Study of Bioaccumulation and Depuration of Pb Metal Ions in Green

Mussels (Perna viridis)

Riska Tamala

, Budiawan

and Sri Handayani

Departement of Chemistry, FMIPA Universitas Indonesia, Kampus UI Depok, 16424, Indonesia

Keywords: Green mussel, Depuration, Lead, Atomic Absorption Spectroscopy (AAS).

Abstract: In this study, bioaccumulation and depuration studies of Pb in green mussels were performed. The

bioaccumulation process was carried out using flowing water method for 7 days. The Pb ion concentration

used was 1.225 ppm. During bioaccumulation, Pb contained in mussels was determined every 24 hours.

Two depuration methods were applied in this study, flowing clean water method for 7 days and immersing

in acid solution for 2 hours. Two variations of acid solutions used were acetic acid and citric acid with

variations concentration of 0.75%; 1.5%; and 2.25%. Pb contained in mussels was analyzed using AAS. The

research showed that the highest value of Pb contained was reach after 7 days exposure with concentration

of 41.92 mg/kg and concentration factor (CF) value of 32.15 L/kg. The lowest content of Pb was reached

after depuration by immersing in 2.25% citric acid for 2 hours. Pb content after depuration was 16.96 mg/kg

with the decrease of Pb by 59.5%. Bioaccumulation ability was expressed by Concentration Factor (CF).

Based on this experiment, green mussel can be classified as low category bioindicator biota for Pb

accumulation.

1 INTRODUCTION

The development of industrial sector around Jakarta

impact on pollution in the region. This is due to the

Jakarta Bay area that accommodate the waste

generated by 13 rivers that bring millions of waste

every year to the sea. So level of polution from year

to year increase. (Hutagalung, 1991). Water

pollution in this area causes a decrease in water

quality, so the water can no longer be used as

intended. Liquid waste is entering the Jakarta Bay

often carries hazardous pollutants, such as heavy

metal. The impact of heavy metal pollution due to its

nature that cannot decompose and easily absorbed

by marine organisms, so it can accumulate in the

body. Heavy metals can also indirectly damage

fisheries and human health (Supriharyono, 2000).

One method that can be used to monitor

pollution in aquatic environments is by using marine

organisms called bioindicators. In choosing the

bioindicator should be based on laboratory research

to obtain a mechanism of pollutant or biokinetic

behaviour. The data acquisition can be used as a

reference for data interpretation in the real aquatic

environment (Suseno, 2006). The phenomenon may

indicate the potential of aspecies as bioindicator in

detecting heavy metals pollution (Hamed, 2006).

One of marine organism that can be used as a bio-

indicator is a green mussel. Green mussels (Perna

viridis) belong to the Bivalvia class that is widely

consumed by humans, because it is rich in protein.

(Ismail, 2006). The edible portion of the shell is all

parts of the body, including the digestive tract. This

aquatic biota is highly susceptible to heavy metal

contamination due to its filter feeder intake and

relatively immobile (sessile) (Gosling, 2004). This

makes it easy for heavy metals to accumulate in

mussels because heavy metals are readily bonded to

particles in the water and difficult to dissolve, thus

settling on the bottom of the waters or feeding

phytoplankton and marine organisms (Siddall,

1980). Aquatic plants and soft animals such as

shells, snails, etc., which are immobile or slow in

mobility, cannot regulate metals like other aquatic

animals (Darmono, 1995). Therefore, it is important

to know how much metal content in an organism

before it is consumed by humans. Prevention or

effort to reduce the level of metal pollution needs to

be done, among others by depuration method.

Tamala, R., Budiawan, . and Handayani, S.

Study of Bioaccumulation and Depuration of Pb Metal Ions in Green Mussels (Perna viridis).

DOI: 10.5220/0009843400002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

In this research, bioaccumulation and depuration

study of lead (Pb) was conducted using green

mussels as bioindicator. Two depuration methods

were applied, i.e by continuous flowing clean water

method and immersing in acid solution. Pb

contained in mussels were then analysed by using

Atomic Absorption Spectroscopy.

2 MATERIAL AND METHODS

2.1 Material

Materials used in this reasearch were Green Mussel,

Aquabidest, Boiling Stones, Arthemia sp., Sea

Water, Aquadest, HNO

65%, H

97%, Lead

Solution 1000 μg/mL, CH

COOH

(l)

25%, Citric

Acid, Selenium, NaOH

(l)

30%, H

5%,

Phenolpthalein, BCG-MR Indicator, MR Indicator,

Hydrocholric Acid 1,0 N, Na

.10H

2.2 Methods

2.2.1 Acclimatization

The acclimatization was performed for seven days in

an aquarium of clean seawater. Green mussels were

fed with Artemia sp. every day. During feeding, the

filtration system was switched off for 1 hour.

Research can be further conducted if the number of

dead green mussels were less than 20%.

2.2.2 Process of Bioaccumulation

Lead (Pb) exposure was carried out in an aquarium

with a capacity of 80 L. The concentration of Pb

ions presented is 1.225 ppm, which is half of the

value (Dobson, 1991). Bioaccumulation was

conducted for 7 days.

2.2.3 Process of Depuration

Two variation depuration methods were performed.

The first method was the recycling of water using

aquarium filled with seawater and equipped with

fitration and aeration system. Previously also carried

out measurements of temperature, pH, salinity, and

concentration in seawater were measured.

Depuration was performed for 7 days.. Samples of

green mussels were taken daily to determine the

level of Pb. The second method was immersion in

acid solution. Two variations of acid solution used

were acetic acid and citric acid. The variations of

concentration of the two acid solutions used were

0.75%, 1.5%, and 2.25%, with variations of

sampling time 24, 48, 72, 96, and 120 minutes.

2.2.4 Pb Content Analysis

The determination of Pb content, the green mussels

meat were destructed using 5 mL of HNO

and 1 mL

of H

. Pb levels were then analyzed using

Atomic Absorption Spectrometer (AAS).

3 RESULT AND DISCUSSION

3.1 Process of Bioaccumulation

The process of accumulation on green mussels body

can occur because heavy metal ion entering into the

body of the green mussel form a complex with the

cell follows several steps, including metal diffusion

from solution to the biological surface, metal

adsorption/complexation on the passive side of the

bond in a protective layer or spesific binding side of

the outer surface of the plasma membrane and

internal metal picking transported along the plasma

membrane. One of the passive diffusion processes

experienced by metals passes through epithelial

tissue located in the green mussel’s tissue, especially

in the gills that are the most significant limbs

associated with the outflow of substances derived

from the aquatic environment. Heavy metals

entering the gills will tend to form complexes with

proteins in glycoprotein constituents of gill mucus

(Palar, 1994). In mussels, heavy metal like Pb can

replace the essential metal and induce changes in

protein conformation that caused protein

denaturation. Heavy metals may bind to sulfhydryl

(-SH), carboxyl (-COOH), hydroxyl (-OH), or amino

groups of proteins. One of the ligands present in the

green mussels body is the sulfihydryl (-SH) group of

cysteine (Grant, 2008).

The formation of metal-protein complexes can be

attributed to hard of acid base (HSAB) concept

which describes the tendency of hard or soft an acids

and base. The metal which is the Lewis acid will act

as an electron acceptor and a protein that is Lewis

base will act as electron donor. The -SH group of

cysteine belongs to the soft-base group. Therefore

heavy metals have a tendency to form complexes

with soft base groups as well. The complex forming

reaction is presented in Figure 1.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Figure 1. Reaction of Complex Formation

The form of equilibrium equation is a bond with

heavy metal deprecating the bonding amide of a

nitrogen atom of a peptide. The metal is electrophile

with d orbitals which having number of unfilled

electrons, while the thiol (sulfhydryl) group in the

amino acid cysteine is nucleophile, so that between

heavy metals and thiols have a tendency to form

bonds. In the process of exposure is used metal in

nitrate salts, it allows nitrate ions in the water to

form nitric acid compounds that can reduce the pH

of the solution, there by triggering in increased

toxicity of the green mussel, so the toxicity of heavy

metals increasingly great (Hutagalung, 1990). In

bioaccumulation process will get the value of CF

(Concentration Factor). This value proves the ability

of accumulation of green mussel with contaminants.

To obtain the value of CF by comparing the metal

ion content in green mussel with the metal ion

content in water using equation 1.

Figure 2. Pb Content in Green Mussels During

Bioaccumulation

The value of CF continues to increase and tend

to reach the state of steady state was started from the

7 days was 32.15 L/kg. Condition based on the data,

indicating that green mussel can accumulate as much

as 32 times the concentration of lead ion in the sea

water. The implementation of the experimental

resuts, if there is lead ion pollution in Jakarta bay

then after 1 day the concentration of lead reaches 13

times compared with concentration in sea water. If

the pollution is still going on, then in 7 days the

concentration wil increase to 32 times compared

with concentration in sea water. Then a 30-days was

modeled in Figure 3.

Figure 3. Modeling of CF values if observed until 30 days

When the bioaccumulation is done for 5 days,

there is steady state and it can use to get CF

value.

It is a maximum capability of green mussel when

accumulate lead ion. It is 32.15 mL/g. The

bioaccumulation capasity of the green mussel was

also represented by the rate of taking the

contaminant (k

). In a single compartement, value of

is assumed as a contaminant uptake mechanism

by the entire body of the green mussel. However, the

speed of distribution into various type of organs was

neglected. Value of k

(mL/g.day) was an uptake

rate calculated based on the slope of the CFt curve to

t (from t = 0 to t in steady state (Umbara, 2007). The

value of k

was the influence of physiological

factors of green mussel, chemical species of

contaminants and the interaction between

physiological factors and chemical species. The rate

constant is determined by converting equation (2) to

a liniear equuation so that (3) is obtained:

= CF

(1-e

-ku.t

) (2)

ln(CF

-CF

) = -k

.t (3)

Then pass the equation (3) in the graph where the x

axis is the duration of exposur to the contaminant

(day) and the y axis is ln(CFss-CFt). The slope value

obtained from the line equation in the graph

represents the constant value of the uptake constanta

) by the green mussel. Based on the experiment,

the uptake constanta (k

) was 0.403 mL/g.day.

0 2 4 6 8

Concentration Factor (L/kg)

time (days)

0 5 10 15 20 25 30 35

research modeling until 30 days

Study of Bioaccumulation and Depuration of Pb Metal Ions in Green Mussels (Perna viridis)

3.1 Process of Depuration

In this method is done by transferring samples

derived from the bioaccumulation aquarium to a

contaminant free aquarium to allow the green mussel

to continue the filter feeding process. Therefore, it’s

important to keep the green mussel to stay alive. The

process is carried out for 7 days and for 7 days also

done replacement of seawater. Factors that can

weaken the complex bonds between metals and

protein is in the presence of other ligands that can

form more stable complexes with metals, such as

O. Therefore, a water solvent is used as a provider

of H

O ligands in the depuration process which will

disupt the stability of the metal complex with –SH

group in the amino acid cysteine protein constituent.

In this water-treatment is done with the replacement

of seawater media everyday with the speed of flow

from water made constant.

Figure 4. % Retention Value on Depuration Using

Continuous Flowing Water

From figure 4, it can be seen that the percentage

(%) retention of metal ion of lead retained on the

green mussel body decreases during depuration time

as the replacement of water is repeated. Retention

rate is percentage in the body of the green mussel

against the predicted time (Sari, 2005). The decrease

in metal ion of lead content may be due to the H

ligand which will form a complex with metal ion of

lead to [Pb(H

]

and it will cause the bond

between the –SH group in sistein and it cause the

metal bond to be disturbed. The H

O ligand is a

group of monodentic ligands which can donate one

pair of free electrons to fill the empty d orbitals from

metal ion of lead. The process of release or excretion

from metal ion of lead is one of the processes to

maintain electrolyte balance in the body of green

mussel. The ability to release contaminants by the

green mussel bodies is represented by the value of

the realease constant (t

). The value to be obtained

by changing the equation of model depuration (eq.

4) into linear equation (eq. 5).

= A

.e – k

.t (4)

ln (A

-A

) = k

.t (5)

Equation 5 is plotted into graph and a line

equation of the graph is determined. The slope of the

line equation represent the value to the green mussel.

The value obtained is 0.129 day

-1

. This may indicate

a decrease in the ability of the green mussel to

eliminate contaminants from the body. In the other

depuration method by using acid solution, that is

asectic acid and citric acid. The use of immersion

media with that acid is expected to interfere with the

bond between metal and protein. The observed

results are presented in Figure 5-a and Figure 5-b.

(a)

(b)

Figure 5. % Retention on Depuration Using (a) Acetic

Acid, (b) Using Citric Acid

From figure 5 is seen that retention (%) of Pb

metal ions retained on the green mussel body

decrease during depuration time along with the

addition of cencentration to the acid. The

determination of heavy metals in green mussel can

be caused of insoluble acetat salts from the metals

(Suprapti, 2016). The ability to realease

contaminants by the biota bodies is represented by

100

0 20 40 60 80 100 120 140

Retention (%)

time (minutes)

0,75% 1,50% 2,25%

0 20 40 60 80 100 120 140

Retention (%)

time (minutes)

0,75% 1,50% 2,25%

0 2 4 6 8

Retention (%)

Time (days)

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

the value of the realease constant (k

). The gain of

the value to be derieved from equation 5 is plotted

into a graph and the line equation of the graph is

determined. The slope of the line equation represents

the k

value of the green mussel. This may indicate a

decrease in the ability of the green mussel to

eliminate contaminants from the body. The values to

those obtained are presented in Tabel 1.

Table 1. Biokinetic of Lead In Green Mussel

(day

)

(day

)

BCF

1/2(b)

(day)

Depuration

Using Water

Drainage

32.15

0.41

0.13

3.12

5.37

Depuration

Using Acetic

Acid 0,75%

32.15

0.41

0.06

6.88

11.75

Depuration

Using Acetic

Acid 1,5%

32.15

0.41

0.05

8.83

15.07

Depuration

Using Acetic

Acid 2,25%

32.15

0.41

0.02

27.07

46.20

Depuration

Using

Citric Acid

0,75%

32.15

0.41

0.07

5.64

9.63

Depuration

Using

Citric Acid

1,5%

32.15

0.41

0.06

6.34

10.83

Depuration

Using

Citric Acid

2,25%

32.15

0.41

0.06

6.66

11.36

Biokinetic data is a mode that can be

implemented in sea water conditions. The rate of

taking lead by green mussel is 0.41 times per-day

from the concentration in seawater.release rate of

0.02-0.13 perday from the animal cody with several

different realease methods. Biological residence

time so that the concentration become half time

1/2

). The lead ion in the body of green mussel is

5.37 to 46.20 days. The bioaconcentration factor

(BCF) lead ion the animal is 3.12 to 27.07 times

compared to the concentration in seawater with

several different dischange methods.

4 CONCLUSIONS

Heavy metal ion of Lead can accumulate in green mussels

seen from CF (Concentration Factor) value obtained that is

equal to 32.15 L/kg.day, the value of k

(Uptake

Constanta) obtained from treatment of exposure of heavy

metal ion of Lead foor 7 days equal to 0.403 L/Kg.day, the

value of k

(Elimination Constanta) for depuration

treatment with a recurrent water-draining method equal to

0.129 day

-1

, the smallest value of depuration with

immersion of acetic acid is 0.0015 day

-1

when the

variation of concentration in 2.25%, and the smallest value

of depuration with immersion of citric acid is 0.0061 day

-1

when variation of concentration in 2.25%, the value of

%retention obtained by 44% in reccurent water drainage,

49.87% when immersion of acetic acid with variation of

concentration in 2.25% and 40.46% when immersion of

acetic acid with variation of concentration in 2.25%.

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