Vignati Davide
email: david-anselmo.vignati@univ-lorraine.fr

Martin Loïc

Poirier Laurence

Zalouk- Vergnoux Aurore

Fouque Chantal

Bojic Clément

Hissler Christophe

Cossu-Leguille Carole

Behaviour and speciation of lanthanides during standardized ecotoxicity tests with Daphnia magna: implications and recommendations for hazard and risk assessment

Keywords: aquatic ecotoxicity, elemental speciation, Daphnia magna, critical raw materials, hazard assessment

Lanthanides (LNs) are a group of 14 elements with steadily increasing economical importance due to their multiple uses in technologies essential for sustainable ecological, digital and energetic transitions. Although knowledge on LN ecotoxicology has greatly improved over the last decade, uncertainty persists with regard to their actual hazard and risk in freshwater environments. In particular, only limited information is available on i) the actual relationships between LN speciation vs. ecotoxicological responses in standardized laboratory tests and ii) the existence of regular and predictable patterns in LN ecotoxicity along the LN series. The present paper provides the first report on the ecotoxicity of all lanthanides (except Pm) for the freshwater crustacean Daphnia magna along with an unprecedented level of detail on LN speciation in the exposure medium.

Experimental data show that exposure concentrations can decrease by up to 95 % over the test duration, with the percentage decrease being inversely related with LN atomic mass. Thermodynamic speciation calculations confirm the possible formation of insoluble species, mainly LN carbonates. However, the corresponding theoretical solubility limits do not fully agree with measured concentrations at the end of the tests. Experimental verification of exposure concentrations (as a minimum at the beginning and end of laboratory tests) remains therefore mandatory to reach proper conclusions as to the ecotoxicity of each LN. A decreasing trend in ecotoxicity can actually be observed along the LN series when temporal changes in the exposure concentrations are properly accounted for. However, this trend remains dependent on exposure time and selected exposure metrics. This and other caveats must be considered in future research to reach a community-based consensus for the proper hazard and risk assessment of LN towards daphnids and other aquatic organisms.

Introduction

The transition from a global economy dominated by fossil fuels to one based on renewable energy sources and the increasing digitalization of our society will both lead to a more intensive use of several mineral resources that are collectively termed critical raw materials [START_REF] Bobba | Critical raw materials for strategic technologies and sectors in the EU : a foresight study[END_REF]IEA, 2021IEA, , 2023)). Rare Earth Elements (REY), which include lanthanides (LN), Yttrium and Scandium, are needed in a large number of strategic, interlinked technologies and sectors such as wind energy generation, electric mobility (fuel cells and traction motors), robotics, drones, information and communication technologies at large, and medical applications [START_REF] Bau | Anthropogenic origin of positive gadolinium anomalies in river waters[END_REF][START_REF] Bobba | Critical raw materials for strategic technologies and sectors in the EU : a foresight study[END_REF]. The increasing demand for REY has raised a) geopolitical issues in relation to supply chains [START_REF] Bobba | Critical raw materials for strategic technologies and sectors in the EU : a foresight study[END_REF]IEA, 2021IEA, , 2023)), b) prospections for new resources across the globe [START_REF] Dushyantha | The story of rare earth elements (REEs): Occurrences, global distribution, genesis, geology, mineralogy and global production[END_REF][START_REF] Pell | Towards sustainable extraction of technology materials through integrated approaches[END_REF] ,c) concerns about the disruption of their natural biogeochemical cycles [START_REF] Hissler | Origin and Dynamics of Rare Earth Elements during Flood Events in Contaminated River Basins: Sr-Nd-Pb Isotopic Evidence[END_REF][START_REF] Kulaksız | Anthropogenic dissolved and colloid/nanoparticle-bound samarium, lanthanum and gadolinium in the Rhine River and the impending destruction of the natural rare earth element distribution in rivers[END_REF][START_REF] Lerat-Hardy | Rare Earth Element fluxes over 15 years into a major European Estuary (Garonne-Gironde, SW France): Hospital effluents as a source of increasing gadolinium anomalies[END_REF][START_REF] Louis | Spatial distribution of Rare Earth Elements in a transnational watershed: The case of the Danube River[END_REF][START_REF] Martin | Contrasting distribution of REE and yttrium among particulate, colloidal and dissolved fractions during low and high flows in peri-urban and agricultural river systems[END_REF], and d) concerns about the insufficient knowledge as to their potential environmental and human health risks [START_REF] Blinova | Potential Hazard of Lanthanides and Lanthanide-Based Nanoparticles to Aquatic Ecosystems: Data Gaps, Challenges and Future Research Needs Derived from Bibliometric Analysis[END_REF][START_REF] Gonzalez | Environmental fate and ecotoxicity of lanthanides: Are they a uniform group beyond chemistry?[END_REF][START_REF] Malhotra | An Updated Review of Toxicity Effect of the Rare Earth Elements (REEs) on Aquatic Organisms[END_REF].

Specific challenges arise when assessing REY ecotoxicity. The test media and experimental conditions (notably pH values) used in (standardized) ecotoxicity testing cause lanthanides to precipitate as insoluble chemical species such as carbonates, phosphates and hydroxides [START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF][START_REF] Gonzalez | Environmental fate and ecotoxicity of lanthanides: Are they a uniform group beyond chemistry?[END_REF][START_REF] Kang | Assessment of the biotoxicity of lanthanides (La, Ce, Gd, and Ho) on zebrafish (Danio rerio) in different water environments[END_REF][START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF]. The formation of such precipitates leads to variable exposure conditions during ecotoxicity tests, which makes the choice of exposure metrics particularly critical when deriving concentration vs. response relationships for hazard and risk assessment. Proposed solutions include modification of standard media to avoid, in principle, the formation of precipitates [START_REF] Aharchaou | Lanthanum and Cerium Toxicity to the Freshwater Green Alga Chlorella fusca: Applicability of the Biotic Ligand Model[END_REF][START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF][START_REF] El-Akl | Linking the chemical speciation of cerium to its bioavailability in water for a freshwater alga[END_REF][START_REF] Ma | Toxicity of cerium and thorium on Daphnia magna[END_REF][START_REF] Revel | Determination of the distribution of rare earth elements La and Gd in Daphnia magna via micro and nano-SXRF imaging[END_REF][START_REF] Vukov | Acute dysprosium toxicity to Daphnia pulex and Hyalella azteca and development of the biotic ligand approach[END_REF] or the choice of exposure metrics based on analytically measured concentrations at the end of the tests or based on geometric means between initial and final filterable concentrations [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF][START_REF] Vukov | Acute dysprosium toxicity to Daphnia pulex and Hyalella azteca and development of the biotic ligand approach[END_REF]. However, modifications in standard procedures should be consensus-based not to sacrifice reliability, sensu [START_REF] Roth | A critical review of frameworks used for evaluating reliability and relevance of (eco)toxicity data: Perspectives for an integrated eco-human decision-making framework[END_REF], for relevance (note that we mean no criticisms to the scientific validity of the studies having adopted this approach). Furthermore, modification of test medium does not necessarily avoid the formation of colloidal-sized precipitates at circumneutral pH values [START_REF] El-Akl | Linking the chemical speciation of cerium to its bioavailability in water for a freshwater alga[END_REF][START_REF] Revel | Determination of the distribution of rare earth elements La and Gd in Daphnia magna via micro and nano-SXRF imaging[END_REF]. Meaningful and informative comparisons among studies on LN ecotoxicity must therefore give the utmost consideration to LN behavior and speciation in the different test media and experimental conditions [START_REF] Gonzalez | Environmental fate and ecotoxicity of lanthanides: Are they a uniform group beyond chemistry?[END_REF].

Daphnia magna is a well-studied model filter-feeder used in standardized ecotoxicity testing [START_REF] Iso | norm 6341)[END_REF]OCDE, 2004). However, information on its responses following exposure to REY remains quite limited with a few acute ecotoxicity studies on selected LN [START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF][START_REF] González | Lanthanide ecotoxicity: First attempt to measure environmental risk for aquatic organisms[END_REF][START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF][START_REF] Ma | Toxicity of cerium and thorium on Daphnia magna[END_REF][START_REF] Revel | Determination of the distribution of rare earth elements La and Gd in Daphnia magna via micro and nano-SXRF imaging[END_REF] and a handful of works on chronic ecotoxicity, transgenerational effects and life-history traits [START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF][START_REF] Blinova | Long-Term Toxicity of Gadolinium to the Freshwater Crustacean Daphnia magna[END_REF][START_REF] Deng | Toxic Effects of Rare Earth Element Ytterbium and Neodymium on Daphnia magna[END_REF][START_REF] Galdiero | Cerium and erbium effects on Daphnia magna generations: A multiple endpoints approach[END_REF][START_REF] Lürling | Effects of lanthanum and lanthanum-modified clay on growth, survival and reproduction of Daphnia magna[END_REF][START_REF] Shu | Two-phase effects of environmentally relevant lanthanum on life-history traits of Daphnia magna and transgenerational bioenergetic profiles: Implications for nutritional and environmental consequences[END_REF]. Studies on the effects of selected LN mixtures are also available and suggest that LN mixtures could follow a concentration addition model for mixture ecotoxicity [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF][START_REF] Romero-Freire | Assessment of the toxic effects of mixtures of three lanthanides (Ce, Gd, Lu) to aquatic biota[END_REF]. However, to the best of our knowledge, there is no peer-reviewed investigation published to date that compares the effects of the full LN series including its 14 elements on D. magna.

Because lanthanides exhibit regular variation in their physico-chemical properties from Lanthanum (La) to Lutetium (Lu), ecotoxicologists have been looking for similar predictable patterns in ecotoxicity along the LN series or for ecotoxicological predictions based on physico-chemical properties [START_REF] Le Faucheur | Development of Quantitative Ion Character-Activity Relationship Models to Address the Lack of Toxicological Data for Technology-Critical Elements[END_REF]. The presence of ecotoxicological trends along the LN series implies that the effects of individual LN and their mixtures could be predicted from studies on selected LN. Apart from its fundamental importance, this scenario would result in considerable time and money savings without compromising reliability and relevance of ecotoxicological results; a highly desirable situation for both fundamental and applied research in this field. The few studies examining at least ten LN have identified both increasing and decreasing trends in their ecotoxicological effects along the LN series [START_REF] Blaise | Ecotoxicity responses of the freshwater cnidarian Hydra attenuata to 11 rare earth elements[END_REF][START_REF] Borgmann | Toxicity of sixty-three metals and metalloids to Hyalella azteca at two levels of water hardness[END_REF][START_REF] Lin | Lanthanides Toxicity in Zebrafish Embryos Are Correlated to Their Atomic Number[END_REF][START_REF] Manusadzianas | Ecotoxicity responses of the macrophyte algae Nitellopsis obtusa and freshwater crustacean Thamnocephalus platyurus to 12 rare earth elements[END_REF][START_REF] Weltje | Bioavailability of lanthanides to freshwater organisms. Speciation, accumulation and toxicity[END_REF]. Except for [START_REF] Weltje | Bioavailability of lanthanides to freshwater organisms. Speciation, accumulation and toxicity[END_REF], these observations require further confirmation because of missing or incomplete information on LN speciation in the exposure medium, a key factor when looking for trends along the LN series [START_REF] Gonzalez | Environmental fate and ecotoxicity of lanthanides: Are they a uniform group beyond chemistry?[END_REF].

The present study provides the first report on the acute ecotoxic effects of all LN to the model species D. magna according to the norm ISO 6341 (2012). The behavior and speciation of LN in the exposure medium are examined with an unprecedented level of detail to i) provide reliable and relevant data for hazard and risk assessment, ii) verify the existence of ecotoxicity trends along the complete LN series for this model organisms, and iii) summarize evidence-based recommendations to ensure maximum reliability and relevance in future research on the ecotoxicity of LN.

Experimental results show that a statistically significant linear relationship exists between the atomic mass and the solubility of LN in the standard ISO 6341 test medium, implying that reliable ecotoxicological endpoints must explicitly account for changes in LN concentration over the test duration. On the other hand, ecotoxicological trends along the LN series appear dependent on exposure time and the selected exposure metrics, highlighting the need for the definition of shared experimental and reporting procedures across the research community on this aspect. The most critical and pressing issues include further research on LN speciation in ecotoxicological test media and, for risk assessment purposes, in natural waters along with understanding the bioavailability and ecotoxicological potential of different LN chemical species.

Methods

Chemicals

All lanthanides salts (chloride or nitrates) were purchased from Sigma-Aldrich and had a minimal purity of 99% (Table S1). Chemicals for preparing the test medium for acute tests with Daphnia magna were also purchased from Sigma-Aldrich. Concentrated hydrochloric acid (HCl Suprapur 30%) and concentrated nitric acid (HNO 3 67%, Prolabo) were from VWR. Ultrapure water for washing/preconditioning of plasticware and preparing test medium and LN stock/working solutions was obtained from a Veolia Purelab® Chorus apparatus.

Ecotoxicity testing with Daphnia magna

Parthenogenetic specimen of D. magna were reared in Lefebvre-Czarda (LC)-Volvic medium (Table S2) at 20 °C and under a 16h light : 8 h dark photoperiod (light intensity 35 µmol/s/m²). The growth medium was changed three times a week and daphnids were fed an algal mixture of Chlorella vulgaris, Pseudokirchneriella subcapitata and Scenedesmus subspicatus (2.5, 5, and 2.5 × 10 6 cells/day/daphnia, respectively) after each medium renewal.

Acute immobilization tests (48 h) were performed according to international guideline NF EN ISO 6341 (2012) at 20 ± 2 °C in the dark. Daphnids less than 24 h old were used in all tests and were obtained from parent organisms aged from two to six weeks. The composition of the standardized ISO 6341 medium was: CaCl 2 × 2H 2 O (294 mg/L), MgSO 4 × 7H 2 O (123 mg/L), NaHCO 3 (64.7 mg/L), and KCl (5.75 mg/L).

In range-finding tests (data not shown), daphnids were exposed to a series of seven dilutions of all LN (except the radioactive Pm) at concentrations ranging from 0.05 to 45 mg/L of the corresponding salt (see Table S3 for details and the equivalent elemental concentrations). This range had to be extended up to 1,750 mg/L of LN salts to identify the appropriate test concentrations for the definitive tests with La, Nd, Sm, Tb, Dy, Ho, Er and Tm (Table S3). Preliminary tests were performed at an initial pH of 7.8 units, as recommended by the ISO 6341 norm, and of 6.5 units to reduce the visible precipitation of lanthanides carbonate species without compromising organisms' survival. In the preliminary tests performed at the initial pH value of 7.8, observed mortality did not span the 0-100% range required for optimal EC x estimations. Definitive tests were therefore carried out only at an initial pH of 6.5 units.

In definitive tests, minimum and maximum nominal exposure concentrations (expressed as elemental values) were 0.7 to 15 mg/L and 20 to 70 mg L -1 , respectively. A dilution series of nine or ten concentrations plus one control were used for each element (Table S4). Test tubes were preconditioned for 24 h with the corresponding test solutions to minimize adsorption losses on test vials. The preconditioning solutions were discarded before starting the experiments. Four replicate exposures (10 mL total volume) with five organisms each were used for all test concentrations. Immobilization of daphnids was evaluated after 24 and 48 h of exposure. Two independent definitive tests were performed for each element. The pH of exposure solutions was measured in control tubes at t = 0 (assuming homogenous initial pH for all exposure concentrations) and again in control tubes and selected exposure concentrations at t = 48 h (see section R.1). Potassium dichromate was used as a positive control with EC 50 24 h values between 0.6 and 2.1 mg/L of K 2 Cr 2 O 7 as recommended by the ISO 6341 norm (2012).

Analytical verification of exposure concentrations in the test medium

Analytical determinations were carried out by Inductively Coupled Plasma -Mass Spectrometry (ICP-MS, Nexion 350x, Perkin Elmer). The following isotopes were measured: 139 175 Lu. Rhenium ( 187 Re) was used as internal standard. Limits of detection (LOD) and quantification (LOQ) ranged from 0.1 to 0.8 µg/L (Table S5). LOD Analytical and procedural blanks were prepared and analyzed using the same analytical procedure as for samples. The accuracy of the whole analytical protocol was validated using international certified material (BCR-668-mussel tissues) from LGC standards. The recovery of LN ranged from 105 to 116 % (Table S5).

Actual exposure concentrations were analytically verified at t = 0 and 48 h for total (unfiltered) concentrations and at t = 0, 5, 24 and 48 h for filterable concentrations. Filtrations were performed with 0.45 µm syringe filters (Millex 33 mm diameter, mixed cellulose esters -MCE; Millipore, reference SLHA033SB, lot R7HA27577). The day before use, filters were washed with 10 mL of diluted (10% v/v) HCl, rinsed three times with 10 mL of ultrapure water and stored at 4 °C in the dark in a zip-bag with a few drops of MilliQ water to prevent drying of the membranes. Filtered and unfiltered solutions were collected in 15 mL falcon tubes previously preconditioned in two baths of diluted 10% v/v HNO 3 followed by two baths of milliQ water. The soaking time was 24 h in each baths. Except for t = 0 h, all solutions for analytical measurements were incubated in the presence of daphnids as described for ecotoxicity tests. The first 2 mL of filtered solutions were discarded and any living daphnids or visible debris were eliminated from the unfiltered samples before transfer into the falcon tubes. All samples were acidified at 1% v/v with concentrated nitric acid and stored at 4 °C in the dark pending analysis. Analyses at t = 0 and 48 h were performed in duplicate for both total and filterable concentrations. Measurement precision was estimated using the formula: [START_REF] Campbell | Biotic Ligand Model[END_REF] where m 1 is the first replicate measurement, m 2 is the second replicate measurement and abs is the absolute value of the difference.

Analytical verifications were carried out on solutions initially amended with a concentration corresponding to the 24 h EC 50 , hereinafter C EC50 , determined in the first round of definitive test. Tubes for the analytical verification of LN concentrations were incubated in parallel with the second definite tests and pH values were verified at t = 0 and 48 h for selected concentrations (see sections R.1 and R.2). For selected elements Lanthanum (La), Cerium (Ce), Gadolinium (Gd) and Ytterbium (Yb), total and filterable concentrations were measured also in solutions amended with the minimum (C min ) and maximum (C MAX ) test concentrations (Table S4). These additional measurements were performed in duplicate at t = 0, 24 and 48 h using solutions where daphnids were initially added (except at t = 0 h). The pH was checked in all the C min and C MAX solutions immediately before filtration and/or transfer to falcon tubes for analysis. Procedures for filter cleaning and removal of debris from unfiltered solutions were as previously described.

Speciation of lanthanides in the test medium

Speciation calculations were performed using VisualMinteq (VM) version 3.1. The precipitation of carbonate and hydroxide complexes was allowed as 'possible solid phases'. The default VM database specifies values for carbonate solubility products only for La, Neodymium (Nd), Samarium (Sm), Europium (Eu), Gd, Dysprosium (Dy) and Yb. Additional calculations were therefore performed using the carbonate solubility products determined by [START_REF] Firsching | Solubility products of the rare-earth carbonates[END_REF] who provided values for all lanthanides except Ce (Table S6). The value reported by [START_REF] Li | Preparation of nano-sized CeO 2 by mechanochemical reaction of cerium carbonate with sodium hydroxide[END_REF] was used for Ce 2 (CO 3 ) 3 solubility. Input temperature was fixed at 20 °C, medium alkalinity was specified as 0.831 meq/L CaCO 3 and the Debye-Hückel equation was used for activity correction. Calculations were performed in pH range 5 to 9 using increments of 0.2 units and for nominal concentrations from 10 ng/L (5.7-7.2 × 10 -11 M) to 50 mg/L (2.9-3.6 × 10 -4 M). The upper and lower limit of the molar concentration ranges refer to La and Lu, respectively. The nominal concentration range used for speciation calculation covered the range of tested and measured concentrations in the present study. For each element, theoretical solubility limits were estimated from speciation calculation by subtracting the percentage of precipitated concentrations from the corresponding nominal values.

Speciation was also modelled for solutions amended with concentrations corresponding to the 24 h EC 50 obtained in the first round of definitive tests for each element. In this case, the elemental concentration for VM runs was entered as the time-weighted mean (TWM) of the 24 h C EC50 to account for the concentrations decreases observed over the test duration (see the Results section for details). The TWM was calculated from the experimentally measured concentrations at 0 and 24 hours using the formula (OECD, 2008):

(2) where: Conc t(x) is the measured concentration at t = 0; Conc t(x=1) is the measured concentration at 24; Ln is the Napierian logarithm of the measured concentrations at any time 't'.

Because TWM concentrations are mathematical estimates, the corresponding pH values to specify for VM calculations were estimated as follows. Changes in pH were monitored between 0 and 48 h for several concentrations of all tested elements (Table S7). Except for Praseodymium (Pr), linear relationships existed between pH at 48 h and LN nominal concentrations. These relationships were used to estimate the expected pH values for the calculated TWM concentrations and for the concentrations measured at t = 24 and 48 h (Table S8). In the case of Pr, the pH value of the concentration closer to the TWM EC 50 was used. It will be shown later that pH changes were minor between 24 and 48 h.

Data analysis and statistics

The presence of statistically significant linear relationships between variables was verified using the standard Pearson least squares method and a robust MM estimation method that is less sensitive to outliers and non-normally distributed errors [START_REF] Susanti | M estimation, S estimation, and MM estimation in robust regression[END_REF][START_REF] Venables | Modern Applied Statistics with S-plus[END_REF]. The letters MM indicate that the estimation is of the maximum likelihood type, but do not form an acronym. Rather, they refer to the type of estimation procedure as explained in [START_REF] Susanti | M estimation, S estimation, and MM estimation in robust regression[END_REF]. Linear relationships were obtained with R version 3.6.0 (R Core Team 2019 R Core Team, 2019. R: A Language and Environment for Statistical Computing. R Foundation for statistical computing, Vienna. https://www.Rproject.org). Graphs were prepared using Sigma Plot (version 14).

RESULTS

Total concentrations in the test medium

Measured levels of LN in control solutions were always below the corresponding analytical detection limits (Table S5). The relative standard deviation (RSD) for replicate measurements of total LNs concentrations was less than 10% at t = 0 and usually within 30% at t = 48h. Total concentrations at t = 0 were within 20% of the expected nominal values except for solutions amended with Dy at C EC50 where measured values were 60% of the expected ones (Fig. 1 and Table S9). This discrepancy, possibly due to dilution errors, does not affect the objectives of the present paper nor the data interpretation presented below. At the end of the tests (48 h), total concentrations in solutions initially amended with C EC50 were within 25% of initial values for Nd, Tb, Ho, Tm, Sm and Lu, but had decreased by more than 40% for the other elements (Fig. 1A and Table S9). Total concentrations also decreased by more than 50% in medium aliquots spiked with C min of La, Nd, and Gd (6.55, 4.44 and 2.67 mg/L, respectively), but remained relatively constant for Yb (2.0 mg/L). In solutions spiked with C MAX , total concentrations (La = 53.5, Nd = 47.0, Gd = 39.4 and Yb = 29.6 mg/L) showed little variation over 48 h (Fig. 1B, Table S9).

Filterable concentrations in the test medium

In solutions amended with C EC50 , filterable concentrations decreased by 50-98% over the test duration according to three temporal patterns as described in figure 2A for selected elements and in table S9 and Figure S2 for the entire LN series. In pattern one (La and Ce), filterable concentrations decreased by more than 80% within 5 h from the start of the incubation and fell below 5% of the initial values after 48 hours. In pattern two (Pr, Sm, Eu, Gd, Er, Dy, Yb), the concentration decrease was less rapid and reached 70-95% after 48 hours. In pattern three (Nd, Tb, Tm, Ho, Lu), the overall concentration decrease was less than 50% with little changes between 24 and 48h. After 48 h, the differences between total and filterable concentrations were less than 2-fold for Nd and the heavier REE (Tb, Dy, Ho, Er, Tm, Yb, Lu), but between 3-and 34-fold for the lighter REE. The small differences for heavier REE suggest that these elements were either more soluble than the lighter REE or formed precipitates that were not retained by the 0.45 µm filters. At C min , the behavior of La, Nd, Gd and Yb essentially agreed with the observations at C EC50 , while filterable concentrations remained within 30% of the initial values for C MAX scenarios (Fig. 2B, Table S9).

Theoretical solubility of lanthanides in the test medium

The present section will focus on speciation results obtained using the carbonate solubility product (K SP ) established by [START_REF] Firsching | Solubility products of the rare-earth carbonates[END_REF], except for La and Ce. In the case of La, using the K SP of [START_REF] Firsching | Solubility products of the rare-earth carbonates[END_REF] clearly overestimated La solubility compared with experimental results. The default K SP in the VisualMinteq (VM) database was therefore preferred (Table S6). An indicative K SP for Ce 2 (CO 3 ) 3 was taken from Li et al. (2003), because no value for Ce was available in [START_REF] Firsching | Solubility products of the rare-earth carbonates[END_REF].

In the pH range from 6.0 to 7.8 (covering the values measured in ecotoxicity tests, Table S7), formation of insoluble carbonate species was predicted for all LNs and for concentrations as low as 10 µg/L (Fig. 3). Formation and precipitation of hydroxides, LN(OH) 3 , was also predicted for Er, Tm, Yb and Lu.

Lanthanides ecotoxicity

In the first round of definitive test, nominal ECs 50 ranged from 6.84 to 30.5 mg/L and from 4.70 to 15.7 mg/L after 24 and 48 h of exposure, respectively (Fig. 4a; Table S10). To account for the decrease in LN concentrations over time (section 3.1), the TWM EC 50 were calculated as follows. For the 24h TWM EC 50 , the experimental data presented in section 3.1 (i.e., the filterable concentrations at t = 0 and t = 24 h) were directly entered into equation 2. For the 48h TWM EC 50 , the LN concentrations at 48h were estimated using equation 5 assuming initial concentrations equal to the nominal 48h EC 50 (see Table S11 for a detailed explanation of the procedure and for calculation results). Note that the experimental measurements at 48h (section 3.1) could not be used for this purpose, because they refer to medium aliquots initially spiked with concentrations equal to the 24h EC 50 . For comparison purposes, the same procedure was also performed for the 24h TWM EC 50 estimated using equation 4 (Table S11).

The TWM 24h EC 50 calculated using experimental values differed from the nominal EC 50 by more than 2-fold for La, Ce, Pr, Sm, Eu, Gd and Dy (Fig. 4A). The 24h TWM EC 50 estimated using equation 4 were within 20 % of those obtained using experimental data for La, Pr, Eu, Tb, Ho, Tm, Yb, Lu and within a factor of two for the reaming elements except Nd (Table S11). Differences between nominal and TWM 48h EC 50 were more than 2-fold for La, Ce, Pr, Nd, Sm and Eu (Table S11). The observations confirm that TWM correction would avoid underestimating the ecotoxicity of La, Ce, Pr, Sm, Dy and, to a lesser extent, Eu and Gd (Fig 4A). For the second round of definitive tests, 24h and 48h EC 50 ranged from 10.5 to 35.6 S10, Fig. 4B). The TWM corrections were performed using equations 6 and 7 (Table S11), leading to results and conclusions equivalent to those discussed for the first round of tests.

DISCUSSION

Temporal changes in total LN concentrations

In the present study, total LN concentration at t = 0 h were in good agreement with expeceted values, but showed an unexpected decrease at the end of the test for several LN (Fig. 1 and Table S9). We cannot exclude that the decrease in total LN concentrations could partly originate from adsorption losses onto the tubes walls despite preconditioning of the test tubes. However, daphnids can actively ingest LN precipitates forming into the test medium and contribute to the observed losses, although the extent of such ingestion is element-dependent [START_REF] Revel | Determination of the distribution of rare earth elements La and Gd in Daphnia magna via micro and nano-SXRF imaging[END_REF]. The effect of LN addition on the pH of the test medium (Table S7) also determines the observed behavior at C EC50 and C min . Because test tubes were open to the atmosphere, medium pH increased by 0.5-1.0 pH units in most tubes over the test duration (Table S7). A similar situation was observed for tubes amended with C min of La, Nd, Gd and Yb, while addition of C MAX of the same elements resulted in pH values remained close to the initial value of 6.5 (Table S7). An inverse relationship between LN concentrations and pH was observed (Table S8) and reflects the formation of LN 2 (CO 3 ) 3 complexes in test solutions along with the release of protons according to the following equilibrium [START_REF] Luo | Carbonate complexation of yttrium and the rare earth elements in natural waters[END_REF]):

(3) Ln 3+ + nHCO 3 -= Ln(CO 3 ) n 3-2n + nH + where n = 1 or 2

As for alkaline waters in equilibrium with atmospheric CO 2 [START_REF] Luo | Carbonate complexation of yttrium and the rare earth elements in natural waters[END_REF], carbonate complexes are expected to dominate LN speciation regardless of the added concentrations. The formation and subsequent precipitation of such complexes consume the HCO 3 -/ CO 3 2-ions present in the test medium, reduce its buffering capacity, shift its pH toward more acidic values via the release of protons (equation 3) and eventually increase LN solubility at high concentrations (see section 3.3). At the end of the test, pH differences can exceed one unit across the exposure range (Table S7), indicating that LN speciation is not homogenous for all exposure concentrations. The ecotoxicological significance of such variability remains to be ascertained and will be further discussed in section 3.4.

Temporal changes in filterable LN concentrations

The decrease in filterable concentrations was always more marked than the corresponding total ones, suggesting the presence of insoluble LN particles large enough to be retained by the pores of 0.45 µm filters (see also section 3.3). Inverse linear correlations (equations 4 to 7) existed between the decrease in filterable concentrations at 24 and 48 h (expressed as % of initial concentrations) and LN atomic masses in solutions amended at C EC50 and C min (Fig. 5). Robust MM regressions showed a better fit compared with conventional partial least-squares regressions for the C EC50 scatterplots, while the performance of the two regressions was equivalent for C min (data not shown). For the sake of clarity, only robust MM regressions will be presented and discussed. The better goodness-of-fit at 24 h reflects the presence of elements showing little concentration decrease between 24 and 48 h (i.e. pattern three in Figs. 2 andS2). Regression slopes were higher for solutions amended with C min (equations 6 and 7) than with C EC50 (equations 4 and 5). These results indicate that i) the concentrations of lanthanides decreased faster in solutions amended with C EC50 than with C min and ii) the differences in concentration decrease between C min and C EC50 progressively became more important from La to Lu (see Fig. S1 for details and examples). In solutions amended with C EC50 , the observed percentage decrease in Nd concentrations (atomic mass = 144.24) was markedly lower than expected at both 24 and 48h (Fig. 5). We do not have a definite explanation for Nd behavior, but we notice that [START_REF] El-Akl | Linking the chemical speciation of cerium to its bioavailability in water for a freshwater alga[END_REF] reported the formation of Nd-colloidal particles with an approximate size of 60 nm in a non-complexing medium test medium for freshwater algae. Particles of such sizes can probably pass through the pores of standard 0.45 µm filters. However, it remains unclear while only Nd would form such small-sized particles compared with other LN. Some modelled % losses fall outside the 0-100 % range; i.e. the actual boundaries for experimental observations. These model artifacts do not invalidate the clear trend in increasing LN solubility from La to Lu in the ISO 6341 test medium for acute ecotoxicity testing with D. magna. The proposed linear models can be used to optimize analytical efforts to check for exposure concentrations in ISO test medium for D. magna over the test duration (at least for concentration up to C EC50 and with the possible exception of Nd).

Although decreasing total or filterable LN concentrations have been previously reported in standardized test media for D. magna or Daphnia spp. [START_REF] Barry | The acute and chronic toxicity of lanthanum to Daphnia carinata[END_REF][START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF][START_REF] González | Lanthanide ecotoxicity: First attempt to measure environmental risk for aquatic organisms[END_REF][START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF][START_REF] Ma | Toxicity of cerium and thorium on Daphnia magna[END_REF][START_REF] Vukov | Acute dysprosium toxicity to Daphnia pulex and Hyalella azteca and development of the biotic ligand approach[END_REF], the present study provides the first data of the different kinetics in concentration decrease across the whole LN series. Overall, LN solubility appears to be time and concentration dependent and a detailed follow up of changes in exposure concentrations over test duration is needed to correctly interpret LN ecotoxicity data. Caution is also required to understand the impact of adding large quantities of LN to the test medium. Indeed, LN concentrations remained within ± 30 % of the initial values in solutions amended with C MAX for La, Nd, Gd and Yb; contrary to what observed for C EC50 and C min (Figure 2A,B). Furthermore, the pH in solutions amended with C MAX remained around the initial value of 6.5 units or slightly decreased over time, again in contrast with observations of C EC50 and C min where pH increased to about 7.5 units over the test duration (Table S7 andS9). To the best of our knowledge, these are the first results considering the changes in pH during standardized ecotoxicity test with D. magna following protocol ISO 6341 (2012). These findings could also bear relevance for correct interpretation of chronic ecotoxicity tests such as the Daphnia magna reproduction test (OCDE, 2012). Depending on the tested LN, exposure concentration can decrease by up to 80% of initial values in five hours; a span much shorter than the 48 h among medium renewal usually adopted in chronic tests with D. magna.

Theoretical solubility of LN

At the initial pH for the definitive ecotoxicity tests (range from 6.4 to 6.6, Table S7), all LN concentrations were above the corresponding theoretical solubility except for the C min of Er, Tm, Yb and Lu (Table S12). The decrease in total and filterable LN concentrations over time (Fig. 1, Table S9) therefore agreed with the modelling results. However, the agreement between modelled and observed LN solubility was far from perfect, reflecting the limitations of thermodynamic speciation models for our experimental matrix and conditions.

In solutions spiked with C EC50 , measured filterable concentrations at t = 24 and 48 h were consistently higher than the corresponding theoretical solubility values in the pH range from 6.6 to 7.4 (Table S12). The pH range 6.6-7.4 covers the corresponding values estimated from concentration-pH relationships in solutions initially spiked with C EC50 (Table S8). The discrepancies between theoretical and measured concentrations could simply indicate that the system had not yet reached equilibrium at the end of the experiments. This explanation holds for La, Ce, Pr, Sm, Eu, Gd, Dy, Er, Tm and Yb. The filterable concentrations of these elements continuously decreased between 0 and 48 h (Fig. 2) and the trend could have continued beyond 48 h. On the other hand, the non equilibrium explanation does not apply to Nd, Tb, Ho, Tm and Lu whose filterable concentrations plateaued after 24 h while remaining above the corresponding theoretical solubility limits (Fig. 2). One possibility is that the measured filterable concentrations of these elements included colloidal-sized precipitates small enough to pass through the filter pores. The presence of colloids could have increased the apparent solubility of these elements compared with VM calculations that do not consider the particle size of the precipitates. Finally, the conditional nature of VM modelling parameters could also contribute to the observed differences for all LNs (Rodríguez de San [START_REF] De San | Conditional Equilibrium Constants Reviewed[END_REF].

The reasoning developed for solutions amended with C EC50 also applies to experiments using C min for La, Nd, Gd and Yb. In solutions amended with C MAX for the same elements, all daphnids died within 24 h and pH values at t = 48 h had dropped below the initial value of 6.5 (Table S9). Both factors likely contributed to the stability of filterable concentrations in these solutions (Fig. 2) even if the added LN levels initially exceeded the corresponding theoretical solubilities and precipitation could be expected (Fig. 3 and Table S12). Besides low pH values, the death of daphnids likely released different organic molecules into test solutions, possibly increasing LN solubility [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF].

Comparison of different exposure metrics for lanthanides ecotoxicity

The two rounds of tests gave equivalent results (i.e., overlapping 95% confidence intervals of nominal EC 50 ) for Pr, Nd, Gd, Tb, Ho, Tm, Yb, Lu and, at 24 h only, Sm (Table S10). In all other cases (La, Ce, Eu, Dy, Er, and, at 48 h only, Sm), EC 50 were higher in the second round of definitive tests than in the first one (Table S10, Fig. 4). Except for Eu (see below), we surmise that these differences were caused by the higher pH values of the exposure solutions during the second round of tests (Table S7).

In both experiences, pH values were measured at t = 0 h in control solutions (assumed to be representative of all exposure solutions at the beginning of the tests) and at 48 h for selected concentrations (Table S7). Measurements of pH after 24 h of exposure were not systematically performed during definitive tests to avoid additional stress to daphnids. However, available data showed limited pH variability between 24 and 48 h (Table S9).

Statistically significant inverse linear relationship were found between the measured pH values at 48 h and the initial LN concentration in the test medium (Table S8). These linear relationships were used to estimate the pH value in hypothetical aliquots of test media spiked with the corresponding nominal EC 50 (Table S8). Because pH controls the solubility of Ln2(CO3)3, a higher pH value at the end of the test (data in Table S7) implies i) a larger and probably faster decrease in the actual exposure concentrations over the test duration (see sections 3.2 and 3.3) and hence ii) higher EC 50 values in the second round of definitive tests. The pH explanation does not hold for Eu whose exposure solutions showed lower pH values for the higher EC 50 in the second test series (Table S7 andS8). We acknowledge that the proposed simple explanation does not account for kinetics processes and for confounding processes linked to the presence of daphnids in the exposure solutions.

Besides the formation of insoluble precipitates, expressing the EC 50 in terms of free Ln 3+ concentrations in the dissolved fraction can provide further insight into the ecotoxicity of LNs and increase the reliability and relevance of concentration-response relationships. Estimation of Ln 3+ concentrations was done for TWM EC 50 values using the same assumptions described in section 3.3 for the choice of stability constants for Ln 2 (CO 3 ) 3 precipitation. These estimations will be presented only for the first series of definitive tests.

In absolute terms, Ln 3+ based EC 50 were between 1 to 10 µg/L for La, Ce, Sm, Eu, Gd, Tb and Dy and in the tens to hundreds of µg/L for the remaining elements. Differences between Ln 3+ and TWM-based EC 50 were between 1 and 3 orders of magnitude at both 24 and 48h of exposure (Fig. 6). The actual ecotoxicological significance of such estimations depends on how well the constants used in the thermodynamic models are representative of the true LN speciation in the test medium. For example, estimates of free La 3+ concentrations were three orders of magnitude higher when using the La2(CO3)3 solubility product reported by Firshing et al. (1986) compared with the default value in the VisualMinteq database (Fig. 6). Further work is needed to experimentally measure the free Ln 3+ concentration in exposure medium. Considering the important quantities of LN salts added to the test medium (Table S3) and the tendency of test medium to get more acidic at high levels of added LN salts, it is important to verify the possible contribution of these two factors (pH and counterions added along with LNs) onto the survival of D. magna. Available reports [START_REF] Chen | Influences of Temperature, pH and Turbidity on the Behavioral Responses of Daphnia magna and Japanese Medaka (Oryzias latipes) in the Biomonitor[END_REF] and online article abstracts [START_REF] El-Deeb | Effects of pH on Survival, Growth and Reproduction Rates of The Crustacean, Daphnia Magna[END_REF][START_REF] Zhuang | The effects of low pH value on survival, growth and reproduction of Daphnia magna (Crustacea: Cladocera)[END_REF] suggest that effect of low pH become visible at values below 5.5 units, lower than those observed in the present study. In the case of the NO3-and Cl-ions, the maximum concentration added along with LN were 76 mg/L and 46 mg/L, respectively (information on the specific counterion for each LN salt can be found in section 2.1). In the case of nitrates, LC50 reported in the literature for D. magna are in the range 323-611 mg/L N-NO3-, i.e., 1,430-2,705 mg/L NO3 [START_REF] Scott | Acute and chronic toxicity of nitrate to fathead minnows (Pimephales promelas), Ceriodaphnia dubia, and Daphnia magna[END_REF]. In the case of chlorides, LC50 depends on the specific salt added to the test medium. Potassium chloride (KCl) has the lowest reported 48h LC50 of 660 mg/L, which corresponds to 314 mg/L of Cl- [START_REF] Mount | Statistical models to predict the toxicity of major ions to Ceriodaphnia dubia, Daphnia magna and Pimephales promelas (fathead minnows)[END_REF]. Based on this information, the toxic effects observed in the present study can be ascribed to exclusively to the added LN.

The EC 50 values based on nominal values (Fig. 4 and Table S10) agree with those observed in previous studies with daphnids and other crustaceans (see the recent data compilation reported in Table S5 of [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF]. Comparison based on measured LN concentrations or on modelled free ion concentrations are less straightforward because of the methodological differences among the studies containing such information. In the case of Nd, Gd, and Yb, our TWM EC 50 at 48 h (Table S11) are within the 95% confidence intervals of the values reported by [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF] who used the same test medium and analogous methodologies. However, our estimations for EC 50 based on modelled free-ion concentrations (Fig. 6) are three orders of magnitude lower than those of [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF] because of different assumptions in model calculations. In particular, we allowed the precipitation of Ln 2 (CO 3 ) 3 solids in the test medium and subtracted the precipitated concentrations from the total ones before calculating the free-ion concentrations. Both approaches can be acceptable depending on the experimenter's choice as to the complexation and solubility constants in the speciation model (see sections 2.x and 3.9 for further discussion). [START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF] did observe a marked decrease in La, Ce, Pr, No, and Gd concentrations in standard artificial freshwater (OCDE, 2004), but did not attempt to calculate 48h EC 50 based on measured concentrations. [START_REF] Vukov | Acute dysprosium toxicity to Daphnia pulex and Hyalella azteca and development of the biotic ligand approach[END_REF] reported a 48 h EC 50 of 0.49 mg/L Dy for D. pulex based on the concentration measured at the end of the text. This value is 10-to 20-fold lower than the 48h TWM EC 50 of the present study (Table S11). However, it compares favorably with the filterable Dy concentration of 2 mg/L that remained in our exposure medium after 48 hours (Table S9). [START_REF] Shu | Two-phase effects of environmentally relevant lanthanum on life-history traits of Daphnia magna and transgenerational bioenergetic profiles: Implications for nutritional and environmental consequences[END_REF] reported 24 h and 48 h EC 50 of 16.2 and 2.3 mg/L, respectively, for D. magna exposed to La according to OCDE guideline 202 (OCDE, 2004). These values referred to analytically measured filterable La concentrations at t = 0 (after 2h of equilibration following the initial La spike). They are higher than our EC 50 based on TWM (Table S11), but difficult to compare with nominal values considering the tendency of La concentration to rapidly decrease upon addition to the test medium (Fig. 1). Finally, [START_REF] Ma | Toxicity of cerium and thorium on Daphnia magna[END_REF] substituted NaHCO 3 with NaOH in the test medium for D. magna and successfully avoided carbonate precipitation when testing the ecotoxicity of Ce. As a result, the filterable (0.22 µm) Ce concentration showed little variation between the beginning and the end of the test. Their 48h EC 50 of 1.5 mg/L (based on measured concentration) is in excellent agreement with our 48h TWM EC 50 of 1.11 mg/L (1st series) and 1.64 mg/L (2nd series) (Table S11), showing that correction for concentration decrease can improve data comparability across studies. We are not aware of other studies providing EC 50 based on free ion concentrations for D. magna or other daphnids, apart from the mentioned comparison with [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF].

Trends in EC 50 along the lanthanides series

Ecotoxicologists are constantly seeking for the presence of regular, predictable variations in EC 50 along the LN series, in analogy with the predictable changes in physico-chemical properties such as ionic radius and ionization potential. Regular ecotoxicological patterns would allow to focus research efforts on a few representative lanthanides from which relevant information could then be derived for the whole series.

In the present study, nominal EC 50 values did not show any clear trend along the LN series after 24 or 48h of exposure (Fig. 4). On the other hand, linear relationships could be derived between the atomic mass of lanthanides and the EC 50 values expressed as TWM (equations 8 to 12, see Table S13 for the statistical significance of regression coefficients and calculation details). For 24 h exposure in the first series of experiments, two relationships were derived; one using TWM values calculated from equation 2 with analytically measured concentration at 0 and 24 h as inputs (equation 8) and one using TWM estimated from equation 4 (equation 9). On average, the predicted values differed by 30-40 % from the input TWM values, with differences larger than 50% for many elements (Table S14).

(8) R 2 = 0.29 (9) R 2 = 0.48 (10) R 2 = 0.48 (11) R 2 = 0.62 (12) R 2 = 0.51
Expressing TWM EC 50 in terms of free ion concentrations resulted a clear M-shaped pattern at both 24 and 48 h (Fig. 6). This pattern resembles the tetrad effect linked to the progressive filling of f orbitals along the lanthanides series [START_REF] Mclennan | Rare earth element geochemistry and the "tetrad" effect[END_REF] and is also similar to the changes in the energy of electronic configuration from La to Lu [START_REF] Schinzel | Structural and Electronic Analysis of Lanthanide Complexes: Reactivity May Not Necessarily Be Independent of the Identity of the Lanthanide Atom -A DFT Study[END_REF]. In particular, La, Gd and Lu have the largest differences between the energy of the highest occupied and lowest occupied molecular orbital and form the most stable complexes, which agrees with their higher ecotoxic potential (lower Ln 3+ EC 50 ) compared with adjacent elements (Fig. 6). However, the solubility products used in the present study (Fig. S3) also showed a M-shaped trend when plotted against the atomic mass of the corresponding elements.

Otherwise stated, the pattern observed in Ln 3+ EC 50 may simply be a model artifact linked to speciation calculation. Methodological developments are clearly necessary to obtain experimental measurements of LN speciation in ecotoxicological test media and verify the true meaning of this M-shaped pattern in Ln 3+ EC 50 .

Few studies examined the ecotoxic effects of the whole LN series and they reached contrasting results (Table 1). Data from [START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF] provide the most relevant comparison for the present study and suggest a linear decreasing trend for 48 h LC 50 to D. magna based on nominal values (La > Ce > Pr > Nd ≈ Gd). However, a strong decrease in LN concentration was reported over the test duration and the trend based on measured concentration could not be validated based on the information provided in the paper. Similarly, 21 d chronic LC 50 did not show any trend (La ≈ Gd > Ce ≈ Pr ≈ Nd). Decreasing ecotoxicity along the LN series has been reported for Hyalella Azteca, Hydra attenuata and Danio rerio based on nominal concentrations (Table 1). However, the heaviest lanthanides did not conform to the observed trends for H. azteca and H. attenuata (see footnotes in Table 1). These findings (i.e., a decrease in ecotoxicity along the LN series) agree with the observations of the present study, but the comparisons cannot be considered fully conclusive due to the missing analytical verification of exposure concentrations in these previous works (Table 1).

At the opposite, increasing ecotoxicity along the LN series was reported for the charophyte alga Nitellopsis obtusa [START_REF] Manusadzianas | Ecotoxicity responses of the macrophyte algae Nitellopsis obtusa and freshwater crustacean Thamnocephalus platyurus to 12 rare earth elements[END_REF] and the bacterium Aliivibrio fischeri [START_REF] Weltje | Bioavailability of lanthanides to freshwater organisms. Speciation, accumulation and toxicity[END_REF] (Table 1). In the very simple medium (0.355 M NaNO 3 ) used for testing with A. fischeri, free Ln 3+ were predicted to be the dominant species and showed the same trend as total dissolved concentrations (nominal values). However, total dissolved and free Ln 3+ concentration will likely have different patterns in more complexing media, especially those containing phosphates or carbonates. [START_REF] Gonzalez | Environmental fate and ecotoxicity of lanthanides: Are they a uniform group beyond chemistry?[END_REF] warned about establishing trends in the absence of detailed and complete information on LN speciation in the various test media used for different organisms. Until minimum analytical requirements are adopted by the whole ecotoxicological community, it will remain elusive to establish meaningful and generally valid ecotoxicity trends along the LN series. $, The existence of trends has been examined by the authors of the present study, but was not mentioned in the original publication.

‖, only for 24 d LC 50 (not for shorter exposure time); #, if Er and Lu excluded from the regression; §, if Tm, Yb and Lu excluded. §, Tm, Yb, Lu do not follow the trend

Proposal for a unified working framework for hazard and risk assessment of lanthanides

The results of the present study highlight a number of caveats that should be addressed to improve the reliability and relevance of laboratory studies on LN ecotoxicity. The proposed list of caveats (Table 2 and text below) considers only issues directly linked to the results and approaches used in the present study and does not claim to be exhaustive.

Caveat # 1. Formation of insoluble LN species during ecotoxicity testing

The ecotoxicological community is already well aware of the tendency of LN to form insoluble precipitates in ecotoxicological test media, especially in the presence of carbonates or phosphates [START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF][START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF][START_REF] Ma | Toxicity of cerium and thorium on Daphnia magna[END_REF]. Formation of Ce-bearing colloidal particles has been reported even in a non-complexing medium (0.01 M NaHEPES + 10 -5 M Ca(NO 3 ) 2 ) spiked with approx. 5 µg/L of Ce at pH between 6 and 8, with 40 to 70 % of the total concentration being retained by a 3kDa centrifugal ultrafiltration device [START_REF] El-Akl | Linking the chemical speciation of cerium to its bioavailability in water for a freshwater alga[END_REF]. It is therefore advisable to consider that insoluble LN species will form in any medium and take the necessary analytical steps to verify this hypothesis (Table 2, point #1). The use analytical verifications based on total concentrations (i.e., unfiltered samples) should however be discouraged because they do not distinguish between soluble and (nano)particulate LN species [START_REF] Vukov | Acute dysprosium toxicity to Daphnia pulex and Hyalella azteca and development of the biotic ligand approach[END_REF]. Furthermore, soluble species and precipitates do not necessarily contribute to ecotoxicity in the same way (Table 2, point # 3). The norm ISO14442 (ISO, 2006) provides useful recommendations that are applicable to the hazard assessment of elements tending to form insoluble precipitates such as LN.

Further research should also better characterize the particle size distribution of LN precipitates, because filtration at 0.45 or 0.22 µm will not detect the presence of colloidal precipitates smaller than the filter cut-off. Based on Single-Particle ICP-MS, El-Akl et al. ( 2015) estimated an average size of 60 nm for Ce particles formed in an exposure medium for short-term uptake experiments with Chlamydomonas reinhardtii. It is highly likely that part of the filterable concentrations reported in the present and previous studies actually include a certain proportion of particles having passed through the filter pores.

Measurements of pH values (ideally at multiple times during exposures) provide valuable additional information to i) ensure that LN speciation is similar across the chosen range of exposure concentrations in individual studies and ii) facilitate comparisons across studies. Even in a complexing medium such as the ISO 6341 used in the present study, the decrease in filterable LN concentrations was almost suppressed when the quantity of added LN salt was high enough to keep the pH at values below 6.5 units over the duration of the test (Fig. 1). Finally, modifications of exposure media are a possible alternative to avoid the formation of LN precipitates and even increasing the environmental relevance of the results [START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF][START_REF] Ma | Toxicity of cerium and thorium on Daphnia magna[END_REF]. These options would not eliminate the need for a thorough preliminary verification of LN precipitation in the chosen matrix across the range of exposure concentrations. Indeed, substituting NaCl for NaHCO 3 in ISO medium 6341 (i.e., the medium used in the present study without modification) did not prevent La and Gd filterable concentrations from decreasing by 50-98 % (La) and 40-70% (Gd) over the exposure period of 48 h [START_REF] Revel | Determination of the distribution of rare earth elements La and Gd in Daphnia magna via micro and nano-SXRF imaging[END_REF]. On the other hand, [START_REF] Ma | Toxicity of cerium and thorium on Daphnia magna[END_REF] apparently avoided the formation of Ce precipitates larger than 0.22 µm by substituting NaOH for NaHCO 3 in the same medium. Medium modifications should therefore be standardized among research groups to ensure that data across studies are comparable. A similar reasoning should ideally apply also to the procedures to determine filterable LN fractions [START_REF] Horowitz | A Review of Selected Inorganic Surface Water Quality-Monitoring Practices: Are We Really Measuring What We Think, And If So, Are We Doing It Right[END_REF][START_REF] Vignati | Comparative performance of membrane filters vs. high-surface area filtration cartridges for the determination of element concentrations in freshwater systems[END_REF][START_REF] Weyman | Aquatic toxicity tests with substances that are poorly soluble in water and consequences for environmental risk assessment[END_REF].

Caveat # 2. Variable exposure conditions for test organisms over the test duration

The results of analytical verification (see caveat # 1) will determine the choice of the appropriate exposure metric to calculate ecotoxicological endpoints. In principle, evaluation of LN ecotoxicity should not be based on i) nominal or measured concentrations at the beginning of the test, or ii) total concentrations that may contain a mixture of soluble and precipitated LN species [START_REF] Vukov | Acute dysprosium toxicity to Daphnia pulex and Hyalella azteca and development of the biotic ligand approach[END_REF]; present study). Both choices can lead to a marked underestimation of ecotoxic effects (Fig. 4). Measured concentrations at the end of the test would be a conservative alternative when exposure concentrations decrease over time. However, we recommend either TWM or geometric mean values as the best options. In the specific case of the test medium ISO 6341 (2012) for D. magna, future studies may rely on equations 4 to 7 to estimate LN concentrations at the end of the tests and provide EC 50 estimates as TWM 654

(present study) or geometric mean concentrations [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF]. 655 Ecotoxicity trends along the LN series may be artefacts related to methodological limitations and experimental choices Do not establish trends in the absence of minimum verification of exposure concentrations (see # 1 and 2) and Table 1.

True trends require determination of the actual LN bioavailable fraction.

7

Inappropriate comparison between exposure metrics from ecotoxicity testing and environmentally measured levels

Clearly report what is being compared. Consider the implications of the different exposure metrics from ecotoxicity testing in terms of possible underestimation or overestimation of the risk.

Matrix effects on LN speciation remains to be fully understood. Field-based studies using DGTs may provide hints on LN bioavailability in natural conditions. Reporting conventions to be defined.

From a fundamental point of view, the simultaneous presence of particulate and soluble LN species (and the changes in their relative abundances during the tests) deserve further investigations as to their respective contributions to the observed ecotoxic effects. In particular, it is not possible to establish beyond doubt if biological responses are caused solely by the free ionic LN species whose concentrations will vary during the tests and may be only a tiny fraction of total or filterable ones (Fig. 6). Using Diffusive Gradients in Thin Films (DGTs) could help in measuring the time-averaged labile LN species that are considered good proxies of the corresponding bioavailable fraction [START_REF] Gu | Rare earth elements in sediments from a representative Chinese mariculture bay: Characterization, DGT-based bioaccessibility, and probabilistic ecological risk[END_REF][START_REF] Paller | Assessing effects of dissolved organic carbon and water hardness on metal toxicity to Ceriodaphnia dubia using diffusive gradients in thin films (DGT)[END_REF]. This approach would not address the possible contribution of LN precipitates to the observed ecotoxic responses, though.

Caveat # 3. Unknown ecotoxicological role of precipitates

Revel et al. ( 2023) recently observed that particle formation was accompanied by different effects and tissue distributions in daphnids exposed for 48 h in medium aliquots initially amended with soluble La and Gd in the same concentration range. As discussed in caveat #1, both elements formed insoluble precipitates albeit La did so to a greater extent. Micro-and nano-XRF analysis showed that La concentrated almost totally in the digestive tract, while Gd was apparently taken up and distributed across different tissues. Only Gd caused a clear ecotoxicological response in the exposed organisms with 48h EC 50 of 13.9 mg/L (nominal value) or 8.22 mg/L (filterable concentration at the end of the test); comparable with the present study. The absence of La ecotoxicity suggests that precipitates were not available for uptake by internal organs, while the observed Gd effects cannot be univocally ascribed to soluble or precipitated forms.

Identifying the possible role of precipitates in LN ecotoxicity is related, but not equivalent, to understanding the ecotoxicity of nanoparticular LN forms. LN-bearing nanoparticles are engineered materials of known composition and primary particle-size distribution. This contrasts with the formation of precipitates following addition of soluble LN salts into the test medium. LN precipitates have variable composition, i.e., different types of precipitates can be present at the same time, and unknown particle size distribution. Their concentration may also change over the test duration depending on the stability of exposure conditions and on the solubility of the precipitates. Specific experimental approaches are required to identify the presence of precipitates and their possible contribution of precipitates to LN ecotoxicity in standard laboratory tests (Table 2).

Alternatively, exposure media could be aged after spiking with soluble LN forms and the precipitates removed by filtration, ultrafiltration or centrifugation before ecotoxicity testing in analogy to approaches proposed in the ecotoxicity of engineered nanoparticles [START_REF] Sørensen | Controlling silver nanoparticle exposure in algal toxicity testing -A matter of timing[END_REF]. Results from the present study suggest that formation of particles larger than 0.45 µm (nominal cut-off) becomes progressively less important along the LN series (Figs. 1 and2). However, the time to reach stable filterable concentrations varies among LN (Fig. 1), which may complicate studies on the effects of LN mixtures. The choice of the appropriate filter cut-off also requires further information on the particle size distribution of the precipitates. Such consideration may equally apply to chronic ecotoxicity testing because theoretical calculations suggest that the solubility limits could be as low as a few tens of micrograms per liter depending on specific elements and medium pH (Fig. 3 and Table S12). These solubility limits are comparable to, or lower than, the lowest chronic ecotoxicity values reported by [START_REF] Shu | Two-phase effects of environmentally relevant lanthanum on life-history traits of Daphnia magna and transgenerational bioenergetic profiles: Implications for nutritional and environmental consequences[END_REF] and [START_REF] Lürling | Effects of lanthanum and lanthanum-modified clay on growth, survival and reproduction of Daphnia magna[END_REF] for La (59 µg/L and 100 µg/L, respectively in artificial media), but higher than Ce and Er concentrations of 1 µg/L used by [START_REF] Galdiero | Cerium and erbium effects on Daphnia magna generations: A multiple endpoints approach[END_REF] to study multigenerational effects. Comparison with the chronic values determined by [START_REF] Blinova | Evaluation of the potential hazard of lanthanides to freshwater microcrustaceans[END_REF] for La, Ce, Pr, Nd and Gd (range 300 -490 µg/L) is hindered by the use of a natural lake water at pH 7.5 and containing 10.6 mg/L of dissolved organic carbon.

Caveats # 4 and 5. Using modelled free ion concentrations as an improved exposure metric

According to the scientific underpinnings of the biotic ligand model (BLM), ecotoxicological responses are controlled by the bioavailable elemental fraction that binds to specific biological ligands. The bioavailable fraction is mostly controlled by the the free ion concentration although contributions from other elemental species are possible [START_REF] Campbell | Biotic Ligand Model[END_REF]. While suitable analytical techniques are being explored [START_REF] Janot | Free Eu(III) Determination by Donnan Membrane Technique with Electrochemical Detection: Implementation and Evaluation[END_REF][START_REF] Leguay | Determination of the free-ion concentration of rare earth elements by an ion-exchange technique: implementation, evaluation and limits[END_REF][START_REF] Nduwayezu | Speciation of a lanthanide (Sm) using an ion exchange resin [10.1039/C6AY02018F[END_REF], free ion concentrations for LN are usually estimated using thermodynamic speciation models. This approach facilitates comparisons across studies using different exposure media and provides ecotoxicity estimation more relevant for risk assessment. However, it has some conceptual limitations that researchers should understand to avoid overstretching the environmental significance and implications of experimental results (Table 2, points # 4 and 5).

The choice of Ln 3+ as a metric of exposure assumes that only free ions contribute to the observed ecotoxic effects. In support of this assumption, [START_REF] Macmillan | Environmental Drivers of Rare Earth Element Bioaccumulation in Freshwater Zooplankton[END_REF] found good overall relationships between the modelled ∑REE free ion concentrations and REE accumulation by indigenous zooplankton in Canadian lakes. On the other hand, studies with a variety of model organisms provide contrasting evidence as to the bioavailability of Ln-containing particles formed in ecotoxicological media following the addition of soluble LN forms [START_REF] Aharchaou | Lanthanum and Cerium Toxicity to the Freshwater Green Alga Chlorella fusca: Applicability of the Biotic Ligand Model[END_REF][START_REF] El-Akl | Linking the chemical speciation of cerium to its bioavailability in water for a freshwater alga[END_REF][START_REF] Revel | Determination of the distribution of rare earth elements La and Gd in Daphnia magna via micro and nano-SXRF imaging[END_REF][START_REF] Vukov | Acute dysprosium toxicity to Daphnia pulex and Hyalella azteca and development of the biotic ligand approach[END_REF]. The problem is exacerbated when comparing Ln 3+ concentrations in ecotoxicological media and in natural waters.

Although insoluble precipitates can dominate LN speciation in the laboratory, LN in natural waters can be strongly associated with colloids and occurring as complexes with dissolved natural organic matter [START_REF] Janot | Free Eu(III) Determination by Donnan Membrane Technique with Electrochemical Detection: Implementation and Evaluation[END_REF][START_REF] Marsac | Modeling rare earth elements binding to humic acids with model VII[END_REF][START_REF] Martin | Contrasting distribution of REE and yttrium among particulate, colloidal and dissolved fractions during low and high flows in peri-urban and agricultural river systems[END_REF][START_REF] Pinheiro | Electroanalytical Trace Metal Cations Quantification and Speciation in Freshwaters: Historical Overview, Critical Review of the Last Five Years and Road Map for Developing Dynamic Speciation Field Measurements[END_REF]. Thermodynamic speciation calculations in the presence of natural organic matter still suffer from conceptual shortcomings that could lead to erroneous estimations of Ln 3+ in natural waters [START_REF] Tesfa | Absolute and Relative Positioning of Natural Organic Matter Acid-Base Potentiometric Titration Curves: Implications for the Evaluation of the Density of Charged Reactive Sites[END_REF][START_REF] Town | Rigorous Physicochemical Framework for Metal Ion Binding by Aqueous Nanoparticulate Humic Substances: Implications for Speciation Modeling by the NICA-Donnan and WHAM Codes[END_REF]. Similarly, speciation calculation in a given standard ecotoxicological medium (or across ecotoxicological media) will yield different results depending on the stability constants chosen for modelling by the experimenters as in the case of [START_REF] Lachaux | Implications of speciation on rare earth element toxicity: A focus on organic matter influence in Daphnia magna standard test[END_REF] vs. the present study. The choice to allow or not for the precipitation of insoluble species during speciation modelling (provided that the necessary solubility products are available) can also affect the comparability of free ion based metrics across different studies. Theoretical solubility limits can also vary markedly depending on the choice of solubility values for the possible precipitate species. In the present study, solubility limits could differ by over 10-fold when comparing calculations using the 'Firsching' and the 'Default VM' databases for carbonate solubility (Table S12), notably in the case of La. Finally, estimation of free ion concentrations based on nominal or total concentrations and filterable concentrations at the beginning of the test should be avoided for the same reasons discussed in caveat #1.

Caveat # 6. Identifying meaningful trend along the LN series

The previous caveats show that defining a generally valid ecotoxicological trend along the LN series remains elusive at the current state of knowledge, but also provide some hints to ensure that future research provides valid results for verifying the actual existence of such trends. In particular, results from studies not providing minimum analytical information on exposure concentrations (Table 1) should be considered of limited reliability for hazard and risk assessment purposes until further verification. Trends based on average, measured exposure concentrations (or free ion concentrations derived from such measurements) provide better information, but are also subject to further screening as more knowledge on LN speciation and bioavailability becomes available in both standardized test media and natural waters.

The results of the present study do not end the quest for the presence or absence of a regular, generally valid ecotoxicological patterns along the LN series in relation to their acute ecotoxicity toward Daphnia magna. They do however show a clear trend in the concentration decrease (Fig. 5), suggesting the best approaches to avoid underestimating LN ecotoxicity, especially in the case of light LN. The speciation calculation and discussion of the corresponding caveats provide a new starting point for future research on these topics. The necessary improvements in the hazard and risk assessment frameworks for LN are the subject of forthcoming work.

Caveat # 7. Comparing laboratory-derived exposure metrics with natural concentrations

Risk assessment is based on the comparison between the EC 50 derived from laboratory studies (hazard assessment) with the measured ambient concentrations in the case of site-specific investigations or probable environmental concentrations to establish environmental quality standard on a wider scale (European Commission, 2019). At the current state of knowledge, the utmost care is required to avoid both overestimation and underestimation of the potential risks arising from (increasing) concentrations of LN following anthropogenic impacts (Table 2). Laboratory exposure metrics derived from nominal concentrations (or concentrations measured at the beginning of the tests) would clearly underestimate risk. At the opposite extreme, comparing properly derived EC 50 with total elemental concentrations in natural water would likely overestimate the actual risk because LN bound to environmental particles will be considered equivalent to the dissolved (bioavailable) fraction. Studies attempting risk assessment should therefore be germane as to the analytical details of the concentrations being compared. An intriguing possibility would be to base risk assessment on comparisons of time-averaged, DGT-labile concentrations measured in both laboratory media and natural freshwater by extending approaches proposed for in situ ecological risk in sediments [START_REF] Gu | First attempt to assess ecotoxicological risk of fifteen rare earth elements and their mixtures in sediments with diffusive gradients in thin films[END_REF][START_REF] Gu | Application of diffusive gradients in thin films to determine rare earth elements in surface sediments of Daya Bay, China: Occurrence, distribution and ecotoxicological risks[END_REF].

Concluding remarks and way forward

Reliable exposure vs. response relationships are the foundation of hazard and risk assessment. Understanding the dynamic and complex speciation of LN is therefore a missing cornerstone in standardized ecotoxicity tests for these elements. In the absence of such information, it is not possible to choose the appropriate metric to link exposure to LN with the measured biological even in controlled laboratory conditions. Given the difficulties in obtaining stable exposure concentrations of LN in many test media, one may even question the ability of standardized methods to provide reliable results on the ecotoxicity of LN to aquatic organisms. However, similar issues would equally apply to New Approach Methodologies (NAM) such as in vitro screening, and to any test method unless they adopt test media allowing a thorough control of LN speciation. In our opinion, adressing the caveats discussed in section 4.6 will provide stronger basis for mutual acceptance of data across studies and help to reconcile standardized tests with the much more complex environmental reality [START_REF] Vignati | Reconciling standard procedures and environmental realism in ecotoxicology: conceptual and practical challenges[END_REF]. Similary, ecotoxicity trends along the LN series will remain conditional (i.e., dependent on the chosen exposure metric and expsure time, among the other variables) until precise information becomes available as to the LN form(s) capable of interacting with test organisms.

Figure 1 .

 1 Figure 1. (A) Total concentrations of lanthanides (LN) measured at 0 and 48 hours in solutions initially amended with LN concentrations equal to the corresponding EC 50 for D. magna. (B) Same as panel A for solutions initially amended with the maximum (LN M) and the minimum (LN m) concentrations of La, Nd, Gd and Yb used in ecotoxicity tests. Nominal values expected at t = 0h are indicated for comparison purposes in both panels.

Figure 2 .

 2 Figure 2. (A) Temporal changes in the filterable concentrations (< 0.45 µm) of La, Gd, Nd, Yb for solutions initially amended with lanthanides concentrations equal to the corresponding EC 50 at 24 h. The selected elements are representative of the three different behaviors across the whole lanthanides series (see text for details and Table S9 and Fig. S2 for full results). (B) Same as (A), but for solutions initially amended with maximum (MAX) and minimum (min) tested concentrations.

Figure 3 .

 3 Figure 3. Theoretical solubility of La, Nd, Gd and Yb as a function of pH in ISO 6341 medium for acute toxicity testing with Daphnia magna. Model calculations were performed for theoretical concentration from 1 µg/L to 50 mg/L. No precipitation was predicted in theoretical solutions amended with 10 and 100 ng/L (plots not shown).

  and 5.4 to 19.7 mg/L, respectively (Table

Figure 4 .

 4 Figure 4. Nominal and Time-Weighted Mean (TWM) EC 50 values for specimen of Daphnia magna exposed to lanthanides for 24 or 48 h. (A) first series of definitive tests and (B) second series of definitive tests. Bars represent 95% confidence intervals for nominal EC 50 (estimation not possible for TWM). Values for 24 and 48 h have been slightly offset for graphical representation purposes.

  Figure 5. (A) Scatterplots of percentage decrease in lanthanides concentrations vs. atomic mass at 24 and 48 h in solutions initially amended with LN concentrations equivalent to the corresponding C EC50 and (B) to the minimum tested concentration C min . Dotted lines are the corresponding linear fits (robust MM regressions -equations 4 to 7). Note that a 100% decrease means a filterable concentration equal to zero after 24 or 48h, while a 0% decrease means that concentrations did not change over time. See text and Figure S1 for the actual meaning of modelled values above 100% or below 0.

Figure 6 .

 6 Figure 6. Comparison between 24h and 48h Time Weighed Mean (TWM) EC 50 expressed as total filterable concentrations and free Ln 3+ concentrations. Free Ln 3+ concentrations were estimated using VisualMinteq (see section 3.3 for details). Calculations for free lanthanum (La 3+ ) are presented using two different La 2 (CO 3 ) 3 solubility products to invite caution when making ecotoxicological inference using modelled La 3+ values (see text for details).

  La, 140 Ce, 141 Pr, 146 Nd, 147 Sm, 153 Eu, 157 Gd, 159 Tb, 163 Dy, 165 Ho, 166 Er, 169 Tm, 173 Yb,

Table 1 .

 1 Summary of studies examining trends in ecotoxicity along the lanthanides series to aquatic organisms. ↓ or ↑ indicate a decreasing or increasing trend in ecotoxicity along the lanthanides series, respectively. [LN] measured refers to analytical verification of exposure concentration; Y, yes; N, no.

	Elements	Model organism	Trend (Y/N)	[LN] measured	Medium composition	Reference
	La, Ce, Pr,	Daphnia magna	Y $ (acute, nominal), ↓	Y	Acute test: OECD (2004)	Blinova et al. (2018)
	Nd, Gd		N (chronic)		Chronic test: Lake Water
					(pH 7.5 ± 0.2, DOC 10.6 mg/L C)
	All LN	Danio rerio	Y, ↓	N	According to OECD 203 (2019)	Lin et al. 2022
	La, Ce, Pr, Nd, Sm,	Hydra attenuata	Y # , ↓	N	Hydra medium	Blaise et al. (2018)
	Gd, Tb, Dy, Er, Lu				CaCl2, TES, pH 7
	(and Y)
	All LN	Hyalella azteca	Y, ↓ §	Y	Soft water	Borgmann et al. (2005)
	All LN except Pr, Ho,	Vibrio fischeri	Y, ↑	N	0.355 M NaNO3, pH 5.5	Weltje (2002)
	Yb
	All LN except	Nitellopsis obtusa	Y, ↑ ‖	N	Artificial Pond Water	Manusadžianas et al. (2020)
	Ho, Tm, Yb				KCl, NaCl, CaCl2 and HEPES (pH 7.5)
	All LN except	Thamnocephalus platyurus	N	N	Standard freshwater	Manusadžianas et al. (2020)
	Ho, Tm, Yb				NaHCO3, CaSO4, MgSO4 and KCl
	All LN	Cell lines of Danio rerio and	N	Y	Mixed medium	Fleurbaix et al. (2022)
		Onchorynchus mykiss
	La, Ce, Gd, Ho	Danio rerio	N $	Y	Standard dilution water	Kang et al. 2022
					CaCl2, MgSO4, NaHCO3, KCl
	All LN	Hyalella azteca	N	N	Tap water	Borgmann et al. (2005)
	All LN	Chlorella vulgaris	N	N	K2HPO4, KH2PO4, (NH4)2SO4, KNO3 (pH just below 7)	Den Dooren de Jong (1965)
	All LN	Skeletonema costatum	N	N	f/2 medium without EDTA: NaNO3, NaH2PO4, trace elements and	Tai et al. (2010)
					vitamins (pH 8.0)

Table 2 .

 2 Caveats, recommendations and open issues for properly testing LN ecotoxicity to Daphnia magna and aquatic organisms in general. The recommendations are applicable to other ecotoxicological media where LN precipitation may occur. Open issues indicate possible priorities for future research and consensus-building (also see TableS15for a more detailed presentation of each caveat). TWM, Time-Weighed Mean; LN, lanthanides.

	#	Caveats

Acknowledgments

This work was partly done with resources from the Pôle de compétences en biologie environnementale ANATELo, LIEC laboratory, UMR 7360 CNRS -Université de Lorraine. We are indebted to and Bénédicte Sohm (LIEC) for supervising routine laboratory functioning. We thank Armand Maul (LIEC) for statistical advice. We acknowledge the assistance of Danièle Pauly in maintainance of daphnids, reagent preparation and test organization.

This work is licensed under a CC BY 4.0 license (2023).

Data, scripts, code, and supplementary information availability

Data and supplementary information are available online: https://doi.org/10.57745/NW0GF9 (Vignati et al., 2023)

Funding

This work is part of the French National Research Agency (ANR) ECOTREE project (contract no. ANR-16-CE34-0012) and was partly supported by the Luxembourg National Research Fund (FNR) and the in the framework of the FNR/INTER/ANR research program (contract no. INTER/ANR/15/11209808/ECOTREE).

Conflict of interest disclosure

The authors declare that they comply with the PCI rule of having no financial conflicts of interest in relation to the content of the article. Davide A.L. Vignati is a recommender for PCI Ecotoxicology and Environmental Chemistry.