Rates of reduction of N-chlorinated peptides by sulfite: Relevance to incomplete dechlorination of wastewaters

Article abstract of DOI:10.1021/es9707365

Biologically induced fragmentation of proteins during wastewater treatment produces peptides, which form long-lasting organic chloramines when the water is disinfected with Cl₂. To protect aquatic wildlife from residual chlorine, including chloramines, wastewaters are often treated with sulfur dioxide or sulfite salts. This strategy incompletely eliminates residual chlorine species. Here we report that dechlorination rate constants of N- chloropeptides are 1-2 orders of magnitude smaller than those for NH₂Cl and some aliphatic organic chloramines. Slow rates explain the prevalence of N- chloropeptides in dechlorinated wastewaters after faster reacting chlorine species have been eliminated. Dechlorination is subject to general acid catalysis. For N-chlorinated leucylalanine, the rate law above pH 6 in phosphate buffer at 25 °C and / ? 0.1 M is as follows: rate = (9.92 ± 0.41 x 10³[H₂PO₄^-] + 5.70 ± 0.52 x 10⁸[H₃O⁺] + 5.3 ± 0.2)[SO₃^2-][Cl- Leu-Ala] (concentrations in M, time in s). Rate constants for other peptides appear to be of similar magnitude; variations in the acid-catalyzed terms among different hydrophobic peptides correlate with solvation energies of side chains. The kinetic data suggest that reducing N-chloropeptides in wastewaters by 75% or more will require reaction times generally >0.5 h at environmentally acceptable S(IV) doses and pH values. Biologically induced fragmentation of proteins during wastewater treatment produces peptides, which form long-lasting organic chloramines when the water is disinfected with Cl₂. To protect aquatic wildlife from residual chlorine, including chloramines, wastewaters are often treated with sulfur dioxide or sulfite salts. This strategy incompletely eliminates residual chlorine species. Here we report that dechlorination rate constants of N-chloropeptides are 1-2 orders of magnitude smaller than those for NH₂Cl and some aliphatic organic chloramines. Slow rates explain the prevalence of N-chloropeptides in dechlorinated wastewaters after faster reacting chlorine species have been eliminated. Dechlorination is subject to general acid catalysis. For N-chlorinated leucylalanine, the rate law above pH 6 in phosphate buffer at 25 °C and I≈0.1 M is as follows: rate = (9.92±0.41×10³[H₂ PO₄^- ]+5.70±0.52×10⁸[ H₃O⁺]+5.3±0.2) [SO₃^2-][Cl-Leu-Ala] (concentrations in M, time in s). Rate constants for other peptides appear to be of similar magnitude; variations in the acid-catalyzed terms among different hydrophobic peptides correlate with solvation energies of side chains. The kinetic data suggest that reducing N-chloropeptides in wastewaters by 75% or more will require reaction times generally >0.5 h at environmentally acceptable S^IV doses and pH values.

Full text of DOI:10.1021/es9707365

Environ. Sci. Technol. 1998, 32, 516-522
(6), we report chromatographic evidence that N-chlorinated
Rates of Reduction of N-Chlorinated
Peptides by Sulfite: Relevance to
Incomplete Dechlorination of
Wastewaters
peptides and protein fragments constitute a large fraction of
the slowly reduced residual chlorine in treatment plant
effluents.
Amino nitrogen compounds in municipal wastewaters
include a proteinaceous fraction (7, 8). During treatment,
extracellular enzymatic degradation (proteolysis) of this
material produces amino acids and peptides, compounds
that react with chlorine to form chloramines even more
rapidly than NH3 (9-11). The N-chlorinated free amino acids
undergo first-order decomposition, effectively a self-dechlo-
rination process; half-lives range from seconds to hours (12-
14). On the other hand, peptides that become chlorinated
at their terminal N atoms appear to be more stable and hence
possibly of greater concern in the environment unless
eliminated at the treatment plant. For example, half-lives
of N-chloroalanylalanine and N-chloroleucylglycine are 9
and 25 h, respectively, at physiological pH and temperature
(i.e. 37 °C, ref 15); the half-life ofN-chloroalanylphenylalanine
is 111 h at pH 7 and 23 °C (16). N,N-dichloropeptides, which
may also form, decompose more rapidly than N-monochlo-
ropeptides (15, 17) and thus are possibly of less concern in
the environment.
†
J A M E S S . J E N S E N A N D
G E O R G E R . H E L Z *
Chemistry and Biochemistry and Water Resources Research
Center, University of Maryland, College Park, Maryland 20742
Biologically induced fragmentation of proteins during waste-
water treatment produces peptides, which form long-
lasting organic chloramines when the water is disinfected
with Cl2. To protect aquatic wildlife from residual chlorine,
including chloramines, wastewaters are often treated with
sulfur dioxide or sulfite salts. This strategy incompletely
eliminates residual chlorine species. Here we report that
dechlorination rate constants of N-chloropeptides are
In this paper, we examine the kinetics of dechlorination
of peptides that have been monochlorinated at the terminal
N. This paper constitutes a reconnaissance of an inherently
vast subject. One goal is to evaluate the prospect for curtailing
the amount of SIV-refractory residual chlorine in wastewaters
through modification of dechlorination practices.
1-2 orders of magnitude smaller than those for NH Cl and
2
some aliphatic organic chloramines. Slow rates explain
the prevalence of N-chloropeptides in dechlorinated
wastewaters after faster reacting chlorine species have
been eliminated. Dechlorination is subject to general
acid catalysis. For N-chlorinated leucylalanine, the rate
law above pH 6 in phosphate buffer at 25 °C and I ≈ 0.1
M is as follows: rate ) (9.92 ( 0.41 × 103[H PO -] + 5.70
Experimental Section
The purity of commercially available peptides (Sigma Chemi-
cal) was confirmed by thin-layer chromatography. Reagent
grade NaOCl (Johnson Matthey) was used to chlorinate the
peptides. The N-chloropeptide solutions were prepared less
than 10 min before an experiment by adding NaOCl to dilute,
buffered solutions of peptides. By keeping the peptide in
∼10-fold excess to NaOCl and by rapid mixing, unwanted
dichloro products were avoided. Water for making solutions
was obtained from distilled water that was filtered through
a Milli-Q water system (Millipore Corp.). To avoid metal
contamination, glassware was washed with PC-54 cleaning
solution and then washed in HNO3. Reagent-grade Borax or
phosphate salts were used to prepare buffers. To ensure
that buffers were reductant-free, they were treated with ∼2.5
mg/ L NaOCl, stored for approximately 24 h, and then
dechlorinated with a UV lamp. Fresh sulfite solutions were
made each day from reagent-grade Na2SO3 (J. T. Baker) and
N2-purged Milli-Q water; they were standardized iodometri-
cally. Reagents used for the amperometric titration method
(iodine and pH 4 acetate buffer) were prepared according to
standard methods (18).
An automated titrator (Metrohm 716 DMS Titrino) was
used to standardize chlorinated reagents amperometrically.
The reductive titrant was 0.00564 N phenylarsine oxide (PAO)
solution (Fisher), which was standardized against I2 produced
from weighed amounts of KIO3 in acidic KI solution.
A photodiode array spectrometer (Hewlett-Packard model
8452A) fitted with a 10-cm cuvette provided the UV/ Vis
spectra and kinetic data. Rate constants were produced by
the kinetics software supplied with the instrument. Absor-
bance could be read every 0.5 s. Reagent solutions were
thermostated in a circulating water bath prior to mixing in
the cuvette. We relied on thermal inertia to hold the
temperature during the few minutes needed to establish
initial rates in the spectrometer. To start an experiment,
2
4
( 0.52 × 108[H O+] + 5.3 ( 0.2)[SO 2-][Cl-Leu-Ala]
3
3
(concentrations in M, time in s). Rate constants for other
peptides appear to be of similar magnitude; variations
in the acid-catalyzed terms among different hydrophobic
peptides correlate with solvation energies of side chains.
The kinetic data suggest that reducing N-chloropeptides in
wastewaters by 75% or more will require reaction times
generally >0.5 h at environmentally acceptable SIV doses
and pH values.
Introduction
Chlorine-disinfected municipal wastewaters contain toxic
agents, many of which are organic and inorganic N-
chloramines (1, 2). To protect aquatic wildlife, especially
fish, increasing numbers of wastewater treatment plants
now include a dechlorination step after disinfection, just
prior to discharge. Most often, SO2 or sulfite salts are applied
for this purpose (3, 4). Here, these dechlorinating agents
will collectively be called SIV compounds because they con-
tain sulfur in the +IV oxidation state. The goal of dechlo-
rination is to reduce all oxidative forms of residual chlorine
to Cl-, including that in N-chloramines. However, field
and laboratory evidence shows that dechlorination with SIV
is incomplete on a <1 h time scale, and many treatment
plants relying on dechlorination may be failing to meet
discharge requirements for residual chlorine (5). Elsewhere
* Telephone 301-405-1797, email: GH17@umail.umd.edu.
† Current address: Pharmaceutical Delivery Systems, Parke-Davis,
170 Tabor Rd., Morris Plains, NJ 07950-2536.
9
5 1 6 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 4, 1998
S0013-936X(97)00736-0 CCC: $15.00
1998 Am erican Chem ical Society
Published on Web 02/15/1998

standardized sulfite was delivered by a calibrated, adjustable
autopipet to a cuvette containing the N-chloropeptide
solution. The cuvette was agitated rapidly for 6-8 s and
then placed in the spectrometer. Reduction of the ClHN
group was monitored at 266 nm rather than at the absorption
maximum near 250 nm. This minimized a slight interference
from unreacted sulfite.
Results
The first peptide investigated was N-chloroalanylalanine
(henceforth, Cl-Ala-Ala). In a series of pH 8 solutions
containing 1 mM total alanylalanine and 0.1-0.25 mM Cl-
Ala-Ala, the molar extinction coefficient of Cl-Ala-Ala at
250 nm was found to be 333 ( 5 M-1 cm-1. Using this value,
the stoichiometry of dechlorination of this peptide was
determined by spectrophotometric titration in 0.05 M
phosphate buffer at pH 6.97. A 10-cm cell was used with a
small magnetic stir bar to ensure rapid mixing. Aliquots of
standardized sulfite solutions were injected into a cell
containing 1 mM total peptide and 0.158 mM initial Cl-Ala-
Ala. Spectra were taken until no further changes occurred.
Two such titrations yielded a mean molar ratio of Cl-Ala-Ala
reduced per SIV added of 0.97. Additional titrations of
solutions containing 0.134 mM initial Cl-Ala-Ala yielded a
mean ratio of 0.98.
Consistent with other studies on chloramines (19-21),
these neutral pH results imply that dechlorination involves
reaction of one molecule of SIV with one molecule of the
monochloropeptide, implying that SIV loses two electrons,
producing sulfate:
ClNH-peptide + SO32- + 2H2O f
FIGURE 1. Dechlorination of N-chloroalanylalanine. (A) Variation
of initial rate with sulfite concentration and (B) with N-chloro-
alanylalanine concentration. (C) observed first-order rate constant
(i.e., rate/[N-chloroalanylalanine]) that is found to be independent
of borate buffer concentration along two pH isopleths. Conditions:
25 °C, I ) 0.06.
NH2-peptide + Cl- + SO42- + H3O+ (1)
Side reactions appear to be minor, confirming that on a time
scale of minutes the monochloropeptide is stable against
self-decomposition.
The order of the dechlorination reaction of Cl-Ala-Ala
was determined with respect to total SIV at pH 8.6. The ionic
strength (adjusted to 0.06 with KCl) and the concentration
of Cl-Ala-Ala were held constant while varying the initial
quantity of sulfite. The logarithm of the absorbance varied
linearly with time over several half-lives, indicating a 1st-
order dependence on Cl-Ala-Ala. Plotting the log of the initial
rate for each dechlorination reaction vs the log of the sulfite
concentration (Figure 1A) yields a line with the slope equal
to 0.97 ( 0.02, implying that the reaction is probably 1st-
order with respect to SIV.
practical limit for our manual mixing technique is encoun-
tered at t1/ 2 ≈ 10 s. Therefore, we embarked instead on a
survey of other dipeptides, seeking examples that react
less rapidly with SIV, these being of chief interest in this
study.
Only peptides containing amino acids with side chains
that are unreactive with chlorine on the time scale of minutes
were considered. The results are shown in Table 1. All
compositional variables (pH, ionic strength, total SIV, total
B, total peptide, and N-chloropeptide) were made nearly
identical in this series of experiments, so the observed
rate constants, while dependent upon solution composi-
tion, are nonetheless measures of the relative intrinsic
reactivities of the N-chloropeptides. A range in kobs of slightly
less than 1 order of magnitude is observed. A similar
comparison was made between dialanine, tetraalanine, and
hexaalanine (Table 2). The kobs values in this case display
negligible dependence on chain length for these short
chains.
Figure 1B shows a plot of the log of the initial rate vs the
log Cl-Ala-Ala at constant pH, buffer concentration, and initial
SIV concentration. A slope of 1.06 ( 0.02 is observed. In a
similar manner, the effect of borate buffer concentration
and pH were investigated. Figure 1C shows kobs (i.e., initial
rate/ [Cl-Ala-Ala]) for a set of experiments in which the borate
concentration was varied at constant pH. In the 0.05Ms0.02
M total B range, it was possible to hold the pH to (0.02.
Figure 1C demonstrates two points: the rate is accelerated
by increasing H3O+ and the rate is unaffected by H3BO30 (up
One of the slower peptides from Table 1, N-chloroleucyl-
alanine (henceforth Cl-Leu-Ala), was investigated over a wider
pH range in order to better understand the reaction. With
SIV in 10-fold excess, kobs values were obtained in near-neutral
and alkaline 0.02 M phosphate buffers. These rate constants
are plotted in Figure 2A. In contrast to the alanylalanine
case (Figure 1C), here the acceleration in the rate with
decreasing pH is clearly resolved. Furthermore, a distinct
relationship between total phosphate and kobs is demon-
strated at constant, near-neutral pH in Figure 2B. On the
other hand, no dependence was found between total
phosphate and kobs at pH 11.8.
0
to 0.05 M). H3BO3 is the predominant form of total B at
these pH values. The latter behavior contrasts with observa-
tions on dechlorination of NH2Cl (19) and N-chloropiperidine
(20); in both cases H3BO30 behaved as a general acid catalyst.
In the pH range covered by Figure 1C, the reaction order
with respect to H3O+ is less than 1, suggesting that these
experiments were done in a pH region where both first- and
zeroth-order pathways are significant.
Pursuing the kinetics at lower pH to resolve these path-
ways is of interest, but at pH 8.56 the half-life corresponding
to the kobs values in Figure 1C is already under 1 min. A
9
VOL. 32, NO. 4, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 5 1 7

a
TABLE 1. Observed Rate Constants for Dechlorination of Dipeptides with 10 Times Excess Sulfite(total)
a
Conditions: 0.05 M borate(total) buffer, 25 °C, I ) 0.06 with KCl, pH 8.58 ( 0.03, peptide total 1.0 m M, N-chloropeptide 0.10 m M, dechlorination
m onitored at 266 nm , phenylalanine-glycine dechlorination m onitored at 275 nm . *kobs is a pseudo-first-order rate constant where -dC/dt )
k[sulfite]total[N-chloropeptide] ) kobs[N-chloropeptide].
Discussion
TABLE 2. Dechlorination of Alanine Oligomers in 0.05 M
Borate Buffera
Kinetic Interpretation. A minimum of three reactions is
needed to account for the kinetics of dechlorination of Cl-
Leu-Ala based upon the observations in Figure 2:
oligomer
kobs (s-1 b
)
t1/2 (s)
pH
dialanine
tetraalanine
hexaalanine
0.0157 ( 0.005
0.0184 ( 0.007
0.0169
44
38
41
8.06
8.11
8.08
-
H2PO4- + Cl-Leu-Ala + SO32- + 2H2O f H2PO4
H3O+ + H-Leu-Ala + SO42- + Cl-
+
k1 (2)
a
b
I ) 0.06M, 0.10 m M N-chloropeptide, 1 m M SIV, 25 °C. Quoted
uncertainty ) half the range of two m easurem ents.
H3O+ + Cl-Leu-Ala + SO32- + 2H2O f 2H3O+
H-Leu-Ala + SO42- + Cl-
+
Thus, like NH2Cl and N-chloropiperidine (19, 20), dechlo-
rination of Cl-Leu-Ala appears to be subject to general acid
catalysis. The previous finding (above) of no observable
buffer effect in the case of Cl-Ala-Ala (Figure 1C) can be
explained by the weaker acidity of H3BO30 vs H2PO4- and the
faster uncatalyzed dechlorination rate of the latter peptide.
Strength of acidity also explains why HPO42- (at high pH) is
k2 (3)
Cl-Leu-Ala + SO32- + 2H2O f H3O+ + H-Leu-Ala +
SO42- + Cl-
k3 (4)
All 3 reactions conform to the stoichiometry of reaction 1;
the first two reactions involve respectively H2PO4- and H3O+
as acid catalysts.
-
ineffective, whereas H2PO4 (at neutral pH) is effective in
catalyzing Cl-Leu-Ala reduction.
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5 1 8 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 4, 1998

TABLE 3. Comparison of Rate Constants for Monochloramines
(Constants Defined by Eqs 5 and 7 in Text)
k1
(M-2 s-1
k2
k3
k4
chloramine
)
(M-2 s-1
)
(M-2 s-1
)
(M-2 s-1
)
N-chloro-Ala-Ala
N-chloro-Leu-Ala
N-chloro-triAlaa
NH2Cl (19)
N-chloropiperidine
(20)
1.9 × 109
1.2 × 109
8.0 × 1010
1.1 × 1011
6.9
5.3
9.9
7.7
4.1
9.92 × 103 5.7 × 108
1.3 × 104
1.3 × 106
2.1 × 107
3.6 × 108
a
From refitting data in ref 21; constants are approxim ate due to
paucity of data.
FIGURE 2. Dechlorination of N-chloroleucylalanine. (A) Variation
of observed first-order rate constant with pH in 0.02 M phosphate
buffer. (B) Variation of observed rate constant with total phosphate
at pH 7.44. Conditions: 25 °C, 0.1 mM Cl-Leu-Ala, 1 mM total
SIV. Curves calculated from eq 5.
FIGURE 3. Dechlorination of N-chlorotrialanine at 25 °C in 0.02 M
phosphate buffer (data from ref 21). The curve has been calculated
from a model discussed in the text. The second-order rate constant,
k′′, plotted on the vertical axis has units of M-1 s-1 and is defined
by
2-
All reactions have been written with SO3 rather than
-
HSO3- as the kinetically active SIV species. In general, HSO3
δ[Cl-triAla]
is a much poorer reducing agent than SO32-. For example,
in the reduction of O3, the rate constant for HSO3- is 4 orders
of magnitude smaller than the rate constant for SO32- (23).
A similar but less extreme contrast exists in the rate constants
for reduction of NH2Cl by SO3 and HSO3 (19). For this
reason, SO32- is expected to control dechlorination rates in
the near-neutral to alkaline pH realm where it is the
predominant SIV species.
-
) k′′[Cl-triAla][SIV] ) ((k1[H2PO4-] +
-[Cl-triAla][SIV
δt
k2[H3O+] + k3)RSO 2-) + k [H O+]R
HSO3
]
4
3
3
2-
-
Although these values reflect the slightly faster dechlorination
rate of Cl-Ala-Ala vs Cl-Leu-Ala, consistent with expectations
based on Table 1, the values for the two dipeptides are
nonetheless very similar.
Based on reactions 2-4, the empirical rate law would be
Table 3 compares the constants for Cl-Leu-Ala and
Cl-Ala-Ala with corresponding constants for the other
monochloramines that have been studied (19-22). The k2
values for both peptides lie roughly 2 orders of magnitude
below those observed for NH2Cl and N-chloropiperidine. In
contrast, values for k3 vary by less than 3-fold for all
monochloramines so far studied.
δ[Cl-Leu-Ala]
-
) kobs[Cl-Leu-Ala] ) (k1[H2PO4-] +
k2[H3O+] + k3)[SO32-][Cl-Leu-Ala] (5)
δt
Quantities in square brackets are molar concentrations and
kobs ) (k1[H2PO4-] + k2[H3O+] + k3)[SO32-].
The only previous study of the rate of dechlorination of
peptides was the work of Stanbro and Lenkevich (21, 22),
who investigated the rate of dechlorination of N-chlorotri-
alanine at a fixed phosphate buffer concentration of 0.02 M.
Having no information on the variation of rate with phos-
Fitting the data in Figure 2 with this empirical rate law
produces the following rate constants at 25 °C and I ≈ 0.1:
k1 ) 9.92 ( 0.41 × 103 M-2 s-1, k2 ) 5.70 ( 0.52 × 108 M-2
s-1, and k3 ) 5.3 ( 0.2 M-1 s-1; quoted uncertainties are
standard deviations. In the fit, the following ionization
constants for H3PO4 were used (I ) 0.1): pK1 ) 2.1, pK2 )
6.7; the second ionization constant of H2SO3, needed to
partition total SIV into SO32- and HSO3-, was pK2 ) 7.2. The
curves shown in Figure 2 are calculated from eq 5 using
k1-k3 and indicate the high quality of this three-parameter
model.
Fitting the same rate law without the k1 term to the pH
8-9, phosphate-free Cl-Ala-Ala experiments described earlier
yields k2 ) 1.9 ( 0.1 × 109 M-2 s-1 and k3 ) 6.9 ( 0.6 M-1
s-1. A significant contribution from both a H3O+-catalyzed
and a H3O+-independent pathway accounts for the previously
mentioned reaction order between 0 and 1 in this pH range.
phate, these workers interpreted their results in terms of
-
three parallel reactions involving respectively H2SO3, HSO3
,
2-
-
and SO3 as reductants. They obtained kH
> kHSO
>
SO
2
3
3
2-
kSO , an order opposite to that observed in other systems
3
in which SIV is a reducing agent (19, 23). As a general matter,
protonation is expected to diminish the facility with which
a reducing agent will surrender electrons to an oxidizing
agent.
Figure 3 shows that a reasonable fit can be obtained to
the data in ref 21 with a model similar to eq 5. However, to
fit the data below pH 6, it is necessary to propose an additional
reaction:
9
VOL. 32, NO. 4, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 5 1 9

H3O+ + Cl-triAla + HSO3- + 3H2O f 3H3O+
H-triAla + SO42- + Cl-
+
k4 (6)
δ[Cl-triAla]
-
-
) k4[H3O+][Cl-triAla][HSO3
]
(7)
δt
The following constants were used to calculate the curve in
Figure 3: k1 ) 1.3 × 104 M-2 s-1, k2 ) 1.2 × 109 M-2 s-1, k3
) 9.9 M-1 s-1 and k4 ) 2.1 × 107 M-2 s-1. The values of k1-k3
are similar in magnitude to those in Table 3 for Cl-Ala-Ala
and Cl-Leu-Ala. The rate constant for the H3O+-catalyzed
reaction involving HSO3- as reductant is less than 2% of the
2-
rate constant involving SO3 as reductant (i.e., the triAla
analogue of reaction 3). Roughly the same relationship
between the analogous constants for NH2Cl has been reported
(19).
In view of the similar kinetic behavior during dechlori-
nation of all five monochloramines in Table 3, it seems
reasonable that the reactions all operate by a similar
mechanism. The mechanism of Yiin et al. (19) for NH2Cl
dechlorination also accounts for the new information here
on N-chloropeptides. In this mechanism, a ternary inter-
FIGURE 4. Relationship between kobs for dipeptides (Table 1) and
hydrophobicity of the side chains of dipeptides. Free energy of
transfer is the free energy reduction caused by transferring the side
chain of an amino acid from water to an organic solvent; the scale
is based on glycine which is assigned ∆G ) 0. Values on the
horizontal axis are sums for both side chains in each dipeptide.
2-
mediate is formed in which SO3 binds to Cl on the
chloramine and a proton donor (H2PO4-, H3O+, H2O) binds
to the lone pair on N. This intermediate then dissociates,
Aspartylglycine is not plotted because no ∆G value is
available.
2-
releasing chlorosulfate, which hydrolyzes rapidly to SO4
+
Cl- (24). Proton donors of greater acidity speed the reaction
The implication of Figure 4 is that the extent of a peptide’s
solvation, which will affect its molecular conformation, is an
important factor in its reactivity with SIV. This may be of
practical importance in view of Helz and Nweke’s (5) evidence
that much of the SIV-refractory residual chlorine in wastewater
can be extracted into octanol.
by withdrawing electron density to a greater extent from the
N-Cl bond. Hence the rate constant for H3O+ is much larger
-
than the rate constant for H2PO4
. Rate constants for
dechlorination of NH2Cl in the presence of a series of proton
donors obey the Bronsted relationship (19).
While the range of observed rate constants encountered
among the different dipeptides in Table 1 is not large, it is
significant. All the dipeptides have an identical core structure
and differ only in the side chains of the N- and C-terminal
amino acids. It is reasonable to look to inductive and steric
effects caused by these side chains to explain the variations.
The inductive effect would influence rates by affecting the
basicity of the amino N. However, the inductive effect should
have only a small influence among the peptides in Table 1.
Except for glycine, the differences in the side chains occur
only after the second C atom from the amine. As expected,
when the pKa values of the unchlorinated N-terminal amino
acid in each dipeptide were compared to the rate constants
in Table 1, the correlation was poor. The same was true
when pKa values of the amino N in the dipeptides, themselves
were compared.
The steric effect influences rates through restriction of
access of reactants to the reactive site on a molecule. It is
plausible that steric effects could be particularly important
in the Yiin et al. (19) mechanism, which requires formation
of a three-component intermediate. However, comparisons
of the rate constants in Table 1 with molecular volumes of
the side chains (25) produced insignificant correlations; this
was true whether the comparison was made to the volume
of the N-terminal side chain or to the total volume of the N-
and C-terminal side chains.
On the other hand, there appears to be a relationship
between the observed rate constants (Table 1) and hydro-
phobicity of the side chains in the case of dipeptides
composed of amino acids with poorly solvated side chains
(Figure 4). As a measure of hydrophobicity, the free energy
of transfer of the side chain components between water and
a nonpolar solvent (26) was used. Figure 4 shows that the
dipeptides with the most hydrophobic side chains possess
the slowest rate constants. Serylleucine and phenylalanylg-
lycine, which have side chains that participate in H-bonding,
react more rapidly than anticipated from this relationship.
While the inductive effect apparently does not account
for differences in reaction rate among the dipeptides in Table
1, it probably does account for the broader differences among
the compounds in Table 3. For example, the pKa for NH2Cl
is 1.44 and for N-chloroglycylglycine is -0.67 (11). If the
value for N-chloroglycylglycine is representative of peptides,
then the ∼2 order of magnitude pKa difference between NH2-
Cl and N-chloropeptides nicely explains the similar differ-
ences in the k1 and k2 values in Table 3.
Prospects for Im proving Dechlorination Treatm ent.
The observation that SIV-refractory residual chlorine in
wastewaters includes a prominent contribution from N-
chloropeptides (6) can be understood from the comparison
in Table 3. In ordinary wastewaters, which have a near-
neutral pH and total P usually below 10-4 M, the H3O+-
catalyzed term will dominate. Under these conditions,
peptides can compete much less effectively than inorganic
and aliphatic monochloramines for SIV, due to their much
lower rate constants. Furthermore, as Helz and Nweke (5)
have noted, other inorganic forms of residual chlorine (HOCl,
OCl-, NHCl2, NCl3) will be reduced much faster than any of
the monochloramines. Thus, N-chloropeptides will be
survivors of the dechlorination process and after several
minutes will become prevalent constituents of total residual
chlorine, as is observed.
As shown in Table 3, rate constants for the peptides studied
here are all of similar magnitude. Because of this and because
SIV-refractory residual chlorine at wastewater treatment plants
includes a prominent contribution from moderately hydro-
phobic N-chloropeptides (6), it is reasonable to explore ways
of enhancing the efficiency of dechlorination treatment by
considering peptide dechlorination rates. We will use Cl-
Leu-Ala as a model for peptides generally, because it is the
one for which we have the most accurate data above pH 6.
Two simplifying assumptions will be made. First, we
assume that dechlorination of individual peptides in waste-
water is a pseudo-first-order process. This will often be the
9
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5 shows that dechlorination is favored by acidic conditions.
However even at pH 6, 50% reduction of Cl-Leu-Ala can be
accomplished in 1 min only with SxIV of approximately 20
ppm; 97% reduction (5 half-lives) would require either waiting
5 min at 20 ppm SxIV or raising SxIV to 100 ppm and waiting
1 min. In either case, such high SxIV concentrations would
have environmentally unacceptable consequences (28). At
higher pH, longer reaction times or higher SxIV concentrations
are necessary. For example at pH 8, dechlorination of Cl-
Leu-Ala requires approximately 4 times longer than at pH 6.
To achieve 50% reduction of Cl-Leu-Ala with a SxIV concen-
tration of 1 ppm, reaction times of 0.3-2 h (depending on
pH) are needed.
Figure 5 does not take into account temperature variations.
Evidence in ref 21 suggests that k3 may depend strongly on
temperature whereas the other constants may be less
sensitive. If this preliminary indication is supported in future
work, then cold weather would lengthen dechlorination times
at high pH but have less effect near neutrality.
It is of interest that the observed 26-min half-life for
reduction of dechlorination-resistant residual chlorine at a
wastewater treatment plant (5) is predicted reasonably well
by Figure 5. This wastewater had a pH of ∼6.5 and a SxIV of
∼1 ppm.
The residual chlorine in real wastewaters consists of a
mixture of compounds and cannot be expected to conform
exactly to Figure 5. Nonetheless, because the best informa-
tion now available (6) suggests that hydrophobic N-chlo-
ropeptides comprise a major fraction of dechlorination-
resistant residual chorine, Figure 5 may serve as a useful
guide to how real wastewaters behave during dechlorination.
FIGURE 5. Time needed to achieve 50% reduction of N-chloro-
leucylalanine at 25 °C as a function of pH and SxIV. (SxIV is the HSO -
3
+ SO 2- remaining after rapidly reduced residual chlorine has been
3
Acknowledgments
U.S. Geological Survey support to the Maryland Water
Resources Research Center made this work possible.
destroyed.) Time needed to achieve 97% reduction (5 half-lives)
IV
would be 5-fold larger for the same pH and Sx conditions.
Calculations assume no catalysis by acids other than H O+ and
3
IV
N-chloroleucylalanine , Sx
.
Literature Cited
case in treatment situations, where it is common practice to
add a SIV dose that is about 50% greater on a molar basis than
the total residual chlorine in the water to be dechlorinated.
More than 90% of the residual chlorine reacts within 1 min
or so (5). After this initial phase, the ratio of excess SIV
(henceforth, SxIV) to the remaining residual chlorine will be
>5, and subsequent changes in [SxIV] due to dechlorination
reactions will be small. Second, we will neglect general acid
catalysts, which in general are much less important in natural
waters and wastewaters than they are in laboratory experi-
ments involving high buffer concentrations (27). For ex-
(1) Brungs, W. A. J. Water Pollut. Control Fed. 1973, 45, 2180-2193.
(2) Hall, L. W.; Helz, G. R.; Burton, D. T. Power Plant Chlorination,
A Biological and Chemical Assessment; Ann Arbor Science: Ann
Arbor, MI, 1981; 237 pp.
(3) Helz, G. R.; Kosak-Channing, L. Environ. Sci. Technol. 1984, 18,
48A-55A.
(4) White, G. C. The Handbook of Chlorination, 3rd ed.; Van
Nostrand Reinhold: New York, 1992.
(5) Helz, G. R.; Nweke, A. C. Environ. Sci. Technol. l995, 29, 1018-
1022.
(6) Jensen, J. S. Chemical Studies to Understand the Dechlorination
Process Used at Wastewater Treatment Plants. Ph.D. Disserta-
tion, University of Maryland, College Park, MD, 1997, 145 pp.
(7) Parkin, G. R.; McCarty, P. L. Water Res. 1981, 15, 139-149.
(8) Hureiki, L.; Croue´, J. P.; Legube, B. Water Res. 1994, 28, 2521-
2531.
-
ample, the maximum influence of H2PO4 on the rate of
dechlorination of Cl-Leu-Ala, according to eq 5, occurs near
pH 7. In order for the H2PO4--catalyzed term to exceed the
H3O+-catalyzed term at pH 7, H2PO4 must exceed 6 mM
-
(200 ppm as P). Such a concentration would be unusual,
even in a wastewater.
(9) Armesto, X. L.; Canle, M.; Santaballa, J. A. Tetrahedron 1993, 49,
275-284.
(10) Morris, J. C. In Principles and Applications of Water Chemistry;
Faust, S. D., Hunter, J. V., Eds; John Wiley & Sons: New York:
1967; pp 23-53.
(11) Margerum, D. W.; Gray, E. T.; Huffman, R. P. In Organometals
and Organometalloids, Occurrence and Fate in the Environment,
Bellama, J. M., Brinckman, F. E., Eds.; ACS Symposium Series
82; American Chemical Society: Washington, DC, 1978; pp 264-
291.
(12) Hand, V. C.; Snyder, M. P.; Margerum, D. W. J. Am. Chem. Soc.
1983, 105, 4022-4025.
On the basis of these two assumptions, the concentration
of SxIV needed to produce 50% reduction in Cl-Leu-Ala within
a given time interval at a given pH and 25 °C can be calculated
IV
from the following equation. Here, Sx is in ppm as SO2,
-
time is in minutes, pKa is for HSO3 (i.e., 7.2 at 25 °C), and
the k values are as given in Table 3; the constant, 741,
reconciles the various units of measurement:
741[1 + 10(pK -pH)
]
a
IV
(13) Tan, H.; Sen, A. C.; Wheeler, W. B.; Cornell, J. A.; Wei, C. I. J.
Sx
)
(8)
[k210-pH + k3]t1/ 2
Food Sci. 1987, 52, 1706-1711.
(14) Stanbro, W. D.; Smith, W. D. Environ. Sci. Technol. 1979, 13,
446-451.
Figure 5 is a plot of eq 8 and shows that achieving high
degrees of N-chloropeptide reduction is unlikely without
holdup tanks to provide reaction time. Such tanks are not
currently included in most dechlorination systems. Figure
(15) Stelmaszynska, T.; Zgliczynski, J. Eur. J. Biochem. 1978, 92, 301-
308.
(16) Fox, T. C.; Keefe, D. J.; Scully, F. E., Jr. Environ. Sci. Technol.
1997, 31, 1979-1984.
9
VOL. 32, NO. 4, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 5 2 1

(17) Stanbro, W. D.; Lenkevich, M. J. Int. J. Chem. Kinet. 1985, 17,
401-411.
(18) Standard Methods For The Examination of Water and Waste-
water, 18th ed.; Greenberg, A. E., Clesceri, L. S., Eaton, A. D.,
Eds.; American Public Health Association: Washington, DC,
1992.
(25) Creighton, T. E. Proteins: Structures and Molecular Properties;
W. H. Freeman: New York, 1993; 507 pp.
(26) Nozaki, Y.; Tanford, C. J. Biol. Chem. 1971, 246, 2211-2217.
(27) Perdue, E. M.; Wolfe, N. L. Environ. Sci. Technol. 1983, 17, 635-
642.
(28) Kijak, P. J.; Helz, G. R. Environ. Sci. Technol. 1988, 22, 1171-
1177.
(19) Yiin, B. S.; Walker, D. M.; Margerum, D. W. Inorg. Chem. 1987,
26, 3435-3441.
(20) Jensen, J. S.; Helz, G. R. Water Res. In press.
(21) Stanbro, W. D.; Lenkevich, M. J. Int. J. Chem. Kinet. 1984, 16,
251-258.
Received for review August 18, 1997. Revised manuscript
received November 25, 1997. Accepted November 28, 1997.
(22) Stanbro, W. D.; Lenkevich, M. J. Science 1982, 215, 967-968.
(23) Hoffmann, M. R. Atmos. Environ. 1986, 20, 1145-1154.
(24) Yiin, B. S.; Margerum, D. W. Inorg. Chem. 1988, 27, 1670-1672.
ES9707365
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