Assessment of Antibiotics Contained on the Wastewater

of Hospitals in Tuguegarao City

A Research Proposal presented to the

Faculty of Chemical Engineering Department

Cagayan State University-Carig Campus

Tuguegarao City

In Partial Fulfilment of the Requirements of the Degree

Bachelor of Science in Chemical Engineering

By:

Allauigan, Maria Louiza Victoria A.

Capili, Rochelle A

Doquilo, Kimberly C.

Omnes, Preciously U.

2019
 TABLE OF CONTENTS

CHAPTER I: THE PROBLEM AND ITS BACKGROUND

1.1 Background of the Study .......................................................................................... 1

1.2 Statement of the Problem ......................................................................................... 2

1.3 Objectives of the Study ............................................................................................. 2

1.4 Significance of the Study........................................................................................... 3

1.5 Locale of the Study .................................................................................................... 3

1.6 Scope and Limitations............................................................................................... 3

1.7 Definition of Key Terms ........................................................................................... 4

CHAPTER II: REVIEW OF RELATED LITERATURE

2.1 Antibiotics .................................................................................................................. 6

2.1.1 Uses of Antibiotics .............................................................................................. 6

2.1.2 Properties of Some Antibiotics ......................................................................... 7

2.1.3 Antibiotics Resistance ........................................................................................ 8

2.2 Effects on living thing ............................................................................................... 9

2.2.1 Aquatic Life ........................................................................................................ 9

2.2.2 People .................................................................................................................. 9

2.2.3 Plants ................................................................................................................. 10

2.3 Antibiotics Safe Level.............................................................................................. 10

2.4 Antibiotics in Rivers ................................................................................................ 11

 2.5 Sources of Antibiotics.............................................................................................. 13

2.5.1 Hospitals............................................................................................................ 14

2.6 Liquid Chromatography – Mass Spectrometry (LC-MS) ................................... 16

2.7 Conceptual Framework .......................................................................................... 16

CHAPTER III: METHODOLOGY

3.1 Sampling Sites.......................................................................................................... 18

3.2 Parameters ............................................................................................................... 18

3.3 Sample collection ..................................................................................................... 18

3.4 Pre – Treatment ....................................................................................................... 19

3.5 Sample Analysis ....................................................................................................... 19

3.5.1 Solid phase extraction (SPE) ........................................................................... 19

3.5.2 Liquid Chromatography – Mass Spectrometry (LC-MS) ........................... 19

3.6 Statistical Analysis................................................................................................... 20

APPENDIX

Appendix A

A.1. List of Predicted No-Effect Concentrations (AMR Industry Alliance, 2018) ....... 21

A.2. Various antibiotics source and action (Shikha Goyal, 2016) ................................. 22

A.3. Some antibiotics which are developed through various Biotechnologies (Shikha

Goyal, 2016). ...................................................................................................................... 23

Appendix B ........................................................................................................................ 24

BIBLIOGRAPHY ................................................................................................................. 25
 CHAPTER I

THE PROBLEM AND ITS BACKGROUND

1.1 Background of the Study

Antibiotics are widely used to treat range of infections caused by bacteria,

parasites and fungi for humans and animals. Modern animal agriculture utilizes

antibiotics as therapeutic drugs to combat infectious diseases and as growth promoters to

enhance feed efficiency for livestock (Aga & Mojica, 2011). The broad use of antibiotics

is due to ease of use, affordability and availability of it compared to alternatives

(Rahamat-Langendoen, 2008).

The continuous rise of antibiotic consumption imposes natural threat since large

portion of antibiotics administrated to humans or animals are excreted to original form

and are discharged from the hospital or communal effluents to environmental water

bodies (Kummerer, 2004). A recent study quantifying concentration of pharmaceutical

contaminants shows that two-thirds of river sites tested contain unsafe levels of

antibiotics (Boxall & Wilkinson, 2019). The presence of antibiotics in the environment

contribute to the development of antibiotic resistant genes increasing the potential of

compromising the effectiveness of antibiotic therapy which can risk human health (Kim

et al., 2004). Exposure to antibiotic residues on the environment can lead to carcinogenic

reactions to people and can endanger soil and aquatic organisms (Göbel et al., 2004).

Hospital wastewaters are mixture of chemical and biological substances which are

anthropogenic pollutants (Boillot, 2008; Verlicchi et al., 2010). Antibiotics passing

through the wastewater treatment facility are subsequently discharged in considerable

amounts into bodies of water (Czekalski et al., 2012).

1
 There are only few comprehensive investigations on the characteristics of

antibiotics as pollutants due to the difficulty of determination of sampling points that

could establish idea of the occurrence, magnitude and spatial distribution of antibiotics on

natural bodies of water (Marshal & Roy, 2016). Establishing correlation on the antibiotic

distribution between sampling points is challenging because of the varibility on reactive

nature, patterns input and concentration of antibiotics (Boy-Roura et al.,2017). There’s

also a lack of information on antibiotic residue level in hospital effluent. Therefore, the

overall goal of the study is to establish data and basic information that could be used to

characterize the behaviour and amount of antibiotics in effluents of Saint Paul Hospital

Tuguegarao and Cagayan Valley Medical Center.

1.2 Statement of the Problem

Literatures have shown that approximately ninety- five percent of the total amount

of antibiotic taken into the body is discharged in the wastewater through excretion and

this results in the contamination of the water from antibiotic residue. In this study, water

samples from the effluent of Saint Paul Hospital and Cagayan Valley Medical Center

(CVMC) in Tuguegarao City, Cagayan will be investigated. Specifically, the study will

answer the following questions:

1. What are the most common antibiotics found in the hospital wastewater?

2. Is the concentration level of antibiotics found in the hospital wastewater

comparable with the international standard?

1.3 Objectives of the Study

Generally, this study aims to:

1. Identify the top five most common antibiotics found in the hospital

wastewater;

2
 2. Characterize the concentration levels whether they fall on the safe level or

overcome the international standard level of concentration for antibiotics

present in wastewater.

1.4 Significance of the Study

This study regarding the assessment of antibiotics contained in the wastewater of Saint

Paul Hospital and Cagayan Valley Medical Center will identify the top five common

antibiotics found in the effluent. It presents a quantitative evaluation on how high or low

is the concentration level of these antibiotics hence, will improve the existing knowledge

on the relation of antibiotics and its resistance activity.

Moreover, this study can be beneficial for future researchers who have the desire to do

further study on the antibiotic level of wastewater from hospitals and other sources

including its potential impact among humans and in the aquatic environment, and on

antibiotic resistance.

1.5 Locale of the Study

This study will be carried out at the waste water discharge of Saint Paul Hospital

Tuguegarao and Cagayan Valley Medical Center (CVMC). The tests will be administered

at the analytical laboratory of University of the Philippines - Diliman. Data analyses will

be conducted at Cagayan State University – Carig Sur, Tuguegarao City, Cagayan.

1.6 Scope and Limitations

This study aims to assess the amount of antibiotics of waste water discharge of

Saint Paul Hospital and CVMC. This study will be limited in the determination of

antibiotics for water samples only. Antibiotics from sediments, soil, and fishes are not

3
 included. Also, its effects on the environment and public health are not on the scope of

the study. The study will be conducted from November 2019 to February 2020.

1.7 Definition of Key Terms

Analyte. Any chemical or biological substance for which concentrations in a sample will

be determined.

Antibiotics. Include range of powerful drugs and are used to treat diseases caused by

bacteria.

Contaminant. Biological or chemical substances added to the medium of concern,

commonly through human activity.

Contamination (of water). Change of ambient water composition by the addition of

biological or chemical substances as a result of human activity or natural processes.

Addition of such substances can be detrimental to the quality of the water resource.

Ethylenediaminetetraacetic acid (EDTA). An aminopolycarboxylic acid and a

colorless, water-soluble solid and is widely used as a chelating agent.

Liquid Chromatography – Mass Spectroscopy (LC–MS) analysis of antibiotics. An

exceedingly sensitive and specific analytical technique that can precisely determine the

identities and concentration of compounds within sample. A method using on-line solid

phase extraction directly coupled to liquid chromatography/ tandem mass spectrometry

which offers more environmentally friendly, method for and costs while reducing

hazardous waste and potential environment pollution as compared to offline SPE

methods.

pH. Maintaining the pH of the water sample at a certain level can help to inhibit

microbial activity.

4
Solid-phase extraction (SPE). A sample preparation process by which compounds that

are dissolved or suspended in a liquid mixture are separated from other compounds in the

mixture according to their physical and chemical properties.

SPE cartridge. Available with a variety of silica bonded to a specific functional group

including hydrocarbon chains of variable length (for reversed phase SPE), quaternary

ammonium or amino groups (for anion exchange), and sulfonic acid or carboxyl groups

(for cation exchange), each of which can separate analytes according to different

chemical properties.

Temperature. Monitoring and maintaining the temperature of the water sample at a

certain level can help to inhibit microbial activity.

Turbidity. Turbidity will be the basis if there are any solid suspended on the water

samples. This can indicate the possibility of having antibiotic residues on the water.

QuEChERS. QuEChERS came from the word formed from "quick, easy, cheap,

effective, rugged, and safe". It is a solid phase extraction method commonly used for the

detection of pesticide residues in food but it can also be used in the detection of antibiotic

residues on solids or liquids.

5
 CHAPTER II

REVIEW OF RELATED LITERATURE

According to the study led by the University of York which spanned over 70 countries

and six continents, Rivers worldwide are polluted with antibiotics that exceed environmental

safety thresholds by up to 300 times.

2.1 Antibiotics

Antibiotics have been extensively and effectively used for several decades not

only to relieve symptoms and treat human and animal diseases, but also to promote

growths in the livestock, aquaculture and plant agriculture (Yihan Chen et al., 2018).

Wise (2002) estimated antibiotic consumption worldwide to lie between 100,000 and

200,000 ton per annum, with approximatively 50% used for veterinary medicine and as

growth promoters. Global antibiotic consumption by humans alone increased by 36%

between 2000 and 2010 which illustrates that antibiotic pollution is an ever-growing

problem (Van Boeckel et al., 2014).

According to World Health Organization report on surveillance of antibiotic

consumption: 2016-2018 early implementation, Philippines consumed about 260.55

metric tons of Antibiotics in the year 2015. Tetracyclines (J01A) and penicillins (J01C)

were the most frequently consumed antibiotics in the Philippines, each contributing to

about 30% of total consumption.

2.1.1 Uses of Antibiotics

2.1.1.1 Medical Uses

Antibiotics are medication use that destroy or slow down the growth of bacteria.

(Adam Felman, 2019). It is also used to treat a wide variety of infections or diseases

caused by bacteria, such as respiratory tract infections (eg, pneumonia and whooping

6
 cough), urinary tract infections, skin infections and infected wounds (NPS

MedicineWise, 2019)

2.1.1.2 Agriculture Uses

Antibiotics are employed in livestock farming, where antibiotics can be used for

disease treatment of animals, and in sub-therapeutic levels in concentrated animal feed

for growth promotion, improved feed conversion efficiency, and for the prevention of

diseases (You Y. et al., 2014).

2.1.1.3 Domestic Uses

In the home, numerous products are available to kill potentially harmful

bacteria. Many such as soaps and shampoos contain triclosan, a synthetic compound

with antibacterial, antifungal, and antiviral properties. As well as cleaning products, it

has also been incorporated into solid products such as fabrics and children’s toys.

(Richard William Meek, 2015).

2.1.1.4 Aquaculture Uses

Antibiotics, in and of themselves, do not cure fish. Antibiotics merely control

the population growth of bacteria in a fish long enough for its immune system to

eliminate them (Yanong, Roy, 2016).

2.1.2 Properties of Some Antibiotics

Tetracyclines: they are the most frequently used antibiotics in veterinary medicine.

Tetracyclines have three pKa values, although at pH between 7 and 8 they are negatively

charged in water and are expected to be sorbed to sediment. They tend to form stable

complexes and as a result, they are more likely to remain on the topsoil or bind to

suspended organic matter. Therefore, their presence in surface or groundwater is not

prevalent, and they have just been detected in a few studies (Boxall et al., 2002).

7
Quinolones: Quinolones are polar, mostly amphoteric and exhibit poor water solubility at

pH 6–8. Quinolones are likely to persist in the environment and they are considered as

immobile in soils (Boxall et al., 2002). Several studies detected quinolones at

concentrations in the lower hundreds of ng/L in river water (Carvalho and Santos, 2016;

and references herein).

Sulfonamides: they are one of the oldest antibiotic groups that are still currently in use

and the most extensively studied group. Occurrence of sulfonamides in groundwater has

been reported in several studies (Sui et al., 2015), and it is important to highlight the high

frequency of detection of these antibiotics in water, in particular sulfamethoxazole. In

fact, sulfonamides is the most frequently reported group of pharmaceuticals in

groundwater in Catalonia (Jurado et al., 2012), and sulfamethoxazole was among the

most prevalent antimicrobial contaminant detected in a nationwide groundwater survey

conducted by the U.S. Geological Survey (Underwood et al., 2011). The widespread

occurrence of sulfonamides in groundwater might be a result of their weak sorption to

soil, low biodegradability and fast migration (Strauss et al., 2011), which makes these

compounds likely to leach to groundwater bodies.

2.1.3 Antibiotics Resistance

Antibiotic resistance is the ability of bacteria or other microbes to resist the

effects of an antibiotic. Antibiotic resistance occurs when bacteria change in some way

that reduces or eliminates the effectiveness of drugs, chemicals, or other agents

designed to cure or prevent infections. The bacteria survive and continue to multiply

causing more harm (National Center for Immunization and Respiratory Diseases, 2006).

According to (Vanessa Ngan, 2005) about 70% of bacteria that cause infections

in hospitals are resistant to at least one of the antibiotics most commonly used to treat

infections. Methicillin (meticillin) resistant Staphylococcus aureus (MRSA) is a

8
 particular problem for patients with skin diseases, ulcers and surgical wounds (Vanessa

Ngan, 2005).

2.2 Effects on living thing

The greatest concern about antibiotics in the environment is their potential role in

promoting resistance development in human and animal pathogens (Martinez JL, 2009).

Another concern is the potential effect of antibiotics on the function and services of

ecosystems. Micro-organisms in farmland soil are crucial for e.g. nitrogen fixation and other

nutrient fluxes, and there is a potential for antibiotics to disrupt such processes (D. G. Joakim

Larsson, 2014).

2.2.1 Aquatic Life

Antibiotic “pollution,” in which excess antibiotics enter natural systems and

influence the bacteria living there, helps speed along the development of resistant

strains. It also disrupts the delicate ecological balances in rivers and streams, changing

the makeup of bacterial communities (Alejandra Borunda, 2019).

Sub-inhibitory concentrations of antibiotics can have potential effects on species

interactions such as changes in population dynamics and bacterial community

composition which can lead to changes in trophic interactions but we are still lacking a

population-level perspective (Grenni et al., 2018; Hiltunen et al., 2017).

The individual concentrations of antibiotics, which are measured in the

environment, can be low but the combined concentrations could result in significant

toxicity for aquatic organisms (Marie-Claire Danner et al., 2019).

2.2.2 People

According to the study of (Gothenburg; 2011), there are two potential distinct

threats where environmental contamination with antibiotics and antibiotic resistance

9
 elements can have severe consequences for human health (not considering potential

direct toxic effects): Spread of resistant bacteria or resistance genes and Emergence of

new multidrug-resistant pathogenic bacteria.

A 2016 report found that each year around 700,000 people worldwide die of

infections that are resistant to the antibiotics we have today. Scientists, medical experts,

and public health officials worry that number could skyrocket as resistance to

commonly used medicines increases (Alejandra Borunda, 2019).

2.2.3 Plants

Soil and water-containing antibiotics constitute a potential route of human

exposure to antibiotic resistance genes through their uptake by plants (Grote M. et al,

2007) (Kumar K et al, 2005). Uptake by plants can also have other effects, such as the

accumulation of nitrofuran-type antibiotics in the edible parts of spring onions, and the

subsequent metabolism of these into genotoxic and potentially carcinogenic hydrazine-

containing metabolites (Wang Y et al, 2017).

Main effects were delayed germination or reduced plant biomass. These effects

varied markedly depending on the plant species concerned, but were most pronounced

in the two herb species, particularly by penicillin and sulfadiazine (Herbblurb, 2017).

In the study of (Michelini et al. 2012; Li et al. 2011), it showed that antibiotics

can alter biomass production, number of leaves, branching patterns, shoot length,

internode length, root/shoot ratio, fresh/dry weight, C/N and K:Ca ratio etc.

2.3 Antibiotics Safe Level

The PNEC table contains two values. PNEC‐Environment (PNEC‐ENV) values

are based on eco-toxicology data generated by Alliance member companies and relevant

peer reviewed literature. These values are intended to be protective of ecological species

10
 and incorporate assessment factors consistent with standard environmental risk

methodologies (Brandt et al., 2015; Le Page et al., 2017). The PNEC‐Minimum

Inhibitory Concentration (PNEC‐MIC) values are based on the approach published in

Bengtsson‐Palme and Larsson (2016) and are intended to be protective of resistance

promotion. The table can be found in appendix.

2.4 Antibiotics in Rivers

In the study conducted on May 2019 at University of York, concentrations of

antibiotics found in some of the world’s rivers exceed “safe” levels by up to 300 times the

first ever global study has discovered. The researchers looked for antibiotics in rivers in

72 countries across six continents and found the 14 commonly used antibiotics at 65% of

the sites monitored. In the River Thames, the researchers detected a maximum total

antibiotic concentration of 233 nanograms per litre (ng/L), whereas in Bangladesh the

concentration was 170 times higher. Sites in Bangladesh, Kenya, Ghana, Pakistan and

Nigeria were determined to have exceeded ‘safe’ levels. The study revealed that high-risk

sites were typically adjacent to wastewater treatment systems, waste or sewage dumps

and in some areas of political turmoil.

According to (Danner, et.al., 2010), worldwide antibiotic usage is more than

100,000 tons per year and there is an increasing concern over the fate of these substances.

In their study, bioassays show that some of the antibiotics found in surface waters affect

microbes at concentrations below 10 μg/L. Ciprofloxacin is one of the most commonly

used antibiotics that is mostly in streams and rivers in these concentrations. Sub-lethal

concentrations contribute to increase bacterial resistance and change the composition of

single-celled communities, as demonstrated in laboratory experiments. This has

implications for the microbial food web which will also affect the larger organisms and

ecosystem health.

11
 Up to 11 antibiotics were found by (Roura, et.al., 2017) in groundwater

corresponding to 4 different chemical groups: Fluoroquinolones, macrolides, quinolones

and sulfonamides. Fluoroquinolones and sulfonamides were quantified in surface waters

and the most frequent antibiotics were sulfamethoxazole and ciprofloxacin. The absence

of clear continuous spatial concentration trend of antibiotics observed in the aquifer

supported by the short spatial correlation found in the variograms, indicates that the

physical-chemical properties and processes of each antibiotic (mainly, sorption and

degradation), and other environmental issues, such as a patchy diffuse input and the

manure antibiotic content itself, play an important role in their spatial distribution in

groundwater.

In the study of (Chen, et. al., 2018), the occurrence, distribution, and ecological

risk assessment of 17 common antibiotics in a vital drinking water source represented as a

river-reservoir system in South China were determined. At least 15 antibiotics were

detected once in the watershed. Environmental risk assessment revealed that tetracycline

and ciprofloxacin could pose high risks in the aquatic environment. They suggested that

further investigations should be performed to elaborate the environmental behaviors of

antibiotics in the river-reservoir system, especially in drinking water sources.

(Borunda, 2019) found that in Danube, the second-longest river in Europe, seven

different types of antibiotics prevails. They found one—clarithromycin, which is used as

a treatment for respiratory tract infections like bronchitis—in concentrations four times

higher than ‘safe’ levels.

In the study of (Hwang, et. al., 2019) where twelve antimicrobial agents were

prepared in microdilution trays for storage at 4, -10, -25, and -70 degrees C and for

weekly susceptibility testing. They found out that 12 drugs had stable biological activity

12
 when stored at -70 degrees C for 1 year. They found out that all but ampicillin and

aztreonam were stable at -25 degrees C. Storage at -10 degrees C was least satisfactory.

Desiccation occurred at 4 degrees C, but short-term storage at this temperature is possible

since the antimicrobial agents are stable for up to several months.

On a study, they evaluated the effects of pH control strategy on cell growth and

the production of antibiotic by Xenorhabdus nematophila and enhance the antibiotic

activity. They adjusted the pH pf each sample by by adding 1 mol/L NaOH or 1 mol/L

HCl. Experiments showed that the optimal initial pH for cell growth and antibiotic

production of X. nematophila YL001 occurred at 7.0. Under different constant pH, a pH

level of 7.5 was found to be optimal for biomass and antibiotic activity. (Wang, et.al.,

2011).

2.5 Sources of Antibiotics

Antibiotics, despite its benefits, its continuous release into the environment and its

potential adverse impact on living organisms is of great concern leading to the term

antibiotic pollution (Brandt, et al., 2015). Majority of antibiotics are not completely

absorbed and utilized in the bodies of humans and animals, a high percentage of

administered drugs is discharged into water and soil through municipal wastewater,

animal manure, sewage sludge, and biosolids that are frequently used to irrigate and

fertilize agricultural lands (Bouki, et al., 2013). The study reported that 75–80, 50–90,

and 60% of the intake doses of tetracyclines, erythromycin, and lincomycin respectively,

are excreted in urine and feces (Kumar, et al., 2005a; Sarmah, et al., 2006). Reported

antibiotic concentrations in wastewater vary significantly and range from nanograms to

micrograms per mL (Kulkarni, et al., 2017). Though some wastewater treatment

processes can degrade antibiotics, there is notable variability in antibiotic removal rates.

This can be attributed to differences in treatment processes, such as nature of influent,

13
 treatment plant capacity, and the type of technology used (Forsberg, et al., 2012; Wu et

al., 2014).

During the last two decades, occurrence of antibiotics into water bodies and

subsequent development of resistance in microorganisms (referred to as antibiotic

resistance) have come into scientific and public focus as an issue of potential concern.

The continuous increase in resistance, as established today, is considered to be closely

linked with the widespread misuse and overuse of antibiotics in humans, animals and

agriculture (Zaman, et. al., 2017).

The first comprehensive study which demonstrates an alarming usage and

emission of various antibiotics in China was done by (Zhang, et.al., 2015). They studied

the national consumption, emissions and multimedia fate of 36 frequently detected

antibiotics in China by market survey, data analysis and level III fugacity modelling tools.

Based on their survey, the total usage for the 36 chemicals was 92700 tons in 2013, an

estimated 54000 tons of the antibiotics was excreted by human and animals, and

eventually 53800 tons of them entered into the receiving environment following various

wastewater treatments. The fugacity model successfully predicted environmental

concentrations (PECs) in all 58 river basins of China, which are comparable to the

reported measured environmental concentrations (MECs) available in some basins. The

bacterial resistance rates in the hospitals and aquatic environments were found to be

related to the PECs and antibiotic usages, especially for those antibiotics used in the most

recent period.

2.5.1 Hospitals

For many antibiotics, urine and feces from users can contain considerable amounts

of active residues (Larsson, 2014)

14
 In general, 50–80% of total parent compounds are excreted through urine and

faeces: higher excretion rates are observed for ciprofloxacin (50 to 80%) and tetracycline

(80 to 90%), while lower excretion rates are observed for erythromycin (5 to 10%),

sulfamethoxazole (15 to 30%) or clarithromycin (25%) (Mompelat et al., 2009).

The proportion of the parent compound excreted via WWTP effluents can differ

greatly among antibiotics and the process used (Monteiro and Boxall, 2015) but

Kümmerer and Henninger (2003) found that approximately 70% of the consumed amount

of antibiotics was excreted unchanged from hospitals and households into effluents in

Germany.

When we use an antibiotic, typically between 30–90% of the active compound

will get excreted and flushed down which means that sewage plants are full of a city’s

medicines (King, 2018). For many antibiotics, urine and feces from users can contain

considerable amounts of active residues. In most high-income countries with well-

developed sewage infrastructure, discharge to the environment is reduced, but microbial

communities within the treatment plants can nevertheless be exposed to µg/L

concentrations of selected antibiotics. Much of the antibiotics accumulate in sludge that

subsequently may be utilized on farmland with the intent to recycle nutrients. In surface

waters receiving municipal waste-water, it is found that the concentrations of antibiotics

rarely exceed 1 µg/L, but are more regularly in the low ng/L range. Also, antibiotic

residues have been found in marine environments (Larsson, 2014).

About 20% of antibiotics are used in hospitals. However, the major role of health

care institutions in the spread of bacterial resistance is recognized due to the broad

spectrum of antibiotics used and the high risk of human-to-human transmission of

selected resistant bacteria. Some of the antibiotics prescribed to infected people are not

15
 used and are therefore released and added with our daily waste into the environment

(Max, 2019).

In the study done by (Larsson, 2014), she highlighted the importance of waste

disposal especially in hospitals and households. Since a portion of prescribed medicines

that we buy are not used it will eventually be discarded and be considered as a waste.

Many countries do not have well-established take-back programs and still dump their

household waste in landfills. In such situations, environmental exposure from unused

medicines should not be neglected. On the other hand, some countries who have a solid

take-back program in place for are very unlikely to contribute to an appreciable extent to.

2.6 Liquid Chromatography – Mass Spectrometry (LC-MS)

There are several methods to determine antibiotic residues including bioassays,

immunoassay, thin layer chromatography, fluorescence, ultraviolet (UV) or

radioimmunoassay (RIA), but the use of Liquid chromatography (LC) has become the

technique of choice for multiclass analysis, especially when coupled to mass spectrometry

(LC-MS) and tandem MS (LC-MS2) (Moreno-Bondi M. et al., 2009). LC/MS/MS is

much more compelling due to its higher specificity and sensitivity, which leads to better

quantitation and identification. (Zhou, 2010).

2.7 Conceptual Framework

The conceptual framework of the study was shown in Figure B.1. This model

displays the underlying processes including the input and output of the research. The

samples will be investigated according to the following parameters: pH, temperature and

turbidity, prior to liquid chromatography- mass spectroscopy (LC-MS) determination.

The tests and analysis will be primarily derived and adopted from literatures that

effectively studied the characterization of antibiotics from hospital effluents. Statistical

analysis and graphic visualizations will be evaluated by using one- way analysis of

16
variance (ANOVA), means and standard deviations. Eventually, the output of this study

is to quantify and characterize the concentration level of antibiotics present in the effluent

of Saint Paul Hospital and CVMC basing from the parameters that will be investigated.

17
 CHAPTER III

METHODOLOGY

3.1 Sampling Sites

Two of the nearest hospitals in the Cagayan River was the Saint Paul Hospital and

the Cagayan Valley Medical Center. Their wastewater discharge will direct on to the

Cagayan river. In this study, the following sampling stations were selected for water

sample collection:

Table 2. Study sampling sites in Cagayan River

Station Wastewater Discharge Location

1 Saint Paul Hospital Ugac Highway, Tuguegarao City

2 CVMC Carig Sur, Tuguegarao City

3.2 Parameters

The study was limited to analyze the amount of the following top 5 antibiotics
prescribed to in-patients of the two hospitals:
a. Amoxicillin
b. Cephalexin
c. Ciprofloxacin
d. Doxycycline
e. Erythromycin

3.3 Sample collection

A total of 2 samples, including raw water samples from the sampling sites will be

collected per month, monthly from January 2020 to March 2020. Samples of 1 liter will

be collected from the raw wastewater. The samples will be collected from the center of

the stream using a water grab sampler. Samples will be collected in a 1liter amber glass

bottles, which will be stored at 4°C and will be kept on ice during transportation. Samples

must be extracted within 5 days of collection.

18
3.4 Pre – Treatment

All samples will be tested for their turbidity, temperature and pH prior to LC-MS

determination. The pH of samples will be measured using pH meter, and the temperature

of will be measured throughout the study period and taken into consideration the

variability of temperatures within the season. Turbidity will be measured to indicate the

existing of suspended particles such as clay, organic particles, or microorganisms. The

sample pH will be adjusted at 4 or 7 using NaOH or HCl.

3.5 Sample Analysis

The samples will be taken and analyze in University of the Philippines - Diliman.

All water samples will be filtered and stored at 4oC in an insulated ice chest “cooler” until

they are extracted, typically within 1 week. Chelating agent is used to reduce antibiotic

binding to major cations, thereby promoting analyte retention on the solid phase

extraction cartridge. Filtered samples will be analyzed within 3 days if stored at 4oC and

within 3 weeks if stored at −18oC after filtration the following procedures were done:

3.5.1 QuEChERS

QuEChERS is a solid phase extraction method to be used for the detection of

antibiotic residues. The solid-phase extract is prepared according to the laboratory’s

protocol with some modifications. The water sample is passed through the cartridge at

flow rate using a vacuum extraction manifold. The extracts are filtered and transferred to

auto sampler vials, stored at a low temperature.

3.5.2 Liquid Chromatography – Mass Spectrometry (LC-MS)

The LC system is a system with a variable wavelength UV detector. The mass

spectrometer is a duo ion trap equipped with an Electrospray ionization (ESI) source

19
 operated in positive ion mode. The LC column temperature will be kept at 15oC. The

investigated antibiotics were then eluted.

3.6 Statistical Analysis

Kruskal Wallis Test and Mann-Whitney-Wilcoxon tests will be used to analyze

the data gathered. Kruskal Wallis Test is a non-parametric method for testing whether

samples originate from the same distribution. This test will be used to compare the

parameters of the samples in every collection. While Mann-Whitney-Wilcoxon Tests

essentially calculates the difference between each set of pairs and analyses these

differences, thus, this test will be used to compare the results of CVMC and Saint Paul

Hospital.

20
APPENDIX

Appendix A

A.1. List of Predicted No-Effect Concentrations (AMR Industry Alliance, 2018)

Active PNEC-ENV PNEC-MIC Lowest Value

Pharmaceutical (μg/L) (μg/L) (μg/L)

Ingredient

Amoxicillin Testing On-Going 0.25 0.25

Cephalexin 0.08 4.0 0.08

Ciprofloxacin 0.45 0.06 0.06

Doxycycline Testing On-Going 2.0 2.0

Erythromycin 0.50 1.0 0.50

Lincomycin 1.8 2.0 1.8

Metronidazole N/A 0.13 0.13

Norfloxacin 120 0.50 0.50

Ofloxacin 10 0.50 0.50

Oxytetracycline 18 0.50 0.50

Sulfadiazine 720 N/A 720

Sulfadimethoxine 50 N/A 50

Sulfadoxine 0.60* N/A 0.60

Tetracycline 3.2 1.0 1.0

Tylosin 0.82 4.0 0.82

* PNEC-ENV (Predicted No‐Effect Concentrations – Environment)

*PNEC-MIC (Predicted No‐Effect Concentrations - Minimum Inhibitory Concentration)

21
A.2. Various antibiotics source and action (Shikha Goyal, 2016).

Antibiotics Source Action

Pencillin Penicillium chrysogenum, Tonsilitis, Sore Throat,

P.notatum + Phenyl Gnonorrhea, Rheumatic

Acetic Acid Fever, some Pneumonia

types.

Tetracyclines/Aureomycin Streptomyces Viral pneumonia,

aureofaciens Osteomyelitis, Whooping

Cough and Eye infections.

Oxytetracycline/Terramycin Chlorotetracycline- Intestinal and Urinary

Hydrogenation Infections (Spirochaetes,

Streptomyces rimousus Rickettsia and Viruses)

22
A.3. Some antibiotics which are developed through various Biotechnologies (Shikha

Goyal, 2016).

Some Antibiotics Developed through Biotechnology

Antibiotic Microbial Source

Penicillin Penicillium notatum and P chrysogenum

Bacitracin Bacillus subtilis

Cephalosporin Cephalosporium acremonium

Griseofulvin Penicillium griseofulvum

Streptomycin Streptomyces griseus

Tetracycline S erythraeus

Erythromycin S aureofaciens

Chloramphenicol S venezuelae

23
Appendix B

INPUT PROCESS OUTPUT

 Raw water  Sample  Concentration

samples Collection level of
 Chelating  Pre- treatment antibiotics
from the
Agent  Sample Analysis
wastewater of
 Solid- phase
Saint Paul
Extraction
Hospital and
 Liquid
CVMC.
Chromatograph
y- Mass
Spectroscopy
 Statistical
Analysis

Figure B.1: A Model for the Determination of Concentration Level of Antibiotics contained

in the Wastewater of Saint Paul Hospital and Cagayan Valley Medical Centre

24
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