edited by MALCOLMM. RENFREW University of Idaho MOSCOW, Idaho 83843
A Less Hazardous Chromic Acid Substitute for Cleaning Glassware Paul L. Manskel, Teresa M. Stimpfel, and Edward L. G e r s h e y The Rockefeller University, 1230 York Avenue, New York, NY 10021 Using chromic acid is one of the most dangerous laboratory activities, and, if the chromium concentration exceeds 5 m g n , the acid must be disposed of as hazardous waste (I). We evaluated several common cleaning solutions for their ability to remove known amounts of baked-on lipid or protein from glassware. All of the solutions removed over 99.7% of the lipid. An EDTA-organosulfonate detergent was as efficient (99.98%) as acid solutions for removing protein. This detergent does not present serious physical or toxicological hazards or leave residues that interfere with standard molecular tecbniques or cell culture. I t is economical and may he disposed via the sanitary sewer. The immediate hazards of working with chromic aeid are numerous. Mixing concentrated sodium (or potassium) dichromate solutions or chromium trioxide with concentrated sulfuric acid produces a strongly exothermic reaction. The large quantities of concentrated acid typically handled make preparation accident-prone. Most lahoratory personnel prepare and use the solution a t a sink, not in a fume hood, which allows the oxidation products to escape into room air. The toxicity of chromium is well documented. Chromium(V1) is more toxic (2,3) and mutagenic (4) than chromium(III), due perhaps to the stability of chromium(II1) in biological systems (5) and inability to cross membranes readily (3,6). Chromium(V1) usually forms strong oxidizing chromate and dichromate ions, which readily cross biological membranes and are easily reduced to chromium(II1) under physiological conditions (7). This may explain why chromic acid residues are toxic to cells in culture (6.7). Even after 15 washes with distilled water, glassware must he further treated so as not to inhibit cell attachment culture (8). Chromium(V1) compounds cause mutations and allied effects in a wide range of prokaryotic and euksryotic systems both in vitro and in vim (4). A recent review of the genetic toxicity of chromium compounds (9) has shown that occupational exposures to chromium(V1) compounds have resulted in an increased frequency of chromosomal aberrations and risk of lung cancer (4,lO). NIOSH has recommended limits for occupational exposure to both chromic acid (11) and ehromium(V1) (12). The oxidizing and
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corrosive characteristics of chromic acid pose definite, known risks including the explosive potential of stored chromate-sulfuric aeid mixtures (13-171, and ignition of the solvent vapor upon additionof acetone (17), alcohols (17,18), acetic acid, naphthalene, glycerin, or flammable liquids (18). Nonglass containers frequently leak, and spills are all too frequent during use and transfer. Once spilled, the solution is virtually impassible to remove from furniture, floors, crevices, and ceilings, and contaminated surfaces often will not accept paint. The spill kits for cleanup of simple mineral acids are inappropriate for chromic acid. Its neutralization is a two-stage process: reduction of chromium(V1) to chromium(1II) by bisulfite followed by acid neutralization. Sewer disposal requires that the heavy metal he precipitated from the neutralized acid before dilution of the neutralized effluent (19). Disposal of chromic acid as a solution of heavy metals is expensive, $164.00/gal due to the required precipitation and encapsulation of the chromium. As landfill becomes a less attractive alternative, the only disposal outlets for chromium solutions will be the few existing recovery and recycling plants. We evaluated several common acid-hased and detergent-based cleaning solutions (Fig. 2) for their ability to remove known amounts of protein and lipid from lahoratory glassware. To assess both the capacity and thoroughness of the cleaning agents, two pratocols were used. In the first, test tubes were heavily coated with bovine seN m albumin (BSA) (Fig. 1).In the second, glass rods were coated with 100 times less BSA, hut '"1-BSA was added to increase the analytical sensitivity (Fig. 2). Tubes and rods with a haked-on coating of BSA were soaked in the cleaning solutions for different time intervals and rinsed with distilled water. To measure the amounts of BSA remaining in heavily coated test tubes, protein assays were conducted in situ, and the results were measured directly in a spectrophotometer. The radioactivity remaining on the washed rods was measured by counting the rods in a gamma scintillation counter before and after cleaning. To assess lipid cleaning, scintillation vials with a coating of egg lecithin and L4C-phosphatidylchaline were soaked in the test solutions and then
rinsed with water and methanol. Vials and rinses were monitored by liquid scintillation counting. After rinsing with water alone, the approximate number of BSA molecules remaining on the lightly protein-coated glassware corresponds to the number of m d e eules that would be present in a monolayer of BSA. This suggests that cleaning agents are essential for overcoming the proteinglass interactions. The efficiency of protein ;ernoval varred over the treatment Gmes 31)min to 24 h (Figs. 2 and 3,. All cleaning solurionseace~tPHOS were over 999'0efficient in removing protein after 4 h a n d lipid after 1h. Even after 24 h, PHOS removed only 98% of the protein. With all the cleaning agents, maximum lipid removal was achieved in the first hour of treatment (Table). The phosphate-hased detergent (PHOS) was mare efficient at removing lipid than protein, but the differences between the cleaning agents do not appear significant. Removing residual lipids may require rinsing the glassware with methanol, chloroform-methanol. or other lipid solvent. Llpld Removal from Coaled Glasswarea Treatment
Percent 99.78 i 0.11 99.78 i 0.13 99.72 =t 0.23 99.98 =t0.24 99.89 i 0.04
3:l CHROM EOSULF PRSULF PHOS
s s nx lat on rwe~zom.. *heaton, ucre coated r m a s o ~ ~ t o n c o n np ~in 10t.c 'Y-~hownatmo,cno na r- ,o mlm 10" t. h e ~Ena an0 ~ L aar. C NEG682 rnec he acti&, 153 m ~ i / m ~ j [ sci l O in 0.5 ~ L Iand , egg ~ecimin
.
10 & I ~ L (sigma) in chloroform as ripreaentative lipids dry. A coating ~olvtionof and then allowed to drain activitv 99.95 10.5 mL contained 0.100 a of lioid . fsoeclllc .. nciimgl. After treatment 15 mL of scintillation cocktail
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A survey of cleaning agents in use at this university confirmed that most investigators usrd a c h n n i r acid concentrate mixed with suliuric acid (CHHOW, a few used an EDTA-organusulionate.based detergent
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Figure 1. Coating glassware with BSA. Borosilicate glass test tubes (12 X 75 mm, Coming) were filled with 0.1 M phosphate buffer (PB) or PB containing either 0.1. 0.5. 1. 5. 10. 50, or 100 mg/mL of BSA (Sigma) (Xaxis), drained. and dried in an oven at 850 OC. The amount of protein remaining on the glassware (Yaxis) was dstennined lnsituby the Lowry assay (20.2 1). Foiinand Ciocaiteu'~phenol reagent was purchased from SIGMA (F9252). Assay voiumes were adjusted so that only the lower poRion of the tube was assayed. corresponding to en area of 1320 mmz. Development time was 2 h. and absorption readings at 700 pm were obtained on a visible spectrophotometer (Coleman Junior), using the experimental tubes as cuveltes. Protein concentrations were derived from a calibration curve. Ail measurements were performed in triplicate and were highly reprcducible. As expected, more protein coated the glassware exposed to higher concentretians of BSA. but the deposition did not appear linear with regard to concentration. The lack of linearity at high coating concentrations (50 and 100 mg/mL) is primarily a reflection of exceeding the upper range (100 pg/mL) for the Lowry assay. The concentration of BSA coating the glass. 2 pglmm2, was estimated from the specific activlty of radiolabeled BSA (100 mg/mL), and was 100 times the costing from an iodinated BSA solution of 1 mg/mL (see legend to Fig. 3).
1.00 :
/O
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CI
C
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im2
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/O
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100.0
BSA C o a t i n g Solution (rng/ml)
IU
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0
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Figure 2. Cleanlng lubes heavily coated with protein.'~istiiied water was compared with the following cleaning agents: CHROM = a chromic acid concentrate mixed with sulfuric acid (Chromerge. Manostat inc.): PRSULF = a preparation of ammonium penulfate (3.4%) in sulfuric acid (NOCHROMIX, oodax Laboratories. Inc.); 3:l = a 3:l solution of suifuric acid to nitric acid; EOSULF = an EDTA-wganosulfonate-based detergent (MICRO, Internall. Rod. Cap.):andPHOS = a phasphafebeseddeWent(7X. Linbro Scientific). CHROM, open circle; PRSULF, ciosed circle; 31, open triangle; PHOS, ciosed triangle; EOSULF, opsn square; distilled water, closed square. Glassware was coated witha solution of100 mg/mL BSA as described in the legend for Figure 1. The surface contained approximately 2pg/mm2 BSA. BSA-coated tubes were then laced in beakers filled with the various cleanino solutions lorepared according to manufacturers' recommendations)or distilled water as a 20
r
.
control. Figure 2a: The percent of protein remaining on Uw glass (Yaxis) aner the tubes were removed in replicate sets otthree. 0.5, 1. 4. 6, and 24 h aner treatment IXaxis). washed five times with distilled waterand then analvzed for , prole n as oescrlbea m F i g ~ r e1 Tne oata reDreren! the average of love repirmtes and the erra oars represent 95% Cconfldence levels The re at veiy o m l performance of PHOS may h due lo lne lnlerference of the clean ng agent residues whh lhe Lowry assay. Even distilled waterremoved over 99.9% of the BSA. Also. estimation of the protein remaining by radiochemical rather than colorimetric technique indicated that PHOS had removed over 99.9% of the BSA. Amplification of thevsRica1 Scale (Fig. 2b) shows that the cleaning efficiencies of the acid-based agents and EOSULF are equivalent and removal is complete by 4 h. The amount af protein remaining is approximately 2 no/mm2.
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i
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A
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0 0
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1 0
Time (hrs) Figure 3. Cleaning lightly protein-coated rcds. CHROM, open circle; PRSULF. ciosed circle; $1, open biangle; PHOS, closed triangle; EOSULF, open square; distilledwater, ciosed square. The data represent three replicates, and the error bars represent 95% confidence levels. Borosilicategiass rcds 8 mm diameter X 10 cm length were coated by immersion to a depth of 1.5 cm in a sol~tion of 1 mg/mL '211-iabeled BSA (New EnglandNuclear NEX-076, specific activity in 1 rng/mL BSA solution, 0.024 ~Ci/eg).The coated surface measured 679 mmz. Rods were then withdrawn and dried. The coated sumce contained approximately 21 ng/mm2 BSA and 500 pCi/mm2 '25i. The lower level of detection was determined to be 3.5 pCi/mm2. For Cleaning. the rods Were piaced in beakers fiiied withthevarious cieaningsolutions and withdrawn
5
10
15
25
20
Time (hrs) in replicate sets of three. 0.5, 1.4.6, and 24 h after exposure (Xaxis). The rods were dipped sequentially in five beakers containing distilled water, by which time no more radioactivity was removed. The washed rods were placed in vial carriers and counted to a 95% confidence level in a oamma counter iPackard instrument Tricarb 85221) standardized for counting lZSi at 50% efficiency. The percent of protein remaining on the glass (Yaxis) is based on a comparison of activity before and aner treatment. Water alone removed 84-89% of the BSA, PHOS only 98%. and the rest greater than 99.9% (Fig. 3a). As bith me heavily coated tubes. by 4 h the cleaning agents, with lhe exception of PHOS, had removed 99.98% of the BSA, at which pointapproximately4.2 pg/ mm2of BSA remained (Pig. 3b).
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(KOSULF) or a 3 1 solut~onuf ,ulturir to nirric acid C3:1,. The rest ured a variety of detergents. PHOS was used primaril) hy laboratories engaged in culturing cells and viruses. Although phosphates work as surfactants and the phosphate ions may assist in solubilizing materials in a polar solvent such as water, the ions cling tenaciously to glassware. Consequently, glassware for culture is frequently segregated exclusively for this purpose. The phosphate residues may not be cytotoxic hut do interfere with some sensitive biochemical assays, and the disposal of phosphate-based detergents is an environmental concern. EOSULF is nonacidic, has no toxic ions, and is biodegradable. The organic sulfonate acts as a surfactsnt, while the EDTA is effective at chelating and weakening the charge between contaminants and glassware. However, as the analytical sensitivity of various techniques increases, the picogram residues of protein left by even the most effective cleaning solutions will be of concern. EOSULF was as effective as any of the solutions tested and has relatively few risks associated with its preparation, use, storage, or disposal. For special applications, cleaning withEOSULF can be followed by arinse with 1 N HCI. Use of EOSULF has greatly reduced the amounts of acid stored in laboratories, as well as reduced daily risks to investigators and to safety personnel who respond to spill emergencies, and has lowered hazardous chemical disposal costs.
Acknowledgment The authors are grateful to Moises Eisenberg for contributing his enumeration ofthe approximate number of BSA molecules in a monolayer, and they gratefully acknowledge the editorial skills of Amy Wilkerson.
Literature Cited
1979: p 383. 3. MerLz, W. Physiol. Rsu. 1969,19,163. 4, International Agency for Research on Cancer, IARC Monopiophs 1980,23,205. 5. National Academy of sciences. Chromium: National Academy: Washingtan. DC, 1974. 6. Whiie.L. R.:Jakobnen. K.;O~tgsard,K.Enuiron.Re6. 1971.20.166 ~~
7. Levis. A. J.;Majone, F. B r J. Cancer 1979.40.323. 8. Monahsn, J. J.Maihods Cell Bid. 1976,14,105. 9. Vennitt. S. Excerpts Med. 1986,249,249. 10. International Agenw for Research on Cancer, IARC Monopmphs Suppl. I987.6,167. 11. NationallnstituteforOccupationalSafefyandHealfh. NIOSH 1973,73-11021. 12. Netionslln~tiluteforO~~"p~tii"dSsffty~"dH~~Ith. NIOSH 1976,76-129. 13. Bryson, W. R. Chrm.Brit. 1975.11.377, 14. Pitt, M. J. Chem.Brit. 1975. 11,456. 15. Downing, S. Chem. Brit. 1975,ll. 456. 16. Baker. P.B. Chrm.Brit. l975,11,456. 17. Bretherick, L. Handbook of Reoaiue Chemirol Hol~ ords: Butllnuorth~:London, 1985: p 1019. 18. Manufaduring Chemists Association. Guide /or SoleL.v in the Chemirol Lobaralory: Place, 1972; p 216. 19. Sittig, M. Handbook of Toxic and Hozordous Chemicola: Noyes: Park Ridge, NJ. 1981: p 176. 20. Folin. O.;Cioeslteu,V. J.Bio1. Chrm. 1927. 73.627. 2,. Lowry, 0. n.;Rosebrough. N. J ; Farr, A. L : Randall. R. J. J. Bid. Cham. 1951.193.265-275.
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