DISQUIETING EFFECT OF ZNS NANOPARTICLES ON AN INDIAN MINOR CARP, LABEO BATA (HAMILTON, 1822) AND THE ASIAN DWARF STRIPED CATFISH MYSTUS VITTATUS (BLOCH, 1794) WITH RESPECT TO SOME OF THEIR VITAL ORGANS: A COMPARATIVE STUDY

Nilanjana Chatterjee 1 and * Baibaswata Bhattacharjee 2 . 1. Department of Zoology, Ramananda College, Bishnupur-722122, Bankura, India. 2. Department of Physics, Ramananda College, Bishnupur-722122, Bankura, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


Experimental:-Preparation and Characterization of ZnS Nanoparticles
The ZnS NPs are prepared by simple wet chemical method using zinc nitrate hexahydrate [Zn(NO 3 ) 2 ⋅6H 2 O] as zinc precursor and sodium sulphide (Na 2 S) as sulphur precursor [12,25].The as precipitated nanoparticles were filtered out and were washed for several times in distilled water and absolute alcohol (100% ethanol) and then were dried at 30∘C in a vacuum oven. The nanoparticles were characterized using X-ray diffraction study (XRD), Transmission Electron Microscopy (TEM), Particle Size Analysis (PSA), Energy dispersive X-ray study (EDX), and X-ray Photoelectron Spectroscopy (XPS). The synthesis process and characterization results have been discussed in detail elsewhere [12,25].

Measurement of dissolved oxygen content and pH value of water
Properly calibrated electronic lab meters with probes having accuracy up to one decimal point were used to measure the dissolved oxygen content and pH of water. A calibration curve was drawn and used to obtain the dissolved oxygen content in water under different experimental conditions.

Fish Husbandry
Matured live L. bata and M. vittatus of both sex groups were collected from local fishermen. After collection, fish were kept in water tight containers (capacity of 100 litres) containing tap water that has been allowed to stand for a few days. Fish are maintained at 25 ∘ -30 ∘ C of temperature to ensure the natural environment. Small, regular supplies of food were provided to the fish.

Toxicity test
Both the fish species were exposed to five concentrations (100, 250, 500, 750 and 1,000 μg/L) of the ZnS NPs of different sizes (3, 7, 12 and 20 nm) for different times of exposure (6, 12, 18 and 24 days) to discern the disquieting effects on liver, kidney and gills of the fish.

Histological and histometric study:-
After the controlled and treated fish were sacrificed; the liver, kidney and gills were dissected out and subjected to routine histological procedures. Upgrading or dehydration of hepatic, renal and branchial tissues was done by putting them for 10 minutes each (2 changes) in distilled water, 30%, 50%, 70%, and 90% ethanol, and finally absolute alcohol (100% ethanol). Dehydration using upgraded alcohol was followed by xylene treatment, paraffin embedding (melting point 56 ∘ -58 ∘ Celsius), section cutting (4 m), and staining using Delafield's Haematoxylin and Eosin (HE) before they were observed under a compound light microscope at different desired magnifications (X 400, X 600, X 800) and photographed with a digital camera.
For histometric analysis, hepatocyte and glomelular diameters were measured with reticulomicrometer and ocularmicrometer attached to the compound light microscope. Each measurement was made four times and their mean value was used for any analysis.
Statistical analysis and curve fitting:-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. Curve fitting to the experimentally obtained data was done using the software Origin 9.

ZnS NP induced hypoxia and environmental acidification
In the present study, the dissolved oxygen content in water (DO 2 ) was measured to be 8.9 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 2.9 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 photo-oxidation process of ZnS NPs, The S atoms exposed to the ZnS surface got oxidized and an increase in concentration of chemisorbed SO 2 at ZnS surface with increasing exposure time was observed in the samples [12]. The oxide leaves the surface as a molecular species (SO 2 ), leaving Zn and a freshly exposed layer of ZnS behind. Water may dissolve a part of the SO 2 released in the process causing reduction in the pH value of the water [12,26]. 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 [11,12,[20][21][22][23][24][25][26].
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.

Impact of ZnS nanoparticle exposure on 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 [27]. Therefore, liver histology can be used as an indicator to show the harmful effect of ZnS nanoparticles on L. bata and M. vittatus. Figures 1a, 2a show the histomorphology of L. bata and M. vittatus liver respectively in controlled condition portraying the liver cells in normal and healthy states. In these figures, liver cells are found to be large with regular outlines and dominated by 1602 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. For exposure to ZnS concentration of 100μg/L (Figure 1b, 2b), 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 1c, 2c). Therefore, 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 1c, 1d; 2c, 2d). Under high concentration exposure of smaller ZnS nanoparticles, some of the livers also show disruption of hepatic cell cords and apoptotic changes such as chromatin condensation and pyknosis as indicated by the green arrows in figures (Figure 1d, 2d).The histological alterations are found to be more pronounced in case of L. bata compared to M. vittatus. Figure 1: Photomicrographs showing the liver histology of L. bata under (a) controlled condition, (b) exposure to ZnS NP concentration of σ = 100 μg/L for 6 days, d = 3 nm, (c) exposure to ZnS NP concentration of σ = 500 μg/L for 6 days, d = 3 nm and (d) 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)].
These observations are indicative of degradation of liver cells under nanoparticle exposure. Due to the minimization of food intake under nanoparticle exposure, the hepatic cells of the fish are found to shrink and empty spaces generate in between them as they use the storage in the hepatocytes and fat vacuoles to maintain the metabolic activities of the fishes in this adverse condition. These effects can be associated directly with the changing feeding behavior, which in turn makes a detrimental effect on growth, maturity and spawning of the fish. Lakani et al. [28] reported reduced food intake and growth in Huso huso under hypoxia treatment. The present observation is also in agreement with previous findings that hypoxia affects both the appetite and growth rate of fish [29]. Decreased feed 1603 utilization might be an indicator of the higher levels of stress [30] and it could be an indirect mechanism by which prolonged hypoxia reduces growth and may be a way to reduce energy and thus oxygen demand [31].
The quantitative study on decreasing hepatocyte diameters for both fish species has been conducted to compare the adverse effect of ZnS NP on these fish. Figure 3 and figure 4 show the change in the values of hepatic cell diameter (δ) of L. bata and M. vittatus respectively with increasing nanoparticle concentration (σ). δ values are found to decrease with increase in σ value up to 500 μg/L for every size of the nanoparticles (d) used. Beyond this concentration, this value remains nearly constant. Data for both the fish species are fitted well with first order exponential decay curves expressed by the equation where δ 0 is the average hepatocyte size without any nanoparticle exposure, α is a parameter and inverse of τ determines the slope of the curve. Therefore smaller values of τ corresponds to the steeper curves. The curve fitting parameters under different experimental conditions are shown in the figure 3 (inset) and figure 4 (inset) for L. bata and M. vittatus respectively. An investigation on the slopes of the curves establishes undoubtedly that the detrimental effect is stronger for particles with smaller sizes in both the cases. From the τ values of tables it is also clear that for a fixed nanoparticle size; the detrimental effect is greater for L. bata compared to M. vittatus. These observations are indicative of degradation of liver cells under nanoparticle exposure. Due to the minimization of food intake under nanoparticle exposure, the hepatic cells of the fish are found to shrink and empty spaces generate in between them as they use the storage in the hepatocytes and fat vacuoles to maintain the metabolic activities of the fishes in this adverse condition. These effects can be associated directly with the changing feeding behavior, which in turn makes a detrimental effect on growth, maturity and spawning of the fish. Lakani et al. [28] reported reduced food intake and growth in Huso huso under hypoxia treatment. The present observation is also in agreement with previous findings that hypoxia affects both the appetite and growth rate of fish [29]. Decreased feed utilization might be an indicator of the higher levels of stress [30] and it could be an indirect mechanism by which prolonged hypoxia reduces growth and may be a way to reduce energy and thus oxygen demand [31].
The quantitative study on decreasing hepatocyte diameters for both fish species has been conducted to compare the adverse effect of ZnS NP on these fish.  When the fish are exposed to relatively lower concentration of ZnS NPs (σ ≤ 200 μg/L), the kidneys of the fish show shrinkage in glomerulus. In addition to that, dilution of tubular lumen is also observed. 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. 5b; 6b) 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. 5c; 6c). 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. 5d; 6d)   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). Similar type of qualitative variations is found in case of M. vittatus (data not shown). Strong negative correlation (r = -0.892 for L. bata and r = -0.826 for M. vittatus) 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 different concentrations of nanoparticle exposures in case of both the fish species. The lumen diameter of the collecting tubules are found to decrease (r = -0.704 for L. bata and r = -0.662 for M. vittatus) and increase in muscular wall thickness (r = 0.801 for L. bata and r = 0.752 for M. vittatus) are observed with increasing exposure time for a fixed concentration of ZnS NP for both the species. 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. In case of M. vittatus these effects become noticeable when the exposure time exceeds 12 days.

Impact of ZnS nanoparticle exposure on branchial histology:-
The fish gill is the basic site of respiration, ionic regulation, acid-base regulation, and excretion of nitrogenous wastes [32][33][34]. Three main cell types are present on the gill surface. The freshwater gill is known to alter its morphology in response to environmental variations, thereby adapting its structure and function to suit its environment [33,[35][36][37][38]. Hypoxia is considered as one of the stressors that cause rapid morphological change in fish gills. Histomorphological changes in fish gills under hypoxia can be attributed to the acclimatization process of the fish to encounter hypoxia by gradual elongation of the respiratory lamellae, expansion of their respiratory surface area and reduction of water-blood diffusion distance. The increase in respiratory surface area and reduction in water-blood diffusion distance should facilitate oxygen uptake during hypoxia. The changes in gill histomorphology occur more 1609 rapidly in the L. bata but the degree of morphological change of the gills is found to be lower relative to that observed in the M. vittatus. This observation may be a reflection of the more active lifestyle of the M. vittatus.

Conclusion:-
Two economically important fish species of different phylogenic group, Labeo bata and Mystus vittatus, has been exposed to ZnS NPs to study and compare the disquieting effect of the NP with respect to some vital organs viz. liver, kidney and gills of the fish. Exposure of ZnS nanoparticles in water results in significant depletion of dissolved oxygen content associated with reduction in pH value of water owing to the enhanced photo-oxidation property of the NPs. Both the fish species exposed to ZnS nanoparticles, responded to hypoxia with varied behavioural, physiological and cellular responses in a dose dependent manner in order to maintain homeostasis and organ function in an oxygen-depleted environment. Experimental observations suggest that the species L. bata is more vulnerable compared to the species M. vittatus against ZnS nanoparticle exposure when vital organs like liver, kidney and gills are concerned.