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The determination of metals in aquatic organisms has become an important tool in ecotoxicological studies. In the present study, silver and copper were determined for the first time in the haemolymph of Parhyale hawaiensis, a millimetric marine crustacean. Silver (Ag) was selected because this metal has become of environmental concern due its intense use in nanomaterials. Copper (Cu) was included in this study as a possible biological internal standard. The procedures for haemolymph collection, quantification, and preparation were optimized. The adult organisms were exposed to silver in concentrations from 0 to 100 μg L-1 in a saline water medium for 96 hours. For quantification and comparison of the method, GFAAS and ICP-MS analysis was used and all operational conditions were optimized. It was found that the Ag concentrations in the haemolymph increased in relation to the increase in Ag concentration in the water, reaching 11 ng mg-1. However, a plateau was observed at the two highest concentrations. More studies are required to verify the usefulness of copper as an internal standard. Both GFAAS and ICP-MS provided satisfactory results in the quantification of Ag and Cu in the haemolymph of these small animals and are an excellent tool for ecotoxicological studies, especially for the evaluation of silver from nanomaterials in the aquatic environment. with marine amphipods is still scarce, although a large quantity of effluents containing toxic substances is constantly discharged directly and indirectly into the estuarine and marine regions (5, 6). The marine amphipod Parhyale hawaiensis is an epibenthic small organism (less than 2 cm), lives in the rocky shores with tidal fluctuations associated with algae and other organisms. It is a versatile model organism amenable to experimental manipulations (7), easy to cultivate in the laboratory, and tolerates wide variations in salinity and temperature (8). Several authors have evaluated the metal concentrations in the whole body of amphipods (9–14), but not in haemolymph, probably due to the difficulties to collect it from these tiny animals. For example, an average male of an adult Parhyale hawaiensis reaches no more than 2 cm and weighs less than 0.008 g. The determination of metals in the whole body can be sufficient for some studies, but to prove that a contaminant was in fact absorbed or for understanding its toxicokinetics, internal concentration studies are required (15–17). Evaluation of the haemolymph in amphipods is an interesting tool to help in evaluating metal exposure, such as silver. Today, the most important and applicable property of silver is its antimicrobial broad spectrum (18, 19) and one of the main reasons for its widespread use in nanomaterials (19–21). As a result of its many INTRODUCTION The determination of metals in the tissues and fluids of aquatic organisms is an important tool for ecotoxicological studies. The haemolymph is the circulatory fluid of various invertebrates and is functionally comparable to the blood and lymph of vertebrates. It fills the interior of arthropods and molluscs in an open circulatory system (1). When a contaminant is absorbed by an aquatic organism and distributed to the different organs through the haemolymph it is possible to measure the concentrations. This type of study has been performed in relatively large animals such as decapod crustaceans, e.g., the freshwater crayfish Cambarus diogenes (weight from 19 to 39 g) (2) and fishes, e.g., the common carp Cyprinus carpio (weight of 28.1 ± 12.6 g) (3), but not in smaller aquatic organisms. Amphipods are invertebrates that live close to the sediment, thus they are an interest group for studies of metal contamination and exposure. The analysis of fluids and organs of this group is still a challenge due to their minimal size. They are appropriate for ecotoxicological studies because they are abundant, widespread, ecologically relevant, and an important link between the producers and the secondary consumers in the food chain (4). The number of studies

GFAAS and ICP-MS Determination of Ag and Cu in the Haemolymph of a Millimetric Marine Crustacean (Parhyale hawaiensis) as a Tool in Ecotoxicology