Fatty acids as biomarkers of planktonic inputs in the stratified estuary of the Krka River, Adriatic Sea: relationship with pigments

Marine Chemistry, 32 ( 1991 ) 299-312 Elsevier Science Publishers B.V., Amsterdam

299

Fatty acids as biomarkers of planktonic inputs in the stratified estuary of the Krka River, Adriatic Sea: relationship with pigments P. Scribe, J. Fillaux, J. Laureillard, V. Denant and A. Saliot Laboratoire de Physique et Chimie Marines de l'Universitb Pierre et Marie Curie, UA CNRS 353, Tour 24, 4 Place Jussieu, 75252 Paris Cbdex 05 (France) (Received October 12, 1989; revision accepted May 10, 1990 )

ABSTRACT Scribe, P ' Fillaux, J., Laureillard, J., Denant, V. and Saliot, A., 1991. Fatty acids as biomarkers of planktonic inputs in the stratified estuary of the Krka River, Adriatic Sea: relationship with pigments. Mar. Chem., 32:299-312. Fatty acids (FA) associated with particles were quantitativelydetermined at several stations in the Krka Estuary in March 1987 and May 1988. Particles were sampled from riverine stations (Eo anti E2) down to the open sea (C2). Two intermediate stations (E3 and E~ ) were sampled on both sides of the freshwater-seawater interface (PSI) and as close as possible to the interracial layer. In winter (March 1987), total FA concentrations were highest at station E~, located off the city of gibenik (75.9/zg 1- ~at the surface). Total FA concentrations found at stations E0 (7.0 gg 1- ~) and C2 (2.6/~g 1- ~) were low and of the same range as usually encountered in non-productivewaters. A net decrease of terrigenous inputs was observed between riverine station Eo and marine station C2 (FA>C24=9.7% and 0.3% of total FA, respectively). In spring (May 1988), the total FA maxima were found at the FSI at stations E~ and E3 (26.6 and 11.2 gg l- ~). Polyunsaturated fatty acids (PUFA) were predominant compared with the monounsaturated fatty acids (MUFA) at stations Eo and E~ (surface and interface), indicatingfreshly biosynthesized planktonic material. The terrigenous fatty acids (FA > C24) were totally absent at this period. The microbially derived fatty acid contribution is still low, except at station E~ interface (9.2% of total FA). In May 1988, FA and pigment taxonomic information was qualitativelyin good agreement, and in some cases a significant linear correlation was observed, e.g. the ratio of sum C16/sum C~s, known to be very much higher in diatoms than in other algae, was correlated with the fucoxanthin percentage of chlorophyll a. The concentration of 18:4oJ3 was also positively correlated with the alloxanthin concentration and 18:3to3 with chlorophyll b. These results are in good agreement with a phytoplanktonic production generally dominated by diatoms, with ubiquitous Cryptophyceae and Chlorophyceae present to a smaller extent throughout the estuary. In contrast, in winter conditions (March 1987 ) FA and pigments do not follow the same general trends.

INTRODUCTION

Marine coastal areas receive through estuaries large amounts of organic matter that originates from autochthonous riverine algae, detrital naturally 0304-4203/91/$03.50

© 1991 w Elsevier Science Publishers B.V.

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derived material stemming from drainage basins, and anthropogenic compounds that result from human activities (Hedges and Parker, 1976; Mantoura and Woodward, 1983; Groupe de Grochimie du GRECO ICO, 1984; Marchand et al., 1986; Saliot et al., 1988). The total stock of organic suspended matter is constituted of a complex mixture of biological, physical and chemical species (phytoplankton/zooplankton/microorganisms, living/dead cells, and particles (organic compounds adsorbed onto mineral; colloids), that are transported and discharged to the sea through estuaries. What are the major sources of organic matter in the Krka Estuary? What biogeochemical description of a stratified estuary could fatty acids provide? Is such information consistent with the general features described by other biomarkers such as pigments, and by biological studies? In connection with a study of pigments (Denant et al., 1991 ), we performed a total fatty acid analysis to assess source signatures of the particulate organic matter in fresh and marine waters and to investigate the biochemical processes that occur at the boundary layer of the stratified system of the Krka Estuary. MATERIALSAND METHODS

Sampling sites Water samples were collected in the Krka River Estuary in winter (March 1987 ) and in spring (May 1988). Sampling sites were distributed from the waterfalls through the estuary to marine waters off the north coast of Zlarin Island (Fig. 1 ). Water was sampled in the surface freshwater layer (Eo, E3 and Eaa surf), at the boundary layer (E 3 and Eaaint ) and in the bottom saltwater layer (E3 and E4abot). Surface water was sampled at station C2 as the marine end-member. Sampling depths were as follows: in March 1987, Eosurf=0.5 m, E4asurf= 1 m, E4aint=3.5 m, E4abot=4 m and C2=4 m. In May 1988, Eosurf=0.2 m, E3surf=0.5 m, E3int=2.4 m, E3bot-10 m, E4asurf=0.2 m, E4~int = 1.8 m, E4~bot = 30 m and C2=6 m.

Sampling and filtration Surface water samples (20 1) were collected by in situ pumping, and bottom water was collected with a 20-1 Niskin bottle. Interface water samples were taken by a diver holding a 10-1 Niskin bottle horizontally. Water was filtered, a few hours after sampling, through a glass fibre filter ( W h a t m a n GF/F, 0.7-am pore size, 142-mm diameter). Filters were cleaned by rinsing with methylene chloride for 24 h in a Soxhlet apparatus. Filters were always kept in a freezer ( T < - 2 0 ° C ) until laboratory analysis.

FATTY ACIDS AS BIOMARKERS

301

SKRADIN

WATER FALLS T f

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~IBENIK

~C2.

ADRIATIC

Fig. 1. Sampling sites.

Analysis offatty acids Extraction of the filters was performed in a Soxhlet (2 × 12 h ) with a mixture of methylene chloride-methanol (3: 1, v / v ) . The extracts were concentrated in a Biichi rotary evaporator at a temperature < 40 ° C. Each extract was spiked with a known amount of deuterated methyl tricosanoate as interhal standard for gas chromatography (GC) quantitation. The extracts were saponified for 2 h in a solution of 1 N KOH in a mixture of methanol-toluene (1: 1, v / v ) under argon. After cooling, addition of distilled water and acidification to pH 2 with HC1 (4 N), the lipids were extracted three times with a hexane-ether mixture (9: 1, v/v). The extracts were evaporated to dryness under a stream of pure nitrogen. Fatty acids were then isolated by adsorption chromatography on a small column (4-mm i.d. ) filled with 2 g of SiO2 (Merck G6o, 5% deactivated). The first fraction, eluted by 6 ml of hexane, contained aliphatic hydrocarbons; the second fraction, eluted by 6 ml of hexane-ethyl acetate (50: 1, v : v ) , contained aromatic hydrocar-

302

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bons; and the third fraction, eluted by 20 ml of ethyl acetate, contained fatty acids and alcohols. The fatty acids were converted to their methyl esters by a solution of 14% of BF3 in methanol and the esters were purified by adsorption chromatography on SiO2 under the same conditions as previously described. Methyl esters (FAME) were found in the second fraction.

Gas chromatography analysis (GC) FAME-GC analyses were performed with a Girde13000 gas chromatograph equipped with a flame ionization detector (FID) and a Ross-type injector. Samples were analysed using a non-polar fused silica capillary column ( 30-m length, 0.25-mm i.d.) coated with DB5 (Chromoptic, France). The oven temperature was programmed from 100 to 300°C, at a rate of 2 °C min -1. A polar fused silica capillary column (25-m × 0,32-mm i.d. ) coated with Silar 5CP (Chrompack, France) was also used for fatty acid identification. The oven temperature was programmed from I00 to 195°C, at a rate of 2 °C m i n - ~. Helium was used as carrier gas (flow rate 2 ml m i n - ~). The detector temperature was 320 ° C. Components were quantitated by using the FID response.

Gas chromatography-mass spectrometry analysis (GC-MS) G C - M S analyses were performed with R 10-10C Nermag quadrupole spectrometer coupled to a Girdel 32 gas chromatograph. The chromatographic conditions (non-polar column, injector, oven temperature, carrier gas) were as described above. The operating conditions for the mass spectrometer were: transfer line temperature 310°C; electron impact energy 70 eV, 1 scan per second, mass range 31-550 a.m.u. Electron impact mass spectra were acquired and processed using an on-line P D P 11/23 computer with a Sidar 111 data system. Fatty acid identifications were confirmed by comparing mass spectra and retention times with those obtained from commercial standards. Dimethyldisulfide adducts of MUFA were formed to determine the double bond position o f unusual 15: 1¢ol 1, 17: lo96, 17: lto8, and 19:loJ8 fatty acids, using methods previously described by Dunkelblum et al. ( 1985 ) and Scribe et al. (1988). Fatty acids are designated for example as 16:1091 O: 16 is the total number of carbon atoms, 1 is the number of double bonds and 10 its position in the carbon chain from the terminal methyl group as indicated by thegreek letter omega. For PUFA, the last number indicates the position of the ultimate double bond.

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TABLE 1 (continued)

X SAFA XBrFA XMUFA XPUFA -rTotal FA (#g I-~ )

Eosurf

E4asurf

E4~int

E4abot

C2

46.8 4.6 34.6 11.2

43.0 2.4 33.1 21.5

44.3 1.9 26.6 27.3

5 t.4 2.0 29.6 16.q

53.9 1.7 31,7 12.7

7.0

75.9

28.4

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2.6

RESULTS

The sampling strategy can be summarized as follows: ( l ) surface water was sampled from riverine station E0 to the marine coastal station C2 (Fig. 1 ); (2) intermediate stations with a stratified and stable freshwater-salt water interface (E3 and E4,) were chosen to focus on biochemical processes at the boundary layer. Tables l and 2 give, for the stations of the estuary, the total fatty acid concentrations and the percentage compositions of individual components, including linear saturated (SAFA), branched saturated (BrFA), monounsaturated ( MUFA ), and polyunsaturated (PUFA) fatty acids. SAFA include fatty acids from C~4 to C36. MUFA are mainly 16: lo)7, 18:1(o9 and 18: lo)7, and PUFA are 16: 2o)4, 18: 20)6, 18: 3o)3, 18: 4o)3, 20: 4o)6, 20: 5o)3 and 22: 6o)3. BrFA are also present as the 14: 0 and 16: 0 iso and the 15 : 0 and 17 : 0 iso and ante-iso. These fatty acids have been recognized as bacterial markers in sediments (Perry et al., 1979) and in estuarine particles (Saliot et al., 1988). Concentrations in surface water, bottom water and at the interface have been compared. It should be noted that fatty acids with 16 carbon atoms are dominant especially during spring, whereas the C24-C36 series is present only in winter. Some other unusual odd-carbon-numbered unsaturated fatty acids such as 15: ltol l, 17: lo)6, 17: lo)8, 19:lo)6 and 19:lo)8 were identified. DISCUSSION

The general features of the particulate fatty acid pattern in the Krka inner estuary and coastal water offthe river mouth can be summarized as follows: (1) In winter (March 1987) and in spring (May 1988) conditions, the fatty acid concentrations at the riverine station Eo (7.0 and 6,6 #g 1-~ respectively) and marine station C2 (2.6 and 4.9 gg l-~ respectively) indicate low productive conditions. (2) The highest fatty acid concentration was observed at station E4, at the surface during winter (75.9 #g 1-1 ) and, to a lesser extent, at the boundary layer of the same station (E4,int), where the concentrations were similar in winter and spring. Whereas the suspended matter load decreases regularly from Eo to C2, the

305

FATTY ACIDS AS BIOMARKERS TABLE 2

Percentage composition of fatty acids associated with particles in May 1988

14:0 15:0 16:0 17:0 18:0 19:0 20:0 21:0 22:0 23:0 24:0

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Easurf

E3int

E3bot

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E4.int

E~bot

C2

19.03 0.76 35.01 0.30 7.46 0.30 . 0.30 . 0.30

7.1 8.8 28.8 0.5 3.3 0.5

9.6 0.7 32.5 0.4 2.7 0.4 . 0.4

12.3 1.0 27.2 1.0 7.2 0.5 4.6 . 0.5

7.0 0.7 32.2 0.4 3.3 0.1 0.4

6.6 2.6 26.5 1.5 14.8 0.5 1.0

2.5 1.1 31.0 0.9 5.9 0.8

0.4

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14.1 0.8 24.1 0.2 4.0 0.3 0.6 . 1.8 0.2 2.5

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15:1o911 16:1o97 16:1o910 18:1o97 18:lo99 20:1o99 22:lo99

5.02 1.07 1.52 5.78 0.15 0.15

0.5 6.0 4.7 13.7 0.3 -

0.3 6.2 0.7 4.7 12.8 0.3 -

9.2 1.5 4.6 11.8 0.5 -

0.5 7.3 0.9 9.9 0.4 0.2

0.2 17.0 0.3 2.9 3.2 0.4 0.2

0.5 6.6 7.1 2.6 8.2 0.5 -

19.8 1.5 4.6 11.8 0.9 -

16:2o94 18:2o96 18:3o93 18:4o93 20:4o96 20:5o93 22:6o93

0.61 8.07 2.44 1.52 2.28 2.59 2.13

0.3 5.8 4.7 3.6 0.3 3.3 2.2

0.4 5.4 5.1 3.7 0.4 3.0 8.0

1.0 3.1 1.5 2.6 1.5 4.6

0.4 4.5 8.3 7.2 0.5 5.6 5.5

0.7 2.0 1.6 4.7 0.5 7.2 2.4

0.5 1.5 1.0 1.0 1.0 0.5

0.4 4.0 1.7 1.2 0.5 5.1 3.4

14:0i 15:0i 15:0a 16:0i 17:0i 17:0a

0.46 1.22 0.46 0.46 0.30 0.30

0.3 2.2 0.5 0.5 0.3 0.3

0.2 1.2 0.4 0.3 0.2

1.0 0.5 0.5 1.0

0.3 1.9 0.7 0.5 0.4 0.4

0.6 0.7 0.4 6.5 -

0.5 0.5 1.0 1.5 0.5 0.5

1.6

63.47 3.20 13.70 19.63

50.5 4.1 25.3 20.1

47.0 2.1 24.9 26.0

54.9 3.1 27.7 14.4

45.0 4.0 19.1 31.8

48.6 8.2 24.2 19.0

64.3 4.6 25.5 5.6

43.1 1.6 38.6 16.3

3.6

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1.9

10.6

26.6

1.9

4.9

27 SAFA 27 BrFA 27MUFA 27 P U F A 27 Total FA (/tgi - l )

6.6

particulate organic carbon (POC) pattern shows a clear increase at the surface at station E4a (Cauwet, 1991 ). First reports indicate that the ~ibenik bay surface layer receives periodically high-phosphorus inputs of anthropogenic

306

P SCRIBE ET AL.

origin (Gr~eti6 et al., 1991 ). The question raised by the particulate fatty acid winter maxima is whether it is due to direct sewage input or the result of nutrient inputs fertilizing the surface water, thus leading to an irregular and local autochthonous phytoplankton production. The fatty acid distribution at station E4asurf (Table 1 ), and, particularly, the proportion of PUFA as well as the percentage of individual plankton markers such as 18:1oJ9, 20:5093 and 22 : 6093, probably supports the latter hypothesis, although 16: 0, 18 : 0, 16: 1o97 and 18: 2096 are frequently encountered in sewage effluents (Farrington and Quinn, 1973 ). Furthermore, a local plankton bloom is consistent with a high chlorophyll a concentration (Eaasurf: 3.5/tg 1-1). If surface waters of riverine stations Eo and Eaasurf are compared, total fatty acid Concentration increases by a factor of 10 whereas chlorophyll a increases by a factor of five. It should also be noted that the maximum of phaephorbide a-like compound, interpreted as an indicator of the degradation state of chlorophyll a, is found at this station (E4~surf) (Denant et al., 1991 ).

Continental plant-derived inputs Even-carbon-numbered saturated fatty acids from 24 to 36 carbon atoms are commonly used as indicators of allochthonous detrital inputs in sediments (Kolattukudy and Walton, 1973; Cranwell, 1981; Leenheer and Meyers, 1983 ). At the riverine station Eo, except in March 1987 when the 25-34 FA contents reached 10% of total fatty acids, long-chain FA represented < 2% during the winter, as they did also at marine station C2, which is nevertheless clearly under the influence of continental input. These continental plant-derived markers were totally absent in the particles during the spring sampling cruise.

Microbial imprints Branched fatty acids (BrFA) are often used as specific markers of the bacterial contribution to the stock of organic matter associated with suspended particles (Saliot et al., 1982; Tronczynski et al., 1985 ) or sediments (Perry et al., 1979; Volkman et al., 1980; Gillan and Sandstrom, 1985 ), because they are the main constituents of bacteria in cultures, where they account for up to 70% of the total fatty acids (Gillan et al., 1983), and are not involved in either the vegetal or animal metabolisms of marine or freshwater organisms. In May 1988, total BrFA represented from 1.6% at the marine station C2 to 8.2% at the boundary layer (E~aint). During both cruises (March 1987 and May 1988 ), at the marine station (C2) the percentages of BrFA were stable, in contrast to the riverine stations, where a complex situation was observed: in winter, at station E4a, the concentrations of BrFA at the three sampled lev-

FATTYACIDSASBIOMARKERS

307

els were similar but the BrFA concentration relative to the POC was higher at the interface, where the highest number of bacteria and specific growth rate were observed (Fuks et al., 1991 ). In spring, the BrFA maximum was observed at the boundary layer (E4aint). However, the question of whether the increase of BrFA concentrations at the boundary layer is due to an accumulation of active bacterial populations or dead bacterial cells cannot be answered at present. In recent studies, a film formed at the freshwater-seawater interface by accumulation of biologically derived organic matter has been clearly identified, by direct visual observation of divers and surface-active organic material measurements. This film contains a potential food source for heterotrophic organisms (Zuti6 and Legovir, 1987; Svetli~i6 et al., 1991 ). Moreover, BrFA, as saturated fatty acids, are particularly stable relative to other biomarkers (namely PUFA, MUFA and pigments). In oxic conditions, for instance, selective degradation of unsaturated compounds could also lead to accumulation of BrFA in the lipophilic boundary layer. The fatty acid 16: 1co10 has been detected in particles at every station and in every season. Thus MUFA has been suggested as a bacterial marker (Sicre et al., 1988). Interestingly, analyses of individual lipid classes in a range of freshwater, seawater and boundary layer samples in the Krka Estuary (unpublished results) strongly suggest that 16: 10910 might also originate from zooplankton or invertebrates, as proposed by Nichols et al. (1990).

Planktonic imprints Even-carbon-number fatty acids were predominant, from C14 to C22 with maxima at C~6. If linear saturated major fatty acids (C14-C22) cannot be attributed to a specific biological origin, MUFA such as 16:1097, 18:1099, 20:1099 and 22: 1099, and PUFA 18: 3093, 18:4093, 20:5093 and 22:6093, can be confidently assigned to a planktonic origin (Morris and Culkin, 1976; Goutx and Saliot, 1980; Kattner et al., 1983; Bourdier and Amblard, 1987; Volkman et al., 1989). In the Krka Estuary, the proportions of MUFA and PUFA vs. SAFA are rather stable regardless of station and season, except at E4abOt in May 1988, where a clear depletion in PUFA was observed. In contrast, the variations of individual fatty acid compositions reflect the patchy vertical and longitudinal distributions of phytoplankton. Some interesting features appear when the ratio of the sum of C~6 FA vs. the sum of C ~8 FA is considered as a signature of omnipresent diatoms. The sums include all the linear, saturated and unsaturated FA except 16: 0 iso and 18:1097, which are considered as bacterial markers, and 16:1 col 0, which is absent in diatoms. The plot of this ratio vs. the fucoxanthin content expressed as a percentage of chlorophyll a (chlorophyll a equivalent) shows a significant correlation (r = 0.9 8 5, n = 5 ) during the spring period, in contrast to the

308

P S C R I B E ET AL.

• f~J

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large deviation observed in winter (Fig. 2 ). A satisfactory relationship is found in spring because diatoms are known to biosynthesize predominantly 16:0 and 16:lo97 (Opute, 1974; Volkman et al., 1989), and are the dominant species all along the Krka Estuary at this period. Another interesting feature appeared when 18:4o93 and alloxanthin contents were considered. During the spring period, a linear correlation was observed (r=0.98, n = 6 ) , which can be interpreted as the consequence of the omnipresence of Cryptophyceae throughout the inner estuary (Fig. 3 ). However, 18:4o93 is not a specific indicator of Cryptophyceae; Xanthophyceae (Ackman et al., 1968) and Haptophyceae (Chuecas and Riley, 1969) also biosynthesize substantial amounts of 18: 4o93. Nevertheless, this finding suggests that 18: 4o93 here probably originates mainly from Cryptophyceae, as a good correlation between 18:4o93 and alloxanthin is observed. A general relationship was also found between 18: 3o93 and chlorophyll b (r= 0.96, n = 5 ) (Fig. 4). 18:3o93 is produced abundantly in Chlorophyceae and forms from 8% (Chuecas and Riley, 1969) to 43% (Volkman et al., 1989) of the total fatty acids, according to species. Chlorophyll b was considered by Jeffrey and Hallegraeff ( 1987 ) to be a specific marker of green algae. In contrast, no correlation between pigments and fatty acids was observed

309

FATTY ACIDS AS BIOMARKERS

0,80

0 E4ai

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310

P. SCRIBEET AL.

in winter (Figs. 2-4). This might be attributed to several causes, such as faster degradation of pigments relative to fatty acids, as well as the distribution and the physiologic state of the phytoplankton population. Too few data are available on pigment and lipid rates of decomposition in such an environment, or on the micro- and nanoplankton distribution in the inner estuary, to permit further interpretation of this discrepancy. CONCLUSIONS

Particulate fatty acid concentrations vary over a large range along the Krka Estuary. During spring (May 1988 ) and winter (March 1987 ), the river itself shows rather low productivity, of the same order of magnitude as seawater, except in the inner estuary close to ~ibenik, where human activity may fertilize the surface water. The planktonic origin of the main stock of organic matter in the estuary can be clearly recognized. Long-chain fatty acids reveal fluctuations and a low content of terrigenous organic matter inputs, probably as a result of the karstic drainage area. Fatty acid and pigment distributions are qualitatively in good agreement, and in some cases (e.g. in May 1988 ) significant linear correlations are observed. One of these indicates the predominance of diatoms. In winter, pigment and fatty acid concentrations do not follow the same trend. The fatty acid concentration in the interfacial layer at the most productive station (E4a) and the high proportion o f branched fatty acids suggest that the lipophilic boundary may be an efficient zone of accumulation and degradation of organic matter. ACKNOWLEDGEMENTS

We thank Dr. V. Zuti6, Dr. G. Cauwet and Dr. J.M. Martin, coordinators of the French-Yugoslavian program on the Krka River. This research was supported by CNRS, France, as part of the GRECO Interactions ContinentOcean program.

REFERENCES Ackman, R.G., Tocher, C.S. and McLachlan, J., 1968. Marine phytoplankton fatty acids. J. Fish. Res. Bd. Can., 25: 1603-1620. Bourdier, G. and Ambtard, C., 1987. Evolution de la composition en acides gras d'un phytoptancton lacustre (Lac Pavin, France). Int. Rev. Ges. Hydrobioi., 72: 81-95. Cauwet, G., 199 I. Carbon inputs and biogeochemical processes at the halocline in a stratified estuary: Krka River Yugoslavia. Mar. Chem., 32: 269-283. Chuecas, L. and Riley, J.P., 1969. Component fatty acids of the total lipids of some marine phytoplankton. J. Mar. Biol. Assoc. U.K., 49:97-116.

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