most widely used insecticides , 
 with global production valued at us$2.5 billion and registrations 
 in more than 120 countries for commercial use on more than 140 crops . these insecticides are used for control of aphids , 
 whiteflies , planthoppers , lepidoptera , and some coleopteran and other 
 pests , where they function as powerful neurotoxins . in december , 
 2013 , the european commission introduced a two year 
 moratorium on clothianidin , imidacloprid , and thiamethoxam , following 
 reports by the european food safety authority ( efsa ) of these substances 
 posing an  acute risk  to honeybees essential to farming 
 and natural ecosystem . temporal and expired 
 restrictions allow for potential current and future uses of neonicotinoids 
 in these settings . adverse effects from widespread use of neonicotinoids 
 have been reported recently for many nontarget organisms like phloem - feeding 
 insects , pollinators and bees , and aquatic invertebrates . median lethal dose values ( ld50 ) of neonicotinoids 
 for bees vary from 5 to 70 ng / bee . sublethal 
 doses have been shown to cause atp synthesis inhibition , resulting in weakening of foraging success , memory 
 and learning , damage to the central nervous system , and increased susceptibility to diseases . a recent review based on 214 toxicity tests of 48 species 
 suggested that average individual environmental concentrations of 
 greater than 35 ng / l may severely affect sensitive aquatic invertebrates 
 populations . another study indicated that 
 aquatic macrofauna populations dropped sharply at concentrations between 
 13 and 67 ng / l . a study in the netherlands observed a decline in the insectivorous 
 bird population after the introduction of imidacloprid , the highest 
 production volume insecticide in the world ; imidacloprid concentrations 
 of greater than 20 ng / l correlated with 3.5% average annual declines 
 in bird populations . imidacloprid is 
 moderately toxic to fish communities ; oxidative stress and dna damage have been reported in zebrafish . furthermore , co - occurrence of multiple neonicotinoids 
 is known to impart synergistic toxic effects . during the past decades global contamination with neonicotinoids 
 has been observed in surface waters , many of which receiving treated 
 effluent from wastewater treatment plants . in a nationwide assessment 
 of united states streams , at least one neonicotinoid was detected 
 in 53% of the samples analyzed ( n = 38 ) . in california , imidacloprid was detected in 
 89% of surface water samples collected from agricultural regions ( n = 75 ) in which 19% of the samples exceeded concentrations 
 of 1.05 g / l , the chronic invertebrate aquatic life benchmark 
 value established by the united states environmental protection agency 
 ( usepa ) . in canadian wetlands of the 
 central - eastern region of saskatchewan , neonicotinoids were detected 
 frequently in 20122013 ( 48% ; n = 440 ) at 
 a total average concentration of 51.8 ng / l , with higher detection 
 frequencies being observed in spring and higher mean concentrations 
 in summer . in several rivers around sydney , 
 australia , ng / l ; 
 imidacloprid was the most common neonicotinoid , detected in 93% of 
 samples ( clothianidin 
 was detected with a detection frequency of 46.6% in groundwater and 
 surface water samples ( n = 58 ) collected in germany . wastewater constitutes a potential source 
 of neonicotinoids in 
 the environment that has not received much attention yet . neonicotinoids 
 are widely used in nonindustrial agricultural applications such as 
 pet flea treatment , horticulture , and household pest control products . a few studies have detected imidacloprid in wastewater , 
 showing that treated effluent can inadvertently contribute to neonicotinoid 
 discharge into receiving water bodies . indeed , a nationwide assessment 
 of united states streams showed a positive correlation between neonicotinoid 
 occurrence and urban land usage but not with agricultural use . in oregon , effluent samples from 52 wastewater 
 treatment plants ( wwtps ) analyzed for imidacloprid showed detections 
 in 9.8% samples ( n = 102 ) , with an average concentration 
 of 270 ng / l . in spain , imidacloprid was 
 detected in wastewater influent and effluent samples at concentrations 
 ranging from 1.4165.7 ng / l ( 59.4% ; n = 32 ) . in another related spanish study , imidacloprid was detected in river water receiving wwtp effluent at a maximum concentration 
 of 19.2 ng / l , identifying sewage treatment facilities as a source 
 of neonicotinoids in the environment . in beijing , china , imidacloprid was detected in wwtp influent and 
 effluent at concentrations of 45100 and 45106 ng / l , 
 respectively , with no further information being provided on the removal rate . with wastewater 
 representing a likely source of neonicotinoids 
 in the united states aquatic freshwater environment , the goal of the 
 present study was to conduct a first mass balance assessing the fate 
 of six neonicotinoids ( listed in order of decreasing global annual 
 turnover : imidacloprid , thiamethoxam , clothianidin , acetamiprid , thiacloprid , 
 and dinotefuran ) during conventional wastewater 
 treatment and wetland treatment and to obtain through a nationwide 
 reconnaissance a first national emission estimate by monitoring additional 
 treatment facilities from across the united states . organic solvent of high performance 
 liquid chromatography ( hplc ) grade and formic acid of american chemical 
 society ( acs ) grade ( 98% ) were purchased from sigma - aldrich corp . , 
 st . ultrapure lc - ms grade water was purchased from 
 thermo fisher scientific , waltham , ma , u.s.a . analytical standards 
 for six neonicotinoids , an acetamiprid degradate , and deuterated labeled 
 standards for imidacloprid ( imidacloprid - d4 ) , acetamiprid ( acetamiprid - d3 ) , and 
 clothianidin ( clothianidin - d3 ) were obtained 
 from sigma - aldrich corp . , st . stock solutions of analytical 
 standards ( 1 ppb to 10 ppm ) and their mixtures were prepared in acetonitrile 
 and stored at 20 c . sampling for this mass balance assessment one plant and wetland were studied in 
 great detail to obtain general information on the fate of neonicotinoids , 
 and then additional plants were sampled to see whether the information 
 obtained is more broadly applicable to treatment facilities in the 
 united states . in early december 2014 for a period of five consecutive 
 days ( thursday through monday ) , a large activated sludge sewage treatment 
 plant with an engineered wetland downstream was sampled extensively . the plant is located in the southwestern region of the united states 
 and designed to serve a population of up to 2.5 million with design 
 capacity of 870 million l / d ( mld ) , receiving sewage comprised of 94% 
 domestic wastewater and 6% industrial wastewater . the treatment facility 
 produces class b+ reclaimed water discharged into a river and class 
 b sludge used for land application . the highest - flow treatment train 
 was selected for detailed studies on plant performance . unit processes 
 performed at the wwtp include screening , grit removal , primary sedimentation , 
 activated sludge biological treatment , secondary clarification , disinfection 
 treatment by chlorination , thickening of primary sludge , waste - activated 
 sludge by centrifugation , anaerobic sludge digestion , and dewatering 
 of digested sludge by centrifugation . primary sludge and waste activated 
 sludge are digested at 35 c , with an average solids retention 
 time of 21 days . effluents from a total of five parallel treatment 
 trains are combined , and a portion of this total flow is directed 
 into an engineered wetland located immediately downstream and featuring 
 a hydraulic retention time ( hrt ) of about 4.7 days , an average water 
 depth of about 1.5 m , total suspended solids ( tss ) concentrations 
 in wetland influent and effluent of 1015 mg / l , and average 
 wastewater flow received and discharged around 280 and 250 mld , respectively . average values of carbonaceous biochemical oxygen demand ( cbod ) for 
 plant influent and wetland effluent were 288  23 and 7  
 1 mg / l , respectively , demonstrating cbod removal of approximately 
 98% . average tss values in plant influent and wetland effluent were 
 437  160 and 14  3 mg the treatment train selected for sampling received 
 wastewater at a flow rate averaging 230 mld . seven portable automated 
 samplers ( 6712 full - size portable sampler , teledyne isco , lincoln , 
 ne , u.s.a . ) were programmed based on three - week average hourly daily 
 flow rate data to collect 2.5 l of flow - weighted composite samples 
 of primary influent , primary effluent , secondary effluent , waste activated 
 sludge , disinfection basin effluent , wetland influent , and wetland 
 effluent over a period of 24 h for 5 consecutive days . detailed information 
 on the sample programming and flow diagram of wwtp ( figure s1 ) is provided in the supporting information ( si ) . samples were collected in precleaned ( acetone 
 washed and heated at 500 c for 5 h ) amber 2.5 l wide - mouth glass 
 bottles . grab samples of primary sludge and dewatered sludge were 
 collected in precleaned amber 1 l glass bottles and amber 40 ml volatile 
 organic analysis ( voa ) glass vials , respectively . after collection , 
 samples were placed into coolers and shipped 
 to the laboratory , where 600 mg / l of kathon cg - icp preservative and 
 80100 mg / l of sodium thiosulfate were added to disinfect and 
 dechlorinate the samples and to prevent biological and chemical degradation 
 of analytes to take place during storage ( see si for additional information ) . then , 500 ml of aliquots of 
 water were fortified with 200 ng of the deuterated surrogate standards 
 to account for losses during storage , extraction , and analysis . solid 
 samples were dried and fortified with labeled standards to a nominal 
 concentration of 400 ng / g ( dry weight solids ) . for the expanded nationwide 
 reconnaissance , 12 additional united 
 states wwtps voluntarily collected 24 h flow - adjusted samples that 
 were provided to the study team in the year of 2015 as a composited 
 sample . the wwtps , who requested anonymity as a prerequisite of study 
 participation , are located in different regions of the country as 
 described in the discussion section . typically , 
 only one composite each was provided of raw influent and treated effluent 
 collected simultaneously on a random workday . four facilities provided 
 effluent only ; three facilities performed tertiary treatment by filtration , 
 three facilities performed uv disinfection instead of chlorination ; 
 all other facilities performed conventional treatment ( secondary treatment 
 followed by chlorine disinfection ) . an automatic solid - phase 
 extraction instrument ( dionex autotrace 280 , thermo scientific , waltham , 
 ma , u.s.a . ) was used to concentrate and elute analytes from water 
 samples from the sorbent bed for analysis . following screening of 
 extraction efficiency of a combination of sorbents and sample volumes , 
 reverse phase functionalized polymeric styrene divinylbenzene sorbent 
 ( strata x and xl , 500 mg/3 ml , phenomenex , torrance , ca , u.s.a . ) was 
 selected and loaded with 500 ml of wastewater sample . before loading , 
 cartridges were conditioned with 3 ml of methanol , followed by 3 ml 
 of water . then , 500 ml of wastewater samples spiked with 200 ng of 
 the deuterated surrogate standards were loaded onto the cartridges 
 at a flow rate of 2 ml / min , washed with water , and dried with nitrogen 
 gas for 5 min . two consecutive elutions were performed , each with 
 4 ml of a mixture ( 95:5 , v / v ) of methanol and formic acid . equal volumes 
 of serial eluates were combined , evaporated , and reconstituted to 
 half the volume of water and methanol solution ( 80:20 , v / v ) in 0.1% 
 formic acid for lc - ms analysis . waste activated sludge and primary 
 sludge samples featuring a tss content of approximately 2% and 6% , 
 respectively were spun in a centrifuge at 7500 g for 
 10 min . resultant supernatants were extracted as described above for 
 water samples , whereas the solids separated from the samples were 
 extracted separately as described below . solid samples were dried 
 under nitrogen using an evaporator ( reacti - therm ts-18821 , thermo 
 scientific , waltham , ma , u.s.a . ) . one gram aliquots of solids samples 
 ( dry weight ) spiked with 400 ng of the deuterated surrogate standards 
 were transferred into 40 ml voa vials , extracted with 10 ml acetone , 
 placed on a shaker for 24 h , and sonicated for 1 h. extracts were 
 spun in a centrifuge at 3000 g for 5 min , and the 
 supernatants were transferred into new vials . the solids were extracted 
 a second time with acetone , vortexed for a minute , and centrifuged , 
 and the supernatants were combined with the first extracts . after 
 two extractions in sequence , the resultant acetone extracts were dried 
 under a stream of nitrogen , and analytes were reconstituted in 6 ml 
 of hexane , following which the resultant extract was cleaned up by 
 solid phase extraction ( similar to usepa method 3620c ) with a sorbent 
 bed featuring a blend of magnesium oxide and silica gel ( sep - pak vac 
 florisil cartridge 6 cc containing 1 g of sorbent , waters corporation , 
 milford , ma , u.s.a . ) . before loading , the sorbent was conditioned 
 successively with 6 ml of dichloromethane ( dcm ) , 6 ml of acetone , 
 and 6 ml of hexane . extracts in hexane were loaded onto the cartridges , 
 the resin bed washed with 6 ml of hexane , and analytes eluted subsequently 
 with 4 ml of dcm and 4 ml of acetone . aliquots of 1 ml of each serial 
 extract ( acetone and dcm ) were transferred and combined into 2 ml 
 lc analysis vials , dried under a gentle stream of nitrogen , and reconstituted 
 with 1 ml of a solution of water , methanol , and formic acid ( 80/20/0.1 , 
 v / v / v ) for analysis . separation was carried out using a shimadzu ultra 
 performance liquid chromatography ( uplc ) system , equipped with the 
 sil-20ac autosampler and 20-ad solvent delivery system ( shimadzu scientific 
 instruments , inc . , simultaneous chromatographic 
 separation of the six neonicotinoids plus one degradate was performed 
 by reverse phase liquid chromatography using a 4.6 mm  150 mm 
 c8 column ( xbridge , waters corporation milford , ma , u.s.a . ) 
 with 3.5 m bridged ethylene hybrid ( beh ) particles . a binary 
 gradient with 0.1% formic acid in water and methanol at a total flow 
 rate of 0.5 ml / min was applied . the injection volume was 100 l , 
 and the mobile phase consisted of 20% organic with an initial 1 min 
 ramp of 10% solvent content increased per min , followed by a 6 min 
 ramp of 10.8% per min to 95% organic , where it was held for 3.5 min , 
 for a total run time of 14 min . identification and quantitation were 
 performed using an api 4000 tandem mass spectrometer ( absciex , framingham , 
 ma , u.s.a . ) in positive electrospray ( esi+ ) mode by monitoring the 
 first and second most abundant ion transitions for quantification 
 and confirmation , respectively . mass spectrometry was performed at 
 a source heating temperature of 700 c , ion spray voltage of 
 4500 v , curtain gas ( nitrogen ) pressure of 50 psi , nebulizer gas pressure 
 of 90 psi , heater gas pressure of 75 psi , and dwell time of 70 ms . analyst software , version 1.5 ( absciex , framingham , ma , u.s.a . ) was 
 used for lc - ms / ms system control and data analysis . information on 
 calibration curves , method validation , quality assurance , and quality 
 control can be found in the si . an analyte mass balance was 
 performed for the full - scale wastewater treatment train over a period 
 of 5 consecutive days ( to account for the hydraulic residence time ) , 
 combining primary , activated sludge , and disinfection treatment , using 
 the following equation:1where , transformed = mass input 
 of neonicotinoids lost to transformation or unaccounted 
 for ( g / day ) , qinf = flow rate of influent 
 to primary clarifier ( l / day ) , cinf = concentration 
 of neonicotinoids in influent entering primary clarifier ( g / l ) , qeff = flow rate of effluent after chlorine disinfection 
 ( l / day ) , ceff = concentration of neonicotinoids 
 in effluent leaving treatment plant ( g / l ) , mdws = mass of dewatered sludge produced ( kg / day ) , and cdws = concentration of neonicotinoids in dewatered 
 sludge ( g / kg ) . individual mass balance for primary treatment , 
 activated sludge treatment , disinfection treatment , and constructed 
 wetland were calculated similarly ( see si ) . a paired two tailed t - test was performed (  
 = 0.05 ) to compare mean daily masses between treatment streams . differences 
 were determined at the p < 0.05 significance level . to determine 
 the sorption affinity of analytes onto sludge particulates , a partitioning 
 study was conducted . ten milliliters 
 of aliquots of water having 1 , 10 , and 100 ppm of all six neonicotinoids 
 was added to 1 g of dewatered sludge , and after 10 days of shaking 
 in the dark at 22 c , water and solids were analyzed to establish 
 the partitioning behavior . sludge was inactivated prior to shaking 
 by addition of 600 mg kathon cg / icp and 300 mg of sodium azide to 
 prevent any possible biotransformation . to determine kd values , the sorbed concentration was plotted against 
 bulk concentration remaining after sorption , and eq 2 was used:2where kd = distribution 
 coefficient , l / kg dry weight ; cs = sorbed 
 concentration on the solid particulates , mg / kg dry weight of dewatered 
 solids ; cd = bulk concentration remaining 
 after sorption , mg / l . the tandem mass spectrometry method 
 developed for this study targeted six neonicotinoids and one degradate 
 simultaneously at part - per - trillion levels by monitoring two ion transitions 
 via multiple reaction monitoring ( mrm ) . mass spectrometry parameters 
 optimized for detection are summarized in table s2 of the si . limits of detection 
 of analytes in different matrices are shown in table 1 ( see si for information 
 on data analysis and reporting methods ) . to ensure the quality and 
 validity of results , each analysis batch of environmental samples 
 contained a field blank , method blank , and check samples . no false 
 positive values suggesting postsample collection contamination were 
 detected during the analysis of all samples . values of relative percent 
 deviation ( rpd ) were in an acceptable range for imidacloprid ( 25  
 17% ) , acetamiprid ( 20  17% ) , acetamiprid - n - desmethyl 
 ( 28  22% ) , and clothianidin ( 18  22% ) , as summarized in table s3 . 80% detection 
 frequency ; n / a , not 
 applicable ( as isotope - labeled surrogate standard was not available ) ; 
 bdl , below detection limit ; dw , dry weight ; kow , n - octanol water partition coefficient ; kd , sludge  water partition coefficient ; 
 loq , limit of quantification . over the sampling period of 5 consecutive days ( thursday 
 through monday ) with 3 workweek days and 2 weekend days , consistent 
 loading with imidacloprid ( 4555 ng / l ; 100% df ) and acetamiprid 
 ( 35 ng / l ; 100% df ) , and erratic loading of clothianidin ( < 1666 
 ng / l ; 80% df ) was observed ( table 1 ) . also detected was acetamiprid - n - desmethyl ( 12 ng / l ; 100% df ) , a degradate of acetamiprid 
 formed here as a result of activated sludge treatment . neonicotinoids 
 not detected in process streams included thiacloprid , thiamethoxam , 
 and dinotefuran , with their corresponding method detection limits 
 summarized in table 1 . during the 5 day sampling period , the average concentrations ( mean 
  standard deviation ) of imidacloprid , acetamiprid , and clothianidin 
 detected in plant influent were 54.7  9.3 , 3.7  0.8 , and 
 149.7  273.1 ng / l , respectively . these neonicotinoids entered 
 the primary clarifier in which settling occurred , diverting 1% of 
 the total volumetric flow away as sludge featuring a tss content 17 
 times higher than that of the clarifier effluent . resultant daily 
 composite effluent samples of primary treatment contained similar 
 levels to those found in raw sewage ( influent ) during the 5 day sampling 
 period . secondary treatment consisted of an activated sludge unit 
 operation , a biological process aimed at breaking down organic compounds 
 primarily by microbial degradation . average concentrations of imidacloprid 
 and clothianidin in secondary effluent were 48.6  7.8 and 131.3 
  170.8 ng / l , implying no discernible removal by processes including 
 microbial degradation , hydrolysis , and oxidation in the aeration basin . prior lab studies also had shown insignificant transformation of imidacloprid 
 in both acidic and neutral water . however , 
 acetamiprid undergoes relatively fast dissipation in a neutral environment 
 having an aqueous dissipation half - life of 4.7 days , and corresponding results were observed during secondary 
 treatment , with effluent concentration of acetamiprid ( 1.8  
 0.4 ng / l ) cut in half compared to influent , and the formation of acetamiprid - n - desmethyl being observed , thereby confirming transformation 
 of acetamiprid in the aeration basin , presumably mediated in part 
 by aerobic microorganisms . secondary effluent showed average daily 
 concentrations of acetamiprid - n - desmethyl of 1.3 
  0.3 ng / l . concentrations of acetamiprid - n - desmethyl 
 in primary influent and primary effluent were below the detection 
 limit ( < 0.5 ng / l ) . to meet microbial removal criteria , the wastewater 
 facility examined herein uses chlorination at a chlorine dosage of 
 2.5 mg / l . although chlorine has the potential to oxidize organic compounds , 
 no change in concentrations of imidacloprid , acetamiprid , acetamiprid - n - desmethyl , and clothianidin concentration ( table 1 ) data on neonicotinoids in 
 aqueous process streams were used in conjunction with corresponding 
 flow rate ( table s4 ) information to compute 
 pesticide mass flow through 
 the facility . on the basis of the daily average flow received by the 
 treatment train , the total mass of analytes passing through the facility 
 during the monitoring period was determined ( figure 1 ) . error values on the total mass are derived 
 from maximum and minimum values of detected concentrations from two 
 experimental replicates . total mass of imidacloprid ( a ) , acetamiprid 
 ( b ) , and clothianidin 
 ( c ) in wastewater unit operation flows over a 5 day period . mass in raw sewage of imidacloprid , 
 acetamiprid , and clothianidin 
 corresponded to 66.7  3.0 , 4.5  0.4 g , and 183.0  
 7.3 g/5 days , respectively . after primary treatment , the total mass 
 leaving the primary clarifier in effluent was similar to those in 
 influent ( figure 1 ) , 
 implying insignificant sorption of neonicotinoids onto sludge particulates , 
 with the analytes persisting during primary treatment . the mass of 
 imidacloprid and clothianidin leaving the secondary clarifier was 
 58.3  6.3 and 159.6  8.9 g/5 days , respectively . these 
 data indicate persistence of both compounds during secondary treatment . 
 the mass of acetamiprid leaving the secondary clarifier in the form 
 of the parent compound was 2.1  0.1 g/5 days , indicating a 53 
  3% loss of acetamiprid in the aeration basin . the acetamiprid 
 degradate , acetamiprid - n - desmethyl , accounted for 
 1.6  0.2 g/5 days in effluent , which reduced the total mass 
 removal estimate for acetamiprid and its major degradate to 18  
 4% . whereas relevant information on the toxicity of acetamiprid - n - desmethyl is unavailable , in theory this degradate could 
 still impart toxicity to nontarget organisms via its cyano group , 
 which is known to interact with the nachr receptor of insects . similarly , a mass balance on chlorination treatment of imidacloprid , 
 acetamiprid , acetamiprid - n - desmethyl , and clothianidin 
 showed resistance of each of these compounds to oxidation under real - world 
 conditions . paired t - tests were performed to 
 compare the influent 
 and effluent concentrations of the three analytes . the mean and standard 
 deviation for imidacloprid for influent and effluent were 13.3  
 2.4 and 11.7  2.1 , respectively . the mean daily influent and 
 effluent mass loadings of imidacloprid detected over the sampling 
 period were statistically indistinguishable ( t = 
 1.88 , p = 0.09 , ci = 95% ) . the mean 
 and standard deviation for acetamiprid for influent and effluent were 
 0.90  0.21 and 0.73  0.09 , respectively . a mass balance 
 over the wwtp showed total acetamiprid removal of 18  4% ( t = 3.31 , p = 0.01 , ci = 95% ) , with 45 
  4% of the initial mass being discharged as acetamiprid and 
 37  4% as its degradate , acetamiprid - n - desmethyl . strong variations in the loading of clothianidin during the sampling 
 period stood in the way of conducting a firm mass balance ; nevertheless , 
 notable persistence ( > 70% ) of the compound during treatment was 
 firmly 
 established . as primary 
 sludge and waste activated sludge represented 2% of the total facility 
 flow , the mass of neonicotinoids accumulated in sludge was assessed 
 as part of the mass balance analysis . partitioning of neonicotinoids 
 to wastewater solids was not a major factor for their fate during 
 treatment , however . levels of imidacloprid , acetamiprid , acetamiprid - n - desmethyl , and clothianidin were all below their respective 
 mdls of 1.1 , 0.7 , 1.9 , and 1.4 g / kg dry weight sludge . despite 
 these nondetect values , refined concentration estimates were obtained 
 by analyzing the decanted liquid of sludges and using the established 
 partition coefficients ( table 1 ) to calculate the approximate neonicotinoid concentrations 
 on dry weight solids . as shown in table s6 , the resultant concentrations 
 were low and inconsequential for the mass balance analysis ( < 1% 
 of total mass ) . availability 
 of sunlight , an average water depth of only about 1.5 m , low tss concentrations 
 ( 1015 mg / l ) , and a hrt of about 4.7 days made the constructed 
 wetland a location of potential photolysis of neonicotinoids . imidacloprid 
 concentrations entering and leaving the engineered wetland after 5 
 days were 54.4  3.4 and 49.9  14.6 ng / l , respectively , 
 with corresponding mass loading and discharge of 15.1  0.9 and 
 11.4  3.3 g / day . though lab studies have shown that the photolysis 
 half - life of imidacloprid in water is less than 1 day , no significant removal of imidacloprid was observed . 
 during the sampling period ( 5 days ) , average concentrations of imidacloprid 
 entering and leaving the engineered wetland were 48.2  4.8 and 
 41.5  11.5 ng / l , respectively ; the corresponding average daily 
 mass loading and output values were 13.6  1.1 and 10.2  
 2.7 g / day . thus , no significant removal of imidacloprid was observed 
 in the wetland regardless of whether average concentrations or daily 
 concentrations off set by the hrt were compared . similar results were 
 found for acetamiprid and acetamiprid - n - desmethyl 
 ( figure s3 ) . notable changes in loading 
 of clothianidin made it impossible to draw any firm conclusions about 
 potential losses in the wetland ( table s3 ) . considering the high toxicity of neonicotinoids 
 to aquatic communities at low concentrations , it is necessary to consider 
 wwtp effluent as a source of pesticides to the environment . therefore , 
 to better define the discharge of neonicotinoids into united states 
 surface waters nationwide and to confirm that the observed behavior 
 is not plant specific , composite wastewater samples were collected 
 from 12 united states wwtps between january and december 2015 and 
 analyzed . the wwtps analyzed were located in the western ( n = 4 ) , southern ( n = 6 ) , and midwestern 
 ( n = 2 ) regions of the united states , featuring diverse 
 microbial communities , suspended solids , sludge age , and hydraulic 
 retention time . influent and effluent concentrations ( figure 2a ) of neonicotinoids coincided 
 with the conducted mass balance . facilities 2 , 5 , and 12 performed 
 tertiary treatment by filtration , and facilities 2 , 6 , and 12 performed 
 uv disinfection instead of chlorination . all other facilities performed 
 conventional treatment , i.e. , secondary treatment followed by chlorine 
 disinfection . regardless of treatment strategy investigated , neonicotinoids 
 persisted in each case without notable differences . the average concentrations 
 discharged ( including information on minimum , maximum , and median 
 values in ng / l as well as detection frequency ) were 62.6 ng / l ( 18.5 , 
 146.4 , 52.7 , 100% ) for imidacloprid , 1.9 ng / l ( 0.6 , 5.7 , 1.3 , 67% ) 
 for acetamiprid , and 12.1 ng / l ( 9.9 , 13.4 , 12.5 , 33% ) for clothianidin . thiamethoxam , thiacloprid , and dinotefuran were not detected in any 
 of the samples examined , with mdls of 0.3 , 0.1 , and 32.6 ng / l , respectively . on the basis of the detected concentration of neonicotinoids in influent 
 and the population served by the studied treatment facilities , the 
 total neonicotinoid annual loading in sewage is estimated to range 
 from 3.1 to 10.7 mg / person / y , a value reflecting both known domestic 
 and unknown agricultural insecticide uses in the respective sewersheds . accordingly , the mass of neonicotinoids discharged into united states 
 surface waters nationwide is estimated to be on the order of approximately 
 1.03.4 t of imidacloprid [ united states population is considered 
 318.9 million ( 2014 ) ( source : united states census bureau ) ] . no estimates 
 are provided for acetamiprid and clothianidin here because of low 
 concentrations ( < 10 ng / l ) and relatively low detection frequencies . 
 the nationwide estimate provided here could be improved upon by future 
 studies featuring a larger number of seasonal samples taken at a greater 
 number of plants . imidacloprid concentrations detected in 12 united states 
 wastewater 
 treatment plants ( a ) ; for wwtps 710 (  ) , only effluent 
 was analyzed . also shown is a comparison of published ecological toxicity 
 benchmark values for chronic and acute exposure ( red dotted lines ) 
 with discharged effluent concentration of imidacloprid at different 
 times of year ( b ) . appropriate in - stream dilution factors for receiving 
 surface water bodies need to be considered for risk assessment and 
 may be as small as unity in effluent - dominated streams . the international regulatory framework for neonicotinoids 
 is still 
 immature . in the united states , there currently are no binding regulations 
 in place for neonicotinoid residues in treated wastewater . the dutch 
 government has established maximum permissible risk threshold levels 
 for ecosystems ranging from 8 to 13 ng / l , and other published ecological reference values for aquatic invertebrates are 
 about 3040 ng / l . the imidacloprid concentrations in discharged 
 treated wastewater established in this study ( 18.5146.4 ng / l ) 
 exceed the above - mentioned thresholds ( figure 2b ) . risk posed by wastewater - borne neonicotinoids 
 will be most pervasive in situations where the discharge receiving 
 stream is effluent - dominated , as is the case in the southern locations 
 examined here . fate of discharged neonicotinoids will be influenced 
 by vegetation downstream , water depth , and ph , among other factors . in this study , the fate of the discharged neonicotinoids 
 was traced with a comprehensive sampling campaign at one wwtp only , 
 and significant persistence was observed . whether wwtp effluent - borne 
 neonicotinoids pose related threats to plants and wildlife in wetlands 
 and aquatic ecosystems downstream of wwtp discharge locations is currently 
 unknown and deserves further study . aside from posing direct toxicity 
 to aquatic species , these systemic pesticides also can be taken up 
 by plants and circulated throughout the plant tissues ; this represents a potential pathway for exposure of pollinator 
 species and other susceptible , nontarget organisms upon accumulation 
 of insecticide mass in pollen and nectar . during this one time sampling event at each facility , it was observed 
 that relatively higher concentrations were discharged in the period 
 of june to november when compared to the december to may time frame ; 
 however , regional time series analysis is required to confirm and 
 elucidate this phenomenon . ng / l ) during sampling for 5 consecutive days ( not coinciding 
 with seasonal pesticide applications in the region ) and 100% detection 
 frequency at various locations throughout the year suggest that nonagricultural 
 neonicotinoid uses also should be considered as contributors . neonicotinoids 
 have been detected in urine samples of japanese adults and children 
 without occupational spraying histories , suggesting exposure from 
 daily lives and consumables . in recent years , nonagricultural 
 applications of neonicotinoids have expanded . some of the best selling 
 canine and feline flea control products in united states contain around 
 10% imidacloprid as an active ingredient . neonicotinoids also are being used 
 in household sectors as fly bait , roach bait , and ant bait , and to 
 eradicate bed bugs . however , lack of inventory and application 
 rate for such nonagricultural usage of these active ingredients is 
 a major knowledge gap to study their contribution , transport , and 
 impact on nontarget organisms . in summary , the present work adds much needed data to the occurrences 
 and fates of neonicotinoid pesticides in the built water environment . imidacloprid , thiamethoxam , clothianidin , and acetamiprid are frequently 
 detected neonicotinoids in global surface waters . according to 
 a recent study , 74% of global surface waters exhibited concentrations 
 of individual neonicotinoids exceeding 35 ng / l ( n = 17 ) . yet , the role of real - world , conventional 
 pollution control infrastructure in attenuating sewage - borne neonicotinoids 
 was until now ill defined . this study adds to the present state 
 of knowledge by furnishing 
 the first mass balance for three neonicotinoids  namely , imidacloprid , 
 acetamiprid and clothianidin  in a full - scale , conventional 
 wastewater treatment plant and constructed wetland in the united states , 
 using previously established methods to obtain reliable data . adding to prior fate studies including a recent nationwide assessment 
 of neonicotinoids in united states streams , we here provide the first nationwide reconnaissance on the occurrence 
 and fate of neonicotinoid insecticides during wastewater treatment . acetamiprid - n - desmethyl was identified as a major 
 degradate formed during activated sludge treatment . the present work 
 establishes the presence of neonicotinoids in urban sewersheds , demonstrates 
 significant recalcitrance of these compounds during conventional and 
 advanced wastewater treatment , and indicates risk to the effluent - dominated 
 ecosystems . an order - of - magnitude estimate of the discharge load to 
 surface waters in the united states indicates that successful management 
 of risks posed by neonicotinoid compounds will have to take sewage 
 sources into consideration , even for urban , nonagricultural geographical 
 settings .
occurrence 
 and removal of six high - production high - volume neonicotinoids 
 was investigated in 13 conventional wastewater treatment plants ( wwtps ) 
 and one engineered wetland . 
 flow - weighted daily composites were analyzed 
 by isotope dilution liquid chromatography tandem mass spectrometry , 
 revealing the occurrence of imidacloprid , acetamiprid , and clothianidin 
 at ng / l concentrations in wwtp influent ( 60.5  40.0 ; 2.9  
 1.9 ; 149.7  289.5 , respectively ) and effluent ( 58.5  29.1 ; 
 2.3  1.4 ; 70.2  121.8 , respectively ) . 
 a mass balance showed 
 insignificant removal of imidacloprid ( p = 0.09 , 
 ci = 95% ) and limited removal of the sum of acetamiprid and its degradate , 
 acetamiprid - n - desmethyl ( 18  4% , p = 0.01 , ci = 95% ) . 
 clothianidin was found only intermittently , whereas 
 thiamethoxam , thiacloprid , and dinotefuran were never detected . in 
 the wetland , no removal of imidacloprid or acetamiprid was observed . 
 extrapolation of data from 13 wwtps to the nation as a whole suggests 
 annual discharges on the order of 10003400 kg / y of imidacloprid 
 contained in treated effluent to surface waters nationwide . 
 this first 
 mass balance and first united states nationwide wastewater reconnaissance 
 identified imidacloprid , acetamiprid , and clothianidin as recalcitrant 
 sewage constituents that persist through wastewater treatment to enter 
 water bodies at significant loadings , potentially harmful to sensitive 
 aquatic invertebrates .