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Abstract

The use of silver nanoparticles (Ag-NPs) is rapidly increasing, but there are limited data on their effects on the aquatic environment. The present study aimed to determine the acute toxicity and evaluate the effect of subacute concentrations of Ag-NPs (Nanocid®: average particle size of 61 nm) on hematological and plasma biochemical indices of silver carp, Hypophthalmichthys molitrix, after 3, 7 and 14 days. The 24-, 48-, 72- and 96-h median lethal concentration (LC₅₀) values of Nanocid for silver carp were estimated at 0.810, 0.648, 0.383 and 0.202 mg/L, respectively; 20% and 10% of the 96-h LC₅₀ values (0.04 and 0.02 mg/L) were selected for subacute study. Red blood cell (RBC) count, hemoglobin (Hb) count and hematocrit (Hct) level were significantly reduced at both concentrations tested (p < 0.05). White blood cell (WBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), cortisol and glucose levels in Nanocid-treated groups were significantly higher than the controlled group at experimental periods (p < 0.05). In conclusion, Ag-NPs intoxication resulted in erythrocyte reduction, hematological disturbances, leucocytosis and stress response in silver carp and offered a simple tool to evaluate toxicity-derived alterations.

Keywords

nanotoxicology fish Ag-NPs Nanocid®toxicity test

Introduction

Nanoparticles are particles that have one dimension that is 100 nm or less in size. Nanoparticles are increasingly being used, or being evaluated for use, in many fields. Silver (Ag) is one of the most commonly used nanoparticles due to its bactericide effect.¹ Products based on Ag nanoparticles (Ag-NPs), such as odor-resistant textiles, food packaging, cosmetics, household appliances and medical devices may release Ag particles (nanoparticles or aggregates) or Ag+ ions via effluent discharge into the aquatic environment.² Aside from interests in the potential applications of Ag-NPs, little information is available regarding their potential harmful effects on the environment and fish. Some studies showed that Ag-NPs can affect the physiology of different aquatic organisms such as fish, polychaete and freshwater alga.^1,3

–7 However; the toxicological effects of Ag-NPs on fish blood remain to be investigated.⁸

Hematological values are progressively used in fish as indicators of the physiological or sublethal stress response to endogenous or exogenous alterations and are more quickly reflected in the poor condition of fish than in other commonly measured.⁹ Silver carp, Hypophthalmichthys molitrix, is one of prime cultured fresh water fish in polyculture and of great economic importance. Moreover, Silver carp has been widely introduced in aquaculture ponds, lakes, reservoirs and sewage lagoons for biological control of plankton. So, the purpose of the current study was to determine the Median lethal concentration (LC₅₀) values (24, 48, 72 and 96 h) of Ag-NPs on silver carp and evaluate short-term effects (3, 7 and 14 days) of two subacute concentrations (20% and 10% of 96-h LC₅₀) on some hematological and plasma biochemical indices.

Materials and methods

Experimental animals

Live specimens of the silver carp (71.68 ± 5.87 g, 20.34±3.12 cm) were purchased from Bony Fish Propagation and Rearing Center of Sijeval (Bandar Torkaman, Gorgan, Iran). Fish were transferred to Aquaculture Research Center of Gorgan University of Agricultural Sciences and Natural Resources and acclimated to the laboratory conditions for 2 weeks. Fishes were parceled in 12 fiberglass tanks (500 L). The average values for aerated and dechlorinated tap water used during the both acclimation period and experiments were pH 7.79 ± 0.50, dissolved oxygen 7.93 ± 0.25 mg/L, temperature of 20.50 ± 1°C and total hardness 298 ± 2.35 mg/L as CaCO₃. Water was renewed daily, and the water quality parameters mentioned above were measured twice a week during the acclimation period and subacute toxicity test. Moreover, water quality parameters were monitored daily during acute toxicity test, with no substantial changes recorded throughout acclimation and experiments period (n = 4, post hoc, Tukey, p > 0.05). Water pH, temperature and dissolved oxygen were determined by Wagtech portable pH/temp meter and oxygen meter (Berkshire, UK). Water total hardness was determined using portable photometer with commercial kits provided by the manufacturer (Wagtech Portable Photometer 7100, Berkshire, UK). Throughout the acclimation and experiment periods, fish were held under a natural photoperiod conditions (11:13 light–dark).

Ag-NP characterization

In this study water-soluble form of colloidal, brown Ag-NPs with the commercial name of Nanocid® was used. It was concentrated at 4000 mg/L (Ag-NPs) with an average nanoparticle size of 61 nm. This was a P-series powder product by Nano Nasb Company, Tehran, Iran, for antimicrobial purposes. Size distribution of AgNPs in stock solution was analyzed by dynamic light scattering (Figure 1), and the particle form in exposure study was photographed by transmission electron microscopy (TEM) in Nano Nasb Pars Company, Tehran, Iran (Figure 2). The stock solution was added to the test water to achieve the target nominal Ag-NPs concentrations specific for each dose. Test solutions were prepared daily from the stock solution.

Figure 1.

Size distribution of nanoparticles in stock solution analyzed by dynamic light scattering.

Figure 2.

Form of nanoparticles used in exposure study photographed by transmission electron microscope (TEM).

Acute toxicity test

Only healthy fish, as indicated by their activity and external appearance, were maintained alive on board in a fiberglass tank. Fish were transferred to a 500-L aerated tank equipped with aeration with 200 L of test solution. The acute toxicity test was conducted following the Organization for Economic Cooperation and Development guideline under static renewable test conditions. Groups of 21 fish were exposed to 0, 0.01, 0.1, 0.5, 1, 2.5 and 5 mg/L Nanocid for 96 h. Values of mortalities were measured at 24, 48, 72 and 96 h, and dead fish were immediately removed by dip net to avoid possible deterioration of the water quality. The LC₅₀ values were calculated by EPA Probit Analysis V. 1.5 for 24, 48, 72 and 96 h.¹⁰

Clinical chemistry and hematology

Two concentrations (20% and 10% of 96-h LC₅₀) were selected for the assessment of the hematological and biochemical effects of Ag-NPs on fish. Just before experiment, eight randomly selected fish from acclimation tanks were used for hematological and biochemical analysis (day 0). For subacute toxicity assay, 108 silver carp were randomly distributed in nine 500-L fiberglass tanks. Every tank containing 12 fish were exposed to test solutions with the following concentrations of Nanocid: 0.0 (control), 0.02 and 0.04 mg/L, respectively. The test water in the fiberglass tanks was renewed daily and freshly prepared solution was added to maintain the concentration of Ag-NPs at a constant level.

After 3 days of exposure to subacute dose of Ag-NPs, eight fish from control and treatment groups were taken from one tank for hematological and biochemical study. Fish were anesthetized with clove oil at 300 mg/L and blood samples were taken from caudal vein, using nonheparinized syringes. Four fish remaining in each group after these procedures were moved in to clean water and removed from study. Similar methodology was followed for 7 and 14 days.

For the hematology tests, the blood samples were placed into tubes containing 1% ethylenediaminetetraacetic acid (EDTA) as the anticoagulant. The erythrocyte and leukocyte counts were determined by an improved Neubaeur hemocytometer, with Hayem and Turck diluting fluids, and hemoglobin (Hb) measurement was determined by the cianometahemoglobin method.¹¹ Capillary tubes containing blood were centrifuged for 3 min at 1000g and the hematocrit (Hct) was determined as percentage of packed cell volume. The corpuscular indices, including the mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), were calculated using standard formulas, and values were also determined for Hct, blood Hb percentage, and red blood cell (RBC) count.¹²

For the biochemical tests, the blood was placed in tubes and allowed to clot at room temperature (∼22°C), for 30 min. Serum was removed from the clotted sample after centrifugation at 3500 g for 5 min and frozen at −80°C until analysis. Glucose was measured using a spectrophotometric method (WPAS 2000-UV/VIS, Biochrom, Cambridge, UK) with reagents provided in standard analyses kits (Pars Azmon, Tehran, Iran). Cortisol was determined directly from serum using an ELISA kit (DRG Diagnostics, Mountainside, NJ, USA), as described by Shaluei et al.¹³

Statistical analyses

All results are expressed as mean ± SD. Statistical analyses were carried out using the computerized package PASW 18.0 (SPSS, Chicago, IL, USA) for Windows. Normality of data was first estimated using a Kolmogorov–Smirnov’s test and homogeneity of variance was assessed with Levene’s test. Based on these tests, all data were found to be normally distributed. To evaluate the effect of subacute exposure to Nanocid on hematological and biochemical indices, all data were subjected to one-way ANOVA followed by Tukey’s test at a 5% significance level (p < 0.05).

Results

Acute toxicity test

The results of the acute toxicity test for the Nanocid on H. molitrix are presented in Table 1. No mortality was observed in the control group during the experiment. Fish mortality increased significantly when the concentration and the time of exposure were increased. One hundred percent (100%) mortality was observed in fish exposed to 5, 2.5 and 1 mg/L dose of Nanocid for 96 h. No observed effect concentration (NOEC) at 96 h for Nanocid was 0.01 mg/L. The 96-h LC₅₀ value obtained for H. molitrix was 0.202 mg/L (confidence interval 0.142–0.281 mg/L). As expected, the 96-h LC ₅₀ values decreased with increase in exposure time. This indicates an increase in toxicity with exposure duration. Prior to death, fish exhibited rapid gill movement, nervous manifestations, erratic swimming, loss of equilibrium and inability to remain upright.

Table 1.

Median lethal concentrations (LC ₅₀) of Nanocid® (mg/L) at 24, 48, 72 and 96 h in silver carp, H. molitrix.

Time of exposure (h)	LC₅₀ (mg/L)	Confidence interval
Time of exposure (h)	LC₅₀ (mg/L)	Lower limit	Upper limit
24	0.810	0.664	1.001
48	0.648	0.484	0.810
72	0.383	0.259	0.505
96	0.202	0.142	0.281

H. molitrix: Hypophthalmichthys molitrix; LC₅₀: median lethal concentration.

Subacute toxicity test

No mortality was recorded during subacute exposure for all treatments. Results of blood profile of the controls and experimental fish under subacute exposure are given in Table 2. The value of total RBC count decreased significantly in 0.04 mg/L Nanocid at 3, 7 and 14 days and in 0.02 mg/L Nanocid at 7 and 14 days. Hct content decreased in 0.04 mg/L Nanocid at 7 and 14 days and in 0.02 mg/L Nanocid at 14 days. During the experimental period, Hct and Hb levels decreased significantly in both fish groups exposed to Nanocid at 7 and 14 days. However, WBC count was increased in both fish groups exposed to Nanocid at 7 and 14 days. Moreover, Nanocid exposure induced significant increase in MCH and MCHC at 7 and 14 days. Among the hematological indices, MCV value was comparable in all groups (Table 2). Effects of subacute Nanocid exposure on serum cortisol and glucose levels are presented in Figures 3 and 4. Serum cortisol levels in experimental fish were significantly higher than that in the control group. After exposure to Nanocid, the levels of glucose increased significantly after 3 days. However, the value of glucose decreased after the increment (Figure 4).

Figure 3.

Serum cortisol levels in silver carp exposed to sublethal concentrations of Nanocid (control: black circle, 0.02 (mg/L): black squares and 0.04(mg/L): black triangles) for 3, 7 and 14 days. Data are presented as mean ± SD (n = 8). Asterisks (*) indicate a significant difference (p < 0.05) between the control (day 0) and Nanocid-exposed animals (p < 0.05) and plus signs (+) indicate a significant difference (p < 0.05) from the same day controls.

Figure 4.

Serum glucose levels in silver carp exposed to sublethal concentrations of Nanocid (control: black circle, 0.02 (mg/L): black squares and 0.04 (mg/L): black triangles) for 3, 7 and 14 days. Data are presented as mean ± SD (n = 8). Asterisks (*) indicate a significant difference (p < 0.05) between the control (day 0) and Nanocid-exposed animals (p < 0.05) and plus signs (+) indicate a significant difference (p < 0.05) from the same day controls.

Table 2.

Derived hematological indices in silver carp, H. molitrix, exposed to subacute concentrations of Nanocid®.^a

Parameters	0 Days	3 Days	7 Days	14 Days
RBC (×10⁶ cells/mL)
Control	2.243 ± 0.179	2.200 ± 0.152	2.23 ± 0.147	2.304 ± 0.127
Nanocid (0.04)	2.243 ± 0.179	1.973 ± 0.082^*,+	1.514 ± 0.169^*,+	1.248 ± 0.180^*,+
Nanocid (0.02)	2.243 ± 0.179	2.097 ± 0.117	1.662 ± 0.144 ^*,+	1.563 ± 0.149^*,+
WBC (×10³ cells/mL)
Control	6.53 ± 0.23	6.43 ± 0.25	6.13 ± 0.19	6.64 ± 0.34
Nanocid (0.04)	6.53 ± 0.23	7.56 ± 1.16⁺	8.61 ± 0.95^*,+	9.18 ± 1.07^*,+
Nanocid (0.02)	6.53 ± 0.23	6.73 ± 0.77	7.75 ± 0.98^*,+	8.20 ± 0.71^*,+
Hb (g/dL)
Control	7.74 ± 0.51	7.78 ± 0.44	7.65 ± 0.56	7.99 ± 0.48
Nanocid (0.04)	7.74 ± 0.51	7.04 ± 0.48	6.36 ± 0.61^*,+	6.24 ± 0.90^*,+
Nanocid (0.02)	7.74 ± 0.51	7.04 ± 0.45	6.94 ± 0.40⁺	6.47 ± 1.05^*,+
Hct%
Control	27.87 ± 2.08	28.29 ± 2.32	26.52 ± 1.22	26.75 ± 2.16
Nanocid (0.04)	27.87 ± 2.08	24.63 ± 2.55⁺	19.18 ± 5.00^*,+	15.96 ± 2.81^*,+
Nanocid (0.02)	27.87 ± 2.08	26.48 ± 2.73	21.09 ± 2.29^*,+	19.22 ± 1.55^*,+
MCV (fL)
Control	124.88 ± 12.77	129.16 ± 13.76	119.03 ± 7.20	116.42 ± 11.64
Nanocid (0.04)	124.88 ± 12.77	125.29 ± 16.31	126.84 ± 47.40	129.77 ± 15.91
Nanocid (0.02)	124.88 ± 12.77	126.49 ± 14.13	127.69 ± 17.10	123.89 ± 14.63
MCH (pg)
Control	34.57 ± 1.92	35.45 ± 1.77	34.33 ± 2.73	34.76 ± 2.29
Nanocid (0.04)	34.57 ± 1.92	35.79 ± 3.28	42.38 ± 5.63^*,+	45.52 ± 8.87^*,+
Nanocid (0.02)	34.57 ± 1.92	33.68 ± 3.04	41.95 ± 3.04^*,+	41.68 ± 7.51^*,+
MCHC (%)
Control	27.91 ± 2.96	27.73 ± 3.33	28.90 ± 2.48	30.01 ± 2.23
Nanocid (0.04)	27.91 ± 2.96	28.87 ± 3.48	29.07 ± 4.17	35.00 ± 4.71^*
Nanocid (0.02)	27.91 ± 2.96	26.91 ± 3.71	33.24 ± 3.81	34.02 ± 7.61^*

H. molitrix: Hypophthalmichthys molitrix; RBC: red blood cell; WBC: white blood cell; Hb: hemoglobin; Hct: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration.

^a Effects of subacute concentrations of Nanocid (0.02 and 0.04 mg/L) on hematological indices were determined in silver carp (n = 8) after 3, 7 and14 days of exposure. Data are presented as mean ± SD. Asterisks (*) indicate a significant difference (p < 0.05) between the control (day 0) and Nanocid-exposed animals (p < 0.05) and plus signs (+) indicate a significant difference (p < 0.05) from the same day controls.

Discussion

Acute toxicity

The use of nanosilver is growing exponentially because of its strong antimicrobial properties. Ag-NPs may be released into aquatic environments from factory waste discharges, through leaks or spills during transportation and via materials containing Ag-NPs, while only little information is available on their possible ecotoxicological effects.¹⁴ LC₅₀ is the most widely accepted basis for acute toxicity test, and it is the concentration of a test chemical, which kills 50% of the test organisms after a particular period of exposure, usually 96 h. Results of the present study indicate that Ag-NPs is highly toxic to silver carp. Only a few acute toxicity tests have been conducted using Ag-NPs in fish. The acute toxicity results of the present work are also in agreement with the results of other workers. Griffitt et al. studied the acute toxicity Ag-NPs (particle size of 26.6 ± 8.8 nm) to zebrafish (Danio rerio) and reported 48-h LC₅₀ value as 7.07 mg/L in adult fish and 7.20 mg/L in larvae.¹⁵ Chae et al. found the 96-h LC₅₀ value of Ag-NPs (49.6 nm mean particle size, 99% purity) for Japanese medaka (Oryzias latipes) as 34.6 ± 0.9 µg/L.¹⁶ Examining Ag-NP toxicity with two different commercially available Ag-NPs, the work of Laban et al. reported the Ag-NPs 96-h LC₅₀ values of 1.25 and 1.36 mg/L for fathead minnow (Pimephales promelas).¹⁷ In addition, the 96-h LC₅₀ value of Ag-NPs (Nanocid) in Caspian roach (Rutilus rutilus caspicus) was found to be 0.028 mg/L.¹⁰ Review of toxicity of nanoparticles on fish by Shaw and Handy indicates that lethal concentrations are in the milligram per liter (mg/L), rather than the microgram per liter (µg/L) range.⁸ Toxicity of nanoparticles is influenced by several factors, such as their size, shape, chemical composition, aggregation characteristics, surface area, surface charge, and organism species. Among the large number of nanoparticles available, Ag-NPs are of specific concern for fish and the aquatic environment. Silver ions can be released from the surface of Ag-NPs. Dissolved silver ions in the environment are the most toxic metals to fish and other aquatic organisms, being lethal even at low concentrations (µg/L).¹⁶

Subacute toxicity test

Hematological and plasma biochemical indices can be useful for the measurement of physiological disturbances in stressed fish and thus used as reliable indicators for toxicological research and environmental monitoring and as indicators of disease and stress. There are no data on the effects of Ag-NPs on hematological indices in fish. In this study, a significant (p < 0.05) decrease in RBC count, Hb and Ht levels was recorded in the groups exposed to Nanocid compared to the control groups. Similar results were also reported in fish exposed to metals,¹⁷ pesticides¹⁸ and other toxicants.^19,20 The decrease in RBC count, Hb and Hct levels observed in this study may be indicators of acute anemia. Reduction in RBC count, Hb and Hct levels in toxicant-treated fish may be due to erythropoiesis disorder and the formation of RBCs. Some authors suggested that in toxicological experiments the decrease in RBC count, Hb and Ht levels could be related to the conditions of confinement or stress induced by the lack of food.²¹ Moreover, lysing of RBC due to toxicant stress may also lead to a reduction in Hb and Hct values in the fish.²² Lysing of RBC occurs in response to toxicity that leads to alteration in the selective permeability of the membrane.²³ Following these alterations, MCV, MCH and MCHC levels might change because they are calculated based on Hct, Hb and RBC values.

An increase in the number of white blood cells ((WBCs) leucocytosis) is a normal reaction of fish to substances that alter their normal physiological processes.²⁴ In the present study, significant increase in WBC count was observed in Nanocid-exposed groups as compared to the control group. The observed leucocytosis in H. molitrix indicated that Ag-NPs had an immediate stimulatory effect on the immune system of the fish. Similar increases in WBC count were found when fish were exposed to various pollutants, including metals^24,25 and pesticides.¹⁸ In vitro assays with macrophage culture system indicated that carbon nanotubes had immunotoxicity effect on rainbow trout.²⁶ Cortisol is the major corticosteroid produced by teleostean fish. Different stressors activate the hypothalamus–pituitary–interrenal (HPI) axis, resulting in a cortisol release, which causes secondary stress responses. In the present study, serum cortisol level increased significantly in Nanocid-exposed groups, and a parallel increase in WBC count were observed in a time-dependent manner (Figure 1 and Table 1). Conversely, some authors reported that stress induced immunosuppression in large part due to this corticosteroid.²⁷ Cortisol can affect distribution of leukocytes, result in lymphocytopenia and suppress the growth of lymphocytes.²⁷ It has been known for many decades that the changes in WBC counts after exposure to toxicant may be associated with a decrease in nonspecific immunity of the fish. Also increase in WBCs due to toxicological experiments could be related to infection that might have been caused by toxicant and also secondary infections that may be contracted after the weakening condition of the fish.

Increases in plasma levels in glucose reflect release of catecholamines and glucocorticoids from adrenal tissues of fish under stressful conditions.²⁸ In our study, the glucose level increased significantly in fish exposed to Nanocid, but the value of glucose decreased after the increment (Figure 4). Initial increase in glucose level was due to glycogenolysis to provide energy for the increased metabolic demands imposed by Nanocid stress. In the present investigation, the decrease in plasma glucose after 7 days might be due to the hypoxic conditions caused by Nanocid, leading to an excess utilization of stored carbohydrates.²⁹ Our own unpublished results indicated that Nanocid induced various pathological alterations in H. molitrix gills and might cause hypoxic conditions for animals. An increase in cortisol and glucose levels was observed in rainbow trout exposed to low µg/L concentrations of ionic silver (e.g. AgNO₃).¹⁶ Similar changes in the stress indicators were also reported by Farmen et al. in juvenile Atlantic salmon (Salmo salar) exposed to low µg/L concentrations of Ag-NPs.¹

In conclusion, our results indicate that inducing stress due to subacute exposure to Ag-NPs (Nanocid) causes erythrocyte reduction, hematological disturbances and leukocytosis in silver carp. Based on the results of this study, it is suggested that industrial and commercial applications of Ag-NPs should be more carefully and thoroughly assessed as to their potential toxic effects to the aquatic environment and fish.

Footnotes

Funding

We are grateful to the Gorgan University of Agriculture & Natural Resource for providing technical and financial facilities.

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Effect of subacute exposure to silver nanoparticle on some hematological and plasma biochemical indices in silver carp ( Hypophthalmichthys molitrix )

Abstract

Keywords

Introduction

Materials and methods

Experimental animals

Ag-NP characterization

Acute toxicity test

Clinical chemistry and hematology

Statistical analyses

Results

Acute toxicity test

Subacute toxicity test

Discussion

Acute toxicity

Subacute toxicity test

Footnotes

Funding

References