Effects of freeze-drying and use of artificial seawater on phosphorus exchange isotherms in brackish water sediments

This study reports results on the estimated magnitude of changes in P sorption isotherms in clayey mud sediments when performed using (1) freeze-dried instead of fresh sediment samples and (2) artificial sea water (ASW) instead of ambient near-bottom water. The sediments used differed in their P sorption. For the isotherms, sediments were equilibrated for 48 h in solutions of varying P concentration whereafter the amount of P sorbed or desorbed was determined. We adjusted the modified Freundlich equation to the isotherm data and assessed differences in the isotherms between the treatments by comparing equilibrium P concentrations and P buffering capacities determined from the isotherm equation. Freeze-drying decreased the P sorption in all investigated sediments, but the magnitude of the changes varied depending on the properties of the sediments. The effect was minor in the sediment abundant in P sorption sites and low in easily exchangeable P, while it was clearer in the sediments originally high in P or low in sorption surfaces. ASW and ambient water produced similar isotherms suggesting that ASW is a suitable equilibrium solution in P exchange experiments with muddy clay sediments. This study enlightens the processes occurring in freeze-drying. The results highlight that when examining the effects of dissimilar treatments or experimental conditions on the P exchange isotherms the magnitude and significance of the observed changes should be evaluated in a proper context considering the precision of the method. The effects of freeze-drying on P sorption isotherms are minor in sediments abundant in unoccupied sorption surfaces. Artificial sea water is a suitable background matrix for the P exchange isotherms. The precision of the method should be considered when comparing the effects of differing isotherm treatments. The effects of freeze-drying on P sorption isotherms are minor in sediments abundant in unoccupied sorption surfaces. Artificial sea water is a suitable background matrix for the P exchange isotherms. The precision of the method should be considered when comparing the effects of differing isotherm treatments.


Introduction
Phosphorus (P), one of the key nutrients regulating primary production in aquatic ecosystems, is directly bioavailable as dissolved inorganic phosphate (PO 4 -P) anions. An excess of anthropogenic P causes eutrophication, leading to an accumulation of particulate P in sediments from which it may return to the aquatic P cycle or be buried during sedimentation. In clayey mud sediments, important sorption components for P are iron (Fe) and aluminium (Al) oxyhydroxides [1,2], but associations with metal cations in organic matter (OM) are also possible [3].
Adsorption of P onto Fe and Al oxyhydroxides is a ligand exchange reaction in which P replaces coordinated − OH or − OH 2 groups on the oxyhydroxide surfaces [4,5]. This two-way reaction aims to maintain an equilibrium between the P concentration on the oxyhydroxide surface and in the surrounding solution and proceeds in two steps. In the first one, P is sorbed onto a solid surface within a timescale of minutes to hours. The second step, a slow diffusion process of P towards the interior of the porous oxyhydroxide material, can take months or even years [6]. Correspondingly, desorption of P from oxyhydroxide surfaces starts as a response to a decrease in the P concentration in the surrounding solution in order to maintain (or reach) a given P equilibrium between solid and solution phases.
Desorption-sorption isotherms, introduced by Beckett and White [7], elucidate the equilibrium nature of P exchange, particularly the first step of desorption/ sorption, without taking any stand on the reaction mechanism. They predict the change in P desorption from or sorption onto the sediments at changing P concentrations in the solution. Isotherms have widely been applied in studies of the P exchange properties of agricultural soils [8][9][10][11] and lake [12], fluvial [13], estuarine [14,15] and marine [16,17] sediments. They are prepared by equilibrating solids in solutions of varying P concentration, usually for 24 − 48 h. Thereafter, the P concentration of the solution is measured and used to calculate the amount of P sorbed onto or desorbed from the sediment, which is then plotted as a function of the equilibrium P concentration in the solution phase.
Information of the isotherm can be summarized by parameters that are determined from the equation fitted to isotherm data at certain points of the graph. The parameters often used are the equilibrium P concentration at zero net P sorption (EPC 0 ) and the slope of a tangent at this point [13,16,18], denoted in this study as k EPC0 . EPC 0 represents the P concentration in the solution at which the P desorption turns to sorption. k EPC0 indicates the P buffering capacity of the sediments, i.e. the ability of sediment to resist a change in the P concentration of the solution phase. With increasing P concentration in the solution, the P sorption sites gradually become saturated and, subsequently, the P buffering capacity decreases [19].
Isotherm experiments are sensitive to experimental conditions. When investigating marine sediments collected from wide study areas over extended periods of time, storage of samples is often unavoidable. For instance, to ensure equal solid-to-solution ratios, comparative isotherm studies require accurate weighing, which is not possible on-board. Furthermore, in the case of large sample sets, the storage times are inevitably long. Thus, storage of fresh samples requires shielding with inert gas, such as nitrogen (N 2 ) or argon, particularly to minimize oxidization of reduced Fe compounds to Fe oxyhydroxides, which act as sorbents for P e.g. [20]. Maintaining the gas shielding during a long-term storage may, however, be laborious and uncertain.
Freeze-drying is often used as a sample pre-treatment in P exchange isotherm studies [21,22]. It is considered to maintain the sample quality during storage due to a drying-induced decrease in microbial activity [23][24][25]. Removal of the liquid phase in freezing and drying presumably slows down all chemical reactions in the samples [26]. However, similarly as in storing of fresh sediment samples, freeze-drying may change the partitioning of Fe in chemically different fractions [27,28]. It is, nevertheless, superior to air-and oven-drying due to the minimal loss of volatile compounds and the lower degree of aggregation [24].
Also, ionic strength and composition of the solution are known to affect the P exchange. Lehr and Van Wesemael [29] showed that solutions containing divalent cations released less P than those containing monovalent cations. Furthermore, P sorption increases with increasing molarity of the background solution [30,31]. In the absence of ambient water, the use of artificial solutions is unavoidable in the P exchange studies. Artificial seawater (ASW) is tailored to coarsely mimic the ionic composition of seawater [32]. Thus, it could be considered a good choice to be used in P exchange studies of marine and brackish water sediments.
The present study investigates the effects of freeze-drying on the P exchange isotherms with brackish Baltic Sea sediments expected to differ in their ability to sorb P. The objective is to gain perspective on the magnitude of the possible freeze-drying-induced changes in the P exchange compared with fresh subsamples (shielded from oxidation). In addition, we compared the P exchange isotherms produced using ASW and ambient water solutions to find out whether the ASW could replace the ambient water in the P exchange experiments. We hypothesize that 1) the isotherms of the freeze-dried sediments differ from those produced with the fresh sediments (especially from those shielded with N 2 ) and the differences are detected in the P exchange parameters; 2) the response to freeze-drying is related to sediment properties and 3) the P exchange isotherms produced using ASW do not differ markedly from those produced with ambient water. The results of this study increase information on the effect of freeze-drying and the use of ASW on the P exchange parameters. The impact of these factors is important to recognize when planning P exchange experiments and interpreting their results.
In the following, we describe the research material and the methods used in the experiments. In Sect. 3, we first present the results showing the effects of freeze-drying on the P exchange isotherms in different sediments and discuss how the sediment properties may explain the observed changes. Then we present the results on the comparison of P exchange isotherms produced using ASW and ambient water and discuss the role of the characteristics of equilibrium solution in P sorption. Further, we discuss the importance of considering the scale of observed changes and the effects of pre-treatment for improved planning and interpretation of results of P exchange experiments.

Sampling and storage of sediments
Recent sediments in the study area consist mostly of siltclayey muds and are typically low in CaCO 3 [33]. Sediments S1 − S7 were collected from the northern Baltic Proper and the Gulf of Finland, Baltic Sea, during cruises of R/V Aranda in 2010 and 2012 (Fig. 1). Sampling was carried out using a Gemax gravity twin-corer (Ø 90 mm, length 60 cm). The topmost 2-cm layers from two or three replicate sediment cores were sectioned and pooled in a plastic container. For sediments S2 and S3, this was performed under N 2 shielding to protect them from atmospheric O 2 (see [20] for details). The containers were sealed in gas-tight plastic bags filled with N 2 and stored at ca + 5 °C prior to the use of a portion of the samples in the experiments on P exchange and reducible metals on-board. The rest of the samples S2 and S3 were frozen on-board (− 20 °C), freezedried (− 60 °C) and sieved (Ø 2 mm) in the laboratory. Sediments S4 − S7 were frozen immediately after slicing and freeze-dried and sieved similarly as sediments S2 and S3. The dry samples were stored in plastic containers in the dark at room temperature until analysis.
In addition, three cores from site S1 were stored unsealed (in the dark, at + 5 °C) for a few months to allow oxygenation of their surfaces. They were considered oxygenated when the colour of the topmost 0 − 2 cm surface layer was light brown. Therefore, the oxygenated 0 − 2 cm layers were sliced and pooled in a plastic container. The sample was homogenized by mixing gently with a spatula and then stored overnight in the dark at + 5 °C prior to the use of a portion of the sample in the P exchange experiment. For further analyses, the rest of the sediment was frozen, freeze-dried, sieved and stored as described in the previous paragraph for the dried samples.

Background analyses of the sediments and the near-bottom waters
The water content of sediments S1 − S3 was measured using a moisture analyser (Ohaus MB45) and that of sediments S4 − S7 as weight loss during freeze-drying.
To ensure the homogeneity of the sample material, the total content of various elements in the sediments was analysed from the freeze-dried and pulverized (Pulverisette 5, Fritsch) samples. Total carbon (TC) and nitrogen (TN) were determined with a Vario MAX CN-analyser (two replicates deviated < 2% from their average values). The sediments in the study area are poor in carbonate minerals [34], wherefore, TC was used as a proxy for OM content. Total phosphorus (TP), iron (TFe), aluminium (TAl), manganese (TMn), calcium (TCa) and sulphur (TS) were analysed after digestion of the sediment in a microwave oven with a mixture of aqua regia, boric acid and hydrofluoric acid, using inductively coupled plasma optical emission spectroscopy (ICP-OES) ( [35]; modification from Loring and Rantala [36]). The specific surface area (SSA) of the freezedried and sieved sediment samples was determined using the N 2 adsorption method (PANK 2401). The deviation between two replicates was < 2% from their average value. The O 2 concentration and salinity in the near-bottom water (ca 5 cm above the sediment surface) was determined in the samples taken from Gemax cores using Winkler titration [37] and a salinometer (Guildline autosal 8400B), respectively. Sediments with near-bottom O 2 concentrations > 2 ml l −1 were considered oxic and those with < 2 ml l −1 hypoxic, see e.g. [38]. Ambient water above the surface of sediments S1 and S4 − S7 in the Gemax cores was siphoned, filtered (0.40 µm, Whatman Nuclepore, polycarbonate, PC, membrane filter) and frozen (− 20 °C). Prior to use in the P exchange experiments, the ambient water of each sample was thawed at room temperature for approximately 14 h and thereafter analysed for phosphate (PO 4 -P), nitrate (NO 3 -N), ammonium (NH 4 -N), silicate (SiO 4 -Si) and the total concentration of dissolved P and N after acid persulphate digestion [39] using a Lachat Quick-Chem 8000 autoanalyser.

Phosphorus exchange isotherms of fresh and freeze-dried sediments
Comparison of the P exchange in the fresh and freezedried sample pairs was carried out with sediments S1 − S3 ( Table 1). The sample portioning and the isotherm experiments of the fresh samples S2 and S3 were performed on board under N 2 shielding within 12 h from the sampling. Since accurate weighing on board was impossible, portioning of the fresh samples was done on a volume basis using plastic syringes with cut tips in a glove bag.
The 3 ml subsamples of the fresh sediment were inserted into 100 ml plastic centrifuge tubes (n = 3/each P addition level). Three similar 3 ml subsamples were taken to determine their dry weight (DW) later in the laboratory. Thus, due to the volumetric sample portioning on board, the solid-to-solution ratios of the isotherm experiments with the fresh samples S2 and S3 were revealed afterwards (see Table 1). Potassium phosphate (KH 2 PO 4 ) was added to ASW solutions bubbled with N 2 (composition described in the following section) in the glove bag to obtain solutions containing 0, 5, 10, 15, 20, 50, 75 or 100 µmol P l −1 . A 60 ml volume of each solution was pipetted into the centrifuge tubes containing the fresh sediment samples and into the empty ones (n = 2/each P addition level). All tubes were flushed with N 2 , sealed with caps, tightened with Parafilm and placed in a gas-tight plastic bag filled with N 2 before removing from the glove bag. The suspensions were equilibrated on an orbital shaker for 48 h (in dark clima room, + 5 °C, 200 rpm). Thereafter, the tubes were centrifuged (3000 rpm, 15 min, 22 °C) and the supernatants were filtered (0.40 µm, Nuclepore, PC membrane filter) in the glove bag under N 2 shielding. The filtrates were analysed for P using the molybdenum blue method [40] and a spectrophotometer (Genesys 10 UV, Thermo Spectronic, equipped with a 5 cm flow-injection cuvette, detection limit 0.05 µmol l −1 ). The isotherm experiments were repeated later in the laboratory with the freeze-dried portions of samples S2 and S3 using solid-to-solution ratios (1:50 and 1:300, respectively) corresponding to the real ratios in the experiments with the fresh samples. In the case of freeze-dried sediment S2, the five smallest P additions (0, 5, 10, 15 and 20 µmol P l −1 ) were used due to the limited amount of sample material. Furthermore, to elucidate the impact of the differing solid-to-solution ratio on the isotherms, the P exchange test was repeated with freeze-dried sediment Table 1 Experimental conditions for the comparisons of P exchange of the fresh (treated with or without N 2 shielding) and the freeze-dried sediments (ASW = artificial seawater, fd = freeze-dried, salinity of water determined ca 5 cm above the sediment surface) a lower P addition range than that used in the other tests due to a limited amount of sample material S2 using solid-to-solution ratio 1:200 (Table 1). The isotherm experiments with sediment S1 were performed under indoor air and the samples for both treatments were portioned by weighing (0.3 g DW). Otherwise, the experiments were carried out similarly as described with other sediments. In all experiments, the P concentration in the ASW solutions differed between the treatments by 1.3 ± 1.2% (mean ± standard deviation, SD) from the target concentrations.

Phosphorus exchange isotherms in ambient water and in ASW solutions
The freeze-dried sediments S1, S4, S5, S6 and S7 were used to compare the P exchange isotherms produced using ASW and ambient water solutions. The salinity of the ambient waters ranged from 7.7 to 10.8, and the ASW solutions were adjusted to correspond them separately for each sediment. ASW solutions were prepared by diluting a stock ASW solution (210‰) with Milli-Q water. The stock solution (modified from [37]) consisted of sodium chloride (NaCl; 190 g), magnesium sulphate (MgSO 4 ; 61 g) and sodium bicarbonate (NaHCO 3 ; 1.2 g) dissolved in 1 l of Milli-Q water. For the isotherms, 0.3 g of sediment (DW) was weighed and transferred into 100 ml plastic centrifuge tubes (n = 2 − 3/each P addition level). The sediments were rewetted to their ambient water content with Milli-Q water and left to moisten overnight on an orbital shaker in the dark (50 rpm, + 5 °C). ASW or ambient water (60 ml) with added P concentrations of 0, 5, 10, 15, 20, 50, 75 or 100 µmol l −1 were pipetted into the sample and the blank tubes without samples (n = 2/each concentration level). The sediments were equilibrated and centrifuged, and the supernatants were filtered and analysed for P as in the experiments with fresh and freeze-dried samples. Prior to the filtration, the supernatants of the equilibrium solutions from the samples of 0 and 100 µmol l −1 of added P were analysed for pH (i.e. pH-P 0 and pH-P 100 , respectively).

Isotherm fitting, determination of phosphorus exchange parameters and statistical tests
The amount of P desorbed from or sorbed onto the sediment (Q, µmol g −1 ) during the equilibration was calculated as a change in the P concentration of the solution. The Q value of each replicate was plotted against its equilibrium P concentration (I, µmol l −1 ) to form two or three replicate isotherm graphs. A modified Freundlich equation (Eq. 1) was fitted to the isotherm data separately for each replicate graph for each treatment, except for fresh sediment S2 where the linear equation (Eq. 2) was used. In the modified Freundlich equation, Q 0 stands for P potentially desorbed from the sediment [7], while a and b are fitting parameters.
In the linear equation, k and b are constants.
EPC 0 (µmol l −1 , I when Q = 0) and the slopes of the tangents at EPC 0 (k EPC0 ; l g −1 ) and at each data point resulting from the P additions 5, 20 and 100 µmol l −1 (i.e. k 5 , k 20 , k 100 ; l g −1 ) were calculated from the isotherm equations. EPC 0 was calculated by solving I in the Freundlich equation when Q = 0 (Eq. 3). The different k values were calculated by determining the derivative at each measured data point of the isotherm (Eq. 4). For the fresh sample S2, EPC 0 and k EPC0 were determined by solving I (when Q = 0) and the slope k from the linear equation (Eq. 2), respectively. The P exchange parameters were determined separately for each replicate of the isotherm graphs.
where, k x = k EPC0 , k 5 , k 20 or k 100 , I x = I at EPC 0 or I resulting from P addition 5, 20 or 100 µmol l −1 EPC 0 describes the P concentration above which the P desorption turns to sorption and vice versa. In other words, the lower the EPC 0 , the lower the P concentration in the solution above which the sediment begins to sorb P. The k values describe the P buffering capacity of the sediments at different points of the isotherm graph, i.e. in increasing P sorption. The higher the k value, the steeper the isotherm and the higher the buffering capacity of the sediment against the change in P concentration in the surrounding solution [18].
The differences in the P exchange isotherms between the treatments were investigated by comparing the amount of P sorbed at each P addition level (Q values), the EPC 0 values and the P buffering capacities k EPC0 , k 20 , k 50 and k 100 using a paired t-test (when applicable). The isotherm graphs and box plots were created, and the statistical analyses performed, using R (version 5.2., 2018-12-20, R Core Team 2018).

Ascorbate-extractable Fe and Mn in the sediments
Ascorbate-extractable Fe and Mn were determined in freeze-dried sediment S1 and fresh and freeze-dried sediments S2 and S3 to describe the amount of potential adsorbents for P in them and to elucidate the effect of freeze-drying on their contents. Ascorbate solution (50 g  [41]. It is shown to extract Fe from easily reducible, poorly crystalline Fe oxyhydroxides [41,42]. For fresh sediments S2 and S3, two replicate 3-ml subsamples were portioned into 100 ml centrifuge tubes under a N 2 atmosphere. The tubes were flushed with N 2 , sealed tightly and stored for a few days (+ 5 °C) under a N 2 atmosphere prior to the extraction. In the extraction, 30 ml of ascorbate solution was added to the centrifuge tubes in a glove bag filled with N 2 for extraction. The tubes were flushed with N 2 , closed with caps (tightened with Parafilm) and placed in a gas-tight plastic bag filled with N 2 . The suspensions were extracted on an orbital shaker (200 rpm, 2 h, ca 22 C°) and centrifuged (3000 rpm, 15 min, 22 C°). The supernatants were separated and filtered (0.40 µm, Nuclepore, PC membrane filter) in the glove bag under N 2 atmosphere. For the freeze-dried samples, the extraction procedure was repeated similarly, except without the N 2 shielding and by weighing the samples (0.3 g for S1). The filtrates were acidified to pH < 2 with concentrated nitric acid (65% HNO 3 , Suprapur). The Fe and Mn in the extracts were analysed using ICP-OES.

Physico-chemical properties of the sediments
Physico-chemical properties in the sediment-water interface have an essential impact on the P binding of the sediments. Based on the O 2 concentrations in the near-bottom water, the surface layers of sediments S2 and S7 were considered oxic and those of S3, S4, S5 and S6 hypoxic/reduced ( Table 2). At the time of sample sectioning, sediment S1 was visually evaluated as oxic due to its light brown 0-2 cm surface layer. In oxic sediments the redox-sensitive Fe oxyhydroxides are assumed to be capable of binding P. Organic rich muddy sediments have often fluffy surface layers and high water contents.
As common in the investigated sea area, all sediments were relatively high in TFe (> 700 µmol g −1 ) and TAl (1635 − 2614 µmol g −1 ) but clearly low in Ca [33] ( Table 2). TP in the sediments ranged typically from 36 to 52 µmol g −1 . However, sediment S2 differed from others by its clearly higher TP, TFe and TMn contents (see Table 2). In the portioning of the fresh sample, sediment S2 was found to contain abundantly small Fe-Mn concretions (diameter range of 0.5 − 3 mm). The concretions were avoided during the portioning. However, although the sieving of the freeze-dried sample removed the biggest concretion fragments (Ø > 2 mm), it was impossible to exclude them completely as the dry concretions were very fragile and broke during sample handling. The presence of the concretions explains the high TP, TFe, TMn and SSA in this sediment. The concretions also explain the freeze-drying-induced elevation in ascorbate-extractable Fe in sediment S2 from 29.5 to 94.9 µmol g −1 and Mn from 5.7 up to 89.2 µmol g −1 . Correspondingly, in sediment S3, freeze-drying increased the ascorbate-extractable Fe from 8.3 to 22.6 µmol g −1 , while Mn increased only from 0.3 to 0.4 µmol g −1 . In this originally hypoxic sediment, the low easily reducible Mn indicates its reduction-induced dissolution. The freezedried sediment S1 was the highest in ascorbate-extractable Fe, 92.2 µmol g −1 .

Effects of freeze-drying on phosphorus exchange
In the three investigated sediments, freeze-drying increased the desorbable P and the EPC 0 and decreased the P sorption, as reflected in lower Q values. These findings are supported by earlier studies with soils and sediments reporting similar responses both to freeze-drying [28,43] and air-drying [10,44]. The freeze-drying-induced changes resulted in more gently bending isotherm graphs (Fig. 2) pointing to a lowered P buffering capacity (Table 3, k EPC0 , k 5 , k 20 and k 100 ) as compared with the corresponding fresh samples. In the investigated sediments, the freezedrying-induced changes were parallel but differed in their magnitude. The sediments varied in their physico-chemical properties allowing us to detect possible reasons for the observed changes. We assessed the differences in P sorption between the treatments at various stages of the isotherm graphs, which is not usually done in the isotherm studies.
The originally oxic sediment S1 was least affected by the freeze-drying. Freeze-drying increased desorbable P only slightly and resulted in similar increase in EPC 0 (Fig. 3). Sediment S1 had the steepest isotherms and thus the greatest ability to sorb P (Fig. 2). The high P sorption can be explained by the abundance of easily reducible Fe (Fe Asc 92.2 µmol g −1 ) reflecting presence of poorly crystalline Fe oxyhydroxides in the sediment. Freeze-drying decreased the P sorption significantly only at the highest P addition (Q P100 ) (see Online Resource Table 1). However, the buffering capacities in lower P additions were ca four to five times higher in the fresh than in the freeze-dried samples (k EPC0 , k 5 and k 20 ; Table 3) and the difference was even greater at the highest P addition (k 100 ). However, the k values of the replicates deviated most in fresh sediment S1 since its isotherm was almost linear while the Freundlich fitting usually describes well particularly bending isotherms [45].
In originally hypoxic sediment S3, the isotherm graphs were more gently bending than in originally oxic sediment S1 (Fig. 2), presumably due to the lower amount of Fe oxyhydroxides (Fe Asc 22.6 µmol g −1 ). The Fig. 2 Phosphorus exchange isotherms of fresh and freezedried (fd) sediments S1 − S3 drawn by using the average Q (amount of P desorbed from or sorbed onto the sediment) and I (equilibrium P concentration) values a, and Q and I values of the three replicates, separately b (triangles = replicate fresh samples, crosses = replicate fd samples). Solid-to-solution ratios were 1:200, 1:50 and 1:300 for sediments S1, S2 and S3, respectively. Note the different scales on the x-and y-axes Table 3 k EPC0 , k 5 , k 20 and k 100 values (mean ± standard deviation, SD; l g −1 ) of fresh and freeze-dried (fd) sediments S1, S2 and S3 (n = 3, except for fd sediment S2 n = 2) a k values determined from linear equation * Significant differences between the fresh and freeze-dried k EPC0 and k 5 values (t = 18.096, df = 2, p = 0.003 and t = 6.748, df = 2, p = 0.021, respectively) freeze-drying-induced changes were also more marked than in sediment S1 and seen in significantly lower P sorption (e.g. the Q values) nearly at all P additions (see Online Resource Table 1). The differences in the P buffering capacities between the treatments were clear at the beginning of the isotherm (k EPC0 and k 5 ) being ca seven times higher in the fresh than in the freeze-dried samples (t = 18.096, df = 2, p = 0.003 and t = 6.748, df = 2, p = 0.021, respectively), but they decreased with increasing P additions (Table 3). Also, in sediment S3, desorbable P and EPC 0 increased only slightly upon freeze-drying (Fig. 3). Sediments S1 and S3 were similar in their OM but differed in their original O 2 status and Fe oxyhydroxide content. Since in both sediments freeze-drying lowered the P sorption it is likely that the drying resulted in changes in their sorption surfaces. Raiswell et al. [42] showed with synthetic ferrihydrite that freeze-drying and the following storage of the dry sediments (at room temperature) may lead to aggregation of poorly crystalline Fe oxyhydroxides. Poorly crystalline Fe oxyhydroxides have a high affinity for P [46], but their structural changes, for instance along with ageing, weakens the P sorption [47]. The originally oxic sediment S1 was four times higher in Fe oxyhydroxides than the originally hypoxic sediment S3. Thus, the decrease in the P sorption was less pronounced in sediment S1 having more Fe oxyhydroxides available for the P sorption than sediment S3, regardless of the possible Fe aggregation during freeze-drying. Therefore, in sediment S1, the weakening sorption of P was not clearly seen until at the highest P additions. In fact, the notably high P buffering capacity k 100 in fresh sediment S1 indicates the abundance of unoccupied adsorbents and a high P sorption potential left even after the highest P addition. This additional information highlights the benefit of investigating the buffering capacities at different stages of the isotherm graph. It is also noteworthy that the freeze-dried samples had been stored for different periods prior to the isotherm experiment: S1 for 1.5 and S3 for 4 months. Thus, we cannot exclude the potential effect of the storage time on the results of the dried samples, although the changes are presumably slow [26].
Previous studies have reported that air-drying causes rupture of OM with consequent release of P [10,43,48]. In sediments S1 and S3, the slight increases in desorbable P (and the concomitant increase in EPC 0 ) can originate from ruptured OM (Fig. 3). It is also noteworthy that the isotherm experiments with these sediments were performed using dissimilar solid-to-solution ratios (1:200 in S1 and 1:300 in S3) as explained in the Materials and methods section. It is possible that the smaller solid-to-solution ratio (i.e. the lower particle concentration) solely explains the greater increase in desorbable P and EPC 0 in freeze-dried sediment S3 than in sediment S1. Furthermore, Hjort [28] found freeze-drying to promote dissolution of Fe from fractions representing OM in anoxic OM-rich sediments. Thus, the freeze-drying-induced increase in the ascorbateextractable Fe in sediment S3 may originate from changes in the sediment OM. However, since the freeze-drying decreased the P sorption, it suggests that the released Fe did not participate in the P sorption. Fig. 3 Box plots illustrating the differences in the desorbable P (a, P DES ; µmol l −1 ) and in equilibrium P concentration at zero net P sorption (b, EPC 0 ; µmol l −1 ) of the fresh and the freezedried (fd) sediments (n = 3, except for the fd S2 n = 2). Note the different scale in S2 | https://doi.org/10.1007/s42452-022-05116-7 Research Article The originally oxic concretion-rich sediment S2 showed the lowest P sorption and, thus, had the most gently bending isotherm graphs (Fig. 2). The Q values were consistently lower in the freeze-dried than in the fresh samples, but the buffering capacities were similar in both treatments ( Table 3). The difference in Q values between the treatments was greater (ca 0.2 µmol g −1 ) than in sediment S1 (0.01 − 0.08 µmol g −1 ) but smaller than in sediment S3. However, the freeze-drying increased markedly desorbable P (t = 19.399, df = 2, p = 0.003), resulting in a corresponding increase in EPC 0 (Fig. 3). Presumably, this released P was occluded in the Fe-Mn concretions prior to their disruption during drying and sieving of the sample. This conclusion is supported by the drying-induced threeand 15-fold increases in ascorbate extractable Fe and Mn, respectively, and by the exceptionally high content of TP, TFe and TMn in the sediment. According to Winterhalter [34], some surface sediments in the Gulf of Finland contain concretions high in Fe, Mn and P. Furthermore, part of the P in the concretions is found to be dithionite extractable (Lukkari pers. comm.) assumed to be bound onto reducible Fe compounds, mainly Fe oxyhydroxides [49].
Air-drying of originally submerged sediments has been reported to elevate EPC 0 . For instance, in the study of Qiu and McComb [50] the increase was 0.4 µmol l −1 , whereas Kerr et al. [51] recorded an increase as high as 5-5.8 µmol l −1 . Simpson et al. [52] compared the effects of air-drying and freeze-drying with the corresponding fresh fine (< 2 mm) stream sediments having originally low EPC 0 (< 0.65 µmol l −1 ). In their study, the freeze-drying either increased or decreased EPC 0 by 0.3 µmol l −1 , the effects being rather modest compared with those recorded in the air-drying. In our previous study with 22 freeze-dried surface sediments collected from the coastal northern Gulf of Finland, the EPC 0 values ranged from 0.2 to 38 µmol l −1 [53]. Thus, the freeze-drying-induced increase of ca 0.5 µmol l −1 recorded in the present study (i.e. in the sediments that did not contain concretions) is relatively small. This highlights the importance of evaluating the impact of freeze-drying on the P exchange in the context of the observed scale of the investigated parameters in the given research area.

Phosphorus exchange in artificial seawater and ambient water solutions
Another goal of the present study was to find out whether ASW would produce similar P exchange isotherm as ambient water. The ambient water of sediments S1 and S4 − S7  Table 2). The highest PO 4 -P and NH 4 -N concentrations were recorded in the ambient water of sediment S4. The pH is known to affect the P sorption. Therefore, we determined pH of the equilibration solutions at the lowest and the highest P additions (pH-P 0 and pH-P 100 ) and found out that they were generally higher in the ambient water than in the ASW (Online Resource Table 2). The biggest differences (0.4 − 0.5) in pH values between the ASW and the ambient water were recorded in sediments S4, S5 and S7, but their impact was not clearly visible in the isotherms (Fig. 4).
In sediments S1, S5, S6 and S7, the P exchange isotherms produced with ambient water and ASW were similar, while in sediment S4 they differed to some extent (Fig. 5). Generally, the P sorption (Q values) was slightly higher in the isotherms produced when using the ambient waters (supplementary data Table 3), probably due to the higher P concentration (1.3 − 3.5 µmol l −1 ) in the ambient water than in ASW. Also, the ionic composition being more diverse in the ambient water than in ASW solutions, presumably resulted in a higher ionic strength in the former, even though the salinities of the ASW solutions were adjusted to correspond to those of the ambient water. Increasing ionic strength enhances the P sorption [54]. Nevertheless, the variation in the Q values (on average 0.2 − 1.0 µmol g −1 ) between the two different solutions was close to that recorded between the replicate samples and in most of the sediments the isotherm graphs almost equalled.
In sediment S4, ASW produced a steeper P exchange isotherm than the ambient water. This was reflected in P buffering capacities being somewhat higher in the ASW (≤ 2.7 l g −1 ) ( Table 4). Sediment S4 was higher in OM (TC 8.7%) than the other investigated sediments (TC 3.6 − 7.5%). This was presumably reflected in the properties of its ambient near-bottom water and, thus, in the lower P buffering capacities in the sediment. Dissolved organic matter (DOM) can contain organic anions that can disturb the P sorption by competing for the same sorption sites [4] and, thus, lower the P buffering capacity of the adsorbent. The highest PO 4 -P and NH 4 -N concentrations in the ambient water of sediment S4 can refer to the presence of mineralization products, which supports this conclusion. In the other sediments, however, the k values were rather similar. Also, the EPC 0 values as well as the isotherm graphs produced with the ambient water and the ASW were almost equal (Fig. 6). Our results suggest that ASW can be used to replace ambient water in the P exchange studies without significant effects on P sorption. In fact, in the P exchange studies emphasizing the comparison of Fig. 5 Equilibrium P concentrations at zero net P sorption (EPC 0 ) values of sediments S1 and S4 − S7 equilibrated in ambient water (amb) and artificial seawater (ASW). Note the different scales on the y-axes Table 4 k EPC0 , k 5 , k 20 and k 100 values (mean ± SD; l g −1 ) of isotherms executed with artificial seawater (ASW) and ambient water (amb) solutions with sediments S1 and S4 − S7 (n = 2, except for ASW S1 n = 3) the sediment properties, ASW may even be a better option than ambient water, as ASW minimizes the interference of the unknown solution matrix in the experiment.

Noteworthy matters regarding phosphorus exchange experiments
The results of this study highlight that when examining the effects of dissimilar treatments or experimental conditions on the P exchange isotherms, it is important to assess the magnitude of the changes in a proper context. For instance, differences that are close to the analytical detection limit of P can be considered minor. Thus, when considering the detection limit of the P analysis (0.05 µmol l −1 ; [40]) and the range in EPC 0 values in brackish water sediments (up to 38 µmol l −1 ; [53]), it is justified to state that the freeze-drying-induced changes on the easily exchangeable P and on EPC 0 (0.1 − 0.5 µmol l −1 ) in sediments S1 and S3 were practically quite small. Furthermore, the analytical uncertainty assessed by means of repeated measurements (n = 20) of the commercial control solutions containing 0.42, 4.2 and 10.5 µmol P l −1 was on average 13, 6 and 5%, respectively. This should be considered, particularly when examining the differences in EPC 0 values and the equilibrium P concentrations at low P additions. We also assessed the impact of the solid-tosolution ratio on P exchange by executing an additional experiment using freeze-dried sediment S2 with the same experimental setup with a wider solid-to-solution ratio (1:200) than used when comparing the fresh and the freeze-dried subsamples of sediment S2 (1:50) (Fig. 6).
Decreasing the solid-to-solution ratio from 1:50 to 1:200 elevated EPC 0 by 1.5 µmol l −1 and the P desorbed from the sediment (i.e. Q 0 ) by 1.5 µmol g −1 . This result highlights that also the solid-to-solution ratio used should be considered when interpreting or comparing the results of the isotherm experiments. Furthermore, also the possible influence of the isotherm fitting on the calculated P exchange parameters should be taken into account. As shown in the present as well as in some previous studies, freeze-drying and storage of fresh samples affect sediment properties [20,28]. In particular, originally reduced sediments are subjected to fast changes when in contact with atmospheric O 2 [20,55]. Thus, they are expected to exhibit chemical changes if stored unprotected from oxidation. When storing of sediments prior to P exchange experiments cannot be avoided, the practices to be used should be carefully planned and preferentially tested according to the goals of the research. Furthermore, the storage period of the samples to be compared should be kept equal. The use of fresh sediment samples is preferable if accurate weighing, according to known dry matter content, is possible and the experiments can be carried out in N 2 atm soon after the sampling and if the sampling and the storage can be performed shielded from atmospheric O 2 . As can be seen in the isotherms of fresh sediments S2 and S3, the volumetric portioning resulted in more marked deviation among the replicates than weighing (see Fig. 2b).

Conclusions
This study investigated the effects of freeze-drying on the P exchange of brackish water sediments differing in their ability to sorb P. Freeze-drying lowered the P sorption in all sediments, but the magnitude of the change depended on the properties of the sediments. Based on our results we conclude that the sediments high in free sorption sites, particularly Fe oxyhydroxides, are likely to overcome freeze-drying with minor changes. The suggested reason for this is that the impact of the possible freezedrying-induced aggregation of sorbents does not appear so strongly when the sediment possesses abundantly unoccupied surfaces available for P sorption. On the other hand, the sediments low in Fe oxyhydroxides are at higher risk to freeze-drying-induced changes since they already possess a limited amount of unoccupied oxyhydroxides. In the sediments rich in Fe-Mn concretions, freeze-drying promotes the release of occluded P from the concretions and, subsequently, elevates EPC 0 . Thus, sieving of fresh samples can be recommended before freeze-drying to diminish the amount of concretions in the experiments. The results also suggest that when freeze-dried sediments are used in the P exchange studies, the potential impact of the sample treatment should be taken into account when interpreting the results. Furthermore, the solid-to-solution ratio, differences among the replicates and the analytical limitations should be considered when the magnitude of the changes caused by sample treatments are assessed. ASW was shown to be a practical alternative to the ambient water in the P exchange studies with muddyclay brackish water sediments. As the composition of the ASW solution is known, unlike that of ambient water, it is suitable especially when the study is focused on sediment properties. Further studies are needed to unravel the potential changes in the P sorption properties of the dry samples during long-term storage as well as the mechanisms involved in the treatment-induced changes in the P exchange of various sorbents.