Larval Phthalate Soup

Samuel Daughenbaugh, DePauw University

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The Approach: In my previous post, I described a group of chemical additives called phthalates and their potential impact on the development of sea urchin larvae. The plastic industry uses several phthalates that vary in chemical structure and toxicity levels. One way phthalates differ in structure is by their size. I am studying the effects of three phthalates with different molecule sizes — DMP (small), DBP (medium), and DEHP (large) — on mortality (lethal effect) and larval skeletal growth (sublethal effect).

My first major challenge was to dissolve the chemicals in seawater. As hydrophobic liquids, phthalates only mix with water molecules at very low concentrations; larger types (longer side chains) are less soluble. By dissolving each chemical in acetone, I am able to get DMP into seawater at 1000 parts per million (ppm), or 0.01%, and DBP and DEHP at 1 ppm. I am testing 5 concentrations of each chemical in addition to an acetone control (no phthalate), and a seawater control (no phthalate or acetone).

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Experimental jars with stirring paddles

Once the chemicals are in solution, I spawn male and female sea urchins via electric voltage and collect their sperm and eggs. Then, I fertilized the eggs and introduce them to experimental jars where they then begin to develop into larvae. Small paddles stir the water to increase the oxygen level and keep the larvae suspended. After growing the larvae for two days, a period before they start to depend on food, I transfer them into small tubes, preserve and store them in a freezer.

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Normal 4-arm pluteus larvae (Photo taken by Jaclyn Caruso)

 

To measure and categorize larvae into different stages of development, I observe them under a microscope that can record landmark points on the larval body in three dimensions. After determining the proportion of individuals that failed to develop to the normal 2 or 4-arm pluteus stage (pictured below), I use the landmarks to calculate the lengths of different skeletal features to determine how much the larvae had grown. At the end of each trial, I will have observed hundreds to thousands of dead larvae and once all of them have been counted and measured, I can begin to analyze the data and learn whether the phthalates are having a significant effect on their development.

Acknowledgements

This project is supported by Dr. Robert Podolsky and the Fort Johnson REU Program, NSF DBI-1757899.

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Life in Plastic, It’s not Fantastic

Samuel Daughenbaugh, DePauw University

2DA71FE7-975A-4AA8-8A78-DF3D1E545F05The Problem: We live in a plastic world. Plastics have saturated all aspects of our daily lives and, as a consequence, have also entered the natural world.  About 8.3 billion metric tons have been produced in the past 60 years, playing a pivotal role in the advancement of modern society (Parker, 2018). Although they are used to create many things we enjoy and benefit from, there are serious consequences for the health of humans and the environment that are associated with their use.

We have found plastics in unexpected places, everywhere from human guts to the most remote locations on earth (Schwabl, 2018; Woodall, 2014). Plastics have a long list of negative effects on living organisms, but their impact in the ocean is of special concern. Pictures of turtles with straws up their noses, bottle caps spilling out of dead bird stomachs, and penguins strangled in plastic beverage rings are often posted on social media sites. Less widely known are the chemical additives that leach from plastics. Phthalates are one such group of additives that pose threats to the health of humans and marine life.

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Current Fort Johnson REU Interns (Julianna Duran not pictured) collecting plastic and sand dollars on Otter Island. (Photo credit: R. Podolsky)

Phthalates have been valuable to the plastic industry because they promote flexibility and durability in many plastics (EPA, 2017). An astounding 470 million pounds of phthalates are used in the United States every year (EPA, 2017). This presents a significant problem because phthalates interfere with the production of important hormones that regulate growth and metabolism in humans and other animals (Boas et al., 2012).

This summer I am exploring the effects of three different phthalates– dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEHP)–on the larval development of marine invertebrates, using the purple-spined sea urchin (Arbacia punctulata) as a model. Sea urchin larvae float freely in the water column for an extended period of time and, therefore, are vulnerable to many marine pollutants.

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Purple-spined sea urchin (Arbacia punctulata)

Sea urchins are an important model because they are closely related to humans. Both humans and sea urchins use a signaling hormone called thyroxine, which is especially important for growth in early developmental stages (Heyland et al., 2006). Exposure to phthalates can disrupt the production of thyroxine. Additionally, larvae are very important to study because they form the base of food webs. Being at the bottom of the food chain means they feed animals at higher levels, many of which humans rely on for protein. Therefore, understanding how phthalates affect sea urchin growth and metabolism can lead to new insights into how these pollutants directly and indirectly impact human health.

 Acknowledgements

I would like to thank my mentor, Dr. Robert Podolsky, for his continued support, guidance, and encouragement. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

References

Boas, M., Feldt-Rasmussen, U., & Main, K. M. (2012). Thyroid effects of endocrine disrupting chemicals. Molecular and Cellular Endocrinology, 355(2), 240-248. 

Environmental Protection Agency (Ed.). (2017). Phthalates. America’s Children and the Environment, 3, 1-19.

Heyland, A., Price, D. A., Bodnarova-Buganova, M., & Moroz, L. L. (2006). Thyroid hormone metabolism and peroxidase function in two non-chordate animals. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 306B(6), 551-566.

Parker, L. (2018, December 18). A whopping 91% of plastic isn’t recycled. Retrieved from  http://www.nationalgeographic.com

Schwabl, P. (2018, October). Assessment of Microplastic Concentrations in Human Stool. Conference on Nano and microplastics in technical and freshwater systems, Monte    Verità, Ascona, Switzerland.

Woodall, L. C., Sanchez-Vidal, A., Canals, M., Paterson, G. L., Coppock, R., Sleight, V., . . . Thompson, R. C. (2014). The deep sea is a major sink for microplastic debris. Royal      Society Open Science, 1(4), 140317-140317. doi:10.1098/rsos.140317

Playing with Plutei

Hailey Conrad, Rutgers University

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Me! Photo Credit: Kady Palmer

Ocean acidification is known as climate change’s evil twin. When the pH of ocean water drops, carbonate ions in the water form carbonic acid instead of calcium carbonate. Calcium carbonate is the form of calcium that marine animals that have calcium-based skeletons (like us!) and shells use to build their bones and shells. Having smaller and weaker skeletons or shells impacts their ability to survive. However, some individuals within certain species or populations of species have genes that make them more resistant to ocean acidification. If these individuals are able to pass on these genes to their offspring, then the species has the ability to evolve in response to ocean acidification instead of going extinct. This summer I’m working with Dr. Bob Podolsky in College of Charleston’s Grice Marine Field Station to study the extent to which ocean acidification affects Atlantic purple sea urchins, Arbacia punctulata. We are specifically trying to see if any individuals within a population from Woods Hole, Massachusetts, have any heritable genetic resistance to the negative impacts of ocean acidification. We hypothesize that there will be genetic resistance given that the northern Atlantic coast naturally has lower levels of saturated calcium carbonate, so a population that has evolved to live in that type of environment should have some resistance to lower calcium carbonate levels already (Wang et al 2013). We’re using a basic cross breeding technique to rear Arbacia punctulata larvae to their plutei stage, when they have four main body rods. At this stage they look less like sea urchins than they do like Sputnik!

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A sea urchin pluteus larvae with four body rods

Then, we will look to see if any of the male parents consistently produce male offspring that are more resistant to ocean acidification.  If males like these exist within this population, then the species has the capacity to evolve in response to ocean acidification, instead of going extinct! This is a very big deal, and could potentially be very hopeful. Even if we don’t get the results that we are hoping for, the results of this research could inform policy and management decisions.

Literature Cited:

Wang, Z. A., Wanninkhof, R., Cai, W., Byrne, R. H., Hu, X., Peng, T., & Huang, W. (2013). The marine inorganic carbon system along the Gulf of Mexico and Atlantic coasts of the United States: Insights from a transregional coastal carbon study. Limnology and Oceanography, 58(1), 325-342. doi:10.4319/lo.2013.58.1.0325

Thank you to the National Science Foundation and College of Charleston’s Grice Marine Laboratory for funding my project. And, special thanks to Dr. Bob Podolsky for being a wonderful and supportive mentor!

 

Invasive Species: Friend or Foe

Melanie Herrera,  University of Maryland – College Park

Invasive species…. Haunting, domineering, and downright evil. Or are they? Unlike the infamous Zebra Mussels, dominating the Great Lakes, or Fire Ants, constantly wreaking havoc, Gracilaria Vermiculophylla, are giving invasive species a good name. Don’t get me wrong, invasive species infuriate me just as much as the next guy; but Dr. Tony Harold and I are here to draw out the benefits of this invasive sea grass to baby fish.

Unlike the native, simpler sea grass previously occupying Charleston Harbor, Gracilaria is characterized by coarse branching structures that appeal to many species of fish as protective homes. We are particularly interested in fishes in the larval and juvenile stages (the young ones) that associate with these complex habitats. Having access to more protective sea grass, such as this invasive, in these vulnerable life stages can help determine how many of these little guys make it into adulthood. Similar macro-algae to Gracilaria, such as seaweeds, have been known to be preferable hideouts for larvae and juveniles, reducing the pressures of predation. Since Gracilaria is on the rise in our local estuary, the Charleston Harbor, it’s important to find out the role they play in keeping our fish alive and well.

Our project is designed to better understand the level of association of local fish such as Gobies, Atlantic Menhaden, Atlantic Silversides, and other estuary-occupying fishes, with Gracilaria. We will compare abundance and distribution of young fish in dense patches of Gracilaria to sparse patches. Maybe these young fishes prefer the familiarity that native sea grass and open water brings. Or maybe Gracilaria’s “new and improved” design is too advantageous to resist. After we figure this out, we can go on sustainably managing local fish critical to commercial and recreational use and condemning the rest of the invasive species.

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An example of a collection site characterized as a “dense” habitat of Graclaria vermiculophylla.  Photo Credit: Melanie Herrera

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An example of a collection site characterized as a “sparse” habitat of Gracilaria vermiculophylla. Photo Credit: Melanie Herrera

 

Thank you so much to my mentor Dr. Tony Harold and his lab for his advice and guidance. Thank you to Mary Ann McBrayer for helping me facilitate this project. This research is funded through the National Science Foundation and College of Charleston’s Grice Marine Lab.

 

Works Cited

Munari, N. Bocchi & M. Mistri (2015) Epifauna associated to the introduced Gracilaria vermiculophylla (Rhodophyta; Florideophyceae: Gracilariales) and comparison with the native Ulva rigida (Chlorophyta; Ulvophyceae: Ulvales) in an Adriatic lagoon, Italian Journal of Zoology, 82:3, 436-445, DOI: 10.1080/11250003.2015.1020349