m f e t y in the chemkal laboratory
edited bv MALCOLM M. RENFREW university01 Idaho Moscow, Idaho 83843
Validated Methods for Degrading Hazardous Chemicals: Some Alkylating Agents and Other Compounds George Lunn and Eric 6. Sansone Program Resources, Inc., Environmental Control and Research Program, NCI-Frederlck Cancer Research and Development Center, P. 0 . Box B, Frederick, MD 21702
Many hazardous chemicals are found in teaching and research laboratories, yet there are few validated procedures for degrading them to less toxic products. The compounds investigated were: dimethyl sulfate (DMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), 1,3-propane sultone (PS), butadiene diepoxide (BDE), urethane (UT), methyl csrbamate (MC), N-methylurethane (MUT), N-ethylurethane (EUT), Ppropiolaetane (BPL), and cycloserine (CS). DMS and DES have been widely used aa alkylating agents, the sulfanic acid esters (MMS, EMS, and PS) have been used as experimental mutagens, BDE has been used fur cross-linking pulgmere and as a prrservative, the rarbamic acid esters (UT, MC. MIIT, and EUTj have been used in organic synthesis, BPL has been used in organic rvnthesis and as a sterilant. and CS has been ,~~~ used as an antibrotic. Even if there rompuunds are no longer used in the lahuratory, rt i.i not uncommon tocome across hlrttles of them in the stockroom or in laboratories that are being cleaned out. We wish to report the results of a study aimed a t developing validated procedures for degrading these compounds. We investigated the use of strong hase to degrade these compounds, and we also looked at the degradation of P-propiolactone and cycloserine using potassium permanganate in sulfuric acid when strong hase did not work. Many of these compounds are very hazardous, and they should all be handled with great care in a properly functioning chemical fume hood. Dimethyl sulfate is quite volatile and is highly toxic (I), causing severe burns and injury to the lungs, kidneys, and liver. I t may be a human carcinogen (2),and it is known to eause cancer in laboratory animals (3).DES is known to cause cancer in experimental animals, and it may he a human earcinoeen (4). MMS (5). EMS (6). BDE (7), ~ ~ ~ (&d 8 0BPL , (9)k carciho: genic in experimental animals. ~
~
Materials and Methods Warning! Several of these compounds have been shown to cause cancer in laboratory animals; they should all be treated as potential human carcinogens. These compounds should be regarded as hazardous and should only he used in a properly functioning chemical fume hood. Reagents N-Methyl urethane was obtained from Pfaltz and Bauer, Stamford, CT. All other compounds and reagents were obtained from Aldrich Chemical Co., Milwaukee, WI. Ana/yt/cal Procedures Colorimetry (for the Analysis of DMS, DES, MMS, EMS, BDE, PS, and BPL). One-hundred uL of the solution to be analyzrd wna a d d h 1 mL of a solution eon. taining 2 mL of acetic acid in 98 ml. of 2methoxyethanol. (N.B. If reaction mixtures containing 5 M sodium hydroxide solution are to be analyzed, an aliquot of the reaction mixture should he diluted with four volumes of water and 100 pL of this solution analyzed.) This mixture was swirled, and l mL of a solution of 5 g of 4-(4-nitrabenzyl)pyridine (4-NBP) in 100 mL of 2-methoxyethanol was added. The mixture was heated at 100 OC for 10 min, then cooled in ice for 5 min. Piperidine (0.5 mL) and 2-methoxyethanol (2 mL) were added, and the violet color was determined at 560 nm using 10mm disposable plastic cuvettes in a Gilford 240 UVIvis spectropbotometer. To validate the analytical procedure, add a small quantity of one of these analytes to the solution to he analyzed after the acetic acidI2-methoxyethanol has been added but before the 4-NBP has been added. A violet color will indicate that the analytical technique is satisfactory. Using this analytical procedure, thelimib of detection were: DMS 10 m g L , DES 27 ~
~
Volume 67
m g L , MMS 21 m g n , EMS 275 m g L , BDE 90 m g L , P S 66 m g L , and BPL 12 m g L A just noticeable violet color corresponded to a concentration that was about twice the detection limit. Gas Chromatography Vor the Analysis of UT, MC, MUT, and EUT). For analysis by gas chromatography a Hewlett Packard HP5880 gas chromatograph equipped with a 1.8-m X 2-mm4.d. packed column was used with !%me ionization detection. The iniectian temoerature was 200 'C. the detector temoerature was 300,OC.. and the carrirr gas waa nifrogen flou,in~ at 30 rnl./rnin. A cdumn packed with ;la( Carbowax 20 M on RO IUD Chnmosorb H HP war used. The oven temperature was 120 'Cfor MUT and EUT and 140 "C for U T and MC. We found that injecting reaction mixturea containing sodium hydroxide solution onto the hot GC column degraded any residual compound and gave unreliable results. Accordingly, 2 mL of each reaction mixture was acidified before analysis with 0.2 mL (for solutions containing 1 M NaOH) or 1 mL (for salutions containing 5 M NaOH) of eoncentrated hydrochloricaeid (caution:exothermic), and these mixtures were neutralized by adding solid sodium bicarbonate until the effervescence of carhon dioxide ceased. Spiking experiments showed that residual eompound would be detected. The column was fitted with a precolumn that was changed when the response to standards decreased. Under these conditions the retention times were approximately 2.4 min for MUT, 2.3 min for EUT, 4.3 min for UT, and 3.6 min for MC. The limits of detection were apnroximatelv,~~ 30 me&. .. Thp pruducrs from these degradation reactions were conveniently d~terminedby gas chromatogrnphy. We wed a column packed with 10D; Carhoaax 20 M + 2'1 KOH on 8011011Chromowrh \\' A W at I50 'C to determine methylamine (0.4 min), ethyl~~~
~~~
.~ ~
.~ ~
~~
~
~~~
~~~
~
~~~~
(Continued on page A250)
Number 10
October 1990
A249
RecommendedDegradation Proced~re~ Note: The reaction times given below gave good results in our tests. However, the reaction time may be affected by such factors as the size and shape of the flask and the rate of stirring. If two phases are apparent, this is an indication that the reaction is amine (0.5 min), methanol (0.5 m i d , and not complete. Stirring should be continued ethanol (0.6 m i d . until the reaction mixture is homogeneous. The GC conditions given above are only a Destruction of Dimethyl Sulfate and Diguide and the exact conditions would have ethyl Sulfate. Ten mL of DMS or DES was to be determined experimentally. added a t once to a flask containing 500 mL Speetrofluorirnetry (for the Analysis of of rapidly stirred 1M sodium hydroxide soCS). Cycloserine was determined by taking lution. Fifteen minutes after the last of the 100 of the neutralized reaction mixture DMS had gone into solution, no DMS could and adding 4 mL of buffer (prepared by be detected, and 3 h after the last of the DES dissolving 11g of sodium tetraborate decahad gone into solution, no DES could be hydrate in 950 mL of water and adding 42 detected. mL of 1 M hydrochloric acid (pH 8.1)) folThis procedure may also be adapted for lowed by 100 FL of a 0.0% solution of 1,4the destruction of larger quantities. Thus, benzoquinone in 95% ethanol. This mixture 100 mL of DMS was added to 1 L of 5 M was heated at 100 'C for 30 min, then alsodium hydroxide solution, and the reaclowed to cool for 10 min. Fluorescence was tion mixture was stirred. Fifteen minutes determined by using a spectrophotofluoafter the last of the DMS had gone into rimeter set a t an excitation wavelength of solution, no DMS could be detected. Similar 381 nm and an emission wavelength of 502 results were obtained for DES, hut dissolunm (11). Thelimit of detection was 20mgL. tin" wna much slower. After a total reaction ..-.. time of 24 h no DES could be detected in Mutageniciv Assays solution. The reaction mixture should he checked for completeness of destruction To test the reaetion mixtures from the and discarded with the nonhazardous aquedestruction procedures for mutagenicity, ous waste. the plate incorporation technique of the Destruction of Methyl MethanesulfoSalrnonello/mammalian microsome mutanate, Ethyl Methanesulfonate, Butadiene genicity assay was performed essentially as Diepoxide, and 1,3-Propane Sultone. (A) recommended by Ames e t al. (12) with the One mL of the compound was added to 50 modifications of Andrews e t al. (13). Tester mL of 1M sodium hydroxide solution, and strains TA98, TA100, TA1530, and TA1535 the reaction mixture was stirred for 1 h were used with and without S9 rat liver mi(PS), 6 h (MMS), 20 h (BDE), or 48 h erosomal activation. Pure compounds were (EMS). At the end of this time the reaction generally tested by applying 1 mg of the mixture should be checked for completeness compound, in DMSO solution, to each plate. of destruction and discarded with the nonNeutralized reaction mixtures were tested hazardous aqueous waste. Although BDE by applying 100 pL to each plate. If the was completely degraded, the final reaction number of revertants was more than twice mixture was slightly mutagenic (see below). the mean of the number of revertants proAccordingly,ProeedureB maybe preferable duced by the controls, then the mixture befor this compound. ing tested was deemed to be significantly (B) One mL of the compound was added mutagenic. Degradation ol DMS, DES, MMS, EMS, BDE, PS, MC, VT, MUT, and EUT by Hydrolysis wlth Aoueour Sodlum Hvdroxide Solution'
mfety
~
Molarity of Sodium Hydroxide
Reaction Time
DMS
1 5
15min'a 15 min'
RT RT
