Objective: to Find a Relation Between Hard Water and Strength of Hair

1 TITLE: 2 To evaluate and compare, changes in strength of hairs after treating with hard water 3 and de-ionized water. 4 5 ABSTRACT: 6 Objective: To find a relation between hard water and strength of hair. 7 Design: Experimental Study with Randomized Control Trials (RCT). 8 Place and Duration: Duration of study was 6 months from Sept 2014 to Dec 2015 and war 9 carried out in Kohat and Peshawar, KP, Pakistan. 10 Methodology: Water samples were collected from 10 main districts of KP, Pakistan. 11 Hardness levels of all samples were determined, in order to find the water sample of 12 maximum hardness that hair would encounter in KP, Pakistan; and was considered our 13 sample hard water. 14 Hair samples of 70 male individuals, from district with minimum hardness levels, were 15 collected. Each hair sample was divided into three equal parts and three groups (Group A, B 16 & C) of hair were established, each containing 70 hairs. Group A was considered control. 17 Group B was treated with de-ionized water and group C was treated with hard water. 18 Tensile strength of all three groups was measured using the universal testing machine and 19 compared, using paired t-test. 20 Results: The mean values of tensile strength for hairs of group A, B, and C were 255.49, 21 254.84 and 234.16 with a standard deviation of 57.55, 58.74 and 56.25 respectively. 22 Significant decrease was observed in strength of hair when treated with hard water (P<0.05) 23 as compare to hair treated with de-ionized water (p> 0.05). 24 Conclusion: 25 Hard water decreases strength of hair and thus increases breakage. 26 Key Words: 27 Hard water, hair, magnesium sulphate, calcium carbonate, tensile strength. 28 29 INTRODUCTION: 30 Hair is valued as a most important part of human body as they portray the extent of 31 personality and confidence. Therefore; great care is taken in handling hair to prevent hair 32 problems. 33 But hair problems are usually observed in all communities and commonly hair loss (16-96%) 34 and breakage.1-3 It is a well established fact that there are underlying genetic causes of hair 35 loss,4 but hair breakage, on the other hand, is mostly thought to be due to the use of hard 36 water.3 37 Hardness represents salts of calcium carbonate and magnesium sulphate in water that results 38 in temporary and permanent hardness respectively and is expressed as equivalent of calcium 39 carbonate.5 Though hard water is thought to have negative effect on hair, yet it is found to 40 have positive effects on health as it serves as a fine source of magnesium and calcium. 6, 7 41 United States Geological Survey (USGS) classifies water into four main types based on its 42 water hardness contents (table 1).8

1 43 The main constituent of hair is a very stable protein, which is rich in cysteine and is resistant 44 to proteolytic enzyme activity, known as keratin.9, 10 The stability is due to the existence of 45 different kind of bonds i.e. covalent bonds, ionic bonds and hydrogen bonds, which may also 46 act as a site for chemical process thus making hair reactive as well.10-12 A property of hair to 47 swell up in a medium with pH greater than 5.5; thus allowing entry of the metal ions from the 48 medium into the hair structure and a pH below 5.5 stops this process.13 A chemical reaction 49 results in oxidation of the disulphide bonds in hair that further results in the creation of 50 sulfonic acid and sulfonate that causes the ionization (de-protonation) of the side chains of 51 hair proteins forming a negatively charged resin that assist in dragging the cations from the 52 solution in to the hair structure (e.g. in hair dyeing).10 In a very same manner when hair 53 interact with hard water, the cations; magnesium and calcium are absorbed from the water by 54 the anion sites (negatively charged resin) of hair and results in its oxidation.10, 14 The more 55 exposure to the cations may result in oxidative damage (similar to oxidative damage in hair 56 dyeing), which is directly proportional to both levels of hardness and pH of the medium.10, 14 57 The regular interaction between hair and hard water during bathing, washing etc., lead to this 58 study, to evaluate the effect of hard water on strength of hair in men and create awareness. 59 METHODOLOGY: 60 Two types of samples were used in this experimental study i.e. hard water and hair. This 61 study included a purposive, non-probability sampling technique and Randomized Control 62 Trials (RCT). This study was approved by the institutional review board (IRB) and advance 63 studies and research board (AS&RB), Khyber Medical University, Peshawar, Pakistan. 64 Hard Water Sample: 65 From 10 main districts, of Khyber Pakhtunkhwa (KP), Pakistan, tap water samples were 66 collected from 10 different places in each district. 3 samples per station, one each day for 67 consecutively 3 days were collected leading to a total of 30 samples per districts. pH values 68 of all the samples were calculated using pH meter and their relative hardness was evaluated 69 with help of ethylendiaminetetraacetate (EDTA) assay, according to International 70 Organization for Standardization (ISO) standards, 15, 16, in terms of standard unit for hardness

71 i.e. mg/L of CaCO3. Average hardness was calculated from all the 30 samples per district 72 (Table 2) 73 From table 2 we concluded that average maximum water hardness is found in district Kohat 74 and is considered as our sample hard water that will be used in the experiment. The purpose 75 of finding maximum hardness was to establish the amount of maximum hardness a hair can 76 encounter in Khyber Pakhtunkhwa, Pakistan. Table 3 gives us complete profile of average 77 hardness and pH of water sample from district Kohat (table 3). 78 79 Hair Samples: 80 Hair samples were collected from Peshawar, the area with minimum average water hardness. 81 The reason behind is to observe the effect on hair that has been exposed to minimum 82 hardness in an area after it is exposed to water with more hardness i.e. water from Kohat with 83 almost double the hardness as that in Peshawar (table 2). 84 70 young and healthy male individuals, aged 20-30, with straight and at least 24 cm long 85 straight hair with no history of cardiac disease,17 skin infection (fungal),18 no hair loss 86 patterns, autoimmune disease (alopecia), chemotherapy, smoking,19 diabetics,20 were selected

2 87 after a written informed consent. Hair achieve maximum diameter during age 20-30 years 88 (thus has maximum strength at this age) and the contents of hair are mostly proteins, whereas 89 straight hair has a uniform diameter.13 90 The minimum length was kept 24 cm as each hair had to be divided in to three equal halves. 91 Thus each hair of the 70 samples was divided in to three equal parts and thus three groups 92 were made namely Group A, Group B and Group C, with 70 hairs in each group. Each hair of 93 all three groups was tied to a glass rod (figure 1), labeled for the sake of identification and 94 stored in a controlled temperature room at 22˚C±3.

95 96 Figure 1: Hair tied up to glass rods

97 98 Hairs in group A were left as such to keep it as a control Group. Hairs of group B were 99 treated with de-ionized water whereas hairs of group C were treated with hard water from 100 district Kohat. Each hair of group B and group c was treated with de-ionized water and hard 101 water respectively, for 10 minutes on alternate days and for 3 months. The experiment was 102 designed to give hair an exposure to hard water and de-ionized water as it gets during 103 bathing, washing etc. The whole experiment was performed at 22˚C ±3 and fresh samples of 104 both de-ionized and hard water were provided. 105 106 Tensile strength of each hair, of all three groups, was then measured with the help of 107 Universal Testing Machine (UTM) (M500-1000KN, United Kingdom), available at the 108 Centralized Resources Laboratory (CRL), Department of Physics, Peshawar University, 109 Pakistan and results were stored in a preformed proforma. (Figure 2) 110

3 111 112 Figure 2: UTM machine (Left), Hair in clamps of UTM machine (Right)

113 114 Tensile strength is the measure of tensile stress applied to a material. In UTM each hair was 115 stretched at a speed of 1 mm/second. 116 117 STATISTICS: 118 Tensile strengths of hairs in Group A represented actual strength of hair where those of group 119 B and group C represented tensile strengths of hairs after being treated with hard water and d- 120 ionized water respectively. To find whether strength of hair is altered because of the 121 application of de-ionized water and hard water, first tensile strengths of hairs in group A and 122 Group B were compared and then tensile strengths of group A and Group C were compared. 123 This comparison was done by Students’ paired t-test using Statistical Package for Social 124 Sciences (SPSS, Version 20, Inc, Chicago, IL, USA). P<0.05 was regarded to be statistically 125 significant. Results were tabulated as means and ± Standard Error of Mean (SEM). 126 127 RESULTS: 128 United States Geological Survey (USGS) classifies water into four main types based on its 129 water hardness contents.8 (Table 1) 130 131 Table 1: Hard Water classification 8

Classification of Hard Water Hardness in mg/L of CaCO3 Soft 0-60 Moderately Hard 61-120 Hard 121-180 Very Hard >180 132 133 Hardness level and pH values of water samples, collected from 10 different main districts of 134 Khyber Pakhtunkhwa, Pakistan, were measured (Table 2). The unit used was hardness as

135 CaCO3. 136

4 137 Table 2: Average hardness of tap water samples from different districts of KP and their pH Values.

Sample Area Total Hardness as CaCO3 (mg/L) pH 1 Abbottabad 250.88 7.44 2 Haripur 361.58 6.26 3 Swat 302.89 6.22 4 Swabi 280.31 6.05 5 Mardan 281.43 6.31 5 Charsada 293.20 6.45 6 Peshawar 241.98 6.58 7 Kohat 487.47 6.85 8 Karak 423.52 6.51 9 D. I. Khan 403.36 6.78 138 139 Table 3: Total hardness level of water sample collected from Kohat.

Parameters Results pH 6.85 Total Magnesium Compound as CaCO3 361. 58 mg/L Total Calcium Compound as CaCO3 129.95 mg/L Total Hardness as CaCO3 487.47 mg/L 140 141 Significant decrease was observed in strength of hair when treated with hard water (P=0.001) 142 as compare to hair treated with de-ionized water (p= 0.609) (Table 4) 143 144 Table 4: Comparison of Mean Tensile Strength of Group A (Control), Group B and Group C compared using 145 students paired t-test (N=70)

Mean Std. Deviation Minimum Maximum SEM P Value (N/mm2) (S.D) (N/mm2) (N/mm2) Tensile Strength 255.49 57.55 135.23 371.26 6.88 (Group A (control)) 0.609 Tensile Strength 254.84 58.74 111.13 348.58 7.02 (Group B) Tensile Strength 255.49 57.55 135.23 371.26 6.67 (Group A (control)) 0.001 Tensile Strength 234.16 56.25 113.18 355.38 6.72 (Group C) 146 Values = Mean ± SEM. 147 148 149 150 DISCUSSIONS: 151 The importance of hairs cannot be denied in an individual’s life and extra care is taken in 152 their handling in order to avoid any damage to them. Use of oils, conditioners and shampoos 153 are in routine practices. Interaction of hair with water is also very common e.g. during 154 bathing and washing etc., but the contents of water can have a negative effect on hair. This 155 study was conducted to evaluate the relation of hard water on strength of hair and it was 156 shown that hard water has a significant effect in decreasing the strength of hair that was 157 similar to work done previously by Luqman et al.21 In this study hair were divided into 3 158 groups and tensile strength of control hair was compared with tensile strength of hairs treated

5 159 with de-ionized water and hard water (from district Kohat). Similar study was carried out by 160 Srinivasan et al3 and Evans et al14 and revealed no significance of hard water on strength of 161 hair. The reason perhaps is that Srinivasan collected hair samples from women and in our 162 study hairs were taken only from male individuals, pointing towards the role of gender.

163 Besides the hardness level of hard water used in both Srinivasan (212.71mg/L of CaCO3) and

164 Evans (272mg/L of CaCO3) study was almost half that of hard water used in our study (i.e. 165 486.7mg/L of CaCO3. Furthermore, the hair samples in our study were exposed for a longer 166 period of time and the sample size was much larger than either study. Also the use of de- 167 ionized water (which is more pure form of water) in our study, rather than distilled water may 168 have played a role in different results. There was no specific relation or pattern observed, 169 when age and strength of hair were compared. 170 Normally, we observe a frequent interaction between water and hair, but the damage is not 171 that evident. It is because, the process metal uptake by hair (oxidation) and thus hair damage 172 (oxidative damage) is slowed down by the use of different chelants in form of shampoos, hair 173 conditioners etc. The chelants lead to removal of metal ions from water by surrounding them 174 10 and thus decreases the chances of interaction between hair and those metal ions. 175 The addition of acids, e.g. citric acid in to shampoos and conditioners and the also the use of 176 topical application of yogurt and lemon (containing lactic acid and ascorbic acids 177 respectively) in some areas helps decrease the action of metal ions on hair. The acidic 178 environment (especially pH less than 5.5) has a number of implication that include desorption 179 of metal ions, side chains re-protonation and shrinking of hair scales; thus, prevents the entry 180 of metal ions in to the hair structure 10 and thus minimizing the hair damage. 181 In our study we observed a significant statistical decrease in strength of hair after treating 182 them with hard water. However, the results may also vary with different hardness levels and 183 sample size. The mechanism, of how this happens, is much debatable and need further 184 comprehensive studies, especially on molecular levels. 185 CONCLUSIONS: 186 Our study helped us in concluding that hard water plays a significant role in 187 decreasing strength of hair; after treatment with hard water and de-ionized water and thus 188 results in increase hair breakage. 189 AUTHORS’ CONTRIBUTIONS:

190 1. Dr. Muhammad Waqas Luqman: Study conception, design, sample collection, experimental work, and data collection. 2. Dr. Muhammad Haris Ramzan: Helped in experimental work and critical revision. 3. Dr. Usama Javaid: Helped acquisition of data, sample collection. 4. Dr. Roshan Ali: Supervised the study and final approval of the manuscript to be published. 5. Dr. Muhammad Shoaib: Helped in drafting of manuscript. 6. Muhammad Ayyas Luqman: Helped in analysis and interpretation of data.

191 192 CONFLICT OF INTERESTS: 193 Authors declare no Conflict of interest. 194 195 FINANCIAL SUPPORT:

6 196 It was self supported and no financial favors were taken from any company or institution. 197 198 ACKNOWLEDGMENTS: 199 All Authors present immense gratitude to Khyber Medical University-Institute of Basic 200 Medical Sciences (KMU-IBMS), Peshawar, Pakistan, Centralized Resource Laboratory 201 (CRL), Physics Department, University of Peshawar, Pakistan and Pakistan Council of 202 Scientific and Industrial Research (PCSIR) Laboratories, Peshawar, Pakistan and its staff for 203 their help and support in carrying out different experimental procedures involved in our 204 study. 205 206 207 BIBLIOGRAPHY: 208 1. Ghanaat M. Types of hair loss and treatment options, including the novel low- 209 level light therapy and its proposed mechanism. Southern medical journal. 210 2010;103(9):917-21. Epub 2010/08/07. 211 2. Ellis JA, Sinclair RD. Male pattern baldness: current treatments, future 212 prospects. Drug discovery today. 2008;13(17-18):791-7. Epub 2008/07/12. 213 3. Srinivasan G, Srinivas CR, Mathew AC, Duraiswami D. Effects of hard water 214 on hair. International journal of trichology. 2013;5(3):137-9. Epub 2014/02/28. 215 4. Kiso M, Tanaka S, Saba R, Matsuda S, Shimizu A, Ohyama M, et al. The 216 disruption of Sox21-mediated hair shaft cuticle differentiation causes cyclic alopecia 217 in mice. Proceedings of the National Academy of Sciences of the United States of 218 America. 2009;106(23):9292-7. Epub 2009/05/28. 219 5. Calcium and Magnesium in Drinking-water: Public Health Significance: World 220 Health Organization; 2009. 221 6. Sengupta P. Potential Health Impacts of Hard Water. International journal of 222 preventive medicine. 2013;4(8):866-75. Epub 2013/09/21. 223 7. Momeni M, Gharedaghi Z, Amin MM, Poursafa P, Mansourian M. Does water 224 hardness have preventive effect on cardiovascular disease? International journal of 225 preventive medicine. 2014;5(2):159-63. Epub 2014/03/15. 226 8. Briggs JC, Ficke JF. Quality of rivers of the United States, 1975 water year; 227 based on the National Stream Quality Accounting Network (NASQAN). USA: 1977. 228 9. Tobin DJ. Hair in Toxicology : An Important Bio-Monitor. London, UK: Royal 229 Society of Chemistry; 2005. 230 10. Evans AO, Marsh JM, Wickett RR. The uptake of water hardness metals by 231 human hair. J Cosmet Sci. 2011;62(4):383-91. 232 11. Chen N, Bhushan B. Morphological, nanomechanical and cellular structural 233 characterization of human hair and conditioner distribution using torsional resonance 234 mode with an atomic force microscope. Journal of microscopy. 2005;220(2):96-112. 235 12. Quadflieg JM. Fundamental properties of Afro-American hair as related to 236 their straightening/relaxing behaviour: Bibliothek der RWTH Aachen; 2003. 237 13. Robbins CR. Chemical and Physical Behavior of Human Hair: Springer; 2012. 238 14. Evans AO. Investigation of the Interaction between Water Hardness Metals and 239 Human Hair: University of Cincinnati; 2011.

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