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The Influence of Moisture Content and Temperature on the Long-Term Storage Stability of Freeze-Dried High Concentration Immunoglobulin G (IgG)

Arnold duralliu.

1 Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, SW7 2AZ, UK; [email protected]

Paul Matejtschuk

2 Standardisation Science, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK; [email protected]

Paul Stickings

3 Bacteriology Division, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, UK; [email protected] (P.S.); [email protected] (L.H.); [email protected] (R.T.)

Laura Hassall

Robert tierney, daryl r. williams.

High protein concentration products for targeted therapeutic use are often freeze-dried to enhance stability. The long-term storage stability of freeze-dried (FD) plasma-derived Immunoglobulin G (IgG) from moderate to high concentrations (10–200 mg/mL) was assessed. Monomer content, binding activity and reconstitution times were evaluated over a 12-month period under accelerated and real-term storage conditions. In the first case study it was shown that FD IgG from 10 to 200 mg/mL had minimal monomer/activity losses at up to ambient temperature after 12 months of storage. However, at 45 °C the sucrose-to-protein ratio played a significant impact on IgG stability above 50 mg/mL. All IgG concentrations witnessed moisture ingress over a 12-month period. The impact of moisture ingress from environmental exposure (between 0.1% and 5% w / w moisture) for IgG 50 mg/mL was assessed, being generated by exposing low moisture batches to an atmospheric environment for fixed time periods. Results showed that at −20 °C and 20 °C there was no significant difference in terms of monomer or antigen-binding activity losses over 6 months. However, at 45 °C, there were losses in monomer content, seemingly worse for higher moisture content samples although model binding activity indicated no losses. Finally, the difference between a low moisture product (0.1–1% w / w ) and a moderately high moisture (3% w / w ) product generated by alternative freeze-drying cycles, both stoppered under low oxygen headspace conditions, was evaluated. Results showed that at −20 °C and 20 °C there was no difference in terms of binding activity or monomer content. However, at 45 °C, the low moisture samples had greater monomer and binding activity losses than samples from the highest moisture cycle batch, indicating that over-drying can be an issue.

1. Introduction

There is rising demand in the biopharmaceutical industry for increasingly high concentrations of therapeutic proteins, such as monoclonal antibodies (mAbs). However, with this requirement there comes a host of challenges, including an increased instability, degradation and viscosity, as well as unwanted protein–protein interactions in the liquid state [ 1 , 2 ]. There is no precise standardized definition of what constitutes a “high concentration” protein formulation but generally it has been considered to fall between 50 and 150 mg/mL [ 3 ]. Freeze-drying (FD) is a common technique used to enhance shelf stability by delivering a product in a solid-state format. [ 4 , 5 ]. It consists of three stages: (1) Freezing (solidification), (2) primary drying (sublimation of frozen ice) and (3) secondary drying (desorption of bound water). Sugar-based excipients are usually added to these formulations to enhance the stability of the active pharmaceutical ingredient (API) during prolonged storage. Other studies have focused their investigations into the slow reconstitution times of high concentration protein formulations and ways to improve them [ 6 , 7 ]. However, in addition to the continuing development of higher protein concentration formulations in FD formats, there are challenges with regard to selecting the appropriate moisture content for long-term storage stability. Typically, after FD the resulting solid freeze-dried “cake” would be expected to have a low moisture content. For FD intravenous Immunoglobulin G formulations, the acceptable moisture contents would be at or below 3% w / w [ 8 ]. Plasma-derived immunoglobulin has long been used as a therapeutic product and intravenous immunoglobulin concentrations of 50–100 mg/mL are common [ 9 , 10 ]. Previous studies have also looked into the stability of mAbs with regard to higher moisture content ranges of between 1% and 8% w / w ; however, these studies focussed on the impact of the higher end of the moisture range. Breen et al. [ 11 ] was able to show that while increasing the moisture content up to 8% w / w might decrease chemical stability in FD rhuMAb, it might in fact actually increase the physical stability as long as storage was below the dry state glass transition (T g ). The range of moisture content that is desirable in FD cakes has long been a matter of debate. Hsu et al. showed that an optimum moisture content range might exist, whereby both over and under drying could be detrimental to stability [ 12 ]. Long ago Greiff hypothesised that the relationship between moisture content and stability could correspond to a “bell shaped” distribution [ 13 ]. Alternatively, Pikal et al. noted that for FD Human Growth Hormone at 40 °C, there was a linear relationship between moisture content and aggregation rather than a bell-shaped distribution [ 14 ]. Guidelines for use when developing stressing strategies for biotechnology products have been published [ 15 ] and industrial stressing conditions have also been reviewed [ 16 , 17 ]. During long-term storage, the sugar excipient used in the formulation, moisture content and temperature can all affect the physical and chemical stability of FD cakes. Plasma-derived IgG was chosen to be investigated here as a substitute model for a typical therapeutic mAbs because of its relative ease of availability at high concentrations. A comprehensive set of long-term case studies investigating the stability of FD IgG was assessed over a 6–12-month period. The monomer content, retention of anti-diphtheria/tetanus antibody titres and change in moisture content were evaluated under accelerated and real-time storage conditions (−20 °C, ~ <5% RH; 20 °C, ~40% RH; and 45 °C, ~10% RH, though the incubators used did not directly control the RH) to assess FD storage stability.

2. Materials and Methods

2.1. materials and formulation characterisation.

The bulk of the 50 mg/mL IgG (2.2 L) was obtained by dialysis from time-expired clinical grade standard product (NIBSC, Potters Bar, UK) and maintained sterile at 2–8 °C. A quantity, 150 mL, was dialysed into 1% w / v sucrose, 10 mM citric acid adjusted at pH 6.6 with 5 M NaOH and 0.01% Tween 20 against 3 × 5 L over 20 h using a Spectrapor 8 kDa cut-off dialysis membrane (Sigma-Aldrich, Gillingham, UK). A portion from this bulk was diluted down to 10 mg/mL. The remaining bulk pool was then ultrafiltered to get nominal target concentrations of 100 and 200 mg/mL IgG. All the preparations were then dialysed in dialysis membrane sacs against the citrate Tween 20 sucrose buffer. Target concentrations were achieved by diafiltration using the Vivaflow 200 crossflow system and with a 50 kDa PES (polyethyl sulfone) membrane cartridge (Sartorius Stedim Biotech, Gottingen, Germany). Concentrations were confirmed by OD 280 nm (E1 % for IgG = 13.5 AU) using triplicate 1:100 dilutions measured with a UV spectrophotometer (Pharmacia Biotech, GE Healthcare, Little Chalfont, UK) blanked on sucrose citrate buffers. Average readings were 0.98 ± 0.02%, 5.43% ± 0.19%, 9.86% ± 0.13% and 20.4% ± 0.97% w / v . These were approximated to 1%, 5%, 10% and 20% w/v, which is equivalent to 10, 50, 100 and 200 mg/mL, respectively.

2.2. Modulated DSC

Solid sample or liquid aliquots of IgG were loaded into large volume hermetically sealed pans (part number 900825.902 TA Instruments, Elstree, UK). Samples were evaluated on Q2000 DSC (TA Instruments) against an empty pan as reference. A heating ramp rate at 5 °C/min up to 200 °C was used. Instrument calibration was performed using an indium sample of known mass. Data analysis was performed using Universal Analysis Software (TA Instruments).

2.3. Case Study 1: Freeze Drying of a High Concentration IgG

A batch using a “low” moisture cycle run was included in all the IgG concentrations (10–200 mg/mL). Sample filling was completed with an automated multipette stream (Eppendorf, Stevenage, UK) into 5 mL volume screw capped vials (41.5 × 18 mm i.d. Schott VC005, Adelphi Tubes, Haywards Heath, UK). The fill volume for all concentrations was 1 mL. The vials were loaded onto the shelves of the Telstar LyoBeta 15 (Azbil-Telstar SpA, Terrassa, Spain). After the cycles had finished the vials were backfilled with dry N 2 and were stoppered down with 14 mm diameter igloo halobutyl stoppers (Adelphi Group, Haywards Heath, UK). Afterwards the vials were screw-capped, labelled and stored at −20 °C, 20 °C and 45 °C until further testing.

2.4. Case Study 2: Optimum Moisture Content of IgG

A total of 50 mg/mL IgG was lyophilised with the same conditions and same “low” FD cycle as described in Section 3.2 . After FD, samples were laboratory-stored at 20 °C (with a relative humidity of 57% ± 2% RH, as measured with a TFH 620 Hygrothermometer (Ebro-Xylem, Ingoldstat, Germany). The stoppers were removed temporarily, and vials were exposed to the moisture in the external atmosphere for different time intervals (0, 30, 90 and 180 min) to result in different moisture contents in the cakes. A total of 4 different moisture contents were prepared and an average confirmed using Karl Fisher titration in triplicate ( n = 3).

2.5. Case Study 3: Comparison of Low and High Moisture FD Cycles

The “high” moisture cycle and “low” moisture cycle batches of FD IgG (50 mg/mL) were prepared to allow comparison between these two different cycles that induce moisture content as shown in Table 1 . After FD cycles, samples were once again backfilled from vacuum with dry N 2 and were stoppered down with 14 mm diameter igloo halobutyl stoppers (Adelphi Group, Haywards Heath, UK). Afterwards the vials were screw-capped, labelled and stored at −20 °C, 20 °C and 45 °C until further testing.

Freeze drying cycle for IgG formulations.

2.6. Diphtheria and Tetanus ELISA

Anti-diphtheria or anti-tetanus IgG binding activity levels are commonly determined using the ELISA technique as an alternative to in vivo toxin neutralization tests [ 18 ]. In brief, 96-well plates were coated with either diphtheria toxoid (NIBSC product code 13/212) or tetanus toxoid (NIBSC product code 02/126) as coating antigen, sealed and left to incubate overnight at 4 °C. The next day plates were washed with PBS containing Tween-20 0.05% v / v (PBST) and then blocked with blocking buffer (PBST + 5% skimmed milk powder). Plates were incubated at 37 °C for 1 h and washed as before. Sample and reference antitoxin dilutions were prepared in sample buffer (PBST + 1% skimmed milk powder). WHO International Standard antitoxins for diphtheria (NIBSC product code 10/262) and tetanus (NIBSC product code TE-3) were included on each plate to allow specific IgG concentrations to be expressed in IU/mL. A total of 200 mL of diluted sample or reference were added to the top row of wells and titrated down the plate using a manual multichannel pipette set to 100 µL. After serial dilutions, plates were once again incubated at 37 °C for 2 h and washed as before. Anti-human IgG HRP-conjugate (Sigma A8792, Sigma-Aldrich) was diluted 1/2000 in sample buffer and 100 µL was added to each well before incubation at 37 °C for 1 h. Substrate solution containing citric acid buffer and ABTS (2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) tablets was added to all wells for colour to develop. Absorbance was read at 405 nm on a Molecular Devices Vmax plate reader running Softmax Pro 6.5.1 (Molecular Devices, Wokingham, UK). Data analysis was performed in CombiStats (version 5.0, 2013, EDQM, Strasbourg, France).

2.7. SEC-HPLC

Size-exclusion high performance liquid chromatography (SEC-HPLC) is a commonly applied chromatographic method used for determining of aggregation or monomer content of biotherapeutic proteins in solutions via size or molecular weight differences [ 19 ]. SEC-HPLC was used to determine the monomer content of the FD IgG samples after reconstitution. Analysis was performed using the Thermo Scientific UltiMate 3000 HPLC System (Thermo Fisher Scientific, Loughborough, UK) with a TSKgel G3000SWXL HPLC column (300 × 7.8 mm, Sigma-Aldrich). Ultraviolet (UV) detection was measured at 280 nm in triplicate ( n = 3). The mobile phase was prepared from the formula as outlined in European Pharmacopeia 9.0: Human Normal Immunoglobulin for intravenous administration (01/2012:0918) [ 8 ], consisting of disodium hydrogen phosphate dihydrate (0.49% w / v ), sodium dihydrogen phosphate monohydrate (0.17% w / v ), sodium chloride (1.17% w / v ) and sodium azide (0.01% w / v ). The flow-rate on the apparatus was set at 0.5 mL/min. Peak analysis was performed using Chromeleon 7.2SR software (Thermo Fisher Scientific, Loughborough, UK).

2.8. Residual Moisture Content

The moisture content of the FD cakes within the vials was measured using an automated coulometric Karl Fischer instrument (Mitsubishi CA-200, A1-Envirosciences Ltd., Blyth, UK). Samples were transferred into HPLC autosampler vials within a pyramid dry bag (Captair pyramid, 2200A Cole Palmer, London, UK). Nitrogen gas was used to purge the pyramid air bag to ensure that a humidity < 5% RH was achieved. All samples were tested in triplicates ( n = 3), i.e., 3 vials were tested.

3. Results and Discussion

3.1. case study 1: effect of moisture ingress and excipient ratio on storage stability of high concentration igg.

Polyclonal human immunoglobulin is derived from many thousands of donors from the population, and so a dimer content of between 8% and 10% can occur naturally and is of itself not an indicator of aggregation [ 20 ]. Usually a protein-to-sugar weight ratio of 1:1 would be suitable for optimal stabilisation. However, in this study the IgG molar/weight ratio was intentionally varied as the sucrose excipient concentration of 10 mg/mL (1% w / w ) was maintained in order to investigate the destabilizing effect of moisture ingress and/or storage temperature conditions during long-term storage. As such the sucrose:protein molar ratio for these investigations were as follows: 1% sucrose to 1% IgG is 439:1; 5% IgG is 88:1; 10% IgG is 44:1; and 20% IgG is 22:1. The moisture content before and after 12 months storage is displayed in Figure 1 .

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Average moisture content (%) for low to high concentration freeze-dried (FD) IgG before and after 12 months of storage at −20, 20 and 45 °C. Error bars are 95% confidence intervals ( n = 3 vial replicates).

After 12 months of storage, for all IgG concentrations there was an increase in moisture content, especially at the higher storage temperatures of 20 °C and 45 °C. The 10 mg/mL samples had the highest moisture ingress at 20 °C and 45 °C at around 3–4% w / w moisture content ( n = 3). With increasing IgG concentration there was less moisture uptake at these elevated temperatures of between 1% and 2% w / w moisture. The most common sources of moisture ingress are likely due to either trapped moisture in the headspace of the vials or moisture actually emanating from the rubber stoppers [ 14 , 21 ]. Past studies and research have shown the negative consequences of elevated moisture and temperature on FD biologics stability and structure [ 22 , 23 ]. Increasing moisture content can lead to a reduction of the T g due to the its plasticizing effect [ 24 , 25 ]. Table 2 shows the T g of the IgG after 12 months of storage as measured with DSC ( n = 2). The difference between storage temperature and the T g (denoted as T g -T) is largest at the lowest temperature of −20 °C storage and becomes smaller as the storage temperature is increased through 20 °C to 45 °C.

Comparison of difference in storage temperature T and glass transition temperature T g after 12 months of storage.

The impact of the storage conditions and moisture on the monomeric (%) content and binding activity of the IgG are shown in Figure 2 and Figure 3 . Storage at −20 °C showed that there was minimal monomeric loss (less than 1%) for IgG at all concentrations. Increasing temperature resulted in greater loss of monomer and higher IgG concentrations suffered greater losses in monomer, especially at 45 °C. The order from least monomer loss to highest at 45 °C storage was 10 mg/mL < 50 mg/mL< 100 mg/mL < 200 mg/mL. The binding activity (IU/mL normalised into IU/mg) was measured by ELISA for specific IgG (anti-diphtheria andante- tetanus) over the course of the 12-month storage. Anti-tetanus and anti-diphtheria antibodies were chosen as markers for specific IgG since the majority of donors used in production of the IgG products will have received diphtheria and tetanus immunisation as part of routine immunisation programmes. It is interesting to note that while the monomer content was decreasing throughout storage at elevated temperature, the binding activity remained relatively stable until the last time point (T = 12) and only for the 50 and 100 mg/mL samples. It is important to note that the specific antibodies being measured here represent only a small proportion of the total IgG in the formulation. However, for the 200 mg/mL samples there was high variability in activity, due to fact that the 200 mg/mL samples did not fully reconstitute, thus giving variable ELISA results.

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Average monomer content (%) over time for low to high concentration IgG with contour stability maps as measured by SEC-HPLC over 12 months stored at (from top to bottom) −20 °C, 20 °C and 45 °C ( n = 3 vial replicates). Error bars at 95% confidence intervals are too small to be seen.

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ELISA Diphtheria and Tetanus normalized binding activity (IU/mg) for IgG over the course of 12 months stored at −20 °C, 20 °C and 45 °C.

The 10, 50 and 100 mg/mL IgG had a clear visual appearance after being fully reconstituted. However, the 200 mg/mL samples stored at 45 °C were slightly cloudy/viscous with tiny clumps of particulates still undissolved ( Figure 4 ). In addition, on average the 10–50 mg/mL samples took up to 5 min to reconstitute while the 200 mg/mL samples took over 2 h to dissolve. Long reconstitution times have been commonly reported for high concentration protein solutions [ 26 , 27 ], and strategies for addressing this have been shared [ 7 ].

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Visual appearance of IgG 200 mg/mL dissolved in water up to 0, 60 and 120 min for the T = 12-month-old sample aged at 45 °C.

The results presented in this study are slightly unusual in that the highest concentration protein was shown to be the least stable. These results are counter to the general trend expecting higher concentrations tending to be more stable. It is possible that at these very high concentrations of FD IgG they are more temperature labile compared to lower concentrations due to a lack of excipient protection from the formulation, as the ratio of IgG:sucrose falls from 400:1 to 22:1. Table 2 shows that the 10 mg/mL IgG had a smaller difference (T g -T) value than for the higher concentrations, yet demonstrated lower monomer loss at 45 °C. Even though the lowest concentration IgG had the highest moisture content and reduced T g , it was shielded because it had a higher excipient-to-protein ratio protection while the higher concentration did not, thus supporting a case for water replacement theory. However, the moisture content was similar at both 20 °C and 45 °C, yet the largest drops were only observed at 45 °C for samples of 50–200 mg/mL. One possible explanation for this is that of the T g being closer to the high storage temperature and hence increasing molecular flexibility—supporting a role for the glass dynamics hypothesis (T g -T). The question of optimum moisture content and mechanisms of stabilization (be it water substitution theory or glass vitrification hypothesis) has been a matter of debate [ 28 ]. These results provide a case for both water replacement theory and vitrification hypothesis in terms of stabilization. Overall, moisture ingress during storage (from as high as 1% to 4% w / w ) occurs for all FD IgG concentrations up to 200 mg/mL and needs to be considered, although for these high concentration proteins the moisture appeared to have minimal impact on the stability at −20 °C and 20 °C.

3.2. Case Study 2: Optimum Moisture Content of IgG

The second investigation considered the acceptable “moderate” moisture content of FD IgG at 50 mg/mL over the course of a 6 months stability study. Samples were all prepared initially in a low moisture cycle producing moisture contents of 0.05% ± 0.01% w / w ( n = 3), (A). Vials were exposed to the humid room atmosphere at several different time intervals of 30, 90 and 180 min, respectively (57% RH at temperature of 20 °C). The increased moisture contents before storage were measured as (B) 0.67% ± 0.14% w / w , (C) 3.02% ± 0.06% w/w and (D) 4.95% ± 0.40% w / w ( n = 3). Figure 5 shows the moisture ingress over 6 months from different initial starting moisture contents. All samples showed constant moisture content at −20 °C; however, the drier the sample had started out then the more moisture ingress was observed after 6 months at elevated storage temperatures. Sample A (with the lowest initial moisture content) had a significant increase in moisture content after 6 months at 20 °C and 45 °C up to 1% w / w . For Sample A, this uptake is most likely to reflect moisture transfer from inside the vial stopper. For very low moisture content FD materials, there is a diffusion-driven water migration from the stopper interior to the relatively dry cakes. The other samples have moisture contents within an experimental error of each other and exhibited no similar major moisture ingress. The effect of different moisture content on the monomer content (%) and binding activity (IU/mL) is shown in Figure 6 and Figure 7 . Table 3 shows the comparison between moisture content of samples and the resulting measured T g . Over the course of 6 months, storage at −20 °C to 20 °C increased the moisture content up to 5% w / w and had no impact on monomer content or binding activity. However, at 45 °C, just after 6 months storage, the samples with higher moisture (above 1% w / w moisture) began to see around a 10% drop in monomer content, whereas for sample A (below 1% w / w moisture) there is no losses. This is in stark contrast with the binding activity results, which show that at no point is there any loss for all moisture content samples (A–D) up to 5% w / w . One possible explanation for this behaviour is that in the previous case study moisture content ingress naturally increased over time, whilst for this study the samples were exposed to atmosphere and hence possible oxygen trapped in headspace. Oxidation might have occurred in this study, which could have led to more protein–protein aggregation.

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Change in moisture content (% w / w ) after 6 months of storage starting at different initial starting moisture samples of IgG 50 mg/mL (A = 0.05% ± 0.01% w / w , B = 0.67% ± 0.14% w / w , C = 3.02% ± 0.06% w / w and D = 4.95% ± 0.40% w / w ) at −20 °C, 20 °C and 45 °C. Error bars represent 95% confidence intervals ( n = 3 vial replicates).

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Change in monomer (%) after 6 months of storage at 45 °C for different initial moisture samples of IgG 50 mg/mL (A = 0.05% ± 0.01% w / w , B = 0.67% ± 0.14% w / w , C = 3.02% ± 0.06% w / w and D = 4.95% ± 0.40% w/w ). Monomer content was stable for −20 and 20 °C at 84.0% ± 0.5% Error bars represent 95% confidence intervals ( n = 3 vial replicates).

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ELISA Diphtheria binding activity (IU/mL) after 6 months of storage for different initial moisture samples of FD IgG 50 mg/mL (A = 0.05% ± 0.01% w / w , B = 0.67% ± 0.14% w / w , C = 3.02% ± 0.06% w / w and D = 4.95% ± 0.40% w / w ) at −20 °C, 20 °C and 45 °C. Error bars represent 95% confidence intervals.

Comparison of difference in storage temperatures and glass transition temperatures after 6 months of storage.

These monomer and binding activity results from the induced exposure moisture seemed to suggest that modest moisture content (≈3% w / w ) was perfectly acceptable for 50 mg/mL IgG when stored at up to 20 °C. However, from the results in this study there was no Gaussian-shaped distribution in terms of moisture and stability observed. In fact, a more linear correlation with moisture was observed for higher temperature storage, which was more in line with what Pikal et al. had observed in a moisture-based stability study [ 14 ]. It is worth noting that Chang et al. in a study on a Mab also reported that there was better stability at 2–3% moisture content than for drier samples [ 29 ]. This supports our finding here that an optimum moisture content exists and should be investigated for each formulation.

3.3. Case Study 3: Stability of Induced “Low” Versus “High” Moisture FD Cycles

In Case Study 3, we looked at the effect of FD cycle-induced moisture content between a very low moisture content and a moderately high one for a single IgG concentration (50 mg/mL). Residual moisture content is controlled by the shelf temperature used in secondary drying. Post-drying exposure to controlled humidity was used in Case Study 2. Cycle-induced moisture might provide different data results with regard to moisture content. The low moisture cycle produced cakes with 0.11% ± 0.01% w / w , while the high moisture cycle produced cakes with 3.02% ± 0.8% w / w ( n = 3). Previously it has been described that a much higher moisture contents(8% w/w) decreased the stability of a MAbs dried at a 40 mg/mL concentration [ 11 ]. However, here, more representative moisture contents (0.1% w / w for low cycle and 3% w / w for high cycle) within the acceptable moisture range for intravenous immunoglobulin [ 8 ] were engineered by control of the secondary drying temperature conditions. The moisture content changes from the initial starting points over 12 months of storage. The samples from the low moisture cycle batch had an increase to about 1.5% w / w moisture at 20 °C and 45 °C storage. The samples from the high moisture batch, however, exhibited no such moisture ingress across the temperature range. This may illustrate an equilibration of the moisture in the closure and the hygroscopic dry FD product. Matejtschuk et al. [ 30 ] had observed similar equilibrating moisture effects in vials in a study with a model HSA formulation. Once again, a moisture increase over time is seen for vials at or above 20 °C, especially for FD samples that initially had a very low moisture content. It was most likely that a significant portion of moisture ingress could be arising from the stoppers themselves ( Figure 8 ). Stopper drying has been effectively used in the past to maintain a low moisture content during storage [ 31 ], and can help reduce water ingress and potential degradation at higher storage temperatures.

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Difference in moisture content for FD IgG (50 mg/mL) between vials with vacuum-oven-dried stoppers and vials with untreated stoppers straight out of packaging after 3 months of storage at −20 °C and 45 °C ( n = 3 vial replicates).

Figure 9 and Figure 10 shows the binding activity and monomer (%) content and compared for IgG (50 mg/mL) at both a high and low moisture content. After 12 months, there is no major difference or drop in monomer content at −20 °C and 20 °C for vials in either a low cycle or high cycle batch moisture content (remains steady above 80% monomer content). Similarly, the binding activity does not change greatly for either low and high cycle batch vials at −20 °C and 20 °C storage. However, at 45 °C, samples from the low moisture cycle were observed to have a significantly greater drop in monomer content compared to samples from the high moisture cycle batch. This was also seen for the binding activity data, where the low moisture sample vials saw a drop with activity at 45 °C, whereas the high moisture vials did not. This is also in contrast to the previous Case Study 2, where the samples with highest moisture saw greatest monomer drop at elevated temperature of 45 °C. As stated previously, one possible explanation for this is that the vials in this study were stoppered under vacuum and nitrogen gas, while in the other study the vials were exposed to the environment and had oxygen trapped in the headspace. Oxidation might have occurred in the previous case study, which would have induced pathways to lead to more protein–protein aggregation. However, in a previous study we did not observe any detriment in antibody activity on storage from a higher oxygen content in the headspace [ 32 ]. This might also be further evidence of over-drying and for water replacement theory with some moisture being essential for the FD cake stability in allowing hydrogen bonds to form.

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Diphtheria Binding Activity determined by ELISA for FD IgG 50 mg/mL in “low” or “high” moisture cycle batches over 12 months of storage. Error bars represent 95% confidence intervals.

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Change in monomer content (%) of FD IgG 50 mg/mL vials made under “low” or “high” moisture cycles over 12 months of storage. Error bars represent 95% confidence intervals ( n = 3).

Previous reported research from Wang [ 33 ], Liu et al. [ 34 ] and Greiff [ 13 ] have all described an increase in aggregation with higher moisture content (over the optimum level). However, some past studies have also shown that acceptable moisture contents may be permissible in FD cakes without effecting stability [ 5 ]. The results here showed evidence that over drying can also have a detrimental effect on stability. In another alternate study, Wang showed for a different formulation that over-drying had greater loss in activity and increased aggregation. Over-drying of FD insulin has also showed to cause damage and increased product degradation [ 35 ]. Hubbard et al. noted how a FD reference plasma, with an optimised formulation and a modest moisture content, had greater stability than one with a very low moisture content [ 36 ]. These results show some confirmation of the water replacement theory. If the FD IgG samples are over dried, the hydrogen bonds are lost between the protein and the water, hence you have a change in conformational structure. Some moisture may be crucial to maintaining the stability of the FD biologics.

These results suggest that in this model system the higher moisture (3% w / w ) samples are as good as the extremely low moisture samples (<1% w / w ) and might even show reduced monomer/activity loss at elevated temperatures. Upon visual inspection, there was no colour change between the high and low moisture vials at −20 °C and 20 °C for the entire storage duration. However, at 45 °C a visible yellow discolouration had appeared on the cakes with the higher moisture content ( Figure 11 ). The discolouration present in the higher moisture batch vials was probably due to Maillard browning (caused by protein free amino group reaction with sugars). Schüle et al. also saw a brown discoloration with lactose in their dried IgG1 stabilization study, which they attributed to Maillard reactions [ 37 ]. Kanojia et al. found that after accelerated thermal treatment a chemical modification had occurred in their dry sucrose-dextran IgG formulations, which they too postulated was due to Maillard reaction products [ 38 ]. One explanation then might be that there was reducing sugar impurities present in the formulation; however, this is very unlikely due to using a pharmaceutical-grade material. This study also used sucrose in the formulation, which is not a reducing sugar. However, while sucrose is not a reducing sugar, it can break down to fructose and glucose, which are reducing sugars; so, this may explain why this discoloration was only observed for samples stored at higher temperature [ 39 ]. Matejtschuk et al. spotted similar discolouration for formulations containing sucrose at elevated storage temperatures [ 40 ]. The presence of high moisture content further facilitates these reactions and results in a change in the appearance. It is also a possibility that at the formulated sucrose concentration, buffer crystallisation could have occurred during the freezing, thereby leading to a change to low pH at which sucrose could have broken down to fructose and glucose [ 41 ]. Of course, when it comes to commercialisation, product appearance is a key factor and acceptable and unacceptable product appearance defects have recently been reviewed [ 42 ]. Although it may be possible to illustrate that a relatively higher moisture content is beneficial in terms of product stability, the risk of developing a yellow cake appearance on storage would be a drawback for customers as most would be familiar with a white homogeneous product appearance. The potential immunogenicity effects would also need to be addressed.

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Comparison of the visual appearance between vials FD in either “low” or “high” moisture cycles for FD IgG (50 mg/mL) after 12 months storage at 45 °C.

4. Conclusions

Freeze-drying can provide excellent long-term stability for high concentration IgG proteins but needs to be considered in the context of factors such as moisture content and intended storage temperature. FD IgG from 10–200 mg/mL had minimal monomer/activity losses at up to 12 months of storage at 20 °C. IgG 10 mg/mL with the 1:1 protein:sucrose ratio had greater stability shielding at elevated temperatures compared to concentrations above 50 mg/mL, but this may reflect the need for a higher sucrose content at a higher protein composition. There may be an optimum moisture content range for FD IgG, and this needs to be informed with reference to the storage temperature compared to the glass transition temperature. From −20 °C to 20 °C moisture content up to 5% w / w does not seem to have any impact on monomer or binding activity during prolonged storage. It is only at elevated temperatures, such as at 45 °C storage, that high moisture seemed to affect the monomer for IgG at 50 mg/mL, although this might be because of oxygen ingress during the exposure trial study. In the comparison between “high” versus “low” FD cycle-prepared materials the opposite was seen, in that the higher moisture content vials performed better at 45 °C than the extremely dry moisture samples. While some moisture content might be permissible and in fact even beneficial, Maillard reactions can occur (discolouration), which could affect commercial prospects for high moisture content samples aged at 45 °C. Moisture ingress into the FD cakes was observed to occur in every trial reported here, and is especially the case for drier samples and at more elevated storage temperatures, although one potential way of counteracting this effect could be by vacuum heat treating the rubber stoppers [ 31 ].

Acknowledgments

The research was performed at Imperial College Department of Chemical Engineering in collaboration with National Institute for Biological Standards and Control (NIBSC, UK). We also thank the Bacteriology Division (NIBSC, UK) for ELISA testing assistance and Kiran Malik, Chinwe Duru and Ernest Ezeajughi (Standardisation Science) for providing training in some of the analytical techniques.

Author Contributions

The studies were devised by A.D. in conjunction with D.R.W. and P.M. with input from P.S. on biological assays. The practical laboratory work was performed by A.D. and in terms of the biological activity assays in cooperation with R.T. and L.H. Data analysis and interpretation involved all authors. The manuscript was written by A.D. and developed and revised in conjunction with P.S., D.R.W. and P.M. All authors have read and agreed to the published version of the manuscript.

This work was funded by a PhD grant from Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training in Emergent Macromolecular Therapies, Department of Biochemical Engineering, at University College London to DRW to cover AD’s PhD studies.

Conflicts of Interest

The authors declare no conflicts of interest.

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  • Published: 17 August 2021

Analysis of the need for soil moisture, salinity and temperature sensing in agriculture: a case study in Poland

  • Lech Gałęzewski 1 ,
  • Iwona Jaskulska 1 ,
  • Dariusz Jaskulski 1 ,
  • Arkadiusz Lewandowski 2 ,
  • Agnieszka Szypłowska 3 ,
  • Andrzej Wilczek 3 &
  • Maciej Szczepańczyk 4  

Scientific Reports volume  11 , Article number:  16660 ( 2021 ) Cite this article

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  • Environmental sciences
  • Environmental social sciences

Efficient use of scarce water resources is both a marketing objective and an environmental obligation for sustainable agriculture. In modern agricultural production, which is intensive and should at the same time be environmentally friendly, there is a need to monitor soil moisture, salinity and temperature. The aim of the study was to determine the demand of producers of agricultural and horticultural plants for equipment and systems for monitoring soil properties at an individual farm level in regions with highly developed agriculture. A questionnaire survey was conducted among 1087 respondents, also direct interviews in Poland were undertaken. According to the producers' responses, it is important to know soil moisture, salinity and temperature, although currently only about 4% of the surveyed farmers have the equipment to evaluate these soil parameters. In their view cost is not the most important obstacle to the purchase of the necessary probes. More important is that the devices should be easy to install and use, and have an easy to use application for data collection, processing and transfer. The current market does not offer solutions that meet these producers expectations. The demand for suitable probes is very high as over 80% of the farmers declared their willingness to purchase such probes. Technical problems related to the operation and servicing of such equipment were the most frequently mentioned impediments in their use. However, farmers and horticulturists believe that knowledge of their soil properties would allow them to optimize the elements of cultivation technology, including the use of plant irrigation systems, the use of mineral fertilizers and plant protection products.

Climatic conditions shape plant production- not only the yields, but also the area sown, production intensity and selection of technologies 1 . Climatic elements, especially the rainfall and the air temperature are necessary for the proper functioning of the physiological processes of crops 2 , their development and productivity 3 , as well as for shaping the broadly understood properties of soil 4 . Crop plants are sensitive to climate change: deficiency or excess water, or suboptimal temperatures may cause severe abiotic stresses 5 , 6 . Drought stress causes molecular, biochemical, physiological and morphological changes in plants 7 . Plants then grow smaller organs, e.g. roots, leaves, and also their productivity, biomass and yield are lower 8 . According to Daryanto et al. 9 , a shortage of about 40% of water in relation to the needs of wheat and maize may reduce the yield of these plants by around 20% and 39%, respectively. The results of research conducted on a global scale also indicate the negative impact of an increase in temperature on the yields of the crops most important for feeding humanity. An increase in temperature by one degree Celsius reduces the yield of maize by 7.4%, wheat by 6.0%, rice by 3.2% and soy by 3.1% 10 .

Climate change, limited water resources and the high demand for water in agriculture require an increase in the efficiency of water use 11 , 12 . This can be possible through interdisciplinary research and activities in the field of plant genetics and biology, as well as in measurement technology and agrotechnology 13 , 14 . The first condition is the monitoring of rainfall and air temperature and other climatic elements that strongly affect the water balance in the soil available to plants 15 , 16 . There are many sources of data on that topic in the literature 17 , 18 , 19 , however, they are often not very accurate and vary in terms of their measurement methodology 20 . The spatial and temporal monitoring of soil moisture is even more difficult. There are many methods for making direct and indirect evaluations of soil moisture: thermogravimetric direct method, electrometric method, capacitance method, frequency domain reflectometry, time domain reflectometry and the neutron method 21 , 22 . However, there are few systems for monitoring and collecting data that use a standardized method for soil moisture measurements, as well as for probe data registration, collection and transmission from multiple stations located over a large area. One example of such a system is the International Soil Moisture Network (ISMN) that collects data on soil moisture world-wide 23 .

The lack of accurate information concerning the spatial differentiation of soil water resources available to plants greatly limits the effectiveness and economic legitimacy of sustainable and precision agriculture. The idea of precision agriculture is the application of production means in accordance with the conditions resulting from soil variability, including its moisture, temperature and salinity 24 , 25 .

In regions with highly developed agriculture and horticulture, producers eagerly use the benefits of technical and technological progress, although in the case of precision agriculture their implementation in Europe is smaller than it is in American or Australian agriculture 26 . In the literature, however, there are few reports on the practical use of devices and systems for the monitoring of spatial and temporal variability of soil properties at a field or farm level. Also, not all modern technologies are accepted among producers, which may be due to a lack of confidence in their effectiveness 12 , 27 , 28 . It was thus hypothesized that despite the large market demand for measurement solutions, their popularization in agricultural and horticultural production would require universal, durable, technically uncomplicated, easy-to-use and inexpensive devices. Such requirements can be met by dielectric probes. The knowledge on soil moisture, temperature and salinity is needed by producers to make many decisions regarding not only plant irrigation 29 , 30 , but also for optimization of fertilization, as well as of dates and methods of tillage, sowing, cultivation measures and plant harvesting.

The purpose of the present work was to determine the demand of producers of agricultural and horticultural plants in regions with highly developed agriculture for devices and monitoring systems of soil properties at the farm level. The research was aimed at understanding the expectations of farmers and horticulturists regarding the functional features of such solutions and their expected usefulness in making production decisions. A critical analysis of the global market of available and easy-to-use dielectric solutions for assessing the continuous changes in soil properties during the vegetation period was also performed.

Material and methods

The research was carried out in the following stages:

Formulation of the research problem

Research method—survey preparation

Surveying the farmers

Characterizing respondents' farms (location, acreage, crop structure)– Tables 1 , 2 , 3

Assessment of farmers' (respondents) knowledge about the need for and possibilities of soil properties monitoring – Tables 4 , 8

A review of the state-of-the-art in relation to probes for assessing moisture, temperature and salinity of soils – Table 5

Analysis of the needs and expectations of farmers in relation to the probes depending on the actual farming conditions– Tables 6 , 7 , 9 , Figs.  1 , 2 , 3

Multivariate analysis of the features that farmers would expect from soil properties monitoring probes’– Figs.  4 , 5 , 6 , 7 , 8 , 9

The source material consisted of the results of the survey and face-to-face interviews. The respondents consisted of owners or managers of farms in Poland. The respondents were selected randomly during the most important national agricultural meetings in the summer of 2018. 1087 correctly completed questionnaires were obtained, which is a representative sample. The respondents had farms located in 246 communes in 10 voivodeships (provinces) that have the most developed agricultural and horticultural production in Poland. The selection of the research area, the diversity of respondents and the economic potential of their farms enables a generalization of the obtained results to many agricultural regions in Europe and in the world.

The survey questionnaire and the interview scenario included questions about the location of the farm, its area, the species structure of cultivated plants and the use of crop irrigation. The questionnaire also included questions about the farmers’ knowledge of the impact of soil properties on the applicability and effectiveness of cultivation elements. The resulting data was subjected to mathematical and statistical analysis. By grouping results according to a given criterion, incorrectly filled questionnaires were removed. Excel 2016 spreadsheet (Microsoft Corporation) and the statistical package Statistica 12.5 (StatSoft Inc.) were used to analyse the results. Multivariate analyzes were also performed. The results of the surveys and interviews were analyzed in three groups of respondents: territorial - voivodship, area - farm size, and crop structure - species and groups of crops. In the first group, there were 7 cases (with over 10 respondents in each voivodeship), in the second group-11 cases, and in the third - 7 cases. The variables were the functional and technical features (16 traits) of probes for monitoring soil moisture and salinity as well as the agronomic functionalities of these probes that were expected by farmers to be useful for optimization of plant production. The percentages of respondents' positive responses were treated as values for individual features and were standardized prior to cluster analysis and principal component analysis. The result of the cluster analysis is presented as a dendrogram after using Ward's method for grouping cases. The results of the principal component analysis (two principal components) are presented as a projection of primary variables on the plane.

Characteristics of the surveyed population

The largest number of surveys were carried out in regions with well-developed field agricultural and horticultural production—vegetable growing, fruit growing, i.e. in Kuyavian-Pomeranian voivodship - 55.0% of surveys, Greater Poland - 23.4%, Pomeranian—9.8%, as well as in West Pomeranian, Lublin, Lubusz and Łódź—each more than 1% (Table 1 ).

The surveys covered farms diversified in terms of their area, the type of agricultural land and the crop structure. The test sample included both small farms and large agricultural enterprises with several thousand of ha of agricultural land. The largest group was family farms with an area of 20–40 ha, which represented close to 30% of the tested sample (Table 2 ). The percentage of large (> 100 ha) and very large (> 1000 ha) farms was higher than it would have been if based on their share in the area structure of agricultural holdings in Poland. This is a consequence of the data collection methodology. The survey questionnaire and direct interviews were carried out mainly among the owners and users of farms with a high production potential and, to a lesser extent, in small farms producing for self-supply of the family and not having a commercial character. On the other hand, such a disproportion in the surveyed area groups of farms is justified, since farms with larger areas constitute more of a potential market for devices for monitoring the soil moisture, temperature and salinity.

A particularly important group of the respondents interested in soil moisture monitoring were the owners of farms who grow commodity crops while applying irrigation. Thus, farms were grouped based on the presence (Yes, No) of irrigation systems and cultivated plants. Only 11 farms (1.01%) did not grow basic cereals. These were farms specialized in the cultivation of vegetables, maize or other crops (Table 3 ). Irrigation was used mainly in horticultural crops—in vegetables—on 29% of farms, orchards - 44% of farms and potatoes - 17%. In Poland, however, farms cultivating basic agricultural crops, such as cereals, oilseed rape and sugar beet, dominate. In the study group, these crops constituted 99.0%, 56.5% and 69.9% of holdings, respectively, and about 10% of them apply irrigation.

Direct interviews were conducted in 15 locations with producers of agricultural, horticultural and special crops. These were farms of various sizes and production types: three large-area farms (450–1400 ha) mainly engaged in the production of agricultural crops; three large-area (600–1100 ha) and three smaller (20–80 ha) farms focused on the production of vegetables; four fruit farms; as well as two farms growing hops.

All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects—the questionnaires were anonymous. All respondents were of legal age. None of the experimental protocols required approval by licensing committees or institutions.

Results and discussion

The survey results indicate that for most farmers and horticulturists in Poland, i.e. for 96.7% of respondents, the knowledge of soil properties, including moisture, is important, although currently only 4.3% of them monitor soil moisture in their farms (Table 4 ). It follows from this data that farmers are aware of the impact of soil moisture on the conditions and effects of plant cultivation, but they have no possibility to make an ongoing assessment and analysis. Such a situation exists not only in Poland, but also in other countries. Even innovative and well-developed soil moisture monitoring systems on a global scale have limited application in individual farms 23 , 29 . Farmers expect easy-to-use devices that monitor soil moisture for the purpose of making optimal decisions when irrigating arable crops 30 . Similar expectations of Polish farmers were confirmed by the high percentage of respondents - 83.2%, who declared the willingness to test such devices in their farms prior to purchasing them.

Our analysis of the available commercial solutions on the market indicates that the currently available monitoring systems are imperfect and difficult in direct use on a farm, e.g. soil moisture measurement is dependent on its salinity and temperature, measuring probes require connections using a cable and have limited wireless connection options available and no battery power supply. Only one of the solutions cited is completely wireless (HYDRA 100 Scout). It uses, however, capacitive probes, whose measurements are affected by a systematic error resulting from the influence of salinity and soil texture (Table 5 ).

Despite an awareness of the present technical limitations, the majority (88.4%) of respondents declared their willingness to purchase and use one or more probes to assess the moisture, salinity and temperature of the soil in their farms. A condition was that the probe manufacturers eliminate current design imperfections (Fig.  1 ). In total, the sample of surveyed farmers declared a demand for 2905 probes, and most of them said they would build a system for monitoring soil properties in their farm in the future. The largest groupof the respondents, 31%, declared an interest to purchase only one probe, but 18.7% said two probes and about 10% of respondents declared an interest to purchase 10 or more probes. This number of devices could make it possible to build a measurement network for monitoring soil properties in a farm. The obtained declarations of purchase and use indicates the farmers' desire for simple, easy-to-use devices for assessing soil moisture, salinity and temperature. According to the results of studies conducted by Jury and Vaux 31 and Regan et al. 32 , such a high demand for soil moisture monitoring systems may also be a result of the growing economic and ecological awareness of agricultural producers. The consumption of water in agricultural production amounts to approximately 75% of the available global freshwater resources, and it is increasing. Therefore, conserving water is a duty of everyone, including farmers regardless of their type and size of production.

figure 1

Number of respondents declaring willingness to buy and use a certain number of probes.

Although a statistically significant relationship was found between the surface area of the farm and the declared number of devices for purchase and use, the correlation coefficient r = 0.223 was low (Fig.  2 ). Therefore, the size of the farm was not a very important premise that farmers followed when declaring their willingness to purchase equipment for the ongoing assessment of soil properties. Such results confirm, therefore, a high awareness of farmers on the impact of soil moisture, salinity and temperature on production efficiency and the environment. This is also indicated by a stronger relationship between the irrigated area within the farm and the declared number of probes to be used. There the correlation coefficient was r = 0.352 (Fig.  3 ). This is justified because irrigation is a high-cost element of cultivation technology, and its effectiveness depends on the properties of the soil. Therefore, the knowledge of its moisture, but also temperature and salinity, allows one to optimize the time of irrigation, the dose of water and coexisting fertilizing. The spatial variability of soil also contributes to the need for accurate monitoring of soil properties in an irrigated field 33 .

figure 2

Relationship between the size of the farm and the declared number of purchases and use of probes.

figure 3

Relationship between irrigated area on the farm and the declared number of purchase and use of probes.

The characteristics of an agricultural holding, i.e. the structure of the cultivated plants and the use of an irrigation system, did not have a major impact on farmers' interest in the use of equipment for an ongoing assessment of soil properties. This interest was high and varied from 65.1 to 84.5%, depending on the farm characteristics (Table 6 ). In the group of farmers using irrigation systems, as many as 83% indicated the willingness to have an average of 4.15 probes in their farms. Also, in the group of farms not currently using irrigation, the interest in these devices was high with 74% of farmers declaring a potential average use of 3.51 probes. In farms not cultivating cereals, and thus having other intensive crops, the interest in these devices was the highest with an average of 7.5 probes per farm. The data in Table 6 show that a relatively high interest in devices for monitoring soil moisture, salinity and temperature was demonstrated by farmers growing various groups of plants. However, the largest number of such declarations were made by farmers growing vegetables - 84.5%. Farmers growing oilseed rape were also more interested in using probes than those who do not grow oilseed rape. It follows, therefore, that those most interested in the use of these devices were farmers growing crops generating potentially large profits, but requiring favourable soil conditions the correction of which is possible by agricultural practices when the soil moisture, salinity and temperature are known.

The declarations of purchase and use of devices for the assessment of soil properties were regionally diversified (Table 7 ). All surveyed farmers and horticulturists from the Mazovian voivodeship expressed a need to have such devices in their farms. It should be noted that this is the region of Poland with the largest concentration of orchards and with many vegetable farms. In the West Pomeranian voivodeship, in turn, there are large-scale agricultural farms, whose users have a high awareness of spatial and temporal diversification of soil properties and their impact on the effectiveness of agricultural practices 34 . These conditions were also emphasized by farmers in direct interviews.

More than 90% of farmers in the surveys declared that the knowledge of soil moisture, temperature and salinity is helpful in determining the starting date of field works and sowing (Table 8 ). In their opinion, the influence of soil properties on other cultivation practices, such as fertilization, plant protection and irrigation, is also high. Such reasoning is fully justified, because the soil properties determine its bearing capacity and traction capacity for tractors and agricultural machinery without adversely affecting the soil structure 35 , 36 . Soil temperature and moisture, in turn, are the basic factors of seed germination and plant growth 37 , 38 , 39 .

Most farmers have recognized that the most important features that characterize a good soil monitoring device are measurement accuracy and reliability. Also important are the ability to assess soil properties at various depths, wireless data transmission and estimation of the water dose during irrigation based on the obtained soil moisture and temperature (Table 9 ). According to farmers, the price of equipment is also important although it is not the most important.

Farmers' expectations regarding the performance features of the probes and the possibility of using them in the optimization of agrotechnical treatments were most similar in the Kuyavian-Pomeranian (KP), Greater Poland (WP), and Pomeranian (PM) voivodships. These are regions of Poland with typical family farms conducting commercial crop and livestock production with the inclusion of horticultural production. Farmers from the Lubusz (LU) and West Pomeranian (ZP) voivodships—regions of north-western Poland with large-scale farms focused mainly on field crop production—had the most different expectations for the probes (Fig.  4 ).

figure 4

Clusters of voivodeships with similar farmers' expectations regarding the features of probes for the assessment of soil moisture and salinity. Voivodeship: KP-Kuyavian-Pomeranian, LE-Lublin, LU-Lubusz, LÓ-Łódź, PM-Pomeranian, WP-Greater Poland, ZP-West Pomeranian.

According to the expectations of farmers from their respective voivodeships of Poland, the most important features are the possibility of using the probes to optimize the method and dose of fertilization 5 and the choice of the date of fertilization of plants 7 , which is not correlated with the aforementioned characteristic—it is the greatest contribution to the first component. According to the respondents, the operational features of the probes are also important, such as their reliability, ease of use, transfer of results to a smartphone/computer, and using the results for irrigation of plants 12 , 13 , 14 , 15 , 16 —it is the second main component (Fig.  5 ).

figure 5

Main components of farmers' expectations in voivodeships with regard to the possibility of using probes to optimize agrotechnical treatments: 1-date of commencement of field works, 2-sowing date, 3-sowing depth, 4-method of soil cultivation, 5-method and dose of fertilization, 6-application of plant protection products, 7-dates of fertilization. With regard to performance characteristics of the probes: 8-price, 9-measurement accuracy, 10-measurement range 0–15 cm, 11-measurement range 0–30 cm, 12-reliability and resistance to damage, 13-easy installation and operation, 14-sending results to a smartphone/computer, 15-information about the dose of water for irrigation, 16-measurement data used to automate irrigation.

Farmers' expectations regarding the functionality of the probes were related to the size of their farms (Fig.  6 ). The expectations of the farmers of the smallest farms (up to 20 ha) and large farms (of 800–1000 ha) were the most divergent. The requirements of owners of farms with a smaller area, up to 400 ha, differed from the requirements of farmers running production on farms with a large area.

figure 6

Clusters of farms of different areas (ha) with similar expectations of farmers in terms of the features of the soil moisture and salinity probes.

Farmers expected, above all, that the probes would facilitate the use of plant protection products 6 and that they would be reliable, easy to use, and reported results online 12 , 13 , 14 —these are the first main component (Fig.  7 ). Probes should also optimize the choice of the date of commencing field works 1 and fertilization of plants 5 , 7 with the ability to measure soil moisture and salinity both in the 0–15 cm and 0–30 cm layer 9 , 10 —these are the second main component.

figure 7

Main components of expectations of owners of farms with different areas with regard to the possibility of using probes to optimize agrotechnical treatments: 1-date of commencement of field works, 2-date of sowing, 3-sowing depth, 4-method of soil cultivation, 5-method and fertilization dose, 6-application of plant protection products, 7-dates of fertilization. With regard to functional features of the probes: 8-price, 9-measurement accuracy, 10-measurement range 0–15 cm, 11-measurement range 0–30 cm, 12-reliability and resistance to damage, 13-easy installation and operation, 14-sending results to a smartphone/computer, 15-information on the dose of water for irrigation, 16-measurement data used to automate irrigation.

The dendrogram (Fig.  8 ) shows that farmers' expectations as to the performance of the probes depended on the crops they were growing. The first group were farmers growing agricultural crops such as cereals, beetroot, rape and others, and the second group were potato growers. Horticulturists growing vegetables and fruit trees are separate groups.

figure 8

Clusters of groups of plants cultivated by farmers with similar expectations regarding the features of soil moisture and salinity probes.

Farmers cultivating various groups of plants declared that probes monitoring soil moisture and salinity should help in choosing the optimal date of field works 1 , including the date of sowing 2 , the method of soil cultivation 4 and the method and dose of fertilization 5 —these are the first main component. In addition, the probes should optimize the fertilization date 7 and plant irrigation 15 , 16 on the basis of information sent to a smartphone/computer 14 —these are the second component (Fig.  9 ).

figure 9

Main components of the expectations of farmers cultivating various groups of plants with regard to the possibility of using probes to optimize agrotechnical treatments: 1-date of commencement of field works, 2-sowing date, 3-sowing depth, 4-soil cultivation, 5-method and fertilization dose, 6-application of plant protection products, 7-dates of fertilization. With regard to functional features of the probes: 8-price, 9-measurement accuracy, 10-measurement range 0–15 cm, 11-measurement range 0–30 cm, 12-reliability and resistance to damage, 13-easy installation and operation, 14-sending results to a smartphone/computer, 15-information about the dose of water for irrigation, 16-measurement data used to automate irrigation.

Direct interviews conducted with a representative sample of farmers confirmed the results of the survey. Regardless of the production sector (agriculture, horticulture), farm size or region, farmers emphasized the need for ongoing monitoring of soil properties, mainly moisture, temperature and salinity. This knowledge would allow them to optimally use the natural fertility of the soil, increase the efficiency of the use of means of production and make agricultural production more environmentally friendly. In their opinion the monitoring of rainfall and air temperature is no longer sufficient. Agricultural producers in Poland believe that nowadays soil properties should be assessed not only on the farm scale, but also in fields and even in fragments of a field. Creating monitoring systems would allow for rational and precise application of water, mineral fertilizers or plant protection products. It would also be easier to make decisions on scheduling work in fields and in orchards. The interviewees pointed to such solutions already functioning in Poland, but adapted to drought monitoring throughout the whole country 40 . Widespread monitoring of soil properties in the farm, especially of monitoring systems, requires a technical improvement in the devices for assessing soil moisture, salinity and temperature. Jones et al. 41 indicate a problem that may explain why such a small percentage of farmers use soil moisture probes: the growing number of new sensors across the globe is creating a market filled with confusing choices for consumers and decreasing market share for producers. Without informed consumer choices a product price point may be controlled more by advertising advantage than by product performance and quality. Technical problems connected to the operation and servicing of such equipment are the most frequent reasons for discontinuation of their use by respondents. In the farmers' opinion, these devices must be easy to use and reliable. However, as in drought monitoring 42 , they should contain advanced computer applications for optimizing and verbalizing cultivation recommendations.

Conclusions

Currently, only 4.0% of the surveyed agricultural and horticultural farms use soil moisture, temperature and salinity probes, but as many as 80% of the respondents declare their willingness to purchase and use them. Most of the respondents report the need to purchase one probe for point measurements of soil parameters, but about 10% of respondents declared their willingness to purchase the probes in quantities that would allow for the creation of farmland monitoring systems.

The price of the probes in question is not the most important purchase criterion. Farmers and horticulturists expect probes that are easy and practical to use, reliable and durable. Agricultural producers are also interested in applications that facilitate making agrotechnical decisions.

The obtained results prove the insufficient supply of probes meeting the expectations of agricultural producers and show a high demand for equipment with appropriate utility features. The analysis of currently available solutions shows that the market does not offer such devices. This is also confirmed by the opinions of the respondents.

The vast majority of farmers are aware of the importance of assessing soil moisture, temperature and salinity in optimizing irrigation, fertilization and pesticide application as well as other agrotechnical procedures.

Farmers using irrigation systems, as well as producers of vegetables and rape, showed a greater interest in purchasing probes. A territorial differentiation in this respect was also shown.

The presented results could be used by the manufacturers of soil moisture, salinity and temperature probes in order to provide devices more suited to the farmers’ needs and expectations and to adjust their marketing strategies.

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This work was funded by the Polish National Centre for Research and Development in the framework of Project No. TANGO2/340132/NCBR/2017.

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Gałęzewski, L., Jaskulska, I., Jaskulski, D. et al. Analysis of the need for soil moisture, salinity and temperature sensing in agriculture: a case study in Poland. Sci Rep 11 , 16660 (2021). https://doi.org/10.1038/s41598-021-96182-1

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Wireless powered moisture sensors for smart agriculture and pollution prevention: opportunities, challenges, and future outlook.

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Performance Tests of Loadcell as Real-Time Moisture Content Sensor: Case Study Moringa Oleifera Leaves Drying

E K Pramono 1 , A Taufan 1 , Novrinaldi 1 , S A Putra 1 , M A Karim 1 , A Haryanto 1 and S I Kuala 1

Published under licence by IOP Publishing Ltd IOP Conference Series: Earth and Environmental Science , Volume 1024 , The 3rd International Conference on Agricultural Postharvest Handling an Processing 12/10/2021 - 14/10/2021 Bogor, Indonesia Citation E K Pramono et al 2022 IOP Conf. Ser.: Earth Environ. Sci. 1024 012018 DOI 10.1088/1755-1315/1024/1/012018

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Drying agricultural products with solar power to maintain quality and extend the shelf life of agricultural products has been done by many farmers because it is easy and cheap to do. However, the availability of solar heat may be different every day and is greatly influenced by weather conditions causing different drying times for the product to reach the desired moisture content. This research aimed to develop a real-time water content measurement system during drying process. Measurement of water content was carried out using a weight measurement approach by a loadcell sensor. Data processing was carried out using the internet of things (IoT) -based digital data processor so that the measurement results could be seen remotely. Tests were carried out by drying the moringa ( Moringa oleifera ) leaves for 6 hours. A comparison measurement of water content was also done every hour by a gravimetric method using Halogen moisture meter Toledo Metler HB43-S. The results showed a good match between these two measurements in which the experiment resulted in R 2 and MAE of 0.9815 and 3.78%, respectively. The results indicate this design can be used as a real-time water content measurement instrument with considerable results.

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Measuring water activity in food production - a case study

Dr Brady Carter explains why water activity, not moisture content is the key for safer food production

Moisture content is commonly measured on food products for the purpose of making sure it will have the desired safety and quality. However, water activity, not moisture content, determines the safety of food products. Recently, a major food manufacturer became painfully aware of the importance of releasing product based on water activity and not moisture content. The company has been producing high quality product for over 20 years with little to no complaints. Then, out of nowhere, it started receiving multiple complaints about mouldy product. How could this happen? It had released product based on a set moisture content for 20 years with no problems. Without making any changes to its moisture content specification, it suddenly had mould. The cost of the subsequent recalls, public relations efforts, and causal discovery activities were in the millions of dollars.

How a water activity-based release specification would have prevented the problem

The first mistake made by this company was using a moisture content specification to control mould growth instead of water activity. Water activity is the energy status of water in the food and microorganisms don’t care how much water is present, only if the water activity (energy) is high enough for them to access it. Mould growth will be prevented if the water activity is less than 0.70 and any problems with mould growth would have been avoided if the company was using 0.65 aw as their release specification instead of moisture content.

The other contributing factor that led to the sudden appearance of mould was a change in the instrument being used to measure moisture content. This new method was unknowingly giving lower moisture content readings than the actual moisture content (Fig. 1). This means that although the company thought it was making product to the same moisture content specification; the actual moisture content was 3-4% higher. This higher moisture level now corresponded with a water activity higher than the growth limit for mould. If the company had been tracking water activity, it would have known immediately that something had changed, and that the product was now being made to water activities higher than 0.70 aw. A water activity-based release specification would have prevented any problems and saved millions in lost revenue.

How a water activity-based release specification proved to be the solution

The first change to be implemented was to immediately implement a water activity release specification below 0.70 aw and reject any product that does not meet this specification, regardless of its moisture content. This immediately stopped all complaints about mould. The second change was to correct the moisture content measurement to make sure it was giving results consistent with historical values. In so doing, the company realised that current formulations limit the amount of moisture that can be in its products and still maintain a safe water activity level. Consequently, the next step was to adjust the formulations to maximise the achievable moisture level while still meeting the newly implemented water activity release specification (Fig. 2). This will allow the food manufacturer to maximise profitability while having the security of knowing that the painful days of mouldy product are behind it.

Dr Brady Carter i9s application scientist with Neutec Greoup

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case study on moisture content

ORIGINAL RESEARCH article

Moisture content online detection system based on multi-sensor fusion and convolutional neural network.

Taoqing Yang

  • 1 College of Mechanical and Electrical Engineering, Shihezi University, Shihezi, China
  • 2 Key Laboratory of Northwest Agricultural Equipment, Ministry of Agriculture and Rural Affairs, Shihezi, China
  • 3 Key Laboratory of Modern Agricultural Machinery Corps, Shihezi, China
  • 4 College of Engineering, China Agricultural University, Beijing, China
  • 5 College of Food, Shihezi University, Shihezi, China

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To monitor the moisture content of agricultural products in the drying process in real time, this study applied a model combining multi-sensor fusion and convolutional neural network (CNN) to moisture content online detection. This study built a multi-sensor data acquisition platform and established a CNN prediction model with the raw monitoring data of load sensor, air velocity sensor, temperature sensor, and the tray position as input and the weight of the material as output. The model's predictive performance was compared with that of the linear partial least squares regression (PLSR) and nonlinear support vector machine (SVM) models. A moisture content online detection system was established based on this model. Results of the model performance comparison showed that the CNN prediction model had the optimal prediction effect, with the determination coefficient (R 2 ) and root mean square error (RMSE) of 0.9989 and 6.9, respectively, which were significantly better than those of the other two models. Results of validation experiments showed that the detection system met the requirements of moisture content online detection in the drying process of agricultural products. The R 2 and RMSE were 0.9901 and 1.47, respectively, indicating the good performance of the model combining multisensor fusion and CNN in moisture content online detection for agricultural products in the drying process. The moisture content online detection system established in this study is of great significance for researching new drying processes and realizing the intelligent development of drying equipment. It also provides a reference for online detection of other indexes in the drying process of agricultural products.

Keywords: convolutional neural network1, Prediction model2, multi-sensor fusion3, moisture content4, online detection5

Received: 06 Sep 2023; Accepted: 15 Feb 2024.

Copyright: © 2024 Yang, Zheng, Xiao, Shan and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Xia Zheng, College of Mechanical and Electrical Engineering, Shihezi University, Shihezi, China Hongwei Xiao, College of Engineering, China Agricultural University, Beijing, China Chunhui Shan, College of Food, Shihezi University, Shihezi, China Jikai Zhang, College of Mechanical and Electrical Engineering, Shihezi University, Shihezi, China

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Moisture content vs water activity: Choosing the right measurement

Moisture content (mc) and water activity (a w )  both have to do with water, but the distinction between the two isn’t always clear. is one better than the other , it doesn’t have to be complicated. these are the facts., moisture content 101: what it is moisture content and how it’s used.

Water is everywhere. In the food we eat, the air we breathe, and the objects around us – and it significantly influences the physical characteristics of all of the above. It’s no wonder that workers from carpenters to petroleum scientists want to know: How much water is in this material?

It’s a simple question. It seems like it should have a simple solution. Unfortunately, it’s  very difficult to get a clear and precise answer .

The primary challenge (though there are many) has to do with measuring only moisture while ignoring the rest of the material. Once water makes its way into a material, it often doesn’t come out as easily – which makes it difficult to remove and measure it. Other methods, like measuring moisture without removing it from its host material, present a host of other challenges.

Which method of measurement you use comes down to two factors: how precise you need to be and what tradeoffs you’re willing to make.

  • If you’re a petroleum scientist studying infinitesimal amounts of water in oil or plastic, you may be willing to spend the hours (and dollars) necessary for titration.
  • If you’re a carpenter or contractor on-site trying to decide whether a piece of wood is suitable, you may be willing to trade lab-grade precision for the ease of a quick handheld device that approximates moisture levels.
  • If you work in food or cannabis production, you might want  something in between , like loss-on drying – a relatively high precision measurement within a reasonable amount of time.

The examples above are far from all the uses for moisture content measurements, but the tradeoffs are similar no matter the industry. Whichever method you choose, beware the many misconceptions about what moisture content can and can’t tell you. A simple rule to remember is that moisture content is a “quantity” measurement.

For example, moisture content is a valuable measure of yield. Many products are sold by weight, so since water is such an inexpensive (and heavy) ingredient, measuring and managing moisture content can have an  outsized impact on profits .

Moisture content also provides information about texture, since increasing levels of moisture provide mobility and lower the glass transition temperature.

What won’t moisture content tell you? Much at all about “quality” metrics like consistency, microbial safety, moisture migration, and nutrient deterioration. Those key factors are better left to another measurement – water activity.

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Water activity 101: What it is water activity (Aw) and how is it used?

The simple amount of water in a substance doesn’t give a complete picture of the impact that water will have on the product. That’s where water activity ’s strengths lie.

Viewed too closely, water activity can appear to be a very complex concept. Fortunately, you don’t need to know the thermodynamics and obscure equations that prove how it works. The following high-level principles are enough for most people’s purposes.

  • Water activity measures how energetic and available water is.  Some substances hold a great deal of water, but the water is locked in place with molecular bonds. That makes it less able to seep out or interact with the world around it – we’d call that low water activity, even though there could be a great deal of water present. The inverse can happen, too: Some materials hold very little water that is also very accessible.
  • Water wants equilibrium.  If you leave a soft cookie outside in a dry climate, the low water activity atmosphere draws the moisture from the high water activity cookie – trying to create equilibrium. On the other hand, two materials with the same water activity level – i.e. a cake and the frosting that coats it – won’t exchange moisture with one another. In essence, water activity can tell you which direction water wants to move.
  • Water’s energy levels factor into many valuable predictions.  Like which microorganisms will grow on a substance, how its texture will change over time, when it will decompose, how it will react with other substances, and plenty more.

Water activity can be measured in a few different ways, with the two most popular being capacitance sensors and chilled mirror instruments. Capacitance sensors are often inexpensive, though at the cost of accuracy and precision. Chilled mirror devices are more accurate and are generally considered the food industry standard. Other more niche methods include resistive electrolytic sensors and tunable diode lasers.

Unlike moisture content, water activity is most helpful for managing the quality and safety of a substance or product, not quantity. As such, it has been widely adopted in the food, pharmaceutical and cannabis industries.

Which one should you use?

It depends on what you’re trying to accomplish. Neither measurement is simply better than the other. They measure very different concepts, and each offers big benefits in different environments.

If you’re trying to nail the right product weight to maximize profit, moisture content should be your go-to. If your goal is to keep a snack food crispy or crunchy for months on the store shelf, or how to put jelly inside a snack cake without the cake getting soggy, choose water activity.

For a deeper dive into the science behind these two measurements, explore our knowledge base —>

Since moisture content does affect texture, some may tell you that it’s the only measurement needed to track both quantity and quality. However, those who try that approach soon learn that it’s inefficient and nearly impossible to manage microbial growth and other quality-related issues using only moisture content.

Moisture sorption isotherms: How moisture content and water activity work together

Water activity and moisture content are valuable separately, but linking the two can reveal exactly how to solve a long list of moisture-related mysteries.

Sometimes called “moisture maps,” moisture sorption isotherms graph how moisture content and water activity levels change as moisture is adsorbed and desorbed from a material held at a constant temperature.

Stated more simply: an isotherm shows changes in both moisture content and water activity on the same graph.

Every substance has a different isotherm, and the relationship between moisture content and water activity isn’t linear and can be complex and unpredictable – until you see it mapped on an isotherm, that is.

In the past, isotherms weren’t particularly practical, due to the month or more of lab work needed to create a single graph – the process involved putting samples into desiccators then weighing them repeatedly for days or weeks to get a single data point. Newer technologies have automated the process and helped make isotherms more accessible and practical.

Today, they’re frequently used in the food industry to pinpoint the exact levels where changes like caking, clumping, and loss of texture occur, predict how a product will respond to formulation changes, estimate shelf life, and plenty more. They’re also frequently used to analyze wood, building materials and textiles.

Drawing your own conclusions

Moisture content and water activity have different strengths. Visualizing them together as an isotherm reveals more valuable insight than either one provides alone.

In many cases, neither is a one-size-fits all measurement – it all depends on the application. It’s up to the user to decide.

If you need  help deciding which is right for you  or what instrument you need, contact us. Our experts are eager to hear from you.

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Soil moisture information can improve shallow landslide forecasting using the hydrometeorological threshold approach

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  • Volume 17 , pages 2041–2054, ( 2020 )

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  • Pasquale Marino   ORCID: orcid.org/0000-0003-0801-5410 1 ,
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  • Roberto Greco 1 &
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Empirical thresholds indicating the meteorological conditions leading to shallow landslide triggering are one of the most important components of landslide early warning systems (LEWS). Thresholds have been determined for many parts of the globe and present significant margins of improvement, especially for the high number of false alarms they produce. The use of soil moisture information to define hydro-meteorological thresholds is a potential way of improvement. Such information is becoming increasingly available from remote sensing and sensor networks, but to date, there is a lack of studies that quantify the possible improvement of the performance of LEWS. In this study, we investigate this issue by modelling the response of slopes to precipitations, introducing also the possible influence of uncertainty in soil moisture provided by either field sensors or remote sensing, and investigating various soil depths at which the information may be available. Results show that soil moisture information introduced within hydro-meteorological thresholds can significantly reduce the false alarm ratio of LEWS, while keeping at least unvaried the number of missed alarms. The degree of improvement is particularly significant in the case of soils with small water storage capacity.

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Acknowledgements

The authors acknowledge the Civil Protection Agency of Campania and Servizio Informativo Agreometeorologico Siciliano (SIAS) for providing rainfall data. The research is part of the Ph.D. project “Modelling hydrological processes affecting rainfall-induced landslides for the development of early warning systems” within the Doctoral Course “A.D.I.” of Università degli Studi della Campania “L. Vanvitelli”. Most of the work was developed during Marino’s 6-month stay as a visiting researcher at the Section Water Resources of Delft University of Technology. The research has been also funded by Università degli Studi della Campania ‘L. Vanvitelli’ through the programme “VALERE: VAnviteLli pEr la RicErca”.

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Department of Civil Engineering and Architecture, University of Catania, 95123, Catania, Italy

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Marino: bibliographic research, numerical experiments, writing (original draft)

Peres: rainfall generation, writing (original draft and review)

Cancelliere: rainfall generation, writing (review)

Greco: analysis of uncertainty, methodology, supervision, writing (original draft and review)

Bogaard: methodology, supervision, writing (original draft and review)

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Marino, P., Peres, D.J., Cancelliere, A. et al. Soil moisture information can improve shallow landslide forecasting using the hydrometeorological threshold approach. Landslides 17 , 2041–2054 (2020). https://doi.org/10.1007/s10346-020-01420-8

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Received : 12 December 2019

Accepted : 21 April 2020

Published : 13 May 2020

Issue Date : September 2020

DOI : https://doi.org/10.1007/s10346-020-01420-8

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