The induction of biochemical changes in Daphnia magna by CuO and ZnO nanoparticles

Aquatic Toxicology 150 (2014) 201–209

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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

The induction of biochemical changes in Daphnia magna by CuO and ZnO nanoparticles Phenny Mwaanga a,d,∗ , Elizabeth R. Carraway a,b , Peter van den Hurk a,c a

Clemson Institute of Environmental Toxicology, Clemson University, Pendleton, SC, USA Department of Environmental Engineering and Earth Sciences, Clemson University, Anderson, SC, USA Department of Biological Sciences, Clemson University, Clemson, SC, USA d Department of Environmental Engineering, Copperbelt University, Kitwe, Zambia b c

a r t i c l e

i n f o

Article history: Received 15 October 2013 Received in revised form 8 March 2014 Accepted 11 March 2014 Available online 20 March 2014 Keywords: Oxidative stress Biomarkers NOM Metal oxide nanoparticles Daphnia magna

a b s t r a c t Whilst a considerable number of studies have been reported on the acute toxicity of nanoparticles (NPs) on invertebrates such as Daphnia magna, few studies have been reported on the biochemical change (biomarkers) induction on these species by NPs, especially metal oxide NPs. The aim of this study was to investigate some biomarkers in D. magna induced by copper oxide (CuO) and zinc oxide (ZnO) NPs under controlled laboratory conditions. We exposed the 5 day old D. magna for 72 h to sublethal concentration of CuO and ZnO NPs in synthetic moderately hard water (MHW) with and without dissolved natural organic matter (NOM) and estimated the glutathione-S-transferase (GST) activity, formation of oxidized glutathione (GSSG), and amounts of thiobarbituric acid reacting substances (TBARS) and metallothionein (MT). Additionally, complementary short term dissolution studies on CuO and ZnO NPs were conducted. The results showed inactivation of GST enzyme by both metal oxide NPs. The results also showed increased production of oxidized GSH, increased generation of TBARS and increased induction of MT. In the presence of NOM, significant reduction (p < 0.05) in these biochemical changes was observed. These results indicated that oxidative stress is one of the toxicity mechanisms for these metal oxide NPs. Furthermore, the results suggest that these metal oxide NPs compromise the health of D. magna, and possibly other aquatic organisms, and therefore have potential to affect ecosystem stability. The short term dissolution studies showed that the proportion of dissolved NPs is higher (1.2% and 70% of initial concentration for dissolved Cu and Zn, respectively) at low particle concentration and is lower (0.4% and 17% of initial concentration for dissolved Cu and Zn, respectively) at higher particle concentration. These results suggest that the observed toxicity may be caused by both metal oxide nanoparticles and metal ions dissociated from the nanoparticles. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The production, use and commercialization of nanoparticles (NPs) has rapidly increased in recent years (Nowack and Bucheli, 2007; Baun et al., 2008; Farre et al., 2009; Auffan et al., 2009; Hotze et al., 2010; Lin et al., 2010). It is projected that this will inevitably lead to an increase of the release of NPs into aquatic systems (Lovern and Klaper, 2006; Klaine et al., 2008; Wiench et al., 2009; Keller et al., 2010). This will lead to concomitant increase in the exposure of aquatic organisms to NPs, which may result into adverse

∗ Corresponding author at: Copperbelt University, Environmental Engineering, Jambo Drive, Riverside, Kitwe, Zambia. Tel.: +260 969156065. E-mail addresses: [email protected], [email protected] (P. Mwaanga). http://dx.doi.org/10.1016/j.aquatox.2014.03.011 0166-445X/© 2014 Elsevier B.V. All rights reserved.

effects on these biota (Nowack and Bucheli, 2007; Baun et al., 2008; Sharma, 2009; Lin et al., 2010). Several NPs have been shown to induce a variety of toxic effects in aquatic organisms (Brayner et al., 2006; Lovern and Klaper, 2006; Franklin et al., 2007; Heinlaan et al., 2008; Aruoja et al., 2009). Exposure of organisms to sublethal concentrations of toxic NPs has been observed to result in changes at cellular and biochemical levels that manifested before any physiological effects were observed at the whole organism level (Moore, 2006; Klaper et al., 2009). These biochemical changes (biomarkers) can help explain the underlying mechanisms of toxicity (Forbes et al., 2006; Jemec et al., 2008; Klaper et al., 2009), and have potential for use as early warning signals for ecologically relevant effects (Forbes et al., 2006). When NPs gain entry into an organism, their interaction with target sites or receptors can trigger an increase in oxidative stress, resulting in proteomic and non-proteomic responses (Wong et al.,

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2010; Radwan et al., 2010). These responses could be in the antioxidant defense system aimed at protecting the organism against reactive oxygen species (ROS), and may include enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) (Gill and Tuteja, 2010; Loro et al., 2012). The responses could also involve the induction of general detoxification enzymes such as the glutathione-S-transferases (GSTs) (Gill and Tuteja, 2010; Loro et al., 2012) and stress proteins such as metallothioneins (MT) (Wong et al., 2010). Additionally, non-enzymatic molecules such as ascorbic acid (ASC), glutathione (GSH) and some amino acids could also be involved as antioxidants (Gill and Tuteja, 2010; Ortega et al., 2011). Both the enzymatic and non-enzymatic responses are used as biomarkers for oxidative stress (Radwan et al., 2010). However, the extent to which these biomarkers are induced or expressed can vary considerably in different species (Van den Hurk et al., 2012). The generation of ROS is a consequence of aerobic life (Gill and Tuteja, 2010; Yu et al., 2011) and the ROS molecules are usually scavenged by the antioxidant systems under normal conditions. The presence of toxicants (like NPs) can disturb the steady-state conditions, leading to increased production of ROS, which could overwhelm the defense capacity of the organisms over time, resulting in the inactivation of some enzymes, oxidation of proteins, damage of DNA and other essential biomolecules such as lipids (Barata et al., 2005; Gill and Tuteja, 2010). Furthermore, the destruction of lipids could lead to production of several electrophilic molecules such as malondialdehyde (MDA), 4-hydroperoxy-2-nonenal, 4-oxo-2-nonenal, or 4-hydroxy-2-nonenal that can further impair membrane functions, inactivate membrane-bound receptors and enzymes, and increase membrane permeability (Ortega et al., 2011). Some of these products of lipid peroxidation, such as MDA, can easily be measured through reaction with thiobarbituric acid to form fluorescent compounds called thiobarbituric acid reacting substances (TBARS) (Barata et al., 2005; Loro et al., 2012; Gill and Tuteja, 2010). CuO and ZnO NPs are among the metal oxide NPs with a wide application. For example ZnO NPs besides being among the ingredients in toothpaste, beauty products, sunscreens, they can also be used in textile (Wang et al., 2009) and in solar driven self-cleaning coatings (Cai et al., 2006). CuO NPs have been used in wood preservation and antimicrobial textiles (Gabbay et al., 2006) and have potential for use as catalysts for carbon monoxide oxidation and as heat transfer fluid in machine tools (Aruoja et al., 2009). The toxicity of both metal oxide NPs has been reported (Karlsson et al., 2008). Interestingly, there is unresolved question of whether the effects observed when organisms are exposed to CuO and ZnO NPs are due to dissolved metal ions or NPs. Whilst some studies have reported that the toxicity of both CuO and ZnO NPs on aquatic organisms were due to dissolved Cu2+ ions and Zn2+ ions (Aruoja et al., 2009; Heinlaan et al., 2008; Blinova et al., 2010), others have reported that the observed toxicity is due to the metal oxide NPs. For example, Zhu et al. (2009), reported that the toxicity of ZnO NPs on Daphnia magna was due to NPs. There is need therefore to conduct more studies to establish whether toxicity by CuO and ZnO NPs on D. magna is due to metal ions or NPs or the combination of both, as suggested by Ponyton et al. (2011). Furthermore, the bulk of the work in assessing the effects of NPs on D. magna has largely been carried out at organism level using mortality as endpoint (Lovern and Klaper, 2006; Adams et al., 2006; Heinlaan et al., 2008; Griffitt et al., 2008; Strigul et al., 2009; Wiench et al., 2009; Zhu et al., 2009; Blinova et al., 2010; Zhu et al., 2010). The use of biochemical changes in observing the effects of NPs on D. magna still requires further investigation. The few studies that have investigated the cellular or biochemical changes in D. magna have mainly been carried out with TiO2 and carbon based

NPs (Klaper et al., 2009; Kim et al., 2010). Other NPs such as CuO and ZnO NPs, which have equally wide application, are yet to be extensively studied with regards to induction of biochemical changes in D. magna. The aim of this study was to investigate some of the oxidative stress related biomarkers that are induced in D. magna when exposed to CuO and ZnO NPs in synthetic moderately hard water under controlled laboratory conditions. We investigated the induction of GST enzymes, the depletion of glutathione (GSH) by measuring the amount of oxidized GSH (GSSG), the production of TBARS, which indicates the extent of lipid breakdown in organisms, and the amount of MT in D. magna when exposed to these metal oxide NPs. Short term dissolution studies were also conducted in moderately hard water under similar conditions as the ones for toxicity tests, in order to ascertain the possible contribution of metal ions to the observed toxicity.

2. Materials and methods 2.1. Materials and chemicals Both CuO and ZnO NPs were purchased from Sigma-Aldrich Co. and were used without any purification. The particle sizes were indicated by the manufacturer as <50 nm for CuO and <100 nm for ZnO. Dynamic laser light scattering (DLS) measurements in Milli-Q water indicated presence of particle sizes of 190 nm and 250 nm for CuO and ZnO, respectively. Other particle characteristics are shown in Table 1. The standard Suwannee River Humic Acid (SRHA), reverse osmosis isolate (ROI) was purchased from International Humic Substances Society (IHSS). A DIONEX high performance liquid chromatography system, fitted with Diox auto sampler AS50, and 175 Diox PDA-100 detector was used for the measurement of metallothionein (MT). A size exclusion chromatographic column (SEC), the Protein-pakTM 125 10 ␮m, 7.8 × 300 mm HPLC column was purchased from Waters. A YMC-pack Diol—120, 300 × 8.0 mm ID, S—5 ␮m, 12 nm DL12S05-3008WT HPLC column was purchased from YMC. A Spectra Max Gemini fluorescent plate reader and a Spectra Max190 absorbance plate reader (Molecular Devices) were used in the determination of several biomarkers. Dithiothreitol (DTT), phenylmethylsulphonyl fluoride (PMSF), 1-chloro-2,4-dinitrobenzene (CDNB), reduced glutathione (GSH), rabbit liver metallothionein (MT-1) standard, ethylenediaminetetra acetic acid (EDTA), 5 ,5 dithio-bis-2-nitrobenzoicacid (DTNB), butylatedhydroxytoluene (BHT), 1,1,3,3-tetramethoxypropane (97% purity), HPLC protein standard mixture SLBB6450V and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Co. The bicinchoninic acid (BCA) protein assay reagents were purchased from Pierce. The glutathione fluorescent detection kit was purchased from Arbor Assays, and all other chemicals were of analytical grade and were purchased from VWR.

2.2. Test organisms The fresh water cladoceran D. magna was used as a test organism in the investigation of biomarkers induction in this study. It was chosen as a test organism because it is a good sentinel species for aquatic organisms. This species is sensitive, easy to culture and has a short generation time in the laboratory of approximately 8–10 days. The test organisms were cultured in the laboratory at the Clemson Institute of Environmental Toxicology, in artificially moderately hard water (Table S1) as specified by EPA (2007). The culture medium was renewed three times a week and the daphnids were fed a diet of an algal suspension and YCT (Yeast Cereal Leaves, by Tetramin) food mixture.

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Table 1 Summary of metal oxide nanoparticle characteristics in Milli-Q water. NP Name

Manufacturer’s reported size (nm)

CuO ZnO

<50 <100

DLS sizes (nm) (2 h)

(24 h)

171 ± 4 273 ± 25

190 ± 9 >3000

Percent purity (%)

Zeta Potential (mV)

Surface area (m2 /g)

99.99 99.9

30 ± 6 −16 ± 5

16.1 19.1

Surface area, DLS and zeta potential data were experimentally determined; other data were provided by the manufacturer (Sigma-Aldrich Co.).

2.3. Test suspensions For each metal oxide NPs two types of stock suspensions were prepared at 200 mg/L: one in distilled and deionized (DDI) water and the other in MHW. The stock suspensions of NPs were prepared in both DDI water and MHW due to differences in dissolution and aggregation in these media (see details later). From the DDI water stock suspensions of CuO and ZnO NPs, two types of test suspensions were prepared. The first test suspension type was prepared by pipetting appropriate volumes of the stock suspensions of each metal oxide NP into 30 mL plastic test vials and diluted to 25 mL with MHW to give the concentrations of 0.0, 0.3, 0.8 and 1.1 mg/L for each metal oxide NP. These concentrations were chosen after preliminary study showed that no significant mortality would be observed at 1.1 mg/L for both metal oxide NPs in moderately hard water. The second test suspension type had similar metal oxide concentrations as the first type, but also had NOM concentrations of 0.5 mg C/L NOM. The metal oxide NPs test suspensions with NOM were allowed to stand for 24 h before being used for the toxicity tests. Prior to being used in the toxicity tests, these test suspensions were sonicated for 10 min. From MHW stock suspensions of CuO and ZnO NPs, one type of test suspension was prepared for each metal oxide NPs. The test suspensions were prepared by pipetting appropriate volumes of the stock suspensions of each metal oxide NPs into 30 mL plastic test vials and diluted to 25 mL with MHW to give the concentrations of 0.0, 0.3, 0.8 and 1.1 mg/L for each metal oxide NP. 2.4. Sublethal toxicity bioassay D. magna sublethal toxicity bioassays were performed in 30 mL test vials with 25 mL of test suspensions prepared as described above. Five individual daphnids (≈5 day old) were placed in each test vial for 72 h under static non-renewal conditions. Nine replicates per test concentration were used for GST, oxidized glutathione (GSSG) and TBARS; for MT 14 replicates were used. At the end of the 72 h exposure period, any dead organism was excluded and the live organisms in each replicate for each concentration were pooled together to yield enough mass for the determination of both the total protein content and the appropriate biomarkers. For GST and TBARS, all the three types of test suspensions were used. For the oxidized GSH and MT, the test suspensions from the MHW stock were not used. Additionally, for MT, positive controls for copper (Cu2+ ) and zinc (Zn2+ ) metal ions were used at concentrations of 0.0, 5.0, 10.0, 25.0 and 50.0 ␮g/L metal ions. 2.5. Glutathione-S-transferase (GST) The GST enzyme analysis was carried out according to the procedure of Barata et al. (2005) with some modifications. The pooled juvenile daphnids for each metal oxide NP test suspension concentration were homogenized at 4 ◦ C in 250 ␮L of 100 mM phosphate buffer at pH 7.4, containing 100 mM KCl and 1 mM EDTA using a glass Potter-Elvehjem tissue homogenizer. The homogenates were centrifuged at 10,000 × g for 10 min at 4 ◦ C and the supernatants were used for the determination of enzyme activity and protein content. The reaction was carried out in a 96-well microplate and

the reaction mixture in each well consisted of sample, 1 mM CDNB and 1 mM GSH. Enzyme activity (Vmax over 2 min) was then read on the Spectra Max 190 absorbance plate reader. The protein content was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, USA). The protein concentrations were determined by using a standard curve of bovine serum albumin. The GST enzyme activity was normalized for the protein content in each sample. 2.6. Oxidized glutathione (GSSG) The pooled juvenile daphnids for each metal oxide NP test suspension concentration were homogenized at 4 ◦ C in 250 ␮L of 100 mM phosphate buffer at pH 7.0 using a glass homogenizer. The homogenates were centrifuged at 14,000 rpm for 10 min at 4 ◦ C. Then an aliquot of the supernatant was removed for protein determination. The protein content was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, USA). The protein concentrations were determined by using a standard curve of bovine serum albumin. The remaining supernatant was deproteinized using 5-sulfo salicylic acid and the GSSG was determined using a glutathione fluorescent detection kit (catalog number K006F1) from Arbor Assays (Michigan, USA). The amount of oxidized GSH was normalized to the total protein content of the sample. 2.7. TBARS Lipid peroxidation was measured as the amount of thiobarbituric acid reactive substances, such as malondialdehyde. The TBARs were measured by the procedure described by Barata et al. (2005). The pooled juvenile daphnids for each metal oxide NP test suspension concentration were homogenized at 4 ◦ C in 250 ␮L of 100 mM phosphate buffer at pH 7.4 containing 100 mM KCl and 1 mM EDTA using a glass homogenizer. The homogenates were centrifuged at 10,000 × g for 10 min at 4 ◦ C and the supernatants were used for the determination of the biomarker and the protein content. The protein content was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce, USA). The protein concentrations were determined by using a standard curve of bovine serum albumin. The amounts of TBARS were normalized using the total protein content of the sample. 2.8. Metallothionein (MT) For the determination of MT, a combination of protocols described in Lobinski et al. (1998), Stulik et al. (2003) and Alhama et al. (2006) were used with modifications to suite the available column and other high performance size exclusion chromatography (HPSEC) accessories. The pooled juvenile daphnids for each metal oxide NP test suspension concentration were split into two portions in a ratio 1:1. One portion was homogenized at 4 ◦ C in 250 ␮L of 100 mM phosphate buffer at pH 7.4 containing 100 mM KCl and 1 mM EDTA using a glass homogenizer. The homogenates were centrifuged at 10,000 × g for 10 min at 4 ◦ C and a subsample of the supernatants was used for the determination of the protein content. The protein content was measured by the bicinchoninic acid (BCA) protein assay kit (Pierce, USA). The other portions of the samples were prepared according to the procedure adopted and

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modified from Viarengo et al. (1997). Each portion was homogenized at 4 ◦ C in 250 ␮L 0.5 M sucrose and 20 mM tris–HCl buffer, pH 7.4, containing 6 ␮M leupeptine and 0.5 mM PMSF as antiproteolytic agents, and 0.01% ␤-mercaptoethanol as a reducing agent. The homogenates were then centrifuged at 14,000 rpm at 4 ◦ C for 24 min to obtain a supernatant containing metallothionein. The supernatant samples were then analyzed by HPSEC, using the rabbit liver metallothionein (MT-1) as a standard. 2.9. Complementary dissolution studies Complementary dissolution studies were conducted in moderately hard water test medium at concentrations similar to those commonly used in toxicity tests. Freshly prepared stock suspensions of CuO and ZnO NPs in DDI water, at 200 mg/L were used in this study (stock suspensions were used within 30 min of preparation). Test suspensions of both metal oxide NPs were prepared in duplicate by pipetting appropriate volumes of the stock suspensions of each metal oxide NP into 30 mL plastic test vials and diluted to 25 mL with MHW to give the concentrations of 0.0, 1.0, 2.0, 5.0 and 10 mg/L for each metal oxide NPs. These suspensions were then left to stand in the culture chamber for 48 h under 16 h light:8 h dark at temperature of 25 ± 1 ◦ C (same conditions as those used for toxicity tests). After 48 h, the amount of dissolved metal ions was determined in each plastic test vial. The dissolved metal ions were separated from NPs using 50 nm polycarbonate membrane filters. The dissolved metal ions (Zn+2 and Cu+2 ) in suspensions were quantified using ICP–MS. The ICP–MS method was used as described by Talbot and Weiss (1994) with minor modifications. The dissolved zinc and copper species and free metal ions concentrations in MHW (pH 7.8) were calculated using Visual Minteq version 3.0. Similarly, the effect of NOM on free metal ions was simulated using the dissolved organic carbon (DOC), Stockholm Humic Model (SHM) in the Visual Minteq. When SHM model was selected the default parameters and constants for the generic fulvic acid were used without modification. 2.10. Statistics All measurements were performed in triplicates (unless stated otherwise) and the data are expressed as mean± standard deviation. All the data were tested and exhibited normality. Comparisons among mean values were performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significance difference test (LSD) to verify and confirm differences among controls and treatments at different test suspension concentrations of the NPs with and without NOM. Furthermore, the differences between the control and each test suspension were assessed by using student’s paired t-test. Significance differences were established at p < 0.05. All these statistical tests were performed using the Origin pro 8.6 student version software. 3. Results and discussion 3.1. Metal oxide NPs characteristics The metal oxide NPs used in this study and their critical characteristics are shown in Table 1. The particle sizes advertised by the manufacturer were found to differ significantly from DLS measured sizes in Milli-Q water. Interestingly, the ZnO NPs were forming aggregates at such a high rate that within 24 h the average aggregate sizes were outside the nano-range. However, this aggregation did not appear to significantly impact the biomarker responses (see below). The CuO NPs on the other hand appeared to have relatively smaller and stable aggregates. The DLS measurements were equally conducted in MHW; however, for both CuO and ZnO NPs,

the average aggregate sizes obtained were quite large and outside the nano-range. In both media (Milli-Q and MHW), the DLS measurements were carried out at the particle concentration of 1 mg/L. 3.2. GST enzyme activity The results for CuO and ZnO NPs on GST enzyme activity indicated that there was a concentration dependent decrease in the GST enzyme activity for both metal oxide NPs (Fig. 1). These results are quite remarkable, especially because several studies have reported an increase in GST activity after NP exposure in D. magna (Klaper et al., 2009; Kim et al., 2010), although in those studies titanium and carbon based NPs were used. Other researchers have reported inactivation of GST isoforms when organisms were exposed to metal ions (Salazar-Medina et al., 2010; Loro et al., 2012). Our results may therefore indicate that the toxicity of CuO and ZnO NPs exposure for the GST enzyme activity is mostly driven by oxidative stress comparable to the action of dissolved metal ions. It is known that GST enzymes have a variety of functions and different isoforms have separate mechanisms of actions (Sheehan et al., 2001; Salazar-Medina et al., 2010), including peroxidase activity (Barata et al., 2005). The decrease in GST activity observed in this study could be explained in the following way: initially when reactive oxygen species (ROS) are generated by the NPs, the GST enzymes are engaged to detoxify these ROS through their peroxidase activity. But this detoxification mechanism involves the oxidation of thiol groups on the enzyme (destruction of disulfide bonds), which makes the enzyme lose its functionality (Letelier et al., 2006). In order to restore its functionality, the GST enzyme requires GSH to chemically change the oxidized thiol groups back into its original reduced state (Klaassen, 2008). However, GSH is also susceptible to oxidation (Gill and Tuteja, 2010), and is likely to be simultaneously oxidized when also the GST isoforms are oxidized. When this happens, both GST and GSH will be inactivated. This particularly could be the case when ROS production is increased and the anti-oxidative defense capability of the organism is overwhelmed (Barata et al., 2005), leading to complete enzyme inactivation and protein destruction. For this reason both oxidized GSH (GSSG) and lipid peroxidation (measured as TBARs) were also measured in this study (see details later). The inactivation of GST enzymes could also proceed through the non-specific binding of metal ions to the thiol groups of the GST molecules, forming metal thiolate clusters whose conformation is different from non-bound thiol groups and this change in structure would affect function (Letelier et al., 2006; Salazar-Medina et al., 2010). The latter mechanism of direct GST enzyme inactivation could probably be due to the substantial dissolution of metal ions from the metal oxide NPs (Mwaanga, 2012; Li et al., 2013). The effect of NOM on the metal oxide NPs’ ability to inactivate GST enzyme was compared in the suspensions of both CuO and ZnO NPs made from DDI and MHW stocks suspensions with and without NOM. The data demonstrated that the presence of dissolved NOM reduced significantly (p < 0.05) the GST inactivation at higher test concentrations (0.8 and 1.1 mg/L metal oxide NPs) for both CuO and ZnO NPs. The reduction in the toxic effects of NPs in the presence of dissolved NOM could be attributed to the sorption of NPs to NOM thereby making the NPs less bioavailable (Li et al., 2011; Chen et al., 2011). In this study, it was shown by the short term dissolution experiment that both ZnO and CuO NPs dissolve substantially (Table 2). The reduction in the toxic effects in the presence of NOM could therefore partly be attributed to the sorption of dissolved metal ions to NOM, thereby making them less bioavailable (De Schamphelaere et al., 2004). Visual Minteq modeling of free metal ions in the presence of NOM appeared to corroborate this conclusion. For example, the model results showed that about 90% of the

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Fig. 1. The GST activity response in D. magna exposed to metal oxide NPs with and without dissolved NOM: (a) CuO NPs test suspensions, (b) ZnO NPs test suspensions. Error bars indicate ± 1 standard deviation from mean, n = 3. Significant differences (p < 0.05) among exposure treatments after Fisher’s LSD multiple comparisons tests are followed by different letters.

dissolved copper (from CuO NPs) was complexed with dissolved organic matter as shown in Table S2 (Supplementary information) and less than 1% was in the free metal ions (free Cu2+ ions). For the zinc oxide NPs, the free Zn2+ ions proportion was relatively high at 72% of the total dissolved metal (Table S3) and this reduction in free metal ions could be enough to cause a substantial reduction in the GST induction. The results further showed that the test suspensions made from the MHW stock suspensions had reduced effect on GST inactivation when compared to the suspension made from the DDI water stock suspensions. This observation was interesting, especially that in literature researchers often prepare their stock suspensions of their NPs in different media of different ionic strengths, where dissolution and aggregation is different (Ponyton et al., 2011; Li et al., 2013). The reduction in the GST inactivation in suspensions made from MHW stock in comparison to those made from DDI stock suggests differences in dissolution and aggregation of metal oxide NPs and hence differences in concentrations of NPs and metal ions to which organisms are exposed (Klaine et al., 2008). Modeling of metal oxide dissolved species distribution using Visual Minteq showed that in MHW, 81% of the dissolved copper species are in the form of carbonates and only 6% is in the free Cu2+ ions (Table S4). For zinc, the free metal ions were 74% of the total dissolved (Table S5), while the carbonate form accounted for only about 10%. On the other hand, Visual Minteq species distribution in DDI showed higher proportions of dissolved free metal ions from CuO and ZnO NPs of 75% and 98%, respectively (Tables S6 and S7). Therefore, the reduced effect of GST inactivation by the test suspensions made from the MHW stock suspensions could partly be attributed to the increased aggregation of these metal oxide NPs in the MHW (as was shown by DLS measurements), and partly due to increased

competitive binding by Ca and Mg ions or by the association of metal ions, especially Cu to carbonate in MHW (Santore et al., 2001). 3.3. Oxidized glutathione (GSSG) As mentioned above, GSH plays an important role as antioxidant, preserving protein and enzyme integrity, and serves as co-factor for the GST enzyme. Given the observed inhibitory effects of CuO and ZnO NPs on the GST enzyme, curiosity was raised to investigate whether the metal oxide NPs, through oxidative stress, would affect the levels of reduced glutathione in D. magna. We therefore determined the oxidized forms of glutathione (GSSG) and used this as an index of oxidative stress (Gill and Tuteja, 2010). The same metal oxide NPs concentrations as those used for the study of GST were used. However, only test suspensions from DDI water stock both with and without dissolved NOM were used. The results, consistent with our expectation, showed that both CuO and ZnO NPs caused a concentration dependent increase in the amount of oxidized glutathione (Fig. 2). The observed increase in GSSG would correspond to a decrease in GSH (Radwan et al., 2010), although, the decrease in GSH should not necessarily corresponds directly to an increase in GSSG, as some GSH may be used by GST for conjugation of electrophilic compounds during exposure (Sheehan et al., 2001; Letelier et al., 2006; Klaper et al., 2009; Salazar-Medina et al., 2010). In addition, some GSH may be used directly in the formation of specific thiol complexes with metal ions released from the NPs (Radwan et al., 2010). The increase in the amount of GSSG appears to confirm that the ROS generated by the exposure of D. magna to metal oxide NPs led to the oxidation of the thiol groups of compounds (GST and GSH) that have peroxidase activity (Sheehan et al., 2001). The method of determining GSSG used in this study, as described

Table 2 Metal ions in suspension and dissolved for CuO and ZnO NPs used for complementary dissolution studies in MHW. Nominal concentration (mg/L)

Initial concentrations metal ions in suspensions

Final concentrations metal ions in suspensions

Dissolved concentrations metal ions in suspensions

% Dissolved of the initial concentrations metal ions in suspensions

ZnO

1.0

0.787 ± 0.031

0.634 ± 0.020

0.557 ± 0.040

70.77

CuO

2.0 5.0 10.0 1.0 2.0 5.0 10.0

1.522 ± 0.114 3.391 ± 0.500 6.513 ± 0.294 0.562 ± 0.030 1.333 ± 0.151 3.107 ± 0.330 6.867 ± 0.180

0.966 ± 0.060 1.076 ± 0.020 2.470 ± 0.280 0.141 ± 0.030 0.309 ± 0.010 0.560 ± 0.070 0.718 ± 0.060

0.850 ± 0.201 0.938 ± 0.070 1.102 ± 0.480 0.007 ± 0.002 0.013 ± 0.003 0.015 ± 0.002 0.026 ± 0.030

55.85 27.66 16.92 1.25 0.98 0.48 0.39

Nominal concentrations are in mg/L metal oxide NPs, the initial and final metal ions concentrations are in mg/L metal ions. The standard deviations shown were calculated from two replicates (n = 2).

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Fig. 2. The formation of oxidized GSH (GSSG) in D. magna when exposed to metal oxide NPs with and without dissolved NOM: (a) CuO NPs test suspensions; (b) ZnO NPs test suspensions. Error bars indicate ± 1 standard deviation from mean, n = 3. Significant differences (p < 0.05) among exposure treatments after Fisher’s LSD multiple comparisons tests are followed by different letters.

in the Arbor Assays kit manual, is very specific for GSH (reduced or oxidized) as it involves the removal of all proteins, and hence only non-protein thiol groups (from GSH) is determined. There is little information in published literature that has reported on the induction, depletion or oxidation of GSH in D. magna when exposed to metal oxide NPs. Some studies have reported depletion of GSH after exposure to metals ions in aquatic organisms (Loro et al., 2012). In general, both the depletion of GSH and increase of GSSG could be used as indicators of oxidative stress (Gill and Tuteja, 2010; Radwan et al., 2010). In this study, the extent of GSH depletion due to its use for GST facilitated conjugation of electrophilic compounds formed through ROS generation (though not measured) was considered minimal based on the inactivation of GST as seen above. The formation of specific bonds with metal ions could have contributed to the depletion of GSH (Radwan et al., 2010), but this could not be estimated using the Arbor Assays method used herein. The emphasis was therefore placed on investigating the conversion of the reduced to the oxidized form of GSH as confirmation of oxidative stress. Once the reduced GSH has been converted into the oxidized form, its ability to reduce oxidative stress is lost and hence this could lead to increased ROS levels, and eventual increase in lipid peroxidation. The mitigative role of dissolved NOM during GSH oxidation by the metal oxide NPs in D. magna was also investigated. The results showed that both CuO and ZnO NPs in the presence of 0.5 mg C/L NOM had reduced effects of GSH oxidation (Fig. 2), which is consistent with the observations for GST activity reported above. Given that there was substantial dissolution of both CuO and ZnO NPs, these results would therefore further suggests that metal ions dissociated from the NPs may have played a role in the observed effects. 3.4. Lipid peroxidation (TBARS) As a result of oxidative stress, membranes can be damaged through peroxidation of the lipid tails in the membrane structure, and form reactive compounds like malondialdehyde (MDA) (Barata et al., 2005). These TBARS were measured in D. magna juveniles after exposing them to sublethal concentrations of metal oxide NPs. For this part of the study, three test suspensions were used, as was the set-up for the GST activity measurements. The results indicated that both CuO and ZnO NPs caused a concentration dependent increase in the amount of TBARS generated at all the test suspension concentrations used in this study (Fig. 3). When ROS, such as superoxide anions, hydroxyl radicals and peroxide radicals are generated, they are, under normal conditions, detoxified by antioxidant pathways such as GSH and GST scavenging, followed by catalase and glutathione peroxidation (Gill and Tuteja, 2010). However, when

these antioxidant pathways are overwhelmed or inactivated (as seen above), the ROS eventually cause lipid peroxidation. Lipid peroxidation leads to production of organic hydroperoxides, which can breakdown into a variety of organic substances including MDA (Barata et al., 2005). This often creates a cyclic destructive pathway, because some of the breakdown products are strongly electrophilic in nature (Ortega et al., 2011), and therefore could lead to further production of ROS and hence increased lipid peroxidation (Gill and Tuteja, 2010) with consequential death to the organism. The observed increase in the amount of TBARS in this study is consistent with the observed increase in the GSSG and increased inactivation of GST, some of the key antioxidant moieties in organisms, thus reaffirming that oxidative stress is one of the mechanisms by which metal oxide NPs cause toxicity. There is paucity of data in the literature about NPs causing lipid peroxidation; some studies involving exposure of metal ions to aquatic organisms have made similar observations as presented here (Radwan et al., 2010; Loro et al., 2012). The effect of dissolved NOM on metal oxide NPs’ ability to cause lipid peroxidation was equally investigated, and the results were compared to the test suspensions of metal oxide NPs made from DDI water and MHW stocks suspensions without NOM. The data indicated that the presence of dissolved NOM reduced the generation of TBARS in all treatments for both CuO and ZnO NPs. Given that there was both dissolution and aggregation in these metal oxide suspensions (as explained above) in this study, the contribution of the observed effects could be from both NPs and free metal ions. This was consistent with data shown in Tables S8 and S9, which indicated lower free metals ion concentrations in MHW with NOM than in MHW without NOM for zinc and copper, respectively. Whilst the differences in these free metal ion concentrations for zinc were small, large differences with an order of magnitude for copper were observed (see Tables S8 and S9). The results further showed that there were significant differences (p < 0.05) between the effect of NPs test suspensions from DDI water and MHW stock suspensions (Ponyton et al., 2011), which strengthens the suggestion that bioavailability of dissociated metal ions from the NPs is a crucial component in the toxicity of metal oxide NPs. 3.5. Metallothionein (MT) Only few studies have investigated the induction of MT in organisms by NPs (Dua et al., 2012; Farmen et al., 2012; Gomes et al., 2012). In this study the possibility of MT induction by CuO and ZnO NPs was investigated in D. magna. A variety of methods that could be used to estimate and quantify MT have been described (Haase and Maret, 2004; Stulik et al., 2003; Alhama et al., 2006).

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Fig. 3. The TBARS generation in D. magna when exposed to metal oxide NPs with and without dissolved NOM: (a) CuO NPs test suspensions; (b) ZnO NPs test suspensions. Error bars indicate ± 1 standard deviation from mean, n = 3. Significant differences (p < 0.05) among exposure treatments after Fisher’s LSD multiple comparisons tests are followed by different letters.

However, each of these methods has its own challenges. In this study, an HPSEC method was used as a hybrid of several other methods, as described in the methods section. In trying to ascertain that the selected method would work, a series of MT standards were run using two different brands of size exclusion columns, the Waters column and the YMC column, and their resolutions for the MT standards were compared (Fig. S1). Additionally, an HPLC standard protein mixture purchased from Sigma-Aldrich was run using both the Waters and YWC column. The main objective was to see if the columns would be able to separate complex protein samples, and the results obtained (Fig. S2) were encouraging. However, it was observed that for satisfactory separations of protein samples, the columns should be relatively new, because in more used columns the protein separation is declining. Based on the satisfactory separations of the protein standard mixture, we proceeded to analyze the exposed daphnid samples. The results showed that both CuO and ZnO NPs were able to induce MT in D. magna (Fig. 4). Other researchers working with CuO NPs, though with different organisms (the mussel Mytilus galloprovincialis), were also able to observe MT induction by metal oxide NPs (Gomes et al., 2012). The

induction of MT in organisms is known to be caused by metals, but also by growth hormones, temperature changes, and oxidative stress (Dallinger et al., 2004; Shaw-Allen et al., 2005). The results presented here demonstrate that CuO and ZnO NPs do increase the internal concentrations of MT in D. magna. However, it could not be determined to what extent metal ions (due to dissolution of NPs) contributed to this phenomenon. To address the extent to which Cu2+ and Zn2+ metal ions would differ from metal oxide NPs in the induction of MT, D. magna were exposed to ionic metal solutions in a separate experiment. The metal ions were used as positive controls for MT induction. The choice of the concentration of the metal ions used as positive controls was based on the need to have low concentrations that would not result in excessive mortality of the test organisms (since any dead organism was excluded from MT determination) (Barata et al., 2005) and at the same time have high enough concentrations to induce significant MT in the test organisms. Visual Minteq modeling of dissolved metal ions (though the model assumed equilibrium) from the concentration of the metal oxide NPs used in this study suggests that the concentration range of Zn ions from dissolved ZnO NPs was higher

Fig. 4. Induction of metallothionein (MT) in D. magna when exposed to metal oxide NPs with and without dissolved NOM: (a) CuO NPs test suspensions; (b) ZnO NPs test suspensions. Error bars indicate ± 1 standard deviation from mean, n = 3. Significant differences (p < 0.05) among exposure treatments after Fisher’s LSD multiple comparisons tests are followed by different letters.

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than the range used for the positive control. But the model suggests that the concentration range of dissolved Cu ions from CuO NPs was lower than that for the positive control. The exposure results showed that the induction of MT by metal ions was generally higher than that for metal oxide NPs. Taken together; these results suggest that that both metal ions and NPs elicits toxic effects in D. magna. Based on the dissolved ions, these results also suggest that for ZnO NPs, the contribution to the observed toxicity from Zn ions is much more significant than that for Cu ions. Thus our results appear to be consistent with the study by Ponyton et al. (2011) who made similar observations for ZnO NP suspensions and ZnSO4 solutions on D. magna, that both metal ions and NPs were causing the observed effect, though through different mechanisms. Furthermore, the effect of NOM on the metal oxide NPs and metal ions induction of MT was compared with metal oxide NPs and metal ions without NOM. However, in this study, unlike other biomarkers described above, the mitigative role of dissolved NOM in the induction of MT was not clearly demonstrated (see Fig. 4). 3.6. Complementary dissolution studies In previous toxicity studies, especially acute toxicity studies involving metal oxide NPs, some researchers concluded that the dissolution of NPs was responsible for the observed toxic effects on organisms (Gojova et al., 2007; Franklin et al., 2007; Aruoja et al., 2009; Xia et al., 2008). As a result, this study sought to establish whether the release of Cu and Zn metal ions could have contributed to the induction of biochemical changes in D. magna. The results (Table 2) showed that the proportion of the dissolved metal ions of these metal oxide NPs is much higher at lower particle concentration than at higher concentration. Similarly, the results in Table 2 showed big losses of metal ions in the final suspension with respect to the initial suspensions. The percentage loss of metal ions in the final suspension increased with increasing particle concentration, from 19% to 62% for ZnO NPs and from 75% to 90% for CuO NPs. The DLS measurements in MHW indicated massive aggregation for both metal oxide NPs (at 1 mg/L metal oxide NPs), such that the average aggregate sizes were outside the nano-range. Therefore, this loss was attributed to aggregation and eventual sedimentation of formed aggregates. The increase in the percentage loss with increase in particle loading was consistent with observations made by others (Amal et al. (1990), who observed that aggregation increased with increase in nanoparticle concentration in suspensions and could be attributed to the increased collision frequency and hence high aggregation rate. Considering the fact that the concentrations of NPs used in this whole study were relatively low (0.3 mg/L to 1.1 mg/L), it was expected that the proportions of the dissolved metal ions for both metal oxide NPs were even higher. This therefore, would suggest that metal ions play some significant role in the induction of biochemical changes observed in this study. This observation is similar with other observations that have attributed the contribution of metal ions to toxicity based on dissolution (Gojova et al., 2007; Franklin et al., 2007; Aruoja et al., 2009). However, more specific studies targeting the use of gene expression analysis are needed to distinguish metal ions exposure from that of metal oxide NPs. Ponyton et al. (2011) were able to demonstrate using gene expression analysis that both Zn ions and ZnO NPs contribute to toxic effects observed in D. magna, with distinct modes of action. 4. Conclusion The results of our study indicated that both CuO and ZnO NPs at sublethal concentrations can cause oxidative stress related biochemical changes to D. magna. The exposure caused a

dose-dependent decrease in GST activity, and dose-dependent increases in GSH oxidation, TBARS formation and MT induction. Modification of water hardness and organic matter concentrations appeared to influence the observed biomarker responses. The short-term complementary dissolution studies showed a concentration-dependent decrease in the proportion of dissolved metal ions from the metal oxide NPs. All these observations would point at the possibility that the observed toxicological effects are induced by both metal ions released from the metal oxide NPs and NPs themselves. However, more studies targeting the use genomic tools are needed to discriminate between toxicity due metal ions and NPs. The extent and nature of the induction of these biochemical changes can vary considerably between or among species. Thus, while in some cases up-regulation can be observed, in other cases inactivation would occur, as was demonstrated in this study. This calls for caution in the interpretation of cellular/biochemical results. The results suggest that the presence of the metal oxide NPs will interfere with the health of aquatic organisms by destabilizing and saturating their defense systems for natural oxidative stress, thereby making the organisms susceptible to opportunistic biotic and abiotic factors. The ultimate consequence would be a reduction in the stability of aquatic ecosystems. The observation that the presence of dissolved NOM drastically reduced some of these effects, has interesting positive ecological ramifications, especially because dissolved NOM is ubiquitous in aquatic systems. 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.2014.03.011. References Adams, L.K., Lyon, D.Y., Mcintosh, A., Alvarez, P.J.J., 2006. Comparative toxicity of nano-scale TiO2 , SiO2 and ZnO water suspensions. Water Toxicol. 54 (11–12), 327–334. Alhama, J., Romero-Ruiz, A., Lopezi-Barea, J., 2006. Metallothionein quantification in clams by reversed—phase high performance liquid chromatography coupled to fluorescence detection after monobromobimane derivatisation. J. Chromatogr. A 1107, 52–58. Aruoja, V., Dubourguier, H.C., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ., 1461–1468. Auffan, M., Rose, J., Bottero, J., Lowry, G.V., Jolivet, J., Wiesner, M.R., 2009. Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective. Nat. Technol. 4, 634–641. Amal, R., Rapper, J.A., Waite, T.D., 1990. Fractal structure of hematite aggregates. J. Colloid Interface Sci. 140 (1), 158–168. Barata, C.T., Varo, I., Navarro, J.C., Arun, S., Porte, C., 2005. Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox cycling compounds. Comp. Biochem. Physiol. C: Pharmacol. Toxicol. Endocrinol. 140, 175–186. Baun, A., Hartman, N.B., Grieger, K., Kusk, K.O., 2008. Ecotoxicity of engineered nanoparticles to aquatic invertebrates: a brief review and recommendations for future toxicity testing. Ecotoxicology 17, 387–395. Blinova, I., Ivask, A., Heinlaan, M., Mortimer, M., Kahru, A., 2010. Ecotoxicity of nanoparticles of CuO and ZnO in naturalwater. Environ. Pollut. 158, 41–47. Brayner, R., Ferrari-lliou, R., Brivois, N., Djediat, J., Benedetti, M.F., Fievet, F., 2006. Toxicological impact studies based on Escherichia coli bacteria in ultrafine ZnO nanoparticles colloidal medium. Nanotechnol. Lett. 6 (4), 866–870. Cai, R., Van, G.M., Aw, P.K., Itoh, K., 2006. Solar driven self-cleaning coating for a painted surface. R. R. Chimie 9, 829–835. Chen, J., Xiu, Z., Lowry, G.V., Alvarez, P.J.J., 2011. Effect of natural organic matter on toxicity and reactivity of nano-scale zero-valent iron. Water Res. 45, 1995–2001. Dallinger, R., Chabicovsky, M., Lagg, B., Schipflinger, R., Weirich, H.G., Berger, B., 2004. Isoform-specific quantification of metallothionein in the terrestrial gastropod helix pomatia. II. A differential biomarker approach under laboratory and field conditions. Environ. Toxicol. Chem. 23 (4), 902–910. De Schamphelaere, K.A.C., Vasconcelos, Tack, F.M.G., Allen, H.E., Janssen, C.R., 2004. Effect of dissolved organic matter source on acute copper toxicity to Daphnia magna. Environ. Toxicol. Chem. 23 (5), 1248–1255. Dua, P., Chaudhari, K.N., Lee, C.H., Chaudhari, N.K., Hong, S.W., Yu, J., Kim, S., Lee, D., 2012. Evaluation of toxicity and gene expression changes triggered by oxide nanoparticles. Bull. Korean Chem. Soc. 32 (6), 2051–2057.

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