Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Environmental Toxicology


Klaine, Stephen J

Committee Member

Tomasso , Joseph

Committee Member

Lee , Cindy

Committee Member

Diamond , Jerry

Committee Member

van den Hurk , Peter


The US EPA utilizes the Biotic Ligand Model (BLM) when developing site-specific water-quality criteria for copper. The BLM was calibrated using toxicity data from fixed duration continuous exposures (48 or 96h LC50). However, exposures of copper in natural aquatic systems typically vary in concentration, duration, and may occur multiple times over the life-span of an organism. Additionally, organisms will experience periods in copper free water, potentially allowing them to detoxify and recover homeostasis. Yet, the BLM framework does not consider brief exposures (<24h), multiple exposures, latent responses, or organism recovery, potentially resulting in an inaccurate assessment of exposure toxicity. A recently proposed physiologically-based addition to the BLM will extend the applicability of the framework to brief, multiple pulse copper exposures. Thus, the overall goal of this research was to characterize the recovery of larval Pimephales promelas following episodic copper exposure at a biochemical, physiological, individual, and population level.
The response of whole-body sodium, whole-body Na+/K+-ATPase, and whole-body carbonic anhydrase in larval P. promleas was quantified during 21d bioassays where organisms were exposed to single or double pulses of 50, 100, or 200µg/L Cu for 3 or 9h durations with various recovery intervals (0, 24, 48, 96, and 144h). Organisms required 48 to 96h post exposure termination to recover sodium homeostasis. Researchers have proposed 30% sodium loss as the critical threshold for the onset of mortality in adult freshwater fish; however, larval P. promlas recovered sodium homeostasis and survived even when exceeding this threshold. Recovery interval allowed between exposures affected the response of organisms to subsequent exposures. Following a second pulse, organisms that were allowed short recovery intervals (<48h) exhibited resistance to any further sodium loss, and organisms allowed longer recovery intervals (>96h) displayed an independent response, but were able to recover faster. Whole-body Na+/K+-ATPase and carbonic anhydrase had a poor correlation with whole-body sodium (r2=0.0001 and 0.1025, respectively), but the carbonic anhydrase correlation to whole-body sodium was significant (p=0.0004). Na+/K+-ATPase was not significantly affected by copper exposure, which may have been due to the dilution effect of the whole organism Na+/K+-ATPase concentration and brief exposure durations (<24h). Carbonic anhydrase was likely involved in the inhibition of sodium uptake and the subsequent recovery post exposure; however, it did not assist in the protection to the second exposure.
Lethal and sublethal (growth, reproduction, whole-body sodium, whole-body copper) responses of P. promleas exposed for 24h to 50µg/L Cu every 4 or 9d over their entire life-span were observed using a life-cycle reproductive toxicity test. Latent mortality was observed for 48-72h post exposure termination for larval organisms. Organisms were able to recover by depurating accumulated copper and compensating for sodium losses within 4d. There were no significant effects on the growth of larval organisms; however, significant effects on juveniles and adult growth and weight were observed (p<0.05). Reproduction in both copper treatments was reduced compared to controls, but this difference was not significant (p=0.1647). There was no significant effect on population fitness when given at least 4d to recover between pulses; however, any deviation (i.e. increased concentration, longer duration, shorter recovery interval) will increase the probability of effects to the population. The results of this research stress the importance of incorporating latent responses and organism recovery when modeling the toxicity of multiple pulse copper exposures.