The Properties Of Arsenic As A Poison Biology Essay

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According to Newman (2005), arsenic is known as "poison for king" and "king for all poison". It is because arsenic has the all appropriate features such as colourless and odourless to be utilize as poison (Newman 2005). In fact, centuries long, arsenic has been used as homicide poison (Reilly 1980). It is said that, there are possible that Napoleon Bonaparte, who died in year 1821 was poisoned with arsenic. This hypothesis existed after a toxicologist analysed Napoleon's hair sample (Reilly 1980).

In term of the usage, arsenic was long introduced to medicine field. In the 5th century, arsenic was used to heal ulcers (Newman 2005). Arsenic was also one of the ingredients in Fowler solution, which was produced in 1786 to treat all type of diseases from asthma to cancer (Newman 2005). Fowler solution flourish in terms of usage for more than 150 years (Newman 2005) and in 1965, a publication shows that most of the patients treated with this solution for long term have developed skin cancer (Reilly 1980). Until now, arsenic is used as an effective chemotherapy agent. Besides its usage in medical fields, arsenic is also being used as an ingredient on wood preservatives, agriculture chemicals (especially in pesticides and desiccants, non-steel alloy smelting, glass industry and other usages (Codex 2007).

Two main chemical form of arsenic is inorganic arsenic which combines with elements such as oxygen, chlorine and sulphur; and organic arsenic which combines with carbon and hydrogen (Hymer & Caruso 2004). There are few differences in inorganic and organic arsenic form. Table 2.1 shows some of the forms of arsenic.

Table 2.1 Forms of inorganic and organic arsenic

Name

Abbreviation

Chemical Form

Inorganic arsenic, trivalent

Arsenite

As III

As(OH)3

Arsenic (III) oxide

As2O3

Inorganic arsenic, pentavalent

Arsenate

As V

AsO(OH)3

Arsenic (V) oxide

As2O5

Organic arsenic

Monomethylarsinic acid

MMAIII

CH3As(OH)2

Monomethylarsonic acid

MMAV

CH3AsO(OH)2

Dimethlyarsinous acid

DMAIII

(CH3)2AsOH

Dimethlyarsinic acid

DMAV

(CH3)2AsO(OH)

Arsenobetaine

AsB

(CH3)3As+CH2COO¯

Arsenocholine

AsC

(CH3)3As +CH2COO¯

Trimethylarsine oxide

TMAO

(CH3)3AsO

Tetramethylarsonium

ion

Me4As+ or TETRA

(CH3)4As+

Arsenic-containing ribosides

Arseno-sugar

Various sugar structure

Source: Hymer & Caruso 2004

The most important commercial form of arsenic is arsenic oxide. It is obtained as by-product in copper production. This is because oxide exists naturally in arsenide metal and arsenic sulphite that is foamed and oxidised in smelting process (Lao et al. 1974). Important factor in distribution and transportation of arsenic in the environment depends on the vapour pressure at ambient temperature produced in this process (Lao et al. 1974).

According to ASTDR (2007), inorganic arsenic form is more dangerous than organic arsenic. This fact is always confirmed dose-response effect study. One study on dose-response effect shows the ingestion of inorganic arsenic causes respiration tissue damage in liver and kidney of a rat (WHO 1981b). Further researches provide evidence that suggest the inorganic arsenic in trivalent form is more toxic than pentavalent inorganic arsenic (Hymer & Caruso 2004). Arsenic (III) oxide fatal dose for human through ingestion includes from 1 to 2.5 mg As/kg body weight (WHO 1981b). Since the ingestion quantity is low to cause fatal, thus, a conclusion can be drawn that trivalent arsenic is pose more toxic that pentavalent arsenic. This information is strengthen by mutagenicity study, where a study carried out on human chromosome by in vivo and in vitro (WHO 1981b). There is an increase in chromosome abrasion when it is exposed to arsenic (III) oxide (WHO 1981b). However, no adverse effect was observed in rat that was ingested with food containing "seafood arsenic", also known as organic arsenic (WHO 1981b). The rat on this study was fed with prawn that contained 14 mg As/kg for 12 months. Monomethylarsinic acid, organic arsenic, found to go through placenta of the studied rat (WHO 1981b). This methyl form arsenic is presumed as harmless as its level of acute toxicity is considerably low (Codex 2007).

When arsenic is ingestion, inorganic arsenic will go through a methylation process, which is said to be an important detoxification process in human body (Hymer & Caruso 2004) and it is also a major metabolic pathway for inorganic arsenic (Sirot 2008). In this process, arsenate, arsenic (V) is reduced to arsenite, arsenic (III) form, followed by addition of methyl group to form monomethylarsonic acid and dimethlyarsinic acid (MMA and DMA) which will be excreted through renal pathway (Sirot 2008). Minimal excretion is observed in faeces, nails and hair (Sirot 2008).

2.2 Arsenic contamination source and cycle in aquatic environment

In this 21st century, arsenic is an element that is widely distributed in the surface of earth. Arsenic existence in the environment is due to natural causes such as rocks, sediments and human activity for example coal burning, copper smelting and mineral ore processing (FSA 2005; Hymer & Caruso 2004; Reilly 1980). Volcanic activity has become a source of arsenic distribution to the environment through air. In addition of geological source, usage of excessive pesticides (which contains arsenic component) can contribute to high level of arsenic in the soil (NZFSA 2005). Other sources of arsenic contamination are commercial usage of fungicides, herbicides, insectides and wood preservatives (Reilly 1980).

Arsenic cannot be destroyed in the environment, it can only change form. When arsenic being released into the environment, it settles down to earth or transport out into the space via rain (Reilly 1980). Most of the arsenic compound is water soluble and aquatic organisms have the tendency to absorb arsenic from its habitat (Reilly 1980). Since the metabolic process occurs naturally in the biosphere, arsenic (inorganic and organic) is easily found in food chain (Codex 2007). Aquatic organism bio accumulates arsenic from its food. This bioaccumulation demonstrates the metabolic transformation chain when arsenic enters the aquatic organism's food chain (Borak & Hosgood 2007). Besides that, wood burning also releases arsenic especially inorganic pentavalent, to the atmosphere, due to its present in wood preservatives (WHO 1981b).

In general, arsenic is circulated into the environment through water (WHO 1981b). Arsenite is more predominate in oxygenated aquatic area, while arsenate is widely distributed in deep waters (WHO 1981b). Aquatic area such as sea can be divided into 5 zones, which are epipelagic, mesopelagic, bathypelagic, abbyssopelagic and hadopelagic as shown in figure 2.1.

Epipelagic or suface zone is the area where the photosynthesis occurs because of sufficient sunlight and oxygen supply. In this zone, main domain of aquatic organism are tuna fish, shark and jelly fish

At mesopelagic zone, sunlight penetration is not sufficient for photosynthesis process. Aquatic organism that lives in this area usually has efficient gills and limited movement such as squid, sword fish and woffish.

Bathypelagic zone ia dark zone. There is no vegetation here and the aquatic animals that live in this zone are giant octopus and whale.

No light can penetrate abbyssopelagic zone and this area is known as bottomless zone.

Hadopelagic zone is assumed to have depth of 6000 m. No species is believed to live in this area.

Figure 2.1: Zones in aquatic area

Inorganic methylation process often related with biological activities in the water (WHO 1981b). Some of the aquatic organism is said have the capabilities to transform inorganic arsenic to complex organic arsenic compound such as arsenobetaine and arsenocholine (WHO 1981b). Arsenic biomethylation started to be known when arsine was produced from Scopulariopsis brevicaulis fungi culture. Arsenate methylation mechanism is as shown in the pathway below:

2e CH3+ 2e CH3+

AsVO43- --->ÄsIIIO33- --->CH3AsVO32- --->CH3ÄsIIIO22- --->

--O2- --O2-

2e CH3+ 2e

(CH3)2AsVO2- --->(CH3)2ÄsIIIO- --->(CH3)3AsVO --->(CH3)3ÄsIII

--O2- --O2-

Figure 2.2 : Arsenic methylation process

Mechanism in figure 2.2, shows AsV is reduced to AsIII before methylation. The discovery of methylarsenic acid in sea water and fresh water proves that arsenic compound have undergone other processes besides reduction and oxidation (WHO 1981b). Methylation process is related with plankton, whether in sea or fresh water (WHO 1981b). This is because plankton or algae can easily uptake arsenic in arsenate form and during detoxification process, arsenate is reduced and methylated which produce arsenic sugar and DMA and other methylated arsenic (Borak & Hosgood 2007). Bioaccumulation occurs in food chain, when lower level organism which contains arsenic is consumed by higher level animal in food chain. Figure 2.3 shows arsenic distribution and biomethylation in the environment.

Figure 2.3 : Arsenic distribution and biomethylation in the environment

2.2 Contamination level of arsenic in fish

In Malaysia, fish is largely consumed because of its abundances, inexpensive and healthy protein source (Hajeb et al. 2009). Food consumption statistics of Malaysia 2002/2003 for adult population, aged 18 to 59 years indicates prevalence percentage for marine fish intake is more than 90 % with estimated intake of 60.67 g/day as shown in table 2.2. Marine fish shows highest level of estimated intake(g/day) and prevalence(%) as compared of other type of seafoods.

Table 2.2 : Food consumption statistic for fish and fish products in Malaysia

Type of food

Estimate intake (g/day)

Prevalence (%)

Marine fish

60.67

94.66

Fresh water fish

12.11

53.13

Anchovies

4.90

85.64

Canned fish

4.30

75.56

Cockles

4.70

58.81

Fresh prawn

3.00

67.51

Fresh squid

12.94

72.45

Dried squid

5.65

25.95

Crab

3.94

62.79

Dried fish

2.93

67.77

Fish ball/cake

10.70

63.76

Keropok Lekor

4.17

55.10

Source : Ministry of Health 2006

Though consuming fish promotes good health benefits, there were numerous reports on contamination of fish by heavy metals in the environment, particularly, arsenic (Hajeb et al. 2009). Malaysia standards have set maximum permitted proportion (MPP) for arsenic for fish and fish product as 1 mg/kg (Malaysia Food Act and Regulation 1985.).

Two food safety activities were conducted by Ministry of Health, which clearly shows the contamination level of arsenic in Malaysia. First activity is Total Diet Study (TDS), conducted in 2006 (KKM 2006). It was found in this study, that 80% of fish and fish product analysed contains total arsenic exceeding maximum permitted proportion for arsenic for fish and fish product stated in Schedule Fourteen, Regulation 38, Food Regulation 1985, which is 1 mg/kg (KKM 2006).

In continuation of the above mentioned study, in 2007, a national monitoring program was conducted to determine the contamination level of arsenic in fish and fish product that is retail around Federal States, Selangor, Negeri Sembilan and Melaka. The type of sample studied are sea fish (ikan kerisi, ikan kembung, ikan sardin, ikan susu, ikan bawal putih dan ikan bawal hitam), fresh water fish (ikan talapia merah, ikan tilapia hitam, ikan keli, ikan lampan jawa, ikan patin, ikan siakap), squid, crustaceans (crabs and prawns) and seafood products (dried shrimps and anchovies). From 96 sample analysed, it was found that almost 20 % of the samples violate arsenic contamination level as stated in Schedule Fourteen, Regulation 38, Food Regulation 1985. However, this level refers to total arsenic. At the present, there is no data obtained for inorganic arsenic or arsenic species contamination in fish in Malaysia.

Even though, arsenic level is high in aquatic organism, according to Food Standard Agency, who conducted a survey on total arsenic and inorganic arsenic in fish and fish product in 2005 shows that about 1-3% of total arsenic is inorganic arsenic (FSA 2005). Most of the arsenic form found in the fish sample are arsenobetaine and arsenocholine, which sometimes refered as "fish arsenic".

In the year of 2003, dietary exposure study for total arsenic in seafood was conducted in Europe. The contribution of arsenic in seafood exceeds 50% (Sirot et al. 2008). Where, organic arsenic is dominant to the contribution especially AsB, AsC, MMA dan DMA, as compared to inorganic arsenic (Sirot et al. 2008). According to Sirot et al. (2008), scientific research reported the percentage of inorganic arsenic in seafood is between 0.4 to 5.3% and the rest is in organic form especially AsB. In the 20th Total Diet Study conducted in Australia, the inorganic arsenic level in fish, prawn and canned tuna fish was found to be lower than limit of reporting that 0.05 mg/kg.

Even though, some of the study shows that inorganic arsenic level is low in aquatic organism, research on arsenic species is constantly conducted by scientist. This is because, in methylation process, which is a detoxicification mechanism is a metabolism chain, arsenic produce intermediates in the form of active metabolites such free radicals which releases oxygen, which can be a catalyst for toxic effect (Gong et al. 2002). This reaction is observed in toxicology study conducted on rats, where carcinogen metabolite in rat erythrocyte was found as a product of arsenic metabolic activity (Borak & Hosgood 2007). According to Gong et al. (2002), this metabolite is more toxic than inorganic arsenic.

2.4 Health affect

International Agency for Research on Cancer (IARC) has classified arsenic as carcinogenic agent for human (category 1) (Sirot 2008). This is because arsenic has the capability to exploit certain cell pathway, bind with protein and distrupt molecule system (Newman 2005). In general, 75 % of arsenic is excreted through kidney and little percentage through faeces (WHO 1981b). Arsenic excretion rate in human is slow as compared with animals and is being stored in certain organs (WHO 1981b). A study conducted on rat and human confirms that inorganic arsenic can be transferred from mother to fetus through placenta (WHO 1981b). DMA also have the capability to pass through the placenta barrier, this has been proved by comparison test of mother and fetus blood (WHO 1981b).

Both pentavalent and trivalent form of arsenic is easily absorbed in digestion system (Reilly 1980). However, trivalent arsenic is considered more toxic than pentavalent arsenic (Reilly 1980). When arsenic is being absorbed, it spreads to organs and tissue in the form of complex protein, probably with α-globulin (Reilly 1980). It binds with sulphyryl group in protein and inhibits enzymatic activity, especially those related to metabolism and mitochondria respiration (WHO 1981b). Below is the pathway of how arsenic compound binds with sulphyryl group enzyme.

SR'

/

R - As = O + 2R'SH <==> R - As + H2O

\

SR'

After 24 hours exposure, arsenic concentration in most organs will be reduced, however, accumulation in skin, nails and hair may increase after few days of digestion (Reilly 1980). The most sensitive indicator is skin peeling for systematic toxicity due to chronic arsenic exposure. Mainly, skin cancer cases reported through medical treatment exposure is due to trivalent arsenic compound (WHO 1981b).

Arsenic have no biological function and prolong exposure may be hazardness to health (FSA 2005). Prolong exposure or intake of arsenic will cause some adverse effect to some organs (Newman 2005). Classical signs of acute arsenic poisoning are nausea, vomit, diarrhoea, low blood pressure and finally death (Newman 2005). Chronic arsenic poisoning symptoms are lost appetite, weight lost, distrupt to digestion system, eye disorder and others diseases. Exposure to inorganic arsenic can cause various effects to health such as colon and stomach disease, reduction of red and white blood cell production, changes to skin which is related to skin that can lead to skin cancer and lung disease (Hung et al. 2004; Reilly 1980).

Arsenic in the form of inorganic can cause DNA distruption and is known to cause cancer such as skin, lungs, liver and spleen (FSA 2005; Hung et al. 2004). This is because DNA contains protein with sulphyryl bond and arsenic have the potential to bind with this bond, subsequently, disturpt DNA mechanism.

There are few incident related to arsenic poisoning. In the late 19th century, thousands of United Kingdom citizens experience arsenic poisoning caused by starch that is hydrolysed by arsenic tainted acid and used in beer fermentation process (Reilly 1980). Another serious case occurs in 1955 in Japan, involves more than 12 000 babies given milk that was contaiminated with arsenic trioxide. This compound was used unintentionally in sodium phosphate to stabilize the milk powder (Reilly 1980). A profound case occurred in Bangladesh around 1970s, where a massive arsenic poisnoning took place when well water was contaminated with arsenic.

Total Arsenic Analysis

Mineralization procedure in biological sample is vital in obtaining desirables results. Various sample preparation procedures can be employed such as dry ashing, wet digestion and microwave digestion. In dry ashing procedure, high temperature is applied to the sample and digested for long hours. In some cases, the temperature can be rised up to 500°C and digested for more that 16 hours (Demirel et al. 2008). Wet digestion is basically an open digestion system, which requires high volume of acid mixture with digestion time of 4 to 6 hours. Microwave digestion aids digestion with microwave radiation usually provides a closed digestion system. This procedure requires short time and small volume of acid for digestion.

According to Soylak et al. (2004), wet and dry ashing procedures are time-consuming as compared to microwave digestion. Microwave digestion has the features for fast and efficient sample decomposition method for trace metals determination (Tuzen 2002). Study conducted by Soylak et al. (2004) in comparing microwave, dry and wet digestion, proves the recoveries of the trace metals using microwave digestion were in the range of 95-103% with standards deviations less than 10%. The author concluded that dry and wet digestion procedures are slow, complicated and less efficient than microwave digestion.

In another study by Reyes et al (2009), microwave digestion was employed to digest DOLT-3 (Dogfish Liver) and BCR-627 (Tuna Fish Tissue) with recovery of total arsenic was 98% and 100% respectively. According to Demirel et al. (2008), microwave digestion procedure was preferred to dry ashing and wet digestion procedures because it provides more accurate results, shorter digestion time and achieve better recovery in the samples. Microwave digestion method is suitable for digestion of volatile element such as arsenic (Demirel et al. 2008), because it provides closed digestion system which minimize lost as compared to dry or wet digestion.

There are many spectrometry instrumentation techniques that can be employed to detect total arsenic in fish. Some of the techniques are hydride generation-atomic absorption spectrometry (HG-AAS), graphite furnace atomic absorption spectrometry (GFAAS), inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS) (EFSA 2009). ICP-MS is the best technique because the analytical performance is stable and robust, with low LoQ and wide dynamic linear range (EFSA 2009).

2.6 Speciation Technique

International Union for Pure and Applied Chemistry (IUPAC) suggested definition for speciation analysis as activity for determining and measuring quantity of one or more individu chemical species in sample. Elemental speciation concept in differentiating total element fraction, exist gradually late 1950s. Only in 1980s, analytical instrumentation achieved required determination rate to compute trace element fraction in the environment and biological sample.

Speciation technique requires instrumentation with separation device and detection instrumentation (Gong et al. 2002). Coupling of this analytical technique is needed to achieve sensitivity and selectivity in individual arsenic species determination (Gong et al. 2002). Usually, speciation technique involves coupling of separation chromatographic and determination spectrometry (Gong et al. 2002).

High performance liquid chromatography (HPLC) often used as separation tool in arsenic speciation (Gong et al. 2002). While, instrument such as gas chromatography, supercritical fluid chromatography and capillary electrophoresis also used in arsenic speciation, though, the usage is not wide. Separation modes utilized in HPLC are ion-pairing, ion-exchange and size exclusion (Gong et al. 2002). In arsenic species analisis, ion-exchange is commonly used, followed by ion-pairing (Hymer & Caruso 2004).

In the basic design of chromatography, ion-exchange is employed to separate ion and easily ionized compound (Hymer & Caruso 2004). It utilizes equilibrium exchange mechanism between stationary phase which contains surface ion and ion with reverse charge mobile phase (Hymer & Caruso 2004). Ion-exchange chromatography can applied in two separation mode, that is, anion kation exchange (Hymer & Caruso 2004). Ionic strength in solution, mobile phase pH, flow rate, concentration and ionic strength of buffer solution and temperature influences the separation and retention time in ion-exchange HPLC (Hymer & Caruso 2004). Anion exchange chromatography typically use for separation of arsenite arsenit(AsIII), arsenate (AsV), monomethylarsonic(MMAV) dan dimethlyarsenic (DMAV), where else, kation exchange is emplyed in separation of AsB, AsC and trimethylarsine oxide (TMAO) (Hymer & Caruso 2004).

Ion exchange chromatography system utilizes isocratic and gradient system in separation of arsenic compound (Hymer & Caruso 2004). Most study is base on isocratic system, though; gradient separation allows a better resolution time and peak performance (Hymer & Caruso 2004). According to Reuter et al. (2003), gradient system is employed to determine AsB, AsIII, DMA, MMA dan Asv; it require time for column re-equilibration; long analysis time however, gradient system produce better resolution time as shown in figure 2.4.

Figure 2.4 : Chromatograhy of five arsenic species at five difference concentration using gradient system

Source : Reuter et al.(2003)

Study conducted by Cao et al. (2009) and Hymer et al. (2001) explains chromatography technique of anion exchange with gradient system could separate both anion and kation arsenic. This is achieved by modifying flow rate and buffer solution concentration that is ammonium carbonate (Hymer et al. 2001). Flow rate at 0.8 , 1.0 and 1,5 with concentration range of ammonium carbonate(buffer) at 5.00, 10.0, 15.0, 20.0 dan 30.0 mmol-1 is used to evaluate the separation between AsB, AsC, AsIII, Asv , DMA dan MMA (Hymer et al. 2001).

Isocratic system has advantage in term of time, where total run time is less than gradient system (Reuter et al. 2003). Even though, this system is time saving, resolution time is less satisfactory as compared to gradient system (Reuter et al. 2003) a can be observed in figure 2.5 that shows chromatography of arsenic species at single concentration and blank using isocratic system. The resolution time diference between gradient system and isocratic system is predominantly observed in AsB AsIII, DMA and Asv.

Figure 2.5 : Chromatography of arsenic species at single concentration and blank using isocratic system

Source: Reuter et al. (2003)

Buffer system that is normally used in ion exchange chromatography (to separate polar arsenic compound), including phosphate, carbonate, trimethylammonia hydroxide and formate (Hymer & Caruso 2004). Potassium phosphate is not suitable to be employed as buffer because it leave residue on skimmer and sampler cone in ICP-MS, where else carbonate does not leave much trace (Hymer & Caruso 2004).

ICP-MS has become an efficient detection technique in arsenic speciation (Gong et al. 2002). It is ultra sensitive and has the capability to determine various elements (Hymer & Caruso 2004). This ability allows simultaneous determination of various elements such as arsenic species (Hymer & Caruso 2004). Arsenic is prone to spectral disturbance in certain conditions during detection in ICP-MS (Hymer & Caruso 2004). This is indirectly related to arsenic of being monoisotopic with mass of 75 amu (Hymer & Caruso 2004). Argon gas from plasma and chlorine from sample matric have the ability to combine to form argon chloride, 40Ar35Cl, with similiar mass-to-charge ratio to arsenic, which is 75 amu (Hymer & Caruso 2004). In monitoring the signal at 75 m/z, the source of signal is from 2 origins which are arsenic compound and argon chloride disturbance (Hymer & Caruso 2004). Introduction to reaction cell or collision cell at mass spectrometry quadrapole will resolve this polyatomic reaction (Hymer & Caruso 2004). Reuter et al. (2003) suggest the dynamic reaction cell (DRC) technology can "transfer" arsenic ion from Ar+Cl- by reaction of arsenic ion with oxygen to form arsenic oxide, 75As16O+, which can be determine at 91 m/z.

Figure 2.6 : HPLC-ICP-MS coupling operation scheme

HPLC-ICP-MS coupling mechanism is illustrated in figure 2.6. The components in the scheme are HPLC with mobile phase to carry the sample to column with the aid of pump (Reuter et al. 2003). Sampel that pass through the column will proceed to ICP plasma in aerosol form (Hymer & Caruso 2004). After the ionization process in plasma, arsenic ions will continue to mass spectrometry (Hymer & Caruso 2004). Then, data can be interpretated with specific software employed accordingly to the instrument model.

According to Hymer & Caruso (2004), there are 2 approaches in sample extraction for arsenic speciation that is traditional technique and enhanced technique. Traditional techniques are:

Liquid-liquid or solid-liquid extraction, also know as sonification technique - this technique utilizes solvent such as methanol, acetone and chloroform.

Solid phase extraction(SPE)

Solid-phase microextraction

Enhance technique consist of:

Pressurized liquid extraction(PLE) or also known as automatic extraction using Automated Solvent Extraction (SPE)

Microwave-assisted extraction (MAE) and Supercritical fluid extraction (SFE)

The summary of extraction method from other studies in table 2.2 shows that in traditional extraction, the sonification technique exhibit highest recovery percentage (100.9%) as compared to other method. While, recoveray rate for MAE extraction method is too wide.

Table 2.3: Summary of extraction method from other studies

Sample

Extraction method

Arsenic species

Recovery range

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