Reza A. Ghiladi 1

Benthic Survey of Bald Head Island PI: Reza A. Ghiladi Benthic Survey of Bald Head Island: Identifying Polychaete Diversity and Salt Marsh Organic Sampling PI: Reza A. Ghiladi (NC State University) in collaboration with the Bald Head Island Conservancy Brief Overview This Innovation Fund proposal is to request support for conducting the initial year of a 3-year benthic survey of Bald Head Island (BHI), with the overarching goal of providing a baseline of polychaete diversity that can be correlated with the levels of polyaromatic hydrocarbons present in the organic layer in which the polychaetes reside. The support requested will enable research that is of mutual benefit to the Bald Head Island community, the BHI Conservancy and the Ghiladi research lab in that: i) the community will gain more specific knowledge of the levels of organic compounds that may flow into BHI from upstream sources, providing a point of reference for monitoring potential pollutants, ii) the BHIC will have an established baseline of the infaunal organisms present in the benthic ecosystems of BHI, thereby establishing polychaete diversity as a means of assessing the health of the benthic layer, and iii) the Ghiladi lab will gain the knowledge and expertise to conduct field collections of polychaetes along the North Carolina coast, thereby enabling their lab to document polychaete biodiversity based upon both morphological and molecular traits. Summary of the Research Problem Background. Recently, the Ghiladi lab has identified four classes of organic compounds as potential toxins for marine annelids. Specifically, we have shown that benzimidazole, benzotriazole, indazole, and imidazole all have a high binding affinity to the hemoglobin of the terebellid polychaete Amphitrite ornata (see Figure, left). The binding of these compounds to A. ornata represents a potentially new mechanism by which organic compounds may be X-ray crystal structures recently solved by the Ghiladi toxic to marine organisms, and suggests that there lab of the Amphitrite ornata hemoglobin bound with may be a much larger number of compounds than benzotriazole (left, 1.08 Å), and benzimidazole (right, have been identified by the EPA that pose a serious 1.14 Å). threat to the health and biodiversity of benthic ecosystems. We highlight two examples as background for this proposal: 1) benzimidazole compounds have two avenues by which they may enter benthic ecosystems. Benzimidazole fungicides and their derivatives are used to protect several types of crops, both during field and post-harvest treatments. The majority of these fungicides are either applied directly to the soil, or are sprayed over fields, and hence released into the environment. In this manner, the benzimidazole fungicides contaminate watersheds either directly or through drainage of agricultural lands. Benzimidazole anthelmintics are a class of drugs that are used against parasitic worms, both in humans as well as in veterinary medicine. Anthelmintics administered to animals (cows, sheep, hogs) enter into environment primarily through excretion (urine or feces). Following excretion, these substances may persist in the environment and impact non-target organisms. Moreover, studies have shown that some benzimidazole metabolites are more toxic than the parent drug. 2) Benzotriazoles have been used as ultraviolet light absorbers to protect polymers from photochemical deterioration, and more recently have been widely used as anticorrosive additives in cooling and hydraulic fluids, antifreeze products, aircraft de-icer and anti-icer fluid, and in dishwasher detergents for silver protection. These benzotriazole corrosion inhibitors are frequently detected in rivers, lakes and groundwater as they are not completely removed during wastewater treatment. Importantly, neither benzimidazoles nor benotriazoles are on the EPA priority pollutant list. As such, these and similar compounds are not assessed as environmental contaminants, yet may be present in what are considered ‘pristine’ environments. Moreover, their toxicities to marine organisms are unknown. 1 Benthic Survey of Bald Head Island PI: Reza A. Ghiladi Rationale and Motivation. Our rationale for the proposed research is built upon the knowledge recently gained by the Ghiladi lab that compounds such as benzotriazole and benzimidazole are likely highly toxic to the terebellid polychaete Amphitrite ornata as they bind the organism’s hemoglobin with relatively high affinity. As these compounds are not yet considered by the EPA to be pollutants given this relatively recent result, previously performed organic samplings would not look for these compounds as contaminants in the mud layer. As such, there is a lack of baseline information regarding the presence of benzotriazole, benzimidazole, indazole, and imidazole in the benthic ecosystems of BHI, and more broadly, of related polycyclic aromatic hydrocarbons. If these compounds are indeed found on BHI, the community may be better able to identify the upstream activities that contribute to the contamination, and potentially shut them down or propose alternatives. Conversely, the awareness of these compounds to the BHI community will enable them to proactively respond to proposed upstream activities that may lead to an accumulation of these organic compounds, thereby preventing future contamination of BHI. Moreover, a benthic survey has not been performed over the past 10-15 years, and as such, the health and biodiversity of the benthic layer with respect to polychaetes needs to be meaningfully assessed and benchmarked now in order to enable future comparisons to better understand how the diversity of the infaunal species evolves over time as a function of both natural and anthropogenic forces. The Ghiladi lab is motivated to conduct the studies proposed below in the research plan to provide these meaningful baseline measurements and surveys to the BHI community and the BHIC, but also to gain access to additional polychaete hemoglobins from which they can expand their studies of these proteins. Research Plan (to be performed annually for 3 years, contingent upon Year 1 results) Specific Aim 1: To Identify and Collect Polychaetes from A Different Region on Bald Head Island Annually. The polychaetes will be collected from a different benthic region each year that will enable a broad survey of marine annelids. Polychaetes will be identified following known protocols (Zootaxa 2015, 4019, 240–274; Zootaxa Special Volume 2015, 4019, 1-801; Marine Biology 2015, 162, 1319–1327) in conjunction with our Benthic technical expert Cindy Huggette. Particular attention will be paid to identify polychaetes with known genomes (such as Capitella teleta). Field samples will be collected and cryo- preserved for cataloging offsite. Outcome 1: Our goal for Specific Aim 1 is to provide a baseline for polychaete biodiversity on Bald Head Island as a benthic survey can be used as a means to monitor the long-term health of the ecosystem. Specific Aim 2: To Purify and Screen the Polychaete Hemoglobins for Pollutant Binding. Up to 3 species of polychaete will be bled, and the hemoglobin proteins will be purified to homogeneity using FPLC in a similar fashion as to how A. ornata hemoglobin was initially purified (J. Biol. Chem. 1996, 271, 4609- 4612). UV/visible binding assays to determine the impact of benzimidazole and benzotriazole binding on the polychaete hemoglobins will be performed in a similar fashion as has been published for indole (J. Am. Chem. Soc. 2014, 136, 7914−7925). Hemoglobin samples that exhibit pollutant binding will be submitted for sequence analysis (tryptic digest combined with LC-MS/MS analysis) to the NCSU proteomics facility. Outcome 2: Our goal for Specific Aim 2 is to document polychaete biodiversity on Bald Head Island based upon both morphological and molecular (pollutant binding) traits. Specific Aim 3: To Determine Concentration Levels of Hemoglobin-Binding Contaminants. Solid phase extraction (SPE) coupled with liquid chromatography (fluorimetric or UV/vis detection) will be used to determine the levels of contaminants present in the organic mud layer from the regions where polychaetes will be collected annually (see Anal. Methods 2010, 2, 739-745). Examples of the annelid hemoglobin- binding contaminants that will be assessed initially include imidazole, benzimidizole, benzotriazole, and indazole. In years 2 & 3, additional screening of polycyclic aromatic hydrocarbons will be examined, such as acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzoanthracene, chrysene, benzofluoranthene, benzofluoranthene, benzopyrene, dibenzoanthracene, benzoperylene, and indenopyrene. Outcome 3: Our goal for Specific Aim 3 is to monitor the organic contaminants present, either as EPA priority pollutants (known) or the hypothesized annelid hemoglobin-binding compounds, in the mud layer in which the polychaetes are found, thereby providing a baseline for future comparisons. 2 Benthic Survey of Bald Head Island PI: Reza A. Ghiladi Potential for Future External Support In the NSF CAREER grant awarded to PI Ghiladi (CHE-1150709), it was posited that the hemoglobin from the terebellid polychaete Amphitrite ornata is the archetype of multifunctional globins, and having been found in a relatively under-investigated family of organisms (i.e., marine annelids/polychaetes) when compared to more traditional (terrestrial) organisms, that it is plausible that such marine organisms harbor a new subfamily of these enzymes. To

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