Article pubs.acs.org/est
Tracing Maternal Transfer of Methylmercury in the Sheepshead Minnow (Cyprinodon variegatus) with an Enriched Mercury Stable Isotope Emily S. Stefansson, Andrew Heyes,* and Christopher L. Rowe Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, P.O. Box 38, Solomons, Maryland 20688, United States ABSTRACT: Maternal transfer of methylmercury (MeHg) to eggs is an important exposure pathway for developing offspring. However, our understanding of this process is limited, particularly in estuarine fish. We conducted a 91-day chronic laboratory exposure of Cyprinodon variegatus to four concentrations of dietary MeHg ranging from 0.04 to 9.90 μg g−1 dw. Juvenile fish were fed a preoogenesis MeHg diet for 28 days, after which the diet was switched to a diet enriched with Me199Hg, spanning the period of oogenesis, allowing us to differentiate between mercury stored in female tissues and mercury assimilated from the maternal diet during oogenesis. We found that both maternal body burden and clutch size were strong predictors of egg Hg content. A constant percentage of preoogenesis Hg was transferred to eggs in each treatment. Additionally, preoogenesis Hg and during-oogenesis Hg were transferred proportionally to eggs, suggesting that both female tissues and the maternal diet during oogenesis are significant sources of Hg.
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INTRODUCTION
reflection of the maternal diet during oogenesis, rather than Hg stored in female tissues.14 Dynamics of methylmercury transfer from parent to offspring are poorly understood yet could significantly affect the reproductive fitness of offspring. Given the potential importance of the maternal diet in Hg transfer to eggs,14 we used an enriched stable mercury isotope to differentiate between mercury stored in female tissues and mercury assimilated from the maternal diet during oogenesis. Stable mercury isotopes are powerful tools to trace the fate of mercury species and investigate processes such as methylation, bioaccumulation, and adsorption onto particles.15,16 Effects of methylmercury exposure on lower trophic levels have implications for higher trophic level species, including important commercial species. Furthermore, compared to freshwater species, accumulation and effects of MeHg in estuarine and marine organisms are understudied. Therefore, we chose to focus on the estuarine sheepshead minnow, Cyprinodon variegatus. Cyprinodon variegatus inhabits shallow, coastal waters along the Atlantic coast of North America, as well as the Gulf of Mexico.17 Sheepshead minnows hatch after 4 to 7 days, remain as larvae for approximately 28 days, and metamorphose to the juvenile stage which lasts approximately 35 days prior to sexual maturation.18 Sheepshead minnows are ideal for laboratory studies because of their small size, rapid
Pollutants transferred from mother to offspring have the potential to significantly affect survival and development of embryos,1,2 as well as adversely affect offspring later in life.3,4 The effects of maternally transferred compounds ultimately have implications for population viability.2,5 Mercury is a contaminant of concern in many ecosystems, particularly in its methylated form (methylmercury, MeHg), which readily accumulates in fish and other biota.6 Recent studies have shown endocrine disruption and reproductive effects in fish as a result of dietary MeHg exposure.7,8 Effects of MeHg observed in freshwater fathead minnows (Pimephales promelas) include changes in sex hormone levels7 and decreases in the production of vitellogenin,8 as well as eggs.9 As both an endocrine disruptor and potent neurotoxin, MeHg has the potential to impair developing offspring. However, our current understanding of maternal transfer of MeHg and its effects remain limited, particularly in estuarine fish. While diet is the major source of MeHg for adult fish,10,11 maternal transfer has been shown to be a significant route of exposure for larval and juvenile fish.1,12 A recent study on the cellular mechanisms by which MeHg moves through the body showed that MeHg readily complexes with cysteine.13 This structure mimics that of methionine and can therefore be transferred across cell membranes to developing oocytes via methionine transporters.13 It was originally thought that MeHg partitioned from stores in female tissues into developing oocytes. However, a more recent study indicated that egg mercury content for the freshwater fathead minnow was a © 2014 American Chemical Society
Received: Revised: Accepted: Published: 1957
September 27, 2013 January 8, 2014 January 9, 2014 January 9, 2014 dx.doi.org/10.1021/es404325c | Environ. Sci. Technol. 2014, 48, 1957−1963
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Husbandry. This husbandry protocol was approved by the University of Maryland Center for Environmental Science IACUC. Fish were housed in 76 L static aquaria at Chesapeake Biological Laboratory (Solomons, MD, USA). All tanks were equipped with individual filters, heaters, and aeration to maintain water quality. Water temperature was maintained at approximately 26 °C for the duration of the experiment. Filtered ambient river water (Patuxent River, MD, USA) was used, thus salinity varied naturally between 7 and 15 ppt over the course of the experiment. A 14:10 h light/dark cycle was maintained throughout. Each tank received 1 g of food daily, which was confirmed to be an ad libitum regimen, as excess food remained prior to subsequent food additions. Tanks were cleaned weekly by siphoning out excess food and waste. Water quality was monitored by measuring dissolved oxygen, temperature, salinity, conductivity, and pH weekly. Ammonia levels were also measured colorimetrically (API, Mars Fishcare Inc.) in a random subset of tanks periodically. About half of the aquaria were eliminated from the experiment due to high ammonia levels after biological filters failed, causing high mortality in affected tanks. Sheepshead minnows were acquired from Aquatic Biosystems (Fort Collins, CO, USA) at 14 days of age. All individuals were fed control food and acclimated to laboratory conditions for two weeks prior to the start of dosing. At the end of this period, individuals (28 days old) were randomly distributed among 76 L aquaria at a density of 12 fish per tank and dosing was initiated. Treatments and Sampling Design. Treatments consisted of a control diet and three different methylmercury diets (Table 1). Each treatment consisted of six replicate tanks, totaling 24 aquaria. Treatments were randomly distributed among tank positions. Fish were fed a diet spiked with MeHg for 28 days (Table 1), which contained a natural abundance of Hg isotopes (preoogenesis Hg). When fish were 56 days old, diets were changed to a diet enriched with Me199Hg, although concentrations were not exactly the same (Table 1). To confirm the diet change occurred before the onset of oogenesis, 3 females were sacrificed from one tank per treatment and dissected to evaluate reproductive status. No eggs were found in individuals at the beginning of isotope dosing, therefore it was assumed that the enriched isotope diet spanned the period of oogenesis. Although no histological analysis was performed to verify this, this assumption was supported by other studies, which found evidence of sexual maturity in laboratory-reared C. variegatus between 65 and 107 days old.18,23,24 Fish were fed the enriched 199Hg isotope diet for 63 days, for a total Hg dosing period of 91 days. At the end of the experiment, fish were held for 48 h in separate aerated buckets to clear gut contents prior to euthanasia via cervical dislocation, measurement of total length and wet mass, and storage at −80 °C for subsequent Hg analysis. Prior to freezing, all female fish were dissected to remove eggs. Eggs were lightly rinsed with deionized water, counted, and weighed. Eggs from each female were pooled into 5 mL Teflon vials and frozen at −80 °C for subsequent digestion and mercury analysis. Because sheepshead minnows have asynchronous ovaries, eggs varied in development and size within the gonad. Therefore, only eggs that were large enough to be accurately removed and counted were included in egg analyses. Sample Digestion. Fish carcasses (without eggs) were freeze-dried for 24 h and digested in 5 mL of 50:50
development, and tolerance of laboratory conditions. Due to its abundance in estuarine marshes,19 C. variegatus serves as an important food source for other vertebrates. We conducted a 91-day chronic laboratory exposure of C. variegatus to four concentrations of dietary MeHg ranging from 0.04 to 9.90 μg g−1 dw (Table 1). These concentrations Table 1. Target MeHg Concentrations and Corresponding Measured Concentrations ± SE in Diets for Each Treatmenta treatment
target MeHg dose (μg g−1 dw)
control low medium high
0.00 1.00 5.00 10.00
preoogenesis MeHg dose (μg g−1 dw) 0.04 0.84 4.40 7.01
± ± ± ±
0.005 0.18 0.34 1.71
enriched isotope MeHg dose (μg g−1 dw) 0.03 1.30 5.34 11.35
± ± ± ±
0.002 0.42 0.85 1.31
a
Measured concentrations were averaged for pre-oogenesis and enriched isotope diet values.
spanned from levels of MeHg reported in prey species in relatively uncontaminated systems20,21 to levels unlikely to occur in natural environments. Dosing consisted of a MeHg spiked diet (hereafter referred to as preoogenesis Hg), followed by a diet enriched with Me199Hg which spanned the period of oogenesis. The objectives of this study were to (1) quantify maternal transfer of mercury and determine if it occurs in a dose-dependent nature and (2) use a stable mercury isotope to test recent findings from another species that suggest maternally transferred mercury is largely derived from the maternal diet during oogenesis.14
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EXPERIMENTAL SECTION Food Preparation. Methylmercury diets were prepared with methylmercury(II) chloride (Alfa Aesar) for the first period of dosing (28 days), and with laboratory synthesized Me199Hg for the remainder of dosing (63 days). An enriched (83.8%) stable isotope of mercury (199Hg) was purchased from Trace Science (Pilot Point, TX). Me199Hg was synthesized by methylation of 199Hg with methylcobalamin and subsequent extraction using methylene chloride.15 Me199Hg solutions were analyzed for both total Hg (THg) and MeHg to confirm that all mercury in solution was MeHg. MeHg was incorporated into TetraMin Tropical Flake fish food (Tetra) via an agar/gelatin matrix. Gelatin, agar, flake food, and deionized water were combined in a mass ratio of 0.7:1:20:100, respectively. Agar and gelatin were added to boiling deionized water and mixed with preground flake food in a Pyrex baking dish. Aqueous MeHg was then added to the mixture at calculated volumes to achieve the following nominal concentrations: 1, 5, and 10 μg g −1 dw (measured concentrations of Hg in food are provided in Table 1). Control diets were prepared using the same procedure, but without a MeHg spike. After setting in a refrigerator overnight, food was freeze-dried and ground to produce a fine, dry, flake mixture. Fish diets were stored at −4 °C between feedings to minimize degradation.22 Each batch of food made was subsampled and analyzed in triplicate, in order to measure actual methylmercury doses. Since significant MeHg breakdown in food was not found in previous experiments,22 each batch of food was measured once. 1958
dx.doi.org/10.1021/es404325c | Environ. Sci. Technol. 2014, 48, 1957−1963
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Pearson product moment correlation analysis was used to determine correlations between egg THg concentration and (1) the number of eggs in the mother and (2) Hg in the maternal diet. To calculate the percent of Hg transferred from a female to her eggs, a preoogenesis fish body burden was estimated by adding fish and egg Hg burdens. The percent of this total found in eggs was then calculated. The percentages of 199Hg and preoogenesis Hg maternally transferred were log transformed prior to correlation analysis (Pearson product moment). These percentages were also compared among treatments with oneway ANOVA.
concentrated nitric acid/sulfuric acid on a hot plate at 120−150 °C for 6−9 h.25 Samples were diluted to 50 mL with DI water, oxidized with 1 mL of bromine monochloride (BrCl), and analyzed the following day. Eggs from each female were freezedried in 5 mL Teflon vials and digested with 2 mL of 50:50 nitric acid/sulfuric acid for 24 h in a 60 °C oven. Mercury Analysis. Because transformation of some ingested MeHg within the fish is possible, but unlikely, we analyzed fish for THg rather than MeHg. A previous study with similar MeHg dosing methods found that nearly all of the mercury in maternal fathead minnows was MeHg.14 Therefore, THg is a good estimate of MeHg in this experiment. Analysis of THg was perfomed using a Tekran model 2600 cold vapor atomic fluorescence (CVAF) system (Tekran Instruments, Canada) and quantified according to EPA method 1631.26 For analysis of Hg isotopes, this instrument was interfaced with an ICP−MS (Agilent 4500) and concentrations of 199Hg and preoogenesis Hg were calculated based on previously published methods.15 This calculation utilizes the concentrations of known impurities measured in the enriched isotope to correct for their presence in all samples to which it is added. This correction means that any background Hg measured is not from impurities in the enriched isotope but from that which is naturally occurring, or in our case, from the 28 day preoogenesis dosing phase, as well as Hg naturally occurring in the food. Total Hg standards were prepared from a NIST stock solution and quality control included calibration blanks, replicate standards and samples, duplicate dilutions, and duplicate SRMs (DORM-2, National Research Council Canada). We analyzed food samples for MeHg via distillation, ethylation with sodium tetraethylborate,27,28 and purge and trap (Tenax) cold vapor atomic fluorescence detection (Tekran model 2500). Quality control included blanks, sample replicates, and SRMs (DORM-2, National Research Council Canada). Statistical Analyses. Statistical analyses were conducted using Minitab (version 13.1, Minitab Inc.). Mean values for each replicate were calculated and treatments were compared by analysis of variance (ANOVA). Because of a water quality issue in several aquaria, about half of the replicates were lost (two control, three low, three medium, and four high treatment tanks). Therefore, sample sizes for ANOVA are lower than the original number of replicates stated in the experimental design. We excluded the high MeHg treatment from all ANOVA due to the low n value (n = 2). For linear regressions and correlation analysis relating females to their eggs, values for individual fish from all replicates were used. This was thought to be the most biologically relevant approach, in order to capture the specific relationship between each individual and her offspring. As a result, sample sizes are larger in linear regressions and correlation analyses than in ANOVA. Assumptions of normality and homoscedasticity were tested prior to each analysis and data were transformed if necessary. Statistical significance was evaluated at α = 0.05 in all cases. Total Hg, preoogenesis Hg, and enriched Hg isotope concentrations in fish and eggs were log transformed and analyzed for treatment differences using one-way ANOVA. THg concentrations were calculated by summing preoogenesis Hg and 199Hg. Control fish and eggs were eliminated from these analyses because they were never exposed to enriched 199 Hg. Linear regression analysis was used to determine if egg THg concentration was dependent on maternal Hg burden.
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RESULTS AND DISCUSSION Female egg production did not significantly differ between control, low, and medium Hg treatments, which had an average of 74 ± 8, 49 ± 26, and 69 ± 28 eggs per female, respectively. Females fed the high MeHg diet had the lowest number of eggs (mean = 10); however, this treatment was excluded from ANOVA due to the low n value. Total Hg concentrations in eggs were significantly dependent on maternal THg body burden and showed a strong positive linear relationship (p < 0.001, R2 = 0.914; Figure 1). Egg THg
Figure 1. Linear regression analysis of mass specific THg concentrations in female bodies versus eggs. Points represent individuals (n = 39). All treatments are included.
concentrations were negatively correlated with the number of eggs in the mother (p = 0.023, r = −0.443; Figure 2). Total Hg concentrations in eggs were significantly different between treatments (F(3,8) = 88.58, p < 0.001) and egg Hg content was significantly positively correlated with Hg concentration in the maternal diet (p = 0.003, r = 0.894). Overall, both preoogenesis Hg and the enriched Hg isotope (199Hg) increased with dose in fish and eggs (Figure 3). Fish in the medium treatment accumulated significantly more 199Hg than individuals in the low treatment (F(1,4) = 18.72, p = 0.012; Figure 3). However, while fish tissue concentrations of preoogenesis Hg differed, this difference was not significant between treatments at the 95% confidence level (F(1,4) = 7.04, p = 0.057). Eggs from the medium treatment had significantly more preoogenesis Hg than eggs from the low treatment (F(1,4) = 12.21, p = 0.0250; Figure 3), while enriched Hg isotope concentrations in eggs were statistically similar between treatments (F(1,4) = 3.20, p = 0.148). Additionally, 1959
dx.doi.org/10.1021/es404325c | Environ. Sci. Technol. 2014, 48, 1957−1963
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Table 2. Mean Percent Pre-oogenesis Hg ± 1 SE in Fish and Eggs for the Low (n = 3), Medium (n = 3), and High (n = 2) Treatmentsa mean % preoogenesis Hg treatment
fish
eggs
low med high
13.4 ± 0.9 10.3 ± 2.6 10.3 ± 1.0
7.9 ± 2.1 14.8 ± 2.9 8.6 ± 0.5
a
Values represent a percentage of the total mercury concentration. Percentages for low and medium treatments are statistically similar. High treatment values were not included in ANOVA due to low n value.
199
Hg and percent of preoogenesis Hg transferred (p < 0.001, r = 0.779; Figure 4), suggesting that both preoogenesis Hg and Figure 2. Correlation between the number of eggs in a given female and the mass specific THg concentration of her eggs. Points represent individuals (n = 26). Controls were excluded due to low concentrations (12.3 ± 8.2 ng g−1 dry wt).
concentrations of preoogenesis Hg in eggs in medium and low treatments were significantly higher than control concentrations, indicating these levels were above background mercury from the flake food diet alone and therefore the result of the preoogenesis dosing phase. Although significant amounts of both preoogenesis Hg and the enriched Hg isotope were transferred to eggs, a majority of Hg in eggs was 199Hg. In eggs from the low treatment, 94% of mercury was 199Hg, 84% was 199 Hg in eggs from the medium treatment, and 92% was 199Hg in eggs from the high treatment. In addition to comparing tissue concentrations, it is important to look at the relative percentages of both pools of Hg transferred to eggs in order to trace sources of maternally transferred Hg. The percent of total mercury that was preoogenesis Hg did not significantly differ between fish and eggs (F(1,10) = 0.188, p = 0.674; Table 2) or between low and medium treatments (F(1,10) = 0.470, p = 0.509; Table 2). The percent of mercury transferred from a female to her eggs averaged 0.36% of preoogenesis Hg and 0.44% of 199Hg. There was a significant positive correlation between the percent of
Figure 4. Relationship between the percent of preoogenesis Hg and the percent of 199Hg transferred from female to eggs. Points represent individuals (n = 26).
the enriched Hg isotope were transferred proportionally. The percent of Hg transferred to eggs did not significantly differ
Figure 3. Mean concentrations of preoogenesis Hg and 199Hg in eggs (A) and fish carcass (B), for low (n = 3), medium (n = 3), and high (n = 2) treatments. Different letters above bars indicate significantly different values within a series. High treatment values were excluded from statistical analysis due to low n value. 1960
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among treatments for either preoogenesis Hg or (F(1,10) = 0.734, p = 0.412; Table 3).
199
Table 3. Mean Percent Pre-oogensis Hg and Percent Transferred from Female to Eggs ±1 SE for Each Treatmenta
199
treatment
% preoogensis Hg transferred
low medium high
0.31 ± 0.14 0.60 ± 0.01 0.17 ± 0.04
%
199
treatment. In this study, preoogenesis Hg represents historical mercury exposure and the enriched Hg isotope (199Hg) represents recent Hg exposure, including the period of oogenesis. Higher preoogenesis mercury in eggs from higher dietary treatments is evidence that a significant portion of maternally transferred Hg is from the burden stored in female tissues. It is clear from our study that recently ingested MeHg is not the only source of maternally transferred mercury. If this were the case, we would have found almost entirely 199Hg in eggs. Preoogenesis Hg concentrations in eggs from Hg-exposed females were significantly higher than in control eggs. Therefore the presence of preoogenesis Hg in eggs cannot be attributed to background mercury levels in flake food. Preoogenesis Hg and the enriched Hg isotope were transferred proportionally to eggs, further suggesting that both female tissues and the diet during oogenesis are sources of maternally transferred Hg. While it appears both mercury sources are important, a majority of the Hg found in eggs was from the diet during oogenesis. This is similar to the distribution of Hg found in laboratory-dosed fathead minnow eggs.14 However, it must be noted that in our study the enriched isotope dosing phase was much longer than the preoogenesis dosing phase (63 days vs 28 days), which may have contributed to the larger percentage of 199 Hg found in eggs. On the basis of these findings, it appears that both historical and more proximate (relative to oogenesis) mercury exposure can be important in the context of maternal transfer. For example, if a fish experiences low Hg exposure during early life stages, but feeds on a highly contaminated diet during oogensis, maternally transferred mercury will largely reflect recent dietary exposure, as found by Hammerschmidt and Sandheinrich.14 However, if a fish accumulates high mercury concentrations early in life, but feeds on a relatively uncontaminated diet during oogenesis, maternal mercury transfer may still be high due to historical exposure. In this case, the source of maternally transferred Hg would be largely from the burden stored in female tissues. This may be especially relevant for migratory species (e.g., salmon and striped bass), which hatch and spend early life stages in rivers, but would likely undergo oogenesis in an ocean environment before returning to spawn upriver. If juveniles spend time in a highly contaminated river or estuary prior to migrating to open ocean, historical Hg exposure may play a larger role in maternal Hg transfer. Maternal transfer of Hg may affect larger scale ecological processes, such as larval recruitment and Hg trophic transfer. Laboratory experiments have demonstrated that embryonic exposure to MeHg causes behavioral changes in larval fish that impair survival, such as decreased foraging ability and increased susceptibility to predation.1,31,32 Additionally, predation is a major source of mortality for larval fish,33 making these populations an important link in the trophic transfer of MeHg. Species feeding on larval or juvenile fish are affected by maternally transferred MeHg, as this constitutes a large portion of Hg contamination in young fish. The technique of using enriched isotopes as tracers offers the ability to more directly trace a contaminant from source to parent to offspring, as well as connect source to effect. Understanding maternal transfer of contaminants is key to assessing the potential effects of contaminants on offspring. There is limited research on the effects of maternally transferred MeHg on offspring health, particularly in fish. Although the maternal contribution of MeHg is relatively small,
Hg
Hg
Hg transferred
0.64 ± 0.01 0.52 ± 0.15 0.17 ± 0.12
a
Percentages for low and medium treatments are statistically similar. High treatment values were not included in ANOVA due to low n value.
Because eggs were stripped directly from females after euthanasia, eggs were not exposed to aqueous MeHg. Therefore we can assume all Hg found in eggs was maternally transferred. We found an average of 0.4% of female total Hg body burden was transferred to eggs. This percentage is similar to the THg transfer measured in five different species: 0.3% (white bass), 0.4% (smallmouth bass), 0.6% (rainbow trout), 1.8% (white sucker), and 2.3% (yellow perch).29 A study by Hammerschmidt et al. (1999) also found 1.9% transfer in fieldcollected yellow perch. The percentage of female Hg burden transferred to eggs was relatively low, compared to observations of transfer of organic contaminants, such as PCBs, pesticides, and fungicides. For organic compounds, studies have found that 5−30% of the maternal burden is transferred to fish eggs, depending on the specific compound and species.29 Mercury transfer was also lower than that observed for some inorganic contaminants in other organisms. One study found that female frogs transfer approximately 50% of their total selenium burden and 3−8% of their strontium burden into eggs.2 The low percentage of Hg transferred from mother to egg suggests that spawning is not a significant mercury depuration route for female C. variegatus, although it may be for other contaminants. We found a significant negative correlation between the number of eggs in a female and the Hg concentration in her eggs (Figure 2). Additionally, egg mass did not differ between treatments, nor did the percent of Hg body burden transferred (Table 3). This suggests that a specific proportion of Hg is partitioned from the female to developing eggs. If this burden is distributed among a larger number of eggs, a lower mercury concentration would be expected in offspring. Therefore, it appears the clutch size of an organism during a particular reproductive event may have important implications for the amount of Hg transferred and subsequent effects on offspring. This has also been suggested for maternal transfer of selenium in frogs.2 We found that maternal Hg body burden was a strong predictor of Hg content of eggs. Mercury concentrations in eggs increased linearly with female Hg concentration, as observed elsewhere in both field-collected yellow perch 30 and laboratory-dosed fathead minnows.14 This linear model could be determined for a specific species and then applied to Hg tissue concentrations of wild fish populations, in order to predict exposure and potential risk to offspring. The most striking result of this study was the increase of preoogenesis Hg in eggs, with increasing dose administered over the 28 days prior to egg formation (Figure 3). A constant percentage of preoogenesis Hg was transferred to eggs in each 1961
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(12) Latif, M. A.; Bodaly, R. A.; Johnston, T. A.; Fudge, R. J. P. Effects of environmental and maternally derived methylmercury on the embryonic and larval stages of walleye (Stizostedion vitreum). Environ. Pollut. 2001, 111, 139−148. (13) Simmons-Willis, T. A.; Koh, A. S.; Clarkson, T. W.; Ballatori, N. Transport of a neurotoxicant by molecular mimicry: the methylmercury−L-cysteine complex is a substrate for human L-type large neutral amino acid transporter (LAT) 1 and LAT2. Biochem. J. 2002, 367, 239−246. (14) Hammerschmidt, C. R.; Sandheinrich, M. B. Maternal diet during oogenesis is the major source of methylmercury in fish embryos. Environ. Sci. Technol. 2005, 39, 3580−3584. (15) Hintelmann, H.; Ogrinc, N. Determination of stable mercury isotopes by ICP/MS and their application in environmental studies. ACS Symp. Ser. 2003, 835, 321−338. (16) Harris, R. C.; Rudd, J. W.; Amyot, M.; Babiarz, C. L.; Beaty, K. G.; Blanchfield, P.J...; Tate, M. T. Whole-ecosystem study shows rapid fish-mercury response to changes in mercury deposition. Proc. Natl. Acad. Sci. 2007, 104 (42), 16586−16591. (17) Murdy, E. O.; Birdsong, R. S.; Musick, J. A. Fishes of Chesapeake Bay; Smithsonian Institution Press: New York, 1997. (18) Raimondo, S.; Hemmer, B. L.; Goodman, L. R.; Cripe, G. M. Multiple generation exposure of the estuarine sheepshead minnow, Cyprinodon variegatus, to 17β-estradiol II: population effects through two life cycles. Environ. Toxicol. Chem. 2009, 28 (11), 2397−2408. (19) Rowe, C. L.; Dunson, W. A. Individual and interactive effects of salinity and initial fish density on a salt marsh assemblage. Mar. Ecol.: Prog. Ser. 1995, 128, 271−278. (20) Allard, M.; Stokes, P. M. Mercury in crayfish species from thirteen Ontario lakes in relation to water chemistry and smallmouth bass mercury. Can. J. Fish Aquat. Sci. 1989, 46, 1040−1046. (21) Hall, B. D.; Rosenberg, D. M.; Wiens, A. P. Methylmercury in aquatic insects from an experimental reservoir. Can. J. Fish Aquat. Sci. 1998, 55, 2036−2047. (22) Stefansson, E. S.; Heyes, A.; Rowe, C. L. Accumulation of dietary methylmercury and effects on growth and survival in two estuarine forage fish: Cyprinodon variegatus and Menidia beryllina. Environ. Toxicol. Chem. 2013, 32 (4). (23) Zillioux, E. J.; Johnson, I. C.; Kiparissis, Y.; Metcalfe, C. D.; Wheat, J. V.; Ward, S. G.; Liu, H. The sheepshead minnow as an in vivo model for endocrine disruption in marine teleosts: A partial lifecycle test with 17α-ethynylestradiol. Environ. Toxicol. Chem. 2001, 20 (9), 1968−1978. (24) Lytle, T. F.; Manning, C. S.; Walker, W. W.; Lytle, J. S.; Page, D. S. Life-cycle toxicity of dibutyltin to the sheepshead minnow (Cyprinodon variegatus) and implications of the ubiquitous tributyltin impurity in test material. Appl. Organomet. Chem. 1993, 17 (9), 653− 661. (25) Bloom, N. S. On the chemical form of mercury in edible fish and marine invertebrate tissue. Can. J. Fish Aquat. Sci. 1992, 49, 1010−101. (26) Method 1631: mercury in water by oxidation, purge and trap and cold vapor atomic fluorescence spectrometry; EPA-821-R-96−001; U.S. Environmental Protection Agency, Office of Water: Washington, DC, 1996; (27) Bloom, N. S. Determination of picogram levels of methylmercury by aqueous phase ethylation followed by cryogenic gas chromatography with cold vapor atomic fluorescence detection. Can. J. Fish Aquat. Sci. 1989, 46, 1131−1140. (28) Horvat, M.; Liang, L.; Bloom, N. Comparison of distillation with other current isolation methods for the determination of methyl mercury compounds in low level environmental samples Part II. Water. Anal. Chim. Acta 1993, 282, 153−68. (29) Niimi, A. J. Biological and toxicological effects of environmental contaminants in fish and their eggs. Can. J. Fish Aquat. Sci. 1983, 40 (3), 306. (30) Hammerschmidt, C. R.; Wiener, J. G.; Frazier, B. E.; Rada, R. G. Methylmercury content of eggs in yellow perch related to maternal exposure in four Wisconsin lakes. Environ. Sci. Technol. 1999, 33, 999− 1003.
exposure to MeHg during sensitive early life stages could have effects on an individual’s reproductive success later in life. Studies have demonstrated that such effects are possible in fish,34 even at the population level.35 Therefore crossgenerational effects of maternally transferred MeHg remain an important area of future study in order to better understand population-level responses.
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AUTHOR INFORMATION
Corresponding Author
*Phone: (410) 326-7439; fax: (410) 326-7341; e-mail: heyes@ umces.edu. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS A graduate fellowship from Chesapeake Biological Laboratory Graduate Education Committee supported E. Stefansson during this study. We appreciate the assistance of E. Jenny and L. Lockard in fish care and laboratory analyses. We are also grateful to E. Orlando for valuable input over the course of the experiment. Contribution number 4855 of the University of Maryland Center for Environmental Science.
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REFERENCES
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