600 J 00
Ascorbic acid reduction of active chlorine prior to- determining Ames
mutagenicity of chlorinated natural organic matter (NOM)|
Edward T. Urbansky* and Kathleen M. Schenck
United States Environmental Protection Agency (EPA), Office of Research and Development,
National Risk Management Research Laboratory, Water Supply and Water Resources
Division. Cincinnati, Ohio 45268 USA. E-mail: Urbanskv.Edward@EPA.gov;
Schenck.Kathleen@EPA.gov; Fax: +1 513 569 7658; Tel: +1 513 569 7655
Received 15th November 1999, Accepted 28th January 2000
Published on the Web 29th February 2000
M2P>y potable water disinfection byproducts (DBFs) that result from the reaction of natural organic matter
(NOM) with oxidizing chlorine are known or suspected to be carcinogenic and mutagenic. The Ames assay is
routinely used to assess an overall level of mutagenicity for all compounds in samples from potable water
supplies or laboratory studies of DBF formation. Reduction of oxidizing disinfectants is required since these
compounds can kill the bacteria or react with the agar, producing chlorinated byproducts. When mutagens are
collected by passing potable water through adsorbing resins, active chlorine compounds react with the resin,
producing undesirable mutagenic artifacts. The bioanalytical and chemoanalytical needs of drinking water DBF
studies required a suitable reductant. Many of the candidate compounds failed to meet those needs, including
2,4-hexadienoic (sorbic) £cid, 2,4-pentanedione (acetylacetone), 2-butenoic (crotonic) acid, 2-butenedioic (maleic
and fumaric) acids and buten-2-ol (crotyl alcohol). Candidates were rejected if they (1) reacted too slowly with
active chlorine, (2) formed ftiutagenic byproducts, or (3) interfered in the quantitation of known chlorination
DBFs. L-Ascorbic acid reacts- rapidly and stoichiometrically with active chlorine and has limited interactions
with halogenated DBFs. ID this work, we found no interference from L-ascorbic acid or its oxidation product
(dehydroascorbic acid) s£i mutagenicity assays of chlorinated NOM using Salmonella typhimurium TA100, with
or without metabolic activation (S9). This was demonstrated for both aqueous solutions of chlorinated NOM
and concentrates derived from the involatile, ether-extractable chlorinated byproducts of those solutions.
Aim of investigation
The study of disinfection byproducts (DBFs) in water intended
for human consumption continues to be a concern for the
EPA. In particular, the potential presence of carcinogens and
mutagens remains a focus in the assessment of the risks
associated with chlorinated potable water supplies. To measure
this potential, the Ames assay1'2 for mutagenicity has been
applied to field studies of DBF formation in drinking water and
laboratory studies of chlorinated natural organic matterf
(NOM). Many halogenated byproducts have been identified
and may be directly quaatitated.3"5 However, because of the
nature of NOM, a considerable number of uncharacterized and
potentially genotoxic compounds may result from its reaction
with oxidizing (active) chlorine compounds used for disinfec-
tion. It is neither possible nor practical to identify and
quantitate all of these. Consequently, the Ames assay provides
a convenient assessment of mutagenicity as a bulk property and
allows for whatever synergy or additivity naturally exists.
This laboratory has generally extracted DBFs from potable
.waters using polymeric adsorbent resins, specifically, Amber-
• 'tlite® XAD-2 and XAD-8 (Rohm and Haas, Philadelphia, PA,
USA).2'4'6 This approach has been criticized .because the
fThis paper is the work product of United States government
employees engaged in their official duties. As such, it is in the public
domain and exempt from copyright restrictions. © US government.
Jin keeping with the current convention, we have chosen the term
natural organic matter as opposed to such terms as humic acid, humate,
fulvic acid, or fulvate. NOM is often poorly characterized and
substantially variable from one source water to another. Because
thiols, alcohols, amines, and other moieties can contribute significantly
to the composition of NOM, such terms as humic acid are misleading
and no more descriptive.
residual active chlorine reacts with the resin and produces a
small contribution to the overall mutagenicity of the sample.7
While this contribution is small, it is nonetheless a source of
debate since the artifactual mutagenic compounds remain
unidentified.
Because DBFs continue to form in the distribution system,
the kinetics of DBF formation and thus variation of
mutagenicity with time are of interest. However, attempts to
monitor the Ames mutagenicity as a function of time have been
hampered by the obvious complication that oxidizing chlorine
compounds are bactericidal. Dechlorination with common
reductants, e.g., S2Osz~ or SOs2", caused measurable loss of
chlorinated organic compounds and mutagenicity.7"10 Experi-
mentally, it is difficult to neutralize residual oxidant without
adding a small excess of reducing agent, and residual reductant
concentrations below 30 uM were shown to affect results.8 In
1989, Croue and Reckhow showed that sulfite rapidly attacked
trichloroethanenitrile (Cl3CC=N), dibromoethanenitrile
(Br2CHC=N), and trichloronitromethane (chloropicrin,
C13CNO2).8 In 1985, Wilcox and .Denny demonstrated that
thiosulfate, sulfite, weta-bisulfite, sulfur dioxide, and biotin all
reduced the Ames mutagenicity of chlorinated water sample,
although biotin did not exert this effect when added to a solvent
extract.9 As early as 1980, Cheh et al. found that adding sulfite
to reduce residual chlorine led to a loss of mutagenicity and
suggested that post-chlorination addition of sulfite might be
used to degrade mutagenic compounds in the treatment
process.10
Due to the lack of a satisfactory reducing agent, our
laboratory was resigned to accepting a mutagenic artifact
resulting from reaction of halogen oxidants with organic
matter, i.e., XAD® resins or agar. Attempts to find workable
chlorine-scavenging agents focused on a variety of alkenes,
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such as 2,4-hexadienoic (sorbic) acid, butenoic (crotonic) acid,
(jE)-butenedioic (fumaric) acid, (Z)-butenedioic (maleic) acid,
buten-2-ol (crotyl alcohol), and 2,4-pentanedione (acetylace-
tone, better represented as the enol, 4-hydroxypent-3-ene-2-
one), or on carboxaldehydes, such as dextrose. Nonetheless, all
of these were rejected on account of either insufficiently facile
reaction with active chlorine, mutagenicity of reaction
products, or both.
L-Ascorbic acid, better known as vitamin C, is known to
react stoichiometrically with iodine1' and rapidly with hypo-
chlorous acid.'2 In reaction with halogens or hypohalous acids,
ascorbic acid is oxidized to dehydroascorbic acid, which exists
in several forms and can itself decompose.13 Accordingly, L-
ascorbic acid was deemed a possible choice for scavenging
chlorine in potable water samples or quenching large excesses
of active chlorine in studies on DBF formation.
The characteristics ascribed to an ideal reducing agent were
described previously.14 It has been shown that L-ascorbic acid
interacts minimally with halogenated hydrocarbons (EPA
Method 551.1AB analytes)14 and halocarboxylates (EPA
Method 552.1 analytes).15 Both of these classes of compounds
are found as DBFs and can be determined directly as opposed
to relying on a bioassay for evidence of mutagenic activity. In
drinking water, one of the mutagens of greatest activity is (Z)-
2-chloro-3-dichloromethyl-4-oxo-2-butenoic acid (Mutagen
X),6 and so any interaction with Mutagen X would proscribe
use of a particular reducing agent. Fortunately, Fukui et al.
demonstrated that exposure to L-ascorbic acid at 5 mM for up
to 1 h resulted in no loss of MX.16 L-Ascorbic acid had no effect
on mutagenicity of a number of materials, including coal dust,
tobacco, diesel emissions, and fried beef.17 A number of
investigations have demonstrated a reduction in Ames
mutagenicity due to L-ascorbic acid; however, all of these
involved nitric oxide, nitro- or Af-nitrosp-compounds,18"21
aflatoxin,22'23 or peroxides.24 These compounds do not result
from chlorination except for trichloronitromethane. Peroxides
would not be expected to survive in a raw water, and certainly
not after chlorination. If the other compounds were in fact
present in a potable water supply, we would expect to find them
in both raw and finished water as they are not DBFs. Any
reduction in mutagenicity due to interactions should therefore
be observed in tests of both raw and finished water and the
effects should cancel out.
Experimental procedure§
Reagents
Standard chlorine solutions. Commercially available solu-
tions of 5.0% w/w aqueous sodium hypochlorite (NaOCl) were
standardized by iodometric analysis, kept under refrigeration
at 7+2°C, and used without modification.
Standard thiosulfate solution. A solution of 0.10 M Na2S2O3
was prepared by dissolving the pentahydrate salt in doubly
deionized water. The solution was preserved with 0.1 % w/w
Na2COa to prevent disproportionation and a few drops of
HPLC grade CHC13 to prevent growth of Thiobacillus
thiopants.
Standard iodine solution. Into a 1000 ml volumetric flask,
2.85 g KIO3, 600ml of doubly deionized water, and 170ml
0.25 M H2SO4 were added. A mass of 37.7 g KI was dissolved in
a minimum of doubly deionized water. While stirring
magnetically, the KI solution was slowly added to the>iodic
acid solution. Doubly deionized water was added to volume.
The following comproportionation [eqn. (1)] leads to a
§Mention of specific manufacturers or brand names should not be
construed as endorsement by the United States government.
standard solution that contains primarily triiodide, I3 :
IOJ- + 81 -+6H •*•->• 3Ij- + 3H2O (I)
The excess iodide (~0.1 M) ensures the solubility and limits the
volatility of the I2 by forming the triiodide ion. Some
precipitation of I2 was initially observed, but the iodine
dissolved with 2h of continuous stirring. Total oxidant:
Mr = M + [V] = 0.0400 M.
Stock NOM solution. A mass of 120 g humic acid (Fluka,
Buchs, Switzerland) was combined with 800 ml doubly
deionized water with constant stirring. The pH was adjusted
to 7.0 with dropwise addition of 50% w/w sodium hydroxide,
and the mixture was stirred for 3 h. The mixture was divided
into 250ml polypropylene tubes and ce'ntrifuged for 20min.
The centrifugates were recombined, and the pH was readjusted
to 7.0. Double deionized water was added to bring the final
volume to 1000 ml, and the mixture was permitted to settle
overnight. The mixture was divided and centrifuged. again. The
clear liquid portion was decanted into a low actinic glass bottle
and stored at 4 °C.
Triplicate 3 g samples of stock NOM solution were weighed
into tared vials and dried overnight at 11,0°C; the remaining
solid was 8.63% (by mass) of the initial sample mass. The dried
solid was collected and sent, to Microanalysis, Inc. (Wilming-
ton, DE, USA) for elemental analysis. Found: 49.7 % C, 3.4 %
H, 3.4 % N, 16.0% ash. Calculation of the total organic
carbon concentration from these values gives [C]org=
42.9 gl"1 =3.57 M.
Working NOM solution. Stock NOM solution was diluted
with doubly deionized water to produce a solution containing
2.1 gdl"1 C. The chlorine demand of this solution was
determined using five replicates as follows: Into 40 ml PTFE-
lined screw-cap glass vials, a 10.00 ml aliquot of working NOM
solution was dispensed. Subsequently, 3.00 ml of standard
chlorine solution and 3.0 ml of 0.25 M H2SO4 were added. The
vials were capped and shaken for several minutes, then placed
into a constant temperature bath at 45 °C for 3.5h. When
cooled to room temperature, a 10.00ml portion (excess) of
standard thiosulfate solution was added to each vial. Vials were
recapped and repeatedly inverted for about 10 min. Vial
contents were quantitatively transferred to Phillips beakers and
diluted to about 70 ml with deionized water. To each, 7 ml of
9M H2SO4 and 2g of KI were added. While stirring
magnetically, the excess thiosulfate was back-titrated with
standard iodine solution to a starch-iodide end point. To
reduce foaming and improve visibility of the end point, a few
drops of propanone were added just prior to equivalence.
Chlorine demand is calculated as the ratio of chlorine
consumed relative to organic carbon (molar basis): A[Cl->]r/
A[C]org = A([C12] + [HOC1] + [CIO -])/A[C]org =0.99.
Preparation of mutagenic samples
Chlorinated NOM. To a 1 000 ml volumetric flask, the
following were added: 60.0 ml of stock NOM solution,
100ml of phosphate buffer (0.50 M NaH2PO4 + 0.50 M
Na2HPO4), and 141 ml of stock chlorine solution (0.708 M);
this represents 94% of the calculated chlorine demand. Doubly
deionized water was added to bring the volume to 1000 mi. The
mixture was permitted to react for 24 h; iodometric titrimetry
showed -90% of the active chlorine (HOC1) had been
consumed. Consequently, most of the DBFs should have
been formed. The post-chlorination solution pH was 6.59.
The solution was divided into 100ml portions. Some were
reserved without further treatment; however, within another
24 h, no chlorine could be detected in the unreduced aliquots by
starch-iodide. Although this means that additional chlorinated
byproducts were formed, we assume that the major contribu-
162 J. Environ. Monit., 2000. 2, 161-163
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tors to mutagenicity are derived from the 90% of the active
chlorine that had already reacted. To other portions, ascorbic
acid was added at 2.0 gdl~'; this resulted in immediate
reduction of the remaining 10% of the active chlorine (0.07 M
HOC1) as indicated by starch-iodide. Volumes of both
solutions (unreduced and reduced) were collected and frozen
at —15 °C pending mutagenicity testing.
Extraction and concentration. Three unreduced and three
ascorbic acid-reduced (100ml) portions of the aqueous
solution were extracted with equal volumes of pesticide residue
analysis grade tert-butyl methyl ether (MTBE). Extracts were
rotary evaporated under house vacuum at 60 °C to remove the
bulk of the MTBE. Solid residues were dried for 72 h at 45 °C
to remove any traces of MTBE and then redissolved in 10.0 ml
of HPLC grade dimethyl sulfoxide (DMSO). Assuming that
partitioning strongly favors the MTBE, this step should
concentrate the involatile extractable constituents by a factor
of ~10 relative to the aqueous solutions above. The DMSO
extracts were collected and frozen at — 15°C pending
mutagenicity testing.
Ames mutagenicity assay
Mutagenic activity was determined hi Salmonella typhimuriwn
strain TA100 with and without metabolic activation, using the
standard plate method of Maron and Ames.1 Genetic markers
for TA100 were verified before use. Spontaneous and positive
control (l.Oug NaN3 plate"1 and l.Oug 2-aminoanthracene
plate"1) responses and solvent controls were incorporated in
each assay. L-Ascorbic acid and unhalogenated NOM were
tested separately and found to be non-mutagenic. The samples
were assayed using five dose levels, with duplicate plates per
dose. A repeat assay was done on a separate day. For the
aqueous solutions, the highest dose was 1000 ul; for the DMSO
concentrates, the highest dose was 200 \ii. The liver homo-
genate (S9) was prepared from Aroclor 1254-induced male,
Sprague-Dawley rats (Molecular Toxicology, Inc., Boone, NC,
USA) and the S9 cofactor mix was prepared as described in
Maron and Ames.1 The S9 concentration in the S9 mix was 5%
v/v, and 500 ul of the mix was added per plate.
Results and discussion
Mutagenicity (revertants per unit volume dose) was expressed
as the average slope, calculated by covariance analysis. There
are no significant differences between the slopes of the aqueous
chlorinated NOM with and without ascorbic acid (P=0.1069)
or between the slopes of the DMSO concentrates with and
without ascorbic acid (P- 0.7486) based on covariance analysis
of the without S9 data (Table 1). These results therefore suggest
that ascorbic acid does not reduce the observed mutagenicity of
chlorinated NOM, whether the aqueous solution is tested
directly or a concentrated extract is tested.
Taking into account the intrinsic variability associated with
the Ames assay, the level of mutagenicity observed in the
Table 1 Ames mutagenicity (revertants uF1) for chlorinated NOM
samples
Sample type
HAsc added? Without S9 With S9
Aqueous solutions
DMSO concentrates
N
Y
N
Y
0.20±0.01"
0.17 + 0.01
1.53 + 0.08
1.50 + 0.06
0.07 + 0.01''
0.09 + 0.01A
0.25 ±0.05''
0.46 + 0.04''
"Standard error of the average slope of the dose-response data deter-
mined by covariance analysis. ''The highest dose used in the assays
did not produce a two-fold increase above background (solvent con-
trol value), although there were marginal dose-related increases in
the number of revertants observed.
concentrates is about ten-fold that of the aqueous solutions.
This would seem to confirm our assumption that the mutagenic
compounds are both involatile and MTBE-extractable. In the
presence of S9, the levels of activity in each of the samples are
reduced, consistent with results from previous studies of
chlorination byproducts.4
It appears that ascorbic acid can satisfactorily meet both the
chemoanalytical and bioassay needs associated with monitor-
ing DBF formation, especially in a laboratory study. None-
theless, this work does not demonstrate that ascorbic acid is
suitable under all conditions and for all water supplies.
Investigators wishing to use this compound to scavenge
residual chlorine are cautioned to carefully evaluate the
potential matrix interactions. Certainly, ascorbic acid is the
most promising candidate we have found thus far, and we
anticipate it will prove useful hi future investigations of
chlorination byproduct formation.
Acknowledgements
We acknowledge Mano Sivaganesan for assistance in statistical
analyses and Betty Merriman for assistance in preparing the
stock NOM solution and in carrying out the bioassay.
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Paper a909046k
J. Environ. Monit.. 2000. 2, 161-163 163
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