Effects of petroleum on estuarine bacteria

TABLE 2 Effects of short periods of contact with carcinogenic agents on subsequent growth of Antithamnionplumula (Ellis) Thur. Compound Concentration Time of % Growth (mg/l) contact stimulation (h) compared with control material 20-Methylcholanthrene*

3,4-Benzpyrene

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* Data from Boney and Corner (1962). TABLE 3 Effects of various aromatic hydrocarbons on the growth of sporelings of the red algae Antithamnion plumula (Ellis) Thur. (all applied at concentrations of 0.3 mgll), Compound

Acenaphthene Acetylaminofluorene Anthracene Azobenzene Benzidine Chrysene (1,2 benzpbenanthrene) 2-Methylanthracene 9-Methylanthracene Perylene (dibenzanthracene) 2 Pbenylnapththalene Pyrene (benzophenanthrene) Triphenylene (9,10 benzphenanthrene)

% Growth stimulation compared with control material --3 10 --20 0 12 58 --12 14 0 0 0 --0.2

Ishio et al. (1972) have pointed to the suitability of Porphyra as a test organism for carcinogenic agents. Whilst this is true, of equal significance is their observation that compounds occur in marine muds which can induce tumour-like proliferations on Porphyra similar to those produced by known carcinogenic agents of nonmarine origin. These cancerous growths were induced after a single period of contact with the tumour-inducing agent. It seems evident that the meristematic cells of the red algae are noticeably sensitive to growth stimulatory compounds of this nature. Short-term periods of contact in nature could result f r o m the temporary release from the mud of aromatic compounds of potential carcinogenic properties, either accumulated in the mud or synthesised on the sea bed. In their enhancement of the growth of filamentous algae they could induce morphological changes and the premature onset of fruiting. Aberrant growth of the nature described for Porphyra tenera would seem to be a feature of the membranaceous plant forms. All reports so far on cancer-like growths appear to have come from Japan, where Porphyra tenera is cultivated on a massive scale as a source of food. These results would generally seem to indicate further implications of the sub-lethal effects of certain (man-made ?) compounds in inshore waters. A . D . BONEY Department o f Botany, University o f Glasgow, Glasgow, W.2., Scotland.

Methods of application as described in Boney & Corner, 1962. A number of other aromatic compounds, some with known toxic properties, have also been tested with sporelings of Antithamnion (Table 3). In these results, whilst a small growth stimulus is shown following application of benzidine (of known carcinogenic activity), a similar effect is also shown by 9-methylanthracene. Chrysene induced a marked growth stimulation. Neither of these compounds is as yet reported to have any carcinogenic action. Hence it would seem possible that stimulatory effects on algal growth may be obtained with aromatic compounds not yet reported as being carcinogenie in tests involving applications to the skin of small mammals.

Arasaki, S., Inouye, A. & Kochi, Y. (1960). A disease of the cultured Porphyra, with special reference to the cancerdisease and the chytrid-disease which occurred at the culture field in Tokyo Bay during 1959-1960. Bull. Jap. Soc. Sci. Fish., 26: 1074-1081. Boney, A. D. & Corner, E. D. S. (1962). On the effects of some carcinogenic hydrocarbons on the growth of sporelings of marine red algae. J. mar. biol. Ass. U.K., 42: 579-585. Ishio, S., Yano, T. & Nakagawa, H. (1970). Algal cancer and causal substances in wastes from the coal chemical industry. Presented at 5th International Water Pollution Research Conference, July-Aug. 1970, San Francisco. Ishio, S., Yano, T. & Nakagawa, H. (1972). Cancerous disease of Porphyra tenera and its causes. Proc. Seventh International Seaweed Symposium, 373-376. Katayama, T. & Fujiyama, T. (1957). Studies on the nucleic acid of algae with special reference to desoxyinbonucleic acid contents of crown gall tissues developed on Porphyra tenera Kjellm. Bull. Jap. Soc. Sei. Fish., 23: 249-254.

Effects of Petroleum on Estuarine Bacteria The effects of petroleum on the natural microbial flora of a given environment, that is, on the bacteria, yeasts, filamentous fungi, and achlorophyllous algae, have largdy been ignored. This paper represents the first recorded investigation of the impact of petroleum on ecologically important groups of bacteria. A recent b o o k (Cowell, 1971) described the m a n y and varied effects of oil on macroorganisms. F r o m the work of Mitchell, et al. (1972), it is known that hydrocarbons have an effect on bacterial chemotoxis. W o r k in our laboratory (Walker & Colwell, 1974) showed that there is a growth-limitation of petroleum hydrocarbons 186

on bacteria in the natural estuarine and marine environments. The present study was undertaken to delineate further the effects of petroleum on ecologically important estuarine bacteria.

Materials and Methods Water and sediment samples were collected aseptically in a marsh in Chesapeake Bay, using a Niskin sampler and a Ponar grab. One litre Erlenmeyer flasks containing 400 ml marsh water and 100 g marsh sediment were supplemented with 0.1% of a South Louisiana crude oil or a No. 2 fuel oil. A control flask contained no

TABLE 1 Composition of the media used in this study, Chitin NH4NO3 1g Purified chitin 10 g 10% SolutionKH2PO4 3 ml 10% SolutionK2HPO4 10 ml Purified agar 20 g Estuarine salts solution* 1000 ml Lipid** I. NI-I4NO3 1g Yeast extract 1g Purified agar 20 Estaurine salts solution 1000gml II. Tween20 10 ml *See Walker and Colwell .1973). **I and II autoclavedand added after cooling to 44°C.

petroleum. Sample aliquots were withdrawn at 24 h, 7, 14, 21 and 28 days and plated on the casein medium of Sizemore and Stevenson (1970) and on the media listed in Table 1. Each of the media was prepared by pouring over a layer of water agar. The percentage of chitin-, lipid- or protein-hydrolysing bacteria was calculated by dividing the number of hydrolysers by the total number of colonies appearing on the plate.

It was interesting to note that, although both the crude oil and Fuel Oil No.2 are lipophilic, their addition to the water and sediment samples did not result in an increase in the number of lipolytic bacteria above that of the control. The work described in this paper is being continued, with longer incubation times. Results of the work in progress should be informative, since fatty acids resulting from petroleum degradation may not be formed in significant quantities to promote increased lipolytic activity during 4 weeks, the period of study employed in the work reported here. The percentage of chitinolytic bacteria in the control oscillated between 5 and 10% during the 4 week period (Fig. 3). The portion of chitinolytic bacteria in the populations present in water and sediment samples exposed to South Louisiana crude oil remained constant over the 4 Week period, compared with the samples exposed to No. 2 fuel oil, which showed a slight decrease followed by a return to the initial level, which was only slightly below the control (Fig. 3). In summary, throughout all the experiments reported here, the physiological groups of bacteria in the samples of water and sediment exposed to oil did not reach the

30 Results and Discussion The area selected for study is a sub-estuary marshland in Chesapeake Bay. A marsh ecosystem was selected for study because of the high productivity of marshes and their well-known susceptibility to oil pollution (Blumer et al., 1971). Because bacteria are important as decomposers, particularly in marshlands, we chose to focus our efforts on these microorganisms. The oils selected for study provide information on the different effects produced by crude oil and a refined product. Proteolytic bacteria were found to increase by 30% during the first week of growth. An oscillation in population number was noted after 3 weeks (Fig. 1). The percentage of proteolytic bacteria in the natural population which had been exposed to South Louisiana crude oil decreased by 15 % during the first week, then increased by about 25 %, but remained lower than the maximum in the control population (Fig. 1). The proteolytic bacteria exposed to the No. 2 fuel oil increased about 15 % during the first 24 h then decreased 30 ~o to a level of 20 % (which, in general, stayed fairly constant from 1 to 4 weeks) lower than the control population (Fig. 1). Similar effects were noted with lipolytic bacteria (Fig. 2). After an increase from 15 to 25 % during the first week of growth in the control, the percentage of lipolytic bacteria dropped to about 3% and remained at this low level for the duration of the experiment. The addition of No. 2 fuel oil very nearly eliminated the lipolytic bacteria until 28 days, at which time 2 % of the population comprised lipolytic species. South Louisiana crude oil caused a decrease in the lipolytic bacteria, with only a slight increase after 14 days. The percentage of petroleum-degrading bacteria was not monitored in these experiments; however, fuel oil and crude oil concentration was found to decrease with time, with the assumption being that the percentage of petroleumdegraders increased with time.

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population numbers found in the controls, i.e., samples not exposed to oil. These results substantiate earlier observations that petroleum can limit bacterial growth (Walker & Colwell, 1974). In addition, we conclude that there are measurable effects of oil on ecologically important bacterial groups, since the crude and refined oils employed in our work demonstrated a significant limiting effect on total viable numbers and, probably, the activity of these micro-organisms.

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Blumer, M., Sanders, H. L., Grassle, J. F. & Hampson, G. R. (1971). A small oil spill. Environment, 13: 2-12. Cowell, E. B. (1971). The ecological effects of oil pollution on 10 littoral communities, 250 p., Elsevier, London. Mitchell, R., Fogel, S. & Chet, I. (1972). Bacterial chemoreception inhibited by hydrocarbons. Water Res., 6: 1137-1140. i .~ = I t Sizemore,R. K. & Stevenson, L. H. (1970). Method for the iso0 / 14 21 28 lation of proteolytic marine bacteria. Appl. Microbiol., 20: 991-992. Time (days) Walker, J. D. & Colwell, R. R. (1974). Some effects of petroleum on estuarine and marine microorganisms.Jan. Can. Microbiol. (submitted). Walker, J. D. & Colwell, R. R. (1973). Microbial ecology of Fig. 3 Fluctuation in percentage of chitinolytic bacteria: absence petroleumutilization in Chesapeake Bay, pp. 685-690, In: of added substrate (©); with Fuel Oil No. 2 ([-1); and with APZ/EPA/USCG,Conference on Prevention and Control of Louisiana crude oil (A). Oil Spills, American Petroleum Institute, Washington, D. C. D

Intertidal Organisms of an Industrialized Estuary A study of the intertidal organisms of the Clyde Estuary is being undertaken to assess the effects of changing levels of pollution and to relate to these and other changes the distribution of important winter flocks of waders and ducks. The Clyde sea area may be subdivided into the Firth of Clyde, the sea lochs and the Clyde estuary. While the first two categories have in the past received much attention from biologists based at Millport and elsewhere, the last has only recently begun to receive the study it deserves. In this paper the estuary is defined as extending from the tidal weir in Glasgow westwards to a line between Gourock pier and Kilcreggan, and excluding the Gare Loch north of a line from Rhu to Roseneath (Fig. 1). The estuary was one of those described by Porter (1973) in her case studies of polluted estuaries, and much of what is now known about it is the product of work by the Clyde River Purification Board's staff (see for example papers by Mackey et al. 1969, 1971, 1972). The present authors have become concerned with two main aspects of the estuary. The first is the relationship between the distribution of organisms and the pollution load with special reference to changes which are anticipated during the next few years, a study with which the River Purification Board is associated. The second aspect relates to the food and feeding distribution of birds in the estuary, a matter which has acquired special importance recently, as in other estuaries, from pro188

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posals for land reclamation to provide industrial building sites; in this we have collaborated with the Nature Conservancy Council and other interested bodies.

Distributions of intertidal organisms The upper estuary between the tidal weir and Erskine is relatively narrow and enclosed between steep walls. Under conditions of high river flow, low salinites may