The perspective of the logarithmic field sprayer technology.

A logarithmic sprayer was suggested about 70 years ago, but it has not yet been seriously used in research and development, and subsequent registration of plant protection products. Logarithmic sprayers have resorted to mere demonstration experiments to show end users and others how plant protection products work. Fitting dose-response curves in field experiments, however, generates much essential information, e.g., extraction of various effective field rate levels (e.g., ED20, ED50, and ED80). One of the reasons for it rarely being used in the registration of plant protection products is that the dose-response curve regression was hitherto difficult to fit; the registration requirement solely focuses on analyses of variance. Another alleged obstacle is that the logarithmic plots have systematically, not randomly distributed field rates. This paper goes through some of the problems of how to non-randomly analyze field rates by taking autocorrelation into account to make the logarithmic sprayer palatable as registration documentation by assessing efficacy, selectivity, environmental side effects, general toxicity of plant protection products, and cost-effectiveness. The development in precision agriculture, drone technology, and automation of data capture and subsequent analysis could make the logarithmic sprayer a cost-effective alternative to numerous ANOVA experiments with very few fixed field rates to aid the precision spraying of pesticides and thus reduce unnecessary environmental side effects. © 2024 Society of Chemical Industry.

How to use statistics to claim antagonism and synergism from binary mixture experiments.

We review statistical approaches applicable for the analysis of data from binary mixture experiments, which are commonly used in pesticide science for evaluating antagonistic or synergistic effects. Specifically, two different situations are reviewed, one where every pesticide is only available at a single dose level and a mixture simply combines these doses, and one where the pesticides and their mixture are used at increasing doses. The former corresponds to using factorial designs whereas the latter corresponds to fixed-ratio designs. We consider dose addition and independent action as references for lack of antagonistic and synergistic effects. Data from factorial designs should be analyzed using two-way analysis of variance models whereas data from fixed-ratio designs should be analyzed using non-linear dose-response analysis. In most cases, independent action seems the more natural choice for factorial designs. In contrast, dose addition is more appropriate for fixed-ratio designs although dose addition is not equally compatible with all types of dose-response data. Fixed-ratio designs should be preferred as they allow validation of the assumed dose-response relationship and, consequently, provide much stronger claims about antagonistic and synergistic effects than factorial designs. Finally, it should be noted that, in any case, simple ways of summarizing pesticide mixture effects may come at the price of more or less restrictive modeling assumptions. © 2021 Society of Chemical Industry.

bmd: an R package for benchmark dose estimation.

The benchmark dose (BMD) methodology is used to derive a hazard characterization measure for risk assessment in toxicology or ecotoxicology. The present paper's objective is to introduce the R extension package bmd, which facilitates the estimation of BMD and the benchmark dose lower limit for a wide range of dose-response models via the popular package drc. It allows using the most current statistical methods for BMD estimation, including model averaging. The package bmd can be used for BMD estimation for binomial, continuous, and count data in a simple set up or from complex hierarchical designs and is introduced using four examples. While there are other stand-alone software solutions available to estimate BMDs, the package bmd facilitates easy estimation within the established and flexible statistical environment R. It allows the rapid implementation of available, novel, and future statistical methods and the integration of other statistical analyses.

Dose-Response Analysis Using R.

Dose-response analysis can be carried out using multi-purpose commercial statistical software, but except for a few special cases the analysis easily becomes cumbersome as relevant, non-standard output requires manual programming. The extension package drc for the statistical environment R provides a flexible and versatile infrastructure for dose-response analyses in general. The present version of the package, reflecting extensions and modifications over the last decade, provides a user-friendly interface to specify the model assumptions about the dose-response relationship and comes with a number of extractors for summarizing fitted models and carrying out inference on derived parameters. The aim of the present paper is to provide an overview of state-of-the-art dose-response analysis, both in terms of general concepts that have evolved and matured over the years and by means of concrete examples.

Tolerance of two Bifora radians Bieb populations to ALS inhibitors in winter wheat.

BACKGROUND: Bifora radians, an annual weed in winter wheat, is distributed mainly in the Mediterranean area, Asia Minor and the Caucasus. It infests winter-sown crops of the Central Anatolia and Middle Black Sea regions of Turkey. Field experiments in heavily B. radians-infested fields were conducted over 3 years in Samsun, Turkey, to determine the response of B. radians to ALS-inhibiting herbicides, because growers had complained of a decrease in herbicide effect. RESULTS: The efficacy of ALS inhibitors on a putatively tolerant population sprayed annually with ALS inhibitors and an adjacent allegedly sensitive population was estimated at the ED(50) and ED(90) response levels. The recommended rates of herbicides controlled 90% of the weed (ED(90)) in the sensitive population at the early stage of B. radians development, but not in the tolerant population. The relative potencies (ED(x(tolerant))/ED(x(sensitive))) of herbicides on the two populations were estimated by assuming years as being random effects. The relative potency was on average about 1.7, irrespective of the ED(x) levels. CONCLUSION: Although the relative potencies were not large, they were large enough to be noted by growers. In field experiments it would be important to establish tools demonstrating when farmers recognise loss of herbicide efficacy. There has been no indication of evolution of resistant biotypes so far, but continuous spraying favours biotypes with increased levels of tolerance.

Using a selectivity index to evaluate logarithmic spraying in grass seed crops.

BACKGROUND: Grass seed crops are minor crops that cannot support the development of selective herbicides for grass weed control in grass seed crops. An option is to screen for selective herbicides with the use of logarithmic spraying technology. The aim of this paper is to assess selectivity of various herbicides in grass seed crops by using dose-response curves. RESULTS: Six grass species were subjected to logarithmic spraying with 11 herbicides and with Poa pratensis L. as a weed. The ratio between the doses that caused 10% of damage to the crop and 90% of damage to the weed was used as a selectivity index. Compounds with selectivity indices above 2 can be safely used in a crop. The two ACCase herbicides clodinafop-propargyl and fenoxaprop-P-ethyl and a mixture of the two ALS herbicides mesosulfuron and iodosulfuron could be used selectively to control P. pratensis in Festuca rubra L., although the selectivity indices in no instances were greater than the desired 2.0. CONCLUSION: The logarithmic sprayer can be a rapid screening tool for identifying compounds with favourable selectivity indices. Good experimental design is needed to alleviate rates being systematically distributed and confounded with growth rate and soil fertility gradients.

Functional regression analysis of fluorescence curves.

SUMMARY: Fluorescence curves are useful for monitoring changes in photosynthesis activity. Various summary measures have been used to quantify differences among fluorescence curves corresponding to different treatments, but these approaches may forfeit valuable information. As each individual fluorescence curve is a functional observation, it is natural to consider a functional regression model. The proposed model consists of a nonparametric component capturing the general form of the curves and a semiparametric component describing the differences among treatments and allowing comparisons of treatments. Several graphical model-checking approaches are introduced. Both approximate, asymptotic confidence intervals as well as simulation-based confidence intervals are available. Analysis of data from a crop experiment using the proposed model shows that the salient features in the fluorescence curves are captured adequately. The proposed functional regression model is useful for analysis of high throughput fluorescence curve data from regular monitoring or screening of plant growth.

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.

Monitoring the efficacy and metabolism of phenylcarbamates in sugar beet and black nightshade by chlorophyll fluorescence parameters.

Desmedipham, phenmedipham and a 50% mixture of the two decreased the maximum quantum efficiency of photosystem II (F(v)/F(m)) and the relative changes at the J step (F(vj)) immediately after spraying in both sugar beet and black nightshade grown in the greenhouse. Sugar beet recovered more rapidly from phenmedipham and the mixture than from desmedipham. Desmedipham and the mixture irreversibly affected F(v)/F(m) and F(vj) in black nightshade at much lower doses than in sugar beet. Black nightshade recovered from phenmedipham injury at the highest dose in the first experiment (120 g AI ha(-1)) but not in the second experiment (500 g AI ha(-1)). The dry matter dose-response relationships and the energy pipeline presentation confirmed the same trend. There was a relatively good correlation between F(vj) taken 1 day after spraying and dry matter taken 2 or 3 weeks after spraying. The differential speed of herbicide metabolism between weed and crop plays an important role in herbicide selectivity and can be studied by using appropriate chlorophyll a fluorescence parameters.

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.

Is mixture toxicity measured on a biomarker indicative of what happens on a population level? A study with Lemna minor.

For plants, pigment content has shown to be a remarkably consistent biomarker across chemicals with different modes of action. In this study, we evaluated the use of pigment content as endpoint in binary mixture toxicity studies compared to three growth endpoints on the floating plant Lemna minor. Six binary combinations of six herbicides with different mode of action were used. Data were tested against both the concentration addition (CA) and independent action (IA) reference models. For CA, two statistical approaches were used. The study showed that for some herbicide combinations the mixture toxicity measured on pigment content did not reflect the results measured on plant population growth, emphasizing the importance of measuring growth in parallel with biomarkers. CA explained the data just as well as IA, and the two different statistical models used to test the data in relation to CA showed very similar results.

Is prochloraz a potent synergist across aquatic species? A study on bacteria, daphnia, algae and higher plants.

Fungicides inhibiting the biosynthesis of ergostrol, such as the triazoles and imidazoles, have been shown to enhance the effect of insecticides on birds, mammals and invertebrates in the terrestrial environment. The synergy is proposed to be due to an effect on P450 monooxygenase enzymes active in pesticide metabolism in these organisms. Fungicides often enter the aquatic environment jointly with other pesticides. It is therefore possible that they could act as synergists also in the aquatic environment. In this study we tested the joint effect of the imidazole fungicide prochloraz together with the herbicides acifluorfen, diquat and terbuthylazine, the fungicide azoxystrobin and the insecticides chlorfenvinphos, dimethoate, and pirimicarb on the bacteria Vibrio fischeri (six binary mixtures), the crustacean Daphnia magna (four binary mixtures), the algae Pseudokirchneriella subcapitata (four binary mixtures) and the floating plant Lemna minor (three binary mixtures). All the binary mixtures were evaluated both in relation to the model of concentration addition (CA) and independent action (IA) using isobolograms. The study showed strong synergy in relation to CA between prochloraz and azoxystrobin, diquat and esfenvalerat on D. magna with sums of toxic units for the 50:50% effect mixture ( summation TU(50:50)) as low as 0.25. The mixture with dimethoate was however antagonistic with summation TU(50:50) of 2.04. Four out of the six mixtures testes on V. fisheri showed synergy in relation to CA, but for three of the mixtures the response could be explained by IA. Only the mixture with diquat showed synergy in relation to both IA and CA with summation TU(50:50) around 0.50. There was no significant synergy for any of the combinations tested on the plant and the algae species in relation to CA and only for diquat in the algae-test in relation to IA. Hence, prochloraz does synergise the effect of some pesticides in the aquatic environment, but not consistently across species. The organism most susceptible to synergy by prochloraz in this study was D. magna. Especially the combination with insecticides such as esfenvalerate, where the concentration needed to immobilize 50% of the daphnia was reduced from >3microgL(-1) to less than 0.5microgL(-1) when prochloraz was added, could be problematic as these concentrations are environmentally realistic. Furthermore, insecticides and ergostrol-biosynthesis-inhibitors (EBI-fungicides) are often applied together, and are therefore likely to co-exist in surface waters, enhancing the problem of the already very potent insecticides.

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.

The toxicity of herbicides to non-target aquatic plants and algae: assessment of predictive factors and hazard.

Widely used herbicides sometimes inadvertently contaminate surface waters. In this study we evaluate the toxicity of herbicides to aquatic plants and algae and relate it to environmental herbicide concentrations and exposure scenarios, herbicide formulation and mode of action. This was done experimentally for ten herbicides, using the aquatic macrophyte Lemna minor L. and the green alga Pseudokirchneriella subcapitata (Korshikov) Hindak, supplemented with a database study comprising algae toxicity data for 146 herbicides. The laboratory study showed that herbicide formulations in general did not enhance herbicide efficacy in the aquatic environment. The Roundup formulation of glyphosate proved to be the only exception, decreasing the EC(50) of the technical product for both L. minor and P. subcapitata approximately fourfold. Comparison of the sensitivity of L. minor and P. subcapitata revealed up to 1000-fold higher sensitivity of L. minor for the herbicides categorized as weak acids (pK(a) < 5), emphasizing the importance of higher plants in hazard assessment. Database analyses showed that no herbicide group, categorized by site of action, was significantly more toxic than another. Synthetic auxins were the exception as they are virtually non-toxic to unicellular algae. There was no strong correlation between toxicity to algae and K(ow) of the herbicides, not even within groups having the same site of action. Evaluating all data, few herbicides were toxic at concentrations below 1 microg l(-1), which is the 99.9th percentile of the herbicide concentrations measured in the Danish surveillance programme. Joint action of several herbicides cannot however be excluded.

Can the choice of endpoint lead to contradictory results of mixture-toxicity experiments?

Theoretically, the effect of two independently acting compounds in a mixture will depend on the slope of the dose-response curves of the individual compounds if evaluated in relation to the model of concentration addition (CA). In the present study, we explored development of the shape of the dose-response relationship for four different recommended endpoints (surface area, frond number, fresh weight-specific, and dry weight-specific relative growth rates [RGRA, RGRF, RGRFW, and RGRDW, respectively]) and for two differently acting herbicides (metsulfuron-methyl and terbuthylazine) over time (3-15 d) on the standard test plant Lemna minor to identify endpoints and experiment times for which predictions of independent action (IA) would depart the most from those of CA. After a test time of 6 d, predictions of IA based on RGRA and RGRFW showed antagonism in relation to CA. Based on RGRDW, synergy was predicted, whereas IA based on RGRF was indistinguishable from CA. To test the prediction of choice of endpoint giving different results in mixture-toxicity experiments, three endpoints and six combinations of independently acting herbicides were evaluated using isobolograms. The experiments showed that in four of six herbicide combinations, different conclusions were reached depending on endpoint. The contradictory isoboles did not follow the theory of IA and, therefore, are more likely to be related to differences in susceptibility of the physiological processes affecting each endpoint than to the shape of the dose-response curve.

Species-specific sensitivity of aquatic macrophytes towards two herbicides.

The s-triazine herbicide terbutylazine, an inhibitor of photosystem II, is often found in surface waters in concentrations < 1 microg L(-1), but concentrations up to 13 microg L(-1) have been measured. To study the effect on the aquatic flora, we tested the sensitivity of 10 aquatic macrophyte species and a natural epiphyte community in a 2-week laboratory multispecies test at constant terbutylazine concentrations and two irradiance regimes. The data were described by a log-logistic concentration-response model and species sensitivity distributions (SSDs) were created from the EC50 and EC10 values. The 5% hazard concentration (HC5) of the EC10-based SSD for terbutylazine was 1 and 3 microg L(-1); hence the low chronic terbutylazine concentrations measured in the environment are not likely to affect the macrophyte community. To compare the species sensitivity between different groups of herbicides, SSDs were constructed from a published study on the sulfonylurea metsulfuron-methyl, an inhibitor of acetolactate synthase. There was no correlation between species-specific sensitivity to the two herbicides; hence, the combined exposure of different herbicides might affect the macrophyte community more broadly rather than seriously affecting a few susceptible species. Evaluating the standard procedure of leaving at least a factor of 100 between the EC50 of standard tests on Lemna sp. and the predicted environmental concentration seems to be protective for at least 95% of the macrophyte species for both terbutylazine and metsulfuron-methyl.

Sensitivity of aquatic plants to the herbicide metsulfuron-methyl.

The sensitivity of 12 aquatic plant species to the herbicide metsulfuron-methyl was tested in microcosm experiments under two growth conditions. As reference species, barley (Hordeum vulgare) and oil-seed rape (Brassica napus) were grown with their roots submerged in the microcosms. Two response variables were chosen: relative growth rate and specific leaf area (SLA). SLA was the most sensitive response variable, with 11 of the 12 aquatic species responding to the herbicide. EC(50) values varied 56-fold between species, with the commonly used aquatic test species Lemna minor being one of the most sensitive. Fast-growing species with a small exposed leaf area proved to be more sensitive to the herbicide than slow growing species with a large exposed leaf area, which was believed to be primarily due to variations in growth rates rather than to variations in exposed leaf area. The aquatic plants displayed high tolerance in growth to metsulfuron compared with the sensitive crop oil-seed rape. Hence, possible spray-drift events and leaching of the herbicide applied at agricultural rates are not considered to have a large impact on the growth of the aquatic flora tested.

Linking fluorescence induction curve and biomass in herbicide screening.

A suite of dose-response bioassays with white mustard (Sinapis alba L) and sugar beet (Beta vulgaris L) in the greenhouse and with three herbicides was used to analyse how the fluorescence induction curves (Kautsky curves) were affected by the herbicides. Bentazone, a photosystem II (PSII) inhibitor, completely blocked the normal fluorescence decay after the P-step. In contrast, fluorescence decay was still obvious for flurochloridone, a PDS inhibitor, and glyphosate, an EPSP inhibitor, which indicated that PSII inhibition was incomplete. From the numerous parameters that can be derived from OJIP-steps of the Kautsky curve the relative changes at the J-step [Fvj = (Fm - Fj)/Fm] was selected to be a common response parameter for the herbicides and yielded consistent dose-response relationships. Four hours after treatment, the response Fvj on the doses of bentazone and flurochloridone could be measured. For glyphosate, the changes of the Kautsky curve could similarly be detected 4 h after treatment in sugar beet, but only after 24 hs in S alba. The best prediction of biomass in relation to Fvj was found for bentazone. The experiments were conducted between May and August 2002 and showed that the ambient temperature and solar radiation in the greenhouse could affect dose-response relationships. If the Kautsky curve parameters should be used to predict the outcome of herbicide screening experiments in the greenhouse, where ambient radiation and temperature can only partly be controlled, it is imperative that the chosen fluorescence parameters can be used to predict accurately the resulting biomass used in classical bioassays.

PSII inhibitory activity of resorcinolic lipids from Sorghum bicolor.

Resorcinolic lipids were isolated from the root extracts of Sorghum bicolor and identified as 4,6-dimethoxy-2-[(8'Z,11'Z)-8',11',14'-pentadecatriene]resorcinol (4), 4-methoxy-6-ethoxy-2-[(8'Z,11'Z)-8',11',14'-pentadecatriene]resorcinol (5), and 4-hydroxy-6-ethoxy-2-[(10'Z,13'Z)-10',13',16'-heptadecatriene]resorcinol (6). Compounds 4 and 5 inhibited photosynthetic oxygen evolution (IC50 0.09 and 0.20 microM, respectively). Compound 4 could not be enzymatically converted to a quinone, suggesting that the quinone moiety is not required for its photosystem II inhibitory activity. Compounds 5 and 6 are reported for the first time.

Joint action of phenolic acid mixtures and its significance in allelopathy research.

Although the ecological significance of mixtures of phytotoxins is recognized in research on chemical plant interference (allelopathy), few studies convincingly demonstrate the joint action of phytotoxin mixtures, key to understanding the ecological impact of these materials, using established models from other biological disciplines, e.g. toxicology and pharmacology. Addressing this need, the present study investigates the joint action of the phenolic acids, p-hydroxybenzoic, p-coumaric and ferulic acids on root growth inhibition of perennial ryegrass (Lolium perenne L). The Additive Dose Model (ADM) isobole and estimated concentration of phenolic acid mixture were calculated on ED20, ED50 and ED80 from the dose-response curves for the phenolic acids applied alone or in mixtures of fixed ratios. The binary combination of three selected phenolic acids is generally antagonistic relative to the ADM. No evidence for synergistic activities of phenolic acids in the mixture was noted. Since allelopathic activities in nature are largely due to the presence of several compounds in a mixture, the present study advances understanding of the joint action of binary combination of allelochemicals in a mixture.