Consultant's Report - Minnesota State Legislature

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Appendix 1. Development of D. polymorpha Culturing and Larval Toxicity Testing Protocol Overview The purpose ofthe study was to evaluate the toxicity ofCutrine®-Ultra and Cu (as CUS04) on D. polymorpha. Because of the potential differences in sensitivity at various life stages and levels of development, exposure methods were developed for fertilized eggs, trochophore, and veliger stage organisms. Materials and Methods Test organisms The zebra mussel, Dreissena polym01pha, is an exotic freshwater bivalve that was likely introduced to North America in the mid-1980s in Lake Saint Claire near Detroit, MI (Ram et al. 1993; Hincks and Mackie 1997). The life history of D. polym01pha consists ofa ciliated trochophore/pre-veliger stage (57-121/lm), aD-shaped veliger stage (70-160 /lm), an umboned veliger stage (120-280 /lm), a settling pediveliger (167-300 /lm), plantigrade (158 - 500 /-lm), juvenile (500 - 5000 /lm) and adult (Ackerman 1995). Although adults are considered sedentary, smaller individuals were observed to use byssal threads to climb substrate, such as the sides of the aquaria in this study. Juvenile/adult mussels range from 1-50 mm, although most are
vigorously using air stones and filtered ufiing a charcoal filter/jet. To keep mussels from adhering to one another, inert rocks were scattered along the bottom of aquaria as in Stoeckel and Garton (1993). Aquarium water temperature was held at 12 ± 1 °C in a water bath to inhibit spawning (Ram et al. 1993, Fisher et al. 1994). Mussels in each aquarium were fed daily with 30 mL of concentrated Raphidocelis subcapitata (formerly Selenastrum capricornutum) from a laboratory-maintained culture (US EPA 2002). Conductivity, temperature and ammonia levels were monitored daily. A 50 - 100 % water exchange was conducted if total ammonia levels exceeded 2 mg/L. Dead mussels were removed from holding tan1es daily, although only 2 mussels died during the holding period (20 April- 12 July 04). Table 1. Properties ofLake Ossawinnamakee and reconstituted water. Properties HRW 1 Lake Ossawinnamakee pH 8.2 8.4 Hardness 171 mg L- 1 (as CaC0 3 ) 163 mg L- 1 (as CaC03) Alkalinity 110 mg L- 1 (as CaC0 3 ) 126 mg L- 1 (as CaC0 3 ) June Temperature 18 ± 1 0C 2 18 - 21°C 3 1 Hard reconstItuted water (US EPA 2002) 2 Temperature used in exposure experiments 3 Approximate temperature range ofLake Ossawinnamakee, June (Montz, pel's. comm.). Spawning induction Spawning was induced by 30-minute exposure of 10 - 20 mm* mussels (acclimated for 18-h at 18 ± 1°C) to a 5x 10- 4 M solution of serotonin creatinine sulfate monohydrate (CAS# 61-47-2), as conducted in previous study (Ram et al. 1993, Fisher et al. 1994, John Lynn, LSD, per. comm.). Males were observed to spawn within 15 - 30 minutes, at which point they were transferred to clean HRW. Mussels that did not spawn in 30 minutes were assumed to be female and were transferred to clean HRW. Viable ·females spawned in clean HRW after 60 - 90 minutes as observed in previous study (Ram et al. 1993, Fisher et al. 1994). Gametes were used in subsequent trials within 1.5 h of sperm production. After 15 minutes, to allow fertilization to occur, HRW containing fertilized eggs was gently aerated (2-3 bubbles/sec) to provide flow to embryos, and maintained at 17 ± 1°C. Larval exposure protocols Studies have demonstrated that the pelagic larval stages ofD. polymorpha may be the most sensitive stage for control measures (Vanderploeg et al. 1996). In addition, Fisher et al. (1994) showed that the sensitivity of zebra mussel life stages to different molluscicides varied, although in most cases the veliger stages were more sensitive than later stages. Therefore TRV data were derived for D. polymorpha larvae across a range of developmental stages. Previous studies have shown that laboratory-reared D. * Smaller mussels did not praduce a suitable stock of gametes, while larger mussels produced gametes of inconsistent quality. 8
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List ofTables MINNESOTA DEPARTMENT
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I. Introduction Purpose MINNESOTA D
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Feasibility Study To Limit the Spre
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Hydrological Characteristics MINNES
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Outlet Structure MINNESOTA DEPARTME
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lBhsl Nllrtq Osso'winnomakee Pelica
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Age and Growth MINNESOTA DEPARTMENT
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Works Cited MINNESOTA DEPARTMENT OF
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Table V-1. Methods Available for Co
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Cavitation MINNESOTA DEPARTMENT OF
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Potassium Salts MINNESOTA DEPARTMEN
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Works Cited MINNESOTA DEPARTMENT OF
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Combined Treatments MINNESOTA DEPAR
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Feasibility Study To Limit the Spre
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METHOD OF INVESTIGATION MINNESOTA D
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VII-B. Treatment Alternatives Expan
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Message Page 3 of4 3/21/2005 best.
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enougl1 to eV8Juatc £1'
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I \ I iV!1';UJIOOiS for Estimaling
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FishPro Consulting Engineers and Sc
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Page 190 and 191: VII-G. List of Abbreviations Used
Page 192: VII-H. Glossary of Terms
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Consultant's Report - Minnesota State Legislature

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