APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1976, Copyright © 1976 American Society for Microbiology
p.
522-535
Vol. 31, No. 4 Printed in U.S.A.
Oil Degradation in Soil R. L. RAYMOND,* J. 0. HUDSON, AND V. W. JAMISON Sun Ventures, Inc., Marcus Hook, Pennsylvania 19061
Received for publication 30 October 1975
The environmental effects of adding certain selected petroleum products to field soils at widely separated geographical locations under optimum conditions for biodegradation were studied. The locations selected for study of soil biodegradation of six oils (used crankcase oil from cars, used crankcase oil from trucks, an Arabian Heavy crude oil, a Coastal Mix crude oil, a home heating oil no. 2, and a residual fuel oil no. 6) were Marcus Hook, Pennsylvania, Tulsa, Oklahoma, and Corpus Christi, Texas. The investigative process, covering a period of 1 year at each location, was conducted in 14 fields plots (1.7 by 3.0 m) to which the oils were added in a single application at a rate of 11.9 m3/4 x 103 M2. Onehalf of the plots at each location were fertilized, and the incorporation of the oils and fertilizers was accomplished with rototillers to a depth of 10 to 15 cm. Concentrations of all oils decreased significantly at all locations. The average reduction ranged from 48.5 to 90.0% depending upon the type of oil and location. Rates of degradation did not exceed 2.4 m3/4 x 103 ml per month. Compositional changes in the oil with time were investigated using silica gel fractionation, gas chromatography, and ultraviolet absorbance. With the possible exception of the two fuel oils, the compositional changes were generally in the same direction for all of the oils. The silica gel fractionation and gravimetric data on residual oils show that all classes of compounds were degraded, but the more polar type degrade more slowly. Analysis of runoff water, leachate, and soils indicated that at the concentration applied no oil loss was observed from these plots via water movement. No significant movement of lead compounds added to the soils in the used crankcase oils was observed. Significant increases in hydrocarbon-utilizing microorganisms were demonstrated in all treated plots using either the pure hydrocarbon, n-hexadecane, or the applied oils as the growth substrate. These increases were usually sustained throughout the year. Significant increases in hydrocarbon-utilizing fungi were not demonstrated by the plating technique used. The concentrations of residual oils or their oxidation products were of sufficient magnitude in the treated plots, 9 months after application, to cause significant inhibition of plant growth. From the data obtained, it was not possible to determine the type of compounds causing this inhibition or their long-term environmental effects. The removal of oil that has been accidently or purposely spilled into the environment is of great concern to the petroleum industry. The present study was undertaken to develop ecological background data which would give a better understanding as to ways in which biodegradation might be enhanced and to determine what might be the environmental consequences of oil disposal in soils in the United States. *The literature on the subject of microbial degradation of petroleum and its products is quite voluminous. Because it has been reviewed so frequently in the past few years, we have taken the liberty of citing only a few recent papers which we consider most pertinent.
The most extensive study of application of oils to soil was that reported by C. B. Kincannon (5) at the Shell Oil Company refinery at Deer Park, Texas. He reported rates of degradation of the order of 8.3 m3/4 x 103 m2 per month for crude oil tank bottoms, a fuel oil (Bunker C), and a waxy raffinate. Bacterial assimilation was assumed to be responsible for the disappearance of the oils, but the increased number of microbial populations observed were not shown to be hydrocarbon-utilizing bacteria. Very high initial concentrations of oil (10%) in the test plots and lack of data on quantities of the various oils added make it difficult to draw any conclusions as to levels of non-biodegradable residues that remained at the conclusion of this study. 522
VOL. 31, 1976
In a more recent publication, Francke and
Clark (4) state that "a biological assimilatory process for the disposal of plant oil waste products has been successfully demonstrated." The process to which they refer involves adding used crankcase and vacuum pump oils to fairly large experimental field plots (3.6 x 102 M2) in Oak Ridge, Tenn. They, like the Shell study, depended upon the natural soil flora to degrade the oil. They did not demonstrate, with the microbiological techniques used, a direct relationship between oil degradation and hydrocarbon-utilizing bacteria. For a 3-month period they calculated a degradation rate of approximately 11.9 m3/4 x 103 m2 per month, with approximately 40% of the applied carbon being degraded at the end of that period. The length of study period reported does not permit any evaluation of possible residue levels. Studies in colder climates have been reported by Cook and Westlake (2) and Parkinson (8). Parkinson, working with Arctic terrestrial ecosystems, found that spilling a crude oil (Norman Wells, Northwest Territories) stimulated the microflora but he did not distinguish between nonhydrocarbon- and hydrocarbon-utilizing microorganisms. It was concluded that addition of nitrogen and phosphate speeded up the utilization of the alkane fraction of the crude oil that had been spilled on the soil. The overall degradation of the crude oil cannot be deduced from the data reported. In more extensive studies, Cook and Westlake determined the influence of degradation by such factors as fertilizer and oil-utilizing bacteria application to field plots in the Norman Wells area of the Northwest Territories and in the Swann Hill area of north central Alberta (2). Applications of the crude oils obtained from wells in these areas were made at a rate of 60 liters/9 M2. This rate produced significant increases in the microflora, but no effort was made to assess what fraction of the total were hydrocarbon-utilizing types. Chromatographic separations of recovered oils indicated that extensive degradation of the alkane fractions was taking pl'ace, but no data were given for quantities of residual oil remaining with time. They concluded from the chromatographic data that fertilizer speeded up degradation. They also suggested that application of oil-utilizing bacteria to the natural flora was beneficial. More recently, Cook and Westlake (3) have reported the results of additional studies on their 1972 field applications, 1973 field plots, and the Nipisi oil spill. They determined that a significant increase in microbial populations (which they equated to increased biodegradation) occurred in those oil-treated plots that had
OIL DEGRADATION IN SOIL
523
at least 3.7 x 122 kg of nitrogen added per 4 x 103 M2. The microbial numbers and changes in chemical composition of the recovered oil were not significantly influenced by the addition of hydrocarbon-utilizing bacteria to these oiltreated plots if fertilizer was present. As in previous studies, no attempt was made to determine the actual quantity of oil degraded or the levels of hydrocarbon-utilizing flora. They conclude from data obtained on soils taken at the site of the Nipisi oil spill that populations were present that could utilize Nipisi oil. Unfortunately, the data presented do not include the gravimetric data, which would substantiate this conclusion. The authors point out that the soil type in this spill area was representative of others in the Mackenzie Valley in that it was a sphagnum bog. If these soils are very high in organic matter, as might be deduced from the description, the significance of the increase in microbial numbers obtained as the result of fertilizing where oil is present is questionable in light of the absence of controls that contain only fertilizer. It would also greatly assist in evaluation of the analysis of the chemical changes observed if the efficacy of the pentane extraction method in sphagnum bogs and other high organic-containing soils were given. Odu (7) reported that the microbiological activity of a sandy, sedimentary, well-drained soil in Nigeria was enhanced by an oil spill that resulted from a blowout of an oil well. No indication was given as to proportion of hydrocarbon-utilizing flora present in those enumerated. In the work to be described below we have attempted to define the magnitude of the stimulation of the specific hydrocarbon-utilizing flora in the soil after the addition of six different oils to small field plots. Since the ultimate goal was to determine if residues did remain and, if so, what effect they had on the ecosystem, nematode and plant populations were used as the criteria to measure this effect. Free-living nematode concentrations in soils reflect to some degree the availability of bacteria and fungi, which are major sources of food. They are also extremely sensitive to drought conditions and hydrocarbons of the naphthalene series. The effects of soil adjuncts have been reported for both nematodes and plants, but the prolonged effects of petroleum or biodegradation products on these flora and fauna have not been reported. MATERIALS AND METHODS Field sites. A location on or near refinery property was chosen in Marcus Hook, Pa., Tulsa, Okla.,
524
APPL. ENVIRON. MICROBIOL.
RAYMOND, HUDSON, AND JAMISON
and Corpus Christi, Tex. The soil type at Marcus Hook was identified as a Glenville silt loam (pH 5.4); at Tulsa, sandy loam (pH 6.3); and at Corpus Christi, black clay loam (pH 7.6). The layout of the experimental plots (Fig. 1) was the same at each location. After plowing and disking, soil samples were taken and submitted to the Pennsylvania State University Soil and Forage Testing Merkle Laboratory, or the Texas A&M University Agricultural Extension Service for determination of soil nutrient levels. Limestone and gypsum were added at Marcus Hook to correct a low pH. The fertilized plots at Marcus Hook received a 10:5:5 (N-P-K) mixture at a rate of 1.4 x 103 kg/4 x 103 M2. At Tulsa the fertilized plots received a 10:20:10 mixture at a rate of 2.7 X 103 kg/4 x 103 M2. The Corpus Christi plots had sufficient phosphorus and potassium and a satisfactory pH, necessitating only the addition of 2.7 x 103 kg of ammonium sulfate per 4 x 103 m2 to the fertilized plots. All sites were chosen with the intent of using land that probably had in the past been under cultivation or grazing and had not been exposed to intentional petroleum hydrocarbon contamination. It was recognized, however, that air contamination from automobile and industrial sources could influence the microbial populations at all three locations. In the year prior to the test, the Marcus Hook and Corpus Christi locations had very heavy weed cover. The Tulsa site had a very heavy Bermuda grass cover. Oil characterization, application and tilling. Six
C-I
CC
| CC-F
DC
DC-F | HAC
oils were selected for study (Fig. 1). Properties of these oils are given in Tables 1 and 2. The oils were poured from 20-liter cans onto the leveled plots, lightly raked into the surface, and incorporated into the top 10 to 15 cm of soil with a rototiller. The equipment used at Tulsa was a Howard Rotovator manufactured by Rotary Hoes, Limited, West Horn, England. It was necessary to use smaller but similar equipment at Corpus Christi and Marcus Hook for incorporation and tilling. All plots at Tulsa and Corpus Christi were tilled once a month for the first 3 months, followed by tilling at 3-month intervals for the remainder of the project life. At Marcus Hook it was not possible, due to frozen soil, to till in January and February. Additional tilling was carried out in March, April, and May so that the amount of cultivation was equal at all three sites. Runoff and leachate water collection. Before application of oils, a hole was drilled in the center of each plot to accommodate a l-quart (ca. 0.95-liter) bottle holding a funnel filled with glass wool. This collector was positioned and covered with soil so that the top of the funnel was approximately 0.3 m below the soil surface. Water was removed from these bottles with a syringe or hand vacuum pump attached to a long small-diameter tube. The runoff water collectors were 20-liter plastic tubs positioned about 1.5 m from the center of the plot, 15 cm below grade. Analytical methods. Residual oil remaining in the soil was determined on a composite sample
THAC-FIT CLC |CLC-F11 022
*2-F
_1*6-C-2
-RUN -OFF WATER COLLECTORS 10 FEET
FIG. 1. Layout of field plots used to study oil degradation at Marcus Hook, Pa., Tulsa, Okla., and Corpus Christi, Tex. C-1, Control no. 1; CC, used crankcase oil from cars; DC, used crankcase oil from trucks; HAC, Arabian Heavy crude oil; CLC, Coastal Mix crude oil; no. 2, home heating oil no. 2; no. 6, residual fuel oil no. 6; F, fertilizer added; C-2, control 2.
TABLE 1. Characterization of oils applied to experimental plots Parameter
Gravity, API/60 F Viscosity, SUS/77 F Viscosity, SUS/100 F Viscosity, KV/100 F cs Viscosity, SFS/122 F Flash PM (F) Pour point (F) Silica gel fraction (%) Heptane CCl4 Benzene Methanol Sulfur
a, Not determined.
Used crankcase oils -a
-
70.5a 9.5 8.6 11.3
0.3
Heavy crude
Arabian oil
Gulf Coast Mix crude oil
Homeoilheating fuel (no. 2)
Residual fuel oil (no. 6)
27.4 164.0 103.0
23.6 324.0 161.0
35.0
16.0
-
-
-
-
2.4
p.
522-535
Vol. 31, No. 4 Printed in U.S.A.
Oil Degradation in Soil R. L. RAYMOND,* J. 0. HUDSON, AND V. W. JAMISON Sun Ventures, Inc., Marcus Hook, Pennsylvania 19061
Received for publication 30 October 1975
The environmental effects of adding certain selected petroleum products to field soils at widely separated geographical locations under optimum conditions for biodegradation were studied. The locations selected for study of soil biodegradation of six oils (used crankcase oil from cars, used crankcase oil from trucks, an Arabian Heavy crude oil, a Coastal Mix crude oil, a home heating oil no. 2, and a residual fuel oil no. 6) were Marcus Hook, Pennsylvania, Tulsa, Oklahoma, and Corpus Christi, Texas. The investigative process, covering a period of 1 year at each location, was conducted in 14 fields plots (1.7 by 3.0 m) to which the oils were added in a single application at a rate of 11.9 m3/4 x 103 M2. Onehalf of the plots at each location were fertilized, and the incorporation of the oils and fertilizers was accomplished with rototillers to a depth of 10 to 15 cm. Concentrations of all oils decreased significantly at all locations. The average reduction ranged from 48.5 to 90.0% depending upon the type of oil and location. Rates of degradation did not exceed 2.4 m3/4 x 103 ml per month. Compositional changes in the oil with time were investigated using silica gel fractionation, gas chromatography, and ultraviolet absorbance. With the possible exception of the two fuel oils, the compositional changes were generally in the same direction for all of the oils. The silica gel fractionation and gravimetric data on residual oils show that all classes of compounds were degraded, but the more polar type degrade more slowly. Analysis of runoff water, leachate, and soils indicated that at the concentration applied no oil loss was observed from these plots via water movement. No significant movement of lead compounds added to the soils in the used crankcase oils was observed. Significant increases in hydrocarbon-utilizing microorganisms were demonstrated in all treated plots using either the pure hydrocarbon, n-hexadecane, or the applied oils as the growth substrate. These increases were usually sustained throughout the year. Significant increases in hydrocarbon-utilizing fungi were not demonstrated by the plating technique used. The concentrations of residual oils or their oxidation products were of sufficient magnitude in the treated plots, 9 months after application, to cause significant inhibition of plant growth. From the data obtained, it was not possible to determine the type of compounds causing this inhibition or their long-term environmental effects. The removal of oil that has been accidently or purposely spilled into the environment is of great concern to the petroleum industry. The present study was undertaken to develop ecological background data which would give a better understanding as to ways in which biodegradation might be enhanced and to determine what might be the environmental consequences of oil disposal in soils in the United States. *The literature on the subject of microbial degradation of petroleum and its products is quite voluminous. Because it has been reviewed so frequently in the past few years, we have taken the liberty of citing only a few recent papers which we consider most pertinent.
The most extensive study of application of oils to soil was that reported by C. B. Kincannon (5) at the Shell Oil Company refinery at Deer Park, Texas. He reported rates of degradation of the order of 8.3 m3/4 x 103 m2 per month for crude oil tank bottoms, a fuel oil (Bunker C), and a waxy raffinate. Bacterial assimilation was assumed to be responsible for the disappearance of the oils, but the increased number of microbial populations observed were not shown to be hydrocarbon-utilizing bacteria. Very high initial concentrations of oil (10%) in the test plots and lack of data on quantities of the various oils added make it difficult to draw any conclusions as to levels of non-biodegradable residues that remained at the conclusion of this study. 522
VOL. 31, 1976
In a more recent publication, Francke and
Clark (4) state that "a biological assimilatory process for the disposal of plant oil waste products has been successfully demonstrated." The process to which they refer involves adding used crankcase and vacuum pump oils to fairly large experimental field plots (3.6 x 102 M2) in Oak Ridge, Tenn. They, like the Shell study, depended upon the natural soil flora to degrade the oil. They did not demonstrate, with the microbiological techniques used, a direct relationship between oil degradation and hydrocarbon-utilizing bacteria. For a 3-month period they calculated a degradation rate of approximately 11.9 m3/4 x 103 m2 per month, with approximately 40% of the applied carbon being degraded at the end of that period. The length of study period reported does not permit any evaluation of possible residue levels. Studies in colder climates have been reported by Cook and Westlake (2) and Parkinson (8). Parkinson, working with Arctic terrestrial ecosystems, found that spilling a crude oil (Norman Wells, Northwest Territories) stimulated the microflora but he did not distinguish between nonhydrocarbon- and hydrocarbon-utilizing microorganisms. It was concluded that addition of nitrogen and phosphate speeded up the utilization of the alkane fraction of the crude oil that had been spilled on the soil. The overall degradation of the crude oil cannot be deduced from the data reported. In more extensive studies, Cook and Westlake determined the influence of degradation by such factors as fertilizer and oil-utilizing bacteria application to field plots in the Norman Wells area of the Northwest Territories and in the Swann Hill area of north central Alberta (2). Applications of the crude oils obtained from wells in these areas were made at a rate of 60 liters/9 M2. This rate produced significant increases in the microflora, but no effort was made to assess what fraction of the total were hydrocarbon-utilizing types. Chromatographic separations of recovered oils indicated that extensive degradation of the alkane fractions was taking pl'ace, but no data were given for quantities of residual oil remaining with time. They concluded from the chromatographic data that fertilizer speeded up degradation. They also suggested that application of oil-utilizing bacteria to the natural flora was beneficial. More recently, Cook and Westlake (3) have reported the results of additional studies on their 1972 field applications, 1973 field plots, and the Nipisi oil spill. They determined that a significant increase in microbial populations (which they equated to increased biodegradation) occurred in those oil-treated plots that had
OIL DEGRADATION IN SOIL
523
at least 3.7 x 122 kg of nitrogen added per 4 x 103 M2. The microbial numbers and changes in chemical composition of the recovered oil were not significantly influenced by the addition of hydrocarbon-utilizing bacteria to these oiltreated plots if fertilizer was present. As in previous studies, no attempt was made to determine the actual quantity of oil degraded or the levels of hydrocarbon-utilizing flora. They conclude from data obtained on soils taken at the site of the Nipisi oil spill that populations were present that could utilize Nipisi oil. Unfortunately, the data presented do not include the gravimetric data, which would substantiate this conclusion. The authors point out that the soil type in this spill area was representative of others in the Mackenzie Valley in that it was a sphagnum bog. If these soils are very high in organic matter, as might be deduced from the description, the significance of the increase in microbial numbers obtained as the result of fertilizing where oil is present is questionable in light of the absence of controls that contain only fertilizer. It would also greatly assist in evaluation of the analysis of the chemical changes observed if the efficacy of the pentane extraction method in sphagnum bogs and other high organic-containing soils were given. Odu (7) reported that the microbiological activity of a sandy, sedimentary, well-drained soil in Nigeria was enhanced by an oil spill that resulted from a blowout of an oil well. No indication was given as to proportion of hydrocarbon-utilizing flora present in those enumerated. In the work to be described below we have attempted to define the magnitude of the stimulation of the specific hydrocarbon-utilizing flora in the soil after the addition of six different oils to small field plots. Since the ultimate goal was to determine if residues did remain and, if so, what effect they had on the ecosystem, nematode and plant populations were used as the criteria to measure this effect. Free-living nematode concentrations in soils reflect to some degree the availability of bacteria and fungi, which are major sources of food. They are also extremely sensitive to drought conditions and hydrocarbons of the naphthalene series. The effects of soil adjuncts have been reported for both nematodes and plants, but the prolonged effects of petroleum or biodegradation products on these flora and fauna have not been reported. MATERIALS AND METHODS Field sites. A location on or near refinery property was chosen in Marcus Hook, Pa., Tulsa, Okla.,
524
APPL. ENVIRON. MICROBIOL.
RAYMOND, HUDSON, AND JAMISON
and Corpus Christi, Tex. The soil type at Marcus Hook was identified as a Glenville silt loam (pH 5.4); at Tulsa, sandy loam (pH 6.3); and at Corpus Christi, black clay loam (pH 7.6). The layout of the experimental plots (Fig. 1) was the same at each location. After plowing and disking, soil samples were taken and submitted to the Pennsylvania State University Soil and Forage Testing Merkle Laboratory, or the Texas A&M University Agricultural Extension Service for determination of soil nutrient levels. Limestone and gypsum were added at Marcus Hook to correct a low pH. The fertilized plots at Marcus Hook received a 10:5:5 (N-P-K) mixture at a rate of 1.4 x 103 kg/4 x 103 M2. At Tulsa the fertilized plots received a 10:20:10 mixture at a rate of 2.7 X 103 kg/4 x 103 M2. The Corpus Christi plots had sufficient phosphorus and potassium and a satisfactory pH, necessitating only the addition of 2.7 x 103 kg of ammonium sulfate per 4 x 103 m2 to the fertilized plots. All sites were chosen with the intent of using land that probably had in the past been under cultivation or grazing and had not been exposed to intentional petroleum hydrocarbon contamination. It was recognized, however, that air contamination from automobile and industrial sources could influence the microbial populations at all three locations. In the year prior to the test, the Marcus Hook and Corpus Christi locations had very heavy weed cover. The Tulsa site had a very heavy Bermuda grass cover. Oil characterization, application and tilling. Six
C-I
CC
| CC-F
DC
DC-F | HAC
oils were selected for study (Fig. 1). Properties of these oils are given in Tables 1 and 2. The oils were poured from 20-liter cans onto the leveled plots, lightly raked into the surface, and incorporated into the top 10 to 15 cm of soil with a rototiller. The equipment used at Tulsa was a Howard Rotovator manufactured by Rotary Hoes, Limited, West Horn, England. It was necessary to use smaller but similar equipment at Corpus Christi and Marcus Hook for incorporation and tilling. All plots at Tulsa and Corpus Christi were tilled once a month for the first 3 months, followed by tilling at 3-month intervals for the remainder of the project life. At Marcus Hook it was not possible, due to frozen soil, to till in January and February. Additional tilling was carried out in March, April, and May so that the amount of cultivation was equal at all three sites. Runoff and leachate water collection. Before application of oils, a hole was drilled in the center of each plot to accommodate a l-quart (ca. 0.95-liter) bottle holding a funnel filled with glass wool. This collector was positioned and covered with soil so that the top of the funnel was approximately 0.3 m below the soil surface. Water was removed from these bottles with a syringe or hand vacuum pump attached to a long small-diameter tube. The runoff water collectors were 20-liter plastic tubs positioned about 1.5 m from the center of the plot, 15 cm below grade. Analytical methods. Residual oil remaining in the soil was determined on a composite sample
THAC-FIT CLC |CLC-F11 022
*2-F
_1*6-C-2
-RUN -OFF WATER COLLECTORS 10 FEET
FIG. 1. Layout of field plots used to study oil degradation at Marcus Hook, Pa., Tulsa, Okla., and Corpus Christi, Tex. C-1, Control no. 1; CC, used crankcase oil from cars; DC, used crankcase oil from trucks; HAC, Arabian Heavy crude oil; CLC, Coastal Mix crude oil; no. 2, home heating oil no. 2; no. 6, residual fuel oil no. 6; F, fertilizer added; C-2, control 2.
TABLE 1. Characterization of oils applied to experimental plots Parameter
Gravity, API/60 F Viscosity, SUS/77 F Viscosity, SUS/100 F Viscosity, KV/100 F cs Viscosity, SFS/122 F Flash PM (F) Pour point (F) Silica gel fraction (%) Heptane CCl4 Benzene Methanol Sulfur
a, Not determined.
Used crankcase oils -a
-
70.5a 9.5 8.6 11.3
0.3
Heavy crude
Arabian oil
Gulf Coast Mix crude oil
Homeoilheating fuel (no. 2)
Residual fuel oil (no. 6)
27.4 164.0 103.0
23.6 324.0 161.0
35.0
16.0
-
-
-
-
2.4
