Document VK2Q3J0E7aN6xen44mm4pazQg

P-1 "McCrea, Deborah" <mccrea@taftlaw.com> 0 3 /0 6 /2 0 0 9 02:34 PM To N C IC O P P T @ E P A cc "Bilott, R obert A." < bilott@ taftlaw .com > bcc S ubject Subm ission to E P A -H Q -O P P T -20 03-0 01 2 Taft / Deborah McCrea / Legal Assistant Taft Stettinius & Hollister LLP 425 Walnut Street, Suite 1800 Cincinnati, Ohio 45202-3957 Tel: 513.381.2838 Fax: 513.381.0205 www.taftlaw.com / mccrea@taftlaw.com A d -^ -Q - Internal Revenue Service Circular 230 Disclosure: As provided for in Treasury regulations, advice (if any) relating to federal taxes that is contained in this communication (including attachments) is not intended or written to be used, and cannot be used, for the purpose of (1) avoiding penalties under the Internal Revenue Code or (2) promoting, marketing or recommending to another party any transaction or matter addressed herein. This message may contain information that is attorney-client privileged, attorney work product or otherwise confidential. 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If you received this transmission in error, please notify the sender by reply e-mail and delete the message and any attachments. 1570_001.pdf CONTAINS NO CP' >r i ' j * , .. *' t t P-2 0oN Sn IV1N00 P-3 Taft/ Taft Stettmius & Hollister LLP 425 W alnut Street, Suite 1 8 0 0 /Cincinnati, OH 4 5 2 0 2 -3 9 5 7 /T e l: 5 1 3 .3 8 1 .2 8 3 8 /F ax: 5 1 3 .3 8 1 .0 2 0 5 /w w w .taftlaw .com Cincinnati / Cleveland / Columbus / Dayton / Indianapolis / Northern Kentucky / Phoenix / Beijing Ro b erta Bilott 513- 357-9638 '.com March 6 ,209 FEDERAL EXPRESS EPA Docket Center, MC 2822T U.S. Environmental Protection Agency EPA West, Room 3334 1301 Constitution Avenue, NW Washington, D.C. 20004 Re: Submission to IRIS and AR-226 Database For PFOA/PFOS: EPA-HQO R D -2003-0016 To IRIS Database for PFOA/PFOS: In response to the Notice issued by USEPA on February 23, 2006, regarding USEPA's efforts to consider perfluorooctanoic acid ("PFOA") and perfluorooctane sulfonate ("PFOS") within the Integrated Risk Information System ("IRIS"), 71 Fed. Reg. 9333-9336 (Feb. 23 ,2 0 06 ), we are submitting the following additional information to USEPA for inclusion in that review, and for inclusion in the AR-226 database: 1. Costa, G., et a!., "Thirty Years of Medical Surveillance in Perfluorooctanoic Acid Production Workers," 51 J.O.E.M . 1-9 (March 2009); and 2. Joensen, U.N., et al., "Do Perfluoroaikyl Compounds Impair Human Semen Quality?," Environ. Health Persp. (doi: 10.1289/ehp.0800517) (online March 2 ,2 0 0 9 ). RAB:mdm Enclosures /y ' Robert A. Bilott 11361467.1 CONTAINS N O OP March 6, 2009 Page 2 cc: Gloria Post (NJDEPXw/ enei.) (via U.S. Mail) Heien Goeden (M DH)(w/ enei.) (via U.S. Mail) Lora Werner (ATSDR)(w/ enei.) (via U.S. Mail) {W 1405808.1} JOEM Volume 51, Number 3, March 2009 i P-5 Thirty Years o f Medical Surveillance in Perfluooctanoic Acid Production Workers Giovanni Costa, MD Sam antha Sartori, BS Dario Consonni, M D Objective: To report health outcomes o f 30 years (1978-2007) of medical surveillance of workers engaged in a perfluooctanoic acid (PFOA) production plant. Methods: Fifty-three males workers (20 to 63 years) were submitted every year to medical examination and blood chemical chemistry tests, and serum PFOA dosage. Results: In the latest survey PFOA serum levels rangedfrom 0.20 to 47.04 xg/mL in currently exposed workers, and from 0.53 to 18.66 xg/mL in thoseformerly exposed. No clinical evidence ofany specific trouble or disease has been recorded over the 30 years, and all the biochemical parameters, including liver, kidney and hormonalfunc tions, turned out to be within the reference ranges, but a significant association of total cholesterol and uric add with and PFOA serum level was evidenced. Conclusions: A probable interference o fPFOA on interme diate metabolism deservesfurther investigations. (J Occup Environ Med. 2009;51:000-000) From the Department of Occupational and Environmental Health (Mr Costa. Mr Sartori), University of Milano. Italy; Unit of Epidemiology (Mr Consonni). IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milano. Italy; and Institute of Medical Statistics (Mr Sartori) and Biometrics "G.A. Maccacaro," University of Milano, Italy. Address correspondence to: Prof. Giovanni Costa, Department of Occupational and Environmental Health, University of Milano, Clinica del Lavoro "L. Devoto," Via S. Bamaba 8, 20122 Milano; E-mail: giovanni.costa@unitni.it Copyright 2009 by American College of Occupational and Environmental Medicine DOI: 10.1097/JOM.0b013e3181%Sd80 erfluorooctanoic acid (PFOA, C7F l5COOH, CAS No. 335-67-1) is a fully fluorinated carboxylic acid, widely used as emulsifier in fluoropotymer polymerization. It is solu ble in water, where it forms a mixture with its dissociated perfluorooctanoate anion (C7F15COO_); it is absorbed by oral, dermal and inhalation routes, and mainly bound to serum albumin. It is mostly found in liver, serum and kid ney, and is excreted, not metabolized, mainly in urines, with large differences in elimination speeds between species and sexes. It is a mild irritant for skin and mucoses, it is not a sensitizer and has neither teratogenic nor fetotoxic effects, but it delays sexual maturation in animals.1"3 It is not a genotoxic, but in two chronic studies in rats it has been reported to increase the incidence of tumors (adenomas) of liver, pancre atic acinar cells, and testicular Ley dig cells,6-7 like other peroxisome proliferators, as it acts mainly as a peroxisome proliferator-activated re ceptor alpha (PPARa) agonist.8-9 In recent years, many studies have been carried out with the aim of better understanding the toxico kinetics and mode of actions, al though with not clear and definite conclusions.10-17 Its half-life of elimination in se rum markedly differs between spe cies and sexes, being 1 to 9 days in rats, 18 days in mice, 20 to 40 days in dogs, 20 to 32 days in monkeys, and 3.8 years in humans.18-20 Hence, its biopersistence in the human blood, its detection in the general population21-24 and its presence in several biota and animals all over the f P-6 2 Medical Surveillance of Workers Exposed to PFOA Costa et al world25-27 are a concern for possible although this association was con chronic effects.28,29 founded by body mass index. Epidemiological studies on large Extending the survey in the same cohorts of occupationally exposed production plant to 265 workers for a workers, mainly of US plants (3M longer period,37 a negative associa and DuPont) in the period 1947-- tion was recorded between serum 2002, could not find any association between PFOA exposure and signif icant adverse health effects in terms of morbidity and mortality.30-34 For mortality in particular, neither Gilli land and Mandel31 nor Alexander32 and Leonard et al33 found excess of death for cancers of any type in quite large 3M and DuPont cohorts. Also the slight excess of prostate cancer signaled by Gilliland and Mandel,31 and of bladder cancer found in the first study by Alexander,32 were not confirmed by subsequent more spe cific surveys both in 3M34 and Du Pont33 workers. However, some contrasting and somewhat inconsistent findings, mainly related to lipid metabolism, have been reported as concerns per turbation o f biochemical variables. The first study, made by Ubel et al30 in 1980 in about 300 workers of 3M Cottage Grove plant, having total serum organic fluorine (90% PFOA) up to 71 p.g/mL, did not report any significant deviations from the nor mal values as concerns hematology, liver enzymes, total cholesterol, glu cose, and uric acid. In a subsequent study on 115 workers o f the same plant,35 after adjusting for age, smoking, alcohol intake and BMI in a multivariate regression model, a total serum flu orine level above 10 ppm was asso ciated with slight increases in hepatic PFOA and plasma Cholecystokinin (although inside the normal range), and no effect on hepatic enzymes, bilirubin, triclycerides, HDL, and LDL cholesterol. However, in a subsequent survey concerning 518 workers of both sexes from two US and European plants,38 the same authors found a modest positive association between PFOA and cholesterol and triglycer ides in both cross sectional and lon gitudinal (over a 6 year time period) analysis, after adjusting for potential confounders, such as age, BMI, smoking, alcohol, and location. More recently, Olsen and Zobel39 have re-examined the same parame ters in 506 3M workers from three US and European 3M plants, by multiple and logistic regressions and analysis of covariance: serum PFOA concentrations were negatively asso ciated with HDL and bilirubin, and positively with triglycerides, after adjusting for possible confounders. Very recently two reports, one lon gitudinal and another cross-sectional, on DuPont production workers have been published. In the retrospective study of 454 production workers, with repeated measurements con cerning liver function and lipids over a period of 25 years,40 a positive association between serum PFOA and total cholesterol and AST was enzymes through an interaction with found, whereas a negative associa obesity. A slight interference with tion with bilirubin, after adjustment HDL cholesterol was also observed, for age, BMI, gender, and decade of when in association with alcohol hire, but without control for lipid consumption. lowering medications. In other two cross-sectional stud In the other cross-sectional study ies on 191 3M production workers,36 on 1025 active workers,41 including serum PFOA was not significantly hematology, liver and renal function, associated with any of the 11 mea lipid and purine metabolism, and sured hormones, except for a 10% hormones, a modest but statistically increase in mean estradiol level significant positive relationship be among employees having the highest tween PFOA and total cholesterol, levels of serum PFOA (>30 ppm), LDL, VDLD, GGT and uric acid was recorded, after excluding people tak ing lipid lowering medications. The present study is aimed at providing a further epidemiological contribution to the understanding of possible effects on humans. The data refer to the workers of a chemical plant (Miteni, Trissino, It aly), where PFOA has been produced by electrochemical fluorination since 1968. Since 1978 all the workers engaged in PFOA production have been submitted every year to the medical surveillance program by an expert Occupational Health Physi cian, according to the Italian legisla tion concerning health and safety at work. Materials and Methods Subjects As a whole, 53 workers, all males, engaged in the PFOA production de partment have been examined every year from 1978 to 2007, for a total of 919 persons-years. Subjects' age ranged from 20 to 63 years and length of work exposure varied from 0.5 to 32.5 years in the period. At the latest PFOA biomonitoring, in 2007, 37 were active workers in the PFOA department, whereas 16 were no longer exposed being retired or transferred to other departments in the meantime. All the other male workers, 12 executive clerks and 95 blue collars from the other departments of the company, who have never been ex posed to PFOA but submitted to the same periodical medical surveil lance, were considered as "controls." Table 1 summarizes the main characteristics of the examined workers. Methods All the workers (exposed and not exposed) have been submitted eveiy year to physical examination, includ ing recording of blood pressure, height and weight, and to blood chemical chemistry tests, including hematology (Ht, Hb, WBC, RBC, PLTS), liver (albumin, a r a r |3-y p .7 JOEM Volume 51, Number 3, M ardi 2009 3 globulins, bilirubin, AST, ALT, ALP GGT) and renal (urea nitrogen, cre atinine) functions, glucose and lipid metabolism (total and HDL Choles terol, triglycerides), and uric acid. All the variables have been measured over the years by autoanaiysers (SMA) in the same laboratory of the nearby regional public general hos pital, undergoing a periodical quality control program. In addition, in the workers of the PFOA department, also Apo-A and Apo-B lipoproteins, protein C reac tive, immunoglobulins IgA-IgGIgM, Testosterone, Estradiol, FSH, TSH, FT3, FT4, and Prostatic Serum Antigen (PSA) were tested in blood in 2002 and 2006. The biological monitoring of se rum PFOA started in April 2000 and was repeated in May 2001, Decem ber 2001, June 2002, September 2003, September 2004, May 2006, and May 2007. It included not only the workers of the PFOA department currently exposed in those years, but also those formerly exposed who had been transferred to other departments or had retired in the meantime, as well as some non-exposed workers (clerks), taken as controls. PFOA serum levels were deter mined by HPLC-Electrospray-Tandem Mass Spectrometry42 in two internationally accredited laborato ries: the first one carried out the analysis in 2000,2001, and 2002, the second one in the following years. A double blind comparison between the two laboratories carried out in 2003 gave a correlation index equal to 0.90. In 2000 and 2001, the method had an upper scale detection limit fixed at 45.5 pg/mL, thereafter no detection limit was present. Statistical Analysis ANOVA. t test and multiple linear regression models were used to eval uate the relationships between PFOA exposure and hematochemical ef fects while adjusting for relevant co variates. Natural log transformation of the dependent variables was per formed where appropriate. General TABLE 1 Demographic Characteristics of the W orkers Examined PFOA Currently PFOA Previously W orkers Exposed W orkers No Sex Age (mean and SO)* Years of work (mean and SO)* Years elapsed since PFOA exposure (mean and SD)* Person-years of observation BMI (mean and SO) Smoking (%) Alcohol consumption (%) 37 All males 4 1 .6 (8 .3 1 ) 1 4 .3 (9 .1 ) 487.2 26.2 (2.9) 38.2 73.5 16 All males 52.0 (8.7) 16.3(9.9) 9.5 (8.3) 431.8 25.7 (3.1) 47.6 71.4 At the latest medical check. C o n tro l Group 107 An males 4 1 .9 (9 .2 ) 14.0(9.3) 1499.3 25.1 (2.8) 36.5 62.6 ized Estimating Equations (GEE) models with an exchangeable corre lation matrix were fitted when ana lyzing repeated measurements and relationships between PFOA serum level and the different (continuous or dichotomous) health outcomes, while accounting for within-subject correlations.43 In the regression models the following covariates were included: age, years of expo sure, year of PFOA sampling, BMI, smoking, and alcohol consumption. Stata 1044 software was used to perform all the statistical analyses. TABLE 2 PFOA Serum Level (pg/mL), Recorded in the Year 2007, in Currently and Formerly Exposed Workers Currently Form erly Exposed Exposed No. Min 25th percentile Median Mean arithmetic Mean geometric 75th percentile Max Standard deviation 39 0.20 2.25 5.71 12.93 4.02 23.55 47.04 14.43 11 0.53 2.61 4.43 6.81 3.76 9.24 18.66 6.06 Results From die clinical point of view, all the workers w oe in good health and no excess of morbidity for any disease has been recorded over the years. In the latest survey, carried out in May 2007, the PFOA serum levels ranged from 0.20 to 47.04 pg/mL (ppm) in the currently exposed work ers, and from 0.53 to 18.66 pg/mL in the formerly exposed workers (Table 2): in the seven non-exposed subjects (clerks/staff) examined in 2006, the PFOA serum levels ranged from 0.05 to 0.181 pg/mL. The highest level recorded was 91.9 pg/mL in 2002. Table 3 shows the PFOA serum levels recorded over the years in the production workers. As above mentioned, in 2000 and 2001 the method had an upper scale detec tion limit fixed at 45.5 pg/mL; conse quently, in order to compare the data over the years the maximum level in 2000 and 2001 was set by default as the highest level record! in 2002, when the method had no upper scale detection limit. A significant decrease of both peak and mean levels was recorded after 2002, following a renovation of the plant carried out in that year, including a partial automation of the process, and the adoption of more strict working procedures and use of suitable protective devices. In the 27 currently production workers who had their serum PFOA measured in all the four last occa sions, median value decreased from 13.6 pg/mL in 2002 to 7.1 pg/mL in 2007 ( --47.8%), mean value de creased from 22.2 pg/mL to 14.0 pg/mL (-37.0% ), and peak level decreased from 86.3 pg/mL to 47.0 pg/mL (-45.5% ), respectively. AH the biochemical parameters, turned out to be on average within p .8 4 Medical Surveillance of Workers Exposed to PFOA Costa et al the laboratory reference ranges, while some subjects had some pa rameters above the upper reference values, as shown in Table 4, which refers to the results of the latest check carried out in 2007. As com pared with the control group some differences, both in terms of mean TABLE 3 Serum PFOA Levels (pg/m L) Recorded in Production W orkers Over the Year 2000 2001 2002 2003 2004 2006 2007 No. Min 25th percentile Median Mean arithmetic Mean geometric 75th percentile M ax Standard deviation 25 1.54 5.53 11.92 18.8 11.7 32.0 86.3* 20.0 42 0.73 4.35 11.07 19.7 10.2 19.72 91.9* 23.6 46 0.34 4.57 10.15 19.3 9.3 20.80 91.9 23.0 41 0.38 4.11 6.25 13.7 6.9 14.20 74.7 16.6 34 0.54 2.84 6.82 11.4 6.5 18.97 46.3 12.0 49 0.54 2.36 5.27 10.8 5.8 16.31 41.9 11.8 50 0.20 2.18 3.89 11.6 5.4 18.66 47.0 13.3 ` Upper scale detection mit at 45.5 m j/m L (discussed in text). values and number of persons above the upper references limits, were noted for some parameters such as uric acid, cholesterol, triglycerides, and liver enzymes. As the crude correlations between PFOA and the biochemical variables, recorded in the last 7 years, gave some indication on a possible asso ciation between PFOA serum levels and some lipids and uric acid (data not shown), three more in-depth analyses in this respect were per formed. The aim was clarifying this possible association by controlling the most important confounding fac tors, and after preliminary exclusion TABLE 4 Haem atology: Results of the Last Check in the Currently Exposed Workers and Control Group Exposed Control Group Glucose (mg/dL) Urea nitrogen (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Cholesterol total (mg/dL) Cholesterol HOL (mg/dL) Triglycerides (mg/dL) Bilirubin total (mg/dL) Bilirubin conjugated (mg/dL) AST (U/L) ALT (U/L) 7GT (U/L) ALP (U/L) Proteins total (g/dL) Albumin (%) a1 globulins (%) a2 globulins (%) globulins {%) y globulins (%) WBC (X 109/L) RBC <X10,2/L) Hb (g/dL) H t (%) Platelets (X10/L) Protein C reactive (mg/L) Apo A (g/L) Apo B (g/L) igG (g/U IgA (g/U IgM (g/L) PSA (ng/mL) Testosterone (ng/mL) Estradiol (pg/mL) TSH (U I/m U FT3 (pg/mL) FT4 (ng/dU 0 CO 1 d Reference Values M in-M ax 7 0 -1 1 0 7 -2 2 0 .8 0 -1 .3 0 3 .5 -7 .2 191-240 >39 3 0 -1 8 0 < 1.0 < .S 0 <50 <50 8 -6 1 4 0 -1 2 9 6 .4 -8 .2 5 2 -6 7 3 -7 5 -9 5 -1 2 1 0 -2 2 4 .0 -1 0 .9 4 .5 -5 .9 13.5-17.5 4 1 -5 3 1 4 0 -4 4 0 0 .0 -5 .0 1 .1 0 -2 .0 5 0 .5 5 -1 .4 0 7.0 -1 6 .0 0 .7 0 -4 .0 0.4 0-2 .3 0 < 4.0 1 0 -5 0 0 .2 7 -4 .2 0 2 .0 -4 .4 0.8 0-1 .7 0 M ean <SD) 92.7 (8.7) 15.2 (3.1) 0 .9 2 (0 .1 2 ) 6.2 (1.1) 239.7 (50.9) 55.9 (14.7) 178.9(118.6) 0.58 (0.22) 0 .1 5 (0 .0 5 ) 32.4 (8.8) 47.8 (22.3) 53.8 (46.7) 71.8 (22.8) 7.71 (0.50) 6 1 .6 (3 .0 ) 4.1 (0.9) 8.9 (1.1) 5.9 (0.9) 1 4 .7 (2 .6 ) 7.02 (2.13) 5.06 (0.37) 15.6(0.88)1 45.9 (2.2) 2 4 1 .3 (5 5 .0 ) 2 .5 9 (4 .2 1 ) 1 .4 3 (0 .2 4 ) 1.25(0.31) 1 0 .6 (2 .4 ) 3.1 (1.2) 1 .2 (0 .7 6 ) 0.80 (0.94) 5.88 (0.57) 3 0 .8 (1 .3 ) 1.99(5.48) 3.62 (0.67) 1.31 (13.5) % Outside Reference Range 2.6 0 0 17.9 35.9 0 7.7 0 2 .6 2.6 17.9 28.2 0 1 0 0 12.8 0 0 2 .6 0 2.6 0 0 5.1 5.1 5.1 2.6 12.8 5.1 2.6 5.1 2.6 2.6 2.6 2.6 M ean (SO) 89.3 (16.5) 15.2(3.0) 0 .9 3 (0 .1 1 ) 5.7 (1.2) 2 1 3 .5 (3 9 .7 ) 56.7 (12.3) 146.3 (97.9) 0.67 (0.27) 0 .1 8 (0 .1 1 ) 29.7 (10.7) 4 0 .6 (2 1 .8 ) 44.5 (41.4) 6 7 .7 (1 9 .6 ) 7.81 (0.56) 62.3 (3.9) 5.5 (9.4) 8.8 (1.4) 5.8 (0.5) 14.7 (2.3) 6 .9 6 (1 .8 8 ) 5.11 (0.39) 15.7 (0.86) 46.0 (2.4) 237.1 (54.4) % Outside Reference Range 6.5 0.9 0 4.7 26.2 6.6 27.1 10.5 7.6 4.8 26.2 17.9 0 0 0 0.9 14 0 0 5.6 1.8 0.9 0 0 P-9 JOEM Volume 51, Number 3, March 2009 o f people under treatment of primary hyperiipidemias and with a history of chronic hepatitis. In the first analysis. 34 currently exposed workers were matched with 34 workers from the other depart ments with the same age ( l year) and job seniority ( l year) in the company, never exposed to PFOA, having the same working hours (shift work or daywork), and being resi dent in the same area with similar housing and living conditions. Table 5 shows that the two groups signifi cantly differ only for total choles terol and uric acid mean levels, being slightly higher in workers exposed to PFOA. In the second analysis, the same 34 currently, exposed workers were compared with all the other 107 male workers o f the company. The multi ple regression analysis, adjusted by age, job seniority, body mass index, smoking and alcohol consumption, confirmed a significant effect of PFOA exposure as concerns an in crease in mean total cholesterol and uric acid (Table 6). The third analysis considered the 56 subjects (currently, formerly and never exposed) who had serum PFOA assessed concurrently with the biochemical parameters in the last 7 years. Multivariate GEE mod els (including age, job seniority, body mass index, alcohol consump tion, and years of observation as potential confounders) show that to tal cholesterol and uric acid levels were weakly but significantly corre lated to serum PFOA (Table 7) as well as some liver enzymes and alpha2 globulins; however, total biliru bin appears to be inversely related to PFOA. As expected, also body mass in dex was found to be significantly correlated to total (Coef. = 0.028, P < 0.01) and HDL (Coef. = -.029, P < 0.001) cholesterol, triglycerides (Coef. = 0.065, P < 0.001) and uric acid (Coef. = 0.022, P < 0.001), as well as with some liver enzymes (ALT, ALP), suggesting a possible interaction, like in previous surveys.35 TABLE 5 Comparison Between 34 Exposed and Non-Exposed Workers, M atched By Age, W ork Seniority, Day/Shiftwork, and Living Conditions N o n -E x p o s e d Exposed f Test_______P No. BMI Glucose (mg/dL) Urea nitrogen (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) Triglycerides (mg/dL) Total bilirubin (mg/dL) AST (U/L) ALT (U/L) GGT (U/L) ALP (U/L) Total proteins (g/dL) Albumins (%) ot1 globulins (%) 2 globulins (% ) globulins (%) 7 globulins (%) WBC (X10/L) RBC (X 1012/L) Haemoglobin (g/dL) Haematocrlt (%) Platelets (x 10A) 34 25.94 89.41 14.82 0.93 5.73 206.4 56.68 150.03 0.58 31.62 42.74 48.52 69.41 7.72 62.87 3.82 8.76 5.68 14.44 6.64 5.08 15.69 45.86 233.03 34 26.16 93.56 19.47 0.94 6.29 237.0 57.82 155.35 0.55 31.44 35.29 43.88 67.65 7.67 61.51 3.94 8.90 5.76 14.47 7.53 5.00 15.37 45.36 238.97 0.34 1.30 1.14 0.19 2.10 3.06 0.21 0.77 0.18 0.41 1.35 0.05 0.24 0.22 0.99 059 0.20 0.49 0.10 1.86 0.60 0.40 0.74 0.84 NS NS NS NS 0.039 0.003 NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS TABLE 6 Multiple Regression Analysis Comparing the 34 Exposed Workers (E) W ith All the Other 107 Workers (ALL) o f the Plant Param eter Coef. (E vs M L ) 95% C .I. P Glucose (mg/dL) Urea nitrogen (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) Total cholesterol (mg/dL) HDL cholesterol (mg/dL) Triglycerides (mg/dL) Total bilirubin (mg/dL) AST (U/L) ALT (U/L) GGT (U/L) ALP (U/L) Total proteins (g/dL) Albumin (%) a1 globulins (%) 2 globulins (%) 8 globulins (%) y globulins (%) WBC (X109/L) R B C (X 1012/L) Haemoglobin (g/dL) Haematocrlt (%) Platelets (X10*/L) 3.53 3.92 0.004 0.50 21.7 2.42 -.1 5 -0 .0 9 155 -5 .1 8 0.32 -0 .7 8 -0 .2 0 -0 .7 3 -1 .8 2 0.27 -0 .0 0 3 -0 .5 3 0.58 -0 .0 8 -0 .2 7 -0.5 1 1.31 -2 .2 1 /9 .2 8 -0 .9 5 /8 .7 9 -0 .0 4 /0 .0 5 0.06/0.94 6.83/36.6 -2 .3 0 /7 .1 3 -3 4 .6 /3 4 .3 -0 .1 9 /0 .0 1 -2 .7 2 /5 .4 1 -1 3 .7 /3 .3 2 -1 7 .5 /1 8 .1 -8 .5 1 /6 5 5 -0 .5 7 /0 .1 7 -3 .4 4 /1 .9 7 -8 .1 8 /4 .5 4 -0 .7 5 /1 .2 8 -0 .3 7 /0 .3 6 -2 .2 9 /1 .2 4 -0 .1 9 /1 .3 5 -0 .2 3 /0 .0 7 -0.6 0 /0 .0 7 -1 .4 2 /0 .4 0 -1 8 .8 /2 1 .4 NS NS NS 0.027 0.005 NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS h ' a::. V.?,3 ! p. 10 6 Medical Surveillance of Workers Exposed to PFOA Costa et al TABLE 7 Results o f the Multivariate Analysis (GEE Model) in the 56 Subjects (Currently, Formerly, and Never Exposed) W ho Had Serum PFOA Assessed Concurrently W ith the Biochemical Parameters in the Last Six Y r (Controlled for Age, Job Seniority, Body Mass Index, Alcohol Consumption, and Yr of Observation) C oef. Pfa 95% C .I. P Glucose Urea nitrogen Creatinine Uric acid Total cholesterol HOL cholesterol Triglycerides Total bilirubin Conj. bilirubin AST ALT GGT ALP Apo A Apo B igG igM Estradiol Prot C reactive PSA Testosterone Total proteins Albumins a t globulins <x2 globulins globulins y globulins WBC RBC Haemoglobin H aem atocrit Platelets 0.005 0.017 -0.0 1 1 0.026 0.028 -0 .0 1 8 0.055 -0 .0 8 0 -0 .0 3 4 0.038 0.116 0.177 0.057 -0 .0 0 1 0.023 -0 .0 1 7 0.048 -0 .0 1 2 -0 .0 2 0 -0 .0 3 2 -0 .0 0 5 -0.0 0 1 -0 .0 0 9 0.026 0.026 0.011 0.013 0.029 -0 .0 0 2 0.008 0.122 1.987 -0.0 1 1 /0 .02 0 -0.0 1 5 /0 .04 9 -0.0 2 5 /0 .00 4 0.001/0.053 0.002/0.055 -0.0 4 7 /0 .01 2 -0.0 3 6 /0 .14 7 --0 .1 3 7 /--0.024 -0 .0 9 9 /0 .0 3 1 -0.0 0 3 /0 .08 0 0.054/0.177 0.076/0.278 0.007/0.107 -0.0 3 8 /0 .03 5 -0.0 4 1 /0 .08 7 --0 .1 1 5 /--0.080 --0.093/0.190 -0.0 8 0 /0 .05 7 -0 .2 6 8 /0 .22 8 -0.2 8 6 /0 .22 2 -0 .0 2 1 /0 .0 1 1 -0.0 1 0 /0 .00 8 -0 .0 1 7 /0 .0 0 1 -0.0 0 1 /0 .05 3 0.007/0.045 -0.0 0 8 /0 .03 0 -0 .0 0 5 /0 .0 3 1 -0 .0 1 1 /0 .0 7 1 -0.0 0 9 /0 .00 6 --0.113/0.129 -0.1 3 2 /0 .37 6 -7.3 6 /1 1 .34 NS NS NS < 0.05 < 0.05 NS NS <0.01 NS NS <0.01 <0.01 < 0 .0 5 NS NS NS NS NS NS NS NS NS NS NS <0.01 NS NS NS NS NS NS NS 60 -1 0 1 2 3 4 5 6 7 9 10 11 12 13 14 IS 16 17 18 19 20 21 22 23 24 2S 26 2 7 28 29 30 years after exposure Fig. 1. PFOA serum level (jig/mL) in the 16 formerly exposed workers, who retired or were transferred to other departments. Discussion In our cohort neither clinical evi dence of specific disturbances nor health disorders have been recorded over 30 years of medical observa tions of workers exposed to PFOA, having serum levels ranging from 0.20 to 91.9 p.g/mL. A significant decrease in PFOA blood levels (--37% in mean level and --45.5% in peak level) was re corded in the 4 last years after plant renovation and improvement of working conditions. However, in evaluating this trend it is necessary to take into account the long biolog ical half-life of the substance, so that the present blood levels largely re flect the exposure conditions of the previous years. In fact, contrary to the rather rapid rates of elimination reported in lab oratory animals, PFOA appears to be slowly eliminated in humans.3,30 Very recently Olsen et al20 have determined the serum elimination half-life of PFOA in 26 retired fluorochemicals production workers over a 5 year span; the arithmetic mean half-life was 3.8 years (95% C l = 3.1 to 4.4) and the geometric mean half-life of 3.5 years (95% Cl = 3.0 to 4.1), with a range from 1.5 to 9.1 year. A similar trend can be found also in our data (Fig. 1). In the 16 for merly exposed workers, who retired or were transferred to other depart ments, the decrement of PFOA blood concentration over die last 7 years appears to be more or less steeper depending on both extent and dura tion of the past exposure, and years elapsed since leaving the job. Al though the data are not complete and homogeneous, in terms of PFOA level, number and years of measure ments, time elapsed since exposure, the observed trend in the decrement suggests an estimated half-life of 5.1 (sd 1.7) years, varying from 2.6 to 9.7 years (geometric mean 4.8). It is noteworthy that even after 20 years from exposure some people still have 1 to 3 (xg/mL of PFOA in their blood. i p. 11 JOEM Volume 51, Number 3, March 2009 7 As regards biological effects, no significant perturbation of liver and renal functions, immunology, and hormonal secretion (including estra diol, testosterone, thyroid, and pros tate hormones) were recorded in our workers, except for a significant in terference with lipids (cholesterol) and purines (uric acid) metabolism, beside the influence of BMI. As mentioned in Introduction, some previous cross sectional studies in 3M workers did not rind any consistent association between se rum PFOA and liver, renal and lipid functions, and hormonal secretion. As concerns liver function and lipids metabolism in particular, Gilli land and Mandel35 did not record any significant difference between ex posed and non-exposed workers in any biological parameter of liver function, and no significant correla tion between total serum fluorine (above 10 (jug/mL) and cholesterol, LDL or HDL, after adjusting for age, BMI, alcohol, and smoking. The same negative results were obtained in three successive cross-sectional investigations in workers with serum PFOA up to 114.1 pg/mL.37 More recently, in two other 3M groups, including both European and US occupationally exposed workers, Olsen et al38 found no significant effect on liver function but a posi tive, although modest, association between serum PFOA (range: 0.01 to 12.7 (j-g/mL) and cholesterol as well as triglycerides, both in a cross sectional and in a 6-year longitudinal analysis. Moreover, in a further study in workers with serum PFOA ranging from 0.007 to 92.03 pg/mL, Olsen and Zobel39 recorded a weakly negative association with HDL and total bilirubin, and a positive one with triglycerides, after excluding people taking cholesterol lowering medications; however, these findings were explained by demographic dif ferences across the three locations. However, Sakr et al in a cross sectional study on DuPont work ers,41 having scrum PFOA from 0.005 to 9.55 pg/mL, reported a modest, but significant positive cor destruction in vitro when added to a relation between serum PFOA and pure bacterial enzyme (CYP102 or total cholesterol, LDL, and VLDL, BM3) specialized in fatty acid me whereas in another longitudinal tabolism. By analogy, it is possible study40 they found a positive associ that a similar effect might take place ation of serum PFOA (range: 0 to in humans on interaction of PFOA 22.66 pg/mL) with total cholesterol with a fatty acid metabolizing CYP and AST, and a negative association enzyme.51 with total bilirubin, after adjusting Moreover, we recorded a positive for potential confounders (age, BMI, significant association between se gender, and decade of hiring). rum PFOA and uric acid, that was However, it is worth mentioning also reported by Sakr et al in DuPont that serum cholesterol appears to workers.41 This may be suggestive of move in the opposite direction than some interference in liver purines observed in experimental studies in metabolism, which deserves specific rodents, in which PFOA has a signif investigations. icant hypolipidemic effect-in serum, In our study, we tried to control as being a strong PPARa agonist.45 No many confounding variables as pos study reported any hypolipidemic ef sible, in particular age, BMI, smok fect in exposed workers, whereas ing, alcohol consumption, and shift three studies including the present work. The first four factors were one recorded a significant increase in taken into consideration in the GEE cholesterol levels, besides other non analysis, whereas the possible inter consistent increases of triglycerides ference of shift and night work on and liver enzymes. Also in cynomol- metabolic parameters, as evidenced gus monkeys, after 6 months of oral in literature,52-53 was controlled by dosing of PFOA from 3 to 30/20 mg/ comparing exposed workers and kg/die (with serum PFOA ranging controls matched for (shift) work from 20 to 467 pg/mL) no decrease of seniority (Table 5). cholesterol, but a significant increase So, beside some inconsistent find of triglycerides was observed.18 This ings among the studies, it is probable might be due to different mechanisms that PFOA exerts some interference of action and/or receptor agonisms, ie, on intermediate metabolism also in PPARot/y and/or CAR20 in rodents humans. Owing to the small number rather than in primates, which deserves of exposed subjects it is not possible further investigation. For example it is to draw any firm conclusion, though well known that there are different the long time span of the careful expressions and properties of PPAR medical surveillance may allow to isoforms (a, (3, and y) in animals and exclude a severe impact o f PFOA un humans9,13-15-46-48 as well as there human health. However, the possi might be different bindings with serum ble interference on the intermediate proteins (ie, albumin in animal, CETP) metabolism should not be underes and in renal readsorption (Oatpl and timated and deserves further inves Oat3),49-50 or interference with hepatic tigations on mechanisms for action, metabolism of fatty acids, ie, via and as a possible risk factor for CYP450. metabolic disorders and/or CVD As to the latter, it is worth men diseases. tioning that three studies (Olsen and Zabel,39 Sakr et al,41 and the present one), also recorded a significant neg References ative association between serum 1. Kennedy GL, Butenhoff JL Jr, Olsen PFOA and total bilirubin, that was not documented in rodents and pri mates. It is worth noting in this regard that perfluorolauric acid proved to cause a marked bilirubin GW, et al. The toxicology o f perfluorooctanoate. Crit Rev Toxicol. 2004;34: 351-384. . 2. Lau C, Anitole K, Hodes C, Lai D, Pfahles-Hutehens A, Seed J. Perfluoroalkyl acids: a review of monitoring and p. 12 8 Medical Surveillance of Workers Exposed to PFOA Costa et al toxicological findings. Toxicol Sci. 2007; 99:366-394. 3. Butenhoff JL, Kennedy GL, Frame SR, O'Connor JC, York RG. The reproductive toxicology of ammonium perfluorooctanoate (APFO) in the rat. Toxicol ogy. 2004;196:95-116. 4. Lau C, Thibodeaux JR, Hanson RG, et al. Effects of periluorooctanoic acid expo sure during pregnancy in the mouse. Toxicol Sci. 2006;90:510-518. 5. Abbott BD, W olf CJ, Schmid JE, et al. Periluorooctanoic acid (PFOA)-induced developmental toxicity in the mouse is dependent on expression of peroxisome proliferator activated receptor-alpha (PPARa). Toxicol Sci. 2007;98:571-581. 6. Sibinski Li, Allen JL, Erickson EE. Two Year Oral (diet) Toxicity/Carcinogenicity Study o f Fluorochemicat FC-143 in Rats. S t Paul, MN: Riker Laboratories, Inc., E xpt No. 0281CR0012. U.S. Environ mental Protection Agency Administrative Record 226-0437-0440; 1987. 7. Cook JC, Murray SM, Frame SR, et al. Induction of Leydig cell adenomas by ammonium perfluorooctanate: a possible endocrine related mechanism. Toxicol Appl Pharmacol. 1992;192:209-217. 8. Bicgel LB, H unt ME, Frame SR, O 'Connor JC, Cook JC. Mechanisms of extrahepatic tumor induction by peroxi some proliferators in male CD rats. Toxi col Sci. 2001;60:44-55. 9. Maloney EK, Waxman DJ. Transactiva tion of PPARa and PPARy by structur ally diverse environmental chemicals. Toxicol Appl Pharmacol. 1999;161:209218. 10. Butenhoff JL, Gaylor DW, Moore JA, et al. Characterization of risk for general population exposure to perfluorooctanoate. Regul Toxicol Pharmacol. 2004;39: 363-380. 11. Andersen ME. Clewell HJ. Tan YM. Butenhoff JL. Olsen GW. Pharmacokinetic modeling of saturable, renal resorption qf perfluoroalkyiacids in monkeys--probing the determinants of long plasma half lives. Toxicology. 2006;227:156-164. 12. Klaunig JE, Babich MA, Baetcke KP, et al. PPARalpha agonist-induced ro dent tumors: modes of action and hu man relevance. Crit Rev Toxicol. 2003; 33:655-780. 13. Palmer CN, Hsu M-H. Griffin KJ. Raucy JL. Johnson EF. Peroxisome proliferator activated receptor-a expression in human liver. Mo! Pharmacol. 1998;53:14-22. 14. Cheung C, Akiyama TE, Ward JM, et al. Diminished hepatocellular proliferation in mice humanized for the nuclear recep tor peroxisome proliferator-activated re ceptor alpha. Cancer Res. 2004;64: 3849-3854. 15. Moriinura K, Cheung C, Ward JM, Redd JK, Gonzalez FJ. Differential susceptibil ity of mice humanized for peroxisome proliferator-activated receptor alpha to Wy-!4,643-induced liver tumorigenesis. Carcinogenesis. 2006;27:1074-1080. 16. Rosen MB, Lee JS, Ren H, et al. Toxicogenomic dissection of the perfluorooctanoic acid transcript profile in mouse liver: evidence for the involve ment of nuclear receptors PPARalpha and CAR. Toxicol Sci. 2008;103:46-56. 17. Kudo N, Kawashima Y. Toxicity and toxicokinetics of perfluorooctanoic acid in humans and animals. J Toxicol Sci. 2003;28:49-57. 18. Butenhoff J, Costa G, Elcombe C, et al. Toxicity o f ammonium perfluorooctanoate in male cynomolgus monkeys after oral dosing for 6 months. Toxicol Sci. 2002;69:244-257. 19. Harada K, Inoue K, Morikawa A. Yoshinaga T, Saito N, Koizumiet A. Renal clearance of perfluorooctane sulfonate and perfluorooctanoate in humans and their species-specific excretion. Environ Res. 2005;99:253-261. 20. Olsen GW, Burris JM, Ehresman DJ, et al. Half-life of serum elimination of perfluorooctanesulfonate, perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemicat production work ers. Environ Health Perspect. 2007;115: 1298-1305. 21. Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Tully JS, Needham LL. Se rum concentrations of 11 polyfluoroalkyl compounds in the U.S. population: data from the National Health and Nutrition Examination Survey (NHANES) 1999 2000. Environ Sci Technol. 2007;4I: 2237-2242. 22. Emmett EA, Shofer FS. Zhang H, Free man D, Desai C, Shaw LM. Community exposure to perfluorooctanoate: relation ships between serum concentrations and exposure sources. J Occup Environ Med. 2006;48:759-770. 23. Kannan K, Corsolini S, Falandysz J, et al. Perfluorooctanesulfonate and related fluorochemicals in human blood from several countries. Environ Sci Technoi. 2004;38:4489-4495. 24. Olsen GW, Huang H-Y, Helzlsouer KJ, Hansen KJ. Butenhoff JL. Mandel JH. Historical comparison of perfluorooclanesulfonale, perfluorooctanoate, and other fluorochemicals in human blood. Environ Health Perspect. 2005; 113:539-545. 25. Giesy JP, Kannan K. Global distribution of perfluorooctane sulfonate and related perfluorinated compounds in wildlife. Environ Sci Technol. 2001;35:13391342. 26. Nakata H, Kannan K, Nasu T, Cho H-S, Sinclair E. Takemura A. Perfluorinated contaminants in sediment and aquatic organisms collected from shallow water and tidal flat areas of the Ariake Sea, Japan: environmental fate of perfluo rooctane'sulfonate in aquatic ecosystems. Environ Sci Technol. 2006;40:49164921. 27. Tao L, Kannan K, Kajiwara N, et al. Perfluorooctanesulfonate and related fluorochemicals in albatrosses, elephant seals, penguins, and polar skuas from the Southern Ocean. Environ Sci Technol. 2006;40:7642-7648. 28. US Environmental Protection Agency. Perfluorooctanoic Acid. 2008. Available at: http://www.epa.gov/oppt/pfoa. Ac cessed October 3, 2008. 29. UK Committee on Toxicity o f Chemicals in Food, Consumer Products and the Environment- Second Draft Working Pa per on the Tolerable Daily Intake fo r Perfluorooctanoic Acid. 2006. Available at: http://cot.gov.uk/pdfs/tox-2006I8.pdf. Accessed October 3, 2008. 30. Ubel FA. Sorenson SD, Roach DE. Health status of plant workers exposed to fluorochemicals--a preliminary report. Am bid Hyg Assoc J. 1980;41:584-589. 31. Gilliland FD, Mandel JS. Mortality among employees of a perfluorooctanoic acid production plant. J Occup Med. 1993;35:950-954. 32. Alexander BH. Mortality Study o f Work ers Employed at the 3M Cottage Grove Facility. St. Paul, MN: 3M Company; W ashington DC; U.S. EPA Public Docket AR-226-1030a018; 2001. 33. Leonard RC, Kreckmann KH, Sakr CJ, Symons JM. Retrospective cohort mor tality study of workers in a polymer production plant including a reference population o f regional workers. Ann Epi demiol. 2008;18:15-22. 34. Alexander BH, Olsen GW. Bladder can cer in perfluorooctanesulfonyl fluoride manufacturing workers. Ann Epidemiol. 2007;17:471-478. 35. Gilliland FD, Mandel JS. Serum perfluo rooctanoic acid and hepatic enzymes, li poproteins and cholesterol: a study of occupationally exposed men. Am J Ind Med. 1996;29:560-568. 36. Olsen GW. Gilliland FD, Burlew MM, Burris JM, Mandel JS, Mandel JHL. An epidemiologic investigation of reproduc tive hormones in men with occupational exposure to pefluorooctanoic acid. J Oc cup Environ Med. 1998;40:614-621. 37. Olsen GW, Bums JM, Burlew MM, ofthis anfcis is pfuHibilisC. JOEM Volume 51, Number 3, March 2009 9 Mande! JH. Plasma cholecystokinin and hepatic enzymes, cholesterol and lipopro teins in ammonium perfluorooctansulfonate production workers. Drug Chem Toxicol. 2000;23:603-620. 38. Olsen GW. Burris JM, Burlew MM, Mande! JH. Epidemiologic assessment of workers serum perfluorooctansulfonate (PFOS) and pefluorooctanoate (PFOA) concentrations and medical surveillance examinations. J Occup Environ Med. 2003;45:260-270. 39. Olsen GW, Zobel LR. Assessment of lipid, hepatic, and thyroid parameters with serum periluooctanoate (PFOA) concentrations in fluochemical produc tion workers, hit Arch Occup Environ Health. 2007;81:231-246. 40. Sakr CJ, Leonard RC, Kreckmann KH, Slade MD, Cullen MR. Longitudinal study of serum lipids and liver enzymes in worker with occupational exposure to ammonium perfluooctanoate. J Occup Environ Med. 2007;49:872-879. 4L Sakr CJ, Kreckmann KH, Green JW, Gilles PJ, Reynolds JL, Leonard RC. Cross-sectional study of lipids and liver enzymes related to a serum biomatker of exposure (ammonium perfluooctanoate or APFO) as part of general health survey in a cohort of occupationally exposed workers. J Occup Environ Med. 2007;49: 1086-1096. 42. Hansen KJ, Clemen LA, Ellefson ME. Johnson JHO. Compound-specific, quanti tative characterization of organic fluorochemicals in biological matrices. Environ Sei Technol. 2001;35:766-770. 43. Twisk JWR. Applied Longitudinal Data Analysis fo r Epidemiology. Cambridge: Cambridge University Press; 2003. 44. StataCorp. Stata Statistical Software: Re lease 10. College Station, TX: StataCorp LP; 2007. 45. Xie Y, Yang Q, Nelson BD, DePierre JW. The relationship between liver per oxisome proliferation and adipose tissue atrophy induced by peroxisome protiferator exposure and withdrawal in mice. Biochem Pharmacol. 2003;66:749-756. 46. Michalik L, Desvergne B, Dreyer C, DeGavillet M. Laurini RN, Wahl W. PPAR expression and function during vertebrate development, fnt J Dev Biol. 2002;46:105-114. 47. Rosen ED, Spiegelman BM. PPAR-y: a nuclear regulator of metabolism, differ entiation, and growth. / Biol Chem. 2001; 276:37731-37734. 48. Toth B, Homung D, Scholz C. Djalali S, Friese K, Jeschke U. Peroxisome proliferatoractivated receptors: new players in the field o f reproduction. Am J Reprod Immunol. 2007;58:289-310. 49. KatakuraM, K udoN .T sudaT. HibinoY. Mitsumoto A. Kawashima Y. Rat organic anion transporter 3 and organic anion transporting polypeptide 1 mediate perfluotooctanoic acid transport J Health Sci. 2007;53:77-83. 50. Kudo N, Katakura M, Sato Y, Ka washima Y. Sex hormone-regulated renal transport of perfluorooctanoic acid. Chem Biol Interact. 2002;139:301-316. 51. Pons N, Pipino S, De Matteis F. interaction of polyhalogenated compounds of appro priate configuration with mammalian or bacterial CYP enzymes. Increased bilirubin and uropofobylinogen oxidation. Biochem Pharmacol. 2003;66:405-414. 52. Karlsson B, Knutsson A, Lindahl B, Alfredsson L. Metabolic disturbances in male workers with rotating threeshift work. Results o f the WOLF study. fnt Arch Occup Environ Health. 2003; 76:424 -430. 53. Biggi N, Consonni D, Galluzzo V, Sogliani M, Costa G. Metabolic syndrome in permanent nightworkers. Chronobiol Int. 2008;25:1-12. ehponline.org ENVIRONMENTAL HEALTH PERSPECTIVES Do Perfluoroalkyl Compounds Impair Human Semen Quality? Ulla Nordstrom Joensen, Rossana Bossi, Henrik Leffers, Allan Astrup Jensen, Niels E. Skakkebaek, and Niels Jorgensen doi: 10.1289/ehp.0800517 (available at http://dx.doi.org/) Online 2 March 2009 A NIEHS NT ^ N a tio n a l In s titu te s o f H e a lth U .S . D e p a rtm e n t o f H e a lth and H um an Services Page 1 of.26 Do Perfluoroalkyl Com pounds Im pair Human Sem en Q uality? r p. 15 U lla Nordstrom Joensen1, Rossana Bossi2, H enrik L effers1,A llan Astrup Jensen3, N iels . Skakkebaek1, N iels Jorgensen1. 1University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark 2National Environmental Research Institute, University of Aarhus, Roskilde, Denmark 3FORCE Technology, Br0ndby, Denmark A ddress o f institution where work was done: University Department of Growth and Reproduction Rigshospitalet, section 5064 Blegdamsvej 9 2100 Copenhagen Denmark C orresponding author: Ulla Nordstrom Joensen, MD University Department of Growth and Reproduction, Rigshospitalet Blegdamsvej 9 2100 Copenhagen Denmark Telephone: +45 3545 5064 Fax: +45 3545 6054 E-Mail: ullanordstroem.ioensen@rh.regionh.dk 1 p. 16 Page 2 o f26 Acknowledgements/grant support: The authors greatly appreciate the support from the European Union (contract no. QLK4CT- 2002-00603), the Danish Agency for Science, Technology and Innovation (grant nos. 9700833 and 271070678), the Danish Ministry of Health (ISMF) (Grant no. 7-302-02-9/3), the Danish Environmental Protection Agency, and the University of Copenhagen. The authors declare they have no competing financial or non-financial interests. Running title: PFAAs and Human Semen Quality. EHP article descriptor: Reproductive Health Key words: Endocrine disrupters, male reproductive health, perfluoroalkyl compounds, PFAA, PFC, semen quality, sperm morphology, testosterone. Abbreviations: BMI: body mass index Cl: confidence interval ESI: electrospray ionization FAI: free androgen index FSH: follicle stimulating hormone HPLC: high performance liquid chromatography LC-MS-MS: liquid chromatography-tandem mass spectrometry LH: luteinizing hormone LOD: limit of detection PFAA: perfluoroalkyl acids PFC: polyfluorinated compounds PFDA: perfluorodecanoic acid PFDoA perfluorododecanoic acid PFHpA: perfluoroheptanoic acid PFHxS: perfluorohexane sulfonic acid PFNA: perfluorononanoic acid PFOA: perfluorooctanoic acid PFOS: perfluorooctane sulfonic acid PFOSA: perfluorooctane sulfonamide PFTrA: perfluorotridecanoic acid PFUnA: perfluoroundecanoic acid SHBG: sex hormone binding globulin TDS: testicular dysgenesis syndrome 2 Page 3 of. 26 O utline o f section headers Abstract Introduction Materials and methods Study population PFAA analysis Reproductive hormone analysis Semen analysis Statistical analysis Results Levels ofPFAAs in serum PFAAs and semen variables PFAAs and reproductive hormones Smoking and BMI Discussion and conclusions References Tables Figure legends Figures 3 p. 17 p. 18 Page 4 of 26 Abstract Background: Perfluoroalkyl acids (PFAAs) are found globally in wildlife and humans, and are suspected to act as endocrine disruptors. There are no reports of PFAA levels in adult men from Denmark, and no reports of a possible association between semen quality and PFAA exposure. Objectives: To investigate possible associations between PFAAs and testicular function. The hypothesis was that higher PFAA levels would be associated with lower semen quality and lower testosterone levels. Methods: We included 105 Danish men (median age 19 years) from the general population, and analyzed serum samples for levels o f 10 different PFAAs and reproductive hormones, and assessed semen quality. Results: Considerable levels of PFOS, PFOA and PFHxS were found in all young men (median 24.5, 4.9 and 6.6 ng/mL, respectively). Men with high combined levels of PFOS and PFOA had a median of 6.2 million normal spermatozoa in their ejaculate in contrast 15.5 million among men with low PFOS-PFOA (p=0.030). In addition we found non-significant trends with regard to lower sperm concentration, lower total sperm counts and altered pituitarygonadal hormones among men with high PFOS-PFOA levels. Conclusion: High PFAA levels were associated with fewer normal sperms. Thus, high levels of PFAAs may contribute to the otherwise unexplained low semen quality often seen in young men. However, our findings need to be corroborated in larger studies. 4 1 p. 19 Page 5 of 26 Introduction Perfluoroalkyl acids (PFAAs) are degradation products of many man-made polyfluorinated compounds (PFCs) used in consumer and industrial products, for example for impregnation of carpets, textiles and paper (Jensen et al. 2008; Jensen and Leffers 2008; Kissa 2001). Studies of environment, wildlife and humans suggest widespread presence and exposure, as well as persistence in the environment and bioaccumulation (Giesy and Kannan 2001; Kannan et al. 2004). For perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS) and perfluorohexane sulfonic acid (PFHxS), three of the most abundant PFAAs, half-lives for humans have been estimated as 3.8, 5.4 and 8.5 years, respectively (Olsen et al. 2007). Some studies suggest that men may have higher serum concentrations of PFAAs than women, and younger individuals may have higher levels than older (Calafat et al. 2006). Thus, young men may have particularly high levels of exposure and may therefore be a group at risk for potential adverse effects of PFAAs. PFAAs can cross the placental barrier and therefore have the potential to affect the fetus. In humans, levels of PFOS and PFOA in umbilical cord blood have been inversely related to birth weight (Apelberg et al. 2007). In addition, PFOS, PFOA and PFHxS have been detected in human seminal plasma samples (Guruge et al. 2005). However, data on effects in humans are sparse, and most come from studies of occupationally exposed individuals. These studies have not given conclusive evidence of adverse effects. A recent study, however, measured PFAA levels in early pregnancy found that higher levels of PFOS and PFOA was associated with significantly longer waiting time to pregnancy (Fei et al. 2009). Animal studies provide some evidence for adverse reproductive effects on animals exposed as adults or in utero. Exposure of adult male rats to PFOA reduced their testosterone levels and increased their estradiol levels, which may partly explain earlier 5 p. 20 Page 6 of 20 findings of induction of Leydig cell hyperplasia and/or adenomas in the testes of exposed animals (Biegel et al. 1995; Cook et al. 1992). Our objective was to investigate the associations between PFOS, PFOA, PFHxS and other PFAAs and testicular function. Our primary hypothesis was that high concentrations of PFAAs would be associated with low testosterone levels and secondly that high PFAA levels are negatively associated to semen quality variables. M aterials and m ethods Study population. Since 1996, semen quality of young men in Denmark has been surveyed in a cross-sectional study (Andersen et al. 2000; J0rgensen et al. 2002). All young Danish men must report for military draft, and annually new cohorts of approximately 300 men from the Copenhagen area in Denmark have been included. They each provided one semen sample and had a venous blood sample drawn. Of the 546 men examined in 2003, we selected 105 for the investigation of associations between PFAAs and testicular function. The 105 men included the 53 men (group 1) with the highest testosterone levels (median 31.8 nmol/1, range 30.1 - 34.8), and the 52 men (group 2) with the lowest testosterone levels (median 14.0 nmol/1, range 10.5 -15.5). We chose the men examined in 2003, as this was the latest year from which we had completed analyses of reproductive hormones. The median age of men in group 1 was 18,9 years (range 18,2 - 24,6), median age in group 2 was 19,0 years (range 18,2 - 25,1), and median age for all 105 young men was 19.0 years (range 18.2 - 25.2) years. Information on ejaculation abstinence period and hour of blood sampling was recorded. All samples of semen and blood were collected between 8.30 a.m. and 1.15 p.m. Serum was stored at -20C until chemical analysis. The Danish National Committee on Biomedical Research Ethics, Copenhagen Region, approved the research, and all young men gave written informed consent. 6 Page 7 of 26 p. 21 PFAA analysis. Thawed serum samples were analyzed in January 2008 for ten different perfluorinated chemicals, with carbon chain length from C6 to C l3: PFHxS (perfluorohexane sulfonic acid), PFHpA (perfluoroheptanoic acid), PFOA (perfluorooctanoic acid), PFOS (perfluorooctane sulfonic acid), PFOSA (perfluorooctane sulfonamide), PFNA (perfluorononanoic acid), PFDA (perfluorodecanoic acid), PFUnA (perfluoroundecanoic acid), PFDoA (perfluorododecanoic acid), and PFTrA (perfluorotridecanoic acid). One mL of serum was spiked with the surrogate standards 13CsPFOA, i3C2-PFDA and l3C4-PFOS and extracted according to the ion pairing method described previously (Hansen et al. 2001). Matrix-matched standards were prepared by spiking rabbit serum (Sigma Aldrich, Schnelldorf, Germany) with the analytes and the surrogate standards. Blank samples consisted of rabbit serum spiked with only surrogate standards. Standards and blanks were extracted together with each batch of samples. Instrumental analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS-MS) with electrospray ionization (ESI). The extracts (20 pL injection volume) were chromatographed on a C 18 Betasil column (2.1 x 50 mm, Thermo Hypesil-Keystone, Bellafonte, PA) using an Agilent 1100 Series HPLC (Agilent Technologies, Palo Alto, CA). The high performance liquid chromatography (HPLC) was interfaced to a triple quadrupole API 2000 (Sciex, Concorde, Ontario, Canada) equipped with a Turboion Spray source operating in negative ion mode. Chromatographic conditions and transition MS-MS ions have been described in details previously (Bossi et al. 2005). The limits of detection (LODs) ranged from 0.1 to 0.5 ng/mL. Reproductive hormone analysis. Thawed serum samples were analyzed for the levels of testosterone, estradiol, SHBG (sex hormone binding globulin), LH (luteinizing hormone), 7 ! p. 22 FSH (follicle stimulating hormone) and inhibin B as described previously (Paasch et al. 2008). Free androgen index (FAI) was calculated as [testosterone x 100 / SHBG]. Ratios between hormones were calculated by simple division. Page 8 o f26 Semen analysis. Semen volume was assessed by weight, and the sperm concentration by use of a Brker-Trk haemocytometer. Total sperm count was calculated as semen volume x sperm concentration. The percentage of motile spermatozoa (WHO class A+B+C) was assessed on fresh samples. Sperm morphology slides were fixed, Papanicolaou stained and all assessed according to strict criteria (Menkveld et al. 1990) by one trained technician over a period of one week. Further details of the semen analysis can be seen in previously published work (J0rgensen et al. 2002). Statistical analysis. Medians and 5-95thpercentiles were used to describe the levels of PFAAs in group 1 and 2. Mann-Whitney U-test was used to compare the groups 1 and 2 with respect to PFAA levels, BMI (body mass index) and smoking status. Samples with values below LOD were set to 0 ng/mL. We used Pearson's correlation coefficients and related p-values to describe correlations between levels of different PFCs. Univariate regression analysis was done for comparison of hormone levels between group 1 and 2, and to describe associations between PFAAs and hormones or semen variables. Sperm concentration, semen volume and total sperm count were adjusted for the effect of ejaculation abstinence period. Sperm motility was adjusted for time between ejaculation and assessment of motility. Sex hormone concentrations were adjusted for hour of blood sampling. Semen variables and hormone levels and ratios, except sperm morphology and total testosterone, were In transformed to obtain normality of the residuals. Smoking and 8 BMI were tested for confounding effects but were found to be non-significant, and therefore not included in the final analyses. We proceeded to analyze for associations between PFAAs and testicular function (hormone levels and semen quality) for the whole group of 105 men. We singled out PFOS and PFOA, and results were calculated as estimated changes in endpoint (reproductive hormones and semen variables) with a change in serum concentration of 1 ng/mL of PFOS, PFOA concentrations, and the summed concentration of PFOS and PFOA. We divided the men into three groups from the combined concentrations of PFOS and PFOA. Each sample was given a quartile score of 1 to 4 for PFOS and PFOA levels separately. Score 1 was given to samples with levels within die lowest quartile, and score 4 within the highest quartile. We then summed the quartile scores for PFOS and PFOA, giving each sample a possible score from 2 to 8. Samples were then divided into 3 quartile groups for PFOS and PFOA combined: "Low PFAA" group (N=29) with summed quartile score from 2 to 3, "intermediate PFAA" (N=48) group with score 4 to 6, and "high PFAA" (N=28) group with score 7 to 8. Analysis for association between quartile group and hormone levels or semen variables was done using univariate regression analysis, adjusted for the abovementioned confounders. Statistical analysis was performed using SPSS statistical software version 16.0 (SPSS Inc., Chicago, IL, USA). R esults Levels o f PFAAs in serum. The serum levels and number of samples above LOD for all PFAAs are shown for the low- and high testosterone groups and the entire group of 105 men (Table 1). Except for PFOSA, which was detected in only 56 men, there was no significant difference in levels of any PFAA between group 1 and 2. The median PFOS, PFOA and PFHxS concentrations for the whole group of men were 24.5, 4.9 and 6.6 ng/mL, respectively, and only these were included in the final regression analyses. The remaining PFAAs were found in much lowqr concentrations, and therefore these results are not discussed further. PFOS levels were positively correlated with PFOA (r = 0.594, p < 0.0005) and PFHxS (r = 0.304, p = 0.002) levels. PFOA and PFHxS levels were positively correlated, but not statistically significantly (r = 0.136, p = 0.2). PFAAs and semen variables. In the whole group of 105 men, there was a tendency toward reduced levels of all semen variables in the "high" PFAA quartile group compared to the "low" group (groups constructed to include PFOS and PFOA levels, see statistical analysis), Table 2. The difference in percentage of morphologically normal spermatozoa as well as in the total number of normal spermatozoa (total sperm count x % morphologically normal sperms) was statistically significant (p = 0.037 and 0.030, respectively). In the high PFOS-PFOA group the median number of normal spermatozoa in the ejaculate was 6.2 million vs. 15.5 million in the low group (Figures 1 and 2). When analyzing associations of semen variables to PFOS and PFOA separately, as well as the simple summed concentration of PFOS and PFOA, estimated changes in semen variables with a change in serum PFAA concentration of 1 ng/mL indicated negative but non-significant associations between the PFAAs and semen variables (Table 3). PFAAs and reproductive hormones. There was no significant association between testosterone levels and PFAA levels, and no significant difference in PFAA levels between the high- and low-testosterone groups. For the whole group of 105 men, adjusted medians for the hormones point to a negative association to PFAA levels - however, none of these tendencies were statistically significant (Table 2). 10 i Page 11 of 26 Estimated associations between reproductive hormones and PFOS and PFOA separately and the summed concentration of PFOS and PFOA, showed no significant associations (Table 4). p. 25 Smoking and BMI. 35% of the 105 men were smokers, and there were more smokers in the high testosterone group compared to the low testosterone group (49% and 21% smokers, respectively, p - 0.003). Smoking status was significantly associated to higher testosterone, lower estradiol and higher SHBG when entered as a confounder in univariate analyses for these three variables only (p = 0.004 to 0.009). Smoking was not associated with any semen quality variables (p = 0.1 to 0.4), or levels of any PFAA (p = 0.1 to 1.0). Including smoking as a confounder did not considerably change the presented results or significance levels. Therefore, smoking was not included in the final analyses. BMI was not associated to levels of any PFAA (p = 0.1 to 1.0), nor was there any confounding effect on any semen variables (e.g. p = 0.5 for morphologically normal sperms, and p = 0.9 for total number of morphologically normal sperms in analyses for difference between high and low PFAA groups). D iscussion and conclusions This study examined PFAA levels in young adults. High serum concentrations of PFAAs were significantly associated with reduced numbers of normal spermatozoa. In addition, sperm concentration, total sperm count and sperm motility showed some tendency towards lower levels in men with high PFAA levels, although not at a statistically significant level. A tendency toward lower inhibin B/FSH ratio with high PFAA levels was in agreement with these findings, as these hormones reflect the spermatogenetic activity. 11 p. 26 Testosterone, FAI, testosterone/LH ratio and testosterone/estradiol ratio could suggest a poorer Leydig cell function in the "high" compared to the "low" PFAA quartile group. However, the associations between reproductive hormones and PFAAs were not completely consistent, and all were non-significant. This study therefore cannot demonstrate an adverse effect of PFAAs on Leydig cell function. Page 12 of 26 We singled out PFOS and PFOA, as there are no data that specifically support PFHxS as an endocrine disruptor, and divided the men into three groups from the combined concentrations of PFOS and PFOA to account for a potential different effect at same concentration levels. As we could find no significant association between testosterone levels and PFAA levels, and no significant difference in PFAA levels between high- and low-testosterone groups, we could analyze associations between PFOS-PFOA levels and reproductive hormones or semen variables for the whole group. Controlling for confounding effect of smoking or BMI did not change estimates or significance levels. To our knowledge, there have been no consistent reports of associations between PFAA levels and smoking or BMI. Our material included only 105 men, and in addition, we had selected the men based on their serum testosterone values. The selection of two groups with high and low testosterone was done to test our primary hypothesis and therefore affects the homogeneity of the group when correlations are analyzed for the group as a whole. This could potentially influence the subsequent analysis of semen quality by bias or confounding, and may affect the general applicability of the results. A larger follow-up study should preferably include randomly selected men from the general population. Our study is, to our knowledge, the first report of a correlation between semen quality and PFAAs. Very few studies of other endocrine disruptors (e.g. phthalates and pesticides) have previously demonstrated such an association (Duty et al. 2003; Hauser et 12 p. 27 Page 13 of 26 al. 2003; Hauser et al. 2007; Meeker et al. 2008; Swan et al. 2003). If the results from our preliminary study of an association between high levels of PFAAs and decreased number of normal sperms are confirmed, then high levels of PFAAs may be regarded as another endocrine disrupting factor contributing to the low semen quality seen among many young men. However, the importance of mixture effects of low-dose exposure to multiple compounds is becoming evident from animal studies but remains to be studied in humans (Hass et al. 2007; Kortenkamp et al. 2007). The mode of action by PFAAs is not clear and only a few animal studies have explored mechanistic issues. These show decreased testosterone levels and reduced expression of steroidogenesis genes associated with Leydig cell hyperplasia in adult animals (Biegel et al. 1995; Shi et al. 2007), suggesting a direct testicular effect Recent studies have indicated that the fetal gonad is particularly sensitive to exogenous factors (Skakkebaek et al. 2001). However, our results could indicate that exposures later in life may contribute to impairment of semen quality, in line with other recent studies (Hauser et al. 2007). We speculate that morphology is perhaps more susceptible to this than sperm concentration or total sperm count. Sperm morphology has proven an important indicator of semen quality and fertility in a clinical setting, even in men with normal sperm concentration (Guzick et al. 2001). Interestingly, a recent study showed higher levels of maternal PFOS and PFOA levels in early pregnancy was associated with significantly longer waiting time to pregnancy (Fei et al. 2009). We speculate that men and women living together may have similar exposure to PFAAs and that decreased semen quality caused by high PFAA levels may contribute to the longer waiting time to pregnancy found in that study. The use and emission polyfluorinated compounds continue to increase, and they are not readily cleared from the environment (Jensen et al. 2008; Prevedouros et al. 2006). 13 p. 28 Page 14 of 26 Therefore, humans and wildlife worldwide will be exposed for years to come. We found positive correlations between levels of different PFAAs, as has been found previously (Apelberg et al. 2007; Calafat et al. 2007; Fei et al. 2007), suggesting common sources of exposure. The PFAAs levels we have detected are comparable to those found in other countries like Sweden and the Faroe Islands (Krrman et al. 2007; Weihe et al. 2008), but lower than earlier results from Denmark from 1996-2002 (Fei et al. 2007). Thus, the effects we have indicated may also be true for other than the Danish population. In conclusion, our results indicate that higher PFAA levels were associated with lower numbers of normal sperms. In addition, we found non-significant negative associations between PFAA levels and other semen variables and reproductive hormones. Thus, high levels of PFAAs may contribute to the otherwise unexplained low semen quality seen in many young men. However, results from this first and preliminary study should be corroborated in larger studies. 14 R eferen ces Andersen AG, Jensen TK, Carlsen E, Jorgensen N, Andersson AM, Krarup T et al. 2000. High frequency of sub-optimal semen quality in an unselected population of young men. Hum Reprod 15:366-372. Apelberg BJ, Witter FR, Herbstman JB, Calafat AM, Halden RU, Needham LLet al. 2007. Cord serum concentrations of perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) in relation to weight and size at birth. Environ Health Perspect 115:1670-1676. Biegel LB, Liu RC, Hurtt ME, Cook JC. 1995. Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies. Toxicol Appl Pharmacol 134:18-25. Bossi R, Riget FF, Dietz R. 2005. Temporal and spatial trends of perfluorinated compounds in ringed seal (Phoca hispida) from Greenland. Environ Sci Technol 39:7416-7422. Calafat AM, Kuklenyik Z, Caudill SP, Reidy JA, Needham LL. 2006. Perfluorochemicals in pooled serum samples from United States residents in 2001 and 2002. Environ Sci Technol 40:2128-2134. Calafat AM, Wong LY, Kuklenyik Z, Reidy JA, Needham LL. 2007. Polyfluoroalkyl chemicals in the U.S. population: data from the National Health and Nutrition Examination Survey (NHANES) 2003-2004 and comparisons with NHANES 1999 2000. Environ Health Perspect 115:1596-1602. Cook JC, Murray SM, Frame SR, Hurtt ME. 1992. Induction of Leydig cell adenomas by ammonium perfluorooctanoate: a possible endocrine-related mechanism. Toxicol Appl Pharmacol 113:209-217. Duty SM, Silva MJ, Barr DB, Brock JW, Ryan L, Chen Z et al. 2003. Phthalate exposure and human semen parameters. Epidemiology 14:269-277. 15 Fei C, McLaughlin JK, Lipworth L, Olsen J. 2009. In press. Maternal levels of perfluorinated chemicals and subfecundity. Hum Reprod. Fei C, McLaughlin JK, Tarone RE, Olsen J. 2007. Perfluorinated chemicals and fetal growth: a study within the Danish National Birth Cohort. Environ Health Perspect 115:1677-1682. Giesy JP, Kannan K. 2001. Global distribution of perfluorooctane sulfonate in wildlife. Environ Sei Technol 35:1339-1342. Guruge KS, Taniyasu S, Yamashita N, Wijeratna S, Mohotti KM, Seneviratne HR et al. 2005. Perfluorinated organic compounds in human blood serum and seminal plasma: a study of urban and rural tea worker populations in Sri Lanka. J Environ Monit 7:371-377. Guzick DS, Overstreet JW, Factor-Litvak P, Brazil CK, Nakajima ST, Coutifaris C et al. 2001. Sperm morphology, motility, and concentration in fertile and infertile men. N Engl J Med 345:1388-1393. Hansen KJ, Clemen LA, Ellefson ME, Johnson HO. 2001. Compound-specific, quantitative characterization of organic fluorochemicals in biological matrices. Environ Sei Technol 35:766-770. Hass U, Scholze M, Christiansen S, Dalgaard M, Vinggaard AM, Axelstad M et al. 2007. Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environ Health Perspect 115 Suppl 1:122-128. Hauser R, Chen Z, Pothier L, Ryan L, Altshul L. 2003. The relationship between human semen parameters and environmental exposure to polychlorinated biphenyls and p,p'-DDE. Environ Health Perspect 111:1505-1511. Hauser R, Meeker JD, Singh NP, Silva MJ, Ryan L, Duty S et al. 2007. DNA damage in human sperm is related to urinary levels of phthalate monoester and oxidative metabolites. Hum Reprod 22:688-695. 16 p. 31 Page 17-of 26 Jensen A.A., Poulsen P.B., Bossi R. 2008. Survey and environmental/health assessment of fluorinated substances in impregnated consumer products and impregnating agents. Survey of chemical substances in consumer products no. 99. Danish Agency for Environmental Protection. H ttp ://www.mst.dk/Udgivelser/Publications/2008/10/978-87-7052-845-0.htm [accessed 10 February 2009]. Jensen AA, Leffers H. 2008. Emerging endocrine disrupters: perfluoroalkylated substances. IntJAndrol 31:161-169. Jprgensen N, Carlsen E, Nermoen I, Punab M, Suominen J, Andersen AG et al. 2002. EastWest gradient in semen quality in the Nordic-Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland. Hum Reprod 17:2199-2208. Kannan K, Corsolini S, Falandysz J, Fillmann G, Kumar KS, Loganathan BG et al. 2004. Perfluorooctanesulfonate and related fluorochemicals in human blood from several countries. Environ Sci Technol 38:4489-4495. Karrman A, Ericson I, van Bavel B, Damerud PO, Aune M, Glynn A et al. 2007. Exposure of perfluorinated chemicals through lactation: levels of matched human milk and serum and a temporal trend, 1996-2004, in Sweden. Environ Health Perspect 115:226-230. Kissa E. 2001. Fluorinated surfactants and repellents, 2nd edition. Surfactant science series, Vol. 97. New York: Marcel Dekker. Kortenkamp A, Faust M, Scholze M, Backhaus T. 2007. Low-level exposure to multiple chemicals: reason for human health concerns? Environ Health Perspect 115 Suppl 1:106-114. 17 p. 32 Page 18 of 26 Meeker JD, Barr DB, Hauser R. 2008. Human semen quality and sperm DNA damage in relation to urinary metabolites of pyrethroid insecticides. Hum Reprod 23:1932 1940. Menkveld R, Stander FS, Kotze TJ, Kruger TF, van Zyl JA. 1990. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 5:586-592. Olsen GW, Burris JM, Ehresman DJ, Froehlich JW, Seacat AM, Butenhoff JL et al. 2007. Half-life of serum elimination of perfluorooctanesulfonate.perfluorohexanesulfonate, and perfluorooctanoate in retired fluorochemical production workers. Environ Health Perspect 115:1298 1305. Paasch U, Salzbrunn A, Glander HJ, Plambeck K, Salzbrunn H, Grnewald S et al. 2008. Semen quality in sub-fertile range for a significant proportion of young men from the general German population: a co-ordinated, controlled study of 791 men from Hamburg and Leipzig. Int J Androl 31:93-102. Prevedouros K, Cousins IT, Buck RC, Korzeniowski SH. 2006. Sources, fate and transport of perfluorocarboxylates. Environ Sei Technol 40:32-44. Shi Z, Zhang H, Liu Y, Xu M, Dai J. 2007. Alterations in gene expression and testosterone synthesis in the testes of male rats exposed to perfluorododecanoic acid. Toxicol Sei 98:206-215. Skakkebk NE, Rajpert-De Meyts E, Main KM. 2001. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 16:972-978. Swan SH, Kruse RL, Liu F, Barr DB, Drobnis EZ, Redmon JB et al. 2003. Semen quality in relation to biomarkers of pesticide exposure. Environ Health Perspect 111:1478 1484. 18 ! Page 19of 26 Weihe P, Kato K, Calafat AM, Nielsen F, Wanigatunga AA, Needham LL et al. 2008. Serum concentrations of polyfluoroalkyl compounds in Faroese whale meat consumers. Environ Sci Technol 42:6291-6295. p. 33 19 .i p. 34 Page 20 of 26 Table 1 PFAA median concentrations (5-95* percentiles) for high and low testosterone groups and all study subjects (ng/mL), and p-values for difference between the two groups. Samples High testosterone > LOD N=53 PFHxS PFHpA PFOA PFOS PFOSA PFNA PFDA PFUnA PFDoA PFTrA 105 98 105 105 56 105 104 101 102 7 6.6 (4.0-13.0) 0.2 (0.01-0.9) 4.4 (2.6-7.0) 25.5 (14.2 - 39.6) 0.1 (0.00-3.7) 0.8 (0.4-1.8) 0.9 (0.2-1.1) 0.1 (0.04-0.3) 0.08 (0.04 - 0.8) 0.00 (0.000-0.4) Low testosterone N=52 6.6 (3.5-12.1) 0.3 (0.00-1.3) 5.0 (2.7-7.5) 23.9 (12.8 - 45.2) 0.00 (0.00 - 3.5) 0.8 (0.4-2.0) 0.8 (0.4-1.2) 0.2 (0.00-0.4) 0.08 (0.02 - 0.8) 0.00 (0.00 - 0.06) Whole group N=105 6.6 (4.0-12.1) 0.2 (0.00-1.1) 4.9 (2.7-7. 2) 24.5 (14.2 - 42.1) 0.06 (0.00 - 3.5) 0.8 (0.4-1.8) 0.9 (0.3-1.1) 0.1 (0.02-0.4) 0.08 (0.04 - 0.8) 0.00 (0.00 - 0.2) Pvalue 0.8 0.8 0.1 0.4 0.008 0.6 0.9 1.0 0.8 0.2 20 ! p. 35 Page 21 o f 26 Table 2 Adjusted means (95% Cl) for PFAA quaitile groups, and p-value for difference between the low and high PFAA quartile groups. Sex hormones8 Low PFAA Intermediate PFAA High PFAA N Testosterone (nmol/L) Estradiol (pmol/L) SHBG (nmol/L) LH (IU/L) FSH (IU/L) Inhibin-B (pg/mL) FAI Testosterone/LH FAI/LH Estradiol/Testosterone Inhibin/FSH N = 29 25.2 (21.7 - 28.7) 77.6 (70.3 - 85.8) 27.8 (24.0 - 32.2) 3.4 (2.8-4.1) 2.7 (2.1-3.5) 181 (142-232) 84.1 (74.1-95.5) 6.9 (5.6-8.4) 24.7 (19.9 - 30.7) 3.3 (3.0 - 3.7) 66.8 (42.1 - 106.0) N = 48 22.3 (19.6 - 25.0) 72.4 (67.0 - 84.7) 25.4 (22.6 - 28.5) 2.9 (2.5-3.4) 2.7 (2.2-3-3) 175 (144 - 212) 80.2 (72.6-88.7) 7.0 (5.9-8.2) 27.4 (23.1-32.5) 3.6 (3.2-3.9) 66.0 (45.9 - 94.8) N = 28 22.3 (18.8 - 25.8) 76.6 (69.3 - 84.7) 26.1 (22.5-30.3) 3.7 (3.0-4.4) 3.0 (2.3-3.8) 152 (119- 195) 77.8 (68.5-88.3) 5.5 (4.5-6.8) 21.2 (17.1-26.4) 3.8 (3.4 - 4.2) 51.3 (32.3-81.4) Semen qualityb p-value 0.2 0.9 0.5 0.6 0.6 0.3 0.4 0.1 0.3 0.1 0.4 Volume (mL) 4.0 (3.2-5.0) Concentration (mioVmL) 59 (36 - 96) Total count (mio.) 228 (134-389) Motile sperms (%) 73 (69 - 77) Morphologically normal (%) 8.8 (7.2-10.4) Total morphologically normal (mio.) 15.5 (7.3-33.0) 3.4 (2.9-4.1) 51 (35-74) 172 (114-261) 70 (66 - 73) 7.7 (6.4-9.0) 10.0 (5.6 - 17.9) 3.5 (2.9-4.4) 40 (25 - 64) 143 (86-237) 71 (66-75) 6.3 (4.6 - 8.0) 6.23 (3.0 -12.8) 0.3 0.2 0.1 0.4 0.037* 0.030* " Hormone levels are adjusted for time of blood sampling. b Volume, concentration and total count are adjusted for duration of abstinence. Motility is adjusted for time between ejaculation and semen analysis. Morphology is not adjusted for confounders. 3 21 p. 36 Page 22 of 26 Table 3 Estimated change in semen variables with a change in PFAA of 1 ng/mL (95% Cl). All 105 men included. PFOS PFOA PFAA sumb In Volume 0.000 (-0.012 - 0.011) In Concentration -0.020 (-0.044 - 0.005) In Total count -0.018 (-0.045 - 0.010) In Motility -0.006 (-0.019 - 0.007) Morphology -0.085 (-0.200 - 0.026) -0.002 -0.080 -0.074 -0.027 -0.540 (-0.070 - 0.066) (-0.230 - 0.066) (-0.230 - 0.086) (-0.110-0.053) (-1.200 - 0.110) 0.000 -0.018 -0.016 -0.006 -0.082 (-0.010 - 0.010) (-0.040 - 0.004) (-0.041 - 0.008) (-0.018 - 0.007) (-0.181-0.018) a Volume, concentration and total count are adjusted for duration of abstinence. Motility is adjusted for time between ejaculation and semen analysis. Morphology is not adjusted for confounders. b"PFAA sum" is PFOS and PFOA mass concentrations summed (ng/mL). 22 Page 23-of 26 Table 4 Estimated change in reproductive hormones3with a change in PFAA concentration of 1 ng/mL (95% Cl). All 105 men included. p. 37 PFOS PFOA PFAA sumb Testosterone -0.087 (-0.32-0.15) In Estradiol -0.001 (-0.008 - 0.005) ln SHBG 0.002 (-0.007 - 0.012) ln LH 0.000 (-0.014-0.012) ln FSH 0.004 (-0.13 - 0.22) In Inhibin-B -0.004 (-0.21 - 0. 12) ln FAI -0.006 (-0.015 - 0.002) In Testosterone/LH -0.003 (-0.017-0.011) ln FAI/LH -0.006 (-0.020 - 0.009) In Estradiol/ Testosterone 0.003 (-0.005 - 0.010) In Inhibin/FSH -0.009 (-0.039 - 0.022) -0.98 (-2.33-0.37) -0.012 (-0.051 - 0.027) -0.009 (-0.067 - 0.048) -0.010 (-0.084 - 0.064) -0.037 (-0.14 - 0.064) 0.012 (-0.084-0.11) -0.038 (-0.087 - 0.011) -0.037 (-0.12 - 0.045) -0.028 (-0.114 - 0.058) 0.035 (-0.010 - 0.081) 0.049 (-0.13-0.23) -0.093 (-0.303 - 0.116) -0.001 (-0.007 - 0.005) 0.002 (-0.007 - 0.011) 0.000 (-0.012 - 0.010) 0.003 (-0.013-0.018) -0.003 (-0.018 - 0.012) -0.006 (-0.014 - 0.001) -0.003 (-0.016 - 0.009) -0.005 (-0.018 - 0.008) 0.003 (-0.004-0.010) -0.006 (-0.034 - 0.022) a Hormone levels are adjusted for time of blood sampling. b"PFAA sum" is PFOS and PFOA mass concentrations summed (ng/mL). Figure legends p. 38 Page 24 of 26 Figure 1. Morphologically normal spermatozoa (%) and PFAA quartile groups (adjusted means and 95% Cl). AH 105 men included. * p = 0.037 for difference compared to Low PFAA quartile group. Figure 2. Total morphologically normal spermatozoa (mio.)a and PFAA quartile groups (adjusted means and 95% Cl). All 105 men included. a Total morphologically normal spermatozoa is adjusted for duration of abstinence. * p = 0.030 for difference compared to Low PFAA quartile group. 24 Page 25 of 26 Figure l. Morphologically normal sperm loro* (%). LowPFAA to(erM4iatr FRAA High PFAA p. 39 M orphologically n orm al sperm atozo a ( % ) and PFAA q uartile groups (ad ju s te d m eans and 9 5 % C l) , All 1 05 m en included. * p = 0 .0 3 7 fo r difference com pared to Low PFAA q uartile group. 201x285m m (150 x 150 DPI) F igure 2. T otal nwpfeelogkaHy n o ratti sperm atozoa (ado.). p. 40 Page 26 of 26 Total m orphologically norm al sperm atozoa (m io .)a and PFAA q u a rtlle groups (ad justed m eans and 9 5 % C l). All 105 m en included. a Total m orphologically norm al sperm atozo a is adjusted fo r d uration of abstinence. * p = 0 .0 3 0 fo r difference com pared to Low PFAA q uartile group. 201x285m m (1 5 0 x 150 DPI)