Palmer_299-314.indd INTRODUCTION Outbreaks of pest blackflies (Diptera: Simuliidae) have been one of the most serious problems affect- ing agriculture and tourism along the middle and lower Orange River following the completion of the Gariep and Vanderkloof Dams in the late 1970s (Palmer 1997; Myburgh & Nevill 2003). The blackfly problem is attributed mainly to high winter flows, which provide suitable habitat for over-wintering blackfly larvae. The main pest species is Simulium chutteri, although there are times when Simulium damnosum and Simulium impukane are problem- atic. Adult female blackflies usually need a blood meal to complete the development of eggs, and their large numbers and tendency to crawl into ears, noses and eyes, make them problematic to livestock and people. All outdoor activities are seriously af- fected, particularly stock farming, irrigation farming, river rafting and other tourist activities. Chemical control of pest blackfly in South Africa be- gan in the 1960s with the use of DDT, a wide-spec- trum larvicide (Howell & Holmes 1969). In 1991 the 299 Onderstepoort Journal of Veterinary Research, 75:299–314 (2008) Evaluation of larvicides in developing management guidelines for long-term control of pest blackflies (Diptera: Simuliidae) along the Orange River, South Africa R.W. PALMER1* and N.A. RIVERS-MOORE2 ABSTRACT PALMER, R.W. & RIVERS-MOORE, N.A. 2008. Evaluation of larvicides in developing management guidelines for long-term control of pest blackflies (Diptera: Simuliidae) along the Orange River. Onder- stepoort Journal of Veterinary Research, 75:299–314 In 2000 and 2001 Orange River levels were higher than normal: associated serious outbreaks of blackfly had a substantial detrimental impact on the local economy. The poor control was attributed to the suspected development of larval resistance to temephos. A long-term solution to blackfly control, through the identification of a suitable replacement to temephos for use during high flow conditions, was proposed. This study, however, failed to identify or register a suitable larvicide for use during high flow conditions. Although permethrin was highly effective against blackfly larvae, it was rejected be- cause of its detrimental impacts on non-target fauna. Various formulations of locally produced dry Bacillus thuringiensis var. israelensis (B.t.i.) were tested, but these were ineffective against blackflies. The study also confirmed that resistance to temephos has developed among Simulium chutteri in the middle and lower Orange River. The feasibility of “reversing” the resistance to temephos through the use of the synergist piperonyl butoxide (PBO) was investigated, but the results were not favourable. Furthermore, PBO was highly toxic to blackflies and non-target organisms, and was not recommend- ed for further testing. This means that B.t.i. currently remains the only symptomatic measure of treat- ment currently applied. Although resistance to B.t.i. has not been reported for blackflies elsewhere in South Africa, there is a need to remain vigilant and to implement an operational strategy that mini- mizes the risks of resistance developing. Keywords: Larvicide trials, Orange River, pest blackflies, resistance, Simuliidae ∗ Author to whom correspondence is to be directed. E-mail: rob@nepid.co.za 1 Nepid Consultants, P.O. Box 4349, White River, 1240 South Africa 2 Institute for Water Research, P.O. Box 94, Rhodes University, Grahamstown, 6140 South Africa Accepted for publication 14 August 2008—Editor 300 Larvicides in developing management guidelines for control of pest blackfl ies (Diptera: Simuliidae), South Africa Department of Agriculture initiated a programme to control these pests along the middle and lower Orange River (Palmer 1997). The programme was initially based on aerial applications of two larvi- cides, namely temephos, an organophosphate, with Abate® 200EC being the only product registered for use in South Africa, and the bacterium Bacillus thur- ingiensis var. israelensis (B.t.i.), with Teknar® HP-D and Vectobac® 12AS being two products registered for use in South Africa. The bacterial larvicides, which contain protein toxins produced by the naturally occurring soil bacterium B.t.i. are generally the preferred method of blackfly control because of their high target specificity and low impact on the environment. However, these products are relatively bulky and are suitable for use when flows in the Orange River are moderate to low (< 100 m3/s). At river levels exceeding 300 m3/s it becomes difficult to airlift the volumes needed to treat the Orange River effectively. Under these con- ditions it is preferable to use Abate® 200EC, an or- ganophosphate that is more concentrated and eas- ier to airlift than B.t.i. Between 1991 and 1999 the control programme was supported by research conducted by the Onderste- poort Veterinary Institute with funding from the Water Research Commission and the Red Meat Producers Organization. The research aimed to ensure that the control programme was both effective and envi- ronmentally safe (Palmer 1997; Myburgh 1999). The con trol programme was highly effective during this period, but research was discontinued in 1999. Serious outbreaks of blackflies were experienced in 2000 and 2001 as a result of river levels being high- er than normal, and this led to appeals from organ- ized agriculture for the problem to be rectified, and for a long-term solution be found. The aim of this study was to find a suitable larvicide for use against blackflies in the Orange River during high flow con- ditions. The findings of the dispersal properties of various products and their toxicity to blackfly larvae and common non-target organisms, using flow- through gutters and field trials are described. MATERIALS AND METHODS Study area The main study area for this project was the Orange River downstream of Vanderkloof Dam (Fig. 1). The Great Fish River near Grahamstown was also used to conduct trails to investigate larval resistance to temephos, as the river contains populations of the main pest species, S. chutteri, which had not previ- ously been exposed to temephos. Alternative larvi- cides were tested in the Orange River at Upington, and in various rivers near Grahamstown, in the Eastern Cape Province. The latter were chosen be- cause of the availability of mean daily flow data, and their small size which reduced the volume of larvi- cide needed. Criteria for evaluation The criteria used to evaluate the larvicides were based on a hypothetical ideal larvicide which should have the following characteristics: • Effective against blackflies and target specific • Easy to airlift and apply • Disperses rapidly and evenly in water FIG. 1 Map of the middle and lower Orange River downstream of Van der Kloof Dam (formerly PK le Roux Dam), with the main blackfly problem area highlighted in grey 301 R.W. PALMER & N.A. RIVERS-MOORE • Carries for long distances downstream • Good shelf life • Cheap • Safe to handle • Without problems of cross resistance with teme- phos. Larvicides selected for evaluation This study was limited to investigating currently avail- able commercial products for blackfly control, as it was well beyond its budget and scope to investigate new products for development. The study relied largely on the experience of the World Health Or- gan ization’s Onchocerciasis Control Programme in West Africa (OCP). The OCP has tested over 50 lar- vicides and hundreds of formulations for use against pest blackflies since it inception in 1974 (Kur tak, Back, Chalifour, Doannio, Dossou-Yove, Duval, Guil- let, Meyer, Ocran & Wahle 1989). Most of the tests were conducted during the early phases of the OCP. The OCP was discontinued in 2002 (Lévêque et al. 2003), and since then very few investigations of new products for blackfly control have been under- taken. The larvicides that were most commonly used by the OCP are listed in Table 1. This provided a good starting point for identifying a suitable larvi- cide for use in the Orange River. The use of alterna- tive organophosphates was not considered because of the possible cross-resistance with temephos, while the use of carbamates was not considered be- cause of the high impacts on non-target fauna. The most suitable candidate was therefore one of the synthetic pyrethroids, a group of powerful broad- spectrum insecticides (Mueller-Beilschmidt 1990) which act as neurotoxins. The two pyrethroid com- pounds that were commonly used in West Africa were permethrin and etofenprox. Although permeth- rin is more toxic to non-target aquatic fauna than etofenprox, it has a lower mammalian toxicity. Per- methrin is particularly suited for large rivers and was considered to be the most suitable candidate larvi- cide for testing in South Africa. While synthetic pyre- throids generally degrade to varying degree by ex- posure to light, permethrin is a newer, light-stable pyrethroid (Mueller-Beilschmidt 1990). Several agrochemical companies were contacted for samples of permethrin, or similar products to re- place temephos, for trial purposes. The only com- pany to express an interest in supplying alternative products to temephos was Wefco Marketing. The other companies approached indicated that they were not prepared to take the risk, mainly because of legal implications and public concerns about en- vironmental safety. Wefco Marketing suggested us- ing piperonyl butoxide (PBO) in combination with temephos, as piperonyl butoxide combined with te- mephos or permethrin has the ability to “reverse” the TABLE 1 Main characteristics of the blackfly larvicides used by Onchocerciasis Control Programme in West Africa. [Data from Dr Jean-Marc Hougard, Institut de Recherche pour le Développement, France] Family Organophosphates Pyrethroids Carbamate Bacteria Common name Temephos Phoxim1 Pyraclofos Permethrin Etofenprox Carbosulfan B.t. H-14 Formulation EC2 EC EC EC EC EC WD3 % active ingredient 20 50 50 20 30 25 < 2 Toxicity class4 III II II II III II III Toxicity against NTF5 Low Medium Medium High Medium High Low Dose6 1507 150 120 45 60 120 500 Average carry at (km) 10 m3/s 12 3 Unused Unused Unused Unused 1.5 100 m3/s 16 5 18 7 6 9 5 300 m3/s 20 Unused 23 8 8 Unused Unused 1 Chlorphoxim until 1991 2 Emulsion concentrate 3 Water dispersible 4 According to the WHO (1988) classification of active ingredient: II, moderately hazardous; III, slightly hazardous 5 Toxicity against non-target fauna according to the criteria of the Ecological Group 6 In mℓ of formulation per m3/s 7 300 mℓ in clear water 302 Larvicides in developing management guidelines for control of pest blackfl ies (Diptera: Simuliidae), South Africa development of resistance (Jones 1998). Larvicide synergy is a useful approach since the combined exposure of two or more larvicides causes more ad- verse effects than the sum of their individual effects (Cox 1998). This opened the possibility of continu- ing with the operational use of temephos, but trials were needed on blackflies from non-resistant pop- ulations, such as the Great Fish River, as well as on presumed resistant populations in the Orange River. Wefco Marketing supplied small quantities of four formulations of permethrin and three formulations of PBO for initial trials, while BASF supplied small quantity of Abate® 200EC. Another possible option was the use of a powdered formulation of B.t.i., which would be lighter and there- fore more easily air lifted than the standard liquid concentrate, particularly during high flow conditions. Although the operational application of powdered formulations of B.t.i. could be a problem because of wind and equipment available, the powder could be mixed with water prior to application. Plant Health Products supplied small quantities of two locally pro- duced powdered and granular B.t.i. formulations for initial testing, which were toxic to mosquitoes (M. Mor ris, personal communication 2005). The products and formulations tested during this study are listed in Table 2. Dispersal properties The dispersal properties of the various products were observed by mixing the product with various volumes of water and applying this mixture to a jar of standing clear water. This simple test provided a rapid visual indication of how the product is likely to behave when applied in a river and was used as the first step in screening potential larvicides. For m u la- tions that were buoyant or sank to the bottom were immediately rejected. Viscosity Viscosity of B.t.i. products can be a serious prob- lem, so the relative viscosity of the B.t.i. was meas- ured by filling a small (125 mℓ) cup containing a small hole (approx. 3 mm in diameter), and timing the cup to empty. This was performed four times for each of two B.t.i. concentrations (8 and 24 g/ℓ) plus a control of 0 g/ℓ. Gutter trials Gutter trials were conducted in the Great Fish River at the Pigott’s Bridge gauging weir (Q9H012), 40 km from Grahamstown (33°05’53.5” S; 26°26’42.2” E). This site was chosen because of accessibility, the weir provided sufficient head that allowed water to be gravitated into the gutters, and there were high populations of S. chutteri within close proximity to the trial site. The trials were conducted using a flow- through four-gutter system in which river water gravitated from the gauging weir. The gutters were 3.45 m long and 0.15 m wide and were made of a galvanized tin alloy (Fig. 2). Blackfly larvae were ob- tained from the main river (5 min walk away) by cut- ting lengths of Cyperus reeds which were trailing in fast current. Twenty reeds were placed in each gut- ter and wedged in position using 0.20 m lengths of dowels. Larvae were given at least 1.5 h to settle TABLE 2 Products and formulations tested during this study for potential use for blackfly control Active ingredient Class Formulation Suppliers B.t.i. Bacterial toxin Dry powder 500:5001 Dry powder 500:2502 Slow-release granules Plant Health Products Permethrin Pyrethroid Larvex™ 0.5 % w/v RD 95/A 200 g/ℓ RD 95/B 200 g/ℓ Permethrin (unlabelled) Wefco Marketing Piperonyl butoxide Synergist RD 96A RD 96/B PBO (unlabelled) Wefco Marketing Temephos Organophosphate Abate® 200EC SA Cyanamid BASF Wefco Marketing 1 Formulation consists of 500 g B.t.i. fermentation broth plus 500 g carrier, mixed and dried down 2 Formulation is twice as concentrated as the 500:500 dry powder, and consists of 500 g B.t.i. fermentation broth plus 250 g carrier, mixed and dried down 303 R.W. PALMER & N.A. RIVERS-MOORE before exposure to chemical larvicides, whereas for B.t.i. trials larvae were given an 1.5 h to settle. The only species of blackfly that was present was S. chutteri. Flow in each gutter was determined prior to each application by holding a container (11.8 ℓ) at the exit of each channel and timing it to fill. Larvicide was applied over 10 min in the header chamber at the top end of each gutter to ensure homogenous mix- ing, using a 60 mℓ syringe. Water temperature at the time of each application was recorded. Larval mor- tality was assessed 1 h after application for chemi- cal products and 24 h for B.t.i. products. The as- sessment was based on the abundance of live larvae in an untreated control gutter and compared with the abundance of live larvae in treated gutters. Abundance of larvae was based on a 10-point visu- al ranking method described by Palmer (1994). The abundance ratings were converted to population densities to determine efficacy, although this meth- od is unlikely to detect mortalities less than 50 %. At least 15 counts were made per gutter. Field trials Belmont Valley Field trials were undertaken at two sites in the Bel- mont Valley near Grahamstown (Site 1: 33°19’25.2” S; 26°36’00.8” E; Site 2: 33°19’21.4” S; 26°36’49.7” E). The stream was chosen because of its small size, close proximity to Grahamstown and high populations of blackflies. A road bridge crosses the stream and culverts were used to measure the stream flow using Equations 1a–c (Gordon et al. 1992). Q = 1000vA [1a] A = 0.5r2(θ–Sinθ) [1b] θ = 2 cos–1(r – d) [1c] r where Q = discharge in ℓ/s; v = average current speed in m/s; A = cross-sectional area in m2; θ is in radians. The only species of blackfly that was present at Site 1 was the pest species Simulium nigritarse, but Si- mulium adersi was also present further downstream at Site 2. Larvicide was applied with a 1 ℓ hand-held garden sprayer over a period of 8 min. Water tem- perature at the time of the application was recorded. Larval mortality was assessed 3 h after application for chemical trials and 24 h after application for B.t.i. trials. The assessment of efficacy was based on the abundance of live larvae on the stones-in-current in an untreated stretch of stream compared to the abundance of live larvae in the treated section, be- fore and after application. Abundance of larvae and assessment of efficacy was based on the same method as used for the gutter trials, and sample size was also at least 15 stones or reeds. Buffalo River A field trial was undertaken in Eastern Cape Province in the Buffalo River at a gauging weir near King Will- i am’s Town (R2H010 – 32°56’26.5” S; 27°27’41.3” E). The site was chosen because of the high populations of blackflies and close proximity to an accurate gauging weir. The most common species of blackfly that was present was Simulium hargreavesi, but other species present were Simulium vorax, S. nigri- tarse and S. damnosum. Larvicide was again applied with a 1 ℓ hand-held garden sprayer over a period of 8 min. Water temperature at the time of the applica- tion was recorded. Larval mortality was assessed 3 h after application. The assessment of efficacy was based on the abundance of live larvae in the stones- in-current in before and after application. Abundance of larvae was based on the same method as used in the gutter trials. At least 15 counts were made. Orange River Field trials were conducted in the Orange River at Upington and Kanoneiland in August 2005 and October 2006. For the earlier trials, the same meth- ods as those described above were used. The most common species present was S. chutteri, although S. damnosum was also present. In the latter field trials, two formulations of Abate® 200EC were ap- plied by boat, one to each of two channels on either side of the Orange River at Kanoneiland. FIG. 2 Flow-through gutter system used for larvicide trials on S. chutteri larvae 304 Larvicides in developing management guidelines for control of pest blackfl ies (Diptera: Simuliidae), South Africa Impacts on non-target organisms Impact of permethrin on non-target organisms was evaluated during selected field trials. Population densities of aquatic invertebrates were estimated before and after application using the visual method of assessment developed for estimating blackfly pop ulations (Palmer 1994). RESULTS Dispersal properties Initial screening of the physical behaviour of the products in water showed that permethrin and pip- eronyl butoxide formulations dispersed well. Of the formulations tested, permethrin RD95B dispersed TABLE 3 Viscosity (mℓ/s) for tap water and two concentrations of B.t.i. (500: 250) Sample Flow time (s): 0 g/ℓ (125 mℓ tap water) Flow time (s): 8 g/ℓ (1 g in 125 mℓ) Flow time (s): 24 g/ℓ (3 g in 125 mℓ) 1 2 3 4 37.9 38.5 36.8 38.5 39.4 37.3 37.5 36.8 38.1 37.9 37.6 37.4 Mean 37.9 37.8 37.8 Flow rate (mℓ/s) 3.3 3.3 3.3 TABLE 4 Conditions of application and results of flow through gutter trials to test the toxicity of permethrin RD 95/B to blackfly larvae at Pigott’s Bridge, Great Fish River Trial no. Date Water temp. (°C) Gutter channel Flow (ℓ/s) Dosage (mg/ℓ) Efficacy (%) 1 05.06.05 11.5 1 2 3 4 1.23 1.07 1.28 1.01 0.01626 0.03738 0.04688 0.09901 98 99 99 100 2 05.06.05 11.5 1 2 3 4 1.76 1.64 1.60 1.54 0.00201 0.00366 0.00833 0.01732 62 74 94 99 TABLE 5 Conditions of application and results of field trials to test the toxicity of permethrin RD95B to blackfly larvae Trial no. Date Site Water temp. (°C) Flow (ℓ/s) Dosage (mg/ℓ) Efficacy (%) 1 03.05.05 Belmont Valley Site 1 11.5 69 0* 0.024 0.048 0 0 63 2 03.06.05 Belmont Valley Site 2 12.0 69 0* 0.024 0.048 0.072 0 25 88 88 3 06.06.05 Buffalo River 12.0 279 0.050 99 4 07.06.05 Belmont Valley Site 1 11.0 91 0 0.02 0 82 5 08.06.05 Belmont Valley Site 1 10.0 70 0 0.040 0 83 6 08.06.05 Belmont Valley Site 2 10.2 70 0 0.030 0 56 * Control 305 R.W. PALMER & N.A. RIVERS-MOORE the best by far. The Abate® 200EC dispersed well. The density of Larvex™ 0.5 % w/v was very low and floated on the surface, so this product was excluded from further testing. The standard formulation of powdered B.t.i. mixed well in water, but the more concentrated formulation was dense and settled rapidly when applied to water. Viscosity The powdered formulation of B.t.i. mixed well with small quantities of water, but the concentrated for- mulation formed sticky lumps when mixed in larger volumes. The viscosity of the supernatant was low and not significantly different from that of tap water (Student’s t-test; P < 0.05; d.f. = 3) at concentrations of 8 and 24 g/ℓ (Table 3). By contrast, the viscosity of the lumps of the concentrated formulation was exceedingly high. To undertake the viscosity trials the formulation was dissolved in a separate beaker prior to draining through a cup so as to prevent the drainage hole from being blocked with lumps. Larvicide trials PERMETHRIN Gutter trials Gutter trials conducted at Pigott’s Bridge confirmed that permethrin RD95B is highly effective against S. chutteri at dosages as low as 0.016 mg/ℓ (Table 4). River trials River trials conducted at two sites in the Belmont Valley indicated that permethrin achieved between 63 % and 88 % mortality of S. nigritarse at a dosage of 0.0483 mg/ℓ (Table 5). The trials assumed that flows at the two sites were the same, but the results suggest that flows at the downstream site were slightly lower, and therefore dosages higher, than upstream. A subsequent trial in the Buffalo River achieved 99 % mortality at a slightly higher dosage of 0.0502 mg/ℓ (Table 5). Mortality curves for Simulium using permethrin RD95B were constructed for both gutter trial and river trial experiments. The mortality curve for the gutter trials was significant (P < 0.05; d.f. = 8; inter- cept = 9.80 ± 0.75; slope = 1.50 ± 0.35), while the mortality curve for the river trials was not significant (P < 0.05; d.f. = 5; intercept = 21.83 ± 3.64; slope = 11.38 ± 2.47). The LC50 values for gutter trials (0.001 mg/ℓ) were, however, 30 times lower than for the river trials (0.033 mg/ℓ), suggesting that the ef- fects of higher flow rates may have an impact on effective concentrations of permethrin (Fig. 3). Sim- ilar findings were made in West Africa, and the re- searcher recommended the use of a closed-circuit trough system for screening conventional larvicides for blackfly control (Ocran 1989). Impacts on non-target organisms A series of trials were conducted to assess the tar- get specificity of permethrin in local rivers. Initial tri- als were conducted in the Bloukrans River near Grahamstown, and the Buffalo River near King Wil- liams Town, because of their small size, ease of lo- gistics and ability to measure stream flows. Invertebrate diversity in the Bloukrans River was low prior to a trial application of permethrin in June 2005. Individuals of the caddisfly Macrostemum ca- pense and Gyrinidae beetles were absent from these aquatic invertebrate communities, while Chironomids were present in low numbers only. The aquatic in- vertebrate communities were largely unaffected by applications of permethrin at concentrations of 0.02, 0.03 and 0.04 mg/ℓ (Tables 6A and B). Invertebrates which were affected negatively by the application of permethrin were limpets (Ancylidae) and flatworms (Turbellaria). The diversity of invertebrates in the Buffalo River, near King Williams Town, was very low prior to appli cation because the site is highly polluted from domestic and industrial wastes. This meant that the fauna that were present were highly tolerant spe- cies. Despite this, the application of permethrin RD95B at a concentration if 0.05 mg/ℓ had major impacts on non-target organisms, particularly water boatmen and mayflies, followed by caddisflies and FIG. 3 Mortality curves for Simulium spp. based on applica- tions of permethrin RD95B at different concentrations, for gutter and river trials 306 Larvicides in developing management guidelines for control of pest blackfl ies (Diptera: Simuliidae), South Africa flatworms (Table 6C). The only taxon that appeared to be unaffected was non-biting midges. The num- bers of non-biting midges appeared to increase af- ter application, but this was probably because they were small and must have been largely overlooked prior to application, whereas after application they were about the only fauna left alive, so they were more visible. The intention was to use permethrin for blackfly control in the Orange River, so a trial was conducted to investigate its impacts on non-target fauna in the Orange River. Permethrin was applied at a concen- tration of 0.1 ppm from a road bridge to the left channel of the river at the Kanoneiland. The compo- sition of aquatic invertebrates was assessed before and after application at Blaauwskop, 5.4 km down- stream of the bridge. The application had a devas- tating impact on non-target fauna: 100 % mortality was recorded for various families of mayflies, dam- selflies, water boatmen and hydroptilid caddisflies (Table 6D). Prior to application the population of blackfly larvae was very high, but very few (3 %) survived the application. Other taxa that were detri- mentally affected included non-biting midges, lim- pets and two species of caddisfly that are important TABLE 6A Relative abundance of invertebrate taxa in the Bloukrans River (Belmont Valley Site 2) before and after application of permethrin at 0.03 mg/ℓ Taxa Application Rating Efficacy (%) Turbellaria (Flatworm) Before After 221412121432211 111111112132124 99 Ancylidae (Limpets) Before After 221212212212121 111212111111112 99 Cheumatopsyche afra (Caddisfly) Before After 322333131341122 214122323211233 0 Chironomidae (non-biting Midge) Before After 111111111111111 111111111111111 0 Macrostemum capense (Caddisfly) Before After 111111111111111 111111111111111 0 Baetidae (Mayfly) Before After 232212422322223 422234322422223 0 Gyrinidae (Water Boatmen) Before After 111111111111111 111111111111111 0 TABLE 6B Relative abundance of invertebrate taxa in the Bloukrans River (Belmont Valley Site 1) before and after application of permethrin at 0.04 mg/ℓ Taxa Application Rating Efficacy (%) Chironomidae (non-biting midge) Before After 111111111111111 211111111111111 n/a Ancylidae (Limpets) Before After 133322221222232 123111322122222 0 Cheumatopsyche afra (Caddisfly) Before After 121211112221232 121211112121212 0 Turbellaria (Flatworm) Before After 221232222232222 222211112112221 0 Macrostemum capense (Caddisfly) Before After 111111111111111 111111111111111 0 Baetidae (Mayfly) Before After 112324222224232 212232222222332 0 Gyrinidae (Water boatmen) Before After 111111111111111 111111111111111 0 307 R.W. PALMER & N.A. RIVERS-MOORE TABLE 6C Relative abundance of invertebrate taxa in the Buffalo River before and after application of permethrin at 0.05 mg/ℓ Taxa Application Rating Efficacy (%) Gyrinidae (Water Boatmen) Before After 211212222222222 111111111111111 100 Baetidae (Mayfly) Before After 322323434433353 111211111111111 99 Macrostemum capense (Caddisfly) Before After 321221211411112 111211111112111 89 Turbellaria (Flatworm) Before After 221231212462132 111213211212124 74 Cheumatopsyche afra (Caddisfly) Before After 333433334443443 212321131223232 69 Ancylidae (Limpets) Before After 324112222222323 212214122122122 37 Chironomidae (non-biting Midge) Before After 221121221222213 222232232223222 0 TABLE 6D Relative abundance of invertebrate taxa in the Orange River River before and after application of permethrin at 0.10 mg/ℓ at Kanoleiland, left channel, 5.4 km upstream, at discharge of 12.92 m3/s, on 14/08/2005 Taxa Application Rating Efficacy (%) Baetidae (Mayfly) Before After 111224123231112 111111111111111 100 Heptageniidae (Mayfly) Before After 112331112134111 111111111111111 100 Leptophlebiidae (Mayfly) Before After 113411113114121 111111111111111 100 Gyrinidae (Water Boatmen) Before After 211112221111121 111111111111111 100 Coenagrionidae (Damselfly) Before After 112121111111111 111111111111111 100 Hydroptilidae (Caddisfly) Before After 112211111121111 111111111111111 100 Simuliidae (Blackfly) Before After 81567767716677(10) 502513143331121 97 Chironomidae (non-biting Midge) Before After 89688818877188(10) 664555555177777 82 Ancylidae (Limpets) Before After 111241323231122 112111311111111 80 Amphipsyche scottae (Caddisfly) Before After 331132241531312 221113211112122 63 Cheumatopsyche thomasseti (Caddisfly) Before After 231121233331322 321311213221221 35 Turbellaria (Flatworm) Before After 113111235311225 203411113451111 18 Ecnomidae (Caddisfly) Before After 111111111121211 121112111111111 n/a Elmidae (Beetle) Before After 211111111111121 111111121111111 n/a 308 Larvicides in developing management guidelines for control of pest blackfl ies (Diptera: Simuliidae), South Africa predators of blackflies, Cheumatopsyche thomas- seti and Amphipsyche scottae. The results were conclusive evidence that permethrin is unsuitable for use in the Orange River because of its detrimen- tal impact on non-target fauna. Temephos and piperonyl butoxide Gutter trials Gutter trials using the Abate® 200EC formulation of temephos showed that the product was effective against S. chutteri in the Great Fish River, but inef- fective in the Orange River (Table 7). Gutter trials conducted at Pigott’s Bridge, Great Fish River, indicated that piperonyl butoxide RD95B is highly effective against S. chutteri at dosages as low as 0.01 mg/ℓ (Table 8). However, temephos (Abate® 200EC) combined with PBO in various combina- tions showed no mortality (Table 8). Field trials Two field trials using Abate® 200EC (obtained from Wefco Marketing) were conducted in the Great Fish River, on 13 December 2005, one at Coniston and one at Carlisle Bridge. These trials were undertaken TABLE 7 Conditions of application and results of flow through gutter trials to test the toxicity of various formulations of temephos to blackfly larvae Trial no. Date Water temp. (°C) Gutter channel Flow (ℓ/s) Dosage (mg/ℓ) Efficacy (%) Abate® 200EC (Great Fish River) 2* 19.07.05 12.5 1 2 3 4 0.97 1.23 1.31 1.29 0 0.010 0.050 0.100 0 0 0 50 3*** 12.12.05 22.0 1 2 3 4 0.53 0.68 0.81 0.81 0 0.050 0.100 0.300 0 40 77 90 4*** 13.12.05 25.0 1 2 3 – 1.14 0.97 0 0.080 0.150 0 22 68 5*** 14.12.05 27.0 1 2 3 1.08 1.11 1.04 0 0.05 0.50 0 65 92 Abate® 200EC (Orange River) 6** 02.10.06 20 1 2 3 4 1.89 1.74 1.82 1.56 0.000 0.050 0.100 0.500 0 0 0 0 7*** 03.10.06 17 1 2 3 4 1.73 Dry 1.72 1.67 0.050 0 0.100 0.500 0 – 0 0 8** 03.10.06 18 1 2 3 4 1.67 1.78 Dry 1.71 1.000 5.000 0 12.000 0 0 0 0 9*** 04.10.06 19 1 2 3 4 1.68 1.85 1.85 1.91 5.000 10.00 20.00 30.00 0 0 0 0 * Old product supplied by SACyanamid ** New product supplied by BASF *** New product supplied by Wefco Marketing 309 R.W. PALMER & N.A. RIVERS-MOORE to confirm the results of the gutter trials, that teme- phos is effective in the Great Fish River. Larval numbers prior to application were assessed at 0.2, 0.5, 1.0 and 4 km downstream of Carlisle Bridge, and 5.5, 6.8 and 10.8 km downstream of Coniston. Larval numbers were consistently high at all sites (rating between 9 and 10). Water temperature at the time of application was moderate (20 °C), and the water was turbid (Secchi depth 7 cm). The flow at the Pigott’s Bridge gauge was estimated at 5.2 m3/s, whereas the flow at Carlisle Bridge was assessed at 4.5 m3/s. Larvicide was applied at a concentration of 0.1 ppm at Coniston and 0.05 ppm at Carlisle Bridge. Larval counts conducted the following day indicated highly successful control at both concen- trations. The 0.05 ppm application from Carlisle Bridge achieved 97 % blackfly mortality at 4 km downstream. The 0.1 ppm application achieved 99 % mortality at Cranford, 10.8 km downstream of the point of application, and larval counts at the farm Mowbray, 17.7 km downstream, indicated mortality of 45 %. The results of these trials confirmed that the Abate® 200EC formulation was highly effective against blackflies in the Great Fish River. The next step was to conduct a similar test in the Orange River. A field trial using Abate® 200EC was conducted in the Orange River at the top end of Kanoneiland on 4 October 2006. Flows in the two channels on either side of Kanoneiland were esti- mated at 62 and 26 m3/s for the right and left chan- nels, respectively. Larval numbers prior to applica- tion were very high (rating of 10). Two formulations of Abate® 200EC were applied by boat, both at 0.1 ppm. The left channel received 8 ℓ of Abate® 200EC, obtained from Wefco Marketing, and the right channel received 18.5 ℓ of Abate® 200EC from BASF. Larval counts conducted the following day at the road bridge, 7 km downstream of the point of application, showed no indication of mortality. The results of these trials confirmed that larval resist- ance to temephos has developed in the Orange River. Powdered and granular B.t.i. Preliminary results from the orbital shaking table indicated that the powdered formulation of B.t.i. (500:500) is toxic to S. chutteri, but results were in- TABLE 8 Conditions of application and results of flow through gutter trials to test the toxicity of Piperonyl Butoxide combined with temephos (Abate® 200EC) Trial no. Date Water temp. (°C) Gutter channel Flow (ℓ/s) Dosage (mg/ℓ) Efficacy (%) PBO only (Great Fish River) 1 09.06.05 10.0 1 2 3 4 1.83 1.72 1.52 1.72 0 0.01 0.05 0.10 0 82 97 99 PBO plus temephos (Great Fish River) 20.07.05 13.2 1 2 3 4 1.60 1.61 1.34 1.66 0 0.01 ppm (80 % temephos; 20 % PBO) 0.05 ppm (80 % temephos; 20 % PBO) 0.1 ppm (80 % temephos; 20 % PBO) 0 0 0 0 PBO plus temephos (Orange River) 1 15.08.05 14.6 1 2 3 4 1.84 1.76 1.91 2.08 100 % PBO (0.5 ppm PBO only) 80 % PBO; 20 % temephos (0.5 ppm) 20 % PBO; 80 % temephos (0.5 ppm) 0 % PBO (0.5 ppm temephos only) 0 0 2 5 2 15.08.05 14.6 1 2 3 4 1.84 1.75 1.91 2.08 100 % PBO (0.1 ppm PBO only) 80 % PBO; 20 % temephos (0.1 ppm) 20 % PBO; 80 % temephos (0.1 ppm) 0 % PBO (0.1 ppm temephos only) 0 0 0 0 2* 04.10.06 18.0 1 2 3 4 1.68 1.85 1.85 1.91 10 % PBO (0.019 PBO + 0.2 temephos) 20 % PBO (0.038 PBO + 0.2 temephos) 50 % PBO (0.095 PBO + 0.2 temephos) 50 % PBO (0.095 PBO only) 0 0 0 0 * PBO was applied first and temephos was applied 2 h later 310 Larvicides in developing management guidelines for control of pest blackfl ies (Diptera: Simuliidae), South Africa conclusive. A mortality of 98 % was obtained for lar- vae exposed for 1 h at a dosage of 2 mg/ℓ, com- pared to 78 % mortality at half the dosage (Table 9). However, it took a long time to record the results and by the time the control group was counted, 3 h had passed and mortality was 86 %. Clearly, the closed- system shaking table is unsuitable for trials exceed- ing 1 h in duration. Gutter trials Gutter trials conducted at Pigott’s Bridge, Great Fish River, indicated that the powdered formulation of B.t.i. (500:250) is ineffective against S. chutteri, even at dosages of 19.38 mg/ℓ (Table 10). A possi- ble source of error was that walking past the gutters sometimes cast a shadow on the gutters and this would temporarily stop larvae from feeding. If this occurred during larvicide application, mortality would be reduced. However, this did not occur and larvae were feeding during the time of application, so the poor results could not have been caused by a lack of larvicide ingestion. Flow volumes in the gutter tri- als ranged between 0.75 and 0.91 ℓ/s and this cre- ated average current speeds of between 0.5 and 0.8 m/s, which is within the flow preference for S. chutteri. The poor results are therefore considered to be un- related to inadequate experimental design, and were probably caused by inadequate toxicity. The formation of sticky lumps which remained behind in the syringe, could partly explain the poor results at lower concentrations, but this formulation problem is unlikely to have affected the results at higher con- centrations. Gutter trials conducted at Upington on the Orange River using granular B.t.i. also showed that this formulation was ineffective against S. chut- teri (Table 10). River trials A field trial conducted in Belmont Valley, Grahams- town, on 4 June 2005 indicated that the powdered formulation of B.t.i. (500:250) is ineffective against S. nigritarse at a dosage of 4.57 mg/ℓ (water tem- perature 11.5 °C; flow 62 ℓ/s; 170 g applied; and 0 % mortality). Higher dosages were not undertaken be- cause such concentrations would be impractical for aerial application, even if the product is applied as a dry powder. A possible reason for the poor efficacy was that the active ingredient could have settled in the sticky mass at the base of the sprayer. However, it is unlikely that all active ingredient remained in the sprayer. TABLE 9 Conditions of application and results of orbital shaking table to test the toxicity of Bacillus thuringiensis var. israelensis (500:500) to blackfly larvae at Upington Date Water temp. (°C) Container no. Dosage (mg/ℓ) No. dead No. alive % dead 07.09.04 Not recorded 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0* 0.000000 0.000001 0.000002 0.000004 0.000008 0.000015 0.000031 0.000061 0.000122 0.000244 0.000488 0.000977 0.001953 0.003906 0.007813 0.015625 0.031250 0.062500 0.125000 0.250000 0.500000 1.000000 2.000000 38 26 23 14 30 23 28 29 8 24 15 21 12 30 23 18 47 34 39 30 48 26 41 56 6 10 5 8 12 15 14 10 40 4 22 37 13 36 34 26 29 26 31 11 11 4 11 1 86 72 82 64 72 61 67 74 17 86 40 36 48 45 40 41 62 57 56 73 81 87 78 98 * Control 311 R.W. PALMER & N.A. RIVERS-MOORE DISCUSSION Permethrin The results of this study indicate that the permethrin formulation RD95B is highly effective against black- flies at a concentration of 0.043 mg/ℓ, and was by far the most promising larvicide tested. However, the chemical has two problems, namely potential for re- sistance and impacts on the environment. Resistance to permethrin has been reported for a wide variety of insects and cross-resistance to a range of synthetic pyrethroids has been reported (Cox 1998). The use of permethrin, or any addition- al replacement larvicide for temephos, in the Orange River Blackfly Control Programme would have to be used within a careful management framework to avoid resistance developing again. Permethrin decomposes rapidly in water and, while it is known to be highly toxic to aquatic organisms, including fish, it is relatively safe to people. Muirhead- Thomson (1977) noted that permethrin was 40 times more toxic than Abate®, but raised concerns on its effects on non-target organisms. Wide spectrum lar- vicides may result in the undesirable eradication of most aquatic invertebrates and a change in ecosys- tem equilibrium and functioning. Kreutzweiser & Kings bury (1987) noted that river systems took 1–18 months to recover from applications of permethrin. Impacts on non-target organisms may often be de- tected through increases in the density of drifting invertebrates. Kreutzweiser & Kingsbury (1987) re- ported a major increase in downstream drift of non- target organisms following an application of per- methrin in forest streams in Canada. Such impacts would be exacerbated through further exposure to aquatic invertebrates as they drift downstream with the “slug” of larvicide (Muirhead-Thomson 1977). While synthetic pyrethroids have been shown to have moderate acute toxicity to birds (LD50 = 1 000 mg/kg) (SPIOP 1986), the US Environmental Protection Agency has classified permethrin as a carcinogen, since it causes cancerous tumours in lung and liver tissue of mice (Cox 1998). Permethrin has been widely reported as being highly toxic to aquatic invertebrates (Mueller-Beilschmidt 1990; Cox 1998), with an LC50 of less than 1.0 ppb, which is similar to that used for pest blackfly control (Smith & Stratton 1986). The aquatic invertebrate groups most sensitive to permethrin are mayfly, damselflies and zooplankton (Anderson 1982; Smith & Stratton 1986; SPIOP 1986). Such impacts could potentially TABLE 10 Conditions of application and results of flow through gutter trials to test the toxicity of various dry formulations of Bacillus thuringiensis var. israelensis to blackfly larvae Trial no. Date Water temp. (°C) Gutter channel Flow (ℓ/s) Dosage (mg/ℓ) Efficacy (%) Standard powder formulation (500:250) (Great Fish River) 03.06.05 11.5 1 2 3 4 0.88 0.86 0.75 0.83 0* 0.9 2.2 6.0 0 0 0 0 Double strength powder formulation (500:500) (Great Fish River) 04.06.05 11.2 1 2 3 4 0.85 0.91 0.82 0.86 0* 3.7 8.1 19.4 0 0 0 0 04.06.05 11.2 1 2 3 Granular formulation (Orange River) 03.10.06 18 1 2 3 4 1.62 1.79 1.79 1.85 0.5 0.9 1.9 9.0 0 0 0 0 * Control 312 Larvicides in developing management guidelines for control of pest blackfl ies (Diptera: Simuliidae), South Africa have a significant indirect effect on fish, through di- minished food supplies (SPIOP 1986). Permethrin is highly toxic to fish (Cox 1998), partic- ularly at lower water temperatures, and to smaller fish (Hill 1985), since fish lack the enzymes to break permethrin down (Haya 1989). The LC50 values of many fish species tested is less than 1.0 ppm (World Health Organization 1990), the same concentration recommended for pest blackfly control. Permethrin shows intermediate toxicity to fish within the spec- trum of pyrethroid toxicities, although it is also noted that pyrethroids in general are highly toxic to fish, with about 40 % of LC50 values for fish being less than 1 ppb (Smith & Stratton 1986). Differences in mortalities between gutter trials and river trials at the same concentrations recorded in this study may be attributed to differences in expo- sure time and/or interactions between permethrin and suspended organic matter. Muirhead-Thomson (1987) reported that pyrethroids were more toxic to fish in the laboratory than in natural water because they adhere to suspended organic matter in the wa- ter and sediment. Temephos Formulations of temephos (Abate® 200EC) were highly effective against blackfly in the Great Fish River, but the same products were ineffective in the Orange River. The results indicate clearly that larval resistance to temephos has developed in the Orange River, but the mechanisms of resistance were not investigated in this study. Temephos has a number of chemical bonds that are available for metabolic attack by oxidases or es- terases. Laboratory studies in West Africa have shown that resistance to temephos among the S. damnosum complex is associated mainly with de- toxication by esterases, but oxidase enzyme sys- tems are also involved in some members of the complex (Kurtak 1990). Cross-resistance tests showed no cross-resistance to carbamate insecti- cides with these mechanisms, but negative correla- tions with most pyrethroids (Kurtak 1990). The feasibility of “reversing” the resistance through the use of piperonyl butoxide was investigated. The results showed that piperonyl butoxide on its own is highly toxic to blackfly larvae. This contradicts the generally held view that piperonyl butoxide is non- toxic on its own. Various combinations of piperonyl butoxide and temephos showed no enhanced toxic- ity as predicted. B.t.i. formulations The physical properties of the standard dry powder concentrated formulation of B.t.i. (500:250) were un- suitable for blackfly control, firstly because of poor vertical dispersion and rapid settling in water. The implication of this is that downstream carry is likely to be limited. Secondly, the product forms sticky lumps when mixed with water and this is likely to cause clogging problems with application equip- ment, should the product be applied as a wet formu- lation. A possible solution to this problem would be to apply the product as a dry powder, but this is like- ly to restrict applications to periods when wind is not blowing. The slow-release granular formulation, by contrast, showed excellent dispersal properties. A more serious problem was that gutter and field trials found that all formulations tested were ineffec- tive against blackflies. This was unlikely to have been due to low water temperatures, since water temper- atures always exceeded the 10 °C temperature threshold of efficacy found by De Moor & Car (1986). Concentrations of B.t.i. applied in these trials were also within the range used by Parkes & Kalff (2004), who applied B.t.i. to larval simuliids in rivers in south- ern Quebec at concentrations of 1 g/ℓ/s and achieved in excess of 80 % mortality. Similarly, De Moor & Car (1986) achieved equivalent mortalities in S. chut teri in the Orange River, at concentrations of 1.6 ppm per 10 min. CONCLUSIONS Various potential larvicides for use during high flow conditions were investigated in laboratory, gutter and river trials, but none was found to be suitable. This study therefore failed to identify or register a suitable larvicide for use during high flow conditions. Permethrin was highly effective against blackfly lar- vae, but was rejected because of its detrimental im- pacts on non-target fauna. Various formulations of locally produced dry B.t.i. were tested, but these were ineffective against blackflies. Trials with 20 % EC formulations of temephos in the Great Fish River found that the product is effective, but gutter and river trials in the Orange River showed no efficacy, even at dosages that were 300 times the recommended dose. The results confirmed that larval resistance to temephos has developed in the Orange River. The feasibility of “reversing” the resistance to teme- phos through the use of piperonyl butoxide was in- 313 R.W. PALMER & N.A. RIVERS-MOORE vestigated. Various combinations of piperonyl bu- toxide and temephos were tested, but none showed enhanced toxicity, as predicted. The results showed that piperonyl butoxide alone is highly toxic to black- fly larvae and non-target organisms, and is there- fore not recommended for use in blackfly control. The natural recovery from resistance will depend on the rate at which the population mixes with non-re- sistant populations. The middle and lower Orange River is geographically isolated, therefore the resist- ant population of blackflies is likely to remain so for some time. How long reversal to resistance will take, is unknown, but findings elsewhere have shown that the development of resistance is much more rapid than its reversal. The risks of larval resistance to temephos were well known when the programme started in 1991, and nothing was done to monitor resistance or test the development of resistance when it was first sus- pected in 2000. This underscores the need for radi- cal improvements in the management of the control programme. Resistance to B.t. toxins has for many years been considered remote because of the complexity of the toxin, containing multiple toxins with multiple target sites (McGaughey & Whalon 1992; McGaughey 1994). However, resistance to B.t.i. has been docu- mented in the laboratory for at least eight species of pest, and the diamond backed moth (Plutella xylos- tella) has developed widespread resistance in the field (Tabashnik 1994). Possible physiological mech- anisms of resistance include changes in the gut pH or enzymes that would deactivate the toxic protein (McGaughey & Whalon 1992). In some moths, re- sistance is due to changes in the binding sites in the insect mid gut (McGaughey & Whalon 1992; Ta- bashnik 1994). Although resistance to B.t.i. has not been reported for blackflies (Kurtak et al. 1989), there is a need to remain vigilant and to implement an operational strategy that minimizes the risk of re- sistance developing. ACKNOWLEDGEMENTS We thank the Water Research Commission, Agri- Noord Kaap, the Orange River Producers Alliance and Plant Health Products for financial support. The WRC Steering Committee, and in particular Steve Mitchell (Water Research Commission) and Dirk Steenkamp (national Department of Agriculture), are thanked for their input comments over the course of this research. Nkosinathi Mtwa and Vivian McPher son are thanked for field assistance. Keith Craig (Kwan dwe Private Game Reserve, Eastern Cape Prov ince) and Mike Palmer (Strowan Farm, Grahamstown) are thanked for facilitation in the Great Fish River trials. Two anonymous reviewers are thanked for their comments and suggestions. REFERENCES ANDERSON, R.L. 1982. Toxicity of fenvalerate and permethrin to several nontarget aquatic invertebrates. Environmental Entomology, 11:1251–1257. COX, C. 1998. Insecticide factsheet: Permethrin. Journal of Pest- icide Reform, 18:14–20. DE MOOR, F.C. & CAR, M. 1986. A field evaluation of Bacillus thuringiensis var. israelensis as a biological control agent for Simulium chutteri (Diptera: Nematocera) in the middle Orange River. Onderstepoort Journal of Veterinary Research, 53:43– 50. GORDON, N.D., MCMAHON, T.A. & FINLAYSON, B.L. 1992. Stream hydrology: An introduction for ecologists., Chichester, England: John Wiley & Sons. HAYA, K. 1989. Toxicity of pyrethroid insecticides to fish. En vi- ronmental Toxicology and Chemistry, 8:331–391. HILL, I.R. 1985. Effects on non-target organisms in terrestrial and aquatic environments, in The pyrethroid insecticides, ed- ited by J.P. Leahey. London, UK: Taylor & Francis. HOWELL, C.J. & HOLMES, G.W. 1969. The control of Simuliidae in the Vaalharts irrigation complex. Journal of the South Afri- can Veterinary Medical Association, 40:59–67. JONES, D.G. 1998. Piperonyl butoxide: The insecticide synergist. San Diego, California: Academic Press. KREUTZWEISER, D.P. & CAPPEL, S.S. 1992. A simple stream- side test system for determining acute lethal and behavioural effects of pesticides on aquatic insects. Environmental Toxi- cology and Chemistry, 11:993–999. KURTAK, D.C., BACK, C., CHALIFOUR, A., DOANNIO, J., DOS SOU-YOVO, J., DUVAL, J., GUILLET, P., MEYER, R., OCRAN, M.R. & WAHLE, B. 1989. Impact of B.t.i. on black fly control in the Onchocerciasis Program Control in West Africa. Israel Journal of Entomology, 23:21–38. KURTAK, D.C. 1990. Maintenance of effective control of Sim u- lium damnosum in the face of insecticide resistance. Acta Leiden, 59:95–112. LÉVÊQUE, C., HOUGARD, J.M., RESH, V., STATZNER, B. & YAMÉOGO, L. 2003. Freshwater ecology and biodiversity in the tropics: what did we learn from 30 years of Onchocerciasis control and the associated biomonitoring of West African riv- ers? Hydrobiologia, 50:23–49. MCGAUGHEY, W.H. 1994. Problems of insect resistance to Bacillus thuringiensis. Agriculture, Ecosystems and Environ- ment, 49:95–102. MCGAUGHEY, W.H. & WHALON, M.E. 1992. Managing insect resistance to Bacillus thuringiensis toxins. Science, 258: 1451–1455. MUELLER-BEILSCHMIDT, D. 1990. Toxicology and environ- mental fate of synthetic pyrethroids. Journal of Pesticide Re- form, 10:32–37. MUIRHEAD-THOMSON, R.C. 1977. Comparative tolerance lev- els of black fly (Simulium) larvae to permethrin (NRDC 143) and temephos. Mosquito News, 37:76; 172–179. 314 Larvicides in developing management guidelines for control of pest blackfl ies (Diptera: Simuliidae), South Africa MUIRHEAD-THOMSON, R.C. 1987. Pesticide impact on stream fauna with special reference to macroinvertebrates. Cam- bridge, UK: University Press. MYBURGH, E. 1999. The control of blackflies in South Africa. Internal report prepared for the National Department of Agriculture, Directorate of Agricultural Resource Conser va- tion, by the Onderstepoort Veterinary Institute for the con- tract year 1998/1999. MYBURGH, E. & NEVILL, E.M. 2003. Review of blackfly (Diptera: Simuliidae) control in South Africa. Onderstepoort Journal of Veterinary Research, 70:307–317. OCRAN, M.H. 1989. Larvicide screening methodology for clas- sifical chemical compunds in the Onchocerciasis Control Pro- gramme (OCP). World Health Organization. WHO/VBC/89. 969. PALMER, R.W. 1994. A rapid method of estimating abundance of immature blackflies (Diptera: Simuliidae). Onderstepoort Journal of Veterinary Research, 61:117–126. PALMER, R.W. 1997. Principles of integrated control of black- flies (Diptera: Simuliidae) in South Africa. Pretoria: Water Re- search Commission (WRC Report No. 650/1/97). PARKES, A.H. & KALFF, J. 2004. Feeding by black fly (Diptera: Simuliidae) larvae causes downstream losses in phytoplank- ton, but not bacteria. Journal of the North American Ben tho- logical Society, 23:780–792. SIBLEY, P.K. & KAUSHIK, N.K. 1991. Toxicity of microencapsu- lated permethrin to selected non-target aquatic invertebrates. Archives of Environmental Contamination and Toxicology, 20:168–176. SMITH, T.M. & STRATTON, G.W. 1986. Effects of synthetic py- rethroid insecticides on nontarget organisms. Residue Re- views, 97:93–120. SPIOP 1986 [Subcommittee on Pesticides & Industrial Organic Pesticides, Associate Committee on Scientific Criteria for Environmental Quality, National Research Council of Can- ada]. Pyrethroids: Their effect on aquatic and terrestrial eco- systems. NRCC No. 24376. Ottawa, Canada: Environmental Secretariat, National Research Council of Canada. TABASHNIK, B.E. 1994. Evolution of resistance to Bacillus thur- ingiensis. Annual Review of Entomology, 39:47–79. WHO 1988. The WHO recommended classification of pesticides by hazard and guidelines to classification. Geneva: World Health Organization. WHO 1990. Environmental health criteria. Vol. 94, Permethrin. Geneva: World Health Organization. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (Europe ISO Coated FOGRA27) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts false /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages false /ColorImageDownsampleType /Bicubic /ColorImageResolution 600 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages false /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Bicubic /GrayImageResolution 600 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages false /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. Stvoreni PDF dokumenti mogu se otvoriti Acrobat i Adobe Reader 5.0 i kasnijim verzijama.) /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken die zijn geoptimaliseerd voor prepress-afdrukken van hoge kwaliteit. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.) /NOR /POL /PTB /RUM /RUS /SKY /SLV /SUO /SVE /TUR /UKR /ENU (Use these settings to create Adobe PDF documents best suited for high-quality prepress printing. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.) >> /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ << /AsReaderSpreads false /CropImagesToFrames true /ErrorControl /WarnAndContinue /FlattenerIgnoreSpreadOverrides false /IncludeGuidesGrids false /IncludeNonPrinting false /IncludeSlug false /Namespace [ (Adobe) (InDesign) (4.0) ] /OmitPlacedBitmaps false /OmitPlacedEPS false /OmitPlacedPDF false /SimulateOverprint /Legacy >> << /AddBleedMarks false /AddColorBars false /AddCropMarks false /AddPageInfo false /AddRegMarks false /ConvertColors /ConvertToCMYK /DestinationProfileName () /DestinationProfileSelector /DocumentCMYK /Downsample16BitImages true /FlattenerPreset << /PresetSelector /MediumResolution >> /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ] >> setdistillerparams << /HWResolution [2400 2400] /PageSize [595.276 841.890] >> setpagedevice