Chemical Characterization of a Legacy Aqueous Film-Forming Foam Sample and Developmental Toxicity in Zebrafish (Danio rerio)

A legacy AFFF, a 3% application formula, was acquired through the MA-DEP. The formulation and manufacturer of this mixture were unknown. A serial dilution of 0.003% AFFF was prepared for quantification analyses, and a serial dilution, starting at 0.00352% AFFF was prepared for toxicity testing. Perfluorooctanesulfonic acid (PFOS) (Sigma; Product 77282, Lot: BCBX5798) was prepared as a $282.30\text{-mg}/\mathrm{L}$ stock in 100% dimethylsulfoxide (DMSO). Perfluorohexanesulfonic acid (PFHxS) (Sigma; Product 50929, Lot: BCBX0925) was prepared as a $45\text{-mg}/\mathrm{L}$ stock. Additionally, a PFOS/PFHxS mixture was prepared containing $282.30\mathrm{mg}/\mathrm{L}$ PFOS and $45\mathrm{mg}/\mathrm{L}$ PFHxS. Sodium dodecyl sulfate (SDS) (Fisher Scientific, Product BP166-100, Lot: 136458) and sodium tetradecyl sulfate (TDS) (Alfa Aesar, Product B21941, Lot: U20F005) were prepared as $\mathrm{3,200}\text{-mg}/\mathrm{L}$ stocks in deionized water.

The nontarget screening of PFAS was carried out using an Orbitrap Fusion™ Tribrid™ Mass Spectrometer equipped with UltiMate™ 3000 UHPLC (Thermo Fisher Scientific), in negative ionization mode. The mass range was set at m/z 100–1,000 with the Orbitrap resolution power of 30,000. For ionization, a spray voltage of $3\text{\hspace{0.17em}kV}$ and an ion transfer tube temperature of 300°C were used. Chromatographic separation was performed on a Waters ACQUITY BEH C18 column ($2.1\mathrm{mm}\times 100\mathrm{mm}$ and $1.7\text{-}\mathrm{\mu }\mathrm{M}$ particle size). The column oven temperature was set at 40°C. The mobile phase consisted of a) $2\text{\hspace{0.17em}mM}$ ammonium acetate; and b) $2\text{\hspace{0.17em}mM}$ acetonitrile. The mobile phase flow rate was $0.15\mathrm{mL}/\text{min}$, and the following gradient program was used: initial conditions were 5% B held for $0.5\text{\hspace{0.17em}min}$, then increased to 95% B over $4.5\text{\hspace{0.17em}min}$ where it was held for $2\text{\hspace{0.17em}min}$. The ratio of B was restored to 5% over $0.5\text{\hspace{0.17em}min}$ and maintained for $2.5\text{\hspace{0.17em}min}$. A total analytical running time was $10\text{\hspace{0.17em}min}$, and the injection volume was $5\mathrm{\mu }\mathrm{L}$.

The Xcalibur™ (version 4.1; Thermo Fisher Scientific) with Qualbrowser was used for the peak detection, and Compound Discoverer software (version 3.0.0; Thermo Fisher Scientific) with mzCloud was used for the nontarget screening of PFAS compounds. Reported in Table 1 are tentative identities of PFAS compounds beyond the 24 tested using a modified U.S. EPA Method 537.1. Relative peak intensities are provided for non-PFAS compounds in Table 2.

A sample of AFFF (1:1,000 serial dilution of the 3% sample) was sent to Eurofins TestAmerica, a commercial laboratory, for quantification of PFAS compounds in the AFFF formulation. Eurofins provides PFAS testing in water through a propriety modified method based on U.S. EPA Method 537.1. Table 1 contains the 26 PFAS and other fluorinated alkyl substances, with their method detection limits (MDL), that were identified using the Orbitrap Fusion™ Tribrid™ Mass Spectrometer.

The surfactants SDS and TDS were prepared in $1.0\text{-mg}/\mathrm{mL}$ stocks in water and acetonitrile, respectively. These concentrations were based on the active ingredient, excluding the sodium ion. One milliliter of the AFFF sample was added to a volume of $9\mathrm{mL}$ water/acetonitrile solution (1:1, v/v). Serial dilutions of the solution were prepared to reach a final AFFF sample dilution of 1:10,000. Analysis was completed on a Waters ACQUITY UPLC H-class system equipped with Waters Xevo TQD Triple Quadrupole Mass Spectrometer having an electrospray ionization source. The quantitation of target compounds was performed in the negative ionization mode with the optimized multiple reaction monitoring transitions. Chromatographic separation was performed on a Phenomenex Kinetex PFP column ($2.1\mathrm{mm}\times 100\mathrm{mm}$ and $2.6\text{-}\mathrm{\mu }\mathrm{M}$ particle size). The mobile phase consisted of a) 0.1% formic acid in water and b) 0.1% formic acid in acetonitrile. The mobile phase flow rate was $0.25\mathrm{mL}/\text{min}$, and the following gradient program was used: initial conditions were 5% B held for $0.5\text{\hspace{0.17em}min}$, then increased to 95% B over $2.5\text{\hspace{0.17em}min}$, where it was held for $4\text{\hspace{0.17em}min}$. The ratio of B was restored to 5% over $0.5\text{\hspace{0.17em}min}$ and maintained for $2.5\text{\hspace{0.17em}min}$. A total analytical running time was $10\text{\hspace{0.17em}min}$, and the injection volume was $5\mathrm{\mu }\mathrm{L}$. These analyses were repeated in triplicate.

Adult zebrafish, Danio rerio, were housed in Aquaneering stand-alone systems. In the colony, fish were maintained in $6\text{-L}$ tanks containing 30–40 male and female fish. Fish were maintained on a 14:10h light:dark cycle and fed twice daily a diet of GEMMA Micro 300 (Skretting). Water quality was monitored daily to ensure pH was within pH 7.3–7.4, conductivity was within $600–800\mathrm{\mu }\mathrm{S}$, and temperature was held at $28\pm 1°\mathrm{C}$. Glass containers with mesh lids were placed into tanks with decreased water levels to encourage breeding. Breeding occurs roughly as the lights turn on in the facility, and embryos were collected from the containers at approximately 1 h post fertilization (hpf). Embryos were cleaned to remove dead embryos and debris. Mortality and morphometric assessments were conducted in the wildtype AB strain obtained from Zebrafish International Resource Center (ZIRC). Liver morphology was assessed in the Tg(gut:GFP) strain obtained from University of Massachusetts Medical School (UMass Medical), which fluoresce in the liver through the ef1a promoter (Field et al. 2003). Pancreas morphology was examined in Tg(ins:GFP), which fluoresce in the insulin-producing beta cells of the endocrine pancreas, and Tg(ptf1a:GFP), which fluoresce in the exocrine pancreas, also obtained from UMass Medical (diIorio et al. 2002; Godinho et al. 2005). All experiments were conducted following protocols of the University of Massachusetts Amherst Institutional Animal Care and Use Committee (A3551-01).

Determination of ${\text{LC}}_{50}$ values and examination of morphological effects were accomplished using embryos of wildtype AB strain of fish. Embryos were screened under light microscopy, and healthy embryos staged at 3 hpf, the 1,000-cell stage, were selected for treatment. Fifteen embryos were assigned at random to each treatment or control group, with each embryo placed into an individual glass vial containing $1\mathrm{mL}$ solution. Exposure solutions were prepared in $0.3\times$ Danieau’s solution, $17.4\text{\hspace{0.17em}mM}$ NaCl, $0.21\text{\hspace{0.17em}mM}$ KCl, $0.12\text{\hspace{0.17em}mM}$ ${\text{MgSO}}_4$, $0.18\text{\hspace{0.17em}mM}$ $\mathrm{Ca}{\left({\mathrm{NO}}_3\right)}_2$, $1.5\text{\hspace{0.17em}mM}$ HEPES buffer, pH 7.2 (Westerfield 2000). The 3% AFFF formulation was diluted so that the relative PFOS concentration of the dilution matched PFOS concentrations previously examined by our lab, $16–32\mathrm{\mu }\mathrm{M}$ PFOS (Sant et al. 2017). However, embryos were not viable at these AFFF concentrations, necessitating additional dilutions. Serial dilutions of the AFFF sample were prepared so that 8 concentrations from $4.40\times {10}^{-6}$ to $3.52\times {10}^{-3}\%$ AFFF, defined in Table 3, were examined for mortality assessment. Each vial contained $1\mathrm{mL}$ total volume, and solutions were prepared and refreshed daily. Embryos were reared at $28\pm 1°\mathrm{C}$, and embryonic health was monitored daily through 96 hpf, as outlined in OECD 236, Fish Embryo Acute Toxicity Test (OECD 2013). End points such as craniofacial malformations, spinal curvature, yolk malformations, swim bladder inflation, and hatch rate were noted. There was $>90\%$ health and survival in the control group. This experiment was replicated a total of 4 times ($n=60$ larvae per treatment group).

To provide comparison with the most prevalent PFAS, PFOS and PFHxS, individual compound exposures and coexposures were completed following the same paradigm. Stock concentrations of PFOS, PFHxS, and a PFOS/PFHxS mixture were prepared in DMSO as described above. Therefore, control embryos received a 0.01% DMSO control exposure. Dilutions were prepared in 0.3× Danieau’s solution to mimic PFOS and PFHxS doses present in the AFFF sample, and therefore, PFOS/PFHxS mixture held at a ratio of 6.27:1 PFOS:PFHxS. The dilutions are summarized in Table 4. These experiments were run at the same time and replicated thrice ($n=45$ embryos in total).

The use of DMSO was necessary in stock preparations of the PFAS compounds, therefore necessitating the use of DMSO as a solvent control in the toxicity testing of the individual PFAS compounds but not the AFFF sample. Dilutions of the stock were prepared such that DMSO concentrations were at 0.01% DMSO, which is previously reported to have no impact on zebrafish morphological development (Hallare et al. 2006; Turner et al. 2012; Kais et al. 2013).

This exposure paradigm was replicated for the surfactants, SDS and TDS. Stocks of each surfactant were prepared in distilled water at concentrations of $\mathrm{3,200}\mathrm{mg}/\mathrm{L}$. Stocks were diluted in 0.3× Danieau’s solution to reach test dilutions of 32, 16, 8, 4, 2, 1, and $0.5\mathrm{mg}/\mathrm{L}$. An additional dose of $0.25\mathrm{mg}/\mathrm{L}$ was added to the TDS exposures. Each experiment was replicated thrice ($n=45$ embryos in total).

A smaller range of AFFF concentrations (2.20 to $4.40\times {10}^{-4}\%$ AFFF) were examined in transgenic strains to assess impacts of exposure on development of endoderm-derived tissues and in wildtype fish with exposures carried to 120 hpf to examine swim bladder inflation. The transgenic strains examined for target organ assessments included the Tg(gut:GFP) for liver development, Tg(ins:GFP) for endocrine pancreatic beta cell islet development, and Tg(ptf1a:GFP) for exocrine pancreas development. The wildtype strain AB was replicated to examine swim bladder inflation at 120 hpf. For these target organ assessments, 10 embryos at 3 hpf were group housed in $20\text{-mL}$ glass scintillation vials containing $10\mathrm{mL}$ of AFFF dilutions. Solutions were prepared and refreshed daily. Transgenic experiments were carried until 96 hpf, and the swim bladder inflation was assessed at 120 hpf. Three vials were prepared to test each AFFF dilution, and each experiment was replicated 2–3 times. Average measurements are reported for each vial ($n=6–9$ vials).

Our lab has previously reported that developmental exposure to $16–32\mathrm{\mu }\mathrm{M}$ PFOS reduced the growth of both the exocrine and endocrine pancreas in 96 hpf larvae (Sant et al. 2017). In the present study, the same a paradigm was followed to assess liver morphology in the Tg(gut:GFP) strain. Because the PFOS stocks were prepared in DMSO, an 0.01% DMSO control exposure was necessary. Embryo were exposed from 3 hpf to 96 hpf to 16 or $32\mathrm{\mu }\mathrm{M}$ PFOS, or 0.01% v/v DMSO. Solutions were prepared and refreshed daily. This experiment was replicated thrice ($n=9\text{\hspace{0.17em}vials}$).

Following exposures, larvae were thoroughly washed and imaged live. Larvae were anesthetized using 2% v/v MS-222, tricaine mesylate, and positioned in 3% methyl cellulose. Trans images for the morphological assessment of the nontransgenic larvae were captured using a Zeiss Axiozoom v16 microscope. Fluorescent tissues of the transgenic larvae were imaged using a customized Olympus upright microscope by Kramer Scientific and equipped with an Axiocam 503 camera (Carl Zeiss Inc.) and an 89 North® PhotoFluor® II light source. Images were analyzed using ZEN2 software, and larval length and yolk sac area were measured for all larvae.

Liver morphology was assessed through measurements of dorsal-ventral organ length which was normalized to individual body length. Similarly, exocrine pancreas anterior caudal length, which was measured from the depressed fluorescence of the endocrine beta cell islet to the pancreas tail, was normalized to individual body length. Stunted and severely stunted pancreatic phenotypes were determined using the thresholds of 10th and 1st percentiles of control pancreas lengths, respectively, as defined in Sant et al. (2019). The area of the primary endocrine pancreatic beta cell islets was measured, and qualitative assessment of islet structure was completed to stratify normal, tight structure from fragmented islets. The percentage of normal and fragmented morphologies was determined for the population of larvae in each vial, and averages of these are reported. All endoderm measurements and assessments were completed on blinded images.

Data are presented as $\text{mean}\pm \text{SEM}$, and figures were produced using either Excel or GraphPad Prism 8.2.1 software. Statistical analyses were completed using JMP14 software (SAS Institute Inc.), and $p<0.05$ was defined as significant. ${\text{LC}}_{50}$ values and 95% confidence intervals (CI) were determined through Probit analysis. Mortality curves were compared using parallelism tests of the Probit analysis. Statistical significance for developmental measurements was determined through one-way analysis of variance (ANOVA) with Tukey’s HSD post hoc test. A Fisher’s Exact test was used to determine significance of aberrant pancreas morphologies.

Of the 24 perfluoroalkyl compounds assessed using a modified EPA Method 537.1, 10 compounds were detected at quantifiable levels (Table 1). PFOS made up 79.2% of the detected PFAS, followed by PFHxS (12.6%) for a subtotal of 91.8% between these two compounds. Perfluorobutyl sulfonate (PFBS) and perfluoropentane sulfonate (PFPeS) (both at 1.9%), perfluoroheptane sulfonate (PFHpS; 1.3%), perfluorohexanoic acid (PFHxA; 1.1%) PFOA (0.9%), perfluoropentanoic acid (PFPeA; 0.4%), perfluoroheptanoic acid (PFHpA; 0.4%), and perfluorobutanoic acid (PFBA; 0.3%) made up the remaining 8.1% of the total quantified PFAS content. These 10 PFAS were also identified using the nontargeted Orbitrap HRMS method (Table 1). PFNS and PFDS were identified by the Orbitrap HRMS method but were not quantified by the modified EPA method because they were below the MDL. The dilution of the AFFF sample analyzed by Eurofins was too great to quantitate PFNS and PFDS because a 1:1,000 dilution was necessary to bring the PFOS and PFHxS concentrations within the working range of the instrument (Table 1). Perfluoro-1-hexanesulfonamide and N-(3-(dimethylamino)propyl)tridecafluorohexanesulfonamide were also identified by the Orbitrap HRMS method. None of the remaining 13 compounds of the 24 PFAS listed in the EPA method were detected by either approach.

An additional 100 PFAS with relative intensity percent values greater or equal to 0.1% of PFOS were detected using exact mass by Orbitrap HRMS (Table S1). Of these 100 PFAS, there were 21 unidentified fluorinated compounds with relative intensity percent values greater or equal to 1.0% of PFOS, representing the first 21 compounds in Table S1. However, the compounds were not assigned structures because the mzCloud software was not able to detect fragmentation patterns of known compounds in the library.

There were also 14 nonfluorinated compounds identified in the AFFF sample using the Orbitrap HRMS method (Table 2). SDS and TDS were the most abundant in the AFFF mixture.

Concentrations of the two most prevalent non-PFAS compounds found in the nontarget analysis, SDS and TDS, were determined through HRMS. SDS was present in the AFFF sample at a concentration of $574.8\mathrm{mg}/\mathrm{L}$, and TDS was present at a concentration of $496.4\mathrm{mg}/\mathrm{L}$.

Figure 1 depicts a breakdown of the composition of the 3% AFFF sample, assuming that the sample represents an accurate preparation of $3\mathrm{g}$ solids per $100\mathrm{mL}$ water. PFOS was the most abundant compound at $\mathrm{9,410}\mathrm{mg}/\mathrm{L}$ (31% of all the analytes measured in the AFFF). PFHxS was the second most abundant PFAS at $\mathrm{1,500}\mathrm{mg}/\mathrm{L}$ (5% of all the analytes measured in the AFFF). Eight other PFAS were quantified through the EPA Method 537.1, together at a concentration of $965.4\mathrm{mg}/\mathrm{L}$ (3% of all the analytes measured in the AFFF). Assessment of the relative peaks of the compounds detected in the nontarget analysis revealed SDS and TDS to be abundant non-PFAS compounds in the AFFF sample. These were quantified at $574.8\mathrm{mg}/\mathrm{L}$ SDS and $496.4\mathrm{mg}/\mathrm{L}$ TDS. Each surfactant comprised roughly 2% of the AFFF sample. The remaining 57% of the AFFF sample consisted of other PFAS and non-PFAS compounds identified, but not quantified, through nontarget analysis, as well as other compounds not detected in our methods.

The ${\text{LC}}_{50}$ of the AFFF mixture at 96 hpf was calculated to be $7.41\times {10}^{-4}\%$ AFFF (Figure 2A). The 95% CI were $6.88\times {10}^{-4}$ to $8.03\times {10}^{-4}\%$ AFFF. The relative concentrations of each compound in the AFFF ${\text{LC}}_{50}$ are summarized in Table 5.

From exposures to PFOS alone, an ${\text{LC}}_{50}$ of $31.03\mathrm{mg}/\mathrm{L}$ PFOS was calculated (95% CI: $\mathrm{23.15,42.99}\mathrm{mg}/\mathrm{L}$, Figure 2B). Test concentrations of PFHxS were too low to derive an ${\text{LC}}_{50}$ value. An ${\text{LC}}_{50}$ value of $29.63\mathrm{mg}/\mathrm{L}$ PFAS was determined for the PFOS/PFHxS mixture (95% CI: $\mathrm{24.18,36.14}\mathrm{mg}/\mathrm{L}$; Figure 2B). Although this value was slightly lower than the ${\text{LC}}_{50}$ of the mixture, the CI of the PFAS mixture and PFOS alone overlapped. However, exposures to PFOS and the PFOS/PFHxS mixture produced mortality curves that were statistically different from the AFFF mortality curve (PFOS: $p<0.0001$ for growth rate, inflection point, and upper asymptote; PFOS/PFHxS mixture $p=0.021$ for growth rate, $p=0.0001$ for inflection point, and $p=0.0004$ for upper asymptote).

Developmental toxicity was also assessed for the non-PFAS surfactants, TDS and SDS, (Figure 2C). The ${\text{LC}}_{50}$ of TDS was $0.66\mathrm{mg}/\mathrm{L}$ (95% CI: $0.59,0.74\mathrm{mg}/\mathrm{L}$), and the ${\text{LC}}_{50}$ of SDS was $3.67\mathrm{mg}/\mathrm{L}$ (95% CI: $3.09,4.38\mathrm{mg}/\mathrm{L}$). Neither compound produced a mortality curve statistically different from the mortality curve of AFFF ($p>0.05$ for growth rate, inflection point and upper/lower asymptote). These ${\text{LC}}_{50}$ values are summarized in Table 5. Taken together, the AFFF mixture was observed to be more toxic to the developing zebrafish than were exposures to any of the four individual constituents found in the highest concentrations in the AFFF sample.

Exposure to AFFF affected the development of the wildtype larvae, specifically in larval length (Figure 3). Larvae exposed to the highest concentration of $4.40\times {10}^{-4}\%$ AFFF had significantly reduced body lengths compared to control larvae ($p=0.027$). It is interesting to note that larvae exposed to the concentration of $4.40\times {10}^{-5}\%$ AFFF were significantly longer in body length than were control larvae ($p=0.020$). There was no significant difference in yolk sac area following AFFF exposure (Figure 3B). Additional exposures to AFFF were completed to 120 hpf to examine whether AFFF exposure impacted swim bladder inflation, yet no significant effects were observed (Figure S1).

Larvae exposed to the PFOS/PFHxS mixture had a significantly shorter body length at 96 hpf after exposure to $10.23\mathrm{mg}/\mathrm{L}$ PFAS (equivalent to the amount of combined PFOS and PFHxS in $2.81\times {10}^{-3}\%$ AFFF, $p=0.0043$) and $20.46\mathrm{mg}/\mathrm{L}$ PFAS (equivalent to $5.63\times {10}^{-3}\%$ AFFF, $p=0.0013$) (Figure 4A and Figure S2). These larvae also had a significantly larger yolk sac area after exposure to $10.23\mathrm{mg}/\mathrm{L}$ PFAS ($p=0.035$) and $20.46\mathrm{mg}/\mathrm{L}$ PFAS ($p=0.0001$) (Figure 4B).

Single exposures to PFOS and PFHxS were also completed to test their relative contribution to toxicity. Exposure to PFOS alone at $17.64\mathrm{mg}/\mathrm{L}$ (equivalent to the amount of PFOS in $5.63\times {10}^{-3}\%$ AFFF) resulted in significantly smaller larval length ($p<0.0001$) and larger yolk sac area ($p=0.0014$) (Figure 5 and S2). However, PFHxS up to $22.50\mathrm{mg}/\mathrm{L}$ (equivalent to the amount of PFHxS found in $4.50\times {10}^{-2}\%$ AFFF) was insufficient to affect either morphometric end point.

Larvae exposed to $0.5–2.0\mathrm{mg}/\mathrm{L}$ SDS and $0.25–0.5\mathrm{mg}/\mathrm{L}$ TDS, doses below their respective ${\text{LC}}_{50}\mathrm{s}$, appeared to not have different body lengths or yolk sac areas compared with control larvae (Figure 6 and Figure S3).

In the present study, developmental PFOS exposures were completed in Tg(gut:GFP) embryos through 96 hpf to provide a comparison with the morphological effects with AFFF exposure on liver development. Liver lengths were normalized to body length to account for the changes in growth observed in PFOS- and AFFF-exposed larvae. Developmental exposure to PFOS did not appear to affect the elongation of the liver (Figure 7A). In contrast, larvae exposed to AFFF had significantly smaller liver lengths at doses of $2.20\times {10}^{-4}\%$ AFFF ($p=0.050$) and $4.40\times {10}^{-4}\%$ AFFF ($p=0.0028$) in comparison with control larvae (Figure 7C). The liver length measurements were consistent with liver area measurements, where mean liver areas normalized to body length were 0.027, 0.026, and $0.021\mathrm{\mu }\mathrm{m}²/\mathrm{mm}$ for 0, $2.20\times {10}^{-4}$, and $4.40\times {10}^{-4}\%$ AFFF, respectively.

Morphological assessment of the exocrine pancreas and endocrine beta cell islets was completed in Tg(ptf1a:GFP) and Tg(ins:GFP) larvae, respectively, at 96 hpf. AFFF exposure did not result in any significant differences in beta cell islet area ($p=0.24$) or the morphology of the islet ($p=0.61$) (Figure 8A–C). Raw values are located in Table S2. Additionally, mean exocrine pancreas length did not differ with AFFF exposure in comparison with control larvae ($p=0.12$) (Figure 8D). However, at $4.40\times {10}^{-4}\%$ AFFF, there was a significant increase in the incidence of stunted ($<10\%$) and severely stunted ($<1\%$) exocrine pancreas phenotypes ($p=0.0073$, Figure 8E–F).