The Effect of seaweed (Eucheuma cottonii) extract and duration of soaking on reduction of Copper (Cu) level in Freshwater Mussel (Pilsbryoconcha exilis)

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 iota carrageenan (65%) which is a sulfated polysaccharide containing hydroxyl groups and carboxyl groups capable of retaining and accumulating heavy metals. In addition E. cottoni as a biosorbent in the aquatic environment. 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%.


Introduction
Eucheuma cottonii is a type of Rhodophyceae seaweed that can absorb heavy metals because it has a chemical content such as iota carrageenan (65%) [1].Their cell wall polysaccharides contain sulfate groups capable of retaining and accumulating heavy metals [2].The content of phytochelatin compounds contained in seaweed serves to accumulate and depurate heavy metals [3,4].Based on research by [5] the 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%.In addition Algae and seaweed biomass has emerged as a sustainable solution for the production of environmentally friendly adsorbents [6].
Already known that Freshwater mussels are as filter feeders and are populated abundantly in the Barumun River.The Barumun River has tributaries, namely the Simangayat River in Asam Jawa Village, Torgamba, Labuhan Batu Selatan, and Samak Reservoir.Around the river and reservoir are oil palm plantations, factory waste disposal, and anthropogenic activities.The utilization of Freshwater mussels is limited to community consumption as daily food.The community widely uses the Barumun River 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 [7].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 these backgrounds, research is needed on the effect of seaweed (E.cottonii) extract and duration of soaking on the reduction of Copper (Cu) levels in Freshwater mussels (P.exilis).

Research site and sample collection
The research was carried out in April until 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 o 33'16.823"LS and 119 o 25'27.412"BT because this seaweed grows abundantly, and the waters are not polluted (atoxic).Preparation of seaweed extracts and phytochemical screening in Plant Physiology Laboratory, Faculty of Mathematics and Natural Sciences, University of Sumatera Utara, Medan, Indonesia.Protein test was undertaken in the Biochemistry Laboratory, Faculty of Mathematics and Natural Sciences, University of Sumatera Utara, Medan, Indonesia, and Testing for Copper (Cu) was conducted in the Regional Health Laboratory Technical Implementation Unit of North Sumatra Province, Medan, Indonesia.

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.

Sampling of seaweed (Eucheuma cottonii).
Before seaweed sampling, in situ and ex-situ 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.(Cu) testing on freshwater mussels.Initial Cu heavy metal testing was carried out on mussel samples as controls (without seaweed extract immersion) which will be used as comparison data.

Preparation of Eucheuma cottonii extract.
The preparation of Eucheuma cottonii extract was made by the maceration method.Maceration is a simple method of extraction that involves soaking plant-prepared material in crude or powder form in a solvent [8].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 were extracted using the maceration method 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 being maceration for 3 x 24 hours the ethanol solvent was evaporated with a rotary evaporator for 6 hours to form a thick extract of seaweed [9].

Phytochemical screening test of Eucheuma cottonii extract.
Phytochemical screening is the first step to identify the compound content in the simplisia or plants that will be tested [10] as follows: Alkaloid Test, 1 ml of extract sample is added with a little 1% HCl, then 1 ml Mayer reagent.The presence of a precipitate or turbidity indicates an alkaloid compound.The Flavonoid analysis was carried out with 1 ml of extract sample mixed with Mg powder and some drops of HCl concentrated.The emergence of pink, magenta, and orange colors indicates flavonoid compounds.Saponin Test, 1 ml of extract sample is added to 10 ml of aquadest and shaken 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.For the Tannin Test, 1 ml of extract sample is added to 10 ml of aquadest and boiled.Add a few drops of FeCl3.The presence of brownish green or bluish-black color indicates a tannin compound.

Determination of Eucheuma cottonii seaweed extract concentration.
The concentrations used were 6%, 10%, 14% b/v, and without soaking seaweed extract.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% weigh 14 grams of seaweed extract and then dissolve it with aquadest as much as 100 ml.

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 finishing the soaking process, the mussels were drained with filter paper.The drained mussel samples were immediately subjected to organoleptic tests first and then tested for heavy metal Copper (Cu) content.[11].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 in reducing heavy metal levels in Freshwater Mussels.Samples that have been mashed are weighed as much as 5 grams in a porcelain cup, then ashes in the furnace, gradually increasing the furnace temperature by 100 o C every 30 minutes until it reaches 450 o C and maintained for 18 hours.After the ash is formed completely white, cool the sample at room temperature, add 5 mL of 6 M HCl to the sample, and homogenize, then evaporate on a hot plate at 100 o C until dry.After drying, add 10 mL of 0.1 M HNO3 homogenize and cool at room temperature for 1 hour, then transfer the solution into 50 mL measuring flask (polypropylene), the finished solution was read on a flame atomic absorption spectrophotometer (AAS) with the wavelength of 324.8 nm.According to [13] After obtaining the actual concentration, the percent reduction of heavy metals is then calculated using the formula: × N × 14.007 × 100%

Copper (Cu) heavy metal content test
After obtaining %N, the protein content is then calculated by multiplying a factor.
% Protein = % nitrogen × conversion factor (6.25) (4) Description: N : Normalcy of standard HCl used 14.007 : Nitrogen atomic weight 6.25 : Protein conversion factor value for fish 2.4.4.Organoleptic test.Organoleptic testing was carried out after soaking according to [15] the method using a fresh mussel organoleptic score sheet with 30 untrained panelists.Organoleptic uses a scoring test with the criteria of score 1 as its lowest value and score 9 as its highest value.

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.

Phytochemical screening of Eucheuma cottonii extract
The results of the screening test of phytochemicals from Eucheuma cottonii in this study is presented in Table 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 [16] secondary metabolite compounds flavonoids, alkaloids, and tannins can bind metal ions.Some flavonoids can chelate metal ions by forming stable complexes through multiple hydroxyl groups (-OH) and carbonyl groups.In addition, the opinion of [17] is that the antioxidant mechanism of triterpenoids is by capturing/scavenging reactive species, such as superoxide, and chelating Fe 2+ and Cu 2+ metals.

Initial copper (Cu) and effectiveness of copper (Cu) metal content reduction
The test result of copper (Cu) levels in freshwater mussels (Pilsbryoconcha exilis) in every treatment is presented in Table 2.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 higher the concentration given and the longer the soaking time can reduce heavy metal levels.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 [18] Absorbent of Gracilaria fisheri red algae pre-treated for cadmium (Cd) and copper (Cu).The highest uptake values were 0.63 and 0.72 mmol/g, respectively.The absorption rate of cadmium and copper was highly rapid with 90% of biosorption finished in 30 min.
Surface adsorption has been the most widely used method to sorb pollutants due to its benefit-cost relationship which is determined by the lower cost of maintaining lifeless biomass and high adsorption results gained by algae biomass related to its complex cell walls properties ( high alginate, fucoidan, and high carboxyl group content), which enhance its ability to adsorb pollutants [19,20].The future trend shows the enhancement of biosorption nature through developing large-scale surface modification methods to improve metal sorption.Biomass or polysaccharides mobilization and a hybrid of biosorption mechanisms using a chemical and biological approach are needed to improve the selectivity of a specific pollutant with a high capacity of biosorption [21].

Correlation of protein content and Cu heavy metal
Based on the results of research on heavy metal levels and protein content in mussels, show 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 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.The correlation value between Cu levels and protein content is r = 0.557 which means a moderate correlation.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 [22] that the more the presence of heavy metal Cu in fish meat is followed by a decrease in fish protein content.In addition, The protein content is significantly impacted by the total concentrations of Pb, Fe, Cr, Cu, and Cd in the fish and muscle [23].The content of protein was linked to the chemical quality of the water.

Organoleptic test of freshwater mussel meat
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.Based on the variance analysis results (ANOVA) test on the appearance, odor, and texture values, 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 does not affect 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 by soaking times 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 fish meat.According to [15], the organoleptic value of mussels is still acceptable for consumption 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% by 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.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 [9], 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 by soaking time of 45 minutes, namely 8.04 with elastic, compact, and dense specifications.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 [24], 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.

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.The utilization of seaweed as a heavy metal reduction agent in mussels can also be used as a natural biosorbent in the aquatic environment and the biosorbent from a natural waste-based approach provides promising results compared to other methods in terms of low cost, operational cost, and cost of treatment.

Figure 1 .
Figure 1.Correlation of protein content and Cu heavy metal.

Table 2 .
Decrease in copper (Cu) heavy metal levels in mussel meat.