Solvejg K. Mathiassen

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.

The Occurrence of Hormesis in Plants and Algae

This paper evaluated the frequency, magnitude and dose/concentration range of hormesis in four species: The aquatic plant Lemna minor, the micro-alga Pseudokirchneriella subcapitata and the two terrestrial plants Tripleurospermum inodorum and Stellaria media exposed to nine herbicides and one fungicide and binary mixtures thereof. In total 687 dose-response curves were included in the database. The study showed that both the frequency and the magnitude of the hormetic response depended on the endpoint being measured. Dry weight at harvest showed a higher frequency and a larger hormetic response compared to relative growth rates. Evaluating hormesis for relative growth rates for all species showed that 25% to 76% of the curves for each species had treatments above 105% of the control. Fitting the data with a dose-response model including a parameter for hormesis showed that the average growth increase ranged from 9±1% to 16±16% of the control growth rate, while if measured on a dry weight basis the response increase was 38±13% and 43±23% for the two terrestrial species. Hormesis was found in >70% of the curves with the herbicides glyphosate and metsulfuron-methyl, and in >50% of the curves for acifluorfen and terbuthylazine. The concentration ranges of the hormetic part of the dose-response curves corresponded well with literature values.

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.

Herbicide impact on non-target plant reproduction: what are the toxicological and ecological implications?

Declining plant diversity and abundance have been widely reported in agro-ecosystems of North America and Europe. Intensive use of herbicides within cropfields and the associated drift in adjacent habitats are partly responsible for this change. The objectives of this work were to quantify the phenological stages of non-target plants in in-situ field situations during herbicide spray and to compare plant susceptibility at different phenological stages. Results demonstrated that a large number of non-target plants had reached reproductive stages during herbicide spray events in woodlots and hedgerows, both in Canada and Denmark where vegetation varies considerably. In addition, delays in flowering and reduced seed production occurred widely on plants sprayed at the seedling stage or at later reproductive periods, with plants sprayed at reproductive stages often exhibiting more sensitivity than those sprayed as seedlings. Ecological risk assessments need to include reproductive endpoints.

Herbicidal effects of soil-incorporated wheat.

The hydroxamic acid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and the benzoxazolinones benzoxazolin-2-one (BOA) and 6-methoxybenzoxazolin-2-one (MBOA) have been identified as important allelochemicals in wheat. This study examines the possibility of exploiting the allelopathic properties of wheat as a weed control strategy by cultivating wheat as a precrop and incorporating plant residues into the soil before the next crop is sown. Different wheat varieties were cultivated in field plots during two seasons in both conventional and organic farming systems. Plants were sampled at various growth stages, and their contents of DIMBOA, MBOA, and BOA were determined by chemical analyses. The wheat samples were incorporated into soil, and the effect on germination and growth of 12 different weed species was examined in pot experiments under controlled conditions. In some cases significant effects were obtained, but the results were inconsistent and the effects were not correlated to the content of DIMBOA, MBOA, and BOA in the incorporated wheat plants. ED50 doses of the pure compounds were estimated in dose-response experiments in Petri dishes, and these turned out to be much higher than the predicted maximum concentrations of DIMBOA, MBOA, and BOA in the soil water following incorporation. The study shows that a prerequisite for exploiting the incorporation of wheat residues as a weed control strategy is the development of wheat varieties with an increased content of allelochemicals.

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.

Decision Support System for Optimized Herbicide Dose in Spring Barley

Abstract Crop Protection Online (CPO) is a decision support system, which integrates decision algorithms quantifying the requirement for weed control and a herbicide dose model. CPO was designed to be used by advisors and farmers to optimize the choice of herbicide and dose. The recommendations from CPO for herbicide application in spring barley in Denmark were validated through field experiments targeting three levels of weed control requirement. Satisfactory weed control levels at harvest were achieved by a medium control level requirement generating substantial herbicide reductions (∼ 60% measured as the Treatment Frequency Index (TFI)) compared to a high level of required weed control. The observations indicated that the current level of weed control required is robust for a range of weed scenarios. Weed plant numbers 3 wk after spraying indicated that the growth of the weed species were inhibited by the applied doses, but not necessarily killed, and that an adequate level of control was reached later in the season through crop competition. Nomenclature: Spring barley; Hordeum vulgare L. Resumen Crop Protection Online (CPO, Protección de Cultivos en Línea) es un sistema de ayuda para la toma de decisión, el cual integra algoritmos que cuantifican el requerimiento de control de malezas y un modelo de dosis de herbicidas. CPO fue diseñado para ser usado por asesores y productores para optimizar la selección de herbicidas y dosis. Las recomendaciones de CPO para la aplicación de herbicidas en cebada de primavera en Dinamarca fueron validadas mediante experimentos de campo enfocados a tres niveles de requerimientos de control de malezas. Niveles satisfactorios de control de malezas al momento de la cosecha se alcanzaron con un nivel de requerimiento de control medio, lo que generó reducciones sustanciales de herbicidas (∼60% medido como el índice de frecuencia de tratamiento (TFI)) al compararse con el nivel de requerimiento de control de malezas alto. Las observaciones indicaron que el nivel actual de requerimientos de control de malezas es robusto para un rango amplio de escenarios de malezas. Los números de plantas de malezas, 3 semanas después de la aplicación, indicaron que el crecimiento de las especies de malezas fue inhibido por las dosis aplicadas, pero estas no necesariamente murieron, y que un nivel adecuado de control fue alcanzado después en la temporada debido a la competencia del cultivo.

Multiple Herbicide Resistance in Lolium multiflorum and Identification of Conserved Regulatory Elements of Herbicide Resistance Genes.

Herbicide resistance is a ubiquitous challenge to herbicide sustainability and a looming threat to control weeds in crops. Recently four genes were found constituently over-expressed in herbicide resistant individuals of Lolium rigidum, a close relative of Lolium multiflorum. These include two cytochrome P450s, one nitronate monooxygenase and one glycosyl-transferase. Higher expressions of these four herbicide metabolism related (HMR) genes were also observed after herbicides exposure in the gene expression databases, indicating them as reliable markers. In order to get an overview of herbicidal resistance status of L. multiflorum L, 19 field populations were collected. Among these populations, four populations were found to be resistant to acetolactate synthase (ALS) inhibitors while three exhibited resistance to acetyl-CoA carboxylase (ACCase) inhibitors in our initial screening and dose response study. The genotyping showed the presence of mutations Trp-574-Leu and Ile-2041-Asn in ALS and ACCase, respectively, and qPCR experiments revealed the enhanced expression of HMR genes in individuals of certain resistant populations. Moreover, co-expression networks and promoter analyses of HMR genes in O. sativa and A. thaliana resulted in the identification of a cis-regulatory motif and zinc finger transcription factors. The identified transcription factors were highly expressed similar to HMR genes in response to xenobiotics whereas the identified motif is known to play a vital role in coping with environmental stresses and maintaining genome stability. Overall, our findings provide an important step forward toward a better understanding of metabolism-based herbicide resistance that can be utilized to devise novel strategies of weed management.

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.

The effect of nitrogen and glyphosate on survival and colonisation of perennial grass species in an agro-ecosystem: does the relative importance of survival decrease with competitive ability?

The ecological success of a plant species is typically described by the observed change in plant abundance or cover, but in order to more fully understand the fundamental plant ecological processes, it is necessary to inspect the underlying processes of survival and colonization and how they are affected by environmental conditions. A general ecological hypothesis on the effect of environmental gradients on demographic parameters is proposed and tested. The hypothesis is that decreasing fitness or competitive ability along an environmental gradient is associated with an increasing importance of survival for regulating the abundance of the species. The tested hypothesis is related to both the stress gradient hypothesis and whether the importance of competition increases along productivity gradients. The combined effect of nitrogen and glyphosate on the survival and colonization probability of two perennial grass species, Festuca ovina and Agrostis capillaris, which are known to differ in their responses to both glyphosate and nitrogen treatments, is calculated using pin-point cover data in permanent frames. We found that the relative importance of survival increased with the level of glyphosate for the glyphosate sensitive A. capillaris and decreased for the glyphosate tolerant F. ovina. Likewise, increasing levels of nitrogen increased the importance of survival for the relative nitrophobic F. ovina. Consequently, the proposed hypothesis was corroborated in this specific study. The proposed method will enable predictions of the effects of agricultural practices on community dynamics in a relatively simple setup eliminating the need to quantify all the interaction among the species in the plant community. The method will be immediately useful for the regulation of non-cultivated buffer strips between agricultural fields and semi-natural and natural biotopes such as hedgerows and waterways.