Doctor of Philosophy
Department of Chemistry
Angel, Brad M, Trace metal behaviour in an industrialised estuarine system and the toxicity of pulsed copper exposures, PhD thesis, Department of Chemistry, University of Wollongong, 2009. http://ro.uow.edu.au/theses/798
The concentrations of metal contaminants may fluctuate in estuarine waters due to the erratic nature of sources and various physico-chemical parameters that influence concentrations. Standard toxicity tests use continuous contaminant exposure to assess organism toxicity even though organisms may respond differently when exposed to fluctuating concentrations. An investigation was made of the spatial distribution and short-term temporal fluctuation trace metals in an industrialised estuarine system and the influence of short-term fluctuations in metal concentrations on the toxic effects elicited to aquatic organisms. Copper was determined to be the metal of greatest concern due to the elevation of this metal above concentrations representative of regional waters and because copper is known to be relatively toxic to aquatic organisms. The toxic effects and mechanisms of toxicity in a marine algae and amphipod elicited by pulsed copper exposures were thoroughly investigated in laboratory bioassays.
Spatial and temporal sampling and analysis of trace metals in waters and sediments was undertaken in the highly industrialised central Queensland harbour of Port Curtis, Australia, and surrounding waters. The trace metals measured in Port Curtis were significantly higher (p
Various aspects of pulsed copper exposure (concentration and duration, the duration between pulses, and the frequency and timing of pulses, delayed toxicity, and recovery following pulses) were investigated using new bioassay procedures that were developed in the laboratory specifically for the marine amphipod, Melita plumulosa and the marine alga, Phaeodactylum tricornutum. Mortality and biomass inhibition endpoints wereused for M plumulosa and P. tricornutum, respectively. The pulses were generated by spiking with dissolved copper sulfate into seawater and terminated by replacing the copper-spiked water with clean seawater.
The bioassays with M. plumulosa employed dissolved concentrations in the range 301200 ug CulL and exposure durations of 2-240 h. This species exhibited delayed mortality, of which, the majority of effects occurred 48-96-h post-exposure. The results from the current study indicated that short (e.g. 4-d) bioassays may underestimate toxicity, as individuals may be counted as unaffected at the end of tests, but would eventually succumb to effects in the post-exposure period if tests lasted 10-d. The copper pulse concentration and duration were both important parameters that affected the mortality ofM plumulosa. The lack of significant difference in the mean morality of M. plumulosa exposed to multiple pulses separated by different durations in clean water, or to pulses of different frequency but similar time-averaged concentration (TAC), indicated the organism was not able to recover in the period between pulses.
The bioassays with P. tricornutum employed dissolved concentrations in the range 4600 ug CulL and exposure durations of 0.3-72 h. Short-term pulses were observed to elicit significant biomass inhibition of P. tricornutum.
However, the rate of cellular division of P. tricornutum was similar to that of controls after 24-48-h post-exposure, indicating the alga recovered from exposure after this duration in clean water. The timing of single pulsed exposure in the 72-h bioassay did not generally effect the biomass inhibition ofP. tricornutum. Different exposure scenarios with equivalent TACs of dissolved copper generally elicited similar toxic responses in M. plumulosa and P. tricornutum, indicating that toxicity was related to the TAC of dissolved copper exposure over the duration of the bioassays. This research supports the use of standard toxicity tests that utilise continuous contaminant exposure to predict toxicity in the field, as negative effects elicited by continuous exposure were similar to those elicited by pulsed exposures with similar TACs. The findings suggest single time-point measurements in the field may not adequately assess eco-system contamination, because this only reveals the exposure concentration at the instant the sample was collected, when it is the time-averaged concentration that is related to organism response.
A mechanistic study of the effects of copper exposure on P. tricornutum was performed using exposure concentrations in the range 10-50 ug CulL for durations in the range0.3-72 h. The P. tricornutum cells were observed to increase in size and clump together following copper exposure. Cell surface-bound (extra-cellular) copper was measured by extraction with a solution of EDTA, and internalised (intra-cellular) copper was measured by digesting the EDTA-washed algal cells with nitric acid. Copper rapidly bound to the surface of P. tricornutum cells and then surface-copper remained relatively constant. A linear rate of copper internalisation within cells was measured over the exposure duration, indicating copper internalisation was the rate limiting step of metal uptake into P. tricornutum cells. The concentrations of intra- and extra-cellular copper were only observed to increase significantly (p<0.05) above control cells when significant (p<0.05) biomass inhibition was observed. The copper bound to the surface of P. tricornutum cells decreased rapidly when cells were placed in clean seawater. However, the internalised copper per cell did not decrease significantly (p<0.05) until 18-h post-exposure, after which, it exhibited a linear decrease with exposure time. Although the intra-cellular copper concentration per P. tricornutum cell decreased, the total copper measured within all cells in the test population remained relatively constant during the post-exposure period. This indicated that the decrease in intra-cellular copper per cell was due to dilution of copper when divided between daughter cells produced by cellular division, and was not the result of copper efflux from cells.
For short-term pulsed copper exposures to P. tricornutum, surface-bound copper increased rapidly, but the short durations did not allow significant internalisation. Between pulses, copper rapidly desorbs from cell surfaces. The short pulsed exposures of 0.3-4 h employed in Chapter 5 were not expected to result in significant copper being internalised within cells, but these exposures still resulted in significant (p<0.05) biomass inhibition. It is hypothesised that the toxicity of short-term copper pulses to P. tricornutum cells is an acute response exerted by copper bound to the surface of cells, rather than a chronic response to internalised copper.
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