OTC 21794

New Biocide Formulations for Oil and Gas Injection Waters with Improved
Environmental Footprint
Michael Enzien/The Dow Chemical Company, Bei Yin/The Dow Chemical Company

Copyright 2011, Offshore Technology Conference

This paper was prepared for presentation at the Offshore Technology Conference held in Houston, Texas, USA, 25 May 2011.
This paper was selected for presentation by an OTC program committee following review of information contained in an abstract submitted by the author(s). Contents of the paper have not been
reviewed by the Offshore Technology Conference and are subject to correction by the author(s). The material does not necessarily reflect any position of the Offshore Technology Conference, its
officers, or members. Electronic reproduction, distribution, or storage of any part of this paper without the written consent of the Offshore Technology Conference is prohibited. Permission to
reproduce in print is restricted to an abstract of not more than 300 words; illustrations may not be copied. The abstract must contain conspicuous acknowledgment of OTC copyright.

Abstract
Combinations of various biocides with Glutaraldehyde or THPS (tetrakis hydroxymethyl phosphonium sulfate), the
two most commonly used injection fluid biocides in the oil and gas industry, showed both improved performance and lower
environmental aquatic toxicity profiles. Treatment conditions included temperatures up to 80C, salinities as high as 15%,
and contact times for as long as 7 days. Combination tests were evaluated against both Sulfate Reducing Bacteria (SRBs) and
Acid Producing Bacteria (APBs). Many of the new formulations tested had a lower environmental aquatic toxicity profile
than either glutaraldehyde or THPS alone because of the favorable aquatic toxicity profiles of the complimentary
combination actives. Therefore, these new formulations not only improved or equaled performance of incumbent biocides but
did so with a better environmental footprint. The new biocide formulations were developed for treatment of injection waters
used in water flooding or hydraulic fracturing applications. In both applications, improved heat stability and thus longer
preservation were goals for new product development.

1. Introduction
Microbial control in the oil and gas industry is primarily practiced to prevent the detrimental effects of microbial
growth on production equipment, pipelines, and the reservoir. These effects are well known in the industry and include:
biofouling (Videla et al., 1995), microbiologically influenced corrosion (Pope, 1991; Videla, 1991), microbial souring
(McInerney and Sublette, 1997; McInerney et al., 1993), i.e. biogenic production of H2S; and reservoir permeability
reduction (Khazipov et al., 1993). The impact of the detrimental effects of poor microbial control results in lost production
time, poor quality crude oil and gas, and occasionally dramatic pipeline failures.
While it is well known that thermophilic bacteria exist and predominate under reservoir conditions (Leu et al., 1998;
Mueller and Nielsen, 1996; Nilsen et al., 1996; Pedersen, 2000; Sanchez et al., 1993), most microbial screening tests are still
performed on bacteria grown at temperatures well below reservoir temperatures (Johnson et al., 2008; Pope et al., 1990;
Ruseska et al., 1982). In addition, most screening tests rarely use contact times longer than 24 hours and if longer contact
times are used they do not incorporate re-challenge testing. It is important to provide microbial re-challenges in microbial
control screening tests conducted over extended time periods because normal reservoir conditions will contain endemic
microorganisms that persist throughout the formation, not just at the near well-bore areas. If re-challenge testing is not used,
extended contact times simply represent initial kill which will favor quick acting control products which generally do not
provide any extended performance attributes. Tests of this kind really are more of a disinfection study which is not a
practical endpoint for oil and gas operations.
Thermal degradation profiles of biocides are well known and many biocides used in the oil and gas industry degrade
very quickly under extreme temperatures of deep reservoirs. Many of the biocides that have fast thermal degradation profiles
also happen to be the biocides with the fastest mode of action. Likewise, biocides with slow thermal degradation profiles or
heat stabile biocides are relatively slow acting. Therefore, in order to meet the performance metrics of both quick-kill and
then long-term preservation from extended microbial burdens, the development of combination products was pursued.

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2. High Throughput Microbial Screening

2.1 Materials and Methods.
A patent pending (Yin 2009) method for rapidly screening antimicrobial treatments under anaerobic conditions was used
to identify promising treatments for microbial control under high temperature and high salinity conditions. This method
allows for rapid screening of a higher number of treatments and combinations and with more precision (Yin et al., 2007).
Table 1. Biocide products tested in this study
Chemical Names

Abbreviation

Glutaraldehyde
Tetrakis hydroxymethyl phosphonium sulphate

Glut
THPS

Tris (hydroxymethyl) nitromethane (Tris Nitro)

cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride
Dimethyl oxazolidine
Alkyl dimethyl ammonium chloride

TN
CTAC
DMO
ADBAC

Didecyl dimethyl ammonium chloride

DDAC

2.2 Biocidal Efficacy against SRB and APB.

Biocide treatment solutions and combinations (Table 1) were prepared in a defined salt solution (3.1 g of NaCl, 1.3
NaHCO3, 47.7 KCl, 72.0 CaCl2, 54.5 MgSO4, 172.3 Na2SO4, 43.9 Na2CO3 all mg, in 1 L deionized water) and then were heat
treated at 60 or 80C for 7 days, with daily challenges of SRB inoculums (~105 CFU/mL). Some studies had Na2S (10 ppm
as S2-) added to matrix for additional challenge testing. The concentration of NaCl was adjusted to achieve various salinties
from 0.3 to 15% as NaCl in the test matrices. The efficacy of these combinations was evaluated at different time intervals and
was determined by the minimum tested biocide dosage required to achieve bacterial control for both short time (2 hours) and
extended time (7 days) periods. Enumerations were performed using a triplicate serial dilution method using a Modified
Starkeys medium (3.5 g of sodium lactate, 1.0 g of NH4Cl, 0.5 g of K2HPO4, 2.0 g of MgSO47H2O, 0.5 g of Na2SO4, 0.1 g
of CaCl22H2O, 0.5 g of yeast extract, 0.1 g of sodium thioglycolate, 0.1g of (NH4)2SO4FeSO46H2O, in 1 L deionized water)
for Sulfate Reducing Bacteria (SRB) and modified Phenol Red Dextrose media (BD, Phenol Red Dextrose Broth with 15%
NaCl) for halophilic Acid Producing Bacteria (APB). All tests were conducted under anaerobic conditions using a Bactron
3000 anaerobic chamber, Sheldon Laboratories.

3. Results and Discussion

3.1 Biocidal Efficacy at High Temperatures
Several studies were conducted to identify biocide combinations that could provide both quick kill and longer persistent
microbial control under higher reservoir temperature conditions. Data are presented as concentrations of total biocide actives
required to achieve complete kill for both the short term and 7 day time points. In addition, the following charts plot adjusted
use concentrations of biocide actives that have been normalized based on the aquatic toxicity values for the combined
biocides. Table 2 lists the aquatic toxicity values used to make these normalizations. While there are many types of aquatic
toxicity tests, the two listed in this table are two of the most commonly used when evaluating toxicity for marine pollutants.
Concentrations of biocide combinations were normalized as follows:
ND = BD / bD
NS = BS / bS
N P = N D * NS
where,
BD = EC50 tox value against Daphnia magna for the benchmark biocide in the combinations being evaluated, either
glutaraldehyde or THPS.
bD = EC50 tox value against Daphnia magna for the secondary biocide in the combinations being evaluated.
BS = EC50 tox value against Selenastrum capricornutum for the benchmark biocide in the combinations being evaluated,
either glutaraldehyde or THPS.
bS = EC50 tox value against Selenastrum capricornutum for the secondary biocide in the combinations being evaluated.
ND = normalization factor of biocide aquatic toxicity for Daphnia magna
NS = normalization factor of biocide aquatic toxicity for Selenastrum capricornutum
NP = product of correction factors for both Daphnia magna and Selenastrum capricornutum

OTC 21794

Table 2. Aquatic Toxicity Values and Normalized Calculations

Aquatic
Invertebrate
Acute Toxicity

Aquatic Plant
Toxicity

THPS Normalized

Glut Normalized

48

EC50 S.
capricornutum
2
(mg/L)
4.2

CTAC (Dowicil)

18.2

0.82

glutaraldehyde

0.81

Tris Nitro

50

0.65

0.30

0.31

0.092

0.10

1.25

THPS

15

0.2

1.00

1.00

1.000

0.33

4.05

1.35

DDAC

0.377

0.13

13.26

6.23

82.64

ADBAC

0.52

0.09

9.62

9.00

86.54

EC50 D. magna
1
(mg/L)

Biocide
DMO (oxazolidine)

ND

NS

NP

ND

NS

NP

0.31

0.05

0.015

0.10

0.19

0.02

0.82

0.24

0.200
1.00

1.00

1.00
0.12

EC50, water flea Daphnia magna, Effective concentration of immobilization (mg/L) of 50% of test population.
2
EC50, green alga Selenastrum capricornutum, Effective concentration for growth inhibition (cell density reduction) within 96 h for
50% of test samples.
3
This value is actually for a similar quaternary ammonium compound, hexadecyltrimethyl-ammonium bromide (CTAB) rather than
ADBAC as no value for this algae could be found for ADBAC. CTAB is very similar in hydrophobicity and efficacy to ADBACs.

Figure 1 shows the efficacy of several THPS combinations versus treating with THPS alone. In these tests, the matrix was
similar to seawater in chemical composition and the solutions were heated to 80C for 7 days. Values plotted represent the
total actives concentration required to achieve a complete kill of the bacteria inoculum at both 2 hr of contact as well as 7
days following repeated re-challenges with microbial inocula. This metric therefore tests not only quick-kill of bacteria but
also the persistence of microbial control under high temperature conditions up to 7 days. As can be seen in Figure 1, THPS
alone performed as well as any of the THPS combinations based on total actives used to achieve desired endpoint. THPS is
considered one of the top performing biocides in oil and gas applications for microbial control and even though it is promoted
as a green biocide, it has come under increasing environmental regulatory scrutiny as it relates to offshore uses. While
combination treatments of THPS with TN and DMO did not lower the total actives required, these combinations did lower
the aquatic toxicity adjusted concentrations by as much as 65% (Figure 1). Aquatic toxicity adjusted values were calculated
by mulitplying the NP values for the respective biocide concentrations in the combinations. This calculation shows how
combination treatments can effectively lower the environmental footprint of a microbial control program.
In Figure 2, a similar effect is shown for combinations of THPS and CTAC with an even greater improvement of the
environmental footprint with these combination treatments. THPS and CTAC combinations have the added benefit of

New THPS Combos: 4% salt, 80C, 2hr and 7 day SRB kill

Total Actives

Aquatic Tox Adjusted

45
40
Total Actives (ppm)

35
30
25
20
15
10
5
0
2:1
THPS

1:2
THPS:TN
Biocide

1:2
THPS:DMO

Figure 1. THPS combination treatments. Biocide ratios in X-axis are based on active levels. Aquatic Tox
adjusted values are described in text. Treatment conditions include 4% NaCl matrix, exposure
temperatures of 80C and contact times up to 7 days. Endpoints for effective control include complete
kill of inoculated SRB culture at both 2 hrs and 7 days.

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New THPS Combos: Seawater, 80C, SRB kill, Na2S Challenge

90

Total Actives

Aquatic Tox adjusted

Total Actives (ppm)

80
70
60
50
40
30
20
10
0
2:1
THPS

1:1

1:2

1:4

THPS:CTAC
Biocide

Figure 2. THPS plus CTAC combination treatments. Biocide ratios in X-axis are based on active levels.
Aquatic Tox adjusted values are described in text. Treatment conditions include 4% NaCl matrix,
exposure temperatures of 80C and contact times up to 7 days. In these tests and additional challenge of
2Na2S (10 ppm as S ) was added to the test matrix each day. Endpoints for effective control include
complete kill of inoculated SRB culture at both 2 hrs and 7 days.

showing synergy based on the efficacy of the combined actives. Therefore, when the aquatic Tox normalizations are
calculated the adusted concentrations are lowered by as much as 90% compared to using THPS alone. THPS and CTAC
combinations where also subjected to a more stringent challenge and were spiked with Na2S (10 ppm as S2-) each day of the
exposure period. The improved performance of these combinations under these stringent test conditions provides an excellent
screening for new microbial control treatments that meet the challenging environments of deep and sour reservoirs.
Glutaraldehyde is another commonly used biocide in the oil and gas industry for microbial control. Several combinations
of Glut plus other biocides were shown to improve efficacy over the Glut/ADBAC product used as the benchmark for our
glutaraldehyde tests (Figure 3). The best combination based on actives was Glut plus TN combinations. Similar to the THPS
combination experiments, these tests were conducted in a seawater matrix heated to 80C for 7 days. Glutaraldehyde combos
seem to show greater enhancement in performance or synergies than the THPS combos. Glutaraldehyde is not as heat stabile
as THPS therefore it will take a larger initial concentration of glutaraldehyde to provide the 7 day complete kill endpoint.
When glutaraldehyde is combined with a more heat stabile biocide, such as TN, an improved synergistic treatment is
achieved. Also interesting to note in these experiments, was the improvement of Glut/DDAC over the benchmark
Glut/ADBAC product (Figure 3). It is difficult to determine from this data if this effect is a result of the different quaternary
ammonium compounds (Quats) used in the formulations or the ratios of Glut:Quats. Quats are known to have very good heat
stability so a formulation with more Quats would certainly expect to have better heat stability also.
Several intersting observations can be made of the Aquatic Tox adjusted concentrations of the glutaraldehyde
combinations. First, the combinations with quaternary ammonium compounds, including the benchmark product, have higher
adjusted values because of the high aquatic toxicity for Quats (Table 2). Second, the best performing glut combo based on
total actives is not the same as the best based on the Aquatic Tox adjusted values (Figure 3). The Glut plus DMO combos
have the lowest environmental footprint with respect to aquatic toxicity compared with the Glut plus TN combinations. This
is mainly due to the low aquatic toxicity values for DMO which adjusts the concentration downward. While these adjusted
values are theoretical in nature, they do show how biocide combinations can lower the environmental impact without losing
the target performance requirements.

OTC 21794

New Glut Combos: 4% salt, 80C, 2hr and 7 day SRB kill
200

Total Actives

Aquatic Tox adjusted

180

Total Actives (ppm)

160
140
120
100
80
60
40
20
0
5.6:1

4:1

Glut:ADBAC

Glut:DDAC

2:1

1:1
Glut:TN

1:2

1:4
Glut:DMO

1:5
Glut:THPS

Biocide

Figure 3. Glutaraldehyde combination treatments. Biocide ratios in X-axis are based on active levels. Aquatic
Tox adjusted values are described in text. Treatment conditions include 4% NaCl matrix, exposure
temperatures of 80C and contact times up to 7 days. Endpoints for effective control include complete kill of
inoculated SRB culture at both 2 hrs and 7 days.

3.2 Biocidal Efficacy at High Salinities

While initial injection water chemistry for oil and gas recovery may consist of salinities at or below seawater, once these
waters come in contact with reservoir rock minerals and formation water, they become extremely saline. Injection water
salinities can easily reach levels as high as 15-25%. Few biocide efficacy studies are conducted at very high salinities,
because it is often difficult to isolate and grow bacteria under these conditions. Figure 4 shows results of high salinity
microbial efficacy studies using THPS and combinations with TN. A mixed culture of a halophilic Acid Producing Bacteria
(APB) was used in these studies. The microorganisms in this culture actually require greater than 10% salinity for their
growth. Studies were conducted at 15% salinities, 35C, and under anaerobic conditions. The results show that combinations
of THPS plus TN improve performance compared to THPS alone under high salinity conditions (Figure 4). These results
have important ramifications for treating reservoirs and their corresponding produced waters containing high salinities. Again
another advantage with the THPS/TN combinations is the lowered environmental footprint that can be achieved by
combining two actives (Figure 4).

4. Conclusions
Using more rigorous screening methods which include both high temperature exposure (80C) of test matrix
solutions, and extended contact times up to 7 days, several unique patent pending biocide combinations were identified that
meet these new more stringent microbial control metrics. These combinations include several biocides commonly used in the
oil and gas industry such as tetrakis hydroxymethyl phosphonium sulfate (THPS) and glutaraldehyde in combination with
complementary actives. These combinations were not only able to provide long-term efficacy under repeated microbial
challenges but also met the standard 2 and 24 hour contact time metrics at equal to or lower total actives than the standard
biocides used alone. Two other stretch performance metrics were also screened: 1) presence of 10 ppm sulfide in the test
matrix and 2) high salinity matrices (15% NaCl). In addition, the development of combination treatments also lowered the
apparent environmental footprint compared with standard actives by as much as 90%. These analyses were made by using
a calculation based on aquatic toxicity values of individual actives to normalize total actives.
These results show that improved microbial control performance metrics needed to increase oil and gas production from
the challenging deep reservoirs can be achieved more effectively when combination treatments are practiced and the resulting
combinations provide improved environmental footprints of chemicals.

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New THPS Combos: High Salt 15%, APB kill

Total Actives

Aquatic Tox adjusted

70

Total Actives (ppm)

60
50
40
30
20
10
0
2:1
THPS

1:2
THPS:TN

Biocides

Figure 4. THPS plus TN combination treatments. Biocide ratios in X-axis are based on active levels. Aquatic
Tox adjusted values are described in text. Treatment conditions include 15% NaCl matrix, exposure
temperatures of 35C and contact times up to 7 days. Endpoints for effective control include 3 Log10 kill of
inoculated halophilic APB culture at both 2 hrs and 7 days

5. References
JOHNSON, K., FRENCH, K., FICHTER, J. K., and ODEN, R., "USE OF MICROBIOCIDES IN BARNETT SHALE GAS WELL
FRACTURING FLUIDS TO CONTROL BACTERIA RELATED PROBLEMS," presented at the CORROSION 2008, New Orleans
LA, 2008.
KHAZIPOV, R. K., SILISHCHEV, N. N., KRITSKII, I. R., ILYUKOV, V. A., KAMALOV, M. M., and DAVYDOV, S. P.,
"Improvement of petroleum production in the Urshak field by biocides," Neft. Khoz., pp. 37-9, 1993.
LEU, J.-Y., MCGOVERN-TRAA, C. P., PORTER, A. J. R., HARRIS, W. J., and HAMILTON, W. A., "Identification and Phylogenetic
Analysis of Thermophilic Sulfate-Reducing Bacteria in Oil Field Samples by 16S rDNA Gene Cloning and Sequencing," Anaerobe,
vol. 4, pp. 165-174, 1998.
MCINERNEY, M. J. and SUBLETTE, K. L., "Petroleum microbiology: Biofouling, souring, and improved oil recovery," Man. Environ.
Microbiol., pp. 600-607, 1997.
MCINERNEY, M. J., SUBLETTE, K. L., BHUPATHIRAJU, V. K., COATES, J. D., KNAPP, R. M., and EUGENE, T. P. A. A. W.,
"Causes and Control of Microbially Induced Souring," in Developments in Petroleum Science. vol. Volume 39, ed: Elsevier, 1993, pp.
363-371.
MUELLER, R. F. and NIELSEN, P. H., "Characterization of Thermophilic Consortia from Two Souring Oil Reservoirs," Appl. Envir.
Microbiol., vol. 62, pp. 3083-3087, September 1, 1996 1996.
NILSEN, R. K., BEEDER, J., THORSTENSON, T., and TORSVIK, T., "Distribution of Thermophilic Marine Sulfate Reducers in North
Sea Oil Field Waters and Oil Reservoirs," Appl. Environ. Microbiol., vol. 62, pp. 1793-1798, May 1, 1996 1996.
PEDERSEN, K., "Exploration of deep intraterrestrial microbial life: current perspectives," FEMS Microbiology Letters, vol. 185, pp. 9-16,
2000.
POPE, D. H., "Mechanisms of microbiologically influenced corrosion of carbon steels," Gas, Oil, Coal, Environ. Biotechnol. 3, [Pap.
IGT's Int. Symp.], 3rd, pp. 499-509, 1991.
POPE, D. H., ZINTEL, T. P., ALDRICH, H., and DUQUETTE, D., "Efficacy of biocides and corrosion inhibitors in the control of
microbiologically influenced corrosion," Mater. Perform., vol. 29, pp. 49-55, 1990.
RUSESKA, I., ROBBINS, J., COSTERTON, J. W., and LASHEN, E. S., "Biocide testing against corrosion-causing oil-field bacteria helps
control plugging," Oil & Gas Journal, pp. 253-264, 1982.
SANCHEZ, G., MARIN, A., VIERMA, L., and EUGENE, T. P. A. A. W., "Isolation of Thermophilic Bacteria from a Venezuelan Oil
Field," in Developments in Petroleum Science. vol. Volume 39, ed: Elsevier, 1993, pp. 383-389.
VIDELA, H. A., "Microbially induced corrosion: an updated overview," Biodeterior. Biodegrad. 8, [Proc. Int. Biodeterior. Biodegrad.
Symp.], 8th, pp. 63-88, 1991.

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VIDELA, H. A., GUIAMET, P. S., FREITAS, M. M. S., and CANALES, C. G., "Assessing biocorrosion in oil recovery systems by means
of updated monitoring techniques," Biodeterior. Biodegrad. 9, Int. Biodeterior. Biodegrad. Symp., [Proc.], 9th, pp. 500-6, 1995.
YIN, B. A high throughput test method for evaluation of biocides against anaerobic microorganisms, Pat: WO2009039004A1, 2009.
YIN, B., YANG, J., BERTHEAS, U., ADAMS, J., High throughput evaluation of biocides for biofouling control in oilfield, In: Chemistry
in the Oil Industry X, Manchester, UK: Royal Society of Chemistry and the European Oilfield Speciality Chemicals Association,
2007.