Article pubs.acs.org/est
Potential Impact of Low-Concentration Silver Nanoparticles on Predator−Prey Interactions between Predatory Dragonfly Nymphs and Daphnia magna as a Prey Lok R. Pokhrel† and Brajesh Dubey*,‡ †
Department of Environmental Health, College of Public Health, East Tennessee State University, Johnson City, Tennessee 37614-1700, United States ‡ Environmental Engineering, School of Engineering, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada S Supporting Information *
ABSTRACT: This study investigated the potential impacts of lowconcentration citrate-coated silver nanoparticles (citrate−nAg; 2 μg L−1 as total Ag) on the interactions of Daphnia magna Straus (as a prey) with the predatory dragonfly (Anax junius: Odonata) nymph using the behavioral, survival, and reproductive end points. Four different toxicity bioassays were evaluated: (i) horizontal migration; (ii) vertical migration; (iii) 48 h survival; and (iv) 21 day reproduction; using four different treatment combinations: (i) Daphnia + citrate−nAg; (ii) Daphnia + predator; (iii) Daphnia + citrate−nAg + predator; and (iv) Daphnia only (control). Daphnia avoided the predators using the horizontal and vertical movements, indicating that Daphnia might have perceived a significant risk of predation. However, with citrate−nAg + predator treatment, Daphnia response did not differ from control in the vertical migration test, suggesting that Daphnia were unable to detect the presence of predator with citrate−nAg treatment and this may have potential implication on daphnids population structure owing to predation risk. The 48 h survival test showed a significant mortality of Daphnia individuals in the presence of predators, with or without citrate−nAg, in the test environment. Average reproduction of daphnids increased by 185% with low-concentration citrate−nAg treatment alone but was severely compromised in the presence of predators (decreased by 91.3%). Daphnia reproduction was slightly enhanced by approximately 128% with citrate−nAg + predator treatment. Potential mechanisms of these differential effects of low-concentration citrate−nAg, with or without predators, are discussed. Because silver dissolution was minimal, the observed toxicity could not be explained by dissolved Ag alone. These findings offer novel insights into how exposure to low-concentration silver nanoparticles could influence predator−prey interactions in the fresh water systems.
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concentrations, thus minimizing11 or potentially replacing the use of bulk silver in material applications. On the other hand, limited understanding of nanotoxicology has hindered risk assessment of AgNPs in aquatic environments. An absence of standard analytical technique and/or protocol to quantify ENMs in the test matrix or in vivo has been the main hurdle to understand whether the response(s) displayed by an organism is primarily due to NPs or ions released from NPs or is the combined effects of both.6,12 Furthermore, as nanotoxicity literature generally accounts for exceedingly higher concentrations,13,14 it offers less information about the potential risks that nanomaterials might have on natural aqueous systems. Acute toxicity studies of ENMs using Daphnia species as test organisms are abundant,15−17 but literature accounting for
INTRODUCTION With better understanding of the physicochemical properties of engineered nanomaterials (ENM) in recent years, it has been possible to tune nanomaterials with desired novel functionalities.1,2 Hence, these novel nanomaterials have become integral components of hundreds of consumer products available in the market, today.3 Increasing applications of nanoproducts could lead to environmental contamination due to leaching of chemicals, possibly in ionic- and/or nanoform, as these products age and weather during their life cycle.4,5 Increasing number of both in vitro and in vivo studies suggest that ENMs could potentially pose risk to the living systems.6−8 Among a wide variety of ENMs, silver nanoparticles (AgNP) have found applications in more than 300 products as documented by the Project on Emerging Nanotechnologies (PEN) inventory.3 Historically, Ag (as ions or compounds) is known for its antimicrobial properties;6,9,10 but with the progress in nanotechnology, AgNPs incorporated into products seem to offer the same end result at relatively low © 2012 American Chemical Society
Received: Revised: Accepted: Published: 7755
November 13, 2011 June 9, 2012 June 14, 2012 June 14, 2012 dx.doi.org/10.1021/es204055c | Environ. Sci. Technol. 2012, 46, 7755−7762
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chronic toxicity on Daphnia is rather scarce.18,19 More importantly, studies investigating long-term exposure of daphnids (as a prey) to low-concentration NPs, with special focus on potential interactions among the model NPs, predators (dragonfly nymphs), and prey, could enable better understanding of the potential toxicity of NPs to daphnids in the aquatic environments. Toxicological studies of conventional toxicants at sublethal doses suggest that changes in the behavior and/or physiology of zooplanktons, such as daphnids, could elicit long-term impacts in the population structure and ecosystem dynamics.20,21 More specifically, altered behavioral responses of daphnids on exposure to a pesticide have been shown to promote predation risk.21 For example, daphnids exposed to a carbaryl pesticide exhibited abnormal swimming patterns and were preferentially preyed upon by predatory bluegills (Lepomis macrochirus) compared to the unexposed daphnids that exhibited normal swimming behavior.21 Literature suggests that the behavioral end points, such as horizontal and vertical migrations, can serve as sensitive indicators for lowconcentration xenobiotic exposure.20−22 Horizontal migration plays a crucial role by allowing daphnids to migrate from pelagic (open water) to shallow waters and thus avoid fish predation by retreating against the vegetation.23,24 On the other hand, vertical migration enables daphnids to acquire planktonic food via filter feeding,25 which is essential for their survival and reproduction.26 Recently, a few studies that explored behavioral and physiological responses of daphnids on exposure to carbon-based NPs (i.e., fullerenes)27,28 or nTiO227 showed potential effects on their survival, reproduction, and food web dynamics in the aquatic ecosystems.27,28 Brausch et al.28 further explored potential interactions of the predator + fullerene NPs on the behaviors of D. magna using water conditioned with predatory bluegill (Lepomis macrochirus) and used as a predatory cue. However, an application of visual predators, such as the live dragonfly nymphs or fish, in the test environment has not yet been realized in the nanotoxicology research. Physical presence of the predators in the laboratory experiment should provide both the visual29 and chemical cues to Daphnia,28 which could incur stress via predation risk as in the natural environment.27,28,30 This critical knowledge gap is addressed here by investigating the potential impacts of low-concentration citratecoated silver nanoparticles (citrate−nAg; 2 μg L−1 as total Ag) on the interactions of D. magna (as a prey) with the predatory dragonfly (Anax junius: Odonata) nymphs. We evaluated the spatial migratory patterns, survival, and reproduction in D. magna using four different toxicity bioassays: (i) test for horizontal migration; (ii) test for vertical migration; (iii) 48 h survival test; and (iv) 21 day reproduction test; and complementary treatments using the ensuing combinations: (i) Daphnia + citrate−nAg; (ii) Daphnia + predator; (iii) Daphnia + citrate−nAg + predator; and (iv) Daphnia only (control).
electrical conductance (EC) has been associated with lower impurity fraction in similar NP suspensions, our citrate−nAg suspension (as retentate) with EC of 5 μS cm−1 indicated near absence of ions and/or impurities in the purified suspension (19 mg L−1 of total Ag) (Supporting Information, Table S1).31,32 A low-concentration of 2 μg citrate−nAg L−1 (as total Ag) was used in this study for all four toxicity bioassays tested. This concentration value was adapted from the 48 h acute toxicity data collected previously in our laboratory for the same Daphnia species and the laboratory conditions.33 Additionally, a recent study by Kennedy et al. that reported median lethal concentration (LC50 for 48 h test) of 0.3 to 2.2 μg Ag L−1 (as dissolved Ag) for D. magna34 and the revised USEPA national recommended water quality criteria of 1.9 and 3.2 μg Ag L−1 for salt and fresh water systems, respectively, also provided bases for this study.35 Nanoparticle Characterization. Purified citrate−nAg suspension was characterized using dynamic light scattering (DLS), UV−Vis spectrophotometry, and transmission electron microscopy (TEM). Average hydrodynamic diameter (HDD, volume weighted), electrophoretic mobility, and zeta (ζ) potential of citrate−nAg samples were estimated with a PSS Nicomp 380ZLS particle sizer/zeta potential unit (Particle Sizing Systems, CA, USA) using the DLS method. The unit was calibrated at 23 °C with Duke 500 (491 nm) NIST 3490A standard (PSS Nicomp, FL, USA) prior to the measurements. The stability of NPs in moderately hard water was evaluated for a period of 21 days, measuring average HDD, ζ potential, and electrophoretic mobility. The surface plasmon resonance (SPR) spectra were recorded using a HACH DR5000 UV−Vis spectrophotometer (HACH Company, CO, USA). By using a Philips EM 420 transmission electron microscope, citrate−nAg suspension was imaged in the bright-field mode at 120 kV to visualize NP morphology. An aliquot of sample was dropped onto a carbon-coated copper Formvar grid (Ted Pella, cat. no. 01813-F) and was air-dried before acquiring TEM images. Using ImageJ 1.44 particle size distribution (PSD) of the sample was estimated from a representative TEM imagery.36 Graphite furnace- or flame-atomic absorption spectroscopy (AAS, Varian 220Z/220FS) was used to quantify total Ag concentrations in the nanosuspension digested using HNO3 (trace element grade) following the standard USEPA method 3050B.37 Daphnia magna Toxicity Bioassays. A year old established culture of Daphnia magna Straus was used for this study. The stock culture of D. magna was originally procured from Aquatic Biosystems, Inc., Fort Collins, CO. Four different toxicity bioassays were conducted: (i) test for horizontal migration; (ii) test for vertical migration; (iii) 48 h survival test; and (iv) 21 day reproduction test. The aim of these fourpronged tests was to understand the potential impacts of lowconcentration (2 μg L−1 as total Ag) citrate−nAg on the interactions of D. magna (as a prey) with the predatory dragonfly (A. junius) nymphs, with focus on the spatial migratory patterns, survival, and reproduction in D. magna, but not that of predatory nymphs as they were tied with thread restricting their locomotion (detail presented in the following sections; schematics shown in the Supporting Information, Figures S1 and S2). All tests were run in triplicates in 9.4 L (30.7 L × 15.5 W × 21 H cm3) aquaria, each containing 5 L of moderately hard reconstituted water (MHRW), 2 × 103 algal (Selenastrum capricornutum) cells mL−1 test media, and 1 mL
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MATERIALS AND METHODS Nanomaterial Preparation and Dosimetry. Citratecoated silver nanoparticles (citrate−nAg) were synthesized and stabilized in the laboratory following a previously described procedure.31 By using the protocol described in our previous work,6 citrate−nAg was cleaned using a Kros Flo Tangential Flow Filtration (TFF) System equipped with polysulfone (PS) 10 kD hollow fiber membranes (P/N: X31S-300-02P, surface area = 145 cm2; Spectrum Laboratories, CA). Because lower 7756
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trout food L−1 test media, unless noted otherwise. The following four different treatment combinations were used for all bioassays: Daphnia + citrate−nAg; Daphnia + predator; Daphnia + citrate−nAg + predator; and Daphnia only (control). Test for Horizontal Migration. A total of 30, 7−8 days old D. magna were released into the center of each aquarium using a sterile pipet and were allowed to acclimate for 2 h in the test medium (with or without nanoparticles and/or with or without nymphs) before the test was initiated. Live dragonfly nymphs were collected from the nearby Brush Creek (vicinity of East Tennessee State University, Johnson City, TN, USA) and were cleaned several times with water prior to their placement in the aquaria. A 2 ft long wide-spectrum fluorescent tube was used as a light source. Two dragonfly nymphs (body length 2−3 cm) tied to a thread were introduced into the water at one side of an aquarium in a way such that they could swim to a distance of not more than 5 cm from that region of the aquarium. Hence, the area beyond 5 cm from that corner of an aquarium where the nymphs were located was defined as an “open area” (Supporting Information, Figure S1). Daphnids present in the open area were counted at the end of every 30 min for four consecutive time periods with an hour interval between the readings as a response to the presence (or absence) of the predatory nymphs. Whether this response was altered (if so, to what extent) in the presence of low-concentration citrate−nAg was of special interest. This simple microcosm experiment may simulate interaction between prey and predator in the aqueous environment contaminated with AgNPs. Test for Vertical Migration. Test for vertical migration was conducted as described previously for D. magna by Brausch et al.,28 with ensuing modifications. The experimental setup was manually designed, which was composed of a 25 mL plastic pipet glued to one end at 90° onto the center of a Petri dish, while the top-end was cut wide open to facilitate efficient cleaning and transfer of test solution, including two 7−8 days old D. magna, into the tube. After erasing the milliliter scale that was on the outer surface of the pipet with acetone, the 250 mm long tube was divided into eight 30 mm sections and a 10 mm section at the top end. This setup was placed inside a 1 L beaker, containing MHRW with 10 unexposed daphnids to mimic the natural population.28 The beaker was wrapped with a sheet of black plastic from all sides including the bottom (Supporting Information, Figure S2), and the daphnids were acclimated for an hour in dark in the respective test suspensions. Soon the highest vertical position of Daphnia was recorded under light for a period of 5 min, with 30 min intervals between each of the four repeated measures. A 2 ft long wide-spectrum fluorescent tube provided light for this test. One dragonfly nymph that was placed at the topmost part of the tube represented a predator and was tied to a thread, restricting its movement within the topmost 3 cm of the tube. Animals were fed during the entire acclimation and test periods as described earlier for horizontal migration. Tests were run in five replicates. 48 h Survival Test. The 48 h acute toxicity test was conducted following the standard USEPA guidelines,38 with some modifications as noted. Thirty 0.5), and no significant interaction between time and treatment on Daphnia migration toward open area (GLM: F = 1.405, p > 0.1). This suggests that the response of Daphnia to different treatments was not influenced by time in our experiment. On average, with low-concentration citrate−nAg + predator treatment, Daphnia response was highest among the treatments (Figure 1A). However, Tukey’s posthoc test showed that this combined effect was not significantly different with predator only (p > 0.5) or citrate−nAg only treatment (p > 0.1). These results are consistent with the literature on predator−prey interactions.41 Impact on Vertical Migration. Daphnia displayed negative taxis to the presence of live predator and remained furthest away from the predator, which was present on the topmost part of the experimental setup, compared to other treatments (Figure 1B). Prey individuals descending downward or residing at the bottom of the experimental setup when predatory nymph and light were present on the surface water is
p 0.001
mean difference
std error
p
−2.0 −2.833 −3.666 −0.833 −1.666 −0.833
0.859 0.859 0.859 0.859 0.859 0.859
0.107 0.010 0.001 0.767 0.227 0.767
As expected, daphnids showed negative taxis to the presence of predatory nymphs, with or without citrate−nAg, in the test environment (Tukey’s posthoc, p < 0.01 in both cases; Table 1). However, with low-concentration citrate−nAg treatment alone, daphnids’ response was not different to that observed when predators were present in the test chamber (p > 0.5). This suggests that either the chemical cue(s) due to citrate− nAg in the test chamber was indistinguishable to that of 7758
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Figure 2. Variation in horizontal (A) and vertical (B) migrations in Daphnia magna over time upon exposure to citrate−nAg (2 μg L−1 as total Ag), or predatory dragonfly nymphs (NY), or a combination of both (NPNY). Daphnids count in the open area represented horizontal migration response. Data are presented as mean ±1 standard error of the means. For horizontal migration, each data point represents means of triplicate runs; for vertical migration, each data point represents means of five replicates. NP, 2 μg L−1 citrate−nAg; NPNY, citrate−nAg + predatory dragonfly nymphs combined; Control indicates D. magna only (no NPs, no nymphs).
in good agreement with the visual predator avoidance hypothesis.42 ANOVA followed by Tukey’s posthoc test showed that citrate−nAg treatment did not significantly influence daphnids’ response compared to control (p > 0.5; Supporting Information, Table S7). However, with citrate−nAg + predator treatment, it appeared that the NPs somehow reduced or perhaps neutralized the chemical/visual stimuli from the predator, allowing daphnids to react in the way similar to when there was no predator in the test environment, and migrated vertically upward in the water column reaching closest the predator that was confined to the top (Figure 1B). In one instance, a daphnid that almost reached the water surface was instantly preyed upon by the predatory nymph. Tukey’s posthoc test revealed a significant difference between predator only and citrate−nAg + predator treatments (p = 0.01). These data suggest the possibilities that (i) the sensory system of daphnids might have been compromised on exposure to citrate−nAg, and/or (ii) the nymph was unable to release any chemical cue into the test environment as citrate−nAg may have also impacted it, which might have led daphnids to mistaken for the absence of predator when it was actually present. Average variability in vertical migration of Daphnia among treatments over a time period of approximately 2 h is presented in Figure 2B. No significant main effect of time (F = 0.272, p > 0.5) and no significant interaction between time and treatments (F = 1.425, p > 0.1) on vertical migration in Daphnia were shown by the GLM. This suggests that the response of Daphnia to different treatments was not influenced by time during our experimental period of ∼2 h. Impact on 48 h Survival. Results of the 48 h survival bioassay showed that, in the presence of predatory nymphs, with or without citrate−nAg, Daphnia survival was significantly compromised (Figure 3; p < 0.05 in both cases). Furthermore, this result also suggests that the combined effect of citrate−nAg + predators was not different to that when only predators were present (p = 1.0). With low-concentration citrate−nAg treatment alone, average survival of Daphnia decreased by 50% in the 48 h test period (Figure 3); however, this was not significantly different from control (p > 0.1; Supporting Information, Table S8).
Figure 3. Variation in Daphnia magna survival as shown by 48 h survival test upon exposure to citrate−nAg (NP; 2 μg L−1 as total Ag), or predatory dragonfly nymphs (NY), or a combination of both (NP + NY). Y-axis represents average percent survival of the triplicate runs, each run consisting of 30 daphnids. Error bars represent ±1 standard error of the means. * denotes p < 0.05 compared to control (C).
Impact on 21 Day Reproduction. Average reproduction in Daphnia was significantly reduced by 91.3% in the presence of predators (without citrate−nAg) compared to control (Figure 4). On the other hand, reproduction was significantly enhanced, on average, by 185% with low-concentration citrate− nAg treatment alone (Figure 4; Table 2). Whether hormesis was triggered at this low concentration remained to be tested, which would require multiple doses of citrate−nAg treatments. Reproduction was also higher with citrate−nAg + predator treatment (Figure 4), which increased by an average of approximately 128% of control. This might be due to lower threat perceived by Daphnia of the predators as the latter movement was restricted and/or the potential interference with the chemical cue released by predators due to citrate−nAg, thereby reducing the predation risk on the prey individuals. Predation risk is known to be energetically costly,43 and given that the predation risk was lowered with citrate−nAg, daphnids’ investment of energy into foraging might have promoted its reproduction.44,45 Potential adaptation of Daphnia to the fixed predators in a 21 day period might also explain in part the observed increase in reproduction. In nature, this may not likely 7759
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Phototaxis involves vertical migration of animals in response to the light stimuli,47 and this migration is crucial for Daphnia for foraging or escaping predation from littoral predators such as the dragonfly nymphs.25,48,49 Laboratory experiments assessing vertical migration in Daphnia are found to resemble the patterns observed in the field; hence, shorter vertical distance experiments generally preferred in the laboratory settings are sensitive enough to understand the potential behavioral changes in zooplanktons in the fresh water systems.50,51 In the presence of the predatory nymph, Daphnia significantly lowered their vertical distance (Figure 1B), supporting the predator avoidance tactics.21,42 Our observation of Daphnia showing relatively higher vertical movement toward predator with low-concentration citrate−nAg treatment suggests that these daphnids would be vulnerable to predation (Figure 1B). This is consistent with the result of a previous study showing that Daphnia increased their vertical position with PCB153 (polychlorinated biphenyl congener) treatment in the presence of fish kairomone.46 Changes in daphnids’ migratory behaviors with citrate−nAg treatment, as our results indicate, could potentially affect their survival with associated changes in their ability to recognize the presence of predators in natural habitats.21 Because potential interactions of this planktonic prey with multiple predators in the ecosystem might be complex, single predator−prey interaction models as described in this study and one other28 should provide a basis to further our understanding on nanoecotoxicity. How would low-concentration AgNPs affect the predator behavior and how changes (if any) in the latter behavior would in turn affect the prey response to AgNPs remained to be tested. Unlike in this study, different experimental design(s) might be necessary to assess both the predator and prey responses to low-concentration AgNPs. As similar studies utilizing other nanomaterials at low concentrations should decrease the current uncertainty of potential risks to the environment, future studies should also address how environmental heterogeneity, such as the presence/absence of vegetation stands (as refuge), would affect the population and associated community structure when the aquatic systems are contaminated with different kinds of ENMs. Furthermore, better understanding of the fate of nanomaterials in aqueous systems should enable researchers to study interactive effects of nanomaterials on the predator−prey behaviors and life history traits in the field environments. Daphnia species are important grazers of phytoplanktons, while they are preferentially preyed upon by fish and invertebrates such as Chaoborus, Bythotrephes, Leptodora or the Odonata nymphs (e.g., dragonfly, damselfly, etc.).25 Thus, Daphnia is recognized as a keystone genus in the freshwater ecosystems, providing a major link in the energy flow between primary producers and secondary consumers in these food webs.52 Besides occupying the trophic position of primary consumer,26 daphnids’ significance in maintaining water quality via filter feeding organic detritus from the water column has also been recognized to be crucial.21 Results of this study indicate that the behavior, survival, and reproduction in D. magna could be altered upon exposure to low-concentration citrate−nAg, one of the most dominant ENMs3, in the presence of its common natural predator, A. junius nymphs. Notably, our study not only extended and supported the previous findings of Lovern et al.27 and Brausch et al.,28 our experimental design also took the traditional ecotoxicity studies a step ahead by investigating the potential impact of low-concentration citrate−
Figure 4. Variation in Daphnia magna reproduction in a 21 day period upon exposure to citrate−nAg (NP; 2 μg L−1 as total Ag), or predatory dragonfly nymphs (NY), or a combination of both (NP+NY). Y-axis represents average number of daphnids (both surviving adults and newborn neonates) counted on the 21st day of the experiment. Error bars represent ±1 standard error of the means. ** indicates significant difference with control at p < 0.01; *** indicates significant difference at p ≤ 0.001.
Table 2. ANOVA Followed by Tukey’s Posthoc Comparisons for Daphnia magna 21 Day Reproduction Test under Different Treatments sum of squares
mean square
F
between groups 207394.917 69131.639 30.802 Tukey’s Posthoc Test for Multiple Comparisons comparison between control
citrate-nAg nymph
citrate−nAg nymph citrate−nAg + nymph nymph citrate−nAg + nymph citrate−nAg + nymph
p
