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THE F A T E OF PHENOLICS IN WASTEWATER -

D E T E R M I N A T I O N BY DIRECT-INJECTION GLC
AND WARBURG RESPIROMETRY
R. B. BAIRD, C. L. KUO, J. S. SHAPIRO,and W. A. YANKO
The C o u n t y Sanitation Districts o f
Los Angeles County,
San Jose Creek Water Quality Laboratory
1965 South Workman Mill R o a d
Whitt~er, California 90601

The effects of phenol, cresol isomers, and chlorinated phenols on the respiration
of activated sludges have been studied using Warburg techniques. The bio-
degradability of these materials under simulated treatment conditions in activated
sludge was also determined. It was found that less than I0 mg/liter concentrations
of phenol and chlorophenols, while being biodegraded, proved toxic to sludges
unacclimated to phenolics. The toxic effects of the cresols were not so severe for
the sludges tested.
Phenolic analysis in these studies was accomplished employing GLC techniques
with a new column packing, dinonylphthalate on Chromosorb G. This chromato-
graphic technique was shown to be effective in rapidly identifying phenolic
materials in activated sludge, treatment plant effluents, and industrial discharges:
the separation of all isomers tested, except m- and p-chlorophenol, was possible
using this technique.

Introduction
The contamination of natural and wastewater by phenolic materials results from a
number of sources, including petroleum and coking industry discharge, chemical spills,
biological degradation of wastes, and agricultural run-off. The presence of these materials,
even in trace amounts, presents a variety of problems in the environment, the most
notable of which are the abnormal taste and odor characteristics imparted to the water
(Hoak 1956). In addition, the toxicity of some phenolics can be a problem in the
domestic use of surface waters, or may be the cause of upsets of biological waste
treatment processes.

The control of phenolic pollution requires the ability to identify and measure
individual phenols in aqueous systems. The classic technique employed in phenol
analysis is the 4-aminoantipyrine colorimetric procedure (Dannis 1951; Ettinger e t al.
1951; Mohler and Jacob 1957). However, it is widely known that this method is both
non-specific and insensitive to some common phenolics (Taras e t al. ed. 1971), thus
inadequate in the control of phenolic contamination of waters.

The use of gas-liquid chromatography (GLC) in the detection of various phenols has
been reviewed and studied extensively (Baker and Malo 1967): a variety of steric-type

Archives of Environmental Contamination 165


and Toxicology,Vol. 2, No. 2, 1974, © 1974
by Springer--Vertag New York Inc.
166 R.B. Baird et al.

substrates on different solid supports was evaluated for suitability in the direct injection
GLC determination of phenolics in aqueous systems. Comparative studies with the
aminoantipyrine method have also been performed using direct injection techniques
(Baker 1966). Of the then available materials tested, Carbowax 20 M and FFAP sub-
strates were recommended on Chromosorb T; these mixtures, while yielding sharp peaks,
minimizing tailing, and being more selective than any colorimetric procedure, could not
separate phenol and o-cresol. No recovery' data were given. The isolation and concentration
of phenolic contaminants has been accomplished by freeze concentration (Baker 1965)
or carbon-chloroform extraction (Eichelberger e t at. 1970) with the ultimate analysis
being performed by GLC techniques employing either electron capture (EC) or flame
ionization detectors (FID). Although the sensitivity of the former was excellent, the
techniques appeared to lack good precision, and the recovery of some constituents was
poor. The extraction techniques suffer from possible loss of volatile phenolics, possible
chemical alteration of the substances, and selective adsorption-desorption on the carbon
filter.

"Standard Methods for the Examination of Water and Waste-water" (Taras et al. ed.
1971) describes a direct injection GLC technique for the determination of phenols based
upon the method of Baker (1966). The column packings employed in this method suffer
from the inability to separate many of the phenolics tested, notably, phenol and o-cresol,
as well as exhibiting excessive retention times for the polychlorinated phenols. It became
desirable, therefore, to develop a rapid direct-injection method for separating and
quantitating a series of common alkyl- and chloro-phenols in addition to phenol itself.

This paper describes the studies of the fate of various phenolic materials in an aerobic
biological treatment system as well as a rapid direct-injection GLC method for the
identification of different phenolic materials commonly found in industrial and domestic
wastes.

Experimental
Warburg respirometry. Toxicity studies. Conventional Warburg bioassay techniques
(Umbreit et al. 1949) employing activated sludge samples dosed with various concentra-
tions of phenolic standards were used to determine the toxic effects of these materials on
the respiration of organisms found in typical activated sludges of secondary treatment
plants accepting combined domestic and industrial discharges. An 18-unit Precision
Scientific Warburg apparatus was employed for all runs at a constant temperature of
25°C. Nine pairs of manual double capillary manometers (PS66665), connected to
calibrated 50-ml Pyrex reaction vessels having centerwells and one sidearm each
~$66697) were employed in each run. M1 samples were set up and run in duplicate for
each experiment. Fluted, KOH-soaked filter papers were used for CO2 absorption in the
centerwell of each vessel. In each case 3.0 ml return sludge samples were tested. Return
sludges were 24-hour composites taken from either of two treatment facilities. Endogen-
ous respiration was measured during each run with the addition of 3.0 ml of distilled
water to the seed. Sludge samples were dosed with 3.0 ml of phenolic standards to
contain final concentrations of 1.0, 10.0, and 100 rag/liter of the toxic medium. Phenol,
The Fate of Phenolics in Wastewater 167

o-cresol, m-cresol, p-cresol, o-chlorophenol, and p-chlorophenol standards were prepared


from the Eastman Organic chemicals, without further purification, by dilution in phenol-
free Pyrex-distilled water. Thermobars were run using 6.0 ml of distilled water for each
run. Microliters of oxygen uptake (/21 02) was measured for each unit every 20 minutes
for six-hour runs. It had previously been demonstrated that oxygen uptake readings on
this unit were correct with a relative precision of -+ four percent. Microliters of oxygen
per mg of volatile suspended solids (return sludge) was plotted versus time for each set of
flasks. These curves were compared to the endogenous respiration curves for each batch of
sludge to graphically compare the toxic effects of each phenolic.

Biodegradation studies. The biodegradability of the aforementioned phenolic materials


was determined by dosing activated sludge samples as described above. The initial con-
centrations, and three- and six-hour concentrations at the t.0, 10.0, and 100 rag/liter
level of each phenolic standard were determined by the direct injection GLC techniques
described below. Biodegradability was expressed in terms of the amount of phenolic con-
stituent lost in six hours. To demonstrate that the degradation was bacterial, an activated
sludge sample was sterilized by autoclave and spiked with 10.0 rag/liter of phenol.The
initial and four-hour phenol concentrations were measured by GLC and found to be
identical.

Gas-liquid chromatography. All GLC work was performed on a Perkin-Elmer Model


900 chromatograph equipped with a flame ionization detector. The sensitivity of the
FID was optimized at a hydrogen pressure of 20 psi and an air pressure of 30 psi. A
Perkin-Elmer Model 165 recorder was operated with a one millivolt (mV) span at a
speed of ten mm/min. A 50-/21 Hamilton syringe with a stainless steel needle (16 rail id)
was used for all aqueous injections. Typical injection volumes were ten/21.

Initially, several column packing materials which had been previously described in
the literature (Baker 1967) for direct-injection aqueous phenol analysis were studied. In
addition, a few other columns were experimented with, none of which have been de-
scribed in earlier reports (ten percent Bentone and five percent Carbowax 20M-TPA on
Chromosorb W; Chromosorb 102; four percent Celanese Ester on Chromosorb G; four
percent diisodecylphthalate on Chromosorb G). Comparison studies led to the selection
of a new column packing which exhibited the resolution, peak shape and retention
times desired for routine wastewater analysis. The columns used in the study described
here consisted of four percent dinonylphthalate (Applied Science Inc.) on Chromosorb G,
80/100 mesh. Two sets of column dimensions were used: two m × three m m i d and four
m × three mm id Pyrex glass. Columns were pre-conditioned for 24 hours at a tempera-
ture of 140°C. The maximum operating temperature of the dinonylphthalate liquid phase
is 150°C.

Injector port, manifold, and column temperatures were t80°C, 180°C, and 125°C,
respectively. Ultra high purity nitrogen (Linde) was used as the carrier gas at a pressure of
40 psi and a flow rate of 50 ml/min.
168 R.B. Baird et al.

Stock solutions (1000 mg/liter) of phenol, o-cresol, m-cresol, p-cresol, o-chlorophenol,


m-chlorophenol, p-chlorophenol and the dichlorophenols (2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and
3,5-isomers) were prepared by dissolving the Eastman Organic Co. chemicals (without
further purification) in quartz-distilled, deionized water. Dilute standards were prepared
fresh daily by serial dilution of the stock solutions. Mixed standards were prepared by
serial dilution of the desired mixture of stock solutions.

Aqueous samples were analyzed by direct injection, although some samples containing
high solids were homogenized with a mechanical/ultrasonic mixer before injection. The
effect of solids on the recovery of phenolic materials was checked by spiking raw and
treated sewage with I0.0 mg/liter concentrations of phenol, the cresol isomers, and the
chlorophenol isomers, then filtering and analyzing by direct injection on a GLC.

The detector response to individual phenolics was determined to be linear through a


range of 1-1000 mg/liter by plotting the peak area obtained at 1.0, 10.0, 100, and 1000
mg/liter concentrations. This permitted the determination of calibration factors for each
component. Peak areas were measured with a planimeter, and calculations were per-
formed taking into account the instrumental attenuation and individual calibration
factors (picograms per square in.). The limit of detection for the various phenolics was
estimated to be between 0.2 and 0.5 mg/liter based on peak areas which could be re-
produced within ten percent for a given set of instrumental parameters on the two
m column. These limits are reflected in the calibration factors which are presented in
Table I along with the relative retention times of the various phenolics.

Injection of pure water following injection of phenolic standards has been used to
eliminate "ghost" peaks or column residuals (Baker and Malo 1967) and is recommended
in the routine analysis of wastewater samples.

Results and discussion


Toxicity and biodegradation studies. There seems to be a widely-held opinion in
sanitary waste-treatment circles that activated sludge is capable of metabolizing up to 500
mg/liter of phenol without exhibiting toxic effects. This concept, though technically true,
is quite misleading when used out of the context of the actual research from which it was
drawn. It has been shown (McKinney et al. 1956) that an activated sludge can, with pain-
staking work, be conditioned to metabolize industrial phenolic wastes in concentrations
up to 500 rag/liter. Biological slimes have also been cultured (Reid e t al. 1956) which
can accept up to 1500 rag/liter of phenol providing a proper nutrient substrate is also
administered. Some cultures have even been used to treat industrial phenol wastes. How-
ever, it was stressed in the reports that long acclimation periods are necessary to achieve
these capabilities in bacterial populations.

The Warburg respirometry data obtained from the current study indicate that adminis-
tration of even relatively low concentrations of certain phenolic materials to sludges
which are unaccustomed to significant levels of phenolics can have a deleterious effect on
respiration. The activated sludges from the two treatment plants used in this study were
The Fate of Phenolics in Wastewater t 69

Table I. Relative Retention and Calibration Factors.for


Phenolic Standards, 4% Dinonylphthalate/Chromosorb G,
Two-M Pyrex Column

Compound r f b

phenol 1.00 70 182


o-cresol 1.33 61 192
p-cresol 1.65 63 202
m-cresol 1.71 63 203
o-chlorophenol 0.68 87 176
p-chlorophenol 5.48 98 217
m-chlor ophenol 5.72 98 214
2,3-dichlorophenol 2.65 97 -
2,4-dichtorophenot 2.65 95 210
2,5-dichlor ophenol 2.74 97 210
3,5-dichtorophenol 26.7 - 233
3,4-dichlorophenol 30.8 - 254

r = Relative retention.
f = Calibration factors, reported as picograms/square inch based
on a chart speed of 10 mm/min, 1 mV span, attenuation
10X2.
b = Boiling point, °C.

conditioned to continuous incoming phenolic concentrations in the range of 0.01-0.35 mg/


liter for the previous 12-month period. Although none of the seven phenols tested ex-
hibited any toxic effects at one mg/liter concentrations, phenol and the chlorophenol
isomers began to induce reduced respiration at levels of 10 mg/liter for six-hour contact
times. These effects were greatly accentuated at 100 mg/liter. Figure 1 depicts the
respiration curve of an activated sludge at 10 and 100 mg/liter concentrations of phenol.
The toxicity at 10 and 100 mg/liter levels of the chlorophenols is demonstrated in Figure 2
using m-chlorophenol as an example. Cresol isomers on the other hand seemed not to
inhibit respiration in the sludges tested until 100 mg/liter concentrations were used.
Figure 3, using m-cresol as an example, exhibits typical respiration curves obtained at
concentrations of 10 and 100 mg/liter.

It is of significant interest to compare these results with other Warburg studies per-
formed in this laboratory (Lehner 1970) which showed a domestic activated sludge
capable of metabolizing up to 120 mg/titer phenol with no apparent toxic effects. This
ability to utilize higher phenol concentrations is probably due to the fact that this
particular activated sludge had been conditioned to a constant influent concentration of
approximately 20 rag/liter of phenols from petroleum industry discharge for the pre-
ceeding 12-month period.
170 R.B. Baird et al.

50- C

40-

>
~' 30
E
('4
'0

E 20-

o
lO-

I I 1 I I I
0 60 120 180 240 300 360
Time (min)
Fig. 1. Warburg respiration of phenol at (a) 10 mg/1 and (b) 100 mg/1, compared with
endogenous respiration (c).

McKinney et al. (1956) studied the metabolism of several phenolics at high concentra-
tions in activated sludges acclimated to feed levels of 500 rag/liter phenol, and showed
that phenol and its simple substitution products (cresols, catechol, etc.) were oxidized to
approximately the same extent by similar mechanisms in conditioned sludge. However,
since it has been shown here that relatively low levels of phenolic compounds may be
toxic to unconditioned sludges, degradation studies were performed to determine the
fate of phenolics under these conditions. Since the sludges used were not laboratory
cultures grown in phenolic substrates, but were actual treatment plant activated sludge
samples containing residual organic material, theoretical oxidation data could not be
calculated from Warburg oxygen uptake data. Instead, initial, three-hour, and six-hour
phenolic concentrations in samples subjected to Warburg conditions were determined
experimentally in order to calculate percent biodegradation.

Table II exhibits this data for seven phenolics at 1.0, 10.0 and I00 mg/liter doses. Al-
though this method gives no indication of the subsequent oxidation intermediates, earlier
work (McKinney et al. 1956) suggests that enzymatic cleavage of the aromatic ring
structure leads to intermediates which are oxidized to CO2 and H20 by beta-oxidizing
enzymes, rather than by a mechanism of simultaneous adaptation proceeding through
the citric acid cycle. Comparison of the data in Table II with the toxicity curves shows
The Fate of Phenolics in Wastewater 171

50- C

a
40-

30-

20-

10-

I I I I I I
0 60 120 180 240 300 360
Time {rain)

Fig. 2. Warburg respiration of m-chlorophenol at (a) 10 mgfl and (b) 100 mg/1 compared
with endogenous respiration (c).

that, while the inteerity of the original phenolic is destroyed, respiration is inhibited at
concentrations as low as 10 rag/liter. This observation suggests that McKinney's explana-
tion is reasonable: bacterial adaptation to phenolics does not imply adaptation to all
oxidative intermediates. Therefore, in a sludge not acclimated to high levels of phenolic
wastes, certain levels of the phenol may be enzymaticatly degraded initially while
oxidative intermediates subsequently prove toxic to the bacterial population.

Since domestic sludges exhibit such a sensitivity to phenolics, it becomes quite im-
portant that a rapid method for the isolation and quantitative identification of these
materials in the wastewaters of areas likely to receive sporadic discharges of industrial
phenol wastes be available for monitoring and control purposes.

Gas-liquid chromatography. Any direct-injection technique employed in the analysis


of wastewaters must demonstrate good recovery of the analate from the aqueous system.
Table III exhibits the average recovery data obtained for eight phenolics in various types
of wastewaters. The recovery range of 95-99 percent indicates that very little of these
phenolics is lost by adsorption onto particulate matter in the samples. Therefore, sample
filtration prior to injection is a feasible step to promote precision and lengthen column
life.
172 R.B. Baird et at.

50-- ac

40-

>

"~ a0-
o

10

I 1 I I ! I
0 60 120 180 240 300 360
Time (min)

Fig. 3. Warburg respiration of m-cresol at (a) 10 mg/1 and (b) 100 mg/1 compared with
endogenous respiration (c).

A two m Pyrex glass column packed with four percent dinonylphthalate on 80/100
mesh Chromosorb G proved to be quite satisfactory in the routine analysis of phenolic
wastes in industrial and domestic discharges. The usual criteria used in judging column
performance, namely, peak symmetry, resolution, lack of tailing, absence of base line
noise, and elution interval are demonstrated for eight phenolics on a two m column in
Figure 4. The elution of phenol, cresols, chlorophenols, and dichlorophenol (except the
3,4- and 3,5-isomers) is complete 20 rain after injection, although the m- and p-isomers of
cresol and chlorophenol are not resolved. Should the resolution of m- and p-cresol be
desired, a four m column will perform the task within t 5 rain. The longer column is in-
capable of resolving the m- and p- isomers of chlorophenol, however. These phenomena
were also observed by Baker (1966) for both cresols and chlorophenols. Additionally,
polychlorinated phenols will not elute as sharp peaks from the dinonylphthalate column,
probably due to the temperature limitation of the liquid phase.

As with all steric-type substrates, the effects of ortho-substitution with respect to the
hydroxyl group promote faster elution (Baker 1966) on the dinonylphthalate column.
Thus the earlier elution of o-chlorophenol respective to phenol and the other chloro-
The Fate of Phenolics in Wastewater 173

Table II. Percent Biodegradation of Various Phenolics

Concentration (mg/l)
Compound 1 10 100

phenol 100 a 100 a 20 b


o-cresol 100a 100 a 17b
m-cresol 100a 100 a 4b
p-cresol 100 a 100 a 3b
o-chlorophenol 100 a 97 b 20 b
m-chlorophenol 100 b 40 b 0b
p-chlorophenot 100 b 80 b 16b

aAfter 3 hours.
bAfter 6 hours.

Table III. Recovery of Phenols from Spiked


Wastewater Samples

Compound Recoverya (%)

phenol 96
o-cresol 96
m-cresol 99
p-cresol 99
o-chlorophenol 98
m-chlorophenol 99
p-chlorophenol 99
2,4-dichlorophenol 95

aRecovery based on average of 12 wastewater


samples spiked with mixed standard.

phenols, o-cresol respective to the m- and p-isomers, and 2,6-dichlorophenol compared to


other dichlorophenols is explained.

Figures 5 and 6 depict phenol analyses of samples taken from within two petroleum
plants. Typical levels of phenolic wastes found in various trunk sewer and industrial
samples are depicted in Table IV. Note the wide range of phenol and cresol isomers and
the lack of chlorophenols detected. The incidences of chlorophenol detection in samples
obtained from the Los Angeles Basin area have been rare since the incorporation of the
GLC techniques into routine analysis. This observation probably indicates that the
I74 R . B . Baird et al.

o
0,.

"0

I I I I I I I I I I I I I I I I I
16 15 14 13 12 11 10 9 8 7 6 6 4 3 2 I 0

Fig. 4. Chromatogram of 8 phenolics on a 2 m, 4% dinonylphthalate/Chromosorb G


column: (a) o-chlorophenol; (b) phenol; (c) o-cresol; (d) m- and p-cresol; (e) 2,4-
dichlorophenol; (f) m- and p-chlorophenol. 10 pl injection; 100 rag/1 @ 10 X 2
attenuation.
The Fate of Phenolics in Wastewater 175

I 1 I I '1 I I I 1 I I
10 9 8 7 6 5 4 3 2 1 0

Fig. 5. Chromatogram of a petroleum industry discharge: (a) phenol; (b) o-cresol; (c) m-
and p-cresol. 10 ml sample, 10 X 8 attenuation.
176 R.B. Baird et al.

O
0..

r,t"

10 9 8 7 6 5 4 3 2 1 0

Fig. 6. Chromatogram of phenolic discharge from a petroleum industry: (a) phenol;


(b) o-cresol; (c) m- and p-cresol. 10 #1 sample, 10 × 4 attenuation.
The Fate of Phenolics in Wastewater 177

Table IV. Typical Industrial Phenol Analysis

Constituent concentration, mg/l


m- & p- Chlorophenol
Sample No. Phenol o-cresol cresol isomers

8967 310 110 290 0


8970 240 14 70 0
9056 12 1.4 1.2 0
9058 0.2 0.0 0.4 0
9084 11 1.1 3.5 0
9085 15 2.6 5.7 0
9098 3t 0 50 240 0

nature of phenolic waste problems may be regional or area-related as well as being the
result of increased efforts to curb industrial discharges.

Summary
Phenolic materials in concentrations as low as ten rag/liter can be deleterious to
bacterial respiration in activated sludge which has not been conditioned to the presence
of these compounds. Since the phenolics seem to be readily degraded under these
conditions, however, the toxic effects may be tile result of the formation of toxic oxida-
tion intermediates rather than the parent compound itself.

The sensitivity of domestic sludges to these materials necessitates a rapid method for
identifying individual phenotics. A direct-injection technique employing a steric liquid
phase, four percent dinonylphthalate, on 80/100 mesh Chromosorb G has been shown
effective in separating all the phenolics tested except m- and p-chlorophenol isomers.
When a two m column is used a routine analysis may be completed in as little as 20 rain
without any appreciable interference from solids or other organics ordinarily found in
sewage. The sensitivity exlfibited by the analyses of standards under these conditions
was in the sub-milligram per liter range, while the detector response was shown to be
linear up to 1000 mg/liter concentrations of those compounds tested.

References

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raphy. J. Water PoUn. Contr. Fed., 37, 1166 (1965).
Baker, R. A.: Phenolic analyses by direct aqueous injection gas chromatography. J. Amer.
Water Works Assoc. 751, (June 1966).
178 R.B. Baird et al.

Baker, R. A., and B. A. Malo: Phenolics by aqueous injection gas chromatography. En-
viron. Sci. Technol. 1,997 (1967).
Dannis, M.: Determination of phenols by the aminoantipyrine method. Sewage and Ind.
Wastes 23, 1516 (1951).
Eichelberger, J. W., R. C. Dressman, and J. E. Longbottom: Separation of phenolic com-
pounds from carbon chloroform extract for individual chromatographic identifica-
tion and measurement. Environ. Sci. Technol 4, 576 (1970).
Ettinger, M. B., C. C. Ruchhoft, and R. J. Lishka: Sensitive 4-aminoantipyrine method
for phenolic compounds. Anal. Chem. 23, 1783 (1951).
Hoak, R. D.: Sources of odorous materials in industrial wastes. Ind. Waste Conf. Proc.
229 (1956).
Lehner, J. D.: Unpublished data (t970).
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pounds by activated sludge. Sewage and Ind. Wastes 28, 547 (1956).
Mohler, E. F., and L. N. Jacob: Determination of phenolic-type compounds in water and
industrial waste waters: comparison of analytical methods. Anal. Chem. 24,
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Reid, G. W., R. Wortman, and R. Walker: The use of biological slimes in the treatment of
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Manuscript received July 5, 1973; accepted October 9, 1973