global research vol ii iss ii 8

8/10/2019 Global Research Vol II Iss II 8

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Global Research Journal of Global Research Computer Science & TechnologyResearcher Forum (JGRCST)

Vol.-II, Issue-II, October 2014ISSN: 2349 - 5170

58

Separation and Characterization of Biological Degradation of selectedPharmaceuticals in Water

Rajesh Kumar

OPJS University, Rajasthan, India

1 IntroductionThe organic pollutants like pharmaceutical drugs and pesticides are group of persistent

contaminants of environmental and toxicology with great social concern. The difference

between pharmaceuticals and pesticides with respect to environmental release is that

pharmaceuticals have the potential for ubiquitous direct release into the environment due

to different humans activities. Due to worldwide use of pharmaceutical products in large

quantities, they have been identified in a wide variety of environmental media and biota

(1-8). The persistent and bio-accumulative nature of pharmaceutical products have been

recognized particularly in aquatic ecosystems, where the stepwise accumulation in soils,

sediment, fish and humans and degraded and concentrated through food-chain is rather

common (9-11).

The distribution of pharmaceuticals is a large function of their production volumes,

which can rival those for many pesticides. The pharmaceutical drugs with their

respective metabolites and transformation products will collectively referred to as

pharmaceuticals. The pharmaceuticals are continually enter into the environment

through sewage water treatment (23, 24) which cause to contaminate the ground

water and surface water(25, 26) . The presence of numerous drugs in aquatic

environment sharing the specific mode of action could lead to significant effects on

humans (26, 27) and marine organisms. There is a little literature on the occurrence


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and effects of degraded drugs in the environment more data exist for antibiotics than

for any other therapeutic class. This is a result of their extensive use in both human

(23, 24) therapy and animal husbandry, their morrow easily detected effects and

points and their grater chances of introducing into the environment, not just by

sewage treatment plants.

Pharmaceuticals are designed to target specific metabolic pathways in humans and

domestic animals, they can have numerous often unknown effects on metabolic

systems of non-target organisms especially invertebrates. Although many non-target

organisms share certain receptors with humans, effects on non-target organisms are

usually unknown. It is important to recognize that many drugs, their specific modes

of action even in the target species are also unknown without knowing to mode of

action of the degraded product, it is impossible to assess the toxicity tests.

Pharmaceuticals will refer to non-biologic drug. The number of biologics approved

by USFDA is growing and their fate in the environment is unknown. Pharmaceutical

drugs are chemicals used for diagnosis treatment, alteration or prevention of disease

health condition of the human body. The drugs are usually designed with specific

mode of action in mind, they can also have numerous side effects on non-target

organisms. The world combines literature has addressed only a very small

percentage of degraded pharmaceuticals compounds.

Pharmaceuticals are continually released into the environment in enormous

quantities as a result of their manufacture, use (via excretion, mainly in urine and

feces) and disposal of unused / unwanted drugs those that have disposed both

directly into the domestic sewage system and via burial in landfill. Although largely

unknown there is evidence that large quantities of prescription and nonprescription

over-the-counter drugs are never consumed (28) and many of these are undoubtedly


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eventually disposed down toilets or via domestic refuse. The possibility that

pharmaceuticals can enter the environment from a number of different routes and

possibly cause untoward effects in biota has been in the literature for several decades

(1,2,3,4,6) . The evidence support the case that drugs refractory to degradation and

transformation (1) do indeed have the potential to reach the environment. But study

on the degradation product pharmaceuticals in the environment is limited.

Pharmaceutical enter into the environment are degraded and forum a new compound

due to environmental condition. The degraded product- metabolites and conjugates

from eukaryotic and prokaryotic metabolism and from physicochemical alteration-

add to the already complex picture of thousands of highly bioactive chemicals. The

concentrations of degraded products are increased through food chain (10). The

degraded products can give more side effects on marine organisms and humans.

Compounds surviving the various phases of metabolism and other degradative or

sequestering actions (environmental persistence) can then pose an exposure risk for

organisms in the environment. Even the less/ nontoxic conjugates can later be

converted back to the original bioactive compounds via enzymatic or chemical

hydrolysis. Some degradation products can even be more bioactive than the parent

compound. Therefore conjugates can essentially act as storage reservoirs from whichthe free drugs can alter be released into the environment (6, 12, and 29).

Several authors reported the distribution of pharmaceuticals in ecosystem in

Germany (26, 30) and in other countries (19). But there is not much literature on the

degradation and metabolite products of pharmaceuticals in the environment in India

and in other countries.

Therefore it is important to understand that fate of biological degradation and its

metabolites products of pharmaceutical in water from sewage treatment plant(STP) and


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drinking water and soils Therefore identifying the metabolic and biological degradation

products of pharmaceuticals is essential to understand the impact on humans and marine

organisms by using instruments like LC-MS and Gas Chromatograph equipped with Mass

spectrometer(GC-MS). And also this study may be helpful to remove the pharmaceuticals

in water from sewage treatment plants and the effluents from pharmaceutical industries.

2 Literature Survey

a) Rationale of the study supported by cited literature.

b)

Hypothesis the organic pollutants like pharmaceutical drugs and pesticides are

group of persistent contaminants of environmental and toxicology with great

social concern. The difference between pharmaceuticals and pesticides with

respect to environmental release is that pharmaceuticals have the potential for

ubiquitous direct release into the environment due to different humansactivities.

Due to worldwide use of pharmaceutical products in large quantities, they have

been identified in a wide variety of environmental media and biota (1-8). The

persistent and bioaccumulative nature of pharmaceutical products have been

recognized particularly in aquatic ecosystems, where the stepwise accumulation

in soils, sediment, fish and humans and degraded and concentrated through food-

chain is rather common (9-11).

The distribution of pharmaceuticals is a large function of their production volumes,

which can rival those for many pesticides. The pharmaceutical drugs with their

respective metabolites and transformation products will collectively referred to as

pharmaceuticals. The pharmaceuticals are continually enter into the environment

through sewage water treatment (23,24) which cause to contaminate the ground


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water and surface water (25,26) . The presence of numerous drugs in aquatic

environment sharing the specific mode of action could lead to significant effects on

humans (26,27) and marine organisms.

There is a little literature on the occurrence and biological degradation and

metabolites of pharmaceuticals in the environment in India and other countries. This

is the result of attempt to carry out this research problem.

b) Current status of research and development in the subject (both international and

national status)Several authors reported the distribution of pharmaceuticals in

ecosystem in Germany (1-5) and in other countries (6,7). But there is not much

literature on the degradation products, Metabolites of pharmaceuticals in the

environment in India and in other countries. Therefore it is important to understand

that fate of biological degradation, Metabolites of pharmaceutical products in water

from sewage treatment plants (STP), water and soil and further need to study the

characterization.

3 Methodology

3.1 Materials

The selected pharmaceuticals were procured from Sigma Aldrich chemicals.

1) Sodium diatrizoate dehydrate, acetamidophenol sigmaultra (paracetamol),

cetrizine, ciprofloxacin, Meclofenomic acid, Bezafibrate, sulfamethoxazole.

2) Methanol, hexane, benzene, ethyl acetate and chloroform are glass distilled

and use for extraction of degraded pharmaceutical product from samples by solid

phase extraction method (SPE).


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3) Solid phase extraction column C-18 (SPE).

3.2 Source

1) Sewage water was collected in sterile jar and used for isolation of different type

of drug tolerant bacteria and fungus.

2) Water effluent from juggat pharma was collected in sterile jar and used for

isolation of different type drug tolerant bacteria and fungus.

3.3

Isolation

3.3.1 Isolation of bacteria: effluent from pharmaceutical industries and from pesit

drainage were taken and inoculated on agar plate containing known concentration of

standard drug.

Nutrient Agar preparation

For 1 L of nutrient agar

Component Amount(g)

Beef extract 3

Peptone 5

Nacl 5

Agar for bacterial culture 15

Distilled water 1000(ml)

Bacterial plate preparation


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o Agar was autoclaved and cooled down to 600c then Standard (0.2 gm/100ml

media) was mixed well.

o It was poured in sterilized petriplate in LAF and left for solidifying

o 1ml of 10-6diluted sample water was poured and using spread plate technique

inoculation was performed.

o It was kept for 48 hour in incubator at 370c.

o Bacterial culture were obtained

Pure culture of bacteria

Pure culture of bacteria was done using streaking method in slant culture.

3.3.2 Isolation of fungus: effluent from pharmaceutical industries and from pesit


standard drug.

MRBA preparation

For 1 L of MRBA

Component Amount(g)

Peptone 5

KH2pPO4 5

Dextrose 10

Agar 15

Streptomycin 0.03

Rose Bengal 0.013



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Fungal plate preparation

o MRBA was autoclave and cooled down to 600c then Standard (0.2 gm/100ml

media) was mixed well.

o It was poured in sterilized petriplate in LAF and left for solidifying

o 1ml of 10-6diluted sample water was poured and using spread plate technique

inoculation was performed.

o It was kept for 48 hour in incubator at 370c.

o Fungal culture were obtained

Pure culture of fungus

Pure culture of bacteria was done using streaking method in test tube on MRBA.

3.4 Drug sensitivity method

o Eight Nutrientagar plate were prepared with each having 8 wells,

o Drug tolerance bacteria were taken and inoculated on these plates.

o 7 micro liter of each standard were dropped in these wells and kept for

incubation for 48 hour.

o

Clearance zone was observed and drug sensitivity test was performed for

checking bacterial tolerance.

3.5 Bacterial physical characterization

Color: From basic observation.

Gram stain:


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o Smear preparations: Smear was prepared using aseptic technique.

Gram Staining Procedure:

o Cover with Crystal Violet for 20 seconds (Primary Stain).

o Gently rinse off the stain with water and shake off the excess.

o Cover with Gram's Iodine for one minute (Mordant).

o Pour off the Gram's iodine.

o Run 95% Ethyl Alcohol down the slide until the solvent runs clear (about 10-20)

(Decolorizing Agent).

o

Rinse with water to stop the action of the alcohol. Cover With Safranin for 20

second (Counter Stain).

o Gently rinse off the stain with water and clean off the bottom of the slide with

95% alcohol.

o Slides were viewed under microscope and bacteria were characterized.

3.6 Bacterial Biochemical characterization

I) Indole test

It detects ability of bacteria to breakdown tryptophan to indole.

II)Methyl red test

It detects ability of bacteria to produce and maintain stable acid end product from glucose

fermentation.

III)Vogues proskauer test

Use to detect production of acetylmethylcarbinol (acetoin) from pyruvic acid during

glucose fermentation.

IV)Simmonsscitrate test

It detects capability to utilize citrate as carbon source.


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3.7 TLC

Optical density bacterial( 0.06) culture were taken and centrifused to form pelet and

supernatant.palet was taken and mixed with standard drug . Then degradation was studied

with help of TLC method.

3.8 Bioagumentation

Known concentration of pharmaceutical standard sodium ditrizoate dihydrate and

acetaminophin sigmaultra (paracetamol), citrizine, ciprofloxacin were inoculated with 0.6

OD of bacterial prime culture of known volume. The degradation was analyzed by with

cod and degradation product was extracted through SPE column for analysis by HPLC.

Prime culture

For bacterial growth studies

o Prime culture media was prepared

For 1 L of Prime culture

Component Amount(g)Beef extract 3

Peptone 5

Nacl 5


o Tolerant bacteria were inoculated and kept for 48 hour of incubation.

o OD was found by spectrometer.


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4 Analysis

4.1 Analysis by COD method

o Reagents

o Standard potassium dichromate solution, 0.04167M: Dissolve 12.259 g

K2Cr2O7, primary standard grade, previously dried at 150C for 2 h, in

distilled water and dilute to 1000 mL. This reagent undergoes a six-

electron reduction reaction; the equivalent concentration is

6 X 0.04167M or 0.2500N.

o Sulfuric acid reagent: Add Ag2SO4, reagent or technical grade, crystals

or powder, to conc H2SO4 at the rate of 5.5 g Ag2SO4 /kg H2 SO4. Let

stand 1 to 2 d to dissolve. Mix.

o Ferroin indicator solution: Dissolve 1.485 g 1,10-phenanthroline

monohydrate and 695 mg FeSO47H2O in distilled water and dilute to

100 mL. This indicator solution may be purchased already prepared.*

o Standard ferrous ammonium sulfate (FAS) titrant, approximately

0.25M: Dissolve 98 g Fe(NH4)2(SO4)26H2O in distilled water. Add 20

mL conc H2SO4, cool, and dilute to 1000 mL.

o

Standardize this solution daily against standard K2Cr2O7 solution.o Add all reagents to the refluxing flask open to the atmosphere without

the condenser attached for 2 hour. Find dichromate utilized by titration

with FAS.

4.2 Extraction of degraded compound by SPE column

o Column conditioning: One column of acetone, one column of methnol,

one column of distilled water was passed through SPE column.


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o 20 ml of each solution was passed through SPE column after

conditioning and then it was extracted with 10ml of methanol.

4.3 Analysis by HPLC method

Samples were analyzed.

5. Result and Discussion5.1 Isolation of Fungus

Isolation of fungus: effluent from pharmaceutical industries and from pesit drainage were

taken and inoculated on MRBA plate containing known concentration of standard drug

Plate prepared by spread plate technique

Fungi on Diclofinac fungi on glaciphage fungi on Nimuslide fungi on Ranitidine

These four fungi were isolated on MRBA media which contains different drugs.

5.2 Isolation of bacteria


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Isolation of bacteria and fungus: effluent from pharmaceutical industries and from pesit


standard drug.

Isolation from Pesit Drainage

DICLOFINAC GLACIPHASE NIMUSULIDE

RANITIDINE MIXTURE OF MEDIA WITHOUT DRUG


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DICLOFIONAC GLACIPHAGE,

NIMUSULIDE RANITIDINE

These bacteria were isolated from PESIT drainage and pharmaceutical industrial effluent

Isolation of Bacteria from Industrial Effluent

Ciprofloxacin acetaminophin ditrizoate dehydrate Cetrizine sigmaultra

(paracetamol)

5.3 Bacterial physical and biochemical characterization

Bacterial physical and biochemical characterization were performed .bacterial tolerance

were checked by drug sensitivity method.

CHARACTERIZATION OF BACTERIA ISOLATED FROM PESIT DRAINAGE

Tolerance

bacteria

symbol Color Gram

test

Indole

test

Methyl red

test

Voges

proskauer test

Citra


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Test

DICLOFIONAC D Creamy +vecocci

+ve -ve +ve -ve

GLACIPHAGE G White +vecocci

+ve -ve +ve -ve

NIMUSULIDEN

Brown +vecocci

+ve +ve +ve +ve

RANITIDINER

Brown +ve

cocci

+ve -ve +ve -ve

MEDIA

WITHOUT

DRUG

MW

Creamy + cocci + ve -ve + ve -ve

MIXTURE OF

ALL ABOVE

DRUG

MA

Brown + cocci +ve -ve +ve -ve

D G N R MW MA D G N R

MW MA

Indol test Methyl Red Test

D G N R MW MA D G N R MW MA


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Voges Test Simmons Citrate Test

Bacterial Biochemical characterization result

I) Indole test :all bacteria were breaking down tryptophan to indole.

II)Methyl red test: nimuslide bacteria produce and maintain stable acid end product

from glucose fermentation , other do not.

III)Voges proskauer test : all bacteria are producing acetylmethylcarbinol (acetoin)

from pyruvic acid during glucose fermentation.

IV)Simmonss citrate test: bacteriag rown on nimesulide media have the capability to

utilize citrate as carbon source, other are not.

CHARACTERIZATION OF BACTERIA ISOLATED FROM PESIT DRAINAGE

Tolerance

bacteria

symbol Color Gram

test

Indole

test

Methyl

red test

Voges

proskauer

test

Citrate

Test

Cetrizine CT Creamy +ve

cocci

-ve -ve -ve -ve

Cifrofloxacin CF White +ve

cocci

-ve +ve -ve -ve

acetamidophenol

sigmaultra

(paracetamol)

P Creamy +ve

cocci

-ve -ve -ve -ve


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Sodium

ditrizoate

dehydrate

D White +ve

cocci

-ve +ve -ve -ve

Bacterial Biochemical characterization result

I) Indole test :no bacteria were breaking down tryptophan to indole.

II) Methyl red test: ciprofloxacin and ditrizoate bacteria produce and maintain stable

acid end product from glucose fermentation, other do not.

III) Voges proskauer test: all bacteria are producing acetylmethylcarbinol (acetoin)

from pyruvic acid during glucose fermentation.

IV)Simmonss citrate test : none bacteria have the capability to utilize citrate as carbon

source.

P D CT CF P D CT CF

Indol test Vouges test

CF D P CT CF D P CT


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Methyl Red Test Simmons Citrate Test.

5.3 Drug sensitivity method

Fig. known concentration of all eight standards

1. Cetrizine 3. Ciprofloxacin 5. Methyl propionic acid 7. Paracetamol2. Bezafibrate 4. Sulfamethoxazole 6. Meclofenomic acid 8. Sodium ditrizoate


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Observation:Clear zone large :4Clear zone small :3

Clear zone verysmall:2

Observation:Clear zone large:4Clear zone small :3

Clear zone verysmall:2

Observation:Clear zone large:noClear zone small :5

Clear zone verysmall:8,1

Observation:Clear zone large:5Clear zone small :4,3Clear zone verysmall:1,7,6

Observation:Clear zone large:4Clear zone small :3Clear zone very small:2

Observation:Clear zone large:4Clear zone small :3,2Clear zone very small:no


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Inferences : all the bacteria were recheked for all eight standards.

5.4 Bioagumentation

Known concentration of pharmaceutical standard sodium ditrizoate dehydrate and

acetaminophin sigmaultra (paracetamol), citrizine, ciprofloxacin were inoculated with 0.6

OD of bacterial prime culture of known volume. The degradation was analyzed by with

cod and degradation product was extracted through SPE column for analysis by HPLC.

OPTICAL DENSITY OF PRIME CULTURE (at 540nm)

TIME 24 hour 48 hour 72 hour 96 hour 120 hour

Observation:

Clear zone large:4Clear zone small :3,2,1Clear zone very small: 6

Observation:Clear zone large:4Clear zone small :3,1Clear zone very small:2,8


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Cetrizine 0.34 0.54 0.68 0.75 0.78

Cifrofloxacin 0.25 0.60 0.66 0.72 0.80

acetamidophenol

sigmaultra

(paracetamol)

0.32 0.64 0.70 0.74 0.76

sodium ditrizoate

dehydrate

0.28 0.50 0.60 0.66 0.67

The bacteria grown in presences of selected pharma

Inferences : Maximum growth was shown at around 48 hour so bacteria were in log phase

so 0.60 OD bacterial culture were taken for bioaugumentation.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

24 hour 48 hour 72 hour 96 hour 120 hour

cetrizine

ciprofloxacin

paracetamol

ditrizoate


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COD of Acetaminophin standard solution with Acetaminophin tolerance bacteria

DAY 0 4 14 18 22 32

SAMPLE 3420 792 460 300 200 180

CONTROL 3420 1528 1320 840 480 290

COD of Sodium Ditrizoate Dihydrate standard solution with Sodium Ditrizoate

Dihydrate tolerance bacteria

0

500

1000

1500

2000

0hour 24hour 48hour 72hour 96hour

SAMPLE

CONTROL

0

500

1000

1500

2000

2500

3000

3500

0day 4 14 18 22 32

SAMPLE

CONTROL


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DAY 0 12 16 20 26 32

SAMPLE 980 464 320 210 200 190

CONTROL 980 598 498 300 240 210

Fig. SPE setup

0

200

400

600

800

1000

0 12 16 20 26 32

SAMPLECONTROL


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Extraction: SPE C18 cartridges were used for the extraction of standard drug from water

after conditioned the column with ethyl acetate, methanol and water and residue eluted

with methanol.

6. Conclusions

We have isolated the bacteria (9 types) and fungi (4 types) from the domestic waste and

also from the pharmaceutical industrial effluents. We have used two bacterial from the

isolated and used for the degradation of the pharmaceuticals. We have studied

biochemical charectistics of the isolated Bactria. The rate of the degradation of the

pharmaceutical compounds was observed by measuring the chemical oxygen demand andbacterial growth in the selected medium was recorded. We have established the SPE

extraction method for the extraction of the pharmaceutical compounds. The samples are

used for the analysis of pharmaceutical compounds by HPLC(the work is in progress).

(Based on the outcome of this work, I have submitted detailed major research project to

the DST for the financial support).

7. Acknowledgements

We thank to the PESIT Management, Principal, R& D Director for the financial support

and also we thank to Head of the department of Biotechnology for the encouragement and

support to carry out the project work in the department.

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