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Mississippi State Chemical Laboratory (MSCL) Laboratory Methods

Method Code

Method Title

001

Analysis or Organochlorine Pesticides and PCBs In Animal and Plant Tissue

002

Analysis For Organochlorine Pesticides and PCBs In Soil and Sediment

003

Analysis For Aliphatic and Polynuclear Aromatic Hydrocarbons In Animal and Plant Tissue

004

Analysis For Organochlorine Pesticides and PCBs, Aliphatic and Polynuclear Aromatic Hydrocarbons In Soil and Sediment

005

Analysis For Organochlorine Pesticides and PCBs, Polynuclear Nuclear Aliphatic and Hydrocarbons In Water

006

Analysis For Chlorinated Hydrocarbon Pesticides And Related Compounds - Micro Method

007

Analysis for Organochlorine Pesticides, N/P Pesticides, Aliphatic and Polynuclear Aromatic Hydrocarbons, and Chlorophenoxy Acid Herbicides in Water

008

Analysis for Organochlorine Pesticides, Aliphatic and Polynuclear Aromatic Hydrocarbons, and Chlorophenoxy Acid Herbicides in Soil and Sediment

009

Determination of Organochlorine Pesticides, Arochlors, Aliphatic Hydrocarbons and Polynuclear Aromatic Hydrocarbons in Hexane Extracts From Passive In-Situ Samplers

010

Analysis For Oil and Grease In Soil and Sediment

011

Analysis for Organochlorine Pesticides and PCBs in Blood Serum, Plasma or Whole Blood

012

Analysis for Amitrol-T in Soil and Sediment

013

Analysis For Oil and Grease In Water

014

Elution Profiles for Florisil, Silica Gel and Silicic Acid Column Separations

015

Analysis for Purgeable Organic Compounds by Capillary Column GC/MS.

016

Analysis for AHH Active PCB Congeners in Animal Tissue

017

Analysis For Carbamates In Sediment

018

Analysis For Carbamates In Water

019

Grain Size

020

Total Organic Carbon

021

Semivolatile Organic Compounds

022

Analysis For Selected Organophosphate and Synthetic Pyrethroid Pesticides In Animal and Plant Tissue.

023

Analysis For Selected Organophosphate and Synthetic Pyrethroid Pesticides and Triazine Herbicides in Soil and Sediment.

025

Analysis For Selected Organophosphate and Synthetic Pyrethroid Pesticides and Triazine Herbicides in Water

026

Analysis For PCP, Carbaryl, 4-nonylphenol, and Permethrin by GC/MS

027

Method for Substituted Urea Herbicides in Water

028

Analysis For AHH active PCB Congeners In Soil and Sediment
Analysis For AHH active PCB Congeners - Micro Method
DETERMINATION OF STARLICIDE IN WATER
Accelerated Solvent Extraction (ASE) and Analysis of Synthetic Pyrethroid Pesticides in Filter Disks.
Accelerated Solvent Extraction (ASE) and Analysis of Synthetic Pyrethroid Pesticides in Plant Tissue.
Accelerated Solvent Extraction (ASE) and Analysis of Disyston, Disyston Metabolites, and Oxygen Analoges in Filter Disks.
Analysis for Chlorophenoxy Acid Herbicides in Liver
Analysis for Organochlorine Pesticides and PCBs in Soil and Sediment by ASE.
Analysis of Soil and Vegetation for Glyphosate
Dioxins
Analysis For Selected Organophosphates in Soil and Sediment by ASE and GPC.

040

Analysis for PAHs in Soil/Sediment and plant tissue by ASE and GPC.
041 Analysis of Water -- Special for E. Snyder-Conn
042 Analysis for Organochlorine Pesticides, N/P Pesticides, Aliphatic and Polynuclear Aromatic Hydrocarbons, and Chlorophenoxy Acid Herbicides in Water
043 Analysis For Triazine Herbicides in Soil, Sediment and Tissue.
Analysis of Synthetic Pyrethroid Pesticides in Bat Guano.
045 Analysis for PCB Congeners In Animal Tissue or Soil/Sediment
046 Dioxin and Furan in Tissue
047 Synthetic Pyrethroid Pesticides / Micromethod
048 Analysis For Carbamates In Tissue
049 Analysis for Pyrethroids in Animal Tissue
050 Analysis For Organochlorines Pesticides and PCBs in Animal Tissue using soxhlet/GPC.

051

Analysis For Organochlorines Pesticides and PCBs, Aliphatic and Polynuclear Aromatic Hydrocarbons In Soil and Sediment

052

Benomyl in Water

053

Ammonia Procedure

054

Nitrite and Nitrate Procedure
056 Analysis For Organophosphate In Soil and Sediment, GPC cleanup.
057 Analysis for TPH (total petroleum hydrocarbon) in Soil and Sediment
Analysis for Paraquat and Diquat in Water
060 Analysis for Aliphatic and Polynuclear Aromatic Hydrocarbons in Animal Tissue
Analysis for Atrazine metabolites in Water
Analysis of Water for Glyphosate
Toxicity Characteristic Leaching Procedure (TCLP)
Analysis for Organophosphate Pesticides and Triazine Herbicides in Animal Tissue using ASE/GPC
Analysis for Pirate and Metabolites Tissue using ASE/GPC.
Analysis for Organochlorine Pesticides and PCBs in Animal Tissue using ASE/GPC.
Analysis for Organochlorine Pesticides and PCBs, Aliphatic and Polynuclear Aromatic Hydrocarbons in Soil and Sediment
Analysis for Organochlorine Pesticides and PCBs in Soil and Sediment, ASE, Dridisk.
Imadacloprid in Tissue
Imidacloprid in Soil and Sediment
Anticoagulant Rodenticides in Blood and Liver
Analysis of Water for Imazapic
Analysis for TPH (total petroleum hydrocarbon) in Water
% Moisture

 

 

 

 

 

 

Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 001

Analysis For Organochlorine Pesticides and PCBs In Animal and Plant Tissue

Ten gram tissue samples are thoroughly mixed with anhydrous sodium sulfate and soxhlet extracted with hexane for seven hours. The extract is concentrated by rotary evaporation; transferred to a tared test tube, and further concentrated to dryness for lipid determination. The weighed lipid sample is dissolved in petroleum ether and extracted four times with acetonitrile saturated with petroleum ether. Residues are partitioned into petroleum ether which is washed, concentrated, and transferred to a glass chromatographic column containing 20 grams of Florisil. The column is eluted with 200 ml 6% diethyl ether/94% petroleum ether (Fraction I) followed by 200 ml 15% diethyl ether/85% petroleum ether (Fraction II). Fraction II is concentrated to appropriate volume for quantification of residues by packed or capillary column electron capture gas chromatography. Fraction I is concentrated and transferred to a Silicic acid chromatographic column for additional cleanup required for separation of PCBs from other organochlorines. Three fractions are eluted from the silicic acid column. Each is concentrated to appropriate volume for quantification of residues by packed or megabore column, electron capture gas chromatography. PCBs are found in Fraction II.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 002

Analysis For Organochlorine Pesticides and PCBs In Soil and Sediment

Twenty-five gram soil or sediment samples are extracted with acetone followed by hexane, by allowing to soak one hour in each with intermittent shaking. The combined extracts are centrifuged and decanted into a separatory funnel containing sufficient water to facilitate partitioning of residues into hexane portion. The hexane is washed twice with water and concentrated to appropriate volume for transfer to a 1.6 gram Florisil mini-column topped with 1.6 grams sodium sulfate. Residues are eluted from the column in two elution fractions. Fraction I consists of 12 milliliters hexane followed by 12 milliliters of 1% methanol in hexane, and Fraction II consists of an additional 24 milliliters of 1% methanol in hexane. If additional cleanup is required to separate PCBs from other organochlorines in Fraction I, further chromatography on a Silicic acid column is performed. Quantification of residues in the two Florisil fractions and three Silicic acid fractions is by packed or megabore column, electron capture gas chromatography.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 003

Analysis For Aliphatic and Polynuclear Aromatic Hydrocarbons In Animal and Plant Tissue

A sample of appropriate size (i.e. 15 grams animal or plant tissue, 2 grams adipose, 5 grams eggs) is digested in 6N aqueous potassium hydroxide for 24 hours at 35 degrees C. Cool digestate thoroughly in an ice bath and carefully neutralize with glacial acetic acid. Extract the neutralized reaction mixture three times with methylene chloride; concentrate the combined extracts to near dryness and reconstitute in petroleum ether for transfer to a 20 gram 1% deactivated silica gel column, topped with 5 grams neutral alumina. Aliphatic and polynuclear aromatic hydrocarbon residues are separated by eluting aliphatics from the column with 100 ml petroleum ether (Fraction I) followed by elution of aromatics using first, 100ml 40% methylene chloride/60% petroleum ether, then 50 ml methylene chloride (Combined eluates, Fraction II). If needed, Fraction I containing aliphatics is subjected to additional cleanup by concentration and transfer to a deactivated (2% water) Florisil column. Aliphatic residues are eluted from the Florisil column using 200 ml 6% diethyl ether/94% petroleum ether. The eluate is concentrated to appropriate volume for quantification by capillary column, flame ionization gas chromatography. The silica gel Fraction II containing aromatic hydrocarbons is concentrated, reconstituted in methylene chloride, and subjected to gel permeation chromatography (GPC) cleanup prior to quantification by capillary, flame ionization gas chromatography and fluorescence HPLC.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 004

Analysis For Organochlorine Pesticides and PCBs, Aliphatic and Polynuclear Aromatic Hydrocarbons In Soil and Sediment

Twenty gram soil or sediment samples are extracted with acetone, followed by petroleum ether, by allowing to soak one hour in each with intermittent shaking. A final acetone/petroleum ether extraction is done, and the extracts are combined, centrifuged, and transferred to a separatory funnel containing sufficient water to facilitate partitioning of residues into petroleum ether portion. The petroleum ether is washed twice with water and concentrated by Kuderna-Danish to appropriate volume. An aliquot of the concentrated extract for pesticide determination is transfered to a 1.6 gram Florisil mini-column topped with 1.6 grams sodium sulfate. Residues are eluted from the column in two elution fractions. Fraction I consists of 12 milliliters hexane followed by 12 milliliters of 1% methanol in hexane, and Fraction II consists of an additional 24 milliliters of 1% methanol in hexane. If additional cleanup is required to separate PCBs from other organochlorines in Fraction I, further chromatography on a Silicic acid column is performed. Quantification of residues in the two Florisil fractions and three Silicic acid fractions is by packed or megabore column, electron capture gas chromatography.

A second aliquot of the concentrated extract for hydrocarbon determination is transfered to a 20 gram 1% deactivated silica gel column, topped with five grams neutral alumina. Aliphatic and polynuclear aromatic hydrocarbon residues are fractionated by eluting aliphatics from the column with 100 ml petroleum ether (Fraction I) followed by elution of aromatics using first, 100 ml 40% methylene chloride/60%petroleum ether, then 50 ml methylene chloride (Combined eluates, Fraction II). If needed, Fraction I containing aliphatics is subjected to additional cleanup by concentration and transfer to a deactivated (2% water) Florisil column. Aliphatic residues are eluted from the Florisil column using 200 ml 6% diethyl ether/94% petroleum ether. The eluate is concentrated to appropriate volume for quantification by capillary column, flame ionization gas chromatography. The silica gel Fraction II containing aromatic hydrocarbons is concentrated, reconstituted in methylene chloride, and subjected to gel permeation chromatographic (GPC) cleanup prior to quantification by capillary, flame ionization gas chromatography and fluorescence HPLC.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 005

Analysis For Organochlorine Pesticides and PCBs, Polynuclear Nuclear Aliphatic and Hydrocarbons In Water

A 500 milliliter water sample is extracted four times by shaking with 50 milliliter portions of methylene chloride. The four extracts are combined and concentrated by Kuderna- Danish to near dryness, then reconstituted in 5 milliliters petroleum ether. An appropriate aliquot is removed for organochlorine and PCB analysis and transfered to a 20 gram Florisil column. The column is eluted with 200 ml 6% diethyl ether/94% petroleum ether (Fraction I) followed by 200 ml 15% diethyl ether/85% petroleum ether (Fraction II). Fraction II is concentrated to appropriate volume for quantification of residues by packed or capillary column electron capture gas chromotography. Fraction I is concentrated and transferred to a silicic acid chromatographic column for additional cleanup required for separation of PCBs from other organochlorines. Three fractions are eluted from the silicic acid column. Each is concentrated to appropriate volume for quantification of residues by packed or megabore column, electron capture gas chromotography. PCBs are found in Fraction II. The remainder of the petroleum ether from the above methylene chloride extraction is transferred to a 20 gram 1% deactivated silica gel column, topped with 5 grams neutral alumina. Aliphatic and polynuclear aromatic hydrocarbon residues are separated by eluting aliphatics from the column with 100 ml petroleum ether (Fraction I) folllowed by elution of aromatics using first, 100 ml 40% methylene chloride/60% petroleum ether then 50 ml methylene chloride (combined eluates, Fraction II). If needed, Fraction I containing aliphatics is subjected to additional cleanup by concentration and transfer to a deactivated (2% water) Florisil column. Aliphatic residues are eluted from the Florisil column using 200 ml 6% diethyl ether/94% petroleum ether. The eluate is concentrated to appropriate volume for quantification by capillary column, flame ionization gas chromotography. The silica gel Fraction II containing aromatic hydrocarbons is concentrated, reconstituted in methylene chloride, and subjected to gel permeation chromatographic (GPC) cleanup prior to quantification by capillary, flame ionization gas chromatography and fluorescence HPLC.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 006

Analysis For Chlorinated Hydrocarbon Pesticides And Related Compounds - Micro Method

This method is necessary when sample size is limited (below 4 g, approximately) and in case of organ tissue as substrate and is a modified version of the method described in "Manual of Analytical Methods for the Analysis of Pesticides in Humans and Enviromental Samples", EPA-600/8-80-038, June 1980, Section 5, A (2). It is suitable for adipose, kidney, liver, muscle, brain, and other tissues:

1. Weigh 0.5 g or less of well-mixed tissue into a size 22 Duall tissue grinder.

2. Extract tissue by grinding three times with acetonitrile; the first time being with 4 ml followed by two 2.5 ml portions.

3. Remove the pestle after each grinding and centrifuge, decanting the extract into a 50 ml glass stoppered graduated mixing cylinder.

4. Combine all extracts and record the total volume of the three extracts.

5. Add a volume of PRQ water equivalent to 3.3 times the extract volume. Then add 2 ml saturated NaCl solution.

6. Extract the aqueous acetonitrile mixture with 5 ml hexane by vigorous shaking for 1 minute.

7. Allow layers to separate, and remove the hexane layer with a Pasteur pipet into a 15 ml screw-capped culture tube.

8. Re-extract twice with 2 ml hexane each time, combining the extracts into the culture tube.

9. Concentrate the combined hexane extracts under nitrogen to approximately 0.5 ml volume.

10. Clean-up on a florisil mini-column as described in Method 2, Steps 8, 9, 10. and 11.

Note: For brain tissue additional treatment is necessary before column clean-up:

11. Proceed through Steps 1-9 above, add 0.3 ml acetic anhydride and 0.3 ml pyridine, cap tightly and incubate for 30 minutes in a water bath at 60-65 degrees C.

12. Add 8 ml PRQ water and 1 ml saturated NaCl and extract three times with 2 ml hexane, combining the extracts into a clean tube.

13. Concentrate the combined extracts under nitrogen to about 0.3 ml and proceed with florisil mini-column clean-up. (Step 10)

Note: The following changes in sample handling, particularly column clean-up, should be observed for Kepone analysis:

14. Maintain the integrity of the analyte in sample extracts by insuring that the samples are not allowed to reach dryness during concentration steps. Kepone easily adheres to glass, but the use of polar solvents such as methanol and acetonitrile within the analysis will provide better recoveries of this analyte.

15. Modifications to florisil mini-column clean-up are as follows:

* Following addition of sample to the column, apply a 1ml rinse of 1% methanol in hexane to the sample tube. This rinse should be added after the first phase of of the first fraction (12mls hexane) and will insure removal of trace quantities of kepone adhered to glass. Decrease the total volume of the second phase of the first fraction (12mls 1% methanol/hexane) to 11mls.

* Modify the total volume of the second fraction from 24mls to 36mls 1% methanol/hexane. This fraction contains Kepone.

* Concentrate column fractions on N-EVAP and transfer with 1% methanol/hexane to calibrated test tubes. Adjust sample volume to calibrated level and proceed to determination by gas chromatograph.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 007

Analysis for Organochlorine Pesticides, N/P Pesticides, Aliphatic and Polynuclear Aromatic Hydrocarbons, and Chlorophenoxy Acid Herbicides in Water

This procedure was developed to allow for sample extraction to be performed in the same container in which the sample is collected, thus eliminating the need to transfer the sample to other glassware and eliminating the possibility of incomplete extraction of any analyte that may adhere to glass surfaces. Five pint acid bottles, calibrated at two liters, are convenient for sample collection/extraction.

This procedure may be used with any volume of sample with the appropriate volume reduction of solvents, reagents, and glassware. However, if transfer from the collection container is necessary, this container should be rinsed with the extraction solvent to insure complete recovery of all analytes.

1. Reduce sample volume to 2 l or record volume if < 2 l.

2. Add PRQ 6N KOH to sample, seal and shake vigorously 30s. Test pH with glass rod (pH = 8). Adjust to proper pH by dropwise addition of 6N KOH. Allow to stand one hour at pH 8.

3. Add 100 ml CH2Cl2 and shake 2 min. with periodic venting. Remove CH2Cl2 into 500 ml French Square bottle using glass suction device attached to aspirator system. Reverse suction device to return water in CH2Cl2 layer to the bottle. Repeat 2 X 100 ml CH2Cl2. After final step, assure return of all water to bottle using Pasteur pipet. This combined extract contains organochlorine, nitrogen- /phophorous-containing pesticides, aliphatic hydrocarbons and polynuclear aromatic hydrocarbons.

4. Acidify water with PRQ 12N H2SO4. Shake 30s and test pH with glass rod. Adjust pH to ó 2 with dropwise addition of acid if necessary.

5. Extract water with 200ml Ethyl ether (EtoEt) by shaking two minutes. Remove either layer with suction device to 1000ml French Square. Return excess water to bottle. Repeat 2 x 100ml EtoEt. Extract with a final aliquot of 100ml petroleum ether. Remove all water from French Square with Pasteur pipet. This combined extract contains chlorophenoxy acid herbicides.

6. Concentrate acid and neutral extracts with Kuderna-Danish evaporators and reduce volume to adequate size for column clean-up.

7. Column Clean-Up:

* NEUTRAL FRACTION (N/P and organochlorine pesticides, Aliphatic and Polynuclear aromatic hydrocarbons) - adjust sample extract to exact volumn and remove an appropriate aliquot for column clean-up techniques specific to analyte; for pesticides use Mini-florisil (described in Method 2), for hydrocarbons use 1 % deactivated silica gel (described in Method 4).

* ACID FRACTION (Chlorophenoxy acid herbicides) - Derivitization: Reduce sample volume to approximately 0.5ml and ethylate using diazoethane (15 min.). Exchange to hexane (N-EVAP) and reduce volume to 0.2ml.

Column clean-up: Place 2.0g of 1% deactivated silica gel in a 7mm i.d. chromatography column (#22 Kontes). Top with 1cm Na2SO4 and prewetcolumn with 10ml hexane. Collect sample eluents in three fractions as follows:

Fraction A: add sample and rinse container with two 0.5ml washes of 20% benzene in hexane. Elute with 9ml of the same solution.(Conta ins PCP.)

Fraction B: add 10ml 40% benzene in hexane. Add 10ml 60% benzene in hexane.(Contains Dalapon, PNP, Silvex, Dinoseb, portion of Dicamba.) Fraction C: add 10ml 80% benzene in hexane. Add 10ml 100% benzene.(Contains remaining Dicamba, Dichlorprop, 2,4-D, 2,4,5-T, 2,4-DB, Bentazon, Blazer.)

[ Reference for column clean-up for acid herbicides:Shafik, T. A.,H. C. Sullivan, H. R. Enos, 1973." Multiresidue Procedure for Halo- and Nitrophenols. Measurement of Exposure to Biodegradable Pesticides Yielding These Compounds as Metabolites." J. Agr. Food Chem. 21:295-298. ]

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 008

Analysis for Organochlorine Pesticides, Aliphatic and Polynuclear Aromatic Hydrocarbons, and Chlorophenoxy Acid Herbicides in Soil and Sediment

1. Weigh 20 g soil into a PRQ centrifuge bottle. (Add 10 ml PRQ H2O to dry samples) Adjust pH to ó 2 with PRQ 12N sulfuric acid (about 1 ml). Add 50 ml acetone and shake 6 times over a one and one-half hour period (about every 15 mins.). Add 50 ml of a 1:1 petroleum ether/ ethyl ether mixture and repeat shaking. Centrifuge and decant liquid into a 500 ml separatory funnel containing 200 ml PRQ water. Re- extract soil by shaking one minute with 50 ml 1:1 PE:EtoEt (may need to add 10 ml H2O & adjust to pH < 2), then centrifuge and decant liquid into sep. funnel.

2. Using PRQ 6N KOH (5 ml), adjust contents of sep. funnel to pH ò 12. Shake vigorously 2 min, then allow to stand 30 min. with intermittent shaking. Drain water layer and reserve ether layer. Re-extract H2O layer with 100 ml 1:1 PE:EtoEt. Cap and reserve combined ether extracts.(This contains organochlorine pesticides, aliphatic and polynuclear aromatic hydrocarbons.)

3. Adjust aqueous layer to pH ó 2 using 3 ml of PRQ 12 N sulfuric acid and extract with 100 ml 1:1 PE:EtoEt. Reserve this extract and re-extract H2O with 100 ml 1:1 PE:EtoEt. Combine extracts (these extracts contain chlorophenoxy acid herbicides).

4. Concentrate acid and basic extracts with Kuderna-Danish evaporators and reduce volume to adequate size for column clean-up.

5. Column Clean-up:

* BASIC FRACTION (N/P and Organochlorine pesticides, Aliphatic and Polynuclear aromatic hydrocarbons) - adjust sample extract to exact volume and remove an appropriate aliquot for column clean-up techniques specific to analyte; for pesticides use Mini-florisil (described in Method 2), for hydrocarbons use 1% deactivated silica gel (described in Method 4).

* ACID FRACTION (Chlorophenoxy acid herbicides) - Derivitization: Reduce sample volume to approximately 0.5ml and ethylate using diazoethane (15 min.). Exchange to hexane (N-EVAP) and reduce volume to 0.2ml. Column clean-up: Place 2.0g of 1% deactivated silica gel in a 7mm i.d. chromatography column (#22 Kontes). Top with 1cm Na2SO4 and prewet column with 10ml hexane. Collect sample eluents in three fractions as follows:

Fraction A: add sample and rinse container with two 0.5ml washes of 20% benzene in hexane. Elute with 9ml of the same solution.(Contains PCP.)

Fraction B: add 10ml 40% benzene in hexane. Add 10ml 60% benzene in hexane.(Contains Dalapon, PNP, Silvex, Dinoseb, portion of Dicamba.)

Fraction C: add 10ml 80% benzene in hexane. Add 10ml 100% benzene.(Contains remaining Dicamba, Dichlorprop, 2,4-D, 2,4,5-T, 2,4-DB, Bentazon, Blazer.)

[ Reference for column clean-up for acid herbicides: Shafik, T. A.,H. C. Sullivan, H. R. Enos, 1973." Multiresidue Procedure for Halo- and Nitrophenols. Measurement of Exposure to Biodegradable Pesticides Yielding These Compounds as Metabolites." J. Agr. Food Chem. 21:295-298. ]

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 009

Determination of Organochlorine Pesticides, Arochlors, Aliphatic Hydrocarbons and Polynuclear Aromatic Hydrocarbons in Hexane Extracts From Passive In-Situ Samplers

1. Measure and record the total extract volume; transfer to a 1 l Kjeldahl- shaped flask.

2. Concentrate the hexane extract to about 1 ml (45øC bath temp.).

3. Transfer the extract with petroleum ether through a 9" Pasteur pipet containing about 1 inch of anhydrous Na2SO4 into a 16 mm X 150 mm screw- capped culture tube.

4. Carefully concentrate to about 0.5 ml on the N-Evap.

5. Transfer to an appropriate size calibrated screw-capped tube with hexane and adjust volume to 2.0 ml (or other appropriate volume).

6. Determine residues using appropriate glc techniques.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 010

Analysis For Oil and Grease In Soil and Sediment

Fifty gram soil or sediment samples are extracted with acetone, followed by petroleum ether, by allowing to soak one hour in each with intermittent shaking. The samples are centrifuged, and the supernatant is decanted into a separatory funnel containing sufficient water to facilitate partitioning of residues into petroleum ether portion. Two further acetone/petroleum ether extractions are done, and the extracts are sequentially centrifuged, and transferred to the separatory funnel. The aqueous portion is extracted with petroleum ether and the combined ether extracts are washed twice with water and concentrated by Kuderna-Danish to appropriate volume for transfer. The sample is transferred with petroleum ether rinsing through a bed of sodium sulfate into a tared glass tube. Solvent is removed under nitrogen ( N-EVAP ), and tube weights are allowed to equilibrate prior to the determination of oil and grease values.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 011

Analysis for Organochlorine Pesticides and PCBs in Blood Serum, Plasma or Whole Blood

Reference: Manual of Analytical Methods for the Analysis of Pesticides in Humans and Enviromental Samples, Section 5, A, (3), (a)

Modified as follows:

A 2-ml aliquot of serum, Plasma or whole blood, is extracted with 6 ml of hexane in a 16 X 125 mm screw-capped(teflon liner) culture tube. The extraction is conducted for 2 hours at 50 RPM on a rotating mixer. Alternatively, the extraction may be accomplished by vigorous mixing on a vortex mixer for 2 minutes. The formation of emulsion is unlikely, but if it should occur, centrifugation may be used to effect separation of the layers. In most instances, quantification by ec/gc can be performed on the hexane extract without further treatment; otherwise a suitable aliquot of the hexane layer may be quantitatively transferred to an evaporative concentrator tube to which is affixed a modified micro-Snyder column. The extract is concentrated in a water or steam bath, and the final volume is adjusted to correspond to the expected concentration of the pesticide residue. A suitable aliquot is analyzed by electron capture gas chromatography.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 012

Analysis for Amitrol-T in Soil and Sediment

1. Weigh 20 g well-mixed, homogeneous soil/sediment into a 200 ml screw-capped centrifuge bottle. Add 50 ml 80 % ethanol/H2O and allow to soak 1 1/2 hour with frequent intermittent shaking.

2. Centrifuge appropriately and transfer the aqueous extract to a 250ml separatory funnel.

3. Repeat the extraction with 80 % ethanol/H2O twice more as before, reducing the extraction time to 30 minutes. Combine the aqueous extracts in the separatory funnel.

4. Wash the aqueous extract with 20 ml hexane, discarding the hexane.

5. Repeat hexane wash of aqueous extract twice more, discarding hexane each time.

6. Transfer the extract to a 300 ml, 24/40 pear-shaped flask using PRQ H2O, and concentrate to ca 2 ml on a rotary evaporator with the waterbath set at 60 degrees C.

7. Transfer the sample with PRQ H2O to a screw-capped, calibrated culture tube and adjust volume to the calibration mark (10 ml normally used) with H2O. Quantitate by capillary gas chromatography (TSD optimized for N2 detection).

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 013

Analysis For Oil and Grease In Water

Sample volume and pH are determined, and an 800 ml aliquot is extracted with 75 ml methylene chloride three times. The combined ether extracts are washed twice with water and concentrated by Kuderna-Danish to appropriate volume for transfer. The sample is transferred with petroleum ether rinsing through a bed of sodium sulfate into a tared glass tube. Solvent is removed under nitrogen ( N-EVAP ), and tube weights are allowed to equilibrate prior to the determination of oil and grease values.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 014

Elution Profiles for Florisil, Silica Gel and Silicic Acid Column Separations

A. Florisil Column:

1. Fraction I - (6% ethyl ether containing 2% ethanol, 94% petroleum ether) HCB, alpha-BHC, beta-BHC, gamma-BHC, delta-BHC, oxychlordane, heptachlor epoxide, gamma-chlordane, trans-nonachlor, toxaphene, PCB's, o,p'-DDE, alpha-Chlordane, p,p'-DDE, p,p'-DDT, cis-nonachlor, o,p'-DDT, p,p'-DDD, p,p'-DDT, mirex, dicofol, endosulfan I (Split with FII).

2. Fraction II - (15% ethyl ether containing 2% ethanol, 85% petroleum ether) dieldrin, endrin, dacthal, endosulfan I (split with FI), endosulfan II (split with FIII), endosulfan sulfate (split with FIII).

3. Fraction III - (50% ethyl ether containing 2% ethanol, 50% petroleum ether) endosulfan II (split with FII), endosulfan sulfate (split with FII), malathion.

B. Florisil Mini-Column:

1. Fraction I - (12 ml hexane followed by 12 ml 1% methanol in hexane) HCB, gamma-BHC (25%), alpha-BHC (splits with FII), trans-nonachlor, o,p'-DDE, p,p'-DDE, o,p'-DDD, p,p'-DDD (splits with FII), o,p'-DDT, p,p'-DDT, mirex, cis-nonachlor, cis-chlordane, trans-chlordane, PCB's, Photomirex and derivatives.

2. Fraction II - (24 ml 1% methanol in hexane) gamma BHC (75%), beta-BHC, alpha-BHC (splits with FI), delta-BHC, oxychlordane, heptachlor epoxide, toxaphene, dicofol, dacthal, endosulfan I, endosulfan II, endosulfan sulfate, octachlorostyrene, Kepone (with additional 12mls 1% methanol in hexane).

C. Silica Gel:

1. SG Fraction I - (100 ml petroleum ether) n-dodecane, n-tridecane, n-tetradecane, ocylcyclohexane, n-pentadecane, nonycyclohexane, n-hexadecane, n-heptadecane, pristane, n-octadecane, phytane, n-nonadecane, n-eicosane.

2. SG Fraction II - (100 ml 40% methylene chloride in petroleum ether followed by 50 ml methylene chloride) napthalene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, 1,2-benzanthracene, chrysene, benzo [b] fluoranthene, benzo [k] fluoranthene, benzo [e] pyrene, benzo [a] pyrene, 1,2:5,6-dibenzanthracene, benzo [g,h,i] perylene.

D. Silicic Acid:

1. SA Fraction I - (20 ml petroleum ether) HCB, mirex

2. SA Fraction II - (100ml petroleum ether) PCB's, p,p'-DDE (splits with SA III)

3. SA Fraction III - (20 ml mixed solvent: 1% acetonitrile, 80% methylene chloride, 19% hexane) alpha-BHC, beta-BHC, gamma-BHC, delta-BHC, oxychlordane, heptachlor epoxide, gamma-chlordane, trans-chlordane, toxaphene, o,p'-DDE, alpha-chlordane, p,p'-DDE (splits with SAII), o,p'-DDT, cis-nonachlor, o,p'-DDT, p,p'-DDD, p,p'-DDT, dicofol.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 015

Analysis for Purgeable Organic Compounds by Capillary Column GC/MS.

This method is applicable to all types of samples (Water, Soil/Sediment, Animal Tissues, and Plant Tissues). GC/MS CONDITIONS

1) The MS must pass spectrum criteria for 4-bromofluorobenzene as stated in EPA methods 524.2 and 8260.

2) Generate a five point standard curve that passes the EPA criteria.

WATER

Measure ten (10) ml of water in a glass syringe. Add four-hundred (400) ng of internal standards (dibromotetrafluoroethane, d-6 benzene, and d-5 chlorobenzene) and four- hundred (400) ng of surrogates (bromofluorethane, bromofluoromethane, bromofluorobenzene). Load the purge and trap unit (Tekmar LSC2000 & ALS2016) with the sample and purge with He using the needle sparger. Trap the compounds on a Vocarb 3000 Trap (Supelco) and determine the compounds using a Varian Saturn ion trap with a Sixty (60) m 0.32 RTX-Volatile capillary column.

SOIL/SEDIMENT

Weigh up to five (5) grams soil into test tube. Add internal standards and surrogates to ten (10) ml reagent water. Add this to the soil and purge with needle sparger. Follow the water procedure from here.

TISSUES

Weigh up to two (2) grams of frozen tissue into the test tube. Follow the above procedure for Soil/Sediment.

REFERENCES:

Method 524.2 Measurement of Purgeable Organic Compounds in Water by Capillary Column Gas Chromatography/Mass Spectrometry Rev.3.0, 1989, Environmental Monitoring Systems Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268.

Method 8260 Volatile organic compounds by Gas Chromatography/Mass Spectrometry (GC/MS): Capillary column technique, 1990. Test Methods for Evaluating Solid Wastes Volume 1B: Laboratory Manual Physical/Chemical Methods, U.S. Environmental Protection Agency.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 016

Analysis for AHH Active PCB Congeners in Animal Tissue

Five gram tissue samples are thoroughly mixed with anhydrous sodium sulfate and soxhlet extracted with hexane for seven hours. The extract is concentrated by rotary evaporation; transferred to a tared test tube, and further concentrated to dryness for lipid determination. The weighed lipid sample is dissolved in petroleum ether and extracted four times with acetonitrile saturated with petroleum ether. Residues are partitioned into petroleum ether which is washed, concentrated, and transferred to a glass hromatographic column containing 20 grams of Florisil. The column is eluted with 200 ml 6% diethyl ether/94% petroleum ether (Fraction I). Fraction I is concentrated and transferred to a Silicic acid chromatographic column for additional cleanup required for separation of PCBs from other organochlorines. Three fractions are eluted from the silicic acid column. Fraction I is 20 ml of Petroleum Ether. Fraction II is 150 ml of Petroleum ether and contains the PCB's. Fraction III contains most of the other organochlorine pesticides.

Fraction II is concentrated to one ml and fractionated on a carbon column. This column consists of 2 grams of 1:20 AX-21 Carbon on activated silica gel. (AX-21 Activated Carbon, Anderson Development Co., Adrian, MI, vacuum washed with 200 ml each water, methanol, toluene, acetone, methylene chloride, 2X pet ether) (Silica gel EM Science Silica gel 60-230 mesh activated @ 130 C more than 10 days) Fraction A consists of 100 ml Pet Ether followed by 50 ml 10% methylene chloride in Pet Ether. Fraction B is 100 ml of 50% methylene chloride in Pet Ether. Fraction C is 50 ml toluene. Fraction B contains PCB# 105, 114, 118, 156. Fraction C contains PCB# 077, 126, 169. The PCB fractions were chromatographed on a Varian 3400 GC using a 60m 0.25 DB-5 capillary column. Confirmation was done on a 30m 0.53 DB-608 megabore column.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 017

Analysis For Carbamates In Sediment

Analysis of samples for carbamates was performed on the waters Carbamates analysis system equipped with a WISP model 712 Autosampler. Sediment samples (20g) were extracted with 20ml methanol by sonication for 30 minutes, then allowed to sit one hour. The samples were filtered through 0.45u nylon filters. A dilution in water ( 1:10) was made, and 500ul injected. The separation of the analytes was at room temperature, and the column used was a 3.9mm x 150mm waters nova-par C10 (4u). The solvent gradient used was as follows:

TIME
FLOW
%WATER
%METHANOL
%ACETONITRILE
CURVE
INIT
1
88
12
0
*
4
1
88
12
0
1
4.1
1
68
16
16
5
16.1
1
30
35
35
10
25
1
88
12
0
9

The post-column derivatization was done at 80.0 C. The samples were hydrolyzed with 0.05m NaOH, then reacted with o-phthaldialdehyde and 2- mercaptoethenol in 0.05m sodium borate decahydrate to yield a highly fluorescent isoindole product. The flow from the post-column pumps was 0.5ml/min. The detector was a waters 470 scanning fluorescence detector at 338nm and at 400nm.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 018

Analysis For Carbamates In Water

Analysis of samples for carbamates was performed on the waters Carbamates analysis system equipped with a WISP model 712 Autosampler. All samples were filtered through 0.45u nylon filters. Water samples (500ul) were directly injected.

The separation of the analytes was at room temperature, and the column used was a 3.9mm x 150mm waters nova-par C (4u). The solvent gradient used was as follows:

TIME
FLOW
%WATER
%METHANOL
%ACETONITRILE
CURVE
INIT
1
88
12
0
*
4
1
88
12
0
1
4.1
1
68
16
16
5
16.1
1
30
35
35
10
25
1
88
12
0
9

The post-column derivatization was done at 80.0 C. The samples were hydrolyzed with 0.05 m NaOH, then reacted with o-phthaldialdehyde and 2-mercaptoethenol in 0.05m sodium borate decahydrate to yield a highly fluorescent isoindole product. The flow from the post-column pumps was 0.5ml/min. The detector was a waters 470 scanning fluorescence detector at 338nm and at 400 nm.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 019

Grain Size

Disperse sample of soil or sediment to pass 2 mm sieve and place a weighed 40 gram in a 600 ml beaker. Take additional 30 gram sample for moisture determination so that air-dried weight may be corrected to oven-dried weight. Add 50 ml 10% "Calgon" solution (sodium meta- phosphate with sufficient sodium carbonate to give a pH of approximately 8.3 in a 10% solution) to 40 gram sample and allow to soak for at least 10 minutes. After soaking, quantitatively transfer sample with distilled water to Waring blender cup so that cup is approximately half full. Blend for four minutes and transfer suspension to sedimentation cylinder adjusting liquid level to 1000 ml mark with distilled water. Place cylinder in constant temperature water bath (approximately 38oC). Prepare sample "blank" by adding 50 ml 10% "Calgon" solution to second sedimentation cylinder and add distilled water to the 1000 ml mark. When the suspension reaches water bath temperature, the mixture is thoroughly stirred prior initiation of sedimentation. The time that stirring ceases is noted as the zero settling time. At the end of eight hours, lower the hydrometer (ASTM 152 H) gently into the suspension and read the scale at the end of the meniscus. Record the time of hydrometer reading, the hydrometer reading, and water bath temperature. After thorough mixing, record the hydrometer reading in the sample "blank" solution of water and "Calgon". After hydrometer readings are recorded, pour the suspension onto a 270 mesh, 53 micron sieve and wash all silt and clay out with the water. Transfer sample material remaining on the sieve into an evaporating dish; place in 110oC oven and allow to dry for 24 hours. After cooling, weigh the sample to determine the weight of oven-dry sand left on the sieve. Using moisture data determined, correct sample air-dry weight to oven-dry weight. Calculate the concentration of suspension in grams per liter from the following equation:

C = R - R< where:

  • C = concentration (gm/liter)
  • R = hydrometer reading in suspension
  • R< = hydrometer reading in "Calgon" solution.

Calculate the Clay percentage, PC from the following:

  • Pc = 100 * C/Co; where Co represents the oven-dry weight Co of soil per liter of suspension.

Calculate the Sand percentage, Ps from the following:

  • Ps = 100 * S/Co; where S is the weight of the oven-dry sand Co left on screen and Co is as in the Clay formula.

Silt percentage (S) is:

  • S = 100 - Pc – Ps

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 020

Total Organic Carbon

Weigh approximately 0.35 g sample into a numbered glazed ceramic boat. Record the weights and boat numbers.

In an acid safe hood, add 1:1 HCl dropwise to each sample until completely moistened, usually 5 to 10 drops. Observe the samples for any bubbling, and note this. Heat the samples on a hot plate until dry. The addition of HCl and hot plate drying must be repeated until no further bubbling occurs. Dry samples in a drying oven at 100oC for 1 hours.

Samples were analyzed using a Leco CR-412 Carbon Analyzer. The instrument was calibrated using CaCO3.

The right anhydrone tube of the furnace was replaced with a chlorine trap before TOC samples were analyzed.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 021

Semivolatile Organic Compounds

Semivolatiles were analyzed using EPA SW-846, Method 8270; Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry/Capillary Technique.

Ten grams of soil/sediment was dried with 50 grams of Na2SO4 and soxhlet extracted for 16 hours with methylene chloride. The extract was concentrated to 1 ml and shot on a Finnigan Incos 50 mass spec.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 022

Analysis For Selected Organophosphate and Synthetic Pyrethroid Pesticides In Animal and Plant Tissue.

This method is suitable for the extraction and quantitation of the following analytes:

1) Organophosphate pesticides- chlorpyrifos, diazinon, EPN, ethyl parathion, malathion, methyl chlorpyrifos, and methyl parathion,

2) synthetic pyrethroids- cypermethrin and fenvalerate, Ten gram tissue samples are thoroughly mixed with anhydrous sodium sulfate and soxhlet extracted with hexane for seven hours. The extract is concentrated by rotary evaporation; transferred to a tared test tube, and further concentrated to dryness for lipid determination. The weighed lipid sample is dissolved in petroleum ether and extracted four times with acetonitrile saturated with petroleum ether. Residues are partitioned into petroleum ether which is washed, concentrated, and transferred to a glass chromatographic column containing 20 grams of Florisil. The column is eluted with 200 ml 6% diethyl ether/94% petroleum ether (Fraction I) followed by 200 ml 15% diethyl ether/85% petroleum ether (Fraction II). For organophosphates, an additional Fraction III (200 ml 50% diethyl ether/50% petroleum ether) is required for complete recovery of requested analytes. Fraction II (and Fraction III, if required) is concentrated to appropriate volume for quantification of residues by capillary column electron capture gas chromatography. Fraction I is concentrated and transferred to a Silicic acid chromatographic column for additional cleanup required for separation of PCBs from other analytes. Three fractions are eluted from the silicic acid column. Each is concentrated to appropriate volume for quantification of residues by megabore column, electron capture gas chromatography. PCBs are found in Silicic acid Fraction II.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 023

Analysis For Selected Organophosphate and Synthetic Pyrethroid Pesticides and Triazine Herbicides in Soil and Sediment.

This method is suitable for the extraction and quantitation of the following analytes: 1) Organophosphate pesticides- chlorpyrifos, diazinon, EPN, ethyl parathion, malathion, methyl chlorpyrifos, and methyl parathion, 2) synthetic pyrethroids- cypermethrin and fenvalerate, 3) Triazine herbicides- atrazine, metribuzin, propazine, simazine, 4) Other herbicides- alachlor and metolachlor.

Twenty gram soil or sediment samples are extracted with acetone, followed by petroleum ether, by allowing to soak one hour in each with intermittent shaking. A final acetone/petroleum ether extraction is done, and the extracts are combined, centrifuged, and transferred to a separatory funnel containing sufficient water to facilitate partitioning of residues into petroleum ether portion. The petroleum ether is washed twice with water and concentrated by Kuderna-Danish to appropriate volume. An aliquot of the concentrated extract for pesticide determination is transfered to a 1.6 gram Florisil mini-column topped with 1.6 grams sodium sulfate. Residues are eluted from the column in two elution fractions. Fraction I consists of 12 milliliters hexane followed by 12 milliliters of 1% methanol in hexane, and Fraction II consists of an additional 24 milliliters of 1% methanol in hexane. Quantification of residues in the two Florisil fractions is by 30M DB-608 and DB-5 megabore columns, electron capture gas chromatography and by TSD (thermionic specific detector), to detect N and P containing compounds.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 025

Analysis For Selected Organophosphate and Synthetic Pyrethroid Pesticides and Triazine Herbicides in Water

This method is suitable for the extraction and quantitation of the following analytes: 1) Organophosphate pesticides- acephate, azinphos methyl, chlorpyrifos, coumaphos, dementon, diazinon, dichlorvos, dicrotophos, dimethoate, disulfoton, ethoprop, EPN, famphur, fensulfothion, fenthion, malathion, methylparathion, mevinphos, monocrotophos, parathion, phorate, terbufos and trichlorfon 2) synthetic pyrethroids- cypermethrin and fenvalerate, and permethrin 3) Triazine herbicides- atrazine, metribuzin, propazine, simazine.

A 800 milliliter water sample is extracted four times by shaking with 75 milliliter portions of methylene chloride. The four extracts are combined and concentrated by Kuderna-Danish to near dryness, then exchanged into iso-octane, and reconstituted to appropriate volume in hexane. Quantification of residues is by megabore column, electron capture gas chromatography.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 026

Analysis For PCP, Carbaryl, 4-nonylphenol, and Permethrin by GC/MS

A 800 milliliter water sample or all the sample if less than 800 ml is extracted four times by shaking with 50 milliliter portions of methylene chloride. The four extracts are combined and concentrated by Kuderna-Danish. Sample volume is adjusted to one milliliter by N-evap and quantified by Gas Chromatography - Mass Spec.

Standard solutions are diluted with an appropriate solvent and determined by direct injection on GC/MS.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 027

Method for Substituted Urea Herbicides in Water

A measured volume (1000ml) of water is placed into a 2-L separatory funnel and surrogate standard is added. The pH of the sample is adjusted to 7 by the addition of phosphate buffer, then 100g NaC1 is added. The sample is shaken to dissolve the salt. Methylene chloride (60ml) is added and the sample is extracted by shaking for 2 minutes with periodic venting. After the layers have been allowed to separate, the organic layer is drained into a flask, and the extraction repeated for a total of three times. The methylene chloride extracts are combined, then drained through a small amount of sodium sulfate to remove water, into a Kuderna-Danish concentrator. The solvent is concentrated to a small volume, then taken just to dryness with a stream of dry nitrogen. One ml of acetonitrile containing the internal standard is added, and the sample mixed 30 seconds on a Vortex mixer. After filtration, the samples are analyzed on a high performance liquid chromatograph using UV detection at 254 nm.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 028

Analysis For AHH active PCB Congeners In Soil and Sediment

Twenty gram soil or sediment samples are weighed into a pesticide residue quality (PRQ) centrifuge bottle. If the samples are less than 10% moisture, then 10 ml water is added. Fifty ml B & J high purity acetone is added and the sample shaken well six times over a ninety minute period (about every 15 minutes). Fifty mls redistilled petroleum ether is added to the sample and the shaking repeated. The sample is centrifuged and the liquid decanted into a 500 ml separatory funnel containing 200 ml PRQ water and 15 ml saturated sodium chloride solution. Fifty ml of a 1:1 acetone/pet ether mixture is added to the sample and it is shaken, centrifuged, and the liquid added to the separatory funnel. The separatory funnel is shaken vigorously for two minutes and the layers allowed to separate. The pet ether is removed, and the water fraction extracted again with 50 ml petether. The combined pet ether is washed twice with water and concentrated by Kuderna-Danish to appropriate volume. The sample is transferred to a glass chromatographic column containing 20 grams of Florisil. The column is eluted with 200 ml 6% diethyl ether/94% petroleum ether (Fraction I). Fraction I is concentrated and transferred to a Silicic acid (Mallinckrodt SillicAR CC-4, activated at least 7 days in a 1300C oven) chromatographic column for additional cleanup required for separation of PCBs from other organochlorines. Three fractions are eluted from the silicic acid column. Fraction I is 20 ml of Petroleum Ether. Fraction II is 150 ml of Petroleum ether and contains the PCBs. Fraction III is 20 mls of 1:19:80 acetonitrile:hexane:methylene chloride and contains most of the other organochlorine pesticides.

Fraction II is concentrated to one ml and fractionated on a carbon column. This column consists of 2 grams of 1:20 AX-21 Carbon on activated silica gel. (AX-21 Activated Carbon, Anderson Development Co., Adrian, MI, vacuum washed with 200 ml each water, methanol, toluene, acetone, methylene chloride, 2X pet ether) (Silica gel: EM Science, Silica gel 60-230 mesh activated @130oC more than 10 days). Fraction A consists of 50 ml Pet Ether. Fraction B is 50 ml 10% methylene chloride in Pet Ether. Fraction C is 120 ml of 50% methylene chloride in Pet ether. Fraction D is 50 ml toluene. Fraction B contains PCB# 128, 138, 158, 166, and 170. Fraction C contains PCB# 105, 114, 118, 123, 156, 157, 167,and 189. Fraction D contains PCB# 077, 126, and 169. The PCB fractions were chromatographed on a Varian 3400 GC using a 60m 0.25mm DB-5 capillary column. Conformation was conducted on a 30m 0.53mm DB-608 megabore column.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 029

Analysis For AHH active PCB Congeners - Micro Method

This method is necessary when sample size is limited (below 4 g, approximately) and in case of organ tissue as substrate and is a modified version of the method described in Manual of Analytical Methods for the Analysis of Pesticides in Humans and Enviromental Samples, EPA-600/8-80-038, June 1980, Section 5, A (2). It is suitable for adipose, kidney, liver, muscle, and other tissues:

Weigh 0.5 g or less of well-mixed tissue into a size 22 Duall tissue grinder. Extract tissue by grinding three times with acetonitrile; the first time being with 4 ml followed by two 2.5 ml portions. Remove the pestle after each grinding and centrifuge,decanting the extract into a 50 ml glass stoppered graduated mixing cylinder. Combine all extracts and record the total volume of the three extracts. Add a volume of PRQ water equivalent to 3.3 times the extract volume. Then add 2 ml saturated NaCl solution. Extract the aqueous acetonitrile mixture with 5 ml hexane by vigorous shaking for 2 minutes. Allow layers to separate, and remove the hexane layer with a Pasteur pipet into a tared 20 ml screw-capped culture tube. Re-extract twice with 4 ml hexane each time, combining the extracts into the culture tube. Concentrate the combined hexane extracts under nitrogen just to dryness. Weigh the tube to determine the lipid.

The weighed lipid sample is dissolved in petroleum ether and transferred to a glass chromatographic column containing 20 grams of Florisil. The column is eluted with 200 ml 6% diethyl ether/94% petroleum ether (Fraction I). Fraction I is concentrated and transferred to a Silicic acid (Mallinckrodt SillicAR CC-4, activated at least 7 days in a 1300C oven) chromatographic column for additional cleanup required for separation of PCBs from other organochlorines. Three fractions are eluted from the silicic acid column. Fraction I is 20 ml of Petroleum Ether. Fraction II is 150 ml of Petroleum ether and contains the PCBs. Fraction III is 20 mls of 1:19:80 acetonitrile:hexane:methylene chloride and contains most of the other organochlorine pesticides.

Fraction II is concentrated to one ml and fractionated on a carbon column. This column consists of 2 grams of 1:20 AX-21 Carbon on activated silica gel. (AX-21 Activated Carbon, Anderson Development Co., Adrian, MI, vacuum washed with 200 ml each water, methanol, toluene, acetone, methylene chloride, 2X pet ether) (Silica gel: EM Science, Silica gel 60-230 mesh activated @130oC more than 10 days). Fraction A consists of 50 ml Pet Ether. Fraction B is 50 ml 10% methylene chloride in Pet Ether. Fraction C is 120 ml of 50% methylene chloride in Pet ether. Fraction D is 50 ml toluene. Fraction B contains PCB# 128, 138, 158, 166, and 170. Fraction C contains PCB# 105, 114, 118, 123, 156, 157, 167,and 189. Fraction D contains PCB# 077, 126, and 169. The PCB fractions were chromatographed on a Varian 3400 GC using a 60m 0.25mm DB-5 capillary column. Conformation was done on a 30m 0.53mm DB-608 megabore column.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 030

DETERMINATION OF STARLICIDE IN WATER

Standard Preparation The concentrated standard solution was made by dissolving 25 mg of 3-chloro-p-toluidine hydrochloride in 25 ml distilled water. Serial dilutions were made from this stock to the appropriate levels for analysis. After trials on the HPLC, 1.0, 0.5, 0.1, and 0.05 ppm were determined to be the best working concentrations. All samples and standards were stored and extracted under as low light conditions as practical to prevent photodegradation of the Starlicide.

HPLC Conditions Separate reservoirs of filtered (0.45 um) degassed water and acetonitrile were delivered at 20% water / 80% acetonitrile. An Alltech Econosil C-18, 5-um, 4.6x250 mm column was used for the separation. The flow rate was 1 ml/min at room temperature. The detector was 241 nm UV.

Method Validation Direct injections of the water samples determined the concentration of Starlicide to be too low to accurately quantitate. The limit of detection of Starlicide in a 100 ul injection was determined to be 0.05 ppm. Three 200 ml 0.01 ppm water spikes were concentrated by passing through 360 mg C-18 Sep-Paks then eluding with 4 mls 100% acetonitrile. This resulted in an increase in detection limit to 0.001 ppm.

Sample
Fortification Level (ug/ml)
Observed Level (ug/ml)
#1
0.01
0.0073
#2
0.01
0.0072
#3
0.01
0.0082
#4
0.001
0.00074

References PM Resources Laboratory Method #840-1 U.S.D.A. Denver Wildlife Research Center Method #28A U.S.D.A. Denver Wildlife Research Center Method #36A

Sample Perparation

Two hundred mls of each sample was passed through a 360 mg C-18 Sep-Pak and eluded with 4 mls 100% acetonitrile. Each sample was injected on the HPLC using the conditions described above.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 031

Accelerated Solvent Extraction (ASE) and Analysis of Synthetic Pyrethroid Pesticides in Filter Disks.

This method is suitable for the extraction and quantitation of synthetic pyrethroids, cypermethrin, and fenvalerate in filter disks. The filter disks were folded and each placed in a clean 11 cm extraction cell. Each cell was extracted with 100% hexane using the following ASE conditions: 5 min. heating cycle, 2x2 min. static cycles, 60% flushing volume, and 2000 psi @ 100oC. The resulting ~18 ml of extract was evaporated to one ml under nitrogen and quantitated on a megabore column with an electron capture gas chromatograph.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 032

Accelerated Solvent Extraction (ASE) and Analysis of Synthetic Pyrethroid Pesticides in Plant Tissue.

This method is suitable for the extraction and quantitation of synthetic pyrethroids, cypermethrin, and fenvalerate in plant tissue. Two grams of chopped plant tissue was mixed with 4.5 grams of Hydromatrix and placed in a clean 33 cm extraction cell. An additional 1 to 2 grams of Hydromatrix was add to each cell to fill the remaining void. The cells were extracted with 50:50 hexane:acetone using the following ASE conditions: 5 min. heating cycle, 2x2 min. static cycles, 60% flushing volume, and 2000 psi @ 100oC. The resulting ~ 55 ml of extract was rotovaporated @ 45oC to near dryness and transfered through sodium sulfate with hexane. The extract was then evapotated under nitrogen to ~5 ml and transfered to a cleanup column containing 25 grams of Florisil topped with 1 cm of sodium sulfate. The column was prewashed with 50 ml hexane and the hexane extract transfered with 3 small hexane rinses. Fraction I was eluted with 200 ml methylene chloride:hexane (20:80) @ ~5 ml/min. This fraction contained cis-permethrin. Fraction II was eluted with 200 ml of acetonitrile:methylene chloride:hexane (0.35:50:50) @ ~5 ml/min. This fraction contained the remaining pyrethroids. Both fractions were combined in a 1000 ml round bottom flask and rotovaped to near dryness @ 45oC. The extract was concentrated to the appropriate volume for quantitation on a megabore column with an electron capture gas chromatograph.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 033

Accelerated Solvent Extraction (ASE) and Analysis of Disyston, Disyston Metabolites, and Oxygen Analoges in Filter Disks.

This method is suitable for the extraction of Disyston, Disyston sulfoxide, Disyston sulfone, and the oxygen analoge of each in filter disks. The filter disks were folded and each placed in a clean 11 cm extraction cell. Each cell was extracted with 50:50 (hexane:acetone) using the following ASE conditions: 5 min. heating cycle, 2x2 min. static cycles, 60% flushing volume, and 2000 psi @ 20oC. The resulting ~18 ml of extract was evaporated to one ml under nitrogen and the water removed by passing sample in petroleum ether through sodium sulfate. The samples were injected on a megabore column with an electron capture gas chromatograph. Only Disyston and Disyston sulfone could be quantitated on the ECD because of thermal break down and conversion to Disyston sulfone of all the other compounds. Disyston sulfoxide and the oxygen analoges can be quantitated if only that single compound is present, but when combined with the Disyston or Disyston sulfone they appear to convert to the Disyston sulfone making quantitation impossible.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 034

Analysis for Chlorophenoxy Acid Herbicides in Liver

Weigh 0.5 gm of liver into a size 22 Duall tissue grinder. Add 2 ml PRQ water and 6 drops 6N HCl. Grind the liver. Extract the liver with 4 ml diethyl ether by grinding. Centrifuge and remove ether to a screw top tube. Extract the liver 2X with 2 ml diethyl ether and once with 2 ml pet. ether. This combined extract contains chlorophenoxy acid herbicides.

Derivitization: Reduce sample volume to approximately 0.5ml and ethylate using diazoethane (15 min.). Exchange to hexane (N-EVAP) and reduce volume to 0.2ml.

Column Clean-Up:

Column clean-up: Place 2.0g of 1% deactivated silica gel in a 7mm id. chromatography column (#22 Kontes). Top with 1cm Na2SO4 and prewet column with 10ml hexane. Collect sample eluents in three fractions as follows:

Fraction A: add sample and rinse container with two 0.5ml washes of 20% benzene in hexane. Elute with 9ml of the same solution. (Contains PCP.)

Fraction B: add 10ml 40% benzene in hexane. Add 10ml 60% benzene in hexane. (Contains Dalapon, Silvex, Dinoseb, portion of Dicamba.)

Fraction C: add 10ml 80% benzene in hexane. Add 10ml 100% benzene. (Contains remaining Dicamba, Dichlorprop, 2,4-D, 2,4,5-T, 2,4-DB, Bentazon, Blazer.)

[Reference for column clean-up for acid herbicides: Shafik, T. A.,H. C. Sullivan, H. R. Enos, 1973." Multiresidue Procedure for Halo- and Nitrophenols. Measurement of Exposure to Biodegradable Pesticides Yielding These Compounds as Metabolites." J. Agr. Food Chem. 21:295-298. ]

[Reference for extraction: A modification of Determination of Pentachlorphenol in Blood, Manual of Analytical Methods for the Analysis of Pesticides in Humans and Environmental Samples, Section 5,A,(3),(b).EPA June 1980.

Recoveries: N=3
Dicamba
93%
Dichloroprop
101%
24D
115%
PCP
96%
Silvex
105%
245T
114%
24DB
98%
Blazer
90%

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 035

Analysis for Organochlorine Pesticides and PCBs in Soil and Sediment by ASE.

Weigh 10-gram soil or sediment sample and 5-grams of Hydromatrix into a PRQ (SOP 1.105) beaker. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. The sample can be stored in a desiccator over night. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 33-ml cell with a 1.91-cm cellulose filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spatula, and beaker with no more than 6-ml total of pet ether (SOP 1.81) and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 60% solvent flush, 120 sec purge cycle, 100 degrees C @ 2000psi, 1:1 pet ether:acetone (SOP 1.255). Prepare a 500-ml separatory funnel with 200-ml PRQ water (SOP 1.255) and 15-ml PRQ saturated sodium chloride (SOP 1.255). Rinse the sample extract into the separatory funnel with 50ml of 1:1 acetone: pet ether. The separatory funnel is shaken vigorously for one minute and the layers allowed to separate. The pet ether is removed, and the water fraction extracted again with 50-ml pet ether. The combined pet ether is washed twice with 50-ml water and concentrated by Kuderna-Danish (SOP 1.261) to appropriate volume. An aliquot of the concentrated extract representing 2 grams of sample is transferred to a column containing 20 grams of Florisil. The column is eluted with 200 ml 6% diethyl ether/94% petroleum ether (Fraction I) followed by 200 ml 15% diethyl ether/85% petroleum ether (Fraction II). Fraction II is concentrated to appropriate volume for quantification of residues by a Varian 3600 gas chromatograph equipped with dual 30M DB-5/DB-608 megabore columns and electron capture detectors. Fraction I is concentrated to 5 ml and transferred to a silicic acid (SOP 1.255) chromatographic column (custom columns 1 cm OD x 40 cm with a 100 ml reservoir on top, Ace Glass) for additional cleanup required for separation of PCBs from other organochlorines. Five grams of hot silicic acid is put into the column, which already has a glass wool plug and about 3-mm sodium sulfate in the bottom. The silicic acid is topped with 10-mm sodium sulfate and prewashed with 10-ml hexane. Three fractions are eluted from the silicic acid column. The sample in 5-ml solvent is added to the column and rinsed into the column with 3,1,1-ml hexane. Then the sample is eluted with 20-ml pet ether (fraction SAI). Fraction SAII is 150-ml pet ether. Fraction SAIII is 20 ml of a mixed solvent consisting of 1 part acetonitrile: 19 parts hexane: 80 parts methylene chloride (SOP 1.255). Each is concentrated to appropriate volume for quantification of residues by megabore column, electron capture gas chromatography. HCB and Mirex are in SAI. PCBs are found in SAII. The rest of the compounds are in SAIII.

GC determinations for SAI and SAIII were run on a Varian 3600 GC with a Varian Star Data System and a Varian 8200 Autosampler. All GCs were equipped with dual DB-608 (0.83u film thickness, J & W Scientific # 125-1730) and DB-5 (1.5u film thickness, J & W Scientific # 125-5032) 30M megabore columns. All compounds were calculated using a three point standard curve forced through the origin using external standards (SOP 1.267).

PCB's (SAII) are shot on a Varian 3350 with a 60M DB-5 capillary column and an EC detector and a Varian Star Data System, version 4.5. All the mixture standards are at 0.5 ng/ul with one ul shot.

Starting with Arochlor 1260, 4 peaks that are unique to this mixture are located. The areas of the standards are summed and the same peaks located in the sample and also summed. Arochlor 1260 is calculated by the following formula to obtain PPM 1260.

(Area sample) (weight of std shot in ng)

(Area 1260 std) (basis shot in mg)

Arochlor 1254 is calculated by locating the major peaks in the mixture that are normally found in samples. The areas of these peaks are summed. Because some of this area comes from Arochlor 1260 and not all from Arochlor 1254, the contribution from the 1260 has to be subtracted from the total area. Arochlor 1254 is calculated by using the formula:

{(Area sample)-[((PPM 1260) (basis) (area from 1260))/ng 1260 std]}
(wt 1254 std in ng)
----------------------------------------------------------------------------------------------------------------------------------------

(Area 1254 std) (Basis shot in mg)

Results are in PPM.

Arochlor 1248 and Arochlor 1242 are calculated in a similar fashion, subtracting the contribution from 1254 in the 1248 and the 1248 in the 1242.

Total PCBs are calculated by adding the sum of Arochlor 1242, 1248, 1254, and 1260.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 036

Analysis of Soil and Vegetation for Glyphosate

The samples were analyzed using the method of Moye, A. et al., Validation of An Analytical Residue Method for Glyphosate and Metabolite: An Interlaboratory Study. J. Agri. & Food Chem., 1986. 34:955-960. A Partisil-10 SAX column was used as the primary column and a Hamilton PRP X-400 column used for conformation.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 038

Dioxins

Dioxins were analyzed using EPA Method 8290; Analysis of Samples for the Presence of Polychlorinated Dibenzo-p-Dioxins and Dibenzofurans by High-Resolution Chromatography/High-Resolution Mass Spectrometry.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 039

Analysis For Selected Organophosphates in Soil and Sediment by ASE and GPC.

This method is suitable for the extraction and quantitation of the following analytes:

1) Organophosphate pesticides- chlorpyrifos, diazinon, EPN, malathion, methyl parathion, ethyl parathion, phorate, azinphos-methyl, coumaphos, demeton, ethoprop, dimethoate, fensulfothion, fenthion, mevinphos, terbufos, and famphur.

2) Other herbicides- alachlor.

Weigh 10-gram soil or sediment sample and 5-grams of Hydromatrix into a PRQ (SOP 1.105) beaker. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. The sample can be stored in a desiccator over night. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 33-ml cell with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spatula, and beaker with no more than 6-ml total of pet ether (SOP 1.81) and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA Method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 60% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, 1:1 pet ether: acetone (SOP 1.255). Prepare a 500-ml separatory funnel with 200-ml PRQ water (SOP 1.255) and 15-ml PRQ saturated sodium chloride (SOP 1.255). Rinse the sample extract into the separatory funnel with 50ml of 1:1 acetone: pet ether. The separatory funnel is shaken vigorously for one minute and the layers allowed to separate. The pet ether is removed, and the water fraction extracted again with 50-ml pet ether. The combined pet ether is washed twice with 50-ml water and concentrated by Kuderna-Danish (SOP 1.261) and exchanged into methylene chloride and adjusted to 4 ml. Two ml of the sample injected on a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap and then exchanged into iso-octane. Quantification of residues is by electron capture gas chromatography with both 30M DB-608 and DB-5 megabore columns, and by TSD (thermionic specific detector) with a 30M DB-608 megabore column to detect N and P containing compounds.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 040

Analysis for PAHs in Soil/Sediment and plant tissue by ASE and GPC.

Weigh 10-gram soil/sediment or plant tissue sample and 5-grams of Hydromatrix into a PRQ (SOP 1.105) beaker. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. The sample can be stored in a desiccator over night. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 33-ml cEll with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spatula, and beaker with no more than 6-ml total of pet ether (SOP 1.81) and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA Method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 60% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, 1:1 pet ether: acetone (SOP 1.255).

Prepare a 500-ml separatory funnel with 200-ml PRQ water (SOP 1.255) and 15-ml PRQ saturated sodium chloride (SOP 1.255). Rinse the sample extract into the separatory funnel with 50ml of 1:1 acetone: pet ether. The separatory funnel is shaken vigorously for one minute and the layers allowed to separate. The pet ether is removed, and the water fraction extracted again with 50-ml petether. The combined pet ether is washed twice with 50-ml water and concentrated by Kuderna-Danish (SOP 1.261) to appropriate volume. The sample is dissolved in 4 ml of methylene chloride and 2 ml injected into a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap and then exchanged into hexane. The sample is transferred to a column containing 20 grams of 1% deactivated silica gel (SOP 1.255) column (silica gel is added to the column in a pet ether slurry) topped with five grams neutral alumina (SOP 1.255). Aliphatic and polynuclear aromatic hydrocarbon residues are fractionated by eluting aliphatics from the column with 100 ml petroleum ether (Fraction I) followed by elution of aromatics using first, 100 ml 40% methylene chloride (SOP 1.255)/60% petroleum ether, then 50 ml methylene chloride (Combined elutes, Fraction II). The silica gel Fraction II containing aromatic hydrocarbons is concentrated, reconstituted in methylene chloride to a known volume, and quantified by gas chromatography - mass spec (SOP 1.276).

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 041

Analysis of Water -- Special for E. Snyder-Conn

1. Record sample volume. Add 800 ml of sample to a 1000-ml separatory funnel. Add 100 grams of sodium chloride and 50 mls phosphate buffer to the funnel and shake to mix.

2. Adjust to pH 8 by adding PRQ 6N KOH (SOP 1.255) or 6N sulfuric acid to sample, and shake vigorously 30 sec. Allow sample to stand one hour at pH 8. Rinse the sample container with 25-ml CH2Cl2 (SOP 1.255) and add this to the funnel. Pour the remainder of the sample back into the sample container and rinse the graduated cylinder 2 X 25 ml CH2Cl2.

3. Shake 2 min. with periodic venting. Drain CH2Cl2 through a funnel of CH2Cl2 washed sodium sulfate into a 500 ml French Square bottle if there is no emulsion. Otherwise, drain lower layer into centrifuge bottle and centrifuge to separate layers. Repeat 2 X 60 ml CH2 Cl2. This combined extract contains organochlorine and nitrogen/phosphorous containing pesticides.

4. Adjust pH to less than 2 with drop wise addition of PRQ 6N H2SO4 (SOP 1.255).

5. Extract water with 100ml Ethyl ether (SOP 1.255) by shaking two minutes. Drain the water from the separatory funnel and pass the ether layer through acidified sodium sulfate into a 500-ml French square bottle. Repeat 2 x 60ml EtoEt. Extract with a final aliquot of 100ml petroleum ether (SOP 1.81). This combined extract contains chlorophenoxy acid herbicides.

6. Concentrate acid and base/neutral extracts separately with Kuderna-Danish evaporators (SOP 1.261).

7. Acid extract (chlorophenoxy acid herbicides) derivitization: Reduce the sample volume to approximately 0.5ml and ethylate using diazoethane (15 min.)(SOP 1.10). Add acid and base/neutral extracts together and concentrate to 1 ml for GC-MS analysis.

8. GC-MS analysis was conducted using a Saturn Ion Trap with a 3400 GC. A SPI 1078 injector was used. Instrument conditions as follows: Injector temperature 220oC, 30M DB-5ms column, 80 o C start temp, 1 min, ramp to 290 o C @8o/min, then hold for 2.75 min. Multiplier 1750 volts, A/M amplitude 3.0 volts, trap temp 190oC, Manifold 50oC, Transfer 285oC. 2ul injection using an 8200 autosampler.

Phosphate Buffer:

  • 29.6 ml 0.1N hydrochloric acid & 50 ml 0.1M dipotassium phosphate
  • 0.1N hydrochloric acid = 2.5 ml HCl in 296 ml H2O
  • 0.1M dipotassium phosphate = 8.7g dipotassium phosphate to 500 ml H2O

(This makes approximately 800 ml buffer.)

References:

  • E.P.A. Method 507
  • E.P.A. Method 508
  • E.P.A. National Survey of Pesticides in Drinking Water Wells (11/90) Method 4

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 042

Analysis for Organochlorine Pesticides, N/P Pesticides, Aliphatic and Polynuclear Aromatic Hydrocarbons, and Chlorophenoxy Acid Herbicides in Water

1. Record sample volume and pH. Add 800 ml of sample to a 1000-ml separatory funnel. Add 100 grams of sodium chloride and 50 mls phosphate buffer to the funnel and shake to mix.

2. Adjust to pH 8 by adding PRQ 6N KOH (SOP 1.255) or 6N sulfuric acid to sample, and shake vigorously 30 sec. Allow sample to stand one hour at pH 8. Rinse the sample container with 25-ml CH2Cl2 (SOP 1.255) and add this to the funnel. Pour the remainder of the sample back into the sample container and rinse the graduated cylinder 2 X 25 ml CH2Cl2.

3. Shake 2 min. with periodic venting. Drain CH2Cl2 through a funnel of CH2Cl2 washed sodium sulfate into a 500 ml French Square bottle if there is no emulsion. Otherwise, drain lower layer into centrifuge bottle and centrifuge to separate layers. Repeat 2 X 60 ml CH2Cl2. This combined extract contains organochlorine, aliphatic and aromatic hydrocarbons, and nitrogen/phosphorous containing pesticides.

4. Adjust pH to less than 2 with drop wise addition of PRQ 6N H2SO4 (SOP 1.255).

5. Extract water with 100ml Ethyl ether (SOP 1.255) by shaking two minutes. Drain the water from the separatory funnel and pass the ether layer through acidified sodium sulfate into a 500-ml French square bottle. Repeat 2 x 60ml EtoEt. Extract with a final aliquot of 100ml petroleum ether (SOP 1.81). This combined extract contains chlorophenoxy acid herbicides.

6. Concentrate acid and neutral extracts separately with Kuderna-Danish evaporators (SOP 1.261).

7. Column Clean-Up:

* NEUTRAL FRACTION (N/P and organochlorine pesticides, Aliphatic and polynuclear aromatic hydrocarbons). Adjust sample extract to exact volume and remove an appropriate aliquot for column clean-up techniques specific to the analyte, for pesticides use florisil (SOP 1.194), for hydrocarbons use 1% deactivated silica gel (SOP 1.203), for organophosphates, shoot directly on a NP-detector.

*ACID FRACTION (Chlorophenoxy acid herbicides) - Derivitization: Reduce the sample volume to approximately 0.5ml and ethylate using diazoethane (15 min.)(SOP 1.10). Exchange to hexane (SOP 1.68) and reduce volume to 0.2ml.

* Column clean-up (CPA): Place 2.0g of 1% deactivated silica gel (SOP 1.255) in a 7mm id. chromatography column (#22 Konte s). Top with 1cm Na2SO4 (SOP 1.255) and prewet column with 10ml hexane (SOP 1.255). Collect sample eluents in three fractions as follows:

Fraction A: add sample and rinse container with two 0.5ml washes of 20% benzene (SOP 1.255) in hexane. Elute with 9ml of the same solution.(Contains PCP.)

Fraction B: add 10ml 40% benzene in hexane. Add 10ml 60% benzene in hexane. (Contains Dalapon, Silvex, Dinoseb, portion of Dicamba.)

Fraction C: add 10ml 80% benzene in hexane. Add10ml 100% benzene. (Contains remaining Dicamba, Dichlorprop, 2,4-D, 2,4,5-T, 2,4-DB, Bentazon, Blazer.)

Reference for column clean up for acid herbicides: Shafik, T. A., H. C. Sullivan, H. R. Enos, 1973." Multiresidue Procedure for Halo- and Nitrophenols. Measurement of Exposure to Biodegradable Pesticides Yielding These Compounds as Metabolites." J. Agr. Food Chem. 21:295-298.

Phosphate Buffer: 29.6 ml 0.1N hydrochloric acid & 50 ml 0.1M dipotassium phosphate

  • 0.1N hydrochloric acid = 2.5 ml HCl in 296 ml H2O
  • 0.1M dipotassium phosphate = 8.7g dipotassium phosphate to 500 ml H2O (This makes approximately 800 ml buffer.)

References:

  • E.P.A. Method 507
  • E.P.A. Method 508
  • E.P.A. National Survey of Pesticides in Drinking Water Wells (11/90) Method 4

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 043

Analysis For Triazine Herbicides in Soil, Sediment and Tissue.

This method has been found suitable for the extraction and quantitation of the following triazine herbicides- atrazine, metribuzin, and cyanazine.

Weigh 10-gram soil / sediment or 5-gram tissue sample and 5-grams of Hydromatrix into a PRQ (SOP 1.105) beaker. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. The sample can be stored in a desiccator over night. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 33-ml cell with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spatula, and beaker with no more than 5-ml total of methylene chloride and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA Method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 60% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, 10% methanol in methylene chloride. The sample is concentrated by Turbovap to appropriate volume (SOP 1.298). The sample is dissolved in 4 ml of methylene chloride and 2 ml injected into a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap and then exchanged into hexane.

Quantification is by Varian 3400 GC with a 30M RTX-200 meg abore column and a TSD (thermionic specific detector), to detect N containing compounds.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 044

Analysis of Synthetic Pyrethroid Pesticides in Bat Guano.

This method is suitable for the extraction and quantitation of the following synthetic pyrethroids-, cis and trans permethrin, cypermethrin and fenvalerate.

A five-gram guano sample is weighed into centrifuge bottle. If the samples are less than 40% moisture, then 5 ml of pet ether extracted water is added to each sample.

The samples are extracted with 50-ml acetone, followed by 50-ml petroleum ether, by soaking samples for 1 1/2 hour in each with intermittent shaking. The samples are centrifuged, and transferred to a 500-ml separatory funnel containing 200-ml pet ether extracted water and 15-ml saturated sodium chloride solution. A final 50 ml 1:1 acetone/petroleum ether extraction is done. The samples are centrifuged and the extracts combined in the separatory funnel. The petroleum ether is washed twice with water and concentrated by Kuderna-Danish to 5-ml. The sample is transferred to a 300 ml glass chromatographic column (Kontes # 420280-0242) containing 20 grams of Florisil topped with 1 cm of sodium sulfate and the sample tube rinsed three times with about 2-ml pet ether. The column is eluted with 200-ml 6% diethyl ether /94% petroleum ether followed by 200-ml 15% diethyl ether/85% petroleum ether. The diethyl ether used in this analysis contains 2% ethanol. The samples are concentrated to an appropriate volume for quantification of residues by megabore column electron capture gas chromatography (DB-608 and DB-5 dual columns). GC determinations were run on a Varian 3600 GC with a Varian Star Data System and a Varian 8200 Autosampler. All GCs were equipped with dual DB-608 (0.83u film thickness, J & W Scientific # 125-1730) and DB-5 (1.5u film thickness, J & W Scientific # 125-5032) 30M megabore columns. All compounds were calculated using a three point standard curve forced through the origin using external standards.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 045

Analysis for PCB Congeners In Animal Tissue or Soil/Sediment

Five-gram tissue samples or ten-gram sediment samples are weighed into a 150-ml beaker then thoroughly mixed with 5-grams of Hydromatrix. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 33-ml cell with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample. Rinse the funnel, spatula, and beaker with no more than 5-ml total of methylene chloride and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 60% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, methylene chloride (SOP 1.255).

If organochlorine pesticides and PCB congeners are requested on the same sample and the sample size is too small to run both analysis then the following modification is used. The samples are extracted as in method 1. The remainder of the sample not shot on GPC is used for PCB congeners starting with clean-up on the H4IIE columns. The lipid tube is rinsed with 20 ml of methylene chloride and the GPC vial is rinsed with 30 ml for a total of 50 ml of methylene chloride on column. This is added at step 6 below.

Column Chromatography Clean-Up of Tissue for H4IIE Bioassay

1. PRQ 2-cm i.d. column. Place glass wool in bottom of each column and cover with ca1-cm anhydrous sodium sulfate.

2. Add 10 ml KS (potassium silicate) to each column and gently tap to settle and level column.

3. Next, add 10-ml 40/60 SA/SG (sulfuric acid/silica gel), again gently settling and leveling.

4. Place a 1-cm layer of sodium sulfate over the SA/SG. Then add 15 ml of 30/70 SA/SG and settle.

5. Presaturate each column with 25-30 ml dichloromethane, allowing the solvent to descend just to the top of the absorbent. Close the stopcocks and place 60 ml I-Chem vial under each column. Have a second vial ready for each sample.

6. Pour the sample extract from the ASE extraction on to the top of the column and open the stopcock. Allow the sample to flow into the SA/SG until the extract is about 3 to 4 cm above the SA/SG. Close the stopcock and stir the top layer of SA/SG with a metal rod to remove the gas bubbles. Disturb the sodium sulfate as little as possible. Rinse the rod with dichloromethane back into the sample.

7. Open the stopcock and allow the sample solution to descend into the absorbent. Rinse the sample vial with three 5-ml portions of dichloromethane, sequentially adding these rinses to the column. Wash the column walls with three 3-ml portions of dichloromethane. When all the washes have descended into the absorbent, close the stopcock and add approx. 1 cm of sodium sulfate to the top of the column. Change the receiver vial.

8. Gently pour 50 ml of dichloromethane into the column reservoir and open the stopcock. Flow at 3 ml/min until all effluent is collected.

9. Turbovap extracts to about 10-ml, combine extracts into one vial per sample,add 6-ml of isooctane to each flask and turbovap to 3-ml. Hold extracts for addition to next column.

10. Pack a 1-cm i.d. column as follows: a plug of glass wool, 1-cm sodium sulfate, 10-ml SG-60 silica gel (washed with dichloromethane), 3-ml KS, 5-ml 40/60 SA/SG, and 1-cm sodium sulfate. Presaturate column with 3% dichloromethane: 97% hexane. Place 60 ml I-Chem vial under column. Have a second vial ready for each sample. Transfer sample to top of column and rinse sample flask with three 3-ml portions of eluant, sequentially applying the rinses to the column. Pour 70-ml of the eluant into the column and adjust flow rate to 3-ml/min. Change the vial when the column is about half finished.

11. Add 1-ml of hexane to each vial as a "keeper" and turbovap to about 10-ml. Combine extracts and concentrate to about 1 ml. The sample is now ready for the carbon column.

The sample is fractionated on a carbon column. This column consists of 2 grams of 1:20 AX-21 Carbon on activated silica gel (SOP 1.255). Fraction A consists of 200-ml methylene chloride and contains most of the PCB's. Fraction B is 75-ml redistilled toluene (SOP 1.255) and contains PCB# 77, 81, 126, and 169.

Use a 200ml chromatography column fitted with a stopcock (such as Kontes 420280-0213). Pack the column as follows: a plug of glass wool, 1 cm sodium sulfate, 2 grams of carbon/silica gel, and 1 cm sodium sulfate. The carbon/silica gel needs to be added to the 200 ml column through a long glass funnel that will reach the bottom of the column. If PCB#52 is requested, then pre-wash the column with 200 ml CH2Cl2. If this analyte is not requested, then pre-wash the column with 25 ml CH2Cl2.. Do not let the column go dry. Add the sample and rinse 3X 1ml methylene chloride. Elute the first fraction (C-1) with 200 ml methylene chloride. Elute the 2nd fraction (C-2) with 75 ml redistilled toluene. Concentrate and exchange C-1 to hexane, with the volume dedending on the concentration of PCB's in the sample as determined in the OC analysis. C-2 is concentrated to 1 ml.

The PCB fractions were chromatographed on a Varian 3400 GC using a 60m 0.25mm DB-XLB capillary column (SOP 1.265) and calculated by the software using a three point standard curve forced through the origin. Confirmation was done on a 60m 0.25mm DB-5 capillary column (SOP 1.265). Mass spec confirmations were run on a Saturn 2000 ion trap with a 60m DB-XLB capillary column and a 1078 injector.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 046

Dioxin and Furan in Tissue

Add 5-g hydromatrix to a 10g portion of ground tissue. Load into ASE cell. Add 20ul of the surrogate mixture to the sample. A 10-g sample requires the addition of 1000 pg. 13C12-2,3,7,8-TCDD to give the required 100-ppt fortification level.

ASE extract with methylene chloride, 60% flush Column Chromatography Clean-Up of Fish for H411E Bioassay

1. PRQ 2-cm id. column. Place glass wool in bottom of each column and cover with ca 1-cm anhydrous sodium sulfate.

2. Add 10 ml KS (potassium silicate) to each column and gently tap to settle and level column.

3. Next, add 10-ml 40/60 SA/SG (sulfuric acid/silica gel), again gently settling and leveling.

4. Place a 1-cm layer of sodium sulfate over the SA/SG. Then add 15 ml of 30/70 SA/SG and settle.

5. Presaturate each column with 25-30 ml dichloromethane, allowing the solvent to descend just to the top of the absorbent. Close the stopcocks and place 60-ml vial under column.

6. Pour the sample extract on to the top of the column and open the stopcock. Allow the sample to flow into the SA/SG until the extract just reaches the sodium sulfate between the SA/SG layers. Close the stopcock and stir the top layer of SA/SG with a metal rod to remove the gas bubbles. Disturb the sodium sulfate as little as possible. Rinse the rod with dichloromethane back into the sample.

7. Open the stopcock and allow the sample solution to descend into the absorbent. Rinse the sample vial with three 5-ml portions of dichloromethane, sequentially adding these rinses to the column. Wash the column walls with three 3-ml portions of dichloromethane. When all the washes have descended into the absorbent, close the stopcock and add approx. 1 cm of sodium sulfate to the top of the column.

8. Gently pour 50 ml of dichloromethane into the column reservoir, change collection vial, and open the stopcock. Flow at 3 ml/min until all effluent is collected.

9. Turbovap both vials of extract to about 10-ml. Combined both fractions into one vial using hexane. Turbovap to about 5 ml of hexane. Hold extracts for addition to next column.

10. Pack a 1-cm id. column as follows: a plug of glass wool, 1-cm sodium sulfate, 10-ml SG-60 silica gel (washed with dichloromethane), 3-ml KS, 5-ml 40/60 SA/SG, and 1-cm sodium sulfate. Presaturate column with 3% dichloromethane: 97% hexane. Place 60-ml vial under column. Transfer sample to top of column and rinse sample flask with three 3-ml portions of eluant, sequentially applying the rinses to the column. Pour 70-ml of the eluant into the column and adjust flow rate to 3-ml/min. Change vial so that each vial contains about half of the column eluant (approx. 50-ml).

11. Add 1-ml of hexane to each flask as a “keeper” and turbovap evaporate to about 1-ml.

Alumina Column Cleanup

Pack a column (glass, 30 cm X 10.5 mm) fitted with a Teflon stopcock, with alumina as follows:

Glass wool plug, 4 g sodium sulfate, 4 g activated neutral alumina, 4 g sodium sulfate. Elute with 10-ml hexane.

Apply sample to top of alumina column. Rinse flask 2X 2ml hexane. Elute with 20 ml hexane, save. Elute with 15 ml of 60% methylene chloride/hexane. Concentrate to 2 ml.

Prepare an AX-21/Celite 545 column as follows: Mix 5.4-g active carbon AX-21 and 62.0 g Celite 545 to produce an 8% (w/w) mixture. Activate the mixture at 130oC for 6 hours and store in a desiccator.

Cut off both ends of a 10 ml disposable serological pipet to give a 10 cm long column. Fire polish both ends and flare. Insert a glass wool plug at one end, the pack the column with 1 cm Celite 545, then 1-g carbon mixture, then 1-cm Celite 545 and another glass wool plug.

Calibration of carbon: add 50ul of the continuing calibration solution to 950-ul hexane. Run this through the column, concentrate to 50ul and analyze. If the recovery of any analytes is less than 80%, discard the carbon.

Rinse the carbon column with 5 ml toluene, then 2 ml 75:20:5(v/v) methylene chloride/methanol/toluene, then 1 ml 1:1 (v/v) cyclohexane/methylene chloride, and 5 ml hexane. Flow rate less than 0.5 ml/min. Add sample to the column, rinse tube with 2X 1ml hexane. Elute with 2X2ml hexane, 2-ml cyclohexane/methylene chloride (50:50) and 2-ml methylene chloride/methanol/toluene (75:20:5). Turn the column upside down and elute the PCDD/PCDF fraction with 20-ml toluene. Concentrate to 0.1ml; transfer to calibrated 0.2- ml autosampler insert and concentrate to 50 ul. Rinse container 3X 300ul of 1%toluene in methylene chloride and add to insert, evaporating sample as necessary. Add 20ul nonane internal standard solution. (13C12-1,2,3,4-TCDD at 200pg/ul per compound) Store samples in the dark, in a freezer until ready for MS. Just before shooting, adjust the sample to 100ul final volume. The samples are shot on a Varian Saturn 2000 ion trap in MS/MS mode. A 50ul sample is shot using an 8200 autosampler with a 100-ul syringe. The ion trap has a programable 1078 split / splitless injector operated in splitless mode. A 60M DB-XLB capillary column with a 0.25 film thickness was used.

For fish tissue:
(2,3,7,8-TCDD)
Lower MCL
1.0ppt
Upper MCL
200ppt
Weight (g)
10
IS Spiking level (pg/ul)
40
Final Extract Vol.(ul)
100

Reagent Preparations Preparation of Washed Silica Gel

Place 70-230 mesh silica gel in a large glass fritted funnel and wash with dichloromethane (pesticide residue grade). Let dichloromethane flow by gravity. Use 500-ml dichloromethane per 300 ml (150 grams) of silica gel. When funnel is no longer dripping, apply a vacuum until all liquid solvent is removed and silica gel begins to dry. Pour silica gel into shallow PRQ glass pan and air dry in hood overnight. Cover the pan with PRQ aluminum foil and activate in 130 C oven.

Preparation of Sulfuric Acid / Silica Gel (SA/SG)

Determine the amount of each type of SA/SG needed for sample clean up since the shelf life of these products is only six months. (If these products appear lumpy before the six months have elapsed, discard and make a fresh batch.) Weight washed silica gel into 4-liter glass bottle with Teflon lined cap. Calculate 2/3 of the weight of the silica gel and add this amount of concentrated sulfuric acid for 40/60 SA/SG or 3/7 of the weight for 30/70 SA/SG. Shake until there are no lumps and the silica gel appears dry and powdery. Label each bottle with contents and expiration date. Store at room temperature.

Preparation of Potassium Silicate (KS)

Pour 750 ml of PRG methanol into a 2 liter round bottom flask (be sure to use Teflon sleeve in flask joint). Weigh out 168 grams of KOH pellets and add to flask. Place flask on Rotovap with full water bath (water will dissipate the heat). Rotate flask until KOH dissolves. Remove flask from Rotovap heat water bath to 55 C. Slowly add 300 grams of washed silica gel, swirling the flask to prevent the formation of lumps. Return flask to the Rotovap and rotate for 90 mins.

Set up a large column plugged with glass wool and two 600-ml beakers to catch the column effluent. Swirl the flask and quickly pour as much of the slurry as possible into the column. Exchange beakers and resuspend the solid material in the flask with the methanolic KOH effluent until all of the silica gel is in the column. Allow the liquid level to descend to the top of the KS, then wash 3X with 100 ml of methanol. When the methanol level has reached the top of the KS after the last wash, add 175-ml dichloromethane. Wash with dichloromethane twice more and allow column to drain after the final wash. Apply approx. 2 psi of nitrogen to the top of the column and continue blowing until column is no longer cold (approx. 4 hours). Pour KS into shallow glass pans and air dry in hood overnight. Cover pan with PRQ aluminum foil and activate at 130 C overnight. Pour all KS into 4 liter bottle and roll to mix.

Weigh 1.00 gram of KS into 125 ml Erlenmeyer flask and add 25 ml distilled water.

Stopper flask and shake for 30 min. Prepare 1.21 mM/ml HCl solution (5.0 ml conc. HCl + 45.0 ml distilled water). Calibrate pH meter at pH 10 and pH 7. Add a stirring bar to the KS flask and slowly titrate until pH 6.9 to 7.1 has been attained. The solution is slow to equilibrate after each addition of acid, because of the KOH leaching out of the pores of the silica gel. Determine the moles of KOH / gram of KS from the amount of HCl needed to neutralize the KS. The KS should contain between 4.8 and 5.3 mMoles / gram to be suitable for use. Pour KS into smaller bottles, label, and store at 130 C until needed.

Reference:

Sample Preparation for fish in the BEST Mississippi Basin Study-H411E Bioassay, Standard Operating Procedures of Midwest Science Center, 4200 New Haven Road, Columbia, MO, 65201.

EPA Method 8290: Polychlorinated Dibenzodioxins (PCDDs) and Polychlorinated Dibenzofurans (PCDFs) by High-Resolution Gas Chromatography / High-Resolution Mass Spectrometry (HRGC/HRMS)

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 047

Synthetic Pyrethroid Pesticides / Micromethod

This method is suitable for the extraction and quantitation of the following analytes: cis and trans permethrin, cypermethrin and fenvalerate. One gram of tissue (or the entire sample if smaller) is weighed into a size 22 Duall tissue grinder. The sample is extracted three times with methylene chloride. The extracts are filtered through sodium sulfate into a screw top tube. The Methylene chloride is evaporated and the residue brought up to 1 ml with toluene. The samples are shot directly into a GC containing dual megabore columns with no clean-up.

Note: This procedure is hard on the GC, but necessary for very small samples.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 048

Analysis For Carbamates In Tissue

Ten-gram samples are weighed into a 150-ml beaker then thoroughly mixed with 5-grams of Hydromatrix. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 33-ml cell with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spatula, and beaker with no more than 5-ml total of methylene chloride and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 60% solvent flush, 60 sec purge cycle, 40 degrees C @ 1500psi, 100% methylene chloride. The extract is concentrated by Turbovap; transferred to a 4-ml calibrated test tube through a Pasteur pipette containing sodium sulfate using methylene chloride, and concentrated to the 4-ml mark. The fat is removed by injecting 2 ml (5 gram basis) on a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap to 1 ml in methylene chloride. Transfer sample solution onto aminopropyl Bond-Elute extraction cartridge(100 mg/1 ml volume) prewashed with 1 ml methylene chloride. Rinse sample tube with 1 ml 1.5% methanol in methylene chloride and add to column. Elute carbamate residues with 5 ml 1.5% methanol in methylene chloride. (Note: If recovery of aldicarb sulfoxide drops below 80% increase wash volume by 5 ml.) N-evap to dryness at no more than 30 degrees Centrigrade. Do not over dry. Dissolve residue in 1 ml of MeOH. Add 4-ml water for a 1gram/ml basis. Filter samples through 0.45 um teflon filters before injection on HPLC.

Analysis of samples for carbamates was performed on the Waters Carbamates analysis system equipped with a WISP model 712 autosampler (SOP 1.102). 500ul of sample was injected. The separation of the analytes was at room temperature, and the column used was a 3.9mm x 150mm waters Nova-pak C18(4u). The solvent gradient used was as follows:

TIME
FLOW
%WATER
%METHANOL
%ACETONITRILE
CURVE
INIT
1
88
12
0
*
4
1
88
12
0
1
4.1
1
68
16
16
5
16.1
1
30
35
35
10
25
1
88
12
0
9

The post-column derivatization was done at 80.0oC. The samples were hydrolyzed with 0.05m NaOH, then reacted with o-phthaldialdehyde and 2-mercaptoethenol in 0.05m sodium borate decahydrate to yield a highly fluorescent isoindole product. The flow from the post-column pumps was 0.5ml/min. The detector was a Waters 470 scanning fluorescence detector with ex at 338nm and em at 400nm.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 049

Analysis for Pyrethroids in Animal Tissue

This method has been found suitable for the extraction and quantitation of the following Pyrethroids: Cis and trans permethrin, cybermethrin and fenvalerate.

Weigh 5-gram tissue sample and 5-grams of Hydromatrix into a PRQ (SOP 1.105) beaker. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. The sample can be stored in a desiccator over night. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 33-ml cell with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spatula, and beaker with no more than 5-ml total of methylene chloride and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA Method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 60% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, 1:1 hexane acetone. The sample is concentrated by Turbovap to appropriate volume (SOP 1.298). The sample is dissolved in 4 ml of methylene chloride and 2 ml injected into a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap and then exchanged into hexane. Quantification is by Varian 3400 GC with a 60M DB-XLB capillary column and a ECD. A secondary detection system is a Varian 3600 with dual 30M megabore columns, DB-608 and DB-5.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 050

Analysis For Organochlorines Pesticides and PCBs in Animal Tissue using soxhlet/GPC.

Five-gram fish samples or two-gram egg samples are weighed into a 250 ml beaker then thoroughly mixed with 150 grams (5g samples) or 75 grams (2g samples) of anhydrous sodium sulfate (SOP 1.255). The samples are stored in a desiccator overnight. The samples are then soxhlet extracted (SOP 1.259) with 600 ml hexane (SOP 1.255) for seven hours. The extract is concentrated by rotary evaporation (SOP 1.129); transferred to a tarred test tube through a Pasteur pipette containing sodium sulfate, and further concentrated to dryness for lipid determination (SOP 1.264). The weighed lipid sample is dissolved in 4 ml of methylene chloride and the fat removed by injecting 2 ml on a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap and then exchanged into iso-octane. The sample is transferred to a 300 ml glass chromatographic column (Kontes # 420280-0242) containing 20 grams of Florisil (SOP 1.255) topped with 1 cm of sodium sulfate and the sample tube rinsed three times with about 2 ml pet ether. The column is eluted with 200 ml 6% diethyl ether (SOP 1.255)/94% petroleum ether (Fraction I) followed by 200 ml 15% diethyl ether/85% petroleum ether (Fraction II). If Endosulfan II and/or Endosulfan Sulfate analysis is required, then 200 ml of 50% diethyl ether/petroleum ether (Fraction III) is required. The diethyl ether used in this analysis contains 2% ethanol (SOP 1.255). Fractions II and III are concentrated to an appropriate volume for quantification of residues by megabore column electron capture gas chromatography (SOP 1.265)(DB-608 and DB-5 dual columns) Dieldrin and Endrin are in Fraction II, and some delta BHC. Fraction I is concentrated to 5 ml and transferred to a Silicic acid (SOP 1.255) chromatographic column (custom columns 1 cm OD x 40 cm with a 100 ml reservoir on top, Ace Glass) for additional cleanup required for separation of PCBs from other organochlorines. Five grams of hot Silicic Acid is put into the column, which already has a glass wool plug and about 3-mm sodium sulfate in the bottom. The Silicic acid is topped with 10-mm sodium sulfate and prewashed with 10-ml hexane. Three fractions are eluted from the silicic acid column. The sample in 5-ml solvent is added to the column and rinsed into the column with 3,1,1-ml hexane. Then the sample is eluted with 20-ml pet ether (fraction SAI). Fraction SAII is 150-ml pet ether. Fraction SAIII is 20 ml of a mixed solvent consisting of 1 part acetonitrile, 19 parts hexane and 80 parts methylene chloride (SOP 1.255). Each is concentrated to appropriate volume for quantification of residues by megabore column, electron capture gas chromatography. HCB and Mirex are in SAI. PCBs are found in SAII. The rest of the compounds are in SAIII. GC determinations were run on a Varian 3600 GC with a Varian Star Data System and a Varian 8200 Autosampler. All GCs were equipped with dual DB-608 (0.83u film thickness, J & W Scientific # 125-1730) and DB-5 (1.5u film thickness, J & W Scientific # 125-5032) 30M megabore columns. All compounds were calculated using a three point standard curve forced through the origin using external standards (SOP 1.267).

PCB's were determined by shooting SAII fractions on a Varian 3400 GC with a Varian Star Data System and a Varian 8200 Autosampler. This GC is equipped with a 60M DB-5 0.25 ID capillary column. Another 3400 equipped with a 60M DB-XLB 0.25 ID capillary column is also used as a second system for PCB's. The compounds were calculated in the following manner. All the aroclor standards are at 0.5 ng/ul with one ul shot.

Starting with Aroclor 1260, 4 peaks that are unique to this mixture are located. The areas of the standards are summed and the same peaks located in the sample and also summed. Aroclor 1260 is calculated by the following formula to obtain PPM 1260.

(Area sample) (weight of std shot in ng)
(Area 1260 std) (basis shot in mg)

Aroclor 1254 is calculated by locating the major peaks in the mixture that are normally found in samples. The areas of these peaks are summed. Because some of this area comes from Aroclor 1260 and not all from Aroclor 1254, the contribution from the 1260 has to be subtracted from the total area. Aroclor 1254 is calculated by using the formula:

  • {(Area sample)-[((PPM 1260) (basis) (area from 1260))/ng 1260 std]} (wt 1254 std in ng) (Area 1254 std) (Basis shot in mg)

Results are in PPM.

Aroclor 1248 and Aroclor 1242 are calculated in a similar fashion, subtracting the contribution from 1254 in the 1248 and the 1248 in the 1242.

Total PCBs are calculated by adding the sum of Aroclor 1242, 1248, 1254, and 1260.

Basis = (weight of the sample mg/final volume of sample ul)(ul of sample shot)

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 051

Analysis For Organochlorines Pesticides and PCBs, Aliphatic and Polynuclear Aromatic Hydrocarbons In Soil and Sediment, GPC cleanup

Weigh a ten-gram soil or sediment samples into a PRQ (SOP 1.105) centrifuge bottle. Add 50-ml acetone, and shake for 1 minute every 15 minutes, for 11/2 hours. Add 50-ml petroleum ether and repeat the shaking. Centrifuge the sample and decant the liquid into a 500-ml separatory funnel containing 200-ml PRQ water (SOP 1.255) and 15-ml PRQ saturated sodium chloride (SOP 1.255). Extract the sample again for 1 minute with 50 ml 1:1 acetone/petroleum ether (SOP 1.255), centrifuge and decant the liquid into the separatory funnel. The separatory funnel is shaken vigorously for one minute and the layers allowed to separate. The pet ether is removed, and the water fraction extracted again with 50-ml pet ether. The combined pet ether is washed twice with 50-ml water and concentrated by Kuderna-Danish (SOP 1.261) to appropriate volume. The sample is dissolved in 4 ml of methylene chloride and 2 ml (5 gram equalivant) injected into a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). This fraction is analyzed for PAHs. The remainder of the sample is diluted to 4 ml with methylene chloride, and 1.6 ml (2grams) shot on the Waters high pressure GPC. This fraction is analyzed for OCs.

PAH

The fraction is concentrated by Turbovap and then exchanged into hexane. The sample is transferred to a column containing 20 grams of 1% deactivated silica gel (SOP 1.255) column (silica gel is added to the column in a pet ether slurry) topped with five grams neutral alumina (SOP 1.255). Aliphatic and polynuclear aromatic hydrocarbon residues are fractionated by eluting aliphatics from the column with 100 ml petroleum ether (Fraction I) followed by elution of aromatics using first, 100 ml 40% methylene chloride (SOP 1.255)/60% petroleum ether, then 50 ml methylene chloride (Combined elutes, Fraction II). The silica gel Fraction II containing aromatic hydrocarbons is concentrated, reconstituted in methylene chloride to a known volume, and quantified by gas chromatography - mass spec (SOP 1.276) on a Varian Ion Trap GC/MS with a 30M DB-5MS capillary column. Fraction I is concentrated and shot on a Varian 3400 GC with a 30M RXT-1 megabore column and a FID detector. Aliphatics from C-10 to C-34 are identified.

OCs

The 2-gram aliquot of the extract is transferred to a 1.6 gram Florisil mini-column topped with 1.6-gram sodium sulfate. Residues are eluted from the column in two elution fractions. Fraction I consists of 12 milliliters hexane followed by 12 milliliters of 1% methanol in hexane, and Fraction II consists of an additional 24 milliliters of 1% methanol in hexane. If additional cleanup is required to separate PCBs from other organochlorines in Fraction I, further chromatography on a Silicic acid column is performed. Quantification of residues in the two Florisil fractions and three Silicic acid fractions is by megabore column, electron capture gas chromatography.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 052

Benomyl in Water

Midwest Research Institute Pesticide Analysis Method Manufacturing Site: DuPont, Texas Pesticide Class: Urea (Benomyl)

Benomyl is known to be slowly hydrolyzed to carbendazim in water. Since neither the rate nor the extent of conversion is predictable, it was decided to develop the method around the total conversion to carbendazim. This method involves a procedure to complete the hydrolysis process and an analysis for Benomyl as carbendazim using the method previously developed for carbendazim. Included in this method are the parameters developed for the determination of carbendazim.

1. Application: Analysis of wastewater samples for Benomyl

2. Chemistry

  • CAS Nomenclature: Methyl [1-[ (butyl- Amino) carbonyl]-1H-benzimidazol-2-yl]carbamate
  • Trade Names: Benlate, Tersan
  • Molecular Weight: 290
  • Molecular Weight carbendazim = 191.2
  • Conversion to benomyl: ppm x 290/191.2 or ppm x 1.52

3. Apparatus

a. Instrumentation: Waters Associates dual Model 6000/A pumps, Model 660 solvent programmer, U6K injector and Model 440 absorbance detector.

b. Operating Parameters:

(1) Detector: Set at a wavelength of 280 nm.

(2) Column: Waters Associates uBondapak C18 (10 um, 4 mm x 30 cm ID)

(3) Guard Column: Whatman CO:PELL ODS (30-38 um, 4 mm x 7 cm ID)

c. Glassware:

  • Kuderna-Danish (K-D) 3 Ball Snyder column 250 ml evaporator 10 ml receiver
  • Chromatographic column: 2.5 x 15 cm
  • Sample filtration apparatus:
  • 10 ml. Syringe, Luer slip;
  • Stainless steel Swinney, 13 mm;
  • Millipore Fluoropore filter, 13 mm
  • Separatory funnel: 2 liters
  • Solvent filtration apparatus:
  • 1 liter suction flask
  • Millipore filter holder, 47 mm
  • Millipore Fluoropore filter, 47 mm
  • Syringes: 10 1, 25 1, 50 1, 100 1
  • Screw cap vials: 7 ml

d. Chemicals:

  • Carbendazim Standard: Supplied by EPA/HERL, No. 1071
  • Benomyl Standard: Supplied by EPA/HERL, No. 0500
  • Distilled in Glass Solvents: Supplied by Burdick and Jackson
  • methylene chloride, hexane, petroleum ether, diethyl ether, acetonitrile, methanol.
  • Florisil (PR)
  • Sodium sulfate, anhydrous

4. Standards

a. Stock: A 25 mg/25 ml stock solution was prepared in acetonitrile.

b. Working: The stock was diluted to prepare a 100 ug/ml standard. The 100 ug/ml standard diluted to prepare 20, 10, 5, 1 and 0.5 ug/ml standards.

5. Procedures

a. Sample handling: One liter of neutral wastewater and 10 ml of HC1 are stirred for 24 hr to assure the complete hydrolysis of Benomyl to carbendazim. The pH was then raised to 7 for sample extraction.

b. Extraction: One hundred milliliter portions of each sample were extracted with three 35 ml volumes of methylene chloride. The sample was shaken each time for 2 min. The methylene chloride was passed through a 5 cm long column of anhydrous sodium sulfate and drained into a 250 ml K-D. A 60 ml methylene chloride final wash of the sodium sulfate column was added to the K-D evaporator.

The extract was concentrated to 10 ml in a K-D evaporator on a steam bath. The solvent remaining was evaporated under a stream of nitrogen and the residue redissolved into 5 ml acetonitrile.

c. High Pressure Liquid Chromatography: The sample extracts and carbendazim standards were analyzed using as a mobile phase: 80/19.8/0.2 acetonitrile/methanol/acetic acid.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 053

Ammonia Procedure

Samples were analyzed using a meter (capable of reading millivolts) equipped with an ion-selective electrode designed to measure the amount of ammonia as N in water and wastewater samples. Samples were analyzed without a pre-distillation step and were evaluated against a standard curve obtained from standards prepared from a stock solution of standard Ammonium Chloride. Samples and standards were analyzed by immersing the electrode in 100 mL of sample to which 1 mL of 10N sodium hydroxide was added. All standards and samples were continuously stirred at a slow rate with a magnetic stirrer during measurement readings.

Samples were analyzed on an Orion meter, research microprocessor ionalyzer model 901, equipped with a Corning Combination Ammonia Electrode. The detection limit of the electrode is 0.14 mg/L and has a working range up to 140 mg/L. The amount of ammonia as N for each sample was reported as ammonia by multiplying by a conversion factor of 17/14.

Reference:

Method 4500-NH3 F. Ammonia Selective Electrode Method: Standard Methods for the Examination of Water and Wastewater (1992) 18th Edition, pp 4-81 to 4-82, Edited by Arnold E. Greenberg, Lenore S. Clesceri and Andrew D. Eaton.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 054

Nitrite and Nitrate Procedure

Samples were analyzed by ion chromatography using suppressed conductivity detection. Equipment includes a Dionex Ion Chromatograph, model DX-100, an autosampler, an advanced computer interface, and a computer operating with Windows 95 and employing the PeakNet (version 5.1) Ion Chromatography software by Dionex. Columns used are the IonPac AS4A-SC analytical and AG4A-SC guard columns. Eluent used is 1.8 mM sodium carbonate/1.7 mM sodium bicarbonate with a flow rate of 2.00 mL/minute.

Detection is achieved with suppressed conductivity using the ASRS-ULTRA 4 mm self-regenerating suppressor by Dionex. Normal operating conditions yield detection limits of 0.1 g/L.

Samples were evaluated against a 4-point standard curve generated from multi-ion standards (20, 10, 5 and 1 mg/L) containing each of the following: Fluoride, Chloride, Nitrite, Nitrate, Phosphate and Sulfate. Upon calibration, samples were analyzed and diluted accordingly to fall within the calibration range.

References:

EPA Method 300.0: The Determination of Inorganic Anions in Water by Ion Chromatography (1991), U.S. Environmental Protection Agency, Environmental Monitoring and Systems Laboratory, Cincinnati, OH, Authors: John D. Pfaff, Carol A. Brockhoff and James W. O'Dell.

Method 4110 B. Ion Chromatography with Chemical Suppression of Eluent Conductivity: tandard Methods for the Examination of Water and Wastewater (1992) 18th Edition, pp 4-1 to 4-5, Edited by Arnold E. Greenberg, Lenore S. Clesceri and Andrew D. Eaton.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 056

Analysis For Organophosphate In Soil and Sediment, GPC cleanup.

Weigh a ten-gram soil or sediment samples into a PRQ (SOP 1.105) centrifuge bottle. Add 50-ml acetone, and shake for 1 minute every 15 minutes, for 1-1/2 hours. Add 50-ml petroleum ether and repeat the shaking. Centrifuge the sample and decant the liquid into a 500-ml separatory funnel containing 200-ml PRQ water (SOP 1.255) and 15-ml PRQ saturated sodium chloride SOP 1.255). Extract the sample again for 1 minute with 50 ml 1:1 acetone/petroleum ether (SOP 1.255), centrifuge and decant the liquid into the separatory funnel. The separatory funnel is shaken vigorously for one minute and the layers allowed to separate. The pet ether is removed, and the water fraction extracted again with 50-ml pet ether. The combined pet ether is washed twice with 50-ml water and concentrated by Kuderna-Danish (SOP 1.261) to appropriate volume. The sample is dissolved in 4 ml of methylene chloride and 2 ml (5 gram equalivant) injected into a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A).

The extract is concentrated, and exchanged into hexane. The sample is shot on a Varian 3400 GC with a 30M megabore column and a Thermionic Specific Detector (N and P detector) and on a Varian 3600 GC with dual 30M megabore DB-608 and DB-5 columns and electron capture etectors.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 057

Analysis for TPH (total petroleum hydrocarbon) in Soil and Sediment

1. Weigh 20 -g soil into a PRQ centrifuge bottle (SOP 1.105). (Add 10 ml PRQ H2O (SOP 1.255) to dry samples) Adjust pH to < 2 with PRQ 12N sulfuric acid (SOP 1.255). Add 50 -ml acetone (SOP 1.255) and shake 6 times over a one and one-half hour period (about every 15-min). Add 50 ml of a 1:1 petroleum ether (SOP 1.81)/ ethyl ether (SOP 1.255) mixture and repeat shaking. Centrifuge (SOP 1.100) and decant liquid into a 500 -ml separatory funnel containing 200 -ml PRQ water and 15-ml PRQ saturated NaCl.

Re-extract soil by shaking one minute with 50 ml 1:1 petroleum ether: ethyl ether (may need to add 10 ml H2O & adjust to pH < 2), then centrifuge and decant liquid into separatory funnel.

2. Using PRQ 6N KOH (SOP 1.255), adjust contents of separatory funnel to pH >12. Shake vigorously 2 min, then allow to stand 30 min. with intermittent shaking. Drain water layer and reserve ether layer. Re-extract H2O layer with 100 ml 1:1 petroleum ether: ethyl ether. Cap and reserve combined ether extracts. (This contains Total Petroleum Hydrocarbons.)

3. Concentrate the extract with Kuderna-Danish evaporators (SOP 1.261) and reduce volume to adequate size for column clean up.

4. Column Clean-up: The concentrated extract for TPH determination representing 20 grams of sample is transferred to a 20 gram 1% deactivated silica gel (SOP 1.255) column (silica gel is added to the column in a pet ether slurry) topped with five grams neutral alumina (SOP 1.255). TPH residues are fractionated by eluting the aliphatics from the column with 100 ml petroleum ether. The extract is concentrated and a known aliquot is air dried in an aluminum weighing dish to determine the weight of oil.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 059

Analysis for Paraquat and Diquat in Water

A known volume of water is made acidic by the addition of 15ml concentrated (18M) sulfuric acid. The entire sample is percolated through a cation - exchange resin (Dowex-50W, 3.5g) that retains the Paraquat and Diquat. The herbicides are eluted with saturated ammonium chloride at a flow rate of one ml/min. Fifty mls of eluent are collected in a 50ml volumetric flask.

20 ml of the eluent is pipetted into a 25ml test tube. 4ml of 0.2% sodium dithionate solution is added to develop the color. The paraquat and diquat are determined by using a HP 8453 UV-Vis spectrophotometer which is operated in the 2nd derivative. Paraquat is determined at 396 nm and 1.2 ug/ml of Paraquat and 0.001 to 0.2 ug/ml for Diquat.

Calculations:

  • Volume of eluent(ml) X concentration in eluent(ug/ml) = ppm
  • Original Volume of sample(ml)

Reference:

Modification of: The Determination of Residues of Paraquat in Crops -- A spectrophotometric method; Company method from ICI Plant Protection Division, Residue Analytical method No. 1B.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 060

Analysis for Aliphatic and Polynuclear Aromatic Hydrocarbons in Animal Tissue

Extraction

A sample of appropriate size (i.e. 5 grams animal tissue, 2 grams adipose, 5 grams eggs) is digested for 24 hours in 25 ml 6N aqueous potassium hydroxide (SOP 1.255). Shake once an hour at least 7 times during the digestion process. Cool digestate thoroughly in an ice bath and carefully neutralize with 15 ml glacial acetic acid, agitate slowly to cool. Add 100 ml PRQ H20 to a 500-ml separatory funnel. Pour sample into a separatory funnel, rinse 3 times with a total of 100 ml PRQ H20, then rinse the sample bottle with 25 ml CH2Cl2. Shake separatory funnel vigorously for 1 minute. Let the sample settle, then drain the CH2Cl2 into a PRQed 250-ml wide mouth French square bottle. Centrifuge if necessary. Add 25 ml CH2Cl2 to the separatory funnel again; shake vigorously for 1 minute and drain. Repeat this procedure once more, so that 75 ml of extract has been collected in the sample jar.

Backwash

Discard the H20 in the separatory funnel, rinse funnel with tap water, then with PRQ H20. Add 100 ml PRQ H20 to the separatory funnel, and then add 50 ml of 6N potassium hydroxide to the separatory funnel resulting in 150 ml of 2N KOH. Shake to mix. Add the 75 ml of extract to the funnel, rinsing the collection jar 3 times with CH2Cl2. Shake the sample gently for 1 minute. After settling, drain the sample and add 25 ml CH2Cl2 to the funnel. Shake gently for 1 minute, drain again, and repeat this procedure twice more, until the expected 150 ml of sample extract has been collected in the sample jar.

KD

Pour sample from the jar to the KD flask and rinse 3 times using hexane, then add 10 ml hexane to each flask. KD the samples.

N-evap

Evaporate the samples gently, until the solvent level reaches approximately 3mls. The samples should be in hexane at this point. If you smell methylene chloride in the samples, add 5 ml more of hexane, mix and blow down again to about 3 ml.

Silica Gel

PRQ silica gel columns. Pack with 20 grams 1% deactivated silica gel by making a slurry with 50-ml petroleum ether and washing the silica gel into the column with an additional 50ml pet ether. Top each column with 5-ml of neutral alumina (SOP 1.255). Rinse the columns with 100 ml more Pet Ether if aliphatic fraction is to be analyzed. Transfer the sample to the column with a Pasteur pepette, rinsing the tube 3 times with petroleum ether. Pour 100mls of petroleum ether into the column, adjusting the drip rate to 5mls a minute (SG-1, aliphatics). When the first fraction is complete, change the sample bottle and pour 100-ml of 40% CH2Cl2/PE (methylene chloride/petroleum ether) onto the column. Just before the column goes dry, add 50-ml of CH2Cl2 and collect in the same sample bottle as 40% (SG- 2, PAH).

Concentration

SG-1 is transferred to the KD flask using petroleum ether to rinse the collection jars. SG-2 is transferred to the KD flask using CH2Cl2. The samples are concentrated to about 5 ml. The samples are further concentrated by n-evap. The samples are transferred to calibrated tubes so that the final concentration is 5g/ml using the above solvents. Do NOT let these samples go dry.

Analysis

Both fractions are analyzed using a Varian Saturn 2000 GC/MS ion trap with a 30M DB-5MS or equivalent 0.25mm capillary column. Methylated aromatics are calculated against the unsubstituted parent compound, or against a named methylated compound in this list. C1-naphthalenes are the sum of the 2-methyl and 1-methyl naphthalene. C2-naphthalenes are calculated against 2,6-dimethylnaphthalene. C3 and C4-naphthalenes are calculated against 2,3,5-trimethylnaphthalene. Methylated phenanthrenes are calculated against 1-methyl phenanthrene. C1-fluoranthenes + C1-pyrenes are calculated against fluoranthene. All other methylated aromatics are calculated against their unsubstituted parent compound.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 061

Analysis for Atrazine metabolites in Water

1. Record sample volume and pH. Add 1500 ml of sample to a 2000-ml separatory funnel. Add 200 grams of sodium chloride and 100 mls phosphate buffer to the funnel and shake to mix.

2. Adjust to pH 8 by adding PRQ 6N KOH (SOP 1.255) or 6N sulfuric acid to sample, and shake vigorously 30 sec. Allow sample to stand one hour at pH 8. Rinse the sample container with 50-ml CH2Cl2 (SOP 1.255) and add this to the funnel. Pour the remainder of the sample back into the sample container and rinse the graduated cylinder 2 X 50 ml CH2Cl2.

3. Shake 2 min. with periodic venting. Drain CH2 Cl2 through a funnel of CH2Cl2 washed sodium sulfate into a 1000 ml French Square bottle if there is no emulsion. Otherwise, drain lower layer into centrifuge bottle and centrifuge to separate layers. Repeat 2 X 100 ml CH2Cl2. This combined extract contains the compounds of interest.

4. Concentrate the extract by turbo-vap.

5. Exchange sample extract into hexane and adjust to 2 ml.

6. Shoot the extract on two columns, a 60m 0.25mm x 0.25u DB-XLB and a 60m 0.25mm x 0.25u DB-5 with electron capture detector.

  • Phosphate Buffer: 29.6 ml 0.1N hydrochloric acid & 50 ml 0.1M dipotassium phosphate
    • 0.1N hydrochloric acid = 2.5 ml HCl in 296 ml H2O
    • 0.1M dipotassium phosphate = 8.7g dipotassium phosphate to 500 ml H2O (This makes approximately 800 ml buffer.

References:

  • E.P.A. Method 507
  • E.P.A. Method 508
  • E.P.A. National Survey of Pesticides in Drinking Water Wells (11/90) Method 4

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 062

Analysis of Water for Glyphosate

The samples were analyzed using the method of Moye,A. et.al., Validation of An Analytical Residue Method for Glyphosate and metabolite: An Interlaboratory Study. J. Agri. & Food Chem., 1986. 34:955-960.

Measure 100 ml water

Adjust pH to 2.0 plus-minus 0.4 with 6N HCL Add sample to the following chromatography columns: (2 columns)

1. Pour sample through a Chelex column. Elute with 15ml 6N HCL. Collect HCL, and make up to 25ml with concentrated HCL and add to next column.

2. AG-1X8 column , catch sample then elute with 6N HCL (2ml then 8ml) for a total of 35 ml. Concentrate by rotovap and re-dissolve in 10ml water

Inject on HPLC

Conditions:

  • Fluorescence detector Emission wavelength 455 Excitation wavelength 339
  • Mobile Phase: 0.05% Phosphoric acid in Deionized Water 1.0 ml/min
  • Oxidative solution 0.3 ml/min
  • Derivatization solution (OPA-Borate) 0.4ml/min
  • Post-column derivatization at 38 degrees C.
  • Waters IC-Pac Ion-Exclusion 7 micrometers 7.8x150mm column.

If Glyphosate is found, the confirmation column a Hamilton PRP X400: 7 micrometer 4. 1X250mm column

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 063

Toxicity Characteristic Leaching Procedure (TCLP)

Summary: Solid waste is extracted with an amount of extraction fluid equal to 20 times the weight of the solid phase.

Apparatus: Agitation apparatus: The agitation apparatus must be capable of rotating the extraction vessel in an end-over-end fashion at 30 +-2 rpm.

Bottle extraction Vessel: For a nonvolatile extraction, a jar with sufficient capacity to hold the sample and the extraction fluid is needed. Headspace is allowed in this vessel. It is recommended that borosilicate glass bottles be used.

Filtration device: Any filter holder capable of supporting a glass fiber filter and able to withstand the pressure needed to accomplish separation may be used. Wastes should be filtered using positive pressure filtration. Filters should have an effective pore size of 0.6 to 0.8 um.

Reagents: Extraction fluid.

Add 5.7 ml of glacial acetic acid to 500 ml of reagent water, add 64.3 ml of 1N sodium hydroxide and dilute to 1 liter. The pH of this fluid is 4.93 +-0.05.

Procedure:

Weigh out a subsample of the waste (100 gram minimum). Transfer the solid material into the extractor bottle. Slowly add 20 times the weight of sample in extraction fluid (2000 ml for a 100 gram sample) to the extractor vessel. Close the extractor bottle tightly, secure in the rotary agitation device, and rotate at 30 rpm for 18 +- 2 hours at an ambient temperature of 23 C. Following the extraction, separate the material in the extractor vessel into its component liquid and solid phases by filtering through a glass fiber filter in a pressure filter. The liquid obtained is the TCLP extract.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 064

Analysis for Organophosphate Pesticides and Triazine Herbicides in Animal Tissue using ASE/GPC

Four-gram samples or two-gram fat samples are weighed into a 150-ml beaker then thoroughly mixed with 4-grams of Hydromatrix. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. The sample can be stored in a desiccator over night. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 22-ml cell with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spa tula, and beaker with no more than 4-ml total of hexane and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 100% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, 1:1 hexane: acetone (SOP 1.255).

The extract is concentrated by Turbovap; transferred to a tarred test tube through a Pasteur pipette containing sodium sulfate using methylene chloride, and further concentrated to dryness for lipid determination (SOP 1.264). The weighed lipid sample is dissolved in 4 ml of methylene chloride and the fat removed by injecting 2 ml on a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap and then exchanged into hexane.

Quantification is by Varian 3400 GC with a 30M RTX-200 megabore column and a TSD (thermionic specific detector), to detect N and P containing compounds.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 065

Analysis for Pirate and Metabolites Tissue using ASE/GPC.

Ten-gram samples are weighed into a 150-ml beaker then thoroughly mixed with 6-grams of Hydromatrix. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 33-ml cell with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spatula, and beaker with no more than 4-ml total of hexane and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 60% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, 1:1 hexane: acetone (SOP 1.255).

The extract is concentrated by Turbovap; transferred to a tarred test tube through a Pasteur pipette containing sodium sulfate using methylene chloride, and further concentrated to dryness for lipid determination (SOP 1.264). The weighed lipid sample is dissolved in 4 ml of methylene chloride and the fat removed by injecting 2 ml on a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap and then exchanged into hexane. A drop of methylene chloride is added to the sample.

The sample is transferred to a 300 ml glass chromatographic column (Kontes # 420280-0242) containing 20 grams of Florisil (SOP 1.255) topped with 1 cm of sodium sulfate and the sample tube rinsed three times with about 2 ml pet ether. The column is eluted with 200 ml 6% diethyl ether (SOP 1.255)/94% petroleum ether (Fraction I) followed by 250 ml 15% diethyl ether/85% petroleum ether (Fraction II). The diethyl ether used in this analysis contains 2 % ethanol (SOP 1.255). Fractions II contains the Pirate and metabolites. This fraction is concentrated to an appropriate volume for quantification of residues.

GC determinations were run on a Varian 3400 GC with a Varian Star Data System and a Varian 8200 Autosampler. The GC is equipped with a 60m DB-XLB (0.25u film thickness) capillary column. All compounds were calculated using a three point standard curve forced through the origin using external standards (SOP 1.267).

  • Pirate, also known as chlorfenapyr, CAS # 122453-73-0

Metabolites

  • CL303267, CAS# 122454-23-3
  • CL303268, CAS# 122454-29-9

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 066

Analysis for Organochlorine Pesticides and PCBs in Animal Tissue using ASE/GPC.

Two-gram samples are weighed into a 150-ml beaker then thoroughly mixed with 4-grams of Hydromatrix. Stir the sample with a PRQ spatula until the mixture becomes a flowable powder and leaves the sides of the beaker clean. The sample can be stored in a desiccator over night. Pour the sample through a PRQ powder funnel into a PRQ Accelerated Solvent Extractor (ASE) 22-ml cell with a 2-cm glass fiber filter in the bottom cell cap. Tap the ASE cell to settle the sample and add more Hydromatrix if needed to fill the cell. Rinse the funnel, spatula, and beaker with no more than 4-ml total of methylene chloride and add the rinses to the cell. Place the top cap on the cell and tighten both caps hand tight. Record the cell number, the position number, and the number of the sample placed in the cell in the ASE logbook as the cell is placed in the ASE for extraction. Place the sample number on the collection vial. Operate the ASE according to SOP 1.260 (EPA method 3545) with the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 100% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, 100% methylene chloride. (SOP 1.255).

The extract is concentrated by Turbovap to about 20 ml. The water is removed by pouring the sample through a Horizon drydisk apparatus. The extract is transferred to a tarred test tube using methylene chloride, and further concentrated to dryness for lipid determination (SOP 1.264). The weighed lipid sample is dissolved in 4 ml of methylene chloride and the fat removed by injecting 2 ml on a Waters high pressure GPC (Gel Permeation Chromatography)(EPA Method 3640A). The fraction is concentrated by Turbovap and then exchanged into hexane.

The sample is transferred to a 300 ml glass chromatographic column (Kontes # 420280-0242) containing 20 grams of Florisil (SOP 1.255) topped with 1 cm of sodium sulfate and the sample tube rinsed three times with about 2 ml pet ether. The column is eluted with 200 ml 6% diethyl ether (SOP 1.255)/94% petroleum ether (Fraction I) followed by 200 ml 15% diethyl ether/85% petroleum ether (Fraction II). If Endosulfan II and/or Endosulfan Sulfate analysis is required, then 200 ml of 50% diethyl ether/petroleum ether (Fraction III) is required. The diethyl ether used in this analysis contains 2% ethanol (SOP 1.255). Fractions II and III are concentrated to an appropriate volume for quantification of residues by megabore column electron capture gas chromatography (SOP 1.265)(DB-608 and DB-5 dual columns) Dieldrin and Endrin are in Fraction II, and part of the delta BHC.

Fraction I is concentrated to 5 ml and transferred to a Silicic acid (SOP 1.255) chromatographic column (custom columns 1 cm OD x 40 cm with a 100 ml reservoir on top, Tutor Glass) for additional cleanup required for separation of PCBs from other organochlorines. Five grams of hot silicic acid is put into the column, which already has a glass wool plug and about 3-mm sodium sulfate in the bottom. The Silicic acid is topped with 10-mm sodium sulfate and prewashed with 10-ml hexane. Three fractions are eluted from the silicic acid column. The sample in 5-ml solvent is added to the column and rinsed into the column with 3,1,1-ml hexane. Then the sample is eluted with 20-ml pet ether (fraction SAI). Fraction SAII is 150-ml pet ether. Fraction SAIII is 20 ml of a mixed solvent consisting of 1 part acetonitrile, 19 parts hexane and 80 parts methylene chloride (SOP 1.255).

Each is concentrated to appropriate volume for quantification of residues by megabore column, electron capture gas chromatography. HCB and Mirex are in SAI. PCBs are found in SAII. The rest of the compounds are in SAIII.

GC determinations were run on a Varian 3600 GC with a Varian Star Data System and a Varian 8200 Autosampler. All GCs were equipped with dual DB-608 (0.83u film thickness, J & W Scientific # 125-1730) and DB-5 (1.5u film thickness, J & W Scientific # 125-5032) 30M megabore columns. All compounds were calculated using a three point standard curve forced through the origin using external standards (SOP 1.267).

PCB's were determined by shooting SAII fractions on a Varian 3400 GC with a Varian Star Data System and a Varian 8200 Autosampler. This GC is equipped with a 60M DB-5 0.25 ID capillary column. Another 3400 equipped with a 60M DB-XLB 0.25 ID capillary column is also used as a second system for PCB's. The compounds were calculated in the following manner. All the aroclor standards are at 0.5 ng/ul with one ul shot.

Starting with Aroclor 1260, 4 peaks that are unique to this mixture are located. The areas of the standards are summed and the same peaks located in the sample and also summed. Aroclor 1260 is calculated by the following formula to obtain PPM 1260.

(Area sample) (weight of std shot in ng)
(Area 1260 std) (basis shot in mg)

Aroclor 1254 is calculated by locating the major peaks in the mixture that are normally found in samples. The areas of these peaks are summed. Because some of this area comes from Aroclor 1260 and not all from Aroclor 1254, the contribution from the 1260 has to be subtracted from the total area. Aroclor 1254 is calculated by using the formula:

(Area sample) -
(PPM 1260)(Basis shot in mg)(area from 1260)
_____________________________________
ng 1260 std
(ng 1254 std)
--------------------------------------------------------------------------------------------------------------------------------------------------
(Area 1254 std)(Basis shot in mg)

Results are in PPM.

Aroclor 1248 is calculated in a similar fashion, subtracting the contribution from 1254 in the 1248. Aroclor 1242 is calculated using the area of five early peaks.

Total PCBs are calculated by adding the sum of Aroclor 1242, 1248, 1254, and 1260.

Basis = (weight of the sample mg/final volume of sample ul)(ul of sample shot)

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 067

Analysis for Organochlorine Pesticides and PCBs, Aliphatic and Polynuclear Aromatic Hydrocarbons in Soil and Sediment

A 10-gram soil or sediment sample is weighed into a PRQ (SOP 1.105) centrifuge bottle. If the sample is less than 10% moisture, then 10- ml water (SOP 1.255) is added. Fifty-ml acetone (SOP 1.255) is added and the sample shaken well six times over a ninety-minute period (about every 15 minutes). Fifty mls petroleum ether (SOP 1.81) is added to the sample and the shaking repeated. The sample is centrifuged (SOP 1.100) and the liquid decanted into a 500-ml separatory funnel containing 200 ml PRQ water and 15 ml saturated sodium chloride solution (SOP 1.255). Fifty mls of a 1:1 acetone/pet ether mixture is added to the sample and it is shaken, centrifuged, and the liquid added to the separatory funnel. The separatory funnel is shaken vigorously for two minutes and the layers allowed to separate. The pet ether is removed, and the water fraction extracted again with 50-ml pet ether. The combined pet ether is washed twice with water and concentrated by Kuderna-Danish (SOP 1.261) to appropriate volume. An aliquot of the concentrated extract representing 2 grams of sample is transferred to a 1.6 gram Florisil (SOP 1.255) mini-column topped with 1.6 grams sodium sulfate (SOP 1.255) for pesticide determination. A Kontes #7 column is used for this. Residues are eluted from the column in two elution fractions. Fraction I consists of 12 milliliters hexane (SOP 1.255) followed by 12 milliliters of 1% methanol (SOP 1.255) in hexane, and Fraction II consists of an additional 24 milliliters of 1% methanol in hexane. If additional clean up is required to separate PCBs from other organochlorines in Fraction I, further chromatography on a Silicic acid (SOP 1.255) column is performed. Quantification of residues in the two Florisil fractions and three Silicic acid fractions is by dual megabore column (DB-608 and DB-5), electron capture gas chromatography (SOP 1.265).

A second aliquot of the concentrated extract for hydrocarbon determination representing 8 grams of sample is transferred to a 20 gram 1% deactivated silica gel (SOP 1.255) column (silica gel is added to the column in a pet ether slurry) topped with five grams neutral alumina (SOP 1.255). Aliphatic and polynuclear aromatic hydrocarbon residues are fractionated by eluting aliphatics from the column with 100 ml petroleum ether (Fraction I) followed by elution of aromatics using first, 100 ml 40% methylene chloride (SOP 1.255)/60% petroleum ether, then 50 ml methylene chloride (Combined elutes, Fraction II). Both fractions are concentrated, reconst chromatography - mass spec (SOP 1.276) using a Varian Saturn 2000 ion trap mass spec with a 30M DB-5MS capillary column.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 068

Analysis for Organochlorine Pesticides and PCBs in Soil and Sediment, ASE, Dridisk.

Extract a two-gram dry weight samples using a Dionex Accelerated Solvent

Extractor (ASE) with 22-ml cells under the following extraction conditions: 5-min heating cycle, 2x2-min static cycles, 100% solvent flush, 60 sec purge cycle, 100 degrees C @ 1500psi, 100% methylene chloride.

Concentrate the extract by Turbovap to about 10 ml. Remove the water by pouring the sample through a Horizon drydisk apparatus. Exchange the sample into hexane and transfer to a 7mm ID column containing 1.6 gram Florisil topped with 1.6 grams sodium sulfate.

Elute the residues from the column in two fractions. Fraction I consists of 12 milliliters hexane followed by 12 milliliters of 1% methanol in hexane, and Fraction II consists of an additional 24 milliliters of 1% methanol in hexane. If additional cleanup is required to separate PCBs from other organochlorines in Fraction I, further chromatography on a Silicic acid column is performed. Adjust the samples to an appropriate volume. Quantification of residues in the two Florisil fractions and three Silicic acid fractions is by dual megabore column (DB-608 and DB-5), electron capture gas chromatography (SOP 1.265).

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 069

Imadacloprid in Tissue

ASE

  • 100% methylene chloride
  • 22 ml cells-100% flush
  • 5 min heat 2X 2 min static cycle
  • 100 degrees C
  • 4 grams Hydromatrix

PROCEDURE

  • Weigh 2g of homogenized tissue into beaker.
  • Add 4g of Hydromatrix and mix to a flowable powder.
  • Load ASE cells and extract.
  • Turbovap to ~ 15 mls at 40 degrees C

Dry Disk

  • Turbovap and transfer to tube with methylenechloride
  • N-evap to dryness and pipet 2 mls of 20/80 acetonitrile/water into tube
  • Filter into HPLC vial and inject

Reference

S. Baskaran, R.S. Kookana, R. Naidu, J. Chromatography, A, 787 (1997) 271-275Bayer-Environmental Fate Group, Analytical method

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 070

Imidacloprid in Soil and Sediment

Solvent

  • 80/20 acetonitrile/water
  • 20/80 acetonitrile/water
  • Glassware and Other Materials
  • Soil shake bottle
  • Thin tipped funnel

ASE tube

  • 10 ml calibrated tube
  • Pipette
  • Glass wool

Procedure

  • Weigh 10g soil into shake bottle
  • Add 20ml 80/20 acetonitrile/water
  • Shake 15 min
  • Centrifuge 5 min
  • Decant thru funnel plugged with glass wool into ASE tube
  • Put extract in Turbo and concentrate
  • Repeat 2 more time putting extract in same ASE tube Put concentrated extract in10 ml tube and adjust with 20/80 acetonitrile/water
  • Shoot on HPLC

Reference

S. Baskaran, R.S. Kookana, R. Naidu, J. Chromatography, A, 787 (1997) 271-275Bayer-Environmental Fate Group, Analytical method

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 071

Anticoagulant Rodenticides in Blood and Liver

EQUIPMENT

1) HPLC with absorbance and fluorescence detectors

2) Tissue grinder (e.g., Tissue-mizer)

3) Centrifuge

4) Silica and C18 Sep-Paks (Waters)

5) Disposable syringes, Monoject

6) Filter papers, ½ in, Schleicher and Schuell (S & S)

7) C18 HPLC column, 4.6 mm X 15 cm; e.g. Vydac or Luna

8) Glass Luer-Lok syringe, 25 or 50 ml

9) Syringe adapters (e.g., Baker)

10) 16 X 100 test tubes

11) Large test tubes (e.g., 25 X 150)

12) HPLC sample vials

REAGENTS AND CHEMICALS

1) Standards (from EPA or other sources): HYDROXYCOUMARINS: Racumin (coumatetralyl), warfarin, bromodiolone, difenacoum and brodifacoum; INDANEDIONES: pival (Pindone), diphacinone and chlorophacinone and potentially others;

2) Acetic acid, ACS grade

3) Phosphoric acid, ACS grade

4) Aluminum oxide, basic (50-200 mcm), Brockman activity grade I

5) Acetonitrile, HPLC grade

6) Methanol, HPLC grade (mobile phase A)

7) Methylene chloride, HPLC grade

8) Florisil, 60-100 mesh

9) Potassium phosphate, ACS grade

10) Triethylamine

11) Formic acid (0.25%) in methylene chloride

12) Distilled water

13) Helium

14) Triethylamine

15) Acetic acid

16) Ammonium acetate

17) Mobile phase B, for hydroxycoumarins: 752 ml water, 2 ml triethylamine, 2 ml acetic acid and 2.89 g ammonium acetate, all mixed thoroughly; plus 250 ml acetonitrile; degas with helium

18) 0.05 M phosphate buffer, pH = 6.9

19) Mobile phase B for indanediones: 500 ml phosphate buffer (0.05 M, pH = 6.9) plus 500 ml methanol; degas with helium

PREPARATION OF STANDARDS

1) For each compound, prepare 1,000 ppm stock solutions in acetonitrile

2) Prepare dilutions as necessary for standard curves, spiking, etc.

CONTROL SAMPLES

1) Prepare duplicate extractions of serum or liver and spike to 5 ppm with the appropriate stock

2) Extract as regular samples

PREPARATION OF CLEAN UP COLUMNS

1) Aluminum oxide/ Florisil clean-up column: Place 1 S&S filter in a 6 ml syringe barrel. Add 1 ml (ca.0.5 g) of Florisil, 1 S&S filter and then 2 ml (ca. 2 g) basic aluminum oxide and 2 S&S filters Before use wash with 4 ml each of methanol, water and acetonitrile, in that order.

2) Aluminum oxide/ C18 clean-up column: Place 1 S&S filter in a 6 ml syringe, add 3 g (ca. 3 ml) basic Alumina and 2 S&S filters.

Connect a C18 Sep-Pak to the outlet. Before use wash with 4 ml each methanol, water and acetonitrile, in that order.

SERUM EXTRACTION

1) Place 2 ml serum in test tube

2) Add 4 ml acetonitrile

3) Vortex

4) Centrifuge at medium speed for 10 min

5) Decant supernatant into 2nd test tube

6) Wash protein precipitate with additional 4 ml acetonitrile

7) Combine wash with 1st supernatant

8) Attach glass syringe to a washed Florisil/ aluminum oxide clean-up column with an adapter.

9) Pass extract through clean-up column, collecting eluate in a large test tube

10) Wash clean-up column with 4 ml acetonitrile, combining wash with 1st eluate

11) Dry eluates at ca. 55 C under nitrogen

12) Dissolve residue in 0.15 ml methanol and add 0.05 ml water

13) Transfer sample extract to HPLC sample vial

14) Proceed to HPLC analysis of hydroxycoumarins or indanediones

LIVER EXTRACTION: HYDROXYCOUMARINS

1) Weigh 2 g liver into a large test tube

2) Add 6 ml acetonitrile

3) Homogenize with tissue grinder

4) Centrifuge at medium speed

5) Decant supernatant into a 2nd large test tube

6) Wash pellet with additional 6 ml acetonitrile

7) Combine wash with initial supernatant

8) Attach washed aluminum oxide/ C18 clean-up column to a glass syringe with an adapter

9) Pass extract through the clean-up column, collecting eluate in a 2nd large test tube

10) Wash clean-up column with additional 6 ml acetonitrile, combining eluates

11) Dry at ca. 55 C under nitrogen

12) Dissolve residue in 0.15 ml methanol and add 0.05 ml water

13) Transfer extract to an HPLC vial

14) Proceed to HPLC of hydroxycoumarins

ADDITIONAL LIVER EXTRACTION CLEAN-UP FOR INDANEDIONES

1) Attach glass syringe to silica Sep-Pak

2) Apply 0.1 ml liver extract

3) Wash Sep-Pak with 3 ml methylene chloride, discarding wash

4) Elute indanediones into a large test tube with 30 ml 0.25% formic acid in methylene chloride

5) Dry at ca. 55 C under nitrogen

6) Dissolve residue in 0.15 ml methanol and add 0.05 ml water

7) Transfer extract to an HPLC vial

8) Proceed to HPLC of indanediones

HPLC OF HYDROXYCOUMARINS (FLUORESCENCE DETECTION)

1) Equilibrate HPLC column with 30% A (methanol) and 70% B (for hydroxycoumarins), total flow rate = 1.5 ml/ min

2) Fluorescence detection: EX = 318 nm and EM = 390 nm

3) Inject 0.01 ml or more of extract or standard

4) Ramp rate = 2.5 %/ min

5) Integrate area under peaks

6) Proceed to calculations

HPLC OF INDANEDIONES

1) Equilibrate C18 column with 55% A and 45% B

2) Absorbance detection at 285 nm

3) Inject 25 ml extract or standard

4) Ramp rate = 2.5 %/ min

5) Integrate area under peaks

6) Proceed to calculations: Method Code 071

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 072

Analysis of Water for Imazapic

Record sample volume and pH. Add 300 ml of sample to a 500-ml separatory funnel. Add 25 grams salt and 15 ml phosphate buffer. Adjust pH to less than 2 with drop wise addition of PRQ 18N H 2SO 4 (About 8 drops) (SOP 1.255).

Extract water with 50 ml methylene chloride by shaking two minutes. Drain the lower organic layer from the separatory funnel through acid sodium sulfate into a 200-ml turbovap cup. Repeat 2 x 50 ml methylene chloride. This combined extract contains imazapic.

Imazapic - Evaporate samples just to dryness. Reconstitute the samples in 20% acetonitrile / 80% water (HPLC grade) at a 100 ml/ml concentration. Samples are injected on a HPLC for quantitation.

HPLC conditions:

Waters 2690 HPLC system with a Waters 2487 UV detector
Column: Phenomenex Lichrosphere 5 RP-Select-B 4.0mm x 250mm
UV wavelength 240
Flow rate 1 ml/min

Gradient:

min

%A

 

0

80

 

14

60

 

15

60

 

16

80

 

20

80

Mobile phase:

A Water (0.1% Acetic acid)
B Acetonitrile

This procedure was developed for water samples that had no sediment. Dirty water samples might require clean-up.

mazapic CAS # 104098-48-8

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 073

Analysis for TPH (total petroleum hydrocarbon) in Water

  1. Mark the level of the water in the bottles. After extraction determine the volume of the samples.
  2. Pour the entire sample into a separatory funnel. Rinse the sample bottle with 1:1 petroleum ether: ethyl ether using 100 ml of solvent per 500 ml of sample. Add to the separatory funnel. Shake 2 minutes. Drain water layer and reserve ether layer. Re-extract H 2O layer with 100 ml 1:1 petroleum ether: ethyl ether. Combine ether extracts. (This contains Total Petroleum Hydrocarbons.)
  3. Concentrate the extract by KD and reduce volume to adequate size for column clean up (3 to 5 ml).
  4. Column Clean-up: The concentrated extract is transferred to a 20 gram 1% deactivated silica gel column (silica gel is added to the column in a pet ether slurry) topped with five grams neutral alumina. TPH residues are fractionated by eluting the aliphatics from the column with 100 ml petroleum ether. The extract is concentrated by KD and air dried in an aluminum weighing dish to determine the weight of oil (weighed to 5 places).
Spike: Use 1 ml of a 5 mg/ml TPH std in 1000 ml of water. Make a co-spike of 1 ml of the solution in a weighed Al dish.

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Lab Name: Mississippi State Chemical Laboratory (MSCL) Laboratory                     Method Code 099

% Moisture

Performed at other laboratory.

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