Dynamic Selenium Assimilation, Distribution, Efflux, and Maternal

Feb 4, 2014 - Dynamic Selenium Assimilation, Distribution, Efflux, and Maternal Transfer in Japanese Medaka Fed a Diet of Se-enriched Mayflies. Justin...
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Dynamic Selenium Assimilation, Distribution, Efflux, and Maternal Transfer in Japanese Medaka Fed a Diet of Se-enriched Mayflies Justin M. Conley,† AtLee T. D. Watson,† Lingtian Xie,‡ and David B. Buchwalter*,† †

Environmental and Molecular Toxicology Program, Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina 27695, United States ‡ Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China ABSTRACT: Selenium (Se) trafficking in oviparous species remains understudied and a major source of uncertainty in developing sound Se regulations. Here, we utilized 75Se to follow Se through a simulated natural food chain (water, periphyton, mayflies (Centroptilum triangulifer), fish (Japanese medaka)). We specifically examined Se assimilation efficiency, tissue distribution, efflux rate, and maternal transfer in medaka. Selenium assimilation efficiency (AE) averaged 63.2 ± 8.8% from mayfly diets and was not affected by mayfly [Se] across a dietary range of 5.6−38.7 μg g−1 (dry wt). However, AE decreased significantly as mayfly larva size increased. Efflux rate constants (ke) were consistent between reproductively inactive (0.066 d−1) and spawning females (0.069 d−1). Total Se loss rate constant (ke+egg; efflux and egg deposition) was 0.17 d−1 in spawning females. Interestingly, medaka appeared to rapidly shuttle Se to their eggs directly from their diet via the ovary, as opposed to mobilization from surrounding tissues, resulting in dynamic egg [Se] that was more attributable to recent dietary Se ingestion than female whole body [Se] in this asynchronous spawning fish. Spawning strategy likely plays a large role in the process of fish egg Se deposition and requires further attention to understand risk and toxicity of Se to fish.

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INTRODUCTION Selenium (Se) is an essential element that can be particularly toxic to oviparous species via dietary exposures.1−3 A wide variety of human activities introduce elevated Se into aquatic ecosystems including mining (e.g., coal, phosphate, uranium), inappropriate storage and/or spills of coal combustion wastes, irrigation of Se-rich soils, and fossil fuel refining.4,5 Fish and birds, which are either permanent or transient residents in these systems, are at risk of ingesting Se-enriched prey and may suffer reproductive failure and/or teratogenic defects in offspring.4,6 Understanding the dietary incorporation and maternal transfer of Se across a range of species is critical to properly assessing the risk to fish and wildlife populations from Se exposure. Regulatory entities struggle with Se, in part because the determination of acceptable exposure levels is complicated by highly variable and dynamic processes.7 Environmental Se cycling, foodweb incorporation, trophic transfer, and maternal transfer each play important roles in determining Se risk.1,8 The available scientific literature that has addressed these processes remains surprisingly limited given the prevalence of elevated Se in aquatic systems and is skewed toward observational field studies (as opposed to controlled laboratory studies). In particular, we currently know very little about the biological processes that govern tissue-specific Se distribution and trafficking in fish (and its variation across species), including © 2014 American Chemical Society

the deposition into eggs (i.e., are tissue Se stores mobilized to the ovary for egg deposition or is Se more directly shuttled from the diet?). Further, the influence of fish reproductive strategies (synchronous, group synchronous, asynchronous) on Se partitioning into eggs remains poorly understood. These processes are extremely important from a regulatory perspective as entities attempt to create defensible fish tissue based criteria (e.g., refs 9 and 10). In order to begin addressing these questions, the goal of the current study was to investigate the biodynamic nature of dietary Se in female fish via a simulated natural food chain (dissolved 75Se to periphyton to mayfly larvae to fish to eggs). While we previously reported on the speciation and dynamics of Se at the base of our laboratory food chain,11,12 here we focused on the kinetics and partitioning of Se within the highest level consumer, Japanese medaka. We utilized environmentally relevant dissolved Se exposures (3−10 μg L−1)7,13−15 to generate Se-enriched periphyton diets, which were then fed to newly hatched Centroptilum triangulifer mayfly larvae. Once larvae were large enough to handle, we fed them to both female Received: Revised: Accepted: Published: 2971

November 5, 2013 January 27, 2014 February 4, 2014 February 4, 2014 dx.doi.org/10.1021/es404933t | Environ. Sci. Technol. 2014, 48, 2971−2978

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(grown in October 2011) was used to rear mayfly prey for measuring medaka Se AE, tissue distribution, and efflux rate constants, while the other (grown in November 2011) was used for medaka Se maternal transfer experiments. In both instances periphyton was loaded with Se by exposing it to dissolved Se for 7 days in 2.0 L glass jars containing 1.8 L of synthetic soft water (48 mg L−1 NaHCO3, 30 mg L−1 CaSO4·H2O, 30 mg L−1 MgSO4, and 2 mg L−1 KCl; pH 7.6 ± 0.4).20 Periphyton exposure solutions were spiked with both H275SeO3 and Na2SeO3, each dissolved in 0.1 N HNO3 (specific volumes and concentrations below). Water samples were collected both prior to adding periphyton to the solutions and at the end of loading to characterize dissolved [Se]. Periphyton subsamples were collected from each plate at the end of loading to characterize periphyton [Se]. After loading and sampling, periphyton was moved to clean water and newly hatched C. triangulifer larvae were reared until they were large enough to collect and feed to medaka (∼15−25 d). Before feeding to medaka, mayfly larvae were rinsed in clean water, and a subsample was weighed wet and assayed for radioactivity to determine mayfly [Se]. Medaka Se Assimilation Efficiency, Tissue Specific Distribution, and Efflux Rate. Periphyton was loaded with Se at three treatment levels: low (background), medium, and high. All three periphyton treatments received 1000 μL of H275SeO3 (2.64 μCi mL−1; 2.77 μg Se L−1). The medium (3 μg Se L−1) and high (10 μg Se L−1) treatments also received 27 and 90 μL of a 200 μg Se mL−1 solution of Na2SeO3, respectively, whereas the low treatment received no stable Se in addition to the radiotracer. In the low treatment the Se radiotracer was used to follow the movement of background Se. At the end of the loading phase, and after accounting for background [Se], the resulting periphyton Se concentrations were 2.1 ± 0.3 (low), 4.9 ± 0.7 (medium), and 15.5 ± 4.4 (high) μg g−1 (dry wt). Medaka Se AE was measured by feeding each of 22 fish a single radiolabeled, Se-enriched C. triangulifer larva and assaying each fish for radioactivity both after engulfing the mayfly and again after defecation of the remaining undigested mayfly carcass. The use of γ-emitting isotopes allowed for the in vivo measurement of Se mass in medaka. Assimilation efficiency was taken as the % of retained Se following defecation by dividing the total mass of Se (μg) in the fish after defecation by the total mass of Se (μg) in the fish immediately after consuming the mayfly prey. Mayfly larval masses ranged from 0.9 to 5.4 mg (wet wt) and average mayfly whole body [Se] was 1.6 ± 0.1 μg g−1 (wet wt; low), 4.2 ± 0.4 μg g−1 (wet wt; medium), and 8.4 ± 0.8 μg g−1 (wet wt; high). Assimilation efficiency was assessed in six female fish per treatment level. Two male fish were assessed at the medium and high treatment levels for basic comparative purposes only as Se is known to be a female reproductive toxicant, and therefore females were the primary focus of our experiments. Following AE measurement, fish were fed 2 additional radiolabeled C. triangulifer larvae (from their respective treatment level) per day for 4 days. Fish were then fed newly hatched Artemia nauplii for one day to evacuate gut contents of radioactive food. Fish were then euthanized with MS-222 (0.015% w/v), assayed for radioactivity, blotted dry, weighed, and individually dissected for collection of digestive tract, liver, ovary/testes, and remaining carcass tissue. For ovary dissections, no attempt was made to remove previtellogenic or early vitellogenic follicles. Each tissue was weighed wet and

(non-egg-bearing and egg-bearing) and male medaka. We report on the assimilation efficiency (AE), tissue-specific distribution (digestive tract, liver, ovary/testis, and carcass), efflux rate constants, and maternal transfer of Se in medaka.

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MATERIALS AND METHODS Test Animals. The mayfly Centroptilum triangulifer (Ephemeroptera: Baetidae; WCC-2 clone) was obtained from culture at Stroud Water Research Center (SWRC, Avondale, PA) and maintained at North Carolina State University (NCSU, Raleigh, NC) in culture as described previously.11,12,16−18 Japanese medaka (Oryzias latipes) were obtained from a stock population maintained under recirculating freshwater aquaculture conditions at NCSU and fed a diet newly hatched Artemia f ranciscana nauplii (INVE Aquaculture, Inc., Salt Lake City, UT) as described previously.19 Animal care and use were in conformity with protocols approved by the NCSU Institutional Animal Care and Use Committee in accordance with the National Academy of Sciences Guide for the Care and Use of Laboratory Animals. Background [Se] Measurement. Background [Se] was analyzed in newly hatched Artemia nauplii, male and female medaka, and fish eggs. Artemia were collected on a 47-mm glass fiber filter via vacuum filtration and dried overnight at 80 °C. Medaka and eggs were freeze-dried at −50 °C overnight. All samples were weighed (dry) and digested whole in a Mars Xpress microwave digester (CEM Corporation, Matthews, NC) in 40-mL PTFE bombs according to USEPA method 1052 using ultrapure HNO3 (OmniTrace Ultra High Purity, Darmstadt, Germany). Digests were then transferred to 10mL PTFE tubes and analyzed by ICP-MS at the Environmental and Agricultural Testing Services Lab (Department of Soil Sciences, NCSU). Quality control was monitored via sample blanks (n = 11, no detection of Se above method detection limit of 0.3 μg L−1) and standard reference material (n = 5, NIST 2976 freeze-dried mussel tissue; analytical determination 1.78 ± 0.06 μg g−1 (dry wt), expected 1.80 ± 0.15 μg g−1 (dry wt)). Radioactivity Measurement. 75Selenium was obtained from the Missouri University Research Reactor as selenous acid (H275SeO3; i.e., Se(IV)). All measurements of radioactivity in water, periphyton, mayflies, fish, and eggs were performed using a Wallac Wizard 1480 automatic gamma counter (PerkinElmer, Shelton, CT). All radioactive samples were counted for 3 min, and all counting errors were ≤5%. All water samples were collected and analyzed in duplicate, and all periphyton samples were collected and analyzed in triplicate. Selenium concentrations herein are reported incorporating appropriate correction for background (stable) [Se] in periphyton as received from SWRC and reported on a wet weight (wet wt) or dry weight (dry wt) basis. Mayfly Se Enrichment and Radiolabeling. All experiments involved feeding Se-enriched, radiolabeled C. triangulifer larvae to medaka. Mayflies were raised on a natural periphyton diet grown at SWRC and shipped to NCSU as previously described.11,12,16 Upon arrival each batch of periphyton plates was randomly sampled (n = 3) and analyzed for background [Se] using ICP-MS. This analysis allowed us to account for the background Se naturally found in periphyton in our calculations of the movement of Se through the simulated food chain using the radiotracer. Two different sets of periphyton plates (and mayflies grown on those plates) were utilized. One set of periphyton plates 2972

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end of the experiment. Therefore, we used the female body mass on day 6 to calculate the whole body [Se] on days 0, 1, 2, and 3. We suspect that female body mass did not change appreciably over the spawning period; however, we acknowledge that it may have slightly decreased from days 0−6 due to egg release, in which case our estimates of whole body [Se] in relation to egg [Se] are conservative. We were also interested in the pattern of Se movement from the mayfly diet to the eggs. We estimated the average total mass (ng) of Se consumed by females by multiplying the number of larva consumed each day by the average mass of larvae (1.5 mg wet wt) and the average [Se] concentration of the larvae (4.1 ± 1.0 μg g−1 (wet wt)). Egg [Se] was calculated as the mean egg [Se] of all egg clutches oviposited on a given day. Statistical Analyses. All data reported represent mean ± standard deviation unless otherwise noted. Further, all data analyses were performed using Prism (v6.02, GraphPad Software, Inc., La Jolla, CA) at α = 0.05. Background medaka [Se] in females and males were compared using t test. Assimilation efficiency was compared in females and males using t test and assessed as a function of mayfly [Se] and mayfly mass using simple linear regression. Efflux rate constants (ke) were calculated as the slope of the natural log of the proportion of metal retained in the fish tissue relative to the initial Se burden as a function of depuration time using the following equation:

assayed individually for radioactivity to determine distribution of newly acquired dietary Se. We further report the percent mass of each tissue out of the fish whole body mass (wet wt basis) for comparison to the Se burden of those tissues. Se efflux rate constants (ke) of both male and female medaka were determined by feeding a separate cohort of medaka radiolabeled mayfly larvae and following the loss of assimilated radiotracer. For this experiment only mayfly larvae from the high treatment level (8.4 ± 0.8 μg g−1 (wet wt)) were utilized as prey items. Six female fish and four male fish were each fed a single radiolabeled mayfly larva. Fish were individually assayed for radioactivity following defecation of the undigested mayfly carcass, returned to clean water, and fed a control diet of Artemia nauplii twice daily. Fish were serially assayed daily for 7 days to follow the loss of 75Se with daily water refreshment to prevent unintended absorption of excreted Se. Medaka Se Maternal Transfer. Medaka Se maternal transfer was measured by feeding female fish radiolabeled mayfly larvae and following the movement of tracer into eggs. Medaka spawn asynchronously, typically producing a daily clutch of eggs for several days. Periphyton was loaded with Se at a single treatment level using 489 μL of H275SeO3 (specific activity, 2.22 μCi mL−1; [Se], 2.7 μg Se L−1) and 27 μL of 200 μg mL−1 Na2SeO3 (final dissolved [Se], 3 μg L−1). Following loading, periphyton [Se] was 4.7 ± 1.7 μg g−1 (dry wt). Newly hatched C. triangulifer larvae were exposed to treatment diets for ∼25 d then removed and rinsed for fish feeding. The average mayfly whole body [Se] was 4.1 ± 1.0 μg g−1 (wet wt). Twelve female fish were individually housed and fed radiolabeled mayflies ad libidum for 4 days. In general, three mayflies were continuously available for consumption throughout the day. Fish consumed 18−30 larvae (mean larval mass, 1.5 ± 0.6 mg wet wt) over the 4-day period with an average of 6.2 larvae consumed per fish per day. Fish were given ∼20 h to purge their guts of radiotracer by feeding Artemia nauplii, and then two male fish were introduced to each female tank to stimulate egg production and fertilize spawned eggs. During egg laying fish were fed a control diet of Artemia nauplii twice daily. Each morning tanks were checked, and if a given female produced a clutch of eggs the female was removed, eggs were collected, and both were assayed for radioactivity. Only complete egg clutches that were adherent to the females were collected and assayed. Following radioactivity determination females were returned to their tanks for a total of 6 days of possible egg laying. After collecting eggs on day six, female fish were euthanized in MS222 (0.015% w/v), weighed, and assayed for radioactivity. Ovaries were dissected (as above), weighed, and assayed for radioactivity. Fish [Se] and egg [Se] were calculated based on the mass of newly acquired Se from days 0, 1, 2, and 3 only because egg radioactivity counts decreased below the level of acceptable counting error (i.e., >5%) by day 4 of the experiment. In order to estimate egg dry mass we subsequently collected eggs from stock medaka females (n = 10) and determined the relationship between clutch size and total egg dry mass:

Ct = C i e−ket

where Ct is the mass of Se in the fish at time t (μg), Ci is the initial mass of Se (μg), and t is the time (d). The efflux rate constant (ke) only considers physiological excretion, as defined by Luoma and Rainbow,21 and does not include egg deposition. To calculate ke in reproductively active females we added the Se mass (μg) in daily egg clutches back to the Se mass in the female (μg) and found the slope of the proportion of Se retained using the above equation. We further calculated the total Se loss rate constant (ke+egg) in reproductively active females, which combined the Se loss due to physiological excretion and egg deposition. To calculate the total loss rate constant (ke+egg) we simply took the slope of the proportion of Se retained using only the Se mass in the female (μg) each day assayed after oviposition (identical equation to ke above). Fish whole body [Se] and ovary [Se] (both wet wt basis) were compared using simple linear regression for both reproductively active females (post egg spawning, from the maternal transfer experiment) and reproductively inactive females (from the AE and tissue distribution experiment). Comparing slopes of these regressions allowed us to determine if the ovary becomes depleted in newly acquired Se relative to the whole body due to the process of Se deposition into eggs. Fish whole body [Se] and egg [Se] (both dry wt basis) were compared based on egg:whole body [Se] ratios for each day (0, 1, 2, and 3 of egg production). Egg:whole body [Se] was calculated for each individual fish by dividing the egg [Se] by the fish whole body [Se] and calculating the mean ratio across replicate fish.

Y = 0.2719x − 0.0129

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where x is the number of eggs in a clutch and Y is the total estimated dry mass (mg) of those eggs (r2 = 0.997, p < 0.0001). Similarly, fish whole body dry mass was estimated by determining the whole body moisture content of stock females (72.5 ± 1.3%, n = 6). We assayed for radioactivity daily; however, we only measured the wet weight of the females at the

RESULTS

Background Se Concentrations. Selenium concentrations in medaka males and females, Artemia nauplii, and medaka eggs were measured in order to determine background levels and provide a reference for the radiotracer experiments.

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Figure 1. Medaka Se assimilation efficiency (AE) as a function of dietary (C. triangulifer larva) [Se] (a) and C. triangulifer larval size (b). Each data point represents a single female medaka fed a single radiolabeled mayfly larva. Assimilation efficiency did not change as a function of dietary [Se]; however, there was a significant decrease in AE as larval size increased.

Female [Se] was nearly identical between those that were actively producing eggs (1.85 ± 0.22 μg g−1 (dry wt)) and those that were not (1.85 ± 0.20 μg g−1 (dry wt)). Male [Se] (1.66 ± 0.06 μg g−1 (dry wt)) was marginally lower than female [Se] on average; however, neither this difference (t test, p = 0.210) nor moisture content (overall mean, 72.5 ± 1.3%; t test, p = 0.553) were significantly different. Medaka cultured on Artemia nauplii with Se concentrations of 1.74 ± 0.15 μg g−1 (dry wt) had trophic transfer factors (TTF) that ranged from 0.84 to 1.24. Finally, the medaka egg [Se] (1.84 ± 0.11 μg g−1 (dry wt); moisture, 79.5 ± 1.6%) was remarkably consistent with the female whole body [Se] (and the average Artemia [Se]) producing an egg:whole body [Se] of 1.0-fold. Medaka Se Assimilation Efficiency. Medaka assimilated 63.2 ± 8.8% (range 45.6−76.7%) of the Se in their mayfly diets, on average, with no significant difference between male and female AE (t test, p = 0.307). There was also no significant effect of mayfly [Se] on medaka AE (linear regression, p = 0.157; Figure 1a) across the dietary range used here (1.4−9.7 μg g−1 (wet wt basis) or 5.6−38.7 μg g−1 (dry wt basis)). The only factor that significantly affected medaka Se AE was the size of the larva ingested, where larger larvae yielded lower AEs (y = −3.5x + 74, where x = mayfly mass (mg wet wt) and y = medaka AE; r2 = 0.38, p = 0.0064, Figure 1b). Medaka Tissue-Specific Se Distribution. Tissue-specific Se distribution was assessed in both female and male medaka after consuming radiolabeled mayfly larvae followed by a chase (purging of radiolabeled gut contents) of Artemia nauplii. Overall, the mass of Se in each tissue compartment increased with treatment level; however, the proportional distribution was not significantly different across treatments. Female and male medaka distributed nearly identical proportions of Se to the digestive tract (18 ± 4% and 17 ± 5% in females and males, respectively) and liver (15 ± 3% and 14 ± 3% in females and males, respectively) (Figure 2). These tissue compartments also comprised a similar proportion of the overall body mass (5 ± 1%, 4 ± 1% for digestive tract and 3 ± 1%, 3 ± 1% for liver in females and males, respectively). The major difference in tissue-specific Se distribution between female and male medaka was in reproductive tissue. Female medaka partitioned 24 ± 6% of their newly acquired Se to ovary tissue, while males partitioned only 0.8 ± 0.3% to their testes. This was largely explained by tissue mass where the ovary comprised 10 ± 3% of the body mass in females, whereas the testes comprised only 0.3 ± 0.1% of the body mass in males. The difference in reproductive tissue Se distribution

Figure 2. Tissue-specific Se distribution (large pie charts) and relative tissue mass recovered from (small pie charts, wet wt basis) female (upper pie charts) and male (lower pie charts) medaka. Values represent mean ± SD of the proportion of Se in each tissue compartment and the proportional weight of each tissue out of the whole fish Se and fish mass, respectively. Proportions do not add up to 100% due to some loss of body fluid during dissections.

between females and males resulted in a large difference in Se partitioning to the remaining carcass. Females partitioned 42 ± 7% of their Se to the remaining carcass, and males partitioned 64 ± 7%, which was also largely explained by the difference in proportional body mass of the carcass (75 ± 4% in females and 87 ± 1% in males). Medaka Se Efflux. Selenium efflux was determined in males and both reproductively active and reproductively inactive females. There was no significant difference in ke between males (0.069 ± 0.011 d−1) and reproductively inactive 2974

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females (0.066 ± 0.010 d−1) (p = 0.591). Females were further assessed comparatively between those that were reproductively active (producing eggs; new cohort of female fish) and those that were not (same females from comparison with males above). After correcting for loss of Se to the eggs, the ke in reproductively active females was 0.069 ± 0.013 d−1, which was not significantly different than the ke of reproductively inactive females (0.066 ± 0.010 d−1; p = 0.848). Reproductively active females displayed a total Se loss rate constant (ke+egg, i.e., excretion and egg deposition) of 0.17 ± 0.019 d−1, which was ∼2.5-fold greater than the ke alone. Medaka Se Maternal Transfer. Because of the importance of reproductive tissue in the assessment of fish populations impacted by Se contamination we examined the relationships between whole body [Se] and ovary [Se] as well as whole body [Se] and egg [Se]. Ovary [Se] increased as whole body [Se] increased in both reproductively inactive females and females that had been spawning eggs for 6 d (Figure 3). Reproductively

Figure 4. Time course of Se ingestion (total mass basis) from mayfly diet (left y-axis, gray bars) and subsequent change in egg [Se] (right yaxis, white bars). It appears that the pulse of Se in the diet is reflected in the pulse of Se in egg clutches, displaying the connection between dietary Se ingestion and Se deposition into eggs for medaka (asynchronous spawning strategy). Bars with different capital or lower case letters (separate ANOVAs for Se ingestion mass and egg [Se]) are significantly different. Bars represent mean ± SE.

the greatest deposition of newly acquired Se into eggs (1.0 ± 0.4 μg g−1 (dry wt), day 1), which corresponds to the period of vitellogenesis and oocyte maturation in medaka.22−24

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DISCUSSION Assimilation efficiency is a key determinant in Se bioaccumulation. Here, we observed medaka AEs that ranged from 45.6% to 76.7% with no trend across a wide range (5.6−38.7 μg g−1 (dry wt)) of dietary [Se]. The mean AE from our study (63.2 ± 8.8%) was within the range of fish Se AEs from the literature, which can be as low as 12.7 ± 2.7% (grunt (Terapon jarbua) fed barnacles (Balanus cirratus))25 and up to 76.6 ± 15.8% (Mediterranean sea bream (Sparus auratus) fed Artemia salina nauplii).26 However, the majority of fish Se AEs tend to be >50%.27 To our knowledge, no other studies have investigated the effect of prey size on Se AE, which seems relevant because fish feeding behavior is dominated by engulfing whole prey (i.e., no mastication).28 Here, medaka Se AE decreased at a rate of ∼3.5% (95% confidence interval, 5.7−1.1%) per gram increase in mayfly mass (wet wt). We speculate that the reduction of medaka AE as a result of increasing larval size was due to incomplete digestion of the intact (nonmasticated) larvae. It is evident from this study and the literature that fish Se AE is highly variable and dependent on a number of factors including fish species,29−32 prey species,33 prey [Se],34 prey size, ingestion rate,33 gut passage time,35 fish size,36 and fish age.35 There is a noticeable lack of consistency in the tissue compartments chosen for assessing fish Se distribution. Compartments range from a variety of major internal organs (e.g., mix of liver, kidneys, gills, digestive tract, ovaries/ testes)26,29,31,33,37−39 to generic body sections (e.g., head, tail).35 Ni et al.31 chose similar compartments to those investigated here in their study of the intertidal mudskipper (Periophthalmus cantonensis). Mudskippers partitioned Se roughly similar to medaka with ∼25% digestive tract, ∼7% liver, and ∼60−65% carcass. Tissue Se distributions are also highly dynamic, though. Mathews et al.26 displayed the variability of fish Se tissue distribution as a function of time where the proportion of Se decreased in the gill and digestive tract and increased in the liver over a 2-week period. In regard to Se water quality criteria, which is focused on protection of fish reproduction, the ovary is a primary organ of

Figure 3. Newly acquired dietary Se (i.e., radiotracer-associated) in medaka ovary [Se] as a function of whole body [Se] in reproductively inactive females (gray squares) and reproductively active females postegg laying (white circles). Each data point represents a replicate female with concentrations reported on a wet weight basis.

inactive females had ovaries that were ∼2.5-fold greater in [Se] (wet wt basis) than the whole body concentration, whereas the ovaries of females that had been laying eggs were only ∼1.5fold greater in [Se] (wet wt basis) than the whole body. Further, the ovary tissue comprised a similar proportion of the total body mass whether the female was reproductively inactive (10.3 ± 3.2%) or had been spawning eggs (10.1 ± 3.5%), yet the ovary contributed a smaller percentage (15.8 ± 4.6%) of the total Se body burden in post-spawning females than in reproductively inactive females (23.8 ± 5.8%; p = 0.0004). It appears that the ovary is a preferential site for Se distribution (along with gut and liver tissue) and that the ovary becomes depleted in Se during the process of egg production (relative to the whole body) presumably due to egg Se deposition. Egg [Se] was variable day-to-day, whereas fish whole body [Se] tended to decrease for days 0−3 of egg production resulting in a range of egg:whole body [Se]: 1.8 ± 0.3 (day 0), 2.9 ± 0.7 (day 1), 2.4 ± 0.8 (day 2), 2.2 ± 0.5 (day 3). Interestingly, the pulse of Se (total mass basis) from the mayfly diet was mimicked in the pattern of egg [Se] across days (Figure 4). The greatest consumption of dietary Se from mayflies (60.8 ± 14.4 ng Se, day −2) occurred ∼72 h prior to 2975

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[Se] increased to ∼2.8-fold. Bluegill are synchronous spawners, which may explain the greater range in egg:whole body [Se] in our study (1.0−2.9-fold) relative to the range observed by Coyle et al.37 (2.1−2.8-fold). Overall, it appears medaka may deposit Se directly from the diet to the eggs (via the ovary), producing egg [Se] that is determined by recent ingestion of dietary Se more than the maternal whole body [Se]. Spawning strategy is a potentially important process to consider when assessing risk to fish populations in Secontaminated systems. Depending on the home range of a given fish species, multiple stream reaches of a watershed may be traversed and dietary items consumed across a range of contamination. With asynchronous spawning fish even transient shifts in dietary [Se] may have a large impact on egg [Se] and overall reproductive success. Further, fish species that utilize synchronous (or group synchronous) spawning strategies may also be prone to shifting egg:whole body [Se] ratios depending on changes in dietary [Se],37 albeit potentially less dramatically. These intraspecific shifts in egg:whole body or ovary:whole body [Se] increase variability and weaken our ability to accurately predict egg [Se] based on other tissues, potentially leading to either over- or underestimation. For example, if we use the egg:whole body [Se] from spawning day 0 (1.8-fold) to predict the average egg [Se] on the following day, the result is a significant underestimation of the actual average egg [Se] (predicted, 0.7 ± 0.1; actual, 1.2 ± 0.4 μg g−1 (dry wt); p = 0.04). Conversely, a fish with an elevated whole body [Se] transiently consuming low dietary [Se] during egg production may skew the prediction in the opposite direction (i.e., overestimation of egg [Se]). Mechanistically Se is known to largely be deposited in fish eggs during the process of vitellogenesis (yolk protein development).1,7,41 Medaka are estimated to complete vitellogenesis and oocyte maturation within 72 h of oviposition.22−24 Here, medaka exhibited a bell-shaped pattern of Se acquisition from the diet with a relatively similar bell-shaped pattern of subsequent egg [Se]. It appears that a substantial portion of Se incorporated into the eggs during vitellogenesis was derived from recent dietary consumption based on this pattern and the rapid appearance of Se radiotracer in eggs. Therefore, the timing of oogenesis in relation to dietary Se consumption may dictate the deposition of Se into eggs. It is unclear whether this observation is unique to medaka (although a similar pattern has been reported in mallards42), typical of asynchronous spawners, or typical of Se trafficking in fish overall. Our findings suggest that, within medaka, egg:whole body [Se] is not fixed but instead linked to recent dietary Se consumption. Thus, the application of a universal ratio for estimating egg [Se] based on other tissues (whole body, muscle, or ovary [Se]) should appropriately incorporate the inherent variability both within and among fish species. For example, here we found egg:whole body [Se] ranged from 1.0to 3.8-fold (absolute range; range of means, 1.0−2.9-fold) in medaka, whereas other researchers have found egg:muscle [Se] ranging from 1.0- to 10-fold across 8 different species.1,40 If Se water quality criteria are to be based on fish tissue, it may be prudent to characterize the distribution of egg:whole body [Se] both within and among a wide range of species in order to capture that variability. More fully understanding the range of uncertainty that is inherent to this biological process could then allow for the application of appropriate safety factors and/or the selection of an appropriately conservative ratio based on a well characterized distribution.

interest for assessing Se distribution. We observed a change in Se ovary distribution in response to oviposition. Ovary contribution to the total Se body burden was significantly greater in females before laying eggs (23.8 ± 5.8%) than postegg laying (15.8 ± 4.6%). Alquezar et al.29 determined the female toadfish (Tetractenos glaber) ovary contained 8.7−11% of the total Se burden, which was lower than we found in both non-ovipositing and post-ovipositing medaka. The majority of Se radiotracer studies we found did not examine ovary distribution, however. Similar to Se AE, it is clear that tissue Se distribution is highly dynamic with variability reported in regard to dissolved versus dietary Se exposure,29 fish species,40 fish sex, and timing of tissue sampling.26 Our results indicate that the ovary may become depleted in Se during the spawning process relative to the whole body as a result of Se deposition into eggs producing variable egg:ovary [Se] ratios that depend on the timing of the female reproductive cycle as opposed to a constant ratio for a given species. Selenium efflux was more consistent than AE, tissue distribution, and egg deposition. Here, we found that both male and female medaka had similar ke’s and that females had similar ke’s regardless of whether they were also actively depositing Se into eggs. In comparison, fish Se ke’s in the literature range from as low as 0.019 d−1 (intertidal mudskipper (Periophthalmus cantonensis))31 to as high as 0.13 d−1 (juvenile Mediterranean sea bream (Sparus auratus)).26 These rate constants can be affected by fish age35 (reduced in older fish), dietary versus dissolved Se exposure31 (reduced following dietary Se exposure), and fish species.26 Overall though, ke’s for Se from the literature tend to fall in the range of 0.03−0.05 d−1.29−31,33,36 In contrast, the total Se loss rate constant (ke+egg, 0.17 d−1) we calculated for reproductively active females was ∼2.5-fold greater than the ke alone. To our knowledge, no other published studies have identified the discrepancy in ke versus the total loss rate constant due to excretion and egg deposition (ke+egg). On the basis of our results, egg deposition appears to play a major role in the trafficking and loss of newly acquired dietary Se in medaka. One potential modifying factor in fish egg Se deposition is the variety of spawning strategies.1 Generically, fish tend to fall into one of three spawning strategy categories: synchronous, group synchronous, and asynchronous.23 Synchronous spawners ovulate all oocytes simultaneously, whereas group synchronous spawners have at least two populations of maturing oocytes to be spawned discretely. Asynchronous spawners have oocytes present in all stages of development during the reproductive season. Medaka are asynchronous and spawn daily clutches of eggs (typically 20−40 eggs) for up to 3 months during the reproductive season.23,24 We were able to exploit this reproductive strategy for a short time to observe the movement of radiolabeled Se from the diet to the eggs. In cultured medaka, background egg [Se] was nearly identical to the whole body [Se] and the Artemia diet [Se] (i.e., egg:whole body [Se] of 1.0-fold). However, when we transiently increased dietary [Se] to ∼16 μg g−1 (dry wt) the egg [Se] increased (up to 2.9-fold, egg:whole body [Se]) in relation to the mass of Se consumed. A similar pattern was described by Lacoue-Labarthe et al.41 in the cuttlefish (Sepia off icinalis), which is also an asynchronous spawning fish. In comparison, Coyle et al.37 reported that bluegill fed a background Se diet (0.8 μg g−1 (dry wt)) produced eggs that were ∼2.1-fold more concentrated than the whole body, but when the diet was increased to 16.8 μg g−1 the egg:whole body 2976

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(7) Fan, T. W.-M.; Teh, S. J.; Hinton, D. E.; Higashi, R. M. Selenium biotransformations into proteinaceous forms by foodweb organisms of selenium-laden drainage waters in California. Aquat. Toxicol. 2002, 57, 65−84. (8) Stewart, R.; Grosell, M.; Buchwalter, D.; Fisher, N.; Luoma, S.; Mathews, T.; Orr, P.; Wang, W.-X. Bioaccumulation and Trophic Transfer of Selenium. In Ecological Assessment of Selenium in the Aquatic Environment; Chapman, P. M., Adams, W. J., Brooks, M. L., Delos, C. G., Luoma, S. N., Maher, W. A., Ohlendorf, H. M., Presser, T. S., Shaw, D. P., Eds.; CRC Press: Boca Raton, FL, 2010; pp 93−139. (9) Draft Aquatic Life Water Quality Criteria for Selenium-2004; United States Environmental Protection Agency: Washington, DC, 2004. Available online: http://water.epa.gov/scitech/swguidance/ standards/criteria/aqlife/selenium/ upload/complete-2.pdf. (10) Payne, R. G. Update to Kentucky Water Quality Standards for Protection of Aquatic Life: Acute Selenium Criterion and TissueBased Selenium Chronic Criteria. Available online: http://water.ky. gov/Documents/Regulations/ Proposed%20Se%20Criteria%204%202%202013.pdf. Frankfort, KY, 2013. (11) Conley, J. M.; Funk, D. H.; Buchwalter, D. B. Selenium bioaccumulation and maternal transfer in the mayfly Centroptilum triangulifer in a life-cycle, periphyton-biofilm trophic assay. Environ. Sci. Technol. 2009, 43, 7952−7957. (12) Conley, J. M.; Funk, D. H.; Hesterberg, D. H.; Hsu, L.-C.; Kan, J.; Liu, Y.-T.; Buchwalter, D. B. Bioconcentration and biotransformation of selenite versus selenate exposed periphyton and subsequent toxicity to the mayfly Centroptilum triangulifer. Environ. Sci. Technol. 2013, 47, 7965−7973. (13) Lindberg, T. T.; Bernhardt, E. S.; Bier, R.; Helton, A. M.; Merola, R. B.; Vengosh, A.; Di Giulio, R. T. Cumulative impacts of mountaintop mining on an Appalachian watershed. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 20929−20934. (14) Pond, G. J.; Passmore, M. E.; Borsuk, F. A.; Reynolds, L.; Rose, C. J. Downstream effects of mountaintop coal mining: Comparing biological conditions using family- and genus-level macroinvertebrate bioassessment tools. J. North Am. Benthol. Soc. 2008, 27, 717−737. (15) Muscatello, J. R.; Belknap, A. M.; Janz, D. M. Accumulation of selenium in aquatic systems downstream of a uranium mining operation in northern Saskatchewan, Canada. Environ. Pollut. 2008, 156, 387−393. (16) Conley, J. M.; Funk, D. H.; Cariello, N. J.; Buchwalter, D. B. Food rationing affects dietary selenium bioaccumulation and life cycle performance in the mayfly Centroptilum triangulifer. Ecotoxicology 2011, 20, 1840−1851. (17) Xie, L.; Funk, D. H.; Buchwalter, D. B. Trophic transfer of Cd from natural periphyton to the grazing mayfly Centroptilum triangulifer in a life cycle test. Environ. Pollut. 2010, 158, 272−277. (18) Kim, K. S.; Funk, D. H.; Buchwalter, D. B. Dietary (periphyton) and aqueous Zn bioaccumulation dynamics in the mayfly Centroptilum triangulifer. Ecotoxicology 2012, 21, 2288−2296. (19) Dong, W.; Hinton, D. E.; Kullman, S. W. TCDD disrupts hypural skeletogenesis during medaka embryonic development. Toxicol. Sci. 2012, 125, 91−104. (20) Dilution Water. In Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms; United States Environmental Protection Agency: Washington, DC, 2002; pp 40−45. (21) Luoma, S. N.; Rainbow, P. S. Why Is metal bioaccumulation so variable? Biodynamics as a unifying concept. Environ. Sci. Technol. 2005, 39, 1921−1931. (22) Shibata, N.; Nakamoto, M.; Shibata, Y.; Nagahama, Y. Endocrine Regulation of Oogenesis in the Medaka, Oryzias Latipes. In Medaka: A Model for Organogenesis, Human Disease, and Evolution; Naruse, K., Tanaka, M., Takeda, H., Eds.; Springer: Tokyo, 2011; pp 269−285. (23) Kinoshita, M.; Murata, K.; Naruse, K.; Tanaka, M. Reproduction of Medaka. In Medaka: Biology, Management, and Experimental Protocols; Wiley-Blackwell: Ames, IA, 2009; pp 67−99.

Alternatively, our results suggest that quantifying the [Se] of common prey items within a given aquatic system may be a reasonable approach to determining the potential for toxic effects of Se on fish. However, it remains uncertain to what degree the deposition of dietary Se in eggs (via the ovary) is a function of medaka physiology or is attributable to spawning strategy. Overall, the aquatic biogeochemistry, food web incorporation, and physiological dynamics of Se in fish are extremely complex2,7 and variable, making it difficult to adopt generalizations about the movement of Se through aquatic systems and potential for toxic effects in biota. Despite the intensity of debate over the appropriate way to regulate Se and controversy regarding an adequate criterion level for protection, there is still a significant shortfall in our understanding of Se dynamics in aquatic organisms and the degree to which these processes vary.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 919-513-1129. Fax: 919-515-7169. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Tom Augspurger (USFWS), Allison Camp (NCSU), Seth Kullman (NCSU), Gerald LeBlanc (NCSU), Monica Poteat (NCSU), Joseph Skorupa (USFWS), and three anonymous reviewers for revising earlier drafts of the manuscript; Seth Kullman for supplying medaka; Dave Funk (SWRC) for providing periphyton plates and initial mayfly culture material; Kim Hutchison (NCSU) for analytical determinations; and Dave Hinton (Duke) for assistance in the preliminary pilot experiments. Funding for J.M.C. was provided by a USEPA STAR Fellowship (GAD no. FP917322) and the SETAC/ICA Chris Lee Award for Metals Research. The USEPA has not formally reviewed this publication, and the views expressed herein may not reflect the views of the USEPA.

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REFERENCES

(1) Janz, D. M.; DeForest, D. K.; Brooks, M. L.; Chapman, P. M.; Gilron, G.; Hoff, D.; Hopkins, W. A.; McIntyre, D. O.; Mebane, C. A.; Palace, V. P.; Skorupa, J. P.; Wayland, M. Selenium Toxicity to Aquatic Organisms. In Ecological Assessment of Selenium in the Aquatic Environment; Chapman, P. M., Adams, W. J., Brooks, M. L., Delos, C. G., Luoma, S. N., Maher, W. A., Ohlendorf, H. M., Presser, T. S., Shaw, D. P., Eds.; CRC Press: Boca Raton, FL, 2010; pp 141−231. (2) Luoma, S. N.; Presser, T. S. Emerging opportunities in management of selenium contamination. Environ. Sci. Technol. 2009, 43, 8483−8487. (3) Simmons, D. B. D.; Wallschläger, D. A Critical review of the biogeochemistry and ecotoxicology of selenium in lotic and lentic environments. Environ. Toxicol. Chem. 2005, 24, 1331−1343. (4) Hamilton, S. J. Review of selenium toxicity in the aquatic food chain. Sci. Total Environ. 2004, 326, 1−31. (5) Young, T. F.; Finley, K.; Adams, W. J.; Besser, J.; Hopkins, W. A.; Jolley, D.; McNaughton, E.; Presser, T. S.; Shaw, D. P.; Unrine, J. M. Selected Case Studies of Ecosystem Contamination by Se. In Ecological Assessment of Selenium in the Aquatic Environment; Chapman, P. M., Adams, W. J., Brooks, M. L., Delos, C. G., Luoma, S. N., Maher, W. A., Ohlendorf, H. M., Presser, T. S., Shaw, D. P., Eds.; CRC Press: Boca Raton, FL, 2010; pp 257−292. (6) Lemly, A. D.; Smith, G. J. Aquatic Cycling of Selenium: Implications for Fish and Wildlife; U. S. Department of the Interior Fish and Wildlife Service: Washington, DC, 1987; Fish and Wildlife Leaflet 12 2977

dx.doi.org/10.1021/es404933t | Environ. Sci. Technol. 2014, 48, 2971−2978

Environmental Science & Technology

Article

(24) Iwamatsu, T.; Ohta, T.; Oshima, E.; Sakai, N. Oogenesis in the medaka Oryzias Latipes - Stages of oocyte development. Zool. Sci. 1988, 5, 353−373. (25) Zhang, L.; Wang, W.-X. Significance of subcellular metal distribution in prey in influencing the trophic transfer of metals in a marine fish. Limnol. Oceanogr. 2006, 51, 2008−2017. (26) Mathews, T.; Fisher, N. S. Trophic transfer of seven trace metals in a four-step marine food chain. Mar. Ecol.: Prog. Ser. 2008, 367, 23− 33. (27) Luoma, S. N.; Rainbow, P. S. Trace metal bioaccumulation. In Metal Contamination in Aquatic Environments: Science and Lateral Management; Cambridge University Press: Cambridge, 2008; pp 126− 168. (28) Hunter, J. R. The Feeding Behaviour and Ecology of Marine Fish Larvae. In Fish Behaviour and Its Use in the Capture and Culture of Fishes; Bardach, J. E., Magnuson, J. J., May, R. C., Reinhart, J. M., Eds.; ICLARM: Manila, Phillipines, 1980; pp 287−334. (29) Alquezar, R.; Markich, S. J.; Twining, J. R. Comparative accumulation of 109Cd and 75Se from water and food by an estuarine fish (Tetractenos Glaber). J. Environ. Radioact. 2008, 99, 167−80. (30) Creighton, N.; Twining, J. Bioaccumulation from food and water of cadmium, selenium and zinc in an estuarine fish, Ambassis jacksoniensis. Mar. Pollut. Bull. 2010, 60, 1815−1821. (31) Ni, I.-H.; Chan, S. M.; Wang, W.-X. Influences of salinity on the biokinetics of Cd, Se, and Zn in the intertidal mudskipper Periophthalmus cantonensis. Chemosphere 2005, 61, 1607−1617. (32) Reinfelder, J. R.; Fisher, N. S. Retention of elements by juvenile fish (Menidia menidia, Menidia beryllina) from zooplankton prey. Limnol. Oceanogr. 1994, 39, 1783−1789. (33) Xu, Y.; Wang, W.-X. Exposure and potential food chain transfer factor of Cd, Se and Zn in marine fish Lutjanus argentimaculatus. Mar. Ecol.: Prog. Ser. 2002, 238, 173−186. (34) Guan, R.; Wang, W.-X. Dietary assimilation and elimination of Cd, Se, and Zn by Daphnia magna at different metal concentrations. Environ. Toxicol. Chem. 2004, 23, 2689−2698. (35) Baines, S. B.; Fisher, N. S.; Stewart, R. Assimilation and retention of selenium and other trace elements from crustacean food by juvenile striped bass (Morone saxatilis). Limnol. Oceanogr. 2002, 47, 646−655. (36) Zhang, L.; Wang, W.-X. Size-dependence of the potential for metal biomagnification in early life stages of marine fish. Environ. Toxicol. Chem. 2007, 26, 787−794. (37) Coyle, J. J.; Buckler, D. R.; Ingersoll, C. G.; Fairchild, J. F.; May, T. W. Effect of dietary selenium on the reproductive success of bluegills (Lepomis macrochirus). Environ. Toxicol. Chem. 1993, 12, 551−565. (38) Kleinow, K. M.; Brooks, A. S. Selenium compounds in the fathead minnow (Pimephales promelas)–I. Uptake, distribution, and elimination of orally administered selenate, selenite and l-selenomethionine. Comp. Biochem. Physiol., Part C: Pharmacol., Toxicol. Endocrinol. 1986, 83, 61−69. (39) Mauk, R. J.; Brown, M. L. Selenium and mercury concentrations in brood-stock walleye collected from three sites on Lake Oahe. Arch. Environ. Contam. Toxicol. 2001, 40, 257−263. (40) Tissue Selection Criteria. In Selenium Tissue Thresholds: Tissue Selection Criteria, Threshold Development Endpoints, and Potential to Predict Population or Community Effects in the Field. Submitted to North American Metals Council − Selenium Working Group, Washington, DC,2008; pp 1−30. (41) Lacoue-Labarthe, T.; Warnau, M.; Oberhänsli, F.; Teyssié, J.-L.; Jeffree, R.; Bustamante, P. First experiments on the maternal transfer of metals in the cuttlefish Sepia officinalis. Mar. Pollut. Bull. 2008, 57, 826−831. (42) Heinz, G. H. Selenium accumulation and loss in mallard eggs. Environ. Toxicol. Chem. 1993, 12, 775−778.

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