Is your baby immune to Nitrite?

By

Ashogbon Habib Biology 2107k lab report

Abstract Experiments were conducted to find the nitrite concentration of four different sites (A brand new subdivision of high-priced McMansion, complete with golf course, tennis courts, and swimming pool, a textile plant that produces Braves T-shirts, an organic farm with a herd of 50 cows and a 10 acre field of zucchini, and a mountain lake that has become overgrown with algae) that may have contaminated a drinking well. This experiment was carried out due to a case of four babies having excess nitrite concentration in their body causing the blue baby syndrome. Sample from all four sites were gotten and tested using spectrophotometer. Optical densities of all the samples were derived using the spectrophotometer and plotted on a standard graph in order to find the nitrite concentration in parts per million (ppm). A ppm above 10 is considered harmful, so any site(s) that registers a ppm over 10 would be a site that has contributed to the blue baby syndrome. The ppm of each sites textile, new subdivision, organic farm, and Mountain Lake are 10, 97, 3, and 2, respectively.

Introduction Understanding the nature of how the world works the way it is as a whole is one fascinating story to tell with science. The atmospheric composition of the earth as of today has well over 75% nitrogen which says a lot about the presence of nitrogen as regards to other elements (5). It is essential to note that nitrogen is a major component of our bodys nucleic acid (DNA and RNA) and also a component of the amino acids which creates our proteins (5). So many living organisms depend on the action of nitrogen fixing bacteria to convert nitrogen into ammonia (NH3), and later alters it into nitrates (NO3) and nitrite (NO2) (5). Nitrogen as nitrate is

relatively nontoxic, but nitrogen in terms of nitrite has a greater chance of causing serious health problems especially to infants. It is so important to know about nitrates or nitrites because significant amount of both can be found in vegetables such as lettuce, spinach etc. we humans having the luxury to eat both plants and animals makes it highly possible to consume large amount of nitrogen based compounds (5). Nitrite in excess reacts with iron component of our hemoglobin (the protein that carries oxygen in blood to tissues in the body) and results in the chemically altered hemoglobin known as the methemoglobin - a compound that is repulsive of oxygen, leading to the change in skin color to blue (1,2,5). Methemoglobin is of more concern to infant and children than adults because adults have enzymes that convert the compound back to hemoglobin while infants have little amount of that enzymes, thus when baby consume excessive amount of nitrite it results in what is called the blue baby syndrome (5). The coloration begins with the lips, nose, and ears during the early stage of the infection (4). Reduced oxygen in the tissue can lead to adverse implication in a child and in most extreme cases leads to coma or death (4). An appropriate nitrite concentration would be 10 parts per million (ppm), so anything above that would be considered harmful to infants. In the village of Gopher Hollow, CDC reported a case of four infants with blue baby syndrome. The connecting fact between all these infants is the consumption of water from the same well. After testing the water for nitrate/nitrite contamination, it showed an optical density level (OD) of 50ppm. There were four sites that happen to be prime suspect due to be being situated along the rivers and streams upstream of the contaminated well. A brand new subdivision of high-priced McMansion, complete with golf course, tennis courts, and swimming pool, a textile plant that produces Braves T-shirts, an organic farm with a herd of 50 cows and a 10 acre field of zucchini, and a mountain lake that has become overgrown with algae

are the four sites to be tested for the nitrate/nitrite contamination and the OD level. If any site registers an OD level of over 10ppm, then theres a high probability that the site(s) is a contributing factor to the blue baby syndrome. The surprising thing to be learned is Nitrates have been detected in breast milk, (3), which means a baby can be affected by the mother having excess nitrite concentration in her system rather than the baby drink contaminated water.

Materials Test tubes were used to collect sample from all four sites, and also used to dilute some samples that were above 1 OD because samples over 1 OD cant be accurate to read with the spectrophotometer available. Spectrophotometer is the instrument used to obtain data of specific wavelength at different ppm. Accuracy is essential in every experiment so in achieving close accurate result a micropipette was used to measure precise volume of sample, solution, or reagent needed. 1-naphthylethylenediamine sulfanilamide is an indicator reagent that helps prove the presence of nitrogen in samples. The cuvette is a small plastic that samples are poured in and placed in the spectrophotometer for readings. Distilled water was used to blank the spectrophotometer making sure it runs fine.

Methods (procedure) Firstly, plotting a standard graph (see graph 2) was necessary in achieving nitrite concentration result from the four sites experimented on. In getting the graph, 1ml of nitrite standard solution at 9 different concentrations (0.5ppm, 1ppm, 2ppm, 3ppm, 4ppm, 6ppm, 10ppm, 20ppm, 40ppm, into 9 different test tubes) was added to 20ml of sulfanilamide into all 9

solutions using a micropipette. The mixed solution was gently wobbling in the test tube and allowed for full color development by letting it sit for 5 minutes before measuring the absorption level. Spectrophotometer was switched on and allowed to warm up for 15 minutes. Small portion of each sample (using a disposable pipette) was placed in the cuvette and into the spectrophotometer to measure the absorption of light at different wavelength. When all data has been collected and accurately written down, the standard graph was plotted using the data to achieve what is called standard line (see graph 2 in result). The line tends to be the main component in finding the concentration of the four sites. Secondly, 2ml of samples from the four different sites were placed into 4 different test tubes. Added was 40microliters of sulfanilamide reagent to each tube and allow it to sit for 5 minutes till color develops. Transfer a little portion of those solutions (one after the other) into the cuvette and place the cuvette in the spectrophotometer to measure the absorption of the four different sites. Any site that registers above 1.00 at 550nm absorption was diluted to 1:10 (1ml of site sample to 9 ml of water). None of the site after the 1:10 dilution was above 1 above one, but if any was above 1 then a 1:50 dilution (1ml of 1:10 to 4ml of water) would have been needed. When the data was achieved, the OD of the four sites was plotted on the standard graph to get the sites concentrations.

Result Table1. Nitrite Concentration (ppm) Absorption at 550nm

Distilled water 0.5 1.0 2.0 3.0 4.0 6.0 10.0 20.0 40.0

0.000 0.038 0.058 0.145 0.066 0.379 0.404 1.054 1.339 2.004

The above table is the data to be used to plot the standard graph. This data was gotten from the first experiment done when 9 different solution was made while adding nitrite concentration in each of them and recording the absorption using a spectrophotometer. The absorption seems increasingly consistent with an exception at 3ppm. Factor that could have lead to that might have been the adding of little nitrite in the solution when making the nitrite solution. The suspected site(s) should it be in a range less than 1.054 must have a ppm less than 10ppm given that it wasnt diluted.

Graph 1

2.5

Absorption at 550nm vs Nitrite Concentration (ppm)

Absorbance (OD) at 550nm

1.5

0.5

0 0 5 10 15 20 25 30 Nitrite Concentration (ppm) 35 40 45

Graph 2

Absorption at 550nm vs Nitrite Concentration (ppm)

1.2 Absorbance (OD) at 550nm 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 Nitrite Concentration (ppm)

Graph 1 contains all the data collected and some were above 1 OD. Graph 2 is where the OD would be plotted because if the OD is above 1, it makes it less likely for the spectrophotometer to get an actual accurate OD. Pretty much if the reading is above 1 OD it cant be trusted. Table 2 Sample Textile New subdivision Organic farm Mountain lake Absorbance 2.270 2.266 0.369 0.272 1:10 0.147 0.956 Concentration 10ppm 97ppm 3ppm 2ppm

The concentration data above was derived from the plotting of the OD gotten of the samples of the four sites. All OD that was greater than 1 was diluted and the plotted ppm would be multiplied by dilution factor. See graph 3 below.

Graph 3

Absorption at 550nm vs Nitrite Concentration (ppm)

1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 Nitrite Concentration (ppm)

The above graph is the plotting of the suspected sites OD at 550nm.

Absorbance (OD) at 550nm

Discussion All the experiment done right up to this moment was in effectively identifying the site(s) that may have contaminated the drinking water the led to four cases of infected babies. The four sites were accurately analyzed and the conclusion drawn from the experiment renders the new subdivision as the site that most likely caused the outbreak. This is due to having a ppm over 10. Two of the sites tested were less than 10ppm while one registered exactly 10ppm (see table 2). When creating the standard graph, there was a change in the consistency of the data at 3ppm. Suspected cause would be less nitrite concentration in the solution because it was too much the

value should be high not low, the value derived was low (see table 1). There are so many factors leading up to this point that highlighted the new subdivision site has the tendency of being the culprit because from the onset due to chemical that may have been spilled when the subdivision was been made could have been a factor. They had to dig the ground to lay a strong foundation for the house or structure theyre about to make. The wood definitely has some chemicals on it to prevent it from getting eaten up by rodent of some kind. Also the people working in the new subdivision site might not have disposed their waste product in a healthy manner. So these underlining factors may have caused the nitrite concentration to a rise significantly. The prediction made was, if the OD is less than 1.054 then the ppm of that site should be less than 10. The prediction was significantly accurate. The rest of the sites didnt show sign of environmental degradation, so this can be attributed to a healthy disposal of their wastes, but as for the mountain lake, nature is doing what it does best. Improvement can still be made to help limit the risk of high nitrite concentration by testing the water reservoir from time to time. Some do think by boiling/filtrating water it reduces the nitrite concentration, but In fact, boiling water that contains high nitrates can actually increase the nitrate concentration, (6). In conclusion, the site with the highest ppm is the prime suspect of the contamination of the drinking water. In this case, the site is the brand new subdivision of high-priced McMansion, complete with golf course, tennis courts, and swimming pool.

Reference

1. Knobeloch, L., et al. 2000. "Blue babies and nitrate-contaminated well water." Environ.Health Perspect. 108(7):675-678. 2. U.S. Agency for Toxic Substances and Diseases Registry. 2001. "Case Studies in Environmental Medicine: Nitrate/Nitrite Toxicity." http://www.atsdr.cdc.gov/HEC/CSEM/nitrate/docs/nitrate_nitrite.pdf. 3. Dusdieker, L.B., et al. 1996. "Does increased nitrate ingestion elevate nitrate levels in human milk?" Arch.Pediatr.Adolesc.Med. 150(3):311-314. 4. U.S, EPA. "Nitrates and Nitrites." Teach Chemical Summary (2007): 1-14. US EPA, 22 May 2007. Web. 18 Oct. 2012. http://www.epa.gov/teach/chem_summ/Nitrates_summary.pdf 5. Gutzler, Stephanie J. "Exercise 6." Principles of Biology Laboratory Manual. By Matthew W. Brewer. 1st ed. Plymouth: Hayden-McNeil, 2012. 39-45. Print. 6. Benton Franklin Health District. 2002. Nitrites and Drinking water: What to do if you have high nitrates in your drinking water. http://www.bfhd.wa.gov/info/nitrate-nitrite.php
Accessed by 2012 Oct 15.