Mechanistic toxicodynamic model for receptor-mediated toxicity of diazoxon, the active metabolite of diazinon, in Daphnia magna

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.     

Uptake and elimination routes of inorganic mercury and methylmercury in Daphnia magna

Mercury (Hg) is an important environmental pollutant due to its highly toxic nature and widespread occurrence in aquatic systems. The biokinetics of Hg in zooplankton have been largely ignored in previous studies. This study examines the assimilation, dissolved uptake, and efflux of inorganic mercury [Hg(II)] and methylmercury (MeHg) in a freshwater cladoceran, Daphnia magna, and models the exposure pathways of Hg(II) and MeHg in the daphnids. The assimilation efficiencies (AEs) of both Hg species decreased significantly with increasing algal carbon concentrations. The dissolved uptake of Hg(II) and MeHg was proportional to the ambient concentration (ranging from environmentally realistic to high concentration over a 3-4 orders of magnitude variation), whereas MeHg had a slightly higher uptake rate constant (0.46 L g(-1) h(-1)) than Hg(II) (0.35 L g(-1) h(-1)). Surprisingly, the efflux rate constants of Hg(ll) and MeHg were rather comparable (0.041 -0.063 day(-1)). The release of both Hg(II) and MeHg via different routes (excretion, egestion, molting, and neonate production) was further examined at different food concentrations. It was found that regeneration into the dissolved phase was important for D. magna to eliminate both Hg species, but maternal transfer of Hg(II) (11-15%) and MeHg (32-41%) to neonates represented another important pathway for the elimination of Hg(II) and MeHg from the mothers. Modeling results suggest that food is an important source for MeHg exposure (47-98%), but water exposure represents 31-96% of Hg(II) accumulation in D. magna, depending on the variation of Hg bioconcentration factor in ingested food. Furthermore, MeHg predominates the bioaccumulation of Hg in D. magna even though MeHg constitutes only a small percentage of the total Hg in the water. The results strongly indicate that maternal transfer of Hg(II) and MeHg in freshwater zooplankton should be considered in manytoxicity testings and risk assessment in aquatic food chains.     

Two-Compartment Toxicokinetic-Toxicodynamic Model to Predict Metal Toxicity in Daphnia magna

Relating the toxicity of metals to their internal concentration is difficult due to complicated detoxification processes within organisms. Only the metabolically available metals are potentially toxic to organisms, while metals in the detoxified form are toxicologically irrelevant. Accordingly, we developed a two-compartment toxicokinetic-toxicodynamic model for metals in a freshwater cladoceran, Daphnia magna. The toxicokinetics simulated the bioaccumulation processes, while the toxicodynamics quantitatively described the corresponding processes of toxicity development. Model parameters were estimated for D. magna and three metals, i.e., cadmium, zinc, and mercury, by fitting the literature data on metal bioaccumulation and toxicity. A range of crucial information for toxicity prediction can be readily derived from the model, including detoxification rate, no-effect concentration, threshold influx rate for toxicity, and maximum duration without toxicity. This process-based model is flexible and can help improve ecological risk assessments for metals.     

Mechanisms of action of selective serotonin reuptake inhibitors in Daphnia magna

Selective serotonin reuptake inhibitors (SSRI) are known to increase offspring production in Daphnia magna. This study tested the hypothesis that the increase of serotonin postsynaptic activity by SSRI changes the perception of the food environment and switches life-history responses toward higher food level: females reproduced earlier, producing more but smaller offspring. D. magna reproduction tests, respiration, feeding, and survival-starvation assays and studies of lipids, proteins, and carbohydrate levels of unexposed and exposed females to the SSRI fluoxetine and fluvoxamine and the 5-HT serotonin receptor antagonist cyproheptadine were conducted. Factorial life-history experiments and reproductive assays showed that exposure to SSRI increased juvenile development rate, clutch size, and decrease offspring size at low and intermediate levels of food rations. These effects were reversed by the presence of cyproheptadine, indicating that 5-HT function was essential to the SSRI effects on Daphnia and linking them to the pharmacological effects of SSRI in humans. Respirometry and survival assays and biochemical analyses of lipids, proteins and carbohydrate levels showed that exposure to SSRI increased oxygen consumption rates and decreased carbohydrate levels in adult females. These changes did not affect survival under starving conditions, but they significantly affected the capacity of the exposed animals to survive under anoxic conditions. These results suggest that SSRI increased aerobic catabolism in D. magna making exposed individuals apparently more able to exploit food resources under normoxic conditions, but at the cost of being more sensitive to low oxygen levels, a common situation in natural environments.     

Assessment of chemical mixtures and groundwater effects on Daphnia magna transcriptomics

Small organisms can be used as biomonitoring tools to assess chemicals in the environment. Chemical stressors are especially hard to assess and monitor when present as complex mixtures. Here, fifteen polymerase chain reaction assays targeting Daphnia magna genes were calibrated to responses elicited in D. magna exposed for 24 h to five different doses each of the munitions constituents 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, 2,6-dinitrotoluene, trinitrobenzene, dinitrobenzene, or 1,3,5-trinitro-1,3,5-triazacyclohexane. A piecewise-linear model for log-fold expression changes in gene assays was used to predict response to munitions mixtures and contaminated groundwater under the assumption that chemical effects were additive. The correlations of model predictions with actual expression changes ranged from 0.12 to 0.78 with an average of 0.5. To better understand possible mixture effects, gene expression changes from all treatments were compared using high-density microarrays. Whereas mixtures and groundwater exposures had genes and gene functions in common with single chemical exposures, unique functions were also affected, which was consistent with the nonadditivity of chemical effects in these mixtures. These results suggest that, while gene behavior in response to chemical exposure can be partially predicted based on chemical exposure, estimation of the composition of mixtures from chemical responses is difficult without further understanding of gene behavior in mixtures. Future work will need to examine additive and nonadditive mixture effects using a much greater range of different chemical classes in order to clarify the behavior and predictability of complex mixtures.     

In vivo biomodification of lipid-coated carbon nanotubes by Daphnia magna

This study examined the interactions between Daphnia magna and a water-soluble, lysophophatidylcholine coated single-walled carbon nanotube. D. magna were able to ingest the nanotubes through normal feeding behavior and utilize the lysophophatidylcholine coating as a food source. D. magna were able to modify the solubility of the nanotube, likely through digestion of the lipid coating. This study provides evidence of biomodification of a carbon-based nanomaterial by an aquatic organism. The modification significantly altered the physical properties of the nanomaterial in freshwater. Acute toxicity was observed only in the highest test concentrations. These are important findings related to determining the behavior and potential toxicity of coated nanomaterials released into the environment.     

Acute toxicity of mercury to Daphnia magna under different conditions

We investigated the variations of acute toxicity of mercury (Hg) in Daphnia magna under different temperatures, population origins, body sizes, and Hg pre-exposures. We measured Hg concentrations in the water and in the surviving daphnids, and used the subcellular fractionation approach to determine Hg in the metal-sensitive fraction (MSF) to predict Hg toxicity. The 24-h median lethal concentrations and 24-h lethal body burden were 12-55 microg L(-1) and 10-26 mg kg(-1) wet wt, respectively. High Hg tolerance accompanied by reduced Hg uptake occurred in the daphnids under extreme conditions (low temperature and high pre-exposure to Hg). Correlating Hg levels in different compartments and daphnid survival resulted in the following order of sequence: aqueous Hg > whole body Hg > Hg in the MSF. However, the threshold lethal concentration of Hg (concentration causing 1% mortality) based on the concentration of Hg in the MSF was the best indicator of Hg toxicity. Therefore, the subcellular fractionation approach is less useful in explaining acute toxicity than is sub-lethal Hg toxicity. The number of Hg binding sites in the animals varied under different conditions but the affinity of the transporter to Hg generally decreased as the animals' tolerance increased. Mercury tolerance under different conditions could be enhanced by reducing the Hg uptake, enhancing the intrinsic tolerance, and/or increasing the detoxification activity.     

Population-level modeling to account for multigenerational effects of uranium in Daphnia magna

As part of the ecological risk assessment associated with radionuclides in freshwater ecosystems, toxicity of waterborne uranium was recently investigated in the microcrustacean Daphnia magna over a three-generation exposure (F0, F1, and F2). Toxic effects on daphnid life history and physiology, increasing over generations, were demonstrated at the organism level under controlled laboratory conditions. These effects were modeled using an approach based on the dynamic energy budget (DEB). For each of the three successive generations, DEBtox (dynamic energy budget applied to toxicity data) models were fitted to experimental data. Lethal and sublethal DEBtox outcomes and their uncertainty were projected to the population level using population matrix techniques. To do so, we compared two modeling approaches in which experimental results from F0, F1, and F2 generations were either considered separately (F0-, F1-, and F2-based simulations) or together in the actual succession of F0, F1, and F2 generations (multi-F-based simulation). The first approach showed that considering results from F0 only (equivalent to a standard toxicity test) would lead to a severe underestimation of uranium toxicity at the population level. Results from the second approach showed that combining effects in successive generations cannot generally be simplified to the worst case among F0-, F1-, and F2-based population dynamics.     

Temperature-dependent effects of cadmium on Daphnia magna: accumulation versus sensitivity

Standard toxicity tests are performed at one constant, optimal temperature (usually 20 degrees C), while in the field variable and suboptimal temperatures may occur. Lack of knowledge on the interactions between chemicals and temperature hampers the extrapolation of laboratory toxicity data to ecosystems. Therefore, the aim of this study was to analyze the effects of temperature on cadmium toxicity to the waterflea Daphnia magna and to address possible processes responsible for temperature-dependent toxicity. This was investigated by performing standard toxicity tests with D. magna under a wide temperature range. Thermal effects on accumulation kinetics were determined by estimating uptake and elimination rates from accumulation experiments. To study temperature dependency of the intrinsic sensitivity of the daphnids to cadmium, the DEBtox model was used to estimate internal threshold concentrations (ITCs) and killing rates from the toxicity and accumulation data. The results revealed that increasing temperature lowered the ITC and increased the killing rate and the uptake rate of the metal. Enhanced sensitivity of D. magna was shown to be the primary factor for temperature-dependent toxicity. Since temperature has such a major impact on toxicity, a temperature correction may be necessary when translating toxicity data from the laboratory to the field.     

Cd and Zn uptake kinetics in Daphnia magna in relation to Cd exposure history

The uptake kinetics of Cd and Zn in a freshwater cladoceran Daphnia magna after exposure to different concentrations of Cd for various durations was quantified. The accumulated Cd concentrations increased with ambient Cd concentration and exposure duration. As a detoxification mechanism, metallothioneins (MTs) were induced when the Cd preexposure condition was beyond the noneffect threshold. The MT induction was dependent on both Cd concentration and duration of preexposure. Increasing the Cd exposure concentration to 20 microg L(-1) for 3 d caused a 44% reduction in Cd assimilation efficiency (AE, the fraction assimilated by the animals after digestion) by the daphnids from the dietary phase, but a 2.4-fold increase in Zn AE. Generally, the dissolved metal uptake rate was not significantly affected by the different Cd preexposure regimes, except at a much higher Cd concentration (20 microg L(-1)) when the Zn influx was enhanced. Significant effects from Cd exposure on the ingestion rate of the daphnids were also observed. When the MT synthesis was not coupled with the accumulated Cd tissue burden (e.g., a delay in MT synthesis), apparent Cd toxicity on the feeding behavior and the Cd AE was observed, thus highlighting the importance of MTs in modifying the metal uptake kinetics of D. magna. Overall, daphnids responded to acute Cd exposure by reducing their Cd AE and ingestion, whereas they developed a tolerance to Cd following chronic exposure. The bioavailability of Zn was enhanced as a result of Cd preexposure. This study highlights the important influences of Cd preexposure history on the biokinetics and potential toxicity of Cd and Zn to D. magna.     

Mechanisms of acute and chronic waterborne nickel toxicity in the freshwater cladoceran, Daphnia magna

We present evidence that Mg2+ antagonism is one mechanism for acute toxicity of waterborne Ni to Daphnia magna. Acutely, adult D. magna were exposed to either control or 694 microg Ni L(-1) as NiSO4 in moderately soft water (45 mg L(-1) as CaCO3; background Ni approximately 1 microg Ni L(-1)) for 48 h without feeding. Chronically, adults were exposed to either control or 131 microg Ni L(-1) for 14 days (fed exposure). These concentrations were approximately 65% and 12%, respectively, of the measured 48-h LC50 (1068 microg Ni L(-1)) for daphnid neonates in this water quality. The clearest effect of Ni exposure was on Mg2+ homeostasis, as whole-body [Mg2+] was significantly decreased both acutely and chronically by 18%. Additionally, unidirectional Mg2+ uptake rate (measured with the stable isotope 26Mg) was significantly decreased both acutely and chronically by 49 and 47%, respectively, strongly suggesting that Ni is toxic to D. magna due at least in part to Mg2+ antagonism. No impact was observed on the whole-body concentrations or unidirectional uptake rates of Ca2+ during either acute or chronic Ni exposure, while only minor effects were seen on Na+ and Cl- balance. No acute toxic effect was seen on respiratory parameters, as both oxygen consumption rate (MO2) and whole-body hemoglobin concentration ([Hb]) were conserved. Chronically, however, Ni impaired respiratory function, as both MO2 and [Hb] were significantly reduced by 31 and 68%, respectively. Acutely, Ni accumulation was substantial, rising to a plateau between 24 and 48 h of approximately 15 microg g(-1) wet weight--an increase of approximately 25-fold over control concentrations. Mechanisms of acute toxicity of Ni in D. magna differ from those in fish; it is likely that such mechanistic differences also exist for other metals.     

Toxicogenomic responses of nanotoxicity in Daphnia magna exposed to silver nitrate and coated silver nanoparticles

Applications for silver nanomaterials in consumer products are rapidly expanding, creating an urgent need for toxicological examination of the exposure potential and ecological effects of silver nanoparticles (AgNPs). The integration of genomic techniques into environmental toxicology has presented new avenues to develop exposure biomarkers and investigate the mode of toxicity of novel chemicals. In the present study we used a 15k oligonucleotide microarray for Daphnia magna, a freshwater crustacean and common indicator species for toxicity, to differentiate between particle specific and ionic silver toxicity and to develop exposure biomarkers for citrate-coated and PVP-coated AgNPs. Gene expression profiles revealed that AgNO(3) and AgNPs have distinct expression profiles suggesting different modes of toxicity. Major biological processes disrupted by the AgNPs include protein metabolism and signal transduction. In contrast, AgNO(3) caused a downregulation of developmental processes, particularly in sensory development. Metal responsive and DNA damage repair genes were induced by the PVP AgNPs, but not the other treatments. In addition, two specific biomarkers were developed for the environmental detection of PVP AgNPs; although further verification under different environmental conditions is needed.     

Role of titanium dioxide nanoparticles in the elevated uptake and retention of cadmium and zinc in Daphnia magna

Titanium dioxide nanoparticles (nano-TiO(2)) are now widely applied in consumer products, and the dispersion of nano-TiO(2) may adsorb metals and modify their behavior and bioavailability in the aquatic environment. In the present study, the aqueous uptake, dietary assimilation efficiency (AE), and efflux rate constant (k(e)) of two toxic metals (cadmium-Cd, and zinc-Zn) adsorbed on nano-TiO(2) in a freshwater zooplankton Daphnia magna were quantified. The biokinetics was then compared to daphnids that were exposed only to dissolved metals as controls. The aqueous uptake of Cd and Zn involved an initial rapid uptake and then an apparent saturation, and the uptake of metals was accompanied by an ingestion of nano-TiO(2). The AEs of Cd and Zn adsorbed on nano-TiO(2) were 24.6 ± 2.4-44.5 ± 3.7% and 30.4 ± 3.4-51.8 ± 5.0%, respectively, and decreased with increasing concentrations of nano-TiO(2). Furthermore, the difference between the AEs of Cd and Zn indicated that the desorption of Cd and Zn from nano-TiO(2) may have occurred within the gut of daphnids. With the use of algae as carrier, the AEs of Cd and Zn adsorbed on nano-TiO(2) were significantly higher than those of Cd and Zn directly from nano-TiO(2). The efflux rate constants of Cd and Zn adsorbed on nano-TiO(2) in the zooplankton were significantly lower than those of Cd and Zn not adsorbed on nano-TiO(2). Our study shows that the uptake and retention of toxic metals is enhanced when they are adsorbed on nano-TiO(2), and suggests more attention be paid to the potential influences of nano-TiO(2) on the bioavailability and toxicity of other contaminants.     

Biology-based modeling to analyze uranium toxicity data on Daphnia magna in a multigeneration study

Recent studies have investigated chronic toxicity of waterborne depleted uranium on the life cycle and physiology of Daphnia magna. In particular, a reduction in food assimilation was observed. Our aims here were to examine whether this reduction could fully account for observed effects on both growth and reproduction, for three successive generations, and to investigate through microscope analyses whether this reduction resulted from direct damage to the intestinal epithelium. We analyzed data obtained by exposing Daphnia magna to uranium over three successive generations. We used energy-based models, which are both able to fit simultaneously growth and reproduction and are biologically relevant. Two possible modes of action were compared - decrease in food assimilation rate and increase in maintenance costs. In our models, effects were related either to internal concentration or to exposure concentration. The model that fitted the data best represented a decrease in food assimilation related to exposure concentration. Furthermore, observations of consequent histological damage to the intestinal epithelium, together with uranium precipitates in the epithelial cells, supported the assumption that uranium has direct effects on the digestive tract. We were able to model the data in all generations and showed that sensitivity increased from one generation to the next, in particular through a significant increase of the intensity of effect, once the threshold for appearance of effects was exceeded.     

Population developmental stage determines the recovery potential of Daphnia magna populations after fenvalerate application

This study investigated the responses of Daphnia magna populations to pulsed exposures of the pyrethroid insecticide Fenvalerate applied during an early and a late stage of population development, and analyzed the dynamics of the subsequent recovery. A novel digital observation technique was used to describe the size and numbers of animals. High Fenvalerate concentrations caused high mortality rates during exponential population growth as well as during the food-limited stationary phase. However, recovery of populations took considerably longer in the stationary phase than in populations growing exponentially. The poor nutritional and reproductive state of food-deprived adults was indicated as the main cause of the slow recovery of populations. It is argued that populations operating at the carrying capacity of their environment are vulnerable to toxicant-induced disturbances to an extent not predictable from observations on exponentially growing populations such as are commonly used in ecotoxicology.     

Acute toxicity of cadmium in Daphnia magna under different calcium and pH conditions: importance of influx rate

Water chemistry is generally thought to influence metal toxicity via affecting metal bioavailability and bioaccumulation, but its effects on tissue residue-based toxicity are poorly known. We conducted toxicity tests in parallel with uptake kinetics experiments of cadmium (Cd) in waters of different calcium (Ca) concentrations and pH levels using acclimated Daphnia magna as a model organism. Both the acute toxicity and uptake of Cd were reduced by higher Ca concentration and lower pH. Strikingly constant median effective influx rates (EJ(50), 1.3-1.6 μg g(-1) h(-1)) of Cd were observed when the concentration of Ca varied from 0.5 to 200 mg L(-1), indicating that acclimation to different Ca levels did not affect the tissue residue-based toxicity. The EJ(50) values increased consistently with decreasing pH level, showing that acclimation to acidic water decreased the tissue residue-based toxicity. With the use of calcium uptake inhibitors, we demonstrated that both Ca channel and Ca(2+)/Na(+) exchanger were involved in Cd uptake in daphnids, but there were also other possible pathways with higher affinity. The relative importance of different pathways was clearly dependent on the ambient Ca availability. Our findings are helpful for the development of a more accurate biotic ligand model in predicting the acute toxicity of Cd to daphnids.     

Differential gene expression in Daphnia magna suggests distinct modes of action and bioavailability for ZnO nanoparticles and Zn ions

Zinc oxide nanoparticles (ZnO NPs) are being rapidly developed for use in consumer products, wastewater treatment, and chemotherapy providing several possible routes for ZnO NP exposure to humans and aquatic organisms. Recent studies have shown that ZnO NPs undergo rapid dissolution to Zn(2+), but the relative contribution of Zn(2+) to ZnO NP bioavailability and toxicity is not clear. We show that a fraction of the ZnO NPs in suspension dissolves, and this fraction cannot account for the toxicity of the ZnO NP suspensions to Daphnia magna. Gene expression profiling of D. magna exposed to ZnO NPs or ZnSO(4) at sublethal concentrations revealed distinct modes of toxicity. There was also little overlap in gene expression between ZnO NPs and SiO(x) NPs, suggesting specificity for the ZnO NP expression profile. ZnO NPs effected expression of genes involved in cytoskeletal transport, cellular respiration, and reproduction. A specific pattern of differential expression of three biomarker genes including a multicystatin, ferritin, and C1q containing gene were confirmed for ZnO NP exposure and provide a suite of biomarkers for identifying environmental exposure to ZnO NPs and differentiating between NP and ionic exposure.     

Determining genetic variability in the distribution of sensitivities to toxic stress among and within field populations of Daphnia magna

To extrapolate credibly from individuals in the laboratory to field populations, it is essential to account for genetic differences in susceptibility to toxic stress and thus incorporate genetic variability into ecological risk estimates. In this study, the distribution of sensitivities across two toxic chemicals among and within field populations of Daphnia magna were used to quantify genetic variability. The study employed 30 D. magna clones from three geographically separate European populations. The sensitivity of each population studied and its constituent clones was estimated in terms of the concentrations of lambda-cyhalothrin and cadmium impairing individual fitness by 10 and 50% (EC10-50). Results revealed that differences in tolerance among clones within populations were large when compared with differences between populations and that the genetic range of sensitivities to toxic stress within populations was log-normally distributed. Furthermore, reported variation in sensitivity values to toxic stress among different laboratory species, populations, and clones was similar to that observed among and within field populations of Daphnia. These results suggest that it is possible to estimate genetic variability by estimating the tolerance distribution of laboratory populations and clones and that extrapolation approaches currently used in ecological risk assessment should explicitly incorporate genetic variability in tolerance into risk estimates.