Journal of Life Sciences Volume 8 Number (2)
J LS
Journal of Life Sciences
Volume 8, Number 6, June 2014 (Serial Number 74)
Contents
Physiology and Biochemistry
481 The Changes of Water Quality in Space and Time in the Mekong River, Bassac River and Adjacent Waterways
Luu Duc Dien, Ma Tu Lan and Nguyen Dinh Hung 489
Analysis Method for Pesticide Residues in Biological Matrices: Gas Chromatography-mass Spectrometry
François Xavier Nshimiyimana, Abdellah El Abidi, Mohamed Fekhaoui, Bouchaib Benbakhta, Nezha Barakate, Hind Hami and Abdelmajid Soulaymani
Contribution to the Bio-ecological Study of Date Palm Entomofauna in the Region of Saoura (South Algeria)
Ali Boulanouar, Mohammed Anouar Khelil, Ahmed Makhlouf and Larbi Benlarbi 504
Applying Natural Fertiliers to Herbaceous Crops
Disciglio G., Frabboni L., Tarantino A. and Tarantino E.
Botany and Zoology
Propagation of Grevillea banksii Affects the Dynamic of Mycorrhizal Fungi Communities Associated with Native Tree Species of Madagascar
Martial Doret Andrianandrasana, Rondro Harinisainana Baohanta, Herizo Randriambanona, Marson Raherimandimby, Damase Khasa, Robin Duponnois and Heriniaina Ramanankierana
517 Pheomelanin Formation and Low Tyrosinase Activity in Fading Body Color Variant BdlR Strain Oryzias latipes
Nobuhiko Asada, Aoi Kedamori, Yumiko Kusano and Tetsuro Takeuchi
522 Time Course of Changes in Trabecular Bone Microstructure in Rats with Spinal Cord Injury
Akira Minematsu, Yasue Nishii, Hidetaka Imagita and Susumu Sakata
529 A Primary Hepatobiliary Neoplasia in a Persian Cat
Mohamed Shokry, El-Sayed Berbish and Iman Shaheed
Interdisciplinary Researches
533 Perception of Agricultural Science /Home Economics as a Career among Senior Secondary School Students in Abia State, Nigeria
Nathaniel Chika Ezebuiro, Kenneth Chikwado Ekwe, Ekwuruchi Ogbonna Mbanaso, Flora Ngozi Nwakor, Godwin Ndubuisi Asumugha and Justin Enyinnaya Ewuziem
544 Flavonoïds from Euphorbia guyoniana Boissier & Reuter
Ouanissa Smara, Audrey Julia, Cécile Moral-Salmi, Claire Vigor, Joseph Vercauteren and Belgacem Legseir
552 Induced Immunity of Response to Some Allergens Towards Response of Other Skin Test Allergens and Its Correlation with Immediate Hypersensitivity Reactions
Younus Jasim Abdullah
558 Granulomatous Inflammatory Reaction in Breast Silicone Implants
Tammaro A., Giulianelli V., Narcisi A., Abruzzese C., Cortesi G., Parisella F.R., Persechino S. and Grippaudo F.R.
June 2014, Vol. 8, No. 6, pp. 481-488
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
The Changes of Water Quality in Space and Time in the Mekong River, Bassac River and Adjacent Waterways
Luu Duc Dien, Ma Tu Lan and Nguyen Dinh Hung Research Institute for Aquaculture No. 2, 116 Nguyen Dinh Chieu Street, District 1, Hochiminh City 70000, Vietnam
Received: February 18, 2014 / Accepted: June 18, 2014 / Published: June 30, 2014.
Abstract: Catfish (Pangasianodon hypophthalmus) farming along Mekong river and Bassac river depends heavily on water quality of the two rivers, whereas water quality of these rivers are affected by the waste of aquaculture activities, agricultural production, industrial and municipal waste. This report analyzes the monitoring data on Mekong river, Bassac river and adjacent waterways in the period of 2011-2012, focusing on parameters of organic pollution to assess the current quality of these two rivers. Based on the results, the water quality in the river-head was generally better than in the middle and at the end of the river, and the quality of water of the Mekong river was better than Bassac river. In terms of time, water quality in July was considered the best in all the basins. At adjacent natural rivers and canals, ammonia levels increased and exceeded the Vietnamese standard in April, and BOD values were also much higher compared to two major rivers. The results of the model also showed that the levels of pollution index of the Mekong and Bassac river were very low (1.33 and 1.47), and the values (Y) in the canals were higher (1.63-1.67) but still in permitted level. Therefore, the water quality of the Mekong and Bassac river in the period 2011-2012 was generally still quite good.
Key words: Bassac river, catfish, Mekong river, model, water quality.
1. Introduction observation of two lines of Mekong and Bassac river and neighboring canals in the period of one year,
Catfish has been the object of intensive farming in based on water quality criteria, especially parameters freshwater fish ponds mainly in the Mekong Delta indicated pollution (BOD, phosphate, nitrate, with an annual output of 1 million ton, bringing ammonia, coliform...) to assess the status of water exports from 1.4-1.6 billion USD [1]. The most quality on the Mekong and Bassac river in the context concerned issue of farmers and local governments of fish farming developing. The results of this study has been the water quality in the rivers and canals in were the scientific basis for assessing the current state the region [2]. Water quality is also affected by of the environment and predict changes in the future upstream water quality, discharge of living and other and helping managers to find appropriate solutions for economic activities in this area. Therefore, the sustainable catfish planning for provinces along the current trend of rapid development, the strength of
Mekong and Bassac river.
the economic sectors, in the near future, if the effluent monitoring and management of
2. Materials and Methods
environmental impacts are not conducted properly,
2.1 Time and Place of Sampling and Analysis Criteria pollution will be unavoidable. Following this, the
reverse of its impact on people’s production and life Survey time: from April 2011 to January 2012, could cause the damage.
made four 3-month frequencies survey/time. The paper focused on the analysis of survey data,
Sampling location: taking water samples in 40 points (30 points on the main line and 10 Corresponding author: Luu Duc Dien, research field:
measurement points in the channel/main rivers environmental engineering. E-mail: dienld.ria2@mard.gov.vn.
The Changes of Water Quality in Space and time in the Mekong River,
Bassac River and Adjacent Waterways
flowing into the Mekong and Bassac river). Each assessment model in this report. Specifically, the tributaries and the canal/ditch into the Mekong and
model was applied to 10 parameters having limited Bassac river were divided into five areas from
value, including chloride, pH, TSS, total iron, DO, upstream to downstream (Table 1).
BOD, nitrate, phosphate, ammonia and coliform. Analysis: 16 parameters [Salinity, temperature, pH,
With the overall objective assessment of water dissolved oxygen (DO), total suspended solids (TSS),
quality monitoring parameters, methods of ecological biochemical oxygen demand (BOD), chlorophyll a,
risk assessment was applied to calculate. Overall ammonia (N-NH4), nitrate (N-NO3), total nitrogen
water quality was calculated according to the formula: (TN), total phosphorus (TP), phosphate (P-PO4),
Y= Wi * Xi [4-7]. Where, Y was the level of water chloride, coliform, total iron, aluminum metal] in the
pollution index, Xi was the level of water pollution cross section and over time.
monitoring parameters i, and Wi was the ratio of the Water samples were collected by using bathometer,
number of observation i.
0.5-1.0 m water deep. Water samples were then fixed Determine the level of water pollution (X): The and stored following standard procedures and limit values monitoring of the parameters used in this transported to the laboratory to be analyzed less than
paper were referenced at Vietnamese standard
24 h from the time of sample collection [3]. 08:2008/MNRE (column A2). To calculate the level of water pollution (X), the value of monitoring
2.2 Methods of Assessing the Current State of the parameter at 75% of the limit value, then X had a Water Environment value of 1; from 75%-100% of the limit value of X
Although 16 water quality parameters were analyzed, was 2; limit value, the value of X equaled to 3 only the typical indicators selected for inclusion in the
(Table 2).
Table 1 Water quality assessment partition by region.
Area
Symbol 1. Upstream
Mekong river
Symbol
Bassac river
River: ST1, ST2, ST3, ST4
ST_V1
River : SH1, SH2, SH3
SH_V1
KSH1 2. Upper part of the river River: ST5, ST6, ST7, ST8
Canal: KST1
KST1
Canal: KSH1
ST_V2
River : SH4, SH5, SH6, SH7
SH_V2
KSH2 3. Middle of the river
Canal: KST2
KST2
Canal: KSH2
River: ST9, ST10, ST11
ST_V3
River : SH8, SH9, SH10
SH_V3
KSH3 4. Lower part of the river River: ST12, ST13
Canal: KST3
KST3
Canal: KSH3
ST_V4
River : SH11, SH12, SH13
SH_V4
Canal: KST4
KST4
Canal: KSH4
KSH4
SH_V5 5. Downstream
River: ST14, ST15
ST_V5
River : SH14, SH15
Canal: KST5
KST5
Canal: KSH5
KSH5
Table 2 The value of monitoring parameters and indicators of water pollution X.
Freshwater fish
No. Parameter Unit
Limit values
< 6.0 or > 8.5 2 chloride mg/L 6.0-8.5 300
7.0-8.0
6.0-7.0 or 8.0-8.5
300-400 > 400 3 TSS
22-30 > 30 4 Total iron mg/L 1
mg/L 30
0.75-1.00 > 1.00 5 DO
5.0-5.5 < 5.0 6 BOD
mg/L 5
4.5-6.0 > 6.0 7 N-NO 3 mg/L 5
mg/L 6
3.75-5.00 > 5.00 8 P-PO 4 mg/L 0.2
0.15-0.20 > 0.20 9 Coliform MPN/mL 0.2
37.5-50.0 > 50.0 10 N-NH 4 mg/L 50
0.15-0.20 > 0.20
The Changes of Water Quality in Space and time in the Mekong River,
Bassac River and Adjacent Waterways
Table 3 The group parameters, and the weight W of each monitoring parameter.
No. Parameter Parameter’s group weight coefficient W 1 chloride
Normal and iron alum
2 TSS
Normal and iron alum
3 pH
Normal and iron alum
4 Total iron
Normal and iron alum
7 N-NO 3 Pollution 2 2/17 8 P-PO 4 Pollution
9 Coliform Pollution
10 N-NH 4 Highly toxic organic pollution
Sum 17 17/17
Table 4 Overall assessment of water based on the index value Y.
No. The level of water pollution index Y
Overall assessment of water quality
1 1<Y 1 + 2/3
No signs of pollution
2 1 + 2/3 < Y 1 + 4/3
Signs of pollution
3 1 + 4/3 < Y 3
Pollution alert
Determination of W for each monitoring parameter: pH between basins changed insignificantly. BOD estimated by the method of grouping parameters based
concentrations ranged appropriately from 3.6 to 4.6 on the criterion of the influence of groups of
mg/L on the Mekong and Bassac river, respectively, environmental parameters in aquaculture. The report
proportion to the DO values in the suitable range of defined the influence of each parameter into three
4.89-6.51 mg/L.
levels of impact parameter corresponding to the three Results also showed that P-PO4 and N-NO3 levels groups [8-10]. Table 3 showed the correlation between
in water were quite low, in the respectively range of the parameter’s group and the weight W of each
0.03-0.14 mg/L and 0.17-0.42 mg/L. This result was monitoring parameter.
relatively equivalent to the water quality during Assess the level of water pollution index (Y): Y is
2005-2010 [10]. Although concentrations of N-NO3 continuous range of values from 1.00-3.00. The
were low, most TN values were higher than 4.0 mg/L. overall assessment of water quality at each location as
Based on some international standards (such as well as the whole river was correlated with three about
American standard: TN < 0.9 mg/L, Chinese standard: the value of Y (Table 4).
TN < 0.5-1.0 mg/L for freshwater fish farming), the
3. Results and Discussion TN content in the water were almost in excess of
regulations for aquatic conservation purposes and
3.1 Assessing the Current Water Quality of Mekong
potentially cause eutrophication [11].
and Bassac River The ratio of TN/TP in the Mekong and Bassac river
3.1.1 According to space in each survey area (Fig. 1) in the range of
3.1.1.1 Mekong and Bassac river considerably narrows from 5.1 to 7.3 (Bassac) and The pH values of both basins properly ranged from
5.2-6.2 (Mekong). In general, the limiting factor
7.1 to 7.4 and relatively stable from upstream to eutrophication depended on space and time, in which downstream, as these are the two largest main
the majority of P was the limiting factor.
freshwater rivers of the Mekong Delta, flow was high, The average total coliform density in the Mekong
The Changes of Water Quality in Space and time in the Mekong River,
Bassac River and Adjacent Waterways
The ratio of TN/TP in the Mekong and Bassac river
3.1.2 According to time
TN/TP
3.1.2.1 Mekong and Bassac river
pH values on the Mekong river were in the range of
7.0 to 7.5, and these were broader range 6.9-7.7 in the
Bassac river. BOD contents of the Mekong and
6.0 Mekong river
Bassac river
Bassac river fluctuated below 5.0 mg/L in the
beginning months of the year and tended to increase towards the end of the rainy season, reaching the
highest point 8.0 mg/L in October (Figs. 2 and 3).
4.0 area
0 1 2 3 4 5 Therefore, at the end of the rainy season the amount of
Fig. 1 TN/TP in Mekong and Bassac River.
water increased due to heavy rain in September-October, and the pouring of water from
river was not so high (32-86 MPN/mL) while in the upstream carried more silt, sediment creating a flow Bassac river the coliform levels were recorded that contains many impurities and suitable for the (194-756 MPN/mL) exceeding many times compared development of microbial pathogens, resulted in the
to the permitted level (< 50 MPN/mL) in all basins.
increasing of BOD.
The high coliform density can be attributed to the concentration of boats, the poultry farming and
BOD (mg/l)
BOD changes in the Bassac river
grazing activities, the strong development of aquaculture
activities, crowded residential areas, markets... 8.0
3.1.1.2 In the channel/adjacent canal linking to the 7.0 Mekong and Bassac river 6.0
Total nitrogen and total phosphorus content on the
channel/canal were as the same rate as in the main two
lines (TN: 2.97-5.34 mg/L; TP: 0.24-1.47 mg/L). In
terms of the ratio TN/TP in the Bassac river channel
points, both N and P were limiting factors, whereas
0.0 Month
phosphorus was more dominant the limiting factor in SH_V5
SH_V1
SH_V2
SH_V3
SH_V4
the Mekong river channel points. Fig. 2 The changes of BOD in the Bassac river based on
time.
In the canal, level of microbial contamination was
quite high because the presence of high coliform was BOD changes in Mekong river BOD (mg/l)
several times higher than the permissible limits. In 9.0 addition, total coliform of Bassac’s channels was 8.0
higher than that in Mekong’s channels. For example,
in KSH1 (Thanh My Tay An Giang), KSH4 (Nhon
Nghia_Can Tho) levels of pollution were 20 times
higher than the limits of Vietnamese standard. 3.0 Meanwhile the highest pollution level in the Mekong 2.0 channel was just 6 times higher than Vietnamese 1.0
Month
standard (My Duc Dong_Tien Giang). This implied
ST_V1
ST_V2
ST_V3
ST_V4 ST_V5
the level of microbial contamination of the Bassac
Fig. 3 The changes of BOD in the Mekong river baesd on
channels was quite alarming.
time.
The Changes of Water Quality in Space and time in the Mekong River,
Bassac River and Adjacent Waterways
In contrast to the evolution of BOD, the variety of the value of 0.58 mg/L (compared with the permited ammonia in the Mekong and Bassac river depended
level of 0.2 mg/L). Moreover, at the end of the dry on each region and time. However, the concentration
season average ammonia values were also recorded of ammonia in both rivers was maintained at lower
higher than the rainy season.
levels as opposed to the limit of Vietnamese standard. High bacteria concentrations recorded at the canals In the upstream of the Bassac river flow, ammonia
linking to Bassac river with the highest value of 4,600 levels were high in April and gradually decreased to
MPN/mL in January in Thanh My Tay_An Giang less than 0.02 mg/L in July, while the water
(KSH1) and October in Nhon Nghia_Can Tho (KSH4) downstream of the Bassac river tended to rise at the
(Fig. 6), whereas the river flows into wages, the end of year (0.06 mg/L). In the Mekong river, highest
highest value was 1,100 MPN/mL in January in My ammonia fluctuated in January in the region between
Duc Dong Tien Giang (KST4) (Fig. 7). ending areas (ST_V4) and decreased in July.
3.2 Model Assessment of Water Pollution Results recorded coliform density in Mekong river
has much lower than that in Bassac river. The
3.2.1 Regarding to space
presence of total coliform in Mekong river was less Water quality in Mekong and Bassac river was variable in terms of time on the whole river, only the
pretty good, represented by the value level of water average range of 100-120 MPN/mL and the most
Change of Coliform in Bassac river
polluted river downstream of Mekong river was 200
Coliform (MPN/ml)
MPN/mL in January (Fig. 4). On the contrary, in the 20
Bassac river, coliform levels fluctuated extremely
differently in every phase of the survey. In the rainy 1600
season, coliform density of the Bassac river was 10
times higher than that of Mekong river, with the 1000
presence in the end source (SH_V5) 2100 MPN/mL in
July (Fig. 5). 400
3.1.2.2 In the channel/adjacent canal linking to the
0 Month
1 4 7 Mekong and Bassac river 10
The rising of ammonia levels exceeded the threshold
SH_V1
SH_V2
SH_V3
SH_V4 SH_V5
Fig. 5 The change of Coliform in Bassac river based on
in Tan Phu Dong_Dong Thap (KST2) in April with
time.
Change of Coliform in Mekong river Change of Coliform in canals (Bassac river) Coliform (MPN/ml)
Coliform (MPN/ml)
1 4 7 10 0 1 Month 4 7 10 ST_V1
0 Month
ST_V2
ST_V3
ST_V4
ST_V5
KSH4 KSH5
Fig. 4 The change of Coliform in Mekong river based on Fig. 6 The change of Coliform in canals linking to Bassac time.
river.
The Changes of Water Quality in Space and time in the Mekong River,
Bassac River and Adjacent Waterways
Change of Coliform in canals (Mekong river) Coliform (MPN/ml)
often observed in low concentrations (< 0.90 mg/L for
N-NO3, and < 0.20 mg/L for P-PO4), the value of TN
was usually higher than 3.5 mg/L. Therefore, the
concentration of TN in the Mekong and Bassac river
were at potentially cause eutrophication, especially in
the dry season [12]. However, when considering
factors limit eutrophication in the Mekong and Bassac
river and canals/ditches through the ratio of TN/TP,
phosphorus can be seen as the major limiting factor.
0 Month
1 4 7 10 BOD parameter in Mekong and Bassac river were
KST1 KST2
almost in the allowed range except for October
Fig. 7 The change of Coliform in canals linking to
(recorded in the 8.0 mg/L). Hence, the BOD values of
Mekong river.
the Mekong and Bassac river reached good quality for pollution index (Y) was very low during the survey
aquaculture, but they tended to increase in the region period of one year (Fig. 8). In addition, the water
and in the rivers/canals/ditches flowing into the quality in Mekong river was better than branches of
Mekong and Bassac river where aquaculture the Bassac river. In the canal/ditch into the Mekong
development such as An Giang, Dong Thap, Can Tho and Bassac river, the Y values were higher, and some
province, especially in the last months of the rainy Y values in the basin “signs of pollution” (as KSH1,
season. This can be due to the impact of the flood and KSH5; KST2, KST4) but generally indicators of
one direction water flow from upstream, carrying high organic pollution were in the appropriate range for
organic contents. The increasing of BOD and freshwater aquaculture.
decreasing of DO levels, especially in the
3.2.2 In tems of time canals/ditches showed an increase in organic pollution In Bassac river, source of water was relatively good
due to the development of more productive activities except for a few basins (SH_V3 and SH_V5) in
(industry, agriculture, aquaculture...) over the year. October, the Y value was 1.71. In Bassac’s channels,
Contamination by pathogens (total coliform except in July, the rest have recognized the value of Y
bacteria) was one of the most concerns about water “signs of pollution” in a few areas. Among them,
quality because they can cause intestinal disease to October was considered the highest pollution levels.
humans and animals. Bacteria pollution often In Mekong river, all four time surveys recorded
originated from municipal waste, urban and industrial water quality in the Mekong river was very good. In
wastes from human and animal waste into the Mekong’s channels, water quality was also quite
canal/ditch and into the Mekong and Bassac river. similar to that in Bassac’s channel: “signs of
Total coliform concentrations in the river exceeded pollution” in many basins (except in July).
the permitted level many times [6]. It proved that the water in the Bassac river and the channel/canal into
3.3 General Discussions the Mekong/Bassac river has been contaminated by
Most of the water quality parameters in the Mekong coliform bacteria-a potential cause disease for aquatic and Bassac river such as chloride, pH, ammonia, total
animals in the basins.
iron... were achieved Vietnamese standard In the period 2005-2010, the pollution level in the 08:2008/MNRE applied to water to protect aquatic life.
Mekong and Bassac rivers was only 1.62 [10], the Although the concentrations of P-PO4 and N-NO3
period of 2011-2012 water quality of Mekong and
The Changes of Water Quality in Space and time in the Mekong River,
Bassac River and Adjacent Waterways
Fig. 8 Water quality map of the areas along the Mekong and Bassac river according to space.
Bassac river was considered even better with Y value time, at the end of the dry season, lower DO
1.40 (only for own Mekong and Bassac river) and concentrations in organic content, such as ammonia,
1.52 (including the canals/ditches into the Mekong total nitrogen, total phosphorus was usually higher and Bassac river). This was quite reasonable when the
than the rainy season. However, at the end of the rainy recorded values were lower for indicators: pH, total
season, levels of coliform in water were very high and iron, DO, ammonia... Therefore, there are still many
many times exceeds permitted level. July can be seen concerns about water pollution problems in Mekong
as the best water quality in all basins. In October, and Bassac river and sometime had also recorded
indicators of organic pollution were lower than that in pollutant levels exceeded standard, especially in the
April and July, but the amount of microbial channel/canal, but in overview picture water quality of
contamination was the highest. At the canals, the the Mekong and Bassac river was quite good to
increasing of ammonia levels exceeded the standard in facilitate the planning of catfish (Pangasianodon
April. BOD was also much higher than that in two hypophthalmus) farming areas.
major rivers. The results of the model also showed that the level of pollution of the Mekong and Bassac
4. Conclusions
river was very low (1.33 and 1.47), and on Upstream water quality was considered better than
neighboring canals flowing into the Mekong and that between the source and downstream, and water
Bassac river, the value Y was higher (1.63-1.67) but quality in Mekong river in 2011-2012 was still
still in permitted level. Consequently, the overall considered better than the Bassac River. Following
picture of water quality in the Mekong and Bassac
488
The Changes of Water Quality in Space and time in the Mekong River,
Bassac River and Adjacent Waterways
river in the 2011-2012 period was still pretty good.
Vietnam. [7] Lang, T. T. 2008. Study to assess water quality index and
Acknowledgments
partition the water quality of Bassac river. Scientific report, University of Natural Resources and Environment
This research was financed by MARD (Ministry of
of Ho Chi Minh City.
Agriculture and Rural Development, Vietnam). [8] Bao, T. Q., Truc, N. T., Thuy, D. N., Tuan, T. T., Vuong, D.Q.T., Bao, T.N., Dien, L.D., and Phuoc, L.H. 2010.
References
Monitoring for aquaculture environment and epidemic in some areas of the provinces of the Mekong Delta and the
[1] Tung, N. T. 2008. Development planning, Production southeast of Vietnam in 2010. Scientific report, Research and consumption of fish in the Mekong Delta in 2010 and
Institute for Aquaculture No. 2.
2020 orientation. Scientific report, Research Institute for [9] Tuan, T. T. 2010. Construction of warning simulator to Aquaculture No. 2.
model the environment and diseases for intensive shrimp [2] Trung, N. D. 2004. “Water Quality Management in
and catfish farming. Scientific report, Research Institute Aquaculture.” Agricultural Publishing House.
for Aquaculture No. 2.
[3] Boyd, C. E., and Tucker, C. S. 1992. Water Quality and [10] Dien, L. D., Tuan, T. T., and Vuong, D.Q.T. 2012. Pond Soil Analyses for Aquaculture Alabama: Auburn
“Assessment of the current water quality in the Mekong University.
and Bassac river for catfish (Pangasius hypophthalmus) [4] Smith, D. G. 1990. “A better water quality indexing
farming.” Journal of Agriculture and Rural Development system for rivers and streams.” Water Resources 24:
(7): 68-76.
1237-1244. [11] Hop, N. V., Tung, T. Q., Long, T.H., Phong, N.H., [5] Vuong, D. Q. T., Truc, N. T., Tuan, T. T., Thuy, D. N.,
Marconi M., Tuan, L.C., Hue, N.V., To, T.C., and Bang, Bao, T. N., Tien, D. V., and Phuoc, L. H. 2012.
T.H. 2008. Assessment of the quality of water and Construction of parameters, indicators and monitoring
sediment in Tam Giang-Cau Hai, Integrated project processes of the aquaculture environment. Scientific
management activities lagoon in Thua Thien Hue report, Research Institute for Aquaculture No. 2.
province, Vietnam. Scientific report, Hue University. [6] Vietnamese standard 08:2008/MNRE. 2008. National
[12] Chapman, D., ed. 1996. Water quality assessments: A technical regulation on surface water quality. The
guide to use of biota, sediments and water in Ministry of Natural Resources and Environment,
environmental monitoring . Cambridge University Press.
June 2014, Vol. 8, No. 6, pp. 489-495
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Analysis Method for Pesticide Residues in Biological Matrices: Gas Chromatography-mass Spectrometry
1 2 3 François Xavier Nshimiyimana 2 , Abdellah El Abidi , Mohamed Fekhaoui , Bouchaib Benbakhta , Nezha
Barakate 2 , Hind Hami 1 and Abdelmajid Soulaymani 1 1. Genetics and Biometry Laboratory, Faculty of Sciences, University Ibn Tofail, BO. 133 Campus Universitaire, Kénitra 14000,
Morocco
2. Hydrology and Toxicology Laboratory, National Institute of Hygiene, 27 Avenue Ibn Battouta, BO 769, Rabat 10000, Morocco 3. Ecotoxicology Laboratory, Scientific Institute of Rabat, Avenue Ibn Batouta, 1014, Rabat 10000, Morocco
Received: April 27, 2014 / Accepted: June 03, 2014 / Published: June 30, 2014.
Abstract: Pesticides have done a great service to human, but their use is not safe for public health. Apart from pesticides acute toxicity, their chronic toxicity can cause various problems for human health. The objective of this work was to validate a liquid-liquid extraction method, which allows a fairly reliable analysis of pesticides using gas chromatography-spectrometry mass (GC/MS) in toxicology laboratory at National Institute of hygiene, Rabat Morocco. The equipment required to perform these analyzes are the biological matrices (blood, gastric fluid), in which the authors have doped the Organophosphorus pesticides such as Chlorpyrifos, Dichlorvos and Organochlorine pesticide: dichlorodiphenyltrichloroethane (DDT) and Heptachlor. After extracting the mixture with toluene, the supernatant was collected after centrifugation and concentrated in a small volume of 1.5 mL and then analyzed in GC/MS. After analyzing, the authors found that the yields of each pesticide in samples are significant; respectively they represented 73.4% of Chlorpyrifos, 70.8% of Dichlorvos, 47.8% of DDT and 71.6% of Heptachlor. The blood has a strong link with the most pesticides, where it’s important to use the GC/MS to identify these products. The extraction with toluene was effective, especially to OP, but it’s also sensitive to OC.
Keys words: Validation methods, organochlorine pesticides, organophosphatus pesticides, health, GC/MS.
1. Introduction toxicities (accidental or suicide), chronic toxicities (long term exposure) may be related to various human
Pesticides are defined as any substance or mixture health issues such as infertility and cancer etc. [4]. of substances intended to protect the plants and Organochlorine and organophosphorus pesticides in animals against all king of harmful organisms. In a particular are effective agents commonly pulverized to broad sense, they are intended to destroy, deter and counter pests and both have been mostly applied in render harmless these organisms [1]. The pesticides agriculture in the world. Due to their high chemical are classified in several groups depending on the stability and lipid solubility, these groups of pesticides species to fight (insecticides, herbicides, fungicides, are environmental pollutants. As a consequence, these rodenticides to mention only the main groups), or by pesticides are often detected in the food chain exposed chemical functions (organochlorines, organophosphates, to animals and humans, such as in fish, wildlife, carbamates, etc.). Pesticides have certainly promoted adipose tissue and breast milk [5, 6]. The reference agricultural resources, but their use is not without method established in this study will allow them to danger to public health [2, 3]. Beside their acute evaluate human exposure to some pesticides.
Corresponding author: Abdelmajid Soulaymani, Ph.D., The objective of this study was to develop a professor, research fields: genetics, biometry and epidemiology. E-mail: asoulaymani@yahoo.fr.
specific and simple method to detect the baseline of a
Analysis Method for Pesticide Residues in Biological Matrices:
Gas Chromatography-mass Spectrometry
series of organochlorines and organophosphates in organophosphorus pesticides standards (chlorpyrifos biological matrices. The method described here is a
and dichlorvos) and organochlorine pesticides standards simple one requiring minimal preparation of the
(heptachlor and Dichlorodiphenyltrichloroethane: sample. The method is intended to provide DDT). Firstly, 2 mL of blood was doped by 1 mL of exploratory and confirmatory data, although focused
pesticides (5 ppm each one) and 2 mL of toluen were on the analysis blood. The pesticides used have been
added. The mixture was slowly vortexed for 2 min, selected based on their occurrence, persistence,
and centrifuged at 5000 rpm for 15 min. The layer toxicity and human prevalence in biological matrices.
recovered of supernatant of each sample doped was evaporated and concentrated in small volume by
2. Materials and Methods
liquid nitrogen [8].
As pesticides or their residues are not directly The organochlorines (OC) are persistent pollutants assayable in biological matrices, except by few
from the environment, very often dosed simultaneously. immunochemical methods according to Koivunen et
Because of their resistance to degradation and their al. [7]. It is necessary to perform the sample
high solubility in organic solvents and lipids, these pretreatment and pre-concentrate the compounds. This
compounds accumulate in the tissues and biological is intended to identify chemical compounds from the
fluids. They are, as the authors have said earlier complex biological matrix. Therefore, the various
responsible for adverse effects on human health [4]. procedures have applied such as: solid phase
3. GC/MS
extraction (SPE), solid phase micro extraction (SPME) and liquid-liquid extraction (LLE). The third method
The technique used for pesticides is often Gas was used in their study. This method, they are
Chromatography (GC) coupled to mass spectrometry essentially aimed to simplify and ensure the efficiency
(MS). Methods rather describe the dosage given a of the extraction. The liquid-liquid extraction (LLE) is
family of pesticides. The authors note that such a based on a difference of solubility between the
process is difficult to use, because these chemical analytes and biological matrices relative to the solvent
compounds have the different physicochemical of extraction. In the case the samples are doped with a
parameters such as volatility, pKa, thermostability, series of pesticides. The first step was an erythrocyte
polarity, solubility in solvents, etc.. This complicates lysis and the precipitation of proteins during vortex
both the extraction and analysis. But there are still agitation, was followed by centrifugation of the
many compounds that can’t be analyzed directly by samples. Toluene was used as extraction reagent. The
GC because of their low volatility, their high polarity supernatant was then collected and concentrated in a
and/or their thermal instability. GC-MS is now a small volume by evaporation under liquid nitrogen.
complementary technical to quantify pesticides in the Therefore, equipment necessary to perform these
environment and in biological matrices [9]. analysis in order to highlight the pesticides problems
4. Extraction
to human health, are the biological matrices such as viscera blood, gastric fluid, urine or hair. The
The analysis of samples transferred into the vials biological matrices (blood and gastric fluid) were used
were carried out by gas chromatography (GC-QP2010 in their study. They were obtained at the Department
Shimadzu) coupled to mass spectrometer (MS
of Toxicology, National Institute of Hygiene, Rabat, QP-2010 model). The column used for the separation Morocco. Therefore, the authors doped to their
of compounds is a capillary column DB5MS (5% samples (blood and gastric fluid), the series of
phenyl, 95% methyl polysiloxane) 30 m long, 0.25
Analysis Method for Pesticide Residues in Biological Matrices:
Gas Chromatography-mass Spectrometry
Sample doped + 2 mL of toluene (solvent extractants)
2 min vortexing
Centrifugation at 5000 rpm, 15 min, 4 °C
Recovery of the supernatant of each sample doped
Final volume in the vial
Analysis by GC/MS
Fig. 1 Liquid–Liquid Extraction Procedure.
Table 1 Extraction of pesticides doped in biological matrices.
Pesticides names Pest. families
Qr (ppm) Yield (%) Dichlorvos OP
RT
Std area
Sample area
Qi (ppm)
9.8 476291 224825 5 3.54 70.8 Chloropyriphos OP
3.67 73.4 heptachlor OC
22.5 918270 438669 5 3.58 71.6 DDT OC 31.95 2271035 724233 5 2.39 47.8
RT: retention time, Std: standard area, Qi: doped concentration, Qr: concentration recovered, OP: organophosphate, OC: organochlorine, DDT: dochlorodiphenyltriethane.
mm internal diameter and 0.25 µ of film thickness. 1 µ the authors use mass spectrometry to better identify of sample is injected in mode of splitless pulsed (20
compounds that contain samples injected. psi for 60 s), with a purge flow of 50 mL/min for one
5. Results
minute, the injector temperature is 220 °C. The temperature program used for the analysis is as
After analyzing, the quantitative results have been follows: 50 °C/min, 20 °C/min to 180 °C, 10 °C/min
presented [concentration recovered (Qr), and Yield to 190 °C, 3 °C/min to 240 °C, followed by a
(%) ] in (Table 1).
temperature gradient to 300 °C at 10 °C/min, this As the dichlorvos chromatogram has showed in Fig. temperature being maintained for 5 min. The carrier
2, the compound that the authors injected, it was gas used is helium (8.4 psi Helium N55) to 1.7
detected at 9.75 min. And then after molecular mL/min (constant flow). The temperature of the
fragmentation of the compound by mass spectrometry, interface between the GC and the mass spectrometer
it has confirmed by mass of the molecular ion of the was maintained constant (200 °C). The ionization of
same compound doped in Fig. 3.
the compounds was performed by electron impact at The molecule doped was detected at 25.092 min, as
70 eV. The ions separated by a quadrupole filter has shown on the chlorpyrifos chromatogram (Fig. 4). according to their mass/charge ratio (m/z). The
The mass of the molecular ion in the spectrum do not detection performed by ion selection mode (SIM
correspond with the molecular mass of the compound Selected Ion Monitoring) with the following parameters:
really doped (Fig. 5). Therefore, it means that the dwell time = 80 ms, 1.16 cycles per second and the
molecule has lost the chloride ion in position 3 of all electron multiplier voltage 400 V. autotune. Generally,
molecules. It’s due from the sulfur approximity which
Analysis Method for Pesticide Residues in Biological Matrices:
Gas Chromatography-mass Spectrometry
the number of electronegativity close to chlorine. But sulfur is much more stable with phosphorus by their double bond. This chlorine ion losing gives the molecular mass ion which is present in the spectrum.
As the DDT chromatogram has showed (Fig. 6), the compound with was injected, it was detected at 31.96 min. And then after molecular fragmentation of the compound by mass spectrometry, it has confirmed by mass of the molecular ion of the same compound doped (Fig. 7).
In this case, the bond of C-Cl is broken faster than C-H bond. It’s due to their steric hindrance, and then the molecular ions have lost chlorine ion. Therefore,
Fig. 5 Chlorpyfos structure [11].
Fig. 2 Dichlorvos Chromatogram.
Fig. 3 Dichlorvos structure [10].
Fig. 6 Dichlorodiphenyltriethan chromatogram.
Fig. 4 Chlorpyrifos chromatogram.
Fig. 7 (DDT) structure [12].
Analysis Method for Pesticide Residues in Biological Matrices:
Gas Chromatography-mass Spectrometry
organisms but also to non-target organisms. The human is one of them. Because he is applicator of these substances but he also consumes the food contaminated by residues [14]. The risk of toxicity depends on the exposure mode, acute toxicity or chronic toxicity [15]. The human contamination by pesticides can be done in different ways: contamination by ingestion, skin contact or by breathing (inhalation). The risk associated with contamination by ingestion is much more dominant than any other type of contamination, followed by contamination by inhalation [16, 17].
Once in the body, pesticides accumulate in adipose tissue, never to dislodge. However, the toxicity varies according to the active substances they contain. Organochlorines for example, by their persistence in the environment pose a significant animals risk.
Fig. 8 Heptachlor chromatogram.
Especially as humans accumulation is mainly in adipose tissue but also in the liver and muscles [16,
17]. Different studies [15, 18, 19] showed that human exposure to pesticides can manifest diseases long and short term. They also showed the degree of toxicity and effects in the human body. Despite this, it appears that the toxic dose in humans varies from one group to other groups of pesticides. Therefore, it is very
Fig. 9 Heptachlor Structure [13].
difficult to establish a toxic dose “unique”. It is the the molecular mass of the heptachlor compound doped
same, hence the concentrations lethal blood or serum is equal to the sum of the molecular mass ion in
which justifies sometimes looking for a very low limit spectrum (Fig. 8),and the atomic mass of chloride ion
of quantification [2, 20].
lost. As organophosphates are toxic chemicals, that act by inhibiting cholinesterase [21]. These are the most
used in the world. Because of their easy availability in Generally, pesticides are a group of chemical
6. Discussion
rural areas, their high toxicity and rapid action, they compounds very different from one class to another
cause many cases of accidental poisonings estimated 3 with physico-chemical characteristics, which will
million cases per year and are responsible for 220,000 require the development of varied assays and deaths in the world according to W.H.O, according to consequently highly diversified analytical equipment;
the many studies [22, 23]. They are also sometimes this can also be a handicap for many laboratories [2].
used for suicide. So, today, apart from the As shown in the various studies, the pesticides are
measurement of cholinesterase activity, what is lacks ubiquitous in environment. They are biologically
selectivity and sensitivity for low exposures, the active and therefore intentionally toxic to target
diagnosis of OP poisoning is done by direct
Analysis Method for Pesticide Residues in Biological Matrices:
Gas Chromatography-mass Spectrometry
measurement of the compound in the blood. extraction (LLE) it’s very important to measure them In general, according to a study published by
by instrumental analysis that uses the methods of gas Lacassie and Coll. [24], an assay of OP in blood and
chromatography (GC) for volatile compounds or high serum by GC-MS capillary column PTE5, ionization
pressure liquid chromatography (HPLC) coupled to is produced by electron impact and SIM acquisition
the mass spectrometry (MS). These methods allow mode, revealing Limits of Detection (LOD) ranging
characterizing them.
from 5 to 25 ng/mL depending on the pesticide.
Acknowledgments
Limits of quantification (LOQ) ranged from 10 to 50 ng/mL. The authors observed excellent response
The study was performed at National Institute of linearity from LOQ up to 1 mg/L [24].
Hygiene (NIH) of Rabat, Morocco. The authors In fact the work of Barr and Lopez and colleagues,
acknowledge the research team of NIH, particularly they describe techniques for the determination of OC
Ms. Mama Idamine for her kind Help for laboratory pesticides and their metabolites. In serum after
analysis. The authors also thank Dr. Elom Kouassive extraction by SPE column DB-5MS [25] and SPME
Aglago of the research in Nutrition and Food Sciences capillary column DB-XLB [26] after dosing by
of Ibn Tofail University, Morocco, for his pertinent Chomatographie gas coupled to high resolution mass
remarks.
spectrometry (GC-HRMS) both, they show that the
References
limits detections are the order of pg/mL. The pesticide most frequently observed is pp DDE, metabolite of pp
[1] Nations, U. 2003. International Code of Conduct on the Distribution and Use of Pesticides (Revised Version)
DDT, the average grade of 2.1 ng/mL. adopted by the Hundred and Twenty-third Session of the In order to highlight the adverse effects of
FAO Council in November 2002.
pesticides on human health, in this study, the authors [2] Anger, J., Kintz, P., Rennes, U. De, and Cedex, R. 2009. used samples of blood and gastric fluids which are “Difficultés analytiques de la caractérisation des pesticides dans le sang, Analytical Difficulties in the
often used in analyzes of human exposure. The blood Characterization of Pesticides in Blood.” Ann. Toxicol. has a strong link with the most toxic pesticides. It is
Anal. 21 (3): 131-141.
also considered as the main compartment, where high [3] Aprea, C., Colosio, C., Mammone, T., Minoia, C., and concentrations of residues are likely to be expected. Maroni, M. 2002. “Biological monitoring of pesticide exposure: A review of analytical methods.” Journal of
Therefore, because of the complexity of the blood, the Chromatography B. 769 (2): 191-219. quantification of these residues is a difficult task. It is
[4] Alavanja, M. C. R., Hoppin, J. A., and Kamel, F. 2004. important to use GC/MS to identify these products.
“Health effects of chronic pesticide exposure: Cancer and neurotoxicity.” Annual Review of Public Health 25:
The authors found that this extraction method used is
155-197.
much more sensitive to the OP for the OC [27]. [5] Liu, S., and Pleil, J. D. 2002. “Human blood and environmental media screening method for pesticides and
7. Conclusions
polychlorinated biphenyl compounds using liquid extraction and gas chromatography.” Mass Spectrometry
The dosage of pesticides and their metabolites in
Analysis 769: 155-167.
the blood allows both the diagnosis of intoxication [6] Liberda, E. N.,Tsuji, L. J. S., Martin, I. D., Cote, S., and the supervision of a professional or environmental
Ayotte, P., Dewailly, E., and Nieboer, E. 2014. “Plasma exposure. Blood is much more complex, which
concentrations of persistent organic pollutants in the Cree explains the great difficulty of assaying multitudes of northern Quebec, Canada: results from the multi-community environment-and-health study.”
chemical compounds that are dangerous to human Science of The Total Environment 470-471: 818-828. health. Indeed, after treatment of the sample liquid
[7] Koivunen, M. E., Gee, S. J., Nichkova, M., Chang, K.
Analysis Method for Pesticide Residues in Biological Matrices:
Gas Chromatography-mass Spectrometry
and Hammock, B. D., 2007. Monitoring human exposure Rendus Biologies 330 (2): 143-147. to pesticides using immunoassay.
[19] Masri, W., Belwaer, I., Brahmi, N., Ghorbal, H., [8] Kudo, K., Nagamatsu, K., Umehara, T., Usumoto,Y.,
Hedhili, A., and Mouldi, A. 2011. “Incidence et Sameshima, N., Tsuji, A. and Ikeda, N., 2012. “Rapid
caractéristiques des intoxications aux inhibiteurs de and reliable screening method for detection of 70
cholinestérases.” Revue Francophone des Laboratoires pesticides in whole blood by gas chromatography–mass
429: 41-46.
spectrometry using a constructed calibration-locking [20] Wang, H. S., Chen, Z. J., Wei, W., Man, Y. B., Giesy, J. database.” Legal Medicine 14 (2): 93-100.
P., Du, J., Zhang, G., Wong, C. K. C., and Wong, M. H. [9] Hernández, F., Sancho, J. V., and Pozo, O. J. 2005.
2013. “Concentrations of OCPs (organochlorine “Critical review of the application of liquid
pesticides) in human blood plasma from Hong Kong: chromatography/mass spectrometry to the determination
Markers of exposure and sources from fish.” of pesticide residues in biological samples.” Analytical
Environment International 54: 18-25. and Bioanalytical Chemistry 382 (4): 934-946.
[21] Cochran, R. C., Kishiyama, J., Aldous, C., Carr, W. C., [10] Wright A. S., Huston D. H., and Wooder M. F., 1979.
and Pfeifer, K. F. 1995. “Chlorpyrifos: Hazard "The chemical and biochemical reactivity of Dichlorvos.”
assessment based on a review of the effects of short-term archives of Toxicology 42: 1-18.
and long-term exposure in animals and humans.” Food [11] Han, X. L., Tian, F. F. , Ge, Y. S., Jiang, F. L., Lai, L., Li,
and Chemical Toxicology 33 (2): 165-172. D. W., Yu, Q. L., Wang, J., Lin, C., and Liu, Y. 2012.
[22] Chaudhary, S., Vora, S. G. M. D. H., Modi, P., Chauhan, “Spectroscopic, structural and thermodynamic properties
V., and Chotaliya, D. 2013. “An Epidemiological Study of chlorpyrifos bound to serum albumin: A comparative
of Fatal Aluminium Phosphide Poisoning At Rajkot.” study between BSA and HAS.” Journal of
IOSR Journal of Pharmacy pp. 17-23. Photochemistry and Photobiology B 109: 1-11.
[23] Konradsen, F., Hoek, W. van der, Cole, D. C., [12] Abad, A., Manclu, J. J., Mojarrad, F., Mercader, J. V.,
Hutchinson, G., Daisley, H., Singh, S., and Eddleston, M. Miranda, M. A., Primo, J., Guardiola, V., and Montoya,
2003. “Reducing acute poisoning in developing A. 1997. “Hapten Synthesis and Production of
countries—options for restricting the availability of Monoclonal Antibodies to DDT and Related
pesticides.” Toxicology 192 (2-3): 249-261. Compounds.” Journal of Agricultural and Food
[24] Lacassie, E., Gaulier, J. M., Marquet, P., Daguet, J. L., Chemistry pp. 3694-3702.
and Lachatre, G. 2001. “Multiresidue Determination [13] Suwalsky, M., Benites, M., Villena, F., Aguilar, F., and
Method for Organophosphorus Pesticides in Serum and Sotomayor, C. P. 1997. “The organochlorine pesticide
Whole Blood by Gas Chromatography—Mass-selective heptachlor disrupts the structure of model and cell
Detection.” Journal of Chromatography B: Biomedical membranes.” BBA Biomembranes 1326 (1): 115-123.
Sciences and Applications pp. 109-116. [14] Soleas, G. J., Yan, J., Hom, K., and Goldberg, D. M.
[25] Barr, J. R., Maggio, V. L., Barr, D. B., Turner, W. E., 2000. “Multiresidue analysis of seventeen pesticides in
Sjödin, A., Sandau, C. D., Pirkle, J. L., Needham, L. L., wine by gas chromatography with mass-selective
and Patterson, D. G. 2003. “New high-resolution mass detection.” Journal of Chromatography A 882: 205-212.
spectrometric approach for the measurement of [15] Beaulieu, C. De, and Multigner, L. 2005. Effets retardés
polychlorinated biphenyls and organochlorine pesticides des pesticides sur la santé humaine 4: 187-194.
in human serum.” Journal of Chromatography B 794 (1): [16] Wyk, E. van, Bouwman, H., Bank, H. van der, Verdoorn,
137-148.
G. H., and Hofmann, D. 2001. “Persistent organochlorine [26] López, R., Goñi, F., Etxandia, A., and Millán, E. 2007. pesticides detected in blood and tissue samples of
“Determination of organochlorine pesticides and vultures from different localities in South Africa.”
polychlorinated biphenyls in human serum using Comparative Biochemistry and Physiology Part C:
headspace solid-phase microextraction and gas Toxicology & Pharmacology 129 (3): 243-264.
chromatography-electron capture detection.” Journal of [17] Furst, P., Furst, C., and Wilmers, K. 1994. “Human Milk
Chromatography B-Analytical Technologies in the as a Bioindicator for Body Burden of PCDDs , PCDFs ,
Biomedical and Life Sciences 846 (1-2): 298-305. Organo- chlorine Pesticides , and PCBs.” Environ. Health
[27] Tarbah, F. A., Mahler, H., Temme, O., and Daldrup, T. Perspect. 102: 187-193.
2001. “An Analytical Method for the Rapid Screening of [18] Rezg, R., Mornagui, B., Kamoun, A., El-Fazaa, S., and