Bluegill (Lepomis macrochirus) as a Chemical Warfare Agent Respiratory Exposure Model
IAAAM Archive
Scott Willens1; James B. Kelly1; Todd M. Myers1; Kimberly A. Whitten1; Steven I. Baskin1; Mark V. Haley2; Brian A. Logue3
1United States Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD, USA; 2Edgewood Chemical Biological Facility, Aberdeen Proving Ground, MD, USA; 3South Dakota State University, Brookings, SD, USA


Fish are important biomonitor sentinel species for aquatic pollutants. Bluegill (Lepomis macrochirus) were used to establish an in vivo model to characterize respiratory exposure to sodium cyanide and its cysteine conjugate, 2-amino-2-thiazolidine-4-carboxylic acid (ATCA).8 ATCA is a promising biomarker for cyanide exposure because of the greater stability of ATCA and the limitations of direct determination of cyanide. Studies have shown that ATCA is an excitotoxin responsible for spastic paresis and glutamate antagonist-sensitive seizures and hippocampal lesions in mice injected intracerebroventricularly.1,2 The effects of ATCA in fish and its relative toxicity to cyanide are not known. The even temperament of bluegill make them ideally suited for monitoring of respiratory physiology parameters after exposure to cyanide.7 Gill rate, depth, and purge can be determined by amplified, filtered electrical signals generated by opercular movements and recorded by carbon block electrodes, using the real-time automated biomonitoring portable ventilatory unit.

Each experimental run of the fish biomonitor accommodated 2 groups of 8 fish in 16 individual monitoring chambers. Fish were acclimated to the ventilatory chambers for 24 hours, during which time baseline data were recorded. One group served as an unexposed control, and the other was exposed to either cyanide or ATCA at 0.2 (EPA maximum contaminant level for cyanide in drinking water), 0.1 (96-h LC50 for cyanide in bluegill), or 0.05 mg/L (chronic sublethal) dissolved in well water.3,5,6,7 Control fish were used as exposed fish in the subsequent run to reduce the number of animals. Measurements were taken at 15-s intervals and summed into 15-min bins over the 24-h exposure period. An alarm was triggered when 6 of 8 fish in a group displayed a significantly different reaction beyond baseline in gill rate, depth, purge, or total body movement relative to baseline. Periodic water samples were taken to determine cyanide (sodium cyanide exposures only) and ATCA (all exposures) concentrations.

Upon completion of the 24-h exposure period, exposed animals were euthanized with MS-222. The brain, gills, and liver were preserved in 10% neutral buffered formalin and submitted for histopathologic examination (necrosis, inflammation, degeneration). Tissue concentrations of cyanide (sodium cyanide exposures only) and ATCA (all exposures) were also obtained from fresh/frozen samples. Cyanide concentrations in well water and tissues were determined by a new spectrophotometric technique using metmyoglobin, and ATCA concentrations were determined by gas chromatography/mass spectrometry.4

Another component of our study used an automated photobeam activity monitors to record motion tallied over 15-min intervals over a 4-h period. Groups of 8 fish in 20-L tanks were exposed to either cyanide or ATCA at 0.2, 0.1, or 0.05 mg/L for 4 hours. Visual observations were made over a 4-h period including gill rate, depth, and coloration, activity level, posture, grouping, buoyancy, location, flashing, navigation, and mortality.

The data collected by the multiprobe can determine whether or not fish responses are due to the presence of toxicants. Applications for biomonitoring include watershed protection, wastewater treatment plant effluent, or source water for drinking water protection.


1.  Bitner RS, A Kanthansamy, GE Isom, GKW Yim. 1995. Seizures and selective CA-1 hippocampal lesion induced by an excitotoxic cyanide metabolite, 2-iminothiazolidine-4-carboxylic acid. Neurotoxicology. 16, Pp. 115-122.

2.  Bitner RS, GKW Yim, GE Isom. 1997. 2-Iminothizolidine-4-carboxylic acid produces hippocampal CA1 lesions independent of seizure excitation and glutamate receptor activation. Neurotoxicology. 18, Pp. 191-200.

3.  Eisler R, DR Clark Jr., SN Wiemeyer, CJ Henny. 1999. Sodium cyanide hazards to fish and other wildlife from gold mining operations. In: Azcue, J.M. (ed.). Environmental impacts of mining activities: emphasis on mitigation and remedial measures. Springer-Verlag, Berlin. Pp. 55-67.

4.  Logue BA, NP Kirschten, I Petrikovics, MA Moser, GA Rockwood, SI Baskin. Determination of the cyanide metabolite 2-amino-2-thiazoline-4-carboxylic acid in urine and plasma by gas chromatography-mass spectrometry. Journal of Chromatography B. 819, Pp. 237-244.

5.  U.S. Environmental Protection Agency. 1980. Ambient water quality criteria for cyanides. United States Environmental Protection Agency Report. 440, Pp. 1-72.

6.  U.S. Environmental Protection Agency Website. Accessed 2005. Consumer factsheet on: cyanide.

7.  van der Schalie WH, TR Shedd, MW Widder, LM Brennan. 2004. Response characteristics of an aquatic biomonitor used for rapid toxicity detection. Journal of Applied Toxicology. 24, Pp. 387-394.

8.  Xuan R, W Hu, Z Yang. 2003. One-pot synthesis of DL-2-amino-2-thiazoline-4-carboxylic acid. Synthetic Communications. 33, Pp. 1109-1112.

Speaker Information
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Scott Willens
Mystic Aquarium, Mystic, CT, USA

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