# Seasonal Monitoring of Volatiles and Antioxidant Activity of Brown Alga _Cladosteplus spongiosus_
## 1 Introduction  
The genus _Cladosteplus_ currently consists of five taxonomically recognised species. These species are cosmopolitan brown algae that grow on rocks in the intertidal zone and at depths of up to six metres, mainly in temperate seas [1]. _Cladosteplus spongiosus_ occurs in the Adriatic Sea [2]. Its thalli are usually between 3 and 20 cm long, with the longest thallus present in summer, during its growing season, when the sea temperature is the highest.  
In winter, when the sea temperature is at its lowest, only small specimens 3 cm in length are observed [3].  
Several studies have confirmed the bioactivity of compounds obtained from this species. The extracts from _C. spongiosus_ have been reported to exhibit antioxidant, antibacterial, anticandidal, cytoprotective, insecticidal, and amoebicidal activities [4; 5; 6; 7; 8]. It has been previously reported that the bioactivity of the extracts could be affected by the season of algal harvest and extraction method [9; 10; 11]. However, to our knowledge, no studies on these effects have been performed for _C. spongiosus_. Air-dried samples of _C. spongiosus_ harvested from the Algerian coast were extracted and the extracts were analysed by HPLC and GC-MS. Fucoxanthin, apo-9\({}^{\prime}\)-fucoxanthinone, apo-13\({}^{\prime}\)-fucoxanthinone, and lolioliolid were identified in the samples [12]. The major compounds identified in the acetone extracts of _C. spongiosus_ were 4-hydroxy-4-methylpentan-2-one, \(n\)-hexadecanoic acid, and propenylguaiacol, while octadeca-9,12,15-trienoic acid ethyl ester, hexadecanoic acid ethyl ester, and 3,7,11,15-tetramethylhexadec-2-en-1-ol were the major compounds in the ethanol extracts [8].  
[PERSON] et al. [13] isolated volatile compounds from _C. spongiosus_ harvested in Australia using combined steam distillation solvent extraction with pentane/diethyl ether. Overall, 2,6-dibromophenol, 2-bromophenol, 2,4,6-tribromophenol, 4-bromophenol, and 2,4-dibromophenol were identified in the samples using gas chromatography and mass spectrometry (GC-MS). In addition, the lipid phase of freeze-dried samples of _C. spongiosus_ collected off the Algarve coast in Portugal was extracted and analysed by GC-MS. The authors reported 31.74% of saturated fatty acids, 12.15% of monounsaturated fatty acids, and 56.11% of polyunsaturated fatty acids [14]. [PERSON] et al. [15] determined the profile of volatile compounds in _C. spongiosus_ harvested in Spain using headspace solid-phase microextraction (HS-SPME). The authors tentatively identified pent-1-en-3-one, pent-2-en-1-ol, hexanal, hexan-1-ol, tribromomethane, 1-iodo-3-methylbutane, and oct-1-en-3-one. To our knowledge, there are no studies on the changes of volatile organic compounds (VOCs) during the growth period of _C. spongiosus_. In the present study, VOCs were separated and analysed from air-dried and fresh _C. spongiosus_ samples from the Adriatic Sea using HS-SPME and hydrodistillation (HD). These techniques were chosen because they allow the isolation headspace, volatile and semi-volatile compounds. Algal samples were collected during the growing season from May to August. The study objectives were: (1) to isolate VOCs from _C. spongiosus_, (2) to determine the chemical composition of obtained VOCs, (3) to determine the effects of air drying and growing season on VOCs composition, (4) to determine the seasonal antioxidant potential of _C. spongiosus_ ethanolic extracts using different assays, (5) to compare the ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) to produce the extracts with higher antioxidant activity, and (6) to identify the non-volatile compounds in the most potent ethanolic extract.