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In this study, long-term effects of Ni, a widespread heavy metal in the aquatic ecosystems, have been determined on growth and lethality of the clam Ruditapes philippinarum, a known bioindicator of the marine environment. Three/four-month-old bivalves have been exposed to different concentrations of Ni dissolved in synthetic seawater. Growth and lethality as endpoints after 28 days of treatment have been observed. Obtained results are the following: EC = 3.97 ± 0.94 and 9.45 ± 1.59 mg/L and NOEC = 1.56 and 6.25 mg/L for growth and mortality, respectively. Moreover, this study can be considered a new tool for the evaluation of fitness of bivalve clam, together with other biological responses following to the biological impacts of metal pollution.

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Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/ brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded 1,2 . Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes 3 . The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation 4 . This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol. Video LinkThe video component of this article can be found at http://www.jove.com/video/3790/ Protocol Method StandardOur testing involves exposing Artemia larvae to an interval of substance concentrations or a test sample for the purpose of determining the concentration or dilution levels that would cause the death of 50% of the organisms (LC 50 ) exposed over 14 days and meeting the conditions defined by this method. If necessary and possible, the following may also be determined: a) the concentration level that causes the death of 20 % of organisms exposed (LC 20 ); b) the highest analyzed concentration level that does not determine a higher mortality rate than that of the negative control (NOEC); the lowest tested concentration level that determines, after 14 days, a higher mortality rate than that of the negative control (LOEC).

The availability of standardized protocols for both organism culture and bioassay with ecologically relevant species is of great concern in ecotoxicology. Acartia tonsa represents an important, often dominant, member of zooplankton communities and meets all the practical criteria suggested for model species. New standardized procedures for laboratory culturing of the copepod A. tonsa and standardized methods for acute (24- and 48-h) and semichronic (7-d, static-renewal) toxicity tests with the nauplius stage are described. In both cases, eggs are the starting stage, and nauplius immobilization is the endpoint. The methods were the object of an intercomparison test involving nine laboratories, and nickel was the reference toxicant. Relative reproducibility was 24, 25, and 34% for 24-h, 48-h, and 7-d tests, respectively.

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Acute heavy metal toxicity was compared in nauplii of an autochthonous Artemia parthenogenetica (Branchiopoda, Anostraca) from Saline di Cervia (Ravenna, Italy) and of a non native commercially available A. franciscana. No significant difference in sensitivity was detected between two species.