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Very little is known about the toxicity of an array of PFAs that can occur in the environment. The key challenge 455

in the risk assessment of PFAS lies in understanding the exposure to PFAS mixtures and the effect of those 456

mixtures. It is essential to identify as many PFAS to assess their toxicity in a high throughput manner. A key 457

factor in the assessment is whether the analytical methods used are sufficient to capture all the PFAS that are 458

likely to occur in the environment. A study confirmed the effects of 113 unique structures with novel 459

bioactivities, particularly RXRβ agonism [123]. In another study, developmental zebrafish are used to 460

evaluate and compare the toxicity of 139 PFAs libraries across 13 morphological endpoints and 2 behavioural 461

assessments [124]. The assessment of PFA toxicity can be done from targeted analyses, non-targeted analyses, 462

and surrogate indices or nonspecific methods. Targeted analysis always screens for known, specified species, 463

and new structures are absent from mass spectral libraries. In the targeted approach, the method is set up 464

depending on what is to be known and the analysis is done accordingly. In the non-targeted approach, 465

everything is analysed and then an assessment is done depending on the specific data extracted. The 466

assessment is done based on data extracted from the LC-ESI MS, MS/MS, HPLC MS/MS in negative ESI 467

mode quantifies f negatively charged acidic PFAS analytes, and positive mode ESI can be used for assessing 468

the neutral, cationic, or zwitter ionic PFAS chemicals [125]. Precursors of PFOS and PFOA can be assessed 469
by HPLC-MS/MS, both liquid and gas chromatography coupled with mass spectrometry. Neutral and volatile 470

PFAS in solids can be analysed by targeted GC/MS. GC–MS/MS can also be applied for determining volatile 471

telomeres [126]. Suspects (both known and unknowns) and non-targeted unknowns of novel molecular 472

structures require high-resolution mass spectrometry (HRMS) equipment such as quadrupole time-of-flight 473

(QToF) or orbitrap instruments. Fingerprinting MS signals is particularly applicable to PFAs families that 474

differ by a repeating chemical unit [127], for example, −CF2− for PFAS homologs and – CF2 CF2 for 475

fluorotelomers [128]. Non-targeted analyses by LC/ToF MS can identify per and polyfluoroalkyl ether acids 476

(PFEAs) in drinking water, and subsequent incorporation into a targeted analytical LC-MS/MS method for 477

assessment. Very low concentrations of complex PFAS mixtures are expensive and time-consuming to analyse; 478

nonspecific methods, sum parameters, surrogate indices, or total indices are used to identify the PFAS family. 479

For PFAS, the total oxidizable precursor assay (TOP or TOPA) and the total organic fluorine assay (TOF or 480

TOFA) are the most common surrogate indices. TOP and TOF reveal the presence of unidentified PFAS [129]. 481

The TOP assay is an indirect measure of the total oxidizable PFAA precursor resulting from unavailable or 482