fipronil is a phenylpyrazole 
 insecticide used in a variety of pest 
 control products , including seed coatings , roach and ant bait , flea 
 and tick topical treatments , and various termiticide formulations . 
 incomplete transformation of fipronil is known to yield several degradation 
 products of similar or higher toxicity . fipronil is known to undergo biotic oxidation to yield fipronil 
 sulfone and reduction to form fipronil sulfide ; these are generally 
 the two most common environmental fipronil degradates . a pair of minor 
 environmental transformation products are fipronil amide formed during 
 hydrolysis and fipronil  desulfinyl produced during photolysis . fipronil application to rice paddies has been 
 directly implicated in the sharp decline in crawfish populations in 
 southern louisiana . fipronil and its degradates also 
 are toxic to nontarget vertebrates , including fish and gallinaceous 
 birds . fipronil has been implicated as 
 a potential contributor to colony collapse disorder of honeybee populations . with lethal dosages ( ld50 ) of 413 ng / bee , fipronil is extremely toxic to honeybees , which play a critical 
 ecosystem function and also provide an added economic value to the 
 united states crop industry estimated at $ 514 billion per 
 year . due in part to its likely role 
 in pollinator poisoning and its effects on aquatic wildlife , china 
 placed heavy restrictions on use of fipronil starting in 2009 , and the european union followed suit in 2013 . as a result of its widespread use , fipronil 
 has been detected in 
 urban waterways and in rural rivers . in a survey 
 of urban waters in orange county , california , fipronil and fipronil . in another 
 study of fipronil contamination in the mermentau and calcasieu river 
 basins in the united states , fipronil , fipronil sulfide , and fipronil 
 sulfone were detected in 78.0 , 90.0 , and 81.7% of surveyed samples , 
 respectively . these compounds were also 
 shown to have accumulated in sediments in the same area ( 100% detection 
 frequency ) . fipronil , like other neurotoxic 
 insecticides ( e.g. , the neonicotinoid compound imidacloprid ) , has 
 been linked to wildlife population declines , with a notable impact 
 on biological diversity . numerous studies 
 have investigated fipronil impacts on copepods , fish , gallinaceous birds , and reptiles . among 
 the suspected sources of fipronil contamination are agricultural runoff , urban runoff , and 
 treated wastewater . mass - spectrometry and mass - balance assessments 
 along with flow data logging are valuable tools 
 for studying the fate of recalcitrant anthropogenic pollutants , including 
 pesticides , herbicides , and biocides of agricultural and domestic 
 uses . prior to the present work , only 
 a single study employed a mass - balance approach to investigate the 
 fate of fipronil during wastewater treatment , reporting a removal 
 efficiency of 18  22% ; the large margin of error prevented any 
 firm conclusions as to whether fipronil was removed at all , and major 
 transformation products were not monitored in this prior work . it is interesting to note that partial or complete 
 loss of fipronil during wastewater treatment does not necessarily 
 imply a reduction of the total toxicity of the sum of fipronil - related 
 compounds , given that transformation of the parental pesticide may 
 give rise to equally or even more potent toxic degradates . the 
 primary objective of this study was , therefore , to assess the 
 fate of parental fipronil and its major degradates ( i.e. , fipronil 
 sulfide , fipronil sulfone , fipronil amide , and fipronil  desulfinyl ) 
 in a large wastewater - treatment plant ( wwtp ) by performing mass balances 
 for various conventional treatment unit operations and for a constructed 
 wetland located immediately downstream . analytical grade solvents ( water 
 and acetonitrile ) were obtained from thermo fisher scientific ( waltham , 
 ma ) and emd millipore ( billerica , ma ) . neat analytical standards of 
 fipronil and fipronil  desulfinyl were purchased from sigma - aldrich 
 ( st . louis , mo ) , and neat standards of fipronil sulfide , sulfone , 
 and amide were produced by bayer and basf ( ludwigshafen , germany ) . isotopically labeled fipronil ( c2n2-fipronil ) was 
 purchased from toronto research chemicals , incorporated ( toronto , 
 ontario canada ) . we systematically assessed 
 the fipronil compound reduction capability of one representative treatment 
 train as well as the entire treatment plant and a constructed wetland 
 located downstream . automatic samplers were deployed at the following 
 locations along the treatment train to capture primary influent , primary 
 effluent , secondary effluent , return - activated sludge , disinfection - basin 
 effluent , wetland influent , and wetland effluent . sampling was carried out in mid december 
 over five consecutive days , from 12 pm on thursday through 12 pm the 
 following tuesday . the isco 6700 and 6712 samplers ( teledyne technologies , 
 thousand oaks , ca ) were programmed for flow - weighted composite sampling . 
 to obtain flow - weighted composites the fraction of the total composite 
 volume sampled any given hour was proportionate to the deviation from 
 daily average flow into the plant ( as determined by hourly flow data 
 over a period of 21 days ) . more details on sampler programming can 
 be found in the figure s1 . at 12 pm each 
 day , the composite from the prior day was replaced with an empty 2.5 
 l amber bottle . primary sludge was sampled once per day at 9 am using 
 a 1 l bottle . biosolids were taken as grab samples in 40 ml glass 
 vials , starting 21 days after the first day of the water sampling 
 campaign , to account for the solids retention time in the anaerobic 
 digesters . aliquots ( 10 ml ) of refrigerated , 
 homogenized water samples were transferred to 15 ml centrifuge tubes 
 and were subsequently centrifuged at 3500 g . the remaining solids were dried , 
 weighed , spiked with 20 ng labeled fipronil , extracted with 10 ml 
 of acetone at room temperature via placement on a rotary shaker operated 
 at 60 rpm for 24 h. the extraction mixture was centrifuged again , 
 and the solvent was collected in a glass vial . after a second extraction 
 with 10 ml of acetone , the serial extracts were combined , evaporated 
 under nitrogen to near dryness , and reconstituted with 6 ml of hexane . sample cleanup was done using 1g/6 ml sep - pak ( waters corporation , 
 milford , ma ) cartridges containing florisil . the cartridges were conditioned 
 with 6 ml dichloromethane , 6 ml acetone , and 6 ml of hexane before 
 the samples were loaded . once loaded , the cartridges were dried under 
 vacuum and exhaustively eluted with dichloromethane and acetone ( 1:1 
 v / v ) . total suspended solids ( tss ) for each stream was determined 
 by dividing the solids mass of the samples described above by the 
 10 ml wet volume . fipronil 
 compounds were 
 extracted from 500 ml aliquots of wastewater and wetland water ( in 
 duplicate for all streams except primary sludge ) using automated , 
 high - volume solid - phase extraction . extraction was carried out using 
 cartridges containing polystyrene divinylbenzene resin modified with 
 pyrrolidone ( 500 mg/3 ml strata x and strata xl , phenomenex , torrance , 
 ca ) installed on an autotrace 280 ( thermo scientific dionex , sunnydale , 
 ca ) . the resin was eluted with 5% formic acid in 
 methanol , and then aliquots of these extracts were reconstituted to 
 either 50% methanol in water ( for lc analysis ) or 100% hexane ( for 
 gc analysis ) . water samples with high tss , such as waste - activated 
 sludge ( was ) and primary sludge ( ps ) were centrifuged at 7500 g , and 500 ml of the supernatants was decanted and extracted 
 as described . analyte mass on the solid fraction of those streams 
 was determined as described in the previous section , and the weighted 
 mass contribution of the solids was added to that of the water to 
 determine the total mass of fipronil compounds in was and ps . all analytes except fipronil desulfinyl 
 were separated by liquid chromatography and detected and quantified 
 by negative electrospray ionization  tandem mass spectrometry 
 ( lc  esi desulfinyl displayed 
 a significantly lower detection limit by gas chromatography electron 
 impact  tandem mass spectrometry ( gc  ei  ms / ms ) 
 and was therefore analyzed using a gc liquid 
 chromatography mass spectrometric analyses were done using a shimadzu 
 prominence hplc ( shimadzu scientific , kyoto , japan ) controlled by 
 analyst 1.5 software ( applied biosystems , framingham , ma ) coupled 
 to an absciex api-4000 ms / ms ( applied biosystems , framingham , ma ) . liquid chromatographic separation was achieved by an xbridge c8-column ( 3.5 m particle size , 4.6  150 mm ; waters 
 corporation , milford , ma ) . the mobile phase consisted of 50% acetonitrile 
 ( acn ) and 50% water flowing at a rate of 1 ml / min with a total runtime 
 of 10 min , and a gradient profile of 10% acn / min to 95% . analytes 
 were introduced into the mass spectrometer using an electrospray ionization 
 probe operating in negative mode , and multiple reaction monitoring 
 ( mrm ) was used for qualitative analysis . optimized conditions for 
 the ionization and a fragmentation of the analytes are specified in 
 the supporting information . quantitation 
 of fipronil was done using isotope dilution and an eight - point calibration 
 curve , with matrix spikes using c2n2fipronil . quantitation of other 
 analytes was done using the standard addition method with four analysis 
 sample spike levels . gas chromatographic mass spectrometric analyses 
 were performed on an agilent 7890 gc coupled to an agilent 7000 triple 
 quad ms ( agilent technologies , santa clara , ca ) operating in positive 
 mode , and mrm was used for qualitative analysis . more details on analytical 
 instrument parameters and quality control , including limit of detection 
 determination , can be found in the supporting information . method 
 detection limits 
 were determined by analyzing seven spiked surrogate matrix replicates 
 and employing the usepa s recommended analysis for determination 
 of limits of detection . solid and water aliquots were spiked with 
 authentic standards for fipronil and its major degradates prior to 
 extraction . spiking levels for each analyte were chosen within a ratio 
 of 3:1 and 10:1 relative to background levels , and the detected concentrations 
 were estimated using a six - point calibration curve . the standard deviation 
 using six degrees of freedom was multiplied by the appropriate student s t99 value , providing an estimate of the lowest 
 concentration detectable and identifiable with 99% confidence . because all samples of wastewater and archived sludge exhibited peaks 
 reflective of the presence of fipronil , surrogate matrices void of 
 fipronil compounds were obtained in the form of peat moss and peat 
 moss slurry . this selection was made in accordance with usepa method 
 1694 , which recommends use of this surrogate matrix as a proxy for 
 biosolids for quality assurance in the absence of an analyte - free 
 reference matrix . automatic samplers 
 were programmed to 
 take a number of 20 ml incremental samples within the first few minutes 
 of a given hour . the number of 20 ml increment samples taken in a given 
 hour was calculated using eq 1.1where n20  ml(t ) is the number 
 of 20 ml increments in the first 
 few minutes of a given hour t , q(t ) is the measured flow rate at hour t , and q is the average daily flow rate over the course 
 of 21 days . mass loads for fipronil compounds in process streams 
 were determined by multiplying determined concentrations with the 
 flow rates for corresponding days . a combination of daily average 
 flows ( 12 am to 12 am ) and monitored hourly flows is reported ( see supporting information ) . applying a steady - state 
 assumption ( accumulation = 0 ) , we calculated 
 the mass balance over the treatment train as shown in eq 2.2 the bracketed terms 
 ( primary influent , etc . ) represent the total 
 mass load through each respective stream over a five day period , where q is flow rate ( l / d ) , c is concentration 
 ( ng / l ) , t is time ( day ) , f is the 
 mass fraction of solids in a stream ( gsolid / gwastewater ) , and mconverted is the mass not accounted 
 for in all influent and effluent streams , assumed to be transformed 
 ( ng ) . the notations 1inf , dieff , ps , and was refer , respectively , 
 to primary influent , disinfection basin effluent , primary sludge , 
 and waste activated sludge . individual analyte 
 masses were first converted to mmol before being added together to 
 compute a total concentration expressed in fipronil equivalents . the 
 flow rate of was was not directly measured but was instead obtained 
 by subtracting the return activated sludge ( ras ) flow rate from the 
 secondary effluent flow rate . eq 3 illustrates the 
 method for performing a mass balance over the wastewater - treatment 
 plant , accounting for total influent and effluent streams , and biosolids . the total plant - influent mass was estimated using the product of the 
 treatment train primary influent concentration ( c1inf ) and total plant influent flow rate ( qtot ) . the effluent streams from the plant were 
 directed to the downstream wetland ( wl ) and a power plant ( pp ) . the 
 biosolids , or dewatered sludge ( dws ) , concentrations were given in 
 units of g / g , and dws production rates were expressed in units 
 of mass per day ( g / d ) . the total mass of fipronil compounds converted 
 ( mtotal_converted ) represents the mass 
 of fipronil compounds that presumably reacted or degraded during treatment 
 through the entire plant.3 eq 4 was used to calculate 
 the conversion of fipronil compounds in the wetland.4 it should be noted that an 
 effluent stream feeding a nearby power 
 plant was not directly sampled , but because it was split off from 
 the plant effluent , the concentration in that stream was assumed to 
 be the same as the concentration in the plant effluent . total analyte 
 masses and concentrations were converted to fipronil equivalents by 
 multiplying them by the relative molar mass of fipronil . eq 5 was used to calculate 
 the species - specific hazard quotient ( hqx ) of the influent and effluent wastewater streams , 
 using methods established in literature . ( stark and banks ) there are 
 three fipronil degradates ( fipronil , fipronil sulfone , and fipronil 
 sulfide ) accounted for in the calculation , indicated in the equation 
 by i. influent or effluent stream concentrations 
 are indicated by cstream . the hqs of the 
 influent and effluent streams were then compared to determine whether 
 treatment affects the toxicity of wastewater.5 toxicity indices were calculated for two arthropod 
 species , hyalella azteca and chironomus dilutus , using the half - maximal effective 
 concentrations ( ec50 ) for the various analytes . these species 
 were chosen due to their 
 relative sensitivities to fipronil ; h. azteca is moderately sensitive , and c. dilutus is highly sensitive . the c. dilutus ec50 values used in this calculation were 32.5 and 10 
 ng / l for fipronil and its degradates , respectively . the h. azteca ec50 values used in this calculation were 0740 , 161 , and 0.5 ng / l for 
 fipronil , fipronil sulfone , and fipronil sulfide , respectively . detection limits in surrogate wastewater 
 ranged from 0.05 to 0.77 ng / l , while for surrogate biosolids , they 
 ranged from 0.02 to 0.24 ng / g ( dry weight ) . relative recovery of fipronil 
 was 116  14% in water and 120  13% in solids . absolute 
 recoveries of individual analytes ( prior to normalization with the 
 respective isotope - labeled surrogate standards ) from water samples 
 ranged from 60  14% to 101  195% ( overall average recoveries 
 for all analytes was 78  20% ) , while absolute recoveries of 
 individual analytes from solid samples ranged from 48  18% to 
 90  21% ( overall average recoveries for all analytes was 73 
  28% ) . all water and solids samples were spiked with 20 
 ng of labeled fipronil prior to extraction , and final fipronil concentrations 
 were quantified using the isotope - dilution method . other analyte concentrations 
 were assessed using standard addition with either three or four calibration 
 points generated from sample extracts spiked just prior to instrument 
 analysis . method development indicated that nearly all losses were 
 due to matrix effects , and standard addition and isotope dilution 
 proved to mitigate the quantitative effects of these losses . all samples 
 were quantified by background subtraction of method blank controls . in the wastewater treatment 
 train selected for extensive monitoring , fipronil was present in raw 
 sewage at an average daily concentration of 17 to 31 ng / l and exited 
 in disinfected treated effluent at levels of 13 to 21 ng / l . fipronil 
 sulfone was detected in all process streams at concentrations ranging 
 from 0.5 to 40 ng / l . the sulfide , amide , and desulfinyl degradates 
 were detected in most wwtp process streams at low levels : 0.13.8 a mass balance of total fipronil through the treatment 
 train indicated that as a group , fipronil and its immediate degradates 
 were conserved throughout . a 5 day mass load of total fipronil entering 
 and exiting the treatment train yielded 77  11 and 69  
 6 mmol/5 days , respectively ; mass loads in primary and secondary effluent 
 were similar to those in the primary influent stream , suggesting conveyance 
 of the contaminants through the treatment train ( figure 1 ) . overlapping error bars and 
 a two - tailed t - test ( 95% confidence level ) revealed 
 that the mean daily influent and effluent masses of total fipronil 
 compounds were statistically indistinguishable ( p = 0.29 , n = 10 ) , implying that conventional wastewater 
 treatment is ineffective at converting fipronil beyond the four immediate 
 degradates studied herein ( sulfone , sulfide , amide , and desulfinyl ) . limited settling of fipronil compounds occurred in the primary and 
 secondary clarifiers , despite their considerably high logarithmic n - octanol  water partitioning coefficients ( log kow  4.05.4 ) . while total fipronil compounds experienced no appreciable mass loss 
 during passage through the treatment train , the parent compound fipronil 
 was transformed at a rate of approximately 25% ( p = 0.00025 , n = 10 ) , with about 1% being removed 
 from water by the solids in waste activated and primary sludge . the 
 decreased fipronil mass in the treatment train was accounted for in 
 the total mass of the degradates , primarily fipronil sulfone ( figure 2 ) . this result is 
 in agreement with and refines prior estimates from a 2009 study , in 
 which fipronil was found to be removed from a similar u.s . conventional 
 wastewater - treatment plant at a rate of 18  22% ; the considerable 
 analytical error in that study did not allow the unambiguous identification 
 of differences between influent and effluent concentrations , and unlike 
 in the current study , neither a detailed analysis of fipronil s 
 degradates nor the effectiveness of individual unit operations was 
 undertaken . flow diagram of the wastewater - treatment 
 train . labeled streams 
 i , ii , iii , iv , v , and vi indicate primary influent , primary effluent , 
 primary sludge , waste - activated sludge , secondary effluent , and disinfection 
 basin effluent , respectively . total fipronil - related compound mass 
 loads ( in mmol/5 days ) for the sampled streams are given ( n = 2 experimental replicates per composite ) . the primary 
 sludge ( stream ( iii ) was taken as a 1 l grab sample each day during 
 the 5 day sampling period , which yielded only one experimental replicate 
 and only one 5 day sum , so no error is given . biosolids were sampled 
 21 days after the water sampling campaign began to account for the 
 solids retention time in the anaerobic digesters . hw , headworks ; gc , grit chamber ; pc , primary clarifiers ; ab , aeration 
 basins ; sc , secondary clarifiers ; di , disinfection basin ; ad , anaerobic 
 digesters , centrifuges , and dewatering systems . ( a ) mass loads ( in mmol ) in wastewater streams over the course 
 of 5 days . direction of water flow is from left to right ( primary 
 influent to disinfection basin effluent ) . ( b ) enlarged portions 
 of the histogram in panel a to make visible the masses of fipronil  desulfinyl . sludge streams ( n = 2 ) are omitted because 
 their mass contributions are negligible . approximately 58% of the flow and 48% of the total 
 fipronil mass discharged by the wastewater facility was directed to 
 an engineered wetland located immediately downstream , whereas 43% 
 of total fipronil mass was distributed to a power plant , and 9% was 
 sequestered in biosolids . the average daily mass loads of total fipronil 
 in the wwtp inputs and outputs were 33.2  5.6 mmol / day and 37.6 
  7.3 mmol / day , respectively ( see figure 3 , panel a ) . similar to the individual treatment 
 train , the daily mean input and output masses of the entire wwtp were 
 not significantly different ( n = 10 , p = 0.14 ) , indicating a complete lack of , or only insignificant removal 
 of , total fipronil . the computed error in reported masses is cumulative , 
 accounting for variability of calibration in flow meters used to measure 
 flow rates , of recovery rates during extraction , of estimated solids 
 retention time of anaerobic digesters , and of instrument response . ( a ) average 
 daily mass loads over 5 days , where error bars represent 
 standard deviations ( n = 10 ) . ( b ) daily mass loads 
 of wetland ( wl ) influent and effluent streams on days 1 and 5 , respectively , 
 where error bars represent maximum and minimum values ( n = 2 ) ; the average hydraulic retention time of the wetland was 4.7 
 days . the right - hand y - axis is expressed as grams 
 of fipronil per day . relative abundance of fipronil - related compounds in input 
 and output 
 streams underwent little change . the input stream composition was 
 approximately 75% fipronil , 1% fipronil sulfide , 2122% fipronil 
 sulfone , 04% fipronil amide , and 12% fipronil  desulfinyl . however , the mass ratio of sulfone degradate to parental fipronil 
 in waste - activated sludge was about 0.74 , whereas in primary influent , 
 the same ratio was much lower at about 0.3 ; this implies that fipronil 
 sulfone was formed in either the aeration basins or in the secondary 
 clarifiers . if the solids retention time in the clarifiers enabled 
 the conversion of fipronil to fipronil sulfone , then this pattern 
 should also be seen in the primary sludge , but it was not ( sulfone / parent 
 ratio = 0.14 ) . considering that fipronil sulfone is an oxidative byproduct 
 of fipronil , the evidence suggests that the sulfone degradate was 
 formed during aerobic digestion . aerobic transformation is the most 
 likely mechanism for sulfone formation , given that the only unit operations 
 in secondary treatment are aerobic digestors and secondary clarifers ; 
 however , anaerobic microenvironments also may exist within this treatment 
 unit . the primary sedimentation step did not appear to contribute 
 to the formation of fipronil sulfone , and aerobic basins are more 
 prone to facilitate enzymatic oxidation . fipronil sulfone did 
 not appear to form at appreciable levels during 
 chlorination , a result running counter to findings from a prior study 
 examining the chemical oxidative removal of fipronil in water - treatment 
 plants ( using permanganate , chlorine , etc . ) ; the latter study identified 
 fipronil sulfone as the predominant byproduct of fipronil transformation 
 by chlorine . however , the chlorination 
 basin in the wastewater treatment train examined here had a contact 
 time of less than a minute , with a dose of 2.5 mg / l , and a residual 
 concentration of about 1 mg / secondary - treated wastewater has a 
 high chlorine demand , so it is not surprising that fipronil was not 
 easily oxidized . in biosolids , the proportions of the individual 
 anaytes were roughly 
 as follows : 15% fipronil , 65% fipronil sulfide , 9% fipronil sulfone , 
 1% fipronil amide , and 9% fipronil  desulfinyl . the dominant 
 species in biosolids was the sulfide degradate , a result that is consistent 
 with a study examining the degradation of fipronil in anaerobic sediment 
 porewater . in all water streams , 
 the primary photodegradate fipronil desulfinyl 
 accounted for less than 3% of the total fipronil compound mass . in 
 the wetland , fipronil  desulfinyl accounts for less than 5% 
 of the fipronil compound mass loss . this might seem surprising because 
 fipronil has been shown to readily photodegrade , but usgs surface 
 water screenings confirm that fipronil desulfinyl is considerably 
 less abundant in environmental waters than the parent , the sulfone , 
 or the sulfide transformation products . the wetland downstream of the 
 wwtp had a hydraulic retention time ( hrt ) of about 4.7 days , and so 
 the mass load into the wetland on the first day of sampling should 
 correspond with the mass load out of the wetland 4 to 5 days later . a mass balance on the wetland ( figure 3b ) using the first day s influent mass load 
 and the fifth day s effluent mass load indicates that fipronil 
 was reduced at a rate of 44  4% , and total fipronil was attenuated 
 in the wetland at a rate of 47  13% . over the five - day period , the average effluent concentrations of total fipronil were about 24% 
 lower than the influent concentrations ( n = 10 , p = 210 ) . the discrepancy between 
 mass and concentration changes can be accounted for by evapotranspiration 
 ( the effluent flow rate is about 87% of the influent flow rate ) and 
 daily mass - load deviations over the five - day period not captured by 
 the mass balance ( the wetland mass balance only uses the first and 
 fifth day mass loads to account for the wetland s hydraulic 
 retention time , while the average concentration over 5 days accounts 
 for all 5 days of sampling , wherein concentration fluctuations occurred ) . total fipronil compound levels detected in the wetland ranged from 
 6141 ng / l . these concentrations are much lower than concentrations 
 detected in california urban waterways , where median total fipronil 
 concentrations ranged from 204440 ng / l and 90th percentile 
 concentrations ranged from 3401170 ng / l . data sets from the united states geological survey in several 
 states indicate that concentrations of fipronil - related compounds 
 in urban and agricultural runoff are generally similar and typically 
 less than 200 ng / l . in louisiana rice - field 
 runoff , losses of 2444% of total fipronil mass during 
 passage of water through the wetland suggests that further attenuation 
 may take place in the effluent receiving stream ; however , a deeper 
 water column and higher turbidity may hinder the effectiveness of 
 some loss mechanisms , such as photolysis . to assess whether the fipronil - related 
 toxicity was affected by treatment , we calculated hazard quotients 
 for process streams in the studied treatment train , including primary 
 influent , disinfection basin effluent , wetland influent , and wetland 
 effluent . for the moderately sensitive species , h. 
 azteca , these values were 0.072  0.014 , 0.23 
  0.14 , 0.18  0.07 , and 0.22  0.09 , respectively . the mean hq ( h. azteca ) of the primary 
 influent stream was compared with the effluent from disinfection , 
 the wetland influent , and the wetland effluent using a two - tailed t - test ( n = 10 ) assuming equal variances ; p values for these analyses were 0.002 , 0.0001 , and 0.00006 , 
 respectively . for the highly sensitive organism c. 
 dilutus , the primary influent , disinfection basin 
 effluent , wetland influent , and wetland effluent hqs were 1.4  
 0.3 , 1.3  0.5 , 1.3  0.4 , and 1.0  0.3 , respectively . testing for statistical differences in the means of the hqs of disinfected 
 effluent , wetland influent , and wetland effluent process streams relative 
 to the primary influent stream yielded p values of 
 0.8 , 0.5 , and 0.007 , respectively . thus , conventional wastewater treatment 
 did not significantly affect the overall toxicity posed by fipronil 
 related compounds to c. dilutus , but 
 the fipronil - associated toxicity was increased toward h. azteca due to the formation of fipronil sulfide , 
 to which h. azteca is highly sensitive . passage of water through the wetland reduced the threat posed by fipronil 
 related compounds for c. dilutus but 
 not for h. azteca . these hq values 
 indicate that treated and untreated wastewater streams are probably 
 toxic to highly sensitive organisms and potentially toxic to moderately 
 sensitive organisms . the wastewater 
 treatment plant in this study discharges an estimated 7.9 g / day of 
 pheneylpyrazole pesticide mass ( as fipronil ) into the wetland , with 
 3460% estimated to be attenuated there . to what extent fipronil 
 and its degradates are taken up by plant and animal life is not well 
 understood and likely varies by degradation and exposed species . fipronil 
 manufacturers recommend no more than 0.050 lb ( 23 g ) of active ingredient 
 to be applied annually per acre of land for varied uses such as mole 
 cricket control . the water exiting the wetland discharges an estimated 
 total fipronil load of 5.2 g / day . biosolids produced by the treatment 
 plant contribute a total fipronil - related compound load of 1.4  
 0.7 g / day , mostly in the form of fipronil sulfide . considering that 
 the toxic load inherent to total fipronil compounds was left essentially 
 unattenuated upon conventional wastewater treatment , the next best 
 opportunity to control harmful exposures of aquatic biota and ecosystems 
 is to limit use and loading of raw wastewater with the parent pesticide , 
 fipronil . although mechanisms of fiprole toxicity to ecosystems were 
 not evaluated here , it has been demonstrated that fipronil can be 
 taken up by angiosperms , transported through their xylem , and deposited 
 on pollen and seedlings . bees and other pollinating insects 
 may be exposed to fipronil upon direct application via treated seeds 
 and potentially upon application of biosolids on land used to grow 
 flowering plants . supportive of risks stemming from direct pesticide 
 application , one survey in treated sunflower fields in france showed 
 levels of total fipronil - related compounds in pollen as high as 8.3 
 ng / g . it is unclear how wastewater 
 contributes to fipronil loads detectable in angiosperm pollen , body 
 burdens of aquatic organisms , or to toxicological effects in other 
 nontarget organisms . further research is needed to determine whether 
 and to what extent fipronil related compounds contained in wastewater 
 effluents can impact plants and nontarget organisms . fipronil is among 
 the most potent insecticides on the market , with a toxicity to honeybees 
 over 6000 times greater than that of the banned pesticide ddt ( 27  000 
 versus 4.2 ng / bee ) . acute lethal doses ( ld50 ) for numerous nontarget invertebrates also are in the ng range per 
 individual organism . some studies 
 have shown that indirect exposure to certain insecticides may have 
 adverse effects on vertebrate organisms , as well . a study in madagascar 
 indicated that insectivorous lizards and birds are exposed to fipronil 
 compounds through the food chain , due to the fact that their diets 
 consisted largely of the target organism ( termites ) , and that they 
 experienced sublethal effects . to fill 
 information gaps , it would be necessary to evaluate the bioaccumulative 
 and toxic effects of fipronil at various levels of the food chain . in addition , the plants in environments impacted by sources containing 
 fipronil can be evaluated for uptake and xylem transport by extracting 
 and analyzing pollen and leaves , as described by one study in france , 
 wherein fipronil related residues were detected in 13% of randomly 
 selected pollen - load samples in honeybee hives . it is currently uncertain whether the levels released 
 into the 
 environment via wastewater effluent may cause accumulation of fipronil - related 
 compounds in sediments and aquatic flora and elicit acute toxic effects 
 in foragers or benthic organisms . the present study showed that fipronil 
 and its immediate transformation products are remarkably resilient 
 to degradation in wastewater treatment plants despite their passage 
 through multiple unit operations that potentially could facilitate 
 their removal ( e.g. , aerobic digestion , anaerobic digestion , chemical 
 oxidation ) . considering that the half - life of fipronil in water and 
 sediments is typically on the order of several days ( more than 200 
 days in the case of the sulfone , sulfide , and desulfinyl degradates ) and considering the paucity of knowledge about the ecological impacts 
 of both direct and indirect discharge of fipronil into the environment , a more extensive longitudinal study of the transport of fipronil in 
 surface waters and their fate in sediments , combined with biomonitoring 
 studies , may help to illuminate potential associations between wildlife 
 population changes and the presence of fipronil in the environment .
attenuation 
 of the pesticide fipronil and its major degradates 
 was determined during conventional wastewater treatment and wetland 
 treatment . 
 analysis of flow - weighted composite samples by liquid and 
 gas chromatography 
 tandem mass spectrometry showed fipronil 
 occurrence at 1231 ng / l in raw sewage , primary effluent , secondary 
 effluent , chlorinated effluent , and wetland effluent . 
 mean daily loads 
 of total fipronil related compounds in raw sewage and in plant effluent 
 after chlorination were statistically indistinguishable ( p = 0.29 ; n = 10 ) , whereas fipronil itself was partially 
 removed ( 25  3% ; p = 0.00025 ; n = 10 ) ; the associated loss in toxicity was balanced by the formation 
 of toxic fipronil degradates , showing conventional treatment to be 
 unfit for reducing overall toxicity . 
 in contrast to these findings 
 at the municipal wastewater treatment , both parental fipronil and 
 the sum of fipronil - related compounds were removed in the wetland 
 with efficiencies of 44  4% and 47  13% , respectively . 
 total fipronil concentrations in plant effluent ( 28  6 ng / l as fipronil ) were 
 within an order of magnitude of half - maximal effective concentrations 
 ( ec50 ) of nontarget invertebrates . 
 this is the first systematic 
 assessment of the fate of fipronil and its major degradates during 
 full - scale conventional wastewater and constructed wetland treatment .