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Abstract Background In order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe. Results Our studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (~μm), single point (~1 mm2) and macroscopic (~8 × 6 mm2 of a leaf). Multiphoton excited fluorescence lifetime imaging of Triticum aestivum leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC—a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 ± 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 ± 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620–710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For all these in vivo plant autofluorescence lifetime measurements, the plants were not dark-adapted. Conclusions We demonstrate that the local impact of a photosystem II herbicide on living plant leaves can be conveniently mapped in space and time via changes in autofluorescence lifetime, which we attribute to changes in chlorophyll fluorescence. Using portable fibre-optic probe instrumentation originally designed for label-free biomedical applications, this capability could be deployed outside the laboratory for monitoring the distribution of herbicides in growing plants.

Toxicokinetic and toxicodynamic models are a promising tool to address the effects of time-variable chemical exposure. Standard toxicity tests usually rely on static concentrations, but these chemical exposure patterns are unlikely to appear in the field, where time-variable exposure of chemicals is typical. In the present study, toxicodynamic processes were integrated into an existing model that includes the toxicokinetics and growth of the aquatic plant Myriophyllum spicatum, to predict the impact on plant growth of 2 iofensulfuron short-term exposure patterns. To establish a method that can be used with standard data from risk assessments, the toxicodynamics of iofensulfuron were based on effect data from a 14-d standard toxicity test using static concentrations. Modeling showed that the toxicokinetic and toxicodynamic growth model of M. spicatum can be successfully used to predict effects of short-term iofensulfuron exposure by using effect data from a standard toxicity test. A general approach is presented, in which time-variable chemical exposures can be evaluated more realistically without conducting additional toxicity studies., (© 2015 SETAC.)

Effects of chemicals are, in most cases, caused by internal concentrations within organisms which rely on uptake and elimination kinetics. These processes might be key components for assessing the effects of time-variable exposure of chemicals which regularly occur in aquatic systems. However, the knowledge of toxicokinetic patterns caused by time-variable exposure is limited, and gaining such information is complex. In this work, a previously developed mechanistic growth model of Myriophyllum spicatum is coupled with a newly developed toxicokinetic part, providing a model that is able to predict uptake and elimination of chemicals, as well as distribution processes between plant compartments (leaves, stems, roots) of M. spicatum. It is shown, that toxicokinetic patterns, at least for most of the investigated chemicals, can be calculated in agreement with experimental observations, by only calibrating two chemical- specific parameters, the cuticular permeability and a plant/water partition coefficient. Through the model-based determination of the cuticular permeabilities of Isoproturon, Iofensulfuron, Fluridone, Imazamox and Penoxsulam, their toxicokinetic pattern can be described with the model approach. For the use of the model for predicting toxicokinetics of other chemicals, where experimental data is not available, equations are presented that are based on the log (P oct/wat) of a chemical and estimate parameters that are necessary to run the model. In general, a method is presented to analyze time-variable exposure of chemicals more in detail without conducting time and labour intensive experiments., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Laboratory toxicity tests are a key component of the aquatic risk assessments of chemicals. Toxicity tests with Myriophyllum spicatum are conducted based on working procedures that provide detailed instructions on how to set up the experiment, e.g., which experimental design is necessary to get reproducible and thus comparable results. Approved working procedures are established by analyzing numerous toxicity tests to find a compromise between practical reasons (e.g., acceptable ranges of ambient conditions as they cannot be kept completely constant) and the ability for detecting growth alterations. However, the benefit of each step of a working procedure, e.g., the random repositioning of test beakers, cannot be exactly quantified, although this information might be useful to evaluate working procedures. In this paper, a growth model of M. spicatum was developed and used to assess the impact of temperature and light fluctuations within the standardized setup. It was analyzed how important it is to randomly reassign the location of each plant during laboratory tests to keep differences between the relative growth rates of individual plants low. Moreover, two examples are presented on how modeling can give insight into toxicity testing. Results showed that randomly repositioning of individual plants during an experiment can compensate for fluctuations of light and temperature. A method is presented on how models can be used to improve experimental designs and to quantify their benefits by predicting growth responses.

The suitability of two in vitro oxidation systems as chemical models for the biological degradation of plant protecting agents has been investigated. As representative herbicides diclofop, fenoxaprop, isoproturon, linuron and monolinuron have been oxidised by two systems, the Fentons' reagent and the ascorbic acid oxidation system (AAOS) and the results compared to those of the known metabolic pathways of these compounds. The herbicides have been oxidised by Fentons' reagent (hydroxy radicals). The main products were isolated by preparative scale HPLC and identified with (1)H-NMR and MS. Some of the products have been identified by comparing their retention times and UV/Vis-spectra to those of standard compounds. Several products known from biological degradation are also found after chemical oxidation, however, notable differences between the two pathways have been observed, for instance in the case of diclofop. Oxidation by the AAOS leads to comparable results. Reaction rates for the oxidation with the AAOS have been studied and compared with data known from degradation studies of the herbicides in soil. Compounds which are slowly degraded in soil are oxidised more slowly in the biomimetic process than those with a fast degradation in soil.