Mechanistic basis of the reduced aqueous bioavailability of highly hydrophobic perfluoroalkyl phosphinic acids in fish

Perfluoroalkyl phosphinic acids (PFPiAs) are a persistent, understudied class of per- and polyfluoroalkyl substances (PFAS). In the first aqueous bioconcentration study of PFPiAs in fish, Chen, Gong and Kelly (2016) exposed adult zebrafish for 24 days and found extraordinarily large steady-state bioconcentration factors and uptake rate constants for most PFPiA homologs, but a marked attenuation of both parameters for the most hydrophobic homologs (C8/C10 and C6/C12, 18 perfluorinated carbons, log D_mw near 9). The authors could not determine whether this attenuation reflects reduced freely dissolved concentrations from sorption to dissolved or particulate organic matter, or steric/molecular-size limitation of gill membrane permeation, and the study determined neither PFPiA-specific partitioning coefficients under the exposure conditions nor biotransformation rate constants in fish. No subsequent study has measured PFPiA sorption to organic matter under exposure conditions, gill membrane permeation for PFPiAs, or PFPiA metabolic rate constants in fish, and no toxicokinetic model for fish covers the 18-perfluorinated-carbon PFPiAs at issue.

Background

Per- and polyfluoroalkyl substances (PFAS) are synthetic fluorinated chemicals; the best-known subgroups are the perfluoroalkyl carboxylates (PFCAs, e.g. PFOA) and perfluoroalkane sulfonates (PFSAs, e.g. PFOS), which are anionic, persistent, and proteinophilic surfactants. Perfluoroalkyl phosphinic acids (PFPiAs) are a less-studied PFAS subclass: dialkylated molecules bearing two perfluoroalkyl chains joined by a phosphinic acid head group, used since the 1970s as wetting and leveling agents in consumer products and as anti-foam additives in pesticide formulations. They have been detected in surface waters, wildlife, house dust, and human blood. The structurally related mono-chain perfluoroalkyl phosphonic acids (PFPAs) are their environmental degradation and biometabolic products.

For regulatory screening of aquatic contaminants, the bioconcentration factor (BCF, the ratio of the chemical concentration in fish to that in water at steady state) is the central bioaccumulation metric, and it is commonly predicted from octanol-water partitioning ($K_{ow}$). That framework fails for ionized, amphiphilic PFAS because these anions partition preferentially into proteins and phospholipids rather than neutral storage lipids. PFAS-specific descriptors were therefore introduced, notably the membrane-water distribution coefficient ($D_{mw}$, the ratio of a chemical's concentration in a phospholipid membrane phase to that in water) and the protein-water partition coefficient ($K_{pw}$, the analogous ratio for a protein phase). Uptake from water occurs across the fish gill; for extremely hydrophobic ('superhydrophobic') neutral organics it has long been known that bioconcentration can decline at very high hydrophobicity because membrane permeation becomes diffusion-limited and because sorption to dissolved organic matter (DOM, the humic and other macromolecular material in natural water) lowers the freely dissolved concentration available for uptake. Whether analogous mechanisms control ionogenic PFAS uptake is far less established.

Against this background, Lee, De Silva and Mabury (2012) showed in juvenile rainbow trout that PFPiAs undergo dietary exposure with biomagnification factors (BMF, the fish-to-food concentration ratio) below 1 and evidence of metabolic conversion. Chen, Gong and Kelly (2016) then performed the first aqueous (flow-through, 24-day uptake, 24-day depuration) bioconcentration study of PFPiAs and PFPAs in adult female zebrafish, alongside long-chain PFCAs and PFSAs. They reported extremely large steady-state BCFs and the largest uptake rate constants ($k_u$) among the tested compounds for PFPiAs such as C6/C10, positive log-log correlations of BCF with $D_{mw}$ and $K_{pw}$, and in vivo biotransformation of PFPiAs to PFPAs that was nevertheless insufficient to offset uptake. Crucially, for the most hydrophobic homologs tested - the 18-perfluorinated-carbon C8/C10 and C6/C12 PFPiAs, with estimated log $D_{mw}$ near 9 - both BCF and $k_u$ fell markedly below the monotonic trend established by the less hydrophobic homologs, breaking the linear log BCF versus log $D_{mw}$ relationship that held up to log $D_{mw}$ of about 8.

The authors interpreted this attenuation as most consistent with reduced aqueous bioavailability (sorption to dissolved or particulate organic matter in the exposure water, or solute-solute interactions lowering the freely dissolved concentration) and/or reduced gill membrane permeability from molecular size or steric hindrance, explicitly excluding increased depuration or enhanced metabolism as explanations on the basis of their kinetic data. However, the study measured none of the quantities needed to discriminate among these mechanisms: no DOM sorption coefficients, no size-resolved particle partitioning, no membrane permeability assays, and no metabolic rate constants ($k_m$) were determined. In addition, PFPiAs had to be quantified semi-quantitatively (using the response of a C10 PFPA relative to a $^{13}$C$_2$-PFDoA surrogate) because isotope-labeled PFPiA standards were unavailable, and existing physiologically based toxicokinetic (PBTK) models for fish - which describe uptake, distribution, metabolism, and excretion as physiological flows and partitioning processes - rely on $K_{ow}$-based partitioning relationships unable to represent amphiphilic PFAS partitioning into phospholipids, protein binding, or organic anion transporters. The mechanistic question therefore stands: what physically causes the most hydrophobic PFPiAs to be less bioavailable from water than their hydrophobicity would predict?

Problem Statement

Determine, with quantitative measurements under aqueous exposure conditions, the mechanism or combination of mechanisms responsible for the reduced bioavailability and attenuated uptake rate constants and bioconcentration factors of the most hydrophobic perfluoroalkyl phosphinic acids (the 18-perfluorinated-carbon homologs C8/C10 and C6/C12, with estimated log $D_{mw}$ near 9) in fish, relative to the monotonic increase of bioconcentration with hydrophobicity displayed by less hydrophobic PFASs. Specifically: (i) measure PFPiA sorption to dissolved and particulate organic matter and the resulting freely dissolved fractions in exposure water, so that reduced aqueous availability can be separated from organism-side limitation; (ii) measure gill membrane permeation of these homologs and its steric or molecular-size limitation, so that the membrane-resistance hypothesis can be quantified; and (iii) quantify metabolic transformation rate constants ($k_m$) and tissue-specific pathways of PFPiA-to-PFPA conversion in fish, confirming or refuting the exclusion of enhanced metabolic elimination as the cause. Because log $D_{mw}$ near 9 lies outside the validated domain of existing predictor equations, these equations do not yield values of the partitioning descriptors ($D_{mw}$, $K_{pw}$) for these homologs.

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