The sublethal biochemical actions of copper in live, intact red abalone (Haliotis rufescens) were characterized by in vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy. This non-invasive technique is ideal for examining cellular respiration since critical metabolite concentrations, including phosphoarginine ([PA]), inorganic phosphate ([Pi]) and [ATP], and the arginine kinase (AK) rate constant, can be monitored in real time. Both metabolite concentrations and enzyme rate constants were measured in abalone during 8-h exposures to 66 mg L^-1 (1.04 mM) and 126 mg L^-1 (1.98 mM) copper (as CuCl₂). Significant decreases in [PA] and corresponding increases in [Pi] resulted, while [ATP] remained constant. In controls [PA], [Pi] and [ATP] all remained unchanged. Furthermore, both copper concentrations induced a significant elevation in the forward AK rate constant over the basal value of 0.020 ± 0.002 s^-1.

Metabolite levels and enzyme rate constants were also measured during 8-h, 66 mg L^-1 copper exposures both before and after a 2 week subchronic exposure to 36 mg L^-1 (0.57 mM) copper. Unlike before the subchronic exposure, no significant changes in [PA], [Pi] or [ATP] were observed after the 36 mg L^-1 copper treatment, compared to controls. This induced tolerance was also evident from the forward AK rate constant data. Finally, copper accumulation was determined in gill, digestive gland and foot muscle samples. Whereas acute exposure only led to significant accumulation in the gill, copper levels in subchronically-exposed abalone were significantly elevated in both the gill and digestive gland, and marginally so in foot muscle.

Overall, the gill appears to be the primary site of copper accumulation and toxicity, while the foot and adductor muscles maybe secondarily impacted. The observed metabolic changes may result from insufficient oxygen delivery to the muscles, resulting from mucus accumulation or cytological damage at the gill. In conclusion, abalone acutely exposed to copper pollution may develop asphyxial hypoxia. Since their survival is dependent upon adherence to rock surfaces, such a reduction of muscle function could ultimately prove fatal.

The sublethal actions of the piscicide, 3-trifluoromethyl-4-nitrophenol, were characterized in two marine molluscs using in vivo ³¹P nuclear magnetic resonance spectroscopy. This non-invasive technique is ideal for examining cellular respiratory toxicity since inorganic phosphate, phosphoarginine, and adenosine 5'-triphosphate levels, and the arginine kinase rate constant, can be measured. These parameters were characterized in both red abalone (Haliotis rufescens) and owl limpets (Lottia gigantea) during 5-h exposures to 3 mg L-1 (14.5 mM) 3-trifluoromethyl-4-nitrophenol, followed by 5-h recovery periods. In exposed abalone, phosphoarginine decreased by 50%, inorganic phosphate increased by 900%, and the arginine kinase rate constant quadrupled - all compared to controls and consistent with an uncoupling of oxidative phosphorylation. Limpets were less severely impacted, showing no change in phosphoarginine, an increase of only 200% in inorganic phosphate, and an approximate doubling of the arginine kinase rate constant. Adenosine 5'-triphosphate levels remained constant in all control and exposed molluscs. In addition, at the cessation of dosing, all biochemical parameters in abalone returned to pre-exposure levels within 3-4 h, consistent with rapid depuration, while the limpet recovery data was inconclusive. We suggest that the owl limpet's greater tolerance towards the pesticide could result from an increased capability for anaerobic glycolysis, which is required to survive the periodic anoxia associated with residing higher in the intertidal zone. Thus, the natural histories of non-target organisms should be considered when determining the effects of pesticide exposure.

In vivo nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for characterizing the sublethal actions of physical and chemical stressors in live, intact organisms. In particular, ³¹P NMR is ideal for observing perturbations to cellular energetics since critical metabolite concentrations, including phosphagens, ATP, and inorganic phosphate, can be measured both non-invasively and in real time. This technique's versatility will be demonstrated using recent studies of diverse species, ranging from adult marine molluscs to embryonic fish.

Spectra of relatively large organisms, including red abalone (Haliotis rufescens) and sea anemone (Anthopleura elegantissima) were obtained using a wide-bore Biospec-7T spectrometer and a custom-built NMR probe. A 10x7.5x3.75 cm fluoropolymer animal chamber, housed within the probe, received a continuous flow of temperature-controlled, aerated seawater to which toxicants could be added. Illustrative studies will include the actions of copper in abalone, and the effects of physical stressors on sea anemone energetics. Spectra of larvae and embryos can be obtained using a high-field Avance 500 spectrometer with commercial NMR probes and a novel flow-through perfusion system. Results from a study of medaka embryo (Oryzias latipes) energetics, including decreases in phosphomonoesters and phosphodiesters concurrent with increases in phosphocreatine and ATP throughout development, will be presented.