In this study, we investigated the establishment of natural bacterial degraders in a sand filter treating groundwater contaminated with the phenoxypropionate herbicides (RS)-2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) and (RS)-2-(2,4-dichlorophenoxy)propanoic acid (DCPP) and the associated impurity/catabolite 4-chlorophenoxypropanoic acid (4-CPP). A pilot facility was set up in a contaminated landfill site. Anaerobic groundwater was pumped up and passed through an aeration basin and subsequently through a rapid sand filter, which is characterized by a short residence time of the water in the filter. For 3 months, the degradation of DCPP, MCPP, and 4-CPP in the sand filter increased to 15 to 30% of the inlet concentration. A significant selection for natural bacterial herbicide degraders also occurred in the sand filter. Using a most-probable-number (MPN) method, we found a steady increase in the number of culturable phenoxypropionate degraders, reaching approximately 5 ؋ 10 5 degraders per g sand by the end of the study. Using a quantitative PCR targeting the two phenoxypropionate degradation genes, rdpA and sdpA, encoding stereospecific dioxygenases, a parallel increase was observed, but with the gene copy numbers being about 2 to 3 log units higher than the MPN. In general, the sdpA gene was more abundant than the rdpA gene, and the establishment of a significant population of bacteria harboring sdpA occurred faster than the establishment of an rdpA gene-carrying population. The identities of the specific herbicide degraders in the sand filter were assessed by Illumina MiSeq sequencing of 16S rRNA genes from sand filter samples and from selected MPN plate wells. We propose a list of potential degrader bacteria involved in herbicide degradation, including representatives belonging to the Comamonadaceae and Sphingomonadales.G roundwater is a valuable resource that is used for drinking water in many countries all over the world. The quality of the water is vital to our health, and regulations have been made to control the concentration of contaminants and protect the groundwater (1). A major concern is leaching of pesticides mainly from agricultural areas but also from urban areas or from point sources (2). In the European Union, a legal threshold limit has been set at 0.1 g/liter for any given pesticide or 0.5 g/liter for a total concentration of mixed pesticides in drinking water (3).In Denmark, monitoring of the groundwater status has detected pesticides in almost 40% of the investigated wells, and the concentrations exceed the allowed threshold limit in 12% of the wells (4). Phenoxypropionate herbicides, including the compounds (RS)-2-(2,4-dichlorophenoxy)propanoic acid (known as DCPP or dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy)propanoic acid (known as MCPP or mecoprop), are among the pesticides frequently found in contaminated wells (4). The herbicides are, in particular, a problem in old landfill areas, from where they are leaching into the groundwater as a point source contamination (5). Phe...