Developmental toxicity and DNA damaging properties of silver nanoparticles in the catfish (Clarias gariepinus)

Accepted Manuscript Title: Developmental toxicity and DNA damaging properties of silver nanoparticles in the catfish (Clarias gariepinus) Authors: Alaa El-Din H. Sayed, Hamdy A.M. Soliman PII: DOI: Reference:

S1383-5718(16)30411-9 http://dx.doi.org/doi:10.1016/j.mrgentox.2017.07.002 MUTGEN 402835

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Mutation Research

Received date: Revised date: Accepted date:

28-11-2016 5-7-2017 10-7-2017

Please cite this article as: Alaa El-Din H.Sayed, Hamdy A.M.Soliman, Developmental toxicity and DNA damaging properties of silver nanoparticles in the catfish (Clarias gariepinus), Mutation Research/Genetic Toxicology and Environmental Mutagenesishttp://dx.doi.org/10.1016/j.mrgentox.2017.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Developmental toxicity and DNA damaging properties of silver nanoparticles in the catfish (Clarias gariepinus) Running title: Ag-NPs induce embryonic malformations in fish

Alaa El-Din H. Sayeda*; Hamdy A. M. Solimanb

a

Zoology Department, Faculty of Science, Assiut University, 71516 Assiut, Egypt

b

Zoology Department, Faculty of Science, Sohag University, 8562 Sohag, Egypt

*Corresponding author Tel.: +20 882411444 Fax: +20 882342708 E-mail address: [email protected] [email protected] (A.H. Sayed). Highlights    

We evaluated the toxicity of silver nanoparticles on early developmental stages of Clarias gariepinus. The mortality rates of the embryos increased in a dose- and embryonic stagedependent manner. Different types of malformation and histopathological alterations were observed in exposed larvae. Silver nanoparticles induced oxidative stress and genotoxicity in exposed larvae

Abstract Although, silver nanoparticles (AgNPs) are used in many different products, little information is known about their toxicity in tropical fish embryos. Therefore, this study evaluated the developmental toxicity of waterborne silver nanoparticles in embryos of Clarias gariepinus. Embryos were treated with (0, 25, 50, 75 ng/L silver nanoparticles) in water up to 144 h postfertilization stage (PFS). Results revealed various morphological malformations including notochord curvature and edema. The mortality rate, malformations, and DNA fragmentation in embryos exposed to silver nanoparticles increased in a dose- and embryonic stagedependent manner. The total antioxidant capacity and the activity of catalase in embryos exposed to 25 ng/L silver nanoparticles were decreased significantly while the total 1

antioxidant capacity and the activity of catalase were insignificantly increased with increasing concentrations in the embryos from 24 to 144 h-PFS exposed to 50 and 75 ng/L silver nanoparticles. Lipid peroxidation values showed fluctuations with doses of silver nanoparticles. Histopathological lesions including severely distorted and wrinkled notochord were observed. The current data propose that the toxicity of silver nanoparticles in C. gariepinus embryos is caused by oxidative stress and genotoxicity.

Keywords: Embryo; nanoparticles; Clarias gariepinus; histopathology; stress; DNA

1. Introduction Nanosilver has been consolidated into consumer products including materials, contraceptives, beauty care products, youngsters' toys, medical equipment, air filters, water filters, and residential washing machines [1]. The usage of consumer products containing silver nanoparticles (AgNPs) indicates that AgNPs may be released into the aquatic environment [2]. Aquatic ecosystems are often impacted by chemical pollution, originating from consumer products; therefore, fish provide a good experimental model for monitoring toxicity in aquatic systems because they are extremely sensitive to pollutants [3-5]. AgNPs can cause defects in the spinal cord, heart, and eye [6]. Asharani et al. [7], Griffitt et al. [8], and Yeo and Kang [9] have also observed an increase in mortality and hatching delay as well as various types of malformations. Moreover, some authors have that AgNPs could cause cellular and DNA damage in addition to roles as carcinogens and oxidative stress agents [10-12]. Different types of morphological abnormalities are reported in fish exposed to environmental pollutants, such as fin erosion, skull deformation, jaw deformities, and skeletal deformities such as lordosis, scoliosis, and kyphosis; opercular deformity; fin deformity; lower lip protrusion; gill deformity; ocular disorders; scale deformity and disorientation; and neoplasia or hyperplasia [13-16]. It is well established that during toxicity studies, stressors essentially target energy metabolism which is of prime concern in fish physiology [17]. Energy metabolism is a complex that includes a range of enzymes. Modifications to these enzymes or their substrates reflect changes in energy metabolism, which can then be used as significant indicators of stress factors [18, 19]. Oxidative stress has been linked with some pathological processes in fish diseases caused by pollution [20]. Environmental pollutants can cause the production of reactive oxygen species (ROS) in living organisms and accordingly may result in oxidative stress, which is a possible explanation for the toxicity of nanoparticles [21, 22]. It has been reported that AgNPs cause DNA fragmentation in adult catfish [22] and that can cause defects 2

in offspring. Furthermore, catfish have been regarded as a model organism for studies of embryology and genotoxicity because artificial spawning is easy to perform [15, 22, 23]. Therefore, the aim of this study is to investigate oxidative stress in embryos of the African catfish C. gariepinus in response to AgNPs using antioxidant enzymes as biomarekers. 2. Materials and methods 2.1. Preparation and characterization of silver nanoparticles:AgNPs were purchased from Sigma-Aldrich, St. Louis, MO, USA. The physical characteristics of the particles according to the manufacturers data were: size (≤100 nm), purity (99.5%), trace metal basis, surface area (5.0 m2/g), and density (10.49 g/cc). The preparation and description of AgNPs were reported by Sayed [22]. 2.2. Gamete collection Criteria for selection and rearing of mature C. gariepinus were described by De Graaf and Janssen [24]. The catfish specimens were kept in 100-L glass tanks and acclimatized for a two-week period at 27- 29 ºC, PH=7.56, and dissolved oxygen 88- 94% saturation. The photoperiod was a 12L:12D cycle and the catfish specimens were fed on a commercial pellet diet (3% of body weight/day). For the collection of semen, male fish were anesthetized with 200 mg/L Ms-222 (tricaine methane sulfonate, Crescent Research Chemicals, Phoenix, AZ, USA) buffered with 800 mg/L sodium bicarbonate and the testes were removed surgically. Blood was cleaned from the testes by surgical towels. The sperm from the testes were pressed through a mesh fabric into a sterile dry Petri dish and used directly for dry fertilization. For the collection of eggs, ovulation was artificially induced. Females were injected intraperitoneally with pellets (gonadotropin-releasing hormone analogue, D-Ala6, Pro9-NEt) containing 2.5- 3.0 mg of water-soluble dopamine antagonist metoclopramide (Interfish Ltd, Hungary) dissolved in 0.65% NaCl. One pellet was used per kg body weight. After injection (10- 11 h), the fish were stripped and the eggs were collected in clean dry plastic containers; dry fertilization was then performed [15].

2.3. Experimental setup and sampling The fertilized eggs were incubated in dechlorinated tap water (pH= 7.56, dissolved oxygen 8894% saturation, temperature 27- 29 C°, and photoperiod 12L:12D). Exposure started 2 h postfertilization (2 h-PFS). Fertilized eggs were divided into four groups: one control and three groups exposed to AgNPs once with 25, 50 and 75 ng/L according to Govindasamy and Abdul Rahuman [25] and the OECD [26]. Exposure took place in a 30x30x30 cm glass tank (10 L water level), three replicates for each group (300 samples). Sampling was at intervals of 24, 48, 3

72, 96, 120, and 144 h-PFS after AgNP exposure. Ten embryos were collected and anesthetized with 200 mg/L Ms-222 buffered with 800 mg/L sodium bicarbonate at each sampling point and fixed at -80 °C until Measurements of enzyme activities and other samples fixed for 24 h in Bouin’s solution for gross and minor external malformation and histopathology. The hatching process started at 22 h-PFS. Mortality was recorded as in the Karber method [27]. Morphological malformations were observed and reported according to Mahmoud et al. [15]. 2.4. Measurements of enzyme activities Measurements of lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G6PDH) were according to a modified protocol [19]. The catalase level was estimated according to a method described by Aebi [28]. Total antioxidant capacity was determined according to Abu Khalil et al. [29] using a commercial kit (Antioxidant Assay Kit, Cat. No. MAK187; Sigma-Aldrich, Cairo, Egypt). 2.5. Lipid peroxidation Lipid peroxidation (LPO) in the embryos was determined by a procedure previously described [30]. The absorbance of each aliquot was measured at 535 nm. The rate of lipid peroxidation was expressed as nmol of thiobarbituric acid reactive substance formed per hour per milligram of protein using a molar extinction coefficient of 1.56 M-1 cm-1 [31]. 2.6. DNA fragmentation measurement DNA fragmentation was determined by a procedure using a spectrophotometer (Micro Lab 200 Vital Scientific, Dieren, The Netherlands) at 575 or 600 nm against a reagent blank [32]. The percentage of fragmented DNA was estimated by the following formula: % of fragmented DNA = fragmented DNA/ (fragmented + intact DNA) x 100. 2.7. Comet assay The comet assay was performed according to a modified protocol based on Jarvis et al. [33] and Mekkawy et al. [19]. 2.8. Histopathology Fixed specimens were dehydrated and subsequently embedded in paraffin. Transverse serial sections 5- 7μm thick were cut and stained with hematoxylin and eosin. Sections were studied using an Omax advanced trinocular biological microscope with a 14MP USB Digital Camera (CS-M837ZFLR-C140U). 2.9. Determination of Ag bioaccumulation:4

Embryos (144 h-PFS) and water samples were used for the determination of Ag ions. Embryos (0.5 g of each) and water media (500 µ1) were added to 1.5 mL of concentrated HNO3 and then boiled for 45 min at 100 °C to break down the embryonic tissue and dissolve all the silver content. The mixture was cooled, diluted with 500 mL of deionized water, and the silver quantified using graphite furnace AA (GFAA) spectroscopy [34]. 2.10. Statistical analysis The basic statistical means and standard errors were estimated. One-way analysis of variance in the SPSS package [35] was used to determine patterns of variation at the 0.05 significance level. The Tukey’s HSD test was considered for multiple comparisons. 2.11. Ethical statement All experiments were carried out in accordance with Egyptian laws and university guidelines for the care of experimental animals. All procedures of the current experiment have been approved by the Committee of the Faculty of Science of Assiut University, Egypt. Most larvae used in this study were under 6 days postfertilization (dpf) and according to the current European animal directive (2010/63/EU), larvae (5 dpf) are defined as nonanimals. 3. Results 3.1. Mortality and malformation rate To evaluate the possible toxicity of AgNPs (0, 25, 50, and 75 ng/L) on C. gariepinus embryos, the percentage of mortality rate and the percentage of malformations were measured. Table 1 represents the mortality of embryos exposed to AgNPs, which increased in a dose and embryonic stage-dependent manner. At the lowest concentration (25 ng/l), there was no significant variation in mortality. With increasing dosages, the mortality increased remarkably compared with that of the control. In addition, the percentage of malformations, as displayed in Table 1, increased significantly at high concentrations (50 and 75 ng/L) from 24 to 72 h-PFS; however, there was no significant difference in the percentage of malformations from 96 to 144 h-PFS. 3.2. Morphological abnormalities Exposure of C. gariepinus embryos to AgNPs induced a set of deformations in larvae. Abnormalities included notochordal curvature (lordosis, kyphosis, scoliosis, and C-shape curvature), finfold defects (reduction, blistering, and necrosis of the fin), and yolk sac edema (bag shaped, balloon shaped, and oval shaped odema). Representative photographs displaying these deformations are shown in Figs.1 and 2. 3.3. Enzymes and oxidative stress biomarkers

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In embryos from 24 to 72 h-PFS exposed to AgNPs (0, 25, 50, and 75 ng/L), the activity of G6PDH was significantly decreased in embryos from 96 to144 h-PFS; activity significantly increased then decreased with increasing concentrations (Table 2). In the instance of LDH, the activity was significantly decreased in a dose- and embryonic stage-dependent manner (Table 2). In embryos from 24 to 144 h-PFS exposed to 25 ng/L AgNPs, the total antioxidant capacity and the activity of catalase were significantly decreased in embryos from 24 to 144 h-PFS exposed to 50 and 75 ng/L AgNPs; the total antioxidant capacity and the activity of catalase activity increased with increasing concentrations but not significantly (Table 2). In embryos 24 to 144 h-PFS exposed to AgNPs (50 and 75 ng/L), lipid peroxidation was significantly decreased in the embryos at 48, 72, and 120 h-PFS; lipid peroxidation significantly increased then decreased with increasing concentrations (Table 2). 3.4. DNA damage As shown in Table 2, the percentage of DNA fragmentation of embryos exposed to AgNPs increased in a dose -and embryonic stage-dependent manner. At the lowest concentration (25 ng/L), there was a statistically significant difference in the percentage of DNA fragmentation except at 72 h-PFS and 144 h-PFS. With increasing dosages, the percentage of DNA fragmentation increased remarkably compared with that of control. In the present study, the cells of the control group give comets composed of a compact head with or without a very short tail, indicating intact DNA. Comet assays of AgNP-exposed embryos displayed a concentration-dependent increase in tail length as compared with cells of the control group. The tail moment scores were significantly different between groups (Fig. 3). 3.5. The notochord histopathology The embryos from 24 to 144 h-PFS exposed to AgNPs (0, 25, 50, and 75 ng/L) showed severely distorted and wrinkled notochord with a decrease in vacuolated cell size compared with the notochord of the control group, which have a regular shape composed of a notochordal sheath, notochordal epithelium, and vacuolated cells (Fig. 4). In AgNPs-exposed embryos, the somites appear compacted and disorganized compared with control embryos, which appear organized. 3.6. Ag-ions bioaccumulation:The pooling of embryos and water media was used to measure the concentration of Ag ions as Ag bioaccumulates during the period of exposure. The mean Ag level in the embryos and water media exposed to AgNPs for 6 days was found to be concentrationdependent (Table 3). 4. Discussion

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The increasing production, use, and consequent emission of AgNPs into the aquatic environment make it necessary to assess the environmental and health hazards that these compounds could exert. In the current study, the toxic effects of AgNPs were evaluated in embryos of a model organism C. gariepinus. The results reported in the present study show that the mortality and malformation rate of the embryos treated by AgNPs increased in a dose- and embryonic stage-dependent manner. An increased mortality rate and hatching retardation were also observed in zebrafish embryos [68, 36-39], larval fathead minnows [40], and Oryzias latipes embryos [41] trated with AgNPs. Odema may be the result of osmoregulation disruption, which could lead to malformations in fish [42]. In addition, malformations in the vertebral column could be caused by disturbances in osteogenesis [43]. The role of selenoprotein N (Sel N1) gene expression has been reported to be associated with notochord abnormalities and heart disease in zebrafish treated with AgNPs [9, 44]. The pattern and shapes of malformations were recorded in the embryos of C. gariepinus after exposure to UVA [15], lead nitrate [14], and 4-nonylphenol [45], in medaka embryos after UVA exposure [16], and in Bufo regularis tadpoles [46]. All of those malformations were dependent basically on the developmental stages and stressor dose. The toxicity of AgNPs is induced by the generation of ROS: the dissolution of metals from nanoparticles enhances the creation of free radicals causing oxidative damage [47-49]. All the biomarkers used in this study for health status and antioxidant evolution were affected by AgNP exposure. Fluctuations in those biomarkers values after exposure to AgNPs in different developmental stages were recorded in the current study, indicating the role of development mechanism, developmental stage, and gene switch on/off during embryogenesis. Similar results were reported in C. gariepinus after exposure to UVA [15], lead nitrate [14], and atrazine [50]. Wu and Zhou [41] showed a reduction in ROS concentrations in embryos treated by the induction of SOD and inhibition of GSH (total antioxidant capacity) or the oxidation of nanoscale zerovalent Ag with H2O2 [51]. Catalase activities were increased in zebrafish exposed to silver and ZnO nanoparticles due to free radical production [9, 52]. However, the inhibition of catalase by AgNPs may also lead to the accumulation of oxyradicals to cause oxidative damage [11, 53]. In contrast, Massarsky et al. [39] observed silver ions and AgNPs did not alter catalase and SOD activities. Leopold et al. [54] reported that the overexpression of G6PDH protects cells against the oxidative damage caused by higher levels of ROS. LDH plays a key role in metabolism and therefore, its inhibition may be due to ion imbalance or plasma membrane damage [55] and may also be due to the formation of enzyme inhibitor complexes [56]. This is one possibility of high lipid peroxidation currently with high level of TAC and catalase enzymes. In addition, Wu and Zhou [41] observed AgNPs increased the activity of LDH in medaka eggs, proposing that AgNPs impaired aerobic/anoxic balance and thereby inhibited hypoxia tolerance; thus, embryogenesis of medaka may be deteriorated [57]. In contrast, Reddy et al. [58] found no significant changes in the activity levels of LDH and G6PDH in selected tissues of AgNPexposed fish. The percentage of DNA fragmentation of embryos exposed to AgNPs increased in a dose and embryonic-stage manner. Sayed [22] and Arora et al. [59] indicated the presence of apoptotic DNA fragmentation after exposure to AgNPs, indicating cell death by apoptosis. Furthermore, it has been found that AgNPs can damage DNA (human lung fibroblasts, human 7

glioblastoma cells, human testicular embryonic carcinoma cells, and cultured cortical neurons) either by enhancing ROS generation or lowering ATP creation [7, 60, 61]. Moreover, AgNP treatment increased DNA fragmentation, the length of tail and percentage of DNA in the tails of Neries diversicolor coelomocytes [62] human liver and lung cells [63, 64] and Artemia [65]. Ag+ ions bind with DNA by covalent bonds and cause DNA fragmentation [66]. This is the mechanism by which we can explain the increase of DNA damage in dose and embryonic stage manner and fluctuations of other oxidative stress biomarkers. Tissue damage induced by AgNPs in fish were observed in catfish [67] but little information is available on the effects of nanoparticles on the histology the of whole larvae. In our study, the embryos showed severely distorted and wrinkled notochord with a decrease in vacuolated cells size and the somites appear compacted and disorganized compared with control embryos. Lashein [68] observed that a higher concentration of lead and cadmium caused distortion of notochord and disorganization of the peripheral red myogenic cells in the teleost fish Ctenopharyngodon idellus. Additionally, copper-exposed silver carp larvae (0.3 mg/L, at 26 °C) showed external lordosis, deflected spinal axis, and inhibition of skeletal ossification, which might have resulted from impaired ionic regulation [69]. Moreover, malformed notochord was recorded in the embryos of C. gariepinus and medaka exposed to lead nitrate and UVA, respectively [14-16]. In the current study, the mean Ag level in the embryos exposed to AgNPs for 6 days was found to be concentration-dependent. AgNPs were also reported to be taken up by the liver of rainbow trout [70] Danio rerio larvae before and after evacuation of the gut content [71], zebrafish embryos [6-8, 11, 72], serum of catfish [22], and the brain of medaka larvae [37]. 5. Conclusion AgNPs induced mortality, morphological malformation, antioxidant enzymes, and histopathological changes indicating cytotoxicity probably by oxidative stress. DNA fragmentation and comet assay denoted the genotoxicity of AgNPs. The developmental stage affects the biomarkers trends indicating the physiological, biochemical, and genetic changes during fish embryogenesis. 6. Acknowledgment This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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12

Fig. 1: Notochordal abnormality (body curvature) in the embryos of Clarias gariepinus after exposure to silver nanoparticles showing (a) control (30 h-PFS), (b) kyphosis (30 h-PFS exposed to 50 ng/L ), (c) C-shaped (30 h-PFS exposed to 75 ng/L) and (d) lordosis (30 h-PFS exposed to 75 ng/L). PFS = postfertilization stage, scale bar = 1 mm.

13

Fig. 2: Yolk sac odema and fin blistering in the embryos of Clarias gariepinus after exposure to silver nanoparticles showing (a) control (60 h-PFS), (b) oval-shaped odema (60 h-PFS exposed to75 ng/L), (c) bag-shaped odema (60 h-PFS exposed to75 ng/L) and (d) balloonshaped odema (60 h-PFS exposed to 50 ng/L). PFS = postfertilization stage, scale bar = 1 mm.

14

Fig. 3: Comet assay of embryos of Clarias gariepinus, exposed to silver nanoparticles showing comparison between the values of the mean tail moment score in different groups. PFS = postfertilization stag.

15

Fig. 4: Sections of the embryo of African catfish Clarias gariepinus following waterborne exposure to silver nanoparticles. (a) Control (72 h-PFS), (b) 25 ng/L (24 h-PFS) (c) 50 ng/L (24 h-PFS) and (d) 75 ng/L (96 h-PFS). PFS = postfertilization stage, N = notochord, SC = spinal cord, VC = vacuolated cells, NE = notochordal epithelium, CN = collapsed notochord, S= somites and CS= compacted somites.

16

Table 1: Effect of different concentrations of silver nanoparticles on the percentage of mortality and malformation rate during early developmental stages of the African catfish Clarias gariepinus, N = 3/treatment. Parameter

24-PFS#

72-PFS

96-PFS

120-PFS

144-PFS

5.66±0.88** 1.66±0.33

1.33±0.33

0.67±0.33

1±0.00

0.33±0.33

25 ng/L

5.33±0.88

2.66±0.67

1.33±0.33

1.33±0.33

3.67±0.88

2.67±0.67

50 ng/L

6±0.58

3±0.58

3.67±0.88

1±0.58

5±0.58*

3.67±0.88*

75 ng/L

8.66±1.02

5±0.58*

5±0.58*

3±0.58*

4±0.58*

2.67±0.67

Malformation

Control

1.33±0.33

0.67±0.33

0.33±0.33

1±0.58

3.66±0.88

2.33±0.88

rate %

25 ng/L

2.33±0.33

2.33±0.88

3±0.58

2.33±0.88

3.66±0.88

2.66±0.88

50 ng/L

9.33±0.88*

4±0.58*

4±0.58*

4±0.58

5.66±0.88

3.66±2.02

75 ng/L

14±1.16*

8.33±0.88*

2.33±0.88

3.33±1.20

3.33±0.67

2.66±0.88

Mortality

Group rate Control

%

#

PFS= postfertilization stage

48-PFS

* statistically significant difference (ANOVA, p < 0.05)

** The numbers as mean ± SE

17

Table 2: Effect of different concentrations of silver nanoparticles on the activity of G6PDH, LDH, total antioxidant capacity, catalase, lipid peroxidation, and DNA fragmentation during early developmental stages of the African catfish Clarias gariepinus, N = 3/treatment. Biomarker

Group

24-PFS#

G6PDH

Control

50.15±0.58* 13.38±0.26

5.54±0.29

(U/g)

25 ng/L

*

6.21±0.23*

50 ng/L 75 ng/L

48-PFS

72-PFS

96-PFS

120-PFS

144-PFS

0.98±0.02

0.48±0.01

1.01±0.06

5.25±0.20

1.12±0.06

0.54±0.03

1.29±0.09

31.96±0.89* 3.45±0.25*

6.03±0.08

1.38±0.10*

0.84±0.03*

1.82±0.1*

22.36±0.78* 3.12±0.08*

4.08±0.09*

0.44±0.03*

0.33±0.03*

0.48±0.03*

21.91±0.94* LDH

Control

1197±3.00

1322±16.59

1836±38.21

2343±31.27

1725±17.52

1638±4.01

(U/g)

25 ng/L

1165±18.39

1323±8.58

1735±18.49

2243±15.52

1589±4.60*

1484±3.34*

50 ng/L

1063±26.46

1087±1.60*

1656±7.68

2090±4.01*

1423±6.34*

1285±12.18*

75 ng/L

916.2±2.00* 848±16.7*

1236±22.66* 1744±33.71* 1213±3.00*

843.7±3.37*

Total

Control

54.23±3.73

55.5±3.73

53.53±4.63

antioxidant

25 ng/L

capacity

55.17±3.74

54.87±4.01

54.83±4.26

39.83±3.04* 40.57±2.90* 40.57±2.87*

40.8±3.27

39.97±2.84* 40.53±2.90

50 ng/L

48.46±0.87

48.63±0.99

47.93±0.87

47.16±0.69

49.5±0.57

48.4±1.14

75 ng/L

53.93±0.85

54.33±1.13

54.43±0.71

53.33±0.60

55.4±1.00

53.6±1.52

Catalase

Control

11.80±1.20

12.71±0.85

12.21±1.16

12.66±0.78

12.26±0.93

12.91±0.61

(U/ml)

25 ng/L

8.50±0.35

8.67±0.09*

8.82±0.07

9.00±0.25*

8.89±0.04

9.10±0.36*

50 ng/L

9.59±0.24

9.69±0.15

9.46±0.19

9.82±0.21

9.18±0.65

9.38±0.25*

75 ng/L

12.84±1.10

12.76±1.14

12.67±1.11

12.73±1.11

12.79±1.06

12.78±1.09

Lipid

Control

35.33±0.88

17.33±0.88

42.33±0.88

31.67±1.20

29.67±0.88

32.33±0.88

peroxidation

25 ng/L

35.33±0.88

53.33±0.88* 47.67±0.88*

35.67±0.88

45.33±0.33* 33.33±0.67

(nmol/mg)

50 ng/L

31.33±0.88* 47±0.57*

44.67±0.33

30.67±0.66

45±1.00*

75 ng/L

24.67±0.67* 44±1.00*

32.33±0.33*

28±0.58

33.67±0.88* 27.67±0.67*

DNA

Control

7.51±0.26

10.64±0.46

17.88±0.41

17.45±0.41

fragmentatio

25 ng/L

20.66±0.30* 31.91±0.47* 27.14±0.55

31.44±0.47*

24.04±0.51* 25.74±0.38

n%

50 ng/L

31.21±0.46* 40.47±0.60* 51.33±0.76*

45.41±0.67*

34.43±.051* 36.55±0.54*

75 ng/L

33.04±0.49* 51.24±0.76* 70.94±1.05*

48.11±0.71*

41.22±0.61* 38.55±0.57*

14.43±0.36

#

PFS= postfertilization stage

32±1.00

14.92±0.37

* statistically significant difference (ANOVA, p < 0.05)

** The numbers as mean ± SE

18

Table 3: Silver bioaccumulation concentration (ng/L) in embryo pooling and water media of 144 h-PFS African catfish exposed to different AgNPs concentrations (25, 50, and 75 ng/L), N = 3/treatment. Control

25 ng/L

50 ng/L

Embryo pooling

0 ± 0**

8±1.15*

12.33±0.88*

16.66±1.76*

Water media

0±0

13.3±0.66*

17±0.57*

21±1.52*

* Statistically significant difference (ANOVA, p < 0.05) ** The numbers as mean ± SE

19

75 ng/L