The Effect of Fertilizer Runoff Conditions on the Development

of Danio rerio EmbryosErica Gillespie

Senior Research Advisor: Dr. GibbsStetson University

What is Eutrophication?

http://www.ecodetail.net.au/wp-content/uploads/2012/07/eutrophication1.jpg

Danio rerio (Zebrafish) Transparent embryos Short developmental period

(72 hours) Development of Zebrafish is

well-known. Near completion of of the zebrafish genome allows scientists to identify end points of toxicity (Hill et al., 2005)

Freshwater Fish are less tolerant of nitrite toxicity than marine animals

http://fc06.deviantart.net/fs39/i/2008/348/9/c/Zebra_Fish_Embryo_by_IbieMonstah.jpg

Environmental Protection Agency (EPA) Guidelines

Natural levels of nitrate in surface waters seldom exceed 0.1 mg/l of N, but waters influenced by human activity normally contain up to 5 mg/l of N (Chapman, 1992)

National drinking water standards are 10 µg N/L of nitratesAfter reviewing existing nutrient criterions directed by the

Everglades Forever Act (Payne et al., 2012), The EPA and the Florida Department of Environmental Protection (FDEP) set a total phosphorous (TP) and total nitrogen (TN) limit of 10 µg/L. areas of Lake Okeechobee exceed 92 µg/L (Payne, 2012).

Studies show nitrate concentrations of 10 mg NO3 – N/L (USA federal maximum level for drinking water) can affect sensitive aquatic animals when exposed long term (Camargo &Alonso, 2006)

What are the Implications?

http://inhabitat.com/aquatic-dead-zones-produce-greenhouse-gas-300x-more-potent-than-co2/

Implications for Biota Study conducted by Thomas &

Rahman (2010) focused on testicular development and sperm production in the northern Gulf.

Due to low oxygen levels production of sperm declined, the fish began to have smaller lumens, and even experience a 50% decrease in testicular growth.

Impact population abundance if they cannot reproduce enough to replenish the population http://www.chartingnature.com/img/fish-

illustration-prints/atlantic-croaker-2085.jpg

Nitrogen in Reclaimed WaterReported by Institute of Food and Agricultural Sciences

http://edis.ifas.ufl.edu/ae479

Deland Water Utilities Water Quality Report 2011 & 2013

http://www.deland.org/Pages/DeLandFL_PSUtilities/index

HypothesisZebrafish embryos exposed to fertilizer runoff

conditions would have a dose-dependent response to fertilizer that will include retarded growth.

Methods

Control15 ml Spring Water

24 Hour Exposure

12.5x 25x

50x 100x0.001

g/L

150x0.0015

g/L

Control15 ml Spring Water

48 Hour Exposure

12.5x 25x

50x 100x0.001

g/L

150x0.0015

g/L

24/7 Exposure

20Control15 ml Spring Water

2000x=0.02

g/L

4000x = 0.04

g/L

5000x=0.05

g/L

10000x=0.1 g/L

Methods

ResultsThere was no effect of

fertilizer concentration on zebrafish mortality

http://s0ftpedia.net/files/zebrafish%20embryo&id=mix

Figure 1. Average Head Width (mm)

There was not a dose-dependent change in head width after exposure to fertilizer

Head Width T-Test ResultsControl vs. 2000x

0.00231Control vs. 4000x

1.88E-09Control vs.5000x

1.85E-09Control vs.10000x

0.448

4000x vs. 5000x 0.4614000x vs. 10000x

0.143

2000x vs. 4000x

0.00644

2000x vs. 5000x

0.00746

2000x vs. 10000x

0.264

5000x vs. 10000x

0.146

Table 1. P Values from T-Test for Head Width (mm) comparing the control to upper solution

concentrations

Table 2. P Values from T-Test for Head Width (mm) comparing 2000x to upper solution

concentrations

Table 3. P Values from T-Test for Head Width (mm) comparing upper solution

concentrations

Figure 2. Average total length (mm) from tip of head to end of tail.

There was a significant threshold response of body length to fertilizer concentration

Total Length T-Test Results

Table 4. P Values from T-Test for Total Length (mm) comparing the control to solution

concentration

5000x vs 10000x

0.421

4000x vs 5000x 0.1424000x vs 10000x 0.202

Table 5. P Values from T-Test for Total Length (mm) comparing upper solution

concentrations

Figure 3. Average body width (mm) from widest point.

There was a significant threshold response of body width to fertilizer concentration

Body Width T-Test Results

4000x vs. 5000x

0.387

4000x vs. 10000x

0.4665000x vs 10000x

0.356

Control vs. 2000x

0.00242

Control vs. 4000x

1.01E-07

Control vs. 5000x

3.94E-08

Control vs. 10000x

1.8E-07

Table 6. P Values from T-Test for Body Width (mm) comparing the control to solution

concentrations

Table 7. P Values from T-Test for Body Width (mm) comparing upper solution

concentrations

ConclusionsThere was no statistically significant difference in

zebrafish mortality exposed to varying fertilizer concentrations

There were no dose-dependent responses to the fertilizer, however, there were strong threshold responses (reductions) of body length and width starting at 4000x.

Concentrations used here exceeded EPA safe levels but with additional research can compare to local values in reclaimed water/irrigation.

Future ResearchWider ranges to determine a specific threshold.Have adult zebrafish spawn in fertilizer conditions so

embryos continuously exposed to run-off conditions. Would reproducing in these conditions cause greater growth deformities?

Further morphological measurements (head density, top of head to bottom, etc.)

These measurements focused on external growth, possible internal issues with development?

Replicate Local Values/ Reclaimed water

AcknowledgementsStetson UniversityBiology DepartmentDr. GibbsFriends and Family

ReferencesCamargo J. & Alonso A. (2006). Ecological and

toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: A global assessment. Environment International. 32(6): 831-849.

Chapman, D. 1992. Water quality assessments. Chapman and Hall, London, UK.

Payne G., Weaver K., Xue S. (2012). Chapter 2C: Status of Phosphorous and Nitrogen in the Everglades Protection Area. 2012 South Florida Environmental Report. 1 (2): 2C-1 – 2C-30).

Questions?