Valutazione della Ecotossicità del PFBA con Spirodela polyrhiza e Phaeodactylum tricornutum

Perfluoroalkyl substances (PFASs) are anthropogenic compounds known for their capacity of being persistent in both aquatic and terrestrial environments. Due to their resistance to degradation, they are often referred to as “forever chemicals”. These substances can accumulate in the tissues of living organisms and enter the food web through biomagnification processes, allowing them to be detected far from areas of production or emission. PFASs are widely used daily in household items, including non-stick cookware coatings (e.g. Teflon®), and waterproof fabrics (e.g. Goretex®). A noteworthy case of environmental contamination is that of the second-largest European aquifer, which was polluted by industrial discharges of PFASs, including chemicals such as C604 and GenX, from the Miteni plant located in Trissino (Vicenza, Italy). PFAS are also recognized for their adverse effects on human health. They act as endocrine disruptors and have been associated to testicular and kidney cancers, thyroid dysfunction, diabetes and various other pathologies. Furthermore, they may have potential impacts on ecosystems, assessed through targeted ecotoxicology studies on model organisms. This thesis investigates the effects of PFBA (perfluorobutanoic acid) on two model aquatic species: Spirodela polyrhiza (a freshwater macrophyte) and Phaeodactylum tricornutum (a marine microalga). Following the OECD 221 protocol (originally developed for Lemna minor), S. polyrhiza was exposed over a period of 7 days to five different concentrations of PFBA, plus a control. For each flask, growth inhibition indicators, including total frond number, total frond area, and total root length, were determined using the image analysis software ImageJ. Moreover, a colorimetric analysis of the fronds was performed, to determine an “greenness index”. Values at the end of the test (T1) were compared to those at the beginning (T0) to obtain the average growth rate (μ) for each tested concentration. This rate was then used to calculate the percentage of growth inhibition, which was analysed in relation to the PFBA concentrations. A statistically significant p-value (p = 0.02) was obtained for total root length, suggesting that PFBA may negatively affect the root growth in S. polyrhiza. The toxicity test on Phaeodactylum tricornutum was based on the US-EPA 96h Acute Toxicity Test protocol (OCSPP 850.4500), with appropriate modifications. P. tricornutum in exponential growth phase was exposed for 96 h to seven different PFBA concentrations, including a negative control. At the end of the exposure period, cell counts were performed to determine any potential growth inhibition effects. A parametric one-way variance analysis was performed in Microsoft Excel (© Microsoft 2025), which yielded a p-value of 0.01, indicating that at least one tested concentration sample was significantly different from the control group. To identify which concentrations were significantly different from the control, a two-tailed t-test with Bonferroni correction was applied as post-hoc test. However, all p-values obtained were well above the significance threshold adjusted using the Bonferroni correction, indicating that none of the tested PFBA concentration groups were significantly different from the control group.

I PFAS (molecole perfluoroalchiliche) sono sostanze di sintesi conosciute per la loro capacità di essere persistenti in ambiente, sia acquatico che terrestre, in quanto non biodegradabili, e sono definiti contaminanti perenni o eterni. Sono in grado di bioaccumularsi nei tessuti, e quindi di entrare ad ogni nodo della rete trofica, grazie ai fenomeni di biomagnificazione. Queste sostanze sono utilizzate quotidianamente in casa, come nei rivestimenti antiaderenti (Teflon®), e nei tessuti impermeabili (Goretex®). Non va dimenticata inoltre la contaminazione della seconda falda acquifera più grande d’Europa, in cui sono state riversate per decenni sostanze come C604 e Gen X, residui di produzioni industriali dell’azienda Miteni di Trissino (Vicenza). I PFAS sono conosciuti anche per i loro effetti nella salute umana: sono distruttori endocrini, possono essere correlati a tumori testicolari e renali, patologie tiroidee, e diabete. Inoltre, possono avere effetti diretti o indiretti sugli ecosistemi, valutati tramite studi mirati di ecotossicologia su organismi modello. In questo progetto in particolare, sono stati indagati gli effetti del PFBA su due specie: Spirodela polyrhiza (pianta di acqua dolce) e Phaeodactylum tricornutum (microalga marina). Basandosi sul protocollo OECD 221, S. polyrhiza è stata esposta per 7 giorni a 5 diverse concentrazioni, ottenute da diluizioni seriali di una soluzione di partenza di 1 g/L. Sono stati valutati diversi indicatori di inibizione della crescita sulla piantina, quali l’area delle fronde totale, e la lunghezza media delle radici per ogni flask (tramite software di analisi d’immagine ImageJ). Inoltre, è stata effettuata un’analisi colorimetrica delle fronde (tramite ImageJ), determinando un indice che può correlarsi al contenuto dei pigmenti fotosintetici. I valori alla fine del test (T1) sono stati confrontati con gli stessi all’inizio del test (T0). Per quanto riguarda Phaeodactylum tricornutum, il protocollo di riferimento utilizzato è stato il US-EPA 96 h Acute Toxicity Test protocol - OCSPP 850.4500, con opportune modifiche. P. tricornutum in fase esponenziale di crescita è stato esposto all’interno di pozzetti per 72 h a 6 diverse concentrazioni, ottenute da diluizioni seriali di una soluzione di partenza di 1 g/L. Alla fine del test, è stata effettuata una conta cellulare per determinare l’eventuale inibizione della crescita.