Abstract

Nanoparticles have been applied on different organisms to reveal their effect on them. A study was conducted to record the effect of silver nanoparticles (AgNPs) on gonadal (testes) histopathology indices of Indian minor carp Labeo bata (Hamilton, 1822). Gonadosomatic index (GSI) was calculated in control and treated male fishes (100µg/liter) for 14 days, 28 days, 42 days intervals and the histopathological alterations were correspondingly observed. Results showed that AgNPs cause significant reduction in GSI value of treated fish, indicating subsidence in their reproductive status. Further histological observation of testes in treated fishes showed definite alternation of structure in early stages of reproductive cells, disruption of Sertoli cells, Leydig cells, etc. Thus, AgNPs were found to exert significant effect on the reproductive structure and reproduction process in Indian minor carp Labeo bata (Hamilton, 1822) which may be a concern for their viability and sustenance in future.

1. Introduction

Recently human civilization is going through an era of nanotechnology by augmented utilization of different engineered nanoparticles in various fields. Among them silver nanoparticle (AgNP) is a vanguard member, well known by its multi-potential properties and extensive use in different fields like- electrical, health care, food and textile products ^{1, 2}. It has been reported that AgNP performs better in electric and electrical application and consequently global consumption is likely to increase in the near future ^{3, 4}. Globally, around 320-420 tons of AgNPs are produced annually which comprise one fourth (435 nano products) of total nanomaterials produced in recent times ^{5, 6}. Among its multiple applications, AgNPs role as antibacterial agents ⁷, use in bioremediation processes ^{8, 9}, wastewater treatment ^{10, 11} are noteworthy. Thus, increment in the field of application of AgNPs around human civilization along with increased release into the environment, particularly aquatic environment in an indiscriminate manner, has augmented the chances of adversely affecting aquatic fauna, particularly fishes ^{12, 13}. The adverse effect of AgNPs on fishes have already been recorded in rainbow trout (Oncorhynchus mykiss) ¹³, Japanese rice fishes (Oryzias latipes) ¹⁴ and zebra fishes (Danio rerio) ¹⁵. Till date, majority of studies on toxicity of AgNP on aquatic organisms have focused on the physiological, histological and behavioral changes of fishes, however, little attention has been paid to the effect of AgNP on the gonadal structure of Indian fishes. Hence, a study was designed to highlight the adverse effect of AgNPs on gonadal development of male Labeo bata (Hamilton 1822). Effect of varying concentration of AgNP on gonadal (testes) development of male Labeo bata (Hamilton 1822) under laboratory condition after 14, 28 and 42 days of exposure in pre-spawning phase of fish in terms of the GSI value and histopathological alteration of testes was also aimed to be studied.

2. Materials and Methods

AgNPs were synthesized in ecofriendly way by using bile which was collected from freshly sacrificed Labeo rohita (local name Rohu fish) weighing approximately 1 kg from the local market at Paschim Medinipur, West Bengal, India. The collected bile was then centrifuged and the supernatant was collected and diluted to 5% concentration using double distilled water. 5 ml 0.1 mM aqueous solution of silver nitrate was then mixed with 5ml aliquot of bile. The mixture was exposed to a microwave of 700W with 2450 MHz frequency for 30 sec. A precipitation of yellow colored AgNP was found to be developed which was used for further characterization ¹⁶.

The newly formed bile mediated AgNPs were examined using different techniques like, high resolution electron Microscopy (HRTEM), UV-Visible spectroscopy (UV-Vis), X-Ray diffraction (XRD), Field Emission Electron Microscopy (FESEM) and FTIR for complete characterization ¹⁶.

For experimentation, Indian minor carps, adult male Labeo bata (Hamilton, 1822) were collected from some selected fish-ponds at Midnapore town, West Midnapore district. The fishes were taken weighing between 75 gm to 90 gm. and sized between 20 cm to 25 cm. Fishes were acclimatized at laboratory condition over a period of one week prior to being transferred to the experimental set up. Four numbers of tanks were taken, each of which had been maintained with 12 males Labeo bata. In the first tank, fishes were maintained under controlled conditions without application of AgNP, while, the second, third and fourth tanks fishes were treated with AgNPs for 14, 28, and 42 days respectively with the concentration of 100 µg/L.

After specific intervals (i.e., 14, 28, 42 days) the total body weight and testicular weight of the 12 experimental fishes were taken to calculate the mean GSI using the following formula:

GSI= testes weight / total body weight x 100 (Parameswaram, 1974). The weight of the gonads was measured using monopan balance in the gram unit.

After weighing, the testes were cut into small pieces for better penetration of fixative and kept for 18 hours for fixation by using Bouin’s fixative. The fixed tissues were dehydrated through an upgraded series of ethanol, cleared by xylene and then embedded in paraffin (56°C-58°C). Sections of testes (4 µm thick) were stained by using hematoxylin- eosin (HE) and Mallory triple (MT) stains.

The stained tissue slides were mounted permanently using DPX and observed under compound microscope and photographed using a camera (Leica) attached to it.

3. Results

A pair of elongated testes of Labeo bata, situated within the abdominal cavity hanging from its dorsal wall usually becomes prominent during spawning season. In our study it was found that the normal GSI value of the pre-spawning phase of control fishes ranged from 0.478 ± 0.038. After exposure to AgNPs for 14, 28 and 42 days respectively the GSI values declined and were 0.385±.032, 0.323±.036 and 0.318±.034 respectively.

In controlled group of fishes, the testes were found to be enlarged with creamy white color. Seminiferous tubules were enlarged with the proliferation of spermatids (St) and spermatozoa (Sz) andZ leydig cells and connective tissues were seen between seminiferous tubules. Numerous clusters of primary and secondary spermatocytes appeared and spermatids and spermatozoa were also seen. Many interstitial cells occupied the intercellular space.

After 14 days of exposure, seminiferous tubules and lumens were seen roughly the same in size similar to the controlled set up. The overall structure of testes did not look well-ordered compared to the controlled group. There were slight signs of connective tissue splintering or breakage and the lumen was more dilated. The primary spermatocytes were the same in number but were found to be smaller than the control group.

After 28 days of exposure the size of the seminiferous tubules and the lumens increased and slightly disrupted some damaged Sertoli cells. Due to breakdown of seminiferous tubules spermatogonia and primary spermatocytes were found to be in close proximity. The spermatids were damaged structurally and the proportion of spermatogonia decreased. Increase in vascular or interstitial proteinaceous fluid and disrupted Leydig cells were distinctly documented.

After 42 days of exposure, the seminiferous tubules and connective tissue were much harder to see. The spermatozoa showed great clumping, seemed damaged and appeared fewer in number. Cytoplasmic coagulation was also seen. Leydig cells aggregated and occupied more space. Damaged sertoli cells were observed as well.

4. Discussion

Reproduction is a very essential physiological process in fishes which is controlled by internal physiological conditions as well as ambient external environmental factors ¹⁷. Different nanoparticles, as external factors, have toxic effects on different physiological processes including reproduction. Studies have shown a number of adverse effects of AgNP on Zebrafish ¹⁸, Danio rerio ¹⁹ and Rainbow Trout ²⁰. Likewise, AgNP also has a negative impact on renal physiology of Labeo bata ¹⁶. The silver nanoparticles, as it gets added into the aquatic environment, delays the normal reproduction process in male Labeo bata and when it occurs, the fishes try to skip the reproduction during that season. In our last study, it was seen that AgNPs affected the normal development of ovaries and hampered the oogenesis of female Labeo bata ²¹. GSI is a very essential parameter to determine the reproductive status and breeding period of fishes ²². In the present study the GSI value of controlled and AgNP treated fishes were determined. Labeo bata have five reproductive phases- a) Resting phase (November-December), b) Preparatory phase (January-March), c) Pre-Spawning phase (April-May), d) Spawning phase (June -July), e) post-spawning phase (August-October). The most active phase of the reproductive cycle (i.e., pre-spawning phase during April to May) when most of the developments occur within testes was targeted for study. In controlled fishes, GSI values were found to increase with the maturation of testes and reached the highest level on spawning phase. After treating with AgNP, GSI gradually decreased which indicates that the maturation of the testes was complete. In controlled fishes, the normal mean GSI value was 0.478±0.036, while, in treated fishes the value at 14, 28, and 42 days lowered to 0.385±0.036, 0.323±0.034, 0.318±0.034 respectively. Thus, the GSI values drastically decreased after 14 days and 28 days of exposure, but the change was very slight after 42 days of exposure. This indicated that the fish tried to adjust to the prevailing conditions, although the development of testes was hampered and skipped the annual reproduction phase and just survived in that environment.

The histology is another important factor to show the internal architecture of any organ. Dutta et al. ²³ showed the effect of endosulfan altering the testes structure of Lepomis macrochirus by histological observation. Kotil et al. ²⁴ also showed similar results by studying the histological alterations. Histological examination revealed marked effect of nonylphenol on testes structure in adult Zebrafish. Vajargah et al ²⁵ particularly pointed out that 3 ppm of AgNP solution is comparatively more harmful than 2 ppm and 1 ppm solution in long term on gonadal development of male gold fishes (Carassicus auratus gibelia). In a similar study, it was also shown that AgNP exposures cause concentration dependent bio-accumulation of Ag in gonads ²⁶. The present study inferred that the AgNPs entered into the fish through gills as the main entry point, passing through blood vessels and gradually reached the testes and hampered the normal structure of testes as well as the spermatogenesis process [Figure 2-3, Figure 2-4]. After 14 days, the lumen became dilated as the structure of the seminiferous tubules was not intact [Figure 2-2]. After 28 days similar structural dissociation continued [Figure 2-3]. The above-mentioned results revealed that the AgNP have negative impacts on the process of spermatogenesis. With mitotic division the spermatogenesis began and lead to new stem cells and primary spermatogonia. Through meiosis the spermatogonia differentiate into spermatozoa ²⁷. As mentioned, after 14 days, and 28 days size of spermatogonia reduced in treated fishes. The spermatids were also not found to be well developed. The total spermatogenesis process occurs under the influence of testosterone hormone levels which is secreted by Leydig cells ²⁸. In present study disorganized leydig cells were seen in treated fishes, which indicated that the testosterone level was insufficient causing the early stage of more reproductive cells. Presence of damaged sertoli cells is also seen. The damaged sertoli cells could have limited their effectiveness in providing nourishment.

5. Conclusion

AgNPs, when present in aquatic environments, hamper the normal development process of male reproductive system and disrupt the structure of testes of Labeo bata. Consequently, Indian minor carp, male Labeo bata, may face disturbance in sexual maturation which may directly decrease their number in future. Therefore, more attention should be provided before release of AgNP into the environment and their levels should be monitored regularly.

Acknowledgements

The authors are thankful to the Principal, Ramananda College, Bishnupur, Bankura, India for providing laboratory facilities to conduct the histological work. The authors are also thankful to the Principal, Sabang Sajanikanta Mahavidyalaya, Paschim Medinipur, India for AgNP study and also to the Head, Department of Zoology, Vidyasagar University, Midnapore. The author is deeply thankful to Souraditya Chakraborty for overall support.

References