The Effect of seaweed (Eucheuma cottonii) extract and

duration of soaking on reduction of Copper (Cu) level in

Freshwater Mussel (Pilsbryoconcha exilis)

A F Dewinta*, A Susanti, and I E Susetya

Aquatic Resources Management Program Study, Faculty of Agriculture, Universitas

Sumatera Utara, Medan, North Sumatera 20155, Indonesia.

*Email: astridfd@usu.ac.id

Abstract. Freshwater Mussels (Pilsbryoconcha exilis) are filter-feeder animals that have eating
habits by filtering food in the water, making it possible that heavy metal copper (Cu) will
accumulate in the body of the mussel. One type of seaweed that can absorb heavy metals is
Eucheuma cottonii because it contains keraginan (65%) which is a sulfated polysaccharide
containing hydroxyl groups (-OH) and carboxyl groups (-COOH) and is an active site where a
metal interacts with seaweed. This research aims to determine the effect of extract
concentration and the best soaking time in reducing copper (Cu) levels. This research was
conducted from April to May 2023. In this study, the concentrations used were 6%, 10%, and
14% with a period of 45 minutes, 90 minutes, and 135 minutes. The results showed that the
concentration of 14% extract with a soaking time of 135 minutes showed the highest decrease
in copper (Cu) levels of 1.298 mg/kg or 87.10% and the lowest decrease in copper (Cu) metal
levels at a concentration of 6% with a soaking time of 45 minutes of 0,442 mg/kg or 29.68%.

1. Introduction
Eucheuma cottonii is a type of Rhodophyceae seaweed characterized by cylindrical thallus with a
smooth surface. E. cottonii can absorb heavy metals because it has chemical content such as iota
carrageenan (65%), sulfur (9.5%), carbohydrates, protein, crude fiber, fat, ash, and water. Keraginan is
a sulfated polysaccharide where the sulfate ester content is 28-35%. Sulfur (S) and oxygen (O) atoms
in sulfate esters, -OH and -COOH in polysaccharides, are active sites where a metal interacts with
seaweed [1]. The content of phytochelatin compounds contained in seaweed Eucheuma cottonii serves
to accumulate and depurate heavy metals by binding and forming complex metal phytochelatin and
stored by organelles such as vacuoles and kroloplas [2]. Based on researched by [3] that seaweed
species Eucheuma cottonii has the ability as a biosorbent, where E. cottonii has an average absorption
of heavy metal cadmium (Cd) of 0.1561 mg/L and has a percentage of heavy metal cadmium (Cd)
absorption of 97.4%.
Freshwater Mussels is known as filter feeders and their populations are abundant in rivers in North
Sumatra Province, one of which is the Barumun River. The Barumun River has a tributary, the
Simangayat River, which is in Asam Jawa Village, Torgamba, South Labuhan Batu. Asam Jawa
Village also has a reservoir that was formed due to oil palm plantation activities which then formed a
large basin. Around this river and reservoir there are oil palm plantations, palm oil mill waste disposal,

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and anthropogenic activities. The muddy substrate of rivers and reservoirs is a habitat favored by
mussels.
The utilization of Freshwater Mussels is limited to community consumption as daily food. The
Barumun River is widely used by the community as a fishing ground. The number of fishing boats
located downstream and industrial ships upstream that cross the Barumun River can result in heavy
metal contamination and settle in aquatic sediments [4].
Based on the results of preliminary tests of heavy metal levels that have been carried out on
Chinese Pond Mussel (Anodonta woodiana) from the same habitat, it shows that the levels of heavy
metal copper (Cu) in Chinese Pond Mussels meat have the highest levels when compared to heavy
metals Pb, Cd, and Hg which have exceeded the quality standards for heavy metals in fish with an
average Cu heavy metal level of 1,936 mg/kg in Samak Reservoir and 1,430 mg/kg in Simangayat
River. Based on this background, it is necessary to research the effectiveness of seaweed extract (E.
cottonii) and the length of immersion in reducing heavy metal levels of copper (Cu) in freshwater
mussels (P. exilis).

2. Materials and methods

2.1. Research site

This research was conducted in April-May 2023. Sampling was carried out in different places.
Freshwater mussels were taken in Simangayat River and Samak Reservoir, Asam Jawa Village, South
Labuhan Batu, North Sumatra. While sampling of Eucheuma cottonii seaweed in the waters of Punaga
Village, Takalar Regency, South Sulawesi with coordinates 5 o33'16.823 "LS and 119o25'27.412" BT
because this seaweed grows abundantly, and the waters are not polluted (atoxic). Preparation of
seaweed extracts and phytochemical screening of seaweed extracts were carried out at the Plant
Physiology Laboratory, Faculty of Mathematics and Natural Sciences, University of North Sumatra,
protein tests were carried out at the Biochemistry Laboratory, Faculty of Mathematics and Natural
Sciences, University of North Sumatra Medan, and copper (Cu) heavy metal tests were carried out at
the Regional Health Laboratory Technical Implementation Unit of North Sumatra Province, Medan.

2.2. Tools and materials

The tools used in the research include a refractometer, meter, pH meter, thermometer, stationery, label
paper, cool box, knife, blender, glass jar, Whatman 40 filter paper, funnel, metal spatula, beaker glass,
erlenmeyer, beaker glass, ziplock plastic size 8 x 5 cm, furnace, a set of Atomic Absorption
Spectrophotometer (AAS) tools, kjeldahl deconstruction apparatus, test tube, hot plate, porcelain cup,
furnace, dropper, measuring flask, refrigerator or freezer, volumetric pipette 5 ml and 10 ml,
desiccator, plastic spoon, measuring cup ml, evaporator flask, rotary evaporator, desiccator,
micropipette, an analytical digital scale with accuracy of 0.0001 gram, and camera. The materials used
in this study were Eucheuma cottonii seaweed, Freshwater Mussels 96% ethanol, distilled water, 65%
nitric acid (HNO3) 0.1 M, Cu standard solution, hydrochloric acid (HCl) 6 M, H 2SO4 98%, selenium
mix, NaOH 30%, H3BO3 3%, HCl 0.1 N, Tashiro indicator, phenolphthalein (PP) indicator, Mayer
reagent, HCl 1%, H2SO4, FeCl3, H2O2, Mg powder, tissue, and stationery.

2.3. Research procedure

2.3.1. Sampling of freshwater mussels (Pilsbryoconcha exilis). The sampling method was carried out
by purposive sampling. Mussel samples were taken from the Simangayat River and Samak Reservoir
in Asam Jawa Village, Torgamba District, South Labuhan Batu. The samples taken were still in one
piece (still shelled), then the samples were put into a cool box to keep them fresh and weighed and
measured for length as supporting data.

2.3.2. Sampling of seaweed (Eucheuma cottonii). Before seaweed sampling, in situ and ex-situ

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measurements of water quality parameters were conducted. Measurement of physical parameters
consisted of temperature using a thermometer and salinity using a refractometer, while chemical
parameters consisted of pH using a pH meter and DO using the Winkler method.

2.3.3. Initial heavy metal (Cu) testing on freshwater mussels. Initial Cu heavy metal testing was
carried out on mussel samples as controls (without seaweed extract immersion) by [5] which will be
used as comparison data.

2.3.4. Preparation of Eucheuma cottonii extract. Preparation of Eucheuma cottonii extract was carried
out using the maceration method. Maceration is an extraction method with the process of soaking the
material with a solvent that is by the active compounds to be taken with low heating or in the absence
of a heating process [6]. Seaweed samples were air-dried without direct sunlight for ± 14 days. The
dried samples were cut into pieces and pulverized using a blender until they became powder. Samples
that have been finely extracted using the maceration method (immersion in the extractor solution) with
96% ethanol solvent for 3 x 24 hours with periodic stirring. The seaweed-solvent ratio used was 1:8
(b/v) seaweed as much as 360 grams and 96% ethanol as much as 2880 ml. After maceration for 3 x
24 hours then the ethanol solvent was evaporated with a rotary evaporator for 6 hours to form a thick
extract of seaweed [7]. Then proceed with a phytochemical screening test of Eucheuma cottonii
seaweed extract.

2.3.5. Phytochemical screening test of Eucheuma cottonii extract. Phytochemical screening tests on
seaweed according to [8] are as follows: Alkaloid Test, 1 ml of extract sample is added with a little 1%
HCl, then add 1 ml Mayer reagent. The presence of a precipitate or turbidity indicates an alkaloid
compound. Flavonoid Test, 1 ml of extract sample is mixed with Mg powder and a few drops of
concentrated HCl. The emergence of pink, magenta, and orange colors indicates flavonoid compounds.
Saponin Test, 1 ml of extract sample is added to 10 ml of distilled water and then shaken vigorously
for 30 seconds. The presence of stable foam indicates saponin compounds. Steroid/Triterpenoid Test, 1
ml of extract sample is added a little anhydrous acetate and 1 drop of H 2SO4 (Lieberman Buchard
reagent). The presence of a blue-green color indicates a steroid compound, and the brownish red color
or brownish-pink ring indicates a terpene compound compound. Tannin Test, 1 ml of extract sample is
added to 10 mL of distilled water and then brought to a boil. Add a few drops of FeCl 3. The presence
of brownish-green or bluish-black color indicates tannin compound.

2.3.6. Determination of Eucheuma cottonii seaweed extract concentration. The concentrations used
were 6%, 10%, 14% b/v, and without soaking seaweed extract. For a concentration of 6% weighed 6
grams of seaweed extract then dissolved with distilled water as much as 100 ml, a concentration of
10% weighed 10 grams of seaweed extract then dissolved with distilled water as much as 100 ml. For
a concentration of 14% weighed 14 grams of seaweed extract and then dissolved it with distilled water
as much as 100 ml.

2.3.7. Immersion of mussels with seaweed extracts. The soaking of mussels was carried out by taking 8
grams of mussel meat in one piece and then immersed in seaweed extracts of 30 ml each in a ziplock
plastic measuring 8 x 5 cm, each repeated three times with a concentration of 6%, 10%, and 14%
Eucheuma cottonii extract and the length of soaking was 45 minutes, 90 minutes, and 135 minutes.
After completing the soaking treatment, the next mussel is drained in a colander. The drained mussel
samples were immediately subjected to organoleptic tests first and then tested for heavy metal Cu
content.

2.3.8. Copper (Cu) heavy metal content test [5]. Testing of heavy metal Cu levels was carried out
before and after soaking seaweed extracts so that it could be seen the effectiveness of Eucheuma
cottonii extract soaking on reducing heavy metal levels in Freshwater Mussels. Samples that have been

3
mashed are weighed as much as 5 grams in a porcelain cup, then ashes in the furnace, gradually
increasing the furnace temperature by 100 oC every 30 minutes until it reaches 450 oC and maintain for
18 hours. After the ash is formed completely white, cool the sample at room temperature, then add 5
mL of 6 M HCl to the sample and homogenize, then evaporate on a hot plate at 100 oC until dry. After
drying, add 10 mL of 0.1 M HNO3 then homogenize and cool at room temperature for 1 hour, then
transfer the solution into a 50 mL measuring flask (polypropylene), the solution is ready to be read on
a flame atomic absorption spectrophotometer with a wavelength of 324.8 nm.

2.4. Test parameters

2.4.1. Calculation of Cu heavy metal content. According to [9], To obtain the actual heavy metal
concentration using AAS (Atomic Absorption Spectrophotometry), the calculation of copper (Cu)
heavy metal content using the formula:

C ×V
Cu (mg/kg) ¿ (1)
W

Where:
C : Concentration of AAS sample solution (mg/kg)
V : Solvent volume (ml)
Ws : Sample weight (mg)

2.4.2. Effectiveness of Cu heavy metal reduction. According to [10] After obtaining the actual
concentration, the percent reduction of heavy metals is then calculated using the formula:

K 1−K 2
% Decrease ¿ ×100 % (2)
K1

Where:
% Decrease : Metal reduction rate (%)
K1 : Initial levels of heavy metals or control levels (mg/kg)
K2 : The final level to be sought (mg/kg)

2.4.3. Protein test. According to [11]

(mL HCl sampel−mL blanko)

% Nitrogen¿ × N ×14.007 × 100 % (3)
mg sampel

After obtaining %N, the protein content is then calculated by multiplying a factor.

% Protein ¿ % nitrogen× conversion factor (6.25)

(4)

Where:
N : Normality of the standard HCl used
14.007 : Atomic weight of nitrogen.
6.25 : Protein conversion factor for fish

2.4.3. Organoleptic test. Organoleptic testing was carried out after soaking according to [12] method

4
using a fresh mussel organoleptic score sheet with 30 untrained panelists. The parameters observed in
the organoleptic test were appearance, odor, and texture. The test method used is the scoring test using
the criteria number 1 as the lowest value and number 9 as the highest value.

2.5. Data analysis

The research data were analyzed descriptively quantitatively, namely explaining, or concluding data in
the form of numbers using Analysis of Variance (ANOVA). Data from the test results of the effect of
seaweed extract on reducing heavy metal levels and organoleptic test were analyzed using the SPSS
Statistics 25 application, while the results of the correlation test between Cu heavy metal levels to
protein content were analyzed using Microsoft Excel 2021.

3. Result and discussion

3.1. Phytochemical screening of Eucheuma cottonii extract

The results obtained from the phytochemical screening test of Eucheuma cottonii in this study can be
seen in Table 1.

Table 1. Secondary metabolite compound of Eucheuma cottonii extract.

Secondary metabolite Reagent Result
Alkaloid Maeyer +
Flavonoid Mg powder + concentrated HCl +
Saponin Aquadest +
Triterpenoid Lieberman-Buchard +
Tannin FeCl3 1% +
The results of phytochemical screening tests on Eucheuma cottonii seaweed extract contain several
types of secondary metabolite compounds including alkaloids, flavonoids, saponins, triterpenoids, and
tannins which are indicated by positive results (+) by testing the reagents used. According to [13]
secondary metabolite compounds flavonoids, alkaloids, and tannins can bind metal ions, due to the
presence of hydroxyl groups and amine groups that play a role in binding metal ions. In addition, the
opinion of [14] that the antioxidant mechanism of triterpenoids is by capturing/scavenging reactive
species, such as superoxide, and chelating Fe2+ and Cu2+ metals.

3.2. Initial Copper (Cu) and Effectiveness of Copper (Cu) Metal Content Reduction
The results of the examination of copper (Cu) levels in freshwater mussels (Pilsbryoconcha exilis) in
each treatment are presented in Table 2.
Table 2. Decrease in Copper (Cu) heavy metal levels in mussel meat.
Cu Level Decrease
Treatments
(mg/kg) mg/kg %
Control 1.4907 0 0
A1B1 1.048±0.34b 0.442 29.68 %
A1B2 0.672±0.26b 0.819 54.92 %
A1B3 0.566±0.16b 0.925 62.0 %
A2B1 0.375±0.20a 1.115 74.82%
A2B2 0.339±0.19a 1.152 77.26 %
A2B3 0.319±0.19a 1.172 78.60 %
A3B1 0.306±0.24a 1.185 79.48 %
A3B2 0.216±0.13a 1.275 85.53 %

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 A3B3 0.192±0.03a 1.298 87.10 %
Note: - (A1:6%, A2:10%, A3:14%, B1: 45 min, B2: 90 min, B3 :135 min)
- Data is the average result of 3 replicates standard ±deviation

The results of testing the levels of heavy metals Cu using AAS in Freshwater Mussel meat
(Pilsbryoconcha exilis) after soaking seaweed extracts with different concentrations and soaking times
are presented in table 2. Table 2 shows the percentage decrease in Cu metal levels, based on these data
the greater the concentration given and the longer the soaking time can reduce heavy metal levels
greater. Based on this research, the best concentration, and the best time to reduce heavy metal levels
of Cu in mussel meat is in the A3B3 treatment (14% seaweed extract concentration and 135 minutes of
soaking) with a decrease in Cu heavy metal levels of 1.298 mg/kg or 87.10%. According to [15] that
the highest reduction in heavy metal levels in seaweed concentrations with the highest concentration
and the longest soaking.

3.3. Correlation of protein content and Cu heavy metal

Based on the results of research on heavy metal levels and protein content in mussels, it shows that
each treatment shows a negative correlation with the equation y = -0.8334x + 2.9644 or y = 2.9644-
0.8334x, meaning that the lower the level of heavy metal Cu, the protein content will increase. The
coefficient of determination (R-Square) on the results of the correlation test between Cu levels and
protein content is 0.3104, which means that only 31.04% of the Cu heavy metal factor affects the
protein content in the meat and 68.96% is influenced by other factors. While the correlation value
between Cu levels and protein content is r = 0.557 which means a moderate correlation.

3.5

3
Protein (%)

f(x) = − 0.853650301348812 x + 2.99353081695604

2.5 R² = 0.346578666779679

1.5

0.5

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Cu level (mg/kg)

Note: - (A1:6%, A2:10%, A3:14%, B1: 45 min, B2: 90 min, B3 :135 min)
Figure 1. Correlation of protein content and Cu heavy metal.
Figure 1 The lowest protein levels were found in the control treatment while the highest levels were
found in soaking with seaweed extract at 14%. The correlation test results between heavy metal levels
and protein content showed negative correlation results, this is to the research of [16] that the more the
presence of heavy metal Cu in fish meat is followed by a decrease in fish protein content.

3.4. Organoleptic test of freshwater mussel meat

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Organoleptic parameters are assessed based on an assessment using the five senses. Organoleptic
testing is carried out by looking at several parameters: appearance, odor, and texture. Observations
were made on mussel meat that had been soaked with extract concentrations of 6%, 10%, and 14%,
and soaking times of 45 minutes, 90 minutes, and 135 minutes. Determination of the organoleptic
physical deterioration of mussel meat is carried out using a score sheet that has been determined by
[13].

6
Organoleptic value

4 Appreance

Odor
3
Texture
2

0
Control A1B1 A1B2 A1B3 A2B1 A2B2 A2B3 A3B1 A3B2 A3B3

Treatment

Figure 2. Organoleptic value of freshwater mussel meat.

Based on the results of the analysis of variance (ANOVA) test on the appearance, odor, and texture
values, it is known that the P-value of appearance is 0.057, the P-value of odor is 0.444, and the P-
value of texture is 0.770. This value shows that the P-value > 0.05 indicates that the provision of
seaweed extract treatment with different concentrations and soaking times has no effect on the
organoleptic values of appearance, odor, and texture of the meat.
The appearance value of freshwater Mussel meat after soaking in 6%, 10%, and 14%
concentrations of seaweed extract with a soaking time of 45 minutes, 90 minutes, and 135 minutes has
the highest average value in soaking with 10% seaweed extract for 45 minutes, namely 8.44 and the
lowest value in soaking 14% extract for 135 minutes, namely 7.69 which is presented in Figure 2. This
shows that the longer the soaking of the meat with seaweed extract shows a decreased appearance but
is still within the criteria for intact appearance, type-specific meat color, rather bright, and clean
because the average organoleptic value for appearance is 7.92 and this value still meets the quality
value of shark meat. According to [13], the organoleptic value of shellfish that is still suitable for
consumption is at least 7. Seaweed has antioxidant compounds that can inhibit the oxidation process in
cells to prevent or reduce damage.
The odor of freshwater mussel meat after soaking seaweed extract concentrations of 6%, 10%, and
14% with a soaking time of 45 minutes, 90 minutes, and 135 minutes has the highest average value of
14% extract treatment with a soaking time of 90 minutes, namely 7.98 with fresh odor specifications.

7
While the lowest average value of the odor of mussel meat is found in the control treatment, which is 7
with a fresh odor specification. According to [7], seaweed extract is sufficient to prevent biochemical
changes in the body of fish for a long time to prevent spoilage, especially fish with a lot of water in
their bodies.
The texture of freshwater mussel meat after soaking in seaweed extract concentrations of 6%, 10%,
and 14% with a soaking time of 45 minutes, 90 minutes, and 135 minutes has the highest average
value in the treatment of 14% extract with a soaking time of 45 minutes, namely 8.04 with elastic,
compact, and dense specifications. While the lowest average value of the texture of the meat was
found in the treatment of 14% extract with a soaking time of 135 minutes, namely 7.58 with elastic,
compact, and less dense specifications. This shows that seaweed extract can maintain the texture of the
meat well. According to [17], seaweed contains carrageenan which is one of the ingredients of
seaweed that plays a role in texture formation, in fish or meat products, the use of carrageenan to
maintain texture and prevent the release of fat from tissues and can absorb water. This shows that
carrageenan in seaweed can control the texture of blood clam meat.

4. Conclusion
The best concentration and length of soaking to reduce heavy metal Cu levels in Freshwater Mussel
meat (Pilsbryoconcha exilis) was obtained in the treatment of 14% seaweed extract with a soaking
time of 135 minutes, resulting in a reduction value of 1.298 mg/kg or 87.10%, the concentration of
seaweed extract gives an effect on the addition of protein levels in the meat found in soaking with 14%
seaweed extract and the correlation value of the correlation between Cu and protein is r = 0.557 which
means a moderate correlation, and the organoleptic test (appearance, odor, and texture) the P-value >
0.05 which means that the treatment does not have a significant effect on the organoleptic meat of
mussels.

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