IDENTIFICATION OF PHYTOCHEMICAL COMPOUNDS OF ETHYL ACETATE EXTRACT OF BULUNG ANGGUR ( Caulerpa sp.) BY GC-MS AND TOXICITY TEST ON Artemia salina Leach SHRIMP LARVAE

Sea grape ( Caulerpa sp.) is one of the potential types of seaweed. Sea grape potential as a natural ingredient for making medicine. Balinese people use sea grape for consumption. The content of bioactive compounds in sea grape is suspected to be toxic at certain doses. The purpose of this study was to determine the compound content of ethyl acetate extract of sea grape ( Caulerpa sp.) and the toxicity on Artemia salina Leach. The identification of the bioactive compounds was carried out by Gas Chromatography-Mass Spectrophotometry and the toxicity test of extracts was carried out using the Brine Shrimp Lethality Test (BSLT) method. The results showed that the ethyl acetate extract of sea grape ( Caulerpa sp.) contained 27 chemical compounds, 7 of which had a quality value > 70%, namely Propanoic acid, ethyl ester ; n-Propyl acetate ; sec-Butyl acetate ; Toluene ; Acetic acid, butyl ester ; Bicyclo [4.2.0] octa-1, 3, 5-triene ; and Styrene . The results of the toxicity test of Artemia salina Leach are toxic which had an LC 50 value of 44,070 ppm so that they can be used as a larvicide


INTRODUCTION
Indonesia is known as a country which has a sea area with a high biodiversity county.
Seaweed is one of biological resources there are available abudant in Indonesian waters.In generally, seaweed well-known source of important food hydrocolloids, such as agar, alginates, and carrageenan.In addition, secondary metabolites found in seaweed potential for activity antimicrobial such as antiviral, antibacterial, and antifungal (Suptijah, 2002).Several species of seaweed is potentially developed as a medicine.Sea grape (Caulerpa sp.), locally known as bulung anggur in Bali has potensial nutrient such as carbohydrates, crude fiber, high ash, and low fat content (Tapotubun, 2018).Balinese people have been consuming sea grape as a fresh vegetable and salad.
The previous study reported several beneficial phytochemicals components in the ethyl acetate extract of Caulerpa sp., which exhibited strong antibacterial activity and antioxidant (Marraskuranto et al, 2021).

Preparation of Sample
Samples of fresh bulung anggur (Caulerpa sp.) macroalgae used in this study was collected at Serangan, Bali.Bulung anggur was cleaned from impurities and washed under running water.The sample bulung anggur is thoroughly rinsed with water, dried with air at room temperature for 4 days, then oven-dried at a temperature of 45 0 C for 3 days until a constant weight was obtained.

Extraction with Macerated Method
The dried sample was blended and sieved resulting in powder.250 g of sample powder was macerated by 3 L of ethyl acetate for a period of three days with regular shaking.
The extract is roughly filtered with filter paper and funnel.The total filtrate with extraction carried out in double, then evaporated at 35 0 C until the viscous extract obtained.

Identification Bioactive Compounds Using
Gas Chromatography-Mass Spectrometry

(GC-MS)
Analysis of sample bulung anggur was performed by using GC-MS machine in Denpasar Police Forensic Denpasar.A volume of 1µl was injected in injector with temperature was set at 290 0 C for 27 minutes.
Nitrogen was used as a carrier gas at a constant flow of 1.0 ml/min.

Identification of the bioactive compounds in the ethyl acetate extract of Caulerpa
The chromatogram of gas chromatography analysis is shown in Figure 1  Bicyclo [4.2.0] octa-1, 3, 5-triene can be used as medicine mixture (Firdouse, 2019).
Table 2 shows the result of Caulerpa extract on A. salina.
A. Salina Synthetic sea water was prepared by dissolving 40 g of salt without iodine in 2000 mL of water which was irradiated with 25 watt fluorescent lamp.At the same time, aeration is regulated by utilizing an aerator.Then enter the amount of approximately 1 mg of A.salina eggs into the vessel that already contains seawater.The eggs hatch after 24-36 hours.A period of 2 to 3 days, larvae into instar level 2 and larvae can be used as test animals.Toxicity Test The test solution is made with a concentration of 500 ppm, 250 ppm, 125 ppm, 50 ppm, 10 ppm, and 0 ppm as a control or without added extract.The sampel was added Dimethyl sulfoxide (DMSO) as much 10 µl to dissolve in 5 mL of a suitable solvent into each test tube.Put 10 larvae of A. salina into the test tube and add seawater to a final volume of 5 ml so that the final results of the test solution are obtained with concentrations of 500 ppm, 250 ppm, 125 ppm, 50 ppm, and 10 ppm.The control group was only given 5 ml of seawater and added DMSO without added extract.Each concentration had five replications.The total number of A. salina was used 300 larvae.The percentage of dead A. Salina observed after 24 hours an was calculated.The standard criteria for assessing the mortality of A. salina larvae is that the larvae do not show movement of observation.The toxicity test was assessed by determining the LC 50 score.Eq. 1 shows the % larvae mortality equation...........................................................................(1) The LC50 score is defined as the concentration of a compound causing 50% mortality of larvae A. Salina.Data were analyzed by probit in linear regression y = mx + b was carried out using the Microsoft office excel.The level of toxicity of a compound was classified according to Hamidi et al., 2014.It was toxic with high to low intensity when the LC50 of < 1000 mg/L and it was non toxic when the LC50 > 1000 mg/L.
Total larva mortality was obtained by adding up the larvae that died at each concentration.The highest number of deaths occured at a concentration of 500 ppm an the lowest occured at a concentration of 10 ppm.Meanwhile in the control batch there is no the death of the larvae shrimp, it means caused by substance contained in extract, not from factors beyond control such as temperature, humidity, light intensity and the lack of other food sources.The result of each organic solvent partitioning revealed a different effect on the mortality rate of the tested Artemia salina larvae shrimp.This was caused by the different extractive substance content in each solvent.Toxicity evaluation via BSLT method required high precision, because many factors can affect the mortality of larvae shrimp Artemia salina L. The shrimp larvae are very sensitive to any substance presence within their habits.Their skin is a thin membrane circumstances which allow diffusion of substances from environment, affecting their metabolism.In addition to their sensitivity to the environment.Figure 2 shows the graph

Figure 2 .
Figure 2. Graph of the log relationship between the concentrations of bulung anggur extract

Table 2 .
The result of Caulerpa extract on A. salina