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Presented By: MUJEEB ADEYEMI BALOGUN

DECEMBER 2022

Total Mercury Concentration in Selected Tissues of Bottle Nose Dolphins

(Tursiops truncates ) Stranded along the Northern Gulf of Mexico

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OBJECTIVES

Determine Total mercury (THg) concentration in the selected tissue (skin & blubber) of bottlenose dolphins in the study locations

INTRODUCTION

Mercury (Hg) is a non-essential trace element and a pollutant of great concern for both human and ecosystem health due to its toxicity and persistence in the environment

Origin: Mercury originates from natural and anthropogenic sources and enters the ocean primarily from atmospheric deposition and runoff

Due to their long-life span and top trophic position, bottlenose Dolphins can accumulate high concentrations of mercury (Hg) in their tissues

Binding of Hg & Se toxicologically inert HgSe

Influence of body length, sex & age on THg & Se concentration in the skin & blubber of bottlenose dolphins

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Hg:

Natural element: •Natural deposit in cinnabar (HgS) • In volcanic rocks and hot springs • In fossil fuels

Anthropogenic emission sources: Coal, Oil, Wood, Waste Incineration, Non-ferrous metal industry (Non-ferrous metals are alloys or metals that do not contain any appreciable amounts of iron)

Dolphins are active predators and eat a wide variety of fishes, squids, and crustaceans such as shrimps. The foods available to a dolphin vary with its geographic location. Dolphins show strong preferences for certain species of food fish. Coastal dolphins tend to eat fishes and bottom-dwelling invertebrates.

However, it has been hypothesized that odontocetes may be able to mitigate/detoxify the effects of Hg toxicity through two processes: 1) the demethylation of MeHg to inorganic Hg, a less toxic form of Hg, and 2) the binding of Hg with selenium (Se) which results in the formation of toxicologically inert mercury selenide (HgSe) compound or sequestering of Hg in toxicologically inert mercury selenide (HgSe) compounds.

In marine systems, sulfate-reducing bacteria convert inorganic Hg (Hg2+) to methylmercury (CH3Hg+; MeHg) which enters the food web via phytoplankton, bioaccumulates in organisms, and biomagnifies up marine food webs (Hammerschmidt and Fitzgerald, 2006; Fitzgerald et al., 2007)

What are non-essential metals?

Non-essential metals (NEMs) are priority pollutants that pose potential risks to human health and the environment. Exposure to NEMs such as arsenic, cadmium, lead and mercury has been associated with a significant number of adverse health effects in humans.

Non-essential trace metals such as Cadmium (Cd), Mercury (Hg) and Lead (Pb) have no known essential role in living organisms; exhibit extreme toxicity even at very low (trace) exposure levels and have been regarded as the main threats to all forms of life especially human health

Trace elements refer to any chemical element that is present in the human body in very small amounts, usually less than 0.1% by volume. Most trace elements can be classified as nutritionally essential, probably essential, or potentially toxic.

The key difference between bioaccumulation and biomagnification is that bioaccumulation refers to the build-up of a toxic chemical in the body of a living organism while biomagnification is the increase of the concentration of a toxic chemical when going along a food chain.

Bioconcentration is the accumulation of a chemical in or on an organism when the source of chemical is solely water.^[1] Bioconcentration is a term that was created for use in the field of aquatic toxicology.^[1] Bioconcentration can also be defined as the process by which a chemical concentration in an aquatic organism exceeds that in water as a result of exposure to a waterborne chemical.^[2]

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MATERIALS AND METHODS

Collection of Stranded Dolphins

in Florida (FL) & Louisiana (LA)

277 individuals (2011-2016)
Condition Code: 1-4

(Geraci & Lounsbury, 2005)

Selected Tissues

Skin and blubber samples were collected & stored at - 20°C

until THg Analysis

Analysis: Instrumentation

Direct Mercury Analyzer (DMA)

using thermal decomposition,

gold amalgamation, & atomic

absorption spectrometry

ICPMS: Se concentrations

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The THg concentration in blubber (10–15 mg) and skin (10–20 mg) was determined using a Direct Mercury Analyzer….

(Smithsonian Institution Coding System; Geraci and Lounsbury, 2005).

Inductively Coupled Plasma Mass Spectrometry- ICPMS

Rshp btwn Selenium and Mercury

code 1 (alive) to code 4 (advanced decomposition)

In FL=> Code 2 (fresh dead) with 44% was the highest

In LA=> Code 3 ( moderate decomposition)

In FL, the condition of the stranded animals varied, ranging from condition code 1 (alive) to code 4 (advanced decomposition) (Geraci and Lounsbury, 2005) with 18% from code 1 individuals, 44% from code 2 individuals, 29% from code 3 individuals, and 9% from code 4 individuals. Odontocetes that were alive at the time of examination (code 1) were euthanized, or died naturally on the beach, during transportation to the rehabilitation facility, or while at the rehabilitation facility.

In LA, all odontocetes sampled were of condition code 3 (moderate decomposition). Samples from less decomposed individuals [e.g., condition code 2 (fresh dead)] are preferable, as decomposition may affect trace element concentrations; however, other studies have found that samples from more heavily decomposed individuals (e.g., condition code 3 and 4) can be utilized for trace element analyses if condition code is considered as a potential confounding variable (Martínez-López et al., 2019a; McCormack et al., 2020b).

What is a confounding variable?

A confounding variable, also called a confounder or confounding factor, is a third variable in a study examining a potential cause-and-effect relationship.

A confounding variable is related to both the supposed cause and the supposed effect of the study. It can be difficult to separate the true effect of the independent variable from the effect of the confounding variable.

In your research design, it's important to identify potential confounding variables and plan how you will reduce their impact.

Mercury in Skin and Blubber

Condition codes ranged from 2 to 4 (code 2 = fresh, code 3 = moderate decomposition, code 4 = advanced decomposition; Smithsonian Institution Coding System; Geraci and Lounsbury, 2005). In FL, 43%, 31%, and 26% of blubber samples came from code 2, 3, and 4, individuals, respectively. In LA, 4%, 9% and 87% of blubber samples came from code 2, 3, and 4, individuals, respectively.

In FL, 19%, 19%, and 62% of skin samples came from code 2, 3, and 4, individuals, respectively. In LA, 4%, 7%, and 89% of skin samples came from code 2, 3, and 4 individuals, respectively.

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Fig. 1. THg concentration (mean + SD) in blubber & skin of Florida (FL) & Louisiana (LA) bottlenose dolphins.

Fig. 2. Relationship between body length & age in Florida (FL) & Louisiana (LA) bottlenose dolphins with growth curves fitted using the Gompertz model.

RESULTS

McCormack et al., 2020 a,b

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Fig 1: Mean blubber THg concentrations were significantly less than mean skin THg concentrations in both FL (ANOVA; p < 0.001) and LA (ANOVA; p = 0.005)

There were significant differences in mean THg concentrations between FL and LA dolphins for both blubber (ANOVA; p < 0.001) and skin (ANOVA; p < 0.001).

Lower THg concentrations in dolphins from LA may be a result of indirect influences from the Mississippi River. The Mississippi River which drains 41% of the neighbouring United States delivers large amounts of sediments and nutrients to the central northern Gulf of Mexico (Presley et al., 1998; Apeti et al., 2012). Large amounts of sediments could dilute atmospherically deposited Hg with material that is lower in Hg concentration and large influxes of nutrients support higher phytoplankton productivity which can reduce THg concentrations in fish; it is possible that lower concentrations in fish may be reflected in dolphins

In FL dolphins, the mean ± SD blubber THg concentration was 2.36 ± 2.71 μg/g dry wt (range: 0.0378–13.9 μg/g dry wt) or 1.25 ± 1.21 μg/g wet wt (range: 0.0295–5.69 μg/g wet wt) and the mean ± SD skin THg concentration was 4.36 ± 3.55 μg/g dry wt (range: 0.562–14.7 μg/g dry wt) or 2.25 ± 2.41 μg/g wet wt (range: 0.225–12.3 μg/g wet wt).

In LA dolphins, the mean ± SD blubber THg concentration was 1.33 ± 2.78 μg/g dry wt (range: 0.0163–24.9 μg/g dry wt) or 0.679 ± 1.18 μg/g wet wt (range: 0.0125–8.96 μg/g wet wt) and the mean ± SD skin THg concentration was 1.94 ± 3.51 μg/g dry wt (range: 0.0531–25.2 μg/g dry wt) or 1.06 ± 1.81 μg/g wet wt (range: 0.0305–9.98 μg/g wet wt)

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What is the meaning of significant difference in statistics?

A statistically significant difference is simply one where the measurement system (including sample size, measurement scale, etc.) was capable of detecting a difference (with a defined level of reliability). Just because a difference is detectable, doesn't make it important, or unlikely.

What does it mean if there is a significant difference between two groups?

A “statistically significant difference” simply means there is statistical evidence that there is a difference; it does not mean the difference is necessarily large, important, or significant in terms of the utility of the finding.

What does mean plus standard deviation tell you?

It tells you, on average, how far each score lies from the mean. In normal distributions, a high standard deviation means that values are generally far from the mean, while a low standard deviation indicates that values are clustered close to the mean.

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Age determination

-Teeth were collected from the left lower mandible (generally teeth positioned at numbers 13–16 in the row), stored in 10% neutral-buffered formalin for up to 48 h, rinsed in tap water, and archived in 70% ethyl alcohol. Teeth were then prepared for sectioning using standard procedures

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Fig 2:

Overall, bottlenose dolphins in this study had a mean ( ± SD) body length of 212 cm ± 53 (range: 74–285 cm).

Dolphins ranged from < 2 months to 35 years old with the mean ( ± SD) being 14.7 ± 10.0 years.

In both locations, there was a rapid increase in growth through age 5 followed by a slowing of growth until an asymptotic body length was reached (243 cm and 252 cm for combined male and female FL and LA dolphins, respectively).

A Gompertz growth curve provided a good fit to the data for both FL and LA dolphins (Fig. 2).

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Fig. 3. Total Hg (THg) concentrations in blubber & skin of bottlenose dolphins from (FL) & (LA) in relation to body length (left column) & age (right column).

Fig. 4. Relationship between Se concentration (μg/g dry wt) & body length (cm) in the blubber, & skin of stranded bottlenose dolphins.

RESULTS

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Sex was not an influential predictor of THg for either tissue > for Mercury in Skin and Blubber paper

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The present study found no difference in THg concentration between sexes, consistent with the findings of previous studies

In some cases, condition code positively influenced THg concentrations. This may be a result of lipid loss due to poor body condition and/or decomposition of the sample. The concentration of THg has been shown to decrease with lipid content. Since Hg does not preferentially bind to lipids, greater lipid concentrations can dilute THg concentrations

The present study also found significant positive relationships between THg concentration and body length/age in both the blubber and skin, supporting the findings of previous studies

In both tissues, THg concentrations increased with body length, but there was greater variation in THg concentrations among larger (> 225 cm) individuals compared to smaller individuals (≤ 225 cm)

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Selenium

Body length positively influenced Se concentrations in the blubber, kidney, lung, and skin.

Sex significantly influenced Se concentrations in blubber, lung, and skin, and in all cases, males had lower Se concentrations than females

Unlike Hg, within mammalian systems, Se, like other essential trace elements, is regulated within the body (Khan and Wang, 2009; CáceresSaez et al., 2018). However, the coaccumulation of Hg and Se in marine mammal tissues is well documented, and as a result, Se concentrations can exceed the limit of homeostatic control.

In addition, Se concentrations were greater in dolphins that stranded along the FL coast compared to those that stranded along the LA coast. Differences in tissue Se concentrations may be due to differences in Se concentrations between dolphin prey, but may also be due to the coaccumulation of Hg and Se

Se is an essential element (Schomburg et al., 2004). Serving as a vital component of some proteins (selenoproteins) and enzymes (selenoenzymes), Se contributes to normal metabolic function in addition to playing an important role in Hg detoxification.

Furthermore, the coaccumulation of Hg and Se in multiple odontocete tissues (e.g., blubber, brain, heart, kidney, liver, lung, muscle, and spleen) has also been reported, providing indirect evidence of an interaction between these two elements

As a result, across a variety of marine vertebrates, including teleosts, sharks, and cetaceans, it has been suggested that if present in molar excess (i.e., Se:Hg molar ratio >1:1), Se may reduce the likelihood of Hg toxicity.

In the present study, . On average, in bottlenose dolphins, Se:Hg molar ratios were approximately 1:1 in the liver and >1:1 in blubber, kidney, lung, and skin, suggesting that Se likely protects against Hg toxicity.

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CONCLUSION

LIMITATIONS & FUTURE RESEARCH DIRECTION

The source stock of the stranded dolphins was unknown.

THg concentrations were determined in stranded animals which may not be representative of healthy free-ranging populations.

It would be beneficial in future studies to differentiate between source stocks either isotopically, genetically, or through photo-identification to determine if THg concentrations vary among source stocks population.

THg concentration was highest in the skin tissue

Explanatory variables positively influenced THg concentrations in the stranded Dolphins

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The source stock of the stranded dolphins was unknown and THg concentrations may vary among inshore (bay, sound, and estuarine) stocks within FL and LA and between inshore and nearby coastal stocks.

The potential for stranded dolphins to serve as a tool for monitoring free-ranging populations would be enhanced if their stocks of origin were known

a genetic stock identification (GSI) method that is based on the detection of genetic variability with gel electrophoresis. The method uses naturally occurring genetic differences between stocks to provide estimates of the composition of mixed-stock fisheries.

What is genetic stock identification?

Genetic stock identification (GSI) estimates stock proportions and individual assignments through comparison of genetic markers with reference populations. It is used widely in anadromous fisheries to estimate the impact of oceanic harvest on riverine populations.

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REFERENCES

Geraci, J. R., & Lounsbury, V. J. (2005). Marine mammals ashore: a field guide for strandings. National Aquarium in Baltimore.

McCormack, M. A., Battaglia, F., McFee, W. E., & Dutton, J. (2020a). Mercury concentrations in blubber and skin from stranded bottlenose dolphins (Tursiops truncatus) along the Florida and Louisiana coasts (Gulf of Mexico, USA) in relation to biological variables. Environmental research, 180, 108886.

McCormack, M. A., Jackson, B. P., & Dutton, J. (2020b). Relationship between mercury and selenium concentrations in tissues from stranded odontocetes in the northern Gulf of Mexico. Science of The Total Environment, 749, 141350

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Thank you

for your attention