CURRENT RESEARCH Analytical Method for Nonionic Surfactants in Laboratory Biodegradation and Environmental Studies Stanton L. Boyerl*, Kathryn F. Guin2, Richard M. Kelley3, Marvin L. Mausner4, Howard F. Robinson5,Thomas M. Schmitt6, Charles R. Stah17, and Eugene A. Setzkorn8 Analytical Subcommittee of the Chemical and Environmental Research Committee of the Soap & Detergent Association, 475 Park Avenue South, New York, N.Y. 10016
An analytical method for nonionic surfactants is developed and recommended for biodegradation and environmental studies. A foaming procedure for concentrating the surfactant and an ion-exchange step for its isolation make the method applicable to virtually any aqueous sample. The nonionic surfactant concentration is determined by the colorimetric response of a cobalt thiocyanate-nonionic surfactant complex. In 1972 the Soap and Detergent Association (SDA) Analytical Subcommittee renewed its evaluation of methods for quantitating nonionic surfactants in biodegradation media and environmental samples. In 1969 the SDA published a technical report ( 1 ) in which it concluded the analytical methods then available contained serious deficiencies. Reports of method improvements, e.g., Wickbold's procedure ( 2 ) , cobalt thiocyanate ( 3 ) ,and iodide-iodine complexation ( 4 ) encouraged a reevaluation. A method was sought which had applicability to a wide range of polyether nonionic surfactant types, including alkyland alkyl phenol-ethoxylated alcohols. Besides being sensitive to the trace levels of nonionics commonly found in biodegradation and environmental samples, the method needed to have a high degree of inter- and intralab reproducibility. The method should have the capacity for a large number of Samples and be amenable to automation. A surfactant concentrating step would be necessary to collect the surfactant from several hundred milliliters of sample. In this way the surfactant concentration could be increased to a level sufficient for analytical measurement. Further separation of the concentrated surfactants would depend upon the variety of surfactants in the sample, particularly for environmental samples, and the selectivity of the concentrating step. The analytical measurement should be sensitive to milligram quantities of nonionic surfactants. In 1972 Wickbold published a method ( 2 ) for trace levels of nonionic surfactants which involved isolation of the nonionic by foaming (sublation). The surfactant was then precipitated with Dragendorff's reagent (barium-bismuth-iodide solution), followed by potentiometric titration of the bismuth Procter & Gamble Co., Cincinnati, Ohio 45217. Shell Development Co., Houston, Tex. 77001. ,'j Colgate-Palmolive Co., Piscataway, N.J. 08854. Witco Chemical Co., Paterson, N.J. 07524. Lever Brothers Co., Edgewater, N.J. 0'7020. BASF Wyandotte Corp., Wyandotte, Mich. 48192. 'i GAF Corp., Wayne, N.J. Continental Oil Co., Ponca City, Okla. 74601.
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in the precipitate. Surfactants responding to this reagent are called Dragendorff active substances (DAS). After considerable study, the Organization for Economic Cooperation and Development (OECD) appeared ready to adopt this procedure as an official method for measuring nonionic surfactants in biodegradation media. The Analytical Subcommittee agreed that an evaluation of the applicability of this approach for use in the USA was advisable. Of the colorimetric techniques, the cobalt thiocyanate measurement (5-1 0) has been successfully applied to biodegradation and environmental studies. In fact, several SDA companies have used this procedure extensively. The nonionic surfactant was determined by complexation with cobalt thiocyanate and subsequent colorimetric measurement. Its simplicity and fast operation made it an attractive alternative to the bismuth potentiometric measurement. Based on the subcommittee's initial evaluation of the Wickbold method, the Wickbold sublation procedure was found to be an effective and a relatively simple concentration step for surface active agents. The presence of foamable ionic surfactants however, particularly in environmental samples, made Osburn's ion-exchange procedure ( 3 ) a necessary separation step after sublation. Because of the acceptability of these procedures, the subcommittee decided to concentrate its efforts on the various quantitative determinations which could follow the cleanup steps. The subcommittee chose to evaluate the acceptability of the cobalt thiocyanate colorimetric response and the bismuth potentiometric response. As a result of this investigation, the measurement of cobalt thiocyanate active substances (CTAS) is recommended along with sublation for biodegradation studies; an improved sublation procedure, ion exchange, and measurement of CTAS are recommended for environmental studies. The new method is considered the best available for the determination of nonionic surfactants in biodegradation and environmental studies. The cobalt thiocyanate and bismuth potentiometric measurements were shown to yield comparable analytical data. This allows for mutual acceptance of data collected by either method, and provides a proven alternate method. The cobalt thiocyanate measurement is recommended because of greater simplicity, ease of automation, and faster operation. The improved sublation and ion-exchange procedures will be beneficial to the OECD and others about to embark on environmental monitoring programs.
Method The recommended method combines the concentration and isolation steps of sublation and ion exchange with the quantitation by cobalt thiocyanate. Acceptance of this method resulted from comparable CTAS and DAS measurements on Volume 11, Number 13, December 1977
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samples split after ion exchange. This section contains the recommended method; excluded are sample splitting and DAS steps. (The DAS procedure can be found in ref. 2.) Reagents. Anion-exchange resin, hydroxide form, 50-100 mesh such as Bio-Rad AG1-X2: converted from chloride to hydroxide by eluting with 1N NaOH, followed by a methanol wash until water is displaced. Cation-exchange resin, acid form, 50-100 mesh such as Bio-Rad 50W-X8. Cobaltothiocyanate solution, 30 g Co(N03)2.6H20, 200 g NH4SCN dissolved and diluted to 1 L with distilled water (stable for a t least one month @ 25 "C). All reagents and chemicals must be analytical reagent grade. Procedure. All samples are preserved with 1%formalin. Nonionic Concentration S t e p . The sample must be made homogeneous by gently swirling. A portion of the sample is taken so that 0-2 mg nonionic can be determined. A 500-mL portion is recommended for all environmental samples. The sample (diluted if necessary) is used to dissolve 100 g NaCl and 5 g NaHC03. This solution is quantitatively transferred to the sublator (Figure 1)and diluted with distilled water until the level is brought to the drain cock. One hundred milliliters of ethyl acetate (EtOAc) is layered on the aqueous phase, and a nitrogen stream is passed through the sublator a t the fastest rate possible, while avoiding mixing a t the phase interface. The sample is sublated until 4 L of nitrogen have passed through, e.g., 20 L/h for 12 min. The EtOAc layer is then drained into a 500-mL separatory funnel; any aqueous phase is returned to the sublator. This procedure is repeated until a total of four such 100-mL EtOAc portions are collected in this manner and combined in the 500-mL separatory funnel. (The number of sublations depends on the suspended solids content of the sample. The higher the solids level, the greater the adsorption of CTAS; more sublations are necessary to desorb the CTAS for quantitative recovery. Two sublations have been adequate for biodegradation samples and river water samples. Four sublations were necessary for samples from sewage treatment plants.) About 20 mL of EtOAc are used to rinse the top of the sublator and are then combined with EtOAc portions in the separatory funnel. Any aqueous fraction in the separatory funnel should be removed. The EtOAc is dehydrated with 10-15 g of anhydrous Na2S04and then decanted into a 600-mL beaker. I t is evaporated to dryness on a steam bath, aided by a gentle stream of clean, dry nitrogen. The sample should be removed from the steam bath as soon as the solvent has evaporated. Nonionic Surfactant Isolation. (If ionic surfactants are
1
F (
ETHYL ACETATE LAYER
2 TEFLOhi S T O P C X K
,//No
+
,-AQUEOUS
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SAMPLE LAYER
MEDIUM POROSITY FRIT
NITROGEN INLET
Figure 1. Sublator 1168
Environmental Science & Technology
-
, IOC
INFLUEN' EFFLIIEh'
I000
E
300
tl
f ? 3 8 % R S D . 95 % I4C RECOVERED
-7[
P, ? 4 0 % R S D , 90% I 4 C RECOVERED , $ t 2 7 % R S D , 9 0 % I4C RECOVERED
loo
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f
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06001
2
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? 4 I % RSD, 91 % 14C RECOVERED
6 5 % R S D , 93 2
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Figure 2. Standard addition of
70
I4C
4 ADDED
C72-,8E7
RECOVERED 6
8
to environmental samples
known to be absent, this step can be omitted.) The ionic surfactants are removed by ion exchange. The anionic and cationic exchange resins are slurried with methanol and placed in the same glass column (1 cm diameter). Each resin bed should be about 10 cm long. The cationic resin is placed above the anionic resin, separated by a plug of glass wool. The same column may be used for up to six samples before repacking. The residue from the sublation step is quantitatively transferred in 5-10 mL of methanol to the column and eluted with methanol a t a rate of 1 drop/s into a clean, dry 150-mL beaker until a volume of 125 mL is collected. The methanol is evaporated to dryness on a steam bath aided by a gentle stream of clean, dry nitrogen. Again, the sample should be removed from the bath as soon as the solvent is completely evaporated. Measurement of Cobalt Thiocyanate Active Substance (CTAS). The nonionic residue is dissolved in 15.0 mL of methylene chloride (CH2C12). Exactly 10.0 mL are pipeted into a 125-mL separatory funnel. (Note that this is an aliquot: CH2C12 evaporation is to be avoided.) (Do not further dilute.) Five milliliters of the cobaltothiocyanate solution is added, and the phases are vigorously mixed by shaking for 60 s. After the layers have separated, the CH2C12 layer is drained into a 12-mL centrifuge tube and centrifuged a t about 4000 rpm for 3 min. A Pasteur pipet is used to transfer the CHzCl2 to a 2.0-cm cell, and the absorbance of the cobalt thiocyanatenonionic surfactant complex vs. CH2C12 is measured a t 620 nm. Nonionic Concentration. The concentration of nonionic surfactant in parts per million (ppm) is determined from a calibration curve derived with the same nonionic surfactant as is suspected (known) in the sample. An alkyl ethoxylated alcohol with a 12-18 range of carbon atoms and an average ethoxylate number of seven was chosen as the reference material for environmental samples; the calibration curve has a slope of 0.224 absorbance unitdmg. Scope of Method During the development and evaluative stages of the method, several features were observed to demonstrate its applicability. Standard addition of an alkyl ethoxylated alcohol ( A E ) ( C ~ ~ - Iand ~E~ a radiotagged ) AE (C14E7) to environmental samples gave AE recoveries consistently above 90% with relative standard deviations consistently below f 7 %
~~
~
Table I. CTAS and DAS Response of Various Nonionic Surfactants CTAS, ~ 6 2 nm/rng 0
DAS, mL of titrantlmg
c 12-1 EE7
0.224
20.0
(312
0.362 0.216 0.208 0.258 0.328 0.238 0.204
29.4 23.0 21.4 25.8 27.8 22.6 22.0
Surfactant a
c 1 2-15E9 c 16-lEE9 c 16-lEE 10 Cl6-18EZO
NPEio OPE10
-?Thesubscripts following the C designate the range of carbon atoms in the alkyl chain length; NP and OP stand for nonyl phenol and octyl phenol, respectively; the subscript following the E designates the average ethylene oxide units per mole.
(Figure 2 ) . These data were obtained over a 0.5-8 ppm CTAS concentration range. The limit of detection of this method is 0.1 ppm. Environmental samples were analyzed for CTAS (and DAS) with and without the ion-exchange step. Omission of this isolation step resulted in a doubling of the CTAS value. For example, the CTAS level of a sewage treatment plant influent increased from 4.0 to 8.5 ppm. The increase is attributable to anionic surfactants of the sulfated AE type. Accordingly, the ion-exchange step is necessary for analysis of environmental samples. In fact, in this investigation the ion-exchange step was used with environmental samples and not used with biodegradation samples. The method is applicable to alkyl- and alkyl phenolethoxylates with a linear or branched carbon chain length Cs-Cls and an ethoxylate chain length E4-E20. Examples of nonionic surfactant response to the CTAS and DAS measurements are presented in Table I. Although the CTAS (and DAS) procedures are useful for environmental monitoring, the objectives of any monitoring program must fall within the limitations imposed by the nonspecificity of the methods. CTAS yields an integrated response to many nonionic surfactants of current interest and perhaps to some unidentified interferences. When applied to environmental monitoring, the CTAS response would include intact nonionic surfactants. But the method establishes neither the identity nor the environmental consequences of the measured species.
Results and Discussion of Method Application
The evaluation of the method resulted from the three round robin experiments among SDA members: analysis of standard reference samples, biodegradation studies, and environmental monitoring. Standard Reference Samples. The purpose of this experiment was to examine interlab and intralab accuracy and precision. T o ensure sampling homogeneity, one lab provided all other labs with three solutions. Sample a was a 100-mg/L stock solution of the nonionic surfactant NPElo, a nonyl phenol with 10 ethylene oxide adducts. This solution was diluted 1 : l O before analysis. Sample b was a lO-mg/L NPElo solution taken at time “zero” in a biodegradation study. Sample c was a sample taken from the lO-mg/L NPElo biodegradation study after 7 days of permitted degradation plus 1m g L NPElo added. Triplicate samples of a-c were subjected to the entire method. Data are presented in Table 11. The statistical evaluation (11) of the data indicated that there was no significant difference between the methods at the 5% confidence level, but therg was a significant difference between labs. The agreement between labs was not acceptable and was readdressed in later round robins. Treatability Studies. River water die-away samples were generated to evaluate the two analytical methods. Each lab prepared in triplicate its own die-away solutions of NPElo and analyzed each sample in triplicate. The data are presented in Table I11 and further support CTAS and DAS comparability. T o assess method comparability with various nonionic surfactants, four surfactants were examined under four test conditions. The following surfactants were chosen because they were either assumed to be commonly present in environmental samples or they correspond to OECD test materials: NPElo-nonyl phenol with an average of 10 ethylene oxide adducts (OECD test materials); OPElo-octyl phenol with an average of 10 ethylene oxide adducts; C12-15E9-primary alcohol ethoxylate (OECD environmental reference material); and C12-18E7-primary alcohol ethoxylate (SDA environmental reference material). The following biodegradation test methods were chosen and performed in cooperation with the SDA Treatability Subcommittee: SDA ( I ) and OECD Shake Flask (IZ),SDA semicontinuous activated sludge (SCAS) ( I ) , and OECD continuous activated sludge (CAS) (12). The statistical evaluation of the data (unpublished SDA data) for starting samples demonstrated that CTAS and DAS were significantly different a t the 5% confidence level, but
Table II. CTAS and DAS Measurements of Standard Reference Samples of 10 ppm NPElo Sample a
Sample c
Sample b
Lab
CTAS
DAS
CTAS
DAS
CTAS
DAS
1
10.3 10.4 10.1 10.0 10.3 10.9 11.2 11.2 11.2 10.6 10.6 10.3 10.0 9.9 10.1 10.5 0.2
10.2 10.1 10.0 10.1 10.2 11.3 12.3 12.2 12.3 9.8 10.5 9.5 8.3 9.2 8.7 10.3 1.3
9.0 9.8 9.8 10.0 10.3 9.9 10.7 10.5 10.4 9.5 8.9 9.2 8.8 9.1 10.7 9.8 0.5
9.9 10.0 9.4 9.7 10.0 9.2 10.5 10.5 10.4 9.8 9.1 8.3 9.5 8.3 9.5 9.6 0.5
1.8 1.8 2.0 1.9 1.9 1.9 2.1 2.2 2.1 2.8 2.9 3.1 2.0 2.0 2.0 2.2 0.2
1.9 1.9 1.9 2.1 2.1 2.2 2.0 2.0 2.1 2.1 2.1 2.0 1.6 0.8 1.5 2.0 0.4
2
3
4
5
Av SD (of all data)
Volume 11, Number 13, December 1977
1169
~~~~~~~~~
~
Table 111. CTAS and DAS Measurements (ppm) of Days “0” and ‘‘8” of River Die-Away of NPElo Solution 1 Day
Lab
0
1
2
3
4
5
8
1
2
3
4
5
Solution 3
Solutlon 2
CTAS
DAS
CTAS
DAS
CTAS
DAS
9.2 9.1 9.7 10.0 10.0 9.5 11.7 10.9 10.9 12.7 13.4 13.2 6.9 9.2 9.2 3.5 3.3 10.6a 1.3 1.6 1.2 0.5 0.5 0.5 1.5 1.4 1.5 1.1 1.1 1.o
8.5 8.7 9.7 10.3 10.1 9.9 10.2 10.0 10.2 12.4 12.3 12.4 7.3 7.9 7.8 3.4 3.0 8.7a 1.3 1.7 1.4 0.9 0.9 0.9 1.4 1.3 1.4 1.o 0.9 1.1
9.1 9.5 9.2 9.6 9.6 9.6 8.5 10.4 8.5 13.5 13.6 14.0 8.1 7.6 7.9 2.8 2.7 2.7 1.o 1.4 0.8 1.4 1.4 1.4 1.9 2.7 2.2 1.o 0.7 0.7
9.0 8.7 9.0 11.5 11.8 11.8 10.0 9.9 10.0 13.1 13.2 13.2 7.9 8.0 8.3 2.4 2.3 2.4 1.o 1.4 0.5 1.2 1.3 1.3 1.9 2.0 1.9 1.4 1.o 2.2
9.3 10.0 9.9 11.4 11.6 11.6 9.3 9.6 9.1 14.6 14.3 14.3 9.5 8.5 9.7 4.9 5.0 5.1 1.o 1.1 1.o 0.9 0.9 0.9 1.2 0.9 1.2 0.9 1.o 0.9
9.5 9.4 9.4 11.3 11.7 10.7 9.8 9.6 9.8 14.5 14.6 14.9 8.2 8.0 8.2 4.5 4.5 4.7 1.1 1.1 0.8 1.3 1.3 1.3 0.8 1.o 1.1 1.o 1.o 0.9
Bunexplaineddata.
were not significantly different a t the 1%level. But the analyses were comparable at the 5% level for degraded samples. This was true of all labs and all surfactants. However, when the data are transformed into percent biodegradation, the same conclusions result from both methods. Environmental Monitoring. International acceptance of a method requires the method to be applicable to environmental samples, for example, sewage treatment plant influents and effluents and river waters. The presence of unknown species, possible interferences, together with variable solid levels is a more rigorous test of applicability. An acceptable method must be as insensitive to these parameters as possible. Improvements of the nonionic sublation step necessary to meet the above criteria are reported in this section. CTAS and DAS comparability for environmental monitoring was established in a round robin among six SDA member labs. The same sewage treatment plant influent, ef-
fluent, and receiving water samples were analyzed in each lab. CTAS and DAS comparability and good intralab precision are demonstrated by the data in Table IV. Again, the data indicate significant variability among labs. Having satisfactorily demonstrated CTAS and DAS comparability, the subcommittee concentrated on interlab precision. Note that data in Table IV were collected from samples sublated twice as stipulated by Wickbold ( 2 ) .It was suggested that four sublations rather than two and a careful standardization of sampling techniques, such as gently swirling the sample before pouring a portion for analysis, could potentially increase interlab precision. To test this hypothesis, a sewage treatment plant influent was allowed to settle so that “light solids” and “heavy solids” solutions resulted. (This was done to examine the effect of solids on analytical precision.) Each solution was mixed until homogeneous, and portions were sent to each lab for analysis.
Table IV. CTAS and DAS Measurements (ppm) of Common Environmental Samples Influent CTAS
DAS
CTAS
DAS
1
4.2 3.7 4.7 4.4 2.9 2.9 1.2 1.1 3.4 2.5 2.3 2.2 2.4
4.2 3.5 4.4 4.2 2.8 2.7 1.4 1.3 3.5 2.4 2.1 2.2 2.1
1.4 1.9 1.6 1.4 1.7 1.7 1.o 0.8 2.3 1.3 1.3 1.3 1.4
1.8 2.1 2.0 1.8 1.4 1.5 1.0 1.o 1.8 1.2 1.2 1.2 1.2
2 3 4
5 6
1170
Effluent
Lab
Environmental Science & Technology
Receiving waters CTAS
DAS
0.7 0.6 0.4 0.3 0.6 0.8 0.3 0.4 0.5 0.5 0.5 0.5
0.5 0.6 0.3 0.4 0.3 0.4 0.3 0.3 0.6 0.5 0.5 0.4
Table V. Nonionic Surfactant Concentrationa (ppm) of Sewage Treatment Plant Influent Lab
Llght rolldr
Heavy rolldr
1
4.92 4.98 5.16 4.18 3.90 3.76 4.88 5.13 5.53 4.94 4.94 5.10 5.09 5.52 5.37 4.89 f 0.53
6.30 6.24 6.42 5.88 4.26 3.96 5.19 5.08 5.17 6.30 5.50 5.74 6.96 7.03 6.96 5.79 f 0.91
2
3
4
5 A v f l
(r
9econd decimal figures are presented for statistical considerations only. In no way does it indicate the number of analyticallysignificant figures. One figure after the decimal point is recommended. The results of this investigation are presented in Table V. The interlab relative standard deviations were f l l %for the “light solids” solution and f 1 6 % for the “heavy solids” solution. The intralab relative standard deviation was better than f10% in almost all cases. Recovery of nonionics was 90% with four sublations. This observation is based upon an additional 10% recovery with two more sublations (a total of six). Results with this precision and accuracy are very acceptable for environmental monitoring and for biodegradation studies. The
concentration differences noted for “light” and “heavy” solids demonstrate the loss in CTAS that occurs if one filters a sample before analysis. Conclusions An improved analytical method for nonionic surfactants has been developed and is recommended for biodegradation and environmental studies. An improved sublation procedure and an ion-exchange step have expanded the method applicability to virtually any aqueous sample. The CTAS analysis was demonstrated to be a reasonable and acceptable alternative to the DAS analysis. The method possesses acceptable accuracy and precision and has the proven advantage of simplicity and applicability to biodegradation and environmental samples.
Literature Cited (1) SDA Scientific and Tech. Rep. No. 6. “The Status of the Biodegradability Testing of Nonion
