Bioremediation of Petroleum Contaminated Water Using Oil Spill Dispersant and Lemna minor in Laboratory Scale of Constructed Wetland

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Yani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5885729/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Petroleum pollution due to industrial activities is a significant environmental problem, especially when polluting water resources. This study aims to evaluate the effectiveness of using oil spill dispersant (OSD), constructed wetlands, and phytoremediation using Lemna minor in improving the quality of petroleum-polluted water. The experiment was conducted using a group randomized design with a combination treatment of petroleum-based commercial OSD (Non-Bio-OSD) and environmentally friendly palm oil-based OSD (Bio-OSD) in a laboratory-scale constructed wetland system. The results showed that Bio-OSD significantly reduced COD and BOD₅ levels to meet water quality standards. The highest COD reduction efficiency of 39.78% was achieved when Bio-OSD DOR 0.1:1 treatment was implemented. Under this treatment, BOD reduction efficiency was 27.60%. GC-MS analysis showed the degradation of long-chain hydrocarbons such as n-hexadecane and nonadecane. The highest COD Reduction efficiency by Non-Bio-OSD was 27.17% with DOR 0.25:1. This result showed that Bio-OSD performed better in reducing COD than Non-Bio-OSD. The weight of Lemna minor biomass decreased slightly during the process, indicating that OSD is toxic to the plant. Regardless of the successful construction of wetlands in reducing COD and BOD 5 , it is recommended that the growth condition of the aquatic plant be improved for a sustainable phytoremediation process. Environmental Engineering Constructed wetland crude oil Lemna minor oil spill dispersant phytoremediation water quality Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Environmental pollution caused by oil spills remains a significant global challenge, particularly in resource-rich countries like Indonesia, which has abundant oil and gas reserves. Oil spills frequently result from tanker accidents, leaks in subsea pipelines, and industrial activities related to oil extraction and refining, all producing hazardous waste. These incidents lead to substantial economic losses and severe ecological damage, disrupting marine ecosystems and threatening biodiversity (Prince 2015 ). Petroleum contains toxic organic compounds such as benzene, toluene, ethylbenzene, and xylene (BTEX), posing significant risks to aquatic and terrestrial ecosystems. In aquatic environments, spilled oil forms a thick layer on the water's surface, blocking the penetration of oxygen and sunlight. This disrupts the photosynthesis process in aquatic plants and harms aquatic organisms, leading to ecological imbalances and declining biodiversity. On land, oil contact with soil alters its structure and fertility, degrading terrestrial habitats, impairing nutrient cycling, and inhibiting plant growth (Yavari et al. 2015 ). In Indonesia, crude oil production has reached 386,395.79 barrels, with oil spills accounting for 9,110.20 barrels, underscoring the environmental impact of oil pollution. Oil spill dispersants (OSD) are commonly used to manage oil spills. OSDs break down oil into smaller droplets, increasing their surface area and promoting microbial degradation. These formulations combine chemicals and surfactants to enhance dispersal efficiency and mitigate environmental damage (Brakstad et al. 2018 ). The Surfactant and Bioenergy Research Centre (SBRC-IPB) has developed a more effective, environmentally friendly Bio-OSD derived from palm oil, surpassing commercial performance products (Elvina et al. 2016 ). Oil pollution can be managed through constructed wetlands, which harness plants to purify water naturally. These systems use physical, chemical, and biological processes inherent in wetland ecosystems to remove contaminants. Constructed wetlands are cost-effective, energy-efficient, and have minimal environmental impact, making them ideal for long-term remediation (Mustapha and Lens 2018 ). Phytoremediation, an environmentally friendly method that utilizes plants to absorb, degrade, and stabilize contaminants, can restore polluted environments. Constructed wetlands, as a form of phytoremediation, replicate natural processes to treat petroleum-contaminated water by leveraging the actions of plants, microorganisms, and substrates to remove and degrade pollutants (Ansari et al. 2019 ). Lemna minor , or duckweed, is an effective plant for phytoremediation due to its rapid growth and ability to accumulate contaminants (Gupta and Prakash 2013 ). It plays a crucial role in hydrocarbon phytoremediation, as it can absorb and biodegrade petroleum hydrocarbons, contributing to the purification of polluted water and the restoration of environmental quality (Ekperusi 2019). This study aims to assess the impact of OSD application, constructed wetlands, and Lemna minor phytoremediation on improving petroleum-polluted water quality. Methods Simulation of Petroleum-Polluted Water Petroleum polluted water simulation was conducted using medium crude oil at a concentration of 1% of the water volume. The constructed wetland using 45L containers sized 55 cm x 36 cm x 29 cm. The containers were filled with a substrate consisting of 10 cm of gravel and 10 cm of sand, and the groundwater well was 15L. Crude oil is added to the water at 150mL (Fig. 1 ). L. minor used in the study were acclimatized for seven days, and plants selected for the experiment were uniform in size, fresh, and structurally intact. This study was conducted at the Greenhouse. Experimental design Details of the reactor treatment design, including variations in Dispersant Oil Ratio (DOR), Bio-OSD, and Non-Bio-OSD combinations, are provided in Table 1 . This study used a Completely Randomized Block Design (CRBD) with treatment combinations applied to constructed wetlands conducted in 3 replications. Table 1 Reactor Treatment Design with DOR, Bio-OSD, and Non Bio-OSD Variations Reactor Code DOR Plant Treatment P0 Bio-OSD DOR 0.1:0 L. minor P1 Bio-OSD DOR 0.25:0 L. minor P2 Bio-OSD DOR 0.5:0 L. minor P3 Bio-OSD DOR 1:0 L. minor P4 Bio-OSD DOR 0.1:1 L. minor P5 Bio-OSD DOR 0.25:1 L. minor P6 Bio-OSD DOR 0.5:1 L. minor P7 Bio-OSD DOR 1:1 L. minor P8 Non Bio-OSD DOR 0.1:0 L. minor P9 Non Bio-OSD DOR 0.25:0 L. minor P10 Non Bio-OSD DOR 0.5:0 L. minor P11 Non Bio-OSD DOR 1:0 L. minor P12 Non Bio-OSD DOR 0.1:1 L. minor P13 Non Bio-OSD DOR 0.25:1 L. minor P14 Non Bio-OSD DOR 0.5:1 L. minor P15 Non Bio-OSD DOR 1:1 L. minor Phytoremediation Performance Test Each treatment's contaminant level was tested through daily observations (DO, pH, TDS, and temperature) at the beginning and end of treatment (BOD 5 , COD, hydrocarbons). The DO, pH, TDS, and temperature were measured using AZ instrument 86031 water quality meter during the study. BOD 5 and COD were analyzed at the experiment's initial and end (14 days). BOD₅ is APHA 5210 B, and for COD is APHA 5220 C (APHA 2023). The contaminant removal efficiency of BOD 5 and COD are calculated with the following equation: $$\:\varvec{R}\:\left(\varvec{\%}\right)=\:\frac{{\varvec{C}}_{0}-{\varvec{C}}_{\varvec{t}}}{{\varvec{C}}_{0}}\:\times\:100$$ 1 R = contaminant removal efficiency (%) Co = initial contaminant value (mg/L) Ct = end of study contaminant value (mg/L) At the end of the research, water samples were analyzed for oil hydrocarbons from phytoremediation using gas chromatography-mass spectrometry (GCMS) brand Thermo Scientific and compared with crude oil chromatograms. Observations of the plants were made daily to assess their freshness and growth ability. The biomass (wet and dry bases) of L. minor was observed initially and at the end of the experiment (14 days). Data analysis The data obtained were analyzed using SPSS 27 to examine the Analysis of Variance (ANOVA). If treatments have a significant effect at the actual level of 5%, they are analyzed using Duncan's Multiple Range Test. Result and Discussion Water-Quality Changes Table 2 shows the range of test results for BOD 5 , COD, TDS, pH, and DO parameters from all treatments at the initial and end of the study. BOD 5 and COD levels decreased, although they did not meet the water quality standards. The pH and TDS levels complied with the water quality standards by Indonesian Regulation Number 21 of 2021 concerning Implementing Environmental Protection and Management. However, DO levels dropped from 5.65–6.90 mg/L to 1.35–5.80 mg/L, thus meeting water quality standards. Table 2 Test results of water pollutant levels before and after phytoremediation of petroleum-polluted water. Parameter Unit Analysis Result Quality standards Initial (0 Days) End (14 Days) BOD 5 mg/L 11–41 8–25 2–12 COD mg/L 72–424 62–348 10–80 TDS mg/L 97–150 105–158 1000–2000 pH - 7,23–8,95 7,24–7,78 6–9 DO mg/L 5,65–6,90 0,85–5,80 1–6 a Indonesian Regulation Number 21 of 2021 concerning the Implementation of Environmental Protection and Management Chemical Oxygen Demand (COD) Chemical Oxygen Demand (COD) indicates the level of water pollution by reflecting the amount of organic matter present. A decrease in COD occurs as organic matter in the water decreases, which can be achieved by aquatic plants that absorb the organic matter. A high COD value indicates a large amount of oxidized organic matter, which reduces dissolved oxygen levels in water (Boguniewicz-Zablocka et al. 2020 ). COD reduction data were analyzed using the ANOVA test with a significance level of α 5%, showing significant differences in each treatment. The COD value reduction and COD reduction efficiency are shown in Fig. 2 . COD reflects the level of water pollution by indicating the amount of oxidized organic matter, which contributes to a decrease in dissolved oxygen (DO). Significant COD reduction occurred in all treatments, with statistical test results showing significant differences. On day 0, the lowest COD was found in P4 Bio-OSD DOR 0.1 :1 (208 mg/L), while the highest was in P11 Non-Bio-OSD DOR 1:0 (432 mg/L). On day 14, the lowest COD was found in P0 Bio-OSD DOR 0.1:0 (127 mg/L) and the highest in P15 Non Bio-OSD DOR 1: 1 (348 mg/L). The decrease in COD reflects the reduction of organic matter, with L. minor playing a role in COD removal through organic matter absorption. COD tended to increase with the addition of OSD. On day 14, the decrease in COD was supported by the photosynthesis of L. minor , which increased dissolved oxygen, creating aerobic conditions that accelerated the activity of microorganisms in reducing COD (Aziz et al. 2020 ). The COD reduction was influenced by the absorption of organic matter by L. minor and phytoremediation activities. COD removal efficiency in handling petroleum-polluted water was found in the P4 Bio-OSD DOR 0.1: 1 (39,78%) and lowest in P13 Non-Bio-OSD DOR 0.25:1 (13.67%). The significant decrease in COD value indicates that L. minor can stimulate the growth of microorganisms that play a role in the degradation of organic compounds in wastewater. These microorganisms utilize organic compounds as an energy source, thus increasing the effectiveness of L. minor in reducing organic load and improving water quality (Ugya 2015 ). The COD reduction efficiency achieved in this study is consistent with the findings of (Li et al. 2012 ), who reported a COD reduction of 52–67% using annual grass vegetation such as Geophila herbacea and Lolium perenne L. as phytoremediation agents. These results also align with the research of (Mustapha and Lens 2018 ), which showed a 66–91% reduction in COD using Typha latifolia . The findings confirm that the presence of plants in a phytoremediation system is essential in improving treatment efficiency. This is due to plant metabolic activity, the rhizosphere zone's microbial environment enhancement, and the roots' role in providing contact surfaces for contaminant uptake or transformation. Biological Oxygen Demand (BOD 5 ) Biochemical Oxygen Demand (BOD 5 ) measures the need for dissolved oxygen to oxidize the waste material in water, reflecting the oxygen demand for the decomposition process. High oxygen consumption indicates the presence of waste materials with significant oxygen demand. BOD 5 reduction data were analyzed using the ANOVA test with a significance level of α 5%, showing significant differences in each treatment. The reduction in BOD 5 value and the efficiency of BOD 5 reduction are shown in Fig. 3 . BOD 5 measurements were conducted on days 0 and 14 to assess the efficiency of phytoremediation. On day 0, the lowest BOD 5 was found in the P0 Bio-OSD DOR 0.1:0 treatment (19.61 mg/L), while the highest was in the P15 Non-Bio-OSD DOR 1:1 treatment (40.90 mg/L). On day 14, BOD 5 was lowest in the P0 Bio-OSD DOR 0.1:0 treatment (12.96 mg/L) and highest in the P15 Non-Bio-OSD DOR 1:1 treatment (31.28 mg/L). The highest BOD 5 concentration on day 0 indicates that the addition of 1% Non-Bio-OSD increases biological oxygen demand due to the addition of new organic matter, such as oil spills or industrial waste (Atlas and Hazen 2011 ). The efficiency of BOD 5 reduction in this study ranged from 11.29–37.69%, with the highest efficiency value achieved in the P5 Bio-OSD DOR 0.25:1 treatment. The analysis showed that using Bio-OSD tended to produce a more significant increase in efficiency than Non-Bio-OSD. This can be seen from the graph showing that the Bio-OSD treatment's efficiency is consistently higher than the Non-Bio-OSD treatment. Phytoremediation using L. minor effectively reduces BOD 5 , supported by the activity of microorganisms that decompose organic matter into simple elements that plants absorb. Significant reductions in BOD 5 occurred based on the growth of microorganisms and parameters such as COD, DO, and pH. Microorganisms secrete enzymes that break down complex organic compounds into simple nutrients that are absorbed by macrophytes, thereby reducing organic compound levels and dissolved oxygen use, lowering BOD 5 in petroleum-polluted wastewater (Bhutiani et al. 2019 ). The results of this study are consistent with the findings of (Agarry et al. 2018 ), who reported that using Eichhornia crassipes plants in a phytoremediation system achieved a BOD 5 reduction efficiency of up to 94.6%. In addition, the research of (Rehman et al. 2018 ) This study also supported the results; combining Typha domingensis and Leptochloa fusca plants with bacterial consortium resulted in a BOD 5 reduction efficiency of up to 93%. Comparison of Wastewater Treatment Efficiency Using Constructed Wetland and Alternative Techniques Table 3 compares the treatment efficiencies of petroleum-contaminated water using various constructed wetland methods alongside other treatment techniques, incorporating different plant species. The summarized studies include varying treatment durations, the plant species utilized, and the observed reductions in pollutant parameters such as Chemical Oxygen Demand (COD) and Biological Oxygen Demand (BOD 5 ). This comparison offers valuable insights into the effectiveness of these methods for the remediation of petroleum-contaminated water, highlighting the specific advantages associated with each approach. Table 3 Comparison of petroleum-polluted water treatment efficiency using constructed wetland method and other techniques with various plant types No. Author Methods Plants Result 1 This Research Constructed Wetland using OSD for 14 days L. minor COD efficiency up to 40%, BOD 5 38% 2 (Ugya 2015 ) Constructed Wetland for 3 weeks in wastewater from the refinery L. minor The BOD 5 decreased by 68%, and the COD decreased by 91.6%. 3 (Azeez and Sabbar 2012 ) Constructed Wetland for 1 month L. minor The BOD 5 decreased by 49.6%, and the COD decreased by 32.7% 4 (Li et al. 2012 ) Floating beds Geophila herbacea and Lolium perenne L. The efficiency of COD reduction ranged from 52–67%. 5 (Mustapha and Lens 2018 ) Constructed Wetland Typha latifolia The reduction in COD values ranged from 66–91%. 6 (Agarry et al. 2018 ) Constructed Wetland Eichhornia crassipes The BOD 5 decreased by 80.2%, and the COD decreased by 92.6% 7 (Rehman et al. 2019 ) Floating treatment wetland Typha domingensis and Leptochloa fusca plants with bacterial consortium The efficiency of BOD 5 reduction reached up to 93%. This study made a significant contribution by demonstrating that the constructed wetland system, utilizing Oil Spill Dispersant (OSD) and L. minor for 14 days, achieved a COD reduction efficiency of 40% and a BOD 5 reduction of 38%. The primary advantage of this approach is the relatively short treatment time compared to other studies, highlighting its potential for operational-scale application with faster and more efficient treatment processes. In comparison, studies by (Ugya and Imam 2015 ) and (Azeez and Sabbar 2012 ) Employed L. minor for three weeks and one month, respectively, leading to higher reductions in BOD 5 and COD. However, the longer treatment durations in these studies may present limitations for field applications, particularly in situations that demand a swift response. As such, this research provides value by optimizing treatment efficiency within a shorter timeframe, making it more feasible for field implementation. The combination of OSD with L. minor offers an innovative approach to wastewater treatment, setting it apart from more conventional techniques, such as those employed by (Li et al. 2012 ) using floating beds or (Mustapha and Lens 2018 ) Who utilized constructed wetlands without additional modifications. Incorporating OSD provides an effective solution for treating petroleum-contaminated water, enhancing the overall treatment efficiency and addressing challenges associated with petroleum pollution. Although other studies, such as those by (Agarry et al. 2018 ) and (Rehman et al. 2019 ) Reported excellent efficiencies in BOD 5 and COD reduction; their methods involved longer treatment durations or combinations of plants and microorganisms that require additional management. This study presents a more straightforward alternative with competitive results, making it a promising option for practical applications due to its ease of management. Overall, this study emphasizes the time efficiency and integration of innovative technologies such as OSD and L. minor as a phytoremediation plant, offering a sustainable and practical solution for wastewater treatment. The findings provide a strong foundation for further development and potential large-scale applications. Influence of Environmental Conditions on Water Quality in Constructed Wetland In this study, Dissolved Oxygen (DO) levels are crucial in assessing water quality, with a standard value set at ≥ 1 mg/L according to Indonesian Regulation 21 of 2021. On day 0, the highest DO was found in the P1 DOR 0:0 treatment (6.90 mg/L), while the lowest DO was found in the P2 DOR 0:1 treatment (5.65 mg/L). On day 14, the highest DO was found in the control group (P0) at 5.8 mg/L, and the lowest was in the P5 Bio-OSD DOR 0.5:1 treatment (1.35 mg/L). The addition of surfactant resulted in a significant decrease in DO, reflecting increased microbial activity and consuming more oxygen (Arora et al. 2022 ). DO is essential for aquatic life, with optimal levels for fish between 5–6 mg/L (Ali et al. 2022). High DO levels indicate lower pollution; low levels indicate higher organic contamination. DO also plays a key role in increasing the efficiency of constructed wetlands to treat petroleum-contaminated water and supports more effective microbial degradation processes, especially under aerobic conditions (Al-baldawi et al. 2013 ). Regarding pH, a decrease in water pH was observed over time, with the highest pH recorded on day 0 at 8.95. By day 14, the pH ranged from 7.23 to 7.78. This decrease was likely due to plant nutrient absorption, where plant roots release H + or OH- ions, affecting pH changes (Ali et al. 2024 ). While pH fluctuations can impact microbial degradation, the pH levels in this study remained within the optimal range for plant growth and organic compound degradation, between 7 and 8.5. L. minor effectively reduced pH towards neutral, enhancing bioremediation (Baruah et al. 2016 ). L. minor is known to reduce pH to near neutral through interactions with microorganisms that utilize organic compounds. Previous research by (Reema et al. 2011 ) showed that L. minor grows optimally at pH 6-7.5, and by the end of this study, the pH was within this range. This decrease in pH towards neutral creates more favorable conditions for the bioremediation process. Measuring pH before and after the phytoremediation process can be used as an indicator of success while emphasizing the importance of pH management to improve the remediation efficiency of petroleum-contaminated water (Jones et al. 2023 ). Total Dissolved Solid (TDS), which includes dissolved minerals, salts, metals, and ions, showed fluctuations without significant decreases, with increases caused by the accumulation of dissolved organic and inorganic materials. The efficiency value of TDS reduction by L. minor tested can be attributed to the ability of macrophyte roots to filter and retain fine suspended particles. In addition, L. minor also plays a role in stimulating microbial growth, which then supports the decomposition process of these particles. This interaction between roots and microbes creates a more optimal condition for water purification through absorption, physical filtration, and biological degradation (Saha et al. 2015 ). The water temperature ranged between 26.5°C and 27.8°C, favoring microbial activity and organic matter decomposition, enhancing the treatment process. Temperature affects microbial dynamics and biochemical reactions, influencing the efficiency of contaminant degradation in constructed wetlands. While higher temperatures can increase microbial activity, extremes in temperature may inhibit microbial growth, reducing the overall effectiveness of the bioremediation process (Saeed and Sun 2012 ). Temperature is an important factor affecting the efficiency of contaminant reduction in the treatment process of petroleum-polluted wastewater using constructed wetlands. Higher temperatures can increase the activity of microorganisms by accelerating the rate of biochemical reactions that favor the degradation of organic compounds. Under certain conditions, increased temperature can also improve oxygen solubility in water, although DO solubility generally decreases at high temperatures. In addition, temperature affects microbial dynamics, including the balance between aerobic and anaerobic microorganisms, which contribute to nitrification and denitrification processes. However, low or high-temperature extremes can inhibit the growth of microorganisms, thus decreasing the efficiency of contaminant degradation (Li et al. 2017 ). Plant Weight after Phytoremediation The wet weight of L. minor was measured as the weight of the whole plant after harvest, while the dry weight was measured after the oven to remove the moisture content, as shown in Fig. 4 . The observation results showed that the P0 Bio-OSD DOR 0.1: 0 treatment produced the highest wet weight of 11.38 g, and the P1 Bio-OSD DOR 0.25: 1 treatment produced the highest dry weight of 3.82 g. In contrast, P11 Non-Bio-OSD DOR 0:1 and P15 Non-Bio-OSD DOR 1:1 produced a weight of 0 g, indicating plant death due to high OSD concentration. The dry weight reflects the results of photosynthesis and nutrient absorption. Plants with optimal uptake showed good growth with high wet and dry weights, while poor uptake affected photosynthesis (Tang and Angela 2019 ). High concentrations of crude oil and surfactants can cause plant death. The addition of OSD, which improves the distribution of crude oil in water, can exacerbate toxic effects by increasing the bioavailability of dispersed harmful compounds. This is in line with (Grifoni et al. 2020 ) Research states that the higher the concentration of crude oil, the greater the impact on the survival of L. minor . It also found that adding dispersants to oil contamination can cause the death of aquatic organisms, including aquatic plants. High concentrations of dispersants can produce enough toxicity to cause aquatic plant mortality (Lewis and Pryor 2013 ). Although dispersants are designed to speed up the cleanup of oil spills, their impact on aquatic ecosystems, particularly aquatic plants, needs further investigation. Root and Leaf Cross Sections of L. minor Cross-sectional observations of the roots and leaves of L. minor aimed to determine the response of plant roots to petroleum contamination and the effect of OSD on plants. These observations were made at the base of the roots of L. minor treated with crude oil and OSD compared to L. minor that was not treated with crude oil and OSD. This observation was done with a wet preparate, i.e., fresh plants were cut as thin as possible and then observed under a microscope with 100x magnification. Root and leaf cross sections of L. minor are shown in Fig. 5 . This study evaluated the response of Lemna minor to crude oil and surfactant contamination through a cross-sectional analysis of roots and leaves. The results showed that crude oil penetrated the epidermis, cortex, and endodermis layers in the crude oil treatment (Figs. 5 b and 5 f). In the P5 Bio-OSD DOR 0.25:1 treatment, crude oil and Bio-OSD reached the epidermis, cortex, endodermis, and xylem layers (Fig. 5 c), as indicated by the discoloration of the tissues. In contrast, in the P13 Non-Bio-OSD DOR 0.25:1 treatment (Figs. 5 d and 5 h), plant tissue damage was found in response to the high concentration of crude oil and surfactant. In contrast, the untreated plants (Fig. 5 a) remained fresh without discoloration or tissue damage. Petroleum contamination can cause significant damage to plant root cells and tissues. Hydrocarbons in oil damage the cell membranes of growing roots, inhibiting the uptake of water and nutrients. The oil also forms a hydrophobic layer around the roots, limiting water and nutrient absorption. In addition, the structure of the root tissue is disrupted, with damaged epidermal cells and disorganized subepidermal tissue patterns. As a result, roots become stunted and lose their physiological functions, impairing plant survival under contaminated conditions (da Silva Correa and Maranho 2021 ). Leaf damage in petroleum-contaminated plants occurs through chlorosis, a change in leaf color to yellow due to impaired photosynthesis. Oil also causes necrosis of the leaves, reducing the plant's ability to absorb sunlight and produce energy. In addition, oil increases stomatal density, inhibiting transpiration, and gas exchange, which worsens plant conditions (Hussain et al. 2018 ). Changes in hydrocarbon compounds after the phytoremediation process Gas chromatography-mass spectrometry (GCMS) analysis showed significant changes in the composition of hydrocarbon compounds in water samples before and after phytoremediation. Crude oil initially contains hydrocarbon compounds from C-7 to C-44, and their degradation occurs during phytoremediation through microorganisms and non-biological processes such as evaporation, photooxidation, and chemical oxidation. Microorganisms initiate degradation from simple to complex hydrocarbon compounds, with long-chain compounds such as n-tetracosane and n-hexacosane being more difficult to degrade. The Bio-OSD treatment degraded significantly, while the Non-Bio-OSD contained the long-chain hydrocarbon compound C44. The addition of OSD helped to accelerate hydrocarbon degradation and reduce COD. Based on the GC-MS analysis presented in Fig. 6 , the results of crude oil and phytoremediation analysis after 14 days showed that in the P4 Bio-OSD DOR 0.1:1 treatment, there was a decrease in peak area, indicating the presence of hydrocarbon compounds at certain retention times. The hydrocarbon compounds were no longer detected. In contrast, in the P12 Non-Bio-OSD DOR 0.1:1 treatment, peak areas indicating the presence of hydrocarbon compounds were still detected at certain retention times. These results suggest that treatment with Bio-OSD is more effective in reducing and degrading hydrocarbon compounds than Non-Bio-OSD. In this study, Bio-OSD was more effective in degrading long-chain hydrocarbons such as n-Hexadecane and Nonadecane, as well as fatty acid compounds and esters such as Hexadecanoic acid. These compounds were completely undetectable after phytoremediation using Bio-OSD, indicating complete degradation. In contrast, Non-Bio-OSD was more effective in degrading short to medium-chain hydrocarbons, such as n-Dodecane and Naphthalene, 1,7-dimethyl-. Root exudates from plants such as L. minor support microbial activity in the rhizosphere, creating a conducive environment for hydrocarbon degradation by providing organic matter that favors microbial growth (Kösesakal et al. 2015 ). This study is in line with the research of (Ekperusi et al. 2020 ) L. paucicostata showed this plant's ability to effectively degrade hydrocarbon compounds up to 99.84% within 120 days. Research by (Moreira et al. 2011 ) Rhizophora mangle was reported to be able to degrade hydrocarbons with an efficiency of up to 82% compared to several other bioremediation methods in contaminated sediments. General Discussion This study evaluated the effectiveness of phytoremediation using L. minor to reduce petroleum-contaminated water pollution by utilizing surfactants such as Bio-OSD and Non-Bio-OSD. The results showed significant reductions in major pollutant parameters, such as BOD 5 , COD, and total hydrocarbons. However, not all of them met the quality standards by Indonesian Regulation No. 21 of 2021. The highest decrease in pollutant levels in the Bio-OSD treatment was in the Bio-OSD DOR 0.1:1 treatment, with a reduction in COD value from 218 mg/L to 131 mg/L (efficiency of 37.35%), a decrease in BOD value from 28.59 mg/L to 15.19 mg/L (efficiency of up to 27.60%), and a plant dry weight of 3.34 g. The highest decrease in the Non-Bio-OSD treatment occurred in the Non-Bio-OSD DOR 0.5:1 treatment. Meanwhile, the highest decrease in the Non-Bio-OSD treatment happened in the Non-Bio-OSD DOR 0.5:1 treatment, with a reduction in COD value from 344 mg/L to 277 mg/L, a decrease in BOD value from 34.19 mg/L to 25.82 mg/L (efficiency of 21.66%), and a plant dry weight of 1.46g. These findings demonstrate that Bio-OSD outperforms Non-Bio-OSD in lowering COD and BOD₅ levels. In addition, the plant dry weight parameter showed that plant biomass also influenced the effectiveness of the remediation process. In Lemna minor plants, wet and dry weights varied due to exposure to surfactants and crude oil, where high concentrations caused plant stress and death. Cross-sectional analysis of leaves and roots showed the penetration of oil droplets facilitated by surfactants, which supports the role of microbes in the hydrocarbon degradation process. Compared to Ugya ( 2015 ) This study's results are consistent with previous findings on the effectiveness of phytoremediation using aquatic plants. However, the degradation efficiency is still below that of some studies using surfactant formulations or more sophisticated methods. The advantage of Bio-OSD over Non-Bio-OSD in lowering COD and BOD₅ levels lies in its active components, derived from natural palm oil-based surfactants, which are more environmentally friendly and better suited to hydrocarbon-degrading microorganisms. The surfactants in Bio-OSD break the oil into smaller and more stable droplets, thus increasing the surface area of the oil for microbial degradation. At the same time, Non-Bio-OSD based on synthetic chemicals tends to have a higher toxic impact on microbes and plants, inhibiting the biodegradation process. In addition, Bio-OSD more effectively facilitates oil droplet penetration, as seen from the cross-sectional analysis of Lemna minor leaves and roots, favoring synergistic interactions between surfactants, plants, and microbes. This makes Bio-OSD superior as an environmentally friendly and sustainable solution to Non-Bio-OSD. The benefits of this research include contributing to the development of environmentally friendly technology-based oil pollution mitigation methods. The combination of L. minor and Bio-OSD offers a solution that is efficient and has the potential to be applied on an industrial scale, supporting the restoration of contaminated water ecosystems. This research also reinforces the importance of innovation in hazardous waste management through sustainable technologies. Further research is recommended to test this technique under field conditions to evaluate more complex environmental dynamics. Specific microorganisms that can degrade aromatic and long-chain hydrocarbons should be explored to improve remediation efficiency. In addition, developing enzyme-based surfactants or natural materials can reduce environmental impact and production costs. Long-term ecotoxicological studies are also needed to ensure the safe use of surfactants in aquatic ecosystems. Conclusion This study showed that oil spill dispersant (OSD), constructed wetlands, and phytoremediation with Lemna minor effectively improved the quality of petroleum-polluted water. Bio-OSD showed higher efficiency than Non-Bio-OSD in reducing pollution parameters such as COD and BOD₅. The results showed that Bio-OSD significantly reduced COD and BOD₅ levels to meet water quality standards. The highest COD reduction efficiency of 39.78% was achieved when Bio-OSD DOR 0.1:1 treatment was implemented. Under this treatment, BOD reduction efficiency was 27.60%. GC-MS analysis showed the degradation of long-chain hydrocarbons such as n-hexadecane and nonadecane. The highest COD Reduction efficiency by Non-Bio-OSD was 27.17% with DOR 0.25:1. This result showed that Bio-OSD performed better in reducing COD than Non-Bio-OSD. The weight of Lemna's biomass decreased slightly during the process, indicating that OSD is toxic to the plant. Regardless of the successful construction of wetlands in reducing COD and BOD5, it is recommended that the growth condition of the aquatic plant be improved for a sustainable phytoremediation process. References Agarry SE, Oghenejoboh KM, Latinwo GK, Owabor CN (2018) Biotreatment of petroleum refinery wastewater in vertical surface-flow constructed wetland vegetated with Eichhornia crassipes: lab-scale experimental and kinetic modeling. Environ Technol (United Kingdom) 41(14):1793–1813. 10.1080/09593330.2018.1549106 Al-baldawi IA, Rozaimah S, Abdullah S, Suja F (2013) Effect of aeration on hydrocarbon phytoremediation capability in pilot sub-surface flow constructed wetland operation. Ecol Eng 61:496–500. 10.1016/j.ecoleng.2013.10.017 Ali B, Mishra A A (2022) Effects of dissolved oxygen concentration on freshwater fish: A review. 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Environ Exp Bot 153:80–88. 10.1016/j.envexpbot.2018.05.012 Jones G, Scullion J, Dalesman S, Robson P, Gwynn-Jones D (2023) Lowering pH enables duckweed (Lemna minor L.) growth on toxic concentrations of high-nutrient agricultural wastewater. J Clean Prod. 395 July 2022:136392. 10.1016/j.jclepro.2023.136392 Kösesakal T, Ünlü VS, Külen O, Memon A, Yüksel B (2015) Evaluation of the phytoremediation capacity of Lemna minor L. In crude oil spiked cultures. Turkish J Biol 39(3):479–484. 10.3906/biy-1406-85 Lewis M, Pryor R (2013) Toxicities of oils, dispersants and dispersed oils to algae and aquatic plants: Review and database value to resource sustainability. Environ Pollut 180:345–367. 10.1016/j.envpol.2013.05.001 Li H, Hao H, Yang X, Xiang L, Zhao F, Jiang H, He Z (2012) Purification of refinery wastewater by different perennial grasses growing in a floating bed. J Plant Nutr 35(1):93–110. 10.1080/01904167.2012.631670 Li J, Sun S, Yan P, Fang L, Yu Y, Xiang Y, Wang D, Gong Yejing G, Yanjun, Zhang Z (2017) Microbial communities in the functional areas of a biofilm reactor with anaerobic–aerobic process for oily wastewater treatment. Bioresour Technol 238:7–15. 10.1016/j.biortech.2017.04.033 Lipps WC, Braun-Howland EB, Baxter TE (2023) Standard methods for the examination of water and wastewater. APHA Press. https://engage.awwa.org/PersonifyEbusiness/Bookstore/Product-Details/productId/162167531 Moreira ITA, Oliveira OMC, Triguis JA, dos Santos AMP, Queiroz AFS, Martins CMS, Silva CS, Jesus RS (2011) Phytoremediation using Rizophora mangle L. in mangrove sediments contaminated by persistent total petroleum hydrocarbons (TPH’s). Microchem J 99(2):376–382. 10.1016/j.microc.2011.06.011 Mustapha HI, Lens PNL (2018) Constructed Wetlands to Treat Petroleum Wastewater. Nanotechnology in the Life Sciences. Springer Science and Business Media B.V., pp 199–237 Prince RC (2015) Oil spill dispersants: Boon or bane? Environ Sci Technol 49(11):6376–6384. 10.1021/acs.est.5b00961 Reema RM, Saravanan P, Kumar MD, Renganathan S (2011) Accumulation of methylene blue dye by growing Lemna minor . Sep Sci Technol 46(6):1052–1058. 10.1080/01496395.2010.528503 Rehman K, Imran A, Amin I, Afzal M (2018) Inoculation with bacteria in floating treatment wetlands positively modulates the phytoremediation of oil field wastewater. J Hazard Mater 349:242–251. 10.1016/j.jhazmat.2018.02.013 Rehman K, Imran A, Amin I, Afzal M (2019) Enhancement of oil field-produced wastewater remediation by bacterially-augmented floating treatment wetlands. Chemosphere 217:576–583. 10.1016/j.chemosphere.2018.11.041 Saeed T, Sun G (2012) A review on nitrogen and organics removal mechanisms in subsurface flow constructed wetlands: Dependency on environmental parameters, operating conditions and supporting media. J Environ Manage 112:429–448. 10.1016/j.jenvman.2012.08.011 Saha P, Banerjee A, Sarkar S (2015) Phytoremediation potential of duckweed (Lemna minor L.) on steel wastewater. Int J Phytorem 17(6):589–596. 10.1080/15226514.2014.950410 da Silva Correa H, Maranho LT (2021) The potential association of Echinochloa polystachya (Kunth) Hitchc. with bacterial consortium for petroleum degradation in contaminated soil. SN Appl Sci 3(1):1–12. 10.1007/s42452-020-04070-6 Tang KHD, Angela J (2019) Phytoremediation of crude oil-contaminated soil with local plant species. IOP Conf Ser Mater Sci Eng 495(1). 10.1088/1757-899X/495/1/012054 Ugya AY (2015) The efficiency of Lemna minor L. in the phytoremediation of Romi Stream: A case study of kaduna refinery and petrochemical company polluted stream. J Appl Biol Biotechnol 3(1):11–14. 10.7324/jabb.2015.3102 Ugya AY, Imam T (2015) The efficiency of Eicchornia crassipes in the phytoremediation of waste water from Kaduna Refinery and petrochemical company. IOSR J Environ Sci Toxicol Food Technol 9(1):43–47. 10.9790/3008-10147680 Yavari S, Malakahmad A, Sapari NB (2015) A review on phytoremediation of crude oil spills. Water Air Soil Pollut 226(8). 10.1007/s11270-015-2550-z Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5885729","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":405975450,"identity":"115dbcbb-302b-40b5-9871-a344e2a7706b","order_by":0,"name":"Muhammad Ridho Fitrisyaah","email":"","orcid":"https://orcid.org/0009-0008-6257-9982","institution":"IPB University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Ridho","lastName":"Fitrisyaah","suffix":""},{"id":405975451,"identity":"ca470dcb-ec77-4043-b64b-4a7945d4f2d5","order_by":1,"name":"Anas Miftah Fauzi","email":"","orcid":"https://orcid.org/0000-0001-5158-3996","institution":"IPB University","correspondingAuthor":false,"prefix":"","firstName":"Anas","middleName":"Miftah","lastName":"Fauzi","suffix":""},{"id":405975452,"identity":"c72c4593-4126-4bfc-baac-200d52d39485","order_by":2,"name":"Moh. Yani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYFACHgjJD+FJMBiAaQNmwlokG0jVwmBwAMqHaGHArUW3vfcAM0/NHRnj42ePPfzBYJFnzsD88ANDgTVOLWZnziUw8xx7xmN2Ji/dmIdBotiygc0Y6Lx03Fpu5Bgw87Ad5jE7kGMmDfRL4oYDDGZA5x0moOXfYR7j/jdmkj/AWti/EdbC23aYx0Aix0yCB6yFh4AtZ84YHJzbd5hH4sY7oF8MgH5p5imWSMDnl+M9hg/efDtsz9+fCwyxiro8c/b2jR8+/MEdYiBwCBqbbKBISQDHSAJeDQwMjD/gWggrHgWjYBSMghEIAB9OS/jIgeSEAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-2231-8143","institution":"IPB University","correspondingAuthor":true,"prefix":"","firstName":"Moh.","middleName":"","lastName":"Yani","suffix":""}],"badges":[],"createdAt":"2025-01-23 06:52:39","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5885729/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5885729/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74898156,"identity":"7f57860b-3b47-4b4d-ad1f-fdd5515ff7c1","added_by":"auto","created_at":"2025-01-28 06:51:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":234722,"visible":true,"origin":"","legend":"\u003cp\u003eConstructed wetland design\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5885729/v1/7ec2eafbe1d3e67365a07bf5.png"},{"id":74897957,"identity":"6f0539bc-eb09-4d10-8f63-009ccb207272","added_by":"auto","created_at":"2025-01-28 06:43:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27138,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance of decrease in chemical oxygen demand (COD) value in constructed wetland for crude oil contaminated water using OSD and Bio-OSD treatments for 14 days\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5885729/v1/e565959c88d35c3aa9230d5c.png"},{"id":74897958,"identity":"7cec384d-d474-4e74-b599-10cc249847bc","added_by":"auto","created_at":"2025-01-28 06:43:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34200,"visible":true,"origin":"","legend":"\u003cp\u003ePerformance of decrease in biological oxygen demand (BOD\u003csub\u003e5\u003c/sub\u003e) value in constructed wetland for crude oil contaminated water using OSD and Bio-OSD treatments for 14 days\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5885729/v1/f19ad1f0de558d07d1064151.png"},{"id":74898157,"identity":"5f4911f5-c2d6-4b8c-b642-ff018998b117","added_by":"auto","created_at":"2025-01-28 06:51:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24926,"visible":true,"origin":"","legend":"\u003cp\u003eWeight of phytoremediation plants after 14 days.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5885729/v1/b3c3d482cd9d87c0cc0cefa4.png"},{"id":74898158,"identity":"3fbe3aed-e89e-43d0-98ad-0080cad79b03","added_by":"auto","created_at":"2025-01-28 06:51:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":378674,"visible":true,"origin":"","legend":"\u003cp\u003eRoot cross-section of \u003cem\u003eL. minor\u003c/em\u003e (a); No treatment (b); Crude Oil only (c); P5 (Bio-OSD DOR 0.25:1); (d) P13 (Non Bio-OSD 0.25:1). Cross section of \u003cem\u003eL. minor\u003c/em\u003eleaf (e); No treatment (f); Crude Oil Only (g); P5 (Bio-OSD DOR 0.25:1); (h) P13 (Non Bio-OSD 0.25:1).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5885729/v1/a22d8500e76339ebb4c1052f.png"},{"id":74898165,"identity":"5a843e84-5d27-42e0-a2bd-c2a93aa233e7","added_by":"auto","created_at":"2025-01-28 06:51:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52303,"visible":true,"origin":"","legend":"\u003cp\u003eGCMS Chromatogram a. Crude Oil; b. P4 Bio-OSD DOR 0.1:1; c. P12 Non-Bio-OSD DOR 0.1:1\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5885729/v1/eb51c9b1c57a4910208030e5.png"},{"id":74899182,"identity":"44c74507-b76b-476a-98eb-a7014532de67","added_by":"auto","created_at":"2025-01-28 07:07:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1578174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5885729/v1/c32a7248-ce44-45e5-b957-14a802e47268.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eBioremediation of Petroleum Contaminated Water Using Oil Spill Dispersant and \u003cem\u003eLemna minor\u003c/em\u003e in Laboratory Scale of Constructed Wetland\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEnvironmental pollution caused by oil spills remains a significant global challenge, particularly in resource-rich countries like Indonesia, which has abundant oil and gas reserves. Oil spills frequently result from tanker accidents, leaks in subsea pipelines, and industrial activities related to oil extraction and refining, all producing hazardous waste. These incidents lead to substantial economic losses and severe ecological damage, disrupting marine ecosystems and threatening biodiversity (Prince \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePetroleum contains toxic organic compounds such as benzene, toluene, ethylbenzene, and xylene (BTEX), posing significant risks to aquatic and terrestrial ecosystems. In aquatic environments, spilled oil forms a thick layer on the water's surface, blocking the penetration of oxygen and sunlight. This disrupts the photosynthesis process in aquatic plants and harms aquatic organisms, leading to ecological imbalances and declining biodiversity. On land, oil contact with soil alters its structure and fertility, degrading terrestrial habitats, impairing nutrient cycling, and inhibiting plant growth (Yavari et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Indonesia, crude oil production has reached 386,395.79 barrels, with oil spills accounting for 9,110.20 barrels, underscoring the environmental impact of oil pollution. Oil spill dispersants (OSD) are commonly used to manage oil spills. OSDs break down oil into smaller droplets, increasing their surface area and promoting microbial degradation. These formulations combine chemicals and surfactants to enhance dispersal efficiency and mitigate environmental damage (Brakstad et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The Surfactant and Bioenergy Research Centre (SBRC-IPB) has developed a more effective, environmentally friendly Bio-OSD derived from palm oil, surpassing commercial performance products (Elvina et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOil pollution can be managed through constructed wetlands, which harness plants to purify water naturally. These systems use physical, chemical, and biological processes inherent in wetland ecosystems to remove contaminants. Constructed wetlands are cost-effective, energy-efficient, and have minimal environmental impact, making them ideal for long-term remediation (Mustapha and Lens \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Phytoremediation, an environmentally friendly method that utilizes plants to absorb, degrade, and stabilize contaminants, can restore polluted environments. Constructed wetlands, as a form of phytoremediation, replicate natural processes to treat petroleum-contaminated water by leveraging the actions of plants, microorganisms, and substrates to remove and degrade pollutants (Ansari et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLemna minor\u003c/em\u003e, or duckweed, is an effective plant for phytoremediation due to its rapid growth and ability to accumulate contaminants (Gupta and Prakash \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It plays a crucial role in hydrocarbon phytoremediation, as it can absorb and biodegrade petroleum hydrocarbons, contributing to the purification of polluted water and the restoration of environmental quality (Ekperusi 2019). This study aims to assess the impact of OSD application, constructed wetlands, and \u003cem\u003eLemna minor\u003c/em\u003e phytoremediation on improving petroleum-polluted water quality.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSimulation of Petroleum-Polluted Water\u003c/h2\u003e \u003cp\u003ePetroleum polluted water simulation was conducted using medium crude oil at a concentration of 1% of the water volume. The constructed wetland using 45L containers sized 55 cm x 36 cm x 29 cm. The containers were filled with a substrate consisting of 10 cm of gravel and 10 cm of sand, and the groundwater well was 15L. Crude oil is added to the water at 150mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eL. minor\u003c/em\u003e used in the study were acclimatized for seven days, and plants selected for the experiment were uniform in size, fresh, and structurally intact. This study was conducted at the Greenhouse.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eDetails of the reactor treatment design, including variations in Dispersant Oil Ratio (DOR), Bio-OSD, and Non-Bio-OSD combinations, are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This study used a Completely Randomized Block Design (CRBD) with treatment combinations applied to constructed wetlands conducted in 3 replications.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eReactor Treatment Design with DOR, Bio-OSD, and Non Bio-OSD Variations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReactor Code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlant Treatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBio-OSD DOR 0.1:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBio-OSD DOR 0.25:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBio-OSD DOR 0.5:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBio-OSD DOR 1:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBio-OSD DOR 0.1:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBio-OSD DOR 0.25:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBio-OSD DOR 0.5:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBio-OSD DOR 1:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon Bio-OSD DOR 0.1:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon Bio-OSD DOR 0.25:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon Bio-OSD DOR 0.5:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon Bio-OSD DOR 1:0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon Bio-OSD DOR 0.1:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon Bio-OSD DOR 0.25:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon Bio-OSD DOR 0.5:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNon Bio-OSD DOR 1:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePhytoremediation Performance Test\u003c/h3\u003e\n\u003cp\u003eEach treatment's contaminant level was tested through daily observations (DO, pH, TDS, and temperature) at the beginning and end of treatment (BOD\u003csub\u003e5\u003c/sub\u003e, COD, hydrocarbons). The DO, pH, TDS, and temperature were measured using AZ instrument 86031 water quality meter during the study.\u003c/p\u003e \u003cp\u003eBOD\u003csub\u003e5\u003c/sub\u003e and COD were analyzed at the experiment's initial and end (14 days). BOD₅ is APHA 5210 B, and for COD is APHA 5220 C (APHA 2023). The contaminant removal efficiency of BOD\u003csub\u003e5\u003c/sub\u003e and COD are calculated with the following equation:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\varvec{R}\\:\\left(\\varvec{\\%}\\right)=\\:\\frac{{\\varvec{C}}_{0}-{\\varvec{C}}_{\\varvec{t}}}{{\\varvec{C}}_{0}}\\:\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eR\u0026thinsp;=\u0026thinsp;contaminant removal efficiency (%)\u003c/p\u003e \u003cp\u003eCo\u0026thinsp;=\u0026thinsp;initial contaminant value (mg/L)\u003c/p\u003e \u003cp\u003eCt\u0026thinsp;=\u0026thinsp;end of study contaminant value (mg/L)\u003c/p\u003e \u003cp\u003eAt the end of the research, water samples were analyzed for oil hydrocarbons from phytoremediation using gas chromatography-mass spectrometry (GCMS) brand Thermo Scientific and compared with crude oil chromatograms. Observations of the plants were made daily to assess their freshness and growth ability. The biomass (wet and dry bases) of \u003cem\u003eL. minor\u003c/em\u003e was observed initially and at the end of the experiment (14 days).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe data obtained were analyzed using SPSS 27 to examine the Analysis of Variance (ANOVA). If treatments have a significant effect at the actual level of 5%, they are analyzed using Duncan's Multiple Range Test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result and Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWater-Quality Changes\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the range of test results for BOD\u003csub\u003e5\u003c/sub\u003e, COD, TDS, pH, and DO parameters from all treatments at the initial and end of the study. BOD\u003csub\u003e5\u003c/sub\u003e and COD levels decreased, although they did not meet the water quality standards. The pH and TDS levels complied with the water quality standards by Indonesian Regulation Number 21 of 2021 concerning Implementing Environmental Protection and Management. However, DO levels dropped from 5.65\u0026ndash;6.90 mg/L to 1.35\u0026ndash;5.80 mg/L, thus meeting water quality standards.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTest results of water pollutant levels before and after phytoremediation of petroleum-polluted water.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUnit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eAnalysis Result\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eQuality standards\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInitial (0 Days)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEnd (14 Days)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBOD\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u0026ndash;41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u0026ndash;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u0026ndash;12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72\u0026ndash;424\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62\u0026ndash;348\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026ndash;80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTDS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97\u0026ndash;150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105\u0026ndash;158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1000\u0026ndash;2000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7,23\u0026ndash;8,95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7,24\u0026ndash;7,78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u0026ndash;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emg/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5,65\u0026ndash;6,90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0,85\u0026ndash;5,80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u0026ndash;6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003eIndonesian Regulation Number 21 of 2021 concerning the Implementation of Environmental Protection and Management\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cem\u003eChemical Oxygen Demand\u003c/em\u003e (COD)\u003c/div\u003e \u003cp\u003eChemical Oxygen Demand (COD) indicates the level of water pollution by reflecting the amount of organic matter present. A decrease in COD occurs as organic matter in the water decreases, which can be achieved by aquatic plants that absorb the organic matter. A high COD value indicates a large amount of oxidized organic matter, which reduces dissolved oxygen levels in water (Boguniewicz-Zablocka et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). COD reduction data were analyzed using the ANOVA test with a significance level of α 5%, showing significant differences in each treatment. The COD value reduction and COD reduction efficiency are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eCOD reflects the level of water pollution by indicating the amount of oxidized organic matter, which contributes to a decrease in dissolved oxygen (DO). Significant COD reduction occurred in all treatments, with statistical test results showing significant differences. On day 0, the lowest COD was found in P4 Bio-OSD DOR 0.1 :1 (208 mg/L), while the highest was in P11 Non-Bio-OSD DOR 1:0 (432 mg/L). On day 14, the lowest COD was found in P0 Bio-OSD DOR 0.1:0 (127 mg/L) and the highest in P15 Non Bio-OSD DOR 1: 1 (348 mg/L). The decrease in COD reflects the reduction of organic matter, with \u003cem\u003eL. minor\u003c/em\u003e playing a role in COD removal through organic matter absorption. COD tended to increase with the addition of OSD. On day 14, the decrease in COD was supported by the photosynthesis of \u003cem\u003eL. minor\u003c/em\u003e, which increased dissolved oxygen, creating aerobic conditions that accelerated the activity of microorganisms in reducing COD (Aziz et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe COD reduction was influenced by the absorption of organic matter by \u003cem\u003eL. minor\u003c/em\u003e and phytoremediation activities. COD removal efficiency in handling petroleum-polluted water was found in the P4 Bio-OSD DOR 0.1: 1 (39,78%) and lowest in P13 Non-Bio-OSD DOR 0.25:1 (13.67%). The significant decrease in COD value indicates that \u003cem\u003eL. minor\u003c/em\u003e can stimulate the growth of microorganisms that play a role in the degradation of organic compounds in wastewater. These microorganisms utilize organic compounds as an energy source, thus increasing the effectiveness of \u003cem\u003eL. minor\u003c/em\u003e in reducing organic load and improving water quality (Ugya \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe COD reduction efficiency achieved in this study is consistent with the findings of (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), who reported a COD reduction of 52\u0026ndash;67% using annual grass vegetation such as \u003cem\u003eGeophila herbacea\u003c/em\u003e and \u003cem\u003eLolium perenne\u003c/em\u003e L. as phytoremediation agents. These results also align with the research of (Mustapha and Lens \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which showed a 66\u0026ndash;91% reduction in COD using \u003cem\u003eTypha latifolia\u003c/em\u003e. The findings confirm that the presence of plants in a phytoremediation system is essential in improving treatment efficiency. This is due to plant metabolic activity, the rhizosphere zone's microbial environment enhancement, and the roots' role in providing contact surfaces for contaminant uptake or transformation.\u003c/p\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cem\u003eBiological Oxygen Demand\u003c/em\u003e (BOD\u003csub\u003e5\u003c/sub\u003e)\u003c/div\u003e \u003cp\u003eBiochemical Oxygen Demand (BOD\u003csub\u003e5\u003c/sub\u003e) measures the need for dissolved oxygen to oxidize the waste material in water, reflecting the oxygen demand for the decomposition process. High oxygen consumption indicates the presence of waste materials with significant oxygen demand. BOD\u003csub\u003e5\u003c/sub\u003e reduction data were analyzed using the ANOVA test with a significance level of α 5%, showing significant differences in each treatment. The reduction in BOD\u003csub\u003e5\u003c/sub\u003e value and the efficiency of BOD\u003csub\u003e5\u003c/sub\u003e reduction are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBOD\u003csub\u003e5\u003c/sub\u003e measurements were conducted on days 0 and 14 to assess the efficiency of phytoremediation. On day 0, the lowest BOD\u003csub\u003e5\u003c/sub\u003e was found in the P0 Bio-OSD DOR 0.1:0 treatment (19.61 mg/L), while the highest was in the P15 Non-Bio-OSD DOR 1:1 treatment (40.90 mg/L). On day 14, BOD\u003csub\u003e5\u003c/sub\u003e was lowest in the P0 Bio-OSD DOR 0.1:0 treatment (12.96 mg/L) and highest in the P15 Non-Bio-OSD DOR 1:1 treatment (31.28 mg/L). The highest BOD\u003csub\u003e5\u003c/sub\u003e concentration on day 0 indicates that the addition of 1% Non-Bio-OSD increases biological oxygen demand due to the addition of new organic matter, such as oil spills or industrial waste (Atlas and Hazen \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe efficiency of BOD\u003csub\u003e5\u003c/sub\u003e reduction in this study ranged from 11.29\u0026ndash;37.69%, with the highest efficiency value achieved in the P5 Bio-OSD DOR 0.25:1 treatment. The analysis showed that using Bio-OSD tended to produce a more significant increase in efficiency than Non-Bio-OSD. This can be seen from the graph showing that the Bio-OSD treatment's efficiency is consistently higher than the Non-Bio-OSD treatment.\u003c/p\u003e \u003cp\u003ePhytoremediation using \u003cem\u003eL. minor\u003c/em\u003e effectively reduces BOD\u003csub\u003e5\u003c/sub\u003e, supported by the activity of microorganisms that decompose organic matter into simple elements that plants absorb. Significant reductions in BOD\u003csub\u003e5\u003c/sub\u003e occurred based on the growth of microorganisms and parameters such as COD, DO, and pH. Microorganisms secrete enzymes that break down complex organic compounds into simple nutrients that are absorbed by macrophytes, thereby reducing organic compound levels and dissolved oxygen use, lowering BOD\u003csub\u003e5\u003c/sub\u003e in petroleum-polluted wastewater (Bhutiani et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of this study are consistent with the findings of (Agarry et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), who reported that using \u003cem\u003eEichhornia crassipes\u003c/em\u003e plants in a phytoremediation system achieved a BOD\u003csub\u003e5\u003c/sub\u003e reduction efficiency of up to 94.6%. In addition, the research of (Rehman et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) This study also supported the results; combining \u003cem\u003eTypha domingensis\u003c/em\u003e and \u003cem\u003eLeptochloa fusca\u003c/em\u003e plants with bacterial consortium resulted in a BOD\u003csub\u003e5\u003c/sub\u003e reduction efficiency of up to 93%.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComparison of Wastewater Treatment Efficiency Using Constructed Wetland and Alternative Techniques\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e compares the treatment efficiencies of petroleum-contaminated water using various constructed wetland methods alongside other treatment techniques, incorporating different plant species. The summarized studies include varying treatment durations, the plant species utilized, and the observed reductions in pollutant parameters such as Chemical Oxygen Demand (COD) and Biological Oxygen Demand (BOD\u003csub\u003e5\u003c/sub\u003e). This comparison offers valuable insights into the effectiveness of these methods for the remediation of petroleum-contaminated water, highlighting the specific advantages associated with each approach.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of petroleum-polluted water treatment efficiency using constructed wetland method and other techniques with various plant types\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAuthor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMethods\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePlants\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eResult\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThis Research\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConstructed Wetland using OSD for 14 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCOD efficiency up to 40%, BOD\u003csub\u003e5\u003c/sub\u003e 38%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Ugya \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConstructed Wetland for 3 weeks in wastewater from the refinery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThe BOD\u003csub\u003e5\u003c/sub\u003e decreased by 68%, and the COD decreased by 91.6%.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Azeez and Sabbar \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConstructed Wetland for 1 month\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eL. minor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThe BOD\u003csub\u003e5\u003c/sub\u003e decreased by 49.6%, and the COD decreased by 32.7%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFloating beds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eGeophila herbacea\u003c/em\u003e and \u003cem\u003eLolium perenne\u003c/em\u003e L.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThe efficiency of COD reduction ranged from 52\u0026ndash;67%.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Mustapha and Lens \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConstructed Wetland\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTypha latifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThe reduction in COD values ranged from 66\u0026ndash;91%.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Agarry et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConstructed Wetland\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eEichhornia crassipes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThe BOD\u003csub\u003e5\u003c/sub\u003e decreased by 80.2%, and the COD decreased by 92.6%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Rehman et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFloating treatment wetland\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eTypha domingensis\u003c/em\u003e and \u003cem\u003eLeptochloa fusca\u003c/em\u003e plants with bacterial consortium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eThe efficiency of BOD\u003csub\u003e5\u003c/sub\u003e reduction reached up to 93%.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThis study made a significant contribution by demonstrating that the constructed wetland system, utilizing Oil Spill Dispersant (OSD) and \u003cem\u003eL. minor\u003c/em\u003e for 14 days, achieved a COD reduction efficiency of 40% and a BOD\u003csub\u003e5\u003c/sub\u003e reduction of 38%. The primary advantage of this approach is the relatively short treatment time compared to other studies, highlighting its potential for operational-scale application with faster and more efficient treatment processes.\u003c/p\u003e \u003cp\u003eIn comparison, studies by (Ugya and Imam \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and (Azeez and Sabbar \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) Employed \u003cem\u003eL. minor\u003c/em\u003e for three weeks and one month, respectively, leading to higher reductions in BOD\u003csub\u003e5\u003c/sub\u003e and COD. However, the longer treatment durations in these studies may present limitations for field applications, particularly in situations that demand a swift response. As such, this research provides value by optimizing treatment efficiency within a shorter timeframe, making it more feasible for field implementation.\u003c/p\u003e \u003cp\u003eThe combination of OSD with \u003cem\u003eL. minor\u003c/em\u003e offers an innovative approach to wastewater treatment, setting it apart from more conventional techniques, such as those employed by (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) using floating beds or (Mustapha and Lens \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Who utilized constructed wetlands without additional modifications. Incorporating OSD provides an effective solution for treating petroleum-contaminated water, enhancing the overall treatment efficiency and addressing challenges associated with petroleum pollution.\u003c/p\u003e \u003cp\u003eAlthough other studies, such as those by (Agarry et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and (Rehman et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) Reported excellent efficiencies in BOD\u003csub\u003e5\u003c/sub\u003e and COD reduction; their methods involved longer treatment durations or combinations of plants and microorganisms that require additional management. This study presents a more straightforward alternative with competitive results, making it a promising option for practical applications due to its ease of management. Overall, this study emphasizes the time efficiency and integration of innovative technologies such as OSD and \u003cem\u003eL. minor\u003c/em\u003e as a phytoremediation plant, offering a sustainable and practical solution for wastewater treatment. The findings provide a strong foundation for further development and potential large-scale applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of Environmental Conditions on Water Quality in Constructed Wetland\u003c/h2\u003e \u003cp\u003eIn this study, Dissolved Oxygen (DO) levels are crucial in assessing water quality, with a standard value set at \u0026ge;\u0026thinsp;1 mg/L according to Indonesian Regulation 21 of 2021. On day 0, the highest DO was found in the P1 DOR 0:0 treatment (6.90 mg/L), while the lowest DO was found in the P2 DOR 0:1 treatment (5.65 mg/L). On day 14, the highest DO was found in the control group (P0) at 5.8 mg/L, and the lowest was in the P5 Bio-OSD DOR 0.5:1 treatment (1.35 mg/L). The addition of surfactant resulted in a significant decrease in DO, reflecting increased microbial activity and consuming more oxygen (Arora et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). DO is essential for aquatic life, with optimal levels for fish between 5\u0026ndash;6 mg/L (Ali et al. 2022). High DO levels indicate lower pollution; low levels indicate higher organic contamination. DO also plays a key role in increasing the efficiency of constructed wetlands to treat petroleum-contaminated water and supports more effective microbial degradation processes, especially under aerobic conditions (Al-baldawi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding pH, a decrease in water pH was observed over time, with the highest pH recorded on day 0 at 8.95. By day 14, the pH ranged from 7.23 to 7.78. This decrease was likely due to plant nutrient absorption, where plant roots release H\u0026thinsp;+\u0026thinsp;or OH- ions, affecting pH changes (Ali et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). While pH fluctuations can impact microbial degradation, the pH levels in this study remained within the optimal range for plant growth and organic compound degradation, between 7 and 8.5. \u003cem\u003eL. minor\u003c/em\u003e effectively reduced pH towards neutral, enhancing bioremediation (Baruah et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eL. minor\u003c/em\u003e is known to reduce pH to near neutral through interactions with microorganisms that utilize organic compounds. Previous research by (Reema et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) showed that \u003cem\u003eL. minor\u003c/em\u003e grows optimally at pH 6-7.5, and by the end of this study, the pH was within this range. This decrease in pH towards neutral creates more favorable conditions for the bioremediation process. Measuring pH before and after the phytoremediation process can be used as an indicator of success while emphasizing the importance of pH management to improve the remediation efficiency of petroleum-contaminated water (Jones et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTotal Dissolved Solid (TDS), which includes dissolved minerals, salts, metals, and ions, showed fluctuations without significant decreases, with increases caused by the accumulation of dissolved organic and inorganic materials. The efficiency value of TDS reduction by \u003cem\u003eL. minor\u003c/em\u003e tested can be attributed to the ability of macrophyte roots to filter and retain fine suspended particles. In addition, \u003cem\u003eL. minor\u003c/em\u003e also plays a role in stimulating microbial growth, which then supports the decomposition process of these particles. This interaction between roots and microbes creates a more optimal condition for water purification through absorption, physical filtration, and biological degradation (Saha et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe water temperature ranged between 26.5\u0026deg;C and 27.8\u0026deg;C, favoring microbial activity and organic matter decomposition, enhancing the treatment process. Temperature affects microbial dynamics and biochemical reactions, influencing the efficiency of contaminant degradation in constructed wetlands. While higher temperatures can increase microbial activity, extremes in temperature may inhibit microbial growth, reducing the overall effectiveness of the bioremediation process (Saeed and Sun \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTemperature is an important factor affecting the efficiency of contaminant reduction in the treatment process of petroleum-polluted wastewater using constructed wetlands. Higher temperatures can increase the activity of microorganisms by accelerating the rate of biochemical reactions that favor the degradation of organic compounds. Under certain conditions, increased temperature can also improve oxygen solubility in water, although DO solubility generally decreases at high temperatures. In addition, temperature affects microbial dynamics, including the balance between aerobic and anaerobic microorganisms, which contribute to nitrification and denitrification processes. However, low or high-temperature extremes can inhibit the growth of microorganisms, thus decreasing the efficiency of contaminant degradation (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlant Weight after Phytoremediation\u003c/h2\u003e \u003cp\u003eThe wet weight of \u003cem\u003eL. minor\u003c/em\u003e was measured as the weight of the whole plant after harvest, while the dry weight was measured after the oven to remove the moisture content, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The observation results showed that the P0 Bio-OSD DOR 0.1: 0 treatment produced the highest wet weight of 11.38 g, and the P1 Bio-OSD DOR 0.25: 1 treatment produced the highest dry weight of 3.82 g. In contrast, P11 Non-Bio-OSD DOR 0:1 and P15 Non-Bio-OSD DOR 1:1 produced a weight of 0 g, indicating plant death due to high OSD concentration. The dry weight reflects the results of photosynthesis and nutrient absorption. Plants with optimal uptake showed good growth with high wet and dry weights, while poor uptake affected photosynthesis (Tang and Angela \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHigh concentrations of crude oil and surfactants can cause plant death. The addition of OSD, which improves the distribution of crude oil in water, can exacerbate toxic effects by increasing the bioavailability of dispersed harmful compounds. This is in line with (Grifoni et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Research states that the higher the concentration of crude oil, the greater the impact on the survival of \u003cem\u003eL. minor\u003c/em\u003e. It also found that adding dispersants to oil contamination can cause the death of aquatic organisms, including aquatic plants. High concentrations of dispersants can produce enough toxicity to cause aquatic plant mortality (Lewis and Pryor \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although dispersants are designed to speed up the cleanup of oil spills, their impact on aquatic ecosystems, particularly aquatic plants, needs further investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRoot and Leaf Cross Sections of L. minor\u003c/h2\u003e \u003cp\u003eCross-sectional observations of the roots and leaves of \u003cem\u003eL. minor\u003c/em\u003e aimed to determine the response of plant roots to petroleum contamination and the effect of OSD on plants. These observations were made at the base of the roots of \u003cem\u003eL. minor\u003c/em\u003e treated with crude oil and OSD compared to \u003cem\u003eL. minor\u003c/em\u003e that was not treated with crude oil and OSD. This observation was done with a wet preparate, i.e., fresh plants were cut as thin as possible and then observed under a microscope with 100x magnification. Root and leaf cross sections of \u003cem\u003eL. minor\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis study evaluated the response of \u003cem\u003eLemna minor\u003c/em\u003e to crude oil and surfactant contamination through a cross-sectional analysis of roots and leaves. The results showed that crude oil penetrated the epidermis, cortex, and endodermis layers in the crude oil treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). In the P5 Bio-OSD DOR 0.25:1 treatment, crude oil and Bio-OSD reached the epidermis, cortex, endodermis, and xylem layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), as indicated by the discoloration of the tissues. In contrast, in the P13 Non-Bio-OSD DOR 0.25:1 treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), plant tissue damage was found in response to the high concentration of crude oil and surfactant. In contrast, the untreated plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) remained fresh without discoloration or tissue damage.\u003c/p\u003e \u003cp\u003ePetroleum contamination can cause significant damage to plant root cells and tissues. Hydrocarbons in oil damage the cell membranes of growing roots, inhibiting the uptake of water and nutrients. The oil also forms a hydrophobic layer around the roots, limiting water and nutrient absorption. In addition, the structure of the root tissue is disrupted, with damaged epidermal cells and disorganized subepidermal tissue patterns. As a result, roots become stunted and lose their physiological functions, impairing plant survival under contaminated conditions (da Silva Correa and Maranho \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLeaf damage in petroleum-contaminated plants occurs through chlorosis, a change in leaf color to yellow due to impaired photosynthesis. Oil also causes necrosis of the leaves, reducing the plant's ability to absorb sunlight and produce energy. In addition, oil increases stomatal density, inhibiting transpiration, and gas exchange, which worsens plant conditions (Hussain et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChanges in hydrocarbon compounds after the phytoremediation process\u003c/h2\u003e \u003cp\u003eGas chromatography-mass spectrometry (GCMS) analysis showed significant changes in the composition of hydrocarbon compounds in water samples before and after phytoremediation. Crude oil initially contains hydrocarbon compounds from C-7 to C-44, and their degradation occurs during phytoremediation through microorganisms and non-biological processes such as evaporation, photooxidation, and chemical oxidation. Microorganisms initiate degradation from simple to complex hydrocarbon compounds, with long-chain compounds such as n-tetracosane and n-hexacosane being more difficult to degrade. The Bio-OSD treatment degraded significantly, while the Non-Bio-OSD contained the long-chain hydrocarbon compound C44. The addition of OSD helped to accelerate hydrocarbon degradation and reduce COD.\u003c/p\u003e \u003cp\u003eBased on the GC-MS analysis presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the results of crude oil and phytoremediation analysis after 14 days showed that in the P4 Bio-OSD DOR 0.1:1 treatment, there was a decrease in peak area, indicating the presence of hydrocarbon compounds at certain retention times. The hydrocarbon compounds were no longer detected. In contrast, in the P12 Non-Bio-OSD DOR 0.1:1 treatment, peak areas indicating the presence of hydrocarbon compounds were still detected at certain retention times. These results suggest that treatment with Bio-OSD is more effective in reducing and degrading hydrocarbon compounds than Non-Bio-OSD.\u003c/p\u003e \u003cp\u003eIn this study, Bio-OSD was more effective in degrading long-chain hydrocarbons such as n-Hexadecane and Nonadecane, as well as fatty acid compounds and esters such as Hexadecanoic acid. These compounds were completely undetectable after phytoremediation using Bio-OSD, indicating complete degradation. In contrast, Non-Bio-OSD was more effective in degrading short to medium-chain hydrocarbons, such as n-Dodecane and Naphthalene, 1,7-dimethyl-. Root exudates from plants such as \u003cem\u003eL. minor\u003c/em\u003e support microbial activity in the rhizosphere, creating a conducive environment for hydrocarbon degradation by providing organic matter that favors microbial growth (K\u0026ouml;sesakal et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study is in line with the research of (Ekperusi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) L. \u003cem\u003epaucicostata\u003c/em\u003e showed this plant's ability to effectively degrade hydrocarbon compounds up to 99.84% within 120 days. Research by (Moreira et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) \u003cem\u003eRhizophora mangle\u003c/em\u003e was reported to be able to degrade hydrocarbons with an efficiency of up to 82% compared to several other bioremediation methods in contaminated sediments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGeneral Discussion\u003c/h2\u003e \u003cp\u003eThis study evaluated the effectiveness of phytoremediation using \u003cem\u003eL. minor\u003c/em\u003e to reduce petroleum-contaminated water pollution by utilizing surfactants such as Bio-OSD and Non-Bio-OSD. The results showed significant reductions in major pollutant parameters, such as BOD\u003csub\u003e5\u003c/sub\u003e, COD, and total hydrocarbons. However, not all of them met the quality standards by Indonesian Regulation No. 21 of 2021.\u003c/p\u003e \u003cp\u003eThe highest decrease in pollutant levels in the Bio-OSD treatment was in the Bio-OSD DOR 0.1:1 treatment, with a reduction in COD value from 218 mg/L to 131 mg/L (efficiency of 37.35%), a decrease in BOD value from 28.59 mg/L to 15.19 mg/L (efficiency of up to 27.60%), and a plant dry weight of 3.34 g. The highest decrease in the Non-Bio-OSD treatment occurred in the Non-Bio-OSD DOR 0.5:1 treatment. Meanwhile, the highest decrease in the Non-Bio-OSD treatment happened in the Non-Bio-OSD DOR 0.5:1 treatment, with a reduction in COD value from 344 mg/L to 277 mg/L, a decrease in BOD value from 34.19 mg/L to 25.82 mg/L (efficiency of 21.66%), and a plant dry weight of 1.46g. These findings demonstrate that Bio-OSD outperforms Non-Bio-OSD in lowering COD and BOD₅ levels.\u003c/p\u003e \u003cp\u003eIn addition, the plant dry weight parameter showed that plant biomass also influenced the effectiveness of the remediation process. In \u003cem\u003eLemna minor\u003c/em\u003e plants, wet and dry weights varied due to exposure to surfactants and crude oil, where high concentrations caused plant stress and death. Cross-sectional analysis of leaves and roots showed the penetration of oil droplets facilitated by surfactants, which supports the role of microbes in the hydrocarbon degradation process.\u003c/p\u003e \u003cp\u003eCompared to Ugya (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) This study's results are consistent with previous findings on the effectiveness of phytoremediation using aquatic plants. However, the degradation efficiency is still below that of some studies using surfactant formulations or more sophisticated methods. The advantage of Bio-OSD over Non-Bio-OSD in lowering COD and BOD₅ levels lies in its active components, derived from natural palm oil-based surfactants, which are more environmentally friendly and better suited to hydrocarbon-degrading microorganisms. The surfactants in Bio-OSD break the oil into smaller and more stable droplets, thus increasing the surface area of the oil for microbial degradation. At the same time, Non-Bio-OSD based on synthetic chemicals tends to have a higher toxic impact on microbes and plants, inhibiting the biodegradation process. In addition, Bio-OSD more effectively facilitates oil droplet penetration, as seen from the cross-sectional analysis of \u003cem\u003eLemna minor\u003c/em\u003e leaves and roots, favoring synergistic interactions between surfactants, plants, and microbes. This makes Bio-OSD superior as an environmentally friendly and sustainable solution to Non-Bio-OSD.\u003c/p\u003e \u003cp\u003eThe benefits of this research include contributing to the development of environmentally friendly technology-based oil pollution mitigation methods. The combination of \u003cem\u003eL. minor\u003c/em\u003e and Bio-OSD offers a solution that is efficient and has the potential to be applied on an industrial scale, supporting the restoration of contaminated water ecosystems. This research also reinforces the importance of innovation in hazardous waste management through sustainable technologies.\u003c/p\u003e \u003cp\u003eFurther research is recommended to test this technique under field conditions to evaluate more complex environmental dynamics. Specific microorganisms that can degrade aromatic and long-chain hydrocarbons should be explored to improve remediation efficiency. In addition, developing enzyme-based surfactants or natural materials can reduce environmental impact and production costs. Long-term ecotoxicological studies are also needed to ensure the safe use of surfactants in aquatic ecosystems.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study showed that oil spill dispersant (OSD), constructed wetlands, and phytoremediation with \u003cem\u003eLemna minor\u003c/em\u003e effectively improved the quality of petroleum-polluted water. Bio-OSD showed higher efficiency than Non-Bio-OSD in reducing pollution parameters such as COD and BOD₅. The results showed that Bio-OSD significantly reduced COD and BOD₅ levels to meet water quality standards. The highest COD reduction efficiency of 39.78% was achieved when Bio-OSD DOR 0.1:1 treatment was implemented. Under this treatment, BOD reduction efficiency was 27.60%. GC-MS analysis showed the degradation of long-chain hydrocarbons such as n-hexadecane and nonadecane. The highest COD Reduction efficiency by Non-Bio-OSD was 27.17% with DOR 0.25:1. This result showed that Bio-OSD performed better in reducing COD than Non-Bio-OSD. The weight of Lemna's biomass decreased slightly during the process, indicating that OSD is toxic to the plant. Regardless of the successful construction of wetlands in reducing COD and BOD5, it is recommended that the growth condition of the aquatic plant be improved for a sustainable phytoremediation process.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgarry SE, Oghenejoboh KM, Latinwo GK, Owabor CN (2018) Biotreatment of petroleum refinery wastewater in vertical surface-flow constructed wetland vegetated with Eichhornia crassipes: lab-scale experimental and kinetic modeling. 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Water Air Soil Pollut 226(8). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11270-015-2550-z\u003c/span\u003e\u003cspan address=\"10.1007/s11270-015-2550-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"IPB University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Constructed wetland, crude oil, Lemna minor, oil spill dispersant, phytoremediation, water quality","lastPublishedDoi":"10.21203/rs.3.rs-5885729/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5885729/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePetroleum pollution due to industrial activities is a significant environmental problem, especially when polluting water resources. This study aims to evaluate the effectiveness of using oil spill dispersant (OSD), constructed wetlands, and phytoremediation \u003cem\u003eusing Lemna minor\u003c/em\u003e in improving the quality of petroleum-polluted water. The experiment was conducted using a group randomized design with a combination treatment of petroleum-based commercial OSD (Non-Bio-OSD) and environmentally friendly palm oil-based OSD (Bio-OSD) in a laboratory-scale constructed wetland system. The results showed that Bio-OSD significantly reduced COD and BOD₅ levels to meet water quality standards. The highest COD reduction efficiency of 39.78% was achieved when Bio-OSD DOR 0.1:1 treatment was implemented. Under this treatment, BOD reduction efficiency was 27.60%. GC-MS analysis showed the degradation of long-chain hydrocarbons such as n-hexadecane and nonadecane. The highest COD Reduction efficiency by Non-Bio-OSD was 27.17% with DOR 0.25:1. This result showed that Bio-OSD performed better in reducing COD than Non-Bio-OSD. The weight of \u003cem\u003eLemna minor\u003c/em\u003e biomass decreased slightly during the process, indicating that OSD is toxic to the plant. Regardless of the successful construction of wetlands in reducing COD and BOD\u003csub\u003e5\u003c/sub\u003e, it is recommended that the growth condition of the aquatic plant be improved for a sustainable phytoremediation process.\u003c/p\u003e","manuscriptTitle":"Bioremediation of Petroleum Contaminated Water Using Oil Spill Dispersant and Lemna minor in Laboratory Scale of Constructed Wetland","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-28 06:43:37","doi":"10.21203/rs.3.rs-5885729/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4019b0d2-ab2e-4a39-a29d-392c62fb2c86","owner":[],"postedDate":"January 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43288996,"name":"Environmental Engineering"}],"tags":[],"updatedAt":"2025-01-28T06:43:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-28 06:43:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5885729","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5885729","identity":"rs-5885729","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}