Toxicokinetic model describing bioconcentration and biotransformation of diazinon in Daphnia magna

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.     

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.     

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.     

A biotic ligand model predicting acute copper toxicity for Daphnia magna: the effects of calcium, magnesium, sodium, potassium, and pH

The extent to which Ca2+, Mg2+, Na+, K+ ions and pH independently mitigate acute copper toxicity for the cladoceran Daphnia magna was examined. Higher activities of Ca2+, Mg2+, and Na+ (but not K+) linearly increased the 48-h EC50 (as Cu2+ activity), supporting the concept of competitive binding of these ions and copper ions to toxic action or transport sites at the organism-water interface (e.g. fish gill, the biotic ligand). The increase of the EC50 (as Cu2+ activity) with increasing H+, however, seemed to suggest cotoxicity of CuOH+ rather than proton competition. Based on the biotic ligand model (BLM) concept, we developed a methodology to estimate stability constants for the binding of Cu2+, CuOH+, Ca2+, Mg2+, Na+, and H+ to the biotic ligand, solely based on toxicity data. Following values were obtained: log K(CuBL) = 8.02, log K(CuOHBL)= 7.45, log K(CaBL) = 3.47, log K(MgBL) = 3.58, log K(NaBL) = 3.19, and log K(HBL) approximately 5.4. Further, we calculated that on average 39% of the biotic ligand sites need to be occupied by copper to induce a 50% acute effect for D. magna after 48 h of exposure. Using the estimated constants, a BLM was developed that can predict acute copper toxicity for D. magna as a function of water characteristics. The presented methodology can easily be applied for BLM development for other organisms and metals. After validation with laboratory and natural waters (including DOC), the developed model will support efforts to improve the ecological relevance of presently applied risk assessment procedures.     

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.     

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.     

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.     

Waterborne versus dietary zinc accumulation and toxicity in Daphnia magna: a synchrotron radiation based X-ray fluorescence imaging approach

Recent studies have suggested that exposure of the freshwater invertebrate Daphnia magna to dietary Zn may selectively affect reproduction without an associated increase of whole body bioaccumulation of Zn. The aim of the current research was therefore to investigate the hypothesis that dietary Zn toxicity is the result of selective accumulation in tissues that are directly involved in reproduction. Since under field conditions simultaneous exposure to both waterborne and dietary Zn is likely to occur, it was also tested if accumulation and toxicity under combined waterborne and dietary Zn exposure is the result of interactive effects. To this purpose, D. magna was exposed during a 16-day reproduction assay to Zn following a 5 × 2 factorial design, comprising five waterborne concentrations (12, 65, 137, 207, and 281 μg Zn/L) and two dietary Zn levels (49.6 and 495.9 μg Zn/g dry wt.). Tissue-specific Zn distribution was quantified by synchrotron radiation based confocal X-ray fluorescence (XRF). It was observed that the occurrence of reproductive inhibition due to increasing waterborne Zn exposure (from 65 μg/L to 281 μg/L) was accompanied by a relative increase of the Zn burdens which was similar in all tissues considered (i.e., the carapax, eggs, thoracic appendages with gills and the cluster comprising gut epithelium, storage cells and ovaries). In contrast, the impairment of reproduction during dietary Zn exposure was accompanied by a clearly discernible Zn accumulation in the eggs only (at 65 μg/L of waterborne Zn). During simultaneous exposure, bioaccumulation and toxicity were the result of interaction, which implies that the tissue-specific bioaccumulation and toxicity following dietary Zn exposure are dependent on the Zn concentration in the water. Our findings emphasize that (i) effects of dietary Zn exposure should preferably not be investigated in isolation from waterborne Zn exposure, and that (ii) XRF enabled us to provide possible links between tissue-specific bioaccumulation and reproductive effects of Zn.     

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.     

Influence of polyethyleneimine graftings of multi-walled carbon nanotubes on their accumulation and elimination by and toxicity to Daphnia magna

Modifications of carbon nanotubes (CNTs) for different applications may change their physicochemical properties such as surface charge. Assessments of the extent to which such modifications influence CNT ecotoxicity, accumulation, and elimination behaviors are needed to understand potential environmental risks these variously modified nanoparticles may pose. We have modified carbon-14 labeled multi-walled carbon nanotubes (MWNTs) with polyethyleneimine (PEI) surface coatings to increase their aqueous stability and to give them positive, negative, or neutral surface charges. Uptake and elimination behaviors of Daphnia magna exposed to PEI-coated and acid-modified MWNTs at concentrations of approximately 25 and 250 μg/L were quantified. PEI surface coatings did not appear to substantially impact nanotube accumulation or elimination rates. Although the PEI-modified nanotubes exhibited enhanced stability in aqueous solutions, they appeared to aggregate in the guts of D. magna in a manner similar to acid-treated nanotubes. The MWNTs were almost entirely eliminated by Daphnia fed algae during a 48 h elimination experiment, whereas elimination without feeding was typically minimal. Finally, PEI coatings increased MWNT toxicities, though this trend corresponded to the size of the PEI coatings, not their surface charges.     

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.     

Regulatory perspectives in the use and validation of QSAR. A case study: DEMETRA model for Daphnia toxicity

The DEMETRA acute toxicity model toward the water flea (Daphnia magna)was used as a case studyto outline a validation method compatible with regulatory use. Reliability, predictive power, uncertainty, and applicability were verified with an external test set of pesticides. Predictions for this external set using the DEMETRA model, developed ad hoc for pesticides, were compared with the results using ECOSAR and TOPKAT as benchmarks. The evaluation considered statistical parameters and the presence of errors, with their size and sign. DEMETRA gave good statistical predictions, and the maximum error of the outliers was lower than that with the other two models. DEMETRA gave a limited number of false negatives, and the use of defined rules indicated the level of uncertainty was acceptable.     

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.     

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.     

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.     

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.     

Evidence for the Stepwise Stress Model: Gambusia holbrooki and Daphnia magna under acid mine drainage and acidified reference water stress

The Stepwise Stress Model (SSM) states that a cascade of regulative behavioral responses with different intrinsic sensitivities and threshold values offers increased behavioral plasticity and thus a wider range of tolerance for environmental changes or pollutant exposures. We tested the SSM with a widely introduced fish Gambusia holbrooki (Girard) (Pisces, Poeciliidae) and the standard laboratory test species Daphnia magna Straus (Crustacea, Daphniidae). The stress was simulated by short-term exposure to acid mine drainage (AMD) and to acidified reference water (ACID). Recording of behavioral responses with the multispecies freshwater biomonitor (MFB) generated continuous time-dependent dose-response data that were modeled in three-dimensional (3D) surface plots. Both the pH-dependent mortalities and the strong linear correlations between pH and aqueous metals confirmed the toxicity of the AMD and ACID gradients, respectively, for fish and Daphnia, the latter being more sensitive. AMD stress at pH < or = 5.5 amplified circadian rhythmicity in both species, while ACID stress did so only in G. holbrooki. A behavioral stepwise stress response was found in both species: D. magna decreased locomotion and ventilation (first step) (AMD, ACID), followed by increased ventilation (second step) (AMD). G. holbrooki decreased locomotion (first step) (AMD, ACID) and increased ventilation at intermediate pH levels (second step) (AMD). Both species, although from different taxonomic groups and feeding habits, followed the SSM, which might be expanded to a general concept for describing the behavioral responses of aquatic organims to pollution. Stepwise stress responses might be applied in online biomonitors to provide more sensitive and graduated alarm settings, hence optimizing the "early warning" detection of pollution waves.