Triazine Herbicides and Their Metabolites in Urine (8315)
TRIAZINE HERBICIDES and THEIR METABOLITES in URINE FORMULAS: Table 1
MW: Table 1
METHOD: 8315, Issue 1
CAS: Table 1
RTECS: Table 1
EVALUATION: PARTIAL
BIOLOGICAL INDICATOR OF:
8315
Issue 1: 15 March 2003
Exposure to triazine herbicides (1) - (4).
SYNONYMS: See TABLE 1
SAMPLING SPECIMEN:
Urine
VOLUME:
At least 15 mL of sample
PRESERVATIVE:
None
SHIPMENT:
Frozen
SAMPLE STABILITY:
CONTROLS:
Not established. Appear to be quite stable frozen for long (> one year) periods of time Urine from non-exposed persons.
MEASUREMENT TECHNIQUE:
GAS CHROMATOGRAPHY, MASS SELECTIVE DETECTOR
ANALYTE:
s-Triazines (1) - (6)
EXTRACTION:
Two liquid/liquid steps
INJECTION VOLUME:
1 :L
TEMPERATURE -INJECTION: -DETECTOR: -COLUMN:
280 °C 285 °C 50 °C hold for one minute, 50°C/min to 160 °C, 3.5 °C/min to 230 °C, 50°C/min to 280 °C, hold 2 minutes. Total run time, 26.20 min Solvent delay- 5.5 min
ELECTRON MULTIPLIER VOLTAGE: +153 mV from tune setting CARRIER GAS:
Helium, 1.5 mL/min
COLUMN:
Capillary, fused silica, 30 m x 0.20-mm ID; 0.20 :m film SPB-5 or equivalent.
CALIBRATION:
Standard solutions of analytes in ethyl acetate with internal standard.
RANGE:
LOD to ~1900 nmol/L.
ESTIMATED LOD: 20 - 47 nmol/L depending on compound PRECISION ( þ r ):
~20% varies by compound
APPLICABILITY: Triazines are common agricultural herbicides. This method measures the parent compounds and two metabolites simultaneously and specifically. It is applicable to herbicide applicators, farmers, or other occupations with triazine exposure. INTERFERENCES: None identified.
OTHER METHODS: This method is an adaptation of one published by Catenacci, et al. [2]
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition TRIAZINE HERBICIDES and THEIR METABOLITES: METHOD 8315, Issue 1, dated 15 March 2003 - page 2 of 6 EQUIPMENT:
REAGENTS: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Sodium bicarbonate, Certified grade. Ethyl ac etate, HP LC grad e.* Ethyl ether, Sp ectra nalyzed grad e.* Atrazine. Cyanazine. Propazine. Simazine. Desethyl atrazine. Desisopropyl atrazine. Internal standard solution, Phenanthrene-d 10, 100 :g/m L in me thanol. Store in refrigerator. Sodium sulfate, anhydrous, Certified ACS grade. Sodium chloride, reagent grade. Helium, zero grade. Nitrogen, zero grade. Me than ol, HP LC grad e.* Deionized water. A urine pool collected, frozen until use, and then thoroughly mixed
- See SPECIAL PRECAUTIONS
1. Gas chrom atograph with m ass se lective detector,SPB-5 or equivalent capillary colum n and autos am pler (page 8315 -1). NOTE: If large batche s of sam ples are done, chilling the autosampler tray assures that there is no evaporation of the samples 2. Guard column, 1.5 meter x 0.4 mm ID, made of the same m aterial (or SPB-1) as the analytical colum n and joined by a capillary column butt connector and polyamide/graphite ferrule. NOTE: This guard column and the injection port liner should be changed after every 35-40 sam ples. . 3. Analytical balance. 4. Analytical eva pora tor/m anifold fed with nitrogen gas. 5. Centrifuge. 6. “Roto-torque” rotator. 7. Disposable screw-top culture tubes, 16x125 mm , with PTFE-lined caps. NOTE: Caps with rubber liners will not work. 8. Disposable volumetric pipets, 5-mL. 9. Disposable transfer pipets, both short and long. 10. Volumetric flask, 25-mL. 11. Beakers, 50-mL, 5-mL. 12. Dispos able cen trifuge tubes, 15-m L, with caps. 13. Autosampler vials with 100-:L polypropylene inserts and caps. 14. Syringes, 10-:L and 100-:L. 15. Polypropylene bottles, 30-mL and 250-mL. 16. Disposable filtration columns, 10-mL with 20 :m pore frit. 17. Metal scoopula. 18. Repipet dispenser bottles, 1-L.
SPECIAL PRECAUTIONS: Un iversal precautio ns should always be used when dealing with bodily fluids o r extra cts thereo f. Me than ol, ethyl ethe r, and ethyl acetate are a ll highly flam m able; handle with care and use in a hood. Reagents 4-10 come from the manufacturer with expiration dates on them. Be certain to heed these dates.
SAMPLING: 1. Collect the urine in a polyethylene bottle and cap. 2. Ship the s am ple in dry ice in an insulated conta iner, store at -80°C upo n arrival at lab. A rem inder: comm ercial shippers have special labeling requirements for packages containing dry ice.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition TRIAZINE HERBICIDES and THEIR METABOLITES: METHOD 8315, Issue 1, dated 15 March 2003 - page 3 of 6 SAMPLE PREPARATION: 3. W eigh 0.7 g sodium chloride into screw-top culture tubes. 4. W eigh 0.5 g sod ium bicarbon ate into eac h of these tubes. Cap these tubes an d then they can be sto red fo r at least tw o weeks and m ost like ly for as long as necessary. 5. Label two culture tubes (one empty and one containing the two salts) and one centrifuge tube with a unique n um ber for ea ch sam ple. NOTE: The labelling system is unimportant as long as there is no danger of the samples being m ixed-up and s o a record is ke pt of which sam ple correspond s to which tube num ber. 6. Thaw s am ples to roo m tem perature. U sing warm water is oka y. 7. Mix thoroughly as urine becomes inhomogeneous upon freezing. 8. Transfer 5 m L of sam ple to its culture tube conta ining the salts . Leave the caps off th e tubes until samples have been dispensed to every tube and any CO 2 evolved has dispersed. 9. Sp in on R oto -torque for about one m inute on settin g 4 Hig h to dissolve salt. So m e salt w ill rem ain in the tube. 10. Dispense 5 mL ethyl ether into each of the urine tubes. Again leave caps off or on loosely to allow for gas to disperse. 11. Roto-torque for fifteen minutes on setting 4 High. 12. Centrifuge samples for five minutes at 3000 rpm. 13. Rem ove ether layer (top) with short transfer pipet to the second labelled culture tube. Remove all the ether; taking some aqueous phase does not appear to be detrimental to the analyses. 14. Dispense 5 mL ethyl acetate into each sample tube containing the remaining aqueous phase. 15. Roto-torque for fifteen minutes on setting 4 High. 16. Centrifuge samples for five minutes at 3000 rpm. 17. Rem ove organic layer (top) with short transfer pipet and add to the ether layer. Again, remove all the organic layer. 18. Fill the filtration columns about 3/4 full of anhydrous sodium sulfate. 19. Balance the column on top of the labelled 15-mL centrifuge tubes. 20. Using a transfer pipet, transfer the combined extract to the appropriate filtration column. 21. W ash the tube which held the organic phases with one pipetful ethyl ether and add this to the filtration column. 22. W ash the filtration column with another 2 m L ethyl ether. 23. Allow th e filtration colum ns to drain com pletely. 24. Place the centrifuge tubes in the analytical evaporator and gently evaporate the solvent with a stream of nitrogen until the tube is dry. NOTE: A waterb ath helps dissipate the co ld, but it is im porta nt the bath be k ept at room tem pera ture. Raising the bath temperature to 30 oC m ay cause marked decreases in the recoveries of analyte s (5) and (6 ) an d m ay well affe ct th e othe r an alyte s as well. 25. Rinse the sides o f the tubes with about 0.5 m L ethyl ether. 26. Evaporate to dryness again. 27. Add 10 :L internal standard solution to each centrifuge tube. 28. Add 90 :L ethyl acetate to each tube and m ix well. 29. Let the extracts sit for at least thirty minutes. 30. Label autosampler vials and place an insert into each vial. Have the caps ready for the vials. 31. Trans fer the entire e xtrac t to the c orrect autosa m pler vial with a long tran sfer pipet. 32. Cap the vial and place in approp riate tray position in the GC autosam pler.
CALIBRATION AND QUALITY CONTRO L: 33. The stock solution of the six analytes is prepared from the neat solids to about 240 :mol/L by accurately weighing 1.25 mg of each analyte into a 25 mL volumetric flask (larger flasks could be used with a concom itan t increase in the am ount of solid weighed out.) Fill the flas k to the line with ethyl acetate. It is generally necessary to sonicate the flask for complete and quick dissolution, after which the analytes stay in solution. Aliquot into autosampler vials, cap, and store this solution in the refrige rator. Calculate th e ex act c onc entra tion of eac h an alyte from the am oun t of so lid weigh ed o ut. In ethyl acetate these analyte s are quite stab le fo r ex ten ded periods of tim e. A t no point did degradation of the standard solutions become a problem, so they are stable for at least six months under these conditions. NIOSH Manual of Analytical Methods (NMAM), Fourth Edition TRIAZINE HERBICIDES and THEIR METABOLITES: METHOD 8315, Issue 1, dated 15 March 2003 - page 4 of 6 34. The calibration standards are made the day of the analysis. Allow the stock solution and the internal sta ndard solution to w arm to room tem perature before perform ing any pipettin g. A ll the “pip ettin g” in this step is done using a 100 :L LC syringe. Prepare a diluted standard by adding 10 :L of the stock solution to 440 :L ethyl acetate to mak e a secondary solution. Prepare seven standards by first pipetting 10 :L of the internal standard solution into marked autosampler inserts/vials. Then add 10, 45, and 90 :L of the diluted standard to three vials and 10, 20, 50, and 90 :L of the stock solution to four of the vials. Make each vial contain 100 :L fin al volum e by adding the correct a m ounts of e thyl acetate. This will make standards that are about 0.48, 2.4, 4.8, 24, 48, 120, and 216 :m ol/L. Calculate the exact concentrations of each analyte in each standard from the stock solution concentrations. Insert these standards into the autosampler batch prepared in Step 32. 35. The two levels of quality control samples were made by adding 75 :L and 250 :L, of the stock solution to 250 mL of the pooled urine. Calculate the exact concentrations of each analyte in each quality control standard from the stock solution concentrations. Run enough QC samples so that they constitute ~10% of the batch and are equally divided between the two levels. Extract 5 mL of each QC (steps 3 through 32) and run with the samples. 36. Prepare calibration curves of Area std/Area IS vs conc std/conc IS for each of the six analytes.
MEASUREMENT: 37. Set gas chromatograph according to manufacturer’s recomm endations and to conditions on page 8315-1. 38. Set mass selective detector to the following SIM Param eters: Com pounds Dwell (ms) Start time* Ions Metabolites (5) & (6) 100 5.5 173, 158, 145, 172, 187 Parents (1) - (3) 100 14.1 201, 186, 200, 215, 214, 229 100 15.5 188 Phena nthrene-d 10 Cyanazine (4) 100 19.5 172, 225, 240
- These start times m ay change as necessary, and should be checked regularly, especially after
replacing the guard column. A full-scan chromatogram should be performed on a standard to check the retention times and SIM windows. 39. Inject 1 :L ethyl acetate extract from step 32. 40. Measure the peak are as of the sam ples and internal stan dard. Divide the peak a reas of the sam ple by the peak area of the internal standard in the same chromatogram. The following ions are used unless the mass selective detector is capable of adding areas of more than one ion: Desisopropylatrazine 158 Desethylatrazine 172 Simazine 201 Atrazine 200 Propazine 214 Cyanazine 225
CALCULATIONS: 41. Determ ine the concen tration (C c) of the analytes in the extracts (in :mol/L) from the calibration curves. 42. The concen tration (C u) of the analytes in urine is calculated from the equation:
W here 50 is the concentration factorof going from 5 mL urine to 100 :L extracts, and 1000 is the factor for converting :mol/L to nmol/L. Note: These concentrations could be corrected for creatinine level or sample density if desired and if those values were obtained.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition TRIAZINE HERBICIDES and THEIR METABOLITES: METHOD 8315, Issue 1, dated 15 March 2003 - page 5 of 6 GUIDES TO INTERPRETATION: One of the limitations of this method is that different parent triazines are metabolized to the same com pound. Fo r exam ple, de-alkylation of the isopropyl group on a trazine and c yanazine both give des isopropylatra zine, wh ich can a lso be form ed b y the de -alkylation of o ne o f the ethyl groups o f sim azine. Unless exposure is very high the parent compounds are not found in the urine, leaving the analyst with no way of know ing which parent the m etabolite came from in the case of a m ixed expos ure which is very comm on in herbicide applicators. The metabolites and any parent compounds found can be summ ed and reported as total triazine level if desired. There are very few biological monitoring studies of triazines and their metabolites found in the literature on which to develop a good reference range. And to our knowledge there have been no studies or reports on levels of these analytes in the general population. 1. Catenacci (1990) looking at parent atrazine in the urine found maximum excretion rates of 0.14 - 0.42 nmol/hr in workers at an atrazine manufacturing plant and very poor correlation between the concentration in the urine and the concentration in the air. These excretion rates dropped by an order of magnitude or more within twelve hours of the exposure. 2. Ikonen summ ed two metabolites (the desisopropyl atrazine and the bi-dealkylated atrazine, the latter of which was not investigated in this study) and found concentrations of 30-110 :mol/L in railway sprayers. 3. Cate nacc i (19 93) returned to the atrazin e m anufacturing plant w ith an im proved m eth od that co uld measure all three of the de-alkylated metabolites of atrazine and found ranges of 1.1-1.6 :mol/24 hr for the bi-dealkylated metabolite, 0.13-0.21 for desisopropyl atrazine, 0.11-0.20 for desethyl atrazine, and 0.017-0.021 for parent atrazine. To convert to concentration it is possible to assume an average urinary output of 1.1 L/24 hr (Reference 4). Using that assumption and combining the metabolites gives average concentrations ranging from 1.2 - 1.8 :m ol/L. 4. Hines (Reference 5) provides an overview of more recent metabolism studies and results for biomonitoring of the triazines as well as showing the utility of this method (and others) in a study of herbicide applicators. 5. There have been no human studies looking at cyanazine in which to obtain reference ranges of any kind.
EVALUATION OF METHOD: The m ethod was evaluated using 15 spiked urine samples ranging from 5.8 nmol/L - 1878 nmol/L and analyzing these samples on three different days. The recoveries for s-Triazines for compounds (1-5) ranged from 84-88% and fo r com pound (6 ) it was 67%. Precisions and LOD were determ ined from this experiment and are listed on the first page of this method. Sam ple stability was not exhaustively examined. There were, however, samples run after several months that had been stored at -20°C compared to splits of those samples stored at -80°C and no bias was found due to the different storage tem peratures. This wo uld lead one to believe that the samp les would be very sta ble sto red at -80°C for exten ded periods of tim e. T he QC sam ples also tell som eth ing about sa m ple sta bility. T he first batch of these sam ples was stored at -20°C and the control chart sh ow s no trend in these samples even when run over 18 months. There may be some loss of signal on samples that go through multiple freeze/thaw cycles, but conclusive experiments were not done and this may be a function of the am ount of solids that form in the urine during this process an d not becau se the analytes are breakin g dow n. B ecause of this, it is a good idea to aliquot the QC sam ples out into c ontain ers th at w ill only be thawed once or twice and then discarded.
REFERENCES: [1] Catenacci G, Maroni M, Cottica D, Pozzoli L [1990]. Assessment of human exposure to atrazine throu gh the de term ination of free atra zine in urine. Bull. Enviro n. Conta m . Toxicol. 44: 1-7. [2] Catenacci G, Barbieri F, Bersani M, Ferioli A, Cottica D,Maroni M [1993]. Biological monitoring of human exposure to atrazine. Toxicology Letters 69: 217-222.
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition TRIAZINE HERBICIDES and THEIR METABOLITES: METHOD 8315, Issue 1, dated 15 March 2003 - page 6 of 6 [3] Ikonen R, Kangas J, Savolainen H [1988]. Urinary atrazine metabolites as indicators for rat and human exposure to atrazine. Toxicology Letters 44: 109-112. [4] Go rdon Ross, E d.,[1982]. E sse ntials of Hu m an P hysiolog y, 2 nd Edition. Chicago, IL: Year Book Medical Publishers, Inc, p. 382. [5] Hine s C , Dedde ns J , Striley C, B iagini R, Sho em aker D , Brow n K, M acK enzie B, Hull R [20 03]. Biological monitoring for selected herbicide biomarkers in the urine of exposed custom applicators: Application of mixed-effect models. Annals of Occupational Hygiene 47(6): 503-517.
METHOD WRITTEN BY: Dale A. Shoemaker, Ph.D., NIOSH/DART
TABLE 1. STRUCT URAL FORM ULAS, MO LECULAR WEIGHT S, and PROPERTIES Compound
(1) Atrazine
Formula
C8 H1 4 ClN5
Molecular Weight
CAS
215.72
1912-24-9
RTECS
XY5600000
Synonyms
2-Chloro-4-ethylamino-6isopropylamino-s-triazine; 2-Chloro-4-ethylamino-6isopropylamino-1,3,5-triazine; 6-Chloro-N-ethyl-N’-(1- methylethyl)1,3,5-triazine-2,4- diamine
(2) Simazine
C7 H1 2 ClN5
201.69
122-34-9
XY5250000
2-Chloro-4,6-bis(ethylamino)-striazine
(3) Propazine
C9 H1 6 ClN5
229.75
139-40-2
XY5300000
Chloro-4,6-bis(isopropylamino)- striazine; 2,4-Bis(isopropylamino)-6- chloro1,3,5-triazine
(4) Cyanazine
C9 H1 3 ClN6
240.73
21725-46-2
UG1490000
2-Chloro-4-ethylamino-6-(1- cyano-1methyl)ethylamino-s- triazine; 2-[(4-chloro-6-ethylamino-s- triazin2-yl)amino]-2- methylproprionitrile
(5) Desethylatrazine
C6 H1 0 ClN5
187.66
6190-65-4
2-Amino-4-chloro-6- isopropylaminos-triazine; 6-Chloro-N-(1-methylethyl)-1,3,5triazine-2,4-diamine
(6) Desisopropylatrazine
C5 H8 ClN5
173.67
1007-28-9
2-Amino-4-chloro-6-ethylamino- striazine; 6-Chloro-N-ethyl-1,3,5-triazine- 2,4diamine
NIOSH Manual of Analytical Methods (NMAM), Fourth Edition