Acute and chronic aquatic toxicity of ammonium perfluorooctanoate (APFO) to freshwater organisms

ARTICLE IN PRESS Ecotoxicology and Environmental Safety 71 (2008) 749– 756

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Acute and chronic aquatic toxicity of ammonium perfluorooctanoate (APFO) to freshwater organisms Ilaria Colombo a, Watze de Wolf b, Roy S. Thompson c, David G. Farrar d, Robert A. Hoke e,, Jacques L’Haridon f a

Solvay Solexis, Bollate, Italy DuPont, Mechelen, Belgium c AstraZeneca, Brixham, UK d Ineos Chlor, Runcorn, UK e DuPont, Newark, DE, USA f CIT, Evreux, France b

a r t i c l e in fo

abstract

Article history: Received 3 December 2007 Received in revised form 1 April 2008 Accepted 4 April 2008 Available online 5 June 2008

Recent concerns have been raised concerning the widespread distribution of perfluorinated compounds in environmental matrices and biota. The compounds of interest include ammonium perfluorooctanoate (APFO, the ammonium salt of perfluorooctanoic acid, PFOA). APFO is used primarily as a processing aid in the production of fluoropolymers and fluoroelastomers. The environmental presence of perfluorooctanoate (PFO, the anion of APFO) and its entry into the environment as APFO make quality aquatic toxicity data necessary to assess the aquatic hazard and risk of APFO. We conducted acute and chronic freshwater aquatic toxicity studies with algae, Pseudokirchneriella subcapitata, the water flea, Daphnia magna, and embryo-larval rainbow trout, Oncorhynchus mykiss, using OECD test guidelines and a single, well-characterized sample of APFO. Acute 48–96 h LC/EC50 values were greater than 400 mg/l APFO and the lowest chronic NOEC was 12.5 mg/l for inhibition of the growth rate and biomass of the freshwater alga. Un-ionized ammonia was calculated to be a potential significant contributor to the observed toxicity of APFO. Based on environmental concentrations of PFO from various aquatic ecosystems, the PNEC value from this study, and unionized ammonia contributions to observed toxicity, APFO demonstrates little or no risk for acute or chronic toxicity to freshwater and marine aquatic organisms at relevant environmental concentrations. & 2008 Elsevier Inc. All rights reserved.

Keywords: APFO PFOA Perfluorooctanoate Acute Chronic Toxicity Algae Daphnia Fish

1. Introduction Perfluorooctanoic acid (PFOA, CAS no. 335-67-1) has been used in industry primarily as ammonium perfluorooctanoate (APFO, CAS no. 3825-26-1), the ammonium salt of PFOA. PFOA is a member of a broader class of perfluorinated acids that includes both shorter and longer chain perfluorinated carboxylates. Recently, considerable attention has been focused on both PFOA and a sulfonic acid analog, perfluorooctanesulfonic acid (PFOS, CAS no. 1763-23-1) based on their reported presence in the environment including the Arctic. The physical properties, and thus environmental fate, and the toxicological profile of PFOA and PFOS are quite different (USEPA, AR-226, Public Docket). Total global industrial emissions and manufacturing sources of APFO

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E-mail address: [email protected] (R.A. Hoke). 0147-6513/$ - see front matter & 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2008.04.002

from 1999 through 2006 have been discussed by Prevedouros et al. (2006). Most of the existing toxicological studies (aquatic and mammalian) for PFOA have used APFO as the test substance. APFO is fully ionised to the perfluorooctanoate anion (PFO) and NH+4 in water at environmentally relevant pH, has high water solubility, low sorption to soil, and low bioaccumulation potential (Martin et al., 2003a, b; Conder et al., 2008). The observed environmental presence of PFO and the potential for entry into the environment of APFO make high quality aquatic toxicity data necessary in order to facilitate the environmental risk assessment process for APFO. Kennedy et al. (2004) reviewed the potential human health hazards of PFO including pharmacokinetics, toxicity, and the possible mechanisms through which PFO exerts its effects. Studies conducted with APFO have ranged from short- to longterm, encompassed a wide variety of toxicological endpoints, involved several routes of exposure (oral, inhalation, dermal, aqueous), and investigated effects in many species ranging from

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protozoa to non-human primates. In addition, the potential adverse health effects of PFOA have been studied in exposed workers through medical surveillance and epidemiological investigation (Olsen et al., 2003). However, the environmental effects of PFO were not included in the review by Kennedy et al. (2004). The current study presents the first comprehensive compilation of acute and chronic data for APFO toxicity to the green alga, Pseudokirchneriella subcapitata, the invertebrate crustacean, Daphnia magna, and rainbow trout, Oncorhynchus mykiss. Existing toxicity information for PFOA and its ammonium salt are based primarily on studies conducted using the commercial product made from the electrochemical fluorination process, FC143 FLUORADs Brand Fluorochemical Surfactant (US EPA, AR-226, Public Docket). Many of these studies were conducted using methods other than the current standard OECD and US EPA test guidelines and the results show significant variability, which hampers data interpretation. The most important deviations in the existing studies included use of test substance that was not fully characterized for purity and the use of nominal instead of measured test concentrations (Hekster et al., 2003). The objective of the current study was to generate a comprehensive set of toxicity data for algae, Daphnia and fish according to current regulatory guidelines and following Good Laboratory Practice (GLP). This information can be used to inform substance classification and labeling according to European Union Directive 67/548/EEC (European Economic Community, 1967), as well as the Globally Harmonized System on Classification (UNEP, 2003), and for derivation of predicted no effect concentrations (PNECs) for risk assessment (European Union, 2003).

2. Materials and methods 2.1. Test substance A colorless, liquid solution of 19.6% APFO as dry solids (99.7% purity, 3M Laboratories, Minneapolis, MN, USA) in water was used for all testing. The isomer distribution, as determined by 3M using 19F-NMR, is reported in Table 1. The following values have been reported for the aqueous solubility of PFOA: 3.73 g/L at 20 1C (Nakayama, 1967), 4.14 g/L at 22 1C (Prokop et al., 1989) and 9.5 g/L at 25 1C (Kauck and Diesslin, 1951). There are conflicting reports on the aqueous solubility of APFO (Fluoropolymer Manufacturers Group, 2001; United States Environmental Protection Agency, 2002) but it also has relatively high water solubility (4500 mg/l). PFOA is a strong acid, with a pKa of about 2.5–2.8 (Brace, 1962; Ylinen et al., 1990). Dilute aqueous solutions of PFOA or APFO at environmentally relevant pH values will contain almost exclusively the non-volatile anion, C7F15COO (PFO) rather than the un-dissociated form of the compound. PFO is persistent in aqueous matrices in the environment with no evidence of direct or indirect photolysis in aqueous solution when exposed to wavelengths of 290–800 nm at 25 1C. The photolysis half-life in water is reported to be greater than 349 days (Hatfield, 2001) while the hydrolysis half-life in water is reported to be greater than 92 years at 25 1C and pH 5–9 (3M, 2001). Biodegradation of APFO under aerobic conditions does not occur (3M, 2001). The log Kow value cannot be determined since APFO is a surfactant; the log Koc in five soils has been reported to be between 1.7 and 2.4 indicating moderate mobility in soil (Dekleva, 2003).

Table 1 Characterization of isomer content of APFO used for aquatic toxicity testing Description

Isomer

Normal chain Internal monomethyl branch Isopropyl branch t-Butyl branch

CF3(CF2)x–CO2()NH4(+) 77.6 CF3(CF2)x–CF(CF3)–(CF2)yCO2()NH4(+) 12.6 (CF3)2CF–(CF2)x–CO2()NH4(+) (CF3)3C–(CF2)x–CO2()NH4(+) Other isomers

Mole fraction (%)

9.0 0.2 0.3

2.2. Toxicity testing Stock solutions of APFO were prepared by dissolving the test substance directly in the test media or dilution water and then diluting the stock solution to provide a geometric series of test concentrations. The 48-h daphnid toxicity test was conducted according to OECD test guideline 202 and European Commission Directive 92/69/EEC 1992, which are generally similar to the applicable US EPA test methods. D. magna STRAUS-clone 5 neonates between 6 and 24 h old were acclimated in water of the same quality as the test water for 6 h and then assigned to the test vessels. Four replicate test vessels, each with five neonates, were used for each control or test concentration treatment. Culturing and testing conditions were similar with reconstituted M4 water (Elendt and Bias, 1990) used for culturing and testing. Dissolved oxygen was greater than 60% of air saturation and temperature ranged between 18 and 22 1C with a light/ dark cycle of 16/8 h. Test organism loading was one neonate per 20 ml of test solution. Daphnids were not fed during acute testing. Observations were carried out at 0, 24 and 48 h in order to determine the number of immobilized daphnids in each test solution. Rainbow trout, O. mykiss, were tested in a 96-h static test according to OECD test guideline 203 and EU Commission Directive 92/69/EEC. One replicate test chamber containing seven fish was used for the control and each test solution concentration. Test organisms were randomly assigned to the test solutions after a pre-test acclimation period of 12 days. Test organisms loading was 0.76 g/l during the study with fish length ranging from 40 to 50 mm. Dilution water was filtered, dechlorinated tap water that was treated by a softening system to obtain the desired hardness of 150720 mg/l as CaCO3, and pH of 6.0–8.5. A light/dark cycle of 16/8 h, a temperature of 13–17 1C, and dissolved oxygen greater than 60% saturation were used for acclimation and testing. Fish were fed trout chow twice daily during the acclimation period but were not fed during the 24-h pre-test period or during acute testing. Fish were observed for mortality and visible abnormalities at 0, 2, 4, 24, 48, 72 and 96 h. The green alga, P. subcapitata, was tested using OECD test guideline 201 and EU Commission Directive 92/69/EEC. Culturing and test medium was reconstituted water recommended in the French algae test guideline (French Standard, 1990); this media differs slightly from the OECD recommended media with regard to concentrations of P, N and chelators but these differences had no effect on the scientific validity of the study. A temperature of 21–25 1C and constant illumination at approximately 2000 lx were used for culturing and testing. A rangefinding test and two definitive tests were conducted to evaluate toxicity to algae. The rangefinding test used 6 control replicates and 2 replicates per test concentration. The two definitive studies each used 6 control replicates and 3 replicates per test concentration. At test initiation, test solutions were inoculated with 104 cells/ml from an algal culture in log phase growth. Test solutions were agitated to keep algae in suspension during the 96 h test and growth was determined at 24-h intervals over the study by counting an aliquot of test solution from each replicate test chamber. The chronic toxicity of APFO to the cladoceran, D. magna STRAUS clone-5 was assessed in a 21-d static renewal test according to OECD test guideline 211. First instar daphnids, between 6 and 24 h old, were randomly assigned to the test chambers. Ten replicates, with one neonate per replicate, were used per control or test substance concentration. Each test vessel contained 50 ml of control or test solution. During culturing and testing, daphnids were fed daily (until test day 20) with a diet of approximately 6  106 cells of both Chlorella vulgaris and P. subcapitata. The carbon content of this diet corresponded to 0.16 mg of carbon per daphnid per day. The environmental conditions were similar during daphnid culturing and testing. Reconstituted water (M4 medium) was utilized as culture and test dilution water. Water temperature was 18–22 1C, the light/dark cycle was 16/8 h, respectively, with a light intensity of 1200 lx and dissolved oxygen was greater than 60% air saturation. Test solution renewals were typically conducted every 3 days. During test solution renewals, all neonates produced since the last renewal were removed from the test solutions. Observations were made daily to determine the number of immobilized or dead parent daphnids, the number of neonates (alive and dead progeny) for each parent animal and the number of aborted eggs. The length of all surviving parent daphnids was measured at test end. The rainbow trout, O. mykiss, early-life-stage (ELS) test was performed under flow-through conditions and in compliance with OECD test guideline 210. Unfertilized trout eggs and sperm were received from a commercial supplier and the eggs were fertilized in the laboratory. One hundred and eighty newly fertilized eggs were randomly selected and allocated, 60 eggs per replicate, to the three replicate test vessels for each control and test concentration. The number of surviving fish was reduced randomly to 30 per replicate just after the end of the hatching period (day 26) in the control. The number of surviving fish was again reduced randomly to 15 per replicate when swim-up and feeding began on day 50. Actively feeding juveniles were fed trout chow two to four times per day, corresponding to approximately 4% of their body weight per day, from day 50 to the end of the 85-d test. Test solutions were continuously renewed during the study by pumping the stock solutions into flowing dilution water with a peristaltic pump system at a replacement rate of 5.76 times the test vessel volume per day. Dilution water pH

ARTICLE IN PRESS I. Colombo et al. / Ecotoxicology and Environmental Safety 71 (2008) 749–756 was 6.0–8.5, hardness was 150 mg/l as CaCO3, and water temperature was kept between 11.1 and 12.5 1C for embryos and between 11.6 and 14.4 1C for larvae and juvenile fish. The dissolved oxygen concentration was greater than 60% air saturation, the light/dark cycle was maintained at constant darkness until 7 days after hatching, then 16 h light and 8 h dark through test end. Observations were made daily as follows: eggs—marked loss of translucency and change in coloration, white opaque appearance; embryos—absence of body movement or heart beat; larvae and juvenile fish—immobility, absence of respiratory movement or heart beat, white opaque coloration of the central nervous system, lack of reaction to mechanical stimulus, and abnormalities. All animal studies were conducted in accordance with national and institutional guidelines for the protection of animal welfare. 2.3. Chemical analyses Analyses to confirm the APFO test concentrations were not performed during the daphnid and trout acute tests based on the known stability of the test substance in water. Subsequently, both stability and test concentrations were confirmed by direct analysis during the algal inhibition test, the Daphnia chronic test, and the rainbow trout ELS test. Test solutions were analyzed by ion chromatography with electrochemical detection. One dilution was prepared for each sample with one injection of a 25 ml aliquot performed for each final dilution. The concentrations of APFO were determined as PFO from a calibration curve of peak area against APFO concentrations in standard solutions. The limit of quantification (LOQ) of the analytical method was established as 1 mg/l for a standard solution of APFO. Linearity was checked by the analysis of three different sets of five standard solutions containing 1, 5, 10, 50 and 100 mg/l of APFO in MilliQ water with a resulting coefficient of determination for the calibration curve of greater than 0.999 for this range of APFO concentrations in water. The repeatability of injections was validated through 10 analyses of a solution containing 50 mg/l APFO. The analyses gave satisfactory results since the coefficient of variation values were 1% based on both peak height and peak area. Finally, accuracy and precision were demonstrated analysing 6 solutions containing nominally 2.03 and 50.7 mg/l of APFO in Milli-Q water. The mean measured concentrations were 2.02 and 53.7 mg/l, respectively, with calculated precision of 6% and 2% and accuracy of 99% and 106%, respectively. Chemical analyses were performed only in the second definitive algae inhibition test. Measured concentrations in the second test with and without algae (i.e., after centrifugation) were within 720% of the corresponding nominal values at the beginning and end of the test. APFO was not sorbed in a significant manner by either the test vessels or the algae and the study results were based on the nominal test concentrations. In the Daphnia chronic test, some of the measured test concentrations were not within 720% of the nominal values but the maximum coefficient of variation of the measured concentrations was 8.5%. All study endpoints were based on the geometric mean of measured concentrations in the test solutions over the duration of the 21-d test of 4.31 (nominal 6.25), 9.16 (12.5), 20 (25), 44.2 (50) and 88.6 (100) mg/l APFO. In the trout ELS test, the measured test concentrations again were not within720% of the nominal concentrations in some instances. Therefore, the ELS study endpoints also were based on geometric means of the measured test concentrations throughout the test. Geometric mean measured concentrations were 2.18 (3.13), 4.48 (6.25), 10.7 (12.5), 20.9 (25.0) and 40 (50) mg/l APFO. 2.4. Statistical analyses The determination of EC50 (Daphnia), LC50 (trout), and ErC50 (algal growth rate), EbC50 (algal biomass) values was conducted using probit analysis with fiducial limits calculated by Fieller’s method. After checking for normality of the data (w2 and Shapiro–Wilks tests, p ¼ 0.01), as well as the homogeneity of variance (Bartlett’s test, p ¼ 0.01), the NOEC for the algal inhibition test was determined by ANOVA (Bonferroni t-test, p ¼ 0.05 or Dunnett’s test, p ¼ 0.05) using the individual replicates of the area under the curve and the specific growth rate. In the trout early-life stage test, the NOEC was determined based on mortality, weight and length at the end of the test. While mortality data were found to be normally distributed and homogeneous, length and weight data were not normally distributed and the Kruskal–Wallis and Dunn’s tests were used for data analyses. Similar methodologies were applied in the D. magna chronic test data since the length of parent animals at test end was not normally distributed although the variances were homogeneous.

3. Results The results of the acute daphnid and trout tests are presented in Tables 2 and 3. Daphnia magna immobility was 0% in the control and nominal 100, 178 and 316 mg/l APFO concentrations, 35% at 562 mg/l and 100% at the nominal 1000 mg/l APFO concentration

751

Table 2 Immobility in Daphnia magna at 24 and 48-h in a static, acute test with APFO Nominal APFO concentration (mg/l) Replicate

0 100 178 316 562 1000

Number immobile/number at test start 24 h

48 h

A

B

C

D

A

B

C

D

0/5 0/5 0/5 0/5 0/5 5/5

0/5 0/5 0/5 0/5 3/5 5/5

0/5 0/5 0/5 0/5 1/5 5/5

0/5 0/5 0/5 0/5 3/5 5/5

0/5 0/5 0/5 0/5 4/5 5/5

0/5 0/5 0/5 0/5 4/5 5/5

0/5 0/5 0/5 0/5 3/5 5/5

0/5 0/5 0/5 0/5 5/5 5/5

A–D represent replicate test chambers.

during the first 24 h. After 48 h, the highest concentration resulting in no immobilization was 316 mg/l with 80% and 100% immobilization observed at 562 and 1000 mg/l APFO. The calculated 24- and 48-h EC50 values were 599 and 480 mg/l APFO, respectively, based on nominal APFO concentrations and immobility. All trout exposed to the nominal 1000 mg/l APFO concentration died within the first 24 h of the test. No mortality was observed at any other test concentration over the duration of the 96-h test. Therefore, the 96-h LC50, calculated by the binomial method, was 707 mg/l APFO based on nominal APFO concentrations and mortality. The NOEC based on sub-lethal effects (changes in coloration, lethargy) at 96 h was 125 mg/l APFO based on observed sub-lethal effects at test end. The algal ErC50 and EbC50 from the range-finding test at both 72 and 96-h were greater than 100 mg/l. Based on nominal APFO concentrations, the initial definitive test had a 72-h NOEC for growth rate of 200 mg/l APFO, however, the 96-h NOEC for growth rate was significantly lower at 6.25 mg/l although inhibition of the growth rate was only 14% at the nominal 400 mg/l APFO concentration. The 72- and 96-h algal biomass NOECs in the initial definitive test were 400 and 100 mg/l APFO, respectively. Based on the unusual difference in the 72- and 96-h NOEC values for growth rate at 72 and 96 h (200 vs. 6.25 mg/l) and between the NOECs at 96 h for the biomass and growth rate (100 vs. 6.25 mg/l), a second definitive test was conducted with analytical confirmation of test concentrations to determine whether the initial test results were reproducible. The second definitive algae test (Table 4) was conducted using the nominal test concentrations utilized in the initial definitive study. The 72-h growth rate NOEC based on nominal APFO concentrations was 200 mg/l as was observed in the initial test. The 96-h growth rate NOEC of 12.5 mg/l was twice that observed in the initial test but confirmed a significant reduction in the growth rate between 72 and 96 h. The biomass NOEC at 72 h was 200 mg/l but at 96 h the NOEC of 12.5 mg/l was lower than in the initial test but was equivalent to the 96-h growth rate NOEC in the second test. The second definitive test was considered to provide more reliable data due to the better agreement between the growth rate and biomass NOEC values. Therefore these results were considered indicative of the algal toxicity of APFO with 72and 96-h NOEC values, for both growth rate and biomass, of 200 and 12.5 mg/l APFO, respectively. Since the measured APFO concentrations were within 720% required by the OECD test guideline, the study results were based on nominal concentrations. The results of the daphnid chronic reproduction test appear in Table 4. Control mortality was less than 20% during the test and mean control reproduction was greater than 60 neonates per parent daphnid surviving to study termination. Calculation of

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Table 3 Mortality and sublethal effects in rainbow trout, Oncorhynchus mykiss, during a static, acute, 96-h test with APFO Nominal APFO concentration (mg/l)

0 31.3 62.5 125 250 500 1000 a b c

Number dead (sub-lethal effectsa)/number at study start 24-h

48-h

72-h

96-h

96-ha

0/7 0/7 0/7 0/7 0/7 0/7 7/7

0/7 0/7 0/7 0/7 0/7 0/7 7/7

0/7 0/7 0/7 0/7 0/7 0/7 7/7

1a/7 0/7 0/7 0/7 0/7 0/7 7/7

0/6 0/7 0/7 0/7 b,c 7 /7 7b/7 0/0

One fish was accidentally killed during observations. Colour change. Lethargy.

Table 4 Mean percent inhibition of Pseudokirchneriella subcapitata growth (biomass and growth rate) from the second definitive 96-h test with APFO Mean measured APFO concentrations (CV%, n ¼ 3) (mg/l)

Percent inhibition, 72-h growth ratea

Percent inhibition, 72-h biomassa

Percent inhibition, 96-h growth ratea

Percent inhibition, 96-h biomassa

Control 5.76 (4.3) 11.37 (10.1) 22.70 (6.5) 46.33 (1.8) 95.87 (0.9) 180.67 (1.2) 369.67 (3.1)

0 (0.33) (2.3) (2.17) (0.79) (1.33) 1.34 3.78*

0 (0.40)a (4.38) (1.06) (1.57) 3.27 10.55 21.45*

0 0.59 1.8 5.78* 5.44* 6.68* 11.63* 14.79*

0 1.58 2.85 15.47* 15.75* 19.6* 33.66* 43.24*

Values in parentheses represent mean percent stimulation of growth relative to control while an asterisk indicates statistically significant inhibition (p ¼ 0.05) relative to control. a n of replicates ¼ 6 for control treatment, n ¼ 3 for test substance treatments.

study endpoints was based on the geometric mean measured test concentrations of APFO over the duration of the test. No statistically significant mortality was observed in parent daphnids during the test and the 21-d EC50 and NOEC values based on immobility of parent daphnids were greater than 88.6 mg/l APFO. The time to first brood and number of broods of neonates were measured as indicators of reproductive performance. In the control, 4.31 and 9.16 mg/l test concentrations, the first brood occurred between days 8 and 10 of the test. Between the mean measured test concentrations of 9.16 and 88.6 mg/l, APFO induced a delay in the appearance of the first brood, which occurred between days 8 and 16. APFO also caused a decrease in the average number of broods per parent organism surviving to test termination with the mean number of broods decreasing from 4.75 to 1.22 in a concentration-dependent manner. In the same way, the mean number of neonates per surviving parent daphnid at 21 days decreased in a concentration-related manner from 63.3 in the control to 0.9 at 88.6 mg/l APFO. The percentage of dead progeny and aborted eggs at the end of the test was 0%, 0.2%, 0.2%, 1.4%, 12.1% and 99.2% of the total progeny and eggs in the control and 4.31, 9.16, 20, 44.2 and 88.6 mg/l APFO concentrations, respectively. Control reproduction after 21 days was within the range of the historical reproduction data for the laboratory performing the test. Neonate production, as a percentage of the control value, was 112%, 101%, 98%, 38% and 0% at 4.31, 9.16, 20 and 44.2 mg/l and 88.6 mg/l APFO, respectively. The 21-d NOEC based on the number of live neonates per surviving parent daphnid was 20 mg/l APFO while the 21-d EC50 for reproduction was 39.6 mg/l APFO. The effects of APFO on growth of the parent daphnids was also measured based on total length of surviving parent daphnids at

the end of the 21-d test. A statistically significant inhibition of growth of parent daphnids at the end of the test was observed at 88.6 mg/l APFO (Table 5). Growth, as a percentage of the control, was 99% at 4.31 mg/l, 94% at 9.16 mg/l, 95% at 20 mg/l, 94% at 44.2 mg/l and 85% at 88.6 mg/l. Therefore the 21-d EC50 for growth as length was greater than 88.6 mg/l and the 21-d growth NOEC was 44.2 mg/l APFO. Trout chronic ELS study data are summarized in Table 6. OECD test guideline validity criteria for hatching success (466%) and post hatching survival (470%) were exceeded in the test. Since the measured APFO test concentrations were not always within 720% of the nominal values (primarily at the nominal 3.13 and 6.25 mg/l concentrations), the study endpoint calculations were based on the geometric means of the measured concentrations during the 85-d duration of the test. Geometric mean APFO concentrations were calculated as 2.18, 4.48, 10.7, 20.9 and 40 mg/l expressed as APFO for the corresponding nominal values of 3.13, 6.25, 12.5, 25 and 50 mg/l, respectively. APFO did not induce a significant increase in the mortality of trout embryos. At the end of the hatching period, the cumulative mortality at the highest measured test concentration (40 mg/l) was equal to that observed in the control, i.e. 19%. The NOEC for mortality of embryos was 40 mg/l APFO based on Dunnett’s test. Measured APFO concentrations up to 40 mg/l did not delay the hatching period at any concentration relative to control. A slight, although not statistically significant, concentration-related increase was observed in mortality with values of 2%, 3%, 4%, 5% and 7% in the nominal 3.13, 6.25, 12.5, 25 and 50 mg/l test concentrations, respectively. Since this effect was not statistically significant relative to control mortality, the NOEC based on the mortality of larvae was 40 mg/l. Mortality was not related to concentration for

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Table 5 Summary of data from a 21-d Daphnia magna life cycle toxicity test with APFO, an asterisk indicates a statistically significant effect (p ¼ 0.05) relative to controla Geometric mean measured APFO concentrations (CV%, n ¼ 14) (mg/l)

Adult survival (%)

Mean first day of reproduction (SD)

Mean live young (SD)

Mean adult length (SD) (mm)

Control 4.31 (8.5) 9.16 (4.6) 20 (4.5) 44.2 (2.2) 88.6 (7.3)

80 90 100 100 90 90

8.9 (0.7) 8.4 (0.5) 8.5 (0.7) 8.9 (2.2) 10.4* (2.6) 13.6* (1.8)

63.3 (10.4) 70.6 (12.8) 64.1 (9.8) 62.3 (21.6) 24* (26.5) 0.1* (0.3)

3.4 (0.05) 3.3 (0.10) 3.2 (0.12) 3.2 (0.13) 3.2 (0.11) 2.8* (0.17)

a

n of replicates ¼ 10 for all treatments.

Table 6 Summary of data from an 85-day rainbow trout, Oncorhynchus mykiss, early-life stage test with APFO, an asterisk indicates a statistically significant effect (p ¼ 0.05) relative to control Geometric mean measured APFO concentration (CV%, n ¼ 21) (mg/l)

Replicate First day of hatching

Last day of hatching

Percent hatching

Percent survival at Swimup

Percent survival at test end

Mean length (mm) Mean wet wt. (mg) at test end at test end

0

1 2 3 Mean

24 24 24 24

26 26 26 26

83 83 77 81

97 97 100

100 90 80

36.8 36.9 38

619.6 731.1 863.8

2.18 (31)

1 2 3 Mean

25 25 25 25

26 27 27 27

92 74 64 77

97 93 100

95 95 100

34.5 36.3 35.1

646.8 724.3 629.3

4.48 (33)

1 2 3 Mean

26 26 26 26

27 27 27 27

84 75 84 81

97 97 93

95 95 95

35.1 34.7 35.2

644.5 617.2 634.4

10.7 (31)

1 2 3 Mean

26 26 25 26

27 27 27 27

83 78 72 78

100 93 93

90 65 85

35.6 35.5 36.3

663.6 705.8 719.3

20.9 (18)

1 2 3 Mean

25 25 26 25

27 27 27 27

91 75 80 82

100 93 93

100 90 75

35 35.3 35.8

649.2 640 769.7

40 (24)

1 2 3 Mean

26 26 25 26

27 27 27 27

88 91 64 81

90 97 93

85 95 80

35.6 36.2 35.2

707.6 690.2 726.5

juvenile fish with mortality values for the control and five APFO concentrations of 10%, 3%, 5%, 20%, 12% and 13%, respectively. Since the percent cumulative mortality in juveniles in the mean measured 4.48 mg/l (nominal 6.25 mg/l) exposure group was equal in the three replicates (i.e., 5%), the data failed to meet homogeneity of variance and it was not possible to determine a NOEC. When the 4.48 mg/l exposure group was discarded, the data were homogeneous and the NOEC was calculated to be 40 mg/l APFO. These results were confirmed by the Wilcoxon rank sum test when analyzing data for all test concentrations. Therefore, the NOEC value for mortality of all life stages in the trout ELS study was 40 mg/l APFO. Sub-lethal effects, such as abnormalities, immobile organisms, and abnormal respiratory rate were observed in all exposure groups during the study. These transient effects were observed in a limited number of organisms during the test, were not concentration related, and therefore did not appear to result from APFO exposure. The only non-transient sublethal effect was an abnormality observed in one juvenile fish on day 50 in the 40 mg/l exposure group that led to the death of the juvenile on day 60.

Since this effect was observed in a single organism and was not concentration-related, it also appeared to be unrelated to APFO exposure. Weight and length data at test end were not normally distributed and non-parametric analyses were performed using Kruskal–Wallis and Dunn’s tests. These analyses indicated that both length and weight data were non-monotonic. The results of the non-parametric analyses of length and weight indicated the NOEC for both endpoints was 40 mg/l APFO. The mean weights of the fish were not significantly different in the control and five APFO concentrations (738, 669, 632, 696, 686, 708 mg, respectively). The mean lengths also were not significantly different from the control at the mean measured APFO concentrations of 10.7, 20.9 and 40 mg/l but a significant difference was observed at 2.18 and 4.48 mg/l. Since these effects were not concentration-dependent, the maximum reduction in length was less than 6%, and no reduction relative to control was observed at greater test concentrations, these changes were considered to be random occurrences of no biological significance.

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The complete GLP study reports for each of the freshwater aquatic toxicity studies discussed here are available on the Plastics Europe website at http://www.plasticseurope.org/Content/ Default.asp?PageID=1083.

4. Discussion The results of acute and chronic invertebrate and fish testing demonstrate that APFO exhibits low acute and chronic toxicity to aquatic organisms. The reported acute test endpoints are above the European Union and Global Harmonized System (GHS) classification limit of 100 mg/l. In addition, based on the acute endpoints, US EPA would classify APFO as being of low concern for acute hazard in aquatic ecosystems (Smrchek et al., 1995). Similarly, the NOEC values from a chronic life-cycle reproduction study with the cladoceran, D. magna, and from a chronic earlylife-stage study with the rainbow trout, O. mykiss, demonstrate that APFO has low potential hazard for chronic toxicity to aquatic invertebrates and fish (Smrchek et al., 1995). The ammonium salt of PFOA, ammonium perfluorooctanoate (APFO) is expected to be completely dissociated in water to the perfluorooctanoate anion (PFO), pKa ¼ 2.5, and ammonium ion (NH+4). In water, ammonia exists in two forms, un-ionized ammonia (NH3) and ammonium ion (NH+4). The equilibrium between the two forms of ammonia is controlled primarily by pH and temperature. For example, at 20 1C and a pH of 7.5, the percentage of un-ionized ammonia in water is approximately 1%. These speciation relationships are important in determining ammonia toxicity since un-ionized ammonia is generally more toxic to aquatic organisms than ammonium ion (United States Environmental Protection Agency (USEPA), 1999). Analysis of the potential contribution of un-ionized ammonia to the toxicity of APFO suggests it may be an important factor in the overall toxicity of APFO and responsible for some of the variability observed in aquatic toxicity between species and studies based on variations in temperature, pH and form of test substance (e.g., ammonium salt versus lithium salt) during these studies. The estimated concentrations of un-ionized ammonia, based on the indicated temperature and pH values, at the EC50/LC50/NOEC established for APFO in the current tests are presented in Table 7. In marine environments, salinity, temperature and pH are the major factors that affect the concentration of un-ionized ammonia relative to total ammonia. As in freshwater environments, increasing temperature and pH increase un-ionized ammonia while increases in salinity lead to a decrease in un-ionized ammonia (Boardman et al., 2004). Based on existing data from a variety of sources (Nordin, 2001; USEPA, 1989; Boardman et al., 2004), freshwater criteria for ammonia can reasonably be expected to be protective of marine and estuarine species. In the acute Daphnia test, the free NH3 concentration was calculated to be approximately 0.73 mg/l which is within the reported un-ionized ammonia EC50 range of 0.53–4.9 mg/l for Daphnia (US EPA, 1999). A similar observation applies to the acute trout test in which the un-ionized ammonia in the test solution at the calculated 96-h LC50 of 707 mg/l APFO would be approximately 0.38 mg/l. This value is also within the un-ionized ammonia 96-h LC50 range 0.16–1.1 mg/l for rainbow trout (US EPA, 1999). Although few data are available on the chronic toxicity of un-ionized ammonia to daphnids, Gersich and Hopkins (1986) reported a chronic NOEC of 0.42 mg/l based on reproduction. The chronic NOEC established for APFO after 21 d in the D. magna reproduction test was 12.5 mg/l which corresponds to approximately 0.024 mg/l un-ionized ammonia in the test solution. In the trout early-life stage test, the un-ionized ammonia content was calculated to be approximately 0.021 mg/l at the

calculated APFO NOEC of 40 mg/l. This value is almost identical to the reported trout chronic NOEC for un-ionized ammonia of 0.025 mg/l (Calamari et al., 1981). Based on these data, it appears plausible that the presence of un-ionized ammonia in the test solutions could play a substantive role in the observed acute and chronic toxicity of APFO to aquatic organisms. A similar observation was reported by Goleman and Carr (2006) for the toxicity of perchlorate salts to the African clawed frog, Xenopus laevis. Based on their studies of the toxicity of ammonium perchlorate, sodium perchlorate and ammonium chloride to X. laevis, they concluded that ammonium perchlorate was significantly more lethal than sodium perchlorate and attributed the increased lethality to the ammonium ion. Few data exist on the aquatic toxicity of other perfluorocarboxylic acids in comparison to APFO, an 8-carbon chain molecule. The data that are available (Boudreau et al., 2002) suggest that, in both laboratory and mesocosm (semi-field) studies, perfluorinated carboxylic acids with carbon chain lengths from 3 through 10 exhibit toxicity to one or more species of freshwater algae, daphnids and duckweed that is similar to or less than the toxicity of APFO to the test species in the current study. The existing data also suggest that hydrocarbon surfactants are typically more toxic, on both an acute and chronic basis, than APFO. The acute 48-h EC50 of several cationic and non-ionic surfactants to daphnids have been reported to be less than 1 mg/l (Garcia et al., 2001; Morrall et al., 2003). Acute toxicities of several anionic surfactants have been reported to be in the range of 1–20 mg/l (Ying, 2006). Algae also appear more sensitive to hydrocarbon surfactants with reported 72-h EC50 values for four species of algae for a series of anionic, non-ionic, and amphoteric surfactants of 0.14–4.4 mg/l (Pavlic et al., 2005). Based on the acute data, concentrations of hydrocarbon surfactants causing chronic effects on aquatic organisms could be expected to be similar or lower, as has been reported in several studies (Morrall et al., 2003; Lewis, 1991). Therefore, the potential aquatic hazard appears to be greater for traditional hydrocarbon surfactants than for APFO. Various studies have reported the more or less ubiquitous presence of APFO (i.e., PFO) in aqueous matrices in the environment, especially near manufacturing sites, or highly industrialized or urbanized areas. PFO levels in open waters (Atlantic Ocean, Western Pacific Ocean) far from industrial sources have been reported to be in the ng/l–pg/l range (Taniyasu et al., 2004) while surface water concentrations near possible point sources have been reported in the range of ng/l–mg/l and concentrations associated with spills have been reported in the low parts per million (US EPA AR-226 Public Docket). Saito et al. (2004) reported on PFO levels in the Japanese rivers near industrialized areas and in the ocean waters of the coastal Pacific around Japan. PFO levels in these waters varied between 102 and 108 mg/l. This range is similar to that observed for PFO in precipitation to Canadian lakes (Boulanger et al., 2004), as well as the range of 104–107 mg/l reported for precipitation near an industrialized area in the US (Scott et al., 2003). Kallenborn et al. (2004) also measured PFO levels in rainwater, lake water, seawater, sewage and landfill effluent in Norway with reported concentrations between 104 and 106 mg/l while Lange et al. (2004) reported a surface water concentration in Germany of 106 mg/l and McLachlan et al. (2007) reported concentrations of up to 2.04 mg/l from the Po River in Italy. In order to extrapolate a predicted no effect concentration (PNEC), three trophic levels were taken into consideration; the chronic NOEC values measured for fish (40 mg/l), invertebrates (20 mg/l) and algae (12.5 mg/l). Applying a safety factor of 10  to the lowest reported chronic endpoint, as suggested in the European Technical Guidance Document (European Union,

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Table 7 Calculated concentrations of un-ionized ammonia in freshwater acute and chronic aquatic toxicity tests with APFO Daphnia acute test

Temp. (1C) pH % Free NH3 48-h EC50 (mg/l) NH3 (mg/l) 48-h NH3 EC50 (mg/l) (USEPA, 1999)

Trout acute test Range

Calculationa

19.6–20.3 7.7–8.2

20 8.1 4.7 480 0.73 0.53–4.9

Daphnia chronic test Temp. (1C) pH % Free NH3 21-d NOEC (repro) (mg/l) NH3 (mg/l) 21-d NOEC (repro) (mg/l), (Gersich and Hopkins. 1986)

Temp. (1C) pH % Free NH3 96-h LC50 (mg/l) NH3 (mg/l) 96-h NH3 LC50 (mg/l) (USEPA, 1999)

Range

Calculation*

14.8–15.4 7.32–7.88

15 7.8 1.67 707 0.38 0.16–1.1

11.1–14.4 7.37–8.04

13 7.8 1.45 40 0.013 0.025

Trout ELS test 18.5–19.8 7.56–8.26

20 8.2 5.85 25 0.048 0.42

Temp. (1C) pH % Free NH3 NOEC (mg/l) NH3 (mg/l) NH3 NOEC (mg/l), (Calamari et al., 1981)

a Calculations were based on values listed using the following equations: % ammonia-nitrogen (NH3-N) in APFO. MW N  100/MW APFO ¼ 3.248, % free ammonia ðNH3 Þ ¼ 100=ð1 þ 10ðpK a pHÞ Þ, where pKa ¼ 9.245+0.0324 (25T), where T ¼ 1C.

2003), the resulting predicted no effect concentration (PNEC) value is 1.25 mg/l. Using a worst case scenario (PFO predicted environmental concentration (PEC) ¼ 0.02 mg/l), the resulting PEC/PNEC ratio of 0.16 indicates a low risk to aquatic organisms from potential APFO exposure. Sanderson et al. (2003) measured the ecological impact of APFO on the zooplankton community in both indoor and outdoor microcosm experiments. The LOECcommunity from the indoor experiment was in the range of 30–70 mg/l and the LOECcommunity from the outdoor experiment was o70 mg/l. A LOEC for the zooplankton community of 10 mg/l was chosen as a conservative value for the calculation of a PNEC value based on the microcosm data. Applying a safety factor of 100 (10  safety factor for the NOEC, 10  for extrapolation from the LOEC to the NOEC), a PNEC value of 0.1 was calculated from the microcosm data. Based on the data presented above, currently reported concentrations of PFO from surface waters around the globe (other than spill situations) are typically orders of magnitude less than concentrations of concern and as a result PFO (APFO, PFOA) is unlikely to pose a significant risk to aquatic invertebrates and fish under non-spill conditions.

5. Conclusion The acute and chronic studies reported here were conducted according to GLP guidance in conformance with OECD, EU, and US EPA test guidelines. These studies eliminated the variability associated with nominal test concentrations, different PFO counterions, and different batches of inadequately characterized test substance used in historical studies by utilizing a single, wellcharacterized batch of APFO for aquatic acute base set testing, a chronic reproduction study with Daphnia magna, and an early-life stage, embryo-larval study with the rainbow trout, Oncorhynchus mykiss. Although the acute studies were conducted using nominal concentrations, the algal, chronic daphnid and trout ELS studies were conducted with analytical confirmation of test substance concentrations. These studies represent a reliable dataset for evaluation of the acute and chronic hazard and risk of APFO (PFO) to freshwater algae, invertebrates and fish. The results reported here support the conclusion that APFO does not need classification following the European Directives and Legislation (EEC, 1967). Although APFO (PFO) is widely dis-

tributed in aqueous matrices in the environment, concentrations are typically low (e.g., ng/L) and not sufficient to cause significant risk to either freshwater or marine aquatic organisms. Aquatic toxicity test results for freshwater species are likely to be protective of marine species based on the observation that the ammonium counterion (i.e., ammonia toxicity) can be used to account for the observed acute and chronic aquatic toxicity of APFO. Industry has committed to reducing emissions from manufacturing operations. Additional research is needed to better elucidate both historical and existing sources of APFO (Prevedouros et al., 2006) and provide better understanding of the transport and fate processes for APFO/PFOA in the environment.

Disclaimers All animal studies were conducted in accordance with national and institutional guidelines for the protection of animal welfare.

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