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Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment
IMPACT-00024; No of Pages 6 NanoImpact xxx (2016) xxx–xxx
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Research paper
Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment Ning Gong a,b,⁎, Kuishuang Shao c, Guangyao Li a,b, Yeqing Sun a,b,⁎ a b c
Institute of Environmental Systems Biology, Dalian Maritime University, 1 Linghai Road, Dalian 116026, PR China College of Environmental Science and Engineering, Dalian Maritime University, Dalian 116026, PR China National Marine Environmental Monitoring Center, China, 42 Linghe Road, Dalian 116023, PR China
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Article history: Received 1 July 2016 Received in revised form 9 August 2016 Accepted 16 August 2016 Available online xxxx Keywords: Nickel oxide nanoparticles Daphnia magna Aqueous exposure Dietary exposure Algal enrichment
a b s t r a c t Among the emerging literature addressing the biological effects of nickel oxide nanoparticles (nNiO), very little information exists, particularly on aquatic organisms. And the extensive application of nNiO may result in their bio-transfer in a food chain through various routes in a freshwater ecosystem. In this study, the potential effects of nNiO on the water flea, Daphnia magna, were examined with 48-h acute toxicity tests and 21-day chronic assays. In addition, three exposure experiments representing different uptaking mechanisms (aqueous, environmental and dietary exposures) were performed. The acute test indicated that the 48-h LC50 was 36.79 (26.14– 56.72) mg/L and the toxicity increased in a time and dose dependent manner. Meanwhile, the chronic test demonstrated that reproduction was a more sensitive indicator of toxicity than mortality and the adult growth. The LOEC of 0.1 mg/L was obtained by the offspring number at first brood. There was a significant difference between the groups exposed through aqueous and dietary treatments (p b 0.05). Compared to the aqueous exposure, animal survival rates in environmental and dietary exposure groups decreased to 43.3% and 6.7% at 1 mg/L, respectively. Meanwhile, the proportion of animals that survived at 10 mg/L decreased to 23% and 10% for the environmental and dietary exposures, respectively. Dietary exposure group showed the lowest proportion of surviving individuals, indicating that ingestion of food (i.e. algae) contaminated by nanoparticles could be the efficient route for uptake of nNiO. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Rapid developments in the field of nanotechnology and the increasing use of nanoparticles (NPs) have raised concerns about their fate after disposal and their potential hazardous effects to the environment and human health. In the last few years, nickel oxide nanoparticles (nNiO) have been increasingly used for various applications such as a catalyst in gas sensors, alkaline battery cathodes, electro-chromic films, magnetic materials and fuel cells (Rao and Sunandana, 2008; Mu et al., 2011). Moreover, nNiO has been reported to be efficient in the photo catalytic degradation of phenol, removal of metals and metalloids, and in adsorption of dyes from wastewaters (Hayat et al., 2011; Khairy et al., 2012). Unfortunately, its potential effects to wildlife and organisms as it is released into the environment are still poorly understood. A few studies to understand the biological effects of nNiO to mammals and human cells have been done. For example, it has been showed that nNiO enhanced the cytokine gene expression of THP-1 cells (Horie et al., 2015) and induced apoptosis in human bronchial ⁎ Corresponding authors at: Institute of Environmental Systems Biology, Dalian Maritime University, 1 Linghai Road, Dalian 116026, PR China. E-mail addresses: [email protected] (N. Gong), [email protected] (Y. Sun).
epithelial cells (Duan et al., 2015). And recently, Faisal et al. (2013) reported NiO NPs induced apoptosis in tomato roots cells and triggered the release of caspase-3 proteases from mitochondria. However, to date, very few information is available focusing on the effects of nNiO toxicity on aquatic organisms (Gong et al., 2011; Oukarroum et al., 2015), especially the aquatic invertebrates (Nogueira et al., 2015). Daphnia magna, commonly known as a water flea, is a freshwater invertebrate that has been widely used as a model organism to conduct studies on aquatic nano-toxicology (Xiao et al., 2015). They play an important role in linking the primary producers (phytoplankton) as grazers and the higher trophic level as prey to fish and larger vertebrates, thus making them an important indicator of ecosystem change. However, most recent metal-based NPs toxicological studies on water flea primarily focused on the aqueous-phase exposures, and few so far has tested toxic effects by dietary intake of food-associated NPs (Dalai et al., 2014). Hence, the aim of this study is to evaluate the toxicity of nNiO with D. magna by acute immobilization and chronic reproduction tests. In addition, different exposure experiments (aqueous, environmental and dietary exposures) were performed to determine the toxic effects of nNiO through aqueous exposure and food-associated NPs ingestions. In aqueous exposure, the water flea ingested NPs mainly from water.
http://dx.doi.org/10.1016/j.impact.2016.08.003 2452-0748/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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Environmental exposure refers to the animal uptake up NPs from both water and dietary. And for dietary exposure, NPs ingestion was occurred principally through eating algae. The different toxic effects of above exposures indicated which one is the primary NPs uptake route to the water flea. 2. Materials and methods 2.1. Characterization of nickel oxide nanoparticles (nNiO) The powdered form of nNiO was purchased from Sigma-Aldrich, Inc., China (Cat. No. 637130), which had a diameter of b50 nm and 99.8% purity. Morphology and size of nNiO was also examined by a transmission electron microscope. In detail, A drop of the aqueous nNiO suspension was placed onto a carbon-coated copper grid, air-dried and observed with the TEM (JEM-2000FX, Japan) using an accelerating voltage of 120 KV. An initial stock solution of suspended nNiO was prepared by adding the powdered nNiO to Milli-Q water with a final concentration of 1 g/L with the aid of an ultrasonication for 30 min while in an ice bath. This process was repeated every time before the stock solution was used. To determine the size distribution of nNiO in toxic test media, hydrodynamic size of suspended nNiO (10 mg/L) with or without D. magna (10 individuals) was measured by dynamic light scattering (DLS) at 0 h, 24 h and 48 h using Zeta-sizer Nano-ZX90 (Malvern Instruments, Malvern, UK). 2.2. Test organism The freshwater crustacean D. magna was used as the model test organism. The animals were cultured in artificial M4 medium which was prepared according to Organization for Economic Co-operation and Development (OECD) 202 guidelines (OECD, 2004) and incubated at 20 ± 2°C with a 16:8 h light: dark photoperiod. The culture media were renewed twice a week and the daphnids were fed daily with unicellular green algae Pseudokirchneriella subcapitata (6 × 105 cells/mL). Algae were cultured with OECD 201 medium (OECD, 2006). Neonates were removed every 24 h. 2.3. Acute toxicity test Acute toxicity assays, particularly acute immobilization test, on D. magna were carried out following the methods described in the OECD Guideline 202 for testing of chemicals (OECD, 2004). Tests were performed in 60 mL sterile glass beakers. To test the effects of different concentrations of nNiO on Daphnia, culture set-ups or treatments containing 0 (negative control), 5, 10, 20, 40 and 80 mg/L of nNiO were prepared and randomly distributed at 20 ± 2°C with a 16:8 h light: dark photoperiod. Each treatment had 3 replicates. Then, 5 neonates (age b 24 h) were randomly placed in each beaker and not fed for the entire duration of the experiments. Organisms were specifically observed for mortality and/or immobilization after 48 h. Daphnia was considered immobile when they were not able to swim after 15 s of gentle stirring according to the guidelines. Morphologic observation was performed after 48 h with a stereoscopic microscope (Motic SMZ-168).
Immobilization of the parent animals and offspring production were both assessed daily. New neonates were counted and removed from the set-ups immediately after appearance of the first brood. Measured responses included individual mortality, offspring number at first brood, age of first brood, cumulative offspring number and the total number of broods. The body length of the parent animals (excluding the anal spine) was also measured using a stereoscopic microscope (Motic SMZ-168 with Motican 2506 camera) and the Image software (Motic Images Advanced 3.2). 2.5. Influence of algae enrichment To assess the effects of algal enrichment with nNiO on D. magna, the acute toxicity test using two concentrations of nNiO (1 and 10 mg/L) was conducted, each with triplicate and incubated in the same conditions in the preceding section. Specifically, 3 incubation set-ups were tried. The first one was an aqueous exposure (Exp 1), conducted using a modified 48-h acute toxicity test. It was performed in a 60 mL flatbottom glass vial. Each vial was filled with 30 mL M4 medium with different concentrations of nNiO at 0, 1 and 10 mg/L. Then, 10 neonates (age b 24 h) were randomly placed in each vial with 3 replicates. The daphnia were fed daily with the alga P. subcapitata (3 × 105 cells/mL) and individuals that survived were counted after 48-h. In Exp 2 (environmental exposure), the algae were enriched and incubated with the nNiO for 48 h to allow NPs attachment and/or internalization into the algae before potential ingestion of daphnia. Cultures of P. subcapitata with starting density of 3 × 105 cells/mL were cultivated in OECD 201 medium (OECD, 2006) in Erlenmeyer flasks. Final concentrations at 1 and 10 mg/L of nNiO were added to different culture flasks and incubated. Our previous study showed that the algae were not significantly affected by b10 mg/L nNiO and the absorption rate of algae reached the peak with 48-h nNiO incubation (Gong et al., 2011). After 48-h, the algal cultures incubated with nNiO were diluted to 3 × 105 cells/mL and adjusted to a final volume of 30 mL, and were separately added to ten D. magna neonates (age b 24 h) placed in a 60 mL flat-bottom glass. 3 replicates were conducted for each concentration. Animals that survived after 48 h were counted. In Exp3 (dietary exposure), the algal cultures were prepared as in Exp 2, but instead of adding the enrichments to daphnia after 48 h, the algal cells were washed with distilled water for 3 times and filtered with 0.45 μm Millipore membrane filter to remove the unbound nNiO. Algal cells were centrifuged at 1000 rpm for 5 min after every washing and re-suspension. After the final centrifugation, the algae were reconstituted with M4 medium and adjusted to 3 × 105 cells/mL before being added to 10 D. magna neonates (age b 24 h) placed in a 60 mL flatbottom glass vial. Each concentration has 3 replicates. Surviving animals were counted after 48 h. 2.6. Data analyses Data were presented as means ± standard deviations and one-way analysis of variance (ANOVA) was used to test for significant differences in the responses, implemented in Excel software. Values were considered significantly different when the probability (p) was b 0.05. The LC50 was calculated using the probit method in SPSS software (16.0).
2.4. Chronic toxicity test 3. Results The chronic toxicity of the nNiO was determined in a 21-day Daphnia reproduction test following OECD guideline 211(OECD, 2012). Test concentrations included 0, 0.1, 0.2, 0.5 and 1 mg/L of nNiO re-suspended in 20 mL M4 medium in 60 mL beaker with 10 replicates per treatment. Each replicate was added with one neonate (age b 24 h) and incubated at 20 ± 2 °C under a 16:8 h light: dark photoperiod. The test medium were replaced every 3 days and the daphnia were fed daily with the algae P. subcapitata at the culture concentration of 1 × 105 cells/mL.
3.1. Characterization of nNiO Fig. 1 shows the typical TEM image of nNiO. The average particle size of nNiO measured from the TEM images was around 22 ± 6 nm. DLS measurements are summarized in Table 1. Results indicated that the aggregate sizes of nNiO in test medium were N 300 nm after 24 h, and then it increased to 662.8 nm after 48 h. Polydispersity indexes were higher
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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immobilization endpoints (especially at 36-h and 48-h), the mortality rate of D. magna increased as the nNiO concentration also increased, and was significantly affected when the dose was N20 mg/L (p b 0.05). After exposure for 48-h, high nNiO concentrations of 80 mg/L caused nearly 90% mortality. Therefore, the acute toxicity of nNiO on daphnia was influenced by both time of exposure and dosage of the NPs. As a result, LC50 was 36.79 (26.14–56.72) mg/L at 48-h. D. magna observed under a stereoscopic microscope after 48 h exposure showed morphological deformities (Fig. 3). In control and lower dose group (5 mg/L), the bodies of daphnia were clear and the food residue in the digestive tract were almost invisible (Fig. 3A and B). In medium dose groups (10 and 20 mg/L), some of nNiO were found clearly retained in the digestive tract of daphnia but no obvious NPs aggregates adhered to their body (Fig. 3C and D). In higher dose groups (40 and 80 mg/L), however, NPs aggregates appeared clearly on both inside and outside of the body of the daphnia. In 80 mg/L group, residues containing nNiO surrounded the dead animal. These findings suggest that D. magna adsorbed NPs in water column and might fail to excrete all ingested nNiO, and the observed toxicity may be related to the accumulation of nNiO in the body. Fig. 1. The typical TEM image of NiO NPs. Scale bar = 50 nm.
3.3. The chronic toxicity test
than 0.5 in 4 out of 5 samples, which denoted the poor uniformity of particle size distribution of nNiO aggregates. The existence and activity of daphnia increased the hydrodynamic size of nNiO as well as the instability of nNiO in test medium.
No adult mortality occurred in all the groups over the 21-day testing period. Changes in growth measurements and reproductive output as measures of sub-lethal effects are summarized in Table 2. Results suggested that nNiO exposure had no apparent adverse effect on the growth of parent animals but significantly affected their reproduction. The age of first brood was significantly prolonged when exposed to 0.2 mg/L nNiO, while the offspring number of first brood increased significantly even at the low concentration of 0.1 mg/L (p b 0.05). The cumulative offspring number of each animal and the number of broods also decreased remarkably at 0.5 and 1 mg/L. Overall, reproductive capacity was affected in the groups treated with nNiO over 0.5 mg/L. Furthermore, the LOEC was nearly 0.1 mg/L as observed from the changes in the offspring number at first brood. These results revealed that exposure to nNiO even at low concentrations (i.e. 0.1 mg/L), can cause significant effects to D. magna at the population level, and thus, may have significant implications to the entire aquatic ecosystem.
3.2. Acute toxicity test
3.4. Influence of algae enrichment
During the acute toxicity tests, 100% of daphnia in the control survived, meeting the biological validity criterion required in the OECD guideline 202. Effects of the nNiO manifested as mortalities of D. magna in the acute toxicity test are shown in Fig. 2. Based on each
To evaluate acute toxic effects of algal-nNiO enrichment on D. magna, different exposure treatments (i.e. aqueous -Exp1, environmental-Exp2 and dietary exposure-Exp3) were performed and added with 1 and 10 mg/L of nNiO for 48 h. Survival of animals in these treatments is
Table 1 Nickel oxide (NiO) particles characterized by dynamic light scattering (DLS). Results are presented as mean ± SD. PDI⁎
Animals (n)
Time (h)
DLS Mean
SD
Mean
SD
0 0 0 10 10
0 24 48 24 48
349.4 329.4 546.9 406.1 662.8
29.5 17.1 10.9 14.9 14.1
0.67 0.48 0.73 0.65 0.96
0.02 0.02 0.04 0.02 0.06
⁎ PDI, polydispersity index.
Fig. 2. Summary of the mean mortality of Daphnia magna in the acute toxicity exposure of nNiO. Error bars represent standard deviation (SD) of the three replicates.
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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N. Gong et al. / NanoImpact xxx (2016) xxx–xxx
Fig. 3. Morphological observation of D. magna under 48 h nNiO exposures: A-F were 0, 5, 10, 20, 40 and 80 mg/L nNiO, respectively.
shown in Fig. 4. In both control groups (with 0 mg/L nNiO), no animal mortality (100% survival rate) was observed during the test. Interestingly, there was a remarkable difference between Exp1 and the other two groups (p b 0.05). Compared to Exp1 treatments, animal survival rates in Exp2 and Exp3 decreased to 43.3% and 6.7% at 1 mg/L, respectively.
While in 10 mg/L, the survival rates decreased to 23% and 10% for Exp2 and Exp3, respectively. Co-incubation of NPs with the algae also increased the toxicity of nNiO on daphnia dramatically at both dosages. Lastly, dietary exposure group (Exp3) showed the lowest survival in all exposures.
Table 2 Exposure concentrations and toxicity endpoints for Daphnia magna after exposure to nNiO for 21 days. Mean ± standard deviations (SD) are shown for surviving individuals. nNiO conc. (mg/L) 0 0.1 0.2 0.5 1
Body length of parent (mm) a
2.56 ± 0.09 2.52 ± 0.07a 2.53 ± 0.14a 2.58 ± 0.20a 2.53 ± 0.05a
Offspring number at first brood (n) a
2.9 ± 0.99 3.7 ± 1.25ab 4.5 ± 1.27b 4.6 ± 1.07b 3.8 ± 0.79b
Age at first brood (d) a
9.3 ± 0.67 9.8 ± 0.42a 9.8 ± 0.79ab 10.2 ± 0.42b 10.3 ± 0.48b
Cumulative offspring number (n) a
12 ± 1.76 11.5 ± 2.27a 11.7 ± 2.00a 7.6 ± 2.72b 4.2 ± 1.23b
Number of broods (n) 3.6 ± 0.70a 3.3 ± 0.67a 3.1 ± 0.74a 1.9 ± 0.57b 1.1 ± 0.32bc
Different letters in the same column indicate significant differences between treatments (analysis of variance with t-test, p b 0.05).
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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Fig. 4. The mean survival rate of D. magna in different nNiO treatments. Error bars represent standard deviation (SD) of the three replicates.
4. Discussion 4.1. nNiO toxicity to aquatic organisms NiO is a well-known toxic material, along with other nickel compounds, it is classified as Class 1 carcinogenic materials by the International Agency for Research on Cancer (IARC, 1997). In vivo studies (Horie et al., 2011; Morimoto et al., 2016) have demonstrated that nNiO could induce inflammation after intratracheal instillation in rat, and in vitro studies on A549 and JB6 cells (Capasso et al., 2014) showed increased toxicity in nNiO compared to its micro-sized particles. Siddiqui et al. (2012) also observed that nNiO induced oxidative stress in human cells, but could be reversed by the dietary antioxidant curcumin. However, the adverse effects of nNiO on aquatic organisms system have not been extensively studied since only very few data are available concerning their toxicity (Oukarroum et al., 2015). Recently, Nogueira et al. (2015) assessed the potential toxicity of NiO (100 and 10–20 nm) in several aquatic organisms including D. magna. They observed that nNiO appeared acute toxic to the daphnia, with EC50 ranging from 9.74 mg/L (24-h) to 14.6 mg/L (48-h). To date, this is the only published toxicity data of nNiO on daphnia aside from our study. In comparison, our results showed lower toxicity which could be due to differences in the NPs used in the two studies. In the present study, doses lower than 1 mg/L nNiO were used for the 21day chronic test, with 3 response variables including survival, growth and reproduction monitored. Present results indicated that reproduction was the most sensitive response variable relative to nNiO exposure. Our observations were similar to those reported by Nogueira et al. (2015), where they showed that fecundity was significantly affected with LOEC values ranging from 0.045 to 0.14 mg/L when they considered the effects of nNiO with daphnia. 4.2. Effects of nNiO exposure mode on Daphnia The water flea takes in NPs through drinking water and or dietary particulates. In addition, the NPs could also be absorbed by the exoskeleton and setae or through anus if in aqueous form (Ma and Lin, 2013). In this study, three different experiments were performed to determine the uptake routes of nNiO. In aqueous exposure (Exp 1), the algae and daphnia were simultaneously added into the medium with nNiO, which was designed to mimic the natural environmental conditions. In this condition, passive drinking and adsorption through the anus are the principal ways how animals accumulate NPs (Ma and Lin, 2013). Compared to the OECD acute test, the fresh algae were added during the Exp1 experiment. Dalai et al. (2014) believed that in this case, the presence and feeding on fresh algal cells might have helped
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eliminate NPs from the daphnia alimentary canal. In this context, the existence of algae helped reduce the toxicity of NPs. In environmental exposure treatment (Exp 2), the algal cells were initially co-incubated with the NPs for a period of 48 h. In our previous study (Gong et al., 2011), it has been proved that 48 h provided sufficient time for the algal cells to adsorb and/or internalize the NPs. Meanwhile, free NPs not adsorbed by the algae were still present in the medium. Therefore, in this scenario, the animals could ingest the nNiO in two ways, either by uptaking the algae that had adsorbed the NPs or by taking up the free NPs through filtering of the water. After the enrichment of nNiO, the local concentration of algae-NiO complex increased dramatically leading to the increased effective concentration acting on the daphnia when they eating contaminated algae. As a result, the toxic effects in Exp 2 and 3 were much higher than that in Exp1. It also suggested that dietary exposure was the primary route for nNiO adsorption in daphnia. In other words, if the food was contaminated by NPs (nNiO enriched with algae), the toxicity of NPs on water flea would increase remarkably. Dalai et al. (2014) drew similar conclusions when they investigated the toxicity of nano-TiO2 adsorption through different modes in Ceriodaphnia dubia. In order to test the results of Exp-2, the last treatment (dietary exposure- Exp 3) was performed, where the algae washed after enrichment with nNiO before feeding to the daphnia. In this set-up, part of free NPs has been removed and the nNiO was only adsorbed via eating the contaminated algae. Accordingly, the effective concentration of nNiO on the animals reached the highest among the three set-ups, which induced the highest toxicity as expected. It further indicated that dietary exposure is the most efficient way for NPs adsorption and the enrichment of NPs enhanced the effective exposure concentration for daphnia thus caused the highest toxicity. Dalai et al. (2014) also indicated that the dietary uptake of nano-TiO2 (algae-associated) was the primary route for NPs bio-transfer in water flea, with NPs uptake to as high ∼70%. The NPs coated with algal exudates were easily taken up by daphnia as compared to free NPs of the same concentrations, leading to their higher bioaccumulation. Therefore, the dietary pathway is the main mechanism by which NPs could be accumulated in D. magna. 5. Conclusion The current study focuses on the acute and chronic toxic effects of nNiO in daphnia as well as the different modes of nNiO transfer across a food chain from a primary producer (algae as the prey) and a primary consumer (daphnia as the predator). Toxicity of nNiO increased with time of exposure and dosage. Chronic exposure of nNiO also resulted to decreased reproduction capacity of the daphnia. Moreover, the trophic transfer through dietary sources was found to be most efficient, especially when algal cells were enriched with nNiO. It would be worthwhile to study in detail the different modes of trophic transfer of different nanomaterials to evaluate the degree of toxicity as well as their bioaccumulation in the aquatic environment. Acknowledgements The study was financially supported by National Natural Science Foundation of China (41301560) and Regional Demonstration of Marine Economy Innovative Development Project of China (No. 12PYY001SF08). References Capasso, L., Camatini, M., Gualtieri, M., 2014. Nickel oxide nanoparticles induce inflammation and genotoxic effect in lung epithelial cells. Toxicol. Lett. 226 (1), 28–34. Dalai, S., Iswarya, V., Bhuvaneshwari, M., Pakrashi, S., Chandrasekaran, N., Mukherjee, A., 2014. Different modes of TiO2 uptake by Ceriodaphnia dubia: relevance to toxicity and bioaccumulation. Aquat. Toxicol. 152, 139–146. Duan, W.X., He, M.D., Mao, L., Qian, F.H., Li, Y.M., Pi, H.F., Liu, C., Chen, C.H., Lu, Y.H., Cao, Z.W., Zhang, L., Yu, Z.P., Zhou, Z., 2015. NiO nanoparticles induce apoptosis through repressing SIRT1 in human bronchial epithelial cells. Toxicol. Appl. Pharmacol. 286 (2), 80–91.
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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N. Gong et al. / NanoImpact xxx (2016) xxx–xxx
Faisal, M., Saquib, Q., Alatar, A.A., Al-Khedhairy, A.A., Hegazy, A.K., Musarrat, J., 2013. Phytotoxic hazards of NiO-nanoparticles in tomato: a study on mechanism of cell death. J. Hazard. Mater. 250–251, 318–332. Gong, N., Shao, K., Feng, W., Lin, Z., Liang, C., Sun, Y., 2011. Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris. Chemosphere 83 (4), 510–516. Hayat, K., Gondal, M.A., Khaled, M.M., Ahmed, S., 2011. Effect of operational key parameters on photocatalytic degradation of phenol using nano nickel oxide synthesized by sol–gel method. J. Mol. Catal. A Chem. 336 (1–2), 64–71. Horie, M., Fukui, H., Nishio, K., Endoh, S., Kato, H., Fujita, K., Miyauchi, A., Nakamura, A., Shichiri, M., Ishida, N., 2011. Evaluation of acute oxidative stress induced by NiO nanoparticles in vivo and in vitro. J.Occup.Health. 53, 64–74. Horie, M., Nishio, K., Kato, H., Endoh, S., Fujita, K., Nakamura, A., Miyauchi, A., Kinugasa, S., Hagihara, Y., Yoshida, Y., Iwahashi, H., 2015. The expression of inflammatory cytokine and heme oxygenase-1 genes in THP-1 cells exposed to metal oxide nanoparticles. J. Nanopart. Res. 30, 116–127. IARC, 1997. Monographs on the evaluation of carcinogenic risks to humans: Chromium, nickel and welding. World Health Organization International Agency for Research on Cancer, p. 49. Khairy, M., El-Safty, S.A., Ismael, M., Kawarada, H., 2012. Mesoporous nio nanomagnets as catalysts and separators of chemical agents. Appl. Catal. B Environ. 127, 1–10. Ma, S., Lin, D., 2013. The biophysicochemical interactions at the interfaces between nanoparticles and aquatic organisms: adsorption and internalization. Environ. Sci.Process Impacts. 15 (1), 145–160. Morimoto, Y., Izumi, H., Yoshiura, Y., Tomonaga, T., Lee, B.W., Okada, T., Oyabu, T., Myojo, T., Kawai, K., Yatera, K., Shimada, M., Kubo, M., Yamamoto, K., Kitajima, S., Kuroda, E., Horie, M., Kawaguchi, K., Sasaki, T., 2016. Comparison of pulmonary inflammatory
responses following intratracheal instillation and inhalation of nanoparticles. Nanotoxicology 10 (5), 607–618. Mu, Y., Jia, D., He, Y., Miao, Y., Wub, H.L., 2011. Nano nickel oxide modified nonenzymatic glucose sensors with enhanced sensitivity through an electrochemical process strategy at high potential. Biotechnol. Bioeng. 26, 2948–2952. Nogueira, V., Lopes, I., Rocha-Santos, T.A., Rasteiro, M.G., Abrantes, N., Goncalves, F., Soares, A.M., Duarte, A.C., Pereira, R., 2015. Assessing the ecotoxicity of metal nanooxides with potential for wastewater treatment. Environ. Sci. Pollut. Res. Int. 22 (17), 13212–13224. Organization for Economic Cooperation and Development (OECD), 2004D. OECD Guideline for Testing of Chemicals. Daphnia sp., Acute Immobilisation Test. OECD 202, Paris, France. Organization for Economic Cooperation and Development (OECD), 2006D. OECD Guidelinesfor the Testing of Chemicals. OECD 201. France, Paris. Organization for Economic Cooperation and Development (OECD), 2012D. OECD Guidelines for Testing of Chemicals. Daphnia magna Reproduction Test. OECD211, Paris, France. Oukarroum, A., Barhoumi, L., Samadani, M., Dewez, D., 2015. Toxic effects of nickel oxide bulk and nanoparticles on the aquatic plant Lemna gibbaL. Biomed Res Int. 2015, 1–7. Rao, K.V., Sunandana, C.S., 2008. Effect of fuel to oxidizer ratio on the structure, micro structure and epr of combustion synthesized nio nanoparticles. J. Nanosci. Nanotechnol. 8 (8), 4247–4253. Siddiqui, M.A., Ahamed, M., Ahmad, J., Majeed Khan, M.A., Musarrat, J., Al-Khedhairy, A.A., Alrokayan, S.A., 2012. Nickel oxide nanoparticles induce cytotoxicity, oxidative stress and apoptosis in cultured human cells that is abrogated by the dietary antioxidant curcumin. Food Chem. Toxicol. 50 (3–4), 641–647. Xiao, Y., Vijver, M.G., Chen, G., Peijnenburg, W.J., 2015. Toxicity and accumulation of Cu and ZnO nanoparticles in Daphnia magna. Environ. Sci. Technol. 49 (7), 4657–4664.
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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Research paper
Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment Ning Gong a,b,⁎, Kuishuang Shao c, Guangyao Li a,b, Yeqing Sun a,b,⁎ a b c
Institute of Environmental Systems Biology, Dalian Maritime University, 1 Linghai Road, Dalian 116026, PR China College of Environmental Science and Engineering, Dalian Maritime University, Dalian 116026, PR China National Marine Environmental Monitoring Center, China, 42 Linghe Road, Dalian 116023, PR China
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Article history: Received 1 July 2016 Received in revised form 9 August 2016 Accepted 16 August 2016 Available online xxxx Keywords: Nickel oxide nanoparticles Daphnia magna Aqueous exposure Dietary exposure Algal enrichment
a b s t r a c t Among the emerging literature addressing the biological effects of nickel oxide nanoparticles (nNiO), very little information exists, particularly on aquatic organisms. And the extensive application of nNiO may result in their bio-transfer in a food chain through various routes in a freshwater ecosystem. In this study, the potential effects of nNiO on the water flea, Daphnia magna, were examined with 48-h acute toxicity tests and 21-day chronic assays. In addition, three exposure experiments representing different uptaking mechanisms (aqueous, environmental and dietary exposures) were performed. The acute test indicated that the 48-h LC50 was 36.79 (26.14– 56.72) mg/L and the toxicity increased in a time and dose dependent manner. Meanwhile, the chronic test demonstrated that reproduction was a more sensitive indicator of toxicity than mortality and the adult growth. The LOEC of 0.1 mg/L was obtained by the offspring number at first brood. There was a significant difference between the groups exposed through aqueous and dietary treatments (p b 0.05). Compared to the aqueous exposure, animal survival rates in environmental and dietary exposure groups decreased to 43.3% and 6.7% at 1 mg/L, respectively. Meanwhile, the proportion of animals that survived at 10 mg/L decreased to 23% and 10% for the environmental and dietary exposures, respectively. Dietary exposure group showed the lowest proportion of surviving individuals, indicating that ingestion of food (i.e. algae) contaminated by nanoparticles could be the efficient route for uptake of nNiO. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Rapid developments in the field of nanotechnology and the increasing use of nanoparticles (NPs) have raised concerns about their fate after disposal and their potential hazardous effects to the environment and human health. In the last few years, nickel oxide nanoparticles (nNiO) have been increasingly used for various applications such as a catalyst in gas sensors, alkaline battery cathodes, electro-chromic films, magnetic materials and fuel cells (Rao and Sunandana, 2008; Mu et al., 2011). Moreover, nNiO has been reported to be efficient in the photo catalytic degradation of phenol, removal of metals and metalloids, and in adsorption of dyes from wastewaters (Hayat et al., 2011; Khairy et al., 2012). Unfortunately, its potential effects to wildlife and organisms as it is released into the environment are still poorly understood. A few studies to understand the biological effects of nNiO to mammals and human cells have been done. For example, it has been showed that nNiO enhanced the cytokine gene expression of THP-1 cells (Horie et al., 2015) and induced apoptosis in human bronchial ⁎ Corresponding authors at: Institute of Environmental Systems Biology, Dalian Maritime University, 1 Linghai Road, Dalian 116026, PR China. E-mail addresses: [email protected] (N. Gong), [email protected] (Y. Sun).
epithelial cells (Duan et al., 2015). And recently, Faisal et al. (2013) reported NiO NPs induced apoptosis in tomato roots cells and triggered the release of caspase-3 proteases from mitochondria. However, to date, very few information is available focusing on the effects of nNiO toxicity on aquatic organisms (Gong et al., 2011; Oukarroum et al., 2015), especially the aquatic invertebrates (Nogueira et al., 2015). Daphnia magna, commonly known as a water flea, is a freshwater invertebrate that has been widely used as a model organism to conduct studies on aquatic nano-toxicology (Xiao et al., 2015). They play an important role in linking the primary producers (phytoplankton) as grazers and the higher trophic level as prey to fish and larger vertebrates, thus making them an important indicator of ecosystem change. However, most recent metal-based NPs toxicological studies on water flea primarily focused on the aqueous-phase exposures, and few so far has tested toxic effects by dietary intake of food-associated NPs (Dalai et al., 2014). Hence, the aim of this study is to evaluate the toxicity of nNiO with D. magna by acute immobilization and chronic reproduction tests. In addition, different exposure experiments (aqueous, environmental and dietary exposures) were performed to determine the toxic effects of nNiO through aqueous exposure and food-associated NPs ingestions. In aqueous exposure, the water flea ingested NPs mainly from water.
http://dx.doi.org/10.1016/j.impact.2016.08.003 2452-0748/© 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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Environmental exposure refers to the animal uptake up NPs from both water and dietary. And for dietary exposure, NPs ingestion was occurred principally through eating algae. The different toxic effects of above exposures indicated which one is the primary NPs uptake route to the water flea. 2. Materials and methods 2.1. Characterization of nickel oxide nanoparticles (nNiO) The powdered form of nNiO was purchased from Sigma-Aldrich, Inc., China (Cat. No. 637130), which had a diameter of b50 nm and 99.8% purity. Morphology and size of nNiO was also examined by a transmission electron microscope. In detail, A drop of the aqueous nNiO suspension was placed onto a carbon-coated copper grid, air-dried and observed with the TEM (JEM-2000FX, Japan) using an accelerating voltage of 120 KV. An initial stock solution of suspended nNiO was prepared by adding the powdered nNiO to Milli-Q water with a final concentration of 1 g/L with the aid of an ultrasonication for 30 min while in an ice bath. This process was repeated every time before the stock solution was used. To determine the size distribution of nNiO in toxic test media, hydrodynamic size of suspended nNiO (10 mg/L) with or without D. magna (10 individuals) was measured by dynamic light scattering (DLS) at 0 h, 24 h and 48 h using Zeta-sizer Nano-ZX90 (Malvern Instruments, Malvern, UK). 2.2. Test organism The freshwater crustacean D. magna was used as the model test organism. The animals were cultured in artificial M4 medium which was prepared according to Organization for Economic Co-operation and Development (OECD) 202 guidelines (OECD, 2004) and incubated at 20 ± 2°C with a 16:8 h light: dark photoperiod. The culture media were renewed twice a week and the daphnids were fed daily with unicellular green algae Pseudokirchneriella subcapitata (6 × 105 cells/mL). Algae were cultured with OECD 201 medium (OECD, 2006). Neonates were removed every 24 h. 2.3. Acute toxicity test Acute toxicity assays, particularly acute immobilization test, on D. magna were carried out following the methods described in the OECD Guideline 202 for testing of chemicals (OECD, 2004). Tests were performed in 60 mL sterile glass beakers. To test the effects of different concentrations of nNiO on Daphnia, culture set-ups or treatments containing 0 (negative control), 5, 10, 20, 40 and 80 mg/L of nNiO were prepared and randomly distributed at 20 ± 2°C with a 16:8 h light: dark photoperiod. Each treatment had 3 replicates. Then, 5 neonates (age b 24 h) were randomly placed in each beaker and not fed for the entire duration of the experiments. Organisms were specifically observed for mortality and/or immobilization after 48 h. Daphnia was considered immobile when they were not able to swim after 15 s of gentle stirring according to the guidelines. Morphologic observation was performed after 48 h with a stereoscopic microscope (Motic SMZ-168).
Immobilization of the parent animals and offspring production were both assessed daily. New neonates were counted and removed from the set-ups immediately after appearance of the first brood. Measured responses included individual mortality, offspring number at first brood, age of first brood, cumulative offspring number and the total number of broods. The body length of the parent animals (excluding the anal spine) was also measured using a stereoscopic microscope (Motic SMZ-168 with Motican 2506 camera) and the Image software (Motic Images Advanced 3.2). 2.5. Influence of algae enrichment To assess the effects of algal enrichment with nNiO on D. magna, the acute toxicity test using two concentrations of nNiO (1 and 10 mg/L) was conducted, each with triplicate and incubated in the same conditions in the preceding section. Specifically, 3 incubation set-ups were tried. The first one was an aqueous exposure (Exp 1), conducted using a modified 48-h acute toxicity test. It was performed in a 60 mL flatbottom glass vial. Each vial was filled with 30 mL M4 medium with different concentrations of nNiO at 0, 1 and 10 mg/L. Then, 10 neonates (age b 24 h) were randomly placed in each vial with 3 replicates. The daphnia were fed daily with the alga P. subcapitata (3 × 105 cells/mL) and individuals that survived were counted after 48-h. In Exp 2 (environmental exposure), the algae were enriched and incubated with the nNiO for 48 h to allow NPs attachment and/or internalization into the algae before potential ingestion of daphnia. Cultures of P. subcapitata with starting density of 3 × 105 cells/mL were cultivated in OECD 201 medium (OECD, 2006) in Erlenmeyer flasks. Final concentrations at 1 and 10 mg/L of nNiO were added to different culture flasks and incubated. Our previous study showed that the algae were not significantly affected by b10 mg/L nNiO and the absorption rate of algae reached the peak with 48-h nNiO incubation (Gong et al., 2011). After 48-h, the algal cultures incubated with nNiO were diluted to 3 × 105 cells/mL and adjusted to a final volume of 30 mL, and were separately added to ten D. magna neonates (age b 24 h) placed in a 60 mL flat-bottom glass. 3 replicates were conducted for each concentration. Animals that survived after 48 h were counted. In Exp3 (dietary exposure), the algal cultures were prepared as in Exp 2, but instead of adding the enrichments to daphnia after 48 h, the algal cells were washed with distilled water for 3 times and filtered with 0.45 μm Millipore membrane filter to remove the unbound nNiO. Algal cells were centrifuged at 1000 rpm for 5 min after every washing and re-suspension. After the final centrifugation, the algae were reconstituted with M4 medium and adjusted to 3 × 105 cells/mL before being added to 10 D. magna neonates (age b 24 h) placed in a 60 mL flatbottom glass vial. Each concentration has 3 replicates. Surviving animals were counted after 48 h. 2.6. Data analyses Data were presented as means ± standard deviations and one-way analysis of variance (ANOVA) was used to test for significant differences in the responses, implemented in Excel software. Values were considered significantly different when the probability (p) was b 0.05. The LC50 was calculated using the probit method in SPSS software (16.0).
2.4. Chronic toxicity test 3. Results The chronic toxicity of the nNiO was determined in a 21-day Daphnia reproduction test following OECD guideline 211(OECD, 2012). Test concentrations included 0, 0.1, 0.2, 0.5 and 1 mg/L of nNiO re-suspended in 20 mL M4 medium in 60 mL beaker with 10 replicates per treatment. Each replicate was added with one neonate (age b 24 h) and incubated at 20 ± 2 °C under a 16:8 h light: dark photoperiod. The test medium were replaced every 3 days and the daphnia were fed daily with the algae P. subcapitata at the culture concentration of 1 × 105 cells/mL.
3.1. Characterization of nNiO Fig. 1 shows the typical TEM image of nNiO. The average particle size of nNiO measured from the TEM images was around 22 ± 6 nm. DLS measurements are summarized in Table 1. Results indicated that the aggregate sizes of nNiO in test medium were N 300 nm after 24 h, and then it increased to 662.8 nm after 48 h. Polydispersity indexes were higher
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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immobilization endpoints (especially at 36-h and 48-h), the mortality rate of D. magna increased as the nNiO concentration also increased, and was significantly affected when the dose was N20 mg/L (p b 0.05). After exposure for 48-h, high nNiO concentrations of 80 mg/L caused nearly 90% mortality. Therefore, the acute toxicity of nNiO on daphnia was influenced by both time of exposure and dosage of the NPs. As a result, LC50 was 36.79 (26.14–56.72) mg/L at 48-h. D. magna observed under a stereoscopic microscope after 48 h exposure showed morphological deformities (Fig. 3). In control and lower dose group (5 mg/L), the bodies of daphnia were clear and the food residue in the digestive tract were almost invisible (Fig. 3A and B). In medium dose groups (10 and 20 mg/L), some of nNiO were found clearly retained in the digestive tract of daphnia but no obvious NPs aggregates adhered to their body (Fig. 3C and D). In higher dose groups (40 and 80 mg/L), however, NPs aggregates appeared clearly on both inside and outside of the body of the daphnia. In 80 mg/L group, residues containing nNiO surrounded the dead animal. These findings suggest that D. magna adsorbed NPs in water column and might fail to excrete all ingested nNiO, and the observed toxicity may be related to the accumulation of nNiO in the body. Fig. 1. The typical TEM image of NiO NPs. Scale bar = 50 nm.
3.3. The chronic toxicity test
than 0.5 in 4 out of 5 samples, which denoted the poor uniformity of particle size distribution of nNiO aggregates. The existence and activity of daphnia increased the hydrodynamic size of nNiO as well as the instability of nNiO in test medium.
No adult mortality occurred in all the groups over the 21-day testing period. Changes in growth measurements and reproductive output as measures of sub-lethal effects are summarized in Table 2. Results suggested that nNiO exposure had no apparent adverse effect on the growth of parent animals but significantly affected their reproduction. The age of first brood was significantly prolonged when exposed to 0.2 mg/L nNiO, while the offspring number of first brood increased significantly even at the low concentration of 0.1 mg/L (p b 0.05). The cumulative offspring number of each animal and the number of broods also decreased remarkably at 0.5 and 1 mg/L. Overall, reproductive capacity was affected in the groups treated with nNiO over 0.5 mg/L. Furthermore, the LOEC was nearly 0.1 mg/L as observed from the changes in the offspring number at first brood. These results revealed that exposure to nNiO even at low concentrations (i.e. 0.1 mg/L), can cause significant effects to D. magna at the population level, and thus, may have significant implications to the entire aquatic ecosystem.
3.2. Acute toxicity test
3.4. Influence of algae enrichment
During the acute toxicity tests, 100% of daphnia in the control survived, meeting the biological validity criterion required in the OECD guideline 202. Effects of the nNiO manifested as mortalities of D. magna in the acute toxicity test are shown in Fig. 2. Based on each
To evaluate acute toxic effects of algal-nNiO enrichment on D. magna, different exposure treatments (i.e. aqueous -Exp1, environmental-Exp2 and dietary exposure-Exp3) were performed and added with 1 and 10 mg/L of nNiO for 48 h. Survival of animals in these treatments is
Table 1 Nickel oxide (NiO) particles characterized by dynamic light scattering (DLS). Results are presented as mean ± SD. PDI⁎
Animals (n)
Time (h)
DLS Mean
SD
Mean
SD
0 0 0 10 10
0 24 48 24 48
349.4 329.4 546.9 406.1 662.8
29.5 17.1 10.9 14.9 14.1
0.67 0.48 0.73 0.65 0.96
0.02 0.02 0.04 0.02 0.06
⁎ PDI, polydispersity index.
Fig. 2. Summary of the mean mortality of Daphnia magna in the acute toxicity exposure of nNiO. Error bars represent standard deviation (SD) of the three replicates.
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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Fig. 3. Morphological observation of D. magna under 48 h nNiO exposures: A-F were 0, 5, 10, 20, 40 and 80 mg/L nNiO, respectively.
shown in Fig. 4. In both control groups (with 0 mg/L nNiO), no animal mortality (100% survival rate) was observed during the test. Interestingly, there was a remarkable difference between Exp1 and the other two groups (p b 0.05). Compared to Exp1 treatments, animal survival rates in Exp2 and Exp3 decreased to 43.3% and 6.7% at 1 mg/L, respectively.
While in 10 mg/L, the survival rates decreased to 23% and 10% for Exp2 and Exp3, respectively. Co-incubation of NPs with the algae also increased the toxicity of nNiO on daphnia dramatically at both dosages. Lastly, dietary exposure group (Exp3) showed the lowest survival in all exposures.
Table 2 Exposure concentrations and toxicity endpoints for Daphnia magna after exposure to nNiO for 21 days. Mean ± standard deviations (SD) are shown for surviving individuals. nNiO conc. (mg/L) 0 0.1 0.2 0.5 1
Body length of parent (mm) a
2.56 ± 0.09 2.52 ± 0.07a 2.53 ± 0.14a 2.58 ± 0.20a 2.53 ± 0.05a
Offspring number at first brood (n) a
2.9 ± 0.99 3.7 ± 1.25ab 4.5 ± 1.27b 4.6 ± 1.07b 3.8 ± 0.79b
Age at first brood (d) a
9.3 ± 0.67 9.8 ± 0.42a 9.8 ± 0.79ab 10.2 ± 0.42b 10.3 ± 0.48b
Cumulative offspring number (n) a
12 ± 1.76 11.5 ± 2.27a 11.7 ± 2.00a 7.6 ± 2.72b 4.2 ± 1.23b
Number of broods (n) 3.6 ± 0.70a 3.3 ± 0.67a 3.1 ± 0.74a 1.9 ± 0.57b 1.1 ± 0.32bc
Different letters in the same column indicate significant differences between treatments (analysis of variance with t-test, p b 0.05).
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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Fig. 4. The mean survival rate of D. magna in different nNiO treatments. Error bars represent standard deviation (SD) of the three replicates.
4. Discussion 4.1. nNiO toxicity to aquatic organisms NiO is a well-known toxic material, along with other nickel compounds, it is classified as Class 1 carcinogenic materials by the International Agency for Research on Cancer (IARC, 1997). In vivo studies (Horie et al., 2011; Morimoto et al., 2016) have demonstrated that nNiO could induce inflammation after intratracheal instillation in rat, and in vitro studies on A549 and JB6 cells (Capasso et al., 2014) showed increased toxicity in nNiO compared to its micro-sized particles. Siddiqui et al. (2012) also observed that nNiO induced oxidative stress in human cells, but could be reversed by the dietary antioxidant curcumin. However, the adverse effects of nNiO on aquatic organisms system have not been extensively studied since only very few data are available concerning their toxicity (Oukarroum et al., 2015). Recently, Nogueira et al. (2015) assessed the potential toxicity of NiO (100 and 10–20 nm) in several aquatic organisms including D. magna. They observed that nNiO appeared acute toxic to the daphnia, with EC50 ranging from 9.74 mg/L (24-h) to 14.6 mg/L (48-h). To date, this is the only published toxicity data of nNiO on daphnia aside from our study. In comparison, our results showed lower toxicity which could be due to differences in the NPs used in the two studies. In the present study, doses lower than 1 mg/L nNiO were used for the 21day chronic test, with 3 response variables including survival, growth and reproduction monitored. Present results indicated that reproduction was the most sensitive response variable relative to nNiO exposure. Our observations were similar to those reported by Nogueira et al. (2015), where they showed that fecundity was significantly affected with LOEC values ranging from 0.045 to 0.14 mg/L when they considered the effects of nNiO with daphnia. 4.2. Effects of nNiO exposure mode on Daphnia The water flea takes in NPs through drinking water and or dietary particulates. In addition, the NPs could also be absorbed by the exoskeleton and setae or through anus if in aqueous form (Ma and Lin, 2013). In this study, three different experiments were performed to determine the uptake routes of nNiO. In aqueous exposure (Exp 1), the algae and daphnia were simultaneously added into the medium with nNiO, which was designed to mimic the natural environmental conditions. In this condition, passive drinking and adsorption through the anus are the principal ways how animals accumulate NPs (Ma and Lin, 2013). Compared to the OECD acute test, the fresh algae were added during the Exp1 experiment. Dalai et al. (2014) believed that in this case, the presence and feeding on fresh algal cells might have helped
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eliminate NPs from the daphnia alimentary canal. In this context, the existence of algae helped reduce the toxicity of NPs. In environmental exposure treatment (Exp 2), the algal cells were initially co-incubated with the NPs for a period of 48 h. In our previous study (Gong et al., 2011), it has been proved that 48 h provided sufficient time for the algal cells to adsorb and/or internalize the NPs. Meanwhile, free NPs not adsorbed by the algae were still present in the medium. Therefore, in this scenario, the animals could ingest the nNiO in two ways, either by uptaking the algae that had adsorbed the NPs or by taking up the free NPs through filtering of the water. After the enrichment of nNiO, the local concentration of algae-NiO complex increased dramatically leading to the increased effective concentration acting on the daphnia when they eating contaminated algae. As a result, the toxic effects in Exp 2 and 3 were much higher than that in Exp1. It also suggested that dietary exposure was the primary route for nNiO adsorption in daphnia. In other words, if the food was contaminated by NPs (nNiO enriched with algae), the toxicity of NPs on water flea would increase remarkably. Dalai et al. (2014) drew similar conclusions when they investigated the toxicity of nano-TiO2 adsorption through different modes in Ceriodaphnia dubia. In order to test the results of Exp-2, the last treatment (dietary exposure- Exp 3) was performed, where the algae washed after enrichment with nNiO before feeding to the daphnia. In this set-up, part of free NPs has been removed and the nNiO was only adsorbed via eating the contaminated algae. Accordingly, the effective concentration of nNiO on the animals reached the highest among the three set-ups, which induced the highest toxicity as expected. It further indicated that dietary exposure is the most efficient way for NPs adsorption and the enrichment of NPs enhanced the effective exposure concentration for daphnia thus caused the highest toxicity. Dalai et al. (2014) also indicated that the dietary uptake of nano-TiO2 (algae-associated) was the primary route for NPs bio-transfer in water flea, with NPs uptake to as high ∼70%. The NPs coated with algal exudates were easily taken up by daphnia as compared to free NPs of the same concentrations, leading to their higher bioaccumulation. Therefore, the dietary pathway is the main mechanism by which NPs could be accumulated in D. magna. 5. Conclusion The current study focuses on the acute and chronic toxic effects of nNiO in daphnia as well as the different modes of nNiO transfer across a food chain from a primary producer (algae as the prey) and a primary consumer (daphnia as the predator). Toxicity of nNiO increased with time of exposure and dosage. Chronic exposure of nNiO also resulted to decreased reproduction capacity of the daphnia. Moreover, the trophic transfer through dietary sources was found to be most efficient, especially when algal cells were enriched with nNiO. It would be worthwhile to study in detail the different modes of trophic transfer of different nanomaterials to evaluate the degree of toxicity as well as their bioaccumulation in the aquatic environment. Acknowledgements The study was financially supported by National Natural Science Foundation of China (41301560) and Regional Demonstration of Marine Economy Innovative Development Project of China (No. 12PYY001SF08). References Capasso, L., Camatini, M., Gualtieri, M., 2014. Nickel oxide nanoparticles induce inflammation and genotoxic effect in lung epithelial cells. Toxicol. Lett. 226 (1), 28–34. Dalai, S., Iswarya, V., Bhuvaneshwari, M., Pakrashi, S., Chandrasekaran, N., Mukherjee, A., 2014. Different modes of TiO2 uptake by Ceriodaphnia dubia: relevance to toxicity and bioaccumulation. Aquat. Toxicol. 152, 139–146. Duan, W.X., He, M.D., Mao, L., Qian, F.H., Li, Y.M., Pi, H.F., Liu, C., Chen, C.H., Lu, Y.H., Cao, Z.W., Zhang, L., Yu, Z.P., Zhou, Z., 2015. NiO nanoparticles induce apoptosis through repressing SIRT1 in human bronchial epithelial cells. Toxicol. Appl. Pharmacol. 286 (2), 80–91.
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003
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N. Gong et al. / NanoImpact xxx (2016) xxx–xxx
Faisal, M., Saquib, Q., Alatar, A.A., Al-Khedhairy, A.A., Hegazy, A.K., Musarrat, J., 2013. Phytotoxic hazards of NiO-nanoparticles in tomato: a study on mechanism of cell death. J. Hazard. Mater. 250–251, 318–332. Gong, N., Shao, K., Feng, W., Lin, Z., Liang, C., Sun, Y., 2011. Biotoxicity of nickel oxide nanoparticles and bio-remediation by microalgae Chlorella vulgaris. Chemosphere 83 (4), 510–516. Hayat, K., Gondal, M.A., Khaled, M.M., Ahmed, S., 2011. Effect of operational key parameters on photocatalytic degradation of phenol using nano nickel oxide synthesized by sol–gel method. J. Mol. Catal. A Chem. 336 (1–2), 64–71. Horie, M., Fukui, H., Nishio, K., Endoh, S., Kato, H., Fujita, K., Miyauchi, A., Nakamura, A., Shichiri, M., Ishida, N., 2011. Evaluation of acute oxidative stress induced by NiO nanoparticles in vivo and in vitro. J.Occup.Health. 53, 64–74. Horie, M., Nishio, K., Kato, H., Endoh, S., Fujita, K., Nakamura, A., Miyauchi, A., Kinugasa, S., Hagihara, Y., Yoshida, Y., Iwahashi, H., 2015. The expression of inflammatory cytokine and heme oxygenase-1 genes in THP-1 cells exposed to metal oxide nanoparticles. J. Nanopart. Res. 30, 116–127. IARC, 1997. Monographs on the evaluation of carcinogenic risks to humans: Chromium, nickel and welding. World Health Organization International Agency for Research on Cancer, p. 49. Khairy, M., El-Safty, S.A., Ismael, M., Kawarada, H., 2012. Mesoporous nio nanomagnets as catalysts and separators of chemical agents. Appl. Catal. B Environ. 127, 1–10. Ma, S., Lin, D., 2013. The biophysicochemical interactions at the interfaces between nanoparticles and aquatic organisms: adsorption and internalization. Environ. Sci.Process Impacts. 15 (1), 145–160. Morimoto, Y., Izumi, H., Yoshiura, Y., Tomonaga, T., Lee, B.W., Okada, T., Oyabu, T., Myojo, T., Kawai, K., Yatera, K., Shimada, M., Kubo, M., Yamamoto, K., Kitajima, S., Kuroda, E., Horie, M., Kawaguchi, K., Sasaki, T., 2016. Comparison of pulmonary inflammatory
responses following intratracheal instillation and inhalation of nanoparticles. Nanotoxicology 10 (5), 607–618. Mu, Y., Jia, D., He, Y., Miao, Y., Wub, H.L., 2011. Nano nickel oxide modified nonenzymatic glucose sensors with enhanced sensitivity through an electrochemical process strategy at high potential. Biotechnol. Bioeng. 26, 2948–2952. Nogueira, V., Lopes, I., Rocha-Santos, T.A., Rasteiro, M.G., Abrantes, N., Goncalves, F., Soares, A.M., Duarte, A.C., Pereira, R., 2015. Assessing the ecotoxicity of metal nanooxides with potential for wastewater treatment. Environ. Sci. Pollut. Res. Int. 22 (17), 13212–13224. Organization for Economic Cooperation and Development (OECD), 2004D. OECD Guideline for Testing of Chemicals. Daphnia sp., Acute Immobilisation Test. OECD 202, Paris, France. Organization for Economic Cooperation and Development (OECD), 2006D. OECD Guidelinesfor the Testing of Chemicals. OECD 201. France, Paris. Organization for Economic Cooperation and Development (OECD), 2012D. OECD Guidelines for Testing of Chemicals. Daphnia magna Reproduction Test. OECD211, Paris, France. Oukarroum, A., Barhoumi, L., Samadani, M., Dewez, D., 2015. Toxic effects of nickel oxide bulk and nanoparticles on the aquatic plant Lemna gibbaL. Biomed Res Int. 2015, 1–7. Rao, K.V., Sunandana, C.S., 2008. Effect of fuel to oxidizer ratio on the structure, micro structure and epr of combustion synthesized nio nanoparticles. J. Nanosci. Nanotechnol. 8 (8), 4247–4253. Siddiqui, M.A., Ahamed, M., Ahmad, J., Majeed Khan, M.A., Musarrat, J., Al-Khedhairy, A.A., Alrokayan, S.A., 2012. Nickel oxide nanoparticles induce cytotoxicity, oxidative stress and apoptosis in cultured human cells that is abrogated by the dietary antioxidant curcumin. Food Chem. Toxicol. 50 (3–4), 641–647. Xiao, Y., Vijver, M.G., Chen, G., Peijnenburg, W.J., 2015. Toxicity and accumulation of Cu and ZnO nanoparticles in Daphnia magna. Environ. Sci. Technol. 49 (7), 4657–4664.
Please cite this article as: Gong, N., et al., Acute and chronic toxicity of nickel oxide nanoparticles to Daphnia magna: The influence of algal enrichment, NanoImpact (2016), http://dx.doi.org/10.1016/j.impact.2016.08.003