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Dissimilar microbial hydrocarbon transformation processes in the sediment and water column of a tropical bay (Havana Bay, Cuba)
Marine PollutionBulletin
Marine Pollution Bulletin, Volume 20, No. 6, pp. 262-268. 1989. Printed in Great Britain.
0025-326X/89 $3.00 +0,00 © 1989 Pergamon Press pie
Dissimilar Microbial Hydrocarbon Transformation Processes in the Sediment and Water Column of a Tropical Bay (Havana Bay, Cuba) I. RAMOS,* M. FUENTES,* R. MEDEROS,* J. O. GRIMALT* and J. ALBAIGt~S*
*Institute of Chemical Research, Washington, 169, Cerro, Havana, Cuba *Institute of Transport, Ctra. del Asilo, Casablanca, Havana, Cuba *Department of Environmental Chemistry (CID-CSIC), Jordi Girona, 18, 08034-Barcelona, Spain
A three year study on the aquatic and sedimentary hydrocarbons in the Havana Bay, Cuba, has been carried out. Two main microbially mediated processes of hydrocarbon transformation have been observed: one, in the water column, involving the bio-synthesis of C23-C30 n-, /so- and ante~so- alkanes from the spilled petroleum, and the other, in the sediments, consisting on the biodegradation of all n-alkanes. Mixtures of parent polynuclear aromatic hydrocarbons have also been found in the sediments, indicating contributions from high temperature combustion processes. The generally observed lack of correspondence between the hydrocarbon composition of the aquatic and sedimentary compartments seems to question the use of sediments for the assessment of petroleum pollution in semi-enclosed areas receiving high loads of anthropogenic inputs.
Sediments are commonly used for the assessment of petroleum pollution in coastal aquatic systems (Farrington & Tripp, 1977; Gearing et al., 1976; Grimalt et aL, 1986; Keizer et aL, 1978; Venkatesan et aL, 1980). This approach arises from the concept that they act as pollutant sinks and provide an integrated picture of the events taking place in the water column. However, petroleum hydrocarbons undergo diverse microbial and physico-chemical transformation processes before and after sedimentation, which depend on the hydrogeographical characteristics of the area. These processes may result into strong modifications of the initial petroleum mixtures leading to significant differences between the hydrocarbon composition of the water column and sediments. In this sense, biodegradation may be enhanced by the incorporation of nutrients from sewage discharges. 262
The concurrence in Havana Bay of high inputs of both petroleum hydrocarbons and urban sewage waters, prompted us to the investigation of the distribution of petroleum hydrocarbons within the aquatic and sedimentary compartments. Accordingly, we report here the main results of a three year study. This study also represents a contribution to the evaluation of diverse petroleum surveillance concepts developed in cold or temperate coastal areas for their applicability to tropical environments. In this sense, we have previously investigated the use of sediments and sentinel organisms for the assessment of hydrocarbon pollution in, respectively, Coatzacoalcos River, Mexico (Farran et al., 1987) and Todos os Santos Bay, Brasil (Tavares et al., 1988). Material and M e t h o d s Water samples (1 1. each) at different depths were collected with precleaned glass bottles as described by Aminot (1983). 5 ml of cone HC1 and 25 ml of CC14 were added immediately after collection. In the laboratory the samples were extracted with 2 × 25 ml of CCI 4. These extracts were dried over anhydrous sulphate, filtered and concentrated to - 1 5 ml by vacuum rotary evaporation. After reconstitution to 25 ml in CC14 the hydrocarbons were determined by absorption at 2925 cm -1 with a Carl Zeiss IR spectrophotometer Model Specord M-80. Quantitation was performed with respect to a standard mixture containing iso-octane, fuel-oil, diesel-oil and kerosene (25% of each). Surface sediments were collected using a Van Veen grab sampler. Samples were picked from the middle of the grab, wrapped in aluminium foil and stored frozen at --20°C. Before analysis they were freeze-dried and sieved through 250 pxn. Lipids were recovered by Soxhlet extraction with methylene chloride-methanol
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(2 : 1) for 36 h. The extract was vacuum evaporated to 2 ml and hydrolyzed overnight with 35 ml of 6% KOH/ MeOH. Neutrals were separated by n-hexane extraction (3 × 30 ml), concentrated under vacuum to about 0.5 ml, and further fractionated by column chromatography according to a previous established method (Aceves et al., 1988). A column filled with 8 g of each 5% water deactivated alumina (70-230 mesh, Merck) (top) and silica (70-230 mesh, Merck) (bottom) was used. The following fractions were collected: 1. 20 ml of n-hexane (atkanes and alkenes), 2. 20 ml of 10% methylene chloride in n-hexane (monocyclic aromatic hydrocarbons), and 3. 40 ml of 20% methylene chloride in n-hexane (polycyclic aromatic hydrocarbons). The gas chromatographic analysis was performed with a Carlo Erba FTV 4160 GC instrument, equipped with a flame ionization detector and a splitless injector. A column of 25 m × 0.25 mm i.d. coated with SE-54 was used (film thickness 0.15 gm). Hydrogen was the carrier gas (50 cm s-l). The temperature was programmed from 60°C to 300°C at 6°C min -1. Injector and detector temperatures were respectively 3000C and 330°C. The injection was in the splitless mode (solvent iso-octane; hot needle technique) keeping the split valve closed for 35 s. Selected fractions were analyzed by gas chromatography-mass spectrometry (GC-MS) using a HewtettPackard 5995 instrument coupled to an HP-300 datasystem. The chromatographic conditions were the same as described above except that helium was used as carrier gas. Data were acquired in a scanning range of m/z 50-550 at 1 s per decade. MS temperatures were: transfer line 300°C, ion source 200°C and analyser 230°C. The GC profiles were quantitated by comparison with adequate external standard mixtures: n-C14, n-C22, n-C32 and /'/-C36 for the aliphatic hydrocarbons, and phenanthrene, chrysene and benzo(a)pyrene for the aromatics.
tion. Actually, among other facilities, it contains the major harbour of the island, a crude oil refinery and a gas manufacture plant. Furthermore, the bay receives an important part of the untreated residual waters of the City ( - 2 2 0 000 m 3 day-l). These, along the waters of three main rivers: Luyano (56 000 m 3 day-l), Martin Perez (26 000 m 3 day -1) and Tadeo (5000 m 3 day-l), account for most of the freshwater input. These rivers, in turn, act as draining system of several industries, namely ethanol and liquor distilleries (1600 m 3 day -l) and a yeast plant (400 m 3 day-l). The main discharges of pollutants entering into the bay have been summarized in Table 1, according to a report from Mederos et al. (1984). The narrow dimensions of the channel connecting the bay with the Caribbean Sea represent an important limitation for the output of these anthropogenic discharges into the sea. It has been estimated that the residence time of the water in the bay is, on average, 5.5 days (Mederos et al., 1984). With this information an initial estimate of the standing stock of pollufants in the bay can be obtained. Thus, for the petroleum hydrocarbons, the following equation can be formulated: dx/dt -- 34 -- x/5.5
(1)
Results and Discussion Area of study The Havana Bay (Fig. 1) encompasses an area of -5.2 km 2 with an average depth of about 9.2 m (total water volume 4.7 × 107 m3). It is connected to the open sea by a long (1574 m), narrow (140 m) and relatively deep (12-15 m) channel. This bay has undergone intense human activity since the early Spanish coloniza-
Fig. 1 Map of Havana Bay. Lettered (W) and numbered points correspond to sampling stations for water and sediments, respectively.
TABLE 1 Main anthropogenic discharges into Havana Bay (adapted from Mederos et al., 1984). Water flow*
Petroleum Hydrocarbons t
Oils and Greases t
Totai mtrogen t
Totai phosphorous t
Suspended panicles t
Urban draining system
220 000
20 000
8500
1200
1800
18 000
Rivers
100 000
1700
3000
570
860
4000
13 000
11 000
6500
500
750
5700
400
850
330 000
34 000
18 000
2300
3400
28 000
Direct industrial effluents Harbour Total *m 3 day-~; tkg day -~.
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where x is the amount of hydrocarbons present in the bay (in tonnes) and t is the time (in days). In this equation it is assumed that the waters of the bay are well mixed and that the channel is the only output of hydrocarbons. A better estimation requires the consideration of the internal hydrology of the bay as well as the biogeochemical processes taking place, namely degradation, sedimentation and evaporation. The integration of equation 1 gives rise to the following expression: x-- 187. (1 - e x p (-t/5.5))
(2)
where x is the amount of hydrocarbons present in the bay (in tonnes) and t is the time (in days). That is, 187 t of petroleum hydrocarbons will be the standing stock when a steady state is reached. Taking into account the total water volume of the bay, this amount corresponds to an average concentration of about 4 mg 1-t. Similar equations can be formulated for the other major anthropogenic inputs, leading to the following estimates: 100 t of oils and greases (2 mg l-X), 13 t of total nitrogen and 19 t of total phosphorous. Irrespective of the accuracy of the estimates, the order of magnitude of the numbers is indicative of a high anthropogenic influence. Diverse sampling stations were selected within the bay for the study of these anthropogenic inputs (see Fig. 1). Waters were collected monthly at four depth levels (surface, 2 m, 4 m and bottom) over a 3 year period (sites W-I-W-5). Two sediment sampling cruises were performed in 2 successive years (sites 1-11). The corresponding samples were analysed separately.
Nutrients and dissolved oxygen Table 2A reports some chemical parameters for the Havana Bay surface waters. The average values indicate a rather uniform and eutrophic situation which results from the high input of nutrients (Table 1) and the limited exchange of waters between the bay and the open sea. However, the higher dissolved oxygen and the slightly lower concentrations of nutrients in Stn W-1 (the channel) reflect the influence of marine water in this site. Conversely, Stn W-3 (Guasabacoa inlet) appears as the area of higher nutrient load and, therefore, lowest dissolved oxygen. This inlet receives the waters of the Luyano and Martin Perez rivers carrying residues of several industries as well as sewage from the southern area of Havana City. The vertical profiles of these parameters are reported in Table 2B, indicating rather good mixing conditions within the water column. However, despite of this, two water layers can be defined, the shallower ( < 2 m) containing the highest concentrations of nutrients and lowest dissolved oxygen. This difference parallels the salinity gradient, indicating the association of most anthropogenic inputs to freshwater discharges. In turn, such layering is in correspondence with the predominant water mass movements of the bay: exit of brackish-to-salt water by the surface (5-10 cm s-1) and entrance of sea water by the bottom ( - 4 cm s-l). In this respect, the higher levels of dissolved oxygen observed in the deep waters are attributed to the supply provided by the input of marine water. Hydrocarbons in the water column The average hydrocarbon concentrations found in
TABLE 2 Some water quality parameters of five sampling stations at Havana Bay (see Fig. 1). The mean values correspond to data collected monthly. A Horizontal distribution Sampling stations
Dissolved oxygen*
W-1 W-2 W-3 W-4 W-5 Mean
4.4 1.5 1.4 2.0 2.7 2.4
B Vertical distribution Depth
± ± ± ± ± ±
NH4*
1.4 2.2 1.9 1.0 1.3 1.5
3.8 3.4 6.0 4.8 5.6 4.8
Dissolved oxygen*
Surface 2m 5m Depth
1.9 1.8 2.3 3.6
± ± ± +
± + ± ± ± ±
Total phosphoroust
2.8 2.7 2.6 3.3 3.6 3.3
NH4 t
1.8 1.6 1.0 1.6
4.7 5.3 5.1 4.2
± ± ± ±
2.0 2.5 2.6 1.7 1.9 2.1
± ± ± + ± ±
Total nitrogen t
1.6 1.7 1.2 0.9 1.2 1.4
23 46 48 39 39 39
Total phosphorous*
3.2 3.8 3.2 2.9
2.7 2.6 1.7 1.7
± ± ± ±
± ± ± ± ± ±
Total silicon t
13 23 27 42 29 27
5.0 11.0 8.7 5.8 7.7 7.2
Total nitrogen t
1.7 2.0 1.0 1.0
46 41 38 31
+ ± + ±
± ± ± ± ± ±
2.8 6.7 6.5 3.9 4.0 8.0
Total silicon t
27 25 24 32
10.0 8.8 6.4 4.8
+ ± ± ±
6.5 5.7 3.9 3.1
*mg I-t; t p,g-at 1-1.
TABLE 3 Average hydrocarbon concentrations in the water column of Havana Bay (mg 1-1) (sampling stations indicated in l~ig. 1). Depth
0 2m 5m Bottom Mean
264
W-1
W-2
W-3
Samplingstations W-4
W-5
3.0 3.0 2.7 2.7 2.8
3.9 3.7 3.5 3.4 3.6
3.6 3.4 3.3 3.2 3.4
4.0 3.9 3.8 3.7 3.9
3.3 3.0 2.9 2.9 3.0
Mean 3.6 3.4 3.2 3.2 3.3
± ± ± ± ±
1.8 1.7 1.6 1.4 1.6
Volume20/Number6/June 1989 the waters of the Havana Bay are shown in Table 3. In general terms, the concentrations of petroleum derived hydrocarbons in seawater are difficult to compare with previously reported data due to the great variety of sampling and analytical methods used. However, considering cases where also infrared spectroscopy was used for quantitation we may conclude that the Bay supports a high hydrocarbon load. In fact, Ahmed et al. (1974) reported values ranging from 0.19-0.82 mg 1-1 for the Boston Harbour and similar concentrations were found in the Goteborg Harbour (0.47-0.72 mg 1-1) (Carlberg & Skarstedt, 1972). The average concentrations for several semi-enclosed areas of the Mediterranean (harbours, bays, etc.) were slightly higher (0.58-1.50 mg 1-1) (Cuberes & Albaiges, 1975; UNEP, 1980) but below to those found in the Havana Bay. As far as the Havana Bay spatial distribution is concerned, both horizontal and vertical, a trend paralleling
that of nutrients and dissolved oxygen is observed (Table 2). Again, the lowest levels correspond to the waters of Stn W-1 (the channel) but the highest concentrations are found in Stn W-4 (Marimelena inlet), close to the crude oil refinery. In general, the highest hydrocarbon levels correspond to the surface waters, consistently with the above reported trends. Representative chromatograms of the dissolved+ dispersed hydrocarbons in the Havana Bay waters are shown in Figure 2A. Besides the predominant distribution of C23-C30n-alkanes without odd-to-even carbon number preference, an important feature of this profile consists in the series of C23-C30/SO and ante~So alkanes. As it can be observed, this distribution (A) differs considerably from those corresponding to the predominant petroleum discharges occurring in the Bay 03). On the contrary, these hydrocarbon mixtures are similar to those of weathered petroleum tanker washings (Albaigrs & Cuberes, 1980). However, various labora-
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271 25 J d
91
./
I I
17
®
il5
19 23
21
13
Fig. 2 Gas chromatograms of (A) the total extract of a representative water sample from HavanaBay and (B) the predominant petroleum discharges. Numbers refer to n-alkane chain-length. Dots to iso and anteiso alkanes. 265
Marine Pollution Bulletin
tory experiments have shown that these mixtures may also be produced by microbial degradation of petroleum (Walker & ColweU, 1979).. In fact, these distributions of hydrocarbons have been found in cultures of diverse microorganism species (see e.g. Baraud et al., 1967; Davis, 1968; Naccarato et al., 1974). Gassmann (1982) reported the occurrence of these hydrocarbon profiles, including the series of /so- and ante/so-alkanes, in North Sea waters. In agreement with the previous observations and some quantitative considerations he proposed that they were formed from the bio-conversion of oil spillages. In a series of recent experiments, we have also found that these hydrocarbons may be produced by microbial re-synthesis of sedimentary n-alkane mixtures (Grimalt et aL, 1988). At this respect, the similarity between the concentration level predicted by equations 1 and 2 (-4 mg 1-1) and that measured by analytical methods (-3.3 mg 1-1) deserves particular attention. In case that the hydrocarbon profiles displayed in Figure 3A originate from residual products or microbial degradation of the predominant petroleum discharges (Fig. 3B), it would be expected, in view of the strong compositional differences, that the measured hydrocarbon concentrations would be much lower than the values estimated from equations 1 and 2. Since both concentration values are similar the hypothesis of bio-conversion is reinforced. Sedimentary hydrocarbons The concentrations of sedimentary hydrocarbons in Havana Bay are indicated in Table 4. Two values per sampling site are given, corresponding to the two collection cruises. Similarly to the water samples, the spatial distribution of these components is rather uniform. Only the samples located within Marimelena inlet (sites 8 and 10) exhibit slightly higher levels of hydrocarbons, which can be attributed to the local influence of the petroleum refinery. These values can be compared, for instance, with the hydrocarbons levels in several British and Spanish estuaries: 10-100 p.g g-t (Readman et al., 1986) and 1-250 ~tg g-1 (Grimalt et aL, 1986), respectively, or with those from sediments adjacent to effluent discharge sites of a sewage treatment plant in Chesapeake Bay (2-150 ~tg g-l; Brown & Wade, 1984). The levels of sedimentary hydrocarbons in Havana Bay are far higher than those of these samples, which also correspond to petroleum polluted areas.
Two representative examples of the sedimentary aliphatic (A) and aromatic (B) hydrocarbons are displayed in Fig. 3. The aliphatic fraction is largely composed of an unresolved complex mixture (UCM) of alkanes where norpristane, pristane, phytane and a series of C29-C32hopanes constitute the main resolved peaks. As repeatedly reported (Albaigrs & Albrecht, 1979; Farran et al., 1987), the presence of these ClsC20 regular isoprenoid hydrocarbons and the 170~, 2113 (H) stereochemistry of the hopane series, with t w o chromatographically resolved C-22 epimers for the extended homologs, is indicative of a petroleum origin for the UCM. The lack of n-alkanes is due to microbial degradation, as it has commonly been observed in many heavily polluted sedimentary environments (see e.g. Farrington & Quinn, 1973; Giger et aL, 1974; Wakeham, 1976). Accordingly, two different microbial processes related with the occurrence of petroleum hydrocarbons in Havana Bay are oberved. One, in the water column, involving 'bio-synthesis' of long chain n-alkane homologs, and the other, in the underlying sediments, consisting of the elimination of the straight chain aliphatic components. Different environmental conditions must determine their occurrence. It has been previously indicated that despite the high pollutant and nutrient load in the Bay, some small level of oxygen has usually been found in the water column. In contrast, the sediments are anoxic, with hydrogen sulphide emissions being often detected. The aromatic hydrocarbons of these sedimentary samples are predominated by parent polycyclic components such as fluoranthene, pyrene, chrysene, benzopyrenes, etc. (see Fig. 3B). They correspond to contributions of residues from high temperature processes like fossil fuel combustion (LaFlamme & Hites, 1978; Prahl & Carpenter, 1983; Windsor & 1-rites, 1979). Probably the main source is urban run-off and effluents from the gas manufacture and electric power plants located along the Bay. Besides these predominant hydrocarbons, other resolved components are present in high proportion, mostly methyl and dimethyl substituted phenanthrenes, fluoranthenes, pyrenes and chrysenes. These again evidence petroleum contributions (Tissot & Welte, 1984) which, in this aromatic fraction, are also apparent from the presence of an unresolved complex mixture. The dual source origin of the sedimentary hydro-
TABLE4 Hydrocarbonsconcentrationsin ~ e s e d i m e n t s o f H a v ~ a B a y ( ~
g-~).SamplingsitesinFig. 1.
Aliphatics
Station No. 1 2 3 4 5 6 7 8 9 10 11
Aromatics
Waterdepth (m) 15 9 12 9 11 6 8.5 5 3 12 14
Monocyclic 1200" 5500 2400 4900 3800 3800 5000 7500 4100 5900 1500
1000 t 4800 3300 3300 2600 3300 4300 5300 3800 6000 2000
* and * duplicate analysis corresponding to replicate sampling.
266
400* 2100 490 2800 2200 1200 2100 5100 1100 2300 1200
Polycyclic 500 t 3000 200 1500 2100 1200 1800 4000 1400 1900 1000
600* 2400 900 2800 1800 400 600 1600 1600 600 800
300 t 1800 100 1600 1000 300 300 900 1000 1000 600
Volume 20/Number 6/June 1989
carbons isolated in the polycyclic aromatic hydrocarbon fraction is also reflected in their quantitative distribution. In this case, the highest concentrations (Stns 2 and 4; Atares and Guasabacoa inlet) do not correspond to the samples located closer to the refinery, although the levels found in Stn 8 are also above the bay average values.
all n-alkanes. In this situation of heavy pollution stress repeatedly observed during the 3 year period, the hydrocarbon composition of the sediments contrasts strongly with that of the water column and questions the utility of sedimentary samples as geoaccumulators. Financial support from the UNESCO/UNID Project no. CUB/80/001 and Acuerdo de Cooperacion Hispano-Cubano is gratefully acknowledged.
Conclusions The high levels of industrial, urban and petroleum pollution in Havana Bay apparently result into two main microbial hydrocarbon transformation processes: 1. in the water column, with the formation by 'biosynthesis' of distributions of C23-C30n-alkanes without odd-to-even carbon number preference, along with a series of C23-C3o iso and anteiso alkanes; and 2. in the sediments, with the elimination by 'bio-degradation' of
Aceves, M., Grimalt, J. O., Albaig6s, J., Broto, E, Comellas, L. & Gassiot, M. (1988). Analysis of hydrocarbons in aquatic sediments. II. Evaluation of common preparative procedures for petroleum and chlorinated hydrocarbons. J. Chromatogr. 436,503-509. Ahmed, A. M., Beasley, M. D., Efromson, A. C. & Hires, R. A. (1974). Sampling errors in the quantitation of petroleum in Boston Harbor water. In Marine Pollution Monitoring (Petroleum), NBS SP 409, pp. 109-111. National Bureau of Standards, Washington, D.C. Albaig6s, J. & Albrecht, P. (1979). Fingerprinting marine pollutant hydrocarbons by computerized gas chromatography-mass spectrometry. Internat. J. Environ. Anal. Chem. 6,171-190.
@ 6
87 9
2
1"2
® 15 18 16
13
J Fig. 3 Gas chromatograms of the aliphatic (A) and aromatic (B)
hydrocarbons present in the sediments of Havana Bay. Peak identification as follows: A) 1. norpristane; 2. pristane; 3. phytane; 4. 17~2113(H)-30-norhopane; 5. 17ct211~(H)-hopane; 6. 17at21~(H)-homohopane 22S; 7. 170~211~(H)-homohopane 22R; 8. 170,211~(H)-bishomohopane 22S; 9. 17a211~(H)-bishomohopane 22R. B) 10. phenanthrene; 11. fluoranthene; 12. pyrene; 13. benzofluorenes; 14. benz(a)anthracene; 15. chrysene; 16. benzofluoranthenes; 17. benzo(e)pyrene; 18. benzo(a)pyrene; 19. indeno(1,2,3-cd)pyrene; 20. benzo(ghi)perylene.
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Marine Pollution Bulletin Albalgrs, J. & Cuberes, M. R. (1980). On the degradation of petroleum residues in the marine environment. Chemosphere 9,539-545. Aminot, A. (1983). Prrl~vement et prr-traitement des 6chantillons: eau de mer, sddiments et organismes marins. In Manuel des analyses chimiques en milieu marin (A. Aminot & M. Chaussepied, eds), pp. 19-32. CNEXO, Brest. Baraud, J., Cassagne, C., Genevois, L. & Joneau, M. (1967). Prrsence d'hydrocarbons darts rinsaponifiable des lipides des levures. C.R. Acad. Sc. Paris 265(D), 83-85. Brown, R. C. & Wade, T. L. (1984). Sedimentary coprostanol and hydrocarbon distribution adjacent to a sewage ouffall. Water Res. 18, 621-632. Carlsberg, S. R. & Skarstedt, C. B. (1972). Determination of small amounts of non-polar hydrocarbons (oil) in sea water. J. Cons. Int. Explor. Mer 34, 506-515. Cuberes, M. R. & Albalges, J. (1975). Control de la contaminacirn marina por hidrocarburos y su aplicacirn al litoral mediterrfineo espafiol. In Proc. I Congreso Iberoamericano del Medio Ambiente, pp. 937-952. CEMA, Madrid. Davis, J. B. (1968). Paraffinic hydrocarbons in the sulfate-reducing bacterium Desulfovibrio desulfuricans. Chem. Geol. 3,155-160. Farran, A., Grimalt, J., Albaigds, J., Botello, A. V. & Macko, S. A. (1987). Assessment of petroleum pollution in a Mexican River by molecular markers and carbon isotope rations. Mar. Pollut. Bull 18, 284-289. Farrington, J. W. & Quirm, J. G. (1973). Petroleum hydrocarbons and fatty acids in waste water effluents. J. Wat. Pollut. Control Fed. 45, 704. Farrington, J. W. & Tripp, B. W. (1977). Hydrocarbons in western North Atlantic surface sediments. Geochim. Cosmochim. Acta 41, 1627-1641. Gassmann, G. (1982). Detection of aliphatic hydrocarbons derived by recent "Bio-conversion" from fossil fuel oil in North Sea Waters. Mar. Pollut. Bull. 13, 309-315. Gearing, P.. Gearing, J. N., Lytle, T. F. & Lytle, J. S. (1976). Hydrocarbons in 60 northeast Gulf of Mexico shelf sediments: a preliminary study. Geochim. Cosrnochim. Acta 47, 2115-2119. Giger, W., Reinhardt, M., Schaffner, C. & Stumm, W. (1974). Petroleum-derived and indigenous hydrocarbons in recent sediments of Lake Zug, Switzerland, Environ. Sci. Technol. 8, 454. Grimalt, J., Bayona, J. M. & Albaiges, J. (1986). Chemical markers for the characterization of pollutant inputs in the coastal zones. Journ. Etud. Pollut. C.LE.S.M. 7,533-543.
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Grimalt, J. O., Torras, E. & Albaigrs, J. (1988). Bacterial reworking of sedimentary lipids during sample storage. Org. Geochem. 13, 741746. Keizer, P. D., Dale, J. & Gordon, D. C. Jr. (1987). Hydrocarbons in surficial sediments from the Scotian shelf. Geochirn. Cosmochim. Acta 42,165-172. LaFlamme, R. E. & Hites, R. A. (1978). The global distribution of polycyclic aromatic hydrocarbons in recent sediments. Geochim. Cosmochim. Acta 42,289-303. Mederos, R., Jaime, N., Villasol, A., Shabalina, L. & Espinosa, M. A. (1984). Investigaci6n y control de la contaminacirn marina en la Bahia de la Habana. Report of the Ministry of Transport. Project CUB-80-001 -PNUD-PNUMA-UNESCO. Naccarato, W. F., Gilbertson, J. R. & Gelman, R. A. (1974). Effects of different culture media and oxygen upon lipids of Escherichia coli K-12. Lipids 9,322-327. Prahl, F. G. & Carpenter, R. (1983). Polycyclic aromatic hydrocarbon (PAH)--phase associations in Washington coastal sediments. Geochirn. Cosmochim. Acta 47, 1013-1023. Readman, J. W., Preston, M. R. & Mantoura, R. F. C. (1986). An integrated technique to quantify sewage, oil and PAIl pollution in estuarine and coastal environments. Mar. Pollut. Bull 17,298-308. Tavares, T. M., Rocha, V. C., Porte, C., Barcelo, D. & Albaiges, J. (1988). Application of the mussel watch concept in studies of hydrocarbons, PCBs and DDT in the Brazilian Bay of Todos os Santos (Bahia). Mar. Pollut. Bull. 19,572-578. Tissot, B. P. & Welte, D. H. (1984). Petroleum Formation and Occurrence. Springer-Verlag, Heidelberg. UNEP (1980). Summary reports on the scientific results of MED POL I. UNEP/IG. 18/1NF. 3. Venkatesan, M. I., Brenner, S., Ruth, E., Bonilla, J. & Kaplan, I. R. (1980). Hydrocarbon in age-dated sediment from two basins in the Southern California Bight. Geochim. Cosmochim. Acta 44, 789802. Wakeham, S. C. (1976). A comparative survey of petroleum hydrocarbons in lake sediments. Mar. Pollut. Bull 7,206-211. Walker, J. D. & Colwell, R. R. (1976). Long-chain n-alkanes occurring during microbial degradation of petroleum. Can. J. Microbiol. 22, 886-891. Windsor, J. G. & Hites, R. A. (1979). Polycyclic aromatic hydrocarbons in Gulf of Maine sediments and Nova Scotia soils. Geochim. Cosmochim. Acta 43, 27-33.
Marine Pollution Bulletin, Volume 20, No. 6, pp. 262-268. 1989. Printed in Great Britain.
0025-326X/89 $3.00 +0,00 © 1989 Pergamon Press pie
Dissimilar Microbial Hydrocarbon Transformation Processes in the Sediment and Water Column of a Tropical Bay (Havana Bay, Cuba) I. RAMOS,* M. FUENTES,* R. MEDEROS,* J. O. GRIMALT* and J. ALBAIGt~S*
*Institute of Chemical Research, Washington, 169, Cerro, Havana, Cuba *Institute of Transport, Ctra. del Asilo, Casablanca, Havana, Cuba *Department of Environmental Chemistry (CID-CSIC), Jordi Girona, 18, 08034-Barcelona, Spain
A three year study on the aquatic and sedimentary hydrocarbons in the Havana Bay, Cuba, has been carried out. Two main microbially mediated processes of hydrocarbon transformation have been observed: one, in the water column, involving the bio-synthesis of C23-C30 n-, /so- and ante~so- alkanes from the spilled petroleum, and the other, in the sediments, consisting on the biodegradation of all n-alkanes. Mixtures of parent polynuclear aromatic hydrocarbons have also been found in the sediments, indicating contributions from high temperature combustion processes. The generally observed lack of correspondence between the hydrocarbon composition of the aquatic and sedimentary compartments seems to question the use of sediments for the assessment of petroleum pollution in semi-enclosed areas receiving high loads of anthropogenic inputs.
Sediments are commonly used for the assessment of petroleum pollution in coastal aquatic systems (Farrington & Tripp, 1977; Gearing et al., 1976; Grimalt et aL, 1986; Keizer et aL, 1978; Venkatesan et aL, 1980). This approach arises from the concept that they act as pollutant sinks and provide an integrated picture of the events taking place in the water column. However, petroleum hydrocarbons undergo diverse microbial and physico-chemical transformation processes before and after sedimentation, which depend on the hydrogeographical characteristics of the area. These processes may result into strong modifications of the initial petroleum mixtures leading to significant differences between the hydrocarbon composition of the water column and sediments. In this sense, biodegradation may be enhanced by the incorporation of nutrients from sewage discharges. 262
The concurrence in Havana Bay of high inputs of both petroleum hydrocarbons and urban sewage waters, prompted us to the investigation of the distribution of petroleum hydrocarbons within the aquatic and sedimentary compartments. Accordingly, we report here the main results of a three year study. This study also represents a contribution to the evaluation of diverse petroleum surveillance concepts developed in cold or temperate coastal areas for their applicability to tropical environments. In this sense, we have previously investigated the use of sediments and sentinel organisms for the assessment of hydrocarbon pollution in, respectively, Coatzacoalcos River, Mexico (Farran et al., 1987) and Todos os Santos Bay, Brasil (Tavares et al., 1988). Material and M e t h o d s Water samples (1 1. each) at different depths were collected with precleaned glass bottles as described by Aminot (1983). 5 ml of cone HC1 and 25 ml of CC14 were added immediately after collection. In the laboratory the samples were extracted with 2 × 25 ml of CCI 4. These extracts were dried over anhydrous sulphate, filtered and concentrated to - 1 5 ml by vacuum rotary evaporation. After reconstitution to 25 ml in CC14 the hydrocarbons were determined by absorption at 2925 cm -1 with a Carl Zeiss IR spectrophotometer Model Specord M-80. Quantitation was performed with respect to a standard mixture containing iso-octane, fuel-oil, diesel-oil and kerosene (25% of each). Surface sediments were collected using a Van Veen grab sampler. Samples were picked from the middle of the grab, wrapped in aluminium foil and stored frozen at --20°C. Before analysis they were freeze-dried and sieved through 250 pxn. Lipids were recovered by Soxhlet extraction with methylene chloride-methanol
Volume 20/Number 6/June 1989
(2 : 1) for 36 h. The extract was vacuum evaporated to 2 ml and hydrolyzed overnight with 35 ml of 6% KOH/ MeOH. Neutrals were separated by n-hexane extraction (3 × 30 ml), concentrated under vacuum to about 0.5 ml, and further fractionated by column chromatography according to a previous established method (Aceves et al., 1988). A column filled with 8 g of each 5% water deactivated alumina (70-230 mesh, Merck) (top) and silica (70-230 mesh, Merck) (bottom) was used. The following fractions were collected: 1. 20 ml of n-hexane (atkanes and alkenes), 2. 20 ml of 10% methylene chloride in n-hexane (monocyclic aromatic hydrocarbons), and 3. 40 ml of 20% methylene chloride in n-hexane (polycyclic aromatic hydrocarbons). The gas chromatographic analysis was performed with a Carlo Erba FTV 4160 GC instrument, equipped with a flame ionization detector and a splitless injector. A column of 25 m × 0.25 mm i.d. coated with SE-54 was used (film thickness 0.15 gm). Hydrogen was the carrier gas (50 cm s-l). The temperature was programmed from 60°C to 300°C at 6°C min -1. Injector and detector temperatures were respectively 3000C and 330°C. The injection was in the splitless mode (solvent iso-octane; hot needle technique) keeping the split valve closed for 35 s. Selected fractions were analyzed by gas chromatography-mass spectrometry (GC-MS) using a HewtettPackard 5995 instrument coupled to an HP-300 datasystem. The chromatographic conditions were the same as described above except that helium was used as carrier gas. Data were acquired in a scanning range of m/z 50-550 at 1 s per decade. MS temperatures were: transfer line 300°C, ion source 200°C and analyser 230°C. The GC profiles were quantitated by comparison with adequate external standard mixtures: n-C14, n-C22, n-C32 and /'/-C36 for the aliphatic hydrocarbons, and phenanthrene, chrysene and benzo(a)pyrene for the aromatics.
tion. Actually, among other facilities, it contains the major harbour of the island, a crude oil refinery and a gas manufacture plant. Furthermore, the bay receives an important part of the untreated residual waters of the City ( - 2 2 0 000 m 3 day-l). These, along the waters of three main rivers: Luyano (56 000 m 3 day-l), Martin Perez (26 000 m 3 day -1) and Tadeo (5000 m 3 day-l), account for most of the freshwater input. These rivers, in turn, act as draining system of several industries, namely ethanol and liquor distilleries (1600 m 3 day -l) and a yeast plant (400 m 3 day-l). The main discharges of pollutants entering into the bay have been summarized in Table 1, according to a report from Mederos et al. (1984). The narrow dimensions of the channel connecting the bay with the Caribbean Sea represent an important limitation for the output of these anthropogenic discharges into the sea. It has been estimated that the residence time of the water in the bay is, on average, 5.5 days (Mederos et al., 1984). With this information an initial estimate of the standing stock of pollufants in the bay can be obtained. Thus, for the petroleum hydrocarbons, the following equation can be formulated: dx/dt -- 34 -- x/5.5
(1)
Results and Discussion Area of study The Havana Bay (Fig. 1) encompasses an area of -5.2 km 2 with an average depth of about 9.2 m (total water volume 4.7 × 107 m3). It is connected to the open sea by a long (1574 m), narrow (140 m) and relatively deep (12-15 m) channel. This bay has undergone intense human activity since the early Spanish coloniza-
Fig. 1 Map of Havana Bay. Lettered (W) and numbered points correspond to sampling stations for water and sediments, respectively.
TABLE 1 Main anthropogenic discharges into Havana Bay (adapted from Mederos et al., 1984). Water flow*
Petroleum Hydrocarbons t
Oils and Greases t
Totai mtrogen t
Totai phosphorous t
Suspended panicles t
Urban draining system
220 000
20 000
8500
1200
1800
18 000
Rivers
100 000
1700
3000
570
860
4000
13 000
11 000
6500
500
750
5700
400
850
330 000
34 000
18 000
2300
3400
28 000
Direct industrial effluents Harbour Total *m 3 day-~; tkg day -~.
263
Marine Pollution Bulletin
where x is the amount of hydrocarbons present in the bay (in tonnes) and t is the time (in days). In this equation it is assumed that the waters of the bay are well mixed and that the channel is the only output of hydrocarbons. A better estimation requires the consideration of the internal hydrology of the bay as well as the biogeochemical processes taking place, namely degradation, sedimentation and evaporation. The integration of equation 1 gives rise to the following expression: x-- 187. (1 - e x p (-t/5.5))
(2)
where x is the amount of hydrocarbons present in the bay (in tonnes) and t is the time (in days). That is, 187 t of petroleum hydrocarbons will be the standing stock when a steady state is reached. Taking into account the total water volume of the bay, this amount corresponds to an average concentration of about 4 mg 1-t. Similar equations can be formulated for the other major anthropogenic inputs, leading to the following estimates: 100 t of oils and greases (2 mg l-X), 13 t of total nitrogen and 19 t of total phosphorous. Irrespective of the accuracy of the estimates, the order of magnitude of the numbers is indicative of a high anthropogenic influence. Diverse sampling stations were selected within the bay for the study of these anthropogenic inputs (see Fig. 1). Waters were collected monthly at four depth levels (surface, 2 m, 4 m and bottom) over a 3 year period (sites W-I-W-5). Two sediment sampling cruises were performed in 2 successive years (sites 1-11). The corresponding samples were analysed separately.
Nutrients and dissolved oxygen Table 2A reports some chemical parameters for the Havana Bay surface waters. The average values indicate a rather uniform and eutrophic situation which results from the high input of nutrients (Table 1) and the limited exchange of waters between the bay and the open sea. However, the higher dissolved oxygen and the slightly lower concentrations of nutrients in Stn W-1 (the channel) reflect the influence of marine water in this site. Conversely, Stn W-3 (Guasabacoa inlet) appears as the area of higher nutrient load and, therefore, lowest dissolved oxygen. This inlet receives the waters of the Luyano and Martin Perez rivers carrying residues of several industries as well as sewage from the southern area of Havana City. The vertical profiles of these parameters are reported in Table 2B, indicating rather good mixing conditions within the water column. However, despite of this, two water layers can be defined, the shallower ( < 2 m) containing the highest concentrations of nutrients and lowest dissolved oxygen. This difference parallels the salinity gradient, indicating the association of most anthropogenic inputs to freshwater discharges. In turn, such layering is in correspondence with the predominant water mass movements of the bay: exit of brackish-to-salt water by the surface (5-10 cm s-1) and entrance of sea water by the bottom ( - 4 cm s-l). In this respect, the higher levels of dissolved oxygen observed in the deep waters are attributed to the supply provided by the input of marine water. Hydrocarbons in the water column The average hydrocarbon concentrations found in
TABLE 2 Some water quality parameters of five sampling stations at Havana Bay (see Fig. 1). The mean values correspond to data collected monthly. A Horizontal distribution Sampling stations
Dissolved oxygen*
W-1 W-2 W-3 W-4 W-5 Mean
4.4 1.5 1.4 2.0 2.7 2.4
B Vertical distribution Depth
± ± ± ± ± ±
NH4*
1.4 2.2 1.9 1.0 1.3 1.5
3.8 3.4 6.0 4.8 5.6 4.8
Dissolved oxygen*
Surface 2m 5m Depth
1.9 1.8 2.3 3.6
± ± ± +
± + ± ± ± ±
Total phosphoroust
2.8 2.7 2.6 3.3 3.6 3.3
NH4 t
1.8 1.6 1.0 1.6
4.7 5.3 5.1 4.2
± ± ± ±
2.0 2.5 2.6 1.7 1.9 2.1
± ± ± + ± ±
Total nitrogen t
1.6 1.7 1.2 0.9 1.2 1.4
23 46 48 39 39 39
Total phosphorous*
3.2 3.8 3.2 2.9
2.7 2.6 1.7 1.7
± ± ± ±
± ± ± ± ± ±
Total silicon t
13 23 27 42 29 27
5.0 11.0 8.7 5.8 7.7 7.2
Total nitrogen t
1.7 2.0 1.0 1.0
46 41 38 31
+ ± + ±
± ± ± ± ± ±
2.8 6.7 6.5 3.9 4.0 8.0
Total silicon t
27 25 24 32
10.0 8.8 6.4 4.8
+ ± ± ±
6.5 5.7 3.9 3.1
*mg I-t; t p,g-at 1-1.
TABLE 3 Average hydrocarbon concentrations in the water column of Havana Bay (mg 1-1) (sampling stations indicated in l~ig. 1). Depth
0 2m 5m Bottom Mean
264
W-1
W-2
W-3
Samplingstations W-4
W-5
3.0 3.0 2.7 2.7 2.8
3.9 3.7 3.5 3.4 3.6
3.6 3.4 3.3 3.2 3.4
4.0 3.9 3.8 3.7 3.9
3.3 3.0 2.9 2.9 3.0
Mean 3.6 3.4 3.2 3.2 3.3
± ± ± ± ±
1.8 1.7 1.6 1.4 1.6
Volume20/Number6/June 1989 the waters of the Havana Bay are shown in Table 3. In general terms, the concentrations of petroleum derived hydrocarbons in seawater are difficult to compare with previously reported data due to the great variety of sampling and analytical methods used. However, considering cases where also infrared spectroscopy was used for quantitation we may conclude that the Bay supports a high hydrocarbon load. In fact, Ahmed et al. (1974) reported values ranging from 0.19-0.82 mg 1-1 for the Boston Harbour and similar concentrations were found in the Goteborg Harbour (0.47-0.72 mg 1-1) (Carlberg & Skarstedt, 1972). The average concentrations for several semi-enclosed areas of the Mediterranean (harbours, bays, etc.) were slightly higher (0.58-1.50 mg 1-1) (Cuberes & Albaiges, 1975; UNEP, 1980) but below to those found in the Havana Bay. As far as the Havana Bay spatial distribution is concerned, both horizontal and vertical, a trend paralleling
that of nutrients and dissolved oxygen is observed (Table 2). Again, the lowest levels correspond to the waters of Stn W-1 (the channel) but the highest concentrations are found in Stn W-4 (Marimelena inlet), close to the crude oil refinery. In general, the highest hydrocarbon levels correspond to the surface waters, consistently with the above reported trends. Representative chromatograms of the dissolved+ dispersed hydrocarbons in the Havana Bay waters are shown in Figure 2A. Besides the predominant distribution of C23-C30n-alkanes without odd-to-even carbon number preference, an important feature of this profile consists in the series of C23-C30/SO and ante~So alkanes. As it can be observed, this distribution (A) differs considerably from those corresponding to the predominant petroleum discharges occurring in the Bay 03). On the contrary, these hydrocarbon mixtures are similar to those of weathered petroleum tanker washings (Albaigrs & Cuberes, 1980). However, various labora-
® i
271 25 J d
91
./
I I
17
®
il5
19 23
21
13
Fig. 2 Gas chromatograms of (A) the total extract of a representative water sample from HavanaBay and (B) the predominant petroleum discharges. Numbers refer to n-alkane chain-length. Dots to iso and anteiso alkanes. 265
Marine Pollution Bulletin
tory experiments have shown that these mixtures may also be produced by microbial degradation of petroleum (Walker & ColweU, 1979).. In fact, these distributions of hydrocarbons have been found in cultures of diverse microorganism species (see e.g. Baraud et al., 1967; Davis, 1968; Naccarato et al., 1974). Gassmann (1982) reported the occurrence of these hydrocarbon profiles, including the series of /so- and ante/so-alkanes, in North Sea waters. In agreement with the previous observations and some quantitative considerations he proposed that they were formed from the bio-conversion of oil spillages. In a series of recent experiments, we have also found that these hydrocarbons may be produced by microbial re-synthesis of sedimentary n-alkane mixtures (Grimalt et aL, 1988). At this respect, the similarity between the concentration level predicted by equations 1 and 2 (-4 mg 1-1) and that measured by analytical methods (-3.3 mg 1-1) deserves particular attention. In case that the hydrocarbon profiles displayed in Figure 3A originate from residual products or microbial degradation of the predominant petroleum discharges (Fig. 3B), it would be expected, in view of the strong compositional differences, that the measured hydrocarbon concentrations would be much lower than the values estimated from equations 1 and 2. Since both concentration values are similar the hypothesis of bio-conversion is reinforced. Sedimentary hydrocarbons The concentrations of sedimentary hydrocarbons in Havana Bay are indicated in Table 4. Two values per sampling site are given, corresponding to the two collection cruises. Similarly to the water samples, the spatial distribution of these components is rather uniform. Only the samples located within Marimelena inlet (sites 8 and 10) exhibit slightly higher levels of hydrocarbons, which can be attributed to the local influence of the petroleum refinery. These values can be compared, for instance, with the hydrocarbons levels in several British and Spanish estuaries: 10-100 p.g g-t (Readman et al., 1986) and 1-250 ~tg g-1 (Grimalt et aL, 1986), respectively, or with those from sediments adjacent to effluent discharge sites of a sewage treatment plant in Chesapeake Bay (2-150 ~tg g-l; Brown & Wade, 1984). The levels of sedimentary hydrocarbons in Havana Bay are far higher than those of these samples, which also correspond to petroleum polluted areas.
Two representative examples of the sedimentary aliphatic (A) and aromatic (B) hydrocarbons are displayed in Fig. 3. The aliphatic fraction is largely composed of an unresolved complex mixture (UCM) of alkanes where norpristane, pristane, phytane and a series of C29-C32hopanes constitute the main resolved peaks. As repeatedly reported (Albaigrs & Albrecht, 1979; Farran et al., 1987), the presence of these ClsC20 regular isoprenoid hydrocarbons and the 170~, 2113 (H) stereochemistry of the hopane series, with t w o chromatographically resolved C-22 epimers for the extended homologs, is indicative of a petroleum origin for the UCM. The lack of n-alkanes is due to microbial degradation, as it has commonly been observed in many heavily polluted sedimentary environments (see e.g. Farrington & Quinn, 1973; Giger et aL, 1974; Wakeham, 1976). Accordingly, two different microbial processes related with the occurrence of petroleum hydrocarbons in Havana Bay are oberved. One, in the water column, involving 'bio-synthesis' of long chain n-alkane homologs, and the other, in the underlying sediments, consisting of the elimination of the straight chain aliphatic components. Different environmental conditions must determine their occurrence. It has been previously indicated that despite the high pollutant and nutrient load in the Bay, some small level of oxygen has usually been found in the water column. In contrast, the sediments are anoxic, with hydrogen sulphide emissions being often detected. The aromatic hydrocarbons of these sedimentary samples are predominated by parent polycyclic components such as fluoranthene, pyrene, chrysene, benzopyrenes, etc. (see Fig. 3B). They correspond to contributions of residues from high temperature processes like fossil fuel combustion (LaFlamme & Hites, 1978; Prahl & Carpenter, 1983; Windsor & 1-rites, 1979). Probably the main source is urban run-off and effluents from the gas manufacture and electric power plants located along the Bay. Besides these predominant hydrocarbons, other resolved components are present in high proportion, mostly methyl and dimethyl substituted phenanthrenes, fluoranthenes, pyrenes and chrysenes. These again evidence petroleum contributions (Tissot & Welte, 1984) which, in this aromatic fraction, are also apparent from the presence of an unresolved complex mixture. The dual source origin of the sedimentary hydro-
TABLE4 Hydrocarbonsconcentrationsin ~ e s e d i m e n t s o f H a v ~ a B a y ( ~
g-~).SamplingsitesinFig. 1.
Aliphatics
Station No. 1 2 3 4 5 6 7 8 9 10 11
Aromatics
Waterdepth (m) 15 9 12 9 11 6 8.5 5 3 12 14
Monocyclic 1200" 5500 2400 4900 3800 3800 5000 7500 4100 5900 1500
1000 t 4800 3300 3300 2600 3300 4300 5300 3800 6000 2000
* and * duplicate analysis corresponding to replicate sampling.
266
400* 2100 490 2800 2200 1200 2100 5100 1100 2300 1200
Polycyclic 500 t 3000 200 1500 2100 1200 1800 4000 1400 1900 1000
600* 2400 900 2800 1800 400 600 1600 1600 600 800
300 t 1800 100 1600 1000 300 300 900 1000 1000 600
Volume 20/Number 6/June 1989
carbons isolated in the polycyclic aromatic hydrocarbon fraction is also reflected in their quantitative distribution. In this case, the highest concentrations (Stns 2 and 4; Atares and Guasabacoa inlet) do not correspond to the samples located closer to the refinery, although the levels found in Stn 8 are also above the bay average values.
all n-alkanes. In this situation of heavy pollution stress repeatedly observed during the 3 year period, the hydrocarbon composition of the sediments contrasts strongly with that of the water column and questions the utility of sedimentary samples as geoaccumulators. Financial support from the UNESCO/UNID Project no. CUB/80/001 and Acuerdo de Cooperacion Hispano-Cubano is gratefully acknowledged.
Conclusions The high levels of industrial, urban and petroleum pollution in Havana Bay apparently result into two main microbial hydrocarbon transformation processes: 1. in the water column, with the formation by 'biosynthesis' of distributions of C23-C30n-alkanes without odd-to-even carbon number preference, along with a series of C23-C3o iso and anteiso alkanes; and 2. in the sediments, with the elimination by 'bio-degradation' of
Aceves, M., Grimalt, J. O., Albaig6s, J., Broto, E, Comellas, L. & Gassiot, M. (1988). Analysis of hydrocarbons in aquatic sediments. II. Evaluation of common preparative procedures for petroleum and chlorinated hydrocarbons. J. Chromatogr. 436,503-509. Ahmed, A. M., Beasley, M. D., Efromson, A. C. & Hires, R. A. (1974). Sampling errors in the quantitation of petroleum in Boston Harbor water. In Marine Pollution Monitoring (Petroleum), NBS SP 409, pp. 109-111. National Bureau of Standards, Washington, D.C. Albaig6s, J. & Albrecht, P. (1979). Fingerprinting marine pollutant hydrocarbons by computerized gas chromatography-mass spectrometry. Internat. J. Environ. Anal. Chem. 6,171-190.
@ 6
87 9
2
1"2
® 15 18 16
13
J Fig. 3 Gas chromatograms of the aliphatic (A) and aromatic (B)
hydrocarbons present in the sediments of Havana Bay. Peak identification as follows: A) 1. norpristane; 2. pristane; 3. phytane; 4. 17~2113(H)-30-norhopane; 5. 17ct211~(H)-hopane; 6. 17at21~(H)-homohopane 22S; 7. 170~211~(H)-homohopane 22R; 8. 170,211~(H)-bishomohopane 22S; 9. 17a211~(H)-bishomohopane 22R. B) 10. phenanthrene; 11. fluoranthene; 12. pyrene; 13. benzofluorenes; 14. benz(a)anthracene; 15. chrysene; 16. benzofluoranthenes; 17. benzo(e)pyrene; 18. benzo(a)pyrene; 19. indeno(1,2,3-cd)pyrene; 20. benzo(ghi)perylene.
267
Marine Pollution Bulletin Albalgrs, J. & Cuberes, M. R. (1980). On the degradation of petroleum residues in the marine environment. Chemosphere 9,539-545. Aminot, A. (1983). Prrl~vement et prr-traitement des 6chantillons: eau de mer, sddiments et organismes marins. In Manuel des analyses chimiques en milieu marin (A. Aminot & M. Chaussepied, eds), pp. 19-32. CNEXO, Brest. Baraud, J., Cassagne, C., Genevois, L. & Joneau, M. (1967). Prrsence d'hydrocarbons darts rinsaponifiable des lipides des levures. C.R. Acad. Sc. Paris 265(D), 83-85. Brown, R. C. & Wade, T. L. (1984). Sedimentary coprostanol and hydrocarbon distribution adjacent to a sewage ouffall. Water Res. 18, 621-632. Carlsberg, S. R. & Skarstedt, C. B. (1972). Determination of small amounts of non-polar hydrocarbons (oil) in sea water. J. Cons. Int. Explor. Mer 34, 506-515. Cuberes, M. R. & Albalges, J. (1975). Control de la contaminacirn marina por hidrocarburos y su aplicacirn al litoral mediterrfineo espafiol. In Proc. I Congreso Iberoamericano del Medio Ambiente, pp. 937-952. CEMA, Madrid. Davis, J. B. (1968). Paraffinic hydrocarbons in the sulfate-reducing bacterium Desulfovibrio desulfuricans. Chem. Geol. 3,155-160. Farran, A., Grimalt, J., Albaigds, J., Botello, A. V. & Macko, S. A. (1987). Assessment of petroleum pollution in a Mexican River by molecular markers and carbon isotope rations. Mar. Pollut. Bull 18, 284-289. Farrington, J. W. & Quirm, J. G. (1973). Petroleum hydrocarbons and fatty acids in waste water effluents. J. Wat. Pollut. Control Fed. 45, 704. Farrington, J. W. & Tripp, B. W. (1977). Hydrocarbons in western North Atlantic surface sediments. Geochim. Cosmochim. Acta 41, 1627-1641. Gassmann, G. (1982). Detection of aliphatic hydrocarbons derived by recent "Bio-conversion" from fossil fuel oil in North Sea Waters. Mar. Pollut. Bull. 13, 309-315. Gearing, P.. Gearing, J. N., Lytle, T. F. & Lytle, J. S. (1976). Hydrocarbons in 60 northeast Gulf of Mexico shelf sediments: a preliminary study. Geochim. Cosrnochim. Acta 47, 2115-2119. Giger, W., Reinhardt, M., Schaffner, C. & Stumm, W. (1974). Petroleum-derived and indigenous hydrocarbons in recent sediments of Lake Zug, Switzerland, Environ. Sci. Technol. 8, 454. Grimalt, J., Bayona, J. M. & Albaiges, J. (1986). Chemical markers for the characterization of pollutant inputs in the coastal zones. Journ. Etud. Pollut. C.LE.S.M. 7,533-543.
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Grimalt, J. O., Torras, E. & Albaigrs, J. (1988). Bacterial reworking of sedimentary lipids during sample storage. Org. Geochem. 13, 741746. Keizer, P. D., Dale, J. & Gordon, D. C. Jr. (1987). Hydrocarbons in surficial sediments from the Scotian shelf. Geochirn. Cosmochim. Acta 42,165-172. LaFlamme, R. E. & Hites, R. A. (1978). The global distribution of polycyclic aromatic hydrocarbons in recent sediments. Geochim. Cosmochim. Acta 42,289-303. Mederos, R., Jaime, N., Villasol, A., Shabalina, L. & Espinosa, M. A. (1984). Investigaci6n y control de la contaminacirn marina en la Bahia de la Habana. Report of the Ministry of Transport. Project CUB-80-001 -PNUD-PNUMA-UNESCO. Naccarato, W. F., Gilbertson, J. R. & Gelman, R. A. (1974). Effects of different culture media and oxygen upon lipids of Escherichia coli K-12. Lipids 9,322-327. Prahl, F. G. & Carpenter, R. (1983). Polycyclic aromatic hydrocarbon (PAH)--phase associations in Washington coastal sediments. Geochirn. Cosmochim. Acta 47, 1013-1023. Readman, J. W., Preston, M. R. & Mantoura, R. F. C. (1986). An integrated technique to quantify sewage, oil and PAIl pollution in estuarine and coastal environments. Mar. Pollut. Bull 17,298-308. Tavares, T. M., Rocha, V. C., Porte, C., Barcelo, D. & Albaiges, J. (1988). Application of the mussel watch concept in studies of hydrocarbons, PCBs and DDT in the Brazilian Bay of Todos os Santos (Bahia). Mar. Pollut. Bull. 19,572-578. Tissot, B. P. & Welte, D. H. (1984). Petroleum Formation and Occurrence. Springer-Verlag, Heidelberg. UNEP (1980). Summary reports on the scientific results of MED POL I. UNEP/IG. 18/1NF. 3. Venkatesan, M. I., Brenner, S., Ruth, E., Bonilla, J. & Kaplan, I. R. (1980). Hydrocarbon in age-dated sediment from two basins in the Southern California Bight. Geochim. Cosmochim. Acta 44, 789802. Wakeham, S. C. (1976). A comparative survey of petroleum hydrocarbons in lake sediments. Mar. Pollut. Bull 7,206-211. Walker, J. D. & Colwell, R. R. (1976). Long-chain n-alkanes occurring during microbial degradation of petroleum. Can. J. Microbiol. 22, 886-891. Windsor, J. G. & Hites, R. A. (1979). Polycyclic aromatic hydrocarbons in Gulf of Maine sediments and Nova Scotia soils. Geochim. Cosmochim. Acta 43, 27-33.