• Introduction
• Objectives
• Methods

Effects of Chlorfenapyr on Adult Birds      

INTRODUCTION

Chlorfenapyr is the first commercial pesticide to be derived from a class of microbially-produced compounds known as halogenated pyrroles.  Synthesized in 1988 from a naturally-produced chlorinated pyrrole, chlorfenapyr (AC 303,630 Technical: 4-bromo-2-(4-chlorophenyl)-1-(ethosymethyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile) is being used in at least 32 countries, including the United States.  Chlorfenapyr is a 'proinsecticide', i.e., it requires activation through metabolism. The parent compound is converted to a metabolite, which functions as an uncoupler of oxidative phosphorylation at mitochondria.The primary, and most toxic, metabolite is the N-dealkylated compound AC 303,268 (Fig. 1).Chlorfenapyr has low volatility and water solubility; is lipophilic; binds strongly to soil particles; and degrades slowly in soil (avg. half life of 1 yr), sediment (avg. half life of 1.1 yr), and water (avg. half life of 0.8 yr). Biological evidence presented by the manufacturer (BASF) indicates that chlorfenapyr is rapidly metabolized and excreted by mammals, birds, and fish; hence, unlikely to bioaccumulate in individual organisms or biomagnify between trophic levels.

Figure 1.  Molecular structure of chlorfenapyr and  the consequence of metabolism.

Emergency exemptions to use chlorfenapyr on cotton have been granted by EPA for at least 11 states since 1995.  In March 2000, the registrant withdrew its application for use on cotton in the U.S. because of a very negative review by EPA.  Critical scientific input from government and non-government scientists and environmental groups, supported an EPA risk assessment that revealed excessive risk to wildlife; the results of laboratory avian toxicity tests were central to the EPA argument.  In January of 2001, technical chlorfenapyr was unconditionally registered with EPA as a pesticide and a formulated product (PYLON) was registered as a miticide for use in commercial greenhouses.  In December 2001, a formulated product (PHANTOM) was conditionally registered as a termiticide.  

Chlorfenapyr has high acute, sub-acute, and chronic (reproductive) toxicity in birds; and poses an acute poisoning hazard to aquatic organisms (definitive data are lacking for chronic effects).  Results of experiments with laboratory mice and rats revealed a number of histological abnormalities, including vacuolation of brain, spinal cord, and optic nerve tissue in mice and vacuolation of spinal nerves and myelin sheath swelling in rats.  As a consequence, the EPA health (human) effects risk characterization contains a recommendation for a developmental neurotoxicity study.  Because the registrant is expected to continue to pursue  registration for outdoor uses, it is important to increase our knowledge of the biological effects of chlorfenapyr on wildlife.

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1. Describe the pathological effects (macro and micro) of chronic exposure to chlorfenapyr. 

2. Identify biochemical responses that can be used to diagnose exposure to chlorfenapyr. 

Adult mallards are being fed diets containing technical or formulated chlorfenapyr in concentrations that cause effects ranging from a moderate incidence of death to no deaths.  The duration of the feeding trial is 10 weeks, beginning in early July and ending in mid-September.  Mallards that die during the study and mallards that are euthanized at study termination will be necropsied and tissues will be removed for histological examination, biochemical evaluation, and chemical analysis for chlorfenapyr and the primary metabolite.  A preliminary study is being performed in 2002 to determine appropriate concentrations in feed, the best tissues for histological examination and chemical analysis, and possible biochemical measures of chlorfenapyr exposure; and to compare the effects of technical and formulated chlorfenapyr.  The primary study will be performed in 2003. 

The preliminary study consists of 55 adult mallards purchased from a commercial source.  Ducks are in elevated outdoor pens, one bird per pen.  Each pen has a food container, flowing water, and shade.  Ducks receive experimental diets consisting of chlorfenapyr mixed into pelleted duck food (Fig. 2).  Mallards receive diets containing either 0 ppm (15 birds), 2 ppm (10), 5 ppm (10), or 10 ppm (10) technical chlorfenapyr; or 5 ppm formulated chlorfenapyr (10).  Samples of mixed diet for all nominal concentrations from the first mixed batch and a later batch are being saved for chemical analysis.  Food consumption is being estimated during weeks 1, 2, 3, 5, 7, and 9 by subtracting feed left in the food container from the food added (Fig. 3). 

Figure 2.  Chlorfenapyr being mixed into the diet.	Figure 3.  Food containers being weighed and feed being added.

Mallards were weighed at study onset and are being weighed at weekly intervals.  Necropsies are performed within 24 hrs of death (Fig. 4).  Tissues being removed for histological examination are the brain, sciatic nerve, spinal cord (neck and lumbar), skeletal muscle, heart, proventriculus, duodenum, liver, kidney, pancreas, and thyroid.  Tissues being removed and retained for possible histological use are ventriculus (gizzard), testes, lung, spleen, thymus, jejunum, ileum, cloaca, and adrenal.  A portion of the liver is being frozen and saved for chemical analysis for chlofenapyr and the primary metabolite.  At termination of the study, all ducks will be euthanized and processed, as were birds that died during the study.   
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Figure 4.  Necropsy of a mallard that died during the study [ back to the top]

Biomarkers have not been identified for the exposure of wildlife to chlorphenapyr.  Because most toxic agents impact proteins, such as enzymes, examination of the suite of proteins present in a tissue or an organism is an attractive approach for identifying a biomarker for chlorphenapyr.  Such protein studies are part of the field called proteomics.  We are using this as a very broad approach to look for changes in the protein composition in selected tissues from chlorphenapyr-treated animals as compared to controls.  Basically, this involves subjecting tissue protein extracts to two-dimensional polyacrylamide gel electrophoresis, followed by visualization and use of a computer program to compare the resulting protein patterns from treated and control animals.  Protein patterns could be used as biomarkers or they might provide leads for specific proteins, such as enzymes, that could serve as biomarkers by direct assay.  If this approach proves unproductive in the preliminary study, we will consider using a suite of assays of enzyme response in the primary study.