The following is a example of a EXCELLENT lab.  Be sure to label all figures and tables and refer to them in the text.  You are emulating a published article in peer-review journal such as Analytical Chemistry

Kim Trout

 Instrumental Methods of Analysis Lab #3

Dr. Ashley

 Lab Conducted April 4, 2006

“Mercury Levels in Hair Related to Fish Consumption”

Abstract

            Mercury can be very toxic at high levels. It can enter the environment through many different processes; once in the environment, it can accumulate in the water and fish. Most people are exposed to mercury by eating fish. Mercury can cause a variety of neurological defects, as well as cause birth and mental defects of unborn fetuses. This study used a Flow Injection Mercury System using cold vapor atomic absorption to test mercury levels in 36 hair samples of students and faculty at Philadelphia University. The subjects were asked to complete a survey about their lifestyle and fish-eating habits. Mercury concentrations of the hair samples fell into the range of 0.1 μg/g to 3.6 μg/g with a mean (+ standard deviation) of 0.912 + 1.108 μg/g. Even though women and children are the major concern with mercury toxicity, the EPA suggests that both men and women have less than 1 μg/g in their hair. Approximately one fifth of the total participants in the study had levels exceeding this limit. There were no strong correlations found between mercury levels and frequency of fish eating, nor were there any with age.

Introduction

            Mercury, a heavy metal, acts as a neurotoxin when it enters the human body. It has been known to damage the nervous system and heart in adults, while causing learning and developmental disabilities in young children (Cotel). Exposure to mercury can happen in many ways; however, one of the most common ways is by eating fish. When mercury is released into the air or water, whether by industry or waste combustion, it becomes methylated and accumulates in animal tissues; fish is currently the primary source of methylmercury. Methylmercury is especially harmful to women of child-bearing age because the fetus is highly susceptible to the effects (Cotel).

            The U.S. Department of Health and Human Services (HHS) and the U.S. Environmental Protection Agency (EPA) have compiled a list of the mercury levels in fish. This list states that King Mackerel, Shark, Swordfish, and Tilefish from the Gulf of Mexico have the highest levels and should be avoided (“Mercury”). However, the Natural Resources Defense Council has also compiled a list of fish to avoid eating because the mercury levels are so high. In addition to the four previously listed, they suggest avoiding Grouper, Marlin, and Orange Roughy (“Consumer”).

            Mercury testing can be done in many ways: either in the saliva, urine, blood, skin, stool, or hair. Out of these techniques, hair testing is the most non-invasive. The subject simply gives a hair sample and they’re done; this method was chosen for this particular study. The EPA has set a guideline of 1 ppm or 1 μg/g of mercury in hair as the safe limit for both men and women (Maas).

            The purpose of this study was to compare the levels of mercury in hair to the frequency of consumption and other lifestyle factors and identify any correlation. It was hypothesized that older participants and those who consumed a large amount of fish would have higher levels of mercury in their hair. This would support the findings of a previous study done by Tom Clarkson in Canada in 1998 (“Methylmercury”).

Methods and Materials

            Approximately 0.5 g of each of 36 hair samples was weighed out and placed in a tube for microwaving. Next, 10 mL of nitric acid was added to each of the tubes and their weights were recorded. Three duplicates (samples analyzed twice) were performed such that analytical precision could be assessed. In addition, a Mercury analyzer kit was obtained and one sample was sent in to assess analytical accuracy. Blanks were prepared using 10 mL nitric acid. Blanks are prepared in order to asses how much mercury is in the lab and/or reagents. The instrument is “zeroed” with these blanks; it judges the absorption of the samples from the blank. The tubes were then digested in the microwave for approximately half an hour. When the tubes were taken out of the microwave, they were depressurized and the solutions were brought up to 100 mL with Deionized water. The solutions were then placed in clean glass bottles and 3 mL of potassium permanganate was added to each bottle to digest any hair that may not have fully been digested. The solutions would now be ready for analyzing; however, the potassium permanganate is purple in color and would not be suitable for the instrument to properly analyze. Therefore, 1 mL of 12% sodium chloride-hydroxylamine hydrochloride was added to reduce the excess potassium permanganate and return the solution to colorless (Kiry “Tissue”).

            The mercury analysis was performed by a Perkin-Elmer Flow Injection Mercury System-400 (FIMS-400) Mercury Analyzer, which measures the absorption of radiation at 253.7 nm by mercury vapor. The calibration curve was prepared by the instrument, which is used to determine the amount of mercury in each sample. The sample weight and ID information were entered into the computer program associated with the instrument, and the analysis method was selected. The instrument then analyzed each sample, re-running a standard every ten samples to make sure the instrument is still calibrated. Using the calibration curve previously created, the instrument determined the concentration of mercury and gave an output in μg/g/L (Kiry “Mercury”).

Results

Table 1 gives the sample ID information, final concentration in μg/g, age, and frequency of fish consumption.

Table 1. Mercury concentrations compared to age and frequency of consumption

21% of the participants in the study exceeded the recommended limit of 1 μg/g. The mean concentration was found to be 0.912 μg/g, while the standard deviation was found to be 1.108 μg/g.

There was a correlation found between the final concentration and age. A Pearson’s correlation coefficient (r) was calculated between age and final Hg concentration (μg/g).  The alpha level was set to .05 using a one-tailed test.  A significant, positive correlation was discovered(r (32 df) = .30, one-tailed p < .05).  Because this p is less than .05, it is acceptable to conclude that it is not just due to chance that older people really did have more Hg than younger people. Figure 1 shows a plot of concentration vs. age. Table 2 contains the correlations found.

Figure 1. Significant Relationship of final mercury concentration and age (r = .30,

p < 0.05)

Table 2. Correlations found between final concentration and age.

There was no significant correlation found between final mercury concentration and frequency of fish consumption (servings/month). Figure 2 shows the relationship.

Figure 2. Relationship between final mercury concentration and frequency of fish consumption.

There was no direct correlation found between previous knowledge of the consequences of mercury exposure and high mercury levels. However, the mean mercury level of those who knew about exposure (n=15) was 1.167 μg/g while the mean level of those who were not aware of the consequences of mercury exposure (n=18) was 0.606 μg/g. Figure 3 shows a comparison of the data.

Figure 3. A comparison of the final mercury concentration levels of those who had previous knowledge about mercury exposure and those who had no previous knowledge.

Cigarettes contain mercury, which could affect the results of a study such as this one. Figure 4 represents a graph of the mean mercury concentrations and standard deviations of smokers (n=8) and non-smokers (n=24).

Figure 4. Comparison of average mercury levels of smokers and non-smokers.

Dental amalgams have been known to contain mercury and therefore raise the concentration of mercury in the system; this information was provided on the questionnaire. Figure 5 represents a graph of the mean mercury concentrations and standard deviations of those with (n=18) and without dental amalgams (n=16).

Figure 5. A comparison of mean mercury concentrations and standard deviations of those with and without dental amalgams.

Another portion of the questionnaire dealt with the specifics of the fish: cooked or raw, bought or caught, and processed or unprocessed. There were not enough samples in both categories to accurately make comparisons between cooked and raw or between bought or caught (one group in each comparison had only 2 samples). Figure 6 shows a comparison of those who eat processed (n=11) and unprocessed fish (n=14) as well as those who eat both (n=6). Four people did not respond to that portion of the questionnaire.

Figure 6. Comparison of mean mercury levels and standard deviations of those who eat processed and unprocessed fish, as well as those who eat both.

 Discussion

             The participants of this study were mostly in their early 20’s, which could have skewed that data somewhat. It would be suggested to repeat the study with more variation in age to see if the mercury concentration and age correlation can be replicated. It was found, however, that the older subjects did generally have higher mercury levels, which correlates with a recent study which found a correlation between age and mercury concentrations (Maas). In the 36 samples analyzed, only 7 contained levels of mercury above the recommended limit. These elevated levels could be due to many things besides just eating fish. For instance, one of the samples that had high levels had a small sample size. Had there been a larger sample, the concentration probably would have gone down. There could also be exposure to mercury in other areas besides fish. For instance, a sample containing high levels was reportedly exposed to mercury in grade school; however, it is highly unlikely that it is still in their system more than 10 years later. The study showed that frequency of fish consumption does influence mercury levels in the body. These findings correlate with two other studies, one by the Dept. of Hygiene & Medical Institute of Environmental Hygiene at Heinrich-Heine University and one by the Environmental Quality Institute (Cotel and McDowell). In a study done by Greenpeace, those with a frequency of 0-4 servings/month had a mean concentration of 0.35 μg/g; those eating 5-9 servings/month had a mean concentration of 0.89 μg/g; and those eating 10+ servings/month had a mean concentration of 1.35 μg/g (Maas). The mean concentrations for this study were found to be 0.927 μg/g for 0-4 serving/month, 0.8195 μg/g for 5-9 servings/month, and 1.035 μg/g for 10+ servings/month. Figure 7 shows a comparison of the mean levels of the two studies.

Figure 7. Comparison of the mean levels grouped by frequency of fish consumption of this study and the Greenpeace study.

There was also no significant correlation found with smoking and mercury levels, however, the mean level of the eight people who smoked was higher with 0.832 μg/g while the mean level on the non-smokers was .742 μg/g. These finding correlate with a current study that found exposure to cigarette smoke increases mercury levels (Cotel). There was also no correlation found between mercury concentrations and dental amalgams; the mean mercury level of those with amalgams was .757 μg/g while the mean level for those without fillings was 1.064 μg/g. These findings go against a current study which found that dental amalgams increased mercury levels (Cotel).

References

Clarkson, Tom. “Methylmercury and fish consumption: Weighing the risks.” Proquest. Canadian Medical Association. Journal. Ottawa: 2 Jun 1998. Vol. 158, Issue 11.

“Consumer Guide to Mercury in Fish.” Natural Resources Defense Council. 8 Feb. 2006. <http://www.nrdc.org/health/effects/mercury/guide.asp>.

Cotel, Orli. “A Hair-Raising Lesson.” Proquest. Sierra. Nov/Dec 2005, Vol. 90, Issue 6.

Kiry, Paul and Adam Boettner. “Mercury Analysis on FIMS-400.” Academy of Natural Sciences Patrick Center for Environmental Research. Aug. 2000. Procedure # P-16-103.

Kiry, Paul and Adam Boettner. “Tissue and Sediment Microwave Digestion for Metals Analysis.” Academy of Natural Sciences Patrick Center for Environmental Research. Aug 2000. Procedure # P-16-XX.

Mass, Richard P; Steven C. Patch, and Kimberly R. Sergent. “A Statistical Analysis of Factors associated with Elevated Hair Mercury Levels in the US Population: An Interim Progress Report. UNC-Asheville Environmental Quality Institute Technical Report #04-136. October 2004.

McDowell, Margaret A., Charles F. Dillon, et al.  “Hair Mercury Levels in U.S. Children and Women of Childbearing Age.”  NHANES.  27 May 2004.

“Mercury Levels in Commercial Fish and Shellfish.” US Department of Health and Human Services and US Environmental Protection Agency. May 2001. Jan 2006. 8 Feb. 2006. <http://www.cfsan.fda.gov/~frf/sea-mehg.html>.