Exposure of phytoplankton to ekofisk crude oil

Marine Environmental Research 11 (I 984) 183-200

Exposure of Phytoplankton to Ekofisk Crude Oil Kjetill t stgaard, Ingvar Eide & Arne Jensen Institute of Marine Biochemistry, University of Trondheim,

N--7034 Trondheim--NTH, Norway (Received: 23 February, 1982) ABSTRACT Solutions of Ekofisk crude oil in sea water were prepared by slow stirring .]'or 21h in a closed system. Headspace and GC/MS techniques were applied to establish dose composition and levels. The standardized test medium produced in a closed system contained approximately 14 mg oil per litre and was dominated by low molecular weight aromatic hydrocarbons and phenols. The toxicity of the standarized oil-containing medium to three marine diatoms was studied in a cage culture turbidostat and by conventional batch culture technique. The three algal species differed in sensitivity to the oil compounds, but showed identical ranking in both test systems. Standardized test medium diluted to 50 % with respect to oil content stopped the growth of the most sensitive alga, Skelctonema costatum. For comparison naphthalene was applied and gave 50 °//ogrowth reduction at a concentration of 4001tg litre -z The growth of Chaetoceros ceratosporum was only slightly affected by the full strength standard test medium, which had no influence on the growth of Phaeodactylum tricornutum, the least sensitive organism. The presence of an oil layer on the surface of the standard test medium during the growth test greatly increased its toxicity and blocked completely the photosynthesis even of P. tricornutum after 4 days.

INTRODUCTION The toxicity of crude oils and petroleum fractions to phytoplankton has been investigated frequently during recent years. Quantitative data on the toxicity of dissolved compounds are, however, still sparse. 183 Marine Environ. Res. 0141-1136/84/$03.00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain

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Kjetill Ostgaard, Ingvar Eide, A rne Jensen

In most cases the toxicity has been related to the total amount of petroleum added to the medium (Nuzzi, 1973; Hsiao, 1978; Kusk, 1978) or to a dilution of a presumably equilibrated solution, the real concentration being unknown (Kauss et al., 1973, Kauss & Hutchinson, 1975; Soto et al., 1975; Giddings & Washington, 1981). In the laboratory the tests were usually carried out in batch culture, and the toxic effect of oil components was to a large extent supposed to be caused by volatile compounds, since the substrates were less toxic when these compounds were allowed to escape (Kauss et al., 1973; Pulich et al., 1974: Kauss & Hutchinson, 1975: Soto et al., 1975: Prouse et al., 1976). The present work is a study of Ekofisk crude oil toxicity to three different diatoms, two of which are common to the North Sea and Norwegian coastal waters. It is also an attempt to relate toxicity to a chronic exposure of dissolved crude oil components. The experiments were carried out in a closed continuous system, namely the cage culture turbidostat described by Skipnes et al. (1980). This system secures constant algal growth conditions throughout the entire experiment. Conventional batch culture experiments were performed for comparison. MATERIALS AND METHODS

Algal cultures The algae used were: Phaeodactylum tricornutum Bohlin (unialgal) (obtained by the courtesy of Drs R.R.L. Guillard and J.H. Ryther, Woods Hole, Mass., USA), Skeletonema costatum (Grev.) Cleve, clone Skel-5 (unialgal) and Chaetoceros ceratosporum (unialgal, axenic) (both isolated by Dr S. Myklestad, Institute of Marine Biochemistry, Trondheim, Norway). The stock cultures were maintained in medium f/10 (Guillard & Ryther, 1962) with 25%0salinity, and kept at 13 °C with a day/night cycle of 14/10. Experiments were performed in continuous light (3565 pE m - 2 s- i) provided by banks of light tubes (Philips type TL 40W/33 and TL 40W/55 mixed).

Culture systems Batch cultures

The algal cultures were grown in 250 ml Ehrlenmeyer flasks, culture

Exposure of phytoplankton to Ekofisk crude oil

~ medium reservoir

(culture)

185

ottle

l collection bottle (medium)

Fig. 1.

Principal sketch of the closed system showing the cage, reservoir- and collection

bottles, and the tubings for transportation of medium and culture. volume 100ml, for a period of 4 days. Exponentially growing cells, concentrated by centrifugation, were used for inoculation (1 ml for each flask) to give initial cell densities between 2 x 104 and 4 x 104 cells mlSamples were taken with a syringe through a silicone rubber cap over a short glass tube opening below the medium surface level of the flask. The culture flasks were illuminated from below to avoid light reduction in cultures under oil layers. One flask in each experiment contained a glass ring on the surface of the medium which restricted the oil slick to approximately one-fourth of the surface area. The turbidostat The cage culture turbidostat is described in detail by Skipnes et al. (1980). Briefly, the growth chamber consisted of a glass cylinder closed at each end by a membrane with cylindrical pores of 2/am diameter. The growth medium was pumped through the cage in a reciprocating manner (3: 2) to prevent clogging of the exit membrane, with a net flow of approximately 50 ml h -x . Analyses of nitrate, phosphate, salinity and pH verified that the composition of the medium was not altered significantly in the cage at this flow-rate. The cell density was kept at 2-5 x 105 cells m l - ~ by the photo-optical system which activated an independent dilution pump (see Fig. I). The activation time of this pump integrated over one hour periods

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Kjetill Ostgaard. lngvar Eide, Arne Jensen

is directly proportional to the growth rate (Skipnes et al., 1980). Samples of the algal culture were taken with a syringe through a silicone rubber cap.

Test media Test media with water-soluble components of Ekofisk crude oil were prepared in a 5 litre glass bottle closed with a stopper of silicone rubber. One part of oil (200 ml) was carefully layered on top of 20 parts of sterile medium (4 litre) and stirred gently by a magnetic stirrer, so that no deformation of the oil-water interface (including oil droplet formation) could be observed. According to Shaw & Reidy (1979), crude oil dispersed in this way can mainly be considered as a solution in sea water. After incubation overnight (approximately 21 h), the medium was removed for experiments/analysis through a silicone rubber tube located below the level of the oil slick. Media containing petroleum components were always kept in closed systems to prevent evaporation (Fig. 1). The naphthalene media were prepared by heating the medium to about 80 °C (the melting point of naphthalene) and adding known amounts of naphthalene. The closed system was mixed for approximately 2 h, and cooled to room temperature. Prior to analyses the sterile samples were kept in closed, completely filled glass bottles in the dark at 4 °C.

Analyses Algae The cells were counted in a haemocytometer. Chlorophyll a and in vivo fluorescence were measured in a Turner 111 fluorometer with a blue RCA F4T5 lamp, two Corning filters, 5-60 (primary) and 2-64 (secondary), and a standard sample holder No. I ! 0 005. The increase in fluorescence after addition of dichlorophenyl dimethylurea (DCMU) was also recorded to monitor the photosynthetic efficiency (Samuelsson & Oquist, 1977). The samples for the determination of chlorophyll a were treated according to Holm-Hansen et al. (1965) using methanol instead of acetone for the extraction. Petroleum components Direct fluorescence analysis of the test media was performed as a rapid

Exposure of phytoplankton to Ekofisk crude oil

187

and simple indicator of the reproducibility of the preparation described above. Emission spectra at excitation wavelength 230 nm, favouring the fluorescence of naphthalenes around 335 nm, and at excitation wavelength 265 nm, favouring the fluorescence of phenols around 300nm (Ostgaard & Jensen, 1983) were recorded using a Perkin-Elmer 3000 fluorescence spectrometer. Samples of the media (l litre) were extracted three times with dichloromethane, and the combined extracts dried with Na2SO 4 before concentration to 300 pl. The content of less volatile components (boiling point higher than that of toluene) was determined by gas chromatography (packed column), using the response of known amounts of Ekofisk crude oil as standards. The fraction thus determined is referred to as the C 7+fraction. One of the samples was analysed more thoroughly with gas chromatography/mass spectrometry (GC/M S) for identification of single components. Quantification was based on a deuterated internal standard (biphenyl-d I 0). The highly volatile components were determined by headspace analysis (Berg et al., 1980). Briefly, purified nitrogen was passed through the sample (held at 60 °C), and the stripped compounds adsorbed on finely granulated active charcoal in a capillary tube. The capillary was transferred to the injector of the gas chromatograph, and the oil components desorbed by heating. The components were identified in the GC/MS instrument, and quantified using benzene-d 0 as internal standard. The naphthalene added to some media was extracted with cyclohexane and determined by high-resolution gas chromatography by comparing the response with that of known amounts of naphthalene. RESULTS Petroleum content in the sea water media

Fluorescence emission spectra of independently prepared sea water media used in the toxicity tests were similar in shape (results not included, cf. ~stgaard & Jensen, 1983), indicating a quantitative reproducibility within + 15 % at the emission maxima. This variability was considered acceptable for the toxicity testing. Analysis of two separate standardized test media gave the values 4.5 mg litre -~ and 4-6 mg litre- ' for the C 7+fraction, determined by gas chromatography.

188

Kjetill Ostgaard, lngvar Fide, Arne Jensen TABLE 1 Content of Ekofisk Crude Oil Components in a Standard Test Medium (Chromatograms are given in Fig. 2) Method[compbund Headspace Compounds lighter than benzene benzene toluene ethylbenzene xylenes C3-alkyl-benzenes

Estimated concentration (rag litre- 1) about 4 4.6 0-6 0.05 0.4 0.2

Extraction & GC total C7 +-fraction

(mg litre- 1) 4.6

Extraction & GC/MS toluene ethylbenzene xylenes C3-alkyl-benzenes phenol indan/alkyl-benzenes methylphenols C4-alkyl-benzenes (probably Ce-alkyl-phenoi) naphthalene sum of C 2- and Ca-alkyl-phenols methylnaphthalenes biphenyl C2-alkyl-naphthalene diethyiphthalate dibutyiphthalate dioctylphthalate

Oaglitre- 1) 700 45 400 132 20 2 145 3 35 60 410 37 5 8 5 l0 20

Data obtained by G C / M S headspace and G C / M S extraction procedures o f standardized test media are given in Table l. The headspace analysis showed that highly volatile hydrocarbons excluded from the CT+-fraction formed a major part o f the total a m o u n t o f dissolved petroleum, as observed by others (McAuliffe, 1979). Otherwise the estimates o f 'overlapping' components agreed well between the two methods in the actual case. The components lighter than benzene (Fig. 2(a)) have not been identified, but were probably alkanes such as pentane, cyclopentane,

m

eb+x

t_ ~

t~

t'5

(a)

retention time (rain)

mph

a~

mn

L ~

| dphtt .....

scan time (mi~ (b)

Fig. 2. Chromatograms of a sea water extract of Ekofisk crude oil. Detailed identification/quantification is given in Table 1. (a) GC-chromatogram of volatile fraction obtained by headspace analysis. (b) GC/MS-chromatogram (total ionic current of MS detector) of less volatile fraction obtained by extraction. Abbreviations: b, benzene; t, toluene: eb, ethylbenzenes; x, xylene: al-b, alkyi- (C3 and C4)-benzenes: ph, phenol: mph, methyl-phenols; n, naphthalene; ran, methyl-naphthalene; IS, internal standard (biphenyl-dto); dpht, dioctylphthalate.

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Kjetill Ostgaard, lngvar Eide, Arne Jensen

methylcyclopentane, butane, etc. (McAuliffe et al., 1980). As well as this fraction, the aromatic compounds benzene, toluene, xylene, naphthalene and alkyl derivatives of these were the dominating compounds. The Ekofisk crude oil itself contains only about 2 ~o aromatics. The chromatogram of the less volatile compounds is illustrated in Fig. 2(b). The observed hydrocarbons were mainly below C1~. As expected from solubility data (McAuliffe, 1979), long chain alkanes were not detected. The majority of the less volatile components were not identified (Table 1) due to instability in the mass spectrometer. In addition to the hydrocarbons, also phenol and its C~-, C 2- and C3-alkylated derivatives were observed (Table 1). The quantitative estimation of these compounds is less accurate, partly due to the extraction method, partly due to the lack of suitable standards, and in part because of imperfect separation. Nitrogen-containing components such as anilines and indoles, found to be important in the water-soluble fraction of some crude oils (Winters et al., 1976) were not detected in the water-soluble fraction of Ekofisk crude. Addition of naphthalene corresponding to the saturation level in distilled water, 35 mg litre -1, gave final concentrations between 1 and 2mg litre -~ in the culture media in independent experiments. All test media with naphthalene were therefore analysed before use. It is known that the solubility of aromatic compounds in water depends on salinity and temperature (Price, 1976). The saturation level for naphthalene in the actual culture medium is approximately 10 mg litre- J at 13 °C (Ostgaard, unpublished results). Batch cultures

The effect of water-soluble crude oil components on the growth of the three test algae is illustrated in Figs 3(a), (b) and (c). All experiments were performed at least in duplicate. Although some differences in growth rates between the controls in different experiments were observed, a similar pattern of the reaction to oil components was seen in comparable tests. Closing the culture flasks with silicone rubber stoppers reduced the growth ofS. costatum, while no such effect was observed in P. tricornutum and C. ceratosporum. The reason for this apparent sensitivity to limited gas exchange in S. costatum is not known. The different algae showed different sensitivities towards petroleum hydrocarbons (Fig. 3). In the undiluted standard test medium cells of S.

w

i

Time (days~

onlrol

%

(b)

Time (days)

Time (daym)

Fig. 3. Growth of diatoms in batch culture with/without exposure to Ekofisk crude oil components. Exposure is indicated as percentages of a standard stock solution, eventually with the addition of a full (F) or partial (P) oil slick, as described in the Materials and Methods section. Closed symbols indicate cells with blocked photosynthesis (less than 5 % increase in fluorescence after addition of DCM U). (a) Skeh, tonema costatum. Values for the 50 % solution were taken from an independent experiment with similar results for control and 100 ~,/, solution. (b) Chaetoceros ceratosporum. (c) Phaeodactylum tricornutum.

(a)

P

100%

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Kjetill Ostgaard, lngvar Eide, Arne Jensen

costatum were lost within the 4th day (Fig. 3(a)), while C. ceratosporum showed only a small reduction in cell growth (Fig. 3(b)). Phaeodactylum tricornutum was even less sensitive, showing the same growth rate in test and control media after a short lag period (Fig. 3(c)). The same ranking in sensitivity was observed when the oil layer was left on the culture media throughout the test. This led however, to a dramatic increase in the toxicity of the medium. Although cells could be observed during the total test period both for P. tricornutum and C. ceratosporum, addition of DCMU did not increase their in vivo fluorescence significantly, as shown in Fig. 3. This indicated a total block of photosynthesis, and these algal cultures were therefore dead or dying.

Turbidostat cultures

The algae were transferred directly from the day/night regime of the inoculum to continuous light in the turbidostat. The growth rate based on turbidity measurement remained rhythmic the first couple of days, the lowest growth rate being about 50~o of the highest. Although the amplitude decreased with time, some fluctuation lasted throughout the total experiment. Average growth rates over 24h of S. costatum and C. ceratosporum were 1.6 and 3.4 divisions/day, respectively. Phaeodactylum tricornutum was cultivated at a higher temperature (16.5°C) and higher light intensity, and the mean growth rate was 2.2 divisions/day. The cultures were exposed to the oil components when the growth had stabilized, i.e. was oscillating systematically (Fig. 4). The load of crude oil components was built up asymptotically, reaching some 50~0 of full strength after 4 h. The effect of this treatment on the growth rate of the test algae is summarized in Table 2. The growth rates are given as mean values over 24 h, in per cent of the control. Both C. ceratosporum and P. tricornutum seemed to tolerate the standard test medium well, whereas the growth of S. costatum was inhibited between 44 and 100~o. Diluting the original medium to 75 ~ caused a 30-60 ~o reduction in the growth ofS. costatum. Upon 50 ~o dilution the dose apparently became non-toxic. From Table 2 it is seen that leaving the oil layer on the surface of the medium reservoir throughout the experiment made the medium more toxic. This stopped the growth of S. costatum after 1-2 days and that of C. ceratosporum after 4 days. The growth of S. costatum was reduced 30-50~o when

F L/~ug oh!

1o%~I~ml

,5O"

(a)

105cells/ml

(b)

8- F L / ~ g chl •

s

_

3" Jg chl/cell 2-

s- ~ceils/ml 4I00"

Growth fate.O/o

5O-

(c) Days

Fig. 4. Growth of diatoms in the turbidostat with/without exposure to a 100 ~o stock solution, as described in the Materials and Methods section. FL, in civo fluorescence; pg chl, amount of chlorophyll a. Growth rate in turbidity is given relative to initial stable value before toxic exposure. (a) Skeletonema costatum. Cell recovery after removal of oil components is also shown. (b) Uhaetoceros ceratosporum. Oil layer present in the medium reservoir. Cell recovery after removal of oil components is also shown. (c) Phaeodactylum tricornutum. Oil layer present in the medium reservoir.

Kjetill Ostgaard, lngvar Eide, Arne Jensen

194

TABLE 2 Turbidostat Growth Rates of Three Diatoms after Exposure to Standard Test Medium or Naphthalene (The growth rates are given as mean values over 24 h in per cent of the control)

Algal

S. costatum

C. ceratosporum

P. tricornutum

Growth rate in % of control Day 1

Day 2

Day 3

0 65 42 81 44 79 94 61 73

0 0 56 38 33 76 33 82

71 39 85

80

64

40

65 91

36 98

0 103

73

46

Exposure

Day 4 1O0 % + layer 1O0 % + layer 100 % + layer

18

6

1oo% ioo% 100 % (Fig. 4(a))

71

0°

75% 75% 50% 100 % + layer 4(b)) 100 % + layer

ioo% 1O0 % + layer

4(c))

S. costatum

87 100

71 100

89

62

62

54

110

80

70

115

70

56

83

30

0

90

20

0

100% + layer

lOO%

" Recovered upon changing to pure medium.

(Fig.

370/~g litre - 1 naphthalene 410~g litre- i naphthalene 410/~g litrenaphthalene 1 1O0/~g litrenaphthalene 1 400 #g litrenaphthalene

(Fig.

Exposure of phytoplankton to Ekofisk crude oil

195

exposed to about 400/zg litre -~ naphthalene. When the concentration was increased to l l00/ag litre -t the growth stopped three day later. Figure 4 shows the growth of the test algae from hour to hour. Cell number, chlorophyll content per cell and the ratio of in vivo fluorescence to chlorophyll content (FL/chl) are also given. The growth rate seems to be the parameter which responded most quickly to the exposure to hydrocarbons. In fact, neither chl/cell, nor FL/chl reacted systematically when the cultures were exposed to oil components in these tests. DISCUSSION Standardized test media

Quantitative studies of the toxicity of petroleum components to aquatic organisms have generally been restricted to homogenous suspensions in order to obtain reproducible dose-response relationships. Although a dispersion without visible oil droplets is often referred to as a 'watersoluble fraction', its actual physical and chemical composition is highly dependent on the mixing procedure. Shaw & Reidy (1979) showed that vigorous stirring led to an emulsion of oil droplets with diameter !-0.03/~m, and thus to an oil dispersion with chemical composition relatively similar to that of intact oil. When formation of oil droplets was avoided by careful stirring, the sea water was found to be highly enriched in the more water-soluble lighter aromatic compounds and in phenols. Since oil emulsions can be relatively stable, dispersions prepared by shaking and separation for some hours (Gordon & Prouse, 1973: Kusk, 1980) may differ considerably from those prepared by careful stirring. The standard method applied in the present study is similar to that used to avoid oil-in-water emulsions (Kauss et al., 1973; Kauss & Hutchinson, 1975: Soto et al., 1975; Shaw & Reidy, 1979: Mahoney & Haskin, 1980; Giddings & Washington, 1981). The chemical composition of aqueous petroleum solutions is also dependent on evaporation (Kauss et al., 1973; Soto et al., 1975), the low molecular weight aliphatic and aromatic hydrocarbons being easily lost. Light-induced oxidation of compounds can also affect the chemical composition with particular importance for toxicity testing (Lacaze & Villedon de Na'ide, 1976). In addition, microbial activity is important in non-sterile systems. These circumstances make comparison of published data on the

196

Kjetiii Ostgaard, lngvar Eide, Arne Jensen

toxicity of different types of oil very difficult, and also call for a chemical description of the actual load of petroleum components. Such descriptions are, however, rare, partly because of the analytical problems involved (Petrakis & Weiss, 1980; Awad, 1981). A complete description of the hundreds of components present, in addition to those possibly formed during the preparation of the test medium, cannot be accomplished today. A more limited description must be accepted, and the data given in Table I could probably illustrate some important aspects of the standard test medium used in the present study. First, the headspace analysis indicated that the highly volatile hydrocarbons not included in the C7+-fraction were of considerable importance for the total composition. This is probably typical for sea water exposed to crude oil in systems with limited evaporation, since these low molecular hydrocarbons also have a relatively high water solubility (McAuliffe, 1979; McAuliffe et al., 1980). The relative enrichment of aromatic compounds in the water phase observed by us (Table 1) agrees with the results obtained by Shaw & Reidy (1979) for petroleum extracts prepared by gentle stirring. Although the water-soluble fraction of petroleum is commonly referred to as hydrocarbons only, highly soluble non-hydrocarbons can be greatly enriched in the water phase. Phenols constituted a significant fraction of the Ekofisk crude oil extract (Table 1), and have earlier been found in quantity in water extracts of other crude oils (Winters et al., 1976: Shaw & Reidy, 1979). The fact that the relative proportions of the petroleumderived components in the standard test medium differed so widely from those of the original crude oil verifies that the test media were dominated by dissolved petroleum components, and contained little or no dispersed crude oil. By combining the data obtained by the two analyses performed, the total content ofoil derived components of the undiluted test medium can be estimated to approximately 14mg litre-'. This value is within the range reported for sea water solutions of other crude oils (Anderson et al., 1974; Price, 1976; Winters et al., 1976; McAuliffe, 1977). It should be noted that the extraction time of 12-24 h generally used (Kauss et al., 1973; Anderson et al., 1974; Soto et al., 1975; Blundo, 1978; Shaw & Reidy, 1979: and present work) was too short to reach a stable level of dissolved oil components, and that a stable level (approximately 17 mg litre-') was only achieved in complete darkness (121stgaard & Jensen, 1983).

Exposure of phytoplankton to Ekofisk crude oil

197

Toxicity tests The growth experiments were carried out in continuous light since the rhythmic nature of growth and biochemical properties in day/night regimes (Sournia, 1974) were expected to conceal partly the toxic effects, especially in the turbidostat where the growth rate was recorded every hour. However, continuous light did not stop completely the rhythmic behaviour of the algae. It should be noted that the turbidity of the culture may not be directly proportional to cell concentration, since the turbidity also depends on additional properties such as biomass of the cells. Cell concentration was therefore recorded at regular intervals as a control (Fig. 4). These circadian rhythms are dealt with in greater detail elsewhere (Ostgaard & Jensen, 1982). The three algae which were tested exhibited different sensitivity to petroleum hydrocarbons. In batch culture the growth of S. costatum ceased in a 50 ~o diluted standard test medium. To stop the growth of C. ceratosporum and P. tricornutum an oil layer had to remain on the standard medium throughout the test. The layer did not have to cover the total surface of the medium to exert its extra toxicity. This indicates that the effect was caused by an increase in the concentration of certain oil components rather than by reduction of the gas exchange between medium and air. The algae seemed to tolerate the exposure to oil components in the turbidostat somewhat better than in batch culture. This may be due to the slow build-up of toxic concentrations in the turbidostat. The ranking of the algae according to sensitivity was, however, the same in both systems. The toxicity increased also in the turbidostat when the oil slick remained on the surface of the medium in the reservoir. This effect was partly related to the sub-optimal extraction time used in the preparation of the standard test solution. It is, however, possible that the phenomenon was due to low molecular weight hydrocarbons which were partly lost to the gaseous phase of the closed system (Fig. 1) in the absence of an oil layer. Low molecular weight hydrocarbons are known to be toxic to algae (Kauss et al., 1973; Pulich et al., 1974; Kauss & Hutchinson, 1975; Soto et al., 1975: Prouse et al., 1976). Photooxidation products might also have interfered (Lacaze & Villedon de Na'ide, 1976). The contribution of these factors will be the topic of future studies. Of the three parameters tested (Fig. 4), growth rate turned out to be more sensitive for detection of exposure to oil components. Considerable

198

Kjetill Ostgaard, lngvar Eide, Arne Jensen

changes in the chl/cell and Fk/chl ratio were observed, but their significance could not be established since large, apparently nonsystematic variations were observed in the absence of toxicants. The tolerance levels of dissolved oil components found in the present work were apparently higher than values reported in the literature (Gordon & Prouse, 1973; Pulich et al., 1974; Mahoney & Haskin, 1980). However, since the dissolved doses are generally so poorly defined, the discrepancies cannot be directly related to differences in the crude oil composition itself. On the other hand, the toxicity found for naphthalene in the present work was apparently higher than that reported by Kauss et al. (1973) and Soto et al. (1975). These authors related, however, the toxicity to the amount of naphthalene added to the medium whereas in the present study, the content measured in the medium was used. In our case the amount of naphthalene in the medium was less than one-tenth of the quantity added. Considering the complexity of crude oil toxicity, it is interesting to note that the ranking in sensitivity of the different species studied was the same for all doses tried (including presence/absence of oil layer) in both experimental systems. This general tendency might indicate a common underlying mode of action, and further make us expect similar differences between the species also under natural conditions.

A C K N O W L E D G E M ENTS We are grateful to Anne Andrewes for skilful technical assistance and for drawing the illustrations, and to Dr R. G. Lichtenthaler, Central Institute of Industrial Research, Oslo, for the oil analysis. This work is part of the Norwegian Marine Pollution Research and Monitoring Programme. REFERENCES Anderson, J.W., Neff, J.M., Cox, B.A., Tatem, H.E. & Hightower, G.M. (1974). Characteristics of dispersions and water-soluble extracts of crude oil and refined oils and their toxicity to estuarine crustaceans and fish. Mar. Biol., 27, 75-88. Awad, H. (1981). Comparative studies on analytical methods for the assessment of petroleum contamination in the marine environment II. Gas chromatographic analyses. Marine Chem., 10, 417-30.

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Berg, N., Gustavsen, K., Gj~s, N., Lichtenhaler, R. G., Oreld, F., Vadum, K. & Dfsti, T. (1980). Chemical analysis of water soluble petroleum fraction and studies of their accumulation in flounders. SI-report No. 780702-1, Oslo, Norway. Blundo, R. (! 978). The toxic effects of the water soluble fractions of No. 2 fuel oil and of three aromatic hydrocarbons on the behaviour and survival of barnacle larvae. Contr. Mar. Sci., 21, 25-37. Giddings, J. M. & Washington, J. M. (1981). Coal-liquefaction products, shale oil, and petroleum. Acute toxicity to freshwater algae. Environ. Sci. Technol., 15, 106-8. Gordon, D. C., Jr. & Prouse, N. J. (1973). The effects of three oils on marine phytoplankton photosynthesis. Mar. Biol., 22, 329-33. Guillard, R. R. L. & Ryther, J. H. (1962). Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microbiol., 8, 229-39. Holm-Hansen, O., Lorenzen, C. J., Holmes, R. W. & Strickland, J. D. H. (1965). Fluorometric determination of chlorophyll. J. Cons. perm. int. Explor. Met., 30, 3-15. Hsiao, S. I. C. (1978). Effects of crude oil on the growth of arctic marine phytoplankton. Environ. Pollut., 17, 93-107. Kauss, P. B. & Hutchinson, T. C. (1975). The effects of water-soluble petroleum components on the growth of Chlorella vulgaris Beijerinck. Enriron. Pollut., 9, 157-74. Kauss, P., Hutchinson, T. C., Soto, C., Hellebust, J. & Gritiiths, M. (1973). The toxicity of crude oil and its components to freshwater algae. Proc. Joh~t ConJ~ Prevention and Control of Oil Spills. Washington, DC, American Petroleum Institute, pp. 703-14. Kusk, K. O. (1978). Effects of crude oil and aromatic hydrocarbons on the photosynthesis of the diatom Nit:schia palea. Physiol. Plant., 43, i -6. Kusk, K. O. (1980). Effects of crude oils and aromatic hydrocarbons on the photosynthesis of three species of Acrosiphonia grown in the laboratory. Bot. Mar., 23, 587-93. Lacaze, J. C. & Villedon de Na'ide, O. (1976). Influence of illumination on phytotoxicty of crude oil. Mar. Pollut. Bull., 7, 73-6. Mahoney, B. M. & Haskin, H. H. (1980). The effects of petroleum hydrocarbons on the growth of phytoplankton recognised as food forms for the eastern oyster, Crassostrea virginica Gmelin. Environ. Pollut. (Series A), 22, 123-32. McAuliffe, C. D. (1977). Evaporation and solution of C z to C Io hydrocarbons from crude oils on the sea surface. In: Fate and effects of petroleum hydrocarbons in marine ecosystems and organisms (Wolfe, D. A. (Ed.)). New York, Pergamon Press, 363-72. McAuliffe, C. D. (1979). Oil and gas migration---chemical and physical constraints. AAPG Bull., 63, 761-81. McAuliffe, C. D., Johnson, J. C., Greene, S. H., Canevari, G. P. & Searl, T. D. (1980). Dispersion and weathering of chemically treated crude oils on the ocean. Environ. Sci. Technol., 14, i 509-18.

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