Can a shift in dominant species of Microcystis alter growth and reproduction of waterfleas?

The bloom-forming species Microcystis wesenbergii and M. aeruginosa occur in many lakes globally, and may exhibit alternating blooms both spatially and temporally. As environmental changes increase, cyanobacteria bloom in more and more lakes and are often dominated by M. wesenbergii. The adverse impact of M. aeruginosa on co-existing organisms including zooplanktonic species has been well-studied, whereas studies of M. wesenbergii are limited. To compare effects of these two species on zooplankton, we explored effects of exudates from different strains of microcystin-producing M. aeruginosa (Ma905 and Ma526) and non-microcystin-producing M. wesenbergii (Mw908 and Mw929), on reproduction by the model zooplankter Daphnia magna in both chronic and acute exposure experiments. Specifically, we tested physiological, biochemical, molecular and transcriptomic characteristics of D. magna exposed to Microcystis exudates. We observed that body length and egg and offspring number of the daphnid increased in all treatments. Among the four strains tested, Ma526 enhanced the size of the first brood, as well as total egg and offspring number. Microcystis exudates stimulated expression of specific genes that induced ecdysone, juvenile hormone, triacylglycerol and vitellogenin biosynthesis, which, in turn, enhanced egg and offspring production of D. magna. Even though all strains of Microcystis affected growth and reproduction, large numbers of downregulated genes involving many essential pathways indicated that the Ma905 strain might contemporaneously induce damage in D. magna. Our study highlights the necessity of including M. wesenbergii into the ecological risk evaluation of cyanobacteria blooms, and emphasizes that consequences to zooplankton may not be clear-cut when assessments are based upon production of microcystins alone.

Rearing and Experimental Uses of Daphnia: Controlling Animal Nutrition and Assessing Lifespan and Life-History Traits.

Caloric restriction has been found to extend the lifespan of many organisms including mammals and other vertebrates. With lifespans exceeding months to years, age-related experiments involving fish and mammals can be overtly costly, both in terms of time and funding. The freshwater crustacean, Daphnia, has a relatively short lifespan (∼50 to 100 days), which makes it a cost-effective alternative animal model for longevity and aging studies. Besides age-specific mortality, there are a suite of physiological responses connected to "healthspan" that can be tracked as these animals age including growth, reproduction, and metabolic rates. These responses can be complemented by assessment of molecular and cellular processes connected to aging and health. Lifespan and metabolism of this model organism is responsive to long studied modulators of aging, such as rearing temperature and nutritional manipulation, but also pharmacological agents that target aging, e.g., rapamycin, which adds to its usefulness as a model organism. Here we describe how to culture Daphnia for aging experiments including maintaining laboratory populations of Daphnia mothers, growing algal food, and manipulating nutrition of these animals. In addition, we provide methods for tracking common physiological and longevity responses of Daphnia. This protocol provides researchers planning to use this model organism with methods to establish and maintain Daphnia populations and to standardize their experimental approaches. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing algae for Daphnia food Basic Protocol 2: General methods for culturing Daphnia Basic Protocol 3: Standardizing and controlling nutrition for experimental Daphnia Basic Protocol 4: Monitoring Daphnia lifespan Basic Protocol 5: Evaluating Daphnia health: Heart rate and respiration, body mass and growth rates, and reproduction.

Thresholds and interactive effects of BPA-gradient and temperature on life history traits of Daphnia magna.

Bisphenol A (BPA), a synthetic organic compound widely used in the production of plastics, is recognized as an emerging contaminant because of its toxicity and the potential risks associated with bioaccumulation in organisms. Despite potential environmental hazards, there is a lack of studies examining BPA toxicity mechanisms and its potential impact on various trophic levels, with even fewer exploring whether global stressors such as temperature can affect the toxicity of BPA in organisms. Our aim was to assess the combined impact of BPA and varying temperature regimes on life-history traits in Daphnia magna. Our results revealed a significant impact of BPA on the growth, reproduction, and accumulated moulting of D. magna, with adverse effects primarily associated with the assimilation of BPA in algae rather than the BPA present in the medium, pointing to a trophic transfer mechanism. The interactive effect between BPA and temperature demonstrated a slight stimulatory effect of low BPA level on D. magna growth rate under warming constant conditions, but an inhibitory under warming fluctuating temperatures. Additionally, a BPA threshold was identified, below which growth became temperature-dependent. This study emphasizes the crucial role of considering temperature in predicting how toxins may affect Daphnia within aquatic food webs.

Rapid communication: effects of cadmium exposure on the growth-related genes of Daphnia magna.

Few data are available regarding the effects of gene expression on growth in Daphnia magna. The aim of this study was to determine the influence of cadmium (Cd) exposure on global gene transcription and growth-related genes in D. magna using RNASeq generated data. Our results demonstrated that Cd exposure decreased gene expression, but did not adversely affect the expression of growth-related genes, suggesting differential allocation of resources to growth avoids the deleterious effect of the toxicant on this trait.

Relationship between oxidative stress and lifespan in Daphnia pulex.

Macromolecular damage leading to cell, tissue and ultimately organ dysfunction is a major contributor to aging. Intracellular reactive oxygen species (ROS) resulting from normal metabolism cause most damage to macromolecules and the mitochondria play a central role in this process as they are the principle source of ROS. The relationship between naturally occurring variations in the mitochondrial (MT) genomes leading to correspondingly less or more ROS and macromolecular damage that changes the rate of aging associated organismal decline remains relatively unexplored. MT complex I, a component of the electron transport chain (ETC), is a key source of ROS and the NADH dehydrogenase subunit 5 (ND5) is a highly conserved core protein of the subunits that constitute the backbone of complex I. Using Daphnia as a model organism, we explored if the naturally occurring sequence variations in ND5 correlate with a short or long lifespan. Our results indicate that the short-lived clones have ND5 variants that correlate with reduced complex I activity, increased oxidative damage, and heightened expression of ROS scavenger enzymes. Daphnia offers a unique opportunity to investigate the association between inherited variations in components of complex I and ROS generation which affects the rate of aging and lifespan.

Acute and chronic toxicity of microcystin-LR and phenanthrene alone or in combination to the cladoceran (Daphnia magna).

Hazardous substances, such as microcystin-LR (MC-LR) and phenanthrene (Phe) are ubiquitous co-contaminants in eutrophic freshwaters, which cause harms to aquatic organisms. However, the risks associated with the co-exposure of aquatic biota to these two chemicals in the environment have received little attention. In this study, the single and mixture toxic effects of MC-LR and Phe mixtures were investigated in Daphnia magna after acute and chronic exposure. Acute tests showed that the median effective concentrations (48 h) for MC-LR, Phe and their mixtures were 13.46, 0.57 and 8.84 mg/L, respectively. Mixture toxicity prediction results indicated that the independent action model was more applicable than the concentration addition model. Moreover, combination index method suggested that the mixture toxicity was concentration dependent. Synergism was elicited at low concentrations of MC-LR and Phe exposure (≤4.04 + 0.17 mg/L), whereas antagonistic or additive effects were induced at higher concentrations. The involved mechanism of antagonism was presumably attributable to the protective effects of detoxification genes activated by high concentrations of MC-LR in mixtures. Additionally, chronic results also showed that exposure to a MC-LR and Phe mixture at low concentrations (≤50 +2 µg/L) resulted in greater toxic effects on D. magna life history than either chemical acting alone. The significant inhibition on detoxification genes and increased accumulation of MC-LR could be accounted for their synergistic toxic effects on D. magna. Our findings revealed the exacerbated ecological hazard of MC-LR and Phe at environmental concentrations (≤50 +2 µg/L), and provided new insights to the potential toxic mechanisms of MC-LR and Phe in aquatic animals.

Short term fluctuating temperature alleviates Daphnia stoichiometric constraints.

In this study, we analysed how short term temperature fluctuation interacts with nutrient limitation in the vertical migrating Daphnia commutata. We hypothesize that short term (daily) temperature fluctuation will alleviate nutrient limitation. We carried out experiments analysing growth rates, phosphorus and RNA content of D. commutate grown under four different temperature regimes and two P-limited conditions. Our experiments showed that individuals grown under fluctuating temperature grew more than at the mean temperature. We estimated the expected sizes for the 15 °C treatment based on the Q10 and for the fluctuating temperature treatment. These expected sizes for both treatments resulted well below the observed ones. The P and RNA content of individuals grown at 10 °C were significantly higher than those at 20 °C, and when individuals grown at 10 °C were translocated to 20 °C they exerted an increased growth rate. Our results suggest that, under a regime of diel vertical migration, the temperature alternation would allow migrating organisms to alleviate the effect of severe nutrient limitation maintaining population growth. Under a scenario of global warming, where epilimnetic temperatures will increase, lake temperature will interact with nutrient limitation for consumers, but, organisms may be able to face these changes if they can still regularly move from a cold hypolimnion to a warmer epilimnion.

Adsorption of bio-organic eco-corona molecules reduces the toxic response to metallic nanoparticles in Daphnia magna.

As the use of engineered nanomaterials increases, so does the risk of them spreading to natural ecosystems. Hitherto, knowledge regarding the toxic properties of nanoparticles (NP's) and their potential interactions with natural bio-organic molecules adsorbed to them, and thereby forming surface coronas, is limited. However, we show here that the toxic effect of NPs of tungsten carbide cobalt (WC-Co) and cobalt (Co) on the crustacean Daphnia magna is postponed in the presence of natural biological degradation products (eco-corona biomolecules). For Daphnia exposed to WC-Co NPs the survival time increased with 20-25% and for Co NPs with 30-47% after mixing the particles with a solution of eco-corona biomolecules before exposure. This suggests that an eco-corona, composed of biomolecules always present in natural ecosystems, reduces the toxic potency of both studied NPs. Further, the eco-coronas did not affect the particle uptake, suggesting that the reduction in toxicity was related to the particle-organism interaction after eco-corona formation. In a broader context, this implies that although the increasing use and production of NPs may constitute a novel, global environmental threat, the acute toxicity and long-term effects of some NPs will, at least under certain conditions, be reduced as they enter natural ecosystems.

Acute toxicity and ecological risk assessment of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC in ultraviolet (UV)-filters.

Ultraviolet (UV) filters are used in cosmetics, personal care products and packaging materials to provide sun protection for human skin and other substances. Little is known about these substances, but they continue to be released into the environment. The acute toxicity of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC to Chlorella vulgaris and Daphnia magna were analyzed in this study. The 96 h-EC50 values of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC on C. vulgaris were 183.60, 3.50 and 0.16874 mg/L, respectively. The 48 h-LC50 of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC on D. magna were 12.50, 3.74 and 0.54445 mg/L, respectively. The toxicity of a mixture of 4,4'-dihydroxybenzophenone and 4-MBC showed addictive effect on C. vulgaris, while the toxicity of mixtures of 4,4'-dihydroxybenzophenone and 2,4,4'-trihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC as well as 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC all showed antagonistic effect on C. vulgaris. The induced no-effect concentrations of 4,4'-dihydroxybenzophenone, 2,4,4'-trihydroxybenzophenone and 4-MBC by the assessment factor (AF) method were 0.0125, 0.00350 and 0.000169 mg/L, respectively.

Reversed evolution of grazer resistance to cyanobacteria.

Exploring the capability of organisms to cope with human-caused environmental change is crucial for assessing the risk of extinction and biodiversity loss. We study the consequences of changing nutrient pollution for the freshwater keystone grazer, Daphnia, in a large lake with a well-documented history of eutrophication and oligotrophication. Experiments using decades-old genotypes resurrected from the sediment egg bank revealed that nutrient enrichment in the middle of the 20th century, resulting in the proliferation of harmful cyanobacteria, led to the rapid evolution of grazer resistance to cyanobacteria. We show here that the subsequent reduction in nutrient input, accompanied by a decrease in cyanobacteria, resulted in the re-emergence of highly susceptible Daphnia genotypes. Expression and subsequent loss of grazer resistance occurred at high evolutionary rates, suggesting opposing selection and that maintaining resistance was costly. We provide a rare example of reversed evolution of a fitness-relevant trait in response to relaxed selection.

Sex Determination and Differentiation in Decapod and Cladoceran Crustaceans: An Overview of Endocrine Regulation.

Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

Methyl farnesoate regulatory mechanisms underlying photoperiod-dependent sex determination in the freshwater crustacean Daphnia magna.

Freshwater zooplankton Daphnia magna has been widely used in ecotoxicology studies. During the last 20 years, it has been demonstrated that the topical application of juvenile hormone (JH) or JH analogs to mother daphnids induce male offspring production. Based on this finding, an in vivo screening validation method for chemicals with JH agonistic effect has developed. Although this screening system successfully identified a number of JH-like chemicals, molecular mechanisms underlying the male sex-determining process remain largely unknown. To address this issue, we established a reliable male- or female-producing system using Daphnia pulex WTN6 strain by changing the rearing photoperiod. Taking advantage of this rearing system, we successfully found several factors involving male sex determination such as ionotropic glutamate receptors, protein kinase C and pantothenate. Here, we used two D. magna strains that can also control the production of female or male offspring by photoperiod differences as model species for ecotoxicology studies. We demonstrated that either treatment of antagonist of ionotropic glutamate receptors or inhibitor of protein kinase C strongly suppressed male offspring production even under male-producing conditions. Moreover, we revealed that male sex-determining processes are likely diverged between D. magna and D. pulex based on the current experiment. This study provides a fine experimental method for in vivo screening not only JH agonists but also JH antagonists. Moreover, using daphnids with photoperiod-dependent sex determination manner will hugely contribute to understanding the mode-of-action of JH in daphnids.

Multiple Screening of Pesticides Toxicity in Zebrafish and Daphnia Based on Locomotor Activity Alterations.

Pesticides are widely used to eradicate insects, weed species, and fungi in agriculture. The half-lives of some pesticides are relatively long and may have the dire potential to induce adverse effects when released into the soil, terrestrial and aquatic systems. To assess the potential adverse effects of pesticide pollution in the aquatic environment, zebrafish (Danio rerio) and Daphnia magna are two excellent animal models because of their transparent bodies, relatively short development processes, and well-established genetic information. Moreover, they are also suitable for performing high-throughput toxicity assays. In this study, we used both zebrafish larvae and water flea daphnia neonates as a model system to explore and compare the potential toxicity by monitoring locomotor activity. Tested animals were exposed to 12 various types of pesticides (three fungicides and 9 insecticides) for 24 h and their corresponding locomotor activities, in terms of distance traveled, burst movement, and rotation were quantified. By adapting principal component analysis (PCA) and hierarchical clustering analysis, we were able to minimize data complexity and compare pesticide toxicity based on locomotor activity for zebrafish and daphnia. Results showed distinct locomotor activity alteration patterns between zebrafish and daphnia towards pesticide exposure. The majority of pesticides tested in this study induced locomotor hypo-activity in daphnia neonates but triggered locomotor hyper-activity in zebrafish larvae. According to our PCA and clustering results, the toxicity for 12 pesticides was grouped into two major groups based on all locomotor activity endpoints collected from both zebrafish and daphnia. In conclusion, all pesticides resulted in swimming alterations in both animal models by either producing hypo-activity, hyperactivity, or other changes in swimming patterns. In addition, zebrafish and daphnia displayed distinct sensitivity and response against different pesticides, and the combinational analysis approach by using a phenomic approach to combine data collected from zebrafish and daphnia provided better resolution for toxicological assessment.

The possible role of toxigenic fungi in ecotoxicity of two contrasting oil-contaminated soils - A field study.

Long-term field experiments were performed to evaluate the phytotoxic properties of fungal metabolites in oil-contaminated soil and to assess the impact of contamination on the allelopathic activity of soil mycobiota. Two contrasting soils of Northwest Russia (sandy and loamy podzols) exposed to oil contamination underwent changes in abundance and allelopathic activities of soil fungi. Shifts within the microbial community caused by oil contamination affected not only oil-decomposition rates but also ecotoxicity of contaminated soil. There were significant differences in soil toxicity dynamics between sandy and loamy podzols. Four years after contamination, ecotoxicity of loamy podzol decreased, whereas sandy podzol remained highly toxic even nine years after contamination. The abundance and allelopathic activity of fungi is correlated with hydrocarbon degradation dynamics. The soil fungal community demonstrated high allelopathic activity which decreased over time in fertile loamy podzolic soil, whereas in poor sandy podzolic soil it remained high over the nine-year monitoring period. The results illustrate how oil contamination may influence allelopathic interactions in soil and demonstrate the advantage of using fungal metabolite toxicity test for testing of oil-contaminated soil samples.

Microplastic-associated trophic transfer of benzo(k)fluoranthene in a limnic food web: Effects in two freshwater invertebrates (Daphnia magna, Chironomus riparius) and zebrafish (Danio rerio).

The continuously growing plastic production and incomplete recycling processes open manifold entry routes for microplastic particles (MPs) into the environment. Since knowledge on trophic transfer of contaminants sorbed to MPs is still insufficient for freshwater systems, the transfer of the model pollutant benzo(k)fluoranthene (BkF) sorbed to polymethyl methacrylate (PMMA) particles in a limnic food web was investigated: Two freshwater invertebrates (Daphnia magna and Chironomus riparius larvae) were selected and either left untreated, exposed to pristine PMMA, PMMA-associated BkF, or exposed to dissolved BkF (BkFaq). As second-level consumers, zebrafish (Danio rerio) were fed twice daily with pre-treated invertebrates over two days. Induction of hepatic cytochrome P450 by BkF was determined as 7-ethoxy-O-resorufin deethylase (EROD) activity. Both invertebrate species readily ingested PMMA particles, tracked via fluorescence microscopy and accumulated BkFaq, measured via GC-MS. Fluorescence signals in gastrointestinal tracts of zebrafish were quantified with confocal laser scanning microscopy (CLSM). The fluorescence signal in gastrointestinal tracts of zebrafish was not altered, whereas, EROD activity was significantly induced when zebrafish were fed with Chironomus riparius, pre-exposed to BkFaq. Trophic exposure scenarios with BkF sorbed to PMMA did not result in any alterations of investigated endpoints in both invertebrate species and zebrafish compared to controls. Given that BkF amounts were in the low ng-range, as detected by GC-MS, the transport of MP-sorbed BkF to zebrafish was less effective than direct exposure to waterborne BkFaq, and the potential threat of trophic transfer of substances such as BkF in limnic food webs may have been overestimated.

Temperature increase altered Daphnia community structure in artificially heated lakes: a potential scenario for a warmer future.

Under conditions of global warming, organisms are expected to track their thermal preferences, invading new habitats at higher latitudes and altitudes and altering the structure of local communities. To fend off potential invaders, indigenous communities/populations will have to rapidly adapt to the increase in temperature. In this study, we tested if decades of artificial water heating changed the structure of communities and populations of the Daphnia longispina species complex. We compared the species composition of contemporary Daphnia communities inhabiting five lakes heated by power plants and four non-heated control lakes. The heated lakes are ca. 3-4 °C warmer, as all lakes are expected to be by 2100 according to climate change forecasts. We also genotyped subfossil resting eggs to describe past shifts in Daphnia community structure that were induced by lake heating. Both approaches revealed a rapid replacement of indigenous D. longispina and D. cucullata by invader D. galeata immediately after the onset of heating, followed by a gradual recovery of the D. cucullata population. Our findings clearly indicate that, in response to global warming, community restructuring may occur faster than evolutionary adaptation. The eventual recolonisation by D. cucullata indicates that adaptation to novel conditions can be time-lagged, and suggests that the long-term consequences of ecosystem disturbance may differ from short-term observations.

Transgenerational reproductive effects of two serotonin reuptake inhibitors after acute exposure in Daphnia magna embryos.

The release of pharmaceuticals and personal care products (PPCPs) into aquatic environments has been a major concern for the health of ecosystems. Transgenerational plasticity is a potential mechanism for organisms to respond to changing environmental conditions, including climate change and environmental contaminants. The purpose of the present study was to determine the long-term transgenerational effects of an abundant freshwater zooplankton, Daphnia magna, to acute embryonic exposures of serotonin re-uptake inhibitors (SSRI - fluoxetine and sertraline). Both SSRIs have been used extensively to treat depression and anxiety disorders for decades and persist in freshwater ecosystems at physiologically relevant concentrations. Our results revealed that even short (72 h) embryonic exposures of D. magna embryos had long lasting consequences on life history and expression of 5HT related genes in the unexposed generation (F3). Moreover, we identified direct effects of SSRIs on heart rate and swimming behavior in the first generation that carried over from embryonic exposure. We also found that SSRI exposure resulted in a transient increase of ephippia formation in the F1 and F2 . Our results suggest that SSRI exposure has transgenerational consequences to the unexposed generation and potentially beyond, even at low concentration (10-100× lower than what can be found in natural ecosystems) and as a result of embryonic exposure. Because of the short reproductive period of D. magna and their integral role in aquatic food webs, these findings have population-level implications and deserve further investigation.

The Effect of Dietary Exposure to Coal Ash Contaminants within Food Ration on Growth and Reproduction in Daphnia magna.

Coal ash contains numerous contaminants and is the focus of regulatory actions and risk assessments due to environmental spills. We exposed Daphnia magna to a gradient of coal ash contamination under high and low food rations to assess the sublethal effects of dietary exposures. Whereas exposure to contaminants resulted in significant reductions in growth and reproduction in daphnids, low, environmentally relevant food rations had a much greater effect on these endpoints. Environ Toxicol Chem 2020;39:1998-2007. © 2020 SETAC.

Toxic microcystis reduces tolerance of daphnia to increased chloride, and low chloride alleviates the harm of toxic microcystis to daphnia.

Salinization of freshwater ecosystems caused by human activities and climate change is a global problem that threatens freshwater resources and aquatic organisms. The aggravation of salinization and the presence of cyanobacterial blooms may pose a serious threat to crustacean zooplankton Daphnia. To test the consequences of these effects, we exposed Daphnia magna to the combined treatments of different chloride concentrations and three food compositions (100% Chlorella pyrenoidosa, 90% C. pyrenoidosa + 10% toxic Microcystis aeruginosa, 80% C. pyrenoidosa + 20% toxic M. aeruginosa) for 21 days, recorded relevant life history indicators, and fitted them using Sigmoidal and Gaussian model if appropriate. Results showed that both increased chloride and the presence of toxic M. aeruginosa in the food had significantly negative effects on key life history traits and clearance rate, and the two factors also had a significant interaction on the survival, development, and reproduction of D. magna. The maximum values of the key life-history traits and clearance rate, the median effect chloride concentrations, and the optimal chloride concentrations derived from the models showed that the survival, reproduction, and clearance rate of D. magna were threatened by high chloride concentrations, which were exacerbated by the presence of toxic M. aeruginosa, but lower concentration of chloride was beneficial to D. magna to resist toxic M. aeruginosa. In conclusion, the combined effects of increasing chloride concentration and cyanobacterial blooms have severely adverse impacts on cladocerans, which may cause cladocera population to decline more rapidly and potentially disrupt the food webs of aquatic ecosystems.

Time-dependent biomolecular responses and bioaccumulation of perfluorooctane sulfonate (PFOS) in Daphnia magna.

Perfluorooctane sulfonate (PFOS) is a persistent pollutant which is potentially harmful and bioaccumulative to aquatic organisms. To evaluate the regulatory alteration of select metabolites with PFOS exposure at early and typical acute exposure periods in an aquatic indicator species Daphnia magna, the hourly abundance of the twenty-three metabolites was investigated over 24 h. To evaluate the bioaccumulation potential of PFOS at a sub-lethal concentration in D. magna, the daily accumulation into D. magna for 16 days was also evaluated. Twenty-three targeted metabolites were quantified over 1 to 4 h and 21 to 24 h of PFOS exposure using liquid chromatography tandem mass spectrometry (LC-MS/MS). Daphnid to water PFOS concentration ratios were monitored separately over different days and life stages at 0 to 76 h and 2 to 16 days of PFOS exposure. The observed metabolite abundance and bioaccumulation in the exposed groups was compared between sampling times. The results reveal that sub-lethal PFOS exposure at 2 mg/L and 20 mg/L alters regulation of arginine, tyrosine and adenosine monophosphate which are directly and indirectly related to energy status. The temporal metabolic responses observed for the early exposure period (4 h), but not for the typical acute exposure period (24 h), suggest the dysregulation potency of PFOS on metabolite regulation of D. magna and the importance of early time-course monitoring approaches. Sixteen days of bioaccumulation monitoring showed that PFOS is more bioaccumulative in younger D. magna. The observation of time-dependent bioaccumulation of PFOS in D. magna requires further studies to define its precise mechanism. Interestingly, the bioaccumulation potential of PFOS was found to be consistent between 72 h and 16 day exposure periods. No difference on the body burden to water concentration ratio during about one third of the life span time (16 days), compared to the 72 h exposure, suggests that the prolonged exposure did not increase the bioaccumulation of PFOS in D. magna. This study demonstrates that the Daphnia metabolites are rapidly responding to sub-lethal PFOS exposure and provides information on life stage and time-dependent bioaccumulation potential of PFOS. As such, metabolite regulation is a sensitive indicator to sub-lethal PFOS exposure and can be informative when combined with other measures of toxicity.