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marinedrugs Marine Drugs Mar. Drugs Marine Drugs 1660-3397 MDPI 10.3390/md10122676 marinedrugs-10-02676 Article Characterization and Cytotoxicity Studies of the Rare 21:4 n-7 Acid and Other Polyunsaturated Fatty Acids from the Marine Opisthobranch Scaphander lignarius, Isolated Using Bioassay Guided Fractionation Vasskog Terje 1 2 Andersen Jeanette H. 3 Hansen Espen 3 Svenson Johan 4 * Norut—Northern Research Institute, PO Box 6434, Tromsø Science Park, N-9294 Tromsø, Norway; Email: Department of Pharmacy, University of Tromsø, Breivika, N-9037 Tromsø, Norway Marbio, University of Tromsø, Breivika, N-9037 Tromsø, Norway; Email: (J.H.A.); (E.H.) Department of Chemistry, University of Tromsø, Breivika, N-9037 Tromsø, Norway Author to whom correspondence should be addressed; Email:; Tel.: +47-776-45505; Fax: +47-776-44765. 26 11 2012 12 2012 10 12 2676 2690 09 10 2012 12 11 2012 15 11 2012 © 2012 by the authors; licensee MDPI, Basel, Switzerland. 2012

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (

The marine opisthobranch Scaphander lignarius has been analyzed in the systematic search for novel bioactive compounds in Arctic marine organisms using bioassay guided fractionation. A number of highly cytotoxic fractions were shown to contain mainly polyunsaturated fatty acids (PUFAs). Selected PUFAs were isolated and identified using both liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). It was shown that the opisthobranch contained unusual PUFAs such as several ω3 fatty acids and the ω7 heneicosa-5,8,11,14-tetraenoic acid (21:4 n-7) not isolated before. The organism was shown to be a very rich source of PUFAs and the activity of the isolated compounds against a range of human cancer cell lines (melanoma, colon carcinoma and breast carcinoma) is further reported. The ω7 PUFA was significantly more cytotoxic in comparison with reference ω6 arachidonic and ω3 eicosapentaenoic acid. A noteworthy non-selective cytotoxicity against normal lung fibroblasts was also established. The paper contains isolation protocols in addition to cytotoxicity data of the isolated compounds. The potential of marine mollusks as a source for rare PUFAs is also discussed.

opisthobranch cytotoxic PUFA Scaphander lignarius ω7 fatty acid
1. Introduction

The marine environment is highly competitive and being able to produce compounds providing an adaptive advantage is highly beneficial for any marine organism. Such compounds are fundamentally interesting to study but they also represent entities with significant potential for several industrial and medical applications as they have been chemically optimized during millions of years of evolution and selection [1]. Marine mollusks represent a particularly promising group of organisms in the search for new bioactive compounds. In fact, the majority (35%) of the 20 marine natural products currently approved as drugs or in clinical trials have their origin in collected mollusks [2,3]. Most marine drugs are used in the treatment of different types of cancer although other disease states such as schizophrenia, Alzheimer’s and hypertriglyceridemia are also represented.

The marine organisms inhabiting the long and twisting coastline surrounding the northern parts of Norway are currently being investigated in a large scale search for novel compounds for drug discovery and development [4]. Numerous mollusks have been gathered and analyzed in the course of the methodical collection and screening of Arctic marine organisms. Bioassay guided fractionation is employed to allow isolation of compounds displaying promising bioactivities. Recently, an organic extract of the opisthobranch Scaphander lignarius generated fractions with high cytotoxic activities (Figure 1).

Results from the primary screening of the Scaphander lignarius acetonitrile extract against human A2058 melanoma cells. The compounds in the current study were isolated from fractions 26–32.

The active components in the fractions were shown to be a group of PUFAs, present in high concentrations. Marine mollusks and invertebrates are rich in lipids and they have been extensively studied over the years and comprehensively reviewed by Bergé and Barnathan [5]. Numerous bioactive fatty acids have been shown to display both antimicrobial [6] and cytotoxic bioactivities in addition to their perhaps more established ability to alleviate the burden and lower the risk of cardiac failure and obesity [7,8,9,10]. In addition to displaying cytotoxic activities on their own, several PUFAs have also been reported to synergistically potentiate the efficacy of established anticancer drugs through several mechanisms [11,12,13]. One of the seven food and drug administration (FDA) approved drugs of marine origin today is a mixture of ω3-acid ethyl esters (Lovaza®/Omacor®) displayed in Figure 2, used to treat hypertriglyceridemia and cardiovascular disease and illustrates the pharmaceutical potential of fatty acids of marine origin.

Chemical structures of the fatty acid ethyl esters of: (A) eicosapentaenoic acid (EPA) and (B) docosahexaenoic acid (DHA), the main active compounds in Lovaza®/Omacor®.

Any biological source able to generate large amounts of unusual, biologically active fatty acids is important to further study as their biological properties are poorly known [14]. In the current investigation, several common PUFAs and an unusual ω7 PUFA (not previously isolated) were identified in a Scaphander lignarius extract. Three representative PUFAs, arachidonic acid (20:4 n-6) (ARA), eicosapentaenoic acid (20:5 n-3) (EPA) and the rare heneicosa-5,8,11,14-tetraenoic acid (21:4 n-7) (HTA) were isolated and selected for dose response cytotoxicity studies against a set of human cancer cell lines and structure activity relationship (SAR) establishment. The current paper describes the isolation and cytotoxic bioactivities of these selected PUFAs against several human cancer cell lines. It represents the first study of the fatty acid composition of Scaphander lignarius and the first assessment of the cytotoxic properties of an oddly numbered ω7 PUFA.

2. Results and Discussion

The opisthobranches represent a large group of marine mollusks belonging to the gastropods with wide geographic distribution and diverse appearances [15]. The opisthobranch Scaphander lignarius in the current study has previously been described as an abundant Mediterranean mollusk [16] but it clearly has a much wider geographical distribution as the biological material used in the current study was collected some 70 degrees north in Arctic waters. It has previously been show to contain a family of conjugated polyketides known as lignarenones, shown in Figure 3, suggested by Cimino to function as alarm pheromones [17,18].

The opisthobranch Scaphander lignarius and the chemical structures of the polyketides: (A) lignarenone A and (B) lignarenone B previously isolated from it.

2.1. Isolation and Structural Determination

Based on what has previously been reported about the content of Scaphander lignarius it was anticipated that potentially new lignarenone derivatives could be accountable for the cytotoxic activity in the extract as polyketides have been shown to display a wide range of biological activities [19,20]. Three of the major constituents (13) found in the active fractions (fractions 26–32 in Figure 1) were isolated from the organic extract. MS-Data was not solely used to unambiguously identify the compounds although the accurate masses of the major compounds could be tentatively assigned to PUFAs. The final identity of the bioactive compounds was established employing NMR analysis. While several 1H signals at a glance could correlate to a lignarenone-like structure this was ruled out during closer inspection of the 1H NMR data which displayed the “classic” set of PUFA signals (Figure 4). 1 and 2 were rapidly assigned to ARA and EPA ([M − H] 303.2339, calculated 303.2324 and 301.2186, calculated 301.2168, respectively). The NMR spectra of 1 and 2 could be used to establish the location of the extra methylene in 3 ([M − H] 317.2497, calculated 317.2481) using 1H and two-dimensional correlation spectroscopy (COSY) experiments by observing differences in the number of “F” methylene protons displayed below in Figure 4 and 3 was subsequently identified as HTA. The NMR spectra of ARA and HTA were, apart from the difference in number of “F” methylene protons, identical. The structure of compounds 13 are presented in Figure 5.

2D COSY spectrum and the atomic labeling of the characteristic PUFA proton signals (arachidonic acid (20:4 n-6) (ARA)) illustrating the correlations between the “F” protons and the terminal “G” methyl group and the “D” protons. Insert from heneicosa-5,8,11,14-tetraenoic acid (21:4 n-7) (HTA) COSY illustrates the differences in “F” intensity allowing for accurate placement of the additional methylene in HTA.

Structure of the isolated PUFAs: (1) EPA; (2) ARA and (3) HTA.

The fatty acid profile of the extract was further established once the isolation and structural elucidation revealed PUFAs as the source of the cytotoxic bioactivity and is compiled in Table 1.

marinedrugs-10-02676-t001_Table 1

Major fatty acid constituents in Scaphander lignarius.

Fatty acid Monoisotopic mass Relative area %
14:1 226.2016 137 0.03
16:1 254.2328 1587 0.31
18:1 282.2640 1026 0.20
20:1 310.2952 1182 0.23
18:2 280.2484 311 0.06
20:2 308.2796 642 0.13
22:2 336.3108 210 0.04
16:3 250.2016 883 0.17
18:3 278.2328 787 0.16
20:3 306.2640 660 0.13
18:4 276.2172 1970 0.39
20:4 n-6 304.2484 88921 17.53
21:4 n-7 318.2640 46741 9.22
22:4 (2 isomers) 332.2796 6986 + 8824 3.12
20:5 n-3 302.2328 201409 39.72
22:5 (3 isomers) 330.2640 31588 + 4406 + 4295 7.95
22:6 328.2484 104560 20.61
Sum 507123 100.00

a Non-Methylene-Interrupted dienoic fatty acids.

The fatty acid distribution profile of the Scaphander lignarius extract corresponds well with that of other marine mollusks [21,22]. The majority of the fatty acids are PUFAs with ARA, EPA and DHA being the most prevalent accounting for nearly 80% of the fatty acid content. A low relative content of the less common NMIDs has been reported in other marine mollusks even though they can be a rich source [22,23]. Of particular interest is the high content of HTA (9.22%) which has mainly only been reported in minute amounts before and not been isolated. It is clear that this rare ω7 fatty acid play an important role in Scaphander lignarius. Unexpectedly, no saturated fatty acids were detected which may be a reflection of the methods used to extract the organism. The semi-automated high-throughput bioassay guided fractionation strategy employed is focused on isolating drug-like compounds of certain molecular mass and polarity [4]. Such a strategy is necessary in order to analyze the vast number of organisms collected, clearly at the potential expense of overlooking interesting compounds not fulfilling the molecular requirements for isolation. These challenges associated with high-throughput isolation of natural products are established [24]. No attempts were thus made at isolating additional fatty acids potentially discarded during the extraction steps and the fatty acid distribution presented in Table 1 is only representative for the aqueous acetonitrile fraction as described in the isolation section. Ion chromatograms of the isolated PUFAs are presented in Figure 6.

Ion chromatograms of the three isolated PUFAs: Top, HTA (m/z 317.2497), middle, ARA (m/z 303.2338) and bottom EPA (m/z 301.2188).

2.2. Cytotoxicity

It is well-established that a regular intake of PUFA rich food can lower the risk of obesity, cardiac failure and certain cancers types [5,25,26]. The association between a high intake of ω3 PUFAs and a reduction of major cardiovascular disease events is nevertheless currently under significant debate [27]. In addition, both saturated fatty acids and PUFAs have been intensively studied as anticancer drugs [28,29]. Several modes of action are suggested in the literature with increased lipid peroxidation, modulation of the cyclooxygenase-2/prostaglandin E2 (COX/PGE2) pathway and inhibition of topoisomerases, crucial for topological DNA processing, being well-established cellular targets [29,30]. A certain group of fatty acids, the non-methylene-interrupted fatty acids (NMI-FA) are particularly common in marine organisms and they have recently been shown to display interesting biological activities [23,31]. These compounds were however only present in minute amounts in the Scaphander lignarius extract which prevented isolation and accurate analysis of double bond placement.

Due to a lowered enzymatic ∆6- and ∆5-desaturase activity, certain tumor types are deficient in PUFAs and this compositional difference can allow cytotoxic PUFAs to be used as selective cytotoxic agents [32]. Yoshida hepatoma cells does for example only contain half the amount of ARA in comparison with normal hepatocytes [30]. Exposure to elevated concentrations of ARA, EPA and γ-linolenic acid have all been shown to induce tumoral apoptosis by increasing the lipid peroxidation and subsequent generation of free radicals such as the highly reactive superoxide anion and hydrogen peroxide [33,34,35].

Tissue selectivity is of major concern for any cytotoxic agent and several studies have shown that PUFAs indeed are toxic to “normal” cells, in addition to tumor cells at sufficiently high concentrations. Das have reported toxicity values (50% dead cells after 7 days incubation) for EPA and ARA against normal human fibroblasts (CCD-41-SK) at ~60 µg/mL and ~30 µg/mL respectively. Those concentrations were some 2–3 times higher than the concentration needed to kill 100% of human breast tumor cells (ZR-75-1) [36]. Stonik et al. further reported significant hemolytic activity at concentrations as low as 1.5 µg/mL for the rare (5Z,9Z)-22-methyl-5,9-tetracosadienoic acid isolated from the marine sponge Geodinella robusta [37]. Nevertheless, most studies suggest an increased sensitivity to PUFAs for tumor cells in comparison with normal cells. In addition, a number of novel tumor delivery strategies, such as intratumoral injections, PUFA/growth factor conjugation and PUFA/cancer drug complexes are under development to allow selective tumoral delivery [30,38] of PUFAs.

Three of the major constituents in the Scaphander lignarius extract, ARA, EPA and HTA were chosen for further cytotoxicity studies against a set of cancer and normal cell lines as described in the methods section. By including an ω3, an ω6 and an ω7 PUFA in the cytotoxicity studies, it is conceivable to establish the potential structure activity relationship (SAR). Despite its abundance, DHA was not isolated as EPA was already included as representative ω3 PUFA. ARA and EPA are well studied PUFAs while cytotoxicity studies of the rare ω7 HTA has not been described in the literature before [5,28]. All the compounds were able to kill cancer cells in a dose dependent manner exemplified below in Figure 7 for A2058 cells. From the dose-response curves it was possible to calculate the IC50 values for the individual PUFAs and those figures are presented in Table 2.

Illustration of the dose dependent PUFA induced killing of A2058 melanoma cells by EPA (-∆-), ARA (-x-) and HTA (-o-).

marinedrugs-10-02676-t002_Table 2

Cytotoxicity of ARA, EPA and HTA against selected cell lines.

Cell line 50% Cytotoxicity (µg/mL)
Normal lung fibroblast (MRC5) 90 90 42
Colon carcinoma (HT29) 93 76 41
Melanoma (A2058) 45 43 31
Breast carcinoma (MCF7) 66 68 44

All the studied PUFAs were shown to be toxic to both tumor and normal cells. The IC50 values were slightly higher than those reported for EPA and ARA by Das against normal fibroblasts and breast tumors [36]. Those studies were however performed with significantly longer cellular exposure times in comparison with the current investigation. The IC50 figures indicate that HTA is nearly twice as cytotoxic in comparison to EPA and ARA. A preference for tumoral cells over normal cells was seen for both EPA and ARA. This cellular selectivity was most pronounced for the melanoma (A2058) and breast carcinoma (MCF7) cells. A lower content of super oxide dismutase in certain cancer cells has been suggested as an explanation of the differences in cellular sensitivity to PUFAs [36]. HTA displayed no obvious cellular selectivity.

The structural differences between the three investigated PUFAs are minor and lie mainly in the number and placement of the double bonds. HTA with its extra methylene contains an additional carbon while EPA contains five double bonds. These differences are reflected in their hydrophobicity and theoretical logP coefficients which range from 5.85, 6.16 to 6.58 for EPA, ARA and HTA, respectively. The calculated logP values (calculated using Chemdraw ultra 10.0) correlate well with their experimental C-18 retention times: 6.63, 6.99 and 7.29 min respectively for EPA, ARA and HTA. The uptake and distribution of fatty acids differ between normal cells and tumor cells and it has previously been shown to influence the extent of cellular death. The increased hydrophobicity appears to provide HTA with augmented cytotoxicity. The lack of distinction between normal cells and tumor cells further implies that the mode of action may be dictated by a feature or mechanism that is mutual in both normal and tumoral cells. A plausible common target could be the cellular membrane. Being an exogenic PUFA, its membrane insertion is likely to yield membrane disruption once a threshold concentration has been reached. Established intracellular events such as increased lipid peroxidation and superoxide radical generation are also possible [29]. The higher hydrophobicity may further allow an increased cellular uptake of HTA compared to EPA and ARA which could be an additional contribution to the apparent higher cytotoxicity. Insufficient amounts of HTA prevented mechanistic investigations such as co-incubation with antioxidants and calcein leakage studies.

HTA has been identified in minute amounts by MS in extracts of cold-seep mussels and in the deep sea-vent crab Shinkaia crosnieri, by Saito [22,39] and in an extract from the sponge Hymeniacidon sanguinea [40] but it has never been isolated and biologically tested. Very recently, HTA was reported to be a main constituent (7.5% of total fatty acids) of an Australian thraustochytrid (Thraustochytrium sp.) from south east Tasmania [41]. The fatty acid composition of 36 different thraustochytrid strains was evaluated as sources for the production of biodiesel and 12 of those where shown to contain HTA. Of those 12, only one contained HTA concentrations comparable to Scaphander lignarius. Being an oddly numbered ω7 PUFA it represents an interesting target for studies as it may yield additional insight into the role and potential of PUFAs as cytotoxic agents. Oddly numbered fatty acids are generally rare in Nature and hence less studied as cytotoxic agents both due to the low abundance but also on the inherent difficulties associated with their detection and identification [14,42].

PUFAs are produced via several biosynthetic pathways depending on substrate availability [41,43]. In addition to the conventional elongation and desaturation pathway they may also utilize the polyketide synthase pathway. Oddly numbered PUFAs have been isolated from crabs, crayfish and bivalves and it has been suggested that they have been accumulated in these organisms from their diet [14]. Oddly numbered PUFAs have for this reason been heralded as useful biomarkers for marine food web studies [43]. That is a likely scenario also for Scaphander lignarius and the high HTA content may be a reflection of a local diet rich in HTA producing microorganisms. The data presented in the current study also indicate that they may be useful cytotoxic agents given their increased cytotoxic activity. Marine mollusks appear to be the richest source so far encountered and further studies will reveal if these compounds are endogenous or a result of their diet and local surrounding conditions.

During the course of the screening process, the PUFA extract was also screened against a set of bacterial strains composed of Escherichia coli, Staphylococcus aureus, Methicillin resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis. No apparent bioactivity against any of the tested strains was seen (data not shown).

3. Experimental Section 3.1. Collection

The biological material was collected by Agassiz dredging at a depth of 204 m in the Porsangerfjord (70°48′N, 26°10′E) in February 2008. The samples were taxonomically identified on board and stored at −23 °C until further used. Voucher specimen deposited at the national biobank Marbank, Norway.

3.2. Extraction and Fractionation

249.5 g of freeze-dried sample of Scaphander lignarius (organism wet weight: 1081.5 g) was ground and extracted twice with ultra-pure water (Milli-Q, Millipore) (24 h and 30 min) at 5 °C in the dark. After centrifugation and supernatant removal, the remaining sediment was freeze-dried, ground, and extracted twice with a 1:1 (v:v) mixture of dichloromethane and methanol (MeOH) (24 h and 30 min) at 5 °C in the dark. The mixture was further vacuum filtered through a Whatman (Ø 125 mm nr. 3) filter (Little Chalfont, UK) and the resulting supernatant was reduced to a concentrated organic extract (27.0 g) under vacuum. 300 mg of the extract was dissolved in 3 mL of hexane and extracted twice with a mixture of 90% acetonitrile and 10% purified water. The acetonitrile phases were collected and reduced to a concentrated liquid which was diluted with 50% aqueous acetonitrile (v:v) and fractionated into 40 fractions on HPLC using a Waters XTerra C18 column (10 µm particle size, 10 × 300 mm) employing a linear gradient from 20% to 100% acetonitrile in water (both containing 0.05% formic acid) over 30 min, and then holding at 100% acetonitrile for 10 min. The flow was set to 6 mL/min, and 40 one-minute fractions were collected. The HPLC-system consisted of a 2767 solvent manager, a 2996 photodiode array UV-detector and 600E pump, and was controlled by MassLynx 4.1 software and the FractionLynx application manager (Waters). The fractions were screened against A2058 cells according to the methodology described in paragraph 3.5 below and shown in Figure 1.

3.3. Isolation

10 g of the organic extract was used for the isolation of the fatty acids which was performed on a Waters (Milford, MA, USA) 2695 separation module employing two different gradients and columns. The initial separation was performed on a Waters Xbridge BEH C-18 OBD preparative column (5 μm particle size, 19 × 250 mm). The mobile phases consisted of A: 0.02% NH3 in purified water, and B: 90% acetonitrile, 10% purified water (containing 0.02% NH3). The fatty acids were collected with a Waters fraction collector II after detection with a Waters 2998 photo diode array detector. Mass spectrometric analysis of the fractions revealed that the fractions contained impurities, and a second clean-up step was necessary. The second preparative HPLC run was performed on a Waters Xbridge BEH C-18 OBD preparative column (5 μm particle size, 10 × 250 mm,). The mobile phases consisted of A: 0.1% trifluoroacetic acid (TFA) in purified water, and B: 90% acetonitrile, 10% purified water and 0.1% TFA. The gradients used for isolation are presented in Table 3. Amount PUFA isolated: EPA, 9.5 mg, ARA, 7.5 mg and HTA, 1.5 mg.

marinedrugs-10-02676-t003_Table 3

Chromatography gradients employed for FA isolation.

Time (min) Flow (mL/min) A (%) B (%)
Gradient 1 a 0 3 90 10
0.5 10 90 10
30 10 40 60
35 10 5 95
45 10 5 95
Gradient 2 b 0 3 50 50
0.5 7 50 50
30 7 10 90
40 7 10 90

a A: 0.02% NH3 in purified water, and B: 90% acetonitrile, 10% purified water (containing 0.02% NH3); b A: 0.1% TFA in purified water, and B: 90% acetonitrile, 10% purified water and 0.1% TFA.

3.4. Structure Determination

The structures of the isolated PUFAs were confirmed using ultra-performance liquid chromatography (UPLC)-HR-MS analysis and nuclear magnetic resonance (NMR) spectroscopy. UPLC-HR-MS analysis was performed on a Waters Acquity I-class UPLC and a Waters Xevo G2 Q-ToF mass spectrometer (see further details below). The data was processed using MassLynx software version 4.1. NMR spectra were obtained on samples dissolved in MeOH-d4 (Sigma) on a Varian 400 MHz Spectrometer (Palo Alto, USA). One-Dimensional 1H and 13C NMR experiments, and two-dimensional 1H-1H COSY experiments were performed to verify the structures. MestreNova 5.2.4 software was used to process the data. EPA: 1H NMR (400 MHz, cd3od) δ 5.35 (m, 10H), 2.84 (m, 8H), 2.29 (t, J = 7.5 Hz, 2H), 2.11 (m, 4H), 1.66 (p, J = 7.4 Hz, 2H), 0.90 (t, J = 6.8 Hz, 3H). ARA: 1H NMR (400 MHz, cd3od) δ 5.36 (m, 8H), 2.83 (m, 6H), 2.30 (t, J = 7.5 Hz, 2H), 2.14 (q, J = 7.4 Hz, 2H), 2.07 (q, J = 7.4 Hz, 2H), 1.67 (p, J = 7.4 Hz, 2H), 1.32 (m, 6H), 0.90 (m, 3H). HTA: 1H NMR (400 MHz, cd3od) δ 5.36 (m, 8H), 2.83 (m, 6H), 2.27 (t, J = 7.5 Hz, 2H), 2.13 (q, J = 7.4 Hz, 2H), 2.07 (q, J = 7.4 Hz, 2H), 1.67 (p, J = 7.4 Hz, 2H), 1.35 (m, 8H), 0.90 (t, J = 6.8 Hz, 3H).

The fatty acid distribution profile was determined by UPLC-MS on a Waters Xevo G2 Q-ToF with negative electrospray ionization. The separation was performed on a Waters Acquity UPLC H-class, fitted with a Waters Acquity UPLC BEH C18 column (1.7 µm particle size, 2.1 × 100 mm) Mobile phase A consisted of H2O + 0.1% formic acid, and mobile phase B of acetonitrile + 0.1% formic acid. A gradient elution was employed, starting at 5% B, with a linear increase to 95% B at 7 min. 95% B was maintained for 3 min and the total runtime was 10 min. A similar ionization efficiency of all the fatty acids is a prerequisite for the given distribution profile. Identification of the fatty acids not isolated was based on retention times, elemental composition calculated by the MassLynx 4.1 software and the characteristic neutral losses of 18, 44, 78 and 98 during MS/MS fragmentation which confirmed the presence of the respective fatty acids.

3.5. Cytotoxicity Assays

The effect of the PUFAs on cell viability was tested against three cancer cell lines: Human melanoma (A2058, ATCC: CRL-1145™), human breast carcinoma (MCF7, ATCC: HTB-22™), and human colon carcinoma (HT29, ATCC: HTB-38™). In addition, non-malignant lung fibroblasts (MRC5, ATCC: CCL-171™) were used as toxicity control. Cell lines, suspended in RPMI-1640 or E-MEM medium (Biochrom AG) with 10% fetal bovine serum medium and 10 µg/mL gentamicin, were seeded in 96-well microtitre plates at 2,000 cells/well. The cell lines were incubated for 24 h before PUFAs (concentration range 6.25–100 µg/mL) were added and then incubated for 72 h at 37 °C in a humidified atmosphere of 5% CO2. The PUFAs were added to the cells in triplicate. Cell viability was determined by a colorimetric [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay. At the end of the exposure time, 10 µL Cell Titer 96® Aqueous One Solution Reagent (Promega, USA) was added to each well, and the plates were incubated for 1 h before absorbance was measured using a DTX multimode detector (Beckman Coulter, CA, USA) at 485 nm. Cells in RPMI-1640 medium were used as negative control, and cells treated with Triton® X-100 (Sigma-Aldrich) reagent was used as positive control. Relative cellular survival was determined by using the measured optical density (OD) and was calculated as follows: Cell survival (%) = (OD treated well − OD positive control)/(OD negative control − OD positive control) × 100.

4. Conclusions

The fatty acid composition in an extract of Scaphander lignarius collected in Arctic waters has been investigated. The mollusk was rich in ARA, EPA, DHA and the unusual ω7 HTA. Selected PUFAs were isolated and their cytotoxic activity was evaluated against a range of adherent human cancer cell lines. HTA was shown to be a significantly more potent cytotoxic agent than ARA and EPA. The cytotoxicity was not selective and normal fibroblasts were equally sensitive to the increased cytotoxicity of HTA. Tumor selective delivery systems would have to be employed to benefit from the cytotoxic properties of HTA without collateral damage to surrounding tissue. This is the first study of the cytotoxic action of an ω7 PUFA isolated from a particularly rich marine source.

Robert Andre Johansen is acknowledged for proving the photograph of Scaphander lignarius. The study was performed at MabCent which is a Centre at the University of Tromsø and supported by the Research Council of Norway, Grant no 174885/130.

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Ponder W. Valdes A. Waren A. Classification and nomenclator of gastropod families Malacologia 2005 47 1 368 Cutignano A. Avila C. Domenech-Coll A. d’Ippolito G. Cimino G. Fontana A. First biosynthetic evidence on the phenyl-containing polyketides of the marine mollusc Scaphander lignarius Org. Lett. 2008 10 2963 2966 Cimino G. Spinella A. Sodano G. Potential alarm pheromones from the mediterranean opisthobranch Scaphander lignarius Tetrahedron Lett. 1989 30 5003 5004 10.1016/S0040-4039(01)80566-9 Cutignano A. Avila C. Rosica A. Romano G. Laratta B. Domenech-Coll A. Cimino G. Mollo E. Fontana A. Biosynthesis and cellular localization of functional polyketides in the gastropod mollusc Scaphander lignarius Chembiochem 2012 13 1759 1766 10.1002/cbic.201200287 Newman D.J. Cragg G.M. Natural products as sources of new drugs over the last 25 years J. Nat. Prod. 2007 70 461 477 10.1021/np068054v Paterson I. Findlay A.D. 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Yanagita T. Conjugated fatty acids in food and their health benefits J. Biosci. Bioeng. 2005 100 152 157 10.1263/jbb.100.152 Rizos E.C. Ntzani E.E. Bika E. Kostapanos M.S. Elisaf M.S. Association between omega-3 fatty acid supplementation and risk of major cardiovascular disease events: A systematic review and meta-analysis JAMA 2012 308 1024 1033 10.1001/2012.jama.11374 Das U.N. Gamma-linolenic acid, arachidonic-acid, and eicosapentaenoic acid as potential anticancer drugs Nutrition 1990 6 429 434 Wendel M. Heller A.R. Anticancer actions of omega-3 fatty acids—Current state and future perspectives Anti-Cancer Agents Med. Chem. 2009 9 457 470 10.2174/1871520610909040457 Das U.N. Essential fatty acids enhance free radical generation and lipid peroxidation to induce apoptosis of tumor cells Clin. Lipid. 2011 6 463 489 10.2217/clp.11.34 Carballeira N.M. New advances in fatty acids as antimalarial, antimycobacterial and antifungal agents Prog. Lipid Res. 2008 47 50 61 10.1016/j.plipres.2007.10.002 Dunbar L.M. Bailey J.M. Enzyme deletions and essential fatty-acid metabolism in cultured-cells J. Biol. Chem. 1975 250 1152 1153 Babior B.M. Kipnes R.S. Curnutte J.T. Biological defense mechanisms—production by leukocytes of superoxide a potential bactericidal agent J. Clin. Invest. 1973 52 741 744 10.1172/JCI107236 Das U.N. Begin M.E. Ells G. Huang Y.S. Horrobin D.F. Polyunstaurated fatty-acids augment free-radical generation in tumor-cells in vitro Biochem. Biophys. Res. Commun. 1987 145 15 24 Huang P. Feng L. Oldham E.A. Keating M.J. Plunkett W. Superoxide dismutase as a target for the selective killing of cancer cells Nature 2000 407 390 395 10.1038/35030140 Das U.N. Tumoricidal action of cis-unsaturated fatty-acids and their relationship to free-radicals and lipid-peroxidation Cancer Lett. 1991 56 235 243 10.1016/0304-3835(91)90008-6 Makarieva T.N. Santalova E.A. Gorshkova I.A. Dmitrenok A.S. Guzii A.G. Gorbach V.I. Svetashev V.I. Stonik V.A. A new cytotoxic fatty acid (5Z,9Z)-22-methyl-5,9-tetracosadienoic acid and the sterols from the far eastern sponge Geodinella robusta Lipids 2002 37 75 80 10.1007/s11745-002-0866-6 Das U.N. Prasad V. Reddy D.R. Local application of gamma-linolenic acid in the treatment of human gliomas Cancer Lett. 1995 94 147 155 10.1016/0304-3835(95)03844-M Saito H. Characteristics of fatty acid composition of the deep-sea vent crab, Shinkaia crosnieri Baba and Williams Lipids 2011 46 723 740 10.1007/s11745-011-3549-x Nechev J. Christie W.W. Robaina R. De Diego F. Popov S. Stefanov K. Chemical composition of the sponge Hymeniacidon sanguinea from the Canary Islands Comp. Biochem. Phys. A 2004 137 365 374 10.1016/j.cbpb.2003.10.016 Chang K.J.L. Dunstan G.A. Abell G.C.J. Clementson L.A. Blackburn S.I. Nichols P.D. Koutoulis A. Biodiscovery of new Australian thraustochytrids for production of biodiesel and long-chain omega-3 oils Appl. Microbiol. 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Transport of Pathogen Surrogates in Soil Treatment Units: Numerical Modeling

Water 2014, 6(4), 1028-1042; doi:10.3390/w6041028

Sorption of Emerging Organic Wastewater Contaminants to Four Soils
Sarah Roberts, Christopher Higgins * and John McCray
Colorado School of Mines, Golden, CO 80401, USA
Author to whom correspondence should be addressed; Tel.: +1-303-384-2002; Fax: +1-303-273-3413.
Received: 3 March 2014; in revised form: 17 April 2014 / Accepted: 18 April 2014 / Published: 24 April 2014

: Conventional onsite wastewater treatment system design relies on a septic tank and soil treatment unit (STU) for treatment of wastewater and integration of the final effluent into the environment. Organic water contaminants (OWCs), chemicals found in pharmaceutical drugs, detergents, surfactants, and other personal care and cleaning products, have been observed in septic tank effluent and the environment. Sorption of OWC mass to soil is a key mechanism in the removal and retardation of many of these chemicals in effluent as it travels through an STU. The primary purpose of this study was to investigate the relationship between the fraction of organic carbon of soil and the equilibrium sorption partitioning coefficient of a selected group of relevant and diverse OWCs. A secondary goal is to evaluate current methods of modeling the sorption of selected OWCs in soil. Five point Freundlich isotherms were constructed from equilibrium sorption batch tests for target OWCs with four different soils. For soils with organic carbon fraction between 0.021 and 0.054, Kd values were calculated between 60 and 185 for 4-nonylphenol, 75 to 260 for triclosan, 115 to 270 for bisphenol-A, 3 to 255 for 17β-estradiol, 40 to 55 for 17α-ethynylestradiol, and 28 to 70 for estrone. An empirically derived, direct relationship between foc and Kd may be a useful approach to estimating sorption for a soil based on organic carbon content.
pharmaceuticals; hormones; sorption; wastewater; soil; organic wastewater contaminants

1. Introduction

Approximately one fifth of the US population is served by onsite wastewater treatment systems (OWTSs), which are a significant and growing means of wastewater treatment in the US [1]. Conventional designs rely on a soil treatment unit (STU) for additional treatment of engineered-unit effluent (such as septic tank effluent) prior to discharge to the environment. Organic wastewater contaminants (OWCs) include a suite of wastewater chemicals such as pharmaceuticals, surfactants, and other personal care products. While the presence of OWCs in OWTS effluent has been well documented [2,3], to date, the factors dictating the efficacy of STUs to remove OWCs have not been comprehensively characterized.

OWCs can have a wide array of ecotoxicological effects and negative impacts on environmental systems. Endocrine disrupting chemicals (EDCs) are a subclass of OWCs that can impair reproductive development and hormone production in some organisms. This effect has been well documented in fish and amphibians, with some populations experiencing skewed gender ratios and/or feminization of males [4]. Other OWCs can affect the renal system, leading to kidney failure [5], while still others are carcinogenic and/or mutagenic [6]. In addition, antimicrobial OWCs may contribute to the evolution of resistant strains of bacteria [7,8,9]. Given these potential adverse impacts and the large and growing fraction of US wastewater treated by STUs, an accurate understanding of the factors controlling the removal of OWCs in STUs is needed. Moreover, this knowledge will aid in the understanding, design and operation of STUs so as to minimize environmental impacts.

One of the major factors controlling the attenuation of OWCs in STUs is removal via sorption to soil media. Building from traditional organic contaminant partitioning paradigms, most sorption models for OWCs assume that the soil-water partitioning coefficients are related to the organic carbon content (i.e., foc) of the soil material (Equation (1)) [10,11,12,13,14,15]:

Kd = Koc × foc
where Koc is the organic carbon-normalized partition coefficient, and Kd is the soil-water partitioning coefficient. These Koc values can in turn be calculated from the octanol-water partition coefficient (Kow) using various linear free energy relationships [16,17,18].

For this study, a representative suite of OWCs was selected to examine whether this modeling paradigm is appropriate for sorption of OWCs to soils typical of those used in STUs. This suite includes triclosan (TCS), 4-nonylphenol (NP), bisphenol-A (BPA), estrone (E1), 17β-estradiol (E2), and 17α-ethynylestradiol (EE2). These target OWCs are ubiquitous in wastewater effluent at significant concentrations [2,19,20] and have demonstrated ecotoxicological effects [4,7,8,21,22,23,24,25].

The overarching goal of this study was to investigate the relationship between the foc of a soil and the Kd of a selected group of relevant and diverse OWCs. In addition, data describing the sorption of these OWCs to a variety of soil types was developed, and current methods of modeling the sorption of the selected OWCs in soil were briefly evaluated.

2. Materials and Methods (or Experimental)

2.1. Chemicals and Soil

Source information and Chemical Abstract Services Registry Number (CASRN) for the six OWCs selected for this study are included in Table 1.

Table 1. Chemical source, Chemical Abstract Services Registry Number (CASRN), Gas Chromatography Mass Spectrometry (GC/MS) retention times, and chemical indicator ions, with qualifying ions used for peak identification in parentheses.
Table 1. Chemical source, Chemical Abstract Services Registry Number (CASRN), Gas Chromatography Mass Spectrometry (GC/MS) retention times, and chemical indicator ions, with qualifying ions used for peak identification in parentheses.
ChemicalSourceCASRNRetention Time (min)Qualifying Ions (m/z)
TriclosanKIC Chem, New Paltz, NY, USA3380-34-516.682289.9 (218.0, 146.0)
4-NonylphenolAldrich, St. Louis, MO, USA25154-52-38.22–12.0135.1 (220.2, 107.1, 121.1)
Bisphenol AAldrich, St. Louis, MO, USA80-05-717.610213.1 (228.1, 119.0)
EstroneAldrich, St. Louis, MO, USA53-16-723.931270.2 (146.1, 185.1, 172.1)
17b-EstradiolAldrich, St. Louis, MO, USA50-28-224.106272.2 (160.1, 213.1)
17a-EthynylestradiolAldrich, St. Louis, MO, USA57-63-624.993213.1 (296.2, 160.1, 133.1)
4-normal-nonylphenolEhrenstorfer, Augsburg, Germany104-40-512.785107.05 (220.2, 77.0)
d6 Bisphenol ACIL, Tewksbury, MA, USA86588-58-117.551216.1 (234.1, 121.1)
d4 17β-estradiolCIL, Tewksbury, MA, USA66789-03-524.084276.2 (161.1, 147.1, 214.1)

Four soils were obtained from locations in Colorado, Nevada, and North Dakota, and bulk soil parameters were measured by a commercial laboratory for each soil type and included: pH, organic carbon content (foc), particle size distribution, and cation exchange capacity (Table 2).

Table 2. Soil characteristics for the four soils considered in sorption batch tests. CEC is the cation exchange capacity.
Table 2. Soil characteristics for the four soils considered in sorption batch tests. CEC is the cation exchange capacity.
CharacteristicSandLoamy SandSandy LoamLoam
Organic Carbon (%)
Soil pH10.
CEC (cmol(+)/kg)
Ca (% of cations)86.654.273.366.2
Mg (% of cations)10.912.519.616.7
K (% of cations)
Na (% of cations)1.410.50.4
% Sand98866530
% Silt0.861544
% Clay1.282026

2.2. Experimental Setup

Sorption isotherms were developed for the six OWCs in the four soils using the aqueous-loss batch approach. Simulated fresh water composed of 0.01 M calcium chloride (CaCl2) was used with a 1:5 soil-water mass ratio in 40 mL glass centrifuge tubes for all replicates and concentrations. This ratio was successfully used in previous experiments involving similar soil [2] and in sorption experiments using the same analytes and similar soil [26]. Prior to initiation of the isotherm experiments, the four soils (sand, loamy sand, sandy loam, and loam) were autoclaved twice for 45 min at 121 °C and 103 kPa to inactive any microbes present and minimize losses due to biodegradation. Preliminary studies comparing sterilization methods found no significant differences in sorption for autoclaved soil when compared to 5% sodium azide biocide solutions (data not shown). While extraction of solids to close the mass balance was not performed, given the short equilibration time and the use of autoclaved soil, minimal biotransformation of the target analytes was expected. Background concentrations of analytes in soils were below analytical detection limits. While pH was not directly measured in the isotherms, the pH of these batch systems was assumed to be similar to the soil pH measured (at an identical 1:5 soil-water ratio; Table 2) by the commercial laboratory.

Batch experiments were conducted in triplicate at five initial concentrations ranging from 60 to 2000 μg/L. This range was chosen based on concentrations of analytes observed in onsite wastewater influent and effluent [2] and analytical method sensitivity. After addition of the approximately 6 g of soil and 30 mL of synthetic freshwater to each tube, samples were mixed with a vortex and then agitated for 36 h on a shaker table (130 rpm). Sorption kinetics were not evaluated: several studies using a similar experimental protocol have reported that equilibrium between the aqueous and sorbed phases was achieved within 22 to 36 h [11,27,28]. After 36 h, glass centrifuge tubes were removed and centrifuged for 20 min at 7000 rpm (3600 rcf) to separate suspended soil particles from solution. The supernatant was decanted and three chemical surrogates (d6-bisphenol-A, 4-normal-nonylphenol, and d4-17β-estradiol) were added to samples at known concentrations to evaluate aqueous analyte concentrations.

2.3. Aqueous Phase OWC Analysis

Target OWCs were extracted from aqueous samples using a method adapted from Barber et al. (2007) [21]. Prepackaged solid phase extraction (SPE) cartridges (Waters Sep-Pak® Plus tC18) were preconditioned with dichloromethane (DCM) using an initial flush of approximately 100 pore volumes. Samples were then loaded on the SPE cartridges under vacuum suction maintaining low flow (approximately 1 mL per minute). Finally, the target OWCs were eluted with 5 mL of DCM under similar low flow conditions. Anhydrous sodium sulfate was used to ensure all water was removed from the vials. The remaining volume was evaporated to 0.5 mL under ultra high purity nitrogen gas and transferred to a 1 mL glass GC vial Sample analysis was conducted in triplicate and two samples spiked with analytes underwent extraction processes for every run to evaluate extraction efficiencies. Recoveries were routinely greater than 99%. Quality assurance and quality control procedures also included triplicate analysis of extractions and use of known concentrations to evaluate the mass recovery capability of the SPE method.

Sample analysis was conducted via electron impact gas chromatography (GC) coupled with selected ion monitoring mass spectrometry (MS). A Hewlett Packard (HP) 6890 GC fitted with an HP Ultra II (5% phenylmethyl silicone) column (25 m by 0.2 mm, 33 μm) was employed with ultra high purity helium as the carrier gas. The injection port was maintained at 300 °C, while the initial oven temperature was 140 °C and increased with a ramp rate of 6 °C per minute up to the final temperature of 300 °C, at which point it was held constant for 15 min. The MS (HP 5793 Mass Selective Detector) was operated with an ionization energy of 70 eV, a source temperature of 250 °C, and an interface temperature of 300 °C. Concentrations of analytes were calculated based on selective ion monitoring abundance data using diagnostic ions for each chemical relative to abundance data for the respective surrogates. These ions are identified by mass and ion retention time determined from standard analysis and a peak signal to noise ratio of 3:1 (Table 1).

2.4. Sorption Coefficients

The Freundlich isotherm model was used to describe the sorption of OWCs from solution [29]. The form of the equation employed was:

Cs = Kf × Cwn
where: Cs is the concentration of the chemical adsorbed to soil particles (μg/kg), Kf is the Freundlich sorption coefficient (L/kg), Cw is the concentration of chemical in the aqueous phase (μg/L), and n is a power function related to the nonlinearity of the isotherm (n = 1 indicates a linear isotherm).When n is unity, the linear isotherm can be described by:
Cs = Kd × Cw
where Kd is the sorption coefficient (L/kg).

If the relationship between the magnitude of sorbed concentration and the magnitude of aqueous concentration is linear (i.e., n = 1), then the Kd value is independent of the concentration. However, if the isotherm is nonlinear and a comparison between different sorbents is desired, interpolation of Kd values from the Kf and n values for a specific concentration is appropriate. In addition, if the partitioning of the OWC aqueous phase to the soil is due to the hydrophobic sorption of the contaminant to the foc of the soil, an organic carbon partitioning coefficient (Koc) can be calculated with Equation (1). Linear regressions were evaluated using the sum of least squares-method to find the straight line of best fit with Microsoft Excel.

3. Results and Discussion

3.1. Sorption of OWCs on Different Soils

The isotherms for the batch sorption isotherms for these experiments were generally well-described by the Freundlich isotherm model (Equation (2), Figure 1), and linear in nature (i.e., the 95% confidence intervals for the Freundlich exponent (n) values included unity). In general, Kd values for all targeted OWCs (Table 4) were much lower in the sand than for all other soils and were generally higher for soils with greater organic carbon content. These values agree with those reported by Karnjanapiboonwong et al. (2010) [30].

Figure 1. Sorption isotherms for TCS, BPA, and NP for soil isotherm data with an R2 value greater than 0.80.
Figure 1. Sorption isotherms for TCS, BPA, and NP for soil isotherm data with an R2 value greater than 0.80.
Water 06 01028 g001 1024

A notable exception to the linear sorption behavior was NP sorption onto the sandy soil, which exhibited highly nonlinear sorption (Figure 2). Due to the high variability of the results for some analytes in some soils, only data sets whose Freundlich model fits had R2 value greater than 0.8 were included in this analysis. Efforts to fit these data with other models (i.e., Langmuir) did not result in better fits. For the sandy soil, the n value for NP was 1.87 ± 0.14 (fitted n ± 95% CI), suggesting that the ratio of sorbed NP mass to aqueous mass increased for higher NP concentrations for this soil. Figure 2 displays equilibrium aqueous concentrations against sorbed concentrations for NP in the sandy soil and also in the sandy loam for comparison. The nonlinear trend is apparent in this plot.

This sorptive behavior has been observed previously for other surfactants, and on a mechanistic level, can be attributed to the hydrophobic self-attraction between the aliphatic carbon chain tails of the NP molecule [31,32]. As is evident in Table 2, the foc in the sandy soil was quite low (0.001). Because sorption of NP increases with increasing aqueous NP levels, it is possible that the chemical is attracted to its own sorbed phase and that this was the dominant sorption mechanism because of the low foc Sorption of NP to the sandy soil was quite low, and the Kf value extrapolated from the Freundlich fit is between 0 and 0.002 L/kg considering the 95% confidence intervals. This is at least three orders of magnitude lower than any other Kf value calculated for any chemical in any soil in the present study. It is possible that in soils where the foc was greater, the sorption to the organic carbon masked this self-sorbing effect, resulting in linear isotherms where the equilibrium between NP in the dissolved and solid phases is primarily dependent on the foc of the soil.

Figure 2. Plots of equilibrium aqueous concentrations and sorbed concentration for 4-nonylphenol in sand.
Figure 2. Plots of equilibrium aqueous concentrations and sorbed concentration for 4-nonylphenol in sand.
Water 06 01028 g002 1024

3.2. Relationship between OWC Sorption and Fraction of Organic Carbon

Kd values appear to generally increase with organic carbon content, and though a clear linear relationship was not evident (Figure 3), this may have been due to the limited number of soil types evaluated in the present study. In addition, the lack of a clear linear relationship may also have been due to the uncontrolled solution effects and/or the potential that sorption mechanisms other than partitioning to organic carbon may have also been important for some soils. Examples of other potential sorption mechanisms include ion exchange, surface adsorption to mineral constituents, and complex formation with metal ions. Solution characteristics, such as dissolved organic carbon content that were not accounted for in this study may influence the degree to which OWCs may sorb. The dissolved organic carbon content of the solution may decrease the sorptive capacity by decreasing the attraction of the hydrophobic molecules to the soil organic carbon.

Figure 3. Plots of Kd values and corresponding foc for soil in which Kd values were measured for analytes.
Figure 3. Plots of Kd values and corresponding foc for soil in which Kd values were measured for analytes.
Water 06 01028 g003 1024

3.3. Influence of pH on Sorption of Anionic forms of Aqueous OWCs

Hydrophobic sorption of organic chemicals to organic carbon is commonly thought to depend on the form of the chemical in solution [18,33,34], which, for ionogenic organic chemicals, is dependent on the pH of the system. For many OWCs, the pKa’s are relatively high and outside the normal pH range for wastewater systems, suggesting that pH effects would be minimal. However, for acidic OWCs, dissociation of the neutral form to form the organic anion may occur within the normal pH range of wastewater. Electrostatic repulsion between the organic anions and the typically negatively charged soil particle surfaces may result in substantially weaker sorption. A simple model for describing the effect of pH on the sorption of acidic organic chemicals to natural organic matter in soil is described by Schwarzenbach et al. (2003) with Equation (4) [29].

Water 06 01028 i001
where Doc is the distribution ratio accounting for sorption only of the protonated (neutral) species to natural organic matter, αa is the fraction of the chemical in the protonated (neutral) form and Water 06 01028 i002 is the sorption coefficient for neutral species. This relationship assumes that the deprotonated (negatively charged) OWC species do not significantly contribute to sorption of the chemical [35,36].

As can be observed in Table 3, the pKa values of most of the target OWCs considered in this study are relatively high. While no pKa values are reported for estrone or 17α-ethynylestradiol, the assumption can be made that they would be similar to that of 17β-estradiol because of chemical similarities between the three chemicals. The reported pKa of TCS (8.14) is lower than the measured pH for one soil. If the pH values measured by the commercial laboratory are truly indicative of the pH of the batch systems and the anionic forms of the OWCs do not sorb to the organic carbon, then the resultant Koc values would be substantially lowered for those soils whose pH values fall above the pKa’s for the target OWCs. However, TCS exhibited a relatively high logKoc (3.12 L/kg) in the sand. While not conclusive, this sorption of presumably anionic TCS agrees with the findings of Tulp et al. (2009), Shareef et al. (2006) and Butler et al. (2012), all of which concluded that anionic forms of organic chemicals are capable of stronger sorption than previously expected [36,37,38].

3.4. Comparison of Experimentally Determined and Literature Reported Koc Values

It is illustrative to compare the range of Koc values calculated from the present data to those reported in the literature for these OWCs (Table 3). Literature reported Koc values are grouped into those calculated using structure-activity models, those derived from single-point equilibrium sorption experiments, and those derived from isotherm equilibrium sorption experiments (which are most analogous to the present study). As is clear from Table 4, the range of Koc values reported in the literature spans more than an order of magnitude for most of the chemicals. While some variability might be expected given the range of approaches from which these data are derived, these data suggest that Koc is not a constant value for these chemicals and may be dependent on the system from which Koc values are calculated. Indeed, the range of values reported for these OWCs was one of the motivations for the present study. Table 4 also highlights the fact that relatively few studies have actually measured Koc values for the target OWCs, indicating a gap in current knowledge.

Table 3. Koc values from isotherm experiments with R2 greater than 0.8 presented in this study compared with Koc values reported in the literature. Chemical parameters, pKa and log(Kow) are also listed for analytes. ND designates a value for which no data was reported.
Table 3. Koc values from isotherm experiments with R2 greater than 0.8 presented in this study compared with Koc values reported in the literature. Chemical parameters, pKa and log(Kow) are also listed for analytes. ND designates a value for which no data was reported.
ChemicalMeasured Log(Koc) ValuesLiterature Reported Log(Koc) ValuesChemical Parameters
Ave ± SD (N)RangeRangePredictedMeasured–Single PointMeasured–IsothermpKaLog (Kow)
4 Nonylphenol3.61 ± 0.73 (4)2.74–4.523.9–5.4ND4.7–5.6 d, 5.3 e, 4.5–5.2 f, 4.7 e3.9 j, 4.6 k10.28 o4.48 t
Triclosan3.56 ± 0.34 (5)3.12–3.923.2–4.63.8–4.0 a, 3.2 b4.6 gND7.9 p4.76 p
Bisphenol–A3.69 ± 0.27 (3)3.40–3.942.8–3.93.2 c, 2.8 bND3.5 i, 3.8 l, 2.9 m, 3.9 n9.6–11.3 q3.40 q
Estrone3.50 ± 0.16 (3)3.34–3.653.2–3.63.6 b3.2 h, 3.3 i3.3 l10.4 r2.95 u
17β-Estradiol3.23 ± 0.16 (3)3.11–3.423.3–3.63.6 b3.3 i3.3 l10.2 s3.86 u
17α-Ethynylestradiol3.25 ± 0.16 (2)3.13–3.362.9–3.3ND3.2 h, 3.3 i2.9 l10.2 s3.67 u

Notes: a: [39]; b: [26]; c: [40]; d: [41]; e: [42]; f: [43]; g: [44]; h: [27]; i: [45]; j: [11]; k: [46]; l: [13]; m: [28]; n: [47]; o: [48]; p: [49]; q: [50]; r: [51]; s: [52]; t: [53]; u: [54]; ND: No Data.

Table 4. Kd and n values reported ±95% CI (confidence interval) calculated from equilibrium sorption isotherm experimental results using Equation (2). ND designates a value for which no data are available or reported due to isotherm variability.
Table 4. Kd and n values reported ±95% CI (confidence interval) calculated from equilibrium sorption isotherm experimental results using Equation (2). ND designates a value for which no data are available or reported due to isotherm variability.
ChemicalSand foc = 0.001Loamy Sand foc = 0.021Sandy Loam foc = 0.031Loam foc = 0.054
NP1.87 ± 0.320.6 ± 0.51.15 ± 0.1552.0 ± 21.70.90 ± 0.14161 ± 44.81.05 ± 0.211783 ± 419
TCS1.06 ± 0.371.3 ± 0.31.11 ± 0.2377.2 ± 46.50.82 ± 0.18259 ± 75.50.83 ± 0.17239 ± 63.9
BPANDND0.99 ± 0.04115 ± 5.90.95 ± 0.07271 ± 16.90.98 ± 0.10135 ± 15.3
E11.22 ± 0.253.2 ± 0.60.90 ± 0.2045.5 ± 23.60.89 ± 0.11255 ± 48.0NDND
E2NDND0.87 ± 0.1954.6 ± 0.60.93 ± 0.1040.1 ± 10.9NDND
EE2NDND0.98 ± 0.2228.6 ± 20.50.85 ± 0.0870.3 ± 12.0NDND

As illustrated in Table 4, the range of Koc values calculated from data in the present study generally agree with those reported previously. The generally large range of reported Koc values suggests that the approach itself may have limited applicability: Koc values tend to range over an order of magnitude for most of the target OWCs. Results from Gerstl et al. (1990) indicate that logKoc values varied by 0.3 in a study of 32 soils and 36 sediments for two OWCs, tetrachloromethane and 1,2-dichlorobenzene [55]. This variability was attributed to the source soil/sediment organic material, which may explain some of the variability in literature reported Koc values. In some cases represented in Table 4, when the foc of the soil was very low, the reported Koc value may have been distorted due to inaccurate measurements of foc. For example, the value measured from isotherm batch tests reported for NP (logKoc = 4.6) was based on sorption to aquifer material with an foc of 0.005 [46]. Conversely, the logKoc values reported by Sekela et al. (1999), ranging from 4.7 to 5.6, were derived from data for sorption to sewage sludge, which has a relatively high foc (approaching 0.5) and facilitates a greater degree of hydrophobic sorption than a soil system may potentially achieve [41]. Although Koc values should not depend on the foc, this comparison shows that the value is clearly not constant for these chemicals. Nevertheless, various fate and transport assessments have utilized a Koc value to reference estimate a Kd value for OWC fate and transport modeling in soil systems [56,57].

The calculation of this partitioning coefficient incorporates a number of assumptions. First, organic carbon is considered as a relatively homogenous phase, essentially meaning that sorption of a chemical does not depend on the type of organic carbon (i.e., root material, leaf litter, decomposing organisms). While studies have shown that the type and geologic age may play a minor role, the soils chosen for the study are analogous to soils used by STUs [58]. Second, the role of dissolved organic carbon in the wastewater stream is not considered in calculation of Kd and Koc. Dissolved organic carbon can serve to reduce the sorption capacity of an OWC to soil and can facilitate solubilization of the compound and aid in transport through the soil. Additionally, because the Koc is calculated using Kd and foc values, the assumption is made that hydrophobic partitioning is the only sorptive mechanism and this partitioning is defined by a linear relationship (Equation (3)). Nevertheless, because foc of a soil is an important factor in the degree of sorption of these chemicals, the Koc of a chemical may be useful in predicting the sorptive capacity of an OWC for a given system. Identifying a useful Koc value from literature studies may incorporate assumptions that lead to a lower degree of confidence in fate and transport modeling results. An empirically determined range of Koc values may be a superior approach to estimating a Kd model input value, but is not always possible for every soil due to resource constraints.

4. Conclusions

Sorption to soil is a key process in the fate and transport dynamics of OWCs as they infiltrate through soil. The Kd value is used to characterize sorption in fate and transport models that consider the advection-dispersion equation incorporating solute transport and, generally, model output is highly sensitive to the magnitude of this parameter. Clearly, effective characterization of a Kd value and the uncertainty surrounding it for a specific system is essential for fate and transport modeling.

To help evaluate this uncertainty, a relationship may be defined to relate a range of Kd values to soil organic carbon content. This range (defined by 95% confidence intervals for a linear regression) incorporates the variability associated with additional soil and solution characteristics by involving a number of various experimentally determined Kd values and not accounting for the variability or effects of soil characteristics other than the foc. Using this approach, a Koc relationship is defined by considering a number of literature-reported sorption studies over an applicable range of organic carbon contents. Figure 4 illustrates an approach for using this empirical relationship. In this plot, Kd values from this study and literature for E1, E2, and EE2 are combined under the assumption that these chemicals, being similar in structure, demonstrate similar sorption characteristics [13,26,27,45]. A regression was fit between the Kd values and corresponding foc values and 95 and 99 percent confidence intervals are included. These confidence intervals may define a range of effective Kd values for an foc based on empirical evidence. It is important to note that the relationship displayed in Figure 4 suggests an approach to identifying a Kd range, but it is unlikely that current Kd information is sufficient to use this approach as a tool for estimating Kd without additional investigation for most OWCs. Rather than requiring extensive numbers of batch studies to characterize Kd values at a variety of soil conditions, a confined range defined by this type of empirical relationship may give a realistic indication as to the effect of sorption on retardation of OWC infiltration.

Figure 4. Plots of the Kd values for associated fraction of organic carbon of soil for isotherms with less than 20% variability in this study and literature reported values [13,26,27,45] for estrogenic hormones (estrone, 17β-estradiol, and 17α-ethynylestradiol). A linear regression fit is included along with the 95% (solid lines) and 99% (dashed lines) confidence intervals.
Figure 4. Plots of the Kd values for associated fraction of organic carbon of soil for isotherms with less than 20% variability in this study and literature reported values [13,26,27,45] for estrogenic hormones (estrone, 17β-estradiol, and 17α-ethynylestradiol). A linear regression fit is included along with the 95% (solid lines) and 99% (dashed lines) confidence intervals.
Water 06 01028 g004 1024

The authors thank Kathryn S. Lowe (CSM) for an initial review of the manuscript. This work was supported by a research grant from the Water Environment Research Foundation (WERF) (Project DEC 1R06).

Author Contributions

Sarah Roberts designed and conducted the experimental work and literature review presented in this paper under the direction, review and guidance of Christopher Higgins and John McCray.

Conflicts of Interest

The authors declare no conflict of interest.

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Topical Collection "Positron Emission Tomography (PET) in Cardiovascular Disease"

A topical collection in Diagnostics (ISSN 2075-4418).

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Dear Colleagues,

Positron emission tomography (PET) is a functional imaging technique with important clinical applications in cardiovascular disease. Multimodality imaging combining PET with CT or MRI yield highly detailed anatomic and molecular information.

Recent advances in PET/CT scanners and specialized software have helped develop an important role for PET/CT imaging in cardiology for diagnosing patients, describing disease and developing treatment strategy.

Commonly used PET radiotracers in clinical practice are F-18 FDG, Rubidium-82, Nitrogen-13 ammonia and Oxygen-15 water. These tracers are used to determine myocardial viability and perfusion. However, the aim of image-guided and personalized preventive vascular medicine may soon be attainable with PET technology. Molecules tagged with a positron emitting isotope enables the visualisation of the cellular function and the follow-up of the molecular process in living organisms without perturbing them. Development of novel molecular probes is essential to the advancement in molecular imaging of cardiovascular disease.

This collection aims to call research and review articles on the latest advancement in the development and application of positron emission tomography in cardiovascular research.

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  • molecular imaging
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Last update: 8 October 2015

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Mean Green Football Looks To Return To Winning Ways On Saturday

DENTON (10/6/06) - After collecting only its seventh Sun Belt loss in six seasons, North Texas returns to conference play against Florida International at Fouts Field on Saturday night. The game will be televised on Comcast/Charter Sports Southeast with the radio call on the Mean Green Radio Network. Kickoff is scheduled for 6:00 p.m. CT.

The Mean Green is coming off its worst home conference loss ever after falling to Middle Tennessee 35-0 last week. The Blue Raiders held North Texas to 19:19 of possession time and 40 total plays, both lows in the Darrell Dickey era. For a second straight week, the Mean Green will face a defense that specializes in tackles for loss. The Golden Panthers rank seventh nationally with 8.2 TFL per game, and also lead the Sun Belt in pass efficiency defense and kick returns.

This will only be the second game between the two schools, with North Texas taking the only meeting 13-10 at FIU Stadium on Oct. 15, 2005.

Florida International ranks first in the Sun Belt Conference in kickoff return average (23.4) while North Texas ranks last (15.2). Florida International ranks first in the league in pass efficiency defense, allowing opponents to complete just 53 percent of their passes while North Texas ranks last in pass efficiency. North Texas and Florida International rank seventh and eighth respectively in rushing offense with averages of 83.6 and 81.2 yards per game.

Click here to read this week's game notes

North Texas Mean Green