A.D. Correia

Age-related changes in antioxidant enzyme activities, fatty acid composition and lipid peroxidation in whole body gammarus locusta (crustacea: amphipoda)

The main aim of this work was to provide baseline data on aspects of pro-oxidant and antioxidant processes in different life-stages of the marine amphipod Gammarus locusta. The activities of antioxidant enzymes and levels of lipid peroxidation were determined in whole body juveniles, subadults, and male and female adults of a laboratory population of G. locusta. Fatty acid composition of individuals at these different stages of development was also characterised in order to examine the contribution that polyunsaturated fatty acids (PUFA) might make to the peroxidation status of animals. The antioxidant enzymes, measured in whole body 100,000 supernatants, comprised catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD; EC 1.15.1.1) and glutathione peroxidase (GPX; EC 1.11.1.9). Fatty acids were analysed as fatty acid methyl esters (FAME). Lipid peroxidation was examined in terms of the levels of lipid peroxides determined as thiobarbituric acid-reactive malondialdehyde equivalents. Age-related changes were seen in antioxidant enzyme status: levels of SOD (p<0.01) and GPX (p<0.001) activities decreased progressively during development from juveniles to adults. Sex-related changes in GPX activity were also seen, the levels being higher in adult males than females (p<0.001). The amount of FAME present in whole body amphipod also changed over the life span. Among PUFA, the eicosapentaenoic (C20:5n-3), arachidonic (C20:4n-6) and docosahexaenoic (C22:6n-3) were the most abundant acids in this species, and both their individual concentrations and total PUFA increased progressively with age (up to 3.3-fold; p<0.001). The latter changes may contribute to the explanation of the observed differences in peroxidation status of the animals with age; thus, levels of lipid peroxides increased up to 40% in adult males compared to other age-classes (p<0.01). Overall, the decline in antioxidant enzyme activities, coupled with increased levels of PUFA, as the individual grows older, may render the older animals more susceptible to lipid peroxidation and oxidative stress.

Effects of water-borne copper on metallothionein and lipid peroxidation in the marine amphipod Gammarus locusta.

The aim of the current work was to determine over 10 days the effects of water-borne exposure of the marine amphipod Gammarus locusta to 4 microgCu l(-1) on the metallothionein (MT; measured by differential pulse polarography) protection system and lipid peroxidation (LP: thiobarbituric acid-reactive malondialdehyde equivalents) as a measure of oxidative damage. MT levels in exposed animals increased significantly at day 2 (36% > control; P < 0.001) and remained high at days 6 and 10 (55 and 38%, respectively, P < 0.001). The maximum level of MT at day 6 coincided with the highest Cu body-burden. LP increased within I day of exposure, indicative of Cu as an oxidative stressor. However, in contrast to MT, the highest LP level was seen at day 4 (68% > control, P < 0.001) before returning to control values by day 6, indicating a protective role of MT against the pro-oxidant effects of Cu.

Effects of sediment geochemical properties on the toxicity of copper-spiked sediments to the marine amphipod Gammarus locusta.

The contribution of some sediment features, namely acid-volatile sulfide (AVS) and total organic carbon (TOC) in order to explain the toxicity of copper-spiked sediments was investigated. A 10-day assay with the marine amphipod Gammarus locusta was performed with three selected sediment types that consisted of different proportions of fine fractions (FF) and TOC (0.5% FF/1% TOC; 25% FF/2% TOC; and 75% FF/4% TOC). Copper toxic effects were observed for 0.5% FF/25% FF sediments when simultaneously extracted copper (SEM(Cu)) minus acid volatile sulfide (SEM(Cu) - AVS) were 0.3 and 3.4 micromol/g, respectively. However, no significant mortality was observed for 75% FF sediment even when the sulfide binding capacity was exceeded (1.1 micromol/g < or = SEM(Cu) - AVS < or = 8.9 micromol/g). These results indicate that mortality could not be described solely as a function of the SEM(Cu) - AVS, suggesting the presence of other binding phases in addition to AVS. Results of the normalized LC50 values to total organic carbon indicated that this component probably also interferes in the amphipod survival, although not completely. The estimated LC50 values were close (2 mg Cu/g C and 8 mg Cu/g C, respectively for 0% FF and 25% FF sediment) although there was still a fourfold difference between them. Considering that none of these sediment features by itself could fully explain copper toxicity, an attempt was made to describe G. locusta mortality as a function of simultaneous SEM(Cu) - AVS and copper concentrations normalized to TOC. Nevertheless, the limited set of data available from this study prevented an attempt to model toxicity as a function of both of these factors. These findings reinforce the need to develop integrated models that include more than one binding phase, sulfide and organic carbon, and also other compartments of the system (e.g. interstitial water) to improve current methodologies for predicting copper toxicity based on sediment geochemical features.

Studies on biomarkers of copper exposure and toxicity in the marine amphipod Gammarus locusta (Crustacea): I. Copper-containing granules within the midgut gland

Sub-lethal exposure of the marine amphipod Gammarus locusta to a range of copper concentrations (Cu) in water and spiked sediments were performed, and the resulting bioaccumulation of Cu into intracellular granules investigated. The presence of Cu-granules was demonstrated histochemically by the rubeanic acid method. The granules were quantified by automated image analyses (expressed as volume fraction, V v ). The metal composition of the granules was characterized by X-ray microprobe analysis. The results showed that granules, rich in copper and sulphur, were formed in response to Cu exposure in water and sediment. These granules appeared in the B-cells of the hepatopancreas. V v values increased over the dose-range of Cu compared with control in water (P<0.001) and sediment (P<0.01) exposures. The abundance of the granules also increased with increasing whole-body Cu content, suggesting that at least part of the increasing Cu level was incorporated into the granules, as a strategy for Cu detoxification along with normal storage of Cu during the moult cycle. The presence of sulphur within the granules is thought to represent an organic detoxification mechanism for Cu. The formation of Cu-granules as a cellular response is a useful biomarker of Cu-exposure in ecotoxicological studies with amphipods. The rubeanic acid method is a useful screening tool for this copper.

QUANTIFICATION OF METALLOTHIONEIN IN WHOLE BODY GAMMARUS LOCUSTA (CRUSTACEA: AMPHIPODA) USING DIFFERENTIAL PULSE POLAROGRAPHY

A polarographic method was optimised for the quantitative determination of metallothionein (MT) in whole body Gammarus locusta (Crustacea: Amphipoda). The identity of the MT-like proteins was examined using electrophoretic analysis (SDS-PAGE) and induction studies (water-borne exposures to copper). The MT-method was then used to examine changes in MT levels over the lifetime (juveniles, sub-adults and adults) of a laboratory population of G. locusta; copper (Cu) and zinc (Zn) body burden were also measured by flame atomic absorption spectrometry. MT was quantified by heat-denaturation of 30 000 g supernatants (to remove the bulk of non-MT proteins) followed by differential pulse polarography (DPP) to measure sulphydryl-group containing proteins. DPP proved to be a sensitive and reliable method to quantify heat-stable total sulphydryl-proteins. The majority of the latter was indicated to be MT from SDS-PAGE and induction studies. SDS-PAGE revealed a putative MT of 23 ± 0.6 kDa app. mol. wt., consistent with the average size of dimeric MTs (10–20 kDa) found in several aquatic invertebrate species. Copper-exposure revealed parallel increases in the amounts of 23 kDa protein and total sulphydryl-proteins. Levels of whole body MT (rabbit pure MT as standard) range between 1.3 and 2.3 mg g−1 dry wt. over the lifetime of G. locusta. Sex-related changes in MT status were observed, the levels in females being 1.6 times higher than in males (p<0.05). Copper and Zn body content changed over the lifetime of G. locusta, the levels in juveniles (p>0.05) and sub-adults (p<0.05) being higher than in adults. It is concluded that the inclusion of MT in ecotoxicological studies with amphipod crustaceans require some caution with respect to the stages of moulting and/or sexual maturation of organisms, but this should not provide any major impediment in its use in metal pollution monitoring.

Functional anatomy of the midgut gland of Gammarus locusta (Crustacea: Amphipoda)

A description of the midgut gland of Gammarus locusta with the aim of using this organ as a complementary tool in ecotoxicological studies was made. The hepatopancreas is composed of two pairs of blind-ending tubular structures. The thickness and length of these tubules were ∼50 μm and 5 mm, respectively, in adult males (10-12 mm length). The distal part of each tubule is characterized by non-vacuolated E-cells typically exhibiting a relatively high nuclear to cytoplasmatic ratio. The epithelium of the remaining tubules is characterized by abundant columnar R- and F-cells, both ∼40 μm long. Large vacuolated B-cells (∼60 μm length) were very common along the midgut gland but seemed to increase in number in the proximal region of each tubule. An SEM examination revealed a delicate network of muscle fibres that limits the organ.