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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All Shrimp Go To Heaven

Carolina Rios, New York University

The Approach: In my previous post, I discussed the negative impact that polychlorinated biphenyls (PCBs), long-lived chemical contaminants, can have on marine ecosystems and human health. Currently, I am working to verify a proposed model to estimate the impact that PCB contamination has on benthic marine invertebrates.

One of the issues with this proposed model is that it is based on data generated from the 1970s; and the analytical methods now available to scientists are much more sensitive and precise. To verify this model, we will generate contemporary data by running a series of short (acute) toxicity tests.

Testing

Test setup for grass shrimp (P. pugio)

In these acute toxicity tests, we measure the response of three species of marine invertebrates to PCBs. The three organisms that we are testing are grass shrimp (Palaemonetes pugio), amphipods (Leptocheirus plumulosus), and mysids (Mysidopsis bahia). We will be measuring mortality from PCB contamination. The standard tests that we are running consists of 6 concentrations, ranging from 6.25 ppb (parts per billion) to 420 ppb. It is important that we also have a control, so that we can understand the response of the organisms unaffected by PCBs. For the grass shrimp and amphipods, the test will run for 96 hours and we will renew the PCB solutions every 24 hours. Samples will be taken for chemical analysis at 0 hours, 24 hours, and 72 hours, so as to measure both the loss of PCBs over the 24 hour period, as well as the consistency of dosing. Loss of PCBs can be attributed to PCBs binding to the glassware and differences in dosing can be attributed to user variability. For the mysids, the test will also run for 96 hours, but the dosing solutions will not be renewed after the initial dosing. Samples will be taken at 0 hours, 48 hours, and 96 hours, so as to measure the loss of PCBs over the 96 hour period. For all tests, mortality will be recorded every 24 hours until the end of the test.

Analysis

Solid Phase Extraction apparatus. Dosed samples are within the large reservoirs at the top of the apparatus. PCBs will be isolated on the nonpolar solid phase, which consists of the smaller columns below the reservoirs.

The PCBs from the collected samples will be isolated through solid phase extraction. Solid phase extraction consists of a nonpolar solid phase and a polar liquid phase; similar to how oil cannot be mixed into vinegar, PCBs are not very soluble in water. As PCBs are nonpolar and hydrophobic, they will bind to the solid phase. The PCBs can then be lifted off of the column by running a nonpolar solvent (ethyl acetate) through the nonpolar solid phase. The sample is then analyzed using gas chromatography-mass spectrometry (GC-MS). The essential concept of GC-MS is that molecules will separate based on differences in size. This is how the amount of each individual PCB is determined, which can then be used to calculate an actual concentration. Analysis of the chromatograph can give more accurate concentrations, allowing us to understand how concentrations vary over time. This will give us a better understanding of the relationship between dose concentrations and the mortality response.

Acknowledgements

I would like to thank Dr. Ed Wirth and Brian Shaddrix for their continued guidance and support, as well as my co-mentor Dr. Paul Pennington. Supported by the Fort Johnson REU Program, NSF DBI-1757899.

America’s Continuing Toxic Legacy: Quantifying the Impact of PCBs

Carolina Rios, New York University

The Problem: Polychlorinated biphenyls (PCBs) are a legacy contaminant that pose a threat to human health. PCBs are classified as possible carcinogens and are known to affect neurological development and contribute to diabetes (Xue et. al 2014). Additionally, PCBs are known to alter liver function, impact immune and thyroid function and effect reproduction, as well as gastrointestinal and respiratory health (Hansen 1987). Humans are largely exposed to PCBs by consuming contaminated animal products, such as contaminated fish or dairy (Xue et. al 2014). Similarly, dolphins sampled near Brunswick, Georgia were found to have elevated levels of PCBs, likely due to the consumption of contaminated fish (Wirth et. al 2014). The hydrophobic properties of PCBs mean that they bioaccumulate and can be found in aquatic organisms in concentrations thousands of times greater than the surrounding environment (Nimmo et. al 1974). PCBs also biomagnify up trophic levels in the web, and can be found in even greater concentrations in predator species, as they consume contaminated prey. Thus, the effects of PCBs can be felt throughout the ecosystem.

As PCBs are still relevant contaminants, it is important that we are able to quantify injury associated with PCB levels found in the coastal environment. It is particularly difficult to assess this risk to benthic marine invertebrates (organisms that live in the interface between the bottom of the ocean and the sediment). Therefore, a model has been proposed that predict rates of injury to benthic marine invertebrates (Finkelstein. et al 2017). This model was created through an extensive literature search. However, the data collected as the basis of this mathematical model dates back to the 1970s. In order to verify this model, it is important that we generate new data to verify the accuracy of the model in predicting benthic marine invertebrate injury.

Biphenyl structure. PCBs consist of a biphenyl structure of varying degrees of chlorination. Created using Chemdraw

PCBs were produced for industrial use, such as dielectric fluids, hydraulic fluids, and heat transfer fluids. From 1929 to 1977, PCBs were produced by the Monsanto Corporation in the US, before being removed from production due to negative effects on human health and the environment. Of the 1.4 billion pounds of PCBs produced in the US, it is estimated that one third has entered the environment (Safe et. al 1987). Though they are no longer being produced, their stability and long half-life means that PCBs are still present and continue to pose a real threat to the environment.

Acknowledgements

I would like to thank Dr. Ed Wirth and Brian Shaddrix for their continued guidance and support, as well as my co-mentor Dr. Paul Pennington. Supported by the Fort Johnson REU Program, NSF DBI-1757899.

References

Finkelstein, K. & Beckvar, N. & Dillon, T. (2016). Benthic injury dose-response models for PCB-contaminated sediment using equilibrium partitioning. Environmental toxicology and chemistry, 36 (5), pp. 1311-1329. doi:10.1002/etc.3662.

Hansen, L. (1987). Polychlorinated Biphenyls: Environmental Occurrence and Analysis. In S. Safe (Ed.), Polychlorinated Biphenyls (PCBs): Mammalian and Environmental Toxicology, pp. 15-48. Berlin, Heidelberg: Springer Berlin Heidelberg.

Nimmo, D. & Forester, J. & Heitmuller, P & Cook, G. (1974). Accumulation of Aroclor 1254 in grass shrimp (Palaemonetes pugio) in laboratory and field exposures. Bulletin of environmental contamination and toxicology. 11 (4) pp. 303-308. 10.1007/bf01684932.

Safe S., & Safe, L., & Mullin, M. (1987). Environmental Toxicology of Polychlorinated Biphenyls. In S. Safe (Ed.), Polychlorinated Biphenyls (PCBs): Mammalian and Environmental Toxicology, pp. 1-13. Berlin, Heidelberg: Springer Berlin Heidelberg.

Wirth, E.F., & Pennington, P.L., & Cooksey, C., Schwake, L., & Hyland, J., & Fulton, M.H. (2014) Distribution and sources of PCBs (Aroclor 1268) in the Salepo Island National estuarine research reserve. Environmental Monitoring and Assessment. 186 (12) pp. 8717-8726. doi:10.1007/s10661-014-4039-4

Xue, J., & Liu, S., & Zartarian, V., & Geller, A., & Schultz, B. (2014). Analysis of NHANES measured blood PCBs in the general US population and application of SHEDS model to identify key exposure factors. Journal of Exposure Science and Environmental Epidemiology. 24 (6) pp. 615-621. doi: 10.1038/jes.2013.91

Cells and Instruments, but no Folsom Prison Blues

Brian Wuertz, Warren Wilson College

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In my previous post, “Hiding in plain sight”, I introduced DOSS, a compound that has been recently identified as a probable obesogen. We are especially concerned about the potential of this compound to cause obesity symptoms in developing children through exposure from their mothers. While DOSS is in many products we use daily, such as homogenized milk and makeup products, it is commonly prescribed to pregnant women in the form of Colace stool softener. I am investigating both how much DOSS is in certain places in the body and how it may promote obesity.

One of the main concerns about obesity is that it elevates the risk of developing other diseases such as diabetes or cancer by causing a state of chronic inflammation (Bianchini 2002).  Chronic inflammation in  adipose tissue is regulated by immune cells, including macrophages. Macrophages are immune cells found throughout the body that help to fight against infection by recognizing invading bacteria and engulfing them in a process called phagocytosis, literally meaning to eat the other cells. In addition to phagocytosis macrophages are important regulators of the larger inflammatory response by secreting proteins that tell other cells to initiate or maintain a state of inflammation (Fujiwara 2005). This inflammatory reaction may be induced by DOSS. We have seen evidence of increased inflammation and obesity in mice treated with DOSS, so in order to figure out what causes that I am focusing on macrophages because of the way they regulate inflammation.

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I am isolating macrophages from breast milk samples under this hood in a sterile environment to make sure they are not contaminated with bacteria.

One way to study the inflammatory response of macrophages is to expose them to DOSS and then see if they produce the inflammatory proteins. Instead of trying to measure the secreted proteins, we can measure how much RNA is made in the cell. The RNA is the translator molecule that takes the plan for the protein from the DNA and makes it available for the cell to read and make the right protein. I identified genes for four different inflammatory proteins to measure the RNA so we can test if DOSS causes the macrophages to make more of any of them. I am testing macrophages that I am isolating from human placenta and breast milk tissue because the developing child is influenced by inflammation in the placenta and breast milk. Macrophages in these tissues could be the source of inflammation that influences how the child develops.

Okay so we have talked about cells, but what about the instruments? In my last post I introduced my instrument of choice, but did not call it that. It is not a guitar or a saxophone, but the HPLC, or high performance liquid chromatograph. This is simply a fancy instrument used to separate chemical compounds by forcing them through a tiny filter column filled with tiny beads. Some compounds stick more to the beads than others, so when you flow a liquid through the column the compounds come out of the column at different times. It is essential to separate the compounds in a sample because then you can measure the amounts of individual compounds.

We want to know where DOSS goes in the body, so we need to be able to measure how much of it is in a sample. I am working to get a system up and running to measure the amounts of DOSS in samples from different cells and tissues. We want to be able to measure DOSS in humans and in marine mammals such as dolphins. Dolphins are exposed to DOSS in the COREXIT oil spill dispersal agent that is applied to large and small scale oil spill issues along coastlines and in harbors. Dolphins are an important sentinel species, meaning that they can provide insight into human health issues.

I have to prepare a column and get the right mixture of solvents to make DOSS come off of the column in a timely fashion and in a way that we can measure it. The measurement is actually done with a mass spectrometer, which measures allows us to identify the compound based on how much it weighs. The number of atoms and types of atoms in the compound determine the mass of the compound. This mass is how the instrument measures the compound. The technique I am using is therefore called liquid chromatography mass spectrometry or LC-MS and the instrument is also referred to by LC-MS. Hopefully by the end of the summer I will be able to find beautiful data with this instrument that will make a coherent tune rather than a jumble of notes.

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This is the MS part. It measures the mass of the compound and then breaks it apart and measures the mass of the pieces of the compounds and the amount of the compounds.

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This is the LC or liquid chromatography part of the LC-MS instrument. Most of the work is figuring out the best solvent system to the sample through the small column with the red tag on it.

Funding for this REU program is generously provided by the National Science Foundation and hosted by the College of Charleston. Dr Demetri Spyropoulos at the Medical University of South Carolina is graciously hosting my research project and providing mentorship.

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References:

Bianchini, F., Kaaks, R., and Vainio, H. (2002). Overweight, obesity, and cancer risk. The Lancet Oncology 3, 565–574.
Fujiwara, N., and Kobayashi, K. (2005). Macrophages in Inflammation. Current Drug Target -Inflammation & Allergy 4, 281–286.

Our complicated relationship with chemicals

Nina Sarmiento, Binghamton University

Chemicals found all around us that have been altered, mimicked, and synthesized to be added to our products, are behind the success of our modern society. They have made our plastics strong, our crops prosperous, and our medicines effective. But I have always wondered about the toxicity of these chemicals.  When you look at their biological activity, a chemical might possess the potential to do harm, like interfere with biological processes. The safety of a potentially harmful chemical is based on exposure and dose. It is important to know if we are touching it, eating it, or breathing it in, and for what period of time. The study of evaluating the harmful effects of substances on exposed organisms is what toxicology is all about. They have such an important job because their findings influence what we know is safe and unsafe, for us and organisms all around us.

I learned early on from pursuing biology that we are exposed to many things we are unaware of. Not only are we exposed to potentially harmful chemicals, but we facilitate exposure to other living organisms that may more sensitive. Take dogs for simple example. The toxic dose of something like chocolate for humans is very high, whereas leaving a small amount of chocolate out for a dog to eat could easily kill it.  Rachel Carson is someone I greatly admire, whose work on the pesticide DDT also exemplifies this reality. Food crops were the target for DDT, but birds were indirectly ingesting it, explaining the decline in the Bald Eagle population.  She is one of the people that sparked my interest in ecotoxicology, looking at the effects of harmful substances on ecology, not just humans.

unnamedThis is an example of some of the questions ecotoxicologists ask when there is a potentially harmful substance found in the environment. Photo credit: globe.setac.org.

Here is a chemical product you may not suspect as a threat, sunscreen. In sunscreens, UV filters protect you from getting burned, but also can act as endocrine disruptors, altering hormones and growth (1). Sunscreens are only meant for human skin, however they end up in our lakes, rivers and oceans through swimming or through waste water treatment effluent (2). Unintentionally, many more organisms become exposed.

Toxic-Sunscreen

Photo credit: thesleuthjournal.com

In my project I will be using sea urchins as a model organism to study the effects sunscreens might be having on coral reefs.  I am learning how to preform toxicity tests on sea urchin sperm and embryos which involve an exposure period with sunscreen formulations and then evaluation of effects. I hope to investigate if the chemicals from sunscreens in the water can have negative impacts on coral reproduction.  My work can potentially help create understanding of how humans are contributing to coral reef decline, and influence others to take action to protect them.

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This is a picture of sunscreen water accomodated fractions (WAFs) I am making. They are a mix of sunscreen and seawater and I will be exposing the sea urchin embryos to each solution!

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This is me in the lab with a microscope I use to look at sea urchin sperm and embryos! Photo by Bob Podolsky

My research is funded by the National Science Foundation and College of Charleston partnered with National Oceanic and Atmospheric Administration

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Works cited:

1 Krause M.,, Klit A., Jensen M., Soeborg T., Fredrickson H., Schlumpf M., Litchensteiger W., Skakkebaek N E., Drzewieck K T. 2012. Sunscreens: are they beneficial for health? An overview of endocrine disrupting properties of UV-filters. International journal of andrology. 35 424-436.

2Kyungho C., Kim  S. 2014. Occurances, toxicities, and ecological risks of benzophenone-3, a common component of organic sunscreen products: a mini review. Environment International. 70 143-157.