The influences of ambient and body calcium on cadmium and zinc accumulation in Daphnia magna

The dissolved uptake rate constant (k(u)), dietary assimilation efficiency (AE), and efflux rate constant (k(e)) of cadmium (Cd) and zinc (Zn) were quantified in a freshwater zooplankton Daphnia magna cultured at different ambient calcium (Ca) concentrations. The animals were first acclimated to different ambient Ca levels for 7 d from birth before the biokinetic measurements at corresponding Ca levels. With increasing ambient Ca level from 0.5 to 200 mg/L, the body Ca content increased from 0.91% (as tissue dry wt) to 3.75%. The k(u) for Cd decreased by nine times; for Zn it decreased by 2.6 times; and the AE decreased from 62 to 19% and from 46 to 24% for Cd and Zn, respectively. In contrast, Ca levels did not affect significantly the efflux rates of Cd and Zn. The effects of ambient and body Ca levels were separated by measuring the biokinetic parameters in both low--(0.5 mg/L) and high--(50 mg/L) Ca environments using the daphnids containing different body Ca levels. Ambient and body Ca levels had synergistic inhibitory effects on the AEs of Cd and Zn; however, the protective effects against dissolved uptake of Cd and Zn were explained fully by the effects of ambient Ca. The body Ca either had no significant effect (for Cd) or stimulative effects (for Zn). Multiphase biokinetic modeling using the measured parameters gave reasonable predictions of the body burdens of Cd and Zn in different Ca environments. Our results better explain the role of ambient and body Ca in the accumulation of Cd and Zn in D. magna.     

Kinetic uptake of bioavailable cadmium,selenium, and zinc by Daphnia magna

Kinetic uptake of Cd, Se(IV), and Zn by Daphnia magna from the dissolved phase was determined using radiotracer techniques in moderately hard water. The metal influx rate and distribution in the soft tissue and the exoskeleton of the daphnids as influenced by metal concentration, inorganic ligands including pH, Ca2+ and SO4(2-), and body size were quantified. When the metal concentrations were <180 nM for Cd and <769 nM for Zn, the concentration factor in daphnids increased linearly within the 12 h of exposure. At a higher concentration, apparent steady state was reached after 3 h of exposure. Cadmium and Zn distribution in the soft tissues was not affected by the total ambient concentrations, whereas Se distribution in the soft tissue decreased by 7 to 10% with increasing Se concentration from 16 to 643 nM. A linear positive power relationship was found between the influx rates of the metals and the ambient concentrations. The concentration factor for Se, however, decreased significantly with increasing Se concentration in water. The influx rate of metals was inversely related to the body size in a power function. When the pH in ambient water increased from 5.0 to 7.0, the influx rate of Cd, Se, and Zn increased by 2.9, 16.6, and 4.1 times, respectively. The influx rates of Cd, Se, and Zn decreased by 6.9, 8.7, and 4.4 times, respectively, with an increase in Ca2+ concentration from 0.6 to 5.1 mM. In contrast, the uptake rates of all three metals were not significantly affected by the SO4(2-) concentration. The majority of accumulated Se was distributed in the soft tissues after 12 h of exposure, whereas Cd and Zn were about evenly distributed in the soft tissue and exoskeleton. Any changes in pH, Ca2+, and SO4(2-) concentrations did not apparently affect their distributions in the daphnids. Our study provides important kinetic data necessary for delineating the exposure routes and for further development of the biotic ligand model in Daphnia. Using a bioenergetic-based kinetic model, we showed that the dissolved uptake is dominant for Zn accumulation (>50%). For Cd and Se, dietary exposure is dominant when the bioconcentration factors of these metals in phytoplankton are at the high end.     

Multiphase biokinetic modeling of cadmium accumulation in Daphnia magna from dietary and aqueous sources

We investigated the Cd accumulation dynamics in a freshwater cladoceran, Daphnia magna, and validated the biokinetic accumulation model under nonsteady-state conditions. All biokinetic parameters were monitored for the animals from birth to the adult stage. During the whole exposure period, two factors were observed to play critical roles in affecting Cd bioaccumulation. One was the Cd partitioning between the dissolved and dietary phases. Although the total Cd loads in the exposure media were kept constant, the increasing Cd partitioning from the water to the food promoted Cd bioaccumulation by approximately twofold. The other factor was the growth rate constant, which was comparable with the Cd efflux rate constant in daphnids and significantly influenced Cd accumulation because of its variation in different developmental stages. A multiphase modeling approach was used to simulate such a nonsteady-state process. This approach was viewed as a success, because the model simulations clearly aided in the interpretation of the experimentally observed temporal Cd accumulation in daphnids. Proper application of the kinetic bioaccumulation model undoubtedly will help us to understand time-dependent responses under the nonsteady state, which may be caused by episodic exposure or seasonal fluctuations in food/particle availability.     

Evolution of cadmium resistance in Daphnia magna

A diverse laboratory population of Daphnia magna Straus was established by combining individuals from eight sources. Artificial selection for increased resistance to the acute toxic effects of cadmium was performed, and after eight generations, the average median effective concentration increased from 61 to 180 microg/L. No differences in life span, offspring production, time to first brood, number of offspring in the first brood, or intrinsic rate of population increase (r) were observed between the cadmium-adapted population and the controls under ideal conditions or under conditions of temperature or feeding ration stress, but cadmium-adapted daphnids were smaller. Control and cadmium-adapted populations were equally sensitive to copper and malathion, but the cadmium-adapted population was less sensitive to lead and more sensitive to phenol. Analysis of amplified fragment-length polymorphisms indicated a significant decrease in genetic diversity in the cadmium-adapted population. Although the evolved cadmium resistance would allow adapted populations to exist in areas where cadmium concentrations would be toxic to unadapted populations, the decreased genetic diversity, smaller size, and increased sensitivity to at least one other toxicant could reduce the probability of long-term survival even in the absence of future cadmium exposure.     

Linking feeding activity and maturation of Daphnia magna following short-term exposure to fenvalerate

Feeding activity and maturation were investigated in Daphnia magna exposed to fenvalerate for 24 h. The feeding activity was monitored by measuring filtration rates and 15N-traced food assimilation. Exposure resulted in individuals with reduced feeding activity and smaller body size at concentrations of 0.3 microg/L or greater as well as delayed maturation at concentrations of 0.61microg/L or greater. Filtration rates recovered within 2 d of the exposure, but long-term effects of reduced feeding activity, such as growth retardation, occurred. Because D. magna needs to reach a minimum size to mature, growth retardation may explain the observed delay in maturity. The proposed causal relationships suggest that exposure to fenvalerate reduces feeding activity, resulting in growth retardation, which leads to delayed maturity. In cases of exposure to such toxicants, the monitoring of feeding activity may predict long-term effects on population parameters.     

Clonal variation in acetylcholinesterase biomarkers and life history traits following OP exposure in Daphnia magna

Two clones of Daphnia magna (Standard and Ruth) were exposed for 7 days to sub-lethal concentrations of acephate (5.0 and 10.0 mg/L). Survivorship, individual growth, reproduction and the population growth rate (lambda) were evaluated over three weeks. Acetylcholinesterase (AChE) activity was measured on days 2, 7 and 21. Acephate exposure inhibited AChE activity but had no direct effect on life history (LH) traits. There was also no effect of clone on AChE activity, LH and lambda. However, a significant interaction between clone and acephate concentration was found on both fecundity and lambda. AChE inhibition at 48 h was associated with a decrease in lambda in the Standard clone and an increase in lambda in clone Ruth. Therefore, our findings show that genotypic variation will influence the link between AChE activity and toxic effects at higher levels of biological organisation in D. magna.     

Modulation of sex ratios in Daphnia magna following multigenerational exposure to sewage treatment plant effluents

The influence of sewage treatment plant effluents on sex ratios in Daphnia magna was investigated. Female daphnids were acclimated for several generations to effluents from a municipal sewage treatment plant and a residential oxidation lagoon and then placed under conditions to maximize male offspring production. Both effluents resulted in a statistically significant decrease in male production and a shift in male broods from earlier broods to later broods near the end of the adult life cycle. For example, sex ratios in control daphnids ranged from 0.43 to 0.67 in broods 3–4 compared with 0.0–0.13 in daphnids exposed to the residential oxidation lagoon. Secondary sexual characteristics of both sexes were statistically significantly increased by the sewage lagoon effluent but not the municipal effluent. These preliminary results suggest that alteration in timing of sexual determination due to exposure to sewage treatment plant effluents could severely impact the survival of daphnid populations.     

Changes in the metabolic elimination profile of testosterone following exposure of the crustacean Daphnia magna to tributyltin

The biocide tributyltin has been found to cause the development of pseudohermaphroditic conditions in some neogastropod species. These abnormalities of the reproductive system have adversely affected the fecundity of some field populations of gastropods, resulting in local population declines. Current evidence suggests that tributyltin elicits these effects by interfering with the biotransformation of testosterone to other steroid derivatives, resulting in an elevation in endogenous testosterone or some of its bioactive derivatives. The purpose of the present study was to determine whether tributyltin altered testosterone metabolism in daphnids (Daphnia magna), a species commonly used in ecotoxicology testing. Exposure of daphnids to 1.2 microg (tin)/L caused a general increase in the rate of elimination of oxido-reduced, hydroxylated, and glucose-conjugated derivatives of testosterone. However, tributyltin exposure had no significant effect on the rate of elimination of the glucose-conjugated forms of the various oxido-reduced and hydroxylated derivatives of testosterone. As a result, the percentage of the oxido-reduced and hydroxylated metabolites of testosterone eliminated as glucose conjugates decreased with increasing tributyltin exposure levels. These results demonstrate that tributyltin causes alterations in testosterone metabolism in daphnids that would result in an increase in the production of oxido-reduced derivatives. These products are preferentially retained in the tissues of daphnids and are variously androgenic in vertebrates. The increased production of oxido-reduced derivatives of testosterone may be mechanically responsible for the masculinizing effects of tributyltin in some species and suggests that daphnids may be a suitable surrogate for evaluating the potential of chemicals to elicit this form of toxicity.     

Development of a chronic zinc biotic ligand model for Daphnia magna

The individual effects of the cations Ca(2+), Mg(2+), Na(+), and H(+) on the chronic toxicity of Zn to the waterflea Daphnia magna were investigated in different series of univariate experiments, resulting in the development of a chronic Zn biotic ligand model (BLM) for this species. Using the mathematical approach based on a linear relationship between cation activity and metal activity at the EC(x) level, the following stability constants for binding of competing cations to the biotic ligand (BL) were derived: logK(CaBL) = 3.22, logK(MgBL) = 2.69, logK(NaBL) = 1.90, and logK(HBL) = 5.77. With the derived constants and a logK(ZnBL) of 5.31, two different BLMs that predict chronic zinc toxicity (EC(50), no observed effect concentration (NOEC)) for D. magna as a function of water characteristics were developed. Fractions of binding sites occupied by Zn at the considered effect levels EC(50) and NOEC were 0.127 and 0.084, respectively. The NOEC-based model predicts the Zn toxicity within a factor of 2, while the chronic EC(50) could be predicted within a factor of 1.5. In the future, these chronic Zn BLMs for D. magna can improve the ecological relevancy of zinc risk assessments by decreasing the bioavailability-related uncertainty of zinc toxicity.     

Influence of duration of exposure to the pyrethroid fenvalerate on sublethal responses and recovery of Daphnia magna straus

This study compares lethal and sublethal responses of Daphnia magna Straus exposed to fenvalerate continuously (21 d) and as a pulse (24 h). Survival was reduced more severely in the continuous- than in the pulse-exposure regime. Complete mortality occurred at 1 microg/L for continuous exposure and at 3.2 microg/L for pulse exposure. Regarding reproductive endpoints, fenvalerate delayed the age at first reproduction. At the beginning of the reproductive phase (day 10), this delay resulted in a reduction of the neonates per living female at similar concentrations in both exposure regimes (0.3 and 0.1 microg/L for continuous and pulse exposure, respectively). The population growth rate was inhibited in continuous and pulse exposure at 0.3 and 0.6 microg/L, respectively. However, the effects of fenvalerate in the pulse exposure were transient. After 21 d, a recovery to values close to the controls occurred with respect to the total neonates per female and the population growth rate over a broad range of concentrations from 0.1 up to 1 microg/L. In contrast, no substantial recovery occurred in the continuous-exposure regime.     

Accumulation and regulation of zinc in Daphnia magna: links with homeostasis and toxicity

Zinc accumulation in Daphnia magna was investigated, and the results were linked to the previously established optimal concentration range for zinc and D. magna. It was observed that organisms cultured in this optimal range (300-600 microg Zn/L) contained 212 +/- 57 to 254 +/- 79 microg Zn/g dry weight. Lower and higher zinc contents were obtained after acclimation to previously established culture concentrations inducing deficiency and toxicity, respectively. The calculation of bioconcentration factors indicated that zinc was actively regulated, at least up to a concentration of 600 microg Zn/L. Zinc uptake and elimination are rapid processes; major increases and decreases in body content occurred within 1 day. Zinc concentrations in daphnids exposed to 600 microg Zn/L fluctuated with 2- to 3-day intervals, suggesting a role of molting in the regulation and elimination of zinc.     

A multivariate biomarker-based model predicting population-level responses of Daphnia magna

A multivariate model is proposed relating short-term biomarker measurements in Daphnia magna to chronic effects (21-d exposure) occurring at the population level (time to death, mean brood size, mean total young per female, intrinsic rate of natural increase, net reproductive rate, and growth). The results of the short-term exposure (48 h-96 h) to eight model toxicants (cadmium, chromium, mercury, tributyl tin, linear alkylsulfonic acid, sodium pentachlorophenolate, lindane, and 2,4-dichlorophenoxyacetic acid) on the following biomarkers were used for the multivariate model: digestive enzymes (amylase, cellulase, beta-galactosidase, trypsin, and esterase), enzymes of the intermediary metabolism (glycogen phosphorylase, glucose-6-phosphate dehydrogenase, pyruvate kinase, lactate dehydrogenase, and isocitrate dehydrogenase), cellular energy allocation (CEA) (protein, carbohydrate, and lipid content and electron transport activity), and DNA damage and antioxidative stress activity. Using partial least squares to latent structures (PLS), a two-component model was obtained with R2 of 0.68 and a Q2 value of 0.60 based on the combined analysis of a limited number of the 48- and 96-h biomarker responses. For the individual population-level responses, the R2 values varied from 0.66 to 0.77 and the Q2 values from 0.52 to 0.69. Energy-related biomarkers (cellular energy allocation, lipid contents, anaerobic metabolic activity--pyruvate kinase, and lactate dehydrogenase), combined with parameters related to oxidative stress (catalase) and DNA damage measured after 48 and 96 h of exposure, were able to predict long-term effects at higher levels of biological organization.     

Toxicity models of pulsed copper exposure to Pimephales promelas and Daphnia magna

Semiempirical models are useful for interpreting the response of aquatic organisms to toxicants as a function of exposure concentration and duration. Most applications predict cumulative mortality at the end of the test for constant exposure concentrations. Summary measures, such as the median lethal concentration, are then estimated as a function of concentration. Real-world exposures are not constant. Effects may depend on pulse timing, and cumulative analysis based only on integrated exposure concentration is not sufficient to interpret results. We undertook a series of pulsed-exposure experiments using standard toxicological protocols and interpreted the results (mortality, biomass, and reproduction) using a dynamic generalization of a Mancini/Breck--type model that includes two compartments, one for internal concentration as a function of exposure and one for site-of-action concentration or accumulated damage as a function of the internal dose. At exposure concentrations near the effects level, the model explained approximately 50% of the variability in the observed time history of survival, 43% of the change in biomass, and 83% of the variability in net reproduction. Unexplained variability may result from differences in organism susceptibility, amplified by the effects of small sample sizes in standard tests. The results suggest that response is sensitive to prior conditions and that constant-exposure experiments can underestimate the risk from intermittent exposures to the same concentration. For pulsed exposures, neither the average nor the maximum concentration alone is an adequate index of risk, which depends on both the magnitude, duration, and timing of exposure pulses. Better understanding about the impacts of pulsed exposures will require use of experimental protocols with significantly greater numbers of replicates.     

Cadmium accumulation and biochemical responses in Sparus aurata following sub-lethal Cd exposure

Cadmium (Cd), a heavy metal with limited biological function, is widely distributed in the aquatic environment as a result of natural and anthropogenic activities. The effect of 4 and 11 days exposure of gilthead sea bream Sparus aurata to sub-lethal concentrations of Cd was evaluated as levels of Cd content and Cd-metallothionein (MT) presence in different organs. The possible genotoxic effect was also evaluated in erythrocytes by using the "comet assay", a promising tool for estimating DNA damage at the single-cell level. The results obtained show that in the controls, Cd content was significantly higher in gills compared to in liver, but the treatment of fish with 0.1mg/l Cd induced a stronger accumulation of metal in liver depending on the length of the exposure period. Cd traces were found in plasma, muscle and kidney. Cd forms complexes in the cytosol with MT only in the liver but Cd-MT content significantly increased after 11 days of exposure to the metal, while after 4 days of treatment the protein level was similar to the control. The "comet assay" performed on S. aurata eryhtrocytes isolated from fish treated for 4 and 11 days with 0.1mg/l Cd, showed that there was no DNA damage at both exposure periods.     

Effect of sublethal doses of cadmium on the phototactic behavior of Daphnia magna

The effect of a sublethal concentration of cadmium (0.06 mg/ L) was tested on the phototactic behavior of a positively phototactic Daphnia magna clone. In experiments lasting 10 min, using animals that had been exposed to cadmium for 1 to 6 h, it was observed that the animals became significantly less positively phototactic after 4 h of exposure to 0.06 mg/L cadmium compared to control animals that had not been exposed to cadmium. In flow-through experiments that lasted for 6 h and during which there were repeated measurements, there was again a significant effect of cadmium exposure on the phototactic behavior of the animals. Irrespective of treatment, time had a significant effect. Results suggest that phototactic behavior can be used to detect sublethal concentrations of pollutant within a few hours, in short-term as well as in longer-lasting experiments with continuous flow-through and repeated stimulation of the animals.     

Importance of acclimation to environmentally relevant zinc concentrations on the sensitivity of Daphnia magna toward zinc

Daphnia magna was acclimated for six generations to an acclimation range of 0.02 to 74 microg/L of Zn2+. This range was determined by combining physicochemical water characteristics of European surface waters with total Zn concentrations in these waters in such a way that they resulted in minimal and maximal free (i.e., assumed bioavailable) Zn ion activities. No significant differences were found in acute Zn tolerance between the different acclimation concentrations: Average 48-h median effective concentration (EC50) values ranged from 608+/-94 to 713+/-249 microg/L of Zn2+. Also, no significant shifts in chronic tolerance were observed: Average 21-d EC50 (based on net reproductive rate) ranged from 91+/-20 to 124+/-22 microg/L of Zn2+. However, at test concentrations less than the 21-d EC50, acclimation significantly increased the reproductive capacity of the offspring produced. This indicates that metal acclimation is not necessarily accompanied by an increase in tolerance but also may manifest in other responses (e.g., reproduction rate). Organisms acclimated to a range from 6 to 22 microg/L of Zn2+ produced significantly more offspring than organisms acclimated to lower and higher Zn concentrations in test concentrations up to 50 microg/L of Zn2+. This range corresponds to a previously established optimal concentration range for D. magna. Bioconcentration factors indicated that Zn was actively regulated in the acclimation range tested.     

Effects of chronic dietary copper exposure on growth and reproduction of Daphnia magna

A matter of current, intense debate with regard to the effects of metals on biological systems is the potential toxicity of metals associated with food particles. Recently developed biotic ligand models (BLM), which predict the toxicity of waterborne metals, may not be valid if the dietary exposure route contributes to metal toxicity. The present study is, to our knowledge, the first that investigates the potential toxicity of dietary copper to a freshwater invertebrate (i.e., Daphnia magna) feeding on a live diet (i.e., the green alga Pseudokircheneriella subcapitata). Algae were exposed for 3 d to different copper concentrations, resulting in algal copper burdens between approximately 6.2 X 10(-16) and 250 x 10(-16) g cell(-1). These algae were then used as food in chronic, 21-d D. magna toxicity tests in which growth, reproduction, and copper accumulation were assessed. Three exposure scenarios were tested: A waterborne exposure, a dietary exposure, and a combined waterborne and dietary exposure. Although exposure to dietary copper resulted in an increased copper body burden of the adult daphnids, it did not contribute to toxicity and did not affect the 21-d effect concentrations expressed as waterborne copper, indicating that the previously established good predictive capacity of the chronic D. magna BLM is not affected. On the contrary, exposure to the highest dietary copper levels resulted in an increase of as much as 75% in growth and reproduction. To our knowledge, this is the first evidence that dietary copper exposure of a freshwater invertebrate feeding on a live diet resulted in a beneficial effect.     

Daphnia magna feeding behavior after exposure to tetradifon and recovery from intoxication.

The feeding behavior of the cladocera Daphnia magna subjected to a short-term exposure to the acaricide tetradifon (4-chlorophenyl 2,4, 5-trichlorophenyl sulfone) was studied. The experiments were performed using the unicellular algae Nannochloris oculata at a density of 5x10(5) cells/ml as food for the organisms. In a first experiment, three generations (F0, F1, and F3) of the daphnids were exposed to sublethal levels of tetradifon (0.1, 0.18, 0.22, and 0.44 mg/l) and the effect of the toxicant on filtration and ingestion rates was determined. Rates of filtration and ingestion of D. magna declined in the three generations studied with increasing toxicant concentrations; however, toxicant effect was greater in daphnids from generations F1 and F3 than in those from the parental generation F0. A second experiment was conducted in order to evaluate whether animals of a first (1) or third (F3) generation coming from parental daphnids (F0) previously exposed to those pesticide concentrations exhibited any alteration in feeding behavior when transferred to clean water (recovery period). The results indicated that the feeding rates of D. magna generations F1 and F3 were still affected during the recovery period but to a less degree. The effective tetradifon concentrations D. magna at which feeding rates were reduced to 50% that of controls (EC(50)) were also calculated.     

The uptake of cadmium from a dietary and soluble source by the crustacean Daphnia magna

Daphnia were exposed to radioactively labeled cadmium in solution and in the presence of Chlorella which had been preloaded with the metal to varying extents. Illuminated algal cells retained the cadmium and greatly reduced its availability to the daphnids. Autoradiographic evidence was obtained which implicated the exoskeleton as a major sink for the cadmium taken up from solution. Cadmium in solution at a concentration close to the 48 hr LC50 level did not affect respiration during the first 6 hr of exposure. Retention patterns were similar, regardless of the source of cadmium, but ecdysis resulted in a considerable loss of body burden provided that this had been acquired via a predominantly soluble route.     

Acute toxicity of cadmium,zinc, and cadmium-zinc mixtures toDaphnia magna

Experiments were performed in flow-through apparatus to determine the acute toxicity of zinc, cadmium, and their mixtures toDaphnia magna in order to compare sensitivity ofDaphnia relative to that of other organisms and to determine if the metal mixture behaves differently than expected on the basis of single metal experiments. Dosage-mortality curves are derived forDaphnia populations exposed to zinc and cadmium for 36, 48, 60, 72, and 96 hr periods. Similar curves are derived forDaphnia exposed to equally potent mixtures of zinc and cadmium as calculated from initial experiments and forDaphnia exposed to equal concentration mixtures of the metals. Cadmium is more toxic than zinc, but zinc-cadmium mixtures are less toxic than expected.Daphnia magna is considerably more sensitive to these metals than are other invertebrates and vertebrates previously studied. This study demonstrates errors which can arise in attempts to set water quality criteria based upon short term studies of organisms with long life spans.This work was supported by a Quebec Minister of Education grant to P. D. Anderson, by a Quebec scholarship to E. N. Attar, and by a National Science and Engineering Council grant to E. J. Maly. We thank G. Leduc and P. D. Anderson for helpful criticism and discussion