Occurrence and patterns of nutritional traits and polycyclic ...

Food Quality and Safety, 2022, 6, 1–23https://doi.org/10.1093/fqsafe/fyac005Advance access publication 13 January 2022Article

Received 24 July 2021; Revised 5 November 2021; Editorial decision 14 December 2021

Article

Occurrence and patterns of nutritional traits and polycyclic aromatic hydrocarbons (PAHs) in sea cucumber (Holothuria polii) tissues: benefits and risk for human healthFrancesca Biandolino, Isabella Parlapiano, Lucia Spada, Antonella Di Leo, Maria Calò, Giovanni Fanelli, Ermelinda Prato*, , and Santina GiandomenicoCNR-IRSA, National Research Council, Water Research Institute–Via Roma 3, Taranto, Italy*Correspondence to: E. Prato, CNR-IRSA, National Research Council Water Research Institute–Via Roma 3, Taranto 74121, Italy. E-mail: [email protected]

Abstract Objectives: The paper evaluates the benefit and risk for human health associated with consumption of sea cucumber Holothuria polii (H. polii) from Italian coasts (Central Mediterranean Sea). Materials and Methods: Body wall (BW), internal tunic (ITu), muscle bands (MBs), alimentary canal (AC), gonad (Gd), and respiratory tree (RT) of H. polii were analyzed for proximate composition. Moreover, amino acids (AAs), fatty acids (FAs) and polycyclic aromatic hydrocar-bons (PAHs) were determined with high-performance liquid chromatography coupled with ultraviolet-visible spectroscopy (UPLC UV/Vis), gas chromatography-flame ionization detector (GC-FID) and gas chromatography mass spectrometry (GC-MS), respectively.Results: Differences in the contents of total amino acids (TAAs) occurred based on tissue and sex, with AC and MB of female and Gd of male showing higher contents (range 47.8–60.2 g/kg we weight (ww)). Glycine and glutamic acid were the most abundant. Polyunsaturated fatty acid (PUFA) was the major class of FAs and arachidonic acid and eicosapentenoic acid (EPA) were the predominant PUFA. n-3 PUFA showed higher content in Gd, AC, and RT, indicating higher quality. A favorable n-3/n-6 in the range of 1.04–1.67 was observed. PAHs showed values ranging from 23 to 207 µg/kg ww with the highest levels in Gd and AC tissues and the lower in BW. Benzo[a]pyrene, the most toxic compound, was detected in all tissues, of both sexes, at levels of 1.5–18 µg/kg ww.Conclusion: All tissues of H. polii, although with differences among them, are valuable food and can contribute for a healthy diet. Excess cancer risk (CR) values for Gd and AC tissues were above the considerable CR threshold of one in 10 000 established by the United States Environmental Protection Agency (USEPA) for high ingestion rate of this seafood.Keywords: Holothuria polii; nutritional quality; polycyclic aromatic hydrocarbons; risk assessment; estimated weekly intake; cancer risk.

IntroductionSea cucumber (Echinodermata: Holothuroidea) represents an important seafood whose consumption is becoming popular around the world (Lovatelli et al., 2004) because of their bio-active compounds (polyunsaturated fatty acids (PUFAs), es-sential amino acids (EAAs), minerals, proteins, etc.), which have many benefits for human health.

In Italy, sea cucumbers are not consumed as food but are used by fisherman as fish bait or fished and exported to Asian countries, where they represent a delicacy for their dietary and curative properties (Çakli et al., 2004; Özer et al., 2004; Aydin et al., 2011).

Sea cucumbers are marketed in different ways such as frozen, cooked–dried, cooked–salted, and cooked–salted–dried products (Çakli et al., 2004). The body wall (BW), usually processed into a dried product and marketed as ‘beche-de-mer’, is valued as an important seafood particu-larly in Asian markets, where they are traded as a luxury seafood. China is the main consuming country followed by Republic of Korea, Indonesia, and Japan (Ferdouse, 2004).

In Japan and Republic of Korea, sea cucumbers are con-sumed raw or brined (cell wall); moreover, products pre-pared from dried gonad (Gd) and fermented or brined intestine are very well appreciated (Ferdouse, 2004). Boiled skin extracts are consumed as a tonic in Malaysia (Çakli et al., 2004). In the USA, their body walls are consumed in the form of dry tablets.

Fresh or frozen longitudinal muscle bands (MBs) of the giant red sea cucumbers (Parastichopus californicus (P. californicus)) harvested in Alaska represent the most ap-preciated export product by consumers, while the BW often much less (Bechtel et al., 2013).

As the market demand increasing, the retail price of dried sea cucumbers is up to 1500 euros per kilogram, which has led to often indiscriminant and exces sive exploitation that re-sulting in a reduction of sea cucumber stocks (Purcell, 2014). In some cases, this has determined the closure of many na-tional fisheries to allow stocks recovery and to establish more sustainable management plans (Roggatz et al., 2016; González-Wangüemert et al., 2018).

© The Author(s) 2022. Published by Oxford University Press on behalf of Zhejiang University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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2 F. Biandolino et al.

The high market price has encouraged the catch and commer-cialization of autochthonous species from Mediterranean Sea, such as Holothuria polii (H. polii). This species is sold as a dried or frozen product to Asian country, where it represents a source of nutraceutical compounds (Ismail et al., 2008; Mokhlesi et al., 2012). Moreover, H. polii is considered a promising species for diversification of aquaculture (Sicuro et al., 2012).

Information on food composition is essential for market exchange and for consumer protection; it has been recog-nized that food security, nutrition, and food safety are inex-tricably linked (WHO, 2015). Jennings et al. (2016) referred that seafood security demands that they should be sustainable for people and provide them with nutritional benefits while posing minimal health risks. H. polii lives on soft sediments and Posidonia meadows, playing a fundamental role in ben-thic dynamics by processing and bioturbating the sediment (Purcell et al., 2016). It is a deposit feeder that ingests a large quantity of sediments to obtain its feeding. However, marine sediments are potentially a sink of chemical contaminants, es-sentially derived from anthropogenic activities. Sea cucum-bers ingesting and accumulating these contaminants in their tissues can pass them on to the next trophic level in the food web, posing a significant threat to human health.

Because these animals are generally 5–6 years old when they reach their commercial size, they are able to accumulate more pollutants than other organisms during that time. For this reason they are considered bioindicators of marine sedi-ment quality (Martin et al., 2017).

Among pollutants, polycyclic aromatic hydrocarbons (PAHs) are of great concern because some are toxics and can cause cancer in humans (IARC, 2010). PAHs are a large group of organic compounds containing two or more fused aromatic rings constituted of carbon and hydrogen atoms. The major route of exposure for non-smoking humans is the consumption of food (SCF, 2002; ESFA, 2008), as it can be contaminated from industrial food processing, cooking prac-tice, and environmental sources (Zelinkova and Wenz, 2015). The sources, natural and anthropogenic, of PAHs in the envir-onment are numerous. Some of these include forest fires and volcanic eruptions, industrial plants, domestic heating, and diesel engines (Howsam and Jones, 1998).

In this context, this work, for the first time, reports the benefit and risk for human health associated with consump-tion of H. polii from Italian coasts (Central Mediterranean). Overall, the specific objectives of this study were: (1) to in-vestigate comparatively the proximate composition, AAs, and fatty acids (FAs) profiles of six tissues of male and female H. polii; (2) to assess PAH distributions in the different tissues examined; and (3) to evaluate non-carcinogenic and carcino-genic risk for human health.

Materials and MethodsSample collectionAbout 20 specimens of H. polii were collected from an area of Mar Grande in Taranto (Ionian Sea, southern Italy) at depths between 10 m and 15 m, by self-contained under-water breathing apparatus (SCUBA) diving, during October–November 2018. In the laboratory, samples were rinsed with distilled water to remove debris and foreign particles. The samples were identified according to identification guidelines described by Aydin and Erkan (2015).

Each sea cucumber was weighed and measured, then they were separated into two groups (male and female), dissected by removing all tissues and placed in separate containers: BW, internal tunic (ITu), MBs, Gd, alimentary canal (AC), and respiratory tree (RT). All tissues were cut into small pieces, finely ground in a blender (Kinematica Polytron™ PT 10/35 GT, Thermo Fisher Scientific, Waltham, MA, USA), frozen at –20 °C for at least 12 h, and freeze-dried. H. polii had a mean length of (16.3±2.7) cm and a mean weight of (115.7±19.8) g. The freeze-dried samples were blended further until a fine powder was produced and stored in a closed, dark bottle before analysis. The storage time was not longer than one month. All analyses were con-ducted in triplicate.

Proximate composition analysisMoisture was determined after drying in an oven at 105 °C until constant weight and ash content by incineration in a muffle furnace at 600 °C for 2 h (AOAC, 2002); protein by the method of Bradford (1976) with blue brilliant of Coomassie (G 250, Merck, Milan, Italy) as reagent and bo-vine serum albumin (Sigma-Aldrich, Milan, Italy) as reference standard; lipid by extraction with chloroform–methanol and gravimetric determination (Folch et al., 1957).

Amino acids analysisInto a vial was added 10 mg of each dry tissue with 1 mL of hydrolyzing solution (HCl 6 mol/L). Then the vial was sealed and placed in an oven at 110 °C for 22 h. After cooling at room temperature, all hydrolysates were filtered on a 0.45-μm Whatman filter (Deerfield, IL, USA). The derivatization procedure was based on the reaction between phenyl isothiocyanate (PITC) and AAs with the formation of a substituted thiourea to be detected at λ=254 nm, fol-lowing the method of Campanella et al. (1999). Shortly, 25 μL of the extract and 25 μL of Norleucine of 10–3 mol/L (Internal Standard (I.S.)) were derivatized with 25 μL of a mixture of ethanol, H2O, Triethylamine (TEA) and phenyl isothiocyanate (PITC) (7:1:1:1, in volume). After the reaction, the sample was dried and 250 μL of mobile phase A (0.7 mol/L CHCOONa+2.5 mL/L TEA+CH3COOH to pH 6.4) were added. For the cysteine analysis, 200 mL of sample were previously added to 25 mL of I.S. and 25 mL of 100 mmol/L iodioacetic acid. After filtration, 50 μL of the solution was analyzed by means of high-performance liquid chromatography (HPLC). The HPLC equipment was a Beckman (System Gold 126; Beckman Coulter, Inc., Brea, CA, USA) Chromatograph equipped with a ultraviolet-visible spectroscopy (UV/Vis) detector set at 15 254 nm. Separations were carried out a LC-18 DB Supelcosil column (25 cm×0.46 cm i.d., 5 μm; Supelco, Bellefonte, PA, USA). Mobile phase flow-rate was 1 mL/min and a ternary gradient was employed where mobile phase A was 0.7 mol/L sodium acetate containing 2.5 mL ad-justed to pH 6.4 with acetic acid; mobile phase B was water; and mobile phase C was acetonitrile–water (80:20, in volume). The gradient was from A–B–C (20:75:5) to A–B–C (20:30:50) in the first 25 min, then from that to A–B–C (10:10:80) in 1 min, and was then held constant for 4 min. A period of 10 min was required to re-equilibrate the initial column condition.

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Nutritional traits and PAHs in sea cucumber 3

Fatty acid analysisTo prepare fatty acid methyl esters (FAME), a 0.1-mL aliquot of the total lipid–chloroform solution fraction and 0.1 mL of methyl nonadecanoate (0.16 mg/mL; i.s.) were evaporated under a stream of nitrogen, and then reacted with 14% (mass concentration) boron trifluoride methanol complex (BF3) for 45 min at 95 °C (Morrison and Smith, 1964). The FAME were extracted with hexane and separated and quantified using an HP 6890 series GC (Hewlett Packard, Wilmington, DE, USA) equipped with a flame ionization detector and a fused-silica capillary column (30 m×0.32 mm, i.d., film thickness 0.25 μm; Omegawax, Supelco, Bellefonte, PA, USA). Helium was used as the carrier gas. The column temperature program was as follows: 150 to 250 °C at 4 °C/min and held at 250 °C. The FA peaks were identified by comparing their retention times to a mixture of FAME standards (Supelco 37 Component FAME Mix). Quantification was made using the technique of internal standardization with methyl nonadecanoate serving as standard (Sigma, St. Louis, MO, USA).

Lipid Nutritional Quality Indices (LNQI)In order to obtain a comprehensive evaluation of the lipid quality of H. polii, atherogenic (AI), trombogenic (TI) indices (Ulbricht and Southgate, 1991), and hypocholesterolemic/hypercholesterolemic (h/H) ratio (Santos-Silva et al., 2002) were used applying following formulas:

AI =[(C12 : 0) + (4× C14 : 0) + (C16 : 0)] /

[∑

n-6 +∑

n-3 +∑

MUFA]

TI =[(C14 : 0) + (C16 : 0) + (C18 : 0)] / [0.5 × +∑

MUFA)

+ (0.5 ×∑

n-6) + (3×∑

n-3) + (∑

n-3/∑

n-6)]

h/H ratio =[(C18 : 1 + C18 : 2 + C18 : 3 + C20 : 3

+ C20 : 4 + C20 : 5 + C22 : 4

+ C22 : 5 + C22 : 6) / (C14 : 0 + C16 : 0)]

PAH analysisFor PAH analysis, 1 g of freeze-dried tissues was extracted with n-hexane/acetone (1:1, in volume) using a microwave system (MARS-X CEM Corporation, Matthews, NC, USA). Prior to extraction surrogate mix standards were added to the sam-ples. The collected extracts, previously concentrated to a small volume by Turbo Vap®II (Biotage, Uppsala, Sweden), were dis-solved in a mixture of cyclohexane/dichloromethane (2 mL; 70:30, in volume) and filtered through PTFE filters of 5µm pore size (Millipore, Bedford, OH, USA). Clean up was car-ried out with AZURA GPC Cleanup system (Knauer, Berlin, Germany) (GPC column: 40 mm×10 mm; phase: Biobeads SX3, about 11 g; mobile phase: cyclohexane/dichloromethane, 70/30 (in volume); flow rate: 1 mL/min; injected volume: 2 mL). Purified samples were analyzed with an Agilent 7890A gas chromatograph equipped with 7693 autosampler and coupled to an Agilent 5975C mass spectrometry (GC-MS) (Agilent Technologies Inc., Santa Clara, CA, USA) according to the modified USEPA (United States Environmental Protection

Agency) method 8270D (USEPA, 1998). Five microliters of analyte was injected into a programmed temperature vapor-izing (PTV) injector in solvent vent. All analyses were separ-ated on a Select PAH column (30m×0.25 mm i.d., 0.15 μm film thickness; Agilent Technologies Inc., Santa Clara, CA, USA). The carrier gas was ultrapure helium, set at a constant flow mode (1.2 mL/min). The oven temperature program was 70 °C (hold 0.7 min), to 180 °C (hold 0 min) at 85 °C/min, to 230 °C (hold 10 min) at 3 °C/min, to 280 °C (hold 20 min) at 28 °C/min, to 330 °C (hold 5.0 min) at 5 °C/min. The mass spectrometer was used in the electronic impact mode (70 eV electron energy) and ion source, quadrupole, and transfer line temperatures were set at of 230, 150, and 280 °C, respectively. PAH quantification was performed in selected ion monitoring (SIM) mode using three ions for each PAH compound. The fol-lowing 18 priority PAHs were determined: naphthalene (NAP), acenaphthylene (ACY), acenaphthene (ACE), fluorene (FLU), phenanthrene (PHEN), anthracene (ANTH), fluoranthene (FLTH), pyrene (PYR), benzo[a]anthracene (B[a]A), chrysene (CHRY), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[j]fluoranthene (B[j]F), benzo[e]pyrene (B[e]P), benzo[a]pyrene (B[a]P), benzo[g,h,i]perylene (B[ghi]P), indeno[1,2,3-c,d]pyrene (IND), and dibenz[a,h]anthracene (D[ah]A). PAHs calibration mix, deuterated internal stand-ards (naphthalene-d8, acenaphthene-d10, phenanthrene-d10, chrysene-d12, and perylene-d12) and surrogate standards (anthracene-d10 and benzo[a]anthracene-d12) were pur-chased from Merck© as well as all chromatographic grade solvents (Merck, Milan, Italy). The recoveries of the method for PAHs were determined by the analysis of spike samples. The recoveries obtained ranged 69%–110% for Holothuria tissues. The method detection limit (MDL) was calculated based on a signal to noise ratio of 3:1. MDLs for PAH com-pounds ranged from 0.6 to 2 μg/kg of dry weight (dw).

Human health risk assessmentThe risk for human health as a result of eating this species was evaluated by calculating the Estimated Daily Intake (EDI) (USEPA, 2000) as follows:

EDI = (C× IR)/Wb,

where C indicates PAH concentration (µg/g wet weight (ww)) in each tissue, Wb is the average body weight (60 kg for adults), and IR is food ingestion rate (g/d each person). The mean IR value was determined according to the Food and Agriculture Organization of the United Nations (FAO) referring to seafood consumption in some Asiatic countries (Korea, Japan, China, Malaysia, Myanmar, Indonesia, and Philippines) in which there is high sea cucumber consump-tion. The mean IR value reported that an adult person eats 106 g/d (FAO, 2021), although as these data referred to a general consumption of all seafood (fish, crabs, molluscs, etc.) and specific sea cucumber intake was not available, in this study, we have considered an IR of 10.6 g/d (IR10) and 106 g/d (IR100) each person, corresponding to 10% and 100% of FAO recommended IR, respectively. In this way, EDI was estimated including the minimum and maximum IR. Moreover, to evaluate possible non-carcinogenic and carcinogenic risk for human health, Target Hazard Quotient (THQ) and Lifetime Excess Cancer Risk (CR), were used according to USEPA risk analysis (USEPA, 2001).

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The THQ is the ratio of the exposure of contaminant and the reference dose (RfD). In general, the RfD is an estimate of daily exposure to the human population that is likely to be without an appreciable risk of deleterious effects during a lifetime and is expressed in units of mg/(kg·d) (USEPA, 2021). If the ratio is above 1, there is a risk of chronic systemic effect. The THQ was calculated on the following equation:

THQ =

ïEF × ED × IR × CRfD×Wb × AT

ò× 10−3

where EF is exposure frequency (365 d a year); ED is ex-posure duration (70 y for adults); IR is food ingestion rate (10.6 g/d and 106 g/d each person); C is the average con-centration of PAHs in holothurians (µg/g ww); Wb is the average body weight (60 kg for adults), AT is the aver-aging time for no carcinogens, which is given by EF×ED. THQs were determined only for NAP, ACE, FLU, ANTH, FLTH, PYR, and B[a]P for which RfD is available. RfD of these compounds, defined by USEPA (2021), is equal to 0.02, 0.06, 0.04, 0.3, 0.04, 0.03, and 0.0003, respectively. Possible additional and interactive effects of contaminants can be calculated by Hazard Index (HI) obtained summing THQ calculated for the single PAHs (USEPA, 1996; Jian et al., 2013). Lifetime Excess CR or CR can be estimated as the incremental probability of an individual developing cancer over a lifetime as a result of exposure to a potential carcinogen and was estimated following the equation (Xia et al., 2010):

CR = [(EF× ED× IR× C× CsF)/(BW× AT)]× 10−3

where CsF is the cancer slope factor expressed in mg/(kg·d). CsF available for PAHs compounds is related only to B[a]P and is equal to 7.3 mg/(kg·d). The estimated CRs were compared to the acceptable guideline value of 10−6 set by USEPA. USEPA establishes a level of risk where there is a CR of one in a million (CR=10−6) over a 70-y lifetime period is considered acceptable, while when CR is one in 10 000 or greater (CR=10−4), it is considerable (USEPA, 1996).

Statistical analysesThe data were expressed as mean values±standard devi-ation (n=11 for females, n=9 for males). Data were ana-lyzed for normality and variance homogeneity through Kolmogorov–Smirnov and Levene tests, respectively. When either assumption was met, all data were examined by two-way analysis of variance (ANOVA) to verify whether there were differences between gender and among tissues. Subsequently, post-hoc Tukey’s LSD multiple comparisons were performed to determine differences between sexes and among tissues.

Results and DiscussionProximate compositionThe proximate composition expressed on ww base, including moisture, ash, protein, and lipid contents of the six tissues of H. polii female and male is shown in Table 1.

Although most previous studies were only carried on BW, the nutrient composition of different tissues can be compared (Drazen et al., 2008; Wen et al., 2010; Aydin et al., 2011; Sicuro et al., 2012; Barzkar et al., 2017; Bilgin and Tanrikulu, 2018; Künili and Çolakoglu, 2019; Zmemlia et al., 2020). There are very limited studies on the proximate composition including different edible portions of sea cucumber, which were investigated in this study.

In general, all sea cucumber tissues showed high moisture and protein contents and low lipid content, a common char-acteristic of marine organisms.

Tissue was found to have significant influence on mois-ture that varied from 78.96% in BW to 85.16% in MBs and from 76.21% in BW to 85.77% in MBs for female and male, respectively (F=6.066, P<0.05). As regards BW, Aydin et al. (2011) found similar result in the same species (81.24%), but higher in Holothuria tubulosa (H. tubulosa) (84.30%) and Holothuria mammata (H. mammata) (85.24%); Rasyid et al. (2020) for Holothuria scabra (H. scabra) also observed higher moisture content (87.12%), as did Barzkar et al. (2017) for Holothuria arenicola (H. arenicola) (93%). Haider et al. (2015) reported lower values for H. arenicola (72.12%), as did Salarzadeh et al. (2012) for Holothuria parva (H. parva) (67.9%) and H. arenicola (68.49%). Bechtel et al. (2013) found similar moisture content (84.50%) in muscle bands of P. californicus from Alaska (USA).

Salunkhe and Deshpande (2012) stated that high moisture content makes products vulnerable to microbial attack and spoilage; on the other hand, the loss of water would cause the concentration of bioactive compounds, increasing the shelf life (Barzkar et al., 2017).

Ash content was significantly higher in BW of both male and female than other tissues (P<0.05) as reported by Bechtel et al. (2013) for P. californicus, which found in BW an ash content twice the value of MBs.

The protein content of H. polii varied among tissues (F=12.38, degree of freedom (df)=5, P<0.05), but not between sexes (F=0.05, P>0.05). No significant interaction of sex and tissue on protein was also observed (F=12.38, P>0.05). AC and Gd of both sexes and BW of male exhibited the highest protein content (P<0.05). Indeed, the gonads are responsible for the development of reproductive cells and, therefore, there is high energy investment in this organ confirmed by the high protein.

As regards BW, H. polii showed a protein content of (11.14±1.00) g/100 g in female and (13.28±0.99) g/100 g in male. Aydin et al. (2011) reported values lower than those of this study, with 8.66% for H. polii, 8.82% for H. tubulosa, and 7.88% for H. mammata from the Aegean region in Turkey. Lower values are also reported by Rasyid et al. (2020) and Özer et al. (2004) (range 5.10–5.78 g/100 g) for H. scabra, and by Barzkar et al. (2017) for H. arenicola (4.40%, ww basis). In addition, González-Wangüemert et al. (2018) found a protein content similar to that of this study in H. mammata (11.1%), but lower in H. polii (7.37%) and H. tubulosa (3.01%). On the other hand, Salarzadeh et al. (2012) reported for H. arenicola and H. parva values higher than those of this study with 24.37% and 17.61%, respectively.

This result confirms sea cucumber as a rich source of protein, although it is mainly eaten as a delicacy and much

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less for its nutritional value. Therefore, such studies will help consumers recognize the high value of each edible part of sea cucumbers in order to include them in their diet.

The total lipids showed significant differences based on sex (F=10.37, df=1, P<0.005), tissue (F=46.92, df=5, P<0.001), and the interaction between sex and tissue (F=14.09, df=5, P<0.001).

Lipid content ranged from 0.49 g/100 g ww (BW) to 3.71 g/100 g ww (Gd) in female. H. polii males exhibited similar results to those of females for all tissues except for Gd, which showed a value significantly lower (1.50 g/100 g ww), i.e. less than half the female content. Similar levels of lipids in BW of both sexes (less than 2.0%) are reported in previous investigations on sea cucumbers, such as H. arenicola (0.6), Stichopus horrens (0.4) (Barzkar et al., 2017), H. polii (0.15), H. tubulosa (0.18), H. mammata (0.09) (Aydin et al 2011), Actinopyga mauritiana (A. mauritiana) (1.4), Thelenota ananas (1.9) (Wen et al., 2010), H. pardalis (1.66) (Svetashev et al., 1991). In a previous study on H. polii, Biandolino et al. (2019b) found in the BW a lower lipid content than that of this study.

Chang-Lee et al. (1989) state that in general sea cucum-bers are characterized by higher moisture and lower protein content than marine fish and shellfish. Moreover, they re-ported for fresh sea cucumbers a high variability with a range of 82.0%–92.6% for moisture, 2.5%–13.8% for protein, 0.1%–0.9% for fat, and 1.5%–4.3% for ash. The data from this study fall within the range of values reported by Chang-Lee et al. (1989). Obviously, the proximate composition of sea cucumber varies from one species to another, with sea-sonal variation, with feeding behavior, life cycle of species and physiological status (Chang-Lee et al., 1989; Özer et al., 2004).

Amino acidsThe AA profile of the different tissues of H. polii female and male is summarized in Table 2. The results are given on a wet weight basis and in order to enable a comparison of the re-sults from this study with international and national research, the data were also expressed as a percentage of AA per total amino acids (TAAs).

Variations in the contents of TAAs occurred based on tissue (F=45.69, df=5, P<0.001), and the interaction be-tween sex and tissue (F=44.66, df=5, P<0.001), but not on

the sex (F=2.27, df=5, P>0.05). AC and MB of female (range 47.8–60.2 g/kg ww) had significantly (P<0.05) higher con-tents of TAAs. On the contrary, Lee et al. (2012) found, for Apostichopus japonicus (A. japonicus) from Korea, a TAAs content of BW significantly higher than that of viscera.

A total of 17 AAs were detected, including 8 EAAs and 9 non-essential amino acids (NEAAs). Recently, the term ‘func-tional AA’ has received more attention because they play an im-portant role in the metabolic pathways, thus affecting immune response, health, reproduction, cell signaling, and welfare.

Both EAAs and NEAAs are important in the balanced diet in order to maximize protein accumulation and optimize health in animals and humans. EAAs are not synthesized by animal cells therefore, and therefore, must be provided from the diet, while NEAAs are synthesized de novo in a species-dependent manner (Wu, 2010). A deficiency and/or imbalance of EAAs or NEAAs damages not only protein synthesis but also whole-body homeostasis (Andersen et al., 2016).

Both sexes had similar contents of EAAs that differs among tissues in a range of 31%–53% and 33%–49% of TAAs in female and male, respectively (P<0.05).

BW and ITu of H. polii exhibited the lowest contents of EAAs (ww base), while MBs and AC of female and MBs, AC, and Gd of male exhibited the highest contents of EAAs (P<0.05). However, Gd of female with 11.86 g/kg ww ac-counted for 53% of TAAs.

The content ratio of EAAs and TAAs is an important ratio reflecting the quality of proteins that should be taken into account in a balanced diet (Wu, 2010).

In this study, both female and male showed similar ratios of EAAs and TAAs, except for Gd and RT that showed a sig-nificantly higher ratio in female than male (P<0.05; Table 2). Moreover, only in Gd and AC of female, the EAAs were higher than the NEAAs (EAAs/NEAAs>1).

As regards BW, this ratio value was comparable with those reported in literature for the same and other species and for different sea cucumbers genera. Sicuro et al. (2012) reported an EAA/NEAAs ratio of 0.37 in H. polii and 0.40 in H. tubulosa. Roggatz et al. (2016) reported an EAA/NEAAs ratio of 0.49 in Holothuria arguinensis (H. arguinensis),and a ratio value of 0.40 in Parastichopus regalis was showed in the study of Roggatz et al. (2018). González-Wangüemert et al. (2018) observed an EAA/NEAAs ratio of 0.27, 0.38 and 0.39 in H. mammata, H. polii, and H. tubulosa, respectively. Wen et al. (2010) found the ratio values of Thelenota ananas (T. ananas), Thelenota anax

Table 1. Proximate composition (wet weight basis) of the six tissues of both female and male Holothuria polii

Proximate composition Sex BW ITu MB Gd AC RT

Moisture (%) Female 78.96±1.77a 84.24±0.24c 85.16±2.72c 83.89±1.99b 81.45±1.17ab 81.86±2.88ab

Male 76.21±1.61a 85.11±1.72d 85.77±0.83d 81.29±0.98b 81.56±1.28b 83.11±1.99c

Ash (%) Female 4.24±0.10d 2.84±0.08a 3.12±0.12b 2.71±0.05a 3.61±0.08c 3.41±0.08c

Male 4.07±0.08d 2.62±0.09a 2.88±0.09b 2.42±0.04a 3.70±0.07c 3.62±0.09c

Protein (g/100 g) Female 11.14±1.00bc 8.91±0.75a 10.04±1.13ab 11.26±0.60c 12.72±0.39d 11.01±0.48bc

Male 13.28±0.99b 8.98±0.63a 9.31±0.68a 13.26±1.25b 13.38±0.89b 10.29±1.02a

Lipid (g/100 g) Female 0.49±0.02a 0.82±0.05a 0.94±0.16a 3.71±0.55cB 2.49±0.12b 2.87±0.54b

Male 0.53±0.05a 0.84±0.02ab 0.74±0.02a 1.50±0.09bA 2.41±0.29c 3.09±0.95c

Data are expressed as mean±standars deviation. BW, body wall; ITu, internal tunic; MB, muscle band; Gd, gonad; AC, alimentary canal; RT, respiratory tree.Different superscript letters in the same row indicate statistical differences among tissues; different superscript letters in the same column indicate statistical differences between sexes (P<0.05).

Dow



ctober 2022


Tab

le 2

. Am

ino

acid

pro

file

of t

he s

ix t

issu

es b

oth

fem

ale

and

mal

e H

olot

huria

pol

ii

Am

ino

acid

Se

x B

W (

g/kg

) IT

u (g

/kg)

M

B (

g/kg

) G

d (g

/kg)

A

C (

g/kg

) R

T (

g/kg

)

His

tidi

ne (

His

)Fe

mal

e0.

46±0

.02

(1.1

1)aB

0.54

±0.0

5 (1

.95)

a1.

11±0

.12

(2.3

2)c

0.54

±0.0

9 (2

.38)

aA1.

11±0

.18

(2.1

5)c

0.80

±0.2

3 (2

.43)

b

Mal

e0.

45±0

.02

(1.4

7)aA

0.57

±0.0

5 (2

.22)

ab0.

88±0

.15

(2.2

6)cd

1.23

±0.1

1 (2

.03)

eB1.

07±0

.00

(2.0

7)de

0.71

±0.0

9 (2

.58)

bc

Isol

euci

ne (

Ile)

Fem

ale

0.98

±0.0

6 (2

.36)

a1.

09±0

.18

(3.8

9)ab

2.24

±0.4

8 (4

.69)

c1.

00±0

.27

(4.3

7)aA

2.97

±0.1

8 (5

.73)

cB1.

59±0

.45

(4.8

3)b

Mal

e1.

03±0

.08

(3.3

5)a

1.14

±0.1

4 (4

.39)

a1.

89±0

.25

(4.8

8)b

2.57

±0.1

5 (4

.26)

cB2.

34±0

.06

(4.5

4)cA

1.22

±0.1

8 (4

.47)

a

Leu

cine

(L

eu)

Fem

ale

1.76

±0.1

0 (4

.24)

a4.

97±0

.83

(17.

81)cB

3.63

±0.8

2 (7

.60)

b4.

40±0

.66

(19.

32)bc

4.15

±0.0

9 (8

.01)

bcA

5.12

±0.3

0 (1

5.51

)cB

Mal

e1.

73±0

.14

(5.6

0)a

1.77

±0.0

8 (6

.80)

aA3.

62±0

.67

(9.3

4)b

3.75

±0.2

1 (6

.22)

b9.

30±0

.28

(18.

07)cB

1.88

±0.1

3 (6

.87)

aA

Lysi

ne (

Lys)

Fem

ale

2.19

±0.3

3 (5

.28)

aB1.

86±0

.25

(6.6

7)aA

5.75

±0.7

3 (1

2.05

)bB1.

84±0

.37

(8.0

9)aA

7.19

±0.7

4 (1

3.87

)cB2.

49±0

.77

(7.5

6)a

Mal

e1.

38±0

.23

(4.4

7)aA

2.39

±0.0

1 (9

.22)

bB4.

13±0

.48

(10.

63)dA

9.02

±0.7

7 (1

4.98

)eB3.

71±0

.13

(7.2

1)cd

A3.

22±0

.18

(11.

77)c

Met

hion

ine

(Met

)Fe

mal

e1.

01±0

.21

(2.4

3)b

0.64

±0.1

4 (2

.31)

aA1.

99±0

.33

(4.1

7)d

0.54

±0.1

5 (2

.39)

aA1.

49±0

.07

(2.8

8)cB

0.58

±0.0

3 (1

.75)

aA

Mal

e0.

79±0

.05

(2.5

6)a

0.84

±0.0

6 (3

.25)

aB1.

91±0

.33

(4.9

2)c

1.23

±0.2

3 (2

.04)

bB0.

98±0

.04

(1.9

1)ab

A0.

71±0

.07

(2.6

1)aB

Phen

ylal

anin

e (P

he)

Fem

ale

2.18

±0.0

2 (5

.25)

bB1.

07±0

.17

(3.8

4)a

1.90

±0.4

4 (3

.99)

b0.

98±0

.29

(4.3

1)aA

3.16

±0.1

9 (6

.10)

cB1.

06±0

.14

(3.2

3)a

Mal

e1.

18±0

.08

(3.8

4)aA

1.14

±0.0

7 (4

.38)

a1.

70±0

.44

(4.3

6)b

1.97

±0.1

0 (3

.28)

bB2.

52±0

.15

(4.8

9)cA

1.24

±0.0

5 (4

.55)

a

Thr

eoni

ne (

Thr

)Fe

mal

e2.

54±0

.03

(6.1

2)bB

1.41

±0.3

9 (5

.07)

a3.

13±0

.28

(6.5

6)c

1.32

±0.2

8 (5

.81)

aA3.

29±0

.21

(6.3

4)cB

2.06

±0.5

5 (6

.24)

b

Mal

e1.

94±0

.16

(6.2

8)bA

1.62

±0.1

6 (6

.24)

a2.

88±0

.25

(7.4

3)c

2.85

±0.1

5 (4

.73)

cB2.

72±0

.01

(5.2

8)cA

1.72

±0.1

1 (6

.30)

ab

Val

ine

(Val

)Fe

mal

e1.

72±0

.01

(4.1

5)ab

B1.

22±0

.18

(4.3

7)a

2.10

±0.4

8 (4

.40)

b1.

22±0

.32

(5.3

3)aA

2.72

±0.1

4 (5

.25)

c1.

82±0

.53

(5.5

3)b

Mal

e1.

55±0

.11

(5.0

1)aA

1.47

±0.1

3 (5

.65)

a1.

37±0

.35

(3.5

4)a

2.67

±0.1

8 (4

.44)

bB2.

54±0

.14

(4.9

4)b

1.41

±0.1

2 (5

.16)

a

Asp

arti

c ac

id (

Asp

)Fe

mal

e0.

57±0

.11

(1.3

7)a

1.39

±0.3

2 (4

.98)

b1.

16±0

.20

(2.4

4)b

1.18

±0.3

5 (5

.16)

bA1.

41±0

.09

(2.7

2)bA

0.99

±0.3

1 (3

.00)

ab

Mal

e0.

42±0

.06

(1.3

6)a

0.89

±0.1

0 (3

.41)

b0.

93±0

.25

(2.4

0)b

3.74

±0.2

6 (6

.21)

dB3.

10±0

.38

(6.0

2)cB

1.16

±0.1

4 (4

.25)

b

Glu

tam

ic a

cid

(Glu

)Fe

mal

e1.

97±0

.42

(4.7

6)aB

3.63

±0.5

1 (1

2.99

)bcB

4.36

±0.6

7 (9

.13)

cB1.

90±0

.36

(8.3

4)aA

3.66

±0.6

4 (7

.06)

cA2.

63±0

.71

(7.9

7)ab

Mal

e1.

00±0

.19

(3.2

4)aA

2.21

±0.1

5 (8

.51)

bA2.

88±0

.31

(7.4

3)cA

7.15

±0.4

2 (1

1.87

)eB6.

40±0

.13

(12.

43)dB

2.59

±0.1

1 (9

.49)

bc

Ala

nine

(A

la)

Fem

ale

4.28

±0.0

3 (1

0.33

)dB1.

29±0

.20

(4.6

1)aA

2.27

±0.3

7 (4

.75)

bc0.

89±0

.26

(3.9

0)aA

2.74

±0.1

0 (5

.29)

cB1.

95±0

.65

(5.9

1)b

Mal

e3.

91±0

.30

(12.

66)cA

1.56

±0.0

7 (6

.00)

aB2.

31±0

.42

(5.9

5)b

2.28

±0.0

2 (3

.78)

bB1.

80±0

.17

(3.5

0)aA

1.60

±0.1

9 (5

.84)

a

Seri

ne (

Ser)

Fem

ale

1.98

±0.0

2 (4

.77)

cB1.

25±0

.33

(4.4

9)ab

2.77

±0.2

3 (5

.80)

dB1.

10±0

.19

(4.8

3)aA

2.94

±0.1

0 (5

.68)

dB1.

59±0

.33

(4.8

3)bc

Mal

e1.

31±0

.13

(4.2

6)aA

1.50

±0.0

3 (5

.77)

ab2.

23±0

.16

(5.7

4)cA

2.41

±0.1

1 (4

.00)

cB2.

37±0

.04

(4.6

1)cA

1.68

±0.1

8 (6

.13)

b

Cys

tein

e (C

ys)

Fem

ale

1.24

±0.0

5 (2

.98)

bB0.

58±0

.07

(2.0

9)aA

1.41

±0.5

3 2.

94)b

0.27

±0.0

5 (1

.20)

a1.

20±0

.17

(2.3

1)b

0.59

±0.1

3 (1

.80)

ab

Mal

e0.

32±0

.02

(1.0

3)aA

0.69

±0.0

3 (2

.63)

bB1.

30±0

.23

(3.3

5)c

0.22

±0.0

1 (0

.36)

a0.

85±0

.13

(1.6

5)b

0.80

±0.0

8 (2

.92)

b

Tyro

sine

(Ty

r)Fe

mal

e1.

84±0

.53

(4.4

4)b

0.97

±0.0

9 (3

.47)

aA1.

94±0

.22

(4.0

6)b

1.50

±0.1

3 (6

.60)

ab2.

83±0

.28

(5.4

5)cB

1.62

±0.5

6 (4

.93)

b

Mal

e1.

17±0

.02

(3.8

0)a

1.24

±0.0

9 (4

.76)

aB1.

92±0

.15

(4.9

5)bc

1.88

±0.2

0 (3

.12)

b2.

14±0

.04

(4.1

5)cA

1.26

±0.1

6 (4

.60)

a

Gly

cine

(G

ly)

Fem

ale

8.70

±0.2

5 (2

0.97

)dB2.

13±0

.35

(7.6

5)b

3.58

±0.3

4 (7

.49)

c1.

16±0

.22

(5.1

0)aA

3.80

±0.2

2 (7

.32)

cB2.

70±0

.56

(8.1

9)b

Mal

e5.

78±0

.81

(18.

72)cA

2.63

±0.1

2 (1

0.13

)ab2.

97±0

.26

(7.6

7)b

6.77

±0.2

8 (1

1.24

)dB3.

26±0

.10

(6.3

4)bA

2.23

±0.3

5 (8

.15)

a

Arg

inin

e (A

rg)

Fem

ale

3.91

±0.2

4 (9

.43)

cB2.

07±0

.30

(7.4

0)ab

4.13

±0.5

3 (8

.66)

cB1.

69±0

.36

(7.4

1)aA

3.97

±0.1

3 (7

.67)

cB2.

80±0

.96

(8.4

8)bB

Mal

e3.

04±0

.36

(9.8

5)bA

2.13

±0.2

2 (8

.21)

a3.

32±0

.25

(8.5

5)bc

A5.

08±0

.34

(8.4

3)dB

3.56

±0.0

6 (6

.92)

cA2.

07±0

.23

(7.5

9)aA

Prol

ine

(Pro

)Fe

mal

e4.

14±0

.18

(9.9

8)dB

1.79

±0.2

5 (6

.40)

abA

4.27

±0.5

4 (8

.94)

dB1.

24±0

.24

(5.4

4)aA

3.18

±0.3

3 (6

.14)

c2.

57±0

.81

(7.7

9)bc

Mal

e3.

85±0

.40

(12.

49)cA

2.18

±0.0

7 (8

.41)

abB

2.56

±0.6

0 (6

.59)

bA5.

42±0

.45

(8.9

9)dB

2.80

±0.1

1 (5

.44)

b1.

83±0

.16

(6.7

1)a

Tota

l EA

AFe

mal

e12

.84±

0.10

(31

.08)

abB

12.8

2±2.

11 (

46.1

0)ab

21.8

7±3.

07 (

45.9

6)c

11.8

6±2.

37 (

52.8

9)aA

26.0

9±0.

09 (

50.7

2)d

15.5

3±2.

39 (

47.5

2)b

Mal

e10

.06±

0.89

(32

.88)

aA10

.95±

0.30

(42

.43)

a18

.38±

2.44

(47

.51)

b25

.30±

1.93

(42

.37)

cB25

.19±

0.73

(49

.33)

c12

.11±

0.27

(44

.43)

a

Dow



ctober 2022


Am

ino

acid

Se

x B

W (

g/kg

) IT

u (g

/kg)

M

B (

g/kg

) G

d (g

/kg)

A

C (

g/kg

) R

T (

g/kg

)

Tota

l NE

AA

Fem

ale

28.6

5±1.

13 (

68.9

2)cB

15.1

0±1.

62 (

53.9

0)b

25.9

0±2.

23 (

54.0

4)cB

10.9

4±0.

65(4

7.10

)aA25

.74±

0.67

(49

.28)

c17

.45±

2.11

(52

.48)

b

Mal

e20

.81±

2.03

(67

.11)

bA15

.02±

0.11

(57

.57)

a20

.43±

1.31

(52

.49)

bA34

.96±

1.14

(57

.62)

cB26

.29±

0.91

(50

.66)

c15

.22±

0.12

(55

.57)

a

TA

AFe

mal

e41

.49±

0.94

cB27

.92±

3.93

ab47

.77±

5.15

cd22

.80±

2.74

aA51

.83±

0.76

d32

.99±

4.56

b

Mal

e30

.87±

3.21

bA25

.97±

0.20

a38

.81±

3.84

c60

.25±

3.27

eB51

.48±

1.45

d27

.33±

0.20

ab

Con

tent

rat

io o

f E

AA

/NE

AA

Fem

ale

0.45

±0.0

1a0.

85±0

.02b

0.84

±0.0

5b1.

08±0

.18cB

1.01

±0.0

2c0.

90±0

.05bB

Mal

e0.

49±0

.02a

0.73

±0.0

2b0.

90±0

.05d

0.72

±0.0

1bA0.

96±0

.01e

0.80

±0.0

2cA

Con

tent

rat

io o

f Ly

s/A

rgFe

mal

e0.

56±0

.11aB

0.90

±0.0

6abA

1.39

±0.0

2c1.

09±0

.02bA

1.80

±0.1

2dB0.

93±0

.25bA

Mal

e0.

45±0

.02aA

1.13

±0.1

1bB1.

25±0

.17b

1.77

±0.0

3cB1.

04±0

.02bA

1.57

±0.2

6cB

The

per

cent

age

of a

min

o ac

ids

(AA

s) (

%)

is r

epor

ted

in t

he b

rack

ets.

Dat

a ar

e ex

pres

sed

as m

ean±

stan

dard

dev

iati

on.

BW

, bod

y w

all;

ITu,

inte

rnal

tun

ic; M

B, m

uscl

e ba

nd; G

d, g

onad

; AC

, alim

enta

ry c

anal

; RT,

res

pira

tory

tre

e; T

AA

, tot

al a

min

o ac

id; E

AA

, ess

enti

al a

min

o ac

id; N

EA

A, n

on-e

ssen

tial

am

ino

acid

.D

iffe

rent

sup

ersc

ript

s in

a s

ame

row

indi

cate

d si

gnifi

cant

dif

fere

nces

am

ong

tiss

ues

(P<0

.05)

; dif

fere

nt s

uper

scri

pts

in a

sam

e co

lum

n in

dica

ted

sign

ifica

nt d

iffe

renc

es b

etw

een

sexe

s (P

<0.0

5).

Tab

le 2

. Con

tinue

d

Dow



ctober 2022


(T. anax), and Holothuria fuscogilva (H. fuscogilva) were 0.60, 0.59, and 0.40, respectively. For Red and Black A. japonicus from different Korean islands, Lee et al. (2012) reported a range of 0.34–0.41 for BW, slightly lower than those of our study, and 0.99–1.15 for viscera, similar to our data.

H. polii from this study contained an interesting combination of valuable AAs. For each AA, statistical ana-lysis (two-way ANOVA) showed an effect of tissue and of sex and tissue interaction (P<0.05), but for most of them, no sexual effect was observed (P<0.05).

In almost all tissues, although with significant differences among them, glycin (Gly) was the dominant AA reported by most literature (Sicuro et al., 2012; Haider et al., 2015; Wen et al., 2010 ; González-Wangüemert et al., 2018; Künili and Çolakoglu, 2019). Gly showed a range of 1.16–8.70 g/kg in female and 2.23–6.77 g/kg in male. Noteworthy is the high content of Gly in BW (21% and 19% of TAAs in female and male, respectively) compared with other tissues (Table 2). Different physiological and immunological activities are at-tributed to Gly that are beneficial for human health (Wang et al., 2013). Gly helps to maintain healthy central nervous and digestive systems, and provides protection via antioxidants from some types of cancer (Wang et al., 2013). Moreover, the richness of glycine favors the reduction of serum total choles-terol level (Ikeda et al., 1993).

Glycine is the most abundant AA in all sea cucum-bers (Actinopyga mauritiana (A. mauritiana), H. scarba, Bohadschia marmorata (B. marmorata), and Holothuria leucospilota (H. leucospilota)) studied by Omran (2013) from the Egyptian coast of Red Sea. Wen et al. (2010) showed that Gly was dominant in Stichopu herrmanni (S. herrmanni), T. ananas, T. anax, H. fuscogilva, Holothuria fuscounctata (H. fuscounctata), Actinopyga caerulea (A. caerulea), and Bohadschia argus.

Among the major AAs were also glutamic acid (Glu), ar-ginine (Arg), and proline (Pro) for NEAAs and leucine (Leu) and lysine (Lys) for EAAs. Considerable amounts of threonine (Thr), phenylalanine (Phe), and alanine (Ala) were also ob-served, overall in MBs and AC. Thr was the dominant EAA in BW, representing 6.1% and 6.3% in female and male, re-spectively. Wen et al. (2010) reported Thr as the major EAA in H. fuscogilva, H. fuscopunctata, A. caerulea, S. herrmanni, T. anax, and T. ananas.

Sicuro et al. (2012) reported similar AA composition for H. polii and H. tubulosa, except for aspartic acid (Asp), which was lower in this study. González-Wangüemert et al. (2018) reported Ala, Arg, Glu, and Gly as the most abundant AAs in H. mammata, H. polii, and H. tubulosa. Lee et al. (2012) found Asp, Glu, Gly, Pro, and Arg to be predominant in BW and Glu, Arg, Asp, Lys, and Leu in viscera in A. japonicus. Bechtel et al. (2013) found Gly and Glu as dominant AAs in the BW of P. californicus, while Arg and Glu dominated AA composition of MBs followed by Lys, which is similar to our results. Other studies reported different AA profiles, probably due to different feeding habits, environmental parameters, and species-specific characteristics (Wen et al., 2010; Bechtel et al., 2013; Haider et al., 2015).

Another important feature of sea cucumber AA compos-ition is its low content ratio of Lys and Arg ratio, which has hypocholesterolemic and anti-atherogenic effects (Bordbar et al., 2011).

In this study, the Lys/Arg ratio showed significant dif-ferences between male and female (P<0.05) in all tissues

examined (Table 2); in female the ratio ranged from 0.56 (BW) to 1.80 (AC), while in males it was 0.45 (BW) to 1.77 (Gd). In P. californicus, Bechtel et al. (2013) found a similar value for BW (0.48) but lower than that for MBs in this study (0.57). Lee et al. (2012), in A. japonicus from different re-gions, reported values of BW and viscera were in the ranges of 0.34–0.41 and 0.57–1.20, respectively, which is similar to this study.

The results found for BW are consistent with those reported in literature for different sea cucumbers spe-cies such as H. arenicola (0.34) and A. mauritiana (0.44) (Haider et al., 2015), H. scabra (0.41), and H. leucospilota (0.43) (Omran, 2013). On the other hand, the ratios of Lys and Arg in this study showed values higher than those of other studies such as 0.27 for H. tubulosa and 0.22 for H. polii (Sicuro et al., 2012), 0.21 for H. arguensis (Roggatz et al., 2016), 0.13 for H. scabra (Sroyraya et al., 2017), 0.11 for Australostichopus mollis (Liu et al., 2017), 0.14 for Stichopus vastus (Rasyid, 2018), 0.09 for H. polii, 0.12 for H. tubulosa, and 0.33 for H. mammata (González-Wangüemert et al., 2018), and values in the range of 0.13–0.39 for eight species of sea cucumber (Wen et al., 2010). Higher values were re-ported by Omran (2013) for B. marmorata (0.9) and A. mauritiana (3.56).

On the other hand, the Lys/Arg ratio in this study was also much lower than that of several other seafoods (e.g. Zuraini et al., 2006; Zhao et al., 2010).

Therefore, based on the above results, all edible tissues of the sea cucumber H. polii from this study can contribute to-wards a healthy diet.

Fatty acid compositionThe FA composition of the six tissues of H. polii female and male, with their absolute amounts (mg/100 g ww) and rela-tive proportions of total FAs (%), is summarized in Table 3.

Additionally, to its highest lipid content, Gd of female and RT of male showed the highest amounts of total FAs. Significant effects of sex, tissue and interaction of these factors on the most FAs were found (two-way ANOVA, P<0.05).

Eighteen FAs exceeding a minimum of 0.1% of total FAs in a minimum of one sample were identified. Despite the low lipid content, overall, in BW, ITu, and MBs, H. polii was an important source of FAs. Indeed, all tissues of both female and male exhibited a favorable FA profile, with a dominance of polyunsaturated fatty acids (PUFAs), that ranged from 156 to 1125 mg/100 g ww (32.5%–59.6% of total FAs), followed by saturated FA (SFA) with a range of 100–835 mg/100 g ww (20.7%–32.5% of total FAs) and monounsaturated FA (MUFA) with 85–920 mg/100 g ww range (17.6%–35.4% of total FAs) of FAs (Table 3, Figure 1). RT of male exhibited an absolute amount of PUFA significantly higher than other tis-sues (P<0.05). In terms of relative proportions, MBs of both sexes, ITu of female, Gd and RT of male exhibited values of PUFA>50% of total FAs. The high proportions of PUFA found in MBs are consistent with research by Svetashev et al. (1991) for shallow-water holothurians. There were significant differences in the PUFA content of H. polii as a result of sex (F=4.38, df=1, P<0.05) and tissue (F=281.4, df=5, P<0.001), and there was also a significant interaction between these fac-tors (F=53.21, df=5, P<0.001). Gd of female contained high amounts of PUFA, when referred to absolute value (mg/100 g

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ww); however, the proportion (of total FAs) was low, ac-counting for 32.5%, while BW of both sexes exhibited the lowest absolute values (P<0.05, Table 3), but high propor-tions (45.8% for female and 41.6% for male).

PUFA content of H. polii was higher than the values found by Wen et al. (2010) for different sea cucumber species, including those belonging to the same or different genera. Ridzwan et al. (2014) reported SFA as dominant in H. scabra, H. leucospilota, and Holothuria atra (H. atra), and a lower amount of MUFA compared to SFA and PUFA.

However, Aydin et al. (2011) reported for H. tubulosa, H. polii, and H. mammata, Sicuro et al. (2012) for H. polii and H. tubulosa, Haider et al. (2015) for H. arenicola, and Bilgin and Tanrikulu (2018) for H. tubulosa, that PUFA to be higher than SFA and MUFA, as observed in this study. González-Wangüemert et al. (2018) reported similar results for H. mammata, while for H. polii they found MUFA to be the dominant FA class. For P. californicus, Bechtel et al. (2013) reported MUFA as dominant in BW and PUFA in MBs and the latter data are very similar to the results of this study. In a similar study on Stichopus japonicus (S. japonicus), Kasai (2003) found PUFA to be the lowest FA class in BW and Gd of both sexes; however, for AC, values of PUFA reported in male and female are very similar to those reported in this study. Biandolino et al. (2019b) reported in Gd, AC, and gills of H. polii a higher amount of SFA and MUFA and lower PUFA (about 24% of total FAs).

The main FAs found in H. polii were arachidonic acid (ARA, 20:4 n-6, range 10.6%–25.6% of total FAs), eicosa-pentaenoic acid (EPA, 20:5 n-3, range 10.0%–19.8% of total FAs), palmitic acid (16:0, range 6.5%–18.4% of total FAs), palmitoleic acid (16:1, range 5.4%–22.2% of total FAs), ste-aric acid (18:0, range 8.6%–15.8% of total FAs), and alfa linolenic acid (ALA, 18:3 n-3, range 5.50%–12.5% of total FAs) (Table 3). However, significant differences between sexes and among tissues were present (P<0.05).

High oleic acid (18:1 n-9) level was also observed in the Gd of H. polii, female and male, accounting for 7.56% and 10.34% of total FAs, respectively. Moreover, there were mod-erate amounts of odd-branched carbon SFA 15:0+17:0 in all tissues that reached 8.31% of total FAs in the ITu of male (Table 3). These are known to be predominant in bacteria, so they are probably produced by bacteria living within the sea cucumber or extracting them from sediment, as they are bottom detritus-feeders.

As mentioned above, the PUFAs n-3 EPA and n-6 ARA were the predominant FAs present in most samples.

Previous studies reported ARA as one of the dominant FAs in almost all species of sea cucumber (Wen et al., 2010). Svetashev et al. (1991) observed values above 20% for tropical sea cucumber species harvested in Vietnamese waters. Mecheta et al. (2020) reported similar values for H. polii (16.5%), H. tubulosa (18.9%), and Holothuria sanctori (15.3%).

The lowest ARA amount was found in BW of both sexes (62.3–70.0 mg/100 g ww), while the highest was in the RT of male (457.72 mg/100 g ww) (P<0.05).

Regarding EPA, the Gd of female had the highest absolute amount (337.6 mg/100 g ww) (P<0.05), while the BW of both sexes had the lowest (36.9–40.0 mg/100 g ww). However, Gd of the male exhibited the highest percentage of EPA (19.8%), while MBs and ITu of both sexes exhibited the highest per-centage of ARA (>20%) (Table 3). Docosahexaenoic acid

(DHA, 22:6 n-3) was also present, but at a much lower con-centration (1.1%–3.7% of the total FAs).

Bilgin and Tanrikulu (2018) reported values of ARA (17.17%) and EPA (10.03%) in BW of H. tubulosa were very similar to those of this study. Bechtel et al. (2013) observed in BW and MBs of P. californicus higher values, with EPA as the predominant FA. Also, Kasai (2003) in S. japonicus found ARA and EPA as dominant PUFA in BW (about 11% of ARA, about 8% of EPA), Gd (about 9% of ARA, about 9.5% of EPA), and AC (about 15% of ARA, about 12.5% of EPA), of female and male, although lower values than those of this study, except for AC which showed values very similar to ours.

High levels of EPA and ARA and low levels of DHA, in the same and other species and genera, are consistent with previous studies (Wen et al., 2010; Lee et al., 2012; Sicuro et al., 2012; Haider et al., 2015; Roggatz et al., 2016; Liu et al., 2017; González-Wangüemert et al., 2018; Biandolino et al., 2019b). On the other hand, Aydin et al. (2011), in H. tubulosa, H. polii¸ and H. mammata, found ARA followed by DHA as major FA.

Differences in FA profiles of different sea cucumber species can be due to nutritional pattern, environmental temperature, geographical conditions, season, sex, and species.

ARA and EPA are both essential FAs because humans, like all mammals, cannot produce them and must obtain them by diet. ARA plays an important role in human health: it is a fundamental constituent of cell membrane phospholipids, making it more fluid and flexible and as a constituent of cell structure, it is particularly needed for development, especially of the central nervous system and retina, and for growth (Carlson and Neuringer, 1999). ARA affects various enzym-atic activities and is involved in cell apoptosis, necrosis, and death, important events during embryogenesis. Moreover, ARA may play an important role in wound healing as it is a precursor of thromboxane, which influences blood clot formation and attachment to the endothelial tissue during wound healing (Esser-von Bieren, 2020).

However, the benefits related to the seafood consumption are due to the high contents of n-3 PUFA, EPA, and DHA, of considerable interest to human nutrition as they exhibit important properties, such as antithrombotic, anti-inflamma-tory, anti-arrhythmic, and vasodilatory (Simopoulos, 2007). They are implied in reducing and treatment of coronary heart disease, cancer, inflammation, rheumatoid arthritis, and psor-iasis (Simopoulos, 2002); in lowering the incidence of diabetes, in fetal neurodevelopment and cognitive development, and in photoreception (vision) (Simonetto et al., 2019). Moreover, Fredalina et al. (1999) hypothesized that the high EPA content is involved in the tissue-repairing ability of sea cucumbers.

ARA and EPA are both precursor compounds for eicosanoid production; however, eicosanoids produced from AA have a pro-inflammatory role, while those produced from EPA have anti-inflammatory properties. Therefore, it is fundamental to maintain the balanced relationship of 1/1 between n-3 (EPA) and n-6 (ARA). However, due to the ex-cessive consumption of n-6 FAs in the Western diet, the ratio of n-6 to n-3 is very high, (15–20)/1 (Simopoulos, 2008). In this way, the eicosanoids from ARA are produced in larger amounts than those derived from n-3 FAs, specifically EPA, promoting the formation of thrombus and atheromas, al-lergic and inflammatory disorders, and the pathogenesis of

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Tab

le 3

. Fat

ty a

cid

profi

le o

f th

e si

x tis

sues

inve

stig

ated

of

both

fem

ale

and

mal

e H

olot

huria

pol

ii

Fatt

y ac

id

Sex

BW

(m

g/10

0 g

ww

) IT

u (m

g/10

0 g

ww

) M

B (

mg/

100

g w

w)

Gd

(mg/

100

g w

w)

AC

(m

g/10

0 g

ww

) R

T (

mg/

100

g w

w)

14:0

Fem

ale

16.0

1±1.

10 (

4.69

)aB9.

26±1

.02

(1.6

0)aA

11.0

2±1.

86 (

1.68

)a71

.94±

11.3

4 (2

.77)

bcB

53.8

7±9.

12 (

3.09

)b78

.55±

11.5

5 (3

.91)

c

Mal

e14

.52±

0.61

(3.

77)aA

13.2

4±0.

73 (

2.25

)aB10

.09±

0.86

(1.

96)a

4.67

±1.1

6 (0

.44)

aA69

.55±

7.24

(4.

12)c

50.5

1±6.

15 (

2.33

)b

15:0

Fem

ale

5.59

±0.7

8 (1

.64)

a4.

90±0

.77

(0.8

5)aA

––

15.9

6±7.

42 (

0.91

)b40

.44±

5.14

(2.

01)cB

Mal

e6.

41±0

.54

(1.6

6)a

8.43

±0.4

2 (1

.43)

aB–

4.13

±0.8

7 (0

.39)

a44

.27±

9.37

(2.

62)c

28.8

8±4.

08 (

1.33

)bA

16:0

Fem

ale

41.4

8±2.

44 (

12.1

6)a

55.3

1±4.

78 (

9.61

)abA

67.3

2±8.

98 (

10.2

7)bB

477.

42±3

2.05

(18

.38)

eB26

4.59

±9.2

2 (1

5.16

)dB15

5.80

±6.0

9 (7

.76)

cA

Mal

e44

.49±

0.37

(11

.55)

a65

.50±

1.41

(11

.12)

bB40

.69±

1.16

(7.

88)aA

68.3

8±9.

21 (

6.52

)bA22

6.79

±8.1

4 (1

3.45

)dA18

7.82

±1.9

8 (8

.67)

cB

17:0

Fem

ale

7.47

±0.6

8 (2

.19)

aA30

.76±

12.3

8 (5

.34)

bc13

.64±

2.88

(2.

08)ab

52.2

3±8.

36 (

2.01

)cB33

.40±

6.04

(1.

91)bc

87.4

8±19

.92

(4.3

6)d

Mal

e9.

78±0

.81

(2.5

4)aB

40.5

3±5.

58 (

6.88

)b11

.87±

0.72

(2.

30)a

40.0

3±8.

30 (

3.81

)bA33

.09±

3.84

(1.

96)b

95.7

2±6.

10 (

4.42

)c

18:0

Fem

ale

29.2

0±1.

01 (

8.56

)aA54

.15±

3.58

(9.

40)a

57.3

6±4.

10 (

8.75

)aB23

3.05

±22.

31 (

8.97

)bB18

8.11

±5.6

3 (1

0.78

)bB20

0.60

±52.

84 (

9.99

)b

Mal

e36

.53±

1.43

(9.

49)aB

56.6

5±1.

58 (

9.62

)b44

.40±

1.23

(8.

60)aA

166.

15±1

0.93

(15

.84)

cA17

4.42

±4.2

1 (1

0.34

)cA21

2.91

±0.9

1 (9

.83)

d

16:1

Fem

ale

40.0

9±1.

10 (

11.7

5)aA

41.9

7±6.

70 (

7.29

)aA45

.22±

2.26

(6.

90)a

576.

65±7

.38

(22.

20)dB

287.

65±1

7.74

(16

.49)

c21

6.50

±16.

64 (

10.7

8)b

Mal

e51

.82±

1.20

(13

.45)

bB52

.22±

1.85

(8.

87)bB

46.3

6±0.

18 (

8.98

)a57

.06±

2.50

(5.

43)bA

270.

36±7

.00

(16.

03)d

201.

78±5

.09

(9.3

2)c

17:1

Fem

ale

7.02

±0.6

0 (2

.06)

b6.

03±2

.40

(1.0

5)a

6.53

±1.8

2 (1

.00)

ab50

.82±

5.20

(1.

96)c

–89

.80±

24.4

9 (4

.47)

dB

Mal

e7.

42±1

.09

(1.9

3)a

––

––

22.6

3±3.

00 (

1.04

)bA

18:1

n-9

cFe

mal

e17

.15±

2.35

(5.

03)aA

24.8

3±9.

56 (

4.31

)a22

.04±

2.83

(3.

36)aA

196.

30±1

4.74

(7.

56)cB

72.8

8±10

.63

(4.1

8)b

95.1

4±21

.06

(4.7

4)b

Mal

e27

.00±

1.70

(7.

01)aB

20.6

8±4.

16 (

3.51

)ab28

.80±

1.27

(5.

58)aB

108.

46±1

6.68

(10

.34)

cA94

.66±

5.01

(5.

61)c

75.7

2±2.

45 (

3.50

)b

18:1

n-7

Fem

ale

11.3

7±0.

42 (

3.33

)aA14

.97±

5.01

(2.

60)ab

A23

.95±

2.60

(3.

65)bc

B39

.24±

4.53

(1.

51)cB

53.4

2±14

.56

(3.0

6)c

96.1

5±5.

06 (

4.79

)d

Mal

e15

.86±

0.92

(4.

12)aB

24.9

8±2.

22 (

4.24

)bB19

.25±

0.27

(3.

73)bA

29.7

0±1.

65 (

2.83

)bcA

36.2

2±6.

11 (

2.15

)c78

.00±

3.18

(3.

60)d

20:1

n-9

Fem

ale

9.33

±0.6

3 (2

.74)

a15

.03±

1.22

(2.

61)ab

17.7

5±0.

95 (

2.71

)b56

.99±

8.11

(2.

19)dB

33.3

9±3.

90 (

1.91

)c82

.46±

11.4

5 (4

.10)

d

Mal

e11

.08±

1.26

(2.

88)a

19.1

9±2.

40 (

3.26

)b14

.47±

0.82

(2.

80)b

20.4

4±2.

43 (

1.95

)bcA

32.5

8±9.

00 (

1.93

)c86

.00±

6.97

(3.

97)d

18:2

n-6

Fem

ale

5.99

±0.4

0 (1

.76)

aA9.

24±2

.28

(1.6

0)a

10.0

8±1.

11 (

1.54

)a42

.47±

3.14

(1.

63)bB

34.6

8±7.

73 (

1.99

)b71

.47±

7.60

(3.

56)cB

Mal

e7.

30±0

.19

(1.8

9)aB

9.32

±2.0

1 (1

.58)

a9.

01±0

.39

(1.7

5)a

25.8

8±6.

25 (

2.47

)bA24

.63±

3.12

(1.

46)b

40.3

6±0.

75 (

1.86

)cA

18:3

n-6

Fem

ale

––

––

8.16

±2.7

3 (0

.47)

–

Mal

e–

––

–11

.11±

2.74

(0.

66)

–

18:3

n-3

Fem

ale

31.5

8±1.

75 (

9.26

)a70

.62±

10.4

5 (1

2.27

)ab76

.34±

8.17

(11

.65)

bB14

2.79

±13.

76 (

5.50

)cB16

2.58

±15.

12 (

9.32

)c17

7.83

±26.

30 (

8.86

)cB

Mal

e38

.76±

1.97

(10

.06)

a62

.28±

6.58

(10

.58)

bc53

.86±

1.97

(10

.44)

abA

78.8

5±5.

98 (

7.51

)cA13

2.18

±7.5

6 (7

.84)

d27

0.24

±23.

87 (

12.4

8)eA

20:3

n-6

Fem

ale

8.25

±0.9

2 (2

.42)

a14

.26±

2.40

(2.

48)b

––

––

Mal

e–

––

––

–

20:3

n-3

Fem

ale

6.70

±1.3

2 (1

.96)

a14

.55±

1.65

(2.

53)b

16.6

1±2.

71 (

2.53

)b9.

72±1

.94

(0.3

7)a

28.1

3±4.

41 (

1.61

)c87

.47±

11.7

3 (4

.36)

dB

Mal

e–

16.7

1±2.

78 (

2.84

)a20

.28±

1.27

(3.

93)a

–30

.03±

7.20

(2.

02)b

44.1

7±1.

51 (

2.04

)cA

20:4

n-6

Fem

ale

62.2

9±1.

02 (

18.2

6)aA

131.

77±5

.08

(23.

09)b

167.

87±1

.07

(25.

62)bB

275.

75±1

3.85

(10

.61)

cB24

1.40

±41.

91 (

13.8

4)c

267.

09±2

5.68

(13

.30)

cA

Mal

e69

.98±

3.34

(18

.17)

aB13

1.01

±13.

98 (

22.2

5)b

130.

79±1

.11

(25.

34)bA

208.

64±2

.39

(19.

88)cA

264.

83±3

6.55

(15

.71)

d45

7.72

±8.4

2 (2

1.14

)eB

20:5

n-3

Fem

ale

36.9

0±0.

64 (

10.8

1)a

66.8

7±7.

31 (

11.8

2)b

99.5

5±3.

13 (

15.2

0)bB

337.

63±2

9.97

(13

.0)eB

233.

66±9

.40

(13.

39)dB

185.

28±1

3.08

(9.

23)cA

Mal

e39

.88±

4.64

(10

.35)

a59

.09±

6.40

(10

.03)

b75

.57±

1.89

(14

.64)

bA20

8.11

±4.8

2 (1

9.83

)cA21

3.15

±8.3

5 (1

2.64

)cA29

3.33

±16.

35 (

13.5

5)dB

22:6

n-3

Fem

ale

4.52

±0.2

6 (1

.32)

a8.

79±2

.95

(1.5

3)a

14.7

9±1.

83 (

2.26

)abB

25.8

6±3.

67 (

0.99

)ab32

.89±

4.42

(1.

88)b

75.0

7±13

.97

(3.7

4)cB

Mal

e4.

25±0

.58

(1.1

0)a

9.04

±1.3

2 (1

.53)

ab11

.15±

1.21

(2.

16)bA

24.1

6±6.

21 (

2.30

)c24

.43±

4.43

(1.

45)c

19.2

5±1.

63 (

0.89

)cA

∑SFA

Fem

ale

99.7

4±1.

93 (

29.2

5)aA

154.

38±1

6.45

(26

.81)

aA14

9.35

±2.6

4 (2

2.79

)aB83

4.63

±81.

79 (

32.1

3)cB

555.

93±1

3.32

(31

.86)

b56

3.86

±25.

06 (

28.0

4)b

Mal

e11

1.73

±3.1

9 (2

9.01

)aB18

4.33

±6.5

6 (3

1.30

)bB10

7.06

±0.6

4 (2

0.75

)aA28

3.37

±14.

68 (

27.0

1)cA

548.

13±3

5.82

(32

.50)

d57

5.85

±9.7

8 (2

5.60

)d

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ctober 2022


Fatt

y ac

id

Sex

BW

(m

g/10

0 g

ww

) IT

u (m

g/10

0 g

ww

) M

B (

mg/

100

g w

w)

Gd

(mg/

100

g w

w)

AC

(m

g/10

0 g

ww

) R

T (

mg/

100

g w

w)

∑MU

FAFe

mal

e84

.97±

2.42

(24

.92)

aA10

2.83

±16.

25 (

17.8

6)a

115.

49±3

.05

(17.

63)a

920.

01±1

3.86

(35

.42)

dB44

7.35

±39.

19 (

25.6

4)b

580.

08±6

3.26

(28

.90)

cB

Mal

e11

3.18

±3.8

0 (2

9.39

)aB11

7.08

±9.0

7 (1

9.88

)a10

8.89

±1.7

1 (2

1.10

)a22

0.11

±16.

99 (

20.9

8)bA

433.

83±1

2.11

(25

.73)

c46

4.14

±14.

98 (

21.4

4)dA

∑PU

FAFe

mal

e15

6.23

±1.6

5 (4

5.82

)a31

8.50

±1.9

0 (5

5.32

)bB39

0.37

±2.8

1 (5

9.58

)bB84

3.16

±92.

46 (

32.4

6)cB

741.

51±3

3.95

(42

.49)

c86

4.23

±45.

03 (

43.0

6)cA

Mal

e16

0.18

±6.7

7 (4

1.60

)a28

7.45

±14.

92 (

48.8

1)bA

300.

66±2

.16

(58.

27)bA

545.

64±5

.33

(52.

01)cA

704.

38±4

5.02

(41

.76)

d11

25.0

7±24

.03

(51.

96)eB

n-3

Fem

ale

79.7

0±0.

50 (

23.3

8)a

162.

04±3

.30

(28.

14)bB

207.

30±5

.55

(31.

64)bB

516.

00±7

7.45

(19

.86)

cB45

7.27

±12.

28 (

26.2

1)cB

525.

66±4

0.78

(26

.18)

cA

Mal

e82

.89±

4.19

(21

.52)

a14

7.12

±3.7

1 (2

4.98

)bA16

0.87

±1.4

7 (3

1.17

)bA31

1.12

±4.8

5 (2

9.65

)cA40

3.80

±22.

00 (

23.9

5)dA

626.

99±1

6.91

(28

.96)

eB

n-6

Fem

ale

76.5

2±1.

83 (

22.4

5)a

156.

47±3

.85

(27.

17)b

183.

08±3

.35

(27.

94)bB

327.

15±1

8.97

(12

.59)

cB28

4.23

±45.

52 (

16.2

9)c

338.

56±3

8.31

(16

.86)

cA

Mal

e77

.29±

3.15

(20

.06)

a14

0.32

±11.

93 (

23.8

3)b

139.

80±0

.72

(27.

09)bA

234.

52±7

.82

(22.

35)cA

300.

58±3

3.24

(17

.83)

d49

8.08

±7.6

7 (2

3.01

)eB

The

fat

ty a

cids

per

cent

age

(FA

s %

) is

rep

orte

d in

the

bra

cket

s. D

ata

are

expr

esse

d as

mea

n±SD

.B

W, b

ody

wal

l; IT

u, in

tern

al t

unic

; MB

, mus

cle

band

; Gd,

gon

ad; A

C, a

limen

tary

can

al; R

T, r

espi

rato

ry t

ree;

SFA

, sat

urat

ed f

atty

aci

d; M

UFA

, mon

ouns

atur

ated

fat

ty a

cid;

PU

FA, p

olyu

nsat

urat

ed f

atty

aci

d; E

PA,

eico

sape

ntae

noic

aci

d; D

HA

, doc

osah

exae

noic

aci

d; w

w, w

et w

eigh

t. n-

3 is

the

sum

of

18:3

n-3

, 20:

3 n-

3, 2

0:5

n-3,

and

22:

6 n-

3; n

-6 is

the

sum

of

18:2

n-6

, 18:

3 n-

6, 2

0:3

n-6,

and

20:

4 n-

6.D

iffe

rent

sup

ersc

ript

s in

the

sam

e ro

w in

dica

ted

sign

ifica

nt d

iffe

renc

es a

mon

g ti

ssue

s (P

<0.0

5); d

iffe

rent

sup

ersc

ript

s in

the

sam

e co

lum

n in

dica

ted

sign

ifica

nt d

iffe

renc

es b

etw

een

sexe

s (P

<0.0

5).

Tab

le 3

. Con

tinue

d

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ctober 2022


many diseases, including cardiovascular disease and cancer. Research has shown that increased dietary intakes of n-3 PUFA exert suppressive effects on ARA-derived eicosanoid production (Calder, 2012).

Lipid Nutritional Quality IndexesThe nutritional value of analyzed tissues of H. polii female and male was also assessed by lipid quality indices, which depend on the relative proportions of some individual sat-urated fatty acids (SFA) and unsaturated fatty acids (USFA) (Table 4).

Ratios of n-3/n-6, n-6/n-3, PUFA/SFA, (MUFA+PUFA)/SFA 18:0, AI, TI, and h/H indices are considered the most useful parameters for evaluating the nutritional value and healthi-ness of foods (Attia et al., 2017).

The n-3/n-6 ratio is the most used index for assessing the nutritional quality of shellfish, such as for fish oil (Piggott and Tucker, 1990). For the reasons explained above, a balanced n-3/n-6 ratio has positive effects on decreasing the risk of car-diovascular disease (CVD) and cancers (Simopoulos, 2008). In this study, all tissues of H. polii female and male showed the higher amount of n-3 than the amount of n-6, thus pro-viding favorable n-3/n-6 ratios, within the nutritional recom-mendations (Simopoulos, 2016).

The n-3/n-6 ratio (Figure 2) ranged from the lowest value of 1.04 in BW and ITu of female, to the highest value of 1.61 in AC of female. Gd, AC, and RT of both sexes showed the highest ratio, although with maximum amounts detected in female (P<0.05).

Despite the differences in the FA profiles, it can be con-cluded that the analyzed tissues of H. polii are good sources of essential n-3 and n-6 PUFAs. Simopoulos (2016) sug-gested an n-3/n-6 ratio in the range of 0.25 to 1.0, while the Food and Agriculture Organization (FAO; FAO and WHO, 2003) recommends an n-3/n-6 ratio in the range of 1:8 to 2:5. Obviously, an n-3/n-6 ratio beneficial for the human diet should be as high as possible. These findings agree with other studies (Gao et al. 2016; Göçer et al., 2018; Biandolino et al., 2019b).

On the other hand, relating to BW, for eight sea cucumber species, Wen et al. (2010) reported the values of n-3/n-6 ratio lower than those of this study, ranging from of 0.25 to 0.61, as well as Künili and Çolakoglu (2019) reporting for H. tubulosa (0.75–0.95), Santos et al. (2017) for H. mammata (0.48), and Svetashev et al. (1991) for tropical species (0.41–0.89). Bechtel et al. (2013) reported for P. californicus BW and MBs values slightly higher than those of this study with 1.83 and 2.61, respectively. Likewise, Drazen et al. (2008), for abyssal species from the North East Pacific Ocean, found values ranging from 1.74 to 2.75.

The n-6/n-3 ratio is also commonly used as an indicator of relative nutritional values of seafood, as a lower ratio in human dietary can protect against coronary heart disease (Simopoulos, 2016). WHO (2003) recommends a ratio of less than 10, and the UK Department of Health (1994) suggests a maximum of 4.0 to achieve a balanced FA intake. In the present study, all tissues of both sexes could be considered beneficial to human health, showing ratios from 0.62 to 0.96, which is similar or even lower than the ratios reported for other species (Aydin et al., 2011; Sicuro et al., 2012; Mecheta et al., 2020).

González-Wangüemert et al. (2018) reported for H. mammata, H. polii, and H. tubulosa from the Mediterranean Sea (Southeast Spain) that the values were higher than those of this study with 2.3, 1.8, and 1.9, respectively. Künili and Çolakoglu (2019) reported values for H. tubulosa that varied between 1.06 and 1.17 in all seasons.

The PUFA/SFA ratio is another used index to assess the nutritional value of dietary foods, such as seafood. Food with PUFA/SFA>0.45 is recommended in human diets (UK Department of Health, 1994), and the higher this ratio, the more positive the effect. PUFAs in the diet can reduce the levels of low-density lipoprotein cholesterol (LDL-C) and lower levels of serum cholesterol, whereas SFAs induce an in-crease of cholesterol in the blood increasing the risk of CVD (Siri-Tarino et al., 2015).

In the current study, the values of PUFA/SFA were far higher than the minimum required value, so very beneficial

Figure 1. Proportions (%) of saturated, monounsaturated and polyunsaturated fatty acids (SFA, MUFA, and PUFA) in six tissues of Holothuria polii female (f) and male (m). BW, body wall; ITu, internal tunic; MB, muscle band; Gd, gonad; AC, alimentary canal; RT, respiratory tree.

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for human health (Table 4). It ranged from 1.01 (Gd of fe-male) to 2.81 (MBs of male), with significant differences among tissues and between sexes (P<0.05). MBs of both sexes exhibited the highest value.

The results of this study were higher compared with the most literature data on the same species and other sea cucum-bers (Wen et al., 2010; Ridzwan et al., 2014; Sroyraya et al., 2017; Göçer et al., 2018; Künili and Çolakoglu, 2019). Kasai (2003) reported the values for BW and Gd of S. japonicus, of both sexes, lower than those of this study, but slightly higher for AC of both sexes.

However, the PUFA/SFA index does not take into account the metabolic importance of MUFAs for preventing CVD and the fact that some SFAs such as stearic acid (18:0) do not increase plasma cholesterol, but appears to be biologically neutral. Therefore, the index of (MUFA+PUFA)/SFA 18:0 was

also used, in which MUFA was added and stearic acid was ex-cluded from the SFA. The values of (MUFA+PUFA)/SFA 18:0 (Figure 2) were significantly higher in MBs of both sexes and in Gd of male (P<0.05) than the other tissues.

AI and TI indices indicate the potential of foods to stimulate platelet aggregation and subsequent thrombus and atheroma formation in the cardiovascular system (Rodrigues et al., 2017). Thus, lower values indicate better nutritional quality of foods because they help to prevent cardiovascular disorders. The SFAs 12:0, 14:0, and 16:0 are considered AI and TI because they have a cholesterol-raising effect that may induce platelet aggregation. The AI value was lowest for Gd of male (0.11), followed by that of the MBs of both sexes (0.20 male and 0.22 female), due to its low content of 14:0 and 16:0 SFAs. The highest values were observed in BW (0.44), Gd (0.43) of female and in AC of both sexes (0.44 male and 0.40 female) (Table 4).

Table 4. Lipid nutritional quality indexes of of the six tissues of both female and male Holothuria polii

Index Sex BW ITu MB Gd AC RT

n-3/n-6 Female 1.04±0.03a 1.04±0.04a 1.13±0.05a 1.58±0.17bB 1.61±0.35b 1.55±0.14bB

Male 1.07±0.04a 1.05±0.08a 1.15±0.00ab 1.33±0.06cA 1.34±0.15c 1.26±0.02bcA

n-6/n-3 Female 0.96±0.03b 0.96±0.04b 0.88±0.04b 0.63±0.07aB 0.62±0.11a 0.64±0.05aA

Male 0.93±0.04c 0.95±0.07c 0.87±0.00bc 0.75±0.03aA 0.74±0.08a 0.79±0.01abB

PUFA/SFA Female 1.57±0.03b 2.06±0.21cB 2.61±0.05dA 1.01±0.20aA 1.33±0.06b 1.53±0.20bA

Male 1.43±0.10a 1.56±0.13bA 2.81±0.01dB 1.93±0.09cB 1.28±0.16a 1.95±0.07cB

(MUFA+PUFA)/SFA 18:0 Female 3.42±0.07b 4.20±0.70cB 5.50±0.36dA 2.94±0.41aA 3.23±0.18ab 3.98±0.44c

Male 3.64±0.14b 3.17±0.20abA 6.54±0.14dB 6.53±1.71dB 3.05±0.35a 4.38±0.15c

AI Female 0.44±0.01cB 0.22±0.01aA 0.22±0.00aB 0.43±0.08cB 0.40±0.04c 0.32±0.04b

Male 0.37±0.01dA 0.29±0.01cB 0.20±0.01bA 0.11±0.04aA 0.44±0.04e 0.24±0.03bc

TI Female 0.27±0.01bc 0.19±0.01aA 0.17±0.01aB 0.35±0.08dB 0.29±0.01c 0.21±0.04ab

Male 0.27±0.02cd 0.23±0.01bcB 0.15±0.00aA 0.20±0.00eA 0.29±0.03d 0.19±0.00ab

h/H Female 2.75±0.09bA 4.87±0.41dB 4.99±0.48dA 1.86±0.32aA 2.44±0.11ab 3.72±0.62cA

Male 3.17±0.13abB 3.70±0.25abA 6.09±0.21cB 8.95±2.62dB 2.54±0.28a 4.85±0.29bcB

Values are means±standard deviation.BW, body wall; ITu, internal tunic; MB, muscle band; Gd, gonad; AC, alimentary canal; RT, respiratory tree; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid, PUFA, polyunsaturated fatty acid; AI, atherogenic index; TI, thrombogenic index; h/H, hypocholesterolaemic/hypercholesterolaemic fatty acids ratio.Different superscripts in the same row tissues indicated significant differences among tissues (P<0.05); different superscripts in the same column indicated significant differences between sexes (P<0.05).

0

1

2

3

4

5

6

7

BW Itu MB Gd AC RT 0 BW Itu MB Gd AC RT

(MUFA+PUFA) / SFA 18:0 n-3/n-6

female male

Figure 2. The ratios of n-3/n-6 and (MUFA+PUFA)/SFA 18:0 in six tissues of Holothuria polii female and male. MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; BW, body wall; ITu, internal tunic; MB, muscle band; Gd, gonad; AC, alimentary canal; RT, respiratory tree. Data are expressed as mean±standard deviation.

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As with AI, TI was significantly lowest in MBs of both sexes (0.17 for female and 0.15 for male), ITu of female and RT of male (both 0.19), while highest in Gd of female (0.35). Zmemlia et al. (2020) reported H. tubulosa from the Bizerta lagoon (Mediterranean Sea) showed higher AI (1.38) and TI (0.55) values compared with this study.

In general, the results are comparable with or lower than those reported for sea cucumbers and other marine organisms (Alexi et al., 2019; Biandolino et al., 2019a, 2019b; Kocatepe et al., 2019; Chen and Liu, 2020; Prato et al., 2020).

The h/H index refers to the specific effects of FAs on chol-esterol metabolism, and in contrast to AI and TI, higher h/H values are considered more beneficial to human health. The h/H value, ranging between the lowest in Gd of female (1.86) to the highest in Gd of male (8.95), indicates that a regular intake of H. polii may lead to a hypocholesterolemic effect (P<0.05). Also, MBs of both sexes, ITu of female and RT of male showed high values. These values are comparable with or higher than with those reported in literature for shellfish and fish (Biandolino et al., 2019a, 2019b; Kocatepe et al., 2019; Chen and Liu, 2020; Prato et al., 2020).

EPA+DHAEPA and DHA are n-3 PUFAs that play essential roles in human biological processes, and they are the most closely as-sociated with lower CVD risk (Nestel et al., 2015).

For these reasons, EPA+DHA is one of the most important lipid nutritional quality indices recognized worldwide. Several dietary guidelines developed by various national and inter-national health scientific agencies report recommendations for the dietary EPA+DHA intake. According to the FAO (2008), the recommended amount is 0.250–2 g/d. EFSA (2015) re-commends a daily intake of 250 mg of EPA+DHA for adults, while for pregnant and lactating women, an increased intake of DHA by an additional 100–200 mg/d is suggested.

A daily intake of each tissue of H. polii female and male was calculated, on the basis of the results obtained in this study, to get the EPA+DHA quantity of 250–500 mg/d as recommended by WHO (Table 5). Significant differences among tissues and species were present (P<0.05). Gd of female appeared to be the most valuable tissue as the essential PUFA source, because it contained the highest EPA+DHA content (referred to 100 g ww). Therefore, in order to obtain a health benefit, a portion of 69–138 g of Gd is enough to satisfy the recommended daily intake of EFA. As regard male, RT had the highest EPA+DHA content, for which a portion of 80–160 g is necessary to meet the daily requirement. In both sexes, BW showed the lowest

EPA+DHA, thus, it has been estimated that the recommended daily intake of EPA+DHA can be satisfied by eating 603–1207 g for female and 566–1133 g for male.

Concentration and composition patterns of PAHsThe average PAHs concentrations for each tissue tested of H. polii female and male are shown in Table 6. The sum of all measured compounds is referred to ∑PAHs while the sum of B[a]P, B[a]A, B[b]F, and CHRY, used as a marker for the oc-currence and effect of carcinogenic PAHs in food, is referred to as PAH4. Because of their physical–chemical and biological properties, 2–3-ring PAHs are called light molecular weight PAHs (L-PAHs) while 4–6-ring PAHs are indicated as high molecular weight PAHs (H-PAHs) (Zhang et al., 2020). The ∑PAHs levels in H. polii ranged between 33–184 µg/kg ww and 23–207 µg/kg ww in female and male tissues, respect-ively. There were significant differences in the ∑PAHs con-tent of H. polii as a result of sex (F=9.691, df=1, P<0.05) and tissue (F=399, df=5, P<0.001), and there was a moderate interaction between these factors (F=5.682, df=5, P<0.05). The ΣPAHs in female and male showed the following order in tissues from high to low: Gd>AC>MB>RT>ITu>BW and Gd>AC>ITu>MB>RT>BW, respectively. The last three tissue concentrations reported above for both sexes were signifi-cantly different from the first three (P<0.05). Gd and AC, in female, displayed 6 to 1 and 5 to 1 times higher PAHs levels than those found in all other tissues, respectively. These ratios ranged from 9 to 1 in Gd and from 8 to 1 in AC for male. PAHs are hydrophobic compounds that tend to accumulate in sediments and consequently into marine organisms. PAHs accumulate mainly in some tissues, such as in the hepatopan-creas or Gd of invertebrates, and in the liver of vertebrates (Angioni et al., 2014; Wang et al., 2020). Because the lipids are one of the key factors influencing the bioaccumulation of PAHs in biota (Sun et al., 2016), the distribution of these hydrocarbons in various organisms and in their organs could be influenced by their content. Although, in this study, the PAHs showed the highest values in lipid-rich tissues such as Gd and AC, their correlation with the lipid level was positive but weak (r=0.3–0.7, P>0.05) in male and female, respect-ively. Therefore, this parameter does not seem to completely affect the distribution of PAHs in the sea cucumber tissues. Differences in PAHs accumulation can depend on the up-take capacity, metabolism and physiology typical of dif-ferent organs. Concerning the composition patterns of PAHs (Figure 3), poor correlations (r=0.6–0.8, P>0.05) were detected in most of tissues except for AC–Gd (r=0.99, P<0.0001) and

Table 5. EPA+DHA content in the six tissues of both female and male Holothuria polii; Tissue portion to satisfy the recommended daily intake of EPA+DHA (250–500 mg/d).

Tissue EPA+DHA content (mg/100 g ww) Tissue portion (g)

Female Male Female Male

BW 41.42±0.4a 44.13±5.19a 603.5–1207 566.5–1133

ITu 75.66±10.1ab 68.13±5.14b 330.74–660.8 367.0–734.0

MB 114.34±4.9bB 86.72±2.78bA 218.7–437.3 288.3–576.6

Gd 363.63±52.5dB 232.27±9.60cA 68.7–137.5 107.6–215.3

AC 266.55±13.5cB 237.58±9.63cA 93.8–187.6 105.2–210.4

RT 260.35±23.9cA 312.58±16.22dB 96.0–192.0 80.0–160.0

EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; BW, body wall; ITu, internal tunic; MB, muscle band; Gd, gonad; AC, alimentary canal; RT, respiratory tree.

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Tab

le 6

. PA

H e

xpos

ure

daily

inta

ke f

rom

inge

stio

n of

bot

h fe

mal

e an

d m

ale

Hol

othu

ria p

olii

PAH

R

ing

No.

B

WM

BIT

uG

dR

TA

C

Fem

ale

Mal

e Fe

mal

e M

ale

Fem

ale

Mal

e Fe

mal

e M

ale

Fem

ale

Mal

e Fe

mal

e M

ale

NA

P2

1.6±

0.16

2.3±

0.26

1.4±

0.14

1.3±

0.13

2.2±

0.2

2.0±

0.21

2.3±

0.26

2.0±

0.18

3.0±

0.31

2.7±

0.34

1.2±

0.15

2.4±

0.28

AC

Y3

0.37

±0.0

3<0

.31

0.46

±0.0

40.

44±0

.04

<0.2

00.

9±0.

051.

7±0.

192.

2±0.

20.

97±0

.10.

86±0

.09

1.4±

0.14

1.7±

0.16

AC

E2

<0.2

70.

5±0.

050.

91±0

.09

0.88

±0.0

9<0

.20

<0.1

9<0

.19

0.5±

0.05

0.55

±0.0

50.

49±0

.05

<0.2

4<0

.24

FLU

22.

6±0.

641.

0±0.

111.

0±0.

10.

98±0

.10.

88±0

.09

0.9±

0.09

4.1±

0.49

0.9±

0.09

3.1±

0.32

2.8±

0.21

3.4±

0.38

4.2±

0.48

PHE

N3

2.7±

0.22

2.0±

0.21

2.6±

0.27

2.5±

0.26

3.1±

0.32

2.0±

0.2

6.4±

0.67

2.3±

0.15

2.8±

0.2

2.5±

0.19

5.2±

0.49

6.5±

0.61

AN

TH

3<0

.19

<0.2

20.

32±0

.03

0.31

±0.0

3<0

.14

0.33

±0.0

31.

7±0.

191.

2±0.

093.

2±0.

32.

8±0.

291.

4±0.

161.

76±0

.18

FLT

H4

1.1±

0.11

1.4±

0.15

1.0±

0.1

0.92

±0.1

11.

2±0.

151.

3±0.

116

±1.5

53.

7±0.

390.

50±0

.03

0.5±

0.03

13±1

.25

16±1

.65

PYR

41.

1±0.

111.

4±0.

141.

2±0.

11.

2±0.

111.

5±0.

152.

4±0.

2118

±1.9

85.

3±0.

581.

4±0.

141.

0±0.

1315

±1.5

518

±1.7

5

B[a

]A4

<0.2

20.

37±0

.03

<0.1

5<0

.14

1.0±

0.15

0.25

±0.0

37.

6±0.

82.

5±0.

22.

3±0.

212.

0±0.

216.

2±0.

617.

7±0.

8

CR

Y4

0.32

±0.0

30.

44±0

.04

0.43

±0.0

40.

41±0

.04

1.6±

0.4

0.52

±0.0

58.

0±0.

852.

5±0.

21.

2±0.

10.

9±0.

096.

7±0.

648.

3±0.

89

B[b

]F5

2.6±

0.29

0.37

±0.0

44.

9±0.

474.

7±0.

473.

6±0.

385.

1±0.

4723

±2.4

128

±2.9

81.

7±0.

191.

5±0.

1519

±1.9

824

±2.5

5

B[k

]F5

0.72

±0.0

81.

2±0.

152.

2±0.

22.

1±0.

211.

7±0.

193.

5±0.

3211

±1.1

212

±1.1

21.

0±0.

10.

89±0

.09

9.9±

0.93

12±1

.15

B[j

]F5

4.7±

0.47

0.59

±0.0

57.

8±0.

757.

5±0.

741.

0±0.

211.

7±0.

1614

±1.2

227

±2.5

83.

9±0.

43.

5±0.

329.

0±0.

9311

±1.1

2

B[e

]P5

4.7±

0.37

3.3±

0.32

7.9±

0.75

7.6±

0.75

5.6±

0.6

8.4±

0.92

14±1

.42

27±2

.65

4.0±

0.44

3.5±

0.35

11±1

.12

14±1

.42

B[a

]P5

1.8±

0.3

1.5±

0.3

2.4±

0.4

2.3±

0.4

2.4±

0.42

2.3±

0.55

17±2

.517

±4.0

42.

9±0.

42.

6±0.

6414

±2.0

418

±4.0

4

D[a

h]A

5<0

.27

<0.3

10.

59±0

.11

0.57

±0.0

6<0

.20

0.5±

0.05

2.2±

0.2

2.8±

0.34

<0.1

4<0

.12

1.8±

0.19

2.2±

0.2

IND

62.

6±0.

262.

0±0.

216.

9±0.

646.

6±0.

613.

1±0.

316.

3±0.

6618

±1.9

833

±32.

3±0.

262.

0±0.

1814

±1.4

418

±1.9

8

B[g

hi]P

65.

8±0.

554.

4±0.

4411

±1.0

210

±1.0

55.

3±0.

4910

±1.0

319

±1.9

838

±3.6

54.

4±0.

443.

9±0.

415

±1.4

519

±1.8

∑PA

Hs

33±0

.56

23±1

.04

52±2

.86

51±4

.54

34±2

.29

48±4

.45

184±

16.4

920

7±15

.339

±0.3

35±0

.314

8±1.

918

5±14

.45

∑PA

H4

4.7±

0.60

2.7±

0.36

7.7±

0.42

7.4±

0.75

8.7±

0.69

8.1±

156

±5.2

550

±6.9

98.

1±0.

177.

1±0.

6446

±2.6

057

±4.2

5

PAH

s, p

olyc

yclic

aro

mat

ic h

ydro

carb

ons;

NA

P, n

apht

hale

ne; A

CY

, ace

naph

thyl

ene;

AC

E, a

cena

phth

ene;

FL

U, fl

uore

ne; P

HE

N, p

hena

nthr

ene;

AN

T, a

nthr

acen

e; F

LTH

, fluo

rant

hene

; PY

R, p

yren

e; B

[a]A

, ben

zo[a

]an

thra

cene

; CH

RY

, chr

ysen

e; B

[b]F

, ben

zo[b

]fluo

rant

hene

; B[k

]F, b

enzo

[k]fl

uora

nthe

ne; B

[j]F

, ben

zo[j

]fluo

rant

hene

; B[e

]P, b

enzo

[e]p

yren

e; B

[a]P

, ben

zo[a

]pyr

ene;

B[g

hi]P

, ben

zo[g

, h, i

]per

ylen

e; I

ND

, ind

eno[

1,2,

3-c,

d]py

rene

; D[a

h]A

=dib

enz[

a, h

]ant

hrac

ene;

∑PA

Hs

is t

he s

um o

f 18

PA

Hs.

∑PA

H4

is t

he s

um o

f B

[a]P

, B[a

]A, B

[b]F

, and

CH

RY

. BW

, bod

y w

all;

MB

, mus

cle

band

; ITu

, int

erna

l tun

ic; G

d, g

onad

; AC

, alim

enta

ry

cana

l; R

T, r

espi

rato

ry t

ree.

Dow



ctober 2022


BW–MB (r=0.9, P<0.001) for female. Despite this, H-PAHs were the dominant hydrocarbons in all tissues accounting in both sexes for 65%–91% of total. Among these, B[ghi]P (10–20% of ∑PAHs), B[e]P (8%–18% of ∑PAHs), B[j]F (6%–16% of ∑PAHs), IND (6%–13% of ∑PAHs), and B[b]F (2%–13% of ∑PAHs) were the most abundant compounds. H-PAHs in various tissues showed a strong correlation with ∑PAHs concentrations (r=0.99, P <0.001) in male and female and a similar trend at those observed for ∑PAHs (in decreasing order Gd>AC>MB>ITu>RT>BW), although it was the same in both sexes. The highest H-PAHs levels in Gd were signifi-cantly different from other tissues (P<0.05) in both sexes. As for L-PAHs, the most abundant compound in all tissues was PHEN except for the RT where FLTH exhibited the highest levels. PAH distribution in marine organisms depends on biota feeding preferences and trophic levels, but also on the anthropogenic pressure of the habitat in which they live. The occurrence of PAHs in sea cucumbers, in our study, effectively

seems to reflect PAHs contamination reported by other au-thors (Cardellicchio et al., 2006) for sediments sampled in the study area. It is recognized that PAHs in the marine en-vironment often result from the combination of pyrolytic and petrogenic sources. Pyrolytic PAHs derive mainly from pyrolysis or incomplete combustion of organic matter while petrogenic PAHs originate from spilled or leaked crude oil and/or their derived products (Neff et al., 2005; Stogiannidis and Laane, 2015; Barhoumi et al., 2016).

Concentration ratios of specific pairs of PAHs, called PAHs molecular diagnostic ratios, can be apply to identify the pos-sible sources of PAHs in environmental media (Baumard et al., 1999). Among these, the following isomeric ratios, in-dicated as ANTH/(PHEN+ANTH) and FLTH/(FLTH+PYR) are commonly used (Yunker et al., 2002; Soliman et al., 2014). In our study, the means of ANTH/(PHEN+ANTH) were 0.3 in both sexes, while FLTH/(FLTH+PYR) varied be-tween 0.6 and 0.5 in female and male, respectively. Because

Figure 3. Box-Plots of PAH compounds in Holothuria polii female (A) and male (B). PAHs, polycyclic aromatic hydrocarbons; NAP, naphthalene; ACY, acenaphthylene; ACE, acenaphthene; FLU, fluorene; PHEN, phenanthrene; ANT, anthracene; FLTH, fluoranthene; PYR, pyrene; B[a]A, benzo[a]anthracene; CHRY, chrysene; B[b]F, benzo[b]fluoranthene; B[k]F, benzo[k]fluoranthene; B[j]F, benzo[j]fluoranthene; B[e]P, benzo[e]pyrene; B[a]P, benzo[a]pyrene; B[ghi]P, benzo[g, h, i]perylene; IND, indeno[1,2,3-c,d]pyrene; D[ah]A=dibenz[a, h]anthracene. .

Dow



ctober 2022


pyrogenic PAHs are generally dominated by H-PAH (Muel and Saguem, 1985) while petrogenic hydrocarbons are char-acterized by L-PAH (Neff, 1979; Wise et al., 1988; Berner et al., 1990), the L-PAH/H-PAH ratio can be applied to get insights on important information on the origin of PAHs. In this study, L-PAH/H-PAH in sea cucumber was always <1. These findings suggest that PAHs in holothuria originated are mostly from pyrogenic sources. The high H-PAH concen-trations found in the environment represent a potential risk for human health because these compounds are persistent and can also be carcinogenic and mutagenic (IARC, 2010). Generally, an increase in the aromatic ring and angularity of a PAH molecule determines an increase in hydrophobicity and electrochemical stability (Zander, 1983; Harvey, 1997). For this reason, H-PAHs are also the top priority PAHs. Among these, B[a]P, the eighth compound in the list from the Agency for Toxic Substances and the Register of Diseases (ATSDR, 2013), is classified as carcinogen (group 1) to humans (IARC, 2010). However, because different H-PAHs such as B[a]A, B[b]F, and CHRY are considered possibly carcinogenic to hu-mans (group 2; IARC, 2010), the EFSA established that the sum of B[a]P, B[a]A, B[b]F, and CHRY (called PAH4) would be the most suitable indicator of PAHs in food. In this way PAH concentrations can also be monitored in those samples in which B[a]P is not detectable, but where other H-PAHs are present (ESFA, 2008).

In this study, B[a]P was detected in all the tissues of H. polii at levels of 1.8–17 µg/kg ww in female and 1.5–18 µg/kg ww in male. Concerning PAH4, concentrations ranged be-tween 4.7–46 µg/kg ww and 2.7–57 µg/kg ww in female and male, respectively. The highest levels were determined, as for ΣPAHs, in Gd and AC tissues of both sexes. Significant dif-ferences were found among these values and those deter-mined in the other tissues (P<0.05). Because there are no specific limits for PAH compounds related to echinoderm tissues, we have compared the B[a]P and PAH4 concentra-tions with the maximum levels set by EC Regulation No. 835/2011 for bivalve mollusks. B[a]P and PAH4, deter-mined in Gd and Ac tissues of both sexes, were above the limit defined for bivalves set to 5.0 µg/kg ww and 30 µg/kg ww, respectively.

Few studies are available to evaluate PAHs concentrations in sea cucumber. Khazaali et al. (2016) have reported PAH concentrations in H. leucospilota and Stichopus hermanni (S. hermanni) from the northern parts of Persian Gulf of 12.49–505.44 µg/kg dw and 8.08–89.39 µg/kg dw, respect-ively. Others authors have assessed PAHs in Bohadschia argus (B. argus) (67 µg/kg ww) and H. atra (35 µg/kg ww) caught in Guam Harbours (Denton et al., 2006). Because the PAH levels reported above are referred only to BW and no infor-mation were found regarding the assessment of these com-pounds in other tissues of H. polii, we have compared only BW concentrations from this study with those previously mentioned studies. Results showed that PAHs determined in BW of both sexes of H. polii were below to those reported for H. leucospilota, S. hermanni, B. argus, and H. atra.

Benefit and risk from H. polii consumptionEDI range, calculated on the basis of IR10 and IR100, corres-ponding respectively to 10% and 100% of IR of 106 g/d each person for all seafood in Asiatic countries, is reported in Table 7.

EDIs of PAH4 and B[a]P from this study were compared with the median dietary exposure of PAHs reported by the European Food Safety Authority (ESFA, 2008) for medium and high European consumers. For different food categories, ESFA reported that B[a]P ranged from 3.9 to 6.5 ng/(kg·d) while PAH4 varied between 19.5 and 34.5 ng/(kg·d). In this study, EDI data for PAH4 and B[a]P relate to GD and AC tissues that were above the ESFA intake range for IR100. These findings underline the importance of contam-inants analysis in all animal tissues to obtain a correct es-timation of potential health risks associated with seafood consumption.

THQ and HI values (Table 8), calculated for NAP, ACE, FLU, PHEN, ANTH, PYR, and B[a]P, were always below the unitary limit for all tissues in both sexes, indicating low chronic systemic effects.

As regard carcinogenic risk, the estimated CR posed by B[a]P was determined considering 10 IR values included in the range IR10–IR100 (Figure 4). Compared to the USEPA screening criteria for carcinogenic risk, all tissues analyzed exceeded the acceptable risk level of 1×10-6. In particular, values found in Gd and AC of both sexes, and consequently those obtained for the whole animals, showed CRs>1×10–4 when IRs were equal to or greater than 45 g/d each person; therefore, the ingestion of these tissues poses considerable carcinogenic risks for higher consumers. However, it is im-portant to underline that, in this study, the potential health risks were based on unprocessed seafood. It is known that PAHs may also be formed during cooking and domestic food preparation, such as barbecuing, smoking, drying, grilling, roasting, and frying foods (Oliveira et al., 2018). In particular, the cooking process is the most imperative con-tributor to the formation of B[a]P in foods (Das and Bhutia, 2018). Therefore, further investigation is needed to complete the risk assessment of this seafood.

ConclusionsThis study showed that nutrients in H. polii were affected significantly according to tissue and sex, relatively to the sea cucumbers with the range of weights and at the time of year in which they were sampled.

In general, H. polii contained a higher protein content compared with lipid level, as commonly reported in marine organisms.

All tissues had adequate amounts of most EAAs, such as Thr, Leu, and Phe coupled with low Lys/Arg ratios that have a hypocholesteremic effect. Based on EAA/NEAA ratio, the quality of proteins was higher in Gd and AC and lower in BW. Glycine was the major component of AA.

All tissues of H. polii, female and male, had high-quality lipids with PUFAs as the dominant class of FAs, except for Gd of female, which showed a prevalence of MUFA. MBs and ITu had the higher proportions of PUFAs, which play an important role in ameliorating the risk of certain diseases; the n-3 EPA and n-6 ARA, very important for nutrition, were the most abundant PUFAs, with Gd of male and MBs of both sexes showing the higher proportion. H. polii had good amount of n-3 FAs with beneficial value of the n-3/n-6 ratio, overall, for Gd, AC and RT of both sexes. It was characterized by favorable values of AI, TI, and h/H, although with differ-ences depending on sex and tissue.

Dow



ctober 2022


Tab

le 7

. Cal

cula

ted

expo

sure

dai

ly in

take

(ED

I) by

inge

stio

n of

Hol

othu

ria p

olii

fem

ale

(f) a

nd m

ale

(m) t

issu

es IR

10 a

nd IR

100 i

ndic

ate

the

IR v

alue

s of

10.

6 g/

d an

d 10

6 g/

d ea

ch p

erso

n, c

orre

spon

ding

to

10%

an

d 10

0% o

f FA

O r

ecom

men

ded

IR fo

r th

e A

siat

ic c

ount

ry, r

espe

ctiv

ely

PAH

B

W-f

BW

-mM

B-f

MB

-mIT

u-f

ITu-

mG

d-f

Gd-

mR

T-f

RT

-mA

C-f

AC

-m

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

NA

P0.

32.

90.

44.

10.

22.

40.

22.

30.

43.

80.

43.

50.

44.

10.

43.

60.

55.

30.

54.

70.

22.

10.

44.

2

AC

Y0.

10.

7–

–0.

10.

80.

10.

8–

–0.

10.

90.

33.

00.

43.

90.

21.

70.

21.

50.

22.

40.

33.

0

AC

E–

–0.

10.

90.

21.

60.

21.

6–

––

––

–0.

10.

90.

11.

00.

10.

9–

––

–

FLU

0.5

4.6

0.2

1.8

0.2

1.8

0.2

1.7

0.2

1.6

0.2

1.6

0.7

7.3

0.2

1.5

0.6

5.5

0.5

4.9

0.6

5.9

0.7

7.4

PHE

N0.

54.

80.

43.

60.

54.

70.

54.

50.

65.

50.

43.

51.

111

.30.

44.

00.

55.

00.

54.

50.

99.

21.

111

.4

AN

TH

––

––

0.1

0.6

0.1

0.5

––

0.1

0.6

0.3

3.1

0.2

2.2

0.6

5.6

0.5

5.0

0.3

2.5

0.3

3.1

FLT

H0.

21.

90.

22.

40.

21.

70.

21.

60.

22.

10.

22.

32.

928

.80.

76.

50.

10.

90.

10.

92.

323

.42.

929

.1

PYR

0.2

1.9

0.2

2.4

0.2

2.1

0.2

2.1

0.3

2.6

0.4

4.2

3.2

31.9

0.9

9.4

0.3

2.5

0.2

1.8

2.6

25.9

3.2

32.3

B[a

]A–

–0.

10.

7–

––

–0.

21.

80.

00.

31.

313

.40.

54.

50.

44.

00.

43.

61.

110

.91.

413

.6

CR

Y0.

10.

60.

10.

80.

10.

80.

10.

70.

32.

80.

10.

91.

414

.20.

44.

40.

22.

10.

21.

61.

211

.81.

514

.6

B[b

]F0.

54.

50.

10.

70.

98.

70.

88.

40.

66.

40.

99.

04.

140

.85.

049

.60.

33.

10.

32.

73.

333

.44.

241

.6

B[k

]F0.

11.

30.

22.

20.

43.

90.

43.

80.

32.

90.

66.

22.

019

.82.

121

.10.

21.

80.

21.

61.

817

.52.

221

.8

B[j

]F0.

88.

20.

11.

01.

41.

71.

313

.20.

21.

80.

33.

12.

424

.34.

747

.10.

76.

90.

66.

21.

615

.92.

019

.7

B[e

]P0.

88.

30.

65.

81.

413

.91.

313

.41.

09.

81.

514

.92.

524

.64.

847

.50.

77.

00.

66.

22.

020

.22.

525

.2

B[a

]P0.

33.

20.

32.

60.

44.

20.

44.

10.

44.

20.

44.

13.

130

.53.

029

.50.

55.

20.

54.

62.

525

.13.

131

.2

D[a

h]A

––

––

0.1

1.1

0.1

1.0

––

0.1

0.9

0.4

3.9

0.5

5.0

––

––

0.3

3.2

0.4

3.9

IND

0.5

4.7

0.4

3.5

1.2

12.1

1.2

11.7

0.6

5.5

1.1

11.2

3.1

31.3

5.8

58.3

0.4

4.0

0.4

3.6

2.6

25.5

3.2

31.7

B[g

hi]P

1.0

10.3

0.8

7.8

1.9

18.6

1.8

17.9

0.9

9.4

1.8

17.7

3.3

32.9

6.7

66.5

0.8

7.7

0.7

6.9

2.7

26.7

3.3

33.2

∑PA

Hs

5.8

57.8

4.0

40.1

9.3

92.6

8.9

89.0

6.0

60.2

8.5

84.9

32.5

325

36.6

366

6.9

69.4

6.1

61.2

26.2

262

32.7

327

∑PA

H4

0.8

8.3

0.5

4.7

1.4

13.6

1.4

13.5

1.5

15.2

1.4

14.3

9.9

98.8

8.8

88.0

1.4

14.3

1.3

12.5

8.1

81.1

10.1

101

The

uni

t fo

r E

DI

is n

g/(k

g·d)

.PA

Hs,

pol

ycyc

lic a

rom

atic

hyd

roca

rbon

s; I

R, f

ood

inge

stio

n ra

te; F

AO

, Foo

d an

d A

gric

ultu

re O

rgan

izat

ion

of t

he U

nite

d N

atio

ns; N

AP,

nap

htha

lene

; AC

Y, a

cena

phth

ylen

e; A

CE

, ace

naph

then

e; F

LU

, fluo

rene

; PH

EN

, phe

nant

hren

e; A

NT,

ant

hrac

ene;

FLT

H, fl

uora

nthe

ne; P

YR

, pyr

ene;

B[a

]A, b

enzo

[a]a

nthr

acen

e; C

HR

Y, c

hrys

ene;

B[b

]F, b

enzo

[b]fl

uora

nthe

ne; B

[k]F

, ben

zo[k

]fluo

rant

hene

; B[j

]F, b

enzo

[j]fl

uora

nthe

ne; B

[e]

P, b

enzo

[e]p

yren

e; B

[a]P

, ben

zo[a

]pyr

ene;

B[g

hi]P

, ben

zo[g

, h, i

]per

ylen

e; I

ND

, ind

eno[

1,2,

3-c,

d]py

rene

; D[a

h]A

, dib

enz[

a, h

]ant

hrac

ene.

∑PA

Hs

is t

he s

um o

f 18

PA

Hs.

∑PA

H4

is t

he s

um o

f B

[a]P

, B[a

]A, B

[b]F

, and

C

HR

Y. B

W, b

ody

wal

l; M

B, m

uscl

e ba

nd; I

Tu, i

nter

nal t

unic

; Gd,

gon

ad; A

C, a

limen

tary

can

al; R

T, r

espi

rato

ry t

ree.

Dow



ctober 2022


Tab

le 8

. TH

Q a

nd H

I by

cons

umpt

ion

of H

olot

huria

pol

ii fe

mal

e (f

) and

mal

e (m

) tis

sues

Tis

sue

Sex

TH

Q-N

AP

TH

Q-A

CE

TH

Q-F

LU

TH

Q-A

NT

HT

HQ

-PH

EN

TH

Q-P

YR

TH

Q-B

[a]P

HI

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

IR10

IR

100

BW

f1.

41E

–05

1.41

E–0

4–

–1.

11E

–05

1.11

E–0

4–

–4.

56E

–06

4.56

E–0

56.

08E

–06

6.08

E–0

51.

04E

–03

1.04

E–0

21.

08E

–03

1.08

E–0

2

m1.

97E

–05

1.97

E–0

41.

42E

–06

1.42

E–0

54.

26E

–06

4.26

E–0

5–

–5.

85E

–06

5.85

E–0

53.

85E

–02

3.85

E–0

12.

44E

–04

2.44

E–0

33.

88E

–02

3.88

E–0

1

MB

f1.

18E

–05

1.18

E–0

42.

62E

–06

2.62

E–0

54.

37E

–06

4.37

E–0

51.

83E

–07

1.83

E–0

64.

11E

–06

4.11

E–0

56.

90E

–06

6.90

E–0

51.

36E

–03

1.36

E–0

21.

39E

–03

1.39

E–0

2

m1.

14E

–05

1.14

E–0

42.

52E

–06

2.52

E–0

54.

21E

–06

4.21

E–0

51.

77E

–07

1.77

E–0

63.

96E

–06

3.96

E–0

56.

65E

–06

6.65

E–0

52.

89E

–04

2.89

E–0

33.

18E

–04

3.18

E–0

3

ITu

f1.

87E

–05

1.87

E–0

4–

–3.

80E

–06

3.80

E–0

5–

–5.

22E

–06

5.22

E–0

58.

34E

–06

8.34

E–0

51.

38E

–03

1.38

E–0

21.

41E

–03

1.41

E–0

2

m1.

72E

–05

1.72

E–0

4–

–3.

83E

–06

3.83

E–0

5–

–5.

57E

–06

5.57

E–0

51.

36E

–05

1.36

E–0

41.

31E

–03

1.31

E–0

21.

35E

–03

1.35

E–0

2

Gd

f2.

00E

–05

2.00

E–0

4–

–1.

78E

–05

1.78

E–0

49.

99E

–07

9.99

E–0

67.

00E

–05

7.00

E–0

41.

03E

–04

1.03

E–0

39.

87E

–03

9.87

E–0

21.

01E

–02

1.01

E–0

1

m1.

75E

–05

1.75

E–0

41.

42E

–06

1.42

E–0

53.

70E

–06

3.70

E–0

57.

02E

–07

7.02

E–0

69.

01E

–03

9.01

E–0

23.

04E

–05

3.04

E–0

49.

57E

–03

9.57

E–0

21.

86E

–02

1.86

E–0

1

RT

f2.

59E

–05

2.59

E–0

41.

57E

–06

1.57

E–0

51.

34E

–05

1.34

E–0

41.

83E

–06

1.83

E–0

52.

15E

–06

2.15

E–0

58.

06E

–06

8.06

E–0

51.

67E

–03

1.67

E–0

21.

73E

–03

1.73

E–0

2

m2.

31E

–05

2.31

E–0

41.

40E

–06

1.40

E–0

51.

19E

–05

1.19

E–0

41.

63E

–06

1.63

E–0

52.

15E

–06

2.15

E–0

55.

72E

–06

5.72

E–0

51.

49E

–03

1.49

E–0

21.

54E

–03

1.54

E–0

2

AC

f1.

02E

–05

1.02

E–0

4–

–1.

44E

–05

1.44

E–0

48.

11E

–07

8.11

E–0

65.

69E

–05

5.69

E–0

48.

40E

–05

8.40

E–0

48.

11E

–03

8.11

E–0

28.

28E

–03

8.28

E–0

2

m2.

02E

–05

2.02

E–0

4–

–1.

80E

–05

1.80

E–0

41.

01E

–06

1.01

E–0

57.

08E

–05

7.08

E–0

41.

04E

–04

1.04

E–0

31.

01E

–02

1.01

E–0

11.

03E

–02

1.03

E–0

1

IR10

and

IR

100

indi

cate

the

IR

of

10.6

g/d

and

106

g/d

eac

h pe

rson

, cor

resp

ondi

ng t

o 10

% a

nd 1

00%

of

FAO

rec

omm

ende

d IR

for

the

Asi

atic

cou

ntry

, res

pect

ivel

y.T

HQ

, Tar

get

Haz

ard

Quo

tien

t; H

I, H

azar

d In

dex;

IR

, foo

d in

gest

ion

rate

; FA

O, F

ood

and

Agr

icul

ture

Org

aniz

atio

n of

the

Uni

ted

Nat

ions

; NA

P, n

apht

hale

ne; A

CE

, ace

naph

then

e; F

LTH

, fluo

rant

hene

; AN

T,

anth

race

ne; P

HE

N, p

hena

nthr

ene;

PY

R, p

yren

e; B

[a]A

, ben

zo[a

]pyr

ene;

BW

, bod

y w

all;

MB

, mus

cle

band

; ITu

, int

erna

l tun

ic; G

d, g

onad

; AC

, alim

enta

ry c

anal

; RT,

res

pira

tory

tre

e.

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ctober 2022


In conclusion, all edible portions of H. polii, particularly Gd, AC, and RT can contribute with different percentage to meet human nutritional needs.

As a result, the consumption of sea cucumber from this geo-graphic location, sampled at this time (October–November), determined a low risk to develop chronic systemic effects for PAHs, but the cancer risk due to the presence of B[a]P could be of health concern, especially for high-frequency consumers of this seafood. Because the risk posed from H. polii investi-gated in this study is related especially to Gd and AC, it would be safe to consume sea cucumber without these tissues or with low consumption rate. On the contrary, from the nutri-tional point of view, Gd and AC were the tissues providing the highest EPA+DHA overall in female, while the maximum value in male was reached in RT. This opposite trend suggests a moderate Gd and AC consumption than other tissue of H. polii in order to balance beneficial properties from EPA+DHA and risk linked to carcinogenic PAHs. In addition, because of the increased market demand for sea cucumber around the world, further investigations on dietary habits and cooking process of this seafood are needed to obtain accurate informa-tion on the carcinogenic risks associated with its consumption.

The findings obtained will make a significant contribution to fill the gaps in existing information, because this is, to the best of our knowledge, the first study documenting the nu-trient and PAHs in tissues of the Mediterranean sea cucumber, H. polii both in female and male.

Author ContributionsFrancesca Biandolino and Santina Giandomenico: Conceptualization, carried out part of the experiments, in-terpreted the results, and wrote the manu script; Ermelinda Prato: Conceptualization, planned the experiments, wrote the manuscript, resources, and project administration; Isabella

Parlapiano and Antonella Di Leo: Contributed to the ex-periment performance; Lucia Spada: Contributed to the data analysis; Giovanni Fanelli: Fields activity and data ana lysis. All authors read and approved the manuscript.

FundingThis research received no external funding.

Conflict of InterestThe authors declare no conflict of interest.

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