Article Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX
pubs.acs.org/est
Dosing, Not the Dose: Comparing Chronic and Pulsed Silver Nanoparticle Exposures Benjamin P. Colman,*,†,‡,# Leanne F. Baker,†,§,∥,# Ryan S. King,†,∥,⊥ Cole W. Matson,†,§,∥ Jason M. Unrine,†,∇ Stella M. Marinakos,†,# Danielle E. Gorka,†,○,# and Emily S. Bernhardt†,‡ †
Center for the Environmental Implications of NanoTechnology, Duke University, Durham, North Carolina, United States Department of Biology, Duke University, Durham, North Carolina, United States § Department of Environmental Science, Baylor University, Waco, Texas, United States ∥ Center for Reservoir and Aquatic Systems Research, Baylor University, Waco, Texas, United States ⊥ Department of Biology, Baylor University, Waco, Texas, United States ∇ Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky, United States ○ Department of Chemistry, Duke University, Durham, North Carolina, United States
Environ. Sci. Technol. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 08/27/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: The environmental impacts of manufactured nanoparticles are often studied using high-concentration pulseadditions of freshly synthesized nanoparticles, while predicted releases are characterized by chronic low-concentration additions of weathered particles. To test the effects in wetlands of addition rate and nanoparticle speciation on water column silver concentrations, ecosystem impacts, and silver accumulation by biota, we conducted a year-long mesocosm experiment. We compared a pulse addition of Ag0-NPs to chronic weekly additions of either Ag0-NPs or sulfidized silver nanoparticles. The initially high water column silver concentrations in the pulse treatment declined such that after 4 weeks it was lower on average than in the two chronic treatments. While the pulse caused a marked increase in dissolved methane in the first week of the experiment, the chronic treatments had smaller increases in methane concentration that were more prolonged between weeks 28−45. Much like water column silver, most organisms in chronic treatments had comparable silver concentrations to the pulse treatment after only 4 weeks, and all but one organism had similar or higher concentrations than the pulse treatment after one year. Pulse exposures thus both overestimate the intensity of short-term exposures and effects and underestimate the more realistic long-term exposure, ecosystem effects, and accumulation seen in chronic exposures.
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Through our field and laboratory studies of the effects of freshly synthesized zerovalent silver nanoparticles (Ag0NPs),7−11 we grew concerned that the effects we measured in single dose experiments would not generate predictions with enough environmental relevance to sufficiently capture expected environmental impacts. In 2014, we reported that the addition of a single exposure of Ag0-NPs to the aquatic compartment of experimental wetlands led to catastrophic mortality of aquatic plants, anoxia, and a 40-fold increase in dissolved concentrations of the potent greenhouse gas methane.9 The impacts we observed occurred at 2.5 mg Ag L−1, a concentration which was on par with historical effluent
INTRODUCTION
In ecotoxicological investigations of manufactured nanomaterials in aquatic ecosystems, there currently exists a disconnect between likely exposure scenarios and the tests that are conducted. Risk assessments suggest exposures in aquatic ecosystems will mostly be chronic exposures to low concentrations of nanomaterials,1 yet the majority of studies examine high-concentration short-term exposures.2,3 Conclusions drawn from high-concentration ecotoxicological studies are often in contrast to the dynamic nature of environmental exposure scenarios and the resulting interactions between nanomaterials, the biota, and their environment at either low or high concentrations.4−6 As such, there are reasons to be concerned that the effects of rapid, high-dose exposures do not capture the unique environmental consequences of chronic exposures for organisms, populations, and ecosystems. © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
April 2, 2018 June 11, 2018 August 3, 2018 August 3, 2018 DOI: 10.1021/acs.est.8b01700 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology concentrations in wastewater from photofinishing facilities12 and is within the range or lower than many AgNP toxicity assays in the literature,2,13−15 but was, nonetheless, more than 4 orders of magnitude higher than predicted environmental releases of silver from nanoparticles.1,16 The environmental relevance of the inferences we could draw from such a study was modest since the magnitude of the impact was a direct result of the one-time pulse addition of a relatively high dose of pristine Ag0-NPs. There were two known limitations of the design of this earlier experiment that we addressed in the follow-on experiment reported here. First, a more realistic exposure scenario would use chronic low-level additions of AgNPs to more closely approximate the rate of release in wastewater effluent or runoff from biosolids applied to agricultural landscapes.1,16 Second, given that both field and laboratory studies9,17−20 indicate AgNPs are sulfidized in wastewater treatment to form Ag2S-NPs, a more realistic exposure scenario would feature chronic additions of Ag2SNPs. This is particularly important given that, whereas lowlevel chronic additions of Ag2S-NPs may be more realistic, laboratory experiments have repeatedly found that Ag2S-NPs are less toxic 21−23 and can be accumulated less by organisms than the precursor Ag0 NPs from which they are formed.22,23 We designed a follow-up experiment to (1) directly compare the effects of the same total mass of Ag0-NPs added either as a one-time pulse addition, or as chronic weekly additions spread over the course of an entire year; and (2) directly compare the effects of chronic and pulse additions of Ag0-NPs with the chronic addition of Ag2S-NPs, the form more likely to be released from wastewater or eroding biosolids. We sought to answer two questions with this one-year field experiment conducted in aquatic mesocosms. First, how does the rate of addition (one-time pulse addition vs chronic weekly additions) of zerovalent silver nanoparticles influence their exposure concentrations, ecosystem-level impacts (e.g., concentrations of dissolved organic carbon, oxygen, and methane), and bioaccumulation in organisms spanning different trophic levels? Second, how do these patterns compare between zerovalent silver nanoparticles used in products and the weathered silver sulfide particles, which are more likely to be the form that silver is entering the environment?
water are in Supporting Information (SI) Table S1). A hoophouse style greenhouse covering was installed in the winter (from the period of 12/11/2013 to 4/29/2014) and heated when temperatures fell below freezing to prevent mesocosms from freezing and damaging the wooden mesocosm boxes. The cover also helped to prevent overflowing of water due to heavy spring rains typical of the region (environmental data are in SI Figure S1). Organisms were either added to or allowed to colonize the mesocosms. Stems of Egeria densa were established in the aquatic zone between 30 and 180 days before the initiation of nanoparticle additions. Zooplankton/phytoplankton and benthic macroinvertebrates were added from several local wetlands. Given their dispersal limitation, small amounts of zooplankton/phytoplankton inocula being added every 2 weeks during the duration of the experiment so long as the source wetlands were not frozen over.25 Addition of larger benthic and pelagic animals occurred 40−90 days before the start of NP additions, and included Physella acuta (pond snails), larval Libellulidae (dragonflies), Gambusia holbrooki (eastern mosquitofish), and Corbicula f luminea (the non-native but naturalized Asiatic clam). Sand trays were used to keep the Corbicula in place, and trays were hung from PVC pipe floats 10 cm below the water surface to ensure adequate oxygen supply and limit predation. The mesocosms were also colonized by Chironomidae (nonbiting midges, hereafter chironomids) and spiders of the Tetragnathidae family (longjawed orb weaver spiders; hereafter spiders). While crayfish were included in the mesocosms (Procambarus sp.) as a bioturbator and detritivore in the food-web, the population sizes, and activity levels were unstable, and so crayfish data are not presented herein. Additionally, attempts were made both before the experiment and throughout the experiment’s duration to minimize crayfish densities. The vertebrate animal research was reviewed and approved by the Duke University IACUC A178−13−07. Nanoparticle Treatments. Gum arabic coated silver nanoparticles were freshly synthesized for this experiment by CEINT. Nanoparticles were synthesized with a mean TEM diameter of 3.9 ± 1.7 nm (mean ± SD, n= 159 particles measured; synthesized as per published methods7). A subset of the Ag0-NPs were then sulfidized through exposure to thioacetamide following published methods.26,27 This yielded spherical particles that were 24.2 ± 6.0 nm (n = 155 particles measured) with an XRD pattern consistent with acanthite (Ag2S). Particles were purified and concentrated by diafiltration (characterization and representative images can be found in SI Table S2 and Figure S2). Nanoparticle suspensions were periodically checked to confirm that no changes in particle size or concentration had occurred during storage. Nanoparticle additions to mesocosms commenced early morning, August 13, 2013, during summer in North Carolina. The dispensing tip of a modified Mariotte bottleprefilled with nanoparticles diluted in mesocosm waterwas submerged to ∼1 cm and moved in a grid to ensure materials were added as uniformly as possible across the entire water surface of each mesocosm. Each treatment consisted of three replicate mesocosms. Control mesocosms received only mesocosm water. Pulse-Ag0 mesocosms received a single dose of a suspension of zerovalent AgNPs equal to 450 mg Ag added to an average volume of 610 L mesocosm water yielding an average aqueous concentration of 0.74 mg Ag/L. Chronic mesocosms received a dose of either zerovalent (Chronic-Ag0) or sulfidized (Chronic-Ag2S) Ag
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MATERIALS AND METHODS Setup. Mesocosms (n = 12) were located in outdoor facilities at the Duke Forest Teaching and Research Laboratory, maintained by the Center for the Environmental Implications of NanoTechnology (CEINT) in Durham, NC. Design and construction of these slant-board wetland mesocosms have been detailed in a previous publication.24 Briefly, they are 3.66 × 1.22 × 0.81 m plywood boxes, constructed with a slope of 13° running for 2.8 m transitioning to a horizontal bottom such that each mesocosm included a permanently flooded aquatic zone in which this work was conducted. Mesocosms were lined with a replaceable highdensity polyethylene material (Permalon; REEF Industries, Inc., Houston, TX), which overlays a fish-safe ethylene propylene diene monomer rubber liner (PondGard; Firestone, Nashville, TN). Wetland soils were created from a blend of topsoils from Durham Sands and Soils (Durham, NC), giving a texture of 63.9% sand, 28.3% silt, and 13.0% clay, with 5.1% loss on ignition. Mesocosms were all filled with well water sourced on site (data on metals, ions, and nutrients in well B
DOI: 10.1021/acs.est.8b01700 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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was done by summing the product of the silver concentration measured at three time points each week (2 h, 3 days, and 7 days after each addition), multiplied by the length of each period until the subsequent measurement, and then divided by 7 days. To avoid introducing bias, when concentration measurements occurred at different frequencies during the week than typical, those weeks were omitted (Weeks 9, 16, 19, 20, 21, 27). With the exception of time-resolved silver, all data from the field experiment were analyzed using generalized linear mixed effects models (GLMM) using the glmer function in the R package, lme4.31 In the case of time-resolved silver, data were analyzed using general linear model (GLM) using the glm function as the differences in sampling frequency for the pulse and chronic treatments made GLMM impracticable. All parameters were analyzed with a Gamma family and log link. For silver in organisms and sediment, the model included the two main effects of treatment (Pulse-Ag0, Chronic-Ag0, Chronic-Ag2S) and ecosystem pool (sediment, periphyton, snail, clam, midge, damselfly, dragonfly, mosquitofish, spider), with treatment and organism nested within sampling date (Day 30 and 371). The model also included mesocosm as a random effect to formally account for mesocosm-level differences in the model. For all other data, the GLMM models differed only in that they did not have the main effect of organisms, had more time points, and for data not dealing with silver concentration, the Control treatment was included in the model. For modeling time-resolved silver data, the random effect of mesocosm was omitted since we used a GLM. Models were first examined by testing different distribution families and link functions. Subsequently, models were examined by analysis of variance (ANOVA) to look for the significance of the interactions and main effects. Alternative models were generated by eliminating nonsignificant interactions and main effects. Alternate models were tested by comparing their sample size corrected Akaike Information Criterion (AICc) using the car package.32 After selecting the most parsimonious model with the best fit, the lsmeans function33 was used to derive least-squares means and generate pairwise comparisons using Tukey HSD, which controls for multiple-comparisons.33
nanoparticles equal to 8.7 mg Ag each week for 52 weeks to reach a final cumulative Ag dose equal to that of the Pulse treatment (8.7 mg × 52 weeks = total 450 mg Ag). Sampling and Measurement. Water and samples of biota were collected before and at regular intervals throughout the experiment. In the first 2 weeks of sampling, water samples were collected frequently (2, 4, 24, and 36 h; 2, 3, 7, 9, 10, and 14 days). From that point forward, water samples were collected three times per week in the chronic treatments with the first sample collected just 2 h after adding nanomaterials, the second sample 3 days later, and the third 7 days later (just before the next nanoparticle addition). Pulse-Ag0 and Control mesocosms were only sampled at the three day and seven day time points. Water was collected by opening sterile 250 mL polypropylene bottles 10 cm below the water surface to avoid sampling the surface microlayer. Subsamples of whole water were collected and acidified to a concentration of 0.15 M HNO3 to dissolve Ag nanoparticles. A broad range of samples were taken at 30 and 371 days, including a range of animals, periphyton, and surficial sediment. Sampled mosquitofish were euthanized in bicarbonate buffered ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma-Aldrich), before flash freezing in liquid nitrogen. Invertebrates with shells were flash frozen in liquid nitrogen; shells were subsequently removed during sample processing. Animals were not depurated before euthanasia and preservation. All animal samples were thawed, homogenized, dried at 60 °C, and digested using a 4:2:1 mixture of 69% HNO3, 35% H2O2, 37% HCl for 120 min at 90 °C. Metals were measured by inductively coupled plasma mass spectrometry (ICP MS; water samples, Agilent 7500cx; biota, Agilent 7900; Santa Clara, CA). For samples with concentrations below the method detection limits (MDL) for water samples (MDL = 0.016 μg L−1) or biota (MDL = 0.006 mg Ag kg−1), we censored the data by substituting 50% of the method detection limit.28 Silver concentration of organisms in control treatments was low, and had an average Ag concentration across all taxa of 0.035 [0.018 to 0.068] mg kg−1 (mean [lower 95% CI to upper 95%CI]), with damselflies having the lowest and snails having the highest mean Ag concentrations (0.005 and 0.37 mg kg−1, respectively). Analytical runs included duplicate samples (1 in 10 samples), reagent blanks (1 in 10 samples), spike recovery samples (1 per run), and intercalibration/cross-calibration verification samples (1 in 10 samples). Spike recovery averaged 94 ± 3%, n = 3. The mean relative percentage difference between duplicate samples of animal tissues was 11%. Periphyton and surficial sediment were collected after all water and animal sampling was completed for a given day and disturbance was kept to a minimum. Periphyton samples were collected by scraping an area on the mesocosm liner from 0− 10 cm below the water surface with a glass microscope slide, and then collecting the resultant material. The surficial sediment was collected from three replicate 5 cm sediment cores. It was operationally defined as being that material which was readily suspended by agitating the overlying water for 30 s, and consisted of a mixture of dead algal and plant material, microbial biomass, and assorted flocculated mineral phases. Statistical Analyses. Statistical analyses were all performed with the R Statistical Computing platform.29 To best represent the average nanoparticle exposures experienced by organisms in a given week, a time-weighted average was also calculated30 for each week using log-transformed data. This
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RESULTS Silver in the Water Column. The highest water column concentrations of Ag measured in the Pulse-Ag0 treatment were observed immediately after the pulsed addition. In this treatment, Ag concentrations initially spiked to 752 [381 to 1480] μg Ag L−1 2 h after addition, and declined with an exponential rate over the course of the following weeks (Figure 1). Our initial dose in both chronic nanoparticle treatments increased water column silver concentrations to 21.5 [10.9 to 42.3] μg Ag L−1 in the Chronic-Ag0 treatment, and 25.2 [12.8 to 49.6] μg Ag L−1 in the Chronic-Ag2S treatment. Silver concentrations in both chronic treatments declined between weekly doses but for the duration of the experiment never fell below 0.4 and 0.38 μg Ag L−1 for Chronic-Ag0 NP and Chronic-Ag2S NP, respectively. By week 3, water column Ag was similar across all three treatments, and starting with the week 4 addition, the chronic treatments Ag concentrations were higher than for the Pulse-Ag0 treatment immediately after additions. By week 11, chronic treatments had higher water column Ag concentrations on average than the pulsed treatment despite having received only 21% of the cumulative C
DOI: 10.1021/acs.est.8b01700 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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had converged after a night of O2 consumption by respiration. Differences in DO between treatments then reemerged by lateafternoon of the second day of the experiment (Pulse-Ag0, 70 [56 to 84] % saturation compared to the Control, 179 [165 to 193] % saturation; p < 0.0001), and was again detected the following morning, 48 h after NP treatment started (p = 0.0053). This was the extent of the initial divergence in DO, and there were no further measured differences in DO between treatments until the Control mesocosms began showing lower DO than one or more silver treatments (Weeks 3, 15, 33, 35− 37, 42, 45−47). We also measured dissolved methane given our past observations, and found evidence in all three nanoparticle treatments of elevated methane concentrations compared with the control treatment, though the timing, magnitude, and duration differed between the Pulse-Ag0 and both chronic treatments (Figure 2). The Pulse-Ag0 treatment had greater
Figure 1. Silver concentration in water column of mesocosms over time shown as (A) time-resolved concentration or as (B) the weekly time-weighted average (TWA) concentrations, with lines representing means and shaded areas representing 95% confidence intervals. Nanoparticle treatments are represented by different colors and line patterns: Pulse-Ag0, black line with dashes; Chronic-Ag0, solid dark gray line; Chronic-Ag2S, blue line with dash-dotted line; Control, green line with long dashes. Red vertical dashed lines denote days 30 and 371, when animals were collected.
Figure 2. Comparison of dissolved CH4 concentrations among treatments. Treatments are represented by different colors and line patterns: Pulse-Ag0, black line with dashes; Chronic-Ag0, solid dark gray line; Chronic-Ag2S, blue line with dash-dotted line; Control, green line with long dashes. Lines are means, and shaded areas represent 95% confidence intervals.
total dose of 450 mg Ag that had been added to the Pulse-Ag0 treatment. Time-weighted average silver concentrations indicative of mean weekly exposure concentrationsshowed similar patterns to the time-resolved data (Figure 1). Much like in the time-resolved data, the pulse and chronic treatments converged by Week 3, but the average higher concentrations in the Chronic-Ag2S NP and Chronic-Ag0 NP in the timeweighted average show that organisms were exposed to a higher aqueous concentration of silver in these treatments than in the Pulse-Ag0 from Week 4 onward. There was a maximum difference in time-weighted average between chronic treatments and the Pulse-Ag0 treatments, where the Chronic-Ag0 had a maximum of 217-fold higher in Week 30, while the Chronic-Ag2S was 170-fold higher in Week 35. Ecosystem Effects. Focusing on ecosystem parameters that were responsive to additions of higher concentrations of pristine AgNP in a previous experiment,9 we found that neither dissolved organic carbon (DOC) nor dissolved CO2 showed differences between treatments at any point in time throughout the entire experiment (DOC, p = 0.45; CO2, p = 0.46). However, the Pulse-Ag0 treatment initially produced a decrease of dissolved oxygen (DO, measured as % saturation) relative to all other treatments, which were not significantly different from one another (SI Figure S3). The early effect in the Pulse-Ag0 treatment was a small signal superimposed on daily DO fluctuations driven by respiration and photosynthesis. On Day 0, just 4 h after dosing, the DO in Pulse-Ag0 treatments was significantly lower than in Control mesocosms (Pulse-Ag0 88 [74 to 102] % saturation vs Control, 118 [104 to 132] % saturation; p = 0.015). The following morning, all treatments
dissolved methane than Control mesocosms (Days 1−7, p < 0.005) with a maximum concentration on Day 2 that was 6fold higher than Control (Pulse-Ag0, 296 [172 to 508] μg CH4−C L−1; Control, 50.1 [29.2 to 86.0] μg CH4−C L−1). The Chronic-Ag0 treatment had elevated methane compared to Control during Weeks 28−34 and 42−45 (p < 0.05) with a subset of those times also exhibiting a significant increase in methane in the Chronic-Ag2S treatment (Weeks 28−29, p < 0.05; Week 30, p = 0.065; and Week 42, p = 0.091). The maximum difference in methane concentrations between either of the chronic treatments and Control mesocosms was during Week 44 when the Chronic-Ag0 NP treatment had 3-fold higher methane concentrations relative to Control mesocosms (Chronic-Ag0, 226 [131 to 389] μg CH4−C L−1; Control, 76 [44 to 130] μg CH4−C L−1). Silver in Biota and Sediment. There was a significant interaction between time, treatment, and ecosystem pool in the analysis of variance (p < 0.0001) indicating that, for any of these three main effects, it is necessary to examine the main effect within each of the combinations of levels of the other two factors. For example, to compare treatments, one must look at the treatments for each ecosystem pool on each date. As such, we compared: (i) differences by treatment for each ecosystem pool for both sampling times (Figure 3); (ii) differences by time for each ecosystem pool in each treatment (same data, redrawn to highlight relevant comparisons; SI Figure S4); and (iii) the differences by ecosystem pool within D
DOI: 10.1021/acs.est.8b01700 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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relative to the levels measured in the same treatment on Day 30 (SI Figure S4). In contrast, in both chronic treatments, surficial sediment, snails, and mosquitofish had Ag concentrations at least 3.3-fold higher after one year than they had after just one month. There were also some differences between the two chronic treatments, as clams increased significantly in Ag content over time in the Chronic-Ag0 treatment but not in Chronic-Ag2S, though it did show a similar trend (p = 0.108). While only chironomids showed decreases in Ag concentration in Chronic-Ag0 treatments, multiple organisms in the Chronic-Ag2S treatment showed declining Ag concentrations over time (periphyton, chironomids, damselflies, and spiders), despite the continued addition of NPs to these mesocosms. There is extensive evidence from this experiment of accumulation of silver in all sampled pools in the mesocosm food web, and possible evidence of increasing concentrations of Ag as we look at trophic levels likely to consume periphyton. Looking across all treatments in the early stages of the experiment (Day 30; SI Figure S5), Ag concentrations in the periphyton were greater than or equal to all other organisms. Concentrations of Ag then decreased when moving from primary producers (periphyton) to primary consumers (snails, clams, and chironomids), and continued to decrease in secondary consumers (damselflies, dragonflies, mosquitofish, and spiders). At the end of the experiment (Day 371) the overall pattern was similar to what was observed for Day 30, but in all treatments, snails had significantly higher Ag concentrations than periphyton, while clams were also higher than periphyton for both Pulse-Ag0 and Chronic-Ag0. Of particular note is the measurement of silver in the terrestrial spider of the genus Tetragnatha representing the trophic transfer of nanoparticle-sourced Ag across ecosystem boundaries into this terrestrial predator from emerging aquatic insects. For all three forms of silver, we see evidence of silver recovery in this spider that lives on the emergent macrophytes and weaves its web over the mesocosms. Silver concentrations in spiders were not significantly different among the nanoparticle treatments at Day 30 (means were from 2.8 to 7.5 mg Ag kg−1; Figure 3), but had diverged according to treatment by Day 371 such that spiders living above the Chronic-Ag0 mesocosms had the highest concentration at 2.35 [0.93 to 5.93] mg Ag kg−1. These were followed by those in ChronicAg2S at 0.097 [0.045 to 0.208] mg Ag kg−1, and finally, the spiders inhabiting the Pulse-Ag0 treatments were lowest at 0.0031 [0.0014 to 0.0066] mg Ag/kg (Figure 3). In some cases, mean Tetragnatha concentrations were as high as or higher than aquatic predators that were not only consuming prey containing silver but also consuming water and exchanging solutes through their gills with the water (SI Figure S5).
Figure 3. Comparison of silver concentration in sediment and organisms between treatments within the two sampling days, Day 30 and Day 371. Individual data points are plotted as small filled circles, while means and pooled 95% confidence intervals are plotted as open circles with error bars. Treatments are represented in each group of three by different colors: Pulse-Ag0, black and on the left; ChronicAg2S, blue and in the middle; Chronic-Ag0, gray and to the right. Concentrations in control mesocosms are not shown, but are described in the methods and were lower than all samples save one dragonfly replicate and several of the spiders. Lowercase letters indicate the results of pairwise comparisons across treatments for each pool on each date.
each treatment at each time point (same data, redrawn; SI Figure S5). Despite the lower cumulative dose in both of the chronic treatments by Day 30 (7.7% of the total added to Pulse-Ag0 as a one time pulse), most of the pools sampled at this time point had similar Ag concentrations across all three nanoparticle treatments. Only surficial sediment, mosquitofish, and clams had the expected higher tissue Ag concentrations in the PulseAg0 treatment (Figure 3). In surficial sediment, the Pulse-Ag0 treatment was 15 and 17-fold higher than the Chronic-Ag0 and Chronic-Ag2S treatments, respectively. Clams were similar to the surficial sediment, with clams from Pulse-Ag0 having silver concentrations five and 10-fold higher than the than the Chronic-Ag0 and Chronic-Ag2S treatments, respectively. The concentration was slightly lower in mosquitofish, which had concentrations of silver that were 3.9- and 7.5-fold higher in the Pulse-Ag0 than in the Chronic-Ag0 and Chronic-Ag2S treatments, respectively. Although we predicted that organisms would take up far less Ag when it was added in the sulfidized form relative to the pristine form, and while there were trends in this direction, we found only dragonflies had significantly lower Ag in the Chronic Ag2S treatment at α = 0.05 at Day 30 (4-fold lower than Chronic-Ag0). In contrast, periphyton had higher Ag concentrations in Chronic Ag2S treatment than in the Chronic Ag0 (Figure 3). After one year, the tissue Ag concentrations in most pools within the Pulse-Ag0 treatment had declined (sediment, periphyton, chironomids, damselflies, mosquitofish, and spiders) or remained the same (snails, clams, and dragonflies)
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DISCUSSION Rate of Addition Drove Exposure. The one-time pulse addition led to higher Ag concentrations initially in the water column; however, by the third week, the time-weighted average Ag concentrations in the Pulse-Ag0 treatment had converged with both the Chronic-Ag0 and Chronic-Ag2S treatments, despite the chronic treatments having received only 5.8% of the total silver added in the Pulse-Ag0 treatment. From the fourth week until the end of the experiment, both chronic silver treatments had higher average concentrations than the Pulse-Ag0 treatment with the chronic treatments E
DOI: 10.1021/acs.est.8b01700 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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trations albeit with a smaller magnitude than in the Pulse-Ag0 treatment in the first week. While the magnitude was lower, the increased methane concentration was more persistent, lasting for a total of 9 weeks in the Chronic-Ag0 treatment and 4 weeks in the Chronic-Ag2S treatment. These are both far longer than the one-week increase observed in Pulse-Ag0. Not only does this suggest that chronic additions can drive changes in methane cycling, but it also suggests that chronic low-level additions of Ag2S-NPs still have the potential to cause changes in greenhouse gas dynamics such as was observed for CH4 despite Ag2S-NPs being less bioavailable (i.e., less available for accumulation in organisms) and less toxic.21,23 The Rate of Addition Drove Accumulation. The concentrations of silver in the periphyton, grazers, filter feeders, and predators demonstrated that the rate of addition had an impact on how Ag accumulated in these organisms. Even though organisms experienced higher exposures initially in the Pulse-Ag0 treatment than in the chronic treatments, by Day 30 only the clams and mosquitofish had higher Ag concentrations in the Pulse-Ag0 treatment; all other organisms were not significantly different between silver treatments. This is important, as the chronic treatments by Day 30 had only received 9.6% of the silver added on Day 0 in the Pulse-Ag0 treatment, yet mean concentrations in most organisms were comparable. This suggests that pulse exposures not only underestimate long-term exposure concentrations, but they may even underestimate uptake and accumulation by organisms on shorter time scales. Conclusions drawn from Pulse-Ag0 treatment underestimate accumulation and trophic transfer at longer time-scales in many organisms relative to the Chronic-Ag0 treatment. Silver concentrations in periphyton, snails, mosquitofish, and spiders in the Chronic-Ag0 treatment were 10 to >100-fold higher than in the Pulse-Ag0 treatment at the conclusion of the study (on Day 371). There were four exceptions to this pattern that showed no significant difference between the Pulse-Ag0 and Chronic-Ag0 treatments for Ag body burdens at day 371: chironomids, clams, damselflies, and dragonflies. This similarity of Ag concentration in both the Chronic-Ag0 and Pulse-Ag0 in these four taxa at the end of the experiment is intriguing; these organisms are very different regarding their longevity in the system. The clams were there for the duration of the experiment, the chironomids likely had two or more generations during the duration of our experiment, and both the damselflies and dragonflies may have been exposed for six months or longer before the final sampling period. It is not surprising that the clams had similar concentrations in the two treatments, given that these individuals were exposed to the initial pulse of silver in the Pulse-Ag0 treatment, as well as the continual addition of silver in the Chronic-Ag0 treatment. Given the much shorter exposure of the chironomids and the more intermediate duration exposures of the damselflies and dragonflies, it is surprising that they do not show a disparity in silver concentrations that many other taxa did, given the disparity in water column concentrations. The diets of these organisms may also drive the lack of differences between the Chronic-Ag0 and Pulse-Ag0 in chironomids, damselflies, and dragonflies at Day 371. Larval chironomids in this experiment likely fed mainly on phytoplankton or periphyton in these mesocosms, giving them exposure through both diet and absorption of silver through their near-constant contact with the high silver concentrations in the periphyton. Measurements of surficial
reaching nearly 5-fold higher concentrations just 7 weeks after the start of exposures (after adding only 13.4% of the amount of silver). The disparity between Chronic and Pulse-Ag0 treatments was even higher during the winter as the PulseAg0 treatment converged on the detection limit, and the Chronic-Ag0 and -Ag2S treatments averaged 90-fold higher than the Pulse-Ag0 between weeks 11 and 38 (September 30th through May 11th of the following year). This is likely due to a combination of environmental (SI Figure S5) and biological factors including a lack of resuspension of sediment by rainfall during the winter due to the greenhouse cover, and cool water temperatures which would decrease bioturbation and silver transformations.34 One could argue from these results that one-time pulse exposures are doubly flawed in that they both overestimate the intensity of short-term exposures, but also are likely to underestimate the more realistic long-term exposure in real-world chronic exposures. Timing and Magnitude of Methane Release Differed by Addition Rate and Speciation. In a previous experiment,9 a one-time pulse addition resulted in a cascade of ecosystem-level effects. These effects included senescence of submersed macrophytes, which led to a 3-fold increase in DOC, a 2-fold increase in CO2, and led to hypoxia in the water column; these factors all contributed to a 40-fold increase in methane concentrations. In that experiment, the silver addition was 900 mg Ag per mesocosm in 360 L of water, which gave a maximum water column silver concentration of 2.5 mg L−1. Given that this concentration was orders of magnitude above expected environmental concentrations in waterbodies receiving wastewater effluent contaminated by silver nanoparticles, this tempered the concerns raised by the observation of these ecosystem-level effects, which were comparable to what was observed with additions of dissolved silver. While that experiment offered keen insights into the mechanism, it left open the question as to what effects might occur in lowerconcentration exposures as one-time or chronic additions of either pristine or weathered AgNPs. When comparing the data from this experiment with those from our previous experiment,9 the dramatic changes in CO2 and DOC observed in the previous experiment were not detected in this experiment, the decline in dissolved O2 was more subtle, yet the timing and duration of the CH4 response in the Pulse-Ag0 treatment mirrored that of the previous experiment. This similar pattern in CH4 is surprising given the vagaries and challenges of replicating experiments in different years in a field setting. The difference in the magnitude is not surprising, given that the water column silver concentrations in the Pulse-Ag0 treatment in this experiment was 3.5-fold lower and water volume was roughly twice that of the previous experiment. As might be expected of a dose−response relationship, we would expect less methane to be produced, with that methane accumulating in a larger volume of water also giving lower concentrations. This much lower concentration may also explain our inability to detect significant increases in DOC and dissolved CO2, and may explain the lower magnitude and shorter duration of the depression in dissolved oxygen. While the Chronic-Ag0 and Chronic-Ag2S treatments had no detected increases in dissolved CH4 in the initial 7 days of the experiment, there was a detectable increase after 28 weeks. By this point, the chronic treatments had received 50% of the total amount of Ag added in Pulse-Ag0, which appears to have been sufficient to lead to increased dissolved methane concenF
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and 3 orders of magnitude lower than spiders in the ChronicAg0 treatment. The disparity in Ag concentration in spiders between the three different treatments at Day 371 is unexpected given that there were no significant concentration differences in their likely prey when analyzed in their larval forms (i.e., mostly chironomids). There are two possible reasons for the differences in spider body burdens. First, if there were lower rates of emergence in the Pulse-Ag0 treatment due to lower abundances of benthic macroinvertebrates, this could lead to the lower transfer of Ag to spiders. Second, if the different treatments led to differences in the incorporation of Ag in different tissues, there may have been differences in the loss of Ag in the different treatments at metamorphosis from wingless larvae to winged adults which could lead to differences in the shedding of body burden.48 Regardless of the specific mechanisms that are responsible for the observed differences in spider Ag concentration, these data provide still one more piece of evidence of how one-time pulse exposures can underestimate the impacts of contaminants, in this case, by underestimating the flux of silver nanoparticle contaminants from aquatic to terrestrial ecosystems. Chronic Additions of Silver Nanoparticles Led to Higher Exposure and Impacts. When these data are taken in total, they reveal that chronic exposures of AgNPs lead to higher overall exposure concentrations due to elevated water concentrations over the course of the experiment when compared to a similar mass added as a one-time pulse (Figure 1). In accord with these higher overall exposures, the ecosystem level effects on CH4 concentrations happened later and were more prolonged in the chronic treatments vs Pulse-Ag0 (4 and 11 weeks in the Chronic-Ag2S and ChronicAg0, respectively, instead of only 1 week in the Pulse-Ag0). While nanoparticle surface modification and passivation through sulfidation can lead to lower uptake of AgNPs and minimal effects with model organisms in laboratory trials,21,23 we saw evidence of ecosystem-level effects (increased dissolved methane), as well as a similar extent of uptake and trophic transfer between Chronic-Ag0 and Chronic-Ag2S treatments. Finally, whereas Ag was accumulated by organisms throughout the food web and transferred into the terrestrial food web through emerging insects, our data were mostly consistent with biodilution, in which concentrations decline from lower to higher trophic levels. The one exception to this was the evidence consistent with biomagnification observed from periphyton to aquatic snails, which is consistent with past studies of dissolved silver.49 While high-concentration single-addition pulse exposures are relevant for examining short-term impacts and mechanisms in model organisms and cell cultures, the findings of this study suggest that such pulse additions are likely to overestimate short-term impacts and underestimate long-term exposure, uptake by organisms, and impacts from the organism to the ecosystem level. To truly address such end points, long-term experiments are required with low-concentration chronic additions in model ecosystems populated by a range of different organisms.
sediment and periphyton show them both to be slightly higher in the Chronic-Ag0 treatment than in the Pulse-Ag0 at Day 371, though chironomids may have been exposed to similar concentrations of Ag in both treatments for much of their life cycle. The patterns in damselflies between Pulse-Ag0 and Chronic-Ag0 treatments may be similar given they likely fed primarily on chironomids in this system given low abundance of ostracods and cladocerans (data not shown).35−37 Similarly, dragonflies were likely changing in tandem with chironomids and damselflies which likely made up the majority of their diet in this system given their relative abundance.38,39 Weathered Nanoparticles Showed Similar Bioavailability to Pristine Nanoparticles. We expected that the Chronic-Ag2S treatment would have lower uptake across all taxa than the Chronic-Ag0 treatment for two reasons. First, sulfidation has been shown to decrease the reactivity and dissolution of Ag+ from AgNPs40 and has been observed to lead to decreased uptake in some organisms.23 Second, smaller particles have been shown to have greater uptake rates in some organisms.41,42 Though the Ag0-NPs that we sulfidized to make the Ag2S-NPs had the same starting size as the particles used in the Ag0 treatments (3.9 ± 1.7 nm), the process of sulfidizing them led to an increase in particle size (24.2 ± 6.0 nm) which would also be expected to contribute to lower uptake. Contrary to our expectations, the majority of organisms in the Chronic-Ag2S treatment had Ag concentrations that were not significantly different from those in the Chronic-Ag0 treatments at both Day 30 and 371. With the exception of dragonflies, all animals at Day 30 had concentrations that were not significantly different between the Chronic-Ag0 and Chronic-Ag2S treatments, whereas periphyton was nearly 4fold higher for the Chronic-Ag2S treatment. At Day 371, periphyton and four out of the seven animal taxa were not significantly different between the Chronic-Ag2S and ChronicAg0 treatment, though for those organisms where the ChronicAg0 treatment was significantly higher, it was roughly an order of magnitude higher (18.6, 7.8, and 24-fold higher in clams, snails, and spiders, respectively) consistent with results published for the submersed macrophyte, Egeria densa.43 Evidence of Aquatic to Terrestrial Trophic Transfer. While emerging macroinvertebrates serve as important subsidies of energy and nutrients to terrestrial food webs, the “dark subsidies”44 of contaminants they carry with them have potentially deleterious effects on terrestrial organisms. Riparian orb-weaver spiders serve as an essential nexus for contaminant transfer from aquatic to terrestrial food webs given they feed almost exclusively on emerging benthic macroinvertebrates and serve as an important component of the diets of many species of songbirds. Given that past studies have focused on their role in aquatic-to-terrestrial trophic transfer of mercury,45 cyanobacterial derived toxins,46 and polychlorinated biphenyls,47 we examined them to see if they would show evidence of aquaticto-terrestrial transfer of AgNPs. Across all treatments, there was evidence of trophic transfer from benthic macroinvertebrates to spiders in the genus Tetragnatha. As early as 30 days into the experiment, the concentration of Ag in spiders was as high as or higher than those in the dominant aquatic predators: damselfly larvae, dragonfly larvae, and mosquitofish. While the concentrations at Day 30 were not significantly different between treatments, by Day 371 spiders in Pulse Ag0 mesocosms were 2 orders of magnitude lower than spiders in the Chronic-Ag2S treatment,
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.8b01700. G
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Manufactured Nanomaterial Environmental Hazards: Are They Relevant? Environ. Sci. Technol. 2014, 48 (18), 10541−10551. (3) Palmqvist, A.; Baker, L.; Forbes, V. E.; Gergs, A.; von der Kammer, F.; Luoma, S.; Lützhøft, H. C. H.; Salinas, E.; Sorensen, M.; Steevens, J. Nanomaterial Environmental Risk Assessment. Integr. Environ. Assess. Manage. 2015, 11 (2), 333−335. (4) Baker, L. F.; King, R. S.; Unrine, J. M.; Castellon, B. T.; Lowry, G. V.; Matson, C. W. Press or Pulse Exposures Determine the Environmental Fate of Cerium Nanoparticles in Stream Mesocosms. Environ. Toxicol. Chem. 2016, 35 (5), 1213−1223. (5) Blakelock, G. C.; Xenopoulos, M. A.; Norman, B. C.; Vincent, J. L.; Frost, P. C. Effects of Silver Nanoparticles on Bacterioplankton in a Boreal Lake. Freshwater Biol. 2016, 61 (12), 2211−2220. (6) Thanh Binh, C. T.; Adams, E.; Vigen, E.; Tong, T.; A. Alsina, M.; Gaillard, J.-F.; A. Gray, K.; G. Peterson, C.; J. Kelly, J. Chronic Addition of a Common Engineered Nanomaterial Alters Biomass, Activity and Composition of Stream Biofilm Communities. Environ. Sci.: Nano 2016, 3 (3), 619−630. (7) Yin, L.; Cheng, Y.; Espinasse, B.; Colman, B. P.; Auffan, M.; Wiesner, M.; Rose, J.; Liu, J.; Bernhardt, E. S. More than the Ions: The Effects of Silver Nanoparticles on Lolium multiflorum. Environ. Sci. Technol. 2011, 45, 2360−2367. (8) Yin, L.; Colman, B. P.; McGill, B. M.; Wright, J. P.; Bernhardt, E. S. Effects of Silver Nanoparticle Exposure on Germination and Early Growth of Eleven Wetland Plants. PLoS One 2012, 7, e47674. (9) Colman, B. P.; Espinasse, B.; Richardson, C. J.; Matson, C. W.; Lowry, G. V.; Hunt, D. E.; Wiesner, M. R.; Bernhardt, E. S. Emerging Contaminant or an Old Toxin in Disguise? Silver Nanoparticle Impacts on Ecosystems. Environ. Sci. Technol. 2014, 48 (9), 5229− 5236. (10) Unrine, J. M.; Colman, B. P.; Bone, A. J.; Gondikas, A. P.; Matson, C. W. Biotic and Abiotic Interactions in Aquatic Microcosms Determine Fate and Toxicity of Ag Nanoparticles. Part 1. Aggregation and Dissolution. Environ. Sci. Technol. 2012, 46 (13), 6915−6924. (11) Bone, A. J.; Colman, B. P.; Gondikas, A. P.; Newton, K. M.; Harrold, K. H.; Cory, R. M.; Unrine, J. M.; Klaine, S. J.; Matson, C. W.; Di Giulio, R. T. Biotic and Abiotic Interactions in Aquatic Microcosms Determine Fate and Toxicity of Ag Nanoparticles: Part 2−Toxicity and Ag Speciation. Environ. Sci. Technol. 2012, 46 (13), 6925−6933. (12) Bard, C. C.; Murphy, J. J.; Stone, D. L.; Terhaar, C. J. Silver in photoprocessing effluents. J. Water Pollut. Control Fed. 1976, 48 (2), 389−394. (13) Orbea, A.; González-Soto, N.; Lacave, J. M.; Barrio, I.; Cajaraville, M. P. Developmental and Reproductive Toxicity of PVP/ PEI-Coated Silver Nanoparticles to Zebrafish. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2017, 199, 59−68. (14) Zou, X.; Li, P.; Lou, J.; Zhang, H. Surface Coating-Modulated Toxic Responses to Silver Nanoparticles in Wolff ia globosa. Aquat. Toxicol. 2017, 189, 150−158. (15) Juganson, K.; Mortimer, M.; Ivask, A.; Pucciarelli, S.; Miceli, C.; Orupõld, K.; Kahru, A. Mechanisms of Toxic Action of Silver Nanoparticles in the Protozoan Tetrahymena thermophila: From Gene Expression to Phenotypic Events. Environ. Pollut. 2017, 225, 481− 489. (16) Keller, A. A.; Lazareva, A. Predicted Releases of Engineered Nanomaterials: From Global to Regional to Local. Environ. Sci. Technol. Lett. 2014, 1 (1), 65−70. (17) Kim, B.; Park, C. S.; Murayama, M.; Hochella, M. F., Jr Discovery and Characterization of Silver Sulfide Nanoparticles in Final Sewage Sludge Products. Environ. Sci. Technol. 2010, 44, 7509− 7514. (18) Ma, R.; Levard, C.; Judy, J. D.; Unrine, J. M.; Durenkamp, M.; Martin, B.; Jefferson, B.; Lowry, G. V. Fate of Zinc Oxide and Silver Nanoparticles in a Pilot Wastewater Treatment Plant and in Processed Biosolids. Environ. Sci. Technol. 2014, 48 (1), 104−112. (19) Kaegi, R.; Voegelin, A.; Ort, C.; Sinnet, B.; Thalmann, B.; Krismer, J.; Hagendorfer, H.; Elumelu, M.; Mueller, E. Fate and
Figures and tables showing nanoparticle characterization, dissolved organic carbon, dissolved carbon dioxide, dissolved oxygen, water temperature, rainfall, mesocosm water volume, and alternative renderings of Figure 3 allowing comparisons by date or organism (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: 406-243-6315; email:
[email protected]. ORCID
Benjamin P. Colman: 0000-0001-6290-3705 Jason M. Unrine: 0000-0003-3012-5261 Emily S. Bernhardt: 0000-0003-3031-621X Present Address #
(B.P.C.) Department of Ecosystem and Conservation Sciences, University of Montana, Missoula, Montana, United States. (L.F.B.) Department of Biology, University of Waterloo, Waterloo, Canada. (S.M.M.) Immucor, Norcross, Georgia, United States. (D.E.G.) Materials Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States.
Author Contributions
B.P.C., L.F.B., R.S.K., C.W.M., and E.S.B. designed the experiment. B.P.C. and L.F.B. contributed to the fieldwork. S.M.M. and D.E.G. adapted the synthesis methods, synthesized, and characterized the nanoparticles used in the experiment. B.P.C., L.F.B., and R.S.K. contributed to statistical analyses. L.F.B. and J.M.U. conducted ICP-MS analyses. All authors contributed to the drafting and revising of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Anna Fedders for her tireless efforts in the field and in the lab which made this work possible. We also thank Benjamin Espinasse for his capable help in getting the experiment set up and helping during emergencies that arose during the year-long experiment. Finally, we thank Brooke Hassett, Medora Burke-Scoll, Mathieu Therezien, Chris Ward, Carly Gwin, Fabienne Schwab, and Steven Anderson for their many contributions in the lab and in the field, and Shristi Shrestha for her ICP-MS analyses of water samples. This work was supported by the National Science Foundation (NSF) and the Environmental Protection Agency (EPA) under NSF Cooperative Agreement EF-0830093 and DBI-1266252, Center for the Environmental Implications of Nanotechnology (CEINT). Any opinions, findings, conclusions, or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF or the EPA. This work has not been subjected to EPA review and no official endorsement should be inferred.
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REFERENCES
(1) Sun, T. Y.; Bornhöft, N. A.; Hungerbühler, K.; Nowack, B. Dynamic Probabilistic Modeling of Environmental Emissions of Engineered Nanomaterials. Environ. Sci. Technol. 2016, 50 (9), 4701− 4711. (2) Holden, P. A.; Klaessig, F.; Turco, R. F.; Priester, J. H.; Rico, C. M.; Avila-Arias, H.; Mortimer, M.; Pacpaco, K.; Gardea-Torresdey, J. L. Evaluation of Exposure Concentrations Used in Assessing H
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Article
Environmental Science & Technology Transformation of Silver Nanoparticles in Urban Wastewater Systems. Water Res. 2013, 47 (12), 3866−3877. (20) Lombi, E.; Donner, E.; Taheri, S.; Tavakkoli, E.; Jämting, Å. K.; McClure, S.; Naidu, R.; Miller, B. W.; Scheckel, K. G.; Vasilev, K. Transformation of Four Silver/Silver Chloride Nanoparticles during Anaerobic Treatment of Wastewater and Post-Processing of Sewage Sludge. Environ. Pollut. 2013, 176, 193−197. (21) Levard, C.; Hotze, E. M.; Colman, B.; Dale, A. L.; Truong, L.; Yang, X.; Bone, A.; Brown, G. E.; Tanguay, R. L.; Di Giulio, R. T. Sulfidation of Silver Nanoparticles: Natural Antidote to Their Toxicity. Environ. Sci. Technol. 2013, 47 (23), 13440−13448. (22) Wang, P.; Menzies, N. W.; Lombi, E.; Sekine, R.; Blamey, F. P. C.; Hernandez-Soriano, M. C.; Cheng, M.; Kappen, P.; Peijnenburg, W. J.; Tang, C.; Kopittke, P. M. Silver Sulfide Nanoparticles (Ag2SNPs) Are Taken up by Plants and Are Phytotoxic. Nanotoxicology 2015, 9 (8), 1041−1049. (23) Starnes, D. L.; Unrine, J. M.; Starnes, C. P.; Collin, B. E.; Oostveen, E. K.; Ma, R.; Lowry, G. V.; Bertsch, P. M.; Tsyusko, O. V. Impact of Sulfidation on the Bioavailability and Toxicity of Silver Nanoparticles to Caenorhabditis elegans. Environ. Pollut. 2015, 196, 239−246. (24) Lowry, G. V.; Espinasse, B. P.; Badireddy, A. R.; Richardson, C. J.; Reinsch, B. C.; Bryant, L. D.; Bone, A. J.; Deonarine, A.; Chae, S.; Therezien, M. Long-Term Transformation and Fate of Manufactured Ag Nanoparticles in a Simulated Large Scale Freshwater Emergent Wetland. Environ. Sci. Technol. 2012, 46 (13), 7027−7036. (25) Hall, S. R.; Leibold, M. A.; Lytle, D. A.; Smith, V. H. Stoichiometry and Planktonic Grazer Composition over Gradients of Light, Nutrients, and Predation Risk. Ecology 2004, 85 (8), 2291− 2301. (26) Djoković, V.; Krsmanović, R.; Božanić, D. K.; McPherson, M.; Van Tendeloo, G.; Nair, P. S.; Georges, M. K.; Radhakrishnan, T. Adsorption of Sulfur onto a Surface of Silver Nanoparticles Stabilized with Sago Starch Biopolymer. Colloids Surf. Colloids Surf., B 2009, 73 (1), 30−35. (27) Stegemeier, J. P.; Avellan, A.; Lowry, G. V. Effect of Initial Speciation of Copper- and Silver-Based Nanoparticles on Their LongTerm Fate and Phytoavailability in Freshwater Wetland Mesocosms. Environ. Sci. Technol. 2017, 51 (21), 12114−12122. (28) Clarke, J. U. Evaluation of Censored Data Methods To Allow Statistical Comparisons among Very Small Samples with Below Detection Limit Observations. Environ. Sci. Technol. 1998, 32 (1), 177−183. (29) R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2017. (30) Finucane, E. W. Concise Guide to Environmental Definitions, Conversions, and Formulae; CRC Press, 1998. (31) Bates, D.; Mächler, M.; Bolker, B.; Walker, S. Fitting Linear Mixed-Effects Models Using Lme4. J. Stat. Softw. 2015, 67 (1), 1−48. (32) Fox, J.; Weisberg, S. An R Companion to Applied Regression, 2nd. ed.; Sage: Thousand Oaks CA, 2011. (33) Lenth, R. V. Least-Squares Means: The R Package Lsmeans. J. Stat. Softw. 2016, 69 (1), 1−33. (34) Dale, A. L.; Lowry, G. V.; Casman, E. A. Modeling Nanosilver Transformations in Freshwater Sediments. Environ. Sci. Technol. 2013, 47 (22), 12920−12928. (35) Pearlstone, P. S. M. Observations on a Natural Population of Damselfly Larvae.; University of British: Columbia, 1971. (36) Thompson, D. J. The Natural Prey of Larvae of the Damselfly, Ischnura elegans (Odonata: Zygoptera). Freshwater Biol. 19788 (4), 377−384. (37) Lawton, J. H. Feeding and Food Energy Assimilation in Larvae of the Damselfly Pyrrhosoma nymphula (Sulz.) (Odonata: Zygoptera). J. Anim. Ecol. 1970, 39 (3), 669. (38) Dillon, P. M. Chironomid Larval Size and Case Presence Influence Capture Success Achieved by Dragonfly Larvae. Freshw. Invertebr. Biol. 1985, 4 (1), 22−29.
(39) Merrill, R.; Johnson, D. Dietary Niche Overlap and Mutual Predation among Coexisting Larval Anisoptera. Odonatologica 1984, 13 (3), 387−406. (40) Levard, C.; Reinsch, B. C.; Michel, F. M.; Oumahi, C.; Lowry, G. V.; Brown, G. E. Sulfidation Processes of PVP-Coated Silver Nanoparticles in Aqueous Solution: Impact on Dissolution Rate. Environ. Sci. Technol. 2011, 45, 5260−5266. (41) Zhao, C.-M.; Wang, W.-X. Size-Dependent Uptake of Silver Nanoparticles in Daphnia magna. Environ. Sci. Technol. 2012, 46 (20), 11345−11351. (42) Meyer, J. N.; Lord, C. A.; Yang, X. Y.; Turner, E. A.; Badireddy, A. R.; Marinakos, S. M.; Chilkoti, A.; Wiesner, M. R.; Auffan, M. Intracellular Uptake and Associated Toxicity of Silver Nanoparticles in Caenorhabditis elegans. Aquat. Toxicol. 2010, 100, 140−150. (43) Yuan, L.; Richardson, C. J.; Ho, M.; Willis, C. W.; Colman, B. P.; Wiesner, M. R. Stress Responses of Aquatic Plants to Silver Nanoparticles. Environ. Sci. Technol. 2018, 52 (5), 2558−2565. (44) Walters, D. M.; Fritz, K. M.; Otter, R. R. The Dark Side of Subsidies: Adult Stream Insects Export Organic Contaminants to Riparian Predators. Ecol. Appl. 2008, 18 (8), 1835−1841. (45) Gann, G. L.; Powell, C. H.; Chumchal, M. M.; Drenner, R. W. Hg-Contaminated Terrestrial Spiders Pose a Potential Risk to Songbirds at Caddo Lake (Texas/Louisiana, USA). Environ. Toxicol. Chem. 2015, 34 (2), 303−306. (46) Moy, N. J.; Dodson, J.; Tassone, S. J.; Bukaveckas, P. A.; Bulluck, L. P. Biotransport of Algal Toxins to Riparian Food Webs. Environ. Sci. Technol. 2016, 50 (18), 10007−10014. (47) Walters, D. M.; Mills, M. A.; Fritz, K. M.; Raikow, D. F. SpiderMediated Flux of PCBs from Contaminated Sediments to Terrestrial Ecosystems and Potential Risks to Arachnivorous Birds. Environ. Sci. Technol. 2010, 44 (8), 2849−2856. (48) Wesner, J. S.; Walters, D. M.; Schmidt, T. S.; Kraus, J. M.; Stricker, C. A.; Clements, W. H.; Wolf, R. E. Metamorphosis Affects Metal Concentrations and Isotopic Signatures in a Mayfly (Baetis tricaudatus): Implications for the Aquatic-Terrestrial Transfer of Metals. Environ. Sci. Technol. 2017, 51 (4), 2438−2446. (49) Ratte, H. T. Bioaccumulation and Toxicity of Silver Compounds: A Review. Environ. Toxicol. Chem. 1999, 18 (1), 89− 108.
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