Assessment of the Possible Inhibitory Effect of PFAS-Containing Aqueous Wastes on Aerobic Biomasses

Abstract

Per- and polyfluoroalkyl substances (PFASs), known as “forever chemicals,” are synthetic organofluorine compounds widely used since the 1940s due to their chemical and thermal stability. However, growing concerns about their environmental and human health risks have emerged. Although the toxicity of PFASs to humans has been extensively researched, their effects on microbial consortia in wastewater treatment plants (WWTPs) have not been as thoroughly investigated. This study evaluates whether aqueous wastes (AWs) containing PFASs inhibit aerobic biomasses from various WWTPs. Approximately 400 respirometric tests showed no acute toxicity. However, biomass tolerance varied based on acclimatization. Biomass from a municipal WWTP was more tolerant to AWs with short-chain PFASs, whereas biomass from a WWTP authorized to receive AWs was less inhibited by AWs rich in long-chain PFASs. These findings highlight the potential role of municipal WWTPs in treating PFAS-contaminated AWs and emphasize the need for tailored treatment strategies to minimize environmental risks.

1. Introduction

Poly- and perfluoroalkyl substances (PFASs) are a broad family of fluorinated organic compounds of anthropogenic origin, characterized by a common aliphatic carbon back-bone, in which hydrogen atoms have been partially (prefix: poly-) or completely (prefix: per-) replaced by fluorine atoms [1,2,3,4]. These synthetic chemicals have molecular structures that include at least one perfluoroalkyl chain –CnF2n+1 (n ≥ 1), which is both hydrophobic and lipophobic, contributing to their high chemical and thermal stability [1,2,3,5] as well as hydrophilic groups like -COOH and SO₃H. PFASs are very stable substances due to their strong, highly polar carbon–fluorine (C–F) bonds. This stability gives them several properties that are highly valued in industrial applications: they are resistant to heat, water, oil, grease, and friction and exhibit anti-adherent and fire-retardant properties [6,7,8]. Following their synthesis in the late 1930s, PFASs have been widely used since the 1940s in the manufacturing of several everyday consumer goods, including non-stick cookware, grease-proof food packaging, waterproof and stain-resistant textiles, adhesives, printing inks, paper products, pharmaceuticals, personal care products, and household products such as cleaning agents and floor wax [9,10]. They have applications in the chemical industry for manufacturing surfactants, emulsifiers, lubricants, coating additives, pesticides, and herbicides, as well as in the metal plating and semiconductor industries [1,2]. Moreover, PFASs are used in the manufacturing of fire-retardant agents and aqueous film-forming foams (AFFFs) for fire-fighting operations [8,9,10]. Finally, they are used as erosion inhibitors in the aviation industry and can also be found in electronic, photographic, and military applications [6].

Due to the remarkable strength and chemical inertness of the C–F bond, PFASs are notable for their lasting presence in the environment and ability to accumulate biologically. This characteristic accounts for their designation as “forever chemicals” [11]. PFOS, PFOA and PFHxS are the three subgroups of PFASs currently listed under the Stockholm Convention as persistent organic pollutants (POPs). Because of the global proliferation of PFASs, governments are becoming more concerned about controlling their exposure and dissemination, which has led to the establishment of PFASs guideline threshold values in a number of nations [12]. The regulatory values of PFASs are influenced by scientific, technical, social, political, and economic aspects, making their regulation complicated and difficult [12,13]. Moreover, since PFASs are emerging contaminants, guidelines are constantly evolving to account for the developing knowledge. As a result, there is considerable variation among the guidelines established by different countries worldwide, making it challenging to define unified standards [12]. However, recent studies indicate that exposure to PFASs leads to a series of adverse health effects, including several cancers (i.e., of the kidney and testes), liver disease, kidney disease, cardiovascular disease, respiratory disease, immune system dysfunction, decreased fertility, endocrine disruption, increased cholesterol levels, diabetes, osteoarthritis, and neurological disorders [9,10,11]. It follows that all PFASs regulations and guidelines have the safety of human health as their primary objective [12].

PFASs enter wastewater treatment plants (WWTPs) through sewer systems, and WWTPs will be collecting more and more PFASs due to their high stability against chemical and biological degradation processes [14,15]. Therefore, industrial and municipal WWTPs are deemed as the most common point sources of the release of PFASs into the environment through both effluent and sludge disposal [16]. It has been demonstrated that PFASs can be adsorbed onto activated sludge [16]. Consequently, the reuse of highly contaminated WWTP sludge in farmlands contributes to soil contamination [17,18]. Over recent years, several technologies have been developed to remediate PFASs from water and wastewater sources. In this context, Phong Vo et al. (2020) observed that a complex PFASs remediation system should include sequential matching of separation/concentration technologies and destruction technologies [19]. The technologies in the first category, which include adsorption, stabilization–solidification, and membrane filtration, are designed to isolate and concentrate PFASs. The second category of technologies, which include Advanced Oxidation Processes (AOPs), as well as thermal, sonochemical, biological processes and plasma, are designed for the defluorination of condensed PFASs solute. Nowadays, the traditional activated sludge process (ASP) is used in most WWTPs. Since a portion of the microorganisms involved in biological wastewater (WW) treatment processes are sensitive to pollutants, certain toxic substances may have adverse effects on them [20]. Unfortunately, the impact of PFASs on mixed microbial consortia has not yet been as broadly studied as the toxic effects on humans [15]. Despite this, some experimental studies on the impact of PFASs on activated sludge have been published in recent years. For example, Yu et al. (2018) found that the long-term exposure—Hydraulic Retention Time was set at 48 h—to a high concentration level (20 mg L⁻¹) of PFOA promoted shifts in microbial community structure towards more PFOA-tolerant species [15]. Moreover, Sheng et al. (2021) found that PFOS caused oxidative damage and destroyed the integrity of microbial cell membranes after 8 h of acute exposure, indicating that PFOS caused cytotoxicity in activated sludge [21].

Respirometry, which is defined as the measurement and interpretation of the rate at which O₂ (or other substrates, such as nitrates) is consumed by a microbial community under well-defined experimental conditions, can provide important information concerning bacterial metabolism, substrate removal, and toxicity issues [22,23,24,25]. It is a very useful tool for managing the biological processes in WWTPs because it makes it possible to assess a possible inhibitory influence on the biomass of the WWTP by characterizing the biodegradability of the influent WW [26,27]. The Oxygen Uptake Rate (OUR) is one of the most important parameters for estimating the microbial activity in biological processes. The use of the OUR measurement for process-monitoring is helpful because it can respond to fluctuations in influent loading rate and disturbance caused by inhibiting/toxic substances [28]. Variations in specific Oxygen Uptake Rate (sOUR), defined as the ratio between dissolved oxygen consumption from biomass and volatile suspended solids (VSS) concentration, can indicate biomass inhibition caused by toxic compounds [27]. More specifically, an exogenous sOUR value lower than the endogenous sOUR value may serve as a warning sign of biomass inhibition due to the presence of toxic substances in the influent sewage. Using respirometric assays is a quick and reliable way to determine whether a wastewater stream is compatible with the biomass that is causing it to degrade. In particular, compared to other potential tests for evaluating biodegradability, OUR (Oxygen Uptake Rate) tests yields results that process engineers can use right away for treatment scheme design and plant operation management. Furthermore, because OUR values are intrinsically respirometric, they are easily compared to the outcomes of standard biodegradability tests (such as the OECD test series).

This work aims to evaluate the potential inhibitory effect of real PFAS-containing aqueous wastes (AWs) on the aerobic mesophilic biomasses taken from real WWTPs of various types located in Italy. To achieve this, approximately 400 OUR tests were conducted using three real aqueous wastes (AWs) consisting of industrial process waters particularly rich in PFASs. Both short-term and medium/long-term toxic effects were investigated through single-OUR tests and multi-OUR tests, respectively. The authors believe that this study will be useful to the scientific community by providing insights for future research and will assist water utilities by providing valuable information to improve understanding of the issue of PFASs in WWTPs. Existing studies have not clarified the differences in the response of activated sludge with different acclimatization backgrounds to short- and long-chain PFASs. This study compares municipal activated sludge (B-1 biomass) and industrial-tolerant activated sludge (B-2 biomass), coupled with single/multiple OUR tests, to reveal the relationship between PFASs types and biomass acclimatization.

2. Materials and Methods

2.1. Oxygen Uptake Rate Test

The laboratory respirometric apparatus employed in this study, shown in Figure 1, is like that used by Borzooei et al. [29] and Capodici et al. [30]. It includes a flask, a magnetic stirrer with a magnetic stir bar, a dissolved oxygen (DO) probe (also called “oximeter”), a personal computer (PC), a mechanical aerator, a porous stone, and sealing film. Since multi-OUR tests measure an entire respirogram made up of several subsequent sOUR measurements [31], the need to provide consecutive aeration periods requires a system automation. DO concentration was measured by the WTW Multi-parameter portable meter Multi-Line^® Multi 3510 IDS thanks to WTW Optical IDS dissolved oxygen sensors FDO^® 925 (Xylem Analytics Germany Sales GmbH and Co, Mainz, Germany) (called “DO probe” in the next sections). The measured DO concentration was transferred to a PC via USB connection and the MultiLab^® Importer for data acquisition via Excel^® software was used.

2.1.1. Single-OUR Test

The speed of execution, low instrumentation requirements, and rapid performance of single-OUR tests represent significant advantages [31]. The single-OUR test execution method is described below [32]. A known volume—400 mL in the presented work—of biomass is poured into a flask, which is placed on a magnetic stirrer in order to keep the sample well mixed during the test. Then, the biomass sample is aerated until DO saturation thanks to the porous stone connected to the mechanical aerator. Only in the case of an exogenous OUR test, after turning off the aeration, an equivalent volume of an external substrate is poured into the flask, together with the biomass. To avoid air exchange with the external environment, the tested volume is isolated with sealing film. DO concentration (mgDO L⁻¹) is measured by the DO feeler, which transfers the data to the PC, where values are recorded on an Excel spreadsheet at 5 s intervals. The decreasing trend of DO concentration over time is plotted in a scatter chart through the calculation program on Excel. The non-specific OUR (mgDO L⁻¹ h⁻¹) is calculated as the slope of the linear regression line which interpolates the measured data [31]. Then, sOUR (mgDO gVSS⁻¹ h⁻¹) is calculated as the ratio of the non-specific OUR to the VSS concentration in the batch reactor (gVSS L⁻¹). sOUR indicates the oxygen consumption rate of the biomass with respect to the degradation of the organic matter contained in the substrate alone, i.e., it is purified by the endogenous portion. If necessary, the non-specific OUR value shall be normalized at a temperature (T) of 20 °C using the following formula (valid for T values between 15 °C and 25 °C):

OUR_{(20 °C)} = OUR ∙ θ^{(20 − T)}

(1)

where θ is equal to 1.05 if T < 20 °C and to 1.07 if T > 20 °C.

In this study, the endogenous OUR tests were conducted with 400 mL of biomass to study endogenous respiration alone. Then, in exogenous OUR tests, 400 mL of biomass have been mixed with 400 mL of a substrate characterized by a certain PFASs concentration. In order to obtain more reliable results, each type of test has been replicated three times, so as to be able to consider the average value between different measurements carried out under the same experimental conditions. Finally, for each experimental condition, the standard deviation and the 95% confidence interval were calculated.

2.1.2. Multi-OUR Test

While single-OUR tests only take less than 20 min, multi-OUR tests offer the possibility of evaluating inhibitory effects during a period of the same order of magnitude of the hydraulic retention time (HRT) of the real scale bioreactor [31]. Therefore, multi-OUR tests provide additional information compared to single-OUR tests, allowing us to evaluate potential medium/long-term toxic effects and pollutants-removal yields.

At the end of a multi-OUR test, a sequence of DO consumption curves is obtained due to the alternation of non-aeration and aeration phases. The same process described for the single-OUR test leads to the calculation of several subsequent sOUR values (mgDO gVSS⁻¹ h⁻¹). The sOUR trend over time is represented by a curve called a “respirogram”. Moreover, the oxygen consumed during the test, which is indicated as “overall ΔDO” (mgDO gVSS⁻¹) in the next sections, can be estimated.

In this study, multi-OUR tests were carried out with a laboratory scale reactor containing 800 mL of biomass and 800 mL of substrate. The aeration was kept between 2 and 5 mgDO L⁻¹ thanks to the connection of the aeration system to an electric mechanism that guaranteed the connection-detachment of the aeration itself. The electrical mechanism was regulated by a software installed on the PC and through the DO concentration measured by the DO probe. The latter was in turn connected to the PC. The data acquisition system was crucial in enabling the PC to obtain the DO data that was measured in the reactor.

2.2. Tested Biomasses

In this study, two different types of aerobic mesophilic biomasses coming from various types of WWTPs located in Lombardy, northern Italy, were tested:

• Biomass B-1: conventional activated sludge (CAS) of a municipal WWTP which treats municipal sewage only (namely: WWTP-1), with a mainly domestic contribution. WWTP-1 capacity: 100,000 PE (Population Equivalent);
• Biomass B-2: CAS of a municipal WWTP which is authorized to receive industrial AWs (namely: WWTP-2). WWTP-2 capacity: 60,000 PE.

The optimal working temperature of these ASPs is approximately 20 °C. In the oxidation tank, the VSS concentration was between 3 and 4 gVSS L⁻¹.

The configuration of the WWTPs from which the biomasses listed above have been sampled is schematically shown in Figure 2. The main difference between these WWTPs is the type of influent WW to be treated: WWTP-1 treats urban WWs only, while WWTP-2 also treats AWs. Moreover, WWTP-1 is characterized by a post-denitrification solution, while in WWTP-2 there is a pre-denitrification scheme.

2.3. Tested Substrates

In this study, three real aqueous wastes (AWs) containing per- and polyfluoroalkyl substances (PFASs) were tested to evaluate their potential inhibitory effects on aerobic biomasses, as described in the previous section. For comparison purposes, tests were also performed using the influent wastewater (WW), to which the biomass is typically acclimated, as well as a reference blank consisting of casein peptone mixed with distilled water (DW). These three AWs were chosen because they represent typical PFAS compositions in industrial wastewater: AW-1 represents the long-chain PFASs (PFOA/PFOS) found in the highest concentrations in the aqueous waste market, while AW-2/3 represent short-chain PFASs (PFBS), ensuring that the results are relevant to the treatment of different types of industrial wastewater. The main characteristics of the AWs and WWs are summarized in

Table 1. PFAS-containing AWs, and influent wastewater characterization.

		PFAS-Containing Aqueous Wastes			Influent Wastewaters
Parameter	U.M. 1	AW-1	AW-2	AW-3	WW-1	WW-2
COD	mgCOD L−1	2333	3300	1350	100–200	800–1500
BOD5	mgBOD5 L−1	400	1730	460	80–100	600–1100
pH	-	8.00	7.80	8.09	6.50–7.50	6.50–7.50
PFBS 2	μg L−1	2.191 × 101	3.757 × 101	1.587 × 101	<0.05	<0.05
PFOA 3	μg L−1	2.870 × 103	2.414 × 101	2.340 × 100	<0.05	<0.05
PFOS 4	μg L−1	1.484 × 102	7.880 × 10−1	8.400 × 10−2	<0.05	<0.05
Sum of PFASs	μg L−1	3.268 × 103	7.870 × 101	2.638 × 101	<0.05	<0.05

¹ Unit of measurement. ² Perfluorobutanesulfonic acid (PFBS) is a PFAS compound with a four-carbon fluorocarbon chain and a sulfonic acid functional group. Chemical formula: C₄HF₉O₃S. CH₄F₉-SO₃H. ³ Perfluorooctanoic acid (PFOA) is a perfluorinated carboxylic acid characterized by an eight-carbon chain structure. Chemical formula: C₈HF₁₅O₂. C₇F₁₅COOH. ⁴ Perfluorooctanesulfonic acid (PFOS) is a perfluoroalkyl substance with an eight-carbon fluorocarbon chain and a sulfonic acid functional group. Chemical formula: C₈HF₁₇O₃S. C₈F₁₇-SO₃H.

. Since the influent WWs were sampled on different days, a range of values is reported for WW-1 and WW-2 instead of a single point value. Liquid wastes, as well as environmental matrices in general, are complex mixtures composed of numerous organic and inorganic substances. This research is based on the application of respirometric assays aimed at evaluating the overall effects exerted by the entire mixture. The attribution of specific effects to individual compounds or substance classes would require an Effect-Directed Analysis (EDA) approach, which involves fractionating the mixture and exposing biological systems (from single cells to whole organisms) to each fraction. Although EDA is occasionally employed for targeted purposes, the present study focuses instead on evaluating the treatability of PFAS-containing leachates in conventional biological treatment systems, assessing their impact at the mixture level rather than isolating individual compound effects.

2.4. Methodological Approach

Figure 3 shows the methodological approach adopted in this study. It consists of three main steps—namely, A, B, and C—which are themselves divided into “sub-steps”, as shown schematically in

Table 2. Details about the methodological approach adopted in this study.

STEP	SUB-STEP	Amount of Performed Tests	Type of Performed Tests	Tested Biomasses	Tested Substrates
A	A(I)	68	Single-OUR tests	B-2	AW-1 WW-2
	A(II)	4	Multi-OUR tests	B-2	AW-1 WW-2
B	B(I)	144	Single-OUR tests	B-1 B-2	AW-1 AW-2 AW-3 WW-1 (only for B-1) WW-2 (only for B-2)
	B(II)	8	Multi-OUR tests	B-1 B-2	AW-1 AW-2 AW-3 WW-1 (only for B-1) WW-2 (only for B-2)
C	C(I)	144	Single-OUR tests	B-1 B-2	AW-1 AW-2 AW-3 Casein peptone
	C(II)	6	Multi-OUR tests	B-1 B-2	AW-1 AW-2 AW-3 Casein peptone

. Within each step, both single-OUR tests and multi-OUR tests were carried out in sub-step X(I) and in sub-step X(II), respectively, where the letter “X” represents the main step A, B, or C.

Table 2 provides a detailed overview of the types and quantities of all tests conducted in this study, including whether each test was single- or multi-OUR, as well as the biomass and raw substrates used. Each single-OUR test lasted less than 20 min. Due to this short duration, single-OUR tests enabled an immediate assessment of the potential short-term toxic effects of the PFAS-containing substrate on the biomass. Multi-OUR tests conducted during steps A and B lasted 12 h, allowing for the evaluation of potential medium-term inhibitory effects. Meanwhile, those performed in step C lasted 5 days, enabling the assessment of long-term inhibitory effects.

This study sequentially verifies the inhibitory effects of PFASs through three experimental steps: Step A preliminarily explores the impact of COD on respiration rate, Step B controls COD to eliminate interference, and Step C applies raw concentration AWs to assess the tolerance of sludge to short- and long-chain PFASs. The results of each step are presented below.

3. Results and Discussion

3.1. Step A

In the first step of the experimental activity, OUR tests were performed by dosing AW-1 mixed with the WWTP influent or with DW, regardless of COD concentration. Only the biomass sampled in the WWTP which is authorized to receive industrial AWs, i.e., biomass B-2, was tested in this step.

3.1.1. Sub-Step A(I)

Within sub-step A(I), single-OUR tests were performed with biomass B-2 and AW-1 mixed with the WWTP-2 influent, i.e., WW-2, or with DW, regardless of COD concentration. Figure 4 shows the results of these tests. All exogenous sOUR values are higher than the endogenous values, indicating that no acute short-term toxicity effects occurred.

Oxygen consumption is related to the COD of the fed substrate [33]. The addition of AW-1 to WW-2 results in increased oxygen consumption kinetics because the high COD concentration of AW-1 increases the overall COD concentration of the mixture consisting of AW-1 and WW-2 together. In more detail, the substrate consisting of 100% WW-2 had a COD of 915 mg L⁻¹, while the substrate consisting of 96% WW-2 and 4% AW-1 had a COD of 972 mg L⁻¹, and the substrate consisting of 90% WW-2 and 10% AW-1 had a COD of 1057 mg L⁻¹. On the other hand, substrates consisting of AW-1 mixed with DW correspond to low sOUR values, due to low values of COD. The substrate consisting of 96% DW and 4% AW-1 had a COD of 93 mg L⁻¹, while the solution consisting of 90% DW and 10% AW-1 had a COD of 233 mg L⁻¹.

3.1.2. Sub-Step A(II)

Table 3. Results of multi-OUR tests carried out within sub-step A(II). These tests were performed with a laboratory scale reactor containing 800 mL of biomass B-2 and 800 mL of substrate (mixture of AW-1 and WW-2 or AW-1 and DW). The BOD₅/COD ratio and PFASs concentration of the tested substrate are also reported.

Tested Biomass	Tested Substrate			Overall ΔDO [mgDO gVSS−1]
	Composition	BOD5/COD [-]	Sum of PFASs [mg L−1]
B-2	10% AW-1 + 90% WW-2	0.65	0.327	114.15
	4% AW-1 + 96% WW-2	0.87	0.131	71.20
	10% AW-1 + 90% DW	0.17	0.327	92.68
	4% AW-1 + 96% DW	0.17	0.131	68.57

shows the results of multi-OUR tests carried out within sub-step A(II). As the percentage of AW contained in the tested substrate decreased, there was a considerable decrease in oxygen consumption, probably due to the lower COD content, as explained above for single-OUR tests. Again, the substrate consisting of DW and AW-1 was found to be less biodegradable compared with the substrate consisting of WWTP-2 influent (i.e., WW-2) mixed with AW-1.

Therefore, it is clear how the COD of the tested substrate plays a fundamental role in respirometric tests, as confirmed by Ziglio et al. [33]. Consequently, in step B, respirometric tests were performed by dosing a substrate consisting of the WWTP influent mixed with the AW pre-mixed with the WWTP effluent so that it had a COD concentration close to that of the WWTP influent, in order to exclude the influence of the variability of the COD of the tested substrate.

3.2. Step B

In the second step of this study, OUR tests were performed by dosing a substrate consisting of the WWTP influent (WW-1 or WW-2) mixed with the aqueous waste (AW-1, AW-2, or AW-3) pre-mixed with the WWTP effluent so that it had approximately the same COD concentration as the WWTP influent. In this step, the biomass taken from the WWTP, which is authorized to receive industrial AWs, i.e., biomass B-2, and the biomass sampled in the municipal WWTP which treats municipal sewage only, i.e., biomass B-1, were both tested for comparison.

3.2.1. Sub-Step B(I)

Within sub-step B(I), single-OUR tests were performed by dosing a substrate consisting of the WWTP influent (WW-1 or WW-2) mixed with the AW (AW-1, AW-2, or AW-3) pre-mixed with the WWTP effluent so that it had a COD concentration close to that of the WWTP influent. The results of these tests can be observed in Figure 5.

Biomass B-2 shows significantly higher oxygen consumption kinetics compared to biomass B-1, which is only exposed to municipal sewage. On the contrary, biomass B-2 is also acclimatized to pre-treated AWs. However, no inhibitory effects are observed, since exogenous sOUR values are always greater than the endogenous values for both biomasses.

With reference to Figure 5b, the substrate consisting of 100% AW is less biodegradable than the mixture consisting of AW and WWTP influent. This is confirmed by the BOD₅ measurements carried out in this step: WW-2 had a BOD₅/COD ratio equal to 0.78, while solutions consisting of 100% AW-1, AW-2, and AW-3 pre-mixed with WWTP-2 effluent were characterized by a BOD₅/COD ratio equal to 0.19, 0.22, and 0.30, respectively. On the other hand, considering Figure 5a, it can be stated that the BOD₅/COD ratio does not vary significantly: WW-1 had a BOD₅/COD ratio equal to 0.70, while solutions consisting of 100% AW-1, AW-2, and AW-3 pre-mixed with WWTP-1 effluent were characterized by a BOD₅/COD ratio equal to 0.96, 0.90, and 0.72, respectively.

3.2.2. Sub-Step B(II)

Table 4. Results of 8 h multi-OUR tests carried out within sub-step B(II). These tests were performed with a laboratory scale reactor containing 800 mL of biomass and 800 mL of substrate. The BOD₅/COD ratio and PFASs concentration of the tested substrate are also reported.

Tested Biomass	Tested Substrate			Overall ΔDO [mgDO gVSS−1]
	Composition	BOD5/COD [-]	Sum of PFASs [mg L−1]
B-1	WW-1	0.70	<5 × 10–5	17.73
	AW-1 1	0.96	0.004	29.44
	AW-2 1	0.90	0.004	36.07
	AW-3 1	0.72	0.002	27.49
B-2	WW-2	0.78	<5 × 10–5	93.82
	AW-1 2	0.19	0.930	57.21
	AW-2 2	0.22	0.020	76.48
	AW-3 2	0.30	0.010	58.83

¹ Pre-mixed with WWTP-1 effluent so that it had a COD concentration close to that of WW-1. ² Pre-mixed with WWTP-2 effluent so that it had a COD concentration close to that of WW-2.

shows the results of 8 h multi-OUR tests carried out within sub-step B(II). As observed for single-OUR tests, biomass B-2 shows a higher oxygen consumption com-pared to biomass B-1: biomass B-1 needs to acclimatize to these substrates. Moreover, it can be noted that there is a link between the BOD₅/COD ratio and the overall oxygen consumption recorded during the test: in general, the latter is higher when the BOD₅/COD ratio of the substrate is higher.

In this step, by diluting the AWs with the WWTP effluent, the concentration of PFASs in the tested substrate was very low, i.e., 0.002–0.004 mg L⁻¹ for tests carried out with bio-mass B-1 and 0.010–0.930 mg L⁻¹ for tests carried out with biomass B-2. Therefore, in order to effectively assess the presence of a potential inhibitory effect caused by PFASs on microorganisms, in step C, raw (i.e., not pre-mixed) AWs were tested.

3.3. Step C

In the third and last step, OUR tests were carried out by dosing AW-1, AW-2, and AW-3 mixed with casein peptone (nutrient) pre-mixed with DW so that it had approximately the same COD concentration as the raw aqueous waste. Even within this step, biomass B-2 and biomass B-1 were both tested for comparison.

3.3.1. Sub-Step C(I)

Within sub-step C(I), single-OUR tests were carried out by dosing AW-1, AW-2, and AW-3 mixed with casein peptone pre-mixed with DW so that it had a COD concentration close to that of the AW. In more detail, AW-1, which had a COD equal to 2333 mg L⁻¹, was compared to a reference blank for biomass feeding, i.e., a solution consisting of casein peptone pre-mixed with DW and characterized by a COD equal to 2500 mg L⁻¹. Analogously, AW-2, which had a COD equal to 3300 mg L⁻¹, was compared to a reference blank for biomass feeding characterized by a COD equal to 2930 mg L⁻¹. Lastly, AW-3, which had a COD equal to 1360 mg L⁻¹, was compared to a reference blank for biomass feeding, characterized by a COD equal to 1950 mg L⁻¹.

The results of these tests can be observed in Figure 6. As previously seen, biomass B-1 shows significantly lower oxygen consumption kinetics compared to biomass B-2, which is already acclimatized to pre-treated AWs. When the substrate consisting of 100% AW was tested, the lowest oxygen consumption values were recorded. However, these exogenous sOUR values were still higher than the endogenous sOUR values, excluding the presence of any acute toxicity effects. Moreover, it can be noted that biomass B-1 seems to be less inhibited by substrates containing up to 60% of AW-2 or AW-3, while B-2 tolerates better those containing up to 80% of AW-1. In this context it is important to focus the attention on the main PFAS compound contained in each AW rather than the overall parameter “Sum of PFASs”. PFASs are frequently referred to as “long-chain” or “short-chain” [3]. The definition provided by the Organisation for Economic Co-operation and Development (OECD) states that:

• Long-chain PFASs include (I) perfluorocarboxylic acids (PFCAs) and their precursors with carbon chain lengths ≥ C7 (including perfluorooctanoic acid (PFOA)) and (II) perfluoroalkane sulfonic acids (PFSAs) and their precursors with carbon chain lengths ≥ C6 (including perfluorohexane sulfonic acid (PFHxS) and perfluorooctane sulfonic acid (PFOS)).
• Short-chain PFASs include (I) PFCAs with carbon chain lengths < C7 and (II) PFSAs with carbon chain lengths < C6.

It should be noted that the classification of PFASs into “long-chain” and “short-chain” categories based on carbon chain length applies specifically to perfluoroalkyl carboxylic acids (PFCAs), perfluoroalkane sulfonic acids (PFSAs), and their precursors [34], and is not applicable to newer-generation PFASs such as HFPO-DA and C6O4. In the case of AW-2 and AW-3, the predominant PFAS compound identified was perfluorobutanesulfonic acid (PFBS), with concentrations of 37.570 μg L⁻¹ and 15.868 μg L⁻¹, respectively. Chemical analyses were focused on target compounds that were expected to be present in the samples. Non-targeted analytical techniques would be needed to generate a more thorough profile of PFAS speciation. These might indicate that degradation byproducts from biotic or abiotic processes are present. The idea that long-term exposure to long-chain PFASs (like PFOA in AW-1) encourages shifts in microbiological communities towards PFAS-tolerant strains is supported by the variations in biomass acclimatization to PFASs. In contrast, B-1 seems to only adapt to short-chain PFASs (like PFBS), which results in metabolic adaptations (Figure 6b). But as was already mentioned, this kind of in-depth description is outside the scope of the current investigation. It should be noted that AW-1 is characterized by very high concentrations of PFOA and PFOS, specifically 2870.060 μg L⁻¹ and 148.380 μg L⁻¹, respectively. This may suggest that different biomasses operating in different types of WWTPs acclimate only to certain PFAS compounds, likely due to microbial community shifts or enzyme adaptation [35]. In the specific case of this study, biomass B-1, which comes from a conventional WWTP, seems to be more tolerant to AWs mainly characterized by short-chain PFASs such as PFBS, while biomass B-2, which comes from an AWTP, appears to be less inhibited by the AW containing mainly long-chain PFASs such as PFOA and PFOS.

3.3.2. Sub-Step C(II)

Table 5. Results of 5 days multi-OUR tests carried out within sub-step C(II). These tests were performed with a laboratory scale reactor containing 800 mL of biomass and 800 mL of substrate. BOD₅/COD ratio and PFASs concentration of the tested substrate are also reported.

Tested Biomass	Tested Substrate			Overall ΔO2 [mgDO gVSS−1]
	Composition	BOD5/COD [-]	Sum of PFASs [mg L−1]
B-1	AW-1 3	0.17	3.268	151.28
	AW-2 3	0.52	0.079	227.00
	AW-3 3	0.34	0.026	236.00
B-2	AW-1 3	0.17	3.268	250.28
	AW-2 3	0.52	0.079	269.85
	AW-3 3	0.34	0.026	369.85

³ Premixed with casein peptone to have a COD concentration close to that of the tested AW.

shows the results of 5 days of multi-OUR tests carried out within sub-step C(II). As observed for single-OUR tests, biomass B-2 shows a higher oxygen consumption compared to biomass B-1. As observed in the previous paragraph, the overall oxygen consumption recorded during the test is higher when the BOD₅/COD ratio of the substrate is higher. Moreover, the effect of PFASs concentration can be noted here: oxygen consumption increases when “Sum of PFASs” decreases. Both biomasses show higher oxygen consumption in the test in which AW-3 was tested, in contrast to the results obtained in single-OUR tests.

4. Conclusions

This study assessed the potential inhibitory effects of real PFAS-containing industrial wastewaters (AWs) on conventional activated sludge (CAS) by means of about 400 respirometric tests. The results confirmed that the COD concentration of the tested substrate plays a crucial role in respirometric responses: sOUR values were generally proportional to COD, underlining the importance of controlling this parameter when investigating PFAS-related inhibition.

When COD was normalized (Step B), PFAS concentrations in the tested mixtures were very low, ranging from 0.002 to 0.004 mg L⁻¹ (with biomass B-1) to 0.010–0.930 mg L⁻¹ (with biomass B-2). In this condition, no clear inhibitory effects could be ascribed to PFASs alone. Conversely, tests with raw AWs (Step C) highlighted distinct responses depending on biomass origin: a municipal WWTP’s biomass (B-1) showed a greater tolerance to AWs dominated by short-chain PFASs like PFBS, while an industrially exposed WWTP’s biomass (B-2) was less inhibited by AWs rich in long-chain PFASs such as PFOA and PFOS. Regardless of the type of PFAS, both biomasses showed decreased oxygen consumption with AW-1 and increased oxygen consumption with AW-3 in 5-day multi-OUR experiments.

Overall, these finding show that neither acclimation effects, nor the inherent properties of specific PFAS compounds, are the main factor governing the tolerance of biomasses to PFASs. The primary factor influencing microbial resistance is acclimation history, and hence, past exposure to industrial inputs. Future research should (i) compare the reported effects to a reference hazardous substance and (ii) look into the adsorption of PFASs onto cell walls, as well as the possibility of biological clearance of these pollutants.