OverviewPurposeTo determine unknown contaminants in water samples during an active fish kill.MethodsA combination of solid phase extraction (SPE ), ion trap LC-MS/MS and high-resolution LC-MS. Results An unknown contaminant was uniquely identified as chloro-e6-trimethyl ester, using both LC-ion trap-MS/MS and high-resolution LC-MS. Introduction On July 9, 2011, a significant fish kill (fish kill I) was observed by the Oklahoma Department of Environmental Quality (OKDEQ) in the Red River, near Ketchum's Bluff, Oklahoma. The Red River, with sources in the Texas peninsula, flows for 917 kilometers, between the borders of Oklahoma (OK) and Texas (TX), before flowing into the Mississippi River. During this fish kill, hundreds of large bottom-feeding fish (e.g., catfish and buffalo) were observed dead, distressed, or actively dying. Nearly two months later, on September 14, 2011, another fish kill (Fish Kill II) occurred farther south along the Red River, approximately 80 miles (130 km) downstream from Ketchum's Bluff near Lake Texhoma. Once again, only 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 toxicants traveling further downstream than 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 occurred (Fish Kill III), also near the confluence area of ​​Ketchum's Bluff and Red Creek. And a final fish kill (fish kill IV) occurred on January 31, 2013, in the same watershed, near the confluence of the Red River and Beaver Creek. Environmental samples (e.g. water, sediment and fish) were collected by OKDEQ and United States Envi...... middle of paper ......undinacea”, Phytochemistry Letters 4(2):79-85Fig 1. Chlorine-e6-trimethyl esterFigure 2a. CID MS/MS LC-ITMS: chloro-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 transformation product of ammonolysis (SPE artefact)Fig. 4 Relationship between mass m/z 639 From and time sequence of fish kill Acknowledgments: Slides of dead fish courtesy of OKDEQ. We would like to thank our students Trevor Nance Jr and Matt Ward for their help in preparing the laboratory samples. We would also like to thank OKDEQ (Chris Armstrong) and EPA Region 6 (Rick McMillin) for their patience. Notice: Although this work has been reviewed and approved by EPA for publication, it may not necessarily reflect official Agency policy. Mention of trade names or commercial products does not constitute approval or recommendation by the EPA for use.