Stress responses of Eisenia andrei and Enchytraeus doerjesi (Oligochaeta) to combined effects of temperature and metal contamination

Otomo, Patricks Voua (2011-03)

Thesis (PhD)--Stellenbosch University, 2011.

Thesis

ENGLISH ABSTRACT: The terrestrial Oligochaete species Eisenia andrei and Enchytraeus doerjesi were exposed to different concentration series of Cd and Zn, both separately and in mixtures for 28 days in artificial OECD soil at 15, 20 and 25°C. At the end of the four week exposure period, survival and reproduction were assessed in E. doerjesi and survival, reproduction, biomass change, metal uptake and biomarker responses (MTT and comet assays) in E. andrei. Survival results for both E. andrei and E. doerjesi indicated that the lethality of Cd increased at higher temperatures, whereas the opposite was observed for Zn.Cadmium LC50 values were the highest at 15°C and the lowest at 25°C. In the Zn exposures LC50 increased with increasing temperature. Mixture results in both test organisms indicated that mixtures were less lethal than the metals separately. Effects of mixtures on survival, nonetheless, increased with increasing temperature. In the Cd experiment, reproduction in E. andrei was only recorded in the control treatments at the three temperatures investigated. However, the deleterious effect of Zn on the reproduction of both E. andrei and E. doerjesi decreased with increasing temperature and Zn EC50 for reproduction increased with increasing temperature. Results for exposures to mixtures indicated in both test organisms that the interaction between Cd and Zn were antagonistic. In both E. andrei and E. doerjesi, the effect of mixture exposures on reproduction decreased with increasing temperatures. The highest mixture EC50 values for reproduction were found at higher temperature. In E. doerjesi (using reproduction results in MixToxModules) Cd and Zn interactions were dose level dependent at the three temperatures investigated. Antagonism was the predominant interaction at lower mixture concentrations whereas synergism occurred at mixture concentrations equal to or higher than the mixtures’ EC50 values. Biomass loss increased with increasing temperature in the Cd exposures (p 9 0.05) but not in the Zn exposures in E. andrei. In this species mixture results indicated antagonistic interactions between Cd and Zn at all temperatures investigated. The deleterious effect of mixtures on the biomass of E. andrei increased with increasing temperature. When Cd and Zn interactions were further investigated in E. andrei (using biomass results in MixToxModules) it was found that they were dose level dependent at the three temperatures investigated. Antagonism was the predominant interaction at lower mixture concentrations whereas synergism occurred at mixture concentrations higher than the mixtures EC50 values. The assessment of metal uptake in E. andrei revealed a temperature dependent Cd uptake with higher Cd body burdens occurring at higher exposure concentrations and temperatures (p ≤ 0.05). In the case of Zn, although uptake was lower at higher temperature, there was no statistical difference in uptake between exposure concentrations and between temperatures. Mixture results however indicated that in mixture exposures less Cd was accumulated by E. andrei than in single Cd exposures (p ≤ 0.05). Inversely, in mixture exposures more Zn was accumulated by E. andrei than in single Zn exposures (p ≤ 0.05). Biomarker studies revealed that Cd and Zn were both cytotoxic and genotoxic whether in single or mixture exposures. Factorial ANOVA analyses of the effects of temperature and metals on the reduction of MTT by E. andrei indicated that temperature rather than the metals was the most important factor controlling mitochondrial activity (p < 0.001). In both Cd and Zn exposures significant deleterious metal effects on mitochondrial processes were found to increase with temperature (p ≤ 0.01). Mixture exposures indicated decreasing cytotoxicity with increasing temperature (p ≤ 0.05) and possible antagonism between Cd and Zn at cellular level. Results of the comet assay showed that the genotoxic profile of Cd was the opposite of the genotoxic profile of Zn. Cd was less genotoxic at lower temperature and increasingly deleterious at higher temperature while Zn was more genotoxic at lower than higher temperature (p ≤ 0.05). The results of mixture exposures indicated decreasing mixture genotoxicity with increasing temperature and suggested that the interactions between Cd and Zn at molecular level were probably antagonistic.

AFRIKAANSE OPSOMMING: Eisenia andrei en Enchytraeus doerjesi is aan verskillende konsentrasiereekse van Cd en Zn, afsonderlik en in mengsels, vir 28 dae in OECD kunsmatige grond onderskeidelik by 15, 20 en 25°C blootgestel Die volgende eindpunte is aan die einde van die vier weke blootstellingsperiode gemeet: oorlewing en voortplanting (by E. doerjesi) en oorlewing, voortplanting, biomassaverandering, metaalopname, MTT en komeettoetse (by E. andrei). Oorlewingsresultate by beide E. andrei en E. doerjesi het getoon dat toenemende temperatuur die letale toksisiteit van Cd laat toeneem terwyl die teenoorgestelde waar was vir Zn. By die Cd blootstellings was die LK50 waardes die hoogste by 15°C en die laagste by 25°C. By die blootstellings aan Zn het die LK50 waardes toegeneem by hoër temperatuur. Resultate by die mengsels by beide toestspesies het aangetoon dat die mengsels minder letaal was as die afsonderlike metale. Effekte van mengsels op oorlewing het nietemin toegeneem met toenemende temperatuur. By die Cd blootstellings is voortplanting slegs by die kontroles en in die geval van E. andrei by die drie onderskeie temperature ondersoek. Die nadelige uitwerking van Zn op voortplanting by beide E. andrei en E. doerjesi het afgeneem met stygende temperatuur en die Zn LK50 vir voortplanting het toegeneem met toenemende temperatuur. Resultate van die blootstellings aan mengsels het getoon dat die wisselwerking tussen Cd en Zn by beide spesies antagonisties was. By beide spesies het die invloed van die mengsels op voortplanting afgeneem met stygende temperatuur. Die hoogste mengsel LK50 waardes vir voortplanting is by hoër temperature gevind. By E. doerjesi was Cd en Zn wisselwerkings by blootstelling aan mengsels (voortplantingsresultate ondersoek deur van MixToxModules gebruik te maak) dosisvlak verwant by die drie temperature wat ondersoek is. Antagonisme was die oorwegende wisselwerking by laer mengsel konsentrasies terwyl sinergisme voorgekom het by mengsel konsentrasies gelyk aan of hoër as die LK50 waardes van die mengsels. In die geval van die Cd blootstellings by E. andrei het biomassaverlies toegeneem met toenemende temperatuur (p ≤ 0.05) maar nie by die Zn blootstellings nie. Resultate van blootstellings aan mengsels het getoon dat die uitwerking van mengsels op die biomassa van E. fetida toegeneem het met toenemende temperatuur. By die verdere ondersoek van Cd en Zn wisselwerkings, waar gekyk is na dosis verhouding of dosisvlak antagonisme (deur van biomassa resultate in MixToxModules gebruik te maak), is gevind dat Cd en Zn wisselwerkings dosisvlak afhanklik was by die drie temperature wat ondersoek is. Antagonisme was die oorwegende wisselwerking by laer mengselkonsentrasies terwyl sinergisme voorgekom het by mengselkonsentrasies hoër as die mengsel EK50 konsentrasies. Die bepaling van metaalopname deur E. andrei het ’n temperatuurafhanklike opname van Cd getoon met hoër Cd liggaamskonsentrasies by hoër blootstellingskonsentrasies en temperature (p ≤ 0.05). Alhoewel Zn opname laer was by hoër temperatuur was daar geen statisties betekenisvolle verskille in opname tussen blootstellingskonsentrasies of temperature nie. Die bepaling van metaalopname by wurms wat aan mengsels blootgestel is, het getoon dat minder Cd deur E. andrei opgeneem is as waneer die wurms aan Cd as enkelmetaal blootgestel is (p ≤ 0.05), Daarteenoor het die teenoorgestelde gebeur in die geval van Zn, Meer van die metaal is opgeneem wanneer E. andrei aan mengsels blootgestel is as aan die enkelmetaal. Biomerkerstudie het getoon dat Cd en Zn beide sito- en genotoksies kan wees ongeag of dit as enkelmetale of in mengsels toegedien is. Faktoriale ANOVA analises van die effekte van temperatuur en metale op die verlaging van MTT by E. andrei het getoon dat temperatuur ’n belangriker faktor was as metaalbesoedeling by die kontrole van mitochondriale aktiwiteit. (p ≤ 0.001). By beide Cd en Zn blootstellings was daar in elk geval statisties betekenisvolle metaaleffekte op mitochondriale prosesse met toename in temperatuur (P ≤ 0.01). By blootstellings aan mengsels is gevind dat sitotoksisiteit afgeneem het met toenemende temperatuur (p ≤ 0.05) asook ’n moontlike antagonisme tussen Cd en Zn op sellulêre vlak. Resultate van die komeettoets het getoon dat die genotoksiese profiel van Cd die teenoorgestelde was as die van Zn. Cd was minder genotoksies by laer temperature en meer en meer skadelik by hoër temperature terwyl Zn meer genotoksies was by laer as by hoër temperature (p ≤ 0.05). Die resultate van blootstlling aan mengsels het laer genotoksisiteit getoon met toename in temperatuur. Dit dui daarop dat wisselwerkings tussen Cd en Zn op molekulêre vlak moontlik antagonisties was.

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