Environ. Sci. Technol. 2010, 44, 3963–3971
Toxicokinetic and Toxicodynamic Modeling Explains Carry-over Toxicity from Exposure to Diazinon by Slow Organism Recovery ROMAN ASHAUER,† ANITA HINTERMEISTER,† IVO CARAVATTI,† ANDREAS KRETSCHMANN,† AND B E A T E I E S C H E R †,‡ Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Du ¨bendorf, Switzerland, and The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 39 Kessels Rd, Brisbane, Qld 4108, Australia
Received November 16, 2009. Revised manuscript received March 17, 2010. Accepted March 20, 2010.
Carry-over toxicity occurs when organisms exposed to an environmental toxicant survive but carry some damage resulting in reduced fitness. Upon subsequently encountering another exposure event stronger effects are possible if the organisms have not yet fully recovered. Carry-over toxicity was observed after exposure of the freshwater amphipod Gammarus pulex to repeated pulses of diazinon with varying intervals. Uptake, biotransformation and depuration kinetics were determined. Metabolites were identified and quantified (diazoxon, 2-isopropyl-6-methyl-4-pyrimidinol, one nonidentified metabolite). Parameters of a process-based toxicokinetic-toxicodynamic modelweredeterminedbyleast-squaresfittingfollowedbyMarkov Chain Monte Carlo parameter estimation. Model parametrization was based on the time-course of measured internal concentrations of diazinon and its metabolite diazoxon in combination with the pulsed toxicity experiment. Prediction intervals, which take the covariation between parameters into account, were calculated for bioaccumulation factors, organism recovery time and simulations of internal concentrations as well as the time-course of survival under variable exposure. Organism recovery time was 28 days (95% prediction interval 25-31 days), indicating the possibility for carry-over toxicity from exposure events several weeks apart. The slow organism recovery and carry-over toxicity was caused by slow toxicodynamic recovery; toxicokinetic processes alone would have resulted in a recovery time of only 1-2 days.
Introduction Background. Exposure of aquatic organisms to environmental pollutants often occurs in fluctuating concentrations or repeated pulses (1-6). Even for constant environmental concentrations of pollutants, a fluctuating concentration profile may be experienced by moving organisms. Mechanistic models have been suggested for the environmental risk assessment of such exposure profiles (7-9) as they can extrapolate between different exposure scenarios. Toxico* Correspondingauthorphone:+41-448235233;fax:+41-448235311; e-mail:
[email protected]. † Eawag. ‡ Entox. 10.1021/es903478b
2010 American Chemical Society
Published on Web 04/16/2010
kinetic-toxicodynamic (TK-TD) models explicitly simulate the time-course of uptake, biotransformation, and elimination of toxicants in the organism (toxicokinetics, TK), which then determines the time-course of damage and organism recovery as the prerequisite for toxic effects (toxicodynamics, TD). The time-course of toxic effects is closely related to the recovery of the organism. Delayed toxicity (1, 10-12) and more recently carry-over toxicity (13-15) have been observed following pulse exposure of aquatic organisms to toxicants. Carry-over toxicity, defined as increased toxic effects compared to organisms which were not prestressed, may occur after the second pulse due to incomplete organism recovery (14-16). Incomplete recovery may be caused either by incomplete elimination (TK) or by mechanisms of toxicity with slow or incomplete reversibility (TD). Hence environmental risk assessment based on standard toxicity tests with constant exposure concentration and fixed duration may underestimate the risk to aquatic organisms as the time course of toxicity and organism recovery is not measured or analyzed. TK-TD models have the potential to elucidate the underlying mechanistic cause of carry-over toxicity, simulate the time-course of toxic effects for variable exposure conditions and quantify ecotoxicological responses in such a way that extrapolations to different exposure patterns are possible. The combination of diazinon and Gammarus pulex is used here to investigate carry-over toxicity and its proposed mechanistic explanation with a TK-TD model while it is also a combination of relevance to current environmental concern and environmental risk assessment. Diazinon is highly toxic to aquatic arthropods such as Gammarus pulex and is frequently detected in surface waters, often exhibiting fluctuating concentration profiles with repeated pulses (3-5). The concentrations used in this study exceeded measured environmental concentrations by a factor of 10-100 and were chosen for a proof of principle, not for realistic exposure. Diazinon is activated by biotransformation to diazoxon (17-19), which acts as inhibitor of acetylcholinesterase with a generally very slow reactivation of the diethylphosphorylated enzyme (20) and is much more potent than diazinon (17, 19, 21). Detoxification occurs via enzymatic hydrolysis to 2-isopropyl-6-methyl-4-pyrimidinol (from here on: pyrimidinol) (17, 18, 22), followed by conjugation of pyrimidinol (18, 20). Our objectives were to quantify (i) carry-over toxicity, (ii) the time-course of toxicokinetics including biotransformation, (iii) the time-course of damage and organism recovery in order to parametrize a TK-TD model, and (iv) the associated uncertainty in model predictions. Two types of experiments were carried out: a toxicokinetic experiment and a pulse toxicity experiment with various intervals.
Experimental methods Chemicals. Diazinon is a hydrophobic insecticide (log Kow 3.81 (23),). The 14C-labeled diazinon ((Pyrimidinyl-6-14C)Diazinon, 99.21% radiochemical purity) used here was supplied by the Institute of Isotopes, Budapest, Hungary. Unlabeled material of diazinon (CAS 333-41-5, 99% purity), diazoxon (CAS 962-58-3, 99% purity) and pyrimidinol (CAS 2814-20-2, 99% purity) was supplied by Sigma-Aldrich (Buchs, Switzerland). Toxicokinetic Experiment. During the toxicokinetic experiment, organisms were first exposed to approximately 30 nmol/L diazinon (carrier acetone
