Collection of Algae and Chemicals

Ulva rigida was collected from Mandapam, Ramanathapuram district,Tamil Nadu, India. Chemicals and reagents with 99.5 % purity and glassware were obtained from Sigma- Aldrich, Mumbai.

Preparation of Aqueous Extract

U. rigida was carefully taken and washed under running tap water to remove the unwanted sand particles in the sample. The excess sample was washed thoroughly and sliced into small pieces. The sliced pieces of the sample were stored in a polythene bag at −20 °C for further use.

Preparation of Hot Extract

5 g of U. rigida was taken and ground using a mortar and pestle using distilled water. Then the extract was filtered through Whatman No. 1 filtered paper. The filtered sample was made up of 100 mL of distilled water and boiled in a water bath for 30 mins at 100 °C.

Preparation of Cold Extract

5 g of U. rigida was taken and ground using a mortar and pestle using 100 mL aqueous extract. It was centrifuged at 5000 rpm for 5 min. The resultant supernatant was collected and filtered through the Whatman No. 1 filter paper. Later, the extract was stored at 4 °C until further use.

Analysis of Heavy Metals

The prepared hot and cold aqueous extract of U. rigida was used to determine the presence of the following minerals: vanadium, chromium, manganese, iron, nickel, cobalt, copper, zinc, cadmium, arsenic, selenium, and lead using inductively coupled plasma mass spectrometry (ICP-MS).

Seed Germination Assay

The seeds of Pennisetum glaucum were collected from Tamil Nadu Agricultural University located in Coimbatore; Tamil Nadu. The seeds of the same size were surface-sterilized in a sodium hypochlorite solution (10 %) for five minutes. Following this, seeds were washed three to five times with distilled water. Ten seeds were sterilized and soaked in U. rigida extract (hot extract), and then placed on filter paper in 9 cm sterile Petri dishes. Different concentrations (20, 40, 60, and 80 μL) of U. rigida extract were poured into the seeds. The positive control was distilled water. Each plate was covered with parafilm to stop infection and solution evaporation. Laminar flow was used for all processes. The Petri dishes were given labels and kept in a germinator with a temperature of 25 °C and 18/6 hours of day and night light. Up until the end of the 5th, the calculation of germinated seeds was noted. The germination percentage and shoot and root length were calculated.

where n is a number of germinated seeds on the 5th day and N is a number of seeds.

Statistical Analysis

All the analysis was carried out with three replications. The obtained results and data were analyzed using the SPSS software and results were expressed as Mean±SD.

2.1 Analysis of Minerals and Heavy Metals in Hot and Cold Extraction of U. rigida by ICP-MS

According to the ICP-MS analysis, elements such as vanadium, chromium, manganese, iron, nickel, cobalt, copper, zinc, cadmium, arsenic, selenium, and lead were detected in U. rigida-hot and cold extract. Results of the concentration of the minerals in U. rigida-hot extract and cold extract are given in Table 1. Generally, a significant difference was noticed between both the extracts of seaweeds and was found that the concentration of heavy metals and minerals in the hot extract of U. rigida was considerably higher than compared to the cold extract process of U. rigida. The concentration of vanadium was higher in hot extract of U. rigida (65.87 mg/kg) than compared to the U. rigida-cold extract (68.55 mg/kg). The hot extract of U. rigida has an increased concentration of chromium (173.19 mg/kg), manganese (108.16 mg/kg), Iron (164.87 mg/kg), nickel (4.20 mg/kg) than compared to U. rigida-cold extract. The trace amounts of cobalt, selenium, and cadmium were present in both hot and cold extracts of U. rigida (Table 1). The content of zinc (71.31 mg/kg) was high in the hot extraction process, and in the cold extraction process, the content of zinc (11.68 mg/kg) was reduced. Moreover, the mineral and heavy metal content of seaweed varied depending on the monsoon and season. Their ability to absorb and accumulate mineral content was 100 times higher than the of terrestrial plants.²³ Čmiková et.al.,²⁴ reported that the content of elements including copper, cobalt, chromium, iron, manganese, selenium, and zinc was analyzed using ICP OES 720 and was found to be higher in seaweed extracts than compared to land plants, and they have high antioxidant, antimicrobial properties. Truus et al.²⁵ analyzed the elemental composition such as Pb, Cd, Zn, Cu, and Cr from the brown algae Fucus vesiculosus. Queiróset al.²⁶ determined that trace elements such as iron (35.7–170 mg/100 g), zinc (0.8–1.9 mg/100 g), manganese (2.6–11.5 mg/100 g) concentration are higher in U. rigida. The difference in the accumulation of these elements between the two extraction processes is due to the conditions such as surrounding water, light, temperature, and uptake capacity of seaweed.²⁷

2.2 Seed Germination Assay

The result presented in Table 2 indicates the application of U. rigida extract on the seed (Pennisetum glaucum) which shows the positive effect by stimulating the germination of the seed (Pennisetum glaucum). It revealed a significant difference between the experimental treatments (20, 40, 60, and 80 μL) with seaweed extracts. 100 % of the seeds were germinated at different concentrations of U. rigida extract and triplicates were maintained. The germination percentage, root length, and shoot length were found to be higher in all the treatments than compared to the control. The root and shoot length on the 5^th day in control was found to be 3±0.2 cm and 8±1.2 cm. The root and shoot length were increased when the concentration of U. rigida extract was increased. The root length was the same in all the treatments, but the shoot length of Pennisetum glaucum was high at 60 μL of U. rigida extract (11±2.5 cm) and it decreased when the concentration of U. rigida extract (80 μL) increased (10±1.8 cm). Mamede et al.²⁸ reported that the seaweed polysaccharides extracted from Saccorhizapolyschides, Gracilariagracilis and Chondruscrispus increased the seed germination (98.67 %) of Brassica napus L. Thriunavukkarasu et al.²⁹ stated that the Padina gymnospora, Gracilaria edulis and Ulvafasciata seaweed aqueous extracts were used as seaweed biofertilizer for the germination of Capsicum annuum. The seeds were soaked in different concentrations of seaweed extract, and the seed germination percentage of Capsicum annum was found to be 75 %. Sasikala et al.³⁰ investigated that the soil fertility, seed germination, growth, and yield of Solanum lycopersicum were increased when Sargassum tenerrimum seaweed extract was used as a biofertilizer.

Heavy metals can have a dual effect on plant growth, acting as both biocatalysts and toxicants, depending on their concentration.^31-33 In low doses, elements such as zinc, copper, iron, manganese and cobalt play a key role in the biochemical processes of plants. They participate in metabolic pathways such as photosynthesis, chlorophyll synthesis, and enzymatic reactions.³⁴ For example, iron is necessary for protein synthesis and the functioning of the electron transport chain in photosynthetic reactions, and zinc is actively involved in protein synthesis and the regulation of enzymatic activity. A number of researches proved this.^35-40 Thus, the presence of these metals in algal extracts such as Ulva rigida can stimulate plant growth by increasing nutrient uptake and root development. However, with increasing dosage of metals, as was noted in the concentration of 80 μL extract, their effects can become toxic to plants. Exceeding threshold levels of heavy metals such as zinc, copper or iron can lead to inhibition of shoot growth, as was observed in experiments with Pennisetum glaucum. High concentrations of these metals cause oxidative stress, damage cell membranes and disrupt normal metabolic processes in plants. For example, excess iron can lead to the formation of reactive oxygen species, causing damage to cellular structures. Also, excessive accumulation of metals can disrupt the balance of macro- and microelements in plant tissues, which leads to weakened growth and reduced productivity. Thus, although metals can act as biocatalysts that promote plant growth, their concentration must be strictly controlled. Exceeding the optimal dosage, as demonstrated with 80 μL of Ulva rigida extract, leads to a decrease in the effectiveness of growth stimulation and deterioration of the plant.

While Ulva rigida extracts show promise as biofertilizers due to their ability to enhance seed germination and plant growth, potential challenges and limitations should also be considered. One significant concern is the accumulation of heavy metals such as cadmium (Cd) and arsenic (As), which are toxic to both plants and soil ecosystems when present above permissible levels. Based on the study, the concentrations of Cd and As in the U. rigida extracts were measured as 0.88 μg/g (hot extract) and 9.17 μg/g (cold extract) for Cd, and 15.88 μg/g (cold extract) for As. These levels highlight the importance of monitoring and controlling their concentrations in agricultural applications. According to environmental guidelines, the maximum permissible concentrations (MPCs) for Cd and As in soil are typically 3 mg/kg and 20 mg/kg, respectively, depending on regional regulations. In plants, the MPCs for Cd and As are usually 0.1 mg/kg and 1.0 mg/kg (dry weight). Exceeding these thresholds can lead to bioaccumulation in crops, posing risks to human and animal health through the food chain. Furthermore, excessive heavy metals can negatively impact soil microbial activity, reducing soil fertility and nutrient cycling. To mitigate these risks, the following measures are recommended: i) conduct routine assessments of Cd and As levels in both U. rigida biomass and treated soils; ii) optimize extract concentrations to ensure heavy metal levels remain within safe limits for agricultural use; iii) harvest U. rigida from uncontaminated waters to minimize the initial heavy metal content. Despite these challenges, careful application and adherence to safety guidelines can allow the benefits of U. rigida extracts to be harnessed sustainably while mitigating potential drawbacks. Further studies on extraction methods and long-term impacts on soil and plant systems are also warranted.

2.3 Recommendations for further Research

Future studies in this area should prioritize addressing the presence of cadmium and arsenic in the extracts to ensure food safety and compliance with regulatory standards. Detailed toxicological assessments and bioaccumulation studies are necessary to confirm that the use of these extracts does not result in harmful accumulation of these elements in edible plant parts. Researchers should explore selective elimination techniques, such as employing microbial biomass, including bacteria like Bacillus subtilis or fungi like Aspergillus niger, as biosorbents to remove cadmium and arsenic. Chelating resins with specific functional groups, such as iminodiacetic acid, could also be investigated for their high selectivity in adsorbing these metals. Cultivating hyperaccumulator plants, such as Pteris vittata for arsenic and Thlaspi caerulescens for cadmium, before planting could help reduce soil contamination. Optimizing pH-controlled extraction processes should be considered, as the solubility of cadmium and arsenic varies with pH, potentially minimizing their extraction while preserving beneficial components. Additionally, the use of nanomaterials, such as iron oxide nanoparticles with high adsorption capacity for these elements, warrants exploration. Blending strategies could be employed to dilute the concentration of cadmium and arsenic by combining Ulva rigida extract with other seaweed extracts containing lower levels of these elements. Throughout this process, rigorous testing and toxicological evaluations at each stage are essential to ensure that the final biofertilizer product is both safe and effective, with cadmium and arsenic levels in target crop plants remaining below regulatory limits. By adopting these approaches, researchers can contribute to the development of sustainable and safe biofertilizer products that align with food safety standards. Future work could also explore the application of machine learning techniques to analyze the large dataset generated from these experiments, allowing for predictive modeling of material performance under various conditions.^41-43 In increasingly complex agro-climatic conditions^44-49 for growing agricultural crops, the use of elements of the circular economy is especially relevant.^50, 51