Meng-Hsien Chen

Mercury, organic-mercury and selenium in small cetaceans in Taiwanese waters

Total Hg (sigmaHg), organic-Hg (O-Hg) and Se bioaccumulations in small cetaceans distributed in Taiwanese waters of the Taiwan Strait and the southwestern Pacific have been investigated for the first time. The results could represent the baseline metal concentrations of marine mammals in the southwestern Pacific, where volcanic activities are possibly the major source of mercury to the environments. Muscle samples of four species of small cetaceans were collected from animals accidentally caught by tuna-longline fisheries from 1994 through 1995. In total, 53 pantropical spotted dolphins, Stenella attenuata, nine spinner dolphins, S. longirostris, five bottlenose dolphins, Turiops truncatus and four Rissos dolphins, Grampus giseus were analyzed. In addition, two stranded pantropical spotted dolphins were investigated. Cold vapour AAS and ICP-MS were used in the analysis of Hg and Se, respectively. Significant species difference was found in the four species of small cetaceans. Among them, the pantropical spotted dolphin showed the highest mean concentration (mg/kg wet wt.) of both sigmaHg (3.64 +/- 2.19) and O-Hg (2.79 +/- 1.23), whereas the Rissos dolphin had the highest mean concentrations of Se (1.77 +/- 1.29). There was no significant sex difference with respect to metal bioaccumulation in the samples of S. attenuata. Significant correlations between body length (BL) and sigmaHg, as well as O-Hg concentrations were observed in pantropical spotted (Sa) and spinner dolphins (Sl). The linear relationships were Sa: sigmaHg = -8.290 + 0.066BL, r = 0.421; Sl: sigmaHg = -2.735 + 0.025BL, r = 0.875; Sa: O-Hg = -3.723 + 0.036BL, r = 0.408; and Sl: O-Hg = -3.017 + 0.025BL, r = 0.870. However, a demethylation phenomenon that decreasing the percentage of O-Hg coupled with increasing levels of Se was observed when the sigmaHg concentrations in the muscle tissues of dolphins reached 4 mg/kg wet wt.

Total and organic mercury concentrations in the muscles of Pacific albacore (Thunnus alalunga) and bigeye tuna (Thunnus obesus).

Muscles of 115 North Pacific albacore (ALB, Thunnus alalunga) and 75 Pacific bigeye tuna (BET, Thunnus obesus), collected from 2001 to 2006, were analyzed. No ALB, but 13 large BET had organic mercury (OH g) concentrations exceeding 1 μg g(-1) wet weight. For both ALB and BET, total mercury (THg) and OH g concentrations were significantly and positively correlated with fork length (FL) and body weight. The muscle Hg bioaccumulation rates of BET were higher than those of ALB, particularly in the adult fish. Moreover, the lines had crossover points among the two species that imply the young BET (FL<110 cm) contains lower muscle Hg concentrations than ALB of the same size. The suggested weekly dietary intake of ALB and small-BET meats is 340 g, and of BET meat it is 150 g for a 60-kg person based on the provisional tolerable weekly intake (PTWI) of methylmercury set by the WHO.

Organic and total mercury levels in bigeye tuna, Thunnus obesus, harvested by Taiwanese fishing vessels in the Atlantic and Indian Oceans.

Muscle samples of 121 and 110 bigeye tuna (Thunnus obesus) caught by Taiwanese long-line fishing vessels in the Atlantic and Indian Oceans, respectively, were used to analyze total mercury (THg) and organic mercury (OHg) content. The overall THg and OHg concentrations were 0.786 ± 0.386 (0.214–3.133) and 0.595 ± 0.238 (0.143–2.222) mg kg−1 wet weight, respectively, similar to the results of previous studies. Our findings, however, reflected the highest THg and OHg concentrations for the species in each ocean among the published data. Mean THg and OHg concentrations in Atlantic tuna were significantly (p < 0.05) higher than those in Indian tuna. Two of 121 samples of tuna from the Atlantic Ocean, but no samples from the Indian Ocean, had levels of OHg above 2 mg kg−1 wet weight set by the Department of Health Taiwan, and 13 of 121 samples of tuna from the Atlantic Ocean and three of 110 samples from the Indian Ocean had levels of OHg above 1 mg kg−1 wet weight set by US FDA and WHO. Accordingly, for adult Taiwanese men and women with average body weight of 65 and 55 kg, respectively, the maximum allowable weekly intake of bigeye tuna is suggested to be 170 and 145 g, respectively.

Investigation of trophic level and niche partitioning of 7 cetacean species by stable isotopes, and cadmium and arsenic tissue concentrations in the western Pacific Ocean.

A total of 24 stranded or bycatch cetaceans, including Balaenoptera omurai, Lagenodelphis hosei, Kogia sima, Stenella attenuata, Grampus griseus, Neophocaena phocaenoides, and Sousa chinensis, were collected from 2001 to 2011 in Taiwan. Using the muscular δ(13)C and δ(15)N data, three ecological groups were identified as the oceanic baleen whale, the neritic, and the coastal toothed whale groups, coinciding with their taxonomy, feeding habits and geographical distribution. A horizontal inshore to offshore distribution was found for the sympatric neritic toothed dolphins, G. griseus, K. sima, S. attenuata, and L. hosei in the outermost offshore waters, accompanying their growth. For the first time we identify Taiwans Chinese white dolphin, S. chinensis, as an exclusive fish eater. Cd and As bioaccumulated in the G. griseus, L. hosei and S. attenuata increase as they grow. Prey-derived As- and Cd-induced health threats were found in L. hosei, and G. griseus.

Tissue concentrations of four Taiwanese toothed cetaceans indicating the silver and cadmium pollution in the western Pacific Ocean

Muscle, lung, kidney and liver tissues of 45 bycatch and stranded cetaceans, including 14 Grampus griseus (Gg), 7 Kogia simus (Ks), 10 Lagenodelphis hosei (Lh), and 14 Stenella attenuata (Sa), were collected in the waters off Taiwan from 1994 to 1995, and from 2001 to 2012. Baseline concentrations (in μgg-1 dry weight) of the cetaceans were lung (<0.05)=muscle (<0.05)<kidney (0.08±0.04)<liver (0.43±0.28) for Ag, and muscle (0.03±0.03)=lung (0.22±0.19)<liver (3.82±3.50)<kidney (16.22±18.81) for Cd. Unhealthy and critically dangerous Ag and Cd tissue concentrations in the toothed cetaceans are suggested. Marked high concentrations of Ag and Cd found in Gg and Lh are highly related to their squid-eating and deep diving habits. The highest ever recorded concentrations of liver-Ag and kidney-Cd were found in two Lh. These Taiwanese cetaceans indicate marked Ag and Cd pollution in the recent two decades in the western Pacific Ocean.

Immunotoxicity of Silver Nanoparticles (AgNPs) on the Leukocytes of Common Bottlenose Dolphins ( Tursiops truncatus )

Silver nanoparticles (AgNPs) have been extensively used and are considered as an emerging contaminant in the ocean. The environmental contamination of AgNPs is expected to increase greatly over time, and cetaceans, as the top ocean predators, will suffer the negative impacts of AgNPs. In the present study, we investigate the immunotoxicity of AgNPs on the leukocytes of cetaceans using several methods, including cytomorphology, cytotoxicity, and functional activity assays. The results reveal that 20 nm Citrate-AgNPs (C-AgNP20) induce different cytomorphological alterations and intracellular distributions in cetacean polymorphonuclear cells (cPMNs) and peripheral blood mononuclear cells (cPBMCs). At high concentrations of C-AgNP20 (10 and 50 μg/ml), the time- and dose-dependent cytotoxicity in cPMNs and cPBMCs involving apoptosis is demonstrated. C-AgNP20 at sub-lethal doses (0.1 and 1 μg/ml) negatively affect the functional activities of cPMNs (phagocytosis and respiratory burst) and cPBMCs (proliferative activity). The current study presents the first evidence of the cytotoxicity and immunotoxicity of AgNPs on the leukocytes of cetaceans and improves our understanding of environmental safety concerning AgNPs. The dose-response data of AgNPs on the leukocytes of cetaceans are invaluable for evaluating the adverse health effects in cetaceans and for proposing a conservation plan for marine mammals.

Investigation of silver (Ag) deposition in tissues from stranded cetaceans by autometallography (AMG)

Silver, such as silver nanoparticles (AgNPs), has been widely used in commercial products and may be released into the environment. The interaction between Ag deposition and biological systems is raising serious concerns because of one health consideration. Cetaceans, as the top predators of the oceans, may be exposed to Ag/Ag compounds and suffer negative health impacts from the deposition of these compounds in their bodies. In the present study, we utilized autometallography (AMG) to localize the Ag in the liver and kidney tissues of cetaceans and developed a model called the cetacean histological Ag assay (CHAA) to estimate the Ag concentrations in the liver and kidney tissues of cetaceans. Our results revealed that Ag was mainly located in hepatocytes, Kupffer cells and the epithelial cells of some proximal renal tubules. The tissue pattern of Ag/Ag compounds deposition in cetaceans was different from those in previous studies conducted on laboratory rats. This difference may suggest that cetaceans have a different metabolic profile of Ag, so a presumptive metabolic pathway of Ag in cetaceans is advanced. Furthermore, our results suggest that the Ag contamination in cetaceans living in the North-western Pacific Ocean is more severe than that in cetaceans living in other marine regions of the world. The level of Ag deposition in cetaceans living in the former area may have caused negative impacts on their health condition. Further investigations are warranted to study the systemic Ag distribution, the cause of death/stranding, and the infectious diseases in stranded cetaceans with different Ag concentrations for comprehensively evaluating the negative health effects caused by Ag in cetaceans.

Th2 cytokine bias induced by silver nanoparticles in peripheral blood mononuclear cells of common bottlenose dolphins (Tursiops truncatus)

Background Silver nanoparticles (AgNPs) have been widely used in many commercial products due to their excellent antibacterial ability. The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect animals at high trophic levels, such as cetaceans and humans, via the food chain. Hence, the negative health impacts caused by AgNPs in cetaceans are of concern. Cytokines play a major role in the modulation of immune system and can be classified into two types: Th1 and Th2. Th1/Th2 balance can be evaluated by the ratios of their polarizing cytokines (i.e., interferon [IFN]-γ/Interleukin [IL]-4), and animals with imbalanced Th1/Th2 response may become more susceptible to certain kinds of infection. Therefore, the present study evaluated the in vitro cytokine responses of cetacean peripheral blood mononuclear cells (cPBMCs) to 20 nm citrate-AgNPs (C-AgNP20) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Methods Blood samples were collected from six captive common bottlenose dolphins (Tursiops truncatus). The cPBMCs were isolated and utilized for evaluating the in vitro cytokine responses. The cytokines evaluated included IL-2, IL-4, IL-10, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. The geometric means of two housekeeping genes (HKGs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M), of each sample were determined and used to normalize the mRNA expression levels of target genes. Results The ratio of late apoptotic/necrotic cells of cPBMCs significantly increased with or without concanavalin A (ConA) stimulation after 24 h of 10 µg/ml C-AgNP20 treatment. At 4 h of culture, the mRNA expression level of IL-10 was significantly decreased with 1 µg/ml C-AgNP20 treatment. At 24 h of culture with 1 µg/ml C-AgNP20, the mRNA expression levels of all cytokines were significantly decreased, with the exceptions of IL-4 and IL-10. The IFN-γ/IL-4 ratio was significantly decreased at 24 h of culture with 1 µg/ml C-AgNP20 treatment, and the IL-12/IL-4 ratio was significantly decreased at 4 or 24 h of culture with 0.1 or 1 µg/ml C-AgNP20 treatment, respectively. Furthermore, the mRNA expression level of TNF-α was significantly decreased by 1 µg/ml C-AgNP20 after 24 h of culture. Discussion The present study demonstrated that the sublethal dose of C-AgNP20 (≤1 µg/ml) had an inhibitory effect on the cytokine mRNA expression levels of cPBMCs with the evidence of Th2 cytokine bias and significantly decreased the mRNA expression level of TNF-α. Th2 cytokine bias is associated with enhanced immunity against parasites but decreased immunity to intracellular microorganisms. TNF-α is a contributing factor for the inflammatory response against the infection of intracellular pathogens. In summary, our data indicate that C-AgNP20 suppresses the cellular immune response and thereby increases the susceptibility of cetaceans to infection by intracellular microorganisms.

Arsenic and five metal concentrations in the muscle tissue of bigeye tuna (Thunnus obesus) in the Atlantic and Indian Oceans

White muscle concentrations of As, Cd, Cu, Fe, Se, and Zn were investigated in Atlantic- and Indian-bigeye tuna (BET) (Thunnus obesus) from 6 regions. As and Cd muscle concentrations were significantly higher in the Indian-BET than in the Atlantic-BET, whereas the Indian-BET caught in the waters off South Africa revealed the highest As, Se, and Zn muscle concentrations. Accordingly, multidimensional scaling separated them into two oceanic groups. Positive linear relationships between muscle Cd concentration and fork length (FL) were established in both oceans. For the other elements, only muscle-Fe and FL relationship was found in the Atlantic-BET. 10.3% of BET > 145 cm FL from both oceans possessed muscle Cd concentrations exceeding the food safety limit (0.1 μg g-1 wet weight) set by the European Commission. Increased Cd, Cu and Zn pollution was found in the Atlantic Ocean compared with previous data, with higher levels found in the Indian Ocean.

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.