EPA/600/A-95/109
Microbial Activity in Subsurface Samples Before and During Nitrate-Enhanced Bioremediation
J. Michele Thomas*, Virginia R. Gordy, Cristin L. Bruce, Stephen R. Hutchins, J'JwM. Sinclair, and
C. Herbert Ward
ABSTRACT
A study was conducted to determine the microbial activity of a site contaminated with JP-4 jet fuel,
before and during nitrate-enhanced bioremediation. Samples at three depths from six different
locations were collected aseptically under anaerobic conditions before and during treatment. Cores
were located in or close to the source of contamination, downgradient of the source, or outside the
zone of contamination. Parameters for microbial characterization included 1) viable counts of aerobic
heterotrophic, JP-4 degrading, and oligotrophic bacteria, 2) the MPN of aerobic and anaerobic
protozoa, 3) the MPN of total denitrifiers, and 4) the MPN of denitrifiers in hydrocarbon-amended
microcosms. The results indicate that the total number of denitrifiers increased by an order of
magnitude during nitrate-enhanced bioremediation in most samples. The number of total
heterotrophs and JP-4 degrading microorganisms growing aerobically also increased. In addition, the
first anaerobic protozoa associated with hydrocarbon-contaminated subsurface materials were
detected.
INTRODUCTION
The effects of in situ bioremedial processes on microbial communities are relatively unknown. At
best, the only indicator of microbial activity that is generally measured during in situ bioremediation
operations is viable cell counts. Although viable cell counts may indicate gross changes in population
size, changes within the microbial community will not be detected. Of importance will be the selection
pressure by the bioremedial process for contaminant-degrading organisms and the fitness traits which
allow these microorganisms to survive and effectively compete with other organisms for nutrients.
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Knowledge of the effects of bioremediation on the microbial community may provide information
that can be used to develop refined methods for process design and enhance the bioremedial process.
Of interest in the present study is the effect of nitrate-enhanced bioremediation on microbial
populations. We assessed changes in the microbial ecology of the site by determining aerobic viable
counts, the MPN of total denitrifiers and JP-4 degrading microorganisms with nitrate as the electron
acceptor, and aerobic and anaerobic protozoa,
EXPERIMENTAL PROCEDURES AND MATERIALS
Core Materials
Core material was collected before treatment at three depths from the following six boreholes; 80AA,
80BA, 80DA, 80EB, 80JB, and 80KB (Fig. 1). Boreholes 80AA, 80BA, SODA, and 80EB were
supposed to represent zones where residual JP-4 was located, borehole JB was supposed to represent a
zone downgradient but influenced by the contamination, and borehole 80KB was supposed to
represent an uncontaminated zone (Table 1). Interim core samples which correspond in depth and
location to the intial samples were collected after 5 months of treatment (80Z, 80ZA, SOW, SOX, 80JC
and 80KC). Boreholes 80Z, 80ZA. 80W2 and 80X were supposed to represent zones containing
residua] contamination. The 80JC borehole is downgradient but influenced by the contamination and
remedial treatment whereas 80KC represents uncontaminated material that will not be impacted by
treatment. The initial and interim samples were collected in March 1993 and August 1994,
respectively. The samples were collected using steam-cleaned drilling equipment and were pared
aseptically under anaerobic conditions (Hutchins et ai, 1991). Core material was subsampled in an
anaerobic glovebox. The samples were kept on ice in the field and during shipping, and then stored at
5°C in the laboratory until used. The pilot demonstration of nitrate-enhanced bioremediation was
conducted by making two treatment cells 100 ft by 100 ft, one which received recharge amended with
10-20 mg/L N03~-N and the second which received the same treatment without nitrate. The recharge
was continuously applied through surface sprinklers at a rate of 11 gpm/cell (Hutchins et al., 1995).
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hi L/uti j^ivyuc^i uuuiiuii uiiuci kjtin 11 iiyui^ conditions
Subsurface material collected before treatment was used to assess the denitrification potential of
compounds found in JP-4 jet fuel. Microcosms were constructed with the Eglin core samples
collected before treatment to evaluate BTEX (benzene, toluene, ethylbenzene, the xylenes) and
trimethylbenzenes removal under strictly denitrifying conditions as described previously (Hutchins,
1991). Selected alkylbenzenes were degraded under denitrifying conditions by indigenous aquifer
microorganisms. The mean zero-order rate constants were 1.2±0.5 mg/L/d alkylbenzene
biodegradation and 2.6 ±1.3 mg/L/d NO3-N removal.
Media and Culture Conditions
Serial dilutions of each sample were prepared in triplicate under aerobic conditions by aseptically
adding 10 grams of subsurface material to dilution bottles that contained 95 mL of 0.1% Na4P207 •
10H2O. The bottles were shaken on a wrist action shaker (Burrell Corporation, Pittsburgh, PA) at a
setting of 10 for 1 hour, after which the rest of the dilution series was prepared using 0.1% Na4P207 •
IOH2O as the diluent. The dilution series were used to determine the number of total heteroptrophs,
JP-4 degraders, oligotrophs, total denitrifiers, microorganisms that denitrify with JP-4 as the sole
carbon source, and aerobic and anaerobic protozoa in each sample. The number of total
heterotrophs, JP-4 degraders, and oligotrophs in each sample was determined under aerobic conditions
on R2A medium (Difco Industries, Detroit, MI), a mineral salts medium incubated in the presence of
JP-4 vapors, and a mineral salts medium incubated without JP-4 vapors, respectively. The colonies
growing on R2A medium were counted after 1-1.5 weeks of incubation, whereas colonies growing on
the other media were counted after 2 weeks of incubation. Counts of aerobic microorganisms are
important because most denitrifiers are aerobic and only switch to anaerobic respiration in the absence
of oxygen. The mineral salts medium (pH 7)contained per liter of deionized water: 0.8 g KH2PO4,
5.58 g Na2HP04 or 6.99 Na2HPC>4 • 2H2O, 1.8 g (NH^SCU, 0.017 g CaSC>4 • 2H2O, 0.123 g
MgS04 • 7H20, 0.5 mg FeS04 • 7H20. 1.54 me MnSOd • HoO. 2.86 me FhBCh O mo mo r,.sn, .
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5H20, 0.021 mg ZnCl2, 0.041 mg C0CI2 • 6H2O, and 0.025 mg Na2MoC>4 • 2H2O. The total
number of denitrifiers was determined using Nitrate Broth (Difco Industries). The number of
organisms that denitrify with JP-4 as the sole carbon source was determined in the mineral salts
medium amended with 1 g/L KNO3" and 200 |iL of JP-4 jet fuel. Vials (40-mL volume) containing
20 mL of sterile aerobic mineral salts medium were amended aseptically with 200 [iL of filter-
sterilized JP-4, inoculated with serial dilutions of the samples, and sealed. Equal numbers of samples
were incubated in the same medium without JP-4 to determine the effect of ambient carbon on
denitrification potential. Because some oxygen was present, the denitrification detected in these
samples could be the result of biodegradation of JP-4 or JP-4 degradation intermediates produced
during the initial aerobic phase of incubation. Denitrifying activity was measured colorimetrically by
testing for NO2" using sulfanic acid and N,N dimethyl-l-naphthylamine. The total number of
denitrifiers and the number of JP-4 degraders that use NO3" as the terminal electron acceptor were
determined after 3 and 6 weeks, respectively.
The number of aerobic and anaerobic protozoa was determined (Sinclair and Ghiorse, 1987) using
subsurface sediment or dilutions of the sediment. Plates containing the protozoan enrichments were
incubated aerobically or anaerobically in an anaerobic glovebox. The aerobic enrichments were
observed at 2 weeks, 1 month, and 2 months. The anaerobic enrichments were observed every 3
weeks for 3 months.
Physical Analysis of Samples
The pH was determined with U. S Environmental Protection Agency method 9045 (U. S.
Environmental Protection Agency, 1986). Texture analysis was conducted by Law Engineering,
Houston, TX. The initial samples were found to consist of at least 92% sand and the rest silt. The
interim samples have not been analyzed yet.
Samples were analyzed for JP-4 at the R.S. Kerr Environmental Research Laboratory, U. S.
Environmental Protection Agency, Ada, OK using the standard operating procedure, RSKSOP-72
(U.S. Environmental Protection Agency, 1991).
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Statistics
The data were compared using the t-test for equal or unequal variances, depending on the samples
(95% confidence). The samples were compared in different ways. The entire depth intervals of initial
and interim samples within the treatment cell (all cores except for JB, JC, KB, KC) were compared to
determine the overall effect of remediation. Proximate cores that could be paired by depth and
compared statistically were 80EB and 80X, 80BA and 80Z, 80JB and 80JC, and 80KB and 80KC.
RESULTS
Denitrification Potential
The average total denitrifier population in the treatment zone (all samples except the J and K cores)
increased from logio 5.8 to 6.9 during the 5 month period of nitrate-enhanced bioremediation
(Table 2). The number of total denitrifiers in the entire interval of the K (control) region did not
increase, suggesting that the treatment stimulated denitrification potential within the nitrate cell.
A comparison of the average number of denitrifiers in the entire depth interval of initial BA core with
the average number in interim Z core indicated that numbers were higher after treatment. When
individual samples within the initial BA core were paired by depth to those in interim core Z, counts
were also higher after treatment. The average number of total denitrifiers in the entire interval of
initial EB and interim X cores was not different. When individual samples within BA and Z, or EB
and X cores were paired by depth and compared, the data indicated that there was a significant
increase in total denitrifiers after treatment in all samples except for the lowest depth, X4, in which
there was a significant decline.
Comparison of the entire depth interval of initial JB and interim JC samples indicated numbers of
total denitrifiers were not different; however, when individual samples within cores were paired by
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depth and compared, stimulation was observed in the upper and lower depths but the middle depth was
not different.
Since the individual samples in cores KB and KC did not correspond directly with depth (Table 1),
the number of total denitrifiers in initial sample KJB6 were compared with the average of those in
interim samples KC2 and KC1 combined. Numbers of total denitrifiers did not increase at this depth
interval.
In contrast to an increase in the number of total denitrifiers, numbers of denitrifiers growing on JP-4
or JP-4 degradation products were lower after 5 months of treatment (Table 2). The MPN of many
samples incubated in the presence of JP-4 could not be calculated because they were below the
detection limit of the assay (logio 2 cell/g dry wt). The results of control samples (no added JP-4)
indicated that ambient carbon could be responsible for some of the denitrification that was detected in
some samples.
Aerobic Viable Counts
When the entire depth interval of every core in the treatment zone was considered, there was an overall
increase in hetrotrophic, JP-4-degrading, and oligotrophic microorganisms after treatment (Table 3).
Numbers of heterotrophs, JP-4 degraders and oligotrophs in the treatment zone increased from logio
5.8 to 6.9, 4.7 to 5.4, and 4.3 to 5.5, respectively; however, numbers of these organisms in the K cores
(control) increased from 5.6 to 5.8, 4.0 to 4.8, and 4.1 to 5.0, respectively, suggesting that some of the
growth resulted from other factors, such as seasonal influences. The K location is downgradient of the
water from the control cell which may have stimulated growth. There were no consistent trends when
pairs of samples were compared by depth.
Protozoa
Numbers of aerobic protozoa in the initial samples ranged from logio 6.2 in shallow samples from
the contaminated zone to less than detection limit (<10 cells/ g) for some KB (control) samples (data
not shown). Of interest is that anaerobic protozoa were detected in low to moderate numbers (not
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greater than 10000 cells/g). Data sets for the interim sampling were not available when this paper was
written.
CONCLUSIONS
Denitrification potential was stimulated in the treatment zone after 5 months of nitrate-enhanced
bioremediation, The number of total denitrifiers was higher in the interim than initial samples and
did not increase in the control core. The increase in the number of viable counts may have been the
result of treatment; however, other factors may have affected growth.
Numbers of viable microorganisms also increased in the control core, which could have been a
seasonal effect. In general, operation of the pilot system resulted in increased pH, nitrate, ammonia,
and orrthophosphate levels throughout the nitrate cell. Total Kjeldahl nitrogen and total phosphate
levels have generally decreased. This probably results from the combined effects of nitrate
assimilation and decomposition, denitrification, and leaching of minerals. This has also resulted in
slightly higher cell counts, as determined by phospholipid fatty acids (PFLA). Other parameters
(TOC, BTEXTMB, JP-4) are too variable in concentrations to generalize. In summary, these data
indicate that the microbial activity at the site has been increased as a result of the pilot operation.
REFERENCES
Hutchins, S.R. 1991. "Optimizing BTEX Biodegradation under Denitrifying
Conditions." Environ. Toxicol. Chem. 10:1437-1448.
Hutchins, S. R., W. C. Downs, J, T. Wilson, G. B. Smith, D. A. Kovaks, D. D. Fine, R. H. Douglass, and
D. J. Hendrix. 1991. "Effect of Nitrate Addition on Biorestoration of Fuel-Contaminated Aquifer:
Field Demonstration." Groundwater. 29:571-580.
Sinclair, J. L. and W. C. Ghiorse. 1987. "Distribution of protozoa in subsurface sediments of a pristine
groundwater study site in Oklahoma." Apol. Environ. Microbiol. 53:1157-1163.
U. S, Environmental Protection Agency. 1986. Testing Methods for Evaluating
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Solid Waste. Laboratory Manual. Office of-Solid Waste and Emergency Response,
U. S. Environmental Protection Agency, Washington, DC.
U. S. Environmental Protection Agency. 1991. Quantitative Analysis of
Aviation Gasoline and JP-4 Jet Fuel in Coarse and Medium Textured Soils by
Gas Chromatography. R. S. Kerr Environmental Research Laboratory, U. S.
Environmental Protection Agency, Ada, OK.
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LIST OF FIGURE AND TABLE CAPTIONS
FIGURE 1. Soil Core Locations- Eglin Air Force Base.
TABLE 1. Physical Characteristics of Initial and Interim Samples
TABLE 2. Denitrification Potential in Initial and Interim Samples
TABLE 3. , Aerobic Viable Counts in Initial and Interim Samples
TABLE 4. Aerobic Protozoa in Initial and Interim Samples
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KEYWORD LIST
Bioremediation
Nitrate
Hydrocarbon
Denitriliens
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FIGURE 1. Soil core locations at Eglin Air Force Base
/I
North
® Initial Soil Core Location
B Interim Soil Core Location
I I Tank
| # | Building (numbered)
50 (f.) 100
80K
80KC
80ZA
.80B
80:
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TABLE 1. Physical characteristics of initial and interim samples
Initial
Sample
Initial
Sample
Depth (ft)
PH
JP4 Cone,
mg/kg
Interim
Sample
Interim
Sample
Depth (ft)
PH
JP4 Cone,
mg/kg
80AA2
2.3-3.4 .
5.46
214
80ZA2
2.3-3.4
7.81
138.0
80AA1
3.4-4.5
5.49
1260
80ZA1
3.4-4.5
8.08
2630.0
80AA7
4.5-5.6
6.77
276
80ZA4
4.8-5.9
8.11
55.4
80BA3
1.0-2.2
4.88
2.8
80Z2
1.3-2.4
7.81
1.1
80BA2
2.2-3.4
6.23
355
80Z1
2.4-3.5
7.13
3750.0
80BA5
4.5-5.6
6.95
8.3
80Z4
4.9-6.0
8.32
34.1
80DA1
2.5-3.2
5.26
34.6
80W2
2.3-3.4
7.77
10.0
80DA5
4.0-5.0
5.77
377
80W1
3.4-4.5
7.76
1310.0
80DA8
6.0-6.8
6.82
54.7
80W4
4.8-5.9
7.75
4.1
80EB2
3.2-4.2
5.29
1160
80X2
2.5-3.8
8.75
4560.0
80EB1
4.2-5.2
5.46
1600
80X1
3.8-5.0
8.46
2620.0
80EB5
6.5-7.5
7.18
6.8
80X4
5.3-6.4
8.38
5780.0
80JB2
2.5-3.5
6.69
8.5
80JC2
2.3-3.4
6.87
11.1
80JB1
3.5-4.5
6.87
4.0
80JC1
3.4-4.5
6.90
1.7
80JB5
6.0-7.0
6.59
ND
80JC3
5.9-7.0
7.87
0.0
80KB2
3.2-4.4
5.07
6.4
80KC2
5.0-6.0
5.63
0.2
80KB1
4.4-5.5
5.80
3.3
80KC1
6.0-7.5
7.01
0.0
80KB 6
5.5-6.7
5.98
3.6
80KC4
7.8-8.9
7.55
0.3
ND means not detected
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TABLE 2. Denitrification potential in initial and interim samples
INITIAL
Sample
Log Denitrifiers
MPN/g dry wt (SD)
Total JP-4 No JP-4
INTERIM
Sample
; Log Denitrifiers
MPN/g dry wt (SD)
Total JP-4 No JP-4
80AA2
7.1 (0.4)
6.8 (0.2)
3.4(0)
80ZA2
6.8 (0.6)
2.0, 1.7, <2
2.7 (0.3)
80AA1
7.2 (0.6)
6.4 (0.1)
<1
80ZA1
6.5 (0.9)
2.0, <2, <2
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TABLE 3. Aerobic viable counts in initial and interim samples
INITIAL
Sample
Viable Counts (LOG io)
R2A JP4 no JP-4
INTERIM
Sample
Viable Counts (LOG 10)
R2A JP4 no JP-4
80AA2
80AA1
80AA7
6.73 (0.05)
6.84 (0.16)
4.68 (0.22)
6.57 (0.08)
5.48 (0.1)
2.43 (0.13)
6.59 (0.07)
5.45 (0.15)
80ZA2
80ZA1
80ZA4
6.97 (0.09)
6.97 (0.07)
6.84 (0.08)
4.88 (0.12)
5.00 (0.17)
4.95 (0.14)
4.99 (0.14)
5.04 (0.1)
5.72 (0.17)
80BA3
80BA2
80BA5
5.69 (0.05)
6.01 (0.08)
4.42 (0.08)
4.53 (0.14)
4.38 (0.18)
3.57 (0.08)
3.64 (0.09)
3.94 (0.08)
3.61 (0.13)
80Z2
80Z1
80Z4
6.12 (0.04)
7.59 (0.05)
7.11 (0.08)
5.12 (0.1)
6.61 (0.2)
6.05 (0.07)
5.47 (0.14)
6.99 (0.05)
6.09 (0.07)
80DA1
80DA5
80DA8
5.86 (0.04)
5.91 (0.03)
5.76 (0.07)
4.87 (0.13)
3.8 (0.12)
5.23 (0.08)
5.07 (0.1)
3.39 (0.1)
5.15 (0.1)
80W2
80W1
80W4
6.55 (0.07)
6.42 (0.04)
7.21 (0.13)
5.16 (0.26)
5.84 (0.17)
5.70 (0.04)
5.04 (0.05)
5.93 (0.1)
5.76 (0.13)
80EB2
80EB1
80EB5
6.80 (0.06)
4.55 (0.06)
5.61 (0.12)
5.65 (0.33)
4.16 (0.10)
5.65 (0.08)
6.18 (0.03)
3.78 (0.09)
5.8 (0.09)
80X2
80X1
80X4
7.57 (0.14)
7.61 (0.09)
6.17 (0.04)
6.93 (0.04)
5.19 (0.56)
3.46 (0.13)
6.79 (0.1)
4.67 (0.37)
3.46 (0.17)
80JB2
80JB1
80JB5
5.58 (0.07)
6.64 (0.06)
4.75 (0.08)
3.33 (0.09)
6.26 (0.08)
4.15 (0.04)
3.32 (0.11)
6.29 (0.05)
4.15 (0.04)
80JC2
80JC1
80JC3
6.92 (0.03)
6.90 (0.04)
5.54 (0.06)
6.00 (0.22)
5.28 (0.14)
4.17 (0.41)
5.99 (0.17)
5.71 (0.16)
4.55 (0.17)
80KB2
80KB1
80 KB 6
5.8 (0.1)
5.2 (0.02)
5.9 (0.2)
4.2 (0.1)
4.4 (0.03)
3.5 (0.1)
4.2 (0.1)
4.9 (0.1)
3.2 (0.1)
80KC2
80KC1
80KC4
5.6 (0.1)
6.0 (0.1)
5.8 (0.3)
5.3 (0.1)
4.8 (0.2)
4.1 (0.1)
5.1 (0.1)
5.5 (0.03)
4.0 (0.1)
(a) Samples were plated on low nutrient agar (R2A), and on a mineral nutrient agar with or without JP-4 vapors
(b) The detection limit for the assay is log 10 2
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TABLE 4. Aerobic protozoa in initial and interim samples
Initial Sample
Logio no.cells/g
dry wt
Interim Sample
Logio no.eells/g
dlry wt
8QAA2
80AA1
80AA7
4.4 (0.2)
3.0, <1, <1
2.9 (0.1)
80ZA2
80ZA1
80ZA4
5.7 (0.5)
2.7 (0.1) !
3 (0.1)
80BA3
80BA2
80BA5
6.2 (0.1)
5.8 (0.4)
2.9 (0.5)
80Z2
80Z1
80Z4
5.9 (0.2)
3.8 (0.2)
2.4 (0.1)
80DA1
80DA5
E0DA8
5.7 (0.4)
6.0 (0.3)
>6
80W2
80W1
80W4
2 (0)
<1, 3.7, 3.0
2.9 (0.1)
80EB2
80EB1
80EB5
2.3 (0.3)
2.7 (0.2)
3.5 (0.1)
80X2
80X1
80X4
3.9 (0.3)
2.7 (0)
2.7 (0)
80JB2
80JB1
80JB5
3.6 (0.7)
2.5 (0.2)
2.8 (0.4)
80JC2
80JC1
80JC3
>6.2
3.5 (0.2)
<1, 2.3, 2.3
80 KB 2
80KB1
80KB6
3.2 (0.2)
<1, <1, <1
2.5 (0.2)
80KC2
80KC1
80KC4
3.3 (0.2)
3.3 (0.1)
>6.2
(a) Detection limit for the assay, logio 1
(b) Replicate Subsamples were averaged unless a replicate was greater or less than the detection limit
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TECHNICAL REPORT DATA
(Please read Iinstructions on ihc reverse before comf
1. REPORT NO. 2.
EPA/600/A-95/109
4. TITLE ANO SUBTITLE
MICROBIAL ACTIVITY IN SUBSURFACE SAMPLES BEFORE
AND DURING NITRATE-ENHANCED BTO.REMEDIATI^I-1
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7* authorisi STEPHEN R. HUTCHIKS (1)
J. MICHELE THOMAS, ET AL., (2)
.TAMES T,. STNCT.ATR Ml
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME ANO AODRESS
US/EPA, RSKERL, ADA, OK (1)
RICE UNIVERSITY, HOUSTON TX (2)
MANTECH ENVIRONMENTAL TECH. INC.,ADA,OK (3)
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
IN-HOUSE RPSH1
12. SPONSORING AGENCY NAME AND ADORESS
U.S./EPA, NRMRL-ADA
SUBSURFACES PROTECTION & REMEDIATION DIVISION
P.O. BOX 1198
ADA, OK 74820
13. TYPE OF REPORT AND PERIOD COVERED
Proceedings
1C SPONSORING AGENCY CODE
F.P A / fiOO / 1 5
15. SUPPLEMENTARY NOTES
16. ABSTRACT
A study was conducted to determine the microbial activity of a site contaminated with JP-4 jet fuel,
before and during nitrate-enhanced bioremediation. Samples at three depths from six different
locations were collected ascptically under anaerobic conditions before and during LreatmenL Cores
were located in or close to the source of contamination, downgradient of the source, oi outside the
zone of contamination. Parameters for microbial characterization included 1) viable counts of aerobic
heterotrophic, JP-4 degrading, and oligotrophic bacteria, 2) the MPN of aerobic and anaerobic
protozoa, 3) the MPN of total denitrifiers, and 4) the MPN of denitrifiers in hydrocarbon-amended
microcosms. The results indicate that the total number of denitrifiers increased by an order ol
magnitude during nitrate-enhanced bioremediation in most samples. iThe number of total
helerotrophs and JP-4 degrading microorganisms growing aerobically also increased In addition, the
first anaerobic protozoa associated with hydrocarbon-contaminated subsurface materials were
detected.
17. key woros and OOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSati field, Croup
AQUIFER
GROUND WATER
ANAEROBIC
BIOREMEDIATION
MICROORGANISM
ECOLOGY
PETROLEUM
HYDROCARBON
DENITRFICATION
PROTOZOA
JET FUEL
AEROBIC
MPN
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19 SECURITY CLASS {Tins Repr.rn
UNCLASSIFIED
2i nc of pages
1 5
20 SECURITY CLASS (This pu?<•
IJNCLASSTFTF.n
77 PRICE
EPA Form 2220 — 1 (R«v. 4 — 77) previous f.oition is obsolete
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