Use of neutral red to assess survival of juvenile freshwater mussels (Bivalvia: Unionidae) in bioassays

Abstract

The effectiveness of vital staining for assessing lethal and sublethal responses of juvenile mussels was examined. Neutral red was used to quantify survival of juvenile Villosa iris and Anodonta grandis after exposures to aqueous copper in 24-hour static bioassays. Live juveniles readily incorporated the stain, but dead individuals did not. Variation in stain intensity was associated with behavioral responses, permitting diagnosis of alive, dead, and sublethal responses of juvenile V. iris. The amber coloration of juvenile A. grandis prevented detection of variations in stain intensity, thus allowing only livingversus-dead determinations to be made. Responding to precipitous declines in populations of freshwater mussels (Unionidae), several workers recently conducted laboratory tests to measure sensitivity of juvenile stages to various pollutants (Johnson et al., 1990; Keller & Zam, 1991; Lasee, 1991). Both Johnson et al. (1990) and Keller & Zam (1991) determined post-exposure mortality from observations of internal anatomy, but did not detail any sublethal effects of the exposures. By contrast, Lasee (1991) assessed both post-exposure mortality and sublethal responses by individual inspection of the juvenile mussels. Juveniles were recorded as alive (active and moving), stressed (no foot movement but cilia beating), or dead (no foot or cilia movement). Toxicity tests depend on an accurate assessment of post-exposure condition and are complicated by the small size (<1 mm) of juvenile mussels. Healthy juveniles are typically active, extruding the foot and gaping (opening) their valves. If immobile or ungaped, their condition is not as apparent. Because juveniles of many species possess transparent valves, with visible internal structure, the reduction or absence of movement by the foot or cilia may be used to assess responses. This requires close, individual inspections, and the effort is time-intensive. A more rapid and equally precise means of assessing postexposure condition of juvenile mussels thus was desirable. Vital staining has been used successfully to distinguish living from dead We thank Mr. Lou Rifici and Ms. Lisa Wolcott for assistance in the laboratory with rearing juvenile mussels and vital staining procedures. This research was supported by a grant from the American Electric Power Company through the American Electric Power Service Corporation, Columbus, Ohio 43216, U.S.A. 2 Address: Department of Biological Sciences, Arkansas State University, State University, Arkansas 72467, U.S.A. 3 Address: U.S. Fish and Wildlife Service, Virginia Cooperative Fish and Wildlife Research Unit, Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, U.S.A. TRANS. AM. MICROSC. SOC., 112(1): 78-80. 1993. ? Copyright, 1993, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.220 on Fri, 02 Sep 2016 04:36:20 UTC All use subject to http://about.jstor.org/terms VOL. 112, NO. 1, JANUARY 1993 invertebrates. Dressel et al. (1972) used vital staining to sort copepods, and Crippen & Perrier (1974) used neutral red to determine mortality among marine plankters. Platter-Rieger & Frank (1987) successfully used neutral red to assess post-exposure effects of tributyltin on mussel larvae (Mytilus edulis). They defined three levels of staining: darkly stained (healthy), lightly stained (stressed and inactive), or not stained (dead). They found increasing percentages of lightly stained larvae in treatments with higher toxicant concentrations and assumed that staining intensity was related to the level of stress. The purpose of our investigation was to evaluate the effectiveness of vital staining with neutral red in determining post-exposure survival of juvenile freshwater mussels. Copper was chosen as the toxicant because it is a known molluscicide, highly toxic to invertebrates, and a common pollutant in riverine systems (U.S.E.P.A., 1985; Van Hassel & Gaulke, 1986). MATERIALS AND METHODS Juveniles of Villosa iris (I. Lea, 1829) and Anodonta grandis Say, 1829 were obtained following metamorphosis of glochidia encysted upon largemouth bass, Micropterus salmoides (Lacepede, 1802). We conducted 24-h, static exposures at eight concentrations ranging from approximately 0 to 200 tg Cu/L. Absolute metal concentration was determined by inductively coupled, argon-plasmaemission spectroscopy. We used two replicates of each toxicant concentration for V. iris and three for A. grandis, with 10 juveniles per replicate. Test containers were held in an incubator at 20?C; after exposure, juvenile response was determined by visual inspection and vital staining. Prior to vital staining, we examined juveniles at 12-50 x magnification with a stereomicroscope. Three classes of response were established: (1) gaped and alive, (2) gaped and dead, or (3) ungaped. Dead juveniles were characterized by their rigid, immobile foot and the absence of beating cilia. We stained juveniles after the initial inspection by the technique of Crippen & Perrier (1974) using a 1-h exposure to a 1:100,000 concentration of neutral red in water. Stained juveniles were stored overnight in a refrigerator at approximately 4?C prior to examination. The degree of staining was assessed using a stereomicroscope at 12-20 x magnification. We defined three levels of staining: (1) brightly stained, (2) lightly or partially stained, and (3) unstained. Classes 2 and 3 were combined to yield a total number affected both by visual inspection and vital