Department Environmental Chemistry

Azole fungicides are known inhibitors of the important enzyme class cytochrome P450 monooxygenases (CYPs), thereby influencing the detoxification of co-occurring substances via biotransformation. This synergism in mixtures containing an azole has mostly been studied by effect measurements, while the underlying mechanism has been less well investigated. In this study, six azole fungicides (cyproconazole, epoxiconazole, ketoconazole, prochloraz, propiconazole, and tebuconazole) were selected to investigate their synergistic potential and their CYP inhibition strength in the aquatic invertebrate Gammarus pulex. The strobilurin fungicide azoxystrobin was chosen as co-occurring substrate, and the synergistic potential was measured in terms of internal concentrations of azoxystrobin and associated biotransformation products (BTPs). Azoxystrobin is biotransformed by various reactions, and 18 BTPs were identified. By measuring internal concentrations of azoxystrobin and its BTPs with high-resolution tandem mass spectrometry in the presence and absence of azole fungicides followed by toxicokinetic modeling, we showed that the inhibition of CYP-catalyzed biotransformation reactions indeed played a role for the observed synergism. However, synergism was only observed for prochloraz at environmentally realistic concentrations. Increased uptake rate constants, an increase in the total internal concentration of azoxystrobin and its BTPs, in vivo assays for measuring CYP activities, and G. pulex video-tracking suggested that the 2-fold increase in bioaccumulation, and, thereby, the raised toxicity of azoxystrobin in the presence of prochloraz is not only caused by inhibited biotransformation but even more by increased azoxystrobin uptake induced by hyperactivity.

Biotransformation is a key process that can greatly influence the bioaccumulation potential and toxicity of organic compounds. In this study, biotransformation of seven frequently used azole fungicides (triazoles: cyproconazole, epoxiconazole, fluconazole, propiconazole, tebuconazole and imidazoles: ketoconazole, prochloraz) was investigated in the aquatic invertebrate Gammarus pulex in a 24 h exposure experiment. Additionally, temporal trends of the whole body internal concentrations of epoxiconazole, prochloraz, and their respective biotransformation products (BTPs) were studied to gain insight into toxicokinetic processes such as uptake, elimination and biotransformation. By the use of high resolution tandem mass spectrometry in total 37 BTPs were identified. Between one (ketoconazole) and six (epoxiconazole) BTPs were identified per parent compound except for prochloraz, which showed extensive biotransformation reactions with 18 BTPs detected that were mainly formed through ring cleavage or ring loss. In general, most BTPs were formed by oxidation and conjugation reactions. Ring loss or ring cleavage was only observed for the imidazoles as expected from the general mechanism of oxidative ring openings of imidazoles, likely affecting the bioactivity of these BTPs. Overall, internal concentrations of BTPs were up to 3 orders of magnitude lower than that of the corresponding parent compound. Thus, biotransformation did not dominate toxicokinetics and only played a minor role in elimination of the respective parent compound, with the exception of prochloraz.

The potential bioaccumulation and distribution of antibiotics in non-target organisms have been inadequately studied in spite of their widespread occurrence in aquatic systems. We investigated the ability of tetracycline to bioaccumulate through aqueous and dietary routes in an aquatic organism, the freshwater crustacean Daphnia magna. D. magna was exposed to algal food (Pseudokirchneriella subcapitata) contaminated with tetracycline for dietary uptake. Tetracycline was transferred to D. magna more through aqueous uptake than through dietary uptake. The uptake rate constant of tetracycline for D. magna was k_in,water = 0.33 ± 0.045 via the aqueous route and k_in,food = 0.16 ± 0.012 via the dietary route for 1.0 mg L⁻¹ tetracycline. Bioconcentration factors of 4.40 ± 0.91 L kg⁻¹ and 3.66 ± 0.50 L kg⁻¹ for 0.1 and 1.0 mg L⁻¹ tetracycline were found for D. magna. The biomagnification factor of 0.19 ± 0.04 indicates that magnification of tetracycline through the food web will not occur. The change in the internal concentration of the target compound was also studied for multigenerational (F1-F4) exposure. The internal concentration in D. magna showed a decreasing trend with increasing generations except for the parent generation. The bioaccumulation tendency showed a biphasic change in multigenerational exposure.

The inhibition of acetylcholinesterase (AChE) activity and energy allocation in the freshwater organism Daphnia magna exposed to carbaryl and potential recovery from the effects was examined. The binding of carbaryl-AChE was characterized through in vitro assays. To evaluate the recovery from inhibition and the alteration in energy budget, in vivo exposure and recovery regime tests were conducted. In comparison to diazoxon, the active metabolite of the insecticide diazinon, the stability of enzyme–carbaryl complex was fifteen times lower and the reactivity toward the active site was two times lower, resulting in approximately 30 times lower overall inhibition rate than for diazoxon. The in vitro reactivation rate constant of the inhibited enzyme and the in vivo recovery rate constant of AChE activity were 1.9 h⁻¹ and 0.12 h⁻¹ for carbaryl, respectively, which are much higher than the corresponding rate constants for diazoxon. The lower AChE inhibition and greater reactivation/recovery rates are in accordance with the lower toxicity of carbaryl compared to diazinon. Carbaryl exposure also altered the profile of the energy reserve: the decrease in lipid and glycogen and the increase in protein content resulted in the reduction of the total energy budget by about 45 mJ/g_ww. This corresponds to 26 percent of the available energy, which might allocate for external stressors. The mechanistic model of AChE inhibition is helpful to get an insight into (eco-)toxicological effects of AChE inhibitors on freshwater crustaceans under environmentally realistic conditions.

Exposure and depuration experiments for Gammarus pulex and Daphnia magna were conducted to quantitatively analyze biotransformation products (BTPs) of organic micropollutants (tramadol, irgarol, and terbutryn). Quantification for BTPs without available standards was performed using an estimation method based on physicochemical properties. Time-series of internal concentrations of micropollutants and BTPs were used to estimate the toxicokinetic rates describing uptake, elimination, and biotransformation processes. Bioaccumulation factors (BAF) for the parents and retention potential factors (RPF), representing the ratio of the internal amount of BTPs to the parent at steady state, were calculated. Nonlinear correlation of excretion rates with hydrophobicity indicates that BTPs with lower hydrophobicity are not always excreted faster than the parent compound. For irgarol, G.pulex showed comparable elimination, but greater uptake and BAF/RPF values than D.magna. Further, G. pulex had a whole set of secondary transformations that D. magna lacked. Tramadol was transformed more and faster than irgarol and there were large differences in toxicokinetic rates for the structurally similar compounds irgarol and terbutryn. Thus, predictability of toxicokinetics across species and compounds needs to consider biotransformation and may be more challenging than previously thought because we found large differences in closely related species and similar chemical structures.

So far, there is limited information on biotransformation mechanisms and products of polar contaminants in freshwater crustaceans. In the present study, metabolites of biocides and pharmaceuticals formed in Gammarus pulex and Daphnia magna were identified using liquid chromatography–high resolution mass spectrometry. Different confidence levels were assigned to the identification of metabolites without reference standards using a framework based on the background evidence used for structure elucidation. Twenty-five metabolites were tentatively identified for irgarol, terbutryn, tramadol, and venlafaxine in G. pulex (21 via oxidation and 4 via conjugation reactions) and 11 metabolites in D. magna (7 via oxidation and 4 via conjugation reactions), while no evidence of metabolites for clarithromycin and valsartan was found. Of the 360 metabolites predicted for the four parent compounds using pathway prediction systems and expert knowledge, 23 products were true positives, while 2 identified metabolites were unexpected products. Observed oxidative reactions included N- and O-demethylation, hydroxylation, and N-oxidation. Glutathione conjugation of selected biocides followed by subsequent reactions forming cysteine conjugates was described for the first time in freshwater invertebrates.

The organothiophosphate diazinon inhibits the target site acetylcholinesterase only after activation to its metabolite diazoxon. Commonly, the toxicity of xenobiotics toward aquatic organisms is expressed as a function of the external concentration and the resulting effect on the individual level after fixed exposure times. This approach does not account for the time dependency of internal processes such as uptake, metabolism, and interaction of the toxicant with the target site. Here, we develop a mechanistic toxicodynamic model for Daphnia magna and diazoxon, which accounts for the inhibition of the internal target site acetylcholinesterase and its link to the observable effect, immobilization, and mortality. The model was parametrized by experiments performed in vitro with the active metabolite diazoxon on enzyme extracts and in vivo with the parent compound diazinon. The mechanism of acetylcholinesterase inhibition was shown to occur irreversibly in two steps via formation of a reversible enzyme–inhibitor complex. The corresponding kinetic parameters revealed a very high sensitivity of acetylcholinesterase from D. magna toward diazoxon, which corresponds well with the high toxicity of diazinon toward this species. Recovery of enzyme activity but no recovery from immobilization was observed after in vivo exposure to diazinon. The toxicodynamic model combining all in vitro and in vivo parameters was successfully applied to describe the time course of immobilization in dependence of acetylcholinesterase activity during exposure to diazinon. The threshold value for enzyme activity below which immobilization set in amounted to 40% of the control activity. Furthermore, the model enabled the prediction of the time-dependent diazoxon concentration directly present at the target site.

A toxicokinetic model for Daphnia magna, which simulates the internal concentration of the insecticide diazinon, its detoxification product 2-isopropyl-6-methyl-4-pyrimidinol, and its active metabolite diazoxon, is presented. During in vivo exposure to diazinon with and without inhibition of cytochrome P450 by piperonyl butoxide, the parent compound as well as its metabolites were quantified with high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) in extracts of D. magna. Rate constants of all relevant toxicokinetic steps were obtained by modeling the time course of the internal concentrations with a multicomponent first-order kinetics model. When cytochrome P450 was inhibited, the kinetic bioconcentration factor (BCF) of diazinon increased from 17.8 to 51.0 mL·g_ww^–1. This clearly indicates that diazinon is biotransformed to a high degree by cytochrome P450 in D. magna. The dominant elimination step of diazinon was shown to be its oxidative dearylation to pyrimidinol (62% of total elimination) with a corresponding rate constant of 0.16 h^–1. In contrast, oxidative activation to diazoxon with a rate constant of 0.02 h^–1 amounted to only 8% of the total elimination. During exposure to diazinon, the active metabolite diazoxon could be detected only in very low concentrations (approximately 0.5% of the parent compound), presumably due to a very fast reaction with the target site acetylcholinesterase. During the exposure experiments (no feeding of daphnids), an exponential decline of the lipid content in D. magna with a first-order rate constant of 0.013 h^–1 was observed. For short exposure times (≤24 h), this had only a minor influence on the determined TK parameters. Such a TK model containing detailed biotransformation processes is an important tool for estimation of the toxic potential of chemicals, particularly, when active metabolites are formed inside an organism.