The Alteration Tissue Structure of Digestive Diverticulum of Anodonta woodiana Induced by Lead Renanda Baghaz

Accumulated the toxic compound such as lead in A. woodiana caused an alteration in the tissue. The aim of the study is observation through histology to determine the alteration of the structure of digestive diverticulum tissue. Anodonta woodiana were maintained in water tub that treated with lead II nitrate exposure (Pb(NO3)2) 0, 15, 25, 35 mg.L-1 for 7 days to determine the alteration of the damaged structure of the digestive diverticulum and determining water quality such temperature, DO and pH. The highest damage occurs in digestive diverticulum which treated with 35 mg.L-1 due to many alteration on the tissue structure such as hyperplasia, edema, atrophy, and necrosis with the percentage of total damage about 35%. The lowest alteration tissue structure occurs in 15 mg.L-1 treatment group of the digestive diverticulum. The temperature was observed about 25.3 – 26.4°C, pH about 6-7, and DO about 6.1-6.7 mg.L-1. Thus, the increment of lead dose treatment induces increment of tissue structure alteration in digestive diverticula tissue, therefore, might promote the death of A. woodiana.


INTRODUCTION 
The common pollutant of heavy metals such as lead (Pb) are considered as toxic pollutants.In addition, the natural aquatic ecology also contaminated with the heavy metals released by domestic activity [1].The organism or biomass contaminated with the heavy metals may have effect on human health risks [2,3].As bivalve, the suspension feeding activity of mussel represents the main pathway for heavy metal uptake and accumulation [4,5].Mussels are suspension feeders both aqueous and dietary; such as material resuspended from sediments.Therefore, it consisted of highmolecular-weight substances, microorganisms, fecal pellets and detritus [6,7].Mussels are organisms that commonly used to assess the ecotoxicological research in nature to analyze the effects of the compound that released from anthropogenic activities [8,9].In the previous study, Mussel was used to evaluate In Situ metal contamination in wastewater effluent and in aquatic ecosystems [10,11].The concentration metal in the tissue of mussel was increased concomitantly with the elevating of metal absorption or uptake and it showed the various metal bioaccumulation levels in different tissues of mussel [12,13].
Lead is a very toxic heavy metal [14] which cumulative [15] in the gills [16] and gastric [17].With histopathological analyses, we could observe the alteration of damaged tissue, such as gills and gastric [18] which affected by the contaminants.Lead penetrated the tissues of the body through the respiratory tract [19] and gastrointestinal [20].It induces the alteration tissue and a functional disorder in A. woodiana [21].
Anodonta woodiana (Kijing Taiwan) is a freshwater mussel found in waters with the muddy substrate.Kijing Taiwan lives in bottom waters and is relatively settled.Kijing Taiwan is a filter feeder organism that can filter water and is able to survive in the polluted environment, so it can be used as a bio-indicator of a water body.Lead induces the tissue damage which caused alteration of tissue structure in A. woodiana.The previous study, the histopathology of A. woodiana used to monitoring the pollution in aquatic environments and alteration structure of gill and kidney tissue observed [22].In another study, they observed enlargement of the gastric wall and diverticulum of A. woodiana due to exposure to pesticides [23].The other research on hyperplasia, atrophy, and necrosis occur in the gills of A. woodiana due to the Cadmium exposure [24].However, the tissue structure alteration of digestive diverticulum in A.

Lead-induced tissue alteration of A. woodiana digestive diverticulum (Putra et al)
woodiana still unknown.The aim of this study was to observed the alteration tissue structure of digestive diverticulum of A. woodiana induced by lead II nitrate exposure treatment for 7 days.

MATERIAL AND METHOD Data Collection
The descriptive qualitative method is used in determining the alteration histology digestive diverticulum of A. woodina and water quality in each treatment and data was collected with 3 times experiment repeatition.Anodonta woodiana in size 8-10 cm obtained from Freshwater Cultivation Management Unit (UP-BAT) Punten Batu, Malang and maintained in Workshop Laboratory in Faculty of Fisheries and Marine Science, University of Brawijaya, Malang.Six of A. woodiana were induced with Lead Nitrate for 7 days in each treatment and maintained in the circulation system water tub (30 liter volume).
Lead II nitrate [Pb(NO3)2] dissolved and diluted in the following concentration 0 mg.L -1 , 15 mg.L -1 , 25 mg.L -1 , and 35 mg.L -1 .Furthermosre, the water quality such as temperature, pH, and DO in each water tub was measured once in a day for 7 days.After 7 days, the stomach of mussels in each water tub was harvested and immediately, preserved in ddH2O containing 10% of formaldehyde.The stomachs were ready for staining and histology analysis.

Histotology analysis
Histology analysis was conducted according to Elsayed et al. [25].Briefly, the stomach of A. woodiana in each treatment was harvested after treatment and immediately fixed in neutral buffered formaldehyde 10%.Further, the stomachs were dehydrated using ascending grades of ethanol (70, 80, 90, and 100% for 1 hour in each concentration).The samples were then cleared in xylene.After blocking using soft paraffin, the stomachs were cut using the microtome to serial sections of 4 µm thickness.Then, sections were stained using hematoxylineosin stain.After staining, the stomachs were observed with microscope Olympus BX41 and scanned using Olympus CX21FS.The percentage of organ damage is calculated based on the following formula.

Statistical Analysis
Data are expressed as mean ± SD.Statistical significance of pairwise differences among three or more groups was determined using one-way analysis of variance (ANOVA) followed by LSD test.P<0.05 was considered statistically significant.The analysis was performed using SPSS for Windows (SPSS Inc., Version 20.0, Chicago, IL, USA).The Graph was performed using GraphPad Prism 7 (GraphPad Software, Inc. USA).

RESULT AND DISCUSSION The Morphology of Digestive Diverticulum Tissue Alteration
The alteration of digestive diverticulum tissue of A. woodiana in each treatment was shown in Figure 1.The digestive diverticulum induced with 15 mg.L -1 of Lead appeared edema and hyperplasia.The epithelia of digestive diverticulum showed swollen and some cells were grown more (Fig 1B).Further, the atrophy and necrosis of digestive diverticulum were found in A. woodiana which induced with 25 mg.L -1 of Lead (Fig. 1C).The digestive diverticulum was narrowed and its epithelia showed damage.In this treatment, it was revealed that the A. woodiana was not resisted to this concentration of Lead.The digestive diverticulum of A. woodiana was then found with more necrosis (Fig. 1D).In this treatment group, many alterations of digestive diverticulum was found.It was suggested that above 15 mg.L -1 of Lead, the A. woodiana was not resisted the toxicity of Lead.It might cause the death because the digestive diverticulum was damage and was not performed normally.Excretion of mucus is a form of A.woodiana self-protection against the toxic lead.The mucus contained the neutrophils that play a role in digesting foreign substances that penetrate to the body of this organism [24].
Previous study explained that the edema occurs as a form of cell adaptation to survive due to the influence of toxic materials [26].Enzymes are out of cells along these substances will inhibit cell metabolism.This will ultimately result in cell death (necrosis) [23].Necrosis is cell death that occurs because of hyperplasia redundant, so tissue no longer intact form or in other words, necrosis occurs accompanied by the death of an organism [27].

The Quantity of Digestive Diverticulum Alteration Analysis
The percentage of alteration on the digestive diverticulum induced by Lead was shown in Figure 2. The hyperplasia was increased significantly in 25 mg.L -1 and 35 mg.L -1 treatment groups about 7.8% and 9.6%, differently compared to 15 mg.L -1 treatment group about 3%.
Moreover, the necrosis was increased significant in 35 mg.L -1 treatment group around 12% compare than 25 mg.L -1 treatment group around 3.1%.In the present study, the edema and atrophy were not significant differences in each treatment group.However, the alteration of digestive diverticulum induced 35 mg.L -1 of Lead showed the highest hyperplasia and necrosis was also observed.
The total percentage of digestive diverticulum alteration was shown in Figure 3.The total alteration of digestive diverticulum was increased significantly in 35 mg.L -1 compare to 15 mg.L -1 and 25 mg.L -1 treatment groups.At this moment, the 35 mg.L -1 treatment group has the highest alteration tissue structure of 36% followed by 25 mg.L -1 for 22% and 15 mg.L -1 for 8%.
The alteration in gastric tissue begins with edema.The gastric has columnar epithelial cells.The Epithelial cells of the digestive organs column are eosinophilic with an elliptical core.Epithelial cells of the stomach started swelling due to growth.Hyperplasia in epithelial cells is a response of the epithelial cells which are eosinophils [28].
Eosinophils are immune properties of the body against substances that are toxic.On the composition of the cilia starting to look irregular.Thickening of cilia results in fusion of the cilium [29].In some parts, the gastric walls look thinner.This is caused by cilia and epithelial cells start to shrink (atrophy).Atrophy of the epithelial cells is a form of adaptive reaction to toxic substances that cause digestive activity is not running perfectly so that the needs of the supply of nutrients and hormones is insufficient

Water Quality Analysis
The treatment of Pb(NO3)2 was not affected the water quality.The present study showed the temperature, pH and dissolved of oxygen (DO) were inappropriate for the ecological life of A. woodiana.A good temperature for the growth of freshwater mussels are temperatures between 15°C-27°C [33].In the observation, pH and DO showed it is good for A. woodiana life.The pH is suitable for the environment for A. woodiana ranging from 6.5 to 7.8.If the pH in the water is more or less, the limit could affect the movement of cilia gills [24].According to Subarijanti, oxygen content which not contained toxic compounds, amounting to a minimum of 2 mg.L -1 is already sufficient to support the normal life of aquatic organisms [34].In general, the oxygen obtained taken through gills that produce currents that go into his coat [35].

CONCLUSION
These result showed that A. woodiana was not resisted with Lead toxicant in the concentration of Lead above 25 mg.L -1 .At concentration up to 25 mg.L -1 , the A. woodiana might death because the digestive diverticulum was not performed normally and it caused by any alteration of digestive diverticulum tissue structure.
[30].Edema and atrophy of the gastric epithelial cells cause the membrane of epithelial cells out of the proponent as well as causing dysfunction organ of the gastric and cause necrosis.Adaptive changes can develop into irreversible characterized by necrosis [31].

86
Lead-induced tissue alteration of A. woodiana digestive diverticulum(Putra et al)

Figure 3 .
Figure 3.The total percentage of digestive diverticulum alteration of A. woodiana after Lead II Nitrate (Pb(NO3)2) exposure for 7 days Results were the mean ± SEM;.*p<0.0001 was significant.Digestive diverticulum in bivalves accumulated pollutants, actively participates in the process of detoxification.Digestive diverticulum sensitive targets such as harmful pollutants.In the digestive diverticulum, occur Hyperplasia as indicated by increasing number of cells basophils seen by the assembly dots.Cells in the vesiculated digestive cell showed dominance in the digestive diverticulum.Depreciation cause the digestive diverticulum basophil cells occurred lyses and necrosis in the digestive diverticulum.Alteration in the gastrointestinal cells, basophils cell mass digestion, the out cells and alteration tissue such atrophy and necrosis of the digestive diverticulum, indicating the occurrence of

Figure 4 .
Figure 4.The water quality analysis in Anodonta woodiana tube after Lead II Nitrate (Pb(NO3)2) exposure for 7 days