The Effects of Glyphosate on the Embryonic Development of Zebrafish. Ali Brodie Pardeeville High School

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1 The Effects of Glyphosate on the Embryonic Development of Zebrafish Ali Brodie Pardeeville High School Abstract This experiment tested the effects of glyphosate, a common household and agricultural chemical, on the mortality and hatch rates of zebrafish embryos. Because zebrafish are model organisms and develop similarly to humans, 240 embryos were spawned and tested for two one week trials to better understand the hazards of glyphosate exposure in human embryos. It was found that glyphosate had little impact on mortality and hatch rates in small concentrations (18 µg/ml, 0.18 mg/ml, and 1.8 mg/ml). In higher concentrations, such as 1.8 mg/ml and 0.18 mg/ml, glyphosate had a large impact on the survival rate and hatch rate of the embryos. Some abnormalities and developmental issues in the zebrafish embryos arose in the presence of glyphosate, and could possibly occur in the development of human embryos. Introduction Glyphosate, the active chemical in Roundup, is a systemic herbicide used in agriculture on many different types of crops, as well as in common circumstances such as weed killing in lawns or gardens. Nearly all corn, cotton and soy fields in the United States are treated with Roundup to prevent weed growth and to kill unwanted weeds. Approximately 300 million pounds of glyphosate were used in 2012 (Grossman, 2015). The production of crops since the beginning usage of glyphosate has increased dramatically, yet, with more toxic glyphosate in the environment, the health of exposed organisms decreases. Glyphosate can pollute freshwater, can cause internal health problems if inhaled, and if eaten, can cause diseases and other issues. Glyphosate residues, found in most commonly consumed foods in the Western diet courtesy of GE sugar, corn, soy and wheat, enhance the damaging effects of other food borne chemical residues and toxins in the environment to disrupt normal body functions and induce disease (Mercola, 2013). The good bacteria found internally in humans is damaged and diseases can be inflicted if glyphosate enters the body (Mercola, 2013). This toxicology experiment tested the effects of glyphosate on 240 developing zebrafish embryos, spawned from fish raised at the University of Wisconsin in Milwaukee and the Pardeeville High School biology classroom. The fish were tested because zebrafish are model organisms, and in the embryonic stage, develop similarly to humans. Because they develop much faster than humans (with a rate of 72 hours), testing takes a week, compared to months in a human pregnancy. It was hypothesized that zebrafish developing in concentrations of glyphosate would have an increased mortality rate and decreased hatch rate. One goal of the procedure was to find the lethal concentration of glyphosate. If zebrafish are exposed to concentrations of 1

2 glyphosate and are affected negatively, then there is a possibility that too much exposure in pregnant women could affect the development of human embryos as well. Materials and Methods The materials needed for this experiment include: 6 stock solutions of glyphosate in 6 different bottles (the solutions are 1.8 mg/ml, 0.18 mg/ml, 18 µg/ml 100 µg/ml, 60 µg/ml, and 18 µg/ml); a beaker for dead embryos and liquid disposal; a Sharpie; Instant Ocean/embryo media solution; a disposable pipette, minimum bore, 1.5 mm for transferring eggs to observation container and manipulating them in the container; a disposable pipette, large bore, 3 ml; 3x4 well plate; a dissecting microscope; gloves to transfer the chemical safely and prevent contact with skin; and goggles to prevent chemical contact in eyes. All of these materials from the first trial were provided by the Wisconsin Inquiry based Scientist Teacher Education Partnership (WInSTEP) Program, which is part of the NIH Science Education Partnership Award (SEPA) Program administered by the University of Wisconsin Milwaukee and the Children's Environmental Health Sciences Core Center. In the second trial, the embryos were provided by the Pardeeville High School biology classroom zebrafish. First, the 3x4 well plate was labeled with the group name using the Sharpie. Column 1 of the well plate was the control, filled halfway with embryo media solution. Column 2 was filled halfway with 18 µg/ml (low) of glyphosate, column 3 with 0.18 mg/ml (medium) of glyphosate, and column 4 with 1.8 mg/ml (high) of glyphosate, using the transfer pipette, goggles and gloves to prevent chemical contact with skin. One hundred and twenty zebrafish embryos were obtained after being spawned on the first day of the experiment. Ten embryos were placed in each well with the large pipette. Each following day of the procedure, the wells were checked for dead embryos using a microscope. If there were dead embryos in the wells, they were removed using the large pipette and disposed in the beaker. The embryo media and concentrations of glyphosate the wells were replaced using the transfer pipette. The old liquids were disposed of in the beaker. Numbers of live zebrafish in each well were recorded, as well as the numbers of dead embryos and observations of deformities and abnormalities. The process of checking wells, observing, and replacing water was repeated for three days. Two weeks later, the whole procedure was repeated, but the concentrations were changed to 18 µg/ml (low) in column 2, 60 µg/ml (medium) in column 3, and 100 µg/ml (high) in column 4. The wells were checked for four days. For data analysis, a t test (GraphPad software, found here: ) was used to calculate the statistical significance of the recorded data. 2

3 Results Glyphosate was placed in wells with ten zebrafish embryos to test whether or not the chemical played a detrimental role in the development of the fish. The independent variables were the concentrations of glyphosate in the wells, and the dependent variables were the mortality rates and hatch rates of the embryos. The control was column 1, with embryo media. The results showed that glyphosate did statistically affect the mortality rate (Table 1) and hatch rate (Table 2) to an extent. The higher concentrations, 1.8 mg/ml and 0.18 mg/ml, were extremely statistically significant and much more fatal than the concentrations of the second trial (18 µg/ml, 60 µg/ml, and 100 µg/ml), but these concentrations had little effect to the embryos in those wells. This means that in high concentrations, zebrafish are unlikely to survive. However, if the concentrations are 100 µg/ml or lower, then the fish are likely to survive but may have developmental deformities (Figure 3). The mortality rate is increased with the highest concentrations tested. Surprisingly, with smaller concentrations (100 µg/ml, 60 µg/ml, and 18 µg/ml), the embryos seemed to have survived better than even the control. The difference in each trial of 18 µg/ml is large, even though they are the same concentration. Treatment Table 1: Final Number of Live Fry in Weeks 1 and 2 Well 1 Well 2 Well 3 Average Standard Deviation Probability Result Control µg/ml p= Not Significant 0.18 mg/ml p = Extremely Significant 1.8 mg/ml p = Extremely Significant Control µg/ml p = Not Significant 60 µg/ml p = Not Significant 100 µg/ml p = Not Significant Table 1: The final number of live fry after week 1 and week 2. These numbers are shown in Figure 1. The first four rows in the table above show data from the first week trial, while the data in rows 5 8 are from the second week trial. 3

4 Figure 1: The averages of live fry compared to exposure of glyphosate concentrations. This graph clearly shows how much an effect is caused by large concentrations 1.8 mg/ml and 0.18 mg/ml. Treatment Table 2: Final Number of Hatched Fry in Weeks 1 and 2 Well 1 Well 2 Well 3 Average Standard Deviation Probability Result Control µg/ml p = Not Significant 0.18 mg/ml p = Extremely Significant 1.8 mg/ml p = Extremely Significant Control µg/ml p = Not Significant 60 µg/ml p = Not Significant 100 µg/ml p = Not Significant Table 2: The final number of hatched fry in week 1 and week 2. These numbers are shown in Figure 2. The first four rows in the table above show data from the first week trial, while the data in rows 5 8 are from the second week trial. 4

5 Figure 2 : The averages of hatched fry compared to the exposure of glyphosate concentrations. This graph shows how in high concentrations, 1.8 mg/ml and 0.18 mg/ml, glyphosate causes fewer embryos to hatch. In lower concentrations, the hatch rate increases. There is a very large standard deviation bar, however, indicating a large variation in the data. Figure 3: Figure 4: Figure 3: The image on the left shows a hatched fry and an unhatched embryo in a control well. Figure 4 : The image on the right shows a deformed hatched fry in well B4, in the high concentration of 100 µg/ml of glyphosate. There is a normal hatched fry near the top of the image for comparison. 5

6 Discussion The hypothesis was that glyphosate would affect the development of zebrafish, including the mortality rate and the hatch rate of the embryos. This experiment did indeed provide evidence to support this hypothesis because in higher concentrations (1.8 mg/ml and 0.18 mg/ml), more embryos died or did not hatch; however, not all concentrations of glyphosate were lethal to the developing embryos. In concentrations 100 µg/ml or less, the presence of glyphosate was not significant to the mortality rate or hatch rate, but rather aided both in the second procedure. This could be due to an embryonic abnormality or human error and this experiment should be repeated to see if it would yield the same results. It is expected that between 100 µg/ml and 0.18 mg/ml is an unknown lethal concentration that would hinder mortality rate and hatch rate similarly. The concentrations used were not accurate enough to find this missing level, but if the experiment was to be repeated, a larger time limit would be beneficial. Other errors observed include not using the same measurements of concentrations in each well, or using dirty beakers and pipettes to transfer liquids and possible contamination of the water or embryo media solution. This experiment shows that, as zebrafish embryos develop similarly to human embryos, glyphosate could affect the developing embryos of pregnant women who are exposed to the chemical. Because glyphosate is such a commonly used chemical, contaminated food eaten by pregnant women or water sources around farms using Roundup on their crops could cause damages to her developing baby while still in the embryonic stage. Birth defects and abnormalities could occur, as well as a miscarriage or stillbirth. Women should be informed as to how much glyphosate they can be exposed to without having the chance of harming their baby. References Dr. Mercola. "The Horrific Truth About Monsanto's Roundup Herbicide."Mercola.com. Mercola.com, 9 June Web. 10 Jan < roundup herbicid e.aspx>. Grossman, Elizabeth. "What Do We Really Know About Roundup Weed Killer?" National Geographic. National Geographic Society, 23 Apr Web. 10 Jan < glyphosate roundup herbicide we eds/>. 6