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A field study on the acute effects of the dispersant Corexit 9527 on glucose uptake by marine microorganisms
Marine EnvironmentalResearch$ (1981) 83-91
A FIELD STUDY ON THE ACUTE EFFECTS OF THE DISPERSANT COREXIT 9527 ON GLUCOSE UPTAKE BY MARINE MICROORGANISMS* R. P. GRIFFITHS, T. M. McN^M^R^, B. A. CALDWELLdk. R. Y. MORITA Department of Microbiology, Oregon State Unirersity. Corvallis, OR 97331, USA (Received: 25 June, 1980)
ABSTRACT
The effects o f the dispersant Corexit 9527 and Corexit with crude oil on the rate o f glucose uptake and mineralisation were studied in Arctic and Subarctic marine waters and sediments. Essentially all o f the 149 water and 95 sediment samples tested displayed decreased glucose uptake rates in the presence of either 15 or 50ppm Corexit. Depresseduptake rates were observed at concentrations as low as l ppm. The mean concentration at which Corexit depressed glucose uptake hi" 50 °//owas 12 ppm. The effect o f Corexit was more pronounced on pelagic than on benthic microbial populations.
INTRODUCTION
There has been growing evidence that dispersants and/or crude oil may have adverse effects on a wide range of aquatic organisms. With the development of offshore oil fields in Alaskan waters, there has been concern over the potential impact the dispersant Corexit 9527 has on marine organisms. Hagstr6m & L6nning (1977) showed that this dispersant interferred with the fertilisation and development of a sea urchin with some adverse effects observed at concentrations as low as 0.0003ppm. Hsiao et al. (1978) showed that crude oil-Corexit mixtures were inhibitory to primary production in Arctic marine phytoplankton. There has been very little information published concerning the impact of dispersants on microbial function. Gunkel (1968) states that 9 0 ~ of marine bacteria were killed in the presence of 10ppm 'emulsifier'. This study, however, was conducted on an early dispersant formulation that is no longer in use. Mulkins-Phillips & Stewart (1974) * Technical Paper No. 5535, Oregon Agricultural Experiment Station.
83 Marine Environ. Res. 0141 - l 136/81/0005-0083/502-50 ~ Applied Science Publishers Ltd, England, 198 l Printed in Great Britain
84
R. P. GRIFFITHS, T, M. M(-NAMARA, B. A. CALDWELL, R. Y. MORITA
showed that two of four dispersants studied slowed initial growth rates. This increased lag phase was more pronounced at an incubation temperature of 10°C than at 25 °C. We are currently conducting a series of studies which suggest that the same phenomenon is taking place in the relatively cold waters of Kachemak Bay, Alaska. The main purpose of this study is to determine if microbial function in the marine environment is altered in the presence of Corexit 9527, a dispersant that could be used in the event of an oil spill in Alaskan waters.
MATERIALS AND METHODS
Study areas and sampling proceth,'es This paper includes data obtained from water and sediment samples collected from four areas along the Alaskan coast. The samples in the Beaufort Sea study were collected in September, 1978 from 35 locations along the Beaufort Sea coast from the Colville River to the US-Canadian border. The location of the furthest sample site was 148 km from land; however, most of the samples were collected within 74 km of the shore. Twelve of the samples were collected from a small boat within ½km of the shore. During the Cook Inlet study, samples were collected from 51 locations which ranged from 167 km southwest of Kodiak Island to 4 km west of Anchorage, Alaska. This study was conducted in May, 1979. The samples in the Norton Sound study were collected in July, 1979 from 47 locations. Most stations were located within Norton Sound (Fig. 1). The samples analysed during the three Kasitsna Bay
0~ D Fig. 1.
The locationof the sample
area relative to
the state of Alaska.
EFFECTS OF COREXIT 9 5 2 7
85
studies were collected from Kachemak Bay in the Cook Inlet. The samples were taken from the same general area during the three studies conducted in February, April and July-August, 1979. The sediments for all studies were collected from depths ranging from I to 2200 m. Most of the sediments were collected at depths less than 100 m. All but 12 of the water samples were collected within 3 m of the surface. The deepest water sample was taken at 35 m. The sampling methodology and sample treatment have been described previously (Griffiths et al., 1978). Analyses were initiated within 3 h of collecting water samples and within 12 h of collecting sediment samples. The storage temperature used between sample collection and analysis was within I °C of the in situ temperature. Proce&tres for uptake studies A detailed description of the techniques used in the uptake studies has been reported by Griftiths et al. (1978). The labelled substrate used in these experiments was D- (U-14C) glucose obtained from Amersham/Searle. Several batches of glucose were used which had specific activities between 304 and 328 mCi/mmol. The final concentration of added glucose ranged from 6 to 7/~g/litre. In the kinetic studies four glucose concentrations were used: 1.8, 3-5, 7.0 and 14 #g/litre. In the Beaufort Sea study, the concentration of added Corexit 9527 (Exxon Chemical Co.) was 15 ppm. In all of the other studies, except the concentration effects study, the final Corexit concentration used was 50 ppm. The crude oil used in the Beaufort Sea study was produced from the Prudhoe Bay oil field. The crude oil used in the other studies was produced from the Cook Inlet oil field. In all cases, 10 plitres of crude oil were added to the 10ml reaction mixture. The reaction vessels were incubated undisturbed in the dark for up to 8 h. The incubation temperature was within 1 °C of the in situ temperature which varied from 2 to 11 °C. In the kinetic experiments, the incubation time varied from 1 to 4 h. The exposure times during the concentration series experiments were 4, 6 and 7 h, respectively for the 0-20, 0-100 and 0-200 ppm concentration ranges. The differences in incubation times used in these studies should not significantly alter the resulting observations since we conducted time course experiments which show that the reduction in uptake rates at a given TABLE 1 EFFECTS OF COREXITON THE KINETICS OF GLUCOSE UPTAKE IN WATERSAMPLES
Sample number
313 314 315
Control Percent respiration 22 29 32
Corexit
Vmax
K, + S .
T,
0.30 0.016 0.11
5 0.2 8
19 13 77
Percent respiration 16 15 22
V,a~
K,+S n
T,
0-90 0.0015 0.011
8 0.03 3
84 19 257
I'm, ` = M a x i m u m potential rate of glucose uptake reported as/~g/litre/h. K, + S , = Transport constant plus the natural glucose concentration reported as pg/litre. 7", = Time in hours required for the microbial population to utilise the naturally occurring glucose.
86
R . P . GRIFFITHS, T. M. McNAMARA, B. A. CALDWELL, R. Y. MORITA
concentration was essentially the same for at least 24 h (unpublished data). The calculation of the kinetic variables presented in Table 1 was made using the equations of Wright and Hobbie (1966). The percent respiration (mineralisation) was calculated by dividing the amount of labelled carbon associated with the CO 2 fraction by the total taken up by the cells (both cell and z4CO 2 radioactivity). The Vmax value is the maximum potential rate at which glucose could be taken up expressed in units of/~g glucose/litre/h. The K,S~value is the transport constant plus the natural concentration of glucose in the sample expressed as/ag/litre. The 7",value is the time in hours required for the microbial population to utilise the naturally occurring glucose.
RESULTS AND DISCUSSION
During six different sampling periods, a total of 149 water samples were studied to determine the effects of the crude oil dispersant Corexit 9527 and Corexit with crude oil on glucose uptake during the initial 8 h exposure (Table 2). With the exception of TABLE 2 PERCENT REDUCTION IN GLUCOSE UPTAKE RATES IN WATER SAMPLES
Sampling location
Treatment Corexit
* Beaufort Sea Cook Inlet Norton Sound Kasitsna Bay, February 1979 Kasitsna Bay, April 1979 Kasitsna Bay, July 1979
58 88 85 68 95 82
Corexit 9527 + crude oil
SD
n
Range
~
SD
n
Range
22 9 11 11 3 19
14 37 60 7 6 25
38-79 59-98 58-98 53-83 92-98 38-98
76 94 . 59 95 91
20 5 . . 20 5 10
14 37 . 7 6 24
54--91 78-100 29-83 85-99 50-99
* These samples treated with 15 p p m Corexit, all other samples treated with 50 ppm Corexit. SD = Standard deviation.
the Beaufort Sea study, all water samples were exposed to 50 ppm Corexit. The mean value for the percent reduction in glucose uptake rates ranged from 58 to 95 ~ in samples exposed to Corexit alone. When the microbial populations were exposed to Corexit and crude oil, the mean reduction ranged from 75 to 95 °,o. In three of the five studies where the effects of both treatments were observed, the mean percent reduction in glucose uptake rates was higher in the samples treated with both Corexit and crude oil. These differences were very significant (p < 0.001) in the Upper Cook Inlet and the July Kasitsna Bay studies. The effect of Corexit and Corexit with crude oil on glucose uptake was also measured in sediment samples collected during the same sampling periods (Table 3).
EFFECTS OF c o R E x I T
87
9527
TABLE 3 PERCENT REDUCTION IN GLUCOSE UPTAKE RATES IN SEDIMENT SAMPLES
Sampling location
Treatment Corexit 9527 + crude oil
Corexit 9527
a Beaufort Sea Cook Inlet Norton Sound Kasitsna Bay, February 1979 Kasitsna Bay, April 1979 Kasitsna Bay, July 1979
15 57 54 38 44 60
SD
n
Range
~
SD
11 17 13 14 24 9
12 6 34 12 Il 20
0-38 39-81 30-84 20-61 17-75 45-80
40 79 . 52 44 74
17 13 . . 14 24 6
n
Range
12 6 . 12 Il 20
0-62 54-93 38-74 6-76 65-85
° These samples treated with 15 p p m Corexit, all other samples treated with 50 p p m Corexit. SD = Standard deviation.
The mean percent reduction in the glucose uptake rates ranged from 15 to 60 ~o in the presence of Corexit and from 40 to 79 ~ in the presence of Corexit and crude oil. In four of the five studies, the latter treatment showed a greater effect. These differences were statistically significant (p < 0.02) in all cases. The effects of Corexit and Corexit with crude oil on glucose uptake rates were greater in water samples than in the sediments. For Corexit alone, the statistical significance of this difference was at thep < 0.001 level and it was at thep < 0-04 level for the Corexit with crude oil treatments. The above-mentioned analyses were conducted using the same concentration of labelled glucose. It is possible that some component of the Corexit produced the same effect as adding non-labelled glucose to the reaction mixtures, thus reducing apparent uptake rates. One method that could be used to determine if this is taking place is to observe the effect of Corexit on the kinetics of glucose uptake using several concentrations and applying the equations of Wright & Hobbie (1966). If Corexit is not acting as a metabolic inhibitor, the turnover time (1",)and the transport constant plus the natural substrate concentration (K,S~)values maychange but the maximum potential velocity (Vma~)of glucose uptake should not change. When this experiment was conducted in three water samples, the Vma~values decreased in the presence of Corexit, suggesting that it is acting as a metabolic inhibitor (Table 1). It should be noted that incubation times of from 1 to 4 h were used in these experiments. This indicates that Corexit probably affects glucose transport soon after exposure. The effect of Corexit on percent respiration in both water and sediment samples was also observed (Tables 4 and 5). If the transport of glucose into the cells were the only function effected by Corexit, one would expect to see no changes in the percent respiration. There was a significant difference between the respiration percentages observed in treated and non-treated samples. For both water and sediment samples, four out of six studies showed significant differences in respiration percentages. Of those studies showing a significant difference in the sediment samples, all mean
88
R. P. GRIFFITHS, T. M. McNAMARA, B. A. CALDWELL, R. Y. MORITA TABLE 4 PERCENT RESPIRATION OBSERVED IN SEDIMENT SAMPLES TREATED WITH COREXIT
Sampling location
Beaufort Sea Cook Inlet Norton Sound Kasitsna Bay, February 1979 Kasitsna Bay, April 1979 Kasitsna Bay, July 1979
Control
31 44 28 20 17 18
Corexit 9527
SD
n
Range
~
SD
n
Range
*p =
9 26 8 5 8 3
18 11 38 15 12 20
22-57 14-78 15-66 13-30 6-34 12-23
34 32 35 24 18 23
9 13 6 6 5 5
18 11 38 15 12 20
23-60 21-60 22-53 9-33 9-25 16-34
0-022 NS 0.00003 NS 0.007 0.000008
° Level of statistical significance between treated and non-treated samples. SD =~Standard deviation. NS ~ Not significant.
values increased in samples exposed to Corexit (Table 4). In the water samples, two showed increases and two decreases (Table 5). In the Norton Sound study, there was a slight increase in the mean percent respiration in the treated samples but the difference was not statistically significant. If, however, the water samples were analysed as two groups, one group showed an increase in the mean and the other showed the reverse trend. The differences in both groups are very significant (p < 0.006). The two sets of samples taken from two different locations in and near the Norton Sound are shown in Fig. 2. The water samples in one group (A) were collected at stations located to the east and samples in another group (B) were collected to the west. Surface salinity and relative microbial activity measurements taken during the same cruise suggest that there are two water masses present in the area which are roughly defined by the line in Fig. 2. The one to the west is more saline and shows lower levels of microbial activity than does the one to the east. These data TABLE 5 PERCENT RESPIRATION OBSERVED IN WATER SAMPLES TREATED WITH COREXIT
Sampling location
Beaufort Sea Cook Inlet Norton Sound Kasitsna Bay, February 1979 Kasitsna Bay, April 1979 Kasitsna Bay, July 1979 Norton Sound Group A Norton Sound Group B
Control
40 28 26 32 37 34 24 34
Corexit 9527
SD
n
Range
~
SD
n
Range
14 15 7 3 7 9 6 8
13 39 62 6 7 24 48 14
18-58 11-70 15-49 28-38 28-46 21-50 11-49 20-46
28 32 28 35 47 26 28 25
I1 13 7 7 10 10 5 10
13 39 62 6 7 24 48 14
15-51 14--67 11-44 29--46 34-62 13-53 18-42 11-44
"Level of statistical significance between treated and non-treated samples. SD -- Standard deviation. NS = Not significant.
ap =
0.0005 0.013 NS 0.040 NS 0-007 0.00001 0.0006
EFFECTS OF COREXIT 9527
89
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-S
-9
.~
.: \ " .
"",:'-'-'-'. . . . .
,.,....:~.~.'v~."-~.~ .:,'.-'
". ".'
~:.: +
3 •
•
-23
X •
-15
~
•
1
6eo4 • ~ S
\o
•
~1
4 NORTON
.8
Do. •
• 3
• 8
t :
1..'i
SOUND
~'" ~-.. ~,.. )-.."
-11
63"
•
....+'!:"'-
s
ST. LAWRENCE I. 1
.
" ..-', *U.ON . , V E .
30NM
I
|
168 °
166"
164 e
162"
Fig, 2. Change in the percent respiration in Norton Sound surface seawater samples exposed to Corexit 9527 at a concentration of 50 ppm.
suggest that different microbial populations may be affected in different ways by the presence of Corexit. The relationship between Corexit concentration and its effect on glucose uptake in nine Kasitsna Bay water samples was studied using three concentration ranges: 00100, 0°50 and 0-20 ppm (Table 6). Figure 3 graphically illustrates the results of one of the three studies. Both this study and that conducted over a range of from 0 to 100 ppm indicate that most of the effect takes place at concentrations below 20 ppm with little further change observed at 50 and 100ppm. In the 0-20 ppm study, the samples showed decreases in uptake rates of 10 and 12 % when exposed to Corexit at l ppm. Using the best-fitting power curve to describe the data, the mean concentration at which 50 % of the original uptake rate was lost was calculated to be 12 ppm. These data suggest that the alterations in microbial activity observed in the 50 ppm studies would represent the maximum effect possible. In the Mulkins-Phillips & Stewart study (1974) it was shown that even though some dispersants caused an increase in the lag phase of growth, growth was not inhibited. When natural populations were exposed to dispersants and crude oil, however, there were qualitative shifts in the microbial population. In one case, the resulting population consisted of 100 % pseudomonads. One of the experiments
90
R. P, GRIFFITHS, T. M. McNAMARA, B. A. CALDWELL, R. Y. MORITA TABLE 6
THE CONCENTRATION OF COREXIT AT WHICH THE GLUCOSE UPTAKE RATE WAS REDUCED TO ONE HALF OF THE CONTROL
Sample number
Goodness of fit to power curve
Corexit concentration (ppm)
Range of concentrations tested (ppm)
0.94 0-96 0.92 0.99 0.99 0-98 0.82 0.98 0.98
15 18 1i 7 7 I0 10 10 10
0-100 0-100 0-100 0-20 0-20 0-50 0-50 0-50 0-50
271 273 275 285A 285B 242 247 250 255
mean = 12 ppm (to reduce uptake 50 ~/o)
conducted during the CEPEX project described by Menzel & Case (1977) consisted of observing the results of adding crude oil and Corexit 9527 to one of the seawater enclosure systems. At the end of the experiment, the microbial population was essentially a pure culture (G. G. Gessey, personal communication). It is quite possible that what we are observing in this study is the initial phase of a selection process which is acting to reduce the diversity of the population. It is very unlikely that the concentrations of Corexit 9527 used in our field studies (15 and 50 ppm) would be realised under recommended application in open waters. However, it must >..
> I00 U
Sample No. • 242 & 247 o 250
~ \
80
'~ 6 0 W
~- 4 0 Z W U
o: 2O W a.
0 0
Fig. 3.
~ I0
i 20
30
~, 40
COREXIT
(ppm)
m
7"1~ 50
The percent remaining microbial activity as measured by glucose uptake rates in seawater samples exposed to Corexit 9527 at various concentrations. Incubation time was 4 h.
EFFECTS OF COREXiT 9527
91
be kept in mind that we did observe a 10-12 ~ reduction in glucose uptake rates at l ppm after 4h exposure and the mean concentration which caused a 50 reduction in glucose uptake rates was 12 ppm. Concentrations within this range could be achieved in shallow inshore areas where tidal and wind energy is low.
ACKNOWLEDGEMENTS
We would like to express our thanks to the personnel on board the USCGS Northwindand the N OAA ship Discoverer for their excellent logistic support during our field studies. The technical help of G. Kurath and P. Yorgey during the Norton Sound cruise is also acknowledged. This research was supported by the Bureau of Land Management through interagency agreement with the National Oceanic and Atmospheric Administration, under which a multi-year programme responding to needs of petroleum development of the Alaskan continental shelf is managed by the Outer Continental Shelf Environmental Assessment Program Office (NOAA contract No. 03-05-022-68).
REFERENCES GRIFFITHS, R. P., HAYASAKA,S. S., MCNAMARA,T, M. & MORITA,R. Y. (1978). Relative microbial activity and bacterial concentrations in water and sediment samples taken in the Beaufort Sea. Can. J. MicrobioL, 24, ! 2 ! 7-226. GUNKEL, W. (1968). Bacteriological investigations of oil-polluted sediments from the Cornish coast following the "Torrey Canyon" disaster. In The Bio/ogica! Effects of Oil Pollution on Littoral Communities. Supplement to Field Studies, Vol. 2, 151-8, Field Studies Council, London. HAGSTR6M,B. E. & L6NStSG, S. (I 977). The effects of Esso Corexit 9527 on the fertilization capacity of spermatozoa. Mar. Po!/ut. Bull., 8, 136-38. HsIAO, S. I. C., KITTLE,D. W. & FOY, M. G. (1978). Effects ofcrude oils and the oil dispersant Corexit on primary production of Arctic marine phytoplankton and seaweed. Environ. Po/lut., 15, 209-21. MENZEL,D. W. & CASE,J. (1977). Concept and design: Controlled ecosystem pollution experiment. Bull. Mar. Sci., 27, I-7. MULKINS-PHILLIPS,G. J. & STEWART,J. E. (1974). Effectof four dispersants on biodegradation and growth of bacteria on crude oil. AppL MicrobioL, 28, 457-552. WmGHT, R. T. & HOBmE, J. E. (1966). Use of glucose and acetate by bacteria and algae in aquatic ecosystems. Ecology, 47, 447-64.
A FIELD STUDY ON THE ACUTE EFFECTS OF THE DISPERSANT COREXIT 9527 ON GLUCOSE UPTAKE BY MARINE MICROORGANISMS* R. P. GRIFFITHS, T. M. McN^M^R^, B. A. CALDWELLdk. R. Y. MORITA Department of Microbiology, Oregon State Unirersity. Corvallis, OR 97331, USA (Received: 25 June, 1980)
ABSTRACT
The effects o f the dispersant Corexit 9527 and Corexit with crude oil on the rate o f glucose uptake and mineralisation were studied in Arctic and Subarctic marine waters and sediments. Essentially all o f the 149 water and 95 sediment samples tested displayed decreased glucose uptake rates in the presence of either 15 or 50ppm Corexit. Depresseduptake rates were observed at concentrations as low as l ppm. The mean concentration at which Corexit depressed glucose uptake hi" 50 °//owas 12 ppm. The effect o f Corexit was more pronounced on pelagic than on benthic microbial populations.
INTRODUCTION
There has been growing evidence that dispersants and/or crude oil may have adverse effects on a wide range of aquatic organisms. With the development of offshore oil fields in Alaskan waters, there has been concern over the potential impact the dispersant Corexit 9527 has on marine organisms. Hagstr6m & L6nning (1977) showed that this dispersant interferred with the fertilisation and development of a sea urchin with some adverse effects observed at concentrations as low as 0.0003ppm. Hsiao et al. (1978) showed that crude oil-Corexit mixtures were inhibitory to primary production in Arctic marine phytoplankton. There has been very little information published concerning the impact of dispersants on microbial function. Gunkel (1968) states that 9 0 ~ of marine bacteria were killed in the presence of 10ppm 'emulsifier'. This study, however, was conducted on an early dispersant formulation that is no longer in use. Mulkins-Phillips & Stewart (1974) * Technical Paper No. 5535, Oregon Agricultural Experiment Station.
83 Marine Environ. Res. 0141 - l 136/81/0005-0083/502-50 ~ Applied Science Publishers Ltd, England, 198 l Printed in Great Britain
84
R. P. GRIFFITHS, T, M. M(-NAMARA, B. A. CALDWELL, R. Y. MORITA
showed that two of four dispersants studied slowed initial growth rates. This increased lag phase was more pronounced at an incubation temperature of 10°C than at 25 °C. We are currently conducting a series of studies which suggest that the same phenomenon is taking place in the relatively cold waters of Kachemak Bay, Alaska. The main purpose of this study is to determine if microbial function in the marine environment is altered in the presence of Corexit 9527, a dispersant that could be used in the event of an oil spill in Alaskan waters.
MATERIALS AND METHODS
Study areas and sampling proceth,'es This paper includes data obtained from water and sediment samples collected from four areas along the Alaskan coast. The samples in the Beaufort Sea study were collected in September, 1978 from 35 locations along the Beaufort Sea coast from the Colville River to the US-Canadian border. The location of the furthest sample site was 148 km from land; however, most of the samples were collected within 74 km of the shore. Twelve of the samples were collected from a small boat within ½km of the shore. During the Cook Inlet study, samples were collected from 51 locations which ranged from 167 km southwest of Kodiak Island to 4 km west of Anchorage, Alaska. This study was conducted in May, 1979. The samples in the Norton Sound study were collected in July, 1979 from 47 locations. Most stations were located within Norton Sound (Fig. 1). The samples analysed during the three Kasitsna Bay
0~ D Fig. 1.
The locationof the sample
area relative to
the state of Alaska.
EFFECTS OF COREXIT 9 5 2 7
85
studies were collected from Kachemak Bay in the Cook Inlet. The samples were taken from the same general area during the three studies conducted in February, April and July-August, 1979. The sediments for all studies were collected from depths ranging from I to 2200 m. Most of the sediments were collected at depths less than 100 m. All but 12 of the water samples were collected within 3 m of the surface. The deepest water sample was taken at 35 m. The sampling methodology and sample treatment have been described previously (Griffiths et al., 1978). Analyses were initiated within 3 h of collecting water samples and within 12 h of collecting sediment samples. The storage temperature used between sample collection and analysis was within I °C of the in situ temperature. Proce&tres for uptake studies A detailed description of the techniques used in the uptake studies has been reported by Griftiths et al. (1978). The labelled substrate used in these experiments was D- (U-14C) glucose obtained from Amersham/Searle. Several batches of glucose were used which had specific activities between 304 and 328 mCi/mmol. The final concentration of added glucose ranged from 6 to 7/~g/litre. In the kinetic studies four glucose concentrations were used: 1.8, 3-5, 7.0 and 14 #g/litre. In the Beaufort Sea study, the concentration of added Corexit 9527 (Exxon Chemical Co.) was 15 ppm. In all of the other studies, except the concentration effects study, the final Corexit concentration used was 50 ppm. The crude oil used in the Beaufort Sea study was produced from the Prudhoe Bay oil field. The crude oil used in the other studies was produced from the Cook Inlet oil field. In all cases, 10 plitres of crude oil were added to the 10ml reaction mixture. The reaction vessels were incubated undisturbed in the dark for up to 8 h. The incubation temperature was within 1 °C of the in situ temperature which varied from 2 to 11 °C. In the kinetic experiments, the incubation time varied from 1 to 4 h. The exposure times during the concentration series experiments were 4, 6 and 7 h, respectively for the 0-20, 0-100 and 0-200 ppm concentration ranges. The differences in incubation times used in these studies should not significantly alter the resulting observations since we conducted time course experiments which show that the reduction in uptake rates at a given TABLE 1 EFFECTS OF COREXITON THE KINETICS OF GLUCOSE UPTAKE IN WATERSAMPLES
Sample number
313 314 315
Control Percent respiration 22 29 32
Corexit
Vmax
K, + S .
T,
0.30 0.016 0.11
5 0.2 8
19 13 77
Percent respiration 16 15 22
V,a~
K,+S n
T,
0-90 0.0015 0.011
8 0.03 3
84 19 257
I'm, ` = M a x i m u m potential rate of glucose uptake reported as/~g/litre/h. K, + S , = Transport constant plus the natural glucose concentration reported as pg/litre. 7", = Time in hours required for the microbial population to utilise the naturally occurring glucose.
86
R . P . GRIFFITHS, T. M. McNAMARA, B. A. CALDWELL, R. Y. MORITA
concentration was essentially the same for at least 24 h (unpublished data). The calculation of the kinetic variables presented in Table 1 was made using the equations of Wright and Hobbie (1966). The percent respiration (mineralisation) was calculated by dividing the amount of labelled carbon associated with the CO 2 fraction by the total taken up by the cells (both cell and z4CO 2 radioactivity). The Vmax value is the maximum potential rate at which glucose could be taken up expressed in units of/~g glucose/litre/h. The K,S~value is the transport constant plus the natural concentration of glucose in the sample expressed as/ag/litre. The 7",value is the time in hours required for the microbial population to utilise the naturally occurring glucose.
RESULTS AND DISCUSSION
During six different sampling periods, a total of 149 water samples were studied to determine the effects of the crude oil dispersant Corexit 9527 and Corexit with crude oil on glucose uptake during the initial 8 h exposure (Table 2). With the exception of TABLE 2 PERCENT REDUCTION IN GLUCOSE UPTAKE RATES IN WATER SAMPLES
Sampling location
Treatment Corexit
* Beaufort Sea Cook Inlet Norton Sound Kasitsna Bay, February 1979 Kasitsna Bay, April 1979 Kasitsna Bay, July 1979
58 88 85 68 95 82
Corexit 9527 + crude oil
SD
n
Range
~
SD
n
Range
22 9 11 11 3 19
14 37 60 7 6 25
38-79 59-98 58-98 53-83 92-98 38-98
76 94 . 59 95 91
20 5 . . 20 5 10
14 37 . 7 6 24
54--91 78-100 29-83 85-99 50-99
* These samples treated with 15 p p m Corexit, all other samples treated with 50 ppm Corexit. SD = Standard deviation.
the Beaufort Sea study, all water samples were exposed to 50 ppm Corexit. The mean value for the percent reduction in glucose uptake rates ranged from 58 to 95 ~ in samples exposed to Corexit alone. When the microbial populations were exposed to Corexit and crude oil, the mean reduction ranged from 75 to 95 °,o. In three of the five studies where the effects of both treatments were observed, the mean percent reduction in glucose uptake rates was higher in the samples treated with both Corexit and crude oil. These differences were very significant (p < 0.001) in the Upper Cook Inlet and the July Kasitsna Bay studies. The effect of Corexit and Corexit with crude oil on glucose uptake was also measured in sediment samples collected during the same sampling periods (Table 3).
EFFECTS OF c o R E x I T
87
9527
TABLE 3 PERCENT REDUCTION IN GLUCOSE UPTAKE RATES IN SEDIMENT SAMPLES
Sampling location
Treatment Corexit 9527 + crude oil
Corexit 9527
a Beaufort Sea Cook Inlet Norton Sound Kasitsna Bay, February 1979 Kasitsna Bay, April 1979 Kasitsna Bay, July 1979
15 57 54 38 44 60
SD
n
Range
~
SD
11 17 13 14 24 9
12 6 34 12 Il 20
0-38 39-81 30-84 20-61 17-75 45-80
40 79 . 52 44 74
17 13 . . 14 24 6
n
Range
12 6 . 12 Il 20
0-62 54-93 38-74 6-76 65-85
° These samples treated with 15 p p m Corexit, all other samples treated with 50 p p m Corexit. SD = Standard deviation.
The mean percent reduction in the glucose uptake rates ranged from 15 to 60 ~o in the presence of Corexit and from 40 to 79 ~ in the presence of Corexit and crude oil. In four of the five studies, the latter treatment showed a greater effect. These differences were statistically significant (p < 0.02) in all cases. The effects of Corexit and Corexit with crude oil on glucose uptake rates were greater in water samples than in the sediments. For Corexit alone, the statistical significance of this difference was at thep < 0.001 level and it was at thep < 0-04 level for the Corexit with crude oil treatments. The above-mentioned analyses were conducted using the same concentration of labelled glucose. It is possible that some component of the Corexit produced the same effect as adding non-labelled glucose to the reaction mixtures, thus reducing apparent uptake rates. One method that could be used to determine if this is taking place is to observe the effect of Corexit on the kinetics of glucose uptake using several concentrations and applying the equations of Wright & Hobbie (1966). If Corexit is not acting as a metabolic inhibitor, the turnover time (1",)and the transport constant plus the natural substrate concentration (K,S~)values maychange but the maximum potential velocity (Vma~)of glucose uptake should not change. When this experiment was conducted in three water samples, the Vma~values decreased in the presence of Corexit, suggesting that it is acting as a metabolic inhibitor (Table 1). It should be noted that incubation times of from 1 to 4 h were used in these experiments. This indicates that Corexit probably affects glucose transport soon after exposure. The effect of Corexit on percent respiration in both water and sediment samples was also observed (Tables 4 and 5). If the transport of glucose into the cells were the only function effected by Corexit, one would expect to see no changes in the percent respiration. There was a significant difference between the respiration percentages observed in treated and non-treated samples. For both water and sediment samples, four out of six studies showed significant differences in respiration percentages. Of those studies showing a significant difference in the sediment samples, all mean
88
R. P. GRIFFITHS, T. M. McNAMARA, B. A. CALDWELL, R. Y. MORITA TABLE 4 PERCENT RESPIRATION OBSERVED IN SEDIMENT SAMPLES TREATED WITH COREXIT
Sampling location
Beaufort Sea Cook Inlet Norton Sound Kasitsna Bay, February 1979 Kasitsna Bay, April 1979 Kasitsna Bay, July 1979
Control
31 44 28 20 17 18
Corexit 9527
SD
n
Range
~
SD
n
Range
*p =
9 26 8 5 8 3
18 11 38 15 12 20
22-57 14-78 15-66 13-30 6-34 12-23
34 32 35 24 18 23
9 13 6 6 5 5
18 11 38 15 12 20
23-60 21-60 22-53 9-33 9-25 16-34
0-022 NS 0.00003 NS 0.007 0.000008
° Level of statistical significance between treated and non-treated samples. SD =~Standard deviation. NS ~ Not significant.
values increased in samples exposed to Corexit (Table 4). In the water samples, two showed increases and two decreases (Table 5). In the Norton Sound study, there was a slight increase in the mean percent respiration in the treated samples but the difference was not statistically significant. If, however, the water samples were analysed as two groups, one group showed an increase in the mean and the other showed the reverse trend. The differences in both groups are very significant (p < 0.006). The two sets of samples taken from two different locations in and near the Norton Sound are shown in Fig. 2. The water samples in one group (A) were collected at stations located to the east and samples in another group (B) were collected to the west. Surface salinity and relative microbial activity measurements taken during the same cruise suggest that there are two water masses present in the area which are roughly defined by the line in Fig. 2. The one to the west is more saline and shows lower levels of microbial activity than does the one to the east. These data TABLE 5 PERCENT RESPIRATION OBSERVED IN WATER SAMPLES TREATED WITH COREXIT
Sampling location
Beaufort Sea Cook Inlet Norton Sound Kasitsna Bay, February 1979 Kasitsna Bay, April 1979 Kasitsna Bay, July 1979 Norton Sound Group A Norton Sound Group B
Control
40 28 26 32 37 34 24 34
Corexit 9527
SD
n
Range
~
SD
n
Range
14 15 7 3 7 9 6 8
13 39 62 6 7 24 48 14
18-58 11-70 15-49 28-38 28-46 21-50 11-49 20-46
28 32 28 35 47 26 28 25
I1 13 7 7 10 10 5 10
13 39 62 6 7 24 48 14
15-51 14--67 11-44 29--46 34-62 13-53 18-42 11-44
"Level of statistical significance between treated and non-treated samples. SD -- Standard deviation. NS = Not significant.
ap =
0.0005 0.013 NS 0.040 NS 0-007 0.00001 0.0006
EFFECTS OF COREXIT 9527
89
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,.,....:~.~.'v~."-~.~ .:,'.-'
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~:.: +
3 •
•
-23
X •
-15
~
•
1
6eo4 • ~ S
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•
~1
4 NORTON
.8
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s
ST. LAWRENCE I. 1
.
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168 °
166"
164 e
162"
Fig, 2. Change in the percent respiration in Norton Sound surface seawater samples exposed to Corexit 9527 at a concentration of 50 ppm.
suggest that different microbial populations may be affected in different ways by the presence of Corexit. The relationship between Corexit concentration and its effect on glucose uptake in nine Kasitsna Bay water samples was studied using three concentration ranges: 00100, 0°50 and 0-20 ppm (Table 6). Figure 3 graphically illustrates the results of one of the three studies. Both this study and that conducted over a range of from 0 to 100 ppm indicate that most of the effect takes place at concentrations below 20 ppm with little further change observed at 50 and 100ppm. In the 0-20 ppm study, the samples showed decreases in uptake rates of 10 and 12 % when exposed to Corexit at l ppm. Using the best-fitting power curve to describe the data, the mean concentration at which 50 % of the original uptake rate was lost was calculated to be 12 ppm. These data suggest that the alterations in microbial activity observed in the 50 ppm studies would represent the maximum effect possible. In the Mulkins-Phillips & Stewart study (1974) it was shown that even though some dispersants caused an increase in the lag phase of growth, growth was not inhibited. When natural populations were exposed to dispersants and crude oil, however, there were qualitative shifts in the microbial population. In one case, the resulting population consisted of 100 % pseudomonads. One of the experiments
90
R. P, GRIFFITHS, T. M. McNAMARA, B. A. CALDWELL, R. Y. MORITA TABLE 6
THE CONCENTRATION OF COREXIT AT WHICH THE GLUCOSE UPTAKE RATE WAS REDUCED TO ONE HALF OF THE CONTROL
Sample number
Goodness of fit to power curve
Corexit concentration (ppm)
Range of concentrations tested (ppm)
0.94 0-96 0.92 0.99 0.99 0-98 0.82 0.98 0.98
15 18 1i 7 7 I0 10 10 10
0-100 0-100 0-100 0-20 0-20 0-50 0-50 0-50 0-50
271 273 275 285A 285B 242 247 250 255
mean = 12 ppm (to reduce uptake 50 ~/o)
conducted during the CEPEX project described by Menzel & Case (1977) consisted of observing the results of adding crude oil and Corexit 9527 to one of the seawater enclosure systems. At the end of the experiment, the microbial population was essentially a pure culture (G. G. Gessey, personal communication). It is quite possible that what we are observing in this study is the initial phase of a selection process which is acting to reduce the diversity of the population. It is very unlikely that the concentrations of Corexit 9527 used in our field studies (15 and 50 ppm) would be realised under recommended application in open waters. However, it must >..
> I00 U
Sample No. • 242 & 247 o 250
~ \
80
'~ 6 0 W
~- 4 0 Z W U
o: 2O W a.
0 0
Fig. 3.
~ I0
i 20
30
~, 40
COREXIT
(ppm)
m
7"1~ 50
The percent remaining microbial activity as measured by glucose uptake rates in seawater samples exposed to Corexit 9527 at various concentrations. Incubation time was 4 h.
EFFECTS OF COREXiT 9527
91
be kept in mind that we did observe a 10-12 ~ reduction in glucose uptake rates at l ppm after 4h exposure and the mean concentration which caused a 50 reduction in glucose uptake rates was 12 ppm. Concentrations within this range could be achieved in shallow inshore areas where tidal and wind energy is low.
ACKNOWLEDGEMENTS
We would like to express our thanks to the personnel on board the USCGS Northwindand the N OAA ship Discoverer for their excellent logistic support during our field studies. The technical help of G. Kurath and P. Yorgey during the Norton Sound cruise is also acknowledged. This research was supported by the Bureau of Land Management through interagency agreement with the National Oceanic and Atmospheric Administration, under which a multi-year programme responding to needs of petroleum development of the Alaskan continental shelf is managed by the Outer Continental Shelf Environmental Assessment Program Office (NOAA contract No. 03-05-022-68).
REFERENCES GRIFFITHS, R. P., HAYASAKA,S. S., MCNAMARA,T, M. & MORITA,R. Y. (1978). Relative microbial activity and bacterial concentrations in water and sediment samples taken in the Beaufort Sea. Can. J. MicrobioL, 24, ! 2 ! 7-226. GUNKEL, W. (1968). Bacteriological investigations of oil-polluted sediments from the Cornish coast following the "Torrey Canyon" disaster. In The Bio/ogica! Effects of Oil Pollution on Littoral Communities. Supplement to Field Studies, Vol. 2, 151-8, Field Studies Council, London. HAGSTR6M,B. E. & L6NStSG, S. (I 977). The effects of Esso Corexit 9527 on the fertilization capacity of spermatozoa. Mar. Po!/ut. Bull., 8, 136-38. HsIAO, S. I. C., KITTLE,D. W. & FOY, M. G. (1978). Effects ofcrude oils and the oil dispersant Corexit on primary production of Arctic marine phytoplankton and seaweed. Environ. Po/lut., 15, 209-21. MENZEL,D. W. & CASE,J. (1977). Concept and design: Controlled ecosystem pollution experiment. Bull. Mar. Sci., 27, I-7. MULKINS-PHILLIPS,G. J. & STEWART,J. E. (1974). Effectof four dispersants on biodegradation and growth of bacteria on crude oil. AppL MicrobioL, 28, 457-552. WmGHT, R. T. & HOBmE, J. E. (1966). Use of glucose and acetate by bacteria and algae in aquatic ecosystems. Ecology, 47, 447-64.