Florence Eustache
email: florence.eustache@inserm.fr

Badria Bennani Smires

Delphine Moison

Raymond Bergès

Marie-Chantal Chantal Canivenc-Lavier

Daniel Vaiman

Jacques Auger

Raymond Berges

Different exposure windows to low doses of genistein and/or vinclozolin result in contrasted disorders of testis function and gene expression of exposed rats and their unexposed progeny

Keywords: endocrine disruption, exposure window, male reproduction, mRNA, multigenerational Funding

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

Introduction

Low doses, mixtures and long-lasting periods characterize environmental exposure to endocrine disrupters (EDs) in humans.

By contrast, the vast majority of experimental schemes were most often based on high unrealistic doses of EDs, generally for a single compound studied with an in utero/perinatal exposure window. One consequence of these studies is that the gestational and lactational periods are more generally considered the prominent developmental periods where EDs induce both contemporary and future physiological dysfunctions, noticeably of the male reproductive system [START_REF] Atanassova | Comparative effects of neonatal exposure of male rats to potent and weak (environmental) estrogens on spermatogenesis at puberty and the relationship to adult testis size and fertility: evidence for stimulatory effects of low estrogen levels[END_REF][START_REF] Christiansen | Low-dose perinatal exposure to di (2-ethylhexyl) phthalate induces anti-androgenic effects in male rats[END_REF][START_REF] Peretz | Bisphenol A and reproductive health: update of experimental and human evidence, 2007-2013[END_REF], this assertion being also one of the main conclusions of human studies [START_REF] Sweeney | Environmental Endocrine Disruptors: Effects on the human male reproductive system[END_REF]. However, continuous EDs exposures encompass the pubertal period where the increase in the production of gonadal steroids underlying change of male sexual behaviour and sperm production may be disrupted [START_REF] Evuarherhe | Organizational role for pubertal androgens on adult hypothalamic-pituitary-adrenal sensitivity to testosterone in the male rat[END_REF][START_REF] Woodman | Reproductive hormones[END_REF]. In addition, possible modulations of the effects due to the continuity of exposure have rarely been considered. In a previous study, using a life-long exposure to the phytoestrogen genistein, a dietary compound present in several legumes, especially soy, and, the antiandrogenic fungicide and food contaminant vinclozolin, alone and in association, we reported various disorders of the male reproductive development and testis function [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF]. In the same study, we also showed that mRNA expression profiles in the adult testis were significantly and differently modified according to the exposure modalities with functional clustering of the genes affected into ontological families.

Besides, a growing number of studies have suggested that EDs could promote the epigenetic transgenerational/multigenerational inheritance of disease and phenotypic variation including male reproductive disorders [START_REF] Doyle | Transgenerational effects of di-(2-ethylhexyl) phthalate on testicular germ cell associations and spermatogonial stem cells in mice[END_REF][START_REF] Skinner | Environmentally induced transgenerational epigenetic reprogramming of primordial germ cells and the subsequent germ line[END_REF]. However, most of these studies rely on high level ED exposure with an exposure window limited to the gestational period. Thus, studies with more realistic exposure modalities are required [START_REF] Brehm | Transgenerational Effects of Endocrine-Disrupting Chemicals on Male and Female Reproduction[END_REF].

The aim of the present investigation was to assess comparatively the effects of a gestational/lactational (GL) versus pubertal and beyond exposure (puberty to adulthood, PA) to low dose genistein and/or vinclozolin using various endpoints related to the male genital tract as well as testicular mRNA expression in the exposed generation as well as their unexposed progeny. We selected the dose of 1 mg/kg bw/d for both compounds according to the reproductive effects reported previously for this dosage [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF]. This dosage corresponds to a level of genistein similar to that present in soya-based diets [START_REF] Tanaka | Relationship between soy and isoflavone intake and periodontal disease: the Freshmen in Dietetic Courses Study II[END_REF]. For vinclozolin, it is similar to the US Environmental Protection Agency NOAEL, no observed adverse effect level (1.2 mg/kg bw/d, based on a combination of chronic toxicity, carcinogenicity and toxicity reproduction in rats; US EPA, 2003) while above the level of human food contamination with vinclozolin residues. The estimated acceptable daily intake according to the European Food Safety Authority, EFSA is 0.005 mg/kg bw/d [START_REF] Food | Reasoned opinion of EFSA prepared by PRAPeR on MRLs of concern for the active substance vinclozolin[END_REF].

Materials and Methods

Chemicals and doses used

Both compounds, genistein (greater than or equal to 99% purity) and vinclozolin (greater than or equal to 95% purity) were obtained as previously reported [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF]. The selected doses were based on the results of Eustache and colleagues (2009) providing evidence that a mixture of genistein and vinclozolin (both at 1 mg/kg bw/d) chronically administered by gavage had a more severe impact on a number of male reproductive endpoints than a markedly higher dose of each compound separately. Therefore, in the present study investigating comparatively the role of exposure windows on male reproductive function as well as the possibly different reproductive consequences in the subsequent unexposed male offspring, we selected the single dose of 1 mg/kg bw/d for both genistein and vinclozolin, administered alone or in association.

Animals and regimens

All animals were treated humanely and suffering was minimized. All procedures involving rats were approved by the Ethics Committee of Burgundy University and conducted in accordance with French guidelines for the care and use of laboratory animals. The selected animals, the acclimatization period, animal facility and feeding conditions were the same as in [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF]. Briefly, specific pathogen-free female and male 8 weeks of age Wistar rats, obtained from Harlan France Sarl (Gannat, France), were maintained under controlled conditions at 22°C ambient temperature with 55% relative humidity and a 12h light and 12h dark cycle. From the acclimatization period, they were fed a soy-free purified diet (18% casein, 46% starch, 23% sucrose, 5% corn oil, 2% cellulose, 5% mineral mixture, and 1% vitamin mixture; Harlan Teklad, Gannat, France) and supplied with water ad libitum. Cages and bottles were made of polypropylene to avoid any contamination by bisphenol A or phthalates and the water was filtered through charcoal to eliminate any pesticide or active endocrine contaminant. Aspen sawdust was used as bedding, to prevent exposure to endocrine-like chemical residues.

Animal husbandry and exposure conditions

Females were mated with male rats as previously described [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF]. They were examined daily and caged separately when a vaginal sperm plug was observed (considered as the day of conception). On the day of parturition (PND, postnatal day 0), pups were weighed and identified by ear and paw tags. All litter sizes were standardized to 12 offspring. Chemicals were dissolved in corn oil (Carrefour, Dijon, France) and administered by gavage (2 mL/kg bw). Control animals were treated with the vehicle alone. Two exposure window modalities were selected (Fig. 1): i) a gestational/lactational exposure window (GL) where dams were exposed daily to genistein or vinclozolin or their association, from conception (gestational day 1, GD1) to weaning (PND21) or, ii) a pre-pubertal to adulthood exposure window (PA) where male rats were exposed daily to genistein or vinclozolin or their association immediately after weaning until PND100.

Fig. 1 Experimental design. A. Gestational-lactational exposure, from GD1 to PND21 (GL). B. Pre-pubertal to adulthood exposure, from PND21 to PND100 (PA). The F1 exposed male rats were mated with naive females to obtain an F2 that was maintained until adulthood without exposure. Necropsies were performed at PND100 in both generations. The 3 exposure modalities consisted in: i) genistein alone (G, 1 mg/kg bw/d) or ii) vinclozolin alone (V, 1 mg/kg bw/d) or, iii) a 1:1 genistein/vinclozolin mixture (GV, 1 mg/kg bw/d genistein + 1 mg/kg bw/d vinclozolin).

Anogenital distance, reproductive abnormalities rate and puberty timing

The anogenital distance (AGD) was measured with a digital caliper at weaning on all male rats as previously described [START_REF] Auger | Environmental levels of oestrogenic and antiandrogenic compounds feminize digit ratios in male rats and their unexposed male progeny[END_REF], and at PND21 on 50 rats per exposure group, by the same observer blind of exposure groups. Relative AGD (AGD/bw) was calculated. All macroscopic developmental anomalies of the reproductive tract (undescended testis, hypospadias…) and anomalies observed after dissection in the adult (i.e., epididymal cysts) were scored in order to determine their overall rate. The time of puberty onset was defined as the post-natal day of complete preputial separation [START_REF] Korenbrot | Preputial separation as an external sign of pubertal development in the male rat[END_REF]. It was assessed on a random subset of 20 animals per exposure group. For all these assays, the observers were blinded to exposure condition to avoid bias.

Mating and fertility

Fertility and reproductive performance were assessed only for F1 male rats. On PND90, 10 males per group were selected for mating with unexposed acclimatized females (Harlan France Sarl, Gannat, France): each male was caged with one female for four days. As described above, females were inspected daily and caged separately until parturition when a vaginal sperm plug was found. The fertility endpoints used were the mating index (number of mated females / number of females cohabited with males x100), the fertility index (number of pregnant females / number of mated females x100), the litter size and mean weight per neonate, the sex-ratio at birth, and the percentage of post implantation loss (number of embryonic scars / number of embryonic buttons and scars x100).

Sacrifice, tissue collection, weighing and epididymal (cauda) sperm number At PND 100 and for each exposure modality, 20 exposed males were anesthetized using isoflurane (2.5%) and killed by thoracic cage opening. All male pups stemmed from exposed fathers were raised until PND100 and sacrificed in the same manner. For each animal (F1 or F2), both testes, epididymis and seminal vesicles, ventral prostate and liver were excised and trimmed of fat, then weighed (Precisa, model 125A; Poissy, France). Relative weights were calculated. Epididymal sperm number was assessed using computer aided sperm analysis (CASA) technology as previously described [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF] in a random sample of 10 animals per exposure group (F1 and F2).

RNA preparation

RNA was prepared from individual testes obtained from six animals in each group, using classical TriZol extraction procedures; briefly, testes were directly put in 1 mL TriZol-containing tubes, the tissue was minced with a sterile steel bead and 2 Hz shaking during 1 minute. Then 200 µL of chloroform was added, the tube was vortexed and centrifuged at 5000 g during 5 minutes. The supernatant (600 µL) was precipitated with 500 µL of cold isopropanol; after centrifugation, the pellet was rinsed with 70% Ethanol, dried 10 minutes under a fume hood and resuspended in ~100 µL of RNAse-free water. RNA was quantified and its quality was evaluated using a Bioanalyzer (Agilent). All RNAs had a RIN (RNA Integrity Number) > 8.

Pools of RNAs from six testes were prepared with strict equilibration of their quantity to ensure equal representation of each individual RNA.

Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)

Reactions were carried out in a 10 µL volume using a Roche Lightcycler (Roche, Meylan, France) and an Invitrogen Sybergreen quantitative PCR kit (Invitrogen, Cergy-Pontoise, France). After 5 minutes at 95°C, each reaction was carried out for 45 cycles (95°C for 10 seconds, 55°C for 15 seconds, and 72°C for 15 seconds when fluorescence was measured). This was followed by progressive melting by increasing the temperature from 65°C to 97°C during 10 minutes, with continuous fluorescence capture. The resulting melting curve was considered satisfactory if one single peak was visible on its derivative.

In addition, PCR products were subjected to electrophoresis on an agarose gel to check for a single product of the expected size. Sdha was used as an endogenous control and for normalization of gene targets. For both generations, six males from each group were evaluated in triplicate. The comparative Ct method was used to calculate relative expression reported as mean relative mRNA expression in arbitrary units. The testicular transcriptome analysis was carried out for a set of 11 genes crucially involved in the meiosis, apoptosis and steroidogenesis steps (see Table 1 reporting the complete primer sequences for the selected genes).

Table1. Primer sets used for quantitative real-time PCR

Gene name

Forward primer (5'-3')

Testosterone Radioimmunoassay

To validate the results on the steroidogenesis genes expression level which was found modified homogeneously in the same direction (decreased overall) only for the GL exposed rats, we measured the intratesticular testosterone level by radioimmunoassay as previously described [START_REF] Habert | Testosterone, dihydrotestosterone and estradiol 17β levels in maternal and fetal plasma and in fetal testes in the rat[END_REF] for a subset of six adult rats exposed to G, V and GV during the gestational lactational period.

Data Analysis

Statistical tests were carried out using the BMDP statistical software [START_REF] Dixon | BMDP Statistical Software Manual[END_REF]. All statistical comparisons between exposure groups were carried out by an analysis of variance (ANOVA). When the null hypothesis was rejected, post hoc Tukey tests using the variance from the experiment as a whole (for the considered variable) were used for pairwise comparisons between different exposures (as shown in the tables). Differences for qualitative variables between treated and control groups were tested using the Pearson chi-square test. The significance level was set at 0.05. However, for some aspects of the study, indicated as appropriate in tables and figures, a threshold at 0.10 was used due to the relatively small size of the groups.

Results

Developmental anomalies of the male reproductive tract in the GL (gestational/lactational period) exposed rats (F1) and their progeny (F2)

Reproductive malformations were observed sporadically in rats gestationally/lactationally exposed to genistein (G) alone or vinclozolin (V) alone or genistein/vinclozolin mixture (GV). Exposure to the mixture significantly induced developmental abnormalities in the urogenital tract in about half of cases (p<0.002), especially, undescended testis observed in one third of animals (p<0.002). The rate of developmental reproductive anomalies was not increased in the unexposed progeny (F2) whatever the exposure condition for the fathers. The relative AGD was significantly modified in the exposed rats and their progeny (Table 2). Other reproductive endpoints at adulthood in the GL exposed rats (F1) and their progeny (F2)

The puberty onset was significantly increased in the vinclozolin and mixture exposure groups (Table 3). It was not significantly modified in the unexposed F2 (Table 3). The body weight and testis, epididymis, seminal vesicles and prostate relative weights were not found modified in the exposed rats as well as in their progeny (data not shown).

Fertility endpoints in the F1

Mating and fertility indexes were not modified whatever the type and period of exposure. Similarly, the litter size, mean pup weight, sex ratio at birth and rate of post implantation loss were unchanged in the exposed groups compared to the controls (data not shown).

Reproductive endpoints in the PA (puberty to adulthood period) exposed rats (F1) and their progeny (F2)

The relative anogenital distance as well as putative developmental anomalies were not evaluated in the F1. The relative AGD was not significantly modified in the progeny. The puberty onset was not delayed in the three exposure groups neither in the progeny. There were no changes in the body weights and relative weights of the reproductive organs in the F1 and F2 except for the relative weight of epididymis that was significantly decreased for the animals exposed to genistein alone (F1; p<0.05).

Similarly, no fertility endpoint was found modified in both F1 and F2 (data not shown).

Epididymal sperm number according to GL and PA exposure modalities (F1 and F2)

Neither each compound alone, nor their combination at the tested dose of 1mg/kg bw/d were found to affect the adult epididymal sperm count in both F1 and F2 when the exposure window was the gestational/lactational exposure (GL) (Fig. 2).

In contrast, the PA exposure window for genistein or vinclozolin decreased the adult epididymal sperm number by almost half in comparison to the values in controls (p<0.01 and p<0.05, respectively). For the mixture GV, only a slight, not significant decrease was found. Finally, a significant decrease of the adult epididymal sperm count was founded in the progeny of the PA animals exposed to V and GV (p<0.01, for both) (Fig. 2)

Fig. 2 Epididymal sperm counts according to the exposure modalities (see, Figure 1). Box plot displaying 10 th , 25 th , 50 th , 75 th , and 90 th percentile values and the mean value (dotted line) of epididymal (cauda) sperm numbers in: A. Rats exposed to genistein (G) or vinclozolin (V) or a 1:1 mixture (GV) from gestation to lactation (GL) and their unexposed progeny and, B.

Rats exposed to the same exposure modalities from pre-puberty to adulthood (PA) and their unexposed progeny.

Measurements at PND100; n = 10 rats per group. The horizontal grey dotted line represents the mean value in the F1 control group (A. exposure modality) for reference. *: p<0.05 and **: p<0.01 compared to control by Tukey post-hoc test.

Figure 3 summarizes the mRNA expression levels in controls and exposed animals for a set of genes playing a major role in the processes of meiosis, apoptosis and steroidogenesis.

Fig. 3 mRNA expression levels (relative fold changes) in controls and exposed animals according to the exposure windows (GL: gestational/lactational; PA: pre-pubertal to adulthood) for a set of genes playing a major role in the processes of meiosis, apoptosis and steroidogenesis (n= 6 per group). *: p<0.05, **: p<0.01 and, p<0.001 compared to control by Tukey post-hoc test.

For both exposure window modalities the selected dose of 1mg/kg bw/d for G, V and their mixture had no or only a weak impact on the level of expression of the genes involved in the meiosis pathway tested in the exposed animals (F1). Only the expression of Daz in G exposed animals was increased compared to the control group (p<0.05), and that of Stra8 was found diminished for V (p<0.01). Interestingly, in the progeny of GL exposed fathers, the levels of expression of Stra8, Spo11 and Sycp3 were increased compared to the control group. Contrasting with these results, the levels of expression of Daz, Stra 8 and Spo11 in the progeny of the rats exposed to G during the PA period were lower than in the control group (p<0.01).

The PA exposure had no impact on the level of expression of the selected apoptosis genes. This contrasted with the impact of the GL exposure window (GL) that was found to modify (not as expected) the levels of expression of Fas for G, V and GV exposures, p53 for GV and Casp3 for G. Concerning the progeny of the rats exposed from gestation to lactation, only the expression of Casp 3 was found reduced when compared to the controls (p<0.01). In contrast, the expression of Fas, p53 and Casp3 mRNAs levels for G was lower than the control group (p<0.01) and the expression levels of p53 and Casp 3 were increased for V compared to the control group (p<0.001) in the progeny of the rats exposed from the pre-pubertal period (PA).

The expressions of the genes of the steroidogenesis pathway were the most affected by both exposure windows, notably, the GL modality. For this exposure window, the expressions of Star, Cyp11a1, Cyp17a1 for G and GV were significantly lower than the control (p<0.01), and the expression of Cyp11a1 and Cyp17a1 for V was also significantly decreased (p<0.001). Hsd3 mRNA levels were significantly lower for GV compared to the controls (p<0.05). This was also observed for the progeny of the GL exposed rats (decreased expression levels of Cyp17a1 and Hsd3 for G and V; p<0.05 and p<0.01, respectively). The level of expression of crucial genes for the steroidogenesis pathway was also modified after the PA exposure modality, for example, the expressions of Star and Cyp11a1 for GV, Cyp11a1 and Hsd3 for V were significantly lower than the control (all, p<0.01). The expression of the genes of the steroidogenesis pathway was also found modified in the progeny of the PA exposed rats, notably a decrease was observed for all the tested genes after exposure of the father to G.

Testosterone Radioimmunoassay

The intratesticular level of testosterone was significantly lower than the control only for the GV exposed rats (Supplementary Fig.).

Discussion

This study shows that an oral exposure to vinclozolin at a dosage below the NOAEL and/or to a realistic dosage of genistein impairs the male reproductive tract and function. It demonstrates clearly how the GL (gestational/lactational) and PA (prepubertal to adulthood) periods of exposure affect differently several reproductive endpoints. Particularly, it provides some substantial evidence that testis function is differentially affected according to the exposure windows studied, with modifications of the expression level of several genes playing a pivotal role in the processes of meiosis, apoptosis and steroidogenesis. In addition, we provide new evidence that exposure to such doses of endocrine active substances impacts the male reproductive tract in the progeny, though not directly exposed, and this in a different way depending on whether the fathers were exposed from gestation to lactation or from prepuberty to adulthood. It should be noted that due to the main purpose and design of our study, we have not investigated here the crucial question of dose effect curves with EDs exposures, rather, we used a single (low) dose of genistein and vinclozolin previously shown to impact several male reproductive endpoints and consistent with human exposure levels [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF].

Only few and controversial data based on in vivo experiments concern the reproductive effects and potential modes of action of alimentary levels of genistein as well as dosages of vinclozolin around the NOAEL or less (for example, after a similar genistein exposure, the relative AGD was found either increased [START_REF] Sherrill | Developmental exposures of male rats to soy isoflavones impact Leydig cell differentiation[END_REF], or unmodified [START_REF] Zhang | Disruption of reproductive development in male rat offspring following gestational and lactational exposure to di-(2-ethylhexyl) phthalate and genistein[END_REF] or, even decreased (Bobert et al., 2013)). Overall, we failed to find in the literature reports on the reproductive effects of genistein or vinclozolin with experimental designs similar to ours and most of them differed in many points (species, strain, modalities of exposure, number of animals, etc…), most being based on high dosages.

Here we did not find any marked effect of the 1mg/kg bw/d selected dosage of genistein on the set of classical reproductive endpoints studied for the GL exposure while the mRNA expression level of the genes of the steroidogenesis pathway was found to be lowered for this exposure window, the testicular level of testosterone being decreased but not significantly. The genistein exposure from the starting puberty only affected the epididymal weight and sperm count. This result is in accordance with [START_REF] Caceres | Isoflavones and their effects on the onset of puberty in male Wistar rats[END_REF] who reported a decreased sperm count after an oral administration of isoflavones in male Wistar rats during a PA period at both 0.56 mg/kg bw/d and 5.6 mg/kg bw/d. In contrast, the dosage of genistein used here was not found to affect the studied testicular mRNA expression levels overall for this exposure window. Regarding the effects of genistein on steroidogenesis, it is also known that exposure to genistein can affect testosterone levels during GL exposure [START_REF] Akingbemi | Exposure to phytoestrogens in the perinatal period affects androgen secretion by testicular Leydig cells in the adult rat[END_REF][START_REF] Meena | Altered spermatogenesis, steroidogenesis and suppressed fertility in adult male rats exposed to genistein, a non-steroidal phytoestrogen during embryonic development[END_REF] but these studies used higher doses of genistein than ours. More precisely, [START_REF] Sherrill | Developmental exposures of male rats to soy isoflavones impact Leydig cell differentiation[END_REF] have shown that a GL exposure to isoflavones (genistein and daidzein) may affect Leydig cell differentiation. Others studies have reported that in turn, isoflavones exposure decreases testosterone production (see for example, [START_REF] Svechnikov | Influence of long-term dietary administration of procymidone, a fungicide with anti-androgenic effects, or the phytoestrogen genistein to rats on the pituitary-gonadal axis and Leydig cell steroidogenesis[END_REF][START_REF] Retana-Márquez | Comparison of the effects of mesquite pod and Leucaena extracts with phytoestrogens on the reproductive physiology and sexual behavior in the male rat[END_REF]. However, the exact mechanisms of genistein action on testosterone production is not fully elucidated and it should be noted that others studies have not shown adverse effects on plasma testosterone levels whatever the doses of isoflavones used (genistein and daidzein) [START_REF] Cederroth | Potential detrimental effects of a phytoestrogen-rich diet on male fertility in mice[END_REF][START_REF] Boberg | Endocrine disrupting effects in rats perinatally exposed to a dietary relevant mixture of phytoestrogens[END_REF].

In the present study, a GL exposure to 1mg/kg bw/d vinclozolin was unexpectedly found to increase the anogenital distance.

This dosage was found to delay the puberty onset and decrease the mRNA expression levels of Cyp11a1 and Cyp17a1 and decrease (as a trend) the intratesticular testosterone level. The vinclozolin exposure from the starting puberty was essentially found to affect the epididymal sperm count. It is interesting to note that [START_REF] Elzeinova | Effect of low dose of vinclozolin on reproductive tract development and sperm parameters in CD1 outbred mice[END_REF] using exposure modalities approaching ours have also reported a decreasing sperm count in mice exposed to 1 mg/ kg bw/d vinclozolin, during the PA window while the decrease was more pronounced for a GL period, a result contrasting with ours. Recently, [START_REF] Flick | Investigations of putative reproductive toxicity of low-dose exposures to vinclozolin in Wistar rats[END_REF] for the same dosage in Wistar rats but using a chronical exposure reported no adverse reproductive effects.

For the doses tested here, the interpretation of the effects observed with the combination of genistein and vinclozolin is not obvious since, depending on the endpoints tested as previously discussed [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF] : i) an effect may be found with the mixture when each compound in isolation has no effect (for example, the rate of developmental anomalies or the level of expression of Hsd3 with GL exposure), ii) the effects observed with only one or both compounds separately are potentiated with the mixture (for example, the drop in intratesticular testosterone for GL exposure) or, iii) the effects with both compounds may be tempered (for example, the anogenital distance) or nullified (for example the sperm count for the PA exposure). It was previously reported that genistein at 1 mg/kg bw/d could antagonize the deleterious impact of a vinclozolin exposure at 1 mg/kg bw/d on the number of foetal/neonatal germ cells [START_REF] Lehraiki | Antagonistic effects of gestational dietary exposure to low-dose vinclozolin and genistein on rat fetal germ cell development[END_REF]. This last result and some of ours are consistent with the report of [START_REF] Zhang | Disruption of reproductive development in male rat offspring following gestational and lactational exposure to di-(2-ethylhexyl) phthalate and genistein[END_REF] showing that genistein may have a protective action against di (2-ethylhexyl) phthalateinduced reproductive effects.

Since our results with the GV mixture are preliminary and obtained with a study design not devoted to investigate the modes of action, further studies are desirable in order to know whether the results with the mixture reported here are reproducible.

Overall and unfortunately, there is still a noticeable gap in our understanding of the mixture in vivo effects with low (realistic) dosages of endocrine active substances, which may act via different modes of action. This contrasts markedly with our knowledge in the field gathered thanks to powerful theoretical approaches devoted to mixtures [START_REF] Kortenkamp | Which chemicals should be grouped together for mixture risk assessments of male reproductive disorders?[END_REF]. For example, it was evidenced that a mixture of high doses of antiandrogens has cumulative effects on androgen-sensitive endpoints in the pubertal male rat [START_REF] Blystone | Cumulative and antagonistic effects of a mixture of the antiandrogens vinclozolin and iprodione in the pubertal male rat[END_REF][START_REF] Christiansen | Combined exposure to antiandrogens causes markedly increased frequencies of hypospadias in the rat[END_REF]. It was also demonstrated in vitro that a mixture of antiandrogenic pesticides has additive effects at very low concentrations [START_REF] Orton | Mixture effects at very low doses with combinations of anti-androgenic pesticides, antioxidants, industrial pollutant and chemicals used in personal care products[END_REF].

It is well documented that foetus or new-born may be more vulnerable to EDs exposure compared to adults. However, the peripuberty years are also a critical period with the setting up of primary and secondary sexual characteristics as well as the initiation of spermatogenesis; of note, this period has been much less studied. A delayed puberty onset was observed after a GL exposure with vinclozolin and mixture. In parallel, a significant decrease in the expression of steroidogenesis genes (Star, Cyp11a1 and Cyp17a1) was observed for rats exposed in utero to weaning with genistein and/or vinclozolin, and, the level of testosterone was decreased for the rats exposed during the gestation/lactation period to G, V and GV (only significant for the GV exposed group). In human, there is few data about effects of neonatal exposure to endocrine disruptors on pubertal timing in boys, and these data are sometimes contradictory. A decrease of testosterone and LH levels was reported in boys of mothers exposed in utero to polychlorinated biphenyls/polychlorodibenzofuranes [START_REF] Hsu | Serum hormones in boys prenatally exposed to polychlorinated biphenyls and dibenzofurans[END_REF], PCB [START_REF] Grandjean | Reproductive hormone profile and pubertal development in 14-year-old boys prenatally exposed to polychlorinated biphenyls[END_REF] and to DDT (Eskenasi et al., 2017). These data suggest a probable central origin of delayed puberty. In our study, when males were exposed in utero, we observed a delayed puberty onset and a decrease of intratesticular testosterone levels in adults. In a recent review [START_REF] Parent | Developmental variations in environmental influences including endocrine disruptors on pubertal timing and neuroendocrine control: Revision of human observations and mechanistic insight from rodents[END_REF], it was reported that a post weaning exposure to EDs (vinclozolin, DDE, DES) in male rodents leads to delayed puberty as opposed to an absence of effects after an in utero/neonatal exposure. The dose of EDs probably also plays a critical role [START_REF] Parent | Developmental variations in environmental influences including endocrine disruptors on pubertal timing and neuroendocrine control: Revision of human observations and mechanistic insight from rodents[END_REF]. Indeed, [START_REF] Monosson | Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat[END_REF] and [START_REF] Yoshimura | Observation of preputial separation is a useful tool for evaluating endocrine active chemicals[END_REF] found that a PA vinclozolin exposure delayed pubertal maturation at 30 and 100 mg/kg bw/d. Thus, the pubertal effects of EDs exposures may depend on the dose but also on the period of exposure.

We failed to find in the literature reproductive studies focusing on EDs exposure windows allowing comparisons because of too different experimental schemes. We have previously reported the reproductive effects of an exposure to genistein and/or vinclozolin from conception to adulthood using the dose of 1 mg/kg for each [START_REF] Eustache | Chronic dietary exposure to a low-dose mixture of genistein and vinclozolin modifies the reproductive axis, testis transcriptome, and fertility[END_REF]. This previous study was carried out using the same animal facilities and experimental conditions than those of the present study allowing us to compare previous results to the results reported here. Table 4 summarizes the effects observed for the different exposure modalities. Clearly, the higher number of disrupted male reproductive endpoints among which, decreased relative AGD, decreased sperm counts, impaired sperm motion parameters, decreased fertility, and increased post-implantation loss, was found for the exposure window encompassing all developmental steps (i.e., the exposure from conception to adulthood, comprising thus the in utero, perinatal, pubertal and adult periods). This reinforces the concept that scrutinizing only the in utero or gestational/lactational periods cannot account for the effects that may arise under realistic conditions.

In the second generation, as for the parental exposure, no marked disruptions of the reproductive endpoints tested were observed except for the sperm count that was found significantly decreased for the rats whose fathers were exposed to vinclozolin and the mixture from the pre-pubertal period. It should be noted that the mRNAs of p53 and Casp3 were overexpressed relative to control only for the offspring of vinclozolin exposed rats. Besides, the Star mRNA under-expression was also found for this group. Recently, EDs have been proved to cause multigenerational and transgenerational effects on both male and female rodent reproductive function [START_REF] Anway | Comparative anti-androgenic actions of vinclozolin and flutamide on transgenerational adult onset disease and spermatogenesis[END_REF][START_REF] Chen | The Mechanism of Environmental Endocrine Disruptors (DEHP) Induces Epigenetic Transgenerational Inheritance of Cryptorchidism[END_REF][START_REF] Zhou | Exposure to an Environmentally Relevant Phthalate Mixture Causes Transgenerational Effects on Female Reproduction in Mice[END_REF]. When the generation F0 female is pregnant and exposed to an ED, then her offspring (F1 generation), are directly exposed to the ED in utero. The F2 generation is indirectly exposed via the germ cells of the F1 generation. Effects of ED observed in the F1 or F2

generations are considered to be multigenerational. The F3 generation is the first generation with no direct exposure to the ED, and effects would be considered transgenerational. In contrast, when F0 males are exposed to ED, effects in the produced F1 are considered multigenerational through exposure in the germline, and effects in the F2, the first generation not directly exposed, are considered transgenerational [START_REF] Daxinger | Understanding transgenerational epigenetic inheritance via the gametes in mammals[END_REF][START_REF] Lim | Bridging the transgenerational gap with epigenetic memory[END_REF]. The transgenerational effects of ED observed on male reproduction are for example: a shorter AGD [START_REF] Manikkam | Transgenerational actions of environmental compounds on reproductive disease and identification of epigenetic biomarkers of ancestral exposures[END_REF], reduced fertility [START_REF] Anway | Seminiferous cord formation and germ-cell programming: epigenetic transgenerational actions of endocrine disruptors[END_REF][START_REF] Song | Transgenerational impaired male fertility with an Igf2 epigenetic defect in the rat are induced by the endocrine disruptor p,p'-DDE[END_REF], and decreased sperm production [START_REF] Anway | Seminiferous cord formation and germ-cell programming: epigenetic transgenerational actions of endocrine disruptors[END_REF][START_REF] Song | Transgenerational impaired male fertility with an Igf2 epigenetic defect in the rat are induced by the endocrine disruptor p,p'-DDE[END_REF][START_REF] Doyle | Transgenerational effects of di-(2-ethylhexyl) phthalate on testicular germ cell associations and spermatogonial stem cells in mice[END_REF]. These transgenerational effects are thought to be mediated via epigenetic mechanisms including DNA methylation, histone modifications and microRNA expression [START_REF] Skinner | Epigenetic transgenerational inheritance[END_REF][START_REF] Larriba | Role of Non-Coding RNAs in the Transgenerational Epigenetic Transmission of the Effects of Reprotoxicants[END_REF][START_REF] Nilsson | Vinclozolin induced epigenetic transgenerational inheritance of pathologies and sperm epimutation biomarkers for specific diseases[END_REF] though the modes of action are not fully elucidated. The vast majority of the transgenerational/multigenerational studies following EDs exposure have used wide dose ranges that are not always relevant to human exposure (for example, 100 mg/kg bw/d for vinclozolin; [START_REF] Anway | Seminiferous cord formation and germ-cell programming: epigenetic transgenerational actions of endocrine disruptors[END_REF] and exposure routes that are not comparable to daily human ones (i. e., intraperitoneal for vinclozolin; [START_REF] Doyle | Transgenerational effects of di-(2-ethylhexyl) phthalate on testicular germ cell associations and spermatogonial stem cells in mice[END_REF][START_REF] Yuan | From the cover: metabolomics reveals a role of betaine in prenatal DBP exposure-induced epigenetic transgenerational failure of spermatogenesis in rats[END_REF]. Here we provide evidence that an oral low dose exposure to EDs from the puberty may impact the reproductive function in the male progeny not directly exposed. The experimental design of our study does not consider a third generation. In addition the possible modes of action and the modest effects observed in the F2 remain to be confirmed. However, the present results indicate that exposure windows, dose ranges and mixtures of chemicals should be more considered in order to use animal studies as proxy for EDs human exposure.

Conclusion

We provide evidence that low dose exposure to endocrine active substances during the crucial developmental period starting from the prepuberty affects the adult testis function at least as much as an exposure during the GL period. This suggests better considering this developmental period in reproductive studies on environmental endocrine compounds. More studies based on relevant dose ranges, mixtures of chemicals, routes and exposure windows are required to better assess human exposure risk as well as for monitoring the possible reproductive effects in the subsequent generations or to better understand the mechanisms involved.

  10

Table 2 .

 2 Relative anogenital distance in rats gestationally/lactationally exposed to genistein (G; 1 mg/kg bw/d) and/or

	vinclozolin (V; 1 mg/kg bw/d), and in F2 rats stemmed from exposed fathers
	AGD/bw measured on PND21 on 50 rats per exposure group, by the same observer blind of exposure groups; ANOVA results
	and * p<0.05 and, **p<0.01for paired comparisons between each exposure group and controls
			GL exposure	p-value
		Control	G	V	GV
	Exposure (F1)	0.273±0.016	0.268±0.019	0.290±0.027** 0.277±0.032** 0.001
	Father exposure (F2)	0.365±0.033	0.375±0.028	0.384±0.027** 0.380±0.026** 0.001

Table 3 .

 3 Puberty onset (day of preputial separation) in rats gestationally/lactationally exposed (GL) to genistein (G; 1 mg/kg bw/d) and/or vinclozolin (V; 1 mg/kg bw/d), and in F2 rats stemmed from exposed fathers. † † p<0.05; ‡ not significant but

	tendency (p<0.10)
			GL exposure		p-value
		Control	G	V	GV
	Exposure (F1)	40.5±2.6 † †, ‡	41.4±2.8	42.8±2.7 † †	42.5±1.9 ‡	0.02
	Father exposure (F2)	39.3±1.3	40.6±1.0	39.9±1.3	40.2±1.0	0.17

Table 4 .

 4 The impact of exposure windows, gestational-lactational vs pre-pubertal to adulthood vs conception to adulthood on

	various reproductive endpoints*
				Present study			Eustache et al., 2009
		Gestational-lactational exposure	Pre-pubertal to adulthood exposure	Conception to adulthood exposure
		G	V	GV	G	V	GV	G	V	GV
	Developmental	-	-	**	ND	ND	ND	#	#	#
	anomalies
	Relative AGD	-			ND	ND	ND	-
	Puberty onset	-		-	-	-	-	-
	Body weight	-	-	-	-	-	-		-	-
	Relative weight of
	testis, epididymis,	-	-	-	***	-	-	***	***	*** ;$
	seminal vesicles
	and prostate
	Epididymal sperm	-	-	-			-	-	-
	number
	Fertility endpoints	-	-	-	-	-	-	Litter	PIL	Mating
								size		index
								PIL		Litter
										PIL
	* From present study and Eustache et al., 2009, both studies being based on G: genistein exposure, V: vinclozolin exposure
	and, GV: mixture exposure at the same dose of 1mg/kg/d for both compounds; arrows represent significantly modified
	endpoints in exposed groups compared to control animals
	** cryptorchidism; *** only for epididymis; # macroscopically irregular, cystic, and/or enlarged epididymis; $ seminal vesicles

PIL: Post implantation loss; ND: not determined.

Acknowledgements

We thank the INRA (Institut National de la Recherche Agronomique) animal facility at Dijon and Sandrine Barbeaux for the English revision of the manuscript.

This work was supported by the French Program on Endocrine Disruption (PNRPE; contract n°CV05000147).

Animal care

All applicable international, national, and institutional guidelines for the care and use of the animals were followed. All animals were treated humanely and suffering was minimized. All

Author Contributions

Edited the manuscript: Jacques


Jiaqi Luo
email: jiaqi.luo@uni-saarland.de

Christina Hein

Jean-François Pierson

Frank Mücklich

Sodium chloride assists copper release, enhances antibacterial efficiency, and introduces atmospheric corrosion on copper surface

Keywords: copper, cuprous oxide, chloride, corrosion, E. coli, antibacterial surface

Sodium chloride (NaCl) is commonly found in the physiological buffers. This study found out its additional but important role in antibacterial efficiency test by better corroding metallic copper surface. Combined with multiple characterisation methods including scanning electron microscope, grazing incidence X-ray diffractometer, and inductively coupled plasma mass spectrometry, 0.9% saline was observed to cause localised corrosion attacks on copper surface, enhancing release of copper content. The 1 h killing rate against Escherichia coli was thus promoted from 22% (when re-suspended in pure water) to 98%. By a long-term observation (3-week), residual NaCl crystals on 0.9% saline treated copper surface were found partially disappeared in atmospheric environment, contributing to an additional Cu2O layer forming above the treated surface. Besides, oxygen-containing species were observed even on fresh copper surface exposed by focused ion beam after saline treatment, suggesting a chloride-assisted atmospheric corrosion process.

Introduction

Antibacterial surfaces are drawing attentions in the recent years [START_REF] Vincent | Contact killing and antimicrobial properties of copper[END_REF][START_REF] Rigo | Nanoscience-Based Strategies to Engineer Antimicrobial Surfaces[END_REF][START_REF] Muller | Antimicrobial surfaces to prevent healthcare-associated infections: a systematic review[END_REF]. They can be applied as touched surfaces, having enormous potential demands in those heavily microbial burdened healthcare environments [START_REF] Dunne | Specialised cleaning associated with antimicrobial coatings for reduction of hospital acquired infection[END_REF][START_REF] Colin | Copper Alloy Touch Surfaces in Healthcare Facilities: An Effective Solution to Prevent Bacterial Spreading[END_REF][START_REF] Chyderiotis | New insights on antimicrobial efficacy of copper surfaces in the healthcare environment: a systematic review[END_REF] as well as in our daily lives [START_REF] Michel | Antimicrobial Benefits of Copper Alloy Touch Surfaces[END_REF][START_REF] Grass | Metallic Copper as an Antimicrobial Surface[END_REF]. In order to verify and assess the antibacterial ability of newly fabricated surfaces, antibacterial efficiency tests were designed and carried out.

Nowadays, there are different types of evaluation methods such as dry plating [START_REF] Warnes | Mechanism of Copper Surface Toxicity in Vancomycin-Resistant Enterococci following Wet or Dry Surface Contact[END_REF], wet plating [START_REF] Molteni | Killing of Bacteria by Copper Surfaces Involves Dissolved Copper[END_REF], agar disk diffusion [START_REF] Sonia | Synthesis of hierarchical CuO nanostructures: Biocompatible antibacterial agents for Gram-positive and Gram-negative bacteria[END_REF], etc. Limited by the incubation of bacteria and transfer process of bacterial suspension, a certain medium must be introduced during these evaluations. This medium is usually called buffer, which provides a suitable aqueous environment for bacterial survival without further growth. Despite the wide selection of buffers, in most of the cases, two main requirements should be satisfied: (1) a manageable pH range and (2) a suitable osmotic pressure for the microbes. Take phosphate-buffer saline (PBS) as an example [START_REF]Phosphate-buffered saline (PBS)[END_REF], it contains a pair of acid and its conjugate base so as to compensate the pH fluctuation around 7.4. For the second purpose, it is sodium chloride (NaCl) that plays the part.

As can be seen, the design of buffers mainly considers the needs of microbes. But when they are to be applied in antibacterial surface research, a question needs to be raised: should the potential interactions between buffers and surfaces also be considered? The answer may be "certainly". For example, copper-containing surfaces have been recently developed for antibacterial applications in various occasions [START_REF] Sun | Technology and Antimicrobial Properties of Cu/TiB2 Composite Coating on 304 Steel Surface Prepared by Laser Cladding[END_REF][START_REF] Steinhauer | Antimicrobial efficacy and compatibility of solid copper alloys with chemical disinfectants[END_REF][START_REF] Hahn | Antimicrobial properties of ternary eutectic aluminum alloys[END_REF]. Their antibacterial capability originates from the multifunctional antibacterial effects of copper ions [START_REF] Solioz | Copper and Bacteria: Evolution, Homeostasis and Toxicity[END_REF]. To strongly interfere the survival of harmful microbes, copper ions should be efficiently released from the surfaces. As metallic surfaces, this release process is, most of the time, achieved by corrosion.

The composition and the properties of buffer could become decisive, as they could facilitate or retard the corrosion process.

Therefore, the solution and the copper surfaces together display how effective the antibacterial activity would be shown in antibacterial efficiency test.

Back to the example of PBS or other saline solutions, without a doubt, NaCl is expected to play an extremely important factor in revealing the antibacterial efficiency of copper surface. Electrochemical study has investigated the roles of chloride ions (Cl -) in formation and transformation of corrosion product (CuCl, Cu2O, Cu(OH)2, etc.) [START_REF] Toparli | State of the Surface of Antibacterial Copper in Phosphate Buffered Saline[END_REF], which could further affect the diffusion of copper ions [START_REF] Webster | Microbiologically Influenced Corrosion of Copper in Potable Water Systems-pH Effects[END_REF]. In general, the presence of Cl -also leads to a relatively corrosive environment for copper, depending on the concentration of Cl - [START_REF] Mayanna | Role of chloride ions in relation to copper corrosion and inhibition[END_REF]. Meanwhile, it easily promotes pitting corrosion and further deterioration on the surface [START_REF] Warraky | Pitting corrosion of copper in chloride solutions[END_REF]. This could, on the contrary, contributes to the copper antibacterial efficiency. Because whenever the corrosion is enhanced, a faster dissolution process of the antibacterial substance (copper ions) is anticipated. For example, a latest study that evaluated antibacterial property of copper-silver surface has discovered the constructive effect of chloride addition in dissolving antibacterial copper ions [START_REF] Ciacotich | Influence of chlorides and phosphates on the antiadhesive, antibacterial, and electrochemical properties of an electroplated copper-silver alloy[END_REF]. Other experiments also suggest that Cu-Fenton chemistry, as one of the antibacterial mechanism, could be accelerated by Cl - [START_REF] Wang | Chloride-accelerated Cu-Fenton chemistry for biofilm removal[END_REF], resulting in a better biofilm removal. On the whole, a different antibacterial efficiency is therefore highly expected in Cl --containing environment.

Other than affecting the corrosion behaviour in media, it is noteworthy that NaCl deposits could induce atmospheric corrosion on dry surfaces. At high humidity levels, for instance, deliquescence of NaCl could happen, initiating micro-droplets formation on metallic surfaces and introducing further corrosion [START_REF] Li | Atmospheric corrosion initiation on steel from predeposited NaCl salt particles in high humidity atmospheres[END_REF]. In lower humidity circumstances (below than 79% RH, deliquescence point of NaCl), where NaCl does not dissolve though, adsorption of water on the surface could form several layers that act as bulk water [START_REF] Schindelholz | Wetting phenomena and time of wetness in atmospheric corrosion: a review[END_REF]. Diffusion of Cl -could thus occur in this quasi-aqueous condition resulting in corrosion attack on copper [START_REF] Chen | Effect of Sodium Chloride Particles on the Atmospheric Corrosion of Pure Copper[END_REF]. Either way, tracking this atmospheric corrosion effect after antibacterial efficiency test may have significant implication for long-term product design [START_REF] Hutchison | Solute Capture and Doping of Al in Cu2O: Corrosion, Tarnish Resistance, and Cation Release of High-Purity Cu-Al Alloys in Artificial Perspiration[END_REF].

Lately, we compared PBS and Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Na-HEPES) in terms of their impacts on evaluating the antibacterial performance of metallic copper and Cu2O surface [START_REF] Luo | Early-stage corrosion, ion release, and the antibacterial effect of copper and cuprous oxide in physiological buffers: Phosphate-buffered saline vs Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid[END_REF]. Corrosion behaviours introduced by PBS caught our attention, therefore further experiments were designed to reveal the roles of bacteria were revealed [START_REF] Luo | Bacteria accumulate copper ions and inhibit oxide formation on copper surface during antibacterial efficiency test[END_REF] and to discuss the corrosion attacks and oxide growth [START_REF] Luo | Localised corrosion attacks and oxide growth on copper in phosphate-buffered saline[END_REF]. Since PBS contains NaCl, this study narrows down to it, to find out if NaCl plays an important factor as expected, in respect to corrosion. The primary objective is to explore how 0.9% saline affects the antibacterial efficiency test on metallic copper surface. Afterwards, the corrosion behaviours were compared among saline with different Cl -or Escherichia coli (E. coli) concentrations. Finally, the following aging effects resulting from the residual NaCl crystals on copper surface were also characterised.

Materials and Methods

Materials

Copper (99.99%, K09, Wieland) coupons were ground with a silicon carbide sandpaper (stepped down to grit number P600), then cleaned with ethanol in an ultrasonic bath, finally dried by air.

Solutions

Saline with various final concentrations (0.45%, 0.9%, and 1.8%) was prepared with NaCl (VWR, Germany) and pure water for analysis (Merck, Germany), sterilised in an autoclave after preparation. PBS was prepared with NaH2PO4. 1H2O (Merck, Germany, final concentration 0.01 M), NaCl (VWR, Germany, final concentration 0.14 M) and pure water for analysis, and its pH value was adjusted by adding NaOH to 7.4. Buffers were sterilised in an autoclave after preparation.

The preparation of bacterial suspension will be described in Section 2.6. Besides, a fivefold concentration E. coli NaCl suspension was simply prepared by re-suspending bacteria in 0.2 mL of NaCl instead of its original volume 1 mL. A tenth fold diluted E. coli NaCl suspension was prepared in the similar manner.

Corrosion protocol

Droplet of 20 µL pure solution or E. coli suspension was applied on ground copper surfaces with a pipette. These copper coupons were placed in a water-saturated atmosphere at room temperature for 3 h. Solutions or suspensions were then withdrawn with a pipette. These steps are chosen to simulate the antibacterial efficiency test further described in Section 2.6.

If not specified, the coupons were then cleaned with ethanol in an ultrasonic bath immediately after withdrawal of droplet. However, for the tests with various NaCl or bacterial concentrations (Section 3.4), the treated surfaces were firstly wiped with detergent-dipped cotton pads in order to remove the adhered bacteria. On the other hand, for some tests designed for atmospheric aging experiments (Section 3.5), the coupons remained unwashed. They were constantly kept in the lab atmosphere at room temperature, except for the time being characterised.

Surface characterization

Different functions of scanning electron microscope (SEM, Helios NanoLab600, FEI) were employed to observe copper surfaces after various corrosion experiments. Secondary electron detector (SE) was applied to examine the top-surface as well as cross-section of the coupons. Meanwhile, with backscatter electron detector (BSE), localised corrosion attacks covered by corrosion products were explored. Acceleration voltages will be further mentioned in the corresponding figure captions. The chemical composition of bacteria was obtained by Energy dispersive X-ray spectroscopy (EDS) at 5 kV.

Optical microscope (OM, OLS4100, Olympus) photos were captured in 3D bright field mode. For further confirmation of the corrosion products covering the surfaces, high resolution grazing incidence X-ray diffractometer (GIXRD, Cu Kα with 1° grazing angle, PANalytical X'Pert PRO-MPD) was applied. In the atmospheric aging experiments, focused ion beam (FIB) was applied to fabricate fresh cross-section of the copper coupon after corrosion treatment, using gallium ions with an accelerating voltage of 30 kV. Images and spectra were obtained within 24 h after the corresponding corrosion treatments, except from the atmospheric aging experiments.

Copper content determination

After corresponding surface treatment, 10 µL samples were withdrawn by repetitive pipetting and diluted in 2.990 mL 1 wt% nitric acid (Merck, Germany). Before being measured by inductively coupled plasma mass spectrometry (ICP-MS, 7500cx, Agilent), 3 µL of 10 mg/L scandium and caesium internal standard solutions were added to the samples. For calibration, standards with 0.1, 0.5, 2.5, 10, 50, 250, and 1000 µg/L of copper were used. Original results with a unit of ppb were converted to the final values with µmol/L (µM), concerning both the fold of dilutions and molar mass 63.55 g/mol for copper. The average values and standard deviations were obtained by three independent experiments.

Antibacterial efficiency determination

Wet-plating test was used, whose details can be referred to a previous publication [START_REF] Molteni | Killing of Bacteria by Copper Surfaces Involves Dissolved Copper[END_REF]. In brief, the E. coli K12 strain was grown aerobically overnight in Lysogeny broth (LB) medium at 37 ⁰C in a water bath with a speed of 220 rpm. The stationary cells from 1 mL culture were collected by centrifugation for 15 min at 5000×g, washed and centrifuged three times with the corresponding solutions, and finally re-suspended in 1 mL of the same type of solution. The test commenced by applying 20 µL of these re-suspended bacteria on the coupons, which were placed in a water-saturated atmosphere at room temperature. After 1 h, 10 µL samples were withdrawn by repetitive pipetting, serially diluted in PBS and spread on LB agar plates. Following incubation at 37 ⁰C for 24 h, the colony-forming unit (cfu) on agar plates was counted and expressed in a way concerning both the fold of dilutions and relative to the original 20 µL samples. The average values and standard deviations were obtained by three independent experiments.

Results and Discussion

Corrosion of copper surface in 0.9% NaCl and pure water

Before analysis of the actual antibacterial efficiency test, this study first focuses on the corrosive effects introduced by 0.9% NaCl (this concentration is hereafter simply referred to as "saline") without bacteria. For instance, Figure 1 (a-c) shows the morphological change and formation of corrosion product on the copper surface after 3 h exposure to this environment.

Uniformly distributed sub-micron corrosion products are evident in SE image. Meanwhile, at some locations, much lower intensity was recorded in BSE (therefore darker), indicating missing of material underneath the corrosion product. These cavities signify where localised deterioration took place. Variation of microstructure definitely has impacts on the initiation of these localised attacks [START_REF] Vinogradov | On the corrosion behaviour of ultra-fine grain copper[END_REF]. The distribution of these attacks can be mostly referred to a recent study [START_REF] Luo | Localised corrosion attacks and oxide growth on copper in phosphate-buffered saline[END_REF], where how electropolished copper surface corroded by PBS droplet was investigated in detail.

Coupon exposed to pure water for the same duration is shown for comparison purposes in Figure 1 (d ande). Fewer features can be summarised: no obvious additional product coverage nor localised corrosion sites. Therefore, it can be preliminarily concluded that, adding NaCl into pure water significantly varies the environmental corrosivity towards copper.

The corrosion product formed on copper was further examined by high resolution GIXRD shown in Figure 1 (f).

Crystalline Cu2O has been identified as the major product after saline treatment, but not in pure water. Similar observation was obtained from potentiostatic polarisation [START_REF] Warraky | Pitting corrosion of copper in chloride solutions[END_REF], highlighting the formation of intermediate product CuCl, followed by the formation of Cu2O and initiation of pits in a chronological sequence. However, CuCl is not identified in the current study.

Meanwhile, the cavities were found being gradually covered by Cu2O films, instead of destroying the films. Therefore, dissolution-precipitation or re-deposition mechanism [START_REF] Luo | Early-stage corrosion, ion release, and the antibacterial effect of copper and cuprous oxide in physiological buffers: Phosphate-buffered saline vs Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid[END_REF] seems to be more appropriate in explaining the oxide growth: it is exceeding amount of copper ions gathering near the copper surface and forming oxide.

Corrosion of copper surface in bacterial suspensions

By adding E. coli (hereafter referred to as "bacteria"), solution becomes suspension, some corrosion behaviours on copper surface thus alter accordingly, as can be found in Figure 2. In saline, localised corrosion attack is still predominant, which can be easily recognised from the BSE images. SE image usually shows more morphological details, but the above-mentioned (Section 3.1) sub-micron corrosion product Cu2O forming in pure solution can hardly be seen. The missing diffraction peak of Cu2O (111) in GIXRD also matches this observation. Highly similar phenomena were recently reported in the case of PBS [START_REF] Luo | Bacteria accumulate copper ions and inhibit oxide formation on copper surface during antibacterial efficiency test[END_REF], which could help interpret the current scenario: it is attributed to the bacterial accumulation effects of copper ions. Copper concentration near surface is thus reduced and oxide growth is inhibited.

Without oxide coverage, a considerable amount of cavities, as a result of localised corrosion attacks, become visible even in SE mode. Those invisible ones, on the other hand, may imply another mechanism of pit initiation: a Cu2O membrane could be found, separating the inner pitting region from the aqueous environment and promoting further pitting corrosion under the uppermost copper surface [START_REF] Sequeira | Inorganic, physicochemical, and microbial aspects of copper corrosion: literature survey[END_REF]. Similar sites have also been observed in the intermetallic particles induced crystallographic pitting grown beneath the surface [START_REF] Kakinuma | Mechanism for the Morphological Change from Trenching to Pitting around Intermetallic Particles in AA1050 Aluminum[END_REF], suggesting galvanic couple could be considered in the current scenario.

In the case of pure water, its bacterial suspension has not introduced distinct morphological or chemical changes on copper surface. These contrasts suggest the important roles of E. coli: although its presence inhibits the formation of Cu2O, it does not affect the major corrosion attacks (especially in saline) or cause additional corrosion effect (especially in pure water). In other words, it is still the properties of solution that determine the corrosion process.

From the microbiological perspective, addition of bacteria introduces not only bacteria themselves, but also their metabolism as well as the products of it. Bacterial respiration, for example, is one of the universal processes frequently discussed, as it could help reducing oxygen content in the aqueous environment [START_REF] Lee | Microbial iron respiration: impacts on corrosion processes[END_REF]. Meanwhile, dissolved oxygen happens to play an indispensable factor in the corrosion of metallic copper in aqueous [START_REF] Hedin | Corrosion of copper in pure O2-free water?[END_REF] or Cl --containing environment [START_REF] Bjorndahl | Copper Corrosion in Chloride Media. Effect of Oxygen[END_REF].

However, in the following sections, this aspect will be further ruled out from the present conditions.

Copper release and antibacterial efficiency

In the above sections, the corrosion behaviours of copper surface were revealed, in terms of how it varies after being exposed to different solutions or suspensions. But apart from this, it is also meaningful to investigate the effects of corrosion on the contacting droplet itself. For instance, the rise of copper content measured by ICP-MS shown in Figure 3 (a) offers valuable information. First of all, it is evident that the copper content measured from the bacterial suspension is higher than its corresponding pure solution. This can be again correlated with the role of bacterial addition summarised above: since bacteria decrease the amount of free copper ions in solutions, it favours a continuous release kinetics. This particularly affects the situation in saline, as it suppresses the formation of Cu2O, which could have formed as a barrier layer reducing the metal/solution interface [START_REF] Luo | Early-stage corrosion, ion release, and the antibacterial effect of copper and cuprous oxide in physiological buffers: Phosphate-buffered saline vs Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid[END_REF] as well as retarding ion diffusion [START_REF] Webster | Microbiologically Influenced Corrosion of Copper in Potable Water Systems-pH Effects[END_REF].

Another marked discrepancy is found between saline and pure water: the amounts of copper presented in saline are, in both cases, evidently higher than that in pure water. This trend matches the SEM observation: copper surface always suffers from more detectable localised corrosion attacks in saline. This is likely attributed to Cl -, which has been regarded as an accelerator of corrosion on metallic surfaces most of the time [START_REF] Prosek | Low-Temperature Stress Corrosion Cracking of Austenitic and Duplex Stainless Steels Under Chloride Deposits[END_REF]. With respect to copper, there was also study conducted on the relationship between Cl -concentration and corrosion rate [START_REF] Mayanna | Role of chloride ions in relation to copper corrosion and inhibition[END_REF]. It shows that the Cl -attachment allows complex formation and thereby promotes a faster ion release.

Additionally, it is still worth noting that neglectable variations of surface topography does not necessarily represent similar copper release level. In the case of pure water, although SEM results do not indicate significant difference, its bacterial suspension does enhance copper release by about three times, from 75.29 µM to 299.15 µM (data listed in the table in Figure 3). This suggests that it is the uniform corrosion that dominates the copper release in pure water, instead of localised corrosion.

Copper content presented in suspension is also positively related to the surface antibacterial efficiency, as presented in Figure 3 (b). Bacteria in both suspensions were grown from the same broths and thence share similar original concentration.

Merely in 1 h, their survival already parted ways: around 98% were killed in the saline suspension, while only 22% was recorded in pure water. Not surprisingly, these numbers match the difference of copper content evaluated in ICP-MS shown above and that accumulated in the bacterium compared in Figure 3 (c): Stronger Cu Lα radiation at 0.930 keV was recorded from the bacteria in saline.

These comparisons demonstrate how corrosion behaviour and antibacterial efficiency are correlated: even though same type of microbe was tested on same type of metallic copper surface, the composition of the solution can dictate the release of antibacterial substance and thus result in a different antibacterial efficiency. Saline, a relatively corrosive solution, causes a higher amount of copper release (at least within a certain period), raising the copper concentration and enabling a faster killing rate against E. coli. A recent study [START_REF] Ciacotich | Influence of chlorides and phosphates on the antiadhesive, antibacterial, and electrochemical properties of an electroplated copper-silver alloy[END_REF] conducted by another research group also found out a similar role of Cl -in enabling sufficient corrosion on not only copper and but also copper-silver alloy to deactivate S. aureus.

Two additional remarks may help further interpreting the antibacterial efficiency results. Since the killing rate follows the same trend of the increasing copper content, it cannot be estimated in the current experiment, how much of the antibacterial efficiency was assisted by the Cl -accelerated Cu-Fenton reaction [START_REF] Wang | Chloride-accelerated Cu-Fenton chemistry for biofilm removal[END_REF] or other combined effects [START_REF] Cassells | Efficacy of a combined system of copper and silver and free chlorine for inactivation of Naegleria fowleri amoebas in water[END_REF]. On the other hand, one may notice that pure water is not a buffer, but a low osmolarity environment in the strict sense. Hypoosmotic shock does not necessarily lead to cell death [START_REF] Booth | Managing hypoosmotic stress: Aquaporins and medianosensitive channels in Escherichia coli[END_REF], but even if it does, the contribution of copper in reaching a 22% killing rate should be considered even lower.

Effects of NaCl concentration and E. coli concentration on copper

Since the effects of NaCl and E. coli in this corrosion system have been preliminarily identified, additional experiments were designed to explore these effects. Figure 4 (a-c) compare the effects of saline concentration on the corrosion behaviours of copper surface. For all these selected concentrations, copper was subjected to a similar type of localised corrosion attacks. As the saline concentration increases, the expansion of cavities is observed, identified by imaging contrast in BSE. Besides, contrast can also be found even within a single cavity. This implies the localised attack inside the cavity is not uniform. On the other hand, oxide growth is ignorable in both 0.45% and 0.9% suspensions. But when it added up to 1.8%, huge areas covered by sub-micron oxides are found. Meantime another striking feature is also observed regarding the distribution of oxide on copper surface: they only grew on specific locations.

According to the re-deposition mechanism discussed (at the end of Section 3.1), oxide growth usually corresponds to excessive copper ions. Therefore, the emergence of oxide could be attributed to a faster corrosion kinetics in a high Cl - concentration scenario, where the amount of copper ions exceeds the accumulation capacity of E. coli. However, as the bacteria presenting in the droplet are supposed to be well-mixed, their copper accumulation effect should be distributed evenly on the surface. That is to say, the localised copper ion concentration is expected similar across the whole near surface.

Therefore, other than copper concentration, there must be another factor governing the preferential locations of oxide growth.

As it can be seen in Figure 4 (d), oxide coverage seems to follow the grinding marks. It strongly suggests its correlation with the state of the original copper surface (e.g. microstructural or morphological difference introduced by the grinding procedure). In other words, variation of the state of the surface might dominate the distribution of cathodic sites, where oxide growth prevails. To design similar experiments on electropolished copper surface may help, as it did reveal the crystallographic tendency of copper corrosion in PBS [START_REF] Luo | Localised corrosion attacks and oxide growth on copper in phosphate-buffered saline[END_REF].

To verify the role of ion accumulation effect introduced by E. coli, another corrosion test was conducted with 1.8% NaCl, whereas fivefold E. coli was re-suspended. After the same duration, oxide coverage can hardly be seen on copper surface, as shown in Figure 5 (a). Surge of the amount of bacteria boosts both the capacity and efficiency of the ion accumulation, compensating the increase of copper release in high Cl -concentration. Besides, higher bacterial concentration did not affect the selective distribution of localised corrosion sites, indicating that the corrosion mechanism stays the same.

However, the size of cavities did shrink, and easy to be found connected altogether. Meanwhile, even on the locations that are free from severe localised corrosion, slight degradation can be observed, as the grinding marks can hardly be found. It is unclear at the current stage, but these phenomena could be related to but not limited to the variation of local chemical concentration (Cl -or chemicals produced by bacterial metabolism), local fluid flow rate (induced by the mobility of bacteria), etc.

On the other hand, with lower bacterial concentration (tenth fold diluted), oxide coverage can always be found in whole series of NaCl concentration (Figure 5 (b-d)). Oxide particles with bigger size are noticed in lower NaCl concentration. That is, NaCl concentration has an impact on the nucleation density of oxide. The trend of how NaCl and bacterial concentration influence oxide growth and corrosion attacks are qualitatively summarised in Figure 5 (e).

Atmospheric corrosion caused by residual NaCl crystals

So far, the impacts of NaCl on copper surface have only been discussed during corrosion test or antibacterial efficiency test.

However, even though the saline will be withdrawn afterwards, some NaCl residues could stay on the copper surface. These NaCl crystals are found to continue playing a decisive part in the copper aging process.

Figure 6 compares the copper surfaces observed 3 weeks after having been exposed to saline. Coupon shown in Figure 6 (a) has been cleaned immediately after the saline was withdrawn in the end of the corrosion test, therefore no NaCl crystal was remained. In this case, the oxide coverage is found unchanged in 3 weeks. However, coupon that has not been cleaned properly experienced significant changes, as shown in Figure 6 (b). Firstly, a secondary layer with a slightly different roughness formed on the previous Cu2O coverage. Moreover, the reason why some uncovered previous Cu2O can be located, is likely related to the NaCl crystals (with brighter colour in SE mode) lying aside: it is assumed that these uncovered zones were also originally covered by the NaCl crystals. Nevertheless, these crystals gradually disappeared during the 3-week atmospheric aging.

From GIXRD results, two facts have been clearly shown. First, NaCl residues were efficiently cleaned by the current protocol, as much weak diffraction signals are found on the cleaned coupon. Second, more Cu2O diffraction peaks with higher intensity appeared from the coupon without cleaning, confirming the newly grown secondary layer is mainly composed of Cu2O as well.

Intriguingly, this kind of aging effect does not occur only on the directly treated copper surface. As shown in Figure 7, a hole on the copper coupon was fabricated by FIB, immediately after the corrosion test without applying cleaning protocol. This exposes some "fresh" areas beneath the original surface. "Fresh" is here defined as having not directly "met" the saline, since they were still inside the bulk copper during the corrosion test. OM image taken straightway after FIB operation (i.e. Day 0) recorded some bright zones, not just in the hole, but also around it, as a result of Ga ion beam bombardment. The areas inside the hole that are darker were due to rougher topography, confirmed by SEM images. However, as the aging experiment proceeds, the bright zones shown in OM turned darker from the boundaries inwardly. This is related to the morphological changes displayed in Figure 8: submicron particles were forming on top of these surfaces. Being continuously exposed to atmosphere, part of this region extended, from the cross-section (edge of the hole) towards the bottom of it. Meanwhile, coarsening can be observed on some of these particles. At the cross-section (invisible in OM owing to observation angle) which is supposed to be relatively smooth at the original state, similar particle formation and coarsening phenomenon are also found. Although the exact chemical or phase composition has yet to be determined, these locations do contain an extra amount of oxygen content (Figure 8 (d&e), obvious O Kα radiation at 0.525 keV), suggesting the formation oxygen-containing species. Served as the control groups, same type of FIB cross-sections was produced on copper coupon exposed to either pure water or E. coli water suspension for the same duration. Neither of these surfaces shows such changes after 3 weeks (data not included), excluding the potential additional effects brought by Ga ion implantation during FIB operation.

All these comparisons help to preliminarily pin down the cause of atmospheric aging: NaCl, as it the only varied environmental parameter. As the solution cannot been withdrawn completely, NaCl crystallised locally on the copper surface after the remained water evaporated. However, this is not yet the final fate of these crystals. Atmospheric corrosion continues to play a role. For example, these salts could go through a process so-called deliquescence [START_REF] Li | Atmospheric corrosion initiation on steel from predeposited NaCl salt particles in high humidity atmospheres[END_REF], thereby absorbs the water from the atmosphere and induce aqueous corrosion. But this process requires a relatively high humidity (79% RH for pure NaCl), which is usually not fulfilled in the lab environment.

In spite of this, NaCl crystals could transform in another way, which is still related to water absorption, not by NaCl itself, but by the general surface. Because it is common to find monolayers of water forming on surfaces [START_REF] Schindelholz | Wetting phenomena and time of wetness in atmospheric corrosion: a review[END_REF]. Therefore, the copper surface could still be regarded as being exposed to a quasi-aqueous environment. In fact, the properties of the water layer could already affect the nucleation and formation rate of Cu2O in a certain range of relative humidity [START_REF] Aastrup | Experimental in situ studies of copper exposed to humidified air[END_REF]. Besides, these monolayers dissolve NaCl and transform itself to electrolyte. Meanwhile, Cl -are able to diffuse through these monolayers, reaching to the other parts of the surface [START_REF] Chen | Effect of Sodium Chloride Particles on the Atmospheric Corrosion of Pure Copper[END_REF]. Considering this circumstance, corrosion related phenomena are not a surprise. In addition, Cl -may not be the only ions that would diffuse through the surface, as copper ions should also participate a similar process. Take the saline corroded surface as an example (Figure 6 (b)), without copper ion diffusion to the top of the existing Cu2O, it is hard to imagine how the secondary layer of Cu2O actually formed on top.

Insight into antibacterial surface research

Firstly, the current results suggest the significance of buffer selection in antibacterial surface research. Buffers are more than a friendly environment designed for microbes. On the contrary, depending on their components as well as the corresponding properties (corrosivity in this case), they may react with the investigated surface in different ways, resulting in an enhanced antibacterial efficiency or vice versa. Evidences for this can also be obtained from other comparisons such as PBS vs Na-HEPES [START_REF] Luo | Early-stage corrosion, ion release, and the antibacterial effect of copper and cuprous oxide in physiological buffers: Phosphate-buffered saline vs Na-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid[END_REF], or nutrient broth (NB) vs minimal media (MM) [START_REF] Javed | Inhibition or acceleration: Bacterial test media can determine the course of microbiologically influenced corrosion[END_REF]. For the same reason, design of materials with weaker corrosion resistance could enhance release of antibacterial substances [START_REF] Tao | Microstructure, mechanical properties and antibacterial properties of the microwave sintered porous Ti-3Cu alloys[END_REF], meanwhile considering that the cytotoxicity might need to be and definitely can be avoided in specific scenarios [START_REF] Fowler | Effect of Copper Ion Concentration on Bacteria and Cells[END_REF].

Secondly, growth and coverage of passive layer as a result of the subsequent atmospheric corrosion, may not be beneficial for the long-term antibacterial property of antibacterial copper surface [START_REF] Hutchison | Solute Capture and Doping of Al in Cu2O: Corrosion, Tarnish Resistance, and Cation Release of High-Purity Cu-Al Alloys in Artificial Perspiration[END_REF]. Therefore, it should particularly inspire the applied research closely related to touched surfaces, as NaCl is also one of the major residues of perspiration. To evaluate its effect on antibacterial efficiency, cyclic test [START_REF] Rtimi | Accelerated Escherichia coli inactivation in the dark on uniform copper flexible surfaces[END_REF] should be one of the effective ways to be included in the future work.

Conclusion

NaCl as a common component in many popular buffers, was examined in this work in view of its roles in copper corrosion. Some concluding remarks are summarised as below:

Ground copper coupon undergoes severe localised corrosion attacks in the environment of 0.9% saline, regardless of whether E. coli is added or not.

A layer of Cu2O grows on copper surface when being exposed to pure 0.9% saline. In the case of its E. coli suspension, formation of Cu2O has been inhibited at least in 3 h.

A faster killing rate of ground copper surface against E. coli (98% in 1 h) is recorded in 0.9% saline, compared to pure water (22%), which is attributed to the accelerated corrosion process in the presence of Cl -.

Increase of Cl -concentration may accelerate copper release, forming Cu2O even in E. coli suspension. However, it can be counteracted by increasing the concentration of E. coli to fivefold, as this enhances the copper ion accumulation effect.

Residual NaCl crystals on copper surface promote the formation of additional Cu2O as corrosion products. A Cl --assisted atmospheric corrosion process is revealed by a FIB-assisted 3-week aging experiment. Table (e) summarises the characteristics of oxide growth and corrosion sites in different conditions: more "+" represents bigger particle size; more "-" represents smaller localised corrosion sites; "X" represents situations where particle coverage was rarely observed.

Figure 1 Typical

 1 Figure 1 Typical SEM images of ground copper surfaces after 3 h exposure to 0.9% NaCl (a-c) or pure water (d, e). Images were obtained by SE detector (a, d) and BSE detector (b, c, e) at 20 kV. The dotted lines in (a, b) surround a typical localised corrosion zone. Images (a, b) correspond to the square dash marked region in (c). High resolution GIXRD results (f) in the range of (111) planes of Cu2O (JCPDS#75-1531) obtained from these surfaces.

Figure 2 Typical

 2 Figure 2 Typical SEM images of ground copper surfaces after 3 h exposure to E. coli 0.9% NaCl suspension (a-c) or E. coli pure water suspension (d, e). Images were obtained by SE detector (a, d) and BSE detector (b, c, e) at 20 kV. The dotted lines in (a, b) indicate two types of localised corrosion site. Images (a, b) correspond to the square dash marked region in (c). High resolution GIXRD results (f) in the range of (111) planes of Cu2O (JCPDS#75-1531) obtained from these surfaces.

Figure 3

 3 Figure 3 Copper content (a) in 0.9% NaCl or pure water, with or without E. coli after 3 h exposed to ground copper surfaces. Antibacterial efficiency test (b) against E. coli in two different suspensions in 1 h. The error bars indicate the standard deviations calculated from three independent measurements. Percentages in (b) above the bars represent the killing rate in 1 h (compared to the original data). Average values from (a) and (b) are listed in the tables. Typical EDS results (c) collected from the bacterium (E. coli) transferred onto silicon wafer after being treated with copper surface in two suspension for 3 h.

Figure 4 Typical

 4 Figure 4 Typical SEM images (BSE, 5 kV) of ground copper surfaces after 3 h exposure to E. coli suspension with different NaCl concentration: (a) 0.45%; (b) 0.9%; (c) 1.8%. The dotted lines in (a, c) indicate typical localised corrosion cavities. Image (c) corresponds to the square dash marked region in (d). Orange arrows in (d) indicate regions with oxide growth, while white arrows for those without.

Figure 5 Typical

 5 Figure 5 Typical SEM images (BSE, 5 kV) of ground copper surfaces after 3 h exposure to (a) fivefold E. coli 1.8% NaCl suspension; (b) tenth fold diluted E. coli 0.45% NaCl suspension; (c) tenth fold diluted E. coli 0.9% NaCl suspension; (d) tenth fold diluted E. coli 1.8% NaCl suspension. Dash line in (a) separates the severe corrosion zone from the almost intact zone.

Figure 6 Typical

 6 Figure 6 Typical SEM images (SE, 5 kV) of ground copper surfaces 3 weeks after 3 h exposure to 0.9% NaCl solution. Coupon shown in (a) has been ultrasonic cleaned by ethanol immediately after the 3 h corrosion experiment, while (b) was kept without further cleaning. Both images share the same scale bars that presented in (a). The dotted region in (b) indicates the missing of NaCl crystal as well as the exposed original oxidised copper surface. High resolution GIXRD results (c) obtained from these surfaces 12 weeks after the original treatment. Cu (JCPDS#04-0836), NaCl (JCPDS#05-0628), and Cu2O (JCPDS#75-1531) were applied to index the patterns. The diffraction peaks marked with hexagonal star ( ) represent diffraction of Cu (111) planes under Cu-Kβ and W-Lα1.

Figure 7 OM

 7 Figure 7 OM images (a-d) and SEM images (e-k) (SE, 5 kV, coupon being 52°tilted) of fresh section of copper surface prepared by FIB after 3 h exposure to 0.9% NaCl. OM images and SEM images share the same scale bars presented in (a) and (e), respectively. This position was repeatedly examined every 7 days. Dark areas surrounded by dotted lines in (a) are the regions pointed out by arrows in (e). Area marked with dotted line in (d) serves as an example for positions that suffered colour change compared to the same positions in (a). High magnification images of the square dash marked regions in (f-h) are further shown in the following Figure 8. A schematic description of the treatment process is shown below the graphs.

Figure 8 High

 8 Figure 8High resolution SEM images (a-c) (SE, 5 kV, coupon being 52°tilted) of fresh section of copper surface prepared by FIB after 3 h exposure to 0.9% NaCl solution. This position was repeatedly examined every 7 days (a-c), whose lower magnification images are already presented in Figure7. Zone covered by light blue colour in (a) highlights the cross-section (edge of the hole). Image (d) (SE, 5 kV) was taken from the Day 21 coupon. The cross shape indicator points out the position where the EDS spectrum (e) was collected.

Figure 8

 8 Figure 8

Acknowledgements

This study was supported by Erasmus Mundus Joint European Doctoral Programme in Advanced Materials Science and Engineering (DocMASE, 512225-1-2010-1-DE-EMJD, European Commission) and the PhD-Track-Programme (PhD02-14, Franco-German University). The ICP-MS experiments were supported by Dr. Ralf Kautenburger from the chair of Inorganic Solid State Chemistry. J. L. particularly thank Prof. Tomáš Prošek from University of Chemistry and Technology Prague, for his inspiring perspective during EUROCORR 2018 and 2019, making this work possible.

Data availability

The data that support the findings of this study are available from the corresponding author on request.

Declarations of interest

None.