Abstract Quality control can de divided in production control, process control, and product control. The first two refer to internal quality control of the production of a microbial pest control product, and ensure a stable production process with a minimum of failures. Product control refers to the quality of the final product that leaves the factory and which needs to perform according to registration and customer satisfaction requirements. Products must meet product specifications, which are set by the manufacturer, until the end of the claimed shelf-life. Registration requirements in terms of quality control are reviewed. Complete quality control procedures and data for validation must be established, although there are no officially recognized criteria. Practical challenges in quality control procedures are reviewed per type of pathogen. Natural variation makes efficacy testing via bio-assays difficult, and setting an internal standard is required. Recommendations for standardization and criteria will be provided. Research needs are identified that may facilitate quality control in the future. Quality control must ensure that end-users receive high quality products. Total quality control covers all aspects of quality, including the field use of a biopesticides. Biocontrol companies should ensure that the whole chain is well aware of quality issues and that those involved act accordingly. A list of benefits of quality control is provided which illustrates that both the biocontrol industry and its customers benefit from proper quality control.
Biopesticides are often criticized for their variable performance and lack of reliability. Quality control (QC) is therefore of paramount importance in order to ensure that products are delivered that comply with pre-determined specifications and deliver the efficacy within the prescribed conditions for use. Quality control does not only refer to the final end-use product, but also to the production and the production processes. In general, quality control objectives are to ensure that:
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(1) properties of incoming raw materials comply with the manufacturer's specifications;
(2) there is consistency between production runs and products;
(3) end-use products meet criteria set by registration authorities; and that
(4) product performance meets the end-user's perception of quality in relation to price, and leads to repeat purchases of the product.
Quality control is performed in all manufacturing fields, but interpreted differently. Thus, each industry must define quality control precisely. In biocontrol, quality control has received much attention in the field of sterile insect technology and in arthropod natural enemies. For an overview of quality control matters regarding mass-reared sterile insects I refer the reader to Boller and Chambers (1977), and for arthropod natural enemies to Van Lenteren (2003a). A brief historical overview on the development of quality control for beneficial insects is provided by Leppla (2008). In quality control of natural enemies, Leppla (2003) distinguished three functions: production, process and product control. In the field of microbial pesticides, however, clear definitions are lacking. Few authors extensively treat quality control in its entirety. Most papers dealing with quality control focus their comments to end-use product control, while aspects of process control are frequently included. Definitions of quality control used for microbials will preferably be similar to the ones used for natural enemies and other beneficial biocontrol agents, for the sake of convenience. I will use the separation of functions as given by Leppla (2003) and use the following slightly modified definitions:
(1) production control is a procedure intended to ensure that incoming raw materials comply with defined specifications, and that production equipment be properly maintained, and that all inputs needed to run the production (materials, labour, energy, etc.) be available at the right time;
(2) process control is a procedure intended to ensure that a manufacturing process runs according to previously determined process parameter profiles so that expected yields and formulated products be delivered;
(3) product quality control is a procedure intended to ensure that a manufactured end-use product adheres to the product specifications, a previously defined set of quality standards, in order to meet the efficacy norms within described conditions for use, and to meet criteria set by registration authorities.
To determine the process parameters and the specifications of incoming raw production materials and formulation ingredients, and the outgoing end-use products, research is needed to establish the appropriate standards and to develop methods to measure these standards. A company can decide itself on specific standards, although for the final product some of these are requirements of registration authorities. As a result, standard operating procedures (SOPs) should be developed. This research is imperative and is a considerable amount of work. It should be conducted within the developmental phase of the production and of the final product.
The results are the basis for a consistent manufacturing process once products are routinely made and quality control is implemented.
The term quality assurance is often used in this context; it covers all activities from designing and developing quality control and the implementation of it during all phases, including reacting to deviations and improving processes and procedures to ensure good quality of the end-use product. It can be described as the continuing process of ensuring good quality. International standards have been developed for manufacturing activities and models are described and certified under ISO 9000 series. Implementation of such a quality management system can ensure consistent production and high quality products. Furthermore, a producer may choose to comply with Good Manufacturing Practices (GMP), although this has been developed for the pharmaceutical industry for which it is more appropriate. ISO and GMP are, however, rarely implemented in the biocontrol industry. The biopesticide companies Andermatt Biocontrol, Futureco and Prophyta are ISO 9001-2000 certified, Valent BioSciences is ISO 9000-2008 certified. The Bio-Fly plant, a subsidiary of Bio-Bee, Israel, operates in compliance with the ISO 9001-2000 quality standards in the production of sterile flies of the Mediterranean fruit fly. Bio-Fly is the only company in the field of mass production of arthropods which works with this certified quality system. Generally, companies find these systems too complicated and too expensive.
The production of MPCAs is a complex system that involves living organisms. This calls for the monitoring of crucial aspects in the whole line of processes, from production to the use of the end-use products. Quality control and feedback from deviations should be an integral part of the development and the use of biocon-trol products within all biocontrol companies. This overall process is called Total Quality Control (TQC). An elaborate view on TQC for natural enemies is given by Leppla (2003); the relevant aspects of TQC apply to microbial control agents as well. Quality control systems for MPCPs are much more comprehensive and specialized than those used for chemical pesticides, where relatively simple analytical techniques can be used. MPCPs contain living organisms, which are inherently variable in performance, and as a result the products will never be as stable as technical products. This variability demands a rigorous quality control system in order to ensure that biological products are delivered within specifications. Manufacturers are required to develop their own systems and regulatory procedures are not harmonized, nor are any standards available. As a result, quality control in biopesticides is neither transparent nor standardized. I will discuss the critical aspects of quality control in the commercial development of biopesticides, focussing on product quality control, and provide recommendations for a more standardized quality control.
2 History of Quality Control in Microbial Pest Control Products
The market of MPCPs is characterized by the frequent appearance and disappearance of products as well as companies (Lisansky 1997). For this, many different explanations have been put forward, including poor performance of products. The lack of effective quality control is often cited as a possible reason for the failure of a product. Some examples are given in the literature for baculovirus products produced in Egypt respectively India (Grzywacz et al., 1997; Battu et al., 2002). Likewise, quality control of EPN products by independent scientists has revealed inconsistent quality. Gaugler et al. (2000) investigated mail-ordered H. bacterio-phora and S. carpocapsae products from cottage industry in the USA and found only 60% of the number of nematodes stated on the label, on average. Also, reduced pathogenicity was often found. Few resources are dedicated to quality control, whose importance is often underestimated in the biocontrol industry. Small companies may not have the knowledge or the facilities for proper quality control checks. To be fair, proper testing methods were lacking in the past.
Registration does require well-described quality control procedures and quality control data, but quality control is seldom enforced following approval. Some countries do not require registrations, or products are sold without formal approvals, and quality control may well be lacking in such situations. I have seen many unregistered MPCPs on the market which did not comply with the label claims. On most of the occasions this concerned the number of infective propagules, which was too low, and sometimes, even infective propagules were lacking. More complicated features such as performance and purity were not checked, but were bound to deliver inferior results. These products damage the image of biocontrol. This underscores the importance of a good quality control programme.
In the field of natural enemies, similar situations have occurred, leading to failures in biocontrol and the disappearance of some companies (Van Lenteren 2003a). The biocontrol industry together with scientists did develop quality control criteria and test guidelines for most natural enemies (Van Lenteren et al. 2003b), and now most of the larger companies do conduct quality control according to these tests and criteria (Bolckmans 2003).
In the field of microorganisms, an attempt was made in 2001 to initiate a similar collaboration between industry and scientists. Scientists of CABI and the International Biocontrol Manufacturers Association (IBMA) initiated a working group on quality control and standards of MPCPs. A joint IBMA - COST 830 workshop was held in 2001 to try to set appropriate standards for microbial products (Migheli and Ruiz Sainz 2003). As a result of a lack of funding, this laudable initiative did not produce many tangible results and the IBMA Working Group is no longer active. Today, there is no mutually agreed set of testing methods and criteria for MPCPs, unlike for natural enemies. Registration procedures for MPCPs generally require quality control methods and data, but standardized guidelines and clear criteria are lacking, even within the EU Directive 91/414 (EC 1991) on registration of plant protection products. Every company is allowed to develop its own methods and to set its own standards with which products should comply. Jenkins and Grzywacz (2003) recognized the lack of standardization and they have suggested a minimum set of parameters for quality control for fungi and baculoviruses.
In the field of EPNs, quality control was first discussed between industry and scientists at the eighth workshop of the IOBC Working Group on "Quality control of mass-reared arthropods" in 1995. All companies agreed that QC criteria needed to be developed for nematodes as with beneficial arthropods (Mason et al., 2002). Ultimately, successful collaboration between scientists and the industry in the EU COST Action 819 led to the development and publication of QC criteria and testing protocols (Grunder 2005). Clear standards, as determined and accepted for natural enemies, however, are still lacking for most quality control parameters for EPNs.
3 Production Quality Control and Process Quality Control
Quality control of production concerns internal procedures for checking whether inputs (purchased raw materials: medium ingredients, formulation compounds and packaging materials) comply with their specifications, whether standard operating procedures (SOPs) are well set-up and followed, and whether production and downstream equipment is maintained properly and is operating as expected. Warrior (2000) emphasized that the quality of raw materials contributes to a large extent to the quality of the final products and that specifications of raw materials must be checked before release for production purposes. He referred mainly to the large scale production of Bacillus thuringiensis (Bt).
Quality control of production processes relates to monitoring vital process parameters, and whether they run according to expected profiles. Conducting these procedures should result in minimum production failures, and in expected yields of good quality. Continuous feedback and improvements should also lead to production stability with minimal extra costs, and to products that match planning. As production systems and processes vary greatly for MPCAs and between companies, details cannot be given here. Nevertheless, quality control is important and should be an integral part of the mass production. Process parameters can be relatively simple such as room temperature and percentage relative humidity in a rearing room for insects in in vivo production of, for example, a baculovirus. On the other hand, it can be a complicated set of parameters that influence each other in liquid production of EPNs. In this case, variables such as pO2, pCO2, pH, temperature, foaming and hydrostatic pressure need to be monitored and adjusted where necessary. Some variables can be adjusted in various ways, as for example oxygen pressure, which can be adjusted by aeration, stirrer speed, temperature and by vessel pressure. This illustrates the complexity of such production processes and computer steered programmes are needed to regulate the process. Process control also includes downstream and formulation processes, packaging and storage. At various steps in the processes, parameters must be monitored and checked. Data should be gathered during all processes and should be used to improve the production on a continuous basis. Production control and process control are often combined in SOPs and performed by production personnel. Production and process control have the greatest effects on the quality of the resulting product and therefore should be taken very seriously within a company.
Quality control on inoculum stability is crucial. A procedure for storage and use of inoculum material for the production is described in Chap. 3 where the chief goal is maintaining inoculum stability. The inoculum needs to be monitored for phe-notypic and genotypic changes and for contaminants. When genetic stability is an issue, specialists may need to be consulted to make sure the inoculum quality is still as desired. Examples of quality problems are loss of plasmids in bacteria, and mutations in baculoviruses. If the original inoculum is no longer of good quality, the strain needs to be recovered anew from a culture collection or from a stored product. Starting with a new isolate is a delicate decision and requires very detailed checking of the strain and its properties before it should replace the older strain and be taken into production. Companies prefer to avoid this as it costs time and money. Long term storage of stable inoculum is the preferred option; if this is not possible new inoculum must be made and tested rigorously. In the case of the production of Green Muscle (Metarhizium anisopliae var. acridum), the standard isolate is passaged through the desert locust every 6 months to avoid loss of virulence (Cherry et al., 1999). Inoculum storage should be taken extremely seriously and measures should be taken to avoid any problems with the stored material as much as possible. For instance, it is wise to keep inoculum in two different places to spread risks and to have an alarm system on the storage facility or deep freezer.
4 Product Quality Control of Microbial Pest Control Products
Quality control generally refers to product control, i.e. to the final formulated product. In manufacturing, however, there may be several in-between products, like the "technical grade active ingredient" (TGAI), that need to be checked. Material from succeeding production batches may be stored to be formulated together. Checks on propagule numbers, microbial contaminants are needed and some batches may be discarded or handled differently, stored longer or shorter. During formulation, propagule numbers may be set to the specification of the final product, thus propag-ule counts are needed before final formulation. The final formulated product is subject to quality control and the product needs to conform to previously determined product specifications ("specs"). These specifications are usually determined with regard to product performance in the field in a broad sense and to registration requirements. Product performance relates to efficacy, obviously, but also to applications characteristics (particle size, emulsion separation, sedimentation in the package) and to shelf-life parameters (moisture content, microbial purity).
The "external" objective of quality control is to deliver a product to the end-user that complies with the specifications at the moment of use. It should also deliver the expected results. This objective focuses at satisfying customers and on complying with registration criteria. Each batch has to be tested prior to sale in order to prevent release of products that do not meet the "specs". The "internal" objective of quality control is to highlight any problem areas which should then be subject to corrective action. This should lead to improved production of products with fewer and fewer discarded batches, and to stability of the production and the products.
A number of papers refer to the overall issue of quality control of MPCAs. Burges (1981) recognized two main aspects of quality control: identity of the pathogen and level of contaminants. In addition he discussed activity, physical features, and safety to vertebrates in an example treating baculoviruses. Jenkins and Grzywacz (2000) reviewed the need for quality control procedures in fungal and viral biocontrol agents in the production as well as for the final products. Product specifications should be determined with regard to physical properties, microbial contaminants, efficacy and storage. They illustrated this by providing recommendations for specifications for fungal and viral products. Lisansky (1985; CPL 2006b) listed five aspects that should be subject to QC: efficacy, microbiological purity, absence of mammalian toxicity, physical characteristics and shelf-life. Quality control methods and specifications are discussed and given in detail for Green Muscle (Jenkins et al. 1998). The need for quality assurance in the industrial production of biopesticides is briefly discussed for bacterial, fungal and viral products by Guillon (1997a). One company, Verdera, Finland, has disclosed some information on their chosen quality criteria for biofungicides. Specifically, they refer to QC during production and to end-use product criteria. Assessed product criteria are viability and efficacy tests, related to storage stability (Palin-Holmberg et al. 2003). In the literature, product quality control generally comprises the features named below.
The organism needs to be properly identified at strain level using the most appropriate scientific techniques. Molecular techniques are needed to do this. The area of taxonomic identification is still developing and species and strain concepts are changing with the development of new technologies. Identification is a difficult topic, but very important with regard to a proper risk assessment. Therefore, the Biopesticide Steering Group (BPSG) of the OECD is developing a guidance document that should help regulators and the industry in assessing the taxonomic identification. Most companies lack the expertise to do this in-house and external laboratories are asked to conduct the identification. The OECD recommends that two laboratories identify the strain to ascertain the identification (OECD 2008). Typically, this identification is done at the beginning of a product development and not as part of the quality control procedure of every batch. Once a master stock culture has been made of the properly identified inoculum, production is started with a specimen of that stock, and batch-wise identification is superfluous. When a new master stock is made, re-identification of the organism should be conducted. Nevertheless, cultures should always be checked visually for deformations or other deviations (genetic or phenotypic changes) to make sure the organism still shows its usual features. At the slightest suspicion, re-identification should be conducted. I consider this aspect to be part of the process control rather than of product control. It cannot be done for every batch, and furthermore, there is no need for it. I recommend re-identifying the inoculum every 3-4 years by a specialist. Then, a recent identity confirmation is available for registration purposes and liability.
A product contains a certain number of infective or virulent propagules. This needs to be specified on the label. This number of infective propagules, when applied according to the recommended methods of application and under the right conditions of use, should achieve the claimed level of control of the pest. Counting of propagules can be done, but is often difficult in technical grade products as well as in formulated products in which remnants of the production medium and/or carrier and particles of formulation ingredients may be difficult to distinguish from spores or virus particles. The number of propagules, however, does not necessarily reflect virulence directly. Accordingly, the actual activity needs to be determined by germination tests or by establishing the number of colony forming units (cfu). This method is used in the case of bacteria and fungi. The active ingredients in Bt's are spores and endotoxins (crystallized proteins), the latter can be measured by analytical techniques. But virulence of the product needs to be assessed in bio-assays. Baculoviruses cannot be counted, usually, because they are hard to distinguish from formulation particles or insect remnants. Even if they can be counted, viable and non-viable particles cannot be distinguished. Since they are obligate parasites that can only replicate in insect cells, a bio-assay is needed to assess the product's virulence. EPNs can usually be counted and living and dead dauer juveniles (DJs) can be distinguished under the microscope. Since yields may vary per batch, counts of infective units needs to be carried out to be able to adjust the number during the formulation process to reach the total number in the end-use product as specified. Next, packaging samples need to be counted again to confirm the "specs". Checking the product on the number of infective propagules is a basic element of product control.
The total number or percentage of microbial contaminants in a MPCP should be limited. These contaminants may negatively influence product quality in terms of shelf-life, efficacy and even physical characteristics. Furthermore, contaminants may pose a risk to the applicator and the consumer. Registration criteria therefore only accept low numbers of contaminants and require absence or near-absence of human pathogens. The total number of contaminating organisms should not exceed a certain number, or, it should not exceed a certain percentage of the number of the active ingredient. The standard depends on what the company decides or on what the registration requirements demand. The standard is often set at 0.1% of the number of infective propagules and is just a pragmatic amount chosen by many companies. There are, however, no obligatory standards within the EU, EPA, OECD and national regulations for MPCPs. For more details, see below at registration requirements. Determination of the level of contaminating organisms, usually bacteria and fungi, is done by cfu testing on microbe-specific media and at certain temperatures.
A total count of mesophiles (fungi, bacteria and yeasts) is often conducted and counts of bacteria, specifically human and mammalian pathogens, by selective tests. Some countries demand animal toxicity tests for Bt products and baculoviruses with every batch to exclude human pathogens. Determination of the level of contaminants needs to be part of process control as well as of product control.
Presence of toxic metabolites is a concern of regulatory bodies and information needs to be presented to the authorities for each specific MPCA where this is considered relevant. The issue of toxins is not well regulated and a discussion between the industry and regulators is ongoing. Clear and appropriate requirements still need to be defined. This topic is dealt with in Chapter 5 in more detail. Standard criteria are not defined and a case by case approach is taken. Harmful effects should be minimal. In the production of some fungi and bacteria, metabolites may be formed and this may depend on the conditions of the production process. Deviations in the process may lead to a higher production of metabolites, and metabolite levels may need to be checked. Toxins are difficult to monitor and biochemical analytical techniques have to be developed to identify them and to monitor them. Routinely monitoring of toxins in the production is only required in some cases following risk assessments by the evaluating authority. An example is the fungus Isaria fumosorosea (=Paecilomyces fumosoroseus) where absence of secondary metabolites must be checked in each fermentation batch. This has subsequently been decided and documented by the EC (2002). When toxins are found, the batch has to be discarded.
4.5 Physical, Chemical and Technical Characteristics
Registration requirements demand product stability with regard to physical and chemical stability, and information on technical properties. The formulation needs to be physically stable during the shelf-life period. This means no clump-forming in wettable powders should occur, nor should irreversible sedimentation of propagules in suspensions or separation of carriers in the case of emulsions take place. Chemical stability (like pH) also needs to be considered and checked according to registration requirements, but in reality this is of lesser importance. Each type of formulation has its particular characteristics that need to be checked. These are referred to as technical properties. Examples are wettability, suspensibility, and particle size distribution in wettable powders and water dispersible granulates. For emulsions it is emulsifiability and stability, for suspensions pourability, etc. These properties have to be tested according to recognized CIPAC methods, which have been developed for chemical pesticides, and comply with standard criteria.
Efficacy is the most significant parameter in quality control. The product's field performance is the most valuable aspect; not only for registration, but also for the company's revenues from the product. If batches of the product do not perform up to expectations, repeated sales will decline. Therefore, every batch should be tested prior to release. This can only be done quickly and in a cost-effective way by using bio-assays. These should reflect the product's performance in the field as much as possible. The relationship between bio-assay results and field results needs to be established from the outset. This study should include batches with poor quality in order to see whether the bio-assay really distinguishes good batches from poor batches. This is often not done and then the test is useless. Bio-assays must be developed in such a way that they give a reliable prediction of how a product will work in the field. Obviously, field studies cannot be conducted for each batch because of time and costs issues.
Bio-assays differ per pathogen and per target. The same bio-assay that was developed for strain selection can also be used in QC tests. There are numerous tests available, see Chapter 2 for more information. Registration requirements demand a description of a QC efficacy test, but specific guidance is not available. Every company is allowed to use its own method as long as it gives reliable data. Bio-assays must be standardized and must be done correctly, otherwise they are meaningless. A major problem in using bio-assays in quality control testing is accuracy and reproducibility. Variations due to insect-rearing fluctuations, assay conditions and insect stage can influence the insect's response to the pathogen. As a result, it is desirable to include a standard product as a reference in the test and to compare the results of the sample with the results of the standard. This is possible in bacteria and bac-uloviruses where material can be stored for years without a change of properties. In fungi this is more difficult. In EPNs this is almost impossible due to difficulties in storing material for along time. Testing for efficacy is clearly the most important aspect of product control.
A product has to comply with its set QC criteria up to the end of its shelf-life period. Usage of a product at the end of its shelf-life should give similar results as does usage of a fresh product. As a consequence, the length of the shelf-life period is determined by the QC criteria and product specifications which it still has to meet at the end of the shelf-life period. For example, if spore germination rate in a fungal product has a minimum set criterion of 80% and after 6 months the germination rate drops under this level, the shelf-life period is determined at 6 months, even when other criteria are still above their minimum criteria. If one parameter does not comply to "specs" anymore, the batch has to be discarded.
There are no clear registration requirements for storage stability for biopesticides in the EU. The company determines the product's shelf-life period in combination with the storage temperature. For instance, a mycoinsecticide has a shelf-life of 6 months when stored between 5 and 10°C. Evidence of product stability should be presented in the registration dossier and then this will be accepted by the authorities. For registration tests, QC parameters need to be tested at initiation and at termination of the storage period. Tolerance levels are, however, only specified for chemical pesticides, and those are not appropriate for biopesticides. Currently, the OECD BPSG (Biopesticide Product Steering Group) and the IBMA are in discussion regarding the establishment of specific testing procedures and tolerance limits for storage stability of MPCPs (OECD 2008). I will discuss this more in detail in Chapter 5. Product quality parameters need to be checked at regular intervals during the shelf-life period to investigate whether product characteristics are stable. This will ensure confidence in the determined specifications. Where deviations are found, corrective measurers need to be taken in the preceding processes. If, in the end, improvements are not possible, the length of the shelf-life period must be reconsidered.
Product quality control needs to be carried out on the formulated product. It may also be done on bulk material just before packaging if this process is known not to influence quality. Testing should require as little time as possible so that no valuable storage time is lost. Parameters subject to testing at this point in time are:
(1) number of infective propagules;
(2) physical-chemical properties;
(3) microbial contaminants;
Once results are conform "specs", the batch can be released for sale. If one of the parameters does not comply with its specification, the batch has to be discarded or send back to production in order to be improved, if possible. Batches where human pathogens or harmful toxins have been detected are to be discarded early in the production process. An identity check is not part of product control.
5 Critical Aspects of Product Quality Control with the Various Types of Pathogens
Product quality control is an integral part of manufacturing biopesticides to ensure sale of high quality products. I believe that today this concept is well recognized in the biocontrol industry and that QC has become a routine procedure. In addition, registration requires detailed description of QC procedures, accompanied by real data to illustrate that products do meet previously determined specifications. Entomopathogenic fungi and bacteria, and baculoviruses are subject to registration, EPNs generally not. Registration requirements with regard to product control of biopesticides are discussed below. In many papers, quality control of MPCPs is discussed. Researchers acknowledge the importance of quality control, but few papers treat this subject systematically and in-depth. I will review the specific aspects of each type of pathogen related to product control. Each type of pathogen and each type of product requires monitoring of specific characteristics related to the number of infective propagules, the formulated product, and its field of application. On the other hand, all products require monitoring of similar parameters that are relevant to all of them such as microbial purity and technical properties. The most determinative parameters are the number of infective propagules, the biological activity of the product, microbial purity, and technical properties. Counting propagules is difficult in bacterial and viral products and generally it does not give reliable information on efficacy. A bio-assay must be conducted to obtain useful information on efficacy. In fungal products, the germination rate and bio-assays produce reliable information on virulence. In nematode-based products counts of dauer juveniles and bio-assays need to be conducted to measure quality.
In bio-assays, standardization of the methods is crucial, as is comparison with a reference standard. This reference ideally is a stored reference standard of the product; if this is not available, then the reference can be a chemical insecticide, or a specific mortality rate. A standard should be developed in the developmental phase of the product and should have a predictable relationship with field efficacy. This must have been established by concurrent testing of the product in the field and in bio-assays. In bacteria and baculoviruses, a reference product can be stored for many years. This is not the case in fungi and nematodes; there a set mortality rate can be the "standard".
Standardization has been investigated and discussed within each group of pathogens. This is, however, still ongoing and appears to be a complex matter. As of today, little standardization has been agreed upon and it is hardly implemented in the industry except for Bt's. For fungal and viral products, standardization of quality control has been proposed by Jenkins and Grzywacz (2003). The authors recommended minimum quality control parameters and methods for each type of product. Research has delivered a good understanding of the critical quality aspects in each type of pathogen. Further investigations are still needed to improve quality control testing methods and to make testing easier and cheaper.
In bacterial insecticides, the essential parameters subject to quality control are the same as for fungal products: the number of active propagules, the biological activity, and the microbial purity. Most of the literature discussing QC in bacterial products refers to Bt products. Warrior (2000) listed five parameters for QC as used by a major producer (Valent BioSciences, formerly Abbott) of Bt products: strain identity, metabolite profile, spore count, biological stability, and physical properties. Potency and physical specifications are the main QC parameters according to Couch (2000). The insecticidal activity of Bt is based on spores and toxins. There are many strains and types of toxins available, each with its specific biological activity. Pathogenicity of Bt's is often primarily due to S-endotoxins (crystallized proteins). Accordingly, spore counts do not correlate well with the biological activity, and hence a bio-assay is a prerequisite for quality product control. Standardization of the bio-assay, the use of a reference standard Bt, and which test insect to use have been the focus of many studies and debates. Detailed bio-assay protocols are needed. Otherwise, reproducibility is poor as was demonstrated by Skovmand et al. (1998) when a B. thuringiensis var. israelensis (Bti) preparation was tested in different laboratories. Factors such as age, stage and strain of larvae used, amount and type of food used and rearing conditions influence bio-assay results. Also the method and duration of sample homogenization may strongly influence results, as well as the method of administration. Analytical biochemical techniques (HPLC and elec-trophoresis) can be used to determine the concentration of the S-endotoxins and this could make QC easier (Bernhard 1992). Within Valent BioSciences this is used in combination with bio-assays (D. Ave, personal communication). If the analytical method is reliable and shows a strong correlation with efficacy, bio-assays may no longer be necessary. This would give quicker and cheaper QC results, but today bio-assays are still required.
Another problem is the lack of standardization when expressing the active ingredients. This can be done in spores/g, but in Bt's this is meaningless in most cases. Endotoxin levels can be expressed in dry weight, but this often includes fermentation products as it is difficult to measure only the crystal proteins, and because other metabolites may also be involved in the pathogenicity process. As a result, standardization in Bt has been a subject of discussion for a long period of time. For two reasons: first, industrial standardization which refers to the need of a producer when checking his product's quality, and second, there is a need for an international standardization to be able to compare products with different strains, and from different producers (Burgerjon and Dulmage 1977). The latter is desirable for scientists, regulators and users. The discussion has resulted in the acceptance of an international reference standard for Bt's where the biological activity is measured in a bio-assay on a single insect species and compared to a reference Bt strain. The biological activity of products, often called potency in Bt's, is expressed in International Units (IU)/mg based on a comparative bio-assay with the product and the standard, assessed against a particular insect. Generally, Trichoplusia ni or Ephestia kuhniella are used as reference insects. Sometimes diamondback moth and Spodoptera units are used. This way of expressing potency of products in order to make them comparable is unique to Bt's within the field of biopesticides. Still, some problems occur with standardization such as the lack of available standard Bt, and the need for more detailed bio-assay and sample preparation protocols (Skovmand et al. 2000).
The increasing number of discovered Bt toxins as well as the number of products warrant suggestions for improvements for bio-assays and internationally accepted standard reference material as given by Skovmand et al. (2000). They suggested the use of three test insect species in the bio-assays because of the differing susceptibilities of lepidopteran species to various endotoxins, and the establishment of a standard based on three Bt strains containing a mix of toxins. Further on, they suggested the combination of bio-assay testing with quantitative protein analysis. Finally, they called for an international podium to further develop and acknowledge the standardization, as was carried out earlier by the USDA and the WHO. A historical overview on the progress in this area over the last 50 years and on the still existing problems is given by Asano (2006).
The above paragraphs are relevant for lepidopteran Bt's. Bti products for mosquito control all contain the same endotoxin which makes comparisons easier. For Bt tenebrionis, for control of Coleoptera, international standards have not been developed (Skovmand et al., 2000). For Bti products, the FAO and WHO have jointly developed specifications for the active ingredient (identity and biological activity), relevant impurities, bacterial contaminants, physical properties and storage stability (FAO/WHO 2006). The document includes detailed physical and biological testing methods. These specifications could be used as a reference for all Bt formulations.
Standardization as discussed above refers to a QC parameter for product efficacy. It does not include other QC parameters such as microbial purity, physical properties and safety.
Registration requires monitoring of these parameters also. Concerning micro-bial purity, Couch (2000) gave a list with acceptable levels of various contaminants based on internationally recognized levels for animal feed. Generally, this is similar for all entomopathogen-based products that need to follow registration requirements. Below these requirements will be discussed in detail. In a Danish post-registration survey on specifications of biopesticides, Bt products were found to be free of contaminants (Winding 2005). For safety reasons, each batch should be tested on toxicity to mice according to Couch (2000). When Bt products were first commercialized, there was concern that possible contamination with B. anthracis would not be detected with conventional testing for contaminants. Therefore, a mouse toxicity test has been required by the EPA in the USA for each batch and companies still perform this test on a regular basis (CPL 2006a).
Specifications for physical properties for Bt products should be developed by a producer and checked for in every batch. The properties depend on the type of formulation. Protocols and specifications as for chemical pesticides should be used as a reference.
Quality control in Bt's has been intensely investigated by many researchers and companies and quality control is well developed. Many organizations and regulatory authorities agree on the methods and reference standards. As a result, Bt products are of high quality and are the most reliable products in the MPCP market.
In a non-Bt product, Invade, based on Serratia entomophila, the quality control system QC has been described by Pearson and Jackson (1995). QC parameters are cell density, purity, virulence and longevity. Purity is defined as confirmation of the production strain by cell growth characteristics on a specific agar plate and the expected phages pattern. Microbial contaminants are not separately tested. Virulence is checked by visualization of plasmids and by a bio-assay confirming pathogenicity on the target insect, i.e. grass grubs. In the longevity test, the decline of cell viability is periodically checked. The authors do not give product criteria or tolerance limits. This demonstrates that QC in bacterial products varies per product and per species and depends on the active ingredient, the mode of action and the target insect(s).
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