Comparing the sensitivity of five earthworm species to cadmium exposure using the comet assay

Fourie, Frana (2006-04)

Thesis (MSc)--University of Stellenbosch, 2006.

Thesis

ENGLISH ABSTRACT: It is known that species differ in their sensitivities to toxicants. This has been exploited to aid in environmental toxicity testing and environmental management. "Sensitivity" in this sense is usually seen as a function of lethality; assessed by determining the toxicant concentration where 50% of the test cohort die. The processes at the sub-organismal or sublethal level are however ignored. Little has been done to combine such sub lethal sensitivities with the concept of species sensitivity differences. The present study therefore focused on the potential use of a cellular biomarker to compare the sensitivities of species. The heavy metal cadmium, which bio-accumulates, is teratogenic, mutagenic and carcinogenic was chosen as toxicant. Earthworms were chosen as experimental animals and species were selected to represent various ecological types that may occur in soils. The species studied were representative of three ecological types: epigeic (Amynthas diffringens, Dendrodrilus rubidus and Eisenia fetida), endogeic (Aporrectodea caliginosa) and anecic (Microchaetus benhami). The alkaline single cell gel electrophoresis assay (SCGE or comet assay), which measures DNA integrity in individual cells, was used as biomarker. Earthworms were exposed to a range of Cd concentrations (2.5, 5, 10 and 20 mg/I Cd) in the form of CdS04, in artificial soil water. A negative control (uncontaminated soil water) and a positive control, nickel (20 mg/I Ni) in the form of NiS04 were used. Exposure to cadmium induced significantly higher levels of DNA damage in the exposed worms than in those exposed to negative controls. Species differed from each other in their sensitivity to Cd. The most sensitive species was E. fetida followed by D. rubidus, A. caliginosa, A. diffringens and M benhami. Ecological type did not predict sensitivity, and it is concluded that physiology and possibly relatedness may provide a possible explanation. All species exhibited a pattern where DNA damage was inhibited at low Cd exposure concentrations, and was increased again at high Cd concentrations. This corresponds with the hormetic dose-response, where a compensatory response is stimulated by low levels of a toxicant, but inhibited at high levels. Two possible compensatory mechanisms are proposed. Firstly, DNA repair could have been upregulated at low Cd concentrations, and inhibited by high Cd concentrations. Secondly, the production of metal-binding metallothioneins, which sequestrate Cd and renders it unavailable to cause toxic responses, could have been increased with low Cd concentrations. At high Cd concentrations, the rate of metallothionein production would not have been high enough to sequestrate Cd before it could cause damage. The exposed earthworms accumulated Cd, but there was no definite relationship between Cd body loads and DNA damage. It is possible that a fraction of the measured Cd in the body was sequestrated, therefore not being available to cause genotoxic effects. It is concluded that the comet assay is a useful biomarker to demonstrate DNA damage and species sensitivity differences in earthworms exposed to cadmium.

AFRIKAANSE OPSOMMING: Dit is bekend dat spesies van mekaar verskil ten opsigte van hul sensitiwiteit vir toksiese stowwe. Hierdie eienskap word gebruik in toksisiteitstoetse en die bestuur van die vrystelling van moontlike toksiese stowwe in die omgewing. Toetse wat die sensitiwiteit van spesies vergelyk word meestal gedoen deur die konsentrasie van 'n toksiese stof te bepaal waar 50% van die toetsorganismes doodgaan. Die prosesse wat op die sub-organismiese vlak inwerk word egter ge"ignoreer. Baie min studies is gedoen waar hierdie sub-letale sensitiwiteit gekombineer word met spesies-sensitiwiteitsverskille. Kadmium is tydens die huidige studie as toksikant gebruik. Kadmium kan in organismes akkumuleer, dit is karsinogenies, veroorsaak mutasies en be"invloed ontwikkeling en groei. Erdwurms was die gekose toetsorganismes, en spesies wat verskillende ekologiese groepe verteenwoordig is geselekteer. Hierdie spesies het drie ekologiese groepe verteenwoordig: Epige"is (Amynthas diffringens, Dendrodrilus rubidus en Eisenia fetida), endoge"is, (Aporrectodea caliginosa) en anesies (Microchaetus benhami). Die komeet-toets (alkaliese-enkelsel-gelelektroforese- toets), waarmee die DNS-integriteit in indiwiduele selle bepaal kan word, is as biomerker gekies. Erdwurms is blootgestel aan 'n reeks kadmiumkonsentrasies (2.5, 5 10 en 20 mg/I Cd) in die vorm van CdS04 in kunsmatige grondwater. 'n Negatiewe kontrole (kunsmatige grondwater sander kadmium) is gebruik, asook 'n positiewe kontrole, nikkel (20 mg/I Ni) in die vorm van NiS04. Erdwurms wat aan kadmium blootgestel is, het hoer vlakke van DNS-skade getoon as die erdwurms in die negatiewe kontroles. Spesies het verskil van mekaar ten opsigte van hulle sensitiwiteit vir kadmium. Die mees sensitiewe spesie was E. fetida, gevolg deur D. rubidus, A. caliginosa, A. diffringens en M benhami. Die ekologiese groepering het nie 'n invloed gehad op die spesie-sensitiwiteitsverskille nie, maar die moontlikheid bestaan dat fisiologiese verskille en selfs filogenetiese verwantskappe dalk 'n rol kan speel om hierdie verskille te verklaar. 'n Patroon waar DNS-skade onderdruk is by Jae kadmium-konsentrasies, en weer hoer was by hoe konsentrasies, kon waargeneem word in al die spesies. Hierdie verskynsel kan moontlik verklaar word deur die teenwoordigheid van 'n kompenseringsreaksie, soos beskryf kan word deur die hormesiese dosis-respons. In die geval van hormesis word 'n kompenseringsreaksie gestimuleer deur Jae konsentrasies van 'n toksikant. By hoe konsenstrasies word hierdie reaksie weer ge"inhibeer. In die geval van die huidige studie word twee moontlike kompenseringsmeganismes voorgestel: eerstens bestaan die moontlikheid dat die herstel van DNS-skade toegeneem het met lae kadmiumkonsentrasies. By hoe konsentrasies is hierdie herstelproses ge'inhibeer. Tweedens kon lae kadmiumkonsentrasies die verhoogde produksie van metaalbindende metallothione'iene stimuleer. Met hoe konsentrasies sou die koers van metallothione'ien-produksie nie hoog genoeg gewees het om al, of meeste van, die kadmium te bind voordat dit skade kon veroorsaak nie. Die blootgestelde erdwurms het kadmium geakkumuleer, maar geen besliste verwantskap het voorgekom tussen vlakke van kadmium in die erdwurms en DNS-skade nie. Dit is egter moontlik dat die gemete kadmium in die erdwurms gesekwestreer is en dus nie beskikbaar was om DNSskade te veroorsaak nie. Die gevolgtrekking kan gemaak word dat die komeettoets 'n bruikbare biomerker is om DNSskade, asook spesies-sensitiwiteitsverskille te toon in erdwurms wat aan kadmium blootgestel is.

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