Australian Dental Journal
The official journal of the Australian Dental Association
Australian Dental Journal 2013; 58: 428–433 doi: 10.1111/adj.12115
Assessment of residual active chlorine in sodium hypochlorite solutions after dissolution of porcine incisor pulpal tissue# RM Clarkson,* TK Smith,† BA Kidd,* GE Evans,‡ AJ Moule* *School of Dentistry, The University of Queensland. †Research and Development, Multitrator Pty Ltd, Brendale, Queensland. ‡School of Mathematics and Physics, The University of Queensland.
ABSTRACT Background: In previous studies, surfactant-containing Hypochlor brands of sodium hypochlorite showed better tissue solubilizing abilities than Milton; differences not explained by original active chlorine content or presence of surfactant. It was postulated that exhaustion of active chlorine content could explain differences. This study aimed to assess whether Milton’s poorer performance was due to exhaustion of active chlorine. Parallel experiments assessed the influence of titration methods, and the presence of chlorates, on active chlorine measurements. Methods: Time required to dissolve one or groups of 10 samples of porcine incisor pulp samples in Milton was determined. Residual active chlorine was assessed by thermometric titration. Iodometric and thermometric titration was carried out on samples of Milton. Chlorate content was also measured. Results: Dissolution of single and 10 pulp samples caused a mean loss of 1% and 3% respectively of active chlorine, not being proportional to tissue dissolved. Thermometric ammonium ion titration resulted in 10% lower values than iodometric titration. Chlorate accounted for much of this difference. Conclusions: Depletion of active chlorine is not the reason for differences in tissue dissolving capabilities of Milton. Thermometric ammonium ion titration gives more accurate measurement of active chlorine content than iodometric titration. Keywords: Dental pulp solubility, iodometric titration, residual active chlorine, sodium hypochlorite, thermometric titration. (Accepted for publication 13 March 2013.)
The widespread use of sodium hypochlorite (NaOCl) as an endodontic irrigant is largely due to its unrivalled antimicrobial and tissue dissolving capabilities.1–3 Studies of the ability of NaOCl to dissolve protein tissues have investigated the effect of the concentration,4–6 the effect of the temperature of the solutions,7,8 and the effect of sonic and ultrasonic agitation.9 The ability of NaOCl to dissolve protein and kill microbes has been shown to vary with changes in the pH of NaOCl solutions.10–12 Experiments by Moorer and Wesselink and Spano et al. measured residual active chlorine levels in NaOCl solutions after the dissolution of various forms of protein.5,6
Invariably, where active chlorine was calculated in the NaOCl solutions tested, iodometric titration was used, there being no specific test for the presence of hypochlorite ion in NaOCl. In a previous investigation, Milton§ brand of sodium hypochlorite, with a nominal active chlorine content of 1% (w/v), performed very poorly in dissolving porcine incisor pulps compared with Hypochlor 1%** brand of sodium hypochlorite, with a nominal active chlorine content of 1.5% (w/v).13 This difference appeared to be greater than could be explained by the difference in concentration. It was suggested, but without evidence, that the difference observed might be due to the presence of a surfactant in the Hypochlor product and/or exhaustion of the
#Editor’s note: This study included Milton solution which has been extensively used in the past as an endodontic irrigant, but currently does not have TGA approval for such use.
§ Milton Australia Pty Ltd, Carole Park, Queensland, Australia. ** Dentalife Pty Ltd, Ringwood, Victoria, Australia.
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Active chlorine in Milton after pulp dissolution active chlorine in the Milton solution. A subsequent investigation appeared to eliminate the presence or absence of surfactant content of Hypochlor as the cause of these differences.14 Overall, the relationship between the ability of various solutions of NaOCl to dissolve tissue and their residual active chlorine content has not been established. One early study did demonstrate that using NaOCl to dissolve a protein hydrolysate caused rapid loss of active chlorine content in the solution.5 Those authors expressed the view that this difference probably reflected ‘reversibly bound chlorine detected by the iodometric technique’, and that the iodometric measurement of remaining active chlorine may not be useful. This ‘reversibly bound chlorine’ is likely to be oxyhalides other than HOCL (H+ and OCl ) and OCl . Oxyhalides are combinations of oxygen and the halogens (chlorine, fluorine, iodine and bromine). Iodometric titration registers all oxyhalides.15 Only the HOCl and OCl content is effective in killing microorganisms and dissolving protein.16 It was considered important to investigate whether the amount of tissue dissolved by a solution affected the residual chlorine content and whether these measurements were useful in assessing the comparative effectiveness of NaOCl solutions. Milton was chosen as the NaOCl solution for this investigation. In a previous study, it exhibited a poor ability to dissolve pig pulp tissue not explained by differences in nominal active chlorine content.13,14 If exhaustion of the active chlorine was the reason for poor pulp dissolution, Milton would be more likely to exhibit this phenomenon than other NaOCl solutions. If there was little loss of residual active chlorine in Milton, then it would be even less likely this would occur in other NaOCl solutions. These findings could then be extrapolated to all other NaOCl solutions. This study aimed to investigate whether the poor ability of some NaOCl solutions to dissolve pulp tissues could be attributed to exhaustion of their residual chlorine content. Presence of chlorate (ClO3 ), chlorite (ClO2 ) and perchlorate ions (and even bromate as a contaminant) have been shown to influence the analysis of available chlorine in NaOCl solutions. This is because iodometric titration for active chlorine registers them along with HOCl and OCl .15 Analysis of the ClO3 , and other oxyhalide concentration in Milton solutions was therefore warranted. Recent developments in thermometric titration allow this type of analysis to be performed by a modified ammonium ion method.17 This technique uses changes in temperature to plot the reaction end point. It was considered desirable to compare the active chlorine values of the same NaOCl products when they were tested by both iodometric and ammonium ion titration. © 2013 Australian Dental Association
MATERIALS AND METHODS Measurement of residual active chlorine Porcine incisor pulps were harvested from the mandibles of young pigs after slaughter. The current investigation did not influence their fate in any way. The use of these animal tissues for research was approved by the Animal Welfare Unit, The University of Queensland. The mandibles were obtained from the abattoir on the day of slaughter and chilled. The teeth were removed within 24 hours by a blow from a mallet and a wooden dowel. Within 20 minutes, they were placed in a sealed plastic envelope then frozen without drying. Within less than seven days, the teeth were thawed briefly, wrapped in rubber dam and compressed in a steel machine vice until the roots cracked. The pulps were gently teased out and placed on a polythene cutting board with mesial or distal surface down. Thirty-three samples were obtained by use of a circular 2.5 mm stainless steel punch. All pulp specimens were dried on filter paper and weighed prior to immersion using a GR-200 analytical digital balance weighing to 0.1 mg.†† The Milton solution used was the Australian commercial product and was purchased fresh less than seven days before use. Six 25 ml aliquots of Milton were prepared for testing the dissolution of the pulp specimens prepared above. A further five identical 25 ml Milton aliquots were prepared for use as controls, making 11 in total. Each of three randomly selected pulp samples was then tested alone in a single 25 ml aliquot of Milton, and the remaining 30 samples were randomly allocated in groups of 10 to each of a further three 25 ml aliquots of Milton. Active chlorine content of the Milton was measured before the experiment and then after the complete dissolution of the samples in each Milton aliquot. Similar measurements were made, each from a single aliquot, at five time intervals over the duration of the experiment for the controls, to ensure that there was no loss of active chlorine content even without immersion of any pulp tissue. Continuous agitation was performed by magnetic stirring apparatus for all test aliquots. The end point for complete dissolution was assessed by a minimum of two observers. Assessment of the active chlorine was performed using a Metrohm 859 Titrotherm thermometric titrimetry apparatus.‡‡ Two 5 mL samples drawn from each 25 ml test aliquot were measured by pipette for assessment of active chlorine content after pulp dissolution. Two titrations were performed on 5 mL samples of the three multiple pulp solutions. Single †† A&D, Thebarton, South Australia, Australia. ‡‡ Metrohm AG, Switzerland. 429
RM Clarkson et al. titrations on 5 mL samples were used to assess active chlorine content in the single pulp aliquots and the five control aliquots. The titrant was a standard solution of ammonium chloride and the hypochlorite was converted to hypobromite solution in situ with a bromide-bicarbonate solution. The reduction in the active chlorine content was analysed using a generalized linear model with the number of pulp samples as a factor. The dissolving time was analysed using a generalized linear model with chlorine percentage as a factor. Comparison of iodometric and ammonium ion assessment of active chlorine The NaOCl solutions Milton, White King,§§ Aldi Power Force Aqua*** and Homebrand,††† were tested for their active chlorine content by the iodometric method and also by the ammonium ion technique described above. Both titration methods used the Metrohm Titrotherm apparatus. In this case, 20 mL aliquots were tested, and the average of six readings was calculated for each solution.
Table 1. Active chlorine values for the Milton control solution at five time intervals during the experiment. Elapsed time (h:mm:ss)
% Active chlorine content (w/v)
0:00:00 0:02:30 0:58:03 2:09:50 2:15:00
1.003 1.004 1.004 1.004 1.006
Table 2. Residual active chlorine values for 25 ml solutions of Milton after total dissolution of either one or ten 2.5 mm diameter porcine incisor pulp samples compared with control solutions. Sample
Control After one pulp dissolved After 10 pulps dissolved
% Average active chlorine content (w/v)
Average weight of each pulp sample (mg)
Average time to dissolution (h:mm:ss)
Mean loss of initial active chlorine content
1.004 0.994
2.37
0:11:19
1%
0.977
3.15
0:15:09
3%
Chlorate and chlorite assay Two further 20 mL aliquots of Milton solutions with batch numbers 345558 and 341424 were submitted to parallel titrations by the iodometric and ammonium ion methods for active chlorine as above. They were also analysed for ClO3 and ClO2 content in 100 mL aliquots by ion chromatography. Chlorate and chlorite ion content was then converted to NaOCl equivalent, for evaluation of their effect on active chlorine assessment by iodometric titration. These assays were performed by a commercial analytical laboratory‡‡‡ using a Dionex ICS-2000 ion chromatography apparatus.§§§ This method determines the concentration of anionic compounds in aqueous samples. The laboratory is accredited by The National Association of Testing Authorities, Australia. Chlorate variation in Milton Seven fresh 1 litre bottles of Milton were purchased from different retailers, all with different batch numbers and well within their expiry dates. A 100 mL aliquot from each bottle was tested for ClO3 ion content within five days of purchase, by the same §§ Pental Products Pty Ltd, Shepparton, Victoria, Australia. *** Aldi Stores, Minchinbury, New South Wales, Australia. ††† Woolworth Safeway, Yennora, New South Wales, Australia. ‡‡‡ Scientific Analytical Services Laboratory, Darra, Queensland, Australia. §§§ Thermo Fisher Scientific, Scoresby, Victoria, Australia. 430
laboratory and the same ion chromatography apparatus as detailed for the ClO3 and ClO2 assay. All statistical analysis was done using R version 2.12.0.**** RESULTS Measurement of residual active chlorine The measured active chlorine content of the Milton control aliquots without immersed pulp samples was essentially unchanged over the nearly two-and-a-half hours of the experiment; small variations in the readings being within the range of accuracy of the pipette used to measure the solution aliquots. Table 1 lists the timing of the five readings and their values. Table 2 details the average dissolution times, active chlorine readings and loss of active chlorine due to dissolution of pulp tissue. Milton aliquots in which single pulp samples (mean weight 2.37 mg) were immersed showed a mean loss of 1% of the initial active chlorine content after complete dissolution of the pulp with a 95% confidence interval (CI) of (0.00, 0.02). The mean time to complete dissolution was 11 minutes and 19 seconds. The Milton aliquots which had each contained 10 samples (mean total weight, 31.53 mg) lost 3% of their initial active chlorine content with a 95% CI of (0.02, 0.04). The six
**** The R Foundation for Statistical Computing. © 2013 Australian Dental Association
Active chlorine in Milton after pulp dissolution active chlorine readings for the three multiple pulp aliquots only varied between 0.973 and 0.980. The mean time to complete dissolution of the 10 samples was 15 minutes and 9 seconds. Statistical analysis of the loss in active chlorine content revealed strong evidence that the mean loss in active chlorine content was less than 5% of the original strength for both the single and multiple pulp samples (p < 0.01). A loss of less than 5% was considered to represent a non-significant loss of active chlorine content in a practical sense. There was also evidence that the mean loss of active chlorine content for the multiple pulp samples was less than 10 times the mean loss of active chlorine content for single pulp samples (p < 0.10).
Values for ClO3 and ClO2 content were converted to NaOCl equivalent by multiplying by the proportion of the relative formula weights. These proportions were 0.89 for ClO3 (74.44/83.45) and 1.10 (74.44/67.45) for ClO2 . Chlorate content variation Chlorate ion content of the seven Milton batches varied between 0.46 g/L and 0.98 g/L with a mean of 0.66 g/L and a 95% CI of (0.45, 0.87) g/L all as NaOCl equivalent. This corresponded to between 4.6% and 9.8% of the nominal active chlorine content (1%, or 10.00 g/L) of the Milton solutions attributable to their ClO3 content. These results are presented in Table 5. The laboratory declared the accuracy of the ion chromatography procedure for the ClO2 and ClO3 measurement to be 10%.
Comparison of titration methods The comparison of the measurement of active chlorine produced by the iodometric and ammonium ion methods reveals that the ammonium ion method gives a near constant reading 10% lower than the iodometric process (Table 3). In the two batches of Milton (#345558 and #341424) tested, the difference in active chlorine between the two methods averaged 1.39 g/L. The same two solutions produced a mean ClO3 content of 0.57 g/L and a mean ClO2 content of 0.024 g/L as NaOCl equivalent when submitted to assay (Table 4). Thus, the combined ClO3 and ClO2 content (0.59 g/L) did not account for the entire active chlorine difference between the iodometric and ammonium ion readings.
DISCUSSION Dental pulp tissue presents unique problems in producing uniform test samples for experimental study. Even with the use of a punch to prepare the pulp samples, there was some inherent variation in sample size due to variation in mesio-distal width of the pulps. Initially, three aliquots with one pulp sample were tested. When these showed a mean active chlorine reduction of only 1%, further testing of single pulp samples was abandoned. Ten randomly assigned pulp samples were then used in each of the
Table 3. Comparison of active chlorine content in a range of NaOCl solutions by iodometric and ammonium ion titration methods; mean of six readings NaOCl g/L Product
Iodometric method
Milton White King Aldi Power Force Aqua Woolworths Homebrand
14.46 39.20 50.11 42.36
0.02 0.04 0.07 0.05
Ammonium ion method
Difference
% Difference between methods
1.44 3.63 4.86 4.02
9.96 9.26 9.70 9.49
13.02 35.57 45.25 38.34
0.04 0.08 0.24 0.13
Table 4. Chlorate and chlorite content of two batches of Milton as NaOCl equivalent,†††† and their contribution to the difference in active chlorine content registered by the iodometric and ammonium ion titration methods Milton Batch #
NaOCl by iodometric method g/L
NaOCl by ammonium ion method g/L
Difference between methods g/L
Chlorate as NaOCl equivalent g/L
Chlorite as NaOCl equivalent g/L
Combined chlorate and chlorite as NaOCl equivalent g/L
345558 341424 Mean of two batches
15.18 14.46 14.82
13.84 13.02 13.43
1.34 1.44 1.39
0.50 0.63 0.57
0.02 0.02 0.02
0.52 0.66 0.59
††††The NaOCl equivalents for chlorate and chlorite were calculated by multiplying by the proportion of their relative formula weights. These were 0.89 for chlorate (74.44/83.45) and 1.10 for chlorite (74.44/67.45). © 2013 Australian Dental Association
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RM Clarkson et al. Table 5. Chlorate ion content of seven different Milton solutions and calculated values as the NaOCl equivalent Milton batch #
Chlorate content g/L
Chlorate content as NaOCl equivalent g/L
326985 326987 326992 326995 329458 341427 349596 Mean
1.10 1.10 0.58 0.70 0.52 0.58 0.61
0.98 0.98 0.52 0.62 0.46 0.52 0.54 0.66
remaining three test aliquots. This provided a much greater challenge to the active chlorine retention abilities of the Milton. Even these Milton aliquots lost only 3% of their active chlorine after dissolving 10 pulp samples. While not conclusive because of some disparity in mean weight, the active chlorine depletion from the dissolution process is not proportional to the mass of pulp dissolved. Single samples took 11 minutes and 19 seconds, compared with 15 minutes and 9 seconds for aliquots with 10 samples for complete dissolution. Therefore, the multiple pulp samples with 13 times the mass of single samples and 10 times the surface area required only about half as long again to be dissolved. This suggests that the speed of the process is principally dependent on the surface area of the individual pieces of pulp rather than total surface area or total mass of pulp tissue. Continuous stirring employed in this study increased the rate of pulp dissolution compared with intermittent stirring in the earlier work of Clarkson et al.13 The dissolution time of 11 minutes and 19 seconds for single pulp samples was not only much lower than for Milton in the 2006 study, but was lower than all solutions except White King 4%. Only the tissue dissolving abilities of Milton were evaluated, and these cannot necessarily be extrapolated to its antimicrobial ability. In 1982, Moorer and Wesselink produced losses approaching 80% of the active chlorine, albeit with up to 100 mg of tissue and small volumes of the NaOCl solutions. For some investigations, those researchers used protein hydrolysate as a substrate. This is partly digested protein more easily dissolved than intact tissue.5 The use of pulp tissue in the current study is more representative of clinical situations. Spano et al. also employed dental pulp to determine residual chlorine in 15 mL of NaOCl, but had a pump circulate the solution. They reported residual active chlorine of 76.75% in a 1% NaOCl solution. Pulp mass and time were not revealed.6 The current study concentrated on explaining an anomaly in the 432
relative performance of Milton in the previous article, and used the same volume of NaOCl as that study.13 The mass to volume ratio of pulp to NaOCl used here, 31.53 mg in 25 mL of solution, is within the range studied by Moorer and Wesselink.5 The differences in pulp dissolution times noted in the earlier study are likely due to factors other than exhaustion of residual active chlorine tested here. These other factors might include Milton’s relatively high salt content and lower than usual pH.13 The active chlorine content of NaOCl solutions diminishes with time,18 and the breakdown products of this deterioration include ClO3 , and perhaps other non-hypochlorite oxyhalides.16 This phenomenon is reflected in the variation in ClO3 content in the seven Milton batches tested separately here, with the highest reading being twice that of the lowest. The active chlorine content of the Milton in the earlier study was not tested. Therefore, it is possible that the Milton used in the earlier work might have been ‘stale’, despite being well within its expiry date. Generally, substantial concentration of non-hypochlorite oxyhalides in Milton could give an unrealistically high reading for active chlorine with iodometric titration, but with no contribution to protein dissolving properties. Such distortion appears unlikely with the ammonium ion titrations used in the current study. It was not possible to test the Milton aliquots used in that previous study of residual active chlorine for their non-hypochlorite oxyhalides. Levels of ClO3 and ClO2 in Milton batches #345558 and #341424 did not account for all of the difference between the iodometric and ammonium ion methods of analysis. The discrepancy is probably due to the presence of perchlorate and bromate ions, which are known inclusions in NaOCl solutions.19 Certainly, the perchlorate concentration of NaOCl solutions is known to rise with increase in ClO3 content.16 The ammonium ion titration method used for this project is an innovative technique, which is only practicable with a device such as the Metrohm thermometric titration unit used here. The ammonium ion titration method produced consistently lower active chlorine readings in testing a variety of NaOCl solutions compared with the iodometric method (Table 3). This indicates that the ammonium ion titration method probably does not register oxyhalide ions other than hypochlorite. It may be a more reliable indicator of performance of NaOCl solutions in dissolving protein than iodometric titration. There is obviously a need for a reliable method of measuring the effective active chlorine in sodium hypochlorite solutions, represented by HOCl and OCl only. This applies not only to domestic use, but also to bulk usage such as in chlorination of reticulated water supplies.16 The current study offers © 2013 Australian Dental Association
Active chlorine in Milton after pulp dissolution persuasive evidence that the combination of thermometric apparatus and ammonium ion titration may fill this need. A major study involving deployment of extensive analytical resources would be required for a definitive understanding of the identity and role of compounds other than OCl in analysing active chlorine in NaOCl solutions. It can be concluded that the depletion of active chlorine is not the reason for differences in the tissue dissolving capabilities of Milton. Thermometric ammonium ion titration gives more accurate measurement of active chlorine than iodometric titration. ACKNOWLEDGEMENTS This study was supported by a grant from the Australian Dental Research Foundation. The authors wish to express their gratitude to The University of Queensland for the provision of laboratory facilities. DISCLOSURE Thomas Smith is a paid consultant to Metrohm Pty Ltd, manufacturers of the 859 Titrotherm thermometric titration equipment used in the experimental work reported here. He is engaged in the development of analytical procedures and demonstration of instrument capabilities. Neither he nor Metrohm Ltd received any remuneration or recompense associated with the work reported here. REFERENCES 1. Clarkson RM, Moule AJ. Sodium hypochlorite and its use as an endodontic irrigant. Aust Dent J 1998;43:250–256. 2. Dutner J, Mines P, Anderson A. Irrigation trends among American Association of Endodontists members: a web-based survey. J Endod 2012;38:37–40. 3. Clarkson RM, Podlich HM, Savage NW, Moule AJ. A survey of sodium hypochlorite use by general dental practitioners and endodontists in Australia. Aust Dent J 2003;48: 20–26. 4. Koskinen KP, Stenvall H, Uitto VJ. Dissolution of bovine pulp tissue by endodontic solutions. Scand J Dent Res 1980;88:406– 411. 5. Moorer WR, Wesselink PR. Factors promoting the tissue dissolving capability of sodium hypochlorite. Int Endod J 1982; 15:187–196.
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6. Spano JC, Barbin EL, Santos TC, Guimaraes LF, Pecora JD. Solvent action of sodium hypochlorite on bovine pulp and physico-chemical properties of resulting liquid. Braz Dent J 2001;12:154–157. 7. Cunningham WT, Balekjian AY. Effect of temperature on collagen-dissolving ability of sodium hypochlorite endodontic irrigant. Oral Surg Oral Med Oral Pathol 1980;49:175–177. 8. Sirtes G, Waltimo T, Schaetzle M, Zehnder M. The effects of temperature on sodium hypochlorite short-term stability, pulp dissolution capacity, and antimicrobial efficacy. J Endod 2005;31:669–671. 9. Al-Jadaa A, Paque F, Attin T, Zehnder M. Acoustic hypochlorite activation in simulated curved canals. J Endod 2009;35:1408–1411. 10. Aubut V, Pommel L, Verhille B, et al. Biological properties of a neutralized 2.5% sodium hypochlorite solution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:e120–e125. 11. Zehnder M, Kosicki D, Luder H, Sener B, Waltimo T. Tissuedissolving capacity and antibacterial effect of buffered and unbuffered hypochlorite solutions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:756–762. 12. Camps J, Pommel L, Aubut V, et al. Shelf life, dissolving action, and antibacterial activity of a neutralized 2.5% sodium hypochlorite solution. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:e66–e73. 13. Clarkson RM, Moule AJ, Podlich H, et al. Dissolution of porcine incisor pulps in sodium hypochlorite solutions of varying compositions and concentrations. Aust Dent J 2006;51:245–251. 14. Clarkson RM, Kidd B, Evans GE, Moule AJ. The effect of surfactant on the dissolution of porcine pulpal tissue by sodium hypochlorite solutions. J Endod 2012;38:1257–1260. 15. Aieta EM, Roberts PV, Hernandez M. Determination of chlorine dioxide, chlorine, chlorite, and chlorate in water. J Am Water Works Assoc 1984;76:64–70. 16. Pisarenko AN, Stanford BD, Quinones O, Pacey GE, Gordon G, Snyder SA. Rapid analysis of perchlorate, chlorate and bromate ions in concentrated sodium hypochlorite solutions. Anal Chim Acta 2010;659:216–223. 17. Kolthoff IM, Stenger VA. Calcium hypochlorite as a volumetric oxidizing agent. Ind Eng Chem Anal Ed 1935;7:79–81. 18. Clarkson RM, Moule AJ, Podlich HM. The shelf-life of sodium hypochlorite irrigating solutions. Aust Dent J 2001;46:269–276. 19. Asami MK, Kosaka K, Kunikane S. Bromate, chlorate, chlorite, and perchlorate in sodium hypochlorite solution used in water supply. J Water Supply Res T 2009;58:107–115.
Address for correspondence: Dr Roger Clarkson School of Dentistry The University of Queensland 200 Turbot Street Brisbane QLD 4000 Email:
[email protected]
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