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Reactive oxygen species (ROS) are subproducts of the oxidative metabolism known to initiate chain reactions with polyunsaturated fatty acids that generate lipid peroxides (LPO). The objective of this work was to adapt the ferrous oxidation/xylenol orange (FOX) assay to measure LPO in invertebrate tissues i.e.: from polychaeta (Laeonereis acuta) and crab (Chasmagnathus granulata) species. Whole polychaetes were homogenized in methanol 100%, being determined the optimal sample volume and the time required for color development. It was tested five sample volumes (8-30 microl), following color development up to 215 min. Absorbance stabilization was observed after 90 min, being linearly related with sample volume. A similar procedure was adopted for crab tissues (anterior gills, posterior gills, and hepatopancreas). Differences between species and between organs of the same species were observed when analyzed nonspecific absorbance increments after adding the standard cumene hydroperoxide (CHP). In polychaeta and crab anterior gills tissue, absorbance increments were lower (21-25%) than samples without tissue extracts (blanks) that received CHP. In crab posterior gills and hepatopancreas, the nonspecific increment was almost negligible. Correction formulae are given to account for these differences and simplified protocols for each tissue and species are also included. Great differences in the lipid peroxides content was detected between worms (127.05 +/- 19.32 nmoles CHP/g of wet tissue) respect to anterior gills, posterior gills, and hepatopancreas from the crab species (52.65 +/- 3.59, 30.54 +/- 4.73, and 48.51 +/- 8.78 nmoles CHP/g of wet tissue, respectively).

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Biomarkers of exposure and effect of pollutants were analyzed in croakers Micropogonias furnieri (Teleostei: Sciaenidae) captured in winter and summer in a polluted and in a non-polluted site at the Patos Lagoon estuary (Southern Brazil). Catalase and glutathione S-transferase activities (exposure biomarkers) and lipid peroxidation (effect biomarker) were analyzed in liver samples. Other two effect biomarkers were also studied: blood cells DNA damage (through comet assay and micronucleus test) and respiratory burst measurements. In a broad view, results point to an important seasonal variation of the biochemical biomarkers analyzed. However, data obtained clearly indicate that croakers collected in winter at the polluted site were subjected to a level of clastogenic agents sufficient to generate irreversible genetic damages (mutations) and impair the fish immune system.

Copepods (Acartia tonsa) were exposed (48 h) to waterborne, diet-borne (non-Cu-equilibrated and Cu-equilibrated food), and waterborne plus diet-borne Cu in either the absence or the presence of food (diatom Thalassiosira weissflogii). Toxicity tests were run in different salinities (5, 15, and 30 ppt) together with measurements of physicochemical parameters and total and dissolved Cu concentrations in the experimental media. Results show that most of the toxic Cu fraction was in the dissolved phase. In general, Cu toxicity was higher in low (5 ppt) than in high salinity (30 ppt), regardless of the pathway of Cu exposure tested. In the absence of food, data clearly indicate that differences in waterborne Cu toxicity can be explained by changes in water chemistry. However, addition of food (either non-Cu-equilibrated or Cu-equilibrated) to the experimental media protected against acute Cu toxicity in salinities 5 and 15 ppt, suggesting that A. tonsa requires extra energy to cope with the stressful condition imposed by Cu exposure associated with the ionoregulatory requirements in low salinities. For diet-borne exposure, a very high Cu concentration was necessary to precontaminate the diatoms to a level resulting in copepod mortality. Therefore, availability of food exerted a more important positive impact in protecting against acute Cu toxicity than its potential negative impact via contamination resulting in toxicity. Findings indicate the need for incorporation of both salinity and food in a future biotic ligand model (BLM) version for Cu in estuarine and marine waters. In this context, the euryhaline copepod A. tonsa would be a suitable model species with which to perform experiments to validate and calibrate any future saltwater BLM.

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