Eur J Nutr DOI 10.1007/s00394-015-0890-5
ORIGINAL CONTRIBUTION
Low‑level mercury, omega‑3 index and neurobehavioral outcomes in an adult US coastal population Caterina Vacchi‑Suzzi1 · Roxanne Karimi2 · Danielle Kruse7 · Susan M. Silbernagel6 · Keith E. Levine3 · Diane S. Rohlman4,5 · Jaymie R. Meliker1
Received: 5 January 2015 / Accepted: 22 March 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Background Neurodevelopmental effects of omega-3 fatty acids and mercury from fish consumption have been characterized in children. In contrast, neurobehavioral outcomes associated with fish are not well studied in adults. Objective This study of avid seafood consumers on Long Island (NY, USA) sought to define associations between mercury, seafood consumption, omega-3 fatty acids and neurobehavioral outcomes. Methods A computer-based test system was used to assess neurobehavioral function. Blood total Hg (Hg) and omega-3 index were measured in 199 adult avid seafood eaters, who also completed the neurobehavioral assessment and an extensive food and fish frequency and demographic questionnaire. Results For most of the outcomes considered, neither Hg nor omega-3 index was associated with neurobehavioral outcomes after adjustment for key confounding variables.
Electronic supplementary material The online version of this article (doi:10.1007/s00394-015-0890-5) contains supplementary material, which is available to authorized users.
Fish consumption, however, was associated with decreased odds of both self-reported fatigue (OR 0.85; 95 % CI 0.72, 1.01) and a constellation of neurologic symptoms (OR 0.79; 95 % CI 0.66, 0.96). Conclusions Results from our study provide little evidence that omega-3 fatty acids or Hg is associated with cognitive function in adult avid seafood consumers. Larger studies are needed to confirm our finding of associations between fish consumption and decreased self-reported fatigue and neurologic impairment. Keywords Mercury · Omega-3 · n-3 fatty acids · Methylmercury · Neurological test · Neurobehavioral test
Introduction Public health organizations and health professionals increasingly recommend that adults consume at least two meals of fish per week [1]. Seafood contains macro- (i.e., unsaturated fatty acids) and micronutrients (i.e., selenium)
* Caterina Vacchi‑Suzzi caterina.vacchi‑[email protected]
4
Occupational and Environmental Health, University of Iowa, Iowa City, IA 52242, USA
Jaymie R. Meliker [email protected]
5
Oregon Institute for Occupational Health Sciences, Oregon Health and Science University, Portland, OR 97239, USA
6
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA 98195, USA
7
School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA
1
Program in Public Health, Department of Preventive Medicine, Stony Brook University, Stony Brook, NY 11794, USA
2
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794, USA
3
Trace Inorganics Department, Technologies for Industry and the Environment, RTI International, Research Triangle Park, NC 27709, USA
13
known for their beneficial effects, and at the same time, mercury (Hg), a known neurotoxicant. Despite a growing body of studies that link consumption of omega-3 fatty acids to favorable cognitive and neurobehavioral outcomes in both animal and human studies [2–8], the benefits of fish consumption can be impaired by the presence of mercury [9], and definitive nutritional guidelines for intake of omega-3 fatty acids (either via seafood consumption or via supplementation) are still lacking [3, 10, 11]. Omega-3 and fish consumption are known to have beneficial effects in the cognitive and neurobehavioral domain in children [12, 13] and at earlier developmental stages [14], while evidence in elderly people (65 years and older) and adults is controversial [15, 16]. Studies examining neurological outcomes associated with dietary intake of omega-3 fatty acids in adults have yielded inconsistent results. A study of middle-aged adults used serum phospholipid fatty acid composition to measure omega-3 intake and found that docosahexaenoic acid (DHA) specifically correlated with improved cognitive results [17]. Studies in elderly adults have found associations between increased fish consumption and a decrease in cognitive decline, but associations with omega-3 levels are mixed [18, 19]. Evidence from randomized clinical trials and blood lipid analyses suggest that higher omega-3 levels (from either diet or supplement) are associated with lower risk of depressive disorder [16, 20]. The omega-3 index, defined as the percent ratio of omega-3 compared to total fatty acids in red blood cell membrane, can be used to assess the presence of fatty acids in the body [21]. A study of deployed US Service members used this index and found associations between higher omega-3 index and increased cognitive function, especially in sleep-deprived individuals [22]. Few studies, however, have examined measures of the omega-3 index, fish intake and mercury in relation to neurobehavioral (computer-based test) and self-reported neurological outcomes. While the neurological consequences of acute Hg poisoning in children are well established [23–30], less is known about neurological effects of low-level chronic exposure to its organic form, methylmercury (MeHg), via fish consumption in adults [9]. A study of fish-eating adults in Korea and elderly adults in the USA found no association between blood Hg and neurobehavioral outcomes [31, 32]; however, the Hg levels were relatively low (max. 16 µg/L blood). Conversely, researchers in Italy and in the USA reported poorer cognitive test results in people with higher blood Hg. However, these studies have methodological limitations. In one case, the sample size was small (10 exposed vs. 6 controls) [33], and in the other, the associations were not adjusted for confounders, such as age [34]. In a study conducted in the Amazon on riverine dwellers, investigators reported worse neurobehavioral test outcomes
13
Eur J Nutr
associated with hair [35], but not with blood Hg levels [36, 37]. Together, these studies provide inconsistent findings for the negative effect of dietary Hg exposure on neurobehavioral function, and more studies are warranted. The benefits of omega-3 fatty acids can mask the neurobehavioral risks associated with MeHg exposure from fish consumption and should be considered as a confounder [38]. The effects of seafood omega-3, combined with the consequences of consuming Hg from seafood, have been investigated in studies involving children [38, 39]; however, most neurobehavioral studies of mercury from fish have not accounted for omega-3 fatty acids in adult subjects [31, 33, 34, 36]. To our knowledge, this is the first study that measured the omega-3 index, allowing for mutual adjustment of mercury in the analysis of adult cognitive outcomes. We conducted a study on the association of blood Hg, omega-3 index and fish consumption with neurobehavioral performance in a cohort of adult men and women from Long Island, NY, who have elevated blood Hg levels due to frequent fish consumption. Cognitive performance/neurobehavioral performance was assessed with a computer-based testing system [40], in addition to self-reported measures of fatigue and neurologic symptoms from a questionnaire.
Materials and methods Long Island Seafood Study cohort We obtained blood samples, demographic, lifestyle, dietary [41] and self-reported health data from 272 adult avid seafood consumers, defined as those who eat seafood 2–4 times per week, which is higher than the estimated consumption for the overall USA [42], and are predicted to be at risk of elevated Hg exposure due to regular fish consumption, from Long Island, NY. For details on procedures and the food frequency questionnaire (FFQ) used to capture fish consumption, see both Karimi et al., 2014 [43, 44]. The self-reported health questionnaire contained questions on Hg-related symptoms, described in a later section. We measured mercury concentrations and omega-3 fatty acid indices in all blood samples. The study was approved by Stony Brook University’s Institutional Review Board for human subjects (IRB# 2010-1179). We calculated intake of seafood by assuming ¼ cup was equivalent to 1.5 oz. This assumption resulted in an average portion size between 3 and 6 oz, depending on the seafood item, which is similar to independent estimates of typical seafood portion sizes [42, 45, 46]. Participants for which age, fish intake or household income were not available were excluded from the analysis. Fish intake was used as a whole, without distinction of species.
Eur J Nutr
Behavioral Assessment and Research System (BARS) The Behavioral Assessment and Research System (BARS) is a computer-based neurobehavioral test battery designed to assess neurobehavioral function in humans. It was developed to provide a series of neurobehavioral tests optimized for the detection of neurotoxicity in human populations, particularly in populations with limited education or literacy, and has been successfully used for assessing the neurobehavioral toxicity of Hg, cadmium and pesticides [47– 50]. The test battery and questionnaire used in our study were comparable to those previously used to investigate the effects of dental amalgam Hg0 exposure on neurobehavioral outcomes and self-reported neurological symptoms in adults [32, 48, 51–59]. Moreover, the tests chosen cover the cognitive domains that have been investigated in previous omega-3 dietary interventions [60]. The tests include simple instructions divided into a series of steps, a 9-button response unit to replace the computer keyboard and spoken instructions [40]. We randomly selected 199 subjects (71 %) to complete a battery of seven tests chosen among the BAR System [61] (Symbol Digit, Reaction Time, Finger Tapping with both Dominant and non-Dominant Hands, Match-to-Sample, Continuous Performance, Digit Span and Reversal Learning). These tests provided nine continuous outcome measures (Symbol Digit Latency, Tapping with Dominant Hand, Tapping with non-Dominant Hand, Simple Reaction Time Latency, Continuous Performance D-Prime, Match-to-Sample Total Latency, Digit Span Forward, Digit Span Reverse and Reversal Learning Total Trials). The Symbol Digit test [80] presents a matrix at the top of the screen that pairs nine unique symbols with the numbers one to nine. A second matrix that contains only the symbols is shown below. Participants are asked to press the corresponding number button for each symbol. Latencies for each button press are recorded. The Tapping test instructs the participant to press a button as many times as they can for a period of time (e.g., 20 s). Performance using each hand and the number of taps was recorded for all trials. In the Simple Reaction Time Test, participants are asked to press a button as fast as they can when a square appears on the screen. Latencies for each button press are recorded. The Continuous Performance Test measures attention. Stimuli are sequentially presented on the screen every 50 ms, when the cue–target stimulus (plus sign followed by a circle) is presented, the participant responds by pressing a button. Three hundred stimuli are presented; 20 % of them are target stimuli. The percent correct, incorrect (false alarms and omissions) and response latencies are recorded.
The Match-to-Sample test measures visual memory. Participants are shown a 10 × 10 matrix with a proportion of squares filled in; after a variable delay (1, 8 or 16 s), they are instructed to choose the correct pattern from among three choices. The number of correct response as well as the latency required to make a correct response is recorded. The Digit Span Test sequentially presents a series of numbers on the screen. The participant is instructed to reproduce the series of numbers by pressing the nine numbered buttons in the same sequence (forward), or, in the second part of the test, in the reverse sequence (reverse). Number sequences are presented in increasing length starting from three numbers. The test is terminated when a participant fails two consecutive trials with the same number of digits, or when they completed a span of nine digits correctly. The Reversal Learning Test presents shapes one at a time until the participant learns the correct response (e.g., press 3 or press 7). Once a performance criterion is reached (e.g., 5 out of 6), the correct response switches. The test measures how long the participant takes to learn the original pattern of responses and then how long to learn the switched or reversed responses. This task is similar to a switching task like the Wisconsin Sorting Task. Self‑reported neurologic and depression outcomes During the study visit, all 272 participants answered 59 questions about their general health conditions and specific diagnoses. We identified participants who reported experiencing symptoms that may be linked to Hg poisoning [62], as those who answered either “Always” or “Usually” to the questions: i. Fatigue: “Over the past year, how often have you felt tired or had little energy”; ii. either of the following: “Over the past year, how often have you felt a loss of balance or coordination?”; “Over the past year, have you experienced numbness or tingling around the mouth?”; and “Over the past year, how often have you had trouble concentrating on things at home or at work (for example, reading the newspaper)?”. Additionally, subjects answered the following question at baseline visit: “Over the past year, how often have you felt down, depressed or hopeless?”. In all cases, the possible answers were: Always; Usually; Sometimes; Rarely; Never; Do not know/Not sure. We generated three dichotomous categorical variables at breaking points that warranted sufficiently powered analyses: The first included only fatigue (1 = Usually or Always, 0 = any other answer), the second was obtained by pooling together reports of loss of balance and coordination, numbness around the mouth and trouble concentrating (1 = Usually or Always in any of the above, 0 = any other answer), and the third included depression (1 = Sometimes, Usually or Always, 0 = any other answer).
13
Blood mercury and selenium levels Hg measured in the blood is known to reflect several weeks to months’ exposure, given its half-life of ~50 days [63, 64]. Blood MeHg represents between 75 and 90 % of the Hg measured in the blood [65, 66] and is highly correlated with Hg measured in the hair, which is considered a long-term exposure marker [36]. Wholeblood Hg and Se were measured as previously described [43, 44]. Briefly, we collected fasting whole-blood specimens for total Hg and Se analyses in trace element blood collection tubes (BD Vacutainer® # 36381). Hg and Se concentration was measured using ICP-MS (Thermo X-Series II) at the RTI Trace Inorganics Laboratory (RTP, NC, USA). Detection limits ranged from 0.10 to 0.70 µg L−1 blood for Hg and 4–15 µg L−1 blood for Se, depending on the batch. Out of the 199 participants, no one had non-detectable Se levels, while three (1.5 %) had non-detectable Hg levels (14 errors in the Simple Reaction Test, total Simple Reaction Time >425 mS,
ORIGINAL CONTRIBUTION
Low‑level mercury, omega‑3 index and neurobehavioral outcomes in an adult US coastal population Caterina Vacchi‑Suzzi1 · Roxanne Karimi2 · Danielle Kruse7 · Susan M. Silbernagel6 · Keith E. Levine3 · Diane S. Rohlman4,5 · Jaymie R. Meliker1
Received: 5 January 2015 / Accepted: 22 March 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Background Neurodevelopmental effects of omega-3 fatty acids and mercury from fish consumption have been characterized in children. In contrast, neurobehavioral outcomes associated with fish are not well studied in adults. Objective This study of avid seafood consumers on Long Island (NY, USA) sought to define associations between mercury, seafood consumption, omega-3 fatty acids and neurobehavioral outcomes. Methods A computer-based test system was used to assess neurobehavioral function. Blood total Hg (Hg) and omega-3 index were measured in 199 adult avid seafood eaters, who also completed the neurobehavioral assessment and an extensive food and fish frequency and demographic questionnaire. Results For most of the outcomes considered, neither Hg nor omega-3 index was associated with neurobehavioral outcomes after adjustment for key confounding variables.
Electronic supplementary material The online version of this article (doi:10.1007/s00394-015-0890-5) contains supplementary material, which is available to authorized users.
Fish consumption, however, was associated with decreased odds of both self-reported fatigue (OR 0.85; 95 % CI 0.72, 1.01) and a constellation of neurologic symptoms (OR 0.79; 95 % CI 0.66, 0.96). Conclusions Results from our study provide little evidence that omega-3 fatty acids or Hg is associated with cognitive function in adult avid seafood consumers. Larger studies are needed to confirm our finding of associations between fish consumption and decreased self-reported fatigue and neurologic impairment. Keywords Mercury · Omega-3 · n-3 fatty acids · Methylmercury · Neurological test · Neurobehavioral test
Introduction Public health organizations and health professionals increasingly recommend that adults consume at least two meals of fish per week [1]. Seafood contains macro- (i.e., unsaturated fatty acids) and micronutrients (i.e., selenium)
* Caterina Vacchi‑Suzzi caterina.vacchi‑[email protected]
4
Occupational and Environmental Health, University of Iowa, Iowa City, IA 52242, USA
Jaymie R. Meliker [email protected]
5
Oregon Institute for Occupational Health Sciences, Oregon Health and Science University, Portland, OR 97239, USA
6
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA 98195, USA
7
School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA
1
Program in Public Health, Department of Preventive Medicine, Stony Brook University, Stony Brook, NY 11794, USA
2
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794, USA
3
Trace Inorganics Department, Technologies for Industry and the Environment, RTI International, Research Triangle Park, NC 27709, USA
13
known for their beneficial effects, and at the same time, mercury (Hg), a known neurotoxicant. Despite a growing body of studies that link consumption of omega-3 fatty acids to favorable cognitive and neurobehavioral outcomes in both animal and human studies [2–8], the benefits of fish consumption can be impaired by the presence of mercury [9], and definitive nutritional guidelines for intake of omega-3 fatty acids (either via seafood consumption or via supplementation) are still lacking [3, 10, 11]. Omega-3 and fish consumption are known to have beneficial effects in the cognitive and neurobehavioral domain in children [12, 13] and at earlier developmental stages [14], while evidence in elderly people (65 years and older) and adults is controversial [15, 16]. Studies examining neurological outcomes associated with dietary intake of omega-3 fatty acids in adults have yielded inconsistent results. A study of middle-aged adults used serum phospholipid fatty acid composition to measure omega-3 intake and found that docosahexaenoic acid (DHA) specifically correlated with improved cognitive results [17]. Studies in elderly adults have found associations between increased fish consumption and a decrease in cognitive decline, but associations with omega-3 levels are mixed [18, 19]. Evidence from randomized clinical trials and blood lipid analyses suggest that higher omega-3 levels (from either diet or supplement) are associated with lower risk of depressive disorder [16, 20]. The omega-3 index, defined as the percent ratio of omega-3 compared to total fatty acids in red blood cell membrane, can be used to assess the presence of fatty acids in the body [21]. A study of deployed US Service members used this index and found associations between higher omega-3 index and increased cognitive function, especially in sleep-deprived individuals [22]. Few studies, however, have examined measures of the omega-3 index, fish intake and mercury in relation to neurobehavioral (computer-based test) and self-reported neurological outcomes. While the neurological consequences of acute Hg poisoning in children are well established [23–30], less is known about neurological effects of low-level chronic exposure to its organic form, methylmercury (MeHg), via fish consumption in adults [9]. A study of fish-eating adults in Korea and elderly adults in the USA found no association between blood Hg and neurobehavioral outcomes [31, 32]; however, the Hg levels were relatively low (max. 16 µg/L blood). Conversely, researchers in Italy and in the USA reported poorer cognitive test results in people with higher blood Hg. However, these studies have methodological limitations. In one case, the sample size was small (10 exposed vs. 6 controls) [33], and in the other, the associations were not adjusted for confounders, such as age [34]. In a study conducted in the Amazon on riverine dwellers, investigators reported worse neurobehavioral test outcomes
13
Eur J Nutr
associated with hair [35], but not with blood Hg levels [36, 37]. Together, these studies provide inconsistent findings for the negative effect of dietary Hg exposure on neurobehavioral function, and more studies are warranted. The benefits of omega-3 fatty acids can mask the neurobehavioral risks associated with MeHg exposure from fish consumption and should be considered as a confounder [38]. The effects of seafood omega-3, combined with the consequences of consuming Hg from seafood, have been investigated in studies involving children [38, 39]; however, most neurobehavioral studies of mercury from fish have not accounted for omega-3 fatty acids in adult subjects [31, 33, 34, 36]. To our knowledge, this is the first study that measured the omega-3 index, allowing for mutual adjustment of mercury in the analysis of adult cognitive outcomes. We conducted a study on the association of blood Hg, omega-3 index and fish consumption with neurobehavioral performance in a cohort of adult men and women from Long Island, NY, who have elevated blood Hg levels due to frequent fish consumption. Cognitive performance/neurobehavioral performance was assessed with a computer-based testing system [40], in addition to self-reported measures of fatigue and neurologic symptoms from a questionnaire.
Materials and methods Long Island Seafood Study cohort We obtained blood samples, demographic, lifestyle, dietary [41] and self-reported health data from 272 adult avid seafood consumers, defined as those who eat seafood 2–4 times per week, which is higher than the estimated consumption for the overall USA [42], and are predicted to be at risk of elevated Hg exposure due to regular fish consumption, from Long Island, NY. For details on procedures and the food frequency questionnaire (FFQ) used to capture fish consumption, see both Karimi et al., 2014 [43, 44]. The self-reported health questionnaire contained questions on Hg-related symptoms, described in a later section. We measured mercury concentrations and omega-3 fatty acid indices in all blood samples. The study was approved by Stony Brook University’s Institutional Review Board for human subjects (IRB# 2010-1179). We calculated intake of seafood by assuming ¼ cup was equivalent to 1.5 oz. This assumption resulted in an average portion size between 3 and 6 oz, depending on the seafood item, which is similar to independent estimates of typical seafood portion sizes [42, 45, 46]. Participants for which age, fish intake or household income were not available were excluded from the analysis. Fish intake was used as a whole, without distinction of species.
Eur J Nutr
Behavioral Assessment and Research System (BARS) The Behavioral Assessment and Research System (BARS) is a computer-based neurobehavioral test battery designed to assess neurobehavioral function in humans. It was developed to provide a series of neurobehavioral tests optimized for the detection of neurotoxicity in human populations, particularly in populations with limited education or literacy, and has been successfully used for assessing the neurobehavioral toxicity of Hg, cadmium and pesticides [47– 50]. The test battery and questionnaire used in our study were comparable to those previously used to investigate the effects of dental amalgam Hg0 exposure on neurobehavioral outcomes and self-reported neurological symptoms in adults [32, 48, 51–59]. Moreover, the tests chosen cover the cognitive domains that have been investigated in previous omega-3 dietary interventions [60]. The tests include simple instructions divided into a series of steps, a 9-button response unit to replace the computer keyboard and spoken instructions [40]. We randomly selected 199 subjects (71 %) to complete a battery of seven tests chosen among the BAR System [61] (Symbol Digit, Reaction Time, Finger Tapping with both Dominant and non-Dominant Hands, Match-to-Sample, Continuous Performance, Digit Span and Reversal Learning). These tests provided nine continuous outcome measures (Symbol Digit Latency, Tapping with Dominant Hand, Tapping with non-Dominant Hand, Simple Reaction Time Latency, Continuous Performance D-Prime, Match-to-Sample Total Latency, Digit Span Forward, Digit Span Reverse and Reversal Learning Total Trials). The Symbol Digit test [80] presents a matrix at the top of the screen that pairs nine unique symbols with the numbers one to nine. A second matrix that contains only the symbols is shown below. Participants are asked to press the corresponding number button for each symbol. Latencies for each button press are recorded. The Tapping test instructs the participant to press a button as many times as they can for a period of time (e.g., 20 s). Performance using each hand and the number of taps was recorded for all trials. In the Simple Reaction Time Test, participants are asked to press a button as fast as they can when a square appears on the screen. Latencies for each button press are recorded. The Continuous Performance Test measures attention. Stimuli are sequentially presented on the screen every 50 ms, when the cue–target stimulus (plus sign followed by a circle) is presented, the participant responds by pressing a button. Three hundred stimuli are presented; 20 % of them are target stimuli. The percent correct, incorrect (false alarms and omissions) and response latencies are recorded.
The Match-to-Sample test measures visual memory. Participants are shown a 10 × 10 matrix with a proportion of squares filled in; after a variable delay (1, 8 or 16 s), they are instructed to choose the correct pattern from among three choices. The number of correct response as well as the latency required to make a correct response is recorded. The Digit Span Test sequentially presents a series of numbers on the screen. The participant is instructed to reproduce the series of numbers by pressing the nine numbered buttons in the same sequence (forward), or, in the second part of the test, in the reverse sequence (reverse). Number sequences are presented in increasing length starting from three numbers. The test is terminated when a participant fails two consecutive trials with the same number of digits, or when they completed a span of nine digits correctly. The Reversal Learning Test presents shapes one at a time until the participant learns the correct response (e.g., press 3 or press 7). Once a performance criterion is reached (e.g., 5 out of 6), the correct response switches. The test measures how long the participant takes to learn the original pattern of responses and then how long to learn the switched or reversed responses. This task is similar to a switching task like the Wisconsin Sorting Task. Self‑reported neurologic and depression outcomes During the study visit, all 272 participants answered 59 questions about their general health conditions and specific diagnoses. We identified participants who reported experiencing symptoms that may be linked to Hg poisoning [62], as those who answered either “Always” or “Usually” to the questions: i. Fatigue: “Over the past year, how often have you felt tired or had little energy”; ii. either of the following: “Over the past year, how often have you felt a loss of balance or coordination?”; “Over the past year, have you experienced numbness or tingling around the mouth?”; and “Over the past year, how often have you had trouble concentrating on things at home or at work (for example, reading the newspaper)?”. Additionally, subjects answered the following question at baseline visit: “Over the past year, how often have you felt down, depressed or hopeless?”. In all cases, the possible answers were: Always; Usually; Sometimes; Rarely; Never; Do not know/Not sure. We generated three dichotomous categorical variables at breaking points that warranted sufficiently powered analyses: The first included only fatigue (1 = Usually or Always, 0 = any other answer), the second was obtained by pooling together reports of loss of balance and coordination, numbness around the mouth and trouble concentrating (1 = Usually or Always in any of the above, 0 = any other answer), and the third included depression (1 = Sometimes, Usually or Always, 0 = any other answer).
13
Blood mercury and selenium levels Hg measured in the blood is known to reflect several weeks to months’ exposure, given its half-life of ~50 days [63, 64]. Blood MeHg represents between 75 and 90 % of the Hg measured in the blood [65, 66] and is highly correlated with Hg measured in the hair, which is considered a long-term exposure marker [36]. Wholeblood Hg and Se were measured as previously described [43, 44]. Briefly, we collected fasting whole-blood specimens for total Hg and Se analyses in trace element blood collection tubes (BD Vacutainer® # 36381). Hg and Se concentration was measured using ICP-MS (Thermo X-Series II) at the RTI Trace Inorganics Laboratory (RTP, NC, USA). Detection limits ranged from 0.10 to 0.70 µg L−1 blood for Hg and 4–15 µg L−1 blood for Se, depending on the batch. Out of the 199 participants, no one had non-detectable Se levels, while three (1.5 %) had non-detectable Hg levels (14 errors in the Simple Reaction Test, total Simple Reaction Time >425 mS,
