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268 C.S. CHARBONNEAU ET AL. vulgaris), water crowfoot (Ranunculus Jlabellaris), lesser duckweed (Lemna minor), and great duckweed (Spirodela polyrhiza). MP contained elodea and pond weed (Polamo- gelOn iIIinoensis) in the deeper areas. Water chemistry char- acteristics on July 10, 1990, were total alkalinity 124 mg/L (as CaC03), pH 9.0, and total hardness 217 mg/L (as CaC03) for LBP and total alkalinity 299 mg/L (as CaC03), pH 8.5, and total hardness 427 mg/L (as CaC03) for MP. The field formulation proposed for use (Vectobac-G) was a corncob granule containing Bti spores and cryslals at 200 inlernationaltoxic units (ITU) per milligram (Abboll Lab- oratories, North Chicago, IL). No previous field studies on the effect of Vectobac-G on benthic invertebrates have been conducted. The recommended application rate (RAR) was 5.6 kg/ha, to be spread on water ranging in depth from 15.0 cm (equaling 3.7 ppm) to 58.0 em (equaling 1.0 ppm) (34). The LBP was used for enclosure test 1 in 1989 and en- closure lests 2 and 3 in 1990. Enclosure test 4 was conducted in MP during 1990 because the population of chironomids in LBP had emerged and MP contained appropriate densi- ties for sampling and testing. The Bass Pond area had not been treated with Bli since 1984 (M.S. Mitchell, Personal Communication). Experimental design. We constructed a number of enclo- sures within a pond for each experiment and used each enclo- sure as a replicate of a particular treatment (35). Preliminary sampling determined that 10 replicate enclosures per treat- ment were required to be 951170 confident that the sample den- sities of common taxa were within 201170 of the true mean (361. Enclosures were designed to isolate a section of sediment, wa- ter, and the associated flora and fauna. The enclosures were cylindrical, constructed of riveted sheets of 5-mm-thick Fi- berglas@) driven into the sediment about 10 em. The other end of the enclosure protruded above the water surface and was stabilized with wooden stakes. The size of the enclosures was different each year. The enclosure seams were closed during application of Bti and opened with wooden wedges to allow water exchange between the pond and enclosures when tests were not being conducted. Each enclosure was numbered and assigned a treatment using a randomized block design. Enclosure test I was conducted in 1989 to determine whether periodic applications of Vectobac-G at the recom- mended rate and timing would affect the benthic fauna. Twenty I m-diameter enclosures, separated by at least 2 m, were placed throughout LBP. Average water depth within en- closures was 61 cm. Ten enclosures were treated at RAR of 5.6 kg/ha of Vectobac-G corncob granules and 10 enclosures with blank corncob granules (purchased locally) serving as controls. Enclosures were treated three times throughout the summer. Invertebrate samples were collected before and af- ter each treatment. The 1990 experiments (enclosure tests 2, 3, and 4) evalu- ated toxic effects of Vectobac-G on the benthic fauna at RAR and at an elevated rate of 5 x RAR. On the basis of 1989 re- sults, we made several modifications. Enclosure size was re- duced to a 53-cm diameter and enclosures were grouped together to reduce variance among samples. Enclosures were located in groups of three, with each group having a control, one RAR, and one 5 x RAR. Enclosures were approximately 8 cm apart with seams facing outward. Groups of enclosures \ were 7 m or more apart in LBP and approximately 20 cm apart in MP. Before enclosures were placed in LBP, approx- imately 80070 of the submersed vegetation was removed with a rake to reduce the dense coverage of vegetation. Water in the LBP enclosures was reduced in 1990 10 a foraging deplh (13.3 cm), within the range of adult dabbling ducks (371. Mean depth of water in MP enclosures was 26.8 em. Vectobac-G was applied in three different tests (enclosure tests 2, 3, and 4), and samples of invertebrates were taken 48 h after each application. Sampling techniques. Invertebrates were sampled with a modified Swanson (38) benthic core sampler (7.6 cm diam- eler). The sampler was driven through the submersed vege- tation and into the sediment to a depth of 10 cm. The core sample was cleaned in a screen-bottom bucket (575-ltm mesh openings) and washed into a labeled plastic bag. Multiple samples were taken within each enclosure. Sam- ple compartments were randomly chosen using a numbered wooden grid placed over each enclosure. A compartment was sampled only once. Samples were transported to the labora- tory, washed through a 425-ltm standard sieve, and sorted while the animals were alive. Samples that could not be sorted the first day were stored at 50C. Sorted invertebrates were preserved in 801170 ethanol. Preserved invertebrates were iden- tified to genus when possible, using the descriptions of Pen- nak (39) and Merritt and Cummins (40). All chironomid larvae were mounted on slides, as described by Coffman and Ferrington (41), and identified to genus. In 1989 (enclosure test I), an emergence trap was attached to the inside of each enclosure. Core samples were not taken from the grid compartment, where the emergence trap was situated. Traps consisted of window screening sewed into a conical shape with a 30.5-cm-diameter bottom and II-cm- diameter top where adult invertebrates were captured with Stickem Special@) on Plexiglas@). The sample (Plexiglas and invertebrates) was placed in 1001170 mineral spirits for easier extraction of invertebrates from the Stickem Special. Adult invertebrates were identified to family. Insecticide application. At least 800 ml pond water was collected from each enclosure in a coded I-L wash bottle. The appropriate preweighed amounts of Vectobac-G at 5.6 kglha (RAR) or 28 kg/ha (5 X RAR) were added to pond water in the wash bottles and mixed for approximately 20 min. The mixture was sprayed from the wash bottle into the enclosure in such a manner that the entire water surface was covered. The wash bottle was rinsed twice within the enclosure to en- sure that 'all granules were removed. Toxicity tests. Verification tests lA, IB, 2, 3, and 4 were conducted at the time of application of larvicide to the en- closures to verify the effectiveness of Vectobac-G as a mos- quito larvicide using mosquito larvae collected, from the Refuge. Toxicity-testing procedures of Peltier and Weber (42) were followed, with some modification to aa:ommodate field conditions. Verification tests used mosquito larvae collected the day before treatment and held for 24 h. After enclosures were treated with Vectobac-G, a grab sample of water was collected from randomly chosen enclosures and 200 ml wa- ler was poured into each test chamber. Mosquito larvae were transferred from the hOlding water to test chambers with an inverted eyedropper. Tests were ended after 48 h. Animals