Environmental Toxicology and Chemistry, Vol. 30, No. 6, pp. 1252–1260, 2011 # 2011 SETAC Printed in the USA DOI: 10.1002/etc.508
OCCURRENCE AND SOURCE APPORTIONMENT OF SULFONAMIDES AND THEIR METABOLITES IN LIAODONG BAY AND THE ADJACENT LIAO RIVER BASIN, NORTH CHINA
AI JIA,JIANYING HU,* XIAOQIN WU,HUI PENG,SHIMIN WU, and ZHAOMIN DONG Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing, China
(Submitted 8 August 2010; Returned for Revision 10 October 2010; Accepted 6 January 2011)
Abstract—The presence of antibiotics in the environment is of great concern because of their potential for resistance selection among pathogens. In the present study we investigated the occurrence of 19 sulfonamides, five N-acetylated sulfonamide metabolites, and trimethoprim in the Liao River basin and adjacent Liaodong Bay, China, as well as 10 human/agricultural source samples. Within the 35 river samples, 12 sulfonamides, four acetylated sulfonamides, and trimethoprim were detected, with the dominant being sulfamethox- azole (66.6 ng/L), N-acetylsulfamethoxazole (63.1 ng/L), trimethoprim (29.0 ng/L), sulfadiazine (14.0 ng/L), and sulfamonomethoxine (8.4 ng/L); within the 36 marine samples, 10 chemicals were detected, with the main contributions from sulfamethoxazole (25.2 ng/L) and N-acetylsulfamethoxazole (28.6 ng/L). Sulfamethoxazole (25.9%), N-acetylsulfamethoxazole (46.6%), trimethoprim (22.9%), and sulfapyridine (1.4%) were the main chemicals from human sources, while sulfamonomethoxine, sulfamethazine, sulfaquinoxaline, sulfaguanidine, sulfadiazine, sulfanilamide, and sulfamethoxypyridazine were dominant in the animal husbandry sources, specifically, swine and poultry farms, and sulfamethoxazole (91%) was dominant in the mariculture source. A principal component analysis with multiple linear regression was performed to evaluate the source apportionment of total sulfonamides in Liaodong Bay. It was found that animal husbandry contributed 15.2% of total sulfonamides, while human sources contributed 28.5%, and combined human and mariculture sources contributed 56.3%. In addition, the mariculture contribution was 24.1% of total sulfonamides into the sea based on mass flux estimation. The present study is the first report that the environmental levels of sulfonamide metabolites were comparable to the corresponding parents; therefore, we should pay attention to their environmental occurrence. Source apportionment showed human discharge (60.7%) significantly contributed to these antibiotics in Liaodong Bay, which provides important information for environ- mental management. Environ. Toxicol. Chem. 2011;30:1252–1260. # 2011 SETAC
Keywords—Sulfonamides Occurrence River water Seawater Source apportionment
INTRODUCTION treatment, while sulfadiazine, sulfadimethoxine, sulfametha- A number of studies have highlighted the environmental zine, sulfathiazole, and sulfachloropyridazine are frequently occurrence of antibiotics after their huge usage throughout the used in veterinary medicine [17]. After oral application, world, and scientists are increasingly concerned about the sulfonamides are excreted via urine and are ultimately released induction and spread of antibiotic resistance and their resistant into the aquatic environment through sewage treatment plant genes in response to an increased selective pressure [1,2]. effluent or agricultural runoff. The removal efficiency of these Sulfonamides are a group of synthetic antibiotics that function compounds during wastewater treatment has proven to be poor as competitive antagonists to p-aminobenzoate in the bacterial [18], and relatively high concentrations of sulfonamides have enzymatic synthesis of dihydrofolic acid [3] and are often been ubiquitously detected in surface water samples [19–24]. administered with trimethoprim to potentiate bactericidal While most of these studies often focused on a narrow range effects [4]. However, some studies had found that continuous of sulfonamides (mainly sulfamethoxazole, sulfamethazine, treatment with sulfonamides such as sulfamerazine and and occasionally sulfapyridine and sulfadiazine), 20 to 30 sulfadimethoxine could induce carcinomas [5–7]. In addition, sulfonamides are commercially available and prevalently sulfonamides resistance genes such as sul1, sul2, and sul3 used around the world [25], which is worth noting because have been found in soils and natural rivers [8,9]. Therefore, of their potential abuse. a gene exchange process known as horizontal transfer, proven In addition to the environmental occurrence of sulfonamides, in the laboratory [10–12], may also happen in the environment their metabolites should get more attention. The main metabolic [8], especially under continuous exposure of sulfonamides pathway for sulfonamides is supposed to be N-acetylation [26] [10,13]. as exemplified by sulfamethoxazole (SMX), of which only 15 to Sulfonamides and trimethoprim are the most frequently 25% is present in unchanged parent form and 43% is present as prescribed antibiotics used in human and veterinary medicine N-acetylsulfamethoxazole (NAcSMX) in the urine [27]. The [14] to treat a variety of bacteria, virus, and protozoan infections retransformation of N-acetylated metabolites into sulfonamides [3,15,16], or as animal growth promoters to improve feed can occur in the environment via microbial activities [1,28]. efficiency [17]. Of these sulfonamides, sulfamethoxazole, This phenomenon has been demonstrated by the transformation sulfapyridine, and sulfamerazine are primarily used in human of NAcSMX to SMX in a water-sediment coexisting test system [29] and N-acetylsulfamethazine to its active parent sulfamethazine during the storage of manure [30]. Thus, sulfo- All Supplemental Data may be found in the online version of this article. namides metabolites are likely to be an extra source of active * To whom correspondence may be addressed sulfonamides within the environment. To our best knowledge, ([email protected]). Published online 23 February 2011 in Wiley Online Library however, only a few studies have focused on the detection (wileyonlinelibrary.com). of NAcSMX in sewage treatment plants [18], while the
1252 Sulfonamides and their metabolites in river and sea samples Environ. Toxicol. Chem. 30, 2011 1253 metabolites of a broad range of sulfonamides in environmental MATERIALS AND METHODS matrices have not been reported. In the present study we developed a liquid chromatography- Chemicals and materials electrospray tandem mass spectrometry (LC-MS/MS) method Sulfaguanidine (SGD), sulfanilamide (SA), sulfathiazole for simultaneously analyzing 19 sulfonamides, five N-acety- (STZ), sulfisomidine (SIM), sulfamonomethoxine (SMM), sul- lated metabolites, and trimethoprim in sewage treatment fisoxazole (SIA), sulfachloropyridazine (SCP), sulfapyridine plant (STP) influent, effluent, and environmental river samples, (SPD), sulfadiazine (SDZ), sulfamethazine (SMA), sulfame- by improving the solid-phase extraction (SPE) cleanup method. thoxazole (SMX), sulfamerazine (SMR), sulfaquinoxaline The method was then applied to investigate the occurrence (SQX), sulfameter (SME), sulfamethizole (SMT), sulfamoxol of target analytes in the Liao River basin, the adjacent Liaodong (SMO), sulfadimethoxine (SDM), sulfamethoxypyridazine Bay, as well as human and agricultural influenced source (SMP), sulfanitran (SNT), and trimethoprim (TMP) were all 13 13 samples in northern China. The parent sulfonamides included obtained from Sigma-Aldrich. C6-sulfamethazine ( C6-SMA) in the present study were chosen because they have potential was obtained from Cambridge Isotope Laboratories. N-acetyl- for use in human medical treatment and animal husbandry in sulfapyridine (NAcSPD), N-acetylsulfadiazine (NAcSDZ), the Liaodong Bay region. Finally, source apportionment N-acetylsulfamethazine (NAcSMA), N-acetylsulfamethoxazole analyses were carried out using principal component (NAcSMX), N-acetylsulfamerazine (NAcSMR), and N-acetylsul- analyses with multiple linear regression based on the profiles famethoxazole-d5 (NAcSMX-d5) were purchased from Toronto of all target compounds to interpret the contribution Research Chemicals (Table 1). from human and agricultural sources to total sulfonamides in The high-performance liquid chromatography (HPLC)- Liaodong Bay. grade methanol, acetone, dichloromethane, ethyl acetate, and
Table 1. Chemical structures and electrospray tandem mass spectrometry (ESI-MS/MS) spectra features of target sulfonamides, their metabolites, and trimethoprim
a b Compound R1 R2 Mass spectra Compound R1 R2 Mass spectra
SGD H 173 (92,156) SQX H 301 (92,156)
SA H H 215 (92,156) SME H 281 (92,156)
STZ H 256 (92,156) SMT H 271(92,156)
SIM H 279 (92,124) SMO H 268 (92,156)
SMM H 281 (92,156) SDM H 311 (92,156)
SIA H 268 (92,156) SCP H 285 (92,156)
SMP H 281 (92,156) SNT 336 (64,156)
SDZ H 251 (92,156) NAcSDZ 293 (134,198)
SPD H 250 (92,156) NAcSPD 292 (134,198)
SMA H 279 (92,186) NAcSMA 281 (92,156)
SMX H 254 (92,156) NAcSMX 321 (134,186)
SMR H 265 (92,110) NAcSMR 307 (134,172)
SAs TMP 291 (110, 123)
a SAs ¼ sulfonamide antibiotics; SGD ¼ sulfaguanidine; SA ¼ sulfanilamide; STZ ¼ sulfathiazole; SIM ¼ sulfisomidine; SMM ¼ sulfamonomethoxine; SIA ¼ sulfisoxazole; SMP ¼ sulfamethoxypyridazine; SQX ¼ sulfaquinoxaline; SME ¼ sulfameter; SMT ¼ sulfamethizole; SMO ¼ sulfamoxol; SDM ¼ sulfadimethoxine; SCP ¼ sulfachloropyridazine; SNT ¼ sulfanitran; SDZ ¼ sulfadiazine; NAcSDZ ¼ N-acetylsulfadiazine; SPD ¼ sulfapyridine; NAcSPD ¼ N-acetylsulfapyridine; SMA ¼ sulfamethazine; NAcSMA ¼ N-acetylsulfamethazine; SMX ¼ sulfamethoxazole; NAcSMX ¼ N-acetylsulfa- methoxazole; SMR ¼ sulfamerazine; NAcSMR ¼ N-acetylsulfamerazine; TMP ¼ trimethoprim. b Multi-selected reaction monitoring transition in ESI-MS/MS: precursor ion (product ions). 1254 Environ. Toxicol. Chem. 30, 2011 A. Jia et al. hexane were purchased from Fisher Chemicals and HPLC grade All water samples were collected in amber glass bottles and formic acid was purchased from Dima Technology. Ethylenedi- filtered with a glass microfiber filter (GF/C, 1.2 mm) (Whatman) amine tetraacetic acid disodium (Na2EDTA) of analytical before being extracted by HLB cartridges on the same day of reagent grade was obtained from Sinopharm Chemical Reagent. collection. These samples were then extracted and prepared for Distilled water was purified by a Milli-Q Synthesis water determination of the target 25 analytes, including sulfonamides, purification system (Millipore). The SPE cartridges, including their metabolites, and trimethoprim. Detailed sample prepara- Oasis hydrophilic-lipophilic balance (HLB) (500 mg/6 ml) and tion and LC-MS/MS conditions are provided in the Supple- Sep-Pak Silica (500 mg/3 ml), were from Waters. Stock solu- mental Data. tions (1000 mg/L in methanol) for all standard substances were prepared and stored at 208C. Quantitation The LC-MS/MS identification was accomplished by com- Sample sites and collection paring the retention time (within 2%) and the signal ratio The Xiaoling River, Daling River, Shuangtaizi River, and (within 20%) of two selected product ions in the environmental Daliao River are the four main rivers flowing into Liaodong samples with standards. To compensate for both variations in 13 Bay, Bohai Sea. Thirty-five water samples from these four the SPE process and instrument response, C6-SMA and rivers and their tributaries and 36 seawater samples from NAcSMX-d5 were used as surrogate standards for parent sul- Liaodong Bay were collected in May 2009 from the subsurface fonamides and N-acetylated metabolites, respectively. Instru- zone (30–50 cm depth) (Fig. 1). ment detection limits (IDLs) were estimated using a signal-to- The river locations cover both urban and agricultural areas. noise approach of the standard dilutions reaching a ratio of Only one sewage treatment plant, which processes approxi- three, and method detection limits (MDLs) were determined by mately two-thirds (100,000 m3/d) of the municipal wastewater calculating the lowest concentration of the target chemicals in of the Xinglongtai area, is in the urban district of Panjin City. various water matrices that yielded an ion signal-to-noise of All untreated sewage is discharged directly into local rivers. three. Recoveries of all target compounds and surrogates were Animal husbandries include approximately 452,000 swine, analyzed by spiking standard solution and the surrogates to the 26,000 cattle, and 14 million poultry and are widespread influent, effluent, river, and sea samples (n ¼ 3), and quality throughout the Panjin area [31]. To identify the human and control was identified by distilled water blanks and duplicate agricultural source apportionment of the target chemicals, samples in every 10 samples. various source samples were collected. In detail, untreated urban sewage from one large canal in the Shuangtaizi area, Source apportionment method the outflow from one drainage station, the 24-h composite Source apportionment analysis was conducted using princi- influents and effluents from the STP, and the discharge from pal component analysis followed by multiple linear regression one local hospital were collected and identified as the human- (PCA-MLR) with SPSS software. Concentrations of variables used sulfonamides source (HS1 to HS5 in Fig. 1). Discharge can be regarded as linear combinations of a number of factors from field streams of intensive swine, duck, chicken, and cattle (or sources), and the purpose of PCA is to reproduce the farms were collected and identified as the animal husbandry correlation matrix in a minimum number of factors [32,33]. source samples (AS1 to AS4 in Fig. 1), and one mariculture Each factor is orthogonal to all others, which results in the sample from a shrimp pond was also collected (MQ in Fig. 1). smallest possible covariance. The first factor represents the
Fig. 1. Study area and sampling locations, China. Sulfonamides and their metabolites in river and sea samples Environ. Toxicol. Chem. 30, 2011 1255 weighted loading of the original variables that account for the Concentration levels greatest variability, and each subsequent factor accounts for less variability than the previous. Multiple linear regression was River. Of the 25 target chemicals, 12 sulfonamides (SMX, than performed on the significant factors to determine the mass SMM, SDZ, SMA, SPD, SGD, SA, STZ, SCP, SQX, SIM, apportionment of each source to total concentration. Stepwise and SDM), four N-acetylated metabolites (NAcSMX, NAcSPD, modeling was used to allow each independent factor to enter NAcSDZ, and NAcSMA), and TMP were detected in the into the regression equation if it could significantly increase the river samples collected from the Liao River basin (Fig. 2; correlation, and a default significance level of 0.05 was used Table 2). The typical MRM LC-MS/MS chromatograms here. After normalization, the MLR equation can finally be obtained from one river sample are shown in Figure S1 (Sup- expressed as: plemental Data). Sulfamethoxazole, TMP, SMM, SDZ, and X SMA were detected in 97 to 100% of the 35 river samples. ^ ¼ Zsum BkFSk (1) The median concentrations of SMX and TMP were 66.6 ng/L and 29.0 ng/L, with a range of 6.7 to 173.2 ng/L and 5.3 to where Z^ is the standard normalized deviate of the sum of sum 121.1 ng/L, respectively. These results are comparable to those the chemical concentrations (i.e., Z^ ¼ðZ mean½Z Þ= sum sum sum reported in surface water in the UK [20], Germany [22], Pearl s; and s is the standard deviation of Z ), B represents the sum k River, China [23], and Hong Kong [24], but lower than those regression coefficients, and FS are factor scores calculated by k in US streams [19,21]. The concentration levels of SMA the PCA analysis. (