Acute and chronic pesticides exposure could cause several health problems and serious diseases, many scientific studies proved a link between pesticides exposure and cancer, leukemia, disruption of the endocrine, immune or neural system (Carrillo et al., 2006). Chemical pesticide, fungicide, and herbicide trace level residues found in food is a concern that has gained international attention. The European Union (EU) adopted regulations in 2001 reducing residual pesticide tolerance levels in tea by one hundred times. The Japanese positive list for maximum residual limits of agricultural chemicals in food was made effective in 2006. The United States also regulates pesticide limits in food, the maximum residue limits for pesticides and extraneous maximum residue limits was also adopted by the Codex Alimentarius Commission.

Rwanda Standards Board Testing laboratory has introduced the testing scope of pesticide residues in Water, Tea, Honey, Cereals, Juice, Fruits and Vegetables. The residues that are currently tested are:  

• The Organochlorine Pesticides (OCP) residues: Chloroneb, Alpha BHC, Beta BHC, Delta BHC, Gamma BHC, Endosulfan ether, Aldrin, 4,4 Dichlorobenzophenone, Fenson, Isodrin, Chlorbenside, 2,4 DDE, Endosulphan alpha, Chlordane, Trans chlordane, Chlorfenson, 4,4 DDE, Dieldrin, 2,4 DDD, Endrin, Perthane, Endosulfan beta, 4,4 DDD, 2,4 DDT, Endrin aldehyde, Methoxychlor olefin, 4,4 DDT, 2,4 Methoxychlor, Endrin ketone and Tetradifon.
• The Organophosphorus Pesticides (OPP) residues:  Diazinon, Isazophos, Chlorpyrifos methyl, Pirimiphos methyl, Fenitrothion, Chlorpyrifos, Pirimiphos ethyl,Quinalphos, Pyridaphenthion,EPN, Phosmet, Phosalone, Aziphos methyl, Pyrazophos, Pyraclophos, Aziphos Ethyl

1.    Analysis of pesticides residues

Gas Chromatography with Mass Spectrophotometer (GC/MS) is utilized to develop trace level calibration curves in samples spiked with 22 OCP/OPP pesticide standards. The GC-MS offers high spectral acquisition rates needed for detection of trace parts-per- billion (ppb) level residual components in complex sample matrices, a sensitive and robust calibration curve is developed from 10 to 500 ppb. Exceptional limits of detection are achieved at or below 10ppb and then different types of samples are qualitatively analyzed with subsequent quantification for the OCP/OPP pesticides. Figure1:  RSB Laboratory Officer operating the GC/MS The GC-MS instrument is equipped with an Agilent 7000 GC/MS Triple Quadrupole featuring a multipurpose auto sampler, a cryogenic inlet system, and GC oven. Mass hunter software is used for all acquisition control, calibration curve development, peak identification, and data quantification. HP-5MS, 30m X 250mm, 0.25µm film thickness capillary column is used for the chromatographic separation. The GC is operated with Helium carrier gas at a corrected constant flow of 1 ml per minute. The OCP/OPP pesticide standards are spiked in various samples at ppb levels from 10 to 500 ppb.  The chromatographic temperature program for oven module was set at different ramps and the MS transfer line temperature is set to 280°C.   Figure 2: Displays the TIC chromatogram for OCP

2. Calibration Curve Development

Pesticide calibration standards are prepared and spiked into samples at ppb levels,  two injections of each extracted standard are made to develop calibration curves at different concentrations from  10 to 500 ppb, The calibration curves are utilized to examine and quantify trace level OCP/OPP pesticide residues in different types of samples. A complete list of all the OCP/OPP pesticides with retention time are in Table 1 for the GC/MS analysis,  shown in Figure 4 is the full range six point calibration curve acquired by GC-MS for Aldrin and developed from two data points for each concentration level. The inset shows the low end linearity for the concentrations from 10 to 500 ppb. The correlation coefficient for Aldrin is 0.9998.

Figure 3: Aldrin calibration curve covering 10 to 500 ppb. Table 1: List of OCP/OPP with retention time (min.) in the GC/MS analysis

Compound Names	Retention time	Compound Names	Retention time
Chloroneb	7.378	4,4 DDD	16.48
Alpha BHC	9.123	2,4 DDT	16.577
Beta BHC	9.646	Endrin aldehyde	16.765
Delta BHC	9.781	Methoxychlor olefin	17.415
Gamma BHC	10.283	4,4 DDT	17.659
Endosulfan ether	10.728	2,4 Methoxychlor	18.059
Aldrin	12.314	Endrin ketone	19.005
4,4 Dichlorobenzophenone	12.543	Tetradifon	20.215
Fenson	12.788	Diazinon	8.233
Isodrin	13.104	Isazophos	8.351
Chlorbenside	13.923	Chlorpyrifos methyl	8.643
2,4 DDE	14.234	Pirimiphos methyl	8.788
Endosulphan alpha	14.448	Fenitrothion	8.821
Chlordane	14.531	Chlorpyrifos	8.953
Trans chlordane	14.68	Pirimiphos ethyl	9.044
Chlorfenson	14.734	Quinalphos	9.264
4,4 DDE	15.176	Pyridaphenthion	10.523
Dieldrin	15.216	EPN	10.614
2,4 DDD	15.441	Phosmet	10.634
Endrin	15.879	Phosalone	10.962
Perthane	15.96	Aziphos methyl	11.023
Endosulfan beta	16.164	Pyrazophos	11.196
Endrin aldehyde	16.765	Pyraclophos	11.328
Methoxychlor olefin	17.415	Aziphos Ethyl	11.353

Reference Cerrillo, I., Olea-Serrano, M.F., Ibarluzea, J., Exposito, J., Torne, P., Laguna, J., Pedraza, V.,    Olea, N. (2006): Environmental and lifestyle factors for organochlorine exposure among women living in Southern Spain. Chemosphere 62(11), pp 1917-1924.