Effects of pesticides used in sugarcane cropping systems ... · 6.5. Soil fauna 14 7. Effects of pesticides on soil biological functions 15 7.1. Soil enzymes 15 7.2. Nitrogen fixation

Effects of pesticides used in sugarcane cropping systems on soil organisms and biological functions associated with soil health Clive Pankhurst 3 Redgum Place, Aberfoyle Park, Adelaide, SA 5159 Telephone: 08 8270 5313 Email: [email protected] A report prepared for the Sugar Yield Decline Joint Venture (April, 2006)

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Contents

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1. Summary 3 2. Introduction 5 3. Pesticide use in sugarcane cropping systems 5 4. Risk assessment requirements for registration of pesticides in Australia 6 5. Environmental properties of selected pesticides 7 6. Effect of pesticides on soil organisms 10 6.1. Soil microbial populations 10 6.2. Microbial biomass 12 6.3. Mycorrhizal fungi 12 6.4. Root pathogens 13 6.5. Soil fauna 14 7. Effects of pesticides on soil biological functions 15 7.1. Soil enzymes 15 7.2. Nitrogen fixation 16 7.3. N mineralization and denitrification 17 7.4. Organic matter decomposition 18 8. Summary of effects of the selected pesticides on soil organisms and soil biological functions

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9. Environmental impact of pesticides following adoption of controlled traffic / minimum tillage / crop rotation farming practices

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10. Conclusions 22 11. References 23 Table 1. Environmental properties of selected pesticides 36 Table 2. Examples of non-target effects of pesticides on soil microorganisms 37 Table 3. Examples of non-target effects of pesticides on soil microbial biomass 38 Table 4. Summary of known effects of selected pesticides on soil organisms and soil biological functions

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1. Summary This report is a review of the scientific literature concerning the impact of selected pesticides (insecticides, nematicides, fungicides and herbicides) used in sugarcane cropping systems, on soil organisms and soil biological functions associated with soil health. The report was commissioned by the Sugar Yield Decline Joint Venture (SYDJV) to address concerns raised by cane growers as to what impact pesticides they currently use have on soil biota and soil health. Nine pesticides used in sugarcane cropping systems were selected for detailed examination. They included 6 herbicides (2,4-D, atrazine, diuron, glyphosate, paraquat, and trifluralin), chlorpyrifos (insecticide), aldicarb (nematicide) and mancozeb (fungicide). These pesticides were found to vary widely in their environmental properties (half-life in soil, mobility and propensity to adsorb to soil particles) with diuron probably posing the greatest environmental risk in terms of its persistence in some soils (eg. Red Ferrosols) and leaching to groundwater. The effects of pesticides generally (with reference where available to the selected pesticides) on soil organisms and soil biological functioning is reviewed. This included separate consideration of the effects of pesticides on soil organisms (microbial populations, microbial biomass, mycorrhizal fungi, root pathogens and soil fauna) and soil biological functions (soil enzymes, nitrogen transformations (N fixation, N mineralization, denitrification) and organic matter decomposition). Generally speaking pesticides have variable effects on soil biology, with the majority of observations in the “no effect” category and with herbicides generally having less impact than insecticides, nematicides and fungicides. However, all of the selected pesticides (herbicides included) were found to have a negative effect on some component of the soil biology. These effects are generally transient, with the affected soil organism group or biological function recovering to pre-treatment levels usually in a matter of days or weeks.

There is evidence that the repeated application of some herbicides (eg. atrazine, 2,4-D, paraquat, trifluralin) over many years may compound a negative impact, change microbial community structure, or build-up biodegradation capacity. However, there has been no report where long-term use of these or other herbicides has resulted in a critical decline in soil organism populations or biological processes associated with soil fertility or crop productivity. A confounding issue with some reports of negative effects of herbicides on soil biology is that the impact may not be due to a direct effect of the chemical per se, but an indirect consequence of the removal of vegetation (killed weeds etc.) and therefore a reduction in organic matter inputs (eg. via root exudates or from above-ground biomass) into the soil. A rough ranking of the selected herbicides in terms of their negative impact on soil biota and soil biological functioning was paraquat > 2,4-D >atrazine > diuron > glyphosate > trifluralin. The long half life of paraquat in soil (~1000 days) may be a contributing factor to it having a greater effect on soil biology than the other herbicides. While all soil organism groups show sensitivity to some pesticides, fungi and actinomycetes appear to be somewhat more sensitive than bacteria with mycorrhizal fungi particularly sensitive to soil applied fungicides. Insecticides and nematicides generally have a greater impact on soil microarthropods (collembola, mites), soil microfauna (nematodes, protozoa) and earthworms than herbicides. It was noted that chlorpyrifos, used widely in the sugar industry as the slow release formulation suSCon® Blue does have a reported “small” negative effect on earthworm survival which may warrant further investigation. Of the soil biological functions examined, soil enzymes are generally not affected by pesticides applied to soil at recommended rates. C and N mineralization is relatively unaffected by pesticides although there is evidence that some herbicides may slow the decomposition of crop

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residues when they are left on the soil surface. In contrast, symbiotic nitrogen fixation appears to be sensitive to most pesticides (including insecticides and nematicides). With the increasing use of legumes as a break crop between sugarcane cropping cycles, the possibility of herbicide residues (contained on the sugarcane trash or on the soil surface) affecting legume root nodule development and nitrogen fixation may need to be investigated. Exacerbation of root disease by herbicide residues (reported in a number of cropping systems) is another potential problem that may need to be monitored if herbicides are used prior to the establishment of a legume break crop or the following sugarcane crop.

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2. Introduction During the latter half of the 20th century the development and use of pesticides to control weeds and animal pests and diseases has increased steadily and pesticide use has now become an integral component of agricultural farming systems in most developed countries. The principal forms of pesticides used in Australia can be categorized into herbicides, insecticides, nematicides and fungicides. Early pesticides were generally broad spectrum in their action and generally more toxic or “hard” compared to those that have been developed in the last 20 years which target pests more specifically, are more efficacious and generally less toxic or “soft” (AATSE report, 2002). Also associated with the early use of pesticides was little appreciation of their potential impact on non target biota or their fate in the environment. These perceptions have since changed and now all pesticides are subjected to a rigorous environment and health risk assessment before they are registered for use. In the last 20 years, Australian farmers have become increasingly concerned about potential impacts of pesticides on soil organisms and the functions they carry out. They have also become more aware of the importance of maintaining soil health as a prerequisite to sustaining crop productivity. Consequently, farmers are seeking more detailed information about the environmental impact of the pesticides they use, including their persistence in the soil, their propensity to move through the soil and into groundwater and their effect on soil organisms and biological processes associated with soil health. Whilst there has been a world-wide concerted research effort (particularly during the period from 1970-1990), into the environmental impact of many of the pesticides in current use, much of this information is contained in scientific publications or reports and is not readily accessible to farmers and land users. In addition there have been few studies carried out in Australia where the information is readily accessible, and there has been no in depth study of the effect of pesticides on soil health in sugarcane farming systems. In this report, which was commissioned by the Sugarcane Yield decline Joint Venture, the environmental impact of nine pesticides used in sugarcane cropping systems is reviewed, with particular attention given to their impact on soil organisms and soil biological functions associated with soil health. The selected pesticides include 6 herbicides (2,4-D, atrazine, diuron, glyphosate, paraquat, and trifluralin), chlorpyrifos (insecticide), aldicarb (nematicide) and mancozeb (fungicide). The first part of the review outlines current pesticide usage in sugarcane cropping systems, current risk assessment requirements for registration of pesticides in Australia and environmental properties of the nine selected pesticides. This is followed by a detailed examination of the effect of these pesticides on soil organisms and soil biological functions, and concludes with a discussion of the environmental impact of pesticides following adoption of controlled traffic / minimum tillage / crop rotation sugarcane farming practices. 3. Pesticide use in sugarcane cropping systems Pesticides (including insecticides, nematicides, fungicides and herbicides) are used to control a variety of pests, diseases and weeds in sugarcane cropping systems in Australia. According to the Cane growers Public Environment Report (2005), the combined costs of controlling major pests and diseases and the subsequent losses in sugarcane production in 1996, was $111M. Soil pathogens accounted for $83M, cane grubs $11M, ratoon stunting disease $6M and sugarcane rust $4M. In production terms, a total of about 3.5M tonnes of sugarcane was lost because of pests and disease. At this time the impact of weeds had not been adequately assessed, but

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subsequent field trials have indicated that failure to control weeds during the early stages of crop growth can reduce yields by 11-34% (Hanlon et al., 2000). Australian cane growers use a diversity of pesticides to grow the sugarcane crop. These include insecticides such as chlorpyrifos (suSCon® Blue) and imidacloprid (confidor) to control canegrubs, the nematicides aldicarb (Temik®) and fenamiphos (Nemacur®) to control nematode pests, and the fungicides shirtan and mancozeb to control a variety of fungal diseases. Herbicides used include pre-emergent and post-emergent residual herbicides to control weeds during sugarcane establishment, and knock down herbicides to control grass and vine weeds in ratoon crops. Among the most widely used herbicides are different registered formulations of 2,4-D, ametryn, atrazine, chlorsulfuron, diuron, flumetsulam, fluroxypya, glyphosate, imazapic, isoxaflutole, metolachlor, metsulfuron methyl, paraquat, pendamethalin, picloram, prometryn, simazine and trifluralin (Callow, 2005). The Australian sugar industry supports the responsible use of all pesticides. This is being achieved by encouraging cane growers to complete a 2 day accredited Chemcert course which covers all aspects of the use and disposal of pesticides. The industry also promotes an integrated approach to pest, disease and weed management by encouraging new initiatives in on-farm management (eg. harvesting and planting times) to combat pest and disease problems, in addition to the use of pesticides. As sugarcane has a long cropping cycle (4-5 years) compared to the brief cropping cycle of other broadacre crops (6-8 months), there are special problems associated with the application and residual action of pesticides in sugarcane cropping systems. There are also certain pests, including many ratoon pests, such as soldier fly, ground pearls and some canegrubs, whose biology ensures that they will always be difficult to control with pesticides (Samson et al., 1998). Integrated pest management strategies are evolving steadily with a new generation of biopesticides such as Biocane, a granular formulation of the fungus Metarhizium anisopliae, showing some success in the control of the greyback canegrub (Samson et al., 1999). Other new tools to help specific on-farm application of pesticides include the risk-based tool called SafeGauge developed by QDNRM (Simpson et al., 2003). This modeling tool takes soil type, weather information and herbicide type as inputs to provide recommendations for the best practice application of herbicides for specific farm blocks. This approach, together with improved farming practice, should assist in minimizing off-farm impacts. 4. Risk assessment requirements for registration of pesticides in Australia Before pesticide products can be used by farmers, they must undergo a rigorous approval process coordinated by the Australian Pesticides and Veterinary Medicines Authority (APVMA). The APVMA administers the National Registration Scheme for Agricultural and Veterinary Chemicals. The process required for registration involves detailed environmental and health based risk assessments leading to the conditions of use being clearly stated on the product label. A number of agencies are involved in assisting the APVMA in this assessment including the Department of the Environment and Heritage which advises on whether products may harm the environment, and how to avoid this. The APVMA also undertakes the review of specific pesticide products that have been on the market for many years to ensure that they meet the latest standards. Its national compliance program also ensures pesticide products continue to meet their registration conditions. Guidelines for the assessment of potential harm of agrochemicals to the environment are contained in the Manual of Requirements and Guidelines for Agricultural chemical products (AgMORAG) (see www.apvma.gov.au). This includes a section on environmental hazard to soil

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dwelling organisms. Little information is provided here with a statement that this is a “developing area, with test protocols now available from various jurisdictions”. Reference is made to a summary of environmental hazard assessment of chemicals in relation to soil fauna provided by Carruthers (1994). This paper discusses the difficulties environmental agencies worldwide have in the assessment of hazard to soil organisms, chiefly due to lack of data on both soil organisms and the fate of chemicals in the soil. This lack of knowledge reflects the low priority the soil habitat has received in the past, with more emphasis being placed on aquatic toxicology (Carruthers, 1994). Both the USEPA (1996) and OECD (2001) provide protocols for agrochemical hazard assessment of soil organisms and soil biological functions. The USEPA provides protocols for testing pesticides for toxic effects against earthworms, the soil microbial community (assessed via measurement of N mineralization (production of NH3 and NO3), soil respiration (CO2 evolution)), Rhizobium – legume nodulation (symbiotic nitrogen-fixation) and a soil microcosm test. The soil microcosm test involves the use of an intact soil core in which plants are grown. The test chemical (preferably radio-labeled) is added to the soil and its environmental fate assessed including its presence in leachate, soil and plant samples. The OECD provides protocols for earthworms, N mineralization, microbial biomass (assessed via substrate-induced respiration) and a plant growth test. The latter involves testing the effect of the chemical on germination and growth of a range of non-target plant species. As the active ingredients of most if not all pesticide products used in Australia are registered for use in Europe and the USA, it is assumed that they will have received some soil biology assessment according the protocols listed above, and that the results of these assessments have been accepted along with other data for registration of the pesticide in Australia. Clearly this remains an area “under development”. While a lot of progress has been made in the general understanding of the role of soil organism groups and biological functions in the maintenance of soil health, more research is required in this area before workable protocols for soil health assessment will be universally accepted. At present there is general agreement that assessment of microbial biomass, earthworm survival, symbiotic nitrogen fixation, and C and N mineralization are suitable parameters (USEPA, 1996; OECD, 2001). These parameters have been listed by others as appropriate biological indicators of soil health (Doran et al., 1994; Gregorich et al., 1997; Pankhurst et al., 1997; Locke and Zablotowicz, 2004). 5. Environmental properties of selected pesticides Environmental properties of 9 selected pesticides used in sugarcane cropping systems (2,4-D, atrazine, diuron, glyphosate, paraquat and trifluralin (herbicides), chlorpyrifos (insecticide), aldicarb (nematicide) and mancozeb (fungicide)) are given in Table 1. The properties include the solubility of the pesticide in water, the half life of the pesticide in soil and the propensity of the pesticide to adsorb to soil particles (the soil adsorption coefficient, SAC). The solubility of a pesticide in water will influence its toxicity and mobility in soil. The half-life (t1/2) of a pesticide in soil is the time taken for half of the initial amount of the pesticide to break down. If the half-life is less than 30 days, the pesticide is considered to have low persistence; if 30-100 days it is moderately persistent; if greater than 100 days, it is highly persistent (AATSE report, 2002). The SAC value of a pesticide will be influenced by its molecular structure and soil properties such as pH, organic matter content and type, particle size distribution and clay mineral composition (Kamrin, 1997). A high SAC value indicates a high propensity for the pesticide to adsorb to soil particles, while a lower value indicates less soil adsorption and a greater propensity for the pesticide to remain in the soil/water solution.

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The impact of a particular pesticide on soil organisms and soil biological functioning will be determined by a number of interacting factors. These include the physicochemical properties of the pesticide (its mode of action and toxicity towards soil organisms) and its behavior in the soil environment, including its bioavailability, susceptibility to abiotic degradation, mobility and propensity to be adsorbed by soil particles. As shown in Table 1 the environmental properties of the selected pesticides vary widely. Consequently, one can expect their impact on soil organisms and soil biological functioning to also vary widely. The following is a brief resumé of the environmental properties of each of the selected pesticides. Atrazine is a selective pre- and post-emergent herbicide providing knockdown and residual action. It has a low rate of volatilization from soil and is moderately persistent (half-life of ~60 days). It is more persistent in neutral to alkaline soils than in soils with low pH (Qiao et al., 1996). It is moderately mobile and able to be leached through soils into groundwater (Kruger et al., 1996). Diuron is a broad spectrum residual herbicide used for pre-emergent control of weeds in agriculture and horticulture. It is non volatile from soil and is more persistent than atrazine (half-life of ~90 days). Organic matter has been shown to significantly influence the adsorption of diuron by soils (Mallawatantri and Mulla 1992). However, whilst considered to be relatively immobile in soil, diuron and its main degradation product (3,4-dichloroaniline) have been detected in groundwater in numerous studies (Giacomazzi and Cochet, 2004). 2,4-D, its salts and esters are broad spectrum systemic herbicides widely used for weed control in agriculture and non-crop situations. 2,4-D has a very short half-life in soils (~10 days) and is readily degraded by soil microorganisms (Smith et al., 1994). It is only weakly adsorbed by soil particles (very low SAC value), a factor related to its short half-life. Glyphosate is a broad spectrum systemic herbicide and is the most widely used herbicide in Australian agriculture (AATSE report, 2002). It is non volatile, is strongly adsorbed by soil particles (high SAC value) and is essentially immobile in soil. Free glyphosate in the soil environment may be rapidly degraded by microorganisms (Nomura and Hilton, 1977). However, it becomes strongly bound to soil particles (analogous to the binding of phosphate) and forms insoluble complexes with iron (III), copper (II), calcium and magnesium ions at or near neutral pH (Subramaniam and Hoggard, 1988). Paraquat is a broad spectrum herbicide that destroys plant tissue by contact action. It is highly soluble in water and because of its ionic properties is strongly adsorbed by soil particles (especially clay minerals) and is essentially immobile in soil (Costenla et al., 1990). The strong binding of paraquat to clay minerals is the factor most likely associated with its long half-life in soils. Trifluralin is a pre-emergence herbicide. It is susceptible to photodecomposition, is reasonably volatile and substantial vapor losses can occur as the soil dries out (Leitis and Crosby, 1974). It can become strongly bound to soil particles and as a consequence has low mobility. Chlorpyrifos is a broad spectrum organo-phosphate insecticide used by the sugar industry as the controlled release formulation suSCon® Blue for the control of canegrubs and other insect pests (Samson et al., 1998). Its persistence in soils is wide ranging depending on soil type and environmental variables with half-lives ranging from 1.9 days to 1575 days recorded (Racke, 1993). Soil pH above 6.2 accelerates the degradation of chlorpyrifos shortening the effective life

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of the product. It is essentially immobile in soil, although its metabolites have some mobility (Racke, 1993; Baskaran et al., 2003). Aldicarb is a soil applied systemic nematicide used to control parasitic nematodes in sugarcane (Stirling et al., 1996) and other insect pests in fruit and vegetable crops. It may be volatilized from soil during soil drying. Aldicarb is readily metabolized in soil to either aldicarb sulfoxide or aldicarb sulfone, with these metabolites generally having a longer half-life than aldicarb itself. Aldicarb and its metabolites are moderately persistent and mobile and able to reach groundwater (Miles and Delfino, 1985). Mancozeb is a dithiocarbamate fungicide used to control many fungal diseases in field crops. It is one of several fungicides used to control orange rust in sugarcane (Magarey et al., 2002) and is also useful for control of fungal root diseases in sugarcane (Magarey and Bull, 2003). It can undergo rapid chemical hydrolysis in soil producing ethylenethiourea and ethyleneurea which are quite mobile and able to reach groundwater (Calumpang et al., 1993). It has moderate persistence at low temperatures and under alkaline conditions. The persistence and fate of several of these pesticides in Australian sugarcane soils has been studied. Hargreaves et al. (1999) examined the soils from three sugarcane properties in the Bundaberg region that had been treated with chlorpyrifos, atrazine, diuron, paraquat and trifluralin (all applied at the recommended rate) for a number of years and found no evidence of a build-up of residues of any of these pesticides in the soils tested. Residues of the 4 herbicides were reduced to less than 10% of their initial concentration within 2-4 months of application with some potential risk for off-site movement during this period. Chlorpyrifos which was applied as controlled release granules (suSCon® Blue) below the soil surface at planting, was present in low or non-detectable concentrations in the surface soil layer (0-25 mm) within days of incorporation into the soil (Hargreaves et al., 1999). In a follow-up study Simpson et al. (2001) reported that field dissipation rates (half-lives) for atrazine, ametryn, diuron and 2,4-D in different soils in the Bundaberg region were quite rapid (1-30 days) even for compounds like atrazine, ametryn and diuron which are normally considered to be moderately persistent. On one soil however, a Red Ferrosol, diuron was highly persistent after an initial phase of rapid decline. Concerns about the environmental impact of pesticides used by the Australian sugar industry have prompted a number of investigations of pesticide residues detectable in groundwater. In a recent report, Beattie et al. (2004) monitored water in 6 drains in 3 mill areas in northern NSW for residues of atrazine, diuron, 2,4-D, glyphosate and chlorpyrifos. During a 15 month monitoring period they failed to detect any residues of glyphosate and chlorpyrifos. Residues of atrazine, diuron and 2,4-D were mostly low or non-detectable and at no time exceeded Australian Drinking Water Guidelines (1996) or recreational use guidelines (ANZECC, 2000). The residue concentration of the pesticides studied was also unrelated to the concentration of nitrate, chloride or phosphate in the drains (Beattie et al., 2004). In another study, Klok and Ham (2004) could not detect quantifiable levels of atrazine, ametryn, diuron, 2,4-D or chlorpyrifos in irrigation water (pumped from groundwater sources) at 6 sites in the Burdekin delta region. However, soil water samples taken over two irrigation seasons at a depth of 1.5 m did show the presence of atrazine (2 of 67 samples) and diuron (14 of 67 samples) at levels above the environmental trigger values provided in the ANZECC (2000) guidelines. This indicates the potential for some pesticides (notably diuron) to move through the soil profile and reach underground aquifers (Klok and Ham, 2004). Studies of sediments from Queensland irrigation channels, drains and streams, has also revealed measureable quantities of diuron, atrazine, ametryn and 2,4-D (Müller et al., 2000; Mitchell et al.,

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2005), indicating the potential for residues of these herbicides to move “off-site” with possible implications for contamination of the great barrier reef. Reported die-back of coastal mangroves in north Queensland has been attributed to high levels of diuron in coastal sediments (Duke et al., 2003). The environmental impact of diuron and its principal degradation product (3,4-dichloroaniline) has been recently reviewed (Giacomazzi and Cochet, 2004) with concerns expressed about its persistence particularly in groundwater. The APVMA is currently reviewing approvals for the use of diuron and diuron containing products in Australia. Its preliminary findings are that environmental exposure from the use of diuron at recommended rates in sugarcane, cotton, citrus and horticultural crops, and general purpose non crop uses (eg. control of weeds in irrigation channels and drain ditches) is likely to have an unacceptable environmental impact. It has indicated that if risk mitigation strategies cannot be found to substantially reduce this environmental impact, approval for the use of diuron and diuron containing products may be withdrawn (APVMA, 2005). Conditions for the continued use of the herbicide atrazine have also been recently reviewed (NRAAVC, 2002). In this case, approval has been granted for the continued use of atrazine containing products in agricultural situations subject to compliance to a range of new regulatory requirements. Atrazine has recently been banned from use in Belgium and France, mainly in an effort to stop the excessive use of atrazine for total weed control (NRAAVC, 2002). 6. Effect of pesticides on soil organisms 6.1 Soil microbial populations Given the large number and diversity of soil microorganisms (bacteria, fungi, actinomycetes, microalgae) that are present in agricultural soils, it is inevitable that any chemical or substrate applied to the soil will perturb the functional dynamics of some component of this microbial community. Generally speaking pesticides when applied at recommended rates cause only slight and usually only transient changes to populations or activities of soil microorganisms (Atlas et al., 1978; Moorman, 1989; Roger et al., 1994; Topp et al., 1997; Richardson, 1998; Pareek and Ladha, 1999; Locke and Zablotowicz, 2004). Additionally, as most modern pesticides have been developed to target specific pests and even individual enzymes of a specific pest (so called soft pesticides (AATSE report, 2002)), there is even less chance that they will cause major long-term damage to total soil microbial populations. Examples of the effect of several pesticides on soil microorganisms in studies ranging from in vitro culture studies to field assessments are summarized in Table 2. These studies illustrate the varied nature of the effect of pesticides on soil microbial populations. The effect is dependent on many factors including the mechanism of action of the pesticide, the bioavailability and physicochemical behavior of the pesticide in the soil (eg. how readily it is absorbed by soil particles), environmental factors (eg. rainfall), and soil management (eg. tillage). Herbicides are less likely to impact adversely on soil microbial populations than fungicides or insecticides. This is particularly the case for herbicides which act primarily at sites unique to the chloroplast. Such herbicides (eg atrazine and diuron) are inhibitory to microorganisms only at concentrations far above those that are toxic to plants (Moorman, 1989). However, some herbicides such as glyphosate and the sulphonylurea group of herbicides that act by inhibiting the synthesis of certain amino acids required for the growth of plants are likely to affect the growth of some microorganisms (eg. certain pseudomonads) because these microorganisms contain a

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similar biosynthetic pathway (Moorman, 1989; Boldt and Jacobsen, 1998). Similarly, the dinitroaniline herbicides (such as trifluralin) disrupt cell-division in plants by inhibiting microtubule formation and consequently may have activity against fungi (Moorman, 1989). Herbicides may also affect soil microbial populations indirectly through changes in the carbon substrate inputs into the soil following the death of targeted plants. For example, Whitelaw-Weckert et al. (2004) found that applications of paraquat and glyphosate to control grass and subterranean clover growth in the inter-rows of vineyards over a 2.5 year period significantly reduced populations of cellulolytic bacteria, Pseudomonas bacteria and fungi compared to untreated plots. However, because the herbicides effectively decreased the vegetation in the inter-rows, it was not possible to determine if the changes in the microbial population were due to a direct effect of the herbicides alone, or in combination with the indirect effect of the removal of the inter-row plant growth and therefore reduction in rhizosphere exudates and other organic material entering the soil. Fungicides which by definition are designed with fungi as targets will inevitably have some impact on non-target microorganisms. For example application of the fungicide mancozeb to sugarcane soils to control fungal root pathogens significantly reduced total fungi, actinomycetes and Pseduomonas bacteria but increased total bacterial populations (Magarey and Bull, 2003). The stimulation of bacteria probably resulted from reduced competition for nutrients following the reduction in the fungal and actinomycete populations. In contrast nematicides and insecticides are unlikely to have a direct impact on soil microbial populations, but their activity may temporarily perturb the functioning of the soil food web resulting in short term fluctuations in microbial populations (Pandey and Singh, 2004). The repeated application of pesticides over many years can have varied effects on soil microbial populations. Generally speaking there are no long-term detrimental impacts (Morman, 1989; Topp et al., 1997), although there have been some reports of persistent changes to the composition of the microbial community. For example cellulolytic bacteria were permanently reduced in an apple orchard kept free from direct vegetative cover by the long-term application of atrazine (Voets et al., 1974), although this is may have been due the reduction in organic matter inputs. Repeated annual applications of paraquat for 18 years was found to have increased populations of aerobic bacteria, cellulolytic bacteria and actinomycetes but decreased fungal populations (Duah-Yentumi and Johnson, 1986) while 15 years of annual applications of 2,4-D was reported to have reduced bacterial, actinomycete and fungal populations (Rai, 1992). In other instances repeated applications of certain pesticides have been shown to promote microbial populations capable of selectively degrading that pesticide (Racke and Coats, 1990). For example microbial degradation was attributed with the rapid loss of chlorpyrifos from sugarcane soils where control of greyback canegrub had failed in parts of the Burdekin canegrowing region (Robertson et al., 1998). Similarly, accelerated microbial degradation was identified as the cause of reduced effectiveness of aldicarb control of nematodes in potato fields following annual applications of the nematicide (Smelt et al., 1987). Sandman and Loos (1984) also reported high populations of 2,4-D degrading microorganisms in the rhizosphere of sugarcane and suggested that this was associated with the rapid degradation of the herbicide in the soil. The potential for pesticides to alter the composition of the soil microbial community without necessarily affecting soil biological functioning has prompted recent calls for (1) inclusion of methods for rapid diagnosis of bacterial diversity in pesticide approval protocols, and (2) consideration of the ecological implications of altered bacterial diversity for soil fertility (Johnsen et al., 2001). For example, El Fantroussi et al. (1999) used Biolog GN fingerprinting and denaturing gradient gel electrophoresis (DGGE) of ribosomal DNA to show significant effects of

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long-term application (10 years) of three phenylurea herbicides (including diuron) on soil bacterial diversity. Detectable changes included the loss of two Pseudomonas phylotypes from the microbial community in the herbicide treated soil. In another study, Seghers et al. (2003) determined the effect of 20 years of atrazine application on different components of the bacterial community using selected PCR primers and DGGE analysis. In this study differences in the methanotrophic community between the atrazine treated and un-treated soil were found with 3 phylotypes being absent in the herbicide treated soil. However, the differences in the methanotrophic community structure were not reflected in methane oxidation capacity, which was similar in the two treatments. 6.2 Microbial biomass Measurement of soil microbial biomass is considered to be a useful tool for assessment of the impact of agricultural practices on soil health (Doran et al., 1994; Gregorich et al., 1997; Pankhurst et al., 1997). Microbial biomass measurements overcome some of the limitations of assessing effects on soil microbial populations, which tend to be compromised by problems associated with whether or not organisms are cultivable or present in the soil as dormant propagules such as spores. Microbial biomass is a standardized component of ecotoxicity assessment in the OECD guidelines for pesticide registration (OECD, 2001). Reported effects of pesticides on soil microbial biomass are varied (Table 3). Hart and Brookes (1996) showed that 19 years of annual field applications of 4 pesticides (glyphosate, benomyl, chlorfenvinphos and triadimefon) applied at the recommended rates had no effect on microbial biomass whereas annual applications of aldicarb caused a significant (16%) increase in microbial biomass. In other field studies, Busse et al. (2001) found no long-term effect of glyphosate on microbial biomass in three California pine plantations while Haney et al. (2002a) reported that glyphosate increased microbial biomass carbon (17%) and microbial biomass nitrogen (76%) in nine agricultural soils 14 days after treatment. Similarly Biederbeck et al. (1987) found microbial biomass levels in several soils after 35 years of annual 2,4-D treatment were no different from levels in untreated soil. In contrast, Rai (1992) reported that 15 years of annual application of 2,4-D decreased microbial biomass by 15-20%, while long-term applications of paraquat (Duah-Yentumi and Johnson, 1986), trifluralin (Dumontet and Perucci (1992) and atrazine (Megharaj, 2002) were all reported to cause a decline in microbial biomass. However, as discussed earlier with regard to microbial populations, it is difficult to be certain that reported long-term negative effects of herbicides on soil microbial biomass are direct effects and not in fact partly due to the stimulatory effects of vegetation on microbial biomass in the non-treated soils. Studies of the effect of herbicides on microbial biomass in dryland and irrigated cropping systems in Australia generally support effects reported in overseas studies (Gupta, V.V.S.R. unpublished). For example, chlorsulfuron, diclofop-methyl, glyphosate, fluometuron and metsulfuron methyl were reported to have no effect on microbial biomass whereas paraquat, pendimethalin and prometryn had a negative impact (Gupta, unpublished). 6.3 Mycorrhizal fungi Not unsurprisingly, some fungicides (including both systemic and non-systemic fungicides) are reported to have an adverse effect on the colonization of the roots of crop plants by arbuscular mycorrhizal (AM) fungi (Trappe et al., 1984; Moorman, 1989; Perrin and Plenchette, 1993; Schweiger and Jakobsen, 1998; Smith et al., 1999). Phosphorus uptake by the hyphae of AM fungi may also be reduced. The nonsystemic fungicides PCNB and thiram were found to be inhibitory to AM fungi while captan had no effect in several studies (Trappe et al., 1984).

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Systemic fungicides including benomyl, carboxin and triademifon reduced AM colonization with some suggestions that the benzimidiazole fungicides (which include benomyl) are selective towards AM fungi (Trappe et al., 1984; Perrin and Plenchette, 1993; Smith et al., 2000). The fungicide carbendazim was shown to inhibit phosphorus uptake by AM fungal hyphae at concentrations lower than recommended field application rates, whereas propiconazole and fenpropimorph, and the insecticide dimethoate had no effect (Schweiger and Jakobsen, 1998). The fungicide mancozeb and the nematicide aldicarb when applied to soil on their own did not affect the colonization of sugarcane roots by AM fungi, but when applied together significantly reduced colonization (Pankhurst et al., 2005). The reason for this synergetic effect is unknown. Herbicides generally have less potential for direct effects on AM fungi but can affect root colonization via herbicidal reduction of photosynthesis and carbon supply to the roots (Trappe et al., 1984). Thus while diuron and trifluralin had no effect on citrus root colonization by Glomus etunicatum, high rates of simazine and paraquat damaged the plants and reduced colonization (Nemec and Tucker, 1983). More recent studies have focused on the potential for herbicides to alter source-sink relationships between herbicide tolerant and susceptible plants growing together, with nutrient transfer from the shoots to the roots of susceptible plants and possible export to the roots of the tolerant plant species mediated via AM fungi common to both (Bethlenfalvy et al., 1996; Rejon et al., 1997). Pesticides have also been reported to stimulate colonization of plant roots by AM fungi. This may be due to a reduction or elimination of competing microorganisms or to a pesticide mediated change in plant metabolism resulting in increased production of materials stimulatory to AM fungi in the rhizosphere. The fungicide fosetyl-Al for example, was shown to stimulate root exudation of soluble sugars in both mycorrhizal and non-mycorrhizal plants and resulted in increased AM colonization of the roots of fungicide treated plants relative to untreated plants (Jabaji-Hare and Kendrick, 1985). The rate of pesticide application and the host plant response to the pesticide probably account for the varied responses observed with AM fungi. Nevertheless the studies do show that pesticides, particularly fungicides, can affect AM fungi sufficiently to affect plant growth. Plant species with a high dependence on AM fungi and/or low soil phosphorus availability are two conditions that could maximize the potential for adverse pesticide effects on AM fungi and plant growth (Moorman, 1989). 6.4 Root pathogens There is some documented evidence that pesticides (in particular herbicides) may exacerbate root disease of crop plants (Neate, 1994). This may occur via a number of possible mechanisms including (1) the weakening of non-target crop plants by the herbicide making it more susceptible to root pathogens, (2) a direct effect of the herbicide on the root pathogen itself or other soil organisms that may normally be suppressive towards the pathogen, and (3) an increase in root pathogen inoculum on killed weed biomass prior to planting the crop. Mekwatanakarn and Sivasithamparam (1987) found that application of diquat + paraquat, glyphosate and trifluralin to field soil increased the incidence of take-all of wheat caused by Gaeumannomyces graminis, and suggested that the mechanism was associated with a herbicide-induced decrease in the number of soil fungi antagonistic towards to take-all pathogen. Similarly, the sulfonylurea herbicides (chlorsulfuron, metsulfuron methyl and triasulfuron) were found to increase root disease of wheat caused by take-all, Rhizoctonia solani (bare-patch), and Heterodera avenae (cereal cyst nematode), particularly in calcareous soils (Neate, 1994). In this

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case, weakening of the host plant by residues of the herbicides was proposed as a possible mechanism. In other studies, trifluralin was reported to increase the incidence and severity of root rot of Medicago truncatula in disease infested soil (Bretag and Kollmorgen, 1986), and diuron, metribuzin and fluazifop were reported to increase the incidence of blackspot of peas (Davidson and Ramsey, 2000). The herbicides pendimethalin, acifluorfen and imazethapyr were also reported to increase in the severity of Rhizoctonia root and hypocotyl rot of soybean in greenhouse experiments (Bradley et al., 2002). In contrast, Ruppel et al. (1988) could find no evidence of increased root disease in a barley-maize-pintobean-sugarbeet cropping sequence following minimum, moderate and intensive use of the herbicides cyanazine, dicamba, pendimethalin, trifluralin and 2,4-D amine. However, in an earlier study, Ruppel and Hecker (1982) reported that the nematicides aldicarb and carbofuran increased the severity of Rhizoctonia root rot of sugar beet, although more recently, Baird et al (2004) found that aldicarb had no effect on pathogenic or saprophytic fungi associated with the roots of cotton. Glyphosate has been found to increase root disease of wheat (caused by Pythium spp.) in a minimum tillage situation when it was used to kill weeds close to the date of sowing. The increased root disease was attributed to the pathogen increasing its inoculum potential on the weed residues prior to sowing (Pittaway, 1995). This probably occurs because of the availability of a nutrient source and a temporary reduction in populations of competing microorganisms. A similar problem, also with wheat, was demonstrated with the pathogen Rhizoctonia and the herbicide mixture paraquat +diquat (Roget et al., 1987). In this case, the problem was overcome by allowing a greater time between applications of the herbicide and sowing (Roget, et al., 1987), presumably to allow competition by soil microorganisms. 6.5 Soil fauna Soil fauna (eg. earthworms, microarthropods, nematodes, protozoans) are important in organic matter transformations and soil structure formation, and appear to be potentially useful bioindicators of the effects of pesticides in soil (Locke and Zablotowicz, 2004). Pesticide toxicity towards earthworms is included in the list of standardized ecotoxicity tests for pesticide registration by both the USEPA (1996) and OECD (2001). Field application rates of common fungicides, insecticides and nematicides can have deleterious effects on earthworms (Parmelee et al., 1990; Potter et al., 1990; Edwards and Bohlen, 1992; Fraser, 1994; Choo and Baker, 1998; Vermeulen et al., 2001). For example, Potter et al (1990) found that a single application of the fungicide benomyl or the insecticides ethoprop, carbaryl, or bendiocarb at labeled rates reduced earthworm populations in turfgrass by 60-90%, with significant effects lasting for 20 weeks. Other insecticides including diazinon and chlorpyrifos caused less severe, but significant earthworm mortality in some tests (Potter et al., 1990). Similarly, Choo and Baker (1998) reported that the nematicide fenamiphos and the insecticide endosulfan reduced the growth and inhibited the reproduction of the earthworm Aporrectodea trapezoides (Lumbricidae) when applied at recommended rates to an Australian pasture soil. The nematicide carbofuran is also well documented as having an adverse effect on earthworm growth and activity (Parmelee et al., 1990; Reddy 1999). In contrast, there is no evidence for any deleterious effect of herbicides (including chlorsulfuron, diuron, 2,4-D, glyphosate and trifluralin) on earthworms when applied in the field at recommended rates (Potter et al., 1990; Fraser, 1994; Dalby et al., 1995; Bauer and Römbke, 1997; Mele and Carter, 1999). Other soil fauna including microarthropods (collembola, mites) and microfauna (nematodes and protozoa) may be affected by soil-applied pesticides with possible flow-on consequences for organic matter mineralization. The nematicide carbofuran for example has been shown in many

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studies to have a negative impact on soil microarthropods particularly collembola and predatory mites (Prostigmata and Mesostigmata) with populations taking up to a year to recover (Reddy, 1999). A similar effect has been noted with the insecticide dimethoate (Martikainen et al., 1998). Long-term use of the fungicide mancozeb in vineyards has also been reported to reduce populations of the predatory mite Typhlodromus pyri (Auger et al., 2004). Herbicides however, have generally been found to have negligible effects on soil microarthropod populations (Edwards, 1989; Martikainen et al., 1998; Cortet et al., 2002), although some early reports have shown that atrazine, simazine, glyphosate and paraquat can cause temporary reductions in microarthropod activity (Popovici et al., 1977; Hendrix and Parmelee, 1985; Edwards, 1989). More recently, Rebecchi et al. (2000) reported that the sulphonylurea herbicide triasulfuron caused a decrease in some collembolan species in an agricultural soil. Effects of insecticides and fungicides on protozoan and nematode populations are also more pronounced than those of herbicides (Edwards, 1989; Gupta and Yeates, 1997). For example, Petz and Foissner (1989) found that mancozeb altered the community structure but not the absolute number of protozoans in a field soil while Ekelund (1999) demonstrated that various groups of protozoa (notably flagellates) were reduced by field application rates of the fungicide fenpropimorph. Similar effects on nematodes were reported by Smith et al. (2000) who showed that long-term application of benomyl in a tall grass prairie had no effect on herbivorus nematodes but reduced populations of certain groups of fungal feeding and predatory nematodes. In another study Wardle et al. (1995) have suggested that evidence for direct negative effects of herbicides on nematode populations were more likely to be indirect effects arising from changes in the quantity and quality of plant inputs (eg. dead organic matter from weeds) to the soil. 7. Effect of pesticides on soil biological functions 7.1 Soil enzymes Soil enzyme activities are useful integrative indicators of soil health and have been used widely to assess the effects of management practices on soil biological functioning (Dick, 1997). Generally speaking, pesticide applications at recommended rates have little or no effect on enzyme activity in soils (Ladd, 1985; Schäffer, 1993; Nannipieri, 1994; Dick, 1997). The status of an enzyme in soil may determine how pesticides affect its activity. Enzymes in soil are either intracellular and present as a component of viable soil organisms (biontic), or extracellular and bound to clay or humic acids (abiontic) (Dick, 1997). The oxidoreductase, dehydrogenase, which has an apparent role in the oxidation of organic matter and can only function within viable organisms, has been widely studied in soils. Short-term studies involving applications of pesticides (including the herbicides glyphosate and 2,4-D) to soils at recommended rates for periods ranging from a few days to 8 weeks have shown slight increases or no effect on dehydrogenase activity (Dick, 1997). Similarly, there appeared to be no short-term effect of pesticide application on phosphatase and urease activity. As both of these enzymes are known to exist in soil as abointic enzymes, an effect would not be expected unless the pesticide had a direct effect on the enzyme reaction (Dick, 1997). Megharaj et al (1999) found no effect of fenamiphos (nematicide) on dehydrogenase, phosphatase and β-glucosidase, whilst Pozo et al (1995) reported that chlorpyrifos caused a temporary reduction (14 days) in dehydrogenase and phosphatase activity. In contrast, when pesticides are applied to soil at higher than recommended rates or over long periods, significant effects on soil enzyme activity have been reported. For example, Voets et al (1974) showed that long-term atrazine applications significantly reduced the activity of

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phosphatase, invertase, β-glucosidase, and urease in soils. However, this was thought to be due to a reduction of biological activity rather than a direct effect on the catabolic behavior of these enzymes. Other similar reports include a decrease in dehydrogenase and urease activity following long-term (15 years) application of 2,4-D (isoctyl ester formulation) (Rai, 1992), a decrease in dehydrogenase and arylsulfatase in South Australian soils following long-term applications of atrazine (Megharaj, 2002), and a decrease in phosphatase activity following long-term applications of glyphosate (Sannino and Gianfreda, 2001). 7.2 Nitrogen fixation Nitrogen fixation by free-living, associative and symbiotic bacteria is a major input of usable nitrogen in most agroecosystems. As a measure of its significance, the USEPA guidelines for pesticide registration provide detailed protocols to assess pesticide effects on symbiotic nitrogen fixation (USEPA, 1996). The effects of pesticides on non-symbiotic nitrogen fixing microorganisms are varied. For example, Martinez-Toledo et al. (1992) reported that chlorpyrifos significantly decreased aerobic nitrogen-fixing bacteria and nitrogen fixation in an agricultural soil, while Gomez et al. (1999) reported that the insecticides diazinon had no effect but profenophos reduced the growth and nitrogen-fixing activity of Azospirillium brasilense on synthetic media. In another study the fungicide mancozeb was found to inhibit nitrogen-fixing bacteria in a natural soil for up to 3 months (Doneche et al., 1993) while captan was found to reduce populations of aerobic nitrogen-fixing bacteria in four soils over a 30-day incubation period with inhibition being highly dose-dependent (Martinez-Toledo et al., 1998). In contrast the herbicides atrazine, simazine and pendimethalin had no effect on the growth of Azotobacter spp. and stimulated nitrogen-fixation (Martinez-Toledo et al., 1991; Jayant et al., 1991). Symbiotic nitrogen fixation by legume root nodules can provide 30-70% of the nitrogen requirements of a legume crop. A range of pesticides are used to grow a legume crop including fungicides applied to the seed at planting. Seed application of fungicides may directly affect colonization of the rhizosphere by the Rhizobium bacteria, or indirectly affect the performance of the either the plant or nitrogen-fixation by the root nodules (Moorman, 1989; Mårtensson, 1992). For example seed application of the fungicides carboxin, captan, pentachloronitribenzene and thiram reduced the survival of Bradyrhizobium japonicum and Rhizobium phaseoli (Curly and Burton, 1975; Graham et al. 1980), while the fungicides benomyl, fenpropopimorph and mancozeb reduced root hair infection and nodule development of red clover by Rhizobium leguminosarum bv. trifolii (Mårtensson, 1992). In contrast, Castro et al. (1997) reported that while mancozeb decreased the growth of Rhizobium sp. in pure culture by 50% it had no effect on root nodulation or peanut seed yields under field conditions. Selection of fungicides with minimal effects on Rhizobium, the use of granular inoculants, or the use of fungicide-resistant Rhizobium strains (Odeyemi and Alexander, 1977) are strategies that can be used to minimize potential adverse effects of fungicides on nodulation. Surprisingly, insecticides and nematicides have been reported to have an adverse effect on symbiotic nitrogen fixation. For example, Smith et al. (1978) reported that while aldicarb, carbofuran, carbaryl, chlorpyrifos and terbufos did not affect the in vitro growth of Rhizobium they reduced nodulation and nitrogen fixation (C2H2-reduction) of alfalfa, sweet clover and red clover to varying degrees. Similarly, Sekar and Balasubramanian (1979) found that aldicarb reduced nodulation and the total nitrogen content of cowpea plants inoculated with Rhizobium sp.

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A large range of herbicides (including bentazone, chlorsulfuron, diuron, glyphosate, MCPA, metribuzin, paraquat and trifluralin), have been found to reduce legume nodulation in laboratory and / or field experiments (Goring and Laskowski, 1982; Edwards, 1989; Clark and Mahanty, 1991; Sandhu et al., 1991; Flores and Barbachano, 1992; Mårtensson, 1992; Yueh and Hansley, 1993; Koopman et al., 1995). Collectively, the evidence suggests that herbicide-induced reductions in nodulation are due to effects upon the plant (eg. reduced photosynthate to the roots, disruption of root hair infection by Rhizobium) and are often accompanied by root stunting or other damage caused by the herbicide. Effects may also be modified by changes in the amount of inorganic nitrogen available to the plant and by the soil moisture content (Mårtensson, 1992). It is clear therefore that care is required when herbicides are used for weed control in legume crops. In more recent examples Gupta et al. (2000) reported that herbicides such as prometryn, cyanazine, diuron + prometryn and imazethapyr + simazine significantly reduced nitrogen fixation by fababeans used as a rotation crop in an Australian cotton cropping system, while King et al. (2001) reported that glyphosate reduced nodulation and nitrogen-fixation of glyphosate-resistant soybeans, with the effects greatest under moisture stress conditions. 7.3 N mineralization and denitrification Standardized testing of pesticides for their effect on nitrogen mineralization (production of NH4

+ and NO3

- from soil organic matter) is required for pesticide registration by both the USEPA (1996) and OECD (2001), emphasizing the importance placed on these processes for the maintenance of soil health. Goring and Laskowski (1982) extensively reviewed the effects of pesticides on N transformations in soil, and determined that most pesticides have no effect or cause less than 25% inhibition of mineralization and nitrification. They found that most pesticides inhibited these processes only at rates above those recommended for field use. Effects are largely mediated via effects on fungal and bacterial populations with nitrification being the more vulnerable process because of the small number of microbial species involved. While general soil fumigants such as methyl bromide have substantial effects on N mineralization, field and laboratory studies with systemic insecticides or nematicides such as oxamyl and fenamiphos had no effect on N mineralization (Ross and Speir, 1985). Similarly, Hart and Brookes (1996) reported that while 19 years of cumulative annual applications of aldicarb resulted in significantly less NH4-N in aldicarb-treated soil than in untreated soil, this was due to immobilization of N by the increased microbial biomass in the aldicarb-treated soil and did not represent a loss of N from the soil system. Some fungicides (including mancozeb) may inhibit N mineralization if applied repeatedly over long periods or if applied above recommended rates (Doneche et al., 1983; Kinney et al., 2005). For example, Kinney et al. (2005) showed that mancozeb and chlorothalonil inhibited nitrification by 5-20% when applied at slightly above the recommended field rate to soil cores from a corn field but by 60-90% when applied at 10 times the recommended rate. In another laboratory study, Chen et al. (2001) showed that captan applied at approximately recommended field rates increased soil NH4-N whereas benomyl and chlorothalonil had little impact. Hart and Brookes (1996) also found that 19 years of annual applications of benomyl had no effect on N mineralization. The available evidence indicates that there are no long-term negative effects of herbicides on the mineralization of N from either soil organic matter or nitrogen fertilizer. Long-term use of herbicides such as paraquat (Smith and Mayfield, 1977), atrazine (Cerevilli et al., 1982), 2,4-D (Biederbeck et al., 1987), glyphosate (Stratton 1990; Olson and Lindwall, 1991; Hart and Brookes, 1996) and triadimefon (Hart and Brookes, 1996) are all reported to have no effect on N

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mineralization. In Australia, Gupta et al. (2000) tested a range of pre- and post-emergence herbicides used in cotton production (including diuron, fluometuron, prometryn and metolachlor) and found that they all caused a temporary reduction (lasting up to 8 weeks) in rates of N mineralization and nitrification. Yeomans and Bremner (1985a, b) tested 33 different pesticides for their effect on denitrification in 3 different soils. The pesticides included the insecticides lindane and malathion, the nematicide carbofuran, the fungicides mancozeb, benomyl, thiram and captan, and the herbicides diuron, trifluralin, 2,4-D, alachlor, atrazine, cyanazine, metribuzin, simazine, dicamba and dinoseb. They were applied at the rate of 10 or 50 µg g-1 soil. None of the pesticides studied significantly affected denitrification when applied at 10 µg g-1 soil. At the higher rate (50 µg g-1 soil) most of the insecticides and fungicides enhanced denitrification whereas the herbicides either enhanced (eg. metribuzin, dinoseb), had no effect (eg. 2,4-D, dicamba) or inhibited denitrification in at least one of the soils (eg. trifluralin, cyanazine). 7.4 Organic matter decomposition The decomposition of crop residues and organic matter is an essential part of the nutrient cycling process in soils. Pesticides come into contact with crop residues during or after incorporation of the residues into the soil, or in the case of herbicides they may become associated with the targeted-plant tissues after application. In the latter situation high concentrations of the herbicide may remain on the residues and potentially impact on their decomposition. The herbicides paraquat and glyphosate, for example have been shown to slow the decomposition of various plant materials (Moorman, 1989). Decomposition was also shown to be slowed more if the crop residues sprayed with these herbicides were left on the soil surface (Hendrix and Parmalee, 1985; House et al., 1987), despite the fact that both of these herbicides are highly absorbed to soil particles and generally have no adverse effect on soil microbial populations. Using litter bag experiments Gupta et al. (2000) reported that application of the herbicides pendimethalin, prometryn and pyrithiobac caused a significant reduction in the rate of cotton stubble decomposition compared to untreated cotton stubble. However, the effects of more widely used herbicides including 2,4-D (Fletcher and Freedman, 1986), trifluralin (Lewis et al., 1978; Boyette et al., 1988) and atrazine (Cortet et al., 2002), on plant residue decomposition appear to be minimal, although in these studies decomposition was measured in herbicide-treated soil. Other measurements of carbon mineralization including the rate of endogenous respiration (CO2 evolution) in pesticide-treated soil are required for registration of pesticides by the USEPA (1996) and OECD (2001). In an early study, Lewis et al. (1978) tested 25 herbicides and herbicide combinations in two soils at rates comparable to those used in agriculture, and reported that none of them affected soil respiration. Similarly Hart and Brookes (1996) reported that 19 years of annual application of 5 pesticides (benomyl, chlofenvinphos, aldicarb, triadimefon and glyphosate), had no effect on soil respiration, and Martikainen et al (1998) found no effect of dimethoate and benomyl on soil respiration in a soil microcosm study. Busse et al (2001) reported that glyphosate did not affect soil respiration when added to soil at recommended field rates, but found that addition of glyphosate at concentrations up to 100-fold greater stimulated respiration, presumably due to utilization of glyphosate as a carbon source. Ghani et al (1996) also found a significant enhancement of CO2 respiration following the application of atrazine to a pasture soil, while Haney et al. (2002b) showed that atrazine and atrazine + glyphosate stimulated both C and N mineralization. In contrast, Gupta and Neate (1998) and Gupta et al. (2000) have shown that a number of pesticides used in Australian agriculture (including endosulfan, paraquat, glyphosate and diuron) have a temporary negative impact on soil respiration which may persist for 6-8 weeks

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after application. In addition, when crop residues were present on the soil surface, herbicide application increased the microbial quotient (qCO2, microbial activity per unit of microbial biomass), indicating a herbicide induced stress on the microbial community (Gupta and Neate, 1998). 8. Summary of effects of the selected pesticides on soil organisms and soil biological functions A summary of the known effects of the selected pesticides on soil organisms and soil biological functions is given in Table 4. There are two main points to note about this table. Firstly, there are many gaps. This could be due to failure to source the relevant information or due to the fact that a comprehensive study of each of the pesticides with respect to all the soil biology measurements included in this review has not been achieved. Secondly, there are many instances where the reported effect of an individual pesticide on a soil organism group or a biological function includes both positive and negative effects. This observation is not surprising given the expected variation in experimental methodology used by researchers. Such variation includes the type of study (laboratory versus field experiments), the duration of experiments, soil type, environmental variables (moisture, pH, temperature etc.) and measurement techniques. Of the observations recorded in Table 4, approximately 70% are either in the “no effect” or “positive” category. Given the fact that most (if not all) of the negative observations are only temporary effects, the body of evidence supports the conclusion made by other reviewers that when pesticides are used at recommended field rates and applied “when” and “how” recommended, there is little or no long-term effect on soil biological functioning or non-targeted soil biota (Atlas et al., 1978; Moorman, 1989; Roger et al., 1994; Hart and Brookes, 1996; Topp et al., 1997; Richardson, 1998; Pareek and Ladha, 1999; Locke and Zablotowicz, 2004). An exception to this (not discussed in this report) is the long-term use of copper-based fungicides to control fungal diseases in pome and stone fruit orchards, vineyards and vegetable crops. In this case Cu residues can build up in the soil to levels that have a long-term significant impact on soil biota and biological functioning (Merrington et al., 2002; Van-Zwieten et al., 2004). Heavy metal contamination of soils (eg. through atmospheric deposition, accidental spills or application of sewage sludge) generally has an adverse effect on soil biology (Pankhurst et al., 1998).

All of the selected herbicides have a negative impact on some soil organism group and / or biological function (Table 4), but again this is generally only minor (and probably no more severe than negative impacts caused by natural environmental stresses) and the affected soil organism group or biological function recovers usually within a few days or weeks. Repeated applications of some herbicides (eg. atrazine, 2,4-D, paraquat, trifluralin) over many years may compound the negative impact, change microbial community structure, or build-up biodegradation capacity. However, in no case has there been any report that long-term use of these or other herbicides has resulted in a critical decline in soil organism populations or biological processes contributing to soil fertility or crop productivity (Moorman, 1989; Roger et al., 1994; Pareek and Ladha, 1999; Locke and Zablotowicz, 2004). In addition, it needs to be stressed that a confounding issue with some reports of negative effects of herbicides on soil biology is the fact that the impact may be due more to the removal of vegetation (killed weeds etc.) and therefore a reduction in organic matter inputs (eg. via root exudates or from above-ground biomass) into the soil than to a direct effect of the chemical. Further inspection of Table 4, reveals that a rough ranking of the selected herbicides in terms of their negative impact on soil biota and soil biological functioning parameters is paraquat > 2,4-D >atrazine > diuron > glyphosate > trifluralin. The long half life of paraquat in soil (~1000 days, Table 1) may be a contributing factor to its tendency to have a longer-term effect on soil biology than the other herbicides.

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While all soil organism groups show sensitivity to some pesticides, fungi and actinomycetes appear to be somewhat more sensitive than bacteria with mycorrhizal fungi particularly sensitive to soil applied fungicides. Insecticides and nematicides generally have a greater impact on soil microarthropods (collembola, mites), soil microfauna (nematodes, protozoa) and earthworms than herbicides. It is worth noting that chlorpyrifos, used widely in the sugar industry as the slow release formulation suSCon® Blue does have a reported “small” negative effect on earthworm survival which may warrant further investigation. Of the soil biological functions examined, soil enzymes appear to be generally unaffected by pesticides applied to soil at recommended rates. Similarly, C and N mineralization appears to be relatively unaffected by pesticides although there is evidence that some herbicides may slow the decomposition of crop residues if they are left on the soil surface. In contrast, symbiotic nitrogen fixation appears to be sensitive to most pesticides (including insecticides and nematicides). Some possible implications of this observation together with the potential for some herbicides to exacerbate root disease will be discussed below. 9. Environmental impact of pesticides following adoption of controlled traffic / minimum tillage / crop rotation farming practices With the gradual adoption of controlled traffic / minimum tillage / crop rotation farming practices by the sugar industry, a brief discussion of how these farming practices, in the particular the adoption of minimum tillage, may influence the environmental impact of pesticides (especially herbicides) is presented. In a recent review of the environmental consequences of the adoption of conservation tillage practices in Europe, Holland (2004) suggests a number of ways the adoption of such practices will influence the environmental impact of pesticides. Firstly, higher infiltration rates and the presence of crop residues associated with minimum tillage will ensure that runoff and sediment loss is reduced and thereby lower the risk that pesticides will be transported directly into surface waters, as may occur with conventional tillage. Secondly, the presence of increased amounts of soil organic matter and associated microbial activity on the soil surface under minimum tillage will facilitate adsorption and breakdown of pesticides. Thirdly, minimum tillage will influence the levels of crop pests, diseases and weeds and thereby the need for pesticides. The introduction of minimum tillage practices is a major component of the new cropping system being developed for the sugar industry by the SYDJV (Bell et al., 2003; Garside et al., 2004). An increase in the use of herbicides will initially be required in the new system, firstly to remove the old sugarcane stool after the final harvest and prior to direct-drill planting the legume break crop, and then in some instances, to remove the break crop prior to direct-drilling the new sugarcane crop (Garside et al., 2004). On the first occasion sugarcane trash will be cover much of the soil surface so that initially very little of the herbicide may in fact come into contact with the soil. Adsorption of herbicide residues onto the trash may slow its decomposition (as discussed above) although this has not been assessed experimentally, and increased residence time of the trash on the soil surface may or may not be a problem. Eventually, herbicide residues retained by the trash will either degrade in situ or be eluted following rainfall events to the soil surface. When herbicide residues reach the soil surface the improved soil structure associated with green cane trash blanketing (GCTB) (Braunack and Ainslie, 2001) including improved rainfall infiltration (Bell et al., 2001), may increase the risk of the residues leaching through the soil profile. This possibility is associated with the increased occurrence of macropores created by decayed plant roots or by soil fauna (eg. earthworms) under minimum tillage practices (Flury, 1996). However, there is some evidence that macropores created by earthworms could actually prevent this from occurring because they are lined with organic matter that retain agrochemicals, while also supporting a diverse and abundant microflora with potential to degrade them (Stehouwer et al.,

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1994). In a recent study Farenhorst et al. (2000) used soil columns with established earthworm burrows to show that while atrazine contained in crop residues on the soil surface was preferentially transported through the burrows following rainfall simulation, the total amount of atrazine leached through the soil column during the experiment was significantly less in the presence than in the absence of earthworms. They concluded that earthworm feeding activity reduced the potential for herbicide leaching by ingesting and transporting herbicide residues away from the soil surface and increasing the amount of non-extractable (non-leachable) herbicide residues in the soil. In addition to the potential benefits associated with increased earthworm activity, one would expect that the increase in soil organic matter and associated microbial activity in the upper soil layers (0-2.5 cm) under GCTB (Sutton et al., 1996; Bell et al., 2001) and under minimum tillage practices generally (Holland, 2004; Dick and Gregorich, 2004), will facilitate the adsorption and breakdown of herbicide residues and reduce the amount potentially moving further into the soil. This has been demonstrated in other cropping systems with a number of different herbicides (Holland, 2004; Locke and Zablotowicz, 2004). Potentially therefore, the environmental impact of herbicide use in the new minimum tillage cropping system could be lower than in the in traditional plough-out / re-plant cropping system. However, this would need to be evaluated at the farm and then at the catchment scale. One concern could be the possibility of run-off occurring via the compacted controlled-traffic zones between the permanent beds in the new system. Another important component of the new cropping system is the use of a legume break crop between successive sugarcane cropping cycles (Garside and Bell, 2001). There are many advantages associated with this practice including reduction in detrimental disease-causing soil organisms associated with yield decline (Pankhurst et al., 2005) and improvements in the nitrogen and organic matter status of the soil (Moody et al., 1999; Noble and Garside, 2000). One potential concern, however, is the possibility that herbicide residues associated with cane trash and the old sugarcane stool at the time of planting the legume crop may affect the establishment and early growth of the legume. As discussed earlier, one soil biological function that is particularly sensitive to pesticide residues is symbiotic nitrogen fixation (Table 4). This apparent sensitivity can result in poor nodule development and nitrogen fixation and hence affect plant growth. While the mechanisms associated with this effect are unclear it seems that perturbations of plant growth mediated either directly (as in the case of a herbicide) or indirectly via some effect on the soil microflora including the Rhizobium bacteria (as in the case of a fungicide, nematicide or insecticide) is involved. As there have been some reports of poor establishment of legume break crops following sugarcane (Garside, personal communication) it may be worth investigating those cases where herbicides have been used to remove the old sugarcane stool prior to planting the legume, to see if herbicide residues are involved in any way in the poor legume establishment. In addition to potential adverse effects of pesticide residues on root nodule development and nitrogen fixation, the reported increased susceptibility of newly planted crops to root disease following the use of herbicides to remove weeds or a previous crop could also be a problem for legume establishment following herbicide removal of the sugarcane stool in the new cropping system. This would most likely involve root pathogens such as Pythium spp. and Rhizoctonia spp. which are know to attack young seedlings. As discussed earlier a number of mechanisms could be involved including an increase in the inoculum levels of the pathogen on the residue of the herbicide-killed sugarcane plants plus a general weakening of the developing seedlings by the herbicide residues. In a similar fashion, herbicide residues could increase the possibility of root disease of the new sugarcane crop if herbicides are employed to remove the legume break crop. 10. Conclusions

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The main conclusions to be drawn from this review that are relevant to the sugar industry are as follows:

1. All pesticides registered for used in Australian agriculture are subject to a rigorous approval process coordinated by the Australian Pesticides and Veterinary Medicines Authority. Currently the Australian guidelines for risk assessment of pesticides does not include tests for effects on soil biota. However, as most (if not all) of the active ingredients of pesticides in common use in Australia are also used in Europe and the USA, it is assumed that they will have been subjected to the soil biota tests included in guidelines for pesticide registration by both the OECD and USEPA.

2. Most (if not all) pesticides have a minor but temporary negative impact on some component of the soil biology.

3. If pesticides are applied at recommended rates and according to label instructions, there should be little or no long-term effect on soil biological functioning or non-targeted soil biota.

4. Repeated application of some pesticides (eg. 2,4-D, atrazine, paraquat, trifluralin) over many years may compound a negative effect on soil biology but there is no evidence that this has any long-term effect on soil fertility or crop production.

5. Diuron is currently under review by the APVMA and may not receive on-going approval because of concerns over off-site impacts.

6. Paraquat because of its long half-life in soils tends to have a longer-term impact on soil biology than other herbicides.

7. Soil fungi and actinomycetes appear to be more sensitive to herbicides than bacteria, with mycorrhizal fungi particularly sensitive to soil applied fungicides.

8. Insecticides and nematicides have a greater impact on soil fauna including microarthropods (collembolan and mites), microfauna (nematodes and protozoa) and macrofauna (earthworms) than herbicides.

9. Chlorpyrifos used in the sugar industry as the slow release formulation suSCon Blue does have a reported negative impact on non-symbiotic nitrogen fixation and a “small” negative effect on earthworms which may warrant further investigation.

10. The mineralization of C and N in soils appears to be relatively unaffected by pesticides.

11. Root nodule development and nitrogen fixation by legumes appears to be sensitive to pesticides. With the increasing use of legume break crops in sugarcane cropping systems a possible effect of pesticide residues on nodulation and nitrogen fixation of the legume crop should be monitored.

12. There is potential for root disease problems to be exacerbated by the use of herbicides. This may need to be monitored during the establishment of both sugarcane and legume break crops if herbicides are used prior to planting.

13. Improved soil structure and organic matter levels and increased soil faunal activity resulting from adoption of minimum tillage practices, may have a beneficial impact on the persistence and rate of movement of pesticide residues through the soil profile. While this requires validation, it could be another potential benefit of the adoption of controlled traffic / minimum tillage / crop rotation farming practices by the sugar industry.

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Table 1. Environmental properties of selected pesticides* Common name Trade name

(examples) Solubility in water (mg/L, 25oC)

Half-life in soil (t1/2) days

Log Soil Adsorption Coefficient

Mobility in soil

Atrazine Atradex 30 60 2 moderate Diuron Diurex 42 90 2.7 low 2,4-D Amicide 620 10 1.3 moderate Glyphosate Roundup 12,000 47 4.4 zero Paraquat Gramoxone 700,000 1000 6 zero Trifluralin Treflan 0.3 60 3.9 low Chlorpyrifos Suscon Blue 1.4 30 3.8 zero Aldicarb Temik 6000 10-70 1.5 moderate Mancozeb Dithane 6-20 50 >3.3 low

(metabolites – moderate)

* Data obtained from Kookana et al. (1998), Simpson et al. (2001), AATSE report (2002) and SafeGauge User Manual (2004).

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Table 2. Examples of non-target effects of pesticides on soil microorganisms Pesticide Organism Effects Reference

2,4-D-iso-octyl ester (H) Culturable soil bacteria,

fungi and actinomycetes Reduced soil populations

Rai (1992)

Aldicarb (N) Culturable soil bacteria and fungi, mycorrhizal infection of plant roots

No effect Pankhurst et al. (2005)

Atrazine (H) Culturable soil bacteria and fungi Cellulolytic bacteria and fungi Methane oxidizing bacteria

No effect Reduction Changed community structure

Voets et al. (1974) Voets et al. (1974) Seghers et al. (2003)

Benomyl (F) Root colonization by mycorrhizal fungi

Reduction Smith et al. (1999)

Captan (F) Culturable soil bacteria, fungi and actinomycetes

Reduced soil populations

Martinez-Toledo et al. (1998)

Chlorpyrifos (I) Culturable soil bacteria and fungi Nitrogen-fixing bacteria

Bacteria reduced, fungi increased Reduction

Pandey and Singh (2004) Martinez-Toledo et al. (1992)

Diazinon (I) Azospirillum brasilense No effect on growth or N-fixation

Gomez et al. (1999)

Diuron (H) Culturable soil bacteria, fungi and actinomycetes Cellulolytic bacteria and fungi

Bacteria increased, others decreased Short term decrease

Bhutani et al. (1984) Gupta et al. (2000)

Fenamiphos (N) Algae and cyanobacteria No effect Megharaj et al. (1999) Fenpropimorph (F) Culturable soil bacteria

and actinomycetes, length of fungal hyphae

Short term decrease in fungi and bacteria, no effect on actinomycetes

Thirup et al. (2001)

Glyphosate (H) Culturable soil bacteria, fungi and actinomycetes Bacterial community structure

Decrease in bacteria, increase in fungi and actinomycetes No effect

Araujo et al. (2003) Busse et al. (2001)

Mancozeb (F) Culturable soil bacteria, fungi and actinomycetes Nitrogen-fixing bacteria

Bacteria increased, others decreased Reduction

Magarey and Bull (2003) Doneche et al. (1993)

Metsulphuron methyl (H)

Pseudomonas sp. Growth inhibition Boldt and Jacobsen (1998)

Simazine (H) Azotobacter chroococcum

No effect on growth, high concentrations increased N-fixation

Martinez-Toledo et al. (1991)

Trifluralin (H) Culturable soil bacteria, fungi, actinomycetes, cellulolytic bacteria, fluorescent Pseudomonas spp.

No effect Moorman and Dowler (1991)

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Table 3. Examples of non-target effects of pesticides on soil microbial biomass Pesticide When measurement

made

Effect on microbial biomass

Reference

2,4-D (H) 3 consecutive applications 25 years (annual application) 15 years (annual application)

No effect No effect Decrease

Olson and Lindwall (1991) Biederbeck et al. (1987) Rai (1992)

Aldicarb (N) 19 years (annual application)

Increase Hart and Brookes (1996)

Atrazine (H) Up to 81 days after single application 3 years after single application

No effect Decrease

Ghani et al. (1996) Megharaj (2002)

Benomyl (F) 5 years (annual application)

Increase Smith et al. (1999)

Chlorpyrifos (I) No effect Adesodun et al. (2005) Diuron (H) 8-10 days and 8-10

weeks after single application

No effect Gupta et al. (2000)

Fenamiphos (N) No effect Ross and Speir (1985) Glyphosate (H) 45 days after treatment

14 days after treatment

No effect Increase

Wardle and Parkinson (1991) Haney et al. (2002a)

Benomyl (F), Glyphosate (H),

19 years (annual application)

No effect Hart and Brookes (1996)

Paraquat (H) Simazine (H) Carbofuran (N)

19 years (annual application) 5 successive monthly applications

Decrease (loss of fungal biomass) No effect No effect

Duah-Yentumi and Johnson (1986)

Trifluralin (H) 1-5 weeks after single application

Decrease Dumontet and Perucci (1992)

39

Table 4. Summary of known effects of selected pesticides on soil organisms and soil biological functions Biological measurement

Atrazine Diuron 2,4-D Glyphosate Paraquat Trifluralin Chlorpyrifos Aldicarb Mancozeb

Soil biota Bacteria 0/+ + – – –/+ –/0 –/+ 0 –/+ Fungi 0/+ – – –/+ – 0 0/+ – Actinomycetes – – – –/+ –/+ 0 – Celluloytic bacteria –/0 – – –/+ 0 Cellulolytic fungi –/0 – – Mycorrhizal fungi 0 – 0 0 0 Root pathogens + 0 + + 0/+ 0/+ – Microbial biomass –/0 0 –/0 0/+ – – 0 + Microfauna – – – – – 0 Microarthropods – – – – Earthworms –/0 0 0 0 –/0 0 – 0 Nitrogen fixation Non-symbiotic N-fixation 0 – – Symbiotic N-fixation – – – – – – –/0 Soil enzymes Dehydrogenase – –/0/+ 0/+ 0 – β-glucosidase – Phosphatase –/0 – – Urease –/+ – + + N transformations Ammonification 0 –/0 0/+ – – Nitrification 0 –/+ –/0 0/+ 0 0 0 0 –/0 Denitrification 0 0/+ 0 –/0/+ 0 0 0 C mineralization CO2 respiration + – 0 0 – –/0 0 0 OM decomposition 0 0 –/0 0 0 – decrease (usually temporary), 0 no effect, + increase (usually temporary).