A Study of the Levels of Some Toxic Substances present in Dry Holi Colours in Kolkata, India

Abstract

‘Holi’ is an Indian festival with a great cultural context, that is celebrated across the world at the onset of spring by applying dry powder of vibrant colours on friends and family. In ancient times holi colours were prepared from different spring flowers, but in modern times, these natural colours have been replaced by commercial industrial dyes prepared by chemical processes. Even products that claim to use organic colours, use synthetic pigments to enhance the brightness of hues. Such synthetic holi colours are sold as herbal colours, in an unregulated manner, in local markets, and no checks can be enforced on the product composition. Also, the quality and the amount of information about the ingredients of the particular packets are missing. These colours sold in the local market often contain hazardous chemicals such as endotoxins, and heavy metals, like lead, potentially causing moderate to severe health problem. Holi colour samples were randomly collected from different sites in Kolkata, India. Red, pink, violet, green and yellow coloured powders were obtained. The powders were prepared and analysed for lead content by Inductively Coupled Plasma-Mass Spectrometric method. Analysis of endotoxin content of different holi colours was also performed by Limulus Amebocyte Lysate test. The lead content was found to be almost 2 times higher in the holi colours, with yellow pigment having the highest concentration, than FDA Standard for maximum permissible limit in cosmetics, which was taken as a reference for safety limit of lead that is dermatologically applicable. The endotoxin levels are alarmingly high, with almost 35 times the FDA reference for dermatological safety limit. Special attention should be given to lead and endotoxin levels in holi colours as their consequences pose serious health threats. Therefore, quality control measures should be recommended for them, in par with products designed for long-term contact with the skin.

Introduction

‘Holi’ is the joyous festival that originated in India and is celebrated across the world at the onset of spring. This symbolises the surrender of instincts, forgivenessfor the past mistakes, symbolised by embracing each other and throwing dry powder of vibrant colours, called ‘Abeer’, ‘Holi powder’ or ‘Gulal powder’ on them. Distinctions like caste, age, sex and social standing tends to be forgotten by thronging holi colour at each other to make everybody and everything seem equal. Gulal has a very strong cultural context in our land.

In ancient times holi colours were prepared from different flowers that blossomed during spring. Due to the urbanisation, the disappearance of trees, and mass manufacturing, these natural colours came to be replaced by commercial industrial dyes prepared by chemical processes. But some products were claimed to have been prepared using flowers as raw materials; though generally, brightness of synthetic colours are higher than the herbal or organic colours. Such synthetic holi colours are sold as herbal colours, in an unregulated manner, in local markets, and no checks can be enforced on the product composition. Also, the quality and the amount of information about the ingredients of the particular packets are mostly poor; although many vendors claim that their products are harmless to health and environment.

Nowadays, holi colours are sold loose, on the roadside, by small traders or seasonal vendors, who often don’t know the source and sometimes these holi colours come in boxes that specifically say ‘For industrial use only’[1]. Most consumers are not aware of the Package Product Act (PPA) that demands the contents, price and proper address of the manufacturer to be mentioned on the packet containing these colours. US and European regulation authorities are not particular about whether the holi colours should be categorised as cosmetic products or general consumer products. The Prevention of Food Adulteration Act (PFA) covers only edible food staff, but offers no protection to consumers who use synthetic holi colours [2]. These colours sold in the local market often contain hazardous chemicals and heavy metals, like lead (Pb) which potentially cause moderate to severe health problems [3].Lead is the most dangerous of all the heavy metals found in holi colours. It can affect the nervous system, kidneys and the reproductive system. Among children, it can affect the physical and mental growth, even in small quantities. If a pregnant woman is exposed to lead, it can be carried to the unborn child and damage its nervous system. It can even result in premature birth, low birth weight, miscarriage or abortion[4].

Even fungal contamination of such holi colours has been seen [5]. As an aftermath of Holi celebration, a significant number of people suffers from skin issues (dermatosis/ dermatitis) [6], respiratory tract infection [7], or eye, including temporary blindness, conjunctivitis, corneal aberration and ocular damage [8]. After holi, methemoglobinemia is very common in children [9]. Being poorly biodegradable holi colours are harmful to the environment too [10].

The present study ventures into the complications caused by unregulated use of such dry holi colours, mainly because of the heavy metals, like lead present in them.

Materials and Methods

Holi colours are mostly adulterated with pigments, which contain high amount of heavy metals, commonly Pb. A myriad of colours – red, pink, violet, green, yellow, etc. are mostly available in powder form. In the present study, 200 holi colour samples were randomly collected from 40 different locations in and around Kolkata, India between 22nd February 2020 and 2nd March 2020. From each location, 50 gm of each of red, pink, violet, green and yellow holi powder were procured, in polyethylene pouches for the analysis. During sampling, special attention was taken to collect holi colours manufactured in different parts of the country in sealed packets of various sizes as well as in loose form also. Product information like origin of country, composition and indications for impact on health on the packaging of the products are depicted in Table 1.

Table 1.

Available information on the different powder form of holi colours in product insert

Colour	Red	Pink	Violet	Green	Yellow
Characterisation of colour	Perfumed herbal holi powder	Perfumed herbal holi powder	Holi powder used at holi festival	Holi gulal	Herbal holi powder
Country Manufactured	India	India	India	India	India
MSDS (Material Safety Data Sheet)	Not Available	Not Available	Not Available	Not Available	Not Available
Regulation	Not Available	Not Available	Not Available	Not Available	Not Available
Ingredients	Herbal	Herbal	Herbal	Herbal	Herbal
Recommended application	Holi festival	Holi festival	Holi festival	Holi festival	Holi festival
Warning	Environmentally friendly, Non toxic	Environmentally friendly, Non toxic	Environmentally friendly, Non toxic	Environmentally friendly, Non toxic	Environmentally friendly, Non toxic
Price printed	Rs. 40 per 100 gm	Rs. 40 per 100 gm	Rs. 40 per 100 gm	Rs. 40 per 100 gm	Rs. 40 per 100 gm
Price in open market	Rs. 20 per 100 gm	Rs. 20 per 100 gm	Rs. 20 per 100 gm	Rs. 20 per 100 gm	Rs. 20 per 100 gm

The powders were prepared and analysed for the Pb content in each of them

Analysis of Pb Content of Different Holi Colours by Inductively Coupled Plasma – Mass Spectrometric (ICP-MS) Method

0.5 gm of each type of holi colour sample was processed in duplicate and then digested using close vessel microwave digestion. All samples were digested with 1(N) nitric acid (HNO₃) and 30% of hydrogen peroxide (H₂O₂) in the ratio 5:1 and are subjected to 3 steps in microwave digestion for 40 min programme [11]. The programme is described in Table 2.

Table 2.

Microwave digestion programme used for holi colours

Steps	Temperature	Time	Pressure	Power
I	130 °C	10 min	29 psi (Pounds per Square inch)	400 W (Watt)
II	150 °C	05 min	29 psi (Pounds per Square inch)	400 W (Watt)
III	180 °C	25 min	29 psi (Pounds per Square inch)	400 W (Watt)

After digestion, the final extract was diluted with 50 ml de-ionised (Milli Q) water in acid washed 50 ml volumetric flask and each sample was filtered through a 0.45 µm Whatmann paper. The samples were digested along with the reagent blank in triplicates. Each sample was analysed with ICP-MS (iCAP RQ Series with Qtegra Intelligent Scientific Data Solution, ISDS Software, Thermo Fisher Scientific, USA) to detect and quantify Pb in holi colour. The operational parameter settings of ICP–MS are shown in Table 3.

Table 3.

The operational parameter settings of ICP-MS for lead (Pb) analysis

Element	Radio frequency power supply	Cool gas flow rate	Intermediate gas flow rate	Nebulizer gas flow rate	Dwell time
Lead (Pb)	1404 W	13 L/min	0.7 L/min	0.98 L/min	10 ms/peak

For blanking, a sample of colour was chosen in a way that it contained negligible metal ion concentration. The analyte, Pb concentration was deducted during every calculation. So, this blank sample was only matrix without any amount of Pb. This blank sample was marked as ‘zero’ matrix sample. This blank sample was further spiked at six points covering the expected lower limit and new processed range, including lower limits of quantification (LLOQ). For the calibration curves these standard solution mixtures were diluted 2% HNO₃ and solution of seven points, excluding blank test solution were prepared. Three replicates were prepared for Pb concentration.

The content of Pb obtained from ICP-MS analyser in mg/L was converted into mg/kg using following formula [12]

$$\text{Lead}\left(\text{Pb}\right)\left(\text{in mg/kg}\right)=\frac{\begin{array}{c}\left[\text{Concentration of Pb in the sample digest}\left(\text{in mg/L}\right)\right)\\ \left(\text{- Concentration of Pb in blank digest}\left(\text{in mg/L}\right)\right]\times \text{Volume of}\\ \text{sample}\left(\text{in ml}\right)\\ \end{array}}{\text{The weight of the holi colour sample}\left(\text{in gm}\right)}$$

Also, Qtegra ISDS software was used for data computing.

Till date, no formulated or standardised guidelines are available for validation of Pb in holi colours. The present study followed a method of validation for tattoo pigments using ICP-MS [13]. The validation schedule was designed according to International Conference on Harmonisation (ICH) guidelines [14].

The instrument detection limit (IDL) was calculated by aspirating the reagent blank ten times and multiply it by three times of the Standard Deviation (SD) of the replicated measurements. The Minimum Detection Limit (MDL) or Limit of Detection (LOD) of Pb in the matrix blank that was measured with statistical certainty. The Limit of Quantification (LOQ) was calculated on ten digested blanks and expressed as tenfold the SD of the replicated measurement divided by the slope of the depicted calibration curve.

For the calibration, a linearity curve was determined, based on

y = a x + b, where y is the signal intensity and x is the known concentration of the Pb, b is the intercept and a is the slope.

The linearity of the calibration curve was acceptable, when the correlation factor R² is within 0.999. The uncertainty of measurement (UM) represents an interval of 95% to the true value.

Accuracy was calculated by conducting a recovery study on nine digested blanks at three spiked levels.

Accuracy was calculated as percentage recovery by the assay of known amount of Pb in the sample and the acceptable criteria is between 70 and 120% for the percentage of recovery.

Inter-day repeatability was determined by analysis the digested colour sample 10 times on three consecutive days. Relative standard deviation (RSD) was calculated for the analysis of precision and the acceptable criteria for RSD is 20%.

The validation data for the analysis of Pb in holi colours by ICP-MS are summarised in Table 4.

Table 4.

Method of validation for lead (Pb) in holi colours

Atomic Mass of Metals	Range (in µg/L)	Calibration curve equation	R2	IDL (in mg/kg)	LOD (in mg/kg)	LOQ (in mg/kg)	Recovery (in %)	RSD (in %)	UM (in %)
208Pb	0.5—60	y = 1x + 0.0001	0.999	0.001	0.10	0.30	102.69	2.47	2.86

Analysis of Endotoxin Content of Different Holi Colours by Limulus Amebocyte Lysate (LAL) Test

Commercially available starch digested as food ingredient was used as a carrier substance of holi colours. Endotoxin or Lipopolysaccharide (LPS) is found in the outer membrane of gram-negative bacteria. We measured the amount of endotoxin in different holi coloursusing commercially available Limulus Amebocyte Lysate (LAL) test kit (Aayur Life Sciences, India) as per manufacturer guidelines. Unit of measurement was set as Endotoxin Units per millilitre (EU/ml), which is explained as one unit of EU, which equals approximately 0.1 to 0.2 ng endotoxin per ml of solutions.

The statistical package (SPSS, Version 17.0) was used for the statistical analysis. Statistical significance was aimed to have been achieved with p-value was equal to or less than 0.001.

Results

The present study is based on the determination of concentration of heavy metal Pb and endotoxin level in holi colours. Their amounts are depicted in Table 5, as the mean concentration of Pb and the standard deviation in the readings. It also reflects the amount of endotoxin in different holi colours as measured by the LAL tests value.

Table 5.

Lead (Pb) content and endotoxin level in the various holi colours

Parameters	Number of Samples	Lead (in mg/kg)	Endotoxin (in EU/ml)
Red	40	29 ± 03	2.31 ± 0.16
Pink	40	30 ± 04	2.46 ± 0.21
Violet	40	31 ± 06	2.01 ± 0.09
Green	40	32 ± 03	2.91 ± 0.14
Yellow	40	37 ± 05	1.96 ± 0.19
FDA Standard for maximum permissible limit in cosmetics		20	0.06

Values are mean ± SD

Table 5 shows the concentration of lead in the holi colours; which is a highly toxic heavy metal harmful even in the smallest amounts for human beings. It has been proven by various studies that one major route of entry of Pb in human body is via absorption through skin [5]. The safe limit of cutaneous absorption of Pb is 20 mg/kg as per FDA [15]. Being in powder form, it also poses significant health hazards if Pb is inhaled through holi colours, specially in infants. Also, Pb is known to adversely affect the functioning of bone, brain and kidney and other organs.

The concentration of Pb is present in the range of 25–42 mg/kg.

The Pb concentrations in various holi colours in decreasing order are:

$$\text{Yellow > Green > Violet > Pink > Red}$$

The presence of endotoxin in Holi colours are much higher than recommended in cosmetics as per FDA, which is 0.06 EU/ml [16]. The endotoxin levels in holi colours are found to be in the range of 1.77 – 2.67 EU/ml.

Both Pb concentration and endotoxin levels are much higher than the recommended level as per FDA for cosmetics Act and significantly higher in them as compared to the FDA recommended value (p< 0.001).

Discussion

Holi colours can potentially be harmful to human health and environment; they can contain a considerable amount of Pb with higher level of endotoxin, which is a potent inducer of various pro-inflammatory cytokines [16]. In holi colours, chemicals are introduced to get different shades as the present study. It was concluded that yellow and violet holi colours showed maximum amount of Pb; interestingly these colours have been reported to cause allergy, skin discolouration, dermatitis, dermatosis and irritation of mucous membrane [17]. In a study of 42 patients with holi dermatoses, it was found that itching was the most common symptom involving almost 60% of sufferers. Itching is followed by burning sensation, pain and oozing. Among the clinical signs eczematous lesions were the commonest, followed by erosions, scaling, and erythema. Even nail fold infections were seen in such patients. Scaling of hands were common in those who prepared such colors [5, 17]. Majority of coloring agents used in ‘holi’ are synthetic dyes of non-food commodities such as textile, paper and leather. Most of the synthetic dye-based ‘holi’ powder contain heavy metals. Scientific tests have verified that these can cause skin and eye irritation, allergy and can even trigger asthma.

Ocular injuries during holi primarily involve the young population, and so, have an adverse effect on the disability adjusted life years and the nation’s productivity [6]. On microscopy, it was found that abnormal extraneous deposits are present in the layers involving the corneal epithelium and stroma. The amount of deposition decreased from epithelium to the deep stroma, corresponding with the depth of diffusion of the toxins, with the maximum being in epithelium. It was accompanied with keratocyte loss [8].

Excessive use of holi colours in this festival will result in a considerable increase in suspended particulate matter concentration in ambient air; which are related to a higher cardiovascular and respiratory disorders [17]. In present study, the presence of endotoxin in different holi colours, which can be responsible for considerable cytokine secretions. It was reported with respect to the eye, the chemicals, their constituent endotoxin and fungal contamination produce ocular surface toxicity by induction of oxidative bursts in granutocytes [2]. Holi colours can potentially be harmful to human health: they contain a considerable number of particles with an aerodynamic diameter smaller than 10 μm and at least in vitro they show a close association with human leukocytes, a pro-inflammatory potential, they can have cytotoxic effects in higher concentration and can induce an oxidative burst in human granulocytes and monocytes [3]. So, now time has come to appreciate how much damage can be caused to the body in a single day, by a single event. These holi colours can potentially damage health in various capacities, even as it has adverse effects on cells and have an inflammatory effect on our body. For children, the damage causing capacity is manifold increased. It’s time for a change, understanding that the essence of the festival remains with organic holi colours from vegetable-based dyes and with effective vigilance of regulatory authority. Medical researchers recommend that use of such toxic colors should be discouraged, and all doctors should caution people against using synthetic dyes. This case report highlights the need to put manufacturing of Holi colors under guidelines of the Food and Drug Cosmetic Act and the Bureau of Indian Standards. Colors which are used during the festival merit examination.

Declarations

Conflict of interest

The authors declare no conflict of interest in the present study.

Human and Animal Rights

No specific permits were required for these described field studies because sample collection doesn’t involve any endangered or protected human, animal or plant species or privately owned locations.