Pest Management Science Pest Manag Sci 62:515–521 (2006) An easy and rapid method using microscopy to determine herbicide effects in Poaceae weed species Maibritt Hjorth, 1,2 Laurence Mondolot, 2 Bruno Buatois, 3 Claude Andary, 2 Sylvie Rapior, 2,3 Per Kudsk, 4 Solvejg K Mathiassen 4 and Helle W Ravn 1∗ 1 Department of Terrestrial Ecology, National Environmental Research Institute, 25 Vejlsøvej, PO Box 314, Silkeborg, Denmark 2 Laboratory of Botany, Phytochemistry and Mycology, CEFE-CNRS UMR 5175, Faculty of Pharmacy, University of Montpellier 1, 15 Avenue Charles-Flahault, F-34093 Montpellier Cedex 5, France 3 Plateforme d’Analyses Chimiques en Ecologie, CEFE-CNRS UMR 5175, 1919 Route de Mende, F-34293 Montpellier Cedex 5, France 4 Department of Integrated Pest Management, Danish Institute of Agricultural Sciences, Flakkebjerg, DK-4200 Slagelse, Denmark Abstract: A new, easy, rapid and relatively inexpensive method using microscopy has been developed for the detection of herbicide effects in leaves of grass weed species displaying no visual signs of damage. The method has potential to be used as a tool to indicate future death of grass species due to herbicide exposure by observing phytochemical effects, i.e. early-warning effects. In the present study, Apera spica-venti (L.) Beauv., Bromus hordeaceus L., Alopecurus myosuroides Huds., Lolium perenne L. and Poa annua L. were exposed to lethal rates of four herbicides with different mode of action. The herbicides investigated were the thiocarbamate: prosulfocarb, the sulfonylurea: iodosulfuron, the aryloxyphenoxypropionate: fenoxaprop-P-ethyl and the organophosphate glyphosate. Autofluorescence of leaves was studied under a microscope using ultraviolet and blue light. The fluorescence of leaves treated to enhance flavonoids was also examined. To confirm the results, microspectrofluorometry was performed. Effects indicating future death of the grasses were observed in visually healthy leaves following treatment with prosulfocarb, glyphosate and iodosulfuron. No changes were detected following treatment with fenoxaprop-P-ethyl. After exposure to glyphosate or iodosulfuron, changes in the content of flavonoids and other compounds with a conjugation system and rigid structure and a decrease in the content of chlorophyll were detected in the leaves. Prosulfocarb treatment resulted in changes in the content of flavonoids and other compounds with a conjugation system and rigid structure and an increase in the content of chlorophyll in the leaves. The results obtained from microspectrofluorometry indicated that exposure to prosulfocarb caused a reduction in the flavonoids quercetin, naringenin and/or naringin.  2006 Society of Chemical Industry Keywords: herbicide metabolism; flavonoids; chlorophyll; microspectrofluorometry; early warning 1 INTRODUCTION In the Poaceae family, 44% of the plant species are among those termed the worst weeds of the world. 1 These include loose silkybent (Apera spica- venti (L.) Beauv.), soft brome (Bromus hordeaceus L.), blackgrass (Alopecurus myosuroides Huds.), perennial ryegrass (Lolium perenne L.) and annual bluegrass (Poa annua L.), which are common in crops in northwestern Europe. In order to control Poaceae weed species in crops, a wide range of herbicides are available. Iodosulfuron, prosulfocarb and fenoxaprop-P-ethyl are selective herbicides that are recommended for control of certain grass weed species, while glyphosate is a non-selective herbicide used for total weed control on non-cropped areas or in transgene glyphosate- resistant crops. The four herbicides represent different modes of action. Iodosulfuron is a sulfonylurea and inhibits the synthesis of branched chain amino acids. 2 Prosulfocarb is a thiocarbamate and inhibits the synthesis of long-chain fatty acids. 3 Fenoxaprop-P- ethyl is an aryloxyphenoxypropionate and inhibits fatty acid synthesis, 4 while the organophosphate glyphosate inhibits the synthesis of aromatic amino acids. 5 The phytochemical changes in composition and content due to exposure are therefore expected to be different for the four herbicides. Phytochemical changes in Poaceae due to herbicide exposure appear before visual signs are observed on the plants in the field. 6 Detection of phytochemical changes can consequently be used to show early- warning signals indicating future death of plants. A wide range of analytical methods is available to detect phytochemical changes, such as chro- matographic techniques coupled online with various detectors. 7–10 These methods are time-consuming ∗ Correspondence to: Helle W Ravn, Department of Terrestrial Ecology, National Environmental Research Institute, 25 Vejlsøvej, PO Box 314, Silkeborg, Denmark E-mail: her@dmu.dk Contract/grant sponsor: Danish Veterinary and Food Administration (Received 11 March 2005; revised version received 2 August 2005; accepted 10 November 2005) Published online 20 April 2006; DOI: 10.1002/ps.1194  2006 Society of Chemical Industry. Pest Manag Sci 1526–498X/2006/$30.00