Effects of oil spill dispersants and drilling fluids on substrate specificity of marine bacteria

Waste Management, Vol. 15, No. 7, pp. 515 520, 1995 Copyright © 1996 Elsevier Science Ltd Printed in the USA. All rights reserved 0956-053X/95 $9.50 + 0.00

Pergamon

0956-053X(95)00045-3

TECHNICAL NOTE

EFFECTS OF OIL SPILL DISPERSANTS AND DRILLING FLUIDS ON SUBSTRATE SPECIFICITY OF MARINE BACTERIA G. C. O k p o k w a s i l i a n d C. N n u b i a Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Nigeria

ABSTRACT. The effects of oil spill dispersants and drilling fluids on the sizes of populations of specific heterotroph subgroups of marine bacteria were monitored in this study. The bacteria were isolated from drill cuttings recovered from Agbara - - an offshore oilfield located some 100 nautical miles off the Atlantic coast of Nigeria. Numbers of cellulolytic, proteolytic, starch-hydrolysing and lipolytic bacteria in the drill cuttings were monitored for 28 days in the presence of oil spill dispersants and drilling fluids. The percentages of these bacterial subgroups within the total heterotrophic population enumerated on tryptic soy agar (10% with 3% NaCI) fluctuated between 3.0 and 17.0%, 0.0 and 27.0%, 4.0 and 25.0% and 3.0 and 18.0% for cellulolytic, proteolytic, starch-hydrolysing and lipolytic bacteria respectively. These results indicate that oil spill dispersants and drilling fluids affect the ability of marine bacteria to metabolize these substrates in the environment. Copyright © 1996 Elsevier Science Ltd INTRODUCTION

viscosifiers. 6 The general functions of drilling mud in the borehole include: lifting of formation cuttings to the surface, control of subsurface pressures, lubrication of drill strings and sides of the hole, bottom hole cleaning and cooling, provision of aid to formation evaluation, and maintenance of stability of uncased sections of the bore hole. 5"7'8 The chemical treatment of oil, dispersing it into the water column before arrival onshore, is, at the moment, a widely recognized alternative to mechanical attempts at controlling offshore oil pollution. 9 Dispersants have been used to ameliorate the effects of spilled oil by distributing it into droplets in the water column to increase the surface area available for microbial attack and indirectly to aid biodegradation. As such, the dispersants are concentrated at the oil-water interface where oil-degrading bacterial populations act. ~° Dispersants have been the subject of serious national and international controversy around the world. Some dispersants are themselves toxic, although the current generation of dispersants is considered to be much less so than in the days of the Torrey Canyon, where the dispersants used were quite toxic. ~1 Toxicity, however, is not the only consideration in the use of dispersants. Of equal significance is their effectiveness12 and biodegradability. ~3'~a The toxicity of crude and refined oils to the natural bacterial population from sediments uncontaminated with oil has been demonstrated by Walker et al. ~5

Biogeochemical processes mediated by microorganisms are responsible for the cycling of carbon and other nutrient elements in the ecosystem. These processes may be at risk from chemical pollution. Global input of petroleum in the marine environment is estimated to be about 6 million tonnes annually. ~ The main portion of this pollution originates not from major disasters but from daily influxes. 2"3 Oil well blowouts notably resulted in greater damage than small spills. 4 Drilling fluids and muds as well as crude oil form the major environmental pollutants during oil well blowouts. Drilling muds are chemical mixtures used for rotary drilling in the extraction of oil and gas from the earth's crust. Essentially, a drilling mud is a suspension of solids (e.g. clays, barite, small cuttings, etc.) in liquids (i.e. water or oil) or in liquid emulsions, with chemical additives as required to modify its properties. 5 These chemical additives include bactericides, surface active agents, dispersants and RECEIVED 26 MARCH 1993; ACCEPTED 5 JULY 1995. Acknowledgements--The authors thank Baroid of Nigeria Ltd for providing the drill mud cuttings used in this study and the Nigerian National Petroleum Corporation (NNPC) for providing the helicopter service to the offshore location. This work was supported by the University of Port Harcourt Senate Research Grant.

515

516

Since heterotrophic bacteria play an important role in the biodegradation of hydrocarbons and other organic matter that enters the marine ecosystems in the overall process of carbon cycling, ~6-~8we designed this study to determine the extent to which subpopulations of heterotrophic marine bacteria with cellulolytic, proteolytic, starch-hydrolysing and lipolytic capabilities were affected by oil spill dispersants and drilling fluids. MATERIALS AND M E T H O D S Sources o f Isolates and Test Chemicals

The drill cuttings used as sources of microorganisms were recovered from an oil well drilling operation at Agbara field located about 100 nautical miles off the Atlantic coast of Nigeria between latitude 4°5'10 '' N and longitude 6 ° 15'15" E. The oil spill dispersants used in this study and their sources were Inhibisol, Ebbclean and Citrikleen (Durafelt Chemicals Ltd, Lagos), Dispolene 36S (SEPPIC, France), Chemiclene (Link Chemical Systems Ltd, Port Harcourt), Corexit 9527 (Shell, Port Harcourt) and Teepol (National Oil and Chemical Marketing Plc, Port Harcourt). The drilling fluids were Clairsol, Enviromul and mineral oil (Baroid of Nigeria Ltd), IDF mineral oil (Worldwide Petroleum Service Ltd), Clean base oil C0110 (Milpark Nigeria Ltd) and the reference chemical diesel oil (National Oil and Chemical Marketing Plc). All the drilling fluid companies are based in Port Harcourt, Nigeria.

G.C. OKPOKWASILI AND C. NNUBIA

distilled water. The medium used for enumerating cellulolytic bacteria was modified from that described by Walker et al. 15 containing in 1 litre of distilled water: cellulose powder, 1.0 g ; NH4NO3, 1.0 g; MgSO4.7H20, 2.3 g; KC1, 0.3 g; NaCI, 10.0 g; purified agar, 20.0 g. It was autoclaved, cooled to 45°C and 3.0 ml of sterile 10% (w/v) KH2PO4 solution and 7.0 ml of sterile 10% (w/v) K2HPO4 solution were added. Total heterotrophic bacteria were obtained using tryptic soy agar (10% with 3% NaC1). Each of these media was used as an overlay for water agar which was previously prepared, poured into plates and allowed to solidify. Culture System

A modified liquid medium of Walker et al. 15 was used. It contained in a 1-1itre Erlenmeyer flask, 400 ml of mineral salts solution and 100 g of drill cuttings. To this was added 0.5 ml of drilling fluid or oil spill dispersant. The control flask had no added toxicant. The cotton-stoppered flasks were incubated on a rotary shaker (flat bed rotator model TKA 0222.100) set at 120 rpm for 28 days at 30°C.

'A

(Drilling Fluids]

(3 0 .Q

.u I0"

Media

_o

The mineral salts medium used in this study was mineral medium C of Mills et al. 19 as modified by Okpokwasili and Okorie. 2° It contained in 1 litre of deionized water: NaC1, 10 g; MgSO4.7H20, 0.42 g; KC1, 0.29 g; KH2PO4, 0.83 g; Na2HPO4, 1.25 g; NaNO3, 0.42 g. The pH was adjusted to 7.4 and the medium sterilized by autoclaving. The medium used for enumerating lipolytic bacteria was modified from that of Walker et al. 21 and Gerhardt et al. 22 It contained in 1 litre of mineral salts solution: NH4NO3, 1.0 g; CaC1.H20, 0.1 g; yeast extract, 1.0 g; purified agar, 20 g. After autoclaving, the medium was cooled and mixed with 10.0 ml of separately sterilized Tween 80. A Davis '23 modified skim milk medium was used to enumerate proteolytic bacteria. It contained 20 g of purified agar in 900 ml of mineral salts solution. After autoclaving, it was cooled to 45°C and 100 ml of sterile 10% (w/v) skim milk (Carnco) solution was added. The medium used for enumerating starch-hydrolysing bacteria was that described by Harrigan and McCance. 24 It contained soluble starch, 15.0 g and nutrient agar, 28.0 g in 1 litre of

~0 s

Time(days) -I 2.~5]

-i

B

(Dispersants)

zol

151 0

7 Time (do/s)

FIGURE I. Fluctuations in percentage cellulolytic bacteria: (A) Drilling fluids: Control (O); Clairsol ( I ) ; Enviromul (~9); Baroid mineral oil (~7); IDF mineral oil (V); Clean base oil (I); and Diesel oil ([S]). (B) Dispersants: Control (©); Inhibisol (0); Dispolene 36S (0); Cherniclene ($7); Ebbclean (V); Citrikleen (I); Corexit 9527 ([]); and Teepol (I-q).

EFFECTS OF OIL SPILL DISPERSANTS AND DRILLING FLUIDS ON MARINE BACTERIA To measure microbial growth, samples from these incubations were withdrawn at times 0, 1, 7, 14, 21 and 28 days and serially diluted in mineral salts solution. Appropriate dilutions in volumes of 0.1 ml were plated in duplicate onto the various media described above. Proteolytic and cellulolytic bacteria were detected by zones of clearing produced after appropriate incubation periods; starch-hydrolysing bacteria were detected by zones of clearing produced and observed after flooding the culture plate with Gram's iodine solution. 24 Lipolytic bacteria were detected by observation of an opaque halo consisting of crystals of calcium soaps 22 that occurred around lipolytic colonies. The percentages of proteolytic, lipolytic, cellulolytic and starch-hydrolysing bacteria were calculated by the formula: ]5 (lytic colonies + total number of colonies) x 100. Inoculated agar plates were incubated aerobically at 30°C for 48 h with the exception of plates for enumeration of cellulolytic bacteria, which were incubated at 30°C for 14 days.

T/

25

A (Drilling

=

RESULTS AND DISCUSSION The restilts from enumeration of cellulolytic, proteolytic, lipolytic and starch-hydrolysing bacteria using (A) drilling fluids and (B) oil spill dispersants as carbon sources are shown in Figs 1 4 . Figure 1 shows fluctuations in percentage cellulolytic bacteria/ml between 2.5 and 17.0% with control fluctuating between 4.0 and 8.0%. The percentage proteolytic bacteria fluctuated between 0.0 and 23.0% for drilling fluid and between 0.0 and 17.0% for oil spill dispersants (Fig. 2) compared with control variation of between 2.0% and 17.0°/,,. Starch-hydrolysing bacteria fluctuated between 5.0 and 27.0%, and 4.0 and 18.0% for drilling fluids and dispersants respectively (Fig. 3) with the control fluctuating between 8.0 and 15%. Similarly, the percentage lipolytic bacteria varied between 3.0 and 18.0%, for both drilling fluids and dispersants (Fig. 4) while the control fluctuated between 6.0 and 20.0%. In all cases, the control had lower overall variation in the percentages of heterotrophic bacte-

Fluids)

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25t

B (Dispersontsl

C ,

_1 ;2 1

B(Dispersantsl

},o

o

r

,,

2,

2s

Time (doys)

FIGURE 2. Fluctuations in percentage proteolytic bacteria: (A) Drilling fluids: Control (O); Clairsol (O); Enviromul (G); Baroid mineral oil (~7); IDF mineral oil (V); Clean base oil (I); and Diesel oil ([]). (B) Dispersants: Control (O); Inhibisol (Q); Dispolene 36S (G); Chemiclene(V); Ebbclean (V); Citrikleen (IS]); Corexit 9527 (l~); and Teepol (El).

o

2,

2s

Time (days)

FIGURE 3. Fluctuations in percentage starch-hydrolysingbacteria: (A) Drilling fluids: Control (O); Clairsol (Q); Enviromul (®); Baroid mineral oil (S7); IDF mineral oil (V); Clean base oil (11); and Diesel oil (F-l). (B) Dispersants: Control (O); Inbibisol (O); Dispolene 36S (0); Chemiclene (V); Ebbclean (V); Citrikleen ([~); Corexit 9527 (~); and Teepol (F-l).

518

G . C . OKPOKWASILI AND C. NNUBIA A

fDrilling Fluids)

25-

o 20

15I0

5.

¢

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Y ......

I~, . . . . . .

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z'l . . . . . .

Ti me (days)

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".3 o ....

÷ ......

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z'8

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FIGURE 4. Fluctuations in percentage lipolytic bacteria: (A) Drilling fluids: Control (©); Clairsol (0); Enviromul (G)); Baroid mineral oil (V); IDF mineral oil (V); Clean base oil ([]); and Diesel oil ([]). (B) Dispersants: Control (C)); Inhibisol (0); Dispolene 36S (@); Chemiclene (xT); Ebbclean (V); Citrikleen (8); Corexit 9527 ([]); and Teepol ([Z).

rial subgroups over the experimental period than the tests with either of the two toxicants. Lipolytic, starch-hydrolysing, proteolytic and cellulolytic bacteria in the absence or presence of drilling fluids and oil spill dispersants fluctuated in numbers with time. For both toxicants, there was an initial slight decrease up to day 7 after which the percentages of cellulolytic bacteria increased slightly between the 14th and 21st days and then decreased with time to the 28th day. The increases and decreases in the control (no toxicant) were lower than with the tests. The results (Fig. 2) show that while some drilling fluids and oil spill dispersants supported mild increases (stimulation) and decreases (inhibition), some showed drastic increases in percentages of proteolytic bacteria by the 7th day. Enviromul, Clean base oil C0110 and diesel oil stimulated increases in percentage proteolytic bacteria by the 7th day, while IDF mineral oil produced an increase in percentages to 22.0% by the 14th day. A mild decrease generally occurred by the 21st and 28th days. Most of the dispersants gave decreased percentages by the 7th day and varied slightly up to the 28th day. However, Corexit 9527 and Teepol showed increased percentages by day 7 while Citrikleen caused a slight increase by day 14. Starch-hydrolysing bacteria showed a general increase by day 7 for both drilling fluids and dispersants (Fig. 3), after which the percentages decreased with time down to the 28th day. The control for these followed the same pattern of increase and decrease but with narrower margins than the test cases. Lipolytic bacteria in the presence of drilling fluids (Fig. 4) generally showed a slight increase up

TABLE 1 Growth of Heterotrophic Bacteria in Presence of (A) Drilling Fluids and (B) Dispersants A. Heterotrophic bacterial counts (colony-forming units/ml) with drilling fluids as main carbon sources Time (days)

0 1 7 14 21 28

Mineral salts broth inoculated with drill cuttings 3.5+0.9x 5.2+1.4x 1.3+0.8 x 9.0 + 2.4 x 5.0+1.1 x 3.7 + 0.8 x

105 106 10s 109 108 108

Clairsol

7.1+2.1 x 5.1+1.6x 9.5+2.9 x 3.0 __.0.7 x 9.8+2.8 x 8.8 + 2.2 x

Enviromul

104 106 108 109 108 10s

4.4+1.1 2.7+0.6 7.2+1.8 3.9 + 1.0 1.5+0.I 5.6 + 1.4

x x x x x x

105 106 108 109 109 108

Baroid mineral oil 1.7+0.1 z 4.1+0.9x 3.7+0.8 x 4.8 + 1.0 x 3.5+0.9x 4.6 + 1.1 x

105 106 108 109 109 109

IDF mineral oil 2.1+0.4x 4.2+0.7x 2.1+0.3 x 1.2 + 0.1 x 2.5+0.3 x 1.2 + 0.1 x

104 l06 108 109 108 109

Clean base oil C0110 2.1+0.2x 4.8+0.9 x 8.9+2.5 x 5.0 _ 1.5 X 6.7-t-1.9 × 5.0 + 1.2 X

104 106 10s 109 108 109

Diesel oil (control) 6.7+2.0x 9.3+3.1 x 3.1+0.1 x 8.1 __.2.3 x 4.1+1.2x 3.5 -I- 0.7 x

104 106 10s 109 10s 109

B. Heterotrophic bacterial counts (colony-forming units/ml) with dispersants as main carbon sources Time (days)

Inhibisol

0 1 7 14 21 28

4.4 + 0.7 x 105 4.6 + 0.8 x 106 1.7+0.1×108 2.2 + 0.2 x 109 1.4+0.1 x 109 1.2:1:0.1 x 109

Dispolene 36S 8.8 + 3.4 2.5 + 0.3 1.1+0.1 1.1 + 0.1 9.9+4.0 1.2 __.0.1

x x x × x x

104 105 109 109 108 109

Chemiclene 1.3 + 0.2 x 2.2 + 0.3 x 1.8+0.3x 7.1 + 2.5 × 1.1 +0.1 x 8.6 + 3.1 x

105 105 106 106 107 105

Ebbclean 4.1 + 2.0 x 105 4.1 + 1.8 x 105 7.2+2.8×108 8.0 + 2.9 x 106 1.7+0.2 x 107 5.5 + 1.3 x 106

Citrikleen 5.6 ± 1.6 x 2.6 + 0.4 x 3.1+0.7x 5.4 + 1.3 x 2.0+0.2 x 3.4 + 0.9 x

105 106 108 108 109 109

Corexit 9527 3.7 + 0.7 x 3.5 __.0.5 x 3.1+0.7x 3.7 + 0.6 x 6.6-t-1.5 X 5.1 + 1.4 x

Teepol (control) 105 106 108 108 107 106

4.5 + 0.8 x 5.4 + 0.8 x 3.5+0.6x 5.4 + 1.6 x 6.6+ 1.4 × 5.0 + 1.2 x

105 106 107 108 106 106

EFFECTS OF OIL SPILL DISPERSANTS

AND DRILLING FLUIDS ON MARINE BACTERIA

to day 14 after which the percentages decreased below 10.0%. In the presence of dispersants, general increases were observed by the 14th and 21st days when a decrease started down to the 28th day. The controls (absence of drilling fluids and dispersants) rather than decrease showed slight increase by the 28th day depicting the absence of any inhibitory substance in the medium. The results obtained indicate a general drop in percentages of cellulolytic and starch-hydrolysing bacteria at the end of the experiment. Walker e t al. ~5 showed that more information may be obtained if the duration of the experiment is extended. The general drop at the end of 28 days is in agreement with the observation of Walker e t al. 21 However, the increase in percentage of lipolytic bacteria obtained in this study does not agree with this report. 2~ This may be due to the source of microorganisms used in this study. The drill cuttings used in this work contained oil and it is expected that the microorganisms in the oil-contaminated cuttings were already adapted to the presence of hydrocarbons. This may explain the variations in results from this work and those of Walker e t al. 15'21 Mills e t al. 19 reported that all petroleum degraders were lipolytic though the converse, however, did not hold; that is, strains which are lipolytic did not all degrade petroleum. This further explains the fact that since drill cuttings already contained hydrocarbon degraders which are lipolytic, the lipolytic activities observed will be different from that reported by Walker e t al. 2~ These workers also suggested that the lipolytic activity of petroleum degraders can be accounted for by the formation of fatty acids. The total aerobic heterotrophic bacteria (those that grew on TSA) monitored for a period of 28 days showed a general increase up to the 14th day after which the numbers fluctuated on a parallel axis till the end of the experiment (Table 1). The observed initial increase is in agreement with the results of Marty e t al. 25 who reported an increase in aerobic heterotrophic bacterial population numbers in the presence of dispersant agents. The later pattern of parallel fluctuations is in accordance with the observation of Traxler e t al. 26 who reported that there was no statistically significant increase in total heterotrophic population but that the percentage of hydrocarbon utilizers within the total population increased with time. Additionally, the near parallel fluctuating pattern of the heterotrophic counts with time is supported by the report of Lizarraga-Partida e t alfl 7

We conclude from the enumeration of cellulolytic, proteolytic, amylolytic and lipolytic bacteria in drill mud cuttings that these heterotrophic microbial processes were negatively affected by drilling fluids and oil spill dispersants. The general trend is that of

519

mild inhibition of geomicrobiological processes by these toxicants in the marine environment.

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London, pp. 175-187 (1959). 24. Harrigan, W. F. and McCance, M. E. Laboratory Methods in Food and Dairy Microbiology. Academic Press, London (1976). 25. Marry, D., Bianchi, A. and Gatellier, C. Effects of three oil spill dispersants on marine bacterial populations. Mar. Pollut. Bull. 10:285-287 (1979). 26. Traxler, R. W., Bhattacharya, L. S., Griffin, P., Garafalo, G., Kulkarni, K. and Wilson, M. P. Jr. Microbial response to dispersant-treated oil in ecosystems. In Biodeterioration 5, T. A. Oxley and S. Barry (eds). John Wiley, London (1983). 27. Lizarranga-Partida, M. L., Rodriguez-Santiago, H. and Romero-Jarero, J. M. Effects of the Ixtoc I blowout on heterotrophic bacteria. Mar. Pollut. Bull. 13:67-70 (1982).

O p e n for d i s c u s s i o n u n t i l 27 S e p t e m b e r 1996