Evaluation of the suitability of responses on various organisational levels in terrestrial Oligochaeta to determine species sensitivity relationships

Fourie, Frana (2011-03)

Thesis (PhD)--Stellenbosch University, 2011.

Thesis

ENGLISH ABSTRACT: Species differ in their sensitivities to toxicants and these differences are exploited in ecological risk assessment methods such as species sensitivity distributions (SSDs). The most commonly used endpoints for ecotoxicity testing and thus to generate data for use in SSDs are on the whole-organismal level, and usually include the evaluation of survival and reproduction. However, suborganismal biomarker responses are in many instances more sensitive than these whole-organismal responses. Therefore, this study investigated and compared responses on various biological organisational levels to determine their suitability for use in SSDs. Five terrestrial oligochaete species (earthworms) were selected as model test organisms, and were exposed to a range of concentrations of a well-studied pesticide, copper oxychloride. The investigated responses included survival, biomass change and reproduction on the whole-organismal level. In order to investigate responses on the suborganismal level, cells (coelomocytes) were extracted non-invasively. The spectrophotometric neutral red retention (NRR) assay was used to determine cell survival and the MTT assay to determine mitochondrial metabolic activity of the coelomocytes. The alkaline single cell gel electrophoresis (comet) assay was used to assess DNA integrity in these cells. The amount of Cu taken up by earthworms was also determined and compared to their responses. Species differences were observed in all responses, and EC50 and EC10 values were calculated for the whole-organismal endpoints and used to generate SSDs. From these SSDs, the hazardous concentrations where 5% of all species would be detrimentally affected (HC5) were calculated, which indicated that the most sensitive whole-organismal endpoint was mass change, followed by reproduction and survival. It was found that earthworms avoided feeding on the contaminated substrate in high copper oxychloride concentration exposures. The concentration where this behaviour occurred could be estimated for each species, and an SSD was constructed with these data. The HC5 value indicated that this response is more sensitive than earthworm survival, but less sensitive than the other responses. It was shown that the earthworms regulated their body Cu concentrations in a species-specific manner. This regulation of Cu was reflected in the suborganismal responses, and the species that had taken up the highest amount of Cu was the most sensitive species for all three suborganismal assays. Due to this regulation of Cu, the resulting dose-responses for the suborganismal endpoints did not allow for the calculation of EC50 values in most of the species and such data could thus not be used to generate SSDs. Sufficient EC10 values were however generated to construct SSDs from the results of the NRR and comet assays. The HC5 values obtained from SSDs constructed with EC10 values for both suborganismal and whole-organismal endpoints indicated that the NRR assay was the most sensitive endpoint, followed by both the comet assay and earthworm mass change, and subsequently the other whole-organismal endpoints. In conclusion, the majority of the responses on the various levels of biological organisation investigated during the present study were shown to be suitable to determine species sensitivity relationships in the terrestrial oligochaete species studied.

AFRIKAANSE OPSOMMING: Spesies verskil van mekaar ten opsigte van hulle sensitiwiteit vir toksikante, en hierdie verskille word in ekologiese risikobepalingsmetodes soos spesie-sensitiwiteitsverspreidings (SSVs) gebruik. Die mees algemene eindpunte vir ekotoksisiteitstoetse, en wat dus gebruik word om data te genereer vir SSVs, is op die heelorganismevlak, en sluit gewoonlik die bepaling van oorlewing en voortplanting van die toetsorganismes in. Hierdie eindpunte is egter in die meeste gevalle minder sensitief as suborganismiese biomerker-response. Hierdie studie het dus die response op verskeie vlakke van biologiese organisasie ondersoek en vergelyk om te bepaal of hulle geskik is vir gebruik in SSVs. Vyf terrestriële spesies van die klas Oligochaeta is gekies as toetsorganismes en is blootgestel aan 'n reeks konsentrasies van die goed bestudeerde pestisied koperoksichloried. Die response oorlewing, massaverandering en voortplanting is op die heelorganismevlak ondersoek. Vir die suborganismiese response is selle (selomosiete) met behulp van 'n nie-ingrypende proses vanuit die erdwurms geïsoleer. Die suborganismese toetse wat op hierdie selle gedoen is, was die neutraalrooi-retensietoets (NRR toets) om sel-oorlewing te bepaal, die MTT toets om mitochondriese metabolisme te bepaal en die alkaliese komeettoets om DNS-integriteit te bepaal. Die hoeveelheid Cu wat die erdwurms opgeneem het, is ook bepaal en met hulle response vergelyk. Verskille is tussen die spesies waargeneem vir al die response. Beide EK50 en EK10 waardes is bereken vir die heelorganismiese eindpunte om SSVs te genereer. Vanaf hierdie SSVs kon die gevaarlike konsenstrasie, waar 5% van alle spesies nadelig beïnvloed kan word (GK5), bereken word. Hierdie GK5 waardes het aangedui dat massaverandering die mees sensitiewe heelorganismiese eindpunt was, gevolg deur voortplanting en oorlewing. Die erdwurms het opgehou vreet aan die gekontamineerde substraat by hoë koperoskichloriedkonsentrasies. Die konsentrasie waar hierdie gedrag plaasgevind het kon vir elke spesie vasgestel word, en 'n SSV is met behulp van hierdie data genereer. Hierdie GK5 waarde het aangedui dat hierdie respons meer sensitief was as oorlewing, maar minder sensitief as die ander response. Die erdwurms kon die konsentrasie van Cu in hulle liggame op 'n spesie-spesifieke manier reguleer. Hierdie regulering van interne Cu is weerspieël in die suborganismiese response, waar die spesie wat die meeste Cu opgeneem het, ook die mees sensitiewe was vir al drie suborganismiese toetse. As gevolg van hierdie regulering van Cu en die gevolglike dosis-responsverhoudings, kon EK50-waardes nie vir al die spesies bereken word nie, en dus was daar geen EK50-data beskikbaar om SSVs mee te genereer nie. Genoegsame EK10 waardes kon egter bereken word vir die NRR- en komeettoetse, en gebruik word om SSVs te genereer. Die GK5-waardes wat bereken kon word vanuit die SSVs met EK10 waardes vir beide suborganismese en heelorganismiese response, het aangedui dat die mees sensitiewe eindpunt die NRR toets was, gevolg deur beide die komeettoets en massaverandering van erdwurms, en daarna die ander heelorganismiese eindpunte. Die gevolgtrekking is dat daar aangetoon kon word dat die meerderheid van die response wat gedurende hierdie studie ondersoek is, geskik is om sensitwiteitsverhoudings van hierdie groep spesies te bepaal.

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