Sensitivity Comparisons of the Insect Centroptilum Triangulifer to Ceriodaphnia Dubia and Daphnia Magna Using Standard Reference Toxicants; Nacl, Kcl

Sensitivity comparisons of the insect Centroptilum triangulifer to Ceriodaphnia dubia and Daphnia magna using standard reference toxicants; NaCl, KCl and CuSO4 A thesis submitted to the Graduate School of the University of Cincinnati on 2/20/12 in partial fulfillment of the requirements for the degree of Masters of Science in the Department of Biology of the College of Arts and Sciences by Katherine Ann Hammer B.S. University of Dayton 2003 Committee Chair: Jodi Shann, Ph.D. Abstract Establishing water quality criteria that is protective of all native biota is a difficult task and often aided by the use of model organisms. Common model organisms may not be the most sensitive or inclusive of all taxa. The US Environmental Protection Agency has cultured a parthenogenetic invertebrate, the insect Centroptilum triangulifer , which potentially has higher sensitivity to certain toxicants. This study established a 48 hr acute and a 14 day chronic testing procedure for C. triangulifer and compared its sensitivity to two model invertebrates, Ceriodaphnia dubia and Daphnia magna . Toxicity bioassays were conducted to determine mortality and growth effects using standard reference toxicants; NaCl, KCl and CuSO 4. Weight was at least 36% more sensitive to the toxicants than body length and head capsule width in C. triangulifer . In 48 hr acute tests, the average LC50 for the mayfly was 659 mg/L NaCl, 1957 mg/L KCl, and 11 µg/L CuSO 4. IC25 values, using weight as the endpoint, were 291 mg/L NaCl, 356 mg/L KCl and 6 µg/L CuSO 4. C. triangulifer was the most sensitive species in NaCl acute and chronic tests. KCl at concentrations tested for the two daphnid species failed to produce mortality in C. triangulifer bioassays, but the species was equally or more sensitive than C. dubia and D. magna for growth measurements. Despite possible food interactions during CuSO 4 testing, C. triangulifer was the most sensitive species during acute testing and for growth parameters in chronic tests. This study determined C. triangulifer has great potential and benefits for use in ecotoxicological studies. ii iii Acknowledgements I would like to thank my committee members; Jodi Shann, Ph.D., Jim Lazorchak, Ph.D. and Eric Maurer, Ph.D. for their wisdom and guidance. I also owe a great deal of gratitude to my coworkers at The McConnell Group for their support throughout my study. In addition, I would like to acknowledge the late Mark Smith for his encouragement to pursue a Masters Degree and his knowledge during the creation of my proposal. Finally I would like to thank my family and friends for their continued support. iv Table of Contents Title Page i Abstract ii Acknowledgements iv Introduction 1 Materials and Methods 4 Results 14 Discussion 34 Bibliography 40 Appendix 43 List of Figures and Tables Figure 1 12 Figure 2 15 Figure 3 16, 17 Figure 4 18 Figure 5 19 Figure 6 22 Figure 7 23, 24 Figure 8 25 Figure 9 26 Figure 10 28 Figure 11 29, 30 Figure 12 31 Figure 13 32 v Table of Contents (cont.) Table 1 20 Table 2 21 Table 3 27 Table 4 27 Table 5 33 Table 6 33 vi Introduction Aquatic laboratory organisms have been utilized for decades to determine the effects of chemicals in freshwater environments and to define water quality criteria. Certain model organisms have been utilized due to their life history traits, ease of culturing in a laboratory and limited variability between individuals. Common model organisms include the fishes Pimephales promelas and Oncorhynchus mykiss , the cladocerans Ceriodaphnia dubia and Daphnia magna , the amphipod Hyalella azteca , and the midge Chironomus tentans , among others. These aquatic species have been used extensively for acute and chronic toxicity testing and are outlined for use by the US Environmental Protection Agency (EPA) (2002a, 2002b). There are many benefits to using a model organism, including the ability to compare results from independent studies and literature, but there may also be disadvantages. Developing water quality criteria that are protective of all native fauna is the main goal of aquatic ecotoxicology risk assessment studies, but the use of model organisms that are restricted to a few taxa may miss some of the more sensitive species. A study of toxicity datasets for metals showed that acute and chronic testing has under-represented aquatic insects and over-represented cladocerans (Brix et al. 2005). Further toxicity testing is needed to be conducted to determine if aquatic insects are more sensitive test organisms than those in current use. The number of toxicity tests on aquatic insects has begun to increase, especially within the order Ephemeroptera (mayflies). A study by Echols et al. (2009) indicates that Isonychia bicolor , a mayfly species, is more sensitive than the cladoceran C. dubia to coal processing 1 effluent. The mayfly Stenonema modestum was utilized in subacute tests by Diamond et al. (1992). Toxicity testing of cadmium and lead has been conducted using the mayfly Baetis tricaudatus . (Irving et al. 2002) (Mebane et al. 2007). B. tricaudatus has also been tested for growth, after exposure to pulp mill effluent (Lowell et al. 1995). The EPA has identified the mayfly Centroptilum triangulifer as a prospective species for use in either mountaintop mining effluent and/or oil and gas extraction effluent studies. C. triangulifer was previously identified by McDunnough (1931) as Cloeon triangulifer and is of the Baetidae family. The species is found in marginal streams and slow flowing aquatic systems throughout eastern North America (Gibbs, 1973). Funk et al. (2006) used genetic and morphological examinations to describe differences between C. triangulifer , which is obligatory parthenogenetic, and Centroptilum alamance , which is a very similar species that reproduces sexually. C. triangulifer females produce clonal eggs and embryonic development is temperature dependent, averaging 6 days until first hatch at 25°C (Sweeney et al. 1984). C. triangulifer feeds by scraping periphyton from stream beds and other surfaces. The relative short life cycle (~30 days) and clonal reproduction makes C. triangulifer a good candidate for toxicology studies. Use of C. triangulifer as a test organism has increased in the last decade. Studies generally indicate this species is sensitive to certain pesticides, metals and non-metals. Work by Sweeney et al. (1993) demonstrated larval survivorship of C. triangulifer was significantly lowered by the pesticide chlordane at a concentration of 4.3 ug/L. Further research indicated that chlordane is also transferred via lipids to mayfly eggs (Standley et al. 1994). Conley et al. (2009) looked at maternal transfer in C. triangulifer and described the detrimental effects of selenium bioaccumulation through ingestion of contaminated 2 periphyton. In addition, bioaccumulation and the trophic transfer of the metal cadmium were examined by Xie et al. (2009). A follow-up study by Xie and Buchwalter (2011) illustrates that dietary cadmium suppressed antioxidant enzymes, leading to increased toxicity, while dissolved cadmium did not. Hassell et al. (2006) determined that survival of Centroptilum sp. was decreased when salinity (as measured by conductivity) was equal or greater than 5.0 mS cm -1 which was lower than the Cloeon sp. tested in the same study. In the past, C. triangulifer bioassays have used field collected or laboratory cultured organisms. Studies that utilized laboratory cultured animals used a non-laboratory method for food (Conley 2009; Xie 2010; Xie 2011; Standley 1994; Sweeney 1993; Funk 2006). This method used local stream water that was pumped over acrylic plates promoting the cultivation of algal colonies. The plates were then placed into containers for feeding during culturing or testing of C. triangulifer . There are problems with this approach. The actual community or species composition and density of the algal slides may vary and utilizing stream water may introduce toxicants present in a local system into the laboratory culture. In addition this method is not practical for those without access to a native stream. An alternate means of maintaining mayfly cultures has been developed (Weaver et al. (unpublished). In this method, diatoms are cultured as a food for the mayfly. Three species of diatoms were selected based on size and availability: Mayamaea atomus var. permitis (Hustedt) Lange-Bertalot, Nitzschia cf. pusilla (Kützing) Grunow emend. Lange- Bertalot, and Achnanthidium minutissimum (K ützing) Czarnecki. These diatoms are added in equal proportions to a container with etched glass microscope slides. After a minimum of 7 days, colonized microscope slides are moved into C. triangulifer culture containers for 3 grazing. This culture method helped standardize culturing techniques, making C. triangulifer more easily cultured for use in testing. Now that culturing techniques have been standardized for this new organism, there is a need for standardized toxicity testing procedures. The establishment of a uniform testing procedure would increase the usefulness of C. triangulifer as a bioassay species. Because studies utilizing mayflies have begun to increase and a convenient culturing method has been documented, this study aims to establish standardized acute and chronic test methods for C. triangulifer . In addition to standardizing bioassays for C. triangulifer , the benefits of using this species as a model system will be evaluated. This study will compare results of C. triangulifer acute and chronic bioassays to parallel tests conducted

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