# Seed responses to change in ambient humidity and the consequences for storability

Shabnam Rezaei, Julia Buitink, Fiona R Hay

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Shabnam Rezaei, Julia Buitink, Fiona R Hay. Seed responses to change in ambient humidity and the consequences for storability. Journal of Stored Products Research, 2024, 109, pp.102477. 10.1016/j.jspr.2024.102477. hal-05079896

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# Seed responses to change in ambient humidity and the consequences for storability

Shabnam Rezaei<sup>a</sup>, Julia Buitink<sup>b</sup>, Fiona R. Hay<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Ambient humidity

Moisture sorption hysteresis

Moisture isotherm

Seed longevity

Seed storage

Water activity

## ABSTRACT

The study of seed moisture sorption isotherms helps us to understand the response of seeds to changes in moisture in their environment and how this might impact their longevity. A seed moisture isotherm shows the relationship between moisture content (MC) and water activity ($a_w$) at a constant temperature. Seed MC is higher when it dries from a hydrated state (desorption) than when it rehydrates from a very dry state (adsorption) to the same RH, an effect known as hysteresis. Seeds on the desorption isotherm will age faster than seeds on the adsorption isotherm at the same RH due to higher MC, resulting in a more rapid decline in seed viability and vigour. In this study, moisture isotherms of seeds from diverse species were determined and modeled using the Guggenheim-Anderson-de Boer (GAB) model; the hysteresis effect was then quantified. The maximum hysteresis magnitude varied between 0.3 and 2.7% MC (occurring between 49 and 57% RH) when seeds were dried over silica gel (below 11% RH) and rehydrated. Cycling seeds between 30 and 50%, 20 and 50%, and 15 and 50% RH, it was found that seeds of barley, lupin, buckwheat, wheat and pea switch to the adsorption isotherm upon rehydration after drying at intermediate moisture levels. Thus, adsorbing seeds of these species have greater longevity than desorbing seeds at the same RH. In contrast, seeds of oilseed rape and yellow mustard do not show a hysteresis effect unless they are dried to very low MC (over silica gel), so the adsorbing seed longevity remains the same as desorbing seeds at a constant RH. It is important to consider the moisture history of seeds depending on species for better seed storage management.

## 1. Introduction

For orthodox (desiccation tolerant) seeds, as soon as there ceases to be a functioning vascular connection between the developing seed and the maternal plant, the seed moisture content (MC) depends on the humidity (relative humidity, RH) of the air surrounding the seed. There will be an initial rapid loss of moisture as the seeds first dry to equilibrium with ambient RH, according to the relationship described by the moisture desorption isotherm. The moisture desorption isotherm shows the relationship between seed MC and corresponding water activity ($a_w$) at a specific temperature for seeds that are losing moisture (Basu et al., 2006; Hay et al., 2022). Thereafter, it is expected that the $a_w$ of most seeds will then fluctuate with the diurnal variation in RH until such time as they are dispersed or taken from the parent plant. An obvious exception to this behaviour of fluctuating MC are seeds with an impermeable seed coat; these seeds lose moisture but do not absorb moisture again unless the barrier of the seed coat is somehow overcome (Baskin

and Baskin, 2004). For seeds which do readily take up moisture, the consequence may be relatively rapid decline in seed quality due to the higher MC; it is well documented that the rate of ageing increases as MC increases (Ellis and Roberts, 1980a, 1980b; Roberts and Ellis, 1989). However, this response may be tempered if the seeds switch to the moisture adsorption isotherm, rather than staying on the desorption isotherm. If seeds switch to the adsorption isotherm, ambient RH must increase over the RH at which the seeds have been dried, for them to take up moisture. This also applies to seeds that have been dispersed and are prone to fluctuating environmental conditions or, more critically, seeds that have been collected or harvested for conservation or commercial reasons. Seed moisture sorption behaviour is therefore relevant from both an ecological perspective and the 'fitness' of seeds in the environment, and from a seed production perspective, where seed quality determines the value of a commercial seed lot or for example, the longevity of a conservation seed lot.

Seed moisture sorption isotherms can be determined by equilibrating

<footnote><sup>a</sup> Department of Agroecology, Aarhus University, Forsøgsvej 1, Slagelse, 4200, Denmark</footnote>

<footnote><sup>b</sup> INRAE, Institut Agro, Université d'Angers, IRHS, SFR QUASAV, 49000, Angers, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: fiona.hay@agro.au.dk (F.R. Hay).</footnote>
seeds in a specific RH environment (for example, by using salt solutions) or by adjusting the MC (based on change in weight change during drying or humidification) (Hay et al., 2023) and then determining the $a_w$ and MC of the seeds (Hay et al., 2008, 2022). The $a_w$ should correspond to the RH of the equilibration environment divided by 100; the $a_w$ scale thus ranges between 0 and 1 whereas the RH scale ranges between 0 and 100%. The isotherm shape and position vary between seeds of different species and are affected by temperature (Hay et al., 2022). Most or- thodox seeds show typical type II isotherm characteristics, being 'reverse sigmoidal' or 'S-shaped' (Brunauer et al., 1940; Vertucci, 1989). Among numerous mathematical models used to describe the sorption isotherms of food products, the Guggenheim-Anderson-de Boer (GAB) model, a kinetic multilayer model, is recommended as a fundamental model and has been used in numerous studies (Prado and Sartori, 2009; Caballero et al., 2015). This model has the advantage of a theoretical basis compared with other isotherm models; it is also suitable for modelling experimental data up to $a_w = 0.9$ (De Oliveira et al., 2017). The GAB model includes a parameter termed "monolayer moisture content" ($M_m$). The $M_m$ is the point below which in theory, ionic-bound water can no longer participate in chemical reactions (Caballero et al., 2015). Above $M_m$, more water becomes available to act as a solvent, resulting in an acceleration of reactions and, in the case of food products, a decline in stability and hence quality (Bell and Labuza, 2000). Below $M_m$, in foods susceptible to unsaturated lipid oxidation such as processed cereals, the rate of shelf-life loss also increases as $a_w$ decreases (Bell and Labuza, 2000; Rahman and Labuza, 2007). Similarly, under open storage conditions, a similar decline in longevity at very low moisture levels has been observed for seeds (Vertucci and Roos, 1990; Vertucci et al., 1994).

Moisture sorption hysteresis is the difference in MC at a given RH when a material is on the adsorption isotherm compared with when it is on the desorption isotherm (Yang et al., 1997). This hysteresis phenomenon has not been systematically studied in seeds. Our recent research showed that if seed lots (oilseed rape and barley) are stored at a specific RH, depending on whether they equilibrate to that RH by de- or adsorption can strongly influence their longevity (Rezaei et al., 2023). In other words, the moisture history of the seeds is an important consideration for seed shelf-life. The difference in the longevity of desorbing vs. adsorbing seeds was attributed to the difference in the MC of the seeds. We also found that seeds of both species respond differently to a subsequent increase in RH (50%) when they have been dried at a lower RH (30%), which could profoundly affect their longevity (Rezaei et al., 2023).

Here, we wanted to further explore the hysteresis phenomenon for seeds from a variety of species, differing in lipid content: oilseed rape and yellow mustard, which have a high seed oil content, and lupin, barley, wheat, buckwheat and pea, which have a low seed oil content. We hypothesized that the magnitude of the hysteresis loop would be greater for seeds with a low oil content and that these seeds would also be more likely to switch to the adsorption isotherm following partial drying at an intermediate RH. We also considered whether contrasting responses to changes in ambient RH could be related to the $M_m$. The potential impact of contrasting moisture sorption behaviour on seed longevity and hence quality is discussed.

# 2. Material and methods

## 2.1. Materials

Seeds of winter oilseed rape (Brassica napus L. var. DK-Exlibris), spring barley (Hordeum vulgare L. var. KWS Irina) and pea (Pisum sativum L.) were received on August 9, 2022 from the experimental fields at the Department of Agroecology, AU-Flakkebjerg. Yellow mustard (Sinapis alba L.), buckwheat (Fagopyrum esculentum Moench), lupin (Lupinus albus L.) and wheat (Triticum aestivum L.) seeds, produced at the same location, were received on August 15, 2022. Immediately after harvest, seeds were threshed and cleaned, and immediately transferred

to the laboratory, minimizing exposure to ambient conditions. Initial water activity ($a_w$) was determined using a Rotronic AwTherm (C.K. Environment A/S, Værloase, Denmark) (Rezaei et al., 2023). The initial $a_w$ of oilseed rape, yellow mustard, lupin, barley, wheat, buckwheat and pea seeds was 0.857, 0.699, 0.693, 0.828, 0.773, 0.817 and 0.646, respectively. A gravimetric seed moisture content (MC) determination was made using the 'low constant temperature oven method' (ISTA, 2021) for oilseed rape, yellow mustard and barley seeds: three replicates of 2–5 g samples of seeds were dried at 103 °C for 17 ± 1 h. The MC of lupin, wheat, buckwheat and pea seeds was determined using the 'high constant temperature oven method' (ISTA, 2021). Samples of seeds were ground in a tube mill (C S000, IKA, Germany) at 24,000 rpm for 5 s and then three replicates of 2–5 g ground seeds were dried at 130 °C for 1 (lupin and pea seeds) or for 2 h (wheat and buckwheat seeds). Immediately after seeds were removed from the oven, all samples were allowed to cool in glass desiccators with silica gel at room temperature for 1 h before reweighing. Seed MCs are calculated and expressed on a dry weight (d.wt.) basis (Hay et al., 2023). To determine seed oil content (d.wt. basis), 100 g samples of seeds of each species were sent to Eurofins Danmark A/S (www.eurofins.dk) in August 2022.

## 2.2. Moisture isotherm determination

Non-saturated LiCl salt solutions giving different relative humidities (between 11 and 95%) were prepared according to Hay et al. (2008). The solutions were placed in air-tight boxes (Ensto Cubo electrical enclosure boxes, Ensto Industry, Finland) at 45 °C a week before the seeds were arranged in dishes on a tray over the solution and allowed to equilibrate. Equilibration of samples of seeds took between 1 and 5 weeks depending on experiment conditions. The moisture isotherm experimental design is summarised in Supplementary Table 1.

## 2.3. Moisture desorption-adsorption cycling

Separate seed samples of oilseed rape, yellow mustard, lupin, barley, wheat, buckwheat and pea (freshly harvested, $a_w > 0.7$) were cycled between (i) 50 and 30% RH; (ii) 50 and 20% RH; and (iii) 50 and 15% RH for two cycles, over LiCl salt solutions at 45 °C (Rezaei et al., 2023). After 2–4 weeks in each equilibration environment, seed water activity ($a_w$) at 45 °C and MC were determined, as before.

## 2.4. Statistical analysis and model fitting

The moisture desorption-1, adsorption-1, desorption-2 and adsorption-2 isotherm data were analysed by non-linear curve fitting in OriginPro 2022 (OriginLab Corporation, Massachusetts, USA) to fit the Guggenheim-Anderson-de Boer (GAB) model (Bell and Labuza, 2000):

$$MC = \frac{M_m Cka_w}{(1 - ka_w)(1 - ka_w + Cka_w)} \quad (Eqn. 1)$$

where $M_m$ represents the MC of the monolayer (expressed as a percentage of dry weight). Constant C corresponds to the water sorption heat of the first layer and constant k is associated with the water sorption heat of the multilayer.

The magnitude of MC hysteresis was calculated as the difference in MC between the GAB model-fits of desorption and adsorption isotherms. The corresponding $a_w$ at $M_m$ was also calculated from the fitted GAB model for each isotherm (Toğrul and Arslan, 2006):

$$a_w = \frac{\sqrt{C} - 1}{K(C - 1)} \quad (Eqn. 2)$$

The $a_w$ at the lower and upper closure points of the hysteresis loop were calculated by solving the equation:
$$f(a_w) = \frac{M_{md}C_dK_d}{(1 - K_d a_w)(1 - K_d a_w + C_d K_d a_w)} = \frac{M_{ma}C_aK_a}{(1 - K_a a_w)(1 - K_a a_w + C_a K_a a_w)} \quad \text{(Eqn. 3)}$$

where $M_{md}$, $C_d$ and $K_d$ are the GAB constants for the desorption isotherm and $M_{ma}$, $C_a$ and $K_a$ are the GAB constants for the adsorption isotherm.

The maximum $\Delta_{Hysteresis}$ MC (%) and $a_w$ at maximum $\Delta_{Hysteresis}$ MC (%) were calculated by taking the derivative of equation (3) ($f'(a_w) = 0$) which gave the maximum $a_w$, where ($f(\text{maximum } a_w) = \max f(a_w)$).

# 3. Results

## 3.1. Moisture sorption isotherms and hysteresis

Fitting the Guggenheim-Anderson-de Boer (GAB) model (Eqn. (1)) to the isotherm data accounted for 99.1–99.9% of the variance for each sorption cycle × species (Table 1). All of the isotherm curves showed the typical sigmoidal shape and can be classified as type II according to the classification scheme of Brunauer et al. (1940). However, the actual shape of isotherms differed for seeds of different species (Fig. 1). The isotherm curves spanned a greater MC range in lupin, barley, wheat, buckwheat and pea compared with oilseed rape and yellow mustard seeds. At $a_w$ between 0.2 and 0.8, the MC for seeds of different species ranged from 2.8 to 11.3% dry weight (range equal to 8.5%, based on fitted isotherms) for oilseed rape; 3.7–14.1% (range 0.4%) for yellow mustard; 5.6–22.2% (range 16.6%) for lupin; 5.4–17.9% (range 12.5%) for barley; 5.7–18.1% (range 12.4%) for wheat; 7.2–19.5% (range 12.3%) for buckwheat; and 6.6–19.3% (range 12.7%) for pea.

For all species, the seed MC consistently maintained higher levels during desorption compared with adsorption (Fig. 1; Supplementary Fig. 1). The magnitude of the hysteresis differed between species and first increased and then decreased with increasing RH for all seed species (Fig. 2A). The $a_w$ at the lower and upper closure points where the adsorption and desorption curves intersected for seeds of different species varied, being 0.116 and 0.768 (yellow mustard), 0.108 and 0.786 (oilseed rape), 0.031 and 0.757 (lupin), 0.064 and 0.869 (barley), 0.136 and 0.751 (wheat), 0.097 and 0.828 (buckwheat), and 0.162 and 0.823 (pea) (Fig. 2A; Table 1). The maximum $\Delta_{Hysteresis}$ MC values were 0.3, 0.8, 1.2, 1.4, 1.7, 2.0 and 2.7% dry weight for yellow mustard, oilseed

rape, barley, lupin, buckwheat, wheat and pea seeds, respectively, occurring at $a_w$ of 0.55, 0.58, 0.55, 0.50, 0.54, 0.49 and 0.57 (Fig. 2B and C). A negative correlation was found for the $\Delta_{Hysteresis}$ MC values and oil content (%) relation. The $a_w$ where hysteresis was maximum was not significantly correlated with oil content ($P = 0.17$).

The $M_m$ values varied between species, increasing with decreasing oil content (Table 1, Fig. 3). Except for the seeds with high oil content, GAB modelling demonstrates that the $M_m$ values are lower for the adsorption curve compared to the desorption curve, being 2.70, 3.61, 6.96, 7.42, 10.87, 9.54 and 11.72% dry weight for oilseed rape, yellow mustard, lupin, barley, wheat, buckwheat and pea seeds, respectively, for the desorption-1 curve and 3.01, 3.30, 5.40, 6.77, 5.77, 6.80 and 6.27% dry weight for the adsorption-1 curve (Fig. 3A). The $M_m$ values of the second cycle of desorption and adsorption-2 were, respectively, 3.1 and 2.4 for oilseed rape and 6.8 and 5.2 for barley seeds, again being lower for desorbing than adsorbing seeds. The $a_w$ at $M_m$ of desorption curves ranged from 0.154 to 0.462 (respectively, desorption-1 of oilseed rape and desorption-1 of wheat) (Fig. 3B). This was higher than the $a_w$ at $M_m$ of adsorption curves, which ranged from 0.122 to 0.293 (respectively, adsorption-2 of oilseed rape and adsorption-1 of barley). The de- or adsorption $M_m$ and corresponding $a_w$ values at $M_m$ were each significantly negatively correlated with seed oil content ($P < 0.05$).

## 3.2. Moisture desorption and adsorption cycling between 50 and 30% RH, between 50 and 20% RH and between 50 and 15% RH

Drying seeds to 11% RH before equilibrating them to high RH led to hysteresis (Fig. 2A). Next, we investigated how much seeds need to be dried (minimum RH) to induce the hysteresis effect. For this, sequential cycles of drying (at 15, 20 and 30% RH) and then rehydration (at 50% RH) were performed. The results differed between seeds of the different species (Supplementary Fig. 1). For oilseed rape, there was no noticeable hysteresis: when the seeds were dried to 0.129 $a_w$ (equivalent to 2.5% MC) and then rehydrated in two cycles at 50% RH, their MC did not significantly differ from that of seeds dried at 50% RH ($P > 0.05$; except for red and blue data points 5 which were higher than the desorption curve) (Fig. 4; Supplementary Fig. 1A). Similarly, for yellow mustard seeds, no hysteresis was observed when seeds were dried to 0.189 $a_w$ (3.5% MC) and then rehydrated at 50% RH ($P > 0.05$) (Fig. 4; Supplementary Fig. 1B; except for black data point 5 which was higher). It

<caption>**Table 1**</caption>

Parameter estimates of the GAB model (Eqn. (1)) for one cycle of desorption and adsorption for yellow mustard, lupin, wheat, buckwheat and pea seeds, and two cycles of desorption and adsorption for oilseed rape and barley (data and fitted isotherms shown in Fig. 1). The $a_w$ at $M_m$ was calculated using Eqn. (2). The $a_w$ at the lower and upper closure points were calculated using Eqn. (3).

<table><thead><tr><th rowspan="2">Species</th><th rowspan="2">Oil content<sup>a</sup> (% d.w.)</th><th rowspan="2">Isotherm cycle</th><th rowspan="2">C (estimate and s.e.)</th><th rowspan="2">K (estimate and s.e.)</th><th rowspan="2">M<sub>m</sub> (estimate and s.e.; % d.w.)</th><th rowspan="2">R<sup>2</sup></th><th colspan="3">a<sub>w</sub></th></tr><tr><th>at M<sub>m</sub></th><th>at lower closure point</th><th>at upper closure point</th></tr></thead><tbody><tr><td rowspan="4">Oilseed rape</td><td rowspan="4">49.8</td><td>Desorption-1</td><td>28.87 (21.30)</td><td>1.02 (0.02)</td><td>2.70 (0.17)</td><td>0.995</td><td>0.154</td><td>-</td><td>-</td></tr><tr><td>Adsorption-1</td><td>20.36 (3.33)</td><td>0.93 (0.01)</td><td>3.01 (0.06)</td><td>0.999</td><td>0.195</td><td></td><td></td></tr><tr><td>Desorption-2</td><td>18.60 (2.02)</td><td>0.91 (0.01)</td><td>3.09 (0.06)</td><td>0.999</td><td>0.207</td><td>0.108</td><td>0.786</td></tr><tr><td>Adsorption-2</td><td>53.42 (25.74)</td><td>0.99 (0.02)</td><td>2.37 (0.10)</td><td>0.992</td><td>0.122</td><td></td><td></td></tr><tr><td rowspan="4">Yellow mustard</td><td rowspan="4">32.1</td><td>Desorption-1</td><td>29.70 (5.62)</td><td>0.93 (0.02)</td><td>3.61 (0.11)</td><td>0.998</td><td>0.167</td><td>0.116</td><td>0.768</td></tr><tr><td>Adsorption-1</td><td>46.10 (9.98)</td><td>0.96 (0.01)</td><td>3.30 (0.08)</td><td>0.999</td><td>0.134</td><td></td><td></td></tr><tr><td>Desorption-1</td><td>17.23 (1.32)</td><td>0.85 (0.01)</td><td>6.96 (0.14)</td><td>0.999</td><td>0.228</td><td>0.031</td><td>0.757</td></tr><tr><td>Adsorption-1</td><td>22.77 (14.78)</td><td>0.95 (0.01)</td><td>5.40 (0.31)</td><td>0.995</td><td>0.182</td><td></td><td></td></tr><tr><td rowspan="4">Lupin</td><td rowspan="4">5.0</td><td>Desorption-1</td><td>9.65 (2.91)</td><td>0.77 (0.04)</td><td>7.42 (0.75)</td><td>0.991</td><td>0.316</td><td>0.064</td><td>0.869</td></tr><tr><td>Adsorption-1</td><td>10.64 (0.89)</td><td>0.80 (0.01)</td><td>6.77 (0.17)</td><td>0.999</td><td>0.293</td><td></td><td></td></tr><tr><td>Desorption-2</td><td>12.58 (4.70)</td><td>0.81 (0.04)</td><td>6.84 (0.36)</td><td>0.990</td><td>0.272</td><td>0.113</td><td>0.785</td></tr><tr><td>Adsorption-2</td><td>22.76 (10.27)</td><td>0.91 (0.01)</td><td>5.22 (0.28)</td><td>0.993</td><td>0.190</td><td></td><td></td></tr><tr><td rowspan="2">Wheat</td><td rowspan="2">2.5</td><td>Desorption-1</td><td>8.59 (0.35)</td><td>0.55 (0.02)</td><td>10.87 (0.41)</td><td>0.999</td><td>0.462</td><td>0.136</td><td>0.751</td></tr><tr><td>Adsorption-1</td><td>21.23 (9.65)</td><td>0.86 (0.03)</td><td>5.77 (0.65)</td><td>0.985</td><td>0.207</td><td></td><td></td></tr><tr><td rowspan="2">Buckwheat</td><td rowspan="2">2.4</td><td>Desorption-1</td><td>14.71 (2.56)</td><td>0.68 (0.03)</td><td>9.54 (0.60)</td><td>0.994</td><td>0.282</td><td>0.097</td><td>0.828</td></tr><tr><td>Adsorption-1</td><td>38.21 (23.11)</td><td>0.82 (0.02)</td><td>6.80 (0.41)</td><td>0.991</td><td>0.170</td><td></td><td></td></tr><tr><td rowspan="2">Pea</td><td rowspan="2">1.7</td><td>Desorption-1</td><td>8.31 (0.54)</td><td>0.60 (0.04)</td><td>11.72 (0.74)</td><td>0.999</td><td>0.429</td><td>0.162</td><td>0.823</td></tr><tr><td>Adsorption-1</td><td>33.68 (16.10)</td><td>0.85 (0.03)</td><td>6.27 (0.39)</td><td>0.991</td><td>0.173</td><td></td><td></td></tr></tbody></table>

<footnote><sup>a</sup> Determined by hydrolyzing the ground samples with hydrochloric acid while boiling. Then, the solution was cooled and filtered. The washed and dried residue was extracted with petroleum ether.</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Moisture desorption and adsorption isotherms at 45 °C, illustrating the hysteresis effect for (A) oilseed rape, (B) yellow mustard, (C) lupin, (D) barley, (E) wheat, (F) buckwheat and (G) pea seeds. The black solid and dashed lines represent desorption-1 and adsorption-1, respectively, corresponding to the first isotherm cycle. The red solid and dashed lines represent desorption-2 and adsorption-2, respectively, indicating the second isotherm cycle for oilseed rape and barley seeds, only. The isotherms were fitted using the GAB model (Eqn. 1) ($R^2 \approx 0.99$; Table 1).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** (A) Moisture sorption hysteresis for seeds of yellow mustard, oilseed rape, barley, lupin, wheat, buckwheat and pea. The curves were calculated based on the fitted GAB models (Table 1). (B) Maximum $\Delta_{Hysteresis}$ moisture content (% dry weight) against oil content (%) and (C) $a_w$ at maximum $\Delta_{Hysteresis}$ moisture content against oil content (% dry weight) for seeds of yellow mustard, oilseed rape, barley, lupin, wheat, buckwheat and pea. The points were estimated from the curves shown in A. Pearson correlation analysis 'r' between the maximum $\Delta_{Hysteresis}$ moisture content and $a_w$ at maximum $\Delta_{Hysteresis}$ moisture content (%) compared with the oil content (%) (dashed lines with corresponding p-values). The x-axes in B and C are logarithmic scales.</figcaption></figure>

should be noted that even drying at 11% RH did not result in a large hysteresis effect in these oily seeds (Fig. 2A). In contrast, for lupin, barley, wheat, buckwheat and pea seeds, hysteresis was observed when seeds were dried at approximately 30% RH; the MC values significantly differed from those approximated by the fitted desorption isotherm ($P < 0.05$). For lupin seeds, a slight shift to the adsorption isotherm was apparent for seeds dried at 30% RH before rehydration at 50% RH, however, a full shift was only seen when seeds were dried to $0.149 a_w$ (5.5% MC) before rehydration at 50% RH (Fig. 4; Supplementary Fig. 1C). For barley and wheat seeds (Fig. 4; Supplementary Figs. 1D and E), hysteresis was observed when seeds dried to 0.295 and $0.328 a_w$ (7.4 and 8.0% MC, respectively), were rehydrated at 50% RH. For buckwheat

seeds, slight shifts to the adsorption isotherm appeared for seeds dried to 0.319, 0.233 and $0.139 a_w$ (9.8, 7.9 and 6.2% MC) before rehydration at 50% RH (Fig. 4; Supplementary Fig. 1F). For pea seeds (Fig. 4; Supplementary Fig. 1G), apparent shifts to the adsorption isotherm appeared for seeds dried to 0.300, 0.205 and $0.157 a_w$ before rehydration at 50% RH; a full shift was seen when seeds were dried to $0.137 a_w$ (6.1% MC).

4. Discussion

Seed longevity is determined by the seed MC, temperature of the storage environment and whether the storage environment is ‘open’ (exchange of air is possible) or ‘closed’ (Ellis and Hong, 2007; Hay et al.,
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** (A) Monolayer moisture content ($M_m$) and (B) water activity ($a_w$) at $M_m$ against oil content for seeds of oilseed rape, yellow mustard, lupin, barley, wheat, buckwheat and pea. Solid down-triangles show desorption-1 data; open up-triangles show adsorption-1 data; half-filled up-triangles show adsorption-2 data. The points were estimated based on the fitted GAB model (Table 1). Negative significant correlations are shown between the de- and adsorption $M_m$ and $a_w$ at de- and adsorption $M_m$ compared with the seed oil content (dashed lines). 'r' is the Pearson correlation coefficient with the corresponding *p*-value. The x-axis is shown in a logarithmic scale.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Seed moisture content upon rehydration at 50% RH depending on the moisture content before rehydration, reached by drying at 30, 20, 15 or <11% RH for two cycles. The solid circle data and empty circle data points show cycles 1 and 2 respectively. The solid and dashed black lines represent the moisture contents at 50% RH for de- and adsorbing seeds, respectively (from Fig. 1). The x- and y-error bars are the standard deviations of three replicates.</figcaption></figure>

2022). If seed MC increases, seeds age more rapidly resulting in declines in seed viability and vigour. Hence, ideally, after seeds have dried, they should be kept dry as far as possible (Guzzon et al., 2024). Nonetheless, knowing the conditions under which seeds will take up moisture, or not, and the extent to which they might increase in MC will help us to understand the potential impact of variable environmental conditions on seed quality, be that for commercial seed lots before or after harvest, or for example, serotinous seeds that remain in the canopy of the plant for a long period of time (Crawford et al., 2011). In the current study, when seeds were dried with silica gel at < 11% RH, the magnitude of the hysteresis loop was found to vary between species, ranging between 0.3 and 2.7% dry weight (Figs. 1 and 2A). The magnitude appeared to be correlated with oil content (Fig. 2B and 4), being smaller for more oily seeds, consistent with the smaller moisture range covered by the isotherms of the oily seeds (yellow mustard, oilseed rape). Silva et al. (2018) described hysteresis magnitudes of up to 3.54% dry weight for ‘Malagueta’ pepper seeds. The RH range of the equilibration

environment over which hysteresis occurs, also depends on the composition (protein, starch) of the material (Okos et al., 2019). In our study, the variable seed oil content (Table 1) could account for some of the differences in the hysteresis plots (Fig. 2A), but for those with similar oil content (e.g. wheat, buckwheat and barley), other compositional factors must also have an impact.

We only measured isotherms and hence hysteresis at one temperature: 45 °C. This temperature was used since we also wanted to study longevity in storage over a reasonable experimental timeframe (Rezaei, 2024). The magnitude of the hysteresis effect increases as temperature decreases, as seen for example for pepper seeds (Silva et al., 2018). Hence, under ambient conditions in nature or under cooled storage conditions, we can expect a greater difference between the de- and adsorption isotherms than observed here. Hysteresis can also be affected by how long the material is held before isotherm determination (Okos et al., 2019). This latter observation relates to the argument that hysteresis is a thermodynamic impossibility and hence must mean that the
true equilibrium state has not been reached (Labuza and Altunakar, 2020). However, in practice, seeds might not be held at a particular RH for long enough for true equilibrium (Rezaei et al., 2023). This is important to bear in mind when comparing seed longevity relations based on MC vs. the RH at which they have been equilibrated (Roberts and Ellis, 1989), especially given the relative rates of ageing at different moisture levels; when seeds are stored at high moisture levels, they are less likely to reach equilibrium before they die compared with seeds stored at low moisture levels. When seeds are stored in air-tight containers, for example, aluminium foil packets or cans, the seed MC will be fixed hence, to reach equilibrium, it is only the $a_w$ that can change. If seeds are on the desorption isotherm when they are packed, the $a_w$ could only increase to come to equilibrium; conversely, if seeds are on the adsorption isotherm, the $a_w$ could only decrease once packed. The extent of hysteresis is also affected by the number of successive cycles of desorption and adsorption (Labuza and Altunakar, 2020). These cycles can cause shifts in the isotherms away from the initial desorption and adsorption patterns (Li et al., 2011; Okos et al., 2019; Rezaei et al., 2023). For barley and oilseed rape seeds, subjecting them to a second desorption cycle resulted in a noticeable shift of the second adsorption isotherm towards lower MCs compared to the first adsorption isotherm (Fig. 1; Supplementary Fig. 1). This finding aligns with previous observations concerning oilseed rape and barley seeds, where the second cycle diverged from the first isotherms at lower RHs (Rezaei et al., 2023). Hence, if seeds are exposed to wide fluctuations in RH, perhaps in *situ* if harvesting is delayed (in the case of shatter-resistant crops), their subsequent sorption behaviour, including response to drying, may differ from seeds that have not been exposed to wide fluctuations in RH.

In fitting the GAB model to the isotherm data, the $M_m$ value at which quality loss in food samples was minimized was determined (Bell and Labuza, 2000). This value was found to be higher for lupin, barley, wheat, buckwheat and pea seeds compared to oilseed rape and yellow mustard seeds (Table 1). Although it is reported that the $M_m$ typically occurs between 20 and 40% RH in foods (Bell and Labuza, 2000), our observations showed a broader range of $M_m$, spanning from 0.154 to 0.462 $a_w$ for desorbing seeds. As well as being a point of maximum stability, it has also been suggested that materials need to be dried below $M_m$ to transition to the adsorption isotherm upon rehydration (Rahman and Labuza, 2007). For oilseed rape and yellow mustard seeds, drying seeds at 30 and 20 RH would mean that they were not below the desorption $M_m$. Accordingly, the seeds did not shift to the adsorption isotherm upon transfer to 50% RH (Fig. 4). In contrast, lupin, wheat, buckwheat and pea seeds partially switched to the adsorption isotherm upon transfer to 50% RH after drying at just 30% RH; in the case of lupin and buckwheat seeds, this was not below the desorption $M_m$ value, although it was for wheat and pea seeds which had very high desorption $M_m$ values (Fig. 4; Table 1). Thus, and in accordance with our earlier research (Rezaei et al., 2023), the monolayer value does not appear to be a good predictor of seed hysteresis behaviour across diverse species. Within each species, significant variation in the $M_m$ values was observed depending on the isotherm series (de- or adsorption). In some cases, the difference between desorption and adsorption $M_m$ exceeded the maximum hysteresis magnitude. Given that $M_m$ is a theoretical parameter, its accuracy and hence utility, as seen here, could be influenced by the quality of isotherm data (Hay et al., 2022).

Harrington's rule of thumb states that seed longevity is doubled for every 1% reduction in MC (Harrington, 1972); conversely then, the rate of decline in viability and vigour is doubled for every 1% increase in MC. Hence, there is an advantage in terms of longevity, of being on the adsorption isotherm rather than the desorption isotherm (Bradford et al., 2015; Rezaei et al., 2023). The $a_w$ of seeds that are sitting in poor or uncontrolled environments, including in nature, will adjust as the RH of the environment varies, but the extent to which MC adjusts will depend on the species and their prior moisture history (Fig. 4). Seeds of some species, for example, as we saw here oilseed rape and yellow mustard, are more likely to remain on the desorption isotherm with

fluctuation in RH, and hence are prone to higher rates of viability loss due to the higher MC. Conversely, seeds of species such as barley more readily shift between the isotherms and hence, there would need to be greater fluctuation in RH for MC to change.

## 5. Conclusions

Seed moisture isotherms exhibited variations across different species. This variability depended on the moisture adsorption or desorption behaviour of the seeds and their specific moisture history. These factors will significantly impact the rates at which seeds lose viability. Seeds cycled between 30 and 50%, 20 and 50%, or 15 and 50% RH also had species-related responses in terms of whether they stayed or shifted between isotherms. This represents the first study, to our knowledge, where distinctive responses to such cycling were evident among seeds of diverse species. The response will variably influence the ageing rates of seeds across different species. Seeds that follow the desorption isotherm will exhibit a more pronounced increase in MC upon rehydration, consequently accelerating the rate of ageing. Considering the moisture history of seeds is essential in understanding the impacts of ambient humidity on their survivability.

### CRediT authorship contribution statement

**Shabnam Rezaei:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Julia Buitink:** Writing – review & editing, Supervision. **Fiona R. Hay:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

### Funding

This work was supported by Aarhus University Research Foundation grant AUFF-E-2019-7-27.

### Declaration of competing interest

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.

### Acknowledgments

We thank Birte Boelt and Palle Kristiansen Ahm for providing fresh seeds and Julie Dybdal Jensen for assistance in the laboratory.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jspr.2024.102477.

### Data availability

Data will be made available on request.

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