Environ. Sci. Technol. 1994, 28, 1278-1284
Identification and Measurement of Food and Cosmetic Dyes in a Municipal Wastewater Treatment Plant Anthony J. Borgerding and Ronald A. Hltes’
School of Public and Environmental Affairs and Department of Chemistry, Indiana University, Bloomington, Indiana 47405 Acid Blue 9, Acid Violet 17, Quinoline Yellow, Acid Red 51, Acid Red 87, and Acid Red 92 along with N-benzylN-ethylaniline sulfonic acid (BEASA), a synthetic precursor, were identified and measured in colored wastewater samples from a municipal treatment plant. Continuousflow fast-atom bombardment mass spectrometry was used to analyze BEASA. Liquid chromatography with ultraviolet detection was used to analyze the other dyes, but its lack of selectivity required prior isolation of the analytes from interfering compounds by solid-phase extraction onto Cl8 extraction disks and onto cartridges packed with strong anion-exchange resins. The xanthene dyes (Acid Red 51, 87, and 92) were found in low parts per billion (ppb) concentrations in the plant influent and were rapidly removed by adsorption to sludge. Acid Red 92 was found to be over 35 times more concentrated on secondary sludge than in the corresponding liquid samples, indicating high levels of accumulation. The other dyes and BEASA were found in hundred ppb concentrations in both the influent and the effluent of the plant, indicating a resistance to both degradation and removal by sorption.
fast-atom bombardment (FAB) from a glycerol matrix, but they do not exhibit such behavior when using such matrices as “magic bullet” (a 4:l mixture of dithiothreitol and dithioerythritol) and thioglycerol. The sensitivity of this method was poor: approximately 100 pg of dye was required for a good spectrum. Because of these difficulties, most analytical studies of food and cosmetic dyes have used liquid chromatography with ultraviolet or fluorescence detection (18-20). This paper reports on our studies of one particular municipal wastewater treatment plant that receives wastewater from a factory that produces xanthene, triarylmethine, and other food and cosmetic dyes. We studied the relative merits of both fast-atom bombardment mass spectrometry and liquid chromatography to identify nontarget analytes and to quantify them in wastewater samples. In addition, the behavior of these compounds in the treatment plant, including their partitioning between the aqueous and solid phases and their degradation, was investigated. Experimental Section
Introduction
Synthetic dyes are often present in wastewater a t concentrations sufficient to give the water a noticeable color. Unfortunately, modern techniques for analyzing organic compounds in water (such as gas chromatographic mass spectrometry) cannot be used for most of these dyes. They are simply too polar and nonvolatile to be measured by vapor phase techniques. Nevertheless, the environmental presence of dyes cannot be ignored. They are produced in large amounts [over lo8kg in 1990 alone ( I ) ] , and several are potentially toxic (2, 3). Most of the analytical chemistry of dyes has focused on azo dyes because they are the most widely used. Liquid chromatography with either ultraviolet (4,5 ) or mass spectrometric (6-13) detection has been used for the analysis of these dyes. Only two studies have measured dyes in ambient environmental samples (14,15). Tincher measured concentrations of 15 acid and disperse azo dyes in the Coosa River basin, which received the treated effluent from several carpet dyeing mills (14). More recently, Camp and Sturrock analyzed Reactive Blue 19 and its derivatives in textile mill wastewater (15). These two studies are unique; all others have analyzed environmental matrices spiked with known dyes. The analysis and environmental behavior of food and cosmetic dyes, such as triarylmethine and xanthene dyes, have received much less attention than azo dyes, despite their direct consumption by humans and despite their genotoxicity (16). The only mass spectrometry study of these dyes was that of Harada et al. (17), who determined that they undergo reduction reactions when ionized by
* E-mail address:
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Materials. HPLC-grade methanol and water were purchased from EM Science (Gibbstown, NJ). Glacial acetic acid and 2 M NaOH were purchased from Fisher Scientific (Fair Lawn, NJ). Hydrochloric acid solutions were made from concentrated HC1 (Fisher). All dyes were purchased from Aldrich Chemical Co. (Milwaukee, WI). Acid Orange 7, Acid Red 92, Acid Red 87, Acid Red 51, and the Quinoline Yellow dyes were used without purification. Acid Violet 17 and Acid Blue 9 were purified by recrystallization from methanol. N-Benzyl-N-ethylaniline sulfonic acid (BEASA) was synthesized by dropping 5 mL of fuming sulfuric acid (Mallinkrodt; Paris, KY) into 5 g of N-benzyl-N-ethylaniline (Aldrich) in 10 mL of hexane in an ice bath and stirring for 30 min. A total of 20 mL of water was added, and the organic fraction was separated and discarded. The aqueous fraction was diluted to 100 mL and divided into five parts, each of which was passed through an Empore Cle extraction disk (Varian; Harbor City, CA). The disks were washed with 20 mL of water to remove residual sulfuric acid, and the BEASA was extracted from the disks with 10 mL of methanol. The extracts were combined, and the BEASA was recrystallized from the methanol solution. Sampling. Samples were collected from the Mill Creek Wastewater Treatment Plant in Cincinnati, OH. Samples of plant influent and effluent were collected, as well as samples from the primary and secondary treatment basins and from the sludge digester. Returned activated sludge from the secondary treatment stage was also collected. The plant effluent is discharged into the Ohio River; thus, river water samples were collected approximately 100 m above and below the point of entry. The samples were collected in polypropylene bottles and immediately re0013-936X/94/0928-1278$04.50/0
@ 1994 American Chemical Society
frigerated. All samples were filtered through Whatman No. 1filter paper within 24 h of collection and extracted within 48 h. Sample Cleanup. Both the filtrates and the solids from the samples were analyzed. Liquid samples were measured using a volumetric flask and spiked with 50 pL of a 1mg/ mL solution of Acid Orange 7. This spiked solution was then passed through a 47-mm Empore C18 extraction disk, which had previously been washed with 20 mL of methanol and 20 mL of water. The analytes were extracted from the disk with 10mL of methanol and evaporated to dryness under a stream of nitrogen. For continuous-flow FAB mass spectrometry analysis, the extract was redissolved in 1 mL of methanol, passed through a 0.45-pm nylon syringe filter (Lida Manufacturing; Kenosha, WI), and analyzed without further cleanup. For liquid chromatographic analysis, the extract was redissolved in 10 mL of water and passed through a 500-mg strong anion-exchange (SAX) BondElut cartridge (Varian). The cartridge was rinsed sequentially with 10 mL of water and with 10 mL of 0.04M HC1. Analytes were extracted in two fractions, the first with 5 mL of 2 M HC1 and the remainder with 5 mL of concentrated HC1. The dyes in these fractions were recovered by passing them through another c18 disk, washing the disks with 20 mL of water, and eluting the analytes with 10 mL of methanol. This extract was evaporated to dryness, redissolved in 1mL of the liquid chromatographic mobile phase, filtered, and analyzed. Solids that were filtered from the samples were separated into three equal portions, each of which was spiked with 100 pL of 1mg/mL Acid Orange 7 and dried for 12 h in an oven at 100 "C. They were then cooled to room temperature and weighed. Analytes were extracted with 30 mL of methanol in a sonicating bath for 3 h. The solids were then filtered, and the filtrate was evaporated to dryness under a stream of nitrogen. The extracts were cleaned up in the same way as the extracts from the liquid samples, again depending on the technique used to analyze them. Liquid Chromatography. The liquid chromatography (LC) system consisted of two Model 6000A reciprocating pumps, which were controlled by a Model 660 solvent programmer (Waters Associates; Milford, MA). The detector was a Waters Model 440 absorbance detector set at 254 nm, which was connected to a Hewlett-Packard 3392A integrator (Avondale, PA). The analytes were separated on a Waters p-Bondapak column (3.9 x 300 mm) packed with 10-pm CU particles using gradient elution. The gradient was 70% mobile-phase A (pH 3 buffer) to 95% mobile-phase B (MeOH) over a 30-min time period at a flow rate of 1mL/min. The buffer solution was made by diluting 20 mL of glacial acetic acid to 1L and raising the pH to 3 with 2 N NaOH. Reproducibility was maintained by equilibrating the column with the initial mobile phase for 20 min prior to sample injection. Mass Spectrometry. A VG 30-250 triple quadrupole mass spectrometer with negative-ion continuous-flowFAB ionization was used (VG Analytical; Altrincham, U.K.). The commercialcontinuous-flowinterface and source were modified as previously described (21). Samples were injected using a Valco CI4W injector (Houston, TX) with a 0.5-pL valve into a flow of a carrier solution made up of methanol (50%), water (45%),and glycerol (5%),which was pumped at 5 pL/min by an Isco pLC-500 syringe pump (Lincoln, NE). Before use, the carrier solution was filtered
and degassed by passing it through a 0.45-pm nylon filter under vacuum and sonicating it for 30 min. For flow injection sample introduction, a 0.5-m fused silica tube with an inner diameter of 75 pm (PolymicroTechnologies, Phoenix, AZ) was directly attached to the valve using a capillary sleeve (Upchurch; Oak Harbor, WA) with a fingertight nut and ferrule. For on-line liquid chromatography/ mass spectrometry (LC/MS) experiments, a Fusica 320 pm X 10cmcapillary column (LC Packings, San Francisco) was attached to the valve. These LC experiments used isocratic elution with a mobile phase consisting of 50% methanol, 45 % pH 3 buffer, and 5 % glycerol. The probe tip was maintained at 50 "C. The FAB gun (Ion Tech; Teddington, UK) was set at 8 kV and 1mA. Xenon was used as the FAB gas. Quantitative analysis of BEASA was done by selected-ion monitoring of rnlz 290. For tandem mass spectrometry experiments, SFs at a pressure Torr was used as the collision gas. The collision of energy was set at 10 eV (laboratory frame of reference).
Results and Discussion Identification of Dyes from Municipal Wastewater. Sample extracts were qualitatively analyzed using negative-ion continuous-flow fast-atom bombardment (FAB) ionization, with the main peaks analyzed by product-ion scans using tandem mass spectrometry. The full-scan, negative-ion FAB spectra of extracts of the influent and effluent from the treatment plant are shown in Figure 1. The influent spectrum shows peaks corresponding to the C1o-C13 homologues of linear alkylbenzene sulfonates (LAS) at r n l z 297,311,325, and 339. LAS are present in most municipal wastewaters because they are popular commercial detergents. Their concentrations have previously been measured in municipal wastewater samples using FAB mass spectrometry and LC techniques (2124). Using a method previously developed in our laboratory (21),the LAS in this influent sample were estimated to have homologue concentrations ranging from 200 (C13) to 700 ppb (C11). The total LAS concentration in the influent was about 1700 ppb, which is well below the average found in a recent survey of treatment plants (22); this may be due to the high dilution of domestic wastewater by industrial wastewater. Abundant ions at rnlz 290 and 170 are present in the spectra of both the influent and the effluent samples. The product ion spectrum of the ion at rnlz 290 showed an abundant (about 90 % of rnlz 290) ion at rnlz 170 and less abundant (about 5 % ) ions at rnlz 80 and 260. Thus, we suspected that rnlz 290 and 170in the influent and effluent samples were from the same compound. The ion at mlz 80 in the product ion mass spectrum corresponds to a sulfonate group (SOa-), and it is highly indicative of sulfonated compounds. These compounds yield even electron anions in FAB, and the even mass of this compound (290 Da) indicated that it probably contained a single nitrogen atom. We had previously seen ions at r n l z 170 in the spectra of azo dyes (251, and we had attributed them to benzene sulfonate substituted either with a nitrogen or with a CH2 group. This latter ion is more stable and much more common (25). Assuming this partial structure, the rest of the molecule needed to have a single nitrogen atom and weigh 120 Da. This corresponded to a structure with four rings and double bonds, indicating another benzene ring. Including the nitrogen Environ. Sci. Technol., Vol. 28, No. 7, 1994
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mi2 Figure 1. Negative-ion, fast-atom bombardment mass spectra of extracts of a municipal wastewater treatment plant influent (top) and its effluent (bottom).
mlz
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BEASA rnlz 260
Flgure 2. Structure and mass spectrometry fragmentation scheme of BEASA.
and the benzene in the structure left 29 Da. Since the possibility of a second nitrogen had been eliminated, we hypothesized that this 29 Da corresponded to an ethyl group. Using this information, we thought that this compound might be N-benzyl-N-ethylaniline sulfonic acid (BEASA); see Figure 2. The structure of BEASA is also consistent with the loss of 30 from the molecular anion, corresponding to the loss of ethane and the reduction of the CN bond to form the ion at mlz 260; see Figure 2. After synthesizing this compound, we obtained its LC retention time and mass spectrum using capillary LC 1280 Envlron. Scl. Technol.. Vol. 28, No. 7, 1994
linked on-line to the continuous-flow FAB mass spectrometer system. Although only one LC peak was observed for the analysis of the standard, the exact position of the sulfonate group on the ring is unknown. In fact, the LC peak may be due to two or more unresolved isomers. The retention times of the BEASA standard and the unknown compound in the wastewater extract were the same, and the product ion spectra of mlz 290 from the BEASA standard and the unknown compound were also identical, further verifying the identification of this unknown compound. While our laboratory and others have determined structural characteristics of dyes and other target compounds using FAB and tandem mass spectrometry (25-29, this is a rare example of the identification of a nontarget, nonvolatile compound directly from an environmental matrix using these methods. BEASA is a precursor to several triarylmethine food dyes, which are produced industrially by the condensation of BEASA with sulfonated benzaldehyde (28). We analyzed several of these dyes using negative-ion continuous-flow FAB and found that they all fragmented in the ion source to form the ion at mlz 170, corresponding to the CHz-substituted benzene sulfonate ion; no molecular ions were observed. Thus, despite the deep color of the extract (which indicated a high concentration of dyes), no mass spectral peaks corresponding to the molecular anions of dyes were observed in the spectra of the wastewater extracts. There was only one company whose outflow entered the municipal wastewater treatment plant and who produced dyes based on BEASA. We obtained samples of wastewater from this company, and liquid chromatography analysis showed that many of the compounds in that sample had the same retention times as those in the municipal wastewater treatment plant, indicating that this company was the source. According to the literature from the dye industry (291,this company made two dyes based on BEASA, Acid Violet 17 and Acid Blue 9 (see Figure 3). We obtained these as well as several quinoline and xanthene dyes that were on the company’s production list (see Figure 3). Several of them had LC retention times that were the same as those in the wastewater samples. In addition, the colors of the collected LC fractions from the wastewater extracts were the same as those of the standards. Based on these qualitative hints, we developed a quantitative method for the analysis of these compounds. Analytical Strategy. Since none of these dyes gave an adequate response to be analyzed by FAB mass spectrometry, we chose to analyze them using LC with ultraviolet detection. While these compounds absorb in the visible range, they absorb more strongly in the UV; therefore, UV detection has been widely used for the sensitive detection of dyes (4, 5, 14). However, because this method of detection is relatively nonselective, we needed an extensive cleanup of the wastewater samples to remove interfering compounds. The analytes possess both ionic and nonionic characteristics, and these features were used in isolating them from other compounds that might interfere with the LC analysis. For the liquid samples, we initially attempted to use a two-step process: First, nonionic compounds were removed by passing the sample through strong anion-exchange (SAX) cartridges, trapping the analytes and other ionic compounds. After elution of the trapped compounds with HCI, analytes were isolated from inorganic ions by extracting them onto CIS
S03Na
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Acid Blua Q
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CI 42650
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Quinoline Yellow,
disulfonata
trisulfonate
CI 47005
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0
NaO
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Er
A c i d Red 51
A c i d Red 07
A c i d Red 92
C I 45380
CI 45430
C I 45410
I
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Br
Figure 3. Structures of the dyes identified from a colored municipal wastewater and their Color lndex numbers.
Table 1. Recovery of Spiked Analytes from 250 mL of Plant Influent Calculated for Different Cleanup Methodsa SAX/ Cis
CIS/ CidSAXt~ciwash)/ SAXICis C18 69h8 63 f 7 31f6 BEASA 95 f 6 46h7 94f5 Acid Blue 9 (AB 9) 96 i 5 33 f 7 97 f 5 Acid Violet 17 (AV 17) 93 f 6 Quinoline Yellow A (QY,) 37 h 9 97 i 6 95 f 7 Quinoline Yellow B (QYb) 39 h 10 96 f 6 93 f 5 88h5 94h5 Acid Red 51 (AR 51) 98 f 5 95h6 95h4 Acid Red 87 (AR 87) 92 f 6 Acid Red 92 (AR 92) 90h 10 93f5 0 All values in percent recovery. Error is the standard deviation based on three measurements.
disks, washing the residual acid and other anions off with water, and extracting the analytes with methanol. Using this method, the recovery of dyes from standard solutions was good, but the recovery from real samples was poor; see the second column of Table 1. We suspected that the problem was in the SAX extraction step. Competition for sites on the SAX material between the analytes and the large amounts of other anions present in the aqueous samples caused the analytes to be incompletely retained. Therefore, removal of the interfering anions was
done using CIS extraction disks before extraction with the SAX cartridges. A final extraction onto Cl8 disks served mainly to remove the residual acid left over from the elution from the SAX cartridges. Recoveries of the dyes from influent samples using this cleanup technique were acceptable, ranging from 93 to 97 % ’ ;see the third column of Table 1. Use of this three-step method actually speeds up the cleanup process. Disks can extract larger volumes of liquid more quickly than cartridges, so extracting the sample onto a disk in the first step saves time. Typically, the complete extraction and cleanup of a 250-mL filtered sample using this three-step method took less than 1 h. While this method was rapid and selective for organic compounds with ionic character, this level of selectivity was not always sufficient to isolate the dyes for quantitative analysis by LC with UV detection. In addition to the dyes, other compounds that are extracted by this method include naturally occurring compounds, such as humic acids and other biological materials, and many types of synthetic anionic surfactants. All of these compounds are found in wastewater samples. We attempted to separate these compounds from the dyes by collecting different fractions in the SAX extraction step. After extracting the sample and rinsing the cartridge with water, 10mL of 0.04 Envlron. Sci. Technol., Vol. 28. No. 7, 1994
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LC retention time (min) Figure 4. Liquid chromatogramsof a mlxture of standard compounds and of influent samples extracted by the following techniques: CIS disks only, C18-SAX (H20rInse)-Cl8, and C18-SAX (0.04 M HCI rinse)CI8. The compound abbreviations are given in Table 1. The chromatogramswere digitally reconstructed.A 0 7 is Acid Orange 7, added as an internal standard.
M HC1 was passed through the cartridge. As shown in Table 1(last column),this additional rinsing did not affect the recovery of the analytes. Figure 4 shows chromatograms of a mixture of standard compounds and of treatment plant influent extracted by using CISdisks alone, CISfollowed by SAX cartridges rinsed with water, and CIS followed by SAX with the 0.04 M HC1 rinse. Note that interfering compounds were removed in each step. The dyes were eluted from the SAX cartridges in two fractions. Five milliliters of 2 M HC1 eluted all but the xanthene dyes, which were eluted with 5 mL of concentrated HC1. The two fractions were combined after the final CISstep. This was done to avoid possible reactions that could occur with the concentrated HC1. Nevertheless, since concentrated HC1was required to elute the xanthene dyes, such reactions could not be prevented completely; Acid Red 92 reacted to give a product that had a slightly lower retention time. However, the reaction was quantitative, allowing us to calculate the concentration of the original dye from the response of its derivatized product. 1282
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Quantitation by LC was based on internal standards. We chose Acid Orange 7 as the internal standard because it is commercially available in high purity, it is quantitatively extracted by the cleanup method, and it does not coelute with any other compounds in the samples analyzed, Response factors between each analyte and Acid Orange 7 were calculated according to their relative peak areas in standard solutions that had been carried through the threestep cleanup process. This was done for solutions containing 10,100, and 1000 ppb of each dye to assure that the response was linear over this concentration range. Concentrations of each dye in the extract were calculated by comparing their LC peak area to that of the internal standard and divided by the volume of the sample. The limit of detection of this method is better than 1ppb based on 250-mL samples. While the extensive cleanup process worked well for isolating the dyes from potentially interfering compounds in the sample, it also removed most of the BEASA, as indicated in Table 1. Fortunately, this analyte responded well to mass spectrometric analysis. Since this method of analysis is very selective, the cleanup process was not required, and the BEASA was analyzed after only a single preconcentration step using CISdisks. Using this method, the recovery of BEASA from the sample was better than 95 % . Cleanup time was reduced since only one extraction was required, and instrumental analysis time was reduced to less than 4 min since no chromatographic separation was necessary. BEASA was quantitated by taking the area of the flowinjection peaks and calculating the mass of BEASA to which that area corresponded from a calibration curve. The equation of this curve was calculated from data obtained for triplicate injections of BEASA standards over a range from 5 to 500 ng. A second set of standards incorporating an equal amount of LAS was used to see if the calibration curve changed due to differences in ionization efficiency in an impure sample; no differences were noted. All calibration curves showed correlation coefficients of at least 0.98 and were verified at the end of each day. The limit of detection of this technique was better than 5 ng injected, which corresponded to an absolute sensitivity of 40 ppb in a 250-mL sample. Behavior of Analytes in Municipal Wastewater Treatment Plant. The treatment facility from which samples were taken is a standard aerobic sludge plant. Approximately 4.5 X 108 L/day of raw sewage is passed through a three-step screening process to the primary treatment tanks, where greases are skimmed from the surface and solids (primary sludge) are allowed to settle. The average retention time for wastewater in this stage is about 2 h. From there, the wastewater flows to the secondary treatment tanks for removal of fine colloidal and soluble organic materials by aerobic biodegradation. The average retention time in this stage is about 6 h. Solids from this process (secondary sludge), consisting mainly of microorganisms, are separated from the wastewater by a clarifier, after which some of the sludge is recycled back to the secondary treatment tanks (returned activated sludge). The remaining secondary sludge and the settled primary sludge are sent to an anaerobic digester prior to disposal by incineration. The treated wastewater is chlorinated and discharged into the Ohio River. Using the LC technique for the dyes and the continuousflow FAB method for BEASA, we measured concentrations
Table 2. Concentration of Analytes from Samples Taken from Wastewater Treatment Plant and Factory Waste Samples. AX 92 AR 87 AR 51 AV 17 BEASA AB 9 QY. 0.35 t 0.04 0.39 f 0.05 0.43 f 0.05 4.7 0.5 8.3 0.7 12 f 1.1 6.3 t 0.8 7.8 f 0.8 factory (ppm) 2.2 1.1 1.9 f 0.9 2.0 1.1 100 20 160 i74 320 f 80 520 f 66 290 f 25 plant influent 39 i 4.2 6.1 i 0.8 8.5 1 . 3 nd nd nd 63 f 17 33 2 primary sludge 78* 10 5.7 1.1 13 2.2 nd nd 140 f 29 nd 15 2 secondary sludge nd nd nd 32f7 100 f 14 250 28 490 72 140 f 13 secondary filtrate nd nd nd 23 3 44 f 8 230 16 530 60 44 f 2.4 effluent 26 2.9 2.3 f 0.4 3.0 0.7 nd nd 38i5 nd 5.2 f 0.5 returned sludge 3.7 A 0.7 1.1 f 0.2 nd nd nd 4.0 0.6 nd 1.0 f 0.3 anaerobic digester
* *
*
*
*
* * *
*
*
* *
*
*
*
a Units are in ppm for factory waste, ppb for all other liquids and for the primary and secondary sludges, which were corrected to the volume of liquid from which they were filtered, and pg/g for returned sludge and solids from the digester. Error is the standard deviation of three measurements. nd is not detected.
Table 3. Concentration of Analytes in Various Samples Calculated Relative to Concentration in Treatment Plant Influent AV 17 QY. QYb AR 51 AR 87 AR 92 BEASA AB 9 primary sludge secondary sludge secondary filtrate effluent
0.11 0.05 0.50 0.15
0 0 0.94 1.02
0.20 0.44 0.78 0.73
(in triplicate) of these compounds in liquid samples and in the associated solids. The average concentrations are given in Table 2 for the various locations in the municipal treatment plant and for the wastewater sampled directly from the dye production facility. Note that these values represent a single day only. Concentrationsof the analytes in the wastewater treatment plant are dependent on the relative volume of wastewater discharged by the source. In addition, the relative amounts of each analyte being discharged may change rapidly. Thus, the relative concentrations of dyes in the factory wastewater may not be the same as the relative concentrations of samples taken on the same day in the treatment plant. We were concerned about degradation of the samples between sampling and extraction. Therefore, in addition to the samples we analyzed immediately upon return to the lab, we analyzed a second set after storing them in a refrigerator for 2 weeks. In all cases, this second measurement fell within the standard deviation of the first set of measurements, indicating that these compounds had not degraded. Table 3 shows the relative concentrations of the eight dyes in the primary and secondary sludges, the secondary liquid, and the effluent; the concentrations have been normalized to the influent concentration, set to unity in all cases. The last row in this table shows that Acid Blue 9 and Acid Violet 17 are present in the effluent at concentrations similar to those in the influent (102 and 73 % , respectively) and that a large fraction of BEASA and the Quinoline Yellow dyes was also present even after treatment (15,28,and 2 3 % ,respectively). Clearly, aerobic sludge treatment does not efficiently degrade these five dyes, a finding which is in agreement with previous studies (1, 30-32). The less ionic xanthene dyes (Acid Red 51,87, and 92) are completelyremoved from the wastewater. Conversely, there are high levels of these dyes in the sludge samples; typically about three to four times the influent for Acid Red 51 and 87 and about 25 times for Acid Red 92 (see last three columns of Table 3). This indicates that these compounds are sorbed to the solid sludge in the systemand are not necessarily degraded. Most of the xanthene dyes
0 0 0.63 0.28
0 0 0.32 0.23
3.0 4.6 0 0
3.2 3.0 0 0
18 36 0 0
were sorbed to solids in the wastewater upon entry into the plant; note that the relative concentrations of the xanthene dyes are much greater than 1 for the primary sludge samples (see the first row of Table 3). The high relative concentration in the secondary sludge might indicate a very high concentration of these dyes from a previous influent, which would allow some of the dye to reach this basin, but more likely this indicates accumulation of these compounds over a longer time period. The dyes partition between the water and the solid sludge to various degrees. Note that the xanthene dyes were found exclusively on the sludge, but that Acid Blue 9 and the Quinoline Yellow dyes were found exclusively in liquid samples. BEASA and Acid Violet 17 are present in both phases (see Table 3). Partitioning between the solid and liquid phases may be dependent on ionic interactions between the dyes and sludge or on simple hydrophobic equilibrium processes. Shaul et al. (31)have studied sorption of sulfonated azo dyes to sludge; they found a general relationship between hydrophobicity and sorption. Hitz et al. (32)noted the same trend, but they found an apparent deviation: Direct dyes were highly sorbed even when multiple sulfonate groups were present; no specific explanation for this behavior was given. Our data also indicate that the hydrophobicity of the dye is the most important parameter controlling its sorption. For example, xanthene dyes, which possess a single weak19 acid carboxylate group (PKa about 4.5) and are otherwise hydrophobic, are completely sorbed to the sludge. Conversely, Acid Blue 9 remains completely in the aqueous phase because it contains a highly acidic (pKa about 1.5) sulfonate group on each of the three aryl groups of the molecule which eliminates most of this molecule’s lipophilic character. BEASA and Acid Violet 17 partition more equally between the liquids and the solids. Both contain sulfonate groups, making then somewhat hydrophilic, but both also have lipophilic branches without sulfonates, causing them to sorb to the sludge. The ultimate fate of these compounds depends on whether or not they sorb to the solids in the wastewatertreatment process. BEASA, Acid Blue 9, Acid Violet 17, and the Quinoline Yellow dyes are discharged into the Envlron. Sci. Technol., Vol. 28, No. 7, 1994 1283
Ohio River with the plant effluent. We analyzed river water 100 m downstream from the discharge point and were unable to detect any of these compounds, most likely because dilution lowered concentrations below the detection limits of the method. The compounds that are removed from the wastewater by sorption to sludge in the plant show evidence of accumulation on these solids. For example, despite the very low concentrations of the xanthene dyes in the secondary treatment tank, the concentrations of these compounds on the secondary sludge are relatively high (see the fourth and fifth rows of Table 2). This indicates that the direct source of these compounds in the sludge is not the wastewater but rather the recycled sludge, which has high concentrations of these compounds (see the seventh row of Table 2). The most obvious example of this is Acid Red 92, which has accumulated in secondary sludge to a level over 35 times higher than the level that would exist if all of the dye in the influent were sorbed (see the second row of Table 3). This particular dye contains four additional chlorines compared to the other xanthene dyes, making it even more hydrophobic and thus more likely to accumulate on sludge. These very high levels of accumulation indicate the possibility of ionic or covalent interactions between the dye and the sludge. Most of the compounds sorbed to solids end up in the anaerobic digesters, and we detected these compounds in solids sampled from there (see the last row of Table 2). However, we were only able to obtain a composite sample of the 12 stages of this digestion process; thus, we cannot unequivocallysay whether or not the analytes are degraded in this process. If not, they are eventually incinerated with the digested sludge.
Conclusions This study used continuous-flow FAB mass spectrometry and LC analysis with ultraviolet detection for measuring in actual wastewater samples a group of compounds with a wide range of hydrophobicities. The mass spectrometric method was selective,which minimized cleanup requirements, and instrumentally rapid since no time was required for chromatographic separation. However, this technique often was insensitive for certain compounds. Of the analytes we identified in our wastewater samples, only BEASA responded well to FAB analysis. A much broader range of compounds could be analyzed by LC with UV detection, making it more applicable to the analysis of large groups of compounds. However, this nonselective method required extensive cleanup of the wastewater samples for the removal of interfering compounds. Our data showed that sorption is much more important than degradation for removing some dyes from wastewater in the municipal treatment plant. Compounds such as Acid Blue 9, which have high ionic character, are not removed from the wastewater at all. Conversely, compounds that are weaker anions, such as the xanthene dyes, are removed entirely and remain attached to the sludge in the plant at least until the sludge is treated in the anaerobic digesters.
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Acknowledgments We thank Amy Minichillo and Chris Hall of the
Cincinnati Metropolitan Sewer District for cooperation and helpful discussions. This work was funded by the U.S.Department of Energy (Grant 87ER-60530). Literature Cited Text. Chem. Color. 1992,24,6. Zollinger, H.Color Chemistry, Synthesis, Properties and Applications of Organic Dyes and Pigments; VCH; New York, 1987;pp 323-324.
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Abstract published in Advance ACS Abstracts, May 15, 1994.
