Determinació simultània de nitrats i antibiòtics de sulfonamides en

dues masses d'aigua subterrània de Catalunya

RESUM

En aquest estudi es va investigar la presència de 19 sulfonamides seleccionades

(incloent-hi un metabòlit acetilat) en mostres d’aigües subterrànies procedents de dues

preses de Catalunya (Plana de Vic i la Selva). Ambdues són àrees designades com a

vulnerables als nitrats d’acord amb la Directiva 91/676/EEC.

El mètode d’anàlisi estava totalment automatitzat i consistia en l’extracció d’una fase

sòlida gràcies a la cromatografia líquida associada a espectrometria de masses en

tàndem, tècnica desenvolupada amb aquest propòsit.

L’elevada sensibilitat i especificitat assolides (límits de detecció al voltant de 0,005-0,8

ng/L) van permetre demostrar la ubiqüitat d’aquests antibiòtics en les aigües

subterrànies analitzades. Els resultats van mostrar un ample rang de concentracions, des

de 0,01 ng/l fins a 3460,57 ng/l . Des que les sulfamides s’utilitzen, es relacionen amb

la ramaderia i les pràctiques veterinàries i, per tant, són un indicador de la contaminació

dels fems. Tot i així, la presència de sulfonamides no sembla que estigui relacionada

amb la concentració de nitrats, ja que en els resultats obtinguts s’han trobat coeficients

de correlació baixos.

Journal of Hydrology 383 (2010) 93–101

Contents lists available at ScienceDirect

Journal of Hydrology

journal homepage: www.elsevier .com/ locate / jhydrol

Simultaneous occurrence of nitrates and sulfonamide antibiotics in twoground water bodies of Catalonia (Spain)

Ma Jesús García-Galán a, Teresa Garrido c, Josep Fraile c, Antoni Ginebreda a, M. Silvia Díaz-Cruz a,*,Damià Barceló a,b

a Dept. Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), CSIC, c/Jordi Girona 18-26, E-08034 Barcelona, Spainb Catalan Institute for Water Research (ICRA), Parc Científic i Tecnològic de la Universitat de Girona, c/Pic de Peguera 15, E-17003 Girona, Spainc Water Catalan Agency (ACA), Provença 204-208, E-08017 Barcelona, Spain

a r t i c l e i n f o

Keywords:Ground water bodyIntensive cattle farmingSulfonamidesOn-line solid-phase extractionLiquid chromatography–quadrupole linearIon trap-mass spectrometry

0022-1694/$ - see front matter � 2009 Elsevier B.V. Adoi:10.1016/j.jhydrol.2009.06.042

* Corresponding author. Tel.: +34 93 4006100; fax:E-mail address: sdcqam@cid.csic.es (M.S. Díaz-Cru

s u m m a r y

In the present work the occurrence of 19 selected sulfonamides, including one acetylated metabolite, wasinvestigated in ground water samples taken from two ground water bodies in Catalonia (Plana de Vic andLa Selva). Both include areas designated as nitrate vulnerable zones, according to Directive 91/676/EEC. Afully automated analytical methodology based on on-line solid-phase extraction–liquid chromatography-tandem mass spectrometry (on-line SPE–LC–MS/MS) was developed for this purpose. The high selectivityand sensitivity achieved (limits of detection between 0.005 and 0.8 ng/L) permitted to demonstrate theubiquity of these antibiotics in both ground water bodies. Results showed a wide range of concentrations,from 0.01 ng/L up to 3460.57 ng/L. Since sulfonamides are related to livestock veterinary practices, theycan be used as a specific indicator of manure contamination. However, the presence of sulfonamidesappeared not to be directly related to the concentration of nitrates, as it is reflected on the low correlationcoefficients found.

� 2009 Elsevier B.V. All rights reserved.

Introduction because once they are contaminated, the effects are often irrevers-

Ground water systems are dynamic and adjust continually toshort-term and long-term changes in climate, ground water with-drawal, and land use. In Catalonia, ground water represents the35% of the water resources, being of high importance in drinkingwater, industry and agriculture supply. Its use in these sectorsequals approximately 900 hm3 per year, only a part of the totalground water resources available; for instance, the average usefor drinkable water equals 200 hm3, corresponding to 30–35% ofthe total ground water supply, a high value considering only thissingle use. The kind of use depends on the location, being the areaswith higher demands (i.e. coastal and touristic areas) not necessar-ily placed in regions with enough ground water resources, and theother way round. It should also be considered that part of the flowin streams, lakes and wetlands is sustained by the discharge ofground water.

When talking about ground water bodies, we are referring to amore protected water matrix. The soil system above acts as ‘pro-tective shield’, providing inertia to quality changes and a slowedpropagation of the contaminants. But for this same reason, how-ever, ground water bodies are especially vulnerable to pollution

ll rights reserved.

+34 93 2045904.z).

ible, or, at least, of difficult and expensive remediation. Pointsources of contamination such as sewage treatment plants dis-charge, industrial facilities and storm water drains contribute witha wide variety of contaminants to rivers and streams which even-tually may also affect ground water quality, especially where riversare the main source of ground water recharge, where ground waterwithdrawals induce seepage from streams and where floods causestream water to become bank storage (www.groundwa-ter.water.ca.gov/other_references/index.cfm).

Application of inorganic fertilizers and manure to cropland canalso result in significant sources of contaminants to ground waterresources. Agriculture is the main cause of diffuse pollution, whichis quite difficult to deal with and prevent, as pesticides and fertil-izers are spread on very extense areas. Intensive cattle farming isanother relevant source of not-point pollution, since a wide varietyof veterinary pharmaceuticals and specially antibiotics, are used inthe prevention and treatment of microbial infections and also asgrowth promoters (Díaz-Cruz and Barceló, 2008). Following treat-ment, livestock will excrete 50–90% of the administered dose, theparent drug making up for 9–30%. These amounts of the un-changed substance vary depending on the form of the drug andthe animal age and species (Parfitt, 1999). Manure is regarded asa very valuable fertilizer, as it contains essential nutrients for plantgrowth such as nitrogen, phosphorous, organic carbon, potassiumetc. The extensive use of manure from medicated animals in crop

94 Ma Jesús García-Galán et al. / Journal of Hydrology 383 (2010) 93–101

fields is among the major routes by which veterinary antibioticsenter the environment (Boxall et al., 2003) and, eventually, theground water systems. Once on the topsoil, the excreted residuesmay percolate and contaminate the aquifers directly or reach sur-face waters during run off and contaminate the ground waterbodies indirectly.

Sulfonamides are one of the most widely used antibiotics in hu-man and especially in veterinary medicine. They have been de-tected in all kind of water matrices (Díaz-Cruz et al., 2008), notonly because of their high consumption rates, but also due to theiramphoteric properties, rather poor chelating ability and low sorp-tion to soils tendency (i.e. partition coefficient values (Kd) of 0.22for sulfamethoxazole, 2.5 for sulfadiazine, 1.6 for sulfapyridineand 1 for sulfamethazine). They are usually classified as short-acting sulfonamides, with half lives (in serum) of 5–10 hours (i.e.sulfisoxazole, sulfadiazine and sulfamethizole); intermediate-act-ing sulfonamides, with half-lives of 10–12 h (i.e. sulfamethoxazole,sulfadimethoxine and sulfamethazine); and finally long-acting sul-fonamides, with half lives from 40 h onwards (i.e. sulfadoxine andsulfamethoxypyridazine) (Scholar and Pratt, 2000). Their relativelylow elimination efficiency during sewage treatment proceduresand the increase in the number of confined animal-feeding opera-tions, which often lack proper waste management practices, areother reasons for greater occurrence of these substances (Carballaet al., 2004). Residues of sulfonamides have been detected inmanure (Kotzerke et al., 2008; Schmitt et al., 2005) and, as theirsorption to soil is weak, they could leach to ground waters. Thispossibility has already been proved in several publications, show-ing the presence of sulfonamides at different concentrations inground water from various sites close to animal farming facilities(Batt et al., 2006; Blackwell et al., 2004; Diaz-Cruz and Barcelo,2008; Karthikeyan and Meyer, 2006; Lindsey et al., 2001; Sacheret al., 2001). A recent study showed sulfamethazine and sulfadime-thoxine in ground water from wells located down gradient and inclose proximity to a confined animal-feeding operation (CAFO)(Batt et al., 2006).

Fig. 1. Location of La Plana de Vic an

A special protection figure is therefore needed for these vulner-able water systems. The so called Ground Water Directive (Direc-tive 2006/118/EC) offspring of the Water Frame Directive (WFD,2000/60/CE) establishes how to manage and protect the differentground water resources from pollution. Under the provisions ofsuch directives, 53 different ground water bodies have been typi-fied in Catalonia in order to evaluate together anthropogenic andnon-anthropogenic pressures on the different water systems andenvironmental impacts, of which 29 are considered under risk ofnon compliance of good status, as it is precluded by the WFD. Sofar, there is no legislation regarding the presence of sulfonamidesor any other antibiotic in any of the environmental compartments.On the other hand, nitrates from animal farming have been thor-oughly studied and regulated in Directive 91/676/EEC and six ni-trate vulnerable zones have been established in Cataloniafollowing this Directive. Nitrogen in its different species (organic,ammonium, nitrite and nitrate) is a major constituent of manure,and, similarly to sulfonamides, is very soluble in water. Increasedconcentrations of nitrate that result from both nitrification ofammonium or direct introduction from mineral fertilizers are com-monly present in both ground water and surface water associatedwith ammended agricultural lands.

The aim of this work is to assess the occurrence of 19 selectedsulfonamides, including one acetylated metabolite, in groundwater samples taken from surveillance and operational monitoringnetworks located in two ground water bodies (Plana de Vic and LaSelva); both of them include areas designated as nitrate vulnerablezones under the provisions of Directive 91/676/CEE.

2. Experimental section

2.1. Chemicals and reagents

HPLC-grade solvents (water, methanol, acetone andacetonitrile) and formic acid (98–100%) were supplied by Merck(Darmstadt, Germany).

0 40 km

N

Groundwater Bodies of CatalanInternal Basins

Plana de VicLa Selva

d La Selva ground water bodies.

Ma Jesús García-Galán et al. / Journal of Hydrology 383 (2010) 93–101 95

High purity standards (>99%) of the 19 selected sulfonamideswere purchased from Sigma (St. Louis, MO, USA). Stock standardsolutions for each of the analytes were prepared in methanol(MeOH) at 1 mg/mL and stored in the dark at �2 �C. Standard solu-tions of the mixtures of all compounds at concentrations rangingbetween 1 ng/mL and 500 lg/mL were prepared by appropriatedilution of the stock solutions in MeOH. The standard mixtureswere used as spiking solutions for preparation of the aqueous cal-ibration standards and in the recovery studies. Aqueous standardsolutions contained <0.1% of MeOH.

Internal standard d4-sulfathiazole (99.9%) was purchased fromToronto Research Chemicals (Ontario, Canada). Stock solutionswere also prepared in methanol and stored at �2 �C until use.

2.2. Sample collection and preparation

Ground water samples were taken in spring 2008 in two groundwater bodies in Catalonia: Plana de Vic and La Selva (see Fig. 1).Water was sampled from 39 sampling sites, including monitoringwells and natural springs. In Plana de Vic, which comprises740 km2, 26 samples were taken from different wells, a naturalspring and a mine. Ground water was sampled from depths rang-ing from 3 to 200 m. In La Selva, with an extension of 291 km2,13 wells were studied. Similarly, ground water was sampled fromdepths going from 10 to 206 m. Land and ground water use in theareas where these two ground water bodies are located are shownin Fig. 2.

Water samples were collected in amber polyethylene tere-phthalate (PET) bottles and transported to the laboratory undercooled conditions (4 �C). Once there, samples were filtered through0.45 lm Nylon filters (Whatman, Maidstone, UK) to eliminate sus-pended solid matter and then stored at 4 �C in the dark until anal-ysis which was always carried out within 48 h of collection toavoid microbial degradation.

2.3. On-line SPE–LC–MS/MS analysis

The fully automated SPE–LC–MS/MS analytical method used todetermine the sulfonamide occurrence in the ground water sam-ples was recently developed by the authors (García-Galán et al.,

HERBACEOUS IRRIGATION NON-

DEPENDANT CROPS23%

EXTENSIVE IRRIGATION DEPENDANT CROPS

44%

URBAN AND INDUSTRIAL AREAS

16%

FOREST MASS5%

FOREST MASS73%

EXTENSIVE IRRIGATIONDEPENDANT CROPS

1%

URBAN AREAS5%

HERBACEOUS IRRIGATION NON-

DEPENDANT CROPS21%

Soil use

DEPENDANT CROPS

A

B

Fig. 2. Soil and ground water use in the areas corresponding to the two

2009) to analyse sulphonamide residues in several aqueous matri-ces. Briefly, the on-line preconcentration of samples, aqueousstandards and operational blanks was performed using an auto-mated on-line SPE sample processor Prospekt 2TM (Spark Holland,Emmen, The Netherlands). 40 mL of ground water samples wereextracted using Oasis HLB cartridges (divinylbenzene and N-vinyl-pyrrolidone polymer, 30-lm particle size) from Waters (Barcelona,Spain).

This system consists of an automated cartridge exchange (ACE)module, which holds two trays of 96 extraction cartridges each,and a high pressure dispenser module (HPD) for handling of sol-vents by a 2-mL high pressure syringe. SPE solvents for condition-ing, equilibration, sample application and clean up are provided bythe HPD. The ACE module has two clamps and two high pressurevalves. An aliquot of the raw sample is introduced by the autosam-pler and, when the SPE is completed, the cartridge is transferred tothe elution clamp and the analytes are eluted from the SPEcartridge directly onto the LC column by the HPLC. DuringLC–MS/MS analysis, the extraction of the next sample is carriedout on a new cartridge in the other clamp. Therefore, SPE runsentirely in parallel with the LC–MS/MS run. This configurationshortens the cycle times (in our case 23 min of sample analysisplus the conditioning and equilibration times only for the firstsample). The Prospekt-2 TM is controlled by means of Sparklinkversion 3.0 (Spark Holland).

LC–MS/MS analyses were carried out in a system consisting ofan HP 1100 chromatograph (Agilent Technologies, Palo Alto, CA,USA) coupled to a 4000 QTRAP mass spectrometer (Applied Biosys-tems, Foster City, CA, USA) equipped with a turbospray electro-spray (ESI) interface. The chromatographic separation wasperformed using an Atlantis C18 (Waters, 150 mm � 2.1 mm,3 lm of particle size) LC-column preceded by a guard column withthe same packing material. Sulfonamides were analyzed in the po-sitive ionization mode (PI). The flow rate was set to 0.2 mL/min,being eluent A HPLC grade water slightly acidified with 0.1% of for-mic acid, and eluent B acetonitrile with 0.1% formic acid. The elu-tion gradient started with 25% of eluent B, increasing to 80% in10 min and 100% in 11 min. During the further 2 min, the columnwas cleaned and readjusted to the initial conditions in 3 min, andequilibrated for 7 min.

DRINKING WATER74%

INDUSTRIAL USE19%

AGRICULTURE7%

AGRICULTURE68%

DRINKING WATER20%

INDUSTRIAL USE12%

Ground water use

ground water bodies studied: (A) La Plana de Vic and (B) La Selva.

96 Ma Jesús García-Galán et al. / Journal of Hydrology 383 (2010) 93–101

The MS/MS experimental conditions were as follows: capillaryvoltage 3.5 kV; source temperature, 700 �C; desolvation tempera-ture, 450 �C; extractor voltage 3 V; and RF lens 0.2 V. Nitrogenwas used as both the nebulizing and the desolvation gas at630 L h�1. For operation in the MS/MS mode, argon was used ascollision gas with a pressure of 2.6 � 10�3 mbar. Instrument con-trol and data acquisition and evaluation were performed withthe Analyst 1.4.2 software package purchased from AppliedBiosystems.

2.3.1. Quantification quality assuranceFor increased sensitivity and selectivity, MS data acquisition

was performed in the selected reaction monitoring (SRM) mode.For each analyte, two transitions between precursor ions and thetwo most abundant product ions were monitored, the most abun-dant transition was used for quantitation and the other one forconfirmation. Table 1 shows the optimized LC–MS/MS conditionsused for the target analytes. A strict criteria has to be followed inorder to ensure positive confirmations of the target analytes inthe samples and avoid false positives. The European Commission

Table 1Optimized time-scheduled SRM transitions [M+H+]. Compound dependent parameters:potential (eV). Retention time is given in minutes.

Compounds [M+H]+ Transitions

Sulfacetamide 215 215/156215/92

Sulfisomidin 279 279/124279/186

Succinyl-sulfathiazole 356 356/256356/192

Sulfathiazole 256 256/156256/92

d4-sulfathiazole 260 260/160260/96

Sulfaguanidina 215 215/156215/92

Sulfadiazine 251 251/156251/108

N4-acetylsulfamethazine 321 321/134321/124

Sulfapyridine 250 250/156250/92

Sulfamerazine 265 265/92265/156

Sulfamethazine 279 279/156279/124

Sulfamethizole 271 271/156271/108

Sulfamethoxypyridazine 281 281/156281/126

Sulfadoxine 311 311/156311/92

Sulfamethoxazole 254 254/156254/108

Sulfisoxazole 268 268/156268/113

Sulfaquinoxaline 301 301/156301/92

Sulfabenzamide 277 277/156277/92

Sulfadimethoxine 311 311/156311/92

Sulfanitran 336 336/156336/198

Decision 2002/657/EC establishes that a minimum of three identi-fication points (IPs) are required for this purpose. Two differentSRM transitions, together with the precursor ion, yield four identi-fication points. Other specific criteria applied in LC–MS/MS is thatthe chromatographic retention time of the analyte in the sampleshould not vary more than 2% in comparison to the calibrationstandards and the relative abundance of the two SRM transitionsmonitored must also be compared to the standards correspondingvalues [22].

The performance of the method applied is shown in Table 2.

2.4. Method validation

The analytical method developed was evaluated in terms of lin-earity, accuracy, selectivity and sensitivity as presented in Table 2.

Quantification was performed based on peak areas and using aninternal standard calibration method, crucial to correct potentialmatrix effects. Concentrations were estimated for the most abun-dant SRM transition selected. d4-sulfathiazole was added to allthe samples at a concentration of 500 ng/L right before analysis.

CE, collision energy (eV); DP, declustering potential (V) and CXP, collision cell exit

Retention time DP CE CXP

3.2 46 21 1046 35 6

3.3 76 33 876 23 14

4.2 71 25 1671 33 16

4.3 40 25 1440 25 10

4.3 71 25 671 25 6

4.3 56 13 1056 31 4

4.5 46 27 1046 30 8

4.6 86 35 486 35 4

4.7 51 28 1251 31 6

5.4 61 47 661 27 8

6.0 26 30 1026 35 10

6.3 36 23 1236 23 8

6.3 66 27 1466 27 12

10.4 46 29 1246 45 4

11.4 56 25 1056 27 10

12.0 71 21 1071 21 8

13.0 76 25 1076 47 12

13.0 56 17 1056 41 6

13.0 76 31 876 31 6

14.7 66 17 1266 29 14

Table 2Performance of the on-line SPE–LC-QqLIT–MS method applied. r2, correlationcoefficient; LOD, method limit of detection; LOQ, method limit of quantificationand RSD, relative standard deviation (%).

Sulfonamide r2 LOQ (ng/L) LOD (ng/L) RSD

Sulfisomidin >0.999 0.012 0.042 3.01Sulfanitran 0.981 0.058 0.195 3.32Sulfaguanidine 0.994 0.796 2.653 5.24Sulfamerazine 0.998 0.086 0.286 2.18Sulfaquinoxaline 0.998 0.016 0.055 1.60Sulfadoxine 0.999 0.019 0.064 1.50Sulfacetamide 0.986 8.876 29.586 6.51Succinyl-sulfathiazole 0.999 0.065 0.218 2.29Sulfabenzamide 0.983 0.019 0.062 8.19Sulfadiazine 0.999 0.021 0.069 3.78Sulfadimethoxine 0.999 0.039 0.131 3.26Sulfamethazine 0.998 0.034 0.113 1.83Sulfamethizole 0.998 0.366 1.221 6.61Sulfamethoxazole >0.999 0.050 0.167 1.41Sulfamethoxypyridazine 0.999 0.036 0.118 4.60Sulfapyridine 0.999 0.023 0.077 2.59Sulfathiazole >0.999 0.005 0.018 2.15Sulfisoxazole 0.997 0.042 0.140 6.02N4-acetylsulfamethazine 0.999 0.049 0.162 1.12

Ma Jesús García-Galán et al. / Journal of Hydrology 383 (2010) 93–101 97

Five to eight point matrix matched calibration curves weremade, using least-squares linear regression analysis at concentra-tions ranging from 0.05 to 1000 ng/L. Correlation coefficients (r2)were equal or higher than 0.999 for the majority of the sulfona-mides. The best limit of detection (LOD) value for the methodwas achieved for sulfathiazole (0.005 ng/L), whereas sulfacetamideand sulfaguanidine were the compounds investigated with thelowest sensitivity.

The intraday precision of the method was evaluated by analyz-ing five consecutive times water spiked with a standard mixture ofthe analytes at 100 ng/L and estimating the relative standard devi-ation (RSD). Values ranged from 1.1% to 8.1% for all thesulfonamides.

3. Results and discussion

3.1. Nitrates

As aforementioned, a total of 38 wells and a natural spring weresampled in two different ground water bodies in Catalonia. Bothare at risk of not reaching WFD environmental objectives in 2015due to nitrate pollution.

Box plots representing the nitrate concentrations measured indifferent aquifers from La Plana de Vic and La Selva ground waterbodies in the last few years are shown in Fig. 3. The boxes havelines at the lower quartile, median and upper quartile values. Thedistribution of the nitrate concentrations hardly shows significantdifferences between seasons or years, with values slightly skewedboth to high and low values in the different sampling dates.

In the case of La Plana de Vic ground water body, high concen-trations of nitrate have been documented during the period1996–2007, with average nitrate concentrations ranging between39.5 and 99.7 mg/L. The highest amounts were detected in 2004and 2006 (Fig. 3A). It should be taken into account that Directive91/676/EEC establishes that a ground water is considered pollutedwhen concentrations of 50 mg/L or higher of nitrates are detectedand that, in this area, a 74% of the ground water extractions is usedfor drinking water uses with the consequent health risks (i.e.gastric problems due to the formation of nitrosamines, blue babysyndrome, etc. Human health risk is out of the scope of this studyand will not be dealt with in this study) as it can be observed inFig. 2, only a 22% of the total soil area is dedicated to agriculture(both irrigated and non-irrigated), being most of the area covered

by forest. On the other hand, this region is one of the most impor-tant of Catalonia regarding livestock, being the 15% of the total pigfarming activity and the 13% of bovine farming located here. There-fore, the pressure derived from cattle droppings is very high, withan estimation of 13.170 tons N/year generated.

Nitrate levels are much lower in La Selva, with peak values nohigher than 100 mg/L, and concentrations between 12.1 and78.5 mg/L have been registered from 1998 to 2007. The only dataavailable for Fig. 3B dated from 2007 onwards. The nitrogen quan-tity from cattle droppings is more moderated than that in Plana deVic, and the pressure on the aquifers is distributed homogeneouslyover the whole area. Agriculture activity, higher than in La Plana deVic (see Fig. 2) may also exert a moderated pressure on the chem-ical quality of the ground waters.

3.2. Sulfonamide concentrations

Table 3 shows the frequencies of detection of each sulfonamidestudied in both ground water bodies, considering the whole dataset and the two sampling sites individually (succinyl-sulfathiazolehas been excluded in this table, as it could not be detected in anysampling site). In the first case (Plana de Vic), frequencies rangefrom 8.16% (sulfamethizole) to 89.74% (sulfadimethoxine and sul-famethazine). In this ground water body, sulfamethizole and sulfa-guanidine are the compounds with the lowest frequency ofdetection (12.12%) and sulfadimethoxine and sulfamethazine arethe two sulfonamides with highest frequencies (69.7%). In La Selva,sulfamethizole was not detected in any of the sampling sites, sul-fisoxazole was detected only in 6% of the cases, and again sulfadi-methoxine and sulfamethazine were the compounds detectedmore frequently (75%). As it can be observed, the frequency valuesfor these two sulfonamides are slightly higher in the area of LaSelva, but this trend does not follow for all the analytes. However,the highest concentrations do not correspond to these two sulfon-amides, but to sulfacetamide (3460.57 ng/L) and sulfamerazine(744.73 ng/L). Whereas sulfacetamide possessed also high frequen-cies of detection (79.49% for all the sampling sites), sulfamerazinehas been detected only in 20.15% of the samples. It should bepointed out that for 14 of the 19 sulfonamides studied; theircorrespondent highest concentration values were detected in LaSelva ground water body.

For the whole data set, the comparison between the mean andthe median values of the concentrations showed that the medianwas lower than the average for all the sulfonamides, indicatingthat concentrations are skewed to low values. Standard deviationvalues (SD) had the same order of magnitude or bigger than themean values for all the sulfonamides, pointing out the big differ-ences of concentration in the different sampling sites.

Box plots shown in Fig. 4A and B have been used to summarizethe results obtained in the sampling points from both groundwater bodies, and show the distribution of the concentration val-ues for each of the sulfonamides in the different sampling points.As mentioned, succinyl-sulfathiazole has been excluded in bothgraphs, as it could not be detected in any sampling site. InFig. 4A, corresponding to La Plana de Vic, the distribution of thevalues was similar for all the sulfonamides except for sulfamera-zine (S4), which showed a higher dispersion of the values (as couldbe expected from its SD value, see Table 2). For all the other com-pounds, concentrations were skewed to very low values, near thelimit of detection. There were also several values out of the boxplot range, all of them above the box plot, highlighting again thebig dispersion of the data. A similar picture could be seen for thebox plot corresponding to La Selva ground water body. Again, con-centration values were generally low and the most disperse con-centration values corresponded to sulfamerazine, and in this casealso to the acetylated metabolite.

23/03/2004 12/04/2005 03/11/2005 19/04/2006 21/02/2007 29/08/2007 18/03/2008 23/10/20080

50

100

150

200

250

300

350

400

Nitr

ates

(mg/

L)

10/07/2007 01/04/2008 01/10/20080

20

40

60

80

100

Nitr

ates

(mg/

L)

Dates

Dates

A

B

Fig. 3. Box plots showing the distribution through time of the nitrate concentrations in: (A) La Plana de Vic (39 samples) and (B) La Selva ground water bodies (13 samples).

Table 3Descriptive univariate statistics of the data. SD, standard deviation and �, municipalities and ground water bodies correspond to the location of the maximum concentration valuedetected for each sulfonamide.

Compound Frequency ofdetection (%)(total)

Frequency ofdetection (%)(Plana de Vic)

Frequency ofdetection (%)(La Selva)

Median(ng/L)

Mean(ng/L)

SD Maximumvalue(ng/L)

Minimumvalue(ng/L)

Municipality* Groundwaterbody

Sulfisomidin 66.67 51.52 56.25 2.80 11.62 15.18 64.40 0.01 Ruidellots de la Selva La SelvaSulfanitran 48.72 42.42 31.25 12.36 44.76 127.7 568.8 0.04 Campllong La SelvaSulfaguanidine 20.51 12.12 25.00 10.61 22.59 29.85 91.78 3.30 Campllong La SelvaSulfamerazine 79.49 66.67 56.25 14.83 49.02 132.9 744.7 0.11 Campllong La SelvaSulfaquinoxaline 74.36 63.64 50.00 1.44 7.13 20.69 112.1 0.01 Campllong La SelvaSulfadoxine 84.62 66.67 68.75 2.33 6.21 12.30 53.63 0.02 Campllong La SelvaSulfacetamide 20.51 15.15 18.75 80.45 631.8 1205 3461 1.77 Campllong La SelvaSulfabenzamide 35.90 33.33 18.75 1.57 1.94 2.56 10.32 0.09 Balenyà La Plana de VicSulfadiazine 23.08 21.21 12.50 0.38 1.25 2.21 6.98 0.14 Malla La Plana de VicSulfadimethoxine 89.74 69.70 75.00 4.18 11.39 19.95 91.48 0.01 Campllong La SelvaSulfamethazine 89.74 69.70 75.00 3.68 8.20 18.74 106.8 0.03 Campllong La SelvaSulfamethizole 8.16 12.12 0.00 0.81 2.78 4.35 9.29 0.22 Les Masies de Roda La SelvaSulfamethoxazole 58.97 51.52 37.50 4.57 23.40 66.40 312.2 0.08 Cassa de la Selva La SelvaSulfamethoxypyridazine 35.90 21.21 43.75 1.00 8.96 18.65 68.70 0.02 Campllong La SelvaSulfapyridine 56.41 48.48 37.50 2.26 6.31 15.33 72.45 0.07 Campllong La SelvaSulfathiazole 56.41 45.45 43.75 2.62 4.50 5.25 16.78 0.01 Balenyà La Plana de VicSulfisoxazole 20.51 21.21 6.25 0.51 1.55 1.75 4.43 0.21 Vic La Plana de VicN4-acetylsulfamethazine 82.05 63.64 68.75 1.59 9.88 16.38 56.95 0.02 Campllong Selva

98 Ma Jesús García-Galán et al. / Journal of Hydrology 383 (2010) 93–101

A

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10S11S12S13S14S15S16S17S18

0

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NC

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/L)

1137 ng/L

B

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10S11S12S13S14S15S16S17S18

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40

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80

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Fig. 4. Box plots for the distribution of the concentrations of the corresponding sulfonamides in the sampling sites of Plana de Vic (A) and La Selva (B). In A, each variable has26 measured values. In B, each variable has 13 measured values. S1, sulfisomidin; S2, sulfanitran c; S3, sulfaguanidine; S4, sulfamerazine; S5, sulfaquinoxaline; S6,sulfadoxine; S7, sulfacetamide; S8, sulfabenzamide; S9, sulfadiazine; S10, sulfadimethoxine; S11, sulfamethazine; S12, sulfamethizole; S13, sulfamethoxazole; S14,sulfamethoxypyridazine; S15, sulfapyridine; S16, sulfathiazole; S17, sulfisoxazole and S18, N4-acetylsulfamethazine.

Ma Jesús García-Galán et al. / Journal of Hydrology 383 (2010) 93–101 99

Sulfonamides commonly used in human medicine, such as sul-fadiazine and mainly sulfamethoxazole, have also been detected inboth ground water bodies and the latter at relatively high fre-quency and concentrations (peak concentration of 312 ng/L and to-tal frequency of detection of 59% for sulfamethoxazole, see Fig. 4and Table 3, respectively). As the origin of these sulfonamides res-idues is not manure application on agricultural practices, alterna-tive pathways for entering this environmental compartmentshould be considered. Waste water treatment plants (WWTPs) dis-

charges into surface waters should be taken into account, as the re-moval efficiency for sulfonamides in these facilities has proved tobe inefficient (Carballa et al., 2004; Miao et al., 2004). There are13 WWTPs in Plana de Vic and nine in La Selva. Despite the factthat sulfonamides are highly soluble substances and possess lowsoil–water partition coefficient (Kd), biosolid applications on agri-cultural soils could also represent the main entrance to the groundwater matrix. In La Selva, biosolids from the nine WWTPs are ap-plied in soils from the different municipalities. High concentrations

Table 4Physico-chemical parameters for each of the ground water bodies studied. TOC, totalorganic carbon.

La La Plana de Vic La Selva

Depth of the sampling sites (m) 3–200 10–206Nitrates (mg/L) 0.60–264 5–60.8Conductivity (lS/cm) 303–1906 290–711TOC (mg/L) 0.6–8.5 1.3–5.2Maximum sulfonamide concentration (ng/L) 3322 1137

100 Ma Jesús García-Galán et al. / Journal of Hydrology 383 (2010) 93–101

detected in the municipality of Casà de La Selva, for instance, couldbe attributed to this practice. However, biosolids are seldom ap-plied in La Plana de Vic, where both sulfadiazine and sulfamethox-azole have also been detected, but at lower concentrations.

3.3. Relationship between the presence of sulfonamides and nitrates

A brief summary of the main physico-chemical parametersthat characterize both ground water bodies studied is shown inTable 4. As nitrates and sulfonamides detected in ground watersmay share a common origin (extensive and intensive cattle farm-ing and manure application in crop lands), the possibility of estab-lishing a correlation between both parameters was worth toconsider. The depth of the sampling site was also considered toplay an important role in the concentration distribution of bothnitrates and sulfonamide concentrations. With this aim, pair wisecorrelations between nitrates concentration, depth of the well andsulfonamides concentration were estimated and are given in Table5. Despite its high solubility and potential to percolate throughsoil, there is a lack of correlation between nitrates and depth. Sim-ilarly, there was no apparent relationship between any sulfon-amide concentration and this parameter, as it is reflected ontheir low correlation coefficients, being sulfisomidin and sulfadox-ine the two compounds showing the highest ones (0.39 and 0.38,respectively). Regarding nitrates and sulfonamides, a clear corre-lation between the occurrence of both could not be establishedfrom the data obtained. Sulfadiazine and sulfamethizole are thetwo sulfonamides with the highest correlation coefficients regard-ing nitrates concentration (0.37 and 0.33, respectively). Howeverboth sulfadiazine and sulfamethizole were the compounds withthe lowest frequencies of detection in both ground water bodies

Table 5Relationship between depth, nitrate and sulphonamide concentrations, expressed as pairwsulfonamides (see legend of Fig. 4). Higher pairwise correlations are marked in bold.

Depth Nitrates S1 S2 S3 S4 S5 S6 S7

Depth 1Nitrates �0.19 1.00S1 0.39 �0.01 1.00S2 0.08 �0.08 0.27 1.00S3 0.20 �0.20 0.29 0.02 1.00S4 0.08 0.01 0.32 0.97 �0.06 1.00S5 0.08 �0.0001 0.30 0.98 �0.06 0.99 1.00S6 0.38 �0.01 0.78 0.69 0.09 0.73 0.73 1.00S7 0.20 �0.15 0.27 0.03 0.97 �0.05 �0.05 0.09 1.0S8 �0.09 0.11 0.14 �0.02 0.06 0.08 0.09 0.16 0.0S9 �0.10 0.37 �0.03 �0.03 �0.06 �0.02 �0.03 �0.05 �0.0S10 0.12 0.24 0.34 0.72 �0.02 0.83 0.83 0.68 �0.0S11 0.11 0.14 0.36 0.91 0.00 0.97 0.96 0.74 0.0S12 0.24 0.33 0.18 �0.03 �0.04 0.13 0.09 0.18 �0.0S13 0.00 �0.11 0.08 0.05 0.25 0.04 0.03 0.04 0.2S14 0.09 0.03 0.35 0.91 �0.04 0.96 0.97 0.75 �0.0S15 0.09 0.01 0.33 0.96 �0.02 0.99 0.99 0.75 �0.0S16 0.20 0.17 0.49 0.46 0.37 0.58 0.56 0.59 0.3S17 0.04 0.27 0.22 �0.04 �0.02 0.07 0.08 0.18 0.0S18 0.19 0.01 0.47 0.54 0.55 0.59 0.57 0.56 0.5

(not detected in any site of La Selva, and only in 12% of the sam-pling sites in La Plana de Vic, see Table 3). Therefore, the relation-ship between both nitrates and these antibiotics should beinvestigated in further detail. Regarding conductivity and total or-ganic carbon (TOC) measured in the sampling sites, no clear rela-tion between those parameters and the concentrations of thesulfonamides detected could be settled, and these data was even-tually not included in Table 5. As an example, TOC seemed to beinversely correlated with the presence of three sulfonamides, sul-fisoxazole, sulfacetamide and sulfabenzamide, although the corre-sponding correlation coefficients were low (�0.40, �0.42 and�0.42, respectively).

Sulfonamides are usually applied in mixtures and not as singleantibiotics. Consequently, correlation amongst them turned out tobe quite high for some of the combinations. For instance, sulfadi-methoxine was detected together with sulfamethoxypyridazine,sulfapyridine and sulfathiazole very frequently, with correlationcoefficients of 0.90, 0.86 and 0.80, respectively. Something similarhappened with the presence of sulfamethazine and the same threesulfonamides. It should be highlighted that N4-acetylsulfameth-azine was detected simultaneously with 12 of the 19 sulfonamidesstudied; the correlation coefficients estimated for the simulta-neous presence of the metabolite and each of the 12 sulfonamideswere higher than 50%.

Conclusions

Contaminants such as sulfonamides and nitrates, highly solublein water, may reach the water table and be transported by theslowly moving ground water, widening its presence through veryextensive ground water systems. 18 out of the 19 target sulfona-mides have been detected in two ground water bodies fromCatalonia, being sulfadimethoxine and sulfamethazine, commonlyused in veterinary practices, those occurring more frequently. Itshould be highlighted, however, the high frequency of detectionfor the acetylated metabolite N4-acetylsulfamethazine, compara-ble to the highest frequencies aforementioned. The need for theinclusion of this and other metabolism products in future monitor-ing studies is unquestionable.

Despite the peak concentration values detected in differentsampling sites, the average detected concentrations of sulfona-

ise correlation coefficients. The variables are the concentrations of the corresponding

S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19

05 1.004 �0.02 1.001 0.42 0.04 1.001 0.17 �0.01 0.91 1.003 �0.04 0.03 0.38 0.24 1.007 0.04 �0.03 0.04 0.08 0.03 1.004 0.26 �0.03 0.90 0.97 0.19 0.04 1.001 0.16 �0.03 0.86 0.98 0.15 0.04 0.99 1.004 0.48 �0.07 0.80 0.71 0.45 0.12 0.68 0.63 1.003 0.64 �0.01 0.36 0.19 0.34 0.09 0.23 0.14 0.47 1.003 0.12 �0.05 0.65 0.66 0.27 0.25 0.60 0.61 0.79 0.17 1

Ma Jesús García-Galán et al. / Journal of Hydrology 383 (2010) 93–101 101

mides are generally below 50 ng/L. Sensitivity is therefore one ofthe most critical parameters in order to obtain unequivocal andreliable determination for he compounds investigated. Whenperforming on-line SPE analysis, its fully automation and the min-imum sample manipulation requirements permits the enhance-ment of sensitivity, as the whole sample volume (40 mL) gets tothe chromatographic system instead of a final reconstituted ex-tract as in off-line procedures, where usually volumes of 200 mLor bigger are reduced to approximately 0.5 mL and only around10 lL will be injected in the mass analyzer. Despite the low sam-ple volumes required in on-line procedures, it has been provedthat sensitivity is not affected but, on the contrary, improved con-siderably, with limits of detection down to the pg/L level. Besides,LC–MS/MS allows for an unequivocal identification of the targetsulfonamides.

From the results obtained, no strong correlation between sul-fonamides and nitrates concentrations could be established.Whereas nitrates in ground water are originated from fertilizersof both animal and mineral origin, sulfonamides could be specif-ically considered as potential indicators of pollution from animalorigin. For this reason, and because data on nitrates is histori-cally richer and more consistent, the presence of sulfonamidesin ground water matrices should be investigated in further detailin order to propose these substances as reference points to indi-cate pollution from animal farm and agriculture practices.

Acknowledgements

This work has been funded by the EU project NOMIRACLE (No.003956-2) and by the Spanish Ministry of Science and Innovationthrough the project CTM2006-26225-E) and reflects the author’sview. The EU is not liable for any use that may be made of theinformation contained in it. M.S.D.C. acknowledges her Ramón yCajal contract from the Spanish Ministry of Science and Innovation.We express our thanks to A. Navarro for her collaboration with thehandling of data, to C. Postigo for her collaboration with the Pros-pekt 2TM instrument and to Spark Holland and Waters for the giftof SPE cartridges.

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