Study of content and bioconcentration of heavy metal (Pb,Hg, and Cd) in green mussels ( Perna viridis ) based on different sizes and ages

Green mussel ( Perna viridis ) has filter-feeding habits and a sessile way of life, with the main food components of plankton and organic matter in the waters. These food components can contaminate green mussels when it accumulates heavy metals. Therefore, this study aims to evaluate heavy metals content and the bioconcentration of lead (Pb), mercury (Hg), and cadmium (Cd) in green mussels based on different sizes and ages. The study was conducted on Pasaran Island, Lampung, Indonesia, and used green mussels aged 3, 5, 7 to 9 months as samples, with 2 replications. The AAS ( Atomic Absorption Spectrophotometry ) method was used in the analysis of Pb, Hg, and Cd, sampling technique used purposive sampling. The result showed that green mussels at different ages and sizes has impact on Pb content, while an increase of one unit of age and size of green mussels has no effect on the metal content of Hg and Cd. Furthermore, the highest bioconcentration of Pb and Hg was obtained at 9 months, while Cd was observed at 3 months. Based on the results of this study it can be concluded that the simple linear regression appropriate a functional relationship between Pb and the different ages and sizes green mussels cultivated on Pasaran Island , while heavy metals Hg and Cd appropriate with a polynomial quadratic approach and almost do not exist in the form of a functional relationship between Hg and Cd with the different ages and sizes green mussels. Even though it contains heavy metals in below the maximum limit, but care needs to be taken in addressing this phenomenon due to its nature heavy metals that can accumulate in living tissue, thus consuming green mussels containing heavy metals even in relatively small amounts is not recommended.


Introduction
The presence of heavy metals in coastal areas can result from natural processes such as rock weathering and volcanic activity (Peng et al., 2018), as well as inputs from the mainland, namely industrial, agricultural, mining, and household activities (Duodu et al., 2017).The impact of these activities increases the amount of waste that flows into water bodies through rivers and empties into the sea, where it is subsequently absorbed by aquatic biota.
This condition has a negative effect on the ecosystem since it is difficult to degrade, undergoes bioaccumulation (Saher and Siddiqui, 2019) and biomagnification (Vandecasteele et al., 2004), as well as toxic when it exceeds certain limits (Rosado et al., 2016), which can then absorbed by aquatic biota, particularly shellfish, such as the green mussel (Perna viridis).
Plankton and organic matter are the main food components for green mussels in the waters due to their eating habits which are filter feeders, and their sessile way of life.These components can contaminate green mussels when they accumulate heavy metals.According to Haryono et al. (2017), the concentration of heavy metals in green mussels in Lekok waters, Pasuruan Regency, is 3.5 times higher than in plankton.This indicates that green mussels can accumulate heavy metals from water and sediments (Maharani et al., 2019), thus, they are recommended as heavy metal biofilters.
The types of heavy metals found in live fish on the coast of Bandar Lampung City are lead (Pb), mercury (Hg), and cadmium (Cd).The Pb metal content found in green mussel meat has a concentration of up to 0.012 mg/kg, which has exceeded the quality standard threshold according to Minister of Environment Decree No. 51/2014, which is 0.08 mg/kg (Sembel, 2012).Furthermore, according to Romandhoni, (2021), the waters of Pasaran Island were contaminated with heavy metal Hg at concentrations of 0.004-0.009mg/l, which surpassed the quality level of 0.001 mg/l set by Ministerial Decree No. 51/2014.This is similar to the observation of Cd metal content in sediments of 7.41 mg/kg in the coastal area of Bandar Lampung City (Pratiwi et al., 2016), where this value has exceeded the quality standard sediment of 1.5 mg/kg set by Minister of Environment Decree No. 51 of 2014.
Based on a study which refers to Sembel (2012) can be concluded that heavy metals Pb, Hg, and Cd have polluted the coastal waters of Bandar Lampung City.Thus, caution is needed in responding to this phenomenon because of the nature of heavy metals that can accumulate in green mussels and impact green mussel consumers.Another study also stated that there was a significant correlation between shellfish and heavy metal concentrations in some coastal areas in Indonesia namely the coast of Makassar City in Metro Tanjung Bunga waters which are polluted by lead (Pb) metal of 0.11 mg/l (Setiawan et al., 2014).Furthermore, Rahayu (2022) reported that the Krueng Sabee river which is located in Aceh Jaya has been polluted by the heavy metal mercury (Hg) of 0.76 mg/l.Cadmium (Cd) heavy metal contamination was also observed in Matras Waters, Bangka Regency of 0.04 mg/l (Nugraha et al., 2022).Singh (2017) reported that the shell size influences the metal content, and the larger the size, the older the age, hence, heavy metals accumulate over time.The ability to absorb and accumulate heavy metals correlates with the reproductive stage.This is consistent with the report of Assaduzzaman et al. (2019) that fish absorbs more plankton as a source of energy and nutrients for gametogenesis during the reproductive phase (aged ± 6 months).However, there is no report on the evaluation of heavy metal contamination based on different sizes and ages.This study is used to evaluate the content of heavy metals, including Pb, Hg, and Cd, based on size and age.It is also used to monitor the environmental pollution and safety of food, as well as heavy metal exposure on green mussels consumed by the public.

Materials and Methods Location and time of research
This study was conducted in the Pasaran Island waters, Lampung, Indonesia, using the raft method to obtain green mussels from floating net cages.(Figure .1)shows the area of Parasan Island water, located within 1 km from the mainland.This study aims to determine the contamination of heavy metals originating from the flow of Lampung Bay on green mussels.The mussels were aged 3, 5, 7 to 9 months, and each was carried out with 2 replications.

Research procedure Sample collection
A Purposive sampling technique was carried out, and the handling referred to the Indonesian National Standard (SNI) 06-241206- -199106- (SNI, 2008)).The sampling was conducted on green mussels cultivation rafts in the southeastern part of Pasaran Island, with 2 replications.Green mussles samples were collected on cultivation rafts in the southeastern part of Pasaran Island.The distance of this point from the mainland is 1 km.The purpose of sampling at that point is because at that point it is usually used as a location for green mussel cultivation.

Parameter analysis Heavy metal analysis
The AAS (Atomic Absorption Spectrophotometry) method was used in the analysis of Pb, Hg, and Cd, which refers to SNI 01-2354.6-2006.

Bioconcentration factor (BCF)
The bioconcentration factors are analyzed by calculating the ratio between the heavy metal content in green mussels' meat and seawater (bio water factor) at the study location (Januar et al., 2019).This value

Data Analysis
The statistical analysis was conducted using simple linear regression, and SPSS software 25.0 was used to conduct all pairwise comparison tests.

Results
(Figure 2) illustrates the fluctuation in heavy metals content and their bioconcentration in green mussels of various sizes and ages using the shell length measurements.(Table 1) depicts the length of green mussels cultivated on Pasaran Island, Lampung, at ages 3, 5, 7, and 9 months.Green mussels cultivated on Pasaran Island, Lampung at ages 3 months was sized (1.5-3.0cm) at thus stage green mussles was started to sticked at the substrate, at 5 months (4.5-6.0cm),7 months (6.0-7.5cm)green mussles at reproductive stage they absorbs more plankton as a source of energy and nutrients for gametogenesis during the reproductive phase (aged ± 6 months).At 9 months (8.0-10.0cm)green mussles has ready to harvested.Based on the data presented in Table 1, it can be seen that every increase age of green mussels will increase the size annually.
Table 2. Results heavy metals content, water quality and bioconsetrations green mussels.
The content of all heavy metals in the waters and in green mussel meat was fluctuated at the beginning of sampling also at the end of sampling, this was caused by various factors such as the type of heavy metal, the ages or size of organisms, the length of exposure, and the conditions of the aquatic environment such as temperature, pH, and salinity.The Pb metal content in green mussels of different sizes and ages is presented in graphical form, as shown in Figure 3, indicating that the metal Pb concentration increased with increasing size at the age of 9 months of cultivation (8.0-10.0cm).This is due to the ability of green mussels to accumulate heavy metals.
The data in (Figure 2) shows The simple linear regression equation of y = 0.0024x+0.019between Pb and the different ages indicates that for every one unit increase in the age of green mussels, the heavy metal content will increase by 0.0024 mg/kg.Coefficient of determination (R 2 ) was obtained for 0.8864 which indicated that the effect of age and size of green mussels on the content of heavy metal Pb was 88.64%.The pattern obtained is that with increasing size and age of green mussels, the heavy metal content of Pb also increases At the medium size (7 months;6.5-7.0cm), the metabolic processes of green mussels peaked and then experienced a decline in development at the large-size stage.Due to the decrease in the metabolic process, the ability to eliminate metals also decreased, hence, the concentration of Pb metal in large green mussels is higher than in small and medium sizes (7 months).Yaqin et al. (2015) stated that the high content of heavy metals is related to the time they accumulate in green mussels.The accumulation occurs because there is a tendency for heavy metals to form complex compounds with organic substances in the body of green mussels.This condition prevents green mussels from immediately excreting heavy metals (Haryono et al., 2017).

Hg Content in Green Mussels Based on Different Ages
Variation of Hg metal concentrations in green mussel meat was observed to be relatively high at 3 and 7 months of age, whereas in green mussels aged 5 and 9 months it was relatively low.This is because heavy metals that enter the waters will experience precipitation, dissolution and dispersion and then be absorbed by organisms that live in the waters.Precipitation of heavy metals occurs due to the presence of carbonate, hydroxyl and chloride anions (Hutagalung, 1984).
Based on (Figure 3) Hg content in green mussels of different sizes and ages was above the allowable threshold for consumption based on Ministerial Decree No. 51 of 2021, namely 0.001 mg/kg.Determination the coefficients of orthogonal polynomials is carried out because age and size almost do not exist in the form of a functional relationship with content of mercury (Hg) with a coefficient of determination (R 2 ) of 0,9558 which means that this data phenomenon is appropriate 95.58% with a polynomial quadratic approach.
Hg levels in sediments generally have higher levels than in water, this is due to the nature of the metal material, in accordance with what Hutagalung (1984) said that heavy metals that enter the waters will experience precipitation, dilution, and dispersion, then be absorbed by biota living in these waters.Hutagalung (1991) said heavy metals have properties that easily bind organic matter and settle to the bottom of the waters and unite with sediments so that the levels of heavy metals in sediments are higher than in water.
The results of research conducted by Rudiyanti (2007) generally, the larger the size of the shells, the concentration of the heavy metal Hg will decrease.The filtering process in bivalves enters through incurrent siphons and gill filters.The gills of green mussels have mucus or mucus, the main constituent of which is glycoprotein, so mercury is bound to become metallothionein because the main constituent is the system, a protein belonging to the sulfhydryl group which is capable of binding metals.Due to the nature of the regenerated gill mucus, Mercury which has been bound to the gill mucus is also released from the body.Related to the mechanism of the filter feeder, seawater flow will continue towards the labial palps where in that part it will go through a filtering process with the cilia.The small particles will escape, while the large particles will be ejected through the siphon-incurrences in the form of pseudofoces Hutagalung (1991) Tan (1977) also said that green mussels can choose (selectively) their food where non-food is released in the form of pseudofaces covered with mucus.This is thought to be one of the factors reducing the mercury content in green mussel meat aged 9 months (8.0-10.0cm).Mussels can undergo a process of elimination of heavy metals by way of  Krismonita et al. (2023) excretion through feces or urine.In addition, factors that affect the level of accumulation of heavy metals are the type of heavy metal, the type or size of the organism, the length of exposure, and the environmental conditions of the waters such as temperature, pH and salinity.

Cd Content in Green Mussels Based on Different Ages
The fluctuating Cd concentration of 0.003-0.008mg/kg on green mussles meat, also on sea water of 0.014-0.04mg/l at Pasaran Island , caused by the flow and intensity of the activities of the surrounding community, is a factor in the decreasing concentration of heavy metals and the increasing size of the shellfish.The Cd content was high at 3 months, then decreased with an increase in age.Based on Figure 4 the results of research on the Cd metal content in green mussels of different sizes and ages was suitable for consumption based on BPOM in 2016 is 0.03 mg/kg.Determination the coefficients of orthogonal polynomials is carried out because age and size almost do not exist in the form of a functional relationship with content of Cadmium (Cd) with a coefficient of determination (R 2 ) of 0,9156 which means that this data phenomenon is appropriate 91.56% with a polynomial quadratic approach.
According to Wardani et al., (2018) the tendency to decrease the value of Cd metal concentrations in large shells indicates the phenomenon of growth delution.The phenomenon of growth delution of heavy metal accumulation is often found in studies related to bivalves.Several reasons related to growth delution, namely that the growth delution mechanism is suspected to be closely related to the way bivalve shells eat, namely filter feeders.The main constituent of the gill membrane layer is a single layer of squamous epithelium and is directly related to the vascular system, and it is suspected that heavy metals that enter together with food particles diffuse through the gill membrane and are carried by the blood stream.Bivalve gills, including green mussels, have mucus or mucus, the main constituent of which is glycoprotein.Then it is suspected that the metal is bound to become metallothienin because its main constituent is cysteine, a protein belonging to the sulfhydryl (-SH) group which is capable of binding metals.Due to the nature of the regenerated gill mucus, the heavy metal cadmium which has been bound to the gill mucus is also released from the body.As for the mechanism of the filter feeder, the flow of seawater will continue towards the labial palp where it will go through several filtering processes with the cilia.Small particles will pass, while large particles will be expelled again through the incurrent siphon in the form of pseudofeces.This is also thought to be a factor in decreasing cadmium concentrations along with increasing body size (Wardani et al., 2018).

Bioconcentration Green Mussels Based on Different Ages
The bioconcentration value of green mussels on lead (Pb) and mercury (Hg) is BCF> 1.This indicates that green mussels can accumulate lead (Pb) and mercury (Hg).Furthermore, the BCF value of green mussels in Pb and Hg metals at 3 months was lower than at 9 months.This shows that the ability of green mussels to accumulate Pb and Hg metals is higher in large shells.This result is consistent with the report of Davies et al. (2006) that the BCF value of shellfish in accumulating Pb metal was higher at large sizes.The greater the number of green mussels, the lower the value of the bioconcentration factor.This is because many individual organisms carry out the absorption process, thus the substance absorbed will be much more evenly distributed among several individuals (Testi et al., 2019).
The Hg content in fish or shellfish depends on the existing geochemical cycles (Fahruddin, 2010).The bioconcentration value of green mussels on cadmium metal (Cd) originating from industrial waste on Pasaran Island was similarly reported to be BCF>1 in relatively younger green mussels (3 months) and BCF1 in older green mussels (9 months).It is believed that the heavy metal Cd in green mussel meat translocates to other bodily tissues.This phenomenon is consistent with the report of Hasani et al. (2021) that the heavy metals contained in water hyacinth (Eichhornia crassipes) translocate to other tissues, namely stems, and leaves, as indicated by the magnitude of the translocation value.
According to Lu (2006), various organisms, including invertebrates, have metallo-thienin (MT), a metal-binding protein in their bodies.The main function of MT is to regulate essential minerals and detoxify toxic metals, such as Hg and Cd. Haryanto et al. (2017) reported that heavy metals can accumulate in the body of organisms through the food chain, which will eventually endanger human health.This condition is referred to as biomagnification.In addition, Rochyatun et al. (2005) asserted that the non-degradability of heavy metals in the environment is the primary cause of their transformation into hazardous pollutants

Conclusion
Based on the data obtained, it can be seen that for every increase of one unit age of green mussels, it will increase the heavy metal content of Pb in green mussel meat by 0.0024 mg/kg, while an increase of one unit of age and size of green mussels has no relationship on the metal content of Hg and Cd.The highest variation content of Pb at green mussles meat was 0,041 mg/kg at the age of 9 months (8.0-10.0cm), Hg and Cd of 0.008 mg/kg at age 3 months (1.5-3.0 cm).The highest bioconcentrations of heavy metals Pb of 6,31 and Hg 3,75 were obtained when green mussel meat was 9 months old, while the highest bioconcentration of heavy metals Cd of 1,21 was found in green mussel meat at a relatively younger age (3 months).

Figure 1 .
Figure 1.Maps showing the location (square) of the mussel farming in Pasaran Island, Indonesia.
Green Mussels Based on Different AgesAll concentrations of Pb metal in green mussels indicate that these shells are not suitable for consumption.This is because the concentration of Pb metal are above the allowable threshold for consumption based on Minister of Environment Decree No. 51 of 2014, namely 0.08 mg/kg.

Figure 2 .
Figure 2. The content of Pb green mussels based on different sizes and ages.

Figure 3 .
Figure 3.The content of Hg green mussels based on different sizes and ages.

Figure 4 .
Figure 4.The content of Cd green mussels based on different sizes and ages.

Figure 5 .
Figure 5.The value of the bioconcentration factor (BCF) of metals Pb, Hg, and Cd in green mussels at the age of 3 and 9 months.Several factors can affect the value of bioconcentration in organisms, including the size or number of organisms, the time that contamination occurred, and the concentration of substances in the media(Arnot and Gobas, 2006;Usman et al., 2019).

Table 1 .
Results of size and age of green mussels.