[Advances in Marine Biology] Advances in Marine Biology Volume 15 Volume 15 || Pollution Studies with Marine Plankton
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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
E. D. S . CORNER The Laboratory, Marine Biological Association,
v m .
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 . . .. .. .. .. .. .. ..
A. Studies using Crude Oils and their Water-soluble Fractions . .
D. Metabolism .. .. .. .. .. .. .. ..
References . . .. .. .. .. .. .. .. .. 289
290 291 293 294 300
317 319 327
335 335 339 340 342 348
351 352 354 356 357
362 362 363 364
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 total alkanes, 0.015-0-043 pg/l for pristane and < 0.001-0.014 pg/l for phytane. Similar to Blumers (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
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 STUDIES WITH 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
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
Dissolved n-alkanes (C14 to 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
0.1 1 0-28
3-34 5.66 0.32
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
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
20.4 0.4 to 0.8
1 to 21
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 Percentage of total
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
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
No phthalenes Biphenyls
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 C84H30 to 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.
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
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
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?) HEH Cryptomonas (old culture) HEH
Haptophyceae Coccolithus huxZeyi (Lohm.)
Kampt. HEH Isochrysis galbana Parke HEH Phaeocystis pouchetii (Hariot)
Euglenaph yceae Eutrepiella sp. HEH, n-C,,, n-C,,
Cyanophyceae Oscillatoria woronichinii
Synechoccus bacillaris Butch.
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
n-alkanes n-alkanes Pristane, n-alkanes
Pristane, n-alkanes Pristane, n-alkanes,
Pristane, n-alkanes, n-C,, : 4
n-C,, : 4
Pristane, n-alkanes Pristane, n-alkanes
Pristane, n-alkanes Pristane, n-alkanes
n -alkanes n-alkanes, other olefins
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 worlds 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 WORLDS OCEANS ~~
Ocean intake ( x 106 metric tom per annum)
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 worlds 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
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
Chaetognath ns 0.02 0.05
0.01 g o . 0 1
Amphipod 0.04 ns
Copepod Copepod Copepod Copepod Copepod Copepod Copepod
Copepod Copepod Copepod
Copepod Copepod Copepod Copepod
Copepod Copepod Copepod
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)
1 ] Neophytadiene Cz0- phytadienes detected by Blumer and Thomas (1965~)
1 CIs-di-and tri-olefins detected by Blumeretul (1969)
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,
//I\\ Metabolic chanqes giving hydrocarbons in zooplankton . .
A H A H 2 0 1
Phytenic acid Di hydrophytol ..... .....
..... L+ ..... A Norphytene .....
(CIS and truns)
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
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
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))
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-
Aromatics Benzene Methylbenzenes Naphthalene Methylnaphthalenes Biphenyl Methylbiphenyls Fluorene Methylfluorenes Dibenzothiophene Phenanthrene Methylphenanthrenes
Total saturates Total aromatics Total hydrocarbons
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
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
0.020 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
0.005 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.
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 value of 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 7-14C was much greater than that a t 16-25C.
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 (195-270C) : 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 (285-385C).
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.
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, which fell by over 90% in 12 days.
One new feature of the work was that growth data for all five test species ~Du~li~lla tertiolecta, Fmgilaria sp., Monochvysis sp., Skele- tonema sp. and Chaetoceros sp.) cultured under axenic conditions were only obtained after the lag phase had finished and the plants were growing exponentially ; another was that the experiments lasted much longer (average 11 days).
An initial concentration of 50 pg/l of a No. 2 fuel oil stimulated the growth of Fragilaria and initial levels of 55 and 106 pg/1 of Kuwait crude oil enhanced that of Dunaliella. However, contrary to previous findings (Gordon and Prouse, 1973), no strong inhibitions were observed and any minor ones that occurred in occasional experiments were short-lived (Fig, 6). Furthermore, the experiments did not show any consistent differences in response between the five test species.
Consistent with the evidence that petroleum hydrocarbons can stimulate photosynthesis by certain species of phytoplankton are data by Dunstan, Atkinson and Natoli (1975) who measured the growth of
324 E. D. 5. CORNER
4 phylogenetically different marine algae exposed to a wide range (0.1 to 100 mg/l) of concentratous of the volatile, aromatic hydrocar- bons benzene, toluene and xylene. The growth rate of Dunaliella tertiolecta was markedly stimulated by all 3 compounds ; smaller effects were observed with Amphidinium carterae Hubert and HymenomonaS cartrterae (Braarud et Paged.) Braarud as Cricosphaera carterae ; no enhancement of growth rate was found with Skeletonema costatum. The
FIG. 6. Growth of Dunaliella tertiolecta in the presence of No. 2 fuel oil. Oil con- centrations: 50 (initial) falling to 2 (final) pg/l (open triangles) : 380 (initial) falling to 45 (final) pg/l (open circles). Dashed line = control. (After Prouse et al., 1976.)
growth rate of Dunaliella was also stimulated and that of Skeletonema reduced by No. 2 fuel oil, both effects depending upon the presence of the volatile fraction.
Winters, O'Donnell, Batterton and Van Baalen (1976) have con- tinued the work of Pulich et al. (1974) with further studies of the effects of WSFs of fuel oils on the growth of individual phytoplankton species. The work was less concerned than that of Prouse et al. (1 976) with using oil concentrations close to those found in the environment. Instead, the main emphasis was on analysing the numerous chemical components of the water-soluble fractions of the oils and attempting to identify those that are particularly toxic. The fuel oils used, referred to by refinery location, were Baytown (Texas), Baton Rouge (Louisiana), Billings (Montana) and Luiden (New Jersey): the algal species were
POLLUTION STUDIES WITH MARINE PLANKTON-I 325
Agmenetlum quadruplicatum, Coccochloris elaabeus (Brkb.) Dr. et D., Dunaliella tertiolecta, Chlorella vulgaris var. autotrophica, Cylindrotheca sp. and Amphora sp.
About half the water-soluble components of each oil were identified by gas-chromatography and mass spectrometry. Included were com- pounds such as naphthalene, alkyl-naphthalenes, benzene and alkyl- benzenes identified earlier by others (e.g. Boylan and Tripp, 1971): particularly interesting, however, was the detection of phenols, methyl- anilines (o-, m- and p-toluidine) and indoles, the methyl-, dimethyl- and trimethyl- derivatives of which were present in relatively high amounts. Phenols accounted for more than half the total identified organic compounds in the WSF of the Baytown fuel oil and were also well represented in that of the Montana fuel oil (Table XI).
TABLE XI. MAJOR CONSTITUENTS OF WATER-SOLUBLE FRACTIONS OF FUEL OILS
Baton Rouge Montana Baytown New Jersey
(PgP) (Pg/l) (PgP)
Total identified organics 8.07 7.90 5.66 3-52 Methylnaphthalenes 0.53 0-81 1.30 0.76 Dimethylnaphthalenes 0.31 0.24 0.55 0.41 Phenols 2.33 4.12 1-96 1-08 Anilines 2.57 0-72 0.27
326 E. D. 9. UORNER
relatively high toxicities of the substituted anilines, particularly p-toluidine, when used against the coccoid blue-green alga Agmenellum (see Table XII). Additional observations showed that other species of blue-green algae are also affected by p-toluidine which may well possess a selective toxicity for these organisms.
TABLE XII. EFFECTS OF HYDROCARBONS AND RELATED COMPOUNDS ON THE GROWTH OF Agmenellum qzcadruplicatum MEASURED BY THE
" ALGAL LAWN " TECHNIQUE
Amount used in teat (mg) 0.5 0.1 0.01 36* 36 36
2, 4, 6-Trimethylphenol Dimethylquinoline 2, 6-Dimethylphenol Dimethylnaphthalene Biphenyl 1, 2, 4-Trimethylbenzene p-Cresol Di-iso-propylbenzene Methylnaphthalene Naphthalene Phenanthrene Tri-ethylbenzene 1, 2, 4, Ei-Tetramethylbenzene Fluorent,
36 36 36
36 36 6 3 1 0
2 36 36 32 23 21 16 16 12 10 2 4 2 0 0
1 36 27 11
6-7 4 0 4 0 0 0
* Numbers are zones of inhibition in mm measured from edge of filter pad. Complete kill of test organism gives 36 mm zone of inhibition. Where 0- , m- and p-derivatives tested isomer with highest toxioity taken.
Data summarized from Pulich et al. (1974) and Winters et al. (1976).
Recent work by Winters, Batterton and Van Baalen (1977) has shown that the hydrocarbon derivative phenalen-1-one (perinaphthe- none) is present in the water-soluble fraction of No. 2 fuel oil and is almost as toxic to green algae as p-toluidine is to blue-green algae. Further evidence of differences in response between algal species was provided by the finding that toluidines were present in water-soluble fractions of Baytown and Montana fuel oils in quantities sufficient to kill Agmenellum and Coccochloris, whereas the benthic diatoms Cylindrotheca and Amphora were not much affected by the water- soluble components of any of the oils tested.
POLLUTION STUDIES WITH MARINE PLANKTON-I 327
B . Studies using naphthalene
Work already described has included studies with specific distillate fractions of oils (Pulich et al., 1974) and individual constituents of the water-soluble fractions (Pulich et al., 1974; Winters et al., 1976, 1977). In addition, detailed studies have been made using the bi-cyclicaromatic compound naphthalene, which has been shown to be present in con- siderable amounts in aqueous extracts of oils (Boylan and Tripp, 1971) and is fairly toxic to a wide range of marine organisms. Thus, as shown in Table XII, in terms of its toxicity to the micro-alga Agmenellum, naphthalene is more active than either phenanthrene or fluorene, but less so than its own alkylated derivatives (methyl- and dimethylnaphthdene) or those of phenol and aniline.
The main difficulty in working with naphthalene is loss of the hydrocarbon by evaporation. For example, Vandermuelen and Ahern (1976), studying its effects on the growth of FTagilaria sp., found that the concentration in the culture medium fell by over 90% in 18 days. This made interpretation of the findings difficult. Thus, a 50% saturated solution of naphthalene in sea water caused a marked inhibition of growth over 14 days, after which the cell population showed a rapid increase: the cells might have recovered through overcoming the inhibitory effects of naphthalene, but there was also the possibility that such recovery was related to loss of the hydrocarbon from the culture medium.
Further studies on the toxicity of naphthalene to algae were those of Soto, Hellebust, Hutchinson and Sawa (1975~) and Soto, Hellebust and Hutchinson (1975a). This work, having been done with the freshwater species Chlamydomows angulosa Dill, lies outside the scope of the present review but deserves brief mention because of its relevance to the important question whether aromatic hydrocarbons can be metabolized by algae. The data from one of several experi- ments are shown in Fig. 7.
Cells treated with 14C-l-naphthalene for several days and then transferred to fresh medium containing no hydrocarbon lost radio- activity rapidly over two days during which there was no increase in the number of cells. Such losses could have been the result of passive diffusion out of the cells, or metabolism, or both. On the other hand, cells pre-treated with W-1-naphthalene and then left in the medium containing the hydrocarbon did not lose radioactivity until they began to divide, the apparent coincidence in time between resumption of cell division and loss of radioactivity per cell suggesting that dilution by cell division alone was probably the main factor affecting the level
328 E. D. 9. CORNER
of naphthalene in the cells. This observation, together with the further finding that no non-volatile radioactive compounds were detected in the medium, was taken to imply that the plant cells were unable to metabolize the hydrocarbon.
The question whether aromatic hydrocarbons remain in the plant cells unchanged or in the form of metabolites is important in the con- text of the transfer of the compounds to higher trophic levels : it there- fore deserves detailed investigation using various unialgal species, both freshwater and marine, as well as with natural populations of
FIG. 7. Uptake and release of naphthalene by C ~ Z a r n ~ d o r n ~ ~ angulosa, together with growth data. Cells initially incubated in naphthaIene-saturated growth medium in e closed system for 6 days (A) or 7 days (B) and then either washed and transferred to fresh medium (open circles) or left in the naphthalene-saturated system (open squares). (After Soto et aZ., 1975a.)
phytoplankton. Many marine micro-organisms such as bacteria and fungi have the ability to degrade petroleum hydrocarbons. So far, however, the only degradation study with an alga seems to be that of Walker, Colwell and Petrakis (1975) who showed that the achloro- phyllous species Prototheca zopjii Kriiger can degrade motor oil and South Louisiana crude oil, in which respect the plant was as efficient as various species of bacteria but not so efficient as the fungi.
The effect of naphthalene on photosynthesis by three marine
POLLUTION STUDIES WITH MARINE PLANKTON-I 329
unicellular algae has been recently studied by Vandermuelen and Ahern (1976). Suppression of photosynthesis was found to be con- centration-dependent : for example, with Pavlova Zzctheri (Droop) Green as Monochrysis 1zLtheri Droop, a concentration of 1 mg naphthalene/l reduced photosynthesis by 40% whereas a concentration of 7 mgfl reduced it by 90%. Cells transferred to uncontaminated medium after pre-incubation for 4 h in a naphthalene concentration of 5 mg/l quickly recovered the ability to photosynthesize, the normal rate being restored within 5 h. Treatment with the hydrocarbon did not reduce levels of chlorophyll a in the cells, but did cause a marked decrease in ATP levels both in the light and the dark. Vandermuelen and Ahern (1976) take this to mean that suppression of photosynthesis could have arisen from a blockage of oxidative phosphorylation. However, although this could well be the case with naphthalene, whole oils seem to have a direct effect on chlorophyll a levels (Mills and Ray: unpublished observations quoted by Anderson, 1975).
V. MECHANISMS OF PRYTOTOXICITY
Although numerous studies have been made of the effects of petroleum hydrocarbons on growth and photosynthesis by marine phytoplankton, little work has been done concerning the mechanisms by which the compounds exert their toxic action. Certain conclusions regarding the effects of hydrocarbons on terrestrial plants, freshwater micro-algae and marine multicellular algae (reviewed by OBrien and Dixon, 1976) do, however, have sufficient relevance to justify inclusion in the following brief account.
Van Overbeek and Blondeau (1 954) suggested that hydrocarbon molecules disrupt the plasma membrane by displacing those of other lipid compounds, so affecting its semi-permeability ; in addition, that the inhibition of photosynthesis could result from hydrocarbons dis- solving in the lipid phase of the grana of chloroplasts and increasing the distance between individual chlorophyll molecules. Baker (1 970) has proposed that a similar disruption could occur in mitochondria1 membranes with inhibition of the tricarboxylic acid cycle and oxida- tive phosphorylation, as noted by Vandermuelen and Ahern (1976) in their studies of naphthalene toxicity to Monochrysis lutheri. Distortion of the lipid in cell membranes by kerosene, with subsequent penetration by toxic agents, was found with different species of marine red algae by Boney and Corner (1959).
The importance of physical factors was emphasized by the work of Currier (1951) who found that the toxicities of benzene, toluene,
330 E. D. S. UORNER
xylene and trimethyl-benzene in aqueous solution were inversely related to solubility. Data for the partition coefficients of these com- pounds between water and paraffin oil indicated that penetration into plant cells would increase with the number of methyl substituents in the benzene ring. Kauss, Hutchinson, Soto, Hellebust and Griffiths (1973) found that toxicity to Chlorella vulgaris increased along the series benzene, toluene, xylene and naphthalene, with water-solubili- ties increasing in the reverse order. However, it should be noted from the work of Pulich et al. (1974) and Winters et al. (1976), summarized in Table XII, that increasing the number of substituent methyl groups in benzene from 3 to 4 causes a marked reduction in toxicity to the micro-alga Agmenellum quadrqlicatum.
Stimulation of both growth and photosynthesis by low concentra- tions of oil have been noted by various workers using unialgal cultures (Galtsoff et al., 1936; Xronov and Lanskaya, 1966; Kauss and Hutchinson, 1975; Soto et al., 1975a) and natural populations of phytoplankton (Gordon and Prouse, 1973; Parsons et al., 1975). It has been suggested that such stimulation may result from the oil components being used as metabolic substrates by the plant cells (Soto et al., 1976a) ; in addition, that it could be caused by the presence in oil of growth regulating compounds (Gordon and Prouse, 1973). Growth stimulation induced by contact with individual PNAH, includ- ing carcinogens found in fossil fuels, has been found with sporelings of multicellular red algae (Boney and Corner, 1962 ; Boney, 1974) : however, the effects of such compounds on growth or photosynthesis by unicellular algae seems not to have been studied.
Another area of investigation has been concerned with the effects of oil components on the chemical composition of marine algae. Thus, Soto, Hellebust and Hutchinson (1975b) studied the effect of naphtha- lene on the levels of pigments, lipid, protein, carbohydrate and total carbon in the freshwater alga Chlamydomonas angulosa and found that high concentrations of the hydrocarbon decreased cellular protein by 34% in seven days. This loss, however, was nearly all recovered within one day after the cells had been transferred to fresh medium. Changes in lipid levels followed the reverse pattern, more than doubling during the seven-day exposure to naphthalene, but falling by roughly the same amount when the hydrocarbon was removed from the growth medium. Carbohydrate also showed a marked increase during naph- thalene treatment, this change probably being associated with thicken- ing of the cell walls: after transfer of the culture to fresh medium, carbohydrate levels fell ~ E J the cell walls regained their normal thick- ness. The observation that a petroleum hydrocarbon may induce an
POLLUTION STUDIES WITH MARINE PLANKTON-I 331
increased conversion of protein into lipid in plant cells has implications in terms of primary and secondary production that deserve more detailed study, particularly with marine species : it is significant, how- ever, that even when high concentrations of the hydrocarbon are used the effects on plant cells are reversible.
The studies of Soto et ab. (1975b) were carried out with a single hydrocarbon. However, Davavin, Mironov and Tsimbal (1975) investigated the effects of whole crude oil, using emulsified suspensions in sea water covering the range 0-1-10 ml/l, and found that it inhibited the biosynthesis and modified the polymerization of DNA and RNA in multicellular algae from the Black Sea. No similar study with unicellular algae seems to have been made.
VI. CONTROLLED EGO-SYSTEM EXPERIMENTS
In the previous sections most of the experimental work described has been carried out in the laboratory with a restricted number of constituents of the marine eco-system. However, because of inter- actions between species in nature it is necessary to discover how whole eco-systems react to pollutants. A recent important development has therefore been to carry out pollution studies using large enclosures containing whole eco-systems set up in inshore areas and subject to natural light and temperature conditions (Takahashi, Thomas, Siebert, Beers, Koeller .and Parsons, 1975). Some of these enclosures are polluted with substances such as crude oil, or mixtures of hydrocarbons, or heavy metals: others serve as controls. Physical, chemical and biological variables are monitored during a preliminary period of stabilization, after which the pollutants are added and their long-term effects on the system followed, usually over several weeks.
This type of approach was first used by Lacaze (1974) in studying the effect of Kuwait crude oil on primary production in an experi- mental eco-system set up in the Rance Estuary (North French coast). Each enclosure, made of rilsan (impermeable to hydrocarbons), contained 560 1 of sea water from which larger zooplankton had been excluded by filtration. In the experiment using Kuwait crude oil 100 ml was added to the enclosure, giving a suspension equivalent to 180 mg/l. Over a period of four weeks during September and October the temperature fell from 17 to 14OC in the open water and primary production steadily decreased : in the control enclosures, however, it fell much more rapidly during the first week and then stabilized at a, level much lower than that of the open water, probably because inside the enclosures the nutrients could not be renewed. Primary production
332 E. D. 9. CORNER
in the samples treated with crude oil underwent an immediate reduc- tion of about 50% during the first day after the oil was added, but by the third day it had regained the same level as that in the controls (see Fig. 8), the rapid initial drop being caused by the presence in the oil of toxic volatile fractions that were quickly lost. Over the next four days primary production in the oil-treated sample fell far more rapidly than that in the controls and stabilized a t a substantially lower level throughout the remainder of the experiment. Clearly the oil caused a
FIG. 8. Primary production in an eco-system polluted by crude oil (open circles, con- tinuous line) compared with that in an untreated system (sled circles, dashed line). Oil added on 25 September. (After Lacaze, 1974.)
significant inhibition of primary production by the natural phytoplank- ton population : however, the effects could have been unnaturally enhanced if the plants were, in fact, in a state of nutrient deficiency. A further complication in this and other long-term studies using whole marine eco-systems exposed to natural conditions of light and tempera- ture is that chemical and photo-chemical oxidations of crude oil com- ponents could proceed rapidly a t the surface of the sea water and lead to the formation of compounds such as hydroperoxides which have considerable biological activity (Burwood and Speers, 1974 ; Frankenfeld, 1973; Larson, Blankenship and Hunt, 1976). Lacaze and Villedon de Naide (1976) examined this possibility using P h u e o ~ ~ ~ y l u m tricornutum as the test organism and Kuwait crude oil. Compared with oil suspensions kept in the dark, those exposed to 10 000 lux for
POLLUTION STUDIES WITH MARINE PLANKTON-I 333
40 h or more under fluorescent light had more than twice the toxicity as measured in terms of inhibition of photosynthesis.
A controlled eco-system experiment of a more extensive kind was that of Lee,' Takaha.shi, Beers, Thomas, Seibert, Koeller and Green (1977). In this study the WSF of No. 2 fuel oil was added to an enclosure of 60000 1 capacity containing sea water from Saanich Inlet (British Columbia), giving an initial hydrocarbon concentration of 40 pgll. The levels of individual compounds were monitored at different depths throughout the duration of the experiment (19 days) and showed a substantial decrease : e.g. those of naphthalene, methyl- naphthalenes and dimethylnaphthalenes combined amounted to 12 pg/l the first day after the WSF was added to the enclosure, but after 3 days had fallen to 5 pgll and by day 16 were below detectable limits. Microbial degradation and metabolism by zooplankton both contributed to the losses of these hydrocarbons. So did removal by sinking particles, concentrations of hydrocarbons increasing in the sediment from the oil-treated enclosure, which included both phyto- plankton and faecal pellets.
When the addition of the WSF took place the standing stock of diatoms in the control enclosure dominated by Cerataulina bergonii (Perag.) Schiitt, now Cerataulina pelagica (Cleve) Hendey, was much higher than that in the oil-treated sample (Fig. 9a). This large diatom population in the control fell sharply during the next four days, being replaced by a micro-flagellate bloom (Fig. 9b) : it then rose again to a peak after ten days. In the treated sample the relatively small diatom standing stock diminished still further after the WSF was added and did not recover : however, micro-flagellates showed a sharp increase (Fig. 9b), though not so great as that in the control enclosure; the population at first being dominated by Chrysochromulina kappa Parke et Manton and later by Ochromonas sp. Associated with the micro-flagellate levels in the treated sample were sharp increases and reductions in the micro-zooplankton populations, particularly tin- tinnids such as Helicostomella subulata (Ehrenberg) (Fig. 9c). However, this close association was not observed in the control enclosure, presumably because diatoms were also available as a food for the herbivores.
The dominant zooplankton species was Pseudocalanus minutus (Kroyer) ; other copepod species were also present and, to a lesser extent, larvaceans, ctenophores and medusae. No major differences were observed between the zooplankton standing stocks of the treated and control enclosures, presumably because even the initial concentration of hydrocarbons used (40 pg/l) was far less than the LC,, values
I l l l l l l l l l l l l l l l l ~ ~ 2 4 6 8 10 12 14 16 18
FIQ. 9. Effects of a water-soluble fraction of No. 2 fuel oil on the production of diatom (a), micro-flagellates ( b ) , and tintinnids ( c ) in a CEPEX study. Control enclosure; filled circles, continuous line. Enclosure to which WSF added on day 6; filled triangles, dashed line. (After Lee et al., 1977.)
POLLUTION STUDIES WITH MARINE PLANKTON-I 335
(590-1 350 pg/l) found for copepods in laboratory bioassays (see Table XV). There was, however, some indication of a slower rate of growth by Pseudocalanus in the enclosure treated with hydrocarbons.
As Fisher and Wurster (1974) have emphasized, aquatic com- munities consisting of herbivores selectively feeding on plants are so interlinked that toxic compounds directly affecting one component of the community can indirectly alter the species composition of the other. The controlled eco-system experiment carried out by Lee et al. (1977) provides a good example of how pollutants causing changes in the composition of a phytoplankton population can lead to alterations in the structure of the micro-zooplankton community representing the next trophic level.
VII. FATE OF HYDROCARBONS IN ZOOPLANKTON
A. Uptake and release Mention has been made earlier of the finding by Blumer et al.
(1964) that the biogenic hydrocarbon pristane is not metabolized by C. hyperboreus. More recent studies have been mainly concerned with the fate in zooplankton of hydrocarbons, such as PNAH, which are found in crude oil and include the carcinogen BP (although the amounts of this compound are small, 0-029-44 mg/kg with an average of 2.0: Pancirov and Brown, 1975).
FIG. 10. Net uptake and release of radioactivity, expressed as equivalents of benzo- [ulpyrene, by Culunus plumchrua. Filled circles, copepods exposed t o 1.0 p g hydrocarbon/800 ml; open triangles, copepods exposed to this same concentration for three days and then transferred to clean sea water. (After Lee, 1976.)
W W Q,
TABLE XIII. RETENTION OF RADIOACTFJITY BY ZOOPLANKTON EXPOSED TO LABELLED HYDROCARBONS
Test species Concn
(P9POO ml) Hydrocarbon
Exposure period (days)
Depuration period (days)
yo Radioactivity retained
Calanus plumchrus Calanus plumchrua Calanus plumchrus Calanus helgolandicus Calanus helgolandicus Calanus helgolandicus Euchaeta japonica Euchaeta japonica Parathemisto paci$ca Cyphocaris challengeri Stebbing
1 -OD BP BP BP
20-MC N BP BP
1.0 0.2 5.0 1.0 1.0 1.0 0.2
80 15 15
3 1 4 2 4 4 1 4 2 2
17 8 3 9 7
28 8 8
U 0-3 1 8.0 40.0 P
0.50 s 0.23 7.7 2.5 0.28 6.7
Q 2.0 0
BP, 8H-benzo[a]pyrene; 20-MC, 3H-20-methylcholanthrene; 1-OD, 14C-l-octadecane; N, 14C-l-naphthalene. Data from Lee (1975).
POLLUTION STUDIES WITH MARINE PLANKTON-I 337
The first of these studies was by Lee (1 975) who examined the net uptake and release of various hydrocarbons by several groups of zooplankton animals collected off the coasts of California, British Columbia and in the Arctic. Copepods were mainly used-although a few observations were also made with euphausiids, amphipods, crab zoeae, ctenophores and jellyfish-and the hydrocarbons were 14C- l- naphthalene, 14C-BP, 3H-BP, 3H-20-methylcholanthrene and 14C-l- octadecane. Typical data are shown in Fig. 10 for the species Calanus plumchrus Marukawa exposed to 3H-BP in sea water at 1.25 pgll, together with 50 pgll of water-soluble hydrocarbons from No. 2 fuel oil.
During the first three days there was an approximately linear increase in the net uptake of hydrocarbon, but no further increase was detected when exposure was continued for another seven days. Animals transferred after three days to clean sea water (containing no added hydrocarbons) lost radioactivity gradually over a further 17-day period, at the end of which a residue equivalent to 0.31% of that originally accumulated still remained in the animals. In studies with various species of copepod different times of exposure and depura- tion were used, but in every case a small residue of the original level of radioactivity remained in the animals after the depuration period (Table XIII). Most notable among these experiments was that with Calanus hyperboreus in which 0.23% of the original level of radioactivity was still present in the animals after a period of depuration lasting 28 days.
Studies by Corner, Harris, Kilvington and OHara (1976b), using the copepod Calanus helgolandicus and the bi-cyclic aromatic hydro- carbon naphthalene, showed that the net uptake of 14C-l-naphthalene from solution in sea water varied with the concentration of hydro- carbon used (Fig. 11). Adult female Calanus showed a daily net uptake of the hydrocarbon from sea water containing very low levels : e.g. 17.8 pglanimal from a sea water concentration of only 0-5 pg/l; this level being an order of magnitude lower than that of 4.8 pg/l calculated from the data of Barbier et al. (1973) for total dissolved bi-cyclic aromatic hydrocarbons in a Channel harbour area (see p. 303).
Using an apparatus designed to provide Calanus with a known ration of Biddulphia sinensis Grev. (Corner, Head and Kilvington, 1972), 14C-1-naphthalene incorporatedin the algal cells was administered to the animals and the subsequent rate of depuration compared with that observed using animals that had accumulated a similar level of hydrocarbon from solution alone. It was found that when the hydrocarbon was taken up by way of the food the subsequent rate of depuration was notably slower (Fig. 12).
t I I I 1 1 o . * n 1 1 # 1 , , , . 1 I ' I I I I 1 J I ' , 1 1 1 1 1 1 ' ' J * ~ ~ d
lo-' 100 10' I02 lo3 Naphthalene concentration in sea water (&I)
Fra. 11. Net uptake of radioactivity, expressed as equivalents of naphthalene, in 24 h by adult female Calanw, helgolandicw, exposed to various concentrations of 1%-I- naphthalene in sea water. Relationship defined by y = 1 . 0 6 6 ~ + 1.67; correlation coefficient = 0.990. (From Corner el al., 197Gb with permission of the Council of the Marine Biological Association.)
Fra. 12. Release of radioactivity by adult female Calanw, helgolalzdicw, that had accumulated 14C- 1-naphthalene either from a sea-water solution (filled circles) or from a diet of Biddulphia cells (open circles). Levels expressed as percentages of the radioactivity originally present in the animals. (From Corner el al. 1976b with permission of the Council of the Marine Biological Association.)
POLLUTION STUDIES WITH MARINE PLANKTON-I 339
In a further study (Harris, Berdugo, Corner, Kilvington and OHara, 1977a) the daiIy net uptakes of 14C-1-naphthalene were measured using seven species of copepod representing oceanic, neritic and estuarine forms. The levels of hydrocarbon used, which ranged from 0.2 to 1000 pg/l, included those for bi-cyclic aromatic hydro- carbons present in the sea under a wide variety of conditions (see Section 11). Combined data from experiments with all the test species showed that total lipid content was a good indicator of the net uptake by copepods of an aromatic hydrocarbon such as naphthalene from solution in sea water during short-term exposures, the regression equation being log y = 0.974 log x +0.61, with r = 0.98 and n = 153, y being pg hydrocarbon/pg copepod body lipid, x the concentration of hydrocarbon in sea water in pg/l, r the correlation coefficient and n the number of determinations. Harris et al. (1977a) confirmed an earlier observation by Lee (1975) that surface adsorption of the hydrocarbon was only a minor factor influencing its net uptake by the animals. They also showed, however, that temperature and degree of starvation were of major importance and that both were negatively correlated with net uptake.
Further experiments by Harris et al. (1977a) verified earlier findings by Lee (1975) that small amounts of hydrocarbons accumulated by copepods can still be detected in the animals after prolonged periods of depuration. In particular they showed that when nauplius I of the estuarine copepod Eurytemora afinis Poppe were immersed in sea water solutions of 14C-l-naphthalene for 24 h, transferred to fresh sea water and reared in the laboratory to adults over a period of 34 days, radioactivity accounting for 10% of the original amount in the nauplius could still be detected in the adults. Such persistence of small quantities of aromatic hydrocarbons, or their derivatives, in zooplankton over long periods implies that although natural processes such as volatilization, photo-oxidation and microbial breakdown of these compounds may occur soon after an oil spill, several weeks later the transfer of an aromatic hydrocarbon like naphthalene from zooplankton to a higher trophic level, such as fish, could still be taking place.
B. Quantitative importance of the dietary pathway
Corner et al. (1976b) found that, in terms of providing the same level of radioactivity in C. helgolandicw, the quantity of 14C-l-naph- thalene needed in solution was much greater than that required as particulate food and concluded that the dietary pathway of uptake
340 1. D. 5. CORNER
was more important quantitatively. Harris et al. (1977a) obtained more direct and detailed evidence of this in 24 h experiments in which levels of radioactivity were measured in animals that simultaneously accumulated lac- 1 -naphthalene from solution alone and from solution supplemented by a known quantity present in algal food. Compared with the amount of labelled hydrocarbon represented by the suspension of algal cells, the amount in solution needed to provide the same increase in radioactivity in the copepods was three orders of magni- tude greater in experiments with females and even two orders of magni- tude greater in those with male animals that capture a relatively small rakion. Further work showed that the quantitative importance of the dietary route was not affected by the levels of hydrocarbon in the sea water or present as food : it did however depend upon the level of food available, greatly increasing at lower cell concentrations.
Earlier it was noted (Section 11) that in some sea areas the amounts of hydrocarbon dissolved in sea water are greater than those present in particulate form. What is not known from such studies is the extent to which the particulate material could be used as a food by zooplankton. Assuming, however, that only a small fraction was present as phytoplankton that could be captured by these animals, this, compared with the much higher levels of hydrocarbon in solution in sea water, could still be a more important source of a compound such as naphthalene in herbivorous copepods.
C. Long-term exposure experiments
The work by Corner et al. (1976b) and Harris et al. (1977a) dealt only with short-term exposure of zooplankton to aromatic hydro- carbons, such as might occur immediately after an oil spill. However, it is also important to know what happens when animals are subjected to long-term exposure to low concentrations of these compounds, a condition characteristic of sea areas subjected to regular small inputs of industrial effluents or natural oil seeps. The amounts of hydro- carbons accumulated by zooplankton after long-term exposure to these compounds is of particular interest because these quantities could be critical in producing sub-acute effects: they are also the amounts likely to be transferred to higher trophic levels.
So far, only two laboratory studies of this problem have been made: one by Lee (1976), already described, and a more recent investigation by Harris, Berdugo, OHara and Corner (1977b). In the latter study adult female Calanus Fvelgolandicus and Eurytemora afinis were exposed to 14C-1-naphthalene over periods of 10-15 days,
POLLUTION STUDIES WITH MARINE PLANKTON-I 341
the animals being maintained in the laboratory on algal diets and the hydrocarbon therefore being taken up from solution and from the food.
Concerning the transfer of hydrocarbons to higher trophic levels, Harris and co-workers found that after a 10-day exposure to 14C-1- naphthalene present at a low concentration (I ,ug/l) a much higher level of radioactivity was accumulated per unit body weight by E. afinis than by C. helgolandicus (Fig. 13) ; and using feeding data for the herring (Blaxter and Holliday, 1958) they calculated that the weekly intake of hydrocarbon by a young fish would be 50 times greater if it fed on E. affinis.
2 4 6 c
FIQ. 13. Net uptake of radioactivity, expressed as naphthalene equivalents, by Calanue helgolnndicus (filled circles), and Eurytemora aflnis (open circles), in terms of dry body weight. (After Harris et al., 1977b.)
As found by Lee (1975) the amounts of I4C-l-naphthalene accumu- lated by both species used in the study by Harris et al. (197713) reached a maximum after multiple-day exposure; in addition most, but not all, of the radioactivity accumulated by the animals during prolonged exposure was rapidly lost after they were transferred to fresh sea water. The maximum level of radioactivity reached during long-term exposure to low levels of I4C-l-naphthalene probably represents a steady-state level in the animals, with hydrocarbon uptake balanced by hydro- carbon release. The higher levels of radioactivity found in animals exposed to higher concentrations of 14C- 1-naphthalene also showed
342 E. D. 9. CORNER
signs of eventually reaching a maximum, however (Fig. 14), and Harris et al. (1977b) suggested that even under conditions where large quantities of hydrocarbon were entering the animals, enzymes involved in the metabolism of hydrocarbons could still be induced or activated sufficiently to restore the balance between uptake and metabolic loss. Evidence for the metabolism of hydrocarbons by zooplankton is considered in the next section.
/%-8 L 0 5 10 Days
FIG. 14. Net uptake of radioactivity, expressed as naphthalene equivalents, b y Euryte- mora afinis (A) and Calanus helgolandicus (B) after multiple-day exposure to hydro- carbon concentrations of 0.2 pg/l (filled circles), 1.0 pg/l (open circles), 10 pg/l (open triangles), 50 pg/l (filled squares), 177 pg/l (open squares) and 992 pg/l (filled triangles). (After Harris el al., 1977b.)
There is evidence from both in vivo and in vitro studies that a mechanism for hydroxylating certain aromatic hydrocarbons is possessed by several species of marine fish (Lee, Sauerheber and Dobbs, 1972b ; Payne, 1976 ; Roubal, Collier and Malins, 1977b ; Stegeman and Sabo, 1976) and crustacean (Corner, Kilvington and O'Hara, 1973 ; Cox, Anderson and Parker, 1975; Burns, 1976; Lee, Ryan and Neuhauser, 1976; Singer and Lee, 1977). On the other hand, at least one group of marine invertebrates, the bivalve molluscs, do not seem able to metabolize hydrocarbons (Carlson, 1972 ; Lee, Sauerheber and Benson, 1972a) but release them from the tissues unchanged (Stegeman and Teal, 1973; Neff and Anderson, 1975).
POLLUTION STUDIES WITH MARTNE PLANKTON-I 343
Although only a few studies have so far been made of hydrocarbon metabolism in zooplankton, there is evidence that certain groups of these animals are able to metabolize several types of hydrocarbon. Thus Lee (1975) found that all the micro-crustaceans used in his study with zooplankton-including copepods, amphipods, crab zoeae and euphausiids-could metabolize naphthalene, BP, 20-methylcholan- threne and octadecane. Of the different species tested the amphipod Parathemisto paci$ca Stebbing showed the most rapid degradation of ingested hydrocarbons, over 50% of each of the four compounds studied being metabolized in 24 h. The main metabolites were hydroxylated derivatives of the hydrocarbons, but more polar com- pounds were also tentatively identified (e.g. octadecanoic acid as a metabolite of octadecane). It is worth noting, however, that not all the species tested possessed the ability to metabolize hydrocarbons : BP was released unchanged by the ctenophore Pleurobrachia pileus (0. F. Miiller) and by an unidentified species of jellyfish.
In his experiments with 3H-BP Lee (1975) showed that Calanus plumchrus which had accumulated this hydrocarbon during prolonged exposure retained metabolites in its tissues for several days; and in similar experiments with Euchaeta japonica, in which 14C- 1 -naph- thalene was used, 88% of the radioactivity retained by the animal after 24 h was still in the form of metabolites. A study of 14C-l- naphthalene metabolism in Calanus helgolandicus by Corner et al. (197613) added to the work of Lee (1975) in two ways : first, by providing evidence for metabolism using animals that had accumulated the hydrocarbon through the diet; secondly, by taking special care to exclude the effects of bacteria, which are known to degrade PNAH (Gibson, 1976; Lee and Ryan, 1976; Harris et al., 1977b), by incorpor- ating the hydrocarbon in autoclaved nauplii of the barnacle Elminius modestus Darwin used as a sterile diet. The ration of autoclaved nauplii captured by the copepods was much smaller than those observed using untreated na,uplii (Corner, Head, Kilvington and Pennycuick, 1976c) but sufficient were taken to ensure a measurable level of radioactivity in the animals after 24 h. The animals were then given a depuration period of 24 h in fresh sea water, after which 86100% of the radio- activity retained in them could still be accounted for as naphthalene, whereas only 25-38% of that excreted into the surrounding sea water during this same period of depuration was present as the unchanged hydrocarbon. Thus, although both studies showed that aromatic hydrocarbons were metabolized by copepods, Lee (1975) found most of the metabolites to be retained by the animals, and therefore available for transfer to a higher trophic level, but Corner et al. (1976b)
344 E. D. 9. CORNER
observed that the major fraction was rapidly excreted. To explain these different findings Corner et al. (1976b) suggested that as Lees (1975) animals were exposed to the hydrocarbons for a much longer
I O O O q - n
WIG. 15. Accumulation (A) and depuration (B) of naphthaleilo and its metabolites (exproused as naphthol equivalents) by Stage V Pandalua platyceroa exposed to 14C-l-naphthalene at a concentration in sea water of 8-12 pg/l. (After Varanasi and Malins, 1977.)
period before depuration began this might favour the retention of metabolites in the tissues. Subsequent work, using multiple-day exposure periods (Harris et al., 1977b) confirmed this view in that 66--77y0 of the radioactivity detected in the tissues of C. helgolandicw
POLLUTION STUDIES WITH MARME PLANKTON-I 345
exposed to 14C-1-naphthalene over periods of 4-6 days could no longer be accounted for as the unchanged hydrocarbon.
Attention is drawn later (Section VIII) to the extreme sensitivity of larvae of the spot shrimp Pandulus phtyMr08 Brandt and the Dunge- ness crab Cancer rnagieter Dana to the hydrocarbon naphthalene (San- born and Malins, 1977). Because of the likelihood that hydrocarbons are complexed with organic macro-molecules (e.g. proteins) in the sea, these workers used naphthalene both in the free state and as a complex with bovine serum albumin (BSA). Experiments with Stage V spot shrimp showed that the hydrocarbon was accumulated much more rapidly when used in the free state, but that naphthalene metabolites (hydroxylated derivatives measured as naphthol equivalents) accumu- lated in the animal whether the hydrocarbon was taken up in the free state or as the complexed form. The metabolites were formed fairly slowly, about 4-50/, of the radioactivity being present in forms other than naphthalene after 10 h (Fig. 16A). Short-term depuration experiments (24 h) showed that there was a rapid initial loss of radio- activity as unchanged hydrocarbon during the first 12 h ; but that radioactivity in the form of metabolites was retained (Fig. 15B). Varanasi and Malins (1977) draw attention t o the possibility that the toxic effects of low levels of naphthalene to Pandaha larvae may be related to the inability of these animals to release toxic metabolites of the hydrocarbon.
The observation by Lee (1975) that micro-crustaceans included among the zooplankton are able to convert aromatic hydrocarbons into hydroxylated derivatives implies that these animals, like fish (Stegeman and Sabo, 1976) and large marine crustaceans (Burns, 1976 ; Philpot, James and Bend, 1976; Singer and Lee, 1978), possess the enzymes known as mixed function oxygenases (MFO), which work with mammals has shown to be involved in the metabolism of steroids and numerous drugs, as well as compounds such as petroleum hydro- carbons. The enzymes are NADPH (reduced pyridine nucleotide) dependent and are sometimes referred to as aryl-hydrocarbon hydroxy- lase (AHH) when working with a specific substrate such ;t9 BP. They operate in conjunction with the electron-transport system cytochrome P-450 and NADPH-cytochrome c reductase and are inhibited by cytochrome c and carbon monoxide. The overall reaction catalysed is :
AH + 2e- + 2H+ + O,+AOH + H,O. With BP as the substrate the assay ie based on measurements of
A full and well-presented account of the distribution of these 3 - h ydr ox ybenzo [alpyrene .
346 E. D. 9. CORNER
enzymes in marine animals is given by Varanasi and Malins (1977). In the context of the present review, however, it is worth stressing that aquatic animals can possess a highly active AHH system: for example, trout liver-microsomes metabolize BP at a rate 5-10 times higher than male rat liver-microsomes when measured per mg of microsomal protein (Ahokas, Pelkonen and Karki, 1975). Furthermore, work by Clark and Diamond (1971) has shown that these high efficiencies for the metabolism of BP are maintained over a wide range of temperatures (5-30C).
A large number of substrates, including " foreign " organic com- pounds (xenobiotics), can induce high levels of MFO in mammals (Conney, 1967) and fish (Payne and Penrose, 1975; Payne, 1976): however, related studies with zooplankton species have not so far been made, although micro-crustaceans, being more metabolically active, might possess MFO systems less sluggish than those normally found in larger crustacean species (Philpot et al., 1976). Payne and Penrose (1975) suggest that the existence of inducible AHH in fish may provide a convenient means of assessing previous exposure of the animals to PNAH although, as they point out, more needs to be known about the decay of this induced activity. There is the further complication that AHH may also be induced by other compounds such as chlor- inated hydrocarbons and aromatic pesticides. At present, nothing is known about the induction of MFO, including AHH, in zooplankton.
The metabolic changes undergone by PNAH in marine animals have been reviewed elsewhere (Corner, 1975; Corner et al., 1976a; Varanasi and Malins, 1977). Briefly, the first stage in the process is the formation of an epoxide which is subsequently converted either into a dihydro-diol or, by conjugation with glutathione, into a premercapturic acid (see Fig. 16). The enzymes involved, epoxide hydrase and gluta- thione-8-transferase, have been detected in marine fish and inverte- brates (James, Fouts and Bend, 1976: cited by Varanasi and Malins, 1977) but no studies have yet been made with planktonic organisms. Compared with the parent hydrocarbon, hydroxylated derivatives such as the dihydro-diol are more water-soluble and, either in the free state or as conjugates with sulphuric acid and either glucuronic acid (mam- mals and fish) or glucose (Arthropoda), are released by the animals. Thus, these metabolic changes would help to reduce the levels of hydrocarbon in the animals and could be regarded as a " detoxifying " process.
Recent work with mammals has shown that dihydro-diols can themselves undergo further oxidation to form " diol-epoxides " (Booth and Sims, 1974) and, in the case of the carcinogenic compound BP, it
POLLUTION STUDIES WITH MARINE PLANKTON-I 341
is this derivative (7,8-&hydro- 7,8-dihydroxybenzo[a]pyrene- 9,lO-oxide) and not the parent hydrocarbon which is mainly responsible for carcino- genic activity (Sims, Grover, Swaisland, Pal and Hewer, 1974). It therefore seems that although some metabolic changes may facilitate the removal of hydrocarbons from the animal, others can increase the carcinogenic potential of these compounds. Stegeman and Sabo (1976)
1 Dihydro- diol Underqo conjugation reactions giving giucuronides (or glucosides) and sulphates
Reactions with cell constituents(eg protein and nucleic acids)
NHCOCH, NADPH NADPH + H+ + H-0
FIG. 16. Metabolism of an aromatic hydrocarbon in mctrine animals. Enzymes involved in main reactions: (1) mixed-function oxygenase, (2) epoxide hydrase, (3) gluta- thione-8- transferase.
have commented on the possible linkage between the activation of potential carcinogens by metabolism and the greater incidence of neoplasia in fish from contaminated regions. It would be interesting to know whether the toxic effects of PNAH in zooplankton are in any way related to metabolic changes undergone by these compounds in the animals, particularly as work with shrimp and crab larvae (Sanborn and Malins, 1977) has shown that naphthalene, which is particularly toxic to
348 E. D. 9. UORNER
these animals, is converted by the spot shrimp into metabolites which are retained in the tissues.
E. Release of hydrocarbons in faecal pellets
Studies by Freegarde, Hatchard and Parker (1 971) with Calanus Jinmarcliicus and by Conover (1971), using this species and Ternora longicornis (0. F. Muller), have shown that when the animals feed in the presence of fine suspensions of crude oil they are able to ingest oil particles and release them in faecal pellets. As these are slightly heavier than sea water they sink out of the surface layer and thus provide a means for transferring a substantial fraction of an oil spill from this region to the benthos: indeed, Conover (1971) concluded that perhaps 20% or more of the particulate Bunker C oil released off the Atlantic coast of Canada by the " Arrow '' disaster was sedimented in zooplankton faeces. Likewise, Elder and Fowler (1977) have estima- ted that faecal pellets released by the euphausiid Meganyctiphanes norvegicca (M. Sars) are an important means by which other organic compounds, in this case polychlorinated biphenyls, are contributed to sediments in the Ligurian Sea.
The transfer of hydrocarbons not only occurs when zooplankton ingest oil particles : water-soluble hydrocarbons directly taken up from solution by algal cells which are then only partially digested by herbi- vorous zooplankton could also be released in faecaI pellets and transfer- red to the benthos in this way. Although the sinlung rates of the faecal pellets ase not so great as to exclude the possibility of the material being removed as a food by animals during its descent (mean values range from 66 to 240 m/day for the faecal pellets released by species of copepods and euphausiid : Turner, 1977) faecal pellet production by these animals can still contribute substantial amounts of material to the benthos in certain sea areas (Seki, Tsuji and Hattori, 1974; Davies, 1975): in the study by Davies (1975), for example, it was found tha t 27% of the primary production as carbon in a Scottish sea loch was transferred to the benthos as faecal pellets released by herbivorous zooplankton. Accordingly, in studying the fate of hydro- carbons in zooplankton it is important to consider the quantities of these compounds that are released in faecal pellets. So far, however, the only study of this problem is that by Harris et al. (1977a) who determined the amounts of radioactivity released in faecal pellets by Calanus helgolandicw ingesting a known ration of Biddulphia previously exposed t o 14C-l-naphthalene. The data, summarized in Table XIV, show that quite a high proportion of the ingested radioactivity
TABLE XIV. ASSIMILATION OF NAPHTHALENE FROM A PLANT DIET BY Calanus helgolandicus
m z A
f - m i d E i 2
yo Ration cuptured Dietury -~
Released in soluble form
Retained faeces in animal Assimilated =
constituent Rejected in Animals
x Females Naphthalene 41.9 58.1 31.2 26.9
Females Nitrogen 65.9 34-1 7.5 26.6 Females Phosphorus 59.6 40.4 0 40.4
Males Naphthalene 39.3 60.7 26-8 23.9
Diet used: Biddulphia sinen-&. Summarized data from Harris et al. (1977a). 3
W tP (D
350 E. D. 9. CORNER
(39.3-41-9y0) was present in the faecal pellets, such radioactivity, in the absence of any evidence for the metabolism of naphthalene by micro-algae (see p. 328), being assumed to represent unchanged hydrocarbon.
Many studies have been made of the assimilation efficiencies of herbivorous zooplankton in terms of basic dietary components (e.g. carbon, nitrogen and phosphorus) and the values usually range from about 50 to 90% (see review by Corner and Davies, 1971). The assimila- tion efficiency obtained by Harris et al. (1977a) for 14C-1-naphthalene (68*1-60-7y0) is near the lower end of this range: however, the Biddulphia used as a diet, compared with other species of diatom, is much less readily digested (Corner et al., 1972) and further data included in Table XIV show that assimilation efficiency in terms of naphthalene was substantially higher than that found for either dietary nitrogen or phosphorus using this particular plant food. It is also worth noting from Table XIV that most of the nitrogen and all the phosphorus assimilated from the diet by the animals was released in soluble form, whereas an average value of only 51.2% was obtained with the hydrocarbon. Thus, compared with natural dietary con- stituents, naphthalene is not only more readily assimilated by the animals: it is less readily lost in soluble form.
As pointed out by Parker, Freegarde and Hatchard (1971), the extent to which copepods can contribute to the immobilization of an oil slick depends upon the amount of oil dispersed as fine droplets suitable for capture, the daily volume of sea water swept clear and the number of animals present in the sea. For a concentration of dispersed oil droplets equivalent to 1.5 mg/l, which can persist near an oil slick for a considerable time in a choppy sea (Parker et al., 1971), and assum- ing a maximal value of 750 ml/day for the volume swept clear by a single Calanus (Paffenhofer, 1971; Corner et al., 1972), the total quantity of oil ingested daily is 1-125 mg. Making the further assump- tion that the value obtained with naphthalene by Harris et al. (1977a) is typical of oil hydrocarbons in general, roughly 40% of this ingested ration, or 0-45 mg, wilI be rejected as faecal pellets. Thus, one female Calanus in one litre of sea water could transfer 30% of the dispersed oil into faecal pellets daily.
Another example of the quantitative importance of the amount of hydrocarbon released as faecal pellets can be assessed from further data by Harris et al. (1977a) presented in Fig. 17. This shows the quantity of radioactivity (as naphthalene equivalents) represented by a suspension of 20 000 Biddulphia cells/l exposed for 24 h to a low concentration of I*C-l-naphthalene (1.37 pg/l) in sea water; the daily
POLLUTION STUDIES WITH MARINE PLANKTON-I 351
ration captured by one female Calanus feeding on this cell suspension for one day ; and the amounts retained and released, either in soluble form or as faecal pellets, by the animal. Radioactivity in the faecal pellets represented 172.8 pg naphthalene/copepod and that present as suspended cells 3 620 pg hydrocarbon/l. Thus, under the experimental conditions, one female Calanus incorporated 4.8% of the total hydrocarbon available as phytoplankton in a litre of sea water into faecal pellets each day.
Naphthalene Naphthalene present Napht ha lene Naphthalene concentration ~ in suspension of ~ captured by released in in sea woter Biddulpim cells oneCa/anus faecal pellets ( 1 3 7 p g / 0 ( 3620 pg/ l ) (3178pgl (172 8 p g l
(Retained ) (Soluble release) ( 6 8 8 ~ 9 ) ( 7 6 2 p g )
FIG. 17. Quantitative aspects of the transfer of an aromatic hydrocarbon from solution in sea water to faeclal pellets released by zooplankton.
VIII. TOXICITY STUDIES WITH ZOOPLANKTON
The toxicities of crude oils, of their total water-soluble fractions and of individual hydrocarbons, to a wide variety of marine animals have been referred to in several reviews (Nelson-Smith, 1970 ; Butler, Berkes and Powles, 1974; Moore and Dwyer, 1974; Anderson, 1975): most of the work described, however, deals with larger marine animals and relatively few studies have been made with zooplankton.
Hyland and Schneider (1976), in considering the possible effects of oil pollution on planktonic communities in the open sea, conclude that although these organisms may be affected by physical coating in a floating oil slick, or by poisoning in the toxic plume immediately beneath, the effects may only be temporary, population densities and age-distributions being rapidly restored because of high reproduction rates and effective dispersal mechanisms such as the immigration of organisms from unaffected areas. Be that as it may, however, they emphasize the much greater susceptibility to oil contamination of local breeding populations of marine organisms, particularly the larval forms of certain fish (ichthyoplankton) and crustaceans and molluscs (meroplankton) in confined coastal areas where recovery from the effects of pollution may take several years. Toxicity data for both ichthyoplanktonic and meroplanktonic animals are therefore included
352 E. D. 9. CORNER
in Table XV, which summarizes the various toxicity studies reviewed in this section.
In early toxicity work, using whole oil, unstable suspensions of this material were prepared by adding it to sea water and shaking the mix- ture. Detailed studies involving multiple-day exposure were carried out by Mironov (1969) with Acartia clausi Giesbrecht, Paracalanus parvus (Claus), Penilia avirostris Dana, Centropages ponticus Karavajev and Oithona nana Giesbrecht collected from the Black Sea. The materials tested were crude oil (Malgobek), Bunker fuel oil F-12 and " solar " oil. In the experiments with adult Acartia a concentra- tion of 0.001 ml crude oil/l (ca. 1 mg/l) produced a slight toxic effect, 50% of the treated population dying in 4-5 days compar6d with 6.5 days for the controls; similar small effects were noted with fuel oil and solar oil used a t the same concentration. However, when animals in better condition were used, 50% of the control samples dying in nine days, no significant effects were observed with either crude oil or solar oil at the concentration of 1 mg/l ; moreover, that found with Bunker fuel oil was only slight, 50% of the test animals dying in eight days compared with nine days for the controls. Definite effects were found with all three oils used a t a concentration of 10 mg/l: at 100 mg/l all three oils killed the whole population of test animals in one day.
Slight toxic effects were found with Bunker fuel oil at a concentra- tion of 1 mg/l in experiments with Penilia, Centropages and Oithona ; but a more marked effect was observed with Paracalanus. As in the tests with Acartia, this oil at a concentration of 10 mg/l produced definite toxic effects with all four species ; and at 100 mg/l all popula- tions were killed within one day.
Mironov (1969) also carried out studies using the naupliar stages of Acartia and Oithona. Slight toxic effects were detected using Bunker fuel oil at a level of 1 mg/l, 50% of the treated population surviving only 3-5 days compared with 4.5 for the controls (Acartia) and 1.5 days compared with 3 (Oithona). The high mortality rates of the controls suggest that the animals were under stress, which may have increased their sensitivity to the oil.
Studies by Barnett and Kontogiannis (1975) using the tidal pool copepod Tigriopus californicus (Baker) showed that crude oil was much less toxic to this species than to those studied by Mironov (1969). In an earlier study, Kontogiannis and Barnett (1973) determined the concentrations of crude oil and its various fractions that represented
POLLUTION STUDIES WITH MARINE PLANKTON-I 353
the critical level of toxicity (i.e. above which the survival of the animal was reduced below that of untreated controls) and found those for diesel oil and kerosene to be 87 and 83 mg/l respectively. As might be expected, the harpacticoid copepod Tigriopus is much more resistant than a pelagic species such as Acartia or Oithona to the effects of crude oil : thus Kontogiannis and Barnett (1973) found that a concen- tration of 25 ml/l took three days to kill a population of Tigriopus, whereas Mironov (1969) found that 0.1 ml/l killed those of Oithona and Acartia within 24 h.
In none of the studies described so far was an even suspension of oil in the test medium maintained throughout the period of the experiment : for example, Barnett and Kontogiannis (1975) initially dispersed the oil using an ultrasonic probe, but the suspensions lasted for only 30 min. However, Spooner and Corlfett (1974) to some extent overcame this problem by placing the test samples on a vertically rotating wheel and by mixing the oil (Kuwait 250C Residue oil: equivalent to one-day weathered) with the dispersant BP 11OOX. They studied the effects of the mixture on faecal pellet production, as a measure of feeding rate, by Calanus helgolandicus feeding on Platymonus suecica Kylin and found that a marked inhibition occurred during a 20h exposure to oil used at a concentration of 10 mg/l. Nevertheless, after the animals were removed from the oil suspension and transferred to fresh sea water the normal feeding rate was recovered within seven days
In a recent study by Wells and Sprague (1976) larvae of the American lobster Homarus americanus Milne-Edwards were used as test organisms and care was taken to monitor the levels of Venezuela crude oil during prolonged exposure, using the U.V. method of Zitko and Carson (1970). There were considerable losses of hydrocarbons during the experiments, some of which lasted 30 days, but the toxicity data refer to initial concentrations only, which therefore represent maximal values. Oil concentrations causing the death of 50% of the test population in four days (4-day LC,,) were 0-86 mg/l for first-stage larvae and 4.9 mg/l for third- and fourth-stage larvae ; by comparison the 30-day LC,, value for first-stage larvae was 0-14 mg/l. As observed in the study by Spooner and Corkett (1974) the presence of oil reduced food consumption. Vaughan (1973) used methods similar to those of Wells and Sprague (1976) to study the effects of three crude oils on larvae of the rock crab Cancer productus Randall: 4-day LC,, values were 3.2 mg/l for No. 2 fuel oil, 220 mg/l for Louisiana crude and 250 mg/l for Kuwait crude. In addition, Wells and Sprague (1976) have calculated from the data of Percy and
354 1. D. 8. OORNER
Mullin (1975) using Venezuela crude oil that 4-day LC,, values for adults of two cold-water species, the amphipod Onisimus afinis H. J. Hansen and the copepod Calanus hyperboreus, were 29 and 82 mg/l respectively. A further contribution from the work of Percy and Mullin (1975) was the use of cell-free homogenates of Onisimus in order to study the effects of petroleum compounds on metabolic processes. Respiration rates of homogenates prepared from animals pre-exposed for 24 h to Norman Wells crude oil were found to be 10-46% greater than those of controls. Testing the effects of petroleum compounds on respiration by whole animals is complicated by the fact that changes in the rate of oxygen consumption could simply reflect differences in locomotory activity : the use of cell-free extracts over- comes this difficulty. The work of Percy and Mullin (1975) has the additional value that it is one of the few studies made using Arctic species.
B. Water-soluble hydrocarbons
The acute effects of the WSF of No. 2 fuel oil (Exxon, Baytown) on both coastal and oceanic zooplankton have been studied by Lee and Nicol (1977) using mixtures of animals collected from the Gulf of Mexico and off the coast of Texas. The 2-day LC,, obtained with coastal zooplankton was 9-5 mg/l (50% WSF). However, oceanic animals were much more sensitive in that the time to kill 50% of the sample a t the same concentration was only 9 h. Among the copepod species calanoids were more sensitive than cyclopoids ; and members of the meroplankton (e.g. barnacle nauplii) were more resistant than holoplanktonic forms (e.g. copepods). In a subsequent study (Donahue, Wang, Welch and Nicol, 1977), a wide range of aromatic compounds found in petroleum were tested for toxicity to nauplii of the barnacle Balanus amphitrite niveus Darwin. Estimated from the effects on larval activity, alkylated benzenes and naphthalenes were more toxic than the parent compounds ; in addition, phenalen- 1 -one, which is particularly toxic to green microalgae (Winters et al., 1977), and naphthalene were found to affect phototactic response. Evidence for the higher toxicities t o zooplankton of alkylated napthalenes compared with that of the parent hydrocarbon has also been found in a recent study by Ott, Harris and OHara (1978). Using Eurytemora afinis as the test organism they found 24h LC,, values of 0-316, 0.852, 1.499 and 3.798 mg/l . for 2,3,5-trimethylnaphthalene, 2,6-dimethylnaphthalene, 2- methylnaphthalene and naphthalene respectively.
Further studies with meroplanktonic animals are those of Byrne
POLLUTION STUDIES WITH MARINE PLANKTON-I 355
and Calder (1977) who tested the toxic effects of the WSFs of six crude oils using larvae of the quahog clam Mercenaria sp. Two-day LC,, values varied considerably with the oil used, that for the WSF of Kuwait crude being 25 mg/l and that for the WSF of used motor oil only 0.10 mg/l. In addition, longer-term exposures (10 days) caused increased toxicities (see Table XV).
Compared with those of adults, the susceptibilities of the larval, meroplanktonic stages of marine crustaceans to the WSF of a crude oil can be markedly greater. Thus, Brodersen, Rice, Short, Macklen- berg and Karinen (1977) found that 4-day LC,, values for Stage I larvae of the King crab Paralithodes camtschatica (Tilesius) vaned from 0.78 to 1-12 mg/l whereas the corresponding values for adults were in the range 3.6-5.0 mg/l: likewise, in experiments with the kelp shrimp Eualus suckleyi (Stimpson) the 4-day LC,, values for Stage I larvae varied from 0.87 to 1.4 mg/l compared with 1.8-2.1 mg/l for adults.
The toxic effects of the WSF of a high aromatic heating oil have been tested by Berdugo, Harris and O'Hara (1977) using the estuarine copepod Eurytemora afiniis. Animals exposed for 24 h to the dissolved hydrocarbons equivalent to a total concentration of 520 pg/l ingested algal food at a rate only 62y0 that of untreated controls. By contrast, the single hydrocarbon naphthalene used a t a higher concentration (1.0 mg/l) caused only an 11% reduction in feeding rate, indicating that compounds other than naphthalene in the WSF were mainly responsible for its toxicity.
Important developments in toxicity work with zooplankton were introduced by Sanborn and Malins (1977) in their studies with larval stages of Pandalus platyceros and Cancer magister. First, a continuous- flow method was used in which the concentration of hydrocarbon was constantly maintained ; secondly, the hydrocarbon was used both in the free state and as a complex with BSA (see p. 345). 1%-l-Naphthalene, used either alone or as the BSA complex, was extremely toxic to the animals, a concentration of only 8-12 pg/l causing 100% mortality in 24-36 h.
A continuous-flow system has also been used by Forns (1977) in studying the effects of unweathered South Louisiana crude oil on larvae of the lobster Homarzts americanzts. A threshold of sensitivity was observed between crude oil concentrations of 0.1 and 1-0 mg/l. Thus, animals exposed to 0-1 mg/l were active feeders with consistent locomotive behaviour and displaying strong aggressiveness : those exposed to 1.0 mg/l were lethargic, active motions were minimal, feeding was depressed and frequently the animals appeared to be dead. In addition, animals exposed to 1 mg oil/l had a slower rate of
356 E. D. S. OORNER
development. Mortality of these animals reached 50% after nine days, whereas survival for the control larvae and those treated with 0.1 mg oil/l exceeded 50% throughout the 17-day period of the experiment.
Compared with static systems, those involving continuous flow have decided advantages : for example, replenishment of dissolved oxygen and nutrients, removal of metabolic waste products, reduction of bacterial contamination and maintenance of constant levels of volatile and unstable test substances such as hydrocarbons. Continuous-flow methodology therefore provides a better approximation to conditions prevailing in the natural environment and is finding increased applica- tion in toxicity studies with corttponents of crude oil (see, for example, Hyland, Rogerson and Gardner, 1977 ; Roubal, Bovee, Collier and Stranahan, 1977a).
C. Possible eflects of hydrocarbons on reproduction by zooplankton
A recent study of the enzyme AHH in the blue crab Callinectes sapidus Rathbun (Singer and Lee, 1977) has focussed attention on the possible interference of xenobiotics, such as aromatic hydrocarbons, with steroid metabolism. Thus, Singer and Lee (1977) noted that the levels of activity of the enzyme, measured by the method of Whitlock and Gelboin (1974) in the green gland, were inversely related to the production of the steroid hormones, ecdysones, which control moulting in these animals (Faux, Horn, Middleton, Fales and Lowe, 1969). It is well known that crustaceans do not carry out de novo sterol bio- synthesis, but convert dietary compounds, such as the phytosterols present in plants, into desmosterol and subsequently cholesterol, the latter conipound then undergoing further changes to give steroids such as the mammalian sex hormones (see review by Goad, 1976). In mammals a further hydroxylation of these sex hormones involves the enzyme system androgen hydroxylase, the activity of which can be greatly stimulated by pre-treatment of the animal with certain drugs (Conney and Klutch, 1963; Lu, Kuntzman, West, Jacobson and Conney, 1972).
Desmosterol and cholesterol have both been detected in the copepod Euchaeta japonica by Lee et al. (1974) and the recent use of a radio- immunoassay technique has indicated the probable presence of the sex hormone oestradiol-l7-/3 in Calanus helgolandicus (OHara, Corner and Kilvington, 1978). Furthermore, work by Lee (1975), referred to earlier (p. 343), has shown that hydroxylated derivatives are formed from BP in copepods, which implies that AHH is present in these animals.
POLLUTION STUDIES WITH MARZNE PLANKTON-I 357
No in vitro studies have yet been carried out to examine the possibility that PNAH might interfere with steroid metabolism in copepods, thereby influencing moulting, sex ratio and reproduction, although there is evidence from the recent in vivo study by Berdugo et al. (1977) that petroleum hydrocarbons might inhibit the rate of egg-production by Eurytemora afinis. The test solution was the WSF of a high aromatic heating oil, with the volatile components benzene and toluene excluded, and ovigerous females immersed in this solution for 4 h showed a subsequent daily rate of egg-production approximately 30% that of untreated controls. This work has recently been extended by Ott, Harris and OHara (1978) who found that multiple-day expo- sure (maximum 29 days) of Eurytemora to each hydrocarbon at a concentration of 10 pg/l in sea water reduced the length of adult life, the total number of nauplii produced, the mean brood size and the rate of egg-production. However, in contrast to the LC,, data (see Table XV), increased alkylation of the hydrocarbon did not lead to greater inhibition of fecundity : thus, dimethylnaphthalene had less effect than the parent hydrocarbon on the total number of eggs produced; and both methyl-and dimethylnaphthalene had less effect than naphthalene on the rate of egg-production. As pointed out by the authors, the hydrocarbons did not necessarily have a specific effect on fecundity, inhibition of which could have resulted from a reduction in feeding rate.
Viability of the eggs is another important factor in reproduction and unpublished work by Lewis and Lee (cited by Lee, 1975), using Euchaeta juponicu, has shown that the hydrocarbons 1- and 2-methyl- naphthalene at a concentration of 80 pg/l cause a 60% reduction in the number of eggs reaching nauplius 11.
D. Summary and general comments Results from the various toxicity studies with zooplankton are
summarized in Table XV. A proper assessment of the possibility that the levels of hydrocarbons in different sea areas are likely to affect the dominant species of zooplankton inhabiting the same regions cannot be made at present : much more work is needed. However, comparison of the data in Table XV and Table I at least indicates that the amounts of crude oil found in the Adriatic (1-40-10.98 mg/l) would affect several species of zooplankton, including the larval stages of certain fish, and that the maximal levels of total hydrocarbons reported for the North Sea (625 pg/l), the Sargasso Sea (559 pg/l) and Goteborg Harbour (710 pg/1) are sufficiently high to affect clam larvae and adult copepods.
w cn 00 TABLE XV. TOXIC EFFECTS ON ZOOPLANKTON OF CRUDE OILS, WATER-SOLUBLE FRACTIONS AND INDMDUAL HYDROCARBONS
Critical level of
Test material Temp* toxicity ("'1 (mq/l or ..
Toxic effect Species Reference
Bunker fuel oil F-12
Bunker fuel oil F-12
Bunker fuel oil F-12
Bunker fuel oil F-12
Bunker fuel oil F-12
No. 2 fuel oil
Holoplankton and meroplankton Treated animals died slightly Paracalanw parvwr (adults)
Treated animals died slightly Centropagea ponticwr (adults)
Treated animals died slightly Penilia aviroatria (adults)
Treated animals died slightly Oithona nanu (adults + young Treated animals died slightly Acartia clauSii (adultsf young
Treated animals died slightly T i g r i o p ~ ~ californicw (adults)
Treated animals died slightly Tigriopw californicw (adults)
4-day LC,, H o m a w americanw (stage I
4-day LC,, Homarw americanus (stage
Homarw americanus (stage 1
Slower rate of development; Nerwnariu sp. (stage I-IV feeding inhibited ; 9-day LC,,
faster than controls
faster than controls
faster than controls
faster than controls stage)
faster than controls stage)
faster than controls
faster than controls
111-IV larvae) 30-day LC,,
larvae) 4-day LC,, Cancer productwr (larvae)
Mironov (1969) P
Mironov (1969) 0 8 2
Kontogiannis and Barnett (1973)
Kontogiannis and Barnett (1973)
Wells and Sprague (1976)
Wells and Sprague (1976)
Wells and Sprague (1976)
Kuwait crude Louisiana crude Venezuela crude Venezuela crude Kuwait crude + B.P. WSF of Kuwait crude WSF of S. Louisiana
WSF of Bunker C WSF of No. 2 fuel oil WSF of Florida Jay
WSF of used motor oil WSF of Kuwait crude WSF of Louisiana crude WSF of Bunker C WSF of No. 2 fuel oil WSF of Florida Jay
WSF of Cook Inlet crude
WSF of Cook Inlet crude
WSF of No. 2 fuel oil WSF of No. 2 fuel oil WSF of high aromatic
heating oil WSF of high aromatic
heating oil Naphthalene Naphthalene 2-Methylnaphthalene
8 8 8 5
25 25 25
25 25 25 25 25 25
15 15 15
250 220 29 82 10
3.2 1-3 0.25
0.10 2.0 2.1 1.6 0.53 0.05
9.5 9.5 0.52
1.0 3.798 1.499
4-day LC,, Cancer productus (larvae) 4-day LC,, Cancer productus (larvae) 4-day LC,, Onisimus a&& (adults) 4-day LC,, Calanus hyperboreus (adults)
Inhibition of feeding Cdanim helgolandicus (adults)
2-day LC,, Mercenaria sp. (larvae) 2-day LC,, Mercenaria sp. (larvae)
2-day LC,, Mercenuria sp. (larvae) 2-day LC,, Mercenaria sp. (larvae) 2-day LC,, Mercenaria sp. (larvae)
2-day LC,, Mercenaria sp. (larvae) 10-day LC,, Mercenaria sp. (larvae) 10-day LC,, Mercenaria sp. (larvae) 10-day LC,, Mercenaria sp. (larvae) 10-day LC,, Mercenaria sp. (larvae) 10-day LC,, Mercenaria sp. (larvae)
4-day LC,, (failure to swim) Eualus suckleyi (Kelp shrimp
4-day LC,, (failure to swim) Paralithodes camtschaticn larvae)
(King crab larvae) 2-day LC,, Mixed coastal plankton 9-hoW LC,, Mixed oceanic plankton
62 ?& reduction in feeding rate Eurytemora afinis (adults)
30% inhibition of egg Eurytemora afinis (adults)
11 % reduction in feeding rate Euryternora a$& (adults) 24h-LC,, Eurytemora a&& (adults) 24h-LC5, Eurytemora a@& (adults)
Vaughan (1973) Vaughan (1973) Percy and Mullin (1975) Percy and Mullin (1975) Spooner and Corkett
Byrne and Calder (1977) Byrne and Calder (1977)
Byrne and Calder (1977) Byrne and Calder (1977) * Byrne and Calder (1977) 2
Byrne and Calder (1977) Byrne and Calder (1977) Byrne and Calder (1977) Byrne and Calder (1977) Byrne and Calder (1977) Byrne and Calder (1977)
Brodersen et al. (1977) Z M
Broderaen et al. (1977) 8 Lee and Nicol (1977) 8 Lee and Nicol (1977) Berdugo et al. (1977) ' Bardugo et al. (1977)
Berdugo et al. (1977) Ott et al. (1978) Ott et al. (1978)
:a 5 a 0
TABLE XV (continued)
Critical level of
Test material Temp. toxicity Toxic effect ("C) (mgllor
Prudhoe Bay crude
Prudhoe Bay crude
15 0.852 24h-LC5,
15 0.316 24h-LC5,
10 0.008- 100% mortality in 24-36 h
10 0.008 - 100% mortality in 24-36 h
NS 0.080 45% reduction in eggs reaching
NS 0.080 46% reduction in eggs reaching
Icthyoplankton 5-11.5 88-110 4-day LC,,
5-11.5 14-16.0 Avoidance effects
Euytemora afinis (adults)
Eurytemora afinis (adults)
Pandalus plutycero8 (larvae)
Cancer naugister (larvae)
Euchaeta japonica (adults) Lee (1975)
Euchueta japonica (adults) Lee (1975)
Ott et al. (1978)
Ott et al. (1978)
Sanborn and Malins
Sanborn and Malins (1977)
Onwrhynchw gorbwchia Rice (1973) (Wdbaum) (Pink salmon fry)
(Pink salmon fry) Onwrhynchw gorbwchia Rice (1973)
Prudhoe Bay crude
362 E. D. 9. CORNER
In addition Table XV draws attention to the enhanced sensitivities of lobster and crab larvae compared with those of adult amphipods and copepods ; and to the high toxicities of the two alkylated naph- thalenes. Concerning this latter finding, mention has already been made of the numerous water-soluble components of crude oils used in the important studies by Winters et al. (1976) which demonstrate the toxicities of a wide range of individual compounds to marine unicellu- lar algae (p. 326). Comparable studies with zooplankton have not been made : yet, in the context of oil pollution there is an obvious need to extend toxicity work with hydrocarbons to include studies with related compounds, particularly the alkylated phenols that can occur in significant quantities in the WSFs of crude oils and which, in com- parison with commonly used hydrocarbons such as naphthalene and its alkyl-derivatives, are less volatile (Winters and Parker, 1977) and could therefore be more persistent in the sea.
IX. CONCLUSIONS Whether further inputs of petroleum compounds into various sea
areas will eventually have serious effects on plankton populations is a matter of conjecture. A strong likelihood, however, is that these compounds will continue to be released into the sea throughout the foreseeable future. It therefore seems worthwhile to conclude by summarizing suggestions for further work, basing these on gaps in knowledge already identified in earlier sections.
A. Chemical analyses As studies with planktonic organisms have shown that certain
groups of hydrocarbons (e.g. PNAH and their alkylated derivatives) and related compounds (alkylated phenols and anilines) present in crude oil possess considerable toxicity, future analyses of petroleum compounds in sea water, both in the dissolved and particulate forms, and especially from areas affected by chronic oil spills, should be pri- marily concerned with identifying and measuring the levels of these more toxic components. Such levels should then be compared with those needed to produce measurable biological effects in short- and long-term toxicity studies.
In addition, there is a need to analyse these compounds and their derivatives in phytoplankton and both herbivorous and carnivorous zooplankton and young fish, particularly in the vicinity of oil spills, in order to assess the extent to which such compounds may be con- centrated in the food web.
POLLUTION STUDIES WITH MARINE PLANKTON-I 363
Concerning the spatial distribution of hydrocarbons in the sea, many previous studies have demonstrated the high levels of these compounds that occur at the surface. Work in various sea areas has shown, however, that large concentrations of phytoplankton occur at frontal areas and in the thermocline region in stratified waters (Lorenzen, 1967 ; Lasker, 1975 ; Savidge, 1976; Pingree, Holligan, Mardell and Head, 1976). The basic importance of phytoplankton in marine production makes such regions especially important : accord- ingly, in determining the levels of petroleum hydrocarbons in the sea, either to obtain background information or to follow what happens immediately after an oil-spill, test samples should be taken from these regions as well as from the surface.
Furthermore, because of the importance of zooplankton faecal pellets in transferring plant material from the euphotic zone to the benthos there is a need, should an oil-spill occur simultaneously with a spring diatom increase, to extend studies concerned with the spatial distribution of petroleum compounds to include analyses of benthic fauna, especially those used as a food by demersal fish.
B. Toxicity studies
Although much of value has already been learned about the effects of individual petroleum compounds on planktonic organisms, more needs to be known about the toxicities of mixtures of these compounds, especially those present in the WSFs of crude oils from areas of relatively recent oil exploration such as the North Sea. Components of these mixtures may behave synergistically in terms of toxic effects : moreover, the uptake and retention of a particular component could also be influenced by the presence of others. In addition, more work is needed on the toxicities of compounds such as hydroperoxides and thiacyclanes (sulphoxides) which result from oxidation processes during the weathering of crude oil on the surface of the sea.
Recent toxicity studies with zooplankton have incorporated con- tinuous-flow methods, or variations thereof, together with monitoring of hydrocarbon levels throughout the experiments. Such techniques, although time-consuming, should also be applied in future work con- cerned with sub-acute long-term effects. Here the main emphasis should be on using mixtures of petroleum compounds at naturally occurring levels ranging from those encountered in the immediate vicinity of an oil-spill, and at different depths in the toxic plume beneath, to those found in estuaries and in-shore waters subject to small but frequent inputs of oil. The possible influence of petroleum
364 E. D. 9. OORNER
compounds on secondary production in the sea should then be assessed in terms of their effects on rates of feeding, moulting, growth, egg- production and sex ratio, using animals maintained over multiple- generations in laboratory cultures.
There is also a need for short-term studies using animals main- tained over a single generation, or even individual stages (particularly with meroplanktonic and ichthyoplanktonic species), to investigate the effects of petroleum compounds on further factors influencing the phytoplankton/zooplankton relationship : important among these are the efficiencies with which plant constituents are assimilated and converted into animal tissue, the process of nutrient regeneration, and food selection by zooplankton in the presence of mixtures of diets occurring naturally in the sea.
Finally, because of interactions between species in nature, there is a need for further studies on the effects of petroleum compounds on complete eco-systems, particularly those found in Arctic regions where, as Percy and Mullin (1975) point out, a marked reduction in the rate of loss of the components of crude oil is likely to produce more pro- longed toxic effects. The use of large plastic enclosures for work of this kind has already led to some interesting findings and it seems desirable for future developments to include additional studies along these lines.
C . Biochemical work
Studies have already been made of the effects of petroleum com- pounds on photosynthesis and the gross chemical composition of unicellular algae (although the algal studies still need t o be done with marine species). Work with fish (Stegeman and Sabo, 1976; Sabo and Stegeman, 1977) has shown that lipid metabolism is affected in animals exposed for long periods to petroleum compounds. The importance of lipids in zooplankton (Lee, Nevenzel and Paffenhbfer, 1971) emphasizes the need for similar studies to be made with these animals, especially the influence of petroleum compounds on the synthesis of particular lipid fractions such as triglycerides, wax esters, phospholipids and sterols. Also needed are studies of the effects of petroleum compounds on the relative amounts of lipid and protein metabolized by planktonic organisms, both plant and animal.
Another biochemical problem is that of ascertaining the extent to which detoxification mechanisms may occur in marine unicellular algae. In addition, present indications that zooplankton animals such as micro-crustaceans possess the enzyme systems involved in convert-
POLLUTION STUDIES WITH MARINE PLANKTON-I 366
ing PNAH into hydroxylated derivatives should be followed up with studies, both in vivo and in vitro, of the induction of these enzymes in animals exposed to various xenobiotics, the rate of decay of any induced activity and the relative levels of such activity in animals from con- taminated and uncontaminated areas. Related to which is the additional need to establish whether the enzymes epoxide hydrase and glutathione-8-transferase are present in planktonic organisms ; and to carry out detoxification and depuration studies, which so far have been made with hydrocarbons only, with related compounds such as phenols and anilines.
A further area of biochemical study is that of establishing mech- anisms for the biosynthesis and metabolism of steroids, particularly sex hormones and moulting hormones, in zooplankton and to assess the possible influence of the structurally related PNAH on these processes.
I am particularly grateful to Dr M. F. Spooner for her many helpful criticisms of the manuscript; and to Drs R. P. Lee and D. C. Malins for sending me advance copies of work that was still in the process of publication and for allowing me to quote it in preparing this review. I am also indebted to several of my colleagues at the M.B.A. : particularly to Mr D. S. Moulder who supplied me with many publica- tions dealing with all aspects of oil pollution, to Dr G. T. Boalch and Miss Elizabeth Roberts for ascertaining the authorities of numerous plant and animal species, to Mr C. C. Kilvington for checking the bibliography, to Miss Linda Carpenter and Miss Marsha Rapson for typing the manuscript and to Mr G. A. W. Battin for re-drawing the figures.
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