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Nanoparticle toxicity in Daphnia magna reproduction studies: The importance of test design
Aquatic Toxicology 126 (2013) 163–168
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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Nanoparticle toxicity in Daphnia magna reproduction studies: The importance of test design Frank Seitz a,∗ , Mirco Bundschuh a,b , Ricki R. Rosenfeldt a , Ralf Schulz a a b
Institute for Environmental Sciences, University of Koblenz-Landau, Germany Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 23 August 2012 Received in revised form 15 October 2012 Accepted 31 October 2012 Keywords: Inorganic nanoparticles Reproduction Growth Flow-through Crustacea
a b s t r a c t The increasing use of titanium dioxide nanoparticles (nTiO2 ) inevitably results in their release into the environment, raising concerns about potential adverse effects in wildlife. By following standard test protocols, several studies investigated the ecotoxicity of nTiO2 among others to Daphnia magna. These studies indicated a large variability – several orders of magnitude – in the response variables. However, other factors, like nanoparticle characteristics and test design, potentially triggering these differences, were largely ignored. Therefore, the present study assessed the chronic ecotoxicity of two nTiO2 products with varying crystalline structure (A-100; P25) to D. magna. A semi-static and a flow-through exposure scenario were compared, ensuring that both contained environmentally relevant concentrations of dissolved organic carbon. Utilizing the semi-static test design, a concentration as low as 0.06 mg/L A-100 (∼330 nm) significantly reduced the reproduction of daphnia indicating environmental risk. In contrast, no implication in the number of released offspring was observed during the flow-through experiment with A-100 (∼140 nm). Likewise, P25 (∼130 nm) did not adversely affect reproduction irrespective of the test design utilized. Given the present study’s results, the particle size, the product composition, i.e. the crystalline structure, and the accumulation of nTiO2 at the bottom of the test vessel – the latter is relevant for a semi-static test design – may be suggested as factors potentially triggering differences in nTiO2 toxicity to D. magna. Hence, these factors should be considered to improve environmental risk assessment of nanoparticles. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The utilization of engineered nanoparticles is still increasing and is expected to become a trillion US-dollar business in the near future (Nel et al., 2006). Titanium dioxide nanoparticles (nTiO2 ), in particular, are frequently used as ingredients in personal care products (Scheringer, 2008) but also for remediation of different media, like air and water (Fujishima et al., 2000). This widespread application suggests their unintended release into environments (Klaine et al., 2011). Although such nanoparticles may pose potential environmental risks (Moore, 2006), their implications in wildlife are largely unknown (Scown et al., 2010). Therefore, researchers have investigated ecotoxicological implications of nTiO2 mainly on standard test organisms using their test guidelines (cp. Griffitt et al., 2008; Hartmann et al., 2010). Guideline-dependent studies, however, indicate a huge variability
∗ Corresponding author at: Institute for Environmental Sciences, University of Koblenz-Landau, Fortstrasse 7, D-76829 Landau, Germany. Tel.: +49 6341 280 31 362; fax: +49 6341 280 31 326. E-mail address: [email protected] (F. Seitz). 0166-445X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2012.10.015
in acute and chronic endpoints on the most frequently investigated species Daphnia magna (Lovern and Klaper, 2006; Heinlaan et al., 2008; Wiench et al., 2009; Zhu et al., 2010). Independent reproduction tests, for instance, revealed 21 d-EC50 values differing by up to three orders of magnitude (Wiench et al., 2009; Zhu et al., 2010). This discrepancy may be explained by differences in nanoparticle characteristics like particle size (Dabrunz et al., 2011). Moreover, the fate of nanoparticles in the test system may determine their ecotoxicological potential. In this context, the accumulation of nTiO2 agglomerates at the bottom of the test vessel may be a driver for adverse effects as displayed for a benthic amphipod (Bundschuh et al., 2011). This pathway may also be relevant for routinely applied (semi-)static exposure scenarios indicated by diminished nTiO2 concentrations in the water column (e.g. Velzeboer et al., 2008; Dabrunz et al., 2011). Hence, the unevaluated application of these standardized test designs, which were originally developed to accommodate traditional chemicals like pesticides, as the foundation of environmental risk assessment of nanoparticles may be questionable (Handy et al., 2012). Hence, the present study assessed potential shortcomings of standardized (semi-)static test protocols by comparing two exposure scenarios: a conventional semi-static test design following
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Table 1 Mean particle size and zeta-potential (n = 3) of the two nTiO2 products (A-100 and P25) measured in the stock suspensions and test medium, respectively. The particle size was assessed over the entire test duration of the 21 d reproduction tests (semi-static and flow-through test design), while zeta-potential was determined exclusively at the start of the experiment. Test-system
Medium
A-100 Mean (±sd; nm)
Semi-static Flow-through
Stock suspension Test medium Test medium
87.3 (±2.4) 326.8 (±20.9) 143.2 (±23.9)
P25 PI
0.132–0.189 0.180–0.281 0.143–0.284
n
Mean zetapotential (±sd; mV)
Mean (±sd; nm)
3 7 22
−32.0 (±1.3) −16.7 (±0.2)
92.4 (±4.1) 156.7 (±18.2) 131.8 (±14.1)
PI
0.152–0.219 0.115–0.230 0.181–0.233
n
Mean zetapotential (±sd; mV)
3 7 22
53.5 (±1.9) −19.4 (±0.4)
PI, polydispersity index.
largely the recommendations of the Organization of Economic Co-operation and Development (OECD, 2008) was compared to a flow-through test design. The latter took nanoparticle-specific properties into account by avoiding the accumulation of nTiO2 agglomerates at the bottom of the test vessels and thus preventing a hypothesized pathway of ecotoxicological effects (Bundschuh et al., 2011). Furthermore, the present study aimed at assessing implications of different crystalline structures of TiO2 . Hence, two nTiO2 products were investigated using both test designs, i.e. A100, purchased as pure anatase, and P25, a mixture composed of approximately 30% rutile and 70% anatase (Bhatkhande et al., 2002). Moreover, the test medium of all experiments was amended by dissolved organic carbon (DOC), usually present in surface waters (Ryan et al., 2009), to increase the environmental relevance of the experiment. In addition, DOC stabilizes nanoparticles in the water column enhancing their availability (Navarro et al., 2008; Hall et al., 2009). Furthermore, nanoparticle characteristics (e.g. particle size, zeta-potential) were monitored over the entire study duration as recommended by Handy et al. (2012). 2. Material and methods
suspensions 50-fold in test medium. This procedure was accomplished since the highest test concentration used in the present study delivered too low intensities to measure the zeta-potential reliably. Finally, the concentration of TiO2 in the water column (Table S1) was measured in the 2.00 mg/L, the 0.20 mg/L and the control treatment once a week during each flow-through test and at the first and third day during each semi-static test (representative for a usual time interval between one medium exchange) by inductively coupled plasma mass spectrometry (Dabrunz et al., 2011; see Supplementary Material for further details). 2.2. Test organisms D. magna were kept in permanent culture at 20 ± 1 ◦ C with a 16:8 h (light:dark) photoperiod using ASTM reconstituted hard freshwater enriched with selenium, vitamins (thiamine hydrochloride, cyanocobalamine, biotine) and seaweed extract (Marinure® , Glenside, Scotland) (=test medium) as recommended for metal toxicity testing (OECD, 2008). Daphnids were fed with the green algae Desmodesmus sp. on a daily basis with an equivalent of 200 g C per organism.
2.1. Nanoparticle characterization
2.3. Reproduction tests
The nTiO2 products assessed in the present study, namely A-100 (Crenox GmbH, Krefeld, Germany) and P25 (Degussa, Essen, Germany), had an advertised primary particle size of 6 nm and 21 nm, respectively, while the advertised surface area (Brunner–Emmett–Teller) was approximately 230 and 50 m2 /g. Both products were applied as dispersant and additive-free, sizehomogenized stable suspensions, obtained by stirred media milling (PML2, Bühler AG, Switzerland). Prior to each application, nTiO2 suspensions were ultra-sonicated for 10 min to ensure a homogeneous distribution of particles. Moreover, particle size distributions as well as zeta-potential of both products were determined via dynamic and electrophoretic light scattering, respectively (DelsaTM Nano C, Beckman Coulter, Germany). A-100 and P25 exhibited in the stock suspension an average particle diameter of <100 nm and a zeta-potential of 53.5 and −32.1 mV, respectively (Table 1). During the semi-static tests, the average particle size in the test medium was monitored every third day and hence between two water exchanges (Table 1), while during both flow-through experiments these measurements were performed daily (Table 1). Therefore, the sample was taken from the center and 2 cm beneath the water surface of one randomly selected replicate of the 2.00 mg nTiO2 /L treatment 1 h after pumping (see Supplementary Material for further details). Additionally, the control test medium was analyzed during all chronic investigations as described above assessing for potential implications of algae and daphnids’ excretions on the particle size measurement. These analyses (data not shown) indicated negligible influence of both factors on the assessed endpoint. Moreover, the actual zeta-potential of both products in the test medium (Table 1) was obtained by diluting the nTiO2 stock
2.3.1. General test design Each of the four reproduction tests was initiated by introducing daphnids younger than 24 h into the testing system. These organisms were subsequently exposed for 21 d to nominal test concentrations of 0.00, 0.02, 0.06, 0.20, 0.60 or 2.00 mg/L nTiO2 irrespective of the product investigated. The range of nTiO2 concentrations was selected on the basis of acute toxicity tests with the same species (Dabrunz et al., 2011). Moreover, the test medium contains a DOC concentration of 8.1 ± 0.80 mg/L, which represents field-relevant levels (Ryan et al., 2009). Dissolved oxygen and pH were measured with a WTW Multi 340i set (WTW Inc., Weilheim, Germany) and met the requirements of the guideline (OECD, 2008; Table S2). Offspring was counted and removed daily. Subsequently, body length – defined as distance from directly above the eyespot to the base of the dorsal spine – of adults and offspring (the latter for the flow-through tests exclusively) was determined at the end of the experiment and within 24 h after their release, respectively, from digital images (Canon, PowerShot SX 200 IS) using AxioVision software (Carl Zeiss, Jena). 2.3.2. Semi-static reproduction tests Semi-static reproduction tests followed the OECD guideline 211 (OECD, 2008). Briefly, 10 juveniles (<24 h) per treatment were placed individually in 50 mL test medium amended by the respective nTiO2 concentration (n = 10). Test organisms were fed daily in an age-dependent manner with Desmodesmus sp. (50–100 g C/test organism). The test medium was changed three times a week, with adult daphnids being carefully transferred to new medium using plastic pipettes.
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nTiO2 agglomerates with an average size below 150 nm in the water column (Table 1). 2.4. Statistical analysis
Fig. 1. Cumulative mean (±sd) offspring per test-organism (n ≥ 8) exposed to (a) A-100 and (b) P25 nTiO2 at different time intervals during a 21 d semi-static D. magna reproduction test. Asterisks denote statistically significant differences to the respective control; p < 0.05 (*), p < 0.01 (**).
For each reproduction test the cumulative mean number of offspring ± standard deviation (sd) was calculated for days 10, 14 and 21. Data were checked for normal distribution (Shapiro–Wilk test) and homogeneity of variances (Bartlett’s test). If requirements for parametric testing were met, repeated measures ANOVAs followed by one-way ANOVAs were performed to assess for statistical significance (p < 0.05) over time and to determine the point in time where statistical significant differences were observed, respectively. If requirements were not met, nonparametric alternatives, namely the Friedman test and the Kruskal–Wallis one-way analysis of variance, were accomplished. Finally, parametric Dunnett’s test or nonparametric Steel’s test, both for multiple comparisons, were applied as post hoc-test. Moreover, repeated measures ANOVAs, followed by Student’s or Welch’s unpaired t-tests were applied to identify statistically significant deviations in the body length of released offspring, while one-way ANOVAs and Dunnett’s post hoc test assessed deviations in adult body length among treatments. In case of multiple comparisons, the alpha-threshold was Bonferroni adjusted. All tests were two-sided. For statistical analyses and figures, the statistics program R version 2.14.0 (R Development Core Team, 2011) and respective extension packages (Helms and Munzel, 2008; Hothorn et al., 2008; Lemon, 2010) were used. Finally, the term significant is exclusively used with reference to statistical significance throughout the present study. 3. Results
2.3.3. Flow-through tests Generally the flow-through system included four replicates per treatment (Fig. S1), while each replicate consisted of a 500 mL funnel with 100 m mesh screen (funnel diameter: 8.5 cm; water column above the mesh screen was approximately 5.5 cm) at the bottom, which prevented a loss of juveniles from the test vessel directly after their release. At the start of each flow-through experiment, five juvenile daphnids (<24 h) were introduced in each replicate. Every second hour throughout the whole test duration fresh test medium was introduced ensuring a whole water exchange per replicate within slightly more than 24 h. Therefore, 148 mL test medium originating from a medium tank and 12 mL nTiO2 stock solution of the respective treatment were transferred in a 500 mL plastic beaker (mixing beaker) using two independent peristaltic pumps (Fig. S1). Following 5 min of mixing, a dosing pump (Dosing Unit SA 4, GHL, Kaiserslautern, Germany) transferred the total volume of 160 mL nTiO2 suspension in equal shares to the four independent replicates. The removal of old medium was accomplished by a passive discharge at the bottom of the funnel utilizing the hydrostatic pressure (Fig. S1). Generally, the test medium in the medium tank was renewed daily and amended in an age-dependent manner with the algae Desmodesmus sp. (see Section 2.3.2). The nTiO2 stock solutions were renewed every three days. The flow-rate of both peristaltic pumps were checked for their pumping performance weekly and adjusted if necessary. Moreover, all pumps were equipped as far as possible with Teflon® -tubes to minimize any loss of nTiO2 . The flow-through system thus avoided the accumulation of agglomerated nTiO2 at the bottom of the test vessel (Velzeboer et al., 2008; Dabrunz et al., 2011), which is a potential pathway of ecotoxicological effects (Bundschuh et al., 2011). At the same time, it ensured a permanent exposure to small
3.1. Semi-static reproduction tests The product A-100 caused after 21 d of exposure with 12% a significant reduced mean number of offspring compared to the control group at concentrations as low as 0.06 mg/L (Dunnett; p = 0.023; n ≥ 8; Fig. 1 a). Furthermore, adults’ body length was significantly reduced with up to 11% at the 0.20 and 2.00 mg/L A-100 treatments (Dunnett; p = 0.003 and 0.026; n ≥ 8; Table S3), while at 0.60 mg/L this endpoint was not significantly affected. Such adverse effects were already indicated after 14 d of exposure by a significantly reduced number of offspring at 0.20 and 0.60 mg/L A-100 (Dunnett; p = 0.038 and 0.045, n ≥ 8; Fig. 1a). A similar effect size was observed at the 2.00 mg/L treatment, which was however, due to the relatively high variability in the response variable not statistically significant. In contrast to A-100, the exposure to the nTiO2 product P25 resulted in only marginal deviations among treatments regarding mean offspring per adult (maximal effect size: approximately 5.0%; one-way ANOVA, p = 0.749; n ≥ 9; Fig. 1b) and adults mean body length (maximal effect size: approximately 3.5%; oneway ANOVA, p = 0.331; n ≥ 9; Table S3). 3.2. Flow-through tests The flow-through experiments displayed for A-100 as well as for P25 no significant differences among treatments, neither regarding the mean body length of adults nor their number of offspring released (Table S3, Table 2). However, the mean body length of juveniles released in the 2.00 mg/L A-100 treatment was between day 19 and 21 by 7.6% significantly reduced if compared to the control (Bonferroni adjusted Student’s t-test p = 0.016, n = 4 means; Table 2).
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Table 2 Treatment related mean body length (n = 4 means) of released juveniles (<24 h) at different time intervals and cumulative mean offspring per test-organism (n = 20) exposed to either A-100 or P25 nTiO2 during a 21 d D. magna reproduction test in a flow-through system. Product
*
Endpoint
Body length
Time interval (d)
7–9
Concentration (mg/L)
Mean (±sd; mm)
A-100
0.00 0.02 0.06 0.20 0.60 2.00
P25
0.00 0.02 0.06 0.20 0.60 2.00
Cumulative offspring 10–12
13–15
16–18
19–21
7–21
0.81 (±0.01) 0.82 (±0.04) 0.83 (±0.02) 0.82 (±0.03) 0.86 (±0.03) 0.80 (±0.08)
1.01 (±0.03) 0.96 (±0.01) 0.96 (±0.01) 0.95 (±0.05) 0.97 (±0.02) 0.97 (±0.04)
1.04 (±0.02) 1.06 (±0.02) 1.06 (±0.02) 1.09 (±0.05) 1.09 (±0.03) 1.06 (±0.01)
1.12 (±0.02) 1.10 (±0.02) 1.08 (±0.03) 1.01 (±0.06) 0.99 (±0.05) 1.07 (±0.05)
1.06 (±0.03) 1.00 (±0.01) 1.05 (±0.07) 1.04 (±0.06) 1.08 (±0.02) 0.98 (±0.02) *
66.50 (±1.71) 73.75 (±1.89) 73.83 (±12.67) 63.05 (±1.17) 64.10 (±5.11) 71.93 (±7.69)
0.86 (±0.03) 0.82 (±0.02) 0.84 (±0.03) 0.82 (±0.01) 0.83 (±0.02) 0.84 (±0.03)
0.91 (±0.02) 0.90 (±0.02) 0.91 (±0.04) 0.92 (±0.03) 0.94 (±0.03) 0.91 (±0.03)
0.95 (±0.01) 0.96 (±0.02) 0.92 (±0.01) 0.94 (±0.02) 0.96 (±0.02) 0.97 (±0.01)
1.01 (±0.02) 1.01 (±0.07) 1.00 (±0.03) 1.04 (±0.02) 1.06 (±0.04) 1.00 (±0.05)
1.05 (±0.02) 1.07 (±0.03) 1.05 (±0.02) 1.03 (±0.03) 1.10 (±0.02) 1.08 (±0.02)
60.55 (±9.27) 67.40 (±4.72) 71.94 (±7.23) 66.70 (±3.96) 69.50 (±4.52) 71.45 (±5.36)
Mean (±sd)
Denotes a statistically significant difference to the respective control; p < 0.05.
4. Discussion Although the results of the semi-static test with A-100 seem not to be entirely concentration-dependent, they indicate adverse effects in the reproduction of D. magna at nTiO2 concentrations only one order of magnitude above those predicted for surface water bodies (Gottschalk et al., 2011). Given the current regulatory framework of the European Union for chemical registration (ECB, 2003), a potential risk for aquatic ecosystems must therefore be assumed. However, the exposure to P25 resulted in only marginal deviations among treatments relative to the control. The chemical analysis during the semi-static test design uncovered that the initial nTiO2 concentration in the water column decreased after three consecutive days by approximately 95% (Table S1) irrespective of the product applied (Velzeboer et al., 2008; Dabrunz et al., 2011). This indicates the formation of a layer at the bottom of the test beakers exhibiting an increased concentration of nTiO2 or agglomerates, which was suggested as a potential pathway of nTiO2 ecotoxicity during bioassays with the benthic amphipod Gammarus fossarum (Bundschuh et al., 2011). As daphnids react negatively phototactically under low UV-irradiation (Storz and Paul, 1998) and low UV-irradiation had recently been indicated for the environmental test conditions in the used experimental unit (Seitz et al., 2012), such a pathway may have also been relevant for the outcome of the semi-static test design with A-100 (Fig. 1a). To further assess the relevance of the bottom-layer for daphnids, a flow-though test design was utilized avoiding this accumulation of nTiO2 agglomerates at the bottom of the test vessels, simultaneously, increasing the availability of nTiO2 in the water column. The success of this procedure was documented by chemical analysis displaying relatively constant nTiO2 concentrations in the water column over the entire study duration of three weeks (Table S1). Moreover, daily particle size measurements displayed an average particle size below 150 nm regardless of the nTiO2 product used (Table 1). Finally, the flow-through experiments indicated a lower toxicity of nTiO2 than the semi-static experiment (Table 2), which suggests the exposure to the bottom-layer as one important pathway of ecotoxicological effects also for experiments with daphnids. Moreover, both test designs indicate a higher ecotoxicity of the product A-100 compared to P25. In this context, the crystalline structure (Handy et al., 2012) of the investigated nTiO2 products may be a trigger. A-100 consists exclusively of anatase, while P25 is a mixture composed of anatase and rutile (Bhatkhande et al., 2002). Pure anatase induces inter alia membrane leakage in dermal mouse
cells inevitably leading to necrosis. Rutile, in contrast, formed under the same conditions exclusively reactive oxygen species, which may be controlled by antioxidants (Braydich-Stolle et al., 2009) making it finally less toxic than anatase. Similarly, Sayes et al. (2006) showed that in contrast to pure rutile or anatase-rutile mixtures (80:20 and 40:60), pure anatase caused meaningfully higher cytotoxicity to human dermal and lung cells, which the authors attributed to the crystalline structure of the investigated nTiO2 and its associated higher potential to generate reactive oxygen species. A comparable effect pathway may be also of relevance for the present study since environmental conditions during all tests allowed for a continuous photocatalytic activity of nTiO2 (Seitz et al., 2012). However, the photocatalytic efficiency of different crystalline structures is controversially discussed in literature (e.g. Bhatkhande et al., 2002; Cong and Xu, 2012), turning the extent the photocatalytic properties of nTiO2 (=formation of reactive oxygen species) relate to the observed effects in the present study into ambiguous. In addition to the crystalline structure, the differences in ecotoxicity between both nTiO2 products during the semi-static test design may be explained by deviations in the average particle size (Dabrunz et al., 2011). Indeed, the particle size measurements in the test medium uncovered an approximately two-fold higher particle size of A-100 agglomerates (Table 1). Although Dabrunz et al. (2011) suggested smaller particles to be more toxic than larger ones, it may be assumed that the larger agglomerates (>300 nm) in the semi-static experiment with A-100 caused an accelerated nTiO2 mass uptake by D. magna into the gut (Gophen and Geller, 1984) when compared to smaller agglomerates observed during the other experiments. This may have reduced the amount of algae consumed indicating adverse effects in their energy budget (Rosenkranz et al., 2009; Zhu et al., 2010) and ultimately affecting daphnids’ reproduction (Glazier and Calow, 1992). Thus, also this effect pathway may partly explain the differences in ecotoxicity of the nTiO2 products investigated. Although the driving factor for the differing ecotoxicological potential of both nTiO2 products used in the present study was not yet uncovered, it became evident that nanoparticle products differ considerably in their ecotoxicological potential. This observation further underlines the necessity of a thorough particle characterization prior to and during each bioassay (Handy et al., 2012). Moreover, the formation of a bottom layer in (semi-)static test designs can be considered as one important effect pathway. At the same time, biological surface coating by nTiO2 , which was suggested as a potential mode of action adversely affecting the molting
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of daphnids and finally their mobility (Dabrunz et al., 2011), was not observed. This may be explained by the presence of DOC in the test medium of the present study, which stabilizes particle size (Navarro et al., 2008; Hall et al., 2009) due to steric or electrostatic repulsion (Hyung et al., 2006) preventing the adhesion of particles onto the exterior surface of daphnids. 5. Conclusion The present study clearly displayed that the exposure scenario – either semi-static or flow-through – substantially affects the ecotoxicity of nTiO2 for D. magna: as a semi-static test design considers besides the exposure via the water column the accumulation of nTiO2 agglomerates at the bottom of the test vessel, it may represent a worst-case scenario for nTiO2 products as well as nanoparticles with similar properties, e.g. insolubility (Belgiorno et al., 2007). The flow-through test design, in contrast, mainly displays implications driven by the exposure via the water column. Hence, it may be more relevant for dissociable nanoparticles releasing toxic ions, which is suggested as important pathway of adverse effects for instance for silver- and copper-based nanoparticles (e.g. Navarro et al., 2008; Kennedy et al., 2010). This hypothesis, however, needs to be further assessed. Moreover, our investigations indicate the importance of considering implications of environmental parameters, like DOC, on the fate, i.e. the formation of a bottom-layer or biological surface coating, and finally the effect of engineered nanoparticles. Hence, understanding the interaction of environmental parameters and nanoparticles seems fundamental to reliably predict the environmental risks associated with the application of nanoparticle products and should urgently be considered during environmental risk assessment of nanoparticles. Acknowledgements The authors are grateful to two anonymous reviewers for their helpful comments on an earlier version of the manuscript, to C. Schilde for the preparation of nTiO2 stock solutions, and J.P. Zubrod for statistical advice. The Ministry of Science Rhineland-Palatinate (MBWJK) funded this study, which is linked with studies conducted within the research group INTERNANO supported by the German Research Foundation (DFG). Moreover, we acknowledge the FixStiftung, Landau for financial support of the research infrastructure. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox. 2012.10.015. References Belgiorno, V., Rizzo, L., Fatta, D., Della Rocca, C., Lofrano, G., Nikolaou, A., Naddeo, V., Meric, S., 2007. Review on endocrine disrupting-emerging compounds in urban wastewater: occurrence and removal by photocatalysis and ultrasonic irradiation for wastewater reuse. Desalination 215, 166–176. Bhatkhande, D.S., Pangarkar, V.G., Beenackers, A., 2002. Photocatalytic degradation for environmental applications – a review. Journal of Chemical Technology and Biotechnology 77, 102–116. Braydich-Stolle, L., Schaeublin, N., Murdock, R., Jiang, J., Biswas, P., Schlager, J., Hussain, S., 2009. Crystal structure mediates mode of cell death in TiO2 nanotoxicity. Journal of Nanoparticle Research 11, 1361–1374. Bundschuh, M., Zubrod, J.P., Englert, D., Seitz, F., Rosenfeldt, R.R., Schulz, R., 2011. Effects of nano-TiO2 in combination with ambient UV-irradiation on a leaf shredding amphipod. Chemosphere 85, 1563–1567. Cong, S., Xu, Y., 2012. Explaining the high photocatalytic activity of a mixed phase TiO2 : a combined effect of O2 and crystallinity. Journal of Physical Chemistry C 115, 21161–21168. Dabrunz, A., Duester, L., Prasse, C., Seitz, F., Rosenfeldt, R., Schilde, C., Schaumann, G.E., Schulz, R., 2011. Biological surface coating and molting inhibition
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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Nanoparticle toxicity in Daphnia magna reproduction studies: The importance of test design Frank Seitz a,∗ , Mirco Bundschuh a,b , Ricki R. Rosenfeldt a , Ralf Schulz a a b
Institute for Environmental Sciences, University of Koblenz-Landau, Germany Department of Aquatic Sciences and Assessment, Swedish University of Agricultural Sciences, Uppsala, Sweden
a r t i c l e
i n f o
Article history: Received 23 August 2012 Received in revised form 15 October 2012 Accepted 31 October 2012 Keywords: Inorganic nanoparticles Reproduction Growth Flow-through Crustacea
a b s t r a c t The increasing use of titanium dioxide nanoparticles (nTiO2 ) inevitably results in their release into the environment, raising concerns about potential adverse effects in wildlife. By following standard test protocols, several studies investigated the ecotoxicity of nTiO2 among others to Daphnia magna. These studies indicated a large variability – several orders of magnitude – in the response variables. However, other factors, like nanoparticle characteristics and test design, potentially triggering these differences, were largely ignored. Therefore, the present study assessed the chronic ecotoxicity of two nTiO2 products with varying crystalline structure (A-100; P25) to D. magna. A semi-static and a flow-through exposure scenario were compared, ensuring that both contained environmentally relevant concentrations of dissolved organic carbon. Utilizing the semi-static test design, a concentration as low as 0.06 mg/L A-100 (∼330 nm) significantly reduced the reproduction of daphnia indicating environmental risk. In contrast, no implication in the number of released offspring was observed during the flow-through experiment with A-100 (∼140 nm). Likewise, P25 (∼130 nm) did not adversely affect reproduction irrespective of the test design utilized. Given the present study’s results, the particle size, the product composition, i.e. the crystalline structure, and the accumulation of nTiO2 at the bottom of the test vessel – the latter is relevant for a semi-static test design – may be suggested as factors potentially triggering differences in nTiO2 toxicity to D. magna. Hence, these factors should be considered to improve environmental risk assessment of nanoparticles. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The utilization of engineered nanoparticles is still increasing and is expected to become a trillion US-dollar business in the near future (Nel et al., 2006). Titanium dioxide nanoparticles (nTiO2 ), in particular, are frequently used as ingredients in personal care products (Scheringer, 2008) but also for remediation of different media, like air and water (Fujishima et al., 2000). This widespread application suggests their unintended release into environments (Klaine et al., 2011). Although such nanoparticles may pose potential environmental risks (Moore, 2006), their implications in wildlife are largely unknown (Scown et al., 2010). Therefore, researchers have investigated ecotoxicological implications of nTiO2 mainly on standard test organisms using their test guidelines (cp. Griffitt et al., 2008; Hartmann et al., 2010). Guideline-dependent studies, however, indicate a huge variability
∗ Corresponding author at: Institute for Environmental Sciences, University of Koblenz-Landau, Fortstrasse 7, D-76829 Landau, Germany. Tel.: +49 6341 280 31 362; fax: +49 6341 280 31 326. E-mail address: [email protected] (F. Seitz). 0166-445X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2012.10.015
in acute and chronic endpoints on the most frequently investigated species Daphnia magna (Lovern and Klaper, 2006; Heinlaan et al., 2008; Wiench et al., 2009; Zhu et al., 2010). Independent reproduction tests, for instance, revealed 21 d-EC50 values differing by up to three orders of magnitude (Wiench et al., 2009; Zhu et al., 2010). This discrepancy may be explained by differences in nanoparticle characteristics like particle size (Dabrunz et al., 2011). Moreover, the fate of nanoparticles in the test system may determine their ecotoxicological potential. In this context, the accumulation of nTiO2 agglomerates at the bottom of the test vessel may be a driver for adverse effects as displayed for a benthic amphipod (Bundschuh et al., 2011). This pathway may also be relevant for routinely applied (semi-)static exposure scenarios indicated by diminished nTiO2 concentrations in the water column (e.g. Velzeboer et al., 2008; Dabrunz et al., 2011). Hence, the unevaluated application of these standardized test designs, which were originally developed to accommodate traditional chemicals like pesticides, as the foundation of environmental risk assessment of nanoparticles may be questionable (Handy et al., 2012). Hence, the present study assessed potential shortcomings of standardized (semi-)static test protocols by comparing two exposure scenarios: a conventional semi-static test design following
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Table 1 Mean particle size and zeta-potential (n = 3) of the two nTiO2 products (A-100 and P25) measured in the stock suspensions and test medium, respectively. The particle size was assessed over the entire test duration of the 21 d reproduction tests (semi-static and flow-through test design), while zeta-potential was determined exclusively at the start of the experiment. Test-system
Medium
A-100 Mean (±sd; nm)
Semi-static Flow-through
Stock suspension Test medium Test medium
87.3 (±2.4) 326.8 (±20.9) 143.2 (±23.9)
P25 PI
0.132–0.189 0.180–0.281 0.143–0.284
n
Mean zetapotential (±sd; mV)
Mean (±sd; nm)
3 7 22
−32.0 (±1.3) −16.7 (±0.2)
92.4 (±4.1) 156.7 (±18.2) 131.8 (±14.1)
PI
0.152–0.219 0.115–0.230 0.181–0.233
n
Mean zetapotential (±sd; mV)
3 7 22
53.5 (±1.9) −19.4 (±0.4)
PI, polydispersity index.
largely the recommendations of the Organization of Economic Co-operation and Development (OECD, 2008) was compared to a flow-through test design. The latter took nanoparticle-specific properties into account by avoiding the accumulation of nTiO2 agglomerates at the bottom of the test vessels and thus preventing a hypothesized pathway of ecotoxicological effects (Bundschuh et al., 2011). Furthermore, the present study aimed at assessing implications of different crystalline structures of TiO2 . Hence, two nTiO2 products were investigated using both test designs, i.e. A100, purchased as pure anatase, and P25, a mixture composed of approximately 30% rutile and 70% anatase (Bhatkhande et al., 2002). Moreover, the test medium of all experiments was amended by dissolved organic carbon (DOC), usually present in surface waters (Ryan et al., 2009), to increase the environmental relevance of the experiment. In addition, DOC stabilizes nanoparticles in the water column enhancing their availability (Navarro et al., 2008; Hall et al., 2009). Furthermore, nanoparticle characteristics (e.g. particle size, zeta-potential) were monitored over the entire study duration as recommended by Handy et al. (2012). 2. Material and methods
suspensions 50-fold in test medium. This procedure was accomplished since the highest test concentration used in the present study delivered too low intensities to measure the zeta-potential reliably. Finally, the concentration of TiO2 in the water column (Table S1) was measured in the 2.00 mg/L, the 0.20 mg/L and the control treatment once a week during each flow-through test and at the first and third day during each semi-static test (representative for a usual time interval between one medium exchange) by inductively coupled plasma mass spectrometry (Dabrunz et al., 2011; see Supplementary Material for further details). 2.2. Test organisms D. magna were kept in permanent culture at 20 ± 1 ◦ C with a 16:8 h (light:dark) photoperiod using ASTM reconstituted hard freshwater enriched with selenium, vitamins (thiamine hydrochloride, cyanocobalamine, biotine) and seaweed extract (Marinure® , Glenside, Scotland) (=test medium) as recommended for metal toxicity testing (OECD, 2008). Daphnids were fed with the green algae Desmodesmus sp. on a daily basis with an equivalent of 200 g C per organism.
2.1. Nanoparticle characterization
2.3. Reproduction tests
The nTiO2 products assessed in the present study, namely A-100 (Crenox GmbH, Krefeld, Germany) and P25 (Degussa, Essen, Germany), had an advertised primary particle size of 6 nm and 21 nm, respectively, while the advertised surface area (Brunner–Emmett–Teller) was approximately 230 and 50 m2 /g. Both products were applied as dispersant and additive-free, sizehomogenized stable suspensions, obtained by stirred media milling (PML2, Bühler AG, Switzerland). Prior to each application, nTiO2 suspensions were ultra-sonicated for 10 min to ensure a homogeneous distribution of particles. Moreover, particle size distributions as well as zeta-potential of both products were determined via dynamic and electrophoretic light scattering, respectively (DelsaTM Nano C, Beckman Coulter, Germany). A-100 and P25 exhibited in the stock suspension an average particle diameter of <100 nm and a zeta-potential of 53.5 and −32.1 mV, respectively (Table 1). During the semi-static tests, the average particle size in the test medium was monitored every third day and hence between two water exchanges (Table 1), while during both flow-through experiments these measurements were performed daily (Table 1). Therefore, the sample was taken from the center and 2 cm beneath the water surface of one randomly selected replicate of the 2.00 mg nTiO2 /L treatment 1 h after pumping (see Supplementary Material for further details). Additionally, the control test medium was analyzed during all chronic investigations as described above assessing for potential implications of algae and daphnids’ excretions on the particle size measurement. These analyses (data not shown) indicated negligible influence of both factors on the assessed endpoint. Moreover, the actual zeta-potential of both products in the test medium (Table 1) was obtained by diluting the nTiO2 stock
2.3.1. General test design Each of the four reproduction tests was initiated by introducing daphnids younger than 24 h into the testing system. These organisms were subsequently exposed for 21 d to nominal test concentrations of 0.00, 0.02, 0.06, 0.20, 0.60 or 2.00 mg/L nTiO2 irrespective of the product investigated. The range of nTiO2 concentrations was selected on the basis of acute toxicity tests with the same species (Dabrunz et al., 2011). Moreover, the test medium contains a DOC concentration of 8.1 ± 0.80 mg/L, which represents field-relevant levels (Ryan et al., 2009). Dissolved oxygen and pH were measured with a WTW Multi 340i set (WTW Inc., Weilheim, Germany) and met the requirements of the guideline (OECD, 2008; Table S2). Offspring was counted and removed daily. Subsequently, body length – defined as distance from directly above the eyespot to the base of the dorsal spine – of adults and offspring (the latter for the flow-through tests exclusively) was determined at the end of the experiment and within 24 h after their release, respectively, from digital images (Canon, PowerShot SX 200 IS) using AxioVision software (Carl Zeiss, Jena). 2.3.2. Semi-static reproduction tests Semi-static reproduction tests followed the OECD guideline 211 (OECD, 2008). Briefly, 10 juveniles (<24 h) per treatment were placed individually in 50 mL test medium amended by the respective nTiO2 concentration (n = 10). Test organisms were fed daily in an age-dependent manner with Desmodesmus sp. (50–100 g C/test organism). The test medium was changed three times a week, with adult daphnids being carefully transferred to new medium using plastic pipettes.
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nTiO2 agglomerates with an average size below 150 nm in the water column (Table 1). 2.4. Statistical analysis
Fig. 1. Cumulative mean (±sd) offspring per test-organism (n ≥ 8) exposed to (a) A-100 and (b) P25 nTiO2 at different time intervals during a 21 d semi-static D. magna reproduction test. Asterisks denote statistically significant differences to the respective control; p < 0.05 (*), p < 0.01 (**).
For each reproduction test the cumulative mean number of offspring ± standard deviation (sd) was calculated for days 10, 14 and 21. Data were checked for normal distribution (Shapiro–Wilk test) and homogeneity of variances (Bartlett’s test). If requirements for parametric testing were met, repeated measures ANOVAs followed by one-way ANOVAs were performed to assess for statistical significance (p < 0.05) over time and to determine the point in time where statistical significant differences were observed, respectively. If requirements were not met, nonparametric alternatives, namely the Friedman test and the Kruskal–Wallis one-way analysis of variance, were accomplished. Finally, parametric Dunnett’s test or nonparametric Steel’s test, both for multiple comparisons, were applied as post hoc-test. Moreover, repeated measures ANOVAs, followed by Student’s or Welch’s unpaired t-tests were applied to identify statistically significant deviations in the body length of released offspring, while one-way ANOVAs and Dunnett’s post hoc test assessed deviations in adult body length among treatments. In case of multiple comparisons, the alpha-threshold was Bonferroni adjusted. All tests were two-sided. For statistical analyses and figures, the statistics program R version 2.14.0 (R Development Core Team, 2011) and respective extension packages (Helms and Munzel, 2008; Hothorn et al., 2008; Lemon, 2010) were used. Finally, the term significant is exclusively used with reference to statistical significance throughout the present study. 3. Results
2.3.3. Flow-through tests Generally the flow-through system included four replicates per treatment (Fig. S1), while each replicate consisted of a 500 mL funnel with 100 m mesh screen (funnel diameter: 8.5 cm; water column above the mesh screen was approximately 5.5 cm) at the bottom, which prevented a loss of juveniles from the test vessel directly after their release. At the start of each flow-through experiment, five juvenile daphnids (<24 h) were introduced in each replicate. Every second hour throughout the whole test duration fresh test medium was introduced ensuring a whole water exchange per replicate within slightly more than 24 h. Therefore, 148 mL test medium originating from a medium tank and 12 mL nTiO2 stock solution of the respective treatment were transferred in a 500 mL plastic beaker (mixing beaker) using two independent peristaltic pumps (Fig. S1). Following 5 min of mixing, a dosing pump (Dosing Unit SA 4, GHL, Kaiserslautern, Germany) transferred the total volume of 160 mL nTiO2 suspension in equal shares to the four independent replicates. The removal of old medium was accomplished by a passive discharge at the bottom of the funnel utilizing the hydrostatic pressure (Fig. S1). Generally, the test medium in the medium tank was renewed daily and amended in an age-dependent manner with the algae Desmodesmus sp. (see Section 2.3.2). The nTiO2 stock solutions were renewed every three days. The flow-rate of both peristaltic pumps were checked for their pumping performance weekly and adjusted if necessary. Moreover, all pumps were equipped as far as possible with Teflon® -tubes to minimize any loss of nTiO2 . The flow-through system thus avoided the accumulation of agglomerated nTiO2 at the bottom of the test vessel (Velzeboer et al., 2008; Dabrunz et al., 2011), which is a potential pathway of ecotoxicological effects (Bundschuh et al., 2011). At the same time, it ensured a permanent exposure to small
3.1. Semi-static reproduction tests The product A-100 caused after 21 d of exposure with 12% a significant reduced mean number of offspring compared to the control group at concentrations as low as 0.06 mg/L (Dunnett; p = 0.023; n ≥ 8; Fig. 1 a). Furthermore, adults’ body length was significantly reduced with up to 11% at the 0.20 and 2.00 mg/L A-100 treatments (Dunnett; p = 0.003 and 0.026; n ≥ 8; Table S3), while at 0.60 mg/L this endpoint was not significantly affected. Such adverse effects were already indicated after 14 d of exposure by a significantly reduced number of offspring at 0.20 and 0.60 mg/L A-100 (Dunnett; p = 0.038 and 0.045, n ≥ 8; Fig. 1a). A similar effect size was observed at the 2.00 mg/L treatment, which was however, due to the relatively high variability in the response variable not statistically significant. In contrast to A-100, the exposure to the nTiO2 product P25 resulted in only marginal deviations among treatments regarding mean offspring per adult (maximal effect size: approximately 5.0%; one-way ANOVA, p = 0.749; n ≥ 9; Fig. 1b) and adults mean body length (maximal effect size: approximately 3.5%; oneway ANOVA, p = 0.331; n ≥ 9; Table S3). 3.2. Flow-through tests The flow-through experiments displayed for A-100 as well as for P25 no significant differences among treatments, neither regarding the mean body length of adults nor their number of offspring released (Table S3, Table 2). However, the mean body length of juveniles released in the 2.00 mg/L A-100 treatment was between day 19 and 21 by 7.6% significantly reduced if compared to the control (Bonferroni adjusted Student’s t-test p = 0.016, n = 4 means; Table 2).
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Table 2 Treatment related mean body length (n = 4 means) of released juveniles (<24 h) at different time intervals and cumulative mean offspring per test-organism (n = 20) exposed to either A-100 or P25 nTiO2 during a 21 d D. magna reproduction test in a flow-through system. Product
*
Endpoint
Body length
Time interval (d)
7–9
Concentration (mg/L)
Mean (±sd; mm)
A-100
0.00 0.02 0.06 0.20 0.60 2.00
P25
0.00 0.02 0.06 0.20 0.60 2.00
Cumulative offspring 10–12
13–15
16–18
19–21
7–21
0.81 (±0.01) 0.82 (±0.04) 0.83 (±0.02) 0.82 (±0.03) 0.86 (±0.03) 0.80 (±0.08)
1.01 (±0.03) 0.96 (±0.01) 0.96 (±0.01) 0.95 (±0.05) 0.97 (±0.02) 0.97 (±0.04)
1.04 (±0.02) 1.06 (±0.02) 1.06 (±0.02) 1.09 (±0.05) 1.09 (±0.03) 1.06 (±0.01)
1.12 (±0.02) 1.10 (±0.02) 1.08 (±0.03) 1.01 (±0.06) 0.99 (±0.05) 1.07 (±0.05)
1.06 (±0.03) 1.00 (±0.01) 1.05 (±0.07) 1.04 (±0.06) 1.08 (±0.02) 0.98 (±0.02) *
66.50 (±1.71) 73.75 (±1.89) 73.83 (±12.67) 63.05 (±1.17) 64.10 (±5.11) 71.93 (±7.69)
0.86 (±0.03) 0.82 (±0.02) 0.84 (±0.03) 0.82 (±0.01) 0.83 (±0.02) 0.84 (±0.03)
0.91 (±0.02) 0.90 (±0.02) 0.91 (±0.04) 0.92 (±0.03) 0.94 (±0.03) 0.91 (±0.03)
0.95 (±0.01) 0.96 (±0.02) 0.92 (±0.01) 0.94 (±0.02) 0.96 (±0.02) 0.97 (±0.01)
1.01 (±0.02) 1.01 (±0.07) 1.00 (±0.03) 1.04 (±0.02) 1.06 (±0.04) 1.00 (±0.05)
1.05 (±0.02) 1.07 (±0.03) 1.05 (±0.02) 1.03 (±0.03) 1.10 (±0.02) 1.08 (±0.02)
60.55 (±9.27) 67.40 (±4.72) 71.94 (±7.23) 66.70 (±3.96) 69.50 (±4.52) 71.45 (±5.36)
Mean (±sd)
Denotes a statistically significant difference to the respective control; p < 0.05.
4. Discussion Although the results of the semi-static test with A-100 seem not to be entirely concentration-dependent, they indicate adverse effects in the reproduction of D. magna at nTiO2 concentrations only one order of magnitude above those predicted for surface water bodies (Gottschalk et al., 2011). Given the current regulatory framework of the European Union for chemical registration (ECB, 2003), a potential risk for aquatic ecosystems must therefore be assumed. However, the exposure to P25 resulted in only marginal deviations among treatments relative to the control. The chemical analysis during the semi-static test design uncovered that the initial nTiO2 concentration in the water column decreased after three consecutive days by approximately 95% (Table S1) irrespective of the product applied (Velzeboer et al., 2008; Dabrunz et al., 2011). This indicates the formation of a layer at the bottom of the test beakers exhibiting an increased concentration of nTiO2 or agglomerates, which was suggested as a potential pathway of nTiO2 ecotoxicity during bioassays with the benthic amphipod Gammarus fossarum (Bundschuh et al., 2011). As daphnids react negatively phototactically under low UV-irradiation (Storz and Paul, 1998) and low UV-irradiation had recently been indicated for the environmental test conditions in the used experimental unit (Seitz et al., 2012), such a pathway may have also been relevant for the outcome of the semi-static test design with A-100 (Fig. 1a). To further assess the relevance of the bottom-layer for daphnids, a flow-though test design was utilized avoiding this accumulation of nTiO2 agglomerates at the bottom of the test vessels, simultaneously, increasing the availability of nTiO2 in the water column. The success of this procedure was documented by chemical analysis displaying relatively constant nTiO2 concentrations in the water column over the entire study duration of three weeks (Table S1). Moreover, daily particle size measurements displayed an average particle size below 150 nm regardless of the nTiO2 product used (Table 1). Finally, the flow-through experiments indicated a lower toxicity of nTiO2 than the semi-static experiment (Table 2), which suggests the exposure to the bottom-layer as one important pathway of ecotoxicological effects also for experiments with daphnids. Moreover, both test designs indicate a higher ecotoxicity of the product A-100 compared to P25. In this context, the crystalline structure (Handy et al., 2012) of the investigated nTiO2 products may be a trigger. A-100 consists exclusively of anatase, while P25 is a mixture composed of anatase and rutile (Bhatkhande et al., 2002). Pure anatase induces inter alia membrane leakage in dermal mouse
cells inevitably leading to necrosis. Rutile, in contrast, formed under the same conditions exclusively reactive oxygen species, which may be controlled by antioxidants (Braydich-Stolle et al., 2009) making it finally less toxic than anatase. Similarly, Sayes et al. (2006) showed that in contrast to pure rutile or anatase-rutile mixtures (80:20 and 40:60), pure anatase caused meaningfully higher cytotoxicity to human dermal and lung cells, which the authors attributed to the crystalline structure of the investigated nTiO2 and its associated higher potential to generate reactive oxygen species. A comparable effect pathway may be also of relevance for the present study since environmental conditions during all tests allowed for a continuous photocatalytic activity of nTiO2 (Seitz et al., 2012). However, the photocatalytic efficiency of different crystalline structures is controversially discussed in literature (e.g. Bhatkhande et al., 2002; Cong and Xu, 2012), turning the extent the photocatalytic properties of nTiO2 (=formation of reactive oxygen species) relate to the observed effects in the present study into ambiguous. In addition to the crystalline structure, the differences in ecotoxicity between both nTiO2 products during the semi-static test design may be explained by deviations in the average particle size (Dabrunz et al., 2011). Indeed, the particle size measurements in the test medium uncovered an approximately two-fold higher particle size of A-100 agglomerates (Table 1). Although Dabrunz et al. (2011) suggested smaller particles to be more toxic than larger ones, it may be assumed that the larger agglomerates (>300 nm) in the semi-static experiment with A-100 caused an accelerated nTiO2 mass uptake by D. magna into the gut (Gophen and Geller, 1984) when compared to smaller agglomerates observed during the other experiments. This may have reduced the amount of algae consumed indicating adverse effects in their energy budget (Rosenkranz et al., 2009; Zhu et al., 2010) and ultimately affecting daphnids’ reproduction (Glazier and Calow, 1992). Thus, also this effect pathway may partly explain the differences in ecotoxicity of the nTiO2 products investigated. Although the driving factor for the differing ecotoxicological potential of both nTiO2 products used in the present study was not yet uncovered, it became evident that nanoparticle products differ considerably in their ecotoxicological potential. This observation further underlines the necessity of a thorough particle characterization prior to and during each bioassay (Handy et al., 2012). Moreover, the formation of a bottom layer in (semi-)static test designs can be considered as one important effect pathway. At the same time, biological surface coating by nTiO2 , which was suggested as a potential mode of action adversely affecting the molting
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of daphnids and finally their mobility (Dabrunz et al., 2011), was not observed. This may be explained by the presence of DOC in the test medium of the present study, which stabilizes particle size (Navarro et al., 2008; Hall et al., 2009) due to steric or electrostatic repulsion (Hyung et al., 2006) preventing the adhesion of particles onto the exterior surface of daphnids. 5. Conclusion The present study clearly displayed that the exposure scenario – either semi-static or flow-through – substantially affects the ecotoxicity of nTiO2 for D. magna: as a semi-static test design considers besides the exposure via the water column the accumulation of nTiO2 agglomerates at the bottom of the test vessel, it may represent a worst-case scenario for nTiO2 products as well as nanoparticles with similar properties, e.g. insolubility (Belgiorno et al., 2007). The flow-through test design, in contrast, mainly displays implications driven by the exposure via the water column. Hence, it may be more relevant for dissociable nanoparticles releasing toxic ions, which is suggested as important pathway of adverse effects for instance for silver- and copper-based nanoparticles (e.g. Navarro et al., 2008; Kennedy et al., 2010). This hypothesis, however, needs to be further assessed. Moreover, our investigations indicate the importance of considering implications of environmental parameters, like DOC, on the fate, i.e. the formation of a bottom-layer or biological surface coating, and finally the effect of engineered nanoparticles. Hence, understanding the interaction of environmental parameters and nanoparticles seems fundamental to reliably predict the environmental risks associated with the application of nanoparticle products and should urgently be considered during environmental risk assessment of nanoparticles. Acknowledgements The authors are grateful to two anonymous reviewers for their helpful comments on an earlier version of the manuscript, to C. Schilde for the preparation of nTiO2 stock solutions, and J.P. Zubrod for statistical advice. The Ministry of Science Rhineland-Palatinate (MBWJK) funded this study, which is linked with studies conducted within the research group INTERNANO supported by the German Research Foundation (DFG). Moreover, we acknowledge the FixStiftung, Landau for financial support of the research infrastructure. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox. 2012.10.015. References Belgiorno, V., Rizzo, L., Fatta, D., Della Rocca, C., Lofrano, G., Nikolaou, A., Naddeo, V., Meric, S., 2007. Review on endocrine disrupting-emerging compounds in urban wastewater: occurrence and removal by photocatalysis and ultrasonic irradiation for wastewater reuse. Desalination 215, 166–176. Bhatkhande, D.S., Pangarkar, V.G., Beenackers, A., 2002. Photocatalytic degradation for environmental applications – a review. Journal of Chemical Technology and Biotechnology 77, 102–116. Braydich-Stolle, L., Schaeublin, N., Murdock, R., Jiang, J., Biswas, P., Schlager, J., Hussain, S., 2009. Crystal structure mediates mode of cell death in TiO2 nanotoxicity. Journal of Nanoparticle Research 11, 1361–1374. Bundschuh, M., Zubrod, J.P., Englert, D., Seitz, F., Rosenfeldt, R.R., Schulz, R., 2011. Effects of nano-TiO2 in combination with ambient UV-irradiation on a leaf shredding amphipod. Chemosphere 85, 1563–1567. Cong, S., Xu, Y., 2012. Explaining the high photocatalytic activity of a mixed phase TiO2 : a combined effect of O2 and crystallinity. Journal of Physical Chemistry C 115, 21161–21168. Dabrunz, A., Duester, L., Prasse, C., Seitz, F., Rosenfeldt, R., Schilde, C., Schaumann, G.E., Schulz, R., 2011. Biological surface coating and molting inhibition
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