Table II. Hydrolysis of RNA by Ceramic-Bound Ribonuclease Reactor“ mode
Aerosol
Solution ‘I
S a m e reactor for b o t h
Hydrolysis,
%
5.7 5.7
Substrate residence time, min
0.5-1.0 20
experiments, 25°C.
ase reactor, a comparison was made of t h e residence time required to achieve t h e same percent hydrolysis of 1% (by weight) R N A solution when t h e substrate was in a continuous aqueous solution a n d when t h e substrate solution had been atomized. Again, t h e aerosol phase behavior indicates a much more rapid reaction rate. It should also be noted t h a t the d a t a in Table I1 show t h a t immobilized RNase is definitely enzymatically active in a n aerosol phase as the hydrolysis products are detected in t h e effluent air stream. It may be argued t h a t the observed effects are due only to a “special absorption” by t h e active enzyme. An argument against this hypothesis is, first, t h a t only some en-
zymes work-e.g., trypsin and lysozyme d o not have this special adsorptive property against influenza. Second, a s discussed above, we have shown t h a t enzymes do function catalytically under t h e experimental conditions. Consequently, we believe t h a t the observations are due to the catalytic properties of t h e enzyme although further work, probably with radio-labeled virus, would be necessary for further proof. It might also be noted t h a t if t h e phenomenon is due to special adsorption effects, it still may be t h e basis for a useful disinfection device. T h e conclusion is t h a t immobilized enzyme systems may be effective disinfection systems for airborne viruses.
Literature Cited (1) Biddle. F . . J Gen’ Virol . 2, 19 (1968). (2) Reginster, M.,Acta Viroi, 10, 111 (1966). ( 3 ) Hirst. G. K.. Pons. M.. Viroion. 17. 546 (19721 (4) Reethal, H . , H., Havewala. ‘%. B.’, Biotech. Bioengr. Symp.. KO.3, p 241, 1973. (.?I) Kirwan. D. J.. Enrieht. .J. T.. Gainer. .J. L.. Biotech Bioenqr , 16, 551 (1974). (6) Schulman. .l.L.. A m J Pub/ Health. 58, 2092 (1968) ( 7 ) Schulman. .J. L . J Exper .Wed, 118, 25’7 (1963). Y
ReceiLed for retieic J a n u a n 17 l 9 i 4 Accepted December 23 1,974 Work cupported under NbF Grant GI 14i72
Gas Chromatographic Determination of Selected Organic Compounds Added to Wastewater Barry M. Austern,* Richard A. Dobbs, and Jesse M. Cohen U.S. Environmental Protection Agency, National Environmental Research Center, Advanced Waste Treatment Research Laboratory, Cincinnati, Ohio 45268
Methods are described for t h e analysis of 11 compounds, styrene, p-xylene, ethylbenzene, nitrobenzene, acetophenone, o-anisidine, anethole, trichloroethylene, turpentine, nonylphenol, and dimethyl phthalate, in spiked wastewater. Extraction with Freon, concentration of t h e extract in a Kuderna-Danish apparatus, a n d gas chromatography are involved. T h e methods are precise and sensitive to t h e low parts-per-billion range. Wastewater treatment systems composed of t h e physical-chemical processes of chemical clarification and carbon adsorption are capable of removing nonbiodegradable organic compounds. At t h e National Environmental Research Center in Cincinnati. t h e capability of these processes to remove certain hazardous organic compounds is being assessed. Compounds for study were selected, in part, on t h e basis of production quantity, potential hazard to health or degradation of water quality, nonbiodegradability, a n d likelihood of presence in wastewater. T o accomplish this objective analytical procedures were needed which could determine t h e selected compounds a t low levels in raw and treated wastewater. This article describes methods developed for l l compounds. These compounds, and t h e reasons they were selected for study are listed in T a b l e I.
Anal$ical M e t h o d Except for t h e gas chromatography, t h e analysis of 10 of t h e 11 compounds was identical. Trichloroethylene, be588
Environmental Science & Technology
cause of its high volatility, was determined slightly differently. Stock solutions of t h e compounds ranging from 20-25 mg/l. were prepared in water or raw wastewater. To aid solubility, a known weight of material was dissolved in a small volume of ethanol, which was t h e n brought up to 1 liter with water or raw wastewater. Appropriate volumes of t h e stock solution were added to wastewater samples t o yield concentration ranges of about 10 wg/l. to 10 mg/l. Except in t h e case of trichloroethylene solutions, t h e sample volumes taken for analysis were 350 ml for concentrations >lo0 pgA. and 1000 ml for lower concentrations. T h e dilute organic wastewater solutions were refrigerated until extracted, generally within 3 hr. T h e spiked samples were transferred to separatory funnels with Teflon stopcocks after p H adjustment with 1N HC1 or KOH solutions, where necessary. (Phenols were acidified to less t h a n pH 6, amines were adjusted to above p H 11, and so forth. T h e solutions were extracted with three volumes of Freon-TF, chosen for reasons of density, volatility, a n d high purity as supplied. For 1000-ml samples, 200ml volumes of Freon were used, a n d 75-ml volumes of Freon were used to extract 350-ml samples. T h e extracts were combined and concentrated in a Kuderna-Danish a p paratus. Final volume after concentration, measured to t h e nearest 0.1 ml, ranged from 4-12 ml. Samples containing trichloroethylene were treated differently. Regardless of concentration, 450-ml samples were extracted with three 5-ml portions of Freon. Because of t h e high volatility of trichloroethylene ( b p 87OC), Freon ex-
tracts containing trichloroethylene were not concentrated. (Trichloroethylene is so volatile t h a t in one preliminary test, 58% of t h e trichloroethylene was lost during concentration in t h e Kuderna-Danish apparatus.) D u e t o t h e m u tual solubility of water a n d Freon, t h e combined volume of t h e trichloroethylene extract was not exactly 15.0 ml, a n d , hence, volume was measured t o t h e nearest 0.1 ml. T h e concentrated Freon extracts were analyzed by gas chromatography with hydrogen flame ionization detection. LJp to 6 - ~ samples 1 were injected using a solvent flush technique consisting of drawing one ~1 of pure solvent into t h e syringe, t h e n a bubble of air, and t h e n t h e sample. T h i s procedure assures t h a t t h e total sample is injected; any m a terial in t h e needle is pure solvent. In all cases t h e column
Table I. Organic Compounds and Criteria for Their Selection Compound
Annual production (US.), 106 Ib (2)
Styrene
4600
p-Xylene
1628
Ethyl benzene Nitrobenzene
4907 484
Acetophenone
1.17
o-Anisidine
2.3
Anethole
2.3
Trichloroethylene
597
Turpentine
258
Nonyl p he no1 Dimethyl phthalate
71.5 7.33c
Environmental concern (reference)
Taste, 0.1 mg/l. (3) Fish toxicity,,I 25 mg/l. (3) Taste, 0.2 mg/l. (3) Fish toxicity, 10 mg/l. (3) Fish toxicity, 50 mg/l. (3) Odor in water, 0.03 mg/l. ( 4 ) Human lethal dose, 10 mg/ kg (5) Taste, 0.17 mg/l. (3) Mouse LD,o,tJ200 mg/kg ( 4 ) Skin irritant, inhaled human toxic concentration, 1.9 mg/m,: (5) Mammalian toxicity, 0.025 mg/kg ( 5 ) Fish toxicity, 55 mg/l. (3) Dog LDx, 5860 mg/kg ( 4 ) Fish toxicity, 100 mg/l. (5) Taste, low concentration Taste, 0.001 mg/l. (.3) Mouse LDjo, 1580 mg/kg (5)
" 9 6 hr TL,,, i.e. concentration that causes 50% mortality in 96 hr. * L e t h a l dose for 5'0% of test animals. CTotal phthalate ester production w a s 884 X 106 Ib a s phthalic acid.
was 3.2-mm (3/s-in.) o.d. stainless steel. T h e carrier gas was nitrogen a t a flow rate of 30 ml/min. T h e hydrogen a n d air flows were 30 ml/min a n d 300 ml/min, respectively. T h e detector a n d injector temperatures were at least 25OC higher t h a n t h e temperature of t h e column. Other chromatographic conditions are given in Table 11. Results were calculated by comparing t h e peak height of t h e injected sample with t h a t of a s t a n d a r d of known mass. All gas chromatographic determinations were made a t least in triplicate. As all t h e compounds did not exhibit a perfectly linear response, t h e standard was chosen such t h a t it was near t h e unknown sample in mass.
Results Minimum Detectable Quantity. T h e minimum detectable quantity of each compound is shown in Table 111. T h i s quantity, arbitrarily chosen, is t h e amount of material, dissolved in pure solvent, t h a t gave gas chromatographic response of two scale divisions with t h e particular instrument operated a t maximum sensitivity. Electronic noise a t t h a t setting was about 1 scale division. Linearity of Chromatographic Response. Because of various factors, many compounds exhibit nonlinear response with some columns, although t h e flame detector itself is linear over a range of 10; (I). T o minimize this problem, columns were chosen to yield a linear response-concentration relationship over t h e widest possible concentration range. Linearity of response for t h e compounds was checked by injecting solutions in pure solvent a t appropria t e dilutions. In all cases, t h e peak height per unit mass did not vary more t h a n 10% between t h e minimum detected, as shown in Table 111. and 400 ng. For most compounds t h e linear range extended much farther. However, linearity much beyond 400 ng was not essential for this study. Recovery of Compounds from Spiked Raw Wastewater. Each compound was added t o raw wastewater, previously filtered through LVhatman No. 1 paper, t o yield a n approximate range of 10 ig/l. to 10 mg/l. ( T h e wastewater was primarily of domestic origin. and typically contained 180 mg/l. BODS, 350 mg/l. COD, a n d 200-250 mg/l. total suspended solids.) For each compound, six individual samples of different concentrations, plus a wastewater blank
Table II. Gas Chromatographic Conditions for Each Compound Column length, f t
Liquid phase
Styrene
6
15% Carbowax 4000
p-Xylene
6
15% Carbowax 4000
Ethylbenzene
6
15% Carbowax 4000
Nitrobenzene
6
15% Carbowax 4000
Acetop he none
6
15% Carbowax 4000
o-Anisidine
6
15% Carbowax 4000
Anethole
6
15% Carbowax 4000
Compound
Trichloroethylene
"
12
10% ov.101
Turpentine
6
10% o v - 1 0 1
Nonylphenol
6
10% o v - 1 0 1
Dimethyl phthalate
4
2% OV-225
Support
80/100 mesh
Chromosorb W(H P) 80/100 mesh C h romosor b W (H P) 80/100 mesh Chromosorb W(HP) 80/100 mesh Chromosorb W(H P) 80/100 mesh C h rom osor b W( H P ) 80/100 mesh Chromosorb W(HP) 80/100 mesh Chromosorb W(HP) 80/100 mesh Chromosorb W(HP) 80/100 mesh Chromosorb W(HP) 80/100 mesh Chrornosorb W(HP) 80/100 mesh G a s Chrom-Q
T e m p , "C
Retention time, m i n
90
5.5
90
3.3
90
2.8
175
4.6
150
4.0
150
13.0
150
7.0
55
4.6
90
200
3.1
135
3.2
Three peaks, retention times, 3.05, 3.9, 5.25 m i n , with relative heights of 75:24:1.
Volume 9, N u m b e r 6, June 1975
589
Table Ill. Minimum Detectable Quantity of Each Compound Compound
Minimum detectable, ng
Styrene pXylene Ethylbenzene Nitrobenzene Acetophenone o.Anisidine
0.5 0.3 0.3 0.7 1.1 0.6
Compound
Minimum detectable, ng
Anethole Trichloroethylene Turpentine Nonylphenol Dimethyl phthalate
0.4 0.7 0.4 2:2 0.4
Table IV. Recovery of Compounds from Spiked Filtered Raw Wastewater Compound
Styrene p-Xylene Ethylbenzene Nitrobenzene Acetophenone o-A n isidi ne Anethole Trichloroethylened Turpentine’ Nonyl p he no1 Dimethyl phthalate
Range of consistent recovery, mg/l.
0.011-9.7 0.011-9.3 0,055-9.4” 0.015-12.9 0.013-11.0 0.069-11.8r 0,012-10.6 0.065-14.7e 0.054-9.28) 0.011-12 .o 0.012-6.80”
Percent recovery ’
+
85.1 3.7 83.3 I= 4 . 3 80.8 z 3.6 96.6 I 3 . 8 87.9 & 2 . 2 86.6 z 0.7 99.7 = 2.9 39.2 1.7 67.3 2.8 96.8 i 5 . 1 51.0 i 1.1
“ M e a n p e r c e n t a n d s t a n d a r d deviation. ‘I Zero recovery f r o m 0.011 m a l l . solution. Zero recoverv f r o m 0.014 mcll. solution. ‘l T w o exoerim z n t s , m e a n = standardidebation for all l? s a m p l e s e S 9 % recovery f r o m 0.016 m g / l . solution. Both 3.05 a n d 3.9 m i n u t e p e a k s were mea. sured Zero recovery f r o m 0.011 mg/l. solution. 11 71.4% recovery f r o m 10.2 m g / l . solution.
Table V. Recovery from Raw and Treated Wastewaters Compound
Styrene p-Xylene Ethyl benzene Nitrobenzene Acetophenone o-Anisidine Anethole Trichloroethylene Turpentine Nonylphenol Dimethyl phthalate
Concentration added, mg/l.
Percent recovery”,’
0.259 0.248 0.252 0.317 0.147 0.157 0.142 1.63 1.24 1.03 0.136
99.6 I 0.9 95.3 T 2.5 100.1 = 3 . 5 99.9 1.8 99.4 = 1.7 9 9 . 1 T 0.8 99.4 i 2.7 103.4 77 6 . 7 1 0 1 . 5 1 1.7 99.9 = 3.5 98.8 z 3.3
*
Percent recovery, corrected for recovery f r o m filtered wastewater, a s shown in Table IV. ‘I Standard deviation.
with no added compound, were extracted. Each extract was injected three or more times. In all cases b u t dimethyl phthalate, none of t h e compounds was found in t h e wastewater blank. T h e raw wastewater blank exhibited a trace of material, eluting a t t h e same retention time as dimethyl phthalate, which was equivalent to less t h a n 0.05 pg/l. In every case but trichloroethylene, a single series of six samples was analyzed for each compound. As t h e low recoveries for trichloroethylene appeared suspect, t h a t experiment was repeated. T o test t h e hypothesis t h a t those low recoveries were due t o poor solubility of trichloroethylene in t h e original stock solution, t h e second experiment was done with a stock solution one t e n t h as concentrated. T h e two experiments, however, yielded identical percent recoveries, which showed t h a t t h e low values were not erroneous. T a b l e IV gives t h e percent recovery from spiked wastewater for each compound. Also shown for each compound is t h e range of consistent recovery. Within t h e range where t h e percent recovery is consistent for each compound, t h e 590
Environmental Science & Technology
scatter of values was random. No trend, with recovery being better (or worse) a t higher concentrations t h a n at lower ones, was seen. Recovery from Different Wastewaters and Effluents. Each compound was added, at a single concentration, to three t o five different wastewaters and effluents, including filtered raw sewage, trickling filter effluent, activated sludge effluent, t a p water, a n d chemically coagulated carbon-treated raw sewage. (Typically, t h e biological effluents contained 80-100 mg/l. COD, and 25-30 mg/l. T S S ; t h e physical-chemical effluents contained about 15 mg/l. COD.) T h e recoveries obtained are shown in Table V. In each case, t h e percent recovery is corrected for t h e mean percent recovery for each compound determined in t h e previous tests. T h e good recoveries, when corrected, a n d t h e good precision indicate t h a t t h e type of sample had little or n o effect on t h e recovery of t h e compounds. T h i s also shows t h e reproducibility from day to d a y of t h e previously determined correction factors.
Discussion When choosing analytical techniques for these compounds, a pragmatic approach was taken. No a t t e m p t was made to optimize t h e procedure once a method was sufficiently useful for t h e study of t h e treatability of these compounds. For t h e purposes of this project, t h e method was considered adequate when concentrations below 100 gg/l. could be determined with good precision. Greater sensitivities t h a n described can easily be obtained by changes in t h e analytical procedure. For example, changes in gas chromatographic temperature or column length may be useful. Some increase in sensitivity could be obtained by reducing t h e final volume of t h e Kuderna-Danish concentrate from t h e 4 to 12 ml used t o some smaller volume. A sample greater t h a n 1 liter can be extracted, although larger volumes become cumbersome. Lastly, peaks smaller t h a n reported can easily be measured. Generally, peak heights for t h e lowest concentration were 15-20 scale divisions; peaks one fourth of this height can be readily measured on a high-quality chromatograph. Variability among individual instruments, gas flow rates, etc., will, of course, affect t h e absolute minimum detectable quantity of each compound. Among compounds, there was a range of precision of recovery, with standard deviations of 0.7-5.1%. T h i s relatively wide range may be due to t h e small statistical sample (a single sample a t each of six concentrations of each compound). Presently this work is proceeding in two directions: methods are being developed for other compounds a n d methods of greater sensitivity are being attempted. Greater sensitivity, making possible t h e elimination of t h e K u derna-Danish concentration step, would greatly simplify t h e procedure a n d allow handling of smaller wastewater sample volumes. Literature Cited (1) Zweig, G., Sherma, J., Eds., “Handbook of Chromatography,”
Cleveland, CRC Press, 1972. (2) “Chemical Statistics Handbook,” 7th ed., Manufacturing Chemists Association, Washington, D.C., 1971. ( 3 ) Dawson, G. W,, Shuckrow, A. J., Swift, W. H., “Control of Spillage of Hazardous Polluting Substances,” U.S. Dept. of the Interior, Water Pollut. Contr. Res. Ser., Rep. No. 15090 FOZ 10/70,1970. (4) Arthur D. Little, Inc., Water Quality Criteria Data Book, “Organic Chemical Pollution of Fresh Waters,” U.S. Environmental Protection Agency, ibid., No. 18010 DPV 12/70. 1970. (5) Christensen, H. E., Ed., Toxic Substances List, 1972 ed., U.S. Dept. HEW, Nat. Inst. Occupational Safety and Health Rep. No. HSM-72-10265 Received for reuieu J u l y 15, 1974. Accepted J a n u a q 15>1975.
