References

Here are some videos about the tragic incident

Disclaimer: disturbing content (view at your own risk!)




"Virtually overnight, my father lost his ability to keep his balance, or to stay afloat in the water once he had fallen off the boat. He could not put on his sandals, walk properly, or understand what others were saying to him. His condition quickly degenerated, and he was hosptialized on the fourth day. There, even tied to his bed with bandages, he "craze-danced," said words that were not words"
                                                                                                                     - Tsuginori Hamamoto

So, what's in the news lately...?

Here are some recent news about the Minamata incident and also on mercury emissions

Targeting the use of mercury

"More than 140 countries negotiating in Geneva on Jan. 19 agreed on a treaty to limit emissions and releases of mercury, which can cause serious environmental pollution and health hazards. This treaty represents the first step in global efforts to prevent environmental and health damage from mercury." Read more about it here

HPLC-ICP-MS Method

Aim: Using HPLC-ICP-MS coupling for mercury speciation that determined it was mercury poisoning.


Sample preparation:


For fish

• Collect 10-20  grams of the edible portion, 
• Place it in polyethylene bags, 
• Store it in a freezer. 
• When collecting fish samples, record the sampling date, location, species, and ages. Also measure the weight and length and the like. 

For shellfish,

• Divide the muscle, digestive tract contents, and adductor muscle 
• Place the portions in polyethylene bags
• Store in a freezer. 
• Since particles of bottom sediment are often contained in the digestive tracts of shellfish, remove these particles before storage. 

• The biological tissue samples are then extracted into a mixture of L-cysteine and 2-mercaptoethanol by microwave digestion. 
• Mercury compounds are separated on a C8-column using the same mixture of L-cysteine and 2-mercaptoethanol.

The HPLC methodology allowed for the separation of MeHg+and inorganic Hg2+ standards.

Quantification of MeHg+and Hg2+species in several samples was achieved using the external calibration curves

Why do we use HPLC-ICP-MS?

Most common methods to quantitate Methylmercury

1) GC-ECD (Gas-Chromatography coupled to Electron Capture Detector)
2) HPLC coupled to Hg detector (fluorescence, photometry, cold vapour atomic absorption and fluoresence spectroscopy).

In this case, why was HPLC-ICP-MS (HPLC coupled to inductively-coupled plasma mass spectrometry) used instead of GC-ECD?



Disadvantages of GC-ECD

1) High limit of detection of 10ng/L despite its long and complicated procedure

2) Low selectivity; Not specific to any element

3) Subjected to interferences, which can result in false positive results or low recoveries

Advantages of HPLC-ICP-MS

1) Compound Independent Calibration

Argon plasma is able to decompose and ionize an element regardless of its chemical structure. This independence of signal to original structure is known as compound independence, and allows the construction of a calibration graph based on the concentration.

2) Low limit of detection

3) Simple sample preparation

4) High sensitivity and selectivity

5) Stationary phase and mobile phase can be adjusted to suit the separation required

6) Sample extracts are directly injected into the system - reduced costs

Speciation of mercury in water at the bottom of Minamata Bay, Japan

Introduction
From the 1930s until the late 1960s, an acetaldehyde-producing factory in Minamata, Japan, has been releasing wastewater contaminated with mercury into the Minamata Bay. The Minamata Bay Pollution Prevention Project (1977-1990) removed sedimentary sludge that had contained high levels of mercury. After the project was over, there were reports and investigations regarding the mercury concentrations in Yatsushira Sea sediments and it was reported that sediments (containing mercury) were being transported from Minamata Bay to Yatsushira Sea. During the re-suspension and transportation of contaminated sediments, some of the mercury present in the sediments may have dissolved into the water.

Investigations on the concentration of  methylmercury in water of the upper and middle layers of Minamata Bay have been conducted. However, as for the layer of water just above the sediment, no previous investigations were done.

In this study, methylmercury levels in the water were determined by the use of HPLC-ICP-MS.



Materials & Apparatus
1) Water samples were taken in at different points as shown in the picture above

Upper layer of the water (clear) was filtered through a 0.45μm Millipore filter
Lower layer of the water (turbid) was centrifuged and the supernatant was filtered through a 0.45μm Millipore filter

2) HPLC-ICP-MS instrument

HPLC conditions
* Column: ZORBAX Eclipse XDB-C18, 2.1mm x 50mm, 5μm
* Mobile Phase: 0.06M ammonium acetate, 5% (v/v) methanol, 0.1% 2-mercaptoethanol (pH 6.8)
* Flow Rate: 0.4mL/min
* Injection volume: 100μL

Pre-conditioning of HPLC column
* HPLC grade methanol was pumped through the column at 0.4mL/min for minimum 2 hours and condition with eluent (at same flow rate) for minumum half an hour
* Without this pre-conditioning, the inorganic mercury will be subjected to contamination in the system leading to poor recovery or peak splitting

3) Reagents & apparatus

All reagents used were of the highest purity available and were of analytical reagent grade
All apparatus were cleaned with detergent, 10% potassium permanganate, 2% hydroxylammonium chloride and water. Glassware were washed and heated before use.

4) Standards

A series of calibration standards (10ng/L to 100μg/L) were prepared by dissolving the appropriate amount of HgCl2 in high purity water  (External Standard)
Bismuth (1.0μg/L) was added to the methanol eluent as an Internal Standard

Methods
1) Both the samples and external standard are injected into the HPLC
2) Obtain peak areas of sample and standards
3) Construct a calibration graph from the peak areas of external standards
4) Determine concentration of samples from the calibration graph

Methods to quantitate mercury in different samples

Here is a list of methods that can be used to identify mercury in different samples.

Natural waters

Chromatographic methods: 

1. Gas chromatography Volatile Hg species are stripped from the sample solution and pre-concentrated, prior to separation. The trapped mercury derivatives are then released thermally and transferred quantitatively to the GC for separation.

2. High performance liquid chromatographic (HPLC) separation 
A reversed phase column based on alkyl-silica and a mobile phase containing an organic modifier, together with a chelating or ion pair reagent (and in some cases a pH buffer) are usually used. 

3. Capillary electrophoresis
Uses charge and frictional forces for the separation of Hg species.


Air

1. Selective adsorption methods

2. Chromatographic methods


Biological samples

1. Gas chromatography

2. HPLC

Introduction to Case Study: Story behind the Mercury Poisoning in Minamata

 Lets go back in time to understand what had happened

1950s

It started in mid 1950s, in Minamata, Japan, when people began to notice strange occurrences in cats. The cats were making strange movements, and falling into the sea. People thought they were committing suicide. In the town itself, the people started noticing unsettling symptoms such as shaking, nervousness and seemed to be going insane as they shouted uncontrollably.

1952

 Around 1952, the production of acetaldehyde boomed and so did the local economy. About the same time, fish began to float in Minamata Bay. Chisso, as it had since 1925, continued to pay indemnity to local fishermen for possible damage to their fishing waters.

1956

In 1956, researchers teamed the disease Minamata disease and they continued studies to find out what caused it. They knew that it affected the nervous system and that the people had a common diet of fish. From the clue that the fish were being poisoned, the reseachers finally determined that the disease was caused by consuming heavy metal poisoned fish and shell fish.

1956 July

By the end of 1956, that was when there was evidence gathered by Kumamoto University researchers that found that organic mercury was the cause of Minamata disease and that the Chisso plant was responsible for dumping the plant's acetaldehye waste water. This caused the symptoms.

Follow up

The Chisso plant continued to refute the information and any link of its mercury waste to the illness. It was later discovered that Chisso Corporation had dumped an estimated 27 tons of mercury compounds into Minamata Bay.

As the mercury dumping continued, babies were born to poisoned mothers. The children were born with severe deformities, including gnarled limbs, mental retardation, deafness, and blindness.

What is Mercury?

Mercury is this shiny, silvery looking liquid that actually looks like it could be something out of this world.

But what is it really?




Mercury is a naturally occurring metal that exists in several forms such as elemental or
metallic mercury, inorganic or organic mercury compounds.

It is the only metal that is liquid at standard room temperature and pressure.

Mercury can bioaccumulate in the environment and pass through the food chain. It also has the ability to penetrate the blood-brain barrier and affect the central nervous system.




Elemental or metallic mercury 
Shiny, silver-white metal that is liquid at room
temperature. It can evaporate to become an invisible, odorless toxic 
vapor.  

Inorganic mercury compounds 
White powder or crystalline which is in mercury salt form. 

Organic mercury compounds 
Methylmercury is the most common organic mercury found in the environment occurs 
when mercury combines with carbon.  Microscopic organisms are responsible for converting  
inorganic mercury into methylmercury