Methylisothiazolone: A New Biocide Product for Closed Loop Systems

Terry M. Williams Rohm and Haas Company

727 Norristown Road, PO Box 904 Spring House, PA 19477-0904

ABSTRACT Closed loop cooling systems provide a unique set of environmental and microbiological conditions for biocides to control microbial growth and fouling. Key factors for biocide selection include in-use stability, solvent content, and materials compatibility. A new biocide product based on the methylisothiazolone active ingredient was recently developed for use in closed loop cooling systems. The new biocide is water-based, effective versus a range of bacteria, stable in high pH and high temperature conditions, and contains no salts, metals, or organic solvents. Results of comparative efficacy and stability studies versus commercial biocides will be presented. Keywords: Isothiazolone, biocide, methylisothiazolone, closed loop, cooling

INTRODUCTION The proper use of industrial biocides is critical to a successful water treatment program to reduce microbial populations on critical surfaces as well as reducing the total microorganism level introduced into the bulk water from external sources. Various biocide technologies have been used successfully in water treatment applications for many years. These include oxidizers, such as chlorine and bromine products, and non-oxidizing biocides, including isothiazolones, quats, organobromines, and glutaraldehyde. The overall efficacy of any given biocide is a function of it’s general spectrum of activity, mechanism of action, stability under environmental conditions, and compatibility with systems components and additives. Closed loop water treatment systems present unique challenges for industrial biocides relative to the open recirculating systems.1,2,3,4,5,6,7 Closed systems may see greater temperature extremes, are more difficult to sample and dose biocides, and often suffer from dead legs in the piping system compared to open recirculating systems. In addition, oxidizers are typically not used due to corrosion issues and the

1

persistence of non-oxidizing biocides in these systems can significantly affect the microbial control program, especially in high pH or high temperature systems.1,3

P

Isothiazolone biocides are widely used in a variety of industrial water treatment applications for control of microbial growth and biofouling.8,9 The most frequently used product is a 3:1 ratio of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-methyl-4-isothiazolin-3-one (MIT). CMIT/MIT has broad spectrum efficacy versus bacteria, algae, and fungi. DCOIT (4,5-dichloro-n-octyl-isothiazolin-3-one) was introduced several years ago for use as an algicide treatment for open recirculating cooling water systems. The most recent isothiazolone biocide developed for industrial water treatment is based on the MIT active ingredient alone and is targeted at long-term preservative applications with higher pH and temperature ranges, such as closed loop systems.10 This report describes a series of chemical stability and antimicrobial efficacy studies to demonstrate the potential of MIT alone as a new biocide product for use in closed loop systems. The studies include chemical stability (persistence) in high pH and high temperature waters (relative to other commercial biocides) and microbiological studies to characterize the mechanism of action and efficacy of MIT as a biocide.

MATERIALS AND METHODS Biocides Evaluated The chemical structures of the isothiazolone biocides described in this paper are shown in Figure 1. CMIT/MIT is composed of a 3:1 ratio of CMIT:MIT at a final concentration of 1.5% total active ingredient.( )1 MIT biocide is a 9.5% active solution in water.( )2 1,2-Benzisothiazolin-3-one (BIT) was included in certain studies for comparison. All biocide concentrations in this paper are reported on an active ingredient basis. The isothiazolone biocides described in this paper are manufactured by Rohm and Haas Company, Philadelphia, PA. Solutions of CMIT and MIT were prepared in distilled water and kept frozen until use. All other chemicals used were reagent grade or the highest available purity, and most were obtained from Sigma Chemical Company, St. Louis, MO, unless stated otherwise. Temperature and pH Stability Studies Biocides were added to various buffer solutions and stability was tested at 22° and 50° C over time. Biocide concentrations tested were 100 ppm MIT, 200 ppm BIT, 44 ppm CMIT. For the elevated temperature studies (70° and 90°°C), biocide was added to a pH 9 buffer solution and stored at in a heat block. The samples were examined for active ingredient concentration at various time points. Initial biocide doses were 166 ppm MIT, 199 ppm BIT, 150 ppm BNPD (bromonitropropanediol), and 1,000 ppm glutaraldehyde.

( ) 1 Kathon™ WT 1.5% Microbicide ( )2 Kordek™ MLX Microbicide

2

The effect of various redox agents on biocide stability was investigated. Oxidizing agents (2 mM) included hydroperoxide (H2O2; 68 ppm), t-butyl hydrogen peroxide (t-BHP; 180 ppm), and potassium persulfate (K2O8S2; 540 ppm). Reducing agents (2 mM) included iso-ascorbic acid (IAA; 352 ppm) and sodium bisulfite (NaHSO3; 208 ppm). Biocides were tested at 1 mM concentrations, representing 115 ppm MIT and 166 ppm BIT in solutions in pH 7.0 and pH 9.0 buffers. The samples were stored at 25° C and analyzed for biocide level by HPLC at 0, 2 and 8 days. The isothiazolone and competitive biocide samples were evaluated for active ingredient concentration via High Pressure Liquid Chromatography (HPLC). Samples were taken from the heating blocks and allowed to cool to room temperature for 30 minutes. The samples were then diluted in deionized water and analyzed by HPLC. Glutaraldehyde levels were estimated using a commercial formaldehyde test kit (EMD Chemicals Inc., Gibbstown , NJ), calibrated to various glutaraldehyde standards.3

SN

CH3Cl

O

5-chloro-2-methyl-4-isothiazolin-3-one CMIT

SN

CH3

O

2-methyl-4-isothiazolin-3-one MIT

1,2-benzisothiazolin-3-one BIT

S

N H

O

Figure 1. Chemical structure of isothiazolone biocides.

Organisms and Culture Conditions

Pure cultures were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Saccharomyces cerevisiae ATCC 4921 was grown in Sabouraud dextrose broth (SDB) at 30° C. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 9027 were grown in M9G minimal salts media with glucose at 37°C (E. coli) or 30°C (P. aeruginosa). 8

� EM Quant™ Formaldehyde Test Kit.

3

Transport and Binding of Isothiazolone Biocides with S. cerevisiae Cells Cell binding studies were conducted to determine the association of the isothiazolone biocide with microbial cells. Chemostat-grown cells of S. cerevisiae were used in these studies and cells were treated with radiolabeled biocide to determine the uptake of the biocide into the cells. Fungal cultures were grown in New Brunswick Scientific BioFlow chemostats at 30° C and an agitation rate of 100 rpm. S. cerevisiae was grown at a generation rate of 5.6 hours. Cells were harvested, washed, and resuspended to an absorbance (660 nm) of 0.8 in 10 mM PO4 buffer (pH 7.2). Samples were frozen and protein content was determined. Protein and optical density (OD at 650 nm) were measured as previously described.9 Determination of Growth Inhibition and Susceptibility

The lowest concentration of biocide required to inhibit growth was determined by a Minimum Inhibitory Concentration (MIC) test. Varying amounts of the test compound were added to media in a 96-well microtiter plate. Ten-fold serial dilutions were performed on a Biomek 2000 Workstation to obtain a range of closely spaced concentrations. A cell suspension, adjusted to provide 106 colony forming units(cfu)/ml in each well, was added to the microtiter plate. Microtiter plates were incubated at 30° C for 48 hours and then were checked for the presence or absence of growth in each well. The concentration of compound in the first microtiter well demonstrating no growth was the MIC for the test compound. The response of P. aeruginosa (ATCC 9027) to MIT inhibition, over the course of the growth cycle, was determined by following increases in absorbance (turbidity) of the culture. Flasks containing M9GY medium were inoculated with 2 ml of an overnight culture (final concentration 106 cfu/ml).8 Cultures were incubated at 30° C with shaking at 200 rpm. Bacterial growth was monitored indirectly by determining the culture absorbance on a Thermomax microplate reader at 660 nm. Speed of Kill Studies versus Bacteria Time-kill studies versus bacteria were conducted versus an active culture of Pseudomonas aeruginosa (ATCC 9027). Flasks containing M9GY medium were inoculated with an overnight culture (final concentration 106-107 cfu/ml). Cultures were incubated at 30° C with shaking at 200 rpm. MIT was added at several concentrations and viable cell counts determined over time using the MPN methods in TSB medium supplemented with 500 ppm sodium thioglycolate to inactivate the biocide. Viable counts were determined after 48 hours incubation at 30°C.8 Effect on Microbial Respiration (Oxygen Uptake). Inhibition of respiration by E. coli was measured for MIT on overnight cell culture. The assay was performed aliquots placed in a water jacketed steel chamber equipped with a Clark a polarographic oxygen electrode (Hansatech LTD, Norfolk, UK). Substrate was added to the sealed chamber to a final concentration of 10 mM, and biocide was added after a steady rate of oxygen consumption was reached (generally within 2 min). All biocide evaluations included an untreated control (no biocide) for comparison.

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Inhibition of Pyruvate Dehydrogenase Biocide activity versus pyruvate dehydrogenase enzyme assay was measured by following the reduction of NAD (nicotinamide adenine dinucleotide) to NADH (reduced nicotinamide adenine dinucleotide) and monitoring absorbance of the reaction mixture at 340 nm. The reaction mixture was initiated by the addition of the enzyme. Additional enzymes were evaluated for inhibition by MIT.

RESULTS AND DISCUSSION Comparative Characteristics of New Methlylisothiazolone Biocide Methylisothiazolone (MIT) biocide is currently formulated at a 9.5% active ingredient concentration in water. No additional salts, solvents, organics, stabilizers, or metals are added. The formulation does not contain any volatile organic compounds (VOC) and is soluble in both water and organic solutions. MIT biocide is a broad spectrum bactericide, with excellent chemical and physical stability under aggressive end use conditions (pH 4-10; 5-60° C). It has excellent compatibility with other water treatment biocides and scale and corrosion chemicals. MIT does not contain or release formaldehyde, biodegrades to non-toxic degradation products, and does not accumulate in the environment.11 Details of the chemical stability are illustrated in the following examples. Biocide Stability in Buffered Water under Extreme pH and Temperatures. Stability studies were conducted versus three isothiazolones and glutaraldehyde in buffered deionized water at two different temperatures (22° and 50° C). BIT was added as a reference biocide, since it is recognized as being very stable under aggressive conditions. MIT was shown to be the most stable of the isothiazolones, followed by BIT, with CMIT the least chemically stable under the aggressive conditions (Tables 1 and 2). MIT showed excellent stability at room temperature up to pH 12. At 50° C, MIT stability decreased slightly, but only at pH 12. BIT showed excellent stability at room temperature and 50° C up to pH 10. Slight degradation occurred at pH 12 at both temperatures. CMIT showed rapid and complete degradation at pH 10-12 at room temperature (22° C) and poor stability at pH 8-12 at the higher temperature. Glutaraldehyde showed poor stability at pH 9 and 25° C with a 50% loss of active (from 1,000 ppm to 500 ppm) after only 3 days. At 50° C, only 10% of the glutaradehyde remained after 3 days. Biocide Stability in pH 9 Water at 70° and 90°C. Studies were conducted to determine the stability of MIT, BIT, glutaraldehyde, and BNPD (bromonitropropanediol) under high pH and high temperature conditions (Figures 2 and 3). MIT demonstrated excellent stability in the pH 9 buffer at 70º after 56 days storage with no loss of MIT and showed only a 35% loss after 35 days at 90° C. BIT showed decreasing stability with 32% loss of active ingredient at day 56 at 70° C and 50% loss at 90° C. BNPD stability was poor with almost immediate total degradation observed after 5 hours at both 70° and 90° C. Glutaraldehyde showed complete degradation in the pH 9 solution when analyzed after 3 days at both elevated temperatures.

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Table 1. Biocide stability at 22° C in buffered water at various pH ‘s.

Percent Remaining at:

Biocide

Day

pH 2 pH 6 pH 8 pH 10 pH 12 0 100 100 100 100 100 21 100 100 100 0 0 43 100 100 91 0 0

CMIT

88 100 100 95 0 0 0 100 100 100 100 100 21 97 100 98 100 69 43 83 86 77 85 55

BIT

88 95 100 84 96 63 0 100 100 100 100 100 21 104 100 100 100 98 43 98 94 95 89 93

MIT

88 100 100 100 100 100

* Glutaraldehyde decreased from 1,000 ppm to 500 ppm at pH 9 and 25°C after 3 days Table 2. Biocide stability at 50° C in buffered water at various pH ‘s..

Percent Remaining at:

Biocide

Day

pH 2 pH 6 pH 8 pH 10 pH 12 0 100 100 100 100 100 21 100 100 15 0 0 43 93 65 0 0 0

CMIT

88 100 30 0 0 0 0 100 100 100 100 100 21 95 100 94 100 68 43 80 88 73 81 55

BIT

90 93 100 89 95 68 0 100 100 100 100 100 21 100 98 100 98 90 43 97 93 95 93 78

MIT

90 100 100 100 100 76

* Glutaraldehyde decreased from 1,000 ppm to 100 ppm at pH 9 and 50°C after 3 days

6

0

20

40

60

80

100

0 7 14 21 28 35 42 49 56Days

% B

ioci

de R

emai

ning

MIT

BIT

BNPD

Glut

Figure 2. Biocide stability at 70° C in buffered water at pH 9.

0

20

40

60

80

100

0 7 14 21 28 35Days

% B

ioci

de R

emai

ning

MIT

BIT

BNPD

Glut

Figure 3. Biocide stability at 90° C in buffered water at pH 9.

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Biocide Stability with Oxidizing and Reducing Agents at pH 7 and pH 9. The stability of MIT and BIT biocides was evaluated in the presence of various redox agents in pH 7.0 and 9.0 buffer after 2 and 8 days (Table 3). BIT was included for comparison due to it’s enhanced stability with aggressive agents. The results showed greater stability of MIT versus BIT under various redox conditions and at elevated pH. MIT was more stable than BIT for all of the oxidants tested and only a minimal pH effect was seen at 8 days with hydrogen peroxide. Very little, if any, BIT remained after 8 days with all oxidants at pH 9. BIT also showed more degradation at higher pH versus pH 7, especially with hydrogen peroxide. MIT showed better stability versus BIT against the ascorbic acid reducing agent and was not affected by pH. Both biocides showed slight degradation with sodium bisulfite at pH 9, but were significantly degraded at pH 7.0 within 2 days. Bisulfite is a known deactivating agent for isothiazolones. Table 3. Comparative stability of MIT and BIT with redox agents

% Remaining after 2 Days

% Remaining after 8 Days

MIT BIT MIT BIT Redox Agent pH 7 pH 9 pH 7 pH 9 pH 7 pH 9 pH 7 pH 9

None

100 100 100 100 100 100 100 100

Hydrogen peroxide (68 ppm)

100 94 39 8 100 88 15 0

t-butyl hydro-peroxide (180 ppm)

99 98 43 38 102 96 8 6

Potassium persulfate (540 ppm)

90 93 0 0 75 73 0 0

Isoascorbic acid (352 ppm)

100 105 89 77 96 103 86 75

Sodium bisulfite (208 ppm)

29 70 38 82 26 78 28 77

* redox agents added at 2mM * biocides added at 1mM (115 ppm MIT and 166 ppm BIT)

Comparative Features of Closed Loop Biocides The following biocides were compared for their chemical and physical properties for use in closed loop systems based on critical functional systems variable for these applications. Key parameters include the presence of salts or metals which may be corrosive, solvents which may serve as food sources for the bacteria or increase the nutrient loading for waste treatment, volatile or foaming materials which produce odors and may cause air pockets in dead legs of piping, environmental and health concerns for formaldehyde releasers, and poor long-term chemical stability which may lead to efficacy failure. The list of non-oxidizers is representative of commonly used biocides, but may not include all products. Oxidizers have been reported to be used in closed loop systems and are included for reference.6

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A comparison of the basic properties of biocides used in closed loop systems is provided in Table 4. Much of the information summarized below was extracted from a recent NACE publication.12 Certain groups of products are clustered together to provide a general characterization. Quats included the cationic alkyldimethylbenzylammonium chloride, tetrakishydroxymethylphosphonium sulfate, and the ionene polyquat. Carbamates included the ethylene bisdithiocarbamates and dimethyldithiocarbamates. Halogens included products which contain or release chlorine (hypochlorite) and bromine (hypobromite). The summary shows that only a few products are both stable at high temperatures and high pH (MIT, quats, and carbamates). Certain products may contain undesirable glycol solvents (BNPD and DBNPA; dibromonitrilopropionamide), salts (CMIT, quats, halogens), volatile organic compounds (glutaraldehyde), or strong odors (glutaraldehyde, carbamates, halogens). Other products may have foaming problems (quats) or compatibility issues with certain additives such as anionic or nitrogen based scale-corrosion inhibitors (quats, halogens). Only a few products (phosphonium quat and BNPD) are known to release formaldehyde in use. Overall, MIT has many desirable features for closed loop system compatibility and performance. MIT is stable in aggressive conditions, is compatible with additives, has no odor, and contains only active ingredient and water. Table 4. Comparative physical and chemical properties of closed loop biocides.

Feature

MIT

CMIT/ MIT

Glutar-

aldehyde

Dithio-carbam-

ates

DBNPA

BNPD

Quats

Halo-gens

pH 9-10 In Use Stability

+ - - + - - + -

>50° C In Use Stability

+ - - + - - + -

Formulated with Water Only

+ - + + - - + +/-

No VOC + + - + - - +/- +

No Salts + - + + + + - -

No Organic Solvents

+ + + + - - + +

No formaldehyde + + + + + - +/- +

Additive Compatibility

+ + + + + + +/- +/-

Non-Foaming + + + + + + +/- +

No/Low Odor + + - - + + + -

(+) = yes; (-) = no; (+/-) = variable feature within the group or condition

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Antimicrobial Efficacy of Methylisothiazolone (MIT) Biocide Minimum Inhibitory Concentration (MIC) Studies. The broad spectrum activity of MIT biocide against a variety of fungi and bacteria was demonstrated using standard MIC efficacy protocols. A summary of the MIC values for the various groups of organisms is shown in Table 5. MIT was shown to be a highly effective antibacterial active ingredient which inhibited growth of various Gram-negative and Gram-positive bacteria (mean MIC’s of 23 and 25 ppm respectively) and was less effective versus yeast and mold (mean MIC’s of 75 and 178 ppm, respectively). The bacteria employed in the testing included aerobes, facultative anaerobes, spore-formers, slime-forming bacteria, and sulfate reducing bacteria. It should be noted that even though these tests covered a wide range of organisms (4 separate groups), media (complex and defined), pH (5.0-8.0), temperature (24-35°C), and incubation periods (1-7 days), the antimicrobial efficacy of MIT was relatively similar and consistent.

Table 5. Minimum inhibitory concentration values for MIT versus microorganisms

MIC (ppm active) Organism And

Group

Number tested Minimum

Value Maximum

Value Mean +

Std. Dev.

Gram Negative Bacteria 56 4 82 23 +14 Gram Positive Bacteria 5 5 49 25 + 20 Yeast 6 40 125 75 + 31 Mold 13 13 500 178 + 159 All Organisms 80 4.3 500 75 + 73

The effect of pH on the MIC efficacy of MIT was evaluated at neutral and alkaline conditions (pH 7 versus pH 9) against BIT, a biocide recognized as very stable in aggressive conditions. The data in figure 4 demonstrates that MIT is equally active at pH 7 and pH 9 whereas, BIT showed reduced efficacy under alkaline conditions (pH 9) with a 2-fold increase in MIC values.

10

0

20

40

60

80

100

120

MITpH 7

MITpH 9

BITpH 7

BITpH 9

MIC

(ppm

act

ive)

Figure 4. Effect of pH on the MIC values for MIT and BIT versus bacteria.

Values represent the mean + std dev. Inhibition of Microbial Growth by MIT. The effect of MIT biocide on the growth of bacteria was evaluated versus P. aeruginosa ATCC 9027. Biocide was added to an actively growing culture and growth (cell density absorbance) was monitored for 8 hours. All biocide levels (25-150 ppm) immediately stopped the growth of the bacteria upon addition (within minutes) for a minimum of 2 hours (Figure 5). After 4 hours, the cultures treated with lower levels of MIT (25-50 ppm) began to show growth (absorbance increases), although the rate of growth was lower versus the control. The samples treated with higher level of MIT (100-150 ppm) displayed no increase in growth after biocide addition.

0.00

0.10

0.20

0.30

0.40

0 1 2 3 4 5 6 7Hours

Abs

orba

nce

(650

nm

)

8

Control 25 ppm MIT 50 ppm MIT100 ppm MIT150 ppm MIT

Biocide Added

Figure 5. Inhibition of bacterial growth by MIT versus P. aeruginosa (ATCC 9027) tested

in M9GY medium at 37° C.

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Speed-of-Kill Activity of MIT versus Bacteria A speed of kill study was conducted with MIT versus P. aeruginosa (ATCC 9027) in M9GY medium (Figure 6). Bacteria in the control samples increased from 107 cfu/ml to 109 cfu/ml over the 72 hour study. Low levels of MIT (25 ppm) reduced the initial level of viable bacteria approximately 90% (1-log) within 6 hours and counts remained constant (inhibited) over the 72 hour period. Treatment with 50 ppm MIT also provided a 1-log (90%) reduction in bacteria after 4 hours and the number of viable bacteria was reduced by two orders of magnitude (99%) at 72 hours. MIT at 100 and 150 ppm showed more rapid killing with greater than a 2- and 3-log kill, respectively, at 4 hours and complete reduction in viable counts within 48 to 72 hours. The results of this study show that MIT is slower killing than the more widely used CMIT/MIT product, despite being used at higher concentrations.8 However, MIT is orders of magnitude more stable under aggressive conditions, which makes it very desirable for long term efficacy in closed loop systems where speed of kill is less important compared to the persistence of the biocide.

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 12 24 36 48 60 7Hours

Viab

le C

ount

s (c

fu/m

l)

25 ppm

100 ppm

50 ppm

Control

150 ppm

2

1010

109

108

107

106

105

104

103

102

101

100

Figure 6. Bacterial kill study with MIT versus P. aeruginosa (ATCC 9027) in M9GY medium. Binding of 14C-Radiolabeled MIT with Microbial Cells Biocide incorporation studies were conducted to better understand the binding and association of MIT into microbial cells and key factors affecting the process. A dose response was observed with the substrate concentration and rate of association (incorporation) of MIT with yeast cells (Figure 7). A linear relationship was observed from 5 -1,000 μM MIT (0.58-120 ppm). The kinetic data were further analyzed using double reciprocal plots to quantify uptake kinetics. The analysis showed a high half-saturation constant (Km ) of 603 μM (70 ppm) and low rate of uptake (Vmax = 10μg/min/mg protein) relative to the other CMIT and DCOIT isothiazolones.9 Previous studies by other investigators have

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demonstrated a 4-5x greater extent of binding by CMIT versus BIT and MIT for both bacteria (E. coli) and fungi (Schizosaccharomyces pombe) and the majority of isothiazolone uptake was recovered within the cytosol (interior) of the cell, versus the outer membranes.13

0

1

2

3

4

5

6

7

0 200 400 600 800 1000

MIT (µM)

µg M

IT /

min

/ m

g pr

otei

n

Figure 7. Rate of association of MIT with S. cerevisiae cells as a function of MIT concentration. Inhibition of Microbial Respiration by MIT The effect of MIT on inhibition of bacterial respiration (oxygen uptake) was evaluated versus E. coli (Figure 8). MIT, tested at 25 and 100 ppm, provided rapid (within 24 minutes) and extensive (90%) reduction in oxygen consumption after biocide addition (at 2.25 hrs). The control sample continued to consume the remaining dissolved oxygen (6.1 mg/l) over the subsequent 4.25 hour period, whereas, the 25 and 100 ppm MIT treatments consumed only 0.55 and 0.65 mg/l of dissolved oxygen, respectively. Based on these studies, MIT is a potent inhibitor of microbial respiration. Previous studies showed that all isothiazolone biocides rapidly inhibit microbial respiration with CMIT more rapid than MIT.9

13

0

1

2

3

4

5

6

7

8

9

0 1 2 3 4 5 6Hours

Dis

solv

ed O

xyge

n (m

g/l)

Control 25 ppm MIT100 ppm MIT

Biocide Added

Figure 8 Inhibition of respiration (oxygen uptake) in E. coli by MIT at 25 and 100 ppm, in

M9GY, compared to control (no biocide) samples. The effect of shutting down microbial respiration in microorganism by isothiazolones means that oxygen uptake is disrupted, thereby affecting the cell’s ability to produce energy for growth and metabolism and efficiently remove waste products. Thus metabolic processes in aerobic and facultative anaerobic organisms are critically disabled and cannot function correctly, including the regulation of enzymatic biosynthetic and catabolic pathways. Inhibition of Enzyme Activity and Thiol Interactions by MIT. A detailed analysis of the inhibitory effects of MIT on enzyme activity was undertaken to determine the degree of specificity and reactivity with this class of biocides. An initial screening was completed with a wide range of catabolic (energy generating) and anabolic (growth related) enzymes for both in vitro and in vivo studies. MIT and the CMIT/MIT combination product react with only a select group of dehydrogenases enzymes. MIT inhibited alcohol and pyruvate dehydrogenases in vitro, and NADH, lactate, and succinate dehydrogenases in vivo. CMIT/MIT and other isothiazolones also showed inhibition of lactate, pyruvate, alcohol, alpha-ketoglutarate, succinate, and NADH dehydrogenases. The effect of MIT versus pyruvate dehydrogenase is shown in Figure 9. MIT concentrations of 29, 58, 115, and 230 ppm demonstrated a clear dose response with regards to the rate and extent of inhibition. The lowest level of MIT tested (29 ppm) showed approximately 20% inhibition after only 27 minutes. Increased inhibition was observed at higher MIT levels with the maximum inhibition (70% inhibition) achieved with 230 ppm MIT. By comparison MIT showed a lower rate of inhibition of the enzyme versus CMIT, as a function of biocide dose (data not shown).

14

0

20

40

60

80

0 5 10 15 20 25 30

Minutes

% I

nhib

ition

29 ppm 58 ppm115 ppm230 ppm

Figure 9. Inhibition of pyruvate dehydrogenase activity by MIT (relative to control). Enzymes inhibition studies have confirmed that isothiazolones inhibit critical sulfur-containing dehydrogenase enzymes on both purified enzyme extracts (in vitro) and whole cell or cell extracts (in vivo). This high degree of specificity results in low use levels for the isothiazolone family of biocides and also means that the biocide is not “used up” reacting with non-essential proteins or enzymes (those not containing thiols). Disabling these dehydrogenase enzymes can result in complete inhibition of critical metabolic functions, including respiration, growth, cell repair, and energy production, by crippling the cells’ central metabolic process, the Krebs (tricarboxylic acid) cycle. MIT and other isothiazolones can block or control multiple sites entering, within, or leaving the Krebs cycle (Figure 10). Therefore, cells cannot process required intermediate compounds for growth or generate energy by electron transport (with oxygen). Disabling this key pathway is central to the growth-inhibiting mechanism of isothiazolone biocides.

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KREBSKREBSCYCLECYCLE

GLUCOSEGLUCOSE

PYRUVATEPYRUVATE

ACETYL CoAACETYL CoA

CITRATECITRATE

KETOGLUTARATEKETOGLUTARATESUCCINATESUCCINATE

OXALOACETATEOXALOACETATE

ELECTRON TRANSPORT SYSTEMELECTRON TRANSPORT SYSTEM

LACTATELACTATE

ETHANOLETHANOL

KREBSKREBSCYCLECYCLE

GLUCOSEGLUCOSE

PYRUVATEPYRUVATE

ACETYL CoAACETYL CoA

CITRATECITRATE

KETOGLUTARATEKETOGLUTARATESUCCINATESUCCINATE

OXALOACETATEOXALOACETATE

ELECTRON TRANSPORT SYSTEMELECTRON TRANSPORT SYSTEM

LACTATELACTATE

ETHANOLETHANOL

Figure 10. Schematic of MIT interaction with the Krebs Cycle and entry to electron transport in microorganisms (red arrows indicate known sites of inhibition). Previous studies have demonstrated that MIT and other electrophilic isothiazolones react with nucleophilic protein thiols (in certain enzymes) destroying both soluble and insoluble types while killing viable cells in the process. This reactivity may also be linked to the killing effect of the biocides such that few survivors remain after contact with the biocides and loss of thiols.9 MIT showed less thiol interaction and less killing than CMIT, which is expected since CMIT is more reactive than MIT. Production of Free Radicals by Cells The cidal or killing mechanism of isothiazolones is linked to the inhibitory actions of the biocide, but inhibition alone does not account for the killing mechanism. Previous studies have shown that MIT and CMIT produce free radicals inside the cells shortly after contact with the biocide.9 Free radicals are produced by microbial cells as a normal function of metabolism, including growth, reproduction, energy generation, and oxygen consumption or detoxification. Healthy cells normally scavenge or detoxify radicals within the cells reducing their toxicity. Excess production of radicals or lack of their control will disable cells resulting in death. Based on these studies, the free radicals produced by MIT are a likely critical event in the cidal reactions of the molecules contributing to cell death. Other biocides such as BNPD are also known to produce excess radicals as a part of their biocidal mechanism. Summary of MIT Antimicrobial Mode of Action MIT biocide utilizes a two-step antimicrobial mechanism of action involving rapid binding (association) to cells and inhibition of growth and metabolism (within minutes), followed by irreversible cell damage resulting in loss of viability (hours). Growth inhibition is the result of rapid disruption of essential metabolic pathways of the cell by inhibition of 5 specific (thiol-containing) dehydrogenase enzymes

16

located in pathways involving the Krebs cycle and electron transport. Critical physiological functions that are rapidly inhibited in microbes include growth (cell numbers) and respiration (oxygen consumption). These processes are critical in bacteria, algae, and fungi which explains why MIT is a broad spectrum biocide. Cell death results from the progressive loss of protein thiols in the cell from one of multiple pathways. As cell metabolism is disrupted free radicals are produced within cells and this is likely a key contributor to the cidal mechanism. Overall, the higher the concentration of biocide, the shorter the contact time required for more complete kill. This unique mechanism results in the broad spectrum of activity of MIT biocide, low use levels for microbial control, and difficulty in attaining resistance. A proposed series of pathways for reactivity of MIT with microbial cells is shown in Figure 11. The initial event is binding of the biocide to protein thiols, resulting in a ring opened disulfide structure. Two primary pathways are proposed to account for the lethal loss of protein thiols leading to cell death: (1) covalent modification via direct electrophilic attack and (2) an intracellular generation of free radical as a result of severe metabolic disruption, which overwhelms the cell’s natural defenses against free radical damage. Unlike the chlorinated isothiazolones, MIT does not form a thioacyl chloride moiety, which has been postulated to provide additional bioactivity to CMIT.13 Overall, the antimicrobial mode of action of MIT is very similar in many respects to the other isothiazolones (CMIT and BIT) relative to enzyme inhibition, respiration inhibition, speed of kill, interactions with thiols, and radical formation.

SN

O

SHN

O

C8H17

S-Protein

+

RSH

SHHN

O

R-S-S-Protein

+ Protein-SH

+

RS.Disruptionof

Balanced Metabolism

Radical Cascade

Cell Damage

Cell DeathCell Death

CH3

O

S

O

C8H17

+

O

+

.

CH3CH3

CH3

SN

O

SHN

O

C8H17

S-Protein

+

RSH

SHHN

O

R-S-S-Protein

+ Protein-SH

+

RS.Disruptionof

Balanced Metabolism

Radical Cascade

Cell Damage

Cell DeathCell Death

CH3

O

S

O

C8H17

+

O

+

.

CH3CH3

CH3

Figure 11. Proposed pathways of isothiazolone biocide lethality for MIT biocide.

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CONCLUSIONS

Methylisothiazolone (MIT) biocide has never been used as the sole biocide in industrial process water systems to date. It has been used alone as a preservative for consumer, household, and industrial products with high organic content and long-term preservation needs. MIT is ideally suited for use in high pH and high temperature industrial water systems, such as closed loop cooling, where long term stability and preservation is required, yet quick kill is not a critical factor, due to the long persistence of the molecule in the system. MIT biocide is currently formulated at a 9.5% active ingredient concentration in water. No additional salts, solvents, organics, stabilizers, or metals are added. The formulation does not contain any volatile organic compounds (VOC) and is soluble in both water and organic solutions. MIT biocide is a broad spectrum bactericide, with excellent chemical and physical stability under aggressive end use conditions (pH 4-10; 5-60° C). It has excellent compatibility with other water treatment biocides and scale and corrosion chemicals. MIT does not contain or release formaldehyde, is biodegrades to non-toxic degradation products. MIT has recently received EPA approval for use in industrial water treatment systems, at 25-150 ppm active ingredient, for closed loop cooling systems, industrial process water, open recirculating cooling towers, fire protection systems, brewery pasteurizers, industrial scrubbing systems, evaporative condenser systems, air conditioner /refrigeration condensate water systems, influent water filtration systems, RO/UF membranes, retort water systems, etc. A key differentiating feature of MIT among the isothiazolone and other commercial biocides is it’s enhanced stability under extreme conditions. Stability studies showed MIT with exceptional persistence in high pH water (pH >10) and high temperatures (>50° C). Other biocides (CMIT, glutaraldehyde and BNPD) rapidly degraded under these conditions. The antimicrobial efficacy of MIT has been demonstrated in minimum inhibitory concentration and speed of kill studies versus various groups of organisms. These included bacteria (gram negative, gram positive, aerobes, facultative anaerobes, spore-formers, slime-forming bacteria, and sulfate reducing bacteria) and fungi (molds and yeasts). MIT was shown to be a highly effective active ingredient which inhibited microbial growth of bacteria with 23-25 ppm biocide. MIT is more effective versus bacteria than fungi; however, fungi are a less important contaminant of closed loop systems. MIT was also shown to be equally effective at pH 7 and pH 9. The antimicrobial mode of action of the electrophilic biocide MIT is complex. MIT utilizes a two-step mechanism involving rapid growth inhibition leading to a loss of viability. MIT rapidly associates (binds) with microbial cells. Growth inhibition is the result of rapid disruption of the central metabolic pathways of the cell by inhibition of 5 selected (thiol-containing) dehydrogenase enzymes located in pathways involving the Krebs (tricarboxylic acid) cycle and electron transport. Key physiological activities that are rapidly inhibited in microbial cells are growth (reproduction) and respiration (oxygen consumption). These processes are critical in bacteria, algae, and fungi which explains why MIT is such a broad spectrum biocide. Inhibition of cellular activity is rapid (within minutes), whereas, cell death (cidal activity) is observed after several hours contact. Cell death results from the progressive loss of protein thiols in the cell from one of multiple pathways. As cell metabolism is disrupted free radicals are produced within cells and this is likely a key contributor to the cidal mechanism.

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