Environ. Sci. Technol. 2005, 39, 3580-3584
Maternal Diet During Oogenesis Is the Major Source of Methylmercury in Fish Embryos CHAD R. HAMMERSCHMIDT† AND M A R K B . S A N D H E I N R I C H * ,‡ Department of Marine Sciences, University of Connecticut, Groton, Connecticut 06340, and Department of Biology, River Studies Center, University of WisconsinsLa Crosse, La Crosse, Wisconsin 54601
Development of the early life stages of fishes can be affected adversely by methylmercury (MeHg) transferred from the maternal parent to the developing egg. We examined maternal transfer of MeHg to eggs of fathead minnows Pimephales promelas and evaluated the role of maternal MeHg burden versus that in the maternal diet during oogenesis on egg concentrations. Juvenile fish were fed one of four diets until sexual maturity (phase 1): A control diet (0.06 µg of Hg g-1 dry weight) and three that were contaminated with MeHg at 0.88 (low), 4.11 (medium), and 8.46 µg of Hg g-1 (high). At sexual maturity, female fish were paired with a male, again fed one of the four diets, and allowed to reproduce (phase 2). To assess the significance of female dietary exposure during oogenesis on MeHg in eggs, some fish were fed diets during phase 2 that differed from those during phase 1. Mean concentrations and burdens of MeHg in eggs from fish fed the same diet throughout the experiment varied with MeHg content of the maternal diet and were related positively to levels in the carcass of the maternal fish. However, MeHg in eggs was not proportional to that in carcasses among dietary treatments; MeHg in eggs from adults fed the control, low, medium, and high MeHg diets averaged 14%, 25%, 32%, and 35% of that in adults. For fish fed the control diet as juveniles and MeHg-contaminated diets after reaching sexual maturity, MeHg in eggs increased rapidly with duration of maternal dietary exposure prior to spawning. Moreover, concentrations of MeHg in eggs from fish fed the same contaminated diet as both juveniles and sexually mature adults were not related to the duration of adult exposure, and they were not appreciably greater than those from fish fed contaminated diets only just prior to spawning. These results indicate that the diet of the maternal adult during oogenesis, and not adult body burden, is the principal source of MeHg in fish eggs. Accordingly, the exposure of embryonic wild fishes to MeHg depends on levels of the contaminant in prey of the adult during oogenesis, which can vary intra- and interannually.
Introduction The toxicological significance of methylmercury (MeHg) to populations of fish is poorly understood (1, 2). Recent * Corresponding author phone: 608-785-8261; fax: 608-785-6959; e-mail:
[email protected]. † University of Connecticut. ‡ University of WisconsinsLa Crosse. 3580
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research, however, has shown that environmentally realistic levels of MeHg in the diet of experimental fish can suppress concentrations of sex hormones (3), alter their gonadal development (4, 5), and ultimately reduce overall reproductive success (5). Additionally, MeHg may affect the survival and development of embryolarval stages. For example, increased mortality of rainbow trout Onchorhynchus mykiss embryos was associated with egg mercury (Hg) levels as low as 0.07-0.10 µg g-1 wet weight (6). These concentrations are only six to nine times those measured in eggs of rainbow trout from Lake Ontario (7) and are within the range measured in eggs of yellow perch Perca flavescens from semi-remote lakes in northern Wisconsin (8). Latif et al. (9) examined the effects of both maternally transferred and waterborne MeHg on embryos and larvae of walleye Stizostedion vitreum from industrially polluted Clay Lake and two remote lakes in Manitoba. In their study, environmentally relevant exposures to waterborne MeHg deleteriously affected the hatching success of eggs and the heart rate of embryos; however, MeHg transferred from the maternal parent did not affect either of these parameters, and the growth of larval walleye was not affected by MeHg exposure from either pathway. Exposure of grayling Thymallus thymallus to waterborne MeHg for 10 days during embryogenesis impaired feeding efficiency and reduced competitive ability of the fish as 3-year-old adults (10). Clearly, much additional work is needed to determine the relative sensitivities of different fish species and life stages to embryolarval MeHg exposure. Diet is the principal source of MeHg in wild adult fish (11, 12), and transfer of MeHg from the maternal parent is the major pathway of exposure for fish embryos (7, 8, 13). Uptake of inorganic Hg and MeHg from surrounding water is inhibited by the chorion (13, 14). It is unclear, however, if MeHg in fish eggs is remobilized from parental somatic tissue or if it is from the maternal diet during oogensis. Here we show that maternal diet during oogenesis is the principal source of MeHg in fish eggs. We fed fathead minnows Pimephales promelas diets containing concentrations of MeHg present in some aquatic food webs, maintained the fish through sexual maturity, and evaluated the relative roles of maternal MeHg burden and MeHg in the maternal diet during oogenesis on levels of the contaminant in fish eggs.
Experimental Section Study Design. The design of this study has been reported previously in our investigation of the effects of dietary MeHg on reproduction of fathead minnows (5). About 1400 juveniles were randomly placed into each of four 500-L flow-through tanks receiving well water, and they were fed ad libitum one of four phase 1 diets, three of which were contaminated with methylmercuric chloride (phase 1). Phase 1 of dietary exposure to MeHg was defined as the period from 90 days posthatch to the onset of sexual dimorphism (about 240 days posthatch; ref 5). Mean concentrations (µg g-1 dry weight; (1 SE) of total Hg in the diets were 0.060 ( 0.003 (control), 0.88 ( 0.02 (low), 4.11 ( 0.08 (medium), and 8.46 ( 0.17 (high). Levels of MeHg in the test diets spanned those in zooplankton, benthic invertebrates, and small forage fish from low-alkalinity lakes in North America (Table 1 in ref 5) and thus reflect potential dietary exposures of fish in many fresh waters with MeHg-contaminated food webs. After fathead minnows became sexually dimorphic (about 240 days posthatch), mature male and female fish were paired randomly, assigned to quadrants in one of fifteen 50-L flowthrough breeding aquaria receiving well water, and allowed to reproduce (phase 2). Phase 2 was defined as the period 10.1021/es0486263 CCC: $30.25
2005 American Chemical Society Published on Web 03/24/2005
TABLE 1. Summary Characteristics ((1 SE) of Eggs and Female Adult Fathead Minnows Fed the Same Diet throughout the Experiment
diet
n
duration of dietary MeHg exposure (days)
control low MeHg medium MeHg high MeHg
17 9 7 6
219 ( 6 238 ( 6 225 ( 6 200 ( 8
total length (mm)
fresh weight (g)
carcass total Hg (µg g-1 dry wt)
egg MeHg (µg g-1 dry wt)
egg MeHg burden (pg egg-1)
57 ( 1 57 ( 1 56 ( 1 62 ( 2
1.93 ( 0.16 2.00 ( 0.09 1.94 ( 0.14 2.38 ( 0.18
0.48 ( 0.04 3.36 ( 0.10 13.85 ( 1.48 19.20 ( 1.47
0.07 ( 0.01 0.84 ( 0.06 4.41 ( 0.27 6.85 ( 0.47
11 ( 1 130 ( 9 730 ( 60 1100 ( 70
from breeding pair assignment to oviposition. Breeding aquaria were arranged randomly within a large water bath, and each aquarium was partitioned into quadrants with plastic screen. One pair of fish inhabited each quadrant with an acid-cleaned spawning substrate (a half-cylinder of PVC pipe). Pairs were fed a phase 2 diet with concentrations of MeHg similar to that of the diet in phase 1. To evaluate the relative contributions of MeHg from maternal fish and their diet during oogenesis on MeHg in eggs, some fish that received the control diet during phase 1 were fed the low, medium, or high MeHg diet during phase 2. Spawning substrates were examined daily for eggs; eggs were transferred from spawning substrates to plastic vials with either a plastic spoon or stainless steel spatula. Females fed the same diet during both phases 1 and 2 were sacrificed within 24 h of spawning. Fish fed the control diet during phase 1 and a MeHg-contaminated diet during phase 2 were allowed to oviposit two or more clutches, and the adults were sacrificed within 24 h of spawning the final clutch. Each adult fish was placed in a food-grade plastic bag, anesthetized in a refrigerator, blotted dry, measured ((1 mm total length), weighed ((0.1 mg), and dissected to remove the gonads. Carcasses (i.e., whole fish minus gonads) and eggs were promptly frozen at e-30 °C until lyophilization. Mercury Determinations. Carcasses and diets of adult fish were analyzed individually for total Hg. We acid-digested whole, lyophilized carcasses of fish weighing < 500 mg, 400mg subsamples of lyophilized carcasses weighing g 500 mg, and 250-mg subsamples of diets following the methods of Hammerschmidt et al. (8). Each digestate was analyzed by flow injection cold-vapor atomic absorption spectroscopy with a Perkin-Elmer FIMS 100. MeHg in fathead minnow eggs and selected fish carcasses (n ) 12) was determined by gas chromatographic cold-vapor atomic fluorescence spectrometry (GC-CVAFS) after extraction with dilute HNO3. Subsamples of lyophilized eggs (4159 eggs; 0.6-23.4 mg) and homogenized carcasses (100150 mg) were transferred to 15-mL centrifuge tubes and digested with 1.4-7.0 mL of 4.57 M HNO3 in a covered 60 °C water bath for 12 h (15). This extraction method quantitatively releases MeHg from biological tissue, and its simple matrix is used easily with conventional purge-andtrap techniques following derivatization of MeHg with sodium tetraethylborate. MeHg in digestates was determined by GCCVAFS with analytical techniques described elsewhere (16, 17). Briefly, a 100-1000-µL aliquot of digestate was transferred to a 250-mL sparging flask containing 100 mL of reagentgrade water, the acidity was neutralized with KOH, and the pH was adjusted to about 4.9 with 2 M acetate buffer. Sodium tetraethylborate (200 µL of 1% (w/v) solution) was added to derivatize the Hg species, and the volatile ethyl-Hg derivatives (i.e., ethylmethylmercury and diethylmercury) were purged from solution with N2 and concentrated on Tenax. Ethylated derivatives of Hg species were thermally desorbed from the Tenax, separated by isothermal gas chromatography, decomposed pyrolytically, and detected with flow-injection CVAFS (18). Quality Assurance. All equipment used to culture fathead minnows was acid-cleaned and rinsed with either reagent-
grade water (nominal resistance g 15 MΩ-cm) or well water. Equipment used for analyses of total Hg and MeHg was cleaned with acid and rinsed with reagent-grade water. Acids and reagents used in digestions and analyses were suitable for use in Hg determinations. Accuracy of total Hg determinations in maternal fish and experimental diets for each analytical batch of samples was quantified by analyses of (1) certified reference materials from the National Research Council of Canada and the U.S. National Institute of Standards and Technology (NIST), (2) replicate subsamples of homogenized fish and test diets, (3) spiked (before digestion) subsamples of homogenized fish and diets, and (4) blanks and standards taken through the digestion procedures. Mean measured concentrations and 95% confidence intervals (CI) of total Hg in the four reference materials analyzed were within the certified ranges, which ranged from 0.27-0.39 to 4.38-4.90 µg g-1 dry weight. Method precision (relative standard deviation) for determinations of total Hg, estimated from analyses of duplicate and triplicate subsamples, averaged 3.3% (range, 0.1-17.9%) for fish and 4.1% (range, 1.3-9.1%) for diets. The mean recovery of total Hg was 98% (95% CI, 97-100%) for adult fish and 96% (CI, 93 -100%) for diets. Our estimated method detection limit (19) for total Hg in a 250-mg sample of homogenized fish was 0.004 µg of Hg g-1. Accuracy of MeHg measurements was assessed with parameters similar to those used for total Hg analyses. Sample MeHg was measured after calibration with procedural standards prepared from a 10 ng L-1 solution, which was calibrated against Hg0 standards taken from the headspace over pure liquid (20) and Hg2+ solutions traceable to the U.S. NIST. Calibrations with procedural standards always yielded standard regressions with coefficients of determination g0.999. All analyses of MeHg in reference materials were within their certified ranges; our mean measured concentration of MeHg in TORT-2 was 0.151 µg g-1 dry weight (certified range, 0.139-0.165 µg g-1), and that in DOLT-2 was 0.715 µg g-1 (certified range, 0.640-0.746 µg g-1). The mean recovery of MeHg from procedurally spiked digestates of egg was 101%, and the precision of methodically replicated subsamples averaged 5.8% RSD. The estimated detection limit (3σ of procedural blanks) for MeHg in a 10-mg sample of lyophilized egg was about 0.0005 µg g-1.
Results and Discussion Nearly all of the mercury in maternal fish was MeHg. Fathead minnow carcasses were analyzed for total Hg, although MeHg was the Hg species measured in fish eggs. We measured MeHg in about 10% of the fish carcasses to verify that it was the dominant form of Hg. MeHg averaged 93% of the total Hg (95% CI, 85-101%) in the 12 carcasses analyzed. Therefore, we consider our measurements of total Hg in fish carcasses to be representative of the MeHg concentration. Mean concentrations and burdens of MeHg in eggs from fish fed the same diet throughout the experiment varied with dietary exposure of the adult fish (Table 1). MeHg in both the VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Relation between methylmercury (MeHg) in eggs and the duration that maternal fish were fed either the control or a MeHg-contaminated diet prior to spawning. NS denotes that the relationship was not significant.
FIGURE 1. Relation between methylmercury (MeHg) in eggs and total Hg in carcasses of maternal fish fed the same diet throughout the experiment. maternal female and its eggs were related positively to that in the maternal diet; mean concentrations of MeHg in eggs from fish fed the control, low, medium, and high diets were 0.07, 0.84, 4.4, and 6.9 µg g-1 dry weight. In addition, the burden and concentration of MeHg in eggs was related positively to total Hg in the carcass of the maternal fish (Figure 1). Similar relationships have been observed for wild populations of fish (8, 21), and they have been interpreted to indicate a direct link between MeHg in the maternal carcass and that partitioned into developing oocytes. However, the concentration ratio of MeHg in eggs to that in the maternal carcass was not constant among dietary treatments (Krukal Wallis, χ2 ) 26.7, df ) 3, p < 0.001) and increased with MeHg in the diet of the parent fish. The concentration of MeHg in eggs from females fed the control, low, medium, and high diets averaged 14%, 25%, 32%, and 35% of that in the carcass. This indicates subtle, but potentially toxicologically significant, differences in the allocation of MeHg to fish eggs. There are few reports in the literature relating Hg in eggs to that in adult fish. Johnston et al. (21) examined MeHg in eggs of walleye from seven geographically distinct populations. Mean concentrations of MeHg in walleye eggs ranged from 0.005 to 0.834 µg g-1 dry weight, which were 1.1-12% of those in the muscle of the parent fish. Hammerschmidt et al. (8) reported that total Hg in eggs of yellow perch from four northern Wisconsin lakes ranged 0.007-0.819 µg g-1 dry weight, and these concentrations were 5-20% of those in the maternal carcass. Most (> 90%) of the Hg in the eggs and carcasses of yellow perch was MeHg (8). The Hammerschmidt et al. data also indicates that MeHg in eggs of yellow perch, as a percentage of that in the adult carcass, increased with MeHg in the adult. For example, eggs of yellow perch from Pallette Lake had a mean concentration of 0.012 µg g-1 dry weight, which was 5.1% of the mean level in the carcass (0.236 µg g-1). The average mercury level in eggs of yellow perch from Little 3582
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Rock Lake was 0.105 µg g-1 dry weight, which was 9.5% of the adult concentration (1.11 µg g-1), and eggs from Max Lake in 1996 had 0.298 µg g-1, which was 19.1% of the adult concentration (1.56 µg g-1). In these two studies, the concentration of MeHg in eggs was related logarithmically to that in the parent fish (8, 21). However, there was no relationship between Hg in eggs and carcasses for five species of fish from Lake Ontario (7). These results indicate that while MeHg in fish eggs is often related to that in the adult carcass for a given fish species, the concentration of MeHg in eggs is not a consistent fraction of that in the carcass. The MeHg concentration of fathead minnow eggs was similar to that in the maternal diet of fish fed the same food during both phases 1 and 2. The mean level of MeHg in eggs of fish fed the control, low, medium, and high diets during both phases of the study averaged 108%, 96%, 101%, and 81% of the concentration in the maternal diet. The concentration ratio of MeHg in the eggs to that in the maternal diet was not significantly different among dietary treatments (Kruskal Wallis, χ2 ) 2.88, df ) 3, p ) 0.41). Moreover, and for all fish combined (n ) 39), egg MeHg averaged 100% of the concentration in the maternal diet, regardless of the level of MeHg in the parental fish or its diet. A strong dietary influence on the MeHg content of fish eggs is consistent with our observation that MeHg in eggs is not a consistent fraction of that in the carcass of fathead minnows and yellow perch (8). Dietary MeHg was transferred rapidly from the adults to the eggs (Figure 2). The MeHg content of eggs increased with the duration of maternal dietary exposure prior to spawning, for fish fed the control diet as juveniles (i.e., phase 1) and MeHg-contaminated diets after reaching sexual maturity (i.e., phase 2; Figure 2). Based on linear regression analyses, eggs from fish fed low and medium diets for only 10 days prior to spawning had predicted MeHg concentrations of 0.25 and 1.36 µg g-1 dry weight. These levels were considerably greater than those in eggs of fish fed only the control diet for 219 days (0.065 µg g-1). Moreover, concentrations of MeHg in eggs of these fish were not appreciably less than those in fish fed the same MeHg-contaminated diets both as juveniles and sexually mature adults (Figure 2). For example, concentrations of MeHg in eggs spawned after 47-, 37-, and 32day exposures of sexually mature adults to the low, medium, and high diets (phase 2 only) were within the 95% CI of mean levels measured in eggs from adults exposed to the same MeHg diets for 159-272 days (phase 1 and phase 2). MeHg in eggs from fish fed MeHg-contaminated diets as both juveniles and as sexually mature adults (i.e., the same
Maternal diet during oogenesis is the principal source of MeHg in fish eggs. Concentrations of MeHg in eggs are commonly related to those in the adult fish; however, the maternal diet is a common source of MeHg in both germ and somatic tissues. MeHg in fish eggs, unlike fish muscle, is highly sensitive to changes in the MeHg content of the maternal diet. Hence, seasonal and annual changes in adult dietary MeHg must be considered when assessing the relative risk of fish embryos to MeHg exposure.
Acknowledgments
FIGURE 3. Relation between methylmercury (MeHg) in carcasses of maternal fish and the duration they were fed either the control or a MeHg-contaminated diet prior to spawning.
We thank Rachel Hoffman for help with determinations of total Hg in fish, and Joshua Duerst, Rilee Stevenson, and Roger Yee for assistance with fish cultures. Support for this study was provided by the University of Wisconsin Sea Grant College Program, the University of WisconsinsLa Crosse River Studies Center, and a STAR graduate fellowship (91591801) to C.R.H. from the U.S. Environmental Protection Agency.
Literature Cited diet in phases 1 and 2) was unrelated to the duration of adult exposure (Figure 2). This is in contrast to what would be expected if maternal burden were the principal source of MeHg to eggs, because Hg in the carcasses of adult fish fed the contaminated diets continued to increase throughout the experiment (Figure 3), although the rate of maternal Hg accumulation was not first-order and decreased with exposure duration. This indicates that extended exposure to the same concentration of dietary MeHg does not enhance levels of the contaminant in fish eggs and that egg MeHg is independent of maternal Hg. This would be expected if diet were the primary source of MeHg in the eggs. This laboratory study demonstrates that the diet of fish during oogenesis, and not adult body burden, is the primary source of MeHg in their eggs. Because fish depurate MeHg slowly (22), concentrations in long-lived wild adult fish do not respond rapidly to changes in the MeHg content of their prey. However, levels in eggs are affected by differences in the MeHg content of the prey of adult fish. For example, Hammerschmidt et al. (8) reported total mercury in carcasses and eggs of yellow perch collected in 1991 from Max Lake, prior to alkalization of the lake by groundwater addition, and in 1996 after 5 years of groundwater addition. Mean levels of total mercury in fish carcasses were not significantly different between years, but concentrations and burdens of MeHg in eggs of fish collected in 1991 were almost twice those of eggs collected in 1996. Accordingly, species of fish occupying the same trophic level, and fractional spawners that lay multiple clutches of eggs over several months, may have substantially different concentrations of MeHg in their eggs if they differ in the timing of oogenesis relative to seasonal or annual changes in MeHg concentrations of the adult prey. MeHg in surface waters can change seasonally due to mixing of MeHg accumulated in low-oxygen hypolimnetic waters (23), variations in both the production and mobilization of MeHg from sediments (24-26), fluctuations in water levels (27), and inputs of MeHg from the watershed (28, 29). Such variations in aqueous MeHg are rapidly reflected in the prey of invertivorous fish (i.e., zooplankton and aquatic insects). Paterson et al. (27), for example, reported that MeHg in zooplankton inhabiting an experimental reservoir increased about 10-fold within 6 weeks of impoundment, and their MeHg content fluctuated with seasonal water levels. In the same reservoir, coincident variations in MeHg also were observed in littoral insects (30) and finescale dace Phoxinus neogaeus, a small species of minnow that preyed upon both zooplankton and aquatic insects (31).
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Received for review September 3, 2004. Revised manuscript received February 12, 2005. Accepted February 16, 2005. ES0486263
