The influence of the chemical behaviour of iodine on the germicidal action of disinfectant solutions containing iodine

Journal of Hospital Infection (1985) 6 (Supplement), 1-11

The influence of the chemical behaviour of iodine on the germicidal action of disinfectant solutions containing iodine Waldemar

Gottardi

Institute of Hygiene, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria

Introduction

In order to u n d e r s t a n d h o w disinfectants containing iodine work, it is necessary to know exactly how iodine behaves chemically in the matrix which serves as a solvent and which forms the basis of the particular preparation. T h e most important iodine-based disinfectants in use today can be divided into the following groups according to their composition: (1) iodine in water or an organic solvent (e.g. drinking water and swimming pool iodination; alcoholic iodine solution); (2) iodine and iodide ( K I or NaI) in water, or alcohol and water mixtures (Lugol's solution, iodine tincture); (3)-iodine, iodide, polymer organic complexing agents (povidone, polyoxy-ethylene etc.) and pharmaceutical mixtures in an aqueous or water and alcohol system (e.g. povidone-iodine (PVP-I) based mucosal disinfectants and washing concentrates). Whereas in pure alcohol solutions iodine is only available in soluble molecular form, in aqueous iodine systems, hydrolysis, dissociation, disproportionation and reactions involving complexes all play a role with the result that, apart from the hydrated iodine molecule (Iz), the H O I , OI , H 2 0 + I , I - , 13- and IO 3- forms are available. T h e equilibrium concentrations of these forms are determined in pure aqueous solutions by the total iodine and total iodide content, the p H value and the temperature, and can be calculated from these parameters. However, this is not the case in aqueous P V P - I solutions, since the povidone molecules (like the other additives) interact with the free iodine through electronic and steric effects in a way not yet fully investigated. This results in a considerable decrease in their equilibrium concentrations. O f the seven free iodine forms, molecular iodine and h y p o i o d o u s acid ( H O I ) are important in disinfection procedures, the latter only in aqueous systems, however, with small quantities of iodide (e.g. drinking water iodination). In pharmaceutical preparations which generally contain iodine O195-6701/85/06A001 + 11 $02,00/0

~'J 1985 The 1 lospital Infection Society

1

2

W.

Gottardi

and iodide, free molecular iodine is almost entirely responsible for the actual microbicidal activity insofar as it can be attributed to the iodine systems 9 T h e content of free molecular iodine ( ~ disinfection kinetics), which can be very easily d e t e r m i n e d potentiometrically, together with the total iodine determined by titration (,-~ disinfectant capacity), are i m p o r t a n t parameters in disinfection performance ( ~ disinfection kinetics + disinfection capacity) of a preparation. Soon after the discovery of the element iodine in 1812, the disinfectant properties of solutions of it in water and in alcohol were recognized (1829 Lugol's solution, U S Pharmacopoeia 1830: 'tinctura iodine') 9 T h e first important studies on a scientific basis appeared in the second half of the last century (Davaine, K o c h quoted in Gottardi, 1983), w i t h - D a v a i n e establishing in 1874 that iodine is one of the most effective antiseptics 9 This statement is still valid more than 100 years later. Since then a considerable n u m b e r of studies have been published on the use of iodine as a disinfectant 9 U n f o r t u n a t e l y , the statements made in them are often of little value since they are based on experiments w h i c h - - i n ignorance of the chemistry of i o d i n e - - w e r e carried out u n d e r conditions which are generally not described in detail but which nevertheless decisively influenced the chemical behaviour of iodine 9 T h i s means that it is not possible to correlate subsequently the disinfection results reported with its chemical behaviour. Iodine chemistry, as with the other halogens, is characterized by a high degree of reactivity with, above all, the ability to substitute covalent h y d r o g e n ( O - H , N - H , C - H and S - H compounds) which is of importance for~ the disinfecting process. Here a distinction m u s t be drawn between (1) the reaction of iodine with the solvent and any additives present, and (2) the reaction of the iodine form so produced with the c o m p o n e n t s of the material to be disinfected (bacteria, dissolved proteins, skin surfaces). R e a c t i o n

w i t h

s o l v e n t s

T h e most important solvents for the administration of iodine in h u m a n medicine are water and alcohol. T h e chemistry of iodine in water can be described by the following reactions: I II III IV V

9 . . hydrolysis I2+H20~HOI +H+ + I 9 . . dissociation HOI~--~OI- +H + 9 . . protonization HOI + H+~--'H20+ I 9 . . complex formation I 2+ I - 7 - - ' I - 3 3HOI~IO3-+2I-+3H + 9 . . disproportionation

T h e s e five linked equilibra mean that, in an aqueous solution, a total of seven different forms of iodine are available: I2, H O I , O I - , H 2 0 + I ,

I 3 , IO3-, I -

As can be f u r t h e r seen from the reaction equations, the H + ion occurs as a

Chemistry and g e r m i c i d a l action of iodine

reaction partner in I, II, III and V and the iodide ion in I, IV and V, with the result that the state of equilibrium depends very greatly on p H value and iodide concentration, and can be easily changed by these parameters. Figure 1 shows this using calculated iodate equilibrium concentrations (Gottardi, 1981). IO0

,n c

,=,

o

80

prig.0

g a0 ,o o

40

u

~ 20 0

0.04

0.08

0-12

Oq6

0'2

0.24

0"28

0.32

I 0'36

Cz-

Figure 1. Course of the iodate equilibrium c o n c e n t r a t i o n (in % oxidation equivalents) of a 0'03 M iodine solution with increasing iodide concentration at various p H levels.

Since, of the seven iodine forms, a bactericidal effect is only attributed to molecular (hydrated) iodine (I2) , h y p o i o d o u s acid ( H O I ) and the iodine cation ( H 2 0 + I ) (see Gottardi, 1978), the state of equilibrium must be adjusted (by regulating the p H value and the iodide concentration) so that the content of non-bactericidal iodine forms, above all IO 3- and I O - , is as low as possible. T h i s can he achieved by a low total iodine concentration (e.g. drinking water iodination: CI2< 10 -5 M/l) and a p H < 8 (Gottardi, 1978) or at a high total iodine concentration by at least an equivalent content of iodide (Lugol's solution: CI2 = 0-19 M/l, CI = 0.64 M/l). Since the molar concentration of H 2 0 + I ( = { H 2 0 + I } ) * can be disregarded with {H +} and {I-} relevant for disinfection procedures, only 12 and H O I are responsible for disinfection in an aqueous system, the latter, however, only in the presence of very small quantities of iodide (e.g. drinking water iodination). Tables I and I I show the equilibrium concentrations in aqueous iodine solutions calculated with and without additional iodide in the p H range 3-9 (Gottardi, 1978). It is striking that the sum of {I2} + {HOI} remains practically constant in the p H range 3-9, that nothing b u t the ratio {I2}:{HOI } changes when a small amount of iodide is added, and only with higher quantities of iodide ({I-}> 10 -4 M/l) does the sum of {I2}+{HOI } decrease. In contrast, in L u g o l ' s solution, o n l y the tri-iodide equilibrium (IV) is of * In the following { } denotes equal molecular concentration.

I I r 16

T 0

9

66~6

'

116&d

-

rll[~

I I I 14~ I [6~4~ 7

T

9

d~

d&&~#

6666

?

I

I I I I~

i

T 0

0

0

6~66~

rlrl I r J I~

L

0

IIII

I

h

9

[llll

N

9~9~9

._.@.

E~

Chemistry and g e r m i c i d a l action of iodine

5

real importance, so that over the total p H range ( p H > 9) only free iodine is available as an active agent ({I2}=6"7x 10-4M/1 resp. 1 7 0 p p m ) , while h y p o i o d o u s acid can be disregarded ({HOI} = < 10 -8 M/1 at p H < 7). In solutions with alcohol, iodine practically only occurs in solvated molecular form: I2"ROH. Since solutions with alcohol used in practice generally also contain water and iodide, equilibria I - V occur. H o w e v e r , because of the iodide content, only the solvate iodine molecules (I2"ROH, I2.aq)--apart from the alcohol-- might be responsible as the active forms for disinfection. A differentiation b e t w e e n the two forms according to relative reactivity should turn out in favour of the hydrate complex because of the greater stability of the I2-alcohol-solvate complex (inductive effect of the alkyl group increases the electron-donating properties of the oxygen). Disinfectant solutions containing iodophors

In disinfectant solutions containing organic polymers with i o d o p h o r properties, the chemistry of iodine is still more complex, since these macromolecules interact with the iodine forms created b y reactions I-V, which can cause a considerable decrease in their equilibrium concentrations. As far as the chemistry of aqueous disinfectant solutions containing iodophors is understood today, both electronic and steric effects are responsible for this interaction. T h u s , taking the known interactions with low molecular oxygen c o m p o u n d s such as amide, ester, ketone, ether etc. ( H a r u k a Y a m a d a & K u n i o Kozima, 1960; S c h m u l b a c h & Drago, 1960) as an analogy, it can be assumed that, b e t w e e n molecular iodine and the iodophor molecules, which without exception contain such functional oxygen-containing groups (e.g. povidone:carbonyl oxygen of the amide function in the pyrrolidone ring), donor-acceptor complexes are formed, with iodine playing the part of the acceptor: \ \ C = O + I2--~C = O~+''' I-P / F u r t h e r m o r e , the iodophors, above all in high concentrations, because of the spatial arrangement of the dissolved p o l y m e r molecules (near regions with helix-like structure) ( H o r n & Ditter, 1984), are obviously able to surround the free iodine form in the manner of clathrate and w i t h d r a w it from the equilibrium ( V I - V I I I). T h i s interaction m u s t be of importance for the iodide ion and above all for the large-mass tri-iodide ion, since in this case the formation of a donor-acceptor complex is not possible because of the negative charge. VI R + I - . ~ R ' I VII R + I 2 ~ R ' I e VIII R + I-s~-R'I J R = structural constituents of the iodophor molecule capable of forming complexe s .

6

W. G o t t a r d i

T h e s e two effects, together with equilibria I-V, result in the content of the free iodine form being greatly reduced in disinfection preparations containing i o d o p h o r (10% aqueous solution of P V P - I : CI2 ~ C l_ ~ 0"04 N/l, {I2}~ 10 -s M/1, resp. 2"54 ppm), in comparison with pure aqueous solutions with the same total iodine and total iodide content (aqueous iodine solution, C I 2 = C l_ =0"04 M/I, p H 5:{I2}=6"8 x 10 -3 M/l, resp. 1727 ppm). T h e high content of free iodide (which varies between 10- 3 and 10-1 M/l, according to the preparation) also means that H O I can be disregarded (see equilibrium I) and only 12 is responsible for disinfection.

/

10-2

10_3

"6

10-4

10-5

10-6

I

).o01

I

LIIIII[

I

O.Ol

I

IIILLI

1

o,I

I

I IIIIIII

I

1.0

t

IIIIII

I0

Concentrotion of PVP-T (%)

F i g u r e 2. T o t a l a n d a v a i l a b l e free i o d i n e in a q u e o u s P V P - I s o l u t i o n s . (1) D e t e r m i n e d i o d o r n e t r i c t i t r a t i o n , (2) d e t e r m i n e d p o t e n t i o m e t r i c a l l y .

by

Its concentration is largely independent of the p H value ( p H 3-6), but changes considerably with the degree of dilution, when it passes through a m a x i m u m of {I2}~10-4M/1 * in the 0"1% solution, as Figure 3 shows. As expected, {I 2} depends on temperature, with a more than 100% increase in {I2} occurring with a rise, for example, from 20 to 35~ (see Figure 3). For this reason, when carrying out in-vitro experiments to test the disinfection properties of a preparation, the temperature at which the preparation is administered (e.g. 35-36~ with musocal disinfectants) should be taken into account. T a b l e I I I shows a s u m m a r y of the most important disinfectant solutions containing iodine. D e p e n d i n g on the composition (solvent, type and quantity of the components), three different forms of iodine responsible for 9 T h e absolute a m o u n t of {12} not only depends on the concentration of PVP-I, but also on its total content of iodine and iodide which, as the specifications of the standard commercially available PVP-I show, undergoes considerable variations. Figure 2 therefore only shows the typical course of 12 as a function of the PVP-I concentration.

Chemistry and g e r m i c i d a l action of iodine soo L 7OO

6OO

=o

50O

400 3OO

Y 2OO

~O0

0

..............

-IOC

0

I

I

I

I0

20

25

I __~

30

J .......

35

40 Temperofure

L. . . . .

50

~_

60

I

70

(~

Figure 3. Relative change w i t h temperature in the content of free iodine at 20~ solutions (measured in I0, 5 and I % solutions of P V P - [ 30/06).*

in P V P - I

microbicidal action can be defined: I f R O H , I2-aq and H O I . Statements can be made in some cases about their concentrations which are based on measurements ({I2} in aqueous systems) or calculations ({HOI}, {I2} in aqueous systems when there are no additives interacting with iodine). I2"ROH should be available quantitatively as such in solutions with pure alcohol, while no statements can be made about the concentration ratios in systems with water and alcohol. R e a c t i o n w i t h t h e m a t e r i a l to b e d i s i n f e c t e d

T h e material to be disinfected generally has a fairly heterogeneous composition: micro-organisms, both living and dead, skin surfaces, dissolved proteins and other body fluid components (sweat, blood, urine, saliva, etc.). Iodine can react with all these substances, while only the reaction with living micro-organisms is desired. All other reactions are undesirable since they lead to iodine being c o n s u m e d (so that it is not available for the actual germicidal process) and, in the case of body surfaces, a too high concentration of free iodine may cause tissue irritation. Despite this heterogeneous composition, the primary reactions which take place can be mainly attributed to the iodination of N - H , C - H and S - H c o m p o u n d s . T h e iodine c o m p o u n d s thus produced are, however, partly unstable (e.g. iodination of S - H compounds) and undergo further reactions. A l t h o u g h * T h e relative change of free iodine is largely independent of the P V P - I concentration (Gottardi & Koller, in preparation)

W. Gottardi

v

8

9

~ =.s

~'~ 'f c~

7

._= -~ =

t

~.~ -~

_~i

"~

._

~

~~

o

.,!

o

o,-

o

.=-

.

Chemistry and g e r m i c i d a l action of iodine

9

iodination of the O H function of the water molecule (iodine hydrolysis, equilibrium I) is of fundamental importance for the chemistry of iodine in an aqueous system, the O H alcohol groups do not react with iodine u n d e r conditions relevant to disinfection procedures. 12 and H O I , which have been shown to play an i m p o r t a n t part in disinfection reactions, do not differ in the iodination of h y d r o g e n c o m p o u n d s with respect to the primary iodination products thus produced: IX

R - H + I 2 ~ R - I + H + + IR - H + H O I ~ R - I + H20

However, in reactions which do not have a quantitative outcome (e.g. iodination of N - H functions), H O I must be at an advantage, since the state of equilibrium is not influenced by the H + and I - concentrations. It can be assumed in the individual case that iodine reacts in the following way (Gottardi, 1983): (1) W i t h basic N - H functions of amino acids (lysine. histidine, arginine) and nucleotides (adenine, cytosine, guanine), the corresponding N-iodine c o m p o u n d s are p r o d u c e d (X). W h e n this happens, i m p o r t a n t positions for h y d r o g e n bonds are blocked, which can result in a lethal change in protein structure. X

R--NHz

~

-H+,-I-

R--NHI

(2.) T h e S - H group of the amino acid cysteine is oxidized, which means that the ability to make disulphide bonds (XI), an important factor in protein synthesis, is lost. _H+,_I_

_ H+,_I _ )

R--SS--R

(3) With the phenol group of the amino-acid tyrosine, w h e r e u p o n the m o n o and di-iododerivatives are produced (XII). In this case the size of the iodine atoms in the ortho position can sterically prevent the formation of hydrogen bonds with the phenol O H group. I

"En

OH - H + - I - R

R

I

OH

R

OH

-H+,-I -

(4) By addition of unsaturated fatty acids to olefinic double bonds ( X I I I ) . T h i s could lead to a change in the physical properties of the lipids and to a decrease in fluidity of the cell m e m b r a n e . y'm

R--CH:Ch--

R'+ I z

)

R--CH--CH--R'

I

I

I

I

10

W. Gottardi F r e e a n d total i o d i n e as i n d i c a t o r s o f d i s i n f e c t i o n p e r f o r m a n c e

In assessing disinfection performance, a differentiation must be made between disinfection rate and disinfection capacity. As has been generally observed with chemical disinfectants, disinfection rate d e p e n d s on the concentration of the active form, and therefore of {I2} and {HOI} in the case of aqueous disinfectants containing iodine. F o r molecular iodine {I2}, which can be determined potentiometrically (Gottardi, 1983), by equilibrium dialysis ( H o r n & Ditter, 1984) or--less p r e c i s e l y - - b y extraction, this was also confirmed b y experiment in the case of P V P - I preparations ( H o r n & Ditter, 1984; Berkelmann, Holland & Anderson, 1982; Pinter, Rackur & Schubert, 1983). However, it was also shown that correlations b e t w e e n {I2} and bactericidal rate are only valid for a defined system and cannot be transferred to other systems differing, for example with regard to composition and the quantity of additives (Gottardi & Puritscher, 1984). 100

8O "6

og

.~ ~

~

I~

ine

m

2o

I 20

I I 40 60 Reduced oxJdofJon equJvolenfs (%)

80

iO0

F i g u r e 4. Relative d e c r e a s e in free a n d total iodine d u r i n g the r e d u c t i o n of a P V P - I p r e p a r a t i o n b y cysteine.

In contrast, disinfection capacity, i.e. the total quantity of iodine which can react with the material to be disinfected, is proportional to the total iodine which can be determined iodometrically. Since the complex forming equilibria described (IV, V I - V I I I ) set in very rapidly, the free iodine used up in the course of the chemical reactions is continuously resupplied. Here it should be noted that, with the decrease in total iodine, the free iodine content also decreases, b u t to a greater degree than the former,* since the tri*This statement concerns the experiment shown in Figure 4, where solid cystein has been added to the PVP-I preparation. If the reducing agent is a solution (e.g. blood) a dilution of the PVP-I system supervenes, with the effect that the relative decrease of free iodine also can be smaller than the one of the total iodine (see Fig. 2) (Gottardi & Koller, in preparation).

C h e m i s t r y and g e r m i c i d a l action of i o d i n e

11

i o d i d e e q u i l i b r i u m ( I V ) is d i s p l a c e d to the d i s a d v a n t a g e o f 12 b y t h e i o d i d e p r o d u c e d (see e q u i l i b r i u m V I I a n d F i g u r e 4). T h e b a c t e r i c i d a l rate t h e r e fore c o n s t a n t l y d e c r e a s e s d u r i n g the p e r i o d in w h i c h the p r e p a r a t i o n takes effect, with the e x t e n t d e p e n d i n g b o t h o n the material to be d i s i n f e c t e d (size o f the p r o t e i n load) a n d o n the d i s i n f e c t i o n s o l u t i o n ( d i s i n f e c t i o n c a p a c i t y , ratio of free iodine to free iodide). References

Berkelmann, R. L., Holland, B. W. & Anderson, R. L. (1982). Increased bactericidal activity of dilute preparations of povidone-iodine solutions. Journal of Clinical Microbiology 15, 635. Oottardi, W. (1983). Iodine. In Disinfection, Sterilization and Preservation (S. S. Block, Ed.). 3rd Edn. Lea & Febiger, Philadelphia. Gottardi, W. (1981). Die Bildung von Jodat als Ursache f/ir die Wirkungsabnahme jodhaltiger Desinfektionsmittel. Zentralblatt ffir Bakteriologie und Hygiene I dbteilungen, Originale B 172, 498-507. Oottardi, W. (1978). W~issrige Jodl6sungen als Desinfektionsmittel. Zentralblattfffr Bakteriologie und Hygiene I dbteilungen, Originale B 167, 206 215. Gottardi, W. (t983). Potentiometrische Bestimmung der Gleichgewichtskonzentrationen an freiem und komplex gebundenem Jod in wfissrigen L6sungen yon PolyvinylpyrrolidonJod (PVP-Jod). Zeitschriftffir analytische Chemic 314, 582-585. Gottardi, W. & Puritscher, M. (1985). Keimt6tungsversuche mit wiissrigen, komplexgebundenes Jod (PVP-Jod, J 3) enthaltenden Desinfektionsl6sungen: Einfluss des Gehaltes an freiem Jod auf das bakterizide Verhalten gegentiber Staphylococcus aureus. Zentralblatt fiir Bakteriologie und Hygiene I Abteilungen, Originale B (in preparation). Haruka Yamada & Kunio Kozima (1960). The molecular complexes between iodine and various oxygen-containing organic compounds. Journal of the American Chemical Society 82, 1543. Horn, D. & Ditter, W. (1984). Physikalisch-chemische Grundlagen der mikrobiziden Wirkung w~issriger PVP-iod-L6sungen. In P V P - I o d in der operativen Medizin (G. Hierholzer & G. G6rtz, Eds). Springer, Berlin-Heidelberg New York-Tokyo. Pinter, E., Rackur, H. & Schubert, R. (1983). Die Bedeutung der Galenik fir die mikrobizide Wirksamkeit yon Polyvidon-Jod-L6sungen. Pharmazeutische Industrie 46, 3-8. Schmulbach, C. D. & Drago Russel, S. (1960). Molecular addition compounds of iodine Ill. An infrared investigation of the interaction between dimethylacetamide and iodine. Journal of the American Chemical Society 82, 4484.