Key Words

ZnS nanoparticles; Photo-oxidation; Hepatocytes; Renal histomorphology; Morphometry

Introduction

Snowballing of nanotechnology and mounting uses of nanoparticles in sundry fields of sciences [1-3] have increased considerably the probability that the nanoparticles would end up in water courses either as chemical, medical, industrial or domestic wastes. ZnS nanoparticles (NPs) are one of such materials that can be found in the wastes of cosmetic, pharmaceutical and rubber industries. Apart from the various physiological disorders due to direct uptake of nanoparticles by the aquatic animals through different parts of their body [4- 10], ZnS nanoparticles are expected to exhibit some passive effects on aquatic environment by changing important physicochemical parameters of water due to its property of surface photo-oxidation [11]. Due to enhanced surface photo-oxidation property of ZnS in its nanoparticle form, the dissolved oxygen content in water is found to reduce in a dose dependent manner from their normal values, when ZnS nanoparticles of different sizes are exposed to the water in various concentrations [8, 11- 12]. This property is more prominent for ZnS nanoparticles with smaller sizes. Consequently under the exposure of ZnS NPs, the aquatic fauna of thatparticular habitat are forced to live in an oxygen depleted atmosphere [8, 11-12]. When living in a habitat with low level of dissolved oxygen, fish respond to hypoxia with varied behavioural, physiological, and cellular responses in order to maintain homeostasis and organ function in an oxygen-depleted environment [13-19].

Fish is one of the major sources of edible protein in India. Therefore, its reproduction has acquired prime importance to the investigators working in this area. Labeo bata is a species of freshwater Indian minor carp, found mainly in the rivers of India, Bangladesh and Myanmar. This species is very common, easy to cultivate and an important target species for the small-scale fishermen. Though this fish species has a high nutritional value in terms of protein and micronutrients, yet it is available in a relatively cheaper price in the fish market compared to some other fishes having equivalent nutritional values. These reasons make L. bata a very attractive candidate for aquaculture in the South East Asia.

The aim of our present study is to monitor systematically the adverse effect of ZnS NPs on histomorphology of liver and kidney of L. bata. This will also help to realise how the growth and maturity of the fish are being hampered when exposed to ZnS NPs. The changing behaviour in growth and maturity of any member of an aquatic environment due to exposure of nanoparticles may cause an adverse effect on the aquatic ecosystem as a whole. In the present case, it also has its detrimental effect on the commercial market of this fish.

Experimental

Synthesis and characterization of ZnS nanoparticles

ZnS NPs were synthesized employing simple wet chemical method as described by Chen et al. [20]. After synthesis the nanoparticles were characterized through Transmission electron microscopy (TEM), Particle size analysis (PSA), X-ray diffraction (XRD) study, Energy dispersive X-ray (EDX) study and X-ray photo electron spectroscopy (XPS) study. The process of synthesis and characterization procedures of the ZnS NPs were described in detail elsewhere [8, 12]. Different characterization techniques ascertained undoubtedly that stoichiometric, spherical ZnS nanoparticles of different sizes (3 nm, 7 nm, 12 nm and 20 nm) were acquired under different experimental conditions of synthesis technique [12].

Fish husbandry

Matured L. bata specimens of both sex groups caught by means of traditional fishing gear cast net and conical trap during daytime (10:00-15:00 hours) in monthly basis from different places of Hooghly and Bankura districts of West Bengal, India, were collected from the local fishermen during the period of September, 2011 to August, 2013. Immediately after collection, fishes were kept in watertight containers containing tap water that has been allowed to stand for a few days. A good supply of necessary oxygen was provided by using a large shallow tank to ensure that a large surface area of water was exposed to the air. Fishes were maintained at 25°- 30°C of temperature to ensure the natural environment. The fishes were fed on natural fish foods. Small, regular supplies of food were provided. The fishes were filtered out in every 10 days and are placed in fresh water

Histology and histometry

To study the hepatic and renal histology, liver and kidney tissues were dissected out and cut into small pieces for preservation in Bouin’s fixative for 18 hours. The tissues were then dehydrated through ethanol, C2H5OH (GR, Merck India) dried over activated molecular sieve zeolite 4A, cleared in xylene and embedded in paraffin of melting point 56°-58°C. Thin sections of 4 µm thicknesses were cut using a rotary microtome machine. The sections were stained with Delafield’s Haematoxylin and Eosin stain and were observed under a compound light microscope of high resolution and eventually photographed with a digital camera attached to the microscope.

The morphometry of hepatic and renal tissues were done using reticulo micrometer and ocular micrometer attached to the compound light microscope. Each measurement was made four times and their mean value was used for any analysis.

Toxicity test

Fish specimens were exposed to six concentrations (σ = 100, 200, 250, 500, 750 and 1000 μg/L) of the ZnS nanoparticles of different sizes (3 nm, 7 nm, 12 nm and 20 nm) for 6 days. Trials were conducted at various concentrations to observe the impact of ZnS nanoparticles on L. bata liver and kidney, comparing the hepatic and renal histomorphology of the exposed fishes to that of the fishes lived in controlled conditions. Electronic lab meters with accuracy up to one decimal point were used to measure the dissolved oxygen content and pH of the water.

Statistical analysis

All data were expressed as means ± SE. One-way analysis of variance was run to compare the differences between groups treated under different experimental conditions and control groups. Differences were considered statistically significant when p < 0.05. Pearson’s correlation coefficients (r) were calculated to determine the correlation, if any, between different hepatic and renal morphometric parameters and nanoparticle concentrations and exposure times at a significance level of 5%. Negative r values prefixed by negative (-) sign andpositive values without any prefix are used in the manuscript. Curve fitting to the experimentally obtained data was done using the software Origin 9.

Results and Discussions

ZnS NP induced hypoxia and environmental acidification

In the present study, the dissolved oxygen content in water (DO2) was measured to be 13.2 mg/L at 15°C before any nanoparticle was introduced in it. This value was found to decrease both with increasing nanoparticle concentration as well as nanoparticle exposure time in water at the same temperature. The value of dissolved oxygen content in water reached to as low as 3.9 mg/L for nanoparticles of size 3 nm at a concentration of 1000 μg/L and exposure time of 6 days.

The photo-oxidation of the surface of ZnS NPs using the dissolved oxygen of water under sunlight and consequent reduction of dissolved oxygen content in water has been confirmed from detailed study of S 2p core level X ray photoelectron spectra of ZnS nanoparticles after different time of exposures [12]. During the surface photooxidation process of ZnS NPs, The S atoms exposed to the ZnS surface got oxidized and an increase in concentration of chemisorbed SO2 at ZnS surface with increasing exposure time was observed in the samples [12]. The oxide leaves the surface as a molecular species (SO2), leaving Zn and a freshly exposed layer of ZnS behind. Water may dissolve a part of the SO2 released in the process causing reduction in the pH value of the water [11]. Subsequently under the exposure of ZnS NPs, the aquatic fauna of that particular habitat were forced to live in an oxygen depleted and acidified atmosphere [8, 11- 12].

In the present study, the pH value of water was found to decrease when exposed to ZnS NPs in a dose dependent manner for a fixed exposure time of 6 days. In controlled condition the pH value of the water used in this experiment was measured to be 7.6. This value was found to decrease both with increasing nanoparticle concentration as well as nanoparticle exposure time in water for a fixed nanoparticle size. The rate of reduction in pH value was found to be higher for the nanoparticles with smaller sizes. In our experiment, the pH value of water dwindled down to 4.8 for nanoparticle concentration (σ) of 1000 μg/L with size (d) 3 nm and exposure time (t) of 6 days. Reduction of water pH and consequent acidification of the environment finally lead the fishes to metabolic acidosis.

After the exposure of the ZnS NPs in the water, the Zn/S ratio in the nanoparticles was found to rise over that of the stoichiometric value of the freshly prepared samples confirming the loss of S from the surface of thenanoparticles. Surfaces of the ZnS NPs, exposed to water and light, were thus effectively destroyed by the redox cycles and resulted in the reduction of the dissolved oxygen content and pH value of water. This property was found to be more prominent for ZnS NPs with smaller sizes. This observation could be explained by the fact that smaller particle size culminated higher surface to volume ratio of the nanoparticles present in the water. Therefore, ZnS NPs having smaller sizes offered greater surface area, making the particles more sensitive to surface photooxidation process. This lead to a faster deficit in dissolved oxygen content and reduction in pH values when exposed to water compared to the samples having larger particle sizes.

Hepatic histology

The liver cell structure of teleosts responds very sensitively to environmental changes, e.g. in temperature, season, feeding conditions or presence of various chemicals in the water [21]. Therefore, liver histology can be used as an indicator to show the harmful effect of ZnS NPs on L. bata. Figure 1(a) shows the in situ position of liver in a female L. bata

Figure 1 (b) shows the histomorphology of L. bata liver in controlled condition portraying the liver cells in normal and healthy states. In this figure, liver cells are found to be large with regular outlines. These cells are dominated by storage deposits. The nuclei are found to be large and centrally located indicating the normal condition of the cells. The cells are found to be in close contact, almost no empty space is found between the cells

Figures 1(c)-1(e) show the effect of increasing nanoparticle concentration on the liver histology of L. bata. For exposure to ZnS concentration of 100µg/L (Figure 1c), few cells are found to be in degenerating states without a prominent nucleus and having diffused cytoplasmic contents. For higher concentration of ZnS nanoparticles (σ = 500 μg/L), decrease in cell sizes due to drastic loss of storage deposits is observed (Figure 1d). Therefore, the relative share of nucleus in cell volume is strongly increased. The cells are found to be in increasing isolated states having no close contact between them (Figure 1d). Under high concentration exposure (σ = 1000 μg/L) of smaller ZnS nanoparticles (d = 3 nm), some of the fish livers also show disruption of hepatic cell cords and apoptotic changes such as chromatin condensation and pyknosis as indicated by arrows in figures (Figure 1e). The histological alterations are more pronounced for exposure to nanoparticles of smaller sizes. This observation can be associated with the increasing surface reactivity of the nanoparticles with decreasing size. The observation is similar for male L. bata.

Figure 1: (a) Exposed thoracic and abdominal cavity of the female Labeo bata, showing the position of liver in situ in the thoracic region. Photomicrographs showing the liver histology of female L. bata under (b) controlled condition, (c) exposure to ZnS NP concentration of σ = 100 μg/L for 6 days, d = 3 nm, (d) exposure to ZnS NP concentration of σ = 500 μg/L for 6 days, d = 3 nm and (e) exposure to ZnS NP concentration of σ = 1000 μg/L for 6 days, d = 3 nm. In this case, livers tissues showed disruption of hepatic cell cords and apoptotic changes such as chromatin condensation and pyknosis as indicated by green block arrows in figure. [hepatocytes (hc), fat vacuoles (fv-white block arrows), blood vessels (Bv), empty space generated due to apoptosis ( ) and blood cells (Bc)]

Figure 2: Variation of the hepatic cell diameters (δ) against increasing nanoparticle concentrations (σ) with correspondingly fitted first order exponential decay curves for nanoparticles of different sizes (d) having fixed exposure time (t) of 6 days in female L. bata

Figure 2 shows the change in the values hepatic cell diameter (δ) for female fishes with increasing nanoparticle concentration (σ) for nanoparticles of different sizes (d) used, when the exposure time is fixed (t = 6 days). δ values are found to decrease with increase in σ value up to 500μg/L for every size of the nanoparticles (d) used and a fixed (t = 6 days) exposure time. Beyond this concentration, this value remains nearly constant. Strong negative correlation (r = – 0.798) is obtained between δ and σ for constant d (3 nm) and t (6 days). Analysis of covariance reveals significant differences between the δ values (p < 0.001) for nanoparticle exposures of different concentrations. A significant negative correlation (r = – 0.902) is revealed between NP exposure time and hepatosite sizes (σ = 500 μg/L, d = 3 nm) during the toxicity test. Also a significant negative correlation (r = – 0.843) can be demonstrated between exposure time and hepatosite density for a fixed nanoparticle concentration (σ = 500 μg/L, d = 3 nm). The percentage of empty space in the hepatic tissue lay out is found to increase (r = 0.712) with increasing exposure time for a fixed concentration of ZnS NP (σ = 500 μg/L, d = 3 nm). These observations become more prominent with decreasing nanoparticle sizes. Similar type of qualitative variation is found in liver histomorphology of male L. bata. Data presented in figure 2 are fitted well to the first order exponential decay curves represented by the following equation

where δ0, α and τ were the fitting parameters for the family of curves shown in figure 2. δ0 corresponded to the extrapolated value of hepatic cell diameter (δ) if the nanoparticle concentration (σ) reached infinity. The inverse of τ values determined the slopes of the fitted curves. Table I portrays the fitting parameters for the curves depicting the changes in the values of hepatic cell diameter (δ) with increasing nanoparticle concentration (σ) for nanoparticles of different sizes (d) having fixed exposure time of 6 days in female L. bata. From the slope of the curves, it can be established undoubtedly that the detrimental effect was stronger for particles with smaller sizes.

Table I

Fitting parameters for the curves depicting the changes in the values of hepatic cell diameter (δ) with increasing nanoparticle concentration (σ) for nanoparticles of different sizes (d) having fixed exposure time of 6 days in female L. bata

Nanoparticle size (d) (nm) δ0 (μm) α (μm) τ (μg/L) Reduced χ 2
3 9.380 11.103 167.134 1.540
7 10.060 10.296 211.027 1.405
12 14.247 6.138 237.224 0.438
20 14.564 5.798 355.829 0.275

These observations of alterations in hepatic histomorphology are indicative of degradation of liver cells under nanoparticle exposure. It has been reported that hypoxia can induce varied behavioural, physiological, and cellular responses among fishes [13-19]. Asian dwarf striped catfish Mystus vittatus is found to minimize their food intake when exposed to ZnS NP induced hypoxia [12]. Similar pattern of altered feeding behaviour can be noticed in L. bata in the present study. Due to the minimization of food intake under nanoparticle exposure, the hepatic cells of the fish are found to shrink and empty spaces generated in between them as they use the storage in the hepatocytes and fat vacuoles to maintain the metabolic activities in this adverse condition. These effects can be associated directly with the changing feeding behaviour, which in turn made a detrimental effect on growth, maturity and spawning of the fish.

Renal histomorphology

The kidney is a complex organ made up of thousands of repeating units called nephrons, each with the structure of a bent tube. Blood pressure forces the fluid in blood to pass a filter, called the glomerulus, situated at the top of each nephron. In L. bata two elongated kidneys

Figure 3: (a) Exposed thoracic and abdominal cavity of the female Labeo bata, showing the position of kidney in situ in the abdominal region. Photomicrographs showing the renal histology of female L. bata under (b) controlled condition, (c) for exposure to ZnS NP concentration of σ= 100 μg/L, (d) for exposure to ZnS NP concentration of σ= 500 μg/L and (e) for exposure to ZnS NP concentration of σ= 1000 μg/L [glomerulus (yellow arrow), Bowman’s capsule (Bc) and collecting tubules (ct)].

are of mesonephric type. Figure 3(a) shows the position of the kidneys in female L. bata. Figures 3(b)-3(e) show the renal histomorphology of L. bata under exposure of ZnS NPs of different concentrations having size (d) of 3 nm for fixed exposure time (t) of 6 days. The histomorphology of the controlled kidney tissues exhibit an ordinary pattern of renal corpuscles (consisting of glomerulus and Bowman’s capsule) and collecting tubules with no abnormalities in any other part of the renal cellular lay out as shown in figure 3(b). When the fishes are exposed to relatively lower concentration of ZnS NPs (σ ≤ 200 μg/L), the kidneys of the fishes show shrinkage in glomerulus anddilution of tubular lumen. For exposure to moderate value of ZnS NPs (σ = 250 μg/L), significant decrease in glomerular size (p < 0.001) and density (p < 0.001) are observed in the renal tissues of the exposed fishes (Fig. 3c) compared to that of the controlled fish. For exposure to relatively higher concentration of ZnS NPs (σ = 500 μg/L), significant decrease in the number density (p<0.001) of collecting tubules was noticed in addition to the previous observations (Fig. 3d). Exposure to even higher concentration of ZnS NPs (σ ≥ 750μg/L), results in vacuolization in renal cell lay out and hyaline degeneration of tubular epithelium. After exposure to the highest ZnS NP concentration (σ = 1000 μg/L) used in the experiment, necrosis and dispersed inter renal cells with pyknosis of some nuclei are observed (Fig. 3e) in L. Bata.

Renal morphometry

Figure 4 shows the change in the values glomerular diameter (D) for female fishes with increasing nanoparticle concentration (σ) for nanoparticles of different sizes (d) used, when the exposure time is fixed (t = 6 days). D values are found to decrease gradually with increase in σ values within the experimental limit for every size of the nanoparticles (d) used and for a fixed exposure time (t = 6 days). Strong negative correlation (r = -0.892) was obtained between D and σ for constant d (3 nm) and t (6 days). Analysis of covariance reveals significant differences between the D values (p < 0.001) for nanoparticle exposures of different concentrations.

a2

Figure 4: Variation of the glomerular diameters (D) with increasing nanoparticle concentrations (σ) with correspondingly fitted first order exponential decay curves for nanoparticles of different sizes (d) having fixed exposure time (t) of 6 days in female L. bata

A significant negative correlation (r = – 0.882) is revealed between NP exposure time and glomerulus size during the toxicity test, but no significant correlation can be demonstrated between exposure time and glomerulus density for fixed nanoparticle concentration. The lumen diameter of the collecting tubules is found to decrease (r = – 0.704) and increase in muscular wall thickness (r = 0.801) is observed with increasing exposure time for a fixed concentration of ZnS NP. Other time dependent histomorphological alterations in renal tissues is not quite prominent for relatively lower concentration of ZnS NPs (σ < 500 μg/L). When the exposure time exceeds 6 days for higher concentrations (σ ≥ 500 μg/L) of ZnS NPs, glomerular vacuolization and hyaline degeneration of tubular epithelium were seen in the renal histomorphology of L. bata. Similar qualitative variation was found for male L. bata.

Data of figure 4 are fitted well to the first order exponential decay curves represented by the equation

Where D0, A and T are the fitting parameters as shown in table II for the family of curves shown in figure 4. D0 corresponded to the extrapolated value of glomerular diameter (D) if the nanoparticle concentration (σ) reached infinity. The inverse of T values determined the slopes of the fitted curves. From the slope of the curves, it can be recognized indisputably that the harmful effect of ZnS NPs was sturdier for particles with smaller sizes.

Table II

Fitting parameters for the curves depicting the changes in the values of glomerular diameter (D) with increasing nanoparticle concentration (σ) for nanoparticles of different sizes (d) having fixed exposure time of 6 days in female L. bata

Nanoparticle size (d) (nm) D (μm) A (μm) T (μg/L) Reduced χ 2
3 49.167 48.063 500.844 0.185
7 57.468 38.327 665.272 0.492
12 59.257 36.869 911.404 0.105
20 56.990 39.477 1393.630 0.071

Ammonia is the primary metabolic waste product of most fishes including teleosts [22, 23]. Teleost freshwater fishes occupy an environment that is hypotonic relative to their tissues and, as a result, experience passive ion loss mainly across the gills [24, 25]. As the loss of ionic homeostasis can lead to severe metabolic impairment [26-28], teleost fishes employ mechanisms to actively take up ions, namely Na+ and Cl- , by reabsorbing ions across the nephron tubules, from the glomerular filtrate back into the blood. In addition, they actively transport ions across their gill surfaces from the surrounding water into the blood.

Hofmann and Butler [29] reported that there exists significant positive correlation between glomerular filtration rate and urine flow rate in rainbow trout, Salmo gairdneri. Glomerular filtration rate also showed linear relationship with oxygen consumption rate of the fishes. In the present work, ZnS NP induced hypoxia forced the fishes to lower their oxygen consumption rate for their metabolic activities. This is supposed to reduce the glomerular filtration rate as well as urine flow of L. bata under exposure to ZnS NPs. This can be attributed to the reduction in glomerular size and density of the exposed fishes as revealed from the histological micrographs

Acidification of the environment due to photo oxidation of ZnS NPs resulted in the enhancement of water H+ levels under experimental conditions. When L. bata were exposed to this water, the existence of H+ gradient from water to blood generated the situation of metabolic acidosis in the fishes reducing the blood pH level. In fish, metabolic acidosis stimulates an elevation in ammonia excretion at both the renal [30-34] and branchial [30, 31] epithelia, presumably as a means of facilitating acid-base regulation. Reduction in water pH had been resulted in a significant decrease in blood pH, a large reduction in plasma HCO3 levels, a severe impairment of swimming ability and an increase in Na+ influx in a teleost fish Oreochromis alcalicus grahami [35]

In the present study changes in plasma acid-base status and ionic composition along with the oxidative stress generated by ZnS NP induced hypoxia are supposed to induce the altered metabolic function in L. bata. This consequently reformed the renal activity leading to the other salient changes in renal histomorphology.

Conclusion

Indian minor carp Labeo bata suffered from salient alterations in hepatic and renal histomorphology owing to ZnS NP induced hypoxia and environmental acidification. Due to the minimization of food intake under nanoparticle exposure, the hepatic cells of the fish were found to reduce in sizes generating empty spaces in between them as they used the storage in the hepatocytes and fat vacuoles to maintain the metabolic activities of the fishes in this hostile condition. Onset of metabolic acidosis in the fishes as a consequence of the environmental acidification due to the photo oxidation of ZnS NPs resulted in the elevation in ammonia excretion at the renal epithelia. Under the combined effect of acidification and oxidative stress generated by ZnS NP in the habitat, L. bata were supposed to induce the altered metabolic function. As a result of this reformed the renal activity, salient changes in renal histomorphology were observed.