(32) Scott, W. D., Hobba, P. V., J . Atmos. Sci., 24,54 (1967). (33) Lindauer, G. S., Castleman, A. W., Jr., Aerosol sei., 2,85 (1971); Nucl. Sci. Eng., 43,212 (1971). (34) Kiang, C. S., Stauffer, D., Mohnen, V. A., Bricard, J., Atmos. Enuiron., 7, 1279 (1973). (35) Timmons, R. B., LeFebre, H. F., Hollinden, G. A., "Reactions of Sulfur Dioxide of Possible Atmospheric Significance", Proc. Symp. on Chemistry Reaction in Urban Atmospheres, Warren, Mich., 1969, C. S. Tuesday, Ed., pp 159-90, Elsevier, New York, N.Y., 1971. (36) Hales, J. M., Sutter, S. L., Atmos. Enuiron., 7,997 (1973).
(37) Murray, F. W., J . Appl. Meteorol., 6, 203-4 (1967). (38) Brown, R., Brookhaven National Lab, Upton, N.Y., private communication, 1975. (39) Evans, V. A., Benkovitz, C. M., Brookhaven National Lab, Upton, N.Y., Dedicated Data Base, 1975. (40) Tanner, R., Newman, L., J . Air Pollut. Control Assoc., 26,737 (1976).
Receiued f o r review March 25, 1977. Accepted September 19, 1977. Research performed under Contract No. EY-76-C-02-0016 with the U.S. Energy Research and Deuelopment Administration.
Preservation of Phenolic Compounds in Wastewaters Mark J. Carter' National Enforcement Investigations Center, Environmental Protection Agency, Box 25227, Building 53, Denver Federal Center, Denver, Colo. 80225
Madeliene T. Huston Central Regional Laboratory, Environmental Protection Agency, 1819 W. Pershing Road, Chicago, 111. 60609
Copper sulfate and phosphoric acid with sample storage a t 4 "C is a common preservative technique used for phenolic compounds in wastewaters. However, there are no data showing its effectiveness. A study was conducted to compare the preservation method with the addition of strong base or acid and sample storage a t 25 and 4 "C. The addition of 2 mL conc H&304/L with sample storage a t 4 "C was most consistently effective in preserving stability for 3-4 weeks. However, the other chemical preservatives were effective for a t least 8 days. Substantial loss of phenolic compounds rapidly occurred in all samples unless the chemical preservative was added immediately after sample collection. A positive correlation was found between loss of phenolic compounds and microbiological activity, suggesting the latter is the dominant factor in determining sample stability. The 1972 Amendments to the Clean Water Act have resulted in limitations on the concentration and loading of pollutants that can be discharged by industries and municipalities ( I ) . The need to monitor these discharges has substantially increased the number of environmental samples requiring analysis, especially for toxic substances such as phenolic compounds. Kelly has reviewed the literature for methods of analyses of phenolic compounds in wastewaters ( 2 ) .The most common methods are the 4-aminoantipyrine (4-AAP) (3-6) and 3methyl-2-benzothiazolinonehydrazone (MBTH) (7, 8) colorimetric procedures and the ultraviolet bathochromic shift method (9).The difficulty and equipment requirements for these methods often result in samples being shipped to a large centralized laboratory for analysis. The shipping process can lead to substantial delays between sample collection and analysis. As a result, the effectiveness of the sample preservation technique will substantially affect the accuracy of the data. I t is also necessary to consider sample stability during the collection process, especially if the 24-h composite method is used ( I O ) . Very little data exist in the literature to give guidance about the best method to stabilize phenolic compounds in wastewaters. The current recommended technique ( I 1) is a modification of work performed over 30 years ago (12). Ettinger e t al. found that copper sulfate effectively preserved river water and river water seeded with sewage for 2-4 days when
the samples were stored a t 25 "C. Subsequent to the work of Ettinger et al., phosphoric acid was added to the copper sulfate preservative to keep the metal ions in solution when added to alkaline samples (11).In addition, it was recommended that all samples be stored a t 4 OC until analysis. No data were presented until 1974 about the effectiveness of the combined phosphoric acid, copper sulfate, 4 "C storage technique for preserving phenolic compounds in water samples. Afghan e t al. showed that either strong acid or base was more effective in retarding bacterial activity and stabilizing phenol in Great Lakes' waters than the combined copper sulfate preservative ( 4 ) . The observation of Afghan et al. raises doubt that the copper sulfate-phosphoric acid preservation method is best for stabilizing phenolic compounds in wastewaters. Therefore, a study was undertaken to determine the most effective and practical preservation method and maximum allowable holding time for phenolic compounds in wastewaters.
Methods Preparation of Samples. Fresh samples were collected in 5-gal high-density polyethylene jugs and immediately brought to the laboratory. The water samples were homogenized with a Tekmar Super Dispax system, preserved and split into 250-mL high-density polyethylene bottles. Some samples were spiked with phenol to raise the starting concentration to a level that could be accurately measured. The samples were preserved and stored as described in Figures 1-4. Chemical Analysis of Samples. The first analysis of each sample was completed within 2 h of sample collection. All samples, standards, and blanks were distilled from acidic solution to separate phenolic compounds from potential interferences (13).The distillates were analyzed by an automated version of the 4-aminoantipyrine method shown in Figure 5. The buffered potassium ferricyanide reagent was prepared by adding 2.0 g potassium ferricyanide, 2.1 g boric acid, 3.75 g potassium chloride, 44 mL of 1 N sodium hydroxide, and 0.5 mL Brij-35 (Technicon Corp. No. T21-0110) to a volumetric flask and diluting to 1 L. The 4-aminoantipyrine reagent was prepared by diluting 0.65 g of the chemical to 1 L. Both reagents were filtered through a 0.45-hm membrane filter before use.
This article not subject to U S . Copyright. Published 1978 American Chemical Society
Volume 12, Number 3, March 1978
309
Two control standards were prepared and preserved with copper sulfate and phosphoric acid by an independent analyst a t the beginning of each study to check on the consistency of the day-to-day standard preparation and instrument calibration. These samples were preserved with copper sulfatephosphoric acid and stored a t 4 "C. Standard Plate Count. Plate count agar was prepared fresh just before use, added to petri dishes, and 1,0.1,and 0.01 mL of each sample were plated in triplicate (13). All samples were incubated a t 35 "C for 24 h. Only those plates having 30-300 colonies were considered valid, and the values reported in Table I are an average of the three replicate dilutions.
Results and Discussion The stability of phenolic compounds in nonwastewater aqueous solutions has been studied by several investigators. Phenolic compounds are good preservatives themselves a t high concentrations (>0.5%)but are readily biodegraded a t lower concentrations (14-1 7). Chambers and Kabler found no detectable nonbiological degradation (15).Extremes in pH (18-22), temperature (23-26), and the use of toxic chemicals (27-29) have been used to reduce microbiological activity in aqueous solution. Strong base (4,30,31),acid ( 4 ) ,and copper sulfate-phosphoric acid (11, 12) in combination with temperature control have been used to stabilize phenolic compounds in surface waters. The stability of phenolics in three different wastewaters preserved with copper sulfate-phosphoric acid and stored a t 4 "C was studied first. The results are shown in Figure 1.The raw sewage was fairly weak with a biochemical oxygen demand (BOD) of only 95 mg/L, and the treated sewage sample was collected after secondary biological treatment but before
1
1
0
I
A
(10
I20
v
u
DAYS
Figure 2. Plot of stability of phenolic compounds in several wastewaters with time; Study 2 All samples preserved with 1.0 g CuS04*5H20/L, pH adjusted to 4.0 with phosphoric acid, and stored at 4 " C . industrial waste, raw and treated sewage samples spiked with phenol to bring their initial concentrations to 110, 165, and 110 pg/L, respectively
Raw Sewage
A \A -A -
O
'"1
Raw Sewage
d A'A-
I
AdA'
I
-A\A -A.
I
LA Treated Sewage
A
04
8 'n
j
F -ATA- 5? . A '
Industrial Wast:
15
XI
'25
DAYS
Figure I . Plot of stability of phenolic compounds in several wastewaters with time; Study 1 All samples with points plotted as "8" preserved with 1.0 g CuS04.5H20/L. pH brought to 4.0 with phosphoric acid, and then stored at 4 O C . Samples plotted as "A" stored at 4 O C with no chemical preservatives. Both Industrial waste, raw and treated sewage samples spiked with phenol to bring their initial concentrations to 50, 100, and 60 pg/L, respectively
310
Environmental Science & Technology
Figure 3. Plot of stability of phenolic compounds in raw sewage sample preserved with several chemicals: Study 3 Aliquots 1 and 2 preserved with copper sulfate and phosphoric acid and stored at 25 and 4 O C . respectively. Aiiquots 3 and 4 preserved with 2 mL conc H2S04/L and stored at 25 and 4 O C , respectively. Aliquot 5 preserved with 2 rnL 10 N NaOH/L and stored at 4 O C . Aliquots 1-4 spiked with phenol to bring their initial concentrations to 125 kg/L. Aliquot 5 spiked with phenol to bring its initial concentration to 130 pg/L
chlorination. The industrial wastewater was collected from the Grand Calumet River, which is essentially a composite from the South Chicago industrial area. Since the samples chosen for study often had low background concentrations of phenolics, each was spiked with phenol as needed so that changes in phenolic concentration would be easier to determine. Phenol was chosen for the spike because Kaplin et al. found phenol to be the least stable of all the phenolic compounds in natural waters (32-35). The most important result of study 1 was the rapid loss of phenolics from the samples a t 4 "C with no addition of any
Control A
1-1-
1
'20
I10
'30
DAYS
Figure 4. Plot of stability of phenolic compounds in raw sewage sample
preserved with several concentrations of sulfuric acid Aliquots 3 and 5 preserved with 2 mL conc HzS04/Land stored at 25 and 4 O C , respectively. Aliquot 4 preserved with 4 mL conc H2SO4/Land stored at 25 OC. All samples spiked with phenol to bring their initial concentration to 130 wg/L
chemical preservative. The percentage loss of phenolics within 24 h for the industrial waste, raw, and treated sewage samples was 85,80, and 4004 respectively. Ettinger et al. reported that an unpreserved river water sample stored at 2 "C lost only 15% of the original phenolic content after 4 days (12). However, the same sample stored a t 25 "C lost all of its phenolic compounds within 2 days. The results from these two studies show that the loss of phenolics from unpreserved samples is variable depending upon sample type, but significant in all cases. The expected precision of sample analysis was determined from daily analysis of the control A and B samples to be f 1 2 pg/L (2 c). There was no statistically significant change in phenolic concentration over the 22-day study for the samples preserved with copper sulfate-phosphoric acid and stored a t 4 OC. However, there were large day-to-day changes in the concentration of phenolics measured. This poor precision was determined to be caused by the problem in taking a representative sample for analysis due to the presence of particulate matter. The problem was solved in later studies by homogenizing all samples before analysis. In the second study (Figure 2), the effectiveness of the combined copper sulfate-phosphoric acid preservative was studied vs. sample type. Activated sludge was added to raw sewage to create a sample organically rich and biologically active. This sample was stable for 12 days, but degraded to 85% of the original phenol concentration after 33 days. The other samples were stable for the duration of the study. The dip in all values on day 1 of the study was attributed to improper calibration. Baylis reported the use of 1.2 mL 1 N NaOH/L of sample to preserve phenolic compounds in potable water samples (30). However, Ettinger et al. found Baylis' procedure to be ineffective for sewage-seeded stream samples (12).Kaplin and Frenko found that a hundredfold increase in base concentration was effective for preserving stream waters (31).Afghan et al. verified the effectiveness of the higher concentration of base for preserving lake waters ( 4 ) .Afghan et al. also showed that 0.1 M HC1 was an effective preservative. The effectiveness of strong base or acid in preserving phenolic compounds was compared with copper sulfate in the third study (Figure 3). The concentration of phenolic compounds was stable in the raw sewage sample studied when stored a t 4 "C regardless of the preservative used. However, the sulfuric acid and copper sulfate preserved samples dete-
Table 1. Effectiveness of Preservatives in Sterilizing Sewage as Indicated by Total Plate Counts
&J
505 nm Yi m m FIC X 1 5 m m ID
Sampler IV
+Waste
20 Turns
10 321 Air
I
116-M89-01 10.231A-Arninoantipyrine
10.231 Potass~urnFerricyanide to FIC lube IL,
11 W1 From FIC
Preservation method, a raw sewage
4OC 2 mL
4
O C
mL conc H2S04, 25 O c mL conc H 2 S 0 4 . 4 O C 4 mL conc H2SO4, 25 O C CuS04,H3P04, 4 O C 2 mL 10 N NaOH, 4 O C 10 mL 10 N NaOH, 4 O C
Automated phenol manifold diagram
Numbers in parentheses correspond to flow rate of pumptubes in mL/min. Numbers adjacent to glass coils and fittings are Technicon Corp. part numbers
>>>30 000
>>>30 000
>>>30 000 2 200 200
conc H2S04, 25 O C 730 3 500 2 mL conc H2S04,4 O C ... 70 4 mL conc H2SO4,25 O C 560 40 CuS04,H3P04, 4 O C 6 300 800 2 mL 10 N NaOH, 4 O C 28 000 110 10 mL 10 N NaOH, 4 O C 230 90 Secondary treated sewage before chlorination 2 2
Figure 5.
Total plate count. colonleslmL Day O D Day 8 Day 20
23000
