) when the four-parameter log-logistic model returns a negative value for the lower asymptote, which is biologically meaningless in most cases, the model should be reduced to its three-parameter version or other types of model should be applied. The mixed-effects model and the meta-analytic approach are suggested as appropriate to average the parameters across repeated dose-response experiments. © 2021 Society of Chemical Industry.

Low Effectiveness of Prosulfocarb and Mesosulfuron-Methyl + Iodosulfuron-Methyl against Vulpia myuros.

Due to natural tolerance to most widely used herbicides for grass weed control, prosulfocarb as pre-emergence or early post-emergence herbicide and mesosulfuron + iodosulfuron as post-emergence herbicide are the mainstays of any chemical control program for Vulpia myuros in Denmark. However, farmers often report variable efficacy of these herbicides on V. myuros compared to other grass weeds. Dose-response experiments were conducted to evaluate the performance of prosulfocarb and mesosulfuron + iodosulfuron on V. myuros. Prosulfocarb was sprayed at different plant growth stages to study the influence of plant growth stage on the performance of prosulfocarb on V. myuros in comparison with the more susceptible grass weed species Apera spica-venti. Doses causing 50% reduction in response variable (ED50) were estimated from the dose-response analysis. The ED50 values revealed a higher tolerance of V. myuros to prosulfocarb and mesosulfuron + iodosulfuron than A. spica-venti. The relative difference in the effectiveness of prosulfocarb between V. myuros and A. spica-venti was constant among plant growth stages studied. The highest levels of V. myuros control were achieved when prosulfocarb was sprayed pre-emergence (BBCH 00), while the control substantially declined at later growth stages. The results from the current study document the tolerance of V. myuros to prosulfocarb and mesosulfuron + iodosulfuron and highlight the importance of optimization of prosulfocarb spray timing for achieving maximum control of V. myuros.

Determination of the Effect of Co-cultivation on the Production and Root Exudation of Flavonoids in Four Legume Species Using LC-MS/MS Analysis.

Flavonoids play a key role in the regulation of plant-plant and plant-microbe interactions, and factors determining their release have been investigated in most of the common forage legumes. However, little is known about the response of flavonoid production and release to co-cultivation with other crop species. This study investigated alterations in the concentration of flavonoids in plant tissues and root exudates in four legumes [alfalfa (Medicago sativa L.), black medic (Medicago polymorpha L.), crimson clover (Trifolium incarnatum L.), and subterranean clover (Trifolium subterraneum L.)] co-cultivated with durum wheat [Triticum turgidum subsp. durum (Desf.) Husn.]. For this purpose, we carried out two experiments in a greenhouse, one with glass beads as growth media for root exudate extraction and one with soil as growth media for flavonoid detection in shoot and root biomass, using LC-MS/MS analysis. This study revealed that interspecific competition with wheat negatively affected legume growth and led to a significant reduction in shoot and root biomass compared with the same legume species grown in monoculture. In contrast, the concentration of flavonoids significantly increased both in legume biomass and in root exudates. Changes in flavonoid concentration involved daidzein, genistein, medicarpin, and formononetin, which have been found to be involved in legume nodulation and regulation of plant-plant interaction. We hypothesize that legumes responded to the co-cultivation with wheat by promoting nodulation and increasing exudation of allelopathic compounds, respectively, to compensate for the lack of nutrients caused by the presence of wheat in the cultivation system and to reduce the competitiveness of neighboring plants. Future studies should elucidate the bioactivity of flavonoid compounds in cereal-legume co-cultivation systems and their specific role in the nodulation process and inter-specific plant interactions such as potential effects on weeds.

Root-Exuded Benzoxazinoids: Uptake and Translocation in Neighboring Plants.

Plants have evolved advanced chemical defense mechanisms, including root exudation, which enable them to respond to changes occurring in their surroundings rapidly. Yet, it remains unresolved how root exudation affects belowground plant-plant interactions. The objective of this study was to elucidate the fate of benzoxazinoids (BXs) exuded from the roots of rye (Secale cereale L.) plants grown with hairy vetch (Vicia villosa). A rapid method that allows nondestructive and reproducible chemical profiling of the root exudates was developed. Targeted chemical analysis with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was performed to investigate the changes in the composition and concentration of BXs in the rye plant, and its root exudate in response to cocultivation with hairy vetch. Furthermore, hairy vetch plants were screened for the possible uptake of BXs from the rhizosphere and their translocation to the shoot. Rye significantly increased the production and root exudation of BXs, in particular 2-ß-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) and 2-ß-d-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), in response to cocultivation with hairy vetch. DIBOA-glc and DIMBOA-glc were absorbed by the roots of the cocultivated hairy vetch plants and translocated to the shoots. These findings will strongly improve our understanding of the exudation of BXs from the rye plant and their role in interaction with other plant species.

Biology and Management of Vulpia myuros-An Emerging Weed Problem in No-Till Cropping Systems in Europe.

Recently, Vulpia myuros has become a problematic grass weed species in parts of Europe. It is most common in no-till cropping systems. The inherent tolerance to several selective grass weed herbicides is of serious concern to the successful management of V. myuros in arable farming. Here, we reviewed the available knowledge about the biology of V. myuros to identify knowledge gaps and assess management efforts to identify best practices for control. V. myuros is a winter-annual species producing seeds with a short dormancy that can germinate at a wide range of conditions. Seed longevity in the soil is short. Little information is available on the influence of V. myuros on crop yield but some results suggest that yield losses can be significant. The findings provide a better understanding of the weedy characteristics of V. myuros and highlight that management strategies in Europe need to be diversified and integrate preventive and cultural control methods. Finally, we identify some of the management tools that should be considered to minimize the impact of V. myuros on European farming and future needs for research to develop sustainable integrated weed management strategies.

Growth and Phenology of Vulpia Myuros in Comparison with Apera Spica-Venti, Alopecurus Myosuroides and Lolium Multiflorum in Monoculture and in Winter Wheat.

Vulpia myuros has become an increasing weed problem in winter cereals in Northern Europe. However, the information about V. myuros and its behavior as an arable weed is limited. Field and greenhouse experiments were conducted in 2017/18 and 2018/19, at the Department of Agroecology in Flakkebjerg, Denmark to investigate the emergence, phenological development and growth characteristics of V. myuros in monoculture and in mixture with winter wheat, in comparison to Apera spica-venti, Alopecurus myosuroides and Lolium multiflorum. V. myuros emerged earlier than A. myosuroides and A. spica-venti but later than L. multiflorum. Significant differences in phenological development were recorded among the species. Overall phenology of V. myuros was more similar to that of L. multiflorum than to A. myosuroides and A. spica-venti. V. myuros started seed shedding earlier than A. spica-venti and L. multiflorum but later than A. myosuroides. V. myuros was more sensitive to winter wheat competition in terms of biomass production and fecundity than the other species. Using a target-neighborhood design, responses of V. myuros and A. spica-venti to the increasing density of winter wheat were quantified. At early growth stages "BBCH 26-29", V. myuros was suppressed less than A. spica-venti by winter wheat, while opposite responses were seen at later growth stages "BBCH 39-47" and "BBCH 81-90". No significant differences in fecundity characteristics were observed between the two species in response to increasing winter wheat density. The information on the behavior of V. myuros gathered by the current study can support the development of effective integrated weed management strategies for V. myuros.

Pathways for advancing pesticide policies.

Numerous pesticide policies have been introduced to mitigate the risks of pesticide use, but most have not been successful in reaching usage reduction goals. Here, we name key challenges for the reduction of environmental and health risks from agricultural pesticide use and develop a framework for improving current policies. We demonstrate the need for policies to encompass all actors in the food value chain. By adopting a multi-disciplinary approach, we suggest ten key steps to achieve a reduction in pesticide risks. We highlight how new technologies and regulatory frameworks can be implemented and aligned with all actors in food value chains. Finally, we discuss major trade-offs and areas of tension with other agricultural policy goals and propose a holistic approach to advancing pesticide policies.

Diversified Resistance Mechanisms in Multi-Resistant Lolium spp. in Three European Countries.

Annual ryegrass species (Lolium spp.) infest cereal crops worldwide. Ryegrass populations with multiple resistance to the acetyl coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors are an increasing problem in several European countries. We investigated the resistance pattern and level of resistance in ryegrass populations collected in Denmark, Greece and Italy and studied the diversity of mechanisms endowing resistance, both target-site and metabolism based. All populations showed high resistance indexes (RI) to the ALS inhibitors, iodosufuron-methyl-sodium + mesosulfuron-methyl (RI from 8 to 70), whereas the responses to the two ACCase inhibitors, clodinafop-propargyl and pinoxaden, differed. The Greek and Italian populations were moderately to highly resistant to clodinafop (RI > 8) and showed low to moderate resistance to pinoxaden (RI ranged from 3 to 13) except for one Italian population. In contrast, the Danish Lolium populations showed low to moderate resistance to clodinafop (RI ranged from 2 to 7) and only one population was resistant to pinoxaden. Different mutant ACCase alleles (Leu1781, Cys2027, Asn2041, Val2041, Gly2078, Arg2088, Ala2096) and ALS alleles (Gly122, Ala197, Gln197, Leu197, Ser197, Thr197, Val205, Asn376, Glu376, Leu574) endowing resistance were detected in the Greek and Italian populations. In several plants, no mutated ALS and ACCase alleles were found showing a great heterogeneity within and among the Greek and Italian populations. Conversely, no mutant ACCase alleles were identified in the four Danish populations and only one mutant ALS allele (Leu574) was detected in two Danish populations. The expression level of nitronate monooxygenase (NMO), glutathione S-transferase (GST) and cytochrome P450s (CYP72A1 and CYP72A2) varied broadly among populations and individual plants within the populations. Constitutive up-regulation of GST, CYP72A1 and CYP72A2 was detected in resistant plants respect to susceptible plants in one Danish and one Italian population. It appears that the mechanisms underlying resistance are rather complex and diversified among Lolium spp. populations from the three countries, coevolution of both target-site resistance and metabolic based herbicide resistance appears to be a common feature in Denmark and Italy. This must be considered and carefully evaluated in adopting resistance management strategies to control Lolium spp. in cereal crops.

Modeling effects of herbicide drift on the competitive interactions between weeds.

Herbicides may drift onto road verges or natural areas adjacent to arable fields and affect nontarget plants. The effect of low doses of mecoprop-P on the competitive interactions and plant community dynamics was investigated in a model system using Capsella bursa-pastoris and Geranium dissectum as test plants. Dose-response experiments on single species showed that compared to G. dissectum, C. bursa-pastoris was more affected by mecoprop-P. Consequently, we expected that G. dissectum would outcompete C. bursa-pastoris when mecoprop-P was applied at a low dose in the competition experiment. Indeed, mecoprop-P had a significant effect on the interspecific competitive ability of both C. bursa-pastoris and G. dissectum. Our previous expectation, however, was not met: The interspecific competitive ability of both species increased significantly with the dose of the herbicide, and it was predicted that C. bursa-pastoris and G. dissectum are more likely to coexist in natural habitats with low concentrations of the herbicide compared to natural habitats with relatively high concentrations. The results from the dose-response experiments on the single species and the more laborious competition experiment approach, which is assumed to mimic the dynamics of plant communities more closely, show considerable discrepancies even though the experiments were performed at the same time and in the same greenhouse. This finding generally reduces the credibility of using single-species tests in ecological risk assessment of herbicide use.

A review of independent action compared to concentration addition as reference models for mixtures of compounds with different molecular target sites.

From a theoretical point of view, it has often been argued that the model of independent action (IA) is the most correct reference model to use for predicting the joint effect of mixtures of chemicals with different molecular target sites. The theory of IA, however, relies on a number of assumptions that are rarely fulfilled in practice. It has even been argued that, theoretically, the concentration addition (CA) model could be just as correct. In the present study, we tested the accuracy of both IA and CA in describing binary dose-response surfaces of chemicals with different molecular targets using statistical software. We compared the two models to determine which best describes data for 158 data sets. The data sets represented 98 different mixtures of, primarily, pesticides and pharmaceuticals tested on one or several of seven test systems containing one of the following: Vibrio fischeri, activated sludge microorganisms, Daphnia magna, Pseudokirchneriella subcapitata, Lemna minor, Tripleurospermum inodorum, or Stellaria media. The analyses showed that approximately 20% of the mixtures were adequately predicted only by IA, 10% were adequately predicted only by CA, and both models could predict the outcome of another 20% of the experiment. Half of the experiments could not be correctly described with either of the two models. When quantifying the maximal difference between modeled synergy or antagonism and the reference model predictions at a 50% effect concentration, neither of the models proved significantly better than the other. Thus, neither model can be selected over the other on the basis of accuracy alone.

Advocating a need for suitable breeding approaches to boost integrated pest management: a European perspective.

Currently, European farmers do not have access to sufficient numbers and diversity of crop species/varieties. This prevents them from designing cropping systems more resilient to abiotic and biotic stresses. Crop diversification is a key lever to reduce pest (pathogens, animal pests and weeds) pressures at all spatial levels from fields to landscapes. In this context, plant breeding should consist of: (1) increased efforts in the development of new or minor crop varieties to foster diversity in cropping systems, and (2) focus on more resilient varieties showing local adaptation. This new breeding paradigm, called here 'breeding for integrated pest management (IPM)', may boost IPM through the development of cultivars with tolerance or resistance to key pests, with the goal of reducing reliance on conventional pesticides. At the same time, this paradigm has legal and practical implications for future breeding programs, including those targeting sustainable agricultural systems. By putting these issues into the context, this article presents the key outcomes of a questionnaire survey and experts' views expressed during an EU workshop entitled 'Breeding for IPM in sustainable agricultural systems'. © 2017 Society of Chemical Industry.

Reproducibility of binary-mixture toxicity studies.

Binary-mixture studies often are conducted with the aim of elucidating the effect of one specific chemical on the biological action of another. The results can be interpreted in relation to reference models by the use of response-surface analyses and isobolograms. The amount of data needed for these analyses is, however, extensive, and the experiments therefore rarely are repeated. In the present study, we investigate the reproducibility of isobole shapes of binary-mixture toxicity experiments in terms of deviation from the reference model of concentration addition (CA), dose-level dependence, and isobole asymmetry. We use data from four herbicide mixtures tested in three to five independent experiments on the aquatic test plant Lemna minor and the terrestrial plant Tripleurospermum inodorum. The results showed that the variation both within and among experiments was approximately half the size for the aquatic test system compared to the terrestrial system. As a consequence, a consistent deviation from CA could be obtained in three of four herbicide mixtures for L. minor, whereas this was only the case for one or two of the herbicide mixtures tested on T. inodorum. For one mixture on T. inodorum, both CA synergism and antagonism were detected. Dose-dependent effects could not be repeated consistently, just as the asymmetry found in some isoboles could not. The study emphasizes the importance of repeating mixture toxicity experiments, especially for test systems with large variability, and using caution when drawing biological conclusions from the test results.

Combination effects of herbicides on plants and algae: do species and test systems matter?

Risk assessment of herbicides towards non-target plants in Europe is currently based solely on tests on algae and floating aquatic plants of Lemna sp. Effects on terrestrial non-target species is not systematically addressed. The purpose of the present study was to compare combination effects of herbicide mixtures across aquatic and terrestrial test systems, and to test whether results obtained in the traditional aquatic test systems can be extrapolated to the terrestrial environment. This was done by evaluating ten binary mixtures of nine herbicides representing the seven most commonly used molecular target sites for controlling broadleaved weeds. Data were evaluated statistically in relation to the concentration addition model, and for selected concentrations to the independent action model. The mixtures were tested on the terrestrial species Tripleurospermum inodorum (L.) Schultz-Bip. (Scentless Mayweed) and Stellaria media (L.) Vill. (Common Chickweed), and on the aquatic species Lemna minor L. (Lesser duckweed) and the alga Pseudokirchneriella subcapitata (Korschikov) Hindak. For the two mixtures of herbicides with the same molecular site of action, the joint effect was additive. For the eight mixtures of herbicides with different sites of action, two of the mixtures were consistently antagonistic across species, while for the remaining six mixtures the joint effect depended on the species tested. This dependence was, however, not systematic, in the sense that none of the species or test systems (terrestrial versus aquatic) had a significantly higher probability of showing synergistic or antagonistic joint effects than others. Synergistic interactions were not observed, but approximately 70% of the mixtures of herbicides with different sites of action showed significant antagonism. Hence, the concentration addition model can be used to estimate worst-case effects of mixtures of herbicides on both terrestrial and aquatic species. Comparing the sensitivity of the species to a 10% spray drift event showed that the terrestrial species were more vulnerable to all herbicides compared with the aquatic species, emphasising the importance of including terrestrial non-target plants in herbicide risk assessment.