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Article
Effects and implications of trophic transfer and accumulation of CeO nanoparticles in a marine mussel 2
Jon Robert Conway, Shannon Kareem Hanna, Hunter Lenihan, and Arturo A. Keller Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es404549u • Publication Date (Web): 13 Jan 2014 Downloaded from http://pubs.acs.org on January 20, 2014
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Environmental Science & Technology
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Effects and implications of trophic transfer and
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accumulation of CeO2 nanoparticles in a marine
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mussel
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Jon R. Conway, Shannon K. Hanna, Hunter S. Lenihan, Arturo A. Keller*
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University of California Center for Environmental Implications of Nanotechnology
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Bren School of Environmental Science and Management
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University of California, Santa Barbara, CA 93106
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KEYWORDS
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CeO2,
Engineered
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galloprovincialis
Nanomaterials,
Trophic
Transfer,
Subchronic
Exposure,
Mytilus
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ABSTRACT
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Bivalves are hypothesized to be key organisms in the fate and transport of engineered
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nanomaterials (ENMs) in aquatic environments due to their ability to filter and concentrate
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particles from water, but how different exposure pathways influence their interactions with
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ENMs is not well understood. In a five week experiment we tested how interactions between
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CeO2 ENMs and a marine mussel, Mytilus galloprovincialis, are affected through two exposure
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methods, direct and through sorption to phytoplankton. We found that phytoplankton sorbed
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ENMs in 99.9% of CeO2 was filtered from the water over a 2 day time
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period. In a previous study using M. galloprovincialis and CeO2 ENMs at a similar concentration
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(1 mg L-1)11 18 MΩcm), and dried for 72 h at 60°C. Tissue samples were weighed and digested
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in a 3:2 mixture of 30% H2O2:HNO3 at 80°C for 2.5 h. Pseudofeces was allowed to settle in
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sample cups after aeration was removed then collected by pipette and triple washed with purified
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water. Identical drying, weighing, and digestion techniques were followed for pseudofeces
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samples. Since mussel feces dissolve in water, it was assumed that in the closed systems used in
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this experiment they were re-filtered by the mussels and eventually the CeO2 ENMs would be
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captured in tissue or pseudofeces. Tissue and pseudofeces samples were analyzed for Ce via
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inductively coupled plasma atomic emission spectroscopy (ICP-AES, iCAP 6300 Thermo
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Scientific, Waltham, MA), with a detection limit of 50 µg L-1. Standard solutions were measured
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every 15 samples for quality assurance. Results are reported as Ce but samples are assumed to
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have contained Ce in the form of nanoparticulate CeO2 before acid digestion.
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Phytoplankton/CeO2 Interactions
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Sorption of CeO2 ENMs to I. galbana was determined by the separation of the fraction of
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phytoplankton with sorbed CeO2 using centrifugal split-flow thin-channel (CSPLITT)
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fractionation (CFS 2000, Postnova Analytics, Salt Lake City, UT). Operational parameters (flow
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rates and centrifuge rpm) were calibrated for this experiment to give a roughly 50/50 split of both
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I. galbana and CeO2 ENMs into the two fractions (lighter and denser) produced by this
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technique, which gave the greatest sensitivity to changes in either component or fraction. The
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cutoff density between the light and dense fractions is approximately 0.5 g cm-3, the average
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density of an I. galbana cell. Sorption of CeO2 ENMs to a cell will increase its density and
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increase the likelihood it will be captured in the dense fraction.
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To perform a separation, 0.5 mL of phytoplankton culture exposed to CeO2 ENMs was
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injected into the channel at 0.5 mL min-1 using a 1:7 filtered seawater:deionized water carrier
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solution with a flow rate of 4.5 mL min-1 and spun at 600 rpm for 15 minutes, followed by a
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second injection of a second 0.5 mL of exposed phytoplankton. Dense and light fractions were
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collected during these steps, which corresponded respectively to phytoplankton with and without
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sorbed CeO2. After an additional 15 minutes, the centrifuge was shut off and collection was
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routed entirely through the dense outflow for 20 minutes before stopping. Phytoplankton
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concentrations were determined through visual counts. Ce concentrations could not be reliably
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detected in either fraction via ICP-AES analysis due to the large dilution necessitated by this
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technique.
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Sorption of CeO2 ENMs to phytoplankton cells was verified by exposing cultures to 1, 10, 20,
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and 30 mg L-1 CeO2 ENMs for 30 min and separating sorbed and unsorbed fractions. Sorption
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kinetics were estimated by exposing phytoplankton cultures to 30 mg L-1 CeO2 ENMs and
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measuring phytoplankton densities in each fraction in a time series, beginning immediately after
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exposure then at 6, 12, 24, 48, 72, and 96 h. Cell weight was calculated by comparing the mass
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of identical volumes of seawater and phytoplankton cultures of a known cell density.
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Dissolved extracellular polymeric substances (EPS) were extracted from I. galbana cultures by
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centrifuging cultures at 1545 g for 30 minutes and collecting the supernatant24. EPS protein and
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carbohydrate content was measured using the modified Lowry Protein Assay Kit (Pierce, 23240,
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USA) and the anthrone method,25 respectively.
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Statistical Analyses
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We tested whether clearance rate, Ce concentrations in pseudofeces, pseudofeces mass, and
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phytoplankton growth varied as functions of time and CeO2 exposure treatment using two-way
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ANOVA. We also tested whether the sorption of CeO2 to phytoplankton varied over time with a
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one-way ANOVA. When ANOVA showed significant differences, Tukey’s tests were conducted
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posthoc. A linear regression was performed to test the dependence of clearance rate of mussels
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exposed to CeO2 ENMs directly on the length of exposure. Levene’s test was used to ensure
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homogeneity of variance between groups. Tissue Ce concentrations did not follow a normal
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distribution, therefore Kruskal-Wallis tests with posthoc Dunn’s test for multiple comparisons
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were used to determine if Ce tissue concentrations varied significantly as functions of time,
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exposure method, and exposure concentration. Hydrodynamic diameters of CeO2 ENMs in
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dissolved EPS solution and phytoplankton growth media were compared using a two-sample t-
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test. All statistical analyses were performed using the statistical software R (v. 2.11.1).
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3. Results & Discussion
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Phytoplankton/CeO2 Interactions
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In I. galbana cultures exposed to 30 mg L-1 CeO2 ENMs for 8 days, we found that the impact
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of CeO2 exposure on cell count depends on exposure time (ANOVA, F2,8 = 2.46, p1 μm) aggregates could
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be seen under a light microscope after 1 h when CeO2 ENMs were suspended in seawater at an
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equivalent concentration. This is most likely a consequence of sorption and not disaggregation
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by EPS because when CeO2 ENMs were suspended at a concentration of 30 mg L-1 in an
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dissolved EPS solution (containing 2.3±0.1 μg mL-1 carbohydrates and 13.3±0.0 μg mL-1
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proteins (±SE)) aggregate sizes measured by DLS were not significantly different (two-sample
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t(2.558)=0.5, p>0.5) from those in growth media only. Additionally, sedimentation of aggregates
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identical to that seen in seawater was observed. Due to the presence of phytoplankton, CeO2
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particle size in exposed cultures could not be determined through DLS to give a direct
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comparison, but together these observations point to the conclusion that all or nearly all ENMs
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sorbed to phytoplankton. This gives a per cell mass load of 27±1 picograms CeO2 (±SE) at 30
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mg L-1 exposure concentration, which corresponds to a non-steady state partitioning coefficient
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(Ks, the ratio of phytoplankton Ce concentration to the concentration of Ce in the surrounding
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media, akin to a bioconcentration factor), of 1.1×104.
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Accumulation & Rejection of CeO2 ENMs by M. galloprovincialis
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Our primary hypothesis in this experiment was that mussels would accumulate more Ce when
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exposed to CeO2 that was sorbed to phytoplankton than free CeO2 in the water column, and that
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they would accumulate more Ce over time. This was refuted by our results as we found that the
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impact of exposure concentration on Ce concentration in mussel tissue varied significantly with
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time (Kruskal-Wallis, H=52.05, p0.5, Figure 3).
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Although there was a difference in accumulation based on exposure method on day 5 (Kruskal-
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Wallis, H=10.58, p0.5) or exposure (ANOVA, F2,3
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= 0.412, p>0.1) corresponding concentration change in pseudofeces over time (Table S2). This
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was due to the increased production of pseudofeces with CeO2 exposure (Figure 4). An estimate
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of the mass balance for each treatment can be seen in Figure 5.
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Based on these data the non-steady state partitioning coefficients (Kt, the ratio of tissue Ce
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concentration to the concentration of Ce in the surrounding media, similar to bioaccumulation
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factors) for each treatment were low, ranging from 8.8 to 18 with a median of 11.
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corresponds well with the results of two other studies using marine invertebrates.14,
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pseudofeces excreted by the mussels, a pseudofeces-water partitioning coefficient (Kp, the ratio
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of pseudofeces Ce concentration to the concentration of Ce in the surrounding media) was also
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determined for each treatment. Expressed as log10Kp, the values we calculated have a range
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between 3.45 and 4.0 with a median of 3.6.
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For
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We found accumulation of CeO2 in mussel tissues over time in all experimental groups, which
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when considered with the constant Ce concentrations in pseudofeces suggests that some ENMs
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were being stored either in the digestive glands or elsewhere in the organism and were not
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immediately captured in pseudofeces and excreted. It is unlikely that a significant amount
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remained in the gut as bivalves have been shown to excrete micron-scale particles after 30 h or
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less with and without additional food.27, 28 In seawater CeO2 ENMs form aggregates near 1 µm
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and so will likely be egested well before the 72 h allowed for here. CeO2 ENMs have been
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shown to disaggregate at lower pH,29 so the presence of Ce in mussel tissues detected by
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elemental analysis could be due either to disaggregation or dissolution caused by the low pH
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encountered in the digestive gland. This would allow for passage of ions or individual ENM
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primary particles through the gut wall. Entry into body tissues of 10 nm amine-coated Au ENMs
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in a freshwater bivalve30 and ionic zinc released by ZnO ENMs in M. galloprovincialis11 has
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been seen, which suggests that Ce ions and/or CeO2 primary particles may behave similarly.
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Physiological Effects of CeO2 Exposure
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A small amount of mortality (
