## 3 Toxicity/Toxicological Effects  
### Toxic Effects of PFAS in Organisms  
In recent decades, major studies on the toxicological effects of perfluoralkyl substances have focused on two of the first legacy compounds discovered, PFOS and PFOA, that are the most representative molecules for perfluoralkyl sulfonic acids (PFSAs) and perfluoralkyl carboxylic acids (PFCAs), respectively [50, 51, 52, 53, 54]. [PERSON] et al. [55] addressed, beside PFOA, PFHxA as a potential ecotoxicological compound for the freshwater microbial community. Statistical analysis showed that both PFOA and PFHxA at elevated doses considerably modified the composition of the microbial community. Moreover, statistical analysis demonstrated the more intense toxicity of PFOA, although unexpected outcomes were also found when PFHxA treatments were contrasted. PFOA reduced the biovolume of the bacteria, but it was unable to show that they reduced activity by monitoring cell respiration. An important factor to consider is the trophic transfer and bioaccumulation of PFAS. The study made by [PERSON] et al. [56] examined many locations in the Belgian portion of the Western Scheldt Estuary and the North Sea, sampling various fish and crustacean species. All matrices included only long-chain PFAS; fish muscle and liver tissue had seven chemicals, whereas crustaceans contained five. Regardless of the kind of fish or the location, liver tissue exhibited considerably higher PFAS levels than muscle tissue overall. The fact that the current study's PFOS concentrations in fish (_P. platessa_) and crustaceans (_C. crangon_ and _crab_ sp.) are lower than those of previous studies is a noteworthy finding. The most likely cause is the PFOS and PFOA phase-out performed by 3M in 2000 [57]. Several studies demonstrate a steady decline in PFOS levels throughout the biota. [PERSON] et al. [58] combined targeted and untargeted approaches to predict toxicity using LC-HRMS, LC-MS, and GC-MS. The study results in a strong positive relationship between body weight and liver damage for 13 PFASs. [PERSON] et al. [59] investigated dietary predictors of prenatal PFAS exposure and discovered seven of them in approximately 65% of subjects. The ingestion of dairy products at least once a week was modestly related to higher levels of PFNA and perfluorodecanoic acid (PFDeA). For dairy milk and cheese, the relationships were shown with PFDeA and PFNA, respectively. The level of PFOA, PFDeA, PFNA, and PFOS were found to correlate with the consumption of fish, chicken, and red meat at least once a week. The work of [PERSON] et al. [60] contributes to a better understanding of children's exposure to PFAS. In particular, 107 raw milk samples and 70 cow feed samples from nine Chinese provinces were analyzed in order to investigate the correlation between the level of PFAS of milk and the contamination of PFASs in milk and feed. The findings showed that children are more vulnerable to the risk of PFAS intake from milk than adults. The findings of the scientific community aim to assist institutions in defining limits and maximum levels of contamination and/or exposure that can be tolerated. The PFOS-induced transcriptional dose-response data were first collected by [PERSON] et al. [61] in both in vitro and in vivo human and animal studies. After the application of statistical models that were demonstrated to be feasible and reliable to perform a human health risk assessment, it was shown that the reference doses for the most sensitive diseases and pathways were below the EPA reference dose, but similar to the tolerable doses settled by EFSA. Cytotoxic effects in humans were also addressed by recent studies. [PERSON] et al. [62] investigated the correlation between structure-activity of linear PFASs and their toxicity, with a focus on their impact on hepatotoxicity in HepG2 cells. The data clearly demonstrate a relationship between the chemical structure of PFAS and their Reactive Oxygen Species (ROS) production and cytotoxic activities in HepG2 cells.