Science of the Total Environment 439 (2012) 136–140

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The antihistamine diphenhydramine is extremely persistent in agricultural soil

Edward Topp ⁎, Mark W. Sumarah, Lyne SabourinAgriculture and Agri-Food Canada, London, ON, Canada N5V 4T3

H I G H L I G H T S

► Diphenhydramine is a widely used antihistamine drug, is found in biosolids, and in runoff from biosolids-fertilized fields.► The persistence of 14C-diphenhydramine was evaluated in soils.► Half lives ranged from 88 to 335 days. Diphenhydramine-N-oxide was the only detected transformation product.► Soil-bound residues was a major sink.

⁎ Corresponding author.E-mail address: ed.topp@agr.gc.ca (E. Topp).

0048-9697/$ – see front matter. Crown Copyright © 20http://dx.doi.org/10.1016/j.scitotenv.2012.09.033

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 August 2012Received in revised form 8 September 2012Accepted 15 September 2012Available online 11 October 2012

Keywords:DiphenhydramineDiphenhydramine-N-oxideDissipationBiosolidsAgricultural soil

The widely used antihistamine diphenhydramine is present in municipal biosolids, and is detected in runofffrom agricultural land fertilized with biosolids. In the present study the kinetics and major pathways ofdiphenhydramine dissipation in a loam, sandy loam, and clay loam soil were determined in laboratoryincubations. The time to dissipate 50% (DT50) of 14C-diphenhydramine residues at 30 °C ranged from 88±28 days in the clay loam to 335±145 days in the loam soil. Mineralization of 14C was insignificant, anddiphenhydramine-N-oxide was the only detected extractable transformation product elucidated by radioiso-tope and HPLC-MS methods. There were no significant effects of municipal biosolids on the kinetics or path-ways of removal. Overall, diphenhydramine is quite persistent in soils, and formation of non-extractablesoil-bound residues is the major mechanism of diphenhydramine dissipation.

Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction

Human medicines are now widely detected at low concentrationin surface freshwater, groundwater, and coastal saltwater; a de-velopment of significant scientific, regulatory, and public concern(Monteiro and Boxall, 2010; Kümmerer, 2009; Focazio et al., 2008;Servos et al., 2007). Potential sources of exposure include pharmaceuti-cal manufacturing facilities, landfill leachate, leaking septic systems,land application of sewage sludge (biosolids) and wastewater treat-ment effluent (Larsson et al., 2007; Carrara et al., 2008; Eggen et al.,2010). We have been evaluating the potential significance of waterdraining from agricultural land fertilized with municipal biosolids as asource of environmental exposure to pharmaceuticals (Gottschall etal., 2012; Sabourin et al., 2009; Edwards et al., 2009; Topp et al.,2008). In recent field experiments we have detected diphenhydra-mine (2-diphenylmethoxy-N,N-dimethylethanamine; Fig. 1) insurface runoff from land receiving dewatered municipal biosolids(unpublished data). Municipal biosolids in Canada contained690–781 μg diphenhydramine kg−1 dry weight (Gottschall et al.,2012; Sabourin et al., 2012). Diphenhydramine is a histamine H-1 an-tagonist, commonly used to treat respiratory and minor skin allergies,

12 Published by Elsevier B.V. All rig

as a sedative, and to alleviate coughing (Berninger et al., 2011). It isone of the most heavily used over the counter medicines, with an esti-mated 6 t consumed annually in Canada, for example (2007 estimate;McLaughlin and Belknap, 2008). Diphenhydramine is subject to hepaticmetabolism with 2–15% of parent compound excreted unchanged(Berninger et al., 2011). The drug is incompletely broken down duringthe wastewater treatment process, and was detected in 25% of surfacewater samples downstream of wastewater treatment plants, with amaximum concentration of about 45 ng L−1 (Stackelberg et al., 2004).A variety of data suggest that it may be persistent in environmentalmatrices. Diphenhydramine is detected in soils for months followingirrigation with reclaimed water (Kinney et al., 2006). There was nodetectable loss of diphenhydramine over a three year period followinga single biosolids application (Walters et al., 2010). Diphenhydraminewas detected in soil up to three years after biosolids application,confirming its persistence (Wu et al., 2010), and it was found in tillagelayer but also deeper in the soil column, indication migration of thedrug through the soil profile (Wu et al., 2010). There is very little dataavailable on potential effects of diphenhydramine at environmental con-centrations. Tissue analysis offish fromeffluent-dominated streams in theUS revealed mean diphenhydramine concentrations ranging from 1.2 to10 ng g−1 (Ramirez et al., 2009). The no observed effects concentration(NOEC) for reproduction in Daphnia magna is 0.8 μg L−1 (Berninger etal., 2011) and in a chronic toxicity test 0.12 μg L−1 (Meinertz et al., 2010).

hts reserved.

Fig. 1. Chemical structures of diphenhydramine and diphenhydramine-N-oxide.[Phenyl-14C (U)]-diphenhydramine was symmetrically labeled in one or the other ofthe phenyl rings.

Days0 20 40 60 80 100

% In

itial

rad

ioac

tivity

rec

over

ed a

s 14

CO

2

0.0

0.5

1.0

1.5

2.0

2.5 Loam Loam + LMB Sandy loam Sandy loam + LMB Clay loam Clay loam + LMB

Fig. 2. Mineralization of [phenyl-14C (U)]-diphenhydramine to 14CO2 in the presence(+ LMB) or absence of liquid municipal biosolids.

Table 2Time to dissipate half (DT ) diphenhydramine in three soils without or with supplemen-

137E. Topp et al. / Science of the Total Environment 439 (2012) 136–140

Overall, given the very large amounts of diphenhydramine used, de-tection in biosolids, detection in runoff from land receiving biosolids,and apparent persistence in environmental matrices, the purpose of thepresent study was to elucidate the kinetics of diphenhydramine dissipa-tion in agricultural soils under controlled laboratory conditions, and bymeans of radioisotope methods, the primary pathways of dissipation.

2. Materials and methods

[Phenyl-14C (U)]-diphenhydramine (Fig. 1; radioactive purity >98%;specific activity 50 mCi mmol−1) was purchased from MoravekBiochemicals (Brea, CA). The molecule is symmetrically labeled inone or the other of the phenyl rings. Unlabeled diphenhydraminewas purchased from Sigma (Oakville, ON). Diphenhydramine-N-oxide(Fig. 1) was purchased from CacheSyn (Mississauga, ON). Stock solu-tions of 14C-labeled (final radioactive concentration of 500k dpm100 μL−1) and unlabeled (1 mg mL−1) diphenhydramine were pre-pared in methanol and stored at −4 °C.

The dissipation of 14C-diphenhydraminewas determined in bulk soillaboratory incubations with three agricultural soils (loam, sandy loam,clay loam) as described in Al-Rajab et al. (2010) and Sabourin et al.(2011). Briefly, microcosms consisted of 150-mL baby-food jars incubat-ed in a sealable glass 1-L Mason jar. A scintillation vial containing 10-mLofwaterwas placed in each jar tomaintain a humid atmosphere and pre-vent desiccation of the soil. A second scintillation vial with 5-mL 1 MNaOH solution for trapping 14CO2 was also placed into each jar. Soilswere supplemented with an initial diphenhydramine concentration of1 μg g−1 and a radioactive concentration of 50,000 dpm g−1.Methanolic stock solutions were added to one-gram portions of pulver-ized air-dried soil, the solvent allowed to evaporate, and the soil thenthoroughly incorporated into 49 g (moist wet) portions of the corre-sponding soil to give a total of 50 g. Soil moisture content was gravimet-rically adjusted to 15%, and microcosms were incubated in the dark at30 °C. Periodically, 5-gram soil portionswere removed from eachmicro-cosm using a spatula and stored at −20 °C until extraction. Three agri-cultural soils (depth 0 to 20 cm) were used in the present study: aloam soil obtained from the Agriculture and Agri-Food Canada researchfarm at London ON (42°59′N, 81°15′W), a sandy loam soil obtainedfrom the Agriculture and Agri-Food Canada research farm at Delhi ON(42°51′N, 80°29′W), and a clay loam soil obtained from the EssexRegion Conservation Authority research farm at Holiday Beach ON(42°2′N, 83°3′W). Key soil properties are described in Table 1. All soilswere obtained from areas that were under sod and had never received

Table 1Key properties of the soils used in this study.

Soil Clay (%) Silt (%) Sand (%) O.M.a (%) CECb (meq 100 g−1) pH

Loam 9.2 27.6 63.2 3.7 13.5 7.5Sandy loam 3.7 7.8 88.5 1.5 5.4 6.1Clay loam 32.8 40.0 27.2 3.7 16.6 5.2

a Organic matter content.b Cation exchange capacity.

biosolids, and were sieved to a maximum particle size of 2 mm. Experi-ments undertaken to determine the effect of liquid municipal biosolids(LMB) on the dissipation of diphenhydramine involved supplementingsoils with 10% (V/V) LMB in addition to diphenhydramine. This volumeis a reasonable estimate for what would be found in the top few cm ofsoil following a commercial biosolids application. Liquid municipalbiosolids were obtained from the Adelaide wastewater treatment plantlocated in London, Ontario and had the following key properties: pH6.70; organic matter 0.2%; total N, 0.02%; total P, 0.01%.

Radioactivity in NaOH traps and solvent extracts was quantifiedwith a Beckman Coulter Model LA 6500 Liquid Scintillation Counter (Ir-vine, CA) after addition of 10 ml of scintillation cocktail. Dissipationrates for parent compounds were estimated on the basis of removal oftotal radioactivity from the extractable phase. A solvent mixtureconsisting of acetonitrilewith 0.1 NNaOH(90:10)was used for diphen-hydramine extraction. The following extraction efficiencies (expressedas % recovery; n=3) were established in preliminary experimentswith 14C-diphenhydramine supplemented soil: loam (71.5±14);sandy soil (64.6±1.3); clay loam (53.0±5.8). Mineralization rates inlaboratory experiments were determined on the basis of radioactivitycaptured in NaOH traps. Raw data analysis was conducted usingMicrosoft Professional Office Plus Excel 2010 (Microsoft Canada, Toron-to, ON). Dissipation and mineralization curves were plotted usingSigmaPlot (Version 10, Systat Software Inc., Chicago, IL). The dissipationrate constants were determined using the first-order exponential decaymodel, and the goodness of fit to a regression expressed with the coef-ficient of determination. Data in figures represent the mean and stan-dard deviation of samples from triplicate microcosms.

Parent compound and potential transformation products in extractswere resolved and detected by high performance liquid chromatographyHPLC (Agilent, Mississauga, ON) with HPLC-UV and HPLC-RD (EG&GBerthold LB509 Radioflow Detector, Berthold GMBH & Co. KG., BadWildbad, Germany). An Agilent Zorbax Eclipse Extended XDB C-18

50

tation with liquid municipal biosolids (LMB). Data are presented as mean+standarddeviation of triplicates. The coefficient of determination (r2) indicates the goodness of fitwith a first order rate model.

Treatment DT50 (days) r2

Loam 335±145 0.8277Loam+LMB 329±121 0.8385Sandy loam 137±53 0.8217Sandy loam+LMB 198±101 0.7702Clay loam 88±28 0.8432Clay loam+LMB 97±22 0.7660

Days0 20 40 60 80 100

Tot

al e

xtra

ctab

le r

adio

activ

ity (

% a

dded

)

0

20

40

60

80

LoamLoam + LMBSandy loamSandy loam + LMBClay loamClay loam + LMB

Fig. 3. Dissipation of extractable [phenyl-14C (U)]-diphenhydramine residues in thepresence (+ LMB) or absence of liquid municipal biosolids.

138 E. Topp et al. / Science of the Total Environment 439 (2012) 136–140

column (4.6 mm×250 mm, 5 μm pore size; Santa Clara, CA) was used,and the UV detector was set at 254 nm. The mobile phase consisted of80:20 methanol:20 mM Na2HPO4. With solvent delivered at1 mL min−1 the retention time of diphenhydramine was 8 min, andthe retention time of the major transformation product was 4 min.

HPLC-MS was used to confirm the purity of the diphenhydramineused for spiking soil, and for the identification of any transformation

Fig. 4. LC–MS total ion current (top panel) of a day 50 DPH soil incubation extract showing tand DPHNO (M+1=272 m/z).

products. The HPLC-MS system consisted of an Alliance 2690 HPLC/autosampler and an LCT orthogonal acceleration time-of-flight massspectrometer (Waters/Micromass). Samples were analyzed using anacetonitrile:water+0.1% formic acid gradient going from 90% H2Oto 90% acetonitrile over 20 min. Analysis was performed using a150×2 mm 4 μm, Synergy Hydro-RP column (Phenomenex) at aflow rate of 0.2 mL min−1 in positive ion mode. The MS conditionsused for analysis were as follows: capillary=3000 V; desolvationtemperature=300 °C; desolvation gas flow=450 L/h; and samplecone=20 V. Mass spectra were acquired over the range of 100to 1000 m/z using MassLynx 4.0 (Waters/Micromass). Under theconditions described, diphenhydramine and the major transforma-tion product had RTs of 13.8 and 14.5 min, respectively. Extractsfor HPLC-MS analysis were obtained from soil incubations withunlabeled diphenhydramine that were processed in parallel withidentical incubations of 14C-diphenhydramine supplemented soil.

3. Results and discussion

The dissipation of diphenhydramine was evaluated in three agri-cultural soils; a loam, a clay loam and a sandy loam, using parallelincubations with 14C-labeled and unlabeled diphenhydramine resi-dues. Following 100 days of incubation, 14CO2 accumulation reacheda maximum yield of approximately 2% in the loam soil, and lessthan 1% in the sandy and clay loam soils (Fig. 2). Given that the nom-inal purity of the 14C-diphenhydramine preparation was 98%, it is notknown if the 14CO2 was produced from the active ingredient or a

he presence of two main peaks (middle and bottom panels) for DPH (M+1=256 m/z)

Table 3Disposition of extractable radioactivity in diphenhydramine and diphenhydramine-N-oxide following 100 days of incubation. Soils were incubated in the presence (+ LMB)or absence of liquidmunicipal biosolids. Data are presented asmean±standard deviationfor three replicates.

% Initial radioactivity recovered as

Diphenhydramine Diphenhydramine-N-O

Loam 47±3 8±0.5Loam+LMB 44±3 11±0.7Sandy loam 41±7 3±0.5Sandy loam+LMB 42±5 1.5±0.2Clay loam 25±6 1±0.2Clay loam+LMB 29±2 0.6±0.1

139E. Topp et al. / Science of the Total Environment 439 (2012) 136–140

contaminant in the commercial preparation, and therefore minerali-zation as measured here is below the method detection limit. TheDT50 of the extractable diphenhydramine in the clay loam soil as de-termined by removal of extractable radioactivity was 88±28 days, inthe sandy loam 137±53 days and in the loam soil 335±145 days(Fig. 3). The r2 values for the fit to a first order rate model wereover 0.82, indicating that the fit to a first order kinetic model was gen-erally good (Table 2). The more rapid dissipation of diphenhydramineand lower efficiency of extraction in the clay loam compared to theother soils are presumably due to sorptive interactions with clay com-ponents. Diphenhydramine is effectively removed from an aqueoussolution by montmorillonite for example, which readily binds di-phenhydramine through interlayer adsorption (Li et al., 2011).

HPLC-RD analysis of the extracts indicated that as the experimentprogressed some portion of the recovered radioactivity did not co-elute with the diphenhydramine standard (RT=8 min), but rather asa new peak with an RT of 4 min. The identity of the transformationproduct was determined by HPLC-MS of extracts from soils incubatedwith only unlabeled residues. Analysis of extracts from the loam soilsampled following 100 days of incubation revealed the presence oftwo peaks with [M+1] ions m/z 256 and 272 respectively, whereas atthe start of the incubation only one peak at m/z 256 was detected.Both fractions had a common fragmentation ion at m/z 167, indicatingthat the second peak was likely a transformation product (Fig. 4).Further HPLC-MS analysis on the fractionated peaks confirmedthat the decrease in diphenhydramine according to the 14C labelin the loam soil (Fig. 2) was accounted for by accumulation ofdiphenhydramine-N-oxide. Comparison of the m/z 272 compound toan authentic standard of diphenhydramine-N-oxide confirmed itsidentity on the basis of RT and fragmentation pattern. The fungusCunninghamella elegans converted diphenhydramine to the productsdiphenhydramine-N-oxide, as well as N-desmethyldiphenhydramine,N-acetyldidesmethyldiphenhydramine, and N-acetyl-N-desmethyl-diphenhydramine (Moody et al., 2000). No products of diphenhydra-mine demethylation or N-acetylation were detected in the presentstudy. Perhaps because they were not simply not produced, or becauseother transformation products were reactive with soil and rapidlyremoved from the extractable phase through processes of sorption orcovalent attachment. The pKa for diphenhydramine is 8.9, and thus atacidic soil pH values it will be cationic, and presumably variation in ion-ization is not a rate-determining factor within the range (5.2 to 7.5) ofpH values of our soils (Table 1; Berninger et al., 2011). The insignificantrates of 14CO2 production (Fig. 2) indicate that parent and any transfor-mation products were not completely biodegradable in these agricul-tural soils, and that formation of non-extractable residues was themajor mechanism of 14C dissipation. After 100 days of incubation,diphenhydramine-N-oxide was recovered at 8% (loam) to less than 1%(clay) of the initial added radioactivity (Table 3). There was no signifi-cant effect of LMBon the amount of diphenhydramine-N-oxide recoveredin any soil. Overall, the wide range of DT50s observed in the present studyindicate that there are rate-controlling soil variables that meritinvestigation.

The impact of LMB addition to soil on rates of diphenhydramine dissi-pationwas evaluated in all three soils (Table 2). TheDT50 value in the clayloam soilwas 97±22 days, in the sandy loam198±101, and in the loamsoil 329±121 days. The r2 values ranged from 0.7702 with the sandyloam to 0.8432 with the clay loam. There was no significant differencein persistence of diphenhydramine in soil without LMB and with LMBat p=0.05. Overall, these results indicate thatmicrobiological and chem-ical components of LMB neither accelerated nor hindered processes thatdissipate diphenhydramine. In contrast, LMB accelerated the dissipationof triclosan and triclocarban in these same soils (Al-Rajab et al., 2009).

In summary, at 30 °C diphenhydramine dissipated in soil with DT50values in the range of about three months to a year, with the shortestdissipation time in a clay soil. Mineralization of ring-labeled materialwas below the detection limit, and diphenhydramine-N-oxidewas a minor transformation product. Formation of non-extractablesoil-bound residues is the major sink for 14C-diphenhydramine.Overall, these results are in agreement with field observations indi-cating that diphenhydramine is persistent in the environment, andmerits further investigation with respect to potential risks fromenvironmental exposure.

Acknowledgments

This study was partially funded by the AAFC SAGES program, andby Health Canada (New Substances Assessment and Control Bureau).

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