OverviewObjectiveTo determine unknown contaminants in water samples during active fish kill.MethodsA combination solid phase extraction (SPE), ion trap LC-MS/ MS and high resolution LC-MS. Results An unknown contaminant was uniquely identified as chlorine-e6-trimethyl ester, using at both ion trap LC-MS/MS and high resolution LC-MS.IntroductionOn July 9, 2011, a significant fish kill (fish kill I) was observed by the Department of Environmental Quality of the Oklahoma (OKDEQ) in the Red River near Ketchum's Bluff, Oklahoma. The Red River, which rises in the Texas Panhandle, flows 917 kilometers between the borders of Oklahoma (OK) and Texas (TX) before emptying into the Mississippi River. During this fish hunt, hundreds of large bottom-feeding fish (i.e. catfish and buffalo) were observed dead, struggling, or dying. Nearly two months later, on September 14, 2011, another fish kill (Fish Kill II) occurred further south along the Red River, approximately 80 miles (130 km) downstream from Ketchum's Bluff near Lake Texhoma. . Again, hundreds of large bottom-feeding fish were observed to be affected by one or more unknown toxins. OKDEQ believed the two fish kills were related, with the unknown toxicant(s) moving further downstream from the first fish kill (July 9, 2011), but causing fish mortality 60 days later downstream. The following year, on June 13, 2012, another fish kill (Fish Kill III) occurred, again near the area of ​​the confluence of Ketchum's Bluff and Red Creek. And a final fish kill IV occurred on January 31, 2013, in the same watershed, near the confluence of the Red River and Beaver Creek. Environmental samples (i.e. water, sediment, and fish) were collected by OKDEQ and US Envi...... middle of paper ......undinacea", Phytochemistry Letters 4(2):79-85Fig. 1. Chlorine-e6-trimethyl esterFigure 2a. CID MS/MS LC-ITMS: Chlorine-e6-trimethyl ester standard, m/z 639.3 (M+H) +2b. CID MS/MS LC-ITMS Unknown m/z 639.3 (M+H)+Fig. 3 Probable ammonolysis transformation product (SPE artifact)Fig. timeline of fish mortalityAcknowledgments: Dead fish slides courtesy of OKDEQ We would like to thank our students Trevor Nance Jr and Matt Ward for their assistance in preparing the laboratory samples. We would also like to thank OKDEQ (. Chris Armstrong) and EPA Region 6 (Rick McMillin) for their patience Note: Although this work has been reviewed and approved by EPA for publication, it does not necessarily reflect official Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the EPA..