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Prévia do material em texto

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,

Outros materiais