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Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Effects of Environmental Temperature Change on Mercury Absorption in Aquatic Organisms with Respect to Climate Warming a
a
b
a
a
Eun Chul Pack , Seung Ha Lee , Chun Huem Kim , Chae Hee Lim , Dea Gwan Sung , Mee Hye c
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a
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Kim , Ki Hwan Park , Kyung Min Lim , Dal Woong Choi & Suhng Wook Kim a
Department of Public Health Science, Graduate School, Korea University, Seoul, Republic of Korea b
College of Medicine, Inha University, Incheon, Republic of Korea
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National Institute of Food and Drug Safety Evaluation, Osong, Chungchungbook-Do, Republic of Korea d
School of Food Science and Technology, Chung-Ang University, Kyunggi-Do, Ansung, Republic of Korea e
College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea
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Department of Biomedical Science, Korea University, Seoul, Republic of Korea Published online: 24 Oct 2014.
To cite this article: Eun Chul Pack, Seung Ha Lee, Chun Huem Kim, Chae Hee Lim, Dea Gwan Sung, Mee Hye Kim, Ki Hwan Park, Kyung Min Lim, Dal Woong Choi & Suhng Wook Kim (2014) Effects of Environmental Temperature Change on Mercury Absorption in Aquatic Organisms with Respect to Climate Warming, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:22-24, 1477-1490, DOI: 10.1080/15287394.2014.955892 To link to this article: http://dx.doi.org/10.1080/15287394.2014.955892
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Journal of Toxicology and Environmental Health, Part A, 77:1477–1490, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.955892
EFFECTS OF ENVIRONMENTAL TEMPERATURE CHANGE ON MERCURY ABSORPTION IN AQUATIC ORGANISMS WITH RESPECT TO CLIMATE WARMING Eun Chul Pack1, Seung Ha Lee1, Chun Huem Kim2, Chae Hee Lim1, Dea Gwan Sung1, Mee Hye Kim3, Ki Hwan Park4, Kyung Min Lim5, Dal Woong Choi1, Suhng Wook Kim6
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1
Department of Public Health Science, Graduate School, Korea University, Seoul, Republic of Korea 2 College of Medicine, Inha University, Incheon, Republic of Korea 3 National Institute of Food and Drug Safety Evaluation, Osong, Chungchungbook-Do, Republic of Korea 4 School of Food Science and Technology, Chung-Ang University, Kyunggi-Do, Ansung, Republic of Korea 5 College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea 6 Department of Biomedical Science, Korea University, Seoul, Republic of Korea Because of global warming, the quantity of naturally generated mercury (Hg) will increase, subsequently methylation of Hg existing in seawater may be enhanced, and the content of metal in marine products rise which consequently results in harm to human health. Studies of the effects of temperatures on Hg absorption have not been adequate. In this study, in order to observe the effects of temperature changes on Hg absorption, inorganic Hg or methylmercury (MeHg) was added to water tanks containing loaches. Loach survival rates decreased with rising temperatures, duration, and exposure concentrations in individuals exposed to inorganic Hg and MeHg. The MeHg-treated group died sooner than the inorganic Hg-exposed group. The total Hg and MeHg content significantly increased with temperature and time in both metal-exposed groups. The MeHg-treated group had higher metal absorption rates than inorganic Hg-treated loaches. The correlation coefficients for temperature elevation and absorption were significant in both groups. The results of this study may be used as basic data for assessing in vivo hazards from environmental changes such as climate warming.
a potent neurotoxin (Ni et al., 2012) prone to bioaccumulation and biological expansion in coastal marine food webs, which are the main routes by which MeHg contacts or enters humans (Sunderland, 2007). Methylmercury is generated by methylation of inorganic Hg, which exists in coasts, deep-water sediments, major rivers, atmospheric deposition, and water columns, by microorganisms such as anaerobic sulfate-reducing bacteria (SRB), iron reducers (FeRP), and methanogens (MPA) (Avramescu et al., 2011). The routes by which Hg absorption occurs are diverse. In fish, cadmium is mostly absorbed in the organ related to ion transport, while
Heavy metals are the primary causes of environmental contamination, and mercury (Hg) is one of the most common contaminants that pose a major hazard by threatening human health globally (Jain, 2013; Carneiro et al., 2014). Mercury ranks third after arsenic and lead on the list of hazardous substances published by the U.S. Environmental Protection Agency (EPA) and the Agency for Toxic Substances and Disease Registry (ATSDR) of the U.S. Department of Health and Human Services. One of the biggest issues with Hg contamination is its trophic transfer and biomagnification in the aquatic food chain (Wang, 2012). In particular, methylmercury (MeHg) is
Address correspondence to Dal Woong Choi, Korea University, Seoul 136-703, Korea. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/uteh 1477
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Hg absorption occurs throughout the gills (Wicklund Glynn et al., 1994). Mercury, when it is absorbed through the gills or intestines, is sometimes absorbed through the channel, carrier, or transport that carries amino acids or other organic compounds. In combination with amino acids in the surrounding area, Hg forms an amino acid analog and is absorbed by passing through the gills or intestines with amino acids (Mason et al., 1996). Methylmercury also forms amino acid analogs by combining with amino acids and is known to be absorbed by forming large analogs similar to amino acids such as methionine or by passing through the cell membrane without being ionized in the form of CH3 HgCl, despite less effect than when MeHg passes through a specific route (Wu, 1997). Although the mechanism through which temperature affects the absorption of heavy metals in aquatic organisms has yet to be explained clearly, empirical data indicate that temperatures in a range typical of the environment exert negligible effects on metal speciation such as free ion activity. Therefore, the biological effects of temperatures need to be examined to explain their impact on metal absorption and accumulation (Sokolova and Lannig, 2008). Various stressors, including climate change, affect aquatic ecosystems (Dijkstra et al., 2011). The rise in water temperatures attributed to climate change may stimulate methylation of Hg. Simulations of ocean warming rates of 0.4◦ C and 1◦ C predicted increases in the mean MeHg concentration of 1.7% (range, 1.6–1.8%) and 4.4% (range, 4.1–4.7%), respectively, resulting in elevated MeHg concentrations in fish (Booth and Zeller, 2005). The toxicity and absorption of Hg are highly dependent on physicochemical factors, such as temperature, as well as biological factors. Therefore, warmer water is expected to affect Hg accumulation in aquatic organisms (Welfinger-Smith et al., 2011). Various studies investigated how absorption of heavy metals such as lead, cadmium, and copper by various species is affected by changes in water temperature (Cember et al., 1978; Tessier et al., 1994a; Khan et al., 2006; Guinot et al., 2012; Dijkstra et al., 2013).
E. C. PACK ET AL.
An analysis of surface water temperatures in East Asia showed a temperature rise of approximately 1–1.9◦ C over the last decade (Jeon, 2010). Methylation of Hg and changes in metal absorption in aquatic organisms, which are induced by warmer water, may in the future affect Hg exposure through the intake of marine food. Most studies on Hg focused on the metal concentration in organisms, primarily aiming to determine the potential risk to humans from consumption of fish (Tsuchiya et al., 2008; Nunes et al., 2014), but only limited data are available on absorption and accumulation of metal (Wang, 2012). In addition, studies that have investigated factors influencing the mechanisms underlying biological absorption and concentration of Hg focused on pH, chloride ions (Cl), dissolved organic carbon (DOC) concentration, and growth rate, while data have been limited related to metal absorption through the natural food chain (Boening, 2000; Li et al., 2008; Phillips et al., 2014). In particular, little is known regarding the effects of temperature on Hg and MeHg absorption. Thus, in this study, the effect of temperature variation was examined on absorption and toxicity of inorganic Hg and MeHg under lab conditions using the Chinese weather loach Misgurnus mizolepis.
MATERIALS AND METHODS Exposure Experiment Loaches used in this study are eurythermal fish that inhabit wide temperature ranges and are acceptable as an experimental model for studies of diverse external temperature gradients (Cho et al., 2012). Among the four genera and eight species known, Misgurnus mizolepis is currently the most frequently used in lab experiments (Nam et al., 2004). Misgurnus mizolepis has many advantages, such as fast and transparent embryonic development, relatively small adult size, short generation time, high fecundity, and well-established chromosomeset manipulation and transgenesis techniques (Liu et al., 2002; Nam et al., 2004) Unlike other
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ENVIRONMENTAL TEMPERATURE CHANGE AND Hg ABSORPTION
fish species, loaches exhibit intestinal breathing when oxygen is insufficient, wherein they inhale air at the water surface, exchange gases in the intestinal epithelia, and discharge air through the anus (Liu et al., 2002). Thus, it is known as a species that can survive well even in brooks, lakes, rice paddies, or swamps with low dissolved oxygen and large amounts of organic matter, and is resilient to water pollution, as it has an excellent ability to adapt even to rapid changes in the environments (Cho et al., 2009). The loaches (Misgurnus mizolepis) were allowed to adapt to the lab environment for approximately 2 wk in order to minimize their stress from environmental changes. Twentyfive fish were placed in each water tank used in the temperature stimulation test. The water tanks were installed with thermometers and heaters, and water temperatures in the tanks were set at 18, 23, or 28◦ C and maintained within an error range of ±0.5◦ C. Inorganic Hg (mercuric[II] chloride, Sigma, USA) or MeHg chloride (Aldrich, Germany) was added to the test water to make appropriate concentrations.
Survival Rate The acute impact presented in the standard test methods of the Organization for Economic Cooperation and Development (OECD), Office of Prevention, Pesticides, and Toxic Substances (OPPTS), European Commission (EC), or Korean Standards (KS) is generally assessed by lethal toxicity tests for 96 h regardless of the species (Santore et al., 2001; Lammer et al., 2009; Carriger et al., 2010). In this study, concentrations suitable for determining survival rates were selected through repeated preliminary experiments. Loaches were considered dead if they stopped breathing and did not swim even after being stimulated. Dead individuals were counted at 12, 24, 48, 72, and 96 h. After washing the specimens with distilled water and homogenization, total Hg and MeHg concentrations were measured.
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Measurement of Total Mercury The total Hg concentration in fish tissue is generally analyzed using the thermal decomposition process method. In this study, total Hg was determined using a Hydra-C mercury analyzer (Teledyne Leeman Labs Co., USA) that quickly and simply analyzes samples, as they are not previously pretreated. The frozen loach samples were thawed and homogenized before analyzing for total Hg. Approximately 0.1 g of homogenized samples was weighed and placed in a nickel boat dedicated to the total Hg analyzer without any separate pretreatment. The apparatus conditions for the measurement of the total Hg were drying set at 70 s at 300◦ C, decomposition set at 150 s at 850◦ C, waiting set at 100 s, and elution set to 30 s. As an Hg standard, a product from Wako Co. (Tokyo, Japan) was used. Measurement of Methylmercury To detect MeHg from food or fish samples, gas chromatography with electrochemical detector (GC-ECD) (Ahmad and Qureshi, 1989), gas chromatography–atomic absorption spectrometry (GC-AAS) (Puk and Weber, 1994) or gas chromatography-atomic fluorescence spectrometry (GC-AFS) (Marusczak et al., 2011) is generally used, and a direct mercury analyzer (DMA) (Maggi et al., 2009) or gas chromatograph–inductively coupled plasmamass spectrometer (GC-ICP-MS) (Yang et al., 2003) is also used. Among the apparatus, the method using GC-ECD is the most generally accepted analytical method; moreover, Japan and the Association of Analytical Communities (AOAC) have adopted GC-ECD as a method to analyze MeHg (AOAC, 1995). In this study, MeHg was determined using the GCECD method registered in the Korean Food Standards Codex of the Korea Food and Drug Administration. As a GC-ECD, a Clarus 500 (Perkin Elmer Co., USA) GC model installed with an ECD detector was used. All test solutions used were GC analysis grade or equivalent, and distilled water used was tertiary distilled water, GC analysis grade, or an equivalent.
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Pretreatment of Methylmercury Approximately 1 g of homogenized sample was added to 10 ml 25% sodium chloride and shaken for 10 min. Four milliliters of hydrochloric acid was added and the mixture was shaken vigorously for 5 min. Then 15 ml toluene (high-performance liquid chromatography [HPLC] grade, J. T. Baker, Phillipsburg, NJ) was added, and shaken for 10 min before extracting the solution. After centrifugation for 10 min at speeds of 4200 × g, the toluene layer was transferred to a funnel. Ten milliliters of 25% sodium chloride and 5 ml L-cysteine solution were added to the solution and shaken for 10 min. After leaving the solution alone for 10 min, the L-cysteine layer was divided with 4 ml 9 N hydrochloric acid solution and 5 ml toluene added to the solution and then shaken for 5 min before extracting the solution. The extracted solution was subjected to centrifugation (8500 × g, 10 min), and the toluene layer was separated and dehydrated employing sodium sulfate anhydrous powder (Junsei, Tokyo, Japan) to use it as test solution for the GC-ECD analysis. Methylmercury Analysis Conditions To efficiently analyze MeHg content using GC, an Ulbon HR-Thermon-Hg column (15 m × 0.53 mm ID, Shimadzu Co., Japan) dedicated to Hg was used. The MeHg dedicated column has disadvantages such as low durability and high costs that require more maintenance compared to general columns. However, it has the advantage of accurately separating MeHg and does not create many interfering peaks. The apparatus analysis conditions were an injector temperature of 200◦ C, a detector temperature of 230◦ C, and nitrogen as a mobile phase gas (flow speed: 8 ml/min). During analysis, oven temperatures were maintained at 50◦ C for 3 min, raised to 130◦ C for 10 min, maintained at 130◦ C for 1 min, raised to 220◦ C for 20 min, and maintained at 220◦ C for 4.5 min. The volume of the injected sample was 2 µl, and injection split rate was set at 5:1. The analysis result was compared to
that of Standard Reference Material 1946 (Lake Superior Fish Tissue, NIST), and MeHg standard stock solution used in the analysis was made by weighing 0.116 g MeHg chloride (Aldrich, Germany) and diluting it with toluene. As loss could occur in the process of pretreatment, the solution was analyzed after it underwent the same pretreatment process as that of the fish samples. Data Analysis Data summary showed the number of samples used in the analysis, means, and standard deviations. To analyze the association between temperatures of individuals administered with inorganic Hg or MeHg and mercury absorption over time, the Pearson correlation coefficient was used in the SPSS statistical program (SPSS version 12.0). The results were compared using a Student’s t-test or one-way analysis of variance (ANOVA) followed by a Newman–Keuls multiple range test. The criterion for significance was set at p < .05.
RESULTS AND DISCUSSION Survival Rates of the Inorganic Mercury-Treated Group To assess the acute toxicity of inorganic Hg within 96 h, the effects on survival rates of water temperature change and time were determined (Figures 1 and 2). No lethality was noted in the individual group treated with 0.1 mg/L or controls (Figure 1). In the 1 mg/L treatment, lethality was not observed at 18◦ C, whereas when temperature was maintained at 23◦ C, the survival rates decreased to 96, 67, 42, and 17% at 24, 48, and 96 h, respectively (Figure 2). When temperature was maintained at 28◦ C, the survival rates were 95, 83, 13, and 3% at12, 24, 48, and 72 h, respectively; all remaining individuals died thereafter. Overall, the 28◦ C group died earlier than the 18◦ C loaches. These results showed a tendency similar to studies conducted on crawfish (Del Ramo et al., 1987) and carp (Rehwoldt et al., 1972).
ENVIRONMENTAL TEMPERATURE CHANGE AND Hg ABSORPTION
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Survival rate of Misgurnus mizolepis (%)
80.00
60.00
40.00
20.00
0.00 HgCl
MeHg 18°C
23°C
MeHg 28°C
FIGURE 1. Survival rate of Misgurnus mizolepis after treatment with inorganic mercury (mercuric chloride; 0.1 mg/L) or methylmercury (MeHg; 0.1 mg/L). 100.00
Survival rate of Misgurnus mizolepis (%)
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80.00
60.00
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0 12 24 48 72 96
0 12 24 48 72 96
0 12 24 48 72 96
0 12 24 48 72 96
0 12 24 48 72 96
0 12 24 48 72 96
0.00
MeHg 18°C
MeHg 23°C
MeHg 28°C
FIGURE 2. Survival rate of Misgurnus mizolepis after treatment with inorganic mercury (mercuric chloride; 1 mg/L) or methylmercury (MeHg; 1 mg/L).
Survival Rates of the Methylmercury-Treated Group Figures 1 and 2 show the effect of water temperatures on survival rates over 96 h with acute MeHg exposure. No lethality was noted in the individual group treated with 0.01 mg/L or control. In the 0.1 mg/L treatment, when the temperature was maintained at 18◦ C, survival
rates decreased to 99, 91, 84, and 63% at 24, 48, 72, and 96 h respectively (Figure 1). At 23◦ C, the survival rates fell to 99, 91, 72 and 45% at 24, 48, 72, and 96 h, respectively. At 28◦ C, the survival rates were 94, 88, 80, 37, and 1% at 12, 24, 48, 72, and 96 h, respectively. In the 1 mg/L treatment, at 18◦ C, the survival rates diminished to 97, 76, 46, and 24%
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at 24, 48, 72, and 96 h (Figure 2). At 23◦ C, the survival rates were 87% at 24 h and 32% at 48 h, and all individuals died after 72 h; at 28◦ C, the survival rates were 79, 62, and 3% at 12, 24, and 48 h, respectively, and all individuals died after 72 h. Data are in agreement with the results of a study conducted on eels (Mallatt et al., 1986).
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Comparison of Survival Rates by Mercury Type The survival rates by Hg species were compared between the 0.1 mg/L and 1 mg/L inorganic Hg- or MeHg-treated groups (Figure 1 and 2). In the 0.1-mg/L treatment, loaches exposed to MeHg had lower survival rates with increasing temperature and time, while the group treated with inorganic Hg did not show any lethality (Figure 1). Comparing the groups treated with a metal concentration of 1 mg/L, at 18◦ C, the MeHgexposed group showed lethality after 24 h while inorganic Hg-treated did not produce any lethality (Figure 2). At 23◦ C, the MeHgtreated group began displaying lethality activities at 24 h, and all individuals died after 72 h. In the inorganic Hg group, although lethality was observed at 24 h, some individuals survived even after 96 h. At 28◦ C, MeHg and inorganic Hg showed lethality at 12 h. The MeHg-exposed group had 16 and 21% lower survival rates than inorganic Hg-treated group at 12 and 24 h, respectively, and all individuals died after 72 h (Figure 2). It is generally known that MeHg exerts greater toxicity and decomposes more slowly than inorganic Hg, thus producing further adverse effects. Methylmercury treatment in this study resulted in lower survival rates than inorganic Hg. Mercury is one of the metals with a potent toxicity in teleosts; 50% of sensitive teleost species were reportedly killed after 96 h of exposure to inorganic Hg with concentrations ranging between 0.036 and 0.098 mg/L (Eisler and Hennekey, 1977; Mayer, 1987). The threshold concentration of Hg chloride was reported to be 0.008 ppm in sticklebacks at a temperature of 15–18◦ C. However, tolerance
to inorganic Hg differs greatly among species (World Health Organization [WHO], 1989), and M. anguillicaudatus, a species similar to the M. mizolepis used in this experiment, was deemed to have a considerably high tolerance to inorganic Hg with a 24-h LC50 of 0.997 mg/L and a 48-h LC50 of 0.813 mg/L (Lin and Tang, 1989). Methylmercury exerts greater toxicity and has a lower lethal concentration than inorganic Hg. Poor egg insemination success (Khan and Weis, 1993), altered blood chemistry (Dawson, 1982), decreased respiration (Armstrong, 1979), and high accumulations (Choi and Cech, 1998) were reported at concentrations lower than the lethal concentration. The LC50 of MeHg for fish is reported to be in the range of 0.004–0.125 mg/L, depending on the species, which is far lower than the LC50 of inorganic Hg (WHO, 1989). The physiological mechanism underlying acute Hg poisoning was suggested to be asphyxia resulting from gill tissue damage and mucus coagulation (Jones, 1964). Amend et al. (1969) noted that death was attributable to necrosis and epithelial separation in the gills. Usually, metal toxicity in organisms rises in proportion to increasing temperatures by accelerating the toxicological mechanism (Rehwoldt et al., 1972; Cairns et al., 1975; McLusky et al., 1986; Heugens et al., 2001; Khan et al., 2006). In fiddler crabs and Uca pugilator, Hg transfer from gills to the hepatopancreas increased at higher temperatures, and the metal concentration was markedly different in each tissue. Vernberg and O’Hara (1972) postulated that such factors may play a role in toxicity of inorganic Hg in fiddler crabs. Quantity of Mercury Absorbed by the Inorganic Mercury-Treated Group The total Hg content in the 0.1 mg/L inorganic Hg-treated group are shown in Figure 3. At 24 h, the 0.1 mg/L inorganic Hg-exposed loaches maintained at 28◦ C possessed 4.2fold and 1.6-fold more total absorbed metal than groups maintained at 18◦ C and 23◦ C, respectively. At 48 h, the loaches maintained at 28◦ C showed 14.4- and 2.1-fold more total
absorbed metal than groups maintained at 18◦ C and 23◦ C, respectively. At 72 h, the group maintained at 28◦ C showed 2.1- and 1.5-fold higher total absorbed Hg than those maintained at 18◦ C and 23◦ C, respectively. The correlation coefficients for temperature increases and absorption were significant. The total Hg content of the group exposed to an inorganic Hg at 0.1 mg/L was compared (Figure 3). At 18◦ C, total Hg content increased to 95.62, 156.36, and 1100.65 µg/kg by 24, 48, and 72 h exposure, respectively, while at 23◦ C total metal content was elevated to 250.46, 1057.55, and 1540.53 µg/kg by 24, 48, and 72 h of exposure, respectively. At 28◦ C, total Hg content increased to 407.57, 2255.64, and 2278.21 µg/kg by 24, 48, and 72 h of exposure, respectively. Thus, total Hg content was elevated markedly over time as shown in Figure 3. Overall, total Hg content absorbed by loaches exposed to inorganic metal at different water temperatures was highest at 28◦ C, followed in order by 23 and 18◦ C. Mercury accumulation over time indicated increase in quantities absorbed associated with elevation in exposure time and water temperatures.
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Methylmercury Contents in the Inorganic Mercury-Treated Group The MeHg content in the experimental groups exposed to inorganic Hg at different concentrations and temperatures is depicted in Table 1. The average values were approximately 26.20 ± 11.21 µg/kg, and no significant differences were observed over time or at different temperatures. This indicates that methylation by enterobacteria in tissues in vivo did not occur (Falter et al., 1999). Differences in the total Hg content and survival rates seemed to be directly affected by inorganic Hg.
TABLE 1. Modification of Methylmercury Concentration After Treatment With Mercury Chloride (0.1 mg/L) Exposure concentration (mg/L) Control
0.1
18°C
23°C
Concentration of methyl mercury (µg/kg)
Temperature (◦ C)
24 h
72 h
18 23 28 18 23 28
27.86 ± 14.16 24.42 ± 13.00 24.87 ± 9.56 26.43 ± 10.52 28.52 ± 13.14 29.44 ± 18.36
24.12 ± 8.41 20.45 ± 11.54 21.42 ± 13.32 30.83 ± 9.51 29.34 ± 15.42 29.96 ± 9.35
Note. Each value is the mean ± SD.
3000
Total mercury concentration (µg/kg)
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ENVIRONMENTAL TEMPERATURE CHANGE AND Hg ABSORPTION
28°C
b
c
2500
2000
a b
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a
1000 c 500
0
a
b
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a 48 Exposure time (hr)
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FIGURE 3. Modification of total mercury concentration after treatment with mercury chloride (0.1 mg/L). Different letters indicate a significant difference at each time point (one-way ANOVA followed by Newman–Keuls multiple range test, p < .05).
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with data for total Hg of fish exposed to inorganic Hg. The total Hg contents were highest at 28◦ C, followed in order by 23◦ C and 18◦ C, and the quantities absorbed continuously rose over time.
The total Hg content in the 0.1 mg/L MeHg-treated groups at different temperatures is presented in Figure 4. At 24 h the group maintained at 28◦ C showed 4.7- and 1.6-fold more total absorbed metal than those maintained at 18◦ C and 23◦ C, respectively. At 48 h, loaches maintained at 28◦ C displayed 1.6and 1.2-fold higher total absorbed Hg than those maintained at 18◦ C and 23◦ C, respectively. At 72 h, the group maintained at 28◦ C showed 2- and 1.6-fold higher total absorbed Hg than groups maintained at 18◦ C and 23◦ C, respectively. The correlation coefficients for temperature increases and absorption were significant. The total Hg content of the groups exposed to MeHg concentration at 0.1 mg/L were compared (Figure 4). By 24, 48, and 72 h exposure, total Hg content increased to, 752.13, 2822.10, and 3417.25 µg/kg, respectively, at 18◦ C; 2157.74, 3728.95, and 4120.82 µg/kg, respectively, at 23◦ C; and 3516.57, 4440.31, and 6723.42 µg/kg, respectively, at 28◦ C. Overall, total Hg contents absorbed by loaches exposed to different concentrations of MeHg at different water temperatures were consistent
Methylmercury Content in the Methylmercury-Treated Group The methylmercury content in the 0.1 mg/L MeHg-treated groups at different temperatures is shown in Figure 5. At 24 h, loaches maintained at 28◦ C showed 4- and 1.7- fold greater absorbed MeHg than those maintained at 18◦ C and 23◦ C, respectively. At 48 h, the group maintained at 28◦ C displayed 1.9- and 1.4-fold more absorbed MeHg than loaches maintained at 18◦ C and 23◦ C, respectively. At 72 h, fish maintained at 28◦ C showed 2.2- and 1.6-fold higher absorption metal than groups maintained at 18◦ C and 23◦ C, respectively. The correlation coefficients for temperature elevation and absorption were significant. The total MeHg content of groups exposed to a metal concentration of 0.1 mg/L were compared (Figure 5). At 24, 48, and 72 h, loach MeHg content were 380.36, 1496.34, and
10000 9000 Total mercury concentration (µg/kg)
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The Quantity of Mercury Absorbed by the Methylmercury-Treated Group
18°C
23°C
b
28°C
8000 7000 6000
b
5000
c
4000 b
3000
b
a a
a
2000 1000 0
a
24
48 Exposure time (hr)
72
FIGURE 4. Modification of total mercury concentration after treatment with methylmercury (0.1 mg/L). Different letters indicate a significant difference at each time point (one-way ANOVA followed by Newman–Keuls multiple range test, p < .05).
ENVIRONMENTAL TEMPERATURE CHANGE AND Hg ABSORPTION
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8000 18°C
23°C
28°C
b
Methylmercury concentration (µg/kg)
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7000 6000 5000 a 4000
b
3000
a
a c
2000
a
b 1000 0
a 24
48 Exposure time (hr)
72
FIGURE 5. Modification of methylmercury concentration after treatment with methylmercury (0.1 mg/L). Different letters indicate a significant difference at each time point (one-way ANOVA followed by Newman–Keuls multiple range test, p < .05).
2550.21 µg/kg, respectively, at 18◦ C; 921.34, 2036.42, and 3525.14 µg/kg, respectively, at 23◦ C; and rose over time to 1536.41, 2857.32, and 5607.11 µg/kg, respectively, at 28◦ C. Overall, the quantities of absorbed MeHg were highest at 28◦ C, followed in order by 23◦ C and 18◦ C. Therefore, bioaccumulation seemed to be dependent on the temperature. Comparison of Quantities Absorbed Between Different Types of Mercury The total Hg absorption rates for fish exposed to 0.1 mg/L MeHg compared to loaches treated using inorganic Hg were higher by 7.9- (18◦ C), 8.6- (23◦ C), and 8.6-fold (28◦ C) at 24 h; by 18- (18◦ C), 3.5- (23◦ C), and 2-fold (28◦ C) at 48 h; and by 3.1- (18◦ C), 2.7- (23◦ C), and 3-fold (28◦ C) at 72 h (Table 2). The MeHgtreated group had higher absorption rates of total Hg than the inorganic metal-exposed fish. These different absorption rates support the differences in lethality between fish treated with inorganic versus organic Hg (Figures 1 and 2). Two major routes of metal absorption in aquatic organisms are ATP-dependent active transport through ion pumps and facilitated diffusion through ion channels (e.g., the Ca2+
channel) (Marshall and Bryson, 1998; Bury et al., 2003). Mercury is known to be absorbed through the transporter, or channel, or carrier (Mason et al., 1996). In particular, MeHg is highly soluble in fat, and therefore, approximately 50% of all of the capacity of intestines is first accumulated in muscle tissue (WHO, 1989), which leads to fast absorption and quick passage through sensitive tissues or neurons (Hodson, 1988). Such an absorption route is heavily influenced by environmental temperatures in poikilothermic animals. Therefore, rising temperatures increase metal absorption, which leads to enhanced metal accumulation in organisms since the elimination rate is not greatly affected by temperature (Denton and Burdon-Jones, 1981; Mubiana and Blust, 2007). Increased metabolism at elevated temperatures may result in metal accumulation in poikilothermic animals due to enhanced energy demand, and warmer temperatures are known to affect fish metabolism in relation to Hg accumulation. The increased demand for energy leads to an enhanced ventilation rate or feeding rate (Harris and Bodaly, 1998; Pörtner, 2002), which results in greater exposure to water or food contaminated with metals. Other studies on bivalves also found that
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TABLE 2. Comparison of Total Mercury Absorption After Treatment With Mercury Chloride (0.1 mg/L) or Methylmercury (0.1 mg/L) Concentration of total mercury (µg/kg) Exposure time (h)
Mercury type
18◦ C
23◦ C
28◦ C
24
HgCl2 MeHg HgCl2 MeHg HgCl2 MeHg
95.62 ± 53.29 752.13 ± 157.54 156.36 ± 48.32 2822.10 ± 417.27 1100.65 ± 209.80 3417.25 ± 526.14
250.46 ± 58.25 2157.74 ± 500.36 1057.55 ± 342.21 3728.95 ± 542.14 1540.53 ± 255.32 4120.82 ± 756.34
407.57 ± 70.34 3516.57 ± 743.17 2255.64 ± 396.04 4440.31 ± 712.41 2278.21 ± 512.36 6723.42 ± 2562.30
48 72
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Note. Each value is the mean ± SD.
the ventilatory activity was the rate-limiting step in metal uptake by such organisms (Massabuau and Tram, 2003). High ventilation rates in fish at elevated temperatures also produce high Hg absorption rates (MacLeod and Pessah, 1973). Mercury absorption has lower sensitivity to temperature than cadmium absorption, which is attributed to the fact that Hg is regulated by a slower biochemical mechanism (Tessier et al., 1994a, 1994b). Other studies also demonstrated the effect of temperature on dietary metal uptake (Hrenchuk et al., 2012; Guinot et al., 2012), a field that requires additional research (Sokolova and Lannig, 2008). This study demonstrated the effects of changes in external temperatures on Hg absorption in fish. Fish were exposed to inorganic Hg or MeHg and water temperatures were maintained at 18, 23, or 28◦ C to observe survival rates and quantities of total Hg or MeHg absorbed at different temperatures over time. The survival rates of fish treated with inorganic Hg or MeHg decreased in proportion to increasing external temperatures and metal concentrations. In addition, the MeHg-exposed groups had lower survival rates than the inorganic Hg-treated fish. The total Hg contents of inorganic Hg-exposed loach rose in proportion to external temperature elevation and exposure times, and all individuals showed significant correlations between warmer temperatures and adsorption. However, no changes in MeHg content in relation to temperature were observed. The total content of Hg and MeHg of methylmercury-treated groups rose in proportion to increasing temperatures and exposure time, and most individuals displayed significant correlations between warmer temperatures and
absorption. Methylmercury demonstrated significantly higher absorption rates than inorganic Hg in all individuals. Data showed that warmer water temperatures increased MeHg and inorganic Hg absorption and that the absorption rates of MeHg were higher than those of inorganic Hg. These results may be used as basic data to predict the potential adverse effects of Hg with water temperature changes resulting from climate warming, heavy metal pollution occurring when industrial wastes are combusted, and rising temperatures generated by waste heat emissions. FUNDING This research was supported by a grant (10162KFDA995) from the Korea Food and Drug Administration, an Institute of Health Science Grant, Korea University and a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0023938). ACKNOWLEDGMENTS The first two authors contributed equally to this work. REFERENCES Ahmad, S. and Qureshi, I. H. 1989. Fast mercury removal from industrial effluent. J. Radioanal. Nucl. Chem. 130: 347–352. Amend, D. F., Yasutake, W. T., and Morgan, R. 1969. Some factors influencing susceptibility of rainbow trout to the acute toxicity
Downloaded by [North Dakota State University] at 04:13 28 October 2014
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of an ethyl mercury phosphate formulation (Timsan). Trans. Am. Fish. Soc. 98: 419–425. Armstrong, F. A. J. 1979. Effects of mercury compounds on fish. In Biogeochemistry of mercury in the environment, J. O. Nriagu, 657–670. New York, NY: Elsevier/North Holland Biomedical Press. Association of Official Analytical Chemists. 1995. Official methods of analysis, ed. W. Horwitz. Washington, DC: AOAC. Avramescu, M., Yumvihoze, E., Hintelmann, H., Ridal, J., Fortin, D., and Lean, R. S. 2011. Biogeochemical factors influencing net mercury methylation in contaminated freshwater sediments from the St. Lawrence River in Cornwall, Ontario, Canada. Sci. Total Environ. 409: 968–978. Boening, D. W. 2000. Ecological effects, transport, and fate of mercury: A general review. Chemosphere 40: 1335–1351. Booth, S., and Zeller, D. 2005. Mercury, food webs, and marine mammals: Implications of diet and climate change for human health. Environ. Health Perspect. 113: 521–526. Bury, N. R., Walker, P. A., and Glover, C. N. 2003. Nutritive metal uptake in teleost fish. J. Exp. Biol. 206: 11–23. Cairns, J., Jr., Heath, A. G., and Parker, B. C. 1975. The effects of temperature upon the toxicity of chemicals to aquatic organisms. Hydrobiologia 47: 135–171. Carneiro, M. F., Grotto, D., and Barbosa, F., Jr. 2014. Inorganic and methylmercury levels in plasma are differentially associated with age, gender, and oxidative stress markers in a population exposed to mercury through fish consumption. J. Toxicol. Environ. Health A 77: 69–79. Carriger, J. F., Hoang, T.C., Rand, G. M., Gardinali, P. R., and Castro, J. 2010. Acute toxicity and effects analysis of endosulfan sulfate to freshwater fish species. Arch. Environ. Contam. Toxicol. 60: 281–289. Cember, H., Curtis, E. H., and Gordon Blaylock, B. 1978. Mercury bioconcentration in fish: temperature and concentration effects. Environ. Pollut. 17: 311–319.
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Cho, Y. S., Kim, B. S., Kim, D. S., and Nam, Y. K. 2012. Modulation of warm-temperatureacclimation-associated 65-kDa protein genes (Wap65-1 and Wap65-2) in mud loach (Misgurnus mizolepis, Cypriniformes) liver in response to different stimulatory treatments. Fish Shellfish Immun. 32: 662–669. Cho, Y. S., Lee, S. Y., Kim, K. Y., and Nam, Y. K. 2009. Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): Gene structure, genomic organization, and mRNA expression analysis. Comp. Biochem. Physiol. B 153: 317–326. Choi, M. H., and Cech, J. J. 1998. Unexpectedly high mercury level in pelleted commercial fish feed. Environ. Toxicol. Chem. 17: 1979–1981. Dawson, M. 1982. Effects of long-term mercury exposure on hematology of striped bass, Morone saxatilis. Fish. Bull. 80: 389–392. Del Ramo, J., Diaz-Mayans, J., Torreblanca, A., and Nunez, A. 1987. Effects of temperature on the acute toxicity of heavy metals (Cr, Cd, and Hg) to the freshwater crayfish, Procambarus clarkii (girard). Bull. Environ. Contam. Toxicol. 38: 736–741. Denton, G., and Burdon-Jones, C. 1981. Influence of temperature and salinity on the uptake, distribution and depuration of mercury, cadmium and lead by the black-lip oyster Saccostrea echinata. Mar. Biol. 64: 317–326. Dijkstra, J. A., Buckman, K. L., Ward, D., Evans, D. W., Dionne, M., and Chen, C. Y. 2013. Experimental and natural warming elevates mercury concentrations in estuarine fish. PLoS One 8: e58401. Dijkstra, J. A., Westerman, E. L., and Harris, L. G. 2011. The effects of climate change on species composition, succession and phenology: A case study. Global Change Biol. 17: 2360–2369. Eisler, R., and Hennekey, R. J. 1977. Acute toxicities of Cd2+ , Cr+6 , Hg2+ , Ni2+ and Zn2+ to estuarine macrofauna. Arch. Environ. Contam. Toxicol. 6: 315–323. Falter, R., Hintelmann, H., and Quevauviller, P. 1999. Conclusion of the workshop on
Downloaded by [North Dakota State University] at 04:13 28 October 2014
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sources of error in methylmercury determination during sample preparation, derivatisation and detection. Chemosphere 39: 1039–1049. Guinot, D., Ureña, R., Pastor, A., Varó, I., Del Ramo, J., and Torreblanca, A. 2012. Longterm effect of temperature on bioaccumulation of dietary metals and metallothionein induction in Sparus aurata. Chemosphere 87: 1215–1221. Harris, R. C., and Bodaly, R. D. 1998. Temperature, growth and dietary effects on fish mercury dynamics in two Ontario lakes. Biogeochemistry 40: 175–187. Heugens, E. H., Hendriks, A. J., Dekker, T., Straalen, N., and Admiraal, W. 2001. A review of the effects of multiple stressors on aquatic organisms and analysis of uncertainty factors for use in risk assessment. Crit. Rev. Toxicol. 31: 247–284. Hodson, P. V. 1988. The effect of metal metabolism on uptake, disposition and toxicity in fish. Aquat. Toxicol. 11: 3–18. Hrenchuk, L. E., Blanchfield, P. J., Paterson, M. J., and Hintelmann, H. H. 2012. Dietary and waterborne mercury accumulation by yellow perch: A field experiment. Environ. Sci. Technol. 46: 509–516. Jain, R. B. 2013. Effect of pregnancy on the levels of urinary metals for females aged 17–39 years old: Data from National Health and Nutrition Examination Survey 2003–2010. J. Toxicol. Environ. Health A 76: 86–97. Jeon, B. Y. 2010. Special lecture: Climate change impacts and risk for human health. J. Vet. Med. Sci. 50: 44–55. Jones, J. R. E. 1964. Fish and river pollution. London, UK: Butterworths. Khan, A. T., and Weis, J. S. 1993. Differential effects of organic and inorganic mercury on the micropyle of the eggs of Fundulus heteroclitus. Environ. Biol. Fish. 37: 323–327. Khan, M., Ahmed, S., Catalin, B., Khodadoust, A., Ajayi, O., and Vaughn, M. 2006. Effect of temperature on heavy metal toxicity to juvenile crayfish, Orconectes immunis (Hagen). Environ. Toxicol. 21: 513–520.
E. C. PACK ET AL.
Lammer, E., Carr, G. J., Wendler, K., Rawlings, J. M., Belanger, S. E., and Braunbeck, T. 2009. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp. Biochem. Physiol. C 149: 196–209. Li, L., Wu, G., Sun, J., Li, B., Li, Y., Chen, C., Chai, Z., Iida, A., and Gao, Y. 2008. Detection of mercury-, arsenic-, and selenium-containing proteins in fish liver from a mercury polluted area of Guizhou Province, China. J. Toxicol. Environ. Health A 71: 1266–1269. Lin, T. S., and Tang, H. C. 1989. Studies on acute toxicities of heavy metals, cyanide, and fluoride to Misgurnus anguillicaudatus and Plecoglossus altivelis. Bull. Taiwan Fish. Res. Inst. 46: 127–138. Liu, Y., Song, L., Li, X., and Liu, T. 2002. The Toxic effects of microcystin-LR on embryolarval and juvenile development of loach, Misguruns mizolepis Gunthe. Toxicon 40: 395–399. MacLeod, J., and Pessah, E. 1973. Temperature effects on mercury accumulation, toxicity, and metabolic rate in rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can. 30: 485–492. Maggi, C., Berducci, M. T., Bianchi, J., Giani, M., and Campanella, L. 2009. Methylmercury determination in marine sediment and organisms by direct mercury analyser. Anal. Chim. Acta 641: 32–36. Mallatt, J., Barron, M. G., and McDonough, C. 1986. Acute toxicity of methyl mercury to the larval lamprey Petromyzon marinus. Bull. Environ. Contam. Toxicol. 37: 281–288. Marshall, W. and Bryson, S. 1998. Transport mechanisms of seawater teleost chloride cells: An inclusive model of a multifunctional cell. Comp. Biochem. Physiol. A 119: 97–106. Marusczak, N., Larose, C., Dommergue, A., Paquet, S., Beaulne, J. S., Maury Brachet, R., Lucotte, M., Nedjai, R., and Ferrari, C. P. 2011. Mercury and methylmercury concentrations in high altitude lakes and fish (Arctic charr) from the French Alps related to
Downloaded by [North Dakota State University] at 04:13 28 October 2014
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watershed characteristics. Sci. Total Environ. 409: 1909–1915. Mason, R. P., Reinfelder, J. R., and Morel, F. M. 1996. Uptake, toxicity, and trophic transfer of mercury in a coastal diatom. Environ. Sci. Technol. 30: 1835–1845. Massabuau, J., and Tram, D. 2003. Ventilation, a recently described step limiting heavy metal contamination in aquatic animals. J. Phys. IV France 107: 839–843. Mayer, F. L. 1987. Acute Toxicity Handbook of Chemicals to Estuarine Organisms, Report No. EPA/600/8-87/017. Environmental Research Laboratory. Gulf Breeze, FL: U.S. EPA. McLusky, D. S., Bryant, V., and Campbell, R. 1986. The effects of temperature and salinity on the toxicity of heavy metals to marine and estuarine invertebrates. Oceanogr. Mar. Biol. Annu. Rev. 24: 481–520. Mubiana, V. K. and Blust, R. 2007. Effects of temperature on scope for growth and accumulation of Cd, Co, Cu and Pb by the marine bivalve Mytilus edulis. Mar. Environ. Res. 63: 219–235. Nam, Y. K., Choi, G. C., and Kim, D. S. 2004. An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach (Misgurnus mizolepis). Theriogenology 61: 933–945. Ni, M., Li, X., Rocha, J. B., Farina, M., and Aschner, M. 2012. Glia and methylmercury neurotoxicity. J. Toxicol. Environ. Health A 75: 1091–1101. Nunes, E., Cavaco, A., and Carvalho, C. 2014. Exposure assessment of pregnant Portuguese women to methylmercury through the ingestion of fish: Cross-sectional survey and biomarker validation. J. Toxicol. Environ. Health A 77: 133–142. Phillips, N. R., Stewart, M., Olsen, G., and Hickey, C. W. 2014. Human health risks of geothermally derived metals and other contaminants in wild-caught food. J. Toxicol. Environ. Health A 77: 346–365. Pörtner, H. 2002. Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular
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hierarchy of thermal tolerance in animals. Comp. Biochem. Physiol. A 132: 739–761. Puk, R., and Weber, J. H. 1994. Determination of mercury (II), monomethylmercury cation, dimethylmercury and diethylmercury by hydride generation, cryogenic trapping and atomic absorption spectrometric detection. Anal. Chim. Acta 292: 175–183. Rehwoldt, R., Menapace, L. W., Nerrie, B., and Alessandrello, D. 1972. The effect of increased temperature upon the acute toxicity of some heavy metal ions. Bull. Environ. Contam. Toxicol. 8: 91–96. Santore, R. C., Toro, D. M. D., Paquin, P. R., Allen, H. E., and Meyer, J. S. 2001. Biotic ligand model of the acute toxicity of metals. 2. Application to acute copper toxicity in freshwater fish and daphnia. Environ. Toxicol. Chem. 20: 2397–2402. Sokolova, I. M., and Lannig, G. 2008. Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: Implications of global climate change. Climate Res. 37: 181–201. Sunderland, E. M. 2007. Mercury exposure from domestic and imported estuarine and marine fish in the U.S. seafood market. Environ. Health Perspect. 115: 235–242. Tessier, L., Vaillancourt, G., and Pazdernik, L. 1994a. Comparative study of the cadmium and mercury kinetics between the short-lived gastropod Viviparus georgianus(Lea) and pelecypod Elliptio complanata(Lightfoot), under laboratory conditions. Environ. Pollut. 85: 271–282. Tessier, L., Vaillancourt, G., and Pazdernik, L. 1994b. Temperature effects on cadmium and mercury kinetics in freshwater molluscs under laboratory conditions. Arch. Environ. Contam. Toxicol. 26: 179–184. Tsuchiya, A., Hinners, T. A., Burbacher, T. M., Faustman, E. M., and Mariën, K. 2008. Mercury exposure from fish consumption within the Japanese and Korean communities. J. Toxicol. Environ. Health A 71: 1019–1031. Vernberg, W. B., and O’Hara, J. 1972. Temperature-salinity stress and mercury uptake in the fiddler crab, Uca pugilator. J. Fish. Res. Board. Can. 29: 1491–1494.
Downloaded by [North Dakota State University] at 04:13 28 October 2014
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Wang, W. 2012. Biodynamic understanding of mercury accumulation in marine and freshwater fish. Adv. Environ. Res. 1: 15–35. Welfinger-Smith, G., Minholz, J. L., Byrne, S., Waghiyi, V., Gologergen, J., Kava, J., Apatiki, M., Ungott, E., Miller, P. K., Arnason, J. G., and Carpenter, D. O. 2011. Organochlorine and metal contaminants in traditional foods from St. Lawrence Island, Alaska. J. Toxicol. Environ. Health A 74: 1195–1214. Wicklund Glynn, A., Norrgren, L., and Müssener, A. 1994. Differences in uptake of inorganic mercury and cadmium in the gills
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of the zebrafish, Brachydanio rerio. Aquat. Toxicol. 30: 13–26. World Health Organization. 1989. Mercury— Environmental aspects. Geneva, Switzerland: WHO. Wu, G. 1997. Effect of probenecid on the transport of methyl mercury in erythrocytes by the organic anion transport system. Arch. Toxicol. 71: 218–222. Yang, L., Mester, Z., and Sturgeon, R. E. 2003. Determination of methylmercury in fish tissues by isotope dilution SPME-GC-ICP-MS. J. Anal. Atom. Spectrom. 18: 431–436.
Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Effects of Environmental Temperature Change on Mercury Absorption in Aquatic Organisms with Respect to Climate Warming a
a
b
a
a
Eun Chul Pack , Seung Ha Lee , Chun Huem Kim , Chae Hee Lim , Dea Gwan Sung , Mee Hye c
d
e
a
f
Kim , Ki Hwan Park , Kyung Min Lim , Dal Woong Choi & Suhng Wook Kim a
Department of Public Health Science, Graduate School, Korea University, Seoul, Republic of Korea b
College of Medicine, Inha University, Incheon, Republic of Korea
c
National Institute of Food and Drug Safety Evaluation, Osong, Chungchungbook-Do, Republic of Korea d
School of Food Science and Technology, Chung-Ang University, Kyunggi-Do, Ansung, Republic of Korea e
College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea
f
Department of Biomedical Science, Korea University, Seoul, Republic of Korea Published online: 24 Oct 2014.
To cite this article: Eun Chul Pack, Seung Ha Lee, Chun Huem Kim, Chae Hee Lim, Dea Gwan Sung, Mee Hye Kim, Ki Hwan Park, Kyung Min Lim, Dal Woong Choi & Suhng Wook Kim (2014) Effects of Environmental Temperature Change on Mercury Absorption in Aquatic Organisms with Respect to Climate Warming, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:22-24, 1477-1490, DOI: 10.1080/15287394.2014.955892 To link to this article: http://dx.doi.org/10.1080/15287394.2014.955892
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Journal of Toxicology and Environmental Health, Part A, 77:1477–1490, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.955892
EFFECTS OF ENVIRONMENTAL TEMPERATURE CHANGE ON MERCURY ABSORPTION IN AQUATIC ORGANISMS WITH RESPECT TO CLIMATE WARMING Eun Chul Pack1, Seung Ha Lee1, Chun Huem Kim2, Chae Hee Lim1, Dea Gwan Sung1, Mee Hye Kim3, Ki Hwan Park4, Kyung Min Lim5, Dal Woong Choi1, Suhng Wook Kim6
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1
Department of Public Health Science, Graduate School, Korea University, Seoul, Republic of Korea 2 College of Medicine, Inha University, Incheon, Republic of Korea 3 National Institute of Food and Drug Safety Evaluation, Osong, Chungchungbook-Do, Republic of Korea 4 School of Food Science and Technology, Chung-Ang University, Kyunggi-Do, Ansung, Republic of Korea 5 College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea 6 Department of Biomedical Science, Korea University, Seoul, Republic of Korea Because of global warming, the quantity of naturally generated mercury (Hg) will increase, subsequently methylation of Hg existing in seawater may be enhanced, and the content of metal in marine products rise which consequently results in harm to human health. Studies of the effects of temperatures on Hg absorption have not been adequate. In this study, in order to observe the effects of temperature changes on Hg absorption, inorganic Hg or methylmercury (MeHg) was added to water tanks containing loaches. Loach survival rates decreased with rising temperatures, duration, and exposure concentrations in individuals exposed to inorganic Hg and MeHg. The MeHg-treated group died sooner than the inorganic Hg-exposed group. The total Hg and MeHg content significantly increased with temperature and time in both metal-exposed groups. The MeHg-treated group had higher metal absorption rates than inorganic Hg-treated loaches. The correlation coefficients for temperature elevation and absorption were significant in both groups. The results of this study may be used as basic data for assessing in vivo hazards from environmental changes such as climate warming.
a potent neurotoxin (Ni et al., 2012) prone to bioaccumulation and biological expansion in coastal marine food webs, which are the main routes by which MeHg contacts or enters humans (Sunderland, 2007). Methylmercury is generated by methylation of inorganic Hg, which exists in coasts, deep-water sediments, major rivers, atmospheric deposition, and water columns, by microorganisms such as anaerobic sulfate-reducing bacteria (SRB), iron reducers (FeRP), and methanogens (MPA) (Avramescu et al., 2011). The routes by which Hg absorption occurs are diverse. In fish, cadmium is mostly absorbed in the organ related to ion transport, while
Heavy metals are the primary causes of environmental contamination, and mercury (Hg) is one of the most common contaminants that pose a major hazard by threatening human health globally (Jain, 2013; Carneiro et al., 2014). Mercury ranks third after arsenic and lead on the list of hazardous substances published by the U.S. Environmental Protection Agency (EPA) and the Agency for Toxic Substances and Disease Registry (ATSDR) of the U.S. Department of Health and Human Services. One of the biggest issues with Hg contamination is its trophic transfer and biomagnification in the aquatic food chain (Wang, 2012). In particular, methylmercury (MeHg) is
Address correspondence to Dal Woong Choi, Korea University, Seoul 136-703, Korea. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/uteh 1477
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Hg absorption occurs throughout the gills (Wicklund Glynn et al., 1994). Mercury, when it is absorbed through the gills or intestines, is sometimes absorbed through the channel, carrier, or transport that carries amino acids or other organic compounds. In combination with amino acids in the surrounding area, Hg forms an amino acid analog and is absorbed by passing through the gills or intestines with amino acids (Mason et al., 1996). Methylmercury also forms amino acid analogs by combining with amino acids and is known to be absorbed by forming large analogs similar to amino acids such as methionine or by passing through the cell membrane without being ionized in the form of CH3 HgCl, despite less effect than when MeHg passes through a specific route (Wu, 1997). Although the mechanism through which temperature affects the absorption of heavy metals in aquatic organisms has yet to be explained clearly, empirical data indicate that temperatures in a range typical of the environment exert negligible effects on metal speciation such as free ion activity. Therefore, the biological effects of temperatures need to be examined to explain their impact on metal absorption and accumulation (Sokolova and Lannig, 2008). Various stressors, including climate change, affect aquatic ecosystems (Dijkstra et al., 2011). The rise in water temperatures attributed to climate change may stimulate methylation of Hg. Simulations of ocean warming rates of 0.4◦ C and 1◦ C predicted increases in the mean MeHg concentration of 1.7% (range, 1.6–1.8%) and 4.4% (range, 4.1–4.7%), respectively, resulting in elevated MeHg concentrations in fish (Booth and Zeller, 2005). The toxicity and absorption of Hg are highly dependent on physicochemical factors, such as temperature, as well as biological factors. Therefore, warmer water is expected to affect Hg accumulation in aquatic organisms (Welfinger-Smith et al., 2011). Various studies investigated how absorption of heavy metals such as lead, cadmium, and copper by various species is affected by changes in water temperature (Cember et al., 1978; Tessier et al., 1994a; Khan et al., 2006; Guinot et al., 2012; Dijkstra et al., 2013).
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An analysis of surface water temperatures in East Asia showed a temperature rise of approximately 1–1.9◦ C over the last decade (Jeon, 2010). Methylation of Hg and changes in metal absorption in aquatic organisms, which are induced by warmer water, may in the future affect Hg exposure through the intake of marine food. Most studies on Hg focused on the metal concentration in organisms, primarily aiming to determine the potential risk to humans from consumption of fish (Tsuchiya et al., 2008; Nunes et al., 2014), but only limited data are available on absorption and accumulation of metal (Wang, 2012). In addition, studies that have investigated factors influencing the mechanisms underlying biological absorption and concentration of Hg focused on pH, chloride ions (Cl), dissolved organic carbon (DOC) concentration, and growth rate, while data have been limited related to metal absorption through the natural food chain (Boening, 2000; Li et al., 2008; Phillips et al., 2014). In particular, little is known regarding the effects of temperature on Hg and MeHg absorption. Thus, in this study, the effect of temperature variation was examined on absorption and toxicity of inorganic Hg and MeHg under lab conditions using the Chinese weather loach Misgurnus mizolepis.
MATERIALS AND METHODS Exposure Experiment Loaches used in this study are eurythermal fish that inhabit wide temperature ranges and are acceptable as an experimental model for studies of diverse external temperature gradients (Cho et al., 2012). Among the four genera and eight species known, Misgurnus mizolepis is currently the most frequently used in lab experiments (Nam et al., 2004). Misgurnus mizolepis has many advantages, such as fast and transparent embryonic development, relatively small adult size, short generation time, high fecundity, and well-established chromosomeset manipulation and transgenesis techniques (Liu et al., 2002; Nam et al., 2004) Unlike other
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fish species, loaches exhibit intestinal breathing when oxygen is insufficient, wherein they inhale air at the water surface, exchange gases in the intestinal epithelia, and discharge air through the anus (Liu et al., 2002). Thus, it is known as a species that can survive well even in brooks, lakes, rice paddies, or swamps with low dissolved oxygen and large amounts of organic matter, and is resilient to water pollution, as it has an excellent ability to adapt even to rapid changes in the environments (Cho et al., 2009). The loaches (Misgurnus mizolepis) were allowed to adapt to the lab environment for approximately 2 wk in order to minimize their stress from environmental changes. Twentyfive fish were placed in each water tank used in the temperature stimulation test. The water tanks were installed with thermometers and heaters, and water temperatures in the tanks were set at 18, 23, or 28◦ C and maintained within an error range of ±0.5◦ C. Inorganic Hg (mercuric[II] chloride, Sigma, USA) or MeHg chloride (Aldrich, Germany) was added to the test water to make appropriate concentrations.
Survival Rate The acute impact presented in the standard test methods of the Organization for Economic Cooperation and Development (OECD), Office of Prevention, Pesticides, and Toxic Substances (OPPTS), European Commission (EC), or Korean Standards (KS) is generally assessed by lethal toxicity tests for 96 h regardless of the species (Santore et al., 2001; Lammer et al., 2009; Carriger et al., 2010). In this study, concentrations suitable for determining survival rates were selected through repeated preliminary experiments. Loaches were considered dead if they stopped breathing and did not swim even after being stimulated. Dead individuals were counted at 12, 24, 48, 72, and 96 h. After washing the specimens with distilled water and homogenization, total Hg and MeHg concentrations were measured.
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Measurement of Total Mercury The total Hg concentration in fish tissue is generally analyzed using the thermal decomposition process method. In this study, total Hg was determined using a Hydra-C mercury analyzer (Teledyne Leeman Labs Co., USA) that quickly and simply analyzes samples, as they are not previously pretreated. The frozen loach samples were thawed and homogenized before analyzing for total Hg. Approximately 0.1 g of homogenized samples was weighed and placed in a nickel boat dedicated to the total Hg analyzer without any separate pretreatment. The apparatus conditions for the measurement of the total Hg were drying set at 70 s at 300◦ C, decomposition set at 150 s at 850◦ C, waiting set at 100 s, and elution set to 30 s. As an Hg standard, a product from Wako Co. (Tokyo, Japan) was used. Measurement of Methylmercury To detect MeHg from food or fish samples, gas chromatography with electrochemical detector (GC-ECD) (Ahmad and Qureshi, 1989), gas chromatography–atomic absorption spectrometry (GC-AAS) (Puk and Weber, 1994) or gas chromatography-atomic fluorescence spectrometry (GC-AFS) (Marusczak et al., 2011) is generally used, and a direct mercury analyzer (DMA) (Maggi et al., 2009) or gas chromatograph–inductively coupled plasmamass spectrometer (GC-ICP-MS) (Yang et al., 2003) is also used. Among the apparatus, the method using GC-ECD is the most generally accepted analytical method; moreover, Japan and the Association of Analytical Communities (AOAC) have adopted GC-ECD as a method to analyze MeHg (AOAC, 1995). In this study, MeHg was determined using the GCECD method registered in the Korean Food Standards Codex of the Korea Food and Drug Administration. As a GC-ECD, a Clarus 500 (Perkin Elmer Co., USA) GC model installed with an ECD detector was used. All test solutions used were GC analysis grade or equivalent, and distilled water used was tertiary distilled water, GC analysis grade, or an equivalent.
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Pretreatment of Methylmercury Approximately 1 g of homogenized sample was added to 10 ml 25% sodium chloride and shaken for 10 min. Four milliliters of hydrochloric acid was added and the mixture was shaken vigorously for 5 min. Then 15 ml toluene (high-performance liquid chromatography [HPLC] grade, J. T. Baker, Phillipsburg, NJ) was added, and shaken for 10 min before extracting the solution. After centrifugation for 10 min at speeds of 4200 × g, the toluene layer was transferred to a funnel. Ten milliliters of 25% sodium chloride and 5 ml L-cysteine solution were added to the solution and shaken for 10 min. After leaving the solution alone for 10 min, the L-cysteine layer was divided with 4 ml 9 N hydrochloric acid solution and 5 ml toluene added to the solution and then shaken for 5 min before extracting the solution. The extracted solution was subjected to centrifugation (8500 × g, 10 min), and the toluene layer was separated and dehydrated employing sodium sulfate anhydrous powder (Junsei, Tokyo, Japan) to use it as test solution for the GC-ECD analysis. Methylmercury Analysis Conditions To efficiently analyze MeHg content using GC, an Ulbon HR-Thermon-Hg column (15 m × 0.53 mm ID, Shimadzu Co., Japan) dedicated to Hg was used. The MeHg dedicated column has disadvantages such as low durability and high costs that require more maintenance compared to general columns. However, it has the advantage of accurately separating MeHg and does not create many interfering peaks. The apparatus analysis conditions were an injector temperature of 200◦ C, a detector temperature of 230◦ C, and nitrogen as a mobile phase gas (flow speed: 8 ml/min). During analysis, oven temperatures were maintained at 50◦ C for 3 min, raised to 130◦ C for 10 min, maintained at 130◦ C for 1 min, raised to 220◦ C for 20 min, and maintained at 220◦ C for 4.5 min. The volume of the injected sample was 2 µl, and injection split rate was set at 5:1. The analysis result was compared to
that of Standard Reference Material 1946 (Lake Superior Fish Tissue, NIST), and MeHg standard stock solution used in the analysis was made by weighing 0.116 g MeHg chloride (Aldrich, Germany) and diluting it with toluene. As loss could occur in the process of pretreatment, the solution was analyzed after it underwent the same pretreatment process as that of the fish samples. Data Analysis Data summary showed the number of samples used in the analysis, means, and standard deviations. To analyze the association between temperatures of individuals administered with inorganic Hg or MeHg and mercury absorption over time, the Pearson correlation coefficient was used in the SPSS statistical program (SPSS version 12.0). The results were compared using a Student’s t-test or one-way analysis of variance (ANOVA) followed by a Newman–Keuls multiple range test. The criterion for significance was set at p < .05.
RESULTS AND DISCUSSION Survival Rates of the Inorganic Mercury-Treated Group To assess the acute toxicity of inorganic Hg within 96 h, the effects on survival rates of water temperature change and time were determined (Figures 1 and 2). No lethality was noted in the individual group treated with 0.1 mg/L or controls (Figure 1). In the 1 mg/L treatment, lethality was not observed at 18◦ C, whereas when temperature was maintained at 23◦ C, the survival rates decreased to 96, 67, 42, and 17% at 24, 48, and 96 h, respectively (Figure 2). When temperature was maintained at 28◦ C, the survival rates were 95, 83, 13, and 3% at12, 24, 48, and 72 h, respectively; all remaining individuals died thereafter. Overall, the 28◦ C group died earlier than the 18◦ C loaches. These results showed a tendency similar to studies conducted on crawfish (Del Ramo et al., 1987) and carp (Rehwoldt et al., 1972).
ENVIRONMENTAL TEMPERATURE CHANGE AND Hg ABSORPTION
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Survival rate of Misgurnus mizolepis (%)
80.00
60.00
40.00
20.00
0.00 HgCl
MeHg 18°C
23°C
MeHg 28°C
FIGURE 1. Survival rate of Misgurnus mizolepis after treatment with inorganic mercury (mercuric chloride; 0.1 mg/L) or methylmercury (MeHg; 0.1 mg/L). 100.00
Survival rate of Misgurnus mizolepis (%)
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100.00
80.00
60.00
40.00
20.00
0 12 24 48 72 96
0 12 24 48 72 96
0 12 24 48 72 96
0 12 24 48 72 96
0 12 24 48 72 96
0 12 24 48 72 96
0.00
MeHg 18°C
MeHg 23°C
MeHg 28°C
FIGURE 2. Survival rate of Misgurnus mizolepis after treatment with inorganic mercury (mercuric chloride; 1 mg/L) or methylmercury (MeHg; 1 mg/L).
Survival Rates of the Methylmercury-Treated Group Figures 1 and 2 show the effect of water temperatures on survival rates over 96 h with acute MeHg exposure. No lethality was noted in the individual group treated with 0.01 mg/L or control. In the 0.1 mg/L treatment, when the temperature was maintained at 18◦ C, survival
rates decreased to 99, 91, 84, and 63% at 24, 48, 72, and 96 h respectively (Figure 1). At 23◦ C, the survival rates fell to 99, 91, 72 and 45% at 24, 48, 72, and 96 h, respectively. At 28◦ C, the survival rates were 94, 88, 80, 37, and 1% at 12, 24, 48, 72, and 96 h, respectively. In the 1 mg/L treatment, at 18◦ C, the survival rates diminished to 97, 76, 46, and 24%
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at 24, 48, 72, and 96 h (Figure 2). At 23◦ C, the survival rates were 87% at 24 h and 32% at 48 h, and all individuals died after 72 h; at 28◦ C, the survival rates were 79, 62, and 3% at 12, 24, and 48 h, respectively, and all individuals died after 72 h. Data are in agreement with the results of a study conducted on eels (Mallatt et al., 1986).
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Comparison of Survival Rates by Mercury Type The survival rates by Hg species were compared between the 0.1 mg/L and 1 mg/L inorganic Hg- or MeHg-treated groups (Figure 1 and 2). In the 0.1-mg/L treatment, loaches exposed to MeHg had lower survival rates with increasing temperature and time, while the group treated with inorganic Hg did not show any lethality (Figure 1). Comparing the groups treated with a metal concentration of 1 mg/L, at 18◦ C, the MeHgexposed group showed lethality after 24 h while inorganic Hg-treated did not produce any lethality (Figure 2). At 23◦ C, the MeHgtreated group began displaying lethality activities at 24 h, and all individuals died after 72 h. In the inorganic Hg group, although lethality was observed at 24 h, some individuals survived even after 96 h. At 28◦ C, MeHg and inorganic Hg showed lethality at 12 h. The MeHg-exposed group had 16 and 21% lower survival rates than inorganic Hg-treated group at 12 and 24 h, respectively, and all individuals died after 72 h (Figure 2). It is generally known that MeHg exerts greater toxicity and decomposes more slowly than inorganic Hg, thus producing further adverse effects. Methylmercury treatment in this study resulted in lower survival rates than inorganic Hg. Mercury is one of the metals with a potent toxicity in teleosts; 50% of sensitive teleost species were reportedly killed after 96 h of exposure to inorganic Hg with concentrations ranging between 0.036 and 0.098 mg/L (Eisler and Hennekey, 1977; Mayer, 1987). The threshold concentration of Hg chloride was reported to be 0.008 ppm in sticklebacks at a temperature of 15–18◦ C. However, tolerance
to inorganic Hg differs greatly among species (World Health Organization [WHO], 1989), and M. anguillicaudatus, a species similar to the M. mizolepis used in this experiment, was deemed to have a considerably high tolerance to inorganic Hg with a 24-h LC50 of 0.997 mg/L and a 48-h LC50 of 0.813 mg/L (Lin and Tang, 1989). Methylmercury exerts greater toxicity and has a lower lethal concentration than inorganic Hg. Poor egg insemination success (Khan and Weis, 1993), altered blood chemistry (Dawson, 1982), decreased respiration (Armstrong, 1979), and high accumulations (Choi and Cech, 1998) were reported at concentrations lower than the lethal concentration. The LC50 of MeHg for fish is reported to be in the range of 0.004–0.125 mg/L, depending on the species, which is far lower than the LC50 of inorganic Hg (WHO, 1989). The physiological mechanism underlying acute Hg poisoning was suggested to be asphyxia resulting from gill tissue damage and mucus coagulation (Jones, 1964). Amend et al. (1969) noted that death was attributable to necrosis and epithelial separation in the gills. Usually, metal toxicity in organisms rises in proportion to increasing temperatures by accelerating the toxicological mechanism (Rehwoldt et al., 1972; Cairns et al., 1975; McLusky et al., 1986; Heugens et al., 2001; Khan et al., 2006). In fiddler crabs and Uca pugilator, Hg transfer from gills to the hepatopancreas increased at higher temperatures, and the metal concentration was markedly different in each tissue. Vernberg and O’Hara (1972) postulated that such factors may play a role in toxicity of inorganic Hg in fiddler crabs. Quantity of Mercury Absorbed by the Inorganic Mercury-Treated Group The total Hg content in the 0.1 mg/L inorganic Hg-treated group are shown in Figure 3. At 24 h, the 0.1 mg/L inorganic Hg-exposed loaches maintained at 28◦ C possessed 4.2fold and 1.6-fold more total absorbed metal than groups maintained at 18◦ C and 23◦ C, respectively. At 48 h, the loaches maintained at 28◦ C showed 14.4- and 2.1-fold more total
absorbed metal than groups maintained at 18◦ C and 23◦ C, respectively. At 72 h, the group maintained at 28◦ C showed 2.1- and 1.5-fold higher total absorbed Hg than those maintained at 18◦ C and 23◦ C, respectively. The correlation coefficients for temperature increases and absorption were significant. The total Hg content of the group exposed to an inorganic Hg at 0.1 mg/L was compared (Figure 3). At 18◦ C, total Hg content increased to 95.62, 156.36, and 1100.65 µg/kg by 24, 48, and 72 h exposure, respectively, while at 23◦ C total metal content was elevated to 250.46, 1057.55, and 1540.53 µg/kg by 24, 48, and 72 h of exposure, respectively. At 28◦ C, total Hg content increased to 407.57, 2255.64, and 2278.21 µg/kg by 24, 48, and 72 h of exposure, respectively. Thus, total Hg content was elevated markedly over time as shown in Figure 3. Overall, total Hg content absorbed by loaches exposed to inorganic metal at different water temperatures was highest at 28◦ C, followed in order by 23 and 18◦ C. Mercury accumulation over time indicated increase in quantities absorbed associated with elevation in exposure time and water temperatures.
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Methylmercury Contents in the Inorganic Mercury-Treated Group The MeHg content in the experimental groups exposed to inorganic Hg at different concentrations and temperatures is depicted in Table 1. The average values were approximately 26.20 ± 11.21 µg/kg, and no significant differences were observed over time or at different temperatures. This indicates that methylation by enterobacteria in tissues in vivo did not occur (Falter et al., 1999). Differences in the total Hg content and survival rates seemed to be directly affected by inorganic Hg.
TABLE 1. Modification of Methylmercury Concentration After Treatment With Mercury Chloride (0.1 mg/L) Exposure concentration (mg/L) Control
0.1
18°C
23°C
Concentration of methyl mercury (µg/kg)
Temperature (◦ C)
24 h
72 h
18 23 28 18 23 28
27.86 ± 14.16 24.42 ± 13.00 24.87 ± 9.56 26.43 ± 10.52 28.52 ± 13.14 29.44 ± 18.36
24.12 ± 8.41 20.45 ± 11.54 21.42 ± 13.32 30.83 ± 9.51 29.34 ± 15.42 29.96 ± 9.35
Note. Each value is the mean ± SD.
3000
Total mercury concentration (µg/kg)
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ENVIRONMENTAL TEMPERATURE CHANGE AND Hg ABSORPTION
28°C
b
c
2500
2000
a b
1500
a
1000 c 500
0
a
b
24
a 48 Exposure time (hr)
72
FIGURE 3. Modification of total mercury concentration after treatment with mercury chloride (0.1 mg/L). Different letters indicate a significant difference at each time point (one-way ANOVA followed by Newman–Keuls multiple range test, p < .05).
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with data for total Hg of fish exposed to inorganic Hg. The total Hg contents were highest at 28◦ C, followed in order by 23◦ C and 18◦ C, and the quantities absorbed continuously rose over time.
The total Hg content in the 0.1 mg/L MeHg-treated groups at different temperatures is presented in Figure 4. At 24 h the group maintained at 28◦ C showed 4.7- and 1.6-fold more total absorbed metal than those maintained at 18◦ C and 23◦ C, respectively. At 48 h, loaches maintained at 28◦ C displayed 1.6and 1.2-fold higher total absorbed Hg than those maintained at 18◦ C and 23◦ C, respectively. At 72 h, the group maintained at 28◦ C showed 2- and 1.6-fold higher total absorbed Hg than groups maintained at 18◦ C and 23◦ C, respectively. The correlation coefficients for temperature increases and absorption were significant. The total Hg content of the groups exposed to MeHg concentration at 0.1 mg/L were compared (Figure 4). By 24, 48, and 72 h exposure, total Hg content increased to, 752.13, 2822.10, and 3417.25 µg/kg, respectively, at 18◦ C; 2157.74, 3728.95, and 4120.82 µg/kg, respectively, at 23◦ C; and 3516.57, 4440.31, and 6723.42 µg/kg, respectively, at 28◦ C. Overall, total Hg contents absorbed by loaches exposed to different concentrations of MeHg at different water temperatures were consistent
Methylmercury Content in the Methylmercury-Treated Group The methylmercury content in the 0.1 mg/L MeHg-treated groups at different temperatures is shown in Figure 5. At 24 h, loaches maintained at 28◦ C showed 4- and 1.7- fold greater absorbed MeHg than those maintained at 18◦ C and 23◦ C, respectively. At 48 h, the group maintained at 28◦ C displayed 1.9- and 1.4-fold more absorbed MeHg than loaches maintained at 18◦ C and 23◦ C, respectively. At 72 h, fish maintained at 28◦ C showed 2.2- and 1.6-fold higher absorption metal than groups maintained at 18◦ C and 23◦ C, respectively. The correlation coefficients for temperature elevation and absorption were significant. The total MeHg content of groups exposed to a metal concentration of 0.1 mg/L were compared (Figure 5). At 24, 48, and 72 h, loach MeHg content were 380.36, 1496.34, and
10000 9000 Total mercury concentration (µg/kg)
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The Quantity of Mercury Absorbed by the Methylmercury-Treated Group
18°C
23°C
b
28°C
8000 7000 6000
b
5000
c
4000 b
3000
b
a a
a
2000 1000 0
a
24
48 Exposure time (hr)
72
FIGURE 4. Modification of total mercury concentration after treatment with methylmercury (0.1 mg/L). Different letters indicate a significant difference at each time point (one-way ANOVA followed by Newman–Keuls multiple range test, p < .05).
ENVIRONMENTAL TEMPERATURE CHANGE AND Hg ABSORPTION
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8000 18°C
23°C
28°C
b
Methylmercury concentration (µg/kg)
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7000 6000 5000 a 4000
b
3000
a
a c
2000
a
b 1000 0
a 24
48 Exposure time (hr)
72
FIGURE 5. Modification of methylmercury concentration after treatment with methylmercury (0.1 mg/L). Different letters indicate a significant difference at each time point (one-way ANOVA followed by Newman–Keuls multiple range test, p < .05).
2550.21 µg/kg, respectively, at 18◦ C; 921.34, 2036.42, and 3525.14 µg/kg, respectively, at 23◦ C; and rose over time to 1536.41, 2857.32, and 5607.11 µg/kg, respectively, at 28◦ C. Overall, the quantities of absorbed MeHg were highest at 28◦ C, followed in order by 23◦ C and 18◦ C. Therefore, bioaccumulation seemed to be dependent on the temperature. Comparison of Quantities Absorbed Between Different Types of Mercury The total Hg absorption rates for fish exposed to 0.1 mg/L MeHg compared to loaches treated using inorganic Hg were higher by 7.9- (18◦ C), 8.6- (23◦ C), and 8.6-fold (28◦ C) at 24 h; by 18- (18◦ C), 3.5- (23◦ C), and 2-fold (28◦ C) at 48 h; and by 3.1- (18◦ C), 2.7- (23◦ C), and 3-fold (28◦ C) at 72 h (Table 2). The MeHgtreated group had higher absorption rates of total Hg than the inorganic metal-exposed fish. These different absorption rates support the differences in lethality between fish treated with inorganic versus organic Hg (Figures 1 and 2). Two major routes of metal absorption in aquatic organisms are ATP-dependent active transport through ion pumps and facilitated diffusion through ion channels (e.g., the Ca2+
channel) (Marshall and Bryson, 1998; Bury et al., 2003). Mercury is known to be absorbed through the transporter, or channel, or carrier (Mason et al., 1996). In particular, MeHg is highly soluble in fat, and therefore, approximately 50% of all of the capacity of intestines is first accumulated in muscle tissue (WHO, 1989), which leads to fast absorption and quick passage through sensitive tissues or neurons (Hodson, 1988). Such an absorption route is heavily influenced by environmental temperatures in poikilothermic animals. Therefore, rising temperatures increase metal absorption, which leads to enhanced metal accumulation in organisms since the elimination rate is not greatly affected by temperature (Denton and Burdon-Jones, 1981; Mubiana and Blust, 2007). Increased metabolism at elevated temperatures may result in metal accumulation in poikilothermic animals due to enhanced energy demand, and warmer temperatures are known to affect fish metabolism in relation to Hg accumulation. The increased demand for energy leads to an enhanced ventilation rate or feeding rate (Harris and Bodaly, 1998; Pörtner, 2002), which results in greater exposure to water or food contaminated with metals. Other studies on bivalves also found that
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TABLE 2. Comparison of Total Mercury Absorption After Treatment With Mercury Chloride (0.1 mg/L) or Methylmercury (0.1 mg/L) Concentration of total mercury (µg/kg) Exposure time (h)
Mercury type
18◦ C
23◦ C
28◦ C
24
HgCl2 MeHg HgCl2 MeHg HgCl2 MeHg
95.62 ± 53.29 752.13 ± 157.54 156.36 ± 48.32 2822.10 ± 417.27 1100.65 ± 209.80 3417.25 ± 526.14
250.46 ± 58.25 2157.74 ± 500.36 1057.55 ± 342.21 3728.95 ± 542.14 1540.53 ± 255.32 4120.82 ± 756.34
407.57 ± 70.34 3516.57 ± 743.17 2255.64 ± 396.04 4440.31 ± 712.41 2278.21 ± 512.36 6723.42 ± 2562.30
48 72
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Note. Each value is the mean ± SD.
the ventilatory activity was the rate-limiting step in metal uptake by such organisms (Massabuau and Tram, 2003). High ventilation rates in fish at elevated temperatures also produce high Hg absorption rates (MacLeod and Pessah, 1973). Mercury absorption has lower sensitivity to temperature than cadmium absorption, which is attributed to the fact that Hg is regulated by a slower biochemical mechanism (Tessier et al., 1994a, 1994b). Other studies also demonstrated the effect of temperature on dietary metal uptake (Hrenchuk et al., 2012; Guinot et al., 2012), a field that requires additional research (Sokolova and Lannig, 2008). This study demonstrated the effects of changes in external temperatures on Hg absorption in fish. Fish were exposed to inorganic Hg or MeHg and water temperatures were maintained at 18, 23, or 28◦ C to observe survival rates and quantities of total Hg or MeHg absorbed at different temperatures over time. The survival rates of fish treated with inorganic Hg or MeHg decreased in proportion to increasing external temperatures and metal concentrations. In addition, the MeHg-exposed groups had lower survival rates than the inorganic Hg-treated fish. The total Hg contents of inorganic Hg-exposed loach rose in proportion to external temperature elevation and exposure times, and all individuals showed significant correlations between warmer temperatures and adsorption. However, no changes in MeHg content in relation to temperature were observed. The total content of Hg and MeHg of methylmercury-treated groups rose in proportion to increasing temperatures and exposure time, and most individuals displayed significant correlations between warmer temperatures and
absorption. Methylmercury demonstrated significantly higher absorption rates than inorganic Hg in all individuals. Data showed that warmer water temperatures increased MeHg and inorganic Hg absorption and that the absorption rates of MeHg were higher than those of inorganic Hg. These results may be used as basic data to predict the potential adverse effects of Hg with water temperature changes resulting from climate warming, heavy metal pollution occurring when industrial wastes are combusted, and rising temperatures generated by waste heat emissions. FUNDING This research was supported by a grant (10162KFDA995) from the Korea Food and Drug Administration, an Institute of Health Science Grant, Korea University and a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0023938). ACKNOWLEDGMENTS The first two authors contributed equally to this work. REFERENCES Ahmad, S. and Qureshi, I. H. 1989. Fast mercury removal from industrial effluent. J. Radioanal. Nucl. Chem. 130: 347–352. Amend, D. F., Yasutake, W. T., and Morgan, R. 1969. Some factors influencing susceptibility of rainbow trout to the acute toxicity
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of an ethyl mercury phosphate formulation (Timsan). Trans. Am. Fish. Soc. 98: 419–425. Armstrong, F. A. J. 1979. Effects of mercury compounds on fish. In Biogeochemistry of mercury in the environment, J. O. Nriagu, 657–670. New York, NY: Elsevier/North Holland Biomedical Press. Association of Official Analytical Chemists. 1995. Official methods of analysis, ed. W. Horwitz. Washington, DC: AOAC. Avramescu, M., Yumvihoze, E., Hintelmann, H., Ridal, J., Fortin, D., and Lean, R. S. 2011. Biogeochemical factors influencing net mercury methylation in contaminated freshwater sediments from the St. Lawrence River in Cornwall, Ontario, Canada. Sci. Total Environ. 409: 968–978. Boening, D. W. 2000. Ecological effects, transport, and fate of mercury: A general review. Chemosphere 40: 1335–1351. Booth, S., and Zeller, D. 2005. Mercury, food webs, and marine mammals: Implications of diet and climate change for human health. Environ. Health Perspect. 113: 521–526. Bury, N. R., Walker, P. A., and Glover, C. N. 2003. Nutritive metal uptake in teleost fish. J. Exp. Biol. 206: 11–23. Cairns, J., Jr., Heath, A. G., and Parker, B. C. 1975. The effects of temperature upon the toxicity of chemicals to aquatic organisms. Hydrobiologia 47: 135–171. Carneiro, M. F., Grotto, D., and Barbosa, F., Jr. 2014. Inorganic and methylmercury levels in plasma are differentially associated with age, gender, and oxidative stress markers in a population exposed to mercury through fish consumption. J. Toxicol. Environ. Health A 77: 69–79. Carriger, J. F., Hoang, T.C., Rand, G. M., Gardinali, P. R., and Castro, J. 2010. Acute toxicity and effects analysis of endosulfan sulfate to freshwater fish species. Arch. Environ. Contam. Toxicol. 60: 281–289. Cember, H., Curtis, E. H., and Gordon Blaylock, B. 1978. Mercury bioconcentration in fish: temperature and concentration effects. Environ. Pollut. 17: 311–319.
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Cho, Y. S., Kim, B. S., Kim, D. S., and Nam, Y. K. 2012. Modulation of warm-temperatureacclimation-associated 65-kDa protein genes (Wap65-1 and Wap65-2) in mud loach (Misgurnus mizolepis, Cypriniformes) liver in response to different stimulatory treatments. Fish Shellfish Immun. 32: 662–669. Cho, Y. S., Lee, S. Y., Kim, K. Y., and Nam, Y. K. 2009. Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): Gene structure, genomic organization, and mRNA expression analysis. Comp. Biochem. Physiol. B 153: 317–326. Choi, M. H., and Cech, J. J. 1998. Unexpectedly high mercury level in pelleted commercial fish feed. Environ. Toxicol. Chem. 17: 1979–1981. Dawson, M. 1982. Effects of long-term mercury exposure on hematology of striped bass, Morone saxatilis. Fish. Bull. 80: 389–392. Del Ramo, J., Diaz-Mayans, J., Torreblanca, A., and Nunez, A. 1987. Effects of temperature on the acute toxicity of heavy metals (Cr, Cd, and Hg) to the freshwater crayfish, Procambarus clarkii (girard). Bull. Environ. Contam. Toxicol. 38: 736–741. Denton, G., and Burdon-Jones, C. 1981. Influence of temperature and salinity on the uptake, distribution and depuration of mercury, cadmium and lead by the black-lip oyster Saccostrea echinata. Mar. Biol. 64: 317–326. Dijkstra, J. A., Buckman, K. L., Ward, D., Evans, D. W., Dionne, M., and Chen, C. Y. 2013. Experimental and natural warming elevates mercury concentrations in estuarine fish. PLoS One 8: e58401. Dijkstra, J. A., Westerman, E. L., and Harris, L. G. 2011. The effects of climate change on species composition, succession and phenology: A case study. Global Change Biol. 17: 2360–2369. Eisler, R., and Hennekey, R. J. 1977. Acute toxicities of Cd2+ , Cr+6 , Hg2+ , Ni2+ and Zn2+ to estuarine macrofauna. Arch. Environ. Contam. Toxicol. 6: 315–323. Falter, R., Hintelmann, H., and Quevauviller, P. 1999. Conclusion of the workshop on
Downloaded by [North Dakota State University] at 04:13 28 October 2014
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sources of error in methylmercury determination during sample preparation, derivatisation and detection. Chemosphere 39: 1039–1049. Guinot, D., Ureña, R., Pastor, A., Varó, I., Del Ramo, J., and Torreblanca, A. 2012. Longterm effect of temperature on bioaccumulation of dietary metals and metallothionein induction in Sparus aurata. Chemosphere 87: 1215–1221. Harris, R. C., and Bodaly, R. D. 1998. Temperature, growth and dietary effects on fish mercury dynamics in two Ontario lakes. Biogeochemistry 40: 175–187. Heugens, E. H., Hendriks, A. J., Dekker, T., Straalen, N., and Admiraal, W. 2001. A review of the effects of multiple stressors on aquatic organisms and analysis of uncertainty factors for use in risk assessment. Crit. Rev. Toxicol. 31: 247–284. Hodson, P. V. 1988. The effect of metal metabolism on uptake, disposition and toxicity in fish. Aquat. Toxicol. 11: 3–18. Hrenchuk, L. E., Blanchfield, P. J., Paterson, M. J., and Hintelmann, H. H. 2012. Dietary and waterborne mercury accumulation by yellow perch: A field experiment. Environ. Sci. Technol. 46: 509–516. Jain, R. B. 2013. Effect of pregnancy on the levels of urinary metals for females aged 17–39 years old: Data from National Health and Nutrition Examination Survey 2003–2010. J. Toxicol. Environ. Health A 76: 86–97. Jeon, B. Y. 2010. Special lecture: Climate change impacts and risk for human health. J. Vet. Med. Sci. 50: 44–55. Jones, J. R. E. 1964. Fish and river pollution. London, UK: Butterworths. Khan, A. T., and Weis, J. S. 1993. Differential effects of organic and inorganic mercury on the micropyle of the eggs of Fundulus heteroclitus. Environ. Biol. Fish. 37: 323–327. Khan, M., Ahmed, S., Catalin, B., Khodadoust, A., Ajayi, O., and Vaughn, M. 2006. Effect of temperature on heavy metal toxicity to juvenile crayfish, Orconectes immunis (Hagen). Environ. Toxicol. 21: 513–520.
E. C. PACK ET AL.
Lammer, E., Carr, G. J., Wendler, K., Rawlings, J. M., Belanger, S. E., and Braunbeck, T. 2009. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp. Biochem. Physiol. C 149: 196–209. Li, L., Wu, G., Sun, J., Li, B., Li, Y., Chen, C., Chai, Z., Iida, A., and Gao, Y. 2008. Detection of mercury-, arsenic-, and selenium-containing proteins in fish liver from a mercury polluted area of Guizhou Province, China. J. Toxicol. Environ. Health A 71: 1266–1269. Lin, T. S., and Tang, H. C. 1989. Studies on acute toxicities of heavy metals, cyanide, and fluoride to Misgurnus anguillicaudatus and Plecoglossus altivelis. Bull. Taiwan Fish. Res. Inst. 46: 127–138. Liu, Y., Song, L., Li, X., and Liu, T. 2002. The Toxic effects of microcystin-LR on embryolarval and juvenile development of loach, Misguruns mizolepis Gunthe. Toxicon 40: 395–399. MacLeod, J., and Pessah, E. 1973. Temperature effects on mercury accumulation, toxicity, and metabolic rate in rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can. 30: 485–492. Maggi, C., Berducci, M. T., Bianchi, J., Giani, M., and Campanella, L. 2009. Methylmercury determination in marine sediment and organisms by direct mercury analyser. Anal. Chim. Acta 641: 32–36. Mallatt, J., Barron, M. G., and McDonough, C. 1986. Acute toxicity of methyl mercury to the larval lamprey Petromyzon marinus. Bull. Environ. Contam. Toxicol. 37: 281–288. Marshall, W. and Bryson, S. 1998. Transport mechanisms of seawater teleost chloride cells: An inclusive model of a multifunctional cell. Comp. Biochem. Physiol. A 119: 97–106. Marusczak, N., Larose, C., Dommergue, A., Paquet, S., Beaulne, J. S., Maury Brachet, R., Lucotte, M., Nedjai, R., and Ferrari, C. P. 2011. Mercury and methylmercury concentrations in high altitude lakes and fish (Arctic charr) from the French Alps related to
Downloaded by [North Dakota State University] at 04:13 28 October 2014
ENVIRONMENTAL TEMPERATURE CHANGE AND Hg ABSORPTION
watershed characteristics. Sci. Total Environ. 409: 1909–1915. Mason, R. P., Reinfelder, J. R., and Morel, F. M. 1996. Uptake, toxicity, and trophic transfer of mercury in a coastal diatom. Environ. Sci. Technol. 30: 1835–1845. Massabuau, J., and Tram, D. 2003. Ventilation, a recently described step limiting heavy metal contamination in aquatic animals. J. Phys. IV France 107: 839–843. Mayer, F. L. 1987. Acute Toxicity Handbook of Chemicals to Estuarine Organisms, Report No. EPA/600/8-87/017. Environmental Research Laboratory. Gulf Breeze, FL: U.S. EPA. McLusky, D. S., Bryant, V., and Campbell, R. 1986. The effects of temperature and salinity on the toxicity of heavy metals to marine and estuarine invertebrates. Oceanogr. Mar. Biol. Annu. Rev. 24: 481–520. Mubiana, V. K. and Blust, R. 2007. Effects of temperature on scope for growth and accumulation of Cd, Co, Cu and Pb by the marine bivalve Mytilus edulis. Mar. Environ. Res. 63: 219–235. Nam, Y. K., Choi, G. C., and Kim, D. S. 2004. An efficient method for blocking the 1st mitotic cleavage of fish zygote using combined thermal treatment, exemplified by mud loach (Misgurnus mizolepis). Theriogenology 61: 933–945. Ni, M., Li, X., Rocha, J. B., Farina, M., and Aschner, M. 2012. Glia and methylmercury neurotoxicity. J. Toxicol. Environ. Health A 75: 1091–1101. Nunes, E., Cavaco, A., and Carvalho, C. 2014. Exposure assessment of pregnant Portuguese women to methylmercury through the ingestion of fish: Cross-sectional survey and biomarker validation. J. Toxicol. Environ. Health A 77: 133–142. Phillips, N. R., Stewart, M., Olsen, G., and Hickey, C. W. 2014. Human health risks of geothermally derived metals and other contaminants in wild-caught food. J. Toxicol. Environ. Health A 77: 346–365. Pörtner, H. 2002. Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular
1489
hierarchy of thermal tolerance in animals. Comp. Biochem. Physiol. A 132: 739–761. Puk, R., and Weber, J. H. 1994. Determination of mercury (II), monomethylmercury cation, dimethylmercury and diethylmercury by hydride generation, cryogenic trapping and atomic absorption spectrometric detection. Anal. Chim. Acta 292: 175–183. Rehwoldt, R., Menapace, L. W., Nerrie, B., and Alessandrello, D. 1972. The effect of increased temperature upon the acute toxicity of some heavy metal ions. Bull. Environ. Contam. Toxicol. 8: 91–96. Santore, R. C., Toro, D. M. D., Paquin, P. R., Allen, H. E., and Meyer, J. S. 2001. Biotic ligand model of the acute toxicity of metals. 2. Application to acute copper toxicity in freshwater fish and daphnia. Environ. Toxicol. Chem. 20: 2397–2402. Sokolova, I. M., and Lannig, G. 2008. Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: Implications of global climate change. Climate Res. 37: 181–201. Sunderland, E. M. 2007. Mercury exposure from domestic and imported estuarine and marine fish in the U.S. seafood market. Environ. Health Perspect. 115: 235–242. Tessier, L., Vaillancourt, G., and Pazdernik, L. 1994a. Comparative study of the cadmium and mercury kinetics between the short-lived gastropod Viviparus georgianus(Lea) and pelecypod Elliptio complanata(Lightfoot), under laboratory conditions. Environ. Pollut. 85: 271–282. Tessier, L., Vaillancourt, G., and Pazdernik, L. 1994b. Temperature effects on cadmium and mercury kinetics in freshwater molluscs under laboratory conditions. Arch. Environ. Contam. Toxicol. 26: 179–184. Tsuchiya, A., Hinners, T. A., Burbacher, T. M., Faustman, E. M., and Mariën, K. 2008. Mercury exposure from fish consumption within the Japanese and Korean communities. J. Toxicol. Environ. Health A 71: 1019–1031. Vernberg, W. B., and O’Hara, J. 1972. Temperature-salinity stress and mercury uptake in the fiddler crab, Uca pugilator. J. Fish. Res. Board. Can. 29: 1491–1494.
Downloaded by [North Dakota State University] at 04:13 28 October 2014
1490
Wang, W. 2012. Biodynamic understanding of mercury accumulation in marine and freshwater fish. Adv. Environ. Res. 1: 15–35. Welfinger-Smith, G., Minholz, J. L., Byrne, S., Waghiyi, V., Gologergen, J., Kava, J., Apatiki, M., Ungott, E., Miller, P. K., Arnason, J. G., and Carpenter, D. O. 2011. Organochlorine and metal contaminants in traditional foods from St. Lawrence Island, Alaska. J. Toxicol. Environ. Health A 74: 1195–1214. Wicklund Glynn, A., Norrgren, L., and Müssener, A. 1994. Differences in uptake of inorganic mercury and cadmium in the gills
E. C. PACK ET AL.
of the zebrafish, Brachydanio rerio. Aquat. Toxicol. 30: 13–26. World Health Organization. 1989. Mercury— Environmental aspects. Geneva, Switzerland: WHO. Wu, G. 1997. Effect of probenecid on the transport of methyl mercury in erythrocytes by the organic anion transport system. Arch. Toxicol. 71: 218–222. Yang, L., Mester, Z., and Sturgeon, R. E. 2003. Determination of methylmercury in fish tissues by isotope dilution SPME-GC-ICP-MS. J. Anal. Atom. Spectrom. 18: 431–436.
