A nationwide survey of the occurrence of melamine and its derivatives in archived sewage sludge from the United States

A nationwide survey of the occurrence of melamine and its derivatives in archived sewage sludge from the United States

^*

Hongkai Zhu

^a

, Rolf U. Halden

^b

, Kurunthachalam Kannan

^a,c,*

aWadsworth Center, New York State Department of Health, and Department of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, United States

bCenter for Environmental Health Engineering, The Biodesign Institute and School of Sustainable Engineering and the Built Environment, Arizona State University, Tempe, AZ 85287, United States

cBiochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, 22254, Saudi Arabia

a r t i c l e i n f o

Article history:

Received 14 October 2018 Received in revised form 23 November 2018 Accepted 26 November 2018 Available online 29 November 2018

Keywords:

Melamine Cyanuric acid Sewage sludge Nationwide survey Risk assessment

a b s t r a c t

Melamine-based resins are used extensively in laminates, plastics, coatings, glues, and dinnerware. Little is known, however, about the occurrence of melamine and its derivatives in the environment. In this study, a nationwide survey of melamine and its derivatives, namely ammeline, ammelide, and cyanuric acid, was conducted, using archived sewage sludge samples collected from 68 wastewater treatment plants in the United States (U.S.). The sum concentrations of four target compounds in sludge ranged from 34 to 1800 ng/g dry weight (dw), with a mean concentration of 240 ng/g dw; melamine (46%) and cyanuric acid (40%) collectively accounted for 86% of the total mass of analytes. No significant geographical variation in the concentrations of melamine and its derivatives in sewage sludge was found.

The estimated emission of melamine and its derivatives via land-application of sludge was approxi- mately 1600 kg/yr in the U.S. The hazard quotient values for melamine in sludge-amended soils ranged from 2.210⁵to 4.410³, indicating that the current levels of melamine in sludge pose a minimal risk to the soil environment.

©2018 Elsevier Ltd. All rights reserved.

1. Introduction

Melamine received worldwide attention in 2007 and 2008 following food scandals that involved the adulteration of pet foods and infant formula (Chen, 2009;Dobson et al., 2008;Gossner et al., 2009). Exposure of pets and infants to the tainted feed and formula, respectively, was linked to kidney stone formation and, in extreme cases, kidney failure and death (Guan et al., 2009; Ingelfinger, 2008). In addition to adulterated foods, melamine exposure can result from its use in a wide range of commercial applications.

Melamine is used in the production of polymer resins (e.g., plastic, kitchenware, dinnerware) and polymeric agents (e.g., laminates, glues, adhesives, coatings,flame retardants) (An et al., 2017). Cya- nuric acid, a structural analogue of melamine, is used in disinfec- tants, chlorine stabilizers, sanitizers, and bleaches (Cantu et al.,

2000). Ammeline and ammelide are not intentionally manufac- tured but are impurities or metabolites of melamine (Ehling et al., 2007). Both legitimate and illegitimate applications of melamine and its derivatives (i.e., ammeline, ammelide, and cyanuric acid) inevitably led to their occurrence not only in foods (Ibanez et al., 2009;Wu et al., 2009;Yan et al., 2009;Zhu and Kannan, 2018a) but also in the environment (Ge et al., 2011;Qin et al., 2010;Zhu and Kannan, 2018b).

Melamine and its derivatives have low octanol/water partition coefficients (logKow:4.08 to1.22 at 25C) and high water sol- ubilities (1.99e1000 g/L at 25C) (Table S1in the Supporting In- formation; SI). Melamine concentrations on the order of several micrograms per liter were reported in a survey of wastewater from China (Qin et al., 2010). Wastewater treatment plants (WWTPs) are generally considered as sinks for many organic compounds, espe- cially those that are used in consumer products (Fu et al., 2017;Li et al., 2015; Zhang et al., 2016). Previous studies have indicated that elevated levels of melamine and cyanuric acid were toxic to certain bacteria in conventional activated sludge treatment sys- tems, which ultimately led to inefficient removal of contaminants

*This paper has been recommended for acceptance by Charles Wong.

*Corresponding author. Wadsworth Center, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509, United States.

E-mail address:Kurunthachalam.Kannan@health.ny.gov(K. Kannan).

Contents lists available atScienceDirect

Environmental Pollution

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m/ l o ca t e / e n v p o l

https://doi.org/10.1016/j.envpol.2018.11.089 0269-7491/©2018 Elsevier Ltd. All rights reserved.

in WWTPs (An et al., 2017;An et al., 2018;Xu et al., 2018;Xu et al., 2013). Nevertheless, little is known about the occurrence of mel- amine and its derivatives in WWTPs. In the U.S., approximately 50e60% of sludge produced in WWTPs is recycled and land applied as biosolids to enhance soil properties and crop production (Venkatesan and Halden, 2013b). Thus, residual melamine and its derivatives present in sludge may enter into terrestrial food chain, and studies have reported on the uptake of melamine by crop plants (Ge et al., 2011;Han et al., 2010;Qin et al., 2010).

Sewage sludge is a suitable matrix for the assessment of mel- amine and its derivatives, as it can provide information regarding the extent of usage of these chemicals in a community and envi- ronmental emissions. To the best of our knowledge, however, no earlier studies have reported on the concentrations of melamine and its derivatives in sewage sludge. To address this knowledge gap, we used the National Sewage Sludge Repository sample archive to determine the occurrence and concentrations of mel- amine, ammeline, ammelide, and cyanuric acid in 76 sewage sludge samples collected from 68 WWTPs by the U.S. Environmental Protection Agency (EPA) in 2006e2007, as a part of the Targeted National Sewage Sludge Survey (TNSSS). Several earlier studies have reported the occurrence of environmental chemicals in sludge collected by the TNSSS program (Venkatesan and Halden, 2013b, 2014b; Venkatesan et al., 2014). On the basis of the measured concentrations in sludge, we evaluated environmental emissions (via sewage sludge and effluent) and potential ecological risks (via land application of sludge) of melamine and its derivatives.

2. Materials and methods 2.1. Sample collection

The TNSSS program was designed by the U.S. EPA to obtain national estimates of the concentrations of emerging contaminants in sewage sludge. Sludge samples collected between August 2006 and March 2007 were analyzed in this study (US EPA, 2009;

Venkatesan et al., 2015). To ensure universality and representa- tiveness of the survey, we selected publicly owned treatment works (POTWs) that met the following criteria: (1) was in full operation in 2002 and/or 2004, (2) treats more than 1 million gallons per day (MGD), (3) employs secondary or better treatment, and (4) is located in the contiguous U.S. A range of final treated sewage sludge, from liquid to solid, was collected by the U.S. EPA for the survey. As a part of the TNSSS program, a total of 84 sewage sludge samples from 74 WWTPs were shipped in solvent-cleaned glass jars to Wadsworth Center, New York State Department of Health, Albany, New York, from the Biodesign Institute at Arizona State University, Tempe, Arizona. These sewage sludge samples covered 35 states across the U.S., with measured concentrations that can be extrapolated to 3337 POTWs. As limited by sample amount, 76 sewage sludge samples from 68 WWTPs were analyzed in this study for melamine and its derivatives. Two (duplicate) samples were collected at eight WWTPs either because the facility had more than one treatment system and produced two types offinal sludge (n¼4) or for quality assurance purposes (n¼4). Only one single- grab sample was collected for the remaining 60 facilities. The in- formation pertaining to the identity of each WWTP was undis- closed by the U.S. EPA, as described elsewhere (Venkatesan et al., 2014), and therefore only general information, such as region, and treatment capacity were available for this study (Table S2). Based on the treatment capacity, the WWTPs were divided into three categories, as 1>MGD<10 (n¼50), 10>MGD<100 (n¼11), and MGD>100 (n¼7), for the purpose of data analysis and interpre- tation. Sewage sludge samples were archived at 20C before analysis.

2.2. Chemicals and reagents

Melamine (99% purity), ammeline (97.9% purity), ammelide (98% purity), and cyanuric acid (98% purity) were purchased from Sigma-Aldrich (Saint Louis, MO). Ammonium formate (99.99% pu- rity) and ammonium hydroxide (28.0e30.0% NH3basis) also were purchased from Sigma-Aldrich. The isotopically labeled internal standards, ¹⁵N₃,¹³C₃-melamine (¹⁵N₃,¹³C₃-melamine; ¹⁵N₃ at 98%

purity,¹³C3at 99% purity),¹³C3-ammeline (¹³C-ammeline; ¹³C3at 99% purity),¹³C3-ammelide (¹³C-ammelide;¹³C3at 99% purity), and

15N₃,¹³C₃-cyanuric acid (¹⁵N₃,¹³C₃-cyanuric acid at 99% purity,¹⁵N₃ at 98% purity,¹³C3at 99% purity), were purchased from Cambridge Isotope Laboratories (Andover, MA). High-performance liquid chromatography (HPLC)-grade solvents methanol, acetonitrile, water, and reagent grade formic acid and hydrochloric acid were supplied by J. T. Baker (Phillipsburg, NJ). Oasis MAX and Oasis MCX solid phase extraction (SPE) cartridges (150 mg, 6 mL, 30m^{m par-} ticles) were purchased from Waters Corp. (Milford, MA).

2.3. Analytical methods

Sludge extraction and cleanup procedures were similar to those described earlier, with some modifications (Andersen et al., 2007;

Braekevelt et al., 2011). Briefly, freeze-dried sewage sludge samples were extracted with a mixture of methanol/water (4:1,v/v) and purified by mixed-mode SPE cartridges (Oasis MAX for cyanuric acid and Oasis MCX for other analytes). The eluates from MAX and MCX cartridges were concentrated to near dryness under a gentle nitrogen stream, reconstituted in 500m^L acetonitrile/5 mM ammonium formate buffer (pH¼4.0) (9:1, v/v), and filtered through a 0.2mm nylon syringefilter (Corning, NY) directly into a glass vial prior to HPLC-MS/MS analysis.

Melamine and its derivatives were analyzed using a Shimadzu LC-30 AD Series HPLC system (Shimadzu Corporation, Kyoto, Japan) connected to an API 5500 triple-quadrupole MS/MS system (Applied Biosystems, Foster City, CA). Chromatographic separations were performed, using a Kinetex HILIC column (100 mm2.1 mm, 2.6mm; Phenomenex, Torrance, CA) serially connected to a Betasil C18 guard column (20 mm 2.1 mm, 5mm; Thermo Scientific, Waltham, MA) with acetonitrile (A) and 5 mM ammonium formate buffer (pH¼4.0; B) as mobile phases. Cyanuric acid and ammelide were analyzed using multiple reaction monitoring (MRM) negative ion electrospray mode (ES-), whereas MRM positive ion electro- spray mode (ESþ) was used for melamine and ammeline detection.

Typical chromatograms of standard and real sewage sample are shown inFig. S1. Further details of the extraction and instrumental analysis are provided in thesupporting information (SI).

2.4. Quality assurance (QA)/quality control (QC)

All disposable glass tubes and Pasteur pipettes were baked at 450C for 4 h prior to use. The HPLC syringe was rinsed twice before and after any injection with a mixture of acetonitrile/water (50:50,v/v). Procedural blanks were analyzed with every batch of 10 samples. No target analytes were present in blanks at concen- trations above the limit of detection (LOD; signal-to-noise ratio of 3). Method quantification limits (MQLs) were 0.80 ng/g for mel- amine, 1.2 ng/g for ammeline, 0.80 ng/g for ammelide, and 1.5 ng/g for cyanuric acid, which were derived from the limit of quantifi- cation (LOQ; signal-to-noise ratio of 10), a nominal sample weight of 0.2 g, and the diluted factor of 5. The recoveries of target com- pounds in spiked sludge matrix (three randomly selected sludge samples spiked at three different levels: 10 ng/g, 100 ng/g, and 200 ng/g; triplicate analysis for each level) ranged from 83% to 118%

with a relative standard deviation of <13%. Matrix-corrected

recoveries of ¹⁵N3,¹³C3-melamine, ¹³C-ammeline, ¹³C-ammelide, and¹⁵N₃,¹³C₃-cyanuric acid spiked into all samples (n¼76) were 87±5.4%, 78±9.0%, 81±14%, and 75±11%, respectively. A 10- point calibration curve at concentrations in the range of 0.05e200 ng/mL (with a regression coefficient of>0.996) was used in the quantification of target analytes.

2.5. Data analysis

A hazard assessment was performed for melamine in sludge- amended soils, using a hazard quotient (HQ) approach, as shown in equation(1):

HQ ¼ PEC_soil=PNECsoil (1)

where PNEC_soil is the predicted no-effect concentration (g/kg).

PNECsoilwas estimated for melamine from an LC50value (median lethal concentration) and an assessment factor (f) of 1,000, using the formula PNEC_soil¼LC₅₀/f (Cristale et al., 2013). A PNEC_soilvalue of 0.0005 g/kg was obtained from short-term growth studies of four terrestrial species (Hordeum vulgare, Tritium aestivum,Raphanus sativus, and Lepidum sativum) (OECD, 1998). Hazard assessment was not performed for three melamine derivatives due to the lack of terrestrial toxicity data. PECsoil was estimated as melamine concentrations in soil, following a single application of sludge. The PEC_soilvalues in soils, to which sludge had been applied, were determined according to the European Commission Technical Guidance Document on Risk Assessment (EU, 2003;Fu et al., 2017), as shown in equation(2):

PEC_soil ¼ C_sludgeAPPL_sludge .

DEPTH_soilRHO_soil (2)

where Csludge(g/kg dry weight; dw) is the concentration of target analytes in sludge, APPLsludgeis the application rate of dry sludge to soil (assumed at 0.50 kg/m²/yr), DEPTH_soil is the mixing depth (assumed to be 0.20 m), and RHOsoilis the bulk density of wet soil (assumed to be 1500 kg/m³for agricultural soils). Statistical ana- lyses (one-way ANOVA and Spearman's correlation) were per- formed, using SPSS 17.0 software packages. Statistical significance was set atp0.05.

3. Results and discussion

A summary of melamine, ammeline, ammelide, and cyanuric acid concentrations determined in sewage sludge is presented in Table 1. It should be noted that three of the 16 relative percentage difference values, calculated from the analysis of duplicate samples (4 field duplicates4 analytes), were above 50%, indicating the heterogeneous nature of sewage sludge samples.

3.1. Concentrations

Melamine and its derivatives were found in >90% of sludge samples. The sum concentrations of melamine and its derivatives (hereafter,PMelamine) varied by 1e2 orders of magnitude among the samples and ranged from 34 to 1800 ng/g dw. The concentra- tions of melamine and cyanuric acid were significantly higher than those of ammeline and ammelide (one-way ANOVA;p<0.05). The mean concentrations of 110 ng/g dw for melamine and 95 ng/g dw for cyanuric acid were approximately 5-fold higher than those of ammeline (19 ng/g) and ammelide (17 ng/g).

No earlier studies have reported concentrations ofP

Melamine in sewage sludge. We compared our data with the reported con- centrations of melamine in other environmental matrices. The

mean/median concentrations of PMelamine in sludge (240/

150 ng/g dw) were similar to those reported for river sediments from Japan (10e400 ng/g dw) (OECD, 1998). Soils collected near melamine factories contained this compound at concentrations in the range of 100e50,000 ng/g dw, whereas it was rarely found in farmland soils>150 km from melamine factories (Qin et al., 2010).

PMelamine concentrations in sludge fell between the concentra- tions found in industrial and farmland soils.

We also compared mean concentrations reported for several environmental chemicals in sludge from the 2001 and 2006e2007 TNSSS programs (Fig. S2). The mean concentration ofPMelamine (240 ng/g dw) was one order of magnitude higher than those of polybrominated dibenzo-p-dioxins and dibenzofurans (10.2 ng/g dw) (Venkatesan and Halden, 2014b) and similar to those of bisphenol A diglycidyl ethers (390 ng/g dw) (Xue et al., 2015), bisphenol analogs (521 ng/g dw) (Yu et al., 2015), perfluoroalkyl substances (mean: 540 ng/g dw) (Venkatesan and Halden, 2013b), andN-nitrosamines (1100 ng/g dw) (Venkatesan et al., 2014); but 2e3 orders of magnitude lower than those of brominatedflame retardants (12,000 ng/g dw) (Venkatesan and Halden, 2014a), pharmaceuticals and personal care products (74,000 ng/g dw) (McClellan and Halden, 2010), and alkylphenol ethoxylates (650,000 ng/g dw) (Venkatesan and Halden, 2013a).

Sewage sludge samples were grouped into four geographic areas as: West (n¼10 WWTPs/11 sludge), Midwest (n¼18 WWTPs/20 sludge), South (n¼24 WWTPs/25 sludge), and Northeast (n¼16 WWTP/20 sludge) for spatial comparison ofPMelamine concen- trations. The mean concentrations of P

Melamine in sludge collected from the Midwestern (280 ng/g dw) and Southern U.S.

(280 ng/g dw) were higher than those of the Western (210 ng/g dw) and Northeastern U.S. (180 ng/g dw) (Table 1), although the dif- ferences were not statistically significant among the four geographic regions (one-way ANOVA; p>0.05). When the data were plotted against the treatment capacity of the WWTPs studied, PMelamine concentrations were the highest in WWTPs that treated>100 MGD (mean: 330 ng/g dw), followed by those with lower treatment capacities of 1e10 (250 ng/g dw) and 10e100 (180 ng/g dw) MGD (Fig. 1,Table S3), although this difference was not statistically significant (one-way ANOVA; p>0.05). Higher concentrations of melamine in larger WWTPs may suggest greater discharges of this compound in urban areas, as large WWTPs are often situated in large cities (Venkatesan and Halden, 2014b;Xue et al., 2015).

3.2. Profiles and correlations of melamine and its derivatives

The percentage composition of melamine and its derivatives in sludge can provide an indication of their sources. Melamine was the major compound, accounting for 45e58% of the total concentra- tions in sludge samples from the Southern, Midwestern, and Western U.S., whereas cyanuric acid predominated in sludge sam- ples from the Northeastern U.S. (56% of the total concentrations) (Fig. 2). Overall, melamine (46%) and cyanuric acid (40%) contrib- uted to 86% of theP

Melamine concentrations, with minor con- tributions from ammeline (7.0%) and ammelide (7.0%). In urine samples collected from cats and dogs, however, cyanuric acid accounted for 70e78% of the total concentrations (Karthikraj et al., 2018). Further, predominance of cyanuric acid in infant formula collected from Canada has been documented (Braekevelt et al., 2011).

The differences in the composition profile of melamine and cyanuric acid between matrices suggest a wide range of applica- tions of these two compounds and/or metabolic/microbial trans- formation of melamine to cyanuric acid (El-Sayed et al., 2006). A significant positive correlation (Spearman correlation;

0.290<r<0.666,p<0.02) was found among melamine, ammeline, and ammelide concentrations in 76 sewage sludge samples (Table S4). This is consistent with the fact that ammeline and ammelide originate mainly from the hydrolysis of melamine or are formed as by-products during melamine production (Braekevelt et al., 2011; WHO, 2009). No significant correlation (Spearman

correlation;r¼0.103,p¼0.370) was found between melamine and cyanuric acid concentrations in sludge (Table S4), which further suggests differences in the sources of these two compounds.

In addition to the occurrence as an impurity (Zhang et al., 2010) or a metabolite of melamine, cyanuric acid has a wide range of commercial applications, including the manufacture of antioxi- dants, decolorizers, herbicides, and drugs, and as a disinfectant stabilizer (Cantu et al., 2000;Karthikraj et al., 2018). Cyanuric acid also is authorized by the U.S. Food and Drug Administration in ruminant fodder as a non-protein nitrogen source (US FDA, 2017).

Moreover, considerable differences in the degradation of melamine Table 1

Concentrations (ng/g dry weight) and detection frequency (%) of melamine and its derivatives in sewage sludge from the United States and the estimated emission (kg/year) through sludge disposal and wastewater discharge.

Region MEL^a AMN^b AMD^c CYA^d P

MELs^e

Northeast n¼16 WWTPs^hn¼20 SSⁱ Mean 45 20 13 100 180

Median 36 9.5 11 80 160

Range 16e120 2.9e160 nd^g-e33 11e300 37e390

df/%^f 100 100 88 100 100

South n¼24 WWTPs n¼25 SS Mean 130 23 22 100 280

Median 37 17 11 83 150

Range 6.5e1300 nde87 nde170 nde450 35e1800

df/% 100 96 92 96 100

Midwest n¼18 WWTPs n¼20 SS Mean 140 22 12 110 280

Median 48 7.0 6.9 48 180

Range 17e810 nde260 nde62 3.2e910 43e1400

df/% 100 83 89 100 100

West n¼10 WWTPs n¼11 SS Mean 120 11 19 58 210

Median 33 9.3 17 62 130

Range 15e600 3.2e23 2.7e47 17e160 67e710

df/% 100 100 100 100 100

All samples n¼68 WWTPs n¼76 SS Mean 110 19 17 95 240

Median 37 8.9 9.1 63 150

Range 6.5e1300 nde260 nde170 nde920 34e1800

df/% 100 90 91 99 100

Emission Estimates Emission through SS/(kg/yr) 720 120 110 620 1600

Emission through WWTPs/(kg/yr) 41,000 6900 6300 29,000 83,000

aMEL: melamine.

b AMN: ammeline.

c AMD: ammelide.

d CYA: cyanuric acid.

e P

MELs: sum concentrations of melamine and its derivatives.

f df: detection frequency.

g nd: not detected.

hWWTPs: wastewater treatment plants.

i SS: sewage sludge. For facilities with duplicate sludge samples, the data were aggregated as arithmetic mean; All concentrations<LOQ were calculated as 1/2 LOQ.

Fig. 1. Sum concentrations of melamine, ammeline, ammelide and cyanuric acid (ng/g dry weight) in sewage sludge collected from the United States stratified by treatment capacity of wastewater treatment plants.MGD stands for million gallons per day. The boxes represent the 25th and 75th percentiles, the whiskers represent the 10th and 90th percentiles, and the dots represent outliers below and above the 10th and 90th percentiles, respectively. The line and square represent the median and mean values, respectively.P

melamine: total concentrations of melamine and its derivatives.

Fig. 2. Composition profiles of melamine and its derivatives in sewage sludge collected from the United States stratified by sample regions.The values within parentheses on the Y-axis represent the number of WWTPs and sewage sludge analyzed. MEL: melamine; AMN: ammeline; AMD: ammelide; CYA: cyanuric acid.

and cyanuric acid in biological wastewater treatment systems have been reported (An et al., 2017;An et al., 2018;Xu et al., 2018;Xu et al., 2013). Melamine was not readily biodegradable, and its removal was mainly by sorption to sludge (An et al., 2017). Even after incubation for 125 d in activated sludge, melamine was shown not to degrade (Xu et al., 2013). In contrast, cyanuric acid was hy- drolyzed and converted to ammonia and CO2 in the activated sludge treatment process (An et al., 2018). The removal efficiencies of both melamine and cyanuric acid decreased by ~60% when their concentrations in influent exceeded 1.00 mg/L (An et al., 2017;An et al., 2018), suggesting antimicrobial activities of these two com- pounds at elevated concentrations.

3.3. Emission estimates through WWTPs and ecological hazard assessment in sludge-applied soils

A nationwide emission rate for melamine and its derivatives through sewage sludge disposal was estimated (Table 1). In the U.S., 6,513,586 tons of sewage sludge (dw) were produced in 2004, and approximately 50e60% of the sludge was land applied (UN HABITAT, 2008). On the basis of the mean concentrations of mel- amine and its derivatives measured in 68 WWTPs, the estimated emission rates of melamine, ammeline, ammelide, and cyanuric acid through sludge disposal were 720, 120, 110, and 620 kg/yr, respectively. Thus, the annual estimated rate of P

Melamine released through sewage sludge to the U.S. terrestrial environment was approximately 800e960 kg/yr.

We also estimated the mass of melamine and its derivatives released through wastewater effluents based on the sorption rates of these chemicals to sludge. The sorption rates of melamine, ammeline, ammelide, and cyanuric acid on sludge, predicted using the U.S. EPA's EPISuite, were 1.75, 1.75, 1.75, and 2.11%, respectively.

Therefore, the estimated emission rates through WWTPs in the U.S.

were 41,000, 6,900, 6,300, and 29,000 kg/yr for melamine, amme- line, ammelide, and cyanuric acid, respectively. The global pro- duction of melamine in 2007 was approximately 1,200,000 tons (Sharma and Paradakar, 2010), with an excess of 50,000 tons in the U.S. (US EPA, 2016). The proportion of melamine and its derivatives emitted through WWTPs was<1% of the production. Thus, sewage sludge and wastewater discharges constitute a small fraction of the environmental emission of melamine. It is important to note that the sorption rates calculated for melamine are crude estimates and may not reflect real-world conditions.

Hazard quotients were calculated for soils amended with sludge containing melamine for the four geographic regions. The calcu- lated HQs ranged from 5.410⁵to 3.910⁴, from 2.210⁵to 4.410³, from 5.710⁵to 2.710³, and from 5.010⁵to 2.010³for the Northeastern, Southern, Midwestern, and West- ern U.S., respectively (Fig. 3). These values were one order of magnitude lower than those reported for melamine in soils from a point source area (with a range from 3.510⁴to 1.110²) but were one order of magnitude higher than those reported for rural soil (<7.010⁵) (OECD, 1998). Overall, the HQs of melamine in sludge-amended soils were far below 1, indicating no significant risk of melamine from soil application of sludge at the currrent levels. Considering the half-life of melamine in soil (75 d) and the persistence of this chemical, however, further studies are needed to assess the long-term risks of melamine in soils.

Although this is the first study to report the occurrence of melamine and its derivatives in sludge, this study has some limi- tations. The archived sewage sludge samples were collected more than 10 years ago (2006e2007). Information regarding trans- formation of melamine and its derivatives during storage at20C is limited. The half-life of these substances in sediments has been predicted to range from 75 to 135 d (Table S1). Melamine and

cyanuric acid were shown to remain stable under freeze-thaw cy- cles (20C to room temperature in urine) and stock stability tests (at 4C for 4 weeks) (Zhang et al., 2010). All of the information above suggests that the concentrations reported in the present study are reliable. The removal efficiency of melamine and cyanuric acid in wastewater treatment processes varied with plant design and treatment types (Tian et al., 2012a;Tian et al., 2012b;Xu et al., 2018;Xu et al., 2013). Information that pertains to treatment type, sources of wastewater, and population served by the WWTPs was not available, except for the treatment capacity. This impedes our ability to describe the sources and fate of melamine and its de- rivatives in WWTPs.

In summary, this study establishes baseline concentrations of melamine and its derivatives in sewage sludge, which can inform potential risks and will enable evaluation of future trends in envi- ronmental releases of these compounds. Further studies on the fate of these chemicals in WWTPs are needed to assess the sources and behavior in the environment.

Notes

The authors declare no competingfinancial interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2018.11.089.

References

An, H.X., Li, X.M., Yang, Q., Wang, D.B., Xie, T., Zhao, J.W., Xu, Q.X., Chen, F., Zhong, Y., Yuan, Y.J., Zeng, G.M., 2017. The behavior of melamine in biological wastewater treatment system. J. Hazard Mater. 322, 445e453.

An, H.X., Liu, J., Li, X.M., Yang, Q., Wang, D.B., Xie, T., Zhao, J.W., Xu, Q.X., Chen, F., Wang, Y.L., Yi, K.X., Sun, J., Tao, Z.L.T., Zeng, G.M., 2018. The fate of cyanuric acid in biological wastewater treatment system and its impact on biological nutrient removal. J. Environ. Manag. 206, 901e909.

Andersen, W.C., Turnipseed, S.B., Karbiwnyk, C.M., Madson, M.R., 2007. Determi- nation of melamine residues in catfish tissue by triple quadrupole LC-MS-MS with HILIC chromatography. US FDA Lab. Inf. Bull. 23 (Food and Drug Admin- istration: Washington, DC).

Braekevelt, E., Lau, B.P.Y., Feng, S., Menard, C., Tittlemier, S.A., 2011. Determination of melamine, ammeline, ammelide and cyanuric acid in infant formula purchased in Canada by liquid chromatography-tandem mass spectrometry. Food Addit.

Contam. Part A-Chem. 28, 698e704.

Fig. 3.Calculated hazard quotients (HQs) for melamine in soils amended with sludge from the United States stratified by sample regions. For hazard quotient calculation, predicted no effect concentrations were derived from plant growth studies.

Cantu, R., Evans, O., Kawahara, F.K., Shoemaker, J.A., Dufour, A.P., 2000. An HPLC method with UV detection, pH control, and reductive ascorbic acid for cyanuric acid analysis in water. Anal. Chem. 72, 5820e5828.

Chen, J.S., 2009. A worldwide food safety concern in 2008-melamine-contaminated infant formula in China caused urinary tract stone in 290 000 children in China.

Chin. Med. J. (Beijing, China, Engl. Ed) 122, 243e244.

Cristale, J., Katsoyiannis, A., Sweetman, A.J., Jones, K.C., Lacorte, S., 2013. Occurrence and risk assessment of organophosphorus and brominatedflame retardants in the River Aire (UK). Environ. Pollut. 179, 194e200.

Dobson, R.L.M., Motlagh, S., Quijano, M., Cambron, R.T., Baker, T.R., Pullen, A.M., Regg, B.T., Bigalow-Kern, A.S., Vennard, T., Fix, A., Reimschuessel, R., Overmann, G., Shan, Y., Daston, G.P., 2008. Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs. Toxicol. Sci. 106, 251e262.

Ehling, S., Tefera, S., Ho, I.P., 2007. High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cerealflours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid. Food Addit. Contam. 24, 1319e1325.

El-Sayed, W.S., El-Baz, A.F., Othman, A.M., 2006. Biodegradation of melamine formaldehyde by Micrococcus sp. strain MF-1 isolated from aminoplastic wastewater effluent. Int. Biodeterior. Biodegrad. 57, 75e81.

European Union, 2003. Technical Guidance Document (TGD) on Risk Assessment of Chemical Substances. Technical Report EUR 20418 EN/2, second ed. Joint Research Centre, European Chemical Bureau, Helsinki.

Fu, L.F., Du, B.B., Wang, F., Lam, J.C.W., Zeng, L.X., Zeng, E.Y., 2017. Organophosphate triesters and diester degradation products in municipal sludge from wastewater treatment plants in China: spatial patterns and ecological implications. Environ.

Sci. Technol. 51, 13614e13623.

Ge, J., Zhao, L.W., Liu, C.Y., Jiang, S.R., Lee, P.W., Lie, F.M., 2011. Rapid determination of melamine in soil and strawberry by liquid chromatography-tandem mass spectrometry. Food Contr. 22, 1629e1633.

Gossner, C.M.E., Schlundt, J., Ben Embarek, P., Hird, S., Lo-Fo-Wong, D., Beltran, J.J.O., Teoh, K.N., Tritscher, A., 2009. The melamine incident: implications for inter- national food and feed safety. Environ. Health Perspect. 117, 1803e1808.

Guan, N., Fan, Q.F., Ding, J., Zhao, Y.M., Lu, J.Q., Ai, Y., Xu, G.B., Zhu, S.N., Yao, C., Jiang, L.N., Miao, J., Zhang, H., Zhao, D., Liu, X.Y., Yao, Y., 2009. Melamine- contaminated powdered formula and urolithiasis in young children. N. Engl. J.

Med. 360, 1067e1074.

HABITAT, U.N., 2008. Global Atlas of Excreta, Wastewater Sludge, and Biosolids Management: Moving Forward the Sustainable and Welcome Uses of a Global Resource. United Nations Human Settlements Programme (UN-HABITAT), Nai- robi, Kenya.

Han, D.F., Wang, D.H., Huang, P.Z., Duan, J.X., Ge, R.S., Liu, M.J., 2010. Effect of the melamine residue in soil on growth of Chinese cabbage. Environ. Sci. 31, 787e792.

Ibanez, M., Sancho, J.V., Hernandez, F., 2009. Determination of melamine in milk- based products and other food and beverage products by ion-pair liquid chromatography-tandem mass spectrometry. Anal. Chim. Acta 649, 91e97.

Ingelfinger, J.R., 2008. Melamine and the global implications of food contamination.

N. Engl. J. Med. 359, 2745e2748.

Karthikraj, R., Bollapragada, R., Kannan, K., 2018. Melamine and its derivatives in dog and cat urine: an exposure assessment study. Environ. Pollut. 238, 248e254.

Li, W.H., Shi, Y.L., Gao, L.H., Liu, J.M., Cai, Y.Q., 2015. Occurrence, fate and risk assessment of parabens and their chlorinated derivatives in an advanced wastewater treatment plant. J. Hazard Mater. 300, 29e38.

McClellan, K., Halden, R.U., 2010. Pharmaceuticals and personal care products in archived US biosolids from the 2001 EPA national sewage sludge survey. Water Res. 44, 658e668.

OECD, 1998. Melamine: screening information dataset (SIDs). Available at:http://

www.inchem.org/documents/sids/sids/108781.pdf. (Accessed 26 August 2018).

Qin, Y.C., Lv, X.W., Li, J., Qi, G.H., Diao, Q.Y., Liu, G.H., Xue, M., Wang, J.Q., Tong, J.M., Zhang, L.Y., Zhang, K.Y., 2010. Assessment of melamine contamination in crop, soil and water in China and risks of melamine accumulation in animal tissues and products. Environ. Int. 36, 446e452.

Sharma, K., Paradakar, M., 2010. The melamine adulteration scandal. Food Secur. 2, 97e107.

Tian, Y.Q., Chen, L.M., Gao, L.H., Michel, F.C., Keener, H.M., Klingman, M., Dick, W.A., 2012a. Composting of waste paint sludge containing melamine resin and the compost's effect on vegetable growth and soil water quality. J. Hazard Mater.

243, 28e36.

Tian, Y.Q., Chen, L.M., Gao, L.H., Michel, F.C., Wan, C.X., Li, Y.B., Dick, W.A., 2012b.

Composting of waste paint sludge containing melamine resin as affected by nutrients and gypsum addition and microbial inoculation. Environ. Pollut. 162, 129e137.

US Environmental Protection Agency, 2009. Targeted National Sewage Sludge Sur- vey Sampling and Analysis Technical Report; EPA-822-r-08-016.

US Environmental Protection Agency, 2016. Chemical data reporting results.

Available at: https://www.epa.gov/chemical-data-reporting/2016-chemical- data-reporting-results#access. (Accessed 26 August 2018).

US Food and Drug Administration, 2017. CFR: code of federal regulations Title 21.

Available at: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/

CFRSearch.cfm?fr¼573.220. (Accessed 26 August 2018).

Venkatesan, A.K., Halden, R.U., 2013a. National inventory of alkylphenol ethoxylate compounds in U.S. sewage sludges and chemical fate in outdoor soil meso- cosms. Environ. Pollut. 174, 189e193.

Venkatesan, A.K., Halden, R.U., 2013b. National inventory of perfluoroalkyl sub- stances in archived US biosolids from the 2001 EPA national sewage sludge survey. J. Hazard Mater. 252, 413e418.

Venkatesan, A.K., Halden, R.U., 2014a. Brominatedflame retardants in U.S. biosolids from the EPA national sewage sludge survey and chemical persistence in out- door soil mesocosms. Water Res. 55, 133e142.

Venkatesan, A.K., Halden, R.U., 2014b. Contribution of polybrominated dibenzo-p- dioxins and dibenzofurans (PBDD/Fs) to the toxic equivalency of dioxin-like dompounds in Archived Biosolids from the US EPA's 2001 national sewage sludge survey. Environ. Sci. Technol. 48, 10843e10849.

Venkatesan, A.K., Pycke, B.F.G., Halden, R.U., 2014. Detection and occurrence ofN- nitrosamines in archived biosolids from the targeted national sewage sludge survey of the US Environmental Protection Agency. Environ. Sci. Technol. 48, 5085e5092.

Venkatesan, A.K., Done, H.Y., Halden, R.U., 2015. United States national sewage sludge repository at Arizona State UniversityeA new resource and research tool for environmental scientists, engineers, and epidemiologists. Environ. Sci. Pol- lut. Res. Int. 22, 1577e1586.

World Health Organization, 2009. Toxicological and Health Aspects of Melamine and Cyanuric Acid. Report of a WHO Expert Meeting in Collaboration with FAO, vol. 2009. Supported by Health Canada. http://www.who.int/foodsafety/

publications/chem/Melamine_report09.pdf. (Accessed 27 August 2018).

Wu, Y.N., Zhao, Y.F., Li, J.G., Grp, M.A., 2009. A survey on occurrence of melamine and its analogues in tainted infant formula in China. Biomed. Environ. Sci. 22, 95e99.

Xu, S.N., Zhang, Y.Y., Sims, A., Bernards, M., Hu, Z.Q., 2013. Fate and toxicity of melamine in activated sludge treatment systems after a long-term sludge adaptation. Water Res. 47, 2307e2314.

Xu, S.N., Sun, M.H., Thompson, A., Hu, Z.Q., 2018. Biodegradation and toxicity of melamine at high activated sludge concentrations in a membrane bioreactor.

Water Sci. Technol. 77, 979e987.

Xue, J.C., Venkatesan, A.K., Wu, Q., Halden, R.U., Kannan, K., 2015. Occurrence of bisphenol A diglycidyl ethers (BADGEs) and novolac glycidyl ethers (NOGEs) in archived biosolids from the US EPA's targeted national sewage sludge survey.

Environ. Sci. Technol. 49, 6538e6544.

Yan, N., Zhou, L., Zhu, Z.F., Chen, X.G., 2009. Determination of melamine in dairy products,fish feed, andfish by capillary zone electrophoresis with diode array detection. J. Agric. Food Chem. 57, 807e811.

Yu, X.H., Xue, J.C., Yao, H., Wu, Q., Venkatesan, A.K., Halden, R.U., Kannan, K., 2015.

Occurrence and estrogenic potency of eight bisphenol analogs in sewage sludge from the US EPA targeted national sewage sludge survey. J. Hazard Mater. 299, 733e739.

Zhang, M.Q., Li, S.J., Yu, C.Y., Liu, G.Y., Jia, J.Y., Lu, C.A., He, J., Ma, Y.H., Zhu, J.M., Yu, C., 2010. Determination of melamine and cyanuric acid in human urine by a liquid chromatography tandem mass spectrometry. J. Chromatogr. B Analyt. Technol.

Biomed. Life Sci. 878, 758e762.

Zhang, T., Xue, J.C., Gao, C.Z., Qiu, R.L., Li, Y.X., Li, X., Huang, M.Z., Kannan, K., 2016.

Urinary concentrations of bisphenols and their association with biomarkers of oxidative stress in people living near e-waste recycling facilities in China. En- viron. Sci. Technol. 50, 4045e4053.

Zhu, H., Kannan, K., 2018a. Continuing occurrence of melamine and its derivatives in infant formula and dairy products from the United States: implications for environmental sources. Environ. Sci. Technol. Lett. 5, 641e648.

Zhu, H., Kannan, K., 2018b. Distribution profiles of melamine and its derivatives in indoor dust from twelve countries and the implications for human exposure.

Environ. Sci. Technol. 52, 12801e12808.