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Adv. mar. B i d , Vol. 15, 1978, pp, 289-380. POLLUTION STUDIES WITH MARINE PLANKTON PART I . PETROLEUM HYDROCARBONS AND RELATED COMPOUNDS E. D. S . CORNER The Laboratory, Marine Biological Association, Plymouth, England I. IT. 1x1. IV. V. VI. VII. v m . IX. X . XI. Introduction . . .. .. .. .. .. .. .. .. Hydrocarbon Levels in Sea Water . . .. .. .. .. .. A. Studies Primarily concerned with Alkanes . . .. .. .. B. " Dissolved " and Particulate Hydrocarbons .. .. .. C. Hydrocarbons in or near the Surface of the Sea . . .. .. D. Comprehensive Analyses . . .. .. .. .. .. Hydrocarbons in Plankton . . .. .. .. .. .. .. A. Phytoplankton .. .. .. .. .. .. .. Phytoplankton and Crude Oil as Sources of Hydrocarbons in the Sea . . .. ., .. .. .. .. .. .. C. Zooplankton . . .. .. .. .. .. .. .. Toxicity Studies with Phytoplankton .. .. .. .. .. B. Studies using Naphthalene . . .. .. .. .. .. Mechanisms of Phytotoxicity . . .. .. .. .. .. .. Controlled Eco-system Experiments . . .. .. .. .. .. Fate of Hydrocarbons in Zooplankton .. .. '. .. .. A. Uptake and Release . . . . .. .. .. .. . . B. Quantitative importance of the Dietary Pathway .. .. C. Long-term Exposure Experiments . . .. .. .. .. E. Release of Hydrocarbons in Faecal Pellets . . .. .. .. Toxicity Studies with Zooplankton . . .. .. .. .. .. A. CrudeOil .. .. .. .. .. .. .. .. B. Water-soluble Hydrocarbons . . .. .. .. .. .. C. Possible Effects of Hydrocarbons on Reproduction by Zooplankton D. Summary and General Comments . . .. .. .. . . Conclusions . . .. .. .. .. .. .. .. .. A. Chemical Analyses . . .. .. .. .. .. .. B. Toxicity Studies .. .. .. .. .. .. .. C. Biochemical Work . . .. .. .. .. .. .. Acknowledgements . . .. .. .. .. .. .. .. B. A. Studies using Crude Oils and their Water-soluble Fractions . . D. Metabolism .. .. .. .. .. .. .. .. References . . .. .. .. .. .. .. .. .. 289 290 290 291 293 294 300 303 305 309 311 317 319 327 329 331 335 335 339 340 342 348 351 352 354 356 357 362 362 363 364 365 365 290 E. D. 5. UOBNER I. INTRODUCTION Marine organisms, including plankton, having been exposed to petroleum hydrocarbons released from submarine seeps throughout geological time, are likely to have evolved physiological and bio- chemical mechanisms allowing them to adapt to the presence of small quantities of these compounds in their natural environment. Never- theless, there is considerable current interest in understanding what might happen to planktonic organisms exposed to the additional and localized inputs of hydrocarbons and related compounds that result from accidental spillages arising from relatively recent industrial activities such as the off-shore production and transport of crude oil. Accordingly, consequent upon incidents such as the wrecking of the tanker " Torrey Canyon ' ) ) a vast and widely dispersed literature has arisen during the past ten years dealing with the effects of petroleum hydrocarbons on numerous marine organisms. The publications on plankton considered in the present review, most of which refer to laboratory studies, are discussed in the context of a simplified food-chain model that begins with sea water and proceeds through phytoplankton to zooplankton. Although such a frame-work serves to carry the main theme of the treatment, several additional but relevant topics have had to be included. For example, in dealing with hydrocarbons in sea water attention has had to be given to matters such as their spatial distribution and the relative amounts in solution and in particulate form. Again, in discussing the levels and types of hydrocarbons in plankton it has been necessary to consider compounds of recent biogenic origin, some of which can also occur in crude oil. Furthermore, as certain studies with zooplankton have shown that the animals do not exclusively accumulate hydrocarbons from phyto- plankton diets, work is also described that deals with the direct uptake of these compounds from solution in sea water. Finally, although the simplified food-chain model is not extended to include fish and benthic animals, consideration is given to factors affecting the retention of hydrocarbons by zooplankton, particularly copepods, which is of key importance in the transfer of these compounds to fish ; as well as to the release of hydrocarbons in faecal pellets, a possible means by which such compounds originally present in the euphotic zone could be eventually transferred to animals that dwell in sediments. 11. HYDROCARBON LEVELS IN SEA WATER When studying the accumulation and fate of hydrocarbons in plankton, and the possible effects of these compounds on the organisms, POLLUTION STUDIES WITH MARINE PLANKTON-I 291 it is necessary to bear in mind the levels of hydrocarbons that plankton normally encounter in various sea areas. Accordingly, a brief review of the available data is attempted by way of introducing the more detailed treatment of studies with plankton that are dealt with in later sections. Although numerous attempts have been made to ascertain the levels and types of hydrocarbons present in sea water under a variety of conditions, the methods used (reviewed by Farrington and Meyer, 1975) have usually provided data for only a particular fraction of the various kinds of hydrocarbons present. More comprehensive analyses have occasionally been made (Barbier, Joly, Saliot and Tourres, 1973; Brown, Searl, Elliott, Phillips, Brandon and Monaghan, 1973), but generally the data still refer to groups of hydrocarbons (e.g. mono- cyclic aromatics) rather than to individual compounds. Data for individual hydrocarbons do exist, but most deal with n-alkanes and the iso-alkanes pristane and phytane (see Figs 2 and 3). A. Studies primarily concerned with alkanes Swinnerton and Linnenbom (1967) detected the simplest n-alkane, methane, at concentrations ranging from 0.025 to 0.283 pg/l at various depths in sampling areas in the Gulf of Mexico and 0.047-0.060 pgll in the North Atlantic. Frank, Sackett, Hall and Fredericks (1970) found somewhat higher concentrations of methane, 0-06-1.25 pg/1, near oil seeps in the Gulf of Mexico: ethane and propane were also present, but at much lower levels. It is known from the work of Blumer (1970) that dissolved organic compounds in coastal waters include a variety of hydrocarbons. Thus, in a qualitative study he identified n-alkanes from C,, to C,, with maximum concentration at C,,-C,, : the compounds included those with odd and others with even numbers of carbon atoms in roughly equal amounts, a distribution different from that in recent marine sediments (where odd-numbered n-alkanes preponderate) but similar to that in marine algae (Clark and Blumer, 1967). Isoprenoid hydro- carbons were represented by pristane ((&), which is also found in marine algae (Clark and Blumer, 1967) and zooplankton (Blumer, Mullin and Thomas, 1963, 1964), as well as phytane (C2,,) which is not commonly detected in marine organisms. Olefinic hydrocarbons were also found, one being identified as squalene which is also present in copepods (Blumer et aZ., 1964) and the liver oils of various species of shark (Heller, Heller, Springer and Clark, 1957 ; Blumer, 1967 ; Corner, Denton and Forster, 1969). Some of the hydrocarbons detected by Blumer (1970) have been 292 1. D. 5. CORNER identified and estimated by Whittle, Mackie, Hardy and McIntyre (1973) in water samples collected from 13 stations off the Scottish coast. Using sub-surface samples (3 m depth) that had been filtered through a 20 pm mesh they found levels of 0.3-1.5 pg/l for totalalkanes, 0.015-0-043 pg/l for pristane and < 0.001-0.014 pg/l for phytane. Similar to Blumer’s (1970) observations the peak levels for individual n-alkanes were usually obtained with C,,-C,, compounds. Hydrocarbon levels vary considerably with sea area. Thus, Mackie, Platt and Hardy (1978), using techniques similar to those of Whittle et al. (1973), found that sea-water samples from King Edward Cove, South Georgia, contained 5.8 pg/l of n-alkanes within the range n-C,5-n-C33, together with 0.18 pg pristane/l; Iliffe and Calder (1974), studying hydrocarbons in the Gulf of Mexico and Caribbean Sea, found an average level of 47 pg/l for non-polar hydrocarbons in the Florida Strait, 12 pg/l in the mid-Gulf region, 12 pg/l in the Yucatan Strait, 5 pg/l in the Cariaco Trench and 8 pg/l in the Caribbean Sea, the samples containing n-alkanes in the range C,, to C,, with peak concentrations in the C,, to C,, region ; Carlberg and Skarstedt (1972), using infrared spectroscopy, obtained values in the range < 50 to 120 pg/l for non-polar hydrocarbons a t ten stations in the Baltic and Kattegat. Hardy, Mackie, Whittle, McIntyre and Blackman (1977) have recently described further data for the amounts of n-alkanes (C15 to CS3) in samples of sea water from various regions surrounding the U.K. The lowest value for n-alkanes in the surface film (mean value 5.7 pg/m2) was found in samples from the open sea (Celtic Sea) ; the the mean value for off-shore samples from sites near urban areas (62.9 pg/m2) was close to that for samples taken near oil refineries (64.2 pg/m2) and greater than that for those collected close to North Sea oil fields (32.8 pg/m2). Mean values for n-alkanes in sub-surface (Im depth) samples ranged from 0.57 pg/l (Celtic Sea) to 4.6 pg/l (North Sea oil fields). Studies described later (Section VII) show that hydrocarbons can enter zooplankton in two different ways: first, by direct uptake from solution in sea water ; second, by assimilation from particulate diets. In considering the quantities of hydrocarbons available to the animals in the sea it is therefore useful to know the relative amounts of the compounds that are present in solution and as particulate material. In addition, as certain species of zooplankton feed near the surface of the sea it is necessary to consider the spatial distribution of hydrocarbons, especially evidence for the presence of high concen- trations in the surface micro-layer. These topics are discussed in the next two sections. POLLUTION STUDIES WITH MARINE PLANKTON-I 293 B. " Dissolved " and particulate hydrocarbons Spillage of Bunker C oil from the grounded tanker " Arrow " in Chedabucto Bay, Nova Scotia, led to several studies of oil levels in that area and along the coast to Halifax Harbour and beyond (Levy, 1971, 1972; Forrester, 1971). Quantitative data were obtained by Levy (1971) for the levels of petroleum residues in the open ocean off Nova Scotia and in the St Lawrence system. Water samples were filtered through a 0.45 pm millipore membrane and the hydrocarbon content of the retained material was determined as equivalents of Bunker C oil using U.V. fluorescence spectroscopy. Similar analyses were made of hydrocarbons that passed through the filter, these being described as " dissolved ". The fluorescence technique is a rapid way of detecting aromatic compounds and allows a large number of samples to be processed in ship-board experiments ; occurring organic material can produce interference that is difficul to quantify (Gordon, Keizer and Dale, 1974), particularly highly con- jugated alkenes (Farrington and Meyer, 1975). The total levels of petroleum residues found in Chedabucto Bay by Levy (1971) were in the range 1fj-41 pg/l (as Bunker C oil equivalents). At several stations substantially higher concentrations of dissolved than particulate compounds were detected. Thus, in surface samples (1 m depth) particulate levels ranged from 5 to 16 pg/l and dissolved from 15 to 90 pg/l. Zsolnay (1971) measured what he terms '' non-olefinic '' hydro- carbons and describes as saturated hydrocarbons and aromatic com- pounds with only one ring in the Gotland Deep, a Baltic basin. Thin- layer chromatography was used to separate the hydrocarbons which were then estimated as total carbon. Average concentrations, based on samples from all depths (20-200 m) and expressed as carbon equiva- lents, were 57-2 pg C/1 for the dissolved hydrocarbons and 1.1 pg C/1 for the particulate, dissolved material in this case being defined as that passing through a pair of Whatman GF/C glass filters. Another study using thin-layer chromatography to separate the hydrocarbons from other lipids was that of Jeffrey (1970), who measured unsaturated hydrocarbons in Baffin Bay (Texas) and found 180 pg/l as dissolved (passing through a 0.3 pm filter) and 70 pg/l as particulate material. The particulate material was mainly phytoplankton, Baffin Bay being a shallow, warm region of high primary production. Nevertheless, the distribution of hydrocarbons between dissolved and particulate forms does not always favour the soluble fractions. Sediments, for example, adsorb levels of these compounds far higher but natur??- 201 E. D. S. CORNER than those found in the associated sea water. Thus, Di Salvo and Guard (1975), studying the hydrocarbons attached to suspended sedi- ments in San Francisco Bay, found them to contain alkanes and aro- matic compounds in concentrations ranging from 190 to 6 188 mg/kg dry weight; by contrast the levels in the associated sea water were o d y 15-450 11.811. Marty and Saliot (1976) have shown that the relative amounts of n-alkanes in particulate and dissolved form depend upon whether the samples are taken from polluted or unpolluted areas. Thus, for coastal waters of the English Channel (Roscoff area) the concentra- tions of total dissolved (i.e. passing through a Whatman GF/C filter) C,, to C,, n-alkanes at 0.5 m depth was 0.11 pg/l compared with 0.28 pg/l for those in particulate form; by contrast, for off-shore waters near the West African coast (2 m depth), the total quantity in solution was 5.66 pg/l but that in particulate form only 0.32 pg/l. One would expect the hydrocarbons detected off the West African coast to be associated with the high primary production in a region of upwelling, for Zsolnay (1973) has described a close correlation between hydrocarbon and chlorophyll a levels in water samples from the same sea area. Likewise Parker, Winters, Van Baalen, Batterton and Scalan (1976) detected higher levels of n-alkanes in spring (0.64 pg/l) than at other seasons (0.13-0.23 pg/l) in sea water samples from the Gulf of Mexico. C. Hydrocarbons in or near the surface of the sea The presence of high concentrations of hydrocarbons in the surface micro-layer of the sea was noted by Garrett (1967) in samples from various Atlantic and Pacific sites near North America, but the com- pounds were not identified. Swinnerton and Linnenbom (1967) measured n-alkanes of low molecular weight (mainly methane) by gas- chromatography in water samples from the Gulf of Mexico (South of Mobile, Alabama) and North Atlantic (500 km west of Ireland). They found higher concentrations a t the surface than a t depth (500 m) in the Gulf of Mexico samples, although peak concentrations occurred a t 30-40m. No significant change in hydrocarbon level with depth was observed in the Gulf of Mexico survey by Frank et al. (1970). Iliffe and Calder (1 974) found higher levels of non-polar hydrocarbons at a depth of 1 m (24 pg/l) than at other depths in the Yucatan Strait, but in the Florida Strait the highest hydrocarbon concentration (75 pg/l) was a t a depth of 144 m. Whittle, Mackie and Hardy (1974), POLLUTION STUDIESWITH MBRINE PLANKTON-I 295 analysing hydrocarbons at different depths in the Clyde, found only 3.21 pg/l in the surface film compared with 7-8 pg/l in the top 15 cm, although at middle depth (10 m) the value obtained was an order of magnitude lower (0.31 pg/l). Duce, Quinn, Olney, Piotrowicz, Ray and Wade (1972) detected three hydrocarbons, tentatively identified as CZ1.,, C,,., and C,,., at a concentration of 8.5 pg/l in the surface micro-layer (100-150 pm) compared with 5.9 pg/l at 20 cm depth. Wade and Quinn (1975) measured the total hydrocarbons present in samples of the surface micro-layer (100-300 pm) from the Sargasso Sea and found the levels to vary from 14 to 559 pg/l (average 155) compared with 13-239 pg/l (average 73) at 20-30 cm depth: n-alkanes from C,, to C,, accounted for 11% of the total hydrocarbons in combined micro-layer and sub- surface samples, being present at an average level of 25.1 pg/l. The authors concluded that a major source of the hydrocarbons was particles of weathered pelagic tar with diameter ranging from 1.0 mm down to 0.3 pm located in the surface micro-layer. Earlier, Morris and Butler (1973) had reported the large amounts of pelagic tar that could be collected by neuston net from the surface of the Sargasso Sea, the average value being 9.4 mg/m2. By comparison, the mean level recovered in the same way from the surface of the North Sea was only 317 pg/m2 (Offenheimer, Gunkel and Gassmann, 1977). The accumula- tion and retention of floating material in the Sargasso Sea is well known. The average level for pelagic tar in the Mediterranean was even greater : thus, Morris and Butler (1973) gave a figure of 20 mg/m2. However, evidence from a more recent study (Morris, Butler and Zsolnay 1975) indicates that the average level of pelagic tar in the Mediterranean has now fallen to 9.7 mg/m2, a value much closer to that for the Sargasso Sea. Conover (1971) has shown that zooplankton are able to ingest small droplets of oil and it seems probable that zooplankton species such as Anomalocera patersoni Templeton that live near the surface of the sea could also ingest small tar particles. Hydrocarbons assimilated from these particles might then be available for transfer to higher trophic levels ; in addition, unassimilated material could eventually reach the benthos as faecal pellets (see p. 348). Tar particles represent a persistent legacy of spilt oil, probably taking years to be degraded because they contain large amounts of high-melting point waxes and asphaltenes (Morris and Bulter, 1973). Further observations on surface enrichment of n-alkanes have been made by Marty and Saliot (1976). The ratio between the con- centration of dissolved compounds in the micro-layer (0.44 mm film) 296 E. D. S. OORNER and that in the underlying water ranged from 6.3 :1 (Etang de Berre : Marseilles) to 161 :1 (Roscoff area) : the corresponding values in terms of particulate hydrocarbons were 170 :1 and 350 :1 respectively. It should be noted that these ratios, if calculated for a micro-layer of only 100 d thickness, would give enrichment factors 104-106 times greater. Marty and Saliot (1 976) concluded that the n-alkanes present in the surface micro-layer were in general of biological origin as they possessed a distribution concentrating on n-C,, to n-C,, which was found by Clarke and Blumer (1967) to be characteristic of marine algae. However, qualitative differences occur between sea areas : thus, Ledet and Laseter (1 974) describe the alkanes at the air-sea interface from off-shore Louisiana and Florida as mainly branched and cyclic com- pounds. Ideally, to establish the biological origin of hydrocarbons in sea-water samples from a particular area i t is necessary to make a direct comparison of these compounds with those present in the plankton: however, no detailed study of this type seems to have been made. Concerning work with aromatic hydrocarbons Levy (1971), in his studies of oil pollution in Chedabucto Bay, found values of 15-90 pgll for dissolved compounds a t a depth of 1 m compared with 7-9 pg/1 at 20 m. On the other hand, Gordon and Michalik (1971)) working in the same sea area, found slightly increasing concentrations with depth : 1.2 pg/l at 5 m, 1.4 pgll at 6-25 m and 1.8pgll at 26-50m. Subsequently, however, in a detailed study of this aspect in the northwest Atlantic Ocean, Gordon et al. (1974)) using Venezuelan crude oil as a reference standard for U.V. fluorescence measurements, obtained concentrations at the surface (0-3 mm) averaging 20-4 pg/l compared with 0.8 pg/l at 1 in and 0.4 pgll a t 5 m. Studies that include measurements of total mineral oil hydrocarbons have given conflicting evidence. Thus, Carlberg and Skarstedt (1972), using samples from Gijteborg Harbour, found values of 0.71 mg/l at the surface compared with 0.47 pg/l at 6 m depth. However, Pavletid, Munjko, Jardas and Matoricken (1975), estimating mineral oil concentrations at different depths in the Adriatic off the Jugo- slaviaii coast, found values of 1-40, 0.65, 1.56 and 10.98 mg/l at depths of 0, 2, 5 and 10 m at Monte Gargano ; but at another station (Pelegrin) surface samples were higher than those at depth, being 4.23, 2.39 and 0.82 mg/l at 0, 5 and 10 m respectively. The various hydrocarbon levels in the sea that have so far been discussed are summarized in Table I. TABLE I. EX~D~PLES OF HYDROCARBON LEVELS IN THE SEA Type of hydrocarbon Concentration Geographic location Reference Methane 0.025 to 0.283 Various depths between 0 and 500 m : Swinnerton and Linnenbom Methane 0.047 to 0.060 Various depths between 0 and 500 m: Swinnerton and Linnenbom Gulf of Mexico (1967) 0 Methane 0.06 to 1.25 Various depths between 0 and 3 742 m : Frank et al. (1970) 2 E n-Alkanes 0.3 to 1-5 3 m depth : Scottish Coast Whittle et al. (1973) e3 Pristane 0-015 to 0.043 3 m depth : Scottish Coast Whittle et al. (1973) 8 2 North Atlantic (1967) Gulf of Mexico (PLgIl) : 8 n-Alkanes (C15 to CJ 0.57 1 m depth: Celtic Sea Hardy et al. (1977) ti n-Alkanes (C15 to C3J 4.5 1 m depth : North Sea Hardy et al. (1977) 2 Non-polar 12 0 to 500 m: Mid-Gulf Iliffe and Calder (1974) c Non-polar 12 0 to 500 m: Yucatan Strait Iliffe and Calder (1974) 3 Phytane <0*001 to 0.014 3 m depth : Scottish Coast Whittle et al. (1 973) n-Alkanes (C15 to C=) 0 to 20 m : King Edward Cove, S. Georgia Mackie et al. (1978) Pristane 0 to 20 m : King Edward Cove, S. Georgia Mackie et al. (1978) z 5-8 0.18 Non-polar 47 0 to 500 m : Florida Strait, Gulf of Mexico Iliffe and Calder (1974) cd +I Non-polar 5 0 to 900 m : Carioco Trench Iliffe and Calder (1974) Non-polar 8 0 to 200 m : Caribbean Sea Iliffe and Calder (1974) Non-polar <50 to 120 0 to 100 m : Baltic and Kattegat Carlberg and Skarstedt (1972) H Total Carlberg and Skarstedt (1972) Non-polar <50 to 200 0 to 31 m : Goteborg Harbour Carlberg and Skarstedt (1972) Total 50 to 710 0 to 31 m: Goteborg Harbour Carlberg and Skarstedt (1972) Dissolved aromatic 1 <50 to 170 0 to 100 m : Baltic and Kattegat 15 to 90 1 m depth : Chedabucto Bay, Nova Scotia Levy (1971) Dissolved aromatic 7 to 9 20 m depth : Chedabucto Bay, Nova Scotia Levy (1971) t.a CD -l t9 a TABLE I-( continued) 0, Type of hydrocarbon Concentration (WlO Geographic location Particulate aromatic Particulate aromatic Dissolved non-olefinic Dissolved non-olefinic Dissolved non-olefinic Dissolved non-olefinic Dissolved non-olehic Particulate non-olefmic Particulate non-olehic Particulate non - olefinic Particulate non-olefinic Particulate non-olehic Dissolved unsaturated Particulate unsaturated Dissolved n-alkanes Particulate n-alkanes Dissolved n-alkanes (C14to C8,) Particulate n-alkanes (C14 to CS7) Dissolved n-alkanes Particu1at.e n-alkanes Dissolved n-alkanes (Cia to C3,) Particulate n-alkanes (C14 to C37) Total hydrocarbons Tot,al hydrocarbons 5 to 16 2 to 11 48 58 58 59 64 0.9 1.0 2.3 1.0 0.5 180 70 17.7 98.0 0.1 1 0-28 3-34 5.66 0.32 1144 14 to 559 13 to 239 1 m depth : Chedabucto Bay, Nova Scotia 20 m depth : Chedabucto Bay, Nova Scotia 20 m depth : Gotland Deep, Baltic Basin 70 m depth : Gotland Deep, Baltic Basin 110 m depth : Gotland Deep, Baltic Basin 150 m depth : Gotland Deep, Baltic Basin 200 m depth : Gotland Deep, Baltic Basin 20 m depth : Gotland Deep, Baltic Basin 70 m depth : Gotland Deep, Baltic Basin 110 m depth : Gotland Deep, Baltic Basin 150 m depth : Gotland Deep, Baltic Basin 200 m depth : Gotland Deep, Baltic Basin (depth not given) : Baffin Bay, Texas (depth not given) : Baffin Bay, Texas Surface micro-layer : Roscoff, English Surface micro-layer : Roscoff, English 0.5 m depth : Roscoff, English Channel 0.5 m depth : Roscoff, English Channel Surface micro-layer : West African Coast Surface micro-layer : West African Coast 2m depth : West African Coast 2 m depth : West African Coast Surface micro-laver : Sareasso Sea Channel Channel Reference Levy (1971) Levy (1971) Zsolnay (1971) Zsolnay (1971) Zsolnay ( 197 1 ) Zsolnay (1971) Zsolnay (1971) Zsolnay (1971) Zsolnay (1971) Zsolnay (1971) Zsolnay (1971) Zsolnay ( 197 1 ) Jeffrey (1970) Marty and Saliot (1976) Marty and Saliot (1976) pl P P Q 0 Ld 3 Jeffrey (1970) a Marty and Saliot (1976) Marty and Saliot (1976) Marty and Saliot (1976) Marty and Saliot (1976) Marty and Saliot (1976) Marty and Saliot (1976) Wade and Quinn (1975) 20 to 30 cm depth : Sarg&o Sea Wade and Quinn (1975) Total aromatic Total aromatic Total saturated Total a.romatics Total hydrocarbons Total hydrocarbons Volatile hydrocarbons (C, to C,) Volatile hydrocarbons (C, to C,) Non-volatile hydrocarbons Non-volatile hydrocarbons Total dissolved hydrocarbons Total dissolved hydrocarbons Total dissolved hydrocarbons Total dissolved hydrocarbons Total dissolved hydrocarbons Total oil Total oil Total oil n-Alkanes n-Alkanes n -Alkanes Total hydrocarbons Total hydrocarbons Total hydrocarbons 20.4 0.4 to 0.8 1 to 21 <1 to 3 0.8 to 5 0.3 to 2 0.33 0.10 14 to 270 25 137 10 19 37 43 1400 to 4 230 Surface : Chedabucto Bay 1-5 m depth : Chedabucto Bay Oto lOmdepth :NorthAtlantictankerroutes Brown et al. (1973) 0 to lOmdepth :NorthAtlantictankerroutes Brown et al. (1973) Surface : Pacific tanker routes Surface : Pacific tanker routes Surface : New York Harbour Gordon et al. (1974) Gordon et al. (1974) Brown and Searl (1976) Koons (1977) Searl et al. (1977) 3 to 10 m depth: Pacific tanker routes Brown and Searl (1976) cd 0 10 m depth : Pacific tanker routes Koons (1977) 2 8 0 to 10 m : Tokyo Harbour Brown et al. (1976) 2 Surface : Brest Harbour Barbier et al. (1973) m 2 000 m depth : West African Coast Barbier et al. (1973) ! 500 m depth : West African Coast Barbier et al. (1973) s 4 500 m depth : West African Coast Barbier et al. (1973) I 50 m deuth : West African Coast Barbier et al. (1973) 1 Surface :*Adriatic 5 m depth : Adriatic 820 to 10 980 10 m depth : Adriatic 0.64 Spring: Gulf of Mexico 0.23 Summer : Gulf of Mexico 0.13 Winter : Gulf of Mexico 10 m depth : North Sea Surface : Gulf of Alaska 1560 to 2 390 17 to 625 2.7 to 14.9 0.95 to 30.5 Subsurface : Gulf of Alaska ci 8 ;1 Parker et al. (1976) td Parker et al. (1976) * Pavletid et al. '(1975) Pavleti6 et al. (1975) Pavleti6 et al. (1975) Parker et al. (1976) E E Offenheimer et al. (1977) Data from Hertz (1974) cited by Myers and Gunnerson (1976) Data from Hertz (1974) cited by Myers and Gunnerson (1976) !I 8 I H 300 E. D. 8. UORNER D. Comprehensive analyses Few data exist covering the whole range of hydrocarbons in sea- water samples ; which is not surprising bearing in mind the difficulties encountered in analysing such a wide range of compounds. Frequently, the concentrations are so low that analytical equipment must be operated at maximum sensitivity ; moreover, the samples can easily be contaminated during collection. A further complication is that hydrocarbons do not maintain a steady concentration. They are constantly being removed or modified by processes such as microbial degradation, accumulation and metabolism by plankton and larger marine organisms, chemical and photochemical oxidation, volatiliza- tion, dissolution and adsorption on particulate material : at the same time they are being renewed by processes such as atmospheric trans- port, oil spills, submarine seeps and release from organisms. Further- more, in coastal areas, industrial effluents, sewage and rivers make a further contribution which can sometimes be substantial. Thus, Hites and Biemaiin (1972), studying organic compounds in the Charles River (Boston), detected the aromatic hydrocarbon naphthalene at a maxi- mum concentration of 3-4 mg/l. Other examples of high levels of oil in the sea are the discharges from off-shore production facilities, such as those in the North Sea, which on average contain a 26 mg/l dispersion of oil in water (C.U.E.P. Pollution Paper No. 6, 1976). Not unexpec- tedly, high concentrations of oil are also found in the immediate vicinity of oil slicks : for example, Cormack and Nichols (1977) give values of 0.79-3.95 mg/l for oil concentrations a t a depth of 2 m beneath the centre of a small slick of “ Ekofisk ” oil. In the study by Brown et al. (1973) ocean water samples were collected by tankers operating along the U.S. Gulf coast to East coast and the Caribbean to East coast. Two samples (3 1) were taken daily a t 12 h intervals, one from the surface using a bucket and the other through the bottom of the ship using a sanitary line (10 m depth) : special precautions were taken to avoid contamination. Approximately 400 samples were examined, concentrations of saturated hydrocarbons (alkanes and 1- to 6-ring naphthenes) varying in the range 1 to 21 pg/l and those of aromatic compounds from 1 to 3 pg/l. As in several studies mentioned earlier, the concentrations in surface samples were greater than in those taken from 10 m depth. Average percentages of the total quantities of hydrocarbons accounted for by the various fractions, based on all the data, are shown in Table 11. Values obtained for aromatic substances showed that the simpler compounds (benzenes, indanes and indenes) were well repre- POLLUTION STUDIES WITR MARINE PLANKTON-I 301 sented and that the levels of tetra-cyclic aromatics were relatively low. This is a distribution similar to that found in many crude oils, but Brown et al. (1973) suggested that sources other than this, such as organic materials released from sediments, could also have contributed. Their data demonstrate the low levels of particular groups of aromatic hydrocarbons found in Atlantic Ocean water. Taking the naphthalenes as an example: the highest value for total hydrocarbons was 50 pg/l and, if it be assumed (Table 11) that naphthalenes accounted for 4.3%, these compounds had a maximum concentration of only 2.2 pg/l. On many occasions total hydrocarbons amounted to only 1.0 pg/l; which gives a concentration of naphthalenes as low as 0.043 pg/L TABLE 11. RELATIVE AMOUNTS OF HYDROCARBON FRACTIONS EXTRACTED FROM ATLANTIC SEA WATER ~ Praction Percentageof total Range Mean Paraffis Naphthenes Benzenes Indanes In d e n e s Naphthalenes Acenaphthenes Fluorenes Phenanthrenes Tetra-aromatics Benzothiophenes Dibenzothiophenes 10 to 27 0 to 25 2 to 13 4 to 13 5 to 13 3 to 8 4 to 9 3 to 12 0 to 15 2 to 7 0 to 24 0 to 9 18.5 9-8 9.5 9.8 8.7 4.3 6.7 8.5 8.3 3-7 7.3 4.8 Adapted from Brown et al. (1973). Recently, Brown and Searl (1976) have measured the total hydrocarbons, both dissolved and particulate, in sea-water samples along tanker routes in the Pacific Ocean. Concentrations had average levels of 2 (0.8-5 pg/l) for surface waters and 0.8 (0-3-2 pg/l) for subsurface (3m and 10m depth) samples. In nearly all cases the hydrocarbons were complex mixtures of paraffins, cyclo-alkanes, and 1,- 2- and 3-ring aromatics. Along the Singapore to San Francisco route aromatics accounted for 36% of the total; but on all other routes the value ranged from 15 to 22%. Taking the values for all samples, both surface and sub-surface, aromatic hydrocarbons in the Pacific range from 0.21 to 0.50 pg/l. Complementing the work of Brown and Searl (1976) are the measure- 302 E. D. 8. CORNER ments by Koons (1977) of volatile hydrocarbons along tanker routes in the Pacific Ocean. The hydrocarbons were in the range C, to C, and included saturated compounds such as n-pentane, cyclopentane, n-hexane, methylcyclopentane, n-heptane, methyl- cyclohexane and n-octane, as well as the aromatic compounds benzene, toluene and xylenes. The average concentration found in samples taken near the surface was 0.33 pg/l compared with 0.10 pgll for those from a depth of 10 m. Another comprehensive analysis of the hydrocarbons in sea water was that of Barbier et al. (1973) who examined samples from the English Channel (Brest and Roscoff ), Mediterranean (Villefranche) and off the west coast of Africa. All the samples were filtered through a 0.45 pm millipore membrane and so the data refer to " dissolved " hydrocarbons only. These were extracted from each sample (100 1) with chloroform, the extracts then being dried and saponified. Kydro- carbons were separated from the unsaponifiable material by thin-layer chromatography, then analysed by gas-liquid chromatography and, as in the study by Brown et al. (1973)) by mass spectrometry. Coastal waters, as well as surface waters, had hydrocarbon contents greater than those found in deep-water samples (500-5 400 m depth). The values ranged from 10 pgfl (open ocean off West Africa) to 137 pg/l (Creek of Poulmic: Brest Harbour) with an average of 40 pg/l. Further evidence for high levels of hydrocarbons in estuarine waters was found by Searl, Huffman and Thomas (1977) in their study of non-volatile hydrocarbons in New York Harbour. Quantities ranged from 14 to 270 pg/l with an average of 39 pg/1, this value being an order of magnitude higher than that found in open Atlantic Ocean waters TABLE 111. RELATIVE AMOUNTS OF HYDROCARBON FRACTIONS EXTRACTED FROM BREST HARBOUR SEA WATER ___ Fraction Percentage of total n- and iao-alkanes 1-ring naphthenes 2-ring naphthenes 3-ring naphthenes 4- and > 4-ring naphthenes Mono-cyclic aromatics Bi-cyclic aromatics Poly-cyclic aromatics 51.5 5.5 9.5 6.5 4-0 18.0 3-5 2.5 Data from Barbier et al. (1973). POLLUTION STUDIES WITH MARINE PLANKTON-I 303 (Brown et al., 1973) but close to the average of 25 pg/l found for Tokyo Harbour (Brown, Sear1 and Koons, 1976). The Brest sample, when analysed in detail by Barbier et al. (1973) using U.V. spectrometry and mass spectrometry, was found to have the percentage composition shown in Table 111. The n-alkanes in both coastal and open-sea waters ranged from C,, to C,,, the most abundant being in the C2, to C,, region, as found in other sea areas by Blumer (1970) and Whittle et al. (1973). There was no predominance of odd-numbered carbon compounds and generally the pattern of distribution resembled that found for marine algae by Clark and Blumer (1967). However, in coastal water the presence of aromatic compounds, such as those found in the Brest sample, indicated pollution. Only the Brest sample was analysed in detail, but assuming the data to apply generally and, again taking naphthalenes (or bi-cyclic aromatic compounds) as an example, the levels of this group of com- pounds varied within the range 0.35-4-9 pg/l, compared with values of 0-043-2.2 pg/l found by Brown et al. (1973). Bearing in mind that the samples analysed by Barbier et al. (1973) and by Brown et al. (1973) were taken from different sea areas, that there were certain differences in the analytical methods used and that Barbier et al. measured dissolved hydrocarbons whereas Brown et al. determined both these and the particulate fraction, the data are sufficiently close, at least at the higher end of the range, to provide a reasonable guide to the background levels of aromatic compounds that should be used in designing laboratory studies concerned with problems such as the uptake and retention of these compounds by plankton and their possible effects on the organisms. 111. HYDROCARBONS IN PLANKTON Determining whether planktonic organisms are contaminated with petroleum hydrocarbons in sea areas prone to oil pollution is com- plicated by the need to recognize that man-made pollution, such as an accidental discharge of crude oil, is not the only source of such com- pounds in these plants and animals : marine organisms are themselves capable of biosynthesizing hydrocarbons. A challenging problem for the analytical chemist has therefore been that of distinguishing between compounds such as hydrocarbons from fossil fuels and those of recent biogenic origin in the organisms. Several studies, discussed in detail in the next two sections, have shown that the hydrocarbons native to marine planktonic organisms 304 E. D. 9. CORNER include only a few representatives of any one group of compounds. Crude oil, however, contains a much more complex mixture (Posthuma, 1977). For example, marine phytoplankton have only a restricted range of n-alkanes, whereas these usually occur in crude oil in a con- tinuous homologous series from C, to C4,,. Likewise, zooplankton contain only a few branched alkanes (iso-alkanes), the major one being pristane ; crude oil, on the other hand, includes a wide range of these Alkanes(n-andiso-o-) Tetra lins Cycloalkanes No phthalenes Biphenyls Benzolol pyrene FIG. 1. Structural formulae of various hydrooarbone found in crude oil. IE represents several types of alkyl substituent. compounds. Cyclo-alkanes (naphthenes), particularly cyclopentane and cyclohexane derivatives with both substituted and unsubstituted rings, aromatic hydrocarbons including 1- to 5-ring compounds, together with their alkylated forms, and naphthenoaromatics such as the tetralins are all well represented in crude oil but are not normally found in planktonic organisms (Koons and Monaghan, 1976). By contrast, the alkenes (olefins), representatives of which have been found in both phytoplankton and zooplankton, are generally absent from crude oil (although they can occur in refinery products). POILUTION STUDIES WITH MARINE PLANKTON-I 306 The structural formulae of some of the hydrocarbons found in crude oil are shown in Fig. 1. A. Ph yloplankton The first investigation of hydrocarbons in phytoplankton using modern analytical methods was that of Clark and Blumer (1967). Cultures of three species of phytoplankton were used : Syracosphacra earterae, now Hymenomm carterae (Braarud e t Fagerl.) Braarud, Skeletonem costatum and an undetermined cryptomonad. Analyses of all the n-alkanes within the range C84H30to C,,H,, showed that the total amounts varied from 34 to 121 mg/kg dry weight; also that in each species one particular n-alkane predominated, i.e. n-C,, in Skeletonem and the unknown cryptomonad and n-C,, in Syracosphaera. The predominance of n-C,, in the latter species was particularly marked in that it accounted for 45.5% of the total n-alkanes. The carbon preference index (i.e. the ratio of compounds containing an odd number to those with an even number of carbon atoms) was 1.1-1.2, so there w&s little evidence of the marked odd-carbon predominance ” found in marine sediments (CPI values of 2 4 4 . 5 : Cooper and Bray, 1963) ; which suggests that the source of a large proportion of the n-alkanes in sediments may not be marine phytoplankton. The isoprenoid hydrocarbon pristane (2, 6, 10, 14-tetrameth~lpentadecane)~ present in recent marine sediments (Blumer and Snyder, 1965), in petroleum (Bendoraitis, Brown and Hepner, 1963) and in zooplankton (Blumer et al., 1963, 1964) was also detected in all three species of phytoplank- ton. No studies of the mechanisms by which hydrocarbons are synthesized in marine unicellular algae seem to have been made. However, as far as the n-alkanes are concerned, work with higher plants indicates that the main mechanism is likely to be one of elongation from palmitic acid (C16) by the addition of C, units from malonyl-CoA, followed by decarboxylation (Kolattukudy, 1976), although recent work by Murray, Thomson, Stagg, Hardy, Whittle and Mackie (1977) indicates that in marine phytoplankton this process of chain-elongation may be limited. Thus, Murray et al. (1977) measured the radioactivity in aliphatic hydrocarbons present in various species of phytoplankton cultured with W-labelled Na,C03, and in mixed zooplankton feeding on the plant cells. Generally, only a few specific hydrocarbons were found to be labelled, compared with the wide array present in the plants and animals. In particular, there was little evidence that long-chain hydrocarbons (C2a to C,.J were synthesized by either micro-algae or zooplankton ; which implies that such compounds, which have been detected in natural plankton samples, are exogenous in origin. A.H.B.-~S 13 306 E. D. 9. CORNER Work by Lee, Nevenzel, Paffenh6fer, Benson, Patton and Kavanagh (1970) identified the C,lH,, olefinic hydrocarbon all-cis-3, 6, 9, 12, 15, 18-heneicosahexaene (HEH) in Skeletonema costatum (see Fig. 2) ; and Blumer, Mullin and Guillard (1970) investigated its distribution in numerous species of marine phytoplankton (Table IV). The presence of the C,, fatty acid docosa-all-cis-4, 7, 10, 13, 16, 19-hexaenoic acid in these algae led Blumer et aE. (1970) to suggest that HEH might arise by decarboxylation of this compound. However, later work (Young- blood and Blumer, 1973) showed that HEH was also present in three species of brown benthic algae that did not contain the C,, fatty acid ; which suggests that the hydrocarbon may also be derived in other ways. TABLE IV. H~~NEICOSAHEXAENE (HEH) CONTENTS or U r m UNICELLULAR ALUAJ3 ~~ No. of teat HEH aa yo species wet weight Bacillariop hyceae Dinophyceae Crypt ophyceae Haptophyceae Euglenaphyceae Prasinophyoeae Cyanoph yceae Rhodophyceae Xanthophyceae Chlorophyceae 2 2 2 3 1 1 1 1 1 1 0.00036 to 0.0027 0.0037 to 0.0040 10.0006 to 0.008 0.0015 to 0.010 0.0035 <0-0009 <0*00001 < 0.00004 <0~00008 <0~000016 Summarized data from Blumer et al. (1970). In a later study (Blumer, Guillard and Chase, 1971) 22 species of marine planktonic algae belonging to nine algal classes were analysed (see Table V). Trace amounts of n-alkanes within the range n-C14H,, to n-CZ5H5, were found in all the species, most of which also contained small quantities of pristane. However, among the groups Bacillariophy- ceae, Dinophyceae, Cryptophyceae, Haptophyceae and Euglenaphy- ceae the predominant hydrocarbon was HEH, the only exception being Rhizosolenia setigera, in which the predominant hydrocarbon was n-heneicosane (C,lH44) ; another centric diatom Tlzalassiosira Jlzcviatilis contained, in addition to HEH, a C,, tetra-olefin, but only as a minor component. Further observations, using species representing the Cryptophyceae (Blumer et al., 1970) and the Dinophyceae (Blumer et al., 1971), showed that HEH was most actively synthesized during POLLUTION STUDIES WITH MARINE PLANKTON-I 307 the logarithmic growth phase : cultures harvested during the stationary phase contained greater amounts of C,, to C,, n-alkanes. By contrast, five algal species representing the Rhodophyceae, Xanthophyceae and Chlorophyceae did not contain HEH : instead, the predominant hydrocarbons were either n-C,,H3, or n-Cl7H3,, or olefins such as an unclassified pentadecene or 7-heptadecene. Two blue-green algae of the class Cyanophyceae were also studied. In one species, Oscillatoria woronichinii, the predominant hydrocarbon was n-C1,H3,, with traces of other n-alkanes ; in the other, Synechowccus bacillaris, the olefin 5-heptadecene predominated, with n-C15H32, n-C,,H,, and n-Cl,H3, also abundant and C,, and C,, mono-olefins present in low amounts. Tornabene, Kates and Volcani (1974), in studies using the non- photosynthetic diatom Nitzschia alba Lewin et Lewin, found that aliphatic hydrocarbons accounted for about 0.1 yo of the total lipids. Pristane, phytane and several long-chain n-alkanes (C,, to C2,) were detected. The presence of phytane in Nitzschia is interesting as this compound is normally regarded as non-biogenic in origin, its source being fossil fuels. The olefin HEH, characteristic of photosynthetic diatoms, was not found : instead, the predominant hydrocarbons were even-numbered C,,, c1, and C,, olehs, in contrast to the odd-numbered compounds found by Blumer et al. (1971). Possibly photosynthetic and non-photosynthetic diatoms have different pathways for the biosynthesis of hydrocarbons. Compared with those of n-alkanes, branched alkanes and olefins, analyses of aromatic hydrocarbons in phytoplankton are few. Smith ( 1954) reported that cycloalkanes and aromatic compounds accounted for more than 0.2% by weight of a dried sample of phytoplankton collected near Woods Hole, Massachusetts. More recent information mainly concerns levels of the carcinogen benzo[a]pyrene (BP), but there are doubts about some of the analytical methods used (Farrington and Meyer, 1975). Mallet and Sardou (1965) detected BP in amounts up to 400 pg/kg dry weight in samples of mixed plankton from the Bay of Villefranche, compared with a value of 5.5 pg/kg dry weight for a sample collected off the west coast of Greenland (Mallet, Perdriau and Perdriau, 1963), an area less prone to man-made pollution. The detection of BP in phytoplankton from another remote area, Clipperton Lagoon in the East Pacific, has been reported by Niaussat (1970) and Ehrhardt (1972). The synthesis of BP and other polynuclear aromatic hydrocarbons (PNAH) by bacteria-free cultures of the freshwater species Chlorella vulgaris Beij. has been demonstrated by Borneff, Selenka, Kunte and 308 1. D. 9. CORNER TABLE V. HYDROCARBONS IN MARINE UNICELLULAR ALGAE Class and species Predominant hydrocarbon Trace hydrocarbons Bacillariophyceae Cyclotella nana* HEH Ditylum brightwellii (West) Van Heurck HEH Lauderia borealis Gran HEH Rhizosolenia setigera Brightw. Skeletonema costatum (Grev.) Cleve HEH Thalassiosira%uviatilis Hust. HEH Thalassiosira sp. HEH HEH, n-C,,, n-C,, Dinophyceae Gonyaulax polyedra Stein HEH Gymnodinium splendens Lebour HEH Peridinium trochoideum (Stein) Lemm. HEH Peridinium trochoideum (old culture) HEH, n-C,,, n-C,, Cryptophyceae Cryptomonas (Rhodomonas?) HEHCryptomonas (old culture) HEH Haptophyceae Coccolithus huxZeyi (Lohm.) Kampt. HEH Isochrysis galbana Parke HEH Phaeocystis pouchetii (Hariot) Lagerh. HEH Euglenaph yceae Eutrepiella sp. HEH, n-C,,, n-C,, Cyanophyceae Oscillatoria woronichinii Synechoccus bacillaris Butch. Rhodophyceae Xanthophyceae Tribonema aequa2e Pascher Anissimova n-c,, n-C,, alkene, n-C,, Porphyridium sp. n-c17 n-C,, alkene, n-C,,, "-el, Undetermined species n-c,,, n-c,,, n-C,, alkene Pristane, n-alkanes n-alkanes n-alkanes Pristane, n-alkanes Pristane, n-alkanes Pristane, n-alkanes, Pristane, n-alkanes, n-C,, : 4 n-C,, : 4 n-alkanes n-alkanes n -alkanes n-alkanes Pristane, n-alkanes Pristane, n-alkanes Pristane, n-alkanes Pristane, n-alkanes Pristane, n-alkanes Pristsne, n-alkanes n -alkanes n-alkanes, other olefins n-alkanes n-alkanes, other olefins n-alkanes, other olefins POLLUTION STUDIES WITH MARINE PLANKTON-I 309 TABLE V (continued) Trace Hydrocarbons Predominant hydrocarbon Class and species Chlorophyceae Dunaliella tertwlecta Butch. Derbesia tenuksima (De Not.) n-C,, alkene, n-C,, n-alkanes, other olefins Crouan frat. n-C,, alkene n-alkanes * Thalassiosira pseudonana Has10 et Heimdal [as Cyclotella nana Hust.]. HEH is 3, 6, 9, 12, 15, 18-heneicosahexaene (presumed all cis) : n-C, is a normal alkane with x carbon atoms : A-C, alkene is a normal alkene with x carbon atoms; nC,,:4 is a tetraoleiin. Adapted from Blumer et al. (1971). Maximos (1968). The alga was grown with 14C-labelled acetate added to the medium and radioactivity was eventually detected in the hydrocarbons, this technique being used to exclude the possibility that the compounds resulted from external contamination. No studies of this kind, however, seem to have been made with marine unicellular algae. It is to be hoped that more detailed data concerning the distribution and levels of aromatic hydrocarbons, particularly PNAH, in plankton will be obtained now that modern methods for analysing these com- pounds are being applied in the marine environment (Giger and Blumer, 1974). B. Phytoplankton and crude oil as sources of hydrocarbons in the sea Interest in the quantitative importance of phytoplankton as a source of hydrocarbons in the sea prompted the following attempt to compare the contribution from marine unicellular algae with that from crude oil. Note, however, that these are not the only sources of hydrocarbons in the sea : Peuerstein (1973) estimates that global emis- sions of hydrocarbons into the atmosphere total 90 x 106 metric tons per annum (mta) of which an average of 0.6 x lo6 mta, or roughly 0.7%, eventually reaches the sea. According to Grossling (1976) the total inputs of crude oil into the world’s oceans, based on 1972 levels of economic activity, are as shown in Table VI. The total, 3.77 x lo6 mta, does not include the con- tribution from on-shore oil seepage that may eventually reach the sea : nevertheless, it comes within the range of values (2.5-4.0 x los mta) previously given by others (e.g. Brummage, 1973 ; Charter, Sutherland 310 E. D. 9. OORNER and Porricelli, 1973). Additional to these inputs is that from natural submarine seepage, for which Wilson (1973) gives a figure of 0.6 x los mta based on the average for high (Southern California) and low (western Canada) seepage areas. The overall total for oil from all sources is therefore about 4.4 x los mta. TABLE VI. INPUTS OF OIL FOR THE WORLD’S OCEANS ~~ Ocean intake ( x 106 metric tom per annum) Source Industrial spent lubricants Automotive spent lubricants Aviation spent lubricants On-shore oil well accidents Off-shore oil well accidents Tanker cleaning operations Tanker accidents Off-shore pipe-line accidents On-shore pipe-line accidents 1.43 0.89 0.04 (0.53 0.33 0.35 0.19 0.01 0.001 Adapted from Grossling (1976). Clark and Blumer (1967) found an average value of 72 mg/kg dry weight for hydrocarbons in marine phytoplankton, a figure that should be regarded as minimal in that it refers only to n-alkanes which are not always the predominant hydrocarbons in marine algae (see p. 308). A feasible estimate for primary production in the world’s oceans is that of Ryther (1969) who gives a value of 20 x los metric tons of organic carbon per year. According to the data of Parsons, Stephens and Strickland (1961), organic carbon accounts on average for 37% of the dry weight of marine phytoplankton : thus, combining this value with that of Clark and Blumer (1967), the average quantity of hydrocarbons in these organisms is 0.195 mg/g organic carbon. The total annual production of hydrocarbons as phytoplankton is therefore 3.9 x lo6 mta, which is similar to that of 4.4 x los mta contributed by crude oil. Such close agreement, bearing in mind the number of assumptions made, is probably fortuitous. Nevertheless, it seems reasonable to conclude that the annual quantity of hydro- carbons released into the sea aa crude oil and that produced as phytoplankton axe of the same order of magnitude. POLLUTION STUDIES WITH HABINBI PLA?SKTON-I 31 1 C. Zooplankton Quantitatively, one of the most important hydrocarbons in zoo- plankton is pristane (2, 6 , 10, 14-tetramethylpentadecane) which is also present in the livers of basking sharks and sperm whales as well aa being a constituent of various crude oils (Blumer et al., 1964). The hydrocarbon was fist identxed in zooplankton by Blumer et al. (1963) who showed that it accounted for 0.46-0.90% of the dry weight and 0.86-2-9% of the total lipids in calanoid copepods collected from the Gulf of Maine. The highest values were obtained with the Boreal- arctic species Calanus hyperboreus and in a later study (Blumer et al., 1964) it was shown that when the animals were starved for 86 days, although all the weight loss was accounted for as a decrease in lipid coptent, pristane actually increased slightly, presumably being slowly formed from precursors. It would be interesting to know how pristane levels vary in calanoid species more active metabolically than C. hyperboreus which, during summer and autumn, enters a non-feeding " diapause " (Conover, 1962). Other species of zooplankton, including representatives of the chaetognaths, pteropods, ostracods, amphipods and euphausiids, were found to possess very little pristane in comparison with the copepods ; and even among these only the calanoids contained substantial quan- tities (Blumer et al., 1964: Bee Table VII). The pathways of biosynthesis of hydrocarbons in marine zoo- plankton have received little study. However, Avigan and Blumer (1968), using tracer isotope methods, showed that the pristane in calanoid copepods could be formed from phytol, a C,,-alcohol present in algal diets as a constituent of chlorophyll. Other phytol-derived hydrocarbons, detected in mixed zooplankton from the Gulf of Maine by Blumer and Thomas (1965a and b) and by Blumer, Robertson, Gordon and Sass (1969), are shown in Pig. 2. All are olefine and are present in amounts much smaller than those of pristane. Moreover, ufike pristane they do not occur in crude oils. Biochemical inter-relation- ships between phytol, pristane, phytane and various olefins are shown in Fig. 3. Blumer et al. (1963, 1964) noted that the copepod Rhincalanus ?uIcButus, although similar in feeding habits to Calanus spp., contained only traces of pristane ; and later work (Blumer et al., 1970) showed the main hydrocarbon in this species to be the C,, polyunsaturated olefin HEH. This hydrocarbon did not, however, occur in R. nusutus to the same extent as did pristane in other calanoid copepods.Thus the amounts of HEH in laboratory cultured animals were in the range 312 E. D; S. CORNER TABLE VII. LEVELS OF PRISTANE IN VARIOUS SPECIES OF ZOOPLANKTON Pristane content Species (Yo total (Yo dry wt) lipid) Group Stage Sagitta elegans Verrill Limacina retroversa (Fleming) Conchoecia sp. Paratherniato gaudichaudii (Guerin) Nematoscelis megalops Hansen Meganyctiphanes norvegica (M. Sara) Calanus finmarchicus Gunnerus Calanus finmarchicus Calanus finmarchicus Calanus finmarchieus Calanus finmarchicus Calanus ghcialis Jaschnor Calanus gbcialia Calanus hyperboreus Calanus hgperboreus Calanus hyperboreus Rhincalaniu nasutus Rhincalanus nasutus Pareuchaeta norvegica Pareuchaela norvegica Pareuchaeta norvegica Pareuchaeto n.orvegica Metridia longa (Lubbock) Metridia lzrcens Boeck Pleuromamma robusta Euchirella Tostrata (Claus) Candacia armata Boeck Kr0yer Giesbrecht (Boeck) (F. Dahl) Chaetognath ns 0.02 0.05 Pteropod Ostracod 0.01 g o . 0 1 t0.01 0.14 0.03 ns ns Amphipod 0.04 ns Euphauviid Euphausiid Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod Copepod <0*01 0.09 ns g0 .01 0.85 0.73 0.77 0-46 0-68 0.45 0.47 0.02 ns IV V V Female Male V V 1.47 1.45 1-69 1.31 0.86 - V V Female 0.92 0.84 0.90 - 1-62 2.94 Female Female 0.03 0.03 0.01 0.01 IV V Female Female Female Female 0.02 0.03 0.05 0.03 0.01 0.01 0.14 0.19 0.08 0.15 - V and adult V and female Female g 0 . 0 1 0.01 I0 .02 0.01 0.04 - < 0.20 Data from Blumer et al. (1964); ns = not stated. POLLUTION STUDIES WITH MARINE PLANKTON-I 313 0.0006-0.22 pg/copepod and accounted for < 0.007 to 0.47y0 total lipid : the levels in " wild " animals were greater, varying from 0.061 to 0.46 pg/copepod and 0.28 to 1.2% total lipid. Blumer et a2. (1970) suggested that the levels of HEH in R. nasutus might vary with the amounts in algal foods used by the animals; Pristane(2,6,10,i4- tetramethylpentadecane) detected by Blumer etul (1973) I 1 ] Neophytadiene Cz0- phytadienes detected by Blumer and Thomas (1965~) isomeric phytadienes 1 CIs-di-and tri-olefins detected by Blumeretul (1969) c~s-heneicosa-3,6,12,15,18- Leeeta/ (1970) - - hexaene (HEH) detected by - - - c FIG. 2. Hydrocarbons detected in zooplankton. they also pointed out that the species seemed exceptional in being able to accumulate HEH from plant diets. Thus, Eucalanus bungii Giesbrecht, belonging to the same taxonomic family as R. nasutus, contained little or no HEH when reared on algal cultures that provided R. nasutus with the olefin. Likewise, Lee et al. (1970) could not detect HEH in Calanus helgolandicus (Claus) fed on Skeletonema, which con- tains considerable amounts of HEH (Blumer et al., 1970). Possibly Note. The top line of Fig. 2 should read Blumer et al. (1963). 314 E. D. 8. (IORNEB these other species are less able to accumulate HEH from algal diets. On the other hand, compared with R. naswtw, they may be more successful in metabolizing the hydrocarbon (Lee et d., 1970). The levels of hydrocarbons, which are normally low, found in various samples of zooplankton are shown in Table VIII. In the study by Lee, Nevenzel and Lewis (1974) with Euchaeta juponica Marukawa pristane was usually the major hydrocarbon, PHYTOL //I\\ Metabolic chanqes giving hydrocarbons in zooplankton . . \A ..... Neo -phytadiene A H A H 2 0 1 Phytenic acid Di hydrophytol ..... ..... I lsornerises ..... L+ ..... A Norphytene ..... (CIS and truns) ..... I L Satul tion Isornerises I Phytonic acid 1 Zooplankton: decarboxylation I accounting for 3&50y0 of the total; HEH was also detected in sub- stantial quantities, representing 30-40% of the total in adults and copepodid V and 7% in the eggs; trace amounts of a series of n-alkanes and n-alkenes were also detected, ranging in chain length from C,, to C26. In the samples examined by Whittle et al. (1974) pristane accounted for 79.7% of the total, most of the remainder being n-alkanes (18-19%) and squalene (0.33%). Pristane was also the main hydro- carbon found in the copepod " slick '' studied by Lee and Williams +d TABLE VIII. HYDROCARBON LEVELS IN ZOOPLANKTON a Species Hydrocarbon levels As yo lipid As yo wet weight Gnathophawk sp. (mysid) Acanthaphyra purpurea Milne Edwards (decapod) Nematobrachion sexspinosis Hansen (euphausiid) A . purpurea (female) A . purpurea (male) Ewhaeta j a p o n k Marukawa Mixed plankton samples from the Clyde Copepod " slick " from N. West Pacific Surface zooplankton from E. Mediterranean Mixed zooplankton from E. Gulf of Mexico (Summer) Mixed zooplankton from E. Gulf of Mexico (Autumn) Mixed zooplankton from E. Gulf of Mexico (Winter) 1-2 2 3 3 33 0.43 0.36 1-44 0.0148 to 0.0299 0.170 0.0294 0.02 to 0.046 0.038 to 0.052 <O-14* 0-0172 0-24* 0.66 to 0-99 0.003 0.002 0.010 r j z Morris and Sargent (1973) a Morris and Sargent (1973) El m Morris and Sargent (1973) (I) Morris (1974) Lee et al. (1974) Whittle et al. (1974) Lee and Williams (1974) 2 Reference z Morris (1974) 8 f Morris (1974) m Calder (1976) # Calder (1976) k Calder (1976) 5 0 x I * Data calculated using wet weight: dry weight ratio of 7-0:l. H 316 E. I). 9. OORNER (1974), accounting for 80% of the total; the remainder was n-alkanes ranging from C,, to C,, with a peak at CZ5. The relatively high levels of hydrocarbons detected by Morris (1974) in surface samples of zoo- plankton from the eastern Mediterranean probably reflect petroleum pollution in this area. The compounds consisted mainly of n-alkanes in the range C,, to C,, with C,, predominating, these being present in greater amounts than those of pristane. Polyunsaturated C,, and C,, hydrocarbons and squalene were also found. Interestingly, compared with the other samples of zooplankton investigated (see Table VIII), those from the Gulf of Mexico (Calder, 1976), a sea area with a relatively long history of oil exploration, had the lowest levels of hydrocarbons. A further finding of interest was that whereas total lipid levels did not vary much with season, that of the hydrocarbons was much greater in winter than in summer or autumn (Table IX). Although no comparative details are given, neither dissolved hydrocarbons nor those associated with particulate material apparently bore any relation to the hydrocarbons in the zoo- plankton, which therefore do not appear to have arisen from exogenous sources such as oil pollution. On the other hand, evidence of an associa- tion between the levels of dissolved hydrocarbons and those present in plankton from the same sea areas has recently been reported by Whittle, Mackie, Hardy, McIntyre and Blackman (1977). Thus, the average total of n-alkanes in plankton collected near oil refineries was 270 pg/g dry weight compared with 71 in samples taken from the open sea (Celtic Sea) : values for dissolved hydrocarbons collected from similar areas were 4.5 pg/l and 0.57 pg/l respectively (Hardy et aE., 1977: see Table 1). The relative amounts of the individual n-alkanes were also determined, but the data provided no clear indication of whether the compounds were endogenous or had been accumulated from the environment.Further evidence that zooplankton from the Gulf of Mexico (South Texas Outer Continental Shelf) possess a hydrocarbon pattern charac- teristically biogenic is that of Parker et al. (1976), included in Table IX, who found particularly high levels of C,, n-alkanes and pristane. Parker et al. (1976) noted the marked difference between the hydro- carbon patterns in samples of zooplankton and neuston collected simultaneously from the same sea area : a third of the neuston samples had n-alkane patterns typical of petroleum, which was attributed to the presence of micro-tarballs in the surface. Concerning the possible biological function of the naturally occurr- ing hydrocarbons in zooplankton, Blumer et al. (1964) proposed that pristane might be used by calanoid copepods as a means of achieving POLLUTION STUDIES WITH MARINE PLANKTON-I 317 TABLE Ix. SEASONAL CHANGES I N HYDROCARBON LEVELS IN ZOOPLANKTON Spring Summer Autumrz Winter (Data for total hydrocarbons : Gulf of Mexico. Summarized from Calder (1976)) Zooplankton biomass (mg dry wt/m3) - 91 18 13 Total lipid content (mg/g dry wt) - 49.9 37.7 135 Total hydrocarbons (pg/g dry wt) - 212 135 719 Total hydrocarbons ( pg/m8) - 19.3 2-4 9.4 (Data for individual compounds (pg/g dry wt) : South Texas Continental Shelf, Gulf of Mexico. Summarized from Parker et al. (1976)) 6.0 0.8 39.6 2.0 2.0 1.2 0.6 3.0 49.1 0.1 1-0 1.8 3.2 0.2 1 -0 8.8 18.6 1.1 3.6 1.6 3.0 0.6 2.0 1.3 0.7 3.3 8.1 17.8 73.9 0.05 0.7 1.4 4.7 buoyancy ; and Youngblood, Blumer, Guillard and Fiore (1971) suggested that HEH might influence sex ratio, drawing attention to the correlation between the percentage of males produced and the degree of predominance of HEH in the algal diets used by the younger stages of C. helgolandicus in studies by Paffenhbfer (1970). However, heavier mortality occurred in the experiments in which fewer males were produced and this may have selectively affected the male animals (Paffenhofer, 1970). The influence of environmental factors on the sex ratio of calanoid copepods and the possible importance of hydrocarbons in this context are topics that obviously deserve further study (see Sections VIII and IX). IV. TOXICITY STUDIES WITH PHYTOPLANKTON An important factor influencing the toxicity of an oil is the size and chemical composition of the water-soluble fraction (WSF), which includes a number of low-boiling aromatic hydrocarbons. Some of these, such as benzene and toluene, are rapidly lost by weathering (Frankenfeld, 1973), but others, notably bi-cyclic aromatic hydro- carbons such as naphthalene and its alkylated derivatives (e.g. 1- and 318 E. D. 9. CORNER 2-methylnaphthalene, dimethylnaphthalenes) are more persistent. Studies by Boylan and Tripp (1971) and by Anderson, Neff, Cox, Tatem and Hightower (1974) have shown that the high proportions of bi-cyclic and tri-cyclic aromatic hydrocarbons in the WSFs of oils TABLE X. HYDROCARBON CONTENTS OF WATER-SOLUBLE FRACTIONS OF FOUR TEST OILS Hydrocarbon colztent (msll) Compound Bunker 0 S. Louisiana Kuwait No. 2 residual crude. oil crude oil fuel oil oil Alkanes It- and wo-alkanes, C, to C, n-alkanes, c,, to C,, Cyclopentane and 2-methyl- Methylcyclopentane Methylcyclohexane pentane Aromatics Benzene Methylbenzenes Naphthalene Methylnaphthalenes Biphenyl Methylbiphenyls Fluorene Methylfluorenes Dibenzothiophene Phenanthrene Methylphenanthrenes Total saturates Total aromatics Total hydrocarbons 8-94 0.089 0-380 0-230 0.220 6-75 6.85 0.12 0.178 0.001 0.002 0.001 0.002 0.001 0.001 0.003 9.86 13.91 23.77 10.76 0.004 0.590 0.190 0.080 3.36 6.60 0.02 0-05 1 0.001 0.002 0.001 0.002 0.001 0.001 0.002 11.62 10.04 21.66 0*424* 0.047 0.020 0.019 0.030 0.550 3.28 0.84 1.09 0.011 0.017 0.009 0.011 0.004 0.010 0.010 0-54 5.73 6-27 0.058 0.012 0.005 0.004 0.002 0.040 0.310 0.210 0.690 0.001 0.002 0.005 0.006 0.001 0.009 0.014 0-081 1.29 1-37 Fractions prepared from 1 pert oil layered on 9 parts 20%, Instant Ocean. * Unresolved GC peaks, probably includes some olefins. Summarized data from Anderson et aZ. (1974). such as No. 2 fuel oil and Bunker C (Table X) could be responsible for the relatively high toxicities of these oils to marine animals. Crude oils, their total WSFs and individual components of them, have all been used in toxicity studies with phytoplankton. POLLUTION STUDIES WITH MARINE PLANKTON-I 319 A. Studies using crude oils and their water-soluble fractions The first laboratory study of the effects of crude oil on phyto- plankton seems to have been that of Galtsoff, Prytherch, Smith and Koehring (1935) who found that a heavy layer of Lake Pelto crude oil over a culture of Nitzschia closterium (Ehrenb.) W. Sm. began to inhibit growth after one week. The WSF of the oil, prepared by dialysis through a collodion membrane, also inhibited growth when used at high concentrations (25 and 50% in sea water) over a period of 13 days. Days FIG. 4. A. Development of Ditylum brightwellii in sea water containing different con- centrations of fuel oil: 1, 0.001 ml/l; 2, 0.01 ml/l; 3, 1.0 ml/l; pecked line = oontrol. B. Development of Meloei~a monilijormia; 1 , O . O O l ml/l; 2,O.l ml/l; 3, 10 ml/l; dashed line = control (After Mironov and Lanskaya, 1966.) Russian work on the effects of crude oil on many species of marine phytoplankton colleoted from the Black Sea (summarized by Mironov, 1968, 1972) showed that species differed considerably in their sensitivi- ties. The effects, however, appeared to vary with the oil concentration used. For example, Mironov and Lanskaya (1966) found that a level of 0.001 ml/l, over a period of three days, stimulated cell division by Ditylum brightwellii (West) Grun. ex Van Heurck but slightly inhibited that of Melosira moniliformis (0. F. Miill.) Agardh; on the other hand, whereas 1.0 ml/I caused a 100% reduction in cell number over 24 h with ~~~~~~~~ 10 mlp used over three days did not signifi- cantly affect the original number of cells in a oulture of Melosira (Fig. 4). 320 E. D. 9. UORNER The oil “concentrations” described in studies such as that of Mironov and Lanskaya (1966) represent oil added to but not neces- sarily dissolved in the sea water ; in fact, suspensions of oil were used and not solutions. Prouse, Gordon and Keizer (1976) refer to oil as being ‘ I accommodated ” in sea water, earlier work (Gordon, Keizer and Prouse, 1973) having shown that oil agitated with an aqueous phase does not all pass into solution : a large fraction (ca. 90%) is pre- sent in particulate form. It is necessary to check the extent to which the amount of oil originally added to sea water might vary during a toxicity experiment. Thus, Gordon and Prouse (1973), studying the effects of three oils (Venezuelan crude, No. 2 fuel oil and No. 6 fuel oil) on the photosynthesis of natural phytoplankton from Bedford Basin, Nova Scotia, measured the amounts of oil (both dissolved and parti- culate) directly before and after the incubation period. The method used was fluorescence spectroscopy (Keizer and Gordon, 1973) which detects aromatic compounds only, but the results were expressed in units of total oil used as a standard. All three oils inhibited photo- synthesis, measured by I4CO, uptake, when present in amounts ranging from 50-300 pg/l, No. 2 fuel oil having a greater effect than the others. However, when lower amounts of oil were used (50 pg/l) Venezuelan crude stimulated photosynthesis. The quantities used in the experiments included the average valueof 20 11.811 found at a depth of 1 m in Bedford Basin a t the same time (Keizer and Gordon, 1973), although much higher levels could occasionally be observed (e.g. 800 pgll at a depth of 25 cm beneath a 2-day old slick of crude oil). By using quantities of oil that included those normally found in field situations the authors were able to conclude that the 1973 levels of oil contamination in Bedford Basin would have had no serious effect on photosynthesis by the natural phytoplankton community. Studies of the toxicities to phytoplankton of the WSPs of crude oils have been made by Lacaze (1969) who detected a 10% reduction in growth of the diatom Phaeodactylum tricornutum Bohlin in a medium containing water-soluble components of Kuwait crude oil used at a level of 10 ml/L In addition, Nuzzi (1973) showed that the WSFs of three different oils varied considerably in toxicity to phytoplankton, that of No. 2 fuel oil being much more toxic than that of either No. 6 fuel oil or an outboard-motor oil when tested with either an axenic culture of Phaeodactylum or a natural population of phytoplankton. In further tests, three algal species showed different susceptibilities to the No. 2 fuel oil, Chlamydomonas sp. being the most resistant and Xkeletonema costatum the least. Certain findings indicate that the effects of petroleum hydrocarbons POLLUTION STUDIES WITH MARINE PLANKTON-I 321 on phytoplankton vary with season. Thus, Gordon and Prouse (1973) found that the effect of Venezuelan crude oil was much more marked in spring than in autumn; and Fontaine, Lacaze, Le Pemp and Villedon de Nayde (1975) observed that the effects of the WSF of Kuwait crude oil on 14C-uptake by natural phytoplankton popula- tions in the Gulf of St Malo (English Channel) were more marked in summer than in spring. At 12"C, the spring temperature, 14C-uptake increased by over 100% at a hydrocarbon concentration of 15 pg/l; but at 17"C, the summer temperature, it was inhibited by over 90%. Changes in species composition could account for the differences in sensitivity to hydrocarbons with season. Another possibility, men- tioned by Fontaine et al. (1975), is that auxins present in crude oil (Gudin and Harada, 1974) might particularly affect spring populations. Temperature effects seem to vary markedly with species, however, for in further experiments by Fontaine et al. (1975), using the single species Phaeodactylum tricornutum, the inhibition of W-uptake at 7-14°C was much greater than that a t 16-25°C. Further studies of the varying degrees of sensitivity to crude oil shown by different phytoplankton species have been made by Pulich, Winters and Van Baalen (1974). Six unialgal species were used: Agmenellum quadruplicatum (Menegh.) BrBb., Nostoc sp, Thalassiosira pseudonana (Hust.) Hasle et Heindal, Dunaliella tertiolecta Butch., Chorella vulgaris var. autotrophica (Shihira et Krauss) Fott et Novakova and ~lenodinium hallii Freudenthal et Lee (referred to as Gymnodinium halli). Growth-rate data were expressed in terms of doubling time and any lag in initiation of growth was measured by comparing the times needed by control and oil-treated samples of algal cells to reach the same point on the growth curve. Photosynthesis was measured as oxygen production (Van Baalen, 1968). Two crude oils (Kuwait and Southern Louisiana) and No. 2 fuel oil were used, a WSF of the oil itself and of various distillates formed a t different temperatures being prepared in each case. Differences in sensitivities of algal species were demonstrated by the finding that the growth of Chlorella was severely inhibited by water- soluble components of the low-boiling fractions (195-270°C) : these, however, had little effect on the growth of either Thalassiosira or Agmenellum which were more susceptible to water-soluble extracts of high-boiling fractions (285-385°C). Experiments using No. 2 fuel oil equilibrated with sea water (15 mg total extractables/l) in various dilutions (0.0075-7-5 mg/l) showed that these had no effects on growth measured as mean generation times for the six test species. However, there was an occasional 322 E. D. 9. CORNER lengthening of the lag phase before growth began, particularly in the experiments with Qlenodinium, Thlassiosira and Agmenellum, the lag phases being substantially increased by exposure to a concentra- tion of 1.5 mg total extractables/l. Studies of the effects of the water-soluble components of a No. 2 fuel oil on photosynthesis (Fig. 5) showed further interesting differences in susceptibility between species : for example, photosynthesis in Thalassiosira was much more readily affected than that in Chlorella, which in turn was more susceptible than that in Agmenellum. Minutes FIG. 6. Effect of water-soluble fraction of No. 2 fuel oil on photosynthesis by 3 species of marine unicellular algae. A, Agmenellum qwdrmplicatum: pecked line = control containing sea water plus growth medium; continuous line = 60% oil: water v/v (i.e. 1-0 ml sea water containing oil solubbs plus 1.0 ml algal suspension). B, Chl'hlorelka autotrophica: pecked line = control; continuous line = 20% oil: water v/v. C, Thalaseiosira p8ewEonana: dashed line = control; continuous line 5 12% oil: water v/v. Algal concentrations for all 3 test species approximately 1 x 107 cellslml growth medium. Temperature, 3OOC. (After Pulich et al., 1974.) Relating to the work by Pulich et aZ. (1974) involving distillate fractions of oils is that of Parsons, Li and Waters (1975) using three different mixtures of hydrocarbons : aromatics (benzene, toluene, m-xylene, o-xylene and p-cymene), n-alkanes (C12 to C16) and n-alkenes (Clo to C14). Laboratory studies were made with natural phytoplankton populations, one dominated by Bkeletonema costatum and the other by Nitzschia sp. Hydrocarbons in low concentrations enhanced photosynthesis by the population dominated by Nitzschia, the effect being greater with aromatic compounds than with either n-alkanes or n-alkenes : thus, POLLUTION STUDIES WITH MdRINE PLANKTON-I 323 at the 5 pg/l level aromatic hydrocarbons enhanced photosynthesis by 70% whereas the corresponding value with n-alkanes was less than 50% and for n-alkenes less than 40%. These effects diminished as the concentrations of hydrocarbons increased, a particularly rapid fall-off being observed with the aromatic compounds. Different trends were observed, however, using the population dominated by Skeletonema : enhancement of photosynthesis by the aromatic compounds was less than 20% at the 5 pgll level but increased to 60% at 500 pg/l ; low levels of n-alkanes slightly suppressed photosynthesis but higher levels (> 100 pg/1) enhanced it ; suppression of photosynthesis by n-alkenes occurred at all concentrations in the range 5 to 500 pgll, higher amounts causing greater effects. Further studies, using unialgal species, have recently been made by Prouse et al. (1976) who, as in their earlier work with natural popula- tions of phytoplankton, paid particular attention to the need to study the toxic effects of crude oil using concentrations similar to those found in the environment. In addition they took care to monitor changes in hydrocarbon composition and level during the experiments, using fluorescence spectroscopy and gas chromatography. During the course of the experiments ( 1 6 1 8 days) they found that the composi- tion of the hydrocarbons " accommodated " in the sea water changed markedly with time, compounds predominant a t the end being, not unexpectedly, the least volatile, most soluble and most resistant to biological alteration : that is, aromatic compounds of medium mole- cular weight. The presence of algae had a marked effect on the levels of oil in the test media,
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