DIETARY RISK EXPOSURE OF HEAVY METAL CONTENT IN COOKED AND RAW

ASIAN CLAMS (CORBICULA FLUMINEA) AMONG FISHERFOLKS ALONGSIDE

LOS BANOS AREA OF LAGUNA LAKE1

Angel Josephine A. Manuel

1
A Research Paper submitted in partial fulfillment of the requirements in HNF 152 (Food and
Nutrition Research) under the supervision of Dr. Wilma A. Hurtada and Prof. Felicito M. Rodriguez 2nd
semester, A.Y. 2016-2017.
 ACKNOWLEDGMENT

This research paper is made possible through the help and support from God and everyone

including my family, friends, professors, colleagues, and my orgmates from Philippine Association

of Nutrition. These people gave me good counsel and supported me throughout the research period.

Without them, this research paper would not be possible. Especially, allow me to dedicate my

acknowledgment of gratitude toward the following important people in the making this study.

First and foremost, I would like to thank Dr. Wilma Hurtada for being the best thesis

professor. She gave encouragement, advice, suggestions and valuable detailed recommendation

for the betterment of my research studies.

Secondly, I would like to thank my thesis professor Professor Felicito M. Rodriguez for

his expertise in the methodology of this study. His advice improved the content of this research

study drastically.

Thirdly, I would like to thank the staff from Laguna State Polytechnic University for

allowing me to utilize their facilities to conduct this study. My gratitude goes to Sir Christian and

Sir Alfon for their assistance during experimentation. I would also like to thank Carmela Jane

Mercado for her advice and assistance all throughout the research period.

Fourth, I would like to thank Gwena Manuel and Anthony Cammayo for the extensive

moral support and wisdom throughout the research process.

Most of all, I would like to thank Our Almighty Father for giving me His graces, blessings,

strengths, peace of mind, good health, and miracles in order to finish my research studies.

2
 ABSTRACT

This study assessed the dietary risk exposure of Los Banos

fisherfolk in relation to the lead and cadmium concentrations

found in Asian Clams (Corbicula fluminea) from Laguna

Lake. Ingestion rate and heavy metal concentrations were

dependent variables obtained via food frequency questionnaire

and wet digestion by nitric-perchloric homogenization before

heavy metal analysis, respectively. Results of the study

revealed that estimated daily intake was lower than the

tolerable daily intake for lead and cadmium. The heavy metal

concentrations between cooked and raw Asian Clams were

compared and were concluded to have lower concentrations

after cooking than analysing the sample raw. It has been

concluded that there are no dietary risks associated with the

consumption of Asian Clams from the lake.

3
 INTRODUCTION

Laguna de Bay is an inland body of water considered to be the largest in the Philippines

and the third largest in Southeast Asia (DENR). The lake is located in an area with an expanding

population as well as a thriving economic development. Through time, it is a valuable source of

raw materials, food, and livelihood for its surrounding civilization.

Through time, wastes from industrial, domestic and agricultural wastes overwhelm the lake

and its underwater ecosystem, including the animals and plants which the locals use on a regular

basis. The accumulation of heavy metals coming from man-made wastes pose a threat and

detriment to the lake’s ecological environment. This will not only cause serious damage to the

aquatic ecosystem but also result to questionable public health security and food safety. For

instance, fishes exposed to polluted water have a tendency to absorb the pollutants directly from

its environment (Molina, 2011). When ingested, they combine with the body’s biomolecules, like

proteins and enzymes to form stable biotoxic compounds, thereby mutilating their structures and

hindering them from the bioreactions of their functions (Duruibe et al, 2007). Aquatic ecosystems

are highly susceptible to heavy metal contamination by biomagnifications over time.

Fish meat has a special nutritive value by being a good source of high quality proteins,

poly-unsaturated fatty accids and various micronutritients. This makes fishes one of the common

food commodities for humans. However, if the aquatic life is contaminated due to

biomagnification of wastes, consumers’ health are threatened. Heavy metals and their compounds

are very hazardous and cancerous for humans. It has also been established that cooking can affect

the mineral content of fish. This is the reason why it is important to determine the concentration

of heavy metals in raw and cooked fish (Diaconescu et al, 2007).

4
Scope and Limitations of the Study

This study will focus on determining the concentration of Cadmium (Cd) and Lead (Pb) in

Asian Clams (Corbicula fluminea) from the Los Banos area of Laguna de Bay. Cooked and raw

samples will be obtained from the flesh of the chosen specie and compared to assess its dietary

risk potential.

Significance of the Study

The utilization of biological organisms (bioindicators) has been proven useful in assessing

the status of pollution in aquatic ecosystems (Gadza³a-Kopciuch et al, 2004). This study aims to

reveal the status of environmental contamination with heavy metals along the Los Banos area of

the Laguna de Bay by determining its accumulation in Asian Clams (Corbicula fluminea).

Moreover, it will be conducted to reveal the difference between the heavy metal (Cadmium,

Chromium, and Lead) content of cooked and raw Asian Clams.

Furthermore, results of this will provide about the health risk of consuming Asian Clams

from this area to the fisherfolk. In this case, this study may help policy makers to formulate

appropriate guidelines and strategies to ensure food safety for consumers.

Objectives of the Study

This research study aims to measure the dietary risk exposure of fisherfolk from Los Banos

to the Cadmium and Lead concentration found in Asian Clams (Corbicula fluminea).

Specifically, this aims to:

5
 1. Estimate the intake of fisherfolk of Los Banos on Asian Clams Corbicula fluminea);

2. Measure the accumulated amount of Cadmium and Lead in Asian Clams from Los

Banos area Laguna Lake;

3. Compare the Cadmium and Lead concentration in Asian Clams from raw and cooked

samples;

4. Assess the estimated daily intake of fisherfolk from Cadmium and Lead found in Asian

Clam using oral reference doses;

5. Determine the non-carcinogenic and carcinogenic potential of ingesting Asian Clams

to the dietary and health risks of exposure to Cadmium and Lead.

Conceptual Framework

Heavy Metal
Concentration

Average Time
Dietary Risk
(for non- from Heavy Ingestion
carcinogenic Metal Rate
potential)
Exposure

Exposure
Frequency
and Duration

Figure 1. Influence of Heavy Metal Concentration, Ingestion Rate, Averaging Time, and

Exposure Frequency and Duration to the Dietary Risks of Heavy Metal Exposure.

6
 This study is focused on determining the dietary risks that Los Banos fisherfolks may

encounter from ingestion of heavy metals found in Asian Clams. Heavy metal concentration and

ingestion rate are dependent variables, one from the seafood itself and the other from the sample

population. While ingestion of heavy metals are considered dangerous, most of these can only be

found in trace amounts in the body. Accumulation over time (exposure frequency and duration)

makes the contaminants possible health threats to the human body. Averaging time is the only pre-

established independent variable as it is based on countless studies to determine the average time

for developing the potential risks from heavy metal exposure. The link between between human

health and health explicitly recognizes that there are multiple sources of exposure from heavy

metals. Since dietary intake is one of the sources of exposure, this study will aid in identifying the

risk that accompany it.

7
 LITERATURE REVIEW

Laguna Lake

Laguna Lake is blessed with an abundance of natural resources, boasting 31 species of

fishes belonging to 16 families and 19 genera. It is considered as a biodiverse pool of a variety of

organisms. The lake is about with fishes, mollusks, crustaceans, plankton, and water lilies. Among

its major contributions includes white goby, mudfish, ayungin, milkfish, catfish, kanduli, tilapia,

common carp and plasalit. Freshwater prawns and clams are also harvested for commercial

purposes. This makes the lake one of the hot sites for fishpens and open fisheries that provide

fishes for local markets and markets in Metro Manila, being the lake’s most dominant utilization.

Not only does it provide food, it is also a means for transportation of oil and oil products;

a reservoir for floodwater in cases of emergency; a source of hydroelectric power; a site for

recreation such as swimming, boating, and sailing; a source of irrigation for agricultural lands; a

source of industrial cooling water; and, a major source of domestic water for nearby towns

surrounding the lake (Laguna Lake Development Authority, n.d.).

Heavy Metals

According to the Laguna Lake Development Authority (n.d.), among the monitored toxic

metals in the LLDA’s program is Lead (Pb). When these harmful substances are suspended in the

water column or accumulated in sediments, they can accumulate in the tissue of fish and shellfish,

which are consumed by humans. Lead may come from the metal pickling and plating livelihoods,

the tanning agent in producing leather, and in the paint and dye production while lead may come

from fuels and production of batteries, paints, pesticides, etc.

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 Lead is a naturally occurring element, making up 0.0013% of the earth’s crust, but is

usually obtained from ores galena (PbS), where majority of lead is obtained

, anglesite (PbSO4), cerussite (PbCO3) and minum (Pb3O4) (Jefferson Lab). It is a soft, malleable

and corrosion resistant material which is known for its uses in paint, ceramics, plumbing materials,

gasoline, batteries, ammunition and cosmetics. Historically, lead is used by ancient Romans to

make water pipes and used to line tanks for storing corrosive liquids. Its high density property

makes it useful in shielding against harmful ultraviolet rays and in X-ray machines and nuclear

reactors. Today, the majority of lead is utilized in lead-acid storage battery production, the type

used in automobiles.

With its many uses, lead has been proven to be toxic to humans and detrimental to their

health. Human can be exposed to lead through the environment itself from its manifestations in

the atmosphere, the soil, the water, and household. It is emitted to the soil from industrial sources

and/or contaminated locations such as mining and smelting sites. Mining, smelting, and refining

are known to increase the natural lead levels in soil. Lead is also released in the atmosphere from

industrial sources or emissions from vehicles, traveling long distances before settling to land or

water. Lead may also seep from contaminated soil to ground water. Other forms of lead found in

household items include lead monoxide (PbO), a yellow solid found in paint; lead arsenate

(Pb3(AsO4)2), used in insecticides; or, lead nitrate (Pb(NO3)2), used to make fireworks and

other pyrotechnics (EPA).

People may become exposed to lead by inhalation and ingestion of contaminated water and

food (WHO). Hutton (1987) claims that the major route of entry for lead is by ingestion and

inhalation with a gastric absorption of 10%. Lead can be excreted in the urine but can also be

accumulated in the bones, kidney and liver. The Center for Disease Control and Prevention (CDC)

9
warns the dangers of lead accumulation such that stores of lead in the body are a source of continual

internal exposure by bone demineralization from aging. Toxic levels of exposure to lead may affect

the neurological development of young children, coma, convulsions and even death.

Compared to other organ systems, the nervous system appears to be the most sensitive and chief

target for lead induced toxicity (Cory-Slechta, 1996). The effects on the peripheral nervous system

are more pronounced in adults while the central nervous system is more prominently affected in

children. Encephalopathy, a progressive degeneration of certain parts of the brain, is a direct

consquenc of lead exposure and major symptoms include dullness, irritability, poor attention span,

headache, muscular tremor, loss of memory and hallucinations. More severe manifestations occur

at very high exposures and include delirium, lack of coordination, convulsions, paralysis, coma

and ataxia. Children who survive severe lead poisoning suffer irreversible mental retardation and

behavioural disorders. It also affects adults by damaging the kidneys and increased risk of high

blood pressure. To pregnant women, heavy exposure can cause miscarriage, stillbirths, premature

delivery, low birth weight babies, and minor malformations. At lower levels of exposure, multiple

body systems injury is known to occur.

Other effects of lead exposure include anemia, hypertension, renal impairment,

immunotoxicity and toxicity to the reproductive organs. Lead directly affects the hematopoietic

system by restraining the synthesis of haemoglobin through inhibition of various key enzymes

(δ­aminolevulinic acid dehydratase (ALAD), aminolevulinic acid synthetase (ALAS), and

ferrochelatase) in the heme synthesis pathway and by reducing the lifespan of circulation

erythroctytes through increasing the fragility of cell membranes; both of which lead to anemia

(Flora et al, 2012). Furthermore, renal functional abnormality rises from the occurrence of nuclear

inclusion of bodies containing lead protein complexes. These bodies degenerates the tubular

10
epithelium and causes acute nephropathy, causing abnormal excessive excretion of glucose,

phosphates, amino acids. Another disease caused by lead exposure is chronic nephropathy, which

is more severe and can potentially cause irreversible impairment. Common

effects seen in men include: reduced libido, abnormal spermatogenesis (reduced motility and nu

mber), chromosomal damage, infertility, abnormal prostatic function and changes in serum

testosterone. On the other hand, women are more prone to inferitility, miscarriage, premature

membrane rupture, pre-eclampsia, pregnancy hypertension and premature delivery. Influences on

the development of the fetus have also been known to occur with lead exposure.

Oral Reference Dose (RfD) is an estimate (with uncertainty spanning perhaps an order of

magnitude) of a daily exposure to the human population (including sensitive subgroups) that is

likely to be without an appreciable risk of deleterious effects during a lifetime. It is based on the

assumption that thresholds exist for certain toxic effects such as cellular necrosis and is expressed

in units of mg/kg-day. Many of lead’s health effects may occur with or without toxicity; however,

there is still no Oral Reference Dose for the element. By comparison to most other environmental

toxicants, Intergrated Risk Information System (IRIS) summarizes that the degree of uncertainty

about the health effects of lead is quite low. It appears that some of these effects, particularly

changes in the levels of certain blood enzymes and in aspects of children's neurobehavioral

development, may occur at blood lead levels so low as to be essentially without a threshold. Lead

body burdens vary significantly with age, health status, nutritional state, maternal body burden

during gestation and lactation, etc. It is for this reason and the continued lack of threshold that lead

has been established as a potential human carcinogen and that it is inappropriate to develop

reference values for. CDC identified 10 µg/dL as a blood lead level of concern.

11
 According to WHO, another heavy metal, Cadmium, is known to induce toxic effects not

only on the kidney, but also on the skeletal and respiratory system. Like lead, cadmium is naturally

occurring at low levels but is increased by human activity. It has various industrial applications

such as the production of alloys, pigments and batteries and can be released to the environment in

ways such as: natural activities (volcanic activity, weathering and erosion), human activities

(smoking, mining, smelting, and refining or non-ferrous metals, fossil fuel combustion,

incineration of municipal waste, manufacture of phosphate fertilizers and recycling of cadmium-

plated steel scrap and electric and electronic waste), or remobilization of historic sources (the

contamination of watercourses by drainage water from metal mines). The use of cadmium in

industrial processes arises from its utilization in electroplating and for manufacturing nickel-

cadmium batteries. From then, cadmium has been used in paint pigments and making polyvinyl

chloride plastics. Workers in non-ferrous smelting industries are exposed more frequently due to

air and water pollution that comes with the occupation.

With the environmental concerns, cadmium has been used less in developed countries.

According to Tchonwou and his co-researchers (2012) in a study about heavy metal toxicity, the

major routes of exposure to cadmium happen through inhalation, cigarette smoking, ingestion and

skin absorption, though rarely. Smoking is a major contributer, followed by ingestion. It has a

gastrointestinal absorption of 5% and is often accumulated in the kidneys and liver (Hutton, 1987).

Foodstuffs, such as liver, mushrooms, shellfish, mussels, cocoa powder, and dried seaweed, rich

in cadmium can contribute to the increase of cadmium concentration in the body. Though some of

this element can be excreted in the urine, it could affect the human body for 10 years and more. It

can also travel long distances through the atmosphere and is readily accumulated in many

organisms, especially mollusks and crustaceans (Tchonwou et al., 2012).

12
 Cadmium is a severe pulmonary and gastrointestinal irritant, which can be fatal if inhaled

or ingested. After acute ingestion, symptoms such as abdominal pain, burning sensation, nausea,

vomiting, salivation, muscle cramps, vertigo, shock, loss of consciousness and convulsions usually

appear within 15 to 30 min. Acute cadmium ingestion can also cause gastrointestinal tract erosion,

pulmonary, hepatic or renal injury and coma, depending on the route of poisoning while chronic

exposure to cadmium has a depressive effect on levels of norepinephrine, serotonin, and

acetylcholine (Tchonwou et al., 2012). Additionally, the kidney is the critical organ of intoxication

after long-term exposure to cadmium. One of the initial signs of renal dysfunction is an increased

urinary excretion of proteins. Cadmium-induced proteinuria is generally considered to be

characterized by the excretion of low molecular weight proteins, This form of proteinuria, caused

by an impaired reabsorption function of the proximal tubules, is not specific for the metal and may

be found in hereditary forms of tubular dysfunction. According to the WHO. high intake of

cadmium can lead to disturbances in calcium metabolism and the formation of kidney stones.

Softening of the bones and osteoporosis may occur in those exposed through living or working in

cadmium-contaminated areas. In an area of Japan where soil has been contaminated with cadmium

from zinc/lead mines, Itai-itai disease used to be widespread and is still seen in women over 50

years of age, characterized by osteomalacia, osteoporosis, painful bone fractures and kidney

dysfunction. Long-term, high-level occupational exposure is associated with lung changes,

primarily characterized by chronic obstructive airway diseases and development of pulmonary

adenocarcinomas. After systemic or direct exposure, it can also cause prostatic proliferative lesions

including adenocarcinomas. There is limited evidence that cadmium may also cause cancers of the

kidney and prostate.

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Dietary Risk Assessment

Dietary Risk Assessment is derived from health risk assessment which a tool used to

determine if a particular mineral poses a significant risk to human health under dietary

consumption. While health risk assessment covers health problems from all kinds of human

exposure, dietary risk assessment evaluates health problems that results from dietary consumption

of contaminated food. Risk assessment help scientists and regulators identify existing health

hazards and set an intervention with optimal benefits and minimum cost to remove a contaminant

like factors such as labor, human safety, etc. A one-in-one million risk of cancer from life-long

exposure is the basis for an acceptable risk since it is significantly low compared to the overall

cancer rate.

According to A Guide to Health Risk Assessment (n.d.), risk assessment undergoes a four-

step process: hazard identification, exposure assessment, dose-response assessment, and risk

characterization. The first step shows the health problems that may result from exposure by

reviewing studies related to heavy exposure. In this, selecting an accurate, timely information is

crucial. The second step determines the amount, duration and pattern of exposure. Next, the third

step estimates the amount of chemical which would cause varying degrees of health effects that

could lead to illnesses. This is where the carcinogenic and non-carcinogenic potential comes to

action. With cancer effects, even a very low exposure may result in the onset of the illness later in

life. Dose-response assessment identifies the levels of exposure which that pose only a low or

negligible risk for non-cancer health effects. Examples of non-cancer effects include neurological

disorders, developmental problems, respiratory illnesses, etc. Lastly, the fourth step evaluates the

risk for cancer and non-cancer potential to the general population.

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 Risk characterization is the final phase of the health risk assessment process. It integrates

the three phases: Hazard Identification, Dose-Response Assessment, and Exposure Assessment.

This phase determines the probability of an adverse effect to a human population by a toxic

substance and outlines permissible exposure levels from which standards of exposure are set.

Estimated Daily Intake (EDI), Target Hazard Quotient (THQ), and Target Cancer Risk (TR) were

the tools used to characterize the risks from heavy metal exposure through consumption of Asian

Clams. EDI is the amount of food item consumed per person in a day. For non-carcinogens, the

reference dose (RfD) must first be determined then compared to the maximum daily dose (MDD)

occurring within the target population, and which is calculated in the exposure assessment phase.

A resulting THQ greater than 1 is considered unsafe while a THQ lower than 1 is considered as

safe. Note that the terms ‘safe’ and ‘unsafe’, although safe exposures, are based on conservatives

estimates. However, these values are used to set regulatory standards for safe drinking water

maximum contaminant levels, permissible exposure limits in the workplace, and pesticide residue

limits in food and feed products. On the other hand, considering that carcinogens pose a risk at any

dose and the probability of developing cancer would increase with dose, the probability can range

from being negligibly low at low dose ranges or unacceptably high at high dose ranges. This means

there is no zero risk for cancer development if there is exposure at all.

Laguna lake is known to be a biodiverse center for livelihood. Through the years, industries

have flourished around the area, making the lake a waste site for these factories. In a journal by

Fang and co-authors (2003), the Pearl Delta River in China was also subjected to the same

condition. For the past twenty years, the rapid economic development in the nearby province

resulted in an over-exploitation of bioresources and excessive release of industrial waste into the

lake. As a result, the heavy metal content of five species of edible shellfish from the lake showed

15
levels higher than tolerable limits and local regulatory levels. In 1991, Vicente-Beckett and

collegues analyzed the levels of trace metals in sediment of Laguna Lake. Total metal analyses

reveal relatively low to moderate pollution in Laguna Lake. Throughout the years, the amount of

trace elements has possibly accumulated in the lake, affecting its aquatic animals and plants.

In 2011, Molina conducted a study assessing the risks of the bioaccumulation of heavy

metal in fish products in Laguna Lake to human health due to exposure. The non-carcinogenic

effect of fish products from the lake show that lead is the pollutant of the highest concern. The

study concludes that fish and fish products coming from the lake are not fit for human consumption

due to lead contamination. Another concern stated in the study is to monitor mercury content of

fishes from Laguna Lake. Chromium was said to only be carcinogenic through inhalation but it is

known that excess consumption of the mineral still results to noncancer effects.

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 MATERIALS AND METHODS

Schematic Diagram

Part 1: Asian Clam Intake of Fisherfolk from Los Banos

Determination of Los Banos Fisherfolk

Sample Size

List of Respondents from Barangay

Malinta, Los Banos, Laguna

Formulation and Pre-testing of

Modified Food Frequency
Questionnaire

Collection of Data via Interview

Statistical Analysis and Interpretation of

Data

Asian Clam Intake of Fisherfolk from Los

Banos

Part 2: Heavy Metal Concentration of Asian Clams from Laguna Lake

17
 Sample Collection (Asian Clams)

Homogenization by Nitric-Perchloric
Acid Digestion

Heavy Metal Analysis by Atomic

Absorption Spectrometer

Analysis and Interpretation of Data

/

Heavy Metal Concentrations in Asian

Clams

Part 3: Dietary Risk Assessment of Cadmium and Lead found in Asian Clams

18
General Methodology

This study used two data gathering tools to to measure the dietary risk exposure of

fisherfolk from Los Banos to the Cadmium and Lead concentration found in Asian Clams

(Corbicula fluminea). Firstly, to estimate the Asian Clam intake of Los Banos fisherfolk, an

interview was conducted using a modified food frequency questionnaire. Secondly, to measure the

accumulated amount of Cadmium and Lead in Asian Clams and compare their concentrations in

from raw and cooked samples, an Atomic Absorption Spectrometer was used to analyse the

Cadmium and Lead concentrations in samples homogenized using Wet Digestion via Nitric-

Perchloric Acid Digestion. Thirdly, to assess the estimated daily intake (EDI) of fisherfolk from

Cadmium and Lead found in Asian Clam using oral reference doses and to determine the non-

carcinogenic and carcinogenic potential of ingesting Asian Clams to the dietary and health risks

of exposure to Cadmium and Lead, the obtained intake and heavy metal concentrations were used

to compute for the EDI, Target Hazard Risk (for non-carcinogenic potential), and Target Cancer

Risk (for carcinogenic potential).

Location of the Study

The survey took place in Barangay Malinta, Los Banos, Laguna, along the coastal areas of

Laguna Lake. Samples were obtained from the same coastal area and were transferred to Laguna

State Polytechnic University for analysis of heavy metal concentration.

Data Collection for Ingestion Rate

The population of the study consists of fishermen who gather their sustenance from Laguna

Lake. Convenience sampling was done near the fishermen’s common area since they are the

respondents most likely to consume Asian Clams from the lake. The data was gathered using a

19
modified food frequency questionnaire and a 24-hour food recall kit. 26 fishermen were

interviewed.

Sample collection of Asian Clams

The Los Banos area of the Laguna Lake was chosen as the collection site. Live samples of

Asian Clams (Corbicula fluminea) were hand-collected from two separate locations at each site.

All samples will be gathered into dark plastic bags containing the ambient water and immediately

transported to the laboratory within an iced chest. All pooled samples weighing approximately 25g

(from 20-25 individuals, necessitated by soft tissue weight) will be stored at freezer temperature

until analyzed for cadmium, CADMIUM and lead content.

AAS Analysis of Heavy Metal Concentration in Asian Clams

Soft tissue samples were processed following the method described by Ferreira et al.

(2016). For cooked samples, boiling at 100 C for 4-6 mins, or until the clams open, was the method

of preparation. Five grams of each sample was accurately weighed in a 100 mL beaker followed

by the addition of 15 mL of nitric acid (HNO3). The beaker was heated on a hot plate at about

100°C until all the solids disappeared and vigorous reaction stopped. The beaker was then removed

and allowed to cool at room temperature. Next, 5 mL of perchloric acid (HClO4) was added to the

beaker and heated until fumes were visible. The temperature was regulated until the fumes

completely disappeared. The beaker was cooled at room temperature afterwards. The mixture was

filtered through a 0.45-µ Millipore membrane filter paper and then transferred quantitatively to a

50 mL volumetric flask by adding de-ionized water and analysed for heavy metals in an atomic

absorption spectrophotometer. Total heavy metal concentration (in mg kg-1) will be determined by

20
flame atomic absorption spectrometry (55B AA, Agilent Technologies) fuelled with air acetylene

using 217 nm spectral wavelength.

Statistical Analysis of Heavy Metal Concentration of Asian Clams

Mean and standard deviations will be used to describe the cadmium and lead in pooled

tissue samples. Significant mean differences of cadmium and lead in samples will be tested in one-

factor ANOVA.

Dietary Risk Assessment

The estimated daily intake (EDI) of the samples was estimated using the following equation

(Giri and Singh, 2015):

[EDI = IR × MC/ BW],

where, IR is the ingestion rate of the samples (amount in grams per person per day) which was

based on interview with local residents, MC is the metal concentration [Cd], [Cr] and [Pb] in

samples (in milligrams per kilogram, fresh weight), and BW is the average body weight (65 kg for

adult Filipinos according to Molina, 2014). A modified food frequency questionnaire was used to

determine amount and part of the aquatic creature consumed per day. To assess the non-

carcinogenic risk due to Cd, Cr, and Pb exposure in fish samples, the target hazard quotient (THQ)

was computed using the equation (Zhuang et al., 2013):

[THQ = [(EFr × ED × IR × MC)/ (RfDo × BW × AT)] × 10-3],

where, EFr is exposure frequency (number of per year for the average and heavy consumer,

respectively, based on local interview); ED is exposure duration (70 years) equivalent to the

average human lifespan, IR is the ingestion rate (amount in grams per person per day); MC is the

21
metal concentration [Cd], [Cr] and [Pb] in samples (in milligrams per kilogram, fresh weight);

RfDo is the oral reference dose. The applied reference doses according to Sipter (2008) are the
1 -1 -1
following: 0.1 µg/kg/bw/day for Hg; 3E-3 mg/kg-day for Cr; 0.05 mg kg day for Pb. BW is the

average body weight (65 kg); AT is the averaging time for non-carcinogens (assuming 30 years or

10,950 days). The potential carcinogenic effect was estimated as the incremental probability of an

individual to develop cancer over a lifetime exposure to potential carcinogen. This was expressed

using the equation (Islam et al., 2015):

[TR = [(Efr × ED × IR × MC × CSFo)/ (BWa × AT)] × 10-3];

where, TR is the target cancer risk. EFr, ED, IR, MC, and BW are assumed the same values in

units given for THQ calculations. AT was adjusted to 365 days per year × number of exposure

years, assuming 70 years or 25,550 days. CSFo is the oral carcinogenic slope factor (0.0085

milligrams per kilogram per day) given in the Integrated Risk Information System database of the

USEPA (Islam et al., 2015).

22
 RESULTS AND DISCUSSION

The results of cadmium and lead contents in cooked and raw samples of Asian Clams

obtained from Los Banos are shown in the Table 1. The concentration of Cd and Pb in the cooked

samples were in the range of 0.02-0.06 (SD=0.0179) and 0.28-0.55 mg/kg (SD=0.1402),

respectively. In the raw samples, the concentration of Cd and Pb were in the range 0.10-0.19

(SD=0.0329) and 0.60-1.32 mg/kg (SD=0.2686), respectively. The maximum concentration of Cd

is 0.06 mg/kg in the cooked samples and 0.19 mg/kg in the raw samples. For lead (Pb), the

maximum concentration is 0.55 mg/kg in the cooked samples while 1.32 mg/kg. The mean

concentration of Cd in cooked and raw samples, respectively, are 0.032 mg/kg and 0.144 mg/kg;

while, the mean concentration of Pb in cooked and raw samples, respectively, are 0.432 mg/kg and

1.012 mg/kg. The metal concentrations for the cooked samples yielded lower values by 57.31%

and 77.78% in Pb and Cd, respectively, as compared to the metal concentrations for the raw

sample. The oral reference doses (RfD) for Pb and Cd are 1x10-3 mg/kg-day and 3.6x10-3 mg/kg-

day, in that order. According to the Integrated Risk Information System, RfD is an estimate of a

daily exposure to the human population that is likely to be without an appreciable risk of

deleterious effects during a lifetime. The mean lead concentration for cooked (0.432 mg/kg) and

raw (1.012 mg/kg) samples are both higher than the RfD for lead. The mean cadmium

concentration for cooked (0.032 mg/kg) and raw (0.144 mg/kg) samples are also higher than the

RfD for the element. Analysis using single-factor ANOVA revealed that F < Fcrit in both lead and

cadmium. Since F > Fcrit means rejecting the null hypothesis that there is no difference between

the pooled samples, the null hypothesis is therefore rejected and thus stating that a difference lies

between cooked and raw cadmium and lead concentrations. Daily consumption of Asian Clams

would induce detrimental health effects from Pb and Cd exposure, especially Pb since it is

23
considered as a carcinogen (Intergrated Risk Information System). However, based on local

interview, fisherfolk would only consume Asian Clam on the average of 2.2 days in a year.

Table 1. Statistical analysis of the Asian Clam samples.

Mean - +
Element Sample N SD F F crit
(mg/kg) (mg/kg) (mg/kg)
Ckd 5 0.432 0.280 0.550 0.1402
Pb 18.314 5.318
Raw 5 1.012 0.600 1.320 0.2686
Ckd 5 0.032 0.020 0.060 0.0179
Cd 44.800 5.318
Raw 5 0.144 0.100 0.190 0.0329

Based on local interview, the ingestion rate (IR) or the amount of Asian Clams consumed by

fisherfolk in Los Banos is 0.363 g/year. The individual ingestion rate ranges from 0.009 g/day to

3.000 g/day. Asian clams were said to be consumed 2.2 days a week on the average, which ranges

from 1-7 days, in the frequency of 1.6 meals/day on the average, which ranges from 1-3 meals/day.

The mean intake of Asian Clams per meal is 0.492 cups or 16.495 g. The per meal intake of Asian

Clams ranges from 0.063-1.250 cups or 2.094-41.875 g. A summary of these value are shown in

Tabl 2. The mean IR was used to compute for the Estimated Daily Intake, Target Hazard Quotient,

and Target Cancer Risk.

Table 2. The mean and range values for the ingestion rate of Los Banos fisherfolk.

Weekly Daily Per meal Per meal Ingestion Rate

Intake:
(day) (meals/day) (cups) (g) (g/year)

2.2 1.6 0.492 16.495 0.363

Mean
1 1 0.063 2.094 0.009
-
7 3 1.250 41.875 3.000
+

24
 The Estimated Daily Intake of Pb found in Asian Clams is 0.0024 mg/kg-day while the EDI

of Cd found in Asian Clams is 0.002. The EDI ranges from 0.0016 to 0.003 mg/kg-day in terms

of lead concentrations and from 0.0001 to 0.0003 mg/kg-day in terms of cadmium concentrations.

The Tolerable Daily Intake for both elements are 0.001 mg/kg-day (Pb) and 0.0036 mg/kg-day

(Cd). Based on the consumption of the fisherfolk, the Pb and Cd concentrations in Asian Clams

are acceptable and poses no detrimental effects to their health. The EDI from the raw concentration

was also computed to serve as comparison, showing that the EDI of both heavy metals in cooked

Asian Clams is lower than the EDI derived from raw Asian Clams. Data is shown in Table 3.

Table 3. The mean and range values for Estimated Daily Intake (EDI) in comparison to the
Tolerable Daily Intake (TDI)

Mean EDI TDI

Mean MC
Element Sample N - + (mg/kg- (mg/kg- Assessment
(mg/kg)
day) day)
Cooked 5 0.43 0.0016 0.003 0.0024 0.001 Below TDI
Pb Above
Raw 5 1.01 0.0033 0.007 0.0056 0.001
TDI
Cooked 5 0.03 0.0001 0.0003 0.0002 0.0036 Below TDI
Cd
Raw 5 0.14 0.0006 0.001 0.0008 0.0036 Below TDI
*TDI=Tolerable Daily Intake; MC=Metal Concentration

Both Cadmium and Lead are heavy metals of concern in terms of their adverse health effects

associated with heavy metal bioaccumulation. A Target Hazard Quotient less than 1 (THQ<1) is

considered as safe; however, a THQ higher than 1 (THQ>1) is considered unsafe and at risk for

developing non-cancer health detriments such as renal diseases, reproductive impairment, or

cardiovascular illnesses, among others. Regardless of the preparation, both raw and cooked

samples yielded a safe Target Hazard Quotient in terms of their potential from lead content of

Asian Clams, as shown in Table 4.. The same observation can be examined from the THQ for

cadmium consumption from Asian Clams. The THQ from cooked Asian Clams is still

25
considerably lower than the THQ from raw Asian Clams (Pb: 0.12374 (ckd) > 0.28988 (raw); Cd:

0.000464 (ckd) > 0.001054 (raw)). Lead and Cadmium are also known carcinogens. Lead-induced

toxicity has been known to induce gene mutrations and sister chromatid exchanges as well as cause

apoptosis in human cancer cells and renal tumors in rats and mice (Tchounwou et al., 2012). The

highest life time cancer risk from Pb in Asian Clams has a risk value of 5.41x10-07. The average

life time cancer risk (TR) for fisherfolks in Los Banos, associated with Pb in Asian Clams, would

result to an excess of 5 cases out of 10,000,000 populations. Studies have shown that high levels

of systematic or direct exposure to cadmium causes pulmonary adenocarcinomas (Waalkes et al.,

1995 in Tchounwou et al., 2012). Even though cadmium is considered as mutagenic enough to

cause pulmonary cancer and DNA damage. The highest life time cancer risk from the Cd in Asian

Clams has a risk value of 4.01x10-08. The average life time cancer risk (TR) for fisherfolks in Los

Banos, associated with Cd in Asian Clams, would result to an excess of 4 cases out of 100,000,000

populations.

Table 4. Risk Assessment Values (non-carcinogenic and carcinogenic) for Pb and Cd

concentrations found in Asian Clams.

Target Target
THQ
Element Sample N Hazard Cancer
Evaluation
Quotient Risk
Cooked 5 0.012374 Safe 5.41x10-07
Pb
Raw 5 0.028988 Safe 12.7x10-07

Cooked 5 0.000464 Safe 4.01x10-08

Cd
Raw 5 0.001054 Safe 18.0x10-08

The heavy metal concentrations in Table 1 defined the rest of the comparisons on the dietary

risks between raw and cooked Asian Clams. Diaconescu and co-authors also observed the effects

26
of cooking on the heavy metal concentration in fish meat. Baking, microwave-cooking and grilling

the fish meat all showed a moderate loss of heavy metals, stating the possibility of reducing the

heavy metal concentration in fish samples by choosing a suitable method of cooking. Pb

concentrations are shown to decrease by cooking fish meat. However, the method of cooking

appeared insignificant in the Cd concentration of fish meat, which is also verified by Gheisari and

co-authors (2016) in the evalution of various cooking methods to the heavy metal concentration

(Pb and Cd, specifically) in more specific species of seafood—shrimp and lobster. Pb

concentrations in shrimp and lobster showed most significant decrease from boiling and steaming.

The study concluded that boiling and steaming are the best cooking method to provide healthful

seafood. Frying seafood appears to increase heavy metal concentrations in fish meat, as further

validated in a study by Kalogeropoulus and researchers in 2012, since frying evaporates water

content of fish meat, thus decreasing the weight of the fish meat.

Heavy metals that has bioaccumulated in the seafood might be due to the industrial and

domestic wastes. The water quality of Laguna Lake has deteriorated because of these activities.

Furthermore, poor treatment from varied perception of water pollution may have caused this.

People with a higher level of education tend to understand the food safety risks of water pollution

and thus make preventive actions to avoid these risks. However, even though the residents are

aware of the polluted status of the lake, over 70% of thm still consume fish from the lake (Gong

et al., 2015)

27
 CONCLUSION

This study measured the dietary risks of heavy metal exposure from Asian Clams

(Corbicula fluminea) among fisherfolks in Los Banos Laguna. The average duration for

consumption of Asian Clams per year was 2.2 days. The average intake of a fishermen in Los

Banos is 0.363 g/year. In evaluating the dietary risk exposure from heavy metals, estimated daily

intake, target hazard quotient and target cancer were used. The EDI in both lead and cadmium

concentration were lower than the tolerable daily intake. Furthermore, the target hazard quotient

was assessed as safe from the heavy metal non-carcinogenic potential of exposure from lead and

cadmium. The average life time cancer risk, or the carcinogenic potential, for fisherfolks in Los

Banos, associated with Pb in Asian Clams, would result to an excess of 5 cases out of 10,000,000

populations whil the TR for the population group associated with Cd in Asian clams would result

to an excess 4 cases out of 100,000,000 populations.

This study concludes that there are no dietary risks associated with the consumption of

Asian Clams from Laguna Lake. Cooking (boiling) has also been observed to decrease the heavy

metal concentration of lead and cadmium in Asian Clams.

28
 REFERENCES CITED

A Guide to Health Risk Assessment, California Environmental Protection Agency. Office of

Environmental Health Hazard Assessment. http://www.oehha.ca.gov/. Accessed March

5, 2010.

DE FLORA, S., CAMOIRANO, A., BAGNASCO, M., BENNICELLI, C., CORBETT, G. E., &

KERGER, B. D. (1997). Estimates of the chromium (VI) reducing capacity in human

body compartments as a mechanism for attenuating its potential toxicity and

carcinogenicity. Carcinogenesis, 18(3), 531-537.

LAGUNA LAKE AUTHORITY. (n.d.). Laguna de Bay. Retrieved from

http://www.llda.gov.ph/index.php

[CDC] CENTER FOR DISEASE CONTROL AND PREVENTION. (n.d.). Lead - Information

for Workers. Retrieved from The National Institute for Occupational Safety and Health

(NIOSH): https://www.cdc.gov/)

CORY-SLECHTA, D. (1996). Legacy of lead exposure: consequences for the central nervous

system. Otolaryngol Head Neck Surg., 114, 224-226.

[DENR] DEPARTMENT OF ENERGY AND RESOURCES. (n.d.). Laguna de Bay [online].

Retrieved from Laguna Lake Development Authority:

http://www.llda.gov.ph/index.php?option=com_content&view=article&id=74&Itemid=4

75

DIACONESCU, C. D. (n.d.). Effects of Cooking Methods on the Heavy Metal Concentrations in

the Fish Meat Originating from Different Areas of Danube River.

29
DURUIBE, J. O. (2007, May). Heavy metal pollution and human biotoxic effects. International

Journal of Physical Sciences, Vol 2.(5), pp. pp. 112-118.

[EPA] ENVIRONMENTAL PROTECTION AGENGY. (n.d.). Learn about Lead. Retrieved

from United States Environmental Protection Agecy: https://www.epa.gov/lead/learn-

aboutlead

FLORA, G. G. (2012, Jun). Toxicity of lead: A review of rcent updates. Interdisciplinary

Toxicology, 2(5), 47-58.

GHEISARI, E. R. (2016). The Effect of Different Cooking Methods on Lead and Cadmium

Contents of Shrimp and Lobster. Journal of Food Biosciences and Technology, 2(6), 53-

58.

GONG, W. S. (2015, April). Undestanding local fish consumption behaviour in Laguna Lak

watershed area, Philippines. African Journal of Food Science, 9(4), 176-181.

HUTTON, M. (1987). Human Health Concerns of Lead, Mercury, Cadmium and Arsenic. In M.

Hutton, Lead, Mercury, Cadmium and Arsenic in the Environment (pp. 53-68). John

Wiley & Sons Ltd.

[IRIS] INTERGRATED RISK INFORMATION SYSTEM. (n.d.). Lead and compounds

(inorganic); CASRN 7439-92-1. US Environmntal Protection Agency .

JEFFERSON LAB. (2017). It's Elemental - The Element Lead. Retrieved from Jefferson Lab:

http://education.jlab.org/itselemental/ele082.html

KALOGEROPOULUS, N. K. (2012, May 22). Heavy metals in raw, fried,grilled Mediterannean

finfish and shellfish. Food and Chemical Toxicoloy(50), 3702-3708.

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MOLINA, V. B. (2011). Health Risk Assessment of Heavy Metals Bioaccumulation in Laguna de

Bay Fish Products. Texas: From the 14th World Lake Conference.

PALLER, V. P. (2016, June). Acanthocephalan Parasites (Acanthogyrus sp.) of Nile Tilapia

(Oreochromis niloticus) as Biosink of Lead (Pb) Contamination in a Philippine

Freshwater Lake. Bulletin of Environmental Contamination and Toxicology, Vol. 96(6),

pp. pp. 810-815.

TCHOUNWOU, P. Y. (2012). Heavy Metals Toxicity and the Environment. EXS(101), 133-164.

Waalkes, M. M. (1995). Handbook on Metal-Ligand Interactions of Biological Fluids (Vol. 2).

New York: Marcel Dekker.

[WHO] WORLD HEALTH ORGANIZATION. (2016, January). Lead poisoning and health.

Retrieved from http://www.who.int/

(2016, January). Mercury and health. Retrieved from http://www.who.int/

31
 APPENDICES

APPENDIX A. Sample preparation

A B

Washing and Pre-preparation (Boiling for Shucking

cooked samples)
C D

Flesh Extration Chopping

32
APPENDIX B. Wet Digestion by Nitric-Perchloric Acid

A B

Weighing Addition of Nitric Acid

C D

Heating Reheating after the addition of Perchloric

Acid
E F

Filtering Storing

33
 APPENDIX C. Modified Food Frequency Questionnaire.

Amount
Frequency of intake
Respondent No.

per intake

HH Count
Name Per week Per day Amount in
Neve Once a 1-2 2-3 3-4 4-5 5-6 6-7 one two three cups
r week times a times a times a times a Times times a meal a meals a meals a
week week week week a week week day day day

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
 APPENDIX D. Sample Consent Form

Dietary Risk Exposure among Fisherfolks alongside Los Banos Area of Laguna Lake in
Relation to the Heavy Metal Content of Asian Clams

Ako, Angel Josephine A. Manuel, isang estudyante ng Unibersidad ng Pilipinas Los Banos, na
may kursong BS Nutrition, ay kumukuha ng HNF 152, isang paksa tungkol sa Food and Nutrition
Research. Ang aking pananaliksik ay tungkol sa Dietary Risk Exposure ng pagkain ng Tulya ng
mga mangingisdang taga-Los Banos base sa Heavy Metal Content ng isdang ito.
Sa pag-uugali ng pag-aaral na ito, ang proseso ng pagtitipon ng data ay maaaring sa anyo ng isang
pakikipanayam.
Ang inyong partisipasyon ay boluntaryo, maaring makilhok o tumanggi sa pagsagot sa
palatanungan.
Ang mga datos na makukuha sa pananaliksik na ito ay lubos na kumpidensyal at naglalayon
lamang para sa pananaliksik na ito.

Baka sakali’t mayroon kayong mga katanungan, mangyaring makipag-ugnay sa mananaliksik:

09162815271/ aamanuel@up.edu.ph

Angel Josphine A. Manuel

Tagapagpananaliksik

Ako ay nabigyan ng isang imbitasyon upang

lumahok sa pananaliksik tungkol sa Kaalaman,
Saloobin at Kasanayan ukol sa Pagpapasuso ng Bata
ng mga Ina sa Mangaldan, Pangasinan. Nabasa ko
lahat ng mga impormasyon Nagkaroon ako ng
pagkakataon na magtanong at ang aking katanungan
ay nasagot ayon sa aking kagustuhan. Hindi ako
sapilitang lumahok sa pag-aaral.
Pangalan (optional)/Pirma:
________________________________________
Pirma: _______________________________
APPENDIX E. Computations for EDI, THQ, and TR

𝑰𝒏𝒈𝒆𝒔𝒕𝒊𝒐𝒏 𝑹𝒂𝒕𝒆 × 𝑴𝒆𝒕𝒂𝒍 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏

𝑬𝒔𝒕𝒊𝒎𝒂𝒕𝒆𝒅 𝑫𝒂𝒊𝒍𝒚 𝑰𝒏𝒕𝒂𝒌𝒆 (𝑬𝑫𝑰) =
𝑩𝒐𝒅𝒚 𝑾𝒆𝒊𝒈𝒉𝒕

For Lead:

 Cooked

𝐼𝑅 × 𝑀𝐶
𝐸𝐷𝐼 =
𝐵𝑊

𝐼𝑛𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝑅𝑎𝑡𝑒 = 0.363

𝐵𝑜𝑑𝑦 𝑊𝑒𝑖𝑔ℎ𝑡 = 65 𝑘𝑔
𝑚𝑔
𝑀𝑒𝑡𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑙𝑒𝑎𝑑) = 0.432
𝑘𝑔
𝑔 𝑚𝑔
0.363 × 0.432
𝑑𝑎𝑦 𝑘𝑔
𝐸𝐷𝐼 =
65 𝑘𝑔

= 0.0024 𝑚𝑔/𝑑𝑎𝑦

 Raw
𝐼𝑅 × 𝑀𝐶
𝐸𝐷𝐼 =
𝐵𝑊
𝐼𝑛𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝑅𝑎𝑡𝑒 = 0.363
𝐵𝑜𝑑𝑦 𝑊𝑒𝑖𝑔ℎ𝑡 = 65 𝑘𝑔
𝑚𝑔
𝑀𝑒𝑡𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑙𝑒𝑎𝑑) = 1.012
𝑘𝑔
𝑔 𝑚𝑔
0.363 × 1.012
𝑑𝑎𝑦 𝑘𝑔
𝐸𝐷𝐼 =
65 𝑘𝑔
= 0.0056 𝑚𝑔/𝑑𝑎𝑦

For Cadmium:

 Cooked
𝐼𝑅 × 𝑀𝐶
𝐸𝐷𝐼 =
𝐵𝑊
𝐼𝑛𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝑅𝑎𝑡𝑒 = 0.363

36
 𝐵𝑜𝑑𝑦 𝑊𝑒𝑖𝑔ℎ𝑡 = 65 𝑘𝑔
𝑚𝑔
𝑀𝑒𝑡𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑙𝑒𝑎𝑑) = 0.03
𝑘𝑔
𝑔 𝑚𝑔
0.363 × 0.03
𝑑𝑎𝑦 𝑘𝑔
𝐸𝐷𝐼 =
65 𝑘𝑔
= 0.0002 𝑚𝑔/𝑑𝑎𝑦
 Raw:
𝐼𝑅 × 𝑀𝐶
𝐸𝐷𝐼 =
𝐵𝑊
𝐼𝑛𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝑅𝑎𝑡𝑒 = 0.363
𝐵𝑜𝑑𝑦 𝑊𝑒𝑖𝑔ℎ𝑡 = 65 𝑘𝑔
𝑚𝑔
𝑀𝑒𝑡𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑐𝑎𝑑𝑚𝑖𝑢𝑚) = 0.14
𝑘𝑔
𝑔 𝑚𝑔
0.363 × 0.14
𝑑𝑎𝑦 𝑘𝑔
𝐸𝐷𝐼 =
65 𝑘𝑔
= 0.0008 𝑚𝑔/𝑑𝑎𝑦

37
 𝑬𝑭𝒓 × 𝑬𝑫 × 𝑰𝑹 × 𝑴𝑪
𝑻𝒂𝒓𝒈𝒆𝒕 𝑯𝒂𝒛𝒂𝒓𝒅 𝑸𝒖𝒐𝒕𝒊𝒆𝒏𝒕 (𝑻𝑯𝑸) = × 𝟏𝟎−𝟑
𝑹𝒇𝑫𝒐 × 𝑩𝑾 × 𝑨𝑻

𝐸𝐹𝑟 = 𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒 𝐹𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦

𝐸𝐷 = 𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒 𝐷𝑢𝑟𝑎𝑡𝑖𝑜𝑛

𝐼𝑅 = 𝐼𝑛𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝑅𝑎𝑡𝑒

𝑀𝐶 = 𝑀𝑒𝑡𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛

𝑅𝑓𝐷𝑜 = 𝑂𝑟𝑎𝑙 𝑅𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝐷𝑜𝑠𝑒

𝐵𝑊 = 𝐵𝑜𝑑𝑦 𝑊𝑒𝑖𝑔ℎ𝑡

𝐴𝑇 = 𝐴𝑣𝑒𝑟𝑎𝑔𝑖𝑛𝑔 𝑇𝑖𝑚𝑒

For Lead:

 Cooked

𝐸𝐹𝑟 = 2.2 𝑑𝑎𝑦𝑠/𝑦𝑒𝑎𝑟

𝐸𝐷 = 30 𝑦𝑒𝑎𝑟𝑠

𝐼𝑅 = 0.363

𝑀𝐶 = 0.43 𝑚𝑔/𝑘𝑔

𝑅𝑓𝐷𝑜 = 0.001 𝑚𝑔/𝑘𝑔

𝐵𝑊 = 65 𝑘𝑔

𝐴𝑇 = 70 𝑦𝑒𝑎𝑟𝑠

𝑑𝑎𝑦𝑠 0.43𝑚𝑔
2.2 × 70𝑦𝑒𝑎𝑟𝑠 × 0.363 ×
𝑦𝑒𝑎𝑟 𝑘𝑔
𝑇𝑎𝑟𝑔𝑒𝑡 𝐻𝑎𝑧𝑎𝑟𝑑 𝑄𝑢𝑜𝑡𝑖𝑒𝑛𝑡 (𝑇𝐻𝑄) = 𝑚𝑔 × 10−3
0.001 × 65𝑘𝑔 × 30 𝑦𝑒𝑎𝑟𝑠
𝑘𝑔

= 0.01237

 Raw

38
𝐸𝐹𝑟 = 2.2 𝑑𝑎𝑦𝑠/𝑦𝑒𝑎𝑟

𝐸𝐷 = 30 𝑦𝑒𝑎𝑟𝑠

𝐼𝑅 = 0.363

𝑀𝐶 = 1.01𝑚𝑔/𝑘𝑔

𝑅𝑓𝐷𝑜 = 0.001 𝑚𝑔/𝑘𝑔

𝐵𝑊 = 65 𝑘𝑔

𝐴𝑇 = 70 𝑦𝑒𝑎𝑟𝑠

𝑑𝑎𝑦𝑠 1.01𝑚𝑔
2.2 × 70𝑦𝑒𝑎𝑟𝑠 × 0.363 ×
𝑦𝑒𝑎𝑟 𝑘𝑔
𝑇𝑎𝑟𝑔𝑒𝑡 𝐻𝑎𝑧𝑎𝑟𝑑 𝑄𝑢𝑜𝑡𝑖𝑒𝑛𝑡 (𝑇𝐻𝑄) = 𝑚𝑔 × 10−3
0.001 × 65𝑘𝑔 × 30 𝑦𝑒𝑎𝑟𝑠
𝑘𝑔

= 0.0290

For Cadmium:

 Cooked

𝐸𝐹𝑟 = 2.2 𝑑𝑎𝑦𝑠/𝑦𝑒𝑎𝑟

𝐸𝐷 = 30 𝑦𝑒𝑎𝑟𝑠

𝐼𝑅 = 0.363

𝑀𝐶 = 0.03 𝑚𝑔/𝑘𝑔

𝑅𝑓𝐷𝑜 = 0.001 𝑚𝑔/𝑘𝑔

𝐵𝑊 = 65 𝑘𝑔

𝐴𝑇 = 30 𝑦𝑒𝑎𝑟𝑠

39
 𝑑𝑎𝑦𝑠 0.03𝑚𝑔
2.2 × 70𝑦𝑒𝑎𝑟𝑠 × 0.363 ×
𝑦𝑒𝑎𝑟 𝑘𝑔
𝑇𝑎𝑟𝑔𝑒𝑡 𝐻𝑎𝑧𝑎𝑟𝑑 𝑄𝑢𝑜𝑡𝑖𝑒𝑛𝑡 (𝑇𝐻𝑄) = 𝑚𝑔 × 10−3
0.001 × 65𝑘𝑔 × 30 𝑦𝑒𝑎𝑟𝑠
𝑘𝑔

= 0.0005

𝐸𝐹𝑟 = 2.2 𝑑𝑎𝑦𝑠/𝑦𝑒𝑎𝑟

𝐸𝐷 = 30 𝑦𝑒𝑎𝑟𝑠

𝐼𝑅 = 0.363

𝑀𝐶 = 0.14 𝑚𝑔/𝑘𝑔

𝑅𝑓𝐷𝑜 = 0.001 𝑚𝑔/𝑘𝑔

𝐵𝑊 = 65 𝑘𝑔

𝐴𝑇 = 30 𝑦𝑒𝑎𝑟𝑠

𝑑𝑎𝑦𝑠 0.14𝑚𝑔
2.2 × 70𝑦𝑒𝑎𝑟𝑠 × 0.363 ×
𝑦𝑒𝑎𝑟 𝑘𝑔
𝑇𝑎𝑟𝑔𝑒𝑡 𝐻𝑎𝑧𝑎𝑟𝑑 𝑄𝑢𝑜𝑡𝑖𝑒𝑛𝑡 (𝑇𝐻𝑄) = 𝑚𝑔 × 10−3
0.001 × 65𝑘𝑔 × 30 𝑦𝑒𝑎𝑟𝑠
𝑘𝑔

= 0.0010

40
 𝑬𝑭𝒓 × 𝑬𝑫 × 𝑰𝑹 × 𝑴𝑪 × 𝑪𝑺𝑭𝒐
𝑻𝒂𝒓𝒈𝒆𝒕 𝑪𝒂𝒏𝒄𝒆𝒓 𝑹𝒊𝒔𝒌 (𝑻𝑹) = × 𝟏𝟎−𝟑
𝑩𝑾 𝒙 𝑨𝑻

𝐸𝐹𝑟 = 𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒 𝐹𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦

𝐸𝐷 = 𝐸𝑥𝑝𝑜𝑠𝑢𝑟𝑒 𝐷𝑢𝑟𝑎𝑡𝑖𝑜𝑛

𝐼𝑅 = 𝐼𝑛𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝑅𝑎𝑡𝑒

𝑀𝐶 = 𝑀𝑒𝑡𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛

𝐶𝑠𝐹𝑜 = 𝑂𝑟𝑎𝑙 𝐶𝑎𝑟𝑐𝑖𝑛𝑜𝑔𝑒𝑛𝑖𝑐 𝑆𝑙𝑜𝑝𝑒 𝐹𝑎𝑐𝑡𝑜𝑟

𝐵𝑊 = 𝐵𝑜𝑑𝑦 𝑊𝑒𝑖𝑔ℎ𝑡

𝐴𝑇 = 𝐴𝑣𝑒𝑟𝑎𝑔𝑖𝑛𝑔 𝑇𝑖𝑚𝑒

For Lead:

 Cooked

𝐸𝐹𝑟 = 2.2 𝑑𝑎𝑦𝑠/𝑦𝑒𝑎𝑟

𝐸𝐷 = 70 𝑦𝑒𝑎𝑟𝑠

𝐼𝑅 = 0.363

𝑀𝐶 = 0.43𝑚𝑔/𝑘𝑔

𝑚𝑔
𝐶𝑠𝐹𝑜 = 0.0085 𝑑𝑎𝑦
𝑘𝑔

𝐵𝑊 = 65 𝑘𝑔

𝐴𝑇 = 70 𝑦𝑒𝑎𝑟𝑠

𝑇𝑎𝑟𝑔𝑒𝑡 𝐶𝑎𝑛𝑐𝑒𝑟 𝑅𝑖𝑠𝑘 (𝑇𝑅)

𝑑𝑎𝑦𝑠 0.43𝑚𝑔 𝑚𝑔
2.2 × 70𝑦𝑒𝑎𝑟𝑠 × 0.363 × × 0.0085 𝑑𝑎𝑦
𝑦𝑒𝑎𝑟 𝑘𝑔 𝑘𝑔
= × 10−3
𝐵𝑊 𝑥 𝐴𝑇

=5.41x10-07

41
 Raw

𝐸𝐹𝑟 = 2.2 𝑑𝑎𝑦𝑠/𝑦𝑒𝑎𝑟

𝐸𝐷 = 70 𝑦𝑒𝑎𝑟𝑠

𝐼𝑅 = 0.363

𝑀𝐶 = 1.01𝑚𝑔/𝑘𝑔

𝑚𝑔
𝐶𝑠𝐹𝑜 = 0.0085 𝑑𝑎𝑦
𝑘𝑔

𝐵𝑊 = 65 𝑘𝑔

𝐴𝑇 = 70 𝑦𝑒𝑎𝑟𝑠

𝑇𝑎𝑟𝑔𝑒𝑡 𝐶𝑎𝑛𝑐𝑒𝑟 𝑅𝑖𝑠𝑘 (𝑇𝑅)

𝑑𝑎𝑦𝑠 1.01𝑚𝑔 𝑚𝑔
2.2 × 70𝑦𝑒𝑎𝑟𝑠 × 0.363 × × 0.0085 𝑑𝑎𝑦
𝑦𝑒𝑎𝑟 𝑘𝑔 𝑘𝑔
= × 10−3
𝐵𝑊 𝑥 𝐴𝑇

= 12.7x10 − 07

For Cadmium:

 Cooked

𝐸𝐹𝑟 = 2.2 𝑑𝑎𝑦𝑠/𝑦𝑒𝑎𝑟

𝐸𝐷 = 70 𝑦𝑒𝑎𝑟𝑠

𝐼𝑅 = 0.363

𝑀𝐶 = 0.03𝑚𝑔/𝑘𝑔

𝑚𝑔
𝐶𝑠𝐹𝑜 = 0.0085 𝑑𝑎𝑦
𝑘𝑔

𝐵𝑊 = 65 𝑘𝑔

42
 𝐴𝑇 = 70 𝑦𝑒𝑎𝑟𝑠

𝑇𝑎𝑟𝑔𝑒𝑡 𝐶𝑎𝑛𝑐𝑒𝑟 𝑅𝑖𝑠𝑘 (𝑇𝑅)

𝑑𝑎𝑦𝑠 0.03𝑚𝑔 𝑚𝑔
2.2 × 70𝑦𝑒𝑎𝑟𝑠 × 0.363 × × 0.0085 𝑑𝑎𝑦
𝑦𝑒𝑎𝑟 𝑘𝑔 𝑘𝑔
= × 10−3
𝐵𝑊 𝑥 𝐴𝑇

= 4.01x10 − 08

 𝑅𝑎𝑤

𝐸𝐹𝑟 = 2.2 𝑑𝑎𝑦𝑠/𝑦𝑒𝑎𝑟

𝐸𝐷 = 70 𝑦𝑒𝑎𝑟𝑠

𝐼𝑅 = 0.363

𝑀𝐶 = 0.14𝑚𝑔/𝑘𝑔

𝑚𝑔
𝐶𝑠𝐹𝑜 = 0.0085 𝑑𝑎𝑦
𝑘𝑔

𝐵𝑊 = 65 𝑘𝑔

𝐴𝑇 = 70 𝑦𝑒𝑎𝑟𝑠

𝑇𝑎𝑟𝑔𝑒𝑡 𝐶𝑎𝑛𝑐𝑒𝑟 𝑅𝑖𝑠𝑘 (𝑇𝑅)

𝑑𝑎𝑦𝑠 0.14𝑚𝑔 𝑚𝑔
2.2 × 70𝑦𝑒𝑎𝑟𝑠 × 0.363 × × 0.0085 𝑑𝑎𝑦
𝑦𝑒𝑎𝑟 𝑘𝑔 𝑘𝑔
= × 10−3
𝐵𝑊 𝑥 𝐴𝑇

= 18.0x10 − 08

43