Life-history responses of Daphnia magna Straus to binary mixtures of toxic substances: pharmacological versus ecotoxicological modes of action

Two prevailing theoretical models: concentration addition (CA) and independent action (IA), predict mixture toxicity on the basis of known toxicities of the mixture components. To date, both models have been in most occasions evaluated using unicellular in vivo responses or biochemical in vitro responses. However, when considering more complex models such as the whole organism physiology or life-history traits, the dominant ecotoxicological mode of action, based on the exposure concentrations at which various toxicological effects become operative at the level of whole organism, should be considered. Offspring production in Daphnia magna is driven by the resources acquired from food, and the number of live offspring produced by an organism is the result of two independent factors: the number of eggs produced and the percentage of eggs that survive egg development. In this study joint toxicity effects on offspring production in D. magna were tested using binary mixtures of toxic contaminants known to specifically impair food acquisition (lambda-cyhalothrin and cadmium) or to cause egg mortality during development (3,4 dichloroaniline). Tests were performed using a simplified 10-day reproduction assay initiated with gravid females. The results obtained indicate that irrespective of their primary pharmacological mode of action, the joint toxicity of cadmium and lambda-cyhalothrin was predicted by the CA model, whereas the joint toxicity of cadmium and 3,4 dichloroaniline was predicted by the IA model. Thus, the results obtained indicate that for life-history traits such as reproduction responses, the dominant ecotoxicological rather than the pharmacological mode of action should be used for predicting joint mixture effects.     

Genetic variability in sublethal tolerance to mixtures of cadmium and zinc in clones of Daphnia magna Straus

To date, studies on genetic variability in the tolerance of aquatic biota to chemicals have focused on exposure to single chemicals. In the field, metals occur as elemental mixtures, and thus it is essential to study whether the genetic consequences of exposure to such mixtures differs from response to single chemicals. This study determined the feeding responses of three Daphnia magna Straus clones exposed to Cd and Zn, both individually and as mixtures. Tolerance to mixtures of Cd and Zn was expressed as the proportional feeding depression of D. magna to Cd at increasing zinc concentrations. A quantitative genetic analysis revealed that genotype and genotype x environmental factors governed population responses to mixtures of both metals. More specifically, genetic variation in tolerance to sublethal levels of Cd decreased at those Zn concentrations where there were no effects on feeding, and increased again at Zn concentrations that affected feeding. The existence of genotype x environmental interactions indicated that the genetic consequences of exposing D. magna to mixtures of Cd and Zn cannot be predicted from the animals' response to single metals alone. Therefore, current ecological risk assessment methodologies for predicting the effects of chemical mixtures may wish to incorporate the concept of genetic variability. Furthermore, exposure to low and moderate concentrations of Zn increased the sublethal tolerance to Cd. This induction of tolerance to Cd by Zn was also observed for D. magna fed algae pre-loaded with both metals. Furthermore, in only one clone, physiological acclimatization to zinc also induced tolerance to cadmium. These results suggest that the feeding responses of D. magna may be related to gut poisoning induced by the release of metals from algae under low pH conditions. In particular, both induction of metallothionein synthesis by Zn and competition between Zn and Cd ions for uptake at target sites on the gut wall may be involved in determining sublethal responses to mixtures of both metals.     

Pharmacological analysis of the pathogenesis of acute poisoning with the synthetic pyrethroid cypermethrin using the hydrobiont Daphnia magna Straus

The results of pharmacological analysis are presented which provide information on the pathogenesis of acute cypermethrin poisoning that involves disturbances in various systems of the organism. These include changes in the system of excitatory amino acids (EAAs) and violation of the free radical generation processes, Na + channel functioning, cholinergic transmission, etc. The screening of drugs belonging to various pharmacological groups influencing the toxicity of pyrethroids (EAA receptor antagonists, antioxidants, Na + channel blockers, M-cholinoreceptor blockers) revealed promising agents for the treatment of cypermethrin poisoning.     

Acute toxicity of 50 metals to Daphnia magna

Metals are essential for human life and physiological functions but may sometimes cause disorders. Therefore, we conducted acute toxicity testing of 50 metals in Daphnia magna: EC50s of seven elements (Be, Cu, Ag, Cd, Os, Au and Hg) were < 100 µg l^?1; EC50s of 13 elements (Al, Sc, Cr, Co, Ni, Zn, Se, Rb, Y, Rh, Pt, Tl and Pb) were between 100 and 1000 µg l^?1; EC50s of 14 elements (Li, V, Mn, Fe, Ge, As, In, Sn, Sb, Te, Cs, Ba, W and Ir) were between 1,001 and 100,000 µg l^?1; EC50s of six elements (Na, Mg, K, Ca, Sr and Mo) were > 100,000 µg l^?1; and. 7 elements (Ti, Zr, Bi, Nb, Hf, Re and Ta) did not show EC50 at the upper limit of respective aqueous solubility, and EC50s were not obtained. Ga, Ru and Pd adhered to the body of D. magna and physically retarded the movement of D. magna. These metals formed hydroxides after adjusting the pH. Therefore, here, we distinguished this physical effect from the physiological toxic effect. The acute toxicity results of 40 elements obtained in this study were not correlated with electronegativity. Similarly, the acute toxicity results of metals including the rare metals were also not correlated with first ionization energy, atomic weight, atomic number, covalent radius, atomic radius or ionic radius. Copyright © 2014 John Wiley & Sons, Ltd.     

Effects of water hardness and humic acid on zinc toxicity to Daphnia magna Straus

The effects of water hardness and humic acid (HA) on the acute and chronic toxicity of zinc to Daphnia magna were evaluated. Increases in either water hardness or HA concentration resulted in proportional decreases in both acute (3-day) and chronic (50-day) zinc toxicity. The effect of either of these variables (hardness or HA) was independent of the other in acute tests. In chronic tests, HA reduced the toxic effect of zinc on reproduction more in soft water than in hard water. In addition to reducing the chronic toxicity of zinc, our data suggest that an increase in water hardness also changes the mode of chronic toxic action of zinc. As previously shown for other metals, this study indicates that a reliance on only reproductive and survival endpoints in short-duration chronic tests may lead to an underestimation of the chronic toxicity of zinc.     

Toxicity of the cyanobacterium Cylindrospermopsis raciborskii to Daphnia magna

The effect of two strains of Cylindrospermopsis raciborskii on the survivorship, somatic growth, and detoxification processes of juvenile Daphnia magna were investigated. Both strains of C. raciborskii (and also Ankistrodesmus falcatus, used as the control) were given to newborn D. magna at equivalent biovolumes. The survival curves for D. magna subjected to the two C. raciborskii treatments differed from those of the starved and fed treatments. After 48 h of exposure, the percentage of D. magna surviving after exposure to Cylin-A (a cylindrospermopsin-producing strain isolated from Australia) and Cylin-P (a non-cylindrospermopsin-producing strain isolated from Portugal) was 10.00% and 93.33%, respectively. The strain that produces cylindrospermopsin caused the greatest toxic effect in juvenile D. magna. Statistically significant differences in D. magna body size between the four treatments (Cylin-A, Cylin-P, A. falcatus, and starved) were detected after 48 h of exposure. The juvenile D. magna that received the two C. raciborskii treatments showed an increase in size (relative to their size at T(0)) of 2.54% and 38.14%, respectively. These values were statistically significantly different than those of the A. falcatus-fed control (55.54%) and the starved control (11.47%). In both C. raciborskii treatments there was a tendency for increased GST enzyme activities after 24 h of exposure. Cylindrospermopsin was detected (HPLC-MS/MS) in D. magna tissues after 24 and 48 h (0.025 and 0.02 ng animal(-)1, respectively). The results of this study indicate that C. raciborskii can affect the fitness and growth potential of juvenile D. magna.     

The relative importance of water and food as cadmium sources to Daphnia magna Straus

Knowledge of the transport pathways of metals into aquatic organisms is paramount in determining the metal's potential mechanism of toxicity. To determine the relative importance of water and food as cadmium (Cd) sources for the cladoceran Daphnia magna grazing on the algae Chlorella vulgaris, we measured cadmium accumulation and toxicity (feeding inhibition and survival) in three genetically different clones of D. magna subsequent to water, food, and water and food exposures. We found that Cd uptake from water and food was independent of source and additive in effect, with D. magna juveniles accumulating twice as much Cd from water than from food (algae). However, the efficiency with which Cd was assimilated by D. magna from its algal diet was much higher (10%) than from water (0.3%). Uptake and toxic responses were inversely related: tolerant clones accumulated more Cd. As a consequence, models based on uptake of metals from the combined routes of water and food may be reliable to predict metal dynamics in the field, but may fail to predict toxic effects since tolerance to metals is not necessarily linked to reduced total uptake of metals.     

Toxicity of soybean rust fungicides to freshwater algae and Daphnia magna

Soybeans are intensively grown over large swaths of land in the Midwestern US. Introduction of the pathogenic fungus responsible for Soybean Rust (Phakopsora pachyrhizi) will likely result in a significant increase in the environmental load of strobilurin and conazole fungicides. We determined the toxicity of six such fungicides to the unicellular algae Pseudokirchneriella subcapitata and the aquatic invertebrate, Daphnia magna. We found that levels of concern of some fungicides were lower than annual average runoff concentrations predicted for Indiana. Our results suggest that pyraclostrobin and propiconazole, and to a lesser extent tebuconazole, may cause impacts to algae and daphnids in areas where soybeans are intensively grown. More studies are needed to describe the ecological effects of sublethal exposures to these fungicides, as well as monitoring environmental concentrations in watersheds where these fungicides are applied to soybeans.     

Toxicity prediction of binary combinations of cadmium, carbendazim and low dissolved oxygen on Daphnia magna

Environmental contamination is often characterised by a combination of stress factors of various sources (biological, physical and chemical). The predictability of their joint effects is an important stage in environmental risk assessment procedures. In this study, the two main conceptual models for mixture evaluation based on the effect of individual compounds, concentration addition (CA) and independent action (IA) and deviations to synergism/antagonism, "dose ratio" and "dose level" dependency were used. The single and combined effects of cadmium, carbendazim and low dissolved oxygen levels were assayed for life-cycle parameters (survival and feeding) of the water flea Daphnia magna Straus. The results of single exposures revealed an increase of acute and chronic toxicity as concentrations of cadmium and carbendazim increases. At low dissolved oxygen levels both survival and feeding parameters were significantly affected (P< or =0.05). In the acute mixture exposure of cadmium and carbendazim a "dose ratio" dependency was observed with a higher toxicity when cadmium was dominant whereas at high concentrations of carbendazim a lower effect on survival was observed. At chronic exposures an antagonistic deviation from IA model was observed for this mixture. The IA model showed to be adequate for toxicity prediction on acute exposure combinations with low DO levels where a synergistic behaviour was observed. However, at sublethal exposures IA and CA models failed by underestimation. Validation from toxicokinetic and toxicodynamic modelling studies should be made in the future as a way to understand toxicological pathways involved in complex mixture/combination exposures.     

Daphnia magna Straus: a new model for evaluating the antioxidant acton of water-soluble preparations in vivo

Hydrobionta species of Daphnia magna Straus were used as a test objects for in vivo evaluation of the antioxidant activity of three hydrophilic thiol compounds: reduced gluthatione, unithiol, and cysteine. These compounds exhibited significant differences in activity under oxidative stress conditions, in the dynamics of observed effects, and in the probability of inversion from anti- to pro-oxidant action. The main advantage of the proposed test objects in comparison to the conventional in vitro experiments (where the antioxidant effect is evaluated over a period of time from several minutes to several hours) is that the development of drug activity (pro- and antioxidant effects) can be monitored over a prolonged period of time (up to several days). In comparison to the tests on mammals, the new method is much simpler and allows the entire antioxidant protection (rather than separate systems) to be evaluated. It is recommended to use Daphnia magna Straus species for comparative evaluation of the antioxidant action of water-soluble preparations in vivo.     

Daphnia magna Straus: a test object for the pharmacological evaluation of GABA-ergic drugs in the whole organism

The toxicity of a series of GABAlytics (11 drugs) representing different pharmacological groups was evaluated in comparative experiments on Daphnia magna Straus and white mice. A high degree of correlation was established between the toxicity of GABA antagonists studied in daphnia and mice. The pharmacological analysis of the interaction of agonists and antagonists of GABA/benzodiazepine/ionophore-receptor complex--the competitive ligands for various binding sites--was carried out. It is suggested that the ability of GABA agonists to prevent the action of GABA antagonists in whole organism is mainly determined by their pharmacokinetic and pharmacodynamic features, rather than by direct competition for binding sites within the receptor complex.     

Tolerance and acclimation to zinc of field-collected Daphnia magna populations.

The zinc tolerance of two Daphnia magna populations collected at a zinc contaminated site was studied. One clone was isolated from each population in order to determine interclonal variation in zinc tolerance. 48hEC50-values, life table parameters, carapace lengths and cellular energy allocation (CEA) were used as test endpoints and compared with the results obtained with a standard laboratory clone. The natural clones were more tolerant to acute zinc toxicity (up to a factor of 4) and exhibited a higher reproduction rate (factor 2) and carapace length (factor 1.2). The optimal zinc concentrations for the natural clones ranged from 80 to 200 microg Zn/l. When cultured without zinc, the natural clones gradually lost their zinc tolerance. Therefore, the environmental relevance of using toxicity data obtained with organisms (natural, as well as laboratory clones) acclimated to culture media containing no or very small amounts of zinc can be questioned.     

Neurotoxicology of pyrethrin and the pyrethroid insecticides.

Natural pyrethrin and synthetic pyrethroid insecticides have been considered among the safest classes of insecticides available. Pyrethrins and pyrethroids are classified on the basis of their chemical structures and their toxicologic, neurophysiologic and pharmacologic effects. Cellular effects of pyrethrin and pyrethroid insecticides have been postulated to involve interactions with sodium channels, receptor-ionophore complexes, neurotransmitters, and ATPases. Toxicity is a function of chemical structure, metabolism, route of exposure, and the presence or absence of synergists. Pyrethroid insecticides are neurotoxic, and the development and severity of clinical signs is proportional to the nervous tissue pyrethroid concentration. Type I pyrethroid poisoning in mice and rats produces a syndrome characterized by tremors, prostration and altered startle reflexes. Type II pyrethroid poisoning in mice and rats causes ataxia, convulsions, hyperactivity, choreoathetosis and profuse salivation. A presumptive diagnosis of pyrethrin/pyrethroid poisoning is based upon history of exposure, development of appropriate clinical signs, and chemical analysis for insecticide residues. Treatment of pyrethrin and pyrethroid toxicosis involves basic life support, seizure control when needed, and the prevention of further insecticide absorption.     

Biomarker-based assessment of the toxicity of the antifungal clotrimazol to the microcrustacean Daphnia magna

Among the vast list of xenobiotics that may promote harmful effects in aquatic ecosystems, pharmaceuticals are currently a prominent class due to their ability to persist in these environments and also due to the lack of information regarding their effects on the different components of the aquatic biota. Antifungals in particular, despite their massive use, are not extensively studied in environbmental terms. The main objective of this study was to characterize the toxicity of the antifungal clotrimazole to the aquatic organism Daphnia magna. To attain this purpose, the effects of this compound were measured, focusing on the determination of acute lethality, and quantification of biomarkers, such as neurotoxicity (soluble cholinesterases, ChEs); and oxidative stress and metabolism (such as catalase, CAT; and glutathione-S-transferases, GSTs). The toxicity assessment with biomarkers was based on animals exposed to concentrations similar to those already found in surface waters in order to increase the ecological relevance of the obtained data. The results showed that exposure to clotrimazole was able to induce significant increases in both CAT amd GSTs activities. ChE activity was not significantly altered after clotrimazol exposure. In view of the above, it is concluded that the drug studied caused adverse effects in terms of oxidative stress, at an ecological relevant levels, showing that the presence of clotrimazol in the wild is not innocuous.     

Bioavailability of sediment-associated Cu and Zn to Daphnia magna

Exposures to mining-impacted, field-collected sediment (Clear Creek, CO, USA) contaminated with Cu (2.4 mg/g) and Zn (5.2 mg/g) were acutely toxic to juvenile Daphnia magna. Dissolved Cu and Zn in the overlying water (sediment+reference water) were at levels that could cause acute toxicity. To reduce dissolved metals below toxic levels, the sediment was repeatedly rinsed to remove any easily mobilized metals. Washing the sediment reduced dissolved Cu by 60% and Zn by 80%. D. magna exposed to washed sediment experienced higher survival (95%) compared to those exposed to the original sediment (<50%). Cu and Zn that remained associated with suspended sediment after washing were not bioavailable, since survival and tissue metal concentrations in D. magna exposed to both filtered (>0.45 microm) and unfiltered overlying water were statistically similar. Multiple regression analysis indicated that only dissolved Cu significantly contributed to mortality of D. magna whereas particulate Cu, particulate Zn, and dissolved Zn did not. Regression analysis on a combined dataset from all Clear Creek exposures (washed and unwashed), revealed a significant (p < 0.0001, r(2) = 0.76) relationship between the concentration of dissolved copper in the overlying water and the mortality of exposed Daphnia, yielding an estimated LC50 of 26 microg/L dissolved copper (hardness approximately 140 mg/L). The results of this study indicate that if the sediment of Clear Creek was subjected to a resuspension event that there would be a significant efflux of metals from the sediment into the water column, resulting in potentially toxic levels in the water column.     

Haem Peroxidase Activity in Daphnia magna: A Biomarker for Sub-lethal Toxicity Assessments of Kerosene-contaminated Groundwater

A novel biomarker was developed in Daphnia magna to detect organic pollution in groundwater. The haem peroxidase assay, which is an indirect means of measuring oxidase activity, was particularly sensitive to kerosene contamination. Exposure to sub-lethal concentrations of kerosene-contaminated groundwater resulted in a haem peroxidase activity increase by dose with a two-fold activity peak at 25%. Reproduction in D. magna remained unimpaired when exposed to concentrations below 25% for 21 days, and a decline in fecundity was only observed at concentrations above the peak in enzyme activity. The measurement of haem peroxidase activity in D. magna detected sublethal effects of kerosene in just 24h, whilst offering information on the health status of the organisms. The biomarker may be useful in determining concentrations above which detrimental effects would occur from long-term exposure for fuel hydrocarbons. Moreover, this novel assay detects exposure to chemicals in samples that would normally be classified as non-toxic by acute toxicity tests.