1. The Effect of Fertilizer Runoff
Conditions on the Development
of Danio rerio Embryos
Erica Gillespie
Senior Research Advisor: Dr. Gibbs
Stetson University
3. 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_Embr
yo_by_IbieMonstah.jpg
4.
5. 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 nitrates
After 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)
6.
7. What are the Implications?
http://inhabitat.com/aquatic-dead-zones-produce-greenhouse-gas-300x-more-potent-
than-co2/
8. 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
9. Nitrogen in Reclaimed Water
Reported by Institute of Food and Agricultural Sciences
http://edis.ifas.ufl.edu/ae479
10. Deland Water Utilities Water
Quality Report 2011 & 2013
http://www.deland.org/Pages/DeLandFL_PSUtilities/index
11. Hypothesis
Zebrafish embryos exposed to fertilizer runoff
conditions would have a dose-dependent response to
fertilizer that will include retarded growth.
18. Results
There was no effect of
fertilizer concentration on
zebrafish mortality
http://s0ftpedia.net/files/zebrafish%20embr
yo&id=mix
19. Figure 1. Average Head Width (mm)
There was not a dose-dependent change in head width after exposure to
fertilizer
20. Head Width T-Test Results
Control vs. 2000x 0.00231
Control vs. 4000x 1.88E-09
Control vs.5000x 1.85E-09
Control vs.10000x 0.448
4000x vs. 5000x 0.461
4000x 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
21. 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
22. 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 0.142
4000x vs 10000x 0.202
Table 5. P Values from T-Test for Total Length
(mm) comparing upper solution concentrations
23. Figure 3. Average body width (mm) from widest point.
There was a significant threshold response of body width to fertilizer
concentration
24. Body Width T-Test Results
4000x vs. 5000x 0.387
4000x vs. 10000x 0.466
5000x 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
25. Conclusions
There 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.
26. Future Research
Wider 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
28. References
Camargo 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).
x-=10 micrograms per liter
Reclaimed levels are around those levels –IFIS UF
Retention ponds->could potentially have concentrated fertilizers if water is all run off – issue that reinforces reclaimed run off takes this level of fertilzer in reasonable range to be concerned.
Eutrophication Process
Cascade Effect: increased algal blooms deflects sunlight which doesn’t reach benthic vegetation to complete photosynthesis to replenish depleted oxygen levels.
-Their transparent embryos make it relatively easy to study deformities during their short developmental period.
-Since the development of zebrafish is well known and their embryos are transparent, one can observe any deformities and focus on the developmental effects caused by the chemicals over another fish species.
-High levels of nitrogen can impair the ability of aquatic animals to survive and grow due to toxicity. Based on Camargo and Alonso’s study, fresh water animals are less tolerant of nitrite toxicity than marine animals
(when converted to grams per liter = 0.01 g/L)
Tried to contact Volusia County
First emailed Dinah Pulver and Environmental Writer for the News Journal
Article on The Indian River Lagoon in Peril back in May 2014 about the algal blooms and issues in regards to water quality
County Parks and Recreation Director, Tim Baylie. I sent him an email May 22, 2014. Still awaiting a response.
So I stayed with the 10 micrograms per liter.
Eutrophication is a growing issue across the United States.
Increased agriculture production leads to an influx of nutrients into our waterways.
The Atlantic croaker testes were compared to reference fish in other locations not experiencing hypoxic conditions. The reference fish did not experience the same reduction in reproductive organs; therefore, this occurs when the fish are exposed to hypoxic conditions. The main consequence to consider is how this may lead to a long-term impact on population abundance if they cannot reproduce enough to replenish the population (Thomas & Rahman, 2010).
-for areas not experiencing a full dead zone, are there still issues to look out for?
My experiment could be extrapolated to different species of concern depending on the area (economically beneficial, keystone ,etc).
Institute of Food and Agricultural Sciences
1 ppm = 1000 micrograms per liter
Set Up:
Spawning conditions include an acclimation period. Initially begin with males and females at a 12 hour day and 12 hour night. Increased the day hours by 30 minutes each day until I reached a 14 hour day and 10 hour night (spawning season conditions).
Measured 150 and 100 since that was measurable amounts then titrated/diluted to get 50 25 and 12.5
To see if they would even survive a long term exposure.
These ranges were not statistically significant
Measured 150 and 100 since that was measurable amounts then titrated/diluted to get 50 25 and 12.5 to see if I could extend to 24/7 exposure
1 Microgram per liter [µg/l] = 0.000 001 Gram per liter [g/l]
X=10 as original (10 micrograms per liter)
Weights in grams were easier than micrograms
10 microgram per liter= 1x10-5 g/L -> I did not take into consideration the fertilizer would be only % of the bag.
To collect eggs I placed marbles in glass dishes so that the zebrafish would not eat the eggs. Once the zebrafish spawned, I collected the embryos and placed them in petri dishes. They were randomly assigned to the various solutions ranging from : control, 2000x, 4000x, 5000x, and 10,000x ? To mix the solution I weighed out the amount of fertilizer and let it sit in the spring water to dissolve. Then added 15 ml to each petri dish. I waited until the zebrafish hatched before sacrificing them with MS222.
I then measured the length of the body from head to tail, width of the head, and width of the body.
Unlike the previous measurements, it is not statistically significant. No dose-dependent relationships or a threshold.
No linear relationship r2 value .65879
There is not a dose-dependent relationship, its more of a threshold concentration that when you exceed it it causes problems. Control and 2000x are not that different it seems to be closer to 4000x where you hit the threshold.
Water quality criteria range from 0.08- 0.35 mg NO2-N/L adequate to protect sensitive aquatic animals for long term exposures (Camargo & Alonso, 2006)
In the future I would also like to know local values to replicate as well.
For funding and supplying the equipment necessary
Guidance and help throughout the process
For support and allowing me to practice in front of you
