To systematically investigate and directly compare, for the first time, the sample stability of a large number of quantitative fecal immunochemical tests (FITs) at different storage conditions.Stool samples were obtained from participants of the German screening colonoscopy program between 2005 and 2010. After an initial FIT-based hemoglobin (Hb) measurement, stool samples were kept frozen at −80 °C until analysis. Twenty randomly selected participants with initial measurements ranging from 10 to 100 μg Hb/g feces were included. Ten quantitative FITs were investigated in parallel. A defined stool amount was extracted using each manufacturer’s brand-specific fecal sampling device and stored at 5 °C, 20 °C, and 35 °C, respectively. After 1, 4, 5, and 7 days, the samples were analyzed blinded. Median fecal Hb concentrations and positivity rates were calculated.Mean age of the participants was 67 years (range: 56–80 years) and 60% were male. The most advanced finding at screening colposcopy was advanced adenoma in five and non-advanced adenoma in eight cases. Hyperplastic polyps were found in two participants and five participants were without any findings. At 5 °C storage temperature, almost all FITs showed fairly stable results throughout the 7-day observation period. At 20 °C, most FITs still showed fairly stable results over 4 days, whereas positivity rates significantly declined from day 4 on for most FITs at 35 °C. Major differences regarding the sample stability between FITs were observed.FIT-specific Hb decay according to ambient temperature and time period between sampling and test evaluation requires careful consideration in the design of FIT-based screening programs. Randomized trials have demonstrated that screening with guaiac-based fecal occult blood tests can reduce CRC mortality by up to 30%4. Even larger effects should be possible with fecal immunochemical tests (FITs) for hemoglobin (Hb), which were shown to enable substantially better diagnostic performance7 and higher adherence rates in routine screening practice9. Therefore, FITs are meanwhile widely recommended and used in many countries as primary CRC screening tests12. Due to the growing market for FIT-based screening, a large number of FITs from diverse manufactures are meanwhile being offered13.Colorectal cancer (CRC) is the third most common cancer with approximately 1.4 million new cases and 700,000 deaths per year worldwide14. However, in this study, we evaluated the fecal samples under equal pre-analytical conditions, including identical sample storage temperature and duration. It is therefore unclear, to what extent diagnostic performance may be affected by potential differences of various FITs in Hb stabilization when the tests are applied under routine conditions, where variation in ambient temperature and time from sampling to analysis are the rule rather than the exception.Each FIT manufacturer uses a brand-specific fecal sampling device (FSD), which is a small vial containing an Hb-stabilizing buffer and a plastic stick for the collection of a defined amount of stool. In a recent publication from our group, we evaluated and directly compared, for the first time, the diagnostic performance of nine quantitative FITs and found very similar diagnostic performance among all tests, after adjusting the positivity thresholds to yield equal specificitiesWe therefore aimed to investigate and directly compare Hb decay and its potential impact on positivity rates (PRs) at various temperatures and sample storing times to be expected under routine screening conditions for the same nine quantitative FITs included in the previous examination plus an additional, previously not included quantitative FIT.15 and the FITTER  checklist16.This article is following the STARD (Standards for Reporting of Diagnostic Accuracy) statementThis project is based on the BliTz  study, an ongoing prospective study conducted among participants of screening colonoscopy. The study is carried out in cooperation with 20 gastroenterology practices in Southern Germany with the aim to collect blood and stool samples for evaluating novel non-invasive CRC screening tests. Participants of the German screening colonoscopy program are informed and recruited at a preparatory visit in the practice, typically 1 week before colonoscopy.20. The study has been approved by the Ethics committees of the University of Heidelberg and of the State Chambers of Physicians of Baden-Wuerttemberg, Rhineland-Palatinate and Hesse.Further detailed information on the BliTz study has been provided elsewheren = 2042). After giving written informed consent, the participants were asked to collect one stool sample from a single bowel movement, without any specific recommendations for dietary or medicinal restrictions, before starting the bowel preparation for colonoscopy. Participants were furthermore asked to keep the stool-filled container in a freezer or, if not possible, in a refrigerator at home until their colonoscopy appointment. Upon receipt, the stool-filled containers were kept  at −20 °C in the practice, then shipped on dry ice to a central laboratory and finally stored at −80 °C at the German Cancer Research Center (DKFZ). Median time from stool collection by the study participants to arrival at DKFZ and storage at −80 °C was 9 days (interquartile range (IQR): 6-14 days) .For this project, we considered study participants who were recruited from 2005 to 2010 and provided stool samples in 60ml-containers  in a previous analysis20. Out of these, seven participants had to be excluded due to an insufficient stool amount. Finally, 20 out of 28 eligible samples were randomly selected and included in this stability analysis.After excluding 530 participants who were already evaluated in the aforementioned previous study from our groupDetailed information on ten quantitative FITs is shown in Table n = 20) and stored at 5, 20, and 35 °C, respectively. Each FSD was a small vial, filled with a defined volume of brand-specific Hb-stabilizing buffer and containing a plastic stick for stool collection. After stabbing the collection stick into three different parts of the fecal sample, it was checked if the collection stick was optimally filled with a sufficient amount of stool. Then the stick with the collected stool was inserted back into the vial. The vials have a tight entrance which removes excess stool, leaving only a defined quantitative amount of stool on the stick. The only exception was the immoCARE-C vial, where a supplied custom-fitted scraper was used to remove excess stool from the collection stick. All FSDs were subsequently mixed on a vortexer, so that the stool could disperse into the preservative buffer to ensure optimal Hb stabilization. Afterwards, the respective FSDs were stored overnight at a temperature of 5, 20, and 35 °C, respectively.Figure n = 240 for each FIT brand) were labeled with a random number to ensure blinded analysis. After aliquoting, the filled tubes were immediately put back in the respective storage temperature  until their assigned day of evaluation.On the following day, all FSDs were homogenized in a vortexer, and the total volume of each FSD was equally divided into four 0.5-ml safe-lock tubes. Each of the four tubes was assigned to a single measurement on one of the chosen 4 days of evaluation. All tubes , only the tubes that were assigned to be measured on that day were taken out to room temperature for test evaluation.Test analyses of all FIT brands were conducted in parallel by laboratory-experienced staff and test calibrators, as well as test controls were performed on a regular basis according to the manufacturers' instructions. The storage temperatures were automatically recorded every 15 min using the temperature data logger.CAREprime Hb and immoCARE-C], Frost Diagnostika [ELISA Test Hb], Immundiagnostik [IDK Hb ELISA and QuantOn Hem], and R-Biopharm [RIDASCREEN Hb]) for immediate test evaluation upon arrival on the same day. The tubes which were stored at 5 °C until the evaluation was transported in a cold-chain, whereas the other tubes were transported without any cooling.Due to limited laboratory space and resources, six tests had to be evaluated externally. On the morning of each of the four measurement days, the test aliquots were packed in a temperature-isolated manner and directly shipped by one logistic company  to the cooperating companies providing the respective tests , the analysis on day 7 at 20 °C storage temperature was based on 19 samples because of a missing test measurement, whereas all other analyses for CAREprime  were based on the total sample size (n = 20).For one test .Exact The 20 participants included in this analysis were recruited between 2006 and 2009. The mean age was 67 years (range: 56–80 years) and the majority of participants were males (60%). The most advanced finding at screening colonoscopy was advanced adenoma in five and non-advanced adenoma in eight cases. Two participants had hyperplastic polyps and five participants were without any findings at screening colonoscopy.The median storage temperature (range) of the samples that were intended to be stored at 5, 20, and 35 °C was 6.1°C (8.4 °C–2.3 °C), 20.0 °C (17.8 °C–20.5 °C), and 34.7 °C (30.6 °C–35.2 °C), respectively. On the day of evaluation, the samples sent for laboratory analysis to the three locally located companies , arrived after a median delivery time of 1 h and 57 min, and the samples sent for laboratory analysis to CARE diagnostica arrived after a median delivery time of 3 h and 45 min. All samples were evaluated in parallel on the same day.QuikRead go iFOBT) was excluded from this analysis because the median Hb concentration was already at the lower detection limit of 15 µg Hb/g feces and therefore no further decrease could be assessed:ELISA Test Hb, IDK Hb ELISA, and SENTiFIT-FOB Gold), statistically significant decrease in Hb concentration was observed   indicated significant decrease in Hb concentration  , a significant decrease in Hb concentration  was observed. For ELISA Test Hb, IDK Hb ELISA, and Eurolyser FOB test, a significant Hb decay in comparison to 5 °C was observable from day 1 on, and for RIDASCREEN Hb and QuantOn Hem, from day 4 on   showed stable Hb concentrations across all combinations of storage conditions in comparison to 1-day storing at 5 °C , the PR decreased significantly by ≥30% units until the seventh day  , the PRs went down significantly by ≥30% units from day 4 on, in comparison to 1-day storing at 5 °C   showed constant PRs among all storage conditions, with differences in PR ranging from −20 to +5% units only   for which it was not possible to adjust the threshold to yield a PR of 85% because of their lower detection limits were not included in this analysis.ELISA Test Hb), the PR remained in the same range up to day 5, but significantly dropped to 20%  at day 7   ranging between 70% (46–88%) and 85% (62–97%)  among all days of storage. For one test  at day 7   ranging between 75% (51–91%) and 85% (62–97%) up to day 5, and between 65% (41–85%) and 80% (56–94%) at day 7, whereas the PR of  95% CI, –38% at dOC Sensor, immoCARE-C, SENTiFIT-FOB Gold, CAREprime Hb, and QuantOn Hem). However, for three tests , the PRs (95% CI) declined significantly to values between 35 (15–59%) and 50% (27–73%) at day 4 and to PRs (95% CI) between 10 (1–32%) and 30% (12–54%) at day 7   went down moderately to levels between 60% (36–81%) and 75% (51–91%) at day 7 for five of the tests , almost all FITs showed fairly stable results over 7 days. At 20 °C, most FITs still showed fairly stable results over 4 days, whereas PRs significantly declined from day 4 on for most FITs at 35 °C. Although all FITs used special FSDs that were filled with a brand-specific preservative buffer to slow down any Hb decay, partly, very large differences between the FITs regarding the sample stability were observed.24 found a significant decrease of the PRs during summer, compared to winter with the OC Sensor . Although the stool-filled FSDs in these studies were partly kept in a refrigerator before analysis, higher ambient temperatures during the shipment were enough to reduce the fecal Hb concentration substantially. In a previous study from our group, Chen et al.19 investigated the SENTiFIT-FOB Gold  and found only a slight and non-significant reduction in the PR according to season of sample collection. Therefore it was essentially unclear whether and to what extent the differences between different FIT brands regarding the sample stability existed.In agreement with our results, several studiesOC Sensor, van Rossum et al.25 found a substantial reduction of the PR, leading to reduced detection rates with a prolonged sample return time. By contrast, a study led by van Roon et al.22 did not observe any substantial reduction in the PR for the OC Sensor, with a prolonged sample return time. Because in these studies, the stool-filled FSDs were also partly kept in a refrigerator; it remained unclear to what extent prolonged storing at higher temperatures had an impact on the PR and if this is also transferable to other FIT brands.In another study also investigating the 26 directly compared three different FITs  and found a superior Hb stabilization ability for OC Sensor in comparison to the two other FITs. However, because the fecal samples from ten initially FIT-negative, healthy and young volunteers were artificially spiked with human blood; it was unclear to what extent these results are transferable to “real life” CRC screening participants, including patients with colorectal neoplasms. In our study, we used samples from 20 participants of screening colonoscopy including participants with advanced and non-advanced adenomas and found similar Hb stabilities between two of these FITs (OC Sensor and SENTiFIT-FOB Gold). In agreement with our findings, Guittet et al found relatively stable fecal Hb concentrations among all three FITs at cool storage temperatures, but a substantial decrease already at temperatures above 20 °C and also with prolonged storage times.In a study from France, Guittet et al.Our study has specific strengths which include the parallel evaluation of a large number of quantitative FITs using exactly the same stool samples of CRC screening participants. The study design essentially precluded any differences in study populations or sample handling as a cause of observed differences for sample stabilization between tests. All FITs were evaluated in exactly the same study participants who were recruited in a CRC screening setting among participants of screening colonoscopy. Stool samples were collected in exactly the same manner, and additional homogenization of the stool samples after thawing and prior to sample extraction for the individual FITs should further have eliminated any variation of fecal Hb concentrations within a single bowel movement. Furthermore, all collected stool specimens, using the brand-specific FSD, were stored in parallel under identical storage conditions (temperature and time) until few hours (during shipment) before conducting the FIT measurements on the respective days.n = 20) was rather low, leading to broad IQRs and 95% CIs, but despite that statistical differences between FITs regarding the sample stability were observed. However, to obtain 20 FIT-positive samples within a fecal Hb concentration range for which a potential Hb decay may turn a positive to a negative test result and therefore be highly clinically relevant, several hundred participants of screening colonoscopy are necessary to recruit. In theory, about 400 participants would be necessary to recruit, with each participant asked to collect three FSDs from ten FITs , of which “only” 20 FIT-positive stool samples (assuming a PR of 5%) would be included in the final stability analysis. Furthermore, despite the limited number of study participants finally included, this study is based on a total number of 20 × 10 (tests) × 4 (days) × 3 (temperatures) = 2400 FITs, and, to the best of our knowledge, the first and only one that provides a comparative evaluation of FIT stability across multiple FITs under fully comparable conditions.However, our study also has limitations. Stool samples were originally collected in stool containers (60 ml) rather than with the FSDs provided by the manufacturers and stored frozen at −80 °C over several years prior to analysis. Our study design though may have been the only way to conduct a parallel Hb stabilization study like this, as it is hard to imagine that screening participants would be willing and able to fill in parallel three FSDs for each of ten different FIT brands , each with different sample collection instructions. Furthermore, it would be almost impossible to ensure exactly the same temperature conditions for all FITs if not performing all the preparation steps at the same place. Although not used for the initial stool collection by the participants, the original FSDs provided by the manufacturers were used when extracting a defined amount of stool from the thawed stool samples, and the samples were stored in their respective preservative buffer systems during the study. Furthermore, the stool-filled FSDs were homogenized on a vortexer so that the stool could completely disperse into the buffer to ensure optimal Hb stabilization in the buffer. Following the manufacturers' instructions, the stool-filled FSDs were stored overnight in their original FSDs to enable a sufficiently long time period for any Hb to move out of the stool into the preservative buffer. Additional homogenization on a vortexer on the next day, just before aliquoting the total volume into the four safe-lock tubes, should have ruled out any variation of Hb concentration within the same FSD. Another limitation is that the number of stool samples  between 10 and 100 µg Hb/g feces. In most settings, the temperature is likely to vary over time, and constant temperatures around 35 °C or even 20 °C would not be expected in higher latitude countries, not even in summer. Therefore the Hb decay and the proportion of false-negative findings due to Hb decay would be expected to be lower in real settings in such countries than observed in our study. Also, false-negative results would be highly unlikely in rare cases of initial Hb concentration > 100 µg Hb/g feces which were not included in our study, but are typical among CRC patientsDespite its limitations, our study provides important information regarding sample Hb stability and its comparability for a large number of quantitative FITs that are increasingly used in CRC screening practice. A constant cool storage of stool-filled FSDs from sampling until analysis was shown to result in fairly constant PRs and fecal Hb concentrations throughout 7 days of observation. At 20 °C, most FITs still showed fairly stable results over 4 days, whereas PRs significantly declined from day 4 on for most FITs at 35 °C. To achieve constant PRs, which reflect the consequent colonoscopy workload, constant detection rates screening programs should aim at minimizing Hb decay by either keeping stool-filled FSDs in a cooled environment from sampling until evaluation, limiting the time period between sampling and test evaluation and/or selecting a FIT brand that combines good diagnostic performance with maximum possible stability even at prolonged sample return time at higher temperature. We hope that our results may help in guiding the design and monitoring of FIT-based screening programs and to stimulate further efforts to optimize FIT buffers in order to enhance and maximize Hb stability.A large number of fecal immunochemical tests (FITs) are meanwhile being offered for colorectal cancer screening.A decrease in FIT positivity rates at higher ambient temperatures has been reported, but it is unclear to what extent different FIT brands are affected.Ten different FITs were directly compared using stool samples from true screening participants.At 5 °C, almost all FITs showed fairly stable results throughout the 7-day observation period. At 20 °C, most FITs still showed fairly stable results over 4 days, whereas positivity rates significantly declined from day 4 on for most FITs at 35 °C.Major differences regarding the sample stability between FITs were observed.FIT-specific sample stability according to ambient temperature and time period between sampling and test evaluation requires careful consideration in FIT-based screening programs.Supplementary Tables


The aim of European water policy is to achieve good ecological status in all rivers, lakes, coastal and transitional waters by 2027. Currently, more than half of water bodies are in a degraded condition and nutrient enrichment is one of the main culprits. Therefore, there is a pressing need to establish reliable and comparable nutrient criteria that are consistent with good ecological status.This paper highlights the wide range of nutrient criteria currently in use by Member States of the European Union to support good ecological status and goes on to suggest that inappropriate criteria may be hindering the achievement of good status. Along with a comprehensive overview of nutrient criteria, we provide a critical analysis of the threshold concentrations and approaches by which these are set. We identify four essential issues: (1) Different nutrients (nitrogen and/or phosphorus) are used for different water categories in different countries. (2) The use of different nutrient fractions  and statistical summary metrics  currently hampers comparability between countries, particularly for rivers, transitional and coastal waters. (3) Wide ranges in nutrient threshold values within shared water body types, in some cases showing more than a 10-fold difference in concentrations. (4) Different approaches used to set threshold nutrient concentrations to define the boundary between “good” and “moderate” ecological status. Expert judgement-based methods resulted in significantly higher (less stringent) good-moderate threshold values compared with data-driven approaches, highlighting the importance of consistent and rigorous approaches to criteria setting.We suggest that further development of nutrient criteria should be based on relationships between ecological status and nutrient concentrations, taking into account the need for comparability between different water categories, water body types within these categories, and countries.  •We review the nutrient criteria used in Europe under the Water Framework Directive.•Different nutrients (nitrogen and/or phosphorus) used in different waters and countries•Different metrics used severely hamper comparability between countries.•A wide range in nutrient criteria values are found even within common types.•Approach to criteria setting has a significant effect on the nutrient criteria. HoweverIn Europe, the Water Framework Directive WFD;  was adopRecently, the focus has shifted from the assessment of ecological status towards identifying the management measures to reach good status . This haThis study aims (i) to provide an overview of nutrient criteria, including thresholds for good ecological status and approaches used to set these; (ii) to identify whether nutrient criteria currently in use actually support good ecological status and are consistent between countries with similar water bodies; and, (iii) to provide recommendations for further work.22.1Information on nutrient , which has resulted in the description of >1500 national types of water bodies across Europe , too manIn the case of coastal and transitional waters (CTW), reported national types were linked to regional ‘common types’ established for the intercalibration of the biological assessment systems (Tables S5 and S6).For rivers, nutrient criteria for 680 national types were reported and 80% of records were matched to 20 broad types; for lakes, nutrient criteria for 369 national types were reported, 73% of which could be matched to broad types, while for coastal and transitional waters 231 national types were reported, of which 60% were allocated to common types.Comparisons of nutrient criteria within broad types were carried out for those N and P parameters assessed by the majority of countries: total phosphorus (TP) and total nitrogen (TN) for rivers and lakes, and TP and dissolved inorganic nitrogen (DIN) for coastal and transitional waters. Most countries use mean (or median) values for nutrient criteria; however several countries use a 90th percentile summary metric. For comparison, the values of these percentiles were halved  as high, good and moderate status ;4.Distribution of nutrient concentrations in all water bodies – using this approach the nutrient criteria are defined from an arbitrary percentile of the distribution of nutrient concentrations from all water bodies .5.−1 in freshwaters is likely to be derived from the guideline value of 25 mg L−1 of nitrate in the Nitrates Directive (91/676/EEC) or now repealed Drinking Water Directive (80/778/EC).Expert judgement, including values taken from the literature or from older European Directives. For example, for nitrate, the common use of the value 5.65 mg-N L6.For coastal and transitional water, the so-called OSPAR Comprehensive Procedure is used widely. In this, a water body is considered to be an ‘Eutrophication Problem Area’ if actual status deviates 50% or more from reference conditions .Each country was asked to summarise information about the way that the good-moderate threshold concentration was set. For both freshwaters and saline waters a wide range of methods were used; for simplification these have been grouped into six main approaches:2.4Statistical analysis of reported threshold values was performed using R 3.5.3. . The sig33.13.1.1For lakes, all countries use TP and three additionally report soluble reactive phosphorus (SRP) , Table 3For rivers, the majority (24 countries out of 29) reported TP thresholds, singly or in combination with SRP. However, four only reported soluble reactive phosphorus (SRP) (Austria and Spain) or TRP (Ireland and United Kingdom). Twenty-two countries reported threshold values for N in rivers, most use nitrate-N (20 countries) rather than TN (13 countries) while four countries do not use N metrics in rivers . More co3.1.2In coastal waters, P is used for assessments by all countries in the Baltic, Mediterranean and the Black Sea, while four countries in the North East Atlantic use only N  . The lowest values were found in highland lakes, siliceous lakes, as well as large deep lakes and mid-altitude lakes, with most of the thresholds being <40 μg TP L−1  and Type 3  (18 to 300 μg TP L−1). This may reflect genuine natural differences in sensitivity or background nutrient concentrations of different lake types not captured within the broad type or different views of the TP concentrations required to support good status. The lake type with the highest boundary values is type 4, which is the lowland, calcareous, very shallow, unstratified lakes (from 20 to 300 μg TP L−1).For several broad lake types, a wide range of TP values was reported for the good/moderate threshold concentrations. The highest range was recorded for Type 6   (−1 (−1.TN good-moderate thresholds range from 0.25 to 4.0 mg TN L TN L−1) . In contL−1) . In general there was a less clear gradation of threshold concentrations across the broad types than for lakes, although the lowest range of threshold concentrations were found in some of the siliceous river types (median ≤ 50 μg TP L−1) and highest in calcareous river types (median ≥ 100 μg TP L−1).The reported TP threshold concentrations in rivers, in addition to being higher than those reported for lakes, were more variable across Europe, ranging from 8 to 660 μg TP L−1 . The medIn comparison to lakes, the majority of countries reported fewer P boundary concentrations, despite having as many or more types for rivers as for lakes. Nine countries have only a single  threshold which is applied to all river types, while five countries have just two threshold concentrations, despite having many river types.−1 to 35 mg TN L−1 (median 2.5 mg TN L−1). As for lakes, there are far fewer national N threshold concentrations than there are national types, with the same concentration often applied to several river types. The lowest TN threshold concentrations (for types with >4 countries) are found in Types 14 (the highland siliceous rivers) and 6 (lowland organic and siliceous), with higher values in the calcareous river types. However, there is more variation than for lakes, with a less obvious gradation from upland siliceous to lowland calcareous river types. Several types have outliers  and Type 9  has a very wide range of values, from 0.43 to 10.0 mg TN L−1.There is a wide range of good-moderate TN threshold concentrations , ranging−1 and 11.3 mg N L−1 by several countries, probably attributable to the guideline value for drinking water (25 mg NO3 L−1) from the now repealed Drinking Water Directive 80/778/EC and from the guideline value of 50 mg NO3 L−1 in the Nitrates Directive 91/676/EEC.The range of nitrate-N boundary concentrations (not shown) was much more variable and there is little indication of any clear relationship with the broad types. There is also a clear influence of the relatively widespread use of criteria values of 5.6 mg N L3.2.3−1; however most values are <25 μg TP L−1 (−1) and from Baltic coastal type BC1 (13–19 μg TP L−1), while the highest values were found in the Baltic type B5 (27–33 μg TP L−1) and the most variable in the Baltic type BC7 (14–38 μg TP L−1). For type descriptions see Table S5.TP threshold concentrations in coastal waters range from 9.3 to 44 μg TP Lg TP L−1 . The low−1), an area of oligotrophic water and several Baltic types, BC1 (0.10–0.18 mg N L−1), BC5 (0.11–0.15 mg N L−1). Four countries sharing common type NEA 1/26a show concentrations in the range 0.25–0.47 mg N L−1).For DIN, the lowest concentrations were reported from the Mediterranean type III E (0.03 N mg N L−1) and the highest in North East Atlantic transitional water type NEA11-140 μg TP L−1. Baltic coastal lagoons BT1 show the greatest heterogeneity of reported threshold concentrations, ranging from 26 to 120 μg TP L−1, with differences probably related to varying influence of freshwaters in the lagoons.For transitional waters, only a few countries have reported their threshold concentrations. The lowest concentrations are from Mediterranean estuaries (19–28 μg TP L−1) but this is not the case for Mediterranean coastal lagoons, shared by Greece and Italy (0.25–0.78 mg N L−1).The countries included in the transitional waters common type NEA 11 show very similar DIN threshold concentrations (0.42–0.49 mg N L3.3a concentration). This approach is less common for rivers, where the most commonly stated method is “expert judgement”. The distribution of nutrient concentrations in water bodies assigned a WFD status was the second most common approach for both lakes and rivers , followed by expert judgement (median 75 μg TP L−1) (−1) and regression (23.8 μg TP L−1) tend to have lower threshold concentrations . The same applies for lake N, with the highest threshold concentrations reported when the distribution of nutrient concentrations in all water bodies (median 4 mg TN L−1) or expert judgement (median 2.5 mg TN L−1) are used, and the lowest when regression techniques or classified water bodies (median 0.73 mg TN L−1) are used.Differences in threshold concentrations were apparent when grouped by the method used to establish the criteria. For lake P, significantly higher (p < 0.001) threshold concentrations are found when the distribution of nutrient concentrations in all water bodies was used (median 100 μg TP L TP L−1) . In cont−1) are found when the distribution of nutrient concentrations in all water bodies is used, followed by expert judgement (median 100 μg TP L−1). Significantly lower (p < 0.001) threshold concentrations are obtained when the distribution of nutrient concentrations in classified water bodies (60 μg TP L−1) or regression (median 45 μg TP L−1) are used.For river TP , the hig−1) is used. Significantly lower (p < 0.001) thresholds were obtained when the ecology was considered: regression method (median 0.68 mg TN L−1) and distribution of classified water bodies (median 1.5 mg TN L−1). No information was provided on the approaches used to set the highest TN threshold concentrations   .It was not possible to carry out this analysis for coastal and transitional waters due to the high heterogeneity of parameters and metrics used reported.44.1There is a widespread belief, arising from ideas developed several decades ago, that P limits primary production in freshwaters e.g. . This isHowever, the assertion that P alone limits primary production in lakes and that reducing P is sufficient to curb eutrophication e.g.  has beenIn coastal ecosystems, N is generally believed to limit primary production , leadingIn the Black Sea, both N and P play a role in the eutrophication processes  so, consIn the Mediterranean region, P is often the limiting nutrient, especially in the Eastern part , althougIn the North East Atlantic region, N is generally assumed to be the limiting nutrient . Some NoIn summary, an increasing body of evidence suggests that there may be drawbacks in relying upon a single limiting nutrient to achieve good ecological status, since limitation can vary both spatially and temporally in all water categories. However, as our study shows, assessment of N is neglected by many countries for inland waters, and P by several countries for coastal-transitional waters.4.24.2.1Our results show that TP is the most widely-used P parameter for lakes, mostly measured as annual or growth season mean. There is less consistency in rivers, with countries using TP, TRP or SRP, either annual or growth season mean or 90th percentile values.Similarly, TN is widely used in lakes while, for rivers, again, there is less consistency, with countries assessing only nitrate, only TN, or both forms together.Science, tradition and pragmatism all play a part in the choice of parameters e.g., to ensure the continuity of long-term water quality datasets, which is important for detecting trends. In lakes, the total nutrient fraction is generally used, as long water retention times typically result in only a very small proportion of P being in the soluble form; the majority being incorporated in planktonic algal cells. In addition, the potential for rapid recycling of this biological nutrient fraction  has meanIn many rivers, planktonic algae are less significant, the exception being large rivers where water retention time is sufficient to allow plankton communities to develop. In rivers, dissolved inorganic fractions of P and N represent a readily bioavailable fraction, in contrast to particulate and organic fractions, and are often a practical measure of nutrient pressure. This can be especially important in agriculturally-loaded rivers where stable particulate P, which is unavailable to support algal growth, may be the dominant fraction . There iWhile dissolved nutrient fractions have long been used for river monitoring and ensure continuity with historical records, there are situations where TP and TN may provide a more robust measure of nutrient supply. This might be case, for example, if low inorganic nutrient concentrations reflected high uptake and turnover rates in a water body that was, in fact, very productive .4.2.2Our study shows many differences in the nutrient criteria used by countries in the assessment of saline waters, both concerning the assessment period , parameters  and the statistical metrics . Not only are there differences between the countries, but there are also differences within the four marine ecoregions and even within countries between transitional and coastal waters.Key eutrophication assessment parameters are winter DIN and SRP concentrations  are used by most countries;•The European broad typology of 15 lake types and 20 river types encompasses most national types ;•Several studies have been carried out demonstrating the nutrient concentrations that support good ecological status .For lakes and rivers, the comparison was made possible by the following factors:4.3.1Comparison of threshold concentrations in lakes shows that differences are partly a result of different lake types: siliceous and upland lakes have lower criteria than lowland and calcareous/mixed or organic lake types, reflecting well-established differences in background P loadings to these lake types .However, there was a wide range of threshold values within lowland calcareous lake types . There is much less literature available for river nutrient criteria than for lakes, linked to numerous difficulties developing pressure-response relationships  drivers which regulate primary production . Moreove4.3.3•differences in the nutrient parameters assessed by countries ;•lack of broad types, leading to the use of common regional sea types, which are mostly shared by only a few countries .For coastal and transitional waters, comparison of nutrient criteria was severely hampered by several factors:Regional Sea Conventions have provided guidelines in an attempt to harmonize assessment among their members ; howeverHELCOM has provided also good environmental status thresholds for open sea sub-regions  but our −1 or 28 μg-P L−1 ; these values correspond to the good-moderate threshold for regional type BC4 (23 μg L−1). −1 for MED Type IA and 13 μg TP L−1 for MED Type II A, which are similar to the TP criteria set by countries sharing these types  presents significant problems that have not yet been fully solved . Our res4.4It is generally recognized that setting of nutrient thresholds should take into account biological responses to nutrient enrichment . Howevera concentration or cyanobacteria abundance  were reported where data-driven (modelling or regression) methods were used to establish criteria values. The highest  thresholds were reported when statistical distributions and expert-based methods were used. This corresponds to previous findings that expert-based nutrient criteria must be used with caution as they may lead to less stringent threshold concentrations and a consequent failure to protect water quality .3-N L−1 are derived from the guideline values of 25 and 50 mg-NO3 L−1 in the Drinking Water Directive (80/778/EC) and the Nitrates Directive (91/676/EC) respectively. However, the standards used for protection of drinking water supplies were not established with the objective of protecting good ecological status.For nitrate, the commonly-used values 5.65 and 11.3 mg NO•the best available information and knowledge are used to derive nutrient criteria;•the BQEs used to derive nutrient criteria are sensitive to nutrients;•the most appropriate statistical techniques are used; if regression is not feasible, categorical methods can be used ;•the resulting thresholds are broadly aligned with the wider body of published literature on nutrient limitation of primary production in different types of water bodies.Given the uncertainty of nutrient/biological relationships and errors associated with determining ecological status, the task of setting threshold concentrations is clearly difficult. However, it is important that:4.51.Where threshold concentrations are too high (=relaxed), the criterion may be achieved without any biological response being observed. In this case, water bodies consequently fail to achieve good ecological status based on the BQE, even though they may meet the prescribed nutrient criterion.2.Where nutrient criteria are based on a single element that is not the limiting nutrient (or which fails to account for P and N co-limitation); or on a nutrient fraction which is only a minor component of the total bioavailable nutrient pool and/or a poor indicator of overall nutrient supply (see above), there may again be no observed biological response. The Redfield ratio  offers a3.Where nutrient thresholds are too low (i.e. too stringent), a water body might be classified as moderate status, despite the BQE corresponding to good ecological status. In such cases, the mismatch may cause measures to be implemented that are not strictly necessary.There are several implications that arise from setting inappropriate nutrient thresholds•Inadequate intensity and targeting of restoration measures;•Legacies of past land use management, which have accumulated nutrient stores within the catchment and can continue to impair water quality over timescales from years to decades and more •Decoupling of algal growth responses to nutrient loadings caused by a variety of factors, such as luxury uptake of phosphorus by algae during periods of high P availability, grazing pressure, physical controls such as flow regime, light availability and temperature, as well as other pressures such as toxic substances and hydromorphological alterations;•Recovery trajectories which are non-linear and characterized by thresholds and alternative stable states.There may also be other reasons why good ecological status has not yet been achieved, despite implementation of restoration measures, but which are not a result of inappropriate nutrient criteria; for example see :•Inadequ55.11.Different nutrients (N and/or P) are used for different water categories, based on prevailing assumptions that P is the most likely to be the limiting nutrient in freshwaters while N is likely to be limiting in coastal waters. Therefore, only P is used to set nutrient thresholds for many river and lake types, and only N for some coastal and transitional water types. Recent research has highlighted that co-limitation is more common than previously assumed so the use of a single nutrient criterion should always be questioned.2.There are many differences in the nutrient parameters assessed , the assessment period , and in the metrics used , especially for coastal and transitional waters. These differences in nutrient criteria may hamper the comparison of threshold concentrations, definition of common objectives and a consistent management approach between countries and water categories.3.In order to ensure good status is attained, nutrient criteria should be derived from biological responses to nutrients. However, a wide range of methods have been used by countries to set nutrient criteria, and their relationship to good ecological status is not always obvious. There is evidence that different approaches to criteria setting are likely to produce different results. For both rivers and lakes, the lowest threshold concentrations are associated with data-driven methods , and the highest with expert judgement based methods .4.−1 and TN > 1.7 mg L−1), reaching as high as 660 μg TP L−1 and 35 mg TN L−1; (iii) countries using N criteria taken from drinking water standards, which were not intended to support good ecological status.There are large variations in the nutrient threshold concentrations used to support good ecological status within shared types, especially for rivers and some types of lakes and transitional waters. Some variation is expected due to natural variation within broad types; however, we identify three major challenges for developing consistent and effective nutrient criteria across European countries: (i) Many countries report a single nutrient threshold concentration to protect good status for all their river types. (ii) Some countries use threshold concentrations that are significantly above known limiting nutrient concentrations  and nutrient sensitive biological variables, taking into account the uncertainty of relationships and thus allowing a range of potential thresholds to be derived for specific water body types and circumstances .The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


BF4− anions were dominant in terms of concentration over TBA+ cations and the anion transfer results in the exclusion of the solvent molecules. TBA+ concentration variation in the electrode was small compared to that of the BF4− counterpart. However, Mw of TBA+ is much higher than BF4− (242.3 vs. 86.6 g·mol−1). Thus, TBA+ cations’ gravimetric contribution to the EQCM response was more significant than that of BF4−. Additional contribution of ACN with an opposite flux direction compared with BF4−, led to a net mass gain/lost during a negative/positive potential scan, masking partially the anion response. Such subtleties of the interfacial ion transfer processes were disentangled due to the complementarity of the EQCM and AC–EG methodologies, which were applied here for the characterization of electrochemical processes at the PEDOT NW electrode/organic electrolyte interface.PEDOT nanowires (NWs) directly grown on the conducting electrode of quartz resonators enable an advanced electrogravimetric analysis of their charge storage behavior. Electrochemical quartz crystal microbalance (EQCM) and its coupling with electrochemical impedance spectroscopy ( These p−1) are lower than those obtained with certain transition metal oxides [Through the doping process, charges are transferred to the polymer chains with an excess of electrons in the case of n–doping and a lack of electrons in the case of p–doping. Then, ions from the electrolyte are inserted within the polymer matrix to maintain electrical neutrality. As the conducting polymer electrode is charged and discharged, the ionic exchanges with the electrolyte should happen very reversibly without damaging the polymer structure and should persist during a long cycling period, leading to suitable material for pseudo-capacitors. The most common ECPs used in literature are poly(pyrrole) (PPy), poly(aniline) (PANi), poly(thiophene) (PTh) and their derivatives such as poly (PEDOT) ,3,4. Evel oxides ,6, extenl oxides ,3.−2 at 1 mA cm−2 and a capacitance retention of 94.3% after 10,000 GCD cycles [Despite the excellent electrochemical performances, one of the most important drawbacks of ECPs relies on their low cycling stability. Nanostructuration of ECPs represents a very promising strategy to overcome this hurdle through, as an example, the elaboration of nanocomposites made of ECPs and carbonaceous structures should be noted. More specifically, the great interest of ECP nanostructuration is also focused on one hand on the large pseudo-capacitance properties associated to their faradaic reactions and on the other hand on the large electrochemical active surface with an optimal ion diffusion path in the ordered nanostructure. According to the literature, various approaches have been already reported in this direction such as nanowires , nanotuD cycles , demonstD cycles ,17,18,19Since the morphology of the ECP nanostructures has a crucial influence to enhance ion transfers at the electrode/electrolyte interface and thus on their electrochemical properties, finding a convenient and high-efficiency synthesis method with suitable nanostructure is required. This nanostructuration can be achieved in various ways: (i) Covering an existing nanostructure , (ii) sy42− in the monomer solution during potentiostatic electrooxidation leads to a 10–20 nm thin non-conducting overoxidized polypyrrole (OPPy) film [42−, PPy NWs grow surrounded by OPPy film. More specifically, the trick consists in using an amphoteric species like hydrogenophosphate or hydrogenocarbonate in the presence of a classical electrolyte. The basic form of the former consumes the protons released during the electropolymerization so that polymerization stops at early stage. As a constant potential is applied at the working electrode, water oxidation takes the relay and produces hydroxyl radicals and oxygen gas bubbles. The hydroxyl radicals overoxidize the already deposited PPy, except where oxygen nanobubbles are produced. These O2 nanobubbles that protect the PPy film against hydroxyl radicals i.e., against overoxidation allows the electropolymerization of Py to continue at these places leading to PPy nanowires [4 salt containing acetonitrile electrolyte. Due to the coupling with impedance spectroscopy, AC–EG has the ability to provide complementary information on the interfacial processes. Besides identifying each species participating in the charge compensation process, it can provide a quantitative picture together with the kinetics of transfer of each species, thereby providing a kinetic and gravimetric deconvolution [Among the synthesis methods, template-free electropolymerization of ECP NW electrodes is becoming a mature process with many recent developments reported in literature ,13,24,25Py) film . It was anowires ,23,27. Aanowires ,29 and ianowires ,32,33,34These nanostructured PEDOT electrodes are of significant interest not only in energy storage  ,10 but a2) of quartz resonators  from an aqueous solution (bidistilled water) containing Electrode preparation and morphological characterization. Nanostructured PEDOT films were electrochemically prepared on the gold electrode (0.2 cmsolution . A Pt gr4), with a platinum grid as counter electrode and an acetonitrile-based Ag/Ag+ as reference electrode. The resonant frequency change, ∆f, of the quartz crystal was then converted into mass changes, ∆m, using the Sauerbrey equation  were calculated from the EQCM data where F is the Faraday’s constant, ∆m and ∆q were obtained from the QCM and the CV data, determined from the oxidation or reduction scan directions [Electrogravimetric characterization and evaluation of capacitive properties. EQCM measurements were performed in a solution of acetonitrile (ACN) containing 0.5 M tetrabutylammonium tetrafluoroborate (TBABFg−1⋅cm2) ). An Agig−1⋅cm2)  in orderrections .f corresponding to the mass response, ∆m, of the WE was measured simultaneously with the ac response, ∆I, of the electrochemical system. The frequency range was between 63 kHz and 10 mHz. The resulting signals were sent to a four-channel FRA, which allowed the electrogravimetric transfer function (TF) ∆m/∆E(ω), and the electrochemical impedance ∆E/∆I(ω), to be simultaneously obtained. For AC–EG , a four–Cs) was calculated using the following equation: Cs = Q/(ΔV × m), where Q is the average voltammetric charge, which is determined by integrating either the oxidation or reduction current of the corresponding CV curve, ΔV is the potential range, m is the active mass of the electrode. A total PEDOT NW mass of 28·μg cm−2 was estimated from QCM measurements. The areal capacitance was calculated taking into account the geometric surface of the electrode (A: 0.2 cm2) from the previous equation, where m is replaced by A.The specific capacitance  [W is the full-width at half-height of the resonance peak and Q, electroacoustic admittance measurements were performed. PEDOT modified quartz resonators were investigated in air and ACN electrolyte (The electrogravimetric behavior was studied in a solution of ACN containing 0.5 M TBABFthe QCM) ,34. HencM sensor ,40,41,42ctrolyte . A high + in 0.5 M TBABF4 in ACN. This media has the advantage of possessing a wide electrochemical window compared to aqueous electrolytes (42− or ClO4− or both) with those of the testing solution (BF4−). Previous X-ray photoelectron spectroscopy (XPS) studies on PPy NWs prepared with a similar method evidenced 96 at% of Cl and 4 at% of P presence, indicating the ClO4− as the main dopant [4− (Mw: 99 g·mol−1) with BF4− (Mw: 86 g·mol−1) can lead to a mass decrease during the stabilization of PEDOT films in TBABF4 containing ACN.After this verification step, EQCM measurements were conducted from −1.5 V to 0.7 V vs. Ag/Agtrolytes b. An impn dopant ,43. Sinc−1  was obtained at a scan rate of 100 mV·s−1. This value was found similar to other PEDOT-based nanowire system  [After this stabilization step, EQCM measurements were performed on PEDOT NWs at different scan rates: 100, 50, 20 and 10 m·Vs TBAClO4 . A large TBAClO4 , which i2 F·g−1) . PeriodiMPE calculated from the EQCM data could provide indications of the nature of the transferred species during cycling [MPE would be its molar mass. The cation and anion contributions lead to negative and positive signs of MPE values, respectively [MPE value of −70 g·mol−1 was calculated right after the oxidation peak for potential from 0 V to 0.7 V vs. Ag/Ag+ . This value was decreased down to around −10 g·mol−1 during the reduction process. The negative sign of the MPE corresponded to a predominant exchange of cations, but these values were lower than the mass of TBA+ cations (242.46 g·mol−1).The  cycling . If onlyectively . A mean 4−) or cations (TBA+) intervene during the doping/de-doping of PEDOT NWs but rather indicate a “mixed behavior”. Different scenarios could explain this result: (i) BF4− anions were expulsed at the same time as TBA+ cations were inserted, leading to a partial cancellation of the observed mass variations or (ii) an indirect participation of the solvent molecules to the charge compensation process. It is also noted that there was a slight change of the total mass variation, as a function of scan rate, , which may support the idea of multi-species contribution to the charge compensation with different kinetics. To unveil the mechanisms occurring in this pseudo-capacitive material and separate the contributions of the exchanged species, AC–EG was proposed to complement the EQCM data.Classically, charge compensation during the doping/de-doping of ECPs occurs with the insertion/de-insertion of the anions. In certain cases, the anions are trapped in ECPs and neutralized by the cations. In the latter case, during the doping (oxidation), these cations can be expulsed for charge compensation purposes. Our EQCM results did not provide a clear indication of whether solely anions , charge/potential TF (Δq/ΔE(ω)); derived from impedance, (ΔE/ΔI(ω)) and mass/potential TF (Δm/ΔE(ω)) were obtained from AC–EG, as described earlier [ac-electrogravimetric data obtained at 0.4V vs. Ag/Ag+. The charge/potential TF, Δq/ΔE(ω) presents one large depressed semi-circle, indicating interfacial transfer of multiple ions (iK represents the kinetics and iG describes the ease/difficulty of each species’ transfer at the electrode/electrolyte interface (fd is the film thickness and F is the Faraday’ constant). This fitting process indeed revealed the involvement of two different ionic species, which resulted in a good agreement between the experimental and the theoretical curves, without the possibility of their identification , was analyzed (q/ΔE(ω) TF in m/ΔE(ω) curve enveloped the transfer of different species in terms of quantity (as well as in terms of frequency domain where each processes occur). Two parameters (iK and iG) previously obtained from the charge/potential TF for each ionic species were used for the fitting of the Δm/ΔE(ω) response with the equation below [To do so, the mass/potential TF, Δanalyzed b, which on below :(2)ΔmΔE). The fitting procedure revealed a configuration involving the transfer of cations (TBA+), anions (BF4−) and solvent (ACN) molecules, which leads to a good agreement between experimental and theoretical data (4−) contribution and analyzing the residual response, which also shows a good agreement of the experimental/theoretical data (6 containing ACN electrolytes [+ appears at higher frequency domain than anions (BF4−), which are followed by free solvent (ACN) molecules. The latter have an opposite flux direction with BF4−, which may indicate that they are excluded from the PEDOT after the anions’ transfer  can be estimated with further calculation, , leading to Equation (4)):By using + cations were exchanged but in smaller quantities than BF4−. ACN contribution was quite significant compared with the ionic species in terms of concentration. As already explained, the solvent molecules were exchanged in the same flux direction with the TBA+ but in opposite flux direction with the BF4−. Since the solvent transfer occurs at low frequencies after the transfer of anions, the exclusion of the solvent molecules with the insertion of anions could be assumed, in agreement with previous reports in the literature, .ac-electrogravimetry). The Δtotalm obtained with ac–electrogravimetry (0.6 to 0 V vs. Ag/Ag+) was 280 ng·cm−2 (56 ng for 0.2 cm−2), which was in the same order of magnitude with the EQCM data obtained in the same potential range (−1), without consideration of the large hysteresis in the EQCM response. This strengthens the validity of the multi-species contribution revealed by AC–EG. In spite of a small concentration variation, TBA+ cations were much heavier than BF4− counterpart, thus TBA+ cations became significant, gravimetrically. Additional contribution of ACN with an opposite flux direction compared with BF4−, led to a net mass gain/lost during a negative/positive potential scan, masking the anions’ response. Using the concentration variations derived above, the mass variations induced by each species were calculated and shown in 4 containing ACN. Although the global EQCM mass response implies the major contribution of cations, the combination of the EQCM with AC–EG revealed TBA+, BF4− and ACN participating, directly or indirectly, in the charge compensation process with different kinetics and quantity. BF4− anions were dominant in terms of concentration over TBA+ and the anion transfer results in the exclusion of the solvent molecules. These results confirm that AC–EG is a very useful tool to operate a deconvolution of the EQCM measurements and unveil the ionic exchange mechanisms at the electrode/electrolyte interface.PEDOT NWs directly grown on the gold electrode of the quartz resonators enabled the electrogravimetric analysis of the charge storage behavior in TBABF


We report the ferroelectric, magnetic and ME properties of PFN/NZFO/PFN trilayer nanoscale heterostructure having dimensions 70/20/70 nm, at room temperature. The presence of only (00l) reflection of PFN and NZFO in the X-ray diffraction (XRD) patterns and electron diffraction patterns in Transmission Electron Microscopy (TEM) confirm the epitaxial growth of multilayer heterostructure. The distribution of the ferroelectric loop area in a wide area has been studied, suggesting that spatial variability of ferroelectric switching behavior is low, and film growth is of high quality. The ferroelectric and magnetic phase transitions of these heterostructures have been found at ~575 K and ~650 K, respectively which are well above room temperature. These nanostructures exhibit low loss tangent, large saturation polarization (Ps ~ 38 µC/cm2) and magnetization (Ms ~ 48 emu/cm3) with strong ME coupling at room temperature revealing them as potential candidates for nanoscale multifunctional and spintronics device applications.Multiferroic materials have attracted considerable attention as possible candidates for a wide variety of future microelectronic and memory devices, although robust magnetoelectric (ME) coupling between electric and magnetic orders at room temperature still remains difficult to achieve. In order to obtain robust ME coupling at room temperature, we studied the Pb(Fe Due to mutual exclusiveness and frequent chemical incompatibility between magnetism and ferroelectricity, few single-phase multiferroic materials exist in nature, with notable examples such as BiFeO3, YMnO3, Pb(Fe0.5Nb0.5)O3, Pb(Fe0.5Ta0.5)O3, Pb(Fe0.67W0.33)O3, TbMnO3, etc10. The most well-studied is the lead-free single-phase multiferroic is BiFeO3 (BFO), having both ferroelectric (Tc = 1143 K) and antiferromagnetic (TN = 643 K) phase transitions above room temperature. However, it is still not envisioned for practical device applications due to high leakage current and low ME coupling. Unfortunately the majority of the other single-phase multiferroic materials are only multiferroic at cryogenic temperatures (magnetic/ferroelectric transitions are below room temperature). The ME coupling is also observed to be weak due to large independency of the magnetic and ferroelectric ordering in single-phase multiferroics9. An alternative route towards the goal of strong room-temperature ME coupling is to explore heterostructures composed of alternatively ferroelectric and ferromagnetic layers, or through development of composites10. The development of sophisticated thin film growth techniques has allowed the fabrication of ultra-high purity atomic-scale thin films free from extended defects, allowing a platform for the exploration of ME coupling in epitaxial heterostructures. Moreover, researchers have proceeded to design and develop artificial composite nanostructures to achieve large polarization and magnetization with strong ME coupling above room temperature9. The obvious choice for a high figure of merit ME nanostructure is a multilayer structure with an atomically smooth interface between the magnetic and ferroelectric layers. Magnetic materials with high magnetostriction, high Curie temperature (Tc), and large magnetization, as well as ferroelectric materials with large polarization, dielectric constant and piezoelectric coefficients, are required to produce strong ME coupling in the heterostructures11. Achieving a large ME coupling in thin films with important implications for device applications is a long-standing scientific challenge9. In heterostructures, the spinel ferrites are widely adopted as the magnetic candidates due to their good thermal, chemical, and structural stabilities along with high resistivity12. The ME coupling may arise due to coupling between electrical and magnetic order parameters or due to cross coupling between magneto- and electro-striction and magnetic order parameters or indirectly via lattice strain and interfacial electronic reconstruction (charge coupling)9. For composites, the ME effect is a product tensor property of the piezoelectric and piezomagnetic coefficient that results from the cross interaction between the two phases11.Multiferroic magnetoelectric materials exhibiting simultaneous ferroelectric and ferromagnetic order parameters with the control and switching of the magnetic order parameter by electric field and vice-versa are the subject of cutting-edge research as they are promising for next generation nanoscale electronic and memory devices22. Strong ME coupling has been observed in different layered composite structures such as: trilayers of BaTiO3/BiFeO3/BaTiO3, bilayers of PbZr0.57Ti0.43O3/NiFe2O4, bilayers and superlattices of Ba0.7Sr0.3TiO3/La0.7Sr0.3MnO3, PbZr0.2Ti0.8O3/La0.8Sr0.2MnO3 heterostructures, PbZr0.57Ti0.43O3/CoFe2O4 bilayer and multilayers and BaTiO3/CoFe2O4 multilayer heterostructures24. The route to understanding the ME coupling has typically proceeded via first-principle approaches which have yielded a wealth of essential information on the complex interplay of spin-dependent interfacial charge transfer, chemical bonding, and electrostatic screening in these systems18. Theoretical calculations of the strain-mediated ME coupling necessitate the determination of three factors: (i) the deformations induced in the magnetic phase by the applied magnetic field; (ii) the degree of strain transmission through the interface between the two ferroic constituents of the hybrid system; and (iii) the response of polarization and dielectric susceptibility of the ferroelectric phase to changes in the lattice strain. In epitaxial heterostructures, the mechanical coupling between the two phases is very strong due to possible perfect mechanical strain transmission25.Several theoretical and experimental investigations have been reported that achieve large ME coupling for various composite bulk and thin film structures0.5Nb0.5)O3 (PFN)/Ni0.65Zn0.35Fe2O4 (NZFO)/Pb(Fe0.5Nb0.5)O3 (PFN) trilayer heterostructure has been chosen for the present study to observe enhanced ferroelectric and magnetic properties with strong ME coupling above room temperature. PFN is a well-known multiferroic material with a ferroelectric Tc between 379–385 K having high dielectric constant (~3000 at RT), low loss tangent (0.01) and high piezoelectric coefficient (d33 ~ 145 pC/N); it also shows strong ME coupling near its TN ~ 150 K29. Recently the existence of ferroelectric, ferromagnetic, and ferroelastic properties in PFN with interesting ferroelectric and magnetic properties have been reported30. Nickel zinc ferrites (NZFO) are soft magnetic materials with high saturation magnetization, low coercivity, high resistivity, reasonable magnetostriction, low dielectric losses, high dielectric constant and high magnetic Curie temperatures. Ni0.65Zn0.35Fe2O4 has been chosen for the present work as this composition exhibits the combination of highest saturation magnetization (86 emu/g), high resistivity (~2 × 106 Ω cm) and high magnetostriction (106 λ100 = −34) in the entire Ni−Zn series with a magnetic Tc of ~663 K33.The Pb buffered (LaAlO3)0.3 (Sr2AlTaO6)0.7 (LSAT) substrate recorded at room temperature. The θ−2θ large angle X - ray scans (20° to 80°) showed only diffraction peaks from the substrate and (00l) pseudocubic reflections from the heterostructure confirming that these films are highly oriented in nature. The peaks with symbols *, # and & correspond to the diffraction peaks of LSAT/LNO (overlapped), PFN and NZFO, respectively. The epitaxial character and the confirmation of the layered nature within the heterostructures were also confirmed by TEM studies (see below). We did not observe any extra reflection peaks that would be indicative of secondary phases and peaks from lead-deficient pyrochlore phases suggesting the high purity of the grown films. All of the observed peaks were assigned to PFN and NZFO, corresponding to perovskite phase with the monoclinic and cubic phase crystal structure respectively34. In order to explore the effect of strain on the ferroelectric, magnetic and magnetoelectric properties of this heterostructure, the effective misfit strain was calculated35. Note that the LSAT substrate has a cubic crystal structure with the lattice parameter a = 3.868 Å, whereas bulk LNO has a rhombohedral crystal structure with the lattice parameter a = 3.838 Å. The LNO electrode has been used in our case as an oxide electrode which is closely lattice-matched with substrate. It was calculated that LNO on LSAT experiences a small in-plane tensile strain of 0.78%. The peak positions of PFN and NZFO in the heterostructures have been compared to the stress free bulk values of PFN and NZFO. The individual peaks of the heterostructures are found to be shifted towards lower angle compared to the stress free bulk samples. The effective misfit strain experiencing by PFN and NZFO layers are found to be −1.02% and −0.91% respectively.In order to obtain knowledge on the phase purity, crystalline quality, interface and structural information in detail, the films were investigated utilizing high-resolution XRD. Figure μm2, lacks granular structure, with average surface roughness (Ra) and root mean squared roughness (Rrms) being ~1.62 and 2.05 nm respectively. It is clearly seen that the upper surface of heterostructure is smooth and very homogeneous, free of microcracks, pores or holes. The smooth surface with low roughness is required for improvement of functional properties. Figure The inset of Fig. 39. Therefore, detailed studies of the interface structure are essential to understand many novel physical phenomena observed in heterostructures. Energy dispersive X-ray (EDX) in conjunction with transmission electron microscopy (TEM) was used to study the crystal structure, strain distribution, and local chemical composition of the heterostructure respectively. Figure l) - oriented PFN/NZFO/PFN heterostructures grown on LNO buffered LSAT substrate. The heterostructure exhibits one NZFO layer of thickness ~20 nm sandwiched between two PFN layers of thickness 70 nm, with total film thickness of ~160 nm. The layers in this heterostructures are distinctly separated from each other; darker bands correspond to the PFN layers and the brighter one to the NZFO layer having sharp and well-defined interfaces as seen from the TEM image. The distinct contrasts of the PFN image represent the domains separated by domain walls. Selected-area electron diffraction (SAED) patterns were collected corresponding to each layer and confirms the epitaxial growth of the layers in this heterostructure. The SAED patterns shown in Fig. l00), as seen in the XRD patterns. The measured lattice spacing from SAED pattern for PFN is 4.065 Å corresponding to the (100) planes whereas measured lattice spacing for NZFO is 2.20 Å corresponding to the (400) planes which are in agreement with the existing literature. Scanning transmission electron microscopy - Energy dispersive X-ray (STEM-EDX) were also used to analyse the existence of all individual elements  with their required stoichiometry of the PFN and NZFO layers in the heterostructure.The multiple interfaces that exist in the heterostructures can lead to carrier confinement effects, magnetic interactions, and lattice strain effects at the interface that play an important role in determining the physical and functional properties of the material40. Of more interest in this case is to probe the local variability of ferroelectric properties, which may be important to understanding the device properties and potential routes towards optimization of the field-induced strain response, Band Excitation PFM (BE-PFM) measurements have been carried out as shown in Fig. 42. It may be noted that PFM is only measuring the top layer of the heterostructure, with resolution typically ~20 nm (the contact radius of the tip) for each grid on the sample. The piezoresponse was measured as a function of tip voltage in the in field (VDC on) and out of field (right after switching VDC off), regimes as shown in Fig. +, R−, V+, V− and work of switching for cycle 2 are found to be 0.095 × 10−3, −0.744 × 10−4, 5.16 V, −4.933 V and 0.00171 respectively. Where R+, R− represent the remanent polarizations and V+, V− represent coercive bias. The on-field response generally includes stronger electrostatic effects, and is noticeable via the change in loop orientation. The off-field response resembles that measured for our pure PFN thin film reported earlier, and shows minimal variations by cycle. As PFM spectroscopy gives the advantage of functional mapping42 of the sample at the nanoscale, the distribution of ferroelectric loops (ave. piezoresponse vs voltage) of a loop area map of a 5 μm × 5 μm area of the sample overlaid with 50 × 50 grids is shown in Fig. Our previous investigations have shown evidence of the ferroelectric behaviour of PFN thin films, most noticeably via the dramatic collapse in the hysteresis loop upon reaching the Curie temperaturec) in our case is found to be 575 (+/− 5) K for 10 kHz and it increases towards higher temperature with increasing frequency which might be due to charge trapping at the interface due to the termination of the ferroelectric polarization or Maxwell-Wagner space charge across the trilayer heterostructure. It may also be noted that pure PFN itself shows a diffuse type of phase transition behavior in bulk, single crystal and in thin film form, so the interface with NZFO is not the only one that can be responsible for this diffuse phase transition behavior30. A modified Curie-Weiss law has been utilized to test the diffusivity of phase transition;lnm). By fitting the data the value of slope γ is found to be 1.6 for measurements at 10 kHz. This exponent suggests that the ferroelectric to paraelectric phase transition is diffuse in nature43. The ferroelectric transition temperature is found to shift towards higher temperature compared to the ferroelectric transition of pure PFN thin film (~ 400 K at 1 kHz) which might be due to the development of the strain at the interfaces of the heterostructure and strong mechanical coupling of ferroelectric phase with the ferromagnetic phase44. The increasing of ferroelectric Tc towards higher temperature with increasing concentration of NZFO in bulk multiferroic composite has already been reported17. As in composite structures, ferroelectric and magnetic order parameters are coupled, so both ferroelectric and magnetic properties are affected by the increase/decrease of ferroelectric and magnetic material content. This type of behavior, i.e., the increase of ferroelectric Tc with increase of magnetic content has also been theoretically predicted for multiferroic composite thin films45. For these heterostructures, the increase of ferroelectric Tc shifts towards higher temperature compared to pure PFN may be explained due to the effective misfit strain and ME coupling. In ferroelectric materials, the quadratic coupling between polarization and strain are particularly strong. This strong polarization-strain coupling is responsible for the changes in the ferroelectric Tc. Due to the strong coupling between strain and ferroelectricity, Tc shifts of hundreds of degrees are theoretically predicted and have been experimentally observed in epitaxial strained ferroelectric thin films47. It is also reported that the development of strain, deforms the crystal in the perpendicular direction which provides space for ionic displacement of B site atoms, hence the Tc is expected to be modified48. In composite structures, the effective misfit strain on ferroelectric material is mechanically transmitted to the magnetic materials through the magnetostrictive effect inducing a change in the magnetic order parameters and Tc45. Another interesting observation is that another anomaly at ~645 K at 100 kHz begins to develop and becomes prominent as the frequency increases; this is near the magnetic phase transition of the heterostructure. While this does not confirm the existence of magnetodielectric coupling, the presence of anomalies in both magnetic and dielectric susceptibilities at a similar temperature is intriguing. Interestingly, we also observed another hump near 400 K in the dielectric spectra, which may be due to onset of the ferroelectric phase transition. The inset of Fig. In order to probe the ferroelectric phase transition, investigations into the temperature dependence of dielectric properties have been carried out. Figure P-E) measurements have been carried out at 10 kHz at room temperature as shown in Fig. s) and remanent polarization (Pr) of 38 and 12 (+/−0.5) µC/cm2 respectively. The coercive field (Hc) of this nanostructure was found to be 0.15 MV/cm. The saturated P-E loops confirm the intrinsic and integral ferroelectric nature of the trilayer films above room temperature.To check the global nature of ferroelectricity and spontaneous polarization of these nanostructures, polarization versus electric field (c) is found to be 650 (+/−10) K with a broad transition until 700 K which reveals the existence of short range spin interactions, which results in spin frustration and phase separation. In the existing literature, the reported antiferromagnetic (AFM) - paramagnetic (PM) phase transition of PFN single crystal and bulk ceramics is 140 to 155 K whereas for thin film it is 170–200 K44. The existence of weak ferromagnetism until 400 K has also been reported49. The magnetic Curie temperature of Ni0.65Zn0.35Fe2O4 ceramic is ~663 K50. The M(H) and M(T) measurements of both parent compounds were also carried out to get more insight to the magnetic properties. PFN thin films grown on LNO/LSAT exhibit weak ferromagnetic like behavior up to 400 K whereas the magnetic phase transition of NZFO thin film is found ~713 K. The saturation magnetization (Ms) of pure PFN and NZFO thin films of 140 nm were found to be 15 and 290 emu/cm3 at RT51. The fact that the magnetic transition temperature of the heterostructures is higher than that of PFN and NZFO might be due to the effective misfit strain experienced by each layer of the heterostructure and strong coupling of electrical and magnetic order parameters. Figure S) of the heterostructure at 300 K is found to be around 48(+/−1) emu/cm3 and gradually decreases with increase of temperature. The magnetization does not completely disappear above the phase transition up to 800 K confirming the presence of a ferro- (ferri) magnetic phase with paramagnetic relaxor background. The monotonic decrease in the magnitude of remnant magnetization (Mr) and coercive field (Hc) were also seen with increase of temperature. The enlarged M-H loops at low field are depicted in the inset of Fig. In order to justify the existence of magnetic ordering above room temperature of PFN/NZFO/PFN heterostructures, dc-magnetization measurements have been carried out as a function of temperature M(T) and magnetic field M(H) as shown in Fig. ME) of the trilayer heterostructure was measured by dynamic method at room temperature and shown in Fig. δH = 1 Oe at 1 kHz and a bias magnetic field H were applied and the change in voltage δV across the trilayer was measured. The ME coupling coefficient was calculated using the empirical formula αME = δE/δH = δV/(t × δH), where δV is the measured output voltage, δH is the applied AC magnetic field and t is the total thickness of the heterostructure. The highest ME coupling coefficient is found to be ~ 383 mV/cm.Oe at a bias field of +80 Oe followed by a sharp decrease with increasing of magnetic field. Similar type of behaviors were also observed in single phase multiferroic ceramics, epitaxial thin films and composite structures56. From the figure it is observed that αME value does not decrease to zero value with increasing H. The ME voltage coefficients (αME) is directly proportional to the product of the piezomagnetic coefficient q = dλ/dH (where λ is the magnetostriction) and the piezoelectric coefficient d. As the ME voltage arises due to magnetic-mechanical-electrical interactions, αME is proportional to q × d, its Hdc dependence is expected to follow the slope of λ vs Hdc of the magnetic material24. The magnetostriction of NZFO is expected to be stronger at lower H in these heterostructures and decreases with increasing H. A similar type of magnetic field dependence of magnetostriction behavior of nickel zinc ferrite has been reported55. In our case the increase or decrease of αME with H is not similar with the application of positive and negative magnetic field. This asymmetry in the ME coefficient might be due to the existence of magnetic anisotropy in this heterostructure and clamping of bottom ferroelectric layer with electrode coated substrate. It can also be from asymmetry in the piezoresponse, which is also noted in Fig. E,31) ~ 383 mV/cm.Oe which is significantly higher than the superlattices of Ba0.7Sr0.3TiO3/La0.7Sr0.3MnO3 , Ni/PZT/Ni trilayer heterostructure  CoFe2O4/BaTiO3 bilayer  NiFe2O4/BaTiO3 multilayer heterostructures , Pb (Zr0.4Ti0.6)O3–Ni0.8Zn0.2Fe2O4 multilayered thin films , (1 − x) BiFeO3- x BaTiO3 composite thin films, BiFeO3/BaTiO3 bilayer 61 We observed strong ME coupling as these heterostructures exhibit strong polarization and saturation magnetization at room temperature and the perfect strain transmission between the ferroelectric and magnetic layers due to epitaxial growth24. The mechanism responsible for the strong magnetoelectric effect in this case might be indirectly via elastic contribution rather than a direct coupling between the magnetic and electric order parameters of both ferroelectric and magnetic layers. By the application of magnetic field, the magnetic phase shows a magnetostriction effect and produces strain in the system. That strain induces a piezoelectric effect in the ferroelectric phase, which changes the electrical order parameter and hence output ME signal60. In the layered thin film composite structures, giant magneto-electric effects can also be induced due to large interfacial strain across two phases, change in oxygen stoichiometry, electronic correlations, interface bonding, exchange bias, etc. The electronic origin of the ME coupling in the nanostructure may arise from a change in the valence states of magnetic ions induced by electrostatic charge modulation. The large ME coupling effect found in these artificial heterostructures might also be due to interfacial magnetic reconstruction driven by charge accumulation62. The exact mechanisms behind the observed magnetoelectric behaviour requires substantial first principles and phenomenological investigations, which are currently underway. The value of such reasonably high ME coupling coefficient observed at room temperature paves the way for the design and development of new spintronic devices. These observations suggest that epitaxial growth of alternative ferromagnetic and ferroelectric layers offers a route towards achieving strong ME coupling. The thickness of the FM may be kept to below the thickness of the FE layer, to minimize leakage currents and dielectric loss. By controlling the strain state via magnetostriction, through changes in anisotropy and magnetic moment the strong coupling to the polarization in the ferroelectric layers can be achieved. Multilayer structure of strongly piezoelectric materials such as lead zirconate titanate (PZT) and other relaxor ferroelectrics with ferromagnetic materials which exhibits higher magnetostriction coefficient than nickel zinc ferrite can be envisioned, although challenges would be in transferring the strain across the interfaces. Such ME multilayer heterostructures and their combination of properties might hold the future for the ultimate multifunctional and spintronics devices.In order to prove the coupling between electrical and magnetic order parameters, the magnetoelectric coupling coefficient  bottom electrode deposited on commercially available (LaAlO3)0.3 (Sr2AlTaO6)0.7 (LSAT) (100) substrates utilizing optimized pulsed laser deposition (PLD) with an excimer laser . Initially 90 nm of LNO bottom electrode was deposited at 700 °C in an oxygen ambient of 200 mTorr, followed by annealing in same oxygen ambient of 300 Torr for 30 minutes at same temperature and then slowly cooled down to room temperature. PFN and NZFO layers were deposited at a fixed temperature of 600 °C under an oxygen pressure of 20 mTorr and 150 mTorr, respectively with a laser energy density ~1.5 J/cm2. Later, the PFN/NZFO/PFN heterosrtructure was annealed in oxygen atmosphere of 300 Torr for 30 minutes at 700 °C and later cooled down to room temperature slowly. The thickness of both PFN layers and the middle NZFO layer were found to be around 70 nm and 20 nm, respectively.The ceramic targets of PFN and NZFO were prepared by a conventional high temperature solid state reaction route. The synthesis conditions and structural properties of each compound are reported elsewhereα radiation with wavelength of λ = 1.5405 Å operated at a scan rate of 1 °/min over the angular range (2θ) of 20–80 at room temperature. The thicknesses of the films were measured utilizing XP-200 profilometer along with TEM. For TEM imaging, the lamellae were cut from the PFN/NZFO/PFN heterostructure, and were lifted using a fine glass needle and micromanipulator under an optical microscope, and transferred onto a carbon coated 3 mm copper-mesh grid. Imaging of heterostructure was then performed using a scanning transmission electron microscopy (STEM-Philips CM 200 (FEG)) on a 200 kV field-emission transmission electron microscope, using a high-angle annular dark-field detector. Specimen imaging was performed with the electron beam perpendicular to the lamellar face. The local chemistry at the PFN and NZFO layers was investigated using STEM-EDS in a Philips CM 200 (FEG) operated at 200 kV. The PFM spectroscopy experiments were performed by moving the tip across a pre-defined coordinate  grid on the sample and perturbing it with a DC bias waveform, and then measuring the strain (piezoresponse) as a function of frequency via Band Excitation (BE)63. BE technique involves the generation of a band of AC frequencies around the contact resonance of the cantilever and measuring the electromechanical response over time with subsequent Fourier transformation back to the frequency domain to yield the frequency-dependent response. This measurement is repeated for several time steps after each DC bias pulse; thus, at each  position, local spectroscopic information is acquired as a function of perturbation voltage (V) and excitation frequency (f), characterizing the system’s mechanical response  R = R. The response R at each  step is fit to a simple harmonic oscillator (SHO) function yielding the amplitude (A), phase(φ), quality factor (Q), and resonant frequency (ω) associated with the response. The thin film was glued onto a sample plate with silver epoxy, which acted as the bottom electrode and was grounded for the PFM experiments. The PFM experiments were performed at room temperature with moderately stiff Budget Sensors ElectriMulti75-G cantilevers (k ∼ 1 N/m) and a free resonance (in air) of ∼75 kHz, on a Multimode (Veeco) AFM equipped with a Nanonis controller. National Instruments DAQ cards were employed for signal generation and acquisition for the band-excitation experiments, which were performed using scripts written in Labview v11 and Matlab version 2011. All analyses were carried out utilizing Matlab version 2011. Temperature dependent magnetic properties of the heterostructure were carried out using a PPMS DynaCool (Quantum design) in a wide temperature range of 300–800 K. The room temperature ME measurements were performed using a magnet with a varying field of up to ±0.5 T with a lock-in amplifier and reference ac magnetic field using Helmholtz coil. Pt top electrodes of area ~10−4 cm2 and thickness ~60 nm were made by dc sputtering utilizing a metal shadow mask for electrical characterization. Dielectric parameters, i.e., capacitance, loss tangent, impedance, and phase angles, were measured from 80 to 700 K in a wide frequency range of 100 Hz to 1 MHz using an impedance analyzer HP4294A with MMR Technologies K-20 programmable temperature controller with fixed ac voltage amplitude of 0.1 V. Ferroelectric (P−E) loops were measured using Precision Multiferroic and Ferroelectric Test System  at room temperature.The epitaxial quality of PFN/NZFO/PFN heterostructures was confirmed by high resolution XRD using CuK