Kohl: A Widely used Eye Cosmetic with Hazardous Biochemical Composition

1Department of Microbiology, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan. 2Department of Environmental Sciences, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan. 3Department of Chemistry, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan. 4Department of Microbiology, University of Karachi, Karachi, Pakistan. 5Department of Botany, Abdul Wali Khan University, Mardan, Pakistan.

Abu DawudTib and TirmidhiTib. The use of Kohl Al-Ethmed before sleeping was recommended by Prophet Muhammad (Peace be upon Him) for enhanced vision and hair growth 4,5 . Initially, the source of Kohl was a black stone "Ithmed", composed of stibnite ore (Sb 2 S 3 ), also known as Antimony which was believed to have beneficial aspects 6 . This stibnite ore was used alone as Kohl or used in combination with other ingredients such as camphor, menthol, almond seeds, wood essence, charcoal or other vegetable ashes. Later, stibnite ore was gradually replaced by the Galena ore (lead sulphide) which owns the same grey black colour and shiny appearance like stibnite 7 . However, the exact composition of Kohl has long been a matter of dispute within the scientific community 8 .
The beneficial potentials of Kohl have been proved by the studies that fully supported the medicinal benefits of the multiple ingredients found in the Kohl 9 . Earlier, Kohl mixed with rose water was also reported to be instilled in the eyes by community people to treat smallpox pustules 10 . Kohl has also been used for keeping eyes cool and clean, also as a prevention from several eye diseases such as cataract, conjunctivitis, chalazion, trachoma and blepharitis. Moreover, there are some places in world with a common practice to apply Kohl on the umbilicus of newborns to help the healing process 11 . The black and shiny particles of galena protected the eyes from the harmful effects of ultra violet radiations from sunlight and dust particles 12 . Most of the ingredients such as different chemicals, herbs and gemstones are also added in Kohl because of their beneficial influence on the eyesight along with astringent, antibacterial and antiviral effects. Some plant extracts such as Coptisteeta, Fennel (Foeniculumvulgare) and Saffron (Crocus sativus) extracts were also used in the composition of Kohl and considered highly beneficial for improving weak eyesight and to fight against many eye diseases such as catarrhal and rheumatic conjunctivitis 2 .
Besides, Kohl has also gained a negative reputation after the use of galena stone as a major constituent rather than antimony ore, as it resulted in lead intoxication due to high content of lead in Kohl samples 13 .Many studies have discouraged the use of Kohl due to its possible health hazards. The possible lead toxicity in eye cosmetic (Kohl) consumers was studied in California, where the users were mainly the children from Pakistan and India as their lead level was observed as 12.9 µg/ dL while it was only 4.3 µg/dL among children not using Kohl cosmetics. 14 However, certain studies also reported no association of increased blood lead level with application of Kohl 15 . The contamination in Kohl samples with pathogenic bacterial and fungal strains has also been reported earlier as a reason of spreading ocular infections among many individuals 10 .
The objective of this study was conceived after the incident reported in Sydney (Australia) where three children got severely ill due to lead toxicity caused by regular use of Kohl.The current study was designed to conduct the microbiological and chemical analysis of the Kohl samples available in different local markets of Karachi City, Pakistan.

Collection of the Kohl Samples
A total of 20 samples (branded and nonbranded) of the traditional Kohls were collected, randomly, from different local markets of Karachi city, and transported to the microbiology research laboratory, Federal Urdu University of Arts, Science and Technology (FUUAST) for further analysis.

Detection of antimicrobial potential of the Kohl samples
Antimicrobial potential of different Kohl samples was detected against different bacterial and fungal reported pathogens of ocular infections, as mentioned below.

Antibacterial potential of the Kohl Samples
The antibacterial effect of the Kohl samples was investigated against different clinical strains of three Gram-negative (K. pneumoniae, P. aeruginosaand P. mirabilis) and two Gram-positive bacteria (S. aureus and S. epidermidis), obtained from Lab of Applied Microbiology and Clinical Mycology, Department of Microbiology, FUUAST. The suspensions of kohl samples were prepared by adding one gram of kohl in 2 ml of sterile distilled water and vortexed well.The antibacterial activity of Kohl samples was assessed by Agar-well diffusion method 16 .

Antifungal potential of the Kohl Samples
Antifungal effect of the Kohl samples was investigated against pathogenic isolates reported for ocular infections, including two yeasts (C. albicansand C. tropicalis) and three molds (A. flavus, F. oxysporum, and Mucorsp.) obtained from Lab of Applied Microbiology and Clinical Mycology, Department of Microbiology, FUUAST. The suspensions of kohl samples were prepared by adding one gram of kohl in 2 ml of sterile distilled water and vortexed well. The antifungal activity of these Kohl samples was determined by Agar-well diffusion method 17 .

Isolation and identification of microorganism from Kohl Samples
For the isolation of microorganisms from the Kohl samples, a serial dilution of each Kohl sample was prepared up to 10 -2 dilution and processed. Identification of the isolated bacterial and fungal colonies was done macroscopically by observing the color, texture and pigmentation of media by microorganisms, while microscopically by staining (Gram staining in case of bacteria and Lactophenol blue staining for fungi isolates), according to established protocol 18 .

Heavy metal analysis of the Kohl Samples
The chemical analysis of Kohl samples was carried out by Flame Atomic Absorption Spectrophotometer (FAAS) (Perkin Elmer-A Analyst 700). The Kohl samples were prepared for this analysis by using wet digestion method, and analyzed, in triplicate, for the estimation of trace metals including arsenic (As), cadmium (Cd), lead (Pb), copper (Cu), iron (Fe) and sodium (Na), using established protocol 19 . The results were expressed    as mean ± SD and compared with BVL standards. According to BVL standards, toxic metals in cosmetic products such as arsenic, cadmium, lead, Mercury exceeding the values 0.5, 0.1, 0.1 and 5 ppm (mg/kg), respectively, should be avoided. 20

RESULTS
In current study, a total of 20 different Kohl (Surma) samples were purchased from different local markets in the Karachi city. Among these Kohl samples, 85% were branded while 15% were unbranded. These Kohl samples were available within the price range of 20 to 90 Pakistani rupees, manufactured locally or imported from India and Saudi Arabia. The antimicrobial potentials of collected Kohl samples were tested against some clinically important pathogens, reported for causing predominant ocular infections. Amongst all, 75% of the total Kohl samples revealed good antibacterial activity. The highest activity was reported against P. mirabilis and S. epidermidis (Fig. 1& 3). Moreover, 30% of the samples demonstrated antagonistic potential against fungal pathogens. The highest activity was observed against Candida and Mucorsp. (Fig 2& 3).
The microbial contamination in collected Kohl samples was also examined by spread plate method. Based on the colony forming unit (CFU), the samples were interpreted as contaminated or free from contamination. Bacterial contamination alone was found in 45% of the Kohl samples while contamination with fungi was observed in 15% of the samples. However, contamination with both bacteria and fungi was detected in 15% of the Kohl samples (Fig.4).
Based on their morphological shape, visualized by Gram's staining, the bacterial isolates were identified as rod shaped (Bacillus) bacteria, while fungal strains were identified as Aspergillusflavus and A. niger, on the basis of their microscopic as well as macroscopic characteristics.
The contamination of different heavy metals such as arsenic, cadmium, lead, copper, iron and sodium in the Kohl samples was analyzed by Flame Atomic Absorption Spectrophotometer (FAAS). The Kohl samples where metals were detected in Below Detection Limit (BDL) were declared as free from heavy metal contamination. Amongst lethal heavy metals (As, Cd and Pb), 90% of the Kohl samples were found contaminated with arsenic metal, however cadmium and lead contamination was detected in 65 and 40% of Kohl samples, respectively. Other than these metals, Cu, Fe and Na metals were also detected from these samples. Both the iron and sodium were detected in 95 to 100 % of the Kohl samples. The results showed complete absence of copper metal in all the Kohl samples (Table1& Fig 5).

Medicinal Importance of Kohl
Kohl, generally known as Surma/Kajal in Indo-Pakistan, has been traditionally used since antiquity in most of the Middle and Far-east countries, having a strong cosmetic, medicinal and religious importance. In respect of medicinal importance, Kohl has been widely used for its therapeutic effects against different eye diseases and for the enhancement of eye vision 21 . Apart from its beneficial importance, detrimental effects of Kohl have also been reported on human health progressively from last few decades due to the presence of microbial contaminants and presence of heavy metals such as lead, arsenic, copper, cadmium etc.in its composition 10 .The present study was focused to investigate both beneficial and injurious aspects of Kohl in various branded and non-branded samples, while the brand names are not mentioned to avoid any possible legal complications. The antimicrobial effect of Kohl samples was investigated against microbial pathogens which are usually involved in ocular infections being well recognized as leading cause of conjunctivitis, blepharitis, dacryocystitis, keratitis and endophthalmitis in the previously reported literature 22 . The study revealed excellent bioactivity of Kohl samples against most of the bacterial strains particularly P. mirabilis and S. epidermidiswhich confirm previous researches, where Kohl showed good antibacterial effect against Staphylococcus and Proteus species 6 . However, previous finings regarding antifungal bioactivity of Kohl is lacking although in the present study many Kohl samples showed antifungal capacity against a number of ocular fungal pathogens. According to current findings, Candida spp.and Mucorspp. were highly inhibited by the Kohl samples. These fungal species are well known ocular pathogens and were recently reported to cause a severe Oculo-rhino-cerebral mucor mycosis in immune-compromised patients 23 . Moreover, Candida and Aspergillus species are also found to be associated with fungal keratitis which has been considered as a common ocular mycosis responsible for blindness in most of the cases 24 .

Microbial Contamination in Kohl Samples
Apart from beneficial importance of Kohl, injurious aspects due to microbial and chemical contamination have also been reported earlier. In current findings, we isolated bacteria and fungi from many Kohl samples. Bacillus and Aspergillus species were found to be the most common contaminants in Kohl samples, which also coincide with a previous study where Kohl, mascara and eyeliner samples were found contaminated with these species 10 . These species are present in environment and therefore their contamination might be due to improper packaging or storage of these kohl samples.

Chemical Contamination in Kohl Samples
The heavy metals are non-essential and non-biodegradable elements and can induce toxicity even at very low doses. Presence of heavy metals in cosmetic products has been associated with many health-related issues due to heavy metals toxicity 25 . However, these toxic metals are now banned or at least restricted by the concerned regulatory authorities of many countries, though permissible limits of metals might be different in many products with respect to various countries 26  Arsenic is one of the most lethal heavy metal having the highest rank in the list of risky substances. Its continuous exposure to humans may lead to severe illnesses such as it reduces the synthesis of erythrocytes and leukocytes, developed skin lesions, neurological problems, pulmonary, peripheral, vascular, cardiovascular diseases, diabetes mellitus and certain types of cancers 27 . In our study, arsenic metal was detected in 90% of the total Kohl samples and 80% of these samples were detected with high metal content (5.912 ppm) according to BVL published standard limits. These findings correlate with the previous studies in which the arsenic content was detected higher within different cosmetic products 28 .
Cadmium is biologically a non-essential heavy metal which is well recognized for its adverse influence on the enzymatic systems of cells 29 . Our results revealed cadmium metal in 65% of the total Kohl samples, in which 30% samples possessed high level of cadmium (1.430ppm) than standard limits. Another study also reported a little lower concentration of cadmium (0.942 ppm) as compared to our findings 30 . Similarly, lead (Pb) is also a non-essential metal, having no nutritional value to living organisms and ranks fifth in the list of metals 31 . Lead is a recognized carcinogen and its minimal exposure to humans can be very toxic causing severe diseases such as headache, hypertension, arthritis, birth defects, paralysis, brain and kidney damages, and even the death 32,33 . Kohl has been reported as one of the major source of lead poisoning mainly in children and it can be absorbed across the conjunctiva by eye rubbing, tearing (lacrimation) while finger licking could further enhance the lead absorption in living systems. Many researchers after laboratory confirmation reported the existence of lead in many Asian Kohls 13 . An analytical study of different types of Kohls in Pakistan also revealed lead concentration within the range of 0.03% to 81.37% 34 . In literature of more than 20 years, many case studies of plumbism (lead poisoning) have been reported, most frequently in infants and children that upon investigation have been attributed to Kohl usage were found with high concentration (199.9 ppm) of lead which is much higher than mandated levels. Another study, however, reported the further higher concentrations (1005 ppm) in Kohl samples 30 .Copper is also known to play a vital role in enhancing and strengthening of eye vision and can inhibit activity of lactate dehydrogenase, which is a sensitive marker of cataract formation 35 . Although this beneficial metalwas not detected in any of our Kohl samples. However, in previous studies, it was detected in high levels from various Kohls. Similarly, another important and beneficial metal Iron was also detected in 95% of the Kohl samples but in very little amount (23.14 ppm) as compared to the previously reported studies 30 .

CONCLUSION
Briefly in this study, we observed that most of the Kohl samples possessing antimicrobial potential against ocular pathogens had high contents of heavy metals. While the Kohl samples with lower content of heavy metals exhibited weak or no inhibitory effect against microbial pathogens. Therefore, the antimicrobial activity of Kohl samples might be due to the presence of high contents of heavy metals which are known to produce bactericidal or fungicidal activities since antiquity 36 . Furthermore, two Kohl samples, SE16 and SE17 demonstrated excellent antimicrobial effect in the absence of toxic metals in their composition. Therefore, the antimicrobial potential of these Kohl suggested their use in ocular infections. However most of the samples were found contaminated with heavy metals particularly samples: SE1, SE2, SE3, SE4, SE5, SE6, SE14 and SE15 were found heavily contaminated with heavy metals such as Lead, Arsenic and Cadmium and all of these Kohl samples were from very famous brands of Pakistan. It was concluded that detection of high contents of heavy metals such as lead, arsenic and cadmium in most of the Kohl samples suggest proper monitoring of locally manufactured or imported cosmetic products in order to ensure the safety of public health.