Haitao Ren

Yue Yuan

Abdelkader Labidi

Qibing Dong

Ke Zhang

Eric Lichtfouse

Ahmed A Allam

Jamaan S Ajarem

Chuanyi Wang
email: wangchuanyi@sust.edu.cn

Green process of biomass waste derived fluorescent carbon quantum dots for biological imaging in vitro and in vivo

Keywords:

In the context of the circular economy, the huge amounts of biomass waste should be converted into value-added materials and energy to diminish pollution, atmospheric CO 2 levels and costly waste disposal. Biological imaging usually uses expensive and toxic chemicals e.g. , organic dyes, semiconductor quantum dots, calling for safer, greener, cheaper fluorescent probes for biological imaging in vitro and in vivo . In these regards, carbon quantum dots (CQDs)-based fluorescent probes using biomass waste as a precursor may have much higher potential. Here we transformed the biomass waste of peach leaves into value-added fluorescent CQDs through a low-cost and green one-step hydrothermal process. The obtained CQDs show excitation-dependent photoluminescence properties with a fluorescence lifetime of 5.96 ns and a quantum yield of 7.71% without any passivation. In addition, the CQDs have a fine size of 1.9 nm with good hydrophilicity and high fluorescent stability over pH 4.0-11.0 range. Fluorescence imaging of in vitro cell cultures and in vivo with zebrafish show that CQDs possess ultra-low toxicity and remarkable performance for biological imaging. Even when CQDs present at a concentration as high as 500 μg/mL, the organism can still maintain more than 90% activity both in vitro and in vivo , and present bright fluorescence. The cheaper, greener, ultra-low toxicity CQDs developed in this work is a potential candidate for biological imaging in vitro and in vivo .

Various organic dyes, e.g. , porphyrins, boron-dipyrromethene, indocyanines, rhodamines and phthalocyanines [1] and semiconductor quantum dots (SCQDs) such as CdSe, ZnS, ZnSe, CdS, PbS [2] have been used as fluorescent probes in vitro and in vivo biological imaging studies. Although those dyes and SCQDs have specific chemical structures with excellent photoluminescence (PL) properties, they may not be suitable for being utilized in real-time long-term imaging or cell multiplication due to security issues [3] . Their main limitations are the low photobleaching thresholds, potential toxicity, poor solubility, high costs and small Stokes shift [4] . In this regard, CQDs emerged as an alternative to fluorescence probes for biological imaging applications owing to their unique PL, low-toxicity, biocompatibility and environmentally-friendly properties [5] . However, as shown in Table S1 (Supporting information), previously the biological imaging of most reported CQDs was mainly used in vitro cell cultures. Compared with intracellular imaging, observing in vivo fluorescence imaging of CQDs is more significant.

CQDs as a new type of carbon nanomaterial have been intensively used in bio-imaging, sensing, anti-counterfeiting, optoelectronic devices, phototherapy, drug release and photo-catalysis [ 6 , 7 ]. The production of CQDs is done by top-down and bottomup methods [8] . Top-down methods break down macroscopic carbon precursors such as graphite, carbon nanotubes, carbon black, candle soot, and activated carbon, into small-sized CQDs by chemical treatment or physical cutting [9] . Bottom-up methods produce CQDs from small organic compounds through thermal decomposition, template-based routes, solvothermal/hydrothermal treatment, and ultrasonic/microwave irradiation [10] . Unfortunately, most of these strategies use expensive or toxic precursors, sophis- ticated equipment, harsh reaction conditions, complicated purification processes, useless by-products, and strong acids or alkali, resulting in manufacturing difficulties and the increase of the production cost. Therefore, there is a need for safer, efficient and costeffective methods to produce CQDs, such as biomass conversion.

Waste biomass is regarded to be low-value or useless and is often discarded, resulting in environmental pollution. About 4.3 billion tons of waste biomass are produced annually in China due to inefficient disposal [ 11 , 12 ]. Waste biomass should therefore be converted into value-added material such as carbon nanomaterials, thus reducing CO 2 emissions and environmental issues [ 13 , 14 ]. Biomasses, such as food waste, vegetables, fruits and plant residues, have been recently used as a carbon source for the production of carbon nanomaterials [ 15 , 16 ]. Peach leaves are a common biomass waste left in the field after harvesting. For instance, China is one of the major agroforestry nations in the world, with an annual production of 2 million tons of peach leaves waste. Thus, the recycling of such waste is a major problem for local farmers. The peach leaf is a renewable and sustainable carbon-rich raw material that is mainly composed of soluble carbohydrates, crude protein, crude fiber and crude fat [ 12 , 17 ]. If this resource can be recycled and effectively used, it may alleviate environmental pollution and create more value-added products. However, few reports have studied the use of peach leaves waste as a precursor for the production of CQDs. Peach leaves waste has great potential for the large-scale and low-cost production of CQDs because they can not only supply abundant carbon sources but also minimize the use of toxic and expensive chemicals.

In this work, we developed a green, facile and low-cost strategy to extract the value-added CQDs from peach leaves waste for the first time, via a simple one-step hydrothermal process ( Scheme 1 ). With this process, the CQDs with graphite-like structure, fine size of 1.9 nm, abundant surface functional groups, good hydrophilicity, high fluorescence stability and fluorescence quantum yield (QY) of 7.71% without any passivation are extracted. To our interest, the CQDs are applicable in cell imaging ( in vitro ) and zebrafish imaging ( in vivo ), demonstrating their ultra-low toxicity in high of the obtained CQDs and remarkable biological imaging performance.

Compared with previously reported methods, as shown in Table S1, our extraction strategy is greener, safer, simpler and more inexpensive, endowing their easier application to the large-scale extraction of fluorescent CQDs, due to the process of using peach leaves waste as a carbon source, only using water as a solvent, without adding any chemical reagents, and avoiding complicated purification processes ( e.g. , high-speed centrifugal, long-term dialysis). Furthermore, compared with other reported CQDs, the ob- tained CQDs are comparable to them in property and biological imaging. Even at a higher CQDs concentration (500 μg/mL), the organism can still maintain more than 90% activity both in vitro and in vivo , and present bright fluorescence.

The microstructure of CQDs extracted from peach leaves waste was characterized by Transmission electron microscopy (TEM) and Raman. The TEM image presented in Fig. 1a displays that CQDs are fully dispersed in a quasi-spherical shape without any agglomeration. The High-resolution TEM (HRTEM) image in Fig. 1b shows that the CQDs are highly crystallized with a lattice distance of 0.21 nm which corresponds to the (100) planes of graphite [18] . The corresponding size distribution histogram ( Fig. 1c ) was obtained by measuring 400 nanoparticles, indicating that the CQDs have a narrow size distribution in the range of 1.1-3.0 nm with an average size of 1.9 nm. Raman spectrum of CQDs exposed in Fig. 1d displays two distinct peaks at 1356 cm -1 and 1582 cm -1 . These peaks correspond to the disordered (D) graphitic band and graphitic (G) band of sp 2 carbon, respectively [ 19 , 20 ]. The ratio of D and G band intensities ( I D / I G ) is equal to 0.86, indicating the relatively low degree of graphitization due to the defects in the sp 2 carbon sites of CQDs arising from the functional groups on the surface [21] . Overall, these results prove that the graphite-like structure of these CQDs can be synthesized from peach leaves waste by applying the hydrothermal process.

The surface functional groups, chemical compositions and elemental states of CQDs were examined by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) and zeta potential. The XPS spectrum ( Fig. 2a ) of CQDs displays the existence of C, O and N in the CQDs, with corresponding contents of 73.29%, 20.13%, and 6.58%, respectively. The high-resolution XPS spectrum of C 1s in Fig. 2b shows three peaks at 284.8, 286. the existence of O-and N-related functional groups on its surface. These results demonstrate the existence of N and O-containing functional groups on the surface of CQDs which improves their hydrophilicity and stability in aqueous systems within the practical application in biological imaging.

The optical properties of CQDs were explored by UV-vis absorption and photoluminescence spectroscopy. The UV-vis absorption spectrum of CQDs in aqueous solutions is shown in Fig. 3a . The CQDs have strong absorption in the UV region, which is consistent with the range of the excitation wavelength of CQDs. It exhibits an intensive peak, at 255 nm, and a shoulder peak at 325 nm. These two peaks are attributed to the π -π * transition of C = C bands and n-π * transition of C = O and C-N bands, respectively [25] . The maximum emission intensity is observed at around 440 nm under the excitation wavelength of 360 nm ( Fig. 3a ). The CQDs are completely scattered in the water and present bright blue fluorescence under 365 nm UV irradiation ( Fig. 3a ,inset). These water-soluble fluorescent CQDs help the application of biological imaging.

The blue fluorescence of CQDs was further confirmed by the Commission Internationale d'Eclairage (CIE) coordinates at (0.1951, 0.2145) obtained from the emission data at 360 nm excitation ( Fig. 3b ). The CQDs exhibit excitation-dependent PL properties ( Fig. 3c ). When the excitation wavelength is tuned from 310 nm to 360 nm, the PL intensity of CQDs increases gradually. The optimal PL peak is centered at 440 nm for 360 nm excitation. This peak decreases with the increase of the excitation wavelength from 360 nm to 400 nm. The normalized PL spectra of CQDs (Fig. S1 in Supporting information) clearly exhibit redshift by increasing the excitation wavelength (310-400 nm). These findings reveal that the PL of CQDs is the excitation-wavelength dependence of the emission wavelength on intensity. The CQDs characteristic excitation-dependent PL property helps to achieve biological imaging in different color channels.

The fluorescence QY of the CQDs, about 7.71%, was measured using quinine sulfate as a reference (Fig. S2 in Supporting information). This value is relatively higher, compared with that of other biomass-derived CQDs [26][27][28][29] . Owing to its excitation-dependent property, the lifetime decay curve was also computed by applying the fitting formula ( Eq. 1 ) [30] written below to acquire the threeexponential fitting curve of CQDs ( Fig. 3d ). The observed lifetimes of CQDs were τ 1 = 0.72 ns (23.39%), τ 2 = 4.14 ns (49.95%) and τ 3 = 13.48 ns (26.66%). Previous reports pointed out that the short-lived component τ 1 , as well as the longlived components τ 2 and τ 3 , were associated with the core states and surface states of CQDs, respectively [31] . According to previous reports [32] , the long lifetime of CQDs in our study demonstrates that the PL results mainly from the surface functional groups. The average lifetime was equal to 5.96 ns, revealing that CQDs can be appropriately used in optronic and biological applications. Furthermore, except that the excitation-dependent down-converted PL characteristic, the CQDs also exhibit an up-converted PL property. Fig. S3 (Supporting information) shows the up-converted PL spectra of CQDs excited by long wavelengths ranging from 600 nm to 900 nm with the emission located in the range from 350 nm to 600 nm. The up-converted emission of CQDs may be attributed to the multi-photon active process, in which the synchronous absorption of two or more photons leads to a shorter emission wavelength [33] . The results indicate that the CQDs may supply potential applications in energy conversion, bioscience, and optoelectronic device.

R ( t ) = B 1 e ( -τ /τ 1 ) + B 2 e ( -τ /τ 2 ) B 3 e ( -τ /τ 3 ) + B 4 e ( -τ /τ 4 ) (1)
To investigate the effect of concentration on PL intensity, the PL spectra of CQDs at different concentrations were measured. As shown in Fig. S4 (Supporting information), the CQDs displayed concentration-dependent PL property with the maximum PL intensity at 1.2 mg/mL. Moreover, the PL stability of the as-extracted CQDs under various pH systems, different ionic strengths and several UV irradiation durations were evaluated. As shown in Figs. S5a-d (Supporting information), the pH values affected considerably the PL intensity. The pH value was adjusted by adding NaOH or HCl. The CQDs exhibit stable PL intensity at pH 4.0-11.0. Then, the PL intensity reduces at lower pH (pH < 4.0) and higher pH (pH > 11.0), which may be due to aggregation of CQDs originated from enhanced non-covalent bond interactions under extreme pH conditions, such as hydrogen bonding between hydroxyl, carboxyl and amino [ 24 , 34 ]. Thus, the excellent properties of pH 4.0-11.0 environment make CQDs ideal candidates in bio-applications as most biological activities happen in this pH range. In addition, CQDs solution had a good photostability since their PL intensities did not significantly change under continuous excitation at 365 nm with a UV lamp for 3.0 h, as indicated in Figs. S5b-e (Supporting information). Moreover, PL intensity was not modified in the concentration of NaCl up to 1.0 mol/L (Figs. S5c-f in Supporting information), which demonstrates that the ionic strength did not affect the PL intensity of CQDs. All these observations reveal that as-extracted CQDs had stable PL over a wide pH range and hard environmental conditions, which makes CQDs of great potential to be used in fluorescence imaging.

Through the detailed analysis above, we found that the peach leaves waste-derived CQDs exhibit fine particle size, good hydrophilicity, and stable fluorescent performance. These findings suggest that the CQDs may be promising for fluorescence imaging, which we will evaluate the biological imaging potential of CQDs in vitro and in vivo . For bio-application of peach leaves waste-derived CQDs, the MTT approach was used to analyze the vitro cytotoxicity of CQDs using A549 (human lung adenocarcinoma) cells ( Fig. 4a ). A549 cells were selected as a model, since lung cancer is the second-most prevalent type of cancer in the world [35] . Re-sults show that, after 24 h of culturing different concentrations of CQDs and A549 cells, the cell viability does not decrease significantly with the increasing concentrations of CQDs. Even if diluted to 500 μg/mL, the cell survival rate remains above 90%. This finding proves that the CQDs possess ultra-low cytotoxicity and can be used in bio-applications including bio-imaging. A549 cells were incubated with 400 μg/mL of CQDs for 24 h then they were imaged by a confocal microscope ( Fig. 4b ). Results show that the confocal image exhibits bright blue fluorescence in the A549 cells. Obviously, the fluorescence is primarily situated in the cytoplasmic region, which means that the CQDs can enter the cell through the cell membrane. In addition, the shape of the A549 cells treated with high concentration CQDs does not change considerably, endorsing the bio-compatibility of CQDs. These results indicate that hydrophilic CQDs derived from peach leaves waste can be used as excellent cell imaging reagents. Compared with intracellular imaging, observing in vivo fluorescence imaging of CQDs is more significant [36] , which we will investigate the potential of CQDs in vivo imaging.

The gene sequence of zebrafish is highly homologous to human and it is widely used in drug delivery and bio-compatibility research [37] . Thus, zebrafish was utilized as a model system to further evaluate the potential of CQDs in vivo imaging. The CQDs can quickly enter the pores of the chorion after simple immersion, because the size of CQDs (1.9 nm) is smaller than that of the nanoscale pores of the zebrafish chorion (0.17 μm 2 ). In the experiment, we observed that when the incubation concentration of CQDs reached 500 μg/mL, zebrafish larvae began to appear abnormal growth of spinal cord flexure. As revealed in Fig. S6 (Supporting information), when zebrafish larvae were exposed to CQDs even up to 500 μg/mL for 3.0 days at 28 °C, the viability of zebrafish larvae increased up to 90%, which indicates the excellent bio-compatibility and ultra-low toxicity of the extracted CQDs.

Fig. 5 shows that zebrafish larvae treated with different concentrations of CQDs exhibit blue fluorescence signal, the fluorescent intensity in the zebrafish larva rise with the increase of CQDs' concentration. In particular, the yolk sac shows the strongest blue fluorescence signal, compared to other tissues, which predominantly accumulated in the intestine and allow the CQDs to enter the digestive system to be metabolizable. In addition, CQDs could accumulate selectively in the eyes, which proves that the CQDs, having wonderful histocompatibility, can enter the eyes through the blood-ocular barrier. These results demonstrate that the CQDs are considered a potential fluorescent probe in biological imaging and other correlative biomedical applications. The biological imaging results demonstrated that the extracted CQDs had ultra-low toxicity and remarkable biocompatibility, which can be attributed to our extraction process is very green, no any chemical reagents are added, and natural biomass waste is used as the precursor.

Finally, Table S2 (Supporting information) compares the properties of biomass waste derived CQDs to SCQDs and organic dyes [ 2,4 ]. It reveals that biomass waste-derived CQDs can be efficiently employed in bio-compatibility, toxicity, synthesis, fluorescence, environmental aspects, hydrophilicity, and cost. Besides, CQDs have been considered as a potential solution to the challenges that SC-QDs and organic dyes have encountered because they are more biocompatible, ultra-low toxicity, and could be extracted from abundant and nontoxic biomass raw materials. Overall, these results confirm that biomass waste-derived CQDs could be considered as a potential replacement for organic dyes and SCQDs in vitro and in vivo biological imaging.

In summary, value-added fluorescent CQDs were successfully extracted from peach leaves waste, sustainable and low-cost natural waste biomass, by adopting a low-cost, green, and simple onestep hydrothermal process using only water as a solvent and without adding any chemical reagents. The obtained CQDs with excellent PL property, fine size, high hydrophilicity, low cost, ultra-low cytotoxicity, and good biocompatibility. They can be considered as a potential candidate for biological imaging reagents, as verified by the in vitro (cell culture) and in vivo (zebrafish) model systems. This work not only provides a meaningful solution to the end-up environmental issues of biomass waste by converting it into valueadded CQDs, but also can supply insights for developing safer and cheaper CQD-based biological imaging agents in vitro and in vivo.

Compared with blue light emissive CQDs, long-wavelength emissive, especially near-infrared CQDs, are more beneficial in biological imaging because of their deeper tissue penetration power. The effect of surface functionalization on the optical properties of biomass waste derived CQDs will be the focus of our future study to achieve near-infrared light emission with higher QY.

Scheme 1 .

 1 Scheme 1. Illustration of the CQDs extraction from peach leaves waste and their use in biological imaging.

Fig. 1 .

 1 Fig. 1. (a) TEM and (b) HRTEM micrograph images, (c) size distribution histogram and (d) Raman spectrum of CQDs.

  3 and 288.2 eV, which are attributed to C-C/C = C, C-N/C-O and C = O, respectively [ 21 , 22 ]. The high-resolution XPS spectrum of O 1s in Fig. 2c contains two peaks at 531.5 and 532.6 eV, which can be assigned to C = O and C-O-C/C-OH/, respectively[ 22 , 23 ]. The high-resolution XPS spectrum of N 1s shown in Fig.2ddisplays two peaks at 399.8 and 401.1 eV, which are ascribed to N-C and N-H, respectively[ 22 , 23 ]. This result was confirmed by the FTIR spectrum of CQDs presented in Fig.2e. The latter shows the characteristic absorption peaks of O-H/N-H at 3221 cm -1 , C-H at 2864 cm -1 , C = O at 1665 cm -1 , C = C at 1462 cm -1 , C-N at 1398 cm -1 , and C-O-C at 1089 cm -1[24] . The zeta potential of CQDs suspended in water is -17.3 mV at pH 6.0 ( Fig.2f), indicating that the CQDs are negatively charged due to

Fig. 2 .

 2 Fig. 2. (a) XPS survey spectrum, (b) C 1s spectrum, (c) O 1s spectrum, (d) N 1s spectrum, (e) FTIR spectrum and (f) zeta potential curve of CQDs.

Fig. 3 .

 3 Fig. 3. (a) UV-vis absorption, PL excitation and emission spectra of CQDs in aqueous solutions ( λex = 360 nm and λem = 440 nm); (inset) appearance of CQDs un- der daylight and 365 nm UV light illumination. (b) CIE coordinates of CQDs. (c) Excitation-emission PL spectra of CQDs. (d) Time-resolved decay and fitting curves of CQDs.

  where τ is time; B 1 , B 2 , B 3 and B 4 are the fractional intensities; τ 1 , τ 2 , τ 3 and τ 4 are PL lifetimes, the parameters gained from iterative re-convolution of the decay with the instrument response function (IRF).

Fig. 4 .Fig. 5 .

 45 Fig. 4. (a) Cell viability of A549 cells after 24 h incubation with various concentrations of CQDs. (b) Confocal fluorescence images of A549 cells after culture with 400 μg/mL CQDs.

Fig. S1 .

 S1 Fig. S1. Normalized photoluminescence (PL) spectra at various excitation wavelengths (310-400 nm).

Fig. S2 .

 S2 Fig. S2. Relationship between the absorbance and the integrated photoluminescence (PL) intensity of quinine sulfate and carbon quantum dots (CQDs)

Fig. S4 .

 S4 Fig. S4. PL spectra of carbon quantum dots (CQDs) (λex = 360 nm) with different concentrations (0.4-1.6 mg/mL).

Fig. S5 .

 S5 Fig. S5. Effect of pH (a, d), UV irradiation time (b, e) and ionic strength (c, f) on the photoluminescence (PL) intensity of carbon quantum dots (CQDs) measured at 360 nm excitation.

Fig. S6 .

 S6 Fig. S6. Viability of zebrafish larvae exposed to different concentrations of carbon quantum dots (CQDs) for 3 days at 28 °C.

Table S1

 S1 Comparison of properties and biological imaging with other reported carbon quantum dots (CQDs).

	Peach leaves waste	One-step hydrothermal	Water	Nano-membrane filtration	1.9	360/440	7.7	5.96	Zebrafish and	500/500	90/90	This
	Precursor	Synthetic method	Solvent	Purification method	Size	Wavelength	QYs (%)	Fluorescence	Biological imaging cells imaging	Concentration	Viability (%)	Refs. work
	NA: not available			(nm)	(nm)		lifetime (ns)	in vitro/in vivo	(µg/mL)	in vitro/in vivo
										in vitro/in vivo
	Bio-waste lignin	Carbonization at 300 o C	NA	Membrane filtration	6.0	280/350	8.1	5.20	Cells imaging/NA	100/NA	96.8/NA	[6]
		and ball-milling
	Mango peel	H2SO4 oxidation	H2SO4	High-speed centrifugal	3.0	310/425	8.5	3.24	Cells imaging/NA	500/NA	90/NA	[7]
				and membrane filtration
	Manilkara zapota	H2SO4 Hydrothermal	H2SO4	Alkaline neutral and dialysis	1.9	350/443	5.7	4.30	Bacteria imaging/NA	300/NA	90/NA	[8]
	fruits
		oxidation method
	Groundnuts	One-step hydrothermal	Water	Centrifugal and filtration	2.5	360/443	7.8	2.76	Cells imaging/NA	1000/NA	83/NA	[9]
	Sugarcane molasses	Hydrothermal	Ethanol	Filtration and centrifugal	2.4	328/390	NA	NA	NA/zebrafish imaging	NA/ 200	NA/80	[10]
	Caulis polygoni	Hydrothermal pyrolysis	Water	High-speed centrifugal and	2.3	380/461	42	3.32	Zebrafish and cells	400/500	80/90	[11]
	multiflora			dialysis					imaging
	Cyanobacteria	Hydrothermal pyrolysis	Water	Filtration and dialysis	2.48	360/439	9.24	3.18	Cells imaging/NA	500/NA	80/NA	[12]
	White rice waste	Carbonization at 250 o C	Acetone and	Filtration and dialysis	5.0	340/432	5.46	7.29	Cells/albino mice	100/100	90/90	[13]
		and HNO3 oxidation	HNO3						imaging
	Cydonia oblonga	Microwave synthesis	Water	High-speed centrifugal	4.85	350/450	8.55	NA	Cells imaging/NA	500/NA	80.5/NA	[14]
	Shiitake mushroom	Hydrothermal	Water	Filtration and dialysis	4.2	330/410	5.5	NA	Cells imaging/NA	400/NA	60/NA	[15]
	Citric acid	Hydrothermal	Ethylenediamin	Dialysis	4.0	340/450	10.7	6.01	Cells imaging/NA	400/NA	95/NA	[16]
			e /boric acid
	Citric acid	Hydrothermal	Urea and	Dialysis	5.0	340/450	15.1	NA	Cells imaging/NA	8/NA	80/NA	[17]
			sodium fluoride
	Reactive Red 2	Hydrothermal	Water	High-speed centrifugal	2.43	360/428	15.7	NA	Zebrafish and cells	400/400	90/90	[18]
				and membrane filtration					imaging
	Fresh mint leaves	Hydrothermal	Water	High-speed centrifugal	6.5	360/441	7.64	5.19	Cells imaging/NA	200/NA	90/NA	[19]
	Glucose and	microwave-assisted	Ethylene glycol	Dialysis	22.3	370/500	11.2	NA	Cells imaging/NA	100/NA	80/NA	[20]
	phenylenediamine	hydrothermal			3

Table S2

 S2 Comparison of the properties of the biomass waste derived carbon quantum dots (CQDs), semiconductor quantum dots (SCQDs) and organic dyes.

	Properties	CQDs	SCQDs	Organic dyes
	Size	< 10 nm and tunable	< 20 nm and tunable	< 0.5 nm protein or small molecule,
		use of natural and green precursors	precursors are toxic and expensive	organic small molecules are used
	Synthesis	low costs, one-step production,	high costs, complicated synthesis,	high costs, tedious synthesis process,
		consume less energy	necessitate the use of special	often with chemicals
			equipment and require higher energy
			consumption
		easy to operate tunable quantum yields	high quantum yields	low quantum yields
	Optical	high resistance to photo-bleaching, narrow	high resistance to photo-bleaching,	low resistance to photo-bleaching
	properties	emission spectra, wide absorption spectra	narrow emission spectra,	broad emission spectra with red-shirt
		extend to the Vis	broad absorption spectra,	tail, narrow absorption spectra
		and near-infrared region	high detection sensitivity	low detection sensitivity
		high detection sensitivity
		emission-dependent tissue penetration	medium size-dependent tissue	high tissue penetration
	Bio-imaging	good hydrophilicity	penetration	high aqueous solubility,
	properties	low toxicity	poor aqueous solubility,	chemistry dependent,
		non-metallic precursors	potential toxicity in-vivo	typically rapid metabolism
			potential for heavy metal leaching
	Key	difficult to near-infrared emission based	poor water solubility and stability,	Prone to photo-bleaching, difficult to
	challenges	on biomass-derived CQDs	difficult to alter biodistribution	achieve in-vivo multiplex imaging

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 21976116 and 52161145409 ), the Shaanxi Science and Technology Program (No. 2020KWZ-005 ), SAFEA of China ("Belt and Road" Innovative Exchange Foreign Expert Project, No. DL2021041001L). The authors acknowledge Researchers Supporting Project number (No. RSP-2021/149), King Saud University, Riyadh, Saudi Arabia.

Declaration of competing interest

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

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.cclet.2022.107998 .

Supporting information

Green process of biomass waste derived fluorescent carbon quantum dots for biological imaging in vitro and in vivo

Haitao Ren a , Yue Yuan b , Abdelkader Labidi a , Qibing Dong a , Ke Zhang a , Eric Lichtfouse c , Ahmed A. Allam d , Jamaan S. Ajarem e , Chuanyi Characterization: High-resolution transmission electron microscope (HRTEM) was executed on an FEI Talos F200i field emission electron microscope operating at a voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were performed on a Thermo Scientific K-Alpha spectrometer using Al Kα X-ray beam of 250 W as the excitation source. Raman spectrum was recorded on a HORIBA LabRAM HR Evolution Raman Spectrometer with a 532 nm laser. Fourier transform infrared (FTIR) spectra were recorded on a KBr medium on a Brucker (Verte70 V) spectrometer. Zeta potential measurements were executed on a Malvern Zetasizer Nano ZS90. The photoluminescence (PL) spectra were carried out using an Edinburgh Instruments FS5 spectrofluorometer; lifetime measurements were obtained on the same FS5 Edinburgh Instruments equipped with an EPLED-340 pulsed source. Ultraviolet-visible (UV-vis) absorption spectrum was recorded with a UV spectrophotometer UV-2600 (Shimadzu).

Environment-friendly production of carbon quantum dots (CQDs):

The value-added fluorescent CQDs were produced from peach leaves waste by a one-step simple hydrothermal process. The whole production process is more efficient and greener, because no useless by-products, only using water as a solvent and without adding any chemical reagents, as well as avoiding complicated purification processes (e.g., high-speed centrifugal, long-term dialysis). A typical synthesis process is shown in Scheme 1, in which peach leaves waste was cleaned with ultrapure water and dried at 80 °C. The dry peach leaves were grounded into a powder. 0.5 g of the latter was mixed with 60 mL ultrapure water. Subsequently, the homogenous mixture was transferred into a 100 mL autoclave and heated at 200 °C for 10 h. The autoclave was cooled naturally. The dark brown suspension was filtered several times by the 0.05 µm membrane filter in order to separate the solution containing CQDs from the solid residue. The small amount of the solid product as a hydrochar was produced after the hydrothermal carbonization process, which can be seen as a high-value by-product in several applications [1][2][3]. Finally, the brownish-yellow CQDs suspension was collected and freeze-dried to obtain the CQDs powder with a yield of ca.64.5 wt% for further experiments.

Calculation of QY: The QY of CQDs was calculated using quinine sulfate (dispersed in 0.1 M H2SO4, QY = 54%) as a reference. The relative QY was calculated applying the following equation [4]:

where "I" represents the integrated emission intensity, "η" is the refractive index of the solvent, "k" denotes the slope of the integrated emission intensity-absorbance linear-regression curve under different concentrations, and "A" refers to the absorbance at the optimum excitation wavelength of 360 nm (usually kept below 0.1 to reduce the re-absorption effects). For all these CQDs solutions, ηCQDs/ηref was approximately equal to 1.

Cellular toxicity test:

The cytotoxicity of CQDs was measured by A549 cells using the MTT experiment [5]. A549 cells were incubated in a 96-well plate with a density of 8×10 3 cells per well. Afterward, the cells were cultured in a medium containing different concentrations of CQDs for 24 h (5% CO2, 37 o C). Subsequently, the medium was removed and the cells were washed three times with pphosphate-buffered saline. 100 µL of MTT (0.5 mg/mL) was added into each well and further incubated at 37 °C for 4 h. Then, the medium was replaced with 100 µL of dimethyl sulfoxide followed by shaking the solution for 10 min. The optical density (OD) of each well was obtained at 570 nm utilizing an enzyme labeling apparatus. The cell viability was calculated applying the formula written below:

Cell Viability(%) = OD B/0CB0D OD EFGB/FH × 100%

The ODtreated and ODcontrol in the formula are the optical density of the well in the presence of CQDs and blank, respectively. All experiments were repeated thrice.

Cellular imaging:

The potential of CQDs in biolabeling was evaluated by the cellular imaging of A549 cells. The latter were seeded in Dulbecco's modified eagle medium with 10% (pH 7.0) phosphate-buffered saline (5% CO2, 37 o C) for 24 h and additional 24 h after addition of 400 µg/mL CQDs. To remove the CQDs, the cells were washed with phosphate-buffered saline thrice and, then, fixed using 4% formaldehyde. Images were obtained employing a confocal laser scanning microscope (Leica TCS SP8) under the excitation of 364 nm.

Zebrafish imaging:

The wild-type Tubingen zebrafish strains, maintained according to the standard experimental protocols set by the laboratory of tissue engineering and stem cell experiment center of Guizhou Medical University, were selected as the experimental fish. They were also maintained according to the guidelines of the experimental animal welfare specified by the Ministry of Science and Technology of the People's Re-public of China (2006). 2 mL of E3 embryo media containing different concentrations of CQDs (0, 100, 200, 300, 400, 500 µg/mL) were placed in a 12-well plate and 20 zebrafish larvae were cultivated at each concentration for 3 days at 28 o C. The viability of zebrafish larvae was observed and recorded at 72 hpf time points to evaluate the toxicity of CQDs. The fluorescent images were taken under an excitation light wavelength of 340 nm with Nikon eclipse 80i microscope. Then, they were recorded by NIS Elements D5 20.00 image analysis software.


domain: sde sdv

text:
Congrès EDSE 2021

ANALYSIS AND MODELLING OF TRANSFER PROCESSES TO PLANTS BY FOLIAR PATHWAY OF RADIONUCLIDES IN PARTICLE FORM #poster

Z. Mawassy 1* , P. Henner 1 , S. Le Dizès-Maurel 1 , J. Rose 2 (1) IRSN, PSE-ENV/SRTE, Laboratoire de recherche sur les transferts de radionucléides dans les écosystèmes terrestres (LR2T), Cadarache, Bâtiment 183, BP 3, 13115 Following normal/incidental releases of nuclear facilities, radionuclides may be released into the atmosphere under different speciations (gaseous, particulate, dissolved in water), then be intercepted and possibly retained by plant foliage and translocated to other organs. In the upcoming years, new release scenarios (ITER, UNGG dismantlement) potentially involving particles will be evaluated, thus increasing the need to assess/ improve the existing tools describing transfers of particles and dosimetric evaluation. Knowledge on foliar transfer is still limited and operational tools do not consider the particulate form leading to possible dose underestimation [START_REF] Dizès-Maurel | Review of existing knowledge and data on deposition of airborne micronic (possibly tritiated) particles and subsequent transfer and internalization in plant biomass[END_REF]. So, this PhD work aims at quantifying and modelling the key processes influencing particulate radionuclide transfers to plants by foliar pathway.

Particles covers a wide range of scenarios depending on their nature, size and reactivity [START_REF] Wang | [END_REF]3]. At leaf interface, radionuclides may be incorporated by stomatal or cuticular pathways, then possibly reaching conductive tissues [4,5,6,7]. The extent of these processes will depend on the possible dissolution of particles on/within foliage, or intact particles transfer. Yet the conditions (particle size?, reactivity) and mechanisms for intact particles transfer are unknown, thus requiring investigation of both incorporation pathways in relation to particle properties [8,9]. The resulting radionuclide stocks and fluxes have to be quantified and compared to non-particulate source terms and finally, models need to be tested and/or specifically parametrized and/or completed to allow global assessment of foliar transfer whatever the transfer pathways.

To address these different questions, the project will consist of:

1) Controlled conditions experiments to obtain data on the plant incorporation-leaf absorption through transcuticular/stomatal pathways and translocation as a function of the main following factors: plants characteristics (e.g. foliage interface physico-chemical and morphological criteria: roughness, cuticle thickness, stomatal density and size), particles characteristics (low/high reactive particles with variety of sizes ranging from nano [20-50 nm] to micrometer [>5 µm]), or type of deposit (dry/wet). Monitoring of contaminant absorption in/translocation out in leaves will be followed qualitatively/quantitatively by different techniques: e.g. assessing particles locations by 2D and 3D imaging (SEM-EDX, micro-X-ray fluorescence, micro and nano-Xray tomography).

2) Development and validation of a simple generic model of radionuclides foliar transfer, potentially including specific treatment for low diameter/low reactivity particles. The data obtained from the experiments (see 1) would be compared to aggregated transfer factor predictions and outputs of other generic models related to non-particle source terms. Based on results of comparisons, existing tools will be completed and improved if needed.

3) Numeric application to a real-case scenario (e.g. ITER/ UNGG-like scenario involving tritiated or 36 Clbearingparticles).

  Saint-Paul-Lez-Durance Cedex, France (2) Centre Européen de Recherche et d'Enseignement des Géosciences de l'Environnement (CEREGE), Bâtiment Pasteur, Europole Méditerranéen de l'Arbois BP80, 13545 Aix-en-Provence cedex 4, France * zeinab.mawassy@irsn.fr


B Bourdiliau

F Onimus

C Cappelaere

V Pivetaud

P Bouffioux

V Chabretou

A Miquet

Impact of the irradiation damage recovery during transportation on the subsequent room temperature tensile behavior of irradiated zirconium alloys

Keywords: Ring tensile test, Transmission Electron Microscopy, internal pressure creep test, heat treatment, deformation mechanisms, Zy-4, Zr-1%Nb, spent nuclear fuel, transportation

Zirconium alloys are commonly used in Pressurized Water Reactor as fuel rod cladding tubes. After irradiation and cooling in pool, the Spent Nuclear Fuel Assemblies are either transported for wet storage to a devoted site or loaded in suitable casks for dry storage on Nuclear Power Plant site. During dry transportation or at the beginning of dry storage, at temperatures around 400°C, the cladding experiences a creep deformation under the hoop stress induced by the internal pressure of the fuel rod. During creep a recovery of the radiation damage can occur that can affect the subsequent mechanical properties. The mechanical behavior of the cladding has been investigated in laboratory on two neutron irradiated cladding materials: fully recrystallized Zr-1%Nb and stress relieved Zircaloy-4. Creep tests under internal pressure were conducted at 400°C and 420°C. After depressurization and cooling, ring tensile tests were carried out at room temperature. In addition, Transmission Electron Microscopy observations have been performed after testing. The post-creep mechanical response exhibited a decrease of the strength compared to the asirradiated material. This decrease is associated with a significant recovery of the ductility, which becomes close to the ductility of the unirradiated material. The Transmission Electron Microscopy examinations, especially conducted on recrystallized Zr-1%Nb ring samples, revealed that the radiation defects have been annealed. It was also observed that, as for the unirradiated material, the deformation occurred homogeneously throughout the grains. No dislocation channel was indeed observed contrary to the as-irradiated material. These observations explain the recovery of the strength and of the ductility after post-irradiation creep that may occur during dry transportation or at the beginning of dry storage.

Introduction

Zirconium alloys are commonly used in Pressurized Water Reactors (PWR) as fuel cladding tubes. After irradiation and cooling in pool, the Spent Nuclear Fuel Assemblies (SNFA) are either transported for wet storage to a devoted site or loaded in suitable casks for dry storage on Nuclear Power Plant site. During dry shipment or at the beginning of dry storage, the cladding experiences creep deformation due both to the fuel rod inner pressure of the fission gas and the significant temperature induced by the residual power of the fuel [START_REF] Cappelaere | Impact of Irradiation Defects Annealing on Long-Term Thermal Creep of Irradiated Zircaloy-4 Cladding Tube[END_REF]. In order to guarantee the integrity of the cladding when retrieving the SNFA both after shipment and storage, it is essential to obtain a good knowledge of the effects of the transportation on the room temperature mechanical properties of the materials.

It has been shown in a previous study [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] that during a creep test, representative of transportation conditions, recovery of the radiation damage occurs in addition to the creep deformation mechanisms. It has also been shown [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF][START_REF] Ribis | Experimental study and numerical modelling of the irradiation damage recovery in zirconium alloys[END_REF] that the recovery of the irradiation damage is associated with the recovery of the hardening induced by irradiation, as demonstrated by Transmission Electron Microscopy (TEM) observations and microhardness tests carried out at room temperature after heat treatments on as-irradiated zirconium alloys.

In order to have a better knowledge and understanding of the effect of post-irradiation creep testing on the subsequent room temperature mechanical properties of zirconium alloys, a thorough study has been achieved. This study is based first on microhardness tests performed on the neutron irradiated zirconium alloys after creep testing but also on tensile tests carried on at room temperature on ring specimens which were machined out of specimens after creep testing. In addition, on some of the tensile tests specimens, TEM observations have been performed after testing in order to obtain a better understanding of the deformation mechanisms in such testing conditions. The experimental results are presented in the first part of the paper. Then, in the last part, the results are discussed and interpreted in terms of microstructure and deformation mechanisms.

Materials and experimental details

Materials studied

Two cladding materials have been studied: the stress relieved Zircaloy-4 and a recrystallized annealed Zr-1%Nb alloy. The chemical composition of the two alloys are given in Table 1.

The materials have been irradiated from four up to six PWR cycles and then cut and defueled in order to perform mechanical tests.

Mechanical testing

Creep specimens

Four stress-relieved Zy-4 specimens irradiated up to four PWR cycles have been tested under internal pressure during creep tests with an applied stress of 130 MPa and at temperatures from 380 up to 420°C, and during 1000 to 17000 hours. The creep tests were performed either in an internal pressure creep furnace with controlled internal gas pressure (hereafter called Controlled Gas Pressure, CGP) or as pre-pressurized samples (with internal gas pressure) in a furnace (called LTC, Long Term Creep). The controlled internal pressure creep test (CGP) is performed at constant pressure and, at the end of the test, the specimen is depressurized before cooling. For the long term creep (LTC) test, the stress in the pre-pressurized specimen remains constant and gradually falls during cooling. The creep test conditions are given in Table 2.

Three recrystallized Zr-1%Nb specimens irradiated from four up to five PWR cycles have also been tested under internal pressure creep test (in CGP or LTC conditions) with an applied stress of 130 MPa and at temperatures from 400 up to 420°C during 900 up to 3300 hours.

The creep test conditions are given in Table 3 as well as the creep strains at the end of the tests.

ZrNb-5a

ZrNb-5b

Hardness measurements

After the creep tests, 10 mm long sections of the specimens have been cut, embedded into a resin and then mechanically polished in hot cell. Vickers microhardness experiments have been carried out using 200 g load along the z-direction of the tube (on the r-θ cross section of the tube). In order to provide correct statistical results, ten hardness measures are performed for each sample at room temperature.

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Tensile ring specimens

Tensile ring specimens have also been spark machined out of the cladding specimens (both Zy-4 and Zr-1%Nb) after creep tests. The rings, cut out in the transverse section of a PWR cladding, were 3 mm long and 2 mm wide in the gauge length. The Fig. 1 shows an example of a tensile ring machined out of a long term creep specimen. Two rings were cut out from a single creep specimen (except the Zy4-4 specimen), from each side of its symetry axis.

Moreover, two as-irradiated specimens, and two unirradiated specimens were machined for both materials. The rings have then been tested on an electromechanical tensile machine fitted with a LVDT sensor and a 50 kN load cell. The specimen was placed on its mandrel so that the sample legs were centered over the loading axis. The tensile test was displacementcontrolled. All the tests were performed at room temperature with a strain rate of 3×10 -4 s -1 .

Several tensile conventional properties are deduced from the tensile curves. The engineering stress is given as a function of the engineering strain: the Yield Stress (YS, stress at 0.2 % plastic strain offset given in MPa), the Ultimate Tensile Strength (UTS, stress corresponding to the maximum force given in MPa), Uniform Elongation (UE, plastic strain measured at maximum force, in %), Total Elongation (TE, plastic strain at fracture, in %). The stressstrain curves have been corrected from the overall testing machine stiffness, which is the result of the elastic deformation in the load line device and the mandrel stiffness. A Young modulus of E = 100 GPa at room temperature has been chosen in agreement with the values given by Northwood et al. [START_REF] Northwood | Elastic constants of zirconium alloys[END_REF].

Pictures of the ring specimens after testing were systematically taken. Some of these pictures are shown hereafter. Concerning the Zr-1%Nb alloy, the non irradiated specimens were 88 ppm gaseous hydrided in order to simulate the hydriding which occurs in-reactor and therefore only measure the effect of irradiation, uncorrelated from the hydriding effect. In addition, in the case of the Zr-1%Nb alloy, under each test condition, one ring was tested up to failure and a second one was stopped at UTS (indicated with a star (*) in Table 3). In these specimens, thin foils have been taken in order to perform TEM observations of the as-deformed microstructure.

Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) observations were carried out on neutron irradiated Zr-1%Nb previously tested only until the maximum load. The tests conditions of these samples are given in Table 3.

After testing, the legs of ring specimens have been cut and mechanically polished in hot cell.

Thin foils have been taken from the polished legs and electro-polished in a glove box.

The TEM observations have then been performed on a Philips EM 430 operating at 300 keV accelerating voltage. The study has been performed using double tilt sample holder, conventional imaging of crystalline defects and diffraction pattern indexing.

Experimental results

Mechanical Tests

Zy-4 specimens

The engineering stress -strain curves corresponding to the tests performed on Zy-4 are shown on Fig. 2. Tensile conventional properties deduced from the stress -strain curves are given in Table 4. When comparing the non irradiated Zy-4 specimens and the as-irradiated specimens, Fig. 2 and Table 4 demonstrate that neutron radiation leads to a strong hardening (increase of the YS and UTS values) as well as to a reduction of the macroscopic ductility (decrease of the UE and TE values).

After creep, it can be seen on Fig. 2 and on Table 4 that the YS as well as the UTS values have decreased compared to the as-irradiated behavior. It is also shown that after the creep test, the UE as well as the TE values have increased proving that the creep test leads to a recovery of the ductility of the material.

However, it can be noticed on Fig. 2 and on Table 4 that after creep tests the room temperature tensile behavior remains different from the non-irradiated mechanical behavior.

Indeed, the UE and TE values remain systematically lower than the values measured for the non-irradiated Zy-4, proving that the macroscopic ductility of the material was not fully recovered even after 4974 hours at 420°C (specimen Zy4-1) or after 17071 hours at 400°C.

On the other hand, the strength (YS and UTS) of the material can be fully recovered after

Zr-1%Nb specimens

The engineering stress -strain curves corresponding to the tests performed on the Zr-1%Nb specimens are shown on Fig. 4 and the conventional tensile properties are given in Table 5. 13/36

Fig. 4 and Table 5 show that, such as for Zy-4 specimens, neutron irradiation leads to a strong hardening, as shown by the increase of the YS and UTS values when comparing the non irradiated and the as-irradiated specimens, and to a reduction of the macroscopic ductility (decrease of the UE and TE values). On the tensile curve of the as-irradiated ring tested up to failure, we can see a load drop just after the UTS. In addition, the tensile curve exhibits a sudden acceleration of the load decrease during the necking.

After post-irradiation creep, it can be seen on Fig. 4 and on Table 5 that the YS as well as the UTS values of the Zr-1%Nb specimens have decreased compared to the as-irradiated behavior, proving that a recovery of the radiation hardening has occurred during the creep test. It is also shown that, after the creep test, the UE values have increased proving that the post-irradiation creep test leads to a recovery of the ductility of the material.

However, it can be noticed, when comparing the behavior after post irradiation creep to the behavior before irradiation, that YS and UTS values after creep are still higher than the values measured before irradiation. The macroscopic ductility also appears to be only partially recovered after creep: the UE and TE values remain lower than the values measured before irradiation. It is also worth noticing that the strain hardening behavior after post-irradiation creep is also very different from the strain hardening behavior before irradiation. Indeed, on the non irradiated Zr-1%Nb specimens, the strain hardening rate is low just after the onset of plastic flow and decreases slowly until reaching the maximum load whereas in the case of the Zr-1%Nb after post-irradiation creep, the strain hardening remains high at the onset of plastic flow and decreases more quickly until reaching the maximum load. This change of strain hardening behavior due to the post-irradiation creep test can be characterized by the difference between UTS and YS values. Table 5 shows, indeed, that the difference between UTS and YS values after post-irradiation creep is nearly twice higher than the difference 14/36 between the UTS and YS values measured on the non irradiated specimens. This proves that the strain hardening after post irradiation creep is higher than the strain hardening before irradiation.

It can also be noticed that when the creep temperature or the creep duration increase, the strength (YS and UTS values) slightly decreases and the ductility (UE and TE values)

increases.

The pictures taken after the tensile test (Fig. 3) show that both the as-irradiated Zr-1%Nb It can be seen on Table 6, when comparing the as-irradiated to the non irradiated Zy-4 specimens, that irradiation leads to a significant increase of the hardness linked to the radiation induced hardening observed during the tensile test. After creep test, the hardness value decreases strongly because of the recovery of the radiation induced hardening. It can be seen that, as for the YS or the UTS values, depending on the post-irradiation creep conditions, the hardness of the Zy-4 specimens can even become lower than the hardness of the non irradiated Zy-4 specimen. The hardness values measured after post-irradiation creep test are also compared on the Fig. 5 to the hardness evolution obtained in a previous study [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] after post-irradiation heat treatments performed on Zy-4 at 350°C, 400°C and 420°C. The empirical recovery law proposed in [START_REF] Torimaru | Changes in Mechanical Properties of Irradiated Zircaloy-2 Fuel Cladding Due to Short Term Annealing[END_REF][START_REF] Dollins | Post Irradiation Recovery of Irradiation Damage[END_REF] and adjusted in [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] is also shown on Fig. 5. The recovered fraction of hardness, f, is defined in Eq. 1.

NI v AsI v v AsI v H H t H H f - - = ) ( (1)
The values AsI v H , NI v H and Hv(t) used in Eq. 1 are respectively the as-irradiated hardness of the material, the non-irradiated hardness of the material and the current hardness of the material all along the heat treatment. According to Dollins [START_REF] Dollins | Post Irradiation Recovery of Irradiation Damage[END_REF], the hardness recovered fraction follows a chemical kinetics empirical law, expressed in Eq. 2.

( )

n f k dt df - = 1 (2)
In Eq. 2, the coefficient k can be written as ( )

T k Q k k B / exp 0 - =
where kB is the Boltzmann constant, T is the temperature of the heat treatment, Q the activation energy of the recovery and 0 k a coefficient. The fitted values are n = 2.3, ko = 1.8×10 8 s -1 and Q = 1.800 eV [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF]. As pointed out in [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF], since in the empirical law the reference hardness value is the hardness before irradiation, the empirical law chosen here is therefore not able to reproduce the decrease of the hardness below its value before irradiation.

It can be seen on Fig. 5

)

After creep tests @420°C Annealing @350°C Recovery law @350°C Annealing @400°C Recovery law 400°C Annealing @420°C Recovery law 420°C Non irradiated (no annealing) The recovery law obtained in [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] is also shown.

Zr-1%Nb Specimens

The hardness values obtained on the Zr-1%Nb specimens after post-irradiation creep performed at 400°C are given in Table 7 and compared to the UTS values. As for the Zy-4 specimens, the hardness values of the Zr-1%Nb specimens follow the same evolution as the YS and UTS values. It can be seen on Table 7 that irradiation induces a strong hardening when comparing the hardness of the as-irradiated Zr-1%Nb and the hardness of the non irradiated Zr-1%Nb specimens. As for the YS and UTS values, it can be seen that after postirradiation creep the hardness decreases due to the recovery of the radiation induced hardening. However, it can be observed that the hardness values after post-irradiation creep of Zr-1%Nb specimens always remain higher than before irradiation.

The hardness values measured after post-irradiation creep tests performed at 400°C were also compared on the Fig. 6 to the hardness evolution obtained in a previous study [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] after postirradiation heat treatments performed on a Zr-1%Nb alloy at 350°C, 400°C and 450°C. The empirical recovery law adjusted in [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] is also shown on Fig. 6. The values of the fitting parameters are n = 3.1, ko = 3.8×10 2 s -1 and Q = 1.075 eV.

It can be observed on Fig. 6 that after post-irradiation creep performed at 400°C during 984 or 3301 hours, the hardness of the Zr-1%Nb specimens remains higher than the hardness measured after a post-irradiation heat treatment at 400°C lasting only 500 hours. This proves that in the case of the Zr-1%Nb specimens, post irradiation creep and heat treatment have different effects on the hardness.

)

After creep test @400°C Annealing @350°C Recovery law @350°C Annealing @400°C Recovery law @400°C Annealing @450°C Recovery law @450°C Non irradiated (no annealing)

Figure 6: Comparison between the hardness evolution after post-irradiation creep tests and post-irradiation heat treatments performed on Zr-1%Nb specimens at 350°C, 400°C and

420°C. The recovery law obtained in [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] is also shown. Non irradiated 498

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Correlation between hardness and UTS values

Tables 6 and7 show that the hardness and the UTS values follow the same evolution for Zy-4

and Zr-1%Nb specimens. The change in UTS (with respect to the non irradiated material) can therefore be plotted in Fig. 7

Results of the TEM observations on Zr-1%Nb samples

As-irradiated sample observed after tensile test

TEM observations have been performed on the as-irradiated sample after tensile testing. TEM pictures of the microstructure obtained after deformation are given in Fig. 8. It can be observed that the microstructure exhibits cleared bands or so-called dislocation channels in numerous grains. The channeling plane has been analyzed using the method described in [START_REF] Onimus | A statistical TEM investigation of dislocation channeling mechanism in neutron irradiated zirconium alloys[END_REF][START_REF] Onimus | Plastic Deformation of Irradiated Zirconium Alloys: TEM Investigations and Micro-Mechanical Modeling[END_REF]. It can be seen on Fig. 8(a) that the channeling plane corresponds to the basal plane.

Deviation of the basal channels on the pyramidal plane can also be observed on Fig. 8(a).

On the other hand, it can be seen that in several grains (Fig. 8(b), 8(c), 8(d)) the observed channels do not correspond to the basal plane. Indeed, the channels can be observed even when the basal plane is nearly perpendicular to the electron beam. The channeling planes have been analyzed and correspond to the prismatic planes. Moreover it appears that contrarily to the basal channels which are only clearly observed when the basal plane nearly contains the electron beam, the prismatic channels can be observed for a wide tilt angle range, even when the electron beam is far from the prismatic plane. This can lead to a more ambiguous determination of the channeling plane.

Since the orientation of the tensile axis is known on the thin foil it is possible to compute the Schmid factors of the various slip systems for each grain observed. The basal and prismatic Schmid factors of the grains observed on Fig. 8 are given in Table 8. It can be seen on Table 8 that, when the basal plane is well orientated for slip (high basal Schmid factor), basal channeling occurs. On the other hand, when the basal plane is not well orientated for slip (low basal Schmid factor) prismatic channeling occurs.

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Table 8: Basal and prismatic Schmid factors for the four grains observed by TEM given Fig. 8. For basal slip the maximum basal Schmid factor is always given. For prismatic slip the prismatic Schmid factor corresponding to the activated slip system is given, when only basal channels are observed the maximum prismatic Schmid factor is given.

Grain observed

Maximum basal Schmid factor

Prismatic Schmid factor

Channeling plane observed On the Fig. 9(a), where the grain is observed with the electron beam close to the <c> axis, two linear dislocation systems are clearly observed at 120° from each other. This shows that in that grain, the prismatic slip is mainly activated. On the Fig. 10(a) and 10(b) the same area is shown using two different diffraction conditions. Nb-rich precipitates under the shape of needles are clearly observed on Fig. 10(b) without any contrast coming from the loops or the dislocations. The microstructure (size and density) of the needle-like precipitates appear to be similar to that observed on the as-irradiated Zr-1%Nb alloy [START_REF] Onimus | A polycrystalline modeling of the mechanical behavior of neutron irradiated zirconium alloys[END_REF]. This suggests that both the creep test and the tensile test did not affect significantly the needle precipitates.

On the other hand it turns out that it is very difficult to clearly distinguish dislocation loops after the tensile tests. As shown on Fig. 9 very few loops are clearly observed and in addition the small loop contrast can be mistaken for the contrast coming from the needle precipitates.

This observation has to be compared to the TEM observations described in [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] after postirradiation creep tests. Indeed, after post-irradiation creep performed at 400°C under a 130

MPa applied stress, small <a> loops could still be observed with size and density in rather good agreement with the microstructure observed after heat treatments at 400°C. This proves that the tensile test performed after the post-irradiation creep test has strongly affected the remaining loops.

It can also be pointed out that the dislocations observed on Fig. 10

Discussion

Discussion on the behavior of the as-irradiated Zr-1%Nb specimens

The TEM observations have shown that in the as-irradiated Zr-1%Nb there is a very high density of small <a> type dislocation loops induced by irradiation [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF]. This very high density of small dislocation loops that act as obstacles against dislocation motion explains the strong irradiation induced hardening measured through tensile and hardness tests.

It has been shown by TEM that the as-irradiated Zr-1%Nb specimen tested at room temperature and interrupted at UTS exhibits dislocation channels. This deformation mechanism consists of the clearing of the irradiation induced loops by gliding dislocations on only one slip system leading to the creation of a defect free zone that becomes a preferred area for further dislocations gliding [START_REF] Onimus | A statistical TEM investigation of dislocation channeling mechanism in neutron irradiated zirconium alloys[END_REF][START_REF] Onimus | Plastic Deformation of Irradiated Zirconium Alloys: TEM Investigations and Micro-Mechanical Modeling[END_REF]. This phenomenon leads to the localization of the plastic strain at the grain scale and to a softening at the microscopic scale. This mechanism, as well as the fact that there is only one activated slip system, can explain the early localization of the plastic strain at the specimen scale which leads to the loss of macroscopic ductility observed after irradiation (decrease of the UE value), the failure mode remaining ductile [START_REF] Fregonese | Failure mechanisms of irradiated Zr alloys related to PCI: activated slip systems, localised strains, and iodine induced stress corrosion cracking[END_REF] .

Moreover it has been shown that the dislocation channels observed correspond both to the basal and prismatic planes proving that during ring tensile tests these two slip systems can be activated at room temperature. This observation has to be compared with the analysis of the post-irradiation deformation mechanisms at 350°C described in [START_REF] Onimus | A statistical TEM investigation of dislocation channeling mechanism in neutron irradiated zirconium alloys[END_REF][START_REF] Onimus | Plastic Deformation of Irradiated Zirconium Alloys: TEM Investigations and Micro-Mechanical Modeling[END_REF]. It has been proven, indeed, throughout many TEM observations that for both transverse tensile tests and internal pressure tests performed at 350°C the deformation occurs by dislocation channeling mechanism. It has also been shown that up to the ultimate tensile strength only basal channeling occurs. Only after the UTS prismatic, pyramidal as well as basal channels are observed on ring specimens tested at 350°C [START_REF] Onimus | Plastic Deformation of Irradiated Zirconium Alloys: TEM Investigations and Micro-Mechanical Modeling[END_REF]. On the other hand it has been shown that at 29/36 350°C when the grains are not properly orientated to allow basal slip, as they are for axial tensile tests, prismatic channels are observed. These deformation mechanisms are very different from the deformation mechanisms observed before irradiation. Before irradiation, indeed, the deformation occurs by the way of an homogeneous gliding of dislocations within the grains mainly by prismatic slip. The change in the principal slip system has been attributed to the difference in junction reactions between the loops and the dislocations gliding in the prismatic plane or the dislocations gliding in the basal plane [START_REF] Onimus | A statistical TEM investigation of dislocation channeling mechanism in neutron irradiated zirconium alloys[END_REF]. Indeed, the irradiation induced loops can be easily swept up by the dislocation gliding in the basal plane since the junctions allow gliding which favors an easy dislocation channeling mechanism in the basal plane. On the other hand the sweeping of loops by dislocations gliding in the prismatic plane is more difficult since the junctions are sessile explaining that the dislocation channeling mechanism in the prismatic plane is more difficult. However, the fact that at room temperature for ring test interrupted at the UTS both basal channels and prismatic channels are observed can be understood if the evolution of the Critical Resolved Shear Stresses (CRSS) of the various slip systems with temperature is taken into account. It has been shown, indeed, by Akthar [START_REF] Akhtar | Basal slip in Zirconium[END_REF] while testing at high temperature pure zirconium single-crystals that the basal CRSS, which is higher than the prismatic CRSS, decreases quickly as a function of the temperature whereas the prismatic CRSS decreases more slowly. A schematic plot of this evolution is proposed on Fig. 11 for polycrystalline Zr-1%Nb at lower temperatures. On the other hand it has been shown by Onchi [12] that the yield stress of both irradiated and unirradiated recrystallized Zy-2 decreases with the test temperature, the decrease being only slightly slower for the irradiated specimens between 20°C and 300°C. This proves that the test temperature has only a small influence on the irradiation induced hardening. It can therefore be assumed that the evolution of both the basal and prismatic CRSS with irradiation only consists of a constant increment of the CRSS independent of the temperature, the increment of CRSS being much higher for prismatic slip than for basal slip [START_REF] Onimus | A statistical TEM investigation of dislocation channeling mechanism in neutron irradiated zirconium alloys[END_REF][START_REF] Onimus | A polycrystalline modeling of the mechanical behavior of neutron irradiated zirconium alloys[END_REF]. As proposed on the schematic plot given in Fig. 11 a reversal of the prismatic and basal CRSS occurs with irradiation at 350°C leading to a change of the easy glide slip system. On the other hand, at 20°C, no reversal of the CRSS occurs but the basal and the prismatic CRSS become very close to each other therefore explaining that both basal and prismatic channels can be observed depending on the grain orientation. However, as shown on Fig. 4 after post-irradiation creep the recovery of the hardening is only partial since the strength (or hardness) remains higher than for the non irradiated material. This phenomenon has several origins. Firstly, after 984 or 3301 hours at 400°C remaining loops can still be present in the material, the loop annealing being slow at this temperature [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF][START_REF] Ribis | Experimental study and numerical modelling of the irradiation damage recovery in zirconium alloys[END_REF]. Secondly, the needle precipitates are also another source of residual hardening. Indeed, even after 960 hours post-irradiation heat treatment performed at 450°C [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF], the hardness of the material is still not fully recovered (Fig. 6). In this condition, only very few large loops remain present in the material [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF][START_REF] Ribis | Experimental study and numerical modelling of the irradiation damage recovery in zirconium alloys[END_REF]. These remaining loops cannot explain the residual hardening. The residual hardening is therefore due to the very small precipitates present in very high density in the material after irradiation [START_REF] Doriot | Microstructural Stability of M5™ Alloy Irradiated up to High Neutron Fluences[END_REF] which can act as obstacles to dislocation motion. These two reasons can explain the residual hardening observed. Nevertheless, as it has been pointed out, after creep performed at 400°C during 984 or 3301 hours, the hardness remains higher than the hardness measured after post-irradiation heat treatment performed at 400°C during 500 hours only. This proves that the residual hardening cannot only be explained by the remaining loops and the needle precipitates that are both present in the case of the heat-treated material.

It has been proven [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF] that during creep tests performed at 400°C with an applied stress of 130 MPa in addition to the recovery of the radiation induced loops the microstructure exhibits a high dislocation density, much higher than the dislocation density observed in the non irradiated material for the same creep strain. It has been proposed that this high dislocation density is the result of dislocation multiplication due to the remaining loops that act as obstacles and therefore decrease the dislocation mean free path. The high density of small needle-like precipitates could also increase the dislocation multiplication. This higher dislocation density observed after post-irradiation creep performed at 400°C with an applied stress of 130 MPa could explain the higher residual hardening than after heat treatment.

The TEM observations made after the ring tensile tests following the post-irradiation creep test have shown that the irradiation induced loops seem to have been fully cleared on the contrary of what occurred with the microstructure observed after post-irradiation creep where remaining loops could still be seen [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF]. This can probably be explained by a homogeneous sweeping of loops by gliding dislocations as it is observed within the dislocation channels on the as-irradiated material.

It has been shown that the deformation occurs homogeneously throughout the grain by dislocation glide, no channel being observed. The easy glide system is the prismatic slip system as shown on Fig. 8(a). The deformation mechanisms are therefore similar to the deformation mechanisms occurring for the non irradiated material. This can explain that after post irradiation creep the material recovers its ductility. However, the dislocation density appears to be very high for this plastic strain level (2.43% and 5.16%). The high dislocation density already observed after creep test could explain these observations. Probably additional dislocation multiplication also occurs during the ring tensile test. Indeed, the high dislocation density, the remaining loops and the high density of needle-like precipitates decreases the mean free path along which the dislocations may glide. This could therefore lead to a high dislocation multiplication during the tensile test. This phenomenon could explain that the strain hardening is higher after post-irradiation creep compared to the non irradiated material.

The fact that the macroscopic ductility (UE and TE values) of the material remains lower than the macroscopic ductility of the non irradiated material could also be explained by the high dislocation density. Indeed the material is already strain-hardened, like a cold-worked material, its remaining strain hardening capability is therefore reduced.

Discussion on the behavior of the Zy-4 specimens after post-irradiation creep

It is well known that non irradiated stress-relieved Zy-4 exhibits a higher strength or hardness than non irradiated recrystallized Zr-1%Nb as confirmed by Tables 6 and7. This can be explained because the two materials show different microstructures. Indeed, Zy-4 is a Stress Relieved alloy with elongated grains that are very small (between 0.1 and 0.5 µm) in the transverse direction and a very high dislocation density, whereas the Zr-1%Nb alloy is a recrystallized alloy with larger equiaxial grains (5 µm) and very low dislocation density. These two microstructural features can explain the higher strength of the Zy-4. This material exhibits also a lower ductility because of its strain-hardened state and therefore its lower strain-hardening capability.

As for the Zr-1%Nb alloy, strong irradiation induced hardening is also observed for Zy-4, the change in strength (YS and UTS) and hardness being similar for the two materials. Zy-4 specimens have not been observed by TEM in this study. The irradiation hardening can also be explained by the high density of small irradiation induced <a> loops. After post-irradiation creep, the strength and hardness of the Zy-4 are recovered probably due to the annealing of the <a> loops. It has indeed been shown [START_REF] Cappelaere | Impact of Irradiation Defects Annealing on Long-Term Thermal Creep of Irradiated Zircaloy-4 Cladding Tube[END_REF] that after 10 days at 470°C, a partial irradiation defects recovery is observed showing a decrease of the number of <a> loops and an increase of their size. Moreover, after creep, the strength (YS and UTS) and microhardness can become lower than the values measured for non irradiated material depending on the creep tests conditions. This phenomenon has already been observed after post-irradiation heat treatment [START_REF] Ribis | Experimental and Modeling Approach of Irradiation Defects Recovery in Zirconium Alloys: Impact of an Applied Stress[END_REF]. The fact that the strength or hardness can decrease below their initial non irradiated values is probably due to the recovery of the dislocation network already present before irradiation and maybe to a start of recrystallization. Indeed it has been observed previously [START_REF] Cappelaere | Impact of Irradiation Defects Annealing on Long-Term Thermal Creep of Irradiated Zircaloy-4 Cladding Tube[END_REF] that after 10 days post-irradiation heat treatment at 520°C the initial structure has evolved towards a structure of equiaxial grains with a low dislocation density. However, according to Dunlop et al. [START_REF] Dunlop | Modelling isothermal and non-isothermal recrystallisation kinetics. Application to Zircaloy-4[END_REF], even after 17071 hours at 400°C the recrystallization might remain very low. The additional softening would be therefore mainly attributed to the annealing of the non irradiated dislocation network. The fact that the effect of post-irradiation creep on Zy-4 is the same as a heat treatment, contrary to Zr-1%Nb, can probably be attributed to the saturation of the dislocation density with plastic strain. Indeed, the dislocation

Figure 1 :

 1 Figure 1: Example of a tensile ring machined out of a long term creep specimen. The second

Figure 2 :

 2 Figure 2: Engineering stress -strain curves obtained during ring tensile tests performed at

Figure 3 :

 3 Figure 3: Pictures of the ring tensile test specimens after failure for : a) as-irradiated Zy-4

Figure 4 :

 4 Figure 4: Engineering stress -strain curves obtained during ring tensile tests performed at

  that the hardness values measured after post-irradiation creep tests performed on Zy-4 specimens at 420°C during 1077 hours and 4974 hours are in good agreement with the hardness measured after post-irradiation heat treatment performed at 420°C for similar duration showing that the effect of post-irradiation creep on the hardness of Zy-4 is the same as a post-irradiation heat treatment.

Figure 5 :

 5 Figure 5: Comparison between the hardness evolution after post-irradiation creep tests and

  as a function of the change in hardness (with respect to the non irradiated material) for both alloys. It can be observed on Fig.7that there is a linear relationship for all specimens between the change in UTS measured in the hoop direction and provided by the ring tensile tests and the change in Vickers hardness measured on the θ r cross section of the tube. The proportionality coefficient is: mm -2 . This analysis is in agreement with the relationship obtained byBusby et al. [8] between hardness and yield stress in irradiated austenitic and ferritic steels.The good correlation obtained between UTS values and hardness values confirm that the two techniques provide a coherent quantification of the recovery state of the material after postirradiation creep testing. In the following, these techniques are both used and compared to the TEM obsvervations.

Figure 7 :

 7 Figure 7: Correlation between the change in UTS and the change in hardness for both Zy-4

Figure 8 :

 8 Figure 8: Pictures of the ZrNb-4a specimen microstructure: the channels appear as clear

Figure 9 :Figure 10 :

 910 Figure 9: a) and b) Pictures of the ZrNb-2a specimen. c) and d) pictures of the ZrNb-3a

Figure 11 :

 11 Figure 11: Schematic plot of the evolution of the prismatic and basal CRSS with temperature

Table 1 :

 1 Chemical composition of the alloys (wt%)

	Alloy	Metallurgical state	Sn	Fe	Cr	Nb	O	Zr
	Zy-4	Stress-relieved	1.4	0.2	0.10	-	0.125 Bal.
	Zr-1%Nb	Recrystallized	-	0.02	-	1.0	0.125 Bal.

Table 2 :

 2

	Creep specimen	Creep test type	Creep temperature, °C	Creep stress, MPa	Creep duration, hours	Tensile specimen
	Zy4-1 4 cycles	LTC	420	130	4974	Zy4-1a Zy4-1b
	Zy4-2 4 cycles	CGP	420	130	1077	Zy4-2a Zy4-2b
	Zy4-3 4 cycles	LTC	380	130	14994	Zy4-3a Zy4-3b
	Zy4-4 4 cycles	LTC	400	130	17071	Zy4-4
	Zy4-5 As-irradiated 4 cycles	-	-	-	-	Zy4-5a Zy4-5b
	Zy4-6					Zy4-6a
	Non-irradiated	-	-	-	-	Zy4-6b

Testing conditions for the creep tests performed on the Zy-4 specimens. References of the tensile tests specimens performed at room temperature are given in the last column.

The following acronyms stand for: LTC: Long-Term Creep, CGP: Controlled Gas Pressure.

Table 3 :

 3 Testing conditions for the creep tests performed on the Zr-1%Nb specimens.

	Creep sample	Creep test type	Creep temperature °C	Creep stress, MPa	Creep duration, hours	Tensile specimen
	ZrNb-1 5 cycles	LTC	400	130	3301	ZrNb-1a ZrNb-1b
	ZrNb-2 4 cycles	CGP	400	130	984	ZrNb-2a* ZrNb-2b
	ZrNb-3 4 cycles	CGP	420	130	912	ZrNb-3a* ZrNb-3b
	ZrNb-4					ZrNb-4a*
	As irradiated	-	-	-	-	ZrNb-4b
	6 cycles
	ZrNb-5
	Non	-	-	-	-
	irradiated

References of the tensile tests specimens performed at room temperature are given in the last column. The following acronyms stand for: LTC: Long-Term Creep, CGP: Controlled Gas Pressure. The star (*) indicates the interrupted tests at UTS for TEM observations after testing.

Table 4 :

 4 Tensile conventional mechanical properties deduced from the engineering stress -

	strain curves obtained for Zy-4 specimens (at room temperature)
	Ring sample	Creep temperature -duration	YS (MPa)	UTS (MPa)	UE (%)	TE (%)
	Zy4-1a Zy4-1b	420°C -4974 h	676 686	731 743	1.71 1.69	17.97 17.32
	Zy4-2a Zy4-2b	420°C -1077 h	719 688	787 764	2.05 2.02	18.80 18.89
	Zy4-3a Zy4-3b	380°C -14994 h	722 722	778 788	1.94 1.49	17.31 15.89
	Zy4-4	400°C -17071 h	674	745	2.16	17.59
	Zy4-5a Zy4-5b	As-irradiated	916 940	966 989	0.84 0.87	9.89 8.68
	Zy4-6a Zy4-6b	Non irradiated	693 689	763 763	2.28 2.69	25.70 26.74

Table 5 :

 5 Mechanical properties obtained for Zr-1%Nb specimens. The star (*) indicates the tests interrupted at UTS in order to provide samples for TEM observations.

	Ring sample	Creep temperature -duration	YS (MPa)	UTS (MPa)	UE (%)	TE (%)	UTS-YS (MPa)
	ZrNb-1a ZrNb-1b	400°C -3301 h	470 484	555 558	5.01 3.91	27.17 29.57	85 74
	ZrNb-2a ZrNb-2b	400°C -984 h	515 525	568 577	2.43 (*) 2.63	-27.59	53 52
	ZrNb-3a ZrNb-3b	420°C -912 h	475 477	544 536	5.16 (*) 4.76	-30.95	69 59
	ZrNb-4a ZrNb-4b	As-irradiated	734 721	741 734	0.41 (*) 0.49	-30.88	7 13
	ZrNb-5a	Non irradiated	460	498	9.26	42.74	38
	ZrNb-5b	hydrided	460	487	6.96	43.07	27

  Table 6 displays the Vickers microhardness obtained on the Zy-4 specimens. Hardness values and measured UTS values were compared.

	3.2 Microhardness measurements
	3.2.1 Zy-4 specimens
	15/36

specimen and the Zr-1%Nb specimen tested after post-irradiation creep show a significant necking. Strong shearing is observed as the crack shows a 45° angle with the tensile axis. The crack is close to 45° on one side of the reduced area, and close to 135° on the other side, due to a change of the crack propagation direction at the reduced area center. It is worth noticing that both specimens shown on Fig.

3

exhibit the same fracture morphology

Table 6 :

 6

	Tensile sample	Creep temperature -duration	UTS (MPa)	Microhardness (HV)
	Zy4-1a	420°C -4974 h	731
				235 ± 5
	Zy4-1b	420°C -4974 h	743
	Zy4-2a	420°C -1077 h	787
				244 ± 3
	Zy4-2b	420°C -1077 h	764
	Zy4-5a	As-irradiated	966
				300 ± 8
	Zy4-5b	As-irradiated	989
	Zy4-6a	Non irradiated	763
				244 ± 3
	Zy4-6b	Non irradiated	763

Microhardness values measured after post-irradiation creep tests on Zy-4 specimens compared to UTS values measured during tensile tests after post-irradiation creep tests.

Table 7 :

 7 Microhardness values measured after post-irradiation creep tests on Zr-1%Nb specimens compared to UTS values measured during tensile tests after post-irradiation creeptests.

	Tensile sample		UTS (MPa)	Microhardness (HV)
	ZrNb-1a	400°C -3301 h	555
				195 ± 5
	ZrNb-1b	400°C -3301 h	558
	ZrNb-2a	400°C -984 h	568
				189 ± 5
	ZrNb-2b	400°C -984 h	577
	ZrNb-4b	As-irradiated	734
				237 ± 6
	ZrNb-4a	As-irradiated	741
	ZrNb-5d	Non irradiated	487
				165 ± 5
	ZrNb-5c

density is already very high in the material, the increase of the dislocation density during creep is therefore not significant. This also explains that the strain hardening behavior of the specimen after post-irradiation creep is similar to the strain hardening behavior of the non irradiated material.

Conclusions

The effect of post-irradiation creep test on the subsequent room temperature mechanical properties of zirconium alloys has been investigated and interpreted in terms of deformation mechanisms observed by TEM. It has been shown that for both stress-relieved Zy-4 and recrystallized Zr-1%Nb alloys post-irradiation creep tests lead to the recovery of the strength of the material (decrease of the YS and UTS values towards its non irradiated values) and to a recovery of the ductility of the material (increase of the UE and TE values towards its non irradiated values). In the case of the Zr-1%Nb it has been proven that the recovery of the strength of the material is due to the irradiation loops annealing that occurs during postirradiation creep test. It has also been shown that the loss of macroscopic ductility of the asirradiated material is due to the dislocation channelling mechanism that leads to an early localization of the plastic strain at the specimen scale.

On the other hand, after post-irradiation creep tests followed by a tensile test no channel is observed, the deformation occurring homogeneously throughout the grains, and the deformation mechanisms are similar to that of the non irradiated material. These observations explain the recovery of the strength and ductility after post-irradiation creep. However, the coupling between the recovery of the mechanical properties and the hydrides reorientation has still to be investigated in order to obtain a full understanding of the mechanical behaviour after dry transportation or dry storage.


Clément Pouget

Gisella Vetere
email: gisella.vetere@espci.fr

Fear memory engram is the mind-killer

Stegemann et al have unraveled a long-lasting role for fear engrams in future pain perception.

Prefrontal pain and fear representations become entangled following a fearful event. As subsequent painful experiences also reactivate the fear network, silencing the fear engram alone can alleviate both tonic pain and hypersensitivity in chronic pain.

MAIN TEXT

In Frank Herbert's novel "Dune", Paul Atreides has to endure a tremendous amount of pain, without flinching, by putting his hand in a mysterious device presented to him by the Bene Gesserit's reverend mother, as a test of his presence of mind. Paul (or Timothée Chalamet, for the more cinematographically-inclined scientists) manages to do so by reciting the 'litany of fear': "I must not fear. Fear is the mind-killer [...]", trying to feel no pain by suppressing his fear of the pain itself. As he was writing those lines, Herbert probably didn't realize how true the litany would eventually ring to neuroscientists. Indeed, pain and fear are inextricably linked. Fear can both powerfully suppress and enhance pain sensation, in different scenarios: sudden fear can temporarily suppress the perception of pain, such as when facing a threat; conversely, previously experienced fear episodes can chronically enhance pain, e.g., in the form of hyperalgesia in chronic pain patients [START_REF] Olango | Neurobiology of stress-induced hyperalgesia[END_REF] . While fear-mediated acute analgesia is well-documented and its mechanism explained 2 , we know very little about the neuronal mechanisms governing the opposite interaction.

In their paper, Stegemann et al. 3 showed that cells active in the mouse medial prefrontal cortex (PFC) during recall of long-term fear memory (LTFM), which form the fear memory 'engram', are involved in subsequent responses to painful events. They demonstrated that, in chronic pain models, the PFC fear-memory network extends to PFC pain-responsive cells and even to PFC touch-responsive cells. Moreover, optogenetic silencing of the LTFM engram cells alleviated hypersensitivity in mouse chronic pain models, providing hope for possible future PFC engram-targeting therapeutic strategies to curb chronic pain in human patients.

In this work, the target was the 'engram', that is, the ensemble of cells representing the physical substrate of a memory trace. These cells are activated when an event occurs and undergo the morphological changes necessary to recruit them again when we remember the same event [START_REF] Josselyn | Memory engrams: Recalling the past and imagining the future[END_REF] . This study focused on the ensemble of cells reactivated at a remote time point in the PFC. As a region known for its implication in both pain [START_REF] Ong | Role of the Prefrontal Cortex in Pain Processing[END_REF] and long-term fear memory [START_REF] Giustino | The Role of the Medial Prefrontal Cortex in the Conditioning and Extinction of Fear[END_REF] , the authors reasoned that the PFC may represent the hub connecting the two. Are pain and fear encoded in overlapping circuits within this region and is it realistic to study one without considering its influence on the other? Here 3 , the authors wished to specifically tag subsets of cells that are active at a particular time (e.g. the engram cells, and pain-responsive cells) using a well-established system called Tet-Off [START_REF] Sakaguchi | Catching the engram: strategies to examine the memory trace[END_REF] . Briefly, the expression of a reporter protein is under the control of both cell activity (regulated by the promoter of the c-Fos protein, a marker of neuronal activity) and the absence of doxycycline (Dox), which is administered to the animals through their food. Hence, the reporter is only expressed in cells that are active during an experimenter-determined time window. To first identify the ensemble of neurons that are active during both long-term fear memory recall and a subsequent tonic pain event (i.e., a hind paw injection of capsaicin 8 ), the LTFM engram was Tet-off-tagged by opening the tagging window during fear recall and using mCherry as a reporter protein. At the same time, cells expressing c-Fos during tonic pain were GFP-immunostained. Fluorescent cell counting revealed a strong, significant overlap between those two populations. Electrophysiological recording of the same PFC cells across the two behaviors confirmed the existence of this overlap.

Proving the existence of this overlap is, in and of itself, interesting. But, perhaps more importantly, it immediately becomes the prime suspect for LTFM-mediated potentiation of future pain. To test it, Stegemann et al. 3 changed the reporter protein so that the tagged engram expressed either an inhibitory or excitatory opsin. This ensemble of cells could then be manipulated during a subsequent tonic painful stimulation (Fig1c). As predicted, but still surprisingly, optogenetically silencing the LTFM engram reduced pain-related behaviors, effectively creating an "optogenetic Litany Against Fear". Conversely, activating the LTFM engram increased such behaviors. These outcomes were specific to the engram since no effect was detected when a similar-sized but random population of cells was targeted and manipulated.

The authors also targeted cells upon initial presentation of TMT, an odor present in urine and feces of foxes, which are natural mouse predators [START_REF] Rosen | The smell of fear: innate threat of 2,5-dihydro-2,4,5-trimethylthiazoline, a single molecule component of a predator odor[END_REF] , hence tagging an innate-fear engram. In this case, optogenetic activation yielded the opposite effect (i.e., suppression of pain) compared to activating a previous remote fear memory. This apparently contrasting result may hide a more complex mechanism, where if an animal experiencing tonic pain is artificially reexposed to a threat, the result is temporal analgesia to the pain. This experiment also confirmed that the effects of engram manipulation on pain-related behaviors were specific to the PFC LTFM engram. Furthermore, the authors tried replacing the tonic pain session with a pain-unrelated white-noise avoidance task or water reward-learning. In both cases, fear engram inactivation failed to modify mouse behavior, demonstrating that the LTFM engram specifically affects behaviors that are directly linked to pain.

Up until this point, the authors were looking at the influence of fear conditioning on acute tonic exposure to a painful stimulus. However, in chronic pain, clinical studies have shown that patients experience extreme, pathological sensitivity to non-painful stimuli. Knowing that fear memory can exacerbate this hyperalgesia [START_REF] Vlaeyen | Learning to predict and control harmful events: chronic pain and conditioning[END_REF] , one could wonder if fear engram manipulation will change subsequent chronic pain sensitivity.

To answer this question, the authors used both a neuropathic and an inflammatory chronic pain model, the former induced by unilateral peripheral spared nerve injury (SNI) and the latter by unilateral paw inflammation with Complete Freund's Adjuvant (CFA) [START_REF] Deuis | Methods Used to Evaluate Pain Behaviors in Rodents[END_REF] . In both models, hypersensitivity was achieved in the contralateral, unaffected paw, as expected. Fear conditioning the mice prior to chronic pain induction significantly heightened hypersensitivity specifically, hyperalgesia in a thermal withdrawal test in the CFA model, and allodynia in a tactile stimulation test in the SNI modelonce chronic pain set in. Similarly to the tonic pain results, fear-responsive cells and thermal or tactile-responsive cells significantly overlapped in mice with chronic pain compared to controls.

Interestingly, the overlap between fear and tactile representations was not confined to the PFC micro-network but expanded across the entire brain. In fact, the authors 3 used Tet-Offtagging during tactile stimulation to study projections of the tactile-responsive cells, and compared it with the fear-responsive cells. In naive animals, the two populations connected to very distinct networks (Fig1a). However, after both fear conditioning and chronic-pain induction (SNI), the tactile cells' connectivity strongly shifted to match that of the LTFM network, including hub regions for fear (Fig1b) such as amygdala, agranular insular cortex, mediodorsal thalamus, and anterior cingulate cortex [START_REF] Shin | The Neurocircuitry of Fear, Stress, and Anxiety Disorders[END_REF] . This suggests a mechanism for fearmediated heightened nociception in chronic pain: the fear network extends itself to include touch-sensitive cells of the PFC.

Considering how touch cells and their connectivity get incorporated into the fear network after chronic pain and fear conditioning, could one imagine reducing hypersensitivity by targeting the PFC LTFM engram? Indeed, when the authors Tet-Off-tagged the LTFM engram cells and optogenetically inactivated them in mice with SNI-or CFA-induced chronic pain, nociceptive hypersensitivity was significantly reduced both shortly (days) and long (weeks) after chronic pain onset (Fig1c).

An important drawback is that optogenetic inactivation only transiently reduced pain hypersensitivity: at the later timepoint, an additional optogenetic inactivation was necessary to reduce the regained hyperalgesia. This result leaves us wondering whether the fear engram can be definitively 'extinguished', in other words, whether its representation can be reverted to separate, untangled representations of fear and pain to achieve a lasting treatment for allodynia and hyperalgesia in chronic pain.

In their experiments, Stegemann et al. 3 consistently tagged the fear engram during memory recall and shortly thereafter presented the pain experience (either tonically or chronically). However, two different events have a higher chance of recruiting overlapping ensemble activity if they happen closer in time [START_REF] Rashid | Competition between engrams influences fear memory formation and recall[END_REF][START_REF] Cai | A shared neural ensemble links distinct contextual memories encoded close in time[END_REF] . This raises another question: would a broader time window between fear memory and pain experiences lead to similar results? Pain can be transiently silenced at critical moments in our lives when necessary. The mechanisms involved are rapidly induced and short-lived. We also know that fear memory can lead to a life-long enhancement of nociception. This opposite effect being long-lasting, it seems reasonable to assume it could be explained by long-term mechanisms such as plasticity or memory. Indeed, Stegemann et al. 3 demonstrate here that an LTFM engram in the PFC is involved in long-term pain heightening following a fearful event. They report evidence that fear-responding cells are not only primed to partially become pain-responding cells in the future, but that, in chronic pain models, fear conditioning results in whole-brain connectivity changes for touch-responding cells: these become entangled in the brain-wide fear network. Furthermore, inactivating the LTFM engram during tactile stimulation after induction of chronic pain greatly reduces hypersensitivity. Hence, the PFC, where fear and pain representations can merge, exchange, and connect, could prove a target of choice for novel therapeutic approaches, where precisely interfering with the mechanisms of fear memory (and not, as one could expect, to pain directly) could prove effective for reducing pain in chronically afflicted patients.

Fig. 1 |

 1 Fig.1 | Silencing the PFC fear memory engram suppresses pathological hypersensitivity in a chronic pain model. a, Tactile and fear-responding cells in the PFC have distinct brain network connectivities. b, However, if a pain-related fear experience precedes pain, the same tactile stimulus will activate touch-responding cells that are strongly connected with the fear network. c, If the fear engram is tagged during long-term fear memory recall, and is subsequently optogenetically silenced during a tonic painful experience such as capsaicin injection, this leads to alleviation of pain behaviors. Similarly,

ACKNOWLEDGMENTS

We would like to thank Colleen Gillon for the helpful comments. This work was supported by grants to G.V. from ANR (ANR-20-CE16-0015-01), Emergence(s) Ville de Paris (2019 DAE 81), a NARSAD Young Investigator Grant from the Brain and Behavior Research Foundation (#29489).