Document Qgo9QDLeQjxebroDBV0oy0aek

een =; ELSEVIER ici Tos3015556 Reproductive Toxicology 33 (2012) 556-562 [r---------- Contents lists available at ScienceDirect RReepprroodduuccttiivvee TTooxxiiccoollooggyy imemmallbemesese: wwwslsevisrcomiiosatalentatos journal hornepage:www.elsevier.comllocate/reprotox EExxppoossuurree aanndd eeffffeeccttss ooff ppeerrfflluuoorrooaallkkyyll ccoommppoouunnddss oonn ttrreeee sswwaalllloowwss nneessttiinngg aatt LLaakkee JJoohhaannnnaa iinn eeaasstt cceennttrraall M Miinnnneessoottaa,, UUSSAA CChhrriissttiinnee MM.. CCuusstteerra,**,, TThhoommaass WW.. CCuusstteerr?a,, HHeeiikkoo LL.. SScchhooeennffuussssb,, BBeetthh HH.. PPooggaannsskkiib,, LLaauurraa SSoolleemmc pre--s --------------------------------" ~ U.S. Gealo~ica~ Sur~ey, UpperMidwestEovironmeata~ Scieaces Center, 2630 fanta Re~ Rd., ~ Crosse, ~ ~3, U~ eeterotaeber eoB h, 5550 ~ A~ua~c T~ico~ ~borat~, De~ent ~ Biolo~cal S~ences, St. Cloud State Univ~si~, St. Cloud, MN 56301, U~ c Minn~ota Pol~u~on Con~ol~en~, gnvironmentaIAnalys~ ~Outcomes, 525 ~keA~nue South, Suite 4~, Duluth, MN 55~2, U~ ARTICLE nro ARTICLE INFO mr Article history: m0 Received 25 October 2010 ay 0 Received in revised form 7 January 2011 Jetty Accepted 21 January 2011 Fo Available online 3 February 2011 Toi ute Keywords: [a PFOS Perfluoroocatane sul fonate fom Reproductive effects I Tachycineta bicolor =PFC5 AvstRACT ABSTRACT Treo cintaBk cles were cold 2 ees rd ey le Tree swallow (Tachycineta bicolor) samples were collected at a reference lake and a nearby lake (Lake ee Remind wih as ed botslo cs. Johanna) in east central Minnesota, USA contaminated with perfluorinated carboylic and sulfonic acids. re of 13prob comput TC) gotscpt Tissues were analyzed for a suite of 13 peffluoroalkyl compounds (PFCs) to quantify exposure and to demi herewa socationbetweecg concentrations of PEC andreproductivescces of determine if there was an association between egg concentrations of PFCs and reproductive success of mows Cention ofsero nt llote 705 ws csi vs sot tree swallows. Concentrations of perfluoroocatane sulfonate (PFOS) were elevated in all t~ee swallow peo iShe LM ou pi ered tissues from Lake Jehanna compared to tissues collected at the reference lake. Other PFCs, except for two, `wereelevated nbod pls LkeJantacompared thereference ake.F708vas tedominant were elevated in blood plasma at LakeJohanna compared to the reference lake. PFOS was the dominant 750 ke horn bo ed 50 dP oe roc Hk oe m3 PFC (>75~;) at Lake Johanna, but accounted for <50% of total PFCs at the reference lake. There was a ee i meat of FRO 5s od Bhd, bo tc negative association between concentrations of PFOS in eggs and hatching success. Reduced hatching wo oo SOR ew success was associated with PFOS levels as low as 150 ng/g wet weight. Pedy vert Published by Elsevier lnc. 1. nroducion 1. Introduction erro compounds FCS) are wokd-wide contaminants Perfluoroalkyl compounds (PFCs) are world-wide contaminants becuse ofthe ered ag 2 ce EST 6 because of their widespread usage as a surface treatment on texsear an etme robo edo oaprepution tiles, leather and carpet, in paper products used for food preparation nd Sora, and mr in oa, mci na To pl. and storage, and in fire fighting foams, insecticides and floor polhes 133 Hered tonenraions of PCs hav be found 1 ishes [1,2]. Elevated concentrations of PFCs have been found in orh otpe od et peat SOB tissues of various species collected near populated, industrialized reahel r RC pe rodacos n eltes]Aflsorochen: areas [3 ] especially near PFC production facilities [2]. A fluorochemCl rodocion unt oad 3 cnr inners Coape ical production unit is located in east central Minnesota (Cottage Grove USA vais wate disposal and arco cletsh Grove), USA; various waste disposal and fabrication facilities that TedPRCateloted nth anounETIeopaooTaMnIn used PFCs are located in the surrounding metropolitan area ofMinpols3nd St Fa, MIN (eater Cries, Cet bo eto neapofis and St. Paul, MN (hereafter Twin Cities). Great blue heron (rier)gcolctodon he Espo RNSCog (Ardea herodias) eggs collected on the Mississippi River near Cottage ov ad aoeh WESPECcontaevie nkorsea Grove had among the highest PFC concentrations ever recorded in hd xs 4]Cope ean concmraonaf efoorockans bird eggs [4]. Geometric mean concentration of perfluoroocatane Sone (ROR) was MORE wet wt. GFWiF 3 max sulfonate (PFOS) was 940 ng/g wet wt. in eggs with a maximum oncentratonol 5s concentration of 1878 ng/g. oe MinnaPollanControlAgrcy MFCA) as ben st The Minnesota Pollution Control Agency (MPCA) has been testing inTiC anhft hepen of RCL 12007 ing fish in Twin Cities' area lakes for the presence of PFCs. In 2007, cpp mcrchi) fon akeoan Ramey Coun, bluegill (Lepomis rnacrochirus) from Lake Johanna, Ramsey County, ai ets osGrmfr IH * Corresponding author.Tel.: +1 608 781 6247; fax: +1 608 783 6066~ E-mail addresses: ecuster@usgs~gov (C.M. Custer )~ tcusrer@usgs.gov hE te OL eve (T.W. Custer), hschoenfuss@stcloudstate.edu (H.L Schoenfus5), ChE Laura.Solem@state.mn.us (L 5olem). 00.23 eo a ety tr. d0doo8ii9:01|400~..1210301816/6$/j-.erepsperreoocftroooxxn2.2t00m111a1.t.t00e11r...0P00u55blished by Elsevier Inc. Fr p-- MN, had concentrations of PFOS, the major chemical constituent IFC hkwee St times Whe tha concerations ound in PFCs that were eight times higher than concentrations found in Sls aloo otf ie thr aks tat eu [Oly bluegills sampled from most of the other lakes that year [5]. Only lel ror kei Washinton Co ake 310 55 of ake bluegill from Lake Elmo, Washington Co., a lake 23 km ESE of Lake baaadstoml.ry hh PROSconenrations 20. Johanna, had similarly high PFOS concentrations in 2007. There ed sone ecto RCSOn Dd ric There are limited studies on the effects ofPFCs on bird reproduconan he susan quvoca Somelaboratorysui sot tion and the results are equivocal. Some laboratory studies suggest tt ot PoChAI loops RC shot that egg hatchability is relatively insensitive to PFC exposure [6], hwess tes esl shoud be sd wih cat because tk however, these results should be used with caution because the Nght dey raiment i at ShyWee ov 10 resdk higher dietary treatments in that study were toxic to some adults ror 03 Cisbig dE con dis are qui: prior to any eggs being laid. Egg-injection studies [7-9] are equivoCl wel Tort wis so patrp been FOS cal as well. There was a dose-response relationship between PFO.S and Ptetingsecs 7.91 to re netHE, Results and hatching success [7,9], but not for three other PFCs [8]. Results Tome con tdsshod ao ee with outionbecane from egg injection studies should also be used with caution because hy me htabe 0 ke to cout pref ehvir es.and theyare notable to take into account parental behavior effects, and reambe munch any of ctors uhscarsolo results can be influenced by a variety of factors such as carrier soluto res recon. mel econ (development ag and tion, area ofinjection, time ofinjection (developmental stage), and heipecenu ReveetaL [oun ho medRC on the species used. Newsted et al. [6] found that elevated PFC concenrationscoed nage ers fel un Cou rman, trations caused enlarged livers in female quail (Colinus virginianus), bt stoi hanes weefound nh its.oleson but no histological changes were found in the birds. No effects on ve 2 of holywee cored mae duo in ars liver size or histology were observed in male quail or in mallards Cras lahore (Anas platyrhynchos) of either sex. ee swallows av ee sed extensively fo mooring xp Tree swallows have been used extensively for monitoring expoure and llcof momenta) comamant (se Mature sure and effects of environmental contaminants (see literature itdsectionsin 10.11] for completes).Treeswallowseedon cited sections in [ 10,11 ] for a complete list). Tree swallows feed on a ree) aquatic insects [ 12] and concentrations of contaminants in eggs and Ein amesecsediment omaminuton 10:11, Teshy nestling tissues reflect sediment contamination [ 10,11 t- The ability Exhibit 3452 33445522..00000011 FO ---- - Custer et aL / Reproductive To~icoIogy 23 ~2072) 556-562 557 i ~ y ja [1 Zen ER A JIiS H suar~ ,pes nsistapi River Asusdtuydlyalkakeess 5 Wie otis 2 0 = TwinGitesmotaroea se km Mississippi River Buffalot, A& oer Green Mountain Lake I Minneapolis a ne A Lake Johanna 25 Twin Cities ~ metro areSat. Paul ! oops i Nr TR, Fig. 1. Map orrwo study lakes in east central Minnesota, USA in 2008 and 2009. Tr ------------ to attract tree swallows to specific sites with the use of nest boxes; Fa their restricted feeding range (within 1 km of nest box [13]) during he breeding sesson; 3nd he abtoSttact sulfcintmumbers Wesonseid 13 or, fae. 3 estingwececefomoh. the breeding season; and the ability to attract sufficient numbers abs el Ee for statistical testing makes the tree swallow a valuable species for a PTR aur studies of contaminant exposure and effects. Finally, organisms at mn lower trophic levels, such as the insectivorous tree swallow, might En be better indicator species for PFC comparisons and effect studIe ec esht hcoer Dnth poemearnSSEtES 20 tO CFSrat ies because PFOS, which covalently hinds to proteins in the liver and blood plasma, may not follow the traditional biomagnification rund iit hy a processes that lipophilic organic chemicals do [ 14]. I) nd The objectives of this study were to document exposure and oR possible effects of PFCs on tree swallows nesting on Lake Johanna are compared to a nearby reference lake and to assess the utility of prem re en TN various avian tissues for quantifying exposure and effects. Because apsBot fr CHa CoB TS of the previously reported high concentrations of PFCs in fish from maa ems bt ey Te ee Se TO Lake Johanna, we expected concentrations to be sufficiently high LS SLE as to test for possible reproductive and .other effects. a I A RE TE see Tree swallow nest boxes (n=33) were positioned along the shoreline of Lake Le Johanna (4502'38"N, 9310'15"W), in 2008 and 2009 (Fig. 1). To increase the spaEE Emin Hale Rmiinls tial coverage of the lake, boxes were split between the north ITony Schmidt Regio nal TE Ts Park, RamseyCo.l and south I Nortbweste ro College I sides of the lake. [n 2008 our refEE Ly Samant erence lake was Square Lake I45o09~16~N~ 92o47~43~W~ Washington Co, MN (n= 19 ee ie nest boxes ), but because no swallows used the nest boxes at that location, our refer ence lake was changed in 2009 to Green Mountain Lake (4510'0D"N, 9347'3WW), Ta retin Wright Co, MN (n= 18 nest boxes). IE ee Nest boxes were placed on metal fence posts approximately 20m apart and EE I Sas protected from predators using metal cylinders. Each nest box was visited approxeer moo, Aw lt imately once per week beginning in early May each year and the number of eggs TE I laid and the number that hatched were recorded for each nest box during each visit. a ns After the eggs hatched, the number of nestlings present was recorded until they i reached 12 days of age (earlyluly~ This wa~ called the nestling period and it started I IDay 0) when the first egg in the nest hatched. AREE. One e~ was randomly collected for contaminant analysis from each clutch Imma SRT after the clutch had been completed. Eggs collected for contaminant analyses were EE ey weighed on an electronic pan balance (0.01 g) and the contents emptied into chemEE Dama ically clean jars. The egg contents were weighed, and the stage of embryonic ST Sor EE development and whether the embryo was alive (viable) or dead was noted. Egg Ee ake: samples were then frozen (<-30 C) until chemically analyzed. Any unhatched eggs I were collected, opened, and the stage at which embryo death occurred was noted. I Imi Eggs that were addled, i.e. with no obvious embryo visible, were not assigned a a stage, but classified as infertile eg~s~ a category which also included those where I Ie the embryo died at a very early age (<a few days old). Eggs that disappeared were os, assumed not to bare hatched. ]n two instances the entire clutch (n =4 and n= 5 eggs) was collected when the adult swallow was found dead and the eggs had little chance of hatching. Each eggwas analyzed individually to examine within clu~h variability. When nestlings reached 12 days of age, 2 nestlings were collected from each brood unless there were only 2 surviving nestlings and in that case only I nestling was collected. T~e nestlings were decapitated and the blood collected in hepariniz ed tubes. The tubes were centrifuged for approximately 10 min and the plasma decanted for PFC analysis. The plasma from the nestlings was pooled by nest box to provide sufficient volume for chemical analysis. The packed red blood cells were discarded. The liver and contents of the stomach were removed from each nestling carcass, pooled by nest box, and frozen later that day for PFC analysis. The carcass remainders were frozen individually and one carcass from each nest box was analyzed for PFCs. 2.I. Analytical chemistry Tree swallow samples were analyzed by Axys Analytical Services, British Columbia, Canada for PFCs including PFOS, perfluorobutane sulfonate (PFBS), periluorohexane sulfonate (PFHxS), perfluorooctane sulfonamide (PFOSA), perflunrooctanuate (PFOA), pertluoroheptannate (PFHph.), perfiuorohexanoate (PR-[xA), perftuorobutanoate (PFBA), perfJuocopentanoate (PFPeA), perfluorononanoate (PFNA~ perfluorodecanoote (PFDA), perfluoroundecanoate (PFUnA), and perfluorododecanoate (PFDoA). Median detection limits (DL) varied by sample matrix and were 0.5 ng~ml in plasma except 1.0 for PFBS, PFHxS and PFOS; 2.43S ng]g wet wt. in carcass remainder except 4.865 for PFBS, PFHxS and PFOS; 7.04 ng/g wet wt. in diet except 14.| for PFBS, PFHxS and PFOS; 5.88 nglg wet wt. in egg except 11.8 for PFBS, PFNxS and PFOS; and 4.95 ng[g wet wt. in liver except 9.9 for PFBS, PFHxS and PFOS. The relatively high detection rates for diet, eggs and livers were because of small sample mass for these matrices (ave. mass = 1.01,1.49, and 2.03 g for the three matrices, respectively). All values provided in tables~ text, and figures are ngjg wet wt. except blood plasma which is expressed as ng]ml. In brief surrogate standards of I3C4-PFOS, t;C4-PFBA, tzC2-PFNx~ ~Cs-PFNA~ t~C~-PFD,K and t~C~-PFDoA (Sigma-AldricK U.S. and Switxerland, and Chiton, Norway) were added and the sample was extracted by shaking with methanolic potassium hydroxide solution. After ozntdfugation an aliquot of the superoatant was diluted with water and cleaned up by solid phase extraction (SPE) containing a weak anion exchange sozbent (Oasis~ Waters~ [reland). The eluate was spiked with two recovery standards, ~C~-2H-pelfluoro-2<lecenoicacid and I~C~-PFOA (see manufacturers above), and analyzed on a high performance liquid chromatug~aph (LC, Waters 2~;90 or Waters 2795) coupled to a triple quadmpole mass spectrometer (MS-MS, Micromass Quattro Ultima). The [ column was a Waters Xtera CIsMS reverse phase Cts, 2.1 mm x 100 mm with 3.5 I~m particles. The MS was operated in negative electrospray ionization ion mode and at unit mass resolution using multiple reaction monitoring. A minimum of six calibration solutions spanning the expected concentrations was used and all were processed through the same SPE procedure./kialyte concentrations were determined by isotope dilution/internal standard metbod~ comparing the area of the quantification ion to that of the t ;C-labelled standards, and correcting for response factors~ The eight carboxylateswereqnantified against ~C;-PFDK PFOA against t~C;-PFOA~ and the sulfonates were quantified against I~C~-PFOS. All samples were analyzed in batches of 15-20 33445522..00000022 = CtCtr R Tcc 012)56-52 558 CM. Custer et al. / Reproductive Toxicology 33 (2012) 556-562 mpl chTote ws os ak ad dpe olin xc 3, Ress samples each. There was one blank and duplicate sample run with each batch for eh de dknel010 quality assurance purposes. Matrix spikes (n = 8) and matrix spike duplicates (n = 6) were also analyzed. Percent recovery for the PFCs ranged from 96.5% for PFBA to ib bcos memgo OS vray 1+ EOS 113% for PFBS. Percent recovery averaged 103% for PFOS. The overall average percent 3. Results 3.1. Exposure recovery te was 102.7%. pr Concentrations were not corrected pre for matrix spike recoversiceonse.: 22 igo P(blEooAd lancd aFBSegw.erveern,tdideeto cecatredasin)a.nyRmOSApwosfladeytecmtaetdriixn 2.2. Histological analyses i somnolencepe ofnolmy L1apkleaoshmaanaand)2anvdePrFOsAam(p2l3eso.f 2P4BsAam(p9loefs)2w4erseamdpelteesc.teadl i9n Five histomorphological endpoints were measured: prominence of hepatocel- rvs oli con,macheSina ago Sc plasmasamples,but no in he oermatrices.PENA. PFDA, PUTA, lular vacuoles containing glycogen, macrophage abundance, adipocyte abundance, reco ed vi ee Fre. Fon and BYKS were detected ih S507 ofpas sample: Dk proteinaceous fluids, and fibrosis of the liver. For these, a small piece was removed from each freshly dissected liver, placed in a histo-cassette, and fixed in 4% neu- TAR ort bn by BS) Ie Ge ws dtd 40%of Samples ofthe ther maiices. Ninety-two percent of he tral buffered formalin. In the laboratory (HLS), the liver tissue was dehydrated {hn Ti ate PR Chr Snipa detections these het matrices were fom Lake Johanna. PROS (Jung TP1050 automated tissue processor, Leica, Germany), embedded in parafAH 3501, Terrsh clo wih ARATE) was detected in 50%ofamps fom all aces. fin (MICROM EC 350-1, ThermoFisher, USA), sectioned with a Reichert-Jung 2030 ne dun kd200tnd on lot hota cause there was no. dfrence in PROS concentrations microtome (3-4 ~m thick and 20 ~m apart), and then stained (Leica Autostainer XL, Leica Germany) with a modified haematoxylin and eosin stain following Carson beCcemamy) ih a odie e rn ia TSEC berweenyears at ake Johann (eggs p=0.37, plasma p07, 3nd. [15]. Between 8 and 12 tissue sections were examined per liver. The slides were in casch Ac Pe CCS 0.73). dat fom bh years wee combined and tested graded using a semi-quantitative severity gradient, which reflects the prominence mop hr er rae 1 id 0 for dereces between lakes PROS was preset in significantly of a histomorphological change in the liver tissue. Grade 0 is assigned to tissue eco iinep merlot nd Gods 4 imdon higher concentrations in blood plas at Like Johanna compared sections not exhibiting the specific histomorphology and Grade 4 to sections domiedly Sty onPeIMNSOGRRGALONE toGrecnMouainLpa<0k001. d= 1.F2- 3290) (30e 1 Sta nated by it. Severity grades were assigned to one spatial histomorphological change (prominence of hepatocellular vacuoles containing glycogen) and the abundance of eo eens Apes Hoe 1h Stlcomparisons wer tat mpie forthe thrFour matrices discrete histological structure (macrophages and adipocytes, following [16]). The ro ss ns ly 1g odes becatse while PFOS wasdetemaclltamepldes fom akeohana, former has been found in vertebrates to change substantially as a response to either krtm FA GR apt orf 14 di samples was ee ot eect (rss direct toxic insult or, more commonly, indirectly as a response of altered body contania hae an icin esiing rom hesires niconithed rganisnby Contaminanp0t5 dition resulting from the stress inflicted on the organism by contaminant exposure [ 17]. Macrophage a bundance is altered in vertebrates as an immunological response, np yam omen ea {Tom Green Mountain ake Table 1) The pater ofhigherconcen- which can be triggered, amongother factors, bycontaminantexposure [ 16]. Thefinal eae so Si bw sp act Uations in Like Johanna compared to Green Mounta Lae was Grade used for summarization and statistical analysis was the average Grade for hr pty Constantin the throumatics weer In agand er sim: the two nestlings from each box. Additional histopathological changes potentially fal preproses spies, the maxim concentrations were 123 and 2.5 mes higher Te froricewrhenegdbcuesvotd indicative ofa toxic insult such as the presence ofproteinaceous fluids or fibrosis of the liver were noted when observed. " {waeirneL8a.k6..3I d1iet manedscahricgahsesres,atgLeaokmeetJroihcanmneaanthcaontcheentarvaetrioangse 2.3. Statistical a analyses ny hd i ics hi ad of ples hove he dr. Lake Only individual PFCs in matrices that had >50% of samples above the detec- mt Sy Ot AGIA samples collected romLake ohanaconcentatoifoFFn05s tion limit were statistically analyzed. One-half the detection limit was substituted or nocd als Ts wilh 50 ot SO GG Tews3 wer OUGY equal 1 IST and C55 amples: however con. for non-detected values for PFCs with >50% but <100% detected. Between year ompaorsweedoo forakeowns la,5.nd esososB0 centration of POS in ver and carcas issues were half or les comparisons were done for Lake Johanna plasma, egg, and nestling tissues indi- vidually using 1-way analysis of variance (ANOVA). Comparisons of individual PFC te corns HPO Lhe concentrations oud in pasa and eggs. Concentrations in congeners between Lake Johanna and Green Mountain Lake were then done using vACocwo t gr ee sendy Dood plasma, ie and carcass were highly coreated (ony 1-way ANOVAs. Concentrations were log transformed to meet the homogeneity of a AAROUAWo ea 5olor nen Lake Jonna data. analyoed) wih ane another (1~086 blood variance assumptions ofANOVAwith geometricmeans and 95% confidence intervals at 4h ber, 7-D74 1 bo and arcas: an 7-074 fn Ir and (CI) provided in tables and text. So AT TC ors wscombsb,es A Carcass), Thre were approximately six times higher concentra The pattern of PFC congeners was compared between lakes with Analysis of Similarity (ANOSIM) which is a multivariate analogue of analysis of variance built on Sn ean om dpic pe ry ton blood and 5s compared o dit, and betwen tree and a simple non-parametric permutation procedure and applied to the rank similarity nag ey ontmgo s1]To a matrix underlying the Bray-Curtis ordination of samples [18]. The test statistic, R, ayo 30120 dn1 te ot her eySs may vary from 1 to +1. An R value close to +1 indicates that there are very clear es an on he rus eg en oe i Av TSM differences in patterns among the groups being tested, in this case lake. A value ee it Gi pte hr ror 0 pr CoofPm Unrilvn piss ll es ddsc near zero means that the distribution of patterns is as similar among the groups as e oneR ea don es AE teeRa 08 hd 0 GA LAT ASN S05 within the groups. An R is considered significant based on its p value (e.g., p < 0.05). ep The proportion that each congener comprised of the total PFCs was used in the ANOSIM; this factored out the influence of concentration differences between lakes. Ra oth es ba Sn To Nemgma) 7 Nemmma) Pearson's correlation coefficient was u sed to assess the relationship among matrices for PFOS concentrations. Te anFOSc concrrstii onineggs e nd he chn incss -------- mn-- ] --n dn) The association between PFOS concentrations in eggs and the hatching success a Oe g oO awe DoT 1 Toes of the remaining eggs in the nest wa s assessed u sing logistic regression [ 10]. Because so een men ann of the small sample size in 2008 and the lack of between year differences, years were Ai To nes . 3 EE combined. Nests that failed because of depredation or were human caused (auto- mobile collision) were excluded from the analyses because these two factors were mt ati t eew Nt hS EP Y Pa LEhaEm presumed not to be contaminant related. Nest success during incubation (probabil- BBeeccaauusseeoofftthhee rreellaattiivveellyy hhiigghh ddeetteeccttiioonn lliimmiittss,, PPFFPPeeAA., PPFFHHXxAA,, PFHpA and PFBS were not detected in any sample of any matrix (blood plasma, egg, liver, diet or carcass). PFOSA was detected in only 1 plasma and 2 liver samples. PFBA (9 of 24 samples, all 9 from Lake Johanna) and PFOA (23 of 24 samples) were detected in plasma samples, but not in the other matrices. PFNA, PFDA, PFUnA, PFDoA and PFHxS were detected in _>5070 of plasma samples, but <4070 of samples of the other matrices. Ninety-two percent of the detections in these other matrices were from Lake Johanna. PFOS was detected in >5070 of samples from all matrices. Because there was no difference in PFOS concentrations between years at Lake Johanna (eggs p = 0.37, plasma p = 0.47, and carcass p =0.73), data from both years were combined and tested for differences between lakes. PFOS was present in significantly higher concentrations in blood plasma at Lake Johanna compared to Green Mountain Lake (p < 0.001, dr= 1,22,F= 639.6) (Table 1). Statistical comparisons were not attempted for the other four matrices because, while PFOS was detected in all samples from LakeJohanna, errexemmceaapiintnddfoeerrr2aannoddf 1dd4iieedtt)i)eootrrsaddmeettpeelccettsee,dditiinwn a<<s5S0e07i%0thooeffrllinivvoeetrr daaenntddeceetggeggd ss(aacmm aprlcpealess.ss from Green Mountain Lake (Table 1 ). The pattern of higher concentrations in Lake Johanna compared to Green Mountain Lake was consistent in the other four matrices, however. In egg and liver sam- aptleLsa,ktehJeomhaanxniamtuhmanctohnecmeantxraitmiounms wcoernec1e2.n3 tanrdaa2t.t5iGtorim eneesns Mhoiguhne-r at Lake Johanna than the maximum concentrations at Green Mountain Lake. In diet and carcasses, geometric mean concentrations wddeeettreeecctt8iio.o6nn allinimmdiitt1i.i6nn ttthihmee ennsoonnh--iddgeehtteeercctateetddLssaaakmempJploleehssanffrnroaommthGGarrneeeetnnheM Moaouvunentrtaaaiginne Lake. In samples collected from LakeJohanna, concentrations of PFOS were roughly equal in plasma and egg samples; however concentrations of PFOS in liver and carcass tissues were half or less the concentrations found in plasma and eggs. Concentrations in blood plasma, liver and carcasses were highly correlated (only Lake Johanna data analyzed) with one another (r=0.86 blood and liver; r=0.74 in blood and carcass; and r=0.74 in liver and carcass). There were approximately six times higher concentrations in blood and eggs compared to diet, and between three and Table 1 Concentrations of PFOS in tree swallow plasma (ng/ml), tissues and diet (ng/g wet wt.) from Lake Johanna (2008 and 2009) and Green Mountain Lake, MN (2009). Tissue Lake Johanna Green Mountain Lake n Blood pla sma 15 Egg 18 Mean (95% CI) n [Min. and max.] 137 A~ 9 (120-157) [75.6-1901 141 10 (112-177) Mean (95% CI) [Min. and max.] 9.66 B (7.91-11.8) [6.11-14.51 6NDb-23.1c ity of at least I egg hatching in the nest) and during the nestling period (probability of at least 1 nestling surviving to 12 days of age) was calculated using Mayfield anal- nS dscn Tsnh em ysis [19-21 ]. This method uses the concept of nest-days, which is the total number at et Toko ed mr of days that the nests are observed. That total is divided by the total number of lo1s0csalceulaste the survival rae (probability)perday. Because the probability losses to calculate the survival rate (probability) per day. Because the probability cn ss pent of ToS, By pl of successive events is the product of those probabilities, the daily survival rate is ih ove f er of pe or wale moms raised to the power of the length of the period. For tree swallows, the incubation Fro gs he oy fe a i a hh, period, which begins the day after the last egg is laid until the first egg hatches, eer dyson Te ig ee dy rg neg Oo Oon averages 13 days long. The nestling period is 12 days long and begins (Day 0) on the i Te aa oes day the first nestling hatches. The hatching and nestling survival probabilities are a ig eens merely the percent of the eggs hatching or nestlings surviving in successful nests. The histological data were analyzed with Fisher's Exact tests. The number of sam- sane Coed soaOo e on hr pies in each Grade as explained above was tested for differences between lakes for Khris cog ros hepatocellular vacuolization and macrophage abundance. Liver (f@sosrtye hn Carcass OFC wop(mioJdes3nw0 I. Diet [57.9-285] 15 70.6 (60.5-82.3) . I32.4-1071 15 41.8 (36.7-47.6) I23.0-62.91 14 22.5 (14.9-34.0) I7.0-41.11 9 8ND-42.2 '9 ~ 9ND 9 9ND TH TR we So TH ~ Means not sharing same letter are significantly different between locations. b Number preceding ND is the number of samples below the detection limit. Dm so LD SO 3 co11d Detection limits are 11.8 ng/g (egg), 9.9 ng/g (liver), 4.87 (carcass), and 14.1 (diet). Te ontramtt eme c The second number is the n~axin~um concentration. 33445522..00000033 CBCrt re oy3201)356-52 = CM. Custer et al. / Reproductive Toxicology 33 (2012) 556-562 559 Lake Johanna Lake Johanna amos <= =-0 ren Green Mountain Lake Green Mountain Lake Sm pron mw -- aon mm Jes ~ 0 pron Seems 2.mPronprio ahacemtPCo conct comprier doof w1o eCSs sos lomosoop d sc o ak oh o nd Gren ocala NUS fn20 30 200.1 Fig. 2. Proportion that each PFC congener comprised of total PFCs in tree swallow blood plasma at Lake Johanna and Green Mountain Lake, MN, USA in 2008 and 2009. The three congeners identified with dashed arrows were below the detection limit in all samples from Green Mountain Lake. {our times higher concentrations in ver and carcass thn in he four times higher concentrations in liver and carcass than in the di,diet. Between lake comparisons for four oer PECs (PFOA, PFDA, Between lake comparisons for four other PFCs (PFOA, PFDA, PENA andPUA)were ade oly or blood lea because most PFNA and PFUnA) were made only for blood plasma because most Sampleswere below thedetection mit aLhe referencearcaGreen samples were below the detection limit at the reference area, Green Mountain Lake (Tbe 2). Tor thee four RCs, PFOA (p-0.453, Mountain Lake (Table 2). For these four PFCs, PFOA (p=0.453, 012122. F-059) and PENA (p01, 4122 F729) did not df=1,22, F=0.59) and PFNA (p=0.013, df=1,22, F=7.29) did not er between he two locations. PFDA and PFUVA were Sgn. differ between the two locations. PFDA and PFUnA were significantly igh at Lake Johanna th3aGrnen Mountain Lake (both cantly higher at Lake Johanna than at Green Mountain Lake (both 75<L001).The 0.05 alpha lve was conected for multiple com: ps<0.001). The 0.05 alpha level was corrected for multiple comPains 5-3). 50 the cllcive p-value was O01 Th geometric parisons (n = 5), so the effective p-value was 0.01. The geometric ream concentation or oa PCS blood pas was 208mg/ml mean concentration for total PFCs in blood plasma was 20.8 ngJml Green MountainLakeand 166.3 nm at ake Johanna. at Green Mountain Lake and 166.3 ngJml at LakeJohanna. When the composition percent hat ach PFCcompriscdofthe When the composition (percent that each PFC comprised of the otal PEC)ofthe ght PCS present nt Jest 50% ofapes nat total PFCs) of the eight PFCs present in at least 50% of samples in at cast one cation was compared in malariae space thre was nearly complete sep(aR=0r95 ap<0t001i) beotwenen the two least one location was compared in multivariate space, there was nearly complete separation (R = 0.99, p < 0.001) between the two ae Table :2 Coemraions of FCslnMod plas(0/01)ad rePC 23065 Concentrations of six PFCs in blood plasma (ng/ml) and one PFC in eggs (ng/g wet wt.) of tree swallows nesting at Lake Johanna (n = 15) and Green Mountain Lake Tees en (n = 9) in 2008 and 2009. Sample size was n = 18 eggs at Lake Johanna. (I=CD =IO]8 200SpeCeowonme(E3D5UI)MiInLs] PFC congener Geometric mean (95% Cl) [Min. and max.] TT aDiicGpbromonocrnmsanie PFDA fraaa]n {seraesn) = PFNA mn ELaryan g7na PFUnA on fgoeomote] wfirtrim), PFOA LakeJohanna 6.81A~ (5.60-8.29 2 [3.43-13.31 3.41A (2.72-4.29 [1.81-7.621 2.67 A (2.14-3.34 [1.18-5.631 2.67 A (2.06-3.47 Green Mountain Lake 1.53 B (1.18-1.98) rr I0.94-2.591 5.71 A (3.85-8.47) [2.22-11.51 1.04 B (0.87-1.23) [0.76-1.231 2.11 A (0.96-4.65) PFDoA os afbirynreay] no PFHxS oes a Gtoroianane no PFDA-eggs 11.52-8.321 1.63 (1.23-2.17 I0.69-4.331 10.37 (8.19-12.87 I4.55-19.21 5.51 (4.01-7.58 [SND-14.01 [1NDb-7.69] 9ND 9ND 9ND ROL A TA Detection limits were 0.5 ng/ml for PFOA and PFDoA, 1.0 ng/ml for PFHxS, and 5.88 for PFDA in eggs. es tn ae nibhchemi doyare ~ Means not sharing same letter within each chemical are significantly different arb ART TO wid ar between locations. A significant p-value of 0.01 was used to accommodate multiple Cont mel so Sp HO comparisons to yield an overall alpha of 0.05. Emi ei ov mes apsek cmt. b Number preceding ND is number of samples below the detection limit. ake, POS accountedfo 76.7 of th total PEC at Lake Johanna. lakes. PFOS accounted for 76.7% of the total PFCs at Lake Johanna, butonly 47.1% atGreen MountainLake (1Th.ecar2box)y a.cids but only 47.1% at Green Mountain Lake (Fig. 2). The carboxylic acids tendedtacomp3rigesr percentage ofthe ta PES a Green tended to comprise a larger percentage of the total PFCs at Green Mountain SKE ha atLakeJohanna.PENAaccountedfo 28%of Mountain Lake than at Lake Johanna. PFNA accounted for 28.9% of the total 3 Green Mountain Lake nd 6.1% a Lake Johanna and the total at Green Mountain Lake and 6.1% at Lake Johanna and RORaccnted for 10.1% t GreenMountain Lake and 30 ake PFOA accounted for 10.1% at Green Mountain Lake and 4.1% at Lake Johanna. Johann& TherewasconsideabevPRaOrS ciontcenotrnatoionfs ngs There was considerable variation ofPFOS concentrations in eggs within clutch. We did nt know he order in whieh the ges within a clutch. We did not know the order in which the eggs wer id, bt thre ws a muasacohld dference nthe con. were laid, but there was as much as a 4-fold difference in the concenration (Table 3) of PROS Among cg wii th same cuch. centration (Table 3) of PFOS among eggs within the same clutch. The arthmetc mean and standard deviation of PFOS for the tus The arithmetic mean and standard deviation for PFOS for the two clutches were 289 2ng]g 1563 (box 730) nd 20ng93373 clutches were 289.2 ngJg 4-156.8 (box ?30) and 209.3 ngJg 4-8?.3 (ox 763) (box ?63). ------ 3.2. "Producten Reproduction ndand feeffects There wee fv re swallow nest ttmpts a Lake johanna in There were five tree swallow nest attempts at Lake Johanna in 2008 and 1312008 No Swallows sid at Square Lake in 2008 2008 and 15 in 2009. No swallows nested at Square Lake in 2008; here were 11 net Aiempt at Green Mountain Lake (reference ae) in 2009. Seventy-nine percentoftheeggs hatched (Table 4). there were 11 nest attempts at Green Mountain Lake (reference lake) in 2009. Seventy-nine percent of the eggs hatched (Table 4), oth years and kes combined: Individual 8 loses were due both years and lakes combined. Individual egg losses were due prmarily diappearance during incubiion and ict. with primarily to disappearance during incubation and infertility, with fewer ss due est abandonment and deathofthe ad bird fewer losses due to nest abandonment and death of the adult bird (lledorphanedin abe4) Fourto fue percentof esis ther (called orphaned in Table 4). Four to five percent of nestlings either diedor disappearedduring the nestingperiod died or disappeared during the nestling period. "The probaiy of est succes durin incubation (probabity The probability of nest success during incubation (probability of at lest 1 gg hatching na uch) rangedbetween G01 (Lake of at least 1 egg hatching in a clutch) ranged between 0.61 (Lake JJoohhaannnnaaiinn 22000088))aanndd 00..9911 ((GGrreeeenn MMoouunnttaaiinn LLaakkee iinn 22000099,, TTaabbllee 55)).. Three ness ere total ares at Lake Johanna, which dos nok Three nests were total failures at Lake Johanna, which does not include the toatl flrs becofadepurdsatoen and automa: include the two total failures because of depredation and automoie collin. 1d on foal are t Green Mountain Lake, The bile collision, and one total failure at Green Mountain Lake. The nest at Green Mountain Lake disppearcd before cg coud be nest at Green Mountain Lake disappeared before eggs could be Caledfo chemical analysis. Facing Success prosabiny (er. collected for chemical analysis. Hatching success probability (perCent of ggs id tht Riche) ranged from 6% 1 98%fbo oth cent of eggs laid that hatched) ranged from 64% to 98% for both nes Table 3 J rr ----r-- Concentrations of PFCs (ng/g wet wt.) in eggs within two tree swallow clutches from orm n3008 Lake Johanna, MN in 2009. WC br tr Gr es wed mes PFC Wo wn wm mm mo PFOS aEmme wok aeom owms omme PFNA _ "WT a TWm3_mwzr_samsWaw PFDA Box # Box 730 Box 763 Box 730 Box 763 Box 730 Box 763 Egg# 1 ~ 96 155 NDb ND ND 4.77 Egg# 2 169 115 4.6 ND 8.87 ND Egg# 3 499 284 24.3 5.37 22.7 7.22 Egg# 4 298 283 12.6 5.49 17.5 8.27 Egg# 5 434 15.9 21.0 oree eeedrwe Egg numbers do not denote the order in which they were laid. Fr ND, not detected. 33445522..00000044 " Ht im s RO CJ~. Custer et all Reproductive Toxieotog~ 33 (2012) 85~- 562 ais [EO Table4 ESrompe--r Egg and nestling summaries for tree swallows nesting at Lake johanna and Green Mountain Lake. MN in 2008 and 2009. "Numberofeggsornestlings Number of eggs or nestlings PPeerrcceennttaaggee. El Se Dem 5fo om LakeJohanna Green Mountain Lake Total Losses calculated from remaining eggs = zoxw x200 ou |JFo r-- \ Eggs 2008 2009 2009 = . x . Laid 27 78 56 161 Collected G 14 10 Remaining 21 134 46 Egg losses En Reason for losses BEC os 1 Missing 3 4 3 Infertile 4 2 0 [=-E Abandoned 4 O 0 EO Dead t 0 0 30 131 oon 10 7.6 6 4.6 4 3.1 1 0.8 o . oscn omTmte v Fig. 3. Logistic regression between hatching success and concentratiun of PFOS in J nr ed A eggs of tree swallows nesting at Lake Johanna and Green Mountain Lake, MN, USA Orphaned~ 0 7 0 7 5.3 in 2008 and 2009. Does not include data from nest~ lost to depredation or human EEgggsThhoaattatccl hhleoeddst 912 9 Nestlingst' 9 BBEe. 8% 2 - Nestling losses . 1: 1% EE rte tres arrse Dead 0 BE iif 8 Eo eres Missing 0 Total lost 0 s5113 o.433 49 36 0 5 2 2 2 7 1W20w 83 7s 21.4 78.6 94 5 5.3 4 4.3 9 g.6 TT TT rs a Includes eggs from Oao nests. In one case the adult was killed by a car and in the ee = other the adult was pecked to death inside the nest box. ARE : TT ~ Nestling period from Day 0 to 12-days of age. ie ring ss stones. T BoT g {1 1 1 lakes. Nestling survival probability was high and ranged between iy ko 0.95 and 1.0. creepers. SE sorry There was a significant negative association between PFOS con- `cceennttrraattiioonniinneeggggssaanndd tthheehhaattcchhiinngg ssuucccceessssooff tthhee rreemmaaiinniinnggeeggggss pyri alybite in the nest (p = 0.003, Fig. 3). Beginning at a0proximately 150 ngfg eT wet wt., total hatching failure became obvious and was present in both years of the study. An almost identical result (p = 0.003) DEE IR ERERID amar was obtained when total PFC concentration, rather than PFOS, was i A regressed against hatching success. oe a Spi deen he fnue oofbe BETmeSs erhly Sendcnaalsetee n me le There was no significant difference in the frequency of his- tological Grades between Lake Johanna and Green Mountain Toke fo iter hemor vacuo (1-04-0720o)r 304Bross ws observed inone mpi omLake Johanna. Lake for either hepatocellular vacuoles (,~2 =0.044, p=0.720) or ios totes macrophages (X2 = 0.030, p = 0.795) (Table 6). For vacuoles, 100% LL of the slides were Stage 2 or less at Green Mountain Lake whereas es only 81% were Stage 2 or less at Lake Johanna. For macrophages, IRI TIDE am 64% were Stage 2 or less, and only 22% were Stage 2.5 or higher at EE Green Mountain Lake. At LakeJohanna 36% was Stage 2.5 or higher. SN ayangresiog eyespnset There was a trend towards more adverse liver histology, i.e. more a livers with higher Stage scores at Lake Johanna, but itwas not statisoo tically significant. The geometric mean PFOS concentration in liver nae tissue at Lake Johanna with Stage >_2.5 was 56.8 ng/g, whereas the BEL E eB geometric mean concentration in livers with Stage <2 was 83.8 ng/g ud disturbance. Table 6 Numbers of broods scored for histomorphological changes in each Stage in tree swallow liver cells for abundance of hepatocellular vacuoles and macrophages from Lake Johanna and Green Mountain Lake, MN in 2009. Metric and Location Stage <1,o 1.5 2 2.5 Hepatocellular vacuoles Lake Johanna Green Mountain 4 2 3 o Macrophages LakeJohanna _ CreaMown Green Mountain 5' 2 1 33 44 111 0 Stage 1 (n=2) combined with Stage 1.5 (n=3) for Fisber's Exact test. which is the opposite of expected ifPFCs were affecting liver histology. Both large sinusoids and proteinaceous fluids were observed, but seemed evenly distributed in samples between the two lakes, six at each location. No adipocytes were observed in either location and fibrosis was observed in one sample from Lake Johanna. 4. Discussion 4.1. Exposure By sampling nestlings, especially songbird nestlings, an assessment of local contamination is possible because nestlings are only fed from the local environment, and for tree swallows that is generally within 0.5-1 km of the nest box [ 13 ]. This contrasts with many other waterbird species which may fly 10-20 km to feed [22,23]. isTable 5 rE Reproductive success including the number of tree swallow nestlings raised to 12 days of age from Lake Johanna and Green Mountain Lake, MN in 2008 and 2009. BE R2o 5T.=BEa ITE, Year Lake No, of nests with eggs and (nestlings) Nest successa Hatching probabilityb (C) Nestling survival probabilityc (D) Mean clutch size (E) No. nestlings to 12 daysof age (A x B x C D x E) Lo Incubation(A) Nestlingperiod Overallnest gs success (a R) Tr rT 2008 Johanna 5 (3)0 0.61 1.00 0.61 0.64 1.0 5A 2.11 BE b= OB 2k EB FB 21)09 Johanna 13 (12)" 0.92~ 1.00 0.92 0.94d 0.96 5A~ 4.48 BTOTER amo 2 o& OB 2 Fo@ e TET IM ee-- e ------ 2009 Green 11 (10) 0.91 0.90 0.82 0.98 0.95 5.1 Mountain 3.89 TT Based on days of nest exposure (see Section 2 and [18,19]). ER Hatching probability (number hatched/total number of eggs in successful nests [at least one egg hatched]). TT or saF " L, Nestling survival probabilitay (number of nestlings surviving to 12 days of ageflotal number of nestlings in successful nests [at least one nestling lived to 12 days of age]). First number is number of nest a~empts and number in parentheses equals the number of soccessful nests (at least one egg hatched). Does not include data from the two nests lost to depredation and human disturbance, so total number of nest attempts= 15. 33445522..00000055 CtCuter ReprodToc 32012) 556-562 on CM. 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Tmvevoheesenntaottbhfhiolotiuhtueygghhtoottthhhheeearvssmeaaammtarppilllcoeeewsmm,eaarassrsdsgeuwwtceaascsstsiccotoornonnnsslgiiimlddyeeirrtfaaofbbrollrypylpssalmmsaamsalmalllaeearr stttahhhmaeannpbleeffsoostrr, coflloronowwcmses,,nttwwhrhehaiitAccihhnontwwwseeearrrpeepprnn[oo2otx4t]iss,maawammtepepllwleeyodd?uaalfdttoLLadaangkktrieeceJJaipotoeahhrtaaenntnthnhaa,a,ntfwwaoooduuuunlldlddt ithhnraaenvveeeesstiwllivviaenelgr-r attmiilssosossuuteegoetnf(o0etrhuuaelsseleoytwwhnhehoretenmndeaPPttFrEeiCcCctededsdee,ttaeeirnccgttuIiioevonesnrsllteoervrvoeencllgassrlyacaarrfeesosraaennpsliaiinsssssmtuuerea.e.eaPsPsFFtwOhOaSeSllbwwoeawassss.t clTioivene`rrcCsseonttnhhctereerarnetet.i.roantsioanpsoprfoPxFimOSateilnyn2e-sftollidnggrbelaotoedr than found in nestling plasmaofbald cagles also generally not detected in liver or carcasses in tree swallows frreoamsoGnr.een MountainLake most kel orth samedetection mit from Green Mountain Lake most likely for the same detection limit 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gwswrpheheeaectnniecerocslotclilolmeeueccltdfteeobddrceacoaocmgmcpeu-pamrareurleleaaddtett0dio;o11ns22oimddnaaetyyhesseaoogcllladdegfenoorsers.swstlwTairnlaogllplsoohwwwiscesp:;roetsthhi7iit0ssiadaolanlllyomoswwaoeelyddd 3ooot-fthhfveeoarrdriooadrrbiggifaaleinntriyiecc,ncchcoooennwitteaavmtmoeitirnna,alawnnPtatCsssBsiisnnwimsswwilaiaallrllootatowwcsstl.h.uheFtFcooarhrmioeefoxxuaatnmrnmetppelsoleewfavtthlhaelerirroaeewtiwwoeanagsassinaa ghaaarveoveaetaneallrssowtoiimpptllheaayyfteoerdrdospashocoicmmcueemproruoosllliaeettiinoionnntthhmieenassyteehidenfiofeetfaregbreleenencsca.eesTs:s;rtohhrpaohiowgichewtvpeoefrso,irvtttwihhoaeeenraadrsmsssf,aoooy.-r 237-)fo.Tlhdeddififsetrreinbcuteiionn total PCBs within a of PEC congeners clutch in tree of tree swallow swallows was eggs sii cPPiniFaCFbtisloCoanosdiwstiptiisitlfhafoostrrmroooatpthheheevirreconorprgaogtasaninotiiuiccorcncoronemntfteaaarymmeinnincnoaeatnnatbtksese[,[a11sg44]e]s.otrPPmaFReiOtOgrSShwitwcafasomsrewddaeeanttreedcccottfeneod.dr 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TwcwoawanssictnteeenCtddriiitsasiepptsoiososaanarlsleaaao,nnfddM9PPN4FF0,CCnUgffSa/aAbbgrrwiiwccehaatttiicioohwnnt.iffsaacclPoiiRllciOtiaiStteeessd,inhhjuaaedsdgtgggsdeeooo4mwm]e.ntestrTrtihircciesammmeweaaaonnsf t?rrahareroeefcllyyo8rddmGeeottrreeeaccenttneetddMca((otooaun,lnlyytPaFi11nHooXfLfSa22kw44ea))spiinndlaessstwmweacaaltllleosdoawwminsspaaallmemlpsplbellwesosh.o.idlIeInnplPccaFoosOnnmtStrarAaasssatwtmatt.oso (ccc3ooonennsosciimeddneeettrrraiaabbctilloymyneshhaiinogg=fhhe1e9rr4410tthhnnaaggnn//gg)ccoowfnnecocteemnnwttrLtra.aatktPiieoFoJnOnossShaiiinnnnnatter.rgeegTeeshsss[ww4a]ha.lillglTohohwewirs ceewoggngga.sss ptpihnlleeessgcgfrorsrooommmfLovLiraaakrnkieteodJJuooashhtawaa,anntnPneaFa.rH.bxiPPSrFRdOOwSSsapvsveaadcsstietllteyeycfddtroeoomdmmiiinsnnaoaatutlteelhddbelttrohhnoeedCPhPpFiEClnCaasssmp2pee8acc]sttraarumnumdm- (cScgteeaennttoturrmsaatteibitouorntincsdccemoouueilladdnshh=aa1vvc4ee1bbnueeageb/nngso)vbbefeesr,ccoaaomuruisbsLeeeoacookaffeunttsJhhoeeehtahhhneeeenrrgoao.ngn'Tssshwhhiesiigrghhheieegcrrhottelrrrlooecppcohhtnieiccd- bttinhuheteegGGPgrFrseeOaaoSttfdvLLiaaadkrkieenossout[[s22dw55]o]amaatiessnritbattiddrediidastpinneGcrtrrieeeeeseenfssrMwwooamaullnllsotoowawuisstnhfferrLoraonmkmeC.LLhaaAikknleeasJJ[oo2ihh8naa]cnnanonnanad,, thwswethahaitilruleees,aPPb.FRCuOOotSSlslaaeennecdddtiirrsoeecnlluasaosttesefdidogccrnheaaehbtmobvielceua,elhmoswerwerbiereorcenceasausintasliedlllttblbhreeeeiiesnnegggswgmmasalawnlnuoeufwrfeaeacgcctgotusulrlreeercddotenimdn Crbthrauaistsnt,P,aFPwPOaFFStUUeRndrAAibdwiwranadsosstattdhnhoeedsmesteicanconaontkndedaeraaditriallGyysrtddeheooenmmiiMinsnatoanountntstPPaFEiCnCeccLooamcnnkeggo.eesAnnoteelsrarobiinnuninnssdocodauountntthh- PrietPhgsigeosslaEvEryeeyaee.tLLhCiaokaleqleuaecrsetkitooinonnsge.oofingagreaaannrtddbrrleeuseseuulhlttesosrfofrrnonomamnhtdghetesrseeeoesststuwudidaiielelsosnwsshheoogguugllsddfhhreoellmpp prCpeaehrnriffknlueauodowrrfiaionntueaarrtttbeehiddridccnsaadrrabbonoomdxxiyyinltlairiccnancaakecieiddadm((aPPosFFntCCghAAe)t)fhniienrtsPtthhoeerRGGserriceenCoaastntwdLLaAaamlkkleoeosSsswts2[a2.5b5)]uS,inbbmdiuualttnatirtt r2e0s0oMM7lovofrerreetohrrmieesccfqeeinuvnettellsyGty,rioehhnaeet.rrrrLiinangkgeggsuullhlla((dLLaaarrruuissthaamrreggteeinncttarmtuuessa)) neeggcggossnccceoolnlllteerccattteeiddonnins toctrooa-hnhedkeroerrmdriiinnfnoggaungruuttlhlPllsiCniiAnndstothh,meePiNGnGArareRneaitacttehLLaeaamkrkeeodssnog[(m22it55h.n]e,awwtPheFhedCerrA(eeGsrPPienFeENnsAwAMaowwlulaonswtooaasnni.eenSoLiofmasftkhtihleeae).r 2btfo0ewd0twee7ceeafernnnoem99s11uflifavaoennnddaG5t5re0e0ca77tonnmLggbai/ikggneeswwdehet(atPdwwFtSat..AriSfft)ohorrm[2PP5e]EFt:HiHcXx5SSm9,,0e%PPaoFFnOOfSSctohaanenncPddeFnSpptAerearsrtffilwoluaonos-s- otcorhorwe-wdaaoapssmpraaionccxaooin--mtddaPootFmmeCilAinynsaa,1nnP0ttF-fNPPoFAFlCCdeAAidtih((LfLeeaarrkkdeeonJJmcooehhinaaiannntnnePaadF.)O.(GAFFirianneanaelldnllyyMP,, FiionUnuMccnAootannctitnorrnaaLcsseattkntt.eoo) rPoPoFfFOdO1SeS5.c.saAAintrereiisttshheummxlfeceotetniieccadteemmdeceaotahmnnebccgiooennnoecmcdeeent(nPtrtFraiaStctAiimoose)nnass[2nii5nnc];ohhn>eec9rre0rrni%intnrggoaftggtuiuhlolleln eeP(gg1Fgg4Ss1sAsa3ntwg11a)44s arttrheaeettiaospinwpsairloionlnxGonGiwrmerbesaalattoetLolLaydakke1pe'0l'ss-afhshoemelrdarrriwdinneifggrfgeegurulesllinlmcegieglggaissnr 2[bP25eF5]tO,]w,AetthheaecnnocdtonhPnceFcseUeennnttAtwrraoactoitPonFinCcoAsenSni,sn- oiiunnfttr1r5eaeleessiosstewwbsaealeclllxaooucwweseeeedggheeggdrssrtppiheneegrrhhggaaeupplolssmsbbeaeetrccreiaaceuuismsteehoeeoafrfntrthcheeoseiinirhdceihengintgohthreraertrsiohttronrorop(tp1hhd4iii1ccstlenevagven/lecg,le), rGtGereleeabebtbibsiviwnnekkalt0eloatPwlaF.lO.bAl[[o22too55]d]enaaprttltairrsicbmbhuumatteeewnddetrtthehfeetshihhmeiiglgilhhoaeenrrrgbecceorotwncnchceeaeennintntrrteaahtdteiisoPoennEssCtwAooSoff iPPPFFFeCUUgAnRgAsAs. bmmGriuiegtgarartaalnsnLotatsskbeaaesnncaddyuehhsaeeernnhrcceoeeruraanirrndeegeegxxupplolosssuuarrreee o levated evel either resident or to elevated levels of PECs in the short distance of PFCs in the cCcreaoollmma.ptpiava[rer2ee5tddo],PtoaoFvnOlediAvresKtro.as.neAnAnsarsindcdeheemmtmoaneol.nsntts[rot1r4afa]tt,theeeddebxlbopyynoWsgWuearran-encgghoafeeitttneaaen.ld. d2[Pi25fF8f]Ce],A,rsGGs eaiebnmbbebioginnngkgks. GPFreISIanntcccLoooannnkctteerrsnaastystrteaat0tori-orttonhhsueeinnrrdeae.lldaauttiliotonnrsshehiiapptbbieetttswwieeneeBnnellligivvieeurrmaawnndd hwwhhoeolvleeerrbbclloooeonod-d saeaorrtueeraaaclss.es[,mm2o5os]s,tt likely because and Kannan et likely because ofal. of diferent exposure. [14], exposure often different exposure pathways and differs among pathways and CocPcneeFcnnOettSnrraatcttroiioanotnncisseonwwntreeairrtneeiobin11os00oidttniimampdeeaussslmtggargreteraeawtateeetrrrteitttthshwaaiinnncBeiinnelgbbhliluooiomogdd,h,wswwhneeefrieffvooleiuurvnnteddirscttsohhunaaet-t, sources. 42. Effcs "TceToxhhpneelcddaeiiinffnfteeerrdareetbinnoyccneetssshiebbneesbttalwwomeoepeedlnneptlthmahasetmrttiiiattxwaa(nnewddrhesostwlwweaaillcbllelooowawosdddhaavigtteaahrsmmausasaiyynpbbllieeavsepprmaaratrit)tsiisaabullulleyty. 4.2. Effects There was a significant negative association between hatching. effnuuxarrpnttlhhaeeecirnraett.deess[btt3iyi]nngtfghoeiiusnsnndaeemteehpddaleeteddcmottonoactfrefiuunxlltllry(ywaetehxixopoplnlleasaiibwnnleottrohhedeess2vee-e5rddsiiutffffiseemrpreeelsnancschmeeissga..h)e,KKrabanuni-n-t STsouutccaTccleehsshessaretaacnnwhddiansccgooannfcscaieieglnnnuttrirrfeaiascttiaboinenotgnsanooneffgPtaPRotFoiOOvcSSecuaiirnnsasssotwwcaia=all1tllio5oow0wnneebgggeglgtsgws PieinnFeOntthShhiisswaetsscttthuudidwnyy..g. nan et al. [3] found that concentrations were 2-5 times higher in Total hatching failures began to occur at >150 ng]g PFOS wet wt. 33445522..00000066 a COCurt Ar olyt 32012e )356-52 562 CM. Custer et al. / Reproductive Toxicology 33 (2012) 556-562 hesasmizpwalssmeall however stheseresults shouldbe sed [2] HT. ndeViperK Die Cons ANv ec The sample size was small, however, so these results should be used HuSiesaatttheatneesssnmaayrbeeconsfoundde--dye--bny--e am--kspoos --wn rof u-- Soret ts epost SR with caution. Additionally, there is always the possibility with field nmea. 1 wiecamnsdoo 1 studies that results may be confounded by unknown or unmeaSed cauTshiesesf.t evel however, was simila0r the dosage [ora Toc. Bvt Wh ch. es, J. sured causes. This effect level, however, was similar to the dosage imine in an cg econ sty of WHE phon chick salemc rot A gb ismd administered in an egg injection study of white leghorn chickens Gallas domestics in whaisgncicahnt eduction percent 1 SomeEos masa CL ens (Gallus domesticus) in which a significant reduction in percent cing began at 100mgwet. (791, Stns (291ml ese ech ek Bs oe hatching began at 100 ng/g wet wt. [7,9]. Bustnes et al. [29] did not find an somo between RC concniraions whole boodal | Br Ue 04 find an association between PFC concentrations in whole blood of dt nd reproductive endpoints na held studyof ese back. 1 MCA(less oon coh Rr) YC Mme am adults and reproductive endpoints in a field study of lesser blackbWerahtgucdlilrse(cktalryuCeofmuspcdaursa),blAeltehotugnhsaTmphliuanlgmtatryiceasnadnthdeacguers. 16] S RoweTCodHSchpk op aGll i . GeMp. cs f backed gulls (Larus fuscus). Although sampling matrices and ages [2] Hoff PT, Van de Vijver K, Dauwe T, Covaci A, Maervoet J, Eens M, et al. Evaluation of biochemical effects related to perfluorooctane sulfonic acid exposure in organohalogen-contaminated great tit (Parus major) and blue tit (Parus caeruleus) nestlings. Chemosphere 2005;61:1558-69. [3] Kannan K, Franson JC, Bowerman WW, Hansen KJ, Jones PD, Giesy JP. Perfluorooctane sulfonate in fish-eating water birds including bald eagles and albatrosses. Environ Sci Techno12001 ;35:3065-70. [4] Custer TW, Kannan K, Tao k Yun SH, Trowbridge A. Perfluorinated compounds and polybrominated diphenyl ethers in great blue heron eggs from three colonies on the Mississippi River, Minnesota. Waterbirds 2010;33:86-95. [5] MPCA (Minnesota Pollution control Agency). PFCs in Minnesota's ambient environment: 2008 Progress Report. Available from: http://www.pca. state.mn.us/publications/c-pfcl-02.pdf. were not directly comparable between that gull study and the cur- ent stutcdemyst.hatexposuinrtehast study wereconsiderably Fetueiocans ontonnalardaodhie icgi xposed rent study, it seems that exposures in that study were considerably Jes thanor ee Swallows nestiong ake Johanna, News etal. pS re ST less than for tree swallows nesting on Lake Johanna. Newsted et al. {51a found es fects o reproduce cng ast of Hake ACA Mn ede [6] also found few effects on reproductive endpoints in a study of owe qual and mallard duck. uc birds a hehigher dosages KL cen(sE ons iyE minToi bobwhite quail and mallard ducks, but birds at the higher dosages ied o were cuthanized before hey cod ay gsa0 ects 3s) SLESkSo22 Sey.,Weta Vergo died or were euthanized before they could lay eggs so effects at higher ex Concentrations could not be assessed. Serum concen: Pip ec HAYSAIC HT CB higher egg concentrations could not be assessed. Serum concen- EE teat osmteieaLi trations of PFOS in the 10/~g/g dosage group (8.7 and 16.6/~g/ml In bloc enumof ene al and matrd) (61 were conser. py Serine LL in blood serum of female quail and mallard) [6] were consider- aby higher than acalculated ive for adults on Lake Johanna. so" 108 yn dose ben Eas ol os cho ie ably higher than a calculated level for adults on Lake Johanna, so Teprodutne acs might hae been pected bused on thatAs rt meme eS pa reproductive effects might have been expected based on that. As in hat same study of mallards nd qual we found few histology SE CogmA n PECT SHOE in that same study of mallards and quail, we found few histolog- iiccaall ppaatthhoollooggiieess iinn lliivveerr ttiissssuuee aaltlhotuhghoaauttrrgeennhdd mmaayyhhaavveeeexxiisstteedd wards more snore stLakJohanna compared tothe Me Cy Msochocs Uk 195-50 ven To en towards more abnormalities at Lake Johanna compared to the ref- ICE ke AR TEN SATIS Of TACTOPMBE OU 50311 Eyth me eb eh erence lake. An increased abundance of macrophages could be a ignof ammationo er sess. [Epi gt op sign of inflammation or liver stress. 5. Conclusions 12 a a F R e Sia t gssvt CRoont. 5. Conclusions Concnearny alltPFCawrieceovtend mtsr sowalflow 1] tS pkey 0. fy si scan by ave sons. Ak Concentrations of nearly all PFCs were elevated in tree swallow issues fomLake Johanna Compared tothe nay reerence lake, | eseatssa 0 SEOE PE tissues from Lake Johanna compared to the nearby reference lake. PFOSwasthe dominant PC (75%)a Lake Johanna, butaccounted Kh 5, 3L0 sJSi. es & SJeFIkme: PFOS was the dominant PFC (>75%) at Lake Johanna, but accounted for SofXol PFC thereference ak, Became hevarition cmd dnt rd HerriEYn for <50% oftotal PFCs at the reference lake. Because ofthe variation insted lect eves rom oratoryand ed sudes andthe | STpnEEios CT in postulated effect levels from laboratory and field studies and the elie Smal sample se. (25 nso fr the SNC logis. 1) SST relatively small sample size (<25 nests) for the significant logisHe egesia in ts St, we recommend further eld uicsto | YADA tic regression in this study, we recommend further field studies to kite th ed cleomtr wat ound the presen 17]FEempl dace validate the field effect levels in tree swallows found in the present ud Srworkls Ongat wallow SY OCHS OPO2 1g) eleAeae ery es study. Similar work is ongoing at swallow study locations on Pool 2 nig EyeLake lon he Missi) River nar putative sources Sie Eko on and Pigs Eye Lake along the Mississippi River near putative sources PPC rin Snof these rs 5 ps EoE n Sets chen A es cs. h--on,--bl of PFCs so further testing of these results is possible. [6] Newsted J, Coady KK, Beach SA, ButenhoffJL, Gallagher S, Giesy JP. Effects of perfluorooctane sulfonate on mallard and northern bobwhite quail exposed chronically via the diet. Environ Toxicol Pharmaco12007;23:1-9. [7] Molina ED, Balander R, Fitzgerald SD, GiesyJP, Kannan K, Mitchell R, et al. Effects of air cell injection of perfluorooctance sulfonate before incubation on development of the while leghorn chicken (Gallus domesticus) embryo. Environ Toxicol Chem 2006;25:227-32. [8] O'Brien JM, Crump D, Mundy LJ, Chu S, McLaren KK, Vongphachan V, et al. Pipping success and liver mRNA expression in chicken embryos exposed in ovo to C8 and C11 perfluorinated carboxylic acids and C10 perfluorinated sulfonate. Toxicol Lett 2009; 190:134-9. [9] O'Brien JM, Carew AC, Chu S, Letcher RJ, Kennedy SW. Perfluorooctane sulfonate (PFOS) toxicity in domestic chicken (Gallus gallus domesticus) embryos in the absence of effects of peroxisome proliferator activated receptor alpha (PPAR~)regulated genes. Comp Biochem Physiol C 2009;149:524-30. [10] cChuesmtiecraClMc,oCntusatmerinTaWno,tnDsutmreme esrwaPlMlo, wMs unensnteiyngaKlL.onEgxpthoesuHreouasnatdoneifcfecRitsvero,f chemical contaminants on tree swallows nesting along the Housatonic River, Berkshire County, Massachusetts, USA, 1998-2000. Environ Toxicol Chem 2003;22:1605-21. [ 11 ] Custer CM, Custer TW, Rosiu CJ, Dummer PM, Melancon MJ, Bickham JW. Expo- sure and effects of dioxins, furans, and other organochlorine chemicals on tree swallows (Tachycineta bicolor) nesting along the Woonasquatucket River, Rhode Island. Environ Toxicol Chem 2005;24:93-109. [ 12 ] Mengelkoch JM, Niemi GJ, Regal RR. Diet of the nestling tree swallow. Condor 2004;106:423-9. [13] Quinney TE, Ankney CD. Prey size selection by tree swallows. Auk 1985;102:245-50. [14] Kannan K, Corsolini S, Falandysz J, Oehme G, Focardi S, Giesy JP. Perfluorooctanesulfonate and related fluorinated hydrocarbons in marine n~an~mals, fishes, and birds from coasts of the Baltic and the Mediterranean Seas. Environ Sci Techno12002;36:3210-6. [15] Carson FL. Histotechnology. Chicago: ASCP Press; 1996. [16] Wolf JC, Wolfe MJ. A brief overview of nonneoplastic hepatic toxicity in fish. Toxicol Patho12005;33:75-85. [ 17] Ferguson HW. Systemic pathology offish: a text and atlas of comparative tissue responses in diseases of teleosts. Ames: Iowa State University Press; 1989. [18] Clarke Pd(, Warwick RM. Nonmetric multivariate analysis in community-level ecotoxicology. Environ Toxicol Chem 1999; 18:118-27. [19] Mayfield H. Nestling success calculated from exposure. Wilson Bull 1961 ;73:255-61. [20] Mayfield H. Suggestions for calculating nest success. Wilson Bull [Tiheeausthors have noconicsof ners that wohvue ndap. CCoonmfilicctooff intteerreessttssttaatteemmeenntt The authors have no conflicts of interest that would have inappropriately influenced the work presented in this manuscript. Aeknawiedgments Acknowledgments WethaMonraky, PjarksoRehcrestnion Dep. RamseyCo: We thankJohn Moriarty, Parks & Recreation Dept., Ramsey Co.; John Elim and eer Mot, public Works Dept. Wasingion Ca John Elholm and Peter Mott, Public Works Dept., Washington Co.; Mat Brow, Northweste CollegeSt,.Paul MN nd HERG Nl. Matt Brown, Northwestern College, St. Paul, MN, and Helen C. NelSn. Bu NN fo allowing u tr Fave eS. bin up on thelr son, Buffalo, MN for allowing us to have nest boxes up on their property: ul Dummer for held and technical assistance Emily property; Paul Dummer for field and technical assistance; Emily Met for histological assistance,Jef Bemardy and Cynthia Tomey Metz for histological assistance, Jeff Bernardy and Cynthia Tomey Tor answeringchemistry Questions, nd Christan Franson, Kevin for answering chemistry questions, and J. Christian Franson, Kevin Keno, nd two anonymous reviewersfocomments on ale raotftshe manUSEript, Useoftrade, produorcHtm, names does. P. Kenow, and two anonymous reviewers for comments on earlier drafts of the manuscript. 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