AN UPDATED WATER QUALITY ASSESSMENT: DETECTION OF AMOXICILLIN

AND PENICILLIN AND THE PRESENCE OF COLIFORMS IN TAMUGAN,

PANIGAN RIVERS, CALINAN AND TALOMO RIVER, DAVAO CITY

An Undergraduate Research Study

Presented to the Faculty of the Biology Program

Davao Medical School Foundation Inc.

In Partial Fulfillment of the Requirements

for the Degree of Bachelor of Science in Biology
____________________________________

Submitted by:

Baricuatro, Nikkolee P.
Diaz, Karl Anthony A.
Patino, Nestor II M.
Senagan, Loise Marie G.

May 2022
 APPROVAL SHEET FOR PROPOSAL DEFENSE

In partial fulfillment of the requirements for the degree of Bachelor of Science in Biology this
RESEARCH entitled:
“AN UPDATED WATER QUALITY ASSESSMENT: DETECTION OF AMOXICILLIN
AND PENICILLIN AND THE PRESENCE OF COLIFORMS IN TAMUGAN,
PANIGAN RIVERS, CALINAN AND TALOMO RIVER, DAVAO CITY”
has been prepared and submitted by Nikkolee Baricuatro, Karl Anthony Diaz, Nestor Patino
and Loise Marie Senagan after passing the Oral Defense.

MA. CATHERINE B. OTERO MARIAN DARA TAGOON

Advise/Mentor Adviser/Mentor

JOHN PAUL BANZON BENITO ANTHONY T. PINGOY

1st Panelist 2nd Panelist

APPROVED in partial fulfillment of the requirements for the degree of Bachelor of Science in
Biology by the Chair of the Department and the Director of Research and Publication Office
(RPO).

_________________________ DR. GENEVIEVE D. TUPAS, FPPS, MMCE

Department Chairman Director

ACCEPTED in partial fulfillment of the requirement for the degree of Bachelor of Science in
Biology.

JOHN PAUL M. BANZON

Dean
 ABSTRACT

Freshwater is of great importance as it provides maintenance, sustenance, source of potable

water, and utilization for anthropogenic activities. However, due to continuous annual population
growth and anthropogenic activities ranging from large-scale industrial and agricultural pursuits,
and personal activities, there has been an ongoing threat in degrading Davao’s water quality. This
study provides updated data regarding the water quality in the major water sources of Davao City
by quantifying coliforms, detecting antibiotics, and assessing physicochemical parameters of the
Tamugan, Panigan, and Talomo Rivers in March, 2022. Flow rate, turbidity, pH, and temperature
were measured during data collection. Water samples were analyzed for enumeration of total and
fecal coliforms by MPN, and the presence of commonly used antibiotics (Amoxicillin and
Penicillin) by HPLC-DAD. The physiochemical parameter analysis shows that Talomo River
exhibits the highest average flow rate having 6.6 lpm, followed by Panigan River (2.43 lpm), and
Tamugan River (0.24 lpm). Panigan and Tamugan River are highly turbid exceeding the
Philippine National Standard for Drinking Water (PNSDW), all rivers have a pH of 7 complying
to WHO and PNSDW (6.5-8.5). The analysis shows that all rivers are highly contaminated with
coliforms and fecal coliforms (170-1,071 MPN/100mL) exceeding the WHO and PNSDW
standard for potable water, however there is a significant decrease in coliform count since 2019
due to different programs executed by government and non-government organizations. No
presence of Amoxicillin and Penicillin were detected. However, water from these rivers requires
annual assessment to ensure that water quality is continuously improving.

Keywords: temperature, high pressure liquid chromatography, amoxicillins, penicillins, World

Health Organization, water quality
 ACKNOWLEDGEMENT

From the creation of our thesis proposal through the completion of our final thesis paper,

we are grateful to Ma'am Maria Catherine B. Otero and Ma'am Marian Dara Tagoon, our thesis

mentors, for their expertise, tireless efforts, and advice. We appreciate their help and valuable

recommendations in helping us complete our research successfully.

We also appreciate Ma'am Glio Florgiley Arabejo's recommendations and suggestions, as

well as Sir Michael Dann Superio, Sir Anthony Pingoy, and Sir John Paul Banzon's support and

presence throughout our sample collecting.

We wish to thank Ma’am Leah Jane Ofima for her help regarding our data analysis.

To the barangay officials of our chosen locations where we conducted our sampling, we

are very thankful for giving us the permission to enter the site.

Finally, we want to express our gratitude to our family, friends, and classmates for their

unwavering support and encouragement throughout the research and as we complete our thesis

successfully.
 TABLE OF CONTENTS

TITLE PAGE i

APPROVAL SHEET ii

ABSTRACT iii

ACKNOWLEDGEMENT iv

TABLE OF CONTENTS v

LIST OF FIGURES vi

LIST OF TABLES vii

INTRODUCTION

Background of the Study

Objectives

Significance of the Study

Review of Related Literature

Definition of Terms

List of Acronyms

METHODOLOGY

Research Design

Setting

Population

Sampling
 Data collection procedure

Ethical considerations

RESULTS

Results

Discussions

Summary/Conclusions

Recommendations

REFERENCES

APPENDICES
 LIST OF FIGURES

FIGURE FIGURE TITLE PAGE

1 Vicinity Map of Davao City 3

2 Method for analyzing samples in HPLC

3 Standardized and water samples of Amoxicillin and Penicillin

 LIST OF TABLES

TABLE TABLE TITLE PAGE

 INTRODUCTION

Background of the Study

Davao City is the prime tourist destination in Mindanao and has been developing its

economy over the years, which resulted in an accelerated increase of population. According to

the Philippine Statistics Authority (2018), there has been an annual population growth rate of

2.30%; from an estimate of only 1.1 million in 2000 to 1.6 million in 2015. Although this is an

improvement for the city’s economy, it also comes with environmental ramifications. As

population rate continues to increase due to urban developments and opportunities, this produces

challenges in the environment. Anthropogenic activities such as large-scale industrial and

agricultural pursuits, as well as personal activities have impacted Davao City’s environment and

ecological system, and as long as species continue to coexist in one ecosystem, this would result

in a cyclic cause-and-effect phenomenon wherein anything that causes biological, chemical or

physiological change in an ecosystem will counteract to the organisms living within the

ecosystem and vice versa (Seebacher & Franklin, 2012). An example of alarming environmental

risk anthropogenic activities initiates is ineffective waste management and water pollution.

Freshwater is of great importance due to its versatility; from providing maintenance and

sustenance in an ecosystem to how it is used on a daily basis as it is a primary necessity that

supports a wide spectrum of species, supply viable source of potable water, and provides

utilization in numerous anthropogenic activities such as agriculture, industrial, and personal use

(Singh, Mishra, Dwivedi &, Mishra, 2017). However, due to the misuse of such an important

contributor to the environment and the needs of numerous species, this has impacted water
quality resulting in water contamination to which water contamination poses a major worldwide

health problem as it gives rise to environmentally-related health threats.

Antibiotics have become the central component for treating different infectious diseases

and it contributed significantly to the global health development during the last 70 years (Wall,

2019). Aside from being mainly used in medical therapeutics, it is extensively used in

conventional agriculture for treatment and prevention from diseases, and also overall therapy

(Kraemer et al., 2019). With antibiotics being primarily seen as a medicine that aids numerous

organisms from infections and diseases, the use of antibiotics also comes with a caveat. While

antibiotics are used to suppress microbial infections, misuse and overuse of such drug may cause

antimicrobial resistance of which the World Health Organization (2020) has deemed

antimicrobial resistance to be one of the most dangerous global public health threats in humanity

as biological fitness of bacteria in response to antibiotics is inevitable and naturally occurring

allowing pathogens to contradict to the principles of antibiotics in which complicates treatments

for infection and disallows the body to respond to antibiotics resulting in life-threatening

situations wherein no drug can alleviate infections. With regards to this, antibiotic residues are

possible sources of antibiotic resistance genes that may contaminate natural environments; not

only leading to environmental pollution, but also potential in spreading antibiotic resistance that

may lead to the use of the wide spectrum of drug compounds for treatment (Mahmood et al.,

2019). Significant existence of antibiotics in the environment is due to multiple factors, including

the release of unabsorbed antibiotics by animals and humans into the water stream, and discarded

residues or unused antibiotics from medicinal practices, laboratories, and factories, are discarded

into the water stream (Kraemer et al., 2019). Thus, makes the presence of these drugs possible in
the environment; to which no studies regarding the detection of antibiotics within the rivers of

Davao city have been conducted.

An ecosystem works in a cyclic biological system wherein chain reactions between

organisms and their environment occur naturally. Waterways, particularly rivers, play a vital role

in disseminating antibiotic resistance genes (Pruden et al., 2013). Waterways that circulate and

are distributed for multiple organisms to use and consume may react to a chain of food webs that

could result in a large-scale effect. For example, a study by Wu et al. (2018) showed the relation

of egret birds carrying antimicrobial resistant genes acquired from contaminated waterways and

spreading AMR genes to its close surrounding through excretion via minimum inhibitory

concentrations method and assessing a variety of antibiotics in opposition to naturally present

Escherichia coli isolates in the environment; to which results show that presence of antibiotic

resistance in near environments to which where egrets circulate, was abundant. Furthermore,

results of the real-time PCR showed that the transportation of antimicrobial resistance genes

could be due to mobile genetic element, together with horizontal gene transfer as colistin

resistant gene mcr-1 was detected from the colistin-resistant E.coli strains of egret feces from the

sample collected from the river. This suggests that the egret birds did, in fact, acquire carrying

AMR from waterways, and are potential contributors to distributing AMR to the environment.

According to Greenpeace Southeast Asia (2007), pollution in natural bodies of water,

specifically the rivers have exposed individuals to environmentally-related diseases. In relation

to this, surface water including rivers in urban areas have become a health threat to the public

while in rural areas become sources of sickness. Furthermore, they also stated that The World

Bank evaluates the exposure of water pollution and poor sanitation of which resulted in one-sixth
of cases reported with around 6,000 premature deaths annually to which thirty one percent of the

diseases in the Philippines are caused by water-borne pathogens.

Apart from detection of antibiotics in specific waterways, this study also focuses on

detecting total coliform bacteria as its presence in waterways serves as an important indicator for

water quality assessment and whether a water supply is contaminated or not (Pepper & Gerba,

2015). Total coliform consists of a wide variety of species that commonly inhabit the

environment and feces of warm-blooded species of which the most common form of coliform

found in drinking water supplies are species of the fecal coliform such as Escherichia coli (E.

coli) (New York State Department of Health, Center for Environmental Health, 2017).

Eschericia coli is an abundant microbial flora and is an important inhabitant of the

gastrointestinal tract of humans and some mammals forming a commensal relationship with

regards to their host (Allocati et al., 2013). Furthermore, these species are capable of transmitting

mild to severe infectious diseases due to its versatility in adapting to different niches, which

enables them to acquire pathogenic and virulence factors (Kapler, Nataro, & Mobley, 2004).

As of 2018, DCWD alongside Apo Agua Infrastructura Inc. initiated a P12-billion

strategic alternative that will change Davao City’s primary source of water from groundwater

wells to surface water due to complications with heavy water consumption from groundwater

sources as statistics have shown that for the last 40 years, the city has been heavily reliant on

groundwater covering 320 million liters of water per day in 2019; suggesting that consumption

of groundwater is used at a more rapid rate causing wells to dry up and land to sink making this a

problem for water quality and allowing contamination in the long run (Llemit, 2019). In addition

to that, if groundwater wells continue to reach a point of degradation at sea level, this will result

in water contamination, which will be an immense problem for the city’s water source. Thus, by
2021, DCWD is aiming for Davao City to rely on the Tamugan watershed as sources of potable

water (Banzon, 2020). Under the Watershed Code (City Ordinance No. 0310-07), the city

prioritizes the quality of raw water in the city by preventing the establishment of large

infrastructures and conventional agriculture using synthetic practices (Ibanez, 2015). However,

human settlement and industrial activities by large companies such as Dole and Highland Banana

Plantations, Davao Fresh Food Corp., Heroes Agriculture venture/Vitarich, and Ana’s Breeding

Farm have caused alarming environmental impacts such as soil erosion and degradation,

flooding in the lowlands, and pollution due to the use of agrochemicals thus, resulting in possible

contamination in the watersheds, which may affect the quality of raw water in the

Panigan-Tamugan watershed (Environmental Management Bureau, 2020). Also, according to the

water assessment of Panigan-Tamugan rivers as stated by Llemit (2019), results showed of high

detection of fecal contamination of fecal coliforms with its most probable number (MPN) was

above the normal scale of detection set by DENR (160,000MPN/100mL > 1.1MPN/100mL).

Similar to the case of Panigan-Tamugan rivers, factories and establishments also surround

Talomo river. Although Davao city is no longer reliant on Talomo river as its primary source of

potable water, the Talomo river still covers 38,000 square meter of land, which serves 53 villages

that gives access to individuals for their personal use making it of greater concern especially

since the water quality assessment of Talomo river as of 2020 stated that the river failed in the

fecal coliform assessment having a result of 135-67005MPN/100mL of water sample throughout

the entire length of the river (Sunstar Davao, 2018; Environmental Management Bureau, 2020).

No further updates have been identified with regards to total coliform assessment and antibiotic

detection therefore, it is only as important to discover such presence of fecal coliforms, and

whether or not changes in MPN have been made over the years to assess whether or not the new
shift in primary water source is indeed safe for consumption and to analyze its correlation to the

possible presence of antibiotics in the rivers.

Objectives

This study aims to assess the water quality of possible water sources in Davao City by

investigating the presence of coliforms, and antibiotics in the Panigan-Tamugan and Talomo

Rivers. Specifically, this study aims to:

1.) Assess water quality of the three rivers with regards to the presence of coliform and

antibiotics and their correlation to physicochemical parameters;

2.) Determine the presence or absence of Amoxicillin and Penicillin in the rivers using

HPLC method; and

3.) Establish the presence of total coliform and fecal coliform by most probable number

(MPN).

Significance of the study

Studies on the presence of antibiotics in our local waterways are lacking. To the

knowledge of the researchers, this research will be the first to explore the presence of antibiotics

in Talomo, Panigan-Tamugan rivers of which the three rivers are situated in upstream river

locations, which mostly cover agricultural, poultry, swamp, brush, and forest lands (Ibanez,

Tampons, Maglinte, & Reazonda, 2012). Therefore, with this surrounding being a cohesive

environment for an abundance of microorganisms to inhabit in due to industrial location and

human inhabitants circulating the said areas that allow for the spread of microorganisms,

presence of total coliform is steadily abundant, contamination of fecal coliform is fairly

common, and detection of antibiotics may be possible. This study will provide baseline

information to the current quality assessment of the rivers in terms of antibiotic pollution and

presence of total coliform; more specifically fecal coliform, and eventually contribute to further

research regarding antibiotics in rivers. In addition to this, Gumba (2020) stated that the

Panigan-Tamugan river’s water quality is clean for drinking consumption. However, due to

environmental factors, the Interface Development Interventions (IDIS) reports that water quality

has degraded its quality from “Class AA” to “Class A” (Cortez, 2017) and as stated by Llemit

(2021) the Panigan-Tamugan watershed faces problems with water pollution and contamination

due to large quantities of garbage wastes found along the river and anthropogenic activities

within the area, which may affect total and fecal coliform count in all three rivers. This would

entail significance in the need for assessing the water quality with regards to the presence of

antibiotics and fecal coliforms especially since surface waters of Tamugan river is set to become

Davao’s new primary water source of which Panigan river is connected to. Furthermore, Talomo

river will also take part in significance since it is adjacent to Panigan-Tamugan rivers and was

Davao’s primary ground water source prior to Panigan-Tamugan rivers to which the shift of

water sources was developed due to rising problems with contamination in Talomo river.

Furthermore, Talomo river covers 38000 square meters of land to which industries and human

settlements making the river readily available for anyone to use, and according to Llemit (2021),

the river is still facing issues with regards to pollution and contamination, similar to

Panigan-Tamugan rivers, due to the abundance of industrial and agricultural companies together

with human settlements surrounding the river making the river’s water quality belonging to

“Class B” to which is a growing concern as the river is a possible carrier of pathogenic

microorganisms that may allow the spread of diseases to individuals that are exposed to the river
(Environmental Management Bureau, 2020). Furthermore, if this study does indicate the

presence of antibiotics in the rivers, the probability of mutated coliforms that are antibiotic

resistant may be possible thus, making the transmission of AMR microorganisms inevitable

since waterways play a vital role to the environment.

Review of Related Literature

Detection of total coliforms and fecal coliforms have been widely used as indicator

organisms that assess microbiological quality of potable water (Mishra et al., 2012). Before so,

detecting factors that affect water quality such as the temperature, pH, turbidity, and flow rate is

as important, since these are possible factors that affect the concentrations of these organisms

(Hayat & Kurniatillah, 2021). Temperature, pH, turbidity, and flow rate are few of the many

factors that participate in influencing water quality; however, for the scope of this study, the

factors mentioned are feasible enough to analyze water quality and how it affects possible

detection of antibiotics and presence of total and fecal coliforms. The WHO standard for

coliform bacteria in water is zero total coliform per 100 ml of water and that any water samples

that contain coliforms must undergo investigation for presence of fecal coliforms (Idibie et al.,

2018). In addition, the Philippine National Standards for Drinking Water (PNSDW) set the

standard total coliform count for potable water at <1.1 MPN/100ml (Department of Health,

2017). The pH of water is an important factor in assessing water quality of which standard pH

for potable water by PNSDW ranges between 6.5-8.5 (Department of Health, 2017) wherein any

level that does not fit within the standard pH values may affect the toxicity of microbial poisons

in water (Choudhury et al., 2016). Turbidity is also a primary factor affecting water quality as it

is caused by sediments suspended or dissolved in water that scatter light making the water appear
cloudy which can have a negative impact on aquatic life and water quality. These sediments may

block light to aquatic plants causing potential environmental risk also, and may carry

contaminants and pathogens (Minnesota Pollution Control Agency, 2008). The PNSDW set the

standard turbidity for potable water at <5 NTU (Department of Health, 2017). Flow rate is an

important factor as well in relation to turbidity as the amount of silt and sediment carried by the

stream is affected by the flow rate to which sediments found in quiet, slow-flowing streams settle

fast to the streambed whereas fast-moving streams keep silt suspended in the water column for

longer (Fleming, 2019). Flow rate measurement is detrimental as to how swiftly a volume of

water is able to discharge throughout the stream as rapidly flowing rivers allow for dilution of

water and degradation of wastes and therefore, prevents water pollution (United States

Environmental Protection Agency, 2012). Lastly, temperature is also of great importance in

assessing water quality as it affects the biological system of the aquatic environment of which

regulates and controls the type of organisms that live in different water forms wherein an

abundance of species inhabit aquatic environments with cooler temperatures as it contains high

rates of dissolved oxygen (U.S. Geological Survey's Water Science School, 2018).

According to Augustyn et al. (2016), the highest number of coliforms were found in the

fall, followed by a slight decrease in the spring and winter, and a large decrease in the summer.

This contradicts the result of Lee et al. (2013), that showed higher levels of TCs and FCs during

hotter months. Aside from the two studies that contradict each other, another study by Singh et

al. (2017), shows that seasonality and thus, temperature had no significant effect on coliform

count throughout the year. Choudhury et al. (2016), results showed that water temperature ranges

from 23-24 C, which complies to the WHO standard for safe drinking water which is < 32C. The

temperature range observed in this study will slow the pace of chemical and biological processes,
as well as the solubility of gasses in water, which may have a negative influence on the water's

taste and odor at higher temperatures. Aside from temperature, Choudhury et al. (2016) results

showed that pH ranged between 6.8-7.6 for all the sites, which is slightly acidic to slightly

alkaline, which comply with the WHO standard. Augustyn et al. (2016), discussed that coliform

bacteria are gram-negative rods capable of performing the fermentation of lactose to form acidic

products group of microorganisms in water of pH≤7.0 demonstrated in this study resulted from

the fact that optimum pH for the growth of these bacteria is 6-7.

The studies of Lee et al. (2013), Augustyn et al. (2016), Choudhury et al. (2016), and

Singh et al. (2017), showed the correlation between temperature and concentration of coliforms

and fecal coliforms in water samples. Lee et al. (2013) stated that during the high seasonal

temperatures of June–September, total coliforms (TC) were detected at a concentration 1.6 times

higher than the average concentration detected. Furthermore, fecal coliforms (FC) were detected

at a concentration 2.5 times higher than the average concentration detected. Also, water sampled

during July–September exhibited high turbidity, suggesting that the presence of detectable TCs

and FCs may be related to turbidity.

According to Mishra et al. (2017) coliform count is highest in areas surrounding urban

settlements and sewage discharge areas. The results of the total coliform count exceeded that of

the WHO standard for coliform bacteria in water which is zero per 100 ml to which the results

suggests that the water may potentially be contaminated by fecal coliforms (Mishra et al., 2015;

Choudhury et al., 2016; Idibie et al., 2018). In addition to that, similar studies conducted by

Singh et al. (2017) and Hayat & Kurniatillah (2021) with regards to coliform pollution showed

that coliform contamination has escalated significantly and that water is neither potable and can

be used for recreational purposes. With large amounts of total coliforms present in bodies of
water, the study conducted by Divya & Solomon (2016) revealed that the increase of bacterial

count can indicate the presence of pathogens in water, which can affect the water quality in the

future.

An article in 2019 discussed the water quality of the Panigan-Tamugan River. In

celebration of the World Water Day, Interface Development Interventions (IDIS), together with

the environmental volunteers of Bantayo Aweg and Bantay Bukid monitored the waters of

Panigan-Tamugan Rivers. Results showed a high presence of fecal coliform of 160.000

MPN/100ml, which is far from the standard set by the PNSDW which is 1.1 MPN/100ml. In

addition, it was also stated that the pH level is at 7 in its neutral range however, continuous

monitoring is needed since results may vary (Llemit, 2019). In 2020, IDIS executive director

Chinkie Peliño-Golle stated that the Tamugan watershed lowered its water quality level from

Class AA to Class A, according to the results of a water quality assessment presented by the

Bantayo Aweg, a group of volunteer researchers. However, she also stated that the water in the

river remained potable. Golle also stated that the Panigan-Tamugan watershed is found to be

polluted with plastic wrappers and residential wastes, and traces of nitrate and phosphate from

fertilizers used in plantations surrounding the area (Gumba, 2020). On the other hand, the

Environmental Management Bureau (2020) presented Talomo river’s water quality data to which

physicochemical parameters suggested that the river’s water quality belonged to “Class B” as

primary parameters such as pH, and temperature passed the assessment wherein the river’s pH

level ranged from 7.1-8.2, and its temperature ranged from 23-29°C. However, physiological

parameters such as turbidity (in some areas throughout the river), and fecal coliform rates failed

in the assessment having 6-217ntu, and 135-67005MPN/100mL, respectively. Rivers that belong
to “Class B” are not potable but are manageable for recreational use such as swimming or

bathing (Environmental Management Bureau, n.d.).

Antibiotics are natural or semi-synthetic compound, that when administered, are partially

metabolized in the body and the rest are excreted unchanged or as metabolite into the ecosystem

(Coman et al., 2017; Carmona-Alvarado et al., 2022). Particularly, antibiotics (as residues) are

pharmaceutical contaminants with the highest use worldwide (Alvarado et al., 2022). The main

sources of antibiotic pollution come from industrial production, hospitals, domestic use and the

excretion of human and animal waste (Coman et al., 2017; Hussain et al., 2016). Unlike human

waste, which is treated via treatment plants or septic systems, livestock waste is oftentimes

directly applied to the land as part of a nutrient management plan. Note that land application of

manure is the main pathway for veterinary antibiotic introduction into the terrestrial and aquatic

environments. Once applied to the land, antibiotics are transported to surface water, via runoff, or

ground waters, through infiltration. Unfortunately, large amounts of administered antibiotics are

not metabolized by animals but instead are excreted in manure (Qualls et al., 2017).

The broad use of pharmaceutical compounds for treatments of diseases can have a

negative impact in human health since its occurrence can lead to the development of antibiotic

resistant bacteria (Hussain et al., 2016; Carmona-Alvarado et al., 2022). It is a basic human

tendency to alleviate infectious diseases with the help of medication such as antibiotics or

antibacterial agents in order for the human race to survive and prevent the spread of deadly

diseases within their system, and the environment. However, as long as coexisting species

continue to compete for survival, adaptation and natural selection will aid in developing

phenotypic or genotypic enhancements to their offspring in order for species to gain reproductive

advantage, a phenomenon known as evolutionary arms race (Morand, 2019). This would imply
how certain microorganisms are able to recuperate from antimicrobial agents, which is due to

genetic mutations and horizontal gene transfer that enables pathogenic microbes to resist

antimicrobial agents as the introduction of antibiotics to prevent bacterial infections enforced the

biological fitness of bacteria making them resistant to antibiotics (Kaplan, 2014). Additionally,

the release of antibiotics into the natural water would cause potential environmental risk,

affecting the evolution of the community structure (Coman et al., 2017; Carmona-Alvarado et al.,

2022). Different studies showed that antibiotics are persistent and pseudo-persistent

contaminants causing toxicological impacts on the fauna of natural water bodies including

synergistic and antagonistic combination effects (Coman et al., 2017).

7 studies had detected the presence of antibiotics in river waters using the HPLC method.

Dang et al., (2016) detected six antibiotics (norfloxacin, ofloxacin, sulfamethoxazole,

trimethoprim, erythromycin and roxithromycin) which revealed that the river water in Hong

Kong has a wide distribution of antibiotics and has different concentrations from different areas.

Soran et al., (2017) detected antibiotics and antibiotic-resistant microorganisms in aquatic

environments, including wastewaters, surface water, ground water, and drinking water.

Specifically, they were found in higher quantities on surface water of rivers near urban

settlements. Coman et al. (2017) looked for the presence of antibiotics that are often used in

human and veterinary applications (penicillin, tetracyclines, and cephalosporins). The results

demonstrate that tetracycline and doxycycline were the most abundant antibiotics in river waters,

indicating that antibiotics in river waters are becoming a rising environmental concern,

necessitating recurrent monitoring. Carmona-Alvarado et al. (2022) monitored the presence of

antibiotics from 5 different families of antibiotics (fluoroquinolones, penicillins, sulfonamides,

trimethoprim and cephalosporins). Results show that trimethoprim and quinolones showed
maximum excitation. It was indicated that the concentration and type of antibiotics may be

influenced by several key factors, such as source of sample, season of the year, prevalence of

diseases, as well as factories located in the vicinity or aquaculture activities. Mirzaei et al. (2017)

monitored the presence of nine antibiotics from different classes: β-lactam (amoxicillin and

penicillin G), cephalosporin (ceftriaxone, Cefixime, and cephalexin), macrolides (azithromycin

and erythromycin), fluoroquinolone (ciprofloxacin) and Nitro-imidazole (metronidazole). The

results show that 7 out of 9 antibiotics are present (Penicillin, Amoxicillin, Azithromycin,

Cephalexin, Ciprofloxacin, Erythromycin, Cefixime). The results also indicated that none of the

antibiotics were present in treated and ground water. Ma et al. (2018) results show that 7 out of 7

antibiotics tested were found and that among the antibiotic families, Tetracycline is the

predominant antibiotic. Correlative analysis showed that there was a positive correlation between

the quantity of many drug-resistant bacteria and the corresponding concentration of antibiotics.

Hussain et al. (2016) monitored the presence of 5 antibiotics (ciprofloxacin, levofloxacin,

ofloxacin [Fluoroquinolones], oxytetracycline, and doxycycline [tetracyclines]) from 2 families.

The results show that five antibiotics were found in all samples. Levofloxacin and

oxytetracycline were found to be highest in concentration. The low presence of amoxicillin was

detected giving an unusual result due to fragility of the lactam ring and susceptibility to

hydrolysis in the environment. However, the presence of the antibiotic in the waste streams and

surface water could be explained if there is a large load of the antibiotic entering the wastewater

treatment plant (WWTP) (Harrower, et al., 2021). The presence of antibiotics may be affected by

certain factors; photolysis, temperature, pH, dilution variables, bacterial populations, hydraulic

residence duration (Liu, et al., 2021) and weather conditions, such as acid rain, could promote

the retention of antibiotics (Polianciuc, et al., 2020).

 Definition of Terms:

This section involves definitions of the unfamiliar terms that will be mentioned in

the research paper that are relevant to the study.

Antibiotics - Are medically used chemicals, which can affect the river’s antibiotic

resistance.

Coliforms - Are water quality indicators in rivers which determine when water is

available as drinkable, or recreational.

High - Performance Liquid Chromatography - A separation technique in compounds

in analytical, and Biochemistry to identify, qualify, or purify individual components of mixture.

Most Probable Number - A method used to estimate viable numbers of bacteria in a

sample by its inoculating broth in 10-fold dilutions.

Acronyms

HPLC

HPLC-DAD

MPN
 METHODOLOGY

Presented in this section is the methodology that will be used to conduct this study

including the research design, setting, research sampling, data gathering procedure, and data

analysis.

Research Design

This study will be utilizing the quantitative descriptive method. The researchers will be

using a descriptive method to be able to determine the antibiotics present in Panigan-Tamugan

and Talomo Rivers. Descriptive methods include identification of characteristics of a certain

event based on an observation, or the correlation that is explored between two or more events

(Williams, 2007).

Setting

Samples were collected from the Panigan, Tamugan, and Talomo rivers, Calinan, Davao

City (Figure 1b). The Panigan-Tamugan watershed shields the upper catchment basin of Panigan

River and Tamugan River which is a larger river downstream into Panigan. The rivers are

lumped with other two watersheds that balance the Davao River water basin. In

Panigan-Tamugan watershed, the Barangay Development plans (BDPs) of 9 barangays reveals

that 72.6% of households have sanitary toilets while Tambobong, its nearby barangay, showed
7% of households that use unsanitary toilets. The difference is that almost half of the households

in Lacson and Tawan-tawan use unsafe toilet sanitation which could then contribute to the

pollution into the watershed. One factor to the contribution of pollution in the watershed is the

banana plantation. A 2007 study showed that the rain leaked into waterways within

Talomo-Lipadas and Panigan-Tamugan watersheds. Factory establishments and agricultural

farms were suspected as major sources of pollutants within watersheds (Ibanez, 2015). In the

report of Compio (2021), Antipolo, Talomo River is located at the back of Calinan Public

Cemetery and has a length of 0.5 km.

Figure 1. Map of Davao City, Philippines (Panigan-Tamugan River & Antipolo, Talomo

River)

Data Collection Procedure

 Before entering the location, we asked prior permission and consent from the barangays

of each river a week before the sample collection.

The collection site was visited on the morning of March 12, 2022. Two samples were

collected on each river. One for total coliform and fecal coliform test, and another for HPLC.

During our site visit, four 1L bottles (2 bottles per collection site) were used to collect samples

for total coliform and fecal coliform test, and HPLC. Three sample tubes were used to collect

samples for the turbidity test, one for each river. Parameters were also measured during this

period such as flow rate, turbidity, pH, and temperature. Flow rate was measured using a flow

meter. The turbidity was measured using a turbidimeter. pH was measured using a pH meter.

Lastly, the temperature was measured using a digital dishwasher thermometer.

Microbial Analysis

The samples collected during the visit were directly transported to the Davao Medical

School Foundation Inc. (DMSFI) Research Center for laboratory analysis. Samples were

analyzed for the presence of coliforms and fecal coliforms by Most Probable Number (MPN)

performed by the laboratory technicians. The testing of bottled drinking water was done in an

aseptic manner. The MPN Method was carried out in three steps: 1) Presumptive test 2)

Confirmed test 3) Completed test (Phyo, Yu, & Saing, 2019).

1) Presumptive test

In this examination, for the presence of the indicator bromocresol purple, MacConkey

broth is widely employed for lactose fermentation. The inverted Durham's tube is used to detect

Gram negative coliform bacteria that produce gas. It's safe to assume that coliform bacteria are
present in these samples because the media turns yellow and gas is collected in Durham's tube.

Each of the 10ml of presumptive broth was inoculated with 5 of the 10ml of water samples

(double strength). In a tube containing 50ml of presumptive broth, 1 of 50ml water sample was

added. After a 48-hour incubation period at 37°C, the number of positive tubes from each set was

counted and compared to a standard chart to determine a presumptive coliform count per 100mL

of water.

2) Confirmatory test

Positive samples from the presumptive test were chosen for the confirmed test to evaluate

whether the coliforms were of fecal origin indicator bacterium Escherichia coli. The

development of a greenish metallic sheen on Eosine Methylene Blue (EMB) media was used to

distinguish Escherichia coli from Gram negative coliform bacteria, confirming the presence of

indicator bacteria E. coli. coli. After 24 hours of incubation at 37°C, streaking loopful samples

from positive tubes reveals the formation of color indications from colonies.

3) Completed test

The bacterial colonies on EMB media from verified tests were subcultured on Mac

Conkey agar plate and inoculated in LB broth at 44.5oC with Durham's tube. The production of

gas and color changes in media confirm the presence of the fecal indicator E.coli.

Experimental Section for HPLC

The samples were also used to analyze the presence of Amoxicillin and Penicillin using

High-performance liquid chromatography with diode array detention (HPLC-DAD) by the

laboratory technicians in the laboratory. The HPLC-DAD method has long been the most
extensively used method for determining antibiotics. Because of its simplicity and robustness,

high-performance liquid chromatography with diode array detector (HPLC/DAD) after solid

phase extraction (SPE) (Ašperger et al, 2009) which is based on the separation of analytes

between a solid phase, which is usually a sorbent contained in a column, and a liquid phase,

which is usually a sample matrix or a solvent containing analytes (Ötles & Kartal, 2016). Before

proceeding to the HPLC-DAD method, solid-phase extraction (SPE) is performed. SPE is a

method for quick, selective sample preparation and purification, prior to chromatographic

analysis. This entails distributing the analyte between a liquid phase sample medium and a solid

phase adsorbent, and dispersing dissolved species between two phases. This technique allows the

enrichment and purification of the analytes from a liquid sample by extracting, partitioning, and

adsorbing onto a solid stationary phase (Keçili et al., 2020). In order to improve the extraction

processes and the quality of the extracted data, a standard combination containing that was

created for qualitative and quantitative examination (Figure 2). Commercial powders for oral

therapy and vials were employed as antibiotic standards specifically, Amoxicillin (500 mg oral

powder) and Penicillin (600 mg vials). For calibration, standard solutions in the concentration

range of 0.65 to 166.7 ng/mL were generated by diluting standard mixture in Milli-Q water. An

HPLC Agilent 1200 Series equipment was used for the chromatographic studies. G1322A

degasser, G1311A quaternary pump, and G1329A quaternary pump. The autosampler, G1315D

DAD detector, and G1316B TCC SL column thermostat were used, as well as an LC-MS system

model Agilent 1200 Series coupled with a 6110 Quadrupole LC/MS detector with AP-ESI

ionization and an LC-MS system model Agilent 1200 Series coupled with a 6110 Quadrupole

LC/MS detector with AP-ESI ionization. ChemStation software was used to gather and process

the chromatographic data. The separation was carried out at a specific flow rate on an Agilent
Poroshell 120 EC-C18 column with a mobile phase consisting of acetonitrile and formic acid in

water under the following gradient. A single wavelength of 194 nm was applied for DAD

detection for penicillin and amoxicillin. The target antibiotics were isolated from the water

samples by solid phase extraction. A 1 L river of water sample was run through the SPE system.

5 mL/min flow rate through cartridges, followed by elimination of residual antibiotics with a 5

mL phosphoric acid + acetonitrile (1:1, v/v) combination. The residue was dissolved in 1 mL

acetonitrile and submitted to HPLC analysis after being evaporated to dryness under nitrogen.

(Coman et al., 2017).

Figure 2. Method for analyzing samples in HPLC.

 RESULTS AND DISCUSSIONS

Results of the HPLC to detect antibiotics, and total and fecal coliform test on water

samples from the Panigan-Tamugan and Talomo Rivers are presented in this section. Table 1

shows the total coliform and fecal coliform count of the rivers. Talomo River has the highest

presence of coliform containing 1,071 MPN/100ml, followed by Panigan River with 403

MPN/100ml, and Tamugan River with 170 MPN/100ml. Fecal coliform tests also show that fecal

coliforms were present and abundant. Panigan River had the highest presence of coliform of 201

MPN/100ml, followed by Talomo River with 172 MPN/100ml, and Tamugan River with 170

MPN/100ml. Regardless of the difference in results, the results indicate that all rivers are

contaminated with high amounts of coliforms and fecal coliforms. Also, the coliform count far

out exceeded the 0 MPN/100ml set by WHO for potable water and the fecal coliform count far

out exceeded the 1.1 MPN/100ml set by the PNSDW for potable water (Idibie et al., 2018).

There is a large significant decrease in coliform count in Panigan-Tamugan watershed

from 2019, during the water monitoring by the Interface Development Interventions (IDIS),
together with the environmental volunteers of Bantayo Aweg and Bantay Bukid in celebration of

the World Water Day, in which results indicate a high presence of fecal coliform of 160,000

MPN/100ml (Llemit, 2019). Furthermore, there is a large decrease of coliform count in Talomo

river from 2020, during the water quality assessment of the Environmental Management Bureau

(2020). In this assessment, physicochemical parameters suggested that the river’s water quality

belonged to “Class B”, and fecal coliform rates failed in the assessment having

135-67005MPN/100mL (Environmental Management Bureau, n.d.).

The significant decrease is presumably due to many different programs carried out by the

government and non-government organizations focused on helping clean and preserve the waters

of Panigan-Tamugan River such as the Upscaling Community Action and Response (UCARE)

project in 2011 by the Interfacing Development Interventions for Sustainability (IDIS) and

Foundation for the Philippine Environment (FPE) recently in 2020. This project led to the

development of the Bantay Bukid Program (BBP), implementing the Davao City Watershed

Code (DCWD), which was later recognized by the city government the following year.

Following the implementation of the program, Bantay Bukid volunteers have apprehended

people violating the code. Violations include improper waste disposal, illegal quarry operations,

illegal poultry farming, illegal fishing ang climbing/trekking at Mt. Tipolog (Foundation for the

Philippine Environment, 2020) in which forest rangers will immediately report to the police if

law violators are witnessed or when they harm the environment (City Government of Davao.,

2019). This group has also organized a monthly water quality monitoring in Panigan-Tamugan

and Talomo Rivers to ensure that the DCWD has been implemented in the rivers (SunStar,

2018). Thus, these results suggest that the programs carried out by these organizations have been

effective.
Table 1. MPN of Panigan River (PR), Talomo River (TL), and TamuganRiver (TM).

MPN Coliform Count/100mL

Samples 12- Mar

Total Fecal

Panigan River 403 201

Talomo River 1,071 172

Tamugan River 170 170

Detection of Antibiotics Through HPLC method

Solid-phase extraction and HPLC-DAD were used to analyze the accuracy for detection

of amoxicillin and penicillin in Talomo River, and Panigan-Tamugan river in which samples

were ran through the same type of column (C18) and solvent system (Gradient elution

Acetonitrile: 0.1% Phosphoric Acid). The test was limited to assessing Penicillin and

Amoxicillin as these antibiotics are commonly used in the Philippines (Barber, 2016). To assess

the presence of antibiotics with regards to the results shown by the HPLC chromatogram,

indication that results are positive should show that retention time of antibiotic samples in

comparison to the water samples should be the exact same otherwise, a difference in retention

time will indicate that there are no antibiotics found in the watersheds.
 The HPLC test of both standard samples (Amoxicillin and Penicillin) as shown in figures

3.1 and figure 3.2 had a clear resolution of wavelengths at 194 and 190 nmu, respectively. With

regards to their presence in the water samples collected from the river, the detection of

antibiotics can be seen through the comparison of retention times from the water samples. Figure

3.1 shows the comparison between the standardized sample (Amoxicillin) and the water sample

from the rivers to which the data signifies that there is no presence of amoxicillin in the rivers as

there is a significant difference in their retention time to which the water sample has a peak of

2.387 while the standard Amoxicillin has a retention time of 2.797. Similar results show for the

detection of Penicillin to which Figure 3.2 shows that there is no presence of Penicillin detected

as seen in the difference of retention time to which the water sample has a retention time of

2.258, and Penicillin has a retention time of 3.288.

Figure 3.1. The standardized sample and water sample data results of Amoxicillin
 Figure 3.2 The standardized sample and water sample and data result of Penicillin

Table 2.1. Peak table for standard sample and water sample for Amoxicillin

Water Sample

Peak # Retention Time Area Height Remarks

1 2.387 300512 9702

Total 300512 9702 No Detection

Amoxicillin Sample

Peak # Retention Time Area Height Remarks

1 1.899 3450 290

 2 2.797 987884 65734 Amoxicillin

Total 991334 66024

Table 2.2. Peak table for standard sample and water sample for Penicillin

Penicillin Sample

Peak # Retention Time Area Height Remarks

1 3.288 1310732 38982 Penicillin

Total 1310732 38982

Water Sample

Peak # Retention Time Area Height Remarks

1 2.258 982526 37117

Total 300512 37117 No Detection

Physicochemical Parameters

In order to assess the quality of the rivers, average flow rate, turbidity, pH levels, and

temperature were noted down as these parameters may affect the quality of the watersheds. Table

1 shows the physicochemical parameters from the three sampling sites of which Talomo River

exhibits the highest average flow rate at 6.6 lpm, followed by Panigan River at 2.43 lpm and

Tamugan River at 0.24 lpm. The Panigan River has the highest turbidity at 20.4 ntu, followed by

Tamugan river at 18.4 ntu, and Talomo river at only 4.2 ntu. The Panigan and Tamugan Rivers

exceeded the standard turbidity set by the Philippine National Standard for Drinking Water
(PNSDW) for potable water which is <5 ntu whereas the Talomo River is within the standard

range. Turbidity is a primary factor affecting water quality as it is caused by sediments

suspended or dissolved in water that scatter light making the water appear cloudy which can

have a negative impact on aquatic life and water quality.

The turbidity in Talomo watershed lowered significantly, during the water quality

assessment of the Environmental Management Bureau (2020), ranging from 6-217 ntu

throughout the river, to a turbidity of 4.2 ntu. High turbidity may indicate high presence of

coliform bacteria, as these sediments may carry contaminants and pathogens (Minnesota

Pollution Control Agency, 2008; Lee et al.,n 2013 ), suggesting that the decrease in turbidity is

correlated to the decrease of total coliform count. In addition, the flow rate of the river may

affect turbidity as it affects the dilution of the water and the degradation of wastes (Fleming,

2019), which would suggest why the total coliform and fecal coliform results of Talomo river

lessened throughout the years alongside monitoring and management activities done by the

government and volunteers.

In contrast, however, results from the Panigan and Tamugan rivers suggest otherwise.

The Panigan and Tamugan rivers exhibit high turbidity with lower coliform count. Whereas the

Talomo river exhibits lower turbidity with higher coliform count which counter the connection of

flow rate and turbidity with respect to presence of total coliform and fecal coliform. The high

turbidity of Panigan and Tamugan Rivers is due to the fact that the area where the samples were

taken is shallow thus, it is closer to the stream bed and that it is a fast flowing water. Turbidity

increases as depth decreases (Faafeng & Mjelde, 1984) and the amount of silt and sediment

carried by the stream is also affected by the flow rate. Sediments found in quiet, slow-flowing

streams settle fast to the streambed. Fast-moving streams on the other hand keep silt suspended
in the water column for longer (United States Environmental Protection Agency). Also, Despite

the fact that the Talomo River exhibits the highest coliform count and flow rate, it exhibits the

lowest turbidity presumably due to our samples being taken in a shallow but concrete area of the

river thus, there is little to no soil sediments present.

All rivers have a pH level of 7 which is the neutral pH level and within the pH range for

potable water set by the PNSDW which is 6.5-8.5 (Department of Health, 2017). The

temperature of all rivers are within the same range (Talomo-25.8°C, Panigan-23.1°C, and

Tamugan-22.6°C) which also complies with the WHO standard for potable water which is

<32°C. With regards to these informations, the pH observed is within the optimal range for the

growth of coliform bacteria (6-7) as they are gram-negative rods capable of performing the

fermentation of lactose to form acidic products in a group of microorganisms in water of pH≤7.0

(Augustyn et al., 2016). In contrast, the temperature range observed in this study will slow the

pace of chemical and biological processes, as well as the solubility of gasses in water, which may

have a negative influence on the water's taste and odor at higher temperatures (Choudhury et al.,

2016). This indicates the temperature range measured in this study alleviates the presence of

coliforms. Furthermore, with these contrasting parameters, this would indicate that coliforms are

within their natural habitat since coliforms are already naturally present in the environment and

as a result of contamination, possibly from human and animal wastes as the source of pathogenic

microorganisms (Divya & Solomon, 2016), to which the results show the presence of high levels

of fecal coliforms. Similarly, Gerba & Pepper (2019) also mentioned that high coliform count

contaminating the waterways, may indicate the presence of various pathogenic microorganisms

that may spread via food and water consumption, and bathing or recreational use, creating a

cyclic transmission of diseases. All three rivers are available for anyone to use and with results
of high total and fecal coliform counts, this remains a concern for water quality of the rivers

especially since Tamugan river is Davao’s primary water source.

The pH and temperature of Talomo watershed has remained in its neutral range since

2020, in which the past assessment shows pH level ranged from 7.1-8.2, and temperature ranged

from 23-29°C.

Table 2. Physicochemical parameters obtained from the three sampling sites.

Rivers Average Flow Turbidity pH Temperature

rate (L/minute) (°C)

Panigan 2.18 lpm 20.4 ntu 7 23.1°C

2.68 lpm

Ave: 2.43 lpm

Tamugan 0.22 lpm 22.7 ntu 7 22.6°C

0.25 lpm

Ave: 0.24 lpm

Talomo 6.42 lpm 4.2 ntu 7 25.8°C

6.78 lpm

Ave: 6.6 lpm

Conclusion
 The abundance of total coliform count in watersheds is a growing concern as it may

allow for the presence of pathogenic microorganisms that may spread and infect individuals

exposed to the river. This study provided an updated water quality assessment with regards to the

presence of total and fecal coliforms by MPN method and a first ever information regarding the

detection of antibiotics (Amoxicillin and Penicillin) using the HPLC-DAD method using water

samples collected from three of Davao’s major rivers, Talomo, Panigan, and Tamugan. In

conclusion, results of the MPN showed that there is an abundance of total and fecal coliform to

which all three rivers have failed the standard coliform count set by PNSW (<1MPN/100mL).

However, more monitoring efforts can be done as the data presented in this paper only

represented one sampling duration. Further studies can be undertaken in order to profile the

physicochemical parameters of the river and their coliform count in order to produce substantial

data that can be used by agencies involved in crafting management policies in maintaining the

qualities of the aforementioned rivers.

In terms of detection of antibiotics, HPLC-DAD data showed that there were no

antibiotics present in all three rivers, detecting Amoxicillin and Penicillin The researchers

suggest continuing on assessing the rivers for the detection of different types of antibiotics as

trends on studies do show the presence of antibiotics aside from Penicillin and Amoxicillin in

water forms. Furthermore, this study also suggests continuing an overall annual water quality

assessment of all the three but not limited to the three rivers especially since Tamugan river is

Davao’s new primary water source, to which is connected to Panigan river.

Recommendations
 After a thorough analysis of data, the researchers have come to recommend situations to

inspire future researchers. Continuation of water assessment between Panigan, Tamugan, and

Talomo rivers to provide the public important information on the status of these waterways and

help the people for management actions. Since the study was only limited to detecting two

standard antibiotics which is Amoxicillin, and Penicillin, it is great for future researchers to

identify other varieties of antibiotics present in between Panigan, Tamugan, and Talomo Rivers.

With the lack of resources, and budget, funding future researchers who are interested in the

rivers for better and more resources, and budget for a wider field of study. Even with the

existence of the city ordinance in Davao City, many people still abide by the laws and this proves

the coliform counts that exist in the researchers data. Stricter implementation of city ordinance

to avoid further contamination between Panigan, Tamugan, and Talomo Rivers, would be great

for the public to follow the rules. Since, the study has not been made for the public to the citizens

of the barangay, it is great to utilize publication to the people, within the area for them to be

informed about the water quality as well as delivering data results to the barangay for them to be

informed about the current quality, to help, and for future government programs. Considering

different seasons occur, the waterways are constantly changing, suggesting future researchers to

gather data on different seasons, and submitting it to barangay, would be a great contribution to

the study, for getting better accurate results for the water quality.

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