Hongbo Liu

Mengting Ye

Xinyi Dong

Zhenxing Ren

Shiping Long

Eric Lichtfouse

Removal of humic substances by the synergistic effect of biochar adsorption and activation of persulfate

Keywords:

Combined adsorption and oxidation using biochar to activate persulfate has been recently applied to remove contaminants, yet most of the experiments are carried out using artificial synthetic water instead of real waters.

Here we tested the removal of humic substances which is abundant and affecting water quality in natural waters, by persulfate combined with corn stalk biochar. Results showed that, compared with biochar prepared at 400 • C, 600 • C and 700 • C, biochar prepared at 900 • C after modified by hydrochloric acid had greater specific surface of 459.260 m 2 /g, richer porosity and higher removal of humic acids of more than 80% at pH 5.0-9.0 in pure water. Treatment of real river water showed decrease of 80.4% and 69.7% of total organic carbon (TOC) and UV 254 , respectively. The addition of tert-butyl-alcohol (TBA) and methanol (MeOH) inhibited the removal to a certain extent suggesting the existence of • OH and SO 4 -• . It was speculated that in addition to the adsorption of biochar, the oxygen-containing functional groups on its surface might act as active sites to promote the production of oxidized species. This work provides a method for corn stalks utilization in accordance with environmental sustainability and resource recovery.

Introduction

Humic substances such as soluble humic acids (HA), fulvic acids and insoluble humin, are major, complex, poorly known organic substances produced by the decay of living organisms in natural waters, wastewaters, soils and sediments [START_REF] Dou | Are humic substances soil microbial residues or unique synthesized compounds? A perspective on their distinctiveness[END_REF][START_REF] Du | Activation of porous magnetized biochar by artificial humic acid for effective removal of lead ions[END_REF][START_REF] Lichtfouse | Temporal pools of individual organic substances in soil[END_REF][START_REF] Schnitzer | Chapter 1 humic substances: chemistry and reactions[END_REF][START_REF] Zhang | Persulfate activation using Co/AC particle electrodes and synergistic effects on humic acid degradation[END_REF]. Although humic substances themselves have few harmful effects on public health, they can induce sensory issues such as odor and bad taste in drinking water indirectly. Humic substances also favor the growth of phytoplankton in reservoirs [START_REF] Herzsprung | Variations of DOM quality in inflows of a drinking water reservoir: linking of van Krevelen diagrams with EEMF spectra by rank correlation[END_REF]. An excess of dissolved organic matter in waters could also induce eutrophication and deaths of aquatic life. Furthermore chlorination of humic substances induces the formation of toxic by-products during water treatment [7], induced by the large number of functional groups in humic functions such as -C=O, -OH and -COOH [START_REF] Imyim | Humic acids removal from water by aminopropyl functionalized rice husk ash[END_REF]. Therefore, reducing the humic load before chlorination could decrease formation potential of disinfection by products.

Physical methods such as coagulation and membrane filtration were commonly used to remove humic substances, but with low efficiency, thus causing secondary pollution [START_REF] Ai | Coagulation mechanisms of humic acid in metal ions solution under different pH conditions: a molecular dynamics simulation[END_REF][START_REF] Hwang | The properties and filtration efficiency of activated carbon polymer composite membranes for the removal of humic acid[END_REF]. Chemical oxidation using advanced oxidation processes (AOPs) that generate reactive oxygen species (ROS) such as hydroxyl radicals, sulfate radicals, and alkoxyl radicals, especially the activation of persulfate (PS), peroxymonosulfate (PMS) and hydrogen peroxide (H 2 O 2 ) are efficient in removing organic pollutants [START_REF] Yang | Enhanced humic acid degradation by Fe3O4/ultrasound-activated peroxymonosulfate: synergy index, non-radical effect and mechanism[END_REF][START_REF] Yousefi | Mineralization of high saline petrochemical wastewater using Sonoelectro-activated persulfate: degradation mechanisms and reaction kinetics[END_REF]. Persulfate is a focusing point of research and application due to its high stability, low cost, broader pH applicability, production of SO 4 -• with wider application, longer half-life time and similar oxidation potential as to • OH [START_REF] Lykoudi | Degradation of sulfamethoxazole with persulfate using spent coffee grounds biochar as activator[END_REF]. Persulfate can be activated by alkali, transition metal, ultraviolet, ultrasound and microwave. Improved humic removal could be achieved by oxidation and adsorption using biochar combined with persulfate. Biochar are adsorbents produced by pyrolysis of organic materials such as agricultural residues, which are typically cheap, easy to obtain, metal-free, rich in oxygen-containing functional groups, porous of high specific surface area (SSA), and resistant to acid and alkali. Organic contaminants can be both adsorbed and oxidized using persulfate and biochar. There are many factors that affect the performance of biochar, including pyrolysis temperature, source of raw materials, modification methods and types of substrates to be removed. For example, 94.2%of norfloxacin was removed by persulfate and corn stalk biochar [START_REF] Wang | Metal-free activation of persulfates by corn stalk biochar for the degradation of antibiotic norfloxacin: activation factors and degradation mechanism[END_REF]. Egg shell biochar with persulfate achieved 90%of 2,4-dichlorophenol

Materials and methods

Reagents

Humic acids (HA), hydrochloric acid (HCl), sodium hydroxide (NaOH), sodium persulfate (PS, Na 2 S 2 O 8 ), tert-butyl-alcohol (TBA) and methanol (MeOH) were purchased from Sinopharm Chemical Regent Co. Ltd., Shanghai, China. All chemicals were of analytical grade and were used as received. Unless otherwise specified, deionized water was used in all experiments.

Preparation and modification of corn stalk biochar

Corn stalks were collected in autumn from a rural area in Anhui, China. Before use, straws were cut to about 100 mesh, cleaned and dried at 60 • C. Dried straw powder was placed in a box furnace and the temperature was raised to 400 • C, 600 • C, 700 • C and 900 • C under dinitrogen atmosphere at a heating rate of 5 • C/min, then kept for 180 min at corresponding temperature. The corn stalk samples were taken out after the pyrolysis and then soaked in 10% (w/w) HCl solution for 12 h. Subsequently, the corn stalk samples were washed with deionized water to neutral pH and dried at 60 • C to obtain the corn stalk biochar. The particle size range of biochar used in the experiment was 0.5-1 mm.

Removal of humic acids in pure conditions

To explore the effect of different factors on humic acids (HA) removal, a batch experiment in pure water was carried out. A measured volume of stock humic solution was mixed with the deionized water to reach the desired HA concentration. The desired amount of persulfate (PS) and biochar was immediately added to a conical flask, which filled with 100 mL of HA solution. Then 5 mL of the solution was collected from the flask placed on a constant temperature shaker (25 • C, 200 rpm) and filtered with a membrane (0.45 μm) at a certain time interval to monitor the removal of humic acid. The effects of biochar dosage of 0.1-0.9 g/L, initial pH of 5.0-9.0, PS concentration of 0.2-2 mM and initial HA concentration of 5-15 mg/L on HA removal were explored. The UV spectrophotometer was used to analyze the concentration of HA at 254 nm. HCl or NaOH (0.1 M) was used to adjust the initial pH of the solution.

To further identify the presence of free radicals in the reaction, a quenching experiment was carried out. Specifically, tert-butyl-alcohol (TBA) and methanol (MeOH) were used to quench • OH and SO 4 -• , respectively. All experiments were carried out for three times.

Removal of organic matter from river water and regeneration tests

To explore the application of biochar combined with persulfate in real water, water samples from the inlet of a reservoir in the Yangtze River were collected to a 2.5 L stainless steel column sampler and put into a collection bottle made of highly density polyethylene. After sampling, the water samples were immediately taken back to the laboratory for storage in a 4 • C refrigerator prior to use. Meanwhile, regeneration tests were used to judge reusability by collecting the used biochar through filtration and dried it in an oven at 60 • C for next use.

Data analysis and processing

The morphological characteristics, crystal structure, surface functional groups, surface pore distribution of biochar were analyzed by scanning electron microscopy (SEM, S4800, Hitachi, Japan), X-ray diffraction (XRD, Rigaku Ultima IV, Japan), Fourier transform infrared spectroscopy (FT-IR, Bruker/tensor 27, Germany) and Brunauer-Emmett-Teller (BET, Autosorb iQ2-up, Quantachrome, America), respectively. And the zero point of charge pH ZPC of the biochar was determined by the salt drop method. Pine needle biochar

[PMS] = 8.0 mM, [1,4-dioxane] = 20.0Мm, [biochar] = 1.0 g/L, T = 25 • C, pH = 6.5.
1,4-Dioxane 84.2% (BC-800, 250 min) [START_REF] Ouyang | Activation mechanism of peroxymonosulfate by biochar for catalytic degradation of 1,4-dioxane: important role of biochar defect structures[END_REF] Lychee branch biochar

[PS] = 8 mM, [BPA] = 5 mg/L, T = 25 • C, BC = 1 g/L, pH = 7.
Bisphenol A (BPA) 82.68% (BC-600, 120 min) [START_REF] Liu | Activation of persulfate with biochar for degradation of bisphenol A in soil[END_REF] Sludgederived biochar (SDBC)

[PDS] = 1.5 mM, [SMX] = 40 μM, SDBC dosage = 2.0 g/L, pH ≈ 5.0, T = 25 • C. Sulfamethoxazole (

Acid orange 7 (AO7)

Over 90% (SBC-700, 180 min) [START_REF] He | Persulfate activation with sawdust biochar in aqueous solution by enhanced electron donor-transfer effect[END_REF] removal via both free-radical and non-radical pathways [START_REF] Liu | Egg shell biochar-based green catalysts for the removal of organic pollutants by activating persulfate[END_REF]. The catalytic performance of biochar materials can be further improved by involving metals; for instance, Fe 3 O 4 coated straw biochar increased the removal of tetracycline hydrochloride to more than 88% over a wide pH range of 3-11 [START_REF] Huang | Efficient activation of persulfate by a magnetic recyclable rape straw biochar catalyst for the degradation of tetracycline hydrochloride in water[END_REF]. Fe/Mn biochar achieved high-efficiency activation of persulfate for the removal of reactive blue 19 [START_REF] Qiu | Degradation of anthraquinone dye reactive blue 19 using persulfate activated with Fe/Mn modified biochar: radical/non-radical mechanisms and fixed-bed reactor study[END_REF]. The specific reaction conditions and the introduction of biochar combined with persulfate to remove different organics in other literature were listed in the Table 1.

Most investigations on persulfate and biochar have been performed on pure compounds such as antibiotics, bisphenol A, and 1,4-dioxane in pure conditions, e.g. dilution in pure water [START_REF] Ouyang | Degradation of 1,4-dioxane by biochar supported nano magnetite particles activating persulfate[END_REF][START_REF] Shen | Catalytic degradation of sulfamethoxazole by persulfate activated with magnetic graphitized biochar: multiple mechanisms and variables effects[END_REF][START_REF] Zhao | Simultaneous removal of bisphenol A and phosphate in zero-valent iron activated persulfate oxidation process[END_REF]. However, the results are not representative of real natural water containing substantial amounts of humic substances. Therefore, here we tested the removal of humic acids using persulfate combined with corn stalk biochar. We used both pure aqueous solutions and real water from the Yangtze River. We monitored fluorescence, UV and total organic carbon (TOC) to follow changes in composition and removal of humic substances, respectively. We also carried out quench experiments to identify involved radical to infer possible mechanisms.

Table 1

The removal efficiency of different biochar combined with persulfate.

HA removal was determined by a UV-vis spectrophotometer (UV-2600, SHIMADZU, China) at 254 nm (UV 254 ). The HA removal efficiency R HA (%) was calculated using Eq. ( 1).

R HA (%) = C t /C 0 (1)
where C 0 and C t represent HA concentration (mg/L) at the initial and time t, respectively. R HA (%) refers to the removal rate (%) of HA. Total organic carbon (TOC) was measured by a TOC analyzer (Muliti N/C 3100, Jena Analytical Instrument Co, Ltd), with Eq. ( 2) to calculate the removal rate:

R TOC = TOC t /TOC 0 (2)
where TOC 0 and TOC t represent TOC concentration (mg/L) at the start time and time t, respectively. EEMs of samples were determined by a fluorescence spectrophotometer (F-7000, Hitachi, Japan) to study the composition ----

Removal of HAs by adsorption and oxidation

We compared the removal of humic acids in water using persulfate, corn stalk biochar, and biochar combined with persulfate by determining the concentration of humic acids in the solution (Fig. 3a). With persulfate alone, the removal rate of humic acids was very low, of less than 5%, indicating that the oxidation activity of persulfate alone at 25 • C is very weak, in agreement with the results of [START_REF] Liu | Activation of persulfate with biochar for degradation of bisphenol A in soil[END_REF]. With corn stalk biochar at 0.5 g/L, 57% of humic acids were removed at pH 8 after 180 min, indicating the high adsorption capacity of biochar. With corn stalk biochar combined with persulfate, humic acids removal reached 88%, indicating the synergic effect between biochar and persulfate.

We further assessed the removal of humic acids by measuring the total organic carbon (TOC) of the solution (Fig. 3b). Overall, our findings showed that humic acid removal was much higher with combined biochar and persulfate treatment. TOC removal was low with persulfate alone, of 5.1%, implying the concurrence of oxidation, with corn stalk biochar removal increased to 45.9% by adsorption. Application of both biochar and persulfate brought about HA removal of 71.9%, which was much higher than the sum of previous runs (51%). The results implied the occurrence of other mechanisms such as activation of persulfate by humic acids and biochar [START_REF] Liu | Activation of persulfate with biochar for degradation of bisphenol A in soil[END_REF][START_REF] Fang | Activation of persulfate by quinones: free radical reactions and implication for the degradation of PCBs[END_REF], thus improved adsorption on biochar surface following the reaction of biochar with persulfate [START_REF] Fan | Effects of chemical oxidation on surface oxygen-containing functional groups and adsorption behavior of biochar[END_REF].

We analyzed the composition of humic acid solutions treated by persulfate, biochar and biochar combined with persulfate by threedimensional fluorescence excitation-emission matrix (3D-EEM) (Fig. 3c, d, e andf). Fluorescence spectra can be divided into four sectors (Table S1), consisting of humic acids, fulvic acids, soluble microbial products (SMP) and protein-like material such as tryptophan and tyrosine [START_REF] Chen | Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter[END_REF]. Results of the initial humic solution showed three main spots corresponding to humic acids, fulvic acids and protein-like materials. Dosing persulfate alone reduced the fluorescence intensity slightly, which strengthened the minor contribution of oxidation of previous results. In contrast, adding either biochar or biochar and persulfate together induced a disappearance of humic and fulvic acid spots, in agreement with the high removal results in Fig. 3a andb. characteristics of HA by scanning the emission spectra from 250 nm to 550 nm at 5 nm wavelength increments and the excitation wavelengths from 200 nm to 500 nm at 5 nm wavelength increments. The scanning speed was set at 12,000 nm/min. The pH values were determined using a pH meter (PXSJ-226, LEICI, China).

Effects of concentrations and pH on the removal of HAs in pure conditions

Results and discussion

Characterization of corn stalk biochar

The morphology of biochar from corn stalks was analyzed by scanning electron microscopy (SEM). Fig. S1 presents biochar produced at different pyrolysis temperatures after soaking in HCl. The shape of biochar is hollow strip structure. The pore structure is being developed with the pyrolysis temperature increasing. After HCl treatment, more pore structures appear on the surface of biochar prepared at 900 • C (Fig. 1a andb). Previous studies have reported that post-treatment of biochar can improve characteristics of biochar more gently [START_REF] Wang | A review on persulfates activation by functional biochar for organic contaminants removal: synthesis, characterizations, radical determination, and mechanism[END_REF][START_REF] Yu | Magnetic nitrogen-doped sludge-derived biochar catalysts for persulfate activation: internal electron transfer mechanism[END_REF]. This morphology is typical of a good adsorbent and catalyst [START_REF] Wang | Preparation, modification and environmental application of biochar: a review[END_REF]. We thus used the HCl-treated biochar for next experiments.

Furthermore, Fig. 1c exhibits X-ray diffraction (XRD) patterns of biochar. Two broad diffraction peaks at around 23 • and 43 • are indexed to (002) C planes and (100) C planes respectively, suggesting thermolytic transformation of corn stalk to amorphous carbon [START_REF] Oh | Insights into the thermolytic transformation of lignocellulosic biomass waste to redox-active carbocatalyst: durability of surface active sites[END_REF]. After carbonization of the biomass to 900 • C, two board peaks became wider and weaker, implying the decomposition of partial amorphous carbon.

FTIR analysis of biochar (Fig. 1d) shows a peak at around 3410 cm -1 , which is assigned to the vibration of O-H in carboxyl and phenol [START_REF] Luo | Unveiling the mechanism of biochar-activated hydrogen peroxide on the degradation of ciprofloxacin[END_REF]. The appearance of peaks at 2969 cm -1 and 2884 cm -1 are due to the stretching of the aliphatic functional group C-H [START_REF] Zhu | Preparation of magnetic porous carbon from waste hydrochar by simultaneous activation and magnetization for tetracycline removal[END_REF]. The existence of the elongation of C O and C C functional groups might be proved by the peak around 1606 cm -1 [START_REF] Warnock | Mycorrhizal responses to biochar in soilconcepts and mechanisms[END_REF]. The distinct peak at about 1081 cm -1 corresponds to the C -O vibration of carboxyl and phenol [START_REF] Zhang | Sustainable mechanisms of biochar derived from brewers' spent grain and sewage sludge for ammonia-nitrogen capture[END_REF]. An aromatic C-H bending deformation is observed at 760 cm -1 and 820 cm -1 [START_REF] Dong | Mechanistic investigation of mercury sorption by Brazilian pepper biochars of different pyrolytic temperatures based on X-ray photoelectron spectroscopy and flow calorimetry[END_REF]. We observed that the oxygen-contained functional groups decreased with the increase of pyrolysis temperature from 400 • C to 900 • C, especially for O -H around 3410 cm -1 . For biochar produced at 900 • C, after HCl modification, the intensities of the functional groups such as -OH, C -O and C=O/C=C increased significantly. After reaction, the intensity of peaks at 3410 cm -1 , 1606 cm -1 and 1081 cm -1 decreased. Pan et al. summarized recent literature and found that the Ocontaining functional groups including -OH, -C=O-, -COOH on the surface of biochar are important active sites for activating persulfate [START_REF] Pan | Preparation of biochar and biochar composites and their application in a Fenton-like process for wastewater decontamination: a review[END_REF]. Based on the above results, we inferred that the oxygen-containing functional groups on the surface of biochar might be involved in the activation of persulfate.

The adsorption-desorption curve of corn biochar prepared at 900 • C shows a type-IV adsorption/desorption isotherm (Fig. 1e), suggesting the existence of mesoporous structure in the biochar [START_REF] Shen | Catalytic degradation of sulfamethoxazole by persulfate activated with magnetic graphitized biochar: multiple mechanisms and variables effects[END_REF]. When the pyrolysis temperature increased from 400 • C to 900 • C, the specific surface area and total pore volume of biochar was 3.05 m 2 /g, 4.42 m 2 /g, 22 m 2 /g, 459.260 m 2 /g, 0.01 cm 3 /g, 0.015 cm 3 /g, 0.026 cm 3 /g and 0.37 cm 3 /g, respectively. The increase of pyrolysis temperature promoted the increase of specific surface area and the total pore volume due to the gradual transformation of corn straw into porous carbon structure and the partial decomposition of amorphous carbon, accompanied by the loss of volatile substances [START_REF] Wang | Characteristics of maize biochar with different pyrolysis temperatures and its effects on organic carbon, nitrogen and enzymatic activities after addition to fluvo-aquic soil[END_REF][START_REF] Zhao | Distribution and evolution of organic matter phases during biochar formation and their importance in carbon loss and pore structure[END_REF]. Analysis of surface-charge properties show that the zeta potential of biochar-900 • C is about 7.0, indicating a pH neutral surface (Fig. 1f).

Fig. 2 shows the biochar produced at different pyrolysis temperature and used to combine with persulfate to remove humic acid. With the increase of pyrolysis temperature, the ability of biochar combined with persulfate to remove humic acid increased. When the carbonization temperature reached 900 • C, the removal rate increased significantly to 89% within 120 min. Therefore, biochar pyrolyzed at 900 • C was used to combine persulfate for subsequent experiments.

47.34% to 86.70% with biochar concentration from 0.1 g/L to 0.9 g/L after 120 min. This trend was probably due to the increase in the number of active sites according to [START_REF] Wang | Metal-free activation of persulfates by corn stalk biochar for the degradation of antibiotic norfloxacin: activation factors and degradation mechanism[END_REF]. When the dosage was above 0.5 gbiochar/L, the increase of humic acid removal was not obvious and the difference of the final removal rate was very small. In terms of cost, 0.5 g/L was used as the dosage of biochar in the subsequent tests.

Fig. 4b demonstrated that humic removal increased from 70.11% at 0.2 mM persulfate to 85.06% at 1 mM persulfate, then slightly decreased to 80.46% at 2 mM persulfate after 180 min. The increase was explained by the generation of more free radicals that react with humic acids [START_REF] Zhang | Persulfate activation using Co/AC particle electrodes and synergistic effects on humic acid degradation[END_REF]. The following decrease was due to the limited biochar active sites and the quenching reaction with an excess of SO 4 -• (Eq. ( 3)) [START_REF] Yang | Activated carbon catalyzed persulfate oxidation of Azo dye acid orange 7 at ambient temperature[END_REF]. Moreover, excess free radicals can react with persulfate and inactivate (Eq. ( 4)) [START_REF] Yu | Free radical reactions involving Cl • , Cl2-• , and SO4-• in the 248 nm photolysis of aqueous solutions containing S2O82-and Cl[END_REF], thus suppressing the removal of humic acids. In addition, persulfate may decompose during the reaction according to [START_REF] Wang | Removal of carbamazepine from aqueous solution using sonoactivated persulfate process[END_REF]. In summary, persulfate should be controlled within an appropriate range. Therefore, 1 mM was used as the PS concentration selected in the subsequent experiments.

SO 4 -+ SO 4 -→S 2 O 8 2- (3)
S 2 O 8 2-+ SO 4 -→SO 4 2-+ S 2 O 8 - (4)
Fig. 4c showed that pH variations from 5.0 to 9.0 had only minor effect on the removal of humic acids, which reached 90.7% at pH 5.0 and 90.1% at pH 9.0 within 180 min. Those trends were explained by the following processes: persulfate treatment induced the release of H + , thus 2-. Humic acids were less charged due to the increase of H + , and were easily adsorbed in turn [START_REF] Yang | Enhanced humic acid degradation by Fe3O4/ultrasound-activated peroxymonosulfate: synergy index, non-radical effect and mechanism[END_REF]. Fig. 4d showed that removal rate of humic acid increased with its initial concentration. This was explained by an excess of humic acids versus active reaction sites. Overall, our findings showed that humic acid removal increased with biochar and persulfate concentration up to a limiting threshold, and that removal was little affected by pH. The final removal rate of HA according to the effect of different key factors was listed in (Tables S2, S3

and S4).

Regeneration of used biochar and treatment of river water with biochar and persulfate

We tested the efficiency of biochar in three consecutive treatments of humic acids with persulfate (Fig. S2). Results showed that humic acid removal dropped from 87.21%at the first treatment to 39.14% at the second treatment and 28.10% at the third treatment after 180 min, revealing the poor recyclability of biochar. This was supported by microscopic analysis that reveals pore destruction and decreasing pore volume (Fig. S3). The specific surface area and the total pore volume of the biochar after reaction decreased from 459.260 m 2 /g to 247.992 m 2 / g and from 0.372 cm 3 /g to 0.256 cm 3 /g, respectively. X-ray diffraction also showed that peaks at 23 • and 43.0 • are weakened (Fig. 1c). These observations were similar to surface coverage by organics observed previously on the surface of biochar [START_REF] Georgi | Interaction of adsorption and catalytic reactions in water decontamination processes: part I. Oxidation of organic contaminants with hydrogen peroxide catalyzed by activated carbon[END_REF]. We also tested the stability of biochar, and we found that humic removal efficiency was still good after 6 months of storage (Fig. S2). Overall, used biochar is poorly recyclable, but unused biochar is stable upon storage.

In order to explore the practical application of biochar, we applied the system of persulfate combined with biochar to the actual water body. The initial concentration of total organic carbon in the solution, of 2.43 mg/L, decreased by 69.7% after synergistic treatment of river water with biochar and persulfate (Fig. S4). 80.4% removal efficiency of UV 254 could be achieved after 180 min. Fluorescence analysis also revealed an almost complete disappearance of the four organic spots (Fig. 5). Our findings evidenced the good application of biochar and persulfate to clean river water containing moderate organic levels. The characteristics of the real water samples were listed in Table 2.

Mechanism of humic acid removal by a combination of persulfate and biochar

Previous researches have shown that SO 4 -• and • OH are the main free radicals reacting with organic substances [START_REF] Huang | Efficient activation of persulfate by a magnetic recyclable rape straw biochar catalyst for the degradation of tetracycline hydrochloride in water[END_REF][START_REF] Qiu | Degradation of anthraquinone dye reactive blue 19 using persulfate activated with Fe/Mn modified biochar: radical/non-radical mechanisms and fixed-bed reactor study[END_REF][START_REF] Zhang | One-step synthesis of biochar supported nZVI composites for highly efficient activating persulfate to oxidatively degrade atrazine[END_REF]. As common quenchers, MeOH is often used as a quencher for both [START_REF] Huang | Mn doped magnetic biochar as persulfate activator for the degradation of tetracycline[END_REF][START_REF] Liu | The promotion effect of biochar on electrochemical degradation of nitrobenzene[END_REF][START_REF] Liu | Role of radical and non-radical pathway in activating persulfate for degradation of p-nitrophenol by sulfur-doped ordered mesoporous carbon[END_REF]. Our results showed that adding TBA decreased the removal of humic acids by 12.8% for a TBA/persulfate molar ratio of 500/1 and by 15.6% for 1000/1 (Fig. 6) after 180 min. By contrast, MeOH addition showed only a minor quenching effect of 4.8% for a 1000/1 molar ratio of MeOH/persulfate within the same time.

Since TBA has much higher affinity for • OH than MeOH [START_REF] Cao | Ascorbic acid induced activation of persulfate for pentachlorophenol degradation[END_REF], our findings suggested that • OH are the dominant free radicals. This hypothesis was supported by similar trends observed in previous reports [START_REF] Lykoudi | Degradation of sulfamethoxazole with persulfate using spent coffee grounds biochar as activator[END_REF][START_REF] Chen | Removal of tetracycline via the synergistic effect of biochar adsorption and enhanced activation of persulfate[END_REF][START_REF] Huang | Activation of persulfates by carbonaceous materials: a review[END_REF]. Overall, the results suggested that both • OH and, to a lesser extent, SO 4 -• , were involved in the reactions.

Biochar played an important role in the whole reaction, participating in the adsorption and oxidation process in the persulfate and biochar system. Pyrolysis temperature can significantly change the composition, structure and functional groups of biochar [START_REF] Song | Preparation of montmorillonite modified biochar with various temperatures and their mechanism for Zn ion removal[END_REF]. With the increase of pyrolysis temperature from 400 • C to 900 • C, biochar generated more pore structure and bigger specific surface area, providing more active sites for the removal of organic matter in the process of adsorption and oxidation [START_REF] Pan | Preparation of biochar and biochar composites and their application in a Fenton-like process for wastewater decontamination: a review[END_REF][START_REF] Zhang | Fabrication of L-cysteine stabilized alpha-FeOOH nanocomposite on porous hydrophilic biochar as an effective adsorbent for Pb(2+) removal[END_REF]. Previous studies have reported that high specific surface area and appropriate pore structure can provide channels for material dispersion and improve mass transfer [START_REF] Li | Performance of lead ion removal by the three-dimensional carbon foam supported nanoscale zero-valent iron composite[END_REF][START_REF] Shukla | Lignocellulosic biomass-based engineered biochar composites: a facile strategy for abatement of emerging pollutants and utilization in industrial applications[END_REF]. After HCl modification, the impurities on the surface of biochar were better removed, and more oxygen-containing functional groups were introduced, improving the adsorption and catalytic properties of biochar.

On the one hand, the adsorption of biochar was significant, which can perform physical adsorption, e.g. encapsulation and electrostatic interactions, or chemical adsorption, e.g. by H and covalent bonds occurred on the surface of biochars to adsorb humic acid and persulfate [START_REF] Huang | Nonnegligible role of biomass types and its compositions on the formation of persistent free radicals in biochar: insight into the influences on Fenton-like process[END_REF][START_REF] Zhang | Efficient phosphorus recycling and heavy metal removal from wastewater sludge by a novel hydrothermal humification-technique[END_REF]. On the other hand, the inhibitory effect of alcohols on the removal rate of the reaction suggested that SO 4 -• and • OH were involved in the reaction process. XRD analysis of biochar showed the presence of sp 2 carbons at 43.0 • (Fig. 1c), facilitating the transfer of electrons [START_REF] Lee | Activation of persulfates by graphitized nanodiamonds for removal of organic compounds[END_REF]. FTIR analysis of biochar showed the presence of O-bearing groups. According to reports, the oxygen-containing functional groups contained in biochar can be used as electron transfer media to promote the generation of free radicals [START_REF] Kimura | Discovery of the activated-carbon radical AC+ and the novel oxidation-reactions comprising the AC/AC+ cycle as a catalyst in an aqueous solution[END_REF]. Therefore, we proposed the main mechanism for the synergistic removal of HA in water with biochar and persulfate (Fig. 7). Firstly, humic acids and persulfate migrate from the liquid phase to the biochar surface and are adsorbed. The oxygen-containing functional groups on the surface of biochar act as the medium of electron transfer, so that persulfate is provided with electrons, leading to the breakage of O-O bond in persulfate; SO 4 -• is produced (Eqs. ( 5), ( 6)), which continues to react in solution to generate other kinds of active free radicals to degrade humic acid and its intermediate products (Eqs. ( 7), ( 8)) [START_REF] Qiu | Degradation of anthraquinone dye reactive blue 19 using persulfate activated with Fe/Mn modified biochar: radical/non-radical mechanisms and fixed-bed reactor study[END_REF].

Biochar

Conclusion

In summary, this work prepared a high-temperature corn stalk biochar that was easy to obtain and synthesize, which had relatively large surface area and abundant oxygen-containing functional groups.

[

  PS] = 9 mmol/L, [SBC] = 1.5 g/L, [AO7] = 50 mg/L, pH 2-10.

Fig. 1 .

 1 Fig. 1. Scanning electron microscopy (SEM) images of biochar-900 • C(a) raw biochar, (b) biochar after HCl treatment; (c) X-Ray diffraction (XRD) and (d) Fourier transform infrared spectroscopy (FTIR) of biochar prepared at 400 • C, 600 • C, 700 • C and 900 • C after HCl treatment, in raw form ('Unactivated') and after three successive treatments ('Used') of a solution of 5 mg/L humic acids (HA) and 1 mM persulfate at pH 8.0 with 0.5 g/L biochar; (e) nitrogen adsorption-desorption isotherms and (f) surface zeta potential of biochar-900 • C.

Fig. 4a demonstrated

  Fig. 4a demonstrated that humic acids removal increased from

Fig. 2 .

 2 Fig. 2. Humic acid removal of biochar at different pyrolysis temperatures in the condition of 5 mg/L humic acids (HA) and 1 mM persulfate at pH 8.0 with 0.5 g/L biochar.

Fig. 3 .

 3 Fig. 3. a) Relative concentration of humic acids and b) aqueous total organic carbon (TOC) removal after 180 min, in solutions containing persulfate (PS), biochar or biochar and persulfate. Fluorescence spectra of solutions containing c) initial humic acids, d) humic acids and persulfate, e) humic acids and biochar, f) humic acids, biochar and persulfate after 180 min of reaction.

Fig. 4 .

 4 Fig. 4. Effects of a) biochar dosage, b) persulfate concentration, c) pH, and d) humic acids concentration on the removal of humic acids. Conditions: a) 5 mg/L humic acids, 1 mM persulfate, pH 8.0; b) 5 mg/L humic acids, 0.5 g/L biochar, pH 8.0; c) 5 mg/L humic acids, 0.5 g/L biochar, 1 mM persulfate, d) 0.5 g/L biochar, 1 mM persulfate, pH 8.0.

Fig. 5 .

 5 Fig. 5. Fluorescence spectra of river water before (a) and after treatment with 0.5 g/L biochar and 1 mM persulfate (b) of 180 min.

Fig. 6 .

 6 Fig. 6. Relative concentration of humic acids after addition of radical scavengers tert-but alcohol (TBA) (a) and methanol (MeOH) (b) in reactions of humic acids with biochar and persulfate.

Fig. S3 .

 S3 Fig. S3. Scanning electron microscopy (SEM) of the biochar surface after reaction of a) biochar and humic acids and b) biochar, persulfate and humic acids.

Fig. S4 .

 S4 Fig. S4. Aqueous total organic carbon (TOC) removal of river water using 0.5 g/L biochar and 1 mM persulfate.

Table 2

 2 The characteristics of the real water samples.

	Parameters	Average value
	pH	8.48
	Conductivity (μS/cm)	285
	Total organic carbon (TOC) (mg/L)	2.43
	Dissolved oxygen (DO) (mg/L)	8.87
	Oxidation-reduction potential (mV)	18.8
	Total dissolved solids (TDS) (mg/L)	216.8

Acknowledgments

We gratefully acknowledged the co-funding of this work by the National Natural Science Foundation of China (No. 52070130) and the Shuguang Project of Shanghai (Education and Scientific Research Project of Shanghai, 18SG45).

Declaration of competing interest

The authors have declared no conflict of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.jwpe.2021.102428.

Supporting Information

Removal of humic substances by the synergistic effect of biochar adsorption and activation of persulfate

Hongbo Liu


Pierre-Henry Gabrielle
email: phgabrielle@gmail.com

Vuong Nguyen

Jennifer J Arnold

Sanjeeb Bhandari

Francesco Viola

Odette A M Tigchelaar-Besling

Gonzaga Garay-Aramburu

Louise O'toole

Ming Gemmy Chui

Daniel Cheung

Barthelmes

Cheung

Daniel Barthelmes

Catherine Creuzot-Garcher

Mark Gillies

Three-Year Outcomes of Neovascular Age-Related Macular Degeneration in Eyes That Do Not Develop Macular Atrophy or Subretinal Fibrosis

Keywords: macular atrophy, subretinal fibrosis, age-related macular degeneration, VEGF inhibitors

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The end-stage sequelae of neovascular age-related macular degeneration (nAMD), macular atrophy (MA), and subretinal fibrosis (SRFi) increase with time, are untreatable, and are associated with poor visual outcomes. [START_REF] Teo | Prevalence and risk factors for the development of physician-graded subretinal fibrosis in eyes treated for neovascular age-related macular degeneration[END_REF][START_REF] Daien | Prevalence and characteristics of macular atrophy in eyes with neovascular age-related macular degeneration. A study from a long-term observational dataset: the Fight Retinal Blindness! project[END_REF][START_REF] Nguyen | Characterization of poor visual outcomes of neovascular age-related macular degeneration treated with anti-vascular endothelial growth factor agents[END_REF][START_REF] Rofagha | Seven-year outcomes in ranibizumabtreated patients in ANCHOR, MARINA, and HORIZON: a multicenter cohort study (SEVEN-UP)[END_REF][START_REF] Okeagu | Principal cause of poor visual acuity following neovascular age-related macular degeneration: AREDS2 Report Number 23[END_REF] Both clinical trials and observational studies tend to find that visual acuity (VA) improves from baseline for six months after starting treatment with vascular endothelial growth factor (VEGF) inhibitors and then progressively declines thereafter in association with the development of foveal MA or SRFi, with final vision depending mainly on the presenting VA at the start of the treatment and the number of injections. [START_REF] Arnold | Two-year outcomes of "treat and extend" intravitreal therapy for neovascular age-related macular degeneration[END_REF][START_REF] Gillies | Long-term outcomes of treatment of neovascular age-related macular degeneration: data from an observational study[END_REF][START_REF] Monés | Undertreatment of neovascular age-related macular degeneration after 10 years of anti-vascular endothelial growth factor therapy in the real world: the need for a change of mindset[END_REF] Older age, presenting VA, and type of choroidal neovascularization (CNV) may predict risk of progression to MA and SRFi under treatment more strongly than treatment strategy and frequency. [START_REF] Teo | Prevalence and risk factors for the development of physician-graded subretinal fibrosis in eyes treated for neovascular age-related macular degeneration[END_REF][START_REF] Spooner | Macular atrophy incidence and progression in eyes with neovascular age-related macular degeneration treated with vascular endothelial growth factor inhibitors using a treat-and-extend or a pro re nata regimen: four-year results of the MANEX Study[END_REF][START_REF] Cho | Progression of macular atrophy in patients undergoing antivascular endothelial growth factor therapy for neovascular age-related macular degeneration[END_REF] Few studies, if any, have investigated whether there is any other mechanism that causes visual loss in eyes with nAMD independently of these features. Here, we tested the hypothesis that eyes with nAMD treated with VEGF inhibitors continue to lose vision through unknown mechanisms, even if they do not develop SRFi or MA.

Methods

Design and Setting

This was a retrospective analysis of treatment-naïve eyes that had received intravitreal VEGF inhibitors for nAMD in routine clinical practice tracked in the prospectively designed observational database-The Fight Retinal Blindness! (FRB!) registry. The details of the FRB! database have been previously published. [START_REF] Gillies | Efficient capture of high-quality data on outcomes of treatment for macular diseases: the fight retinal blindness! Project[END_REF] Analyzed data are 100% completed because all fields must be filled out with in-range values before being accepted by the database. Participants in this analysis included patients from Australia, France, Ireland, Italy, the Netherlands, New Zealand, Singapore, Spain, and Switzerland. Institutional approval was obtained from the University of Sydney, the Royal Australian and New Zealand College of Ophthalmologists, the French Institutional Review Board (IRB) (Société Française d'Ophtalmologie IRB), the Mater Private Hospital IRB in Dublin, Ireland, the Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Milan, Comité de Ética de la Investigación con medicamentos de Euskadi (CEIm-E), and Agencia Española de Medicamentos y Productos Sanitarios (AEMPS), Singhealth Singapore, and the Cantonal Ethics Committee Zurich.

All patients gave their informed consent. Informed consent ("opt-in consent") was sought from patients in France, Ireland, Italy, the Netherlands, Singapore, Spain, and Switzerland. Ethics committees in Australia and New Zealand approved the use of "opt-out" patient consent. This study adhered to the Declaration of Helsinki's tenets and followed the Strengthening the Reporting of Observational Studies in Epidemiology statements for reporting observational studies. [START_REF] Von Elm | The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies[END_REF]

Data Sources and Measurements

We analyzed data from the nAMD module of the FRB! outcomes registry. Data were obtained from each clinical visit, including the VA, the activity of the underlying choroidal neovascularization (CNV) lesion, the presence of SRFi or MA, treatment given, procedures, and ocular adverse events. Distance VA (uncorrected, corrected and pinhole if required) was measured in Snellen chart and converted as the number of letters read on a logarithm of the minimum angle of resolution (logMAR) VA standard ETDRS chart. [START_REF] Dong | Measurement and analysis of visual acuity in multicen-ter randomized clinical trials in the United States: Findings from a survey[END_REF] The activity of the CNV lesion was graded by the treating physician based on findings from clinical examination according to a definition provided in the data collection screen from optical coherence tomography (OCT) and dye-based fundus fluorescein angiography, alone or in combination, at each visit. Physician grading of MA and SRFi was implemented in April 2016 into FRB! to comply with the International Consortium for Health Outcomes Measurements (ICHOM) macular degeneration standard set and was recorded prospectively at each visit from then: these data were retrospectively entered for eyes with data entered before this date (n= 245 eyes). [START_REF] Rodrigues | Defining a minimum set of standardized patient-centered outcome measures for macular degeneration[END_REF] No distinction was made between nonfibrotic scar and fibrotic scar in the grading, [START_REF] Daniel | Development and course of scars in the comparison of age-related macular degeneration treatments trials[END_REF] because the diagnosis was based on the concordance between the appearance of SRFi on clinical examination, color fundus photography, and SD-OCT. At each visit, documentation of MA and SRFi was recorded according to the ICHOM standard set as: "Not present" or if present, based on location: "Extrafoveal" or "Subfoveal." [START_REF] Rodrigues | Defining a minimum set of standardized patient-centered outcome measures for macular degeneration[END_REF] Repeat treatments were at the physician's discretion in consultation with the patient, thereby reflecting routine clinical practice.

Patient Selection and Definitions

Treatment-naïve eyes starting intravitreal injection of VEGF inhibitors of either aflibercept (2 mg Eylea; Bayer Healthcare, Leverkusen, Germany), bevacizumab (1.25 mg Avastin; Genetech Inc/Roche, Basel, Switzerland), or ranibizumab (0.5 mg Lucentis; Genetech Inc/Novartis) for nAMD from January 1, 2010, to September 1, 2017, thereby allowing the possibility of having at least 36 months of observations after the initial treatment, and who did not have SRFI or MA at baseline were tracked in the registry. Eyes were excluded if the grading of SRFI or MA was not entered at baseline.

To ensure that eligible eyes did not have SRFI or MA at presentation, the baseline grading of SRFi or MA was based on multimodal imaging at each visit from the start of the treatment to the three-month visit to detect eyes with undiagnosed SRFi or MA at the beginning of the treatment because of intense exudative signs and exclude other reasons of subretinal hyperreflective material such as fibrin or hemorrhage.

Three groups were defined based on the physician grading of SRFi or MA over 36 months of treatment: absence (i.e., eyes that did not develop SRFi or MA graded over 36 months), subfoveal SRFI or MA (i.e., eyes that developed subfoveal SRFi or MA graded over 36 months) and extrafoveal SRFI or MA (i.e., eyes that developed extrafoveal SRFi or MA graded over 36 months). Eye that developed first extrafoveal SRFi or MA and then progressed to subfoveal SRFi or MA over the period were included in the subfoveal SRFI or MA group for the analysis. Eyes that completed at least 1035 days of follow-up were defined as "completers." Eyes that did not complete 36 months of observations were defined as "non-completers."

To investigate if vision declined after the initial visual improvement, we analyzed the mean change in VA from six months to 36 months. The initial visual improvement was considered as maintained if the VA change from six months to 36 months was more than -5 letters.

Outcomes

The main outcome was the estimated mean change in VA from 6 months at 36 months. Secondary visual outcomes included the estimated mean change in VA from baseline to 36 months, the proportion of eyes who maintained vision (VA change > -5 letters), lost ≥10, and ≥15 letters of vision from six months at 36 months, and the mean final VA. Other outcomes of interest were the median time to the development of MA and SRFi over 36 months, the baseline predictors of the development of MA, and SRFi over 36 months, the proportion of visits over 36 months in which the CNV lesion was graded as active, the median time to first grading of CNV inactivity over 36 months, the median time interval between injections over 36 months, the median number of visits and injections administered over 36 months, the rate of noncompletion, the median time and mean VA change to dropout, and the reason for discontinuation over 36 months.

Statistical Analysis

Descriptive data were summarized using the mean (standard deviation), median (first and third quartiles), and number (percentages) where appropriate. Calculation of crude visual outcomes over 36 months used the last-observation-carried-forward for non-completers.

We compared visual outcomes between SRFi or MA groups over three years using mixed-effects longitudinal generalized additive models with the interaction between the development and location of MA or SRFi during the treatment and time as the main predictor variable. Longitudinal models included all visits from completers and non-completers (all observations until the 36-month visit or dropout). The proportions of eyes who maintained vision, lost 10 letters and 15 letters of vision from six months at 36 months between groups were compared using logistic mixed-effects regression models. Longitudinal and logistic models included age, gender, VA, type of CNV lesion at baseline and lens status during the followup, and nesting of outcomes within practitioners and patients with bilateral disease as random effects. Generalized Poisson regression models were used to compare the number of injections and visits between groups over 36 months. Longitudinal, logistic and generalized Poisson models included age, gender, VA, type of CNV lesion at baseline and lens status during the follow-up, and nesting of outcomes within practitioners and patients with bilateral disease as random effects. Cox-proportional hazards models and Kaplan-Meier survival curves were used to assess and visualize the time to first grading of inactivity, first grading of SRFi and MA, and non-completion rates over 36 months.

A P value = 0.05 was considered statistically significant. All analyses were conducted using R software version 4.0.2 (R Project for Statistical Computing, Vienna, Austria; R Foundation for Statistical Computing; 2020, https://cran.rproject.org) with the mgcv package (V1.8-31) for the generalized additive (mixed) model computation and the survival package (V3. 1-11) for the time to first CNV inactivity, development of SRFi and MA and drop-out analysis.

Results

We identified 2478 treatment-naïve eyes from 2218 patients who were eligible for the present analysis. The number of eyes at each selection criterion is shown in 1).

Visual Outcomes Over Three Years

The 36-month treatment outcomes of the study population are reported in Table 2. The overall crude mean (95% confidence interval [95%CI]) change in VA improved by 6 (5, 6) letters from baseline to six months (mean [SD] six-month VA = 66 [17] letters) and did not differ significantly between groups (P = 0.14) (Table 2). Subsequently, the overall mean change in VA progressively dropped by -2 (-3, -2) letters from 6 months to 36 months (mean [SD] final VA = 64 [20]

letters).

Figure 1 shows the estimated mean VA from longitudinal models in all eyes (including completers and non-completers) and according to the development of SRFi or MA and its foveal involvement. Overall, the estimated mean (95%CI) change in VA was 2 (1, 3) letters from baseline to 36 months (Table 2 and Fig. 1A). The difference in the estimated mean (95% CI) in VA change from baseline at 36 months was significantly in favor of eyes with absence or extrafoveal SRFi or MA than eyes with subfoveal SRFi or MA (14 [13, 16] letters for absence vs. subfoveal, P < 0.01; 12 [10, 14] letters for extrafoveal vs. subfoveal, P < 0.01) (Table 2 and Figs. 1B and1C). Overall, the estimated mean (95% CI) change in VA from six months at 61 (18) 63 (17) 62 (16) 51 (22) VA at 6 months letters, mean (SD) 66 (17) 69 (15) 69 (13) 56 (21) Final VA, mean (SD) 64 (20) 68 (17) 65 (18) 48 (26)

Crude VA change from baseline to 6 months letters, mean (95% CI)

+6 (5, 6) +6 (5, 6) +7 (5, 8) +5 (3, 6)
Crude VA change at 36 months letters, mean (95% CI) a From baseline

+3 (2, 4) +5 (4, 5) +3 (1, 6) -4 (-6, -1)
From 6 months

-2 (-3, -2) -1 (-1, 0) -4 (-5, -2) -8 (-10, -6)
Estimated VA change at 36 months letters, mean (95% CI) b From baseline 36 months was -4 (-5, -3) letters (Table 2). There was a clinically significant drop in the estimated VA from 6 months at 36 months in eyes developing extrafoveal or subfoveal SRFi or MA over 36 months with -3 (-5, -2) and -10 (-11, -8) letters, respectively; while the estimated VA stabilized from six months to 36 months in eyes that did not develop SRFI or GA with -1 (-2, 0) letters at 36 months (Table 2, Fig. 1D).

+2 (1, 3) +6 (5,
Eyes with no or extrafoveal SRFi or MA over 36 months were more likely to maintain their visual improvement from 6 months to 36 months (odds ratio OR [95% CI] = 2.3 [1.5, 3.3] for absence vs. subfoveal SRFi or MA, P ≤ 0.01 and OR = 2.0 [1.2, 3.4] for extrafoveal vs. subfoveal MA or SRFi, P = 0.01) (Table 2). Those eyes were also less likely to lose ≥10 and ≥15 letters of vision from 6 months to 36 months than eyes that developed subfoveal SRFi or MA over 36 months (Table 2). No difference was found between eyes with no SRFi or MA and those that developed extrafoveal SRFi or MA (Table 2).

One hundred sixty-nine eyes underwent cataract extraction, and 15 eyes underwent vitrectomy over three years of treatment. Eyes that did not develop SRFi or MA and developed extrafoveal SRFi or MA tended to have better visual outcomes if they had cataract surgery during the 36 months treatment (See Supplementary Table S1). Crude mean VA change from baseline (-1 [-17, 14] letters vs. 3 [2, 4] letters;

Subretinal Fibrosis or Macular Atrophy Development Over Three Years

Kaplan-Meier estimates the cumulative rate of SRFi and MA and according to their foveal involvement over three years of treatment (Fig. 2). The cumulative rate of SRFi was 11% (8.5% subfoveal vs. 2.5% extrafoveal) at 12 months, 16.5% (12.7% subfoveal, 3.8% extrafoveal) at 24 months, and 21.3% (16.2% subfoveal, 5.2% extrafoveal) at 36 months (Fig. 2A). The cumulative rate of MA was 9.9% (4.9% subfoveal, 5% extrafoveal) at 12 months, 17.9% (8.2% subfoveal, 9.7% extrafoveal) at 24 months, and 24.5% (11.1% subfoveal, 13.4% extrafoveal) at 36 months (Fig. 2B). Eyes with worse baseline VA and type 2 CNV were more likely to develop SRFi over 36 months (hazard ratio [HR] = 0.76 [0.70, 0.83] every 10 letters of increase in baseline VA, P < 0.01 and HR = 1.5 [1.2, 1.8] for type 2 vs. type 1 CNV, P = 0.05 [Tukey adjusted P = 0.047 for multiple comparisons]). Older patients and eyes with worse baseline VA and baseline Type 3 CNV were more likely to develop MA over 36 months of treatment (HR = 1.29 [1.14, 1.45] every 10 years of increase in presenting age, P < 0.01; HR = 0.82 [0.76, 0.89] every 10 letters of increase in baseline VA, P < 0.01; HR = 1.9 [1.5, 2.4] for type 3 vs. type 1 CNV, P = 0.021 [Tukey adjusted P = 0.018]). That is, the incremental likelihood of developing MA over 36 months increased by 18% every 10 ETDRS letters worse the baseline vision and by 29% each additional 10 years in age at the start of the treatment.

Choroidal Neovascularization Activity Outcomes Over Three Years

Overall, the proportion of active visits in eyes completing 36 months was 55%, lower in eyes that developed extrafoveal SRFi or MA (43.3%) than eyes that did not develop (56%) or developed subfoveal SRFI or MA (56%) (P = 0.044). The median (Q1, Q3) time to first grading of inactivity was 119 (82, 385) days and was not significantly different between subgroups (Fig. 3).

Injections and Visits Over Three Years

The median (Q1, Q3) number of injections was 19 (14, 25) over three years in completers with 8 (6, 10), 6 (3, 8) and 5 (3, 8) median (Q1, Q3) injections yearly at first, second, and third year, respectively (Table 2). The adjusted ratio (95% CI) of the number of injections and visits from the generalized Poisson regression model was similar between subgroups according to the development of SRFi or MA in eyes completing 36 months (Table 2). Eyes that did not develop SRFi or MA tended to be more aggressively treated (adjusted ratio third-year injection yearly [95% CI] = 1.2 [1.0, 1.3] for absent vs. extrafoveal, P = 0.011 and 1.1 [1.0, 1.3] for absent vs. subfoveal, P = 0.012) and monitored (adjusted ratio third-year visit yearly [95% CI] = 1.1 [1.0, 1.2] for absent vs. extrafoveal, P < 0.01 and 1.1 [1.0, 1.2] for absent vs. subfoveal, P < 0.01) during the third year than eyes that developed extrafoveal and subfoveal SRFi or MA over 36 months of treatment (Table 2).

Outcomes of Eyes not Completing Three Years

The overall non-completion rate over 36 months was 45.4% (1125 eyes) and was more frequent in eyes that did not develop SRFi or MA than eyes that developed extrafoveal or subfoveal SRFi or MA (50% absence vs. 30% extrafoveal vs. 38% subfoveal, P < 0.01; see Supplementary Fig. S2). The mean VA at drop out was significantly better than the mean baseline VA each year of drop out (Supplementary Fig. S3). The reasons for patients discontinuing treatment were tracked in 141 (14%) eyes. These were mainly not related to a poor outcome (71%, 100 eyes): treatment considered as successful 39% (54 eyes), patient transferred to another doctor 14% (20 eyes), death 14% (20 eyes), and medical contraindication 4% (six eyes).

Discussion

The present study reports that treatment-naïve nAMD eyes receiving VEGF inhibitors maintain their initial six-month visual improvement over three years of treatment in routine clinical practice if they do not develop SRFI or MA. This is not necessarily surprising, because SRFi and MA are well-known associations of poor long-term visual outcomes in treated nAMD eyes. [START_REF] Daien | Prevalence and characteristics of macular atrophy in eyes with neovascular age-related macular degeneration. A study from a long-term observational dataset: the Fight Retinal Blindness! project[END_REF][START_REF] Nguyen | Characterization of poor visual outcomes of neovascular age-related macular degeneration treated with anti-vascular endothelial growth factor agents[END_REF][START_REF] Okeagu | Principal cause of poor visual acuity following neovascular age-related macular degeneration: AREDS2 Report Number 23[END_REF][START_REF] Gillies | Long-term outcomes of treatment of neovascular age-related macular degeneration: data from an observational study[END_REF][START_REF] Daniel | Development and course of scars in the comparison of age-related macular degeneration treatments trials[END_REF][START_REF] Sharma | Macular morphology and visual acuity in the second year of the comparison of age-related macular degeneration treatments trials[END_REF][START_REF] Jaffe | Macular morphology and visual acuity in the Comparison of Age-related Macular Degeneration Treatments Trials (CATT)[END_REF][START_REF] Jaffe | Macular morphology and visual acuity in year five of the Comparison of Age-related Macular Degeneration Treatments Trials[END_REF] The significance of our findings is that there is probably no other disease process that causes loss of the initial gains seen in eyes treated for nAMD. Although it would be helpful for this finding to be consolidated and extended in future studies, it appears that the prevention of MA and SRFi is the final obstacle to achieving better, enduring outcomes in nAMD.

Not surprisingly, eyes in the three groups were not comparable at baseline particularly regarding presenting VA. This may be possibly due to an increased amount of blood or fibrin at baseline in the incident SRFI or MA group. We tried to limit the inclusion of baseline SRFi or MA eyes in the study using multimodal imaging definition to differentiate these features with other causes of subretinal hyperreflective material and defined the status of baseline SRFi or MA on the first three months treatment visits.

Our results emphasize that the development or extension of MA or SRFi in the subfoveal region is associated with poor long-term visual outcomes. [START_REF] Daniel | Development and course of scars in the comparison of age-related macular degeneration treatments trials[END_REF][START_REF] Sharma | Macular morphology and visual acuity in the second year of the comparison of age-related macular degeneration treatments trials[END_REF][START_REF] Jaffe | Macular morphology and visual acuity in the Comparison of Age-related Macular Degeneration Treatments Trials (CATT)[END_REF][START_REF] Jaffe | Macular morphology and visual acuity in year five of the Comparison of Age-related Macular Degeneration Treatments Trials[END_REF][START_REF] Daniel | Risk of scar in the comparison of age-related macular degeneration treatments trials[END_REF] Eyes that developed subfoveal SRFi or MA over 36 months had at least two to three lines difference in the final estimated mean VA change from baseline, were at least half as likely to maintain six-month visual improvement at 36 months and twice as likely to have a two-line or three-line VA loss from six months at 36 months than eyes that developed extrafoveal SRFi or MA over 36 months of treatment.

Approximately 20% and 25% of eligible eyes developed SRFI and MA respectively by 36 months from the start of the treatment. These cumulative rates of SRFi and MA were similar to those reported elsewhere. [START_REF] Spooner | Macular atrophy incidence and progression in eyes with neovascular age-related macular degeneration treated with vascular endothelial growth factor inhibitors using a treat-and-extend or a pro re nata regimen: four-year results of the MANEX Study[END_REF][START_REF] Sharma | Macular morphology and visual acuity in the second year of the comparison of age-related macular degeneration treatments trials[END_REF][START_REF] Jaffe | Macular morphology and visual acuity in the Comparison of Age-related Macular Degeneration Treatments Trials (CATT)[END_REF] The Macular atrophy in Pro re Nata versus Treat-and-Extend (MANEX) study reported that the incidence of new atrophy lesion in consecutive naïve treated nAMD eyes receiving VEGF inhibitors was approximately 19% and 22% at 2 and 3 years of treatment, respectively. [START_REF] Spooner | Macular atrophy incidence and progression in eyes with neovascular age-related macular degeneration treated with vascular endothelial growth factor inhibitors using a treat-and-extend or a pro re nata regimen: four-year results of the MANEX Study[END_REF] In the comparison of AMD Treatments Trials (CATT), non-geographic atrophy and scar rates were estimated to be approximately 12 to 19% and 16 to 20% at 2 years, respectively, depending on the type of drug and treatment pattern. [START_REF] Jaffe | Macular morphology and visual acuity in the Comparison of Age-related Macular Degeneration Treatments Trials (CATT)[END_REF][START_REF] Jaffe | Macular morphology and visual acuity in year five of the Comparison of Age-related Macular Degeneration Treatments Trials[END_REF] The FRB registry has implemented the ICHOM classification grading of SRFi and MA to standardize the diagnosis of these features and compare outcomes between different reports. Our results are derived from an extensive observational database with multiple practitioners grading the clinical features, which may be less precise than in reports from RCTs such as CATT. [START_REF] Sharma | Macular morphology and visual acuity in the second year of the comparison of age-related macular degeneration treatments trials[END_REF][START_REF] Jaffe | Macular morphology and visual acuity in the Comparison of Age-related Macular Degeneration Treatments Trials (CATT)[END_REF][START_REF] Jaffe | Macular morphology and visual acuity in year five of the Comparison of Age-related Macular Degeneration Treatments Trials[END_REF] However, most of the practitioners contributing data are retina specialists who have agreed to use the ICHOM multimodal grading definition of these features. These real-world findings also reflect how diagnosis and treatment decision would be made in daily clinical practice if an effective drug were developed for preventing or treating MA or SRFi.

The three-year visual real-world outcomes of VEGF inhibitors for nAMD were reasonably good (mean +3 letters improvement from baseline) with a median number of injections yearly of eight, six, and five during the first, second, and third year of treatment. Previous retrospective observational studies have reported poorer outcomes at three years. [START_REF]Writing Committee for the UK Age-Related Macular Degeneration EMR Users Group. The neovascular age-related macular degeneration database: multicenter study of 92 976 ranibizumab injections: report 1: visual acuity[END_REF][START_REF] Rasmussen | A 4year longitudinal study of 555 patients treated with ranibizumab for neovascular age-related macular degeneration[END_REF][START_REF] Zhu | Intravitreal Ranibizumab for neovascular Age-related macular degeneration in clinical practice: five-year treatment outcomes[END_REF][START_REF] Arevalo | Intravitreal bevacizumab for choroidal neovascularization in age-related macular degeneration: 5-year results of The Pan-American Collaborative Retina Study Group[END_REF] It is difficult to compare our study to earlier reports because we included only eyes that had been diagnosed early without SRFi or MA when they started treatment.

There was no difference in treatment and visits frequency between the subgroups. However, we found that eyes with no SRFi or MA, which achieved the best visual outcomes, were more aggressively treated and monitored during the third year of treatment. This reinforces the idea that initial VA improvement in nAMD can be maintained with more intensive or proactive treatment approaches in clinical practice. [START_REF] Monés | Undertreatment of neovascular age-related macular degeneration after 10 years of anti-vascular endothelial growth factor therapy in the real world: the need for a change of mindset[END_REF] As previously reported in the literature, presenting VA was a significant predictive factor of MA and SRFi development in our study. [START_REF] Teo | Prevalence and risk factors for the development of physician-graded subretinal fibrosis in eyes treated for neovascular age-related macular degeneration[END_REF][START_REF] Daien | Prevalence and characteristics of macular atrophy in eyes with neovascular age-related macular degeneration. A study from a long-term observational dataset: the Fight Retinal Blindness! project[END_REF][START_REF] Nguyen | Characterization of poor visual outcomes of neovascular age-related macular degeneration treated with anti-vascular endothelial growth factor agents[END_REF] Type 3 CNV was associated with an increased risk of MA and type 2 CNV with an increased risk of SRFi, which has been confirmed in previous reports. [START_REF] Teo | Prevalence and risk factors for the development of physician-graded subretinal fibrosis in eyes treated for neovascular age-related macular degeneration[END_REF][START_REF] Spooner | Macular atrophy incidence and progression in eyes with neovascular age-related macular degeneration treated with vascular endothelial growth factor inhibitors using a treat-and-extend or a pro re nata regimen: four-year results of the MANEX Study[END_REF][START_REF] Cho | Progression of macular atrophy in patients undergoing antivascular endothelial growth factor therapy for neovascular age-related macular degeneration[END_REF][START_REF] Daniel | Risk of scar in the comparison of age-related macular degeneration treatments trials[END_REF] Loss to follow-up may introduce bias because eyes that discontinue may drop out because of poor outcomes or sometimes because of good response to treatment and stabilization of vision. The rate of noncompletion was, in fact, highest in eyes that did not develop SRFi or MA over 36 months of treatment. The mean VA of the eyes that dropped out tended to be better than the presenting VA when treatment had discontinued, suggesting that those eyes tended to have good visual outcomes. Most (70%) of tracked reasons for discontinuation were mainly not related to a poor outcome. Our estimated outcomes, particularly in eyes that did not develop SRFi or MA over the study, may be inferior to the actual results if patients with good vision tended to discontinue follow-up within 36 months.

We acknowledge several limitations that are mostly inherent in retrospective observational studies. Injection decisions in routine clinical practice are made without a guided management protocol, so they may vary among retinal specialists compared to RCTs. The grading of SRFi, MA, lesion type, and lesion activity may have interphysician variability. The FRB! registry receives data from a wide variety of international practices and practitioners. Thus we believe our data are fairly representative of clinicians worldwide, which may reduce potential bias caused by this variability to some extent. We also included nesting of outcomes within practitioners in our models to help account for these effects. This analysis's main strengths are its origi-nality and the large number of eyes that were studied over a long time period in daily clinical practice.

To conclude, our study suggests that SRFi and MA are the main retinal causes of the long-term (three-year) visual decline in vision in nAMD eyes in routine clinical practice. Early diagnosis and appropriate application of treatment regimens to prevent these features and their extension to the subfoveal region stabilize the visual improvement after the start of the treatment. Further research is warranted to determine whether these findings hold for longer periods. There is a need to develop new drugs with potential antifibrotic and neuroprotective effects, combined with VEGF inhibitors, to prevent or even treat these endstage features of nAMD and further improve visual outcomes and the quality of life of our patients.

  quartile; Q3, third quartile. a Last observation carried forward for non-completers. b Calculated from adjusted non-linear mixed-effects regression models. c Odds ratios (95% CI) were calculated from adjusted logistic mixed-effects regression models. d Adjusted ratio (95% CI) of number of injections or number of injections yearly or visits between groups. It was calculated from adjusted generalized Poisson regression models. e Mean differences (95% CI) were calculated from adjusted linear mixed-effects regression models. Pairwise comparison with Holm-Bonferroni adjustment was used for multiple comparisons. Significant p-values are highlighted in bold.

Figure 1 .

 1 Figure 1. Line graphs showing the mean predicted VA (solid lines) in logMAR letters with time (A) in all eyes (black dashed line) and depending on the development of MA or SRFi and its location during treatment (absence [green], extrafoveal [gold], and subfoveal [red]) and the difference in the mean change in VA from baseline between the different subgroups of SRFi or MA (B, C and D) over 36 months irrespective of whether eyes completed or did not complete 36 months of observations from starting treatment. Predictions were made from a generalized additive model for all eyes. In B, C, and D, the gray shaded area represents the 95% CI, and the red dashed lines indicate areas where the 95% CI does not intersect with 0.

Figure 2 .

 2 Figure 2. Kaplan-Meier plots for time from starting treatment to (A) the development of macular atrophy and (B) subretinal fibrosis of any location (black) and according to its location (extrafoveal [royal blue] and subfoveal [orange]) over 36 months.

Figure 3 .

 3 Figure 3. Kaplan-Meier plots for time from starting treatment to the first grading of inactivity in all eyes (black dashed line) and depending on the development of macular atrophy or subretinal fibrosis and its location during treatment (absence [green], extrafoveal [gold], and subfoveal [red]) over 36 months.

Table 1 .

 1 Baseline Characteristics of the Study Population Macular Atrophy or Subretinal Fibrosis Developed Over 36 Months of Treatment

		Overall	Absent	Extrafoveal	Subfoveal	P
	Eyes	2478	1712	291	475
	Patients	2218	1586	268	452
	Females, %	61	61	66	61	0.23
	Age, mean (SD)	78 (9)	78 (9)	78 (9)	79 (9)	0.013
	Lens status (phakic), %	77	79	74	70	<0.01
	Visual acuity, mean (SD)	61 (18)	63 (17)	62 (16)	51 (22)	<0.01
	≥70 letters, n (%)	996 (40)	777 (45)	108 (37)	111 (23)	<0.01
	≤35 letters, n (%)	271 (11)	132 (8)	24 (8)	115 (24)	<0.01
	Angiographic lesion size,	1800 (1000, 3000) 1900 (1000, 3000) 1760 (1000, 2600) 1500 (601, 2500)	0.022
	median μm (Q1, Q3) a
	Angiographic lesion type,					<0.01
	% a
	Type 1	37	39	41	28
	Type 2	14	13	16	17
	Type 3	5	5	11	4
	Polypoidal choroidal	7	8	8	4
	vasculopathy
	Peripapillary choroidal	1	2	1	1
	neovascularization
	Unavailable	35	33	23	46
	Type of VEGF inhibitors, %					<0.01
	Aflibercept	39	36	47	42
	Bevacizumab	28	28	28	26
	Ranibizumab	34	36	25	32
	Q1, first quartile; Q3, third quartile.
	Supplementary Figure S1. There were 1712, 291, and
	475 eyes that did not develop, developed extrafoveal,
	and developed subfoveal SRFi or MA, respectively,
	over 36 months. The mean (standard deviation [SD])
	presenting VA in the subfoveal SRFi or MA group
	was significantly worse than extrafoveal and no SRFI
	or MA groups (51 [22] vs. 62 [16] vs. 63 [17] letters,
	respectively; P < 0.01) with significantly fewer eyes
	with VA ≥ 70 letters and more eyes with VA ≤ 35 letters
	(Table

a Missing values: n = 1068 eyes (angiographic lesion size), n = 856 eyes (angiographic lesion type). Significant P values are highlighted in bold.

Table 2 .

 2 Thirty-Six-Month Visual Outcomes of the Study Population and by Subgroup According to the Development of Macular Atrophy or Subretinal Fibrosis

	Adjusted Group Comparison

Downloaded from tvst.arvojournals.org on 11/12/2021

Acknowledgments

Supported by an educational grant from "La Fondation de France" research fellowship program, a grant from the Macular Disease Foundation Australia, and unrestricted educational grants from Bayer and Novartis. Disclosure: P.-H. Gabrielle, None; V. Nguyen, None; J.J. Arnold, None; S. Bhandari, None; F. Viola, None; O.A.M. Tigchelaar-Besling, None; G. Garay-Aramburu, None; L. O'Toole, None; C.M.G. Cheung, None; D. Barthelmes, None; C. Creuzot-Garcher, None; M. Gillies, None


Thibaut Louise

Arnaud Molin

Nicolas Gruchy

Fontaine Paul

C Francannet

Benjamin Dauriat

Andreea Apetrei

Nicolas Richard

Gruchy

Fontaine

Two novel deletions in the 5'untranslated region of GNAS gene

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION : GNAS LOCUS & PHP1A

Disorder of maternal GNAS locus complex (Figure 1) are causes of pseudohypoparathyroidism type Ia, an autosomal dominant disease, caracterized by organ resistance to these endocrine hormones, variable degrees of intellectual disability and the Albright hereditary osteodystrophy (AHO) phenotype (short stature, round facies, obesity, subcutaneous ossifications and brachydatylie type E).

The complex GNAS locus includes various transcripts of monoallelic expressions under control of differentially methylated regions according to the parental origin. The G⍺s protein, predominantly expressed from the maternal allele is coded by exons 1 to 13 of the GNAS locus, is essential to activate the signalling path of receptors coupled with the G proteins alike the receptor of parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), gonadotropins (LH and FSH), growth hormone-releasing hormone (GHRH), and central nervous system neurotransmitters.

PATIENTS & METHODS

In this study, we investigated two affected individuals, unrelated and without relevant family history, both presenting a typical phenotype of PHP1a (figure 2, table 1).

AHO features Yes Yes

Serum calcium

↓ ↓ Serum phosphate ↑ ↑ Serum PTH ↑ ↑ Serum TSH ↑ ↑ Serum FSH/LH ↑ *

Clinical phenotype PHP1a PHP1a

Table 1 : Clinical features of the two patients. * Not documented.

Family A II 1 2 1 I II 1 2 1 2 3

GNAS locus analysis

A Sanger sequencing of the GNAS locus and a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) analysis were performed for the 2 affected subjects.

A whole genome sequencing (WGS) analysis was performed for the 2 affected patients : the library preparation used a paired-end (PE) with 2x150 bp method.

The sequencing was performed using an Illumina platform, the sequences were aligned at the reference human genome GRCh38 and the data was processed using the GATK pipeline.

Family B

In order to analyse GNAS expression, a qRT-PCR was carried out from RNAs of the probands and one healthy, sex and age paired control.

Primers spanned the coding region of exons 1 and 2.

I Th

There was no gain, deletion or mutation in the GNAS locus detected by the routine analysis.

The epigenetic analysis by MS-MLPA showed normal methylation for the two patients.

The WGS analysis revealed on the two probands two deletions of 466 bp and 1439 bp respectively with both distal breakpoints localized in 5'UTR of GNAS (Figure 3).

RESULTS

A II.1 B II.1 The deletion was confirmed by a PCR analysis of the genomic DNA using primers designed outside of the deleted regions (Figure 4, Figure 5) and by a Sanger analysis.

The qRT-PCR analysis showed a significant reduction of GNAS expression by ≃ 50% and ≃ 90% respectively in comparaison with a normal control (Figure 6).

DISCUSSION & CONCLUSION

We detected two news deletions in the 5'UTR region of the GNAS gene, of 466 bp and 1439 bp respectively, onto two female patients with a typical phenotype of PHP1a using WGS technology.

These two patients had previously benefited from sequencing of a targeted panel of genes involved in phospho-calcium metabolism disorders, carried out in our laboratory and including GNAS gene, as well as an MS-MLPA in search of rearrangements and/or methylation abnormality at the GNAS locus, all non-contributory.

As this GNAS gene region is highly repeated and particularly difficult to sequence, we report here the usefulness of WGS in these cases of undocumented rare diseases.

These observations suggest the possibility that the 5'UTR commun distal breakpoint could be a recurrent hot spot site.

Targeted analysis of this region could be implemented in routine pratice in order to detect such type of rearrangement.

richard-n@chu-caen.fr thibaut-lm@chu-caen.fr Contacts :

Figure 1 :

 1 Figure 1 : Schematic representation of GNAS complex locus, the various transcripts by parental origin and the defect of maternal G⍺s allele (adapted from Pulix M. et al, 2020).

Figure 2 :

 2 Figure 2 : Pedigrees of the two investigated families.

Figure 3 :RESULTSFigure 4 :Figure 6 :

 346 Figure 3 : Use of the Integrative Genome Viewer to show WGS (2 x 150 PE) data of the two deletions found in the same 5'UTR region of GNAS gene.

Figure 5 :

 5 Figure 5 : Schematic representation of PCR primers, their approximate locations in the wild type allle and the expected size of PCR products for the wild type and the mutants alleles.