yt^ REVIEW OF LECONTVIRUS A NUCLEAR POLYHEDROSIS VIRUS WITH SPECIAL EMPHASIS ON THE AQUATIC ENVIRONMENT APRIL 1990 Environment Environnement Ontario Jlm Bradley, Minister/ministre ISBN 0-7729-6452-1 REVIEW OF LECONTVIRUS A NUCLEAR POLYHEDROSIS VIRUS WITH SPECIAL EMPHASIS ON THE AQUATIC ENVIRONMENT Report prepared for: Aquatic Criteria Development Committee Water Resources Branch Report prepared by: Dr. G. A. Surgeoner M. J. Farkas Department of Environmental Biology University of Guelph Guelph, Ontario NIG 2W1 APRIL 1990 Q Copyright: Queen's Printer for Ontario, 1990 This publication may be reproduced for non-commercial purposes with appropriate attribution. PREAMBLE In response to the Ministry of Natural Resources preparation of Class Environmental Assessment for timber management on crown lands, the Ontario Ministry of the Environment commissioned the Environmental Review Of Lecontvirus A Nuclear Polyhedrosis Virus With Special Emphasis On The Aquatic Environment. This review focuses on the aquatic ecosystem effects of this insecticide as they relate to the use of Lecontvirus in the control of the redheaded pine sawfly (Neodiprion lecontei) on crown lands. The review also considers the effects of Lecontvirus to non-target terrestrial biota (wildlife and humans) as they relate to the exposure of these lifeforms through the contact and ingestion of contaminated water and/or biota. The intent was to develop a Provincial Water Quality Objective (PWQO) from the information provided in the review document. However, the lack of an analytical method for enumerating Lecontvirus in water has prohibited the development of a numerical PWQO. Instead, a narrative ENVIRONMENTAL IMPACT STATEMENT has been prepared and is provided on the following pages. ENVIRONMENTAL IMPACT STATEMENT Available toxicological evidence for Lecontvirus and other Baculoviridae to terrestrial and aquatic life, indicates that Lecontvirus would not pose a threat to aquatic life or other beneficial uses of the Province's surface waters from its properly planned, controlled and supervised treatment for redheaded pine sawfly (Neodiprion lecontei) infestations. RATIONALE INTRODUCTION Lecontvirus is a biological insecticide registered in Canada, under the Federal Pest Control Products Act in 1987 (PCP# 17824) for the control of the red-headed pine sawfly Neodiprion lecontei (Fitch) . The insecticide actually consists of diseased N_^ lecontei larvae infected with a nuclear polyhedrosis virus (NPV) . This virus belongs to the family Baculoviridae subgroup A (embedded DNA virus in large numbers of polyhedral inclusion bodies of protein) . Lecontvirus is only effective against the red headed pine sawfly, N_^ lecontei . No organisms outside the family Diprionidae, of which the red-headed pine sawfly is a member, are known to be affected by Lecontvirus. Lecontvirus must be ingested and enter the larval gut where the alkaline pH conditions dissolves the protein inclusion bodies and thereby releases the infectious virions. These virions penetrate the cells of the gut and initiates cellular infection which leads to the death of the target organism in 10 to 12 days. The larvae generally cease feeding well in advance of mortality. The use of Lecontvirus in Canada is restricted for application under the direct supervision of the federal or provincial forestry service. It is used to protect plantations of red pine, jack pine and Scots pine, and is generally only applied to trees less than 15 meters high. ENVIRONMENTAL FATE Little research on the environmental fate of Lecontvirus exists since there is no method to quantify residues. Since the virus exists naturally, it must be capable of overwintering. Baculoviruses can persist for several years in soil and it is reasonable to assume that Lecontvirus can also persist for the same length of time. Corpses of infected larvae adhering to trees and NPV contaminated bird faeces are possible sources and may aid in disseminating the virus in the environment. The most probable route by which Lecontvirus may enter the aquatic environment is through spray drift and surface run-off during and after application. However, plantation sites are typically located on sandy soils generally removed from bodies of water. In addition, the small quantity of polyhedral inclusion bodies applied per hectare would result in minimal exposure of the aquatic environment. EFFECTS ON AQUATIC LIFE Due to the specificity of Lecontvirus, few studies on the toxicity to aquatic organisms exist. Hicks et al . (1981) found no effects in toxicity tests exposing rainbow trout and Daphnia pulex for 21 and 14 days respectively to dosages of Lecontvirus well in excess of amounts of material which could enter streams and water bodies. In addition, Kingsbury et al . (1978) failed to detect any impact on aquatic invertebrates in operational field trials. Tests conducted with a range of aquatic invertebrates and fish to other Baculoviridae (e.g. Heliothis NPV) indicated no effects EFFECTS ON TERRESTRIAL LIFE A range of terrestrial animals have been exposed to Lecontvirus via topical applications, intubation and through inhalation routes to levels well in excess of those that could occur from applications to plantations with no effects observed. The only confirmed effect noted for Baculoviruses on vertebrates, has been slight skin or eye irritation in rabbits with viruses other than Lecontvirus. The effect was believed to be due to the insect material (ground insect parts) associated with the virus, and not the virus itself. HUMAN HEALTH EFFECTS From the available animal data specific to Lecontvirus, human data related to other baculoviruses, and the margin of safety tested, there appears to be no concern for humans from exposure to Lecontvirus. TABLE OF CONTENTS PREAMBLE i ENVIRONMENTAL IMPACT STATEMENT ii TABLE OF CONTENTS iv ACKNOWLEDGEMENTS 1 CONCLUSIONS 2 1 . 0 INTRODUCTION 3 2 . 0 LECONTVIRUS 2 . 1 General Description 4 2 . 2 Production Process 5 2.3 Impurities in Commercial Products 6 2.4 Spraying/Application Practices 7 2 . 5 Environmental Fate 10 2.6 Host Specificity 11 2 . 7 Virulence 15 2.8 Aquatic Toxicity 16 2 . 9 Health Effects (to Humans) 19 2.10 Effects on Non-target Terrestrial Animal 2 0 2.11 Methods for Enumerating Lecontvirus in Aquatic Samples 22 3 . 0 LITERATURE CITED 2 4 4.0 APPENDICES Appendix 1 26 Appendix 2 27 Appendix 3 31 ACKNOWLEDGEMENTS The authors would like to thank Dr. John Cunningham, Forest Pest Management Institute, Sault Ste. Marie, for providing non-published documentation. We would also like to thank the staff of the Ontario Ministry of the Environment: C. Cherwinsky, C. de Barros, E. Leggatt, D. Poirier, J. Ralston and Dr. D. Rokosh for their guidance, review and comments throughout the duration of this project. CONCLUSIONS 1) The scientific literature indicates that all baculoviruses, including Lecontvirus, are specific to the mid-gut epithelial cells of their host insect. The key to safety with the use of Lecontvirus is its inherent host- specificity. 2) In Ontario, use of Lecontvirus is minor. Approximately 2400 hectares of pine plantations have been treated by ground application between 1980-1987. 3) The impact of Lecontvirus to organisms other than Neodiprion lecontei appears to be undetectable, based on all available scientific data. 4) The use of Lecontvirus does not represent a detectable impact to the quality of surface waters in the Province of Ontario. 1.0 INTRODUCTION The Ontario Ministry of Natural Resources has recently completed a draft, Class Environmental Assessment for timber management on crown lands. Under this Class Environmental Assessment, the Ministry of Natural Resources proposes (when required) to use the nuclear polyhedrosis virus, Lecontvirus, for control of the red-headed pine sawfly, Neodiprion lecontei. The potential for this "biological" insecticide to enter surface water during and after application to timber lots is of potential concern to the Ontario Ministry of the Environment. The Ontario Ministry of the Environment has been mandated to develop and, where appropriate, revise the Provincial Water Quality Objectives and Policies to protect the province's water resources. These water quality objectives and policies are designed to assure that "surface waters in the province are of a quality which is satisfactory for aquatic life and recreation." In order to formulate a sound environmental policy for Lecontvirus, the available scientific data must be procured and analyzed. The purpose of this document is to provide the current information known about this compound with particular emphasis on protecting surface waters. 2.0 LECONTVIRUS 2.1 General Description Lecontvirus is a biological insecticide, given temporary registration in 1983 and full registration in 1987 (PCP #17824; Appendix 1), for control of the red-headed pine sawfly Neodiprion lecontei (Fitch) in Canada. The insecticide actually consists of diseased N. lecontei larvae infected with a nuclear polyhedrosis virus. This virus belongs to the family Baculoviridae subgroup A (embedded DNA virus in large numbers of polyhedral bodies of protein) . Larvae infected with virus are frozen, lyophilized and ground to a fine powder. Actual virus represents about 0.05% of the preparation, the rest being milled insect parts (Cunningham et al. 1986) . The material is generally supplied in a suspension in emulsifiable oil which is then diluted in water. The oil formulation is made with Dipel 80 and the formulated product contains 5 X 10' polyhedral bodies/ml. The particles which cause infection (virions) are rod- shaped, contain DNA, and are embedded within rod-shaped proteinaceous crystal bodies known as polyhedral inclusion bodies (PIB's). The PIB's have a mean diameter of 0.72 um (range 0.29-1.44 um) . The insecticide contains a minimum of 5 X 10° PIB's/ml of product. It is the living. replicating virions, and not toxic agents, which cause mortality of larvae. 2.2 Production Process The registrant for Lecontvirus is the Forest Pest Management Institute, Box 490, Sault Ste. Marie, Ontario P6A 5M7 . The actual contact for production of the virus is Dr. John Cunningham at the Institute (705-949-9461) . Unlike most other insecticides, the production of this virus is done in a research facility by Dr. Cunningham and technical staff. The production methods for the virus have been outlined by Cunningham and McPhee (1986; Appendix 2). Basically, healthy N. lecontei larvae (laboratory-reared or field-collected) are infected with NPV virus. Larvae are fed on pine foliage, since no artificial diets exist. The larvae are harvested 10 to 12 days later (allowing time for viral replication) (Anonymous 1987) . The diseased larvae are frozen, gently dried, and ground to a fine powder. This powder contains 0.05% virus particles and 99.95% finely-milled insect parts. The ground larvae form a grey powder whereas purified polyhedras are white. The material is soluble in both oil and water with a pH between 6 to 8 . Ground larvae may be stored for several years without loss of activity at 4°C, but formulated material (i.e. in oil) should be stored no more than 2 months. Each season, Dr. Cunningham formulates new material upon request from forest service personnel . 2.3 Impurities in Commercial Products Commercial Lecontvirus contains virus particles and ground insect parts. These ground insect parts may be considered impurities and obviously contain a wide spectrum of proteins, carbohydrates, lipids, chitin and potential bacterial contaminants. In sterility checks of Lecontvirus preparations, Forsberg et al. (1978) found Enterobacter agqlomerans in healthy larvae and E. agqlomerans , E. cloacae and Escherichia coli in infected larvae. Contamination by bacteria in the 20 ml/ha of actual Lecontvirus used in spray programs would not likely exceed measurable background levels. Toxicological studies of Lecontvirus have included these impurities (ground larvae) within the evaluation, i.e. studies have evaluated the whole spectrum of components, not the purified polyhedral bodies. It is important to realize that this disease does occur in nature (though rare) , being first reported as early as 1912 (see Cunningham and Entwistle 1981, p. 396). However, it was only identified as a NPV in 1951 (Steinhaus 1951) . The disease in N. lecontei was first found occurring naturally in Ontario in 1950 (Bird 1961) . 2.4 Spraying/Application Practices The use of Lecontvirus in Canada is restricted for application under the direct supervision of federal or provincial forestry service personnel. The use of the product is classified as Forest Management (use on greater than 500 ha) or Woodlands Management (use on less than or equal to 500 ha) . The product can be obtained only through request to the Forest Pest Management Institute. This procedure regulates users. The material can be applied both aerially or by ground application (See label, Appendix 1). The volumes of spray mix and dosage are dependent on type of application and life stage of N. lecontei larvae (Appendix 1) . The maximum rate of spray mix applied by ground is 20 litres/ha with an effective dosage of 10 x 10' polyhedral inclusion bodies, i.e. 10 billion inclusion bodies, per hectare. This is formulated from 20 ml of Lecontvirus for each hectare treated. Water used for tank mixes should be in a pH range of 6.0 to 7.2. If acidic, addition of NaOH is recommended (Anonymous 1987) . Ground application would generally be made during mid-July to plantations of either red pine, jack pine or Scots pine, primarily less than five meters in height. Applicators would typically walk along tree rows spraying with a gas-powered mist-blower (Podgwaite et al. 1986) . Applications would normally be conducted when wind conditions were less than 10 km per hour. Applicators should spray in a downwind pattern as they move through the trees. Safety helmet, goggles, gloves and a respirator are recommended, along with standard handling procedures for insecticide use, i.e. clean hands, remove contaminated clothing etc. , but in 20- 30"C weather, reality and comfort dictate safety precautions. Off site biologically significant drift is possible but limited to less than 100 m when doing ground application (Payne et al. 1988) . The majority of red pine and jack pine plantations in Ontario will be on upland sandy soils, well removed from flowing bodies of water, lakes and ponds. At least 3 53 plantations have been treated by ground application by MNR staff (Cunningham et al. 1986) . The small size of most plantations and the spotty distribution of sawflies lends itself to ground applications more than to aerial applications. A summary of spray programs with Lecontvirus in Ontario over the past 5 years has been provided in Appendix 3 (Joe Churcher, Pest Control Section, Ministry of Natural Resources) . Aerial application of Lecontvirus in Ontario has been reviewed (Cunningham et aJ. 1986) . Again, the maximum registered rate is 10 billion PIB per hectare in 9 . 4 litres 8 of water. Application can be made by boom and nozzle or by Micronair atomizers. Between 1976 and 1980, 14 plantations with a total area of 175.5 ha were treated. Since 1980, 2,399 ha have been treated. This is probably a major over- estimate of usage since many plantations are "spot- treated", e.g. 4 trees in a 5 ha plantation (Churcher, per. comm.). Table I of Cunningham et al.'s 1986 paper summarizes the conditions of aerial trials. Applications would typically occur in early morning in mid-July at temperatures greater than 15"C and wind speeds less than 10 km per hour. Off site movement of particles is possible. Again, plantation sites are typically on sandy soils generally not close to bodies of water. If we consider a worst case scenario of direct application to water, we would assume to have 10' PIB per ha/10,000 m or 100,000 PIB's per surface meter^ of water. If we assume aim water depth, this would represent approximately 1 PIB in every 10 ml of water as a worst case direct application to water. The use of Lecontvirus, whether aerially or by ground, is highly effective (Cunningham et al. 1986, Podgwaite et al . 1986) in controlling N. lecontei (greater than 96% reduction in larval numbers) . The control of this insect is critical for proper management of young red pine, Scots pine and jack pine plantations (Benjamin 1955) . Mortality of larvae usually occurs by 15 days post-spray. Larvae generally cease feeding well in advance of mortality. Foliage protection varies with application time but is greater than 95% as measured by centimetres of defoliation per colony of sawflies. In all studies, no re-treatment of plantations was required the year after treatment. The lack of re-treatment may be due to live virus from the previous season or to the poor recolonization capabilities of N. lecontei . 2.5 Environmental Fate Approximately 10 billion PIB's of Lecontvirus are sprayed per hectare. Little research on environmental fate exists since there is no residue assay method. Biological assays suggest that virus particles may exist in plantations for several years after application. With other types of NPV (i.e. the virus used for control of European spruce sawfly) , the half life for the physical presence of polyhedral bodies from either pure suspensions or suspensions including larval material were 38.3 and 55.1 days respectively. Assuming a deposit of 100 PIB's, these would decline to 1.5 and 6.2 PIB's by 240 days when the next larval generation would begin. Decay of PIB's, as measured by biological assays, appears more rapid and is likely due to the viricidal effects of ultraviolet light (see Cunningham and Entwistle 1981, p. 383) . At present, all registered baculoviruses in the U.S. and Canada have been granted exemptions from the requirement of a tolerance 10 for residues (see Shieh and Bohmfalk 1980, p, 359). Since the virus exists naturally, it must be capable of overwintering. Corpses of infected larvae adhering to trees and NPV-contaminated bird faeces are a possible source. Baculoviruses can persist for several years in soil and it is reasonable to assume that Lecontvirus can also (see Cunningham and Entwistle 1981, p. 384). The stability of Lecontvirus in water is unknown. 2.6 Host Specificity An excellent review of safety tests associated with these viruses has been published (Burges et ai. 1980) . Key points of note from the review are: 1. Baculoviruses are unable to replicate in microorganisms, non-insect invertebrate cell lines, vertebrate cell lines, plants and non-arthropod invertebrates and vertebrates. 2. Replication in insects, outside of the particular insect family in which the virus was originally found, is rare. 3. Naturally occurring infections outside this host range have not been found. 4. Medical, veterinary and phytopathology literature 11 contains no reference to Baculoviridae affecting man, animals or plants. 5. At times of natural epizootics of caterpillars, birds have been found to have 18% by weight inclusion bodies in faeces without detrimental effect (see Burges et al. 1980, p. 330). 6. Exposure of 10' to lO' PIB/animal, i.e. shrimp, oysters, water fleas (Daphnia sp.), failed to demonstrate an effect. 7. In vertebrates, challenge studies representing 10 to 100 times field exposure, i.e. 1 x 10' intradermal or 20 x 10' PIB/kg, showed no effect. 8. NPVs have no effect on mammalian chromosomes. In reviev/ing Lecontvirus, the large body of information on Baculoviridae adds supportive evidence to safety tests specific to Lecontvirus. Specific studies related to Lecontvirus are summarized in Table 1. In addition, comparison of restriction enzyme profiles for the original Lecontvirus isolates (ca. 1976) and the virus currently being produced shows no change in the viral genome. This demonstrates the genetic stability of Lecontvirus (Dr. B.M. Arif, per. comm. 1988). Table 1. Species Chickens/ turkeys Safety tests with Lecontvirus involving animals Type of Test Dosage/Exposure Rate Results Intubation 1.4 X 10' PIB's per g body weight No effect (Valli et al.l976; Rats/ rabbits Rabbits Hamsters Rabbits Birds e.g. sparrow grosbeak flicker oriole Honeybees Aquatic inverte- brates Rainbow trout Rainbow trout Daphnia pulex Intubation Multiple dose Dermal exposure Inhalation Eye irritation Aerial application 3 X 10° PIB's per g body weight 3.75 X 10' PIB's over 9 0 days 4.2 X 10° PIB' s per animal 2.72 X 10 PIB's per animal/ 1 hour 4.2 X 10 per eye PIB's 5.5 X 10° PIB/ha Aerial 5.5 x 10' PIB/ha application Aerial 5.5 x 10" PIB/ha application Ingestion 3 x 10' PIB's per g body weight Water-borne 24,000 PIB/ml virus water Water-borne 24,000 PIB/ml virus water No effect (Forsberg et al.l978) No effect (Forsberg et al. 1978) No effect (Forsberg et al. 1978) No effect (Forsberg et al. 1978) No effect (Forsberg et al. 1978) No effect (Kingsbury et al. 1978) No effect (Kingsbury et al. 1978) No effect (Kingsbury et al. 1978) No effect (Hicks et al.l981) No effect (Hicks et al. 1981) No effect (Hicks et al. 1981) 13 Table 1. continued. Species Type of Test Dosage/Exposure Rate Results Guinea Hyper- 10 injections, Initial pig sensitivity 1 every 48 hours flare at 2 X lO'PIB's reaction, per injection no evidence of hyper- sensitivity (Cunningham 1988, personal communi- cation) 14 The only confirmed effect noted from baculoviruses on vertebrates has been slight skin irritation or eye irritation in rabbits with viruses other than Lecontvirus. The effect is believed to be due to the insect material associated with the virus, and not to the virus itself. 2.7 Virulence Lecontvirus is only effective against the red-headed pine sawfly, N. lecontei. Infections of N. sertifer and N. abietis with Lecontvirus will occur but at high concentrations of virus application with little resultant mortality (see Cunningham and Entwistle 1981, p. 381). No organisms outside of the family Diprionidae are known to be affected. All baculoviruses must be ingested by larvae to initiate infection. The alkaline pH of the larval gut dissolves the protein inclusion bodies to release infectious virions. These virions penetrate the cells of the gut and initiate cellular infection. Nucleopolyhedrosis viruses will replicate only in midgut epithelial cells. Polyhedral inclusion bodies are produced and within 10 to 12 days, the larva dies. As the larvae disintegrate, polyhedral bodies are released into the forest. Live virus in bird faeces have been identified and birds feeding on infected larvae may aid in the dissemination of this virus (see Cunningham and Entwistle 1981, p. 383). The infective dose required to kill 50% of the population is extremely difficult to determine, but is believed to range from 20 PIB's per first instar larva to 800 PIB's for early fifth instars (see Cunningham and Entwistle 1981, p. 389). The toxicity of Lecontvirus is measured as the number of PIB's per ml of product, e.g. 5 x 10" PIB's/ml. 2.8 Aquatic Toxicity Due to the specificity of Lecontvirus, few studies on the toxicity to aquatic organisms exist. All available data related to Lecontvirus and baculoviruses in general indicate no toxicity to aquatic organisms. In general, Baculoviridae have been shown not to replicate in grass shrimp, brown shrimp, oysters and water fleas (at dosages of 10' to 10' PIB/animal, see Burges et al. 1980) . Seven species of fish have been exposed to Heliothis NPV without effect (see Burges et al. 1980) . Tests with nuclear polyhedrosis virus affecting Neodiprion sertifer did not affect blue gills or rainbow trout (Cunningham and Entwistle 1981, p. 393). The most extensive aquatic toxicity studies done on Lecontvirus are by Hicks et al. (1981) . The key points are as follows: A. Rainbow Trout Ingestion Study: The materials tested against rainbow trout were: 1) 16 lyophilized, Lecontvirus-infected N. lecontei (sawflies) ; 2) lyophilized, unaffected N. lecontei ; 3) purified polyhedral inclusion bodies of Lecontvirus; and 4) control, A challenge test of 3 million PIB's/g body weight of rainbow trout was used for evaluation. Fish weighed an average of 109 g. Results: 1. no lesions observed in any of the 13 tissues examined. 2. some incidental abnormalities in all groups. 3. no significant differences in weight at any time during study. 4. no abnormalities in behaviour of fish observed. B. Rainbow Trout Water-borne Virus Study: Thirty-six rainbow trout were subjected to the following treatments: 1) lyophilized, Lecontvirus-infected sawfly larvae; 2) purified PIB's of Lecontvirus; and 3) control. The water was treated at 24,000 PIB per ml (i.e. 240,000 X hypothetical field dose) . The aquaria were left as closed systems for 7 days, with the remaining 21 days at a continual flow of 2 . 3 1/min. Results: 1. no lesions observed attributable to treatment. 2. no significant difference in initial, weekly or final weights of fish. 17 3. no abnormalities in behaviour. C. Daphnia pulex Study: A cohort of 60 Daphnia pulex were fed algae, and each treatment group of Daphnia put in 100 ml Hfi. Treatments were as follows: 1) lyophilized Lecontvirus-inf ected N. lecontei ; 2) lyophilized, unaffected N. lecontei; 3) control. Twenty Daphnia received each treatment. Daphnia were exposed to 24,000 PIB/ml water, and samples (consisting of 4 Daphnia) were taken at 0, 1, 3, 7, and 14 days post-inoculation. The progeny of Daphnia were counted, and Daphnia also underwent microscopic examination. Results: 1. no significant difference in brood size or fecundity of Daphnia. 2. no lesions or abnormalities in any of the tissues examined. The overall conclusions were that "under conditions of this study, rainbow trout and Daphnia were unaffected ... at dosages far in excess of amounts of material which could get into ponds or streams." Similarly Kingsbury et al. (1978) failed to detect impact to aquatic invertebrates in operational field trials. 18 2.9 Health Effects (to Hvimans) Specific studies related to human susceptibility of Lecontvirus do not exist. Extrapolations from animal toxicology tests are used to predict potential human health effects. With Lecontvirus, there was no effect in the following studies (refer to Table 1 for dosages) : 1. acute oral tests on rabbits and rats (dosage was equivalent to the amount of product required to spray 40 ha administered to a 70 kg man, i.e. 3 x 10' PIB's/g body weight) . 2. acute dermal tests on rabbits. 3. multiple-dose, 90-day exposure of rats. 4. acute eye irritation of rabbits, 5. inhalation tests in hamsters. In mice, mortality by ingestion was due to the physical effects of forced feeding (see Cunningham and Entwistle 1981, p. 393). They were killed at a rate equivalent to the amount of product required to spray 2400 ha administered to a 70 kg man. Actual human data does exist for other baculoviruses . With Heliothis NPV, studies have been done with humans at dosages from 6 x 10' to 3 x 10" PIB/kg. Skin irritation studies with dosages up to 10' PIB/mm' of skin have been done. Ten humans were fed lO' PIB/day for 5 days (see 19 Burges et aJ. 1980, pp. 331-332). All of these studies showed no effect. If we consider 100 PIB's of Lecontvirus would be in a litre of water, the feeding dosages of Heliothis virus would be equivalent to drinking 10' litres of water. Skin sensitivity tests represent even higher levels. From available animal data specific to Lecontvirus, human data available related to other baculoviruses , and the margins of safety tested, there appears to be no concern for humans from Lecontvirus. 2.10 Effects on Non-target Terrestrial Animals Mammals: The possibility of effect of Lecontvirus to terrestrial animals is predicted by the laboratory toxicology studies already mentioned (see Forsberg et al. 1978 for mammalian studies on rats, mice, rabbits and hamsters). The summary of this study is that: "From these studies it can be concluded that the NPV of Neodiprion lecontei was without effect on laboratory animals housed in a controlled environment and including a group of animals which were immunosuppressed. Since no indication of NPV infection or increased susceptibility to other infections or significant change from accepted values was caused by these preparations by any route we conclude that this NPV is not a hazard to mammals." 20 Birds: Valli and Claxton (1976) evaluated oral toxicity to chickens and turkeys in a laboratory study. The summary of this study is: "In summary, I feel the intensity with which these birds were studied and lack of variation occurring between the test and control groups indicates that the nuclear polyhedrosis virus of the redheaded pine sawfly does not establish infection in these birds or cause any injury. It therefore appears to constitute no hazard whatever to these avian species." During natural invertebrate virus epizootics, the exposure of some vertebrates has been massive, e.g. 18% by weight of inclusion bodies were found in faeces of birds that preyed on infected caterpillars (see Burges et al . 1980, p. 330). There was no indication of bird mortality. Field Studies: A field evaluation of Lecontvirus at 5.5 x 10' PIB/ha has been done by Kingsbury et al. (1978) in Renfrew, Ontario. This study evaluated the impact on birds (many species present), honey bees and aquatic fauna. The conclusions of the study are that: "From the data collected and the field observations, it would appear there were no immediate undesirable effects due to the NPV spray application on the avian or aquatic fauna nor on colonies of honey bees... The honey bees suffered no discernable losses, although further studies would be necessary to 21 determine if there had been any rejection of contaminated pollen by the bees. There were some anomalies in the control stream which made the aquatic study less deterministic than ideal, but except for unexplained decreases in chironomid and mayfly counts the benthic fauna was clearly unaffected." Natural variations found in populations within and between samples would readily explain the benthic observations related to chironomids and mayflies. In addition, results from other tests with baculoviruses indicate no effect to birds, mammals, fish, and honeybees (Burges et al. 1980) . The potential effects to amphibians and reptiles are unknown, but there have been no reported impacts from field studies using baculoviruses. 2.11 Methods for Enumerating Lecontvirus in Aquatic Samples At present, there is no analytical method for enumerating Lecontvirus in water. Biochemical studies on purified PIB^s for Lecontvirus show that the PIB protein has a molecular weight of 28,000 Daltons. The virions have 9 structural proteins ranging from 7,600 to 96,700 Daltons (see Cunningham and Entwistle 1981, p. 392). In the past, microscopic analysis of fresh, dissected gut material has been used to detect NPVs . Viruses can be diagnosed only at 1500x or greater magnification. 22 Enzyme-linked immunosorbent assay (ELISA) will allow for identification of NPV virus in infected larvae. This technique combines the use of immobilised antigens or antibodies on a solid phase with enzyme-labelled antibody or antigen conjugates to yield assays with the sensitivity and specificity of isotopic techniques (Voller and de Savigny 1981) . Ten nanograms of polyhedral protein can be detected, and larvae containing 5 x lO' PIB's or more give positive reactions (see Cunningham and Entwistle 1981, p. 392) . 23 3.0 LITERATURE CITED Anonymous. 1987. Nuclear Polyhedrosis viruses- Technical reference. Forest Pest Management Institute, Box 490, Sault Ste. Marie, Ontario. 4pp. Benjamin, D.M. 1955. The biology and ecology of the red- headed pine sawfly. USDA For. Serv. Tech. Bull. 1118. 57 pp. Bird, F.T. 1961. Transmission of some insect viruses with particular reference to ovarial transmission and its importance in the development of epizootics. J. Insect. Path. 3: 352-380. Burges, H.D., G. Croizier and J. Huber. 1980. A review of safety tests on baculoviruses . Entomophaga 25: 329-39. Cunningham, J.C., P. de Groot and W.J. Kaupp. 1986. A review of aerial spray trials with Lecontvirus for control of redheaded pine sawfly, Neodiprion lecontei (Hymenoptera : Diprionidae) , in Ontario. Proc . Ent . Soc. Ont . 117: 65-72. Cunningham, J.C. and P.F. Entwistle. 1981. Control of sawflies by baculovirus. In: Microbial control of pests and plant diseases 1970-80. H.D. Burges, ed. Academic Press, London, pp. 379-407. Cunningham, J.C. and J.R. McPhee. 1986. Production of sawfly viruses in plantations. Can. For. Serv., Sault Ste. Marie, Tech. Note No. 4. 4 pp. Forsberg, CM., V.E.O. Valli and P. Dwyer. 1978. Mammalian pathogenicity of the nuclear polyhedrosis virus of Neodiprion lecontei . Can. For. Serv., Sault Ste. Marie, Contract Rep. OSU76-00226. 524 pp. + appendices. Hicks, B.D., J.R. Geraci, J.C. Cunningham and B.M. Arif. 1981. Effects of red-headed pine sawfly, Neodiprion lecontei , nuclear polyhedrosis virus on rainbow trout, Salmo gairdneri, and Daphnia pulex. J. Environ. Sci. Hlth. B16: 493-509. Kingsbury, P., B. McLeod and K. Mortensen. 1978. Impact of applications of the nuclear polyhedrosis virus of the red- headed pine sawfly, Neodiprion lecontei (Fitch) , on non- target organisms in 1977. Can. For. Serv., Sault Ste. Marie, Inf. Rep. FPM-X-11. 27 pp. Payne, N. , B.V. Helson, K.M.S. Sundaram and R.A. Fleming. 1988. Estimating buffers for spray applications. Pest. Sci. (in press) . 24 Podgwaite, J.D,, P. Rush, D. Hall and G.S. Walton. 1986. Field evaluation of a nucleopolyhedrosis virus for control of redheaded pine sawfly (Hymenoptera : Diprionidae) . J. Econ. Ent. 79: 1648-1652. Shieh, T.R. and G.T. Bohmfalk. 1980. Production and efficacy of baculoviruses . In: Symposium on microbial agents for the control of insect pests. Biotech. Bioeng. 22: 1357-1375. Steinhaus, E.A. 1951. Report on diagnosis of diseased insects 1944-1950. Hilgardia 20: 629-678. Valli, V.E.O. and M.J. Claxton. 1976. Avian toxicity test of the nuclear polyhedrosis virus of the redheaded pine sawfly Neodiprion lecontei. Can. For. Serv. , Sault Ste. Marie, Contract. Rep. OSS5-0194. 237 pp. Voller, A. and D. de Savigny. 1981. Enzyme linked immunosorbent assay (ELISA) . In: Techniques in clinical immunology. R.A. Thompson ed. Blackwell Scientific Publications, 2nd ed. Oxford, pp. 157-169. Appendix 1 g-5 -D ^ £ -; J) o Q. S g- ro , : o CE o J£ Z2 1° IIctS - „ Q CO D^ Z< 2 5^ ^5 < [L£g cc £r£ uj f r CO s o 08 S| i^S 1l§i lilies £ 5 S- o ^ '" III ^ E i-5^ S , 0 J.UJ I Q. en QC iifl 1 z z 1 I i 1 5.10'' (5 billion polyhedial inclusion bodies) lOxltf' (10 billon polyhedial bod.es) ii 2 S If 2 I suoi.e.ueie 1- Ii; < = if! °: " 0 0 LU m -J y S 2 - - « !? -s I i ?der for 30 seconds in a Waring blender. To obtain a finer powder, which will pass through a 20 mesh sieve when suspended in water or oil, the ground larvae are mixed with an equal amount of crushed dry-ice and re-ground for 30 seconds in a blender. The powdered material is then stored in tightly sealed containers at 4°C until required for bio- control operations. The potency of each batch is deter- mined ty estimating the number of poly- hedral inclusion bodies per gram using the dry coxonting technique described by Wigley (1980). If larvae are virus- killed or heavily diseased when har- vested, the powder contains about 2 x 10^*^ PIB/g. The dead larvae contain some bacteria which are of no concern. How- ever, a quality control check is run to determine if the bacteria are at an acceptable level and that no human patho- gens are present. Quality control pro- cedures are described by Podgwaite and Bruen (1978) . Currently, naterial is shipped to clients as an emulsifiable oil concen- trate. The oil used is Abbott Labora- tories gelled oil vehicle which is the carrier for Bacillus thuringiensis in their product called Dipel 88®. The shelf-life of the NPV in this oil is less than one year and a search is being irade for an alternative vehicle. We only formulate sufficient virus to use for one season and recommend that surplus material be discarded. Conclusions Production costs of Sertifervirus and Lecontvirus are very low; the main factors are salaries, travelling time to production sites, lodging and related expenses. It takes about 50 virus- infected larvae to produce sufficient material to treat 1 ha. If produced close to the laboratory with no ovemi^t accormodation involved, costs range as lew as $.50/ha. Production at a distant site could raise this price to $2.50/ha. Far greater quantities of material can be produced in naturally infested plantations than ty moving colonies of larvae to other sites. In 1984, 6 red and jack pine plantations with a combined area of 9.5 ha yielded 1.0 kg of Lecont- virus which is sufficient to treat 2,000 28 3 - OJ CO C TJ 5 D IT3 4) •M —I U = o 0) -< ■J 5 5^ 0 -I o J £ ^.g 29 ha. In 1985, 200 colonies of European pine sawfly, trcinsferred to suitably located host trees, yielded 50 g of Ser- tifervinos **u.ch is sufficient to treat 100 ha. We have only produced NPVs for con- trol of N. sertifer and N. lecontei, but these methods, could be adopted for other colonial species of DLprionid sawflies knov«i tD be susceptible to NP\fe . These include Swaine's jack pine sawfly, N. swainei, red pine sawfly, N. nanulus nanulus, balscun fir sawfly, N. abietis and the jack pine sawflies N. pratti banksianae and N, pratti paradoxicus. - 4 References Podgveite, J.; Bruen, R.B. 1978. Proce- dures for the microbiological exami- nation of production batches of the nuclear polyhedrosis virus ( Baculo- virus) of the gypsy moth, Lyrmntria dispar L. USDA Ftor. Serv. Qen. Tfech. Rep. NE 38. 8 p. Wig ley, P.J. 1980. Practical: counting micro-organisms. i^ges 29-35 in Microbial ODntrol of Insect Pests (J. Kalmakoff and J.F. iDngworth Oomp.) . N.Z. Dep. Sci. md . Res. Bull. 228. J.C. Cunningham and J.R. McPhee 1219 Queen St. East Sault Ste. Marie P.O. Box 490 Ontario, P6A 5M7 Appendix 3. Applications of Lecontvirus (Red-headed pine sawfly NPV) in Ontario by MNR, 1980-1986. Total Ha Ha Treated Application Year Treated With NPV Rate 1980 1,037 585 5 - 10 X lO' PIB/20L/ha 1981 939 492 5 - 10 X lO' PIB/20L/ha 1982 914 691 5 - 10 X lO' PIB/20L/ha 1983 200 200 5 - 10 X lO' PIB/20L/ha 1984 23 22 5 - 10 X lO' PIB/20L/ha 1985 103 103 5 - 10 X 10' PIB/20L/ha 1986 477 306 5 - 10 X lO' PIB/20L/ha 31