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Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms
JGLR-01247; No. of pages: 7; 4C: Journal of Great Lakes Research xxx (2017) xxx–xxx
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Journal of Great Lakes Research journal homepage: www.elsevier.com/locate/jglr
Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms David M. Wituszynski a,⁎, Chenlin Hu b,1, Feng Zhang b, Justin D. Chaffin c, Jiyoung Lee b,d, Stuart A. Ludsin e, Jay F. Martin a,f a
Department of Food, Agricultural, and Biological Engineering, The Ohio State University, Columbus, OH 43210, USA College of Public Health, Division of Environmental Health Sciences, The Ohio State University, USA F.T. Stone Laboratory, The Ohio State University, USA d Department of Food Science and Technology, The Ohio State University, USA e Aquatic Ecology Laboratory, Department of Ecology, Evolution, and Organismal Biology, The Ohio State University, USA f Ohio Sea Grant, USA b c
a r t i c l e
i n f o
Article history: Received 22 March 2017 Accepted 31 July 2017 Available online xxxx Keywords: Public health Harmful algae HABs Eutrophication Microcystis
a b s t r a c t Microcystin (MC) is a cyanobacteria-produced liver toxin that has been found in fish from Lake Erie, sometimes in excess of World Health Organization (WHO) guidelines for safe consumption. Even so, few studies have quantified MCs in Lake Erie fishes, and these studies have drawn different conclusions concerning the risk that fish consumption poses to public health. To address this gap in knowledge, we used Enzyme-Linked Immunosorbant Assay (ELISA) to evaluate the MC concentration in muscle tissue from three commonly harvested fish in Lake Erie: walleye (Sander vitreus, n = 29); yellow perch (Perca flavescens, n = 52); and white perch (Morone americana, n = 55), collected during summer 2013. Satellite remote sensing was used to compare MC concentrations in fish tissue to bloom conditions in Lake Erie at the time of harvest. We found a significant difference among mean MC concentrations in walleye (71 ng MC/g wet weight), white perch (37 ng MC/g), and yellow perch (8.1 ng MC/g). In addition, MC levels in white perch appeared to depend on local bloom conditions. While few of the fish collected contained MC in excess of WHO guidelines, our results indicate that more toxic blooms could increase MC in fish to levels that pose a greater risk to public health. © 2017 Published by Elsevier B.V. on behalf of International Association for Great Lakes Research.
Introduction Cyanobacterial blooms and their associated health threats are a rapidly growing concern worldwide and are projected to expand due to changes in climate and increases in anthropogenic nutrient loading (Bullerjahn et al., 2016; Kosten et al., 2012; Michalak et al., 2013). These blooms can have substantial ecological and environmental impacts due to shading, hypoxia, and alterations of aquatic food webs (Paerl and Otten, 2013). Additionally, cyanobacteria are known to produce several varieties of toxin, and therefore represent a potential human health threat (Ibelings and Chorus, 2007). The most widespread and best-studied cyanotoxin is microcystin (MC), a potent liver toxin and a suspected tumor promoter (Chorus and Bartram, 1999; Ibelings and Chorus, 2007). This toxin has been found worldwide, and has been implicated in human and animal sickness and death (Chorus and Bartram, 1999). Microcystin exists in the form of more than eighty congeners, differentiated by amino acid substitutions. The most toxic of ⁎ Corresponding author. E-mail address: [email protected] (D.M. Wituszynski). 1 Present address: College of Pharmacy, University of Houston.
these is MC-LR, and this has been the most prevalent congener found in Lake Erie water samples (Brittain et al., 2000; Dyble et al., 2008). The World Health Organization (WHO) has established provisional guidelines for MC content in food, drinking water, and water used for recreational purposes (Chorus and Bartram, 1999). The latter two routes of exposure are regularly controlled in the United States, via beach closures and advanced water treatment. However, few studies have explored the presence of MC in commercially and recreationally harvested aquatic organisms, and currently no national regulation has addressed this concern. Further, the few studies that have been conducted have found widely varying levels of MC in edible muscle tissues (0.5–1960 ng MC/g dry mass) (Kopp et al., 2013; Niedzwiadek et al., 2012; Poste et al., 2011; Schmidt et al., 2013; Wood et al., 2014), suggesting that MC accumulation is strongly species- and ecosystemdependent. The cause of this variability in MC concentrations in fish tissues, however, remains enigmatic for two primary reasons. First, quantifying MCs in animal tissues is a time-consuming process, and the results are often difficult to interpret (Schmidt et al., 2013; Xie et al., 2004). Second, harmful cyanobacterial blooms are highly variable in space and time which could lead to high variability in MC burdens on aquatic organisms
http://dx.doi.org/10.1016/j.jglr.2017.08.006 0380-1330/© 2017 Published by Elsevier B.V. on behalf of International Association for Great Lakes Research.
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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(Hunter et al., 2008; Shi et al., 2015; Stumpf et al., 2012; Wynne and Stumpf, 2015). Therefore, the need exists to establish links between cyanobacterial blooms, which are readily observable, and levels of MC in edible tissues. This knowledge will allow the determination and prediction of health risk via MC exposure through consumption of aquatic organisms. Our study addresses this gap in knowledge by quantifying MC concentrations in walleye (Sander vitreus), yellow perch (Perca flavescens), and white perch (Morone americana), all commercially and/or recreationally important fishes, collected in the western basin of Lake Erie during summer 2013. We also used remotely sensed data to identify linkages between MC levels in edible fish tissues and local bloom intensity for yellow perch and white perch. In so doing, we sought to identify factors that could affect the risk of MC exposure to humans via fish consumption. Methods Study system Lake Erie is the southernmost, shallowest, and most productive of the Laurentian Great Lakes (Ludsin et al., 2001; Michalak et al., 2013). It is important economically and culturally throughout the region, providing numerous ecosystem services such as opportunities for recreation by swimming, boating, and fishing, as well as support for associated tourism activities (Hobbs et al., 2002). The sport fishing industry alone accounts for as much as $1.2 billion per year in economic activity (Lake Erie Improvement Organization, 2012). Additionally, the lake is the source of drinking water for 11 million people. During recent years, however, Lake Erie has been experiencing regular harmful algal blooms (HABs) that have threatened these services (Brittain et al., 2000; Carmichael and Boyer, 2016). The blooms have been dominated by cyanobacteria of the genus Microcystis that can produce MCs (Harke et al., 2016; Michalak et al., 2013), and recent reports indicate that Lake Erie waters experiencing these blooms regularly exceed the 20 μg/L WHO guideline for recreational contact (http://www.glerl. noaa.gov/res/HABs_and_Hypoxia/WLEMicrocystin.html, accessed August 4, 2016). Given that these reports have shown MC concentrations in Lake Erie water regularly reaching 10–20 μg/L, and occasionally as high as 1000 μg/L (Steffen et al., 2014), MC exposure via fish consumption from this lake should be a concern (Poste et al., 2011). Walleye, yellow perch, and white perch were selected for this study, as they are three of the most commonly harvested sport and commercial fish in Lake Erie. While the MC content of all three species has been reported (Dyble et al., 2011; Poste et al., 2011; Wilson et al., 2008), our study is the first to examine large sample sizes of all three species taken from the same year at locations with known bloom intensities. Sample collection and MC extraction and quantification All fish analyzed for this study were captured in western Lake Erie during 2013. White perch (n = 55) and yellow perch (n = 52) were collected as part of an annual bottom trawl assessment program conducted by the Ohio Department of Natural Resources-Division of Wildlife (ODNR-DOW) (ODW, 2014). Individuals used in this study were collected from 13 trawling locations in western Lake Erie during August and September (Fig. 1); these trawls framed the period during which the bloom peaked and then began to senesce (as reported in the Lake Erie Experimental HAB Bulletin: http://www.glerl.noaa.gov/res/HABs_and_ Hypoxia/lakeErieHABArchive/, accessed August 4, 2016). Walleye (n = 43) were collected from various locations within the western basin via charter boats that were participating in water quality monitoring, which was supervised by The Ohio State University's Stone Laboratory. The charter boat captains provided the belly flap (n = 29) or muscle fillets (n = 14) of individual fish caught by anglers. Some charter boat
captains also provided the total length (TL, nearest 1 mm) of the individual fish (n = 22). Fish ages were determined by the ODNR-DOW Sandusky Fish Research Station, using established relationships between age and fish TL for each species (C. Vandergoot, ODNR-DOW, pers. comm.) (Table 1). Details on fish collection and sizes are given in Electronic Supplementary Material (ESM) Table S1 and locations and numbers of white perch and yellow perch collections are in ESM Tables S2 and S3·Fish tissue was analyzed for MCs using methanol extraction followed by Enzyme-Linked Immunosorbant Assay (ELISA), in a manner derived from that of Hu et al. (2008) and Moreno et al. (2005). Fish tissue was dried at 60 °C for 24 h (Poste et al., 2011), homogenized using a mortar and pestle, and then extracted with 75% methanol for 2 h at room temperature while being stirred. Extracts were centrifuged at ~4750 rpm for 15 min, after which supernatant was removed and the solids resuspended in 75% methanol for a second extraction. This process was repeated for a total of three extractions. The supernatant from all extractions was pooled and diluted to one-quarter strength with deionized water and passed through a SepPak® C18 column (Waters corporation, Milford, MA). Microcystin was eluted from the column with 5 mL of 100% methanol. The sample was then diluted to b5% methanol and analyzed using the Microcystins/Nodularins (ADDA) ELISA kit (catalog number PN520011, Abraxis Inc., Warminster, PA) with MC-LR (0.15–5 μg/L) as the working standard and a detection sensitivity of 0.1 μg/L. Data on MC concentrations in collected fish are given in ESM Table S1. Comparison of walleye belly flap and fillets MC concentrations are given in ESM Table S4, and extraction efficiencies are given in ESM Table S5. Lower limits of detection varied by sample, and ranged from 1.27 ng MC/g wet mass to 30.29 ng MC/g wet mass (mean = 9.64 ng MC/g wet mass) for white perch and yellow perch, respectively; for walleye the range was 7.48 ng MC/g wet mass to 86.36 ng MC/g wet mass, with a mean of 26.35 ng MC/g wet mass. Several walleye samples were below the desired wet mass of 0.5 g, which dramatically raised the lower limit of detection for these samples. Only six of the 42 walleye samples did not have detectable MC; of these, the highest lower limit of detection was 41.54 ng MC/g wet mass. As the ELISA was calibrated with MC-LR, results are reported as MCLR equivalents. Doing so assumes that the entire mass of MC discovered is MC-LR, the most toxic of the MCs (Deblois et al., 2011). Therefore, the limits for safe consumption expressed here are conservative. The ELISA test used exhibits good cross-reactivity across all tested MC congeners (Fischer et al., 2001). However, as not all congeners have been tested, it is possible that some species may be present to which the ELISA test is relatively insensitive. These would be under-reported; however, toxicity data is not available for most of these congeners. It should be noted that our method of analysis only extracts free MCs. Studies have found varying amounts of covalently bound MCs in fish tissue, and our results may therefore underestimate the amount of MC actually contained in our samples. Indications are that these bound MCs are not as toxic as free MCs (Ito et al., 2002; Metcalf et al., 2000). Determination of cyanobacterial blooms Bloom intensity at sampling locations for white perch and yellow perch was determined through analysis of remote sensing imagery. Images from the Moderate Resolution Imaging Spectroradiometer (MODIS) were processed to extract the “cyanobacterial index” (CI) developed by Wynne et al. (2013) for the locations and times at which white perch and yellow perch were captured. This index measures the abundance of chlorophyll-a in a cyanobacteria-specific fashion, and it has been shown to be related to field measurements of Microcystis spp. cell counts in western Lake Erie (Wynne et al., 2010). As chlorophyll-a in western Lake Erie has also been shown to be correlated with observed MC concentrations (Stumpf et al., 2016), this index is a good approximation of the potential toxicity of the bloom in any one location. The values of this index were therefore used as a proxy for
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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Fig. 1. Yellow perch and white perch were collected from 13 sampling locations in western Lake Erie during August and September 2013. The numbers next to each site label designate the number of yellow perch and white perch, respectively, caught at that location. Walleye were collected by charter boat captains during September and October 2013. The walleye fishing area is denoted by the white oval. Summaries of the number of white perch and yellow perch collected at each location and at each date are available in Electronic Supplementary Material (ESM Tables S2 and S3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Microcystis spp. bloom intensity. Images used herein were from within one day of the fish sampling dates. Data analysis Methods for all data on fish metrics, MC in fish, associated CI used are reported in the Electronic Supplementary Material (ESM) Table S1 and results of statistical tests of different statistical models are in ESM Table S6. Toxin concentrations in fish tissues were not normally distributed, even after transformation. Thus, we used nonparametric pairwise Wilcoxon tests with Bonferroni correction to test for differences in MC concentration among species. Finding that MC concentrations differed among species (see Results and discussion section), we used speciesspecific linear models to explain fish tissue MC concentration. In all cases, MC concentration was the effect. The following factors were used to predict MC levels in white Perch and yellow Perch: Age, TL (though not in conjunction with age), Bloom Intensity, and Capture Date. Linear models were constructed with ordinary least squares regression, and selection was performed according to corrected Akaike Information Criterion (AICc); models with ΔAICc b 2 were considered equal in explanatory power (Bolker, 2008). Significant models reported here presented normally distributed residuals with no outliers (studentized residuals all b 3). Non-detect values were replaced by a
random number drawn from a uniform distribution between 0 and the lower limit of detection for that sample. Model adequacy was verified by checking the size and distribution of residuals. All statistical analyses were performed in R 3.0.1. An overall alphalevel of 0.05 was used to denote significance. Models constructed for walleye, while significant, explained little of the variance in toxin concentration and so are not reported.
Public health impact Public health exposure was estimated by calculating maximum allowable levels of MC (μg MC/kg body mass) in fish tissues. These thresholds were calculated according to Dyble et al. (2011) using both the WHO lifetime Tolerable Daily Intake (TDI) value of 0.04 μg/kg (Chorus and Bartram, 1999) and the “seasonal TDI” of 0.4 μg/kg proposed by Ibelings and Chorus (2007). The WHO's lifetime TDI value is the amount of MC that an individual could, in theory, consume daily for the remainder of his or her life without suffering negative effects. The TDI was derived from a “No Observable Adverse Effects Level” of 40 μg/kg found in animal studies (Fawell et al., 1999). This value was divided by a safety factor of 100 to account for variability among individuals, as well as among species, and then again by a further factor of 10 to translate the findings of the short-term studies into a lifetime effect level. These
Table 1 Information on the fish analyzed for this study, including fish age and total length statistics, as well as tissue microcystin concentrations. The conditions of the cyanobacteria blooms experienced by fish at the time of capture also is reported. Cyanobacterial Index was transformed to Microcystis cells per mL according to Wynne et al. (2010) to calculate bloom exposure. All fish were collected in the western basin of Lake Erie during August and September 2013. Species
White Perch Yellow Perch Walleye a
Age (years)
Total length (cm)
Bloom Exposure* (Microcystis cells/mL)
MC content (ng MC/g wet mass)
Mean
Range
Mean
Range
Mean
Range
Mean
Range
1.8 3.31 6.86a
1–5 1–7 5–12a
15.24 16.38 22.86a
11.28–20.09 12.07–23.72 20–25a
2.49 ∗ 105 2.20 ∗ 105 NA
1.90 ∗ 105− 3.43 ∗ 105 1.65 ∗ 105− 3.06 ∗ 105 NA
37.5 11.80 84.13
b7.83–91.8 b7.51–73.3 b17.9–303
Ages and lengths were only available for 7 of the 29 Walleye samples.
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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calculations lead to a final TDI of 0.04 μg/kg, which is the accepted WHO guideline (Chorus and Bartram, 1999). Importantly, Ibelings and Chorus (2007) have argued that this TDI should not be applied to situations in which individuals are only at risk of exposure for a short period of time. Because cyanobacterial blooms in temperate regions usually present exposure risk for only a portion of the year (e.g., blooms are generally present during late summer through early fall in Lake Erie; Michalak et al., 2013), Ibelings and Chorus (2007) suggested the use of a “seasonal” TDI of 0.4 μg/kg. Thus, in addition to presenting the lifetime TDI, which is the most commonly used metric in the literature, we also present the seasonal TDI, as it seems more applicable to our study system. We calculate acceptable daily intake levels for different types of (human) fish consumers, assuming the average consumer weighed 70 kg. Using this approach, a person may acceptably take in 2800 ng (lifetime) or 28,000 ng (seasonal) MC per day. We used reference fish consumption rates for five different human populations within the Lake Erie area: the “average” U.S. consumer, the consumer that follows the Ohio Department of Health (ODH) Advisory Level, the typical Lake Erie angler, a typical Native American tribal member, and a “very high” fish consumer (De Rosa and Hicks, 2001; 2016 Ohio Sport Fish Health and Consumption Advisory: http://www.epa.state.oh.us/dsw/ fishadvisory/index.aspx, accessed September 9, 2016). Based on these consumption rates, we then calculated the average MC concentration in fish tissue at which an individual belonging to a given population will exceed the acceptable daily MC intake level (for both lifetime and seasonal TDIs). Finally, these threshold values were used to evaluate the risk to public health posed by the fish MC levels measured in this study. Results and discussion MC levels in fish Microcystin (MC; ng MC/g wet mass) levels differed among fish species (Kruskal-Wallis test: χ2 = 52.9, p b 0.05; Mann-Whitney-Wilcoxon test: all W N 217, all p b 0.05; Table 1, Fig. 2) with mean concentrations in walleye (84 ng/g) being greater than both white perch (38 ng/g) and yellow perch (12 ng/g). White perch MC levels were also significantly higher than yellow perch MC levels (Table 1, Fig. 2). Differences in foraging may partially explain these inter-specific differences in MC concentrations. Age 1 + yellow perch acquire a large
Fig. 2. Microcystin (MC) concentration for yellow perch (n = 53), white perch (n = 55), and walleye (n = 43) collected in western Lake Erie during the 2013 cyanobacteria bloom season. Sample means (ng MC/g wet mass) indicated by the horizontal line are as follows: mean = 11.8 (yellow perch); mean = 37.5 (white perch); and mean = 70.8 (walleye). ND = MC levels below detection limits. NDs are placed at the values used for the data analysis presented above. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
proportion of their diet from benthic sources (Guzzo et al., 2013; Tyson and Knight, 2001). By contrast, zooplankton are a larger component of the diet of age 1+ white perch (Gopalan et al., 1998; Guzzo et al., 2013). The differences in internal MC concentration between these two fish therefore agrees broadly with the literature on MC accumulation in fishes of different feeding guilds. For example, KozlowskySuzuki et al. (2012) conducted a meta-analysis of microcystin in aquatic organisms, finding that zooplanktivorous fish were more likely to concentrate MC than fish of other feeding guilds (e.g., carnivorous and omnivorous). Additionally, during a study in Lake Ijsselmeer (Netherlands), Ibelings et al. (2005) found higher MC concentrations in the livers of European smelt (Osmerus eperlanus), a planktivore, than in the livers of either European Perch (Perca fluviatilis), a carnivore, or Eurasian ruffe (Gymnocephalus cernua), a benthivore. Similarly, in a Chesapeake Bay (USA) tributary, Wood et al. (2014) found that organisms with largely benthic diets accumulated lower concentrations of MC than those organisms with largely pelagic (planktivorous) diets. The fact that adult walleye, as piscivores, had high MC concentrations also was somewhat expected (Xie et al., 2005; Kozlowsky-Suzuki et al., 2012), although Gkelis et al. (2006) did find higher concentrations in planktivorous, zooplanktivorous, and omnivorous fish than piscivorous fish in their study. Xie et al. (2005) found MC concentrations to be higher in piscivores than other feeding guilds, and postulated uptake from sources other than food. MCs may be entering walleye through gill uptake (Cazenave et al., 2005; Malbrouck and Kestemont, 2006). While the use of cyanaobacteria blooms by Lake Erie walleye has not been quantified, walleye may forage in them, owing to their optical properties (sensu Lester et al., 2004) and the potential local abundance of prey species, themselves attempting to reduce predation risk (Engström-Öst et al., 2006). Kozlowsky-Suzuki et al. (2012) suggested that the broader breadth of piscivore diets (e.g., ability to feed on large zooplankton and other invertebrates, in addition to fish), contributed to higher MC concentrations in tissues of these fish relative to other feeding guilds. It is not likely that omnivory is responsible for the high MC levels in Lake Erie walleye, however, as adults are strictly piscivorous, feeding to a large degree on juvenile and adult gizzard shad (Dorosoma cepedianum) (Knight et al., 1984; Knight and Vondracek, 1993). While we can only speculate, perhaps the high MC levels in Lake Erie walleye is due their high consumption of gizzard shad, which is known to be highly phytoplanktivorous during older life stages (Schaus et al., 2002; Yako et al., 1996). An alternative explanation for these inter-specific differences might lie in the depuration mechanisms for MC, which can differ considerably among fish species. For example, Adamovský et al. (2007) found a lower half-life of MC in muscle tissue of silver carp (Hypophthalmichthys molitrix) than in muscle tissue of common carp (Cyprinus carpio), though for both species the value was less than three days. Experiments on the depuration of MC in tilapia (Mohamed and Hussein, 2006), centrarchids (Smith and Haney, 2006), and yellow perch (Dyble et al., 2011) have also shown variable timescales for MC elimination. Because we did not directly measure depuration, and rates have not been measured in walleye or white perch, the role of the depuration process in explaining variation in our results remains uncertain. For this reason, future studies should address this issue by analyzing fish captured both before the bloom begins and after it ends. Microcystin concentration was unrelated to neither fish age nor fish size within all three species (all models tested have p N 0.05). This finding indicates that toxin did not accumulate preferentially in younger or older fish, nor was it likely to accumulate and remain in fish tissues across years. The intensity of the cyanobacterial bloom at the location in which fish were captured was positively related to MC concentration in White Perch (F-test: F = 14.1, p = 0.000431; Table S6), but not in yellow perch (F-rest: F = 0.647, p = 0.425; Table S6), as indicated by effect tests of the Cyanobacterial Index (CI) in linear models. The white perch MC concentration varied with collection date as well, with fish captured
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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in September being significantly more toxic than fish caught during August (F-test: F = 19.67, p b 0. 0.00005). Studies have shown that many fish have efficient MC depuration systems up to a point, but it is possible for them to be overrun by an excess of MC (Deblois et al., 2011; Dyble et al., 2011). Therefore, the increase in MC concentrations in white perch over time may be due to MC accumulation over the bloom season, rather than a response to immediate MC in the environment at the time sampled. The CI for sample sites was higher on average in September than in August, meaning that blooms were more intense during the later period (t-value: 12.05, p-value: b2.2E−16). These two potential explanations of MC concentration in white perch are therefore confounded in our samples. The fact that yellow perch MC levels were unrelated to the CI may indicate that they can rapidly depurate MCs (see Dyble et al., 2011). Risk of MC exposure through fish consumption MC concentrations were below the threshold levels of concern for most potential consumers in the Lake Erie basin, especially when using a seasonal total daily intake (TDI) estimate (Table 2). Whether the lifetime or the seasonal TDI is most appropriate likely varies by the consumer's fishing and eating practices. Cyanobacterial blooms in Lake Erie are only ever present June through October and typically only reach high intensities during August and September (Wynne and Stumpf, 2015). If an individual only consumes freshly-caught fish, the seasonal TDI seems most appropriate. However, anglers may freeze their catch and consume year-round, and restaurants or markets may sell fish that has been previously frozen. MC does not degrade in frozen cyanobacterial tissue (Fastner et al., 2002), and it may likewise not degrade in frozen fish tissue. Therefore, consumers who follow this practice may be exposed to MC year-round, and the lifetime TDI would therefore be the appropriate standard for safe consumption. Using a lifetime TDI, the average fish consumer in the United States —who is estimated to consume 6.5 g of fish per day— could consume fish tissues with a concentration of 431 ng MC/g wet weight for his or her entire lifetime without risk, a concentration that was N40% greater than the highest MC concentration observed in Lake Erie during our study (i.e., 303 ng/g in walleye). The average Lake Erie angler, again using a lifetime TDI, would need to consume fish with lower MC concentrations to avoid risk (no N70 ng/g; (Dyble et al., 2011)), owing to their ~6-fold higher intake of fish than the average US fish consumer (Table 2). 23 (of 43) walleye, five (of 52) white perch, and one (of 55) yellow perch exhibited concentrations above this level of concern (Table 2). However, if the seasonal TDI for MC is used, the risk-free threshold for MC concentration in fish tissues for the average Lake Erie angler is
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much higher (i.e., 700 ng/g), N 130% higher than the highest MC concentration observed in our fish (see Fig. 2). Likewise, when using the seasonal TDI, no fish samples represented a threat to individuals that follow Ohio's established guidelines for sportfish consumption, which were jointly formed by the Ohio Department of Health, Ohio Environmental Protection Agency, and the ODNRDOW. These guidelines, which were implemented to reduce human exposure to other human-derived pollutants (e.g., mercury, PCBs), suggest consuming no more than two yellow perch meals per week (i.e., a total of 226.8 g per week) or no more than one white perch or walleye meal per week (i.e., 113.4 g per week). Given the observed MC concentrations in our fish samples, if Ohio's sportfish consumption guidelines were followed and the seemingly more plausible seasonal TDI values were used to estimate risk (as opposed to Ibelings and Chorus', 2007 lifetime TDI), individuals could continue to consume walleye, white perch, or yellow perch during the cyanobacteria bloom season with minimal risk of over-exposure to MCs. In our study, the only population identified by Dyble et al. (2011) that would potentially receive a regular dose of MC exceeding the seasonal TDI was the population of “Very High Fish Consumers”, which consists of individuals that consumed N 328 g of fish per day. We found 18 of 43 walleye and two of 52 white perch to be above the 85 ng/g seasonal TDI threshold (Table 2), indicating some risk of MC over-exposure to this group; no yellow perch posed a risk when the seasonal TDI was used. Risk also existed for “Tribal Members”, which were estimated to consume above-average quantifies of fish (190 g per day; Dyble et al., 2011). However, this risk only existed for walleye, with six of 43 fish sampled being above the seasonal TDI threshold. It is important to keep in mind that none of the fish examined in this study would have caused a 70-kg individual in any study population to exceed the threshold of 2.5 μg/kg (Ibelings and Chorus, 2007) for acute MC exposure. However, it is equally important to recognize that the safe threshold values presented in this study were calculated for healthy adults of average size (i.e., 70 kg); at-risk populations such as children and the immune-compromised may be susceptible to lower doses of MC (Dyble et al., 2011). As mentioned above, our study only examined free MCs in fish tissues, as bound MCs are not liable to extraction by methanol. It is not yet clear how these bound MCs might be transferred to consumers (Lance et al., 2014), but a study of the theoretical products of bound MC digestion found them half as toxic as free MCs (Smith et al., 2010). As bound MCs may make up the predominant proportion of MCs in an organism (Lance et al., 2010; Williams et al., 1997; but see Pires et al., 2004), this remains an important area of research, and our results should be considered in light of this uncertainty.
Table 2 Daily fish consumption rates, threshold advisory microcystin concentrations (ng MC/g wet weight) in fish tissue (based on a 70 kg person and daily MC limits of 0.04, 0.4, and 2.5 μg MC/ kg), and the percentage of individuals sampled of each species from Lake Erie in 2013 exceeding these values (WP = white perch; YP = yellow perch; WA = walleye). None of the fish had microcystin concentration above the acute threshold. Population type
Average fish consumption (g/d)
Lifetime threshold (ng/g)
Seasonal threshold (ng/g)
Acute threshold (ng/g)
% of WP above MC threshold
# of YP above MC threshold
# of WA above MC threshold
(Sample n = 55)
(Sample n = 52)
(Sample n = 43)
Lifetime Seasonal Lifetime Seasonal Lifetime Seasonal Average U.S. consumer ODH advisory level
Lake Erie Angler Tribal member– low estimate “Very high” fish consumer
6.5a 32.4b (WP, WA)
431 86.4 (WP, WA)
4310 864 (WP, WA)
64.8b (YP) 40c 190c
43.2 (YP) 70 14.7
328c
8.5
0 3.6
0 0
0 1.9
0 0
0 41.9
0 0
432 (YP) 700 147
26,923 5401 (WP,WA) 2701(YP) 4375 921
9.1 83.6
0 0
1.9 19.2
0 0
53.5 81.4
0 14.0
85
533
90.9
3.6
40.4
0
90.7
41.9
Sources of daily fish consumption data: a Derived from Dyble et al. (Dyble et al., 2011). b Derived from the ODH advisory for consumption of Lake Erie sportfish (25). c Quoted in Dyble et al. ((Dyble et al., 2011)).
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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While exposure to unsafe levels of MCs was only prevalent in populations that consumed above-average levels of walleye, and in some cases white perch, our study does not completely rule out risk for the average fish consumer, nor should it be used to suggest that yellow perch may never have unsafe levels of MCs. Our study year (2013), was not an extreme cyanobacteria bloom year when compared to other years (e.g., 2011, 2015; Stumpf et al., 2012; Wynne and Stumpf, 2015; http://www.glerl.noaa.gov/res/HABs_and_Hypoxia/ lakeErieHABArchive/), nor were ambient MC concentrations as high as observed in other years (e.g. a maximum of 191 μg/L in 2015 vs. 56.35 μg/L in 2013; http://www.glerl.noaa.gov/res/HABs_and_Hypoxia/ WLEMicrocystin.html). Thus, the possibility exists that, during years with excessively large, long-lasting blooms or with small, highly concentrated blooms, the depuration systems of fish may become overloaded and edible fish tissues may accumulate unsafe levels of MCs for even the average fish consumer. This may occur with continued climate change, which not only is expected to cause extreme cyanobacteria bloom events, such as those observed in 2011 and 2015, to become more common (Michalak et al., 2013), but also promote white perch, a species of high abundance in Lake Erie that does not tolerate cold temperatures well ((Johnson and Evans, 1990) and that we have shown already has the propensity to accumulate high MC levels. Summary and conclusions While the MC concentration in edible tissues of Lake Erie walleye, white perch, and yellow perch never appeared to exceed unsafe levels for the average Lake Erie angler, when using a seasonal TDI based on 2013 fish samples, we did observe unsafe MC levels in some walleye and white perch (but not yellow perch) for those populations that consume above-average levels of fish (e.g., a typical Tribal Member). Given this latter finding, and the fact that MC levels in white perch were positively related to an index of cyanobacteria bloom intensity, we strongly recommend continued monitoring of MCs both in the water and in the tissues of recreationally and commercially important Lake Erie fishes. Further, we recommend that these assessments be expanded to encompass more than just the cyanobacteria bloom season, especially given that continued climate change is expected to expand the magnitude, frequency, and duration of bloom events (Michalak et al., 2013; Paerl and Huisman, 2009; Paerl and Otten, 2013), as well as promote white perch survival (Johnson and Evans, 1990). By also assessing the preand post-bloom periods, regulatory and management agencies could better understand how the MC burden in edible species varies seasonally, as rates of MC accumulation and depuration are likely to vary through time, owing to species-specific differences in foraging behavior, bloom use (as habitat), and physiology (Guzzo et al., 2013; Knight et al., 1984; Lester et al., 2004). With the knowledge of inter- and intra-annual variation in MC concentrations in fish that would come with expanded long-term monitoring, regulatory agencies would be better positioned to develop consumption guidelines that are based on MC levels, in addition to those based on other pollutants (e.g., mercury, PCBs). These agencies could then provide their constituents a better evaluation of when during the cyanobacteria season fish are safe to consume. Acknowledgments We would like to thank the ODNR-DOW Sandusky Fisheries Research Unit, which provided the yellow perch and white perch used in this study, and also aged our fish. We also want to thank charter boat captains Dave Spangler, Rick Unger, and Paul Pacholski, who provided the walleye samples in coordination with Ohio State's Stone Laboratory. Significant laboratory work was carried out by Nicole Basenback, Megan Levine, Taylor Nesbit, Jeremy Schechter, Kelsey Sikon, and Nathan Stoltzfus. We also thank Ruth Briland and Paul Hurtado for providing essential insights, which helped with the development of this manuscript. This study was supported by a NOAA/Ohio Sea Grant to JFM, SAL, and JL,
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Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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Journal of Great Lakes Research journal homepage: www.elsevier.com/locate/jglr
Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms David M. Wituszynski a,⁎, Chenlin Hu b,1, Feng Zhang b, Justin D. Chaffin c, Jiyoung Lee b,d, Stuart A. Ludsin e, Jay F. Martin a,f a
Department of Food, Agricultural, and Biological Engineering, The Ohio State University, Columbus, OH 43210, USA College of Public Health, Division of Environmental Health Sciences, The Ohio State University, USA F.T. Stone Laboratory, The Ohio State University, USA d Department of Food Science and Technology, The Ohio State University, USA e Aquatic Ecology Laboratory, Department of Ecology, Evolution, and Organismal Biology, The Ohio State University, USA f Ohio Sea Grant, USA b c
a r t i c l e
i n f o
Article history: Received 22 March 2017 Accepted 31 July 2017 Available online xxxx Keywords: Public health Harmful algae HABs Eutrophication Microcystis
a b s t r a c t Microcystin (MC) is a cyanobacteria-produced liver toxin that has been found in fish from Lake Erie, sometimes in excess of World Health Organization (WHO) guidelines for safe consumption. Even so, few studies have quantified MCs in Lake Erie fishes, and these studies have drawn different conclusions concerning the risk that fish consumption poses to public health. To address this gap in knowledge, we used Enzyme-Linked Immunosorbant Assay (ELISA) to evaluate the MC concentration in muscle tissue from three commonly harvested fish in Lake Erie: walleye (Sander vitreus, n = 29); yellow perch (Perca flavescens, n = 52); and white perch (Morone americana, n = 55), collected during summer 2013. Satellite remote sensing was used to compare MC concentrations in fish tissue to bloom conditions in Lake Erie at the time of harvest. We found a significant difference among mean MC concentrations in walleye (71 ng MC/g wet weight), white perch (37 ng MC/g), and yellow perch (8.1 ng MC/g). In addition, MC levels in white perch appeared to depend on local bloom conditions. While few of the fish collected contained MC in excess of WHO guidelines, our results indicate that more toxic blooms could increase MC in fish to levels that pose a greater risk to public health. © 2017 Published by Elsevier B.V. on behalf of International Association for Great Lakes Research.
Introduction Cyanobacterial blooms and their associated health threats are a rapidly growing concern worldwide and are projected to expand due to changes in climate and increases in anthropogenic nutrient loading (Bullerjahn et al., 2016; Kosten et al., 2012; Michalak et al., 2013). These blooms can have substantial ecological and environmental impacts due to shading, hypoxia, and alterations of aquatic food webs (Paerl and Otten, 2013). Additionally, cyanobacteria are known to produce several varieties of toxin, and therefore represent a potential human health threat (Ibelings and Chorus, 2007). The most widespread and best-studied cyanotoxin is microcystin (MC), a potent liver toxin and a suspected tumor promoter (Chorus and Bartram, 1999; Ibelings and Chorus, 2007). This toxin has been found worldwide, and has been implicated in human and animal sickness and death (Chorus and Bartram, 1999). Microcystin exists in the form of more than eighty congeners, differentiated by amino acid substitutions. The most toxic of ⁎ Corresponding author. E-mail address: [email protected] (D.M. Wituszynski). 1 Present address: College of Pharmacy, University of Houston.
these is MC-LR, and this has been the most prevalent congener found in Lake Erie water samples (Brittain et al., 2000; Dyble et al., 2008). The World Health Organization (WHO) has established provisional guidelines for MC content in food, drinking water, and water used for recreational purposes (Chorus and Bartram, 1999). The latter two routes of exposure are regularly controlled in the United States, via beach closures and advanced water treatment. However, few studies have explored the presence of MC in commercially and recreationally harvested aquatic organisms, and currently no national regulation has addressed this concern. Further, the few studies that have been conducted have found widely varying levels of MC in edible muscle tissues (0.5–1960 ng MC/g dry mass) (Kopp et al., 2013; Niedzwiadek et al., 2012; Poste et al., 2011; Schmidt et al., 2013; Wood et al., 2014), suggesting that MC accumulation is strongly species- and ecosystemdependent. The cause of this variability in MC concentrations in fish tissues, however, remains enigmatic for two primary reasons. First, quantifying MCs in animal tissues is a time-consuming process, and the results are often difficult to interpret (Schmidt et al., 2013; Xie et al., 2004). Second, harmful cyanobacterial blooms are highly variable in space and time which could lead to high variability in MC burdens on aquatic organisms
http://dx.doi.org/10.1016/j.jglr.2017.08.006 0380-1330/© 2017 Published by Elsevier B.V. on behalf of International Association for Great Lakes Research.
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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(Hunter et al., 2008; Shi et al., 2015; Stumpf et al., 2012; Wynne and Stumpf, 2015). Therefore, the need exists to establish links between cyanobacterial blooms, which are readily observable, and levels of MC in edible tissues. This knowledge will allow the determination and prediction of health risk via MC exposure through consumption of aquatic organisms. Our study addresses this gap in knowledge by quantifying MC concentrations in walleye (Sander vitreus), yellow perch (Perca flavescens), and white perch (Morone americana), all commercially and/or recreationally important fishes, collected in the western basin of Lake Erie during summer 2013. We also used remotely sensed data to identify linkages between MC levels in edible fish tissues and local bloom intensity for yellow perch and white perch. In so doing, we sought to identify factors that could affect the risk of MC exposure to humans via fish consumption. Methods Study system Lake Erie is the southernmost, shallowest, and most productive of the Laurentian Great Lakes (Ludsin et al., 2001; Michalak et al., 2013). It is important economically and culturally throughout the region, providing numerous ecosystem services such as opportunities for recreation by swimming, boating, and fishing, as well as support for associated tourism activities (Hobbs et al., 2002). The sport fishing industry alone accounts for as much as $1.2 billion per year in economic activity (Lake Erie Improvement Organization, 2012). Additionally, the lake is the source of drinking water for 11 million people. During recent years, however, Lake Erie has been experiencing regular harmful algal blooms (HABs) that have threatened these services (Brittain et al., 2000; Carmichael and Boyer, 2016). The blooms have been dominated by cyanobacteria of the genus Microcystis that can produce MCs (Harke et al., 2016; Michalak et al., 2013), and recent reports indicate that Lake Erie waters experiencing these blooms regularly exceed the 20 μg/L WHO guideline for recreational contact (http://www.glerl. noaa.gov/res/HABs_and_Hypoxia/WLEMicrocystin.html, accessed August 4, 2016). Given that these reports have shown MC concentrations in Lake Erie water regularly reaching 10–20 μg/L, and occasionally as high as 1000 μg/L (Steffen et al., 2014), MC exposure via fish consumption from this lake should be a concern (Poste et al., 2011). Walleye, yellow perch, and white perch were selected for this study, as they are three of the most commonly harvested sport and commercial fish in Lake Erie. While the MC content of all three species has been reported (Dyble et al., 2011; Poste et al., 2011; Wilson et al., 2008), our study is the first to examine large sample sizes of all three species taken from the same year at locations with known bloom intensities. Sample collection and MC extraction and quantification All fish analyzed for this study were captured in western Lake Erie during 2013. White perch (n = 55) and yellow perch (n = 52) were collected as part of an annual bottom trawl assessment program conducted by the Ohio Department of Natural Resources-Division of Wildlife (ODNR-DOW) (ODW, 2014). Individuals used in this study were collected from 13 trawling locations in western Lake Erie during August and September (Fig. 1); these trawls framed the period during which the bloom peaked and then began to senesce (as reported in the Lake Erie Experimental HAB Bulletin: http://www.glerl.noaa.gov/res/HABs_and_ Hypoxia/lakeErieHABArchive/, accessed August 4, 2016). Walleye (n = 43) were collected from various locations within the western basin via charter boats that were participating in water quality monitoring, which was supervised by The Ohio State University's Stone Laboratory. The charter boat captains provided the belly flap (n = 29) or muscle fillets (n = 14) of individual fish caught by anglers. Some charter boat
captains also provided the total length (TL, nearest 1 mm) of the individual fish (n = 22). Fish ages were determined by the ODNR-DOW Sandusky Fish Research Station, using established relationships between age and fish TL for each species (C. Vandergoot, ODNR-DOW, pers. comm.) (Table 1). Details on fish collection and sizes are given in Electronic Supplementary Material (ESM) Table S1 and locations and numbers of white perch and yellow perch collections are in ESM Tables S2 and S3·Fish tissue was analyzed for MCs using methanol extraction followed by Enzyme-Linked Immunosorbant Assay (ELISA), in a manner derived from that of Hu et al. (2008) and Moreno et al. (2005). Fish tissue was dried at 60 °C for 24 h (Poste et al., 2011), homogenized using a mortar and pestle, and then extracted with 75% methanol for 2 h at room temperature while being stirred. Extracts were centrifuged at ~4750 rpm for 15 min, after which supernatant was removed and the solids resuspended in 75% methanol for a second extraction. This process was repeated for a total of three extractions. The supernatant from all extractions was pooled and diluted to one-quarter strength with deionized water and passed through a SepPak® C18 column (Waters corporation, Milford, MA). Microcystin was eluted from the column with 5 mL of 100% methanol. The sample was then diluted to b5% methanol and analyzed using the Microcystins/Nodularins (ADDA) ELISA kit (catalog number PN520011, Abraxis Inc., Warminster, PA) with MC-LR (0.15–5 μg/L) as the working standard and a detection sensitivity of 0.1 μg/L. Data on MC concentrations in collected fish are given in ESM Table S1. Comparison of walleye belly flap and fillets MC concentrations are given in ESM Table S4, and extraction efficiencies are given in ESM Table S5. Lower limits of detection varied by sample, and ranged from 1.27 ng MC/g wet mass to 30.29 ng MC/g wet mass (mean = 9.64 ng MC/g wet mass) for white perch and yellow perch, respectively; for walleye the range was 7.48 ng MC/g wet mass to 86.36 ng MC/g wet mass, with a mean of 26.35 ng MC/g wet mass. Several walleye samples were below the desired wet mass of 0.5 g, which dramatically raised the lower limit of detection for these samples. Only six of the 42 walleye samples did not have detectable MC; of these, the highest lower limit of detection was 41.54 ng MC/g wet mass. As the ELISA was calibrated with MC-LR, results are reported as MCLR equivalents. Doing so assumes that the entire mass of MC discovered is MC-LR, the most toxic of the MCs (Deblois et al., 2011). Therefore, the limits for safe consumption expressed here are conservative. The ELISA test used exhibits good cross-reactivity across all tested MC congeners (Fischer et al., 2001). However, as not all congeners have been tested, it is possible that some species may be present to which the ELISA test is relatively insensitive. These would be under-reported; however, toxicity data is not available for most of these congeners. It should be noted that our method of analysis only extracts free MCs. Studies have found varying amounts of covalently bound MCs in fish tissue, and our results may therefore underestimate the amount of MC actually contained in our samples. Indications are that these bound MCs are not as toxic as free MCs (Ito et al., 2002; Metcalf et al., 2000). Determination of cyanobacterial blooms Bloom intensity at sampling locations for white perch and yellow perch was determined through analysis of remote sensing imagery. Images from the Moderate Resolution Imaging Spectroradiometer (MODIS) were processed to extract the “cyanobacterial index” (CI) developed by Wynne et al. (2013) for the locations and times at which white perch and yellow perch were captured. This index measures the abundance of chlorophyll-a in a cyanobacteria-specific fashion, and it has been shown to be related to field measurements of Microcystis spp. cell counts in western Lake Erie (Wynne et al., 2010). As chlorophyll-a in western Lake Erie has also been shown to be correlated with observed MC concentrations (Stumpf et al., 2016), this index is a good approximation of the potential toxicity of the bloom in any one location. The values of this index were therefore used as a proxy for
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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Fig. 1. Yellow perch and white perch were collected from 13 sampling locations in western Lake Erie during August and September 2013. The numbers next to each site label designate the number of yellow perch and white perch, respectively, caught at that location. Walleye were collected by charter boat captains during September and October 2013. The walleye fishing area is denoted by the white oval. Summaries of the number of white perch and yellow perch collected at each location and at each date are available in Electronic Supplementary Material (ESM Tables S2 and S3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Microcystis spp. bloom intensity. Images used herein were from within one day of the fish sampling dates. Data analysis Methods for all data on fish metrics, MC in fish, associated CI used are reported in the Electronic Supplementary Material (ESM) Table S1 and results of statistical tests of different statistical models are in ESM Table S6. Toxin concentrations in fish tissues were not normally distributed, even after transformation. Thus, we used nonparametric pairwise Wilcoxon tests with Bonferroni correction to test for differences in MC concentration among species. Finding that MC concentrations differed among species (see Results and discussion section), we used speciesspecific linear models to explain fish tissue MC concentration. In all cases, MC concentration was the effect. The following factors were used to predict MC levels in white Perch and yellow Perch: Age, TL (though not in conjunction with age), Bloom Intensity, and Capture Date. Linear models were constructed with ordinary least squares regression, and selection was performed according to corrected Akaike Information Criterion (AICc); models with ΔAICc b 2 were considered equal in explanatory power (Bolker, 2008). Significant models reported here presented normally distributed residuals with no outliers (studentized residuals all b 3). Non-detect values were replaced by a
random number drawn from a uniform distribution between 0 and the lower limit of detection for that sample. Model adequacy was verified by checking the size and distribution of residuals. All statistical analyses were performed in R 3.0.1. An overall alphalevel of 0.05 was used to denote significance. Models constructed for walleye, while significant, explained little of the variance in toxin concentration and so are not reported.
Public health impact Public health exposure was estimated by calculating maximum allowable levels of MC (μg MC/kg body mass) in fish tissues. These thresholds were calculated according to Dyble et al. (2011) using both the WHO lifetime Tolerable Daily Intake (TDI) value of 0.04 μg/kg (Chorus and Bartram, 1999) and the “seasonal TDI” of 0.4 μg/kg proposed by Ibelings and Chorus (2007). The WHO's lifetime TDI value is the amount of MC that an individual could, in theory, consume daily for the remainder of his or her life without suffering negative effects. The TDI was derived from a “No Observable Adverse Effects Level” of 40 μg/kg found in animal studies (Fawell et al., 1999). This value was divided by a safety factor of 100 to account for variability among individuals, as well as among species, and then again by a further factor of 10 to translate the findings of the short-term studies into a lifetime effect level. These
Table 1 Information on the fish analyzed for this study, including fish age and total length statistics, as well as tissue microcystin concentrations. The conditions of the cyanobacteria blooms experienced by fish at the time of capture also is reported. Cyanobacterial Index was transformed to Microcystis cells per mL according to Wynne et al. (2010) to calculate bloom exposure. All fish were collected in the western basin of Lake Erie during August and September 2013. Species
White Perch Yellow Perch Walleye a
Age (years)
Total length (cm)
Bloom Exposure* (Microcystis cells/mL)
MC content (ng MC/g wet mass)
Mean
Range
Mean
Range
Mean
Range
Mean
Range
1.8 3.31 6.86a
1–5 1–7 5–12a
15.24 16.38 22.86a
11.28–20.09 12.07–23.72 20–25a
2.49 ∗ 105 2.20 ∗ 105 NA
1.90 ∗ 105− 3.43 ∗ 105 1.65 ∗ 105− 3.06 ∗ 105 NA
37.5 11.80 84.13
b7.83–91.8 b7.51–73.3 b17.9–303
Ages and lengths were only available for 7 of the 29 Walleye samples.
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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calculations lead to a final TDI of 0.04 μg/kg, which is the accepted WHO guideline (Chorus and Bartram, 1999). Importantly, Ibelings and Chorus (2007) have argued that this TDI should not be applied to situations in which individuals are only at risk of exposure for a short period of time. Because cyanobacterial blooms in temperate regions usually present exposure risk for only a portion of the year (e.g., blooms are generally present during late summer through early fall in Lake Erie; Michalak et al., 2013), Ibelings and Chorus (2007) suggested the use of a “seasonal” TDI of 0.4 μg/kg. Thus, in addition to presenting the lifetime TDI, which is the most commonly used metric in the literature, we also present the seasonal TDI, as it seems more applicable to our study system. We calculate acceptable daily intake levels for different types of (human) fish consumers, assuming the average consumer weighed 70 kg. Using this approach, a person may acceptably take in 2800 ng (lifetime) or 28,000 ng (seasonal) MC per day. We used reference fish consumption rates for five different human populations within the Lake Erie area: the “average” U.S. consumer, the consumer that follows the Ohio Department of Health (ODH) Advisory Level, the typical Lake Erie angler, a typical Native American tribal member, and a “very high” fish consumer (De Rosa and Hicks, 2001; 2016 Ohio Sport Fish Health and Consumption Advisory: http://www.epa.state.oh.us/dsw/ fishadvisory/index.aspx, accessed September 9, 2016). Based on these consumption rates, we then calculated the average MC concentration in fish tissue at which an individual belonging to a given population will exceed the acceptable daily MC intake level (for both lifetime and seasonal TDIs). Finally, these threshold values were used to evaluate the risk to public health posed by the fish MC levels measured in this study. Results and discussion MC levels in fish Microcystin (MC; ng MC/g wet mass) levels differed among fish species (Kruskal-Wallis test: χ2 = 52.9, p b 0.05; Mann-Whitney-Wilcoxon test: all W N 217, all p b 0.05; Table 1, Fig. 2) with mean concentrations in walleye (84 ng/g) being greater than both white perch (38 ng/g) and yellow perch (12 ng/g). White perch MC levels were also significantly higher than yellow perch MC levels (Table 1, Fig. 2). Differences in foraging may partially explain these inter-specific differences in MC concentrations. Age 1 + yellow perch acquire a large
Fig. 2. Microcystin (MC) concentration for yellow perch (n = 53), white perch (n = 55), and walleye (n = 43) collected in western Lake Erie during the 2013 cyanobacteria bloom season. Sample means (ng MC/g wet mass) indicated by the horizontal line are as follows: mean = 11.8 (yellow perch); mean = 37.5 (white perch); and mean = 70.8 (walleye). ND = MC levels below detection limits. NDs are placed at the values used for the data analysis presented above. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
proportion of their diet from benthic sources (Guzzo et al., 2013; Tyson and Knight, 2001). By contrast, zooplankton are a larger component of the diet of age 1+ white perch (Gopalan et al., 1998; Guzzo et al., 2013). The differences in internal MC concentration between these two fish therefore agrees broadly with the literature on MC accumulation in fishes of different feeding guilds. For example, KozlowskySuzuki et al. (2012) conducted a meta-analysis of microcystin in aquatic organisms, finding that zooplanktivorous fish were more likely to concentrate MC than fish of other feeding guilds (e.g., carnivorous and omnivorous). Additionally, during a study in Lake Ijsselmeer (Netherlands), Ibelings et al. (2005) found higher MC concentrations in the livers of European smelt (Osmerus eperlanus), a planktivore, than in the livers of either European Perch (Perca fluviatilis), a carnivore, or Eurasian ruffe (Gymnocephalus cernua), a benthivore. Similarly, in a Chesapeake Bay (USA) tributary, Wood et al. (2014) found that organisms with largely benthic diets accumulated lower concentrations of MC than those organisms with largely pelagic (planktivorous) diets. The fact that adult walleye, as piscivores, had high MC concentrations also was somewhat expected (Xie et al., 2005; Kozlowsky-Suzuki et al., 2012), although Gkelis et al. (2006) did find higher concentrations in planktivorous, zooplanktivorous, and omnivorous fish than piscivorous fish in their study. Xie et al. (2005) found MC concentrations to be higher in piscivores than other feeding guilds, and postulated uptake from sources other than food. MCs may be entering walleye through gill uptake (Cazenave et al., 2005; Malbrouck and Kestemont, 2006). While the use of cyanaobacteria blooms by Lake Erie walleye has not been quantified, walleye may forage in them, owing to their optical properties (sensu Lester et al., 2004) and the potential local abundance of prey species, themselves attempting to reduce predation risk (Engström-Öst et al., 2006). Kozlowsky-Suzuki et al. (2012) suggested that the broader breadth of piscivore diets (e.g., ability to feed on large zooplankton and other invertebrates, in addition to fish), contributed to higher MC concentrations in tissues of these fish relative to other feeding guilds. It is not likely that omnivory is responsible for the high MC levels in Lake Erie walleye, however, as adults are strictly piscivorous, feeding to a large degree on juvenile and adult gizzard shad (Dorosoma cepedianum) (Knight et al., 1984; Knight and Vondracek, 1993). While we can only speculate, perhaps the high MC levels in Lake Erie walleye is due their high consumption of gizzard shad, which is known to be highly phytoplanktivorous during older life stages (Schaus et al., 2002; Yako et al., 1996). An alternative explanation for these inter-specific differences might lie in the depuration mechanisms for MC, which can differ considerably among fish species. For example, Adamovský et al. (2007) found a lower half-life of MC in muscle tissue of silver carp (Hypophthalmichthys molitrix) than in muscle tissue of common carp (Cyprinus carpio), though for both species the value was less than three days. Experiments on the depuration of MC in tilapia (Mohamed and Hussein, 2006), centrarchids (Smith and Haney, 2006), and yellow perch (Dyble et al., 2011) have also shown variable timescales for MC elimination. Because we did not directly measure depuration, and rates have not been measured in walleye or white perch, the role of the depuration process in explaining variation in our results remains uncertain. For this reason, future studies should address this issue by analyzing fish captured both before the bloom begins and after it ends. Microcystin concentration was unrelated to neither fish age nor fish size within all three species (all models tested have p N 0.05). This finding indicates that toxin did not accumulate preferentially in younger or older fish, nor was it likely to accumulate and remain in fish tissues across years. The intensity of the cyanobacterial bloom at the location in which fish were captured was positively related to MC concentration in White Perch (F-test: F = 14.1, p = 0.000431; Table S6), but not in yellow perch (F-rest: F = 0.647, p = 0.425; Table S6), as indicated by effect tests of the Cyanobacterial Index (CI) in linear models. The white perch MC concentration varied with collection date as well, with fish captured
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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in September being significantly more toxic than fish caught during August (F-test: F = 19.67, p b 0. 0.00005). Studies have shown that many fish have efficient MC depuration systems up to a point, but it is possible for them to be overrun by an excess of MC (Deblois et al., 2011; Dyble et al., 2011). Therefore, the increase in MC concentrations in white perch over time may be due to MC accumulation over the bloom season, rather than a response to immediate MC in the environment at the time sampled. The CI for sample sites was higher on average in September than in August, meaning that blooms were more intense during the later period (t-value: 12.05, p-value: b2.2E−16). These two potential explanations of MC concentration in white perch are therefore confounded in our samples. The fact that yellow perch MC levels were unrelated to the CI may indicate that they can rapidly depurate MCs (see Dyble et al., 2011). Risk of MC exposure through fish consumption MC concentrations were below the threshold levels of concern for most potential consumers in the Lake Erie basin, especially when using a seasonal total daily intake (TDI) estimate (Table 2). Whether the lifetime or the seasonal TDI is most appropriate likely varies by the consumer's fishing and eating practices. Cyanobacterial blooms in Lake Erie are only ever present June through October and typically only reach high intensities during August and September (Wynne and Stumpf, 2015). If an individual only consumes freshly-caught fish, the seasonal TDI seems most appropriate. However, anglers may freeze their catch and consume year-round, and restaurants or markets may sell fish that has been previously frozen. MC does not degrade in frozen cyanobacterial tissue (Fastner et al., 2002), and it may likewise not degrade in frozen fish tissue. Therefore, consumers who follow this practice may be exposed to MC year-round, and the lifetime TDI would therefore be the appropriate standard for safe consumption. Using a lifetime TDI, the average fish consumer in the United States —who is estimated to consume 6.5 g of fish per day— could consume fish tissues with a concentration of 431 ng MC/g wet weight for his or her entire lifetime without risk, a concentration that was N40% greater than the highest MC concentration observed in Lake Erie during our study (i.e., 303 ng/g in walleye). The average Lake Erie angler, again using a lifetime TDI, would need to consume fish with lower MC concentrations to avoid risk (no N70 ng/g; (Dyble et al., 2011)), owing to their ~6-fold higher intake of fish than the average US fish consumer (Table 2). 23 (of 43) walleye, five (of 52) white perch, and one (of 55) yellow perch exhibited concentrations above this level of concern (Table 2). However, if the seasonal TDI for MC is used, the risk-free threshold for MC concentration in fish tissues for the average Lake Erie angler is
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much higher (i.e., 700 ng/g), N 130% higher than the highest MC concentration observed in our fish (see Fig. 2). Likewise, when using the seasonal TDI, no fish samples represented a threat to individuals that follow Ohio's established guidelines for sportfish consumption, which were jointly formed by the Ohio Department of Health, Ohio Environmental Protection Agency, and the ODNRDOW. These guidelines, which were implemented to reduce human exposure to other human-derived pollutants (e.g., mercury, PCBs), suggest consuming no more than two yellow perch meals per week (i.e., a total of 226.8 g per week) or no more than one white perch or walleye meal per week (i.e., 113.4 g per week). Given the observed MC concentrations in our fish samples, if Ohio's sportfish consumption guidelines were followed and the seemingly more plausible seasonal TDI values were used to estimate risk (as opposed to Ibelings and Chorus', 2007 lifetime TDI), individuals could continue to consume walleye, white perch, or yellow perch during the cyanobacteria bloom season with minimal risk of over-exposure to MCs. In our study, the only population identified by Dyble et al. (2011) that would potentially receive a regular dose of MC exceeding the seasonal TDI was the population of “Very High Fish Consumers”, which consists of individuals that consumed N 328 g of fish per day. We found 18 of 43 walleye and two of 52 white perch to be above the 85 ng/g seasonal TDI threshold (Table 2), indicating some risk of MC over-exposure to this group; no yellow perch posed a risk when the seasonal TDI was used. Risk also existed for “Tribal Members”, which were estimated to consume above-average quantifies of fish (190 g per day; Dyble et al., 2011). However, this risk only existed for walleye, with six of 43 fish sampled being above the seasonal TDI threshold. It is important to keep in mind that none of the fish examined in this study would have caused a 70-kg individual in any study population to exceed the threshold of 2.5 μg/kg (Ibelings and Chorus, 2007) for acute MC exposure. However, it is equally important to recognize that the safe threshold values presented in this study were calculated for healthy adults of average size (i.e., 70 kg); at-risk populations such as children and the immune-compromised may be susceptible to lower doses of MC (Dyble et al., 2011). As mentioned above, our study only examined free MCs in fish tissues, as bound MCs are not liable to extraction by methanol. It is not yet clear how these bound MCs might be transferred to consumers (Lance et al., 2014), but a study of the theoretical products of bound MC digestion found them half as toxic as free MCs (Smith et al., 2010). As bound MCs may make up the predominant proportion of MCs in an organism (Lance et al., 2010; Williams et al., 1997; but see Pires et al., 2004), this remains an important area of research, and our results should be considered in light of this uncertainty.
Table 2 Daily fish consumption rates, threshold advisory microcystin concentrations (ng MC/g wet weight) in fish tissue (based on a 70 kg person and daily MC limits of 0.04, 0.4, and 2.5 μg MC/ kg), and the percentage of individuals sampled of each species from Lake Erie in 2013 exceeding these values (WP = white perch; YP = yellow perch; WA = walleye). None of the fish had microcystin concentration above the acute threshold. Population type
Average fish consumption (g/d)
Lifetime threshold (ng/g)
Seasonal threshold (ng/g)
Acute threshold (ng/g)
% of WP above MC threshold
# of YP above MC threshold
# of WA above MC threshold
(Sample n = 55)
(Sample n = 52)
(Sample n = 43)
Lifetime Seasonal Lifetime Seasonal Lifetime Seasonal Average U.S. consumer ODH advisory level
Lake Erie Angler Tribal member– low estimate “Very high” fish consumer
6.5a 32.4b (WP, WA)
431 86.4 (WP, WA)
4310 864 (WP, WA)
64.8b (YP) 40c 190c
43.2 (YP) 70 14.7
328c
8.5
0 3.6
0 0
0 1.9
0 0
0 41.9
0 0
432 (YP) 700 147
26,923 5401 (WP,WA) 2701(YP) 4375 921
9.1 83.6
0 0
1.9 19.2
0 0
53.5 81.4
0 14.0
85
533
90.9
3.6
40.4
0
90.7
41.9
Sources of daily fish consumption data: a Derived from Dyble et al. (Dyble et al., 2011). b Derived from the ODH advisory for consumption of Lake Erie sportfish (25). c Quoted in Dyble et al. ((Dyble et al., 2011)).
Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006
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While exposure to unsafe levels of MCs was only prevalent in populations that consumed above-average levels of walleye, and in some cases white perch, our study does not completely rule out risk for the average fish consumer, nor should it be used to suggest that yellow perch may never have unsafe levels of MCs. Our study year (2013), was not an extreme cyanobacteria bloom year when compared to other years (e.g., 2011, 2015; Stumpf et al., 2012; Wynne and Stumpf, 2015; http://www.glerl.noaa.gov/res/HABs_and_Hypoxia/ lakeErieHABArchive/), nor were ambient MC concentrations as high as observed in other years (e.g. a maximum of 191 μg/L in 2015 vs. 56.35 μg/L in 2013; http://www.glerl.noaa.gov/res/HABs_and_Hypoxia/ WLEMicrocystin.html). Thus, the possibility exists that, during years with excessively large, long-lasting blooms or with small, highly concentrated blooms, the depuration systems of fish may become overloaded and edible fish tissues may accumulate unsafe levels of MCs for even the average fish consumer. This may occur with continued climate change, which not only is expected to cause extreme cyanobacteria bloom events, such as those observed in 2011 and 2015, to become more common (Michalak et al., 2013), but also promote white perch, a species of high abundance in Lake Erie that does not tolerate cold temperatures well ((Johnson and Evans, 1990) and that we have shown already has the propensity to accumulate high MC levels. Summary and conclusions While the MC concentration in edible tissues of Lake Erie walleye, white perch, and yellow perch never appeared to exceed unsafe levels for the average Lake Erie angler, when using a seasonal TDI based on 2013 fish samples, we did observe unsafe MC levels in some walleye and white perch (but not yellow perch) for those populations that consume above-average levels of fish (e.g., a typical Tribal Member). Given this latter finding, and the fact that MC levels in white perch were positively related to an index of cyanobacteria bloom intensity, we strongly recommend continued monitoring of MCs both in the water and in the tissues of recreationally and commercially important Lake Erie fishes. Further, we recommend that these assessments be expanded to encompass more than just the cyanobacteria bloom season, especially given that continued climate change is expected to expand the magnitude, frequency, and duration of bloom events (Michalak et al., 2013; Paerl and Huisman, 2009; Paerl and Otten, 2013), as well as promote white perch survival (Johnson and Evans, 1990). By also assessing the preand post-bloom periods, regulatory and management agencies could better understand how the MC burden in edible species varies seasonally, as rates of MC accumulation and depuration are likely to vary through time, owing to species-specific differences in foraging behavior, bloom use (as habitat), and physiology (Guzzo et al., 2013; Knight et al., 1984; Lester et al., 2004). With the knowledge of inter- and intra-annual variation in MC concentrations in fish that would come with expanded long-term monitoring, regulatory agencies would be better positioned to develop consumption guidelines that are based on MC levels, in addition to those based on other pollutants (e.g., mercury, PCBs). These agencies could then provide their constituents a better evaluation of when during the cyanobacteria season fish are safe to consume. Acknowledgments We would like to thank the ODNR-DOW Sandusky Fisheries Research Unit, which provided the yellow perch and white perch used in this study, and also aged our fish. We also want to thank charter boat captains Dave Spangler, Rick Unger, and Paul Pacholski, who provided the walleye samples in coordination with Ohio State's Stone Laboratory. Significant laboratory work was carried out by Nicole Basenback, Megan Levine, Taylor Nesbit, Jeremy Schechter, Kelsey Sikon, and Nathan Stoltzfus. We also thank Ruth Briland and Paul Hurtado for providing essential insights, which helped with the development of this manuscript. This study was supported by a NOAA/Ohio Sea Grant to JFM, SAL, and JL,
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Please cite this article as: Wituszynski, D.M., et al., Microcystin in Lake Erie fish: Risk to human health and relationship to cyanobacterial blooms, J. Great Lakes Res. (2017), http://dx.doi.org/10.1016/j.jglr.2017.08.006