_2000_ Aerobic and Anaerobic Transformations of

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					     Aerobic and Anaerobic Transformations of Pentachlorophenol in Wetland Soils
                                                Elisa M. D'Angelo* and K. R. Reddy
                          ABSTRACT                                             its degradation of chlorinated toxic organic chemicals
   Strategies to enhance biotransformation of pentachlorophenol                (Renner, 1998).
(PCP) in a spectrum of wetland soils were investigated under labora-              Historically, environmental persistence of PCP and
tory conditions, which included manipulations of electron acceptors            less chlorinated phenols has been attributed to the ab-
and donors, and PCP concentrations. Maximum transformation rates               sence of degrading populations of microorganisms.
were found at PCP concentrations <10 jxA/ (methanogenic condi-                 However, increasing numbers of observations in diverse
tions) and >6 \iM to >23 \s,M (aerobic conditions). Differences in             habitats indicate that transformation potential is wide-
PCP toxicity and sorption among soils and treatments were largely              spread, but is manifested only under favorable environ-
governed by the activities of microbial groups. Within this concentra-         mental conditions. Important variables include temper-
tion range, transformation was observed in soils under aerobic and
methanogenic conditions, but was inhibited under denitrifying and
                                                                               ature (Kohring et al., 1989), availability of electron
SOa -reducing conditions. Aerobic PCP transformation initially pro-
                                                                               acceptors (Haggblom et al., 1993), electron donors (Ku-
duced small amounts of pentachloroanisole (PCA). However >75%                  watsuka and Igarashi, 1975; Chang et al., 1996), nutrients
of both chemicals disappeared in 30 d from five soils. Measured soil           (Mileski et al., 1988; Schmidt, 1996), and toxic metals
properties were not significantly correlated to aerobic transformation         (Kuo and Genther, 1996). In wetland and aquatic sys-
rates. Under methanogenic conditions, PCP was reductively dechlori-            tems, these properties are often present as gradients
nated to yield a mixture of tetra-, tri-, and dichlorophenols in eight         resulting in a continuum of microbial activities.
soils, with rates strongly correlated to measures of electron donor                In aerobic soils, transformation of PCP by Flavobacte-
supply (total C, N, organic C mineralization rates) and microbial              rium and Rhodococcus spp., among others, proceeds
biomass. Addition of protein-based electron donors enhanced reduc-             though sequential hydroxylation and reductive removal
tive dechlorination in a soil low in organic matter and microbial              of chlorine substituents yielding poly-hydroxybenzene
biomass. Results demonstrated the widespread occurrence of PCP
transforming microorganisms in soils, which may be promoted by
                                                                               compounds that are eventually mineralized to CO2 (Uo-
manipulating environmental conditions.
                                                                               tila et al., 1995; Xun et al., 1992). Several Rhodococcus
                                                                               spp. also methylate PCP resulting in production of vola-
                                                                               tile PCA (Middelorp et al., 1990). Several species of
                                                                               fungi mineralize PCP via ligninase enzymes (Mileski
C     ONTAMINATION of the environment with polychlori-
      nated phenols (CPs) is of global concern because
of their widespread distribution and universal toxicity
                                                                               et al., 1988) and produce extracellular enzymes that
                                                                                polymerize CPs with humic substances (Bhandari et al.,
                                                                                1996; Ruttimann-Johnson and Lamar, 1997).
to life (Escher et al., 1996; ATSDR, 1998). The most                               In anaerobic soils, the pathway for anaerobic transfor-
common usage of CPs is treatment of wood against fungi                          mation of PCP is sequential replacement of chlorines by
and insects, but other sources include production from                          hydrogen (reductive dechlorination), leading to phenol,
chlorine bleaching of pulp (Kringstad and Lindstrom,                            benzoate, acetate, CO2 and CH4 (Kuwatsuka and Igara-
1984), combustion of organic matter and municipal solid                         shi, 1975; Zhang and Wiegel, 1990). To date, a few
waste (Kanters et al., 1996), and partial transformation                        anaerobic isolates having the capacity for reductive de-
of phenoxy pesticides such as 2,4-D and 2,4,5-T (Mike-                          chlorination of PCP have been discovered, and these
sell and Boyd, 1985).                                                           often gain energy by coupling this process to oxidative
   Chlorophenols that enter nontarget upland, wetland,                          phosphorylation (Mohn and Tiedje, 1991; Loffler et al.,
and aquatic environments associate with colloidal and                           1996). The resultant lesser chlorinated products from
particulate matter and, if not photodegraded, eventually                        reductive dechlorination likely function as carbon and
settle onto surface soils (Shiu et al., 1994). There they                       energy sources for those aerobic and anaerobic microor-
may be biodegraded, depending on whether degrading                              ganisms involved (Haggblom and Young, 1990).
microorganisms are present and whether appropriate                                 Predicting the persistence of microbially transformed
conditions exist for expression of this activity. There is                      toxic organic contaminants is currently hindered by lim-
still much controversy about whether the presence of                            ited knowledge of the influence of environmental vari-
microbial populations or environmental conditions lim-                          ables on degradation rates (Hart, 1996). Previous exper-
                                                                                iments in numerous wetland soils, however, have
E.M. D'Angelo, Soil & Water Biochemistry Lab., Dep. of Agronomy,
                                                                                demonstrated that heterotrophic microbial activities
Univ. of Kentucky, N-122 Agricultural Sci. Bldg. North, Lexington,              were related to soil properties (D'Angelo and Reddy,
KY 40546-0091; and K.R. Reddy, Univ. of Florida Wetland Bio-                    1999), suggesting that similar relationships may also ex-
geochemistry Lab., Soil and Water Sci. Dep., 106 Newell Hall, P.O.              ist for transformation of toxic organic chemicals. The
Box 110510, Gainesville, FL 32611-0510. Florida Agric. Exp. Stn.
Journal Ser. no. R-07269. Mention of a specific product or trade name
does not constitute endorsement of the University of Florida to the            Abbreviations: A, aqueous concentration; CPs, polychlorinated phe-
exclusion of others. Received 11 Mar. 1999. ""Corresponding author             nols; DCP, dichlorophenol; EC;0, effective concentration; Ka, acid
(edangelo@ca.uky.edu).                                                         dissociation constant; Kp, linear sorption coefficient; PCA pentachlor-
                                                                               oanisole; PCP, pentachlorophenol; T, total soil concentration; TCP,
Published in Soil Sci. Soc. Am. J. 64:933-943 (2000).                          trichlorophenol; TeCP, tetrachlorophenol.

                                                                         933
934                                            SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000

Table 1. Origin, classification, and description of organic and mineral wetland soils used in the study.
State                     Soil name (symbol)                          Taxonomic class                      Description (domination vegetation)
Organic
  Michigan           Houghton Lake Peat (HLPI)        euic, mesic Typic Haplosaprists                      Impacted by domestic waste discharge
                                                                                                              (Typha latifolia)
  Michigan           Houghton Lake Peat (HLPU)        euic, mesic Typic Haplosaprists                      Not impacted (Carex spp.)
  Florida            Everglades (W2)                  euic, hyperthermic Typic Medihemists                 Impacted by P-enriched agricultural
                                                                                                              discharge (Typha spp.)
  Florida            Everglades (W8)                  euic, hyperthermic Typic Medihemists                 Not impacted (Cladium spp.)
  Louisiana          Salt marsh (LSM)                 euic, hyperthermic Typic Haplosaprists               (Spartina spp.)
  North Carolina     Belhaven muck (NCB)              loamy, mixed, dysic, thermic Terric Haplosaprists    Subsided organic agricultural soil
  Florida            Lake Apopka muck (LAAF)          euic, hyperthermic Typic Medifibrists                Subsided organic agricultural soil
Mineral
  Alabama            Talladega (TAL)                  loamy-skeletal, mixed, mesic Rupta-Lithic-Entic,     Freshwater sediment (Juncus spp.)
                                                         Haplaudult
  North Dakota       Parnell (PPP)                    fine, smectitic, frigid Vertic Argiaquolls           Prairie pothole
  Louisiana          Crowley (CR)                     fine, smectitic, hyperthermic Typic Albaqualfs       Paddy soil (Oryza saliva)


objectives of this study were to (i) determine whether                   8-wk period. Samples of W8 (5 mL) and TAL (10 mL) were
PCP transformers are commonly found in wetland soils,                    transferred to serum bottles (60 mL) and sealed with teflon-
and (ii) identify chemical and biological conditions that                lined butyl stoppers and aluminum crimps (Wheaton, Millville,
promote this activity. This investigation attempts to                    NJ). Anaerobic bottles were purged with O2-free N2. Bottles
                                                                         were incubated in the dark at 28°C on a rotary shaker at 180
quantify the boundary conditions for microbial transfor-                 rpm. All experiments were conducted in triplicate. Separate
mations of PCP in soils, including effects of concentra-                 EC50 values, defined as the effective concentration of PCP
tion, sorption, electron acceptors and donors, and micro-                that inhibited microbial activity by 50%, were calculated for
bial biomass.                                                            the activities of CO2 production, methanogenesis, and PCP
                                                                         transformation in both soils. Effective concentrations were
             MATERIALS AND METHODS                                       expressed on both PCP concentration dissolved in soil solution
                                                                         (EC50(diss0|Vf!(i), |jJW) and PCP concentration dissolved plus
              Soil Collection and Incubation                             sorbed to the soil (EC5o(lotai), (jimol kg"1).
   Three mineral and seven organic soils were collected from
various wetlands in the continental USA, including soils from                           Influence of Chemical Amendments
freshwater and estuarine, eutrophic and oligotrophic, organic                       on Pentachlorophenol Transformation
and mineral, and natural and constructed wetlands (Table
1). Soils were previously shown to possess a wide range of                  Before initiation of transformation studies, soil slurries were
biogeochemical properties (D'Angelo and Reddy, 1999), and                pre-incubated for 14 d to obtain either aerobic or anaerobic
selected characteristics are summarized in Table 2. These soils          conditions (denitrifying, sulfate-reducing, or methanogenic).
had no known previous exposure to PCP.                                   For aerobic treatments, slurry (100 mL) was incubated in glass
   Samples of the surface soil were collected with a polyvinyl           media bottles (500 mL) fitted with teflon-lined caps (Wheaton,
chloride (PVC) corer (7.5 cm i.d. 15 cm), transferred to a 4-L           Millville, NJ) and opened twice a week to re-aerate the head-
plastic bottle, and returned in an ice chest to the laboratory           space. For anaerobic treatments, slurry (80 mL) was incubated
by overnight mail. When present, surface water samples from              in serum bottles (160 mL) fitted with teflon-lined rubber stop-
each wetland were also collected. Soils were passed through              pers and aluminum crimps (Wheaton) and purged with O2-
a 0.5 cm2 mesh sieve to remove large plant debris, shells, and           free N2. Different anaerobic electron acceptor treatments were
stones. Soils and water were stored in the dark at 4°C for a             imposed by amending soils with a 30-d supply of a given
maximum of 3 mo before being used in experiments.                        electron acceptor, calculated from consumption rates deter-
   Soils were collected either under drained or flooded condi-           mined previously (D'Angelo and Reddy, 1999). Appropriate
tions and, hence, were initially at different water contents and         electron acceptor reducing conditions were confirmed by mea-
redox potentials. To avoid diffusion constraints and develop-            suring pore water for loss of NO3~ in denitrifying treatments,
ment of microsites during transformation experiments, some               loss of SOI" in SO4~-reducing treatments, loss of NH/ and
soils were prepared as slurries with site water. The amount              production of NO^ and SOJ" in aerobic treatments, and heads-
of water used to prepare slurries was arbitrary except to avoid          pace production of CH4 in the methanogenic treatments. Soils
overdilution of solids and microbial biomass. The dry bulk               with high amounts of bioavailable Fe (PPP, CR, TAL) were
densities of slurries were 0.06 to 0.4 kg L~' for organic soils          evaluated for PCP transformation under Fe(III)-reducing con-
and 0.1 to 0.7 kg L"1 for mineral soils. Although there was a            ditions instead of SOl'-reducing conditions. Iron(III) solution
wide range in bulk densities, these were largely attributed to           was prepared as described by Ghiorse (1994). All bottles were
differences in organic matter content. Results are presented             incubated horizontally with shaking. Preliminary experiments
on a soil dry weight basis unless otherwise indicated.                   showed that this shaking protocol did not influence methano-
                                                                         genic activity, and previous work found it to be optimal for
        Pentachlorophenol Toxicity to Aerobic                            aerobic assays (Stark, 1996). All experiments were conducted
                                                                         in triplicate.
          and Anaerobic Soil Microorganisms                                 After appropriate reducing conditions were attained, slur-
   Toxicity was evaluated under aerobic and methanogenic                 ries were spiked with PCP from a stock solution prepared in
conditions in a selected organic soil (W8) and a mineral (TAL)           0.05 M NaOH to give an average final PCP concentration of
soil using a dose-response approach. Five PCP concentrations             0.66 mmol kg"1 dry soil. This PCP concentration was chosen
between 0 and 3.8 mmol kg~' were tested for effects on rates             because it is the median level measured at contaminated sites
of CO2 and CH4 production and PCP transformations over an                in the USA (ATSDR, 1998). Both aerobic and methanogenic
                      D'ANGELO & REDDY: TRANSFORMATIONS OF PENTACHLOROPHENOL IN WETLAND SOILS                                                                         935


sterile controls were included for the most biologically active                                                                                 rH rH
soil (HLPI), and these were prepared by adding HgCl2 to a                             s n .s                      o ooo ooo                     O OO

final concentration of 2% (soil dry weight basis) alone or with                       | +|                        +1 +1 +1 +1 +1 +1 +1          +1 +1 +1
                                                                                                                  oo f> in M ^t in -^
autoclaving at 121°C at 0.1 MPa for 1 h on three consecutive                          Ids •8
                                                                                      ^ y o-
                                                                                                                  TT t' tfi •<*
                                                                                                                  '"'
                                                                                                                                * pi
                                                                                                                                                313
days. Sample sterility was confirmed by monitoring CO2 and
methane production during the experiment.
    On Days 1, 3, 6,10,15,20,25, and 30, PCP and transforma-                                          "3
tion intermediates were extracted from slurries (1-3 mL) with                          OB             §           f> « t»> rH in p»«            m r<
6 mL acetonitrile containing 0.36 M H2SO4 for 16 h on a                                u.2            B           ooooo eo                      0 O 0

                                                                                       •~ ?                       +1 +1 +1 +1 +1 +1 +1          +1 +1 +1
reciprocal shaker. After centrifugation (700 X g), crude ex-                           "o "^                      in f*} f*j QO «\ ^ t-;        1*- in o
tracts were stored in amber glass vials with teflon-lined caps                                                    f*l rH r*5 O CO f> ^          opi-i
                                                                     c                  ^ &                       f> rH rH rH

at 4°C before derivitization and analysis. Preliminary spike         _e
recovery experiments showed this procedure yielded >95%
recovery of mixed CPs from peat and mineral soils. Similar            01
                                                                     •a                  I
approaches for extraction of pesticides from soil matrices have                          a                        rH rH rH O O rH O             rH |H rH

been described (Chang et al., 1996).
                                                                      tn
                                                                      a
                                                                                         .a
                                                                                         jr
                                                                                                                  o ooooeo                      0 O 0

                                                                                          2                       +1 +1 +1 +1 +1 +1 +1          +1 +1 +1
    Additional experiments were conducted to determine               e                                            \o oo N o \o oo in
whether additions of nutrients, vitamins, and electron donors        S                    I                                                     5SS2
 could promote the methanogenic transformation of PCP in a             B
                                                                     e
 soil that previously lacked this capacity. The protocol used was    +1
 similar to that employed to examine transformations under




                                                                     ; replications


                                                                                         Aerobic pH
                                                                                                                  rH O rH P4 rH rH rH
 different electron acceptor reducing conditions, except a lower                                                                                O O0
 PCP concentration was used (0.13 mmol PCP kg"1 equivalent                                                        + 1 +1 +1 +1 +1 +1 +1         +1 +1 +1
 to 5.8 fxA/ in the dissolved phase) to avoid PCP toxicity. The                                                   vo -t in w ON rj p;           rH P4 ^
                                                                                                                  in >n vo vo fj i/i ("*        TT in K
 following deoxygenated solutions (1 mL) were added to sepa-
 rate glass tubes (27 mL) containing PPP soil slurry (3 mL) to
 provide the following 16 treatments: water (control), inorganic     1                   O
 nutrients (Owens et al., 1979), inorganic nutrients + vitamins      CM
                                                                      O                   -J                      p) m in oo N »t >n            VO rH rH
 (Owens et al., 1979), catechol, benzoate, casein, yeast extract,                        2
 peptone, glucose, sucrose, maleic acid, fructose, maltose, acetic     i
                                                                       E
                                                                                                                  + 1 +1 +1 +1 +1 +1 +1
                                                                                                                  SS8S3S8
                                                                                                                                                + 1 +1 +1
                                                                                                                                                rH rH fl
                                                                                                                                                fi P< rH

 acid, ethanol, and propionate. Hydrogen (5 mL) was added               a
                                                                                         1
 to separate tubes (final concentration of 25 kPa). All carbon-
 based electron donor treatments were added at concentrations         'n
                                                                        VI


 of 88 mmol C kg"1 soil and included amendments with inor-             o                  -H-


 ganic nutrients + vitamins. All treatments were conducted in           a
                                                                        a,                3                       2o oooeo
                                                                                                                        O rH f*> O O 00         0 rH 0
                                                                                                                                                0 OO
 triplicate and, at the end of 5 wk, PCP and transformation             2                 .3                      +1 +1 +1 +1 +1 +1 +1          + 1 +1 +1
                                                                                          *S                                                    PI in pi
 intermediates were extracted from soils as described above.            V
                                                                        9
                                                                                                                  J3 o 3 5 P! S. 2              o in o
                                                                                            o                           O       0 9             O O0
                                                                     1                    b
                     Analytical Methods                              •s
                                                                      n
                                                                      H
   Dissolved SOJ" and NO3~ concentrations were determined                                  •H-         »
by analyzing pore water with a Dionex 4500i ion chromato-              'S                 z           —                 n ^i       ^n
                                                                                                                                                0 OO
                                                                        V
                                                                                             o         „           f*) O O fl rH ^ ^
graph (Sunnyvale, CA). Headspace gases were measured us-                 B                 "8          1           +1 +1 +1 +1 +1 +1 +1         +1 +1 +1
ing gas chromatographs equipped with detectors employing
flame ionization for CH4 and thermal conductivity for O2 and
                                                                         o
                                                                          a.
                                                                          X
                                                                                           r
                                                                                           ^
                                                                                                                  rH O I/I O\ GO ^$ ***
                                                                                                                  rH rH r'l C^ rH r-, |fj
                                                                                                                  rH
                                                                                                                                                S«i3
                                                                          V
CO2. Carbon in living, nonresting microbial biomass was esti-
mated using the substrate-induced respiration technique, as           .s
                                                                       •a                                                                                        1
described previously (D'Angelo and Reddy, 1999).                        1                  B.                      ^ ^ rH O CS I/I M            rH rH W
                                                                                                                                                                 4)
                                                                                                                                                                 •c
   Pentachlorophenol and transformation intermediates pres-            £                   3                       +1 +1 +1 +1 +1 +! +1          + 1 +1 +1
                                                                                                                                                                 B
ent in extracts were prepared as acetylated derivatives and            '3                  jS                      ffi M M rH M W rH
                                                                                                                                                O rH "fl
                                                                                                                                                       1/1
analyzed using a gas chromatograph equipped with a 63Ni
                                                                          t/3
                                                                                                                                                                 S
                                                                       •a                                                                                        a
                                                                       B
electron capture detector (Nicholsen et al., 1992). The identity       2                                                                                         B
and concentration of PCP and transformation intermediates                 V
                                                                          ^
                                                                                                                   O\ ("- C\ f> I-- ^ ^*
                                                                                                                   •** fl fl M rH ^ G\
                                                                                                                                                00 PI
                                                                                                                                                rH rH f)
                                                                                                                                                                 £
                                                                                                                                                                 u
                                                                                           E
were determined by comparing retention times of derivitized            tM                                          + 1 +1 +1 +1 +1 +1 +1         + 1 +1 +1
                                                                           e               3
authentic standards of the highest purity available (>98%)                                                         o 9\ co in o i/) f}
(Supelco, Bellefonte, PA, and Ultra Scientific, Hope, RI).                 a                                       rH ("^ M l/> f'l G^ H
                                                                                                                   GO t- rH O 00 ^f m
                                                                                                                   rH rH fM M
                                                                                                                                                             •° +

                                                                        •S                                                                                   "I
                        Data Analysis                                                                                                            rH rH O
                                                                           a                u          OJD         OOOOOHrH                      0 O 0       1 1
                                                                                                                                                             B b
   Transformation rates of PCP and intermediate metabolites                qj                          ^           -H +1 +1 +1 +1 -H +1          +1 +1 +1
were described by zero-order kinetics (p-mol kg"1 d"1) deter-           1
                                                                                            I                                                     -
                                                                                                                                                 1 ^ 00      'S eg

mined by linear regression analysis. Since experiments were
                                                                                                                                                             rH-e
initiated with high concentrations of PCP under well mixed              ~
                                                                        C«                                                                                   V   g

conditions, these rates may be considered maximum velocity               .
                                                                                                                        .3             ^                     •B ' =

rates, or potentials. As experiments progressed, PCP concen-            _a                                                                  •a ^s.           H|


trations decreased, and transformation rates became a func-                                 jt
                                                                                                             ES     l
                                                                                                                   «4   rJ ?J ^ ^5 y ^

                                                                                                             Sii S ffi 1? ? J Z »J          | H§;S
tion of PCP concentrations. Hence, first order (d"1) rates              1                   5                6                              s                II
936                                                                           SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000

        120                  TAL                                  120i

                                               -»                                                                                                                                           Initial NO., •         Final NO, -
                                                                                                                                                                                            392i6 mmol kg'1        232±18 mmol kg'
                                 •4- aerobic
                                 -B- methanogenic
                                                                                                                         'en
                             ————————B                                                                                  ^
                                                                                                                        "3
                0
                    EC
                     SO(lotal)
                         0.75          1.5            2.25
                                                                                      *-SO(total)

                                                                     0 1.25 2.5 3.75 5.0                                                        10           20                                        10        20
                                                                                                                        E
       120                                                               E.                                                    0.8- C.                                              0.8 • D.
                                                                                                                                                                                                              -•-PCP
                                                                                                                                      *———————————— £                                                         -*- 2,3,4,5 TeCP
                                                                                                                               0.6-               1
                                                                                                                                         Initial SO, -            Final SO, *•
                                                                                                                                         254±17 mmol kg-1         86±14 mmol kg-1   „ ,                       -•-3,4,5 TCP
                                                                                                                               0.4-

                                                                                                                               0.2-
                                                                                                                                                                                    0.4 •

                                                                                                                                                                                    0.2-    [\
                                                                                                                                                                                             A
                                                                                                                                                                                                              -«-3,5DCP




                                                                                                                                     0           10          20           30                0          10         20         30

                                                                                                                               0.61 E.

                                                                                                                               0.4
                                                                                                                                                 ^k=
                                                                                                                               0.2-

                                                                                                                               0.0
                                                                                                                                                             20

                                                                                                                                          Time (d)                        Time (d)
                                     l.S            2.25            0 1.25 2.5 3.75 5.0
                                                                                                                      Fig. 2. Microbial transformation of pentachlorophenol (PCP) in
                     Initial PCP concentration (mmol kg"1)                                                               Houghton Lake constructed marsh soil (HLPI) under four electron
                                                                                                                         acceptor reducing conditions, and in aerobic and methanogenic
Fig. 1. Influence of pentachlorophenol (PCP) concentration on activ-                                                     sterile controls: (A) O2; (B) NO3~; (C) S O S ; (D) CO2; (E) O2 +
   ities of aerobic and methanogenic microorganisms in mineral TAL                                                       2% HgCl2; and (F) CO2 + 2% HgCl2. Dotted lines in (E) and (F)
    (A-C) and organic W8 (D-F) wetland soils: (A) and (D) CO2                                                            represent autoclave + HgCl, treatments. Initial and final NO.f and
   production; (B) and (E) CM, production; and (C) and (F) PCP                                                            S(>4~ refer to concentrations at the beginning and end of the experi-
   degradation. Each value represents the mean of three                                                                  ment. Each value represents the mean of three replications ± one
   replications ± one standard deviation. EC5l)(M,i) represents the total                                                standard deviation.
    PCP concentration in the soil (dry weight basis) that inhibits micro-
   bial activity by 50%.
                                                                                                                                                                   RESULTS
were also determined from nonlinear regression analysis. Rate                                                                  Pentachlorophenol Toxicity to Aerobic
constants were calculated from data generated after any lag
                                                                                                                                and Anaerobic Soil Microorganisms
period. The length of the lag period was defined as the period
before a significant (P < 0.05) decrease in concentration was                                                            Aerobic and methanogenic activities (CO2 and CH4
observed between successive time increments, as determined                                                            production and PCP transformation) were typically in-
by t statistic.                                                                                                       hibited when soils were amended with increasing con-
   In separate batch isotherm experiments, linear sorption co-
                                                                                                                      centrations of PCP (Fig. 1). However, specific effects
efficients (Kf, L kg"1) for PCP were determined for all soils
                                                                                                                      differed between aerobic and anaerobic treatments and
examined (D'Angelo, 1998). These were then used to convert
total PCP concentration (T, mmol kg"1) to aqueous concentra-                                                          between mineral and organic soils. In the mineral TAL
tions (A, mmol L"1):                                                                                                  soil, CO2 and methane production, and PCP transforma-
                                                                                                                      tion (under methanogenic conditions only), were signifi-
                            A = T • (Kf                    + 9 • pb"1)-1                                  [1]         cantly reduced at >0.38 mmol PCP kg"1 soil. In contrast,
where 9 is the volumetric water content (L water L"1 soil)                                                            CO2 production and PCP transformation were relatively
and pb is dry bulk density (kg L"1) of soil slurry.                                                                   unaffected at any PCP concentrations tested under aero-

Table 3. Inhibition of aerobic and methanogenic microbial activities by pentachlorophenol (PCP) in mineral TAL and organic W8
  wetland soils.
                                                                                            TAL                                                                                             W8
                                                                  Aerobic                                  Methanogenic                                     Aerobic                                         Methanogenic
Parameter                         Units                    C02                PCP                   CO2         CH4            PCP                    C02                PCP                    CO2             CH4               PCP
                                           1
ECso (total)t              mmol 1kg"                        >1.9              >1.9               0.48            0.68           0.56                  >3.8                 >3.8                  3.0            >3.8              1.2
Kpt                        Lkg" 1                           307               307               32              32             32                     147                  147                  99              99                99
<e • P-')§                 Lkg-                               6                 6                   7            7              7                      17                   17                  18              18                18
ECso (dittolved)H          (unol L '                        >6.0               >6.0             12              17             14                     >23                 >23                   26             >32                10
t PCP concentration on a soil dry weight basis that inhibited microbial activity by 50%; EQoitoui) is effective concentration.
£ Soil:water partition coefficient determined in separate experiments (D'Angelo, 1998). Values are summarized in Tables 4 and 6.
§ Ratio of volumetric water content (6) and bulk density (p) of soil slurries used in the study.
H PCP concentration dissolved in the soil solution that inhibited microbial activity by 50%, calculated from Eq. [1] in text.
                                                                                                                                                                                                                                                                                                                                                                                        Overall loss




                                                                                                                                                                                                                                                                                                                                                                                                                                       75 ± 11
 Chlorophenol concentration (mmol kg'1)




                                                                                                                                                                                                                                                                                                                                                                                                                                       96 ± 1
                                                                                                                                                                                                                                                                                                                                                                                                                                      100 ± 0




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                87 ± 1
                                                                                                                                                                                                                                                                                                                                                                                          in 30 d




                                                                                                                                                                                                                                                                                                                                                                                                                                       95 ± 0
                                                                                                                                   O




                                                                                                                                                                                                                                                                                                                                                                                                                       %




                                                                                                                                                                                                                                                                                                                                                                                                                                         0
                                                                                                                                                                                                                                                                                                                                                                                                                                         0
                                                                                                                                                                                                                                                                                                                                                                                                                                          0




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              0
                                                                                                                                   OC
                                                                                                                                   O\
                                                                                                                                   *•
                                                                                                                                   K»
                                                                                                                                   O




                                                                                                                                                                                                           Table 4. Aerobic transformations of pentachlorophenol (PCP) in wetland soils. Each value represents the mean of three replications ± one standard deviation.




                                                                                                                                                                                                                                                                                                                                                                                                                                                                      0.009
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.042
                                                                                                                                                                                                                                                                                                                                                                                                                                                      0.023
                                                                                                                                                                                                                                                                                                                                                                                                                                                      0.006




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                0.161 ± 0.011
                                                                                                                                                                                                                                                                                                                                                                                        First-order
                                                                                                                                                                                                                                                                                                                                                                                           rate
                                                                                                                                                                                                                                                                                                                                                                                                                      d-'

                                                                                                                                                                                                                                                                                                                                                                                                                                      0.139 ±
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.338 ±
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.224 ±

                                                                                                                                                                                                                                                                                                                                                                                                                                      0.139 ±
                                                                                                                                                                                                                                                                                                                                                                                                                                            0
                                                                                                                                                                                                                                                                                                                                                                                                                                            0
                                                                                                                                                                                                                                                                                                                                                                                                                                            0




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              0
                                              00 o
                                              O\
                                              4*.
                                              s»




                                                                                                                                                                                                                                                                                                                                                                          PCP loss



                                                                                                                                                                                                                                                                                                                                                                                                                    H-mol kg"1 d"1
                                              O




                                                                          JB 0a B - f & J g° t




                                                                                                                                                                                                                                                                                                                                                                                        Maximum




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                45 ± 1
                                                                                                                                                                                                                                                                                                                                                                                                                                                      32 ± 3
                                                                                                                                                                                                                                                                                                                                                                                                                                                      77 ± 7



                                                                                                                                                                                                                                                                                                                                                                                                                                                                      44 ± 6
                                                                                                                                                                                                                                                                                                                                                                                                                                      44 + 8
                                                                                                 *||ti,
                                                               „




                                                                                                                                                                                                                                                                                                                                                                                          rate




                                                                                                                                                                                                                                                                                                                                                                                                                                         0

                                                                                                                                                                                                                                                                                                                                                                                                                                                               0
                                                                                                                                                                                                                                                                                                                                                                                                                                                               0




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   0
                                                                                                                                                      o c/5 c 13 H

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                                                                                                                                                                                                                                                                                                                                                                                        time
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                                                                                                                                                                                                                                                                                                                                                                                                                                                       NA




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 NA

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       NA

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                                                                                                                                                                                                                                                                                                                                                                                                                      d
bic conditions. Therefore, EC50 for aerobic samples
could not be calculated. However, based on the maxi-




                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0002



                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0003
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0002
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0027
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0034

                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0006




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                0.0012 ± 0.0005
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0030
                                                                                                                                                                                                                                                                                                                                                                                                 First-order rate
mum concentration tested, the EC50(totai) (PCP concentra-
tion on a dry soil basis) was >1.9 mmol kg"1 soil for
aerobic activities compared to between 0.5 to 0.7 mmol                                                                                                                                                                                                                                                                                                                                                              d-1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              0
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0017
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0029
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0025
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0099


                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0009
                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0056


                                                                                                                                                                                                                                                                                                                                                                                                                                      0.0030
kg ' soil for methanogenic populations (Table 3).
   For the organic W8 soil, similar trends in toxicity
were observed as for TAL, except that the threshold
                                                                                                                                                                                                                                                                                                                                                                          Methylation




concentrations of PCP for microbial inhibition were sig-
nificantly higher (Fig. 1 and Table 3). For example, the
                                                                                                                                                                                                                                                                                                                                                                                                 Maximum rate
                                                                                                                                                                                                                                                                                                                                                                                                                    nmol kg"1 d"1




                                                                                                                                                                                                                                                                                                                                                                                                                                                      1.1 ± 0.1
                                                                                                                                                                                                                                                                                                                                                                                                                                                      1.1 ± 0.1
                                                                                                                                                                                                                                                                                                                                                                                                                                      1.8 ± 0.2
                                                                                                                                                                                                                                                                                                                                                                                                                                                      1.2 ± 0.3
                                                                                                                                                                                                                                                                                                                                                                                                                                      3.5 ± 1.9
                                                                                                                                                                                                                                                                                                                                                                                                                                                      2.4 ± 1.2




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                0.9 ± 0.3
EC5o(totai) for aerobic activities and methanogenesis were
                                                                                                                                                                                                                                                                                                                                                                                                                                                        0+ 0
>3.75 mmol kg"1, and between 1.2 and 3.0 mmol kg"1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     II Lag time longer than 30-d experimental period; NA, not applicable; ND, not determined.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    0

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              0
for anaerobic PCP transformation and CO2 production.




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     § Initial PCP concentration in the dissolved phase calculated from Eq. [1], see text.
                                                     Influence of Chemical Amendments
                                                           on PCP Transformation
                                                                                                                                                                                                                                                                                                                                                                                                                                                         6±0
                                                                                                                                                                                                                                                                                                                                                                                                                                                         6±0



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                5±2
                                                                                                                                                                                                                                                                                                                                                                                        time




                                                                                                                                                                                                                                                                                                                                                                                                                                                         NDfl
                                                                                                                                                                                                                                                                                                                                                                                         Lag




                                                                                                                                                                                                                                                                                                                                                                                                                                                          <1
                                                                                                                                                                                                                                                                                                                                                                                                                                      <1
                                                                                                                                                                                                                                                                                                                                                                                                                                      <1
                                                                                                                                                                                                                                                                                                                                                                                                                                      <1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                NA
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                NA

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       NA
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              NA
                                                                                                                                                                                                                                                                                                                                                                                                                       d




   The presence or absence of specific types of electron
acceptors had significant effects on rates and mecha-
nisms of PCP transformation in wetland soils (Fig. 2
and 3; Tables 4-6). Transformation was attributed to
                                                                                                                                                                                                                                                                                                                                                                                                                      nmol L-'




                                                                                                                                                                                                                                                                                                                                                                                                                                       2.82
                                                                                                                                                                                                                                                                                                                                                                                                                                       2.28
                                                                                                                                                                                                                                                                                                                                                                                                                                       0.83


                                                                                                                                                                                                                                                                                                                                                                                                                                       0.36



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  2.34
                                                                                                                                                                                                                                                                                                                                                                                        Initial




biological activity since HgCl2 + autoclaved samples
                                                                                                                                                                                                                                                                                                                                                                                        PCP§




                                                                                                                                                                                                                                                                                                                                                                                                                                       1.25


                                                                                                                                                                                                                                                                                                                                                                                                                                       1.29
                                                                                                                                                                                                                                                                                                                                                                                                                                      10.4


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 12.8
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                238




showed no changes in PCP concentration during the
study (Fig. 2). Except for two soils (PPP and CR), PCP
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     I Determined in a separate study (D'Angelo, 1998).




transformation occurred in each soil under at least one
electron acceptor-reducing condition.
                                                                                                                                                                                                                                                                                                                                                                                        coefficient (A'p)i
                                                                                                                                                                                                                                                                                                                                                                                        Linear sorption




   Under aerobic conditions, eight soils transformed
PCP, including all organic soils and one mineral soil
                                                                                                                                                                                                                                                                                                                                                                                                                    Lkg- 1




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  1.3




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     t See Table 1 for soil name symbols.
                                                                                                                                                                                                                                                                                                                                                                                                                                      931



                                                                                                                                                                                                                                                                                                                                                                                                                                                                463
                                                                                                                                                                                                                                                                                                                                                                                                                                                         147
                                                                                                                                                                                                                                                                                                                                                                                                                                      549
                                                                                                                                                                                                                                                                                                                                                                                                                                      225




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                307
                                                                                                                                                                                                                                                                                                                                                                                                                                                                 50


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 49
                                                                                                                                                                                                                                                                                                                                                                                                                                                               2017




(TAL) (Table 4). Pentachlorophenol transformation
proceeded by at least two different mechanisms. Within
the first week after PCP treatment, pentachloroanisole
(PCA) was detected in seven soils as a methylation
product of PCP, with maximum rates of between 0.9
and 3.4 (xmol kg"1 d"1 and first order rates between
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     HLPI + Hg +




0.0009 and 0.01 d"1. Between 4 and 20 d, four of the
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         autoclave
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  Sterile controls
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     HLPI + Hg




organic soils (HLPI, W2, W8, and LAAF) and the min-
                                                                                                                                                                                                                                                                                                                                                                                                                                       HLPU



                                                                                                                                                                                                                                                                                                                                                                                                                                                                        LAAF
                                                                                                                                                                                                                                                                                                                                                                                                                                       HLPI




eral soil TAL additionally exhibited losses of PCA.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                NCB
                                                                                                                                                                                                                                                                                                                                                                                                                                     Organic




                                                                                                                                                                                                                                                                                                                                                                                                                                       LSM



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                TAL
                                                                                                                                                                                                                                                                                                                                                                                                                                                                      Mineral

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                PPP
                                                                                                                                                                                                                                                                                                                                                                                                                                       W8
                                                                                                                                                                                                                                                                                                                                                                                                                                       W2




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                CR
                                                                    !
                                                               9.




                                                                                                                                   o




                                                                                                                                                                                                                                                                                                                                                                                                  Soilt
                                                                                                                                                    3
                                                                          C/3

                                                                                           *1




                                                                                                                     0)
                                                                                           P=




                                                                                                                                                                                             X
                                                                                                                                                                                             fa
                                                                                                                 D.




                                                                                                                                              O.




                                                                                                                                                                                        h+i
                                                                                                 3

                                                                                                           P5
                                                               o>

                                                                    a




                                                                                                                                                                             P
938                                                        SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000

Table 5. Anaerobic transformation rates of pentachlorophenol (PCP) and reductive dechlorination intermediates (tetra-, tri-, and di-
  chlorophenols) in wetland soils in the presence of inorganic electron acceptors. The sequential dechlorination pathway was PCP—>2,3,4,5
  tetrachlorophenol (TeCP)—»3,4,5 trichlorophenol (TCP)—'3,5 dichlorophenol (DCP) unless otherwise indicated. Each value represents
  the mean of three replications ± one standard deviation. Values in parentheses are first-order rates (d ').
                                                                                                        Siilfate
                                                                                                                                                                       Overall loss
Soilt                Nitrate              Lag time                PCP         —
                                                                            —— >          TeCP          ————>          TCP            —— > DCP
                                                                                                                                       —                                in 30 d
                 p.mol kg"1 d~'              d                                   ——————— (xmol kg"1 d"1 ————————
Organic
  HLPI                0                    NAt                        0                     ND                                                                                0
  HLPU                0                    NA                      0                        ND                                                                             0
  W2                  0                   15 ±0              39 ± 2 (0.29)            54 ± 12 (0.20)               21 ±S (0.027)                      ND                33 ± 26
  W8                  0                    NA                      0                        ND                                                                             0
  LSM            8 ± 3 (0.009)              <1               23 ± 3 (0.065)            2±S (0.005)                      trfl                                            13 ± 19
  NCB                 0                    NA                      0                        ND                                                                             0
  LAAF                0                   12 ± 0             40 ± 10 (0.22)#           27 ± 3 (0.25)               23 ± 7 (0.10)               22 ± 9 (0.09)            69 ± 15
Mineral
  TALft                0                   NA                         0                     ND                                                                                0
  PPPtt                0                   NA                         0                     ND                                                                                0
  CRtt                 0                   NA                         0                     ND                                                                                0
t See Table 1 for soil name symbols.
t Lag phase longer than 30 d experimental period.
§ Not detected.
II Trace amounts of 3,4,5-TCP were detected.
# Degradation pathway was PCP-»2,3,4,5-, 2,3,5,6,-, 2,3,4,6-TeCP—23,4,5-, 3,4,5-TCP.
ft These soils were evaluated for PCP reduction under Fe(III)-reducing conditions instead of sulfate-reducing conditions.

order rates between 0.139 and 0.338 d"1. No other chlori-                                transformation of PCP (Table 5). Under SO^-reducing
nated organics were detected. This indicated the pres-                                   conditions, three organic soils (W2, LSM, and LAAF)
ence of either mineralization or chemical binding-                                       showed loss of PCP. After lag periods of between 1 and
attachment to soil. In these five soils, total loss of PCP                               15 d, PCP was transformed at maximum rates between
and PCA ranged between 75 and 100% in 30 d. The                                          23 and 40 (xmol kg"1 d"1 and first-order rates between
remaining five soils showed no loss of PCP during the                                    0.065 and 0.29 d"1. The dominant mechanism of PCP
incubation period.                                                                       transformation was sequential reductive dechlorination
   Under denitrifying conditions, only LSM transformed                                   to tetrachlorophenol (TeCP), trichlorophenol (TCP),
PCP, as indicated by accumulation of trace amounts of                                    and dichlorophenol (DCP), which occurred primarily
the reductive dechlorination product 2,3,4,5-TeCP (Fig.                                  through the pathway PCP-»2,3,4,5-TeCP-^3,4,5-TCP->
2 and 3; Table 5). Of those soils tested under Fe (III)-                                 3,5-DCP, demonstrating a preference for removal of
reducing conditions (PPP, TAL, and CR), none showed                                      chlorines ortho- and para- to the hydroxyl group. The

Table 6. Transformations of pentachlorophenol (PCP) and reductive dechlorination intermediates (tetra-, tri-, and di-chlorophenols) in
  wetland soils under methanogenic conditions. The sequential dechlorination pathway was PCP—>2,3,4,5 tetrachlorophenol
  (TeCP)—>3,4,5 trichlorophenol (TCP)—>3,5 dichlorophenol (DCP) unless otherwise indicated. Each value represents the mean of
  three replications ± one standard deviation. Values in parentheses are first-order rates (d~').
                      Linear sorption            Initial         Lag                                                                                                   Overall loss
Soilt                 coefficient (K,)$          PCP§           time             PCP        - -*• TeCP           — -> TCP                      — *•    DCP               in30d
                                  j                                                                          _ M ,___, j_,                                                   0/
                                             (jimol L"1           d
Organic
   HLPI                        116                 5.52         6±0       70 ± 9 (0.26)        70   ±
                                                                                                    7 (0.24)            65     ±    5 (0.23)     65   ±    5 (0.25)     97 ± 0.6
   HLPU                        314                 2.32        13 ± 4     64 ± 9 (0.18)        77   4 (0.23)
                                                                                                    ±                   59     ±    9 (0.10)     59   ±    9 (0.10)     63 ± 14
   W2                          88                  5.58         6±0       60 ± 4 (0.39)        66   ±
                                                                                                    7 (0.50)            45     ±    2 (0.24)     45   ±    2 (0.24)     99 ± 0.2
   W8                           99                 5.51         9 ± 2     63 ± 4 (0.38)        57   ±
                                                                                                    7 (0.44)            53     ±    9 (0.41)     49   ±    16 (0.39)    99 ± 0.2
   LSM                          66                11.0                    25 ± 2 (0.082)             0                             Irll                   ND#              0
   NCB                         286                 2.89         NAtt            0                   ND                                                                     0
   LAAF                         48                 6.50         4± 0      38 ± 2 (0.21)ft      39 ± 13 (0.23)           38 ± 3 (0.16)            22 ± 8 (0.17)          93 ± 1.4
Mineral
   TAL                          32                19.4           NA               0                     ND                                                                    0
   PPP                          13                50.0           NA               0                     ND                                                                    0
   CR                            1.8             159             NA               0                     ND                                                                    0
Sterile controls
   HLPI + Hg                                                     ND       25 ± 2 (0.15)             (0.15)                     (0.018)                    ND                  0
   HLPI + Hg +                                                   NA               0                  ND                                                                       0
       autoclave
t See Table 1 for soil name symbols.
t Determined in D'Angelo (1998).
§ Initial PCP concentration in the dissolved phase calculated from Eq. [1], see text.
H Trace levels detected.
# Not detected.
ft Lag phase longer than 30 d experimental period.
                          D'ANGELO & REDDY: TRANSFORMATIONS OF PENTACHLOROPHENOL IN WETLAND SOILS                                                        939

Table 7. Relationships between soil properties and maximum rates of pentachlorophenol (PCP) transformation ((unol kg'1 d ') in
  wetland soils. These relationships apply to soils with PCP concentrations <10 |JtM in the dissolved phase.
                                                                  Aerobic transformation rate            Methanogenic transformation ratef
                                                                                         2
Soil property (*)                         Units                   Equation              r                  Equation                     r2
First order rate                    d-i                             230 A:            0.89***            196 x                       0.80**               10
Total C                             mmol g '                        NS|                                  1.7 x - 2.7                 0.58*                 7
Total N                             ixmol N g"                      NS                                   35 In x - 200               0.77**                7
Total P                             |unol N g~                      NS                                   30 Injr - 43                0.49*                 7
Microbial C                         (jimol C g~                     NS                                   35 In x - 80                0.94***               7
Aerobic CO2 + CH,                   (jimol C g" d'1                  NS                                  29 In x - 20                0.66*                 7
Anaerobic CO2 + CH4                 ixmol C g~ d^1                   NA§                                 22 In x + 17                0.83**                7
Inorganic Nil                       ixmol N g~                       NS                                  19 In x - 17                0.71*                 7
Organic N                           (jimol N g~                      NS                                  35 In x - 200               0.77**                7
Unavailable P1[                     ixmol P g~                       NS                                       NS                                           7
*, **, *** Significance at the P =s 0.05, 0.01, and 0.001 levels, respectively.
t Nitrate and sulfate inhibited degradation under anaerobic conditions.
t Not significant at P £ 0.05.
§ Not applicable.
11 Determined as water soluble + KCl-exchangeable nutrients.

agricultural LAAF soil exhibited more complex dechlo-                             peptone (37%), and acetate (5.1%) treatments (data not
rination pathways, in which 2,3,5,6-TeCP, 2,3,4,6-TeCP,                           shown). The dominant pathway was through reductive
and 2,3,5-TCP were also detected, indicating sequential                           dechlorination yielding 2,3,4,5-TeCP and 3,4,5-TCP;
para-, ortho-, as well as meta- dechlorination pathways.                          however, no net loss of CPs was observed.
After 30 d, losses of PCP and breakdown intermediates
were between 13 and 69% for these three soils (Table                                  Relationships between PCP Transformation
5). Monochlorophenols were not detected in any of                                             Kinetics and Soil Properties
the samples.
   Under methanogenic conditions, six organic soils                                  Regression analysis showed that none of the mea-
transformed PCP, but none of the mineral soils did so                             sured soil properties was significantly (P < 0.05) corre-
(Fig. 2 and 3; Table 6). For the transforming soils, there                        lated to the duration of the lag periods before detection
was a lag phase of between 1 and 13 d before loss                                 of PCP transformation. Moreover, none of the mea-
of PCP, after which maximum rates were found to be                                sured soil properties was significantly correlated to aero-
between 25 and 70 (jimol kg"1 d"1 and first-order rates                           bic PCP transformation rates (Table 7).
between 0.082 and 0.39 d"1. As observed under SOi~                                   Pentachlorophenol transformation under anaerobic
-reducing conditions, the dominant mechanism for                                  conditions was typically inhibited by the presence of
transformation was reductive dechlorination, which usu-                           NO3~, Fe(III), SO?- (Table 5) and dissolved PCP con-
ally followed the sequence PCP->2,3,4,5-TeCP—3,4,5-                               centrations >10 to 14 (jiM (Table 3, see discussion).
TCP—>3,5-DCP. Again, LAAF exhibited more complex                                  Hence, to predict anaerobic PCP degradation rates in
dechlorination pathways than other soils, including                               soils, one must ensure that conditions are methanogenic
PCP-»2,3,5,6-TeCP—2,3,5-TCP, 2,3,6-TCP->3,5-DCP                                   and that the dissolved PCP concentration is below the
and PCP—2,3,4,6-TeCP—2,3,6-TCP. The sum of losses                                 toxicity threshold level. After taking these considera-
of PCP and intermediates ranged between none and                                  tions into account, several soil properties were signifi-
99% in 30 d (Table 6).                                                            cantly correlated to PCP transformation, including total
   Rate constants for PCP transformation intermediates                            organic C, N, and P content, microbial biomass, aerobic
were determined by summing the concentrations for a                               and anaerobic carbon mineralization rates, and bioavai-
given intermediate and all preceding products at each                             lable N. Microbial C accounted for more than 90% of
time step, and calculating the least squares fit through
the points (Tables 5 and 6). For soils capable of PCP
transformation under SOij'-reducing conditions, the
rate-limiting steps in overall CP loss were reductive
 dechlorination of TeCP, TCP, and DCP, as indicated                                                                               O HLPI

 by accumulation of these intermediates compared to
 the parent compound PCP. For example in LSM soil,                                                                             y = 35 Ln(x) - 80
 PCP was almost completely transformed to 2,3,4,5-                                                                                 r2 = 0.94
 TeCP, which was not reduced further (Table 5). Under
 methanogenic conditions, most soils (except LSM) dem-
 onstrated similar dechlorination rates for CPs con-                                                 0                            100              150
 taining high and low numbers of chlorine substituents
 (Table 6).                                                                                                        Microbial C (mmol C kg-')

    In PPP soil maintained under methanogenic condi-                               Fig. 4. Relationship between microbial biomass C and maximum
                                                                                      pentachlorophenol (PCP) degradation rate in seven wetland soils
tions and amended with nutrients, vitamins, and elec-                                 under methanogenic conditions. This relationship applies to soils
tron donors, PCP transformation was only observed in                                  where PCP concentration in the dissolved phase was less than the
the water-alone control (8.1%), yeast extract (34%),                                  toxic level of 10 (J.M (see discussion).
940                                      SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000


the variability in rates of reductive dechlorination of           plains why these soils did not transform PCP. In compar-
PCP (Fig. 4).                                                     ison, the TAL and PPP soils transformed PCP in the
                                                                  toxicity and electron donor studies when concentrations
                                                                  were below threshold levels.
                     DISCUSSION                                      Transformation under both aerobic and anaerobic con-
   Our results indicate that most wetland soils contain           ditions was also restricted at concentrations of PCP <
microorganisms with the capacity to transform PCP,                0.3 u,M, at which PCP transformation rates became first
despite no known history of contamination. However,               order (Fig. 2d, 3a, and 3d). At low concentrations, slow
expression of this capacity was regulated by chemical             desorption kinetics plays a major role in regulating
and biological conditions, including contamination                transformation rates (Schlebaum et al., 1998). More-
level, type of electron acceptors and donors, microbial           over, in soils contaminated for long periods, PCP and
biomass, and the co-contaminant (Hg (II)).                        intermediates may become less bioavailable as they dif-
   High (>10 (jiAf) and low (<0.3 jjJVf) PCP concentra-           fuse into microbially inaccessible soil zones. Therefore
tions typically restricted transformation in soils. This          rates in the present study, in which experiments were
inhibition was attributed to toxicity and limited bioavail-       conducted in freshly contaminated soils, may overesti-
ability, respectively. Pentachlorophenol toxicity results         mate actual rates. Our results and those of others (Apa-
from its influence on energy transduction processes car-          jalahti and Salkinoja-Salonen, 1984; Bellin et al., 1990
ried out by the cell (Escher et al., 1996). These effects         Mileski et al., 1988; Mohn and Kennedy, 1992), clearly
were reflected as decreased rates of CO2 production,              show the influence of contaminant concentration on
methanogenesis, and PCP transformation.                           transformation of toxic organics in soils, and the role
   Comparing EC50(totai) values, PCP was apparently more          of sorption in regulating contaminant bioavailability.
toxic to methanogenic activities compared to aerobic              Sorption of PCP may be controlled by manipulating pH,
activities and in mineral soils compared to organic soils.        types of microbial activity (e.g., aerobic and anaerobic),
These results can at least be partially explained by differ-      and organic C content in contaminated soils.
ences in sorption, which was greater under aerobic con-              Availability of electron acceptors was also a key regu-
ditions and in organic soils (Tables 3, 4, and 6). For            lator of both rates and pathways of PCP transformation
example, sorption is known to provide a protective                in soils. In the presence of O2, most soils produced
mechanism by removing PCP from the soluble (bioavai-              pentachloroanisole (PCA) within 1 d after PCP treat-
lable) pool (Apajalahti and Salkinoja-Salonen, 1984).             ment. Methylation is mediated by common aerobic bac-
Sorption of chlorophenols is increased in soils with high         teria and fungi such as Rhodococcus rhodochrous, Pha-
concentrations of H + (pKa of PCP = 4.74), dissolved              nerochaete chrysosporium, and P. sordida (Middelorp
cations, and soil organic matter (Westall et al., 1985;           et al., 1990; Lamar et al., 1990). Subsequently, PCP and
D'Angelo, 1998). Aerobic processes such as nitrification          PCA were lost from many soils without the appearance
and sulfide oxidation tended to acidify soils, while an-          of chlorinated intermediates, indicating either mineral-
aerobic activities produced higher pH values (Table 2).           ization to CO2 or chemical binding-attachment with
Hence, it was expected that PCP transformations would             other pesticide moieties or humic substances. Assuming
proceed at higher concentrations in aerobic and organic           degradation was the dominant mechanism, the maxi-
soils which matched with experimental results (Table 3).          mum PCP loss rates of up to 77 (xmol kg"1 d""1 observed
   Using Eq. [1], average concentrations of PCP dis-              in this study are typically higher than those measured
solved in soil solution ranged between 0.4 and 238 jxM            previously (Briglia et al., 1994; Haggblom and Valo,
(Tables 4 and 6). The EC50(dissoived) for PCP transformation      1995; Laine and Jorgensen, 1997). Higher rates in the
(i.e., concentration of dissolved PCP that decreased PCP          present study perhaps reflected an exclusion of diffusion
transformation by 50%) was 10 to 14 u.M for methano-              constraints by constant shaking.
genic treatments (Table 2). The EC50(diSsoived) for aerobic          Among the soil properties measured, none was signif-
treatments could not be calculated because inhibition             icantly correlated to aerobic transformation rates, indi-
was not observed at any concentration tested (up to               cating that availability of C, inorganic N (ranging be-
6 |xM for the mineral soil and 23 u.M for the organic             tween 1330 and 9770 u,M), and soluble P (ranging
soil). These results suggest that aerobic microorganisms          between 3 and 488 u,M) were not the primary regulators
were less affected by PCP level than methanogenic con-            in the soils tested (Table 7). Schmidt (1996), however,
sortia. These results generally agree with others, with           found a highly significant correlation between aerobic
reported toxicity values ranging from 15 to 1900 jjuM             transformation rates in PCP-contaminated groundwater
for aerobes (Mileski et al., 1988; Stanlake and Finn,             and soluble P between 8 and 90 \iM. As observed in
1982) and 0.45 to 10 |o,M for anaerobes (Mohn and                 our study, Laine and Jorgensen (1997) also found no
Kennedy, 1992; Wu et al., 1993; Uberoi and Bhatta-                correlation between bacterial biomass and aerobic PCP
charya, 1997).                                                    transformation rates in pilot-scale bioremediation ef-
   Knowledge of toxicity threshold concentrations are             forts in Finland.
key to predicting the transformation potential of PCP                Under anaerobic conditions, but not under aerobic
in soils. For example, in the electron acceptor study,            conditions, NO3", Fe(III), and SO^~ inhibited PCP trans-
PCP concentrations in the methanogenic mineral soils              formation. For most soils, lack of transformation under
PPP (50 p,Af), CR (159 |xM), and TAL (19 p,Af) were               intermediate reducing conditions was probably not due
higher than the threshold EC50(dissoived) (l>«M), which ex-       to toxicity, since both NO3~ and SOi" were consumed
                    D'ANGELO & REDDY: TRANSFORMATIONS OF PENTACHLOROPHENOL IN WETLAND SOILS                            941


in anaerobic treatments (Fig. 2 and 3). These results         Recent discovery of toxaphene-, dieldrin-, and DDT-
are in accordance with the paradigm that denitrifiers,        contaminated soils in this former agricultural field
Fe(III)-reducers, and SO4~-reducers outcompeted de-           (SJRWMD, 1999) suggests the occurrence of cross-accli-
halogenators for common electron-donating substrates          mation by PCP dechlorinators.
(Chang et al., 1996; Fennel and Gossett, 1998). The co-           Several soil properties were highly correlated to PCP
occurrence of reductive dechlorination and SC^" reduc-        transformation rates under methanogenic conditions,
tion in LAAF, W2, and LSM soils, however, may reflect         including total C, N, and P, microbial C, aerobic and
the ability of some dehalogenators to compete effec-          anaerobic C mineralization rates, and bioavailable N
tively with SOI'-reducers for electron equivalents. The       (Table 7). Kuwatsuka and Igarashi (1975) also observed
specific identity of electron donors required for reduc-      high correlations with soil organic matter. Microbial
tive dechlorination of PCP, and comparisons of affinity       C showed the highest correlation probably because it
constants between anaerobic microbial groups, have yet        integrated many of the regulators of transformation
to be determined.                                             (e.g., types and amounts of electron donors and enzyme
    Under methanogenic conditions, PCP transformation         systems) into one measurement. Also, since bacteria are
in eight soils proceeded via reductive dechlorination, in     largely composed of protein, it is plausible that dead
which electrons derived from decomposition of organic         microbial cells served as electron donors for reductive
 matter replaced Cl~ atoms of PCP. One can only specu-        dechlorination by the degrading populations. While ad-
 late about the identity of microbial species and enzymes     dition of nutrients and vitamins did not enhance PCP
responsible for anaerobic PCP transformation in this          transformation in PPP soil under methanogenic condi-
 study. However, enhanced transformation in PPP soil          tions, protein-based donors did, demonstrating the pri-
 in response to the addition of the protein-based electron    mary role that electron donors and microorganisms play
 donors yeast extract and peptone indicated the involve-      in regulating reductive dechlorination.
 ment of proteolytic and amino-acid fermenting bacteria.          Methanogenic soils treated with 2% HgCl2 plus auto-
 Clostridium-like species have previously been impli-         claving did not show reductive dechlorination, indicat-
 cated in dechlorination of CPs (Zhang and Wiegel, 1990;      ing a requirement for biological activity. In the absence
 Madsen and Licht, 1992). These results indicate that         of autoclaving, however, reductive dechlorination pro-
 addition of proteinaceous compounds may be a viable          ceeded, albeit at reduced rates, suggesting that dechlori-
 strategy to enrich for PCP-transforming microorganisms       nation activity can be highly resistant to Hg (II) (Fig. 2).
 and to bioremediate chlorophenol-contaminated soils.         In contrast to methanogenic soils, PCP transformation
     The lag time of 1 to 6 d observed prior to initiation    under aerobic conditions was completely inhibited in
 of dehalogenation was not significantly correlated to        Hg (II) treatments with and without autoclaving, indi-
 any of the soil properties. Lag times have been observed     cating a higher microbial sensitivity to Hg (II). Thus co-
 for other microbial activities, including denitrification,   contamination with Hg and possibly other heavy metals
 SO4~-reduction, and methanogenesis (D'Angelo and             may be an important consideration when formulating
 Reddy, 1999), and reductive dechlorination (Linkfield        remediation protocols.
 et al., 1989). Lag times may be explained by limiting            In conclusion, this study has demonstrated the wide-
 environmental (electron donors and redox potential) or       spread geographic distribution of microorganisms capa-
 biological conditions (populations and induction and         ble of PCP transformation, even in systems with no
 synthesis of enzyme systems) (Linkfield et al., 1989). In    known history of contamination. In addition, this study
 our study, however, the latter likely predominated, in       has shown the extent to which PCP transformations are
 view of the fact that soils were preincubated under de-       regulated by selected factors, including PCP concentra-
 sired reducing conditions well before PCP amendment           tion, types of electron acceptors and donors, and micro-
 to the soils.                                                bial biomass. The relationships between these proper-
     Maximum PCP transformation rates under methano-           ties and transformation processes provided in this study
 genic conditions approached 70 jjimol kg~' d"1, which         may be useful in predicting environmental persistence
  is higher than rates reported for many other soils (Ku-      as a function of site specific conditions, as well as provide
 watsuka and Igarashi, 1975; Chang et al., 1996; Mikesell      insight about potential impediments to in situ transfor-
  and Boyd, 1988). However, these reported rates are           mation. Future studies should focus on identifying mi-
  lower than that observed for a methanogenic PCP-accli-       crobial species involved in transformation, and de-
  mated consortia in bioreactors that transformed 10 ^M        termining how chemical and biological factors influence
 PCP at 44000 ixmol kg^1 d~' (Wu et al., 1993).                their transformation of PCP and other toxic organics
     Most soils that transformed PCP under SO;j~-re-           in soils.
  ducing and methanogenic conditions showed preferen-
  tial ortho and para dechlorination, resulting in the pro-                    ACKNOWLEDGMENTS
  duction of 2,3,4,5-TeCP, 3,4,5-TCP and 3,5-DCP. This
  pathway has been shown to be common in unacclimated            This research was partially financially supported by the
                                                              U.S. Department of Agriculture National Research Initiative
 microbial communities (Nicholsen et al., 1992). In the       Competitive Grants Program. We would like to gratefully
 agricultural LAAF soil, however, additional meta de-         acknowledge the cooperation of several researchers who pro-
 chorination pathways were observed, which confirmed          vided soils used in the study, Drs. E. Roden (Univ. of Ala-
 the pathways previously observed in anaerobic soils          bama), C. Lindau and R. DeLaune (Louisiana State Univ.),
 (Kuwatsuka and Igarashi, 1975; Murthy et al., 1979).         C. Crozier (North Carolina State Univ), J. Richardson (North
942                                        SOIL SCI. SOC. AM. J., VOL. 64, MAY-JUNE 2000


Dakota State Univ.), R. Kadlec (Wetland Management Ser-
vices, MI), and Mr. J.R. White and M.M. Fisher (Univ. of
Florida), and the statistical analysis advice of J.M. Harrison
(Senior Statistician, Univ. of Florida). Florida Agricultural
Experiment Station Journal Series no. R-07269.
                                                 NAY & BORMANN: SOIL CARBON CHANGES                                                    943

Wu, W.M., L. Bhatnagar, and J.G. Zeikus. 1993. Performance of            Zhang, X., and J. Wiegel. 1990. Sequential anaerobic transformation
  anaerobic granules for transformation of pentachlorophenol. Appl.        of 2,4-dichlorophenol in freshwater sediments. Appl. Environ. Mi-
  Environ. Microbiol. 59:389-397.                                          crobiol. 56:1119-1127.
Xun, L., E. Topp, and C.S. Orser. 1992. Purification and characteriza-
  tion of a tetrachloro-p-hydroquinone reductive dehalogenase from
  a Flavobacterium sp. J. Bacteriol. 174:8003-8007.

				
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