Toxicity of Wood Smoke from Prescribed Fire Free Essay Example

The use of prescribed fire is expected to increase as more efforts are put in reducing the risks of catastrophic fire especially in the rainforests. Fire is particulate in nature, where it has small particles of less than 2.5 microns, while the small diameter PM are known to impact the climate, the rate of visibility and the health of living organisms ( Adetona et al., 2017) . Most of the studies have focused on the urban particulate and the toxicity of smoke from burning cigarettes. However, this work transcends above the existing literature, where it analyzes the toxicity of fine particles of fire generated in the process of combustion of prescribed fire ( Fernando et al., 2016) . The health effects of the <2.5 micron-sized particles of fire is not only related to the size, but also to the chemical composition ( Samburova et al., 2016) . The best example is the generation of polycyclic aromatic hydrocarbons that have mutagenic and carcinogenic effects during the burning of residential wood of the hard and soft woods, which depicts the chemical composition of the PM 2.5 .The project uses the Chinese Hamster Ovary (CHO) cells in the determination of the cytotoxicity and mutagenicity of extracts taken from particulate generated in the course of combustion of the prescribed fire.

Purpose Statement

The main objective of this project is to analyze cytotoxicity and mutagenicity of PM 2.5 generated during the prescribed fires of the Coconino National Forest around the areas of Flagstaff, AZ ( Halpern, 2016) . There are knowledge gaps in the toxicity of wood smoke generated from prescribed fire, where this research will contribute to this knowledge by analyzing the levels of toxins present in prescribed burns of the Coconino National Forest.

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Overview of the Project

The project involves testing of the levels of toxicity and mutagenicity of PM 2.5 collected during the prescribed fire in the stipulated location. The PM 2.5 is extracted in the form of organic solvents using 1M dichloromethane, and the solvent is exchanged into Dimethylsulfoxide (DMSO). The Chinese hamster ovary (CHO) is used to generate the dose-response curves for evaluation of the levels of toxicity of the extract ( Adetona et al., 2017) . The cells that are considered to be surviving the toxicity are tested for mutagenicity using essays generated from HPRT.

Methodology

Part1: Previous Work

The project is a continuation of the previous work that was carried out by other group members. It does not underscore the efforts that were put into the work as the previous work forms the foundation for the continuity. The previous members had already done vital work such as:

Collection of the PM 2.5 samples during the flaming and smoldering phases of combustion.

The members had also analyzed the field blanks and lab blanks that were collected at the flaming and smoldering fire events as well as the determination of the levels of contamination.

Materials

The battery-operated PM 2.5 was used to sample the ambient, while the chemical speciation monitor equipped with a sharp-cut cyclone were used to remove particles having an aerodynamic diameter of >2.5 microns. The Teflon filters were then used to collect the samples that had previously been preconditioned in the control chamber.

Part 2: Current Work

The current work involves testing for the EOC cytotoxicity, where the fractions of the CHO cells that die due to exposure are determined ( Keir et al., 2017) . In addition, the work involves testing the EOC mutagenicity, where it the fractions of the CHO cells that mutate due to the EOC exposure are determined ( Jones et al., 2016) . Determination of the 24-h cell counts involved incubating the treated cells for 24 hours, where each new cell was counted for each cell. The data from the cell counts was used to create a dose-response curve ( Zu et al., 2016) . On the other hand, testing for the EOC mutagenicity was carried out using the HPRT mutation analysis ( Gaither et al., 2015) . The cytotoxicity tests were used to tell whether or not the organic extracts obtained from the fire generated from the PM 25 presented any toxic properties to the cells ( Gaither et al., 2015) . Their cytotoxicity was determined by the death rate of the untreated cells.

On the other hand, mutagenicity was carried out by determining the CHO cells that underwent mutation considering that these cells have a gene coding for HRPT enzyme used in the process of DNA synthesis ( Dong et al., 2017) . The essay was determined in such a way that of the gene was normal, the 6-TG would engulf and kill the cells ( Johnston, & Bowman, 2014) . On the contrary, the genes that were found to have mutated would survive when inoculated with 6-TG, where they would form colonies ( Zu et al., 2016) . In this project, mutagenicity testing was carried out only in the cell cultures that were treated with the 100 uL dose of EOC and the negative and positive controls as shown in the table below.

Table1: Colony Counts


Treated Cells (1.81 ug/uL EOC)								CYTOXAN	MUTAGEN
			 g in	 g in								24 hr		8 day
Dish	Dose (L)		5 mL	0.1 mL		24 hr cell	8-day colony					% survival		mutants/1E6 cells
4	10		18.1	0.36		8517939	(---)					98%
5	10		18.1	0.36		8408847	(---)					97%
6	10		18.1	0.36		8440886	(---)					97%
										AVE		97%
										SD		1%

7	25		45.3	0.91		7577715	(---)					87%
8	25		45.3	0.91		7373167	(---)					85%
9	25		45.3	0.91		7088138	(---)					82%
										AVE		85%
										SD		3%

10	50		90.5	1.81		6777715	(---)					78%
11	50		90.5	1.81		6673167	(---)					77%
12	50		90.5	1.81		6588138	(---)					76%
										AVE		77%
										SD		1%

13	100		181.0	3.62		5706539	38					66%		18
14	100		181.0	3.62		5800766	41					67%		21
15	100		181.0	3.62		6048063	40					70%		20
										AVE		67%		20
										SD		2%		2

Negative Control
16		50 uL DMSO			8623541			21			99%		1
17		50 uL DMSO			8812302			20			101%		0
18		50 uL DMSO			8659899			21			100%		1
										AVE		100%		1
										SD		1%		1

Positive Control
19		30 uM Cr(VI)			3216445			42			37%		22
20		30 uM Cr(VI)			2897542			43			33%		23
21		30 uM Cr(VI)			3104622			41			36%		21
										AVE		35%		22
										SD		2%		1

Table 1: Colony Counts

The surviving cells were then transferred from each of the harvest tubes into new 100 m dishes containing 9ml of 6TG solution to test for mutagenicity ( Dong et al., 2017) . α-MEM was added until the volume had reached the 100ml label. The dishes were then taken to the incubator at normal body temperature with 5% Carbon dioxide ( Adetona et al., 2017) . The α-MEM was changed every two days with the consistent addition of EDTA to ensure that the cells are dislodged from the sides of the walls ( Keir et al., 2017) . The cells were then harvested and counted, where each living cell was found to be existing in the form of colonies. The mutagenicity was then expressed as mutants per 1 x 10 6 cells. The results were then recorded as shown:

Results and Discussion

Result 1: Results for the treatment of CHO AA8 cells with PM 25 from Rx1 (Ignition/Flaming)

Rx1 (ignite/flam)
 g in	 g in	% survival	SD	mutants per
5 mL	0.1 mL			1E6 cells	SD
18.1	0.36	97%	1%	(---)	(---)
45.3	0.91	85%	3%	(---)	(---)
90.5	1.81	77%	1%	(---)	(---)
181.0	3.62	67%	2%	20	2

(-) control		100%	1%	1	1
(+) control		35%	2%	22	1

Discussion 1

The results above show the percentage between the cells in the treated dish and the average cells in the untreated dishes, which is the cytotoxicity at the ignition/smoldering phase. The results show that there is a correlation between concentration of the EOC in the PM 2.5 and the toxicity levels of the p-prescribed fire ( Adetona et al., 2017) . The results prove the fact that the increase in the levels of EOC exposure results in more deaths of the CHO cells at the ignition and smoldering phase.

Results Table 2: Treatment with CHO AA8 Cells with PM-2.5 from Rx1 (smoldering)

Rx1 (smoldering)
ug EOC	ug EOC	% survival	SD	mutants per
(in 5 mL)	(in 100 uL)			1E6 cells	SD
13.9	0.28			(---)	(---)
34.8	0.70			(---)	(---)
69.5	1.39			(---)	(---)
139.0	2.78

(-) control
(+) control

Discussion 2:

The results above show the percentage between the cells in the treated dish and the average cells in the untreated dishes, which is the cytotoxicity at the smoldering phase alone ( Samburova et al., 2016) . It depicts that there are high numbers of CHO cells that die since EOC content in the PM-2.5 are high at this phase of ignition of the predicted fire ( Gaither et al., 2015) . There are higher levels of death of the CHO cells at the smoldering phase as compared to the ignition phase due to the fact that the EOC content at this phase is relatively higher.

Results 3: Treatment of CHO AA8 cells with PM-2.5 Rx2 (Ig/Flam)

Rx2 (ignit/flam)
 g in	 g in	% survival	SD	mutants per
5 mL	0.1 mL			1E6 cells	SD
22.0	0.44			(---)	(---)
55.0	1.10			(---)	(---)
110.0	2.20			(---)	(---)
220.0	4.4

(-) control
(+) control

Discussion 3:

The results above show the percentage between the cells in the treated dish and the average cells in the untreated dishes, which is the cytotoxicity at the ignition/inflammation phase ( Adetona et al., 2017) . The results are also consistent with the consideration that the high levels of concentration of the OCE levels are high at the ignition and flaming phase considering that the percentage of the total CHO cells that die at this phase is higher as compared to the ignition phase alone.

Data Analysis

Percentage cell survival for each dose

Treated Cells (1.81ug/uL EOC)

Rx1 (ignite/flam)
 g in	 g in	% survival	SD	mutants per
5 mL	0.1 mL			1E6 cells	SD
18.1	0.36	97%	1%	(---)	(---)
45.3	0.91	85%	3%	(---)	(---)
90.5	1.81	77%	1%	(---)	(---)
181.0	3.62	67%	2%	20	2

(-) control		100%	1%	1	1
(+) control		35%	2%	22	1

The results above show the percentage survival for the treated cells using the dosage of 1.81ug/uL EOC. The percentage cell survival can be calculated as