Toxicological impact of cadmium-based quantum dots towards aquatic biota: Effect of natural sunlight exposure

Accepted Manuscript Title: Toxicological impact of cadmium-based quantum dots towards aquatic biota: Effect of natural sunlight exposure Author: B.F. Silva T. Andreani A. Gavina M.N. Vieira C.M. Pereira T. Rocha-Santos R. Pereira PII: DOI: Reference:

S0166-445X(16)30126-6 http://dx.doi.org/doi:10.1016/j.aquatox.2016.05.001 AQTOX 4379

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

3-3-2016 21-4-2016 2-5-2016

Please cite this article as: Silva, B.F., Andreani, T., Gavina, A., Vieira, M.N., Pereira, C.M., Rocha-Santos, T., Pereira, R., Toxicological impact of cadmium-based quantum dots towards aquatic biota: Effect of natural sunlight exposure.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2016.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Toxicological impact of cadmium-based quantum dots towards aquatic biota: Effect of natural sunlight exposure Silva, B.F.1,2, Andreani T.3,4,5, Gavina A.1,2, Vieira, M.N.

1,2

, Pereira, C.M

3,4

, Rocha-

Santos, T.6, Pereira, R.1,2. 1

Department of Biology, Faculty of Science, University of Porto, Rua do Campo Alegre

4169-007 Porto, Portugal 2

CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, Rua dos

Bragas, 289, 4050-123 Porto, Portugal. 3

Centro de Investigação em Química da Universidade do Porto, Rua do Campo Alegre,

687, 4169-007 Porto, Portugal. 4

Department of Chemistry and Biochemistry, Faculty of Science, University of Porto,

Rua do Campo Alegre, 4169-007 Porto, Portugal 5

CITAB - Centre for Research and Technology of Agro-Environmental and Biological

Sciences, University of Trás-os-Montes e Alto Douro, UTAD, Vila Real, Portugal 6

Department of Chemistry and Centre for Environmental and Marine Studies (CESAM),

University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal Highlights

    

Under sunlight exposure, all QDs form particle aggregates in the different media CdSeS/ZnS QDs showed lower toxic effects to V. fischeri before sunlight exposure Sunlight exposure decreased the toxicity of CdS 480 in all organisms Sunlight exposure increased the toxicity of CdS 380 QDs for D. magna Shell of QDs seemed to make them less harmful to aquatic organisms

1

Abstract Cadmium-based quantum dots (QDs) are increasingly applied in existent and emerging technologies, especially in biological applications due to their exceptional photophysical and functionalization properties. However, they are very toxic compounds due to the high reactive and toxic cadmium core. The present study aimed to determine the toxicity of three different QDs (CdS 380, CdS 480 and CdSeS/ZnS) before and after the exposure of suspensions to sunlight, in order to assess the effect of environmentally relevant irradiation levels in their toxicity, which will act after their release to the environment. Therefore, a battery of ecotoxicological tests was performed with organisms that cover different functional and trophic levels, such as Vibrio fischeri, Raphidocelis subcapitata, Chlorella vulgaris and Daphnia magna. The results showed that core-shell type QDs showed lower toxic effects to V. fischeri in comparison to core type QDs before sunlight exposure. However, after sunlight exposure, there was a decrease of CdS 380 and CdS 480 QD toxicity to bacterium. Also, after sunlight exposure, an effective decrease of CdSeS/ZnS and CdS 480 toxicity for D. magna and R. subcapitata, and an evident increase in CdS 380 QD toxicity, at least for D. magna, were observed. The results of this study suggest that sunlight exposure has an effect in the aggregation and precipitation reactions of larger QDs, causing the degradation of functional groups and formation of larger bulks which may be less prone to photooxidation due to their diminished surface area. The same aggregation behaviour after sunlight exposure was observed for bare QDs. These results further emphasize that the shell of QDs seems to make them less harmful to aquatic biota, both under standard environmental conditions and after the exposure to a relevant abiotic factor like sunlight.

Keywords: Quantum dots, sunlight, ecotoxicity, microalgae, bacteria, daphnia

1. Introduction Quantum dots (QDs) are nanocrystals of a semiconducting material, typically chalcogenides (selenides and sulfides) of metals such as cadmium or zinc (CdS, CdSeS, ZnS) (Jamieson et al., 2007). QDs gained a high popularity upon their first use as 2

fluorescent probes in biological staining and diagnostics, as alternative to organic dyes due to their narrow, tunable emission spectra, bright fluorescence and photochemical stability (Bruchez et al., 1998). These exceptional optical and electrical properties allied with their extremely small size but large surface area make them ideal for biotechnological and biomedical applications (Kuzyniak et al., 2014; Niemeyer, 2001; Whitesides, 2005). QDs have been widely used in several existing and emerging technologies, such as optical probes for biological imaging (Jaiswal and Simon, 2004; Liu et al., 2013; Pan and Feng, 2009), LEDs and displays (Jang et al., 2010), cell labelling and diagnostics (Jin and Hildebrandt, 2012), multifunctional nanocomplex integrated targeting (Wang and Chen, 2011), fluorescence resonance energy transfer (FRET) (Qiu and Hildebrandt, 2015), drug delivery and targeting (Probst et al., 2013; Yuan et al., 2010), and as radioand chemotherapy agents (Juzenas et al., 2008). More recently, QDs have been used for the revolutionary development of automated Lab-on-a-Chip (LOC) devices for personalized health care, diagnosis and treatment of several conditions such as cancer (Krishna et al., 2013). There are several types of QDs, including CdS (core-type, cadmium sulphide QDs) and CdSeS/ZnS (core-shell type QDs of cadmium sulphide, cadmium selenium and zinc sulphide shell). In the case of CdS QDs, they have emission spectra near UV light and are less stable than core-shell nanocrystals (Chan et al., 2002; Derfus et al., 2004). On the other hand, they are cheaper and easier to synthesize in comparison to their shell-type counterparts, rendering them useful for basic applications that require single-component nanocrystals. The emission colours of CdSeS/ZnS may span the entire visible spectrum and UV. Alloyed QDs have the special property of being tuned not only by size but also by composition. This makes them perfect for special applications that require very small particles (e.g. in vivo imaging), with similar size but different emission colours (Swafford et al., 2006). This is possible by tuning between the CdSe and CdS band edges while maintaining a similar size (Qin et al., 2012). The growth of a shell with broader band gap such as ZnS on the surface of the QDs is an effective method for improving its spectral properties, stability and for reducing toxicity (Hines and GuyotSionnest, 1996) by passivizing nonradiative recombination sites. Ecotoxicological data about QDs is not easy to find due to the utter number of different core, shell and ligands composition of these particles. The toxicity of 3

cadmium-based QDs is described in the literature mainly due to the liberation of Cd2+ ions upon oxidation of particles surface (Derfus et al., 2004). The exposure to air of TOPO (tri-n-octylphosphine oxide)-capped QDs and to UV-light by these authors showed a change in fluorescence spectra towards the blue end, consistent with the reduction of QDs size due to the removal of surface atoms. Hoshino et al. (2004) described that it was the QDs capping composition that determined the QDs toxicity (Hoshino et al., 2004). The chemical composition of QDs is therefore a key aspect of their toxicity. Previous studies from Su et al. (2009) point towards the fact that a ZnS coating highly improves the biocompatibility of QDs by protecting the inner layers of releasing Cd2+ ions, and are highly effective in reducing QDs toxicity (Su et al., 2009). Kirchner et al. (2005) studied the cytotoxicity of CdSe and CdSe/ZnS with different surface modifications and concluded that besides the release of Cd2+ ions, their surface chemistry (e.g. stability regarding aggregation) has an important role in cytotoxicity (Kirchner et al., 2005). Green and Howman (2005) also noted the formation of free radicals by CdSe QDs, and the enhancement of this process when QDs were activated by light, UV or air-oxidation (Green and Howman, 2005). Although this is the basis for cancer treatment as well as for other therapies using QDs, the effect of light in these systems needs to be thoroughly studied due to the persistence of these particles in tissues and organs (Rzigalinski and Strobl, 2009). Chen et al. (2012) also suggest that the release of Cd2+ ions cannot be the sole reason to the cytotoxicity of QDs in aquatic dispersion, reinforcing the importance of their intracellular distribution and their related nanoscale effects as well (Chen et al., 2012). Lin et al. (2009) reported the adsorption of water-soluble CdSe/ZnS QDs to the cell surface of the algae Chlamydomonas, causing hindrance to their photosynthetic capabilities (Lin et al., 2009). There was no record of QDs inside the cells, probably due to the aggregation of QDs which lose their ability to cross the cell wall. Due to their high toxic potential, it is crucial to do a suitable characterization of QDs as well as a proper ecotoxicological evaluation in order to contribute for the research and synthesis of less toxic QDs for their use in biological applications. Moreover, studies using organisms from different trophic levels, in this case aquatic organisms, can provide deep insight on the impact of these emerging compounds on the final receptor medium, since wastewater from industries or clinical settings are the main source of these materials (Moore, 2006). In fact, we are now becoming aware that existent wastewater treatment plants are not prepared to remove the great majority of 4

emerging compounds, including nanomaterials, emphasizing even more the importance of testing the effects of these compounds to aquatic species after the exposure of QDs to relevant environmental conditions. Based on these considerations, the present study aimed to determine the effect of sunlight exposure on QD’s ecotoxicity, by comparing

the effects caused by their

suspensions not exposed outdoors to sunlight (herein mentioned as pre-exposure suspensions) with those caused by the same suspensions, after have been exposed to sunlight for 24h (herein mentioned as post-exposure suspensions) on aquatic organisms. For this purpose two different types of QDs were selected: core-cadmium QDs (CdS) with different sizes and zinc-shell type QDs (CdSeS/ZnS). This choice also aimed at evaluating the toxicity of QDs with different sizes and surface modifications. These compounds were tested using organisms from different trophic levels, high sensitivity and representativeness, namely, the bacterium Vibrio fischeri, the freshwater microalgae Raphidocelis subcapitata and Chlorella vulgaris and the freshwater cladoceran Daphnia magna. This study is an ecological approach that will give a broader insight on the effects of QDs in the aquatic environments, where these compounds should likely emerge due to their existent and predicted uses in biological detection and imaging.

2. Materials and methods

2.1. QDs tested CdSeS/ZnS alloyed QDs and CdS 380 and 480 from the Lumidot™ CdS-6 QD kit were tested. All QDs were ordered from Sigma-Aldrich®. CdSeS/ZnS QDs, COOH functionalized, core-shell type, with 6 nm estimated diameter and 540 nm fluorescence, were obtained in aqueous suspension (1 mg/L). Lumidot™ CdS-6 core-type QDs were obtained as a dispersion in toluene (5 mg/mL), organically stabilized with oleic acid ligand coating and with a fluorescence emission ranging from 380 to 480 nm. The estimated particle size was 1.6 and 7.3 nm for CdS 380 and CdS 480, respectively. QD suspensions for the toxicity assays were prepared by direct dispersion of the QDs into the proper test media (ATSM, MBL or distilled water for D. magna, algae or Microtox® tests, respectively). The ASTM hardwater is a synthetic medium (160-180 mg/L CaCO3) used for maintenance and toxicity testing of freshwater species, such as 5

Daphnia magna (ASTM, 1980). The Woods Hole MBL medium is a culture medium used for the maintenance of freshwater algae species, such as Raphidocelis subcapitata and Chlorella vulgaris (Stein, 1973). Upon dilution, the tubes were vigorously vortexed for 3 minutes in order to ensure a homogenous distribution of the QDs in an ecologically realistic context, in each test medium (herein mentioned as pre-exposure suspensions). For the sunlight exposure tests, QD suspensions were prepared following the same methodology (herein mentioned as post-exposure suspensions). After preparation, the suspensions were poured into transparent flasks and covered with a perforated film in order to prevent evaporation. The flasks were then placed directly under sunlight exposure outside for 24 h. This time span had 16 h of sunlight and 8 h of darkness (± 1 h). Temperatures varied from 25°C maximum to 19°C minimum (± 4°C) in each exposure period. In the test period, the global solar radiance exposure levels in the Oporto area were 650 MJ/m2 (IPMA, 2013). The organisms were exposed to both pre- and post-exposure QD suspensions at standard laboratory conditions, as described in section 2.3.

2.2. Test species and culture conditions In the present study, Daphnia magna, Raphidocelis subcapitata, Chlorella vulgaris and Vibrio fischeri were used as test organisms. D. magna specimens are continuously reared in laboratory in flasks containing 800 mL of ASTM hard water medium and 30 daphnids each. They are fed every other day with R. subcapitata at the rate of 104 cells/mL and with an organic supplement of Ascophylum nodosum which is added to the flasks, in concentration of 4.8 mL/L of ASTM. Cultures are kept under a 16hL: 8hD photoperiod and at 20±2°C. The date of birth of each brood is registered and females are kept until the 5th brood is released, at this time they are rejected. Neonates, with less than 24 h, from the 3rd, 4th and 5th broods are used either for toxicity tests or for starting a new culture. R. subcapitata and C. vulgaris are maintained in 500 mL Erlenmeyer flasks with 150 mL of fresh autoclaved MBL medium. After the medium cooled down for a day, algae are inoculated in a laminar flow cabinet in the new medium enriched with vitamins. Cultures are kept in a chamber under 16 hL: 8 hD photoperiod and 20 ± 3°C. For D. magna feeding, R. subcapitata is cultured in glass, round-bottom flasks with 4 L of sterilized MBL medium with vitamins, covered with an adequate plug. A continuous 6

air flow is supplied by an air pump with a 0.22 μm syringe filter in order to prevent contamination. Inoculation is performed as described before. All the material and solutions used are autoclaved. R. subcapitata algae used for feeding are centrifuged at 1615 rcf for 3 min in a bench centrifuge. The pellet is re-suspended using ASTM medium and the absorbance is measured at 440 nm in a 1:10 dilution until a final absorbance between 0.4 and 0.8 is obtained. The algae suspensions are stored at 4°C and a previously determined correlation between absorbance and cell number was used to determine the appropriate volume that should be used for feeding daphnids.

2.3. Size and size distribution measurements The average hydrodynamic diameter (Z-Ave) and the polydispersity index (PdI) of all QD dispersions were determined through dynamic light scattering (DLS) using Avid Nano W130i (Avid Nano Ltd., High Wycombe, UK). For the measurements, each QD was dispersed in distilled water and in the different culture media (ASTM or MBL) by vortex for 3 minutes. Both sunlight-exposed and non-exposed suspensions were analysed. The values reported are the mean ± SD of three different measurements performed for each suspension.

2.4. Ecotoxicological assessment of non-exposed and sunlight-exposed quantum dot suspensions D. magna acute immobilization tests were performed in accordance with the Organization of Economic and Cooperation Development Guideline 202 (OECD, 2004) consisting of a 48 h exposure of daphnid neonates (less than 24-h-old) to suspensions with different QD concentrations. The toxicity tests were performed with five D. magna neonates per glass tube in suspensions with different QD concentrations (4 replicates per concentration). For suspensions with no sunlight exposure, the concentrations ranged from 201 to 500 µg/L for CdSeS/ZnS, from 376 to 1000 µg/L for CdS 380 and from 279 to 1000 µg/L for CdS 480. Regarding the suspensions with sunlight exposure, the concentrations tested for CdSeS/ZnS ranged from 282 to 750 µg/L, and for CdS 380 and CdS 480 QDs between 376 to 1000 µg/L. In addition to control with ASTM, a control with toluene (at the maximum concentration of 200 L/L used in the test) was also performed. Test tubes were loosely covered with Parafilm® and placed under 7

controlled conditions in a chamber (20 ± 2°C and 16hL: 8hD). Immobilization was recorded at 24 h and 48 h. Test specimens were considered immobilized if they were not able to swim after 15 seconds of gentle agitation of the test vessel, even if they could still move their antennae (OECD, 2004). Dissolved oxygen and pH were measured at the beginning and end of the tests. Tests were considered valid if the immobilization of daphnids in the control did not exceed 10%, and if dissolved oxygen concentration at the end of the test in all the test tubes was not lower than 3 mg/L. Algae growth inhibition tests were performed according to procedure described in the OECD Guideline 201 (OECD, 2011) and in the International Organization for Standardization (ISO) Guideline 8692 (ISO, 2012), adapted to 24-well microplates. For each test, new cultures of R. subcapitata and of C. vulgaris were started as previously described. Three days after the beginning of the culture, the number of algal cells was estimated by optical cell count with a Neubauer chamber and diluted in order to start the test with a concentration of 104 cel/mL in all the wells. Concentrations tested for CdSeS/ZnS QDs were 209, 251, 301, 362, 434, 520, 625, and 750 µg/L, those for CdS 380 497, 572, 658, 756, 870, and 1000 µg/L, and for CdS 480 279, 335, 402, 482, 579, 694, 833, and 1000 µg/L. Each concentration was tested in triplicate. Microplates were exposed to continuous illumination for 72 h, and the content of each well was mixed every 12 h, with the help of a micropipette. At the end of the test, absorbance at 440 nm was registered for each replicate. The test was considered valid when the biomass in the control increased at least 16 times and when the coefficient of variation of the specific growth rate in the control replicates did not exceed 7% in the tests with R. subcapitata and 10% in the tests with C. vulgaris. V. fischeri (Microtox®) bioluminescence inhibition test was performed following the M500 Microtox manual (Azur Environmental, 1998) for the 81.9% basic test. For this test QD suspensions were prepared in distilled water according to the methodology described previously. For CdSeS/ZnS the maximum concentration tested was 750 µg/L, while for both CdS 380 and CdS 480 the maximum concentration tested was 1000 µg/L (dilution factor 2 for all the QDs).

2.5. Statistical analysis

Growth inhibition of R. subcapitata and C. vulgaris recorded in the different tested concentrations was analysed by one-way analysis of variance (ANOVA). Dunnett 8

Multiple Comparison tests were carried out to look for differences between each treatment and the control. An =0.05 was selected. The effect concentrations (EC50 and EC20 values) were calculated using the non-linear least squares regression procedure supplied by the software STATISTICA® 7 (StatSoft, Inc., Tulsa, OK, USA). For the immobilization test with D. magna, the EC50 and EC20 values were estimated by regression analysis according to the Probit analysis in IBM SPSS Statistics 20 (Finney, 1971). The EC20 and EC50 values and their corresponding 95% confidence intervals for bioluminescence inhibition in V. fischeri were computed using the Software for Microtox Omni Azur (AZUR Environmental, 1998). No correction for colour was performed, since, as stated by the Microtox manual (AZUR Environmental, 1998), no colour was noticed in the suspensions at the concentrations tested.

3. Results

3.1. Characterization of QD suspensions

The Z-Ave and PdI of QDs suspended in both culture media and distilled water are described in Table 1, for the highest concentration tested in each assay, both for nonexposed and sunlight-exposed suspensions. Although, the average particle size of all QDs tested increased considerably in the suspensions not exposed to sunlight, after dispersion into the different test media (comparing with vendor information), CdSeS/ZnS showed a tendency to form smaller aggregates when compared with core type QDs, especially in distilled water and ASTM medium. For the CdS 380 QDs, small aggregates were formed only in the MBL medium. In opposition, for CdS 480 QDs, particle aggregation was less pronounced when QDs were dispersed into ASTM. In the suspensions exposed to sunlight, all QDs showed a tendency to form even larger aggregates. Apart from particle size, PdI is an important parameter to evaluate the physicochemical characteristics of nanomaterials. Analysis of obtained results in the investigated QDs indicated that all formulations are heterogeneously distributed as confirmed by high PdI, except for CdSeS/ZnS dispersed in MBL in suspensions not exposed to sunlight. 9

[Please, insert Table 1 near here]

3.2. Ecotoxicological evaluation of QD suspensions The results obtained in the Microtox® test for QD suspensions are presented in Table 2. The EC50 values were calculated after 5, 15 and 30 min of exposure for each sample. Regarding the suspensions not exposed to sunlight, the core shell QDs (CdSeS/ZnS) were less toxic to the bacteria. The toxicity of suspensions of CdS 380 and CdS 480 QDs not exposed to sunlight was similar with the small QDs slightly more toxic to the bacteria, after 30 min. The slightly higher toxicity of CdS 380 is also confirmed by the EC20 values recorded. For suspensions of QDs tested after sunlight exposure, CdS 380 and CdS 480 QDs showed a remarkable reduction of toxicity, displaying bioluminescence inhibition percentages lower than 50% for all the tested concentrations. These results prevented the estimation of EC50 values. The exception was the core shell QDs, the toxicity of which increased remarkably after their sunlight treatment. [Please, insert Table 2 near here] Daphnia magna immobilization tests were performed for all the QDs suspensions, both before and after exposure to sunlight. All the tests fulfilled the validity criteria described by the OECD 202 guideline (OECD, 2004). Toluene control did not show a significant immobilization after 48 h of exposure (≤10%) at the maximum concentration tested (data not shown). [Please, insert Figure 1 near here] Table 3 displays the EC50 and EC20 values estimated for the non-exposed and sunlight-exposed QD suspensions. In the acute exposure with D. magna, the core shell CdSeS/ZnS QDs and the CdS 480 QDs, displayed similar EC50 values (after 48 h of exposure) (Figure 3A and 1A, respectively). Concerning the core type QDs, CdS 380 suspension was the most toxic one (Figure 2A and 2B). EC50 for 48 h of exposure was not estimated, because an immobilization percentage greater than 50% was recorded for all the concentrations 10

tested, including the lowest one (376 µg/L) (Figure 2A). The sunlight exposure decreased the toxicity of the CdSeS/ZnS and CdS 480 QD suspensions to D. magna (Figure 1B and 3B, respectively). Daphnids exposed to the CdSeS/ZnS suspension exposed to sunlight showed signs of debilitation and partial paralysis at higher concentrations (≥567 µg/L) after 24 h, but no complete immobility was recorded. No signs of paralysis or behavioural dysfunction were registered at lower concentrations. Although it was not possible to estimate EC50 values, figures 2A and 2B demonstrate that sunlight exposure has increased the toxicity of CdS 380 QD suspension, since after 48 h of exposure, all the organisms were immobilized even at the lowest concentration tested (376 µg/L) and the organisms also showed signs of advanced degradation, which are clear signs of an elevated level of toxicity.

[Please, insert Figure 2 near here] [Please, insert Figure 3 near here] [Please, insert Table 3 near here]

The sensitivity of algae to QD suspensions was species- and QD-dependent. R. subcapitata was the most sensitive species to the suspension of CdSeS/ZnS QD in the non-exposed suspensions. While a significant decrease in the growth rate of this species was recorded for the concentration of 625 µg/L, the same effect was recorded for C. vulgaris only for the highest concentration (750 µg/L) (figures 4A and 4C). A highly significant decrease in the growth rate of C. vulgaris was recorded only for the concentration of 251 µg/L of CdSeS/ZnS QDs. However, we consider that this effect has likely occurred by chance. The sunlight exposure of this QD suspension reduced its toxicity to both species (figures 4B and 4D).

[Please, insert Figure 4 near here]

The smallest QDs (CdS 380) were the most toxic to both species of algae, since a highly significant reduction in the growth rate of algae was recorded for all the

11

concentrations tested (Figures 5A and 5C). After the exposure to sunlight, the toxicity of CdS 380 was decreased for both species of algae (Figure 5B and 5D).

[Please, insert Figure 5 near here] R. subcapitata was once again the more sensitive species to the suspension of CdS 480 QDs before sunlight exposure; a significant growth inhibition was recorded for the lowest and highest concentrations tested (Figure 6A). However, after sunlight exposure there was a slight decrease in toxicity, since a significant reduction in the growth rate was recorded only for the QD concentrations of 694 and 1000 µg/L (Figure 6B). C. vulgaris was not sensitive to the CdS 480 suspension for concentrations up to 1000 µg/L (Figure 6C). The exposure to sunlight promoted a slight increase in the toxicity of these QD suspensions, since a significant inhibitory effect was recorded for the highest concentration tested (1000 µg/L) (Figure 6D).

[Please, insert Figure 6 near here]

4. Discussion

4.1. Toxicity of QD suspension QDs have shown several advantages over organic fluorescent compounds, due to their high luminescence, stability against photobleaching, as well as high range of fluorescence wavelengths (Gerion et al., 2001). However, the safety properties of QDs are widely associated to their physicochemical characteristics such as chemical composition, particle size, shape, surface charge and functionalization. The development of QDs for biological and medical applications requires crucial control of particle size, since particle agglomeration/aggregation can affect the kinetics of QDs during in vitro or in vivo analysis, thus influencing their interaction with cells (Ozkan, 2004). In the present study, we assessed how the exposure to sunlight could affect the physical stability and toxicity of cadmium-containing QDs, in order to have a better evaluation of the behaviour and the potential toxicity of these compounds under more realistic environmental conditions.

12

The physical characterization of QDs indicated that these particles showed low physical stability when suspended in the different test media as their size increased dramatically due to high particle aggregation. In fact, several other studies have reported the tendency of QDs to aggregate. For example, Lopes et al. (2012) reported higher average sizes for CdSe/ZnS QDs both in Milli Q water (53513 nm) and in ASTM medium (38215 nm) than those recorded in this study for the CdSeS/ZnS QDs (Lopes et al., 2012). Although the core/shell QDs tested in our study had a surface functionalization with COOH-, for increasing their stability in water, aggregation may occur when these hydrophilic groups are no longer ionized (Moon et al., 2009). This has probably happened when these QDs were dispersed in water or in the test media (MBL and ASTM), but mainly in the growth medium of algae (MBL), which is a nutrient-rich medium with a high ionic strength. Moon et al. (2009) also attributed the aggregation of QDs functionalized with carboxylic groups to the formation of H-bonds between particles (Moon et al., 2009). The tendency to aggregate was also observed for both bare and core/shell QD forms, functionalized or not, when suspended in different aqueous solutions by other authors (Morelli et al., 2013; Feswick et al., 2013; Lee et al., 2010; Navarro et al., 2012; Wang et al., 2013). However, the aggregation of the three tested QDs did not prevent their ability to exert toxic effects on the species tested. From the analysis of the current studies on QDs toxicity, we can divide the main toxicity mechanisms of these compounds into three common processes: First, the oxidation of QDs and the release of Cd2+ ions into the medium, which will to exert toxicity by the common toxicity mechanisms already described for metals (Derfus et al., 2004). Second, the formation of ROS (even when QDs are only adsorbed on cell surface) that can damage several types of molecules such as DNA, proteins and lipids (Gurr et al., 2005; Parak et al., 2005). And, third, the entrance and accumulation of QDs in cells leading to functional disruption of intracellular organelles and of metabolic pathways (Chen et al., 2012). These processes vary greatly depending on core, shell and surface coatings of the QDs and therefore it continues to be difficult to predict their toxicity, and the most relevant mechanism in action. Several authors have reported the role of the shell in preventing the degradation of QDs with subsequent release of Cd2+ ions (Bakalova et al., 2004; Rzigalinski and Strobl, 2009). Nevertheless, and although less toxic than the other bare nanomaterials tested in this work, CdSeS/ZnS-alloyed core/shell QDs showed to be toxic to all tested species in the standard, pre-exposure tests, except to V. fischeri. Since they have a Zn shell, Cd2+-ion release into the medium 13

should not be the main toxicity mechanism, but the generation of ROS is likely a better possibility (Kim et al., 2010). In fact, lipid peroxidation appears to be the main toxicity mechanism in bacteria (Imlay, 2003). However, under stress condition caused by the presence of nanomaterials, bacteria can alter the fatty acid levels in their membrane in order to adapt physiologically (Fang et al., 2007). This ability might explain the lower toxicity of these QD suspensions to V. fischeri. The results obtained from Microtox suggest that CdS 380 as well as CdS 480 QDs showed higher toxicity to bacteria in comparison to core-shell type QDs. The EC50 values for the bioluminescence inhibition assay for these QDs were in fact similar to those obtained by Wang et al. (2010), who reported the accumulation of modified-CdSe nanocrystals inside Photobacterium phosphoreum (presently V. fischeri) (Wang et al., 2010). D. magna was the most sensitive species to the CdSeS/ZnS QD suspension. Some authors demonstrated that the toxicity of core shell QDs in daphnids can be related to their uptake and accumulation in the digestive tract (Lewinski et al., 2010). In fact, COOH functionalized QDs were taken up more efficiently than others by D. magna as shown by Feswick et al. (2013). According to these authors this was due to the establishment of interactions between the lumen of the intestine and the functional carboxylic groups of the QDs (Feswick et al., 2013). If we assume the ZnS shell prevents the release of Cd2+ ions, as mentioned previously, the toxicity mechanism of these QDs is reduced to either the formation of free radicals, or interaction with or transport across the cell wall and cell membrane (Wang et al., 2010). However, the pore diameter of algal wall can range from 5-20 nm (Navarro et al., 2008). Therefore, since these particles showed a considerably larger hydrodynamic diameter due to their shell and COOH functionalization, which was even increased by QD aggregation, it is not expected that they have crossed the cell membrane, unless by an advanced mechanism such as endocytosis (Lin et al., 2009). In the algal growth inhibition tests, only the highest concentrations tested caused a significant inhibition, which corroborates this hypothesis. In fact, Morelli et al. demonstrated by confocal microscopy, that the core shell CdSe/ZnS inhibited Phaeodactylum tricornutum growth, increased ROS production and lipid peroxidation, and also activated antioxidant enzymes only by contact with cell surface (Morelli et al., 2013). Despite aggregation, and comparing the toxicity to algae of bare type QDs, CdS 380 QDs were more toxic than the larger 480 QDs especially for C. vulgaris. Due to their small size, it is expected that these particles were more able to interact with cells 14

causing toxicity. In fact, CdS 380 QDs formed the smallest aggregates in the MBL medium as shown in Table 1. Further, the high PdI values recorded for suspensions not exposed to sunlight demonstrate that particles with different hydrodynamic sizes were present in the suspensions. The effects of QDs are likely related with the smallest particles/aggregates. CdS 380 and CdS 480 core type QDs, unlike CdSeS/ZnS, were obtained as dispersions in toluene. Toluene is a highly toxic compound per se, but usually several QDs are provided stabilized in toluene since they are hydrophobic compounds. At the highest concentrations of QDs, daphnids displayed desynchronized swimming patterns or, ultimately, complete paralysis, though this effect passed over time. Control tests with toluene did not result in significant immobilization by 48 h (≤ 10%). Therefore, it is not expected that the toxicity observed for bare QDs was mainly caused by toluene. However, toluene is toxic for the V. fischeri bacteria, with an estimated 30 min-EC50 of 51 µL/L, expressed as bioluminescence inhibition (Lopes et al., 2012). The highest concentration tested for bare QDs in the Microtox test contained a maximum concentration of toluene of 200 µL/L. Thus, it is possible that the toxicity recorded for these QDs suspensions to V. fischeri may have been caused at least in part by toluene. As far as algae are concerned, Hsieh et al. (2006) reported an EC50 value of 22.2 mg/L (CI: 15.4-39.2) to R. subcapitata, which corresponds to 25.4 L/L of toluene (Hsieh et al., 2006). However, in our study even in highest concentration of bare QDs tested which corresponded to 200 L/L of toluene, no inhibition or an inhibition lower than 50% in growth was recorded, making it difficult to establish the role of toluene in the toxicity of these QDs. Furthermore, and although diluted in corresponding test media, the possible role of toluene in facilitating crossing of cell membranes by bare core QDs cannot be ignored. Even in toluene dispersion, core materials in the presence of oxidating conditions such as air or light, tend to undergo surface oxidation, releasing free Cd2+ into the medium, rendering it toxic (Li et al., 2009). Core-type QDs are also reported to be able to contribute to the formation of ROS (Lovrić et al., 2005) and to entering cells, accumulating in intracellular compartments (Wang et al., 2010). Therefore, cadmium core-type QDs have several toxicity pathways, which depend on several factors as described above. Some of these pathways may have been responsible for the toxicity of these QD suspensions to D. magna and algae. 15

4.2. Effect of light exposure on QD toxicity

The effect of sunlight to the ability of QDs to exert toxic effects is unpredictable, as it is species- and QD-dependent. Light is one of the external stimuli that may result in significant toxicological changes (Lee et al., 2010). The toxicity tests with CdSeS/ZnS are conclusive: sunlight exposure caused a decrease in toxicity of these QDs, at least to algae and daphnids. These results are not coincident with those that documented an increase in toxicity upon UV exposure (Derfus et al., 2004; Kim et al., 2010; Wang et al., 2010) as well as those testing a sunlight exposure (Lee et al., 2010) of trioctylphosphine oxide and mercaptopropionic acid-capped CdSe/ZnS QDs. Previous studies have reported that ZnS shell of QDs prevents the release of Cd2+ by photooxidation, therefore reducing their toxicity (Dabbousi et al., 1997). However, this fact alone may not explain the effective reduction in toxicity upon exposure to sunlight. The irradiation may have also modified the COOH- functional groups, rendering the nanocrystals less stable and therefore more susceptible to aggregation and/or precipitation. Kim et al. (2010) also stated that upon light exposure for several hours (Kim et al., 2010), particle agglomeration can occur, increasing the overall size of particles, as was observed in our study. This affects their toxicity, as they become less available to organisms, by settling and by becoming even less able to cross the cell membrane due to their substantial size. Differences in surface functionalization, in the core chemistry as well as in test procedure may explain the contradictory results obtained between our study and the earlier study of Lee et al (Lee et al., 2010). While in our study, daphnids were exposed to QD suspensions previously treated with sunlight under standard laboratory conditions, in this previously mentioned study the suspensions were exposed to sunlight with the organisms, and only for 4 h. Thus, other abiotic factors, like an increasing temperature, during the exposures of organisms, may explain the differences in toxicity. In R. subcapitata test, both with and without previous sunlight exposure of CdSeS/ZnS QD suspensions, growth stimulation was observed. This is a hormetic response, which is a toxic stimulation resulting from the addition of stimulating growth factors to the medium (OECD, 2011). This means that the exposure of CdSeS/ZnS to radiation or even to air can release Zn2+ from the shell upon oxidation (Zhang et al., 16

2006). Zinc is a trace element component of proteins and enzymes, and therefore essential to organisms. The release of this compound into the medium, due to degradation of the shell, could be the cause of this hormetic response. Lavoi et al. (2012) also demonstrated that the presence of Zn2+ in the medium protects algae from the uptake and toxicity of Cd2+ (Lavoie et al., 2012). According to these authors, this may be explained by the synthesis or by conformational changes of membrane-bound transporters, or simply by the competition with Cd2+ at the metal binding sites. The enhanced aggregation, likely caused by the oxidation of QDs, might be an explanation for the slight reduction in the toxicity of CdSeS/ZnS and of CdS 480 (at least for V. fischeri, D. magna and R. subcapitata) and of CdS 380 (for all the species except for D. magna) after sunlight exposure. The possible oxidation of toluene to benzaldeyde, by sunlight and oxygen (Mao and Bakac, 1996) may also have contributed for the lower toxicity of sunlight-exposed suspensions of bare core QDs. Benzaldehyde has been considered a safe compound due to its low toxicity (Andersen, 2006). Contrary to available data, this work proposes that in an environmentally realistic scenario the exposure of shell-type QDs to sunlight can actually reduce their toxicity, at least to some organisms. Furthermore, it was reinforced that bare QDs are not adequate for biological uses due to their unpredictability and acute toxicity, especially after their release into the environment where they will be subject to different abiotic factors that may interfere with their stability, making changes in their toxicity unpredictable. QDs have the potential to revolutionize medical therapy, as long as low toxicity nanocrystals can be produced. In this work low-sized QDs were the most toxic of the tested compounds. Although the role of toluene cannot be neglected, this fact can be problematic, as low-sized QDs are often preferred for biological applications. Therefore, a throughout chemical and toxicological characterization of particles should be made before use, along with the continuous development of smaller, preferably Zncoated QDs in order to suit both medical and environmental needs.

5. Conclusions In the present study the toxicity of core-type and shell-type, alloyed QDs were assessed with a battery of ecotoxicological tests with aquatic, environmentally relevant test species that cover different functional and trophic levels. The effect of sunlight on QDs 17

toxicity was also evaluated by exposing QD solutions to sunlight and comparing the toxicity results to those obtained with QD solutions not exposed to sunlight. Sunlight exposure effectively decreased CdSeS/ZnS and CdS 480 toxicity in D. magna and R. subcapitata at the conditions tested, and increased CdS 380 toxicity in D. magna test. These results suggested that both the shell and particle aggregation were important factors affecting toxicity. Sunlight exposure may play an important role in the aggregation and precipitation reactions of larger QDs, due changes in functional groups. Nevertheless, the consequences of changes induced by abiotic factor exposures on the toxicity of these QD systems are still unpredictable and species-dependent. The formation of large bulks of nanocrystals, which are less prone to photo-oxidation due to their diminished surface area may lead to a lowered release of Cd2+ ions into the medium, as well as lowered ROS production, therefore reducing their toxicity. However, this was not aobserved after sunlight exposure for all the bare core QDs despite the fact that they all showed increased aggregation. These results emphasize the need of further research on the toxicity mechanisms and chemical interactions of each QD in order to predict their environmental impacts correctly. Nevertheless, core shell QDs seemed to be those that are in fact less harmful to aquatic ecosystems, both under standard environmental conditions, and after the exposure to a relevant abiotic factor, sunlight.

Acknowledgments

The work was partially supported by Fundação para a Ciência e Tecnologia (FCT, Portugal), namely the PhD scholarships (SFRH/BD/94902/2013) attributed to A. Gavina. This research was also partially supported by the Portuguese Government (Programa Ciência — Inovação 2010), by the European Social Fund— COMPETE through the research project REALISE (PTDC/AAC-AMB/120697/2010) and by the Strategic Funding UID/Multi/04423/2013 and UID/AMB/50017/2013 through national funds provided by FCT – Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020.

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Figure Captions Figure 1. Percentage of immobilization of Daphnia magna exposed to CdSeS/ZnS QD suspensions not exposed to sunlight (A) and exposed to sunlight (B) for 24 h and 48 h. Error bars correspond to standard deviation. Figure 2. Percentage of immobilization of Daphnia magna exposed to CdS 380 QD suspensions not exposed to sunlight (A) and exposed to sunlight (B) for 24 h and 48 h. Error bars correspond to standard deviation.

Figure 3. Percentage of immobilization of Daphnia magna exposed to CdS 480 QD suspensions not exposed to sunlight (A) and exposed to sunlight (B) for 24 h and 48 h. Error bars correspond to standard deviation.

Figure 4. Effect of CdSeS/ZnS QD suspensions not exposed to sunlight (A) and exposed to sunlight (B) on R. subcapitata growth and CdSeS/ZnS QD suspensions not exposed to sunlight (C) and exposed to sunlight (D) on C. vulgaris growth. Stimulation of the growth rate of algae was observed for the concentrations indicated by ―i‖. Data are presented as the mean ± SD. Significant difference from control (* p < 0.05 and ** p < 0.01). Figure 5. Effect of CdS 380 QD suspensions not exposed to sunlight (A) and exposed to sunlight (B) on R. subcapitata growth and CdS 380 QD suspensions not exposed to sunlight (C) and exposed to sunlight (D) on C. vulgaris growth. Data are presented as the mean ± SD. Significant difference from control (* p < 0.05 and ** p < 0.01). Figure 6. Effect of CdS 480 QD suspensions not exposed to sunlight (A) and exposed to sunlight (B) on R. subcapitata growth and CdS 480 QD suspensions not exposed to sunlight (C) and exposed to sunlight (D) on C. vulgaris growth. Stimulation of the growth rate of algae was observed for the concentrations indicated by ―i‖. Data are presented as the mean ± SD. Significant difference from control (* p < 0.05 and ** p < 0.01). 24

Figure 1

Figure 2

25

Figure 3

26

Figure 4

27

Figure 5

28

Figure 6

29

30

Table Captions

Table 1. Average hydrodynamic diameter (nm ± SD) and polydispersity index of QDs dispersed in distilled water, ASTM and MBL medium determined by DLS before (pre) and after (post) sunlight exposure. Table 2. Effective concentrations (µg/L) causing a 20 and a 50% bioluminescence inhibition after 5, 15 and 30 minutes of the bacteria V. fischeri exposure to the different QDs (95 % CI, inside brackets) before (pre) and after (post) sunlight exposure. Trend analysis displays the increase or decrease of the toxicity along the exposure time.

Table 3. Effective concentrations (µg/L) causing 20% and 50% immobilization in D. magna upon exposure to the different QDs before (pre) and after (post) sunlight exposure. *100% mortality was observed at all concentrations, for which the lowest concentration is chosen (OECD, 2004).

Table 1.

QDs CdSeS/ZnS Distilled water Z-Ave

PdI

ASTM

MBL

Z-Ave

PdI

Z-Ave

PdI

Pre

197.9±150.4 0.7

75.1±46.4

0.6

314.3±81.4 0.2

Post

large aggregates

large aggregates

large aggregates

Pre

large aggregates

large aggregates

51.8±41.9

Post

211.3±154.3 0.7

large aggregates

large aggregates

Pre

large aggregates

85.5±72.2 0.8

large aggregates

Post

large aggregates

large aggregates

large aggregates

CdS 380 0.8

CdS 480

31

Large aggregates: particles ˃ 1 µm and PdI ˃ 1

Table 2. QDs

Endpoint Exposure

5 min

15 min

Pre

NT

NT

Post

NT

NT

30 min

Trend

NT

CdSeS/ZnS EC50 388.2 (248.9-605.6) ↗

Pre

636.9 (96.8-4185.8) 390.2 (181.5-839.5)

Post

7.4 (2.6-20.5)

Pre

175.7 (160.8-191.9) 219.9 (184.9-261.5)

292.8 (33.5-2555.8) ↗

CdSeS/ZnS EC20

CdS 480

CdS 480

CdS 380

12.4 (9.6-15.9)

↘

270.9 (213.1-344.3)

↘

EC50 Post

NT

NT

Pre

66.6 (58.5-75.7)

77.2 (59.0-101.1)

106.5 (77.5-146.4)

↘

NT

Post

366.5 (294.0-457.0)

602.8 (528.8-687.3)

761.0 (NC)

↘

Pre

202.9 (189.6-217.0)

235.6 (212.2-261.7)

244.1 (232-256-0)

↘

EC20

EC50 Post

CdS 380

12.6 (9.2-17.0)

NT

NT

NT

Pre

62.4 (57.7-67.5)

73.1 (63.0-86.7)

82.8 (71.0-96.6)

↘

Post

375.1 (200.2-702.8)

340.5 (198.0-702.8)

302.6 (152.9-598.3)

↗

EC20

NT: no toxicity.

Table 3

QDs

D. magna immobilization Endpoint

CdSeS/ZnS

COOH 24h EC50

Pre-sunlight exposure

Post-sunlight exposure

n.d.

n.d.

32

functionalized

in 24h EC20

343.0 (278.6 - 397.1)

n.d.

48h EC50

389.3 (351.9 - 441.2)

609.1 (517.4 - n.d.)

48h EC20

262.8 (200.7 - 302.3)

525.4 (346.4 - 604.1)

898.1 (672.2 - n.d.)

n.d.

24h EC20

545.5 (n.d.- 817.2)

n.d.

48h EC50

n.d

˂ 376*

48h EC20

n.d

˂ 376*

703.6 (623.2 - 827.2)

792.8 (729.6 - 879.1)

24h EC20

446.3 (366.5 - 508.3)

560.8 (472.0 - 624.6)

48h EC50

390.6 (n.d.- 627.1)

594.3 (485.9 - 701.9)

48h EC20

n.d

396.3 (n.d.-500.9)

aqueous solution

CdS 380 in toluene 24h EC50 suspension

CdS 480 in toluene 24h EC50 suspension

n.d: not determined

33