N, N Diethyl-P-Phenylene Diamine (DPD) Free/Total Chlorine, Coliform and Heterotrophic Bacterial Profile of Polyethylene Terephthalate (PET) and Sachet Water Sold Within and Around University of Benin Campus.

ater quality, free/total chlorine (Cl 2 ) and bacteriological standard, determine the shelf life of water in the hands of the consumers. This study was carried out to determine the free/total chlorine, coliform and heterotrophic bacterial profile of Polyethylene Terephthalate (PET) and sachet water sold within and around the University of Benin Campus. PET/sachet water was randomly sampled from sales vendors and transported to the laboratory in ice condition for analysis. Standard microbiological methods were followed for the determination of the coliform and heterotrophic bacterial profile of the sampled water. The digital colorimeter methods with N, N Diethyl-P-Phenylene Diamine (DPD) pillow for free chlorine (DPD 1) and total chlorine (DPD 4) determination in water was used. The free chlorine from PET/sachet water samples of UNIBEN, Faithmark, Dandori and Olivia were < 0.1 mg/l, falling short of the recommended World Health Organization (WHO) and Center for Disease and Prevention (CDC) permissible limit (0.2 to 0.5 mg/l). The coliform bacterial counts from this study were recorded as <1.8 MPN, which is less than the WHO and CDC recommended permissible limit. Whereas, the Heterotrophic bacterial counts were well above the recommended limit (> 500 cfu/ml). A proper hygienic regime should be employed during the processing, packing, distribution and sales of PET/sachet water to eliminate high levels of heterotrophic bacteria, which may contaminate the water.


INTRODUCTION
Polyethylene Terephthalate (PET) and sachet water are major sources of water consumed by students, staff and residents of the University of Benin Campus and environ.The quality of these water viz-a-viz free/total Cl2 and bacteriological standard may determine the shelf life or longevity of this water in the hands of the consumers.
According to World Health Organization (WHO) report in 2009, over 80% of diseases in developing countries are water or sanitation-related (Prasanna and Reddy, 2009).Several water treatment methods such as chlorination, ozonation, coagulation, ultraviolet purification and reverse osmosis have been widely used by most PET/sachet water producing companies.Among these methods, chlorination techniques are widely used because it is easy to set up and cost-effective (CDC, 2014).
The process of water treatment using chorine disinfection is the introduction of free chlorine or other oxidizing agents in a gaseous, liquid, or powdered form (Gitis and Hankins, 2018).The greatest undesirable side effect of the introduction of chlorine into water is the formation of trihalomethanes.Trihalomethanes (THMs) are a compound that arises from the reaction of free chlorine with organic matter present in the feed water (Liang and Singer, 2003).
Elevated THMs levels may cause adverse reproductive defects and are a potential carcinogen. Figure 1   The WHO recommended maximum value for free chlorine residual in drinking water is 5 mg/L while the minimum recommended WHO value for free chlorine residual in treated drinking water is 0.2 mg/L (WHO, 2011).The occurrence of chlorine residue in drinking water may account for two major reasons; an indication that a sufficient amount of chlorine was added to the water during the process of treatment to inactivate bacteria and viruses, secondly the water is protected from recontamination during transportation to homes and storage.Center for Disease Control and Prevention (CDC) recommends not exceeding 2.0 mg/L due to taste concerns, and chlorine residual decays over time in stored water (CDC, 2014).
There are three methods to detect free Cl2 in drinking water, such as pool test kits, color-wheel test kits and digital colorimeter methods.However, N, N Diethyl-P-Phenylene Diamine (DPD) Free/Total Chlorine detection method AJHSE 3(1) Okolafor & Idemudia, 2022 | 56 remains a viable method in that DPD method has a 0.035 mg/L detection limit at 5% accuracy and precision (Yang et al., 2007).
This study was carried out to decipher the Free/Total chlorine, coliform and heterotrophic bacterial profile of Polyethylene Terephthalate (PET) and Sachet water sold within and around the University of Benin Campus.

MATERIALS AND METHOD STUDY AREA
The study was carried out within the University of Benin Campus and environ.University of Benin Campus lies between latitude 6.3998° N, and longitude 5.6099° E (Figure 2).

CALIBRATION OF CAMSPEC M501 UV-VIS SPECTROPHOTOMETER/WAVELENGTH SCAN
The calibration followed the manufacturer's instruction in order to determine the absorbance of DPD tablet dissolved in the test samples.A blank reference solution containing distilled water was used to blank the spectrophotometer.
One tablet of DPD 1 and DPD 4 was dissolved in 10 ml test samples and placed into a quartz cuvette for scanning.
After about 2 to 3 minutes routine scanning, absorbance peaks were displayed and interpreted as 530 nm (Figure 3).

DETERMINATION OF THE STANDARD CURVE FOR THE TEST WAVELENGTH
The standard curve provided by the manufacturer was used to determine the concentration of absorbance of the DPD tablet on the test water samples.Following the standard curve of the manufacturer, the absorbance was converted to concentration using the following formula in equation 1 below: Okolafor & Idemudia, 2022 | 57  = 10.00 *  * 1 ----Equation 1C: concertation, r: regression co-efficient (default 1.00), A: absorbance For low and mid-range free Cl2 assay, 1 tablet of DPD 1 pillow tablet weighing about 1 g was added to the already measured 10 ml of the water sample.The content was swirled to mix and allowed to dissolve completely within one minute of addition before being read on the Camspec M501 spectrophotometer at wavelength 530 nm.

QUALITATIVE DETERMINATION OF TOTAL Cl2 CONTENT OF WATER SAMPLES USING DPD TABLE
For low and mid-range total Cl2 assay, 1 tablet of DPD 4 pillow tablet weighing about 1 g was added to the already measured 10 ml of the water sample.The content was swirled to mix and allowed to dissolve completely within 3 to 6 minutes of addition before being read on the Camspec M501 spectrophotometer at optical density (OD) 530 nm.

DETERMINATION OF COLIFORM IN PET AND SACHET WATER
The endpoint dilution method also known as the MPN method was used to determine the presence of coliform bacteria in the PET and Sachet water sold within and around the University of Benin Campus following the method described by Blodgett (2005).The result of the test was read using a McCarthy statistical chart for MPN analysis.

RESULTS AND DISCUSSION
Free chlorine is the amount of available chlorine that is yet to combine with chlorinated water to effectively sanitize contaminants.The WHO, CDC and EU recommended values ranged from 0.2 to 0.5 mg/l.However, the result from this study revealed that the free chlorine from PET/sachet water samples of UNIBEN, Faithmark, Dandori and Olivia waters were < 0.1 mg/l (Figures 4 and 5).This value was less than the recommended free chlorine required to keep the water sanitized during the period of transportation to homes and storage.Most sachet water sold within and around the UNIBEN campus becomes rancid in two months of storage.This common trend in water may be attributed to the low free chlorine levels in the water sampled.CDC recommends not exceeding 2.0 mg/L due to taste concerns, and chlorine residual decays over time in stored water (CDC, 2022).The majority of PET/sachet water-manufacturing companies use the pool test kit and color wheel test kit method for the determination of free and total chlorine in the water.These methods are commonly used owing to their low cost and ease of usage, however, the drawbacks of this test method include the degradation of the orthotolidine (OTO) solution that causes inaccurate readings over time, generally not reliable quantitative results, lack of calibration and standardization and potential for user error.The digital colorimetric method using the DPD tablet remains the best option for free chlorine determination in water because of its highly accurate readings and fast results even though it is quite expensive (CDC, 2022).
The bacteriological quality of PET/sachet water is a major concern because staffs and students consume this water.The coliform and heterotrophic bacterial profiles of PET and Sachet water sold within and around UNIBEN Campus are presented in Table 1.The coliform bacterial counts from this study were recorded as <1.8 MPN, which is less than the WHO, CDC and EU recommended permissible limit.However, the Heterotrophic bacterial counts were way above the recommended limit (> 500 cfu/ml).The result of this investigation showed that UNIBEN, Faithmark, Dandori and Olivia PET/sachet waters sampled were free from all levels of oral-fecal contamination.The high level of heterotrophic bacteria may be attributed to improper safe housekeeping, low hygiene standards in the factory, and rough handling of water by factory workers and distributors.Less than 7% of sachet water contamination occurs during the production of the water while 40 and 45% of contamination happened during distribution sheds and the street hawkers (Omalu et al., 2010).Oladipo et al. (2009) posited that consumption of contaminated water facilitate widespread infections and can ultimately lead to outbreak or epidemic.Bottled water containing a high microbial load may cause significant morbidity to immune-compromised persons (Allen et al., 2004;Pavlov et al., 2004)

CONCLUSION
The quality of PET/sachet water is not limited to the processes involved in the purification, package and distribution rather it also entails meeting with quality standards set by agencies poised with the responsibility to regulate water consumed by the populace.The methods employed by majority of PET/sachet water production companies with regards to free chlorine determination should be reviewed.It has been widely reported by CDC and other regulating agencies that N, N'-Diethyl-P-Phenylene Diamine (DPD) Free/Total Chlorine detection methods remain the best option to accurately determine the free chlorine levels of treated water.PET/sachet water producing companies should ensure that the free chlorine levels of the water sold to consumers maintain the acceptable WHO/CDC/EU levels (0.2 -0.5 mg/l).Anything short of this range may cause the water to lose its organoleptic properties overtime.Proper AJHSE 3(1) Okolafor & Idemudia, 2022 |60 is a flow chart showing the addition of chlorine to water.

Figure 2 :
Figure 2: Google map of sampling location/area

Figure 3 :
Figure 3: Calibration peaks of UV-VIS spectrophotometer against the test sample (Model 501)

Table 1 :
such as those suffering from HIV-AIDS, novel coronavirus, tuberculosis, malnutrition, etc. Coliform and heterotrophic bacterial profile of PET and Sachet water sold within and around UNIBEN Campus