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

Abstract

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.

Figure 1. Experimental design and mean real concentrations of arsenate and/or cadmium tested during the experiments.

Values (µg L⁻¹± s.d.) were measured by graphite furnace atomic - absorption spectrophotometry (AAS Varian SpectrAA-330, detection limits: 2 µg_As L⁻¹ and 0.1 µg_Cd L⁻¹) or by flame atomic – absorption spectrophotometry (AAS PERKIN ELMER AAnalyst 100, detection limits: 20 µg_Cd L⁻¹) Mean real concentrations were based on n = 12 for control and n = 36 for all the combined “temperature x tested conditions”.

Figure 2. Mean mortality (%) in Gammarus pulex exposed to different conditions at three temperatures (5-10-15°C).

The different conditions were “control”, “arsenate”, “cadmium” and four binary mixtures. a) Temperature × condition effect: Mean mortality for each tested condition and temperature. Vertical bars represent standard deviations. b) Condition effect: Weighted mean mortality for each tested condition (results for all the tested temperatures were combined). Vertical bars represent 0.95 confidence intervals. c) Temperature effect: Weighted mean mortality for each temperature (results for all the tested conditions were combined). Vertical bars represent 0.95 confidence intervals. Letters (a to g) were used as labels to illustrate significant differences in mean mortality values (two-way ANOVAs + LSD Fisher post hoc tests; at p<0.05 level of significance).

Figure 3. Comparison of mean mortality (± s.d.) between ‘theoretically expected’ and ‘observed’ combined effects.

Mean mortality obtained for different cadmium/arsenate binary mixtures in Gammarus pulex after 96-hour exposure. An asterisk indicated a significant difference between the observed and expected values for a given binary mixture (Student-t test, p<0.05).

Figure 4. Mean arsenate concentrations in G. pulex body tissues (µg. g dry weight⁻¹).

Concentrations obtained for individuals exposed 96 h to different conditions (control, arsenate, cadmium and four binary mixtures) at three temperatures (5-10-15°C). a) Temperature × condition effect: Mean internal arsenate concentration in G. pulex for each tested condition and temperature. Vertical bars represent standard deviations. b) Condition effect: Weighted mean concentration of internal arsenate for each tested condition (results for all the tested temperatures were combined). Vertical bars represent 0.95 confidence intervals. c) Temperature effect: Weighted mean concentration of internal arsenate concentrations for each temperature (results for all the tested conditions were combined). Vertical bars represent 0.95 confidence intervals. Letters (a to f) were used as labels to illustrate significant differences in mean [AsV] values (two-way ANOVAs + LSD Fisher post hoc tests; at p<0.05 level of significance).

Figure 5. Mean cadmium concentrations in G. pulex body tissues (µg. g dry weight⁻¹).

Concentrations obtained for individuals exposed 96 h to different conditions (control, arsenate, cadmium and four binary mixtures) at three temperatures (5-10-15°C). a) Temperature × condition effect . Mean internal Cd concentrations in G. pulex for each tested condition and temperature. Vertical bars represent standard deviations. b) Condition effect: Weighted mean concentration of internal Cd for each tested condition (results for all the tested temperatures were combined). Vertical bars represent 0.95 confidence intervals. c) Temperature effect: Weighted mean concentration of internal Cd for each temperature (results for all the tested conditions were combined). Vertical bars represent 0.95 confidence intervals. Letters (a to g) were used as labels to illustrate significant differences in mean [Cd] values (two-way ANOVAs + LSD Fisher post hoc tests; at p<0.05 level of significance).

Figure 6. Mean mobility of Gammarus pulex (in %) exposed to different conditions.

Mobility obtained for individuals exposed 96 h to different conditions (control, arsenate, cadmium and four binary mixtures) at three temperatures (5-10-15°C). a) Temperature × condition effect: Mean mobility (%) for each tested condition and temperature. Vertical bars represent standard deviations. b) Condition effect: Weighted mean locomotor activity for each tested condition (results for all the tested temperatures were combined). Vertical bars represent 0.95 confidence intervals. c) Temperature effect: Weighted mean locomotor activity for each temperature (results for all the tested conditions were combined). Vertical bars represent 0.95 confidence intervals. Letters (a to q) were used as labels to illustrate significant differences in mean mobility values (two-way ANOVAs + LSD Fisher post hoc tests; at p<0.05 level of significance).

Figure 7. Mean pleopod beat frequency (MPBF) of Gammarus pulex.

PBF obtained for individuals exposed 96 h to different conditions (control, arsenate, cadmium and four binary mixtures) at three temperatures (5-10-15°C). a) Temperature × condition effect: Mean MPBF for each tested condition and temperature. Vertical bars represent standard deviations. b) Condition effect: Weighted mean MPBF for each tested condition (results for all the tested temperatures were combined). Vertical bars represent 0.95 confidence intervals. c) Temperature effect: Weighted mean MPBF for each temperature (results for all the tested conditions were combined). Vertical bars represent 0.95 confidence intervals. Letters (a to l) were used as labels to illustrate significant differences in MPBF values (two-ways ANOVA + LSD Fisher post hoc tests; at p<0.05 level of significance).

Figure 8. Mean concentrations of haemolymphatic [Na⁺](mmol L⁻¹) in Gammarus pulex.

Concentrations obtained for individuals exposed 96 h to different conditions (control, arsenate, cadmium and four binary mixtures) at three temperatures (5-10-15°C). a) Temperature × condition effect: Mean haemolymphatic [Na⁺] for each tested condition and temperature. Vertical bars represent standard deviations. b) Condition effect: Weighted mean haemolymphatic [Na⁺]for each tested condition (results for all the tested temperatures were combined). Vertical bars represent 0.95 confidence intervals. c) Temperature effect: Weighted mean haemolymphatic [Na⁺] for each temperature (results for all the tested conditions were combined). Vertical bars represent 0.95 confidence intervals. Letters (a to e) were used as labels to illustrate significant differences in values of mean haemolymphatic [Na⁺] (two-way ANOVAs + LSD Fisher post hoc tests; at p<0.05 level of significance).

Figure 9. Mean concentrations of haemolymphatic [Cl⁻](mmol L⁻¹) in Gammarus pulex.

Concentrations obtained for individuals exposed 96 h to different conditions (control, arsenate, cadmium and four binary mixtures) at three temperatures (5-10-15°C). a) Temperature × condition effect: Mean haemolymphatic [Cl⁻] for each tested condition and temperature. Vertical bars represent standard deviations. b) Condition effect: Weighted mean haemolymphatic [Cl⁻] for each tested condition (results for all the tested temperatures were combined). Vertical bars represent 0.95 confidence intervals. c) Temperature effect: Weighted mean haemolymphatic [Cl⁻] for each temperature (results for all the tested temperatures were combined). Vertical bars represent 0.95 confidence intervals. Letters (a to g) were used as labels to illustrate significant differences in values of mean haemolymphatic [Cl⁻] (two-way ANOVAs + LSD Fisher post hoc tests; at p<0.05 level of significance).

Figure 10. Global physiological model of individual and interactive effects of temperature, arsenate and cadmium in Gammarus.

A dotted line represents a lower effect than a full one.