Ecotoxicological effects and mechanism of CuO nanoparticles to individual organisms

Environmental Pollution xxx (2016) 1e9

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Ecotoxicological effects and mechanism of CuO nanoparticles to individual organisms Jing Hou a, Xiangxue Wang a, b, Tasawar Hayat c, Xiangke Wang a, b, c, * a

School of Environment and Chemical Engineering, North China Electric Power University, Beijing 102206, PR China Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, PR China c NAAM Research Group, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2016 Received in revised form 22 November 2016 Accepted 24 November 2016 Available online xxx

Copper oxide nanoparticles (CuO NPs) are used extensively in a variety of applications such as antimicrobial agent, photo-catalyst and gas sensors. The expanding production and widespread utilization of CuO NPs may pose risks to individual organisms and ecosystem. Comprehensive understanding the CuO NPs-induced adverse effects and their underlying mechanism are of great importance to assess the environmental risk of CuO NPs and to expand their use safely. However, toxic effects of CuO NPs to individual organisms and the mechanism of their action are still deficient and ambiguities. To ensure the safely use of CuO NPs, more attention should be paid on the long-term and chronic effects of CuO NPs at low concentration. Efforts should be devoted to develop techniques to differentiate toxicities induced by CuO NPs or dissolved Cu2þ, and to reduce the toxicity of CuO NPs by controlling the particle diameter, modifying surface characteristic, selecting proper exposure route and regulating the release of Cu2þ from CuO NPs. This review provides a brief overview of toxicity of CuO NPs to individual organisms with a broad range of taxa (microorganisms, algae, plants, invertebrates and vertebrates) and to discuss the underlying toxicity mechanisms including oxidative stress, dynamic unbalance and coordination effects. © 2016 Elsevier Ltd. All rights reserved.

Keywords: CuO nanoparticles Toxicity Mechanism Reactive oxygen species Oxidative stress

1. Introduction of CuO nanoparticles With the rapid development of nanotechnology, nanoparticles have received great attention because of their typical physicochemical properties as well as vast applications in catalysts (Liu et al., 2012), photodetectors (Wang et al., 2011a; b; c), biosensors (Rahman et al., 2010), gas sensors (Choi and Jang, 2010), batteries (Zhang et al., 2005), energetic materials (Rossi et al., 2007), super capacitors (Zhang et al., 2011), magnetic storage media (Kumar et al., 2000) and field emission emitters (Zhu et al., 2005). The wide applications of nanoparticles are determined by the following advantages: firstly, they are easily available, biocompatible and nontoxic in most cases; secondly, nanoparticles less than 10 nm in diameter can react with other substances practically without energy supplementation; and thirdly, the surface energy and the share of surface atoms in nanoparticles are significantly higher as compared to that of the bulk, which can result in the appearance of

* Corresponding author. School of Environment and Chemical Engineering, North China Electric Power University, Beijing 102206, PR China. E-mail address: [email protected] (X. Wang).

new chemical properties (Baker-Austin et al., 2006; BhatiKushwaha and Malik, 2014). As one of the most widely used nanoparticles, CuO NPs are being commercially used as antimicrobial agent in wood preservation, antimicrobial textiles, agricultural biocides and antifouling paints (Batley et al., 2012; Kim et al., 2012; Llorens et al., 2012). The high surface area and nanometer size allow the CuO NPs to attach to the negatively charged bacterial cell wall, to interact closely with the cell membrane, to penetrate into the bacteria and bind to deoxyribonucleic acid molecules (Ramyadevi et al., 2012; Mijnendonckx et al., 2013), and thereby causing the helical structure disorganization, cell membrane damage, protein denaturation and cell death (Yu-sen et al., 1998). The antimicrobial properties of CuO NPs have been reported against Escherichia coli, Enterococcus faecalis, Pseudomanas aeruginosa, Klebsiella pneumonia, Staphylococcus aureus and Bacillus subtilis (Azam et al., 2012; Ahamed et al., 2014). Moreover, materials span from subnanometer to several hundred nanometers have high ration of surface area to volume, which is beneficial to provide good catalytic activity and high electronic conductivity (Ko et al., 2012; Wang et al., 2012a; b; Rashad, 2013). CuO NPs have demonstrated efficient catalytic activities in many

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organic reactions such as the degradation of pyronin by urchin-like CuO microspheres (Hong et al., 2013) and the degradation of methylene blue dye by platelet-like CuO nanostructures (Meshram et al., 2012). However, the expanding production and widespread utilization of CuO NPs may pose risks for individual organisms and ecosystem, and have a high degree of toxicity compared to other types of metal oxide nanoparticles (Wang et al., 2011a; b; c). The toxic effects of CuO NPs towards bacteria (Yang et al., 2012), algae (Wang et al., 2011a; b; c), plants (Atha et al., 2012) and human cells (Karlsson et al., 2008) have demonstrated in many previous literature. It was reported that the world-wide production of CuO NPs was estimated to be 200 tons in 2010 and 570 tons in 2014 (Keller et al., 2013). By the year of 2025, the estimated global production of CuO NPs would be 1600 tons. Once they persist for long time in the environment, the chances of exposure to individual organisms and transfer between organisms of different trophic levels will increase (Handy et al., 2008; Ma et al., 2010; Keller et al., 2013). Therefore, comprehensive understanding the CuO NPs-induced adverse effects and the mechanisms of their action are of great importance to assess the environmental risk of CuO NPs. This review will provide a brief overview of toxicity of CuO NPs on individual organisms with a broad range of taxa (microorganisms, algae, plants, invertebrates and vertebrates), and discuss the underlying toxicity mechanisms, which will be of great importance to assess the environmental risk of CuO NPs and to expand their use safely. 2. Toxicity of CuO nanoparticles to individual organisms 2.1. Microorganisms As important decomposers of organic matter, microorganisms play important roles in food chains of terrestrial and aquatic ecosystems. Their movement through food chains may act as contaminant vectors to species at other trophic levels. Thus it is important to understand the toxic effects of CuO NPs on microorganisms from an ecological perspective. Microbial toxicities of CuO NPs have been conducted towards a broad number of microorganisms with respect to their antimicrobial activity and potential applications as antimicrobial agents. These studies focused on evaluating the toxicity of CuO NPs to microorganism by comparing different microbial species or CuO NPs at different particle sizes and different test media. A brief overview of toxicity of CuO NPs to microorganisms is shown in Table 1. Most of those studies focused on comparing the toxicity of nano and bulk CuO to microorganisms. A comparison of growth inhibition of CuO to Saccharomyces cerevisiae found that the 8-h median effective concentration (EC50) were 21.6 mg/L for nano CuO and 2031 mg/L for bulk CuO, which were 94-fold more toxic than their bulk forms. Moreover, the EC50 values calculated by cell viability for nano and bulk CuO were 20.7 mg/L and 1297 mg/L after 8-h exposure, 13.4 mg/L and 873 mg/L after 24-h exposure, which were 63-fold and 65-fold more toxic than their bulk forms (Kasemets et al., 2009). Toxicity of CuO to Vibrio fischeri was found that the 72-h EC50, 20% effective concentration (EC20) and no observed effect concentration (NOEC) of bulk CuO were 48, 38, and 20 times higher than that of nano CuO (Heinlaan et al., 2008). Another study on the same species also found that toxicity of nano CuO to V. fischeri was approximately 57-fold higher than that of bulk forms. The 30-min EC50 for nano CuO was 68 mg/L and for bulk CuO was 3894 mg/L (Mortimer et al., 2008). Exposure of E. coli to nano CuO (20 nm) resulted in increased growth inhibition as compared to bulk CuO (1500 nm) as the higher surface area makes CuO NPs interact with bacteria effectively (Zhao et al., 2013). These

results are enough to conclude that the nano-sized CuO NPs are more toxic than their bulk form in most cases. Besides the particle size, surface characteristic is another key factor influencing the CuO NPs toxicity. Zhao et al. (2013) indicated that membrane damage caused by CuO NPs was reduced by fulvic acid. It can be explained by the increased negative charge of CuO NPs induced by fulvic acid. Direct contact between Escherichia coli and CuO NPs was hindered by fulvic acid as a result of electrostatic repulsion. Therefore, different surface characteristics can cause different toxic actions. Several studies on microorganisms have attempted to investigate the impact of exposure method and exposure medium on toxicity of CuO NPs. For example, CuO NPs in cuvette format was highly toxic to V. fischeri, with a 30-min EC50 of 68 mg/L, whereas the 30-min EC50 for CuO NPs in microplate format was 204 mg/L (Mortimer et al., 2008). E. coli membranes were greatly damaged by CuO NPs in distilled water, but the damage was mitigated by fulvic acid, because the release of Cu2þ from CuO NPs was inhibited and the contact between bacteria cells and CuO NPs was hindered by fulvic acid (Zhao et al., 2013). In addition, a study on the effects of two type soils on toxicity of CuO NPs to bacterial community suggested that bacterial community was more susceptible in sandy loam than in sandy clay loam, which can be explained by the aggregation of CuO NPs into clay fractions and organic matter. The bioavailability of Cu2þ is affected by clay fractions and organic matter within the sandy clay loam (Frenk et al., 2013). These results imply that the toxicity of CuO NPs can be modified by complex chemistry conditions of exposure medium as a result of nanoparticles aggregation, electrostatic repulsion or ion dissolution. Effects of CuO NPs on microbial growth were species-specific. Cell growth inhibition of CuO NPs to E. coli was greater than S. aureus. The minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of CuO NPs were 195 and 180 mg/mL for S. aureus, 160 and 120 mg/mL for E. coli (Chakraborty et al., 2015). Growth inhibition of CuO NPs to E. coli (Zhao et al., 2013) was much greater than Pseudomonas chlororahips (Dimkpa et al., 2011) at the same particles size. E. coli was more sensitive than B. subtilis and S. aureus to CuO NPs (Baek and An, 2011). Bacteria (E. coli) were more sensitive than yeast to CuO NPs. The 4-h and 24-h MBC values of CuO NPs were 1000 mg/L for yeast, while less than 100 mg/L for bacteria (Suppi et al., 2015). A study on the toxicity of CuO NPs to seven microorganisms showed that the MBCs of CuO NPs were 100 mg/L for S. aureus (Oxford), 250 mg/L for E. coli, 2500 mg/L for S. aureus (Golden), Staphylococcus epidermidis, and 5000 mg/L for Proteus spp. and Pseudomonas aeruginosa, demonstrating that the CuO NPs toxicity was species-dependent (Ren et al., 2009). Soil microbial communities are often susceptible to CuO NPs. For example, CuO NPs were found to have strong effects on enzyme activities, microbial biomass carbon and total phospholipid fatty, diversity and composition of soil microbial community, which may be attributed to the bioavailability and dissolution of CuO NPs (Xu et al., 2015). Moreover, the negative effects of CuO NPs on soil microbial community were also indicated by the reduced hydrolytic activities, dehydrogenase oxidative potential and community composition after 2-h exposure (Frenk et al., 2013). Therefore, quantifying the productivity of soil microbial communities and characterizing the changes of community structure and function are necessary for the characterization of CuO NPs toxicities. 2.2. Algae and plants Most toxicity studies on plants have been conducted on algae (Table 2). Toxic effects increased with the increase of CuO NPs concentrations. For example, Cheloni et al. (2016) reported that 24h CuO NPs exposure depressed the growth rate of Chlamydomonas

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Table 1 An overview of toxicity of CuO nanoparticles to microorganisms. Surface area (m2/g)

Hydrodynamic pH Size (nm)

Saccharomyces cerevisiae 30

NA

NA

5.5 30 e5.6

Malt extract medium

Vibrio fischeri

NA

NA

20 6.2 e6.5

Agar medium

Test species

Advertised particle size (nm)

30

Temperature Medium ( C)

Endpoint

Effect

Reference

Ultrastructural changes Growth inhibition Luminescence inhibition

8-h EC50: 20.70 mg/L 24-h EC50: 13.40 mg/L 8-h EC50: 21.60 mg/L

(Kasemets et al., 2009)

30-min EC50: 79 mg/L 30-min EC20: 24 mg/L 30-min NOEC: 16 mg/L 72-h MIC: 200 mg/L

(Heinlaan et al., 2008)

30-min EC50: 68 mg/L 30-min EC50: 204 mg/L 18-h MIC: 0.12 mg/L 18-h MBC: 0.16 mg/L 18-h MIC: 0.18 mg/mL 18-h MBC: 0.20 mg/mL 2-h LD50: 64.50 mg/L

(Mortimer et al., 2008) (Chakraborty et al., 2015) (Chakraborty et al., 2015) (Hu et al., 2009)

Growth inhibition Luminescence inhibition Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes

Vibrio fischeri

30

NA

NA

NA

20

Escherichia coli

50

NA

80 ± 20

7.5

37

Staphylococcus aureus

50

NA

80 ± 20

7.5

37

Escherichia coli

30

NA

NA

NA

37

Escherichia coli

20e30

NA

NA

NA

37

Escherichia coli

20-40 (nano) 1500 (bulk)

165

7.0 30 e7.2

Davis medium Growth inhibition

Bacillus subtilis

20e30

13.31 (nano) 0.52 (bulk) NA

NA

NA

37

Luria-Bertani medium

Ultrastructural changes

Staphylococcus aureus

20e30

NA

NA

NA

37

Luria-Bertani medium

Ultrastructural changes

Staphylococcus aureus (Golden) Staphylococcus aureus (Oxford) Staphylococcus epidermidis SE-51 Staphylococcus epidermidis SE-4 Escherichia coli NCTC 9001 Proteus spp.

60

15.69

NA

NA

37

60

15.69

NA

NA

37

60

15.69

NA

NA

37

60

15.69

NA

NA

37

60

15.69

NA

NA

37

60

15.69

NA

NA

37

Pseudomonas aeruginosa 60 PAOI Escherichia coli MG1655 30

15.69

NA

NA

37

NA

145

7.1

30

30

NA

145

7.1

25

30

NA

145

7.1

25

30

NA

145

7.1

25

30

NA

145

7.1

25

Pseudomonas 30 fluorescens KC-1 Microbacterium 30 testaceum PCSB7 Saccharomyces cerevisiae 30 BY4741

NA

145

7.1

25

NA

145

7.1

25

NA

145

6.6

25

Tryptone soya broth Tryptone soya broth Tryptone soya broth Tryptone soya broth Tryptone soya broth Tryptone soya broth Tryptone soya broth Deionized water Deionized water Deionized water Deionized water Deionized water Deionized water Deionized water Deionized water

Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes Ultrastructural changes

Pseudomonas fluorescens OS8 Pseudomonas aeruginosa DS10-129 Staphylococcus aureus RN4220 Janthinobacterium sp.

Cuvettes Microplates Luria-Bertani medium Luria-Bertani medium Luria-Bertani medium Luria-Bertani medium

24-h EC50: 28.60 mg/L (Baek and An, 2011) 24-h NOEC: 10 mg/L 24-h LOEC: 25 mg/L 2-h decreased by 45.4% (Zhao et al., 2013) at 10 mg/L

24-h 24-h 24-h 24-h 24-h 24-h 24-h

EC50: 61.10 mg/L NOEC: <25 mg/L LOEC: 25 mg/L EC50: 65.90 mg/L NOEC: 25 mg/L LOEC: 50 mg/L MBC: 2.50 mg/mL

(Ren et al., 2009)

24-h MBC: 0.10 mg/mL

(Ren et al., 2009)

24-h MBC: 2.50 mg/mL

(Ren et al., 2009)

24-h MBC: 2.50 mg/mL

(Ren et al., 2009)

24-h MBC: 0.25 mg/mL

(Ren et al., 2009)

24-h MBC: 5.00 mg/mL

(Ren et al., 2009)

24-h MBC: 5.00 mg/mL

(Ren et al., 2009)

4-h MBC:10 mg/L 24-h MBC: 10 mg/L 4-h MBC:10 mg/L 24-h MBC: 1 mg/L 4-h MBC:10 mg/L 24-h MBC: 1 mg/L 4-h MBC:100 mg/L 24-h MBC: 1 mg/L 4-h MBC:10 mg/L 24-h MBC: 10 mg/L 4-h MBC:100 mg/L 24-h MBC: 10 mg/L 4-h MBC:100 mg/L 24-h MBC: 100 mg/L 4-h MBC:1000 mg/L 24-h MBC: 1000 mg/L

(Suppi 2015) (Suppi 2015) (Suppi 2015) (Suppi 2015) (Suppi 2015) (Suppi 2015) (Suppi 2015) (Suppi 2015)

(Baek and An, 2011) (Baek and An, 2011)

et al., et al., et al., et al., et al., et al., et al., et al.,

NA: not available. EC50: the median effective concentration. EC20: the 20% effective concentration. LC50: the median lethal concentration. NOEC: no observed effect concentration. LOEC: lowest observed effect concentration. MIC: minimal inhibitory concentration. MBC: minimum bactericidal concentration.

reinhardtii by 67.7% in the presence of 0.8 mg/L CuO NPs. Melegari et al. (2013) reported the toxicity of CuO NPs to the cell density of C. reinhardtii with a 72-h EC50 near 150 mg/L and a 72-h NOEC less than 150 mg/L. CuO NPs were found to inhibit growth of Landoltia punctata by 50% at 0.8 mg/L CuO NPs after 96-h exposure and decrease chlorophyll content of L. punctata by 40% at 1.0 mg/L CuO NP after 9-d exposure (Shi et al., 2011). CuO NPs at 50 mg/L

inhibited the growth and cell viability of L. punctata by 89.7% and 90.5% as compared with the control (Sankar et al., 2014). In addition, the dissolution of metal ions was one of the key factors determining CuO NPs toxicity. Toxicity of CuO NPs to the growth inhibition of Pseudokirchneriella subcapitata was greater than their bulk forms, with a 72-h EC50 0.71 mg/L, EC20 0.50 mg/L, NOEC 0.42 mg/L for CuO NPs and a 72-h EC50 11.55 mg/L, EC20 9.10 mg/L,

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Table 2 An overview of toxicity of CuO nanoparticles to algae and plants. Test species

Particle size (nm)

Surface area (m2/g)

pH Temperature Medium ( C)

Endpoint

Effect

Reference

Pseudokirchneriella subcapitata

30

NA

NA 24

Algal growth medium

Growth inhibition

(Aruoja et al., 2009)

Pseudokirchneriella subcapitata Chlamydomonas reinhardtii

30

NA

NA 25

Deionized water

30e50

13.1

6.8 NA

Chlamydomonas reinhardtii Landoltia punctata

30e40

NA

NA 23

10e15

141

Microcystis aeruginosa

270

NA

6.0 24 (day) 20 (night) NA 28

Tris-AcetatePhosphate medium High salt culture media Half-strength Hoagland's solution Solid agar medium

Formation of visible spot Growth inhibition

72-h EC50: 0.71 mg/L 72-h EC20: 0.50 mg/L 72-h NOEC: 0.42 mg/L 4-h MBC: >1000 mg/L 24-h MBC: 10 mg/L 24-h decreased by 67.7% at 0.8 mg/L

Triticum aestivum

<50

NA

7.9 28

Sand

Fagopyrum Esculentum

<50

29

6.5 25

1/2 strength Root length Hoagland's solution Seedling biomass Genetic identity

Oryza sativa

<50

NA

NA 25

water soaked cotton Shoot length Shoot weight Root length Root weight

Ultrastructural changes Growth inhibition Chlorophyll content Growth inhibition Ultrastructural changes Shoot length Root length Plant biomass

72-h EC50: 150 mg/L 72-h NOEC: <100 mg/L 96-h EC50: 0.80 mg/L 9-d decreased by 40% at 1 mg/L 96-h decreased by 89.7% at 50 mg/L 96-h decreased by 90.5% at 50 mg/L 14-d reduced by 13.1% at 500 mg/kg 14-d reduced by 58.8% at 500 mg/kg 14-d reduced by 25.0% at 500 mg/kg 7-d decreased significantly at 2000 mg/L 7-d decreased at 4000 mg/L 7-d decreased significantly at 2000 mg/L 7-d decreased by 29.2% 7-d decreased by 24.9% 7-d decreased by 89.5% 7-d decreased by 89.5%

(Suppi et al., 2015) (Cheloni et al., 2016)

(Melegari et al., 2013) (Shi et al., 2011) (Sankar et al., 2014)

(Dimkpa et al., 2012)

(Lee et al., 2013)

(Shaw and Hossain, 2013)

NA: not available. EC50: the median effective concentration. EC20: the 20% effective concentration. NOEC: no observed effect concentration. MBC: minimum bactericidal concentration.

NOEC 8.03 mg/L for bulk CuO, respectively. Comparison of bioavailable Cu2þ in nano CuO and bulk CuO suspensions found that the bioavailability of Cu2þ might be the primary determinant governing the effects of CuO NPs to P. subcapitata (Aruoja et al., 2009). These results suggest that toxicities of CuO NPs to algae are more toxic than bulk CuO, and show dose-dependent responses with increasing CuO NPs concentrations in most cases, confirming that the strongest toxicity driving force for algae is CuO NPs dissolution. A limited number of studies on higher plants are available (Table 2). CuO NPs at 500 mg/kg reduced the root length, shoot length and plant biomass of Triticum aestivum by 58.8%, 13.1% and 25.0% after 14-d exposure. Decreased chlorophyll levels in leaves and increased lipid peroxidation in the root membranes by CuO NPs were also reported. The impaired growth of T. aestivum could be explained by the dissolved Cu2þ from CuO NPs, and the oxidative stress induced by Cu2þ also contributed to the impaired seedling growth (Dimkpa et al., 2012). However, the role of dissolution on nanotoxicity is disputable. It was reported that the average genetic identity and root length of Fagopyrum Esculentum exposed to CuO NPs decreased significantly at 2000 mg/L, and seedling biomass only decreased at 4000 mg/L, which were not related to the toxicity of Cu2þ released from CuO NPs (Lee et al., 2013). Moreover, effects of 1.5 mM CuO NPs on growth of Oryza sativa found that the shoot length, shoot weight, root length and root weight of O. sativa decreased by 29.2%, 24.9%, 89.5% and 89.5%, respectively. CuO NPs were also found to have effects on antioxidant enzymes activity, carotenoids content and proline level, which were indication of production of reactive oxygen species (ROS) and activation of defense mechanism to prevent oxidative stress (Shaw and Hossain, 2013).

2.3. Invertebrates and vertebrates Most toxicity studies on invertebrates were conducted on Daphnia magna (Table 3). Examination on size-related adverse effects of CuO to D. magna found that the EC50, EC20, and NOEC of CuO NPs to D. magna were 3.2 mg/L, 1.2 mg/L and 0.5 mg/L, which were 52-, 95- and 100-fold lower than that of bulk CuO (Heinlaan et al., 2008). Similar results were obtained by observing ultrastructural changes of D. magna: after 48 h of exposure, the EC50 and NOEC of CuO NPs to D. magna were 4.0 mg/L and 0.5 mg/L, respectively (Heinlaan et al., 2011). Another study found that glutathione-Stransferase enzyme activity decreased significantly at 0.8 mg/L and showed a concentration dependent decrease for CuO NPs. On the contrary, CuO NPs caused a concentration dependent increase in oxidized glutathione and malondialdehyde, indicating that oxidative stress is one of the toxicity mechanisms for CuO NPs (Mwaanga et al., 2014). These results suggested that the toxicities of CuO NPs to D. magna were size and concentration dependent in most cases. The size of CuO NPs is related to many essential properties such as solubility. Toxic effects of CuO NPs on many invertebrates could be explained by soluble Cu2þ. For example, Heinlaan et al. (2008) reported that the 48-h LC50, LC20 and NOEC of CuO NPs to Thamnocephalus platyurus were 2.1 mg/L, 1.7 mg/L and 0.5 mg/L, which were 45-, 37-, 50-fold more toxic than that of bulk CuO. In this study, Cu from bulk CuO was only partially bioavailable whereas nano CuO was remarkably more soluble. Pradhan et al. (2012) reported that the mortality of Allogamus ligonifer increased with increasing concentration of CuO NPs. The 96-h median lethal concentration (LC50) and lowest observed effect concentration (LOEC) of CuO NPs were 569 mg/L and 250 mg/L, respectively. Leached

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Test species

Particle size Surface area pH (nm) (m2/g)

Temperature ( C)

Medium

Daphnia magna

30

NA

7.3e7.8

20

Daphnia magna

30e50

26

7.8

20

Daphnia magna

<50

16.1

NA

NA

Thamnocephalus platyurus 30

NA

7.3e7.8

25

Tetrahymena thermophile 30

NA

NA

NA

Hediste diversicolor

197

NA

NA

21

Scrobicularia plana

197

NA

NA

21

Allogamus ligonifer

30e50

NA

NA

NA

Exaiptasia pallida

40e100

NA

7.9

26.1

Mytilus galloprovincialis Xenopus laevis

30e40 23e37

29 25e40

NA 8.1

NA 23

Oreochromis niloticus,

30e40

e

25 ± 1

Chinook salmon

50

e

6.5e7.8, 7.1 e7.3 e

Danio rerio Carassius auratus

51 40

e 20

6.8e7.2 6.8 ± 0.2

Room temperature 28 ± 1 23 ± 2.5

48-h LC50: 3.20 mg/L 48-h LC20: 1.20 mg/L 48-h NOEC: 0.50 mg/L Synthetic freshwater Ultrastructural changes 48-h EC50: 4.00 mg/L 48-h NOEC: 0.50 mg/L 72-h decreased significantly at 0.80 mg/L Distilled and deionized water Glutathione-S-transferase 72-h increased significantly at 1.10 mg/L Oxidized glutathione 72-h increased significantly at 1.10 mg/L Malondialdehyde Synthetic freshwater Mortality 48-h LC50: 2.10 mg/L 48-h LC20: 1.70 mg/L 48-h NOEC: 0.50 mg/L Streptomycin sulphate Fluorescence 4-h EC50: 127 mg/L ATP content 24-h EC50: 97.90 mg/L 4-h EC50: 129 mg/L 24-h EC50: 101 mg/L Natural seawater Catalase 16-d increased by 81.2% at 0.01 mg/L Glutathione-S-transferase 16-d increased by 61.3% at 0.01 mg/L 7-d increased by 79.5% at 0.01 mg/L Natural seawater Catalase 7-d increased by 62.2% at 0.01 mg/L Glutathione-S-transferase 7-d increased by 60.0% at 0.01 mg/L Superoxide dismutase 7-d increased by 16.0% at 0.01 mg/L Metallothionein-like protein Sterile stream water Mortality 96-h LC50: 569 mg/L 96-h LOEC: 250 mg/L 7-d increased significantly at 0.01 mg/L Instant Ocean salt and Catalase activity 7-d increased significantly at 0.01 mg/L reverse osmosis water Glutathione peroxidase 7-d increased significantly at 0.01 mg/L activity Glutathione reductase activity, 7-d increased significantly at 0.05 mg/L Carbonic anhydrase activity Natural seawater Comet assay 15-d increase in the olive tail moment 96-h EC50 >1000 mg/L Deionized water Malformation Malformation 96-h EC10: 2.10 mg/L Growth inhibition 96-h LOEC: 10 mg/L Aerated, dechlorinated Mortality 96-h LC50: 2205 mg/L tap water Cell culture medium CHSE-214 cells 24-h 50% IC50: 19.03 mg/L

Oreochromis mossambicus Oncorhynchus mykiss

100

141

8.25

51

18

7.8

Synthetic freshwater

Effect

Endpoint

Mortality

E3 medium Seawater

Mortality Malondialdehyde content

28 ± 1

De-chlorinated tap water

Opercular ventilation rate

Room temperature

Isotonic medium

Haemolysis

Reference (Heinlaan et al., 2008)

(Heinlaan et al., 2011) (Mwaanga et al., 2014)

(Heinlaan et al., 2008)

(Mortimer et al., 2010)

(Buffet et al., 2011) (Buffet et al., 2011)

(Pradhan et al., 2012) (Siddiqui et al., 2015)

(Gomes et al., 2013) (Nations et al., 2011)

J. Hou et al. / Environmental Pollution xxx (2016) 1e9

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Table 3 An overview of toxicity of CuO nanoparticles to invertebrates and vertebrates.

(Abdel-Khalek et al., 2016) (Srikanth et al., 2016)

48-h LC50: 64 mg/L Dietary exposure: increased significantly at 1.1e1.3 mg/L Waterborne exposure: increased significantly at 1.0 mg/L Increase at 5 mg/L

(Ganesan et al., 2016) (Ates et al., 2015)

1.59 mg/L induced 48.5% haemolysis, 4.77 mg/L induced55.3% haemolysis 7.95 mg/L induced 60.8% haemolysis

(Isani et al., 2013)

(Villarreal et al., 2014)

NA: not available. ATP: adenosine triphosphate. EC50: the median effective concentration. EC10: the 10% effective concentration. LC50: the median lethal concentration. LC20: the 20% lethal concentration. NOEC: no observed effect concentration. LOEC: lowest observed effect concentration. IC50: the 50% inhibition concentration.

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Cu2þ from the CuO NPs accumulated in larval body influenced the growth and feeding behaviour and of A. ligonifer. Toxic effects of CuO NPs to Tetrahymena thermophile evaluated by two endpoints demonstrated that the toxicity of nanoparticles of CuO to T. thermophile was due to increased solubilisation, with the 24-h EC50 of 129 mg/L and 101 mg/L for fluorescence and adenosine triphosphate content (Mortimer et al., 2010). In addition, different mechanisms of action may be mediated through oxidative stress. Biochemical responses of Hediste diversicolor and Scrobicularia plana to CuO NPs found that catalase and glutathione-S-transferase activities increased significantly in both H. diversicolor and S. plana, and superoxide dismutase and metallothionein-like protein increased significantly in S. plana (Buffet et al., 2011). Oxidative stress responses including catalase activity, glutathione peroxidase activity, glutathione reductase activity, and carbonic anhydrase activity of Exaiptasia pallida to CuO NPs were observed to a greater degree (Siddiqui et al., 2015). Genotoxic effects induced by CuO NPs were also reported in the Mytilus galloprovincialis (Gomes et al., 2013). There appears to be very limited toxicity data on vertebrates. Nations et al. (2011) found CuO NPs to be toxic to Xenopus laevis with 96-h malformation EC10 of 2.1 mg/L and 96-h growth LOEC of 10 mg/L. These results suggested that oxidative stress and ion dissolution are important mechanisms for the toxicity of CuO NPs to animals.

3. Mechanism of toxicity The toxic mechanisms of CuO NPs are mainly in two aspects: the oxidative stress induced by intracellular CuO NPs and dissolution of CuO NPs. CuO NPs and extracellular Cu2þ pass through the cell membrane and enter into the cytoplasm via endocytosis and copper transport proteins, respectively. Schematic overview of cellular toxicity induced by CuO nanoparticles is summarized in Fig. 1. 3.1. Oxidative stress ROS generation and oxidative stress had proven to be common and major toxicological mechanism for cell damage induced by NPs (Yang et al., 2009). Small quantities of CuO NPs incorporated into cells could generate large quantities of ROS (Dudev and Lim, 2008). ROS are oxygen derivatives including superoxide anions (O2), hydroxyl radicals ($OH), and hydrogen peroxide (H2O2) originated from the disruption of electron transport chain in the cell (Xia et al., 2008). The mitochondria are the most powerful intracellular sources of ROS (Xia et al., 2007). $OH is usually more reactive than other free radicals and is able to oxidize almost all the organelle (Yamakoshi et al., 2003). The accumulation of ROS is able to culminate with the formation of O2 caused by electron capture on the surface of NPs (De Berardis et al., 2010). Redundant free radicals

Fig. 1. Schematic overview of cellular toxicity induced by CuO nanoparticles.

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may stimulate the anti-oxidant defense system by the oxidation of lipids, denaturation of proteins, and modification of nucleic acids, causing an imbalance between oxidant and anti-oxidant processes (Yang et al., 2009). CuO NPs in lysosomes with acidic conditions inhibit the lysosomal activity and destroy the lysosomal membranes (Nohl and Gille, 2005), which may cause the subsequent translocation of CuO NPs to mitochondria and nucleus (Wang et al., 2012a; b). Once CuO NPs entered into mitochondria, the deposition of CuO NPs in membrane can enhance the membrane depolarization by perturbing the electron transduction in the inner membrane, ultimately triggering membrane permeation and ROS generation (Xia et al., 2007; Zhang and Gutterman, 2007; Xia et al., 2008). ROS in lysosomes can induce DNA double helix destructs or cause DNA point mutations (Singh et al., 2009). ROS in mitochondria can affect mitochondrial respiration and apoptosis, resulting in redox dysequilibrium in the cell, lipid peroxidation in the cell membrane and a range of antioxidant responses (Xia et al., 2007). H2O2 generated in the cytoplasm can freely diffuse across the mitochondrial membranes and produce $OH radical through Fenton reaction, ultimately leading to DNA damage and programmed cell death (Xia et al., 2007). With increased intracellular ROS production, nanoparticles can cause the increase gene expression of the death receptor (Yang et al., 2009) and stimulate the redox-sensitive singling pathways at an intermediate amount of oxidative stress (He et al., 2007). Inflammatory responses are initiated through the activation of mitogen-activated protein kinase (MAPK) and nuclear factor NF-kB signaling cascades, which are closely related to cellular fibrosis and cell death (Nel et al., 2006). Moreover, CuO NPs could be transported to the nucleus by nuclear pore (less than 50 nm) or by direct physical injury of nucleus membrane and interact with nuclear DNA directly (Wang et al., 2012a; b). 3.2. Dissolution Cu2þ released from the surface of CuO NPs had been considered as another major reason for higher toxicity to organisms (Gabbay et al., 2006; Gunawan et al., 2011). Large quantities of Cu2þ were able to release from CuO NPs both in the suspension and in cell medium (Gunawan et al., 2011), and generate large amounts of $OH by catalyzing Fenton reactions, leading to damage of lipids, proteins, and nucleic acids (Festa and Thiele, 2011; Hartwig, 2013). In addition, the intracellular CuO NPs that enter into acidic organelle such as lysosomes or contact acid materials such as nucleic acid can release more Cu2þ from CuO NPs, leading to oxidative damage and DNA damage (Cuillel et al., 2014). The particle dissolution is dependent on both the nature of the particles such as particle size, surface area and chemical composition as well as the environmental parameters such as pH, temperature, organic matter. Cu2þ has the ability to react with electron pair donors to form chelates through the donation of lone electrons from coordination atoms such as N and O atoms to the unfilled orbitals of the outer coordination sphere of Cu2þ. Coordinating with the coordination atoms located in active sites may induce the inactivation of biomolecules, leading to the inhibition of normal physiology processes. The intracellular small CuO NPs can enter into nucleus by nucleopore or gain access with the nucleus lysed during cell division, which may lead to inhibition of transcription and translation machinery, damage of genetic material by interacting with DNA or DNA-related proteins (Singh et al., 2009), activation of signaling cascade by interacting with cellular signal molecules (Miller et al., 2010). Moreover, Cu2þ released from CuO NPs was also able to cause mRNA degradation via direct interaction with mRNA stabilizing proteins (Soenen et al., 2010). Cu2þ plays an important significant role in maintaining cellular

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homeostasis (Galhardi et al., 2005). Metal cation homeostatic mechanisms will be disrupted by the increasing Cu2þ released from CuO NPs once exceeding the physiological tolerance range. Cu2þ can increase the local concentration of intracellular Ca2þ concentration (Xia et al., 2008)，and stimulate Ca2þ influx across the endoplasmic reticulum plasma membrane via Ca2þ channels (Hoyal et al., 1998). Intracellular Ca2þ involved in a broad variety of cellular processes, including the activation of transcription factors such as NF-kB (Dolmetsch et al., 1998), nitric oxide production (Raddassi et al., 1994), superoxide anion generation (Raddassi et al., 1994) and proteins secretion (Berridge et al., 1998), which was able to lead to the mitochondrial perturbation and cell damage (Xia et al., 2008). 4. Conclusion and perspectives Herein we reviewed the toxic effects of CuO NPs on individual organisms with a broad range of taxa (microorganisms, algae, plants, invertebrates and vertebrates), and the corresponding mechanisms including oxidative stress, dynamic unbalance and coordination effects. However, several knowledge gaps need to be filled to gain a comprehensive understanding of CuO NPs ecotoxicity and their corresponding mechanisms. Firstly, the characterization of exposure conditions for the CuO NPs is inadequate. Toxic effects are able to be elicited by different modes of action of CuO NPs such as particle dissolution, particle agglomeration and particle precipitation, which are highly dependent on particle size, agglomerate size, surface characteristics, medium chemistry (pH and temperature), and exposure routes. Therefore, future toxicity study should give a detailed description of the characterization of exposure conditions, which is essential for validating the comparison between different literature and interpreting the toxicity data. Secondly, toxicity data on long term, chronic stress, and low exposure levels are insufficient. Therefore, more attention should be paid on the long-term and chronic effects of CuO NPs at low concentration, which will be more representative for real environment. Thirdly, Cu2þ dissolved from CuO NPs plays an important role in inducing toxicity. Techniques are needed to be developed to differentiate toxicities induced by CuO NPs or dissolved Cu2þ. Fourthly, to ensure the safely use of CuO NPs, efforts should be devoted to reduce the toxicity of CuO NPs and weaken the toxicity mechanisms by controlling particles diameter, modifying surface characteristic, selecting proper exposure route and regulating the release of Cu2þ from CuO NPs. Acknowledgment Financial support from NSFC (21225730, 91326202, 21577032, 21607043), the Fundamental Research Funds for the Central Universities (2016ZZD06, JB2015001), the Open Project of Key Laboratory of Environmental Biotechnology, CAS (Grant No kf2016009), the Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection and the Priority Academic Program Development of Jiangsu Higher Education Institutions are acknowledged. References Abdel-Khalek, A.A., Badran, S.R., Marie, M., 2016. Toxicity evaluation of copper oxide bulk and nanoparticles in Nile tilapia, Oreochromis niloticus, using hematological, bioaccumulation and histological biomarkers. Fish Physiology Biochem. 42, 1225e1236. Ahamed, M., Alhadlaq, H.A., Khan, M.A., Karuppiah, P., Al-Dhabi, N.A., 2014. Synthesis, characterization, and antimicrobial activity of copper oxide nanoparticles. J. Nanomater. 2014, 17e20. Aruoja, V., Dubourguier, H., Kasemets, K., Kahru, A., 2009. Toxicity of nanoparticles of CuO, ZnO and TiO2 to microalgae Pseudokirchneriella subcapitata. Sci. Total Environ. 407, 1461e1468.

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Please cite this article in press as: Hou, J., et al., Ecotoxicological effects and mechanism of CuO nanoparticles to individual organisms, Environmental Pollution (2016), http://dx.doi.org/10.1016/j.envpol.2016.11.066