Review of techniques for the in-situ sterilization of soil contaminated with Bacillus anthracis spores or other pathogens

Abstract

This review summarizes the literature on efficacy of techniques to sterilize soil. Soil may need to be sterilized if contaminated with pathogens such as Bacillus anthracis. Sterilizing soil in-situ minimizes spread of the bio-contaminant. Soil is difficult to sterilize, with efficacy generally diminishing with depth. Methyl bromide, formaldehyde, and glutaraldehyde are the only soil treatment options that have been demonstrated at full-scale to effectively inactivate Bacillus spores. Soil sterilization modalities with high efficacy at bench-scale include wet and dry heat, metam sodium, chlorine dioxide gas, and activated sodium persulfate. Simple oxidants such as chlorine bleach are ineffective in sterilizing soil.

1. Introduction

1.1. Need for soil to be sterilized

The purpose of this review is to summarize the scientific literature on the efficacy of techniques to sterilize soil contaminated with microbiological contaminants. While contamination of soil via enteric pathogens has been an environmental problem for decades [1], there is growing concern over soil contaminated with other pathogens such as Bacillus anthracis. B. anthracis is the bacterium that causes anthrax, a zoonotic disease that affects most mammals but primarily impacts herbivorous livestock and wildlife [2]. Soil may become contaminated with B. anthracis spores from the carcasses of livestock or wildlife that have succumbed to the disease [3–5]. This is a particularly serious issue in Kazakhstan, with over 2,000 anthrax-infected soil foci dispersed throughout the country [6]. B. anthracis spores may survive in soil for decades [7], while others have suggested that Bacillus spores in general may survive for hundreds of years in soil [8]. For this reason, decontamination of the resulting carcasses and remediation of the surrounding soil is recommended [9,10]. Soil may also become contaminated with other zoonotic pathogens that have infected and resulted in the mortality of wildlife, such as with Burkholderia pseudomallei (the bacterium causing melioidosis) [11], Yersinia pestis (plague) [12] and Francisella tularensis (tularemia) [13].

Soil may become contaminated with pathogens in other ways, such as if they’re disseminated intentionally as a biological weapon in a wide area release [14,15]; released accidently (as occurred in Sverdlovsk, Russia in 1979) [16]; or from land disposal of biological materials at a former proving ground [17]. Lastly, soil may need to be sterilized as a result of the Mars Sample Return Earth Entry System landing in the Utah desert [18]. The entire landing site of the Earth Entry System may need to be decontaminated as a precautionary measure. Planetary protection (e.g., protecting Earth from potential biological hazards posed by extraterrestrial matter) standards require treating the Earth Entry System and impact area using protocols consistent with the highest levels of biosafety.

For the above scenarios, soil may be removed and transported for off-site remediation (e.g., incineration) [19], although this may result in further spread of the microbiological contaminant into the environment (e.g., as an aerosol) and is not recommended. Thus, this review focuses on soil decontamination or sterilization techniques that can be implemented in-situ. In the event of a hazardous material (such as a pathogenic microbiological contaminant) release into the environment, prompt containment and remediation are imperative to avoid additional spread of the contaminant [20]. Additionally, the microbial contamination in the soil could potentially increase if suitable growth conditions occur [21]. Please refer to the Supplemental Material for additional background information related to this review.

1.2. Differentiating between soil sterilization and other soil treatments

While McNamara et al. [22] refer to sterilization as the complete elimination of organisms, others specifically suggest that sterilization of materials requires inactivation of both microbial cells and spores [23]. Some practitioners use the terms “disinfection” and “sterilization” interchangeably, although disinfection is a less rigorous antimicrobial process than sterilization. Bacterial spores are among the most resistant life forms to biocidal processes and thus are used as indicators of sterilization efficacy [24].

“Disinfection” refers to the inactivation of vegetative bacteria and viruses on surfaces or materials, but not the inactivation of the more resistant bacterial spores [25]. The spore’s structure and chemical composition play roles in its resistance to heat, chemicals, and radiation – properties that are lacking in growing (vegetative) bacteria [8]. Thus, techniques found to be effective in inactivating bacterial spores in a soil matrix would be expected to be effective as well in inactivating the less resistant vegetative bacteria and viruses.

1.3. Impact of material and soil on decontamination efficacy

Bacterial spores are one of the most resistant microbial forms to inactivate [26], and exacerbating this challenge is that soil is very difficult and costly to effectively sterilize [21]. In general, the material the target microorganism is associated with greatly influences the efficacy of a sterilization technique, with porous and organic materials generally being more difficult to effectively decontaminate [27]. Soils are difficult to remediate for pathogenic contaminants due to their potentially high organic content, porosity, and other variable chemical and physical characteristics [28]. In some cases, even vegetative bacteria and viruses, which would be relatively easily inactivated (compared to spores) on glass or stainless steel (nonporous, inorganic materials), were difficult to fully inactivate when in a soil matrix [29]. Adding to the difficulty of decontaminating soil in-situ is the ability of the decontaminant to maintain efficacy at increasing depths [14].

1.4. Previous reviews of soil sterilization or related studies

The most recent review of the literature for sterilization of soil was provided by Trevors [23] in 1996, which focused on methods that could be used for small amounts of soil (e.g., on the order of a few kg or less) for subsequent bench-scale laboratory experiments. The author noted that chemical and physical properties of soil are usually altered after sterilization, except that physical-based techniques such as gamma irradiation or autoclaving would not leave a chemical residue. Conditions needed for achieving sterilization were summarized, but no efficacy data were provided.

While Gurtler provides an excellent review of full-scale, in-situ decontamination methods for the inactivation of plant and human pathogens in crop soil [30], the focus was on vegetative bacteria and excluded consideration of the more recalcitrant bacterial spores. Nevertheless, some of the chemistries and application techniques discussed in the review do have some relevance for inactivation against bacterial spores in soil.

Wood and Adrion [28] and Campbell et al. [15] provide reviews of decontamination techniques and strategies that could be used following an intentional wide area B. anthracis spore release. These reviews discussed decontamination techniques and approaches that could be used broadly for a variety of materials and environments, and included a short discussion related to the difficulty and complexity of sterilizing soils. The present article focuses specifically on remediation of soil that may be contaminated with pathogenic materials such as B. anthracis spores.

1.5. Scope of this review

In summary, this review focuses primarily on the efficacy of techniques that could be implemented in-situ, and at full-scale, for the sterilization of soil contaminated with pathogens. That is, this review examines how well field-scalable techniques are for inactivating bacterial spores when encompassed within a soil matrix. While the focus of this review is primarily for spores of B. anthracis, data from other Bacillus species are also included since their sensitivity to decontaminants is expected to be similar [31]. Some good overviews of bacterial spore inactivation mechanisms, such as damage to DNA, the inner membrane, or the germination apparatus, may be found elsewhere [32,33]. With respect to damage to the germination apparatus, spores may be only conditionally dead and can be revived with proper treatment, such as with lysozyme [33]. Thus, the soil decontamination method being considered should be tested in the laboratory to ensure that the spores are in fact truly inactivated.

Many soil sterilization-related studies found in the literature focused on the impact of the sterilization technique on subsequent plant growth or impact to soil chemical, physical, or biological properties; see for example [34–36]. In these studies, the efficacy of the sterilization treatment was not evaluated but was assumed to be adequate, and thus no efficacy data were reported. Although impacts to soil are a concern, the efficacy of soil sterilization treatments to inactivate bacterial spores was the primary focus of this review.

When sterilization efficacy is quantified in a study, it may be reported in terms of log₁₀ reduction (LR) (e.g., [37]) of bacterial spores, or it may be reported in terms of microbial counts post-treatment [38]. For this review, a treatment reported as providing a LR ≥ 6, or in which no microbial life was detected post-treatment, is considered “effective” [28].

As a secondary goal of this study, other information (when available) is presented on how these sterilization techniques can be applied to soil at full-scale, following the example of Gurtler, who provided information on application techniques for soil disinfection [30]. For example, Feng et al. [39] provide an overview of in-situ soil remediation options for chemical contaminants and highlight the use of horizontal injection wells to allow chemical treatments to reach the contaminants of concern; in some cases these techniques may be applicable for remediation of soils contaminated with microbial contaminants.

2. Methods

The primary means for searching the scientific literature was the Web of Science^™ Core Collection (Clarivate; London, United Kingdom). Additional information on the literature search may be found in the Supplementary Material.

3. Results and discussion

3.1. Sterilizing soil for laboratory-scale studies

The majority of the literature for soil sterilization focuses on relatively small amounts of soil that may need to be sterilized for the purposes of conducting certain bench-scale laboratory experiments [23]. Examples of lab studies in which sterilized soil is needed include those investigating re-colonization, soil enzymes, chemical degradation [22] and impact on plants/crops [40]. Also, soils used for research may need to be sterilized if they are imported, due to phytosanitary regulations [41].

However, due to differences in scale, some techniques commonly used to sterilize small quantities of soil, such as ethylene oxide gas or gamma irradiation, would not be applicable or available for full-scale, in-situ soil sterilization or remediation. While ethylene oxide and gamma irradiation have demonstrated efficacy in sterilizing soil and minimizing impacts to chemical and physical characteristics of soil [37], their use is confined to commercial facilities equipped and permitted to handle these hazardous materials. For example, although ethylene oxide is widely used for sterilization of medical devices (using spores of Bacillus atrophaeus as the indicator organism) [42], and has been recommended for small quantities of soil, its use is limited to small scale due to its intrinsic hazards including flammability, reactivity, and toxicity [43]. Gamma irradiation is also widely used for sterilization (Bacillus pumilus as the indicator organism) of pharmaceuticals and medical devices [44] and has been recommended for small quantities of soil. But it utilizes cobalt-60, a sealed radioactive source which must be confined to licensed facilities equipped with technical (e.g., shielding and wet source storage) and procedural safeguards such as required under the US Nuclear Regulatory Commission standards [45].

3.2. Recommendations for decontamination of soil where animals have died of anthrax

In an anthrax outbreak involving livestock or wildlife, the World Health Organization (WHO) recommends either burial or incineration of the infected carcass, and if possible, the affected soil should be removed for off-site incineration or heat treatment. If soil removal is not possible, then a 10 % formalin solution should be applied to the contaminated soil at a rate of 50 L per m³ [46]. No efficacy data were presented or referenced to justify these recommendations. Other governmental organizations and researchers also recommend or suggest the use of off-site incineration, or in-situ formalin or formaldehyde solutions, for the decontamination of soil contaminated with B. anthracis spores resulting from anthrax-infected livestock or wildlife [10,47–51]. Others have recommended the use of bleach for the surrounding soil and vegetation if burning is not feasible [4], although this is not recommended (bleach is discussed below). Germany requires field trials and temporary closure for decontamination of soil, but doesn’t specify what chemical to use for decontamination [52]. And while lime (calcium oxide) has been recommended to be added to infected carcasses for burial [48], others suggest the use of lime may actually enhance the environmental persistence of B. anthracis spores [53].

3.3. Soil fumigants

Soil fumigants used for agricultural purposes for disinfestation (control of soil pests such as insects, plant pathogens, and weeds) are potentially good candidates for in-situ sterilization of soils since they have biocidal properties, are widely used [54], and thus have chemical stocks, application equipment, and personnel/expertise readily available. These gaseous decontaminants include methyl bromide (MeBr), chloropicrin, dazomet, 1,3-dichloropropene (1,3-D), dimethyl sulfide, and metam sodium/potassium. These are further discussed below for the few fumigants that have been evaluated for their efficacy in inactivating bacterial spores in soil. For the soil fumigants not yet assessed for sterilization efficacy, this is an area of recommended research.

These soil fumigants are generally applied to the soil as liquids, but then readily volatilize to their gaseous or vaporous form. In particular, MeBr is a gas but compressed into a liquid state for storage and transport, while chloropicrin, dimethyl sulfide, and 1,3-D are considered volatile liquids. These liquids can be injected into, or applied to the surface of, soils using various techniques, including shank injection or drip irrigation (sometimes called chemigation). Dazomet is a solid and applied on the surface with a spreader, and tilled into the soil, which then volatilizes when in contact with soil moisture. After application of a fumigant, the soil is typically sealed using a tarp, mechanical compaction, or the addition of water or another layer of soil, to contain the fumigant to allow for sufficient contact time [55].

3.3.1. Methyl bromide

While the use of MeBr is being phased out under the Montreal Protocol on Substances That Deplete the Ozone Layer, it is still used in some countries under allowed critical use exemptions and for quarantine and pre-shipment uses [56], when no viable alternative is available. MeBr has been shown to be highly effective in inactivating spores of B. anthracis (Ames, NNR1Δ1, and Sterne strains) and Geobacillus stearothermophilus on a number of materials under several bench-scale [57] and full-scale experimental conditions [58]. For soil in particular, MeBr was effective in inactivating B. anthracis spores in small amounts of soil [59]. However, in tests using soil columns with 15 cm depths, MeBr was mostly ineffective [60], even at the surface and with more robust conditions.

In a field study conducted decades ago, Russian researchers demonstrated the use of MeBr for decontaminating soil contaminated with naturally occurring B. anthracis [61,62]. In those studies, tarps were used to contain the MeBr to provide needed contact time, and temperature and dosage requirements (e.g., 1.5 kg/m² for 20 days or 1.5 kg/m² for 7 days, down to 40 cm depth) were elucidated.

3.3.2. Metam sodium

Metam sodium is a widely used pre-emergent soil pesticide, which converts to the bio-active compound methylisothiocyanate when injected into soil [63]. Metam sodium was shown to be effective in inactivating spores of B. anthracis (Ames) and B. atrophaeus in bench-scale studies for both topsoil and a test dust in several test conditions [59].

3.3.3. Chloropicrin

In a decontamination study using small quantities of soil, chloropicrin was found to be ineffective (~1 LR) in the inactivation of B. anthracis as well as Bacillus subtilis spores [64].

3.3.4. 1,3-D

1,3-D is one of the more commonly used soil fumigants in the United States (one brand name being Telone^™) and serves as an alternative to the phased-out MeBr. 1,3-D was shown to have some effect on inactivating naturally occurring Pasteuria penetrans endospores in a study to evaluate 1,3-D’s impact as a nematicide [65]. It was also shown to have moderate effect on soil-borne pathogens (Phytophthora capsici and others) at the conditions tested [66]. Tests to evaluate its efficacy against Bacillus spores in soil are recommended.

3.4. Aldehydes

As previously mentioned, formaldehyde or formalin solutions are recommended by the WHO and other organizations to sterilize soil following natural anthrax outbreaks involving livestock or wildlife. Formaldehyde is a gas, but when dissolved in water, it is referred to as formalin. When fully saturated, formalin has a formaldehyde content of 37 % by weight. The WHO recommends a 10 % formalin solution be used for soil, i.e., a solution of 3.7 % formaldehyde [46]. Formaldehyde is considered carcinogenic by some governmental organizations, and its use may be limited to professionals [67].

Formalin solutions (5 % in seawater) were used to decontaminate tracts of soil on Gruinard Island, Scotland, which was heavily contaminated with B. anthracis spores during biological weapons testing conducted during World War II. Copious amounts of the formaldehyde solution were required (up to 50 L per square meter) and in some heavily contaminated areas required repeated applications. Although there was no quantification of decontamination efficacy, the remediation was declared successful due to negative soil core samples. Sheep were also introduced to the island as sentinel animals and allowed to graze for 5 months, and none of the sheep developed anthrax [68].

Bench-scale studies have quantified the efficacy of formaldehyde solutions in sterilizing soil. In a recent study [69], parametric tests showed liquid formaldehye solutions of 2.5–5% were highly effective in inactivating spores of B. anthracis and B. atrophaeus in soil under several test conditions. Formalin solutions could be applied to soil at full scale in a manner similar to what is used for soil fumigants, i.e., through chemigation or injection into soil. As with the soil fumigants discussed above, formalin is a volatile organic compound and would be expected to readily evaporate to a vapor. As with soil fumigants, containment of the formalin in the soil using a tarp or other means is recommended to ensure sufficient contact time. With this in mind, laboratory tests showed that capping soil samples after formalin was applied, to prevent its loss through evaporation, improved decontamination efficacy [69].

With respect to glutaraldehyde, researchers in Kazakhstan at sites where anthrax-infected cattle were buried reported that a sporicidal solution containing glutaraldehyde (among other active ingredients, such as dimethyl ammonium bromide) was effective in inactivating B. anthracis spores in the soil [70], although no field data were presented. The glutaraldehyde solution was also found to be effective at bench-scale under several conditions: the minimum concentration and contact time found to be effective in soil, based on whether growth of microorganisms occurred following application of the decontaminant, was a 5 % solution of their “Ba-12” disinfectant for 1 h. The authors lamented the limited options available to treat soil contaminated with B. anthracis spores, and so developed their own treatment chemical. In a later field study, this same Ba-12 disinfectant (but at a 20 % concentration) was effective against a vaccine strain of B. anthracis down to 3 m with a contact time of 24 h [6].

3.5. Oxidant-based decontaminants

The advantages of using liquid decontaminants for soil include ease of use and application, as well as the ability to penetrate the soil column. However, as the decontaminant percolates through the soil, this process may quench the active ingredient of the decontaminant prior to reaching the bioagent [29]. This is especially a concern for some of the sterilants that rely on oxidation as the spore inactivation mechanism, such as peracetic acid (PAA), chlorine bleach, and hypochlorous acid. These chemistries have been shown to be effective in sterilizing many types of materials [71,72], but will react with organic material, such as those found in soil, thus consuming the oxidant and rendering them less effective. This was demonstrated in a study in which peracetic acid, hypochlorous acid, and pH-adjusted bleach were evaluated for their ability to sterilize topsoil, and all achieved less than approximately 1 LR [73] against B. anthracis Ames spores. In a subsequent study [59], pH-adjusted bleach was found to be effective in inactivating Bacillus spores in a test dust (with only minimal amounts of organic carbon, <0.5 %), but again was found to be ineffective in topsoil with 9.3 % organic carbon.

Gaseous chlorine dioxide is another oxidative decontaminant known to be a highly effective sterilant gas [28,74] for many materials. In bench-scale column tests, it achieved ≥7 LR of B. anthracis spores in clay and sandy soils, to a 13 cm depth. But in experiments with topsoil, with a higher organic content (5.2 %, compared to 0.2 % organic matter for clay and 0 % for sand), it was only effective against B. anthracis to a 2.5 cm depth [75]. Again, the reduced efficacy in topsoil was most likely due to its higher organic content compared to the clay and sandy soils. Chlorine dioxide gas is used as a biocide (for the inactivation of sulfate reducing bacteria) in hydraulic fracturing for oil and gas production [76] and the methods used for that purpose (injection wells) could potentially be applied for the sterilization of soils.

Alternatively, chemicals referred to as in-situ chemical oxidants (ISCO) that utilize advanced oxidation processes, such as those based on persulfate [77,78] or permanganate [79] chemistry, are designed to withstand the oxidant demand of soil, and are becoming more widely used and studied as remediation tools for soil and groundwater contaminated with organic (e.g., hydrocarbons) pollutants. Persulfate compounds are activated with various catalysts such as ultraviolet light, iron, or hydrogen peroxide, to generate sulfate radicals, which are much more reactive than the parent compound [78]. As such, ISCO’s may be good candidates for sterilization of soil. Indeed, activated sodium persulfate was evaluated in bench-scale tests, which showed it to be effective against B. anthracis spores in topsoil when using several applications of the solution and a 7-day contact time [59]. In soil column tests, sodium persulfate activated with 8 % aqueous hydrogen peroxide prior to use was effective to a depth of 10—13 cm in sandy and clay soils, but was ineffective in topsoil beyond a 2.5 cm depth (similar to results with chlorine dioxide gas discussed above) [60]. The lower efficacy of the activated persulfate with topsoil in the column tests, compared to the bench scale tests, may have been due to a shortened contact time (1 week vs. 48 hr, respectively). Lastly, while potassium permanganate is used for water treatment and disinfection [80], no literature was found for studies that evaluated microbiocidal effects of permanganate compounds in soil. This is an area suggested for further research.

3.6. Heat treatment

Heat used for sterilization purposes may be classified as either dry heat, which uses hot air with a relative humidity less than 100 %, or wet heat, such as steam [81]. Steam used for sterilization of materials is typically applied via an autoclave and is pressurized. Several studies have shown it to be an effective technique to sterilize small quantities of soil, albeit in some cases three autoclave cycles were required [37,82–84].

Although the use of dry heat for sterilization and decontamination purposes has been well studied (see for example [85–88], its use to sterilize soil has not been given a lot of attention. Nevertheless, Wood et al. [14] conducted bench-scale oven and soil column experiments and demonstrated that dry heat is effective in inactivating B. atrophaeus spores in soil, but efficacy varied as a function of time, temperature, soil type, and depth. In small soil column tests using a heat plate set at 141 °C and applied to the surface, B. atrophaeus spores were inactivated at a depth of 5 cm with a 21-day contact time, in two of the three soil types evaluated. No spores were detected at the 2.5 cm depth for all soils at 4 d. Others have provided inactivation kinetics using dry heat for naturally occurring spores in soil [89].

While there is understanding of time and temperature requirements to effectively sterilize soil using steam or dry heat, there are challenges with applying heat at full scale to sterilize soil in-situ. Vidonesh et al. [19] provide a review of techniques to remediate soils contaminated with hydrocarbons using thermal treatment, and some of these techniques may be applicable for soil sterilization. Steam has been applied to soil surfaces using mobile prototype machines for disinfection or disinfestation purposes, and these may be applicable for sterilization; further discussion of applying to heat to soil at full-scale may be found in the Supplementary Material.

3.7. Germinants

One emerging decontamination method is the use of germinants, in which chemicals such as enzymes and nutrients are added to a bacterial spore population to accelerate germination of spores to form vegetative bacteria. As discussed, vegetative bacteria are typically more sensitive to biocides and environmental stressors than spores. In a bench-scale study evaluating the use of germinants in small amounts of unsterile soil [90], a 6 LR of B. anthracis was achieved when spores were successfully converted to the vegetative form, and then the vegetative form of B. anthracis was allowed to naturally attenuate. The tests were conducted over a two-week period at 22 °C and used l-alanine and inosine as the germinants. (This contrasts with the persistence of B. anthracis spores in soil, which showed minimal attenuation in soil after 56 days [91].) Thus, the addition of germinants to soil followed by natural attenuation of the resulting vegetative B. anthracis cells may be a viable soil remediation option without the use of toxic decontaminants [21]. This is an area suggested for further research.

In another study, a field trial was conducted to evaluate the use of germinants followed by the application of peracetic acid, to decontaminate soil contaminated with B. anthracis originating from livestock carcasses. It was found that the germinants did trigger some conversion of spores to the vegetative form, but there was no decrease in B. anthracis (or other naturally occurring soil bacteria) with the addition of peracetic acid [92]. This result exemplifies the difficulty of inactivating even less resistant vegetative bacteria when in a soil matrix, i.e., the soil was the controlling factor in inactivation efficacy and not the form of the bacterium. The authors of the study provided several recommendations to improve decontamination efficacy, such as allowing more time for the spores to germinate, increasing the PAA concentration and volume, and using l-alanine alone.

Lastly, in a novel laboratory test, germinants coupled with nematodes (Caenorhabditis elegans) were evaluated for their efficacy in reducing B. anthracis in soil (nematodes are a natural predator of the bacterium). The germinants alone achieved a 4 LR in spore loading, while the addition of nematodes provided an additional 1 LR [93].

4. Conclusions and recommended research

This is believed to be the first review of the literature for techniques that could be used to sterilize soil in-situ. Several incidents and scenarios were discussed in which large amounts of soil would need to be sterilized, with contamination occurring primarily at the surface. Some techniques commonly used to sterilize small amounts of soil for use in laboratory experiments, such as gamma irradiation and ethylene oxide, were briefly mentioned but cannot be used for in-situ soil remediation due to hazards and other constraints associated with large-scale or off-site use. Conducting soil decontamination in-situ would help to minimize the spread (via aerosolization) of the microbiological contaminant that would occur if the soil was disturbed due to removal and transport for offsite treatment. Techniques demonstrated to be effective in sterilizing soil would be expected to be effective in inactivating the less chemically resistant pathogenic microorganisms such as vegetative bacteria (e.g., Y. pestis and F. tularensis) and viruses.

A summary of the sterilization techniques discussed in this review, for those that have been tested against Bacillus spores in soil and/or are used commercially for other full-scale soil applications (such as soil fumigants), is presented in Table 1. Table 1 provides information related to the technique’s efficacy in inactivating Bacillus spores in soil and whether this has been demonstrated at bench- or full-scale. Methyl bromide, formaldehyde, and glutaraldehyde are the only remediation chemicals that have been demonstrated at full-scale, in-situ, to effectively inactivate Bacillus spores in soil.

Table 1.

Summary of potential full-scale, in-situ techniques to sterilize soil.

Technologya	Commercial full-scale use for soil	Effective in inactivating Bacillus spores in soil?b	Spore-forming test microorganism	Notes	References
Methyl bromide	Agriculture	Effective in bench-scale and full-scale studies	G. stearothermophilus, B. anthracis	Limited availability due to Montreal Protocol	[57,59,61,62]
Metam sodium	Agriculture	Effective in bench-scale study	B. anthracis, B. subtilis		[59]
1,3-D	Agriculture	Not tested against Bacillus spores		Recommend bench-scale efficacy study in soils
Chloropicrin	Agriculture	Ineffective in bench-scale studies	B. anthracis		[64]
Dazomet	Agriculture	Not tested against Bacillus spores		Recommend bench-scale efficacy study in soils
Dimethyl sulfide	Agriculture	Not tested against Bacillus spores		Recommend bench-scale efficacy study in soils
Formalin/formaldehyde	Soil decontamination following natural anthrax cases with livestock or wildlife carcasses	Effective in bench-scale tests and in actual (full scale) remediation for B. anthracis	B. anthracis, B. atrophaeus		[68,69]
Glutaraldehyde	None	Effective in bench-scale tests and in full-scale remediation for B. anthracis	B. anthracis		[70]
Activated sodium persulfate	Remediation of soils contaminated with hydrocarbons	Effective in bench-scale (including column) tests	B. anthracis	Effective to 10–13 cm depth in sandy and clay soils, but was ineffective in topsoil beyond 2.5 cm depth	[75]
ClO2 gas	Biocidal oilfield applications	Effective in bench-scale (including column) tests	B. anthracis	Effective to 10–13 cm depth in sandy and clay soils, but was ineffective in topsoil beyond 2.5 cm depth	[74,75]
Peracetic acid	None	Ineffective in bench-scale tests in topsoil	B. anthracis, B. subtilis		[71–73]
pH-adjusted bleach	None	Effective in bench-scale tests with an inorganic test dust; ineffective in topsoil	B. anthracis, B. subtilis		[59,71–73]
ClO2 aqueous	None	Ineffective in bench scale tests for both test dust and topsoil	B. anthracis, B. subtilis		[72,94]
Steam (wet heat)	Remediation of soils contaminated with hydrocarbons	Effective with small quantities in an autoclave run at several cycles	Naturally-occurring soil microbes	Several prototypes to apply steam to the soil surface at full-scale for disinfestation have been demonstrated	[37]
Dry heat	Remediation of soils contaminated with hydrocarbons	Effective in both bench-scale oven and column tests	B. atrophaeus		[14]
Germinants followed by biocides or attenuation	None	Effective in bench scale tests when allowing for attenuation. Ineffective in field trial	B. anthracis, B. thuringiensis		[90,92]

^aTechniques are listed if they have been tested against Bacillus spores in soil and/or are commercially available, widely used at full-scale for soil applications and with some reasonable expectation of biocidal properties.

^bListed as effective if achieved over 6 log₁₀ reduction of Bacillus spores in at least one test condition in soil, and/or reported that no spores were detected post-decontamination.

Soil is one of the most difficult materials to effectively sterilize and doing so at full-scale and in-situ brings additional challenges. Based on a few bench-scale column studies found in the literature, results showed that efficacy generally diminishes with soil depth. While this may not be an issue for contamination at the surface of soil and not long after an incident, spores are expected to migrate into the soil column over time with rainfall or stormwater infiltration (although this is recommended as an area of further research).

Bench-scale studies to assess inactivation efficacy of Bacillus spores with soil fumigants used for disinfestation, to take advantage of their wide availability, usage, and suspected antimicrobial activity (such as 1,3-D and dazomet), are recommended. For the sterilization techniques that have already shown high efficacy in bench-scale studies (e.g., steam, metam sodium, ClO₂ gas, activated sodium persulfate), full scale demonstrations are needed to assess efficacy as a function of depth and to elucidate potential operational issues associated with application and containment techniques.

Liquid sterilant chemicals utilizing simple oxidation as their spore inactivation modality, such as sodium hypochlorite (chlorine bleach), hydrogen peroxide, PAA, and aqueous chlorine dioxide, are not recommended for soil sterilization unless the soil is confirmed to contain no organic matter. Organic matter can quench the oxidant active ingredient and render such decontaminants ineffective. The exception to this is the advanced oxidation treatments that are used commercially to remediate soil contaminated with hydrocarbons, such as activated sodium persulfate. These advanced oxidants are sufficiently stable in the presence of natural soil organic matter to allow for effective microbial inactivation.