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Assessment of efficacy of biocides in different soil types for use in sorption studies of low molecular weight organic compounds.

Introduction

The presence of low molecular weight organic compounds (LMWOCs), such as amino acids and carbohydrates, in soil is attributed to root exudation and turnover (Farrar et al. 2003; Boddy et al. 2007) and the breakdown of plant and microbial deposits (Fischer and Kuzyakov 2010). Once present, LMWOCs can follow several chemical and biological pathways within the soil solution matrix (Fischer and Kuzyakov 2010). These pathways, critical to the overall C cycle in the soil, occur concurrently and result in the short half-lives and high turnover rates of many LMWOCs in soil. Boddy et al. (2007) and Farrell et al. (2014) showed that sugars and amino acids are cycled extremely rapidly by soil microorganisms and estimated that pools of LMWOCs are turned over -4000 times per year. The competition between microbial uptake and sorption by soil components, including mineral particles and soil organic matter, is of particular concern when targeting the sorption behaviour of specific organic analytes, particularly when abiotic edaphic properties need to be isolated.

There are several methods for overcoming the influence of microbial activity on soil solution concentration in sorption analysis, including short equilibrium times, heat sterilisation and the use of biocides (Fischer et al. 2010; Rousk and Jones 2010). Application of a biocide in sorption experiments is crucial as the activity of the microbial population can not only affect the solution concentration of the target analyte but can also have an effect on composition of the soil matrix (Chefetz et al. 2006). Sterilisation of the soil eliminates changes due to microbial activity and allows an assessment of interactions between the analyte and the soil. Both mercuric chloride (Hg[Cl.sub.2]) and azide salts (K[N.sub.3] or Na[N.sub.3]) have been noted for their effectiveness in soil sterilisation (Wolf and Skipper 1994). The effectiveness of Hg[Cl.sub.2] as a biocide is due to the toxicity of mercury, specifically, its ability to destroy cross-linked thiol groups in proteins, inhibiting their function (Oremland and Capone 1988). Mercury also affects mitochondrial function and cell wall synthesis (Vaituzis et al. 1975). In contrast, Na[N.sub.3] prevents respiration by inhibiting cytochromes, a key component of the respiratory chain (Oremland and Capone 1988), and as a result of the oxidative capacity of the azide radical ([N.sub.3.sup.*]) to inflict damage on cell membranes and inhibit transport of solutes across the membrane (Levy and Chevion 2009).

Despite the effectiveness of Na[N.sub.3] and Hg[Cl.sub.2] as inhibitors of biological activity, the acute and chronic toxicity of these compounds presents a significant hazard to the user. At high concentrations, they add significant ionic background and may affect the pH of the solution, which can influence the results of sorption experiments. It has been reported that Na[N.sub.3] can react with particular analytes and result in a decrease in the concentration of Na[N.sub.3] over time (Chefetz et al. 2006). Sodium azide was shown to form a variety of transformation products when interacting with the target analyte, atrazine, in both anaerobic and aerobic conditions (Ro et al. 1995). As a result, it is necessary to determine the lowest effective concentration of biocide in solution while ensuring efficacy in elimination of microbial activity. Soil type can have a significant effect on the level of biocide required, with clay and organic matter content affecting the inhibitory behaviour of Hg[Cl.sub.2] due to sorption of the biocide onto clay or organic matter surfaces (Wolf and Skipper 1994; Yin et al. 1996; Yang et al. 2008).

We determined the efficacy of increasing doses of Hg[Cl.sub.2], Na[N.sub.3] and a mixture of the two on the microbial degradation of [sup.14]C-labelled glucose, a readily degradable substrate, in a sorption system over a 2-week period. We focused on the use of biocides as opposed to pre-sterilisation via heat, autoclaving or gamma irradiation as the biocide should remain active for the duration of the experiment, negating the possibility of re-introduction of microorganisms during experiment establishment and sampling. We specifically chose glucose as it has previously been shown to have little capacity to be abiotically sorbed to soils (Kuzyakov and Jones 2006), and thus depletion of glucose concentrations should be solely attributable to biological activity. Jones and Edwards (1998) attributed the lack of glucose adsorption to clay minerals to its lack of charge, while Jagadamma et al. (2012) confirmed a lack of specific binding mechanisms of glucose with soil minerals. These results correlate well with data from this study, where loss of glucose from solution was shown to be less than 10% in the first 96 h of shaking (at the highest solution concentration of Hg[Cl.sub.2]). Therefore, the contribution of sorption to the total loss of glucose from solution is small for all soil types tested (Table 1) and is not expected to explain the gradual loss of glucose in soils, which is thus only attributable to microbial utilisation.

Materials and methods

Soils and chemicals

Four different soil types, one belonging to each of the soil groups Arenosol, Luvisol, Ferralsol and Andisol, were selected for this study to represent contrasting physico-chemical properties. The Arenosol was from a cereal cropping enterprise typical of much of the wheatbelt of Western Australia, the Luvisol and Ferralsol both came from high intensity dairy or beef production in the fertile and high-rainfall plains of northern New South Wales. The Andisol was from an area adjacent to a field under dairy production in south-eastern South Australia, and represents one of the few such soils present on the Australian mainland (Lowe and Palmer 2005). A bulk (~50 kg) sample collected from the 0-10 cm layer of these soils (location and characterisation details in Table 1) was air-dried at 40[degrees]C and sieved with a 2 mm sieve before use.

Radiolabelled glucose ([sup.14]C-[U]-glucose with specific activity 0.2 mCi [mL.sup.-1]) was procured from PerkinElmer Australia (Melbourne, Victoria). The biocides (Hg[Cl.sub.2], Na[N.sub.3]) were purchased from Sigma Aldrich Pty Ltd (Sydney, New South Wales). HighSafe 3 scintillation cocktail was also obtained through PerkinElmer Australia.

Glucose mineralisation study

The four soils were analysed in triplicate by shaking in an end-over-end shaker (1:10 soil solution ratio with 2 g soil) over a period of 2 weeks at room temperature (~22[degrees]C) with 1 mM [sup.14]C-[U]-glucose (specific activity 1 kBq [mL.sup.-1]; 20 kBq [sample.sup.-1]) made up in 20 mL 18.2 M[ohm] water. The three biocide treatments (Hg[Cl.sub.2], Na[N.sub.3] and a mixture of Hg[Cl.sub.2] and Na[N.sub.3]) were added to the soil solutions simultaneously with the [sup.14]C-[U]-glucose at final concentrations of 0, 50 and 500 [micro]M and 10mM, based on previous work (Fischer and Kuzyakov 2010). A 250 [micro]L aliquot was removed at intervals of 1, 3, 6, 24, 48, 72, 96, 168, 240 and 336 h, and was centrifuged at 17000g for 5 min. Supernatant was analysed by liquid scintillation counting (HighSafe 3 scintillation cocktail and Tri-Carb 3110 TR scintillation counter: PerkinElmer Australia). The scintillation counter had a working detection limit of 0.083 Bq [mL.sup.-1], with a coefficient of variance of <0.1% on a 1 kBq [mL.sup.-1] standard.

Results and discussion

The effectiveness of the biocides in inhibiting microbial activity varied significantly across the four soils (Fig. 1), with calculated depletion time--10% ([DT.sub.10]) values ranging from <1 h in the Luvisol with no biocide to >336 h in the Luvisol and Arenosol with higher doses of biocide (Table 2; where [DT.sub.10] is the time by which 10% of the added glucose [sup.14]C was lost from solution). The rapid loss of the [sup.14]C label ([DT.sub.10] [less than or equal to] 8h) from the solution in all soils with no biocide highlights how rapidly labile C substrates such as glucose are taken up by microbial activity (Boddy et al. 2007), given that abiotic sorption of glucose is known to be minimal (Jones and Edwards 1998; Kuzyakov and Jones 2006).

While it is possible that [sup.14]C measured in the aliquots of solution could be microbial metabolites of the added glucose, we consider this unlikely because in the control treatments (no biocide), [sup.14]C activity in the solution reduced sharply soon after commencement of the experiment. Sodium azide was least effective in the Luvisol, with the highest solution concentration of lOmM having a [DT.sub.10] of 2h. In the Luvisol, 10 mM Hg[Cl.sub.2] alone and in combination with Na[N.sub.3] was the only effective concentration for which microbial activity was inhibited for the entire experimental duration (Table 2). In contrast, microbial activity in the Arenosol was inhibited by all biocides at the 10 mM concentration as well as the 500 [micro]M application of Hg[Cl.sub.2] and the biocide mixture.

We ascribe the effectiveness of the biocide combinations in the Arenosol to its lower clay content (1.7%) and organic C content (1.5%) than other soils. Clay minerals sorb a variety of compounds from aqueous solutions (Zhao et al. 2010), and at 28.8% clay, the Luvisol likely sorbed the biocides more strongly than the Arenosol, effectively lowering the biocide concentration in solution, possibly due to its higher organic matter content (Chen et al. 2017). This reduces the ability of the biocide to limit microbial activity and results in a higher rate of loss of glucose from solution. Due to lower organic C content of the soil, it is conceivable that the inherent microbial activity was also lower in the Arenosol, as demonstrated in other recent research that included these two soils (Farrell et al. 2015).

All combinations of biocide were least effective in inhibiting microbial activity in the Andisol. However the application of 10 mM Hg[Cl.sub.2] resulted in a ~5-fold increase in [DT.sub.10] when compared with the same application of Na[N.sub.3]. The combination of both biocides at the same concentration resulted in a 32% decrease in the [DT.sub.10] when compared with the Hg[Cl.sub.2] application. For both the Ferralsol and Andisol, the [DT.sub.10] in the highest Hg[Cl.sub.2] concentration was significantly less than for the Arenosol and Luvisol (Table 2), with [sup.14]C not being depleted below 90% of the added concentration within the 336 h experimental period.

Andisols are characterised by high surface area and highly reactive colloidal fractions (Nanzyo 2003), in addition to containing relatively large amounts of organic matter due to the stabilising capabilities of allophanes (Parfitt et al. 1997; Lowe and Palmer 2005). As a result, Andisols are noted for their ability to sorb anions and metals (Huang et al. 2011), with Barton et al. (2005) attributing reduced leaching of P from surface-applied effluent to the relatively high P sorption capacity of Andisols. Stephens et al. (2002) also noted that the single most effective component controlling retention of Hg[Cl.sub.2] by soil is soil organic C, and it is notable here that the [DT.sub.10] was only affected by Hg[Cl.sub.2] at a 10 mM concentration relative to the control. While glucose sorption was very low in all other soils (as indicated by its negligible loss in the presence of 10 mM concentration of two biocides as a mixture), sorption may have also contributed to the gradual loss of glucose with time in the Andisol (Jagadamma et al. 2014).

Both the Ferralsol and the Andisol had a greater soil C concentration (5.17% and 7.86%, respectively) than the Arenosol and Luvisol. As with clay content, soil organic matter has also been correlated with the sorption and desorption of heavy metals (Weng et al. 2001; Bradl 2004; Usman et al. 2008). As such, a significantly higher level of soil C in these soils may impact on the ability of the biocide to reduce microbial activity at lower concentrations. Correlation analysis indicated that the [DT.sub.10] values for glucose in the 10 mM Hg[Cl.sub.2] and 10 mM mixture treatments were strongly negatively correlated with organic C concentration (r=-0.995, P = 0.005 and r=-0.969, P = 0.031 respectively), i.e. efficacy of Hg[Cl.sub.2] decreased as C concentration increased. No correlations were found between organic C and [DT.sub.10] values for the less effective biocide treatments, nor for other soil variables such as clay content or pH. While the mechanisms through which biocides are inhibited in certain soil types were not investigated in this study, this does not weaken the key finding that biocide efficacy does depend on soil type.

The results of this study indicate that soil type has a significant effect on biocide efficacy and the choice of biocide and its concentration for a particular soil type is critical. The efficacy of both biocides used here is most likely affected primarily by sorption to clay, organic matter or both. For all soils in the present study, the application of Hg[Cl.sub.2] alone or in combination with Na[N.sub.3] at a solution concentration of 10 mM proved to be the most effective at inhibiting the activity of the soil microbial population throughout the duration of the experiment. This was most evident in the Luvisol, where low Na[N.sub.3] concentrations alone were least effective at inhibiting microbial activity. In studies currently published in the literature, various biocides seem to be chosen interchangeably; however, this study shows that the optimal choice of biocide is influenced not only by soil type but also by equilibration time, with the potential for less potent and therefore safer applications of biocide to be used when shorter experimental periods are required. It is especially important for studies relying on inhibition of microbial activity to report on the efficacy of inhibition to verify their observations. Further systematic work is required both to ascertain inhibitory concentration indices using a broader range of biocide concentrations, and also to more formally elucidate variation both within a single soil type and between different soil types. As both organic C and clay contents are likely to be the main factors altering biocidal efficacy, comparisons between samples of the same soil soil type, but differ primarily in organic C and clay content should be a high priority for future work. Such an approach would remove any confounding influeces of mineralogy and pH, allowing focus only on these two controlling factors.

https://doi.org/10.1071/SR17282

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This work is a joint output between the National Soil Carbon Program and Biochar Capacity Building Program, and is supported by funding from the Australian Government Department of Agriculture and Water Resources.

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Sheridan Martin (A), Rai S. Kookana (A), Lynne M. Macdonald (B), and Mark Farrell (B,C)

(A) CSIRO Land and Water, Waite Campus, Locked Bag No. 2, Glen Osmond, SA 5064, Australia.

(B) CSIRO Agriculture and Food, Waite Campus, Locked Bag No. 2, Glen Osmond, SA 5064, Australia.

(C) Corresponding author. Email: mark.farrell@csiro.au

Received 17 October 2017, accepted 4 February 2018, published online 19 April 2018

Caption: Fig. 1. Depletion of glucose from soil solution with varied applications of biocide. Values are means [+ or -] s.e.m., n = 3. Horizontal dotted lines represent [DT.sub.10] thresholds at which 10% of the added glucose is degraded (Table 2).
Table 1. Properties of the four soils

                                    Andisol

Location                      37[degrees]56'32"S,
                              140[degrees]45'0"E
Coarse sand (%)                      16.1
Fine sand (%)                        30.3
Silt (%)                             20.1
Clay (%)                             11.5
Organic C (g [kg.sup.-1])            78.6
Total N (g [kg.sup.-1)               6.11
Total P (g [kg.sup.-1)               0.936
Total S (g [kg.sup.-1)               0.289
Total Fe (g [kg.sup.-1)              34.8
Total Al (g [kg.sup.-1)              27.2
pH                                   7.30

                                    Luvisol

Location                      34[degrees]30'34"S,
                              138[degrees]45'7"E
Coarse sand (%)                       5.1
Fine sand (%)                        43.0
Silt (%)                             15.8
Clay (%)                             28.8
Organic C (g [kg.sup.-1])            20.8
Total N (g [kg.sup.-1)               1.91
Total P (g [kg.sup.-1)               0.313
Total S (g [kg.sup.-1)               0.279
Total Fe (g [kg.sup.-1)              19.6
Total Al (g [kg.sup.-1)              37.8
pH                                   8.18

                                   Ferralsol

Location                      28[degrees]48'58"S,
                              153[degrees]23'29"E
Coarse sand (%)                       0.6
Fine sand (%)                        16.1
Silt (%)                             37.7
Clay (%)                             37.2
Organic C (g [kg.sup.-1])            51.7
Total N (g [kg.sup.-1)               5.04
Total P (g [kg.sup.-1)               1.32
Total S (g [kg.sup.-1)               0.638
Total Fe (g [kg.sup.-1)              88.3
Total Al (g [kg.sup.-1)              104.8
pH                                   5.05

                                   Arenosol

Location                      30[degrees]20'38"S
                              115[degrees]36'31"E
Coarse sand (%)                      58.0
Fine sand (%)                        35.8
Silt (%)                              1.8
Clay (%)                              1.7
Organic C (g [kg.sup.-1])            15.0
Total N (g [kg.sup.-1)               1.14
Total P (g [kg.sup.-1)               0.091
Total S (g [kg.sup.-1)               0.133
Total Fe (g [kg.sup.-1)              0.666
Total Al (g [kg.sup.-1)              3.34
pH                                   5.91

Table 2. Mean [DT.sub.10] (degradation time, 10%, h) values for the
Added glucose as affected by biocide and soil type (n = 3)

                            Andisol   Luvisol   Ferralsol   Arenosol

Nil                            5         1          3          8
50 [micro]M Hg[Cl.sub.2]       6         1          7          10
500 [micro]M Hg[Cl.sub.2]      6        26         27         >336
10 mM Hg[Cl.sub.2]            142      >336        225        >336
50 [micro]M Na[N.sub.3]        7         1         16          20
500 [micro]M Na[N.sub.3]       9         1         175        262
10 mM Na[N.sub.3]             30         2         131        >336
50 [micro]M Mixture            5         1          5          31
500 [micro]M Mixture          10        49         199        >336
10 mM Mixture                 96       >336        128        >336
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Author:Martin, Sheridan; Kookana, Rai S.; Macdonald, Lynne M.; Farrell, Mark
Publication:Soil Research
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Geographic Code:8AUST
Date:Aug 1, 2018
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