# Investigating the impact of low-pressure reverse osmosis membrane aging and heterogeneity on performance: A study of virus and microplastic retention

Hugo Taligrot, Vivian Dam Quang, Sébastien Wurtzer, Mathias Monnot, Damien Chaudanson, Laurent Moulin, Philippe Moulin

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## ► To cite this version:

Hugo Taligrot, Vivian Dam Quang, Sébastien Wurtzer, Mathias Monnot, Damien Chaudanson, et al. Investigating the impact of low-pressure reverse osmosis membrane aging and heterogeneity on performance: A study of virus and microplastic retention. Separation and Purification Technology, 2025, 376, 10.1016/j.seppur.2025.133924. hal-05468499

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Investigating the impact of low-pressure reverse osmosis membrane aging and heterogeneity on performance: a study of virus and microplastic retention

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Hugo Taligrot<sup>a</sup>, Vivian Dam Quang<sup>a</sup>, Sébastien Wurtzer<sup>b</sup>, Mathias Monnot<sup>a</sup>, Damien Chaudanson<sup>c</sup>,

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Laurent Moulin<sup>b</sup>, Philippe Moulin<sup>a,*</sup>

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<sup>a</sup> Aix Marseille Univ., CNRS, Centrale Med., M2P2 UMR 7340, Equipe Procédés Membranaires (EPM), Europôle de l’Arbois, BP80, Pavillon Laennec, Hall C, 13545, Aix en Provence Cedex, France

<sup>b</sup> Eau de Paris, Direction de la Recherche du Développement et de la Qualité de l’Eau, 33 Avenue Jean Jaurès, 94200, Ivry-Sur-Seine, France

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<sup>c</sup> Aix Marseille Univ., CNRS, CINAM UMR 7325, 13288 Marseille, France

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* Corresponding author.

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E-mail address: philippe.moulin@univ-amu.fr (Philippe Moulin)

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Abstract

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The increasing demand for freshwater, alongside increasing microplastics and persistent microbiological contaminants, is degrading water quality and highlighting the need for advanced treatment processes with better retention performance. Low-pressure reverse osmosis (LPRO) presents a promising solution for producing drinking water from freshwater, offering an optimal balance between water quality and production costs. However, uncertainties remain regarding the long-term evolution of these performance metrics, which are primarily based on data from reverse osmosis membranes used in seawater desalination. This study investigates the impact of membrane aging on LPRO performance and the role of membrane heterogeneity within a spiral-wound module. New and long-term aged membranes were compared in terms of permeability, salt retention, and virus retention, with additional analysis of microplastic retention. Natural membrane aging led to increased permeability and decreased salt retention, without affecting virus and microplastic retention, for which retention remained total across the tested coupons. Performance heterogeneity was observed depending on the position of the coupons within the module, with a more significant degradation at the beginning of the module and winding, likely due to mechanical effects associated with higher pressure and deformation. In contrast, coupons located at the end of the module and winding showed minimal performance impact. No impact was observed on virus and microplastic retention, which remained complete, confirming that viruses do not pass through an intact membrane but rather through membrane defects and seals. Autopsy of the LPRO modules revealed membrane defects (glue patches, minor and major folds) that affected membrane performance. In particular, the presence of apparent major folds led to significant performance
33 degradation, with lower permeability and salt retention. The folds also caused the passage of viruses
34 into the permeate, while microplastic retention remained unaffected. The virus passing through the
35 permeate of an LPRO pilot would therefore be due to defects in the membrane and O-rings of the
36 modules.

37 Keywords: Low-pressure reverse osmosis, Membrane aging, Virus removal, Microplastics

38 Abbreviations: ADV, adenovirus; CV, Coxsackievirus; EV, enterovirus; LPRO, low-pressure reverse
39 osmosis; LRV, logarithmic reduction value; MPs, microplastics MW, molecular weight; pI, isoelectric
40 point ; PMMA, poly(methyl methacrylate); RO, reverse osmosis; RR, retention rate, (RT)-qPCR,
41 reverse transcription- quantitative polymerase chain reaction; SEM, scanning electron microscopy;
42 TFC, thin-film composite; UF, ultrafiltration; µ-FTIR, micro-Fourier Transform Infrared Spectroscopy

43 1. Introduction

44 Freshwater is a vital resource, essential for life, ecosystem preservation, and industrial activities.
45 However, the increasing demand driven by human, agricultural, and industrial activities is placing
46 growing pressure on water supplies, leading to the degradation of natural water quality. In the context
47 of producing drinking water from natural freshwater sources, some pollutants are not fully removed by
48 conventional treatment processes. The emergence of small-scale chemical pollutants in low
49 concentrations (micropollutants) or microbiological contaminants in large quantities (microorganisms)
50 raises the risk of water contamination at certain times of the year [1,2]. These contaminants, which are
51 responsible for numerous diseases or suspected of causing long-term health effects, pose a threat to
52 public health, making the production of safe drinking water crucial [3]. Ultrafiltration (UF) is a
53 membrane process widely used for freshwater purification. However, UF does not guarantee the
54 complete retention of certain pollutants, such as viruses and microplastics which can also be found in
55 the UF permeate, despite the low pore size of UF membranes [4,5], highlighting the need for more
56 comprehensive treatment processes. Reverse osmosis (RO), a membrane-based process originally
57 developed for seawater desalination, offers a potential alternative due to its higher retention capabilities.
58 Nevertheless, the high pressures required to overcome seawater’s significant osmotic pressure limit its
59 widespread use due to the associated high production costs [6]. Low-pressure reverse osmosis (LPRO),
60 when applied to freshwater for drinking water production [7,8], could provide an interesting compromise
61 between water quality and production costs, with pressure of 10-15 bar [9]. Since the performance of
62 the process can evolve over time, it is essential to study the aging of dense LPRO membranes. However,
63 there is a significant lack of information regarding the aging of LPRO membranes, with most studies
64 focusing primarily on conventional RO membranes. Membrane aging is inevitable in filtration systems
65 and can lead to a progressive decline in performance or complete membrane failure, in both cases
66 reducing the process efficiency [10]. Pype et al. (2016) specifically highlighted a decrease in salt and
organic matter retention as RO membranes age. The aging of RO membranes is primarily driven by
chemical and physical factors. On one hand, the use of chemicals, particularly chlorine, is a major cause
of membrane aging and poses a threat to most polyamide-based membranes, potentially affecting their
performance [12–14]. Indeed, the effects of chlorination on polyamide RO membranes have been widely
reported in the literature [15,16], with studies concluding that chlorine causes irreversible damage to
RO membranes, compromising their integrity. Exposure to other chemical agents can also influence
membrane aging and, consequently, their performance, including ozone (a disinfectant), especially after
prolonged use [17], sodium lauryl sulfate (a surfactant), and EDTA (a chelating agent) [18]. On another
hand, mechanical constraints, such as pressure fluctuations and repeated production cycles, can also
cause physical damage and reduce membrane performance over time. Torii et al. (2019) assessed the
virus removal efficiency (MS2, φX-174, and Aichivirus) of 27 RO membranes based on their age and
estimated total pressurization time. The results showed that more pressurized membranes had a
significantly lower virus removal capacity than less pressurized ones. Antony et al. (2010) investigated
the impact of pressure on the surface modification of a commercial polyamide RO membrane and its
performance. Their research revealed that applied pressure accelerates membrane aging by stretching
the C = C bonds, leading to a decrease in membrane performance [20]. The literature extensively shows
that the aging of RO membranes can lead to their degradation or loss of integrity, influenced by
numerous parameters. However, Fujioka et al. (2013) demonstrated that, although thin-film composite
(TFC) membranes aged in RO and LPRO share the same three-layer structure (a non-woven fabric, a
porous support, and an active/thin layer), these membranes differ in their TFC layer. Positron
annihilation lifetime spectroscopy analysis of an RO membrane used for seawater desalination (2.6 L·h⁻¹·m²·bar⁻¹) compared to an LPRO membrane (5.9 L·h⁻¹·m²·bar⁻¹) revealed smaller values for both the
mean free-volume hole-radius and mean free-volume hole-space, for the RO membrane, measuring
0.259 nm versus 0.289 nm and 0.073 nm³ versus 0.101 nm³, respectively. These findings are consistent
with Hassan et al. (2019), who proposed that the higher permeability of LPRO membranes results from
a thinner TFC layer and larger free volume holes compared to RO membranes used for seawater
desalination. Additionally, operating at lower pressures may help reduce membrane fouling and,
consequently, aging. However, the irregular flow among individual elements within a pressurized
module could be more pronounced in LPRO systems due to their inherently higher permeability [23].
Thus, there may be significant differences in long-term operational stability, particularly concerning
susceptibility to fouling, between RO and LPRO membranes [24]. Undoubtedly, the type of water matrix
to filter (seawater vs fresh water) may also play a key role. In order to improve understanding of the
internal dynamics and aging of a LPRO module, it would be essential to conduct more precise research
by studying the flat membrane that is rolled within spiral modules. This approach would allow the
evaluation of the flat membrane's performance without the influence of the O-rings typically used with
the modules, thereby reducing the risk of micro-leaks mentioned in the literature [25–27].
The aim of this study was to evaluate: (i) The impact of membrane aging on the performance of LPRO membranes by comparing long-term aged membranes with new membranes. This comparison was carried out using various parameters (permeability, retention of monovalent and divalent salts, and virus retention) as well as surface analysis through scanning electron microscopy (SEM). The study of microplastic (MP) retention was also conducted, prompted by the observation of membrane defects measuring several micrometers in size, as revealed by SEM analysis. (ii) The homogeneity of these performances across an LPRO membrane spiral by testing flat membrane samples taken from different positions on the membrane surface. (iii) The effect of filtration with enhanced sealing by comparing the performance of flat membranes with that of spiral-wound LPRO modules, which may vary depending on the presence or absence of O-rings.

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## 2. Materials and Methods

### 2.1 Experimental pilot-plant and membranes

#### 2.1.1 Pilot-plant

A laboratory-scale transverse/tangential flow cell (Sepa CF Cell, Sterlitech Inc., USA) was used to investigate flat membranes. The system was powered by a high-pressure pump (Model 230, Cat Pumps), and the transmembrane pressure was regulated via a micrometric control valve located at the outlet of the retentate stream. This stream was recycled back to the feed tank through a recirculation loop. The temperature was maintained constant using an immersion cooler (Dip Cooler RU-200, Techne). The membrane tests were conducted with a medium-fouling spacer made of polypropylene (Diamant 47 Mil, Sepa CF, Sterlitech) and a permeate support of polytetrafluoroethylene (11-18 Mil, Sepa CF, Sterlitech), as per the manufacturer's instructions. The pilot's PID diagram is illustrated in Figure A.1. The experiments conducted on this LPRO flat membrane device indeed provide improved sealing conditions, as they do not use O-rings like those found in the laboratory- and semi-industrial-scale pilots. This enhanced sealing is important, and contributes to the novelty of this work, because the presence of O-rings in spiral-wound modules has been identified as a significant factor contributing to virus passage into the permeate. Moreover, any leakage from the seals used in this filtration cell would result in visible water escape, making any sealing failure immediately detectable.

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<figure><img src="image_3.png" /><figcaption>Figure A.1: PID diagram of the LPRO flat membrane pilot plant</figcaption></figure>

## 2.1.2 Membranes

The membranes studied were LPRO membranes typically employed for the treatment of brackish water to produce drinking water. Table A.1 presents the main characteristics of the new membranes, which served as a reference for this study.

<table><caption>Table A.1: Main characteristics of the new membranes studied</caption><thead><tr><th>Manufacturer, model</th><th>CSM, RE2540-BE</th><th>Toray, TMH10A</th></tr></thead><tbody><tr><td>Material</td><td colspan="2">Thin film composite polyamide</td></tr><tr><td>Unit membrane area (m²)</td><td>2.5</td><td>8</td></tr><tr><td>Manufacturer's permeability (L h<sup>-1</sup> m<sup>-2</sup> bar<sup>-1</sup>)</td><td>3.3</td><td>6.9</td></tr><tr><td>at T=25°C</td><td></td><td></td></tr><tr><td>Manufacturer's NaCl rejection (%)</td><td>99.7 (2,000 mg L<sup>-1</sup>)</td><td>99.3 (500 mg L<sup>-1</sup>)</td></tr><tr><td>at T=25°C (NaCl concentration)</td><td></td><td></td></tr></tbody></table>

The 2 new membranes were compared to aged membranes of the same type, which were utilized in a previous study involving two LPRO pilots of different scales (Taligrot et al., 2024). One CSM membrane, recovered from a laboratory-scale LPRO pilot, was operated for 950 days and treated approximately 160 m³ of water. This filtered volume may appear low, but as part of research and development pilot studies, these membranes are subjected to intense operational stresses such as prolonged shutdown periods require preservation using sodium bisulfite, temperature variations and repeated pressurization cycles which may induce physical or chemical degradation. For the Toray membranes, six were retrieved from a semi-industrial LPRO pilot with three stages: the first and the penultimate membranes from each stage. These membranes were installed in the pilot for 620 days and treated approximately 21,000 m³ of water for stage 1, 11,000 m³ for stage 2, and 5,500 m³ for stage 3. The nine spiral-wound membranes in total were then cut into coupons measuring 14 cm × 19 cm. These
coupons, sourced from the spiral-wound LPRO modules, were sampled across the entire membrane surface and referenced according to their position within the module (Figure A.2).

<figure><img src="image_4.png" /><figcaption>Figure A.2: Schematic diagram of sampling and numbering of LPRO spiral-wound modules</figcaption></figure>

### 2.1.3 Characterization of membranes

The surface of selected membrane coupons was visualized using a scanning electron microscope (JSM-7900F, JEOL, Japan), equipped with a Schottky field emission electron gun featuring a ZrO/W cathode.

The membranes were examined in “low vacuum” mode, eliminating the need for sample metallization and preventing charge accumulation. This mode employs both a secondary electron detector and a backscattered electron detector, operating at variable pressures between 10 and 300 Pa. For the membranes studied, images were captured at an accelerating voltage of 10 kV and a pressure of 80 Pa.

## 2.2 Filtration experiments and quantification methods

### 2.2.1 Permeability and salt retention

A permeability test was conducted using distilled water, where the permeate flux was measured for different transmembrane pressures. The measured flux was adjusted to 20°C using Equation (A.1) to account for temperature-induced viscosity changes, ensuring accurate permeability calculations.

$$J_{20} (L h^{-1} m^{-2}) = J_{T} \times \frac{\mu_{T}}{\mu_{20}} = \frac{J_{T}}{e^{6.435-\frac{1885}{T}}} \qquad \text{Eq. (A.1)}$$

The retention of monovalent and divalent salts was investigated by sequentially filtering sodium chloride (NaCl) and calcium sulfate (CaSO₄) solutions (at concentrations of 0.5 or 2 g L⁻¹, based on the manufacturer's specifications) under a constant pressure of 12 bar. Conductivity was measured before and after the membrane using a conductivity meter (Cond 3110, WTW, Xylem), enabling the calculation
of the retention rate (RR). It is important to note that permeability and retention tests for monovalent and divalent salts were conducted in triplicate on each membrane coupon.

### 2.2.2 Virus and retention

Three different viruses were simultaneously used in this study: ADV 41, CV-B5, and MS2 phage (Table A.2). These viruses were selected to investigate the potential effects of size and charge on virus retention, as demonstrated in previous studies on UF membranes [4]. All viruses have isoelectric points (pI) within the same range, below the neutral pH value, and are therefore negatively charged under the experimental conditions of this study. They also share a similar shape, characterized by an icosahedral capsid. Notably, CV-B5 and MS2 phage exhibit very similar characteristics: both are single-stranded RNA viruses within a comparable size range (30 nm and 27 nm, respectively) and possess similar molecular weights (MW). In contrast, ADV 41 is a double-stranded DNA virus, approximately three times larger than the other two (90 nm), and has a higher MW. MS2 was chosen as a “standard” virus commonly referenced in studies and sometimes regarded as a model virus, while the other two were selected for their relevance to waterborne diseases. Finally, these three viruses were specifically chosen due to their ability to be produced in sufficient quantities for LPRO filtrations at the targeted concentrations.

<table><caption>Table A.2: ADV 41, CV-B5 and MS2 phage characteristics [4,28,29]</caption><thead><tr><td>Virus</td><td>Size (nm)</td><td>pI</td><td>DNA/RNA virus</td><td>Shape</td></tr></thead><tbody><tr><td>ADV 41</td><td>90</td><td>2-6</td><td>Double stranded DNA</td><td>Icosahedral capsid,</td></tr><tr><td>CV-B5</td><td>30</td><td>3.6</td><td>Single stranded RNA</td><td>non-enveloped virus</td></tr><tr><td>MS2 phage</td><td>27</td><td>3.5-3.9</td><td>Single stranded RNA</td><td></td></tr></tbody></table>

ADV 41 (#ATCC VR-930) and CV-B5 (#ATCC VR-185) were cultivated on monolayer cultures of 293A cells (#ATCC CRL-1573) in Dulbecco's Modified Eagle Medium (#R70507, Invitrogen) supplemented with 2% fetal calf serum (#30-2021, Sigma-Aldrich) at 37°C with 5% CO₂. MS2 phages (#ATCC 15597-B1) were cultivated on K12hFr E. coli cells at 37°C in Luria-Bertani medium. After cell lysis, the supernatants from both eukaryotic and bacterial cultures were mixed with chloroform (10% v/v) and clarified by centrifugation at 8000 g for 15 minutes at 4°C. The resulting supernatants were collected. Virus stocks were aliquoted and titrated by (RT)-qPCR and then stored at -80°C for a few weeks until use. Virus retention was assessed by filtering 4 L of a virus suspension containing 4×10⁶ viruses of each type (AdV 41, CV-B5, and MS2) at a constant pressure of 12 bar, resulting in a concentration of approximately 10⁶ viruses L⁻¹. During the filtration process, 3 L of permeate were collected. Due to the low virus concentrations in the permeate, the samples were concentrated using successive UF devices with very low cut-offs (≤ 18.8 kDa), as described in a previous study [30]. The permeate was firstly concentrated using a UF membrane (Inuvai R180, Fresenius Medical Care,
Germany) through frontal filtration. The retentate, which contained the retained viruses, was eluted by backwashing (in the opposite direction of filtration) with an elution solution composed of 0.5% tripolyphosphate, 0.1% Tween 80, 0.1% antifoam, and 0.05% glycine. This step concentrated 3 L of permeate into an initial elution volume of approximately 200 mL. The 200 mL recovered was further concentrated using a centrifugation based UF device (Vivaspin 20, molecular weight cut-off of 10 kDa, Sartorius) to yield an elution volume of around 200 µL. The virus concentrates were then lysed directly on the membrane of the Vivaspin 20 concentrators by adding lysis buffer (#19076, ATL buffer, QIAGEN). The average virus recovery rates from the concentration method are presented in Table A.3. No concentration was necessary for the concentrate and feed samples, as they were already at sufficient concentrations for detection and quantification by (RT)-qPCR.

<table><caption>Table A.3: Average virus recovery rates (with uncertainties calculated with a 95% confidence interval) of concentration methods of ADV 41, CV-B5 and MS2 phage [30]</caption><thead><tr><th rowspan="2">Virus</th><th colspan="2">Ultrafiltration method</th></tr><tr><th>Low concentration<br>(10<sup>3</sup>-10<sup>4</sup> viruses L<sup>-1</sup>)</th><th>High Concentration<br>(10<sup>6</sup>-10<sup>7</sup> viruses L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>ADV 41</td><td>50 ± 2 %</td><td>84 ± 14 %</td></tr><tr><td>CV-B5</td><td>54 ± 6 %</td><td>83 ± 14 %</td></tr><tr><td>MS2 phage</td><td>55 ± 12 %</td><td>109 ± 15 %</td></tr></tbody></table>

Viral nucleic acids from concentrates were extracted using the EZ2 DNA extraction kit on the EZ2 Connect Fx instrument (QIAGEN), according to the manufacturer's instructions. A blank control was included in each extraction cycle. Nucleic acids were eluted into 100 µL volume, allowing titration in duplicate. Each reaction for each virus was performed using 10 µL of nucleic acid samples, 5 µL of master mix TaqMan® Fast Virus 1-Step (Life Technologies, Carlsbad, CA), and 5 µL of a mix containing primers and probes. Primers and probes designed for ADV 41, CV-B5 and MS2 were used as described previously [4], with final concentrations per reaction as specified by Prevost et al. (2015). All reactions were carried out using a ViiA™ 7 real-time PCR system (Life Technologies, Carlsbad, CA) with the same thermal cycling profile as in a previous work [27]. Each (RT)-qPCR assay included positive and negative amplification controls. Additionally, infectivity tests were conducted to verify the presence of infectious viruses. The quantification of infectivity was performed by counting the lysed wells on monolayer cultures of 293A cells for AdV 41 and CV-B5 according to NF EN 14486 standard, and on WG49 bacteria for MS2 according to ISO10705 standard. Positive and negative controls were systematically carried out to validate the permittivity of target cells and the absence of cross-contamination. For the 293A cells, which are permissive for both AdV 41 and CV-B5, the supernatants from the lysed wells were subsequently analysed by PCR to detect the presence of viral genomes and identify the implicated virus.
## 2.3 Microplastic retention

The retention of MPs was studied by filtering 3 L of a synthetic suspension of poly(methyl methacrylate) (PMMA) beads, with diameters ranging from 38 to 45 µm and a density of 1.2 (Cospheric), at a constant pressure of 12 bar. It is essential to avoid any materials or products composed of plastics or plastic derivatives throughout the experiment to minimize the risk of contamination from other MPs; thus, PMMA plastics are explicitly excluded from the LPRO experimental set-up. Control tests were also performed on ambient air, ultrapure water, and ultrapure water recirculated within the pilot system, confirming the absence of PMMA. The feed suspension was prepared by dispersing 10⁷ PMMA particles (approximately 3×10⁶ particles L⁻¹) in a solution of ultrapure water and Tween 20, following the manufacturer's protocol. Tween 20 is a surfactant designed to prevent the aggregation of PMMA microspheres upon contact with water. Ultrapure water was produced using a purification system (PURELAB® flex 3). A 5 mL feed sample was collected to verify the actual concentration. During crossflow filtration, 2 L of permeate were recovered. The MPs contained in the permeate samples were filtered using a vacuum Büchner funnel (Fisher Scientific) under a laminar flow hood. Filtration was conducted in two stages: first through a 25 µm stainless steel filter, followed by a 13 µm stainless steel filter. The final count of PMMA particles corresponds to the sum of those detected on each filter. The identification and quantification methodology employed for the filters has been described previously [5,32]. This process utilized a Spotlight 400 micro-Fourier Transform Infrared Spectroscopy (µ-FTIR) microscope as part of the µ-FTIR imaging system, along with siMPle (Systematic Identification of MicroPLastics in the Environment) software. In this study, the spectrometer was configured in reflectance mode with a spectral resolution of 16 cm⁻¹ [33]. Background spectrum imaging was collected from a gold filter with a pixel size of 25 µm. Additional identification parameters for the µ-FTIR included 1 scan per pixel, an interferometer speed of 2.2 cm s⁻¹, an infrared spectral range of 690–4,000 cm⁻¹, and an imaging resolution of 25 µm per pixel.

The LPRO retention performance of viruses and MPs is represented in terms of log reduction value (LRV), which allows the representation of the retention rate on a logarithmic scale. Additionally, statistical tests were conducted using a non-parametric paired t-test, specifically the Wilcoxon test. The *p*-value was calculated, with a significance level defined at 5%, to validate the absence of significant differences. All statistical analyses and graphics were generated using GraphPad Prism 9.5 (GraphPad Software, San Diego, CA, USA).
264 **3. Results and discussions**

265 First, it was important to verify the consistency of the replicates obtained for each LPRO module from
266 the two plants (RE2540-BE and TMH10A membranes) in terms of permeability and retention of
267 monovalent salts (NaCl) and divalent salts (CaSO₄). A statistical analysis of triplicates within a single
268 coupon was first conducted (Figure A.3a), followed by an analysis across the entire membrane sampled
269 in nine membrane coupons (Figure A.3b), where the coupon numbers are indicated on the X-axis.

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>Figure A.3: Example of permeability and retention of monovalent and divalent salts in different replicates of a) a membrane coupon and b) a membrane sampled in 9 membrane coupons</figcaption></figure>

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273 Within a single coupon, statistical analysis showed no significant differences, denoted as (ns), among
274 the triplicates for each parameter studied, demonstrating the repeatability of the measurements. On a
275 broader scale, the results highlighted differences in certain parameters measured across the membrane
276 sampled in nine coupons. In very rare instances, one of the measurements may differ from the other two;
277 in such cases, the outlier is excluded. Therefore, for the overall analysis of the sampled membrane, the
278 mean for each parameter was calculated excluding statistically outlying values when present.

279

280 **3.1 Influence of aging on membrane performance**

281 The performance of an aged RE2540-BE membrane (from lab-scale pilot-plant) was compared to that
282 of an identical new membrane in terms of permeability and retention rates for monovalent salts (NaCl)
283 and divalent salts (CaSO₄) at 2 g.L⁻¹. The results are presented in Figure A.4 and represent the average
284 values obtained for the 9 membrane coupons analysed in triplicate.

285
<figure><img src="image_7.png" /><figcaption>Figure A.4: Impact of membrane aging on the performance of new and aged RE2540-BE membranes</figcaption></figure>

The results for the new RE2540-BE membrane were consistent with the supplier's specifications, which indicated a permeability of 3 L.h⁻¹.m⁻².bar⁻¹ and a salt retention rate of 99.7% for NaCl at 2 g.L⁻¹. Similarly, the values obtained for the aged RE2540-BE module were in line with the last measurements taken before its dissection when it was installed on the LPRO pilot, showing a permeability of 5.6 L.h⁻¹.m⁻².bar⁻¹ and a salt retention rate of 99.1%. The aged RE2540-BE membrane exhibited a permeability approximately twice that of the new membrane, probably due to chemical or physical deterioration, correlated with a lower retention rate for each salt studied: an average of 99.4% compared to 98.1% for NaCl and 99.6% compared to 98.7% for CaSO₄. As expected, the retention rate was higher for CaSO₄ than for NaCl [34]. Furthermore, statistically significant differences (p-values < 0.008), indicated by an asterisk (*), were observed between the new and aged membranes for each of the parameters studied, highlighting the impact of aging on membrane performance. This trend aligns with studies on membrane aging in RO systems [10,11,35]. As for the TMH10A membranes (from semi-industrial pilot-plant), the performance of the 6 aged membranes was compared to that of an identical new membrane in terms of permeability and retention rates for monovalent salts (NaCl) and divalent salts (CaSO₄) at 0.5 g.L⁻¹ (Figure A.5). It is important to note that these results represent the average of triplicate measurements from 9 coupons for each of the 6 membranes. The study of the 6 aged membranes, sourced from different locations of an LPRO pilot, also took into account the impact of the module's position within the semi-industrial pilot.
<figure><img src="image_8.png" /><figcaption>Figure A.5: Impact of membrane aging on a) permeability and b) monovalent and divalent salt retention of new and aged TMH10A membranes</figcaption></figure>

The results for the new TMH10A membrane were consistent with the manufacturer's values, indicating a permeability of 6.2 L·h⁻¹·m⁻²·bar⁻¹ and a NaCl retention rate of 99.3% at 0.5 g·L⁻¹. However, the aged TMH10A membranes exhibited a permeability approximately 1.3 to 1.8 times higher than the new membrane, along with a lower retention rate for each salt tested. Furthermore, statistically significant differences (*p*-values < 0.004), were observed between the new and aged membranes for each of the parameters studied. The permeability and retention rates for monovalent (NaCl) and divalent (CaSO₄) salts in the aged modules were 9.3 ± 1.2 L·h⁻¹·m⁻²·bar⁻¹, 99.3 ± 0.3%, and 99.5 ± 0.3%, respectively. Comparing the modules revealed significant differences between the permeability of the first and last aged membrane (*p*-values < 0.004). For example, the permeability between the first module (REX1) and the penultimate module in the semi-industrial pilot (REX6) varied by 2.4 L·h⁻¹·m⁻²·bar⁻¹, more than a 25% difference. A higher transmembrane pressure at the first stage of an RO pilot has often been identified as a major disturbing factor, revealing spatial heterogeneity in fouling across the different stages [36]. Zheng et al. (2018) also reported such heterogeneity, though in the context of municipal wastewater treatment plants, primarily attributing it to significant biological fouling at the first stage, caused by cell adhesion, which could be a major factor in the inter-stage divergence. To eliminate potential variations and confirm the impact of membrane aging, additional experiments were conducted on two aged TMH10A membrane coupons showing no apparent defects: (i) compression of a membrane at 30 bar for 2 hours to eliminate the effect of “pre-pressurization” requested by some manufacturers, and (ii) cleaning the membrane with sodium hydroxide (40°C and pH = 11.5) for 20 minutes. The results showed no significant differences in permeability or retention rates for each tested salt after either compression or cleaning with sodium hydroxide (*p*-values > 0.40). Therefore, experiments on each type of membrane demonstrated that aging under operating conditions has a significant impact on permeability and, consequently, on salt retention rates. These variations can be attributed to repeated pressurization and depressurization cycles, which can affect the membrane in the long term and significantly reduce its performance, as shown by Torii et al. (2019) with virus retention tests on MS2, φX-174, and Aichi virus. Furthermore, the two LPRO pilots on which the modules were used are intended solely for research and development experiments and are therefore frequently subjected to
pressurization and depressurization cycles. Aging could induce changes in the membrane's polymeric structure, including the relaxation of polymer chains, potentially leading to increased permeability [38]. Thus, studying a large number of flat membrane coupons allows testing this hypothesis, as the pressure is not the same on either side of a spiral wound module and varies depending on its proximity to the permeate collector axis [39].

## 3.2 Influence of the position of membrane coupons

To present the results in this section, the position of each membrane coupon was plotted on the X- and Y-axes to highlight differences among the coupons. It is important to note that the permeate collection axis is located on the side of coupons 1, 2, and 3 (at X = 0), with the fluid flow direction moving from coupon 1 to coupon 3, corresponding to increasing Y-coordinates; the Z-axis represents the measured parameter. The performance results for the new RE2540-BE membrane coupons are presented in Figure A.6. For each parameter studied, the results for each membrane coupon represent the average of triplicate measurements conducted per coupon.

<figure><img src="image_9.png" /><figcaption>Figure A.6: Impact of membrane coupon position on a) permeability at 20°C and b) monovalent salt retention of a new RE2540-BE membrane</figcaption></figure>

The results showed that the permeability and retention rates were uniform across all membrane coupons of the new RE2540-BE membrane, highlighting the performance homogeneity throughout the flat-sheet membrane of the module. In contrast, for the aged RE2540-BE membrane, performance variations were observed depending on the position of the membrane coupon (Figure A.7).
<figure><img src="image_10.png" /><figcaption>Figure A.7: Impact of membrane coupon position on a) permeability at 20°C, b) monovalent and c) divalent salt retention of a aged RE2540-BE membrane</figcaption></figure>

This heterogeneity suggested that during the operation of the spiral-wound module, a specific mechanism significantly influenced membrane aging. The data showed similar permeability values, around $5.9 \pm 0.1 \text{ L.h}^{-1}\cdot\text{m}^{-2}\cdot\text{bar}^{-1}$, for coupons 7, 8, and 9 located at the outer edge of the membrane (i.e., the farthest from the permeate collection tube), where membrane deformation due to winding was minimal. Conversely, the highest permeability was observed for coupon 1, at $6.6 \pm 0.1 \text{ L.h}^{-1}\cdot\text{m}^{-2}\cdot\text{bar}^{-1}$, slightly lower for coupon 4, at $6.3 \pm 0.3 \text{ L.h}^{-1}\cdot\text{m}^{-2}\cdot\text{bar}^{-1}$, with permeability progressively decreasing along the feed flow within the module. This trend was explained by the fact that coupons 1, 4, and 7 corresponded to the module's inlet, where pressure was highest and fouling most pronounced [36]. A similar pattern was observed for salt retention rates, which were the lowest for coupon 1, dropping to $95.50 \pm 0.05\%$ for NaCl and $97.34 \pm 0.04\%$ for CaSO$_4$, while the average retention rates across all nine coupons were $98.14 \pm 0.02\%$ and $98.73 \pm 0.02\%$, respectively. The impact on coupons 4 and 7 was less pronounced. These results aligned with the observation that coupon 1 was subjected to the highest pressure and the greatest mechanical deformation from membrane winding. The impact of membrane coupon position was further analysed concerning virus and MP retention. The results showed the absence of viruses in all permeate samples whatever the membrane (aged and new), indicating total retention regardless of membrane aging or coupon position. However, the results of a previous study [27] had demonstrated the presence of viruses in the permeate of a laboratory-scale LPRO pilot using this same aged RE2540-BE membrane. This observation was not well-explained, despite several hypotheses, including the potential roles of O-rings and/or the membrane itself. These new findings absolved the membrane of responsibility for virus passage into the permeate, as the tangential flow cell configuration used in the pilot employed double cylindrical rectangular seals that ensured complete sealing (Figure A.2). Thus, the O-rings were identified as the likely cause of vir passage into the permeate, consistent with various studies on virus retention in LPRO systems [14,26,40–42]. However,
the total retention observed with flat-sheet membranes and the partial retention observed with spiral-wound LPRO modules suggest that micro-leaks along the adhesive lines in spiral-wound modules should also be considered as a plausible hypothesis. For MPs retention, while MPs were occasionally detected in the permeates (ranging from 2 to 50 particles L⁻¹), no PMMA was found, demonstrating the total retention of the LPRO membranes. These results also suggested the inevitability of environmental contamination. For clarity in this section, only the results of the RE2540-BE membranes were presented due to similar findings for the TMH10A membranes, which exhibited: (i) uniform performance across coupon positions for new membrane, (ii) performance heterogeneity depending on coupon position for all six aged membranes, with greater degradation for coupon 1 and less degradation for coupon 9, and (iii) total retention of viruses and MPs, regardless of membrane aging or coupon position. The results for the TMH10A membranes were nevertheless provided in the supplementary data (Figure B.1 and Figure B.2). The results for TMH10A membranes were also compared across different study scales, revealing variations from the coupon scale to the pilot scale, as detailed in the Table B.1.

### 3.3 Impact of membrane defects

During the autopsy of the various membranes, several kinds of membrane defects were highlighted and classified (Table A.4). Notably, these defects were observed exclusively on aged modules and were absent on new modules.

<table><caption>Table A.4: Different types of membrane defects observed during autopsies of different LPRO modules</caption><thead><tr><th>Glued patch</th><th>Minor folds</th><th>Major folds</th></tr></thead><tbody><tr><td></td><td></td><td></td></tr></tbody></table>

The presence of a patch was observed exclusively on one aged spiral-wound module but it probably could has been found in any other module. In contrast, folds were identified on all aged spiral-wound modules, with the total impacted surface areas estimated to 1% in average. Several plausible explanations exist for the origin of these folds: (i) manufacturing defects during the assembly of spiral-
wound modules could lead to fold formation when the membrane is inserted into the module housing, potentially due to alignment errors; (ii) another explanation could involve shear stress and turbulence at the membrane surface caused by feed spacers [43,44]. Damage may occur if the spacer is not fixed and moves relative to the membrane during frequent pressurization and depressurization cycles [45,46], which are often performed in test systems. Additionally, the presence of the observed membrane defects underscored the necessity of evaluating their potential impact on membrane performance. Therefore, two additional coupons, featuring a patch and major folds, have also been studied, regarding their permeability and retention of monovalent, divalent salts (Figure A.8).

<figure><img src="image_11.png" /><figcaption>Figure A.8: Impact of membrane defects on a) permeability and b) monovalent (NaCl) and divalent (CaSO₄) salt retention of an LPRO membrane (REX4)</figcaption></figure>

The results demonstrated that both types of membrane defects affected permeability and salt retention. First, a comparison between an “intact” membrane and one with a patch revealed significant statistical differences in permeability and salt retention rates for all salts studied, except for CaSO₄ at 0.5 g.L⁻¹ (p-values = 0.08). The application of the patch appeared to have caused potential micro-leaks, resulting in a slight increase in permeability and, consequently, a decrease in salt retention rates, particularly at higher salt concentrations: from 0.3% to 3.5% for NaCl and from 0.005% to 3.4% for CaSO₄. Regarding major folds, compared to an “intact” membrane, they seemed to play a critical role in membrane performance, as evidenced by significant statistical differences observed for all parameters studied (p-values < 0.004). Retention rates decreased considerably, with reductions ranging from 14% to 24% for NaCl and 10% to 19% for CaSO₄. Contrary to expectations, despite significantly lower retention rates, permeability also decreased, reaching only 1.7 ± 0.1 L.h⁻¹.m⁻².bar⁻¹ compared to 9.9 ± 0.1 L.h⁻¹.m⁻².bar⁻¹ for an “intact” membrane. The formation of numerous folds under membrane pressurization may have compressed the membrane material around these defects, potentially explaining the substantial reduction in permeability (up to five times lower). Moreover, no significant statistical difference was observed between NaCl and CaSO₄ retention at equivalent concentrations, suggesting that ion size and valence were not determining factors. Furthermore, given the significant degradation in performance observed in the presence of major folds, virus retention as well as MP retention was investigated. Virus retention
performance is presented through two types of results: measurements of viral genomes and infectious particles (Table A.5).

<table><caption>Table A.5: Virus retention of a membrane coupon composed of apparent major folds</caption><thead><tr><th colspan="3">Total number of viral copies (Retention)</th><th colspan="3">Total number of infectious viruses (Retention)</th></tr><tr><th>Adenovirus (AdV 41)</th><th>Enterovirus (CV-B5)</th><th>MS2 phage</th><th>Adenovirus (AdV 41)</th><th>Enterovirus (CV-B5)</th><th>MS2 phage</th></tr></thead><tbody><tr><td>78 (5 log)</td><td>0 (Total)</td><td>946 (3,9 log)</td><td>Not measured</td><td>Not measured</td><td>7 (5,9 log)</td></tr></tbody></table>

The quantification results by (RT)-qPCR confirmed the presence of AdV 41 and MS2 viral genomes in the permeate. Infectivity tests on WG49 bacteria revealed the presence of lysis plaques, indicating the presence of infectious MS2 viruses, estimated to be up to 20 viruses in the total permeate (considering the efficiency of the concentration method). Conversely, infectivity tests on 293A cells did not detect AdV 41 and/or CV-B5 due to excessive cytotoxic effects, that have made the detection or quantification of these two enteric viruses inconclusive. Moreover, the results for MS2 appeared consistent, as the number of infectious viruses was lower than the number of viral genome copies, indicating the presence of a significant proportion of non-infectious viral genomes in the permeate. However, it is noteworthy that the permeate produced by an LPRO flat membrane with visible folds still contained several infectious MS2 viruses. Control samples from infectivity tests ruled out viral contamination, thus confirming the presence of infectious viruses in the permeate samples. Given the configuration of the filtration cell used, it can be concluded that the membrane's major observed folds are responsible for the passage of infectious viruses into the permeate. Additionally, the MP retention results showed no presence of PMMA particles in the permeate, demonstrating complete retention regardless of the presence of major folds. As expected, these results are consistent with the size of the PMMA beads being larger than the fissures observed in the active layer, as determined by SEM analysis, provided in the supplementary data (Figure B.3).

## 4. Conclusion

This study highlighted several factors influencing the performance of LPRO membranes. Aging led to progressive performance degradation, characterized by increased permeability and decreased salt retention, while total retention of viruses and microplastics was maintained for membrane coupons without visible defects. This degradation was heterogeneous, depending on the position of the flat-sheet membrane coupons, with more pronounced degradation observed for coupons subjected to higher pressure and deformation (coupon 1) compared to less impacted coupons (coupon 9). Moreover, the
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presence of membrane defects, mainly visible folds, significantly reduced performance, causing lower
permeability, decreased salt retention, and increased virus passage into the permeate, although
microplastic retention remained total. These findings confirm that virus passage in LPRO permeate is
not caused by the membranes themselves (if fully intact) but rather by membrane defects and the O-ring
seals in the modules.

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Finally, the significant variability in results depending on the scale of analysis (whole module, average
of sampled coupons, individual coupons, or coupons with visible defects) raises important questions
regarding the appropriate protocols for studying membrane aging. This variability underscores the
relevance of the multi-scale approach adopted in this work.

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**Declaration of competing interest**

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The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.

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**Author contributions**

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Hugo Taligrot, Sébastien Wurtzer, Mathias Monnot, Damien Chaudanson, Vivian Dam Quang Laurent
Moulin, Philippe Moulin each contributed to the conception, writing, and editing of this article. All
authors have given approval to the final version of the manuscript.

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**Data availability**

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Data will be made available on request.

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