Occurrence, distribution and fate of pharmaceuticals as chemical markers of contamination from urban sources in the vulnerable area of the Ebro Delta (Spain) Mira Čelić1, Meritxell Gros1, Marinella Farre2, Damia Barceló1,2, Mira Petrović1,3

ICRA-Catalan Institute for Water Research, Water Quality Area1, Girona, Spain

IDAEA-CSIC, Department of Environmental Chemistry2, Barcelona, Spain

Catalan Institution for Research and Advanced Studies3, Barcelona, Spain

OUTLINE

o Introduction

o Sources and routes of entry of pharmaceuticals

o INTEGRA-COAST project

o Objectives of the study

o Study area and sampling sites

o Analysis

o Target pharmaceuticals in water samples

o Extraction of water samples

o Target pharmaceuticals in sediment samples

o Extraction of sediment samples

o Quality parameters obtained during analysis

o Results

o Occurrence of PhACs in water samples

o Removal during wastewater treatment processes

o Occurrence of PhACs in sediment

o Conclusion

INTRODUCTION Sources and routes of entry of pharmaceuticals

sea water

PhACs are the contaminants of anthropogenic origin with the biggest input into the environment.

•After their consumption they are discharged into wastewaters.

•PhACs are partially removed during wastewater treatment processes, being discharged into receiving water bodies.

•Changes in river flow have one of the greatest effects on water quality (point source pollution is controlled by river flow).

•Up to now there have been a lot of studies on their occurrence in waste and surface waters but information about their presence in sea and coastal areas is still sparse.

INTEGRA-COAST PROJECT

INTEGRA-COAST study microplastics, nanomaterials, emerging organic contaminants (pharmaceuticals and personal care products, new pesticides, perfluoroalkyl substances and siloxanes) and marine biotoxins in a vulnerable coastal area.

The main goal of INTEGRA-COAST is to perform an integrated study of the fate, behaviour, and the river transport of emerging pollutants, nanomaterials and microplastics in estuaries, wetlands and coastal waters and to identify specific organic contaminants used as chemical markers of wastewater pollution.

• To assess the impact of WWTP discharges in coastal areas and protected ecosystems, with special focus on the contamination by pharmaceuticals.

• To study the fate and transport of a large number of multiple-class pharmaceuticals in riverine and coastal ecosystems (e.g. occurrence in water and sediments).

• To evaluate possible seasonal fluctuations in the occurrence and behavior of these contaminants in these ecosystems.

• Identify relevant pharmaceuticals as markers of urban pollution in coastal areas with the objective to include them in risk assessment schemes and future monitoring programs.

OBJECTIVES INTRODUCTION

Study area – the Ebro Delta

• wetland area of 320 km2

• the third largest delta in the Mediterranean Sea with high biological productivity

• highly relevant for conservation

• has a typical Mediterranean climate (with rainfall concentrated in autumn and spring (200–300 mm) and intense summer drought (<50 mm))

• c.a. 13% of its total surface is composed of natural lagoons, bays and marshes

• agricultural is one of the main activities such as rice and orchards.

Three sampling campaigns:

• October-November 2015 (autumn)

• February-April 2016 (winter)

• June-July 2016 (spring-summer)

INTRODUCTION

Total number of 213 samples: 87 waters and 71 sediments

Sampling: 29 “hot-spots” sites

WWTP2 WWTP1

•wastewater IN/OUT •emissary •canals •Ebro river •lagoons •sea water (water/sediment)

Wastewater treatment plants (WWTP)

Edar Amposta (WWTP1)

Sant Carles de la Ràpita (WWTP2)

Served population: 19 805 14 262

Population equivalent (PE): 27.500 28.921

Flow (m3 /dan): 5.500 6.310

Type of the treatment: Biological with activated sludge

Biological with elimination of N with tertiary treatment

Analgesics/anti-inflammatories

Ketoprofen

Naproxen

Ibuprofen

Indomethacine

Acetaminophen

Salicylic acid

Diclofenac

Phenazone

Propylphenazone

Piroxicam

Tenoxicam

Meloxicam

Oxycodone

Codeine

Psychiatric drugs Carbamazepine

2-Hydroxycarbamazepine

10,11-epoxycarbamzepine

Acridone

Sertraline

Citalopram

Venlafaxine

Olanzapine

Trazadone

Fluxotine

Norfluxotine

Paroxetine

Diazepam

Lorazepam

Alprazolam

Antibiotics

Erithromycin

Azithromycin

Clarithromycin

Tetracycline

Ofloxacin

Ciprofloxacin

Sulfamethoxazole

Trimethoprim

Metronidazole

Metronidazole-OH

Dimetridiazole

Ronidazole

Cefalexin

To treat asthma Salbutamol

Histimine H2 receptor antagonists Loratadine Desloratadine Ranitidine Famotidine Cimetidine

Β-Blocking agents Atenolol Sotalol Metoprolol Propranolol Carzalol Nadalol

Cholesterol lowering agents

Atrovastatine

Pravastatine

Mevastatine

Antihypertensives Amlodipine Losartan Ibersartan Valsartan

Antihelmintics Albendazole Thiabendazole Levamisole

Diuretic

Hydroclorothiazide

Furosemide

Torasemide

Lipid regulators and cholesterol lowering drugs

Bezafibrate

Gemfibrozil

Pravastatin

Fluvastatin

Atrovastatin

X-ray contrast agents Iopromide

Anticoagulant Warfarin

Prostatic hyperplasia

Tamsulosin

Antiplatelet agent Clopidogrel

Sedation and muscle relaxation

Xylizine Tranquilizer Azaperone Azaperol

Synthetic glucocoticoid

Dexamethasone

Calcium channel blockers Diltiazem Verapamil Norverapamil Antidiabetic Glibenclamide

Target pharmaceuticals in water samples

Total=81 PhACs

ANALYSIS

Extraction of water samples

SAMPLE

PRE-TREATMENT

HPLC-ESI-MS/MS (QqLIT)

FILTRATION 0.7 µm/ 0.45 µm

Addition of 0.1% Na2EDTA

Addition of 34 isotopically labelled standards

500 mL sea water 100 mL river water and canals 50 mL effluent 25 mL influent

Elution with 6 mL MeOH

Analytical Protocol >>

EXTRACTION &

PRE-CONCENTRATION

Surrogate standards

Evaporation to dryness 1mL MeOH/H2O (10:90, v/v)

Gros et al., Journal of Chromatography A, 1248 (2012) 104-121

SPE OASIS HLB®

ANALYSIS

LC colum Purospher ® RP-18 (125x2mm) (5 µm) Quantification of target compounds: • MRM mode: two transitions [M+H]+ > 2 Products in PI [M-H]- > 2 Products in NI

HPLC-MS/MS

liquid chromatography coupled to tandem mass spectrometry

Analgesics/anti-inflammatories Acetaminophen Diclophenac Ibuprofen Indomethacine Ketoprofen Naproxen Mefenamic acid

Lipid regulators and cholesterol lowering drugs Bezafibrate Fenofibrate Gemfibrozil Mevastatin Pravastatin Atrovastatin

Macrolide antibiotics Erytromicin Roxithromycin Clarithromicin Josamycin Tylosin A

Β-blockers Atenolol Sotalol Metoprolol Timolol Nadalol Pindolol

Histimine H2 receptor antagonists Ranitidine Famotidine Cimetidine

Psychiatric drugs Diazepam Lorazepam Carbamazepine

Diuretic Hydrohlorothiazide Furosemide

Antihypertensive Nifuroxazide Enalapril

Sulfonamid antibiotics Sulfamethazine

Β-agonists Clenbuterol Salbutamol

Antidiabetic Glibenclamide

Other antibiotics Trimethoprim Chloramphenicol Metronidazole

Phenazone type of drugs Phenazone

Barbiturates Butalbital

Total=43 PhACs

ANALYSIS Target pharmaceuticals in sediment samples

PLE - ASE 300 Dionex

freeze-drying (-40 ºC, 0.044 bar) store at -20 ºC

Sample pre-treatment

Sample extraction

1g of sediment sample;

T=100ºC

methanol/water, 1/2 v/v

3 static cycles (5 min)

total flush volume 100% of cell

60s of nitrogen purge

SPE – extraction and clean-up

PLE extract (~22 mL) is diluted in 500

mL of HPLC water + 15mL Na2EDTA

SPE OASIS HLB®

Addition of 34 isotopically labelled standards

Elution with 8mL MeOH Reconsituted with 1mL MeOH/H2O (25:75, v/v)

Extraction of solid samples

HPLC-MS/MS (QqLIT)

Jelić et al., Talanta 80 (2009) 363-371

ANALYSIS

liquid chromatography coupled to tandem mass spectrometry

pressurized liquid extraction

Quality parameters obtained during analysis, recoveries (%) and method detection limits (MDL)

Recovery, % (n=3), ± RSD Sea Lagoons Canal Effluent Influent Analgesics/ant-inflammatories

59-114% 50-120% 56-146% 57-112% 59-110%

Lipid regulators and cholesterol lowering statin drugs

58-101% 60-96% 42-117% 70-99% 88-103%

Psychiatric drugs 63-114% 51-124% 41-147% 61-113% 55-115%

Histamine H1 and H2 receptor antagonist

62-124% 52-80% 40-95% 87-119% 50-112%

β-Blocking agents 71-126% 60-90% 57-120% 56-88% 57-109%

Duretic 85-101% 68-113% 53-91% 55-98% 63-76% Antidiabetic 87% 104% 97% 103% 112%

Antihypertensives 73-102% 60-90% 41-101% 51-85% 51-85%

Antiplstelet agent 91% 95% 86% 90% 60% Prostatic hyperplasia 110% 50-97% 100% 112% 56% To treat asthma 72% 82% 73% 117% 108% Anticoagulant 82% 123% 90% 76% 62% X-ray contrast agent 102% 52-85% 100% 94% 70%

Anti helmintics 55-120% 50-110% 50-115% 74-98% 60-112%

Synthetic glucocorticoid 80% 60-118% 82% 74% 65% Sedation and muscle relaxation

85% 123% 88% 83% 87%

Tranquilizer 90-92% 52-85% 93-100% 92-102% 76-92%

Antibiotics 63-121% 57-128% 53-116% 66-116% 61-120%

Calcium channel blocker 86-120% 60-127% 64-96% 83-115% 69-74%

MDL (ng/L) 0.01-7.2 0.01-9 0.03-15.2 0.2-26 0.2-50

ANALYSIS

Sediment Recovery (%)

Analgesics/antiinflammatories 60-118%

Phenayone type drugs 87-110% Lipid regulators and

cholesterol lowering statin drugs 77-115%

Psychiatric drugs 80-105%

Histamine H1 and H2 receptor antagonist 87-104%

Macrolide antibiotics 68-95%

Sulfonamid antibiotics 59-100%

Other antibioptics 75-93%

β-blockers 90-114%

β-agonist 84-111%

Diuretic 71-84%

Antidiabetic 89-116%

MDL (ng/g) 0.01-3.20

% removal WWTP_1st campaign

Elimination efficiency, %

0 10 20 30 40 50 60 70 80 90 100

bezafibrate

ibuprofen

naproxen

salicylic acid

ranitidine

atenolol

gemfibrozil

nadolol

atorvastatin

sulfamethoxazole

trimethoprim

diltiazem

desloratadine

clarithromycin

thiabendazole

oxycodone

hydrochlorothyazide

codeine

furosemide

albendazole

propanolol

valsartan

losartan

diazepam

tamsulosin

citalopram

>80%

(good removal)

Less then < 10 % removal

1st Campaing

RESULTS

Low to moderate removal rates were obtained for most compounds, except for analgesics and anti-inflammatories (>80%).

The pharmaceuticals belonging to the same

therapeutic groups show the similar removal for both

sampling campaing.

0 10 20 30 40 50 60 70 80 90 100

naproxen

bezafibrate

salicylic acid

ranitidine

atenolol

gemfibrozil

nadolol

atorvastatin

ciprofloxacine

sulfamethoxazole

trimethoprim

diltiazem

desloratadine

clarithromycin

thiabendazole

oxycodone

azaperone

hydrochlorothyazide

clopidrogel

codeine

furosemide

propanolol

valsartan

carbamazepine

losartan

diazepam

tamsulosin

Elimination efficiency, %

>80%

(good removal)

% removal _2nd campaign

2nd Campaing

RESULTS

Important - Dilution factor

the natural water bodies receiving effluents discharge

has the same chemical profile as wastewater effluents, detected in µg/L range.

Wastewater effluents are main source of pharmaceuticals in

river waters

0,00

2000,00

4000,00

6000,00

8000,00

10000,00

12000,00

14000,00

WWTP2 effluent Emissary Sant Carles WWTP2 effluent Emissary Sant Carles

1st Campaing 2nd Campaing

Co

nce

ntr

atio

n (n

g/L

)

0,00

2000,00

4000,00

6000,00

8000,00

10000,00

12000,00

14000,00

16000,00

18000,00

WWTP1 effluent DownstreamWWTP1

WWTP1 effluent DownstreamWWTP1

1st Campaing 2nd Campaing

Co

nce

ntr

atio

n (n

g/L

)

Calcium channel blocker

Antibiotics

Tranquilizer

Synthetic glucocorticoid

Anti helmintics

X-ray contrast agent

Anticoagulant

To treat asthma

Prostatic hyperplasia

WWTP2

WWTP1

Concentration of pharmaceuticals in WWTP effluents and in receiving natural water bodies

1,00

10,00

100,00

1000,00

10000,00

1 2 1 2 1 2 1 2 1 2 1 2

Canal A Canal B Canal C Canal D1 Canal D2 Canal D3

Co

nce

ntr

atio

n (

ng

/L)

Analgesics/ant-inflammatories Lipid regulators and cholesterol lowering statin drugs

Psychiatric drugs Histamine H1 and H2 receptor antagonist

β-Blocking agents Duretic

Antidiabetic Antihypertensives

Antiplstelet agent Prostatic hyperplasia

To treat asthma Anti helmintics

Synthetic glucocorticoid Tranquilizer

Antibiotics Calcium channel blocker

Concentration range 1 – 464.2 ng/L

Canals

Canals are used to assist in the growing of agricultural crops, thus water quality

requirements are essential. Water from this canals could be significant route of crop

contamination. dried

Sea water and lagoons

1,0

10,0

100,0

1000,0

10000,0

1 2 1 2 1 2 1 2 1 2 1 2

Bay Alfalcs A(beach)

Bay Alfalcs B(shore)

Bay Alfalcs C(inside)

Bay Fangar A(beach)

Bay Fangar B(shore)

Bay Fangar C(inside)

Co

nce

trat

ion

(n

g/L)

Antibiotics

Tranquilizer

Antihypertensives

Duretic

β-Blocking agents

Psychiatric drugs

Analgesics/ant-inflammatories

Concentration range 1-20 ng/L

ibuprofen acetaminophen salicylic acid phenazone

Atenolo Sotalol Hydrochlorothyazid Ibersartan varsartan

Iopromide tiabendazole

0,1

1,0

10,0

100,0

1000,0

1 2 1 2 1 2 1 2 1 2 1 2

Tancada A (shore) Tancada A (inside) Buda A (shore) Buda B (inside) Encanizada A (shore) Encanizada A (inside)

Co

nce

ntr

atio

n (

ng/

L) Salycilic acid

the most abundant compound with max conc. 252.8 ng/L

Sea water

Lagoons

Frequency of detection in water samples

•acetaminophen

•ibuprofen

•salicylic acid

• phenazone

•valsartan

• irbersartan

•thiabendazole

•iopromide

•atenolol

•clarithromycin

0 20 40 60 80 100

actaminophen

valsartan

thiabendazole

iopromide

venlafaxine

azaperone

ketoprofen

prophyphenazone

10,11-epoxyCBZ

sotalol

clarithromycin

naproxen

diclofenac

bezafibrate

atorvastatin

alprazolam

cimetidine

furosemide

glibenclamide

clopidrogel

levamisol

diltiazemsea water

lagoons

canals

Antihelmintics

Analgesics/anti-inflammatories

Antihypertensives

X-ray contrast agents β-blocking agents

Antibiotics

Sediment samples with influence of WWTPs discharges

0,000

5,000

10,000

15,000

20,000

25,000

Upstream of WWTP1 Downstream WWTP1

Co

nce

ntr

atio

n (

ng/

g)

0,000

2,000

4,000

6,000

8,000

10,000

12,000

14,000

16,000

18,000

20,000

Emissary SCr WWTP2 Canal C (WWTP2)

Co

nce

ntr

atio

n (

ng/

g)

Lipid regulators and cholesterol lowering statin drugs

Β-agonist A

nal

gesi

cs/a

nti

infl

amm

ato

rie

s β

-blo

cke

rs

Antidiabetic

Antibiotics

Sulfonamide antibiotics

Antidiabetic

β-b

lock

ers

An

alge

sics

/an

tiin

flam

mat

ori

es

Lipid regulators

Concentration range 0.09 -7.08 ng/g

Concentration range 0.01 -5.98 ng/g

WWTP1

WWTP2 The total concentration levels were up to 20 ng/g

Sediment samples

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

Bay AlfalcsA (beach)

Bay AlfalcsB (shore)

BayAlfalcs C

(open sea)

Bay FangarA (beach)

BayFangar B(shore)

Bay FangarC (open

sea)

Co

nce

ntr

atio

n (

ng/

g)

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

16,00

Canal A Canal B Canal C Canal D1 Canal D4

Co

nce

ntr

atio

n (

ng/

g)

Canals

0,00

1,00

2,00

3,00

4,00

5,00

6,00

7,00

8,00

9,00

Tancada A(shore)

Tancada A(inside)

Buda A(shore)

Buda B(inside)

EncanizadaA (shore)

EncanizadaA (inside)

Canal VellA (shore)

Co

nce

ntr

atio

n(n

g/g)

Analgesics/antiinflammatories

Phenayone type drugs

Lipid regulators and cholesterol lowering statin

Psychiatric drugs

Histamine H1 and H2 receptor antagonist

Macrolide antibiotics

Sulfonamid antibiotics

Other antibioptics

β-blockers

β-agonist

Diuretic

Antidiabetic

Sea

Lagoons

0.01-4.54 ng/g

0.11-2.81 ng/g

0.06-3.42 ng/g

ketoprofen

ranitidine

ketoprofen

Frequency of detection in sediment samples

0 20 40 60 80 100

diclofenac

bezafibrate

ranitidine

erythromycin

chloramphenicol

ketoprofen

ibuprofen

phenazone

pravastatin

metronidazole

furosemide

Indomethacine

actaminophen

atrovastatin

sotalol

naproxen

gemfibrozil

carbamazepine

cimetidine

sulfamethaxzine

clarithromycin

metoprololsea

lagoon

canal

•Histamine H2 receptor antagonists

ranitidine

diclofenac bezafibrate ibuprofen

erythromycin chloramphenicol

o Levels of PhACs detected in waste, river, canals, lagoons and sea water

indicate that they are widespread pollutants along the Ebro Delta.

o Wastewater treatment plants proven to be an important source of pollution

for water bodies, as well as for sediment.

o The compounds reaching the sea water coming from WWTP discharges

were from the several therapeutical groups: analgesics/anti-inflammatories

(such as acetaminophen, ibuprofen, salicylic acid, phenazone), β-blocking

agents (atenolol, sotalol, ibersartan, valsartan) and diuretics drugs

(iopromide, thiabendazole).

o The compounds most widely detected in sediments were: diclofenac,

bezafibrate, ranitidine, ibuprofen, erythromycin, chloramphenicol.

CONCLUSION

This work is supported by:

The Spanish Ministry of Economy and Competitiveness through the coordinated project TRANSFORM COAST (CGL- 2014-56530-C4-4-R) Generalitat de Catalunya (Consolidated Research Groups 2014 SGR 291–ICRA and 2014 SGR 418 Water and Soil Quality Unit). M.Celic acknowledges for grant for PhD received from the Spanish Ministry of Economy and Competitiveness (BES-2015-072297).

ACKNOWLEDGMENTS

Thank you!