In Vivo and In Vitro Antioxidant Activity of Less Polar Fractions of Dasycladus vermicularis (Scopoli) Krasser 1898 and the Chemical Composition of Fractions and Macroalga Volatilome

The present research is a comprehensive investigation of Dasycladus vermicularis (Scopoli) Krasser 1898 from the Adriatic Sea (Croatia) regarding volatilome–volatile organic compounds (VOCs, mostly nonpolar compounds) and less polar nonvolatile compounds for the first time. Headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were used showing the great volatilome variability among fresh (HS-FrDV and HD-FrDV) and dried (HS-DrDV and HD-DrDV) samples after GC–MS analysis. Aromatic aldehydes were dominant in both fresh and air-dried HS samples with benzaldehyde as the most abundant in fresh samples and decreasing 2.7–3.7 times after drying together with 2-phenylbut-2-enal that was not present after drying. Aliphatic compounds (unsaturated hydrocarbons in HS-FrDV; saturated hydrocarbons in HS-DrDV) were also present. C11-hydrocarbons (dictyopterpene C’ and dictyopterpene D’) were detected in HS-FrDV. (E)-Phytol was the most dominant compound in HD-FrDV and HD-DrDV. Diterpene alcohols (cembra-4,7,11,15-tetraen-3-ol and (Z)-falcarinol) and sesquiterpene alcohol, cubenol, were dominant in HD-FrDV, and their abundance decreased after drying. C13-norisoprenoides (α-ionone and β-ionone) increased after drying. Aliphatic compounds were present in both HD-FrDV and HD-DrDV samples. The less polar nonvolatile compounds in the obtained fractions F3 and F4 were analysed and identified by UHPLC-ESI(+)-HRMS. Identified compounds belonged to a group of pigments (7 compounds), fatty acid derivatives (13 compounds), as well as steroids and terpenes (10 compounds). Porphyrin-based compounds (C55H74N4O5–7), xanthophylls, sphingolipid compounds, fatty acid amides, and phytosterols represented the majority of identified compounds. By implementing both in vitro and in vivo assays for antioxidant activity determination, F3 showed a higher activity than F4. Inhibitory concentrations (IC50) for F3 and F4 were 498.00 ± 0.01 µg/mL and 798.00 ± 0.81 µg/mL, respectively, while a 1.5-fold reduction in the ROS level was observed after pre-treatment of zebrafish larvae with 45 µg/mL of F3.


Introduction
The pharmaceutical industry is in a great rise at the global level, which has caused the necessity for new sources of pharmaceutical compounds such as the potential of marine algae. Cyanobacteria (blue-green algae) are well known for their production of antibiotics and active pharmaceutical compounds (API) [1,2]. Despite increasing interest, the economic utilisation of macroalgae for their antioxidant [3,4], anti-tumour [4], and antiviral properties [5] remains in its infancy. They contain complex structure compounds with a variety of biological activities, such as terpenes (antioxidant, anti-tumour, antifungal, etc.), alkaloids (antioxidant, anti-tumour, antiviral, anti-inflammatory, antibacterial, neuroprotective, etc.), and pigments (antioxidant, anti-tumour, anti-inflammatory, antidiabetic and antiobesity, neuroprotective, etc.) [6,7].
Dasycladus vermicularis (Scopoli) Krasser 1898 (fam. Dasycladaceae) is a green macroalga that inhabits rocky substrates of North Atlantic and Mediterranean littoral zones. To survive under increased UV exposure and temperatures, macroalgae developed antioxidant defence mechanisms that activate under the overproduction of reactive oxygen species (ROS), thus reducing/preventing the occurrence of oxidative stress [8]. Photobiological adaptation is strongly influenced by growth location, harvesting season, and environmental pressure, which can result in the variation in antioxidant molecules even among the same species. The Adriatic Sea is an extremely harsh environment characterised by its high salinity, relatively low depth, and oscillations in temperature, which might reflect in the occurrence of a variety of novel bioactive compounds with beneficial properties [9]. Currently available studies on D. vermicularis mostly focused on phenolic compounds with antioxidant and UV-radiation absorption activities such as coumarins and their sulfated metabolites such as 7-hydroxycoumarin-3,6-disulfate [9] and also isocoumarins [10]. There is a lack of data in the literature concerning volatile organic compounds (VOCs) analysis of D. vermicularis.
Our previous studies [10][11][12][13] showed that there is a great diversity of VOCs (mainly nonpolar compounds) when comparing fresh and air-dried samples of marine algae. This was the reason to analyse both fresh (FrDV) and air-dried (DrDV) samples to determine the VOCs variability of D. vermicularis using gas chromatography-mass spectrometry (GC-MS). In addition to the VOCs analysis, less polar nonvolatile compounds and their in vitro and in vivo antioxidative activities were investigated for the first time in this research. Therefore, full chemical profiles of volatile and nonvolatile polar compounds of D. vermicularis were investigated. The present research, as a continuation of our project Bioprospecting of the Adriatic Sea, has the following key goals: (a) identify and compare VOCs of FrDV and DrDV retrieved by both headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) followed by gas chromatography-mass spectrometry analysis (GC-MS); (b) identify the composition of FdDV (freeze-dried D. vermicularis) less polar fractions using high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionisation (UHPLC-ESI-HRMS); (c) evaluate detailed antioxidant activity of the less polar fractions by four in vitro assays (reduction of radical cation ABTS + , 2,2diphenyl-1-picryl-hydrazyl (DPPH) assay, Folin-Ciocalteu method, and ferric reducing antioxidant power (FRAP)); (d) determine the protective effect of the less polar fractions against hydrogen peroxide-induced oxidative stress in the zebrafish model along with the embryotoxicity assessment.
The abundance of carotenoid degradation products, C 13 -norisoprenoides (α-ionone and β-ionone), increased 7.3 times after the drying mostly because of the increment in β-ionone, most probable due to carotenoids degradation.

Analysis of F3 and F4 Fractions Containing Less Polar Nonvolatile Compounds
The FdDV sample was extracted and fractionated (Section 3.6). F3 and F4 fractions containing less polar nonvolatile compounds were acquired and were analysed by highperformance liquid chromatography-high-resolution mass spectrometry with electrospray ionisation in positive mode (UHPLC-ESI(+)-HRMS). The major compounds according to signal intensity (peak area in counts) from the obtained extracted ion chromatograms (XIC) in positive ion mode were plausibly identified. The identification was based on the compounds' proposed elemental compositions and MS/MS spectra (Table 3). Identified compounds belonged to the group of pigments (7 compounds), fatty acid derivatives (13 compounds), as well as steroids and terpenes (10 compounds). The TICs of the fractions F3 and F4 are shown in Figure 1 and the XICs of the most abundant ions in F3 and F4 are shown in Figure 2.      No chlorophyll was detected, but its derivatives, porphyrin-based compounds (C 55 H 74 N 4 O 5-7 ), were abundant, especially in F4 (Figure 3). Among them, the main component was compound 29 (C 55 H 74 N 4 O 6 ). Two other compounds were less polar, so they were much more abundant in F4. It is well known that the porphyrin ring is an essential part of the chlorophyll structure responsible for the antioxidant activity [18]. Antimutagenic and anti-inflammatory effects of pheophytin a were found in green alga Ulva prolifera (formerly known as Enteromorpha prolifera) [19] and its neuroprotective effect was found in brown alga Sargassum fulvellum [20]. Pheophorbide a was also detected but in a small amount, more in F3 than in F4. It has also shown antioxidant activity in green alga Ulva prolifera [21].  Table 3, the numbers correspond to Table 3.
Xanthophylls, both present in green algae, zeaxanthin (or lutein; the isomers that could not be specified), and the one more characteristic for brown algae, fucoxanthin, were detected and identified ( Figure 3) with a greater abundance in F3. In algae, xanthophylls play an important role to protect photosynthetic apparatus against damage induced by high light intensities. This photoprotective role directly purges ROS (reactive oxygen species) within the thylakoid membrane [22,23]. Similarly, they act as anti-tumour and anti-inflammatory agents and are utilised for the prevention of coronary syndromes, cardiovascular diseases, and they play roles in structural action in neural tissue [24]. Supercritical fluid extraction was used to extract zeaxanthin from green alga Nannochloropsis oculata [25] and lutein was extracted from green alga Scenedesmus almeriensis [26]. The ester of siphonaxanthin, another xanthophyll specific for green algae, siphonein, was detected in both fractions, more abundant in F4.
Fatty acid derivatives in F3 and F4 originate from palmitic, stearic, oleic, and behenic acids (Table 3). Sphingolipid compounds 22 and 25 ( Figure 4) were the most abundant of all detected components of F4, but were also present in F3. Sphingolipids as a part of macroalgae membranes play one of the crucial structural and physiological roles [27]. Recently, studies have shown that sphingolipid, together with associated enzymes and receptors, were involved in the inflammatory process and can provide effective drug targets for the treatment of pathological inflammation [28]. Three fatty acid amides were found: oleamide, erucamide, and palmitamide. They were more abundant in F4 even though oleamide was the most abundant of all detected compounds in F3. Oleamide is an endogenous bioactive signalling molecule found in both marine and terrestrial plants. Acting in various cell types consequently triggers different biological effects, and one of the most recognised ones is its sleep-inducing effect [29]. Isolated from green algae Codium fragile, oleamide also suppressed, among others, the secretion of TNF-α (tumour necrosis factor-α) and interleukins IL-1β (interleukin-1 beta) and IL-6 (interleukin 6), and prevented the translocation of NF-kappa B into the nucleus, which are main features in the anti-tumour process [30]. It was also found in Ericaria sp. [11]. Erucamide and palmitamide were previously identified in Prymnesium parvum [31] and Ericaria sp. [11]. Fatty acid glycerides were also present in both fractions.  Table 3, where the numbers correspond to Table 3.
Steroids and terpenes consisted of 10 identified compounds (Table 3). Four compounds were the major ones ( Figure 5): sterols (compounds 2 and 3) and compound 24 (Table 3), which belong to the class of stigmastenes and derivatives that had a higher abundance in F3; and the steroid, compound 5 ( Table 3) that was more present in F4. Phytosterols, a term referring to plant-derived sterols or stanols, are a group of naturally occurring compounds found in plant cell membranes [32]. Their main therapeutic function is reducing the cholesterol level [33], but they exhibit numerous other health benefits such as anti-diabetes, antiatherosclerosis, anti-obesity, anti-Alzheimer's, anti-tumour, and hepatoprotective [34,35]. Green algae have a relatively heterogeneous sterol composition [36]. Previous research found four sterols in D. vermicularis: cholesterol, 24-methylenecholesterol, brassicasterol, and clionasterol [37].  Table 3, and the numbers correspond to Table 3.

Antioxidant Activity of F3 and F4 Fractions In Vitro
To analyse the antioxidant potential of the obtained D. vermicularis fractions (F3 and F4), and to obtain better insight into their antioxidant behaviour, different assays were used. The results for four assays (Folin-Ciocalteu method, ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and oxygen radical absorbance capacity (ORAC)) are depicted in Figure 6. The results of Folin-Ciocalteu method showed significantly (p < 0.0001) different activity for F3 and F4 fractions, namely, a 50-fold higher activity was obtained for F3. As no polyphenols were found in these fractions (Table 3), this response for F3 could be assigned to the presence of xanthophylls fucoxanthin and zeaxanthin (or lutein) in higher amounts when compared to F4, as it is known that the pigments interact with Folin-Ciocalteu regent [38]. Contrarily, almost the same activity was obtained using the FRAP assay for both fractions, i.e., 3.1 ± 0.2 mmol/g for F3 and 3.2 ± 0.07 mmol/g ferrous equivalents for F4. The scavenging activity using DPPH assay when normalised per gram of the fraction revealed also almost the same activity for both fractions, i.e., for F3, it was 286.79 ± 14.09 mg AAE/g fraction, while for F4, it was 273.42 ± 1.29 mg AAE/g fraction. ORAC assay is based on a fluorescent signal from a probe that is quenched in the presence of reactive oxygen species (ROS). The results shown in Figure 6b revealed a 1.4-fold higher (p > 0.001) activity for F3 than F4. Further on, the detailed reduction of radical cations by implementing ABTS assay was studied. Different concentrations of F3 and F4 fractions were prepared ranging from 0.005 to 5 mg/mL to obtain dose-response curves ( Figure 7) and calculate IC 50 values ( Table 4). As can be seen, two D. vermicularis samples exhibited a similar activity, i.e., IC 50 values were 0.498 and 0.798 mg/mL for F3 and F4 fractions, respectively. A lower IC 50 value for F3 could be ascribed to higher amounts of xanthophylls found in F3. However, one should note that a similar antioxidant behaviour was observed for both F3 and F4 of D. vermicularis, which indicate that, although different compounds were identified in each fraction and in different amounts, a synergistic effect leading to a similar activity can be observed. Additionally, in F4, indole derivatives were found (for example, 1 H-5α-cholest-2-eno [3,2-b] indole) in a higher amount, which probably contributed to the antioxidant responses [39].

Antioxidant Activity of F3 and F4 Fractions In Vivo
The antioxidant potential of F3 and F4 fractions was assessed in vivo in zebrafish Danio rerio exposed to H 2 O 2 -induced oxidative stress. F4 induced a concentration-dependent increase in mortality (half maximal lethal concentration (LC 50 ) = 17.13 ± 1.27 µg/mL) and developmental abnormalities (half maximal effective concentration (EC 50 ) = 29.52 ± 4.27 µg/mL), as presented in Figure 8a. Although no mortality was observed during the exposure to F3, the highest tested concentration (62 µg/mL) increased the incidence of developmental abnormalities (16.67 ± 11.55% of specimens; data not shown). When compared to F3 fraction, F4 fraction contained a higher amount of fatty acid amides such as oleamide, palmitamide, and erucamide that might be responsible for elevated toxicity. This was also the case in our recent study where Ericaria crinita and Ericaria amentacea fractions enriched with mentioned fatty acid amides negatively affected the survival of zebrafish embryos [11]. Considering the observed toxicity, the determination of in vivo antioxidant potential was decided to be conducted on 2.5, 5.0, and 10.0 µg/mL of F4 and 11.3, 22.5, and 45.0 µg/mL of F3. As shown in Figure 8b, the survival rate of the zebrafish larvae was significantly reduced upon exposure to H 2 O 2 . However, pre-treatment with 45 µg/mL of F3 significantly protected zebrafish from H 2 O 2 -induced mortality (survival rate increased by 35.3% compared to the treatment group on H 2 O 2 ; p < 0.05). H 2 O 2 increased ROS production in zebrafish up to 255.8 ± 6.99% compared to the control treatment group on artificial water (100%; Figure 8c), which revealed a high fluorescence of the positive control (Figure 8d). Following the treatment with 45 µg/mL of F3, the ROS levels reduced to 164 ± 6.2% (p < 0.05; Figure 8c,d). The results are expressed as mean ± SD of three replicates; # denotes significant differences (p < 0.05); n.d. = not detected.
These findings are in correlation with the results obtained with UHPLC-ESI(+)-HRMS analysis. Both fractions contain the compounds that have antioxidative properties such as porphyrin-based compounds [40] and xanthophylls [41], as well as sphingolipids [42]. Fucoxanthin and zeaxanthin/lutein (Table 3), molecules well-known for their antioxidant potential, have a greater abundance in F3, which might be the reason of the stronger antioxidant activity of F3 in relation to F4. Nevertheless, one should notice that the determination of the antioxidant potential of F4 in vivo was conducted within the lower concentration range (2.5-10.0 µg/mL) compared to F3 (11.3-45.0 µg/mL). Taken together, the obtained results indicate the pharmaceutical potential of D. vermicularis.

Alga Sample
Dasycladus vermicularis (Scopoli) Krasser 1898 was collected in July 2021 by a singlepoint collection from the Adriatic Sea (Uvala Jasenovo, Ravni Kotari) with the sampling geographical coordinates 44 • 17 01" N; 15 • 12 27" E. The sea depth was 3 m with the sea temperature at 25 • C. The macroalga was transferred to the laboratory in an air-tight plastic box containing both alga and seawater immediately after the collection. It was maintained at 4 • C in the dark for not more than 24 h until further analysis. A part of the collected D. vermicularis was air-dried and placed in the dark at room temperature for 10 days. Both fresh and air-dried samples were sliced into small pieces before further analysis. The identification of the collected alga was performed by marine biology experts Donat Ptericiolli and Dr Tatjana Bakran-Petricioli, professor at the Faculty of Science, University of Zagreb.
A part of D. vermicularis was freeze-dried for fractionation, as described in Section 3.6. Before the freeze-drying, the sample was washed 5 times in tap water and 2 times in deionised water and was sliced into 5-10 mm pieces. Sliced alga was frozen at −60 • C in an ultra-low-temperature freezer (CoolSafe PRO, Labogene, Denmark) for 24 h. The primary and secondary drying temperatures were −30 • C and 20 • C and freeze-drying was performed under a high vacuum (0.13-0.55 hPa) for 24 h.

Headspace Solid-Phase Microextraction (HS-SPME)
The excess seawater from the fresh sample was removed by placing a part of the sample between two layers of filter paper for a few minutes. HS-SPME was performed using an autosampler, PAL Auto Sampler System (PAL RSI 85, CTC Analytics AG, Zwingen, Switzerland), and two SPME fibres of different polarities. One fiber was covered with DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) and the other one with PDMS/DVB (poly-dimethylsiloxane/divinylbenzene). Both fibres were conditioned prior to the extraction according to the manufacturer and were purchased from Supelco Co. (Bellefonte, PA, USA). Prepared samples, with the mass of 1 g, were placed into HS-20 mL glass vials sealed with a polytetrafluorethylene (PTFE)/silicon septa stainless-steel cap. Equilibration of the sample was performed at 60 • C for 15 min and then it was extracted for 45 min. The temperature of the injector was set to 250 • C. Thermal desorption of the sample from the fibre was conducted directly to the GC column for 6 min. HS-SPME was performed in triplicate.

Hydrodistillation (HD)
The hydrodistillation (HD) procedure was obtained in a modified Clevenger apparatus for 2 h. Diethyl ether (J.T. Baker Inc., NJ, USA) and pentane (Fluka, Merck KGaA, Germany) were used as the solvent trap in a v/v ratio of 2:1 (1 mL). HD was performed separately on the prepared samples of fresh and air-dried D. vermicularis. The volatile oil dissolved and trapped in the solvent trap was carefully removed with a pipette avoiding taking the water part. It was then slowly concentrated by the gentle flow of nitrogen until the volume of 0.2 mL was reached. An amount of 2 µL of the sample was used for GC-MS analyses. HD was performed in triplicate.

Gas Chromatography Mass Spectrometry Analysis of VOCs
The GC-MS analyses of isolated VOCs were run on an Agilent Technologies (Palo Alto, CA, USA) gas chromatograph model 8890 tandem mass spectrometer detector model 5977E MSD (Agilent Technologies, Palo Alto, CA, USA). An HP-5MS capillary column with the dimensions of 30 m × 0.25 mm and 0.25 µm film thickness (Agilent Technologies, Palo Alto, CA, USA) was used for the VOCs separation. The injector temperature was set to 250 • C and the detector temperature to 300 • C. The oven temperature was set to 70 • C isothermal for 2 min. The temperature gradient was obtained by temperature increments from 70 to 200 • C at 3 • C/min. After reaching the temperature of 200 • C, it was held isothermally for 15 min. The flow rate of helium as a carrier gas was 1.0 mL/min and the split ratio was 1:50. The MSD (EI mode) was operated at 70 eV with the scanning mass range from 30 to 300 Amu. The identification of the compounds was achieved by comparing their retention indices (RI), which were defined relative to the retention times of n-alkanes (C 8 -C 25 ), with those reported in the literature (National Institute of Standards and Technology) and their mass spectra with the spectra from NIST 17 (D-Gaithersburg) and Wiley 9 (Wiley, New York, NY, USA) mass spectral libraries. The percentage composition of the samples was calculated using the normalisation method without correction factors. The average component percentages in Tables 1 and 2 were calculated from GC-MS analyses of three replicates.

Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-ESI-HRMS) of F3 and F4
The UHPLC-ESI-HRMS analyses were performed using an UHPLC ExionLC AD system (AB Sciex, Concord, ON, Canada), which was equipped with the ExionLC Controller, ExionLC AD Pump, ExionLC AD Degasser, ExionLC solvent delivery system, ExionLC AD Autosampler, and ExionLC AD Column oven tandem quadrupole-time-of-flight (Q-TOF) mass spectrometer TripleTOF 6600+ (AB Sciex, Concord, ON, Canada) having a Duospray ion source. The chromatographic separations were achieved on the analytical column Acquity UPLC BEH Phenyl-Hexyl with dimensions of 2.1 mm × 100 mm and a particle size of 1.7 µm (Waters, Milford, MA, USA). The aqueous mobile phase (A) was water containing 0.1% formic acid and the organic mobile phase (B) was acetonitrile with 0.1% formic acid. The mobile phase flow rate was continuously 0.4 mL/min and the oven temperature was 30 • C. The elution started isocratically for 0.6 min with 2% of B and, later, the gradient program was applied: 0.6-18.5 min (B linear gradient to 100%), 18.5-25 min (100% B). An amount of 4 µL of the sample was injected.
Positive electrospray ionisation (ESI+) was set. Collision-induced dissociation (CID) in information-dependent acquisition (IDA) mode was applied recording the MS/MS mass spectra. The precursor ions only with the signal intensities above the 200 cps threshold were recorded with the maximum number of 15 precursor ions simultaneously subjected to CID. The parameters set in the ion source were as follows: source temperature of 300 • C, nebulising gas (gas 1, air) pressure of 40 psi, curtain gas (nitrogen) pressure of 30 psi, heater gas (gas 2, air) pressure of 15 psi, and ESI capillary voltage of 5.5 kV. Mass spectra were recorded in the m/z range of 100-1000 (MS) with the declustering potential of 80 V and accumulation time of 100 ms. MS/MS data were recorded in the m/z range of 20-1000, and the collision energy of the collision gas (nitrogen) was 40 eV with a spread of 20 eV. The mass scale calibrations (in the MS and MS/MS modes) were performed prior to each run in an automatic regime using an ESI Positive Calibration Solution 5600 (AB Sciex, Concord, ON, Canada).
ACD/Spectrus Processor 2021.1.0. (ACD/Labs, Toronto, ON, Canada) was used for processing data. The accurate masses of the protonated molecules, their isotopic distributions, and the product ions m/z in MS/MS spectra were used for the compounds' elemental compositions determination. Detected components were then identified based on their mass spectra and the elemental compositions linked to search in the Chemical Entities of Biological Interest (ChEBI) database. The choice among the suggested hits was based on MS/MS data matching.

Antioxidant Activity of Tested Fractions by In Vitro Assays
Antioxidant activity determination was performed using five different methods, namely the Folin-Ciocalteu method, ferric-reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, oxygen radical absorbance capacity (ORAC), and the reduction in the radical cation (ABTS) assays. All assays were carried out in triplicates in 96-well plates using a UV/Vis microplate reader (Infinite M200 PRO, TECAN, Switzerland), while all results were expressed as mean ± standard deviation (n = 4). A detailed description of all used methods in this research is described in our recently published manuscript [11].

Embryotoxicity Potential
Zebrafish maintenance and spawning have been described in our recent paper [43]. The zebrafish embryotoxicity test (OECD 236, 2013) was conducted to determine the negative potential of tested F3 and F4 fractions. A wide range of serial dilutions of F3 (115-3.59 µg/mL) and F4 (62-2.88 µg/mL) was tested, at the same time taking care that the concentration of the solvent (MeOH and DMSO) did not exceed 1%. As a negative control artificial water was used, while MeOH and DMSO (1%) were used as a solvent control. Upon 96 h of exposure to tested fractions, mortalities and abnormalities were inspected using an inverted microscope Olympus CKX41, equipped with a Leica EC3 digital camera and LAS EZ 3.2.0 digitising software. The concentrations of fractions that showed no negative impact on zebrafish embryonal development were further used.

Antioxidant Potential In Vivo
To determine the antioxidant potential of DAVE fractions in vivo, the protocol described in Jerković et al. (2021) was followed [12]. Briefly, zebrafish embryos were pretreated with three concentrations of F3 (2.5, 5.0, and 10.0 µg/mL) and F4 (11.3, 22.5, and 45.0 µg/mL) for 2 h and stimulated with 5 mM of H 2 O 2 . At 96 hpf, survived specimens were counted. To detect intracellular ROS formation, larvae were stained with 10 µM of DCF-DA and incubated in the dark for 1 h. Stained larvae were inspected using the fluorescent microscope Olympus ® BX51 and images were taken using the digital camera DP70 and Microsoft ® AnalySIS Soft Imaging System Software 3.1. (Münster, Germany). The fluorescence intensity of images was quantified using ImageJ software.

Ethical Statement
Animal housing and spawning were performed in an aquaria unit approved by the Croatian Ministry of Agriculture and according to the Directive 2010/63/EU. The experiments were conducted on the nonprotected stages of zebrafish development (up to 96 hpf), which do not require permission by animal welfare commissions (Directive 2010/63/EU).

Statistical Analysis
GraphPad Prism software version 8 was used for statistical analysis and graph presentation. A one-way analysis of variance (ANOVA) and Tukey's multiple comparisons were performed to examine the significant differences between the means. A Welch's t-test was used to examine the significant difference between tested samples for each in vitro antioxidant method. Differences were considered significant at p < 0.05. Prior to LC 50 and IC 50 determination, the obtained values were subjected to logarithmic transformation.

Conclusions
Little data are available on D. vermicularis chemical composition in the literature, except for polyphenols and coumarins analysis, and the present research is a comprehensive investigation of D. vermicularis from the Adriatic Sea (Croatia) regarding VOCs and less polar nonvolatile compounds for the first time. VOCs from both fresh (FrDV) and air-dried (DrDV) samples were presented. Both HS-SPME and HD were used, showing a great variability among HS-FrDV and HD-FrDV, as well as among HS-DrDV and HD-DrDV. Utilising these two methods, VOCs of different polarities and volatility were isolated. There is clearly a noticeable difference in fresh (FrDV) and air-dried (DrDV) samples. Aromatic compounds were dominant in both fresh and air-dried HS samples with benzaldehyde and 2-phenylbut-2-enal as the most abundant in fresh samples. C 11 -hydrocarbons dictyopterpene C' and dictyopterpene D' were detected with great abundance in HS-FrDV. Aliphatic compounds were dominant in both HD-FrDV and HD-DrDV samples. In HD-FrDV, diterpene alcohols (cembra-4,7,11,15-tetraen-3-ol and (Z)-falcarinol) and sesquiterpene alcohol cubenol were dominant. Its amount decreased, but C 13 -norisoprenoides (α-ionone and β-ionone) increased during the air-drying process. The less polar compounds in F3 and F4 were analysed and identified by UHPLC-ESI(+)-HRMS. Identified compounds belonged to a group of pigments (7 compounds), fatty acid derivatives (13 compounds), as well as steroids and terpenes (10 compounds). Porphyrin-based compounds (C 55 H 74 N 4 O 5-7 ), xanthophylls, sphingolipid compounds, fatty acid amides, and phytosterols represent the majority of identified compounds.
In vitro analysis of the antioxidant potential of less polar compounds from D. vermicularis by implementing five independent assays (Folin-Ciocalteu, FRAP, DPPH, ORAC, and ABTS) showed a high activity of both F3 and F4 fractions. F3 exerted a higher inhibition of reactive oxygen species than F4, which can be related to the presence of pigments, namely xanthophylls. When compared to the results obtained by in vivo analysis, F3 also showed protective effects against the H 2 O 2 -induced mortality of zebrafish embryos. The first results on D. vermicularis suggest that this green alga might be a potent source of natural antioxidants such as pigments, terpenes, and porphyrin-based compounds, and further research regarding different biological activities is in progress.