An ecotoxicology profile of Metarhizium anisopliae var. acridum IMI 330189 has been prepared for submission as part of registration dossiers.

A full ecotox profile including current data on host range is provided in the report 'Green Muscle Ecotoxicological Profile.'

A full analysis of trials conducted in 1996 is now available for publication (Peveling et al., 1999). Medium-scale experimental field applications of two grasshopper and locust control agents, the mycoinsecticide Metarhizium anisopliae (flavoviride) var. acridum (Deuteromycotina: Hyphomycetes) and the organophosphate fenitrothion, were carried out in August 1996 in Maïne Soroa, Niger. The objective was to evaluate effects and side-effects on target grasshoppers and epigeal non-target arthropods, using simple binary (presence/absence) sampling techniques. Plots (»  50 ha, 3 replicates) were treated at standard recommended dose rates of 5 x 10¹² spores/ha (Metarhizium) and 250 g a.i./ha (fenitrothion). The frequency of occurrence – or total presence – was used as a measure of relative population density. The fauna was monitored at 5 day intervals for 36 days (5 d before and 31 d after treatment) and again in August 1997, using 40 miniature pitfall traps per plot and sampling date. Presence/absence sampling proved appropriate to detect and quantify pesticide effects, and the use of similar techniques in rapid risk assessment programs (RAPs) for locust and grasshopper control is recommended. Both control agents were effective against grasshoppers, reflecting their inherent mode of action and speed of kill. The efficacy of fenitrothion ranged from 75% to 86% over the 31 d post-treatment period. Metarhizium had an efficacy of 76% during the last sampling period (3^rd to 4^th week). Differences between treatments were not significant during this phase. The non-target indicator species or species groups represented four insect families which together made up about 75% of the total catch: Carabidae, Tenebrionidae, Formicidae and Ephydridae. All indicators were found to be reduced by fenitrothion, but statistical significance was only provided in 75% of the cases. The median hazard was 69% (1^st week), 33% (2^nd week) and 51% (3^rd to 4^th week), respectively, and fenitrothion was classified as ‘medium risk’. Most of the non-target fauna had fully recovered after 31 days, but one ant species was still significantly reduced in 1997, one year after application. None of the indicators proved susceptible to the mycopesticide. The median hazard was < 25% and the product was classified as ‘no risk’. The field trials provided further evidence that M. anisopliae var. acridum can be as effective as organophosphates without threatening non-target arthropods other than orthopterans.

Results from the 1997 trials will appear as part of I. Stolz PhD thesis.

The 1998 trial was carried out at Dogo, Niger. Nine 50ha plots were selected, each containing half millet and half pasture. Three each were treated with Metarhizium (50g/ha), Fipronil (2g/ha) or untreated. Very low grasshopper numbers and torrential rain interfered with the smooth conduct of the experiment. Two principal studies were carried out; parasitic hymenoptera in millet (little difference was noted between the three treatments), and ground-dwelling fauna. In the latter study, in the Fipronil plots, ants were significantly reduced, and one species, Cataglyphis diehlii, was no longer found 11 days post-treatment. Two herbivorous coleopterans, the curculionid Dereodus marginellus and the chrysomelid Euryope rubra, were severely affected by fipronil. Even 5 to 6 weeks after treatment, the relative abundance of these species was only 11-16% and 1-2% of the control for E. rubra and D. marginellus, respectively, compared to levels of 143-174% and 70-100% in the Metarhizium treatment.. E. rubra was found on 13% and 22% of the Leptadenia bushes in control and Metarhizium plots, respectively, but only on 2% in the fipronil treatment. The corresponding figures for D. marginellus are 70% (control), 49% (Metarhizium), and 1% (fipronil).

An assessment of risk was prepared for South African registration dossier "Safety statement". Further work on this topic is planned as a separate research programme by the Leverhulme unit in collaboration with the university of Helsinki.

Field work was carried out in Mauritania on Acanthodactylus dumerlii in October/November1996 and 1997. In 1996, a first bioassay (inhalation exposure) with the lizard Acanthodactylus dumerlii did not reveal fungal infections of the respiratory tract, although attempts to recover spores from lungs however were spoiled by contamination of the test plates. The measurement endpoints activity, food consumption and body weight were not different among treatment groups (viable spores, deactivated spores, control).

Trials were conducted in Mauritania October/November 1997. The intended exposure route was intra-nasal exposure, but because of the viscosity of the OF-formulation, oral exposure was used. The LD50 was greater than the maximum challenge dose used in this test (10⁷ spores per g body weight as recommended by EPA).

The methods developed are being incorporated into LOCUSTOX and CILSS standards for the registration of pesticides in the Sahel.

Chemical insecticides were included as toxic standards in the field trials (fenitrothion) and in the lizard bioassays (chlorpyrifos and fipronil). Fenitrothion proved moderately harmful to most arthropods (see 4.A.3). However, the fauna recovered quickly, due to immigration into the relatively small plots (50 ha) in the 1997 trial. Chlorpyrifos was very toxic to A. dumerlii, both via the oral and the dermal route. The oral LD50 was 23.3 mg a.i./kg (95%-CL: 20.9-25.3) and the dermal LD50 was »  40 mg. Levels of brain cholinesterase inhibition in field-collected victims were comparable to those found in test animals (analysis carried out by FAO-Locustox). Further analysis showed that dermal exposure is more important than oral exposure and explains the massive kills which frequently occur in Mauritania following treatment with organophosphates. The oral LD50 of fipronil was estimated at approx. 30 mg a.i./kg. Despite the high toxicity, field mortality is unlikely to occur at current dose rates. Sub-lethal effects on lizards are currently under investigation.

Carried out during 1996 and 1997 trials. The protocols are now being incorporated into LOCUSTOX and CILSS recommendations.

The avian toxicity studies carried out by AgCan have been published (Smits et al. 1999). This study, conducted during June and July, 1997, examined the pathogenic potential of Metarhizium anisopliae to non-target avian species that encounter infected insect prey items or contaminated food sources. Pheasants were exposed to one of three diets, (spore-coated feed, infected insects, or untreated feed), either from 4 to 9 days of age, or from 35 to 40 days of age. Necropsies were conducted on birds 10 days and 46 days old. Neither consumption of infected insects, nor of spore-coated feed, resulted in pathological changes, or significant changes in weight, growth rate, behaviour, or mortality rate. Histological examination of organs indicated either no changes related to treatment, or normal tissue responses to antigenic challenge.

Tests on two aquatic invertebrates, Streptocephalus (fairy shrimp)and Anisops were carried out by LOCUSTOX (Report; 97/1 Marquenie et al). Registration tests to GLP standards on Daphnia and earthworms were contracted to NPP and indicated no effect.

Further collaborative work with LOCUSTOX is planned on development of mammalian safety testing protocols in Africa, and the development of biopesticide regulation guidelines.

Mammalian toxicity studies were carried out under contract early 1997; a ‘toxicity profile’ summarising these studies has been prepared by an independent consultant as part of the registration dossier.

Both BPC and NPP have agreed to respect LUBILOSA's gender policy, and conform to extant health and safety regulations in their respective countries (South Africa and France).