Biomagnification of Cadmium Selenide Quantum Dots in a Simple

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Biomagniﬁcation of cadmium selenide quantum dots in a simple experimental microbial food chain

Article in Nature Nanotechnology · January 2011 DOI: 10.1038/nnano.2010.251 · Source: PubMed

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Biomagnification of cadmium selenide quantum dots in a simple experimental microbial food chain R. Werlin1,2,J.H.Priester2,3,4,R.E.Mielke2,3,4,5,S.Kra¨mer6,S.Jackson2,7,P.K.Stoimenov8, G. D. Stucky2,6,8,G.N.Cherr2,7,E.Orias1 andP.A.Holden2,3,4* Previous studies have shown that engineered nanomaterials can be transferred from prey to predator, but the ecological impacts of this are mostly unknown. In particular, it is not known if these materials can be biomagnified—a process in which higher concentrations of materials accumulate in organisms higher up in the food chain. Here, we show that bare CdSe quantum dots that have accumulated in Pseudomonas aeruginosa bacteria can be transferred to and biomagnified in the Tetrahymena thermophila protozoa that prey on the bacteria. Cadmium concentrations in the protozoa predator were approximately five times higher than their bacterial prey. Quantum-dot-treated bacteria were differentially toxic to the protozoa, in that they inhibited their own digestion in the protozoan food vacuoles. Because the protozoa did not lyse, largely intact quantum dots remain available to higher trophic levels. The observed biomagnification from bacterial prey is significant because bacteria are at the base of environmental food webs. Our findings illustrate the potential for biomagnification as an ecological impact of nanomaterials.

rotozoan grazing of bacteria is ecologically important in water1, To date, no studies have provided direct evidence for bacterial soil2 and in engineered wastewater treatment systems3, and it is transfer of nanoparticles to higher trophic levels, but several Pan efficient way4 by which to provide protozoa with large reports on eukaryotic organisms provide context. Algal cells made amounts of macronutrients4 and dietary metals5. Furthermore, fluorescent using commercially available CdSe QDs were consumed grazing releases nutrients into solution6, stimulating bacterial by Ceriodaphnia dubia, which then demonstrated an acquired fluor- growth and altering the composition of the bacterial community7,8. escence indicating QD trophic transfer25. In another study, functio- Because prokaryotes are major reserves of macronutrients9, micro- nalized CdSe QDs were bioconcentrated (that is, they increased in bially catalysed elemental cycling10 arises in large part from proto- concentration following uptake in the dissolved form16) into ciliated zoan grazing. Bacteria accumulate persistent organic11,12 and protozoa from laboratory media, but rotifer predators that con- metal13 pollutants and, at polluted sites, grazing enhances the sumed the protozoa did not biomagnify the QDs26. It has also growth of biodegrading bacterial populations and contaminant bio- been reported that the internalization of carbon nanotubes by degradation rates14. In nature, grazing transfers the iron accumu- Tetrahymena thermophila impeded subsequent digestion of bac- lated in bacteria to protozoa, and excretion by the protozoa and teria27. Taken together, these reports signal that nanoparticles accu- re-assimilation by the bacteria recycles this necessary nutrient5, mulated by bacteria may affect predators, and therefore higher similar to the process for copper in a freshwater food web15. In con- trophic levels. However, many questions remain unanswered, trast, cadmium can biomagnify (that is, increase in concentration including whether nanoparticles internalized by bacteria can be from primary producers to their predators15,16), suggesting that bac- transferred during predation and whether biomagnification can terial accumulation of toxic compounds could initiate the transfer of occur as a result. toxins into food webs. Previously, it has been shown that P. aeruginosa bacteria accumu- There are general concerns regarding the proliferation of engin- late CdSe QDs and dissolved cadmium (supplied as cadmium acetate) eered nanomaterials (in particular, nanoparticles), including possible during growth21. Here, we show that QDs inside the bacteria are bio- effects on ecological receptors and food webs17. Nanoparticles vary magnified in a protozoan predator, T. thermophila. Bacterial prey cells widely in relation to their core chemistry, morphology, coatings grown with QDs, but not cadmium acetate, inhibited digestion in the and reactivity, and therefore also in their biotic effects18. A diverse food vacuole of the protozoan. Because the poisoned protozoa range of metal nanoparticles associate with bacteria. For example, remained physically intact, an ecological implication of this study is 19 20 CeO2 sorbs onto either activated sludge or Escherichia coli ,and that subsequent predation of QD-containing protozoa could result bare CdSe quantum dots (QDs) are internalized into Pseudomonas in further trophic transfer of largely intact QDs. aeruginosa21 cells. Given the potential22 and documented23 entry into the environment of nanoparticles such as TiO2, which are QD-grown Pseudomonas poison Tetrahymena being produced in ever increasing amounts24, any nanoparticle– An experimental food chain was established that consisted exclu- bacteria association and subsequent nanoparticle mobilization into sively of a protozoan predator, T. thermophila, and a bacterial higher trophic levels should be regarded with concern. prey, P. aeruginosa. The important advantages of both model

1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California 93106-9625, USA, 2UC Center for the Environmental Implications of Nanotechnology (UC CEIN), University of California, Santa Barbara, California 93106-5131, USA, 3Donald Bren School of Environmental Science and Management, University of California, Santa Barbara, California 93106-5131, USA, 4Earth Research Institute, University of California, Santa Barbara, California 93106-5131, USA, 5Jet Propulsion Laboratory, California Institute of Technology – NASA, Planetary Science, Pasadena, California 91109-8099, USA, 6Department of Materials, University of California, Santa Barbara, California 93106-5050, USA, 7Departments of Environmental Toxicology and Nutrition, Bodega Marine Laboratory, University of California, Davis, Bodega Bay, California 94923-0247, USA, 8 Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106-9510, USA. *e-mail: [email protected]

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5 10 a 1.6 10 1.6 10 b 4 2.4 10 5 10 1.4 10 1.4 10

4 5 10 2.0 10 1.2 10 1.2 10 Pseudomonas

5 10 4 1.0 10 1.0 10 1.6 10

4 9 (cells per ml) (cells per ml) 8.0 10 8.0 10 (cells per ml) 4 1.2 10

4 9 6.0 10 6.0 10 3 8.0 10 Tetrahymena 4 9 Tetrahymena 4.0 10 4.0 10

3 4 9 4.0 10 2.0 10 2.0 10

0 0 0 0.0 10 0.0 10 0.0 10 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 Time (h) Time (h)

Figure 1 | Extent and rate of growth of Tetrahymena varies with Pseudomonas prey treatment. a, Tetrahymena population growth (ﬁlled symbols) and Pseudomonas population decline (open symbols) for control (triangles), CdSe QD (squares) and cadmium acetate (circles) treatments. Error bars represent standard error of the mean. For the Tetrahymena data, error bars are masked by the symbols. b, Tetrahymena population increase for three individual cultures in the QD (squares) and cadmium acetate (circles) treatments. Tetrahymena cells that fed upon cadmium-acetate-grown bacteria stopped growing by 6 h, whereas those that fed on QD-grown bacteria continued to grow slowly for up to 14 h.

a b c

25 m25 m25 m

Figure 2 | Tetrahymena cells after 24 h culture with Pseudomonas. a–c, Nomarski bright-field optical micrographs of Tetrahymena cells that have preyed on control (no cadmium) (a), cadmium-acetate-grown (b)andCdSeQD-grown(c) Pseudomonas bacteria. Note the abnormally high accumulation of large food vacuoles (arrow) in c.SomePseudomonas cells can be seen in the media external to the Tetrahymena in all images. organisms for this type of study are summarized in the Methods. unpublished observations), which on a mass basis represents a Starved protozoa were exclusively fed washed, mid-exponential- higher Se concentration than in the CdSe QDs. phase bacteria. The bacteria were cultivated in Luria Bertani (LB) The protozoa that fed upon control bacteria increased their 21 broth without cadmium (control) and with 75 mg l total population with a constant 4 h doubling time (td) throughout the cadmium added as either dissolved cadmium acetate or dialysed first 16 h of the experiment (Fig. 1a; Supplementary Table S3), CdSe QDs. Inductively coupled plasma atomic emission spec- resulting in a yield28 of (1.97+0.3) × 1025 protozoa per bacteria troscopy did not detect any cadmium in either control bacteria or consumed. Observations of the control protozoa culture under dis- control protozoa. secting microscopy showed typical, vigorously swimming cells. Bacteria cultivated with cadmium acetate accumulated 15% of When feeding upon cadmium acetate-grown bacteria, the initial the added cadmium mass, leading to a total cellular cadmium con- protozoan doubling time (td ¼ 5 h) was comparable to that of the centration significantly higher than the media, and a volumetric control (Supplementary Table S3), but growth stopped abruptly concentration factor (VCF)5 of 33 (Supplementary Tables S1 and after the population had doubled once (Fig. 1b). During the first S2). Bacteria cultivated with CdSe QDs accumulated 25% of the 4–6 h, the swimming and physical appearance of the protozoa total administered cadmium, resulting in a VCF of 70 undergoing the cadmium acetate treatment were indistinguishable (Supplementary Tables S1 and S2). The bacteria were then from controls. However, by 8 h none of the treated protozoa were thoroughly washed to remove external cadmium. Previous work21 swimming, and only 1% moving. Abrupt growth cessation with and the observations reported here have shown that Pseudomonas the cadmium acetate treatment was also evident in the significantly bacteria grown with CdSe QDs contain largely intact QDs. lower protozoan yield relative to the control ((3.55+0.9) × 1026; Selenium controls were not included, as the protozoa showed no P ¼ 0.008 by single-factor ANOVA). Complete poisoning of these adverse effects to various forms of Se (Na2SeO4 and Na2SeO3) protozoa was confirmed by their inability to resume growth when even at the highest concentration tested (75 mg l21; R.A. Werlin, transferred to rich growth media without added cadmium.

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Protozoa that fed on QD-grown bacteria grew for 14 h (Fig. 1b). Early time point Late time point This growth period was shorter than the control treatment, but a b .200% longer than that for the cadmium acetate treatment. eFV Growth rates were signiﬁcantly lower than in the control treatments eFV (Supplementary Table S3) and, although growth appeared to be biphasic, growth rates of individual replicate cultures were similar throughout the 14 h period (Fig. 1b). The protozoan yield for the QD treatment was intermediate ((6.20+2.5) × 1026) to the control and cadmium acetate treatments. At 16 h, only 0.3% of pro- OA tozoa that had been fed QD-grown bacteria recovered after transfer IFV to rich growth medium without added cadmium. Protozoan swim- 5 m 2 m ming was similar to that in the cadmium acetate treatment, with few protozoa moving after 10 h. c d The rapid onset of growth (Fig. 1) suggests that the phagocytic IFV ingestion of bacteria was independent of the initial bacterial treatments; in other words, the protozoa fed indiscriminately. m M However, the lower yields for the cadmium acetate and CdSe QD treatments compared to the controls show that the protozoa are eFV extracting less nutrition from the ingested bacteria, perhaps M m because of the energy cost of the cadmium stress response. OA Furthermore, although the end results were similar (death of vir- tually all exposed cells), under QD treatment the protozoa were 5 m 5 m killed more slowly than under the cadmium acetate treatment. Protein carbonylation levels were measured to assay for oxidative damage. For the QD and cadmium acetate treatments, the protozoa e f showed elevated protein carbonylation levels (Supplementary IFV Fig. S1). This oxidative damage could be a direct heavy-metal eFV effect29 or a secondary effect due to damage to the mitochondrial membrane with consequent release of reactive oxygen species to the cytoplasm30.

QD-grown Pseudomonas inhibit Tetrahymena digestion eFV Nomarski bright-field optical microscopy revealed protozoan mor- phological differences between treatments. Control protozoa were 2 m2 m morphologically normal (Fig. 2a), with ciliary rows clearly visible throughout the course of the experiment. At 16 h, protozoa in Figure 3 | Stunted digestion in Tetrahymena cells preying on CdSe cadmium acetate treatments had a pebbly appearance on their QD-grown, but not cadmium-acetate-grown, Pseudomonas bacteria. a–f, surface, which obscured their ciliary rows (Fig. 2b). When focused Dark-field STEM images of Tetrahymena cells that have fed on control through the entire protozoan cell, no other aberrations were appar- Pseudomonas (a,b), cadmium-acetate-grown bacteria (c,d)andCdSe ent. In contrast, protozoa in QD treatments showed abnormally QD-grown bacteria (e,f). a,c,e are taken at 1 h; b is at 16 h, and d,f are at high numbers of food vacuoles (Fig. 2c), averaging 5 mm in diam- 24 h. Triangles, Pseudomonas cells; eFV and lFV, early and late food vacuoles; eter and distributed throughout the cell. No other gross morpho- OA, oral apparatus; M and m, macronucleus and micronucleus. In a,two logical aberrations were apparent at this magnification. Also, we eFV packed with undigested Pseudomonas cells are visible near the oral observed no evidence of protozoan cells undergoing lysis, or rem- apparatus. Note the abundance of Pseudomonas outside the Tetrahymena cell. nants of lysed cells. In b, fine lamellar membranes (arrow) and intact Pseudomonas are seen in The prolonged and slower growth of protozoa that were fed mid- to late-stage food vacuoles. A normal mitochondrion is also visible QD-grown bacteria and the high number of food vacuoles observed (star). In c, the presence of an eFV at 1 h shows that normal phagocytic under optical microscopy could result if the protozoa digested the ingestion has occurred in cadmium acetate-grown Pseudomonas.Ind,intact bacteria at a reduced rate, perhaps because of hitherto unknown Pseudomonas are found outside the Tetrahymena, and amorphous digestion special effects of ingested QDs. Protozoan digestion rates vary products are seen in the lFV. Bright cadmium spots (arrow) appear 31 32 with their prey species , prey morphology and bacterial pro- throughout. In e, eFV shows intact Pseudomonas, and lFV contains cellular 33 duction of a toxic metabolite . Tetrahymena food vacuole lifespans debris, fine lamellar membranes and QDs (arrows). In f,eFV(arrow)shows can differ depending on whether bacterial cells or similarly sized tightly packed, undigested Pseudomonas surrounded by numerous QDs. The 34 inert particles are ingested , suggesting that food quality can signifi- observation of QDs throughout indicates that undigested QDs have crossed cantly affect the digestion and egestion processes. Carbon nanotubes the food vacuole membrane. in Tetrahymena have been reported to interfere with subsequent digestion of phagocytosed bacteria27, and Daphnia have been described as showing signs of digestion interference from CdSe fed upon cadmium-acetate-grown bacteria (Fig. 3c,d) were indistin- QDs in their food vacuoles35. To evaluate if a similar process was guishable from control protozoa throughout the course of the exper- at work here, we examined the ultrastructure of the protozoa in iment. During the first 4 h, the food vacuoles of protozoa that fed on each treatment over the time course of the experiment. QD-grown bacteria (Fig. 3e) were also indistinguishable from those Scanning transmission electron microscopy (STEM) images in the control. However, at later time points, protozoa that had been revealed that, by 1 h, control protozoa had accumulated multiple fed QD-grown bacteria showed an accumulation of food vacuoles food vacuoles, which were usually packed with bacteria (Fig. 3a). packed with undigested bacteria (Fig. 3f). This phenomenon, At subsequent times, food vacuoles with bacteria in all stages of observed under both optical and electron microscopy, implied digestion were observed (Fig. 3b). In this respect, protozoa that that cellular damage from QDs differentially halted intra-food

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ARTICLES NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2010.251

6 a

n = 10 Line scan 5 n = 10 n n = 73 =23 50 nm 4

3 n = 50

Cd:Se (atomic ratio) Cd:Se 500 nm 2

b Se Os Cl Cd Ca 1

6 6 0 CdSe Pa Pa Tt Tt 5 1 QDs 0 h 16 h 16 h FV 16 h CP 10 nm Figure 4 | CD:Se ratios obtained using EDS. Vertical lines represent the 4 ranges of Cd:Se ratios. Boxes are bounded by the 75th and 25th percentiles; intermediate horizontal lines are data medians. The number of observations 3 (100 nm2 spots) is located above each bar. The x-axislabels(lefttoright) Intensity (a.u.) include times relative to the time course of the trophic transfer experiments: as-synthesized CdSe QDs, washed CdSe QD-grown Pseudomonas (Pa) prey, 2 endpoint CdSe QD-grown Pseudomonas outside the predator, endpoint Tetrahymena (Tt) food vacuole (FV) containing undigested CdSe QD-grown 1 Pseudomonas, and endpoint Tetrahymena cytoplasm (CP) outside of food vacuoles (QD treatment). 1,000 2,000 3,000 4,000 5,000 6,000 vacuole digestion and egestion. That some digestion of bacteria had Energy (eV) occurred was indicated by the QD-sized bright spots distributed through the cytoplasm, including around food vacuoles and near Figure 5 | High-resolution STEM image and EDS of Tetrahymena that has mitochondria (Fig. 3f). In combination with the prolonged and fedonQD-grownPseudomonas,after24h.a, High-angle annular dark-field slower growth of the protozoa in the QD treatments (Fig. 1), the micrograph of an EDS line scan (large inset) performed through one bright differential effect on digestion (Figs 2c and 3f) relative to the spot (putative CdSe QD as in Fig. 3f) in a region interior to a mitochondrion cadmium acetate treatments is suggestive evidence for a QD ‘nano- (small rectangle). b, Six EDS spectra acquired in 5 nm steps. Spectra 1, 2 and particle-specific effect’ in the way that the protozoa are intoxicated, 6 are external to, 3 and 5 are near the edge of, and 4 is internal to the although we have not ruled out the unlikely possibility of a very ‘bright spot’ ( 10 nm across) on the line scan axis (inset). Sloping lines specific interaction of some ionic selenium and cadmium generated drawn in spectra 2–5 provide baselines for judging peak magnitudes. The by partial dissolution of QDs. spectra clearly show an enrichment of cadmium from the edges to the Interestingly, at early times only, STEM images of QD-treated interior of the bright spot, consistent with a QD-type particle. Note that the protozoa also revealed the presence of QDs in small (cross-sections, Se peak overlaps the Os peak, precluding reliable identification of Se 10–250 nm), apparently extracellular vesicles in the oral cavity enrichment along this scan. (Supplementary Section 5 and Fig. S2). To our knowledge, such vesicle-like structures have not been reported, and we cannot ratios were not significantly different (Student’s t-test; P . 0.20 explain how the QDs enter the membrane-enclosed space. for all comparisons) and averaged 1.6+0.1 (Fig. 4). This value is consistent with those measured for CdSe QDs freshly synthesized Largely intact QDs are found within the protozoa by similar methods36. In this way, the atom ratio of Cd:Se deter- Strong evidence that QDs remained largely intact after QD-fed bac- mined by STEM–EDS is an effective tracer for stoichiometrically teria were phagocytosed by the protozoa was obtained using energy- intact QDs throughout our trophic transfer experiment. dispersive X-ray spectroscopy (EDS). We measured Cd:Se atom To provide direct evidence that the bright spots represent QDs, an ratios within selected 100-nm2 areas of QD-sized, electron-dense EDS line scan was performed across a single representative bright (bright) spots that, in STEM images of protozoan cells, were seen spot of a 24 h QD-treatment thin-sectioned protozoan cell. The only in the QD treatment. Similar bright spots were not observed scanned spot was located within a mitochondrion separated by in control protozoa (Fig. 3a,b) or in protozoa that were fed three membranes from the food vacuole. The spectrum series unequi- cadmium-acetate-grown bacteria (Fig. 3c,d). Cd:Se ratios were vocally detected cadmium at the edges and interiors of the scanned determined for the as-synthesized QDs, for the QD-grown bacterial bright spot (Fig. 5). (Selenium could not be reliably measured by inoculum, for 16 h bacteria in the media surrounding protozoa from the line scan because, at the higher accelerating voltage, the Se peak the trophic transfer experiment, and for 16 h protozoa, both in food appeared as a small shoulder on a high osmium peak; Fig. 5b). In vacuoles packed with QD-grown bacteria and in the cytoplasm at a summary, given that the bright spots seen in Fig. 3f are similar in substantial distance from food vacuoles. The means of the Cd:Se size to QDs, were not found in osmium-stained protozoa that

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a 40,000 b 120,000

35,000 100,000 30,000 80,000 25,000 g per dry biomass) 20,000 60,000

15,000 40,000 10,000 20,000 5,000 Cadmium concentration ( Cadmium concentration (mg per litre cell volume) 0 0 Pseudomonas Tetrahymena Pseudomonas Tetrahymena

0 h 16 h 0 h 16 h

Figure 6 | Mass- and volume-based cadmium concentrations show biomagnification in the predator relative to the prey. a,b, Cellular cadmium concentrations with standard error bars for CdSe QD (black bars) and cadmium acetate (white bars) treatments plotted as either cadmium mass per cell volume (a) or cadmium mass per dry cell biomass (b). The concentration ratio of the 16 h Tetrahymena to the 0 h Pseudomonas represents the TTF. Volume- and dry mass-based TTFs were, respectively, 4.82 and 5.37 (QD treatment) and 2.97 and 3.54 (cadmium acetate treatment). Ratios greater than 1 reflect cadmium biomagnification during trophic transfer. preyed upon cadmium-acetate-grown bacteria (Fig. 3c,d), have Tetrahymena feed on QD-grown bacteria. Release of QDs from retained the Cd:Se stoichiometry of intact QDs (Fig. 4), and represent the bacteria during the first protozoa doubling should have two con- discrete peaks of cadmium enrichment (Fig. 5), it is reasonable to sequences: exposure of the food vacuole and its membrane to free conclude that QDs, either intact or only partially decomposed, com- QDs and release of cadmium by acid-induced39 dissolution of prise such bright spots. The combined data therefore provide strong QDs40. However, QDs inhibited digestion in the food vacuole evidence for trophic transfer of largely intact QD material from QD- (Figs 2c and 3f). As digestion is required to release bacterial QDs grown bacteria to protozoa. into the food vacuole but free QDs inhibit digestion, this dynamic would tend to set up a self-regulated level of QD release and QDs biomagnify in Tetrahymena relative to Pseudomonas cadmium solubilization. This level may be insufficient to totally Calculations of total cadmium mass per cell on either a volume or overwhelm the cadmium-stress response of the protozoa, so a dry mass basis (Fig. 6, Supplementary Table S1) allow us to calculate small fraction of the cells remain alive and capable of growing the trophic transfer factor (TTF), expressed as the ratio of cadmium slowly throughout the course of the experiment. A high level of concentrations in protozoa at 16 h to those in bacteria at 0 h. The QD biomagnification is attributable to the high initial rates of pha- TTFs, calculated as the dry mass concentration ratio (metal mass gocytic ingestion of QD-grown bacteria. Thus, the inhibition of per dry biomass), are 5.4 and 3.5 for QD and cadmium acetate digestion and perhaps food vacuole acidification may increase bio- treatments, respectively (Supplementary Table S1). A TTF of 1 magnification in the QD treatment. would be indicative of trophic transfer without biomagnification. These dry-mass-based TTFs agree well with TTFs calculated from Conclusions protozoan and bacterial cadmium concentrations on a cell volume Our results show that QDs, once packaged inside bacteria, have the basis (4.8 and 3.0, respectively; SupplementaryTable S1). potential to transfer to higher trophic levels. Cadmium, supplied After 16 h, the cadmium mass ratio between protozoa and bacteria within QD-fed bacteria, served as a relatively stable tracer, and we is greater than 143,000 for the QD treatment (Supplementary were able to infer that more than 100,000 bacteria were ingested Table S1). If cadmium in the QD form was internalized exclusively by phagocytosis for each protozoan cell present at 16 h. This mag- by phagocytosis of QD-grown bacteria, this would suggest that nitude of consumption illustrates the enormous capacity of more than 100,000 bacteria had been phagocytosed to generate Tetrahymena, and probably other ciliated protozoa, for phagocyto- each of these protozoa. These numbers are consistent with reported sis. Taking into account the apparent stability of QDs combined rates of Tetrahymena phagocytosis37,38 and the volumes of with the efficiency of phagocytosis in these protozoa and the Tetrahymena food vacuoles and bacteria (Supplementary Section 6 ciliary adaptations that allow them to filter-feed on bacteria, a and Table S1). potential for biomagnification in ecosystems clearly exists for nano- The diverse and comprehensive set of physical, analytical and particles that penetrate bacterial cells. As these protozoa do not biological observations reported here allows novel insights into lyse in significant numbers after they are killed by ingesting the differential response of Tetrahymena to the phagocytic ingestion QD-contaminated bacteria, largely intact QDs remain potentially of QD-grown and cadmium-acetate-grown bacteria. Cadmium- bioavailable to the next higher trophic level in the food chain. acetate-grown bacteria did not noticeably inhibit digestion at any Because the QD-poisoning of these protozoa is accompanied by time during the experiment (Fig. 2b; Fig. 3c,d), so the metal was loss of motility, the cells most affected by biomagnification are internally bioavailable to the protozoa as digestion proceeded. likely to be preferentially eaten by natural predators, which could Internal cadmium concentrations presumably increased until the accelerate biomagnification of QDs in a food web. The finding finite cadmium-stress response capacity of the protozoa was over- that QDs remained largely intact after transfer to the protozoa whelmed, at which time the protozoa irreversibly stopped growing suggests the possibility of long-term retention. In this respect, the (at 4 h, Fig. 1). A special situation is created when the biomagnification of QDs is reminiscent of DDT biomagnification,

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ARTICLES NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2010.251 a compound that likewise accumulates in bacteria12 and is amplified were acquired using an FEI XL30 ESEM FEG microscope operating at 30 kV intact in successively higher levels of food webs. accelerating voltage with a STEM detector at a working distance of 6.7 mm. EDS analysis was performed in a manner similar to previous reports45 on selected The need to obtain reliable quantitative measurements drove our rectangular areas of 100 nm2 for 15 s at 25.6 mS using an X-ray detector with a experimental design to use high cadmium concentrations and batch sapphire super Ultra-Thin Window (UTW; EDAX) on an FEI Nano600 FEG conditions (rather than steady-state, for example, chemostat con- microscope. Genesis software (EDAX) was used in ZAF mode to analyse the spectra. ditions). Our findings therefore illustrate what could happen. The TEM images and EDS line scans were acquired in high-angle annular dark-field effects of a low-concentration, steady-state influx of toxic nanopar- mode using an ultrahigh-resolution FEI Titan 80-300 (TEM/STEM) operated at 300 kV accelerating voltage, with a spatial resolution in the order of 1 nm2. ticles capable of being internalized by bacteria in a complex natural environment are not directly predictable from our experiments. Nonetheless, the findings do caution that such steady influx may Received 18 August 2010; accepted 17 November 2010; not be risk-free for biomagnification. published online 19 December 2010 Methods Biological materials. Strain SB210E of T. thermophila was used as an apex predator References to test the potential for QD biomagnification. Strain SB210E is a fresh thaw of strain 1. Barbeau, K., Moffett, J. W., Caron, D. A., Croot, P. L. & Erdner, D. L. Role of 41 SB210, the strain for which the macronuclear genome has been sequenced , protozoan grazing in relieving iron limitation of phytoplankton. Nature 380, which is available from the Tetrahymena Stock Centre (http://tetrahymena.vet. 61–64 (1996). cornell.edu/). T. thermophila, a consummate bacterivore, is a freshwater member of 2. Clarholm, M. Interactions of bacteria, protozoa and plants leading to the ciliated protozoa, a major eukaryotic evolutionary group comprising common mineralization of soil-nitrogen. Soil Biol. Biochem. 17, 181–187 (1985). 41,42 inhabitants of all aquatic environments . Tetrahymena cells are among the fastest 3. Gude, H. Grazing by protozoa as selection factor for activated sludge bacteria. growing eukaryotes, actively phagocytise bacteria and represent the ciliated Microb. Ecol. 5, 225–237 (1979). protozoa, which together with other heterotrophic protozoa are vital links in the 4. Ducklow, H. W. Production and fate of bacteria in the oceans. 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G. 81, 293–308 (2002). 21 Pseudomonas cells through uptake into their cytoplasm . 8. Pernthaler, J. Predation on prokaryotes in the water column and its ecological implications. Nat. Rev. Microbiol. 3, 537–546 (2005). Trophic transfer experiment. Chemicals, media and cell preparation methods for 9. Whitman, W. B., Coleman, D. C. & Wiebe, W. J. Prokaryotes: the unseen the trophic transfer experiments are described in the Supplementary Information. majority. Proc. Natl Acad. Sci. USA 95, 6578–6583 (1998). Protozoa were fed bacteria grown in LB broth supplemented in three different ways: 10. Schlesinger, W. H. Biogeochemistry: An Analysis of Global Change (Academic unsupplemented (control), supplemented with cadmium acetate and supplemented Press, 1997). with bare CdSe QDs. Three independent replicates of each experimental 11. Hudson, M. J., Swackhamer, D. L. & Cotner, J. B. Effect of microbes on treatment were established and studied for cell growth by time-course cell counting, contaminant transfer in the Lake Superior food web. Environ. Sci. Technol. 39, endpoint cellular cadmium concentrations, and protozoa viability, morphology, 9500–9508 (2005). ultrastructure and protein carbonyl content (see Supplementary Information). 12. Johnson, B. T. & Kennedy, J. O. Biomagnification of p,p’-DDT and Specific growth rate and yield measurements. The exponential phase specific methoxychlor by bacteria. Appl. Microbiol. 26, 66–71 (1973). growth rates for the protozoa were calculated as described in the Supplementary 13. Mullen, M. D. et al. Bacterial sorption of heavy metals. Appl. Environ. Microbiol. Information. The protozoan yields were calculated for the exponential growth 55, 3143–3149 (1989). periods as absolute values of the ratios of the differences between the initial and final 14. Madsen, E. L., Sinclair, J. L. & Ghiorse, W. C. In situ biodegradation: microbiological patterns in a contaminated aquifer. Science 252, 830–833 (1991). cell concentrations: (CT,1–CT,2)/(CP,–CP,2), where CT and CP are the concentrations of protozoa and bacteria, respectively, and 1 and 2 are the initial and final times of 15. Croteau, M. N., Luoma, S. N. & Stewart, A. R. Trophic transfer of metals along the exponential growth period, respectively. Both types of calculations have been freshwater food webs: evidence of cadmium biomagnification in nature. Limnol. reported previously for predator–prey studies involving protozoan bacterivores28. Oceanogr. 50, 1511–1519 (2005). 16. Luoma, S. N. & Rainbow, P. S. Metal Contamination in Aquatic Environments: Cadmium quantification and biomagnification calculations. Separated protozoan Science and Lateral Management (Cambridge Univ. Press, 2008). and bacterial cells were used to measure total cadmium in each cell fraction. Total 17. Klaine, S. J. et al. Nanomaterials in the environment: behavior, fate, cadmium was quantified by inductively coupled plasma atomic emission bioavailability, and effects. Environ. Toxicol. Chem. 27, 1825–1851 (2008). spectroscopy with a TJA High Resolution IRIS instrument (Thermo Electron 18. Stern, S. T. & McNeil, S. E. Nanotechnology safety concerns revisited. Toxicol. Corporation). The instrument was calibrated against commercial cadmium Sci. 101, 4–21 (2008). standards (Sigma Chemical). Samples were prepared by dissolving in aqua regia. 19. Limbach, L. K. et al. Removal of oxide nanoparticles in a model wastewater Volume-based cellular cadmium concentrations were calculated for bacteria and treatment plant: influence of agglomeration and surfactants on clearing protozoa using an approach from a previous study21. For each cell type and efficiency. Environ. Sci. Technol. 42, 5828–5833 (2008). treatment, cadmium mass, quantified by inductively coupled plasma spectroscopy, 20. Thill, A. et al. 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Environ. Pollut. 156, 233–239 (2008). coupled plasma spectroscopy). Data and intermediate steps in these calculations are 23. Kiser, M. A. et al. Titanium nanomaterial removal and release from wastewater explained in full detail in the text preceding Supplementary Table S1. treatment plants. Environ. Sci. Technol. 43, 6757–6763 (2009). Biomagnification extents were calculated as volume- and dry-cell-mass-based 24. Robichaud, C. O., Uyar, A. E., Darby, M. R., Zucker, L. G. & Wiesner, M. R. 44 TTFs (as by others ), that is, as the ratio of 16 h protozoan cadmium concentration Estimates of upper bounds and trends in nano-TiO2 production as a basis for (volume- or dry-cell-mass-based) to the corresponding 0 h bacterial cadmium exposure assessment. Environ. Sci. Technol. 43, 4227–4233 (2009). concentration (Supplementary Table S1). Calculated volume- and dry-mass-based 25. Bouldin, J. L. et al. Aqueous toxicity and food chain transfer of quantum dots in TTFs fell within 16% of one another. freshwater algae and Ceriodaphnia dubia. Environ. Toxicol. Chem. 27, 1958–1963 (2008). Optical and electron microscopy, and energy dispersive spectroscopy. Swimming 26. Holbrook, R. D., Murphy, K. E., Morrow, J. B. & Cole, K. D. Trophic transfer of behaviour and overall appearance of protozoan cells were observed every 30 min for nanoparticles in a simplified invertebrate food web. Nature Nanotech. 3, 2 h, thereafter every hour for 5 h, and at 2 h intervals for 17 h thereafter under a 352–355 (2008). dissecting scope (×30). The methods for preparing cells for Nomarski imaging and 27. Ghafari, P. et al. Impact of carbon nanotubes on the ingestion and digestion of electron microscopy are provided in the Supplementary Information. STEM images bacteria by ciliated protozoa. Nature Nanotech. 3, 347–351 (2008).

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28. Eccleston-Parry, J. D. & Leadbeater, B. S. C. A comparison of the growth kinetics 45. Clarke, S., Mielke, R. E., Neal, A., Holden, P. & Nadeau, J. L. Bacterial and of six marine heterotrophic nanoflagellates fed with one bacterial species. Mar. mineral elements in an Arctic biofilm: a correlative study using fluorescence and Ecol. Prog. Ser. 105, 167–177 (1994). electron microscopy. Microsc. Microanal. 16, 153–165 (2010). 29. Stohs, S. J. & Bagchi, D. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321–336 (1995). 30. Rikans, L. E. & Yamano, T. Mechanisms of cadmium-mediated acute Acknowledgements hepatotoxicity. J. Biochem. Mol. Toxicol. 14, 110–117 (2000). This research was primarily funded by US Environmental Protection Agency Science To 31. Boenigk, J., Matz, C., Jurgens, K. & Arndt, H. The influence of preculture Achieve Results (STAR) award no. R833323 (to P.A.H. and G.D.S.), and by the National conditions and food quality on the ingestion and digestion process of three Science Foundation and the Environmental Protection Agency under cooperative species of heterotrophic nanoflagellates. Microb. Ecol. 42, 168–176 (2001). agreement no. DBI-0830117 (to P.A.H., G.N.C. and G.D.S.). Any opinions, findings and 32. Corno, G. & Jurgens, K. Direct and indirect effects of protist predation on conclusions or recommendations expressed in this material are those of the author(s) and population size structure of a bacterial strain with high phenotypic plasticity. do not necessarily reflect the views of either the National Science Foundation or the Appl. Environ. Microbiol. 72, 78–86 (2006). Environmental Protection Agency. This work has not been subjected to Environmental 33. Matz, C. et al. Impact of violacein-producing bacteria on survival and feeding of Protection Agency review and no official endorsement should be inferred. Environmental bacterivorous nanoflagellates. Appl. Environ. Microbiol. 70, 1593–1599 (2004). scanning and scanning transmission electron microscopy were partly performed in the 34. Boenigk, J., Matz, C., Jurgens, K. & Arndt, H. Confusing selective feeding with Micro-Environmental Imaging and Analysis Facility at University of California Santa differential digestion in bacterivorous nanoflagellates. J. Eukaryot. Microbiol. 48, Barbara (www.bren.ucsb.edu/facilities/MEIAF/) under National Science Foundation 425–432 (2001). awards nos BES-9977772 and DBI-0216480. Transmission electron microscopy was partly 35. Lewinski, N. A. et al. Quantification of water solubilized CdSe/ZnS quantum performed in the University of California Santa Barbara Materials Research Laboratory dots in Daphnia magna. Environ. Sci. Technol. 44, 1841–1846 (2010). Central Facilities supported by the Materials Research Science and Engineering Centers 36. Lin, Y. W., Hsieh, M. M., Liu, C. P. & Chang, H. T. Photoassisted synthesis of Program of the National Science Foundation under award no. DMR05-20415. The T. CdSe and core–shell CdSe/CdS quantum dots. Langmuir 21, 728–734 (2005). thermophila portion of the work was partially supported by grant no. R01-RR009231 from 37. Nilsson, J. R. & Williams, N. E. in Biochemistiry and Physiology of Protozoa Vol. the National Center for Research Resources of the National Institutes of Health (to E.O.). 2 (Academic Press, 1979). The authors gratefully acknowledge critical comments on the manuscript by T. Klanjscek. 38. Orias, E. & Pollock, N. A. Heat-sensitive development of phagocytotic organelle Thanks also go to anonymous reviewers for valuable suggestions that led to significant in a Tetrahymena mutant. Exp. Cell Res. 90, 345–356 (1975). improvement of the manuscript. 39. Fok, A. K., Lee, Y. & Allen, R. D. The correlation of digestive vacuole pH and size with the digestive cycle in Paramecium caudatum. J. Protozool. 29, Author contributions 409–414 (1982). P.A.H., J.H.P., R.W., E.O. and G.D.S. designed the experiment. P.K.S. and G.D.S. 40. Mahendra, S., Zhu, H. G., Colvin, V. L. & Alvarez, P. J. Quantum dot weathering synthesized and provided the quantum dots. R.W. and J.H.P. executed the trophic transfer results in microbial toxicity. Environ. Sci. Technol. 42, 9424–9430 (2008). experiments. R.E.M. and S.K. performed the electron microscopy and EDS analyses. 41. Eisen, J. A. et al. Macronuclear genome sequence of the ciliate Tetrahymena G.N.C. and S.J. determined protein carbonyl content. All authors contributed to the writing thermophila, a model eukaryote. PLoS Biol. 4, 1620–1642 (2006). of the manuscript. 42. Nanney, D. L. & Simon, E. M. Tetrahmena thermophila (Academic Press, 1979). 43. Steinberger, R. E., Allen, A. R., Hansma, H. G. & Holden, P. A. Elongation correlates with nutrient deprivation in unsaturated Pseudomonas aeruginosa Additional information biofilms. Microb. Ecol. 43, 416–423 (2002). The authors declare no competing financial interests. Supplementary information 44. Xie, L. T., Funk, D. H. & Buchwalter, D. B. Trophic transfer of Cd from natural accompanies this paper at www.nature.com/naturenanotechnology. Reprints and periphyton to the grazing mayfly Centroptilum triangulifer in a life cycle test. permission information is available online at http://npg.nature.com/reprintsandpermissions/. Environ. Pollut. 158, 272–277 (2010). Correspondence and requests for materials should be addressed to P.A.H.

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