1. Introduction
Due to its toxicity, mercury became an international public concern, and it occurs in various forms and bioaccumulates in food webs where it can exert toxic impacts on the organism itself and its prey. Moreover, in some settings, inorganic mercury can be in high concentrations (e.g., some mines including gold artisan mines) and there is a need to know about the toxicity of inorganic mercury to local environments and fish species. The main purposes of this experiment were to reveal the acute toxicity and chronic histopathological effects of Hg(II) on the local fish Oreochromis niloticus. We found that the LC50 of fish for Hg (II) was 0.1435 mg-Hg(II)/L. After exposure to Hg(II), the pathological changes that occurred included pale gills, anemic eyes, and a whitish body color. Our findings confirm that histopathologically, internal organs of gills, liver, and hepatopancreas were greatly affected due to Hg(II) exposure.
The inflow of mercury (Hg) into an aquatic ecosystem occurs naturally as a result of mineral deposits, forest fires, volcanoes, oceanic emission, and crust degassing. The metal can also be released into ecosystems by human activities, such as smelters processing sulfide ores (i.e., in the production of metals such as iron, gold, copper, zinc, and lead), and other industrial activities, such as coal burning [
1,
2,
3]. Because of its considerable potential hazard to public health and the environment, Hg pollution has become an international public health concern, and many studies have reported the presence of Hg in the environment [
4,
5,
6]
The contamination of Hg compounds in marine environments is due to a natural phenomenon relating to anthropogenic discharge that contributes to the Hg flux increase and the alteration of chemical forms. Species of these altered forms may consequently elevate Hg levels in aquatic biota [
7,
8,
9]. An increase in methylation rates will also cause this effect. Ruelas-Inzunza et al., 2004, observed that when elemental Hg (Hg0) is released to the atmosphere, it transforms into a soluble species (HgII) [
10]. This mechanism is a transition from an inorganic compound into an organic form (mainly as methyl Hg), which is the first step in the accumulation process in aquatic biota. This form of Hg conversion can be increased by bacteria that occur under anaerobic and aerobic conditions. After bacterial accumulation and transformation to a neurotoxin, methyl Hg flows through food chains by biomagnification, and it eventually accumulates in other organisms and even humans [
11].
Fish are one of the most important organisms in the aquatic food chain ecosystem. They have the ability to accumulate Hg at 1000 times higher in concentration than their surrounding aquatic environment [
12]. Large-size marine organisms, such as dolphin and tuna, usually contain high levels of Hg. For medium-size fish, the Hg level is relatively small in their muscles. However, their internal organs, such as the liver, may contain much higher levels of Hg [
13].
Ruelas-Inzunza, et al., 2004, reported that the distribution and relative concentrations of Hg in the main tissues of penaeid shrimps mostly accumulated in the hepatopancreas, followed by the muscle, then exoskeleton [
10]. Limbong et al., 2003, reported that there was an increase in emissions of Hg from artisanal gold mining along the watershed of the three main rivers in North Sulawesi [
14]. In Indonesia, the contamination of the rivers with mercury is mostly caused by such mining activities and affects areas like Tawalaan, Bailang, and Kima River in North Sulawesi; Barito, Kahayangan [
14], Kr. Sabe River in Meulaboh, Aceh [
15]; Kr. Sikulat River in Sawang, Aceh [
16]; Wamsait River; and Kayeli Bay [
17]. Almost all gold ore from mining is treated with a direct amalgamation procedure and produces a low gold concentrate, although a relatively high Hg concentration was released to the river. Inadequate technical knowledge and lack of regulation are mostly the major problems in all related gold mining processes occurring in Indonesia [
14,
17].
Human exposure to Hg is largely from the consumption of fish and products from the application of aquaculture. The toxicity risk to ecosystems from contamination of Hg have been increasing and have majorly affected fish toxicity. Thus, it is important to monitor it. However, the extent of toxicity and distribution of Hg in fish organs are poorly understood. This includes its histopathological changes in fish living in the Hg-contaminated tropics and sub-tropics.
Most of the studies on fish exposure to Hg investigated the kinetics of its uptake [
18], and there are no available data on its effects on biochemical, survival, and heavy metal accumulation in Indonesian tropical fish species. Additionally, the histopathological examination has been increasingly recognized as a valuable tool for field assessment in its regard to the impact of environmental pollutants on fish [
19]. Specific lesions, that occur when the organs of a fish are exposed to toxic substances under laboratory conditions, help to identify biomarkers of exposure. The purpose of this work is to understand the acute toxicity (LC
50-72h) and chronic histopathological effects of Hg(II) on the freshwater fish
Oreochromis niloticus after they were exposed to water contaminated with Hg(II). The aim of this experiment is to also improve our knowledge of the tissue and cellular mechanisms of Hg toxicity in fish and to analyze organ distribution of Hg. To determine organ distribution and accumulation of Hg in the main tissues of organisms, levels of Hg in the head, eye, gill, bone, and muscle of fish were measured.
2. Materials and Methods
2.1. Materials
The local freshwater fish species, Oreochromis niloticus, were applied as test organisms and obtained from a stock culture, Fish Seed Breeding and Farming Center, in Jantho, Aceh Besar District, Indonesia. All chemicals (such as HgCl2, NaOH, etc.) were obtained commercially from FUJIFILM Wako Pure Chemical Corporation. Activated alumina (additive B) and a mixture of sodium carbonate and calcium hydroxide (additive M) were purchased from Nippon Instruments Co. (NIC). DOLT3 dogfish liver, as a reference material (Hg 3370 µg/g), was obtained commercially from Canada National Research Council. The experimental equipment used were a Vacuum Oven Eyella VOS 450SD, glassware (PYREX®), muffle furnace, analytical balance (Sartorius), and a desiccator (Pyrex), and heat-vaporization atomic absorption spectrometry (HV-AAS) was performed with a MA2000 automatic mercury analyzer (Nippon Instruments Corporation).
2.2. Toxicity Test
Upon arrival at the laboratory, the fish were immediately allowed to be acclimatized in tap water at room temperature (30 ± 2 °C), with a pH range of 7.6–7.8. A condition of 12:12 light-to-dark cycle of at least two weeks before an in vivo initiation test was applied. Fish were fed twice a day with Hg-free algae dried powder. The toxicity test was conducted based on the OECD Guidelines for Testing of Chemicals (Lammer et al. 2009) [
20]. Fish were exposed to different Hg concentrations for 72 h under controlled conditions using the static method in ponds. The six Hg concentrations used were 0.0012; 0.0049; 0.0141; 0.0524; 0.1126; and 0.5110 mg-Hg(II)/L. Hg-free controlled medium was also prepared with two replications for each. Ten fish (mean weight 80–100 g wet weight) were used for each designed concentration and mortality was thoroughly monitored, with the test being repeated three times. During the toxicity test, observations of the fish test sample were performed based on behavior, mortality, and anatomical pathology. Behavioral observations were carried out during the 72 h exposure period; each treatment group was observed periodically and recorded every 4 h. The behavioral indicators observed were restlessness, sudden jerks, swimming erratically, and lack of appetite. The preparation of the gills, liver, and hepatopancreas was done using a surgical instrument, in which afterwards the organs were processed into histopathological preparations. The changes were observed under the microscope and photographed using a special microscope.
2.3. Ethical Approval
The protocols in this research had ethical approval No. 34/KEPH/I/2018 (date 16 January 2018) from the veterinary ethics committee of the Faculty of Veterinary Medicine, Syiah Kuala University, Banda Aceh, Indonesia. The maintenance and animal application for this research based on the standard method was chosen with the approval of the committee.
2.4. Mercury Accumulation Test
Oreochromis niloticus specimens were exposed to 30 L of dilute medium containing 0.5110 Hg(II) under static conditions for 72 h within the pH range of 7.6–7.8. The fish were fed daily with Hg-free algae dried powder, equivalent to approximately 2% of their body weight. A control medium (Hg-free) was also prepared for two replications. After three days of observation, organs from each organism (bone, head, gill, muscle, and eyes) were dissected from each individual fish, lyophilized (Vacuum Oven Eyella VOS 450SD at −60 °C using liquid nitrogen), and grained.
2.5. Mercury Analysis in Fish Tissue
The total Hg in the tissues was analyzed using a heat-vaporization atomic absorption spectrometry (HV-AAS) and a MA2000 automatic mercury analyzer (Nippon Instruments Co., NIC, Ltd.). Hg(II) standard solutions were adequately diluted and added directly to the ceramic sample boats. According to the manufacturer’s protocol, samples of the organisms (30–40 mg) were added to ceramic boats with two types of additives; one consisted of activated alumina (additive B) and the other was a mixture of sodium carbonate and calcium hydroxide (additive M). All ceramics boats and additives were heated in a muffle furnace at 700–800 °C for 6 h to remove any background Hg. The standard solutions and samples in the boats were placed in the sample loader part of the analyzer to measure the total Hg concentration in the samples. All glasses and ceramic boat wares were cleaned by soaking with a cleaning solution followed by a Milli-Q water rinse before use. Quality control was assured by DOLT3 dogfish liver (Canada National Research Council) with a certified value 3370 µg-Hg/g for total Hg. Our analytical result of total mercury level was 3720.82 µg-Hg/g, and the recoveries of Hg were around 110.4%.
2.6. Histopathology Preparations
Hg-exposed fish were dissected after 96 h to obtain fractions of gills, hepatopancreas, and livers. The gills were fixed in 10% Davidson solution, whereas the hepatopancreas and liver were fixed using 10% formalin solution. Furthermore, the two organs were prepared for histopathological examination by applying the Haematoxylin and Eosin staining technique. Histopathological sample pieces were viewed under a light microscope by applying alternate magnifications to obtain clear images.
2.7. Data Analysis
The concentration used was calculated by the lethal concentration (LC50) using the probit method. Further data analysis was supported with software MiniTab® 16 version 2010. Microscopic examination was performed by looking at the gill and liver histopathology images, and they were analyzed descriptively and qualitatively, based on the changes that occurred. All results were then compared with the control.
