a Not corrected for purity of the standard material. b LOQ Limit of Quanfitation = 0.00910 pg/ mL c LOQ Limit of Quanfitation = 0.0457 pg/ mL d C92987F sample spilled during extraction, no sample remaining for analysis. It is not possible to verify true recovery of endogenous analyte from tissues without radio labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicated ( that the sera data are accurate to ± 30%; liver data are accurate to ± 50%. Group 6 Timepoint Sex 0 ppm 0.5 ppm 2 ppm 5 ppm 20 ppm High Recovery Average ± SD Average ± SD Average ± SD Average ± SD Average ± SD Average ± SD Male < LOQb ( n= 5) 0.907 ± 0.0619 ( n= 5) 4.33 ± 1.16 ( n= 5) 7.57 ± 2.17 ( n= 5) 41.8 ± 7.92 ( n= 5) Week 0 Female 0.0259 ± 0.00663 ( n = 5) 1.61 ± 0.207 ( n = 5) 6.62 ± 0.499 ( n = 5) 12.6 ± 1.73 ( n = 5) 54.0 ± 7.34 ( n = 5)

Male < LOQc ( n = 5) 4.04 ± 0.801 ( n = 5) 17.1 ± 1.22 ( n = 5) 43.9 ± 4.90 ( n = 5) 148 ± 13.8 ( n = 5) Week 14a Female 2.67 ± 4.58 ( n = 5) 6.96 ± 0.993 ( n = 4d) 27.3 ± 2.34 ( n = 5) 64.4 ± 5.48 ( n = 5) 223 ± 22.4 ( n = 5)

Male 0.0249 ± 0.0182 ( n = 5) 146 ± 33.5 ( n = 4) Week 53 Female 0.395 ± 0.777 ( n = 5) 220 ± 44.0 ( n = 5)

Male Day 719 Female 20.2 ± 13.3 ( n = 9)

Male 0.0118 ± 0.0104 ( n = 11) 1.31 ± 1.30 ( n = 10) 7.60 ± 8.60 ( n = 17) 22.5 ± 23.5 ( n = 25) 69.3 ± 57.9 ( n = 22) Week 105 Female 0.0836 ± 0.134 ( n = 24) 4.35 ± 2.78 ( n = 15) 75.0 ± 45.7 ( n = 15) 233 ± 124 ( n = 25)

Male 2.42 ± 5.09 ( n = 10) Week 106 Female 9.51 ± 8.70 ( n = 17) ENV/ JM/ RD( 2002) 17/ FINAL 39 Table 5. Summary of PFOS Concentration Liver ( ug/ g)

Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Timepoint Sex Control Low Mid Mid High High High Recovery Average ± SD Average ± SD Average ± SD Average ± SD Average ± SD Average ± SD Male 0.104 ± 0.0673 ( n = 5) 11.0 ± 2.31 ( n = 5) 31.3 ± 5.84 ( n = 5) 47.6 ± 12.5 ( n = 5) 282 ± 45.3 ( n = 5) Week 0 Female 0.107 ± 0.0486 ( n = 5) 8.71 ± 0.552 ( n = 5) 25.0 ± 6.11 ( n = 5) 83.0 ± 14.1 ( n = 5) 373 ± 44.1 ( n = 5)

Male 0.459 ± 0.0573 ( n = 5) 23.8 ± 3.45 ( n = 5) 74.0 ± 6.16 ( n = 5) 358 ± 28.8 ( n = 5) 568 ± 107 ( n = 5) Week 10 Female 12.0 ± 22.4 ( n = 5) 19.2 ± 3.77 ( n = 5) 69.2 ± 3.46 ( n = 5) 370 ± 22.3 ( n = 5) 635 ± 49.0 ( n = 5)

Male 0.635 ± 1.04 ( n = 10) 435 ± 96.9 ( n = 9) Week 53 Female 0.923 ± 1.77 ( n = 10) 560 ± 180 ( n = 10)

Male Day 719 Female 55.1 ± 31.5 ( n = 9)

Male 0.114 ± 0.148 ( n = 11) 7.83 ± 7.34 ( n = 10) 26.4 ± 20.4 ( n = 17) 70.5 ± 63.1 ( n = 25) 189 ± 141 ( n = 22) Week 105 Female 0.185 ± 0.184 ( n = 24) 12.9 ± 6.81 ( n = 15) 131 ± 61.4 ( n = 15) 381 ± 176 ( n = 25)

Male 3.12 ± 5.97 ( n = 10) Week 106 Female 12.9 ± 10.4 ( n = 17)

It is not possible to verify true recovery of endogenous analyte from tissues without radio labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicated that the sera data are accurate to ± 30%; liver data are accurate to ± 50%.

3.6 Developmental Toxicity Three prenatal developmental toxicity studies of PFOS have been conducted, two studies in rats and one study in rabbits. In addition, preliminary results are available for developmental toxicity studies in rats and mice.

The first study administered four groups of 22 time mated Sprague Dawley rats 0, 1, 5, and 10 mg/ kg/ day PFOS in corn oil by gavage on gestation days ( GD) 6 15 ( Gortner, 1980). Doses were adjusted according to body weight. Dams were monitored on GD 3 20 for clinical signs of toxicity. Individual body weights were recorded on GD 3, 6, 9, 12, 15, and 20. Animals were sacrificed on GD 20 by cervical dislocation and the ovaries, uteri and contents were examined for the number of corpora lutea, number of viable and non viable fetuses, number of resorption sites, and number of implantation sites. Fetuses were weighed and sexed and subjected to external gross necropsy. Approximately one third of the fetuses were fixed in Bouin's solution and examined for visceral abnormalities by free hand sectioning. The remaining fetuses were subjected to a skeletal examination using alizarin red. ENV/ JM/ RD( 2002) 17/ FINAL 40 Signs of maternal toxicity consisted of significant reductions in mean body weights during GD 12 20 at the high dose group of 10 mg/ kg/ day. No other signs of maternal toxicity were reported. Under the conditions of the study, a NOAEL of 5 mg/ kg/ day and a LOAEL of 10 mg/ kg/ day for maternal toxicity were indicated.

Developmental toxicity evident at 10 mg/ kg/ day consisted of reductions in the mean number of implantation sites, corpora lutea, resorption sites and the mean numbers of viable male, female, and total fetuses, but the differences were not statistically significant. In addition, unusually high incidences of unossified, assymetrical, bipartite, and missing sternebrae were observed in all dose groups; however, these skeletal variations were also observed in control fetuses at the same rate and therefore these effects were not considered to be treatment related. The most notable sign of developmental toxicity observed in all dose groups consisted of abnormalities of the lens of the eye, which was not seen in controls. The proportion of fetuses with the lens abnormality in one or both lenses was significantly higher in the high dose group. All eye abnormalities appeared to be localized to the area of the embryonal lens nucleus, although a variety of morphological appearances were present within that location. According to the authors, this abnormality appeared to be an arrest in development of the primary lens fibers forming the embryonal lens nucleus. Secondary lens fiber development progressed normally except immediately surrounding the abnormal embryonal nucleus. Under the conditions of the study, a LOAEL for developmental toxicity of 1 mg/ kg/ day was indicated; a developmental NOAEL could not be established.

In a second prenatal developmental toxicity study, groups of 25 pregnant Sprague Dawley rats were administered 0, 1, 5, and 10 mg/ kg/ day PFOS in corn oil by gavage on gestation days ( GD) 6 15 ( Wetzel, 1983). Sexually mature Sprague Dawley rats, one per sex per cage, were paired until confirmation of mating or until two weeks had elapsed. Mating was confirmed by daily vaginal examinations for the presence and viability of sperm or the presence of a copulatory plug. The day of confirmation of mating was designated as day 0 of gestation. Doses were adjusted according to the most recently recorded body weight measurements. Dams were observed twice daily for signs of mortality and moribundity and once daily for clinical signs of toxicity. Individual body weights and food consumption were recorded on GD 6, 8, 12, 16, and 20. Animals were sacrificed on GD 20 by CO2 asphyxiation and the fetuses were delivered by cesarean section on GD 20. A gross necropsy was performed on all dams. The uterus from each female was excised, weighed and examined for the number and placement of implantation sites, number and of live and dead fetuses, number of early and late resorptions, and any abnormalities and then weighed again after the contents were removed. The ovaries were examined for the number of corpora lutea. Each female was examined by gross necropsy. Each fetus was sexed, weighed, and examined externally. Approximately one third of the fetuses were fixed in Bouin's solution and examined for visceral abnormalities by the Wilson technique, with particular attention to the eyes, palate, and brain. The remaining fetuses were subjected to a skeletal examination that included evaluation of the skull, long bones, vertebral column, rib cage, extremities, and pectoral and pelvic girdles using alizarin red; bone alignment and degree of ossification were also evaluated.

Evidence of maternal toxicity, that was observed at the 5 and 10 mg/ kg/ day dose groups both during and following treatment and considered to be treatment related, consisted of hunched posture, anorexia, bloody vaginal discharge, uterine stains, alopecia, rough haircoat, and bloody crust. Significant decreases in mean body weight gains during GD 6 8, 6 16, and 0 20 were also observed at the 5 and 10 mg/ kg/ day dose groups. These reductions were considered to be treatment related since mean body weight gains were greater than controls during the post exposure period ( GD 16 20). Significant decreases in mean total food consumption were observed on GD 17 20 in the10 mg/ kg/ day dose group, and on GD 7 16 and 0 20 in both the 5 and 10 mg/ kg/ day dose groups. The mean gravid uterine weight in the 10 mg/ kg/ day dose group was significantly lower when compared with controls. The mean terminal body weights minus the gravid uterine weights were lower in all treated groups, with significant decreases at 5 and 10 mg/ kg/ day. High ENV/ JM/ RD( 2002) 17/ FINAL 41 dose animals also exhibited an increased incidence in gastrointestinal lesions. No significant differences were observed in pregnancy rates, number of corpora lutea, and number and placement of implantation sites among treated and control groups. Two dams in the 10 mg/ kg/ day dose group were found dead on GD 17. Under the conditions of the study, a NOAEL of 1 mg/ kg/ day and a LOAEL of 5 mg/ kg/ day for maternal toxicity were indicated.

Signs of developmental toxicity included a dose related trend toward an increased incidence of late resorptions, total resorptions, number of dead fetuses, and fetal loss, although, none of these effects were statistically significantly different from controls. Significant decreases in mean fetal weights for both males and females were observed in the 5 and 10 mg/ kg/ day dose groups. The percent of male fetuses was 52%, 54%, and 60% for 1, 5, and 10 mg/ kg/ day, respectively, compared to 44% in controls. Statistically significant increases in incomplete closure of the skull were observed in the low and high dose groups but not in the mid dose group. Statistically significant increases in the incidences in the number of litters containing fetuses with visceral anomalies, delayed ossification, and skeletal variations were observed in the high dose group of 10 mg/ kg/ day. These included external and visceral anomalies of the cleft palate, subcutaneous edema, and cryptorchism as well as delays in skeletal ossification of the skull, pectoral girdle, rib cage, vertebral column, pelvic girdle, and limbs. Skeletal variations in the ribs and sternebrae were also observed. Under the conditions of the study, a NOAEL of 1 mg/ kg/ day and a LOAEL of 5 mg/ kg/ day for developmental toxicity were indicated.

The developmental eye abnormalities that were seen in the previous study ( Gortner, 1980) were not observed in the 1983 developmental toxicity study even though the study design and doses were the same. Findings of abnormalities in eye development were initially thought to be treatment related but later determined to be artifacts of sectioning ( 3M Company, 1999a).

Lau et al. ( 2001) administered Sprague Dawley rats and CD 1 mice doses of 0, 1, 5 or 10 mg/ kg/ day PFOS in 0.5% Tween 20 by gavage beginning on gestation day 2 and continuing until term. Half of the dams were sacrificed on gestation day 21 ( rats) or gestation day 17 ( mice) and the remaining dams were allowed to deliver. Preliminary results are available. In rats, there was a significant reduction in maternal body weight gain at 5 and 10 mg/ kg/ day. Maternal serum cholesterol and triglycerides were reduced at 10 mg/ kg/ day, but liver weights were comparable to control. At 10 mg/ kg/ day, there was a reduction in fetal body weight and an increase in cleft palate and anasarca. All pups were born alive, but within 4 6 hours after birth all the pups in the 10 mg/ kg/ day group died, and 95% of the pups in the 5 mg/ kg/ day group died within 24 hours. In mice, maternal body weight was unaffected and liver weights were significantly increased at 5 and 10 mg/ kg/ day; serum triglycerides were reduced were elevated at 5 and 10 mg/ kg/ day. The incidence of fetal mortality was slightly increased at 10 mg/ kg/ day and mean fetal body weights were comparable to control. However, neonatal body weights were reduced during the first 3 days of life. Additional studies are underway to further elucidate the dose response relationships and to examine the mechanism for the neonatal death.

Christian et al. ( 1999a) administered pregnant New Zealand White rabbits, 22 per group, doses of 0, 0.1, 1.0, 2.5 or 3.75 mg/ kg/ day PFOS in 0.5% Tween 80 by gavage on gestation days 7 20. A dose volume of 5 mL/ kg was administered, adjusted daily on the basis of individual body weights. The does were observed twice daily for viability, and clinical observations were recorded 1 hour prior to and after dosing during the treatment period and once daily during the post treatment period ( i. e. gestation days 20 29). Maternal body weights were recorded on gestation days 0 and 6 29; food consumption was recorded daily throughout the study. On gestation day 29, the does were euthanized; a gross necropsy of the thoracic, abdominal and pelvic viscera was conducted and the number of corpora lutea in each ovary was recorded. The uteri were examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. The fetuses were weighed, sexed and examined for external abnormalities. All fetuses were examined for visceral and skeletal abnormalities and the brain of one half of the fetuses were free ENV/ JM/ RD( 2002) 17/ FINAL 42 hand cross sectioned and examined in situ.

In addition, a satellite study was conducted in which pregnant New Zealand White rabbits were administered the same doses as in the main study. The number of does was 3, 5, 3, 3 and 5 in the control, 0.1, 1.0, 2.5 and 3.75 mg/ kg/ day groups, respectively. The does were euthanized on gestation day 21, blood samples were collected, and the liver was weighed and sectioned. The fetuses were removed and examined for external abnormalities. Fetuses and placentae were pooled per litter. All samples were sent to the Sponsor ( 3M) for analysis. At this time, only the liver and serum analyses have been reported ( 3M Environmental Laboratory, 2001d).

Maternal toxicity was evident at doses of 1.0 mg/ kg/ day and above. One doe in the 2.5 mg/ kg/ day group and nine does in the 3.75 mg/ kg/ day aborted. All abortions occurred on gestation days 22 28 and were considered treatment related by the study authors. There was a significant increase in the incidence of scant feces in the 3.75 mg/ kg/ day group. Scant feces were also noted in one and three does in the 1.0 and 2.5 mg/ kg/ day groups, respectively. Mean maternal body weight gains were significantly reduced in the 3.75 mg/ kg/ day group on gestation days 10 13, 13 16, 16 19 and 21 24. Mean body weight gains were also calculated for the treatment period ( days 7 21), post treatment period ( days 21 29) and duration of the study ( days 7 29). There was a significant reduction in mean maternal body weight gain during the treatment period in the 1.0, 2.5 and 3.75 mg/ kg/ day groups. Mean body weight gain for the entire study period was also significantly reduced in the 2.5 mg/ kg/ day group. Mean food consumption ( g/ kg/ day) was significantly reduced in the 2.5 mg/ kg/ day group on gestation days 16 19, 19 21 and 21 24, as well as for the entire study period ( days 7 29). Mean food consumption was significantly reduced in the 3.75 mg/ kg/ day group on gestation days 13 16, 16 19, 19 21 and 21 24, as well as the entire treatment period. The LOAEL for maternal toxicity was 1.0 mg/ kg/ day and the NOAEL was 0.1 mg/ kg/ day.

Developmental toxicity was evident at doses of 2.5 mg/ kg/ day and above. The number of corpora lutea, resorptions, live/ dead fetuses, litter size and sex ratio were comparable among treated and control groups. Mean fetal body weight ( male, female and sexes combined) was significantly reduced in the 2.5 and 3.75 mg/ kg/ day groups. There was also a significant reduction in the ossification of the sternum ( litter averages) in the 2.5 and 3.75 mg/ kg/ day groups, and a significant reduction in the ossification of the hyoid ( litter averages), metacarpals ( litter averages) and pubis ( litter and fetal averages) in the 3.75 mg/ kg/ day group. The LOAEL for developmental toxicity was 2.5 mg/ kg/ day and the NOAEL was 1.0 mg/ kg/ day.

In the satellite study of does euthanized on gestation day 21, the liver and serum analyses were reported by 3M Environmental Laboratory ( 2001d). All serum and liver samples ( including those from untreated controls) had detectable levels of PFOS; the values are presented below:

Average Concentration of PFOS in Rabbit Liver and Serum by Dose Group Dose group PFOS conc. PFOS conc. ( mg/ kg/ day) liver ( ug/ g) serum ( ug/ ml)

0.0 0.239 0.0690 0.1 13.1 2.73 1.0 133 23.8 2.5 317 45.8 3.75 416 88.9 Qualitatively, increasing concentrations of PFOS were found in samples of liver and serum as doses of PFOS increased. The levels of PFOS are much higher in the liver than in the serum. ENV/ JM/ RD( 2002) 17/ FINAL 43 These values should be viewed with caution. It was stated that because radio labeled reference material was not available, " it is not possible to verify true recovery of endogenous analyte from tissues." Matrix spike recovery indicates the accuracy of quantitation to be + 30%. It is also noted that liver concentrations may be biased high. The only conclusion presented in the laboratory report is that " PFOS was observed in the liver and serum of all rabbits dosed with the test article" ( 3M Environmental Laboratory, 2001d).

3.7 Reproductive Toxicity A two generation reproductive toxicity study, designed to test for the toxic effects of PFOS on reproductive function in adult animals and on developmental, learning, and reproductive effects in the offspring, was conducted in Sprague Dawley rats ( Christian et al., 1999b). Five groups of 35 rats per sex per dose group were administered PFOS by gavage at doses of 0, 0.1, 0.4, 1.6, and 3.2 mg/ kg/ day for six weeks prior to and during mating. Treatment in male rats continued until one day before sacrifice ( approximately 22 days total); female rats were treated throughout gestation, parturition, and lactation.

F0 Generation:

Parental animals ( F0) were observed twice daily for clinical signs. Body weights and food consumption values were recorded weekly during the treatment period in male rats; and weekly during mating and then daily during gestation, and on lactation days 1, 4, 7, 10, 14, and at sacrifice in female rats. Each dosage group consisted of two sets of female rats. One set consisted of the first ten female rats with confirmation of mating; this group was dosed until gestation day ( GD) 10 and delivered via Caesarean sectioning. The remaining females comprised the second set which delivered naturally. During the 21 day lactation period, the dams were evaluated for clinical signs during parturition and length of gestation, and then each litter was evaluated at least twice daily for size and pup viability at birth. Pup observations during the 21 day lactation period included physical signs, body weights, nursing behavior, surface righting reflex, pinna unfolding, eye opening, acoustic startle response and air righting reflex. Pupil constriction was evaluated only on lactation day 21. On lactation day 4, litters were randomly culled to four male and four female pups. The remaining pups were sacrificed and necropsied. The F0 male rats were sacrificed and necropsied after the end of dosing at the time of parturition ( lactation day 1). The testes, epididymides, prostate, and seminal vesicles were weighed. Evaluations of sperm number, motility, and morphology were not included in the protocol. The F0 generation females that delivered by Caesarean section were sacrificed on GD 10 and necropsied. Pregnancy status was confirmed, the ovaries were examined for the number and distribution of corpora lutea, implantation sites were determined, and embryos were examined for viability. The F0 generation females that delivered naturally were sacrificed on lactation day ( LD) 21 and necropsied. Ovaries were examined as above and the number and distribution of implantation sites was recorded. The liver from each parental rat was removed, weighed and analyzed. Blood samples were collected from 5 male rats that had mated and from 5 female rats on LD 21 for pharmacokinetic analysis; livers from the pups from the litters of these five dams were also collected for analysis.

F1 Generation:

Since F1 generation pup viability was significantly reduced in the 1.6 and 3.2 mg/ kg/ day dose groups, only the 0.1 and 0.4 mg/ kg/ day dose groups were carried into the second generation. Twenty five F1 generation rats per sex per dose group were administered PFOS by gavage at doses of 0, 0.1, and 0.4 mg/ kg/ day beginning on LD 22 and continuing through the day before sacrifice. At 24 days of age, one rat per sex per litter in each dose group was tested in a passive avoidance paradigm. On LD 28, females were evaluated for the age of vaginal patency and on LD 34, male rats were evaluated for the age of preputial separation. One rat per sex per litter were evaluated in a water filled M maze on LD 70. Assignment to cohabitation within each dose group began on LD 90. Females with evidence of mating were considered to be at GD 0 and assigned to individual housing for the remainder of the dosing period. The F1 generation male rats ENV/ JM/ RD( 2002) 17/ FINAL 44 were sacrificed after mating, necropsied and evaluated as described in the F0 generation. All F1 generation females were allowed to deliver naturally. Dams that delivered litters were sacrificed and necropsied on LD 21. All F2 generation pups were sacrificed, necropsied, and examined on LD 21 as previously described for the F1 generation pups.

In the F0 generation male rats, there were no treatment related clinical signs of toxicity, no mortality, and no effects on mating or on any of the fertility parameters evaluated in any dose group tested. Reported effects included reductions in both body weight gains and in absolute and relative food consumption at the 1.6 and 3.2 mg/ kg/ day dose groups during the pre mating period. Following mating, food consumption was significantly reduced in the 0.4. and 1.6 mg/ kg/ day dose groups. Terminal body weights were also significantly reduced in the 1.6 and 3.2 mg/ kg/ day dose groups. Significant reductions in the absolute weights of the seminal vesicles ( with fluid) and the prostate were observed in F0 males at 3.2 mg/ kg/ day; no other organ weight changes were reported. A significant increase in the number of males with brown liver at 3.2 mg/ kg/ day dose group was also reported.

In the F0 generation female rats, no deaths were reported at any dose level. In dams sacrificed on GD 10 for Caesarean sectioning, there did not appear to be any effects on estrous cycling, mating and fertility parameters, the numbers of corpora lutea and implantations, or in the number of viable or non viable embryos. The only findings reported in the F0 dams occurred in the 0.4, 1.6, and 3.2 mg/ kg/ day dose groups and included localized alopecia during pre mating, gestation, and lactation; and reductions in body weight and body weight gain and food consumption values observed during the pre mating period and continuing throughout gestation and lactation.

Reversible delays in reflex and physical development were observed in the F1 generation offspring. The ability to surface right was significantly delayed in the 1.6 and 3.2 mg/ kg/ day dose groups on LDs 3 10 ( delays in the 3.2 mg/ kg/ day dose group were observed on LD 1, after which there were no surviving pups remaining for further observation). By the end of the observation period, however, all surviving pups in the 1.6 mg/ kg/ day dose group had the ability to surface right. There were no delays observed in the ability to surface right in dose groups < 0.4 mg/ kg/ day. Similar responses were seen for pinna unfolding and eye opening. Although there were transient delays seen with these signs of physical development across all dose groups, by the end of the observation period responses in pups were similar to controls. The time of development of the acoustic startle reflex and the ability to air right were both significantly reduced in the 1.6 mg/ kg/ day dose group. No effects on these reflexes were observed in the low dose group of 0.1 mg/ kg/ day and only a transient delay ( on LD 16 only) in the ability to air right was seen in the 0.4 mg/ kg/ day group. At the end of lactation ( LD 21), all live pups in all dose groups ( 0, 0.1, 0.4, and 1.6 mg/ kg/ day) had pupil constriction response.

The most significant finding reported in the offspring was that of reduced pup viability at the two highest dose groups. The reductions in pup viability began to appear on LD 4 postculling in the 1.6 mg/ kg/ day dose group, with over 26% of the pups found dead between LD 2 4. In the 3.2 mg/ kg/ day dose group 45% of the pups were found dead on LD1; no pups survived beyond LD 1. Statistically significant increases in the number of dams with stillborn pups were also observed at 3.2 mg/ kg/ day. As a result, the viability index was greatly reduced in these dose groups ( 0% at 3.2 mg/ kg/ day and 66% at 1.6 mg/ kg/ day). The lactation index was also significantly reduced ( 94.6%) in the 1.6 dose group. In addition, gestation length was significantly reduced in the high dose group and there also was a significant reduction in the number of implantation sites followed by a concomitant reduction in litter size. Statistically significant reductions in pup body weights were also observed at the two highest dose groups. Other adverse signs in the 3.2 mg/ kg/ day dose level associated with reductions in pup viability and maternal care included litters with pups that were not nursing or who had no evidence of milk in the stomach, as well as maternal cannibalization of pups that were stillborn or found dead. ENV/ JM/ RD( 2002) 17/ FINAL 45 Since F1 generation pup viability was significantly reduced in the 1.6 and 3.2 mg/ kg/ day dose groups, only the 0.1 and 0.4 mg/ kg/ day dose groups were carried into the second generation.

Clinical observations in the F1 generation male rats appeared unremarkable. No treatment related deaths were reported and no statistically significant differences were reported for any of the following parameters: body weights/ body weight gains, average day of preputial separation; values for learning, short term retention, long term retention or response inhibition as evaluated by performance in a passive avoidance or watermaze performance paradigm; mating or fertility parameters; necroscopic examinations; absolute or relative weights for the right or left testis, seminal vesicles, right epididymis, or prostate; and terminal body weights. The only reported effects were significant reductions in absolute food consumption on postweaning days 1 8 occurring at the 0.1 and 0.4 mg/ kg/ day dose levels.

Clinical observations for the F1 generation females were likewise unremarkable. Observations at the 0.4 mg/ kg/ day dose group included, reductions in body weights on day 1 postweaning, significant losses in body weight on LDs 1 4, and significant reductions in food consumption on days 1 8 postweaning and during lactation. There were no statistically significant differences reported for any of the following parameters: values for learning, short term retention, long term retention or response inhibition as evaluated by performance in a passive avoidance or water maze performance paradigm; mating and fertility parameters; gestation index; pregnancy rates; and necroscopic examinations.

Evidence of treatment related effects in the F2 generation pups consisted of reductions in mean pup body weights ( on a per litter basis) observed at 0.1 mg/ kg/ day on LD 4 and 7. Body weights were comparable to control levels by LD 14. At 0.4 mg/ kg/ day, statistically significant reductions in mean pup body weights were observed on LDs 7 and 14. Mean body weights on LD21 continued to remain lower than controls, although the difference was not statistically significant ( 46.5 g in 0.4 mg/ kg/ day dose group vs. 50 g in controls). Clinical and necroscopic observations of the F2 generation pups were unremarkable. No other toxicologically significant effects were reported.

Under the conditions of the study, the NOAEL and LOAEL for both the F0 generation male and female parents are 0.1 mg/ kg/ day and 0.4 mg/ kg/ day, respectively, based on reductions in body weight gain and food consumption. The NOAEL for the F1 generation parental males could not be established since treatment related reductions in absolute food consumption values were reported at the lowest dose tested, 0.1 mg/ kg/ day. The NOAEL and LOAEL for the F1 generation parental females are 0.1 mg/ kg/ day and 0.4 mg/ kg/ day, respectively, based on significant reductions in body weights and food consumption. The NOAEL and LOAEL for the F1 generation offspring are 0.4 mg/ kg/ day and 1.6 mg/ kg/ day, respectively, based on significant reductions in the number of implantation sites, litter size, pup viability, pup body weight and survival. The NOAEL and LOAEL for the F2 generation offspring are 0.1 mg/ kg/ day and 0.4 mg/ kg/ day, respectively, based on significant reductions in mean pup body weight.

Liver and sera samples were collected from the F0 and F1 animals at terminal sacrifice ( after cohabitation for males and on lactaton day 21 for females) and analyzed for the presence of PFOS ( 3M Environmental Laboratory, 1999a). The results for the F0 animals are presented below: ENV/ JM/ RD( 2002) 17/ FINAL 46 Dose group Average PFOS conc. Average PFOS conc. ( mg/ kg/ day) in serum ( ug/ ml) in liver ( ug/ g)

0.0 female 0.0307 female 0.171 male 0.0244 male 0.665 0.1 female 5.28 female 14.8 male 10.5 male 84.9 0.4 female 18.9 female 58.0 male 45.4 male 176 1.6 female 82 female 184 male 152 male 323 3.2 female NR* female NR* male 273 male 1360 * samples not received Qualitatively, the F0 results indicate all rats ( including controls) had detectable levels of PFOS in serum and livers. PFOS concentration increased with dose. PFOS concentrations were higher in the liver than in the serum, and males had greatly increased PFOS concentrations in serum and liver when compared with females of the same dose group. Pooled liver samples from the F1 animals sacrificed shortly after birth had lower PFOS concentrations than adults of the F0 generation of the same dose group. The average PFOS concentrations in pooled liver samples from F1 animals shortly after birth were 0.0511, 6.19, 57.6, and 70.4 ug/ g in the 0.0, 0.1, 0.4, and 1.6 mg/ kg/ day dose groups, respectively. These quantitative values for the PFOS concentration in the liver and serum should be viewed with caution. The accuracy of quantitation is + 30%, the purity of the analytical reference substance is unknown, and there were several uncorrected dilution errors.

Two studies were then conducted to further understand the distribution of PFOS in the dam, fetus and neonate. In the first study, Sprague Dawley rats were administered oral doses of PFOS ( 0.1, 0.4, 1.6, and 3.2 mg/ kg/ day) once daily beginning 42 days prior to cohabitation, and continued through day 14 or day 20 of presumed gestation ( 3M Environmental Laboratory, 2001e). Serum, urine, and feces specimens were collected from the dams before mating and at GD 7, GD 15 and GD 21; liver specimens were collected on GD21. A total of 54 pooled serum and liver specimens were collected from fetuses on GD 21. The results are shown below:

Average Results for the Analysis of Serum Samples ( ug/ ml)

0 mg/ kg 0.1 mg/ kg 0.4 mg/ kg 1.6 mg/ kg 3.2 mg/ kg Day 0 0.0723 8.89 40.7 160 318 Day 7 0.126 7.82 40.9 154 105 Day 15 0.0926 8.80 41.4 156 275 Day 21 0.0714 4.24 26.2 136 155 Day 21 Fetal 0.125 9.07 34.3 101 165 ENV/ JM/ RD( 2002) 17/ FINAL 47 Average Results for the Analysis of Liver Samples Dose Group PFOS Conc. ( ug/ g) PFOS Conc. ( ug/ g) ( mg/ kg/ day) Female Adult Fetal Liver 0 0.288 0.169 0.1 29.2 7.93 0.4 107 30.6 1.6 347 86.7 3.2 610 230 In general, there was a dose related increase in the levels of PFOS in the liver and serum of the dams and the fetuses. PFOS was also observed in the control dams, as well as the control fetuses. As observed in the 2 generation reproductive toxicity study described above, the levels of PFOS were much higher in the liver than in the serum for the dams. The levels of PFOS remained fairly steady in the serum of the dams from GD0   GD 15, but the levels dropped at GD21. In the GD21 fetuses, the level of PFOS in the serum was generally comparable to the level observed in the dams, whereas the level of PFOS in the fetal livers was well below that seen in the dams.

In the second study, Sprague Dawley rats were administered oral doses of PFOS ( 0.1 and 1.6 mg/ kg/ day), once daily beginning 43 days prior to cohabitation until confirmed evidence of mating ( 3M Environmental Laboratory, 2001f). Urine and fecal samples were collected from the dams during the following intervals: one day prior to initiation of cohabitation to the following morning, days 6 to 7, 14 to 15, and 20 to 21 of presumed gestation and days 21 22of lactation. Blood samples were collected from each of the dams on the day cohabitation was initiated ( prior to cohabitation), GD 7, 15 and 21, and LD 14 and 22. Day 1 of lactation was defined as the day of birth. On LD 4, litters were culled to five male pups and five female pups per litter, where possible. Sera specimens were collected from pooled litter samples on LD 21. Liver specimens were collected from the dams, and the liver from each pup was collected and pooled per litter. Blood samples were collected and pooled per liter. The results are shown below:

Average Results for the Analysis of Serum Samples ( ug/ ml)

0 mg/ kg 0.1 mg/ kg 1.6 mg/ kg GD 0 0.100 9.21 161 GD 7 0.0796 7.24 129 GD 15 0.0742 5.68 90.6 GD 21 < LLQ 2.58 39.5 LD14 0.0542 1.63 20.6 LD22 0.0492 0.979 14.1 LD 21 Pups 0.0531 1.80 27.1 Average Results for the Analysis of Liver Samples ( ug/ g)

0 mg/ kg 0.1 mg/ kg 1.6 mg/ kg Dam 0.243 6.15 59.7 Pup 0.174 5.00 56.2 In general, there was a dose related increase in the levels of PFOS in the liver and serum of the dams and the levels in the serum decreased with time. The levels of PFOS were much higher in the liver than in the ENV/ JM/ RD( 2002) 17/ FINAL 48 serum of the dams and the pups. The levels of PFOS were similar in the liver of the dams and pups, while the levels in the serum were slightly higher in the pups than in the dams.

Based on the results of the two generation reproductive toxicity study in which significant reductions in pup viability were observed at 1.6 and 3.2 mg/ kg/ day, a cross fostering study was conducted as a means of determining whether the effects observed in pups were a result of in utero exposure to PFOS or as a result of exposure during lactation; thus the potential for a distinction to be made between prenatal and postnatal effects following continuous maternal treatment ( Christian et al., 1999c).

In this study, two groups of 25 female Sprague Dawley rats were administered 0 and 1.6 mg/ kg/ day PFOS in 0.5% Tween 80 by gavage, beginning 42 days prior to mating to untreated ( breeder) males, and continuing throughout gestation and into day 21 of lactation. A dose volume of 5 mL/ kg was administered, adjusted daily on the basis of individual body weight. Parental females were observed twice daily for viability and clinical observations were recorded 1 hour prior to and after dosing during the treatment period. Maternal body weights were recorded once during the acclimation period and then daily during the treatment period and at sacrifice; food consumption was also recorded once during the acclimation period and then daily during gestation and on days 1, 4, 7, 10, and 14 of lactation. During parturition, females were continually evaluated for clinical signs and also for duration of gestation, length of parturition, litter sizes, and pup viability at birth. Maternal behavior was recorded daily throughout lactation. All maternal rats were sacrificed by carbon dioxide asphyxiation on day 22 of lactation and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed; any gross lesions were preserved for future analysis. In addition, the number and distribution of implantation sites were recorded. Rats that did not deliver a litter were not included in the cross fostering procedure and were sacrificed on lactation day 25, examined for gross lesions, and the uteri examined to confirm the presence/ absence of implantation sites. Dams with no surviving pups were sacrificed after the last pup was found dead, missing, or presumed cannibalized.

Following completion of parturition, litters were immediately removed from their respective dams and placed with either a control or PFOS treated dam for rearing. This cross fostering procedure resulted in four groups of 12 13 dams or pups as follows: A) control dams with litters from PFOS treated dams, i. e., in utero exposure only; B) control dams with litters from control dams, i. e., negative control; C) PFOStreated dams with litters from PFOS treated dams, i. e., both in utero and post natal exposure; and D) PFOS treated dams with litters from control dams, i. e., post natal exposure only.

On day 1 of lactation ( birth), each pup was individually weighed and each litter was evaluated twice daily during lactation for viability. Pups were observed once daily for clinical signs and gross external physical anomalies. Pup body weights were recorded on days 1, 4, 7, 14, and 21 of lactation, and then at sacrifice. On day 4 of lactation, each cross fostered litter was culled to 5 males and 5 females. On day 21 of lactation, all pups were sacrificed via decapitation and examined for gross lesions. Pups found dead or sacrificed because of moribundity were examined for gross lesions and for the cause of death or the moribund condition. The lungs, liver, and any gross lesions were collected from selected pups at various timepoints and preserved for possible future analysis. Liver samples were evaluated via electron microscopy. The method of statistical analysis consisted of calculation of averages and percentages; litter values were used where appropriate.

In addition, samples of blood, milk ( including the milk secreting glands), and liver were collected from selected maternal rats and pups ( blood and liver samples were pooled per litter) at various timepoints for analysis of PFOS concentration. Only the analysis of the sera samples are available at this time.

All maternal rats survived to schedule sacrifice. Signs of clinical toxicity observed in the dams during the study period ( e. g., chromorhinorrhea, scaly tail, abrasion on the head, neck, tail and/ or forelimb, missing, ENV/ JM/ RD( 2002) 17/ FINAL 49 broken and/ or misaligned incisors, and localized alopecia, among others) were not considered to be treatment related since they also occurred in the control animals. Mean maternal body weight gains at 1.6 mg/ kg/ day were reduced compared to controls during premating and continuing throughout gestation. Mean maternal body weights and body weight changes in the treated group were comparable to those seen in control animals during lactation. During the premating period, and on into gestation and lactation, food consumption was reduced in treated animals as compared to controls. Reductions in gestation length, the average number of implantation sites, delivered sizes, and live litter size were observed in treated animals. All pregnant animals delivered live offspring.

Following cross fostering on LD 1, live litter sizes were comparable between treated and control groups. Pup mortality was observed in two of the cross fostered groups. On lactation days 2 4, approximately 19% of the pups in group C were either found dead or presumed cannibalized. Pup mortality was also observed in group A at a rate of 9%. In addition, on day 4 of lactation, the number of live pups, numbers of surviving pups per litter, and live litter sizes were also reduced in these two groups. Pup mortality in groups B and D during lactation days 2 4 were at 1.6% and 1.1%, respectively. Reductions in pup body weights were observed in groups A and C on day 1 of lactation. Pup body weights in group D were comparable to controls during that same period. From lactation day 4 21, pup body weights in groups A, C, and D were reduced when compared to group B ( negative control), with the reductions greatest in group C. Two litters in group A and one litter on group C did not nurse. Milk analysis of the stomachs of pups found no milk in the stomachs of 57%, 100%, and 87% of the pups found dead and necropsied in groups A, C, and D, respectively. Sex ratios and the lactation index were comparable among all groups. Signs of clinical toxicity were observed in pups, but were not considered to be treatment related since they also occurred in group B ( negative control) at the same rate.

Electron microscopic examination of the liver revealed an increase in the number of peroxisomes in pups from dams treated with 1.6 mg/ kg/ day PFOS. No significant differences were observed between group B and the other groups following examination of pup lungs.

PFOS concentrations in the serum of untreated dams ranged from below the limit of detection ( 0.05 ug/ ml) to 5.34 ug/ ml. Serum PFOS concentrations in the pups from untreated dams, fostered with untreated dams, were below the limit of detection. Serum PFOS concentrations in the pups from treated dams, fostered with untreated dams, ranged from 47.6 ug/ ml to 59.2 ug/ ml. PFOS concentrations in the serum of treated dams ranged from 59.2 ug/ ml to 157 ug/ ml. Serum PFOS concentrations in the pups from untreated dams, fostered with treated dams, ranged from below the limit of detection to 35.7 ug/ ml. Serum PFOS concentrations in the pups from treated dams, fostered with treated dams, ranged from 79.5 ug/ ml to 96.9 ug/ ml. These data indicate that exposure to PFOS can occur both in utero and via milk from treated dams ( 3M Environmental Laboratory, 1999b). The accuracy of quantitation for the analyses was + 30%.

In conclusion, pups from control dams that were cross fostered with PFOS treated dams ( post natal exposure only) had the same low mortality rate ( 1.1%) as pups from control dams cross fostered with control dams ( 1.6%; negative control). Mortality rates in the remaining two groups, however ( i. e. control dams with litters from PFOS treated dams, i. e., in utero exposure only; and PFOS treated dams with litters from PFOS treated dams, i. e., both in utero and post natal exposure), were much higher at 9.6 % and 19.2%, respectively. Under the limited conditions of the study, the data appear to indicate that reduced pup survival is mainly a result of in utero exposure to PFOS and that post natal exposure via milk in conjunction with in utero exposure may also contribute to reduced pup survival. In contrast, exposure during lactation alone, through milk from exposed dams, does not appear to have any adverse affect on pup viability. Additionally, analysis of PFOS concentration showed that PFOS was observed in the sera of F0 female rats exposed during the in life phase of the study. Additionally, PFOS was observed in sera samples taken from F1 generation pups from female rats exposed to the test substance, and in F1 generation pups exposed via lactation, but not exposed in utero. ENV/ JM/ RD( 2002) 17/ FINAL 50 Several mechanistic studies are being conducted to understand the neonatal death ( 3M Company, 2001c). Preliminary results indicate that reductions in serum lipids and cholesterol synthesis do not appear to play a significant role in the death of the offspring.

3.8 Human Hazard Several occupational studies on the health effects associated with fluorochemical exposure have been conducted. The following studies have all been conducted at the 3M Decatur, Alabama plant where PFOS has been manufactured since 1991, and PFOA since 1998. The studies conducted in 2000 also included the Antwerp, Belgium plant. Only some of the studies examined a possible association between serum PFOS levels in workers and corresponding health effects. The other studies use exposure categories as surrogates for fluorochemical serum levels in workers.

Cross sectional data from medical surveillance conducted at the plants were analyzed to determine if there were any associations between PFOS levels and hematology, clinical chemistries, and hormonal parameters ( Olsen et al., 1998, 1999, 2001a). A group of male volunteers working at the Decatur plant in 1995 and 1997 and volunteers from the 2000 medical surveillance, which also included females and Antwerp employees, were analyzed.

In the 1995 and 1997 analyses, there were no consistent associations between PFOS levels in the workers and the hematology and other clinical chemistry parameters when the data from both plants were combined. Mean serum cholesterol levels remained constant or increased with increasing PFOS serum levels depending on the year; however, those employees with the highest PFOS levels had lower mean HDL values. It should be noted that age was significantly associated with higher PFOS levels. In addition, there were no associations between clinical hepatic enzyme tests and serum PFOS levels in workers.

Eleven hormone values were collected from 88 of the employees who participated in the study in 1995. The employees who participated in the hormone portion of the study were significantly different from the non participants at both locations ( younger, higher PFOS levels, smokers, and higher WBC). After adjusting for confounders, there were no significant associations between PFOS and the hormones analyzed, except estradiol; however, it seems that one employee with high PFOS measurements ( 12.83 ppm) and a large BMI may have influenced these results. Removal of this employee from the analyses resulted in no significant associations with estradiol.

There were several differences between the employees at the two plants, such as body mass index, alcohol consumption, and age. In addition, there were only 61 employees common to both the 1995 and 1997 cohort due to a high rate of turnover at both plants from 1996 1997. Some of the findings were not consistent over the 2 time periods. The participation rate in this voluntary biomonitoring was very low at the Decatur plant ( 35 40%). In addition, the employees at both sites used APFO as a surfactant ( mean PFOA serum levels in 1995 were 1.46 ppm and in 1997 were 1.57 ppm). The number of years that each group worked at the 2 plants ( exposure) were not provided in the analyses, although the youngest employees had the lowest levels of serum PFOS. Also, ninety five percent of the employees across both plants and both time periods had serum PFOS levels less than 6 ppm. These limitations indicate that the results of this study should be interpreted carefully.

The results from the 2000 cross sectional analysis were analyzed for all employees from both plant locations combined and by plant since the Antwerp and Decatur populations were significantly different from each other in several ways. Antwerp employees as compared to Decatur employees had lower PFOS serum levels ( mean 0.96 ppm), were younger, had lower BMIs, worked fewer years, had higher alcohol consumption, higher mean HDL and bilirubin values, lower mean triglyceride, alkaline phosphatase, GGT, ENV/ JM/ RD( 2002) 17/ FINAL 51 AST, and ALT values, and mean thyroid hormone values tended to be higher. The data were also stratified by production status and gender and divided into quartiles of their serum PFOS distribution. However, the PFOS levels are different for each subgroup, making it difficult to compare the results.

Comparisons across quartiles within plant locations did not reveal many differences in thyroid, hematology, or urinanalysis values. The only significant ( p < .05) differences noted were: BUN was significantly higher in male production workers and females in the highest PFOS quartiles at the Antwerp plant, and ALT was significantly higher in the highest quartile in Decatur male production employees.

When the data were analyzed by number of employees who had values above the reference range for hepatic clinical chemistry tests and liver enzyme and bilirubin tests, a higher percentage of male Decatur production workers were in the highest PFOS quartile ( 2.31 10.06 ppm) for ALT, GGT, and total liver panel than the other quartiles. Most notable were the results for ALT where 8% of employees in the lowest PFOS exposure group ( Q1) and 28% in the highest exposure group ( Q4) had values above the reference range, while the percentages for total liver panel ( which includes alkaline phosphatase, AST, ALT, GGT, and total and direct bilirubin) were 18% and 35%, respectively. This trend was not evident in Decatur nonproduction employees, in Decatur females, or in any of the Antwerp employees. However, it should be noted that each sub population had a different serum PFOS quartile distribution.

Mean values for triglycerides, alkaline phosphatase, total bilirubin, and ALT were significantly ( p < .05) higher in Q4 ( mean PFOS level 2.69, range 1.69   10.06 ppm) than in Q1 ( mean PFOS level 0.27 ppm, range 0.04   0.42 ppm) for all male employees at both plants ( n = 421). It should be noted that the number of Antwerp production employees were evenly distributed among the quartiles while this was not the case for Decatur employees. The highest number of Decatur employees was in Q4. These data were analyzed for employees who had values above the reference range for alkaline phosphatase, AST, ALT, GGT, and total liver panel. For male employees for all of these measures, the levels increased from Q1 to Q4; however, statistical significance was not reported. In Q1, 4% of the employees had values above the reference range for ALT and 6% for GGT, while 12% was reported for Q4 for both of these tests. For total liver panel, 14% of the employees had values above the reference range in Q1 as compared to 23% in Q4. The numbers of female employees with values above the reference range was very small ( n = 8). In female employees combined for both plants ( n = 97), alkaline phosphatase and GGT were significantly higher ( p < .05) and total bilirubin significantly lower in Q4 than in Q1.

Thyroid results for male production employees of both plants indicated that T3 was significantly higher ( p < .05) and THBR ( T3 uptake) was significantly lower ( p < .05) in Q4 than in Q1. The range of T3 values was very large for females; however, there were no statistically significant differences between the mean values across the quartiles.

Multivariable regression analyses were conducted to adjust for possible confounders that may affect the results of the clinical chemistry tests. The following variables were included: production job ( yes or no), plant, age, BMI, cigarettes/ day, drinks/ day and years worked at the plant.

A positive significant ( p = .04) association between T3 and PFOS was observed. Plant location was highly significant ( p < .0001) in the model. BMI, cigarettes/ day, alcohol/ day were also significant. In the univariate analyses, Antwerp employees had higher mean T3 levels than Decatur employees overall. However, for each plant ( individually) T3 values increased by quartile as PFOS serum levels increased, although the differences were not statistically significant. THBR, as well as the other thyroid hormones, were not significant in the regression analyses.

A positive statistically significant ( p = .04) association between PFOS and cholesterol was observed. When both PFOS and PFOA were included in the model, neither were statistically significant at p=. 05. ENV/ JM/ RD( 2002) 17/ FINAL 52 PFOS was not significantly associated with HDL, but triglycerides were positively associated with PFOS ( p = .01). No significant associations were observed with PFOS in relation to alkaline phosphatase, GGT, AST or total bilirubin.

A longitudinal analysis of the above data was performed to determine whether occupational exposure to fluorochemicals over time is related to changes in clinical chemistry and lipid results in employees of the Antwerp and Decatur facilities ( Olsen et al., 2001b). The medical surveillance data from 1995, 1997, and 2000 were analyzed using multivariable regression. The plants were analyzed using 3 subcohorts that included those who participated in 2 or more medical exams between 1995 and 2000. When male employees from both plants were combined, no statistically significant ( p < .05) associations were observed over time between PFOS and cholesterol or triglycerides. In addition, there were no significant associations between PFOS and changes over time in HDL, alkaline phosphatase, GGT, AST, ALT, total bilirubin, and direct bilirubin.

There were several limitations to the 2000 cross sectional and longitudinal studies including: 1) serum PFOS levels in these workers have been declining over time, 2) PFOS serum levels were significantly higher at the Decatur plant than at the Antwerp plant, 3) all participants were volunteers, 4) there were several consistent differences in clinical chemistry profiles and demographics between employees of the Decatur and Antwerp plants, 5) PFOA and other perfluorinated chemicals are also present in these plants, 6) plant populations cannot be compared because quartiles are different for each subgroup, and 7) only one measurement at a certain point in time was collected for each clinical chemistry test. In addition, in the longitudinal study only a small number of employees participated in all 3 sampling periods ( 24%), different labs and analytical techniques for PFOS were used each year, and female employees could not be analyzed because of the small number of participants.

A retrospective cohort mortality study was performed on Decatur employees to determine whether plant employees had significantly different death experiences from the general population ( Mandel and Johnson, 1995). There were 1957 employees who had worked at least one year at the Decatur plant between 1961 and 1991. Seventy four deaths were recorded, and there were no significantly elevated SMRs for all major causes of death regardless of the comparison population used ( Alabama state, US, or Alabama counties). When the data were analyzed by job description ( chemical department vs. film plant employees), there was a statistically significant deficit for all causes of death in both job categories.

This study had almost complete follow up of the cohort and ascertainment of causes of death. However, since the workforce was relatively young, only 74 deaths were reported for 1,951 employees. The number of women in the study was very small ( 4 deaths). Additionally, PFOS serum levels were not examined in relation to mortality.

An update of this study was recently conducted to follow the cohort through December 31, 1998, thus adding 7 years of follow up ( Alexander, 2001). This cohort consisted of 2083 employees who had worked at the Decatur plant for at least 1 year. There were 145 deaths reported, almost twice that reported in the last cohort. In this study, employees were placed into 3 exposure categories based on their job descriptions: high, low, and non exposed. Almost half of the employees ( 47%) were placed in the high exposure group, in which there were 65 deaths. Fourteen percent of the cohort was in the low exposure group ( 27 deaths), and 39% were in the non exposed group ( 53 deaths).

SMRs were calculated using state of Alabama reference data. When the entire cohort was analyzed, SMRs were not elevated for most of the cancer types and for non malignant causes. SMRs that were above 1 ( cancer of the esophagus, liver, breast, urinary organs, bladder, and skin) were also elevated when the cohort was limited to any employee ever employed in a high exposure job ( except breast cancer). Only 2 or 3 deaths were reported for each of these cause specific categories and were not statistically significant, ENV/ JM/ RD( 2002) 17/ FINAL 53 except for bladder cancer.

Workers who were employed in high exposure jobs were 13 times more likely to die of bladder cancer than the general population of Alabama ( SMR = 12.77, 95% CI = 2.63  37.35). This effect remained when the data were analyzed using county death rates. Three male employees in the cohort died of bladder cancer ( 0.12 expected). All of these workers had been employed at the Decatur plant for more than 20 years, and all of them had worked in high exposure jobs for at least 5 years ( SMR = 24.49). In the previous cohort mortality study, 1 bladder cancer death was reported.

Two deaths were reported for liver cancer. One was in the low exposure group and one in the high exposure group. The SMR for workers who were employed in either high or low exposure jobs was 3.08 ( 95% CI = 0.37  11.10). Five cases of cirrhosis of the liver were reported in this cohort, 2 in the high exposure group, 1 in the low exposure group, and 2 in the non exposed. The observed did not exceed the expected mortality experience in any of these exposure groups.

Most of the same limitations discussed above for the original cohort study apply to this update. A larger cohort was followed in the update, thus reporting twice as many deaths; however, the cohort is fairly young and the number of deaths is still small ( especially for females in all categories). Death certificates were located for 96% of the cohort, but the 6 not obtained could greatly impact the results of the analyses since the number of deaths in most of the sub cohorts was very small. In addition to fluorochemicals, workers were exposed to other chemicals in the workplace. Biological measurements of fluorochemicals were used to define jobs into exposure categories based on job descriptions; however, there is still a potential for misclassification of exposure.

The high ratio of observed bladder cancer deaths in long time employees to those expected in the general population is troublesome. Although only 3 deaths were reported in high exposure jobs, the expected number in the general population was 0.23. It seems unlikely that this effect would be due to chance, given the magnitude of the risk estimate ( 12.77). Many years of follow up without another death from bladder cancer would have to occur before there would no longer be an appreciable risk. Currently, animal studies on PFOS have not reported a higher incidence of bladder cancer tumors. It is unlikely that tobacco smoking could be solely responsible for the excess in bladder cancer mortality, given the 13 fold increase. Smoking status was not determined for this cohort; however, lung cancer and other smoking related cancers were not elevated. Therefore, it is unlikely that tobacco smoking was any higher in the employees than in the general population.

Given the limitations of this study, it is unclear whether fluorochemicals are responsible for the excess of bladder cancer deaths, or whether other carcinogens may be present in the Decatur plant. At the 3M Cottage Grove facility, where APFO and other fluorochemicals are manufactured, 5 bladder cancer deaths were reported ( SMR = 1.31, 95% CI = 0.42   3.05). Four of these deaths occurred in employees who did not work primarily in the chemical division and therefore were assumed to have little exposure to APFO. The authors of the Decatur study report that the bladder cancer cases worked mostly in maintenance jobs or at the incinerator and wastewater treatment plant and could have been exposed to many chemicals in addition to fluorochemicals. It is important that follow up of this cohort is continued in order to gain a better understanding of the mortality experience of workers exposed to fluorochemicals.

In order to gain additional insight into the effects of fluorochemical exposure on workers' health, an " episode of care" analysis was undertaken at the Decatur plant to screen for morbidity outcomes that may be associated with long term, high exposure to fluorochemicals ( Olsen et al., 2001c). An " episode of care" is a series of health care services provided from the start of a particular disease or condition until solution or resolution of that problem. Episodes of care were identified in employees' health claims records using Clinical Care Groups ( CCG) software. All inpatient and outpatient visits to health care providers, ENV/ JM/ RD( 2002) 17/ FINAL 54 procedures, ancillary services and prescription drugs used in the diagnosis, treatment, and management of over 400 diseases or conditions were tracked.

Episodes of care were analyzed for 652 chemical employees and 659 film plant employees who worked at the Decatur plant for at least 1 year between January 1, 1993 and December 31, 1998. Based on work history records, employees were placed into different comparison groups: Group A consisted of all film and chemical plant workers; Group B had employees who only worked in either the film or chemical plant; Group C consisted of employees who worked in jobs with high POSF exposures; and Group D had employees who worked in high exposures in the chemical plant for 10 years or more prior to the onset of the study. Film plant employees were considered to have little or no fluorochemical exposure, while chemical plant employees were assumed to have the highest exposures.

Ratios of observed to expected episodes of care were calculated for each plant. Expected numbers were based on 3M's employee population experience using indirect standardization techniques. A ratio of the chemical plant's observed to expected experience divided by the film plant's observed to expected experience was calculated to provide a relative risk ratio for each episode of care ( RREpC). 95% confidence intervals were calculated for each RREpC. Episodes of care that were of greatest interest were those which had been reported in animal or epidemiologic literature on PFOS and PFOA: liver and bladder cancer, endocrine disorders involving the thyroid gland and lipid metabolism, disorders of the liver and biliary tract, and reproductive disorders.

The only increased risk of episodes for these conditions of a priori interest were for neoplasms of the male reproductive system and for the overall category of cancers and benign growths ( which included cancer of the male reproductive system). There was an increased risk of episodes for the overall cancer category for all 4 comparison groups. The risk ratio was greatest in the group of employees with the highest and longest exposures to fluorochemicals ( RREpC = 1.6, 95% CI = 1.2   2.1). Increased risk of episodes in long time, high exposure employees also was reported for male reproductive cancers ( RREpC = 9.7, 95% CI = 1.1  458). It should be noted that the confidence interval is very wide for male reproductive cancers and the sub category of prostate cancer. Five episodes of care were observed for reproductive cancers in chemical plant employees ( 1.8 expected), of which 4 were prostate cancers. One episode of prostate cancer was observed in film plant employees ( 3.4 expected).

There was an increased risk of episodes for neoplasms of the gastrointestinal tract in the high exposure group ( RREpC = 1.8, 95% CI = 1.2 3.0) and the long term employment, high exposure group ( RREpC = 2.9, 95% CI = 1.7   5.2). Most of the episodes were attributable to benign colonic polyps. Similar numbers of episodes were reported in film and chemical plant employees.

In the entire cohort, only 1 episode of care was reported for liver cancer ( 0.6 expected) and 1 for bladder cancer ( 1.5 expected). Both occurred in film plant employees. Only 2 cases of cirrhosis of the liver were observed ( 0.9 expected), both in the chemical plant. There was a greater risk of lower urinary tract infections in chemical plant employees, but they were mostly due to recurring episodes of care by the same employees. It is difficult to draw any conclusions about these observations, given the small number of episodes reported.

Chemical plant employees in the high exposure, long term employment group had 2 ½ times more episodes of care for disorders of the biliary tract than their counterparts in the film plant ( RREpC = 2.6, 95% CI = 1.2  5.5). Eighteen episodes of care were observed in chemical plant employees and 14 in film plant workers. The sub categories that influenced this observation were episodes of cholelithiasis with acute cholecystitis and cholelithiasis with chronic or unspecified cholecystitis. Most of the reported episodes occurred in chemical plant employees. ENV/ JM/ RD( 2002) 17/ FINAL 55 Risk ratios of episodes of care for endocrine disorders, which included sub categories of thyroid disease, diabetes, hyperlipidemia, and other endocrine or nutritional disorders, were not elevated in the comparison groups. Conditions which were not identified a priori but which excluded the null hypothesis in the 95% confidence interval for the high exposure, long term employment group included: disorders of the pancreas, cystitis, and lower urinary tract infections.

The results of this study should only be used for hypothesis generation. Although the episode of care design allowed for a direct comparison of workers with similar demographics but different exposures, there are many limitations to this design. Episodes of care are reported, not disease incidence; therefore, this parameter cannot be interpreted in any other manner. The data are difficult to interpret because a large RREpC may not necessarily indicate high risk of incidence of disease. In addition, many of the risk ratios for episodes of care had very wide confidence intervals. The analysis was limited to 6 years. Also, the utilization of health care services may reflect local medical practice patterns. Individuals may be counted more than once in the database because they can be categorized under larger or smaller disease classifications. Episodes of care may include the same individual several times. Not all employees were included in the database, such as those on long term disability. The analysis may be limited by the software used, which may misclassify episodes of care. The software may also assign 2 different diagnoses to the same episode. Certain services, such as lab procedures may not have been reported in the database.

4.0 Hazards to the Environment The 3M Company have reported ecotoxicity test results on five PFOS salts: potassium, lithium, ammonium, didecyldimethylammonium and DEA ( diethanolamine). The majority of testing has however been carried out on the PFOS potassium salt. Only limited data are available for the other salts, but because the salts all dissociate instantaneously at neutral pH to the PFOS ion and the appropriate counterion the potential exists for read across. For the purposes of this review it is assumed that the toxicity of the PFOS anion will be dominant and that read across of toxicity from the potassium salt to the other salts is acceptable as a first approximation, except for the didecyldimethylammonium salt. This is because these counterions are unlikely to be toxicologically significant compared with the contribution of the PFOS ion. Published toxicity data relating to, for example, potassium ( as chloride) to fish and invertebrates show acute and chronic effect concentrations to be > 100 mg/ l ( Dierickx and Bredael Rozen, 1996; Biesinger and Christensen, 1972). Toxic concentrations of the PFOS potassium salt reported for fish and invertebrates in the 3M studies are however all well below 100 mg/ l, suggesting that the potassium ion was not a major contributor to the overall toxicity of the substance. For the present purpose correction for the molecular weight of the counterions is considered to be unnecessary. Didecyldimethylammonium is an exception due not only to its higher molecular weight, but also because high molecular weight amines are known to have the potential for exhibiting significant toxicity to aquatic species. Only two of the studies presented for review related to the salt of this cation   acute studies with the Fathead minnow ( Pimephales promelas) and the water flea ( Daphnia magna). Both were judged to be unacceptable for assessing PFOS toxicity because of the use of an inappropriate method of test medium preparation. The result of the test with the fish did however suggest that the didecyldimethylammonium ion was not a source of significant toxicity in the test medium whilst the result of the test with the water flea suggested that it might have been.

Wherever possible measured exposure concentrations have been used as the basis for expressing the toxicity of the test substance. In some instances the measured concentrations were significantly below nominal. In basing effect concentrations on measured concentrations it is assumed that there is a direct relationship between PFOS concentration and toxicity.

The data summaries in Tables 7 to 18 include columns headed ` Study Standard' and ` Comments', in addition to study information required under the standard OECD column headings. ENV/ JM/ RD( 2002) 17/ FINAL 56 ` Study standard' indicates the overall quality of the reported study taking into account the technical and scientific procedures employed and the information content of the test report. Five standards have been used:

  Good   the study fulfils all requirements in terms of method, reporting and interpretation.   Acceptable   the study is of an overall acceptable standard for use in hazard and risk assessment although there are inadequacies in some elements.   Questionable   there are significant inadequacies in elements of the study that raise concerns about its validity. The results should only be considered supportive of other data.   Unacceptable   there are significant inadequacies in elements of the study that invalidate the data.   Unknown   there is insufficient information to evaluate the standard of the study.

The column headed ` Comments' highlights observations on the study procedures and results that are considered relevant to the assessment. Reasons why procedures or results were considered inappropriate are also given.

The tables also contain a column headed ` Study reference number' that refers to the list of studies in Annex 1. A summary of the lowest acceptable effect concentrations for each trophic level is given in Annex 4.

4.1 Effects on Fish, Invertebrates and Aquatic plants ( Algae and higher plants)

Acute ( short term) data were available from a total of 31 studies covering three species of freshwater fish, one species of saltwater fish, a fish acclimated to saltwater, two species of freshwater and three species of saltwater invertebrate, three species of freshwater and one species of saltwater algae and one species of freshwater higher plant. The studies have been reviewed and this has resulted in 21 being considered to be of an acceptable standard for assessing the acute ( short term) toxic hazard of PFOS to fish, invertebrates and aquatic plants.

Sub chronic/ chronic ( prolonged/ longer term) data were available from a total of 8 studies covering two species of freshwater fish, one species of fresh and saltwater invertebrate and one species of freshwater unicellular algae. A review of the studies has resulted in seven being considered to be of an acceptable standard for assessing the toxic hazard of PFOS to fish and invertebrates. It should be noted that study number 25 with the Fathead minnow ( Pimephales promelas) was considered unacceptable because of a lack information in the report. It is probable that this study was the same as that described more fully in report number 14. Study 14 was judged to be acceptable.

In addition, flow through bioconcentration studies have been conducted in the bluegill sunfish ( Lepomis macrochirus) and carp ( Cyprinus carpio).

The acceptable and unacceptable studies are identified in Table 6. ENV/ JM/ RD( 2002) 17/ FINAL 57 Table 6. Acceptable and unacceptable studies for determining the acute and sub chronic/ chronic ( prolonged/ long term) toxic hazard of PFOS to fish, invertebrates and aquatic plants ( algae and higher plants)

Endpoint Test species Acceptable study reference numbers Unacceptable study reference numbers Pimephales promelas 1, 16, 28 25, 26, 27 Acute ( short term) toxicity to fish ( freshwater) Lepomis macrochirus 20 25 Oncorhynchus mykiss 31, 42 25 Acute ( short term) toxicity to fish ( saltwater) Oncorhynchus mykiss Cyprinodon variegatus 30 43

Pimephales promelas 8, 14 25 Sub chronic/ chronic toxicity to fish ( freshwater) Lepomis macrochirus 41  Daphnia magna 3, 15, 17, 29, 33 23, 25 Acute ( short term) toxicity to invertebrates ( freshwater) Unio complamatus 5  Mysidopsis bahia 4  Acute ( short term) toxicity to invertebrates ( saltwater) Crassostrea virginica 7  Artemia sp 32  Sub chronic/ chronic toxicity to invertebrates ( freshwater) Daphnia magna 9, 15

Sub chronic/ chronic toxicity to invertebrates ( saltwater) Mysidopsis bahia 10

Toxicity to freshwater unicellular algae Selenastrum capricornutum1 2, 13 24 Anabaena flos aquae 36  Navicula pelliculosa 38  Toxicity to saltwater unicellular algae Skeletonema costatum 39

Longer term toxicity to freshwater unicellular algae Selenastrum capricornutum1 13

Toxicity to freshwater higher plants Lemna gibba 37

4.1.1 Fish Acute ( short term) toxicity The results of the acute ( short term) studies are summarized in Table 7. Three species of freshwater fish have been used for testing  Pimephales promelas ( Fathead minnow), Lepomis macrochirus ( Bluegill sunfish) and Oncorhynchus mykiss ( Rainbow trout). The end point assessed in all the tests was mortality during a 96 hour exposure period.

P. promelas was the most susceptible freshwater fish species in acute tests with a lowest 96 hour LC50 for PFOS lithium salt of 4.7 mg/ l2. L. macrochirus, and O. mykiss, were only marginally less susceptible  a 96 hour LC50 value of 7.8 mg/ l was determined with both species for the diethanolamine ( DEA) and potassium salt respectively. A second study on Oncorhynchus mykiss showed an LC50 of 22 mg/ l again using the potassium salt. A very high 96 hour LL50 value of approximately 200 mg/ l determined for the didecyldimethylammonium salt of PFOS was obtained for P. promelas in a test on water accommodated 1 This species has recently been renamed Pseudokirchneriella subcapitata. 2 There are only three acute toxicity reports for Fathead minnow of acceptable or better quality, so the most sensitive value is used in this assessment. ENV/ JM/ RD( 2002) 17/ FINAL 58 fractions of an aqueous mixture containing the substance. However, the actual exposure concentrations of PFOS were not determined and could not be estimated in this test.

PFOS also exhibits acute toxicity to fish in saltwater. A 96 hour LC50 value of 13.7 mg/ l has been determined for the potassium salt in a test with O. mykiss acclimated to saltwater at a salinity of 30 parts per thousand. The data show saltwater acclimated O. mykiss to be of similar susceptibility to PFOS when compared with O. mykiss living in freshwater. However, in the absence of measured exposure concentrations, it should be noted that this study might have been conducted in excess of the substance's salt water solubility ( 2.5 to 20 mg/ l, depending on salinity and purity). A further study using a saltwater fish, Sheepshead minnow ( Cyprinodon variegatus) showed no toxicity up to the water solubility limit, indicated as 15 mg/ l in the test. Loss of concentration during the test, probably due to sedimentation, indicates that this value may be above the true solubility, however.

Sub chronic/ chronic ( prolonged/ long term) toxicity The results of the sub chronic/ chronic ( prolonged/ long term) studies are summarized in Table 8. Studies have been carried out with two species of freshwater fish   Pimephales promelas ( Fathead minnow) and Lepomis macrochirus ( Bluegill sunfish). Tests with P. promelas were designed to determine concentrations affecting early life stages of the fish over exposure periods of up to 42 days. Mortality data for L. macrochirus were obtained from a bioconcentration study in which deaths in the treated and control groups of fish were recorded over the 62 day uptake phase of the study.

The lowest definitive no observed effect concentration ( NOEC) of 0.3 mg/ l was determined for P. promelas for the potassium salt of PFOS. This value was applicable to both survival and growth end points. This NOEC is supported by results from a bioconcentration study with L. macrochirus that showed no significant mortality at an exposure concentration of 0.086 mg/ l over a 62 day uptake phase but 100% mortality at a concentration of 0.87 mg/ l after 35 days. A NOEC of 1.0 mg/ l reported for effects of PFOS potassium salt on early life stages of P. promelas could not be attributed to a more specific end point because insufficient information was contained in the study report.

Bioconcentration A flow through bioconcentration study of PFOS in bluegill sunfish was conducted. Test concentrations of 0, 0.086 and 0.87 mg/ L were used. At the 0.086 mg/ L level, the fish were exposed for 62 days, while at the 0.87 mg/ L level, the fish were exposed for 35 days due to excessive mortality. Fish were collected from the test chambers by random selection at 12 time points during the study. They were euthanized, blotted dry, weighed and measured. They were dissected into edible and nonedible tissue fractions and the fractions were weighed. The head, fins and viscera were considered to be nonedible tissue and the remaining tissue, including skin, was considered to be edible tissue. Whole fish concentrations were calculated from the sum of the edible and nonedible parts. Steady state BCF values were calculated from the tissue concentrations at apparent steady state divided by the mean water concentration. Tissue concentrations were considered to be at steady state if 3 or more consecutive sets of tissue concentrations were not significantly different. The kinetic bioconcentration factor ( BCFK), uptake rate and depuration rate were calculated for the edible, nonedible and whole fish exposed to 0.086 mg/ L using BIOFAC computer software.

In this study, PFOS bioconcentrated in the tissues of bluegill sunfish. Apparent steady state was not clearly attained for the fish exposed to 0.086 mg / L. Although the tissue residue levels of PFOS were not statistically significantly different on days 49, 56 and 62, the concentration of PFOS appeared to be still increasing up to the last day of exposure. BCFK values for edible, nonedible and whole fish tissues were calculated to be 1124, 4013, and 2796, respectively. PFOS depurated slowly. The BIOFAC estimates for ENV/ JM/ RD( 2002) 17/ FINAL 59 the time to reach 50% clearance for edible, nonedible, and whole fish tissues were 86, 116, and 112 days, respectively.

Kurume Laboratory ( 2001) conducted a flow through bioconcentration study of PFOS in carp ( Cyprinus carpio). The fish were checked visually and those demonstrating any abnormality were removed. The fish were reared for 8 days in a flow thorough system following an external disinfection. After rearing, the fish were medicated to eliminate parasites and transferred to an acclimatizing aquarium. After the second external disinfection, they were acclimatized. The fish demonstrating any abnormality during this period were removed and the remainder of the fish were reared for 15 days in a flow through system at temperatures of 25 ± 2o C. The fish were transferred to test tanks and reared at the same temperature in the flow through system for another 27 days. Temperature of the test water was measured with alcohol a thermometer and recorded once a day. Dissolved oxygen concentrations was measured with a dissolved oxygen probe and recorded twice a week. The pH of the test water was measured with a pH meter one a week. Temperature of test water was measured and recorded once a day. Dissolved oxygen in test water was measured and recorded twice a week. During the experimental period, the excreta of carp, dirt on test tanks, were removed once a day. The temperature ranged from 25.0 to 25.4 C; the dissolved oxygen concentration ranged from 7.9 to 8.1 mg/ L; and the pH ranged from 7.6 to 7.8.

Forty carp were exposed to two concentrations of PFOS, 2 and 20 ug/ L respectively for 58 days in a flow though system. The test water of each level was analyzed once before firs analysis of test fish and at the same time as analysis of test fish. Analysis of test fish was performed six times at each level in duration of exposure. Four fish were taken out at each sampling time and divided into two groups, and then both were analyzed individually. Because the stored sample from one fish was too small for the measurement of lipid content, groups of two fish were used. Analysis of control fish was performed before the experimental starting and after the experimental completion. Six fish were taken out at each sampling time and divided into three groups, and then both were analyzed individually.

Analysis of PFOS in the test water and carp was performed using high performance liquid chromatography mass spectrometry ( HPLC MS) analysis. The test water of each level was analyzed once before first analysis of test fish and at the same time as the analysis of the test fish. Steady state was reached when three successive analyses of BCFs made on samples taken at intervals of at least 48 hours were within + 20% of each other. When BCFs were less that 100, it was evaluated that a steady state had been reached after 28 days. The fish which were exposed for 61 days were separated into parts; tegument, head, viscera except liver, liver and remaining matter. BCFs were determined in all the parts.

In this study, PFOS bioconcentrated in the tissues of carp. Test concentrations of 2 and 20 ug/ L were used. The fish were exposed for 58 days to makeup for excessive mortality. Bioconcentration factors were calculated to be 720 for 20 ug/ L and 200  1500 for 2 ug/ L. ENV/ JM/ RD( 2002) 17/ FINAL 60 Table 7. Acute ( short term exposure) toxicity data for PFOS determined in tests with fish Medium Species Protocol Results ( mg/ l) Study Standard Comments Study Ref.

No.

Freshwater Pimephales promelas ( Fathead minnow) OECD 203 &

OPPTS 850.1075 ( Static) 96 hour LC50 = 9.5 96 hour NOEC = 3.3 Good Test substance was PFOS potassium salt.

Measured exposure concentrations 1 Not noted ( Static) 96 hour LC50 = 4.7 ( see comments) Acceptable Test substance was PFOS lithium salt.

Nominal exposure concentrations expressed relative to concentration of test substance ( 24.5% PFOS Li salt and 74.5% water).

Test result divided by 4 to express 96 h LC50 in terms of PFOS Li salt concentration.

Very little information on study protocol;

however, result agrees reasonably well with more other studies ( 1, 25, 26 and 27) 16 Not noted ( Static) 96 hour LC50 = 37.6 and 51 Unknown Test substance was PFOS potassium salt.

Nominal exposure concentrations Standard of studies cannot be judged from information supplied.

However, results are consistent with other studies ( 1, 16, 26 and 27) 25 Not noted ( Static) 96 hour LC50 = 21 Questionable Test substance was PFOS ammonium salt.

Nominal exposure concentrations Isopropanol also present in test samples.

However, results are consistent with other studies ( 1, 16, 25 and 27) 26 Not noted ( Static) 96 hour LC50 = 25 Questionable Test substance was PFOS ammonium salt.

Nominal exposure concentrations Isopropanol also present in test samples.

Results consistent with other studies ( 1, 16,

25 and 26) 27 ENV/ JM/ RD( 2002) 17/ FINAL 61 Table 7 contd. Acute ( short term exposure) toxicity data for PFOS determined in tests with fish Medium Species Protocol Results ( mg/ l) Study Standard Comments Study Ref.

No.

Freshwater Pimephales promelas ( Fathead minnow) OECD 203 ( Static) 96 hour LL50 = 200*

96 hour NOEL =

< 170*

(* calculated assuming 35% of substance in mixture) Acceptable Test substance was a mixture of didecyldimethyammonium salt of PFOS ( approximately 35%) and water with up to 5% residual perfluorochemicals.

Test media were water accommodated fractions ( WAFs).

Nominal exposure concentrations. 28 OECD 203 &

OPPTS 850.1075 ( Static) 96 hour LC50 = 7.8 96 hour NOEC = 4.5 ( see comments) Acceptable Test substance was PFOS DEA salt.

Nominal exposure concentrations expressed relative to concentration of test substance (~ 25% PFOS DEA salt and 75% water).

Results have therefore been divided by 4 to calculate the effect concentrations in terms of PFOS DEA salt concentration based on reported 96 hour LC50 of 31 mg/ l and NOEC of 18 mg/ l. 20 Lepomis macrochirus ( Bluegill sunfish) Not noted ( Static) 96 hour LC50 = 68 Unknown Test substance was PFOS potassium salt.

Nominal exposure concentrations Standard of study cannot be judged from information supplied. 25 Oncorhynchus mykiss ( Rainbow trout) Not noted ( Static) 96 hour LC50 = 11 Unknown Test substance was PFOS potassium salt.

Nominal exposure concentrations Standard of study cannot be judged from information supplied. 25 Standard procedures for testing acute lethality of liquid effluents ( Environment Canada) 96 hour LC50 = 7.8 Acceptable Test substance was PFOS potassium salt.

Sample purity not characterised Nominal exposure concentrations 31 ENV/ JM/ RD( 2002) 17/ FINAL 62 Table 7 contd. Acute ( short term exposure) toxicity data for PFOS determined in tests with fish Medium Species Protocol Results ( mg/ l) Study Standard Comments Study Ref.

No.

Freshwater Oncorhynchus mykiss ( Rainbow trout) OPPTS 850.1075 OECD 203 Static 96 hour LC50 = 22 Acceptable Test substance was PFOS potassium salt.

Sample purity 86.7%; results reported as a. i Based on measured concentrations 42 Saltwater Cyprinodon variegatus ( Sheepshead minnow) OPPTS 850.1075 OECD 203 Semi static ( 24 hour renewal) 96 hour LC50 > 15 mg/ l Acceptable Test substance was PFOS potassium salt.

Sample purity 86.7%; results reported as a. i Based on mean of measured concentrations Concentration losses during each 24 hour period would suggest some sedimentation indicating that the water solubility may have been exceeded. Final concentrations in each 24 hour period varied between 11   16 mg/ l 43 Saltwater Oncorhynchus mykiss ( Rainbow trout) Standard procedures for testing acute lethality of liquid effluents ( Environment Canada) 96 hour LC50 = 13.7 Acceptable Test substance was PFOS potassium salt.

Sample purity not characterised Test fish were acclimated to 30 parts per thousand saltwater Nominal exposure concentrations In the absence of measured exposure concentrations, it should be noted that this study might have been conducted in excess of the substance's salt water solubility ( 2.5 to 20 mg/ l, depending on salinity and purity). 30 ENV/ JM/ RD( 2002) 17/ FINAL 63 Table 8. Sub chronic/ chronic ( prolonged/ long term) toxicity data for PFOS determined in tests with fish Medium Species Protocol Results ( mg/ l) Study Standard Comments Study Ref.

No.

Freshwater OECD 210 &

OPPTS 850.1400 ( Flowthrough 42 day NOECsurv. =

0.30 42 day NOECgrowth =

0.30 5 day NOEChatch > 4.6 Good Test substance was PFOS potassium salt.

Measured exposure concentrations.

Lighting regime not described in summary.

Conclusion ( post hatch survival as the most sensitive endpoint): it is possible that growth was also affected at this concentration, but the test design did not enable this to be determined. 8 Non standard ( Flowthrough 30 day NOECearly lifestages = 1 Acceptable Test substance was PFOS potassium salt.

Measured exposure concentrations.

Results agree with later study Acetone present at 43 µ l/ l 14 Pimephales promelas ( Fathead minnow) Not noted ( Flowthrough NOECearly life stages = 1 Unknown Test substance was PFOS potassium salt.

Standard of study cannot be judged from information supplied. It is however assumed that this study is the same as 14 25 ( Assumed to be same study as 14)

Lepomis macrochirus ( Bluegill sunfish) OECD 305 &

OPPTS 850.1730 62 day NOECmortality =

> 0.086, < 0.87 Good Bioconcentration study Test substance was PFOS potassium salt.

Measured exposure concentrations. 41 ENV/ JM/ RD( 2002) 17/ FINAL 64 4.1.2 Invertebrates The results of acute ( short term) and sub chronic/ chronic ( prolonged/ long term) studies are summarised in Tables 9 and 10.

Acute ( short term) toxicity to freshwater species Two species of freshwater invertebrate have been used for testing  Daphnia magna ( the ` water flea') and Unio complamatus ( fresh water mussel). Immobilisation/ mortality during 48 hours exposure was assessed in the tests with D. magna. Mortality during 96 hours exposure was assessed in the test with U. complamatus.

A lowest acceptable 48 hour EC50 value of 27 mg/ l was determined for PFOS potassium salt in tests with the daphnid. 1 A 48 hour EL50 value of approximately 4.0 mg/ l was also determined for the didecyldimethylammonium salt of PFOS in a daphnid test on water accommodated fractions of an aqueous mixture containing the substance. However, the actual exposure concentrations of PFOS were not determined in this test and, as pointed out in the introduction to this section, it is possible that didecyldimethylammonium may have contributed to the toxicity of the test medium. These values compare with a 96 hour LC50 value determined for mortality of the mussel of 59 mg/ l.

Acute ( short term) toxicity to saltwater species Three species of saltwater invertebrate have been used for testing  Mysidopsis bahia ( Mysid shrimp), Crassostrea virginica ( Eastern oyster) and Artemia sp ( Brine shrimp). Mortality during 96 hours exposure was assessed in the tests with M. bahia and Artemia sp. Reduction in shell deposition was assessed over a 96 hour exposure period in the test with C. virginica. The test substance was PFOS potassium salt in all cases.

A 96 hour LC50 value of 3.6 mg/ l and an associated NOEC of 1.1 mg/ l were determined in the test with the Mysid shrimp. This compares with a 96 hour EC50 value of > 3.0 mg/ l for effects on shell deposition in the oyster and a 48 hour LC50 of 8.9 mg/ l for mortality of the Brine shrimp.

Sub chronic/ chronic ( prolonged/ long term) toxicity to freshwater species Two tests have been carried out on PFOS potassium salt with one species of freshwater invertebrate   the ` water flea', Daphnia magna. The end points assessed in the tests were survival, growth and reproduction measured over exposure periods of up to 28 days.

NOECs of 12 and 7 mg/ l have been determined for D. magna reproduction in 21 and 28 day tests respectively. In the 21 day test the NOECs for survival and growth were also 12 mg/ l, indicating that reproduction was no more sensitive than these two other end points.

1 For Daphnia magna there are five 48 hour acute toxicity studies of acceptable or better quality, although one of these relates to water accommodated fractions. The remaining 48 hour EC50s are 27, 58, 61 and 210 mg/ l. The latter value is based on nominal exposure concentrations, but since the stock solution was prepared at a concentration that exceeded water solubility, exposure concentrations are likely to be lower than nominal. This leaves just three reports with comparable measures of toxicity, and so the most sensitive value is used in this assessment. ENV/ JM/ RD( 2002) 17/ FINAL 65 Sub chronic/ chronic ( prolonged/ long term) toxicity to saltwater species A test has been carried out on PFOS potassium salt with one species of saltwater invertebrate; the Mysid shrimp, Mysidopsis bahia. The end points assessed in the test were survival, growth and reproduction measured over an exposure period of 35 days.

The 35 day NOECs determined for survival, growth and reproduction in this test were 0.55, 0.25 and 0.25 mg/ l respectively. ENV/ JM/ RD( 2002) 17/ FINAL 66 Table 9. Acute ( short term exposure) toxicity data for PFOS determined in tests with invertebrates Medium Species Protocol Results ( mg/ l) Study Standard Comments Study Ref. No.

Freshwater Daphnia magna ( Water flea) OECD 202 &

OPPTS 850.1010 ( Static) 48 hour EC50 = 61 48 hour NOEC = 33 Good Test substance was PFOS potassium salt.

Measured exposure concentrations 3 ASTM 1981 &

OECD 1981 ( Static) 48 hour EC50 = 27 Acceptable Test substance was PFOS potassium salt.

Nominal exposure concentrations.

Result agrees reasonably well with those obtained in studies 3 and 23. 15 Not noted ( Static) 48 hour EC50 = 210 48 hour NOEC = 100 Acceptable Test substance was PFOS lithium salt.

Nominal exposure concentrations.

EC50 is somewhat higher than previous value but stock solution was prepared at a concentration that exceeded water solubility,

therefore exposure concentrations are likely to be lower than nominal. 17 OECD 202 ( Static) 24 hour EC50 = > 42 48 hour EC50 = 14 Questionable Test substance was PFOS potassium salt.

Nominal exposure concentrations.

Data questionable in view of the presence of diethylene glycol butyl ether in the test material ( as part of the formulation).

Result is in reasonable agreement with later study ( 3). 23 Not noted ( Static) 48 hour EC50 = 49.2 Unknown Test substance was PFOS potassium salt.

Nominal exposure concentrations.

Standard of study cannot be judged from information supplied. 25 ENV/ JM/ RD( 2002) 17/ FINAL 67 Table 9 contd. Acute ( short term exposure) and sub chronic/ chronic ( prolonged/ long term) toxicity data for PFOS determined in tests with invertebrates Medium Species Protocol Results ( mg/ l) Study Standard Comments Study Ref. No.

Daphnia magna ( Water flea) OECD 202 ( Static) 48 hour EL50 = 4.0 48 hour NOEL = 2.2 Acceptable Test substance was a mixture of didecyldimethylammonium salt of PFOS ( approximately 35%) and water with up to 5%

residual perfluorochemicals.

Test media were water accommodated fractions ( WAFs).

Nominal exposure concentrations.

48 hour EL50 and NOEL calculated assuming 35% of substance in mixture. 29 ISO, 1982 48 hour EC50 = 58 Acceptable Test substance was PFOS potassium salt.

Sample purity not characterised Nominal exposure concentrations. 33 Unio complamatus ( Freshwater mussel) OECD 203,

OPPTS 850.1075 &

ASTM E 729

88a ( Semistatic 96 hour LC50 = 59 96 hour NOEC = 20 Good Test substance was PFOS potassium salt.

Measured exposure concentrations. 5 Saltwater Mysidopsis bahia ( Mysid shrimp) OPPTS 850.1035 ( Static) 96 hour LC50 = 3.6 96 hour NOEC = 1.1 Good Test substance was PFOS potassium salt.

Measured exposure concentrations. 7 Crassostrea virginica ( Eastern oyster) OPPTS 850.1025 ( Static) 96 hour EC50 = > 3.0 96 hour NOEC = 1.9 Good Test substance was PFOS potassium salt.

Measured exposure concentrations.

Solubility in seawater should have been used to set highest test concentration. 4 Artemia sp.

( Brine shrimp) Draft ISO,

1981 48 hour LC50 = 8.9 Acceptable Test substance was PFOS potassium salt.

Sample purity not characterised Nominal exposure concentrations. 32 ENV/ JM/ RD( 2002) 17/ FINAL 68 Table 10. Sub chronic/ chronic ( prolonged/ long term) toxicity data for PFOS determined in tests with invertebrates Medium Species Protocol Results ( mg/ l) Study Standard Comments Study Ref. No.

Freshwater Daphnia magna ( Water flea) OECD 211,

OPPTS 850.1300 &

ASTM 1193

87E ( Semistatic 21 day NOECrepro = 12 21 day NOECsurv. = 12 21 day NOECgrowth = 12 Good Test substance was PFOS potassium salt.

Measured exposure concentrations.

No effects on reproduction at highest concentration that had no effect on mortality. 9 ASTM 1981 &

OECD 1981 ( Semi static) 21 day EC50repro. = 12 28 day NOECrepro = 7 28 day EC50repro. = 11 Acceptable Test substance was PFOS potassium salt.

Nominal exposure concentrations.

Result agrees well with that obtained in study 9. 15 Saltwater Mysidopsis  bahia ( Mysid shrimp) OPPTS 850.1350 ( Flowthrough 35 day NOECrepro. = 0.25 35 day NOECsurv. = 0.55 35 day NOECgrowth =

0.25 Good Test substance was PFOS potassium salt.

Measured exposure concentrations. 10 ENV/ JM/ RD( 2002) 17/ FINAL 69 4.1.3 Aquatic plants The results of all the short and longer term studies are summarised in Tables 11 and 12 ( algae) and 13 ( higher plants). Note: The species Selenastrum capricornutum has been renamed Pseudokirchneriella subcapitata. For transparency, the name used in the test report has been retained in the discussion below.

Freshwater algae   Short term Three species of freshwater unicellular algae have been used for testing  Selenastrum capricornutum, Anabaena flos aquae and Navicula pelliculosa. The end point assessed in the tests was growth measured in terms of cell density, growth rate and/ or the area under the growth curve over 96 hours. Only studies that assessed toxicity by reference to effects on growth rate are considered here for the purposes of determining the toxicity of PFOS to algae. The results of study number 13 are therefore excluded because they are only expressed relative to cell numbers and cell dry weight. These results could be considered were the EC50 values to be recalculated in respect of growth rate, but since algae were relatively insensitive compared with fish, this is not considered a high priority.

The lowest 96 hour EC50 value for effects on growth rate of 71 mg/ l was determined for S. capricornutum. However there are some uncertainties over the validity of this result given that exposure concentrations were not measured and diethylene glycol butyl ether was present in the test material ( as part of the formulation). A 96 hour EC50 value of 126 mg/ l was obtained for the same species using a test protocol that was judged to meet all the criteria for acceptability of the data. The 96 hour NOECs associated with these two tests were 35 and 44 mg/ l respectively. The tests with the two other species yielded 96 hour EC50 values of 176 mg/ l ( A. flos aquae) and 305 mg/ l ( N. pelliculosa) and respective NOECs of 94 and 206 mg/ l.

Saltwater algae   Short term A 96 hour growth inhibition test has been carried out on PFOS potassium salt with Skeletonema costatum. The test was unable to determine a definitive 96 hour EC50 value because no effects were determined at the highest dissolved PFOS concentration that could be attained under the test conditions ( 3.2 mg/ l).

Freshwater algae   Longer term One species of freshwater unicellular algae has been tested  Selenastrum capricornutum ( study reference no. 13). The test assessed effects on growth rate expressed relative to cell density over 14 days. The results from the test are not discussed further here for two reasons. Firstly, the results are expressed relative to cell density ( see comments in respect of short term tests). Secondly, there are concerns that the 14 day test period and absence of test medium renewal may have resulted in a decline in exposure concentrations over time ( the test media were not analysed for PFOS concentration). The latter is suggested by comparison of the 14 day EC50 value of 95 mg/ l with the 96 h EC50 value of 82 mg/ l determined in the same test.

Freshwater higher plants A growth inhibition test has been carried out on PFOS potassium salt with Lemna gibba ( Duckweed). The test yielded a 7 day IC50 of 108 mg/ l for inhibition of frond production and a 7 day NOEC of 15.1 mg/ l based on the inhibition of frond production and evidence of sub lethal effects. ENV/ JM/ RD( 2002) 17/ FINAL 70 Table 11. Acute ( short term exposure) toxicity data for PFOS determined in tests with algae Medium Species Protocol Results ( mg/ l) Study Standard Comments Study Ref.

No.

Freshwater Selenastrum capricornutum OECD 201,

OPPTS 850.5400 &

ASTM 1218 90E ( Static) 96 hour EC50( cell density) = 71 96 hour EbC50( area under the curve) =

71 96 hour ErC50( growth rate) = 126 96 hour NOEC( growth rate, cell density,

area under the growth curve) = 44 72 hour EC50( cell density) = 70 72 hour EbC50( area under the curve) =

74 72 hour ErC50( growth rate) = 120 72 hour NOEC( growth rate, cell density,

area under the growth curve) = 70 Good Test substance was PFOS potassium salt.

Measured exposure concentrations. 2 OECD 201,

US EPA 600/ 9 78

018 & ASTM E

35.23 ( Static) 96 hour EC50( cell density) = 82 96 hour EC10( cell density) = 10 Acceptable Test substance was PFOS potassium salt.

Result not expressed relative to growth rate Nominal exposure concentrations. Result agrees with those obtained in studies 2 and 24. 13 OECD 201 ( Static) 96 hour EC50( growth rate) = 71 96 hour NOEC( growth rate) = 35 Questionable Test substance was PFOS potassium salt.

Nominal exposure concentrations.

Data questionable in view of the presence of diethylene glycol butyl ether in the test material ( as part of the formulation). 24 ENV/ JM/ RD( 2002) 17/ FINAL 71 Anabaena flosaquae OPPTS 850.5400 96 hour EC50( growth rate) = 176 96 hour NOEC( growth rate) = 94 Good Test substance was PFOS potassium salt.

Measured exposure concentrations. 36 Navicula pelliculosa OPPTS 850.5400 96 hour EC50( growth rate) = 305 96 hour NOEC( growth rate) = 206 Good Test substance was PFOS potassium salt.

Measured exposure concentrations. 38 Saltwater Skeletonema costatum OPPTS 850.5400 96 hour EC50( growth rate) = > 3.2 96 hour NOEC( growth rate) = > 3.2 Good Test substance was PFOS potassium salt.

Measured exposure concentrations. 39 ENV/ JM/ RD( 2002) 17/ FINAL 72 Table 12. Sub chronic/ chronic ( prolonged/ long term) toxicity data for PFOS determined in tests with freshwater algae Species Protocol Results ( mg/ l) Study Standard Comments Study Ref.

No.

Selenastrum capricornutum OECD 201, US EPA 600/ 9 78

018 & ASTM E 35.23 ( Static) 14 day EC50 cell density = 95 14 day NOECcell density = < 26 14 day EC10 cell density = 16 Acceptable Test substance was PFOS potassium salt.

Result not expressed relative to growth rate.

No analysis of exposure but result agrees with those obtained in studies 2 and 24. 13 Table 13. Acute ( short term exposure) toxicity data for PFOS determined in tests with higher plants Medium Species Protocol Result ( mg/ l) Study Standard Comments Study Ref.

No.

Freshwater Lemna gibba G3 ( Duckweed) OPPTS 850.4400 7 day IC50 = 108 Good Test substance was PFOS potassium salt.

Measured exposure concentrations. 37 ENV/ JM/ RD( 2002) 17/ FINAL 73 4.2 Effects on Other Aquatic Organisms 4.2.1 Amphibians Data summarised in Table 14 were obtained in an embryo teratogenesis assay carried out on PFOS potassium salt with Xenopus laevis ( African clawed frog). Exposure of the embryos for 96 hours resulted in an LC50 for mortality of 13.8 mg/ l and an EC50 for malformations of 12.1 mg/ l. The minimum concentration that inhibited growth was 7.97 mg/ l. and the teratogenic index was calculated, as the ratio of the 96 hour LC50 to the 96 hour EC50, to be 1.1. The latter value indicates that PFOS has a low potential to be a developmental hazard in this species. ENV/ JM/ RD( 2002) 17/ FINAL 74 Table 14. Toxicity data for PFOS determined in an embryo teratogenesis assay with the amphibian,

    Medium Species Protocol Result ( mg/ l) Study Standard Comments Study Ref.

No.

Freshwater Xenopus laevis ( African clawed frog) ASTM E1439 91 96 hour LC50 = 13.8 96 hour EC50( malformations) = 12.1 Minimum concentration to inhibit growth = 7.97 Teratogenic index 1.1 Acceptable Test substance was PFOS potassium salt.

Measured exposure concentrations.

In life phases were not subject to GLP. 40 ENV/ JM/ RD( 2002) 17/ FINAL 75 4.2.2 Sediment Dwelling Invertebrates No data are available for effects on sediment dwelling invertebrates. It is possible that a predicted no effect concentration ( PNEC) for sediment dwelling invertebrates could be determined by applying equilibrium partitioning models to the data for water column organisms. However, as discussed in Annex 3, this is unlikely to be feasible at the present time.

4.2.3 Bacteria Data summarised in Table 15 were obtained from 2 reports describing Microtox   studies ( study report reference numbers 18 and 22). The Microtox   procedure assesses light output inhibition from the luminescent marine bacterium Photobacterium phosphoreum, following exposure to a toxicant over a short exposure period ( 30 minutes in the case of these two studies). Both studies are of an unacceptable standard for assessing the hazard of PFOS ( lithium salt) to bacteria because of uncertainty over the true exposure concentrations and in one case because of the presence of diethylene glycol in the test sample. There are also more general concerns over the relevance of the test system and test species for determining effects for this particular group of organisms. No toxic effects were observed in either test at nominal concentrations (> 250 mg/ l) that were significantly in excess of the solubility of PFOS salts in saline medium ( 2.5 to 20 mg/ l, depending on purity/ salinity). ENV/ JM/ RD( 2002) 17/ FINAL 76 Table 15. Toxicity data for PFOS determined in Microtox tests Species Protocol Results ( mg/ l) Study Standard Comments Study Ref.

No.

Photobacterium phosphoreum ( Microtox   ) Microbics Microtox   " BASIC" 15 minute EC50 = > 250 30 minute EC50 = > 250 Unacceptable Test substance was PFOS lithium salt.

Nominal exposure concentrations.

Initial stock solution prepared at concentration that was likely to exceed solubility in saline water ( 2.5 to 20 mg/ l,

depending on purity/ salinity) It is therefore possible that actual exposure concentrations were much lower than nominal.

Result indicates probable absence of toxicity at limit of solubility. 18 30 minute EC50 = > 280 Unacceptable Test substance was PFOS potassium salt.

Nominal exposure concentrations.

Data questionable in view of use of diethylene glycol to aid dosing.

Test concentrations likely to be in excess of solubility in saline medium ( 2.5 to 20 mg/ l, depending on purity/ salinity).

Result is consistent with that obtained in study 18 indicating probable absence of toxicity at limit of solubility. 22 ENV/ JM/ RD( 2002) 17/ FINAL 77 4.2.4 Activated Sludge Microorganisms Data summarised in Table 16 were obtained from 3 reports describing activated sludge respiration inhibition studies ( study report reference numbers 6, 19 and 21). Only study reference number 9 was considered to be of an acceptable standard for assessing the hazard of PFOS to activated sludge. Studies 19 and 21 had significant inadequacies in test procedures related to test conditions, duration of the exposure and/ or absence of a reference substance.

The 3 hour IC50 value for PFOS ( potassium salt) determined in the acceptable study was > 905 mg/ l ( nominal concentration). ENV/ JM/ RD( 2002) 17/ FINAL 78 Table 16. Toxicity data for PFOS determined in Activated Sludge Respiration Inhibition tests Test system Protocol Results ( mg/ l) Study Standard Comments Study Ref. No.

Activated Sludge OECD 209 3 hour IC50 = > 905 ( Nom.) Good Test substance was PFOS potassium salt.

Nominal exposure concentrations of 0.9   905 mg/ l tested. Highest test concentration gave the maximum inhibition of 38.8%. 6 OECD 209 3 hour IC50 = > 245 ( as a nom. 24.5% solution in water) Unacceptable Test substance was PFOS potassium salt.

Nominal exposure concentrations.

Test temperature not controlled and fell well below requirements of standard guidelines.

Respiration rates extrapolated to 20oC.

Oxygenation was inadequate during the first 30 minutes of the 3 hour test.

No inhibitory effect observed. 19 Not noted 7 minute IC50 = > 250 ( as a nom. 25% solution in water) Unacceptable Test substance was PFOS DEA salt.

Nominal exposure concentrations.

Exposure period inadequate by current standards.

No reference substance used.

No inhibitory effect observed. 21 ENV/ JM/ RD( 2002) 17/ FINAL 79 4.3 Effects on Terrestrial Organisms 4.3.1 Soil dwelling Invertebrates No data are available for effects on soil dwelling invertebrates. It is however possible that a predicted no effect concentration ( PNEC) for soil dwelling invertebrates could be determined by applying equilibrium partitioning models to the data for water column organisms. However, as discussed in Annex 3, this is unlikely to be feasible at the present time. It is noted that an earthworm acute toxicity and uptake study is in the planning stages, and a final report should be available in 2002.

4.3.2 Terrestrial Plants No data are available for effects on terrestrial plants. It is however possible that a predicted no effect concentration ( PNEC) for terrestrial plants could be determined by applying equilibrium partitioning models to the data for aquatic plants. However, as discussed in Annex 3, this is unlikely to be feasible at the present time. It is noted that a terrestrial plant toxicity and uptake study should be available in 2002.

4.3.3 Birds Data summarised in Table 17 were from 2 reports describing dietary acute studies with the Mallard duck, Anas platyrhynchos, and the Northern Bobwhite quail, Colinus virginianus ( study report reference numbers 11 and 12). Birds in both tests were exposed to PFOS potassium salt in their diets for 5 days and observations were made after 3 or 17 days. Both studies are considered to be of an acceptable standard for assessing the acute toxic hazard of PFOS to birds via dietary exposure.

The lowest acute dietary LC50 value of 220 mg/ kg of food was determined in the test with the quail. This value was approximately half that obtained in the test with the duck. The lowest NOEC of 37 mg/ kg of food for effects on body weight was, in contrast, obtained in the test with the duck. Once again this value was approximately half that obtained with the other species   the quail.

It is noted that avian reproductive data will be submitted for review in 2002. ENV/ JM/ RD( 2002) 17/ FINAL 80 Table 17. Acute dietary toxicity data for PFOS determined in tests with birds Test species Protocol Results ( mg/ kg of food) Study Standard Comments Study Ref. No.

Anas platyrhynchos ( Mallard duck) OECD 205, OPPTS 850.2200 & FIFRA E 71 2 LC50 = 628 NOECmortality = 146 NOECbody weight = 37 Good Test substance was PFOS potassium salt.

Measured exposure concentration in food.

5 days exposure followed by 3 or 17 days observation 11 Colinus virginianus ( Northern Bobwhite quail) OECD 205, OPPTS 850.2200 & FIFRA E 71 2 LC50 = 220 NOECmortality = 73 NOECbody weight = 73 Good Test substance was PFOS potassium salt.

Measured exposure concentration in food.

5 days exposure followed by 3 or 17 days observation 12 ENV/ JM/ RD( 2002) 17/ FINAL 81 4.3.4 Bees Data summarised in Table 18 were obtained in acute oral and contact toxicity tests carried out with the Honey bee ( Apis mellifera) on PFOS potassium salt. The studies fully meet the required standard for acceptability of the data.

The acute oral test yielded a 72 hour LD50 for ingestion of PFOS of 0.40 µ g/ bee and a 72 hour NOEL of 0.21 µ g/ bee. The contact test yielded a 96 hour LD50 of 4.78 µ g/ bee and a 96 hour NOEL of 1.93 µ g/ bee. The respective LD50 values indicate moderate and high orders of toxicity of PFOS to bees when administered via these routes. ENV/ JM/ RD( 2002) 17/ FINAL 82 Table 18. Acute oral and contact toxicity data for PFOS determined in tests with Honey bees Test species Protocol Results ( µ g/ bee) Study Standard Comments Study Ref. No.

Apis mellifera ( Honey bee) OECD 213,

EPPO 170 ( Oral) 72 hour LD50 = 0.40 72 hour NOEL = 0.21 Good Test substance was PFOS potassium salt.

Nominal exposure following feeding on 50% w/ v sucrose. 34 Apis mellifera ( Honey bee) OECD 214,

EPPO 170,

OPPTS 850.3020 ( draft) ( Contact) 96 hour LD50 = 4.78 96 hour NOEL = 1.93 Good Test substance was PFOS potassium salt.

Nominal exposure following direct application of test substance dissolved in acetone to thorax. 35 ENV/ JM/ RD( 2002) 17/ FINAL 83 5.0 References 3M Reports: " The Science of Organic Fluorochemistry" and " Perfluorooctane Sulfonate: Current Summary of Human Sera, Health, and Toxicology Data", dated February 5, 1999. ( 8EHQ 0299 373).

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