Daphnia as a Sentinel Species for Environmental Health Protection: A Perspective on Biomonitoring and Bioremediation of Chemical Pollution.

Despite available technology and the knowledge that chemical pollution damages human and ecosystem health, chemical pollution remains rampant, ineffectively monitored, rarely prevented, and only occasionally mitigated. We present a framework that helps address current major challenges in the monitoring and assessment of chemical pollution by broadening the use of the sentinel species Daphnia as a diagnostic agent of water pollution. And where prevention has failed, we propose the application of Daphnia as a bioremediation agent to help reduce hazards from chemical mixtures in the environment. By applying "omics" technologies to Daphnia exposed to real-world ambient chemical mixtures, we show improvements at detecting bioactive components of chemical mixtures, determining the potential effects of untested chemicals within mixtures, and identifying targets of toxicity. We also show that using Daphnia strains that naturally adapted to chemical pollution as removal agents of ambient chemical mixtures can sustainably improve environmental health protection. Expanding the use of Daphnia beyond its current applications in regulatory toxicology has the potential to improve both the assessment and the remediation of environmental pollution.

Climate warming restructures an aquatic food web over 28 years.

Climate warming can restructure lake food webs if trophic levels differ in their thermal responses, but evidence for these changes and their underlying mechanisms remain scarce in nature. Here we document how warming lake temperatures by up to 2°C, rather than changes in trophic state or fishing effort, have restructured the pelagic food web of a large European lake (Lake Maggiore, Italy). Our approach exploited abundance and biomass data collected weekly to yearly across five trophic levels from 1981 to 2008. Temperature generally had stronger effects on taxa than changes in fish predation or trophic state mediated through primary productivity. Consequently, we found that, as the lake warmed, the food web shifted in numerical abundance towards predators occupying middle trophic positions. Of these taxa, the spiny water flea (Bythotrephes longimanus) most prospered. Bythotrephes strongly limited abundances of the keystone grazer Daphnia, strengthening top-down structuring of the food web. Warmer temperatures partly restructured the food web by advancing peak Bythotrephes densities by approximately 60 days and extending periods of positive population growth by three times. Nonetheless, our results suggested that advances in the timing and size of peak Bythotrephes densities could not outpace changes in the timing and size of peak densities in their Daphnia prey. Our results provide rare evidence from nature as to how long-term warming can favour higher trophic levels, with the potential to strengthen top-down control of food webs.

Road Salt Impacts Freshwater Zooplankton at Concentrations below Current Water Quality Guidelines.

Widespread use of NaCl for road deicing has caused increased chloride concentrations in lakes near urban centers and areas of high road density. Chloride can be toxic, and water quality guidelines have been created to regulate it and protect aquatic life. However, these guidelines may not adequately protect organisms in low-nutrient, soft water lakes such as those underlain by the Precambrian Shield. We tested this hypothesis by conducting laboratory experiments on six Daphnia species using a soft water culture medium. We also examined temporal changes in cladoceran assemblages in the sediments of two small lakes on the Canadian Shield: one near a highway and the other >3 km from roads where salt is applied in the winter. Our results showed that Daphnia were sensitive to low chloride concentrations with decreased reproduction and increased mortality occurring between 5 and 40 mg Cl-/L. Analysis of cladoceran remains in lake sediments revealed changes in assemblage composition that coincided with the initial application of road salt in this region. In contrast, there were no changes detected in the remote lake. We found that 22.7% of recreational lakes in Ontario have chloride concentrations between 5 and 40 mg/L suggesting that cladoceran zooplankton in these lakes may already be experiencing negative effects of chloride.

Ca2+ levels in Daphnia hemolymph may explain occurrences of daphniid species along recent Ca gradients in Canadian soft-water lakes.

Calcium levels are declining in eastern North American and western European lakes. This widespread issue is affecting the composition of crustacean zooplankton communities, as the presence and abundance of several calcium-rich daphniid species are declining, while two other daphniids, D. catawba and D. ambigua, that apparently tolerate low calcium environments, are prospering. The physiological basis for low calcium tolerance of these daphniids is unknown. In this study the presence of one Ca-rich (D. pulicaria) and one Ca-poor (D. ambigua) daphniid species in Canadian Shield lakes is assessed in relation to lake water Ca levels. The occurrence of D. ambigua was independent of Ca levels in Ontario lakes, whereas D. pulicaria was more likely to occur in lakes with relatively more Ca. In the laboratory, D. ambigua maintained lower levels of hemolymph Ca2+ across a range of low Ca levels (0.7 to 7 mg l-1) compared with D. pulicaria. The hemolymph pH remained steady across this Ca gradient in D. ambigua while it was significantly more acidic in D. pulicaria in the two lowest Ca treatments. While Ca2+ uptake was observed adjacent to the surface of D. ambigua individuals, Ca2+ loss was observed for D. pulicaria assayed under moderately high Ca levels. Based on these observations we propose that D. ambigua is able to survive in low Ca lakes by maintaining low free ionic Ca2+ levels in the hemolymph which minimizes the Ca gradient across the body wall in low Ca water thus limiting overall Ca loss and facilitating Ca2+ uptake.

Predicting chronic copper and nickel reproductive toxicity to Daphnia pulex-pulicaria from whole-animal metabolic profiles.

The emergence of omics approaches in environmental research has enhanced our understanding of the mechanisms underlying toxicity; however, extrapolation from molecular effects to whole-organism and population level outcomes remains a considerable challenge. Using environmentally relevant, sublethal, concentrations of two metals (Cu and Ni), both singly and in binary mixtures, we integrated data from traditional chronic, partial life-cycle toxicity testing and metabolomics to generate a statistical model that was predictive of reproductive impairment in a Daphnia pulex-pulicaria hybrid that was isolated from an historically metal-stressed lake. Furthermore, we determined that the metabolic profiles of organisms exposed in a separate acute assay were also predictive of impaired reproduction following metal exposure. Thus we were able to directly associate molecular profiles to a key population response - reproduction, a key step towards improving environmental risk assessment and management.

Metabolomics confirms that dissolved organic carbon mitigates copper toxicity.

Reductions in atmospheric emissions from the metal smelters in Sudbury, Canada, produced major improvements in acid and metal contamination of local lakes and indirectly increased dissolved organic carbon (DOC) concentrations. Metal toxicity, however, has remained a persistent problem for aquatic biota. Integrating high-throughput, nontargeted mass spectrometry metabolomics with conventional toxicological measures elucidated the mediating effects of dissolved organic matter (DOM) on the toxicity of Cu to Daphnia pulex-pulicaria, a hybrid isolated from these soft water lakes. Two generations of daphniids were exposed to Cu (0-20 µg/L) at increasing levels of natural DOM (0-4 mg DOC/L). Added DOM reduced Cu toxicity monotonically with median lethal concentration values increasing from 2.3 µg/L Cu without DOM to 22.7 µg/L Cu at 4 mg DOC/L. Reproductive output similarly benefited, increasing with DOM, yet falling with increases in Cu. Second generation reproduction was more impaired than the first generation. Dissolved organic matter had a greater influence than Cu on the metabolic status of the daphniids. Putative identification of metabolite peaks indicated that DOM elevation increased the metabolic energy status of the first generation animals, but this benefit was reduced in the second generation, although evidence of increased oxidative stress was detected. These results indicate that Sudbury's terrestrial ecosystems should be managed to increase aquatic DOM supply to enable daphniid colonists to both survive and foster stable populations.

Synergistic interactions of biotic and abiotic environmental stressors on gene expression.

Understanding the response of organisms to multiple stressors is critical for predicting if populations can adapt to rapid environmental change. Natural and anthropogenic stressors often interact, complicating general predictions. In this study, we examined the interactive and cumulative effects of two common environmental stressors, lowered calcium concentration, an anthropogenic stressor, and predator presence, a natural stressor, on the water flea Daphnia pulex. We analyzed expression changes of five genes involved in calcium homeostasis - cuticle proteins (Cutie, Icp2), calbindin (Calb), and calcium pump and channel (Serca and Ip3R) - using real-time quantitative PCR (RT-qPCR) in a full factorial experiment. We observed strong synergistic interactions between low calcium concentration and predator presence. While the Ip3R gene was not affected by the stressors, the other four genes were affected in their transcriptional levels by the combination of the stressors. Transcriptional patterns of genes that code for cuticle proteins (Cutie and Icp2) and a sarcoplasmic calcium pump (Serca) only responded to the combination of stressors, changing their relative expression levels in a synergistic response, while a calcium-binding protein (Calb) responded to low calcium stress and the combination of both stressors. The expression pattern of these genes (Cutie, Icp2, and Serca) were nonlinear, yet they were dose dependent across the calcium gradient. Multiple stressors can have complex, often unexpected effects on ecosystems. This study demonstrates that the dominant interaction for the set of tested genes appears to be synergism. We argue that gene expression patterns can be used to understand and predict the type of interaction expected when organisms are exposed simultaneously to natural and anthropogenic stressors.

Consequences of calcium decline on the embryogenesis and life history of Daphnia magna.

Ambient calcium is declining in thousands of soft-water lake habitats in temperate regions as a consequence of unsustainable forestry practices, decreased atmospheric calcium deposition and acidic deposition. As their exoskeleton is heavily reinforced with calcium, freshwater crustaceans have a high specific calcium requirement relative to other aquatic organisms. Daphnia, in particular, is an ideal crustacean for investigating the consequences of calcium decline because it is an abundant and important member of freshwater zooplankton communities. Although it has been established that adult and juvenile Daphnia have different tolerances to low ambient calcium as a result of their different life stage-specific calcium requirements, the consequences of declining calcium on embryonic development have never been investigated. Here, we describe the distribution of calcium in embryonic stages of D. magna and introduce a novel and easy to use staging scheme. We tested whether calcium can be traced from mothers to their offspring. Finally, we assessed the fitness consequences of maternal provisioning in limiting calcium environments. We found that while embryos require calcium for their development and moulting, they do not equilibrate with environmental calcium levels. Instead, we were able to trace calcium from mothers to their offspring. Furthermore, our data strongly suggest that females are faced with an allocation trade-off between providing calcium to their offspring and using it for growth and moulting. Together, these data provide novel insights into the consequences of calcium decline for freshwater zooplankton.

Food quantity affects the sensitivity of Daphnia to road salt.

Road deicing operations have raised chloride (Cl) levels in many temperate lakes in Europe and North America. These lakes vary widely in trophic status, but to date, no one has quantified the interaction between food quantity and road salt toxicity. We examined the effects of food quantity (particulate algal C concentration (C)) on the chronic toxicity of Cl to Daphnia in soft-water bioassays. There was a strong positive linear relationship (r(2) = 0.92 for NaCl and r(2) = 0.96 for CaCl2) between food quantity and Cl LC50. As food quantity increased from 0.2 to 1.0 mg C/L (levels characteristic of oligotrophic to eutrophic lakes, respectively), the chronic Cl LC50 increased from 55.7 to 284.8 mg Cl/L. Salt type (NaCl or CaCl2) did not affect the Cl LC50, Daphnia life history parameters, or the intrinsic rate of population increase (r). The life history parameter most sensitive to Cl was neonate production. Cl did not inhibit egg production, nor was the maternal lipid investment in eggs changed, but egg viability and the subsequent release of live neonates decreased as Cl levels increased and food decreased. Our results suggest the trophic status of lakes should be considered when assessing ecological threat from Cl.

The jellification of north temperate lakes.

Calcium (Ca) concentrations are decreasing in softwater lakes across eastern North America and western Europe. Using long-term contemporary and palaeo-environmental field data, we show that this is precipitating a dramatic change in Canadian lakes: the replacement of previously dominant pelagic herbivores (Ca-rich Daphnia species) by Holopedium glacialis, a jelly-clad, Ca-poor competitor. In some lakes, this transformation is being facilitated by increases in macro-invertebrate predation, both from native (Chaoborus spp.) and introduced (Bythotrephes longimanus) zooplanktivores, to which Holopedium, with its jelly coat, is relatively invulnerable. Greater representation by Holopedium within cladoceran zooplankton communities will reduce nutrient transfer through food webs, given their lower phosphorus content relative to daphniids, and greater absolute abundances may pose long-term problems to water users. The dominance of jelly-clad zooplankton will likely persist while lakewater Ca levels remain low.

Forests fuel fish growth in freshwater deltas.

Aquatic ecosystems are fuelled by biogeochemical inputs from surrounding lands and within-lake primary production. Disturbances that change these inputs may affect how aquatic ecosystems function and deliver services vital to humans. Here we test, using a forest cover gradient across eight separate catchments, whether disturbances that remove terrestrial biomass lower organic matter inputs into freshwater lakes, thereby reducing food web productivity. We focus on deltas formed at the stream-lake interface where terrestrial-derived particulate material is deposited. We find that organic matter export increases from more forested catchments, enhancing bacterial biomass. This transfers energy upwards through communities of heavier zooplankton, leading to a fourfold increase in weights of planktivorous young-of-the-year fish. At least 34% of fish biomass is supported by terrestrial primary production, increasing to 66% with greater forest cover. Habitat tracers confirm fish were closely associated with individual catchments, demonstrating that watershed protection and restoration increase biomass in critical life-stages of fish.

Metal and proton toxicity to lake zooplankton: a chemical speciation based modelling approach.

The WHAM-FTOX model quantifies the combined toxic effects of protons and metal cations towards aquatic organisms through the toxicity function (FTOX), a linear combination of the products of organism-bound cation and a toxic potency coefficient for each cation. We describe the application of the model to predict an observable ecological field variable, species richness of pelagic lake crustacean zooplankton, studied with respect to either acidification or the impacts of metals from smelters. The fitted results give toxic potencies increasing in the order H(+) < Al < Cu < Zn < Ni. In general, observed species richness is lower than predicted, but in some instances agreement is close, and is rarely higher than predictions. The model predicts recovery in agreement with observations for three regions, namely Sudbury (Canada), Bohemian Forest (Czech Republic) and a subset of lakes across Norway, but fails to predict observed recovery from acidification in Adirondack lakes (USA).

Changes in water chemistry can disable plankton prey defenses.

The effectiveness of antipredator defenses is greatly influenced by the environment in which an organism lives. In aquatic ecosystems, the chemical composition of the water itself may play an important role in the outcome of predator-prey interactions by altering the ability of prey to detect predators or to implement defensive responses once the predator's presence is perceived. Here, we demonstrate that low calcium concentrations (<1.5 mg/L) that are found in many softwater lakes and ponds disable the ability of the water flea, Daphnia pulex to respond effectively to its predator, larvae of the phantom midge, Chaoborus americanus. This low-calcium environment prevents development of the prey's normal array of induced defenses, which include an increase in body size, formation of neck spines, and strengthening of the carapace. We estimate that this inability to access these otherwise effective defenses results in a 50-186% increase in the vulnerability of the smaller juvenile instars of Daphnia, the stages most susceptible to Chaoborus predation. Such a change likely contributes to the observed lack of success of daphniids in most low-calcium freshwater environments, and will speed the loss of these important zooplankton in lakes where calcium levels are in decline.

Application of Biotic Ligand and Toxic Unit modeling approaches to predict improvements in zooplankton species richness in smelter-damaged lakes near Sudbury, Ontario.

Using a 30-year record of biological and water chemistry data collected from seven lakes near smelters in Sudbury (Ontario, Canada) we examined the link between reductions of Cu, Ni, and Zn concentrations and zooplankton species richness. The toxicity of the metal mixtures was assessed using an additive Toxic Unit (TU) approach. Four TU models were developed based on total metal concentrations (TM-TU); free ion concentrations (FI-TU); acute LC50s calculated from the Biotic Ligand Model (BLM-TU); and chronic LC50s (acute LC50s adjusted by metal-specific acute-to-chronic ratios, cBLM-TU). All models significantly correlated reductions in metal concentrations to increased zooplankton species richness over time (p < 0.01) with a rank based on r(2) values of cBLM-TU > BLM-TU = FI-TU > TM-TU. Lake-wise comparisons within each model showed that the BLM-TU and cBLM-TU models provided the best description of recovery across all seven lakes. These two models were used to calculate thresholds for chemical and biological recovery using data from reference lakes in the same region. A threshold value of TU = 1 derived from the cBLM-TU provided the most accurate description of recovery. Overall, BLM-based TU models that integrate site-specific water chemistry-derived estimates of toxicity offer a useful predictor of biological recovery.

An integrated multi-disciplinary approach for studying multiple stressors in freshwater ecosystems: Daphnia as a model organism.

The increased overexploitation of freshwater ecosystems and their extended watersheds often generates a cascade of anthropogenic stressors (e.g., acidification, eutrophication, metal contamination, Ca decline, changes in the physical environment, introduction of invasive species, over-harvesting of resources). The combined effect of these stressors is particularly difficult to study, requiring a coordinated multi-disciplinary effort and insights from various sub-disciplines of biology, including ecology, evolution, toxicology, and genetics. It also would benefit from a well-developed and broadly accepted model systems. The freshwater crustacean Daphnia is an excellent model organism for studying multiple stressors because it has been a chosen focus of study in all four of these fields. Daphnia is a widespread keystone species in most freshwater ecosystems, where it is routinely exposed to a multitude of anthropogenic and natural stressors. It has a fully sequenced genome, a well-understood life history and ecology, and a huge library of responses to toxicity. To make the case for its value as a model species, we consider the joint and separate effects of natural and three anthropogenic stressors-climatic change, calcium decline, and metal contaminants on daphniids. We propose that integrative approaches marrying various subfields of biology can advance our understanding of the combined effects of stressors. Such approaches can involve the measuring of multiple responses at several levels of biological organization from molecules to natural populations. For example, novel interdisciplinary approaches such as transcriptome profiling and mutation accumulation experiments can offer insights into how multiple stressors influence gene transcription and mutation rates across genomes, and, thus, help determine the causal mechanism between environmental stressors and population/community effects as well as long-term evolutionary patterns.

RISING WATER TEMPERATURES ALTER LIPID DYNAMICS AND REDUCE N-3 ESSENTIAL FATTY ACID CONCENTRATIONS IN SCENEDESMUS OBLIQUUS (CHLOROPHYTA)(1).

The biosynthesis of nutritionally important polyunsaturated fatty acids (PUFAs) in phytoplankton is influenced by environmental temperature. We investigated the potential of climate warming to alter lipid dynamics of Scenedesmus obliquus (Turpin) Kütz. by comparing lipid and fatty acid (FA) profiles as well as FA metabolism (using [1-(14) C] acetate) at 20°C and 28°C. We documented an overall decline (53%-37%) in the proportion of n-3 PUFA (in particular, of α-linolenic acid [ALA; 18:3n-3]), and a concomitant increase in saturated fatty acids (SAFAs) in total lipids (TLs) at 28°C, consistent with enhanced incorporation of radioactivity from [1-(14) C] acetate into total 16:0, 18:1, and decreased incorporation into 18:2 and 18:3 FA (from 36% to 22% of the total) at 28°C. Glycerophospholipids were also affected by warming; ALA and stearidonic acids (SDAs; 18:4n-3) both decreased (by 13% and 15%, respectively) in phosphatidylcholine (PC) and (by 24% and 20%, respectively) in phosphatidylethanolamine (PE). The characteristic FA in phosphatidylglycerol (PG; 16:1n-13t) increased (by 22%) at 28°C. The activities of desaturases, which add double bonds to FA moieties, comprised the major suite of reactions affected by the temperature increase in TL and polar lipid (PL) classes. Climate modelers predict an increase in the number of extreme heat days in summer at temperate latitudes, with parallel projected increases in water temperatures of shallow water bodies. Our results suggest that the overall decrease in the essential n-3 FA ALA in S. obliquus at higher water temperatures may lower food quality for higher tropic levels, adding another climate-warming stress.

Accumulated organic debris in catch basins improves the efficacy of S-methoprene against mosquitoes in Toronto, Ontario, Canada.

To control West Nile Virus in the greater Toronto area of Ontario, Canada, S-methoprene (Altosid XRbriquets 2.1% AI) is applied each year to storm water catch basins. Because the efficacy of the XRbriquets to reduce adult mosquito populations had not been evaluated locally and was influenced by organic debris in a pilot study, we compared the efficacy of the briquets in 17 sediment and debris-filled catch basins versus 20 catch basins that were vacuumed free of debris. Emergence rates approached 100% in the 5 untreated control catch basins. Emergence rates were significantly lower, and S-methoprene was detected more often and at higher levels, in debris-filled basins versus cleaned catch basins. Overall, 20% of pupae emerged from clean catch basins versus only 3% from debris-filled ones, the difference between treatments becoming significant after 26 days. S-methoprene and total organic carbon concentrations in the catch basins were positively correlated (P < 0.001). We hypothesize that S-methoprene is binding to the organic fraction in the water and sediment in the debris-filled basins, prolonging S-methoprene doses, which are reflected in lower mosquito emergence rates.

Environmental stability and lake zooplankton diversity - contrasting effects of chemical and thermal variability.

Environmental variability in space and time is a primary mechanism allowing species that share resources to coexist. Fluctuating conditions are a double edged sword for diversity, either promoting coexistence through temporal niche partitioning or excluding species by stochastic extinctions. The net effect of environmental variation on diversity is largely unknown. We examined the association between zooplankton species richness in lakes and environmental variability on interannual, seasonal and shorter time scales, as well as long-term average conditions. We analyzed data on physical, chemical and biological limnology in 53 temperate zone lakes in North America and Europe sampled over a combined 1042 years. Large fluctuations in pH, phosphorus and dissolved organic carbon concentration on different time scales were associated with reduced zooplankton species richness. More species were found in lakes that showed greater temperature variation on all time scales. Environmental variability on different time scales showed similar or, in some cases, stronger associations with zooplankton species richness compared with long-term average conditions. Our results suggest that temporal fluctuations in the chemical environment tend to exclude zooplankton species while temperature variability promotes greater richness. The results indicate that anthropogenic increases in temporal variability of future climates may have profound effects on biodiversity.

Communities contain closely related species during ecosystem disturbance.

Predicting community and species responses to disturbance is complicated by incomplete knowledge about species traits. A phylogenetic framework should partially solve this problem, as trait similarity is generally correlated with species relatedness, closely related species should have similar sensitivities to disturbance. Disturbance should thus result in community assemblages of closely related species. We tested this hypothesis with 18 disturbed and 16 reference whole-lake, long-term zooplankton data sets. Regardless of disturbance type, communities generally contained more closely related species when disturbed. This effect was independent of species richness, evenness, and abundance. Communities already under stress (i.e., those in acidic lakes) changed most when disturbed. Species sensitivities to specific disturbances were phylogenetically conserved, were independent of body size, and could be predicted by the sensitivities of close relatives within the same community. Phylogenetic relatedness can effectively act as a proxy for missing trait information when predicting community and species responses to disturbance.

The widespread threat of calcium decline in fresh waters.

Calcium concentrations are now commonly declining in softwater boreal lakes. Although the mechanisms leading to these declines are generally well known, the consequences for the aquatic biota have not yet been reported. By examining crustacean zooplankton remains preserved in lake sediment cores, we document near extirpations of calcium-rich Daphnia species, which are keystone herbivores in pelagic food webs, concurrent with declining lake-water calcium. A large proportion (62%, 47 to 81% by region) of the Canadian Shield lakes we examined has a calcium concentration approaching or below the threshold at which laboratory Daphnia populations suffer reduced survival and fecundity. The ecological impacts of environmental calcium loss are likely to be both widespread and pronounced.