Investigating the emerging role of comparative proteomics in the search for new biomarkers of metal contamination under varying abiotic conditions.

This study aims at investigating the potential use of comparative proteomics as a multi-marker approach of metal contamination, taking into account the potential confounding effect of water temperature. The major objective was to identify combinations of proteins specifically responding to a given metal, even if included in a metal mixture. The diagnostic approach was performed via the comparative analysis of protein expression on spot mapping provided by adult males of Gammarus pulex (Amphipoda, Crustacea) respectively exposed to arsenate (As), cadmium (Cd) or a binary mixture of these metals (AsCd) at three realistic temperatures (5, 10 and 15°C). Proteomic expression analysis was performed by Differential in-Gel Electrophoresis (2D-DiGE), and completed by an adapted inferential statistical approach. Combinations of under/over-expressed protein spots discriminated the metal identity. However, none of these spots discriminated both the individual metal effect (As or Cd) and its effect in metal mixture (AsCd) whatever the tested temperature. Some limits of the two-dimensional analysis of protein spot maps in G. pulex have been highlighted: (i) the presence of contaminating peptides and/or abundant "déja-vu" proteins which can mask the responses of other proteins of interest or (ii) the presence of post-translational modifications. An optimization of the experimental design (especially during the sample preparation) has been described for future investigations. This study has also highlighted (i) the importance of precisely identifying the protein spots of interest to avoid erroneous interpretations in terms of action mechanisms of chemicals and (ii) the importance of working under controlled laboratory conditions with a temperature close to 10°C. In such conditions, we have demonstrated a higher impact of As than Cd on the energetic metabolism of Gammarus. This As impact is reduced in AsCd mixture confirming the antagonistic interaction of this binary mixture previously observed on G. pulex mortality at 10°C.

Single and combined effects of cadmium and arsenate in Gammarus pulex (Crustacea, Amphipoda): understanding the links between physiological and behavioural responses.

This study aimed at investigating the individual and interactive effects of cadmium (Cd) and arsenate (AsV) in Gammarus pulex (Crustacea, Amphipoda) through the use of several biomarkers. Individuals were exposed for 240 h to two concentrations of AsV or Cd alone, and all the possible binary mixtures of these concentrations of AsV and Cd in a complete factorial design. The pattern of the biomarkers' responses to Cd and AsV alone or in mixture was similar in Gammarus pulex, even if the response intensity varied depending on the tested conditions. G. pulex responded to contamination with increased mobilization of the detoxification systems [i.e. γ-glutamyl-cystein ligase activity (GCL), reduced glutathione content (GSH) and metallothionein concentrations (MT)]. This response seems to imply changes in energy reserve utilization (total lipids and proteins are used prior to glycogen reserves), but also a possible energy reallocation from locomotion to detoxification processes. The observed increase in lipid peroxidation could be relied to the increasing gammarid mortality, despite the higher mobilization of detoxification systems. Even if the outcome of the complex interactions between AsV and Cd remains difficult to unravel, such studies are critically important for better assessing the effects of stressors on organisms, populations and communities in a multi-contamination context of ecosystems.

Effects of sublethal cadmium exposure on antipredator behavioural and antitoxic responses in the invasive amphipod Dikerogammarus villosus.

Amphipods are recognised as an important component of freshwater ecosystems and are frequently used as an ecotoxicological test species. Despite this double interest, there is still a lack of information concerning toxic impacts on ecologically relevant behaviours. The present study investigated the influence of cadmium (Cd), a non-essential heavy metal, on both antipredator behaviours and antitoxic responses in the invasive amphipod Dikerogammarus villosus under laboratory conditions. Amphipod behaviour (i.e. refuge use, aggregation with conspecifics, exploration and mobility) was recorded following a 4-min test-exposure to 500 µg Cd/L with or without a 24-h Cd pre-exposure and in the presence or absence of a high perceived risk of predation (i.e. water scented by fish predators and injured conspecifics). Following behavioural tests, malondialdehyde (MDA) levels, a biomarker for toxic effect, and energy reserves (i.e. lipid and glycogen contents) were assessed. Cd exposures induced (1) cell damage reflected by high MDA levels, (2) erratic behaviour quantified by decreasing refuge use and exploration, and increasing mobility, and (3) a depletion in energy reserves. No significant differences were observed between 4-min test-exposed and 24-h pre-exposed individuals. Gammarids exposed to Cd had a disturbed perception of the alarm stimuli, reflected by increased time spent outside of refuges and higher mobility compared to gammarids exposed to unpolluted water. Our results suggest that Cd exposure rapidly disrupts the normal behavioural responses of gammarids to alarm substances and alters predator-avoidance strategies, which could have potential impacts on aquatic communities.

Behavioural and physiological responses of Gammarus pulex exposed to cadmium and arsenate at three temperatures: individual and combined effects.

This study aimed at investigating both the individual and combined effects of cadmium (Cd) and arsenate (AsV) on the physiology and behaviour of the Crustacean Gammarus pulex at three temperatures (5, 10 and 15 °C). G. pulex was exposed during 96 h to (i) two [Cd] alone, (ii) two [AsV] alone, and (iii) four combinations of [Cd] and [AsV] to obtain a complete factorial plane. After exposure, survival, [AsV] or [Cd] in body tissues, behavioural (ventilatory and locomotor activities) and physiological responses (iono-regulation of [Na(+)] and [Cl(-)] in haemolymph) were examined. The interactive effects (antagonistic, additive or synergistic) of binary mixtures were evaluated for each tested temperature using a predictive model for the theoretically expected interactive effect of chemicals. In single metal exposure, both the internal metal concentration in body tissues and the mortality rate increased along metallic gradient concentration. Cd alone significantly impaired both [Na(+)] and [Cl(-)] while AsV alone had a weak impact only on [Cl(-)]. The behavioural responses of G. pulex declined with increasing metal concentration suggesting a reallocation of energy from behavioural responses to maintenance functions. The interaction between AsV and Cd was considered as 'additive' for all the tested binary mixtures and temperatures (except for the lowest combination at 10 °C considered as "antagonistic"). In binary mixtures, the decrease in both ventilatory and locomotor activities and the decline in haemolymphatic [Cl(-)] were amplified when respectively compared to those observed with the same concentrations of AsV or Cd alone. However, the presence of AsV decreased the haemolymphatic [Na(+)] loss when G. pulex was exposed to the lowest Cd concentration. Finally, the observed physiological and behavioural effects (except ventilation) in G. pulex exposed to AsV and/or Cd were exacerbated under the highest temperature. The discussion encompasses both the toxicity mechanisms of these metals and their interaction with rising temperature.

Antagonistic toxicity of arsenate and cadmium in a freshwater amphipod (Gammarus pulex).

Because toxicants rarely occur alone in the environment, a major challenge in risk assessment is to address the combined effects of chemicals on aquatic organisms. This work is aimed at investigating the joint toxicity action of binary mixtures of cadmium and arsenate on Gammarus pulex. Individuals were exposed during 240 h to four single arsenate or cadmium concentrations and binary mixtures of these metals according to a complete factorial plane. Observed mortality in binary mixtures was compared to observed mortality in single arsenate or cadmium exposures. In addition, interactive effects (antagonistic, additive or synergistic) were evaluated using a predictive model for the theoretically expected interactive effect of chemicals. For all the tested concentration combinations, we observed an antagonist 'between-metals' interaction on G. pulex mortality. This antagonistic effect was more marked for the lowest than for the highest (i.e. 1502.0 µg(AsV) L(-1) and 28.5 µg(Cd) L(-1)) tested concentrations of individual metals in binary mixtures. Metal concentrations in body tissues were evaluated and were significantly lower in binary mixtures than in single metal exposures at similar concentration, especially for combinations corresponding to the highest concentrations of both metals (1502.0 µg(AsV) L(-1) and 28.5 µg(Cd) L(-1)). Results were discussed in terms of (1) mechanisms of uptake and bioconcentration and (2) relationships between metal concentration in gammarid body and observed toxicity.

Comparison of arsenate and cadmium toxicity in a freshwater amphipod (Gammarus pulex).

Cadmium is largely documented on freshwater organisms while arsenic, especially arsenate, is rarely studied. The kinetic of the LC50s values for both metals was realized on Gammarus pulex. Physiological [i.e. metal concentration in body tissues, bioconcentration factor (BCF)] effects and behavioural responses (via pleopods beats) were investigated after 240-h exposure. Arsenate LC50 value was 100 fold higher than Cd-LC50 value after 240-h exposure, while concentrations in gammarids were similar for both metals at their respective LC50s. BCF decreased with increasing cadmium concentration while BCF remained stable with increasing arsenate concentration. Moreover, BCF was between 148 and 344 times lower for arsenate than cadmium. A significant hypoventilation was observed for cadmium concentrations exceeding or close to the 240h-LC50(Cd), while gammarids hyperventilated for the lowest arsenate concentrations and hypoventilated for the highest arsenate concentrations. We discussed the relationships between potential action mechanisms of these two metals and observed results.