The focus in terrestrial ecology is typically on carbon (C) and nitrogen (N), and to a lesser extent on phosphorus (P). But other nutrients such as potassium (K), magnesium (Mg), calcium (Ca), and zinc (Zn) can limit plant growth and influence ecosystem behaviour when in short supply (see e.g. Sardans & Peñuelas, 2015). However, the availability of individual nutrients can be difficult to assess, as, for example, no perfect method exists to determine availability of N or P to plants (Binkley & Hart, 1989; Holford, 1997; Neyroud & Lischer, 2003). Commonly used proxies are N mineralisation (protocol 2.2.5 Nutrient mineralisation), soil NO3 and NH4 concentrations (Keeney & Nelson, 1982), soil C:N ratio (protocol 2.2.4 Soil carbon and nutrient stocks), Olsen P and Bray P (Bray & Kurtz, 1945; Olsen, 1954), and/or ion exchange resin membranes like Plant Root Simulator (PRS) probes (1.4.2 Soil nutrient), but these all have their drawbacks (Binkley & Hart, 1989; Holford, 1997; Neyroud & Lischer, 2003).
Although no perfect method exists to determine nutrient availability, some soil properties are very indicative of the nutrient status of a soil (Vicca et al., 2018). These include bedrock, texture (1.3.5 Soil texture), pH (1.4.1 Soil pH), bulk density (1.3.4 Bulk density), cation exchange capacity, and soil organic matter (SOM). These key soil physical properties are relatively easy to measure and in combination are very indicative of the nutrient status (see e.g. Van Sundert et al., 2018; Vicca et al., 2018). These data on soil properties and nutrients allow disentangling the role of nutrient availability as well as classifying a study site as nutrient-poor, nutrient-rich, or moderately fertile (sensu Vicca et al., 2012; Terrer et al., 2016).
Plant N and P uptake can be calculated from plant N and P concentrations and plant growth:
N uptake = plant growth x [N]
N and P concentrations are important to determine the nutrient status of a study and thus important information for modelling and meta-analysis (see Table 2 in the main paper). Other variables that can be of relevance in some cases are described in stress physiology (Supporting Information S5) and carbon and nutrient cycling (Supporting Information S2).
Below, we present a brief description of complementary measurements that are relatively easy to conduct routinely at any site or study and can provide a robust characterisation of nutrient availability across sites. This list largely corresponds to the measurements recently suggested in Vicca et al. (2018), which we refer to for further reading on the interpretation and relevance of the different measurements.
1.4.1 Soil pH (unitless)
pH is a measure of acidity in the soil and affects many chemical processes, such as plant nutrient availability. Most soil methods are conducted on air-dry soil: however, given that understanding the chemical environment that plants experience is important in ecosystem studies, we propose the use of field-moist soil in preference to dried soil for pH measurements. Soil pH is then carried out on a suspension of fresh field-moist soil in deionised water (DIW), or 0.01 M KCl or CaCl2. Often both DIW and a saline suspensions are measured because both values provide different information. Soils in their natural condition can vary widely in the salt content, also within the same soil the concentration of salts vary with the variation in soil water content, these variations in salinity have an effect in the measurements of pH. The impact of these variations on pH is minimised when measured in 0.01 M of a saline solution and allows valid comparisons of soil pH between seasons and years.
What and how to measure
We focus on measurements in water. The soil:water ratio depends on the amount of organic matter in the soil with a ratio of soil to water of 1:2.5 to 1:5 by weight for mineral soil. The method described here is based upon that employed by the Soil Survey of England and Wales (Avery & Bascomb, 1974) and by the Countryside Survey (Emmett et al., 2010), but measuring soil pH in deionised water using a 1:1 mixture is reported in the NRCS (2014) handbook. Organic soils, however, require a much higher ratio of soil to water of 1:10 or 1:20 by weight
Calibrate the pH meter in buffer solutions. Check pH 4 & 7 buffer calibrations regularly within a sample batch, for example every 10 samples. If either buffer calibration is more than 0.02 of a pH unit from the correct value, repeat calibration. Weigh 10 g of fresh field-moist soil into a 50 ml plastic pH beaker. Add 25 ml of deionised water and stir the suspension thoroughly. Allow it to stand for 30 minutes, stirring occasionally. Measure soil pH electrometrically using the calibrated pH meter.
Include a suitable number of duplicate samples, i.e. carry out the pH measurement twice on approximately one-tenth of the samples. Thoroughly rinse the pH probe between samples with a stream of water from a deionised water wash bottle. Ensure the glass bulb of the pH probe is cleared of soil and be particularly thorough after probes have been immersed in pH buffers. If duplicated samples are not in agreement, repeat the measurements on a small set of samples; from this set of information, determine whether outliers skewed the measurements (remove the outliers if there is good reason, e.g. instrument failure), or whether soil pH was highly variable (report average and standard deviation).
New probes for field measurements of pH are available for instant and fast pH measurements, which enables high-resolution measurement of pH in space and time (e.g. Nielsen et al., 2017).
1.4.2 Soil nutrients
Integrated assessment of soil cation and nutrient availability for plants
Resin membranes like Plant Root Simulator (PRS) probes (Western AG, Saskatchewan, Canada) absorb anions or cations (depending on the type of probe) that are in the soil solution. They thus provide an indication of the nutrient availability as experienced by the biota during the time of burial and are particularly useful for assessing relative differences among treatments and studies. The probes are inserted into the soil for a short period (e.g. 7 days) and are subsequently analysed in the lab for the nutrients of interest (e.g. NO3, NH4, P, K, Ca, Mg, Mn, Fe, Zn). The results indicate the flux of each of these nutrients over the time of burial. Caution is needed to avoid saturation of the probes (i.e. burial time should not be too long) and the absorption of ions is sensitive to soil moisture, which may complicate interpretation in studies where soil moisture differs between the treatments. More information is available from the website of the commercially available PRS probes (https://www.westernag.ca/innov). Instead of buying the commercial product, it is also possible to produce the probes for low cost (see protocol 2.2.5. Nutrient mineralisation).
Cation exchange capacity, exchangeable base cations, and soil electrical conductivity
One of the most important properties of soil colloids (clay and organic matter particles < 0.001 mm diameter) is their ability to adsorb, hold, and release ions. Colloids are generally negatively charged and thus attract primarily positively charged ions, i.e. cations. The more negative charges, the higher the capacity of the soil to bind cations, and thus the higher its cation exchange capacity (CEC, typically expressed as the amount of positive charges that can be exchanged per mass of soil).
For soil fertility, the total exchangeable base cations (Mg2+, Ca2+, and K+ in particular) are especially relevant. These are the base cations bound to the negatively charged colloids. They can be taken up relatively easily by plant roots through exchange for H+. The fraction of CEC that is occupied by exchangeable base cations is termed base saturation. This fraction can be small, especially in acidic and leached soils where many of the negative charges are occupied by (acidic) cations, such as H+, Al3+, or Fe3+.
What and how to measure
Cation exchange capacity and total exchangeable base cations can be determined using the most common method of Brown (1943), for which 1 M buffer ammonium acetate solution (NH4Ac) at pH 7 serves as the extractant. Soil samples are collected and sieved (< 2mm) and air dried.
Soil electrical conductivity (EC; mS m–1)
This is the ability of soil to conduct an electrical current and is commonly expressed in units of milliSiemens per metre (mS m–1). EC estimates the concentration of ions in the soil, namely the anions Cl–, SO42-, and HCO3– and the cations Na+, Ca2+, K+, and Mg2+ (Friedman, 2005; He et al., 2012). Although the relationship between conductivity and salt concentration varies somewhat depending on ionic composition, EC provides a simple and reasonably accurate estimate of solute concentration (Carter & Gregorich, 2006). In addition, as soil EC is affected by several soil properties, its measurement can also be used as a proxy for estimating directly or indirectly the variations in these properties, including soil texture, bulk density, soil water content, water-holding capacity, cation exchange capacity, organic matter, and subsoil characteristics (Corwin & Lesch, 2005a; Grisso et al., 2005). For this reason, over the years, EC has been largely used in agriculture to estimate soil salinity, nutrient availability and loss, soil texture, and available water capacity, being considered a reliable and cost-effective measurement (NRCS, 2014). As an example, high EC values often reflect poor plant growth conditions and the potential for salinity problems (Karlen et al., 2008).
As this variable has been shown to be closely related with distinct soil properties, its measurement assumes special importance under the context of climate change where some properties are expected to alter with consequences for soil quality and its functioning.
What and how to measure
The first measurements of soil EC were made on soil samples, but it was found to be more consistent to measure EC in soil extracts. Hence, the standard laboratory method for determining the EC of a soil is by using an aqueous paste extract of soil and to measure the electrical conductivity of the solution using a conductivity meter (Richard, 1954; Carter & Gregorich, 2006). The determination is carried out to obtain an indication of the content of water-soluble electrolytes in a soil. Because the saturated paste extract method requires time and skill, a fixed soil:water ratio (e.g., 1:1 to 1:5) has been generalised when measuring soil EC and solute concentrations (ISO 11265, 1994). Knowing that EC in soil is dependent on several properties and therefore is highly variable, several samples should be taken from multiple locations.
Besides the methods based on an aqueous paste extract of soil, the apparent EC (ECa, bulk soil electrical conductivity) has become one of the most frequently used measurements to characterise the spatial distribution of soil salinity at field scales. Nowadays, ECa is considered an invaluable tool for the establishment of spatial variation and for identifying the soil properties influencing crop production in precision agriculture (Corwin & Lesch, 2005a, 2005b). ECa has also been used to identify homogeneous areas within a field to implement experiments. Field methods used to measure ECa include the Wenner array or four-electrode, time domain reflectometry (TDR) and electromagnetic (EM) induction (Carter & Gregorich, 2006). The EM method, by using a non-contact sensor, is the most commonly used because measurements can be taken quickly over large areas, the large volume of soil measured reduces local scale variability, and measurements are possible on relatively dry or stony soils because contact is not required between soil and sensor (Hendrickx et al., 1990).
Carbon and nutrient stocks
The soil carbon and nutrient stock is the amount of C, N, P, K, and other nutrients stored in the soil. These stocks are coupled with net primary production and decomposition of above- and belowground material and highly related to climate. For further details and how to measure see protocol 2.2.4 Soil carbon and nutrient stocks.
Soil organic matter (SOM, %)
It is important to determine the soil organic matter (SOM), as changes in environmental factors such as temperature or water inputs may alter the SOM content (directly and indirectly by influencing organic matter inputs), and variation among studies may also be explained by differences in SOM. Soil organic matter can be determined using the Walkley Black method or the loss-on-ignition (LOI) procedure described in Nelson & Sommers (2009). LOI is generally preferred over the Walkley Black method because is less time consuming. One of the aspects to consider when measuring LOI is the choice of combustion temperature in the furnace and the duration of time used for combustion.
What and how to measure
We propose the method of Ball (1964) which is determined by subtracting the weight of a soil after 16 h drying at 105 °C from a soil after placing in a furnace overnight at 375 °C. The amount of soil used to determine SOM is adjustable but should not be lower than 10 g fresh soil to ensure a representative sample. For more details also see Countryside Survey (Emmett et al., 2010).
Soil inorganic carbon (mass fraction: g C g soil-1)
Some soils contain significant amounts of carbonates, especially soils in dry climates. Changes to warming or the precipitation regime may alter the carbonate content. Reporting carbonates is not routine, but may be important if a total carbon balance is required. We refer the reader to the methods described in Nelson & Sommers (2009).
1.4.3 Soil trace metals
Trace metals are a group of metals and metalloids (e.g. arsenic (As) and selenium (Se); hereinafter called “metal”) found in low concentrations (< 100 mg kg–1), in mass fractions of ppm or less, in some specified source, for example, soil, water, plant, or tissue (Duffus, 2002; Hooda, 2010). The most common trace metals are beryllium (Be), aluminium (Al), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), As, Se, molybdenium (Mo), silver (Ag), cadmium (Cd), antimony (Sb), mercury (Hg), thallium (TI), and lead (Pb). Trace metals are important elements in the biogeochemistry of terrestrial ecosystems (Driscoll et al., 1994). The concentration of trace metals in soil directly relates to the growth and development of vegetation and reflects the supply of mineral nutrition to plants by soil (Zhanbin et al., 2013). Depending on the dose, trace metals can become potentially toxic for life (Kabata-Pendias & Mukherjee, 2007; Kabata-Pendias, 2010). Although trace metals are naturally present in soils (Kabata-Pendias & Mukherjee, 2007; Kabata-Pendias, 2010), their concentrations in soils are significantly influenced by anthropogenic activities, which greatly alter the biogeochemical cycles of trace metals and their bioavailability (Driscoll et al., 1994). The observed changes in soil properties could affect soil functioning through their impacts on the composition and activity of microbial communities, which can provoke toxic responses in soil microorganisms, including reducing microbial biomass and decreasing carbon mineralisation and disturbing enzymatic activities. Furthermore, metal stress has been found to change the structure and diversity of microbial communities (Certini, 2005; Hart et al., 2005; Hartmann et al., 2005; Frey et al., 2006). This eventually affects the biogeochemical system’s functions driven by these organisms, since soil microorganisms are important agents in nutrient cycling and energy flow. Assessing the levels of trace metals in soils is crucial to determining the environmental impacts of climate change on soil quality, structure, and functioning (Curran-Cournane et al., 2015).
What and how to measure?
Total concentration of trace metals in soils: sample digestion is often a necessary step before determining total element mass concentration in soils. Various digestion methods are used to determine the mass concentration of trace metals in soils, including different combinations of concentrated acids (Gaudino et al., 2007). The dissolution of soil samples can be obtained by rigorous digestion using the standardised aqua regia extraction protocol which consists of treating a soil sample in a heated 3:1 mixture of hydrochloric (HCl) and nitric (HNO3) acids (ISO 11466, 1995; USEPA 3050B, 1996). This is a partial digestion of the soil solid phase consisting of a very strong acid digestion that dissolves almost all elements that become “environmentally available” (McLean & Bledsoe, 1992; USEPA 3050B, 1996; USEPA 3051A, 2007). Although the aqua regia digestion method is internationally accepted to measure concentrations in soil, fractions of elements extracted by this method are not available for biological uptake (Gaudino et al., 2007). If a total trace metals concentration is required, the soil samples are treated with a mixture of HNO3 + HCl + HF (hydrofluoric acid) using microwave heating with a suitable laboratory microwave system (USEPA 3052, 1996; EN 13656, 2002). After the extraction procedures (ISO 11466, 1995; USEPA 3050B, 1996; USEPA 3052, 1996; EN 13656, 2002; USEPA 3051A, 2007), the extract is filtered through 0.45μm nitrocellulose membrane filters, diluted, and analysed by atomic absorption spectrometry (flame: FAAS or graphite furnace: GFAS) or inductively coupled plasma spectrometry (optical emission: ICP-OES or mass: ICP-MS).
Another commonly used procedure to measure the “total” concentration of trace metals is the digestion with hot HNO3 and hydrogen peroxide (H2O2) procedure also outlined in USEPA 3050B (1996). This method adds H2O2 in order to enhance the destruction of the organic matter in soil.
Sequential extractions of trace metals in soils: chemical extraction is employed to operationally define trace metal fractions, which can be related to chemical species, as well as to potential mobile, bioavailable, or ecotoxicological phases of a sample. Fractionation is usually performed by a sequence of selective chemical extraction techniques, including the successive removal, or dissolution, of these phases and their associated metals (Hlavay et al., 2004). The extraction procedures consist of reacting a soil sample with increasing strengths of chemical solutions. Numerous extraction procedures have been developed for trace metals (Sposito et al., 1982; McLean & Bledsoe, 1992; Singh et al., 1998; Wenzel et al., 2001; Imperato et al., 2003; Hlavay et al., 2004; Hooda, 2010). Supernatants from each fraction will be analysed by FAAS, GFAA, ICP-OES or ICP-MS.
For the Sequential extraction there are numerous methods using different extractants at different concentrations. For example, extraction with specific extracting agents, especially containing chelating agents, allows examination of the distribution of soluble exchangeable forms. Ethylenediaminetetraacetic acid (EDTA), used as extracting agent for many trace elements, has been widely applied in soil science and environmental chemistry (Kocialkowski et al., 1999; Michaud et al., 2007; Komárek et al., 2008).
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