ES202L : Ecology and Environment

Part - 2

Gouriprasanna Roy

Department of Chemistry, IIT Tirupati

 Natural sources of mercury
❖ Mercury is a naturally occurring element and is found throughout the world.

Cinnabar (HgS) Natural Source Metacinnabar (HgS)

(Minerals)

Stable, Insoluble in water Less toxic to humans

Tiemannite (HgSe)

Metacinnabar
[-Hg(II)S]

Cinnabar [α-Hg(II)S]

Trigonal crystal system

(prismatic crystals)
Tiemannite
(HgSe)

Hg(II)Se (zincblende structure)

 Natural sources of mercury
❖ Mercury is a naturally occurring element and is found throughout the world.

Cinnabar (HgS) Natural Source Metacinnabar (HgS)

(Minerals)

Stable, Insoluble in water Less toxic to humans

Tiemannite (HgSe)

Metacinnabar
[-Hg(II)S]

Cinnabar
[α-Hg(II)S]

Tiemannite
(HgSe)

❖ Mercury is also present as an impurity in many other economically valuable minerals, in particular the non-
ferrous metals, and in fossil fuels, coal in particular.
 Methylmercury: A Ubiquitous Environmental Pollutant

Cinnabar [α-HgS]
Ayurveda – ancient India
Natural Source Rasasindur
(Minerals) Metacinnabar [-HgS]
(Rasashastra)
Tiemannite (HgSe)
α-HgS
(Hg:S = 1:1)

❖Biologically inert
❖Less toxic to humans

❖ Anthropogenic Sources:
Thermal Power

Mining areas

Burning of
fossil fuels (Coal, Gold,…)

Inorganic Mercury:
Hg(II), Hg(I), HgCl2
❑ In aquatic systems: Inorganic mercury converts into
methylmercury - accumulate in fish and marine
mammals.

Organomercurials:
(MeHg+, EtHg+, PhHg+)
Barkay et al. Advances in Applied Microbiology, 2005, 57, 1.
 The Geochemical Cycle of Mercury: Source of MeHg+ in Human
❑ The aquatic environment is the main route of exposure of Hg to humans and wildlife.
❑ In aquatic systems, inorganic mercury is transformed into the more toxic form, methylmercury, which can accumulate in fish and
marine mammals consumed by humans.

Anaerobic microorganisms, corrinoid [cobalamin-dependent protein], produce MeHg+ from inorganic Hg(II)].

Adapted from Barkay T et al. Advances in Applied Microbiology, 2005, Vol 57, 1.
 Methylmercury Poisoning
❖ 1956, Minamata city, Japan: The catastrophic epidemics from
environmental MeHg+ contamination - Minamata disease - consumption
of fish from water polluted with Hg/MeHg+ from local industrial discharge.
❖ Subsequently, exposure of MeHg+ was documented in Sweden (1966),
Iraq, Guatemala, Pakistan, and Ghana.

According to the Global Emissions Interpretation

Centre (CGEIC), - India is one of the world’s
mercury hotspots - with mercury being released into
the air uniformly at a rate of 0.1– 2 tonnes per year. Mercury in freshwater fish of Ganges river (WB)

Local Total Hg MeHg

name (μg Hg/g (μg Hg/g
of wet wt) of wet wt)

Rohu 0.18±0.04 0.12±0.03

Catla 0.43±0.15 0.32±0.11

Chital 0.45±0.33 0.25±0.18

Boal 1.48±1.01 0.93±0.61

Vetki 0.35±0.02 0.23±0.01

All contain certain amount of MeHg and some of them contain

above the threatening level given by Prevention of Food
Adulteration (PFA), 0.25 μg/g by wet weight.
Moumita et al. Environ Monit Assess. 2011; DOI 10.1007/s10661-011-2193-5
 Mercury toxicity in India

Thermal Power Areas (NTPC) Mining Areas (Coal, Gold,…)

High Level of Mercury/MeHg+ compounds

7
Mercury Toxicity in India:
Small industrial area:
Toxic mg/L Recommended
Metals (WHO)
As 0.04 0.01 - 0.05

Cd 0.06 0.03 - none

Hg 0.08 (80 fold) 0.001 - none

Pb 0.15 (15 fold) 0.01 - none mugil cephalus

Fe 30 (30 fold) 0.3 – 1.0 (Flathead gray Mullet)
Mercury accumulation in different tissues of fish
from Ulhas River Estuary and Thane Creek
Report (2009)

Report (2013)

Levels of mercury (Hg) from

different locations in Mumbai

Levels of Hg in seafood from Versova dock

Permissible range (FDA, 2003),

0.004 – 0.16 μg/g by wet weight of Hg.
Acceptable range (EU, 2008),
Menon, J. S. Indian J. Mar. Sci. 2013, 42, 812; Report of Centre for Science and Environment 0.5 μg/g by wet weight of Hg.
(CSE), 2012; Deshpande. A et al. Environ Monit assess 2009, 159, 493.
 Mercury Toxicity in Mining Areas

Control Site Paddy (CS-P)

Low soil Hg concentration
THg: 0.29±0.023 mg/kg

Rice with high MeHg levels indeed was the main route of MeHg exposure for the
local residents in the Hg mining area.
artisanal site paddy (AS-P)
High Hg soil concentration
THg: 11±3.4 mg/kg
Small industrial area:
Toxic mg/L Recommended
Metals (WHO)
As 0.04 0.01 - 0.05

Cd 0.06 0.03 - none

Hg 0.08 (80 fold) 0.001 - none

Pb 0.15 (15 fold) 0.01 - none

Rice grown at abandoned Hg mining areas contained levels >100 μg/
kg in the edible portion, which is 10 - 100 fold higher than that of other Fe 30 (30 fold) 0.3 – 1.0
locally grown crop plants, such as corn, rape, tobacco, and cabbage.
 Thimerosal: An Antiseptic and Antimicrobial Agent
❑ Thimerosal – commonly used as a preservative in vaccines and medicines (8 - 50 µg per dose).

❖ 18 months of age, a child undergoing a routine

schedule of immunizations would receive a
cumulative dose of 200 µg of mercury.

Thimerosal
(developed in 1927 and marketed
by Eli Lilly and company)
❑ Flu vaccines: commonly used as a preservative in multiuse vials of
Thimerosal exposure has been directly linked to, vaccines including “influenza vaccines”
❑ Fetal/infant death and birth defects. ❑ Newborns in developing countries are still commonly exposed to
❑ Neurodevelopmental disorders. high levels of EtHg+ during the schedule of immunization.

Autism Spectrum Disorders (ASD): Year 2000 2004 2008 2019

Identified 1 in 150 1 in 125 1 in 88 1 in 59

~ 10 million
prevalence of ASD
children likely to suffer from
autism in India

~ 1 in 100 ~ 1 in 8
children under age 10 children has at least one
has autism in India neurodevelopment ✓ ASD- Neurodevelopmental disorders - impaired
condition in India communication and social interactions.

Source: https://www.cdc.gov/flu/prevent/thimerosal.htm; https://www.spectrumnews.org/news/study-quantifies-prevalence-autism-india/

Arora N.K. et al. PLOS Med. 2018, 15, e1002615.
 Concern: Thimerosal and Vaccines

Mark R. Geier (Father) / David A Geier (Son)

11
 Methylmercury Poisoning
❖ 1956, Minamata city, Japan: The catastrophic epidemics from
environmental MeHg+ contamination - Minamata disease - consumption
of fish from water polluted with Hg/MeHg+ from local industrial discharge.
❖ Subsequently, exposure of MeHg+ was documented in Sweden (1966),
Iraq, Guatemala, Pakistan, and Ghana.

According to the Global Emissions Interpretation

Centre (CGEIC), - India is one of the world’s
mercury hotspots - with mercury being released into
the air uniformly at a rate of 0.1– 2 tonnes per year. Mercury in freshwater fish of Ganges river (WB)

Local Total Hg MeHg

name (μg Hg/g (μg Hg/g
of wet wt) of wet wt)

Rohu 0.18±0.04 0.12±0.03

Catla 0.43±0.15 0.32±0.11

Chital 0.45±0.33 0.25±0.18

Boal 1.48±1.01 0.93±0.61

Vetki 0.35±0.02 0.23±0.01

All contain certain amount of MeHg and some of them contain

above the threatening level given by Prevention of Food
Adulteration (PFA), 0.25 μg/g by wet weight.
Moumita et al. Environ Monit Assess. 2011; DOI 10.1007/s10661-011-2193-5
 Toxicological and Pharmacokinetic Properties of MeHg+ species
Endogenous thiols: Structural Similarity:
MeHgOH, MeHgCl, MeHgI,
MeHgCys, MeHgSG

Stomach

pH ~ 4
MeHgCys Methionine
Small
MeHgCl Dolphin Seals MeHgCys
intestine
Liver

pH ~ 8 MeHgCys – 50% more Hg in brain

MeHgCys
MeHgOH MeHgCys – 250% more Hg in liver
Brain
LAT1

Neurotoxic
MeHgCys Methionine

ACS Chem. Neurosci. 2010, 1, 810; Metallomics 2011, 3, 719; Sci. Rep. 2016, 6, 34361.

Science 2003, 301, 1203; ACS Chem. Biol. 2012, 7, 411.

Science 2004, 303, 763
 Autism Spectrum Disorder (ASD)

❖ Autism is a neurodevelopmental disorder characterized by

impaired social interaction, verbal and non-verbal
communication, and restricted and repetitive behavior.
“Kodaikanal” Story
Helen Margaret, now 39, recalled in a tremulous voice her
10 million days as a worker at the defunct thermometer factory of
Children likely to suffer Hindustan Unilever in Kodaikanal. “In the three years from
1996 when I worked there, I did not know the hazards of
from autism in India mercury. We used to play with the silvery liquid, often
throwing it at each other,” she recollects.
Playing with mercury, recognized as one among top ten
chemicals of major public health concern, came with a price,
she says. Her second son Nitesh Kumar was born with
~ 1 in 66 ~ 1 in 68 mental disability in 2000.
Children between ages children were
2 and 9 are affected in identified with autism
spectrum disorder
-- The Hindu, August 16, 2015
India
(ASD)
Year 2000 2004 2008 2010

Identified 1 in 150 1 in 125 1 in 88 1 in 68

Prevalence of
ASD
Structures of Amino Acids

Look at the end of this presentation

15
 Treatment for Methylmercury poisoning
➢ GSH - plays a very important role in the biliary secretion of several metals, including mercury, copper, silver,
zinc, and chromium.

With increasing the hepatic GSH level, it has been shown to

increase the biliary secretion of methylmercury. Whereas, on the
contrary, the agents that deplete the hepatic content of GSH inhibit
the biliary secretion of methylmercury and simultaneously
decrease the GSH content of bile.
GSH

❑ British anti-lewisite (BAL; 2,3-dimercapto-1-propanol) is historically the best known drug studied in toxicology
research and It has been used since 1951 as a chelating agent for heavy metals such as mercury, lead, and
arsenic.

❑ However, due to severe side effects and low therapeutic index it has been discontinued in chelation therapy.
Currently, two other analog of BAL as sodium 2,3-dimercaptopropanesulfate (DMPS) and meso-2,3-
dimercaptosuccinic acid (DMSA) are widely used as drugs for the treatment of mercury (Hg) poisoning
Ballatori, N. and Clarkson, T. W., Am J Physiol. 1983, 244, 4, 435.
Ballatori, N. and Clarkson, T. W., Biochem Pharmacol. 1984, 33, 7, 1093.
Ballatori, N. and Clarkson, T. W., Toxicol. Sci. 1985, 5, 816.
Holmgren et al. FASEB J. 2011, 25, 370.
 Detoxification of Organomercurials by Bacteria
❖ Several microorganisms have shown resistance to high concentrations of MeHg+/Hg2+

Bacterial mechanisms for organic and inorganic mercury detoxification:

The bacterial mechanism of mercury resistance includes the

uptake and transport of Hg2+ by the periplasmic protein MerP
and the inner membrane protein MerT. MerE is a membrane
protein that probably acts as a broad mercury transporter
mediating the transport of both methylmercury and Hg2+. The
cytosolic mercuric reductase MerA reduces Hg2+ to less toxic
elemental mercury.

In nature, detoxification of MeHg+ is achieved by the combined action of two mercury resistance bacterial enzymes present in
aquatic sediments, organomercurial lyase (MerB) and mercuric ion reductase (MerA).

Organomercurial Mercuric ion

lyase (MerB) reductase (MerA)
Hg(II) Hg(0)

CH4
Bacteria resistant to methylmercury carry “mer
operon” which code for two enzymes, namely,
MerB and MerA.
 Detoxification of Organomercurials by Bacteria
❖ Several microorganisms have shown resistance to high concentrations of MeHg+/Hg2+

1) In nature, detoxification of MeHg+ is achieved by the combined action of two mercury resistance bacterial enzymes present in
aquatic sediments, organomercurial lyase (MerB) and mercuric ion reductase (MerA).

Organomercurial Mercuric ion

lyase (MerB) reductase (MerA)
Hg(II) Hg(0)

CH4
Bacteria resistant to methylmercury carry “mer
operon” which code for two enzymes, namely,
MerB and MerA.

2) On the other hand, several other sulfate reducing bacteria (SRB) show high resistant to MeHg+/Hg2+ by virtue of
a mechanism different from that of broad-spectrum Hg-resistant bacteria.

Gaseous H2S or volatile organosulfur Sulfate-reducing bacterium

compound, dimethyl sulphide (Me2S) Desulfovibrio desulfuricans

2CH3Hg+ + HS- (CH3Hg)2S + H+

Insoluble
MeHg+/ mineral
(CH3Hg)2S (CH3)2Hg + HgS
Hg2+
(CH3)2Hg + H+ CH3Hg+ + CH4 Metacinnabar (HgS)
 Protolytic Cleavage of Hg−C Bonds of Organomercurials by MerB1
Organomercurial Mercuric ion
lyase (MerB1) reductase (MerA)
Hg(II) Hg(0)

R-H

Organomercurials: MeHgCl, EtHgCl, PhHgOAc,

R = Me, Et, Ph Thimerosal, and p-chloromercuribenzoate.

The carboxyl group (-CO2H) of Asp-99 residue is perfectly positioned

in the catalytic triad of MerB1, that helps to donate the proton required
for the proteolytic cleavage of RHg+ to form RH.
Asp99

Cys96
Cys159

(II)

Catalytic triad of MerB1

R = Me, Et, Ph

Lafrance-Vanasse, J et al. J. Biol. Chem., 2009, 284, 938.

Gregory C. B et al. Biochemistry, 2004, 43, 8333. 19
 Active sites of Organomercurial Lyases (MerB)
Two cysteine residues in the active site are conserved in all known variants of MerB - (total 65 complete protein sequences of
organomercurials lyases are identified as of January 2010, UniProtKB database).

Catalytic triad Ser105 Catalytic triad

of MerB1 of MerB2
Aspartic acid is conserved in all but four of the
Asp99
known variants [Bacillus megaterium MerB2,
Bacillus subtillis MerB2, Bacillus sp. (RC607)
MerB2, and Clostridium butyricum MerB2], in which
the Asp99 is replaced with a serine residue (Ser).
Cys96 Cys96
Cys159 Cys159

(In 60 variants of MerB,) (In 4 variants of MerB)

❑ The presence of the serine (Ser) in the active site -- alter both the cleavage activity and the relative substrate specificity.

❑ Bacillus megaterium MerB2 -- has much lower activity and shows a preference for p-chloromercuribenzoate over either
MeHg+ or EtHg+.
Lello et al. “The organomercurial lyase MerB” in Encyclopedia of Inorganic and Bioinorganic Chemistry,
2011, John Wiley & Sons Ltd., New York.
Chien et al. J. Biosci. Bioengin. 2010, 110, 94−98.
Wahba et al. Biochemistry 2016, 55, 1070–1081.

This suggests that Mer2 proteins with a serine in the active site may detoxify organomercurials in different way.

Functional
mimics of MerB

20
21
 Detoxification of Organomercury Compounds by Bacteria
In nature, detoxification of MeHg+ is achieved by the combined action of two mercury resistance
bacterial enzymes present in aquatic sediments, bacterial organomercurial lyase (MerB) and
mercuric ion reductase (MerA).

Organomercurial Mercuric ion

lyase (MerB) reductase (MerA)
[R-Hg(II)]+ Hg(II) Hg(0)

R = Me, Et, Ph
R-H

Organomercury Compounds
 Detoxification of Organomercury Compounds by Bacteria
In nature, detoxification of MeHg+ is achieved by the combined action of two mercury resistance
bacterial enzymes present in aquatic sediments, bacterial organomercurial lyase (MerB) and
mercuric ion reductase (MerA).

Organomercurial Mercuric ion

lyase (MerB) reductase (MerA)
[R-Hg(II)]+ Hg(II) Hg(0)

R = Me, Et, Ph
R-H

Trp95

Asp99
Trp95
Asp99 Cys159

Cys159
Cys96
Cys96
Cys160

His161

His161
Active site
of MerB
 Structural and Functional Mimic of MerB: Demethylation of MeHg+

Trp95 Trp95
Asp99 Asp99

Hg
Cys159 Cys159
+ CH4
Cys96 Cys96
GSH
Cys160 Cys160

His161 His161

Trp95 Trp95
Asp99 Asp99

Hg
Cys159 Cys159
+
Cys96 Cys96
GSH
Cys160 Cys160

His161 His161
25
 Detoxification of Organomercurials by Bacteria
❖ Several microorganisms have shown resistance to high concentrations of MeHg+/Hg2+

1) In nature, detoxification of MeHg+ is achieved by the combined action of two mercury resistance bacterial
enzymes present in aquatic sediments, organomercurial lyase (MerB) and mercuric ion reductase (MerA).

Organomercurial Mercuric ion

lyase (MerB) reductase (MerA)
Hg(II) Hg(0)

R-H

2) On the other hand, several other sulfate reducing bacteria (SRB) show high resistant to MeHg+/Hg2+ by virtue of
a mechanism different from that of broad-spectrum Hg-resistant bacteria.

Sulfate-reducing bacterium
Gaseous H2S or volatile organosulfur Desulfovibrio desulfuricans
compound, dimethyl sulphide (Me2S)

2CH3Hg+ + HS- (CH3Hg)2S + H+

Insoluble
MeHg+/ minerals
(CH3Hg)2S (CH3)2Hg + HgS
Hg2+
(CH3)2Hg + H+ CH3Hg+ + CH4 Metacinnabar (HgS)
Tiemannite (HgSe)
 Detoxification of Organomercurials in Human
❖ Detoxification of Hg (organic or inorganic) is also known to occur in various animals including in human.
❖ Insoluble mercury selenide (HgSe) have been found in different tissues of marine mammals, wild animals and in various organs of
mercury mine workers.
Some marine mammals are known to contain Hg in the liver at
 10000 g/g dry weight. However, no indication of Hg intoxication has
been reported for marine mammals, and thus they seem to have an
ability to tolerate high concentrations of Hg.

The presence of HgSe (tiemannite) in all the tissues (liver, kidney, lung,
spleen, pancreas, muscle and brain) of the striped dolphin was
Striped dolphin Bottlenosed dolphin Seals observed.

❖The presence of HgSe (tiemannite) in all the tissues suggests that Se would be involved in the detoxification
process of Hg in various tissues.

❖ Recently, HgSe was detected in the brains of humans exposed to MeHg+.

X-ray fluorescence images of cerebral cortex (occipital lobe).

The small maps show the individual fluorescence images.

❖ Insoluble HgS(s) and HgSe(s) particles are considered to be less toxic than the soluble MeHg+
species such as MeHgCys.
Korbas et al. ACS Chem. Neurosci. 2010, 1, 810.
Nakazawa et al. Metallomics 2011, 3, 719.
 Detoxification of MeHg+ in Human and Marine Organisms

Dolphin

Hg(SeS) particles
Seals

Brain

Sel P

Se
Se
Se
Bluefin Tuna

❑ 10 selenocysteinyl residues per Sel P

❑ 1,000 units of (Hg-Se) bind to Sel P
❑ Forms water-soluble {(Hg-Se)n}m-Sel P
complex

Selenoneine

Mercury Selenide (HgSe)

zebrafish embryos

ACS Chem. Neurosci. 2010, 1, 810; Metallomics 2011, 3, 719; Sci. Rep. 2016, 6, 34361; Biol Trace Elem Res 2013, 156, 36; Springer
Handbook of Marine Biotechnology 2015, 1059; J. Biol. Chem. 2010, 285, 18134; . Biochem. Biophys. Res. Commun. 1997, 231, 7.
 Detoxification of Organomercurials by intelligent molecules
Angew. Chem., 2015, 127, 9455 – 9459; Angew.
Chem. Int. Ed., 2015, 54, 9323 - 9327.

This work is also highlighted in cover page,

Angew. Chem., 2015, 127(32), 9551; Angew. Chem.
Int. Ed., 2015, 54 (32), 9419.

Intelligent Molecules

Highly Toxic Less Toxic

Volatile/ MeHg+
Soluble R-Hg+

Organomercurials Insoluble
HgS(s)/HgSe(s)
 Detoxification of Organomercurials by intelligent molecules

Highly Toxic Less Toxic

Inorganic
Mercury

MeHg+
Hg2+ R-Hg+
Volatile/
Soluble
Organic Mercury or Insoluble
Organomercurials HgS(s)/HgSe(s)
30
 Mode of Coordination

Through lone pair:

Through double bond:

31
Detoxification of Organomercurials

Concentration: 15 mM
Teperature: 370C

RHgOH

R = Me, Et, Ph, and Ar

NO formation of insoluble
HgS(s) or HgSe(s) particles
 Detoxification of Organomercurials by Intelligent Molecules

Water or
Buffer (pH 7.4) Insoluble HgE(s)
at 37 0C

1 9
11 2 10

11
1/ArHgOH 2/ArHgOH

Degradation of complex 9 & 10

HgS(s) nanoparticles Reaction Time (days) HgSe(s) nanoparticles

Angew. Chem. Int. Ed., 2015, 54, 9323
 Detoxification of Organomercurials by Intelligent Molecules

Water or
Buffer (pH 7.4) Insoluble HgE(s)
at 37 0C

1/ArHgOH 2/ArHgOH
HgS(s) nanoparticles HgSe(s) nanoparticles

EDX analysis of HgS nanoparticles obtained in the reaction of 1 EDX analysis of HgSe nanoparticles obtained in the reaction
with ArHgOH in buffer (pH 7.4). of 2 with ArHgOH in buffer (pH 7.4).
 Role of Counter Anion (X-)

MeHgCys, MeHgCl

George et al. Science 2003, 301, 1203;

Stern et al. Science 2003, 303, 763;
Shade et al. Science 2003, 303, 763.

What about in case of, RHgCl

R = Me, Et, Ph, and Ar

Angew. Chem. Int. Ed., 2015, 54, 9323

 Role of Counter Anion (X-)

What about in case of RHgCl

R = Me, Et, Ph and Ar

Unstable
10
9
14
15

Stable

0 2 4 6 8 10
Reaction Time (days)
 Role of Counter Anion
❖ Interestingly, the decomposition of 1:1 mercury conjugated complexes and the subsequent formation of ketone 11 and the
corresponding HgE nanoparticles were observed under alkaline condition.

15

11

17

Formation of HgE(s) NPs in presence of one equivalent of base such as KOH, NaOMe.

1/EtHgCl/NaOMe
in methanol
Base = KOH or NaOMe
20 nm
20 nm

2/EtHgCl/KOH
(In water/ACN)

HgS NP HgSe NP
 Mechanism of Detoxification of Organomercurials
S Se

O
O

S Crystal Structure of 1 Crystal Structure of 2

C1
O

d (C1• • •O) = 3.858Å

Hg
Se S

C1 C1
O
O

d (C1• • •O) = 3.863Å

(In similar mechanism that sulfate reducing bacteria (SRB) d (C1• • •O) = 3.725Å
is detoxifying MeHg+)
Hg
Se

HRMS m/z (TOF) C1

for [M]+ of C6H9N2O+
Cald.: 125.0709 O
Found: 125.0715

d (C1• • •O) = 3.674Å

 Conclusion
❖ A novel pathway for the detoxification of organomercurials by thiones/selones having N-CH2CH2OH substituent under
physiologically and environmentally relevant conditions has been described.

❖ The reactions produce less toxic insoluble HgS and HgSe

nanoparticles.

❖ The –OH group in these compounds plays a crucial role in

the detoxification process by facilitating the desulfurization
and deselenization processes.

❖ Organomercurials are highly toxic to human both at high levels through poisonings in the environment and at lower levels
through consumption of seafood. Thus, the efficient detoxification of organomercurials is of great importance.

❖ This study may lead to the development of potential compounds to treat patients suffering from organomercury
poisoning.

❖ Such compounds may also be useful in detoxifying other compounds based on toxic heavy metals such as Cu,
Pb, Cd and Sn (**unpublished results**).