Larval Phthalate Soup

Samuel Daughenbaugh, DePauw University

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).

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.

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.

9,000 Larvae Later…

Jaclyn Caruso, Salem State University

Findings: In my previous post, I talked about how counting larval stages and measuring their skeletons could help us determine the lethal and sublethal effects of preservatives used in cosmetics and other personal care products on development. What we found was pretty surprising.

After two days of development in normal conditions, sea urchin larvae should be in the pluteus stage and have 2 or 4 arms. The arms are important because they are surrounded by bands of cilia that help the larva swim and feed. In the controls and at the lowest concentrations that we tested (0.1, 1, and 10 parts per million), the majority of the larvae successfully reached this stage. However, things got interesting at about 32 ppm.

We found that at concentrations at and above 32 ppm, the larvae generally grew shorter arms, had a smaller body size, and were more asymmetric. Any of these abnormalities could potentially have fatal consequences for the larvae. We also found that at high enough concentrations of the preservatives, development will fail completely. At concentrations of 1000 ppm, almost 100% of the fertilized eggs that we added to the jars didn’t develop past the early cleavage stages. This means that development was stopped almost instantly.

If you remember back to my first post, we wanted to test how parabens compared to the newer alternatives they were replaced with. Of the three preservatives we tested, the paraben caused changes in growth and failure of development at the lowest concentrations. The other two preservatives fared slightly better, suggesting that the personal care products industry may have made a good decision by switching. However, because we saw harmful effects in all three preservatives, we can’t say that they are completely safe for marine life at these concentrations.

This summer has taught me a ton about scientific research. I always expected it to require a lot of time, patience, and dedication, and my expectations were absolutely confirmed. In total, we counted and categorized 8,919 larvae and measured 2,224. That’s a lot of long hours at the microscope, and a lot of data to analyze. But, our results were definitely worth it, and I’m greatly looking forward to my next research experience!

Left: normal preserved plutei in the 4- or 2-arm stage. Right: abnormal preserved individuals with incorrectly shaped skeletons or at early stages of development. Jaclyn Caruso, 2018

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Acknowledgements

Thank you to Dr. Bob Podolsky (CofC) for his mentorship, Dr. Cheryl Woodley (NOAA) for providing her procedures and resources, and Pete Meier (CofC) for teaching me the ropes of setting up aquaria. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

Clinking Glasses: Not Just for Toasts

Jaclyn Caruso, Salem State University

The Approach: How can sea urchins tell us whether preservatives used in cosmetics are harmful to the environment? Our approach involves the rhythmic clinking of glasses—but we’ll get to that.

As detailed in my previous post, concerns about the use of parabens as preservatives led to the introduction of new, “safer” alternatives, like phenoxyethanol and chlorphenesin. We are testing the effects of these preservatives on the early development of the purple sea urchin, Arbacia punctulata. Sea urchins are useful for laboratory studies because they are easy to rear and they have free-swimming larvae with distinct morphologies, allowing us to test for both lethal and subtle effects of these chemicals.

Because this is a first test with sea urchins and concentrations that induce a response have varied among studies, the concentrations of chemicals in seawater we are using span a large range—1000 to 0.1 parts per million (ppm). Including controls with no chemicals creates 34 jars per trial. Over the summer we will be able to complete several independent trials using different male-female pairs.

To collect sperm and eggs from male and female sea urchins, we apply a mild voltage with electrodes to the top of the sea urchin close to the gonopores, the small openings where gametes come out. Once we gather the sperm and eggs, we dilute them to specific concentrations, combine them, and let nature take its course! After an hour, we add fertilized eggs to each of the jars, which are stirred by glass paddles. The stirring allows larvae to get plenty of oxygen and to avoid settling at the bottom of the jar. The paddles have to be glass because plastics can potentially alter the results (ASTM, 2015). The whole apparatus has a pleasant clinking sound once running!

After two days, we collect the developing stages, preserve them with methanol, and keep them at ‑20°C until they are measured. Finally, the analysis begins! We load a counting chamber with the larvae and use a microscope to count the number of individuals that are in each stage of development. By this point, a larva should have 4 arms—known as the pluteus stage.

A preserved 4-arm pluteus larva. Scale bar = 0.1 mm. Jaclyn Caruso, 2018.

If larvae in the chemical jars are at earlier life stages compared to the controls, it suggests that the chemicals delay or stop development.

To look at more subtle effects, we are also measuring 20–25 individuals that have reached the larva stage per jar using 10 specific landmarks on the body. The microscope uses a camera lucida to project an image of the larva onto a digitizing pad that can record the location of landmarks in three dimensions. We use these data points to generate 3D models of each of the larvae to measure the size of the skeleton. This technique will allow us to test for sublethal effects of the chemicals that might not be obvious at first glance.

The camera lucida arm of the microscope sits over the digitizing pad. Jaclyn Caruso, 2018.

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Acknowledgements

Thank you to Dr. Bob Podolsky (CofC) for his mentorship, Dr. Cheryl Woodley (NOAA) for providing her procedures and resources, and Pete Meier (CofC) for teaching me the ropes of setting up aquaria. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

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References

ASTM (2015) ‘Standard Guide for Conducting Static Acute Toxicity Tests with Echninoid Embryos’, Astm, 131, pp. 1–2. doi: 10.1520/D7385-13.2.

Is “Paraben-Free” the Way to Be?

Jaclyn Caruso, Salem State University

In the wet lab, we wear a lei to remember to shut off the tank valve. Photo: Jaclyn Caruso, 2018.

The problem: Have you brushed your teeth today? Washed your hair? Put on deodorant, perfume, makeup, or lotion? If you (hopefully) have, you’ve used a cosmetic. According to the FDA, anything that is applied to your body with the intention of cleansing or beautifying it is a cosmetic (FDA, 2018). Because this category covers such a wide variety of products, it’s easy to imagine just how many are used worldwide on a daily basis.

Like anything people use, cosmetics are eventually washed off, and often end up in the ocean from sewage drains and wastewater treatment plants. The problem with this pollution is that cosmetics contain preservatives. Although these components prevent the growth of bacteria and mold, their actions when introduced to natural systems are not tested at great lengths when considering their frequent use. Until a few years ago, the most common preservatives were a group of chemicals called parabens.

But, you’ve probably heard of at least one product that claims to be “paraben-free.” This aversion to parabens followed a landmark study in 2004 which showed that parabens have the potential to accumulate in human breast tumors (Darbre et al., 2004). The authors explicitly stated that the source of the parabens (methylparaben, mainly) was unknown, but many people were shaken by the findings. Cosmetics manufacturers began changing their formulations by using newer, “safer” preservatives like 2-phenoxyethanol and chlorphenesin (Bressy et al., 2016). However, these alternative preservatives have not been extensively tested for their effects on marine animals, which may be at risk when these chemicals enter the ocean.

Collecting sea urchins at Breach Inlet! Photo: Dr. Podolsky, 2018.

My research this summer aims to explore the effects that these alternative preservatives have on marine animal development. We will use the local sea urchin Arbacia punctulata as a model, because it is easily collected in the wild and reared in the lab. Like many marine animals, A. punctulata is a broadcast spawner—males and females release their sperm and eggs into the water column. After fertilization, the embryos develop into free-floating larvae, which are highly sensitive to pollutants.

We will expose the sea urchin larvae to various concentrations of each chemical to test whether larval development is affected negatively by the chemicals. Such negative effects could inhibit the ability of sea urchins to develop properly, leading to death or inability to mature to adulthood. If we see effects in sea urchins, there is a possibility of similar effects in other species that may be more directly important to humans, like fish and crustaceans.

Our ultimate goal is to explore whether products that are safer for people are safer for the marine environment. If they are—great! If not, we need to think critically about the products we use that end up in the ocean, because human and ocean health are inextricably linked. Healthy oceans, for example, provide us with food, medications, recreation, and more (NOAA, 2018).

Left: A beautiful specimen of Arbacia punctulata. Scale bar = 1 cm. Right: Dr. Podolsky demonstrating how to induce spawning in sea urchins using a low voltage across the gonopores. Photos: Jaclyn Caruso, 2018.

 

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Acknowledgements

Thank you to Dr. Bob Podolsky (CofC) for his mentorship and endless patience, Dr. Cheryl Woodley (NOAA) for graciously offering her procedures and resources, and Pete Meier (CofC) for teaching me the ropes of setting up aquaria. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

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References

Bressy, A. et al. (2016) ‘Cosmet’eau—Changes in the personal care product consumptionpractices: from whistle-blowers to impacts on aquatic environments’, Environmental Science and Pollution Research. Environmental Science and Pollution Research, 23(13), pp. 13581–13584. doi: 10.1007/s11356-016-6794-y.

Darbre, P. D. et al. (2004) ‘Concentrations of Parabens in human breast tumours’, Journal of Applied Toxicology, 24(1), pp. 5–13. doi: 10.1002/jat.958.

FDA (U.S. Food & Drug Administration) (2018) ‘Is It a Cosmetic, a Drug, or Both? (Or Is It Soap?)’ https://www.fda.gov/Cosmetics/GuidanceRegulation/LawsRegulations/ucm074201.htm (accessed Jul. 2, 2018).

NOAA (National Oceanic and Atmospheric Administration) (2018) ‘What does the ocean have to do with human health?’ https://oceanservice.noaa.gov/facts/ocean-human-health.html (accessed Jul. 2, 2018).