MD Mathilde Letouzey
email: mathilde.letouzey@gmail.com

E Lorthe

L Marchand-Martin

MD G Kayem

MD C Charlier

MD M Butin

MD A Mitha

M Kaminski

PhD V Benhammou

MD P Y Y Ancel

L Marchand-Martin Msc

M Kaminski Msc

MD P Boileau

MD L Foix-L'hélias

+ Epipage-

Early antibiotic

Keywords: empirical antibiotics, prematurity, preterm birth, neonatal outcome, late-onset sepsis, necrotizing enterocolitis, neonatal death, bronchopulmonary dysplasia, severe cerebral lesions

2) The National Research Agency through the French EQUIPEX program of investments in the future (reference ANR-11-EQPX-0038 and ANR-19-COHO-001).

3) The PREMUP Foundation 4) Fondation de France (reference 11779) 5) Fondation pour la Recherche Médicale (SPF20160936356)

The funding source had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review or approval the manuscript; and the decision to submit the manuscript for publication.

We declare no potential, perceived or real conflict of interest relevant to this article to disclose.

There are no prior publications or submissions with any overlapping information, including studies and patients.

INTRODUCTION

Early-onset sepsis (EOS) remains frequent in very preterm infants with a prevalence of 18 per 1000 live births before 28 weeks, which is 10 to 30-fold higher than in term infants. [START_REF] Stoll | Early-Onset Neonatal Sepsis 2015 to 2017, the Rise of Escherichia coli, and the Need for Novel Prevention Strategies[END_REF] Infection-associated mortality is close to 30% in these vulnerable infants. [START_REF] Stoll | Early-Onset Neonatal Sepsis 2015 to 2017, the Rise of Escherichia coli, and the Need for Novel Prevention Strategies[END_REF] When facing initial clinical instability in preterm infants, neonatologists are trained to suspect EOS and widely prescribe empirical antibiotic treatments upon admission to neonatal intensive care units (NICU).(2) However, the risk of EOS differs according to the cause of preterm birth. At birth, antibiotic administration is widely performed in the context of preterm labor or preterm premature rupture of membranes due to the high risk of EOS. Nevertheless, these antibiotic treatments are questionable in the context of maternal hypertensive disorders or fetal growth restriction associated with a low risk of EOS. [START_REF] Puopolo | Identification of Extremely Premature Infants at Low Risk for Early-Onset Sepsis[END_REF][START_REF] Benitz | Risk factors for early-onset group B streptococcal sepsis: estimation of odds ratios by critical literature review[END_REF] Promoting appropriate use of antibiotics in NICUs is a major concern in neonatology mainly because of their potential adverse impact on antibiotic resistance. [START_REF] Arboleya | Intestinal microbiota development in preterm neonates and effect of perinatal antibiotics[END_REF][START_REF] De Man | An antibiotic policy to prevent emergence of resistant bacilli[END_REF] In addition, antibiotic exposure in early life disrupted the gut microbiota colonization [START_REF] Arboleya | Intestinal microbiota development in preterm neonates and effect of perinatal antibiotics[END_REF][START_REF] De Man | An antibiotic policy to prevent emergence of resistant bacilli[END_REF] and can have long-term effects on health such as obesity, allergic diseases, or inflammatory bowel disease. [START_REF] Bailey | Association of antibiotics in infancy with early childhood obesity[END_REF][START_REF] Hoskin-Parr | Antibiotic exposure in the first two years of life and development of asthma and other allergic diseases by 7.5 yr: a dosedependent relationship[END_REF][START_REF] Kronman | Antibiotic exposure and IBD development among children: a population-based cohort study[END_REF] Studies on early antibiotics treatment in very preterm infants with or without risk factors for EOS have shown that prolonged antibiotic exposure may be associated with neonatal adverse outcomes such as necrotizing enterocolitis (NEC), late-onset sepsis (LOS), bronchopulmonary dysplasia (BPD), and death. [START_REF] Cotten | Prolonged duration of initial empirical antibiotic treatment is associated with increased rates of necrotizing enterocolitis and death for extremely low birth weight infants[END_REF][START_REF] Cantey | Early Antibiotic Exposure and Adverse Outcomes in Preterm, Very Low Birth Weight Infants[END_REF][START_REF] Ting | Duration of Initial Empirical Antibiotic Therapy and Outcomes in Very Low Birth Weight Infants[END_REF][START_REF] Cantey | Antibiotic Exposure and Risk for Death or Bronchopulmonary Dysplasia in Very Low Birth Weight Infants[END_REF] However, these studies did not usually take into account the cause of preterm birth which is associated with both early antibiotic exposure and some adverse outcomes such as death or LOS. [START_REF] Ting | Duration of Initial Empirical Antibiotic Therapy and Outcomes in Very Low Birth Weight Infants[END_REF][START_REF] Esaiassen | Antibiotic exposure in neonates and early adverse outcomes: a systematic review and meta-analysis[END_REF][START_REF] Delorme | Cause of Preterm Birth as a Prognostic Factor for Mortality[END_REF][START_REF] Letouzey | Cause of preterm birth and late-onset sepsis in very preterm infants: the EPIPAGE-2 cohort study[END_REF][START_REF] Li | Early Use of Antibiotics Is Associated with a Lower Incidence of Necrotizing Enterocolitis in Preterm, Very Low Birth Weight Infants: The NEOMUNE-NeoNutriNet Cohort Study[END_REF] Moreover, many of these adverse outcomes occur among critically ill preterm infants who received antibiotics due to their severe clinical status at birth. Therefore, controlling for the initial severity of illness would limit the indication bias. [START_REF] Cantey | Early Antibiotic Exposure and Adverse Outcomes in Preterm, Very Low Birth Weight Infants[END_REF][START_REF] Ting | Duration of Initial Empirical Antibiotic Therapy and Outcomes in Very Low Birth Weight Infants[END_REF] To better understand the impact of early antibiotics treatment and reduce the indication bias, one way is to limit the study to very preterm infants at low risk of EOS, namely born after maternal hypertensive disorders and/or fetal growth restriction, and controlling for severity of illness.

The aim of our study was to assess the association between early antibiotic exposure and adverse neonatal outcomes in very preterm infants at low risk for EOS.

METHODS

Study population

This is a secondary analysis of the EPIPAGE-2 cohort (Etude éPIdémiologique sur les Petits Ages GEstationnels 2), a prospective national population-based cohort of preterm infants that included liveborn and stillborn infants as well as all terminations of pregnancy at 22-31 completed weeks of gestation in all maternity units from 25 regions in France in 2011. [START_REF] Lorthe | Cohort Profile: The Etude Epidémiologique sur les Petits Ages Gestationnels-2 (EPIPAGE-2) preterm birth cohort[END_REF] Participants were recruited over different periods according to gestational age at birth: eight months for births at 22-26 weeks and six months for births at 27-31 weeks. Maternal, obstetric, and neonatal data were collected from medical records following a standardized protocol as previously reported. [START_REF] Ancel | EPIPAGE 2: a preterm birth cohort in France in 2011[END_REF] Recruitment took place at birth after families received information and agreed to participate. During the recruitment period, 8400 births were eligible, among whom 7804 (93%) were enrolled in the study at baseline.( 17 All infants enrolled in the EPIPAGE-2 cohort, born alive between 22 and 31 completed weeks were included in this analysis (figure 1). Exclusion criteria were: death in the delivery room or during the first day of life after antenatal decision of withholding care, severe congenital malformation, and twin pregnancies with twin-twin transfusion syndrome. We also excluded infants with a high risk of EOS having an undisputable indication for early antibiotics administration (infants born after preterm labor, preterm premature rupture of membranes or placental abruption, infants whose mother had clinical chorioamnionitis or received antibiotics during the last 72 hours before birth or at birth). Preterm onset of labor and preterm rupture of membranes are considered a clinical indicator of potential intrauterine infection which is associated with a risk of EOS.(3) Very preterm infants born in these contexts were therefore excluded. Clinical chorioamnionitis was defined as maternal fever (≥ 37.8°C) associated with at least two of the following criteria: maternal leukocytosis (white blood cell count > 15,000 cells/mm3), maternal tachycardia (heart rate > 100 bpm), fetal tachycardia (heart rate > 160 bpm), uterine tenderness and foul-smelling vaginal discharge. [START_REF] Gibbs | Diagnosis of intra-amniotic infection[END_REF] Therefore, very preterm infants included in the present study were born after placental-mediated pregnancy complications (hypertensive disorders during pregnancy or fetal growth restriction) [START_REF] Delorme | Cause of Preterm Birth as a Prognostic Factor for Mortality[END_REF], and had a very low risk for EOS. Fetal growth restriction was defined by an estimated fetal weight below the 10 th percentile (according to the reference curves used by the hospital where the antenatal ultrasound scans were performed), in conjunction with growth arrest and/or relevant fetal Doppler abnormalities.

Finally, we excluded infants with missing data for early antibiotics treatment (n=151).

Maternal and neonatal characteristics of the study population and the population with missing data for early antibiotics treatment were similar except for the rate of multiple pregnancy which was significantly higher in case of missing data (Table 1 -Online).

Main exposure and outcomes

Early antibiotic exposure was defined as antibiotics started at Day 0 or Day 1 of life, regardless of the duration and type of antibiotics.

Neonatal outcomes included death, LOS, NEC, severe cerebral lesions, and moderate-tosevere BPD during initial hospitalization in NICU. LOS was defined as a positive blood culture after 72 hours of life, associated with antibiotic administration for five days or more, or death within five days following positive blood culture. [START_REF] Stoll | Lateonset sepsis in very low birth weight neonates: the experience of the NICHD Neonatal Research Network[END_REF] NEC was defined by the Bell classification of stage ≥ 2.(21) Severe cerebral lesions were defined as intraventricular hemorrhage with ventricular dilatation or parenchymal hemorrhage, [START_REF] Volpe | Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances[END_REF] or periventricular leukomalacia (i.e., periventricular white matter echolucensies at ultrasonography). Moderateto-severe BPD was defined as oxygen requirement for at least 28 days and persistent need for oxygen or ventilatory support at 36 weeks' postmenstrual age (mechanical ventilation or positive pressure). [START_REF] Jobe | Bronchopulmonary dysplasia[END_REF] Severe neonatal complication was a composite outcome including any of the following: LOS; NEC; severe cerebral lesions; and/or moderate to severe BPD.

We defined the main outcome as a composite outcome accounting for the competing risk between death and adverse outcomes, including at least one of the following: death, LOS and/or NEC. We also considered the following composite outcomes: death or severe neonatal complication, death or severe cerebral lesions, death or moderate-to-severe BPD.

Other characteristics considered

Gestational age at birth was determined as the best obstetrical estimate from early ultrasound assessment and/or last menstrual period.(24) Initial severity of illness was assessed for all infants using the Clinical Risk Index for Babies II (CRIB2) score [START_REF] Parry | CRIB II: an update of the clinical risk index for babies score[END_REF]. The CRIB2 score (range 0-27) is based on gestational age, birth weight, neonatal sex, temperature at NICU admission, and initial base deficit. Higher scores of CRIB2 are predictive of higher mortality risk. [START_REF] Parry | CRIB II: an update of the clinical risk index for babies score[END_REF] Newborns were considered small-for-gestational-age if their birth weight was ≤ 10 th percentile according to French EPOPé intrauterine growth curves adjusted for fetal sex and gestational age [START_REF] Ego | Customized and non-customized French intrauterine growth curves. I -Methodology[END_REF].

EOS was defined by positive blood or cerebrospinal fluid cultures before 72 hours of life.

Bacillus and coagulase-negative staphylococci grown on culture of blood <72h were considered contaminants instead of EOS, they were found in 1 and 4 newborns, respectively.(2) Maternity units were considered as type 3 when associated with a NICU. Inborn status was defined as delivery in a type 3 maternity unit. The volume of the NICU was defined by the number of infants included in the EPIPAGE 2 study in the unit, divided into terciles.

Statistical analysis

We first compared maternal, obstetrical, and neonatal characteristics by early antibiotic treatment (yes vs no). Categorical variables were compared using χ 2 or Fisher exact tests as appropriate, and medians of quantitative variables by Wilcoxon's test. All descriptive statistics were weighted according to the duration of recruitment. Infants born at 22-26 weeks were recruited over an eight-month period i.e. the equivalent of 35 weeks. Infants born at 27-31 weeks were recruited over a period of only six months i.e. the equivalent of 26 weeks. We weighted the population of infants born at 27-31 weeks by a factor of 1.34 (35/26) in order to have a population equivalent to what a 35-week recruitment period would have generated. This weighting therefore allowed us to account for the sampling scheme of the cohort and to ensure representativeness. [START_REF] Ancel | EPIPAGE 2: a preterm birth cohort in France in 2011[END_REF] All tests were 2-sided; p<0.05 was considered statistically significant.

We used a propensity score analysis to minimize the indication bias in estimating the antibiotic exposure effect. [START_REF] Austin | An Introduction to Propensity Score Methods for Reducing the Effects of Confounding in Observational Studies[END_REF] The first step in the analysis consisted of estimating the propensity score, defined as the newborn's probability of receiving early antibiotic treatment based on his/her individual observed characteristics at admission to NICU. The score was estimated using a logistic regression model with the administration of early antibiotic treatment as the dependent variable, regressed on baseline characteristics clinically associated with the exposure and/or the outcome, excluding covariates that might be a consequence of the treatment. [START_REF] Garrido | Methods for Constructing and Assessing Propensity Scores[END_REF] We included the following covariates: maternal age (<25, 25-34 or ≥35 years) [START_REF] Klemetti | At what age does the risk for adverse maternal and infant outcomes increase? Nationwide register-based study on first births in Finland in 2005-2014[END_REF], maternal place of birth [START_REF] Howell | Differences in Morbidity and Mortality Rates in Black, White, and Hispanic Very Preterm Infants Among New York City Hospitals[END_REF], overweight (body mass index (BMI) ≥ 25) (31), smoking status during pregnancy [START_REF] Dietz | Infant morbidity and mortality attributable to prenatal smoking in the U.S[END_REF], antenatal corticosteroids administration (at least one injection administered before delivery), single or multiple pregnancy, inborn status, gestational age at birth, birth weight (<1000, 1000-1499, or ≥1500 grams), neonatal sex, CRIB2 score, intubation in the delivery room, surfactant administration in the delivery room and NICU characteristics (level and volume of activity of the unit where the infant was hospitalized during the first 48 hours of life.).

The second step in the analysis involved inverse probability of treatment weighting (IPTW).

IPTW was based on estimated propensity scores, to obtain a synthetic sample (IPTW sample), in which antibiotic treatment assignment was independent of measured baseline covariates. [START_REF] Austin | An Introduction to Propensity Score Methods for Reducing the Effects of Confounding in Observational Studies[END_REF][START_REF] Thomas | Using Propensity Score Methods to Create Target Populations in Observational Clinical Research[END_REF][START_REF] Austin | Moving towards best practice when using inverse probability of treatment weighting (IPTW) using the propensity score to estimate causal treatment effects in observational studies[END_REF] Balance in the observed baseline covariates between treated and untreated patients in the initial and IPTW samples was assessed by examining standardized differences (Figure 2 -Online). Finally, we estimated the association between early antibiotics exposure and outcomes by a multilevel logistic regression within the weighted sample, with NICU as level 2 to account for the wide variation in the proportion of newborns that received early antibiotic treatment by center (Figure 3 -Online). We adjusted for gestational age because it was considered a confounding factor (a common ancestor of both the exposure and outcomes), and was not perfectly balanced in the propensity score (Figure 2 -Online). Results were presented as ORs with a 95% confidence interval (95%CI).

We performed a sensitivity analysis after multiple imputations for missing data. The proportion of missing data ranged from 0.0% to 8.9% for each covariate, and missing data were considered missing at random. Multiple imputations were used including all baseline variables and outcomes of the propensity-score model. Categorical variables were imputed using logistic or multinomial regression as appropriate. We generated 50 independent imputed datasets. A propensity score was estimated for each of the 50 imputed data sets generated, and results were pooled for a final analysis according to Rubin's rules. [START_REF] Rubin | Multiple imputation in health-care databases: an overview and some applications[END_REF] All statistical analyses were performed with STATA statistical software, version 16.1 (StataCorp).

RESULTS

We included 648 very preterm infants at low risk for EOS among which 173 (26.2%) received early antibiotic treatment started at Day 0 or Day 1 of life (figure 1).

Maternal, obstetrical, and neonatal characteristics are presented in Table 2. In this sample of pregnant women with hypertensive disorders and/or with fetal growth restriction, median maternal age was 30 years (interquartile range [IQR] 26-35), 45.5% were overweight at the beginning of pregnancy, 58.5% primipara and 20.3% smoked during pregnancy. Antenatal corticosteroids were widely administered, almost all babies were delivered by cesarean section.

Infants exposed to early antibiotics were more frequently born from multiple pregnancies but less frequently in a level 3 maternity unit. Other maternal and obstetrical characteristics did not differ by early exposure to antibiotics.

The median gestational age at birth was 30.0 weeks (IQR 28.4-31.0) and the median birth weight was 1040 grams (IQR=820-1260). Infants who received early antibiotic treatment had a slightly lower median gestational age at birth (29.6 versus 30.3 weeks) and lower median birth weight (1010 versus 1060 grams). Delivery room intubation and surfactant administration were more frequent in the group of infants with early antibiotics. However, the severity of the initial illness, measured by the CRIB2 score at admission in NICU did not differ significantly (Table 2).

Among the very preterm infants who received early antibiotics, 88.1% had treatment initiated at Day 0. Details about antibiotic treatment received were available for 165 patients. Neonatal outcomes and their association with early exposure to antibiotics are described in table 3. After IPTW, standardized differences between treated and untreated patients were below 0.10 for all covariates, except for a few gestational age groups (Figure 2 -Online).

Mortality was more frequent in the early exposure to antibiotics group (12.0% versus 6.3%).

The frequency of NEC was similar in both groups (5.9% in the early antibiotics group vs 4.9%).

In our population, 25.3% presented at least one episode of LOS at a median age of 12 days (IQR=7-20), with no difference between the two groups. There were 165 episodes of LOS including 41 in the group with early exposure to antibiotics and 124 in the group without exposure to antibiotics. The most common pathogens found in blood cultures collected during LOS were coagulase-negative staphylococci (75.5%), Staphylococcus aureus (15.7%), Enterococcus sp. (3.1%), and Gram-negative organisms (2.6%), with no difference between the two groups. We did not identify any episode of LOS due to a bacterium resistant to the initial early antibiotics administered. Late-onset sepsis characteristics did not differ according to early neonatal antibiotics (Table 4 online). In the complete cases analyses, we did not find any association between early exposure to antibiotics and death (OR=1.78 [95% CI=0.82-3.88]), LOS (OR=0.93 [95%CI=0.64-1.34]) or NEC (OR=1.47 [95%CI=0.65-3.33]). Results were consistent after multiple imputations. The composite outcome (death or LOS or NEC) did not significantly differ between the two groups (35.8% vs 32.5%) and we did not find an association between early exposure to antibiotics and this composite outcome (OR=1.04 [95%CI=0.72-1.50]).

However, severe cerebral lesions, isolated or combined with death, were more frequent when newborns received early antibiotics, (9.1% vs 4.5%, and 16.6% vs 9.9%, respectively). We found an association between severe cerebral lesions and early exposure to antibiotics (OR=2.71 [95%CI=1. .86]). The trend was the same, but at the limit of significance (OR=2.99 [95%CI=0.99-9.04]), after multiple imputations. The associations were less pronounced for the composite outcome death or severe cerebral lesions (Table 3).

Moderate-to-severe BPD was also more frequent in the early exposure to antibiotics group, whether isolated or associated with death (19.7% vs 10.2% and 28.6% vs 15.4%, respectively).

We found an association between moderate-to-severe BPD and early exposure to antibiotics (OR=2.30 [95%CI=1. 21-4.38]). The trend was the same, but at the limit of significance (OR=1.87 [95%CI=0.96-3.65]), after multiple imputations. Results were similar for the composite outcome death or moderate-to-severe BPD (Table 3).

Finally, we studied the composite outcome "severe neonatal complication" (which included any of the following: LOS; NEC; severe cerebral lesions; and/or moderate to severe BPD), and did not find an association with early exposure to antibiotics (OR=1.37 [95%CI=0.94-1.98]).

In a sensitivity analysis after multiple imputations for missing data, results were consistent for the composite outcome (death or LOS or NEC) (Table 5). For severe cerebral lesions and severe BPD, the trend was the same than complete cases analysis, but at the limit of significance (OR=2.99 [95%CI=0.99-9.04] and OR=1.87 [95%CI=0.96-3.65], respectively).

DISCUSSION

Among the 648 very preterm infants at low risk for EOS, 26% were exposed to antibiotics during the first day of life. This early antibiotic exposure was not associated with death, LOS and/or NEC, but was associated with a higher risk of severe cerebral lesions and moderate-tosevere BPD.

The strengths of our study included the use of data from a national prospective populationbased cohort study. Data about antibiotic exposure and neonatal outcomes were prospectively collected using a detailed and standardized questionnaire. We had a relatively small sample, but our study provided a unique opportunity to evaluate the impact of antibiotic prescription on neonatal outcomes, independently of indication biases: firstly, we chose to study a homogeneous sample of very preterm infants at low risk of EOS. We therefore excluded those born following: spontaneous preterm labor, rupture of membranes, maternal antibiotic treatment during the last 72 hours before birth, placental abruption or clinical chorioamnionitis.

These infants had none of the EOS risk factors that would require empirical antibiotic treatment at birth, and only four (in the group without initial exposition to antibiotics) were finally diagnosed with EOS. Among them, three infants were infected with S. aureus. In one case, the infection started on day 2, and in the two other cases on day 3. In this context of preterm birth with cesarean section, vertical transmission is very unlikely and these 3 infections were probably hospital-acquired infection.

Altogether, this empirical administration of antibiotics that involved 26% of our population was probably started out of standard protocols, with wide variations between NICUs. This rate was consistent with previous reports and should be addressed with specific antibiotic stewardship interventions. [START_REF] Puopolo | Identification of Extremely Premature Infants at Low Risk for Early-Onset Sepsis[END_REF][START_REF] Ting | Duration of Initial Empirical Antibiotic Therapy and Outcomes in Very Low Birth Weight Infants[END_REF] Then, we controlled for the initial severity of illness including the CRIB2 score in the propensity score, a validated predictor of mortality in this population. [START_REF] Parry | CRIB II: an update of the clinical risk index for babies score[END_REF] We also added intubation and surfactant administration in the delivery room to account for clinical severity at birth. The initial clinical severity is a confounding factor, associated with both early antibiotics treatment (empirical treatment of possible sepsis) and neonatal adverse outcomes.(2) Finally, to limit the effect of the indication bias, we used a propensity score, based on variables available at the time of decision to initiate or not an early antibiotic treatment. Using IPTW enabled us to estimate the average treatment effect on the whole sample. [START_REF] Austin | Moving towards best practice when using inverse probability of treatment weighting (IPTW) using the propensity score to estimate causal treatment effects in observational studies[END_REF] In contrast, using the matching on propensity score, patients for whom no match existed are excluded, which results in a significant loss of information that ultimately leads to a selection bias, in particular in this population of infants with low risk of EOS at baseline. It provided a way to balance measured covariates across exposed and non-exposed groups independently of the clinical status at admission in NICU that may have influenced the decision to administrate early antibiotics treatment.

Limitations should be kept in mind. We had a limited sample size due to our choice of a low risk of EOS study population. As a part of this epidemiological study, we could not analyze the gut microbiota nor the impact of early antibiotic exposure on subsequent colonization with multi-drug resistant organisms.

In our study, early antibiotic exposure was not associated with a higher risk of death, LOS, and/or NEC. Several studies found an association between early empirical exposition to antibiotics and these outcomes, isolated or combined.(9-11) However, these studies have included preterm infants alive at day 5 [START_REF] Cotten | Prolonged duration of initial empirical antibiotic treatment is associated with increased rates of necrotizing enterocolitis and death for extremely low birth weight infants[END_REF], at day 7 [START_REF] Ting | Duration of Initial Empirical Antibiotic Therapy and Outcomes in Very Low Birth Weight Infants[END_REF] or day 14 [START_REF] Cantey | Early Antibiotic Exposure and Adverse Outcomes in Preterm, Very Low Birth Weight Infants[END_REF], to have sufficient time to be exposed to antibiotics and focused on consequences of duration of antibiotics treatment.

We chose to study mortality and morbidities from the first day of life to obtain a complete assessment of the putative consequences of early antibiotics exposure. In a subgroup of very low birthweight infants with low risk of EOS, Ting et al found an increased risk of a composite outcome (intraventricular hemorrhage, NEC, BPD, severe retinopathy of prematurity, hospitalacquired infections, or death) in case of long antibiotic exposure (4-7 days) versus none (aOR=1.51 [95%CI=1.22-1.87]) but no statistically significant difference in case of short antibiotic exposure (1-3 days) (aOR=1.03 [95%CI=0.84-1.26]). ( 11) More recently, Li et al found a lower incidence of NEC in case of early antibiotic administration in an international cohort of 2831 preterm very low birthweight infants (aOR=0.25 [95%CI=0.12-0.47]).( 16) In this study, almost 90% of infants received antibiotics within the first three days after birth.

Moreover, in the small comparison group that did not receive early antibiotics treatment (269/2831), baseline characteristics differed markedly such as higher gestational age, higher rate of cesarean delivery, and higher proportion of small-for-gestational-age infants. The relatively high NEC incidence in this control group could lead to a potential confounding bias and requires caution in interpretation.

In our study, early antibiotic exposure was associated with a higher risk of moderate-to-severe BPD and severe cerebral lesions, despite adjustment for the severity of initial illness. Our findings were similar to those reported in some studies. [START_REF] Ting | Duration of Initial Empirical Antibiotic Therapy and Outcomes in Very Low Birth Weight Infants[END_REF][START_REF] Cantey | Antibiotic Exposure and Risk for Death or Bronchopulmonary Dysplasia in Very Low Birth Weight Infants[END_REF] Puopolo et al find also similar results: among 5334 low-EOS risk extremely preterm infants, those empirically treated with early prolonged antibiotics in the absence of culture-confirmed infection had a higher adjusted risk of severe cerebral lesions and higher adjusted risk of BPD compared to the infants who were not given early prolonged antibiotics (adjusted for multiple predictors of illness severity). [START_REF] Puopolo | Identification of Extremely Premature Infants at Low Risk for Early-Onset Sepsis[END_REF] We cannot totally rule out a lack of power to explain our observed lack of associations between early antibiotics and death, LOS, and/or NEC. Nevertheless, concerning the association between early antibiotics and severe cerebral lesions and bronchopulmonary dysplasia that we have found, the pathophysiological mechanisms remain to be understood. Consequences of early antibiotics should be different according to the type of antibiotics used or the number of courses during the hospitalization for example. Studying the consequences of early antibiotic treatment in very preterm infants is complex, and requires assessing not only neonatal mortality and morbidity but also effects on microbiota and later health. Indeed, postnatal exposure to antibiotics has been shown to impact the diversity of the gut microbiota. [START_REF] Arboleya | Intestinal microbiota development in preterm neonates and effect of perinatal antibiotics[END_REF] Other concerns also include a potential impact on host immunity and subsequent auto-inflammatory disorders, according to the suggested consequence of microbiota on inflammation and in the pathogenesis of a wide variety of inflammatory disease processes. [START_REF] Underwood | The Microbiota of the Extremely Preterm Infant[END_REF] Tracheal microbiota seems also to be different between infants who develop bronchopulmonary dysplasia and those who do not and postnatal exposition to antibiotics could be involved in these differences. [START_REF] Underwood | The Microbiota of the Extremely Preterm Infant[END_REF] In order to reach a more robust conclusion on the consequences of early antibiotics in preterm infants, a randomized controlled trial would be optimal. This study should include very preterm infants, born between 26 and 31 weeks, without risk factor for early-onset sepsis and the primary outcome will be the composite outcome of LOS, NEC, severe cerebral lesions and BPD or death.

CONCLUSION

Early exposure to antibiotics was not associated with an increased risk of a composite outcome including death, NEC and/or LOS in very preterm infants at low risk for EOS. However, our results question whether exposure to early antibiotics is warranted since it was associated with a higher risk of severe cerebral lesions and moderate-to-severe BPD.

  ) EPIPAGE-2 study was approved by the National Data Protection Authority (Commission Nationale de l'Informatique et des Libertés, CNIL n°911009) and by appropriate ethics committees (Consultative Committee on the Treatment of Data on Personal Health for Research Purposes, reference 10.626) and the Committee for the Protection of People participating in biomedical research (reference CPP SC-2873).

  All children received a combination of at least 2 molecules. Amoxicillin was used in 112/165 patients, third-generation cephalosporin in 129/165 patients, and aminoglycosides in 140/165 patients. Vancomycin was also used in combination in 11 patients, and metronidazole in 5. The median duration of antibiotic administration was 3 days (IQR=2-6). Four infants presented an episode of EOS diagnosed at the second or third day of life. None had received antibiotic treatment within the first day of life. Identified bacteria were Streptococcus agalactiae in one case and Staphylococcus aureus in the three others (including one child who died on Day 3).

Figure 1 :

 1 Figure 1: Flow chart

Figure 3 (

 3 Figure 3 (online): Variations between NICUs in the proportion of very preterm infants at

Figure 1

 1 Figure 1

Figure 2 (Figure 2 Figure 3 8 ( 15 Generalisability 21 1 *

 223815211 Figure 2 (online): Standardized differences in the initial and IPTW samples in complete-

Table 1 (online):

 1 Comparison of maternal and obstetrical characteristics between the study population and the population with missing data for early neonatal antibiotics

	Total	Early neonatal antibiotics	p
	N=799
		Complete cases	Missing data
		N=648 (80.9%)	N=151 (19.1%)

Table 1 ,

 1 online only

Table 2 :

 2 Maternal, obstetrical, and neonatal characteristics according to early neonatal antibiotics administration in very preterm infants

	Total	Early neonatal antibiotics	p
	N=648	No	Yes
		N=475 (73.8%)	N=173 (26.2%)

Table 2

 2 Data are presented as number (percentage) of patients. Percentages are weighted according to the differences between length of recruitment of gestational age groups.

	For each variable, percentages might not sum up to 100%, owing to rounding.
	IQR interquartile range; SGA small-for-gestational-age; CRIB Clinical risk index for babies score
	* Antenatal corticosteroids were defined as at least one injection of corticosteroids before delivery
	** Inborn was defined as birth in a hospital with a neonatal intensive care unit
	*** Characteristics of the unit in which the newborn was hospitalized during the first 48 hours of
	life.

Table 3 :

 3 Neonatal outcomes according to early neonatal antibiotics

	Total	Early neonatal antibiotics	p	Complete-cases analysis
					(N=465)
					aOR (95%CI)
	N=648	No	Yes
		N=475 (73.8)	N=173 (26.2)

Table 3 *

 3 Severe neonatal complication was defined as any of the following outcomes: late-onset sepsis; severe cerebral lesions (intraventricular hemorrhage with ventricular dilatation, parenchymal hemorrhage, and/or cystic periventricular leukomalacia); Bell's stage ≥ 2 necrotizing enterocolitis; and/or moderate-to-severe bronchopulmonary dysplasia (BPD), defined as administration of oxygen for at least 28 days plus need for 21% or more oxygen and/or mechanical ventilatory support or continuous positive airway pressure at 36 weeks' postmenstrual age.

Table 4 (

 4 online): Late-onset sepsis characteristics according to early neonatal antibiotics

	Total	Early neonatal antibiotics	p
	N=648	No	Yes
		N=475 (73.8)	N=173 (26.2)

Data are presented as number (percentage) of patients unless otherwise indicated. Percentages are weighted according to the differences between length of recruitment of gestational age groups.

For each variable, percentages might not sum up to 100%, owing to rounding. LOS Late-onset sepsis; IQR interquartile range;

Table 4 online only

 4only

Table 5 :

 5 Sensitivity analysis: Association between neonatal outcomes and early neonatal antibiotics after multiple imputation.

		Complete-cases analysis	Multiple imputation
		(N=465)	(N=648)
		aOR (95%CI)	aOR (95%CI)
	Death	1.78 (0.82-3.88)	1.51 (0.57-3.99)
	LOS	0.93 (0.64-1.34)	1.12 (0.62-2.03)
	NEC	1.47 (0.65-3.33)	*
	Severe cerebral lesions	2.71 (1.25-5.86)	2.99 (0.99-9.04)
	Moderate-to-severe BPD	2.30 (1.21-4.38)	1.87 (0.96-3.65)
	Composite outcomes
	Any severe neonatal	1.37 (0.94-1.98)	1.54 (0.87-2.72)
	complication
	Death or severe neonatal	1.33 (0.92-1.93)	1.53 (0.85-2.73)
	complication
	Death or severe cerebral	2.16 (1.21-3.85)	1.85 (0.82-4.18)
	lesions
	Death or moderate-to-	2.25 (1.31-3.87)	1.76 (0.91-3.39)
	severe BPD
	Death or LOS or NEC	1.04 (0.72-1.50)	1.28 (0.72-2.28)
	Adjusted odds ratio (aOR) obtained from multilevel analysis with inverse probability of treatment
	weighting, adjusted for gestational age.
	OR Odds ratio; LOS late-onset sepsis, NEC necrotizing enterocolitis, BPD bronchopulmonary
	dysplasia. Severe neonatal complication was defined as any of the following outcomes: late-onset sepsis;
	severe cerebral lesions (intraventricular hemorrhage with ventricular dilatation, parenchymal
	hemorrhage, and/or cystic periventricular leukomalacia); Bell's stage ≥ 2 necrotizing enterocolitis;
	and/or moderate-to-severe BPD

* Convergence not achieve due to the low number of cases of NEC.

Table 5

 5

The EPIPAGE-2 study has been funded with support from 1) The French Institute of Public Health Research/Institute of Public Health and its partners: the French Health Ministry, the National Institute of Health and Medical Research (INSERM), the National Institute of Cancer, and the National Solidarity Fund for Autonomy (CNSA).

*Manuscript (revised),

Abbreviations

BPD bronchopulmonary dysplasia, EOS Early-onset sepsis, IPTW inverse probability of treatment weighting, LOS Late-onset sepsis, NEC necrotizing enterocolitis, NICU Neonatal intensive care unit

CONTRIBUTORS

All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

AKNOWLEDGMENT

We are grateful for the participation of all families of preterm infants in the EPIPAGE-2 cohort study and for cooperation of all maternity and neonatal units in


Menghao Zhang

Pascal Jouquet

Jun Dai

Ling Xiao

Yan Du

Kexue Liu

Mikael Motelica-Heino

Patrick Lavelle

Hesen Zhong

Chi Zhang
email: zhangchi2012@scau.edu.cn

Zhang M H M H Zhang

Assessment of bioremediation

Keywords: Earthworm species, metal, Accumulation, Activation, Soil quality, Earthworm species, Metals, Accumulation, Availability, Soil quality

Highlights:

High survival rates of E. fetida, A. morrisi, and A. robustus were found in a soil polluted by multiple metals (Cu, Cd, Pb and Zn).

Native earthworm species showed high metal accumulation and improved soil quality.

The casts of epigeic and epi-endogeic species had higher available metal concentrations than those of endogeic species.

The native species A. morrisi is recommended for earthworm-assisted bioremediation of metal-polluted soils in South China.

J o u r n a l P r e -p r o o f

Introduction

Earthworms, known as soil ecosystem engineers, have drawn more attention than ever before in soil quality studies [START_REF] Lavelle | Ecosystem engineers in a self-organized soil: A review of concepts and future research question[END_REF][START_REF] Guerra | Tracking, targeting, and conserving soil biodiversity[END_REF]. They not only influence soil organic matter dynamics, nutrient release, and plant growth [START_REF] Lavelle | Soil ecology[END_REF][START_REF] Lavelle | Soil aggregation, ecosystem engineers and C cycle[END_REF], but also play a vital role in the biogeochemical cycling of trace elements [START_REF] Sizmur | Earthworms accelerate the biogeochemical cycling of potentially toxic elements: Results of a meta-analysis[END_REF]. Due to their important role in numerous ecological functions, earthworms have been used in ecological restoration of soils degraded by intensive tillage or excessive agrochemical inputs such as chemical fertilizers (Bedano et al., 2019;Fonte et al., 2019;[START_REF] Noguera | Contrasted effect of biochar and earthworms on rice growth and resource allocation in different soils[END_REF]. However, there is limited research on the potential of earthworms in bioremediation of soils contaminated by potentially toxic elements such as metal(loid)s [START_REF] Zhang | Effects of a native earthworm species (Amynthas morrisi) and Eisenia fetida on metal fractions in a multi-metal polluted soil from South China[END_REF][START_REF] Sizmur | Earthworms accelerate the biogeochemical cycling of potentially toxic elements: Results of a meta-analysis[END_REF]. So far, many studies have been conducted on the exposure of earthworms to metals. Some studies focused on the metal tolerance of earthworm species. For instance, [START_REF] Nahmani | A review of studies performed to assess metal uptake by earthworms[END_REF] investigated the tolerance of Eisenia fetida and some European earthworm species (e.g., Lumbricus rubellus, Lumbricus castaneus, Lumbricus terrestris, Lumbricus friendi, Aporrectodea rosea, Aporrectodea longa, Aporrectodea caliginosa and Allolobophora chlorotica) to metal contaminations. Other studies focused on metal accumulation by earthworms [START_REF] Dai | Heavy metal accumulation by two earthworm species and its relationship to total and DTPA-extractable metals in soils[END_REF][START_REF] Nahmani | A review of studies performed to assess metal uptake by earthworms[END_REF][START_REF] Coelho | Bioavailability and bioaccumulation of heavy metals of several soils and sediments (from industrialized urban areas) for Eisenia fetida[END_REF][START_REF] Xiao | Assessment of earthworm activity on Cu, Cd, Pb and Zn bioavailability in contaminated soils using biota to soil accumulation factor and DTPA extraction[END_REF] or metal mobilization and activation in earthworm-inhabited soil and plant uptake due to passage through the earthworm gut and change in microenvironment (Mondal et al., 2019;[START_REF] Ji | Potential of enhancing the phytoremediation efficiency of Solanum nigrum L. by earthworms[END_REF][START_REF] Sizmur | Earthworms accelerate the biogeochemical cycling of potentially toxic elements: Results of a meta-analysis[END_REF]. However, the key ecological functions of earthworms, such as those associated with C and N cycling, soil structure changes, and microbial activity, have seldom been investigated in these studies (Velasquez et al., 2007;Li et al., 2009a;Dominguez-Haydar et al., 2019).

J o u r n a l P r e -p r o o f

Therefore, a more integrated approach is needed to understand the influence of earthworms on the quality improvement of metal-contaminated soils. This is especially important in China where 16.1% of arable land is polluted by heavy metals (Meng, 2014).

Earthworm-assisted bioremediation technologies have been proposed for metalcontaminated soils due to their low cost, environmentally friendliness, and sustainability [START_REF] Zhang | Application of earthworms on soil remediation in southern China[END_REF][START_REF] Zhang | Effects of a native earthworm species (Amynthas morrisi) and Eisenia fetida on metal fractions in a multi-metal polluted soil from South China[END_REF]. Earthworm, as the stimulators of phytoremediation processes, could tolerate rather high amounts of contamination, enhance metal bioavailability and improve plants growth [START_REF] Zhang | Effects of a native earthworm species (Amynthas morrisi) and Eisenia fetida on metal fractions in a multi-metal polluted soil from South China[END_REF]. However, the tolerance of Asian earthworm species to metals and their influence on metal dynamics and soil quality still remain unknown [START_REF] Xiao | Assessment of earthworm activity on Cu, Cd, Pb and Zn bioavailability in contaminated soils using biota to soil accumulation factor and DTPA extraction[END_REF]. Therefore, our present study was aimed to evaluate the metal tolerance and accumulation of four earthworm species commonly found in China and to assess their influence on soil quality as well.

Materials and methods

Experimental design

Surface soil samples (0-20 cm) were collected from a paddy field (24°30′ N, 113°45′ E) contaminated with Cu, Cd, Pb and Zn in the upstream region of Yang He River near the Dabaoshan opencast mine, Guangdong Province, China. Soils in our study is a granite based soil with low organic C and N contents and acidic pH value but distributed widely in South China [START_REF] Lu | Soil series of China: Guangdong[END_REF]. Low microbial activity and metal availability in polluted soil were also found in this region (Li et al., 2009ab). Samples were homogenized and sieved to 2 mm after air-drying. Soil pH value, contents in total organic C, N and clay-sized particles (<2 µm) and the C:N ratio were 4.18 g kg -1 ±0.03, 15.3 ±0.23 g kg -1 , 1.47 ±0.05 g kg -1 , J o u r n a l P r e -p r o o f 33.6% ±1.0 and 10. 4±0.22, respectively. Total Cu, Cd, Pb and Zn concentrations were 394 ±10.2, 0.639± 0.06, 439±8.44 and 405±29.0 mg kg-1, respectively.

Coco coir and cattle dung were purchased from a flower market in Guangzhou, China.

Coco coir and cattle dung were air-dried, ground, and sieved to 2 mm. Total organic C, total N, and C/N ratio were 50.3%, 0.66%, and 76.0, respectively, for coco coir and 15.7%, 0.62%, and 25.4, respectively, for cattle dung. Contents of total Cd,Pb,Cu and Zn reached 0.8, 0.0, 16.2 and 123.7 mg kg -1 for cattle dung and 0.7, 0.0, 0.0 and 50.7 mg kg -1 for coco bran, respectively. These concentrations were lower than the national quality standard for organic fertilizer of China (NY525-2012). Coco coir and cattle dung were mixed at 4:1 (w/w) for later use as an organic amendment (pH 6.8).

Four earthworm species commonly found in South China were used in this study.

Eisenia fetida (Savigny, 1826), the reference species of several international standard toxicity tests, was collected from a local vermicompost operation. Amynthas corticis (Kinberg, 1867), A. morrisi (Beddard, 1892), and A. robustus (Perrier, 1872), native species in Asia, were collected from uncontaminated soils in Guangdong Province, China.

Of the four, E. fetida and A. corticis are epigeic species, whereas A. morrisi and A. robustus are epi-endogeic and endogeic, respectively. The earthworms were cultured in an uncontaminated soil with organic matter addition under suitable moisture and temperature conditions for one month before use.

The soil and coco coir/cattle dung mixture were mixed thoroughly at 9:1 (w/w) and added to pots (5 cm lower diameter, 11 cm upper diameter, 13.5 cm height). Then, five treatments were set up in triplicates: control, EF, AC, AM, and AR. In control, no earthworm was added, whereas in EF, AC, AM, and AR, approximately 15 g of healthy adult E. fetida (averaging 0.3 g individual -1 ), A. corticis (averaging 0.4 g individual -1 ), A. morrisi (averaging 0.4 g individual -1 ), and A. robustus (averaging 1.5 g individual -1 ) with J o u r n a l P r e -p r o o f clitella were added, respectively. During the experiment, soil moisture was maintained at field capacity and temperature at 25°C. After 60 days of incubation, earthworms in each pot were collected, counted, weighed, and placed on moist filter papers in Petri dishes to allow for excretion of contaminated soil from their guts at 25 o C for 7 days. Then, earthworms were killed in liquid nitrogen and oven-dried at 100 o C for 24 h. Aboveground and belowground casts and non ingested soil were manually separated, air-dried, weighed, ground, and sieved to 2 (for determination of pH, enzyme activities, and available metals) and 0.149 mm (for determination of organic C, total N, and total metals) based on the Chinese state environmental protection standards.

Laboratory analyses

The soil pH was measured using a 1:2.5 solid-to-water ratio. The organic C content was determined by the dichromate digestion method, and total N was determined by Kjeldahl digestion [START_REF] Sparks | Methods of Soil Analysis, Part 3, Chemical Methods[END_REF].

Total Cu, Zn, Pb, and Cd concentrations were determined after digestion with HF, HNO 3 , and HClO 4 (Amacher, 1996). Available Cu, Zn, Pb, and Cd were extracted with diethylene triamine penta acetic acid (DTPA) [START_REF] Lindsay | Development of a DTPA soil test for zinc, iron, manganese, and copper 1[END_REF] and determined by atomic absorption spectrophotometry (AAS).

Soil enzyme activities were considered biological indicators of soil quality [START_REF] Dick | Soil enzyme activities and biodiversity measurements as integrating biological indicators[END_REF]. β-glucosidase and N-acetyl-β-D-glucosaminidase, acid and alkaline phosphatase activities involved in soil organic C, N and P cycles and microbial activities [START_REF] Mora | Functional complement of biogenic structures produced by earthworms, termites and ants in the neotropical savannas[END_REF]Li et al., 2009a). 2 grams of soil or earthworm casts were moistened with 0.5 mL distilled water and incubated during 48 h at 25℃, to which 3 mL of water were added to obtain soil solution. Activities of acid phosphatase and alkaline phosphatase were determined at pH 5 (citrate buffer) and 9 (borate buffer), respectively, using p-nitrophenyl J o u r n a l P r e -p r o o f phosphate as substrate [START_REF] Mora | Functional complement of biogenic structures produced by earthworms, termites and ants in the neotropical savannas[END_REF]. Activities of β-glucosidase and N-acetyl-β-Dglucosaminidase were determined at pH 5 (phosphate buffer) using p-nitrophenyl-β-Dglucopyranoside and p-nitrophenyl-N-acetyl-β-D-glucosaminide as substrate, respectively [START_REF] Mora | Functional complement of biogenic structures produced by earthworms, termites and ants in the neotropical savannas[END_REF]. Overall enzyme activity was evaluated with fluorescein diacetate (FDA) hydrolytic activity which was related with total microbial activities [START_REF] Schnvrer | Flurescein diacetate hydrolysis as a measure of total microbial activity in soil and litter[END_REF].

Metal (Zn, Cd, Cu, and Pb) concentrations of earthworms were analyzed as described by [START_REF] Dai | Heavy metal accumulation by two earthworm species and its relationship to total and DTPA-extractable metals in soils[END_REF]. Earthworm responses to metal pollution were evaluated with survival rate and the biota-to-soil accumulation factor (BSAF). The survival rate I 1 was calculated as:

I 1 = A 60 /A 0  100%
where A 0 and A 60 are earthworm number at the beginning (day 0) and end (day 60) of the experiment, respectively. BSAF was calculated as: BSAF = M worm /M soil where M worm and M soil are metal concentrations of earthworm and soil, respectively.

Cast production was the sum of aboveground casts and belowground casts.

Statistical analyses

Data were analyzed using one-way analysis of variance (ANOVA) in SAS 8.0 (SAS Institute Inc., Cary, NC, USA). Duncan's test was used to analyze the differences between treatments and the significance level was set at P < 0.05. Multivariate analyses, including principal component analysis (PCA) and co-inertia analysis (CIA), were performed using the ADE-4 package in R (R Development Core Team, 2009;[START_REF] Thioulouse | ADE-4: a multivariate analysis and graphical display software[END_REF]. A Monte Carlo permutation test was performed to check the significance of the co-inertia J o u r n a l P r e -p r o o f using the ADE-4 package as well. Quality indices of aboveground casts, belowground casts, and non ingested soil were calculated using the methodology proposed by Velasquez et al. (2007). The pH, organic C and total N contents, cast production, β-glucosidase and N-acetyl-β-D-glucosaminidase, acid and alkaline phosphatase activities and FDA hydrolytic activity, which are associated with C, N and P cycling, soil structure changes, and microbial activity, were chosen as soil quality variables in this study. For the first three axes of the PCA (F2 and F3 were very close in their contributions to the total variance), the variables with the highest contributions were selected. The values of each variable set, adjusted to a range of 0.1 to 1.0 via homothetic transformation, were multiplied by their respective weight factors and summed, giving soil/casts quality index I as:

I = F1  (α 1 a + β 1 b + γ 1 c) + F2  (α 2 a + β 2 b + γ 2 c) + F3  (α 3 a + β 3 b + γ 3 c)
where F1, F2 and F3 are the percentages of variance explained by the PCA on axis 1, axis 2 and axis 3, respectively, α, β, and γ represent the contributions of the variables to the formation of their respective axes, and a, b, and c are the values of the selected variables on their corresponding axes (Velasquez et al., 2007;[START_REF] Rodriguez | Agroforestry systems in the Colombian Amazon improve the provision of soil ecosystem services[END_REF].

Results

Earthworm survival rates and metal accumulation

The survival rates of E. fetida, A. morrisi, and A. robustus reached 94.8, 115, and 99.7%, respectively, significantly higher than that of A. corticis (Fig. 1a). A. morrisi reproduced during the experiment, and its quantity exceeded 100%. After 60 days of incubation, the biomasses of E. fetida, A. corticis, and A. morrisi decreased, while that of A. robustus increased (Fig. 1b). Moreover, the biomasses of E. fetida and A. morrisi were significantly higher than that of A. corticis.

J o u r n a l P r e -p r o o f

Metal accumulation in earthworm tissues varied according to the type of metal. Metal concentration in earthworm tissues ranked as Zn >> Pb and Cu >> Cd (Tab. 1). Compared with E. fetida and A. morrisi, Zn concentration of A. corticis was 33.3-92.1% higher (P < 0.05). Significantly higher Cu and Cd concentrations were found in the native species (i.e., A. corticis, A. morrisi, and A. robustus) as compared with the non-native species E. fetida.

In addition, significantly higher Pb concentration was observed in the endogeic species (i.e., A. robustus) as compared with the epigeic and epi-endogeic species.

In terms of BSAF, the heavy metals ranked as Cd > Zn > Pb and Cu, and the BSAFs of Zn, Cu, and Pb were less than 1 (Tab. 1). The BSAFs of Cd and Cu of the native species (i.e., A. corticis, A. morrisi, and A. robustus) were markedly higher than those of E. fetida, while the BSAF of Pb of endogeic species (i.e., A. robustus) was significantly higher than those of the epigeic/epi-endogeic.

2 Earthworm casts production and casts and non ingested soil properties

Earthworm casts production ranked as A. morrisi > E. fetida > A. robustus > A. corticis (Fig. 2). Eisenia fetida produced the largest amounts of aboveground casts while A. robustus produced the smallest amount. Additionally, A. morrisi and A. robustus produced more belowground casts than E. fetida and A. corticis.

Compared with the control, pH values of the aboveground casts and non ingested soil in EF, AM and AR increased by 0.33, 0.13, 0.09, and 0.12 units, respectively (Tab. 2). In EF, pH value of the aboveground casts was significantly higher than that of the belowground casts. The highest pH values of aboveground and belowground casts and non ingested soil were found in EF, while the lowest pH values of aboveground casts and non ingested soil were observed in AC and AR respectively (P<0.05).

J o u r n a l P r e -p r o o f

The organic C content of casts was 15.4-31.4% higher than that of the soil in control (P < 0.05). Higher organic C contents were always found in the earthworm casts than in the non ingested soil, especially in EF, AC, and AR. Of the four treatments with earthworms, EF showed the lowest organic C content in aboveground casts and in non ingested soil as well (P < 0.05).

Compared with the control, the presence of earthworms in the other treatments increased the total N contents of non ingested soil and casts by 4.74-29.4%. In both EF and AC, the total N content of aboveground casts was significantly higher than that of belowground casts. In EF, the total N contents of both the aboveground and belowground casts were significantly higher than that of the non ingested soil. Of the four treatments, EF displayed the lowest total N content of non ingested soil and the highest total N contents of aboveground and belowground casts.

Earthworm inoculation generally decreased the enzyme activities in soil (Tab. 2). β-Glucosidase activity in non ingested soil was significantly higher in AC than in EF and AM. The enzyme activity in aboveground casts was significantly higher in EF than in AC, and the enzyme activity in belowground casts was significantly higher in EF than in AM and AR. In AC, the enzyme activity was significantly lower in aboveground and belowground casts than in non ingested soil. In general, N-acetyl-β-D-glucosaminidase activity ranked as non ingested soil > aboveground casts > belowground casts. However, in EF, the enzyme activity was significantly higher in aboveground casts than in non ingested soil. The enzyme activity in belowground casts was significantly higher in EF than in AM.

Alkaline phosphatase activity of the four treatments with earthworms ranked as AR > AM > AC > EF but as AC > AR > AM > EF for belowground casts. In AR, the enzyme activity was significantly lower in belowground casts than in non ingested soil and aboveground casts. Acid phosphatase activity in non ingested soil of the four treatments with J o u r n a l P r e -p r o o f earthworms ranked as AC > AR > AM > EF. In AC and AR, the enzyme activity was significantly higher in non ingested soil than in belowground casts. In AC, FDA hydrolytic activity was significantly higher in belowground casts than in non ingested soil and aboveground casts, whereas casts of AR had significantly lower activity than non ingested soil. The activity in non ingested soil and aboveground casts was higher in AR than in the other three treatments with epigeic species. In particular, the differences in FDA hydrolytic activity of non ingested soil were significant between the three treatments with native species.

3 Total and available metals in casts and non ingested soil

Compared with the control soil, total Zn concentration in AC decreased by 10.1% and 5.58% in aboveground and belowground casts, respectively (Tab. 3). In AC, the total Zn concentration of non ingested soil was significantly higher than that of aboveground casts.

In AR, the total Zn concentration of non ingested soil was significantly higher than those of casts. The total Zn concentration of belowground casts was significantly higher in AM than in AC. However, there were no significant differences in total Cd concentration between non ingested soil, aboveground casts, and belowground casts whatever the treatments.

In EF, the total Cu and Pb concentrations of belowground casts were significantly higher than those of non ingested soil. In AC and AR, total Pb concentration was significantly higher in non ingested soil than in aboveground and belowground casts.

Additionally, total Pb concentration of belowground casts was significantly higher in EF and AM than in AC and AR. Earthworm introduction decreased total soil Pb concentration by 0-19.0% (P < 0.05).

J o u r n a l P r e -p r o o f

In general, casts had higher available metal concentrations than the non ingested soil.

The highest metal availability in casts and non ingested soil were found in AC with the epigeic species A. corticis, whereas the lowest values were generally observed in AR with the endogeic species A. robustus (Tab. 3).

In the three treatments with native species, the available Zn concentrations of casts were significantly higher than that of non ingested soil, which was especially true for AC.

Compared with the control, available Zn concentration of aboveground and belowground casts in AC was 13.1% and 6.5%, respectively, higher (P < 0.05), and that of aboveground casts was significantly higher than that of belowground casts.

Compared with the control, available Cd concentrations in EF, AC, and AM were 0.4-41.7% higher. In the three treatments with native species (i.e., AC, AM, and AR), available Cd concentration of aboveground casts was significantly higher than that of non ingested soil. Available Cd concentrations of casts were also significantly higher in AC than in the other treatments.

Compared with the control, available Cu concentration of belowground casts in the treatments with epigeic and epi-endogeic species (i.e., EF, AC, and AM) was 1.4-8.7% higher. In the treatments with native species, available Cu concentration was significantly higher in casts than in non ingested soil. The available Cu concentrations of casts were significantly lower in AR than in EF, AC, and AM.

Compared with the control, the available Pb concentrations of casts in EF and AC were 1.3-13.0% higher. It is also worth noticing that in AR with the endogeic species A. robustus, the available Pb concentration of non ingested soil was significantly higher than those of casts. In terms of the available Pb concentration of casts, the treatments ranked as AC > EF > AM > AR (P < 0.05).

J o u r n a l P r e -p r o o f 3. 4 Relationship between metal concentration in earthworm tissue and available metal in casts A co-inertia analysis was performed to explore the relationship between metal concentration in earthworm tissue and available metal in casts (Fig. 3). Metal concentration of earthworm tissue and available metal of casts were significantly different between treatments (P = 0.005). The first axis explained 71.0% of the total variance. Available Cu and Pb in casts were negatively correlated with the axis, while their total concentrations in earthworm tissue were positively correlated with the axis. The second axis explained 18.3% of the total variance. The total concentrations of Zn and Cd in earthworm tissues were positively correlated with the available Zn and Cd in casts. The Pb-and Cu-activating abilities of epigeic species E. fetida and A. corticis and epi-endogeic species A. morrisi were stronger than those of the endogeic species A. robustus, but their Pb-and Cuaccumulating abilities were lower. There were significant differences in the Zn-and Cdaccumulating and activating abilities between the three epigeic and epi-endogeic earthworm species (P = 0.005). Amynthas corticis had the strongest Zn-and Cdaccumulating and activating abilities, followed by A. morrisi and E. fetida.

Additionally, total metal accumulation in earthworm tissues and total available metal in earthworm casts and their ratios were calculated (Tab. 4). The highest ratios were found in the treatment with the endogeic species A. robustus (P < 0.05). The ratio of Cd was larger than 1.0 for AC, AM, and AR and was considerably larger than those of Zn, Cu, and Pb.

5 Effects of earthworm on soil quality

PCA analysis showed that axis 1 (38.1% variance explained) was mainly related to organic C, cast production, and activities of β-glucosidase, N-acetyl-β-D-glucosaminidase, J o u r n a l P r e -p r o o f acid phosphatase and alkaline phosphatase (Fig. 4a,b,c). Axis 2 (17.5% variance explained) was mainly related to total N and FDA hydrolytic activity. Axis 3 (14.0% variance explained) was mainly related to pH and alkaline phosphatase. Organic C and aggregate production were negatively correlated with enzyme activities (Fig. 4a,b,c), and total N were negatively correlated with the overall enzymatic activity (i.e., FDA hydrolytic activity). The differences in non ingested soil and casts properties between the four treatments of EF, AC, AM, and AR were significant (P = 0.001). In terms of soil quality index, control < non ingested soil < casts (Fig. 4d). The soil quality indices of non ingested soil ranked as: AM (0.41) > AC (0.40) > EF (0.34) > AR (0.30); those of aboveground casts were in the order of: AC (0.90) > AM (0.63) > EF (0.58) > AR (0.51) (P < 0.05); and those of belowground casts were observed as: AM (0.88) > AR (0.85) > EF (0.67) and AC (0.67). Compared with the control (0.12), the average soil quality indices of EF (0.53), AM (0.64), AC (0.66), and AR (0.56) were higher.

Discussion

There is an urgently need for soil comprehensive remediation in polluted soil.

Transfering metals from soil, improving soil quality and increasing plants growth are the three dimensions of soil remediation, which could be achieved by earthworm-assisted bioremediation technologies [START_REF] Zhang | Application of earthworms on soil remediation in southern China[END_REF][START_REF] Zhang | Effects of a native earthworm species (Amynthas morrisi) and Eisenia fetida on metal fractions in a multi-metal polluted soil from South China[END_REF]. When using earthworm for soil bioremediation in the field, it is necessary to choose the most suitable earthworm species and then take several factors into consideration, such as earthworm tolerance to metals, metal accumulation in earthworm, available metals in earthworm casts removed into the aboveground of plants in further, and earthworm effects on soil quality.

Earthworm tolerance to metals

J o u r n a l P r e -p r o o f

High tolerance to pollutants is considered an essential characteristic of organisms that are to be used in bioremediation technologies [START_REF] Macaskie | Microbial metabolism, desolubi-lization and deposition of heavy metals: Uptake by immobilized cells and application to the treatment of liquid wastes[END_REF]. High survival rate in contaminated soil is, obviously, the primary requirement for the selction of earthworm species. In our study, the high survival rates of the four earthworm species in the natural heavy metal-contaminated soil demonstrated their high tolerance to Cu, Cd, Pb, and Zn. Compared with A. corticis, the > 90% survival rates of A. robustus and A. morrisi indicated that the two species may be promising in the field (Fig. 1). Eisenia fetida is commonly used in international standard toxicity tests and readily available from commercial supplies. It is not a natural soil species and inhabits organic-rich habitats [START_REF] Nahmani | A review of studies performed to assess metal uptake by earthworms[END_REF]. In addition, it is less sensitive to contaminants than other species [START_REF] Langdon | Survival, Pb-uptake and behaviour of three species of earthworm in Pb treated soils determined using an OECD-style toxicity test and a soil avoidance test[END_REF]. In our 60-day laboratory experiment with 10% organic matter addition to the soil, a survival rate of 94.8% was found for E. fetida, which was not much lower than those of A. morrisi and A. robustus. Therefore, although not a native species, E. fetida may also be considered for bioremediation of metal-contaminated soils in South China. In further study, some subcellar or molecular experiments should be needed for explaining the tolerant mechanism of earthworms on the long-term with the aim to use earthworms in remediation processes.

Earthworm effects on soil quality

Effects of earthworm on soil quality are also crucial when using earthworm in soil bioremediation. Soil health and sustainability should be maintained [START_REF] Lavelle | Ecosystem engineers in a self-organized soil: A review of concepts and future research question[END_REF].

Soil quality indices reflect soil physical, chemical, and biological processes (Velasquez et al., 2007;Dominguez-Hayda et al., 2019). In this study, soil quality indices were significantly different between treatments with different earthworm species (Fig. 4c). The higher average soil quality indices in AR (0.56) and AM (0.64) as compared with EF (0.53) J o u r n a l P r e -p r o o f may be mainly due to the effects of A. robustus and A. morrisi on soil aggregates, organic C, and microbial activities. Previous studies have shown that soil-dwelling species living in deeper soil layers produce more stable aggregates than epigeic species [START_REF] Blouin | A review of earthworm impact on soil function and ecosystem services[END_REF][START_REF] Lavelle | Soil aggregation, ecosystem engineers and C cycle[END_REF]. Similar results were also found in this study that the endogeic species A. robustus and the epi-endogeic species A. morrisi produced more casts, which are rich in microaggregates, than the epigeic species A. corticis (Fig. 2). The high C contents of aboveground and belowground casts (Tab. 2 and Fig. 4) were related to earthworms' feeding habits that they selectively feed on litter or organic-rich soil [START_REF] Lavelle | Soil ecology[END_REF][START_REF] Van Groenigen | How fertile are earthworm casts? A meta-analysis[END_REF][START_REF] Wu | Impacts J o u r n a l P r e -p r o o f of earthworm species on soil acidification, Al fractions, and base cation release in a subtropical soil from China[END_REF][START_REF] Xiao | Assessment of earthworm activity on Cu, Cd, Pb and Zn bioavailability in contaminated soils using biota to soil accumulation factor and DTPA extraction[END_REF]. Taking their effects on soil quality into account, A. robustus and A. morrisi were better candidate species than E. fetida for bioremediation of metal-contaminated soils.

Metal accumulation in earthworm and available metal in casts and their relationship

Metal accumulation in earthworm and available metal in casts are key to earthworm environmental application. Metal accumulation in earthworm tissue was significantly influenced by earthworm species (Tabs. 1 and 4). The higher metal concentrations in the tissues of the three native species A. corticis, A. morrisi, and A. robustus as compared with E. fetida may be because they ingest larger amounts of soil but excrete smaller amounts of metal [START_REF] Dai | Heavy metal accumulation by two earthworm species and its relationship to total and DTPA-extractable metals in soils[END_REF][START_REF] Suthar | Earthworms as bioindicator of metals (Zn, Fe, Mn, Cu, Pb and Cd) in soils: is metal bioaccumulation affected by their ecological category?[END_REF]. Total metal accumulation and the ratio of metal accumulation in body to available metal in casts of A. robustus were higher than those of the other species (Tab. 4), possibly due to the higher biomass of A. robustus (Fig. 1) [START_REF] Xiao | Assessment of earthworm activity on Cu, Cd, Pb and Zn bioavailability in contaminated soils using biota to soil accumulation factor and DTPA extraction[END_REF]. The higher metal availability in earthworm casts than in non ingested soil, which indicates metal activation in earthworm casts, is attributed to the effects of intestinal microbes according to our previous studies [START_REF] Dai | Effects of earthworm gut on microbial community structure in heavy metal contaminated soils[END_REF][START_REF] Zhang | Earthworm and organic amendment effects on microbial activities and metal availability in a contaminated soil from China[END_REF]. Metal availability in casts was closely associated with earthworm species (Tab.

J o u r n a l P r e -p r o o f 3, Fig. 3). The metal-activating effect of epigeic/epi-endogeic species was stronger than that of endogeic species (Tab. 3). Epigeic and epi-endogeic species have stronger organic C-decomposing ability than endogeic species [START_REF] Lavelle | Soil ecology[END_REF] due to some special microbes in their guts which participate in carbohydrate metabolism regulation [START_REF] Liu | The combined effects of earthworms and Sphingobacterium sp. on soil carbon, nitrogen, microbial activities and diversities[END_REF], resulting in higher availability of elements, including metals. In addition, total available Cd was higher in the casts of A. morrisi than in the casts of the other species (P < 0.05, Tab. 4), demonstrating that A. morrisi is a good Cd activator for Cd bioremediation.

Many studies have focused on metal accumulation [START_REF] Nahmani | A review of studies performed to assess metal uptake by earthworms[END_REF][START_REF] Suthar | Earthworms as bioindicator of metals (Zn, Fe, Mn, Cu, Pb and Cd) in soils: is metal bioaccumulation affected by their ecological category?[END_REF][START_REF] Xiao | Assessment of earthworm activity on Cu, Cd, Pb and Zn bioavailability in contaminated soils using biota to soil accumulation factor and DTPA extraction[END_REF] or metal activation by earthworms [START_REF] Sizmur | Earthworms accelerate the biogeochemical cycling of potentially toxic elements: Results of a meta-analysis[END_REF] and the references in it), but few studies have looked at their relationship. In this study, the ratio values of metal accumulation in earthworm tissue to total available metal in earthworm casts indicated that a large proportion of the metals ingested by earthworms returned to the soil in available forms instead of entered earthworm tissue except Cd (Tab. 4). Therefore, both stabilizing the Zn, Cu, and Pb in earthworm casts and phytoextraction are plausible remediation methods. As Cd is highly mobile [START_REF] Nannoni | Uptake and bioaccumulation of heavy elements by two earthworm species from a smelter contaminated area in northern Kosovo[END_REF] and easily combines with metallothionein (MT) and non-metallothionein proteins (ie. MIP) in earthworm tissue [START_REF] Morgan | A comparison of the cadmiumbinding protein isolated from the posterior alimentary canal of the earthworms Dendrodrilus rubida and Lumbricus rubellus[END_REF][START_REF] Hussain | Metal induced non-metallothionein protein in earthworm: A new pathway for cadmium detoxification in chloragogenous tissue[END_REF], Cd accumulation in earthworm should not be overlooked. Metal BSAFs ranked as Cd > Zn > Cu and Pb (Tab. 1, P < 0.05), which was consistent with the result of [START_REF] Dai | Heavy metal accumulation by two earthworm species and its relationship to total and DTPA-extractable metals in soils[END_REF]. The Cd accumulated in earthworm tissues would re-enter the soil when earthworm dies, posing environmental risk [START_REF] Zhang | Earthworm and organic amendment effects on microbial activities and metal availability in a contaminated soil from China[END_REF]. Therefore, it is important to reduce Cd accumulation in earthworm tissue. [START_REF] Lanno | The bioavailability of chemicals in soil for earthworms[END_REF] pointed out that after ingested, Cd is transported to sites of toxic action and persists in earthworm body for a long time. The co-inertia analysis showed that Cd availability in casts was positively related with Cd accumulation in J o u r n a l P r e -p r o o f epigeic/epi-endogeic species (Fig. 3). Available Cd in earthworm-soil system is influenced by microbe strains, plant roots, and soil properties [START_REF] Dai | Effects of earthworm gut on microbial community structure in heavy metal contaminated soils[END_REF][START_REF] Kaur | Cd induced generation of free radical species in Brassica juncea is regulated by supplementation of earthworms in the drilosphere[END_REF].

Employing phytoextraction technology may decrease Cd in soil and earthworm tissue and remediate Cd pollution in soil.

Above all, the highest total available Cd, Cu, and Pb concentrations in A. morrisi casts demonstrates that A. morrisi is the best option for future application of the three native earthworm species. A. morrisi-assisted phytoextraction may be a suitable remediation method for the Cd/Cu/Pb multi-polluted soils in South China. However, it is worth mentioning that only one type of soil was used in our experiment, which lasted only 60 days. Therefore, more studies are required to confirm the results of this study in other environments and over long-term observations.

Conclusion

When choosing earthworm species for metal-contaminated soil remediation, special attention should be paid to the tolerance of earthworm to metals, metal accumulation in earthworm tissue, metal availability in earthworm casts, and the effect of earthworm on soil quality. Eisenia fetida, A. morrisi, and A. robustus showed higher survival rates than A. corticis in the heavy metal-contaminated soil. Compared with the epigeic species E. fetida, the native epi-endogeic species A. morrisi and endogeic species A. robustus accumulated more metals and had a more beneficial effect on soil quality. In addition, A. morrisi had a strong ability in activating Cd, Cu, and Pb in casts. Based on the relationship between metal accumulation in earthworm tissue and metal availability in casts, A. morrisi is the most promising of the four for application in heavy metal-contaminated soils, and A. morrisi-assisted phytoextraction technology may be exploited for the Cd/Cu/Pb multipolluted soils in South China.

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Fig. 1

 1 Fig.1Survival rate and biomass of the four earthworm species Eisenia fetida, Amynthas

Fig. 3

 3 Fig.3Co-inertia analysis between metal concentration in earthworm tissue and available

Fig. 4

 4 Fig. 4 Principal component analysis of soil properties in treatments with earthworm

J

  o u r n a l P r e -p r o o f Tab. 2 Properties of non ingested soil and aboveground and belowground casts in the different treatments (mean ± S.D., n = 3) J o u r n a l P r e -p r o o f pNP = p-nitrophenol.Different capital letters indicate significant differences (P < 0.05) between non ingested soil, aboveground casts, and belowground casts for and available metals in the aboveground and belowground casts and non ingested soil of all treatments (mean ± S.D., n = 3) J o u r n a l P r e -p r o o f

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Table lists :

 lists Tab. 1 Metal concentration and metal biota-to-soil accumulation factor (BSAF) of the four earthworm species (i.e., Eisenia fetida, Amynthas corticis, A. morrisi, and A. robustus) Metal accumulation in earthworm tissue, total available metal in earthworm casts and the ratio between them Different lower case letters indicate significant differences (P < 0.05) between treatments.Tab. 1 Metal concentration and metal biota-to-soil accumulation factor (BSAF) of the four earthworm species (i.e., Eisenia fetida, Amynthas corticis, A. morrisi, and A. robustus) after 60-day incubation in the Zn/Cu/Cd/Pb multi-polluted soil (mean ± S.D., n = 3)

	after 60-day incubation in the Zn/Cu/Cd/Pb multi-polluted soil (mean ± S.D., n = 3) Different lower letters indicate significant differences between earthworms (P < 0.05). Tab. 2 Properties of non ingested soil and aboveground and belowground casts in the different treatments (mean ± S.D., n = 3) Different capital letters indicate significant differences (P < 0.05) between non ingested soil, aboveground casts, and belowground casts for a same treatment; different lower case letters indicate significant differences (P < 0.05) between treatments for a same component (2016). Tab. 3 Total and available metals in the aboveground and belowground casts and non ingested soil of all treatments (mean ± S.D., n = 3) Different capital letters indicate significant differences (P < 0.05) between non ingested soil, aboveground casts, and belowground casts for a same treatment; different lower case letters indicate significant differences (P < 0.05) between treatments for a same component (i.e., non ingested soil, aboveground casts, or below ground casts). The data for non ingested soil and aboveground casts in EF and AM treatments were from Zhang et al. (2016). species Metal concentration (mg•kg -1 ) Zn Cd Cu Pb Zn E. fetida 95.8±4.80b 5.03±0.117b 30.9±1.67b 56.8±4.86b 0.308±0.043a A. corticis 184±28.3a 15.7±2.62a 72.5±8.74a 62.5±22.7b 0.666±0.099a A. morrisi 138±22.3b 12.0±0.614a 59.9±8.39a 63.6±7.47b 0.508±0.270a A. robustus 150±9.89ab 12.2±4.31a 72.7±5.35a 97.4±13.7a 0.557±0.059a Tab. 4 Earthworm Different lower letters indicate significant differences between earthworms (P < 0.05).	BSAF 0.084± 0.006b 0.138±0.011b Cu Pb 0.194± 0.022a 0.149±0.055b 16.6± 5.58ab 0.156± 0.018a 0.150±0.017b Cd 6.76±1.60b 24.8± 7.03a 24.0± 7.92a 0.196± 0.009a 0.235± 0.034a

(i.e., non ingested soil, aboveground casts, or belowground casts). The data for non ingested soil and aboveground casts in EF and AM treatments were from Zhang et al.

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  Tab. 4 Metal accumulation in earthworm tissue, total available metal in earthworm casts and the ratio between them Different lower case letters indicate significant differences (P < 0.05) between treatments.

	Treatment	Total Zn	Total Cd	Total Cu	Total Pb	Available Zn	Available Cd	Available Cu	Available Pb
	Treatment Metal accumulation in earthworm	mg.kg -1 Total available metal in earthworm casts	The ratio of metal accumulation in earthworm to total available metal
	CK	266.69±5.27	0.65±0.24	382.82±2.86	427.64±2.12	19.9±0.645	0.266±0.009	76.3±1.78	84.3±1.86
	EF					in earthworm casts
	non ingested soil Zn	313.21±27.30Aa Cd Cu mg/pot	0.77±0.19Aa 369.13±8.28Ba Pb Zn Cd J o u r n a l P r e -p r o o f 410.65±8.11Ba 17.1±1.32Aa 0.267±0.012Aa Cu Pb Zn Cd Cu mg/pot	73.6±2.94Aa Pb	79.1±9.69Aa

Acknowledgements

Guangdong Natural Science Foundation (Grant No. 2021A1515011543), National Natural Science Foundation of China (Grant Nos. 41201305 and 41601575), National Science and Technology Fundamental Resources Investigation Program of China (2018FY100300), National Key Research and Development Program of China (2016YFD0201301 and 2016YFD0201200) supported this work, in addition to support from the China Scholarship Council and the scientific section of the French Embassy in Beijing.

indicate significant differences (P < 0.05) in soil quality index of aboveground casts/belowground casts/non ingested soil between different treatments.)

J o u r n a l P r e -p r o o f same treatment; different lower case letters indicate significant differences (P < 0.05) between treatments for a same component (i.e., non ingested soil, aboveground casts, or belowground casts). The data for non ingested soil and aboveground casts in EF and AM treatments were from Zhang et al. (2016).

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Funding acquisition, Project administration. , M.C., 1996. Nickel, cadmium, and lead. In: Sparks, D.L. (Ed.), Methods of Soil Analysis: Part 3. Chemical Methods. Soil Science Society of America Book Series, vol. 5. American Society of Agronomy, Madison, 739-768. Bedano, J.C., Vaquero, F., Dominguez, A., Rodriquez, M.P., Wall, L.G., Lavelle, P., 2019.

Amacher

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Control 5.48±0.00 40.8±0.22 2.11±0.14 209±14.1 212±11.9 85.9±34.0 467±50.3 149±19.5 EF non ingested soil 5.61±0.06ABa 38.4±1.83Bb 2.30±0.05Cb 166±26.1Abc 114±30.5Ba 31±22.2Ab 350±103Ac 80±8.6Ab Aboveground cast 5.81±0.12Aa 47.2±1.62Ab 2.73±0.08Aa 213±28.3Aa 186±45.9Aa 25±17.6Ac 324±68.9Aa 61±13.0Aa Belowground cast 5.45±0.14Ba 48.5±1.59Aa 2.53±0.04Ba 184±49.3Aa 132±8.6ABa 47±33.8Aa 292±47.2Aa 68±19.1Aa AC non ingested soil 5.45±0.08Ab 44.1±2.53Ba 2.59±0.08Aa 293±48.2Aa 201±70.4Aa 72±33.2Aab 615±95.4Aa 22±3.7Bc Aboveground cast 5.23±0.23Ab 53.6±2.96Aa 2.57±0.25Aa 130±13.3Bb 150±41.9Aa 56±15.6Abc 311±51.1Ba 22±2.7Bb Belowground cast 5.44±0.16Aa 50.1±1.63Aa 2.21±0.08Ba 135±11.2Bab 98±45.5Aab 73±14.2Aa 338±74.2Ba 69±6.7Aa AM non ingested soil 5.57±0.11Aab 43.6±3.48Aa 2.51±0.19Aab 136±2.6Bc 175±36.2Aa 106±26.7Aab 438±84.9Abc 81±7.6Ab Aboveground cast 5.31±0.07Ab 48.1±2.28Ab 2.39±0.18Aa 182±24.1Aab 127±56.4ABa 94±37.3Aab 318±61.4Aa 76±18.6Aa Belowground cast 5.28±0.26Aa 47.1±2.92Aa 2.50±0.09Aa 110±12.9Bb 51±38.8Bb 50±20.2Aa 298±77.8Aa 72±8.7Aa AR non ingested soil 5.43±0.04Ab 44.0±1.90Ba 2.56±0.14Aa 250±73.7Aab 180±56.5Aa 142±42.4Aa 521±54.6Aab 117±19.1Aa Aboveground cast 5.60±0.03Aa 49.7±0.95Ab 2.40±0.18Aa 164±42.0ABab 154±18.6Aa 117±8.7Aa 409±51.7ABa 79±3.0Ba Belowground cast 5.31±0.25Aa 50.6±0.66Aa 2.42±0.31Aa 110±36.8Bb 101±36.8Aab 60±8.4Ba 268±98.9Ba 63±5.5Ba

CRediT authorship contribution statement

Different capital letters indicate significant differences (P < 0.05) between non ingested soil, aboveground casts, and belowground casts for a same treatment; different lower case letters indicate significant differences (P < 0.05) between treatments for a same component (i.e., non ingested soil, aboveground casts, or below ground The data for non ingested soil and aboveground casts in EF and AM treatments were from [START_REF] Zhang | Earthworm and organic amendment effects on microbial activities and metal availability in a contaminated soil from China[END_REF]. J o u r n a l P r e -p r o o f

Conflict of interest

The authors declared that they have no conflicts of interest to this work.

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

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